Calmodulin (CaM) is a small protein, encoded by three genes (CALM1-3), exerting multiple Ca²⁺-dependent modulatory roles. A mutation (F142L) affecting only one of the six CALM alleles is associated with long QT syndrome (LQTS) characterized by recurrent cardiac arrests. This phenotypic severity is unexpected from the predicted allelic balance. In this work, the effects of heterozygous CALM1-F142L have been investigated in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) obtained from a LQTS patient carrying the F142L mutation, i.e. in the context of native allelic ratio and potential gene modifiers. Skin fibroblasts of the mutation carrier and two unrelated healthy subjects (controls) were reprogrammed to hiPSC and differentiated into hiPSC-CMs. Scanty I_K1 expression, an hiPSC-CMs feature potentially biasing repolarization, was corrected by addition of simulated I_K1 (Dynamic-Clamp). Abnormalities in repolarization rate-dependency (in single cells and cell aggregates), membrane currents and intracellular Ca²⁺dynamics were evaluated as putative arrhythmogenic factors. CALM1-F142L prolonged repolarization, altered its rate-dependency and its response to isoproterenol. This was associated with severe impairment of Ca²⁺-dependent inactivation (CDI) of I_CaL, resulting in augmented inward current during the plateau phase. As a result, the repolarization of mutant cells failed to adapt to high pacing rates, a finding well reproduced by using a recent hiPSC-CM action potential model. The mutation failed to affect I_Ks and I_NaL and changed I_f only marginally. Intracellular Ca²⁺dynamics and Ca²⁺store stability were not significantly modified. Mutation-induced repolarization abnormalities were reversed by verapamil. The main functional derangement in CALM1-F142L was prolonged repolarization with altered rate-dependency and sensitivity to β-adrenergic stimulation. Impaired CDI of I_CaL underlined the electrical abnormality, which was sensitive to I_CaL blockade. High mutation penetrance was confirmed in the presence of the native genotype, implying strong dominance of effects.

Atrial fibrillation (AF) is the most common type of cardiac arrhythmias, whose incidence is likely to increase with the aging of the population. It is considered a progressive condition, frequently observed as a complication of other cardiovascular disorders. However, recent genetic studies revealed the presence of several mutations and variants linked to AF, findings that define AF as a multifactorial disease. Due to the complex genetics and paucity of models, molecular mechanisms underlying the initiation of AF are still poorly understood. Here we investigate the pathophysiological mechanisms of a familial form of AF, with particular attention to the identification of putative triggering cellular mechanisms, using patient’s derived cardiomyocytes (CMs) differentiated from induced pluripotent stem cells (iPSCs). Here we report the clinical case of three siblings with untreatable persistent AF whose whole-exome sequence analysis revealed several mutated genes. To understand the pathophysiology of this multifactorial form of AF we generated three iPSC clones from two of these patients and differentiated these cells towards the cardiac lineage. Electrophysiological characterization of patient-derived CMs (AF-CMs) revealed that they have higher beating rates compared to control (CTRL)-CMs. The analysis showed an increased contribution of the I_f and I_CaL currents. No differences were observed in the repolarizing current I_Kr and in the sarcoplasmic reticulum calcium handling. Paced AF-CMs presented significantly prolonged action potentials and, under stressful conditions, generated both delayed after-depolarizations of bigger amplitude and more ectopic beats than CTRL cells. Our results demonstrate that the common genetic background of the patients induces functional alterations of I_f and I_CaL currents leading to a cardiac substrate more prone to develop arrhythmias under demanding conditions. To our knowledge this is the first report that, using patient-derived CMs differentiated from iPSC, suggests a plausible cellular mechanism underlying this complex familial form of AF.

Diabetic cardiomyopathy is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD) that precedes the development of systolic impairment. Mechanisms that can restore cardiac relaxation improving intracellular Ca²⁺ dynamics represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime has the dual properties to accelerate Ca²⁺ uptake into sarcoplasmic reticulum (SR) through the SR Ca²⁺ pump (SERCA2a) stimulation and to inhibit Na⁺/K⁺ ATPase (NKA). This project aims to characterize istaroxime effects at a concentration (100 nmol/L) marginally affecting NKA, in order to highlight its effects dependent on the stimulation of SERCA2a in an animal model of mild diabetes. Streptozotocin (STZ) treated diabetic rats were studied at 9 weeks after STZ injection in comparison to controls (CTR). Istaroxime effects were evaluated in vivo and in left ventricular (LV) preparations. STZ animals showed (i) marked DD not associated to cardiac fibrosis, (ii) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, (iii) reduced LV SERCA2 protein level and activity and (iv) slower SR Ca²⁺ uptake rate, (v) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, (vi) increased diastolic Ca²⁺, and (vii) unaltered SR Ca²⁺ content and stability in intact cells. Acute istaroxime infusion (0.11 mg/kg/min for 15 min) reduced DD in STZ rats. Accordingly, in STZ myocytes istaroxime (100 nmol/L) stimulated SERCA2a activity and blunted STZ-induced abnormalities in LV Ca²⁺ dynamics. In CTR myocytes, istaroxime increased diastolic Ca²⁺ level due to NKA blockade albeit minimal, while its effects on SERCA2a were almost absent. SERCA2a stimulation by istaroxime improved STZ-induced DD and intracellular Ca²⁺ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment.

In long QT syndrome (LQTS) patients, modifier genes modulate the arrhythmic risk associated with a disease-causing mutation. Their recognition can improve risk stratification and clinical management, but their discovery represents a challenge. We tested whether a cellular-driven approach could help to identify new modifier genes and especially their mechanism of action. We generated human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) from two patients carrying the same KCNQ1-Y111C mutation, but presenting opposite clinical phenotypes. We showed that the phenotype of the iPSC-CMs derived from the symptomatic patient is due to impaired trafficking and increased degradation of the mutant KCNQ1 and wild-type human ether-a-go-go-related gene. In the iPSC-CMs of the asymptomatic (AS) patient, the activity of an E3 ubiquitin-protein ligase (Nedd4L) involved in channel protein degradation was reduced and resulted in a decreased arrhythmogenic substrate. Two single-nucleotide variants (SNVs) on the Myotubularin-related protein 4 (MTMR4) gene, an interactor of Nedd4L, were identified by whole-exome sequencing as potential contributors to decreased Nedd4L activity. Correction of these SNVs by CRISPR/Cas9 unmasked the LQTS phenotype in AS cells. Importantly, the same MTMR4 variants were present in 77% of AS Y111C mutation carriers of a separate cohort. Thus, genetically mediated interference with Nedd4L activation seems associated with protective effects. Our finding represents the first demonstration of the cellular mechanism of action of a protective modifier gene in LQTS. It provides new clues for advanced risk stratification and paves the way for the design of new therapies targeting this specific molecular pathway.

Background: Sinoatrial node cells (SANC) automaticity is generated by functional association between the activity of plasmalemmal ion channels and local diastolic intracellular Ca²⁺ release (LCR) from ryanodine receptors. Strikingly, most isolated SANC exhibit a “dormant” state, whereas only a fraction shows regular firing as observed in intact SAN. Recent studies showed that β-adrenergic stimulation can initiate spontaneous firing in dormant SANC, though this mechanism is not entirely understood. Methods: To investigate the role of L-type Ca_v1.3 Ca²⁺ channels in the adrenergic regulation of automaticity in dormant SANC, we used a knock-in mouse strain in which the sensitivity of L-type Ca_v1.2 α1 subunits to dihydropyridines (DHPs) was inactivated (Ca_v1.2^DHP−/−), enabling the selective pharmacological inhibition of Ca_v1.3 by DHPs. Results: In dormant SANC, β-adrenergic stimulation with isoproterenol (ISO) induced spontaneous action potentials (AP) and Ca²⁺ transients, which were completely arrested with concomitant perfusion of the DHP nifedipine. In spontaneously firing SANC at baseline, Ca_v1.3 inhibition completely reversed the effect of β-adrenergic stimulation on AP and the frequency of Ca²⁺ transients. Confocal calcium imaging of SANC showed that the β-adrenergic-induced synchronization of LCRs is regulated by the activity of Ca_v1.3 channels. Conclusions: Our study shows a novel role of Ca_v1.3 channels in initiating and maintaining automaticity in dormant SANC upon β-adrenergic stimulation.