The physiologic and genomic relevance of mitoregulin in ischemic heart failure

项目摘要/摘要 心肌梗塞(MI)是世界范围内发病率和死亡率的主要原因。虽然再灌流疗法 改善心肌梗死后的存活率，迅速恢复到心脏的有氧血流会引发氧化应激， 进一步损害组织，称为缺血-再灌注(IR)损伤，加剧一个人发展为心力衰竭的风险 (Hf)。线粒体是IR损伤的中心，它在心肌细胞的钙缓冲和产生 能量和活性氧物种(ROS)。在IR时，线粒体钙超载和ROS触发线粒体 通透性转换(MPT)孔道开放，促进细胞死亡。虽然许多研究强调了 MPT在IR损伤和心肌保护中的作用，导致线粒体钙和ROS改变的多个分子途径 (MPT的关键上游监管机构)仍然定义不清。在最近的研究中，我们发现心脏和肌肉- 富含长的“非编码”RNA实际上编码的是高度保守的单程跨膜微... 定位于线粒体内膜的蛋白质，我们将其命名为“MitoRegin”(MTLN)。在收益方面--和 在功能丧失研究中，我们发现Mtln通过增加线粒体的呼吸超氧化物歧化能力来给线粒体“增压”。 复杂的水平，2)呼吸效率，以及3)钙离子保持能力，同时降低ROS。在这笔赠款中，我们 该提案旨在解决两个中心假设：1)MTLN通过延迟钙离子和钙离子来保护心脏IR损伤 ROS触发MPT，以及2)与心力衰竭患者相关的与mtln表达相关的人类遗传变异 结果。我们的首要目标是在翻译上将MTLN与心脏IR损伤和心衰预后联系起来，以证明需要 以便未来的研究继续定义Mtln的确切行动模式。在目标1中，我们将定义生理学和 Mtln在心肌细胞中的分子功能研究 培养的啮齿动物心肌细胞线粒体呼吸、钙离子和ROS，以及超复合体水平。我们会 还定义了Mtln结构域和其功能所必需的蛋白质相互作用。在目标2中，我们将阐明Mtln的角色 在细胞和啮齿类动物的IR损伤和HF模型中，通过功能获得和丧失的研究来评估MTLN的影响 对模拟IR诱导的心肌细胞死亡以及对心脏IR损伤和随后的反应的调制 对小鼠的心力衰竭。在目标3中，我们将致力于建立遗传变异、心脏mtln表达和心力衰竭患者之间的联系。 结果，重点是36个碱基的3‘非编码区缺失变异体(Mtln-3’UTRdel)的生物学和遗传学相关性 存在于12%的普通人群中，已知与心脏组织中Mtln mRNA水平的降低有关。 我们将识别控制Mtln表达的RNA结合蛋白，并评估3‘UTRdel变体对Mtln表达的影响 这一调节和对人心脏中Mtln蛋白水平的影响。我们还将测试现有的与临床有关的DNA样本 以评估Mtln-3‘UTRdel基因与心力衰竭发病率和预后之间的关系(例如： 死亡率和心律失常事件)。这些多学科研究具有巨大的潜力：1)转变我们的 基础线粒体生物学知识，2)确定mtln是否代表HF治疗的可行靶点，以及3)揭示 新的遗传标记可以转化为改进高危心力衰竭患者的识别和治疗。

PROJECT SUMMARY / ABSTRACT Myocardial infarction (MI) is a primary cause of morbidity and mortality worldwide. Although reperfusion therapy improves post-MI survival, the rapid restoration of oxygenated blood flow to the heart elicits oxidative stress that further damages tissue, known as ischemia-reperfusion (IR) injury, exacerbating one’s risk for developing heart failure (HF). Central to IR injury are mitochondria, which play key roles in cardiomyocyte Ca2+ buffering and production of energy and reactive oxygen species (ROS). Upon IR, mitochondrial Ca2+ overload and ROS trigger mitochondrial permeability transition (mPT) pore opening, which promotes cell death. While many studies highlight the critical role of mPT in IR injury and cardioprotection, many molecular pathways leading to altered mitochondrial Ca2+ and ROS (key upstream regulators of mPT) remain poorly defined. In recent work, we discovered that a heart- and muscle- enriched long, “non-coding” RNA actually does encode for a highly-conserved single-pass transmembrane micro- protein that localizes to the inner mitochondrial membrane; we named this protein “Mitoregulin” (Mtln). In gain- and loss-of-function studies, we found that Mtln “supercharges” mitochondria by increasing their 1) respiratory super- complex levels, 2) respiratory efficiencies, and 3) Ca2+ retention capacities, while reducing ROS. In this grant, we propose Aims to address two central hypotheses: 1) that Mtln protects against cardiac IR injury by delaying Ca2+- and ROS-triggered mPT, and 2) that human genetic variation linked to Mtln expression associate with HF patient outcomes. Our overarching goal is to translationally link Mtln with cardiac IR injury and HF outcomes to justify a need for future studies to continue defining Mtln’s precise modes of action. In Aim 1, we will define the physiologic and molecular functions of Mtln in cardiomyocytes by evaluating the acute effects of modulating Mtln expression on mitochondrial respiration, Ca2+ and ROS, as well as supercomplex levels in cultured rodent cardiomyocytes. We will also define Mtln domains and protein interactions necessary for its function. In Aim 2, we will elucidate a role for Mtln in cell and rodent models of IR injury and HF through gain- and loss-of-function studies to evaluate the impact of Mtln modulation on cardiomyocyte death induced by simulated IR, as well as on responses to cardiac IR injury and ensuing HF in mice. In Aim 3, we will work to establish links among genetic variants, cardiac Mtln expression, and HF patient outcomes, focusing on the biological and genetic relevance of a 36-bp 3’UTR deletion variant (Mtln-3’UTRdel) that is present in 12% of the general population and is known to be linked to decreased Mtln mRNA levels in heart tissues. We will identify RNA-binding proteins that control Mtln expression and assess the impact of the 3’UTRdel variant on this regulation and on Mtln protein levels in human hearts. We will also test available DNA samples linked to clinical data in patient cohorts to assess the association of Mtln-3’UTRdel genotypes with HF incidence and outcomes (e.g. mortality and arrhythmic events). These multi-disciplinary studies have significant potential to 1) transform our knowledge of basic mitochondrial biology, 2) determine if Mtln represents a viable target for HF therapy, and 3) reveal new genetic markers that could translate towards improved identification and treatment of high-risk HF patients.