| Cardiovascular disease is one of the most serious diseases endangering human health in today’s world. At present, the methods of clinical myocardial protection mainly include: to improve the myocardial perfusion fluid composition, perfusion, perfusion temperature, ischemic preconditioning, drug pretreatment, etc. Methods which work well in laboratories lose protection during translation. In addition, myocardial protection researches were carried out in healthy adult animal models. On clinical practice, however, often combined different diseases and conditions, such as aging, which is one of the important factors affects cardioprotection.Due to the differences in morphology, structure and function of elderly and adult myocardium, the same kind of cardioprotection shows variation effect in adult and elderly. Senescent myocardium are easy to be damage during ischemia reperfusion(I/R) injury. Although there are numbers of cardioprotection methods to protect adult myocardium form heart disease, but the impact in elderly has not been confirmed. The current clinical application of myocardial protection methods are proved to be imperfect, and therefore, the study of myocardial protection control strategies of the elderly have important theoretical significance and practical value.In 1986, Murry and colleagues was first proposed ischemic preconditioning(IPC), which refers to repeated transient ischemia and reperfusion can enhance the tolerance of organization to subsequent ischemia and hypoxia. Pharmacological preconditioning(PPC) is based on the IPC mechanism, inspired by drugs or simulating the body’s endogenous substances to reduce the I/R injury. Kerstion et al suggested inhaled anesthetics preconditioning(APC) early protective effect occurs immediately after pretreatment, last 1 ~ 3h disappear(the first window). And the second window of the protection occurs after 24h(delay phase) and last to 72 h. Our work mainly involves the first window protection after preconditioning.We chosen the aged rats as objects, using the model of rat myocardial I/R and myocardial cells from adult heart, to study and compare the different characteristics of myocardial protection mechanism in the early stage after preconditioning. By detecting the interaction relations between activity of Akt/GSK-3β signaling pathway and mitochondrial permeability transition pore(m PTP) of main components, we find that the attenuation of old myocardial preconditioning is related to the increased expression of cyclophilin D(Cyp-D), the main component of mitochondrial m PTP. We find mitochondrial antioxidant treatment reducing the expression of Cyp-D on elderly myocardium and improving the protection of preconditoning for aged rats. Thus we believe this is one of the important reason attenuate cardioprotective of myocardium in aged rats. Our study will provide a novel idea and potential therapeutic target for clinical senescent myocardium protection, and could be translated to other related disease control and prevention.1.Attenuation of myocardial preconditioning protection in the first window is associated with post-transcriptional regulation mechanism in aged rats.By long term oxidative stress, aging myocardium is different from the adult in morphology, function and metabolism, such as increased left ventricular hypertrophy, chronic heart failure, atrial fibrillation. Elderly patients are more susceptible to injury in myocardial I/R and cardiopulmonary bypass, myocardial protection method for the young are not suitable for the aged myocardium.Objective: To investigate the expression changes of myocardial messenger RNA(m RNA) in adult rats and aged rats after preconditioning, to verify the mechanism of transcriptional regulation of pretreatment.Methods: We use rat myocardial I/R model to detection myocardium infarct size and content of ROS in vovo. Microarray was use to detect expression changes in m RNA. We use real-time quantitative PCR and western bloting to confirmed the result from Microarrays.Results: Cardiopretection after APC is attenuated in aged rats during the first wondow. Reactive oxygen species(ROS) increased in adult rat myocardium by APC, while the older rats did not increase. The m RNA expression of Egr1, IL6 and Ccl2 is increased after IPC in adult rat cardiac, but elderly myocardial m RNA did not change significantly.Conclusion: Attenuation of aged myocardial protection by APC is associated with isoflurane failure to increase ROS production in myocardium; Early-phase IPC and APC induced different genomic responses. The absence of detectable changes associated with early-phase APC suggests a posttranscriptional orposttranslational mechanism. The absence of a genomic response in the senescent myocardium(except for IPC-induced Atf3) could underlie the failure of IPC to provide any cardiac protective benefit to older animals.2.The effect of myocardial protection on Akt/GSK-β signaling pathways regulating mitochondrial transition pore in aged rats.Mitochondria are important target organelles of myocardial I/R injury, m PTP is the key factor mediate mitochondrial injury. Akt / GSK-3β signaling pathway plays an important role in myocardial protection, p GSK-3β inhibit m PTP opening to protect mitochondria and maintain cell function and stability.Objective: To investigate the changes of Akt / GSK-3β signaling pathway of myocardial preconditioning in aged rats; Whether can adjust the Akt/GSK- 3 beta signaling pathways, improve the elderly rats myocardial protection; Akt/GSK-3-β signaling pathway and mechanism of m PTP.Methods: Using electron microscopy to compare ultrastructural changes of mitochondria between adult and aged rats after myocardial I / R injury. Measured m PTP opening time by confocal microscopy. Western bloting detect the activity of Akt / GSK-3β signaling pathway and the changes of m PTP components. Using co-immunoprecipitation studies the relationship between GSK-3β and m PTP component proteins.Results: Akt / GSK-3β signaling pathway was been activity by APC in young rat. Mitochondrial edema and swelling reduced by APC in young but not ealerly. However, the activation of pathway is increased in aged myocardium compared with young adults, and precondition can’t further activate the pathway. The distribution of p GSK-3β in mitochondria increased in aged rats, and tend to increase the combination with ANT, the major protein component of m PTP. However, Cyp-D expression is increased in aged rats and combine with ANT, thus the m PTP open time is shortened compared with adult rats.Conclusion: Lack of isoflurane-induced cardioprotective effects, seen in the old animals, can be explained by age-related differences in Akt/Gs K-3bsignaling pathway and the inability to reduce m PTP opening following ischemia/reperfusion injury. Importantly, the level of cyclophilin-D in co-immunoprecipitates with ANT was significantly decreased in the young APC and IPC groups, but not in old rats. We also found that APC or IPC significantly prolonged mitochondrial permeability transition pore opening time in the young cardiomyocytes under oxidative stress, but not in the elderly. Attenuation of APC or IPC protection in the aging heart is associated with failure to reduce ANT–cyclophilin-D interactions and to decreased p GSK-3βresponsiveness of ANT, critical modulators of mitochondrial permeability transition pore.3.Mitochondrial antioxidant can increase protective effect of preconditioning on old rats myocardium by improving the function of mitochondrial permeability transition pore.Cardioprotective effects of anesthetic preconditioning and cyclosporine A(Cs A) are lost with aging. To extend our previous work and address a possible mechanism underlying age-related differences, we investigated the role of oxidative stress in the aging heart by treating senescent animals with the oxygen free radical scavenger Tempol.Objective: the mitochondrial antioxidant treatment in aged rats, observation of the improvement of myocardial and mitochondrial function, and to explore its possible mechanism.Methods: Oldmale Fischer 344 rats(22–24 mo) were randomly assigned to control or Tempol treatment groups for 2 or 4 wk(Tx2wk and Tx4 wk, respectively). Rats received isoflurane 30 min before ischemia-reperfusion injury or Cs A just before reperfusion. Myocardial infarction sizes were significantly reduced by isoflurane or Cs A in the aged rats treated with Tempol(Tx4wk) compared with old control rats. In other experiments, young(4 – 6 mo) and old rats underwent either chronic Tempol or vehicle treatment, and the levels of myocardial protein oxidative damage, antioxidant enzymes, mitochondrial Ca2+ uptake, cyclophilin D protein, and mitochondrial permeability transition pore opening times were measured.Results: Tx4 wk significantly increased Mn SOD enzyme activity, GSH-to-GSSH ratios, Mn SOD protein level, mitochondrial Ca2+ uptake capacity, reduced protein nitrotyrosine levels, and normalized cyclophilin D protein expression in the aged rat heart. Tx4 wk also significantly prolonged mitochondrial permeability transition pore opening times induced by reactive oxygen species in old cardiomyocytes.Conclusion: Our studies demonstrate that 4 wk of Tempol pretreatment restores anesthetic preconditioning and cardioprotection by Cs A in the old rat and that this is associated with decreased oxidative stress and improved mitochondrial function. Our results point to a new protective strategy for the ischemic myocardium in the high-risk older population. |
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