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Background: Brugada syndrome (BrS) is an inherited arrhythmia syndrome caused by loss-of-function variants in the cardiac sodium channel gene SCN5A (sodium voltage-gated channel alpha subunit 5) in ≈20% of subjects. We identified a family with 4 individuals diagnosed with BrS harboring the rare G145R missense variant in the cardiac transcription factor TBX5 (T-box transcription factor 5) and no SCN5A variant. Methods: We generated induced pluripotent stem cells (iPSCs) from 2 members of a family carrying TBX5-G145R and diagnosed with Brugada syndrome. After differentiation to iPSC-derived cardiomyocytes (iPSC-CMs), electrophysiologic characteristics were assessed by voltage- and current-clamp experiments (n=9 to 21 cells per group) and transcriptional differences by RNA sequencing (n=3 samples per group), and compared with iPSC-CMs in which G145R was corrected by CRISPR/Cas9 approaches. The role of platelet-derived growth factor (PDGF)/phosphoinositide 3-kinase (PI3K) pathway was elucidated by small molecule perturbation. The rate-corrected QT (QTc) interval association with serum PDGF was tested in the Framingham Heart Study cohort (n=1893 individuals). Results: TBX5-G145R reduced transcriptional activity and caused multiple electrophysiologic abnormalities, including decreased peak and enhanced “late” cardiac sodium current (I Na ), which were entirely corrected by editing G145R to wild-type. Transcriptional profiling and functional assays in genome-unedited and -edited iPSC-CMs showed direct SCN5A down-regulation caused decreased peak I Na , and that reduced PDGF receptor ( PDGFRA [platelet-derived growth factor receptor α]) expression and blunted signal transduction to PI3K was implicated in enhanced late I Na . Tbx5 regulation of the PDGF axis increased arrhythmia risk due to disruption of PDGF signaling and was conserved in murine model systems. PDGF receptor blockade markedly prolonged normal iPSC-CM action potentials and plasma levels of PDGF in the Framingham Heart Study were inversely correlated with the QTc interval ( P Conclusions: These results not only establish decreased SCN5A transcription by the TBX5 variant as a cause of BrS, but also reveal a new general transcriptional mechanism of arrhythmogenesis of enhanced late sodium current caused by reduced PDGF receptor–mediated PI3K signaling.

Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal cardiac arrhythmia syndrome triggered by catecholamines released during exercise, stress, or sudden emotion. Variants in the calsequestrin-2 gene ( CASQ2 ), encoding the major calcium (Ca) binding protein in the sarcoplasmic reticulum (SR), are the second most common cause of CPVT. Recently, several CASQ2 gene variants, such as CASQ2 -K180R, have been linked to an autosomal dominant form of Casq2-linked CPVT (CPVT2), but the underlying mechanism is not known. Methods: A K180R mouse model was generated using CRIPSR/Cas9. Heterozygous and homozygous K180R mice were studied using telemetry ECG recordings in vivo. Ventricular cardiomyocytes were isolated and studied using fluorescent Ca indicators and patch clamp. Expression levels and localization of SR Ca-handling proteins were evaluated using Western blotting and immunostaining. Intra-SR Ca kinetics were quantified using low-affinity Ca indicators. Results: K180R mice exhibit an autosomal dominant CPVT phenotype following exercise or catecholamine stress. Upon catecholamine stress, K180R ventricular cardiomyocytes exhibit increased spontaneous SR Ca release events, triggering delayed afterdepolarizations and spontaneous beats. K180R had no effect on levels of Casq2, Casq2 polymers, or other SR Ca-handling proteins. Intra-SR Ca measurements revealed that K180R impaired dynamic intra-SR Ca buffering, resulting in a more rapid rise of free Ca in the SR during diastole. Steady-state SR Ca buffering and total SR Ca content were not changed. Consistent with the reduced dynamic intra-SR buffering, K180R causes reduced SR Ca release refractoriness. Conclusions: CASQ2-K180R causes CPVT2 via a heretofore unknown mechanism that differs from CASQ2 variants associated with autosomal recessive CPVT2. Unlike autosomal recessive CASQ2 variants, K180R impairs the dynamic buffering of Ca within the SR without affecting total SR Ca content or Casq2 protein levels. Our data provide insight into the molecular mechanism underlying autosomal dominant CPVT2.

Rationale: The class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive RyR2 (cardiac ryanodine receptor) mediated calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro, reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide’s efficacy in CPVT. Objective: To determine whether RyR2 block independently contributes to flecainide’s efficacy for suppressing spontaneous sarcoplasmic reticulum Ca release and for preventing ventricular tachycardia in vivo. Methods and Results: We synthesized N-methylated flecainide analogues (QX-flecainide and N -methyl flecainide) and showed that N -methylation reduces flecainide’s inhibitory potency on RyR2 channels incorporated into artificial lipid bilayers. N -methylation did not alter flecainide’s inhibitory activity on human cardiac sodium channels expressed in HEK293T cells. Antiarrhythmic efficacy was tested utilizing a Casq2 (cardiac calsequestrin) knockout (Casq2−/−) CPVT mouse model. In membrane-permeabilized Casq2−/− cardiomyocytes—lacking intact sarcolemma and devoid of sodium channel contribution—flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2−/− cardiomyocytes pretreated with tetrodotoxin to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous sarcoplasmic reticulum Ca release, while QX-flecainide and N -methyl flecainide did not. In vivo, flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2−/− mice, whereas N -methyl flecainide had no significant effect on arrhythmia burden, despite comparable sodium channel block. Conclusions: Flecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone did not prevent ventricular tachycardia. Hence, RyR2 channel inhibition likely constitutes the principal mechanism of antiarrhythmic action of flecainide in CPVT.

Background Mutations in cardiac troponin T (TnT) are linked to increased risk of ventricular arrhythmia and sudden death despite causing little to no cardiac hypertrophy. Studies in mice suggest that the hypertrophic cardiomyopathy (HCM)-associated TnT-I79N mutation increases myofilament Ca sensitivity and is arrhythmogenic, but whether findings from mice translate to human cardiomyocyte electrophysiology is not known. Objectives To study the effects of the TnT-I79N mutation in human cardiomyocytes. Methods Using CRISPR/Cas9, the TnT-I79N mutation was introduced into human induced pluripotent stem cells (hiPSCs). We then used the matrigel mattress method to generate single rod-shaped cardiomyocytes (CMs) and studied contractility, Ca handling and electrophysiology. Results Compared to isogenic control hiPSC-CMs, TnT-I79N hiPSC-CMs exhibited sarcomere disorganization, increased systolic function and impaired relaxation. The Ca-dependence of contractility was leftward shifted in mutation containing cardiomyocytes, demonstrating increased myofilament Ca sensitivity. In voltage-clamped hiPSC-CMs, TnT-I79N reduced intracellular Ca transients by enhancing cytosolic Ca buffering. These changes in Ca handling resulted in beat-to-beat instability and triangulation of the cardiac action potential, which are predictors of arrhythmia risk. The myofilament Ca sensitizer EMD57033 produced similar action potential triangulation in control hiPSC-CMs. Conclusions The TnT-I79N hiPSC-CM model not only reproduces key cellular features of TnT-linked HCM such as myofilament disarray, hypercontractility and diastolic dysfunction, but also suggests that this TnT mutation causes pro-arrhythmic changes of the human ventricular action potential.

Rationale: Human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CM) are increasingly being used for modeling heart disease and are under development for regeneration of the injured heart. However, incomplete structural and functional maturation of hiPSC-CM, including lack of T-tubules, immature excitation–contraction coupling, and inefficient Ca-induced Ca release remain major limitations. Objective: Thyroid and glucocorticoid hormones are critical for heart maturation. We hypothesized that their addition to standard protocols would promote T-tubule development and mature excitation–contraction coupling of hiPSC-CM when cultured on extracellular matrix with physiological stiffness (Matrigel mattress). Methods and Results: hiPSC-CM were generated using a standard chemical differentiation method supplemented with T3 (triiodothyronine) and/or Dex (dexamethasone) during days 16 to 30 followed by single-cell culture for 5 days on Matrigel mattress. hiPSC-CM treated with T3+Dex, but not with either T3 or Dex alone, developed an extensive T-tubule network. Notably, Matrigel mattress was necessary for T-tubule formation. Compared with adult human ventricular cardiomyocytes, T-tubules in T3+Dex-treated hiPSC-CM were less organized and had more longitudinal elements. Confocal line scans demonstrated spatially and temporally uniform Ca release that is characteristic of excitation–contraction coupling in the heart ventricle. T3+Dex enhanced elementary Ca release measured by Ca sparks and promoted RyR2 (ryanodine receptor) structural organization. Simultaneous measurements of L-type Ca current and intracellular Ca release confirmed enhanced functional coupling between L-type Ca channels and RyR2 in T3+Dex-treated cells. Conclusions: Our results suggest a permissive role of combined thyroid and glucocorticoid hormones during the cardiac differentiation process, which when coupled with further maturation on Matrigel mattress, is sufficient for T-tubule development, enhanced Ca-induced Ca release, and more ventricular-like excitation–contraction coupling. This new hormone maturation method could advance the use of hiPSC-CM for disease modeling and cell-based therapy.

Ca(2+) leak via ryanodine receptor type 2 (RyR2) can cause potentially fatal arrhythmias in a variety of heart diseases and has also been implicated in neurodegenerative and seizure disorders, making RyR2 an attractive therapeutic target for drug development. Here we synthesized and investigated the fungal natural product and known insect RyR antagonist (−)-verticilide and several congeners to determine their activity against mammalian RyR2. Although the cyclooligomeric depsipeptide natural product (−)-verticilide had no effect, its nonnatural enantiomer [ent-(+)-verticilide] significantly reduced RyR2-mediated spontaneous Ca(2+) leak both in cardiomyocytes from wild-type mouse and from a gene-targeted mouse model of Ca(2+) leak-induced arrhythmias (Casq2(−/−)). ent-(+)-verticilide selectively inhibited RyR2-mediated Ca(2+) leak and exhibited higher potency and a distinct mechanism of action compared with the pan-RyR inhibitors dantrolene and tetracaine and the antiarrhythmic drug flecainide. ent-(+)-verticilide prevented arrhythmogenic membrane depolarizations in cardiomyocytes without significant effects on the cardiac action potential and attenuated ventricular arrhythmia in catecholamine-challenged Casq2(−/−) mice. These findings indicate that ent-(+)-verticilide is a potent and selective inhibitor of RyR2-mediated diastolic Ca(2+) leak, making it a molecular tool to investigate the therapeutic potential of targeting RyR2 hyperactivity in heart and brain pathologies. The enantiomer-specific activity and straightforward chemical synthesis of (unnatural) ent-(+)-verticilide provides a compelling argument to prioritize ent-natural product synthesis. Despite their general absence in nature, the enantiomers of natural products may harbor unprecedented activity, thereby leading to new scaffolds for probe and therapeutic development.

Background— Calmodulin (CaM) is encoded by 3 genes, CALM1 , CALM2 , and CALM3 , all of which harbor pathogenic variants linked to long QT syndrome (LQTS) with early and severe expressivity. These LQTS-causative variants reduce CaM affinity to Ca 2+ and alter the properties of the cardiac L-type calcium channel (Ca V 1.2). CaM also modulates Na V 1.5 and the ryanodine receptor, RyR2. All these interactions may play a role in disease pathogenesis. Here, we determine the spectrum and prevalence of pathogenic CaM variants in a cohort of genetically elusive LQTS, and functionally characterize the novel variants. Methods and Results— Thirty-eight genetically elusive LQTS cases underwent whole-exome sequencing to identify CaM variants. Nonsynonymous CaM variants were over-represented significantly in this heretofore LQTS cohort (13.2%) compared with exome aggregation consortium (0.04%; P 2+ -binding affinity and a functionally dominant loss of inactivation in Ca V 1.2, mild accentuation in Na V 1.5 late current, but no effect on intracellular RyR2-mediated calcium release. Conclusions— Overall, 13% of our genetically elusive LQTS cohort harbored nonsynonymous variants in CaM. Genetic testing of CALM1-3 should be pursued for individuals with LQTS, especially those with early childhood cardiac arrest, extreme QT prolongation, and a negative family history.

Sodium can accumulate in the interstitium and promote inflammation through poorly defined mechanisms. Here we describe a novel pathway by which dendritic cells (DCs) are activated by increased extracellular sodium (190 mM). We show that sodium entry through the epithelial sodium channel (ENaC) and the sodium hydrogen exchanger (NHE) leads to calcium influx via the sodium calcium exchanger, activation of protein kinase C (PKC), phosphorylation of p47phox and association of p47phox with gp91phox. The assembled NADPH oxidase produces superoxide with subsequent formation of immunogenic isolevuglandin (IsoLG)-protein adducts. DCs activated by excess sodium produce increased interleukin 1β (IL-1β) and promote T cell production of cytokines IL-17A and interferon gamma (IFN-γ). When adoptively transferred into naive mice, these DCs prime hypertension in response to a sub-pressor dose of angiotensin II. These findings provide a mechanistic link between salt, inflammation and hypertension involving increased oxidative stress and IsoLG production in DCs.

Intravenous lipid emulsion (ILE), a component of parenteral nutrition, consists of a fat emulsion of soy bean oil, egg phospholipids, and glycerin. Case reports suggest that ILE may reverse hypotension caused by acute poisoning with lipophilic drugs such as verapamil, but the mechanism remains unclear. The methods used are the following: (1) measurement of ILE concentration in serum samples from a patient with verapamil poisoning treated with ILE, (2) measurement of free verapamil concentrations in human serum mixed in vitro with increasing concentrations of ILE, and (3) measurement of murine ventricular cardiomyocyte L-type Ca(2+) currents, intracellular Ca(2+), and contractility in response to verapamil and/or ILE. Maximum patient serum ILE concentration after infusion of 1 L ILE over 1 h was approximately 1.6 vol%. In vitro GC/MS verapamil assays showed that addition of ILE (0.03-5.0 vol%) dose-dependently decreased the free verapamil concentration in human serum. In voltage-clamped myocytes, adding ILE to Tyrode's solution containing 5 μM verapamil recovered L-type Ca(2+) currents (ICa). Recovery was concentration dependent, with significant ICa recovery at ILE concentrations as low as 0.03 vol%. ILE had no effect on ICa in the absence of verapamil. In field-stimulated intact ventricular myocytes exposed to verapamil, adding ILE (0.5 %) resulted in a rapid and nearly complete recovery of myocyte contractility and intracellular Ca(2+). Our in vitro studies indicate that ILE acts as a lipid sink that rapidly reverses impaired cardiomyocyte contractility in the continued presence of verapamil.

Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) are being increasingly used to model human heart diseases. hiPSC-CMs generated by earlier aggregation-based methods (i.e., embryoid body) often lack functional sarcoplasmic reticulum (SR) Ca stores characteristic of mature mammalian CMs. Newer monolayer-based cardiac differentiation methods (i.e., Matrigel sandwich or small molecule-based differentiation) produce hiPSC-CMs of high purity and yield, but their Ca handling has not been comprehensively investigated. Here, we studied Ca handling and cytosolic Ca buffering properties of hiPSC-CMs generated independently from multiple hiPSC lines at Stanford University, Vanderbilt University and University of Wisconsin-Madison. hiPSC-CMs were cryopreserved at each university. Frozen aliquots were shipped, recovered from cryopreservation, plated at low density and compared 3–5days after plating with acutely-isolated adult rabbit and mouse ventricular CMs. Although hiPSC-CM cell volume was significantly smaller, cell capacitance to cell volume ratio and cytoplasmic Ca buffering were not different from rabbit-CMs. hiPSC-CMs from all three laboratories exhibited robust L-type Ca currents, twitch Ca transients and caffeine-releasable SR Ca stores comparable to adult CMs. Ca transport by sarcoendoplasmic reticulum Ca ATPase (SERCA) and Na/Ca exchanger (NCX) was similar in all hiPSC-CM lines, but slower compared to rabbit-CMs. However, the relative contribution of SERCA and NCX to Ca transport of hiPSC-CMs was comparable to rabbit-CMs. Ca handling maturity of hiPSC-CMs increased from 15 to 21days post-induction. We conclude that hiPSC-CMs generated independently from multiple iPSC lines using monolayer-based methods can be reproducibly recovered from cryopreservation and exhibit comparable and functional SR Ca handling.

Familial hypertrophic cardiomyopathy is caused by mutations in genes encoding sarcomere proteins. Among hypertrophic cardiomyopathy–linked disease genes, cardiac troponin T (TnT) mutations are associated with a high incidence of arrhythmic cardiac death [(1)][1], but the underlying mechanism has

Background Genome‐wide association studies have implicated variants in SCN 10A , which encodes Nav1.8, as modulators of cardiac conduction. Follow‐up work has indicated the SCN 10A sequence includes an intronic enhancer for SCN 5A . Yet the role of the Nav1.8 protein in the myocardium itself is still unclear. To investigate this , we use homozygous knockout mice ( Scn10a −/− ) generated by disruption of exons 4 and 5, leaving the Scn5a enhancer intact. Methods and Results We previously reported that pharmacologic blockade of Nav1.8 in wild‐type animals blunts action potential prolongation by ATX ‐ II at slow drive rates (≤1 Hz). Here we present evidence of the same blunting in Scn10a −/− compared to wild‐type ventricular myocytes, supporting the conclusion that Nav1.8 contributes to late sodium current at slow rates. In contrast to earlier studies, we found no differences in electrocardiographic parameters between genotypes. Low‐dose ATX ‐ II exposure in lightly anesthetized animals and Langendorff‐perfused hearts prolonged QT c and generated arrhythmias to the same extent in wild‐type and Scn10a −/− . RNA sequencing failed to identify full‐length Scn10a transcripts in wild‐type or knockout isolated ventricular myocytes. However, loss of late current in Scn10a −/− myocytes was replicated independently in a blinded set of experiments. Conclusions While Scn10a transcripts are not detectible in ventricular cardiomyocytes, gene deletion results in reproducible loss of late sodium current under extreme experimental conditions. However, there are no identifiable consequences of this Scn10a deletion in the intact mouse heart at usual rates. These findings argue that common variants in SCN 10A that affect ventricular conduction do so by modulating SCN 5A .

Background— Calmodulin (CaM) mutations are associated with severe forms of long QT syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT). CaM mutations are found in 13% of genotype-negative long QT syndrome patients, but the prevalence of CaM mutations in genotype-negative CPVT patients is unknown. Here, we identify and characterize CaM mutations in 12 patients with genotype-negative but clinically diagnosed CPVT. Methods and Results— We performed mutational analysis of CALM1 , CALM2 , and CALM3 gene-coding regions, in vitro measurement of CaM-Ca 2+ (Ca)-binding affinity, ryanodine receptor 2–CaM binding, Ca handling, L-type Ca current, and action potential duration. We identified a novel CaM mutation—A103V—in CALM3 in 1 of 12 patients (8%), a female who experienced episodes of exertion-induced syncope since age 10, had normal QT interval, and displayed ventricular ectopy during stress testing consistent with CPVT. A103V modestly lowered CaM Ca-binding affinity (3-fold reduction versus WT-CaM), but did not alter CaM binding to ryanodine receptor 2. In permeabilized cardiomyocytes, A103V-CaM (100 nmol/L) promoted spontaneous Ca wave and spark activity, a cellular phenotype of ryanodine receptor 2 activation. Even a 1:3 mixture of A103V-CaM:WT-CaM activated Ca waves, demonstrating functional dominance. Compared with long QT syndrome D96V-CaM, A103V-CaM had significantly less effects on L-type Ca current inactivation, did not alter action potential duration, and caused delayed afterdepolarizations and triggered beats in intact cardiomyocytes. Conclusions— We discovered a novel CPVT mutation in the CALM3 gene that shares functional characteristics with established CPVT-associated mutations in CALM1 . A small proportion of A103V-CaM is sufficient to evoke arrhythmogenic Ca disturbances via ryanodine receptor 2 dysregulation, which explains the autosomal dominant inheritance.

Introduction: Calmodulin (CaM) is an essential Ca2+ signaling protein in a wide range of biological processes. In the dyadic cleft CaM binds to RyR2, Cav1.2 and CaMKII. CaM mutations are associated with an autosomal dominant syndrome of sudden death that can present with clinical features of catecholaminergic polymorphic ventricular tachycardia (CPVT) or long QT syndrome (LQTS). CPVT-linked CaM mutations activate ryanodine receptor (RyR2) Ca release channels; whereas LQTS-CaMs have no effects on RyR2 channels but prolong the action potential by impairing L-type Ca current (LTCC) inactivation. Here we report a novel CaM mutation – A103V – in CALM3, in a 10 year old female with episodes of exertion-induced syncope and normal QT interval consistent with CPVT. Her mother is mutation positive and also had a positive exercise stress test.Methods: CaM-A103V Ca binding affinity was measured by spectrofluorometry. Ca handling and LTCC were tested in mouse ventricular myocytes dialyzed with WT or the mutant CaM and compared to a published CPVT CaM mutant (N54I).Results: A103V modestly lowered CaM Ca binding affinity (3-fold reduction vs WT-CaM). In permeabilized myocytes, A103V-CaMs at a physiological intracellular concentration (100nM) promoted significantly higher spontaneous Ca wave and spark activity, a typical cellular phenotype of CPVT. Even a 1:4 mixture of A103V-CaM:WT-CaM activated Ca waves, demonstrating functional dominance. In voltage-clamped cardiomyocytes dialyzed with WT or mutant CaM, CaM-A103V had significantly less effects on LTCC inactivation compared to published CaM mutants that cause LQTS.Conclusion: A103V CaM shares functional characteristics with the established CPVT-CaM N54I. A small proportion of A103V-CaM is sufficient to evoke arrhythmogenic Ca disturbances, which explains the autosomal dominant inheritance.

We reported previously that flecainide suppresses Ca waves in MEMBRANE-PERMEABILIZED ventricular myocytes from Casq2-/- mice and that flecainide is more potent (i.e., lower IC50) in Casq2-/-compared to WT myocytes. Other groups reported that in INTACT myocytes flecainide acts only by blocking Na+ channels and has no effect on RyR2. Here we test the effect of flecainide on INTACT ventricular myocytes from Casq2-/- and WT littermates using voltage-clamp. To eliminate any contribution of Na channels, all solutions contained 30 µM TTX. Complete block INa was confirmed for each cell tested. Spontaneous Ca waves were elicited by a pacing train followed by a holding potential of −70mV for 45 s. 6 µM flecainide or vehicle (H2O) was added to both external and pipette solutions. Under such conditions, flecainide had no significant effect on the average number of spontaneous Ca waves in WT myocytes (Vehicle: 3.1±0.6, n=24, Flecainide: 3.0±0.8, n=20, p=NS). In Casq2-/- myocytes, however, flecainide significantly reduced Ca waves (Vehicle: 6.6±1.2, n=23; Flecainide: 2.1±0.4, n=19, p 0.5 vs. Casq2-/- vehicle), suggesting that intracellular dialysis by the patch pipette removed flecainide from the cytosol. We conclude that intracellular concentrations of 6 µM flecainide effectively inhibit arrhythmogenic Ca waves in Casq2-/- but not in WT myocytes, indicating that flecainide action is dependent on the state of RyR2. Our results further demonstrate that flecainide suppression of Ca waves is independent from its Na channel block.

Rationale: Ca binding to the troponin complex represents a major portion of cytosolic Ca buffering. Troponin mutations that increase myofilament Ca sensitivity are associated with familial hypertrophic cardiomyopathy and confer a high risk for sudden death. In mice, Ca sensitization causes ventricular arrhythmias, but the underlying mechanisms remain unclear. Objective: To test the hypothesis that myofilament Ca sensitization increases cytosolic Ca buffering and to determine the resulting arrhythmogenic changes in Ca homeostasis in the intact mouse heart. Methods and Results: Using cardiomyocytes isolated from mice expressing troponin T (TnT) mutants (TnT-I79N, TnT-F110I, TnT-R278C), we found that increasing myofilament Ca sensitivity produced a proportional increase in cytosolic Ca binding. The underlying cause was an increase in the cytosolic Ca binding affinity, whereas maximal Ca binding capacity was unchanged. The effect was sufficiently large to alter Ca handling in intact mouse hearts at physiological heart rates, resulting in increased end-diastolic [Ca] at fast pacing rates, and enhanced sarcoplasmic reticulum Ca content and release after pauses. Accordingly, action potential (AP) regulation was altered, with postpause action potential prolongation, afterdepolarizations, and triggered activity. Acute Ca sensitization with EMD 57033 mimicked the effects of Ca-sensitizing TnT mutants and produced pause-dependent ventricular ectopy and sustained ventricular tachycardia after acute myocardial infarction. Conclusions: Myofilament Ca sensitization increases cytosolic Ca binding affinity. A major proarrhythmic consequence is a pause-dependent potentiation of Ca release, action potential prolongation, and triggered activity. Increased cytosolic Ca binding represents a novel mechanism of pause-dependent arrhythmia that may be relevant for inherited and acquired cardiomyopathies.

Cardiac calsequestrin (Casq2) is the major Ca2+ binding protein in the sarcoplasmic reticulum, which is the principle Ca2+ storage organelle of cardiac muscle. During the last decade, experimental studies have provided new concepts on the role of Casq2 in the regulation of cardiac muscle Ca2+ handling. Furthermore, mutations in the gene encoding for cardiac calsequestrin, CASQ2, cause a rare but severe form of catecholaminergic polymorphic ventricular tachycardia (CPVT). Here, we review the physiology of Casq2 in cardiac Ca2+ handling and discuss pathophysiological mechanisms that lead to CPVT caused by CASQ2 mutations. We also describe the clinical aspects of CPVT and provide an update of its contemporary clinical management.

Using a standard patch-clamp technique in the perforated patch configuration, we studied the effect of a highly specific membrane-permeable inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaM-KII), KN-93, on fast outward A-type potassium current in isolated smooth-muscle cells (SMCs) of an epididymal region of the rat vas deferens. This inhibitor significantly changed the dynamics of the studied current; in particular, it increased the rate of inactivation and considerably slowed down the recovery after inactivation. In the presence of 5 µM KN-93, we observed a moderate (nearly by 20%) decrease in the peak amplitude of fast A-type current. Based on the data obtained, we conclude that voltage-sensitive fast A-type potassium current in SMCs of the epididymal part of the rat vas deferens can be significantly regulated by the activity of CaM-KII. Therefore, by influencing the kinetic characteristics of the above current, this enzyme can be indirectly involved in the control of electrical activity in SMCs.

Aims In cardiac muscle, Ca 2 + release from sarcoplasmic reticulum (SR) is reduced with successively shorter coupling intervals of premature stimuli, a phenomenon known as SR Ca 2 + release refractoriness. We recently reported that the SR luminal Ca 2 + binding protein calsequestrin 2 (Casq2) contributes to release refractoriness in intact mouse hearts, but the underlying mechanisms remain unclear. Here, we further investigate the mechanisms responsible for physiological release refractoriness. Methods and results Gene-targeted ablation of Casq2 (Casq2 KO) abolished SR Ca 2 + release refractoriness in isolated mouse ventricular myocytes. Surprisingly, impaired Ca 2 + -dependent inactivation of L-type Ca 2 + current (I Ca ), which is responsible for triggering SR Ca 2 + release, significantly contributed to loss of Ca 2 + release refractoriness in Casq2 KO myocytes. Recovery from Ca 2 + -dependent inactivation of I Ca was significantly accelerated in Casq2 KO compared to wild-type (WT) myocytes. In contrast, voltage-dependent inactivation measured by using Ba 2 + as charge carrier was not significantly different between WT and Casq2 KO myocytes. Ca 2 + -dependent inactivation of I Ca was normalized by intracellular dialysis of excess apo-CaM (20 μM), which also partially restored physiological Ca 2 + release refractoriness in Casq2 KO myocytes. Conclusions Our findings reveal that the intra-SR protein Casq2 is largely responsible for the phenomenon of SR Ca 2 + release refractoriness in murine ventricular myocytes. We also report a novel mechanism of impaired Ca 2 + -CaM-dependent inactivation of Ca v 1.2, which contributes to the loss of SR Ca 2 + release refractoriness in the Casq2 KO mouse model and, therefore, may further increase risk for ventricular arrhythmia in vivo .

Using a patch-clamp technique in the whole-cell configuration, we studied transmembrane ion currents in isolated single smooth muscle cells of the guinea pig taenia coli. A depolarizing step shift of the membrane potential from −50 mV was accompanied by the appearance of an outward current. Application of d-tubocurarine (d-TK) or a nonselective blocker of voltage-dependent potassium channels, tetraethylammonium (TEA), led to a decrease in the outward current. Application of d-TK against the background of the action of TEA additionally decreased the outward current. Analysis of the current-voltage (I–V) relationships of the d-TK-sensitive current showed that this current is practically voltage-independent. At the same time, an inflection of the I–V curve of the potassium current within the segment of maximum activation of the voltage-dependent potassium current is indicative of the sensitivity of this current to the intracellular Ca2+ concentration. Therefore, the calcium-activated potassium current through small-conductance calcium-dependent potassium channels includes a d-TK-sensitive voltage-independent component. Using depolarizing shifts of the membrane potential, we observed high- and low-amplitude spontaneous outward currents (SOCs) in many studied cells, i.e., the effect of an increase in the conductance of calcium-dependent potassium channels as a result of periodic release of Ca2+ from the intracellular stores. Application of d-TK led to a decrease in the frequency of low-amplitude SOCs and exerted nearly no influence on the high-amplitude SOCs under study.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide a unique tool to evaluate cellular properties in disease models, investigate underlying mechanisms and optimize cell-based therapies. We recently reported that after plating 30 day old single hiPSC-CMs on undiluted Matrigel “Mattress” (thickness ∼0.5 mm) for 5 days, mattress hiPSC-CMs developed a rod-like cell shape, aligned myofilament structure, increased sarcomere length and exhibited robust cell shortening. Here, we compared the electrophysiological properties of mattress hiPSC-CMs to hiPSC-CMs cultured on standard substrates (control) and acutely-isolated adult rabbit CMs. The action potentials of mattress hiPSC-CMs were comparable to rabbit CMs. Compared to control, mattress hiPSC-CMs had significantly increased the action potential upstroke velocity due to two-fold increase in sodium current (INa) (−68.61±11.63 pA·pF-1 in mattress vs. −29.66±7.50 pA·pF-1 in control at −30mV). The inward rectifier current (IK1) is responsible for the maintenance of resting membrane potential and contributes to phase 3 of the action potential. Although resting potential and action potential duration was not different from rabbit CMs, IK1 density in mattress hiPSC-CMs was nine-time smaller than in rabbit CMs, but there was no difference between mattress and control hiPSC-CMs (−4.86±0.66 pA·pF-1 in mattress vs. −43.75±4.13 pA·pF-1 in rabbit vs. −3.98±0.83 pA·pF-1 in control at −120 mV). The funny current (If) is an inward current activating at hyperpolarized membrane potential, which is detectable in the developing heart but absent in the adult ventricle and contributes to the spontaneous beating of hiPSC-CMs. Mattress hiPSC-CMs exhibit robust If that was not significantly different from control (−6.22±1.31 pA·pF-1 in mattress vs.-5.60±1.43 pA·pF-1 in control at −120 mV). In summary, the matrigel mattress method enables the rapid generation of robustly contracting single hiPSC-CMs and increases INa, with negligible effect on IK1 or If.

Voltage-gated components of the outward current in single smooth muscle cells isolated from the epididymal part of the rat vas deferens were studied using amphotericin B perforated patch-clamp techniques. The complex kinetics of the net outward current elicited by positive voltage steps from −80 mV to +40 mV suggested the presence of several components. Bath application of 200 nM charybdotoxin, a potent blocker of large-conductance, Ca2+-dependent K+ channels (BKCa), reduced the current amplitude significantly. When BKCa channels were suppressed, fast-inactivating (I K,f) and delayed rectifying (I K,dr) components of the outward current were identified. I K,f was characterized by fast kinetics of current decay, negative steady-state activation and inactivation dependencies and sensitivity to 4-aminopyridine with an apparent K d of 0.32 mM, properties similar to those of the A-type K+ current. In contrast, I K,dr activated and inactivated at more positive potentials. The time constant of activation of I K,dr was voltage dependent with an e-fold decrease per 21 mV depolarization. I K,dr was inhibited by clofilium, a blocker of voltage-gated K+ channels, with an IC50 of 12 µM and was not blocked by 5 mM 4-aminopyridine. The possible significance of the voltage-gated currents is discussed.

Green tea polyphenolic catechins exhibit biological activity in a wide variety of cell types. Although reports in the lay and scientific literature suggest therapeutic potential for improving cardiovascular health, the underlying molecular mechanisms of action remain unclear. Previous studies have implicated a wide range of molecular targets in cardiac muscle for the major green tea catechin, (-)-epigallocatechin-3-gallate (EGCG), but effects were observed only at micromolar concentrations of unclear clinical relevance. Here, we report that nanomolar concentrations of EGCG significantly enhance contractility of intact murine myocytes by increasing electrically evoked Ca(2+) transients, sarcoplasmic reticulum (SR) Ca(2+) content, and ryanodine receptor type 2 (RyR2) channel open probability. Voltage-clamp experiments demonstrate that 10 nM EGCG significantly inhibits the Na(+)-Ca(2+) exchanger. Of importance, other Na(+) and Ca(2+) handling proteins such as Ca(2+)-ATPase, Na(+)-H(+) exchanger, and Na(+)-K(+)-ATPase were not affected by EGCG ≤ 1 μM. Thus, nanomolar EGCG increases contractility in intact myocytes by coordinately modulating SR Ca(2+) loading, RyR2-mediated Ca(2+) release, and Na(+)-Ca(2+) exchange. Inhibition of Na(+)-K(+)-ATPase activity probably contributes to the positive inotropic effects observed at EGCG concentrations1 μM. These newly recognized actions of nanomolar and micromolar EGCG should be considered when the therapeutic and toxicological potential of green tea supplementation is evaluated and may provide a novel therapeutic strategy for improving contractile function in heart failure.

Interstitial cells of Cajal (ICCs) freshly isolated from rabbit portal vein and loaded with the Ca(2+)-sensitive indicator fluo-3 revealed rhythmical [Ca(2+)](i) changes occurring at 0.02-0.1 Hz. Each increase in [Ca(2+)](i) originated from a discrete central region of the ICC and propagated as a [Ca(2+)](i) wave towards the cell periphery, but usually became attenuated before reaching the ends of the cell. In about 40% of ICCs each rhythmical change in [Ca(2+)](i) consisted of an initial [Ca(2+)](i) increase (phase 1) followed by a faster rise in [Ca(2+)](i) (phase 2) and then a decrease in [Ca(2+)](i) (phase 3); the frequency correlated with the rate of rise of [Ca(2+)](i) during phase 1, but not with the peak amplitude. Rhythmical [Ca(2+)](i) changes persisted in nicardipine, but were abolished in Ca(2+)-free solution as well as by SK&F96365, cyclopiazonic acid, thapsigargin, 2-APB, xestospongin C or ryanodine. Intracellular Ca(2+) stores visualised with the low-affinity Ca(2+) indicator fluo-3FF were found to be enriched with ryanodine receptors (RyRs) detected with BODIPY TR-X ryanodine. Rhythmical [Ca(2+)](i) changes originated from a perinuclear S/ER element showing the highest RyR density. Immunostaining with anti-TRPC3,6,7 antibodies revealed the expression of these channel proteins in the ICC plasmalemma. This suggests that these rhythmical [Ca(2+)](i) changes, a key element of ICC pacemaking activity, result from S/ER Ca(2+) release which is mediated via RyRs and IP(3) receptors and is modulated by the activity of S/ER-Ca(2+)-ATPase and TRP channels but not by L-type Ca(2+) channels.

L-type Ca current (ICa) triggers Carelease from the sarcoplasmic reticulum (SR) by activating RyR2 Ca release channels. Here we probe the role of the SR protein calsequestrin in the regulation of SR Ca2+ release. Voltage-clamped cardiomyocytes loaded with Fluo-4 were stimulated with 1 Hz train (S1 stimuli). Ca2+ release was activated with ICa tail currents to maintain constant trigger. Application of premature extra stimuli (S2) at successively shorter S1-S2 coupling interval resulted in significant depression of Ca2+ release during S2 stimuli in wild-type myocytes. In calsequestrin knockout myocytes (Casq2 KO), the amplitudes of S2 and S1 were the same even at shortest S1-S2 intervals (Figure). This result suggests that calsequestrin is the primary regulator of release refractoriness. To test this hypothesis further, we next used myocytes from calsequestrin overexpressing mice (Casq2 OverX). Casq2 OverX cardiomyocytes exhibit significantly depressed Ca release during S1 transients despite increased SR Ca2+ content. Surprisingly, Ca release amplitudes during the premature S2 stimulus exceeded S1 at short S1-S2 intervals (Figure). Thus, either too little or too much calsequestrin causes loss of SR Ca release refractoriness. Supported by NIH-RO1-HL88635.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Cardiac Ca-release from sarcoplasmic reticulum (SR) is reduced with successively shorter coupling intervals of premature stimuli, a phenomenon known as SR Ca-release restitution. Interestingly, myocytes lacking the SR luminal Ca binding protein calsequestrin (Casq2 KO) lack SR Ca-release restitution. Here we test whether altered L-type Ca current (ICa) responsible for triggering SR Ca-release contributes to the loss of Ca-release restitution.Results: ICa was recorded in voltage-clamped ventricular myocytes isolated from Casq2 KO mice and wild-type (WT) littermates. Cells were pre-treated with ryanodine and thapsigargin to eliminate SR Ca-release. After a 1 Hz train of depolarizing stimuli (S1), ICa restitution was measured by applying premature stimuli (S2) at successively shorter S1-S2 coupling intervals. Both groups have decreased ICa at short S1-S2 intervals, but ICa recovered significantly faster in KO vs. WT myocytes (p

In cardiac excitation-contraction coupling, L-type Ca2+ current (ICa) triggers Ca2+ release from the sarcoplasmic reticulum (SR) via ryanodine receptor (RyR) Ca2+ release channels. It is unclear why SR Ca2+ release cannot be elicited by premature stimuli, even though ICa is fully recovered. Here, we use calsequestrin null mice (Casq2 KO) and wild-type littermates (WT) to test the hypothesis that calsequestrin (Casq2) determines refractoriness of SR Ca2+ release. Ca2+ release refractoriness was measured in voltage-clamped myocytes dialyzed with Fluo-4 by applying premature extrastimuli (S2) at successively shorter S1-S2 coupling intervals following a 1 Hz train (S1 stimuli). To maintain constant trigger, Ca2+ release was activated with ICa tail currents that elicited maximal Ca2+ release during the S1 train. WT S2 Ca2+release was significantly depressed with short coupling interval whereas Casq2 KO cardiomyocytes exhibit no refractoriness of Ca2+ release (Figure, n=11 WT, 12 KO, p = 0.01). At the same time, ICa current density, SR Ca2+ content, and steady-state Ca2+ transients (S1) were not significantly different from WT-myocytes. We conclude that calsequestrin is a critical determinant of SR Ca2+ release refractoriness in cardiac muscle (Supported by NIH-R01HL71670, R01HL88635).View Large Image | View Hi-Res Image | Download PowerPoint Slide

Recent reports identified six calmodulin (CaM) missense mutations associated with three distinct human arrhythmia syndromes: N54I and N98S are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT)-like clinical presentations, D96V, D130G and F142L with long QT syndrome (LQTS) and F90L with idiopathic ventricular fibrillation (IVF). We previously reported that all LQTS-CaMs exhibit reduced Ca affinity and normal or decreased (compared to WT CaM) ryanodine receptor (RyR2)-binding affinity, whereas CPVT-CaMs have normal or modestly lower Ca affinity but increase RyR2 single-channel open probability and show enhanced binding affinity to RyR2, causing significant increase in spontaneous Ca wave and spark activity in cardiac myocytes. Here, we investigate the effect of these mutant CaMs on another target potentially involved in arrhythmogenesis - Cav1.2 channels.Using whole-cell patch clamp, Cav1.2 current (ICa) was measured in freshly isolated murine ventricular myocytes pre-treated with ryanodine (50 µM) and thapsigargin (10 µM) to prevent SR Ca release. Cells were dialyzed with either wild-type or mutant CaM (6 µM) via patch pipette. All LQTS-CaMs and IVF-CaM caused significant impairment of ICa inactivation: τ=254±34 ms (D96V), 251±10 ms (F142L), 187±13 ms (D130G) and 190±8 ms (F90L) vs. 110±5 ms for WT (p

A major concern about using cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) for human disease modeling is their "immature" phenotype which may result in substantial differences in their physiology when compared to native cardiomyocytes. Here we compare intracellular Ca buffering of hiPSC-CM derived from healthy humans to that of acutely-isolated adult rabbit ventricular CM. hiPSC-CM (21 days post cardiac induction) were plated at low density on matrigel-coated dishes and studied 3-5 days after plating. While hiPSC were significantly smaller than rabbit CM (cell capacity 19±2 pF vs 101±12 pF, p

In cardiac muscle, L-type Ca current (CaV 1.2, ICa) induced Ca release from sarcoplasmic reticulum (SR) is reduced with successively shorter coupling intervals of premature stimuli, a phenomenon known as SR Ca release restitution. We previously reported that myocytes lacking the SR luminal Ca binding protein calsequestrin (Casq2 KO) exhibited less SR Ca release restitution and hence largely lack Ca release refractoriness. Here, we test the hypothesis that altered CaV 1.2 channel gating contributes to altered SR Ca release restitution in Casq2 KO myocytes.Methods and Results: ICa was recorded in voltage-clamped ventricular myocytes isolated from Casq2 KO mice and wild-type (WT) littermates. Cells were pre-treated with ryanodine (50 μM) and thapsigargin (10 μM) to eliminate SR Ca release. Compared to WT, ICa peak currents were unchanged, but the inactivation time course was significantly slower in KO myocytes (tau=157 ms vs. 46 ms in WT, p