ABSTRACT
Objective To investigate the effect of Wnt5a on inducing differentiation of Cp15-5a cell to myocardial cell.Methods Recombinant adenovirus wnt5a (Ad-wnt5a) and Ad-GFP was amplified with human embryo kidney 293 cells (HEK293 cells),and then transfected into CP15-5a cells and 3 experiment groups were set up:wnt5a group,GFP group and blank control group.Flow cytometry was used to detect the transfection efficiency of Ad-wnt5a and Ad-GFP.One week after transfection,the expressions of genes GATA binding protein 4 (GATA4) and myocardial enhancement factor 2C (MEF2C) were analyzed by realtime quantitative PCR (qRT-PCR).Two weeks after transfection,the expressions of cardiac-specific connexin 43 (Cx43) and cardiac troponin T (cTnT) in Ad-wnt5a-induced CP15-5a cells were detected by Western blotting and immunofluorescence techniques.The sodium current expression (INa) was detected by whole cell patch clamp techniques.Results The transfection efficiency of Ad-wnt5a and Ad-GFP was 42.8％ and 44.3％,respectively.One week after transduction,the expressions of GATA4 and MEF2C were significantly higher in wnt5a group (1.717 ± 0.220 and 1.847 ± 0.190) than in GFP group (1.003 ± 0.087 and 0.456 ± 0.042,P＜0.05) and blank control group (0.961 ± 0.063 and 0.500 ± 0.095,P＜0.05),while no significant difference existed between GFP group and blank control group.Two weeks after transduction,the expressions of CX43 and cTnT were significantly higher in wnt5a group (1.597 ± 0.267 and 0.727 ± 0.100) than in GFP group (0.723 ± 0.047 and 0.217 ± 0.021,P＜0.05) and blank control group (0.783 ± 0.1333 and 0.253 ± 0.102,P＜0.01),while no significant difference existed between GFP group and blank control group.INa was detected in the wnt5a group compared with GFP group and blank control group.Conclusion wnt5a may induce differentiation of cp 15-5a cell into myocardial cell.
ABSTRACT
Objective To explore whether suberoylanilide hydroxamic acid (SAHA,br.Vorinostat,a kind of histone deacetylase inhibitor) can improve the diastolic function of restrictive cardiomyopathy (RCM) mice by up-regulating the expression of wild type cardiac troponin I (WT-cTnI).Methods Sixteen male cTnI R193H mice were employed and divided into SAHA group (n=6),dimethyl sulfoxide (DMSO) group (n=5) and control groups (n=5).Mice were subcutaneously injected with 50mg/kg SAHA in SAHA group,with 1ml/kg DMSO in DMSO group and with 1ml/kg normal saline in control group,respectively.The total treatment duration lasted for 56 days.Western blotting was performed to determine the levels of acetylated histone H3 (acH3) and the expression of cTnI.The transcription levels of Tnni3 were detected with RT-qPCR.The levels of acH3 in Tnni3 promoter key area were detected with chromatin immunoprecipitation (ChIP).The data of diastolic function measurements were collected using high frequency echocardiography.Results Compared to control group,the acH3 levels in nucleus and in Tnni3 promoter key area increased significantly in SAHA group (P＜0.05).The acH3 level showed no significant difference between SAHA group and DMSO group (P＞0.0S),but the specific acH3 level in Tnni3 promoter key area increased significantly in SAHA group compared to that in DMSO group (P＜0.05).The expression of cTnI protein and Tnni3 mRNA showed no significant difference among the 3 groups.Compared to the control group,Tei index and E/A ratio increased in DMSO group.Left ventricle fractional shortening (LVFS),left ventricle ejection fraction (LVEF),stroke volume (SV),cardiac output (CO),isovolumic relaxation time (IVRT),E peak deceleration time (DT),early diastolic blood flow (E velocity) and late diastolic filling flow (A velocity) showed no significant difference between DMSO group and control group.IVRT,DT and Tei index increased,and E velocity and A velocity decreased in SAHA group compared to that in control group.Furthermore,LVFS,LVEF,SV and CO were decreased in SAHA group compared to that in control group (P＜0.05).Similarly,IVRT increased (P＜0.05) and LVFS,LVEE CO,E velocity and E/A ratio decreased in SAHA group compared to that in DMSO group.Conclusions SAHA may up-regulate the acH3 level in GATA and MEF2 binding site of Tnni3 promoter of RCM mice.However,SAHA may have no effect on the expression and transcription of Tnni3 in heart.Further studies are needed to confirm whether SAHA has any toxic effect on cardiac function.
ABSTRACT
Objective To isolate and identify the proteins that interact with cardiac troponin I (cTnI) in primary mouse cardiomyocytes,and to analyze their biological processes and signaling pathways by use of High throughput proteomics and bioinformatics techniques.Methods The hearts of C57 neonatal mice were used to prepare the primary cardiomyocytes.The cTnI binding proteins were extracted from the whole cell protein by co-immunoprecipitation,and the proteins were analyzed and identified by mass spectrometry.Furthermore,the physicochemical properties,biological processes and signaling pathways of cTnI interacting proteins were predicted by bioinformatics tools.Results A total of 262 proteins were identified by mass spectrometry,cTnI binding proteins were involved in 14 biological processes and 33 KEGG biological pathways (P＜0.05).Conclusion In mouse cardiomyocytes,262 kinds of proteins may interact with cTnI,and participate in regulation of calcium ion influx and PI3K-Akt signaling pathways.