Functional analysis of ryanodine receptor 2 mutations in induced pluripotent stem cell-derived cardiomyocytes from CPVT patients

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an ion channel disorder in the heart, which is characterized by abnormal calcium handling, ventricular arrhythmias, and sudden cardiac death. This inherited disease is predominantly caused by mutations in the ryanodine receptor type 2 (RYR2). Most of the identified mutations are clustered into four distinct domains of the RYR2 channel. Although heterologous expression systems and animal models have brought important insights in the CPVT pathogenesis, the underlying electrophysiological mechanisms have not been completely understood. The aim of the study was to take cells from CPVT patients with specific RYR2 mutations, to create patient-specific induced pluripotent stem cells (iPSCs), to differentiate these cells into cardiomyocytes (CMs) and then to model the disease in vitro for a better understanding of the disease mechanism and for the investigation of novel therapeutic applications for CPVT patients. Somatic cells from skin biopsies of CPVT patients carrying the RYR2 mutation in domain a (R420W), domain b (A2254V), domain c (E4076K) or domain d (H4742Y) were isolated and reprogrammed into patient-specific iPSCs. The CPVT as well as healthy control (Ctrl) iPSC lines were differentiated into CMs. The CPVT- and Ctrl-iPSC-derived CMs were investigated for their biological, electrophysiological, and pharmacological differences between the RYR2 mutations in different domains and healthy controls. The differences in 3',5'-cyclic adenosine monophosphate (cAMP) dynamics were investigated as well. Electrophysiological analyses showed that the CPVT-CMs recapitulated the phenotype of CPVT both by patch-clamp assessment and by multielectrode array assessment. The single CPVTdCMs showed a unique early afterdepolarization (EAD) phenotype in basal condition and isoproterenol- (ISO-) challenged condition. However, CPVTa-, CPVTb- and CPVTc-CMs exhibited delayed afterdepolarization (DAD) and DAD-induced triggered activities (TAs), which were significantly enhanced after the ISO treatment. In the monolayer cultures, all CPVT-CMs revealed a significantly increased number of premature ventricular complex- (PVC-) like events and prolonged duration of ventricular tachycardia- (VT-) like events after the ISO treatment. In contrast, no increased appearance of arrhythmic events (DADs, EADs, DAD- or EAD-induced TAs, and PVC- and VT-like events) was observed in Ctrl-CMs after the ISO treatment. Four antiarrhythmic drugs (flecainide, dantrolene, rycal1 and rycal2) showed antiarrhythmic effects on CPVTa-, CPVTb- and CPVTc-CMs, but no or minor antiarrhythmic effect on CPVTd-CMs. FRET measurement revealed that the contribution of phosphodiesterase 2 (PDE2) to cAMP degradation in all CPVTa- (15.41%), b- (9.48%), c- (15.07%), d-CMs (7.9%) were significantly lower than in Ctrl-CMs (27.5%) in cytosol. The contribution of PDE2 to cAMP degradation at the RYR2 compartment in all CPVTa- (14.19%), b- (25.21%), c- (17.32%), d-CMs (8.6%) were also significantly lower than in Ctrl-CMs (39.98%). Similar to PDE2, the contribution of PDE3 to cAMP degradation in all CPVT-CMs were significantly lower than in Ctrl-CMs both in cytosol and at the RYR2 compartment. However, for PDE4, there were lower activities in cytosol in CPVTb- and CPVTc-CMs when compared to Ctrl-CMs, and lower activities at the RYR2 compartment in CPVTc- and CPVTd-CMs when compared to Ctrl-CMs. Furthermore, the data indicate that PDE4 is the major regulator of cAMP level in CPVT-CMs both in cytosol and at the RYR2 compartment after ISO stimulation and that PDE2 and PDE3 have a smaller contribution to regulate the cAMP level. Taken together, this study reveals that mutation-specific CPVT-iPSCs can be used to model the disease in vitro, to investigate the disease pathophysiological and molecular mechanisms and to optimize drug therapies

Synergistic Adverse Effects of Azithromycin and Hydroxychloroquine on Human Cardiomyocytes at a Clinically Relevant Treatment Duration

Adverse effects of drug combinations and their underlying mechanisms are highly relevant for safety evaluation, but often not fully studied. Hydroxychloroquine (HCQ) and azithromycin (AZM) were used as a combination therapy in the treatment of COVID-19 patients at the beginning of the pandemic, leading to higher complication rates in comparison to respective monotherapies. Here, we used human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to systematically investigate the effects of HCQ, AZM, and their combination on the structure and functionality of cardiomyocytes, and to better understand the underlying mechanisms. Our results demonstrate synergistic adverse effects of AZM and HCQ on electrophysiological and contractile function of iPSC-CMs. HCQ-induced prolongation of field potential duration (FPDc) was gradually increased during 7-day treatment period and was strongly enhanced by combination with AZM, although AZM alone slightly shortened FPDc in iPSC-CMs. Combined treatment with AZM and HCQ leads to higher cardiotoxicity, more severe structural disarrangement, more pronounced contractile dysfunctions, and more elevated conduction velocity, compared to respective monotreatments. Mechanistic insights underlying the synergistic effects of AZM and HCQ on iPSC-CM functionality are provided based on increased cellular accumulation of HCQ and AZM as well as increased Cx43- and Nav1.5-protein levels

Disease Phenotypes and Mechanisms of iPSC-Derived Cardiomyocytes From Brugada Syndrome Patients With a Loss-of-Function SCN5A Mutation

Brugada syndrome (BrS) is one of the major causes of sudden cardiac death in young people, while the underlying mechanisms are not completely understood. Here, we investigated the pathophysiological phenotypes and mechanisms using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) from two BrS patients (BrS-CMs) carrying a heterozygous SCN5A mutation p.S1812X. Compared to CMs derived from healthy controls (Ctrl-CMs), BrS-CMs displayed a 50% reduction of I-Na density, a 69.5% reduction of Na(V)1.5 expression, and the impaired localization of Na(V)1.5 and connexin 43 (Cx43) at the cell surface. BrS-CMs exhibited reduced action potential (AP) upstroke velocity and conduction slowing. The I-to in BrS-CMs was significantly augmented, and the I-CaL window current probability was increased. Our data indicate that the electrophysiological mechanisms underlying arrhythmia in BrS-CMs may involve both depolarization and repolarization disorders. Cilostazol and milrinone showed dramatic inhibitions of I-to in BrS-CMs and alleviated the arrhythmic activity, suggesting their therapeutic potential for BrS patients