Abstract
Extracellular vesicles (EVs) have emerged as key mediators of intercellular communication that have the potential to improve cardiac function when used in cell-based therapy. However, the means by which cardiomyocytes respond to EVs remains unclear. Here, we sought to clarify the role of exosomes in improving cardiac function by investigating the effect of cardiomyocyte endocytosis of exosomes from mesenchymal stem cells on acute myocardial infarction (MI). Exposing cardiomyocytes to the culture supernatant of adipose-derived regenerative cells (ADRCs) prevented cardiomyocyte cell damage under hypoxia in vitro. In vivo, the injection of ADRCs into the heart simultaneous with coronary artery ligation decreased overall cardiac infarct area and prevented cardiac rupture after acute MI. Quantitative RT-PCR-based analysis of the expression of 35 known anti-apoptotic and secreted microRNAs (miRNAs) in ADRCs revealed that ADRCs express several of these miRNAs, among which miR-214 was the most abundant. Of note, miR-214 silencing in ADRCs significantly impaired the anti-apoptotic effects of the ADRC treatment on cardiomyocytes in vitro and in vivo To examine cardiomyocyte endocytosis of exosomes, we cultured the cardiomyocytes with ADRC-derived exosomes labeled with the fluorescent dye PKH67 and found that hypoxic culture conditions increased the levels of the labeled exosomes in cardiomyocytes. Chlorpromazine, an inhibitor of clathrin-mediated endocytosis, significantly suppressed the ADRC-induced decrease of hypoxia-damaged cardiomyocytes and also decreased hypoxia-induced cardiomyocyte capture of both labeled EVs and extracellular miR-214 secreted from ADRCs. Our results indicate that clathrin-mediated endocytosis in cardiomyocytes plays a critical role in their uptake of circulating, exosome-associated miRNAs that inhibit apoptosis.
Highlights
From the Department of Cardiology, Nagoya University School of Medicine, Nagoya, Japan, 65 Tsurumai, Showa, Nagoya 466-8550, Japan
To investigate whether mesenchymal stem cells effect cardiac protection through secretion of paracrine factors, isolated mouse adult cardiomyocytes under hypoxia conditions were treated with the supernatant of bone marrow-mononuclear cell (BM-MNC) or adipose-derived regenerative cells (ADRCs) cultures (Fig. 1A)
ADRCs or BM-MNCs were injected in the heart simultaneously with coronary artery ligation, and infarct changes were determined in triphenyltetrazolium chloride (TTC)-stained sections 2 days after the ligation (Fig. 1B)
Summary
Adipose-derived regenerative cells (ADRCs) protect against ischemia-induced cardiac dysfunction. Mesenchymal stem cells secrete angiogenic factors and enhance angiogenesis in ischemic hind limbs [11], ADRC treatment did not significantly enhance angiogenesis in the heart (Fig. S1C) These findings suggested that ADRC treatment reduces cardiomyocyte apoptosis following coronary artery ligation, preventing MI-induced cardiac rupture. To investigate whether communication between cardiomyocytes and ADRCs affords cardiac protection through clathrin-mediated endocytosis in vivo, male mice were treated with ADRCs alone or in combination with chlorpromazine following coronary artery ligation (Fig. 4C). Chlorpromazine treatment suppressed the ischemia injury–induced cardiac endocytosis (Fig. 4, H and I) These results demonstrated that clathrin-mediated endocytosis is activated during acute MI and plays a critical role in communication between cardiomyocytes and ADRCs in cell-based therapy
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