| Aim:Myocardial I/R injury severely affects the prognosis of patients with coronary heart disease,and there are no effective treatment measures.Calcium overload is one of the mechanisms of I/R injury,and sarcoplasmic reticulum calciun ATPase 2a is an important calmodulin.The results of the previous study showed that I/R injury reduced the expression and activity of SERCA2a SUMOylation.Overexpression of Sumo 1 enhanced SERCA2a SUMOylation and reduced calcium overload,but I/R injury caused the unclear mechanism of SERCA2a SUMOylation reduction.This topic is intended to explore the mechanism of SERCA2a SUMOylationreduction in myocardial I/R injury at both cellular and vivo levels.Method:1)Study the regulation of calcium homeostasis by SERCA2a protein in I/R injury(n=3).SERCA2a KO mice were prepared using SERCA2aflox mice.SERCA2a KO mice and wild-type C57BL/6J mice were used to construct myocardial I/R injury model.Langendoff method is used to isolate ischemic myocardium.The single cell calcium transient and systolic and diastolic function were measured to observe the effect of SERCA2a on the regulation of myocardial calcium homeostasis and on myocardial contraction and diastolic capacity.2)Study the impact of I/R injury on the SUMO system(n=3).C57BL/6J mice,isolated single cardiomyocytes and HL-1 atrial myocytes were used to construct myocardial I/R injury model and sI/R injury model at different time points(Sham,I/ROh,I/R 2h,I/R 6h,I/R12h,I/R3d).RT-q PCR and WB were used to detect the expression levels of Sumo1,Ubc9,Senp 1,Senp2 and protein at each time points after I/R injury.The spatial localization of Sumo 1,Ubc9,Senp1,Senp2,SERCA2a was detected by laser confocal microscopy and immunofluorescence.3)Study the mechanism with which I/R injury led to a reduction in of SERC2a SUMOylation(n=3).Adenovirus and control virus over-expressing SENP1 and SENP2 proteins were constructed to infect myocardium and the HL-1 atrial myocytes.We established s1/R injury model of HL-1 atrial myocytes.Annexin V-APC/7-AAD staining was used to detect the apoptosis rate of cardiomyocytes by flow cytometry.Intramyocardial injection of adenovirus overexpressed SENP1 protein and SENP2 protein,and 3days model was constructed.SERC2a SUMOylation was detected by immunoprecipitation.TTC-Even blue detected myocardial infarct size.Doppler echocardiography was performed to detect cardiac function.GraphPad Prism 5.0 statistical analysis software was used to analyze the experimental data.Measurement data were expressed as mean ± standard error.The pairwise comparison was performed using t test.Comparison between multiple groups was performed using one-way analysis of variance analysis,p<0.05 considered statistically significant.Results:1.Compared with Sham group,the expression of SERCA2a mRNA and protein in I/R group decreased(p<0.01);Compared with WT group,the SERCA2a mRNA decreased to 11.41 ±9.55%in SERCA2a KO group,and SERCA2a protein expression decreased to 5.6±1.38%in WT group.SERCA2a knockout mice were successfully prepared.The results of calcium transients showed that compared with WT group(0.34±0.17),the amplitude of ? FFI in I/R group(0.18±0.09)was decreased(p<0.05),? FFI in SERCA2a KO group(0.27± 0.11)was not significantly decreased;compared with SERCA2a KO group,SERCA2a KO+I/R group(0.06 ± 0.12)decreased,compared with I/R group,SERCA2a KO+I/R group decreased(p<0.001).The results of single cardiomyocyte contractile function test showed that compared with WT group(4.13 ±2.37 ? m),the contraction amplitude of single group I/R cells(1.71 ±0.97 ? m)decreased(p<0.01),SERCA2a KO group(4.23 ±1.84 ? m)did not significantly reduce,and that of SERCA2a KO+I/R group(0.62 ± 1.65 ? m)decreased(p<0.001);compared with SERCA2a KO group,SERCA KO+I/R group decreased(p<0.01);compared with I/R group,SERCA KO+I/R group decreased(p<0.05).2.1n vivo and in cardiomyocytes,Sumo1 mRNA and protein expression decreased from 12h to 3d compared with that in Sham group(p<0.01).The expression of Senpl and Senp2 mRNA was highest at 6h,and it began to decline at 12h and lasted to 3d(p<0.05).There was no statistical difference between Ubc9 groups.In HL-1 atrial myocytes,Sumo1,Senp1,Senp2 mRNA and protein expression increased to the highest level at 12 h after sI/R,and the expression decreased at 3d(p<0.01).There was no statistically significant difference between Ubc9 and control groups.The results of immunofluorescence showed that SENP1 protein and SENP2 protein were distributed in cytoplasm and nucleus of cardiomyocytes,SENP1 and SERCA2a protein colocalized in T tube,SENP2 expresion increase after I/R in cytoplasm,while the expression of SENP1 did not appear to increase after I/R injury.3.1)Adenoviral vectors over-expressing and inhibiting SENP1 and SENP2 were used.WB results showed that compared with Sham group,the expression of SENP 1 and SENP2 protein increased in Ad-Senp1+I/R and Ad-Senp2+I/R groups(p<0.01).The inhibition efficiency was the highest in Ad-Senpl-shRNA3+I/R,Ad-Senp2-shRNA3+I/R groups(p<0.01).2)Co-immunoprecipitation results showed that compared with Sham group,I/R group,GFP+I/R group,RFP+I/R group decreased SERCA2a SUMOylation(p<0.001),Ad-Senpl+I/R group and Ad-Senp2+I/R further reduced SERCA2a SUMOylation(p<0.05).Compared with I/R group,GFP +I/R group,and RFP +I/R group,no increase was observed in the Ad-Senpl-shRNA3+I/R group and SERCA2a SUMOylation was increased in theAd-Senp2-shRNA3+I/R group.3)Annexin V-APC/7-AAD staining combined with flow cytometry showed that compared with Sham group,the apoptosis rate of HL-1 cells in I/R group,GFP+I/R and RFP+I/R increased to 33.43 ±3.30%,37.05 ±3.88%,34.1 ± 1.99%(p<0.001).The apoptosis rate of Ad-Senpl+I/R group was 53.20±2.23%(p<0.001).Compared with Ad-Senpl+I/R group,Ad-Senpl-shRNA3+I/R group decreased the apoptosis rate,reaching 32.6±1.11%(p<0.001);but with I/R group,GFP+I/R group,RFP+I/R group,the Ad-Senpl-shRNA3+I/R group did not reduce the apoptotic rate.The apoptosis rate of Ad-Senp2+I/R group increased further to 79.20±4.91%(p<0.001)Compared with group I/R group,GFP+I/R group and RFP+I/R group,the apoptosis rate of Ad-Senp2-shRNA3+I/R group decreased(p<0.001).4)TTC-Even blue staining showed that compared with Sham group(5.99± 1.25%),AAR/(AAR+ANAR)× 100%of heart in I/R group,GFP+I/R and RFP+I/R increased to 38.72±2.64%,34.02±4.55%,38.99±2.10%(p<0.001).AAR/(AAR+ANAR)×100%of Ad-Senpl+I/R group and Ad-Senp2+I/R were 59.18±0.95 and 64.01±3.83%(p<0.001);but with I/R group,GFP+I/R group,RFP+I/R group,the Ad-Senp1-shRNA3+I/R group did not reduce AAR/(AAR+ANAR)x 100%.The AAR/(AAR+ANAR)x 100%of Ad-Senp2-shRNA3+I/R group decreased 26.20±1.95%(p<0.001).5.LV functions of mice were deteched by echocardiography.Compared with Sham group(66.35±1.728%),EF of heart in I/R group,GFP+I/Rgroup and RFP+I/R group decreased to 39.96 ±6.70%(p<0.001),50.02±4.70%(p<0.05),46.35 ±4.67%(p<0.01).Compared with I/R group,GFP+I/Rgroup and RFP+I/R group,EF of Ad-Senpl+I/R group was 54.18±1.01%(p>0.05),Ad-Senpl-shRNA3+I/R group was 53.85 ± 1.35%(p>0.05).Compared with I/R group,GFP+I/Rgroup and RFP+I/R group,EF of Ad-Senp2+I/R group decreased to 28.96 ±2.22%(p<0.05);Compared with Ad-Senp2+I/R group,EF of Ad-Senp2-shRNA3+I/R group increase to 60.19±7.15%(p<0.01).Compared with Sham group(35.92± 1.36%),FS of heart in I/R group,GFP+I/Rgroup and RFP+I/R group decreased to 19.16±3.54%(p<0.001),25.13.02±3.08%(p<0.05),22.88 ±2.60%(p<0.01).Compared with I/R group,GFP+I/Rgroup and RFP+I/R group,FS of Ad-Senpl+I/R group was 27.03 ±2.25%(p>0.05),Ad-Senpl-shRNA3+I/R group was 27.36±0.74%(p>0.05).Compared with I/R group,GFP+I/Rgroup and RFP+I/R group,FS of Ad-Senp2+I/R group decreased to13.14±1.14%(p<0.05);Compared with Ad-Senp2+I/R group,FS of Ad-Senp2-shRNA3+I/R group increase to31.38±4.80%(p<0.01)(p<0.01).Conclusion:1.I/R injury reduced calcium transients and contraction amplitude of individual cardiomyocytes.SERCA2a knockdown further reduced the calcium transients of cardiomyocytes and the amplitude of contraction of single cardiomyocytes.2.It was confirmed at the level of vivo and cardiomyocytes that I could decrease the expression of Sumo1 mRNA and protein at 12 h and increase Senp1 and Senp2 mRNA and protein expression at 6 h;the mRNA and protein expressions of Sumo 1,Senpl,and Senp2 in HL-1 cells increased to the highest at sI/R 12h and decreased at 3d.Meanwhile,it was confirmed that SERCA2a and SENP1 colocalized in the T-tube of cardiomyocytes,and SENP2 protein in the myocardium after I/R injury.3.Overexpression of SENP1 protein and SENP2 protein confirmed that the protein level of SENP1 and SENP2 could be decreased by I/R injury,and the effect of SENP2 protein was stronger than that of SENP1 protein.Inhibition of SENP2 protein can restore I/R injury-induced SERCA2a SUMOylation,decrease apoptosis,and reduce the area of myocardial infarction,improve cardiac function,while inhibition of SENP1 protein cannot restore the above indicators.Therefore,I/R injury activates SENP1 and SENP2 proteins expression which promote SERCA2a DeSUMOylation,inhibits SENP2 to restore SERCA2a SUMOylation,which can decrease apoptosis rate,reduce myocardial infarct size and improve cardiac function. |
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