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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 (I NaL ) and reduced delayed rectifier potassium current (Ikr). Moreover sarcoplasmic reticulum (SR) Ca 2+ content was reduced because of downregulated SERCA2 and increased RyR2-mediated Ca 2+ leak. Electrical and intracellular Ca 2+ 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 Ca 2+ 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., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Velux Stiftung Background Ageing of cardiomyocytes (CM) involves structural and functional adverse remodelling that finally could result in heart failure (HF) insurgence, which incidence rise along with age (1). Current medical therapies for HF may not always be tolerated in elder patients(2). Having shown that cardiac progenitor cells (CPCs) secrete nanoparticles named exosomes (EXO) enriched of cardioprotective factors(3,4), we are exploring EXO's capacity to ameliorate senescence-derived modification into CMs. However, human models of in vitro cardiac aging are currently missing(5). Aim This study exploits CMs derived from human induced pluripotent stem cells (hiPSCs) as an in vitro model for cardiac senescence, that will be used as platform to characterize mechanisms involved in cardiac ageing and to test protective effect of CPC-derived EXO. Methods Patient-derived CPCs were reprogrammed into hiPSCs and subsequently expanded and differentiated into cardiomyocytes (hiPSC-CMs). Senescence-like phenotype was induced by short exposure (3 hours) to doxorubicin (DOX) at sub-lethal concentration (0.2 µM), followed by washing and medium change. Following DOX exposure, cells were exposed to EXO, derived from the purification of conditioned culture media of CPCs using an ultracentrifugation-based isolation method and quantified and sized using a NTA counter. Senescence induction was highlighted by protein and gene expression analysis and senescence-associated b-galactosidase (SA-β-gal) assay.Electrical activity of hiPSC-CMs was evaluated recording extracellular field potentials through multi-microelectrode arrays (MEA) and by single cell patch clamp. Metabolic features were analysed with western blot, real time RT-PCR and specific biochemical assays. Results DOX treatment in hiPSC-CMs induced senescence, as confirmed by activation of p21 and p16 pathways and increasing of SA-β-gal staining as compared to untreated cells (CTR). Biochemical and gene expression analysis revealed an increased ROS production and a reduction in mitochondrial potential, which drives a strong decrease in the ATP/AMP ratios. Real Time PCR analysis reveal an increased transcription of molecules related to the senescence associated secretory phenotype in DOX-CMs. Moreover, DOX-CMs showed impaired Ca++ handling, prolonged multicellular QTc and single cell APD, with increased APD variability and delayed afterdepolarizations (DADs) incidence in comparison to CTR. EXO treatment mitigated the senescent phenotype induced by DOX, as shown by a decreased ROS induction, higher mitochondrial potential which drives a restored ATP/AMP ratio. Furthermore, DOX-induced QTc prolongation was prevented by EXO treatment. Conclusion Our hiPSC-CMs based cellular model recapitulates the phenotype of aged CMs in terms of senescence markers, electrical and metabolic proprieties. CPC-derived EXOs limit age-related modifications, highlighting the cardioprotective role of small molecules released by EXO. Open in new tab Download slide Graphical Abstract [ABSTRACT FROM AUTHOR]