Mitochondrial connexin43 and mitochondrial KATP channels modulate triggered arrhythmias in mouse ventricular muscle
Mitochondrial connexin43 and mitochondrial KATP channels modulate triggered arrhythmias in mouse ventricular muscle
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DOI：
10.1007/s00424-023-02789-w
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发表时间：
2023-01-28
影响因子：
4.5
通讯作者：
Miura，Masahito
中科院分区：
文献类型：
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作者：
Sato，Haruka;Nishiyama，Masami;Miura，Masahito
连接蛋白43(Cx43)在线粒体膜内以半沟的形式存在。我们研究了线粒体Cx43和线粒体KATP通道如何影响触发的心律失常的发生。为获得心脏特异性Cx43缺陷(Cx43−/−)小鼠，将Cx43Flox/Flox小鼠与α-MHC(MYH6)-Cre+/−小鼠杂交。所产生的后代Cx43Flox/Flox/MYH6-Cre+/−小鼠(CCx43−/−小鼠)及其子代(CCx43+/+小鼠)被用来进行研究。从小鼠右心室解剖出小梁。用酶法从小鼠的心室中分离出心肌细胞。用应变计测量小梁(22℃)的力。为评估心律失常的易感性，测定了小梁细胞电刺激诱发心律失常时的最低细胞外钙浓度([Ca~(2+)]_0，min)。用2‘，7’-二氯荧光素(DCF)、四甲基罗丹明甲酯(TMRM)测定线粒体膜电位，用Fluo-4和共聚焦显微镜观察心肌细胞内钙离子放电频率。用MitoSoxRed法检测线粒体内ROS的生成，用Rhod-2法检测线粒体内钙离子的生成。用二氮嗪激活线粒体KATP。CCx43−/−小鼠大多在8周内猝死。Cx43存在于incCx43+/+小鼠的线粒体膜，而不存在于incCx43−/−小鼠。在Cx43−/−小鼠中，[Ca~(2+)]o，min较低，而Ca~(2+)放电频率、荧光强度斜率、MitoSoxRed荧光和Rod-2荧光较高。在Cx43−/−小鼠中，TMRM荧光强度下降更明显。这些变化大部分被二氮嗪抑制。此外，−/−小鼠、抗氧化肽SS-31和N-乙酰-L-半胱氨酸均可使小鼠脑内[Ca~(2+)]_0，min升高。这些结果提示，Cx43缺乏可能通过线粒体膜电位的去极化、线粒体钙离子的增加和ROS生成的增加而激活SR的钙外泄，从而导致触发的心律失常，Cx43半通道缺乏可能通过激活线粒体KATP通道来补偿。
Connexin43 (Cx43) exits as hemichannels in the inner mitochondrial membrane. We examined how mitochondrial Cx43 and mitochondrial KATPchannels affect the occurrence of triggered arrhythmias. To generate cardiac-specific Cx43-deficient (cCx43−/−) mice,Cx43flox/floxmice were crossed with α-MHC (Myh6)-cre+/−mice. The resulting offspring,Cx43flox/flox/Myh6-cre+/−mice (cCx43−/−mice) and their littermates (cCx43+/+mice), were used. Trabeculae were dissected from the right ventricles of mouse hearts. Cardiomyocytes were enzymatically isolated from the ventricles of mouse hearts. Force was measured with a strain gauge in trabeculae (22°C). To assess arrhythmia susceptibility, the minimal extracellular Ca2+concentration ([Ca2+]o,min), at which arrhythmias were induced by electrical stimulation, was determined in trabeculae. ROS production was estimated with 2′,7′-dichlorofluorescein (DCF), mitochondrial membrane potential with tetramethylrhodamine methyl ester (TMRM), and Ca2+spark frequency with fluo-4 and confocal microscopy in cardiomyocytes. ROS production within the mitochondria was estimated with MitoSoxRed and mitochondrial Ca2+with rhod-2 in trabeculae. Diazoxide was used to activate mitochondrial KATP. Most ofcCx43−/−mice died suddenly within 8 weeks. Cx43 was present in the inner mitochondrial membrane incCx43+/+mice but not incCx43−/−mice. IncCx43−/−mice, the [Ca2+]o,minwas lower, and Ca2+spark frequency, the slope of DCF fluorescence intensity, MitoSoxRed fluorescence, and rhod-2 fluorescence were higher. TMRM fluorescence was more decreased incCx43−/−mice. Most of these changes were suppressed by diazoxide. In addition, incCx43−/−mice, antioxidant peptide SS-31 and N-acetyl-L-cysteine increased the [Ca2+]o,min. These results suggest that Cx43 deficiency activates Ca2+leak from the SR, probably due to depolarization of mitochondrial membrane potential, an increase in mitochondrial Ca2+, and an increase in ROS production, thereby causing triggered arrhythmias, and that Cx43 hemichannel deficiency may be compensated by activation of mitochondrial KATPchannels in mouse hearts.