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BackgroundMitochondrial dysfunction results in an imbalance between energy supply and demand in a failing heart. An innovative therapy that targets the intracellular bioenergetics directly through mitochondria transfer may be necessary.ObjectivesThe purpose of this study was to establish a preclinical proof-of-concept that extracellular vesicle (EV)-mediated transfer of autologous mitochondria and their related energy source enhance cardiac function through restoration of myocardial bioenergetics.MethodsHuman-induced pluripotent stem cell-derived cardiomyocytes (iCMs) were employed. iCM-conditioned medium was ultracentrifuged to collect mitochondria-rich EVs (M-EVs). Therapeutic effects of M-EVs were investigated using in vivo murine myocardial infarction (MI) model.ResultsElectron microscopy revealed healthy-shaped mitochondria inside M-EVs. Confocal microscopy showed that M-EV-derived mitochondria were transferred into the recipient iCMs and fused with their endogenous mitochondrial networks. Treatment with 1.0 × 108/ml M-EVs significantly restored the intracellular adenosine triphosphate production and improved contractile profiles of hypoxia-injured iCMs as early as 3 h after treatment. In contrast, isolated mitochondria that contained 300× more mitochondrial proteins than 1.0 × 108/ml M-EVs showed no effect after 24 h. M-EVs contained mitochondrial biogenesis-related messenger ribonucleic acids, including proliferator-activated receptor γ coactivator-1α, which on transfer activated mitochondrial biogenesis in the recipient iCMs at 24 h after treatment. Finally, intramyocardial injection of 1.0 × 108 M-EVs demonstrated significantly improved post-MI cardiac function through restoration of bioenergetics and mitochondrial biogenesis.ConclusionsM-EVs facilitated immediate transfer of their mitochondrial and nonmitochondrial cargos, contributing to improved intracellular energetics in vitro. Intramyocardial injection of M-EVs enhanced post-MI cardiac function in vivo. This therapy can be developed as a novel, precision therapeutic for mitochondria-related diseases including heart failure.

Endogenous capability of the post-mitotic human heart holds great promise to restore the injured myocardium. Recent evidence indicates that the extracellular vesicles (EVs) regulate cardiac homeostasis and regeneration. Here, we investigated the molecular mechanism of EVs for self-repair. We isolated EVs from human iPSC-derived cardiomyocytes (iCMs), which were exposed to hypoxic (hEVs) and normoxic conditions (nEVs), and examined their roles in in vitro and in vivo models of cardiac injury. hEV treatment significantly improved the viability of hypoxic iCMs in vitro and cardiac function of severely injured murine myocardium in vivo. Microarray analysis of the EVs revealed significantly enriched expression of the miR-106a-363 cluster (miR cluster) in hEVs vs. nEVs. This miR cluster preserved survival and contractility of hypoxia-injured iCMs and maintained murine left-ventricular (LV) chamber size, improved LV ejection fraction, and reduced myocardial fibrosis of the injured myocardium. RNA-Seq analysis identified Jag1-Notch3-Hes1 as a target intracellular pathway of the miR cluster. Moreover, the study found that the cell cycle activator and cytokinesis genes were significantly up-regulated in the iCMs treated with miR cluster and Notch3 siRNA. Together, these results suggested that the miR cluster in the EVs stimulated cardiomyocyte cell cycle re-entry by repressing Notch3 to induce cell proliferation and augment myocardial self-repair. The miR cluster may represent an effective therapeutic approach for ischemic cardiomyopathy.

Introduction: Mitochondrial dysfunction disrupts the balance between energy supply and demand in the failing heart. A novel therapy that promotes cellular energetics by providing autologous mitochondria enhances LVEF and prevents cardiac remodeling after myocardial injury. Hypothesis: Mitochondria-containing extracellular vesicles restore mitochondrial function of the injured human cardiomyocytes. Methods and Results: Conditioned medium (CM) from induced pluripotent stem cells (iPSCs) and iPSC-derived cardiomyocytes (iCMs) improved viability of cardiomyocytes challenged by hypoxia and reoxygenation. 220-nm filtration of CM abolished the protective effects, suggesting that the medium-size extracellular vesicles (mEVs, 220-400nm) conferred therapeutic factors. Flow cytometric analysis revealed that the iPSC- and iCM-CM contained functional mitochondria-positive EVs (13% and 25%, p<0.05, respectively), which were removed by 220-nm filter (Fig.A). Ultra-centrifugation resulted in purification of these mitochondria-containing mEVs. Mitochondria positivity in the purified mEVs was similar between iPSCs and iCMs. However, the iCM-mEVs showed 8 times more ATP production compared to iPSC-mEVs (p<0.05). Moreover PGC-1α mRNA expression, a key molecule in mitochondrial biogenesis, was markedly increased in iCM-mEVs. Both iPSC- and iCM-mEVs were taken up rapidly by the injured cardiomyocytes and the mitochondria were readily co-localized with the host-mitochondria. Treatment with iCM-vs. iPSC-EVs significantly increased intracellular ATP production and mitochondrial DNA copy number, restoring the viability of hypoxia injured iCMS (p<0.05, Fig.B). Conclusions: EVs-mediated transfer of functional mitochondria enhances mitochondrial biogenesis and cellular energetics to restore the failing iCMs, which may enable precision medicine for the failing heart. [ABSTRACT FROM AUTHOR]