We developed a 96-well plate assay which allows fast, reproducible and high-throughput generation of 3D cardiac rings around a deformable optically transparent hydrogel (PEG) pillar of known stiffness. Human induced pluripotent stem cell-derived cardiomyocytes, mixed with normal human adult dermal fibroblasts in an optimized 3:1 ratio, self-organized to form ring-shaped cardiac constructs. Immunostaining showed that the fibroblasts form a basal layer in contact with the glass, stabilizing the muscular fiber above. Tissues started contracting around the pillar at D1 and their fractional shortening increased until D7, reaching a plateau at 25±1%, that was maintained up to 14 days. The average stress, calculated from the compaction of the central pillar during contractions, was 1.4±0.4 mN/mm2. The cardiac constructs recapitulated expected inotropic responses to calcium and various drugs (isoproterenol, verapamil) as well as the arrhythmogenic effects of dofetilide. This versatile high-throughput assay allows multiple in situ mechanical and structural read-outs.

Background: Drug-induced QT prolongation (diLQT) is a feared side-effect as exposing susceptible individuals to fatal arrhythmias. The occurrence of diLQT is primarily attributed to unintended drug interactions with cardiac ion channels, notably the hERG channels that generate the repolarizing current (IKr) and thereby regulate the late repolarization phase. There is an important inter-individual susceptibility to develop diLQT which is of unknown origin but can be reproduced in patient-specific iPSC-derived cardiomyocytes (iPS-CMs). Objective: We aimed to investigate the dynamics of hERG channels in response to sotalol and to identify regulators of the susceptibility to developing diLQT. Methods: We measured electrophysiological activity and cellular distribution of hERG channels after hERG blocker treatment in iPS-CMs derived from patients with highest or lowest sensitivity (HS or LS) to sotalol administration in vivo (i.e., based on the measure of the maximal change in QT interval 3 hours after administration). Specific small-interfering RNAs (siRNA) and CAVIN1-T2A-GFP adenovirus were used to manipulate CAVIN1 expression. Results: While HS and LS iPS-CMs showed similar electrophysiological characteristics at the baseline, the late repolarization phase was prolonged, and IKr significantly decreased after exposure of HS iPS-CMs to low sotalol concentrations. IKr reduction was caused by a rapid translocation of hERG channel from the plasma membrane to the cytoskeleton upon sotalol application. This phenomenon was suppressed by blocking active endocytosis using dynasore. CAVIN1, essential for caveolae biogenesis, was two-times more expressed in HS iPS-CMs and its knockdown using siRNA decreased their sensitivity to sotalol. CAVIN1 overexpression in LS iPS-CMs using adenovirus showed reciprocal effects. Mechanistically, we found that treatment with sotalol promoted trafficking of the hERG channel from the plasma membrane to the cytoskeleton through caveolae and in a manner dependent on CAVIN1 expression. CAVIN1 silencing reduced the number of caveolae at the membrane and abrogated the internalization of hERG channel in sotalol-treated HS iPS-CMs. CAVIN1 also controlled cardiomyocyte responses to other hERG blockers such as E4031, vandetanib, and clarithromycin. Conclusions: Our study identifies unbridled turnover of the potassium channel hERG as a mechanism supporting the inter-individual susceptibility underlying diLQT development and demonstrates how this phenomenon is finely tuned by CAVIN1.

Introduction: Many drugs have been pulled from the market due to their undesirable effect in prolonging the QT interval by altering cardiac ion currents, notably I Kr generated by hERG. This cardiotoxic impact is however extremely variable between individuals and the mechanisms underlying this variability remain undiscovered. Previously, we identified a higher expression of CAVIN1, an essential protein for caveolae biogenesis, in cardiomyocytes (CM) derived from individuals with high susceptibility to develop sotalol-induced long QT. Hypothesis: CAVIN1 can modulate cardiomyocytes’ response to drugs. Methods: We used induced pluripotent stem cells-derived-CM (iCM) from 3 subjects with the highest sensitivity (HS) to sotalol (ie, those with the highest increase in QTc) and 3 others with the lowest sensitivity to sotalol (LS). Results: cardiac repolarization and I Kr were similar between HS-iCM and LS-iCM at baseline. However, HS-iCM displayed a significantly higher prolongation of the repolarization phase along with a decrease in I Kr in response to sotalol and other drugs targeting hERG (E4031, vandetanib, and clarithromycin). CAVIN1 mRNA and protein levels were increased by 2-fold in the HS-iCM. Overexpressing CAVIN1 in the LS-iCM using adenovirus induced higher sensitivity to sotalol and the other hERG blockers. In contrast, CAVIN1 knockdown using siRNA in the HS-iCM switched their phenotype to a lower sensitivity to hERG blockers. We observed that CAVIN1 overexpression in LS-iCM and HEK cells changed hERG distribution within cells with accumulation at the membrane. We applied Methyl-β-cyclodextrin, a detergent that removes caveolae from membranes, and found that caveolae depletion induced prolonged repolarization associated with a decrease in CAVIN1 and hERG sarcolemmal expression, and their accumulation in the cytoskeleton. In contrast with the LS-iCM, sotalol application in the HS-iCM promoted hERG internalization into the cytoskeleton. Conclusions: We identified CAVIN1 as a novel modulator of cardiac repolarization in response to drug by promoting hERG internalization. This represents a novel mechanism sustaining the inter-individual variability in response to drugs.