Hypoxia / Oxygen Reperfusion Induced Myocardial Cell Aging Mechanisms And Intervention Studies

BACKGROUNDCellular senescence has been defined by Hayflick as the ultimate andirreversible loss of replicative capacity occurring in primary somatic cellculture. After Hayflick identifying senescence in human fibroblast firstly,cellular senescence was identified in diverse type cells in human orfibroblasts in other species. Different markers of the senescent phenotypeas, for instance, G0/G1 specificity of cell cycle arrest,senescence-associatedβ-galactosidase activity, mitochondrialdehydration, or senescence-associated gene expression. Cellularsenescence may not only result in the aging of organism, but induceoccurrence of tumors, heart failure, atherosclerosis or arrhythmia.Therefor, it is very important to study the mechanism of cellularsenescence.Hypoxia/reoxygenation (H/R) was the most common injury factorfor cell, tissue or organism. It was also an effective way to induce cellularsenescence. Recetenly, a report has shown that premature senescencecould be induced by H/R in FA bone marrow hematopoietic cells.HMG-CoA reductase inhibitors, i.e. statins, were reversibleinhibitors of the rate-limiting step in cholesterol biosynthesis and weregenerally used for the treatment of hypercholesterolemia. Statins also could reduce mortality for patients with coronary heart disease, increasesurvival for patients with heart transplantation, and improve ventricularremodeling. Besides regulating lipid metabolism, recently, Assmusreported that atorvastatin could reduce senescence and increaseproliferation of endothelial progenitor cells via regulation of modulationexpression of cell cycle genes including upregulation of cyclins anddownregulation of the cell cycle inhibitor p27Kipl. Apart from statinsplay very important role in protection for patients with cardiovasculardiseases, whether pravastatin could inhibit premature senescence ofneonatal SD rat cardiomyocytes induced by H/R or not?In this study, we would investigate whether premature senescencecould be induced by H/R in neonatal SD rat cardiomyocytes. To explorecellular and molecular mechanisms of premature senescence in neonatalrat cardiomyocytes exposed to H/R. To investigate whether pravastatincould inhibit premature senescence of neonatal SD rat cardiomyocytesinduced by H/R and assosiated mechanisms.Partâ… Isolation, culture, purification and identification ofneonatal cardiomyocytes from SD rat heart. Establishmentthe premature senescence model of cardiomyocytes inducedby H/R AIMOn the base of succeeding in isolation, culture neonatalcardiomyocytes from SD rat heart, to explore how to get morecardiomyocytes. To investigate whether premature senescence could beinduced by H/R in neonatal SD rat cardiomyocytes.METHODSTrypsin was used to cut pieces of heart tissue into single cells.Combination diferent time for cells to stain wall and mediumsupplemented with BrdU to remove cardiac fibroblasts.Cardiomyocytes were isolated from neonatal SD rat heart andidentified by a-actin immunohistochemistry. The control cultures wereincubated at 37℃in humidified atmosphere of 5ï¼…CO₂, 95ï¼…air. Thehypoxic cultures were (within a modular incubator chamber filled with 1ï¼…O2, 5ï¼…CO2, and balance N2) for 6 h. The reoxygenated cultures weresubjected to 1ï¼…O2, 5ï¼…CO2 for 6 h then 21ï¼…oxygen for 24 and 48 h,respectively. Cell proliferation was determined using BrdU labelling.Ultrastructure of cardiomyocytes was observed by using electronmicroscope. Flow cytometry were used to investigate alteration of cellcycle.β-galactosidase activity was determined by using Senescenceβ-galactosidase Staining Kit.RESULTSMost cells (＞95%) in the isolated cultures were positive forα-actin antibody. The percentage of BrdU positive cells reduced significantly inH/R treated group (P＜0.01). Under the condition of H/R, mitochondrialdehydration appeared. Most cardiomyocytes resided in G0 and G1 phase inthe group of hypoxia 6 h, reoxygenation 24 and 48 h compared withcontrol one (P＜0.01). Cardiomyocytes did not stain forβ-galactosidasein the group of control, hypoxia 6 h, but did intensely for it inreoxygenation group of 24,48 h, compared with control one (P＜0.01).CONCLUSIONS1. Combination diferent time for cells to stain wall and mediumssupplemented with BrdU to remove cardiac fibroblasts are a good wayto purify cardiomyocytes.2. Premature senescence could be induced in neonatal SD ratcardiomyocytes exposed to H/R.Partâ…¡A study on mechanism of premature senescence ofcardiomyocytes induced by H/RAIMTo explore cellular and molecular mechanisms of prematuresenescence in neonatal rat cardiomyocytes exposed to H/R. METHODSReal-time quantitative PCR and western blot were used to analyzemRNA and protein level of cyclin D1, cyclin dependent kinases (CDK4),inhibitors of the cyclin-CDK complexes (p16IN4Ka, p21WAF1), pRb,p53 in cardiomyocytes of neonatal SD rat in the group of control, hypoxia6h, reoxygenation 24 and 48 h.RESULTSCyclin D1 mRNA and protein levels significantly increased in H/Rtreated group compared with control one (P＜0.05 or P＜0.01,respectively).CDK4 mRNA and protein levels significantly decreased in H/Rtreated group compared with control one (P＜0.05 or P＜0.01,respectively). p21WAF1 inhibited cell cycle through reducing expressionof CDK4. p21WAF1 mRNA and protein levels increased significantly inH/R treated group compared with control one (P＜0.05 or P＜0.01,respectively). Paralleled to the change of p21WAF1, the mRNA andprotein levels of p53 were rosen significantly in H/R treated groupcompared with control one (P＜0.05 or P＜0.01, respectively).Rb mRNA level increased significantly in H/R treated groupcompared with control one (P＜0.05, respectively). But pRb leveldecreased significantly in H/R treated group compared with control one(P＜0.01, respectively), p16IN4Ka mRNA and protein levels, which prevented CDK4/6 to catalyze phosphorylation of Rb, were rosensignificantly in H/R treated group compared with control one (P＜0.01,respectively).CONCLUSIOSH/R inhibited cell proliferation and accelerated prematuresenescence through regulating protein related to cell cycle (cyclin D1,CDK4, p16IN4Ka, p21WAF1, p53).Partâ…¢A study about the effect of pravastatin onpremature senescence of neonatal SD rat cardiomyocytesinduced by H/RAIMTo investigate whether pravastatin could inhibit prematuresenescence of neonatal SD rat cardiomyocytes induced by H/R.METHODSIntervention concentration of pravastatin was 10^-7-10^-5 Mol/L, othermethods were as same as partâ… RESULTSCompared with cells resided in G0/G1 phase in experimental group (72.71ï¼…, 87.45ï¼…, 87.97ï¼…, 89.78ï¼…in control group, hypoxia 6 h,reoxygenation 24 h and 48 h, respectively), pravastatin could not reversecells resided in G0/G1 phase (71.98ï¼…, 86.47ï¼…, 84.98ï¼…, 88.78ï¼…incontrol group, hypoxia 6 h, reoxygenation 24 h and 48 h, respectively) (P＞0.05). Pravastatin also could not reverse cells disappeared in S phase (P＞0.05). Compared with the percentage ofβ-galactosidase positivestaining cells in experimental group (9.13ï¼…, 12.55ï¼…, 42.15ï¼…, 53.48ï¼…in control group, hypoxia 6h, reoxygenation 24h and 48h, respectively)pravastatin could not reduceβ-galactosidase activity, the percentage ofβ-galactosidase positive staining cells is 8.56ï¼…, 12.37ï¼…, 42.34ï¼…,58.28ï¼…, in control group, hypoxia 6 h, reoxygenation 24 h and 48 h,respectively (P＞0.05), different intervetion concertration of pravastatin,with similar outcome.CONCLUSIONSPravastatin could not inhibit premature senescence of neonatal SDrat cardiomyocytes induced by H/R.