Short-term Effects Of PI3K/Akt Signaling Pathway On Cardiomyocytes By Sarcoplasmic Reticulum Calcium ATPase 2a Over-expression

Heart failure is the end stage of various cardiac diseases and is a major cause of mortality. Although progress in conventional treatment has improved survival, its 5-year mortality rate is still high. Molecular biology studies have shown that heart failure is a multi-gene disease. Gene therapy for heart failure provides a new therapeutic approach. Sarcoplasmic reticulum Ca2+ transport impairment is a common pathway for varieties of heart failure. Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) plays a key role in controlling intracellular Ca2+ handling. Previous studies showed that transgenic overexpression of SERCA2a was able to improve the function of myocardial cells of heart failure, correct the abnormal of Ca2+ homeostasis, extend survival time and reverse ventricular hypertrophy and so on. In order to better evaluate the treatment, to explore the molecular mechanisms of the treatment is necessary. Preliminary work in our laboratory shows that transgenic overexpression of SERCA2a can not only increase the expression of related enzymes that provide energy for the myocardial cells, but also correct the abnormal expression of MHC isoforms. In addition, other studies indicate transgenic overexpression of SERCA2a can correct myocardial protein kinase B (Akt1) abnormal expression in heart failure.Anthracycline antibiotics are widely used in the treatment of various malignancies, but they have serious cardiac toxicity. Heart failure is the final performance cardiac toxicity. It was investigated that the drugs impacted on the expression and function of myocardial SERCA2a protein and the change of SERCA2a function of these drugs is a key factor in cardiac toxicity. Anthracycline-induced cardiotoxicity is related to the decrease of Akt phosphorylation. Many facts have proven that a variety of myocardial protection factors through activating Akt pathway restrain the anthracycline cardiac toxicity.Akt is a protein serine/threonine kinase and Aktl is mainly in myocardial cell. Insulin and other growth factors activate Akt via PI3K. Akt activated extensively involved in the regulation of myocardial cell growth, the energy metabolism and resistance to apoptosis and other processes. Akt is the central elements within the cellular signal transduction networks, and the short-term moderate activated Akt has a protective effect on myocardial cells, while long-term activated one may promote heart failure. Transgenic overexpression of SERCA2a can improve the long-term prognosis in animal models of heart failure, while overexpression of SERCA2a can correct the abnormal expression of Aktl in myocardial cell of heart failure, we speculated that long-term beneficial effects of overexpression of SERCA2a may correct the abnormalities of Aktl pathway. In this study, the growth of cardiac cells is in vitro culture, and observed in normal cardiomyocytes PI3K/Akt signaling pathway effects by using recombinant adenovirus gene transient overexpression of SERCA2a. Morever, whether the downstream of GSK3βand GLUT4 molecular being activated is also investigated. First, HEK-293T cells were used to amplification of recombinant adenovirus Ad-GFP and Ad-SERCA2a, and verified that the amplified adenovirus can infect target cells effectively in vitro. Myocardial cell injury is observed by using Ad-GFP infected cardiomyocytes and MTT expremental to determine the optimal MOI in order to provide the basic material for the later experiments. Second, affected Akt pathway is observed througn overexpressing SERCA2a in cultured H9c2 myocardial cell lines and primary myocardial cells. The results showed that overexpression of SERCA2a in vitro can active Akt signaling pathway and result in the elevation of Akt phosphorylation level, which is 2.7 times as high as control group (P<0.05). Then the phosphorylation of downstream substrate GSK3βis also higher and it is 3.1 times as high as control (P<0.05). Although GLUT4 protein expression has on significant changes, the shift from the cytoplasm translocation to the plasm membrane was observed. Further analysis indicated membrane GLUT4 proteins increased in the groups of SERCA2a overexpression, which is 2.1 times as high as control (P<0.05). Overexpression of SERCA2a in primary myocardial cell also can active Akt signaling pathway and result in the elevation of Akt phosphorylation level, which is 2.1 times as high as control group (P<0.05). Then the phosphorylation of downstream substrate GSK3βis also higher and it is 1.6 times as high as control group (P<0.05). PI3K inhibitor (LY294002) blocked the phosphorylation level of the above changes, suggesting that SERCA2a activated Akt through PI3K signaling pathway. Finally, the use of adriamycin induced myocardial injury, the overexpression of SERCA2a observed decreased on the cardiac toxicity of doxorubicin. The results showed that Adriamycin induced myocardial necrosis, increase of cells LDH leakage and intracellular Ca2+ level and decrease of SERCA2a protein expression. Overexpression of SERCA2a could inhibit adriamycin-induced myocardial injury, manifested as myocardial cell death and increased cell survival of myocardial cells (P<0.05), myocardial cells reduce the level of Ca2+ level (P<0.05), accompanied by the activation of Akt signaling pathway. LY294002 only partially blocked by SERCA2a overexpression on adriamycin induced myocardial injury, suggesting that overexpression of SERCA2a inhibition of adriamycin induced myocardial injury via activation of PI3K/Akt signaling pathway, might have other protection mechanisms.