The Molecular Mechanism Of H9C2 Cell Hypertrophy Induced By ERK1/2 Pathway And Autophagy Pathway In Visfatin

BackgroundCardiac hypertrophy is a basic adaptive response of the heart to the various stimuli causinghemodynamic overload, characterized mainly by intracellular protein synthesis and cell size enlargement. In the early stage these cellular responses may be considered compensatory, but eventually lead to cardiac hypertrophy,myocardial remodeling, and congestive heart failure. Currently cardiac hypertrophy has evolved as an independent risk factor for significantly increased cardiovascular morbidity and mortality.In 2005, Fukuhara etc. in animal visceral fat found a cDNA fragment identical to the PBEF(pre-B cell colony-enhancing factor) from the 5 'end sequence of non-coding region. Their subsequent work also demonstrated that such cDNA fragment(which they named Visfatin) is highly expressed in rat as well as human abdominal visceral adipose tissues. A large body of studies to date have shown that expression of Visfatin is closely related to the development of such diseases as metabolic syndrome, coronary artery disease, type II diabetes, various inflammatory diseases, ischemic stroke and even breast cancer. Available literature suggests that very few studies(domestic or international) have focused on the effects of Visfatin on myocardial hypertrophy.In recent years researchers have discovered an important cell proliferation signal regulatory protein-ERK1/2. As one of the main members of the MAPK family, ERK1/2 widely participates in various cellular biological processes, such as cell proliferation and differentiation, cell shape maintenance, cell skeleton building and progression, and apoptosis, etc. Interestingly, many authors also have found that ERK1/2 signaling pathway is extensively involved in the development of cardiovascular diseases, in that ERK1/2 may be activated in response to most hypertrophic stimuli and results in subsequent target protein phosphorylation, although the specific mechanismsremain unclear.Autophagy, initially thought as cell self-destruction, is currently further explainedby a process of enzymatic digestion of some damaged, degenerated or aging intracellular proteins which were transported to the lysosome organelles.Due to its wide range of biological activity, autophagy has attracted great interest by a growing number of research scientists. Studies have established that autophagy is closely involved in the development of many cardiovascular diseases such as coronary atherosclerosis, myocardial ischemia/reperfusion injury, cardiac hypertrophy, myocardial infarction, and heart failure. However, itsspecific role and molecular mechanisms involved in these processes are not clear. Currently available studies seem to suggest that autophagy plays a conflicting or “double-role” during the development of cardiovascular diseases. The exact role autophagy may play in these cardiovascular system diseases, promoting or inhibiting, remains yet to be known.In view of the above research background,we hypothesized that Visfatin might participate in cardiomyocyte hypertrophy. We further asked ifERK1/2signal pathway and autophagy were involved in this process and what roles they might play during Visfatin induced myocyte hypertrophy.To address these questions, we studied the effects of Visfatin on in vitro cultured H9C2 myocardial cells from rat heart. We confirmed that Visfatin could induce myocyte hypertrophy and this effect was likely mediated through ERK1/2 signaling pathway and cell autophagy activity. Our data add to the growing literature exploring the mechanisms and pathogenesis of myocardial hypertrophy, which may eventually lead to new and targeted effective therapy. ObjectiveTo investigate if Visfatin can induce cardiomyocyte hypertrophy by observing its effects on in vitro cultured H9C2 rat cardiac cells and to explore the cellular and molecular mechanisms involved during development of myocyte hypertrophy. MethodsLiterature search suggests that Visfatin at a concentration of 100ng/mL might have the most significant hypertrophic effect on cardiac myocytes at 48 hours of culture. This concentration(100ng/ml) was therefore adopted in our experiment. According to specific instructions, 5ug Visfatin was dispensed and dissolved in 50 ul Milli Q water first, and 450 ul 0.1%BSA was further added to achieve a final concentration of 100ng/ml. Four study groups were included for comparison and analysis:(1)control group;(2)Visfatin-alone group;(3)3-MA+Visfatin group;(4)PD98059+Visfatin group. Cell(H9C2) surface area and morphology were calculated and analyzed by Image-Proplus professional imaging analysis software. Effects of Visfatin on cultured H9C2 cells were further analyzed by quantifying the individual proteins extracted from these myocytes, using the BCA protein quantitative methods. Western blot was also used to analyze intracellular BNP, ?-SMA, LC3 B, ERK1/2, p-ERK1/2 protein expression changes. Results1. Comparing with the control group, Visfatin treated group demonstrated a marked increase in the cell surface area as shown by Image-Proplus imaging analysis software(P<0.05); Either 3-MA or PD98059 treatment resulted in significant decrease in cell surface area(P<0.05). These changes were also statistically different(P<0.05) from Visfatin-alone treatment group.2. Comparing with the control group, Visfatin treated group demonstrated a significant increase(P<0.05) in individual protein synthesis using the BCA protein assay method. Either 3-MA or PD98059 co-culture decreased cell protein synthesiswhich was also significantly different from Visfatin-alone treatment group(P<0.05).3. Western blot analyses demonstrated that Visfatin caused a significant increase in p-ERK1/2, LC3 B, BNP, ?-SMA protein expressions comparing with the control group myocytes(P<0.05). No statistically significant difference was seen for total ERK1/2 protein among the groups(P>0.05). Either 3-MA or PD98059 treatment caused significant decrease in p-ERK1/2, LC3 B, BNP, ?-SMA protein expressions comparing with the Visfatin-alone treatment group(P<0.05). Again there was no statistically significant difference in total ERK1/2 among the group(P>0.05). ConclusionsVisfatin induced H9C2 cell hypertrophy through ERK1/2 pathway and the autophagy pathway.