Enhancing cardiac regeneration: direct reprogramming of fibroblasts into myocardial-like cells using extracellular vesicles secreted by cardiomyocytes.

Abstract

To investigate the promoting effect of extracellular vesicles derived from myocardial cells (CM-EVs) on the reprogramming of cardiac fibroblasts (CFs) into cardiomyocyte-like cells (iCMs) and their therapeutic effect on myocardial infarction (MI) in rats. Cell experiments: The differential adhesion method was used to obtain Sprague Dawley (SD) suckling rat CFs and cardiomyocytes (CMs), while the ultracentrifugation method was used to obtain CM-EVs. Transmission electron microscopy and nanoparticle tracking technology were used to analyze and determine the morphology and particle size of CM-EVs. Western blotting was used to identify the expression of EV markers CD9, CD63, and Alix proteins. Small molecule combination of CHIR99021, Forskolin, Dorsomorphin, SB431542, and Valproic acid (CFDSV) and CFDSV + CM-EVs combination were used to induce CFs to differentiate into cardiomyocytes. The expression of cellular morphological changes, myocardial-specific protein cardiac troponin T (cTnT), and α-actinin were detected on the 3rd, 6th, 9th, and 15th day of reprogramming, respectively. After transfection and inhibition of miRNA-133, immunofluorescence, RT-qPCR, and Western blotting techniques were used to detect the expression of cTnT and α-actinin of induced CFs in the CMs group (CM-EVs), miRNA-133 high expression group (133H), and miRNA-133 inhibition group (133I). Animal experiment: CM-EVs were injected into the margin of myocardial infarction in rats. Cardiac function was detected by echocardiography before and 4 weeks after infarction, and the pathological changes were detected by HE and Masson staining, while Tunel and CD31 fluorescence staining were used to detect myocardial cell apoptosis and angiogenesis. CFs in the CM-EVs group expressed cTnT and α-actinin after induction, and the expression intensity gradually increased with the extension of induction time. On the 15th day after induction, cTnT-positive cells accounted for 85.6% of the total cell count, while the CFDSV group accounted for 48.8%. The majority of cells expressed GATA-binding protein 4 (GATA4), NK2 homeobox 5 (Nkx-2.5), and connexin 43 (Cx43). The RT-qPCR analysis showed the induced CFs expressed mature cardiomyocyte markers, including cTnT, Ryr2, Nkx-2.5, and GATA, which were similar to those of CMs (P < 0.05). Upon induction of CFs into iCMs, iCMs expressed cardiac precursor cell markers, such as source domain transcription factor-1 (Isl-1), mesodermal posterior spiral transcription factor-1 (Mesp-1), GATA4, and fetal liver kinase-1 (Flk-1). RT-qPCR, Western blotting, and immunofluorescence results showed that cTnT and α-actinin were highly expressed in CFs induced by CM-EVs group and 133H group until the 15th day, while the expression levels were low in cont group and 133I group. In animal in vivo experiments, injection of CM-EVs was found to alleviate myocardial fibrosis and reduce apoptosis of myocardial cells in the infarcted area compared to the MI group (P < 0.001). Moreover, there was an increase in capillary density. Results showed a significant improvement in left ventricular ejection fraction and fractional shortening after 4 weeks of CM-EVs injection (P < 0.01). CM-EVs can enhance the reprogramming efficiency of CFs into iCMs, effectively alleviate myocardial fibrosis, resist cell apoptosis, increase angiogenesis, and improve heart function after myocardial infarction. MiRNA-133 plays an important regulatory role in this process.