Effect Of Exercise On Calcium Handling Protein In Normal And Diabetic Rat Heart

Objective:The purpose of this study was to explore the influence of exercise on gene expression of calcium handling protein in normal and diabetic rat heart,and to elucidate possible molecular mechanism of the exercise-induced adaptation and protective effect.Main Methods:Chapter 1 The effects of exercise on gene expression of calcium handling protein in normal rat heartAnimal and groups:4 month-aged Sprague-Dawley rats were divided into three group randomly:sedentary control,acute exercise and chronic exercise.Exercise training protocol:The exercised began a progressive exercise program with treadmill running 5 d·wk-1.Each treadmillsession began with a warm-up period.The endurance exercise program progressed as follows: week 1 began with 5 min·d-1 and ended the week at 25 min·d-1,15 m·min-1; week 2 began at 30 min·d-1 and progressed to 50 min·d-1,20 m·min-1.Not all animals could maintain this pace or time limitation;thus,exercise protocols were individualized.Running scores indicating the quality and the time run were obtained daily for each animal.The chronic exercise rats's running goal for the remaining 16 wk of exercise remained steady at 60 min·d-1,at a pace of 20 m·min-1.The acute exercise(AE) rats were placed on treadmill in the same speed to run to exhausted.The sedentary rats were placed on a stationary treadmill.Animals were provided free access to food and water.The experiments were terminated after 16 wk of chronic exercise and acute exercise rats. Measure transcription of SERCA,PLB,RyR2 calcium channel, semiquantitative RT-PCR was performed.Total RNA was extracted with Trizol Reagent.RNA content was assessed by light absorbance at 260nm, purity was determined according to A260/A280 ratios,and intactness was assessed by intensity of staining of 28S and 18S ribosomal RNA bands following agarose gel electrophoresis.A quantity of 5μlof total RNA was reverse transcribed in a 50μ1 reaction mixture.For PCR 2μ1 of the cDNA mixture was added to 25μ1 of a master mix.PCR products were separated on 1.5%agarose ethidium-bromide gel,visualized under ultraviolet light.Chapter 2 The effects of exercise on gene expression of calcium handling protein in diabetic rat heartAnimal and groups:4 month-aged Sprague-Dawley rats were divided into three group randomly:sedentary control(SC),sedetary diabetic(SD) and exercise diabetic(SE).Induction and Verification of Experimental STZ-Induced Diabetes:Male Sprague-Dawley rats were injected via abdomal with a single dose of STZ in 0.1 M citrate buffer,pH 4.5(65 mg/kg),or citrate buffer only.Three days later,blood glucose levels were determined using a Glucometer II and Glucostix to ensure induction of diabetes.Exercise training protocol:The exercise rats's running goal for the remaining 8 wk of exercise remained steady at 60 min·d-1,at a pace of 20 m·min-1 and with running 5 d·wk-1.Observation of histological sections of myocardium stained with HE under light microscope.Electron microscopic analysis of myocardium.Chapter 3 The possible mechanism of effects of exercise on gene expression of calcium handling protein in rat heartAnimal and group:4 month-aged Sprague-Dawley rats were divided into four groups randomly:sedentary control(SC),exercise conrol(EC),sedetary diabetic(SD) and exercise diabetic(SE).Exercise training protocol:the same to Chapter 2.Measure serum and myocardium MDA,SOD,GSH-Px and Total Anit-oxidative Capacity according the instruction of the reagents.Main Results:1.16-endurance exercise induced changes in mRNAs level of SERCA2,PLB, expression of PLB mRNAs was significantly increased,but RyR2 remain no change.Acute exercise has not affected the mRNA expression of SERCA, PLB and RyR2.2.SERCA2,PLB mRNAs were significantly increase than that in the age-matched control 8 weeks after STZ injection,mRNA expression of RyR2 decreased,and 8-week endurance exercise induced partly decrease of SERCA2,PLB mRNA than that in the age-matched sedentary diabetics and increase of RyR2 mRNA.3.Observation of histological sections of myocardium stained with HE under light microscope didn' t reveal any difference between diabetic and control group.Electron microscopic analysis of myocardium revealed a spectrum of abnormalities indiabetic rats.Swollen,greater clumping, fragmented,distended and disrupted mitochondria were evident upon electron microscopic examination.The cristae in the mitochondria appeared distorted and in some cases were completely lysed.In addition, the sarcoplasmic reticulum was dilated and swollen,space between intercalated disks was widened,and exercise have no significantly affected on these changes.4.Expression of TGF-β1 mRNAs was significantly increased,and exercise result in no changes.MDA in serum and myocardium increased significantly and the activity of GSH-Px in blood and myocardium decreased significantly.Exercise decreased MDA in serum and myocardium and improve the activity of GSH-Px in blood and myocardium. Conclusion:1.16-endurance exercise induced changes in mRNAs level of SERCA,PLB may be one of molecular mechnisms of exercise-induced adaptation.2.8 week exercise reversed partly changes of calcium handling protein in diabetic rat heart,which suggests the protective effec of chronic endurance exercise on diabetic heart disease.3.Reaction and adaptation to oxidant stress may be the common pathway of the change of calcium handling protein gene expression,and the reaction and adaptation is the mechanism of exercise-induced adaptation and protection to rat heart.