| ObjectivesApotosis is one of important pathogenesis of myocardium ischemia-reperfusion injury (MIRI). So far, its exact mechanism remains unclear. Many studies have demonstrated that intracellular calcium plays a vital role in cell apoptosis pathogenesis, particularly the accumulation of nuclear calcium. Recent studies have found that myocardium ischemia-reperfusion caused a continuing elevation of nuclear [Ca2+] with cytosolic [Ca2+], and even myocyte apoptosis both during earlier stages and later stages of myocardium reperfusion. Based on these observations, it is postulated that nuclear calcium overload may be involved in occurrence or developing mechanism of apoptosis during MIRI.In the study, we established a rat model of MIRI to observe the modulation of nuclear calcium signal transduction by IP3R and IP4R in isolated cardiac myocyte nuclear, and the variations of their binding abilities in order to investigate the mechanism of nuclear calcium overload during MIRI.Mehtods120 male rats were randomly divided into two groups: IRI group and sham-operated group. Area of myocardial infarction was measured by TTC staining method; Contents of MDA and NEFA in myocardium and serum were tested by sulfur-barbituric acid method and fatty acid once extracting chromometry. Apoptosis index of myocardial cell was determined using TUNEL assay. After cardiac myocyte nucleus was extracted by saccharose density gradient centrifugation method, the binding abilities of nuclear IPsR and IP4R were determined by radioactivity ligand binding assay.Results1. In IRI group, a rat underwent 30-min regional ischemia and 3h reperfusion, area of myocardial infarction was 26.3?.8%. The contents of MDA and NEFA in myocardium and serum all increased significantly. Apoptosis index of myocardial cell markedly increased, too.2. In IRI group, Bmax of nuclear IP3R markedly increased by 2.6 folds compared to sham-operated group, whereas Kd value of nuclear IP3R did not change.3. In IRI group, the binding ability of phosphorylated nuclear IP3R by activated protein kinase C increased by 1.54 folds, but its binding ability didn't change in sham- operated group.4. In IRI and sham-operated groups, nuclear IP3R were all biphasically regulated by cytosolic free [Ca2+], with increased nuclear IP3R binding ability at Ca2+ concentration of 5M, which was the condition of cytosolic calcium overloaded.and their binding abilities declined at Ca2+ concentration of 100M.5. There are two IP4 binding sites located on the nuclear envelope. They were characterized by specific Kd value and Bmax. As shown in Hill diagram, they were all simple single binding sites.6. In IRI group, Bmax from high affinity binding site of nuclear IP4R significantly increased compared with sham-operated group, whereas Bmax from low affinity binding site didn't change. Kd values of both sites were all significantly decreased by 63% and 55%, respectively.7. Although the binding ability of phosphorylated nuclear IP4R by activated PKC in both IRI group and sham-operated group all markedly increased, it was more apparent in IRI group.8. In sham-operated group,the binding ability of nuclear IP4R increased with increasing free calcium concentrations in cytoplasm, whereas in IRI group its binding ability was biphasically regulated by cytosolic free calcium. Compared to sham-operated group,the binding ability of nuclear IP4R in IRI group increased more markedly at the cytosolic free calcium concentration of 5M than at that of 100nM.Conclusions1. Oxidative injury, apoptosis and infarction of myocardium all appeared in rats underwent 30 min regional ischemia and 3h reperfusion.2. There are two IP4 binding sites located on the nuclear envelope. They were characterized by specific Kd value and Bmax. As shown in Hill diagram, both of them were simple single binding sites.3. When a rat suffered to myocardium ischemia reperfusion injury,the potency ofcardiac myocyte nuclear IP3R in releasing calcium from peri-nuclear into nuclear increased,which was confi... |
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