Effects Of Stress On L-type Calcium Channels Of Rat Ventricular Myocytes And Its Mechanism

Aim: Establishing patch-clamp whole cell recording technique to observe the effects of acute and chronic stress on L-type calcium channels' electrophysiological characters of rat ventricular myocytes , and to explore its its mechnisms and significance in stress-induced cardiomyocyte injury. METHODS: Acute and chronic stress experimental models of rat ventricular myocytes were established. Patch-clamp whole cell recording mode was used to record ion channels' electric movements of ventricular myocytes under acute and chronic stress. The steady-state activation and inactivation characters of L-type calcium channels were observed. The effects of stress on PKA signal transduction pathway of ventricular myocytes were investigated by determining PKA's activity and the experiment of PKI blockage. The expression of L-type calcium channel gene in ventricular myocytes under chronic stress was measured with RT-PCR and Northern blotting techniques. Fura-2/AM fluorometry assay was used to determine [Ca2+]i of ventricular myocytes under Ica-L changed conditions, and the apoptosis rate of cardiomyocytes was determined by FCM and TUNEL assay as well.The ultrastructure of ventricular myocytes was observed by electron microscope. RESULTS:(1) Patch-clamp whole cell recording technique was established, and highly stable whole cell ion channels' current curves of normal ventricular myocytes were ploted. (2)Under acute stress, the INa, IK and Ica-L were significantly increased (P<0.05) and these changes were dose dependent. The steady-state activation plot analysis ofIca-L showed that the V1/2 changed from(-0.69 0.36 )mV to (-14.59 0.24)mV (p<0.05, n=5 ) , indicating that the L-type calcium channels of ventricular myocytes were inclined to be activated under acute stress, while the alteration of steady-state inactivation plot analysis was not found; PKA's activity was markedly increased in acute stress, but was blocked by PKI suggesting that the activation of PKA signal transduction pathway may be the main mechanism of ICB-L'S augmentation. (3) Ica-L was raised obviously under chronic stress, the steady-state activation and inactivation characters were not changed; RT-PCR and Northern blot showed that the expression of L-type calcium channel gene of ventricular myocytes was ascended, which means that the molecular mechanism underlying chronic stress induced increase in Ica-L was due to rising of the amount of corresponding L-type calcium channels' protein. (4) Elevated [Ca2+]i concentration resulted from increase in Ica-L and caused calcium overloading in cytoplasm, and in turn induced ventricular myocytes apoptosis; Changes of myocardial ultrastructure of rats subjected to chronic stress including nuclear membrane shrinkage, perinuclear space widening, mitochondrial swelling, myofibril breakage etc. CONCLUSION: The amplitude of Ica-L was increased under acute and chronic stress, their mechanisms lie in activation of PKA signal transduction pathway or upregulation of the L-type calcium channels expression respectively. The amplitude of Ica-L's abnormal increasing induced intracellular Ca2+ overloading which resulted in cardiomyocyte injury under stress. The results brought forward a new illumination for the mechanism of cardiomyocyte injury induced by stress and will offer some new theoretic foundations for prevention and cure of stress induced cardiovascular diseases.