No abstract

Mammalian voltage-activated L-type Ca 2+ channels, such as Ca(v)1.2, control transmembrane Ca 2+ fluxes in numerous excitable tissues. Here, we report that the pore-forming α1C subunit of Ca(v)1.2 is reversibly palmitoylated in rat, rabbit, and human ventricular myocytes. We map the palmitoylation sites to two regions of the channel: The N terminus and the linker between domains I and II. Whole-cell voltage clamping revealed a rightward shift of the Ca(v)1.2 current–voltage relationship when α1C was not palmitoylated. To examine function, we expressed dihydropyridine-resistant α1C in human induced pluripotent stem cell-derived cardiomyocytes and measured Ca 2+ transients in the presence of nifedipine to block the endogenous channels. The transients generated by unpalmitoylatable channels displayed a similar activation time course but significantly reduced amplitude compared to those generated by wild-type channels. We thus conclude that palmitoylation controls the voltage sensitivity of Ca(v)1.2. Given that the identified Ca(v)1.2 palmitoylation sites are also conserved in most Ca(v)1 isoforms, we propose that palmitoylation of the pore-forming α1C subunit provides a means to regulate the voltage sensitivity of voltage-activated Ca 2+ channels in excitable cells.

Cardiac fibrosis represents a serious clinical problem. Development of novel treatment strategies is currently restricted by the lack of the relevant experimental models in a human genetic context. In this study, we fabricated self-aggregating, scaffold-free, 3D cardiac microtissues using human inducible pluripotent stem cell (iPSC)-derived cardiomyocytes and human cardiac fibroblasts. Fibrotic condition was obtained by treatment of cardiac microtissues with profibrotic cytokine transforming growth factor β1 (TGF-β1), preactivation of foetal cardiac fibroblasts with TGF-β1, or by the use of cardiac fibroblasts obtained from heart failure patients. In our model, TGF-β1 effectively induced profibrotic changes in cardiac fibroblasts and in cardiac microtissues. Fibrotic phenotype of cardiac microtissues was inhibited by treatment with TGF-β-receptor type 1 inhibitor SD208 in a dose-dependent manner. We observed that fibrotic cardiac microtissues substantially increased the spontaneous beating rate by shortening the relaxation phase and showed a lower contraction amplitude. Instead, no changes in action potential profile were detected. Furthermore, we demonstrated that contraction of human cardiac microtissues could be modulated by direct electrical stimulation or treatment with the β-adrenergic receptor agonist isoproterenol. However, in the absence of exogenous agonists, the β-adrenoreceptor blocker nadolol decreased beating rate of fibrotic cardiac microtissues by prolonging relaxation time. Thus, our data suggest that in fibrosis, activated cardiac fibroblasts could promote cardiac contraction rate by a direct stimulation of β-adrenoreceptor signalling. In conclusion, a model of fibrotic cardiac microtissues can be used as a high-throughput model for drug testing and to study cellular and molecular mechanisms of cardiac fibrosis.

The immature electrophysiology of human-induced pluripotent stem cell-derived cardiomyocytes (hiCMs) complicates their use for therapeutic and pharmacological purposes. An insufficient inward rectifying current (IK1) and the presence of a funny current (if) cause spontaneous electrical activity. This study tests the hypothesis that the co-culturing of hiCMs with a human embryonic kidney (HEK) cell-line expressing the Kir2.1 channel (HEK-IK1) can generate an electrical syncytium with an adult-like cardiac electrophysiology. The mechanical activity of co-cultures using different HEK-IK1:hiCM ratios was compared with co-cultures using wildtype (HEK–WT:hiCM) or hiCM alone on days 3–8 after plating. Only ratios of 1:3 and 1:1 showed a significant reduction in spontaneous rate at days 4 and 6, suggesting that IK1 was influencing the electrophysiology. Detailed analysis at day 4 revealed an increased incidence of quiescent wells or sub-areas. Electrical activity showed a decreased action potential duration (APD) at 20% and 50%, but not at 90%, alongside a reduced amplitude of the aggregate AP signal. A computational model of the 1:1 co-culture replicates the electrophysiological effects of HEK–WT. The addition of the IK1 conductance reduced the spontaneous rate and APD20, 50 and 90, and minor variation in the intercellular conductance caused quiescence. In conclusion, a 1:1 co-culture HEK-IK1:hiCM caused changes in electrophysiology and spontaneous activity consistent with the integration of IK1 into the electrical syncytium. However, the additional electrical effects of the HEK cell at 1:1 increased the possibility of electrical quiescence before sufficient IK1 was integrated into the syncytium.

Background and PurposeHuman ether‐a‐go‐go‐related gene (hERG; Kv11.1) channel inhibition is a widely accepted predictor of cardiac arrhythmia. hERG channel inhibition alone is often insufficient to predict pro‐arrhythmic drug effects. This study used a library of dofetilide derivatives to investigate the relationship between standard measures of hERG current block in an expression system and changes in action potential duration (APD) in human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs). The interference from accompanying block of Cav1.2 and Nav1.5 channels was investigated along with an in silico AP model.Experimental ApproachDrug‐induced changes in APD were assessed in hiPSC‐CMs using voltage‐sensitive dyes. The IC50 values for dofetilide and 13 derivatives on hERG current were estimated in an HEK293 expression system. The relative potency of each drug on APD was estimated by calculating the dose (D150) required to prolong the APD at 90% (APD90) repolarization by 50%.Key ResultsThe D150 in hiPSC‐CMs was linearly correlated with IC50 of hERG current. In silico simulations supported this finding. Three derivatives inhibited hERG without prolonging APD, and these compounds also inhibited Cav1.2 and/or Nav1.5 in a channel state‐dependent manner. Adding Cav1.2 and Nav1.2 block to the in silico model recapitulated the direction but not the extent of the APD change.Conclusions and ImplicationsPotency of hERG current inhibition correlates linearly with an index of APD in hiPSC‐CMs. The compounds that do not correlate have additional effects including concomitant block of Cav1.2 and/or Nav1.5 channels. In silico simulations of hiPSC‐CMs APs confirm the principle of the multiple ion channel effects.

No abstract

No abstract

No abstract