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 (I_K1) 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-I_K1) can generate an electrical syncytium with an adult-like cardiac electrophysiology. The mechanical activity of co-cultures using different HEK-I_K1: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 I_K1 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 I_K1 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-I_K1:hiCM caused changes in electrophysiology and spontaneous activity consistent with the integration of I_K1 into the electrical syncytium. However, the additional electrical effects of the HEK cell at 1:1 increased the possibility of electrical quiescence before sufficient I_K1 was integrated into the syncytium.

Conversion and prevention of atrial fibrillation (AF) are essential requirements for its treatment. Abnormal leakage of calcium from the sarcoplasmic reticulum via the ryanodine receptor 2 (RyR2) during diastole is considered a major cause of AF. Although catheter ablation has made it possible to stop persistent AF, it does not address recurrence. In this context, we describe the additional pharmacological and antiarrhythmic effects of the RyR2 inhibitor M201-A, including the results of a phase I study. M201-A inhibited G-protein-gated atrial K⁺ channel at an IC₅₀ value of 0.35 μM, which was associated with a 38% increase in the atrial effective refractory period (ERP) and high atrial selectivity with an atrial ERP/ventricular ERP of 5.7. M201-A inhibited rapid component of the delayed rectifier potassium current (IC₅₀ value of 0.43 μM) but induced only a limited prolongation in the action potential duration in rabbit ventricular cells and human induced pluripotent stem cell-derived cardiomyocytes. M201-A is an R-enantiomer of the active metabolite M201-R of K201. After orally administering 540 mg K201 to patients with AF, the unbound drug concentration of M201-R was 30-fold higher (approximately 15-fold for M201-A) than that of K201, and AF was converted in 6 of the 12 patients (50%) during the first 2 hours following administration. M201-A was the main contributor with a stronger bioactive metabolite than M201-B (S-enantiomer) and M201-R. Furthermore, M201-A was 5-fold more biologically active than K201 in humans due to protein binding rates. These pharmacological properties hold promise as a new therapeutic strategy for AF, particularly the prevention of recurrence after catheter ablation. SIGNIFICANCE STATEMENT: Abnormal leakage of calcium from the sarcoplasmic reticulum via the ryanodine receptor 2 (RyR2) is considered a major cause of atrial fibrillation (AF). M201-A had inhibitory effects on RyR2 and G-protein-gated atrial K⁺ channel. It had high atrial selectivity with limited QT interval corrected by Fridericia's formula prolongation and proarrhythmic risk. It was the most active conversion metabolite when K201 was administered orally in patients with paroxysmal AF. These pharmacological effects of M201-A, combined with its inhibitory actions on RyR2, provide a promising therapeutic option for treating AF.

Cardiac cellular responses to acute exercise remain undescribed. We present a model for mimicking acute aerobic endurance exercise to freshly isolated cardiomyocytes by evoking exercise‐like contractions over prolonged periods of time with trains of electrical twitch stimulations. We then investigated immediate contractile, Ca²⁺, and metabolic responses to acute exercise in perfused freshly isolated left ventricular rat cardiomyocytes, after a matrix‐design optimized protocol and induced a mimic for acute aerobic endurance exercise by trains of prolonged field twitch stimulations. Acute exercise decreased cardiomyocyte fractional shortening 50%–80% (p < .01). This was not explained by changes to intracellular Ca²⁺ handling (p > .05); rather, we observed a weak insignificant Ca²⁺ transient increase (p = .11), while myofilament Ca²⁺ sensitivity increased 20%–70% (p < .05). Acidic pH 6.8 decreased fractional shortening 20%–70% (p < .05) because of 20%–30% decreased Ca²⁺ transients (p < .05), but no difference occurred between control and acute exercise (p > .05). Addition of 1 or 10 mM La⁻ increased fractional shortening in control (1 mM La⁻: no difference, p > .05; 10 mM La⁻: 20%–30%, p < .05) and acute exercise (1 mM La⁻: 40%–90%, p < .01; 10 mM La⁻: 50%–100%, p < .01) and rendered acute exercise indifferent from control (p > .05). Intrinsic autofluorescence showed a resting NAD_state of 0.59 ± 0.04 and FAD_state of 0.17 ± 0.03, while acute exercise decreased NADH/FAD ratio 8% (p < .01), indicating intracellular oxidation. In conclusion, we show a novel approach for studying immediate acute cardiomyocyte responses to aerobic endurance exercise. We find that acute exercise in cardiomyocytes decreases contraction, but Ca²⁺ handling and myofilament Ca²⁺ sensitivity compensate for this, while acidosis and reduced energy substrate and mitochondrial ATP generation explain this.