Department of Pathophysiology, Shanxi Medical University.
Department of Biochemistry, Shanghai University of Traditional Chinese Medicine.
Int Heart J. 2021;62(6):1348-1357. doi: 10.1536/ihj.21-379.
Inward rectifier potassium channels (I, Kir) are known to play critical roles in arrhythmogenesis. Thus, how I agonist affects reperfusion arrhythmias needs to be clarified, and its underlying mechanisms should be determined. Reperfusion arrhythmias were modeled by coronary ligation (ischemia, 15 minutes) and release (reperfusion, 15 minutes). Zacopride (1.5-50 μg/kg in vivo, or 0.1-10 μmol/Lex vivo) was applied in the settings of pretreatment (3 minutes before coronary ligation) and posttreatment (5 minutes after coronary ligation). Hypoxia (45 minutes) /reoxygenation (30 minutes) model was established in cultured H9c2 (2-1) cardiomyocytes. Zacopride or KN93 was applied before hypoxia (pretreatment). In the setting of pre- or posttreatment, zacopride at 15 μg/kg in vivo or 1 μmol/Lin vitro exhibited superlative protections on reperfusion arrhythmias or intracellular calcium overload. Western blot data from ex vivo hearts or H9c2 (2-1) cardiomyocytes showed that I/R (H/R) induced the inhibition of Kir2.1 (the dominant subunit of I channel in ventricle), phosphorylation and oxidation of CaMKII, downregulation of SERCA2, phosphorylation of phospholamban (at Thr17), and activation of caspase-3. Zacopride treatment (1 μmol/L) was noted to strikingly restore the expression of Kir2.1 and SERCA2 and decrease the activity of CaMKII, phospholamban, and caspase-3. These effects were largely eliminated by co-application of I blocker BaCl. CaMKII inhibitor KN93 attenuated calcium overload and p-PLB (Thr17) in an I-independent manner. I-depedent inhibition of CaMKII activity is found to be a key cardiac salvage signaling under Ca dyshomeostasis and reactive oxygen species (ROS) stress. I might be a novel target for pharmacological conditioning of reperfusion arrhythmia, especially for the application after unpredictable ischemia.
内向整流钾通道（I，Kir）在心律失常发生中起着关键作用。因此，需要阐明 I 激动剂如何影响再灌注心律失常，确定其潜在机制。通过冠状动脉结扎（缺血，15 分钟）和释放（再灌注，15 分钟）来模拟再灌注心律失常。Zacopride（体内 1.5-50μg/kg，或离体 0.1-10μmol/L）应用于预处理（冠状动脉结扎前 3 分钟）和后处理（冠状动脉结扎后 5 分钟）。在培养的 H9c2（2-1）心肌细胞中建立缺氧（45 分钟）/复氧（30 分钟）模型。Zacopride 或 KN93 在缺氧前（预处理）应用。在预处理或后处理的情况下，体内 15μg/kg 或离体 1μmol/L 的 Zacopride 对再灌注心律失常或细胞内钙超载表现出卓越的保护作用。来自离体心脏或 H9c2（2-1）心肌细胞的 Western blot 数据表明，I/R（H/R）诱导 Kir2.1（心室中 I 通道的主要亚基）的抑制、CaMKII 的磷酸化和氧化、SERCA2 的下调、磷蛋白（在 Thr17 处）的磷酸化和 caspase-3 的激活。Zacopride 处理（1μmol/L）被发现显著恢复 Kir2.1 和 SERCA2 的表达，并降低 CaMKII、磷蛋白和 caspase-3 的活性。这些作用在共同应用 I 阻滞剂 BaCl 时被大大消除。CaMKII 抑制剂 KN93 以 I 非依赖性方式减轻钙超载和 p-PLB（Thr17）。在钙失调和活性氧（ROS）应激下，发现 CaMKII 活性的 I 依赖性抑制是一种关键的心脏挽救信号。I 可能是再灌注心律失常药理学调节的新靶点，特别是在不可预测的缺血后应用。
