Mitochondria transmit apoptosis signalling in cardiomyocyte-like cells and isolated hearts exposed to experimental ischemia-reperfusion injury

Abstract

Ischemia-reperfusion (I/R) is a condition leading to serious complications due to death of cardiac myocytes. We used the cardiomyocyte-like cell line H9c2 to study the mechanism underlying cell damage. Exposure of the cells to simulated I/R lead to their apoptosis. Over-expression of Bcl-2 and Bcl-x_L protected the cells from apoptosis while over-expression of Bax sensitized them to programmed cell death induction. Mitochondria-targeted coenzyme Q (mitoQ) and superoxide dismutase both inhibited accumulation of reactive oxygen species (ROS) and apoptosis induction. Notably, mtDNA-deficient cells responded to I/R by decreased ROS generation and apoptosis. Using both in situ and in vivo approaches, it was found that apoptosis occurred during reperfusion following ischemia, and recovery was enhanced when hearts from mice were supplemented with mitoQ. In conclusion, I/R results in apoptosis in cultured cardiac myocytes and heart tissue largely via generation of mitochondria-derived superoxide, with ensuing apoptosis during the reperfusion phase.     

Interferons induce the expression of IFITM1 and IFITM3 and suppress the proliferation of rat neonatal cardiomyocytes

Cardiovascular diseases have been one of the leading killers among the human population worldwide. During the heart development, cardiomyocytes undergo a transition from hyperplastic to hypertrophic growth with an unclear underlying mechanism. In this study, we aim to investigate how interferons differentially stimulate the interferon-inducible transmembrane (IFITM) family proteins and further be involved in the process of heart development. The expression levels of three IFITM family members, IFITM1, IFITM2, and IFITM3 were investigated during Sprague-Dawley rat myocardial development and differentiation of H9C2 cardiomyocytes. The effects of interferon-α, -β, and -γ on DNA synthesis in H9C2 cells were also characterized. Up-regulation of IFITM1 and IFITM3 were observed during the heart development of Sprague-Dawley rat and the differentiation of H9C2 cells. Moreover, interferon-α and -β induce the expression of IFITM3 while interferon-γ up-regulates IFITM1. Finally, interferon-α and -β were demonstrated to inhibit DNA synthesis during H9C2 cell differentiation. Our results indicated interferons are potentially involved in the differentiation and cell proliferation during heart development.     

EphB4 Forward‐Signaling Regulates Cardiac Progenitor Development in Mouse ES Cells

Eph receptor (Eph)‐ephrin signaling plays an important role in organ development and tissue regeneration. Bidirectional signaling of EphB4–ephrinB2 regulates cardiovascular development. To assess the role of EphB4–ephrinB2 signaling in cardiac lineage development, we utilized two GFP reporter systems in embryonic stem (ES) cells, in which the GFP transgenes were expressed in Nkx2.5⁺ cardiac progenitor cells and in α‐MHC⁺ cardiomyocytes, respectively. We found that both EphB4 and ephrinB2 were expressed in Nkx2.5‐GFP⁺ cardiac progenitor cells, but not in α‐MHC‐GFP⁺ cardiomyocytes during cardiac lineage differentiation of ES cells. An antagonist of EphB4, TNYL‐RAW peptides, that block the binding of EphB4 and ephrinB2, impaired cardiac lineage development in ES cells. Inhibition of EphB4–ephrinB2 signaling at different time points during ES cell differentiation demonstrated that the interaction of EphB4 and ephrinB2 was required for the early stage of cardiac lineage development. Forced expression of human full‐length EphB4 or intracellular domain‐truncated EphB4 in EphB4‐null ES cells was established to investigate the role of EphB4‐forward signaling in ES cells. Interestingly, while full‐length EphB4 was able to restore the cardiac lineage development in EphB4‐null ES cells, the truncated EphB4 that lacks the intracellular domain of tyrosine kinase and PDZ motif failed to rescue the defect of cardiomyocyte development, suggesting that EphB4 intracellular domain is essential for the development of cardiomyocytes. Our study provides evidence that receptor‐kinase‐dependent EphB4‐forward signaling plays a crucial role in the development of cardiac progenitor cells. J. Cell. Biochem. 116: 467–475, 2015. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.     

Involvement of ubiquitin-proteasome system in icariin-induced cardiomyocyte differentiation of embryonic stem cells using two-dimensional gel electrophoresis

Icariin has been shown to significantly facilitate the differentiation of embryonic stem (ES) cells into cardiomyocytes in vitro. However, the mechanism underlying the icariin-induced cardiomyocyte differentiation is still not fully understood. In the present study, 52 differentially displayed proteins selected from two-dimensional electrophoresis gels were identified by MALDI-TOF mass spectrometry analysis. More than half of proteins could be assigned to six main categories: (1) protein synthesis, metabolism, processing and degradation, (2) stress response, (3) cytoskeleton proteins, (4) energy metabolism, (5) carbohydrate metabolism/transport, and (6) RNA/other nucleic acids metabolisms and transport, nuclear proteins. MALDI-TOF/MS showed that icariin treatment resulted in the induction of five ubiquitin-proteasome system (UPS)-related proteins, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), ubiquitin-conjugating enzyme E2N, proteasome 26S, proteasome subunit-alpha type 6, and proteasome subunit-alpha type 2 in the differentiated cardiomyocytes. These results implied that UPS might play an important role in the control of cardiomyocyte differentiation. Epoxomicin (a proteasome inhibitor) significantly reduced the cardiomyocyte differentiation rate of ES cells and proteasome activities, as well as inhibited NF-κB translocation into the nucleus, which were evidently reversed by presence of icariin. Meanwhile, icariin could significantly reverse the reduction of four proteins (proteasome subunit-alpha type 6, proteasome subunit-alpha type 2, UCH-L1, and ubiquitin-conjugating enzyme E2N) expressions owing to application of epoxomicin. These suggest UPS could be a means by which icariin may regulate expressions of key proteins that control cardiomyocyte differentiation. Taken together, these results indicated that UPS played an important role in ES cell differentiate into cardiomyocytes induced by icariin.