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 1) marked DD not associated to cardiac fibrosis, 2) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, 3) reduced LV SERCA2 protein level and activity and 4) slower SR Ca2+ uptake rate, 5) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, 6) increased diastolic Ca2+, and 7) 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. Translational perspective Deficient sarcoplasmic reticulum (SR) Ca2+ uptake has been identified in cardiomyocytes from failing human hearts with impaired diastolic relaxation (e.g. diabetic hearts) and has been associated with a decreased SERCA2a expression and activity and/or with a higher SERCA2a inhibition by phospholamban. Thus, SERCA2a may represent a pharmacological target for interventions aimed at improving cytosolic Ca2+ compartmentalization into the SR to limit diastolic dysfunction pathologies. In this context, istaroxime is the first-in-class luso-inotropic agent targeting SERCA2a that has already demonstrated its efficacy in clinical trials and may be useful to clarify the relevance of SERCA2a stimulation in controlling cytosolic Ca2+ level.

Background: Heart failure (HF) therapeutic toolkit would strongly benefit from the availability of ino-lusitropic agents with a favorable pharmacodynamics and safety profile. PST3093 is the main metabolite of istaroxime, an agent combining Na+/K+ pump inhibition and SERCA2a stimulation, shown by phase 2 trials to be promising in the acute setting. PST3093 half-life is substantially longer than that of istaroxime; therefore, if it retained the effects of the parent compound, it would allow to exploit istaroxime pharmacodynamics in chronic treatment. Methods and Results: We studied PST3093 for its effects on SERCA2a and Na+/K+ ATPase activities, Ca2+ dynamics in isolated myocytes and hemodynamic effects in an in-vivo rat model of diabetic (streptozotocin (STZ)-induced) cardiomyopathy. At variance with its parent compound, PST3093 is a "selective" (i.e. devoid of Na+/K+ pump inhibition) SERCA2a activator. In in-vivo echocardiographic assessment, PST3093 improved overall cardiac performance (e.g. stroke volume) without decreasing heart rate, and reversed most STZ-induced abnormalities. Modulation of both systolic and diastolic indexes contributed to the improvement. For i.v. administration, PST3093 toxicity was considerably lower than that of istaroxime and its evaluation against 50 targets commonly involved in cardiac and extracardiac side-effects, failed to reveal significant interactions. Conclusions: PST3093 is a "selective" SERCA2a activator, the prototype of a novel pharmacodynamic category with a potential in the ino-lusitropic approach to HF, particularly with prevailing diastolic dysfunction. While PST3093 may actually contribute to the proven clinical efficacy of istaroxime, its pharmacodynamics are peculiar and its pharmacokinetics are suitable for chronic administration.

Heart failure (HF) therapeutic toolkit would strongly benefit from the availability of ino-lusitropic agents with a favorable pharmacodynamics and safety profile. Istaroxime is a promising agent, which combines Na + /K + pump inhibition with SERCA2a stimulation; however, it has a very short half-life and extensive metabolism to a molecule, named PST3093. The present work aims to investigate whether PST3093, still retains the pharmacodynamic and pharmacokinetic properties of its parent compound. We studied PST3093 for its effects on SERCA2a and Na + /K + ATPase activities, Ca 2+ dynamics in isolated myocytes and hemodynamic effects in an in-vivo rat model of diabetic (streptozotocin (STZ)-induced) cardiomyopathy. Istaroxime infusion in HF patients led to accumulation of PST3093 in the plasma; clearance was substantially slower for PST3093 than for istaroxime. In cardiac rat preparations PST3093 did not inhibit the Na + /K + ATPase activity, but retained SERCA2a stimulatory activity. In in-vivo echocardiographic assessment, PST3093 improved overall cardiac performance and reversed most STZ-induced abnormalities. PST3093 i.v. toxicity was considerably lower than that of istaroxime and it failed to significantly interact with 50 off-targets. Overall, PST3093 is a "selective" SERCA2a activator, the prototype of a novel pharmacodynamic category with a potential in the ino-lusitropic approach to HF with prevailing diastolic dysfunction. Its pharmacodynamics are peculiar and its pharmacokinetics are suitable to prolong the cardiac beneficial effect of istaroxime infusion. Significance statementHeart failure (HF) treatment would benefit from the availability of ino-lusitropic agents with a favourable profile. PST3093 is the main metabolite of istaroxime, a promising agent combining Na + /K + pump inhibition and SERCA2a stimulation. PST3093 shows a longer half-life in human circulation compared to parent drug, activates SERCA2a, doesn't inhibit Na + /K + pump and improves cardiac performance in a model of diabetic cardiomyopathy. Overall, PST3093 as This article has not been copyedited and formatted. The final version may differ from this version.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) constitute a mixed population of ventricular-, atrial-, nodal-like cells, limiting the reliability for studying chamber-specific disease mechanisms. Previous studies characterised CM phenotype based on action potential (AP) morphology, but the classification criteria were still undefined. Our aim was to use in silico models to develop an automated approach for discriminating the electrophysiological differences between hiPSC-CM. We propose the dynamic clamp (DC) technique with the injection of a specific IK1 current as a tool for deriving nine electrical biomarkers and blindly classifying differentiated CM. An unsupervised learning algorithm was applied to discriminate CM phenotypes and principal component analysis was used to visualise cell clustering. Pharmacological validation was performed by specific ion channel blocker and receptor agonist. The proposed approach improves the translational relevance of the hiPSC-CM model for studying mechanisms underlying inherited or acquired atrial arrhythmias in human CM, and for screening anti-arrhythmic agents.

The stimulation of sarcoplasmic reticulum calcium ATPase SERCA2a emerged as a novel therapeutic strategy to efficiently improve overall cardiac function in heart failure (HF) with reduced arrhythmogenic risk. Istaroxime is a clinical-phase IIb compound with a double mechanism of action, Na + /K + ATPase inhibition and SERCA2a stimulation. Starting from the observation that istaroxime metabolite PST3093 does not inhibit Na + /K + ATPase while stimulates SERCA2a, we synthesized a series of bioisosteric PST3093 analogues devoid of Na + /K + ATPase inhibitory activity. Most of them retained SERCA2a stimulatory action with nanomolar potency in cardiac preparations from healthy guinea pigs and streptozotocin (STZ)-treated rats. One compound was further characterized in isolated cardiomyocytes, confirming SERCA2a stimulation and in vivo showing a safety profile and improvement of cardiac performance following acute infusion in STZ rats. We identified a new class of selective SERCA2a activators as first-in-class drug candidates for HF treatment.