| Objective: To investigate whether there is dysregulation of SOCE(store-operated calcium entry) and content variation of related proteins(Orai and STIM) in the ventricular myocytes of diabetic rats, and to discuss if SOCE dysregulation is an important factor on the development of diabetic cardiomyopathy.Methods: Male Sprague-Dawley(SD) rats were divided into diabetes group and control group randomly(n=20). Diabetic rats were induced by an intraperitoneal(IP) injection of 60 mg/kg streptozotocin(STZ). Control male SD rats were treated with an equivalent volume of sterile normal saline. The diabetic rats were considered to be successfully induced when caudal vein blood glucose levels were ≥16.0 mmol/L and diabetic rats arose polyuria, polydipsia, polyphagia. All animals were supplied ample food and water for 8 weeks. All experiments were performed after 8 weeks since the animals received the injection of STZ. The blood glucose of animals was monitored continuously every week. 8 weeks later, the animals were recepted an IP injection of 3ml/kg 10%chloral hydrate for anesthesia. After the animals performed muscles weakening, inserted electrode-pins into hypoderm of arms and left leg. ECGs(electrocardiographs) were recorded by BL-420 E multi-channel physiological signal acquisition and processing system. Animals were sacrificed after ECGs were recorded and weighed the hearts. Excised the top of heart for HE(hematoxylin-eosin) staining to ensure whether the heart appeared hypertrophy and fibrosis. In order to investigate the dysregulation of SOCE, chose the left ventricular muscles by culturing, incubating and coloring to measure the content of intracellular Ca2+. The related proteins- Orai(calcium release-activated calcium channel protein) and STIM(stromal interaction molecule) were measured by using Western-blot technology.Results: 1. 8 weeks later: 1) Changes of the living state: the STZ-treated rats appeared polyuria, polydipsia, polyphagia gradually after the injection for 24 h and were in low spirits. The characteristic changed evidently in STZ-induced animals, physiologically. While the control animals showed normal states in eating, drinking and excretion. 2) Changes of body weight: the STZ-treated rats showed the lower weight than the control rats(P < 0.001). 3) The last blood glucose measurement: the blood glucose levels of the STZ-induced animals raised for 8 weeks were much higher than that of control animals(P < 0.001). 4) Changes of heart weight: the heart weight of STZ-treated animals was dramatically lower than that of control group(P < 0.001). 5) The ratio of heart weight/ body weight: the heart/body weight of diabetic animals were heavier significantly, compared to control animals(P < 0.001).2. ECG test: 1) Change of wave shape in ECG: the ECG of control group showed regular heartbeat, equal wave shape, and normal T wave. While the diabetic group showed various kinds of arrhythmias, such as T wave flatness and inversion, ventricular premature beat, and so on. 2) Change of heart rate: the difference of heart rate between the diabetic animals and control animals was not significant.3. HE staing: Myocardial pathological section of control group showed the normal myocardial fibers arrangement and cellular shapes, while the pathological section of diabetic group suggested that the myocardial fibers were disarraged and loosed, what’s more, it also could be seen the myocytes hepertrophy and swelling.4. [Ca2+]i measurement: 1) measurement of Ca2+-store depletion after adding TG(thapsigargin) : the TG-induced [Ca2+]i increase was significantly weakened in the diabetic rats than that in control rats(P < 0.05). 2) Comparison of Ca2+ influx: the [Ca2+]i increase via SOCE was strongly enhanced in the left ventricular muscle cells of the diabetic rats compared with the control rats(P < 0.05).5. Analysis of related proteins expression: 1) Comparison of Orai1: according to half-quantitative analysis, Left ventricular Orai1 expression of diabetic group showed the more quantity than control group(P < 0.05). 2) Comparison of Orai2: left ventricular Orai2 protein of diabetic group is overexpressed compared to control group(P < 0.01). 3) Comparison of Orai3: the difference of Orai3 expression in left ventricular between diabetic and control group wasn’t statistically significant(P > 0.05). 4) Comparison of STIM1:STIM1 expression in left ventricular of diabetic group is significantly more than that of control group(P < 0.01). 5) Comparison of STIM2: In contrast, the STIM2 expression level was markedly decreased in left ventricular of diabetic group compared to that of control group(P < 0.001).Conclusion: 1. SOCE is enhanced in DCM cardiomyocytes, and the relative proteins Ora1, Orai2, STIM1 are upregulated also, but STIM2 decreased. 3. We propose that SOCE plays an irreplaceable role in the development of DCM. |
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