Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, Oslo, Norway; KG Jebsen Centre for Cardiac Research, University of Oslo, Oslo, Norway.
Division of Cardiology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
J Mol Cell Cardiol. 2023 Feb;175:49-61. doi: 10.1016/j.yjmcc.2022.12.002. Epub 2022 Dec 14.
Available evidence suggest that Ca/calmodulin-dependent protein kinase type IIδ (CaMKIIδ) and reactive oxygen species (ROS) are important in early ischemia-reperfusion arrhythmias (IRA). Since ROS can activate CaMKIIδ by oxidation of two methionines at positions 281/282, oxidized-CaMKIIδ (Ox-CaMKIIδ) has been proposed to be important for IRA. However, direct evidence for this is missing.
We exposed Langendorff-perfused hearts and ventricular cardiomyocytes from C57BL/6 mice to global and simulated ischemia, respectively, and recorded arrhythmic events during early reperfusion. Hearts were collected for immunoblotting of key phosphoproteins. We evaluated the effects of beta-adrenoceptor stimulation, inhibition of CaMKII, and reduced ROS levels with isoprenaline, KN93/AIP and N-acetylcysteine (NAC), respectively. We further tested the importance of Ox-CaMKIIδ by using hearts and cardiomyocytes from mice with CaMKIIδ resistant to oxidation of methionines 281 and 282 (MMVV).
Hearts treated with KN93, AIP or NAC had lower incidence of early IRA, and NAC-treated cardiomyocytes had lower incidence of arrhythmogenic events. However, hearts from MMVV mice had a similar incidence of early IRA to wild type mice (WT), and MMVV and WT cardiomyocytes had a similar frequency of Ca waves and Ca sparks. Immunoblotting confirmed high levels of oxidation in early reperfusion, but revealed no significant differences in the phosphorylation levels of Ca-handling proteins in MMVV and WT hearts.
Although CaMKII and ROS both contribute to early IRA, hearts from mice with CaMKII resistant to oxidation at methionines 281/282 were not protected from such arrhythmias, suggesting that oxidation at these sites is not a determining factor.
现有证据表明，钙/钙调蛋白依赖性蛋白激酶 II 型 δ（CaMKIIδ）和活性氧（ROS）在早期缺血再灌注性心律失常（IRA）中很重要。由于 ROS 可以通过氧化位置 281/282 上的两个蛋氨酸来激活 CaMKIIδ，因此氧化型 CaMKIIδ（Ox-CaMKIIδ）被认为对 IRA 很重要。然而，目前还缺乏直接证据。
我们分别将 Langendorff 灌流心脏和来自 C57BL/6 小鼠的心室肌细胞暴露于整体和模拟缺血中，并在早期再灌注期间记录心律失常事件。收集心脏进行关键磷酸化蛋白的免疫印迹分析。我们分别用异丙肾上腺素、KN93/AIP 和 N-乙酰半胱氨酸（NAC）评估β-肾上腺素能受体刺激、CaMKII 抑制和降低 ROS 水平的作用。我们进一步使用对蛋氨酸 281 和 282 氧化具有抗性的 CaMKIIδ （MMVV）的心脏和心肌细胞来测试 Ox-CaMKIIδ 的重要性。
用 KN93、AIP 或 NAC 处理的心脏 IRA 早期发生率较低，用 NAC 处理的心肌细胞心律失常事件发生率较低。然而，MMVV 小鼠的心脏与野生型（WT）小鼠的 IRA 早期发生率相似，MMVV 和 WT 心肌细胞的钙波和钙火花频率相似。免疫印迹证实早期再灌注时氧化水平较高，但 MMVV 和 WT 心脏的钙处理蛋白磷酸化水平没有显著差异。
尽管 CaMKII 和 ROS 均有助于早期 IRA，但对蛋氨酸 281/282 处 CaMKII 氧化具有抗性的小鼠心脏并未免受此类心律失常的影响，这表明这些位点的氧化不是决定因素。
