Mitochondriales Connexin 43: Regulation und Funktion

心肌细胞连接蛋白43(Cx43)在缺血预适应(IP)所致的心肌保护中起一定作用，因为在杂合子Cx43缺陷小鼠心脏和分离的心肌细胞中，IP减少了心肌梗死面积。除了Cx43定位于心肌细胞肌膜外，我们还证明了Cx43存在于小鼠、大鼠、猪和人的心肌线粒体中，并且Cx43在预适应心肌线粒体中的含量高于非预适应心肌线粒体。Cx43在线粒体中的确切定位以及调节其输入或降解的机制尚不清楚。因此，在没有或存在特定的蛋白激酶(A、C、G或MAPK)抑制剂和线粒体Cx43水平的情况下，缺血/再灌流将刺激大鼠心脏和心肌细胞的信号转导通路，并测量其确切的定位(外膜/内膜或基质)。或者，蛋白激酶将被特定的激动剂刺激，线粒体Cx43含量的变化将被评估。为了确定Cx43在线粒体中的功能，将通过注射4-羟基三苯氧胺在转基因小鼠(CRE/FLOX)中诱导其完全消融，并在缺血/再灌注前后测量线粒体膜电位、呼吸、肿胀、通透性转换孔的开放和自由基形成；在后者的实验装置中，将在活体心脏中单独评估梗死面积。

Cardiomyocyte connexin43 (Cx43) plays a causal role in the cardioprotecion by ischemia preconditioning (IP), since infarct size reduction by IP is lost in heterozygous Cx43 deficient mice hearts and isolated cardiomyocytes. Apart from Cx43 being localized at the cardiomyocyte sarcolemma, we have demonstrated that Cx43 is present in mitochondria isolated from mice, rat, pig and human LV myocardium; the Cx43 content is higher in mitochondria isolated from preconditioned than from non-preconditioned myocardium. The exact localization of Cx43 within mitochondria and the mechanisms regulating its import or degradation are unknown. Therefore, signal transduction pathways will be stimulated by ischemia/reperfusion in isolated rat hearts and cardiomyocytes in the absence or presence of specific protein kinase (A,C,G or MAP kinase) inhibitors and rnitochondrial Cx43 levels and its exact localization (outer/inner membrane or matrix) will be measured. Alternatively, protein kinases will be stimulated by specific agonists and changes in mitochondrial Cx43 content will be assessed. To determine Cx43 function in mitochondria, its complete ablation will be induced in genetically modified mice (cre/flox) by the injection of 4-hydroxytamoxifen, and mitochondrial membrane potential, respiration, swelling, opening of the permeability transition pore and radical formation will be measured before and following ischemia/reperfusion; in the latter experimental setup, infarct size will be assessed separately in in vivo hearts.