Gissibl Theresa, Stengel Laura, Tarnowski Daniel, Maier Lars S, Wagner Stefan, Feder Anna-Lena, Sag Can Martin
Department of Internal Medicine II/Cardiology, University Medical Center Regensburg, Regensburg, Germany.
Front Cardiovasc Med. 2024 Jul 15;11:1379930. doi: 10.3389/fcvm.2024.1379930. eCollection 2024.
Acute stimulation of the late sodium current (I) as pharmacologically induced by Anemonia toxin II (ATX-II) results in Na-dependent Ca overload and enhanced formation of reactive oxygen species (ROS). This is accompanied by an acute increase in the amplitude of the systolic Ca transient. Ca transient amplitude is determined by L-type Ca-mediated transsarcolemmal Ca influx (I) into the cytosol and by systolic Ca release from the sarcoplasmic reticulum (SR). Type-1 protein kinase A (PKARIα) becomes activated upon increased ROS and is capable of stimulating I, thereby sustaining the amplitude of the systolic Ca transient upon oxidative stress.
We aimed to investigate whether the increase of the systolic Ca transient as acutely induced by I (by ATX-II) may involve stimulation of I through oxidized PKARIα.
We used a transgenic mouse model in which PKARIα was made resistant to oxidative activation by homozygous knock-in replacement of redox-sensitive Cysteine 17 with Serine within the regulatory subunits of PKARIα (KI). ATX-II (at 1 nmol/L) was used to acutely enhance I in freshly isolated ventricular myocytes from KI and wild-type (WT) control mice. Epifluorescence and confocal imaging were used to assess intracellular Ca handling and ROS formation. A ruptured-patch whole-cell voltage-clamp was used to measure I and I. The impact of acutely enhanced I on RIα dimer formation and PKA target structures was studied using Western blot analysis.
ATX-II increased I to a similar extent in KI and WT cells, which was associated with significant cytosolic and mitochondrial ROS formation in both genotypes. Acutely activated Ca handling in terms of increased Ca transient amplitudes and elevated SR Ca load was equally present in KI and WT cells. Likewise, cellular arrhythmias as approximated by non-triggered Ca elevations during Ca transient decay and by diastolic SR Ca-spark frequency occurred in a comparable manner in both genotypes. Most importantly and in contrast to our initial hypothesis, ATX-II did not alter the magnitude or inactivation kinetics of I in neither WT nor KI cells and did not result in PKARIα dimerization (i.e., oxidation) despite a clear prooxidant intracellular environment.
The inotropic and arrhythmogenic effects of acutely increased I are associated with elevated ROS, but do not involve oxidation of PKARIα.
海葵毒素II（ATX-II）药理学诱导的晚期钠电流（I）急性刺激会导致钠依赖性钙超载和活性氧（ROS）生成增加。这伴随着收缩期钙瞬变幅度的急性增加。钙瞬变幅度由L型钙介导的跨肌膜钙内流（I）进入细胞质以及肌浆网（SR）的收缩期钙释放决定。1型蛋白激酶A（PKARIα）在ROS增加时被激活，并且能够刺激I，从而在氧化应激时维持收缩期钙瞬变的幅度。
我们旨在研究由I（通过ATX-II）急性诱导的收缩期钙瞬变增加是否可能涉及通过氧化的PKARIα刺激I。
我们使用了一种转基因小鼠模型，其中通过在PKARIα调节亚基内将氧化还原敏感的半胱氨酸17纯合敲入替换为丝氨酸，使PKARIα对氧化激活具有抗性（KI）。ATX-II（1 nmol/L）用于急性增强来自KI和野生型（WT）对照小鼠的新鲜分离心室肌细胞中的I。落射荧光和共聚焦成像用于评估细胞内钙处理和ROS生成。破裂膜片全细胞电压钳用于测量I和I。使用蛋白质印迹分析研究急性增强的I对RIα二聚体形成和PKA靶结构的影响。
ATX-II在KI和WT细胞中使I增加到相似程度，这与两种基因型中显著的细胞质和线粒体ROS生成相关。在KI和WT细胞中，就增加的钙瞬变幅度和升高的SR钙负荷而言，急性激活的钙处理同样存在。同样，在两种基因型中，由钙瞬变衰减期间的非触发钙升高和舒张期SR钙火花频率近似的细胞心律失常以可比的方式发生。最重要的是，与我们最初的假设相反，ATX-II在WT和KI细胞中均未改变I的幅度或失活动力学，并且尽管细胞内环境明显促氧化，但并未导致PKARIα二聚化（即氧化）。
急性增加的I的变力和致心律失常作用与ROS升高有关，但不涉及PKARIα的氧化。
