Using the tight-seal voltage-clamp method, the ionic currents in the enzymatically dispersed single smooth muscle cells of the guinea pig taenia coli have been studied. In a physiological medium containing 3 mM Ca 2 § the cells are gently tapering spindles, averaging 201 (length) x 8 #m (largest diameter in center of cell), with a volume of 5 pl. The average cell capacitance is 50 pF, and the specific membrane capacitance 1.15 #F/cm ~. The input impedance of the resting cell is 1-2 Gfl. Spatially uniform voltage-control prevails after the first 400 #s. There is much overlap of the inward and outward currents, but the inward current can be isolated by applying Cs § internally to block all potassium currents. The inward current is carried by Ca 2 § Activation begins at ~-30 mV, maximum/ca occurs at +10-+20 mV, and the reversal potential is ~+75 inV. The Ca ~+ channel is permeable to Sr 2+ and Ba 2+, and to Cs + moving outwards, but not to Na + moving inwards. Activation and deactivation are very rapid at ~33"C, with timeconstants of < 1 ms. Inactivation has a complex time course, resolvable into three exponential components, with average time constants (at 0 mV) of 7, 45, and 400 ms, which are affected differently by voltage. Steady-state inactivation is half-maximal at -30 mV for all components combined, but -36 mV for the fast component and -26 and -23 mV for the other two components. The presence of multiple forms of Ca 2 § channel is inferred from the inactivation characteristics, not from activation properties. Recovery of the fast channel occurs with a time-constant of 72 ms (at + 10 mV). Ca 2 § influx during an action potential can transfer ~9 pC of charge, which could elevate intracellular Ca 2+ concentration adequately for various physiological functions.

Calmodulin is a ubiquitous Ca2+ sensor molecule encoded by three distinct calmodulin genes, CALM1-3. Recently, mutations in CALM1-3 have been reported to be associated with severe early-onset long-QT syndrome (LQTS). However, the underlying mechanism through which heterozygous calmodulin mutations lead to severe LQTS remains unknown, particularly in human cardiomyocytes. We aimed to establish an LQTS disease model associated with a CALM2 mutation (LQT15) using human induced pluripotent stem cells (hiPSCs) and to assess mutant allele-specific ablation by genome editing for the treatment of LQT15. We generated LQT15-hiPSCs from a 12-year-old boy with LQTS carrying a CALM2-N98S mutation and differentiated these hiPSCs into cardiomyocytes (LQT15-hiPSC-CMs). Action potentials (APs) and L-type Ca2+ channel (LTCC) currents in hiPSC-CMs were analyzed by the patch-clamp technique and compared with those of healthy controls. Furthermore, we performed mutant allele-specific knockout using a CRISPR-Cas9 system and analyzed electrophysiological properties. Electrophysiological analyses revealed that LQT15-hiPSC-CMs exhibited significantly lower beating rates, prolonged AP durations, and impaired inactivation of LTCC currents compared with control cells, consistent with clinical phenotypes. Notably, ablation of the mutant allele rescued the electrophysiological abnormalities of LQT15-hiPSC-CMs, indicating that the mutant allele caused dominant-negative suppression of LTCC inactivation, resulting in prolonged AP duration. We successfully recapitulated the disease phenotypes of LQT15 and revealed that inactivation of LTCC currents was impaired in CALM2-N98S hiPSC model. Additionally, allele-specific ablation using the latest genome-editing technology provided important insights into a promising therapeutic approach for inherited cardiac diseases.

The truncation mutations were associated with manifestation of cardiac phenotypes in -related cardiomyopathy, suggesting that genetic analysis might be useful for diagnosis and risk stratification.

IntroductionHuman induced pluripotent stem cells (hiPSCs) offer a unique opportunity for disease modeling. However, it is not invariably successful to recapitulate the disease phenotype because of the immaturity of hiPSC-derived cardiomyocytes (hiPSC-CMs). The purpose of this study was to establish and analyze iPSC-based model of catecholaminergic polymorphic ventricular tachycardia (CPVT), which is characterized by adrenergically mediated lethal arrhythmias, more precisely using electrical pacing that could promote the development of new pharmacotherapies.Method and ResultsWe generated hiPSCs from a 37-year-old CPVT patient and differentiated them into cardiomyocytes. Under spontaneous beating conditions, no significant difference was found in the timing irregularity of spontaneous Ca2+ transients between control- and CPVT-hiPSC-CMs. Using Ca2+ imaging at 1 Hz electrical field stimulation, isoproterenol induced an abnormal diastolic Ca2+ increase more frequently in CPVT- than in control-hiPSC-CMs (control 12% vs. CPVT 43%, p<0.05). Action potential recordings of spontaneous beating hiPSC-CMs revealed no significant difference in the frequency of delayed afterdepolarizations (DADs) between control and CPVT cells. After isoproterenol application with pacing at 1 Hz, 87.5% of CPVT-hiPSC-CMs developed DADs, compared to 30% of control-hiPSC-CMs (p<0.05). Pre-incubation with 10 μM S107, which stabilizes the closed state of the ryanodine receptor 2, significantly decreased the percentage of CPVT-hiPSC-CMs presenting DADs to 25% (p<0.05).ConclusionsWe recapitulated the electrophysiological features of CPVT-derived hiPSC-CMs using electrical pacing. The development of DADs in the presence of isoproterenol was significantly suppressed by S107. Our model provides a promising platform to study disease mechanisms and screen drugs.