Shanna Hamilton

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• Hamilton, S., Terentyeva, R., Veress, R., Perger, F., Nichtova, Z., Bannister, M., Wang, J., Quiggle, S., Battershell, R., Gorr, M. W., Györke, S., Choi, B. R., George, C. H., Belevych, A. E., Csordás, G., & Terentyev, D. (2025). Increased Intermembrane Space [Ca] Drives Mitochondrial Structural Damage in CPVT. Circulation research, 137(12), 1385-1403.
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  Mitochondrial dysfunction caused by abnormally high RyR2 (ryanodine receptor) activity is a common finding in cardiovascular diseases. Mechanisms linking RyR2 gain of function with mitochondrial remodeling remain elusive. We hypothesized that RyR2 hyperactivity in cardiac disease increases [Ca] in the mitochondrial intermembrane space (IMS) and activates the Ca-sensitive protease calpain, driving remodeling of mitochondrial cristae architecture through cleavage of structural protein OPA1 (optic atrophy protein 1).
• Terentyev, D., Glukhov, A. V., Bogdanov, V., Averin, A. S., Veress, R., Choi, B. R., Hamilton, S., & Belevych, A. E. (2025). Small-conductance Ca⁺-activated K⁺ channels in cardiac excitation-contraction coupling: Bridging mitochondria, sarcolemma and antiarrhythmic therapy. The Journal of physiology.
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  Small-conductance Ca⁺-activated K⁺ (SK) channels have emerged over the past decade as compelling antiarrhythmic targets. All three isoforms, SK1, SK2 and SK3, are expressed in both atrial and ventricular cardiomyocytes, where they are exclusively gated by intracellular Ca⁺ via constitutively bound calmodulin. Sarcolemmal SK channels uniquely translate elevations in intracellular Ca concentration into action potential repolarization. In doing so they mitigate pro-arrhythmic disturbances in membrane potential caused by pathological spontaneous Ca⁺ release from sarcoplasmic reticulum, thereby reducing Ca-mediated arrhythmia triggers such as early and delayed afterdepolarizations. However the role of SK channels in arrhythmogenesis is complex. Although their activation can be protective against triggered activity, additional repolarizing force under certain conditions may shorten the action potential excessively and create a substrate for re-entrant arrhythmias. Furthermore SK channels have recently been found in cardiac mitochondria, where they appear to regulate mitochondrial Ca⁺ handling and reactive oxygen species (ROS) production, suggesting a prominent role in cardioprotection. The contribution of mitochondrial SK (mito-SK) channels to cardiac arrhythmogenesis, however, remains incompletely understood. In this review we summarize current advances in understanding the therapeutic potential of SK channels as an antiarrhythmic target, with a particular focus on the contribution of mito-SK channels to cardioprotection and mitochondrial ROS production.
• Veress, R., Terentyeva, R., Belevych, A. E., Perger, F., Nichtova, Z., Pokrass, A., Cheng, Y., Chorna, S., Deschenes, I., Gyorke, S., Knollmann, B. C., Clements, R. T., Singh, H., Liu, B., Csordas, G., Hamilton, S., & Terentyev, D. (2025). Pharmacological Enhancement of Small Conductance Ca2+-Activated K+ Channels Suppresses Cardiac Arrhythmias in a Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia. Circulation Research, 137(Issue 3). doi:10.1161/circresaha.124.325477
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  BACKGROUND: Sarcolemmal small conductance Ca2+-activated K+ channels have the unique capacity to translate intracellular Ca2+ signal into repolarization, while mitochondrial SK channels can link Ca2+ cycling to mitochondrial function. We hypothesize that pharmacological enhancement of SK channels can be protective against malignant cardiac arrhythmias associated with disturbances in Ca2+ handling machinery. METHODS: A mouse CASQ2 KO (calsequestrin type 2 knockout) model of catecholaminergic polymorphic ventricular tachycardia (CPVT) was used for in vivo ECG recordings and for cell electrophysiology, Ca2+, and reactive oxygen species imaging in isolated ventricular myocytes (VMs). RESULTS: Bidirectional and polymorphic ventricular tachycardias in CASQ2 KO mice induced by stress challenge (epinephrine+caffeine cocktail) were attenuated by injection of NS309, a specific SK channel enhancer. Voltage-clamp experiments in isolated VMs treated with β-adrenergic agonist isoproterenol showed a reduction of sarcolemmal SK channel current (ISK) density in CPVT VMs. Application of NS309 to CPVT VMs increased ISK. Current-clamp experiments demonstrated a significant reduction of arrhythmogenic delayed afterdepolarizations and spontaneous Ca2+ waves in isoproterenol-challenged CPVT VMs pretreated with NS309. Importantly, subsequent application of membrane-impermeable SK channel inhibitor apamin did not reverse the protective effects of NS309, suggesting important roles of mitochondrial SK channels in intracellular Ca2+ handling rescue. SK channel enhancement reversed the increased rate of reactive oxygen species production by mitochondria in CPVT VMs. It also reversed increased cardiac RyR2 (ryanodine receptor 2) oxidation measured in samples from CPVT hearts of the animals after the stress challenge. Electron microscopy studies showed a significant widening of mitochondria cristae in the ventricular tissue from CPVT mice, which led to a decrease in quaternary supercomplexes of electron transport chain, resulting in impairment of ATP production in VMs under β-adrenergic stimulation. Application of NS309 facilitated cristae flattening in CPVT ventricular tissue and restored supercomplexes and ATP production in VMs from diseased animals. CONCLUSIONS: Sarcolemmal SK channel enhancement reduces arrhythmic potential by restoring repolarization force in CPVT VMs. Activation of mitochondrial SK channels attenuates mitochondria structural changes in CPVT, restoring more efficient electron transport chain assembly into supercomplexes and reducing mito-reactive oxygen species production. This decreases RyR2 oxidation and thus channel activity, reducing spontaneous Ca2+ waves underlying arrhythmogenic delayed afterdepolarizations.
• Hamilton, S., & Terentyev, D. (2024). The yellow brick road to understanding the RyR2 signalosome. Journal of Physiology, 602(20). doi:10.1113/JP287538
• Pinckard, K., Hamilton, S., Terentyeva, R., Baer, L., Wright, K., Nassal, D., Esteves, J., Abay, E., Shettigar, V., Ziolo, M., Hund, T., Wold, L., Terentyev, D., Stanford, K., & Félix-Soriano, E. (2024). Maternal exercise preserves offspring cardiovascular health via oxidative regulation of the ryanodine receptor. Molecular Metabolism, 82. doi:10.1016/j.molmet.2024.101914
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  Objective: The intrauterine environment during pregnancy is a critical factor in the development of obesity, diabetes, and cardiovascular disease in offspring. Maternal exercise prevents the detrimental effects of a maternal high fat diet on the metabolic health in adult offspring, but the effects of maternal exercise on offspring cardiovascular health have not been thoroughly investigated. Methods: To determine the effects of maternal exercise on offspring cardiovascular health, female mice were fed a chow (C; 21% kcal from fat) or high-fat (H; 60% kcal from fat) diet and further subdivided into sedentary (CS, HS) or wheel exercised (CW, HW) prior to pregnancy and throughout gestation. Offspring were maintained in a sedentary state and chow-fed throughout 52 weeks of age and subjected to serial echocardiography and cardiomyocyte isolation for functional and mechanistic studies. Results: High-fat fed sedentary dams (HS) produced female offspring with reduced ejection fraction (EF) compared to offspring from chow-fed dams (CS), but EF was preserved in offspring from high-fat fed exercised dams (HW) throughout 52 weeks of age. Cardiomyocytes from HW female offspring had increased kinetics, calcium cycling, and respiration compared to CS and HS offspring. HS offspring had increased oxidation of the RyR2 in cardiomyocytes coupled with increased baseline sarcomere length, resulting in RyR2 overactivity, which was negated in female HW offspring. Conclusions: These data suggest a role for maternal exercise to protect against the detrimental effects of a maternal high-fat diet on female offspring cardiac health. Maternal exercise improved female offspring cardiomyocyte contraction, calcium cycling, respiration, RyR2 oxidation, and RyR2 activity. These data present an important, translatable role for maternal exercise to preserve cardiac health of female offspring and provide insight on mechanisms to prevent the transmission of cardiovascular diseases to subsequent generations.
• Clements, R. T., Terentyeva, R., Hamilton, S., Janssen, P. M., Roder, K., Martin, B. Y., Perger, F., Schneider, T., Nichtova, Z., Das, A. S., Veress, R., Lee, B. S., Kim, D. G., Koren, G., Stratton, M. S., Csordas, G., Accornero, F., Belevych, A. E., Gyorke, S., & Terentyev, D. (2023). Sexual dimorphism in bidirectional SR-mitochondria crosstalk in ventricular cardiomyocytes. Basic research in cardiology, 118(1), 15.
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  Calcium transfer into the mitochondrial matrix during sarcoplasmic reticulum (SR) Ca release is essential to boost energy production in ventricular cardiomyocytes (VCMs) and match increased metabolic demand. Mitochondria from female hearts exhibit lower mito-[Ca] and produce less reactive oxygen species (ROS) compared to males, without change in respiration capacity. We hypothesized that in female VCMs, more efficient electron transport chain (ETC) organization into supercomplexes offsets the deficit in mito-Ca accumulation, thereby reducing ROS production and stress-induced intracellular Ca mishandling. Experiments using mitochondria-targeted biosensors confirmed lower mito-ROS and mito-[Ca] in female rat VCMs challenged with β-adrenergic agonist isoproterenol compared to males. Biochemical studies revealed decreased mitochondria Ca uniporter expression and increased supercomplex assembly in rat and human female ventricular tissues vs male. Importantly, western blot analysis showed higher expression levels of COX7RP, an estrogen-dependent supercomplex assembly factor in female heart tissues vs males. Furthermore, COX7RP was decreased in hearts from aged and ovariectomized female rats. COX7RP overexpression in male VCMs increased mitochondrial supercomplexes, reduced mito-ROS and spontaneous SR Ca release in response to ISO. Conversely, shRNA-mediated knockdown of COX7RP in female VCMs reduced supercomplexes and increased mito-ROS, promoting intracellular Ca mishandling. Compared to males, mitochondria in female VCMs exhibit higher ETC subunit incorporation into supercomplexes, supporting more efficient electron transport. Such organization coupled to lower levels of mito-[Ca] limits mito-ROS under stress conditions and lowers propensity to pro-arrhythmic spontaneous SR Ca release. We conclude that sexual dimorphism in mito-Ca handling and ETC organization may contribute to cardioprotection in healthy premenopausal females.
• Clements, R., Terentyeva, R., Hamilton, S., Janssen, P., Roder, K., Martin, B., Perger, F., Schneider, T., Nichtova, Z., Das, A., Veress, R., Lee, B., Kim, D., Koren, G., Stratton, M., Csordas, G., Accornero, F., Belevych, A., Gyorke, S., & Terentyev, D. (2023). Sexual dimorphism in bidirectional SR-mitochondria crosstalk in ventricular cardiomyocytes. Basic Research in Cardiology, 118(1). doi:10.1007/s00395-023-00988-1
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  Calcium transfer into the mitochondrial matrix during sarcoplasmic reticulum (SR) Ca2+ release is essential to boost energy production in ventricular cardiomyocytes (VCMs) and match increased metabolic demand. Mitochondria from female hearts exhibit lower mito-[Ca2+] and produce less reactive oxygen species (ROS) compared to males, without change in respiration capacity. We hypothesized that in female VCMs, more efficient electron transport chain (ETC) organization into supercomplexes offsets the deficit in mito-Ca2+ accumulation, thereby reducing ROS production and stress-induced intracellular Ca2+ mishandling. Experiments using mitochondria-targeted biosensors confirmed lower mito-ROS and mito-[Ca2+] in female rat VCMs challenged with β-adrenergic agonist isoproterenol compared to males. Biochemical studies revealed decreased mitochondria Ca2+ uniporter expression and increased supercomplex assembly in rat and human female ventricular tissues vs male. Importantly, western blot analysis showed higher expression levels of COX7RP, an estrogen-dependent supercomplex assembly factor in female heart tissues vs males. Furthermore, COX7RP was decreased in hearts from aged and ovariectomized female rats. COX7RP overexpression in male VCMs increased mitochondrial supercomplexes, reduced mito-ROS and spontaneous SR Ca2+ release in response to ISO. Conversely, shRNA-mediated knockdown of COX7RP in female VCMs reduced supercomplexes and increased mito-ROS, promoting intracellular Ca2+ mishandling. Compared to males, mitochondria in female VCMs exhibit higher ETC subunit incorporation into supercomplexes, supporting more efficient electron transport. Such organization coupled to lower levels of mito-[Ca2+] limits mito-ROS under stress conditions and lowers propensity to pro-arrhythmic spontaneous SR Ca2+ release. We conclude that sexual dimorphism in mito-Ca2+ handling and ETC organization may contribute to cardioprotection in healthy premenopausal females.
• Terentyev, D., Belevych, A. E., Choi, B. R., & Hamilton, S. (2023). To block or not to block: Targeting SK channels in diseased hearts. Journal of molecular and cellular cardiology, 183, 98-99.
• Terentyev, D., Belevych, A., Choi, B., & Hamilton, S. (2023). To block or not to block: Targeting SK channels in diseased hearts. Journal of Molecular and Cellular Cardiology, 183. doi:10.1016/j.yjmcc.2023.09.005
• Hamilton, S., & Terentyev, D. (2022). ER stress and calcium-dependent arrhythmias. Frontiers in physiology, 13, 1041940.
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  The sarcoplasmic reticulum (SR) plays the key role in cardiac function as the major source of Ca that activates cardiomyocyte contractile machinery. Disturbances in finely-tuned SR Ca release by SR Ca channel ryanodine receptor (RyR2) and SR Ca reuptake by SR Ca-ATPase (SERCa2a) not only impair contraction, but also contribute to cardiac arrhythmia trigger and reentry. Besides being the main Ca storage organelle, SR in cardiomyocytes performs all the functions of endoplasmic reticulum (ER) in other cell types including protein synthesis, folding and degradation. In recent years ER stress has become recognized as an important contributing factor in many cardiac pathologies, including deadly ventricular arrhythmias. This brief review will therefore focus on ER stress mechanisms in the heart and how these changes can lead to pro-arrhythmic defects in SR Ca handling machinery.
• Hamilton, S., Terentyeva, R., Bogdanov, V., Kim, T. Y., Perger, F., Yan, J., Ai, X., Carnes, C. A., Belevych, A. E., George, C. H., Davis, J. P., Gyorke, S., Choi, B. R., & Terentyev, D. (2022). Ero1α-Dependent ERp44 Dissociation From RyR2 Contributes to Cardiac Arrhythmia. Circulation research, 130(5), 711-724.
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  Oxidative stress in cardiac disease promotes proarrhythmic disturbances in Ca homeostasis, impairing luminal Ca regulation of the sarcoplasmic reticulum (SR) Ca release channel, the RyR2 (ryanodine receptor), and increasing channel activity. However, exact mechanisms underlying redox-mediated increase of RyR2 function in cardiac disease remain elusive. We tested whether the oxidoreductase family of proteins that dynamically regulate the oxidative environment within the SR are involved in this process.
• Hamilton, S., Terentyeva, R., Bogdanov, V., Kim, T., Perger, F., Yan, J., Ai, X., Carnes, C., Belevych, A., George, C., Davis, J., Gyorke, S., Choi, B., & Terentyev, D. (2022). Ero1α -Dependent ERp44 Dissociation from RyR2 Contributes to Cardiac Arrhythmia. Circulation Research, 130(5). doi:10.1161/CIRCRESAHA.121.320531
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  Background: Oxidative stress in cardiac disease promotes proarrhythmic disturbances in Ca2+homeostasis, impairing luminal Ca2+regulation of the sarcoplasmic reticulum (SR) Ca2+release channel, the RyR2 (ryanodine receptor), and increasing channel activity. However, exact mechanisms underlying redox-mediated increase of RyR2 function in cardiac disease remain elusive. We tested whether the oxidoreductase family of proteins that dynamically regulate the oxidative environment within the SR are involved in this process. Methods: A rat model of hypertrophy induced by thoracic aortic banding (TAB) was used for ex vivo whole heart optical mapping and for Ca2+and reactive oxygen species imaging in isolated ventricular myocytes (VMs). Results: The SR-targeted reactive oxygen species biosensor ERroGFP showed increased intra-SR oxidation in TAB VMs that was associated with increased expression of Ero1α (endoplasmic reticulum oxidoreductase 1 alpha). Pharmacological (EN460) or genetic Ero1α inhibition normalized SR redox state, increased Ca2+transient amplitude and SR Ca2+content, and reduced proarrhythmic spontaneous Ca2+waves in TAB VMs under β -adrenergic stimulation (isoproterenol). Ero1α overexpression in Sham VMs had opposite effects. Ero1α inhibition attenuated Ca2+-dependent ventricular tachyarrhythmias in TAB hearts challenged with isoproterenol. Experiments in TAB VMs and human embryonic kidney 293 cells expressing human RyR2 revealed that an Ero1α -mediated increase in SR Ca2+-channel activity involves dissociation of intraluminal protein ERp44 (endoplasmic reticulum protein 44) from the RyR2 complex. Site-directed mutagenesis and molecular dynamics simulations demonstrated a novel redox-sensitive association of ERp44 with RyR2 mediated by intraluminal cysteine 4806. ERp44-RyR2 association in TAB VMs was restored by Ero1α inhibition, but not by reducing agent dithiothreitol, as hypo-oxidation precludes formation of covalent bond between RyR2 and ERp44. Conclusions: A novel axis of intraluminal interaction between RyR2, ERp44, and Ero1α has been identified. Ero1α inhibition exhibits promising therapeutic potential by stabilizing RyR2-ERp44 complex, thereby reducing spontaneous Ca2+release and Ca2+-dependent tachyarrhythmias in hypertrophic hearts, without causing hypo-oxidative stress in the SR.
• Ponnalagu, D., Hamilton, S., Sanghvi, S., Antelo, D., Schwieterman, N., Hansra, I., Xu, X., Gao, E., Edwards, J. C., Bansal, S. S., Wold, L. E., Terentyev, D., Janssen, P. M., Hund, T. J., Khan, M., Kohut, A. R., Koch, W. J., & Singh, H. (2022). CLIC4 localizes to mitochondrial-associated membranes and mediates cardioprotection. Science advances, 8(42), eabo1244.
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  Mitochondrial-associated membranes (MAMs) are known to modulate organellar and cellular functions and can subsequently affect pathophysiology including myocardial ischemia-reperfusion (IR) injury. Thus, identifying molecular targets in MAMs that regulate the outcome of IR injury will hold a key to efficient therapeutics. Here, we found chloride intracellular channel protein (CLIC4) presence in MAMs of cardiomyocytes and demonstrate its role in modulating ER and mitochondrial calcium homeostasis under physiological and pathological conditions. In a murine model, loss of CLIC4 increased myocardial infarction and substantially reduced cardiac function after IR injury. CLIC4 null cardiomyocytes showed increased apoptosis and mitochondrial dysfunction upon hypoxia-reoxygenation injury in comparison to wild-type cardiomyocytes. Overall, our results indicate that MAM-CLIC4 is a key mediator of cellular response to IR injury and therefore may have a potential implication on other pathophysiological processes.
• Ponnalagu, D., Hamilton, S., Sanghvi, S., Antelo, D., Schwieterman, N., Hansra, I., Xu, X., Gao, E., Edwards, J., Bansal, S., Wold, L., Terentyev, D., Janssen, P., Hund, T., Khan, M., Kohut, A., Koch, W., & Singh, H. (2022). CLIC4 localizes to mitochondrial-associated membranes and mediates cardioprotection. Science Advances, 8(42). doi:10.1126/sciadv.abo1244
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  Mitochondrial-associated membranes (MAMs) are known to modulate organellar and cellular functions and can subsequently affect pathophysiology including myocardial ischemia-reperfusion (IR) injury. Thus, identifying molecular targets in MAMs that regulate the outcome of IR injury will hold a key to efficient therapeutics. Here, we found chloride intracellular channel protein (CLIC4) presence in MAMs of cardiomyocytes and demonstrate its role in modulating ER and mitochondrial calcium homeostasis under physiological and pathological conditions. In a murine model, loss of CLIC4 increased myocardial infarction and substantially reduced cardiac function after IR injury. CLIC4 null cardiomyocytes showed increased apoptosis and mitochondrial dysfunction upon hypoxia-reoxygenation injury in comparison to wild-type cardiomyocytes. Overall, our results indicate that MAM-CLIC4 is a key mediator of cellular response to IR injury and therefore may have a potential implication on other pathophysiological processes.
• Hamilton, S., & Terentyev, D. (2021). RyR2 Gain-of-Function and Not So Sudden Cardiac Death. Circulation research, 129(3), 417-419.
• Hamilton, S., Terentyeva, R., Clements, R. T., Belevych, A. E., & Terentyev, D. (2021). Sarcoplasmic reticulum-mitochondria communication; implications for cardiac arrhythmia. Journal of molecular and cellular cardiology, 156, 105-113.
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  Sudden cardiac death due to ventricular tachyarrhythmias remains the major cause of mortality in the world. Heart failure, diabetic cardiomyopathy, old age-related cardiac dysfunction and inherited disorders are associated with enhanced propensity to malignant cardiac arrhythmias. Both defective mitochondrial function and abnormal intracellular Ca homeostasis have been established as the key contributing factors in the pathophysiology and arrhythmogenesis in these conditions. This article reviews current advances in understanding of bidirectional control of ryanodine receptor-mediated sarcoplasmic reticulum Ca release and mitochondrial function, and how defects in crosstalk between these two organelles increase arrhythmic risk in cardiac disease.
• Hamilton, S., Terentyeva, R., Perger, F., Hernández Orengo, B., Martin, B., Gorr, M. W., Belevych, A. E., Clements, R. T., Györke, S., & Terentyev, D. (2021). MCU overexpression evokes disparate dose-dependent effects on mito-ROS and spontaneous Ca release in hypertrophic rat cardiomyocytes. American journal of physiology. Heart and circulatory physiology, 321(4), H615-H632.
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  Cardiac dysfunction in heart failure (HF) and diabetic cardiomyopathy (DCM) is associated with aberrant intracellular Ca handling and impaired mitochondrial function accompanied with reduced mitochondrial calcium concentration (mito-[Ca]). Pharmacological or genetic facilitation of mito-Ca uptake was shown to restore Ca transient amplitude in DCM and HF, improving contractility. However, recent reports suggest that pharmacological enhancement of mito-Ca uptake can exacerbate ryanodine receptor-mediated spontaneous sarcoplasmic reticulum (SR) Ca release in ventricular myocytes (VMs) from diseased animals, increasing propensity to stress-induced ventricular tachyarrhythmia. To test whether chronic recovery of mito-[Ca] restores systolic Ca release without adverse effects in diastole, we overexpressed mitochondrial Ca uniporter (MCU) in VMs from male rat hearts with hypertrophy induced by thoracic aortic banding (TAB). Measurement of mito-[Ca] using genetic probe mtRCamp1h revealed that mito-[Ca] in TAB VMs paced at 2 Hz under β-adrenergic stimulation is lower compared with shams. Adenoviral 2.5-fold MCU overexpression in TAB VMs fully restored mito-[Ca]. However, it failed to improve cytosolic Ca handling and reduce proarrhythmic spontaneous Ca waves. Furthermore, mitochondrial-targeted genetic probes MLS-HyPer7 and OMM-HyPer revealed a significant increase in emission of reactive oxygen species (ROS) in TAB VMs with 2.5-fold MCU overexpression. Conversely, 1.5-fold MCU overexpression in TABs, that led to partial restoration of mito-[Ca], reduced mitochondria-derived reactive oxygen species (mito-ROS) and spontaneous Ca waves. Our findings emphasize the key role of elevated mito-ROS in disease-related proarrhythmic Ca mishandling. These data establish nonlinear mito-[Ca]/mito-ROS relationship, whereby partial restoration of mito-[Ca] in diseased VMs is protective, whereas further enhancement of MCU-mediated Ca uptake exacerbates damaging mito-ROS emission. Defective intracellular Ca homeostasis and aberrant mitochondrial function are common features in cardiac disease. Here, we directly compared potential benefits of mito-ROS scavenging and restoration of mito-Ca uptake by overexpressing MCU in ventricular myocytes from hypertrophic rat hearts. Experiments using novel mito-ROS and Ca biosensors demonstrated that mito-ROS scavenging rescued both cytosolic and mito-Ca homeostasis, whereas moderate and high MCU overexpression demonstrated disparate effects on mito-ROS emission, with only a moderate increase in MCU being beneficial.
• Hamilton, S., Terentyeva, R., Perger, F., Orengo, B., Martin, B., Gorr, M., Belevych, A., Clements, R., Gyorke, S., & Terentyev, D. (2021). MCU overexpression evokes disparate dose-dependent effects on mito-ROS and spontaneous Ca2 þ release in hypertrophic rat cardiomyocytes. American Journal of Physiology - Heart and Circulatory Physiology, 321(4). doi:10.1152/ajpheart.00126.2021
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  Cardiac dysfunction in heart failure (HF) and diabetic cardiomyopathy (DCM) is associated with aberrant intracellular Ca2 þ handling and impaired mitochondrial function accompanied with reduced mitochondrial calcium concentration (mito-[Ca2 þ ]). Pharmacological or genetic facilitation of mito-Ca2 þ uptake was shown to restore Ca2 þ transient amplitude in DCM and HF, improving contractility. However, recent reports suggest that pharmacological enhancement of mito-Ca2 þ uptake can exacerbate ryanodine receptor-mediated spontaneous sarcoplasmic reticulum (SR) Ca2 þ release in ventricular myocytes (VMs) from diseased animals, increasing propensity to stress-induced ventricular tachyarrhythmia. To test whether chronic recovery of mito-[Ca2 þ ] restores systolic Ca2 þ release without adverse effects in diastole, we overexpressed mitochondrial Ca2 þ uniporter (MCU) in VMs from male rat hearts with hypertrophy induced by thoracic aortic banding (TAB). Measurement of mito-[Ca2 þ ] using genetic probe mtRCamp1h revealed that mito-[Ca2 þ ] in TAB VMs paced at 2 Hz under b-adrenergic stimulation is lower compared with shams. Adenoviral 2.5-fold MCU overexpression in TAB VMs fully restored mito-[Ca2 þ ]. However, it failed to improve cytosolic Ca2 þ handling and reduce proarrhythmic spontaneous Ca2 þ waves. Furthermore, mitochondrial-targeted genetic probes MLS-HyPer7 and OMM-HyPer revealed a significant increase in emission of reactive oxygen species (ROS) in TAB VMs with 2.5-fold MCU overexpression. Conversely, 1.5-fold MCU overexpression in TABs, that led to partial restoration of mito-[Ca2 þ ], reduced mitochondria-derived reactive oxygen species (mito-ROS) and spontaneous Ca2 þ waves. Our findings emphasize the key role of elevated mito-ROS in disease-related proarrhythmic Ca2 þ mishandling. These data establish nonlinear mito-[Ca2 þ ]/mito-ROS relationship, whereby partial restoration of mito-[Ca2 þ ] in diseased VMs is protective, whereas further enhancement of MCU-mediated Ca2 þ uptake exacerbates damaging mito-ROS emission.
• Hamilton, S., Veress, R., Belevych, A., & Terentyev, D. (2021). The role of calcium homeostasis remodeling in inherited cardiac arrhythmia syndromes. Pflugers Archiv : European journal of physiology, 473(3), 377-387.
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  Sudden cardiac death due to malignant ventricular arrhythmias remains the major cause of mortality in the postindustrial world. Defective intracellular Ca homeostasis has been well established as a key contributing factor to the enhanced propensity for arrhythmia in acquired cardiac disease, such as heart failure or diabetic cardiomyopathy. More recent advances provide a strong basis to the emerging view that hereditary cardiac arrhythmia syndromes are accompanied by maladaptive remodeling of Ca homeostasis which substantially increases arrhythmic risk. This brief review will focus on functional changes in elements of Ca handling machinery in cardiomyocytes that occur secondary to genetic mutations associated with catecholaminergic polymorphic ventricular tachycardia, and long QT syndrome.
• Liu, H., Zhao, Y., Xie, A., Kim, T. Y., Terentyeva, R., Liu, M., Shi, G., Feng, F., Choi, B. R., Terentyev, D., Hamilton, S., & Dudley, S. C. (2021). Interleukin-1β, Oxidative Stress, and Abnormal Calcium Handling Mediate Diabetic Arrhythmic Risk. JACC. Basic to translational science, 6(1), 42-52.
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  Diabetes mellitus (DM) is associated with increased arrhythmia. Type 2 DM (T2DM) mice showed prolonged QT interval and increased ventricular arrhythmic inducibility, accompanied by elevated cardiac interleukin (IL)-1β, increased mitochondrial reactive oxygen species (mitoROS), and oxidation of the sarcoplasmic reticulum (SR) Ca release channel (ryanodine receptor 2 [RyR2]). Inhibiting IL-1β and mitoROS reduced RyR2 oxidation and the ventricular arrhythmia in DM. Inhibiting SR Ca2 leak by stabilizing the oxidized RyR2 channel reversed the diabetic arrhythmic risk. In conclusion, cardiac IL-1β mediated the DM-associated arrhythmia through mitoROS generation that enhances SR Ca leak. The mechanistic link between inflammation and arrhythmias provides new therapeutic options.
• Liu, H., Zhao, Y., Xie, A., Kim, T., Terentyeva, R., Liu, M., Shi, G., Feng, F., Choi, B., Terentyev, D., Hamilton, S., & Dudley, S. (2021). Interleukin-1β, Oxidative Stress, and Abnormal Calcium Handling Mediate Diabetic Arrhythmic Risk. JACC: Basic to Translational Science, 6(1). doi:10.1016/j.jacbts.2020.11.002
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  Diabetes mellitus (DM) is associated with increased arrhythmia. Type 2 DM (T2DM) mice showed prolonged QT interval and increased ventricular arrhythmic inducibility, accompanied by elevated cardiac interleukin (IL)-1β, increased mitochondrial reactive oxygen species (mitoROS), and oxidation of the sarcoplasmic reticulum (SR) Ca2+ release channel (ryanodine receptor 2 [RyR2]). Inhibiting IL-1β and mitoROS reduced RyR2 oxidation and the ventricular arrhythmia in DM. Inhibiting SR Ca2+ leak by stabilizing the oxidized RyR2 channel reversed the diabetic arrhythmic risk. In conclusion, cardiac IL-1β mediated the DM-associated arrhythmia through mitoROS generation that enhances SR Ca2+ leak. The mechanistic link between inflammation and arrhythmias provides new therapeutic options.
• Bronk, P., Kim, T. Y., Polina, I., Hamilton, S., Terentyeva, R., Roder, K., Koren, G., Terentyev, D., & Choi, B. R. (2020). Impact of I Voltage and Ca/Mg-Dependent Rectification on Cardiac Repolarization. Biophysical journal, 119(3), 690-704.
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  Cardiac small conductance Ca-activated K (SK) channels are activated solely by Ca, but the SK current (I) is inwardly rectified. However, the impact of inward rectification in shaping action potentials (APs) in ventricular cardiomyocytes under β-adrenergic stimulation or in disease states remains undefined. Two processes underlie this inward rectification: an intrinsic rectification caused by an electrostatic energy barrier from positively charged amino acids at the inner pore and a voltage-dependent Ca/Mg block. Thus, Ca has a biphasic effect on I, activating at low [Ca] yet inhibiting I at high [Ca]. We examined the effect of I rectification on APs in rat cardiomyocytes by simultaneously recording whole-cell apamin-sensitive currents and Ca transients during an AP waveform and developed a computer model of SK channels with rectification features. The typical profile of I during AP clamp included an initial peak (mean 1.6 pA/pF) followed by decay to the point that submembrane [Ca] reached ∼10 μM. During the rest of the AP stimulus, I either plateaued or gradually increased as the cell repolarized and submembrane [Ca] decreased further. We used a six-state gating model combined with intrinsic and Ca/Mg-dependent rectification to simulate I and investigated the relative contributions of each type of rectification to AP shape. This SK channel model replicates key features of I recording during AP clamp showing that intrinsic rectification limits I at high V during the early and plateau phase of APs. Furthermore, the initial rise of Ca transients activates, but higher [Ca] blocks SK channels, yielding a transient outward-like I trajectory. During the decay phase of Ca, the Ca-dependent block is released, causing I to rise again and contribute to repolarization. Therefore, I is an important repolarizing current, and the rectification characteristics of an SK channel determine its impact on early, plateau, and repolarization phases of APs.
• Bronk, P., Kim, T., Polina, I., Hamilton, S., Terentyeva, R., Roder, K., Koren, G., Terentyev, D., & Choi, B. (2020). Impact of ISK Voltage and Ca2+/Mg2+-Dependent Rectification on Cardiac Repolarization. Biophysical Journal, 119(3). doi:10.1016/j.bpj.2020.06.022
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  Cardiac small conductance Ca2+-activated K+ (SK) channels are activated solely by Ca2+, but the SK current (ISK) is inwardly rectified. However, the impact of inward rectification in shaping action potentials (APs) in ventricular cardiomyocytes under β-adrenergic stimulation or in disease states remains undefined. Two processes underlie this inward rectification: an intrinsic rectification caused by an electrostatic energy barrier from positively charged amino acids at the inner pore and a voltage-dependent Ca2+/Mg2+ block. Thus, Ca2+ has a biphasic effect on ISK, activating at low [Ca2+] yet inhibiting ISK at high [Ca2+]. We examined the effect of ISK rectification on APs in rat cardiomyocytes by simultaneously recording whole-cell apamin-sensitive currents and Ca2+ transients during an AP waveform and developed a computer model of SK channels with rectification features. The typical profile of ISK during AP clamp included an initial peak (mean 1.6 pA/pF) followed by decay to the point that submembrane [Ca2+] reached ∼10 μM. During the rest of the AP stimulus, ISK either plateaued or gradually increased as the cell repolarized and submembrane [Ca2+] decreased further. We used a six-state gating model combined with intrinsic and Ca2+/Mg2+-dependent rectification to simulate ISK and investigated the relative contributions of each type of rectification to AP shape. This SK channel model replicates key features of ISK recording during AP clamp showing that intrinsic rectification limits ISK at high Vm during the early and plateau phase of APs. Furthermore, the initial rise of Ca2+ transients activates, but higher [Ca2+] blocks SK channels, yielding a transient outward-like ISK trajectory. During the decay phase of Ca2+, the Ca2+-dependent block is released, causing ISK to rise again and contribute to repolarization. Therefore, ISK is an important repolarizing current, and the rectification characteristics of an SK channel determine its impact on early, plateau, and repolarization phases of APs.
• Hamilton, S., Polina, I., Terentyeva, R., Bronk, P., Kim, T. Y., Roder, K., Clements, R. T., Koren, G., Choi, B. R., & Terentyev, D. (2020). PKA phosphorylation underlies functional recruitment of sarcolemmal SK2 channels in ventricular myocytes from hypertrophic hearts. The Journal of physiology, 598(14), 2847-2873.
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  Small-conductance Ca -activated K (SK) channels expressed in ventricular myocytes are dormant in health, yet become functional in cardiac disease. SK channels are voltage independent and their gating is controlled by intracellular [Ca ] in a biphasic manner. Submicromolar [Ca ] activates the channel via constitutively-bound calmodulin, whereas higher [Ca ] exerts inhibitory effect during depolarization. Using a rat model of cardiac hypertrophy induced by thoracic aortic banding, we found that functional upregulation of SK2 channels in hypertrophic rat ventricular cardiomyocytes is driven by protein kinase A (PKA) phosphorylation. Using site-directed mutagenesis, we identified serine-465 as the site conferring PKA-dependent effects on SK2 channel function. PKA phosphorylation attenuates I rectification by reducing the Ca /voltage-dependent inhibition of SK channels without changing their sensitivity to activating submicromolar [Ca ] . This mechanism underlies the functional recruitment of SK channels not only in cardiac disease, but also in normal physiology, contributing to repolarization under conditions of enhanced adrenergic drive.
• Hamilton, S., Polina, I., Terentyeva, R., Bronk, P., Kim, T., Roder, K., Clements, R., Koren, G., Choi, B., & Terentyev, D. (2020). PKA phosphorylation underlies functional recruitment of sarcolemmal SK2 channels in ventricular myocytes from hypertrophic hearts. Journal of Physiology, 598(14). doi:10.1113/JP277618
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  Key points: Small-conductance Ca2+-activated K+ (SK) channels expressed in ventricular myocytes are dormant in health, yet become functional in cardiac disease. SK channels are voltage independent and their gating is controlled by intracellular [Ca2+] in a biphasic manner. Submicromolar [Ca2+] activates the channel via constitutively-bound calmodulin, whereas higher [Ca2+] exerts inhibitory effect during depolarization. Using a rat model of cardiac hypertrophy induced by thoracic aortic banding, we found that functional upregulation of SK2 channels in hypertrophic rat ventricular cardiomyocytes is driven by protein kinase A (PKA) phosphorylation. Using site-directed mutagenesis, we identified serine-465 as the site conferring PKA-dependent effects on SK2 channel function. PKA phosphorylation attenuates ISK rectification by reducing the Ca2+/voltage-dependent inhibition of SK channels without changing their sensitivity to activating submicromolar [Ca2+]i. This mechanism underlies the functional recruitment of SK channels not only in cardiac disease, but also in normal physiology, contributing to repolarization under conditions of enhanced adrenergic drive. Abstract: Small-conductance Ca2+-activated K+ (SK) channels expressed in ventricular myocytes (VMs) are dormant in health, yet become functional in cardiac disease. We aimed to test the hypothesis that post-translational modification of SK channels under conditions accompanied by enhanced adrenergic drive plays a central role in disease-related activation of the channels. We investigated this phenomenon using a rat model of hypertrophy induced by thoracic aortic banding (TAB). Western blot analysis using anti-pan-serine/threonine antibodies demonstrated enhanced phosphorylation of immunoprecipitated SK2 channels in VMs from TAB rats vs. Shams, which was reversible by incubation of the VMs with PKA inhibitor H89 (1 μmol L–1). Patch clamped VMs under basal conditions from TABs but not Shams exhibited outward current sensitive to the specific SK inhibitor apamin (100 nmol L–1), which was eliminated by inhibition of PKA (1 μmol L–1). Beta-adrenergic stimulation (isoproterenol, 100 nmol L–1) evoked ISK in VMs from Shams, resulting in shortening of action potentials in VMs and ex vivo optically mapped Sham hearts. Using adenoviral gene transfer, wild-type and mutant SK2 channels were overexpressed in adult rat VMs, revealing serine-465 as the site that elicits PKA-dependent phosphorylation effects on SK2 channel function. Concurrent confocal Ca2+ imaging experiments established that PKA phosphorylation lessens rectification of ISK via reduction Ca2+/voltage-dependent inhibition of the channels at high [Ca2+] without affecting their sensitivity to activation by Ca2+ in the submicromolar range. In conclusion, upregulation of SK channels in diseased VMs is mediated by hyperadrenergic drive in cardiac hypertrophy, with functional effects on the channel conferred by PKA-dependent phosphorylation at serine-465.
• Hamilton, S., Terentyeva, R., Martin, B., Perger, F., Li, J., Stepanov, A., Bonilla, I. M., Knollmann, B. C., Radwański, P. B., Györke, S., Belevych, A. E., & Terentyev, D. (2020). Increased RyR2 activity is exacerbated by calcium leak-induced mitochondrial ROS. Basic research in cardiology, 115(4), 38.
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  Cardiac disease is associated with deleterious emission of mitochondrial reactive oxygen species (mito-ROS), as well as enhanced oxidation and activity of the sarcoplasmic reticulum (SR) Ca release channel, the ryanodine receptor (RyR2). The transfer of Ca from the SR via RyR2 to mitochondria is thought to play a key role in matching increased metabolic demand during stress. In this study, we investigated whether augmented RyR2 activity results in self-imposed exacerbation of SR Ca leak, via altered SR-mitochondrial Ca transfer and elevated mito-ROS emission. Fluorescent indicators and spatially restricted genetic ROS probes revealed that both pharmacologically and genetically enhanced RyR2 activity, in ventricular myocytes from rats and catecholaminergic polymorphic ventricular tachycardia (CPVT) mice, respectively, resulted in increased ROS emission under β-adrenergic stimulation. Expression of mitochondrial Ca probe mtRCamp1h revealed diminished net mitochondrial [Ca] with enhanced SR Ca leak, accompanied by depolarization of the mitochondrial matrix. While this may serve as a protective mechanism to prevent mitochondrial Ca overload, protection is not complete and enhanced mito-ROS emission resulted in oxidation of RyR2, further amplifying proarrhythmic SR Ca release. Importantly, the effects of augmented RyR2 activity could be attenuated by mitochondrial ROS scavenging, and experiments with dominant-negative paralogs of the mitochondrial Ca uniporter (MCU) supported the hypothesis that SR-mitochondria Ca transfer is essential for the increase in mito-ROS. We conclude that in a process whereby leak begets leak, augmented RyR2 activity modulates mitochondrial Ca handling, promoting mito-ROS emission and driving further channel activity in a proarrhythmic feedback cycle in the diseased heart.
• Hamilton, S., Terentyeva, R., Martin, B., Perger, F., Li, J., Stepanov, A., Bonilla, I., Knollmann, B., Belevych, A., Terentyev, D., Györke, S., & Radwański, P. (2020). Increased RyR2 activity is exacerbated by calcium leak-induced mitochondrial ROS. Basic Research in Cardiology, 115(4). doi:10.1007/s00395-020-0797-z
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  Cardiac disease is associated with deleterious emission of mitochondrial reactive oxygen species (mito-ROS), as well as enhanced oxidation and activity of the sarcoplasmic reticulum (SR) Ca2+ release channel, the ryanodine receptor (RyR2). The transfer of Ca2+ from the SR via RyR2 to mitochondria is thought to play a key role in matching increased metabolic demand during stress. In this study, we investigated whether augmented RyR2 activity results in self-imposed exacerbation of SR Ca2+ leak, via altered SR-mitochondrial Ca2+ transfer and elevated mito-ROS emission. Fluorescent indicators and spatially restricted genetic ROS probes revealed that both pharmacologically and genetically enhanced RyR2 activity, in ventricular myocytes from rats and catecholaminergic polymorphic ventricular tachycardia (CPVT) mice, respectively, resulted in increased ROS emission under β-adrenergic stimulation. Expression of mitochondrial Ca2+ probe mtRCamp1h revealed diminished net mitochondrial [Ca2+] with enhanced SR Ca2+ leak, accompanied by depolarization of the mitochondrial matrix. While this may serve as a protective mechanism to prevent mitochondrial Ca2+ overload, protection is not complete and enhanced mito-ROS emission resulted in oxidation of RyR2, further amplifying proarrhythmic SR Ca2+ release. Importantly, the effects of augmented RyR2 activity could be attenuated by mitochondrial ROS scavenging, and experiments with dominant-negative paralogs of the mitochondrial Ca2+ uniporter (MCU) supported the hypothesis that SR-mitochondria Ca2+ transfer is essential for the increase in mito-ROS. We conclude that in a process whereby leak begets leak, augmented RyR2 activity modulates mitochondrial Ca2+ handling, promoting mito-ROS emission and driving further channel activity in a proarrhythmic feedback cycle in the diseased heart.
• Hamilton, S., & Terentyev, D. (2019). Altered Intracellular Calcium Homeostasis and Arrhythmogenesis in the Aged Heart. International journal of molecular sciences, 20(10).
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  Aging of the heart is associated with a blunted response to sympathetic stimulation, reduced contractility, and increased propensity for arrhythmias, with the risk of sudden cardiac death significantly increased in the elderly population. The altered cardiac structural and functional phenotype, as well as age-associated prevalent comorbidities including hypertension and atherosclerosis, predispose the heart to atrial fibrillation, heart failure, and ventricular tachyarrhythmias. At the cellular level, perturbations in mitochondrial function, excitation-contraction coupling, and calcium homeostasis contribute to this electrical and contractile dysfunction. Major determinants of cardiac contractility are the intracellular release of Ca from the sarcoplasmic reticulum by the ryanodine receptors (RyR2), and the following sequestration of Ca by the sarco/endoplasmic Ca-ATPase (SERCa2a). Activity of RyR2 and SERCa2a in myocytes is not only dependent on expression levels and interacting accessory proteins, but on fine-tuned regulation via post-translational modifications. In this paper, we review how aberrant changes in intracellular Ca cycling via these proteins contributes to arrhythmogenesis in the aged heart.
• Hamilton, S., & Terentyev, D. (2018). Proarrhythmic Remodeling of Calcium Homeostasis in Cardiac Disease; Implications for Diabetes and Obesity. Frontiers in physiology, 9, 1517.
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  A rapid growth in the incidence of diabetes and obesity has transpired to a major heath issue and economic burden in the postindustrial world, with more than 29 million patients affected in the United States alone. Cardiovascular defects have been established as the leading cause of mortality and morbidity of diabetic patients. Over the last decade, significant progress has been made in delineating mechanisms responsible for the diminished cardiac contractile function and enhanced propensity for malignant cardiac arrhythmias characteristic of diabetic disease. Rhythmic cardiac contractility relies upon the precise interplay between several cellular Ca transport protein complexes including plasmalemmal L-type Ca channels (LTCC), Na-Ca exchanger (NCX1), Sarco/endoplasmic Reticulum (SR) Ca-ATPase (SERCa2a) and ryanodine receptors (RyR2s), the SR Ca release channels. Here we provide an overview of changes in Ca homeostasis in diabetic ventricular myocytes and discuss the therapeutic potential of targeting Ca handling proteins in the prevention of diabetes-associated cardiomyopathy and arrhythmogenesis.
• Hamilton, S., & Terentyev, D. (2018). Proarrhythmic remodeling of calcium homeostasis in cardiac disease; Implications for diabetes and obesity. Frontiers in Physiology, 9. doi:10.3389/fphys.2018.01517
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  A rapid growth in the incidence of diabetes and obesity has transpired to a major heath issue and economic burden in the postindustrial world, with more than 29 million patients affected in the United States alone. Cardiovascular defects have been established as the leading cause of mortality and morbidity of diabetic patients. Over the last decade, significant progress has been made in delineating mechanisms responsible for the diminished cardiac contractile function and enhanced propensity for malignant cardiac arrhythmias characteristic of diabetic disease. Rhythmic cardiac contractility relies upon the precise interplay between several cellular Ca2+ transport protein complexes including plasmalemmal L-type Ca2+ channels (LTCC), Na+-Ca2+ exchanger (NCX1), Sarco/endoplasmic Reticulum (SR) Ca2+-ATPase (SERCa2a) and ryanodine receptors (RyR2s), the SR Ca2+ release channels. Here we provide an overview of changes in Ca2+ homeostasis in diabetic ventricular myocytes and discuss the therapeutic potential of targeting Ca2+ handling proteins in the prevention of diabetes-associated cardiomyopathy and arrhythmogenesis.
• Hamilton, S., Terentyeva, R., Kim, T. Y., Bronk, P., Clements, R. T., O-Uchi, J., Csordás, G., Choi, B. R., & Terentyev, D. (2018). Pharmacological Modulation of Mitochondrial Ca Content Regulates Sarcoplasmic Reticulum Ca Release via Oxidation of the Ryanodine Receptor by Mitochondria-Derived Reactive Oxygen Species. Frontiers in physiology, 9, 1831.
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  In a physiological setting, mitochondria increase oxidative phosphorylation during periods of stress to meet increased metabolic demand. This in part is mediated via enhanced mitochondrial Ca uptake, an important regulator of cellular ATP homeostasis. In a pathophysiological setting pharmacological modulation of mitochondrial Ca uptake or retention has been suggested as a therapeutic strategy to improve metabolic homeostasis or attenuate Ca-dependent arrhythmias in cardiac disease states. To explore the consequences of mitochondrial Ca accumulation, we tested the effects of kaempferol, an activator of mitochondrial Ca uniporter (MCU), CGP-37157, an inhibitor of mitochondrial Na/Ca exchanger, and MCU inhibitor Ru360 in rat ventricular myocytes (VMs) from control rats and rats with hypertrophy induced by thoracic aortic banding (TAB). In periodically paced VMs under β-adrenergic stimulation, treatment with kaempferol (10 μmol/L) or CGP-37157 (1 μmol/L) enhanced mitochondrial Ca accumulation monitored by mitochondrial-targeted Ca biosensor mtRCamp1h. Experiments with mitochondrial membrane potential-sensitive dye TMRM revealed this was accompanied by depolarization of the mitochondrial matrix. Using redox-sensitive OMM-HyPer and ERroGFP_iE biosensors, we found treatment with kaempferol or CGP-37157 increased the levels of reactive oxygen species (ROS) in mitochondria and the sarcoplasmic reticulum (SR), respectively. Confocal Ca imaging showed that accelerated Ca accumulation reduced Ca transient amplitude and promoted generation of spontaneous Ca waves in VMs paced under ISO, suggestive of abnormally high activity of the SR Ca release channel ryanodine receptor (RyR). Western blot analyses showed increased RyR oxidation after treatment with kaempferol or CGP-37157 vs. controls. Furthermore, in freshly isolated TAB VMs, confocal Ca imaging demonstrated that enhancement of mitochondrial Ca accumulation further perturbed global Ca handling, increasing the number of cells exhibiting spontaneous Ca waves, shortening RyR refractoriness and decreasing SR Ca content. In optically mapped TAB hearts, kaempferol exacerbated proarrhythmic phenotype. On the contrary, incubation of cells with MCU inhibitor Ru360 (2 μmol/L, 30 min) normalized RyR oxidation state, improved intracellular Ca homeostasis and reduced triggered activity in TAB hearts. These findings suggest facilitation of mitochondrial Ca uptake in cardiac disease can exacerbate proarrhythmic disturbances in Ca homeostasis via ROS and enhanced activity of oxidized RyRs, while strategies to reduce mitochondrial Ca accumulation can be protective.
• Hamilton, S., Terentyeva, R., Kim, T., Bronk, P., Clements, R., O-Uchi, J., Choi, B., Terentyev, D., & Csordás, G. (2018). Pharmacological Modulation of Mitochondrial Ca2+ Content Regulates Sarcoplasmic Reticulum Ca2+ Release via Oxidation of the Ryanodine Receptor by Mitochondria-Derived Reactive Oxygen Species. Frontiers in Physiology, 9. doi:10.3389/fphys.2018.01831
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  In a physiological setting, mitochondria increase oxidative phosphorylation during periods of stress to meet increased metabolic demand. This in part is mediated via enhanced mitochondrial Ca2+ uptake, an important regulator of cellular ATP homeostasis. In a pathophysiological setting pharmacological modulation of mitochondrial Ca2+ uptake or retention has been suggested as a therapeutic strategy to improve metabolic homeostasis or attenuate Ca2+-dependent arrhythmias in cardiac disease states. To explore the consequences of mitochondrial Ca2+ accumulation, we tested the effects of kaempferol, an activator of mitochondrial Ca2+ uniporter (MCU), CGP-37157, an inhibitor of mitochondrial Na+/Ca2+ exchanger, and MCU inhibitor Ru360 in rat ventricular myocytes (VMs) from control rats and rats with hypertrophy induced by thoracic aortic banding (TAB). In periodically paced VMs under β-adrenergic stimulation, treatment with kaempferol (10 μmol/L) or CGP-37157 (1 μmol/L) enhanced mitochondrial Ca2+ accumulation monitored by mitochondrial-targeted Ca2+ biosensor mtRCamp1h. Experiments with mitochondrial membrane potential-sensitive dye TMRM revealed this was accompanied by depolarization of the mitochondrial matrix. Using redox-sensitive OMM-HyPer and ERroGFP_iE biosensors, we found treatment with kaempferol or CGP-37157 increased the levels of reactive oxygen species (ROS) in mitochondria and the sarcoplasmic reticulum (SR), respectively. Confocal Ca2+ imaging showed that accelerated Ca2+ accumulation reduced Ca2+ transient amplitude and promoted generation of spontaneous Ca2+ waves in VMs paced under ISO, suggestive of abnormally high activity of the SR Ca2+ release channel ryanodine receptor (RyR). Western blot analyses showed increased RyR oxidation after treatment with kaempferol or CGP-37157 vs. controls. Furthermore, in freshly isolated TAB VMs, confocal Ca2+ imaging demonstrated that enhancement of mitochondrial Ca2+ accumulation further perturbed global Ca2+ handling, increasing the number of cells exhibiting spontaneous Ca2+ waves, shortening RyR refractoriness and decreasing SR Ca2+ content. In ex vivo optically mapped TAB hearts, kaempferol exacerbated proarrhythmic phenotype. On the contrary, incubation of cells with MCU inhibitor Ru360 (2 μmol/L, 30 min) normalized RyR oxidation state, improved intracellular Ca2+ homeostasis and reduced triggered activity in ex vivo TAB hearts. These findings suggest facilitation of mitochondrial Ca2+ uptake in cardiac disease can exacerbate proarrhythmic disturbances in Ca2+ homeostasis via ROS and enhanced activity of oxidized RyRs, while strategies to reduce mitochondrial Ca2+ accumulation can be protective.
• Kim, T. Y., Terentyeva, R., Roder, K. H., Li, W., Liu, M., Greener, I., Hamilton, S., Polina, I., Murphy, K. R., Clements, R. T., Dudley, S. C., Koren, G., Choi, B. R., & Terentyev, D. (2017). SK channel enhancers attenuate Ca2+-dependent arrhythmia in hypertrophic hearts by regulating mito-ROS-dependent oxidation and activity of RyR. Cardiovascular research, 113(3), 343-353.
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  Plasmamembrane small conductance Ca2+-activated K+ (SK) channels were implicated in ventricular arrhythmias in infarcted and failing hearts. Recently, SK channels were detected in the inner mitochondria membrane (IMM) (mSK), and their activation protected from acute ischaemia-reperfusion injury by reducing intracellular levels of reactive oxygen species (ROS). We hypothesized that mSK play an important role in regulating mitochondrial function in chronic cardiac diseases. We investigated the role of mSK channels in Ca2+-dependent ventricular arrhythmia using rat model of cardiac hypertrophy induced by banding of the ascending aorta thoracic aortic banding (TAB).
• Kim, T., Terentyeva, R., Roder, K., Li, W., Liu, M., Greener, I., Hamilton, S., Polina, I., Murphy, K., Clements, R., Dudley, S., Koren, G., Choi, B., & Terentyev, D. (2017). SK channel enhancers attenuate Ca2+-dependent arrhythmia in hypertrophic hearts by regulating mito-ROS-dependent oxidation and activity of RyR. Cardiovascular Research, 113(3). doi:10.1093/cvr/cvx005
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  Aims: Plasmamembrane small conductance Ca2+-activated K+ (SK) channels were implicated in ventricular arrhythmias in infarcted and failing hearts. Recently, SK channels were detected in the inner mitochondria membrane (IMM) (mSK), and their activation protected from acute ischaemia-reperfusion injury by reducing intracellular levels of reactive oxygen species (ROS). We hypothesized that mSK play an important role in regulating mitochondrial function in chronic cardiac diseases. We investigated the role of mSK channels in Ca2+-dependent ventricular arrhythmia using rat model of cardiac hypertrophy induced by banding of the ascending aorta thoracic aortic banding (TAB). Methods and results: Dual Ca2+ and membrane potential optical mapping of whole hearts derived from TAB rats revealed that membrane-permeable SK enhancer NS309 (2 μM) improved aberrant Ca2+ homeostasis and abolished VT/VF induced by β-adrenergic stimulation. Using whole cell patch-clamp and confocal Ca2+ imaging of cardiomyocytes derived from TAB hearts (TCMs) we found that membrane-permeable SK enhancers NS309 and CyPPA (10 μM) attenuated frequency of spontaneous Ca2+ waves and delayed afterdepolarizations. Furthermore, mSK inhibition enhanced (UCL-1684, 1 μM); while activation reduced mitochondrial ROS production in TCMs measured with MitoSOX. Protein oxidation assays demonstrated that increased oxidation of ryanodine receptors (RyRs) in TCMs was reversed by SK enhancers. Experiments in permeabilized TCMs showed that SK enhancers restored SR Ca2+ content, suggestive of substantial improvement in RyR function. Conclusion: These data suggest that enhancement of mSK channels in hypertrophic rat hearts protects from Ca2+-dependent arrhythmia and suggest that the protection is mediated via decreased mitochondrial ROS and subsequent decreased oxidation of reactive cysteines in RyR, which ultimately leads to stabilization of RyR-mediated Ca2+ release.
• Springer, J., White, P. L., Hamilton, S., Michel, D., Barnes, R. A., Einsele, H., & Löffler, J. (2016). Comparison of Performance Characteristics of Aspergillus PCR in Testing a Range of Blood-Based Samples in Accordance with International Methodological Recommendations. Journal of clinical microbiology, 54(3), 705-11.
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  Standardized methodologies for the molecular detection of invasive aspergillosis (IA) have been established by the European Aspergillus PCR Initiative for the testing of whole blood, serum, and plasma. While some comparison of the performance of Aspergillus PCR when testing these different sample types has been performed, no single study has evaluated all three using the recommended protocols. Standardized Aspergillus PCR was performed on 423 whole-blood pellets (WBP), 583 plasma samples, and 419 serum samples obtained from hematology patients according to the recommendations. This analysis formed a bicenter retrospective anonymous case-control study, with diagnosis according to the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (11 probable cases and 36 controls). Values for clinical performance using individual and combined samples were calculated. For all samples, PCR positivity was significantly associated with cases of IA (for plasma, P = 0.0019; for serum, P = 0.0049; and for WBP, P = 0.0089). Plasma PCR generated the highest sensitivity (91%); the sensitivities for serum and WBP PCR were 80% and 55%, respectively. The highest specificity was achieved when testing WBP (96%), which was significantly superior to the specificities achieved when testing serum (69%, P = 0.0238) and plasma (53%, P = 0.0002). No cases were PCR negative in all specimen types, and no controls were PCR positive in all specimens. This study confirms that Aspergillus PCR testing of plasma provides robust performance while utilizing commercial automated DNA extraction processes. Combining PCR testing of different blood fractions allows IA to be both confidently diagnosed and excluded. A requirement for multiple PCR-positive plasma samples provides similar diagnostic utility and is technically less demanding. Time to diagnosis may be enhanced by testing multiple contemporaneously obtained sample types.
• Springer, J., White, P., Hamilton, S., Michel, D., Barnes, R., Einsele, H., & Löffler, J. (2016). Comparison of Performance Characteristics of Aspergillus PCR in Testing a Range of Blood-Based Samples in Accordance with International Methodological Recommendations. Journal of Clinical Microbiology, 54(3). doi:10.1128/JCM.02814-15
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  Standardized methodologies for the molecular detection of invasive aspergillosis (IA) have been established by the European Aspergillus PCR Initiative for the testing of whole blood, serum, and plasma. While some comparison of the performance of Aspergillus PCR when testing these different sample types has been performed, no single study has evaluated all three using the recommended protocols. Standardized Aspergillus PCR was performed on 423 whole-blood pellets (WBP), 583 plasma samples, and 419 serum samples obtained from hematology patients according to the recommendations. This analysis formed a bicenter retrospective anonymous case-control study, with diagnosis according to the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (11 probable cases and 36 controls). Values for clinical performance using individual and combined samples were calculated. For all samples, PCR positivity was significantly associated with cases of IA (for plasma, P0.0019; for serum, P0.0049; and for WBP, P0.0089). Plasma PCR generated the highest sensitivity (91%); the sensitivities for serum and WBP PCR were 80% and 55%, respectively. The highest specificity was achieved when testing WBP (96%), which was significantly superior to the specificities achieved when testing serum (69%, P0.0238) and plasma (53%, P0.0002). No cases were PCR negative in all specimen types, and no controls were PCR positive in all specimens. This study confirms that Aspergillus PCR testing of plasma provides robust performance while utilizing commercial automated DNA extraction processes. Combining PCR testing of different blood fractions allows IA to be both confidently diagnosed and excluded. A requirement for multiple PCR-positive plasma samples provides similar diagnostic utility and is technically less demanding. Time to diagnosis may be enhanced by testing multiple contemporaneously obtained sample types.
• Terentyev, D., & Hamilton, S. (2016). Regulation of sarcoplasmic reticulum Ca release by serine-threonine phosphatases in the heart. Journal of molecular and cellular cardiology, 101, 156-164.
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  The amount and timing of Ca release from the sarcoplasmic reticulum (SR) during cardiac cycle are the main determinants of cardiac contractility. Reversible phosphorylation of the SR Ca release channel, ryanodine receptor type 2 (RyR2) is the central mechanism of regulation of Ca release in cardiomyocytes. Three major serine-threonine phosphatases including PP1, PP2A and PP2B (calcineurin) have been implicated in modulation of RyR2 function. Changes in expression levels of these phosphatases, their activity and targeting to the RyR2 macromolecular complex were demonstrated in many animal models of cardiac disease and humans and are implicated in cardiac arrhythmia and heart failure. Here we review evidence in support of regulation of RyR2-mediated SR Ca release by serine-threonine phosphatases and the role and mechanisms of dysregulation of phosphatases in various disease states.
• Terentyev, D., & Hamilton, S. (2016). Regulation of sarcoplasmic reticulum Ca2+ release by serine-threonine phosphatases in the heart. Journal of Molecular and Cellular Cardiology, 101. doi:10.1016/j.yjmcc.2016.08.020
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  The amount and timing of Ca2+ release from the sarcoplasmic reticulum (SR) during cardiac cycle are the main determinants of cardiac contractility. Reversible phosphorylation of the SR Ca2+ release channel, ryanodine receptor type 2 (RyR2) is the central mechanism of regulation of Ca2+ release in cardiomyocytes. Three major serine-threonine phosphatases including PP1, PP2A and PP2B (calcineurin) have been implicated in modulation of RyR2 function. Changes in expression levels of these phosphatases, their activity and targeting to the RyR2 macromolecular complex were demonstrated in many animal models of cardiac disease and humans and are implicated in cardiac arrhythmia and heart failure. Here we review evidence in support of regulation of RyR2-mediated SR Ca2+ release by serine-threonine phosphatases and the role and mechanisms of dysregulation of phosphatases in various disease states.