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信号传导的各个方面。以前，我们研究了心脏从全球BK-空小鼠在离体Langendorff灌注设置，并发现心肌细胞（CM）的心肌梗死限制因素的mitoBK的证据。在没有BK的情况下，缺氧后活性氧（ROS）的生产从分离的CM线粒体升高，表明mitoBK微调缺氧和复氧时的氧化状态。在与该研究单位的成员合作中，我们进一步证明了在缺血性损伤（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损伤的敏感性降低，但仅当CM中存在NO刺激的鸟苷酸环化酶（NO-GC）和mitoBK时。因为已经充分确定，通常存在于细胞质膜处的典型BK通道直接受到cGMP/cGMP依赖性蛋白激酶I型（cGKI）的刺激，所以iPost和/或cGMP升高化合物提供的心脏保护似乎可能需要CM中的mitoBK来防止体内细胞死亡。我们正在进行的研究现在将揭示局部和全球cGMP的动态之前，期间和缺血后再灌注一段时间。CM和不同非肌细胞细胞类型中的cGMP将在存在和不存在i.）cGMP升高药物和ii.）使用已建立的单细胞测定和源自转基因redDE 5或cGi 500 cGMP指示小鼠模型的完整跳动心脏测定内源性CM NO-GC、cGKI和BK。最后，我们的目标是研究我们的时间和组织特异性突变体缺乏NO-GC，cGKI和BK的CM急性I/R设置和模型，涉及心肌梗死后重塑过程的反应，以揭示如何NO-GC/cGMP和mitoBK反对，例如ROS过度生产和细胞死亡。该子项目还将揭示梗死后心脏的长期存活、功能恢复和/或伤口愈合/再生过程是否需要CM中的功能性cGMP途径。为了回答我们项目的所有主要问题，我们与该研究单位的其他小组进行了密切的互动。通过继续交流工具和技术，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在缺血心脏中的作用，无论是及时再灌注还是在MI后的长期随访期间，这应该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.