BK channels as targets of cGMP signalling in myocardial pre- and postconditioning and survival

该项目涵盖了通过KATP （mitoKATP）和BK型（mitoBK）的线粒体K+通道对抗缺血和再灌注（I/R）损伤的心脏保护性cGMP信号的各个方面。先前，我们在体外Langendorff灌注装置中研究了全球bk缺失小鼠的心脏，发现心肌细胞（CMs）中的mitobk是梗死限制因素的证据。在缺乏BK的情况下，分离的CM线粒体缺氧后活性氧（ROS）的产生升高，表明mitoBK微调了缺氧和再氧化时的氧化状态。在与该研究小组成员的合作中，我们进一步证明了在缺血损伤（iPost）和cgmp升高药物（如西地那非）后直接应用的短暂缺血发作使心脏对I/R损伤的易感程度降低，但仅当no刺激的冠状动脉环化酶（NO-GC）和mitobk存在时。由于已经确定，通常存在于细胞质膜上的典型BK通道直接受到cGMP/cGMP依赖性蛋白激酶I型（cGKI）的刺激，因此iPost和/或cGMP升高化合物提供的心脏保护似乎可能需要CMs中的mitobk来防止体内细胞死亡。我们正在进行的研究将揭示局部和全局cGMP在再灌注缺血之前、期间和之后的动态变化。在存在和不存在cGMP升高药物和ii.内源性CM NO-GC、cGKI和BK的情况下，使用已建立的单细胞试验和来自转基因redDE5或cGi500 cGMP指标小鼠模型的完整跳动心脏，通过FRET成像观察CMs和不同非肌细胞类型中的cGMP。最后，我们的目标是研究cm中缺乏NO-GC、cGKI和BK的时间和组织特异性突变体对急性I/R设置和涉及梗死后重构过程的心肌模型的反应，以揭示NO-GC/cGMP和mitoBK如何对抗ROS过度产生和细胞死亡。该子项目还将揭示心肌梗死后心脏的长期生存、功能恢复和/或伤口愈合/再生过程是否需要CMs中的功能性cGMP途径。为了回答我们项目的所有主要问题，我们与这个研究单位的其他小组密切互动。通过持续的工具和技术交流，我们将提高我们对局部/区室化与全局cGMP信号传导的理解，以及NO-GC、cGKI和mitobk在缺血心脏及时再灌注或心肌梗死后长期随访中的作用，这将验证该途径的不同因素作为潜在的药物靶点。

This project covers various aspects of cardioprotective cGMP signalling opposing ischaemia and reperfusion (I/R) damages via mitochondrial K+ channels of the KATP (mitoKATP) and BK type (mitoBK). Previously we studied hearts obtained from global BK-null mice in an ex vivo Langendorff perfusion setup and found evidence for mitoBKs in cardiac myocytes (CMs) as infarct-limiting factors. In the absence of BK, post-anoxic reactive oxygen species (ROS) production from isolated CM mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and re-oxygenation. In collaboration with members of this research unit we further demonstrated that brief episodes of ischaemia applied directly after an ischemic insult (iPost) and cGMP-elevating drugs such as sildenafil render the heart less susceptible to the I/R injury, but only if NO-stimulated guanylyl cyclase (NO-GC) and mitoBKs were present in the CM. Because it is well established that canonical BK channels usually present at the plasma membrane of cells are directly stimulated by cGMP/cGMP-dependent protein kinase type I (cGKI) it seems possible that cardioprotection afforded by iPost and/or cGMP-elevating compounds require mitoBKs in CMs to prevent cell death in vivo. Our ongoing studies will now reveal the dynamics of local and global cGMP prior, during and upon a period of ischaemia following reperfusion. cGMP in the CMs and in different non-myocyte cell types will be visualized by FRET imaging in the presence and absence of i.) cGMP-elevating drugs and ii.) endogenous CM NO-GC, cGKI and BK using established single cell assays and intact beating hearts derived from transgenic redDE5 or cGi500 cGMP indicator mouse models. Finally, we aim to study the response of our time- and tissue-specific mutants lacking either NO-GC, cGKI and BK in CMs to acute I/R setups and to models involving post-infarction remodeling processes of the myocardium to uncover how NO-GC/cGMP and mitoBK oppose e.g. ROS overproduction and cell death. This subproject will also reveal if long-term survival, functional recovery and/or wound healing/regenerative processes in the post-infarcted heart require a functional cGMP pathway in CMs. To answer all major questions of our project, we strongly interact with the other groups of this research unit. By continuing exchange in tools and techniques we will improve our understanding of local/compartmentalized vs global cGMP signalling and the role of NO-GC, cGKI and mitoBKs in ischemic hearts with either timely reperfusion or during long-term follow up after MI, which should validate the different factors of this pathway as potentially druggable targets.