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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.

Rationale: Aging in the heart is a gradual process, involving continuous changes in cardiovascular cells, including cardiomyocytes (CMs), namely cellular senescence. These changes finally lead to adverse organ remodeling and resulting in heart failure. This study exploits CMs from human induced pluripotent stem cells (iCMs) as a tool to model and characterize mechanisms involved in aging. Methods and Results: Human somatic cells were reprogrammed into human induced pluripotent stem cells and subsequently differentiated in iCMs. A senescent-like phenotype (SenCMs) was induced by short exposure (3 hours) to doxorubicin (Dox) at the sub-lethal concentration of 0.2 µM. Dox treatment induced expression of cyclin-dependent kinase inhibitors p21 and p16, and increased positivity to senescence-associated beta-galactosidase when compared to untreated iCMs. SenCMs showed increased oxidative stress, alteration in mitochondrial morphology and depolarized mitochondrial membrane potential, which resulted in decreased ATP production. Functionally, when compared to iCMs, SenCMs showed, prolonged multicellular QTc and single cell APD, with increased APD variability and delayed afterdepolarizations (DADs) incidence, two well-known arrhythmogenic indexes. These effects were largely ascribable to augmented late sodium current (INaL) and reduced delayed rectifier potassium current (Ikr). Moreover sarcoplasmic reticulum (SR) Ca2+ content was reduced because of downregulated SERCA2 and increased RyR2-mediated Ca2+ leak. Electrical and intracellular Ca2+ alterations were mostly justified by increased CaMKII activity in SenCMs. Finally, SenCMs phenotype was furtherly confirmed by analyzing physiological aging in CMs isolated from old mice in comparison to young ones. Conclusions: Overall, we showed that SenCMs recapitulate the phenotype of aged primary CMs in terms of senescence markers, electrical and Ca2+ handling properties and metabolic features. Thus, Dox-induced SenCMs can be considered a novel in vitro platform to study aging mechanisms and to envision cardiac specific anti-aging approach in humans.