Effect Of Cardiac Hypertrophy On Calcium Transients In Mice Cardiac Myocytes

BackgroundCardiac hypertrophy is an adaptive response of myocardial contraction and cardiac function to this change when the heart is subjected to pressure overload.The symptoms manifested at the level of cardiomyocytes are that the volume of the cells becomes larger and the synthesis is increased in terms of protein.In terms of organizational structure,there is sarcomere remodeling and myocardial fibrosis.Diseases associated with the heart are often accompanied by the development of cardiac hypertrophy,such as heart valvular disease,hypertension,and dilated cardiomyopathy.In addition,physiological emergency such as exercise training and pregnancy can also lead to cardiac hypertrophy.While cardiac hypertrophy greatly aggravates the risk of sudden death,arrhythmia and heart failure,so it can be as a reference indicator for early warning of heart disease.Therefore,studying the cellular and molecular mechanisms that lead to cardiac hypertrophy and its evolution into heart failure helps us better understand the main mechanisms of cardiac stress response to chronic stress,which will help develop new drugs and new treatment modalities,as well as a better guide to prevent heart hypertrophy.Cardiac hypertrophy caused by persistent load,including pressure overload,volume overload,neurohormones,will lead to ventricular muscle structure remodeling,ion channel re-construction?electrical remodeling?along with its pathological progression,and Ca²⁺remodeling,the heart function will also gradually convert to the decompensation period.Ca²⁺remodeling in the myocardium,the abnormal homeostasis of intracellular Ca²⁺,is a core issue involved in the cardiac hypertrophy and the development of heart failure.The maintenance of calcium homeostasis in cardiomyocytes is dependent on a large and complex system.The first of which plays a key role in Ca²⁺transport channels on the cytoplasmic membrane and sarcoplasmic reticulum,such as voltage-gated Ca²⁺channels?VGCCs?,Na⁺-Ca²⁺exchanger?NCX?on the cytoplasmic membrane,ryanodine receptors?RyRs?which is a Ca²⁺sensitive release channel and Ca²⁺ATP ase?SERCA2?on the sarcoplasmic reticulum.Landstrom has found that Ca²⁺transport abnormalities occur in cardiomyocytes of mice with cardiac hypertrophy.When the dynamic balance of Ca²⁺is breaken,the time course of action potentials of cardiomyocytes is prolonged,resulting in myocardial electrical remodeling.Myocardial electrical remodeling predisposes hypertrophic myocardium to early post-and late post-depolarrization,leading to malignant arrhythmias and sudden death.It is thus suggested that cardiac hypertrophy is related to the homeostasis of intracellular Ca²⁺and the Ca²⁺transport channels.However,it is not entirely clear whether the expression of several important Ca²⁺transporter channel proteins associated with calcium homeostasis is altered.In this study,we will bulid mouse model of cardiac hypertrophy by transverse aortic constriction?TAC?,and detect the intracellular Ca²⁺content,sarcomere length and myocardial contractility using cell contraction and ion concentration synchronization measurement system.And we will further observe the expressions of related Ca²⁺transport channels proteins.Methods1.Healthy adult C57BL/6 mice weighing 20-25g?male and female?were selected to establish a pressure overload cardiac hypertrophy model by TAC.2.Four weeks after the TAC operation,the animals were killed and the heart index,lung index,heart weight and tibia length ratio were calculated.Myocardial tissue changes and myocardial fibrosis were detected by HE staining and Masson staining.3.Myocardial cells were isolated from mice by enzymatic decomposition.The concentration of Ca²⁺in myocytes of Sham operated and post-TAC?TAC4W?mice was measured at rest and 1Hz field stimulation by IonOptix myocardial contraction and ion concentration simultaneous measurement system.The contractile force of myocytes was measured by the length of sarcomeres.Additionally,caffeine was added to measure the calcium transient in myocytes and to investigate the effects of Ca²⁺stores in the sarcoplasmic reticulum and RyR receptors on Ca²⁺homeostasis in hypertrophic mice.4.Western blot was used to detect the expression of relevant Ca²⁺transport channel proteins,including Cav.1.2,NCX,RyR2,SERCA2,in the myocardial tissue.Results1.Four weeks after TAC operation,compared with Sham operated mice,the heart index,lung index and the ratio of heart weight to tibia length in TAC4W mice increased significantly,P value was 0.0054,0.0290,0.0002 respectively.These results suggest that the mouse model of pressure overload cardiac hypertrophy has been successfully established.2.Compared with the Sham operated mice,HE staining showed that the volume of cardiac myocytes was larger;Masson staining showed that the interstitial fibrosis and collagen proliferation of cardiac myocytes in the TAC4W mice were disorderly arranged,and the gap between myocytes became wider.It further indicated that the model of cardiac hypertrophy was successfully established.3.The results of Ca²⁺concentration in cardiac myocytes showed that there was no difference between the TAC4W mice and the Sham operated mice at rest.When cardiac myocyets stimulated by the field potential,the transient peak value of calcium decreased about 45%,while the time to reach the peak value of 90%and the time from the peak value to the baseline level of 50%both increased significantly,with P values of 0.0001 and 0.0125,respectively.The calcium transient peaks of myocardial cells induced by 10?M caffeine in cardiac hypertrophy mice were significantly lower than those in the Sham operated mice.4.The initial length of myocardial sarcomeres in the TAC4W mice was significantly shorter than that in the Sham operated mice?P=0.0404?.And the shortening length of myocardial sarcomeres in the TAC4W mice?0.0671±0.0084?m?was significantly smaller than that in the Sham operated mice?0.1249±0.0281?m?.These results suggest that the mice with cardiac hypertrophy have sarcomere remodeling and the contractility of myocytes is significantly reduced.5.Western blot analysis of the expressions of related Ca²⁺transport channels proteins showed that the expression levels of RyR2,SERCA2 and CaV1.2 proteins in the myocardial tissue of the TAC4W mice were significantly lower than that of the Sham operated mice,while the expression levels of NCX protein were significantly higher than that of the Sham operated mice.ConclusionThe model of pressure overload cardiac hypertrophy in mice was successfully established by TAC.There were abnormal calcium homeostasis,sarcomere remodeling and decreased myocardial contractility in cardiac myocytes of the mice after TAC operation,which may be related to the less Ca²⁺storage in sarcoplasmic reticulum and abnormal expression of Ca²⁺transport channels proteins.