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Background: Wnt signaling pathways are involved in cardiomyogenic differentiation. Besides that, nutrients and metabolism may also influence cell differentiation. However, it is still unclear whether the cell lineage commitment could be controlled by the metabolites of its precursor cells. The aim of this study is to examine whether the metabolites of cardiac precursors could determine cardiomyogenic differentiation fate of hiPSC. Methods: hiPSC was maintained in serum-free and feeder-free monolayer condition. After CHIR99021-induced mesendodermal differentiation for two days, IWP4 was added to inhibit Wnt signaling and to induce myogenic differentiation for another two days. After wash, the culture medium was collected during cell differentiation period of day 5-10 (d5-10-diff-medium). ExoQuick-TC kit was used to extract the exosomes in the medium. Cardiomyocytes were identified by immunocytochemistry staining with mouse monoclonal antibody against cardiac sarcomeric actinin alpha, and were counted the percentage by flow cytometry. Results: The exosome-free d5-10-diff-medium was found to be able to dramatically induce cardiomyogenic differentiation only in CHIR99021-induced mesendodermal cells, rather than directly in hiPSC, in the absence of Wnt inhibitor. The metabolic profiling of the exosome-free d5-10-diff-medium was further characterized by LC-MS. After functional validation, the effective metabolites for the induction of cardiomyogenic differentiation were identified and the possible signaling pathways were analyzed. Conclusion: This study demonstrated that the metabolites of cardiac precursors could control cardiac cell commitment by driving the development of cardiac metabolic system.

It was found cardiac precursors secreted exosomes (CPC-exo) may have anti-apoptotic and anti-fibrotic actions to facilitate cardiac repair in the damaged hearts. It remains unclear the effect of CPC-exo in cardiometabolism. The aim of the study was to assess the modulatory action of CPC-exo in cardiometabolism of cardiomyopathic cardiomyocytes. Nkx2.5+ cardiomyoblasts were enzymatically isolated and were purified by FACS sorter from mouse embryonic stem cells under cardiomyogenic differentiation or transgenic mice hearts (2 weeks old) carrying GFP reporter driven by Nkx2.5-enhancer for the purification of the exosomes (mESC-Nkx2.5+exo and postnatal-Nkx2.5+exo) from the culture medium by ExoQuick-TC. The vesicle size was further measured by Nanosight. The exosomal miRNA profile was characterized by miRNA microarray. It was further compared the different effect of mESC-Nkx2.5+exo and postnatal-Nkx2.5+exo in the mitochondrial function of human cardiomyocytes derived from iPSC of left ventricle non compaction (LVNC) patients (LVNC-hiPSC-CM) by Agilent seahorse XF24 analyzer. Cell shortening was measured by Ionoptix system. It was found the defect of cardiac mitochondrial function associated with the weak cell shortening in LVNC-hiPSC-CM on day 70 after differentiation. With the treatment of mESC-Nkx2.5+exo and postnatal-Nkx2.5+exo during day 50-70, the mitochondrial function and cell shortening of LVNC-hiPSC-CM were significantly improved on day 70. The functional exosomal miRNAs were characterized in advance in this study. In conclusion, CPC-exo may exert the benefit in the improvement of cardiometabolism in cardiomyopathic cardiomyocytes.

Using transgenic cardiac Nkx2.5 enhancer-eGFP mice to label Nkx2.5+ cardiomyoblasts, an embryonic Nkx2.5+ cell population representing the common precursor for cardiovascular lineages was identified in the developing heart and a residual small population was also found in the postnatal heart. Our previous study found that A83-01, an inhibitor of TGFb receptor type I (TGFbRI), could improve cardiac function in post-injured adult heart through inducing the proliferation of Nkx2.5+ cardiomyoblasts and enhancing paracrine benefit in the enhancement of myocardial survival. The aim of the present study is to examine whether the exosome secreted from A83-01-primed Nkx2.5+ cell (exo(Nkx-A83)) could increase cardiomyocyte survival and to investigate the cardioprotective mechanism of the exosomal molecules. Adult mice cardiomyocytes were isolated and cultured in IMDM medium in vitro . A83-01 alone did not directly affect myocyte viability. The myocyte viability was 33.3±2.0 % on day 5, and could be prominently increased to be 63.8 ±0.9% when co-cultured with Nkx2.5+ cell in the presence of A83-01. The effect was abolished by GW4869 to block exosome secretion. Moreover, treating cardiomyocyte with the purified exo(Nkx-A83) could markedly increase cell viability in vitro . The miRNA microarray and qPCR validation were performed to identify the different miRNAs in the exosome secreted from Nkx2.5+ cell treated with or without A83-01. It was found the marked increase of miR-30c, miR-489, miR-92b, and miR-98 in exo(Nkx-A83). Functional study demonstrated miRNA mimics of miR-30c and miR-92b could significantly enhance myocyte survival in vitro . Furthermore, administration of the purified exo(Nkx-A83) via intravenous injection in isoprenaline-induced heart injured mice could significantly improve cardiac function monitored by echocardiography. In conclusion, the findings revealed miR-30c and miR-92b in exo(Nkx-A83) could contribute to its cardioprotective action for the enhancement of myocardial survival.

Angiotensin II (Ang II) is involved in the pathogenesis of atrial fibrillation (AF). L-type calcium channel (LCC) expression is altered in AF remodeling. We investigated whether Ang II modulates LCC current through transcriptional regulation, by using murine atrial HL-1 cells, which have a spontaneous calcium transient, and an in vivo rat model. Ang II increased LCC α1C subunit mRNA and protein levels and LCC current density, which resulted in an augmented calcium transient in atrial myocytes. An ≈2-kb promoter region of LCC α1C subunit gene was cloned to the pGL3 luciferase vector. Ang II significantly increased promoter activity in a concentration- and time-dependent manner. Truncation and mutational analysis of the LCC α1C subunit gene promoter showed that cAMP response element (CRE) (−1853 to −1845) was an important cis element in Ang II-induced LCC α1C subunit gene expression. Transfection of dominant-negative CRE binding protein (CREB) (pCMV-CREBS133A) abolished the Ang II effect. Ang II (1 μmol/L, 2 hours) induced serine 133 phosphorylation of CREB and binding of CREB to CRE and increased LCC α1C subunit gene promoter activity through a protein kinase C/NADPH oxidase/reactive oxygen species pathway, which was blocked by the Ang II type 1 receptor blocker losartan and the antioxidant simvastatin. In the rat model, Ang II infusion increased LCC α1C subunit expression and serine 133 phosphorylation of CREB, which were attenuated by oral losartan and simvastatin. In summary, Ang II induced LCC α1C subunit expression via a protein kinase C–, reactive oxygen species–, and CREB-dependent pathway and was blocked by losartan and simvastatin.