The physiologic and genomic relevance of mitoregulin in ischemic heart failure

项目总结/摘要 心肌梗死（MI）是全球发病率和死亡率的主要原因。虽然再灌注治疗 改善心肌梗死后的存活率，快速恢复心脏的含氧血流可增强氧化应激， 进一步损伤组织，称为缺血-再灌注（IR）损伤，加剧了发生心力衰竭的风险 （HF）. IR损伤的中心是线粒体，其在心肌细胞Ca 2+缓冲和产生 能量和活性氧（ROS）。IR后，线粒体Ca 2+超载和活性氧触发线粒体 渗透性转变（mPT）孔开放，促进细胞死亡。虽然许多研究强调了 mPT在IR损伤和心脏保护中的作用，许多分子途径导致线粒体Ca 2+和ROS的改变， (key mPT的上游调节子）仍然不清楚。在最近的研究中，我们发现心脏和肌肉- 富集的长“非编码”RNA实际上编码高度保守的单次跨膜微通道， 定位于线粒体内膜的蛋白质;我们将这种蛋白质命名为“Mitoregulin”（Mtln）。在增益-和 在功能丧失的研究中，我们发现Mtln通过增加线粒体的1）呼吸超负荷， 复合物水平，2）呼吸效率，和3）Ca 2+保留能力，同时减少ROS。在这份协议中，我们 目的是解决两个中心假设：1）Mtln通过延迟Ca 2 +-和 ROS触发的mPT，以及2）与Mtln表达相关的人类遗传变异与HF患者相关 成果。我们的首要目标是将Mtln与心脏IR损伤和HF结局联系起来，以证明需要 未来的研究将继续确定Mtln的确切行动模式。在目标1中，我们将定义生理和 通过评估调节Mtln表达对心肌细胞的急性作用， 线粒体呼吸，Ca 2+和ROS，以及培养的啮齿动物心肌细胞中的超复合物水平。我们将 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的作用 在IR损伤和HF的细胞和啮齿动物模型中通过功能获得和丧失研究来评估Mtln的影响 调节模拟IR诱导的心肌细胞死亡，以及对心脏IR损伤和随后的反应 HF小鼠。在目标3中，我们将努力建立遗传变异、心脏Mtln表达和HF患者之间的联系。 结果，重点是36 bp 3 'UTR缺失变体（Mtln-3' UTRdel）的生物学和遗传相关性， 存在于12%的一般人群中，并且已知与心脏组织中Mtln mRNA水平降低有关。 我们将鉴定控制Mtln表达的RNA结合蛋白，并评估3 'UTRdel变体对Mtln表达的影响。 这种调节和人类心脏中的Mtln蛋白水平。我们还将测试与临床相关的可用DNA样本， 患者队列中的数据，以评估Mtln-3 'UTRdel基因型与HF发病率和结局的相关性（例如， 死亡率和中毒事件）。这些多学科研究具有重大潜力，1）改变我们的 基本线粒体生物学知识，2）确定Mtln是否代表HF治疗的可行靶点，和3）揭示 新的遗传标记，可以转化为提高识别和治疗高风险HF患者。

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.