SERCA2a stimulation by istaroxime improves intracellular Ca2+ handling and diastolic dysfunction in a model of diabetic cardiomyopathy.

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Action potential contour contributes to species differences in repolarization response to ß-adrenergic stimulation.

Aim: Repolarization response to ß-adrenergic (ß-AR) stimulation differs between guinea-pig and canine myocytes and, within the latter, between myocardial layers. Correlative analysis suggests that this may be due to differences in action potential (AP) contour. Here we tested whether AP contour may set the response of current and of repolarization to ß-AR stimulation (10 nM isoproterenol, ISO). Methods and results: The responses of AP and current to ISO were measured under I-clamp and "AP-clamp" in guinea-pig (GP), dog epicardial (DEPI) and dog subendocardial (DENDO) myocytes. Dynamic-clamp (DC) was used to evaluate the impact of AP features on AP response to ISO. ISO prolonged AP duration (APD) in GP myocytes, did not affect it in DENDO and shortened it in DEPI ones. The current induced by ISO (IISO) sharply differed between GP and canine myocytes and, to a lesser extent, between DENDO and DEPI ones. Differences in IISO profile likely important in setting APD response (time-to-peak, time-to-reversal), were minimized when canine myocytes where clamped with GP AP-waveforms and vice versa. Introduction of a "notch" in GP AP (by DC) was alone insufficient to affect the APD response to ISO; nevertheless, when incorporated in a GP AP-waveform, the main "canine" AP features ("notch" and low plateau potential) caused IISO of GP myocytes to acquire canine features. Conclusion: Early repolarization contour and level of plateau potential contribute to species-specificity of IISO profile. Changes in AP contour, also when generated by modulation of ISO-insensitive currents, may be crucial in setting APD response to ß-AR stimulation.

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 ß-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.

IKr Impact on Repolarization and Its Variability Assessed by Dynamic Clamp.

BACKGROUND: Repolarization and its stability are exquisitely sensitive to IKr features. Information on the relative importance of specific IKr abnormalities is missing and would assist in the evaluation of arrhythmogenic risk. METHODS AND RESULTS: In single guinea-pig myocytes, endogenous IKr was replaced by modeled IKr (mIKr) by dynamic clamp (DC) at a cycle length of 1 s. mIKr parameters were systematically modified, and the resulting changes in action potential duration (APD) and its short term variability (SD1) were measured. We observed that (1) IKr blockade increased SD1 more than expected by its dependency on APD; (2) mIKr completely reversed APD and SD1 changes caused by IKr blockade; (3) repolarization was most sensitive to inactivation shifts, which affected APD and SD1 concordantly; (4) activation shifts of the same magnitude had marginal impact on APD, but only when reducing mIKr, they significantly increased SD1; (5) changes in maximal conductance resulted in a pattern similar to that of activation shifts. CONCLUSIONS: The largest effect on repolarization and its stability are expected from changes in IKr inactivation. APD is less sensitive to changes in other IKr gating parameters, which are better revealed by SD1 changes. SD1 may be more sensitive than APD in detecting IKr-dependent repolarization abnormalities.

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.

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 Ca(2+) ATPase (SERCA) 2 activity. We investigated the istaroxime effect on Ca(2+) 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 Ca(2+) release (CICR), sarcoplasmic reticulum (SR) Ca(2+) content, Na(+)/Ca(2+) exchanger (NCX) function, and the rate of SR reloading after caffeine-induced depletion (SR Ca(2+) uptake, measured during NCX blockade) were evaluated by measurement of cytosolic Ca(2+) 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 Ca(2+) content and SR Ca(2+) uptake were strongly depressed. Resting Ca(2+) and the slope of the Na(+)/Ca(2+) exchanger current (I(NCX))/Ca(2+) relationship were unchanged by AoB. Istaroxime effects: CICR gain, SR Ca(2+) content, and SR Ca(2+) 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 Ca(2+) uptake in AoB myocytes. Istaroxime increased resting Ca(2+) and the slope of the I(NCX)/Ca(2+) 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 Ca(2+) 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.

Modulation of sarcoplasmic reticulum function by Na+/K+ pump inhibitors with different toxicity: digoxin and PST2744 [(E,Z)-3-((2-aminoethoxy)imino)androstane-6,17-dione hydrochloride].

OBJECTIVE: To gain some insight on the lesser arrhythmogenic properties of PST2744 [(E,Z)-3-((2-aminoethoxy)imino)androstane-6,17-dione hydrochloride] compared with digoxin, we compared modulation of intracellular Ca2+ dynamics by the two agents. METHODS: SERCA (sarcoplasmic reticulum Ca2+-ATPase) activity and Ca2+ leak rate were measured in sarcoplasmic reticulum (SR) vesicles from guinea pig ventricles. Membrane current, intracellular Ca2+, and twitch amplitude were evaluated in guinea pig ventricular myocytes with or without blockade of the Na+/Ca2+ exchanger. RESULTS: In SR vesicles, PST2744 (30-300 nM), but not digoxin, increased SERCA activity; digoxin only (> or =0.1 nM) increased SR Ca2+ leak. In myocytes with blocked Na+/Ca2+ exchanger, Ca2+ reloading of caffeine-depleted SR was enhanced by PST2744 and slightly inhibited by digoxin. In myocytes with functioning Na+/Ca2+ exchanger, both agents increased diastolic Ca2+, SR Ca2+ content, the gain of Ca2+-induced Ca2+ release, the rate of cytosolic Ca2+ decay, twitch amplitude, and relaxation rate. Consistent with the observations in SR vesicles, the effects on SR Ca2+ content and Ca2+ decay rate were significantly larger for PST2744 than for digoxin. CONCLUSIONS: In isolated SR vesicles, PST2744 and digoxin directly affected SR function in opposite ways; this could be reproduced in myocytes during Na+/Ca2+ exchanger blockade. Under physiological conditions (functioning Na+/Ca2+ exchanger), the two agents affected Ca2+ dynamics in the same direction, as expected by their Na+/K+ pump inhibition; however, differential SR modulation was still expressed by quantitative differences. Thus, the more favorable inotropy-to-toxicity ratio previously described for PST2744 appears to be associated with direct SERCA stimulation and/or lack of enhancement of Ca2+ leak.