Ca2+ and ROS Crosstalk Signaling in Cardiac Mitochondria

维持线粒体的功能对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的通透性。线粒体通透性转换发生在内膜打开一个大电导的非选择性通道--通透性转换孔(PTP)而失去选择性通透性时。众所周知，高浓度的线粒体钙和活性氧物种(ROS)可以打开PTP。PTP开放使线粒体去极化，导致线粒体肿胀，导致线粒体功能障碍和细胞死亡，与许多心血管疾病有关，包括缺血再灌注损伤和心力衰竭。因此，了解PTP是如何被调控的具有重要的临床价值。人们很早就知道，增加线粒体钙浓度可以打开PTP。最近，由分裂和融合介导的线粒体动力学也被认为参与了对PTP的调控。然而，钙离子和线粒体动力学调节PTP的机制仍不清楚。我们的新发现表明，线粒体钙的增加通过激活线粒体中的GSK-3β来诱导亲环素D的磷酸化。此外，我们还发现了一种不同于传统的PTP的瞬时开放的PTP(TPTP)，它受线粒体动力学蛋白的调节。抑制分裂蛋白Drp1增加了这种新的tPTP。重要的是，内膜融合蛋白OPA1被发现是新的tPTP的关键因素。虽然已知抑制Drp1可以减少病理性PTP开放和减少I-R心肌梗死，但这种裂变抑制介导的保护机制尚不清楚。我们推测，线粒体动力学介导的新型tPTP在结构上不同于传统的PTP，因此在病理条件下，它可以作为诱导ROS过度产生的过量基质钙和质子梯度的减压阀，从而防止PTP的病理性开放。在我们的研究结果的支持下，中心假说是基质钙诱导的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，它们之间的相互作用决定了心脏病理结果。我们将通过三个特定的目的来验证这一假说：(1)确定钙诱导的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，以及(3)在病理背景下研究传统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以及新的基于荧光的分析方法。拟议研究的完成将为不同形式的PTP的调节机制及其功能相互作用产生一个新的范式。这一新的发现将为减少心脏I-R损伤和与PTP相关的其他心脏病理的新的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.