Gene Therapy for Catecholaminergic Polymorphic Ventricular Tachycardia by Inhibition of Ca2+/Calmodulin-Dependent Kinase II.

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited cardiac arrhythmia characterized by adrenergically triggered arrhythmias, is inadequately treated by current standard of care. Ca2+/calmodulin-dependent protein kinase II (CaMKII), an adrenergically activated kinase that contributes to arrhythmogenesis in heart disease models, is a candidate therapeutic target in CPVT. However, translation of CaMKII inhibition has been limited by the need for selective CaMKII inhibition in cardiomyocytes. Here, we tested the hypothesis that CaMKII inhibition with a cardiomyocyte-targeted gene therapy strategy would suppress arrhythmia in CPVT mouse models. METHODS: We developed AAV9-GFP-AIP, an adeno-associated viral vector in which a potent CaMKII inhibitory peptide, autocamtide-2-related inhibitory peptide [AIP], is fused to green fluorescent protein (GFP) and expressed from a cardiomyocyte selective promoter. The vector was delivered systemically. Arrhythmia burden was evaluated with invasive electrophysiology testing in adult mice. AIP was also tested on induced pluripotent stem cells derived from patients with CPVT with different disease-causing mutations to determine the effectiveness of our proposed therapy on human induced pluripotent stem cell-derived cardiomyocytes and different pathogenic genotypes. RESULTS: AAV9-GFP-AIP was robustly expressed in the heart without significant expression in extracardiac tissues, including the brain. Administration of AAV9-GFP-AIP to neonatal mice with a known CPVT mutation (RYR2R176Q/+) effectively suppressed ventricular arrhythmias induced by either ß-adrenergic stimulation or programmed ventricular pacing, without significant proarrhythmic effect. Intravascular delivery of AAV9-GFP-AIP to adolescent mice transduced ≈50% of cardiomyocytes and was effective in suppressing arrhythmia in CPVT mice. Induced pluripotent stem cell-derived cardiomyocytes derived from 2 different patients with CPVT with different pathogenic mutations demonstrated increased frequency of abnormal calcium release events, which was suppressed by a cell-permeable form of AIP. CONCLUSIONS: This proof-of-concept study showed that AAV-mediated delivery of a CaMKII peptide inhibitor to the heart was effective in suppressing arrhythmias in a murine model of CPVT. CaMKII inhibition also reversed the arrhythmia phenotype in human CPVT induced pluripotent stem cell-derived cardiomyocyte models with different pathogenic mutations.

Mitochondrial Cardiomyopathy Caused by Elevated Reactive Oxygen Species and Impaired Cardiomyocyte Proliferation.

RATIONALE: Although mitochondrial diseases often cause abnormal myocardial development, the mechanisms by which mitochondria influence heart growth and function are poorly understood. OBJECTIVE: To investigate these disease mechanisms, we studied a genetic model of mitochondrial dysfunction caused by inactivation of Tfam (transcription factor A, mitochondrial), a nuclear-encoded gene that is essential for mitochondrial gene transcription and mitochondrial DNA replication. METHODS AND RESULTS: Tfam inactivation by Nkx2.5Cre caused mitochondrial dysfunction and embryonic lethal myocardial hypoplasia. Tfam inactivation was accompanied by elevated production of reactive oxygen species (ROS) and reduced cardiomyocyte proliferation. Mosaic embryonic Tfam inactivation confirmed that the block to cardiomyocyte proliferation was cell autonomous. Transcriptional profiling by RNA-seq demonstrated the activation of the DNA damage pathway. Pharmacological inhibition of ROS or the DNA damage response pathway restored cardiomyocyte proliferation in cultured fetal cardiomyocytes. Neonatal Tfam inactivation by AAV9-cTnT-Cre caused progressive, lethal dilated cardiomyopathy. Remarkably, postnatal Tfam inactivation and disruption of mitochondrial function did not impair cardiomyocyte maturation. Rather, it elevated ROS production, activated the DNA damage response pathway, and decreased cardiomyocyte proliferation. We identified a transient window during the first postnatal week when inhibition of ROS or the DNA damage response pathway ameliorated the detrimental effect of Tfam inactivation. CONCLUSIONS: Mitochondrial dysfunction caused by Tfam inactivation induced ROS production, activated the DNA damage response, and caused cardiomyocyte cell cycle arrest, ultimately resulting in lethal cardiomyopathy. Normal mitochondrial function was not required for cardiomyocyte maturation. Pharmacological inhibition of ROS or DNA damage response pathways is a potential strategy to prevent cardiac dysfunction caused by some forms of mitochondrial dysfunction.

Drug screening platform using human induced pluripotent stem cell-derived atrial cardiomyocytes and optical mapping.

Current drug development efforts for the treatment of atrial fibrillation are hampered by the fact that many preclinical models have been unsuccessful in reproducing human cardiac physiology and its response to medications. In this study, we demonstrated an approach using human induced pluripotent stem cell-derived atrial and ventricular cardiomyocytes (hiPSC-aCMs and hiPSC-vCMs, respectively) coupled with a sophisticated optical mapping system for drug screening of atrial-selective compounds in vitro. We optimized differentiation of hiPSC-aCMs by modulating the WNT and retinoid signaling pathways. Characterization of the transcriptome and proteome revealed that retinoic acid pushes the differentiation process into the atrial lineage and generated hiPSC-aCMs. Functional characterization using optical mapping showed that hiPSC-aCMs have shorter action potential durations and faster Ca2+ handling dynamics compared with hiPSC-vCMs. Furthermore, pharmacological investigation of hiPSC-aCMs captured atrial-selective effects by displaying greater sensitivity to atrial-selective compounds 4-aminopyridine, AVE0118, UCL1684, and vernakalant when compared with hiPSC-vCMs. These results established that a model system incorporating hiPSC-aCMs combined with optical mapping is well-suited for preclinical drug screening of novel and targeted atrial selective compounds.

MICAL1 constrains cardiac stress responses and protects against disease by oxidizing CaMKII.

Oxidant stress can contribute to health and disease. Here we show that invertebrates and vertebrates share a common stereospecific redox pathway that protects against pathological responses to stress, at the cost of reduced physiological performance, by constraining Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity. MICAL1, a methionine monooxygenase thought to exclusively target actin, and MSRB, a methionine reductase, control the stereospecific redox status of M308, a highly conserved residue in the calmodulin-binding (CaM-binding) domain of CaMKII. Oxidized or mutant M308 (M308V) decreased CaM binding and CaMKII activity, while absence of MICAL1 in mice caused cardiac arrhythmias and premature death due to CaMKII hyperactivation. Mimicking the effects of M308 oxidation decreased fight-or-flight responses in mice, strikingly impaired heart function in Drosophila melanogaster, and caused disease protection in human induced pluripotent stem cell-derived cardiomyocytes with catecholaminergic polymorphic ventricular tachycardia, a CaMKII-sensitive genetic arrhythmia syndrome. Our studies identify a stereospecific redox pathway that regulates cardiac physiological and pathological responses to stress across species.