Ca2+ and ROS Crosstalk Signaling in Cardiac Mitochondria

维持线粒体功能对于心脏的日常运作至关重要。适当的线粒体功能需要维持线粒体内膜对离子和代谢物的调节和选择性渗透性。线粒体通透性转换发生在内膜通过打开大电导非选择性通道而失去其选择性通透性时，即通透性转换孔（PTP）。高浓度的线粒体Ca 2+和活性氧（ROS）是开放PTP的已知因素。PTP开放使线粒体去极化并引起线粒体肿胀;因此，PTP的持续开放导致线粒体功能障碍和细胞死亡，这与许多心血管疾病相关，包括缺血-再灌注（I-R）损伤和心力衰竭。因此，了解PTP是如何调节的具有重要的临床价值。人们早就知道，增加线粒体Ca 2+浓度打开PTP。最近，线粒体动力学介导的裂变和融合也被认为是参与调节PTP。然而，Ca 2+和线粒体动力学调节PTP的机制仍然未知。我们的新发现表明，增加线粒体Ca 2+通过激活线粒体中的GSK-3β诱导亲环素D（CypD）磷酸化。此外，我们已经发现了一个短暂的开放的PTP（tPTP），这是不同于传统的PTP和线粒体动力学蛋白的调节。抑制分裂蛋白Drp 1增加这种新的tPTP。重要的是，发现内膜融合蛋白OPA 1是新tPTP的关键因素。尽管已知Drp 1抑制可减少病理性PTP开放并减少I-R中的心肌梗死，但这种裂变抑制介导的保护机制尚不清楚。我们假设，线粒体动力学介导的新型tPTP在结构上与传统PTP不同，因此在病理条件下，可以作为过量基质Ca 2+和质子梯度的减压阀，诱导ROS过度产生;因此，它可以防止PTP的病理性开放。在我们的研究结果的支持下，中心假设是基质Ca 2+诱导的CypD磷酸化是PTP开放的关键事件，而线粒体动力学调节新的tPTP，它们的相互作用决定了心脏病理结果。我们将通过三个具体目标来验证这一假设：（1）确定Ca 2+诱导的PTP开放的机制，（2）确定线粒体动力学调节的新型tPTP开放的机制，（3）研究病理环境中常规PTP和新型tPTP之间的相互作用。拟议的研究将利用先进的体外和体内细胞和分子生物学方法，沿着新的基于荧光的测定。完成拟议的研究将产生一个新的范式的调节机制的不同形式的PTP及其功能的相互作用。这些新发现将为减少心脏I-R损伤和其他与PTP相关的心脏病理的新治疗策略提供机制基础。

Maintaining mitochondrial function is critical for everyday operation of the heart. Proper mitochondrial function requires maintaining regulated and selective permeability of the mitochondrial inner membrane to ions and metabolites. Mitochondrial permeability transition occurs when the inner membrane loses its selective permeability by opening of a large-conductance nonselective channel, the permeability transition pore (PTP). High concentrations of mitochondrial Ca2+ and reactive oxygen species (ROS) are known to open PTP. PTP opening depolarizes mitochondria and causes mitochondrial swelling; thus, sustained opening of PTP leads to mitochondrial dysfunction and cell death, which is associated with many cardiovascular diseases including ischemia-reperfusion (I-R) injury and heart failure. Therefore, understanding how PTP is regulated has significant clinical value. It has long been known that increasing mitochondrial Ca2+ concentration opens PTP. More recently, mitochondrial dynamics mediated by fission and fusion have also been suggested to be involved in regulating PTP. However, the mechanisms by which Ca2+ and mitochondrial dynamics regulate PTP remain unknown. Our new findings show that increasing mitochondrial Ca2+ induces phosphorylation of cyclophilin D (CypD) through GSK-3β activation in mitochondria. Furthermore, we have found a transient opening of PTP (tPTP) that is distinct from conventional PTP and is regulated by mitochondrial dynamics proteins. Inhibition of the fission protein Drp1 increases this novel tPTP. Importantly, the inner membrane fusion protein OPA1 was found to be a critical factor for the novel tPTP. Although Drp1 inhibition is known to decrease pathologic PTP opening and reduce myocardial infarction in I-R, the mechanism of this fission inhibition-mediated protection is unknown. We postulate that the mitochondrial dynamics-mediated novel tPTP is a structurally distinct entity from conventional PTP, and thus in pathological conditions, can serve as a relief valve for excess matrix Ca2+ and proton gradient that induces ROS overproduction; as such, it could thereby prevent pathologic opening of PTP. Supported by our findings, the Central Hypothesis is that CypD phosphorylation induced by matrix Ca2+ is a key event for PTP opening, while mitochondrial dynamics regulates novel tPTP, and their interplay determines cardiac pathology outcomes. We will test this hypothesis by three specific aims: (1) to determine the mechanism of Ca2+-induced PTP opening, (2) to determine the mechanism of mitochondrial dynamics-regulated novel tPTP opening, and (3) to investigate the interplay between conventional PTP and novel tPTP in the pathological setting. The proposed studies will utilize advanced in vitro and in vivo cell and molecular biological approaches along with new fluorescence-based assays. Completion of the proposed studies will generate a new paradigm for the regulatory mechanisms of different forms of PTP and their functional interplay. The new findings will provide mechanistic basis for a new therapeutic strategy to decrease heart I-R injury and other cardiac pathology associated with PTP.