| BackgroundDiabetic cardiomyopathy is a complication of diabetes mellitus and increases incidence of congestive heart failure,the risk factor is 4> 5-fold higher compare with other peoples without diabetes mellitus.Diabetic cardiomyopathy always could be found in diabetic patients without great vessels lesion or disorder on stratum basale of blood capillary in myocardium and means it may non-ischemia cadiomyopathy . The change of intracellular calcium concentration play a central role in physiological function of cardiacmyocyte and cardiac dysfunction has been linked to Ca2+-cycling defect. It is noted calcium release activities are determined by ryanodine receptor(RyR) and inositol triphosphate (IP3) receptor, activity of the cardiac sarcoplasmic reticulum Ca2+-ATPase(SERCA) is the rate-determining factor of Ca2+reuptake into the SR, and SERCA activity is regulated by phospholamban (PLB), PLB inhibits SERCA activity in its unphosphorylation form, whereas the phospholated form of PLB dissociates from SERCA. It has been made clear that variation of PLB and SERCA paticipate in the pathophysiology of heart failure, but it also have different opinion that how change of two proteins in myocardium. There are a fewinvestigation about variation of PLB and SERCA in diabetic cardiomyopathy. Our experiment observe alteration of quantity of PLB and SERCA in several stage of diabetic cardiomyopathy and investigate the relationship between the protein levels of PLB and SERCA and heart failure, also discuss participation of two proteins in pathophysiology of diabetic cardiomyopathy. Materials and Methods K Study subjectsControl male Sprague-Dawley (SD) rats and diabetis rats which are induced by mesoxalylcarbamide from male Sprague-Dawley. 2 > Equipment and reagentElectrophoresis apparatus (Bio-Rad) ,Monoclonal (Mouse) Anti-Phospholamban Antibody(IgG2a)v Monoclonal (Mouse) Anti-SERCA2 ATPase Antibody(IgG2a) (ABR) ,Goat to Mouse IgG(Beijing zhongshan biotechnology corporation) , ECL Western blotting detection reagent (Molecular biophysics insititure of college of medical,Zhejiang university) , Mesoxalylcarbamide (Sigma) . 3> MethodsDiabetes was induced in male Sprague-Dawley ( SD) rats(200-280g) by administering a single tail-vein injection (40mg/kg) of mesoxalylcarbamide after 12-hour fast,and the control group was injected with distilled water. At the end of two weeks N four weeks and six weeks after the induction of diabetes,animals were killed .Hearts were excised from diabetic and control rats,trimmed free of atria,rinsed in cold 0.9% saline and freeze-clamped in liquid N2,thenpowered in liquid N2 cooled stainless steel mortar pestle.These ventricles were stored in -70癈 until needed for the 'experiment.On day of experiment,frozen ventricles were resuspended in buffer(100mg/1.0ml). The suspension was homogenized for 10-15 minutes,then centrifugated at 14000 rpm at 4癈 for 15 minutes. Protein concentrations were determined by the method of Bradford, using BSA for the standard curve. 20ug cardiac homogenate proteins from rats were loaded on the same SDS-polyacrylamide gel and separated by electrophoresis (5% acrylamide stacking gel and 12% acylamide separating gel ) .Afterwards, the proteins were electrophoretically transferred from the gel at 200mA at 4癈 for 90min onto nitrocellulose membranes with transfer buffer, then the membranes were washed for one minutes in TEST and blocked with 5% defatted milk in TEST for one hour at room temperature, then the membranes were washed three times with TEST and incubated overnight with monoclonal antibody specific for PLB or SERCA at 1:1000 dilution in 5% defatted milk/TBST. The membranes were washed three times with TEST and incubated for one hour with a secondary anti-mouse antibody conjugated to horseradish peroxidase at 1:500 dilution in 5% defatted milk/TBST.Antigen-antibody complexes of all probed were detected by the enhanced chemiluminescence detection system.The intensity of each band wa...
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