| Background CaMKⅡand PKA , which can phosphorylate a couple of calcium handling protein, are the downstream signals ofβ-adrenergic receptor. They plays critical roles in calcium homeostasis in myocytes. Recently several studies from transgenic mice model and normal animals showed that CaMKⅡemerges as a dominant signal pathway for triggered arrhythmias induced by diastolic calcium leak upon adrenergic stimulation compared with PKA. However there are still many controversies and the electrophysiology dates are still missing.Objective To investigate the effect of Isoproterenol and high-frequency stimulation on delayed afterdepolarization (DADs) in heart failure rabbit cardiac myocytes. And explore the role of KN93, a kind of Calcium/calmodulin-dependent protein kinase (CaMKⅡ) inhibitor, and H89, a kind of Protein Kinase A(PKA) inhibitor, in the generation of DADs.Methods Rabbit models of chronic heart failure were prepared. LVEF determined by echocardiogram was lower than 44%. Then Single ventricular myocytes were isolated by enzymatic dissociation method. Action potential (AP) was recorded by using whole cell patch clamp technique. Perfused with isoproterenol (1μmol/L) Tyrode's solution, we monitored the 50%Action Potential Duration (APD50), 90% Action Potential Duration(APD90) and the incidence of DADs under fast frequency electrical stimulation(1-3Hz). On this basis, perfused with KN93 (1μmol/L) and H89 (1μmol/L), the effect of KN93 and H89 on DADs was observed. Result1. 90% action potential duration (APD90) and 50% action potential duration (APD50) in CHF group were longer than Ctl group. APD90 and APD50 in CHF+ISO group were shorter than CHF group.2. Pacing protocols were given with 1 Hz, the incidence of DADs in Ctl group, Ctl+ISO group, CHF group, CHF+ISO group were 15%(3/20),45%(9/20),26.7%(12/45),92.3%(36/39). Compared with Ctl croup, the incidence of DADs in Ctl+ISO group increased significantly (P<0.05). Compared with CHF croup, the incidence of DADs in CHF+ISO group increased significantly (P<0.05). Compared with Ctl croup, the incidence of DADs in CHF group increased significantly (P>0.05). Compared with Ctl+ISO croup, the incidence of DADs in CHF+ISO group increased significantly (P<0.05).3. Pacing protocols were given with 1 Hz to 3 Hz, the incidence of DADs in 1Hz group, 2Hz group, 3Hz group were 26.7%(12/45),76.2%(32/42),83.9%(26/31). Compared with 1Hz croup, the incidence of DADs in 2Hz group increased significantly (P<0.05). Compared with 3Hz croup, the incidence of DADs in 2Hz group increased significantly (P>0.05).4. Perfused with isoproterenol(1μmol/L) Tyrode's solution, pacing protocols were given with 1 Hz, the incidence of DADs in KN93 group was 50%(7/14), the inhibition reached 50%. The incidence of DADs in KN93 group decreased significantly (P<0.05). Pacing protocols were given with 2Hz, amplitude, coupled interval and counts of DADs were recorded. KN93 significantly reduced the amplitude of DADs from 5.83±1.90mV to 1.82±0.78mV (P<0.05). The coupled interval was dramatically prolonged ((250.44±108.36ms Vs 524.62±390.91ms, P<0.05), and counts of DADs were significantly reduced(4.95±0.95 Vs 2.42±0.90, P<0.05).5. Perfused with isoproterenol(1μmol/L) Tyrode's solution, pacing protocols were given with 1 Hz, the incidence of DADs in H89 group was 15.4%(2/13), the inhibition reached 84.6%. The incidence of DADs in H89 group decreased significantly (P<0.05). Pacing protocols were given with 2Hz, amplitude, coupled interval and counts of DADs were recorded. H89 significantly reduced the amplitude of DADs from 5.83±1.90mV to 1.65±0.77mV (P<0.05). The coupled interval was dramatically prolonged ((250.44±108.36ms Vs 837.48±313.53ms, P<0.05), and counts of DADs were significantly reduced (4.95±0.95 Vs 1.83±0.94, P<0.05).6. Perfused with isoproterenol(1μmol/L) Tyrode's solution, pacing protocols were given with 1 Hz, the inhibition in KN93 group and H89 group were 50% and 84.6% and there is no significant difference between the two group(P>0.05).Conclusion APD elongated in chronic heart failure myocytes. The incidence of DADs did not increase significantly until isoproterenol and high-frequency stimulation was given. Both KN93 and H89 can suppress DADs in chronic heart failure myocytes. But in either case the inhibition of DADs was not statistically significant. |
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