Gaspare Mostacciuolo

Elucidating arrhythmogenic mechanisms of long-QT syndrome CALM1-F142L mutation in patient-specific induced pluripotent stem cell-derived cardiomyocytes

Aims 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. Methods and Results Skin fibroblasts of the mutation carrier and two unrelated healthy subjects (controls) were reprogrammed to hiPSC and differentiated into hiPSC-CMs. Scanty IK1 expression, an hiPSC-CMs feature potentially biasing repolarization, was corrected by addition of simulated IK1 (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 ICaL, 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 IKs and INaL and changed If only marginally. Intracellular Ca2+ dynamics and Ca2+ store stability were not significantly modified. Mutation-induced repolarization abnormalities were reversed by verapamil. Conclusion The main functional derangement in CALM1-F142L was prolonged repolarization with altered rate-dependency and sensitivity to &bgr;-adrenergic stimulation. Impaired CDI of ICaL underlined the electrical abnormality, which was sensitive to ICaL blockade. High mutation penetrance was confirmed in the presence of the native genotype, implying strong dominance of effects.

Modulation of sarcoplasmic reticulum function by PST2744 [istaroxime; (E,Z)-3-((2-aminoethoxy)imino) androstane-6,17-dione hydrochloride)] in a pressure-overload heart failure model.

PST2744 [Istaroxime; (E,Z)-3-((2-aminoethoxy)imino) androstane-6,17-dione hydrochloride)] is a novel inotropic agent that enhances sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) 2 activity. We investigated the istaroxime effect on Ca2+ handling abnormalities in myocardial hypertrophy/failure (HF). Guinea pig myocytes were studied 12 weeks after aortic banding (AoB) and compared with those of sham-operated animals (sham). The gain of calcium-induced Ca2+ release (CICR), sarcoplasmic reticulum (SR) Ca2+ content, Na+/Ca2+ exchanger (NCX) function, and the rate of SR reloading after caffeine-induced depletion (SR Ca2+ uptake, measured during NCX blockade) were evaluated by measurement of cytosolic Ca2+ and membrane currents. HF characterization: AoB caused hypertrophy and failure in 100 and 25% of animals, respectively. Although CICR gain during constant pacing was preserved, SR Ca2+ content and SR Ca2+ uptake were strongly depressed. Resting Ca2+ and the slope of the Na+/Ca2+ exchanger current (INCX)/Ca2+ relationship were unchanged by AoB. Istaroxime effects: CICR gain, SR Ca2+ content, and SR Ca2+ uptake rate were increased by istaroxime in sham myocytes and, to a significantly larger extent, in AoB myocytes; this led to almost complete recovery of SR Ca2+ uptake in AoB myocytes. Istaroxime increased resting Ca2+ and the slope of the INCX/Ca2+ relationship similarly in sham and AoB myocytes. Istaroxime failed to increase SERCA activity in skeletal muscle microsomes devoid of phospholamban. Thus, clear-cut abnormalities in Ca2+ handling occurred in this model of hypertrophy, with mild decompensation. Istaroxime enhanced SR function more in HF myocytes than in normal ones; almost complete drug-induced recovery suggests a purely functional nature of SR dysfunction in this HF model.

Ranolazine prevents INaL enhancement and blunts myocardial remodelling in a model of pulmonary hypertension

AIMS Pulmonary arterial hypertension (PAH) reflects abnormal pulmonary vascular resistance and causes right ventricular (RV) hypertrophy. Enhancement of the late sodium current (INaL) may result from hypertrophic remodelling. The study tests whether: (i) constitutive INaL enhancement may occur as part of PAH-induced myocardial remodelling; (ii) ranolazine (RAN), a clinically available INaL blocker, may prevent constitutive INaL enhancement and PAH-induced myocardial remodelling. METHODS AND RESULTS PAH was induced in rats by a single monocrotaline (MCT) injection [60 mg/kg intraperitoneally (i.p.)]; studies were performed 3 weeks later. RAN (30 mg/kg bid i.p.) was administered 48 h after MCT and washed-out 15 h before studies. MCT increased RV systolic pressure and caused RV hypertrophy and loss of left ventricular (LV) mass. In the RV, collagen was increased; myocytes were enlarged with T-tubule disarray and displayed myosin heavy chain isoform switch. INaL was markedly enhanced; diastolic Ca(2+) was increased and Ca(2+) release was facilitated. K(+) currents were down-regulated and APD was prolonged. In the LV, INaL was enhanced to a lesser extent and cell Ca(2+) content was strongly depressed. Electrical remodelling was less prominent than in the RV. RAN completely prevented INaL enhancement and limited most aspects of PAH-induced remodelling, but failed to affect in vivo contractile performance. RAN blunted the MCT-induced increase in RV pressure and medial thickening in pulmonary arterioles. CONCLUSION PAH induced remodelling with chamber-specific aspects. RAN prevented constitutive INaL enhancement and blunted myocardial remodelling. Partial mechanical unloading, resulting from an unexpected effect of RAN on pulmonary vasculature, might contribute to this effect.

IKr Impact on Repolarization and Its Variability Assessed by Dynamic Clamp

In previous work we have shown that action potential duration (APD) and its response to modulation are “intrinsically” proportional. Nevertheless, loss of the rapid delayed rectifier K⁺ current (I_Kr) increases APD short-term variability (BVR) beyond what expected by such proportionality. It is unclear whether this may be explained by known I_Kr gating properties and which among them prevails in limiting BVR. As a preliminary approach to the problem, we investigated whether: 1) BVR changes caused by I_Kr blockade can be reversed by injection of current generated by a deterministic numerical I_Kr model (mI_Kr); 2) modulation of I_Kr maximal conductance (g_max) may affect APD and BVR differentially. In guinea-pig myocytes, native I_Kr was blocked by E4031 and replaced by mI_Kr by Dynamic-Clamp (DC); 2) the effect on APD and BVR of relatively small changes in mI_Kr g_max were assessed. The results thus far indicate that: 1) mI_Kr effectively reversed E4031 effects on both APD and BVR; 2) a 30% decrease in g_max, inadequate to prolong APD, increased BVR significantly. We conclude that 1) the effect of I_Kr changes on BVR may exceed that expected from modulation of APD only; 2) this can be accounted for by the known channel gating features implemented in mI_Kr.

Single- and multi-channel currents recorded with patch electrodes in mouse eggs

Zona-free mouse eggs are amenable to patch recording as well as to whole-cell recording (Bland et al., 1984; Peres, 1986a). Although many patches are silent, others show spontaneous channel opening; the single channel current is 1.5 pA and inwardly directed at resting potential (RP). Current amplitudes distribute normally as a single population at RP, however at RP-50 mV the amplitude histogram indicates two channel populations. Open time distribution is exponential with a mean open time between 4 and 7 msec at RP. In three cases out of thirty-eight, in which depolarizations were given from a holding potential of RP-50 mV, microscopic currents very similar in kinetics and voltage-dependence to the whole-cell Ca2+ current were observed. No single-channel currents could be resolved in these traces. The results reported here indicate that the voltage-dependent Ca2+ current of the mouse egg goes through low-conductance channels located in high density spots on the egg surface.