Reduced Sarcoplasmic Reticulum Ca Research Articles

Toxicity of low dose bisphenols in human iPSC-derived cardiomyocytes and human cardiac organoids – Impact on contractile function and hypertrophy

Bisphenol A (BPA) and its analogs are common environmental chemicals with various adverse health impacts, including cardiac toxicity. In this study, we examined the long term effect of low dose BPA and three common BPA analogs, bisphenol S (BPS), bisphenol F (BPF), and bisphenol AF (BPAF), in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) based models. HiPSC-CMs and human cardiac organoids were exposed to these chemicals for 4–5 or 20 days. 1 nM BPA, BPS, and BPAF, but not BPF, resulted in suppressed myocyte contractility, retarded contraction kinetics, and aberrant Ca2+ transients in hiPSC-CMs. In cardiac organoids, BPAF and BPA, but not the other bisphenols, resulted in suppressed contraction and Ca2+ transients, and aberrant contraction kinetics. The order of toxicities was BPAF > BPA>∼BPS > BPF and the toxicities of BPAF and BPA were more pronounced under longer exposure. The impact of BPAF on myocyte contraction and Ca2+ handling was mediated by reduction of sarcoplasmic reticulum Ca2+ load and inhibition of L-type Ca2+ channel involving alternation of Ca2+ handling proteins. Impaired myocyte Ca2+ handling plays a key role in cardiac pathophysiology and is a characteristic of cardiac hypertrophy; therefore we examined the potential pro-hypertrophic cardiotoxicity of these bisphenols. Four to five day exposure to BPAF did not cause hypertrophy in normal hiPSC-CMs, but significantly exacerbated the hypertrophic phenotype in myocytes with existing hypertrophy induced by endothelin-1, characterized by increased cell size and elevated expression of the hypertrophic marker proBNP. This pro-hypertrophic cardiotoxicity was also occurred in cardiac organoids, with BPAF having the strongest toxicity, followed by BPA. Our findings demonstrate that long term exposures to BPA and some of its analogs cause contractile dysfunction and abnormal Ca2+ handling, and have potential pro-hypertrophic cardiotoxicity in human heart cells/tissues, and suggest that some bisphenol chemicals may be a risk factor for cardiac hypertrophy in human hearts.

Dysfunctional Ca2+ dynamics in primary skeletal myocytes from R2509C-RYR1 mice.

The skeletal muscle contracts when depolarization is transmitted to the dihydropyridine receptor in the T-tubule, and Ca2+ is released through type 1 ryanodine receptor (RYR1) from the sarcoplasmic reticulum (SR). Mutations in RYR1 cause severe muscle diseases, such as malignant hyperthermia (MH), a disorder of Ca2+-induced Ca2+ release (CICR) through RYR1 from the SR. We recently reported that volatile anesthetics (e.g., isoflurane) induce MH-like episodes via enhanced CICR in heterozygous R2509C-RYR1 mice. However, the characterization of Ca2+ dynamics has yet to be investigated in skeletal muscle cells from homozygous mice because these animals die in utero. In the present study, we generated primary cultured skeletal myocytes from R2509C-RYR1 mice. We analyzed morphological and functional changes in myocytes from R2509C-RYR1 mice. In homozygous R2509C-RYR1 myocytes, Ca2+ release by depolarization or caffeine was decreased and resting intracellular Ca2+ concentrations ([Ca2+]cyt) was increased, indicating a Ca2+ leak and a reduced SR Ca2+ content. Also, in homozygous R2509C-RYR1 myocytes, sarcomeres were remarkably shortened to the A-band length, even in the resting condition, which was associated with abnormal Ca2+ homeostasis, particularly an increase in the resting [Ca2+]cyt. In heterozygous R2509C-RYR1 muscle cells, heat sensitivity for intracellular Ca2+ release via the heat-induced Ca2+ release mechanism was increased, and the cellular temperature was elevated in the presence of isoflurane. Our findings suggest that the R2509C mutation in RYR1 causes dysfunctional Ca2+ dynamics in a mutant-gene dose-dependent manner in the skeletal muscles, in turn provoking MH-like episodes and embryonic lethality in heterozygous and homozygous mice, respectively.

Glu-rich N-terminal variable region of troponin T functions as a myofilament Ca2+-reservoir to reduce SR Ca2+-cycling.

The long Glu-rich N-terminal segment of avian pectoral muscle troponin T (TnT) and C-terminal extension of insect TnT are multi-valent Ca2+-binding sites with physiologically relevant affinities. Genetic deletion of the Glu-rich C-terminal extension of insect TnT decreases muscle and heart functions but increases the tolerance of cardiomyocytes to Ca2+ overloading, indicating decreased [Ca2+]i. In contrast, cardiomyocytes from gene-edited mice expressing modified cardiac TnT with N-terminal variable region replaced by a C-terminal segment of insect TnT (51E-cTnT) show higher shortening amplitude and contraction and relaxation velocities than that of wild type (WT) control. Isolated 51E-cTnT cardiomyocytes exhibited shorter resting sarcomeres, which can be corrected by inhibiting myosin ATPase, indicating increased resting [Ca2+]i. A similar gain-of-function model,fast skeletal muscle TnTgene-edited mice with the N-terminal variable region replaced by the N-terminal segment of adult turkey pectoral muscle TnT (Tx9-fsTnT), shows normal baseline muscle functions but slower and less rising of contractile force during caffeine-induced Ca2+ release from sarcoplasmic reticulum (SR) than that of WT control, indicating a decreased SR Ca2+ store. Consistently, 51E-cTnT mouse hearts respond to beta-adrenergic stimulation or Ca2+ agonist with higher than WT ventricular peak pressure and diastolic velocity. The data support a hypothesis that the Glu-rich segments of insect and avian flight muscle TnT function as myofilament Ca2+ reservoirs to tune muscle kinetics and reduce the amount of Ca2+ cycled during contraction and relaxation. The functional effects are prominent in heterozygotes of the genetically modified flies and mice whereas the loss-of-function in Drosophila TnT and gain-of-function in 51E-cTnT and Tx9-fsTnT mice both produced homozygote lethality, indicating sensitive dose effects of the myofilament Ca2+ reservoir for use as a potent mechanism to improve energetic efficiency of cardiac and skeletal muscles.

Abstract 12531: Electrophysiological Mechanisms of Tachycardia-Induced Cardiomyopathy in Human Myocardium

Introduction: Tachycardia-induced cardiomyopathy (TCM) is a reversible form of heart failure. However, the effects of persistent tachycardia leading to TCM in the human are less understood limiting appropriate management of patients with this underrecognized condition. This study investigated the electrophysiological mechanisms of TCM. Methods and Results: Tachycardic in vitro electrical stimulation (120 bpm versus 60 bpm control) of human specimen was performed. In human twitching ventricular trabeculae from patients with preexistent heart failure, tachycardia for 8h compromised systolic force, while diastolic tension and relaxation time were increased (n=6/8 patients each). Human iPSC-cardiomyocytes (iPSC-CM) of healthy donors were utilized as human model suitable for extended electrical pacing. After 24h of tachycardia, systolic Ca 2+ -transient amplitude of iPSC-CM was reduced, while diastolic Ca 2+ levels and cytosolic Ca 2+ elimination were not changed (Fura-2, n tachycardia/control=49/44 cells). 7d of tachycardia resulted in a progressive decline of Ca 2+ -transient amplitude together with an impaired Ca 2+ elimination (n=73/66) thereby demonstrating the progression of TCM. Sarcoplasmic reticulum (SR) Ca 2+ load (caffeine application) and SERCA activity (K sys -K caff ) were reduced after 7d of tachycardia (n=13/13). Besides an impaired SERCA activity, we could identify an increased diastolic SR Ca 2+ spark frequency after 7d of tachycardia (n=76/79) as another mechanism of reduced SR Ca 2+ load. In voltage clamp studies, I CaL density was reduced after 7d of tachycardia (n=11/12), which may further contribute to impaired systolic Ca 2+ -handling. Conversely, action potential duration was prolonged after 7d of tachycardia compared to control (n=19/21). As a potential explanation for prolonged action potentials however, late sodium current (I NaL ) was enhanced after 7d of tachycardia (n=26/19). Conclusions: This study demonstrates for the first time in human myocardium that persistent tachycardia induces early adverse electrophysiological remodeling that may underlie the progression of TCM. These findings may help to understand the pathophysiology of an underrated but prevalent disease

Ablation of Calsequestrin-1, Ca2+ unbalance, and susceptibility to heat stroke.

Introduction: Ca2+ levels in adult skeletal muscle fibers are mainly controlled by excitation-contraction (EC) coupling, a mechanism that translates action potentials in release of Ca2+ from the sarcoplasmic reticulum (SR) release channels, i.e. the ryanodine receptors type-1 (RyR1). Calsequestrin (Casq) is a protein that binds large amounts of Ca2+ in the lumen of the SR terminal cisternae, near sites of Ca2+ release. There is general agreement that Casq is not only important for the SR ability to store Ca2+, but also for modulating the opening probability of the RyR Ca2+ release channels. The initial studies: About 20 years ago we generated a mouse model lacking Casq1 (Casq1-null mice), the isoform predominantly expressed in adult fast twitch skeletal muscle. While the knockout was not lethal as expected, lack of Casq1 caused a striking remodeling of membranes of SR and of transverse tubules (TTs), and mitochondrial damage. Functionally, CASQ1-knockout resulted in reduced SR Ca2+ content, smaller Ca2+ transients, and severe SR depletion during repetitive stimulation. The myopathic phenotype of Casq1-null mice: After the initial studies, we discovered that Casq1-null mice were prone to sudden death when exposed to halogenated anaesthetics, heat and even strenuous exercise. These syndromes are similar to human malignant hyperthermia susceptibility (MHS) and environmental-exertional heat stroke (HS). We learned that mechanisms underlying these syndromes involved excessive SR Ca2+ leak and excessive production of oxidative species: indeed, mortality and mitochondrial damage were significantly prevented by administration of antioxidants and reduction of oxidative stress. Though, how Casq1-null mice could survive without the most important SR Ca2+ binding protein was a puzzling issue that was not solved. Unravelling the mystery: The mystery was finally solved in 2020, when we discovered that in Casq1-null mice the SR undergoes adaptations that result in constitutively active store-operated Ca2+ entry (SOCE). SOCE is a mechanism that allows skeletal fibers to use external Ca2+ when SR stores are depleted. The post-natal compensatory mechanism that allows Casq1-null mice to survive involves the assembly of new SR-TT junctions (named Ca2+ entry units) containing Stim1 and Orai1, the two proteins that mediate SOCE.

Electrophysiological remodeling in tachycardia-induced cardiomyopathy

Abstract Background Tachycardia-induced cardiomyopathy (TCM) is a reversible and likely underrecognized form of heart failure. Thus, a better understanding of the TCM-pathophysiology is warranted as the underlying early mechanisms that mediate the progression of TCM remain unclear. Purpose This study aimed to identify the cellular mechanisms of TCM. Methods and results Human induced pluripotent stem cell cardiomyocytes (iPSC-CM) were utilized as a translational human-based model. We performed chronic tachycardic (120 bpm) or normofrequent (control, 60bpm) cell culture pacing to study cellular changes during TCM progression. Already after 24h of tachycardic stimulation of iPSC-CM, we detected a decrease in Ca transient amplitude compared to control (Fura-2, n=49/44 cells/9 differentiations). Diastolic Ca levels and cytosolic Ca elimination were not affected after 24h of tachycardia (n=49/44/9). We detected no difference in sarcoplasmic reticulum (SR) Ca load (assessed via caffeine application) or SERCA activity (Ksys-Kcaff) after 24h of tachycardia (n=13/15/5). However, demonstrating the progress of TCM, 7d of tachycardia resulted in progressive decline of Ca transient amplitude together with an impaired Ca elimination, while diastolic Ca concentration was unchanged (n=73/66/8). These changes may underlie the reduced systolic force and impaired relaxation in TCM. We could explain these results by a significantly reduced SR Ca load and a diminished SERCA activity after 7d tachycardia (n=13/7 vs. 13/4). Using confocal microscopy (Fluo-4) we detected no difference in SR Ca spark frequency after 24h of tachycardia (n=82/66/8), while 7d of tachycardia caused an increase of Ca spark frequency (n=76/79/7), which is a typical hallmark of maladaptive remodeling in HF and likely underlie the reduced SR Ca load. Voltage clamp data of late Na current (INaL) showed no difference in INaL after 24h of stimulation (n=17/6 vs. 19/7), whereas INaL was increased after 7d of tachycardia (n=26/7 vs. 19/6). Accordingly, whole-cell current clamp experiments revealed a prolongation of the action potential after 7d of tachycardia compared to control (n=21/6 vs. 19/5), while no difference of action potential duration could be detected after 24h (n=37/31/8). Resting membrane potential and action potential amplitude were not changed. Finally, we investigated tachycardia-mediated effects on explanted human failing hearts. 8h of tachycardic stimulation (120 bpm) of human failing ventricular trabeculae already compromised systolic force, and diastolic tension and relaxation time were markedly increased compared to control (60bpm, n=8/6 trabeculae /7/6 human hearts). Conclusion This study demonstrates that persistent tachycardia adversely alters cardiomyocyte excitation-contraction coupling via electrophysiological cellular remodeling. Our translational investigation in human myocardium may help to understand the pathophysiology of an underrated but prevalent disease. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Else Kröner-Fresenius-Stiftung (EKFS)Deutsche Gesellschaft für Innere Medizin

A hydroalcoholic extract of Senecio nutans SCh. Bip (Asteraceae); its effects on cardiac function and chemical characterization

Ethnopharmacology relevanceThe plant Senecio nutans SCh. Bip. is used by Andean communities to treat altitude sickness. Recent evidence suggests it may produce vasodilation and negative cardiac inotropy, though the cellular mechanisms have not been elucidated. PurposeTo determinate the mechanisms action of S. nutans on cardiovascular function in normotensive animals. MethodsThe effect of the extract on rat blood pressure was measured with a transducer in the carotid artery and intraventricular pressure by a Langendorff system. The effects on sheep ventricular intracellular calcium handling and contractility were evaluated using photometry. Ultra-high-performance liquid-chromatography with diode array detection coupled with heated electrospray-ionization quadrupole-orbitrap mass spectrometric detection (UHPLC-DAD-ESI-Q-OT-MSn) was used for extract chemical characterization. ResultsIn normotensive rats, S. nutans (10 mg/kg) reduced mean arterial pressure (MAP) by 40% (p < 0.05), causing a dose-dependent coronary artery dilation and decreased left ventricular pressure. In isolated cells, S. nutans extract (1 μg/ml) rapidly reduced the [Ca2+]i transient amplitude and sarcomere shorting by 40 and 49% (p < 0.001), respectively. The amplitude of the caffeine evoked [Ca2+]i transient was reduced by 24% (p < 0.001), indicating reduced sarcoplasmic reticulum (SR) Ca2+ content. Sodium-calcium exchanger (NCX) activity increased by 17% (p < 0.05), while sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) activity was decreased by 21% (p < 0.05). LC-MS results showed the presence of vitamin C, malic acid, and several antioxidant phenolic acids reported for the first time. Dihydroeuparin and 4-hydroxy-3-(3-methylbut-2-enyl) acetophenone were abundant in the extract. ConclusionIn normotensive animals, S. nutans partially reduces MAP by decreasing heart rate and cardiac contractility. This negative inotropy is accounted for by decreased SERCA activity and increased NCX activity which reduces SR Ca2+ content. These results highlight the plant's potential as a source of novel cardio-active phytopharmaceuticals or nutraceuticals.

Reduced sarcoplasmic reticulum Ca2+ pump activity is antiarrhythmic in ischemic cardiomyopathy

We have described an arrhythmic mechanism seen only in cardiomyopathy that involves increased mitochondrial Ca2+ handling and selective transfer of Ca2+ to the sarcoplasmic reticulum (SR). Modeling suggested that mitochondrial Ca2+ transfer to the SR via type 2a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) is a crucial element of this arrhythmic mechanism. We tested the role of SERCA2a in arrhythmias during ischemic cardiomyopathy. Myocardial infarction (MI) was induced in wild-type (Wt) and SERCA2a heterozygous knockdown (SERCA+/-) mice. Compared with Wt MI mice, SERCA2a heterozygous knockdown (SERCA+/-) MI mice had a substantially lower mortality after 3 weeks of MI without a significant change in MI area. Aside from a significant delay of the cytoplasmic Ca2+ transient decay existed in SERCA+/- compared with Wt, SERCA+/- did not affect cardiac systolic and diastolic function at the whole organ or single cell levels either before or after MI. After MI, SERCA+/- mice had reduced SERCA2a expression in the MI border zone compared with Wt MI mice. SERCA+/- mice had significantly decreased corrected QT intervals and less ventricular tachycardia compared with Wt MI mice. SERCA+/- cardiomyocytes from MI mice showed a reduced action potential duration and reduced triggered activity compared with Wt MI cardiomyocytes. Reduction in arrhythmic risk was accompanied by reduced diastolic SR Ca2+ sparks, reduced SR Ca2+ content, reduced oxidized ryanodine receptor, and increased calsequestrin 2 in SERCA+/- MI mice. SERCA2a knockdown was antiarrhythmic after MI without affecting overall systolic performance. Possible antiarrhythmic mechanisms included reduced SR free Ca2+ and reduced diastolic SR Ca2+ release.

Changes in cellular Ca2+ and Na+ regulation during the progression towards heart failure.

In adapting to disease and loss of tissue, the heart shows great phenotypic plasticity that involves changes to its structure, composition and electrophysiology. Together with parallel whole body cardiovascular adaptations, the initial decline in cardiac function resulting from the insult is compensated. However, in the long term, the heart muscle begins to fail and patients with this condition have a very poor prognosis, with many dying from disturbances of rhythm. The surviving myocytes of these hearts gain Na+ , which is positively inotropic because of alterations to Ca2+ fluxes mediated by the Na+ /Ca2+ exchange, but compromises Ca2+ -dependent energy metabolism in mitochondria. Uptake of Ca2+ into the sarcoplasmic reticulum (SR) is reduced because of diminished function of SR Ca2+ ATPases. The result of increased Ca2+ influx and reduced SR Ca2+ uptake is an increase in the diastolic cytosolic Ca2+ concentration, which promotes spontaneous SR Ca2+ release and induces delayed afterdepolarisations. Action potential duration prolongs because of increased late Na+ current and changes in expression and function of other ion channels and transporters increasing the probability of the formation of early afterdepolarisations. There is a reduction in T-tubule density and so the normal spatial arrangements required for efficient excitation-contraction coupling are compromised and lead to temporal delays in Ca2+ release from the SR. Therefore, the structural and electrophysiological responses that occur to provide compensation do so at the expense of (1) increasing the likelihood of arrhythmogenesis; (2) activating hypertrophic, apoptotic and Ca2+ signalling pathways; and (3) decreasing the efficiency of SR Ca2+ release.

Protective Effects of Allicin on Acute Myocardial Infarction in Rats via Hydrogen Sulfide-mediated Regulation of Coronary Arterial Vasomotor Function and Myocardial Calcium Transport.

Acute myocardial infarction (AMI) is a condition with high morbidity and mortality, for which effective treatments are lacking. Allicin has been reported to exert therapeutic effects on AMI, but the underlying mechanisms of its action have not been fully elucidated. To investigate this, a rat model of AMI was generated by ligating the left anterior descending branch of the coronary artery. DL-propargylglycine (PAG), a specific hydrogen sulfide (H2S) synthetase inhibitor, was used to examine the effects of allicin on H2S production. Isolated coronary arteries and cardiomyocytes were assessed for vascular reactivity and cellular Ca2+ transport using a multiwire myography system and a cell-contraction-ion detection system, respectively. Allicin administration improved cardiac function and myocardial pathology, reduced myocardial enzyme levels, and increased H2S and H2S synthetase levels. Allicin administration resulted in concentration-dependent effects on coronary artery dilation, which were mediated by receptor-dependent Ca2+ channels, ATP-sensitive K+ channels, and sarcoplasmic reticulum (SR) Ca2+ release induced by the ryanodine receptor. Allicin administration improved Ca2+ homeostasis in cardiomyocytes by increasing cardiomyocyte contraction, Ca2+ transient amplitude, myofilament sensitivity, and SR Ca2+ content. Allicin also enhanced Ca2+ uptake via SR Ca2+-ATPase and Ca2+ removal via the Na+/Ca2+ exchanger, and it reduced SR Ca2+ leakage. Notably, the protective effects of allicin were partially attenuated by blockade of H2S production with PAG. Our findings provide novel evidence that allicin-induced production of H2S mediates coronary artery dilation and regulation of Ca2+ homeostasis in AMI. Our study presents a novel mechanistic insight into the anti-AMI effects of allicin and highlights the therapeutic potential of this compound.

Imperacalcin‐Induced Reduction of Sarcoplasmic Reticulum Ca 2+ in Intact Mouse Heart

Introduction Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac channelopathy, that often leads to sudden death due to arrhythmogenic episodes triggered by adrenergic stimulation. Previous studies have shown that CPVT is associated with mutations in the Ca2+ release channel of the sarcoplasmic reticulum (SR), the ryanodine receptor type-2 (RyR2). Loss of function mutations in the RyR2 result in abnormally low Ca2+ release which leads to an increase in SR Ca2+ content. It has been proposed that SR Ca2+ overload precipitates CPVT events by over activating the Na-Ca exchanger (NCX) which favors the development of arrhythmogenic delayed afterdepolarizations (DADs). Previous studies from our group and others have shown that the scorpion-derived peptide, imperacalcin (IpCa) is an effective agent to prevent/revert DADs in CPVT mouse models. The overall goal of this study was to test the hypothesis that the IpCa protects against CPVT by partially reducing SR Ca2+ content, therefore preventing the development of Ca2+ overload. To test this hypothesis, IpCa effect on SR Ca2+ content was evaluated by measuring SR Ca2+ restitution, which is the time-dependent recovery of SR Ca2+ release as a function of the stimulation frequency. If indeed, IpCa reduces SR Ca2+ content due to RyR2-mediated Ca2+ leak, SR Ca2+ restitution would be significantly shorter than in its absence. As a positive control, a known RyR2 agonist like caffeine, was used to define the effects of reducing SR Ca2+ content on SR Ca2+ restitution. Methods To test IpCa effects on SR Ca2+ restitution the pulsed local field fluorescence microscopy was used. Rhod-2AM-loaded hearts were perfused with either Tyrode, caffeine (1 mM), or IpCa (1 µM) and were stimulated at 2, 3, 4, 5, 6, 7, and 8 Hz while action potentials and intracellular Ca2+ transients were simultaneously recorded. SR Ca2+restitution was calculated from the ratio of the Ca2+ transient amplitude before and after the premature stimulation at the tested frequencies. Results At a constant stimulation frequency of 2 Hz, IpCa (1 µM; n=8) induced a 36% reduction of the Ca2+ transients amplitude, without affecting their kinetics or basal Ca2+. In contrast, caffeine (1 mM; n=10) affected basal Ca2+ (~3-fold increase), Ca2+ transients amplitude (~50% reduction), and their kinetics (~65% longer rise time & ~70% shorter decay & half-duration). As a function of the stimulation frequency, the SR Ca2+ exponential recovery (control t = 206±5 ms; n=6) was slightly accelerated by IpCa (t = 198±3 ms; n=4), & by caffeine (t = 183±4 ms; n=6). Conclusion Our results indicate that in intact mouse heart, the effects of IpCa on the intracellular Ca2+ dynamics are similar to those induced by caffeine in terms of reduction of SR Ca2+ content, without affecting the Ca2+ transient kinetics. Consistent with previous observations, our SR Ca2+ restitution results support the notion that IpCa partially depletes SR Ca2+ content, providing a plausible mechanism for its antiarrhythmic effects.

Pathological mechanisms of vacuolar aggregate myopathy arising from a Casq1 mutation.

Mice with a mutation (D244G, DG) in calsequestrin 1 (CASQ1), analogous to a human mutation in CASQ1 associated with a delayed onset human myopathy (vacuolar aggregate myopathy), display a progressive myopathy characterized by decreased activity, decreased ability of fast twitch muscles to generate force and low body weight after one year of age. The DG mutation causes CASQ1 to partially dissociate from the junctional sarcoplasmic reticulum (SR) and accumulate in the endoplasmic reticulum (ER). Decreased junctional CASQ1 reduces SR Ca2+ release. Muscles from older DG mice display ER stress, ER expansion, increased mTOR signaling, inadequate clearance of aggregated proteins by the proteasomes, and elevation of protein aggregates and lysosomes. This study suggests that the myopathy associated with the D244G mutation in CASQ1 is driven by CASQ1 mislocalization, reduced SR Ca2+ release, CASQ1 misfolding/aggregation and ER stress. The subsequent maladaptive increase in protein synthesis and decreased protein aggregate clearance are likely to contribute to disease progression.

JNK2, a Newly-Identified SERCA2 Enhancer, Augments an Arrhythmic [Ca2+]SR Leak-Load Relationship.

We recently discovered pivotal contributions of stress kinase JNK2 (c-Jun N-terminal kinase isoform 2) in increased risk of atrial fibrillation through enhanced diastolic sarcoplasmic reticulum (SR) calcium (Ca2+) leak via RyR2 (ryanodine receptor isoform 2). However, the role of JNK2 in the function of the SERCA2 (SR Ca2+-ATPase), essential in maintaining SR Ca2+ content cycling during each heartbeat, is completely unknown. To test the hypothesis that JNK2 increases SERCA2 activity SR Ca2+ content and exacerbates an arrhythmic SR Ca2+ content leak-load relationship. We used confocal Ca2+ imaging in myocytes and HEK-RyR2 (ryanodine receptor isoform 2-expressing human embryonic kidney 293 cells) cells, biochemistry, dual Ca2+/voltage optical mapping in intact hearts from alcohol-exposed or aged mice (where JNK2 is activated). We found that JNK2, but not JNK1 (c-Jun N-terminal kinase isoform 1), increased SERCA2 uptake and consequently elevated SR Ca2+ content load. JNK2 also associates with and phosphorylates SERCA2 proteins. JNK2 causally enhances SERCA2-ATPase activity via increased maximal rate, without altering Ca2+ affinity. Unlike the CaMKII (Ca2+/calmodulin-dependent kinase II)-dependent JNK2 action in SR Ca2+ leak, JNK2-driven SERCA2 function was CaMKII independent (not prevented by CaMKII inhibition). With CaMKII blocked, the JNK2-driven SR Ca2+ loading alone did not significantly raise leak. However, with JNK2-CaMKII-driven SR Ca2+ leak present, the JNK2-enhanced SR Ca2+ uptake limited leak-induced reduction in SR Ca2+, normalizing Ca2+ transient amplitude, but at a higher arrhythmogenic SR Ca2+ leak. JNK2-specific inhibition completely normalized SR Ca2+ handling, attenuated arrhythmic Ca2+ activities, and alleviated atrial fibrillation susceptibility in aged and alcohol-exposed myocytes and intact hearts. We have identified a novel JNK2-induced activation of SERCA2. The dual action of JNK2 in CaMKII-dependent arrhythmic SR Ca2+ leak and a CaMKII-independent uptake exacerbates atrial arrhythmogenicity, while helping to maintain normal levels of Ca2+ transients and heart function. JNK2 modulation may be a novel therapeutic target for atrial fibrillation prevention and treatment.

Impaired sarcoplasmic reticulum Ca2+ release is the major cause of fatigue-induced force loss in intact single fibres from human intercostal muscle.

Changes in intramuscular Ca2+ handling contribute to development of fatigue and disease-related loss of muscle mass and function. To date, no data on human intact living muscle fibres have been described. We manually dissected intact single fibres from human intercostal muscle and simultaneously measured force and myoplasmic free [Ca2+ ] at physiological temperature. Based on their fatigue resistance, two distinct groups of fibres were distinguished: fatigue sensitive and fatigue resistant. Force depression in fatigue and during recovery was due to impaired sarcoplasmic reticulum Ca2+ release in both groups of fibres. Acidification did not affect force production in unfatigued fibres and did not affect fatigue development in fatigue-resistant fibres. The current study provides novel insight into the mechanisms of fatigue in human intercostal muscle. Changes in intracellular Ca2+ handling of individual skeletal muscle fibres cause a force depression following physical activity and are also implicated in disease-related loss of function. The relation of intracellular Ca2+ handling with muscle force production and fatigue tolerance is best studied in intact living single fibres that allow continuous measurements of force and myoplasmic free [Ca2+ ] during repeated contractions. To this end, manual dissections of human intercostal muscle biopsies were performed to isolate intact single fibres. Based on the ability to maintain tetanic force at >40% of the initial value during 500 fatiguing contractions, fibres were classified as either fatigue sensitive or fatigue resistant. Following fatigue all fibres demonstrated a marked reduction in sarcoplasmic reticulum Ca2+ release, while myofibrillar Ca2+ sensitivity was either unaltered or increased. In unfatigued fibres, acidosis caused a reduction in myofibrillar Ca2+ sensitivity that was offset by increased tetanic myoplasmic free [Ca2+ ] so that force remained unaffected. Acidification did not affect the fatigue tolerance of fatigue-resistant fibres, whereas uncertainties remain whether or not fatigue-sensitive fibres were affected. Following fatigue, a prolonged force depression at preferentially low-frequency stimulation was evident in fatigue-sensitive fibres and this was caused exclusively by an impaired sarcoplasmic reticulum Ca2+ release. We conclude that impaired sarcoplasmic reticulum Ca2+ release is the predominant mechanism of force depression both in the development of, and recovery from, fatigue in human intercostal muscle.

Phosphoproteomics reveals conserved exercise-stimulated signaling and AMPK regulation of store-operated calcium entry.

Exercise stimulates cellular and physiological adaptations that are associated with widespread health benefits. To uncover conserved protein phosphorylation events underlying this adaptive response, we performed mass spectrometry-based phosphoproteomic analyses of skeletal muscle from two widely used rodent models: treadmill running in mice and insitu muscle contraction in rats. We overlaid these phosphoproteomic signatures with cycling in humans to identify common cross-species phosphosite responses, as well as unique model-specific regulation. We identified >22,000 phosphosites, revealing orthologous protein phosphorylation and overlapping signaling pathways regulated by exercise. This included two conserved phosphosites on stromal interaction molecule 1 (STIM1), which we validate as AMPK substrates. Furthermore, we demonstrate that AMPK-mediated phosphorylation of STIM1 negatively regulates store-operated calcium entry, and this is beneficial for exercise in Drosophila. This integrated cross-species resource of exercise-regulated signaling in human, mouse, and rat skeletal muscle has uncovered conserved networks and unraveled crosstalk between AMPK and intracellular calcium flux.

Cardiotoxicity of environmental contaminant tributyltin involves myocyte oxidative stress and abnormal Ca2+ handling

Tributyltin (TBT) is an organotin environmental pollutant widely used as an agricultural and wood biocide and in antifouling paints. Countries began restricting TBT use in the 2000s, but their use continues in some agroindustrial processes. We studied the acute effect of TBT on cardiac function by analyzing myocardial contractility and Ca2+ handling. Cardiac contractility was evaluated in isolated papillary muscle and whole heart upon TBT exposure. Isolated ventricular myocytes were used to measure calcium (Ca2+) transients, sarcoplasmic reticulum (SR) Ca2+ content and SR Ca2+ leak (as Ca2+ sparks). Reactive oxygen species (ROS), as superoxide anion (O2•-) was detected at intracellular and mitochondrial myocardium. TBT depressed cardiac contractility and relaxation in papillary muscle and intact whole heart. TBT increased cytosolic, mitochondrial ROS production and decreased mitochondrial membrane potential. In isolated cardiomyocytes TBT decreased both Ca2+ transients and SR Ca2+ content and increased diastolic SR Ca2+ leak. Decay of twitch and caffeine-induced Ca2+ transients were slowed by the presence of TBT. Dantrolene prevented and Tiron limited the reduction in SR Ca2+ content and transients. The environmental contaminant TBT causes cardiotoxicity within minutes, and may be considered hazardous to the mammalian heart. TBT acutely induced a negative inotropic effect in isolated papillary muscle and whole heart, increased arrhythmogenic SR Ca2+ leak leading to reduced SR Ca2+ content and reduced Ca2+ transients. TBT-induced myocardial ROS production, may destabilize the SR Ca2+ release channel RyR2 and reduce SR Ca2+ pump activity as key factors in the TBT-induced negative inotropic and lusitropic effects.

Sarcoplasmic reticulum calcium leak contributes to arrhythmia but not to heart failure progression.

Increased sarcoplasmic reticulum (SR) Ca2+ leak via the cardiac ryanodine receptor (RyR2) has been suggested to play a mechanistic role in the development of heart failure (HF) and cardiac arrhythmia. Mice treated with a selective RyR2 stabilizer, rycal S36, showed normalization of SR Ca2+ leak and improved survival in pressure overload (PO) and myocardial infarction (MI) models. The development of HF, measured by echocardiography and molecular markers, showed no difference in rycal S36- versus placebo-treated mice. Reduction of SR Ca2+ leak in the PO model by the rycal-unrelated RyR2 stabilizer dantrolene did not mitigate HF progression. Development of HF was not aggravated by increased SR Ca2+ leak due to RyR2 mutation (R2474S) in volume overload, an SR Ca2+ leak-independent HF model. Arrhythmia episodes were reduced by rycal S36 treatment in PO and MI mice in vivo and ex vivo in Langendorff-perfused hearts. Isolated cardiomyocytes from murine failing hearts and human ventricular failing and atrial nonfailing myocardium showed reductions in delayed afterdepolarizations, in spontaneous and induced Ca2+ waves, and in triggered activity in rycal S36 versus placebo cells, whereas the Ca2+ transient, SR Ca2+ load, SR Ca2+ adenosine triphosphatase function, and action potential duration were not affected. Rycal S36 treatment of human induced pluripotent stem cells isolated from a patient with catecholaminergic polymorphic ventricular tachycardia could rescue the leaky RyR2 receptor. These results suggest that SR Ca2+ leak does not primarily influence contractile HF progression, whereas rycal S36 treatment markedly reduces ventricular arrhythmias, thereby improving survival in mice.

Activation of protein phosphatase 1 by a selective phosphatase disrupting peptide reduces sarcoplasmic reticulum Ca2+ leak in human heart failure.

Disruption of Ca2+ homeostasis is a key pathomechanism in heart failure. CaMKII-dependent hyperphosphorylation of ryanodine receptors in the sarcoplasmic reticulum (SR) increases the arrhythmogenic SR Ca2+ leak and depletes SR Ca2+ stores. The contribution of conversely acting serine/threonine phosphatases [protein phosphatase 1 (PP1) and 2A (PP2A)] is largely unknown. Human myocardium from three groups of patients was investigated: (i) healthy controls (non-failing, NF, n = 8), (ii) compensated hypertrophy (Hy, n = 16), and (iii) end-stage heart failure (HF, n = 52). Expression of PP1 was unchanged in Hy but greater in HF compared to NF while its endogenous inhibitor-1 (I-1) was markedly lower expressed in both compared to NF, suggesting increased total PP1 activity. In contrast, PP2A expression was lower in Hy and HF compared to NF. Ca2+ homeostasis was severely disturbed in HF compared to Hy signified by a higher SR Ca2+ leak, lower systolic Ca2+ transients as well as a decreased SR Ca2+ load. Inhibition of PP1/PP2A by okadaic acid increased SR Ca2+ load and systolic Ca2+ transients but severely aggravated diastolic SR Ca2+ leak and cellular arrhythmias in Hy. Conversely, selective activation of PP1 by a PP1-disrupting peptide (PDP3) in HF potently reduced SR Ca2+ leak as well as cellular arrhythmias and, importantly, did not compromise systolic Ca2+ release and SR Ca2+ load. This study is the first to functionally investigate the role of PP1/PP2A for Ca2+ homeostasis in diseased human myocardium. Our data indicate that a modulation of phosphatase activity potently impacts Ca2+ cycling properties. An activation of PP1 counteracts increased kinase activity in heart failure and successfully seals the arrhythmogenic SR Ca2+ leak. It may thus represent a promising future antiarrhythmic therapeutic approach.

Abstract 342: The Absence of S-nitrosoglutathione Reductase (GSNOR -/- ) Reduces Maturation of iPSC-derived Cardiomyocytes

Introduction: Induced pluripotent stem cells (iPSCs) are a potential source of cardiac regenerative therapy. However, incomplete structural and functional maturation of iPSC-derived cardiomyocytes (iPSC-CMs), including lack of T-tubules, immature sarcoplasmic reticulum (SR), and inefficient Ca 2+ -induced Ca 2+ release remain major limitations. We assessed the influence of nitric oxide (NO) on differentiation and maturation of iPSC-CMs. Objective: We hypothesized that enhanced S-nitrosylation impairs SR calcium (Ca 2+ ) handling (storage and uptake) in iPSC-CMs. Methods: iPSCs were derived from fibroblasts from wildtype mice and mice lacking S-nitrosoglutathione reductase (GSNOR -/- ), a denitrosylase that regulates protein S-nitrosylation. iPSCs were differentiated into CMs from embryoid bodies via the hanging-drop method. Analyses of cell morphology and structural changes, gene expression (cardiac markers), and functional tests (intracellular calcium transients) were performed at an early stage of cardiac development (day 21 of cell culture). The involvement of the SR Ca 2+ ATPase (SERCA2) in iPSC-CM contraction was tested by blocking Ca 2+ re-uptake with 1 μM Thapsigargin and SR Ca 2+ stores were estimated by addition of 20 mM caffeine. The role of the cardiac ryanodine receptor (RyR2) was assessed by blocking the channel with 10 μM ryanodine. The dependence of Ca 2+ transients on L-type Ca 2+ channels (LTCC) was demonstrated using 10 μM nifedipine, which eliminated [Ca 2+ ] i transients and cell contraction. Length of sarcomeres within the contractile apparatus was determined by staining cells with α-actinin. Results: Sarcomeres were more organized in WT iPSC-CMs than in GSNOR -/- iPSC-CMs after 21 days in culture. The loss of GSNOR activity reduced SR Ca 2+ content and sarcomere organization in iPSC-CMs. While iPSC-CMs are relatively immature in terms of ultrastructure and Ca 2+ handling; GSNOR –/– iPSC-CMs were less able to recycle intracellular Ca 2+ . Conclusions: Our results provide novel insights into NO-mediated gene regulation and cell proliferation and suggest that the absence of GSNOR affects CM differentiation. These findings have important implications for regenerative medicine and provide new targets for iPSC-based therapy.

Reduction of SR Ca2+ leak and arrhythmogenic cellular correlates by SMP-114, a novel CaMKII inhibitor with oral bioavailability.

Sarcoplasmic reticulum (SR) Ca2+ leak induced by Ca2+/calmodulin-dependent protein kinase II (CaMKII) is centrally involved in atrial and ventricular arrhythmogenesis as well as heart failure remodeling. Consequently, treating SR Ca2+ leak has been proposed as a novel therapeutic paradigm, but compounds for use in humans are lacking. SMP-114 ("Rimacalib") is a novel, orally available CaMKII inhibitor developed for human use that has already entered clinical phase II trials to treat rheumatoid arthritis. We speculated that SMP-114 might also be useful to treat cardiac SR Ca2+ leak. SMP-114 significantly reduces SR Ca2+ leak (as assessed by Ca2+ sparks) in human atrial (0.72±0.33 sparks/100µm/s vs. control 3.02±0.91 sparks/100µm/s) and failing left ventricular (0.78±0.23 vs. 1.69±0.27 sparks/100µm/s) as well as in murine ventricular cardiomyocytes (0.30±0.07 vs. 1.50±0.28sparks/100µm/s). Associated with lower SR Ca2+ leak, we found that SMP-114 suppressed the occurrence of spontaneous arrhythmogenic spontaneous Ca2+ release (0.356±0.109 vs. 0.927±0.216 events per 30s stimulation cessation). In consequence, post-rest potentiation of Ca2+-transient amplitude (measured using Fura-2) during the 30s pause was improved by SMP-114 (52±5 vs. 37±4%). Noteworthy, SMP-114 has these beneficial effects without negatively impairing global excitation-contraction coupling: neither systolic Ca2+ release nor single cell contractility was compromised, and also SR Ca2+ reuptake, in line with resulting cardiomyocyte relaxation, was not impaired by SMP-114 in our assays. SMP-114 demonstrated potential to treat SR Ca2+ leak and consequently proarrhythmogenic events in rodent as well as in human atrial cardiomyocytes and cardiomyocytes from patients with heart failure. Further research is necessary towards clinical use in cardiac disease.