ABSTRACT
ObjectiveExosomes secreted by BMSC overexpressing GATA-4 gene (BMSCGATA-4-exosome) can promote the differentiation of BMSC into cardiomyocyte-like cells, thereby improve cardiac function after myocardial infarction. However, the molecular mechanism of BMSCGATA-4-exosome in cardiomyocyte-like cell differentiation is unknown. The effect of the secretion of BMSCGATA-4 exosome from bone marrow mesenchymal stem cells (BMSC) in the differentiation of stem cells into cardiomyocytes was determined in miRNA-673-5p/Tsc-1 axis dependent manner.MethodsMouse models of myocardial infarction were established and divided into seven groups. Simulation group (BMSCmiR-673-5p-mimic exosome), inhibition group (BMSCmiR-673-5p-inhibitor exosome), GATA-4 group (BMSCGATA-4 exosome), empty vector group (BMSCempty vector exosome）, and BMSC group (BMSC exosome) were injected into the tail vein for 48 h, and the untreated and normal mice were used as the control group. Cardiac ultrasound was used to detect cardiac function in each group. miRNA-673-5p expression in myocardial infarction was detected using real-time polymerase chain reaction (RT-PCR). The myocardial tissues were extracted from the same myocardial infarction site. Myocardial-specific molecules, such as α-actin, Desmin, cTnT, and Cx43, were detected using RT-PCR. Western blot was used to determine the expression of the corresponding target gene of miRNA-673-5p, Tsc-1, Erk1/2, and Mef2c proteins.ResultsThe simulation group wan shown the most significantly improved myocardial function (P<0.05) with an expression peak of miRNA-673-5p in cardiomyocytes (P<0.05). The highest content of myocardial-specific molecules including α-actin, Desmin, cTnT, and Cx43 was found in the simulation group. The simulation group had the lowest expression of Tsc-1 in cardiomyocytes (P<0.05).ConclusionOverexpressed BMSCGATA-4 exosomes inhibit Tsc-1 expression through miRNA-673-5p to improve cardiac function during myocardial infarction.
ABSTRACT
Objective Exosomes secreted from mouse bone marrow mesenchymal stem cells (BMSC) overexpressing the cardiomyocyte transcription factor GATA-4 (BMSCGATA-4-exosome) may play a key role in repairing myocardial injury. This study aimed to investigate the molecular regulatory network of BMSCGATA-4-exosome for inhibiting the apoptosis of cardiomyocytes. Methods Exosomes extracted from GATA-4-overexpressing BMSCs of the mouse cultured with miR-330-3p-mimic were cultured with myocardial cells under hypoxic and serum-free conditions for 24 hours (the experimental group), the overexpressed GATA-4, empty vector and BMSCs were taken as the confounding factor control (CFC), the myocardial cells cultured under hypoxic and serum-free conditions for 24 hours were used as the positive control, and those cultured under the normal condition for 24 hours as the negative control. The apoptosis rates of myocardial cells in different groups were measured by flow cytometry, the expression levels of miR-330-3p in the myocardial cells determined by RT-PCR, and those of the corresponding miR-330-3p target gene Ap2m1 and transcriptional protein Cnot4 detected by Western blot. Results CD29 was expressed in 99.71% of the mouse BMSCs, CD44 in 97.28%, SCA-1 in 99.40%, and CD11b overexpressed in only 0.1%. The early apoptosis rate of myocardial cells was significantly higher in the experimental than in the negative control group ([7.90 ± 0.34]% vs [2.30 ± 0.09]%, P < 0.05) but lower than in the positive control ([51.48 ± 0.40]%), BMSC ([18.32 ± 3.03]%), empty vector ([16.99 ± 2.93]%) and overexpressed GATA-4 groups ([10.22 ± 0.35]%) (P < 0.05). The expression of miR-330-3p in the myocardial cells was markedly higher in the experimental ([396.10 ± 1.02]%) than in the negative control ([1.37 ± 0.33]%), positive control ([0.26±0.32]%), BMSC ([1.40 ± 0.42]%), empty vector ([1.41 ± 0.27]%) and overexpressed GATA-4 groups ([3.80 ± 0.62]%) (P < 0.05). The expressions of Ap2m1 and Cont4 in the myocardial cells were remarkably decreased in the experimental group compared with those in the other five groups (P < 0.05). Conclusion Overexpressed BMSCGATA-4-exosomes suppress the apoptosis of myocardial cells by inhibiting the expression of the Ap2m1 protein via miR-330-3p.