kn-62 and Hypoxia

The present study investigated the possible regulatory mechanisms of redox agents and hypoxia on the K(ATP) current (I(KATP)) in acutely isolated rat ventricular myocytes.. Single-channel and whole-cell patch-clamp techniques were used to record the K(ATP) current (I(KATP)) in acutely isolated rat ventricular myocytes.. Oxidized glutathione (GSSG, 1 mmol/L) increased the I(KATP), while reduced glutathione (GSH, 1 mmol/L) could reverse the increased I(KATP) during normoxia. To further corroborate the effect of the redox agent on the K(ATP) channel, we employed the redox couple DTT (1 mmol/L)/H2O2 (0.3, 0.6, and 1 mmol/L) and repeated the previous processes, which produced results similar to the previous redox couple GSH/GSSG during normoxia. H2O2 increased the I(KATP) in a concentration dependent manner, which was reversed by DTT (1 mmol/L). In addition, our results have shown that 15 min of hypoxia increased the I(KATP), while GSH (1 mmol/L) could reverse the increased I(KATP). Furthermore, in order to study the signaling pathways of the I(KATP) augmented by hypoxia and the redox agent, we applied a protein kinase C(PKC) inhibitor bisindolylmaleimide VI (BIM), a protein kinase G(PKG) inhibitor KT5823, a protein kinase A (PKA) inhibitor H-89, and Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitors KN-62 and KN-93. The results indicated that BIM, KT5823, KN-62, and KN-93, but not H-89, inhibited the I(KATP) augmented by hypoxia and GSSG; in addition, these results suggest that the effects of both GSSG and hypoxia on K(ATP) channels involve the activation of the PKC, PKG, and CaMK II pathways, but not the PKA pathway.. The present study provides electrophysiological evidence that hypoxia and the oxidizing reaction are closely related to the modulation of I(KATP).

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Cells, Cultured; Cyclic GMP-Dependent Protein Kinases; Dithiothreitol; Dose-Response Relationship, Drug; Electrophysiology; Female; Glutathione; Glutathione Disulfide; Heart Ventricles; Hydrogen Peroxide; Hypoxia; Indoles; Male; Maleimides; Myocytes, Cardiac; Oxidation-Reduction; Patch-Clamp Techniques; Potassium Channels, Inwardly Rectifying; Protein Kinase C; Protein Kinase Inhibitors; Rats; Sulfonamides; Time Factors

Calcium plays an important role in the impairment of heart function and arrhythmia under the condition of acute hypoxia, but the mechanism is different from that of chronic hypoxia. This study aimed to evaluate the effect of chronic hypoxia on the expression of calmodulin (CaM) and calcicum/calmodulin-dependent protein kinase II (CaMKII) and the calcium activity in myocardial cells through an animal model of chronic hypoxia in order to get a deeper sight into the mechanism.. A chronic hypoxia model of the rat was prepared by hypoxia exposure (FiO2=10%). The expression of mRNA and protein of CaM and CaMKIIgamma and CaMKIIdelta in myocardial cells were measured by RT-PCR and Western Blot in normal rats and hypoxia rats 1 and the 3 weeks after exposure. The cardiac cells of the rats from the control group and the 3-week hypoxia group were cultured. Then the intracellular calcium activity was detected using laser confocal equipment. The effect of CaMKII on the calcium activity in myocardial cells was evaluated by the application of KN-62 (CaMKII specific inhibitor).. The expression of CaM, CaMKIIgamma and CaMKIIdelta mRNA in myocardial tissues increased in hypoxia rats compared with that in normal controls (P<0.01). The CaM and CaMKIIdelta mRNA expression was different between the 1-week and the 3-week hypoxia groups (P<0.01). The laser confocal demonstrated that the amplitude of calcium wave in hypoxic myocardial cells was not different from that in normal controls, but the duration of calcium wave in hypoxic myocardial cells was longer than that in normal controls (P<0.01). After KN-62 use, the amplitude of calcium wave decreased and the duration of calcium wave prolonged significantly.. The contents of CaM and CaMKII in myocardial cells increased under condition of chronic hypoxia as a compensation to keep calcium homeostasis in a certain time. With more prolonged hypoxia time, abnormal electric activities of heart occurred and the heart function may be impaired.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Blotting, Western; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calmodulin; Chronic Disease; Hypoxia; Male; Myocardium; Norepinephrine; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction