| Background and Objectives Cytosolic calcium overload and alterations of nuclear responses play pivotal roles in the pathogenesis of myocardial ischemia/reperfusion injury. At present it still remains to be elucidated the common pathway of signal transduction from cytosolic calcium overload to alterations of nuclear responses in myocardial ischemia/reperfusion injury. Recent studies have shown that cell nucleus possesses a relatively independent regulatory system which sustains nuclear calcium homeostasis and transduces nuclear calcium signals. It has also been well documented that nuclear calcium homeostasis and nuclear calcium signals regulate various nuclear responses, such as the replication and repair of DNA, the expression of genes, the phosphorylation of nuclear proteins, cell proliferation as well as cell apoptosis. The aim of this study is to reveal the characteristics of changes in free nuclear calcium concentrations([Ca2+]n)and in nuclear ryanodine receptors(RyR)as well as the effects of phosphorylation on the RyR in ischemia/reperfusion-injuried cardiomyocytic nuclei, and to provide experimental evidence for the nuclear calcium mechanism of myocardial ischemia/reperfusion injury. Materials and Methods Healthy adult male Wistar rats were divided randomly into the ischemia/reperfusion injury(IRI)group and the sham-operated(Sham)group. The IRI models were established by ligating the left coronary artery for 30 min and then reperfusing the hearts for 3 h, while the Sham group underwent the same procedure except that the coronary artery was not ligated. The activity of SOD and the content of GSH in myocardial homogenate were determined by spectrometric assay. The expression of c-Fos, Bcl-2 and Bax was observed by immunohistochemical staining, and the average absorbance (AA) of Bcl-2 or Bax positive cells was analyzed by digital image analyzing software. Cardiomyocytic nuclei were isolated and purified by sucrose density gradient ultra-centrifugation, and its purity was confirmed by enzymatic assay. Dual-wavelength fluorophotometry was used to determine [Ca2+]n of isolated cardiomyocytic nuclei. The maximum binding capacity (Bmax) and dissociating ration (Kd) of nuclear RyR were determined by saturation binding of 3H-ryanodine to isolated cardiomyocytic nuclei. Finally, the Bmax and Kd values of nuclear RyR were observed after addition of PMA+PS and Ca2+-CaM to stimulate the phosphorylation of nuclear RyR.Results:(1) Compared with the Sham group, the activity of SOD and the content of GSH in myocardial homogenate of the IRI group were decreased by 18.2% and 15.8% respectively(P<0.05). (2)The ratio of c-Fos positive to negative cells in the IRI myocardium was much higher than that in the Sham group( P<0.01). The expression of Bax and Bcl-2 in IRI myocardium was augmented compared to the Sham group(P<0.01 and P<0.05 respectively). However, the ratio of AA of Bcl-2 positve cells to AA of Bax positive cells in IRI myocardium was lower than that ratio in the Sham myocardium. (3)The contents of proteins in isolated cardiomyocytic nuclei was one 1/35 to 1/40 of that in myocardial homogenate. The ratio of protein to DNA was six to eight. The retrieval rate of cardiomyocytic nuclei was 25% to 35%. The activities of organelles' marker enzymes in isolated cardiomyocytic nuclei were less than 5% of that in myocardial homogenate. (4)In the presence of 10 mmol.L-1 ATP, [Ca2+]n of both the Sham and the IRI cardiomyocytic nuclei increased as the calcium concentration in the incubation buffer increased. When cardiomyocytic nuclei were incubated with 0.1μmol.L-1 calcium, [Ca2+]n in the two groups showed no difference(P>0.05). However, when the calcium concentration in the incubation buffer was 1μmol.L-1 or 10μmol.L-1, [Ca2+]n in the IRI group was much higher than that in the Sham group( P<0.01). (5)In the absence of ATP, no difference in [Ca2+]n was observed between the IRI and the sham cardiomyocytic nuclei in the calcium-free incubation buffer(P>0.05), whereas increased [Ca2+]n was measured in both the IRI and t...
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