Mitochondria-Rich Extracellular Vesicles From Autologous Stem Cell-Derived Cardiomyocytes Restore Energetics of Ischemic Myocardium

Abstract

Background: Mitochondrial 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.

Objectives: The 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.

Methods: Human-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.

Results: Electron 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.

Conclusions: M-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.

Keywords: bioenergetics; heart failure; human stem cells; mitochondria; myocardial infarction.

Figure 1.. Characterization of mitochondria-rich extracellular vesicles (M-EVs).

A. Differential ultracentrifugation was employed to isolate M-EVs. B. Representative flow cytometry (FCM) dot plots and histograms of M-EVs and Large vesicle-depleted EVs (Ld-EVs). C. Transmission electron microscopy of D15 M-EVs. D. The protein expression of the electron transport chain (ETC) in D15 M-EVs. E. M-EVs, but not Ld-EVs, produced extracellular ATP (n=4/group). *P<0.0001 by an unpaired t-test. ATP5A=ATP synthase α-subunit; COX II=cytochrome c oxidase subunit-2; MTDR=mitotracker deep red; MTG=mitotracker green; MTT=3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NDUFB8=NADH dehydrogenase-1β subcomplex subunit-8; SDHB=succinate dehydrogenase complex iron sulfur subunit B; SSA=side-scatter-area; UQCRC2=ubiquinol-cytochrome c reductase core protein-2.

Figure 2.. M-EVs facilitate transfer of their mitochondrial cargo and restore intracellular bioenergetics in hypoxia-injured iCMs.

A and B. Representative microscopic image of iCMs treated with M-EVs containing RFP⁺-mitochondria. Transferred M-EV-mitochondria (red) were observed in the recipient iCMs harboring GFP⁺-mitochondria (green). Some of the transferred mitochondria were fused with the native mitochondrial networks (white arrows). Scale bar: 50 and 20 μm. C to F. ATP levels in iCMs treated with M-EVs or Ld-EVs. (0, 1.0×10⁷, 3.0×10⁷, 1.0×10⁸ or 3.0×10⁸/mL: wedge). Left, 3-hours after treatment. Right, 24-hours after treatment (n=4–6/group). *P<0.05 and **P<0.001 by a one-way ANOVA followed by Tukey’s test.

Figure 3.. Non-mitochondrial cargo in D15 M-EVs restored bioenergetics through activation of PGC-1α-mediated mitochondrial biogenesis in the recipient iCMs.

A. PGC-1α and ERRγ mRNA, normalized to U6, were upregulated in D15 Ld-EVs (n=4/group). *P<0.05 by an unpaired t-test. B. Quantitative gene expression in iCMs, normalized to ACTB shown as fold change relative to normoxic iCMs (n=4–5/group). C. Seahorse extracellular-flux assays measuring oxygen-consumption rate (OCR) (n=5–6/group). *P<0.01, **P<0.001 and ***P<0.0001 by a one-way ANOVA followed by Tukey’s test. ATP5A1=ATP synthase F1 α-subunit; COX6A1=cytochrome c oxidase subunit-6A1; Cs=citrate synthase; CYC1=cytochrome c1; ERRγ=estrogen-related receptor γ; FCCP=carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; NRF1=Nuclear respiratory factor-1; NDUFA1=NADH dehydrogenase-1α subcomplex subunit-1; PGC-1α=peroxisome proliferator-activated receptor γ coactivator 1α; PKM=pyruvate kinase M1/2; SDHA=succinate dehydrogenase complex flavoprotein subunit-A; TFAM=mitochondrial transcription factor-A.

Figure 4.. PGC-1α knockdown compromised the restoration of mitochondrial bioenergetics and biogenesis in the hypoxia-injured iCMs.

A. Relative PGC-1α and ERRγ mRNA expression, normalized to U6, in Ld-EVs from D15 transduced with pLKO.1 expressing control shRNA (shCTRL) or anti-PGC-1α shRNA (shPGC-1α) (n=3/group). *P<0.0001 by an unpaired t-test. B. PGC-1α knockdown reduced restoration of ATP levels in hypoxia-injured iCMs at 24hous (n=5–8/group). C. Representative immunoblots and quantitative analyses of PGC-1α, ETC complexes and β-actin (n=4–6/group). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 by a one-way ANOVA followed by Tukey’s test. Abbreviations as in Figure 1.

Figure 5.. D15 M-EVs improved mitochondrial function, contractile property, and cell survival in hypoxia-injured iCMs.

A. Representative FCM dotplots of iCMs labeled with MTG/MTDR (n=5–6/group). B. D peak and maximum contractile rate in iCMs (n=5–6/group). C. Representative FCM dotplots of iCMs stained with Annexin V/Propidium iodide (n=4–5/group). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 by a one-way ANOVA followed by Tukey’s test.

Figure 6.. The effects of intramyocardial injection of D15 M-EVs on post-myocardial infarction (MI) cardiac remodeling.

A. Representative images of short-axis acquisitions at mid-left ventricle (LV). B to E. LV ejection fraction (LVEF), LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV) and LV mass at weeks-2 and −4 after MI, evaluated by MRI. F. Representative images and quantitative analyses of myocardial viability as visualized by manganese-enhanced MRI (MEMRI). Red arrows: non-viable regions (n=8/group). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 by a two-way ANOVA followed by Tukey’s test.

Figure 7.. D15 M-EV therapy enhances bioenergetics and PGC-1α-mediated mitochondrial biogenesis in the peri-infarct region.

A. MTDR⁺-mitochondria were detected inside the Troponin I⁺-cardiomyocytes, highlighted with white arrows. Scale bar: 50 μm. B. Tissue ATP levels, normalized by tissue protein levels (n=4–6/group). C. Mitochondrial biogenesis-related gene expression levels in the peri-infarct region, normalized to GAPDH, measured by RT-PCR (n=4–5/group). D. Representative immunoblots and quantitative analyses of PGC-1α, VDAC and COX IV, normalized to GAPDH (n=4–5/group). *P<0.05 by a one-way ANOVA followed by Tukey’s test. ATP5ME=ATP synthase subunit-e; COX IV=cytochrome c oxidase subunit-4; COX4I1=cytochrome c oxidase subunit-4 isoform-1; NDUFAB1=NADH ubiquinone oxidoreductase subunit-AB1; VDAC=voltage-dependent anion channels; other abbreviations as in Figure 3.

Central Illustration.. Extracellular Vesicle-Mediated Autologous Mitochondrial Transplantation.

We obtained human-induced pluripotent stem cells (iPSCs)-derived cardiomyocytes (iCMs) and succeeded in collecting mitochondria-rich extracellular vesicles (M-EVs) from iCM conditioned-medium. The autologous M-EVs can be applied to patients with mitochondria-related diseases including heart failure.