Transcriptional Dysregulation Underlies Both Monogenic Arrhythmia Syndrome and Common Modifiers of Cardiac Repolarization.

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 (INa), 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 INa, and that reduced PDGF receptor (PDGFRA [platelet-derived growth factor receptor α]) expression and blunted signal transduction to PI3K was implicated in enhanced late INa. 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<0.001). 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.

Impaired Dynamic Sarcoplasmic Reticulum Ca Buffering in Autosomal Dominant CPVT2.

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.

Thyroid and Glucocorticoid Hormones Promote Functional T-Tubule Development in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

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.

Hypertrophic cardiomyopathy-linked mutation in troponin T causes myofibrillar disarray and pro-arrhythmic action potential changes in human iPSC cardiomyocytes.

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.

Loss of Adult Cardiac Myocyte GSK-3 Leads to Mitotic Catastrophe Resulting in Fatal Dilated Cardiomyopathy.

RATIONALE: Cardiac myocyte-specific deletion of either glycogen synthase kinase (GSK)-3α and GSK-3ß leads to cardiac protection after myocardial infarction, suggesting that deletion of both isoforms may provide synergistic protection. This is an important consideration because of the fact that all GSK-3-targeted drugs, including the drugs already in clinical trial target both isoforms of GSK-3, and none are isoform specific. OBJECTIVE: To identify the consequences of combined deletion of cardiac myocyte GSK-3α and GSK-3ß in heart function. METHODS AND RESULTS: We generated tamoxifen-inducible cardiac myocyte-specific mice lacking both GSK-3 isoforms (double knockout). We unexpectedly found that cardiac myocyte GSK-3 is essential for cardiac homeostasis and overall survival. Serial echocardiographic analysis reveals that within 2 weeks of tamoxifen treatment, double-knockout hearts leads to excessive dilatative remodeling and ventricular dysfunction. Further experimentation with isolated adult cardiac myocytes and fibroblasts from double-knockout implicated cardiac myocytes intrinsic factors responsible for observed phenotype. Mechanistically, loss of GSK-3 in adult cardiac myocytes resulted in induction of mitotic catastrophe, a previously unreported event in cardiac myocytes. Double-knockout cardiac myocytes showed cell cycle progression resulting in increased DNA content and multinucleation. However, increased cell cycle activity was rivaled by marked activation of DNA damage, cell cycle checkpoint activation, and mitotic catastrophe-induced apoptotic cell death. Importantly, mitotic catastrophe was also confirmed in isolated adult cardiac myocytes. CONCLUSIONS: Together, our findings suggest that cardiac myocyte GSK-3 is required to maintain normal cardiac homeostasis, and its loss is incompatible with life because of cell cycle dysregulation that ultimately results in a severe fatal dilated cardiomyopathy.

Troponin I-interacting protein kinase: a novel cardiac-specific kinase, emerging as a molecular target for the treatment of cardiac disease.

Coronary artery disease is the leading cause of death and disability worldwide. In patients with acute coronary syndromes, timely and effective myocardial reperfusion by percutaneous coronary intervention is the primary treatment of choice to minimize the ischemic injury and limit the size of the myocardial infarction (MI). However, reperfusion can itself promote cardiomyocyte death, which leads to cardiac dysfunction via reperfusion injury. The molecular mechanisms of ischemia-reperfusion (IR) injury are not completely understood and new drug targets are needed. Recently, we reported that cardiac troponin I-interacting protein kinase (TNNI3K), a cardiomyocyte-specific kinase, promotes IR injury via profound oxidative stress, thereby promoting cardiomyocyte death. By using novel genetic animal models and newly developed small-molecule TNNI3K inhibitors, we demonstrated that TNNI3K-mediated IR injury occurs through impaired mitochondrial function and is in part dependent on p38 MAPK. Here we discuss the emerging role of TNNI3K as a promising new drug target to limit IR-induced myocardial injury. We will also examine the underlying mechanisms that drive the profoundly reduced infarct size in mice in whichTNNI3Kis specifically deleted in cardiomyocytes. Because TNNI3K is a cardiac-specific kinase, it could be an ideal molecular target, as inhibiting it would have little or no effect on other organ systems, a serious problem associated with the use of kinase inhibitors targeting kinases that are more widely expressed.

Patient-independent human induced pluripotent stem cell model: A new tool for rapid determination of genetic variant pathogenicity in long QT syndrome.

BACKGROUND: Commercial genetic testing for long QT syndrome (LQTS) has rapidly expanded, but the inability to accurately predict whether a rare variant is pathogenic has limited its clinical benefit. Novel missense variants are routinely reported as variant of unknown significance (VUS) and cannot be used to screen family members at risk for sudden cardiac death. Better approaches to determine the pathogenicity of VUS are needed. OBJECTIVE: The purpose of this study was to rapidly determine the pathogenicity of a CACNA1C variant reported by commercial genetic testing as a VUS using a patient-independent human induced pluripotent stem cell (hiPSC) model. METHODS: Using CRISPR/Cas9 genome editing, CACNA1C-p.N639T was introduced into a previously established hiPSC from an unrelated healthy volunteer, thereby generating a patient-independent hiPSC model. Three independent heterozygous N639T hiPSC lines were generated and differentiated into cardiomyocytes (CM). Electrophysiological properties of N639T hiPSC-CM were compared to those of isogenic and population control hiPSC-CM by measuring the extracellular field potential (EFP) of 96-well hiPSC-CM monolayers and by patch clamp. RESULTS: Significant EFP prolongation was observed only in optically stimulated but not in spontaneously beating N639T hiPSC-CM. Patch-clamp studies revealed that N639T prolonged the ventricular action potential by slowing voltage-dependent inactivation of CaV1.2 currents. Heterologous expression studies confirmed the effect of N639T on CaV1.2 inactivation. CONCLUSION: The patient-independent hiPSC model enabled rapid generation of functional data to support reclassification of a CACNA1C VUS to likely pathogenic, thereby establishing a novel LQTS type 8 mutation. Furthermore, our results indicate the importance of controlling beating rates to evaluate the functional significance of LQTS VUS in high-throughput hiPSC-CM assays.

Cardiomyocyte-specific deletion of GSK-3ß leads to cardiac dysfunction in a diet induced obesity model.

BACKGROUND AND RATIONALE: Obesity, an independent risk factor for the development of myocardial diseases is a growing healthcare problem worldwide. It's well established that GSK-3ß is critical to cardiac pathophysiology. However, the role cardiomyocyte (CM) GSK-3ß in diet-induced cardiac dysfunction is unknown. METHODS: CM-specific GSK-3ß knockout (CM-GSK-3ß-KO) and littermate controls (WT) mice were fed either a control diet (CD) or high-fat diet (HFD) for 55weeks. Cardiac function was assessed by transthoracic echocardiography. RESULTS: At baseline, body weights and cardiac function were comparable between the WT and CM-GSK-3ß-KOs. However, HFD-fed CM-GSK-3ß-KO mice developed severe cardiac dysfunction. Consistently, both heart weight/tibia length and lung weight/tibia length were significantly elevated in the HFD-fed CM-GSK-3ß-KO mice. The impaired cardiac function and adverse ventricular remodeling in the CM-GSK-3ß-KOs were independent of body weight or the lean/fat mass composition as HFD-fed CM-GSK-3ß-KO and controls demonstrated comparable body weight and body masses. At the molecular level, on a CD, CM-GSK-3α compensated for the loss of CM-GSK-3ß, as evident by significantly reduced GSK-3αs21 phosphorylation (activation) resulting in a preserved canonical ß-catenin ubiquitination pathway and cardiac function. However, this protective compensatory mechanism is lost with HFD, leading to excessive accumulation of ß-catenin in HFD-fed CM-GSK-3ß-KO hearts, resulting in adverse ventricular remodeling and cardiac dysfunction. CONCLUSION: In summary, these results suggest that cardiac GSK-3ß is crucial to protect against obesity-induced adverse ventricular remodeling and cardiac dysfunction.

Frequency of mononuclear diploid cardiomyocytes underlies natural variation in heart regeneration.

Adult mammalian cardiomyocyte regeneration after injury is thought to be minimal. Mononuclear diploid cardiomyocytes (MNDCMs), a relatively small subpopulation in the adult heart, may account for the observed degree of regeneration, but this has not been tested. We surveyed 120 inbred mouse strains and found that the frequency of adult mononuclear cardiomyocytes was surprisingly variable (>7-fold). Cardiomyocyte proliferation and heart functional recovery after coronary artery ligation both correlated with pre-injury MNDCM content. Using genome-wide association, we identified Tnni3k as one gene that influences variation in this composition and demonstrated that Tnni3k knockout resulted in elevated MNDCM content and increased cardiomyocyte proliferation after injury. Reciprocally, overexpression of Tnni3k in zebrafish promoted cardiomyocyte polyploidization and compromised heart regeneration. Our results corroborate the relevance of MNDCMs in heart regeneration. Moreover, they imply that intrinsic heart regeneration is not limited nor uniform in all individuals, but rather is a variable trait influenced by multiple genes.