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.

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.

Background Repolarization and its stability are exquisitely sensitive to I-Kr features. Information on the relative importance of specific I-Kr abnormalities is missing and would assist in the evaluation of arrhythmogenic risk.Methods and Results In single guinea-pig myocytes, endogenous I-Kr was replaced by modeled I-Kr (mI(Kr)) by dynamic clamp (DC) at a cycle length of 1 s. mI(Kr) 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) I-Kr blockade increased SD1 more than expected by its dependency on APD; (2) mI(Kr) completely reversed APD and SD1 changes caused by I-Kr 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 mI(Kr), 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 I-Kr inactivation. APD is less sensitive to changes in other I-Kr gating parameters, which are better revealed by SD1 changes. SD1 may be more sensitive than APD in detecting I-Kr-dependent repolarization abnormalities.

In canine (D) and human, but not guinea pig (GP), ventricular myocytes, a spike-and-dome profile (SaD), supported by Ito, characterizes ventricular repolarization. β-adrenergic stimulation (by isoprenaline, ISO) shortens action potential (AP) duration (APD) in D (and human) myocytes, but prolongs it in GP ones. Aim: The aim of this work is to clarify whether SaD is the main factor determining the direction of APD response to β-adrenergic stimulation. Methods: AP-clamp with D epicardial, D endocardial, and GP waveforms was applied at different diastolic intervals (DI) to measure ISO-induced current (Iiso) in GP myocytes. Dynamic Clamp was used to test the effect of Ito introduction, and of the resulting SaD, on ISO modulation of GP repolarization. Results: In AP-clamp at DI 1750 ms, Iiso was more inward with both D and GP waveforms. In Dynamic-Clamp, SaD introduction failed to change the direction of ISO-induced APD changes in GP myocytes. Conclusions: SaD profile alone may not account for differences between D and GP in terms of APD response to β-adrenergic-stimulation. Further differences of AP profile and/or diverse contributions of Ca2+ and K+ currents between the two species may be involved.

Aims Pulmonary arterial hypertension (PAH) reflects abnormal pulmonary vascular resistance and causes right ventricular (RV) hypertrophy. Enhancement of the late sodium current (I-NaL) may result from hypertrophic remodelling. The study tests whether: (i) constitutive I-NaL enhancement may occur as part of PAH-induced myocardial remodelling; (ii) ranolazine (RAN), a clinically available I-NaL blocker, may prevent constitutive I-NaL 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 weekslater. 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. I-NaL was markedly enhanced; diastolic Ca2+ was increased and Ca2+ release was facilitated. K+ currents were down-regulated and APD was prolonged. In the LV, I-NaL was enhanced to a lesser extent and cell Ca2+ content was strongly depressed. Electrical remodellingwas less prominent than in the RV. RAN completely prevented I-NaL 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 I-NaL enhancement and blunted myocardial remodelling. Partial mechanical unloading, resulting from an unexpected effect of RAN on pulmonary vasculature, might contribute to this effect.

Background: Pulmonary hypertension (PH) complicates many clinical conditions and may result in right ventricular (RV) hypertrophy and failure. Monocrotaline (MCT) induces PH by direct action on pulmonary microvasculature. Enhancement of the late sodium current (INaL) results from myocardial remodelling and may contribute to its evolution. Aims: To evaluate the effects of chronic INaL blockade in the prevention of PH-induced myocardial remodeling. Methods: PH was induced in adult rats by a single injection of MCT (60 mg/Kg i.p.); animals were studied 3 weeks later and untreated littermates served as controls. The INaL blocker ranolazine (RAN, 30 mg/Kg bid i.p.) was administered to a subset of MCT-treated rats over the 3 weeks period and completely washed-out before study. Results: MCT effects (vs control): MCT increased RV pressure. In the RV, weight, wall thickness, collagen content, myocyte electrical capacitance and cross sectional area were all increased, to indicate hypertrophy. The LV was slightly hypotrophyc instead. Nonetheless, both RV and LV myocytes showed INaL enhancement and signs of remodelling, such as alfa-myosin heavy chain (MHC) and Ito downregulation, prolonged action potential duration (APD) and delayed afterdepolarizations (DADs). IK1 was reduced in RV only. Chronic RAN effects (vs MCT alone): RAN reduced RV pressure and significantly blunted all signs of RV hypertrophy. In both RV and LV, INaL enhancement, DADs induction, alfa-MHC and Ito downregulation were reversed by RAN. However, APD and IK1 abnormalities persisted. Conclusions: MCT caused pressure-induced hypertrophy in the RV, but also remodelled the LV (by indirect strain?). Chronic INaL blockade prevented constitutive INaL enhancement and blunted many aspects of myocardial remodeling in both ventricles. In the RV, but not in the LV, this might partly reflect the unexpected inhibition by RAN of MCT effects on pulmonary vascular resistance.

Theraptosis S.A., Romainville,France.Tumor necrosis factor a (TNFa), a pro-inflammatory cytokine, is associatedwith major cardiomyopathy. In the heart, TNFa binding to the TNF receptor1 (TNFR1) has been implicated in TNFa mediating negative inotropic effectsaswellasapoptosis.TNFa-TNFR1activatescaspase-8whichleadstocaspase-3 activation either directly or following mitochondrial disruption. Here we in-vestigated whether caspase-8-induced mitochondrial dysfunction could lead toTNFa-induced alterations of Ca

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.

Cardiac progenitor cells (CPCs) form three-dimensional structure named cardiospheres (CSs). While CSs are seen as partially committed cardiac precursors, it is unknown whether they already display cardiac-type molecular functions. Whereas IP3-mediated Ca 2+ release is a shared property of many cell types, Ca + release through RyR channels is muscle specific. Aims: To test if cardiac-type Ca 2+ release mechanism is present in CPCs and whether it may develop during the CSs stage. Methods: Cells arose from murine cardiac explants in culture were studied prior to CSs formation (pre-CSs) and after expansion of CSs (post-CSs). Ca 2+ transients were detected in wide optical confocal fields by Fluo4-AM fluorescence. RyR- and IP3-R-mediated Ca 2+ release from intracellular stores was tested by exposures to caffeine (CAF, 10 mM) or ATP (200 μM) respectively in Ca 2+ -free conditions. The expression of the cardiac-specific RyR isoform (RyR2) was tested by immunolabeling and western-blot analysis. Results: In isolated cells CAF-induced response was almost exclusive of post-CSs cells (22.4 % vs. 3.62 %; p<0.05); ATP-induced response was already present in pre-CSs cells and showed only a small increase in post-CSs ones (94.3 % vs 86.1 % p<0.05). The ratio between post-CSs and pre-CSs responses was 6.2 for CAF and 1.1 for ATP. Both CAF and ATP responses were suppressed by the SERCA inhibitor CPA (50 μM), thus confirming intracellular stores as the Ca 2+ source. ATP response only was suppressed by the IP3-R blocker 2APB (10 μM). Ryanodine (20 μM) prevented the response to CAF, but not to ATP. In post-CSs RyR2 protein levels were higher than in pre-CSs and similar to those of adult myocytes. Immunoistochemistry analysis of post-CSs cells results in a distribution of RyR2 expression consistent with immature neonatal cardiomyocytes previously described. Conclusions: At variance with IP3-mediated signaling, RyR mediated Ca 2+ release develops during maturation within the CSs environment, along with expression of the cardiac RyR isoform. Detection of caffeine-induced Ca 2+ responses may be useful in identifying cardio-specific functional maturation in progenitor cell populations.