Kim, Changsung

Introduction: Multifocal ectopic Purkinje-related premature contractions (MEPPC), the most recent addition to the spectrum of SCN5A -mediated cardiac channelopathies, is characterized by frequent premature ventricular complexes (PVCs) originating from the Purkinje system, atrial arrhythmias, and a predilection for PVC-mediated dilated cardiomyopathy. Hypothesis: To investigate the electrophysiology phenotype of MEPPC in a novel, patient-specific, re-engineered cardiomyocyte model. Methods: Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated from a 27-year-old male with a history of frequent, multifocal PVCs and atrial tachycardia, positive for a putative MEPPC-causative SCN5A variant (p.Q419P). A CRISPR-Cas9 Q419P variant-corrected isogenic control (IC) was created to establish the normalized electrophysiology recordings for that MEPPC model. The standard whole-cell patch clamp technique was used to measure Q419P-SCN5A- and IC-derived NaV1.5 sodium currents using voltage clamp configuration. In addition, action potential duration (APD) of the iPSC-CMs were measured using FluoVolt optical voltage-sensing dye. Results: Analysis of the current-voltage relationship revealed that neither peak sodium current densities nor late inward sodium current were impacted by Q419P-SCN5A. However, Q419P-SCN5A significantly right-shifted the V 1/2 in inactivation by 8.3 mV from -81.1±1.7 mV (IC, n=5) to -72.8±0.2 mV (Q419P-SCN5A, n=6, p<0.05 vs. IC) and left-shifted the V 1/2 in activation by -4.1 mV from -39.5±0.3 mV (IC, n=5) to -43.6±0.9 mV (Q419P-SCN5A, n=6, p<0.05 vs. IC) leading to a larger hyperpolarized window current. Optical APD90 measurement revealed no difference between the Q419P-containing (476 ± 23 ms) and the IC (474 ± 46 ms, p=0.867) cardiomyocytes. Conclusions: This is the first in-vitro disease model of MEPPC using patient-specific, re-engineered heart cells. Consistent with the findings in the non-cardiac HEK cell system, this MEPPC-causative variant contributes to increased window current in cardiomyocytes without affecting the cardiac APD. This novel cardiac cell model for MEPPC may facilitate new insights into the pathobiology of MEPPC and enable the testing of novel therapies for it.

Background: Type 2 long QT syndrome (LQT2) is an inherited arrhythmia syndrome caused by pathogenic variants in the KCNH2 gene which result in QT prolongation and confers susceptibility to arrhythmic syncope/seizures and sudden cardiac arrest/death. Oral progestin-based contraceptives increase the risk of cardiac events in women with LQT2 and we previously reported the case of a LQT2 woman with recurrent cardiac events attributed to the use of the progestin-based contraceptive, Medroxyprogesterone Acetate (“Depo-Provera”, Depo). Objective: To evaluate the arrhythmic-risk of Depo in a LQT2-specific, induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model. Methods: An iPSC-CM line was generated from the previously reported woman (KCNH2-G1006A*fs) as well as a control line. Voltage-sensing dye was used to assess the optical action potential duration (APD) with 10 μM Depo. 3-D erratic beating patterns were then assessed using multiple electrode array (MEA) with Depo and isoproterenol (ISO) to mimic an adrenergic surge. Results: Addition of Depo to the G1006A*fs iPSC-CMs decreased the optical APD from baseline 438±14 ms (n=6) to 363±35 ms (n=12; p=0.0001). Despite the APD shortening at baseline, in the setting of ISO, Depo administration induced erratic beating patterns in MEA, characterized as alternating spike amplitudes, alternans, or early after depolarization-like phenomena. Combined Depo and ISO treatment increased the percent of electrodes displaying erratic beating in G1006A*fs [baseline 8%±13 vs. Depo + ISO 38%±40 (p=0.0097)] but not in controls [baseline 2%±5 vs. Depo + ISO 7%±11 (p=0.14)]. Neither Depo nor ISO alone changed beating patterns in G1006A*fs [baseline 8%±13, Depo 30%±41 (p=0.08), ISO 21%±30, (p=0.17)] and controls [baseline 2%±5, Depo 3%±7 (p=0.61), ISO 0%±0 (p=0.4)]. Conclusion: This patient-specific, re-engineered heart cell study provides a potential mechanism for the patient’s clinically documented, Depo-associated episodes of recurrent ventricular fibrillation. This in-vitro data should prompt a large-scale clinical assessment of Depo’s potential pro-arrhythmic effect in women with LQT2.

Abstract Viruses spread between hosts through particles, but within hosts, viral genomes can spread from cell to cell through fusion, evading antiviral defenses and obviating costly infectious virion production1-3. Billions of electromechanically coupled cardiomyocytes (CMs) make myocardium inherently vulnerable to pathological electromechanical short circuits caused by intercellular viral spread 4-6. Beyond respiratory illness, COVID-19 affects the heart7 and cardiac injury and arrhythmias are serious public health concerns8-12. By studying myocardium of a young woman who died suddenly, diagnosed postmortem with COVID-19, we discovered highly focal myocardial SARS-CoV-2 infection spreading from one CM to another through intercellular junctions identified by highly concentrated sarcolemmal t-tubule viral spike glycoprotein. SARS-CoV-2 permissively infected beating human induced pluripotent stem cell (hiPSC)-CMs building multinucleated cardiomyotubes (CMTs) through cell type-specific fusion driven by proteolytically-activated spike glycoprotein. Recombinant spike glycoprotein, co-localizing to sarcolemma and sarcoplasmic reticulum, produced multinucleated CMTs with pathological structure, electrophysiology and Ca2+ excitation-contraction coupling. Blocking cleavage, a peptide-based protease inhibitor neutralized SARS-CoV-2 spike glycoprotein pathogenicity. We conclude that SARS-CoV-2 spike glycoprotein, efficiently primed, activated and strategically poised during biosynthesis, can exploit the CM’s inherent membranous connectivities to drive heart damage directly, uncoupling clinically common myocardial injury from lymphocytic myocarditis, often suspected but rarely confirmed in COVID-19.