In Vitro Rejuvenation of Senescent Human Cardiac Stem Cells Enhances Their Therapeutic Efficacy in Repairing a Mouse Infarcted Heart

Recently our group has demonstrated that human c-Kit-pos Cardiac Stem Cells isolated from explanted failing hearts (E-CSC) are characterized, in vitro, compared to those isolated from healthy donated hearts (D-CSC), by a senescent phenotype: a reduced telomere length and telomerase activity, the occurrence of telomere dysfunction foci and the accumulation of senescent markers, as p16INK4A. The result is an impairment of CSC functional properties in vitro. The aim of this study was to identify molecular mechanisms associated with CSC senescence and to pharmacologically modulate this latter, at the aim to rejuvenate senescent cells in vitro, before transplantation, and to improve the therapeutic potential of CSC in vivo. With this work, we newly demonstrate that senescent E-CSC show an impaired regenerative ability when injected in the peri-infarct region of an infarcted mouse heart. 2 weeks post- Myocardial Infarction, E-CSC treated mice showed, with respect to the D-CSC ones, worst anatomical and functional parameters at echocardiographic analysis. Histologically, E-CSC treated animals displayed a larger scar size, a lower density of capillaries, small arterioles and cycling myocytes, an enrichment in senescent, apoptotic and LC3+ myocytes and a reduced number of cardiac primitive/progenitor cells recruited in the site of injury. In addition, we demonstrated that the E-CSC secretome was strongly enriched in the pro-inflammatory IL-1β and unable to protect rat adult cardiomyocytes - exposed in vitro to SI/RO injury - from apoptosis and senescence, while the D-CSC’s secretome did. The neutralization of IL-1β in cell supernatants restored E-CSC’s secretome protective effect. We then looked for possible mechanisms responsible for E-CSC dysfunction, focusing on molecular pathways associated with cell senescence and ageing. We identified that E-CSC are characterized by an hyper-activation of the canonical NFkB pathway and of Caspase1, an increased activity of the TORC1 complex, an impairment of the authophagic flux and a reduction of AMPK, Akt and CREB activation, moreover leading to a decreased transcription of the cardio-protective microRNA-132. On the basis of these findings, we screened drugs capable to interfere with the above described pathways; we identified that a 3-days treatment with a combination of Resveratrol (0,5microM) and Rapamycin (10nanoM) is able to reduce the fraction of E-CSC affected by cell senescence in vitro and to diminish the secretion of IL-1β, thus restoring the protective effects of CSC’s secretome on cardiomyocytes exposed in vitro to SI/RO injury. At the molecular level, all the alterations in E-CSC could be successfully reverted employing the cocktail of the two drugs: Rapamycin and Resveratrol-treated-E-CSC were characterized by a reduction in TORC1 activity and inflammasome activation, and an increase in AMPK, AKT, CREB, SIRT-1 and miR-132 levels, in addition to the restoration of the autophagic flux. Last, we tested if the pre-conditioning of E-CSC with the combination of the two drugs, prior to the transplantation in the peri-infarct region in a mouse MI-model, is able to restore their reparative ability. The results were positive: mice treated with preconditioned E-CSC showed cardiac functional and dimensional parameters similar to those of D-CSC treated mice; histologically, hearts were characterized by an enhanced arteriolar density, a decreased cardiomyocyte senescence, apoptosis and autophagy and an increased recruitment of host CSCs, that resulted in the reduction of the infarct size. In conclusion, we first demonstrate that senescent c-Kit-pos CSC resident in human failing hearts display an impaired in vivo reparative ability; importantly, the senescent phenotype is not irreversible, and senescent CSC can be rejuvenated in vitro with a short pharmacologic conditioning with a combination of Rapamycin and Resveratrol, finally boosting the in vivo cardiac regeneration. These findings open new avenues to improve autologous CSC therapy in heart failure.