Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease accompanied by structural and contractile alterations. We identified a rare c.740C>T (p.T247M) mutation in ACTN2 , encoding α‐actinin 2 in a HCM patient, who presented with left ventricular hypertrophy, outflow tract obstruction, and atrial fibrillation. We generated patient‐derived human‐induced pluripotent stem cells (hiPSCs) and show that hiPSC‐derived cardiomyocytes and engineered heart tissues recapitulated several hallmarks of HCM, such as hypertrophy, myofibrillar disarray, hypercontractility, impaired relaxation, and higher myofilament Ca ²⁺ sensitivity, and also prolonged action potential duration and enhanced L‐type Ca ²⁺ current. The L‐type Ca ²⁺ channel blocker diltiazem reduced force amplitude, relaxation, and action potential duration to a greater extent in HCM than in isogenic control. We translated our findings to patient care and showed that diltiazem application ameliorated the prolonged QTc interval in HCM‐affected son and sister of the index patient. These data provide evidence for this ACTN2 mutation to be disease‐causing in cardiomyocytes, guiding clinical therapy in this HCM family. This study may serve as a proof‐of‐principle for the use of hiPSC for personalized treatment of cardiomyopathies.

Sex differences exist in the structure and function of human heart. The patterns of ventricular repolarization in normal electrocardiograms (ECG) differ in men and women: men ECG pattern displays higher T-wave amplitude and increased ST angle. Generally, women have longer QT duration because of reduced repolarization reserve, and thus, women are more susceptible for the occurrence of torsades de pointes associated with drugs prolonging ventricular repolarization. Sex differences are also observed in the prevalence, penetrance and symptom severity, and also in the prognosis of cardiovascular disease. Generally, women live longer, have less clinical symptoms of cardiac diseases, and later onset of symptoms than men. Sex hormones also play an important role in regulating ventricular repolarization, suggesting that hormones directly influence various cellular functions and adrenergic regulation. From the clinical perspective, sex-based differences in heart physiology are widely recognized, but in daily practice, cardiac diseases are often underdiagnosed and untreated in the women. The underlying mechanisms of sex differences are, however, poorly understood. Here, we summarize sex-dependent differences in normal cardiac physiology, role of sex hormones, and differences in drug responses. Furthermore, we also discuss the importance of human induced pluripotent stem cell-derived cardiomyocytes in further understanding the mechanism of differences in women and men.

Background: Cardiac repolarization abnormalities in drug-induced and genetic long-QT syndrome may lead to afterdepolarizations and life-threatening ventricular arrhythmias. Human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) should help to overcome the limitations of animal models based on species differences in repolarization reserve. Here, we compared head-to-head the contribution ofI_Ks(long QT1) andI_Kr(long QT2) on action potentials (APs) in human left ventricular (LV) tissue and hiPSC-CM–derived engineered heart tissue (EHT).Methods: APs were measured with sharp microelectrodes in EHT from 3 different control hiPSC-CM lines and in tissue preparations from failing LV.Results: EHT from hiPSC-CMs showed spontaneous diastolic depolarization and AP generation that were sensitive to low concentrations of ivabradine.I_Krblock by E-4031 prolonged AP duration at 90% repolarization with similar half-effective concentration in EHT and LV but larger effect size in EHT (+281 versus +110 ms in LV). AlthoughI_Krblock alone evoked early afterdepolarizations and triggered activity in 50% of EHTs, slow pacing, reduced extracellular K⁺, and blocking ofI_Kr,I_Ks, andI_K1were necessary to induce early afterdepolarizations in LV. In accordance with their clinical safety, moxifloxacin and verapamil did not induce early afterdepolarizations in EHT. In both EHT and LV,I_Ksblock by HMR-1556 prolonged AP duration at 90% repolarization slightly in the combined presence of E-4031 and isoprenaline.Conclusions: EHT from hiPSC-CMs shows a lower repolarization reserve than human LV working myocardium and could thereby serve as a sensitive and specific human-based model for repolarization studies and arrhythmia, similar to Purkinje fibers. In both human LV and EHT,I_Ksonly contributed to repolarization under adrenergic stimulation.

Brugada syndrome (BrS) is a rare inherited disease that can give rise to ventricular arrhythmia and ultimately sudden cardiac death. Numerous loss-of-function mutations in the cardiac sodium channel Na_v1.5 have been associated with BrS. However, few mutations in the auxiliary Na_vβ1–4 subunits have been linked to this disease. Here we investigated differences in expression and function between Na_vβ1 and Na_vβ1b and whether the H162P/Na_vβ1b mutation found in a BrS patient is likely to be the underlying cause of disease. The impact of Na_vβ subunits was investigated by patch-clamp electrophysiology, and the obtained in vitro values were used for subsequent in silico modeling. We found that Na_vβ1b transcripts were expressed at higher levels than Na_vβ1 transcripts in the human heart. Na_vβ1 and Na_vβ1b coexpressed with Na_v1.5 induced a negative shift on steady state of activation and inactivation compared with Na_v1.5 alone. Furthermore, Na_vβ1b was found to increase the current level when coexpressed with Na_v1.5, Na_vβ1b/H162P mutated subunit peak current density was reduced by 48% (−645 ± 151 vs. −334 ± 71 pA/pF), V_1/2 steady-state inactivation shifted by −6.7 mV (−70.3 ± 1.5 vs. −77.0 ± 2.8 mV), and time-dependent recovery from inactivation slowed by >50% compared with coexpression with Na_vβ1b wild type. Computer simulations revealed that these electrophysiological changes resulted in a reduction in both action potential amplitude and maximum upstroke velocity. The experimental data thereby indicate that Na_vβ1b/H162P results in reduced sodium channel activity functionally affecting the ventricular action potential. This result is an important replication to support the notion that BrS can be linked to the function of Na_vβ1b and is associated with loss-of-function of the cardiac sodium channel.

Background: Electrical remodeling in human persistent atrial fibrillation is believed to result from rapid electrical activation of the atria, but underlying genetic causes may contribute. Indeed, common gene variants in an enhancer region close to PITX2 (paired-like homeodomain transcription factor 2) are strongly associated with atrial fibrillation, but the mechanism behind this association remains unknown. This study evaluated the consequences of PITX2 deletion (PITX2 ^−/− ) in human induced pluripotent stem cell–derived atrial cardiomyocytes. Methods: CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9) was used to delete PITX2 in a healthy human iPSC line that served as isogenic control. Human induced pluripotent stem cell–derived atrial cardiomyocytes were differentiated with unfiltered retinoic acid and cultured in atrial engineered heart tissue. Force and action potential were measured in atrial engineered heart tissues. Single human induced pluripotent stem cell–derived atrial cardiomyocytes were isolated from atrial engineered heart tissue for ion current measurements. Results: PITX2 ^−/− atrial engineered heart tissue beats slightly slower than isogenic control without irregularity. Force was lower in PITX2 ^−/− than in isogenic control (0.053±0.015 versus 0.131±0.017 mN, n=28/3 versus n=28/4, PITX2 ^−/− versus isogenic control; P <0.0001), accompanied by lower expression of CACNA1C and lower L-type Ca ²⁺ current density. Early repolarization was weaker (action potential duration at 20% repolarization; 45.5±13.2 versus 8.6±5.3 ms, n=18/3 versus n=12/4, PITX2 ^−/− versus isogenic control; P <0.0001), and maximum diastolic potential was more negative (−78.3±3.1 versus −69.7±0.6 mV, n=18/3 versus n=12/4, PITX2 ^−/− versus isogenic control; P =0.001), despite normal inward rectifier currents (both I _K1 and I _K,ACh ) and carbachol-induced shortening of action potential duration. Conclusions: Complete PITX2 deficiency in human induced pluripotent stem cell–derived atrial cardiomyocytes recapitulates some findings of electrical remodeling of atrial fibrillation in the absence of fast beating, indicating that these abnormalities could be primary consequences of lower PITX2 levels.

Mutations in the HERG gene encoding the potassium ion channel HERG, represent one of the most frequent causes of long QT syndrome type-2 (LQT2). The same genetic mutation frequently presents different clinical phenotypes in the family. Our study aimed to model LQT2 and study functional differences between the mutation carriers of variable clinical phenotypes. We derived human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) from asymptomatic and symptomatic HERG mutation carriers from the same family. When comparing asymptomatic and symptomatic single LQT2 hiPSC-CMs, results from allelic imbalance, potassium current density, and arrhythmicity on adrenaline exposure were similar, but a difference in Ca²⁺ transients was observed. The major differences were, however, observed at aggregate level with increased susceptibility to arrhythmias on exposure to adrenaline or potassium channel blockers on CM aggregates derived from the symptomatic individual. The effect of this mutation was modeled in-silico which indicated the reactivation of an inward calcium current as one of the main causes of arrhythmia. Our in-vitro hiPSC-CM model recapitulated major phenotype characteristics observed in LQT2 mutation carriers and strong phenotype differences between LQT2 asymptomatic vs. symptomatic were revealed at CM-aggregate level.