Study On The Pathogenesis Of Myocardial Depression In Septic Shock

INTRODUCTION AND OBJECTIVESSeptic shock is a serious clinical syndrome, with the highest mortality in intensive care unit. The main clinical features of septic shock is hypoperfusion of the major organs, making the body cells unable to maintain the normal metabolism and normal function , clinically manifested as hypotension, vascular injuries , DIC, eventually leading to irreversible multiple organ failure. Myocardial depression is an important character of septic shock, and heart insufficiency or heart failure may occur in the serious cases. The extent of myocardial depression is related to the prognosis of the patients. The pathogenesis of septic shock is believed to be the cascade reaction caused by proinflammatory cytokines , and TNFa is believed to be the major mediator in endotoxic shock and multiple organ failures. The heart may produce TNFa itself when stimulated by LPS in septic shock, and heart can also express TNFa receptor type I and II. The cardiac contractility is elevated during the early stage of septic shock, and it is decreased only at the late stage of septic shock. TNFa may decrease cardiac contractility both directly and through other mediators. Heart may express IP₃ and its receptors, participating in control of intracellular calcium release from calcium stores, determining the level of free intracellular calcium ion, influencing the myocardial contractility. Calcium is the common way of many factors affecting myocardial contractility. There is a large difference in the calcium ion concentration between inside and outside of the cells in physiological conditions. Calcium influx through cell membrane is a promoting factor for release of intracellular calcium from calcium stores. We established the animal model of endotoxic shock by intravenous injection of different doses of lipopolysaccharide, observed the changes of mean arterial pressure and heart rate, measured the level ofTNFa at different time points, and check the ultrastnictural changes in the four groups in order to investigate the cardiac injuries in septic shock and probe the possible mechanisms. We investigated the changes of IP3R I and IP3R1H expression in heart with endotoxic shock and probe the possible mechanisms of my-ocardial depression. Then we investigate the effect of TNFa on L - type calcium channel of single ventricular myocyte and probe the possible mechanisms of TNFa inhibition on cardiomyocyte contractility.METHODS1. Reagents:tumor necrosis factor a;lipopolysaccharide;TNFa serum detection kit;anti - TNFa receptor type I antibody;SABC immunohistochemstry kit;anti -IP3R I antibody;anti - IP3RIH antibody;FITC marker goat - anti - rabbit -IgG;lysate of tissue protein;electrophoresis buffer solution;transmembrane buffer solution;blocking solution;antibody dilution solution;FITC coloring solution;1 % bovine serum;gold conjugate;calcium Tyrode' s solution;calcium - free Tyrode' s solution;KB solution;collangenase type I } intracellular electrode buffer solution;extracellular electrode buffer solution;TEA;2. Animals:Male Wistar, body weight 220 -250 g3. Methods:(I) Preparation of animal model: Endotoxic shock was made on Wistar by intravenous injection of IPS of different doses, then MAP and heart rate were observed;normal saline was injected to the control group;heart specimens were taken by the end of six hours or death, then were fixed with 10% formalin for later use;(2)The level of serum TNFa was measured at the different time point after LPS injection by ELISA;(DUltrastructural changes of heart were observed under transmission electron microscope;of heart tissue was detected by immunohistochemistry;(DlP3R I and IP3RHI expression of heart tissue were detected by immuno-histochemistry;?IP3R I and IP3R M expression of heart tissue were detected by Western Blot;(7)IP3R I and IP3RHI expression of heart tissue were detected by immuno-labeling of gold conjugate;(^Isolation of single ventricular myocyte;?Detect L - type calcium current by patchclamp, and measure the changes of TNFa on L - type calcium current of single cardiomyocyte;RESULTS(1) LPS may induce the triphasic changes of MAP and heart rate: decreased transiently and then recovered to normal rapidly, decreased gradually till death to normal;(2) Serum TNFa was elevated after LPS injection, peaked at 90min, then decreased gradually;(3) The ultrastructural injuries of heart manifested as mitochondrial injuries , swelling of myofibril;irregular intercalated disc;vacualation of nucleus;congestion;vascular endothelial injuries;and the extent of cardiac injuries is related to the dose of LPS injected;(4) TNFR1 expression in heart tissue were elevated , and related to the dose of LPS and the course of shock;(5)IP3R I and !P3RHI expression were increased in the rat heart with en-dotoxic shock, and ectopic expression IP3R I was observed;(6) TNFa decreased L - type calcium current of single ventricular cardiomyocyte transiently and then increased L - type calcium current till the end of observation;TNFa promote calcium influx;CONCLUSIONS(1) LPS injection can induce triphasic changes of MAP and heart rate, andcause elevation of serum TNFa;(2) LPS can cause ultrastructural injuries in heart, and the extent of injuries is related to the dose of LPS;the extent of increased TNFa receptor type I expression in heart is related to the dose of LPS injected;TNFa participate in the cardiac injuries in endotoxic shock;(3) IP3 R I expression is elevated in the rat heart with endotoxic shock, IP3R I is involved in the regulation of MAP;(4) TNFa can increase L - type calcium current, promoting calcium influx;(5) TNFa increase the IP3 R I expression, inducing calcium influx, and affect cardiac contractility;...