Department of Internal Medicine II, University Medical Center Regensburg, Germany.
Department of Internal Medicine III, University Medical Center Regensburg, Germany.
Am J Physiol Heart Circ Physiol. 2021 Mar 1;320(3):H1199-H1212. doi: 10.1152/ajpheart.00040.2020. Epub 2021 Jan 15.
CaMKII is needed for the recovery of Ca transients during acidosis but also mediates postacidic arrhythmias. CaMKIIδ can sustain its activity following Met281/282 oxidation. Increasing cytosolic Na during acidosis as well as postacidic pH normalization should result in prooxidant conditions within the cell favoring oxidative CaMKIIδ activation. We tested whether CaMKIIδ activation through Met281/282 oxidation is involved in recovery of Ca transients during acidosis and promotes cellular arrhythmias post-acidosis. Single cardiac myocytes were isolated from a well-established mouse model in which CaMKIIδ was made resistant to oxidative activation by knock-in replacement of two oxidant-sensitive methionines (Met281/282) with valines (MM-VV). MM-VV myocytes were exposed to extracellular acidosis (pH 6.5) and compared to wild type (WT) control cells. Full recovery of Ca transients was observed in both WT and MM-VV cardiac myocytes during late-phase acidosis. This was associated with comparably enhanced sarcoplasmic reticulum Ca load and preserved CaMKII specific phosphorylation of phospholamban at Thr17 in MM-VV myocytes. CaMKII was phosphorylated at Thr287, but not Met281/282 oxidized. In line with this, postacidic cellular arrhythmias occurred to a similar extent in WT and MM-VV cells, whereas inhibition of CaMKII using AIP completely prevented recovery of Ca transients during acidosis and attenuated postacidic arrhythmias in MM-VV cells. Using genetically altered cardiomyocytes with cytosolic expression of redox-sensitive green fluorescent protein-2 coupled to glutaredoxin 1, we found that acidosis has a reductive effect within the cytosol of cardiac myocytes despite a significant acidosis-related increase in cytosolic Na. Our study shows that activation of CaMKIIδ through Met281/282 oxidation is neither required for recovery of Ca transients during acidosis nor relevant for postacidic arrhythmogenesis in isolated cardiac myocytes. Acidosis reduces the cytosolic glutathione redox state of isolated cardiac myocytes despite a significant increase in cytosolic Na. Pharmacological inhibition of global CaMKII activity completely prevents recovery of Ca transients and protects from postacidic arrhythmias in MM-VV myocytes, which confirms the relevance of CaMKII in the context of acidosis. The current study shows that activation of CaMKIIδ through Met281/282 oxidation is neither required for CaMKII-dependent recovery of Ca transients during acidosis nor relevant for the occurrence of postacidic cellular arrhythmias. Despite a usually prooxidant increase in cytosolic Na, acidosis reduces the cytosolic glutathione redox state within cardiac myocytes. This novel finding suggests that oxidation of cytosolic proteins is less likely to occur during acidosis.
钙调蛋白依赖性激酶 II（CaMKII）对于酸中毒期间钙瞬变的恢复是必需的，但也介导了酸中毒后的心律失常。Met281/282 氧化后，CaMKIIδ 可以维持其活性。酸中毒期间细胞溶质 Na 的增加以及酸中毒后的 pH 值正常化，应该会导致细胞内产生有利于氧化 CaMKIIδ 激活的促氧化剂条件。我们测试了通过 Met281/282 氧化激活 CaMKIIδ 是否参与酸中毒期间钙瞬变的恢复，并促进酸中毒后的细胞心律失常。从一个经过充分验证的小鼠模型中分离出单个心肌细胞，该模型通过 knock-in 替换两个氧化剂敏感的蛋氨酸（Met281/282）为缬氨酸（MM-VV），使 CaMKIIδ 对氧化激活具有抗性。将 MM-VV 心肌细胞暴露于细胞外酸中毒（pH 6.5）中，并与野生型（WT）对照细胞进行比较。在晚期酸中毒期间，WT 和 MM-VV 心肌细胞均观察到钙瞬变的完全恢复。这与肌浆网 Ca 负荷的明显增强以及 MM-VV 心肌细胞中肌球蛋白轻链磷酸酶 Thr17 的 CaMKII 特异性磷酸化有关。CaMKII 被磷酸化 Thr287，但不是 Met281/282 氧化。与此一致的是，WT 和 MM-VV 细胞中发生的酸中毒后细胞心律失常的程度相似，而使用 AIP 抑制 CaMKII 完全阻止了酸中毒期间钙瞬变的恢复，并减轻了 MM-VV 细胞中的酸中毒后心律失常。使用与谷胱甘肽还原酶 1 偶联的氧化还原敏感的绿色荧光蛋白-2 表达的遗传改变的心肌细胞，我们发现尽管细胞溶质 Na 显著增加，但酸中毒在心肌细胞的细胞溶质中具有还原作用。我们的研究表明，CaMKIIδ 通过 Met281/282 氧化的激活对于酸中毒期间钙瞬变的恢复既不是必需的，也与孤立心肌细胞中的酸中毒后心律失常无关。尽管细胞溶质 Na 显著增加，但酸中毒降低了分离的心肌细胞的细胞溶质谷胱甘肽氧化还原状态。在 MM-VV 心肌细胞中，抑制全球 CaMKII 活性可完全阻止钙瞬变的恢复并防止酸中毒后心律失常，这证实了 CaMKII 在酸中毒情况下的相关性。目前的研究表明，通过 Met281/282 氧化激活 CaMKIIδ 对于 CaMKII 依赖性钙瞬变的恢复既不是必需的，也与酸中毒后细胞心律失常的发生无关。尽管细胞溶质 Na 通常呈促氧化剂增加，但酸中毒降低了心肌细胞中的细胞溶质谷胱甘肽氧化还原状态。这一新发现表明，在酸中毒期间，细胞溶质蛋白的氧化不太可能发生。
