The Effect And Mechanism Of Myocyte Enhancer Factor 2A On Hyperglycemia Induced Cardiac Remodeling

BackgroundDiabetic cardiomyopathy (DCM) is characterized by diffuse cardiomyopathy diabetic cardiovascular complications, independent of coronary artery disease, hypertension, valvular heart disease and alcoholic cardiomyopathy, abnormal tissue morphology mainly for myocardial fibrosis, the collagen deposition in the extracellular matrix, and the change of the extracellular matrix collagen configuration, namely the extracellular matrix (ECM)remodeling.The main pathophysiological changes contributing to ECM include two aspects of myocardial cells and interstitial,recently studies realize that interstitial fibrosisplaya considerable rolein DCM development.The collagen inmyocardial interstitium is synthesized and secreted by fibroblasts, so cardiac fibroblasts (CFs) are the critical mediators of physiological and pathological cardiac remodeling.However, the exact molecular mechanism of DCM is not clear, the study of DCM pathological mechanisms has important significance.Muscle cells enhancement factor 2 (MEF2) is a transcriptional regulation factor, MEF2 family members include four transcription factors MEF2A, MEF2B, MEF2C, MEF2D in vertebrates, in the adult heart main expressed MEF2A and MEF2D.MEF2A gene was on chromosome 15q26 interval, which plays an important rolein regulating myoblastdifferentiation. MEF2A overexpression transgenic mice appeared dilated cardiomyopathy, which was related to ECM regulating genes.MEF2A expression increased in diabetic rat kidney, retina, myocardial cells and heart tissue, which showed that MEF2A played an important role in the pathogenesis of DCM. However, the function of MEF2A in cardiac fibroblasts has not been reported.We hypothesized that an increased level of MEF2A may facilitate hyperglycemia-induced cardiac remodeling via regulating CF proliferation, migration and activation, to affect the DCM pathological development process, which provides new therapeutic targets for DCMtreatment.Objectives1. To investigate the MEF2A expression in cardiac fibroblasts and the effect of high glucose to MEF2A expression.2. To investigate the effect of MEF2A interference onhigh glucose induced fibroblasts proliferation, migration, transformation, collagen synthesis ability and activity of matrix metalloproteinase (MMP).3. To study the mechanism of MEF2A effect cardiac fibroblasts in high glucose state.Materials and methods1. Lentiviral vectors shRNA-MEF2Aconstruction Designed 3 shRNA plasmids of MEF2A gene, analysised of interference efficiency by RT-PCR and western blot, and constructed lentiviral vectors containing green fluorescent protein (GFP).The target sequence for MEF2A (shRNA-MEF2A) was 5’-GCTTGCCACCTCAGAACTTCT-3’and the negative control sequence (shRNA-NC) was 5’-TTCTCCGAACGTGTCACGT-3’.2. Primary mouse CF culture and treatmentsCFs were isolated from ventricular tissues of 1-to 3-day-old neonatal mice.CFs were purified by gradientadherent method and identified by immunofluorescence.The CFs were treated with normal glucose, osmotic control and high glucose at different time points, and observed the expression of MEF2A. The CFs were infected with lentiviruses carrying shRNA-MEF2A and shRNA-NC before high glucose treatment, and observed the effect to CFs action.3. Cell ImmunofluorescenceThe distribution and expression of MEF2A, p-MEF2Aand α-SMA were detected by cell immunofluorescence.4. Western blot analysisCollected cells, extracted the protein and detected the expression of MEF2A, p-MEF2A, α-SMA, collagen Ⅰ, collagen Ⅲ, MMP2, MMP9, TIMP1, TIMP2, TGF-β1, p38 MAPK, p-p38 MAPK, JNK, p-JNK, ERK1/2, p-ERK1/2, Akt, p-Akt and β-actin.5. Quantitative RT-PCRCollected cells, extracted RNA, reverse transcribed and RT-PCR detected the MEF2A mRNA expression in all group cells. 6. Measurement of CF proliferation Proliferation was analyzed by using Cell Counting Kit-8 (CCK-8) and Cell-LightTMEdU assay according to the manufacturer’s directions.7. Measurement of CF apoptosisApoptosis was detected by using a Phycoerythrin (PE)-Annexin V Apoptosis Detection Kit according to the manufacturer’s directions.8. Cell migration assays Cell migration was examined by scratch assay and Transwell migration assay.9. Gelatin zymographyMMP2 and MMP9 activity was detected bygelatin zymography 10. Statistical analysisSPSS v16.0 was used for analysis. The data are expressed as the mean±standard deviation (SD). One-way ANOVA was used to compare differences among groups, followed by Tukey’s test (2-tailed).Results1. HG increases MEF2A expression and translocation in CFsWestern blot analysis showed that MEF2A protein expression in the CFs increased after 6 h of 33 mM glucose treatment and peaked at 48 h as compared with NC and OC. Immunofluorescence andWestern blot analysis revealed that HG made MEF2A diffuse from nucleus to cytoplasm. Further, HG increased only nuclear p-MEF2A expression and did not affect p-MEF2A translocation.2. MEF2A inhibition decreases HG-induced proliferation of CFs and is not associated with apoptosisCCK-8 assay showed that MEF2A silencing significantly reduced HG-induced CF proliferation at alltested time points. The proliferative effect of MEF2A was confirmed by EdU staining. Further, flow cytometric analysis indicated that MEF2A inhibition had no effect on CF apoptosis.3. MEF2A inhibition limits HG-induced CF migration Scratch and Transwell migration assays showed MEF2A inhibition significantly suppressed CF migration to the level of the controls at all time points tested as compared to HG group.4. MEF2 A inhibition attenuates differentiation of CFs into myofibroblasts a-SMA was the markerof CFsdifferentiation into myofibroblasts.Western blot and immunofluorescence analysis showed that MEF2A inhibition significantly suppressedthe increase in a-SMA expression induced by HG.5. MEF2A mediates HG-induced imbalance of MMP-TIMP and collagen synthesis in CFsMEF2A inhibitionsignificantly attenuated HG induced an increase in MMP2 and MMP9 protein expression and activity, and had nomarkedly effect on expression of TIMP1 and TIMP2. In line with the altered MMP activity, collagen I and III protein expression was significantly increased in the HG group, while shRNA-MEF2A treatment inhibited the effect.6. MEF2A inhibition alleviates HG-induced Akt and TGF-β1/Smad signaling pathway activation in CFsCellular levels of activated MAPKs, Akt, TGF-β1, and Smad2/3 were enhanced after HG stimulation as compared with NC or OC treatment.MEF2A inhibition further increased phosphorylation of p38 in CFs, while it reduced HG-induced activation of Akt and TGF-β1/Smad, and had no effect on the activation of ERK1/2 and JNK under HG treatment.Conclusion1. HG increases MEF2A expression and diffuse from nucleus to cytoplasm, increased nuclear p-MEF2A expression in CFs.2. MEF2A inhibition alleviates HG-inducedproliferation, migration, differentiation, MMP and collagen synthesis.3. Inhibition MEF2A improved HG-induced CFs function change via Akt and TGF-β1/Smad signal pathway.BackgroundDiabetes mellitus (DM) can affect cardiac structure and function and lead to heart failure in the absence of atherosclerosis and hypertension, which is called diabetic cardiomyopathy (DCM). DCM is one of the leading causes of increased morbidity and mortality in the diabetic population. Myocardial fibrosis is one of the most common pathological changes, the detailed mechanism responsible for this change remains unclear. Given the increased risk of heart failure in diabetic patients, a better understanding of the underlying mechanisms and additional therapeutic strategies would be of considerable value.Studies have shown that endothelial-to-mesenchymal transition (EndMT) plays a vital role in myocardial fibrosis. EndMT is considered to be a driving process resulting in the trans-differentiation of endothelial cells into mesenchymal cell types, characterized by a loss of cell-cell adhesion and a change in cell polarity and accompanied by a reduction in endothelial marker expression, and an increase in mesenchymal marker expression. EndMT is stimulated by TGF-β2 through Smad, MEK (MAPK [mitogen-activated protein kinase]/ERK [extracellular signal-regulated kinase]), PI3K (phosphoinositide 3-kinase), and p38 MAPK signaling pathways. Inhibitors of these pathways prevent TGF-(32-induced EndMT. Increasing evidence has shown that high glucose levels can induce EndMT. However, the factors regulating EndMT under pathologic conditions of high glucose are not clear and remain to be elucidated.Myocyte enhancer factor 2A (MEF2A) belongs to a family of transcription factors. MEF2A functions during fetal development of the cardiovascular system and controls cell proliferation, differentiation, and death in both the developing fetus and the adult. MEF2A is expressed in endothelial cells and is closely associated with angiogenesis. The overall expression pattern of MEF2A is similar to vascular endothelial growth factor receptor 2(VEGFR2) and Von Willebrand factor.In addition, some studies indicated that bone morphogenetic protein 2 (BMP-2), Smad2, MAPKs p38 and ERK5 interacted with MEF2A. Meanwhile, these proteins are important signal molecules of signaling pathways, which regulate EndMT. Therefore, we hypothesized that administration of MEF2A might achieve cardioprotective effects against fibrosis in diabetic hearts partially by regulating EndMT. To support our hypothesis, we performed a series of experiments both in vivo and vitro.Objectives1. To investigate the influence of MEF2A to high glucose-induced EndMT.2. To investigate themechanism of MEF2A regulating high glucose-induced EndMT.3. To study the effect of MEF2A to cardiac fibrosis and cardiac dysfunction in diabetic mice.4. To study effect of MEF2A to EndMT of cardiac micro vascular endothelial cell in diabetic mice.5. To investigate whether MEF2A influenced cardiac fibrosis and cardiac dysfunction of diabetic cardiomyopathy by regulating EndMT.Methods1. Construction of lentiviral-mediated MEF2A interference vector A lentivirus vector containing a U6 promoter upstream of the cloning site was used for cloning short-hairpin RNAs (shRNAs) of MEF2A interference and a green fluorescent protein (GFP) reporter.2. Cell culture and high-glucosecell modelHuman umbilical vein endothelial cells (HUVECs) were cultured and 33 mmol/L and 5 mmol/Ld-glucosewas used to induce high-glucose reaction in HUVECs in our experiments.3. Interference of cell gene expression:In vitro, lentivector with MEF2A shRNA interference was performed to inhibit MEF2A expression in HUVECs; The inhibitor was performed to inhibit phosphorylation reactionof p38MAPK. Transient transfection with Smad2-siRNAwas used to inhibit Smad2 expression.4. Co-Immunoprecipitation (Co-IP)Co-IP was used to test the interaction between MEF2A and Smad2 under high-glucose state in HUVECs.5. Duolink(?) in situ-proximity ligation assayDuolink(?) in situ-proximity ligation assay also was used to test the interaction between MEF2A and Smad2 under high-glucose state in HUVECs.6. Animal model and RNA interferenceC57BL/6J micewas induced type 1 diabetes by injecting streptozotocin (STZ). Control mice were injected with citrate buffer only. After one week, random glucose>20 mmol/L was determined as diabetes mice. After seventeen weeksinjecting STZ, mice were divided into 3 groups:diabetes (DM), DM+shRNA-NC, and DM+shRNA-MEF2A. In DM+shRNA-MEF2A and DM+shRNA-NC group, lentivector (2×107 UT/30μL) with MEF2A-shRNA or the same volume of lenti-vehicle was injected into three sites of the left ventricle. Keep feeding for 4 weeks, the mice were anesthetized and killed for further studies.7. Cardiac structure and function measurementCardiac structure andfunction was measured under 2.0% isoflurane anesthesia by transthoracic echocardiography. M-mode tracing, pulsed-wave Doppler and tissue Dopplerechocardiography were used to detectsystolic and diastolic function.8. Endothelial cells isolated from the heartMice cardiac cells were dissociated using a gentle MACS Dissociator (MiltenyiBiotec) as described by the manufacturer. Endothelial cells were isolated using positive selection by magnetic affinity cell sorting using a CD146 antibody.9. Histological analysisMasson’s trichrome (MTC) and Picrosirius red staining in heart sections were used to evaluate cardiac fibrosis.10. Immunohistochemistry Immunohistochemistry was used to measure the levels of collagen I and collagen III in myocardial tissues.11. Immunocytochemistry and Immunohistochemistry: Immunocytochemistrywas used to determine the expression and localization of MEF2A, CD31, VE-cadherin, FSP1, a-SMA, p38MAPK and Smad2. Expression and localization of CD31, S100A4 and a-SMA in myocardial tissue were observed using immunohistochemistry methods.12.SYBR Green RT-PCRSYBR Green RT-PCRquantitative assays the mRNA of MEF2A in myocardial tissue and CD31, VE-cadherin, FSP-1, a-SMA in endothelial cells isolated from the heart.13. Western blot analysisWestern blot analyzed the protein expression of MEF2A, CD31, VE-Cadherin, S1004A/FSP-1, vimentin, a-SMA, p38MAPK, phospho-p38MAPK, Smad2, phospho-Smad2 in myocardial tissue and HUVECs.14. Statistical analysisResults are presented as mean±SEM. Statistical analyses were performed one-way ANOVA test for multiple-group comparisons. A value of p< 0.05 was considered statistically significant.Results1. The interference efficiency of Lentivirus transfected HUVECsGFP-labeled transfection efficiency in HUVECs was more than 95%, RT-PCR and Western blot analyzed the interference efficiency, which was 0% he 20%2. MEF2A interference inhibits high glucose (HG)-induced EndMT in HUVECs.HUVECs were treated with 33mmol/L d-glucose with or without ShRNA-MEF2A for 5 days. Fluorescence microscopy revealed that control HUVECs showed a typical rounded or cobblestone shapes, and MEF2A silencing inhibited the change from a cobblestone-like to spindle-shaped feature with HG induction. Immunofluorescence and western blot analysis demonstrated that HG-treatment significantly increased the levels of mesenchymal markers FSP1, a-SMA, and vimentin and caused a reduction in the levels of endothelial markers CD31 and VE-Cadherin as compared with controls. However, the features above were alleviated when MEF2A was knocked down.3. HG-induced EndMT is mediated by the translocation of MEF2A to the cytoplasm and interactions with p38MAPK and Smads in HUVECsImmunofluorescence and western blot analysis revealed HG induced relocation of MEF2A in the cytoplasm of HUVECs, but the phosphorylation state of MEF2A located in the nucleus consistently and HG induced phosphorylation of MEF2A increasing. Conversely, Western blot analysis showed inhibition of phosphorylation of p38 by inhibitor SB203580 was sufficient to prevent the increase of MEF2A and p-MEF2A induced by HG.CO-IP showed there was interaction between MEF2A and Smad2. And PL A analysis revealed that the interaction between MEF2A and Smad2 localized in the nucleus in control group, and localized in the cytoplasm in HG group. Western blot analysis showed that HG increase Smad2 phosphorylation in HUVECs as compared with control group, while MEF2A knockout made Smad2 phosphorylation declined. Conversely, MEF2A expression decreased after smad2 knockdown by si-RNA. FSP-1 and a-SMA decreased, and CD31 and VE-Cadherin increased after smad2 knockdown before HG treatment.4. Injection with LV-shRNA-MEF2A in the myocardium could reduce the expression of MEF2A in the myocardium of diabetic mice.RT-PCR and western blot analysis showed that LV-shRNA-MEF2A interference made MEF2A mRNA and protein reduce 30% and 35% in the myocardium of diabetic mice.5. The influence of MEF2A inhibition to blood glucose, body weight, heart weight, and HW/BW of mice.Blood glucose, body weight, heart weight, and the ratio of heart weight to body weight were measuredin the 4 groups twenty-one weeks post-STZ injection. Body weight (g) and heart weight (mg) were lower in diabetic mice than in control mice. However, the ratio of heart weight to body weight (mg/g) was significantly higher in diabetic mice than in control mice. Heart weight and the ratio of heart weight to body weight were lower in shRNA-MEF2A treatment mice than in vehicle treatment mice. There was’t significantly differ in body weight between ShRNA-MEF2A and vehicle-transfected diabetic mice. These results showed that MEF2A inhibition could reversediabetic mice’s cardiac remodeling.6. MEF2A inhibition attenuates diabetes-induced cardiac structural and functional changesEchocardiogram was performed to detect the cardiac function at 21 weeks after STZ injection. Cardiac function features were worse in diabetic than normal mice: LVEF, FS, E/A ratios, E’/A’ratios and E/E’. As compared with MEF2A-LV interference NC treatment, MEF2A-LV interference treatment improved the attenuated LVEF, FS, E/A ratios, and E’/A’ratios in diabetic mice. LVPWd was significantly increased in diabetic mice, and MEF2A-LV interference treatment attenuated the wall thickness. LVEDD in DM group decreased, whereas there was no statistically significant difference.7. MEF2A inhibition improves diabetes-induced cardiac fibrosisMTC and Picrosirius red staining of heart showed an increase in intramyocardial and perivascularfibrosisof the diabetic mouse myocardial tissue. Quantitative analysis of MTC staining showed that diabetic mice had a 2.60- and 2.05-fold increase in collagen deposition in the intramyocardial and perivascular regions as compared with control mice. ShRNA-MEF2A treatment reduced diabetic induced collagen deposition as compared with vehicle treatment.8. MEF2A inhibition limits diabetes-induced deposition of collagenⅠ and collagenⅢThe immunohistochemistry data and western blot analysis showed that:diabetes enhanced the expression of collagen I and III as compared with the control group, whereas shRNA-MEF2A transfection in diabetic mice significantly reduced collagenⅠ and collagenⅢ levels as compared with vehicle treatment.9. MEF2A knockdown inhibits myocardial fibrosis partially by suppressing EndMT in vivoThe double-positive immunofluorescence staining was examined by z-stack image analysis, which confirmed the specific overlay of CD31+/S100A4+and CD31+/a-SMA+cells, which showed that double-positive cell numbers is significantly increased in the diabetic mice compared with the control mice Moreover, the shRNA-MEF2A transfection in diabetic mice significantly reduced the double-positive cell numbers as compared with vehicle treatment. The immunohistochemistry data were confirmed with RT-PCR analysis results in cardiac endothelial cells by magnetic affinity cell sorting using a CD 146 antibody.Conclusion1. High glucose induced MEF2A expression increase and translocation from nucleus to cytoplasm in HUVECs.2. MEF2A interference inhibited high glucose (HG)-induced EndMT in HUVECs.3. HG-induced EndMT is mediated by the translocation of MEF2A to the cytoplasm and interactions with p38MAPK and Smads in HUVECs.4. MEF2A knockdown inhibited cardiac remodeling, dysfunction and myocardial fibrosis in diabetes mice heart.5. MEF2A knockdown improved cardiac parameters partially by suppressing EndMT.