| Background and objective: Sick sinus syndrome (SSS) is a common cardiovascular disease which is greatly harmful to human health. To elucidate the pathogenesis of SSS and find out the possible measures of prevention and radical cure is always an important part of the angiocardiopathy study. In the previous study, we have found that ischemia/reperfusion(I/R) could cause structural and functional damages to sinoatrial node cells in vivoand in vitro, leading to electrophysiological disturbance by affecting the regular rhythm and frequency. Myocardium ischemic preconditioning(IP), which is considered the most powerful protective inherence found in almost all kinds of animals and human, can relief various kinds of cardiac damages caused by I/R. But the signal transduction of IP is very complicated and redundant, only a little has been known about its protective mechanism and the study of effect of IP on the sinoatrial node is rarely seen. The recent research has assigned an important role of ATP sensitive potassium (KATP) channel in IP signal transduction despite the consequence after different KATP channel opening and it's effects still remain an intense debate. Cytoskeleton has also been given more and more concern about it's contribution to the IP recently due to a key role in the signal transduction. Studies have revealed the integrity of cytoskeleton is essential for the IP except a regulating role in electrical and mechanical change of myocardium. Based on the primary culture of neonatal rat sinoatrial node pacemaker cells, the purpose of our investigation is to observe the effects of IP on the changes of cell viability, homeostasis, cytoskeleton and ion channels after sever I/R, detect the role of mitochondrial KATP channel in IP signal transduction and discuss the mechanism, observe whether depolymerization of cytoskeleton would affect the effects of IP and discuss the significance of maintaining the integrity of cytoskeleton in IP signal transduction, providing experimental evidence for further prevention of sinoatrial node injuries caused by I/R. Methods: 1. Sinoatrial node cells isolated by collagenase Ⅱwere purified by discarding cells adhering earlier and the administration of Brdu. 2. IP procedure was designed on the basis of a tested I/R model and the efficacy was detected with MTT colorimetry and PI staining. 3. To study the effect of IP on the cytoskeleotn of neonatal sinoatrial node cells, Cytoskeletal proteins of F-actin, β-tubulin, vinculin and demin were measured with laser confocal microscopy after fluorescently-labeled. 4. Loaded with Ca2+ fluorescent probe, intracellular Ca2+ was quantitative assayed with laser confocal microscopy to learn the effect of IP on Ca2+homeostasis change. 5. Whole Cell patch clamp technique was used to record the L-tpye calcium current(I-LCa) and hyperpolarization-activated inward current (If) of sinoatrial node cells subjected to normal condition, simulated I/R and IP respectively, the effect of IP on the ion channel was studied. 6. Changes of cytoskeletal proteins, I-LCa, If and tracellular Ca2+ were observed in the circumstance that the sinoatrial node cells were incubated with diazoxide(mitochondrial KATP channel opener) for 5 min before the onset of index ischemia to make sure that whether diazoxide would serve a protective pharmacological preconditioning role. The significance of mitochondrial KATP channel was further detected when sinoatrial node cells subjected to simulated IP were incubated with 5-HD(mitochondrial KATP channel blocker) during the index ishemia. 7. Effects of IP on the I-LCa and If were also detected after severe I/R before which the microfilament and microtubule were selectively depolymerized with cytochalasin D and colchicines to explore the significance of maintaining the integrity of cytoskeleton in IP. Results: 1. The IP procedure made up by different times of simulated ischemia during which the oxygen concentration, pH and ionic constitution were all under a mimic I/R circumstance was proved to be effective in groups with a ischemia between 10min and 20min. The IP plan of two circles of 10min ischemia/10min reperfusion was selected finally due to the most powerful protective effect and convenient operation.2. The simulated IP can significantly augment the cell viability, reduce the intracellular Ca2+ overload, improve the damaged cytoskeleton by preserving F-actin,β-tubulin ,desmin and vinculin, maintain the electrical stability by restraining the activated If and improving the inhibited I-LCa after simulated I/R. 3. Pretreatment of diazoxide also improved the cell viability, maintained the Ca2+ homeostasis, electrical stability and the integrity of cytoskeleton mimicking the protective effects of IP. Administration of 5-HD blocked the protective effects of IP completely by reducing the cell viability, enhancing the intracellular Ca2+ and damaging the cytoskeleton. 4. Selectively depolymerizing microfilament or microtubule by cytochalasin D or colchicines also blocked the protective effects on I-LCa and If provided by IP leading to an electrical instability. Conclutions: 1. The simulated IP can effectively protect the sinoatrial node cells exposed to the simulated I/R, maintaining the cell viability, Ca2+ homeostasis and electrical stability. 2. The simulated IP can prevent the cytoskeleton of sinoatrial node cells from reperfusion injury, which is also an important structural basis for IP developing. 3. Mitochondrial KATP channel plays a key role in IP signal transdunction and mitochondrial KATP channel opener diazoxide can offer a protective pharmacological effect.
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