Intranuclear Cardiac Troponin ? Regulate ATP2a2 Expression In Cardiomyocytes As A Nonclassical Transcription Factor

PARTI THE NUCLEAR TRANSLOCATION OF CTNI IN PHYSIOLOGICAL AND PATHOLOGICAL CONDITIONSObjective:Cardiac troponin I(Cardiac troponin I,cTn?),encoded by TNNI3 has been considered as an cytoplasm protein in the past.However,recently researches have proved that troponin I can enter nucleus.This experiment aims to verify the nuclear translocation of Tn? and to investigate the changes of the subcellular distribution of cTn? in physiological and pathological conditionsMethods:(1)The heart tissue of c57 mice was extracted and separated into nucleoprotein(Nu)and cytoplasm protein(Cy),and the expression of cTn? in nucleoprotein and cytoplasm protein was detected by WB.(2)The heart tissues of 11-week and 14-week human fetal were extracted and separated into nucleoprotein and cytoplasm protein,and the expression of Tn? in nucleoprotein and cytoplasm protein was detected by WB.(3)The subcellular distribution of cTnl in neonatal mice ventricular myocytes(NMVMs)were detected by immunofluorescence.(4)The heart tissue of SD rats were divided into left ventricular heart tissue and right ventricular heart tissue.Total protein,nucleoprotein and cytoplasm protein were extracted from left and right ventricular heart tissue,respectively.WB detected the subcellular distribution of cTn?.(5)SD rats were divided into Sham group(control)and transverse aortic constriction(TAC)group,of which the main symptom was left heart failure.Total protein,nucleoprotein and cytoplasm protein of left ventricular heart tissue were extracted from two group rats,respectively.WB detected the subcellular distribution of cTn?Results:(1)cTn? was detected both in nucleoprotein and cytoplasm protein of c57 mouse heart tissue.(2)Tn? was detected both in nucleoprotein and cytoplasm protein of 11-week and 14-week human fetal heart tissues.(3)In NMVMs,30.43±1.073%cTn? was localized in nucleus(4)The ratio of nuclear/total-cTn? in right ventricle heart tissue was significantly decreased compared with left ventricular heart tissue(p<0.05).(5)The ratio of nuclear/total-cTn? in TAC groups was significantly lower compared with sham groups(p<0.05)Conclusion:(1)The nuclear translocation of cTn?/Tn? was generally found in the heart tissues of mice,rats and human fetuses.(2)The changes of the subcellular distribution of cTn? in physiological and pathological conditions suggested that the intranuclear cTn? may be involved in the regulation of cardiac systolic and diastolic functionPART2 CTNI REGULATES ATP2A2/SERCA2A EXPRESSION AND ITS MECHANISMObjective:Recently,guilt of association analysis showed a strong expression correlation between TNNI3 and ATP2a2,which encodes sarcoplasmic reticulum Ca2+ ATPase isoform 2a(SERCA2a).This part of the experiment aims to verify the regulation of cTnl on ATP2a2/SERCA2a and further explore its regulation mechanismMethods:(1)c57 mice were divided into control group and over-expression group by tail vein injection of TNNI3 gene recombinant adenovirus.cTn?+/-mice and littermate cTn?+/+mice were generated by hybridization of cTnl-knockout mice and wild type mice.The transcription and translation levels of TNNI3/cTn?,ATP2a2/SERCA2a in heart tissues were detected by QPCR and WB.(2)The primary myocardial cells were divided into control group and over-expression group by infecting with TNNI3 gene recombinant adenovirus.And the cells were divided into control group and knockdown group by transfecting with siRNA-TNNI3 The transcription and translation levels of TNNI3/cTn?,ATP2a2/SERCA2a in cells were detected by QPCR and WB.(3)The beating rate and ATP content of primary myocardial cells transfected with siRNA-TNNI3 were detected.To observe the changes of intracellular calcium oscillation frequency and amplitude,we used calcium ion probe ion.Meanwhile,we treated cells with SERCA specific inhibitor Thapsigargin(TG)to detect the calcium releasing by sarcoplasmic reticulum.(4)The binding level of cTnl protein and ATP2a2 promoter region was detected by ChIP-seq technology in the heart tissue of c57 mice,and the specific binding motif of cTn? was predicted.The binding levels of each segment in the promoter region of ATP2a2 were verified by ChIP-QPCR.(5)In 293A cells,pcDNA3.1 plasmid/TNNI3 over-expression plasmid and ATP2a2 promoter luciferase reporter gene plasmid were co-transfected.The effect of cTn? on the transcriptional activity of ATP2a2 was detected by fluorescence spectrophotometerResults:(1)The mRNA and protein levels of ATP2a2 in the heart tissues of the over-expression group were significantly up-regulated compared with the control group(p<0.05,p<0.05).The mRNA and protein levels of ATP2a2 in cTn?+/-mice heart tissue were significantly lower than those in cTn?+/+mice(p<0.01,p<0.01).(2)The mRNA and protein levels of ATP2a2 in the over-expression group cells were up-regulated compared with the control group(p<0.05,p<0.05).The mRNA and protein levels of ATP2a2 in the primary myocardial cells of siRNA-TNNI3 group were down-regulated compared with the control group(p<0.05,p<0.01);(3)At 24h and 48h after transfection,the beating frequency of primary myocardial cells in siRNA-TNNI3 group was significantly lower than that in the control group(p<0.0001,p<0.0001).And total intracellular ATP content increased in siRNA-TNNI3 group(p<0.05).Compared with the control group,the intracellular calcium transient frequency was significantly reduced in siRNA-TNNI3 group,and the increment of intracellular calcium ion concentration was reduced after TG treatment compared with control.(4)Specific binding DNA sequences of cTn? were enriched in ATP2a2 promoter-239-889 region.The specific binding sequence motif of cTnl was analyzed by software as "CCAT".(5)Compared with the control group,the activity of luciferase was significantly increased in the cells transfected with pcDNA3.1-TNNI3 plasmid(p<0.0001)Conclusion:(1)Both in vivo and in vitro,cTn? can regulate the expression of ATP2a2/SERCA2a.(2)cTn? may affect intracellular calcium homeostasis by regulating the expression of ATP2a2/SERCA2a,thereby regulate the diastolic function of cardiac myocytes.(3)The regulation mechanism of cTn? on ATP2a2/SERCA2a is that the nuclear cTn? binds to the promoter region of ATP2a2,stimulating the transcriptional activity of ATP2a2.