Samantha Harris

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• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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
• Bers, D. M., & Harris, S. P. (2011). Translational medicine: to the rescue of the failing heart. Nature, 473(7345), 36-9.
• 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.
• 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., 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.
• 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., 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.
• 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.
• 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.
• 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.

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.