CALCINERUIN-MEF2C Signal Pathway Is Involved In Cardiac Hypertrophy Induced By Endoplasmic Reticulum Stress In Rat Cardiomyocytes

Cardiac hypertrophy is an adaptive response triggered by many physiological and pathological conditions. However, epidemiological data indicate that chronic cardiac hypertrophy takes part in the development of various cardiovascular diseases such as dilated cardiomyopathy, heart failure and sudden death, which severely endanger human lives. So it is very important to interpret the pathogenesis of hypertrophy for clinical prevention and cure of hypertrophy and heart failure.Endoplasmic reticulum (ER) is an organelle regulating intracellular Ca2+, folding of secreted and membrane protein and cell apoptosis. Various stimuli, such as ER-Ca2+depletion, elevated protein synthesis, ischemia and hypoxia, disturb ER homeostasis and result in ER stress. In response to ER stress, ER chaperones such as calreticulin (CRT) and glucose-regulated proteins (GRPs) are upregulated to enhance the ER ability to regulate intracellular Ca2+level and handle with the unfolded proteins, which is cardioprotective. However, when ER stress is excessive and/or prolonged, the ER-related apoptotic process is initiated by induction of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) and caspase-12 activation, which lead to cell apoptosis and tissue injury. Protein kinase R-like ER kinase (PERK) is an important ER transmembrane protein, the relocation of GRP78 from the luminal domain of PERK to misfolded proteins leads to PERK activation, which induces translation of activating transcription factor 4 (ATF4) and its target genes such as CHOP, thus contributing to apoptosis.Accumulating evidence has demonstrated that apoptosis initiated by ER stress is involved in the pathogenesis of neurodegeneration, diabetic cardiomyopathy, and ischemia/reperfusion injury. Moreover, the expression of CRT, GRP78, and GRP94 increases significantly during cardiac hypertrophy, and CHOP-mediated apoptosis contributes to the development from cardiac hypertrophy to heart failure, which indicates that ER stress is involved in the development of hypertrophy, but the roles of ER stress in the development of hypertrophy is still unclear. Intracellular Ca2+ regulated predominantly by ER is a ubiquitous second messenger whose level is elevated significantly in hypertrophied cardiac myocytes. Calcineurin (CaN) is a highly conserved, Ca2+/calmodulin-dependent serine/threonine phosphatase composed of catalytic subunit A and regulatory subunit B, which has important roles in regulating T-cell activation, hypertrophy, cell cycle and so on. Myocyte enhancer factor 2c (MEF2c) is the first gene of the MEF2 family found to be expressed during cardiac development and a substrate for CaN in myocardium. However, whether this pathway is involved in ER stress-induced cardiac hypertrophy is unknown.In this present study, we explored the roles of ER stress in the development of cardiac hypertrophy induced by abdominal aortic constriction in rats. Next, ER stress in cultured neonatal rat cardiomyocytes was induced by two ER stress inducers thapsigargin (TG), which depletes Ca2+from ER, and tunicamycin (TM), which inhibits protein N-linked glycosylation. We explored the effects of TG and TM on ER stress and cardiac hypertrophy in cardiomyocytes, and also investigated the roles of CaN-MEF2c signal pathway in the development of cardiac hypertrophy in cardiomyocytes. The methods and results were as follows:1 ER stress was involved in the development of myocardial hypertrophy induced by abdominal aortic constriction in ratsThis part of work aimed to explore the roles of ER stress response in the development of myocardial hypertrophy induced by abdominal aortic constriction in rats. Healthy male Wistar rats were randomly divided into model group (n=45) and sham group (n=40). The rats in model group were operated on abdominal aortic constriction, while the abdominal aorta in sham group was only separated but not constricted. Hemodynamic changes, whole heart weight-to-body weight ratio. (HW/BW) and left ventricular weight-to-body weight ratio (LVW/BW) were measured at 1 d,3 d,7 d,14 d and 28 d after surgery, respectively.2-D electrophoresis and mass spectrometry were used to identify the proteomic profile in hypertrophic myocardium at 28 d after surgery. The mRNA expression of GRP78, CRT and CHOP, which were important markers of ER stress, were detected by RT-PCR, and western blot was used to assess the protein level of a-actin, GRP78, CRT, CHOP, and apoptosis-associated protein Bax and Bcl-2.It was found that abdominal aortic constriction induced significant myocardial hypertrophy in rats. Compared with sham group, the blood pressure, LVW/BW, and HW/BW in model group increased significantly and cardiac function enhanced compensatively at 7 d after surgery, which increased progressively during the experiment. The expression of myosin light chain increased andα-actin proprotein andα-myosin heavy chain decreased, all of which are the markers of hypertrophy, at 28 d after abdominal aortic constriction. The upregulation ofα-actin expression in model group was also verified by western blot. In addition, as early as 1 d after surgery, the mRNA level of CRT in model group increased by 136%(P<0.01) compared with sham group. The mRNA and protein expression of GRP78 increased at 7 d after surgery, and the expression of CRT and GRP78 sustained high level till to the end of experiment. Prolonged ER stress triggered myocyte apoptosis pathway, compared with sham group, both the expression of CHOP and Bax in model group increased, while the expression of anti-apoptotic protein Bcl-2 decreased at 14 d after surgery. The results above indicated that abdominal aortic constriction induced significant upregulation in ER molecular chaperones at early stage of post-surgery, ER stress response may take part in the development of cardiac hypertrophy. CHOP-mediated cell apoptosis pathway may be involved in the regulation of development in myocardial hypertrophy and heart failure, and control the progression of decompensative hypertrophy.2 ER stress inducers TG and TM induced cardiac hypertrophy and ER stress in neonatal rat cardiomyocytesThe results above indicated that ER stress was involved in the development of hypertrophy in rat heart. To verify ER stress could induce hypertrophy independently, primary cultures from neonatal rats were incubated with the indicated concentration of TG (1,2.5,5,10,20,50,70,100 nmol/L) for 48 h, or with 50 nmol/L TG for 12, 24,36,48,60, and 72 h, respectively; moreover,1,10,100 ng/ml TM, another ER stress inductor, was used to treat cardiomyocytes for 48,72,96 h, respectively.10"7 mmol/L angiotensin (Ang)Ⅱfor 48 h treatment was as positive control. The detections of lactate dehydrogenase (LDH) activity in medium and cell apoptosis rate were used to reflect cell injury in cardiomyocytes. The mRNA expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), which were hypertrophic gene markers, were detected by RT-PCR. Total protein synthesis rate in cardiomyocytes was evaluated by incorporation of 3H-Leucine. F-actin fluorescence staining was employed to view cytoskeleton in cardiomyocytes, and cell surface area in cardiomyocytes was analyzed. In addition, RT-PCR was used to detect the mRNA expression of ER stress molecules, including CRT, GRP78, PERK, ATF4, and CHOP. Western blot was employed to detect the protein expression of ER stress proteins such as CRT, GRP78, CHOP and apoptosis-related Bax and Bcl-2. ER-specific dye Dapoxyl was used to observe ER morph in freshly viable cardiomyocytes, and CRT immunofluorescence in cardiomyocytes was viewed under a confocal laser scanning microscope.The results were as follows:ER stress inducers TG and TM induced cell injury in a time-and dose-dependent manner in cardiomyocytes, as shown as the increase in LDH activity in the medium and cell apoptosis rate. Meanwhile, cardiac hypertrophy in cardiomyocytes was induced by TG and TM in a time-and dose-dependent manner, as characterized by the elevation in the ANP and BNP mRNA expression, in the protein synthesis rate and in the cell surface area. F-actin staining observed that the F-actin fluorescence intensity was augmented significantly in TG-and TM-treated cardiomyocytes. It was found that 50 nmol/L TG for 48 h or 10 ng/ml TM for 72 h treatment was the suitable condition to induce cardiac hypertrophy without severe injury in cardiomyocytes. In addition, it was found that TG induced significant ER stress response in a time-and dose-dependent manner in cardiomyocytes, as characterized by the increase in CRT and GRP78 expression. Of interest, the mRNA expression of PERK and ATF4 in TG-treated cardiomyocytes increased at 24-48 h, and decreased at 60-72 h. Severe or prolonged ER stress triggered CHOP-mediated apoptosis pathway, TG upregulated CHOP expression and downregulated Bcl-2 to Bax ratio in a time-and dose-dependent manner in cardiomyocytes. ER staining in freshly viable cardiomyocytes found that the structure of the ER in TG-induced cardiomyocytes severely destroyed and vacuole appeared. The ER stress profile showed above was confirmed in cardiomyocytes treated with 10 ng/ml TM for 72 h. These results indicated that ER stress inducers induced significant hypertrophy in parallel with ER stress in cardiomyocytes, and CHOP-mediated apoptosis pathway was involved in the development of cardiac hypertrophy in cardiomyocytes.3 CaN-MEF2c signal pathway was involved in ER stress-induced cardiac hypertrophy in cardiomyocytesCytoplasmic Ca2+, which regulates CaN directly, is elevated significantly and plays an essential role in triggering the cardiac hypertrophy response. This part of work was to investigate the roles of CaN-MEF2c pathway in the development of cardiac hypertrophy induced by ER stress in cardiomyocytes. Cardiomyocytes was treated with the indicated concentration of TG (1,2.5,5,10,20,50,70,100 nmol/L) for 48 h, or with 50 nmol/L TG for 12,24,36,48,60, and 72 h, respectively; moreover,10 ng/ml TM, another ER stress inductor, was used to treat cardiomyocytes for 72 h. In addition, to explore the effects of CaN activity inhibition on ER stress-induced cardiac hypertrophy and CaN-MEF2c pathway, cardiomyocytes were pretreated with 5μmol/L cyclosporine A (CsA) for 10 min before 50 nmol/L TG for 48 h treatment, which induced notable cardiac hypertrophy. Fluo-3 AM staining was used to detect the relative Ca2+concentration in cytoplasm. Sarco/ER Ca2+-ATPase (SERCA) activity was measured by a Ca2+-ATPase assay kit according to the manufacturer's instructions, and CaN activity was measured by use of p-nitrophenyl phonphate. Analysis of LDH activity in the medium and cell apoptosis rate was used to evaluate cell injury in TG induced-cardiomyocytes with CsA pretreatment. Cardiac hypertrophy in TG induced-cardiomyocytes with CsA pretreatment was evaluated by analysis of the mRNA expression of ANP and BNP, the protein synthesis rate and the cell surface area. Western blot was used to detect the protein expression of SERCA, phospholamban (PLB), MEF2c, p-MEF2c and ER stress-related proteins such as CRT, GRP78, CHOP, Bax and Bcl-2. Immunofluorescence was performed to observe the change of MEF2c fluorescence in cardiomyocytes.The results were as follows:TG induced significant elevation of intracellular free Ca2+level, CaN activity, and MEF2c/p-MEF2c expression in a dose-and time dependent manner in cardiomyocytes; meanwhile, the SERCA activity decreased in TG-treated cardiomyocytes. The elevation of intracellular Ca+level and CaN activity as well as the decrease of SERCA activity were also verified in TM-treated cardiomyocytes. Immunofluorescence showed that MEF2c localized predominately in cytoplasm in control cardiomyocytes, while MEF2c transferred to nuclei in cardiomyocytes after TG treatment. CaN activity in TG-treated cardiomyocytes was blocked significantly by CsA pretreatment. The cardiac hypertrophy was prevented by CaN activity inhibition with CsA, as shown as the decrease in ANP and BNP mRNA expression, protein synthesis rate and cell surface area. After the inhibition of CaN activity, the MEF2c nuclear translocation was also prevented in TG-treated cardiomyocytes. Meanwhile, the cell injury increased in TG-treated cardiomyocytes with CsA pretreatment, the LDH activity in the medium and cell apoptosis rate increased significantly compared with control cardiomyocytes. In addition, the upregulation of CRT, GRP78 and CHOP in TG-treated cardiomyocytes was not blocked by CsA pretreatment. These results above indicated that the decrease in SERCA activity and the increase of PLB expression may take part in the elevation of intracellular free Ca2+, CaN-MEF2c signal pathway activated by Ca2+was involved in the development of ER stress-induced cardiac hypertrophy in cardiomyocytes. Cardiac hypertrophy was a compensatory response to ER stress-induced injury, the prevention of CaN-MEF2c pathway inhibited cardiac hypertrophy induced by ER stress in cardiomyocytes and resulted in the increase of cell injury, at least in part through an ER stress-mediated cell apoptotic pathway.The conclusions were as followes:ER stress is not only involved in the deyelopment of myocardial hypertrophy induced by abdominal aortic constriction in rats, but also induces cardiac hypertrophy independently in cultured neonatal cardiomyocytes. CaN-MEF2c pathway takes part in the development of ER stress-induced cardiac hypertrophy in cardiomyocytes, and CaN activity inhibition leads to the prevention of cardiac hypertrophy and the increase of cell injury. Our findings suggest that ER stress induces cardiac hypertrophy independently and may be one of pathogenic factors of cardiac hypertrophy, thus providing new insight into the development of novel therapeutic strategies for cardiac hypertrophy.