Samantha Harris

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• Hessel, A. L., Engels, N. M., Kuehn, M. N., Nissen, D., Sadler, R. L., Ma, W., Irving, T. C., Linke, W. A., & Harris, S. P. (2024). Myosin-binding protein C regulates the sarcomere lattice and stabilizes the OFF states of myosin heads. Nature communications, 15(1), 2628.
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  Muscle contraction is produced via the interaction of myofilaments and is regulated so that muscle performance matches demand. Myosin-binding protein C (MyBP-C) is a long and flexible protein that is tightly bound to the thick filament at its C-terminal end (MyBP-C), but may be loosely bound at its middle- and N-terminal end (MyBP-C) to myosin heads and/or the thin filament. MyBP-C is thought to control muscle contraction via the regulation of myosin motors, as mutations lead to debilitating disease. We use a combination of mechanics and small-angle X-ray diffraction to study the immediate and selective removal of the MyBP-C domains of fast MyBP-C in permeabilized skeletal muscle. We show that cleavage leads to alterations in crossbridge kinetics and passive structural signatures of myofilaments that are indicative of a shift of myosin heads towards the ON state, highlighting the importance of MyBP-C to myofilament force production and regulation.
• Rivas, V. N., Crofton, A. E., Jauregui, C. E., Wouters, J. R., Yang, B. S., Wittenburg, L. A., Kaplan, J. L., Hwee, D. T., Murphy, A. N., Morgan, B. P., Malik, F. I., Harris, S. P., & Stern, J. A. (2024). Cardiac myosin inhibitor, CK-586, minimally reduces systolic function and ameliorates obstruction in feline hypertrophic cardiomyopathy. Scientific reports, 14(1), 12038.
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  Hypertrophic cardiomyopathy (HCM) remains the most common cardiomyopathy in humans and cats with few preclinical pharmacologic interventional studies. Small-molecule sarcomere inhibitors are promising novel therapeutics for the management of obstructive HCM (oHCM) patients and have shown efficacy in left ventricular outflow tract obstruction (LVOTO) relief. The objective of this study was to explore the 6-, 24-, and 48-hour (h) pharmacodynamic effects of the cardiac myosin inhibitor, CK-586, in six purpose-bred cats with naturally occurring oHCM. A blinded, randomized, five-treatment group, crossover preclinical trial was conducted to assess the pharmacodynamic effects of CK-586 in this oHCM model. Dose assessments and select echocardiographic variables were assessed five times over a 48-h period. Treatment with oral CK-586 safely ameliorated LVOTO in oHCM cats. CK-586 treatment dose-dependently eliminated obstruction (reduced LVOTOmaxPG), increased measures of systolic chamber size (LVIDs Sx), and decreased select measures of heart function (LV FS% and LV EF%) in the absence of impact on heart rate. At all tested doses, a single oral CK-586 dose resulted in improved or resolved LVOTO with well-tolerated, dose-dependent, reductions in LV systolic function. The results from this study pave the way for the potential use of CK-586 in both the veterinary and human clinical setting.
• Hessel, A. L., Engels, N. M., Kuehn, M., Nissen, D., Sadler, R. L., Ma, W., Irving, T. C., Linke, W. A., & Harris, S. P. (2023). Myosin-binding protein C forms C-links and stabilizes OFF states of myosin. bioRxiv : the preprint server for biology.
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  Contraction force in muscle is produced by the interaction of myosin motors in the thick filaments and actin in the thin filaments and is fine-tuned by other proteins such as myosin-binding protein C (MyBP-C). One form of control is through the regulation of myosin heads between an ON and OFF state in passive sarcomeres, which leads to their ability or inability to interact with the thin filaments during contraction, respectively. MyBP-C is a flexible and long protein that is tightly bound to the thick filament at its C-terminal end but may be loosely bound at its middle- and N-terminal end (MyBP-C). Under considerable debate is whether the MyBP-C domains directly regulate myosin head ON/OFF states, and/or link thin filaments ("C-links"). Here, we used a combination of mechanics and small-angle X-ray diffraction to study the immediate and selective removal of the MyBP-C domains of fast MyBP-C in permeabilized skeletal muscle. After cleavage, the thin filaments were significantly shorter, a result consistent with direct interactions of MyBP-C with thin filaments thus confirming C-links. Ca sensitivity was reduced at shorter sarcomere lengths, and crossbridge kinetics were increased across sarcomere lengths at submaximal activation levels, demonstrating a role in crossbridge kinetics. Structural signatures of the thick filaments suggest that cleavage also shifted myosin heads towards the ON state - a marker that typically indicates increased Ca sensitivity but that may account for increased crossbridge kinetics at submaximal Ca and/or a change in the force transmission pathway. Taken together, we conclude that MyBP-C domains play an important role in contractile performance which helps explain why mutations in these domains often lead to debilitating diseases.
• Lo, S. T., Li, R. H., Georges, C. J., Nguyen, N., Chen, C. K., Stuhlmann, C., Oldach, M. S., Rivas, V. N., Fousse, S., Harris, S. P., & Stern, J. A. (2023). Synergistic inhibitory effects of clopidogrel and rivaroxaban on platelet function and platelet-dependent thrombin generation in cats. Journal of veterinary internal medicine, 37(4), 1390-1400.
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  Dual antithrombotic treatment (DAT) with clopidogrel and rivaroxaban sometimes is prescribed to cats with hypertrophic cardiomyopathy at risk of thromboembolism. To date, no studies have evaluated their combined effects on platelet function.
• Rivas, V. N., Kaplan, J. L., Kennedy, S. A., Fitzgerald, S., Crofton, A. E., Farrell, A., Grubb, L., Jauregui, C. E., Grigorean, G., Choi, E., Harris, S. P., & Stern, J. A. (2023). Multi-Omic, Histopathologic, and Clinicopathologic Effects of Once-Weekly Oral Rapamycin in a Naturally Occurring Feline Model of Hypertrophic Cardiomyopathy: A Pilot Study. Animals : an open access journal from MDPI, 13(20).
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  Hypertrophic cardiomyopathy (HCM) remains the single most common cardiomyopathy in cats, with a staggering prevalence as high as 15%. To date, little to no direct therapeutical intervention for HCM exists for veterinary patients. A previous study aimed to evaluate the effects of delayed-release (DR) rapamycin dosing in a client-owned population of subclinical, non-obstructive, HCM-affected cats and reported that the drug was well tolerated and resulted in beneficial LV remodeling. However, the precise effects of rapamycin in the hypertrophied myocardium remain unknown. Using a feline research colony with naturally occurring hereditary HCM (n = 9), we embarked on the first-ever pilot study to examine the tissue-, urine-, and plasma-level proteomic and tissue-level transcriptomic effects of an intermittent low dose (0.15 mg/kg) and high dose (0.30 mg/kg) of DR oral rapamycin once weekly. Rapamycin remained safe and well tolerated in cats receiving both doses for eight weeks. Following repeated weekly dosing, transcriptomic differences between the low- and high-dose groups support dose-responsive suppressive effects on myocardial hypertrophy and stimulatory effects on autophagy. Differences in the myocardial proteome between treated and control cats suggest potential anti-coagulant/-thrombotic, cellular remodeling, and metabolic effects of the drug. The results of this study closely recapitulate what is observed in the human literature, and the use of rapamycin in the clinical setting as the first therapeutic agent with disease-modifying effects on HCM remains promising. The results of this study establish the need for future validation efforts that investigate the fine-scale relationship between rapamycin treatment and the most compelling gene expression and protein abundance differences reported here.
• Sharpe, A. N., Oldach, M. S., Kaplan, J. L., Rivas, V., Kovacs, S. L., Hwee, D. T., Morgan, B. P., Malik, F. I., Harris, S. P., & Stern, J. A. (2023). Pharmacokinetics of a single dose of Aficamten (CK-274) on cardiac contractility in a A31P MYBPC3 hypertrophic cardiomyopathy cat model. Journal of veterinary pharmacology and therapeutics, 46(1), 52-61.
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  Hypertrophic cardiomyopathy (HCM) is the most prevalent cardiac disease in cats and lacks efficacious preclinical pharmacologic intervention, prompting investigation of novel therapies. Genetic mutations encoding sarcomeric proteins are implicated in the development of HCM and small molecule myosin inhibitors are an emerging class of therapeutics designed to target the interaction of actin and myosin to alleviate the detrimental effects of inappropriate contractile protein interactions. The purpose of this study was to characterize the pharmacodynamic effects of a single oral dose of the novel cardiac myosin inhibitor aficamten (CK-274) on cardiac function in purpose bred cats with naturally occurring A31P MYBPC3 mutation and a clinical diagnosis of HCM with left ventricular outflow tract obstruction (LVOTO). Five purpose bred cats were treated with aficamten (2 mg/kg) or vehicle and echocardiographic evaluations were performed at 0, 6, 24, and 48 h post-dosing. High dose aficamten (2 mg/kg) reduced left ventricular fractional shortening (LVFS%) by increasing the LV systolic internal dimension (LVIDs) and reduced isovolumic relaxation time (IVRT) compared with baseline without significant adverse effects. The marked reduction in systolic function and reduced IVRT coupled with an increased heart rate in treated cats, suggest a lower dose may be optimal. Further studies to determine optimal dosing of aficamten are indicated.
• Sharpe, A. N., Oldach, M. S., Rivas, V. N., Kaplan, J. L., Walker, A. L., Kovacs, S. L., Hwee, D. T., Cremin, P., Morgan, B. P., Malik, F. I., Harris, S. P., & Stern, J. A. (2023). Effects of Aficamten on cardiac contractility in a feline translational model of hypertrophic cardiomyopathy. Scientific reports, 13(1), 32.
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  Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited cardiac disease in humans and cats and lacks efficacious pharmacologic interventions in the preclinical phase of disease. LV outflow tract obstruction (LVOTO) is commonly observed in HCM-affected patients and is a primary driver of heart failure symptoms and reduced quality of life. Novel small-molecule cardiac myosin inhibitors target actin-myosin interactions to alleviate overactive protein interactions. A prospective, randomized, controlled cross-over study was performed to evaluate pharmacodynamic effects of two doses (0.3 and 1 mg/kg) of a next-in-class cardiac myosin inhibitor, aficamten (CK-3773274, CK-274), on cardiac function in cats with the A31P MYBPC3 mutation and oHCM. Dose-dependent reductions in LV systolic function, LVOT pressure gradient, and isovolumetric relaxation times compared to baseline were observed. Promising beneficial effects of reduced systolic function warrant further studies of this next-in-class therapeutic to evaluate the benefit of long-term administration in this patient population.
• Stern, J. A., Rivas, V. N., Kaplan, J. L., Ueda, Y., Oldach, M. S., Ontiveros, E. S., Kooiker, K. B., van Dijk, S. J., & Harris, S. P. (2023). Hypertrophic cardiomyopathy in purpose-bred cats with the A31P mutation in cardiac myosin binding protein-C. Scientific reports, 13(1), 10319.
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  We sought to establish a large animal model of inherited hypertrophic cardiomyopathy (HCM) with sufficient disease severity and early penetrance for identification of novel therapeutic strategies. HCM is the most common inherited cardiac disorder affecting 1 in 250-500 people, yet few therapies for its treatment or prevention are available. A research colony of purpose-bred cats carrying the A31P mutation in MYBPC3 was founded using sperm from a single heterozygous male cat. Cardiac function in four generations was assessed by periodic echocardiography and measurement of blood biomarkers. Results showed that HCM penetrance was age-dependent, and that penetrance occurred earlier and was more severe in successive generations, especially in homozygotes. Homozygosity was also associated with progression from preclinical to clinical disease. A31P homozygous cats represent a heritable model of HCM with early disease penetrance and a severe phenotype necessary for interventional studies aimed at altering disease progression. The occurrence of a more severe phenotype in later generations of cats, and the occasional occurrence of HCM in wildtype cats suggests the presence of at least one gene modifier or a second causal variant in this research colony that exacerbates the HCM phenotype when inherited in combination with the A31P mutation.
• Papadaki, M., Kampaengsri, T., Barrick, S. K., Campbell, S. G., von Lewinski, D., Rainer, P. P., Harris, S. P., Greenberg, M. J., & Kirk, J. A. (2022). Myofilament glycation in diabetes reduces contractility by inhibiting tropomyosin movement, is rescued by cMyBPC domains. Journal of molecular and cellular cardiology, 162, 1-9.
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  Diabetes doubles the risk of developing heart failure (HF). As the prevalence of diabetes grows, so will HF unless the mechanisms connecting these diseases can be identified. Methylglyoxal (MG) is a glycolysis by-product that forms irreversible modifications on lysine and arginine, called glycation. We previously found that myofilament MG glycation causes sarcomere contractile dysfunction and is increased in patients with diabetes and HF. The aim of this study was to discover the molecular mechanisms by which MG glycation of myofilament proteins cause sarcomere dysfunction and to identify therapeutic avenues to compensate. In humans with type 2 diabetes without HF, we found increased glycation of sarcomeric actin compared to non-diabetics and it correlated with decreased calcium sensitivity. Depressed calcium sensitivity is pathogenic for HF, therefore myofilament glycation represents a promising therapeutic target to inhibit the development of HF in diabetics. To identify possible therapeutic targets, we further defined the molecular actions of myofilament glycation. Skinned myocytes exposed to 100 μM MG exhibited decreased calcium sensitivity, maximal calcium-activated force, and crossbridge kinetics. Replicating MG's functional affects using a computer simulation of sarcomere function predicted simultaneous decreases in tropomyosin's blocked-to-closed rate transition and crossbridge duty cycle were consistent with all experimental findings. Stopped-flow experiments and ATPase activity confirmed MG decreased the blocked-to-closed transition rate. Currently, no therapeutics target tropomyosin, so as proof-of-principal, we used a n-terminal peptide of myosin-binding protein C, previously shown to alter tropomyosin's position on actin. C0C2 completely rescued MG-induced calcium desensitization, suggesting a possible treatment for diabetic HF.
• Risi, C. M., Villanueva, E., Belknap, B., Sadler, R. L., Harris, S. P., White, H. D., & Galkin, V. E. (2022). Cryo-Electron Microscopy Reveals Cardiac Myosin Binding Protein-C M-Domain Interactions with the Thin Filament. Journal of molecular biology, 434(24), 167879.
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  Cardiac myosin binding protein C (cMyBP-C) modulates cardiac contraction via direct interactions with cardiac thick (myosin) and thin (actin) filaments (cTFs). While its C-terminal domains (e.g. C8-C10) anchor cMyBP-C to the backbone of the thick filament, its N-terminal domains (NTDs) (e.g. C0, C1, M, and C2) bind to both myosin and actin to accomplish its dual roles of inhibiting thick filaments and activating cTFs. While the positions of C0, C1 and C2 on cTF have been reported, the binding site of the M-domain on the surface of the cTF is unknown. Here, we used cryo-EM to reveal that the M-domain interacts with actin via helix 3 of its ordered tri-helix bundle region, while the unstructured part of the M-domain does not maintain extensive interactions with actin. We combined the recently obtained structure of the cTF with the positions of all the four NTDs on its surface to propose a complete model of the NTD binding to the cTF. The model predicts that the interactions of the NTDs with the cTF depend on the activation state of the cTF. At the peak of systole, when bound to the extensively activated cTF, NTDs would inhibit actomyosin interactions. In contrast, at falling Ca levels, NTDs would not compete with the myosin heads for binding to the cTF, but would rather promote formation of active cross-bridges at the adjacent regulatory units located at the opposite cTF strand. Our structural data provides a testable model of the cTF regulation by the cMyBP-C.
• Walker, A. L., Ueda, Y., Crofton, A. E., Harris, S. P., & Stern, J. A. (2022). Ambulatory electrocardiography, heart rate variability, and pharmacologic stress testing in cats with subclinical hypertrophic cardiomyopathy. Scientific reports, 12(1), 1963.
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  The utility of ambulatory electrocardiography (AECG) to evaluate cats with subclinical hypertrophic cardiomyopathy (HCM) for arrhythmias and heart rate variability (HRV) is not well defined but may provide information regarding risk stratification. This prospective study used AECG to evaluate ectopy and HRV in subclinical HCM cats compared to healthy controls and is the first to implement a pharmacologic cardiac stress test. Twenty-three purpose-bred, Maine coon cross cats (16 HCM, 7 control) underwent 48-h of continuous AECG. Terbutaline (0.2-0.3 mg/kg) was administered orally at 24 and 36 h. Heart rate, ectopy frequency and complexity and HRV parameters, including standard deviation of normal R-R intervals (SDNN), were compared pre-terbutaline and post-terbutaline and across phenotype, genotype and sex. Genotype for an HCM-causative mutation was significantly associated with the frequency of supraventricular (P = 0.033) and ventricular (P = 0.026) ectopy across all cats. Seven HCM cats and zero healthy cats had a sinus arrhythmia. Mean heart rate was significantly higher post-terbutaline (p 
• Harris, S. P. (2021). Making waves: A proposed new role for myosin-binding protein C in regulating oscillatory contractions in vertebrate striated muscle. The Journal of general physiology, 153(3).
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  Myosin-binding protein C (MyBP-C) is a critical regulator of muscle performance that was first identified through its strong binding interactions with myosin, the force-generating protein of muscle. Almost simultaneously with its discovery, MyBP-C was soon found to bind to actin, the physiological catalyst for myosin's activity. However, the two observations posed an apparent paradox, in part because interactions of MyBP-C with myosin were on the thick filament, whereas MyBP-C interactions with actin were on the thin filament. Despite the intervening decades since these initial discoveries, it is only recently that the dual binding modes of MyBP-C are becoming reconciled in models that place MyBP-C at a central position between actin and myosin, where MyBP-C alternately stabilizes a newly discovered super-relaxed state (SRX) of myosin on thick filaments in resting muscle and then prolongs the "on" state of actin on thin filaments in active muscle. Recognition of these dual, alternating functions of MyBP-C reveals how it is central to the regulation of both muscle contraction and relaxation. The purpose of this Viewpoint is to briefly summarize the roles of MyBP-C in binding to myosin and actin and then to highlight a possible new role for MyBP-C in inducing and damping oscillatory waves of contraction and relaxation. Because the contractile waves bear similarity to cycles of contraction and relaxation in insect flight muscles, which evolved for fast, energetically efficient contraction, the ability of MyBP-C to damp so-called spontaneous oscillatory contractions (SPOCs) has broad implications for previously unrecognized regulatory mechanisms in vertebrate striated muscle. While the molecular mechanisms by which MyBP-C can function as a wave maker or a wave breaker are just beginning to be explored, it is likely that MyBP-C dual interactions with both myosin and actin will continue to be important for understanding the new functions of this enigmatic protein.
• Harris, S., Langlais, P. R., Granger, K., Napierski, N., Touma, K., Moran, H., & Strom, J. (2020). A novel “cut and paste” method for in situ replacement of cMyBP-C reveals a new role for cMyBP-C in the regulation of contractile oscillations. Circulation Research.
• Harris, S., Touma, K., Strom, J., Moran, H. R., Langlais, P. R., Granger, K., & Napierski, N. C. (2020). A novel “cut and paste” method for in situ replacement of cMyBP-C reveals a new role for cMyBP-C in the regulation of contractile oscillations. Circulation Research.
• Risi, C. M., Patra, M., Belknap, B., Harris, S. P., White, H. D., & Galkin, V. E. (2021). Interaction of the C2 Ig-like Domain of Cardiac Myosin Binding Protein-C with F-actin. Journal of molecular biology, 433(19), 167178.
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  Cardiac muscle contraction depends on interactions between thick (myosin) and thin (actin) filaments (TFs). TFs are regulated by intracellular Ca levels. Under activating conditions Ca binds to the troponin complex and displaces tropomyosin from myosin binding sites on the TF surface to allow actomyosin interactions. Recent studies have shown that in addition to Ca, the first four N-terminal domains (NTDs) of cardiac myosin binding protein C (cMyBP-C) (e.g. C0, C1, M and C2), are potent modulators of the TF activity, but the mechanism of their collective action is poorly understood. Previously, we showed that C1 activates the TF at low Ca and C0 stabilizes binding of C1 to the TF, but the ability of C2 to bind and/or affect the TF remains unknown. Here we obtained 7.5 Å resolution cryo-EM reconstruction of C2-decorated actin filaments to demonstrate that C2 binds to actin in a single structural mode that does not activate the TF unlike the polymorphic binding of C0 and C1 to actin. Comparison of amino acid sequences of C2 with either C0 or C1 shows low levels of identity between the residues involved in interactions with the TF but high levels of conservation for residues involved in Ig fold stabilization. This provides a structural basis for strikingly different interactions of structurally homologous C0, C1 and C2 with the TF. Our detailed analysis of the interaction of C2 with the actin filament provides crucial information required to model the collective action of cMyBP-C NTDs on the cardiac TF.
• Harris, S., Langlais, P. R., Granger, K., Napierski, N., Touma, K., Moran, H., & Strom, J. (2020). A novel “cut and paste” method for in situ replacement of cMyBP-C reveals a new role for cMyBP-C in the regulation of contractile oscillations. Circulation Research. doi:10.1161/CIRCRESAHA.119.315760
• Napierski, N. C., Granger, K., Langlais, P. R., Moran, H. R., Strom, J., Touma, K., & Harris, S. P. (2020). A Novel "Cut and Paste" Method for In Situ Replacement of cMyBP-C Reveals a New Role for cMyBP-C in the Regulation of Contractile Oscillations. Circulation research, 126(6), 737-749.
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  cMyBP-C (cardiac myosin-binding protein-C) is a critical regulator of heart contraction, but the mechanisms by which cMyBP-C affects actin and myosin are only partly understood. A primary obstacle is that cMyBP-C localization on thick filaments may be a key factor defining its interactions, but most in vitro studies cannot duplicate the unique spatial arrangement of cMyBP-C within the sarcomere.
• Napierski, N. C., Harris, S. P., & Granger, K. (2020). Selective Phosphorylation of cMyBP-C Increases Cross-Bridge Cycling Rates in Permeabilized Cardiomyocytes From SPY-C Mice. Biophysical Journal, 118(3), 591a-592a. doi:10.1016/j.bpj.2019.11.3204
• P, M. B., Stern, J. A., Stern, J. A., P, M. B., Ontiveros, E. S., Ontiveros, E. S., Oldach, M. S., Oldach, M. S., Morgan, B. P., Morgan, B. P., Malik, F. I., Malik, F. I., Hwee, D. T., Hwee, D. T., Harris, S. P., Harris, S. P., Gonzalez, C. E., Gonzalez, C. E., Fousse, S. L., & Fousse, S. L. (2020). Abstract 14254: Pharmacodynamic Effects of a Single Dose of CK-3773274 in Cats With Hypertrophic Cardiomyopathy. Circulation, 142(Suppl_3). doi:10.1161/circ.142.suppl_3.14254
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  Introduction: Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular (LV) hypertrophy in the absence of known causes. HCM can be due to genetic mutations that affect sarcomeric prot...
• Ueda, Y., Ueda, Y., Stern, J. A., Stern, J. A., Rio, C. L., Rio, C. L., Ontiveros, E. S., Ontiveros, E. S., Oldach, O., Oldach, M. S., Oldach, M. S., Napierski, N. C., Napierski, N. C., Harris, S. P., Ferguson, B. S., Harris, S. P., Ferguson, F., & Ferguson, B. S. (2020). Acute effects of a mavacamten-like myosin-inhibitor (MYK-581 in a feline model of obstructed hypertrophic cardiomyopathy: evidence of improved ventricular filling (beyond obstruction reprieve). European Heart Journal, 41. doi:10.1093/ehjci/ehaa946.3713
• Harris, S. P. (2019). Acute Loss of cMyBP-C Induces Auto-Oscillatory Contractions in Permeabilized Cardiomyocytes: Implications for Reverse E-C Coupling?. Biophysical Journal, 116(3), 461a-462a. doi:10.1016/j.bpj.2018.11.2493
• Harris, S. P., & de Tombe, P. P. (2019). Sarcomeric mutations in cardiac diseases. Pflugers Archiv : European journal of physiology, 471(5), 659-660.
• Oldach, M. S., Ueda, Y., Ontiveros, E. S., Fousse, S. L., Harris, S. P., & Stern, J. A. (2019). Cardiac Effects of a Single Dose of Pimobendan in Cats With Hypertrophic Cardiomyopathy; A Randomized, Placebo-Controlled, Crossover Study. Frontiers in veterinary science, 6, 15.
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  Pimobendan has been shown to impart a significant survival benefit in cardiomyopathic cats who receive it as part of heart failure therapy. However, use of pimobendan remains controversial in cats with hypertrophic cardiomyopathy (HCM) due to lack of pharmacodynamic data for pimobendan in cats with HCM and due to theoretical concerns for exacerbating left ventricular outflow tract obstructions. Our objective was to investigate the cardiac effects of pimobendan in cats with HCM. We hypothesized that pimobendan would not exacerbate left ventricular outflow tract obstructions and that it would improve echocardiographic measures of diastolic function. Thirteen purpose-bred cats were studied from a research colony with naturally-occurring HCM due to a variant in myosin binding protein C. Cats underwent two examinations 24 h apart with complete standard echocardiography. On their first day of evaluation, they were randomized to receive oral placebo or 1.25 mg pimobendan 1 h prior to exam. On their second examination, they were crossed over and received the remaining treatment. Investigators were blinded to all treatments. The pimobendan group had a significant increase in left atrial fractional shortening (pimobendan group 41.7% ± 5.9; placebo group 36.1% ± 6.0; = 0.04). There was no significant difference in left ventricular outflow tract (LVOT) velocities between the groups (pimobendan group 2.8 m/s ± 0.8; placebo group 2.6 m/s ± 1.0). There were no significant differences between the number of cats with LVOT obstructions between groups (12 in pimobendan group; 11 in placebo group; = 1.00). There were no detectable differences in any systolic measures, including left ventricular fractional shortening, mitral annular plane systolic excursion, and tricuspid annular plane systolic excursion. Doppler-based diastolic function assessment was precluded by persistent tachycardia. Improved left atrial function in the pimobendan group could explain some of the reported survival benefit for HCM cats in CHF. Pimobendan did not exacerbate LVOT obstructions and thus may not be contraindicated in HCM cats with LVOT obstructions. Future studies are needed to better characterize other physiologic effects, particularly regarding diastolic function assessment, and to better assess safety of pimobendan over a longer time-course.
• Ueda, Y., Stern, J. A., Ontiveros, E. S., & Harris, S. P. (2019). Precision medicine validation: identifying the MYBPC3 A31P variant with whole-genome sequencing in two Maine Coon cats with hypertrophic cardiomyopathy.. Journal of feline medicine and surgery, 21(12), 1086-1093. doi:10.1177/1098612x18816460
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  The objective of this study was to perform a proof-of-concept experiment that validates a precision medicine approach to identify variants associated with hypertrophic cardiomyopathy (HCM). We hypothesized that whole-genome sequencing would identify variant(s) associated with HCM in two affected Maine Coon/Maine Coon cross cats when compared with 79 controls of various breeds..Two affected and two control Maine Coon/Maine Coon cross cats had whole-genome sequencing performed at approximately × 30 coverage. Variants were called in these four cats and 77 cats of various breeds as part of the 99 Lives Cat Genome Sequencing Initiative ( http://felinegenetics.missouri.edu/99lives ) using Platypus v0.7.9.1, annotated with dbSNP ID, and variants' effect predicted by SnpEff. Strict filtering criteria (alternate allele frequency >49%) were applied to identify homozygous-alternate or heterozygous variants in the two HCM-affected samples when compared with 79 controls of various breeds..A total of four variants were identified in the two Maine Coon/Maine Coon cross cats with HCM when compared with 79 controls after strict filtering. Three of the variants identified in genes MFSD12, BTN1A1 and SLITRK5 did not segregate with disease in a separate cohort of seven HCM-affected and five control Maine Coon/Maine Coon cross cats. The remaining variant MYBPC3 segregated with disease status. Furthermore, this gene was previously associated with heart disease and encodes for a protein with sarcomeric function..This proof-of-concept experiment identified the previously reported MYBPC3 A31P Maine Coon variant in two HCM-affected cases. This result validates and highlights the power of whole-genome sequencing for feline precision medicine.
• White, H. D., Risi, C., Harris, S. P., Galkin, V. E., & Belknap, B. (2019). C0 Ig-Domain of Cardiac Myosin Binding Protein-C Interacts with the Regulatory Light Chain of Myosin-S1 Bound to the Native Cardiac Thin Filament. Biophysical Journal, 116(3), 116a-117a. doi:10.1016/j.bpj.2018.11.655
• Ontiveros, E. S., Ueda, Y., Harris, S. P., Stern, J. A., & , 9. L. (2018). Precision medicine validation: identifying the MYBPC3 A31P variant with whole-genome sequencing in two Maine Coon cats with hypertrophic cardiomyopathy. Journal of feline medicine and surgery, 1098612X18816460.
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  The objective of this study was to perform a proof-of-concept experiment that validates a precision medicine approach to identify variants associated with hypertrophic cardiomyopathy (HCM). We hypothesized that whole-genome sequencing would identify variant(s) associated with HCM in two affected Maine Coon/Maine Coon cross cats when compared with 79 controls of various breeds.
• Risi, C., Belknap, B., Forgacs-Lonart, E., Harris, S. P., Schröder, G. F., White, H. D., & Galkin, V. E. (2018). N-Terminal Domains of Cardiac Myosin Binding Protein C Cooperatively Activate the Thin Filament. Structure (London, England : 1993), 26(12), 1604-1611.e4.
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  Muscle contraction relies on interaction between myosin-based thick filaments and actin-based thin filaments. Myosin binding protein C (MyBP-C) is a key regulator of actomyosin interactions. Recent studies established that the N'-terminal domains (NTDs) of MyBP-C can either activate or inhibit thin filaments, but the mechanism of their collective action is poorly understood. Cardiac MyBP-C (cMyBP-C) harbors an extra NTD, which is absent in skeletal isoforms of MyBP-C, and its role in regulation of cardiac contraction is unknown. Here we show that the first two domains of human cMyPB-C (i.e., C0 and C1) cooperate to activate the thin filament. We demonstrate that C1 interacts with tropomyosin via a positively charged loop and that this interaction, stabilized by the C0 domain, is required for thin filament activation by cMyBP-C. Our data reveal a mechanism by which cMyBP-C can modulate cardiac contraction and demonstrate a function of the C0 domain.
• Touma, K., Strom, J., Touma, K. D., Strom, J., Harris, S. P., & Dijk, S. J. (2018). In Situ Replacement of cMyBP-C N'-Terminal Domains using the Novel Spy-C Method. Biophysical Journal, 114(3), 549a-550a. doi:10.1016/j.bpj.2017.11.3002
• White, H. D., Schroder, G. F., Risi, C., Harris, S. P., Glendrange, T., Galkin, V. E., & Belknap, B. (2018). C1 IG-Domain of Myosin Binding Protein-C Activates Cardiac Thin Filament by Means of Thethering Tropomyosin to the Subdomain-1 of Actin. Biophysical Journal, 114(3), 145a-146a. doi:10.1016/j.bpj.2017.11.817
• van Dijk, S. J., Kooiker, K. B., Napierski, N. C., Touma, K. D., Mazzalupo, S., & Harris, S. P. (2018). Point mutations in the tri-helix bundle of the M-domain of cardiac myosin binding protein-C influence systolic duration and delay cardiac relaxation. Journal of molecular and cellular cardiology, 119, 116-124.
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  Cardiac myosin binding protein-C (cMyBP-C) is an essential regulatory protein required for proper systolic contraction and diastolic relaxation. We previously showed that N'-terminal domains of cMyBP-C stimulate contraction by binding to actin and activating the thin filament in vitro. In principle, thin filament activating effects of cMyBP-C could influence contraction and relaxation rates, or augment force amplitude in vivo. cMyBP-C binding to actin could also contribute to an internal load that slows muscle shortening velocity as previously hypothesized. However, the functional significance of cMyBP-C binding to actin has not yet been established in vivo. We previously identified an actin binding site in the regulatory M-domain of cMyBP-C and described two missense mutations that either increased (L348P) or decreased (E330K) binding affinity of recombinant cMyBP-C N'-terminal domains for actin in vitro. Here we created transgenic mice with either the L348P or E330K mutations to determine the functional significance of cMyBP-C binding to actin in vivo. Results showed that enhanced binding of cMyBP-C to actin in L348P-Tg mice prolonged the time to end-systole and slowed relaxation rates. Reduced interactions between cMyBP-C and actin in E330K-Tg mice had the opposite effect and significantly shortened the duration of ejection. Neither mouse model displayed overt systolic dysfunction, but L348P-Tg mice showed diastolic dysfunction presumably resulting from delayed relaxation. We conclude that cMyBP-C binding to actin contributes to sustained thin filament activation at the end of systole and during isovolumetric relaxation. These results provide the first functional evidence that cMyBP-C interactions with actin influence cardiac function in vivo.
• Dijk, S. J., Strom, J., Strom, J., Harris, S. P., & Dijk, S. J. (2017). Reduced Binding of the M-Domain of Cardiac Myosin Binding Protein C to Actin Impairs the Ability of the Heart to Respond to Chronic Stress. Biophysical Journal, 112(3), 5-7. doi:10.1016/j.bpj.2016.11.3004
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  Cardiac myosin binding protein C (cMyBP-C) is a sarcomeric protein that regulates cross bridge cycling to control timing and strength of cardiac contraction. However, the molecular mechanisms by which cMyBP-C influences cross bridge cycling are incompletely understood. The M-domain of cMyBP-C can interact with actin and myosin, and phosphorylation of cMyBP-C in response to β-adrenergic stimulation during cardiac stress diminishes its affinity for both.In this study we determined the functional relevance of cMyBP-C binding to actin on cardiac function. Binding of cMyBP-C to actin can activate the thin filament and we hypothesize that dissociation of cMyBP-C from actin is essential for cardiac relaxation. To test our hypothesis, we created a transgenic mouse model with a mutation in the M-domain of cMyBP-C (E330K) that reduced binding affinity for actin in vitro. We then used echocardiography and pressure-volume recordings to assess cardiac function under sedentary and stress conditions.Both systolic function (e.g. stroke volume, ejection fraction and maximal rate of pressure development (+dP/dt)) and diastolic function (e.g. –dP/dt, isovolumetric relaxation time, E/A) were normal in E330K-Tg mice under sedentary conditions. Acute β-adrenergic stimulation with isoprenaline also led to normal increases in both contractility and relaxation in E330K-Tg mice. However, chronic cardiac stress by increasing preload to the heart by aortocaval fistula was fatal to all E330K-Tg mice within 10 days after surgery.Our results demonstrate that reduced interaction between the M-domain of cMyBP-C and actin did not impair cardiac function in E330K-Tg mice under sedentary conditions, but that this interaction may be essential for the heart to cope with chronic volume overload.This work is supported by NIH HL-080367 and AHA 15POST25700403.
• McNamara, J. W., Li, A., Lal, S., Bos, J. M., Harris, S. P., van der Velden, J., Ackerman, M. J., Cooke, R., & Dos Remedios, C. G. (2017). MYBPC3 mutations are associated with a reduced super-relaxed state in patients with hypertrophic cardiomyopathy. PloS one, 12(6), e0180064.
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  The "super-relaxed state" (SRX) of myosin represents a 'reserve' of motors in the heart. Myosin heads in the SRX are bound to the thick filament and have a very low ATPase rate. Changes in the SRX are likely to modulate cardiac contractility. We previously demonstrated that the SRX is significantly reduced in mouse cardiomyocytes lacking cardiac myosin binding protein-C (cMyBP-C). Here, we report the effect of mutations in the cMyBP-C gene (MYBPC3) using samples from human patients with hypertrophic cardiomyopathy (HCM). Left ventricular (LV) samples from 11 HCM patients were obtained following myectomy surgery to relieve LV outflow tract obstruction. HCM samples were genotyped as either MYBPC3 mutation positive (MYBPC3mut) or negative (HCMsmn) and were compared to eight non-failing donor hearts. Compared to donors, only MYBPC3mut samples display a significantly diminished SRX, characterised by a decrease in both the number of myosin heads in the SRX and the lifetime of ATP turnover. These changes were not observed in HCMsmn samples. There was a positive correlation (p < 0.01) between the expression of cMyBP-C and the proportion of myosin heads in the SRX state, suggesting cMyBP-C modulates and maintains the SRX. Phosphorylation of the myosin regulatory light chain in MYBPC3mut samples was significantly decreased compared to the other groups, suggesting a potential mechanism to compensate for the diminished SRX. We conclude that by altering both contractility and sarcomeric energy requirements, a reduced SRX may be an important disease mechanism in patients with MYBPC3 mutations.
• White, H. D., Virok, B., Harris, S. P., & Galkin, V. E. (2017). Activation and Inhibition of Cardiac Thin Filaments by Single and Multiple Domains Constructs of Human Cardiac Myosin Binding Protein-C (cMyBP-C) at Low Calcium. Biophysical Journal, 112(3), 257a-258a. doi:10.1016/j.bpj.2016.11.1402
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  We have previously shown that the C1 domain of human cardiac myosin binding protein-C (c-MyBP-C) produces a biphasic regulation of cardiac thin filaments at low calcium concentrations. Increasing [C1] first produces activation followed by inhibition of myosin-S1 ATP hydrolysis by cardiac thin filaments at higher [C1]. In contrast, domains C0 and C2 only inhibit and do not activate ATP hydrolysis at any concentration. N’-terminal proteins containing the C1 domain along with C0 or C2 (e.g., C0-PA-C1, C1-M-C2 and C0-PA-C1-M-C2) show a similar pattern of activation and inhibition of ATP hydrolysis at low calcium concentration as the C1 domain alone, but the concentrations required for maximum activation and inhibition are ∼10 fold lower than required using C1 alone.
• Wriggers, W. R., White, H. D., Virok, B., Risi, C., Kovacs, J. A., Harris, S. P., Galkin, V. E., & Eisner, J. (2017). C0 Domain of Cardiac Myosin Binding Protein-C Modulates Interaction of the Neighboring C1 Domain with Tropomyosin Through the Allosteric Interaction with F-Actin. Biophysical Journal, 112(3), 561a. doi:10.1016/j.bpj.2016.11.3023
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  Mutations in cardiac myosin binding protein C (cMyBP-C) are the most common cause of hypertrophic cardiomyopathy affecting millions of people worldwide. Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown. Recently, we demonstrated that despite being structural homologs, C0 and C1 NTDs exhibit different patterns of binding on the surface of F-actin. Moreover, we showed that C1 but not C0 can activate the TF by means of shifting the tropomyosin (Tm) cable from the “closed” to the “open” structural state via direct interaction with Tm. Here we used cryo electron microscopy and image analysis to reveal how C0 and C1 Ig-domains connected by the Proline/Alanine-linker communicate with each other when interacting with the TF in tandem. Our results show that C0 binds to the front of actin subdomain-1 in two distinct modes, and that in these two modes C0 has different effect on the interaction of C1 domain with both actin filament and Tm cable. In one mode C0 enhances the activating effect of C1 domain so that the Tm cable is shifted towards the subdomain-4 of actin to an even larger extent than found in the “open” state of Tm. In the second mode C0 reverses the activation effect of C1 by means of disrupting the interaction of C1 with Tm. Importantly, we demonstrate that the two domains communicate with each other allosterically through the actin filament. Our data suggest that the internal dynamics of the actin molecule is essential for the regulation of the actomyosin interaction in cardiac muscle. We propose a mechanism by which cMyBP-C may modulate actomyosin interactions in cardiac muscle.
• Yang, Y., White, H. D., Hoye, E., Harris, S. P., Belknap, B., & Azimi, V. (2017). Abstract 21304: Calmodulin Modulates the Functional Effects of cMyBP-C on the Thin Filament. Circulation.
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  Introduction: Cardiac myosin binding protein-C (cMyBP-C) is an essential regulator of heart muscle function that is necessary for both normal contraction and for increased contractility in response...
• Harris, S. P., Belknap, B., Van Sciver, R. E., White, H. D., & Galkin, V. E. (2016). C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation. Proceedings of the National Academy of Sciences of the United States of America, 113(6), 1558-63.
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  Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca(2+) have been achieved. We suggest that Ca(2+) modulates the interaction of cMyBP-C with the TF in the sarcomere.
• Kolb, J., Li, F., Methawasin, M., Adler, M., Escobar, Y., Nedrud, J., Pappas, C. T., Harris, S. P., & Granzier, H. (2016). Thin filament length in the cardiac sarcomere varies with sarcomere length but is independent of titin and nebulin. Journal of molecular and cellular cardiology, 97, 286-94.
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  Thin filament length (TFL) is an important determinant of the force-sarcomere length (SL) relation of cardiac muscle. However, the various mechanisms that control TFL are not well understood. Here we tested the previously proposed hypothesis that the actin-binding protein nebulin contributes to TFL regulation in the heart by using a cardiac-specific nebulin cKO mouse model (αMHC Cre Neb cKO). Atrial myocytes were studied because nebulin expression has been reported to be most prominent in this cell type. TFL was measured in right and left atrial myocytes using deconvolution optical microscopy and staining for filamentous actin with phalloidin and for the thin filament pointed-end with an antibody to the capping protein Tropomodulin-1 (Tmod1). Results showed that TFLs in Neb cKO and littermate control mice were not different. Thus, deletion of nebulin in the heart does not alter TFL. However, TFL was found to be ~0.05μm longer in the right than in the left atrium and Tmod1 expression was increased in the right atrium. We also tested the hypothesis that the length of titin's spring region is a factor controlling TFL by studying the Rbm20(ΔRRM) mouse which expresses titins that are ~500kDa (heterozygous mice) and ~1000kDa (homozygous mice) longer than in control mice. Results revealed that TFL was not different in Rbm20(ΔRRM) mice. An unexpected finding in all genotypes studied was that TFL increased as sarcomeres were stretched (~0.1μm per 0.35μm of SL increase). This apparent increase in TFL reached a maximum at a SL of ~3.0μm where TFL was ~1.05μm. The SL dependence of TFL was independent of chemical fixation or the presence of cardiac myosin-binding protein C (cMyBP-C). In summary, we found that in cardiac myocytes TFL varies with SL in a manner that is independent of the size of titin or the presence of nebulin.
• Kooiker, K. B., Dijk, S. J., Kooiker, K. B., Harris, S. P., Dijk, S. J., Harris, S. P., & Dijk, S. J. (2016). Abstract 85: Interactions Between Cardiac Myosin Binding Protein C and Actin Contribute to the Regulation of Cardiac Contraction. Circulation Research, 119(suppl_1). doi:10.1161/res.119.suppl_1.85
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  Cardiac myosin binding protein C (cMyBP-C) is a regulatory muscle protein that is essential for proper cardiac contraction, and mutations in cMyBP-C are commonly associated with hypertrophic cardiomyopathy. cMyBP-C not only interacts with myosin, but with actin as well. In vitro studies have demonstrated that multiple functions of cMyBP-C could readily be explained by an interaction between cMyBP-C and actin, but the in vivo significance of cMyBP-C binding to either myosin or actin is not well understood. Here we created transgenic mice with a single point mutation (L348P) in a key binding domain of cMyBP-C that enhances the binding affinity of cMyBP-C for actin in vitro (Bezold et al , JBC 2013) to gain insights into the relevance of cMyBP-C binding to actin in working hearts. Echocardiograms from 3 month old male L348P-Tg mice (N=23) and non-transgenic (nTg, N=17) controls were used to assess systolic and diastolic function. Results showed significantly prolonged isovolumetric relaxation time (L348P-Tg: 18.5±0.6 vs nTg: 10.2±0.3 ms) and slower movement of the mitral valve annulus (E’: -17.2±1.5 vs -32.2±1.6 and A’: -9.1±1.7 vs -17.6±1.0 mm/s, p
• Li, R. H., Stern, J. A., Ho, V., Tablin, F., & Harris, S. P. (2016). Platelet Activation and Clopidogrel Effects on ADP-Induced Platelet Activation in Cats with or without the A31P Mutation in MYBPC3. Journal of veterinary internal medicine, 30(5), 1619-1629.
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  Clopidogrel is commonly prescribed to cats with perceived increased risk of thromboembolic events, but little information exists regarding its antiplatelet effects.
• McNamara, J. W., Li, A., Smith, N. J., Lal, S., Graham, R. M., Kooiker, K. B., van Dijk, S. J., Remedios, C. G., Harris, S. P., & Cooke, R. (2016). Ablation of cardiac myosin binding protein-C disrupts the super-relaxed state of myosin in murine cardiomyocytes. Journal of molecular and cellular cardiology, 94, 65-71.
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  Cardiac myosin binding protein-C (cMyBP-C) is a structural and regulatory component of cardiac thick filaments. It is observed in electron micrographs as seven to nine transverse stripes in the central portion of each half of the A band. Its C-terminus binds tightly to the myosin rod and contributes to thick filament structure, while the N-terminus can bind both myosin S2 and actin, influencing their structure and function. Mutations in the MYBPC3 gene (encoding cMyBP-C) are commonly associated with hypertrophic cardiomyopathy (HCM). In cardiac cells there exists a population of myosin heads in the super-relaxed (SRX) state, which are bound to the thick filament core with a highly inhibited ATPase activity. This report examines the role cMyBP-C plays in regulating the population of the SRX state of cardiac myosin by using an assay that measures single ATP turnover of myosin. We report a significant decrease in the proportion of myosin heads in the SRX state in homozygous cMyBP-C knockout mice, however heterozygous cMyBP-C knockout mice do not significantly differ from the wild type. A smaller, non-significant decrease is observed when thoracic aortic constriction is used to induce cardiac hypertrophy in mutation negative mice. These results support the proposal that cMyBP-C stabilises the thick filament and that the loss of cMyBP-C results in an untethering of myosin heads. This results in an increased myosin ATP turnover, further consolidating the relationship between thick filament structure and the myosin ATPase.
• Mun, J. Y., Kensler, R. W., Harris, S. P., & Craig, R. (2016). The cMyBP-C HCM variant L348P enhances thin filament activation through an increased shift in tropomyosin position. Journal of molecular and cellular cardiology, 91, 141-7.
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  Mutations in cardiac myosin binding protein C (cMyBP-C), a thick filament protein that modulates contraction of the heart, are a leading cause of hypertrophic cardiomyopathy (HCM). Electron microscopy and 3D reconstruction of thin filaments decorated with cMyBP-C N-terminal fragments suggest that one mechanism of this modulation involves the interaction of cMyBP-C's N-terminal domains with thin filaments to enhance their Ca(2+)-sensitivity by displacement of tropomyosin from its blocked (low Ca(2+)) to its closed (high Ca(2+)) position. The extent of this tropomyosin shift is reduced when cMyBP-C N-terminal domains are phosphorylated. In the current study, we have examined L348P, a sequence variant of cMyBP-C first identified in a screen of patients with HCM. In L348P, leucine 348 is replaced by proline in cMyBP-C's regulatory M-domain, resulting in an increase in cMyBP-C's ability to enhance thin filament Ca(2+)-sensitization. Our goal here was to determine the structural basis for this enhancement by carrying out 3D reconstruction of thin filaments decorated with L348P-mutant cMyBP-C. When thin filaments were decorated with wild type N-terminal domains at low Ca(2+), tropomyosin moved from the blocked to the closed position, as found previously. In contrast, the L348P mutant caused a significantly larger tropomyosin shift, to approximately the open position, consistent with its enhancement of Ca(2+)-sensitization. Phosphorylated wild type fragments showed a smaller shift than unphosphorylated fragments, whereas the shift induced by the L348P mutant was not affected by phosphorylation. We conclude that the L348P mutation causes a gain of function by enhancing tropomyosin displacement on the thin filament in a phosphorylation-independent way.
• Remedios, C. G., Mcnamara, J. W., Li, A., Lal, S., Harris, S. P., Cooke, R., Bos, J. M., & Ackerman, M. J. (2016). Regulation of the Super-Relaxed State of Myosin by Cardiac Myosin Binding Protein-C. Biophysical Journal, 110(3), 293a. doi:10.1016/j.bpj.2015.11.1585
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  Hypertrophic cardiomyopathy (HCM) is one of the most common inherited cardiac diseases, affecting at least 1 in 500 individuals. It is usually characterised by the asymmetric thickening of the ventricular walls, which reduces the ventricular chamber volume. This disease frequently exhibits normal systolic function, but the capacity for relaxation is often diminished. Mutations to the thick filament associated protein cardiac myosin binding protein-C (cMyBP-C) are commonly associated with HCM. Using a well-studied homozygous cMyBP-C knockout (KO) mouse model that exhibits the HCM phenotype, we have examined the role cMyBP-C has in regulating the super-relaxed (srx) state. Using quantitative epifluorescence, we identify a significant reduction in the proportion of super-relaxed myosin heads in skinned left ventricular muscle fibres of homozygous cMyBP-C KO mice compared to wild type and heterozygous knockout mice. This agrees with published electron microscopy of isolated thick filaments that described a more disordered state of the quasihelical arrangement of myosin heads expected from the srx state. Additionally, x-ray diffraction has previously described a shift of myosin heads away from the thick filament in cMyBP-C KO mice, further supporting our results. We will also address the question of whether the srx state of humans with mutations in cMyBP-C mirrors the homozygous or heterozygous mouse model.
• Stern, J. A., Markova, S., Ueda, Y., Kim, J. B., Pascoe, P. J., Evanchik, M. J., Green, E. M., & Harris, S. P. (2016). A Small Molecule Inhibitor of Sarcomere Contractility Acutely Relieves Left Ventricular Outflow Tract Obstruction in Feline Hypertrophic Cardiomyopathy. PloS one, 11(12), e0168407.
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  Hypertrophic cardiomyopathy (HCM) is an inherited disease of the heart muscle characterized by otherwise unexplained thickening of the left ventricle. Left ventricular outflow tract (LVOT) obstruction is present in approximately two-thirds of patients and substantially increases the risk of disease complications. Invasive treatment with septal myectomy or alcohol septal ablation can improve symptoms and functional status, but currently available drugs for reducing obstruction have pleiotropic effects and variable therapeutic responses. New medical treatments with more targeted pharmacology are needed, but the lack of preclinical animal models for HCM with LVOT obstruction has limited their development. HCM is a common cause of heart failure in cats, and a subset exhibit systolic anterior motion of the mitral valve leading to LVOT obstruction. MYK-461 is a recently-described, mechanistically novel small molecule that acts at the sarcomere to specifically inhibit contractility that has been proposed as a treatment for HCM. Here, we use MYK-461 to test whether direct reduction in contractility is sufficient to relieve LVOT obstruction in feline HCM. We evaluated mixed-breed cats in a research colony derived from a Maine Coon/mixed-breed founder with naturally-occurring HCM. By echocardiography, we identified five cats that developed systolic anterior motion of the mitral valve and LVOT obstruction both at rest and under anesthesia when provoked with an adrenergic agonist. An IV MYK-461 infusion and echocardiography protocol was developed to serially assess contractility and LVOT gradient at multiple MYK-461 concentrations. Treatment with MYK-461 reduced contractility, eliminated systolic anterior motion of the mitral valve and relieved LVOT pressure gradients in an exposure-dependent manner. Our findings provide proof of principle that acute reduction in contractility with MYK-461 is sufficient to relieve LVOT obstruction. Further, these studies suggest that feline HCM will be a valuable translational model for the study of disease pathology, particularly LVOT obstruction.
• Ueda, Y., Stern, J. A., Pascoe, P. J., Markova, S., Kim, J., Harris, S. P., Green, E. M., & Evanchik, M. J. (2016). Effects Of A Small Molecule Modulator Of Sarcomere Contractility In Cats With Hypertrophic Cardiomyopathy.. Journal of Veterinary Internal Medicine, 30(4).
• White, H. D., Harris, S. P., Galkin, V. E., & Belknap, B. (2016). C0 and C1 N-Terminal Ig-Domains of Myosin Binding Protein-C Exert Different Effects on thin Filament Activation. Biophysical Journal, 110(3), 1-5. doi:10.1016/j.bpj.2015.11.722
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  Mutations in the genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac Myosin Binding Protein-C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g. C0, C1, M and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remain unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we employed an integral structure-function approach using cryo-electron microscopy, kinetics and force measurements to reveal how the first two Ig-domains of cMyPB-C (e.g. C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) from the closed to the open structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin S1 for binding to F-actin and effectively inhibit acto-myosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres the activating effect of C1 is apparent only once low levels of Ca2+ have been achieved. We suggest that Ca2+ may modulate the interaction of cMyBP-C with the TF in the sarcomere.
• van Dijk, S. J., Kooiker, K. B., Mazzalupo, S., Yang, Y., Kostyukova, A. S., Mustacich, D. J., Hoye, E. R., Stern, J. A., Kittleson, M. D., & Harris, S. P. (2016). The A31P missense mutation in cardiac myosin binding protein C alters protein structure but does not cause haploinsufficiency. Archives of biochemistry and biophysics.
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  Mutations in MYBPC3, the gene encoding cardiac myosin binding protein C (cMyBP-C), are a major cause of hypertrophic cardiomyopathy (HCM). While most mutations encode premature stop codons, missense mutations causing single amino acid substitutions are also common. Here we investigated effects of a single proline for alanine substitution at amino acid 31 (A31P) in the C0 domain of cMyBP-C, which was identified as a natural cause of HCM in cats. Results using recombinant proteins showed that the mutation disrupted C0 structure, altered sensitivity to trypsin digestion, and reduced recognition by an antibody that preferentially recognizes N-terminal domains of cMyBP-C. Western blots detecting A31P cMyBP-C in myocardium of cats heterozygous for the mutation showed a reduced amount of A31P mutant protein relative to wild-type cMyBP-C, but the total amount of cMyBP-C was not different in myocardium from cats with or without the A31P mutation indicating altered rates of synthesis/degradation of A31P cMyBP-C. Also, the mutant A31P cMyBP-C was properly localized in cardiac sarcomeres. These results indicate that reduced protein expression (haploinsufficiency) cannot account for effects of the A31P cMyBP-C mutation and instead suggest that the A31P mutation causes HCM through a poison polypeptide mechanism that disrupts cMyBP-C or myocyte function.
• Dijk, S. J., Harris, S. P., Dijk, S. J., & Bezold, K. L. (2015). The Physiological Relevance of Interactions between cMyBP-C and Actin Studied in a Transgenic Mouse Model. Biophysical Journal, 108(2), 1-3. doi:10.1016/j.bpj.2014.11.737
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  Cardiac myosin binding protein C (cMyBP-C) is a sarcomeric protein that plays an essential role in the regulation of cardiac contraction. Until recently, cMyBP C was thought to hamper cross bridge cycling trough interaction of its N-terminus with the heads of myosin. However, the N-terminal domains of cMyBP-C can also bind with actin and this interaction could well explain the currently known functions of cMyBP-C. For instance, by forming a connection between the thin and the thick filament cMyBP-C could pose a load on the sarcomere and alter its visco-elastic properties. Additionally, cMyBP-C could alter the activation state of the thin filament by intervening with the position of tropomyosin.To study the physiological relevance of an interaction between cMyBP-C and actin, we created a transgenic mouse with a point mutation (L348P) that increases the binding affinity between cMyBP-C and actin in vitro. We hypothesized that stronger binding between cMyBP-C and actin will cause- through afore mentioned mechanisms, which have been demonstrated in different in vitro studies- stiffer cardiomyocytes which would ultimately lead to diastolic dysfunction in the hearts of L348P-Tg mice.Results show that the left atria of L348P-Tg mice are significantly enlarged. Echocardiograms in 12 week old animals demonstrated misshapen E and A waves, making diastolic function difficult to determine. Interestingly, pulse wave and tissue Doppler images were more defined in 24 weeks old mice. Preliminary data show decreased early blood flow over the mitral valve (E) and mitral valve movement (E’) in L348PTg mice, indicative of a stiffer left ventricle. In correspondence with that, isovolumetric relaxation time was prolonged. At neither age we observed systolic dysfunction. Additional in-depth hemodynamic studies to characterize the mechanisms of cardiac function longitudinally in L348P-Tg mice are ongoing.
• Dijk, S. J., Mazzalupo, S., Kostyukova, A. S., Kittleson, M. D., Harris, S. P., Dijk, S. J., & Bezold, K. L. (2015). The A31P Hcm Mutation in cMyBP-C Disrupts the Structure of the C0 Domain But Does Not Cause Haploinsufficiency in a Population of Older Cats Heterozygous for the A31P Allele. Biophysical Journal, 108(2), 200a-201a. doi:10.1016/j.bpj.2014.11.1109
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  Archives of Biochemistry and Biophysics xxx (2016) 1e8 Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi The A31P missense mutation in cardiac myosin binding protein C alters protein structure but does not cause haploinsufficiency Sabine J. van Dijk a, * , Kristina Bezold Kooiker b, 1 , Stacy Mazzalupo a , Yuanzhang Yang a , Alla S. Kostyukova c , Debbie J. Mustacich a , Elaine R. Hoye b , Joshua A. Stern d , Mark D. Kittleson d , Samantha P. Harris a a Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, USA Department of Neurobiology, Physiology and Behavior, University of California, Davis, CA, USA Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA, USA d Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA, USA b c a r t i c l e i n f o a b s t r a c t Article history: Received 27 November 2015 Received in revised form 31 December 2015 Accepted 7 January 2016 Available online xxx Mutations in MYBPC3, the gene encoding cardiac myosin binding protein C (cMyBP-C), are a major cause of hypertrophic cardiomyopathy (HCM). While most mutations encode premature stop codons, missense mutations causing single amino acid substitutions are also common. Here we investigated effects of a single proline for alanine substitution at amino acid 31 (A31P) in the C0 domain of cMyBP-C, which was identified as a natural cause of HCM in cats. Results using recombinant proteins showed that the mu- tation disrupted C0 structure, altered sensitivity to trypsin digestion, and reduced recognition by an antibody that preferentially recognizes N-terminal domains of cMyBP-C. Western blots detecting A31P cMyBP-C in myocardium of cats heterozygous for the mutation showed a reduced amount of A31P mutant protein relative to wild-type cMyBP-C, but the total amount of cMyBP-C was not different in myocardium from cats with or without the A31P mutation indicating altered rates of synthesis/degra- dation of A31P cMyBP-C. Also, the mutant A31P cMyBP-C was properly localized in cardiac sarcomeres. These results indicate that reduced protein expression (haploinsufficiency) cannot account for effects of the A31P cMyBP-C mutation and instead suggest that the A31P mutation causes HCM through a poison polypeptide mechanism that disrupts cMyBP-C or myocyte function. © 2016 Elsevier Inc. All rights reserved. Keywords: cMyBP-C Hypertrophic cardiomyopathy Missense mutation Animal models of cardiac disease 1. Introduction Hypertrophic cardiomyopathy (HCM) is the most common ge- netic cause of cardiomyopathy and is estimated to affect 1 in 500 people [1e3]. Hundreds of mutations in genes encoding sarcomeric proteins are thought to cause HCM [3]. However, the majority of mutations occur in two genes, MYH7 and MYBPC3. MYH7 encodes the b -myosin heavy chain, the primary force generating protein of cardiac muscle, while MYBPC3 encodes cardiac myosin binding protein C (cMyBP-C), an essential regulatory protein that modulates the force and speed of cardiac contraction. Although the majority of mutations in MYH7 cause single amino acid substitutions, most * Corresponding author. Department of Cellular and Molecular Medicine, Uni- versity of Arizona, 1656 East Mabel Street, Tucson, AZ 85724, USA. E-mail address: sjvandijk@email.arizona.edu (S.J. van Dijk). Present address: Division of Pediatric Cardiology, Stanford University, CA, USA. MYBPC3 mutations are non-sense or frameshift mutations that encode premature termination codons and are thus predicted to cause early termination of cMyBP-C. However, truncated cMyBP-C proteins have not been detected in myocardium from HCM pa- tients [4e6], most likely because cell quality control mechanisms either efficiently degrade mRNAs that contain premature termi- nation codons or because truncated or misfolded proteins are rapidly degraded thus preventing their accumulation [4,7,8]. The apparent lack of expression of mutant truncated proteins combined with observations that the total amount of cMyBP-C protein is reduced in some patients with HCM has led to the hypothesis that haploinsufficiency causes sub-stoichiometric amounts of cMyBP-C within the sarcomere which impair contractile function and cause disease. Because reduced cMyBP-C protein expression was also found in myocardium of some patients with cMyBP-C missense mutations that cause single amino acid substitutions [5], both truncation and missense mutations could cause disease through a http://dx.doi.org/10.1016/j.abb.2016.01.006 0003-9861/© 2016 Elsevier Inc. All rights reserved. Please cite this article in press as: S.J. van Dijk, et al., The A31P missense mutation in cardiac myosin binding protein C alters protein structure but does not cause haploinsufficiency, Archives of Biochemistry and Biophysics (2016), http://dx.doi.org/10.1016/j.abb.2016.01.006
• Kittleson, M. D., Meurs, K. M., & Harris, S. P. (2015). The genetic basis of hypertrophic cardiomyopathy in cats and humans. Journal of veterinary cardiology : the official journal of the European Society of Veterinary Cardiology, 17 Suppl 1, S53-73.
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  Mutations in genes that encode for muscle sarcomeric proteins have been identified in humans and two breeds of domestic cats with hypertrophic cardiomyopathy (HCM). This article reviews the history, genetics, and pathogenesis of HCM in the two species in order to give veterinarians a perspective on the genetics of HCM. Hypertrophic cardiomyopathy in people is a genetic disease that has been called a disease of the sarcomere because the preponderance of mutations identified that cause HCM are in genes that encode for sarcomeric proteins (Maron and Maron, 2013). Sarcomeres are the basic contractile units of muscle and thus sarcomeric proteins are responsible for the strength, speed, and extent of muscle contraction. In people with HCM, the two most common genes affected by HCM mutations are the myosin heavy chain gene (MYH7), the gene that encodes for the motor protein β-myosin heavy chain (the sarcomeric protein that splits ATP to generate force), and the cardiac myosin binding protein-C gene (MYBPC3), a gene that encodes for the closely related structural and regulatory protein, cardiac myosin binding protein-C (cMyBP-C). To date, the two mutations linked to HCM in domestic cats (one each in Maine Coon and Ragdoll breeds) also occur in MYBPC3 (Meurs et al., 2005, 2007). This is a review of the genetics of HCM in both humans and domestic cats that focuses on the aspects of human genetics that are germane to veterinarians and on all aspects of feline HCM genetics.
• Lee, K., Harris, S. P., Sadayappan, S., & Craig, R. (2015). Orientation of myosin binding protein C in the cardiac muscle sarcomere determined by domain-specific immuno-EM. Journal of molecular biology, 427(2), 274-86.
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  Myosin binding protein C is a thick filament protein of vertebrate striated muscle. The cardiac isoform [cardiac myosin binding protein C (cMyBP-C)] is essential for normal cardiac function, and mutations in cMyBP-C cause cardiac muscle disease. The rod-shaped molecule is composed primarily of 11 immunoglobulin- or fibronectin-like domains and is located at nine sites, 43nm apart, in each half of the A-band. To understand how cMyBP-C functions, it is important to know its structural organization in the sarcomere, as this will affect its ability to interact with other sarcomeric proteins. Several models, in which cMyBP-C wraps around, extends radially from, or runs axially along the thick filament, have been proposed. Our goal was to define cMyBP-C orientation by determining the relative axial positions of different cMyBP-C domains. Immuno-electron microscopy was performed using mouse cardiac myofibrils labeled with antibodies specific to the N- and C-terminal domains and to the middle of cMyBP-C. Antibodies to all regions of the molecule, except the C-terminus, labeled at the same nine axial positions in each half A-band, consistent with a circumferential and/or radial rather than an axial orientation of the bulk of the molecule. The C-terminal antibody stripes were slightly displaced axially, demonstrating an axial orientation of the C-terminal three domains, with the C-terminus closer to the M-line. These results, combined with previous studies, suggest that the C-terminal domains of cMyBP-C run along the thick filament surface, while the N-terminus extends toward neighboring thin filaments. This organization provides a structural framework for understanding cMyBP-C's modulation of cardiac muscle contraction.
• Mun, J. Y., Kensler, R. W., Harris, S. P., & Craig, R. (2015). The HCM Mutation L348P in cMyBP-C Enhances Thin Filament Activation through Tropomyosin Shift. Biophysical Journal, 108(2), 133a. doi:10.1016/j.bpj.2014.11.738
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  Cardiac myosin binding protein C (cMyBP-C) is a thick filament protein that plays an important role in modulating contraction of the heart. Mutations in cMyBP-C are a major cause of inherited hypertrophic cardiomyopathy (HCM). In addition to binding to myosin, cMyBP-C also interacts with thin filaments via its N-terminal region, mainly the C1 and M-domains, enhancing thin filament Ca2+-sensitivity. L348P is an HCM-causing mutation that occurs in a conserved sequence in the M-domain, enhancing cMyBP-C binding to actin and thin filament Ca2+-sensitization (Bezold et al., JBC 2013). Our goal was to elucidate the structural basis of L348P's gain of function. By EM 3D reconstruction we previously showed that binding of N-terminal cMyBP-C fragments to actin causes displacement of tropomyosin from its blocked (low Ca2+) position to its closed (high Ca2+) position (Mun et al., PNAS 2014), consistent with the increase in thin filament Ca2+-sensitivity. Phosphorylation of the M-domain resulted in a smaller movement of tropomyosin. Here we have investigated the impact of the L348P mutation on tropomyosin shift. Thin filaments were decorated with wild type C1C2 (containing the C1, C2 and M-domains) and with C1C2 having the L348P mutation. F-actin reconstructions showed additional density on actin subdomain 1 with both fragments, but the density was larger with L348P. When thin filaments at low Ca2+ were decorated with C1C2, tropomyosin moved from the blocked to the closed position, as found previously. In contrast, C1C2-L348P showed a significantly larger tropomyosin shift, to approximately the open position, consistent with L348P's greater Ca2+-sensitization of motility and enhanced Ca2+ sensitivity of tension in cardiac myocytes. Preliminary studies showed a smaller shift with phosphorylated than with unphosphorylated C1C2, while phosphorylated C1C2-L348P showed a larger shift, comparable to L348P.
• Walcott, S., Docken, S., & Harris, S. P. (2015). Effects of cardiac Myosin binding protein-C on actin motility are explained with a drag-activation-competition model. Biophysical journal, 108(1), 10-3.
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  Although mutations in cardiac myosin binding protein-C (cMyBP-C) cause heart disease, its role in muscle contraction is not well understood. A mechanism remains elusive partly because the protein can have multiple effects, such as dual biphasic activation and inhibition observed in actin motility assays. Here we develop a mathematical model for the interaction of cMyBP-C with the contractile proteins actin and myosin and the regulatory protein tropomyosin. We use this model to show that a drag-activation-competition mechanism accurately describes actin motility measurements, while models lacking either drag or competition do not. These results suggest that complex effects can arise simply from cMyBP-C binding to actin.
• van Dijk, S. J., Witt, C. C., & Harris, S. P. (2015). Normal cardiac contraction in mice lacking the proline-alanine rich region and C1 domain of cardiac myosin binding protein C. Journal of molecular and cellular cardiology, 88, 124-32.
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  Cardiac myosin binding protein C (cMyBP-C) is an essential regulator of cross bridge cycling. Through mechanisms that are incompletely understood the N-terminal domains (NTDs) of cMyBP-C can activate contraction even in the absence of calcium and can also inhibit cross bridge kinetics in the presence of calcium. In vitro studies indicated that the proline-alanine rich (p/a) region and C1 domain are involved in these processes, although effects were greater using human proteins compared to murine proteins (Shaffer et al. J Biomed Biotechnol 2010, 2010: 789798). We hypothesized that the p/a and C1 region are critical for the timing of contraction. In this study we tested this hypothesis using a mouse model lacking the p/a and C1 region (p/a-C1(-/-) mice) to investigate the in vivo relevance of these regions on cardiac performance. Surprisingly, hearts of adult p/a-C1(-/-) mice functioned normally both on a cellular and whole organ level. Force measurements in permeabilized cardiomyocytes from adult p/a-C1(-/-) mice and wild type (Wt) littermate controls demonstrated similar rates of force redevelopment both at submaximal and maximal activation. Maximal and passive force and calcium sensitivity of force were comparable between groups as well. Echocardiograms showed normal isovolumetric contraction times, fractional shortening and ejection fraction, indicating proper systolic function in p/a-C1(-/-) mouse hearts. p/a-C1(-/-) mice showed a slight but significant reduction in isovolumetric relaxation time compared to Wt littermates, yet this difference disappeared in older mice (7-8months of age). Moreover, stroke volume was preserved in p/a-C1(-/-) mice, corroborating sufficient time for normal filling of the heart. Overall, the hearts of p/a-C1(-/-) mice showed no signs of dysfunction even after chronic stress with an adrenergic agonist. Together, these results indicate that the p/a region and the C1 domain of cMyBP-C are not critical for normal cardiac contraction in mice and that these domains have little if any impact on cross bridge kinetics in mice. These results thus contrast with in vitro studies utilizing proteins encoding the human p/a region and C1 domain. More detailed insight in how individual domains of cMyBP-C function and interact, across species and over the wide spectrum of conditions in which the heart has to function, will be essential to a better understanding of how cMyBP-C tunes cardiac contraction.
• Belknap, B., Harris, S. P., & White, H. D. (2014). Modulation of thin filament activation of myosin ATP hydrolysis by N-terminal domains of cardiac myosin binding protein-C. Biochemistry, 53(42), 6717-24.
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  We have used enzyme kinetics to investigate the molecular mechanism by which the N-terminal domains of human and mouse cardiac MyBP-C (C0C1, C1C2, and C0C2) affect the activation of myosin ATP hydrolysis by F-actin and by native porcine thin filaments. N-Terminal domains of cMyBP-C inhibit the activation of myosin-S1 ATPase by F-actin. However, mouse and human C1C2 and C0C2 produce biphasic activating and inhibitory effects on the activation of myosin ATP hydrolysis by native cardiac thin filaments. Low ratios of MyBP-C N-terminal domains to thin filaments activate myosin-S1 ATP hydrolysis, but higher ratios inhibit ATP hydrolysis, as is observed with F-actin alone. These data suggest that low concentrations of C1C2 and C0C2 activate thin filaments by a mechanism similar to that of rigor myosin-S1, whereas higher concentrations inhibit the ATPase rate by competing with myosin-S1-ADP-Pi for binding to actin and thin filaments. In contrast to C0C2 and C1C2, the activating effects of the C0C1 domain are species-dependent: human C0C1 activates actomyosin-S1 ATPase rates, but mouse C0C1 does not produce significant activation or inhibition. Phosphorylation of serine residues in the m-linker between the C1 and C2 domains by protein kinase-A decreases the activation of thin filaments by huC0C2 at pCa > 8 but has little effect on the activation mechanism at pCa = 4. In sarcomeres, the low ratio of cMyBP-C to actin is expected to favor the activating effects of cMyBP-C while minimizing inhibition produced by competition with myosin heads.
• Chow, M. L., Shaffer, J. F., Harris, S. P., & Dawson, J. F. (2014). Altered interactions between cardiac myosin binding protein-C and α-cardiac actin variants associated with cardiomyopathies. Archives of biochemistry and biophysics, 550-551, 28-32.
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  The two genes most commonly associated with mutations linked to hypertrophic or dilated cardiomyopathies are β-myosin and cardiac myosin binding protein-C (cMyBP-C). Both of these proteins interact with cardiac actin (ACTC). Currently there are 16 ACTC variants that have been found in patients with HCM or DCM. While some of these ACTC variants exhibit protein instability or polymerization-deficiencies that might contribute to the development of disease, other changes could cause changes in protein-protein interactions between sarcomere proteins and ACTC. To test the hypothesis that changes in ACTC disrupt interactions with cMyBP-C, we examined the interactions between seven ACTC variants and the N-terminal C0C2 fragment of cMyBP-C. We found there was a significant decrease in binding affinity (increase in Kd values) for the A331P and Y166C variants of ACTC. These results suggest that a change in the ability of cMyBP-C to bind actin filaments containing these ACTC protein variants might contribute to the development of disease. These results also provide clues regarding the binding site of the C0C2 fragment of cMyBP-C on F-actin.
• Khosa, J. K., Harris, S. P., & Bezold, K. L. (2014). A Gain-Of-Function Mutation in Cardiac Myosin Binding Protein-C Increases Viscoelastic Load and Slows Shortening Velocity in Myocytes from Transgenic Mice. Biophysical Journal, 106(2), 346a. doi:10.1016/j.bpj.2013.11.1973
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  Cardiac myosin binding protein C (cMyBP-C) is a sarcomeric protein involved in the regulation of cardiac muscle contraction. Effects of cMyBP-C on contraction are thought to be mediated in part by limiting the interactions of actin and myosin to slow myocyte shortening velocity and power output. Although interactions with myosin S2 on the thick filament have been proposed as a way in which cMyBP-C could limit shortening velocity (e.g., by creating a drag force on myosin heads), interactions of cMyBP-C with actin could also account for slowed shortening velocity. For instance, cMyBP-C could create a drag that opposes filament sliding by transiently linking thick and thin filaments together. To explore this possibility we created transgenic mice that express a mutant cMyBP-C with a point mutation, L348P (human L352P), located in a conserved sequence within the regulatory M-domain that increases cMyBP-C binding to actin in vitro. We reasoned that if the mutation also enhanced binding to actin in sarcomeres then shortening velocity would be slowed in myocytes from L348P mice. Results show that transgenic mice expressing the L348P mutation are viable and that L348P cMyBP-C is expressed in sarcomeres. Permeabilized myocytes from transgenic mice showed altered force production including reduced maximal force and enhanced calcium sensitivity of tension. Shortening velocity and power output were significantly reduced whereas passive stiffness and myocyte visco-elasticity were significantly increased. Together these data are consistent with the idea that cMyBP-C creates an internal load in the sarcomere by binding to actin.
• van Dijk, S. J., Bezold, K. L., & Harris, S. P. (2014). Earning stripes: myosin binding protein-C interactions with actin. Pflügers Archiv : European journal of physiology, 466(3), 445-50.
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  Myosin binding protein-C (MyBP-C) was first discovered as an impurity during the purification of myosin from skeletal muscle. However, soon after its discovery, MyBP-C was also shown to bind actin. While the unique functional implications for a protein that could cross-link thick and thin filaments together were immediately recognized, most early research nonetheless focused on interactions of MyBP-C with the thick filament. This was in part because interactions of MyBP-C with the thick filament could adequately explain most (but not all) effects of MyBP-C on actomyosin interactions and in part because the specificity of actin binding was uncertain. However, numerous recent studies have now established that MyBP-C can indeed bind to actin through multiple binding sites, some of which are highly specific. Many of these interactions involve critical regulatory domains of MyBP-C that are also reported to interact with myosin. Here we review current evidence supporting MyBP-C interactions with actin and discuss these findings in terms of their ability to account for the functional effects of MyBP-C. We conclude that the influence of MyBP-C on muscle contraction can be explained equally well by interactions with actin as by interactions with myosin. However, because data showing that MyBP-C binds to either myosin or actin has come almost exclusively from in vitro biochemical studies, the challenge for future studies is to define which binding partner(s) MyBP-C interacts with in vivo.
• Bezold, K. L., Shaffer, J. F., Khosa, J. K., Hoye, E. R., & Harris, S. P. (2013). A gain-of-function mutation in the M-domain of cardiac myosin-binding protein-C increases binding to actin. The Journal of biological chemistry, 288(30), 21496-505.
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  The M-domain is the major regulatory subunit of cardiac myosin-binding protein-C (cMyBP-C) that modulates actin and myosin interactions to influence muscle contraction. However, the precise mechanism(s) and the specific residues involved in mediating the functional effects of the M-domain are not fully understood. Positively charged residues adjacent to phosphorylation sites in the M-domain are thought to be critical for effects of cMyBP-C on cross-bridge interactions by mediating electrostatic binding with myosin S2 and/or actin. However, recent structural studies revealed that highly conserved sequences downstream of the phosphorylation sites form a compact tri-helix bundle. Here we used site-directed mutagenesis to probe the functional significance of charged residues adjacent to the phosphorylation sites and conserved residues within the tri-helix bundle. Results confirm that charged residues adjacent to phosphorylation sites and residues within the tri-helix bundle are important for mediating effects of the M-domain on contraction. In addition, four missense variants within the tri-helix bundle that are associated with human hypertrophic cardiomyopathy caused either loss-of-function or gain-of-function effects on force. Importantly, the effects of the gain-of-function variant, L348P, increased the affinity of the M-domain for actin. Together, results demonstrate that functional effects of the M-domain are not due solely to interactions with charged residues near phosphorylatable serines and provide the first demonstration that the tri-helix bundle contributes to the functional effects of the M-domain, most likely by binding to actin.
• Hoye, E., & Harris, S. P. (2013). Calcium-Calmodulin Competes with Actin for Binding to the M-Domain of Cardiac Myosin Binding Protein-C. Biophysical Journal, 104(2), 313a. doi:10.1016/j.bpj.2012.11.1735
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  The regulatory M-domain of cardiac myosin binding protein-C (cMyBP-C) binds to myosin, actin, and to calmodulin when calcium is present (i.e., calcium-calmodulin), but it is unclear whether binding of all three ligands is independent or if binding interactions are competitive. Here we investigated whether calcium-calmodulin (Ca-Cam) binding to the M-domain affected the ability of the M-domain to bind to actin using cosedimentation binding and calmodulin-sepaharose pull-down assays. Results of actin cosedimentation binding assays showed that Ca-Cam significantly reduced specific binding of a recombinant protein containing three N-terminal domains of cMyBP-C (i.e., C1-M-C2, referred to as C1C2) when 10 μM calmodulin was present in the presence of calcium (pCa 3.0). In the absence of calcium (at pCa 10.0) calmodulin had no effect on C1C2 binding to actin. Increasing Ca-Cam concentrations to achieve higher molar ratios with respect to C1C2 further reduced the amount of C1C2 that bound to actin. Conversely, in calmodulin-sepharose pull-down experiments, binding of C1C2 to calmodulin was only modestly reduced in the presence of increasing concentrations of F-actin. Taken together, these results indicate that binding of Ca-Cam can compete with actin for binding to the M-domain. These results suggest a potential mechanism whereby the functional effects of cMyBP-C binding to actin can be regulated by calcium. This work supported by NIH HL080367.
• Karsai, Á., Kellermayer, M. S., & Harris, S. P. (2013). Cross-species mechanical fingerprinting of cardiac myosin binding protein-C. Biophysical journal, 104(11), 2465-75.
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  Cardiac myosin binding protein-C (cMyBP-C) is a member of the immunoglobulin (Ig) superfamily of proteins and consists of 8 Ig- and 3 fibronectin III (FNIII)-like domains along with a unique regulatory sequence referred to as the MyBP-C motif or M-domain. We previously used atomic force microscopy to investigate the mechanical properties of murine cMyBP-C expressed using a baculovirus/insect cell expression system. Here, we investigate whether the mechanical properties of cMyBP-C are conserved across species by using atomic force microscopy to manipulate recombinant human cMyBP-C and native cMyBP-C purified from bovine heart. Force versus extension data obtained in velocity-clamp experiments showed that the mechanical response of the human recombinant protein was remarkably similar to that of the bovine native cMyBP-C. Ig/Fn-like domain unfolding events occurred in a hierarchical fashion across a threefold range of forces starting at relatively low forces of ~50 pN and ending with the unfolding of the highest stability domains at ~180 pN. Force-extension traces were also frequently marked by the appearance of anomalous force drops suggestive of additional mechanical complexity such as structural coupling among domains. Both recombinant and native cMyBP-C exhibited a prominent segment ~100 nm-long that could be stretched by forces
• Khosa, J. K., Harris, S. P., & Bezold, K. L. (2013). Helix Three (H3) of the M-Domain of Cardiac Myosin Binding Protein-C is not Essential for Functional Effects in Permeabilized Rat Trabeculae. Biophysical Journal, 104(2), 186a. doi:10.1016/j.bpj.2012.11.1053
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  Cardiac myosin binding protein C (cMyBP-C) is a sarcomeric protein involved in the regulation of cardiac muscle contraction. Cardiac specific phosphorylation sites in the M-domain regulate binding of cMyBP-C to myosin and actin. Immediately downstream of the phosphorylation sites is a bundle of three α-helixes that are well conserved across all species and isoforms of MyBP-C, however the functional significance of these helices is unknown. Helix 3 (H3) bears high homology to the inhibitory peptide of troponin I, suggesting it as a potential binding site for interactions with actin. To understand the functional significance of H3, we produced a recombinant N-terminal protein containing domains C1, M and C2 (C1C2) with the H3 sequence deleted (H3KO). We then assessed effects of the mutant H3KO protein on contractile forces in permeabilized rat trabeculae and compared them to effects of the wild-type C1C2. Results showed that 5µM C1C2 significantly increased Ca2+-sensitivity of force (ΔpCa50=0.29±0.03), whereas 5µM H3KO had no effect (ΔpCa50=0.02±0.01) and 10µM H3KO only modestly increased Ca2+-sensitivity (ΔpCa50=0.11±0.02,). Similarly, whereas 10µM C1C2 activated force in the absence of Ca2+ (pCa 9.0), H3KO activated force in the absence of Ca2+ only at concentrations >20µM. Together, these results establish that H3 is not absolutely required for the functional effects of the N-terminal domains of cMyBP-C to either increase Ca2+-sensitivity of force or to activate tension in sarcomeres, but that H3 contributes to the overall efficacy of the N-terminus in mediating these effects. Potentially, H3 could dock the N-terminus of cMyBP-C with actin or extend/stabilize cMyBP-C interactions with actin or other regulatory proteins. This work was supported by NIH HL-080367 (SPH), NDSEG and AHA graduate fellowships (KLB), and AHA undergraduate fellowship (JKK).
• Remedios, C. G., Li, A., King, D., Ishiwata, S., Harris, S. P., & Braet, F. (2013). A New Way to Examine the Function of Mutant MYBPC3 Expression in Cardiomyocytes of Mice. Biophysical Journal, 104(2), 3-9. doi:10.1016/j.bpj.2012.11.1718
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  Autosomal dominant mutations in myosin binding protein C (MYBPC3) account for up to 30% of myofibrillar mutations causing Hypertrophic Cardiomyopathy (HCM). We used SPontaneous Oscillatory Contractions (SPOC) to compare the performance of isolated cardiomyocytes from heterozygous MYBPC (+/-) and homozygous MYBPC (-/-) and wild type (+/+) mice. Our aim is to identify changes in their contractile parameters. SPOC is a physiological state that is intermediate to full contraction and relaxation. The stable auto-oscillatory properties of SPOC are well suited to precise measurements of contraction and relaxation.Preliminary evaluations reveal there is: (1) a progressive prolongation in both the relative lengthening and shortening periods from MYBPC+/+ to MYBPC+/- and MYBPC-/-; (2) MYBPC+/- exhibits faster rates of lengthening than MYBPC+/+; (3) MYBPC-/- displays significantly depressed rates of shortening compared to MYBPC+/- and MYBPC+/+. These findings suggest significant systolic dysfunction in MYBPC-/- associated with severe hypertrophic remodelling. Perhaps, more importantly MYBPC+/- exhibits diastolic dysfunction consistent with previous reports examining SPOC in human HCM at the 56th Biophysical Society Meeting. We conclude that SPOC can objectively assess the functional state of heart muscle fibres in this mouse model.View Large Image | View Hi-Res Image | Download PowerPoint Slide
• Sadayappan, S., Lee, K., Harris, S. P., & Craig, R. (2013). Determination of MyBP-C Orientation in the Cardiac Sarcomere by Immuno-EM. Biophysical Journal, 104(2), 309a. doi:10.1016/j.bpj.2012.11.1716
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  Myosin binding protein-C (MyBP-C) is a thick filament accessory protein of vertebrate striated muscle. It is essential for normal cardiac function, and mutations in MyBP-C cause cardiac and skeletal muscle disease. The 40 nm long molecule is composed of 10 or 11 immunoglobulin- or fibronectin-like domains and is located in up to 9 axial stripes 43 nm apart in each half of the A-band. To understand MyBP-C function it is important to know its structural organization in the sarcomere. Several models have been proposed, in which MyBP-C either wraps around the thick filament, or extends radially from or longitudinally along the filament (or some combination of these). To distinguish between these models, we have used immuno-EM of mouse cardiac myofibrils labeled with antibodies to different domains of MyBP-C to determine the relative axial positions of the domains. Myofibrils were obtained from mouse cardiac muscle by homogenizing chemically skinned hearts under rigor conditions. They were labeled with antibodies specific for the N-terminal domain, the middle of the molecule, and the C-terminal domain, and observed by negative stain EM. Labeled myofibrils showed nine stain-excluding stripes in each half of the A-band. The average distance of each stripe from the middle of the M-line was measured. All antibodies labeled axially within 10 nm of each other, at the same axial positions as the unlabeled stripes, suggesting that the bulk of MyBP-C runs radially or circumferentially. A small axial difference between the C-terminal and the central and N-terminal antigenic sites suggests that a short portion of the C-terminus runs longitudinally. Electron tomography of muscle sections (Luther et al., PNAS 2011) and 3D reconstruction of isolated thick filaments (Zoghbi et al., PNAS 2008) support the radial arrangement.
• White, H. D., Harris, S. P., & Belknap, B. (2013). Activation and Inhibition of F-Actin and Cardiac Thin Filaments by the N-Terminal Domains of Cardiac Myosin Binding Protein C. Biophysical Journal, 104(2), 158a-159a. doi:10.1016/j.bpj.2012.11.893
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  Myosin binding protein-C (MyBPC) has been known for over thirty years to inhibit actomyosin ATP hydrolysis and more recently has been shown to affect the calcium sensitivity of force in muscle fibers. The various domains of MyBPC have previously been shown to have complex interactions with other myofibrillar proteins. The C-terminal domains have been shown to bind to the thick filament and the N-terminal domains interact with the S2 region of myosin and with f-actin. The number of mutations in cardiac MyBPC (cMyBPC) are the second most prevalent of sarcomeric proteins in producing cardiomyopathies. We have used steady state kinetics to study the molecular mechanism by which the soluble N-terminal C0C1, C1C2 and C0C2 domains of mouse and human cMyBPC affect the activation of myosin ATP hydrolysis by f-actin and native porcine thin filaments.The N-terminal domains of cMyBPC inhibit the activation of the steady state ATPase myosin by f-actin. However, in the presence of native cardiac thin filaments.the N-terminal domains C1C2 and C0C2 of mouse and human cMyBPC produce biphasic effects on the steady state ATP hydrolysis of cardiac myosin-S1. That is, ATPase is activated at low ratios of cMyBPC-N-terminal domain to thin filament and is inhibited by higher ratios similar to the effects observed with f-actin. These data suggest that low ratios of cMyBPC N-terminal domains activate thin filaments in a mechanism similar to that of rigor myosin-S1 but higher ratios inhibit the ATPase rate by competing with myosin-S1-ADP-Pi binding to actin and thin filaments. Effects also appear species-dependent since the C0C1 domains of human cMyBPC produce similar effects as C1C2 and C0C2 on ATPase in the presence of thin filaments, but mouse C0C1 does not produce significant activation.
• Harris, S. P. (2012). Dynamic Regulation of Contraction by Cardiac Myosin Binding Protein-C. Biophysical Journal, 102(3), 598a. doi:10.1016/j.bpj.2011.11.3255
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  Cardiac myosin binding protein-C (cMyBP-C) is a thick filament associated protein that performs both regulatory and structural roles within cardiac sarcomeres. It is a member of the immunoglobulin (Ig) superfamily of proteins consisting of 8 Ig- and 3 fibronectin (FNIII)-like subdomains along with a unique regulatory sequence referred to as the M-domain whose structure is unknown. Here we used atomic force microscopy (AFM) to probe the structure and mechanical properties of the different subdomains of native and recombinantly expressed cMyBP-C molecules. Results demonstrate that cMyBP-C exhibits complex mechanical behavior under load and contains multiple domains with distinct mechanical properties. The Ig and FNIII-like domains unfold over a range of relatively low forces (50-190 pN), whereas the M-domain is readily extensible at forces
• Harris, S. P., & Bezold, K. L. (2012). Effects of HCM Missense Mutations in the M Domain of Cardiac Myosin Binding Protein C on Calcium Sensitivity of Force and Rate in Rat Trabeculae. Biophysical Journal, 102(3), 558a. doi:10.1016/j.bpj.2011.11.3039
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  Recombinant N-terminal domains of cardiac myosin binding protein C (cMyBP-C) increase calcium-sensitivity of force and the rate of tension redevelopement (ktr) when added to permeabilized rat trabeculae. We previously demonstrated that the regulatory domain of cMyBP-C, referred to as the MyBP-C motif (or “M domain”), is required for these effects. Here we investigated the effects of single amino acid missense substitutions within the M-domain that are associated with human hypertrophic cardiomyopathy (HCM) on force development and ktr in permeabilized trabeculae from rat right ventricles. Individual substitutions (R322Q, E330K, V338D, and L348P) were introduced into a recombinant mouse C1C2 protein (encompassing domains C1-M-C2 of murine cMyBP-C) by site directed mutagenesis and effects were compared to wild-type C1C2. All four of the mutations affected the ability of C1C2 to augment force. Whereas 5 μM wild-type C1C2 induced a pronounced leftward shift in Ca2+ sensitivity of tension (∼0.5 pCa units) and increased ktr at all submaximal Ca2+ concentrations, 3 of the mutations reduced the effects of C1C2. Another variant increased the efficacy of C1C2 and increased passive force independent of Ca2+, but reduced maximal Ca2+ activated force. Together these results indicate that cMyBP-C variants associated with HCM could directly disrupt sarcomere contractile properties through gain or loss of functional effects and that at least some cMyBP-C missense mutations may cause disease through a poison polypeptide mechanism. This work is supported by NIH HL080367 to SPH and a DOD NDSEG graduate fellowship to KLB.
• Kellermayer, M. S., Karsai, A., & Harris, S. P. (2012). Common Mechanical Properties of Recombinant and Native Cardiac Myosin Binding Protein-C by Atomic Force Microscope. Biophysical Journal, 102(3), 5-8. doi:10.1016/j.bpj.2011.11.3040
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  Cardiac myosin binding protein-C (cMyBP-C) is a member of the immunoglobulin (Ig) superfamily of proteins and consists of 8 Ig- and 3 fibronectin (Fn)-like domains along with a unique regulatory sequence referred to as the MyBP-C “motif” or M-domain. Previously we used atomic force microscopy (AFM) to investigate the mechanical properties of the different domains of murine cMyBP-C expressed using a baculovirus/insect cell expression system. To investigate whether the mechanical properties of cMyBP-C are conserved across species, here we used AFM to investigate the mechanical properties of human recombinant cMyBP-C expressed using a baculovirus/insect cell expression system and native cMyBP-C purified from bovine heart. AFM force-extension spectra were obtained from cMyBP-C molecules by randomly adhering individual molecules to the tip of an AFM cantilever and moving the cantilever to impose a load that stretched the molecules. Results show that the spectra for the human recombinant and bovine native proteins are remarkably similar with the first Ig/Fn-like domain unfolding events occurring at low (∼50 pN) forces and the highest stability domains unfolding at ∼190 pN. Experiments also revealed frequent unfolding events that appeared coupled such that lower stability domains would often unfold after higher stability domains. These unexpected force “drops” were highly reproducible and occurred in spectra from both human cMyBP-C and bovine cMyBP-C. In addition, both recombinant and native cMyBP-C exhibited an ∼100 nm long extensible region that could be stretched with less than 50 pN force prior to the unfolding of Ig and Fn-like domains. Combined with our previous results from mouse cMyBP-C, these results establish common mechanical features of cMyBP-C across species. Supported by NIH HL080367.
• White, H. D., & Harris, S. P. (2012). Activation of Cardiac Thin Filaments by N-Terminal Domains of Cardiac Myosin Binding Protein C. Biophysical Journal, 102(3), 4-5. doi:10.1016/j.bpj.2011.11.2382
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  We have used double mixing stopped-flow fluorescence to study the effect of the soluble N-terminal C1C2 domain of cMyBP-C on the activation of product dissociation from myosin-S1-mdADP-Pi by native porcine thin filaments. Product dissociation from cardiac actomyosin is an ordered process in which phosphate dissociation is rate limiting (AM-mdADP-Pi → AM-mdADP → AM) and can therefore be measured by the rate of mdADP dissociation. Myosin-S1 was mixed with an equal concentration of the fluorescent ATP analogue, mdATP, and held in a delay line 2 seconds for the nucleotide to bind and be hydrolyzed (M + mdATP → M-mdATP ↔M-mdADP-Pi). The mixture was subsequently mixed with native cardiac thin filaments, 1 mM MgATP, 1 mM EGTA and expressed mouse cMyBP-C domains C1C2. In control experiments cMyBP-C was either replaced with NEM-S1 (a catalytically inactive form of myosin-S1, modifed with N-ethylmaleimide, which binds to actin in the presence of ATP), rigor myosin-S1 (in the absence of ATP) or 0.1 mM CaCl2. Maximal activation of the rate of product dissociation by cMyBP-C domains C1C2 is the same as observed with saturating rigor-myosin-S1 or 0.1 mM calcium and greater than observed with NEM-S1. These experiments were done at physiological ionic strength (0.18 M KAc, 10 mM Mops, 2 mM MgCl2, pH 7) to eliminate non specific ionic interaction that might occur at lower ionic strength. Cardiac cMyBP-C domains C1C2 not only bind to actin at a similar position to rigor myosin-S1 but substoichiometric concentrations (1 cMyBP-C C1C2 per 7 actin subunits) are as effective as rigor myosin-S1 at activating cardiac thin filament in the absence of calcium.
• Bers, D. M., & Harris, S. P. (2011). Translational medicine: to the rescue of the failing heart. Nature, 473(7345), 36-9.
• Harris, S. P., & Bezold, K. L. (2011). Charged Amino Acids in the N-Terminus of Cardiac Myosin Binding Protein-C Contribute to Contractile Effects in Permeabilized Myocytes. Biophysical Journal, 100(3), 454a. doi:10.1016/j.bpj.2010.12.2674
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  Recombinant N-terminal domains of cardiac myosin binding protein C (cMyBP-C) increase calcium-sensitivity of force (pCa50) and the rate of tension redevelopement (ktr) in permeabilized cardiac myocytes. To identify specific amino acids required for these effects, we investigated functional effects of alanine substitution mutations of charged residues near phosphorylation sites within the regulatory M-domain of cMyBP-C. Mutations were introduced into a recombinant protein, C1C2, comprised of the M-domain and its two flanking Ig domains (i.e, C1-M-C2). Compared to wild-type C1C2, a double mutant containing A for R substitutions at positions 270 and 271 upstream of S273 (phosphorlyation site A), reduced the ability of C1C2 to increase force in permeabilized rat cardiac trabeculae. Another double mutant, the 299AA300 substitution for 299KR300 upstream of S302 (phosphorylation site C), also reduced the efficacy of C1C2. A triple mutant that replaced a cluster of three charged residues 298KKR300 nearly abolished the functional effects of C1C2. These results suggest that positively charged amino acids upstream of protein kinase A phosphorylation sites are important for mediating the contractile effects of C1C2 and that these sites may be involved in the in vivo response to -adrenergic stimulation potentially by contributing to binding of the M-domain to actin and/or myosin S2. This work is supported by NIH HL080367 to SPH and a DOD NDSEG fellowship to KLB.
• Harris, S. P., Lyons, R. G., & Bezold, K. L. (2011). In the thick of it: HCM-causing mutations in myosin binding proteins of the thick filament. Circulation research, 108(6), 751-64.
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  In the 20 years since the discovery of the first mutation linked to familial hypertrophic cardiomyopathy (HCM), an astonishing number of mutations affecting numerous sarcomeric proteins have been described. Among the most prevalent of these are mutations that affect thick filament binding proteins, including the myosin essential and regulatory light chains and cardiac myosin binding protein (cMyBP)-C. However, despite the frequency with which myosin binding proteins, especially cMyBP-C, have been linked to inherited cardiomyopathies, the functional consequences of mutations in these proteins and the mechanisms by which they cause disease are still only partly understood. The purpose of this review is to summarize the known disease-causing mutations that affect the major thick filament binding proteins and to relate these mutations to protein function. Conclusions emphasize the impact that discovery of HCM-causing mutations has had on fueling insights into the basic biology of thick filament proteins and reinforce the idea that myosin binding proteins are dynamic regulators of the activation state of the thick filament that contribute to the speed and force of myosin-driven muscle contraction. Additional work is still needed to determine the mechanisms by which individual mutations induce hypertrophic phenotypes.
• Karsai, A., Kellermayer, M. S., & Harris, S. P. (2011). Mechanical unfolding of cardiac myosin binding protein-C by atomic force microscopy. Biophysical journal, 101(8), 1968-77.
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  Cardiac myosin-binding protein-C (cMyBP-C) is a thick-filament-associated protein that performs regulatory and structural roles within cardiac sarcomeres. It is a member of the immunoglobulin (Ig) superfamily of proteins consisting of eight Ig- and three fibronectin (FNIII)-like domains, along with a unique regulatory sequence referred to as the M-domain, whose structure is unknown. Domains near the C-terminus of cMyBP-C bind tightly to myosin and mediate the association of cMyBP-C with thick (myosin-containing) filaments, whereas N-terminal domains, including the regulatory M-domain, bind reversibly to myosin S2 and/or actin. The ability of MyBP-C to bind to both myosin and actin raises the possibility that cMyBP-C cross-links myosin molecules within the thick filament and/or cross-links myosin and thin (actin-containing) filaments together. In either scenario, cMyBP-C could be under mechanical strain. However, the physical properties of cMyBP-C and its behavior under load are completely unknown. Here, we investigated the mechanical properties of recombinant baculovirus-expressed cMyBP-C using atomic force microscopy to assess the stability of individual cMyBP-C molecules in response to stretch. Force-extension curves showed the presence of long extensible segment(s) that became stretched before the unfolding of individual Ig and FNIII domains, which were evident as sawtooth peaks in force spectra. The forces required to unfold the Ig/FNIII domains at a stretch rate of 500 nm/s increased monotonically from ∼30 to ∼150 pN, suggesting a mechanical hierarchy among the different Ig/FNIII domains. Additional experiments using smaller recombinant proteins showed that the regulatory M-domain lacks significant secondary or tertiary structure and is likely an intrinsically disordered region of cMyBP-C. Together, these data indicate that cMyBP-C exhibits complex mechanical behavior under load and contains multiple domains with distinct mechanical properties.
• Kellermayer, M. S., Karsai, A., & Harris, S. P. (2011). The Motif of Myosin Binding Protein-C is Mechanically Weak and Extensible. Biophysical Journal, 100(3), 453a-454a. doi:10.1016/j.bpj.2010.12.2672
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  Cardiac myosin binding protein-C (cMyBP-C) is a member of the immunoglobulin (Ig) superfamily of proteins and consists of 8 Ig- and 3 fibronectin (Fn)-like domains along with a unique regulatory sequence referred to as the MyBP-C “motif” or M-domain. The structure of the M-domain is not known, but small angle X-ray scattering experiments suggest that it adopts a compact shape in solution and that its overall dimensions are similar to other Ig-like domains. To investigate whether the M-domain behaves similarly to an Ig domain under mechanical stress or load, we used atomic force microscopy (AFM) to investigate single molecule elasticity and mechanical properties of recombinant full-length mouse cardiac cMyBP-C and smaller proteins containing just the M-domain and flanking Ig- sequences. Force-extension curves of full-length cMyBP-C showed unfolding of individual Ig or Fn-like domains at forces between 40-150 pN (500 nm/s pulling speed) which were evident as “saw tooth” peaks in force spectra. Spectra with up to 11 peaks were obtained. The curves also revealed region(s) of very low stability that could be stretched >100 nm prior to any unfolding event. In force extension curves of a shorter three domain construct comprised only of the M-domain and its two flanking Ig domains (i.e., C1-M-C2) an extensible region with a contour length of 43 ± 4 nm and persistence length of 0.42 ± 0.1 nm was also observed before domain unfolding in no more than two sawtooth peaks. The latter data suggest that the M-domain contributes to the extensible segment of cMyBP-C and that the M-domain exhibits mechanical properties that are distinct from those of Ig or Fn-like domains. The M-domain may thus function as a spring-like element within cMyBP-C. Supported by NIH HL080367.
• Kensler, R. W., Shaffer, J. F., & Harris, S. P. (2011). Binding of the N-terminal fragment C0-C2 of cardiac MyBP-C to cardiac F-actin. Journal of structural biology, 174(1), 44-51.
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  Cardiac myosin-binding protein C (cMyBP-C), a major accessory protein of cardiac thick filaments, is thought to play a key role in the regulation of myocardial contraction. Although current models for the function of the protein focus on its binding to myosin S2, other evidence suggests that it may also bind to F-actin. We have previously shown that the N-terminal fragment C0-C2 of cardiac myosin-binding protein-C (cMyBP-C) bundles actin, providing evidence for interaction of cMyBP-C and actin. In this paper we directly examined the interaction between C0-C2 and F-actin at physiological ionic strength and pH by negative staining and electron microscopy. We incubated C0-C2 (5-30μM, in a buffer containing in mM: 180 KCl, 1 MgCl(2), 1 EDTA, 1 DTT, 20 imidazole, at pH 7.4) with F-actin (5μM) for 30min and examined negatively-stained samples of the solution by electron microscopy (EM). Examination of EM images revealed that C0-C2 bound to F-actin to form long helically-ordered complexes. Fourier transforms indicated that C0-C2 binds with the helical periodicity of actin with strong 1st and 6th layer lines. The results provide direct evidence that the N-terminus of cMyBP-C can bind to F-actin in a periodic complex. This interaction of cMyBP-C with F-actin supports the possibility that binding of cMyBP-C to F-actin may play a role in the regulation of cardiac contraction.
• Jia, W., Shaffer, J. F., Harris, S. P., & Leary, J. A. (2010). Identification of novel protein kinase A phosphorylation sites in the M-domain of human and murine cardiac myosin binding protein-C using mass spectrometry analysis. Journal of proteome research, 9(4), 1843-53.
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  Cardiac myosin binding protein-C (cMyBP-C) is a large multidomain accessory protein bound to myosin thick filaments in striated muscle sarcomeres. It plays an important role in the regulation of muscle contraction, and mutations in the gene encoding cMyBP-C are a common cause of familial hypertrophic cardiomyopathy, the leading cause of sudden cardiac death in young people. (1) The N-terminal domains including the C0, C1, cMyBP-C motif, and C2 domains play a crucial role in maintaining and modulating actomyosin interactions (keeping normal cardiac function) in a phosphorylation-dependent manner. The cMyBP-C motif or "M-domain" is a highly conserved linker domain in the N-terminus of cMyBP-C that contains three to five protein kinase A (PKA) phosphorylation sites, depending on species. For the human isoform, three PKA sites were previously identified (Ser(275), Ser(284), and Ser(304)), while three homologous sites exist in the murine isoform (Ser(273), Ser(282), and Ser(302)). The murine cMyBP-C isoform contains an additional conserved consensus site, Ser(307) that is not present in the human isoform. In this study, we investigated sites of PKA phosphorylation of murine and human cMyBP-C by treating the recombinant protein C0C2 ( approximately 50 KDa, which contains the N-terminal C0, C1, M, and C2 domains) and C1C2 (approximately 35 KDa, contains C1, M, and C2 domains) with PKA and assessing the phosphorylation states using SDS-PAGE with ProQ Diamond staining, and powerful hybrid mass spectrometric analyses. Both high-accuracy bottom-up and measurements of intact proteins mass spectrometric approaches were used to determine the phosphorylation states of C0C2 and C1C2 proteins with or without PKA treatment. Herein, we report for the first time that there are four PKA phosphorylation sites in both murine and human M-domains; both murine Ser(307) and a novel human Ser(311) can be phosphorylated in vitro by PKA. Future studies are needed to investigate the phosphorylation state of murine and human cMyBP-C in vivo.
• Shaffer, J. F., & Harris, S. P. (2010). Identification of Amino Acid Residues in the Cardiac Myosin Binding Protein-C Motif Important for Actin Binding. Biophysical Journal, 98(3), 756a. doi:10.1016/j.bpj.2009.12.4150
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  N-terminal domains of cardiac myosin binding protein-C (cMyBP-C) can activate actomyosin interactions in the absence of Ca2+ and bind to actin in a phosphorylation dependent manner. We have previously shown that two N-terminal domains, C1 and the MyBP-C motif (“M”) domain, bind specifically to actin and to thin filaments; however, the sequences or residues that mediate actin binding have not been identified. The goal of this study was to identify residues in the M-domain that contribute to actin binding and to investigate whether interactions between the M-domain and actin mediate the activating properties of cMyBP-C. We therefore used alanine-scanning mutagenesis to target candidate actin binding sites in the M-domain that bear homology to the actin binding motifs in other known actin binding proteins and to assess the effects of mutations on the ability of recombinant proteins to bind actin and activate actomyosin interactions in motility assays. Results demonstrate that mutation of select positively-charged amino acids in the M-domain that are homologous to binding motifs in known actin binding proteins reduced binding of cMyBP-C to actin. The mutations also reduced or eliminated the activating properties of recombinant cMyBP-C in in vitro motility assays. However, mutation of other positively-charged amino acids did not affect actin binding or protein functional properties. These results indicate that specific residues within the M-domain confer actin binding and that interactions with actin contribute to the functional effects of recombinant cMyBP-C N-terminal proteins. Supported by NIH HL080367 to SPH and a NSF Graduate Research Fellowship to JFS.
• Shaffer, J. F., Kittleson, M. D., Hoye, E., Harris, S. P., Gomes, A. V., & Bezold, K. L. (2010). Incorporation of the A31P Cardiac Myosin Binding Protein C Missense Mutation Into Feline Cardiac Sarcomeres. Biophysical Journal, 98(3), 554a. doi:10.1016/j.bpj.2009.12.2999
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  Mutations in cardiac myosin binding protein C (cMyBP-C) are a frequent cause of hypertrophic cardiomyopathy (HCM), a major cause of sudden cardiac death and heart failure. Mutations include single amino acid substitutions and premature stop codons, but it is unclear whether dominant negative effects of mutant proteins, depletion of wild-type protein due to an affected allele (haploinsufficiency), or aberrant protein processing/degradation leads to disease. To distinguish among these possibilities, we investigated the sarcomeric localization and functional effects of a spontaneous cMyBP-C missense mutation in Maine Coon cats, a naturally occurring feline model of HCM. Immunofluorescent localization using an antibody specific for the A31P mutation showed that A31P cMyBP-C was incorporated into the sarcomeres of cats heterozygous and homozygous for the A31P mutation with similar distribution patterns as wild-type cMyBP-C. However, dominant negative effects due to incorporation of the mutant protein were not evident because myofilament Ca2+ sensitivity of tension and rate of tension development were not different in permeabilized myocytes from wild-type versus A31P cats. Actin binding and in vitro motility experiments also showed no difference between wild-type and A31P recombinant feline C0C2 proteins. By contrast, cytosolic proteasomes from a homozygous cat showed elevated β-5 (chymotrypsin-like) proteolitic activity compared to wild-type or heterozygous cats. Additional experiments are necessary to determine whether aberrant protein degradation of A31P cMyBP-C contributes to disease. Supported by NIH HL080367.
• Shaffer, J. F., Wong, P., Bezold, K. L., & Harris, S. P. (2010). Functional differences between the N-terminal domains of mouse and human myosin binding protein-C. Journal of biomedicine & biotechnology, 2010, 789798.
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  The N-terminus of cMyBP-C can activate actomyosin interactions in the absence of Ca2+, but it is unclear which domains are necessary. Prior studies suggested that the Pro-Ala rich region of human cMyBP-C activated force in permeabilized human cardiomyocytes, whereas the C1 and M-domains of mouse cMyBP-C activated force in permeabilized rat cardiac trabeculae. Because the amino acid sequence of the P/A region differs between human and mouse cMyBP-C isoforms (46% identity), we investigated whether species-specific differences in the P/A region could account for differences in activating effects. Using chimeric fusion proteins containing combinations of human and mouse C0, Pro-Ala, and C1 domains, we demonstrate here that the human P/A and C1 domains activate actomyosin interactions, whereas the same regions of mouse cMyBP-C are less effective. These results suggest that species-specific differences between homologous cMyBP-C isoforms confer differential effects that could fine-tune cMyBP-C function in hearts of different species.
• Wong, P., Shaffer, J. F., Harris, S. P., & Bezold, K. L. (2010). Comparative Effects of the Proline-Alanine Rich Regions of Human and Murine Cardiac Myosin Binding Protein-C. Biophysical Journal, 98(3), 555a. doi:10.1016/j.bpj.2009.12.3003
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  The N-terminus of cMyBP-C can activate actomyosin interactions in the absence of Ca2+, but it is unclear which sequences mediate the activating effects. Herron et al. (Circ Res, 98:1290-8, 2006) found that the Pro-Ala rich region (P-A) of human cMyBP-C could activate tension in the absence of Ca2+, whereas Razumova et al. (J Gen Physiol, 132:575-85, 2008) found that murine C1 and M domains activated tension. The different results might be explained by isoform differences, especially in P-A which is only 46% identical between mouse and human cMyBP-C. The goal of this study was to determine if species-specific differences in P-A account for the different activating effects of murine and human cMyBP-C. Recombinant chimeric proteins containing the C0, P-A, and C1 domains (C0C1) from either human or murine cMyBP-C were engineered and their activating effects assessed using in vitro motility and ATPase assays. Consistent with previous observations, human C0C1 activated actomyosin interactions in the absence of Ca2+, whereas murine C0C1 did not. However, substituting human P-A for murine P-A conferred activating properties to murine C0C1, whereas substituting murine P-A for human P-A depressed the activating effects of human C0C1. Activating effects of the chimera proteins were intermediate between those of murine and human C0C1, suggesting that C0 or C1 also contribute to activation properties. Further chimeric substitutions of C0 and C1 demonstrated that the human C1 domain also contributed to activation, whereas the C0 domain did not. These results suggest that the human P-A and C1 domains are sufficient to activate actomyosin interactions in the absence of Ca2+, and that species-specific differences are likely to contribute to functional differences of cMyBP-C. Supported by NIH HL080367 to SPH and a NSF Graduate Research Fellowship to JFS.
• Shaffer, J. F., & Harris, S. P. (2009). Species-specific differences in the Pro-Ala rich region of cardiac myosin binding protein-C. Journal of muscle research and cell motility, 30(7-8), 303-6.
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  Cardiac myosin binding protein-C (cMyBP-C) is an accessory protein found in the A-bands of vertebrate sarcomeres and mutations in the cMyBP-C gene are a leading cause of familial hypertrophic cardiomyopathy. The regulatory functions of cMyBP-C have been attributed to the N-terminus of the protein, which is composed of tandem immunoglobulin (Ig)-like domains (C0, C1, and C2), a region rich in proline and alanine residues (the Pro-Ala rich region) that links C0 and C1, and a unique sequence referred to as the MyBP-C motif, or M-domain, that links C1 and C2. Recombinant proteins that contain various combinations of the N-terminal domains of cMyBP-C can activate actomyosin interactions in the absence of Ca(2+), but the specific sequences required for these effects differ between species; the Pro-Ala region has been implicated in human cMyBP-C whereas the C1 and M-domains appear important in mouse cMyBP-C. To investigate whether species-specific differences in sequence can account for the observed differences in function, we compared sequences of the Pro-Ala rich region in cMyBP-C isoforms from different species. Here we report that the number of proline and alanine residues in the Pro-Ala rich region varies significantly between different species and that the number correlates directly with mammalian body size and inversely with heart rate. Thus, systematic sequence differences in the Pro-Ala rich region of cMyBP-C may contribute to observed functional differences in human versus mouse cMyBP-C isoforms and suggest that the Pro-Ala region may be important in matching contractile speed to cardiac function across species.
• Shaffer, J. F., Harris, S. P., & Bezold, K. L. (2009). Functional Effects of cMyBP-C Phospho-Mimics in Permeabilized Trabeculae. Biophysical Journal, 96(3), 500a. doi:10.1016/j.bpj.2008.12.2581
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  Myosin Binding Protein C (MyBP-C) is a sarcomeric protein that has both structural and regulatory roles in striated muscle contraction. Cardiac (c) isoforms of MyBP-C can be phosphorylated by protein kinase A (PKA) at three to five sites within a unique regulatory region referred to as the MyBP-C motif. We have previously shown, using permeabilized rat trabeculae, that the recombinant protein C1C2, which contains the motif, significantly increased Ca2+ sensitivity of force and increased rates of tension redevelopment (ktr) at submaximal [Ca2+]. To investigate whether these effects are modulated by phosphorylation of the motif, we used the catalytic subunit of PKA to phosphorylate C1C2. In addition, we used site directed mutagenesis to mutate three key serine residues (Ser273, 282, 302) to aspartic acids to mimic phosphorylation at these sites. Results demonstrated that either 10uM phosphorylated C1C2 (C1C2P) or 10uM phospho-mimic C1C2 (C1C23S/D) increased Ca2+ sensitivity of force and increased rates of tension redevelopment (ktr) at submaximal [Ca2+]. However, the phospho-mimic C1C23S/D was more effective than C1C2P in producing these effects. Together these results indicate that the 3 Ser to Asp phospho-mimic does not fully mimic effects of PKA phosphorylation of C1C2 and that the functional effects of C1C2 in permeabilized cardiac trabeculae are mediated at least in part through phosphorylation-independent mechanisms. Supported by NIH HL080367.
• Shaffer, J. F., Kensler, R. W., & Harris, S. P. (2009). The N-terminus of Cardiac Myosin Binding Protein-C Contains Multiple Binding Sites for F-actin. Biophysical Journal, 96(3), 371a. doi:10.1016/j.bpj.2008.12.2000
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  Cardiac myosin binding protein-C (cMyBP-C), long known to interact with thick filaments, also interacts with thin filaments (actin) through its N-terminus. However, a single actin binding site has not been identified and it is unclear whether one or more N-terminal domains of cMyBP-C interact with actin. In this study we aimed to characterize the interaction of the N-terminus of cMyBP-C with actin using recombinant proteins consisting of various cMyBP-C N-terminal domains. Results from high speed cosedimentation binding assays showed that recombinant proteins containing the C1 domain and the MyBP-C motif bound to F-actin at a 1:1 molar ratio with a dissociation constant (Kd) ~ 10 uM. In contrast, proteins containing either C1 or the motif showed reduced binding at a 1:2 molar ratio. Proteins containing both C1 and the motif also bundled actin filaments, suggesting multiple actin interaction sites. Binding of recombinant proteins to Ca2+ regulated thin filaments was similar to binding to F-actin alone. Strongly bound myosin cross-bridges (myosin S1, no ATP) abolished cMyBP-C binding to actin, while weakly bound crossbridges (myosin S1 plus ATP) diminished, but did not abolish, binding. Recombinant myosin ΔS2, which binds to the MyBP-C motif in vitro (~6 uM), did not affect cMyBP-C binding to actin. However, phosphorylation of the motif or alkaline pH both reduced binding. Together, these results suggest that the N-terminus of cMyBP-C contains at least two binding sites for actin and that binding is modulated through electrostatic interactions. Supported by NIH HL080367 to SPH and a NSF Graduate Research Fellowship to JFS.
• Shaffer, J. F., Kensler, R. W., & Harris, S. P. (2009). The myosin-binding protein C motif binds to F-actin in a phosphorylation-sensitive manner. The Journal of biological chemistry, 284(18), 12318-27.
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  Cardiac myosin-binding protein C (cMyBP-C) is a regulatory protein expressed in cardiac sarcomeres that is known to interact with myosin, titin, and actin. cMyBP-C modulates actomyosin interactions in a phosphorylation-dependent way, but it is unclear whether interactions with myosin, titin, or actin are required for these effects. Here we show using cosedimentation binding assays, that the 4 N-terminal domains of murine cMyBP-C (i.e. C0-C1-m-C2) bind to F-actin with a dissociation constant (K(d)) of approximately 10 microm and a molar binding ratio (B(max)) near 1.0, indicating 1:1 (mol/mol) binding to actin. Electron microscopy and light scattering analyses show that these domains cross-link F-actin filaments, implying multiple sites of interaction with actin. Phosphorylation of the MyBP-C regulatory motif, or m-domain, reduced binding to actin (reduced B(max)) and eliminated actin cross-linking. These results suggest that the N terminus of cMyBP-C interacts with F-actin through multiple distinct binding sites and that binding at one or more sites is reduced by phosphorylation. Reversible interactions with actin could contribute to effects of cMyBP-C to increase cross-bridge cycling.
• Shaffer, J. F., Kensler, R. W., Harris, S. P., & Bezold, K. L. (2009). Switching Gears with Myosin Binding Protein-C. Biophysical Journal, 96(3), 4a. doi:10.1016/j.bpj.2008.12.912
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  Myosin binding protein-C (MyBP-C) is a thick-filament protein in vertebrate sarcomeres that limits cross-bridge cycling kinetics and reduces myocyte power output. However, the mechanisms by which MyBP-C influences cross-bridge kinetics are not well understood. The goal of the present study was to investigate the ability of the first 4 N-terminal domains (C0-C1-motif-C2) of cardiac (c) MyBP-C to affect actomyosin interactions and interact with actin. Here we show that recombinant proteins containing the C1 and motif domains increased Ca2+ sensitivity of tension and increased rates of tension redevelopment (ktr) at submaximal [Ca2+] in permeabilized rat trabeculae. Proteins containing these domains also biphasically activated then inhibited Ca2+-activated ATPase rates of heavy meromyosin and myosin S1 in solution. Cosedimentation binding assays demonstrated saturable binding of the 4 N-terminal domains to F-actin at a 1:1 molar ratio (Kd ∼ 10μM). However, more than one interaction site was indicated by turbidity and electron microscope analyses that showed actin bundling in the presence of recombinant proteins. Phosphorylation of the motif or increasing pH reduced binding to a 1:2 molar ratio and abolished actin bundling. Phosphorylation reduced but did not eliminate effects of recombinant proteins to increase Ca2+ sensitivity of tension and ktr at submaximal [Ca2+] in permeabilized trabeculae. Together these results suggest that the N-terminus of cMyBP-C interacts with F-actin through multiple distinct sites, at least one site is modulated by electrostatic charge interactions, and that functional effects of the N-terminus of MyBP-C are mediated in part by phosphorylation independent mechanisms. Supported by NIH HL080367.
• Shaffer, J. F., Leary, J. A., Jia, W., & Harris, S. P. (2009). PKA Phosphorylates Serine 307 of Murine Cardiac Myosin Binding Protein-C In Vitro. Biophysical Journal, 96(3), 500a. doi:10.1016/j.bpj.2008.12.2580
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  Cardiac myosin binding protein-C is a regulatory protein associated with sarcomere A-bands that modulates actomyosin interactions in a phosphorylation dependent manner. The MyBP-C motif, a highly conserved sequence in the N-terminus of cMyBP-C, contains three to five protein kinase A (PKA) phosphorylation sites, depending on species. In the human isoform, three PKA sites have been identified (S275, S284, and S304). Three homologous sites exist in the murine isoform (S273, S282, and S302) along with a potential fourth site, S307, which is not present in human cMyBP-C. In this study, we investigated the effects of PKA phosphorylation of murine cMyBP-C by treating a recombinant protein, C1C2 (which contains the C1, motif, and C2 domains), with PKA and assessing phosphorylation levels using IEF gels, ProQ Diamond staining, and mass spectrometry. The wild-type C1C2 has a pI of ∼8 and PKA treatment (C1C2P) shifted the pI to ∼5-6 as determined by 1-D IEF gels. A mutant C1C2 (3S/D), containing aspartic acid for serine substitutions at S273D, S282D, and S302D, was still phosphorylated upon treatment with PKA as indicated by increased ProQ Diamond staining. However, a mutant 4S/D C1C2 (containing the additional mutation S307D) showed a pI near that of C1C2P and was not further phosphorylated by PKA. Mass spectrometry and MASCOT analysis of C1C2P confirmed that S307 was phosphorylated by PKA. These results suggest that murine S307 can be phosphorylated in vitro. Further studies are needed to investigate the phosphorylation state of murine cMyBP-C in vivo. Supported by NIH HL080367 to SPH and a NSF Graduate Research Fellowship to JFS.
• Jeffries, C. M., Whitten, A. E., Harris, S. P., & Trewhella, J. (2008). Small-angle scattering: the regulatory domains of cardiac C-protein and their complex with F-actin. Acta Crystallographica Section A, 64(a1), 297-297. doi:10.1107/s010876730809051x
• Kensler, R. W., & Harris, S. P. (2008). The structure of isolated cardiac Myosin thick filaments from cardiac Myosin binding protein-C knockout mice.. Biophysical journal, 94(5), 1707-18. doi:10.1529/biophysj.107.115899
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  Mutations in the thick filament associated protein cardiac myosin binding protein-C (cMyBP-C) are a major cause of familial hypertrophic cardiomyopathy. Although cMyBP-C is thought to play both a structural and a regulatory role in the contraction of cardiac muscle, detailed information about the role of this protein in stability of the thick filament and maintenance of the ordered helical arrangement of the myosin cross-bridges is limited. To address these questions, the structure of myosin thick filaments isolated from the hearts of wild-type mice containing cMyBP-C (cMyBP-C(+/+)) were compared to those of cMyBP-C knockout mice lacking this protein (cMyBp-C(-/-)). The filaments from the knockout mice hearts lacking cMyBP-C are stable and similar in length and appearance to filaments from the wild-type mice hearts containing cMyBP-C. Both wild-type and many of the cMyBP-C(-/-) filaments display a distinct 43 nm periodicity. Fourier transforms of electron microscope images typically show helical layer lines to the sixth layer line, confirming the well-ordered arrangement of the cross-bridges in both sets of filaments. However, the "forbidden" meridional reflections, thought to derive from a perturbation from helical symmetry in the wild-type filament, are weaker or absent in the transforms of the cMyBP-C(-/-) myocardial thick filaments. In addition, the cross-bridge array in the absence of cMyBP-C appears more easily disordered.
• Luther, P. K., Bennett, P. M., Knupp, C., Craig, R., Padrón, R., Harris, S. P., Patel, J., & Moss, R. L. (2008). Understanding the organisation and role of myosin binding protein C in normal striated muscle by comparison with MyBP-C knockout cardiac muscle. Journal of molecular biology, 384(1), 60-72.
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  Myosin binding protein C (MyBP-C) is a component of the thick filament of striated muscle. The importance of this protein is revealed by recent evidence that mutations in the cardiac gene are a major cause of familial hypertrophic cardiomyopathy. Here we investigate the distribution of MyBP-C in the A-bands of cardiac and skeletal muscles and compare this to the A-band structure in cardiac muscle of MyBP-C-deficient mice. We have used a novel averaging technique to obtain the axial density distribution of A-bands in electron micrographs of well-preserved specimens. We show that cardiac and skeletal A-bands are very similar, with a length of 1.58+/-0.01 mum. In normal cardiac and skeletal muscle, the distributions are very similar, showing clearly the series of 11 prominent accessory protein stripes in each half of the A-band spaced axially at 43-nm intervals and starting at the edge of the bare zone. We show by antibody labelling that in cardiac muscle the distal nine stripes are the location of MyBP-C. These stripes are considerably suppressed in the knockout mouse hearts as expected. Myosin heads on the surface of the thick filament in relaxed muscle are thought to be arranged in a three-stranded quasi-helix with a mean 14.3-nm axial cross bridge spacing and a 43 nm helix repeat. Extra "forbidden" meridional reflections, at orders of 43 nm, in X-ray diffraction patterns of muscle have been interpreted as due to an axial perturbation of some levels of myosin heads. However, in the MyBP-C-deficient hearts these extra meridional reflections are weak or absent, suggesting that they are due to MyBP-C itself or to MyBP-C in combination with a head perturbation brought about by the presence of MyBP-C.
• Razumova, M. V., Bezold, K. L., Tu, A., Regnier, M., & Harris, S. P. (2008). Contribution of the myosin binding protein C motif to functional effects in permeabilized rat trabeculae. The Journal of general physiology, 132(5), 575-85.
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  Myosin binding protein C (MyBP-C) is a thick-filament protein that limits cross-bridge cycling rates and reduces myocyte power output. To investigate mechanisms by which MyBP-C affects contraction, we assessed effects of recombinant N-terminal domains of cardiac MyBP-C (cMyBP-C) on contractile properties of permeabilized rat cardiac trabeculae. Here, we show that N-terminal fragments of cMyBP-C that contained the first three immunoglobulin domains of cMyBP-C (i.e., C0, C1, and C2) plus the unique linker sequence termed the MyBP-C "motif" or "m-domain" increased Ca(2+) sensitivity of tension and increased rates of tension redevelopment (i.e., k(tr)) at submaximal levels of Ca(2+). At concentrations > or =20 microM, recombinant proteins also activated force in the absence of Ca(2+) and inhibited maximum Ca(2+)-activated force. Recombinant proteins that lacked the combination of C1 and the motif did not affect contractile properties. These results suggest that the C1 domain plus the motif constitute a functional unit of MyBP-C that can activate the thin filament.
• Tu, A., Regnier, M., Razumova, M. V., Harris, S. P., & Bezold, K. L. (2008). The N-terminus of myosin binding protein-C diminishes the Ca2+ dependence of the rate of tension redevelopment in permeabilized cardiac trabeculae. The FASEB Journal, 22.
• Whitten, A. E., Jeffries, C. M., Harris, S. P., & Trewhella, J. (2008). A small-angle neutron contrast variation study of the complex of actin and myosin binding protein C. Acta Crystallographica Section A, 64(a1), 297-297. doi:10.1107/s0108767308090508
• Whitten, A. E., Jeffries, C. M., Harris, S. P., & Trewhella, J. (2008). Cardiac myosin-binding protein C decorates F-actin: implications for cardiac function. Proceedings of the National Academy of Sciences of the United States of America, 105(47), 18360-5.
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  Cardiac myosin-binding protein C (cMyBP-C) is an accessory protein of striated muscle sarcomeres that is vital for maintaining regular heart function. Its 4 N-terminal regulatory domains, C0-C1-m-C2 (C0C2), influence actin and myosin interactions, the basic contractile proteins of muscle. Using neutron contrast variation data, we have determined that C0C2 forms a repeating assembly with filamentous actin, where the C0 and C1 domains of C0C2 attach near the DNase I-binding loop and subdomain 1 of adjacent actin monomers. Direct interactions between the N terminus of cMyBP-C and actin thereby provide a mechanism to modulate the contractile cycle by affecting the regulatory state of the thin filament and its ability to interact with myosin.
• Whitten, A. E., Trewhella, J., Jeffries, C. M., & Harris, S. P. (2008). Small-angle X-ray scattering reveals the N-terminal domain organization of cardiac myosin binding protein C.. Journal of molecular biology, 377(4), 1186-99. doi:10.1016/j.jmb.2008.01.080
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  Myosin binding protein C (MyBP-C) is a multidomain accessory protein of striated muscle sarcomeres. Three domains at the N-terminus of MyBP-C (C1-m-C2) play a crucial role in maintaining and modulating actomyosin interactions. The cardiac isoform has an additional N-terminal domain (C0) that is postulated to provide a greater level of regulatory control in cardiac muscle. We have used small-angle X-ray scattering, ab initio shape restoration, and rigid-body modeling to determine the average shape and spatial arrangement of the four N-terminal domains of cardiac MyBP-C (C0C2) and a three-domain variant that is analogous to the N-terminus of the skeletal isoform (C1C2). We found that the domains of both proteins are tandemly arranged in a highly extended configuration that is sufficiently long to span the interfilament cross-bridge distances in vivo and, hence, be poised to modulate these interactions. The average spatial organization of the C1, m, and C2 domains is not significantly perturbed by the removal of the cardiac-specific C0 domain, suggesting that the interdomain interfaces, while relatively small in area, have a degree of rigidity. Modeling the C0C2 and C1C2 scattering data reveals that the structures of the C0 and m domains (also referred to as the 'MyBP motif') are compact and have dimensions that are consistent with the immunoglobulin fold superfamily of proteins. Sequence analysis, homology modeling, and circular dichroism experiments support the conclusion that the previously undetermined structures of these domains can be characterized as having an immunoglobulin-like fold. Atomic models using the known NMR structures for C1 and C2 as well as homology models for the C0 and m domains provide insights into the placement of conserved serine residues of the m domain that are phosphorylated in vivo and cause a change in muscle fiber contraction by abolishing interactions with myosin.
• Shaffer, J. F., Razumova, M. V., Tu, A., Regnier, M., & Harris, S. P. (2007). Myosin S2 is not required for effects of myosin binding protein-C on motility. FEBS letters, 581(7), 1501-4.
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  The unique myosin binding protein-c "motif" near the N-terminus of myosin binding protein-C (MyBP-C) binds myosin S2. Previous studies demonstrated that recombinant proteins containing the motif and flanking regions (e.g., C1C2) affect thin filament movement in motility assays using heavy meromyosin (S1 plus S2) as the molecular motor. To determine if S2 is required for these effects we investigated whether C1C2 affects motility in assays using only myosin S1 as the motor protein. Results demonstrate that effects of C1C2 are comparable in both systems and suggest that the MyBP-C motif affects motility through direct interactions with actin and/or myosin S1.
• Razumova, M. V., Shaffer, J. F., Tu, A., Flint, G. V., Regnier, M., & Harris, S. P. (2006). Effects of the N-terminal domains of myosin binding protein-C in an in vitro motility assay: Evidence for long-lived cross-bridges. The Journal of biological chemistry, 281(47), 35846-54.
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  Myosin binding protein-C (MyBP-C) is a thick-filament protein whose precise function within the sarcomere is not known. However, recent evidence from cMyBP-C knock-out mice that lack MyBP-C in the heart suggest that cMyBP-C normally slows cross-bridge cycling rates and reduces myocyte power output. To investigate possible mechanisms by which cMyBP-C limits cross-bridge cycling kinetics we assessed effects of recombinant N-terminal domains of MyBP-C on the ability of heavy meromyosin (HMM) to support movement of actin filaments using in vitro motility assays. Here we show that N-terminal domains of cMyBP-C containing the MyBP-C "motif," a sequence of approximately 110 amino acids, which is conserved across all MyBP-C isoforms, reduced actin filament velocity under conditions where filaments are maximally activated (i.e. either in the absence of thin filament regulatory proteins or in the presence of troponin and tropomyosin and high [Ca2+]). By contrast, under conditions where thin filament sliding speed is submaximal (i.e. in the presence of troponin and tropomyosin and low [Ca2+]), proteins containing the motif increased filament speed. Recombinant N-terminal proteins also bound to F-actin and inhibited acto-HMM ATPase rates in solution. The results suggest that N-terminal domains of MyBP-C slow cross-bridge cycling kinetics by reducing rates of cross-bridge detachment.
• Rostkova, E., Moss, R. L., Harris, S. P., & Gautel, M. (2004). Binding of myosin binding protein-C to myosin subfragment S2 affects contractility independent of a tether mechanism.. Circulation research, 95(9), 930-6. doi:10.1161/01.res.0000147312.02673.56
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  Mutations in the cardiac myosin binding protein-C gene (cMyBP-C) are among the most prevalent causes of inherited hypertrophic cardiomyopathy. Although most cMyBP-C mutations cause reading frameshifts that are predicted to encode truncated peptides, it is not known if or how expression of these peptides causes disease. One possibility is that because the N-terminus contains a unique binding site for the S2 subfragment of myosin, shortened cMyBP-C peptides could directly affect myosin contraction by binding to S2. To test this hypothesis, we compared the effects of a C1C2 protein containing the myosin S2 binding site on contractile properties in permeabilized myocytes from wild-type and cMyBP-C knockout mice. In wild-type myocytes, the C1C2 protein reversibly increased myofilament Ca2+ sensitivity of tension, but had no effect on resting tension. Identical results were observed in cMyBP-C knockout myocytes where C1C2 increased Ca2+ sensitivity of tension with the half-maximal response elicited at approximately 5 micromol/L C1C2. Maximum force was not affected by C1C2. However, phosphorylation of C1C2 by cAMP-dependent protein kinase reduced its ability to increase Ca2+ sensitivity. These results demonstrate that binding of the C1C2 peptide to S2 alone is sufficient to affect myosin contractile function and suggest that regulated binding of cMyBP-C to myosin S2 by phosphorylation directly influences myofilament Ca2+ sensitivity.
• Wang, Y., Seidman, J. G., Seidman, C. E., Palmer, B. M., Moss, R. L., Maughan, D. W., Kass, D. A., Janssen, P. M., Harris, S. P., Georgakopoulos, D., Burgon, P. G., Belardi, D. F., & Alpert, N. R. (2004). Role of cardiac myosin binding protein C in sustaining left ventricular systolic stiffening.. Circulation research, 94(9), 1249-55. doi:10.1161/01.res.0000126898.95550.31
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  Despite advances in the molecular biology of cardiac myosin binding protein-C (cMyBP-C), little is understood about its precise role in muscle contraction, particularly in the intact heart. We tested the hypothesis that cMyBP-C is central to the time course and magnitude of left ventricular systolic elastance (chamber stiffening), and assessed mechanisms for this influence in intact hearts, trabeculae, and skinned fibers from wild-type (+/+) and homozygous truncated cMyBP-C (t/t) male mice. cMyBP-C protein was not detected by gel electrophoresis or Western blot in t/t myocardium. cMyBP-C t/t ventricles displayed reduced peak elastance, but more strikingly a marked abbreviation of the systolic elastance time course, which peaked earlier (27.6+/-2.1 ms) than in +/+ controls (47.8+/-1.6 ms). Control hearts reached only 42+/-4% of maximum elastance at the onset of ejection, with substantial further stiffening during ejection. This contrasted to t/t mutants, which reached 77+/-3% of peak elastance before ejection of peak. These unusual findings were not observed in alternative models involving severe cardiomyopathy, but were recapitulated in a cMyBP-C null mouse. The abbreviated elastance time course and lower peak were consistent with earlier time-to-peak trabecular tension, increased unloaded shortening velocity in t/t skinned muscle strips, and dramatically reduced myofilament stiffness at diastolic calcium concentrations. These results provide novel insights into the role of cMyBP-C in myocardial systolic mechanics. Abnormal sarcomere shortening velocity and abbreviated muscle stiffening may underlie development of cardiac dysfunction associated with deficient incorporation of cMyBP-C.
• Moss, R. L., Mcdonald, K. S., Korte, F. S., & Harris, S. P. (2003). Loaded shortening, power output, and rate of force redevelopment are increased with knockout of cardiac myosin binding protein-C.. Circulation research, 93(8), 752-8. doi:10.1161/01.res.0000096363.85588.9a
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  Myosin binding protein-C (MyBP-C) is localized to the thick filaments of striated muscle where it appears to have both structural and regulatory functions. Importantly, mutations in the cardiac MyBP-C gene are associated with familial hypertrophic cardiomyopathy. The purpose of this study was to examine the role that MyBP-C plays in regulating force, power output, and force development rates in cardiac myocytes. Skinned cardiac myocytes from wild-type (WT) and MyBP-C knockout (MyBP-C-/-) mice were attached between a force transducer and position motor. Force, loaded shortening velocities, and rates of force redevelopment were measured during both maximal and half-maximal Ca2+ activations. Isometric force was not different between the two groups with force being 17.0+/-7.2 and 20.5+/-3.1 kN/m2 in wild-type and MyBP-C-/- myocytes, respectively. Peak normalized power output was significantly increased by 26% in MyBP-C-/- myocytes (0.15+/-0.01 versus 0.19+/-0.03 P/Po x ML/sec) during maximal Ca2+ activations. Interestingly, peak power output in MyBP-C-/- myocytes was increased to an even greater extent (46%, 0.09+/-0.03 versus 0.14+/-0.02 P/Po x ML/sec) during half-maximal Ca2+ activations. There was also an effect on the rate constant of force redevelopment (ktr) during half-maximal Ca2+ activations, with ktr being significantly greater in MyBP-C-/- myocytes (WT=5.8+/-0.9 s(-1) versus MyBP-C-/-=7.7+/-1.7 s(-1)). These results suggest that cMyBP-C is an important regulator of myocardial work capacity whereby MyBP-C acts to limit power output.
• Trewhella, J., Moss, R. L., Heller, W. T., Harris, S. P., & Greaser, M. L. (2003). Solution structure of heavy meromyosin by small-angle scattering.. The Journal of biological chemistry, 278(8), 6034-40. doi:10.1074/jbc.m210558200
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  Elucidation of x-ray crystal structures for the S1 subfragment of myosin afforded atomic resolution of the nucleotide and actin binding sites of the enzyme. The structures have led to more detailed hypotheses regarding the mechanisms by which force generation is coupled to ATP hydrolysis. However, the three-dimensional structure of double-headed myosin consisting of two S1 subfragments has not yet been solved. Therefore, to investigate the overall shape and relative orientations of the two heads of myosin, we performed small-angle x-ray and neutron scattering measurements of heavy meromyosin containing all three light chains (LC(1-3)) in solution. The resulting small-angle scattering intensity profiles were best fit by models of the heavy meromyosin head-tail junction in which the angular separation between heads was less than 180 degrees. The S1 heads of the best fit models are not related by an axis of symmetry, and one of the two S1 heads is bent back along the rod. These results provide new information on the structure of the head-tail junction of myosin and indicate that combining scattering measurements with high resolution structural modeling is a feasible approach for investigating myosin head-head interactions in solution.
• Powers, P. A., Moss, R. L., Mcdonald, K. S., Harris, S. P., Hacker, T. A., Greaser, M. L., Douglas, P. S., & Bartley, C. R. (2002). Hypertrophic cardiomyopathy in cardiac myosin binding protein-C knockout mice.. Circulation research, 90(5), 594-601. doi:10.1161/01.res.0000012222.70819.64
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  Familial hypertrophic cardiomyopathy (FHC) is an inherited autosomal dominant disease caused by mutations in sarcomeric proteins. Among these, mutations that affect myosin binding protein-C (MyBP-C), an abundant component of the thick filaments, account for 20% to 30% of all mutations linked to FHC. However, the mechanisms by which MyBP-C mutations cause disease and the function of MyBP-C are not well understood. Therefore, to assess deficits due to elimination of MyBP-C, we used gene targeting to produce a knockout mouse that lacks MyBP-C in the heart. Knockout mice were produced by deletion of exons 3 to 10 from the endogenous cardiac (c) MyBP-C gene in murine embryonic stem (ES) cells and subsequent breeding of chimeric founder mice to obtain mice heterozygous (+/-) and homozygous (-/-) for the knockout allele. Wild-type (+/+), cMyBP-C(+/-), and cMyBP-C(-/-) mice were born in accordance with Mendelian inheritance ratios, survived into adulthood, and were fertile. Western blot analyses confirmed that cMyBP-C was absent in hearts of homozygous knockout mice. Whereas cMyBP-C(+/-) mice were indistinguishable from wild-type littermates, cMyBP-C(-/-) mice exhibited significant cardiac hypertrophy. Cardiac function, assessed using 2-dimensionally guided M-mode echocardiography, showed significantly depressed indices of diastolic and systolic function only in cMyBP-C(-/-) mice. Ca2+ sensitivity of tension, measured in single skinned myocytes, was reduced in cMyBP-C(-/-) but not cMyBP-C(+/-) mice. These results establish that cMyBP-C is not essential for cardiac development but that the absence of cMyBP-C results in profound cardiac hypertrophy and impaired contractile function.
• Patel, J. R., Moss, R. L., Marton, L. J., & Harris, S. P. (2000). Polyamines decrease Ca(2+) sensitivity of tension and increase rates of activation in skinned cardiac myocytes.. American journal of physiology. Heart and circulatory physiology, 279(3), H1383-91. doi:10.1152/ajpheart.2000.279.3.h1383
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  Owing in part to their interactions with membrane proteins, polyamines (e.g., spermine, spermidine, and putrescine) have been identified as potential modulators of membrane excitability and Ca(2+) homeostasis in cardiac myocytes. To investigate whether polyamines also affect cardiac myofilament proteins, we assessed the effects of polyamines on contractility using rat myocytes and trabeculae that had been permeabilized with Triton X-100. Spermine, spermidine, and putrescine reversibly increased the [Ca(2+)] required for half-maximal tension (i.e., right-shifted tension pCa curves), with the following order of efficacy: spermine (+4) > spermidine (+3) > putrescine (+2). However, synthetic analogs that differed from spermine in charge distribution were not as effective as spermine in altering isometric tension. None of the polyamines had a significant effect on maximal tension, except at high concentrations. After flash photolysis of DM-Nitrophen (a caged Ca(2+) chelator), spermine accelerated the rate of tension development at low and intermediate but not high [Ca(2+)]. These results indicate that polyamines, especially spermine, interact with myofilament proteins to reduce apparent Ca(2+) binding affinity and speed cross-bridge cycling kinetics at submaximal [Ca(2+)].

Presentations

• Harris, S. (2019, April). Regulation of cardiac contraction by cMyBP-C: Introducing a novel cut and paste approach for studying sarcomeric proteins. Invited seminar speaker at Cytokinetics, South San Francisco, CA (April, 2019)Cytokinetics, Inc..
• Harris, S. (2019, April/Spring). A cut and paste approach for studyingcardiac myosin binding protein-C. Invited seminar at Cincinnati Children's Hospital, Cincinnati, OH. Cincinnati, OH: Cincinnati Children's Hospital.
• Harris, S. (2019, April/Spring). A cut and paste approach to studyingcardiac myosin binding protein-C. Invited seminar at Tufts University. Boston, MA: Tufts University.
• Harris, S. (2019, April/Spring). What's the Catch? Lessons Marion Taught Me about Mussels, Muscles and Myosin Binding Protein-C. Award recipient and invited lecturer for Marion J. Siegman Lectureship, American Physiological Society, Experimental Biology, Orlando, FL. Orlando, FL: American Physiological Society.
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  Marion J. Siegman Lectureship, American Physiological Society, Experimental Biology, Orlando, FL (April, 2019)
• Harris, S. (2019, August). Stop making waves: A new role for cardiac myosin binding protein-C in damping contractile oscillations. Invited speaker at International Society of Biomechanics/American Society of Biomechanics, Calgary, Alberta, Canada (August, 2019).. Calgary, Canada: International Society of Biomechanics/American Society of Biomechanics.
• Harris, S. (2019, July). Stop making waves: A new role for cardiac myosin binding protein-C in damping contractile oscillations. Invited speaker at American Heart Association, Basic Council on Cardiovascular Sciences (July, 2019). Boston, MA: American Heart Association.
• Harris, S. (2019, March/Winter). Acute loss of cMyBP-C inducesauto-oscillations in cardiac sarcomeres: Implications for reverse EC coupling?. Invited symposium speaker for the annual meeting of the Biophysical Society, Baltimore, MD. Baltimore, MD: Biophysical Society.
• Harris, S. (2019, November). Making waves: A new role for Cardiac myosin binding protein-C (cMyBP-C) in damping contractile oscillations. Invited seminar at the Pennsylvania Muscle Institute. Philadelphia, PA: University of Pennsylvania.
• Harris, S. (2019, November). Making waves: A novel role for cMyBP-C in damping contractile oscillations. Invited speaker at the annual meeting of Scientific Sessions. Philadelphia, PA: American Heart Association.
• Harris, S. (2019, November). Stop making waves: A new role for cardiac myosin binding protein-C in damping contractile oscillations. Invited seminar at Texas A&M University. College Station, TX: Texas A&M University.
• Harris, S. (2019, October). Stop making waves: A new role for cardiac myosin binding protein-C in damping contractile oscillations. Invited seminar at Fralin Biomedical Research Institute. Roanoke, VA: Virginia Tech Carilion School of Medicine.
• Harris, S. (2019, September). Stop making waves: A new role for cardiac myosin binding protein-C in damping contractile oscillations. Faculty seminar in BioMedical Engineering. Tucson, AZ: Biomedical Engineering, U Arizona.
• Harris, S. (2019, September). Stop making waves: A new role for cardiac myosin binding protein-C in damping contractile oscillations. Invited seminar speaker at Eastern Virginia Medical School. Norfolk, VA: Eastern Virginia Medical School.
• Harris, S. (2018, April/Spring). Regulation of cardiac contraction by cMyBP-C. Invited seminar at Novartis. Boston, MA: Novartis.
• Harris, S. (2018, April/Spring). Regulation of cardiac contraction by cardiac myosin binding protein-C (cMyBP-C). Invited departmental seminar at Washington University School of Medicine, Dept of Biochemistry and Molecular Biophysics. St. Louis, MO: Washington University School of Medicine, Dept of Biochemistry and Molecular Biophysics.
• Harris, S. (2018, April/Spring). Regulation of cardiac contraction by cardiac myosin binding protein-C (cMyBP-C). Invited seminar at Ohio State University. Columbus, OH: Ohio State University.
• Harris, S. (2018, Feb/Spring). Hitting a moving (drug) target: cardiac myosin binding protein-C interactions with actin. Invited seminar at Amgen. San Francisco, CA: Amegen, Inc.
• Harris, S. (2018, Feb/Winter). A “cut and paste” approach for modification of cMyBP-C in sarcomeres in situ. Invited seminar at MyoKardia, Inc. South San Francisco, CA: MyoKardia, Inc..
• Harris, S. (2018, Feb/Winter). A “cut and paste” approach for modification of cMyBP-C in sarcomeres in situ. Platform presentation (selected) at the Annual Meeting of the Biophysical Society in San Francisco, CA. San Francisco, CA: Biophysical Society.
• Harris, S. (2018, Feb/Winter). Transgenic mouse models of cMyBP-C interactions with actin. Invited seminar at MyoKardia, Inc. South SanFrancisco, CA: MyoKardia, Inc.
• Harris, S. (2018, June/Summer). Cutting and pasting myofilament proteins (cMyBP-C) with Spy-C mice. Invited talk at Cardiac Regulatory Mechanisms Gordon Conference, Colby-Sawyer College, New London, NH. Colby-Sawyer College, New London, NH: Cardiac Regulatory Gordon Conference.
• Harris, S. (2018, Oct/Fall). New roles for cardiac myosin binding protein-C in cardiac contraction: Bridging the gap between thick and thin filaments. Invited seminar at University of Washington. Seattle, WA: University of Washington.
• Harris, S. (2017, April/Spring). Regulation of cardiac contraction by cardiac myosin binding protein-C. Invited departmental Seminar at UCSD. San Diego, CA: University of California, San Diego Department of Medicine.
• Harris, S. (2017, February/Spring). Regulation of cardiac contraction by cardiac myosin binding protein-C. Invited departmental seminar, University of Miami. Miami, FL: University of Miami, Miller School of Medicine, Dept of Molecular and Cellular Pharmacology.
• Harris, S. (2017, March/Spring). Regulation of cardiac contraction by cardiac myosin binding protein-C. Invited seminar, University of Minnesota, Dept of Integrative Biology and Physiology. Minneapolis, MN: University of Minnesota, Dept of integrative biology and physiology.
• Harris, S. (2017, May/Spring). A case study in hypertrophic cardiomyopathy: from mutation to large animal model using the A31P mutation in cats. Invited seminar presentation to the Animal Genetics group at UC Davis. UC Davis, Davis, CA: UC Davis.
• Harris, S. (2017, Oct/Fall). Hypertrophic cardiomyopathy in cats: A novel large animal model of disease. Invited seminar at MyoKardia, Inc. South San Francisco, CA.
• Harris, S. (2017, October/Fall). Hypertrophic cardiomyopathy in cats: a novel large animal model of disease. Invited seminar at MyoKardia, Inc. South San Francisco, CA: MyoKardia, Inc..
• Harris, S. (2015, November). Myosin binding protein-C: A promiscuous actin binding protein. Invited research seminar at Texas A&M University. Texas A&M University, Temple, TX.

Poster Presentations

• Harris, S. (2018, Sept/Fall). Acute loss of cMyBP-C N’-terminal domains induces spontaneous oscillatory contractions (SPOC) in permeabilized myocytes from Spy-C mice. European Muscle Conference Annual Meeting, Budapest, Hungary.