Experimental Study On Roles Of The Sarcolemmal Calcium Handling Proteins On Myocardial Ischemic Preconditioning And Pharmacological Preconditioning

Background and Objective: Insights into the mechanisms responsible for ischemic preconditioning (IPC) may help in finding novel strategies to treat and prevent myocardial ischemia/reperfusion injuries.Previous studies have demonstrated that intracellular calcium oscillations plays an important role in IPC: elevation of intracellular calcium ( [Ca2+]i ) in the early phase of ischemia is essential for onset of IPC and one of the protective mechanisms of IPC is to remain calcium homeostasis.L-calcium channel were previously considered to be the major sourse of calcium overload.But recent expremental studies demonstrated that sodium-calcium exchanger (NCX) is the primary pathway for calcium influx into cardiac cell and selectively inhibit outward Na+-Ca2+ exchange currents during the early phase of ischemia have a protective effect against ischemia/reperfusion injury.But the precise mechanism of the roles of sarcolemmal calcium handling proteins in ischemia/reperfusion and IPC ,include changes of gene transcription and protein activity ,remain unknown. Although pharmacological preconditioning which induced by active products in IPC simulated the protective effect of IPC in animal experiments, no evident benefit observed in clinic.We investigated the changes of gene transcription and protein activity of membrane calcium handling proteins in IPC and pharmacological preconditoning ,and also investigated the effects of Pd-Ⅰb (an effective component obtained from Bai-hua Qian-hu,a chinese medical plant ) on ischemia/reperfusion rat myocardium. The purpose of this study was to explore the mechanisms of the IPC and investigate myocardiac protection induced by a more effective and more safety remedy against ischemia/reperfusion injuries and explore its mechanism.Methods: 1.Neonatal rat ventricular cardiomyocytes were isolated from 1～2- day-old Sprague-Dawley rats with modified Simpson's method and experimental models of ischemia/reperfusion,IPC and pharmacological preconditioning exerted by adenosine and Pd-Ⅰb were established. To evidence the models,we tested lactate dehydrogenase (LDH) leakage by biochemical autoanalyzer and cell death rate by trypan blue exclusion and changes of myocardial ultrastructure were observed under transmission electron microscopy.2.Cultured neonatal rat cardiomyocytes were grouped randomly into ischemia/reperfusion group,IPC group,adenosine pretreated group,Pd-Ⅰb pretreated group,CaMKⅡ(calmodulin-dependent protein kinaseⅡ) inhibitor KN-93+IPC group,KN-93+adenosine pretreated group,KN-93+ Pd-Ⅰb pretreated group and control group.LDH release in various groups were tested.Semi-quantitative RT-PCR was employed to determine the mRNA levels of NCX.Activity of NCX were indicated by Na+-dependent 45Ca2+ uptake tested by liquid scintillation counting. Activity of plasma membrane calcium atpase were determined by quantifying inorganic phosphorus production.Results: 1. LDH release and cell death rate raised with increasing durations of simulated ischemia and significantly(P<0.001) till 4 h of simulated ischemia.2. In the IPC group induced by 90 min ischemia and 60 min reperfusion,the LDH release and cell death rate were statistically lowered(P<0.01).In pharmacological groups induced by 100μmol/L Pd-Ⅰb or 10μmol/L adenosine for 1 h, LDH release and cell death rate were statistically lowered(P<0.01). Ultrastructure in ischemia/reperfusion group were destructed seriously: cataclastic chromatin and swollen mitochondria and sarcoplasmic reticulum were observed. while IPC,adenosine and Pd-Ⅰb pretreated group maintain the integrity of cell ultrastructure but a little of swollen mitochondria.3. IPC,adenosine and Pd-Ⅰb pretreated group decreased NCX gene transcription and protein activity(P<0.01;P<0.005) along with mild LDH release(P<0.001).These effects were prevented by CaMKⅡinhibitor KN-93.4.No statistically significant difference of PMCA were observed in various groups(P>0.05).Conclusions:1. Neonatal rat cardiomyocytes are inherently less susceptible to ischemia and at least 4 h are required to show significant injury.IPC could be induced by giving 90 min ischemia and 60 min reperfusion.2. Pd-Ⅰb show a significant protection against ischemia/reperfusion injuries in cardiomyocytes similar to that of IPC and adenosine pretreated.3. Decrease NCX gene transcription and protein activity is one of the mechanisms involved in protective effects of IPC,adenosine and Pd-Ⅰb. CaMKⅡmight be involved in the signal transduction.4. PMCA showed no changes to IPC and ischemia/reperfusion progress.