| Objective:Mutant presenilin1(PS1)genes have been identified in patients with dilated cardiomyopathy(DCM),a common cause of heart failure and the most prevalent diagnosis in cardiac transplantation patients.However,the mechanism of PS1 gene in heart is not clear.We have employed PS1 conditional knockout(PS1 KO)mice of heart failure,from structure and function,to study the role of PS1 gene in heart development and in SR calcium homeostasis in myocardial cells,in order to understand effects of PS1 in heart failure.Methods:(1)Establishment of conditional PS1 knockout mice.PS1 Floxp mice were intercrossed with Smo2-Cre mice to obtain cardiac and vascular smooth musclespecific knockout mice.Conditional PS1 knockout mice were analyzed at 20 days.(2)Wild type mice were randomly divided into two groups: sham-operated mouse(n=60)and heart failure mouse(n=60).Conditional PS1 knockout mice were randomly divided into two groups:sham-operated mouse(n=60)and heart failure mouse(n=60).30 days after myocardial infarction(MI),survival rate,heart function and the rate of HW/BW were analyzed.(3)Mouse were deeply anesthetized with ligated left anterior descending coronary.Sham-operated mouse went the same procedure without ligation of left anterior descending coronary.(4)Analyzed of microstructure and ultrastructure of heart tissue.Heart were fixed in 4% paraformaldehyde,followed by conventional paraffin embedding,sectioning,HE staining and the microscopic observation.Heart was fixed by 4% paraformaldehyde,embedding,sectioning,staining and observation.(5)Single cardiomyocyte of the mouse,loaded by Fluo-3/AM or Fluo-5N/AM,were isolated by an enzymatic dissociation method.SR Ca2+ content can be measured by permeabilized myocytes with SR entrapped Fluo-5N/AM directly.CCE was analyzed by Laser Confocal Scanning Microscope.(6)Membrane protein was extracted in left ventricule.The levels of proteins were determined by western blot.(7)Data were expressed as mean ± SD for all the experiments.Statistical analysis were made with ANOVA and followed by LSD test for individual comparisons of means.P-values of <0.05 were considered significant.Results:(1)Establishment of conditional PS1 KO mice.We found that the expression of PS1 protein was significantly decreased in PS1 KO mouse(0.409±0.768)compared with wild type(1.000±0.432,n=3,P<0.01).(2)30 days after MI,we found that the survival rate of mice in KO-HF group is lower than others(log-rank test,P<0.05).(3)The Ejection Fraction(EF)and Fractional Shortening(FS)was significantly decreased in operated group compared with sham-operated group(P<0.01,n=12).EF and FS was significantly increased in PS1 KO group compared with W group(EF:65.568±5.5939 VS 72.411± 5.2133,P<0.05;FS:35.349±4.276 VS 40.900±4.5166,P<0.05).EF and FS was significantly decreased in KO-HF group compared with W-HF group(EF: 17.461±9.8133 VS 28.821±8.3101,P<0.05;FS:8.072±4.790 VS 13.842±4.4160,P<0.05).Compared with PS1 KO mice,IVS was significantly decreased in KO-HF group(1.396±0.1124 VS 0.7267±0.3892,P<0.01).(4)Heart/Body weight ratio were significantly smaller in PS1 KO mice than wild type mice(0.0059±0.0004 VS 0.0063±0.0006,P<0.05).(5)Effects of PS1 on microstructure and ultrastructure.Conditional PS1 knockout leads to heart wall thickening,ventricular chamber dilatation and loose arrangement of ventricular septal cell.After MI,they showed a large number of dead myocardial cell,ventricular wall thinning,increased ventricular chamber and fibrosis in W-HF and KO-HF group.Cardiac remodeling of KO-HF group is more severe.Analysis of heart ultrastructure by transmission electron microscopy revealed that conditional PS1 knockout leads to disorderly arrangment of vascular smooth muscle,swelling endothelial cells and inriched secretory vesicles.In W group,myofilaments were orderly,closely.Mitochondria contain tightly packed cristae.Compared with W group and W-HF group,mitochondria volume increased in KO and KO-HF group(P<0.05,P<0.01).In PS1 KO group,some parts of the myocardial cell appeared intermyofibrillar lysis and sparsing cardiac muscle fibers and widening sarcomere Z lines.In cardiomyocytes,PS1 knockout increased the amount of mitochondria accompanied by irregular shape in KO and KO-HF group(0.0054±0.0013 VS 0.0048±0.0012,P<0.05;0.0083±0.0022 VS 0.0049±0.0017,P<0.01).There are widening intercalated disc,slightly swollen endothelial cells and sparsing cardiac muscle fibers in KO-HF group.In W-HF and KO-HF group,mitochondria were swollen,and mitochondrial cristae were separated.Myocardial cell appeared intermyofibrillar lysis and breakage,fibrosis cells and cell disruption.(6)Isolated cardiomyocytes were loaded with Fluo-5N/AM or Fluo-3AM.The result showed that Conditional PS1 knockout decreased SR Ca2+ content(W:KO 45.417±2.939 VS 36.325±2.866,P<0.01;W-HF:KO-HF 24.908±2.3801 VS 19.342±2.6869,P<0.01,n=12).(7)Measured SR Ca2+ released.Caffeine-induced Ca2+ transients(ΔF/F0)was significantly decreased in PS1 KO group(29.861±7.0094)compared with W group(46.300±5.4057,n=12,P<0.01).Compared with W-HF group,Caffeine-induced Ca2+ transients(ΔF/F0)was significantly decreased in PS1 KO group(26.861±4.0650 VS 18.984±4.0650,n=12,P<0.05).Conditional PS1 knockout significantly decreased calcium release rate and SR Ca2+ content.The amplitude of Ca2+ transients was enhanced in KO,KO-HF and W-HF group(W:KO 9.109±4.990 VS 16.118±5.9169,P<0.01;W-HF:KO-HF 19.6947±4.668 VS 24.742±4.0223,P<0.05,n=12).(8)Analysis of Fluo-3AM fluorescence revealed that CCE was increased in PS1 KO group compared to W group.Conclusion: PS1 protein plays an important role in maintaining normal cardiac function.(1)Conditional PS1 knockout leads to abnormal development of heart,including thickening heart wall,ventricular chamber dilatation,loose arrangement of ventricular septal cell and abnormal Ca2+ handing with cardiac remodeling.(2)After MI,conditional PS1 knockout mice is more likely to develop heart failure.Thinning heart wall,ventricular chamber dilatation,abnormal structure and decreasing SR Ca2+ content weaken heart function and promote the development of heart failure. |
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