Mitochondrial regulation of calcium homeostasis and cell death in muscular dystrophy

肌营养不良症中钙稳态和细胞死亡的线粒体调节

• 批准号: 10475281
• 负责人: Michael Bround
• 金额: $ 9.07万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10475281
• 关键词: Ablation; Acute; Adenine Nucleotides; Adenosine; Ants; Area; Calcium; Cell Death; Cell Membrane Permeability; Cessation of life; Complex; Development; Disease; Disease Progression; Duchenne muscular dystrophy; Dystrophin; Exhibits; Functional disorder; Genes; Genetic; Genetic Diseases; Goals; Heart; Homeostasis; Knock-out; Knockout Mice; Limb-Girdle Muscular Dystrophies; Measures; Membrane; Mitochondria; Modeling; Molecular; Molecular Target; Mus; Muscle; Muscle Mitochondria; Muscle function; Muscular Atrophy; Muscular Dystrophies; Myocardial Ischemia; Myopathy; Necrosis; Nucleotides; Outcome; Pathology; Physiological; Process; Protein Family; Protein Isoforms; Proteins; Publishing; Regulation; Reperfusion Injury; Research; Role; Rupture; SLC25A4 gene; SLC25A5 gene; Sarcolemma; Skeletal Muscle; System; Testing; Up-Regulation; Wasting Syndrome; Work; cyclophilin D; effective therapy; family genetics; gene therapy; inhibitor; insight; mdx mouse; membrane model; mitochondrial membrane; mouse model; new therapeutic target; novel; novel strategies; premature; prevent; targeted treatment; uptake

ABSTRACT PROJECT SUMMARY Muscular Dystrophy (MD) is a family of genetic disorders characterized by progressive muscle wasting, loss of muscle function, and premature death. MD pathology is driven by sarcolemma instability resulting in membrane rupture, necrosis, and myofiber death. Research has demonstrated that MD myofibers have increased cellular Ca2+ levels and that mitochondrial membrane permeability pore (MPTP) dependent myofiber death contributes to MD. There is evidence that MD myofiber mitochondria have elevated Ca2+ levels, but isolated MD mitochondria also have reduced Ca2+ uptake rates and increased expression of MCUb, a negative regulator of Ca2+ uptake. In the heart limiting mitochondrial Ca2+ uptake protects from MPTP-dependent cell death, while genetically promoting Ca2+ overload leads to increased MPTP-activation and cell death. We will therefore test the role of mitochondrial Ca2+ homeostasis in MD (Aim #1). We will disrupt the main mitochondrial Ca2+ efflux mechanism by deleting the myofiber mitochondrial Na+/Ca2+ exchanger (Nclx) in the Mdx model of MD to test the hypothesis that Ca2+ overload promotes MPTP-dependent myofiber death and pathology in MD. We will also disrupt the acute mitochondrial Ca2+ uptake mechanism by deleting the mitochondrial Ca2+ uniporter (Mcu) in myofbers in the Mdx model of MD to test the hypothesis that inhibiting Ca2+ uptake will reduce MPTP-dependent myofiber death pathology in MD. We will delete myofiber Mcub in the Mdx model of MD to test the hypothesis that increased MCUb expression in MD myofibers is protective in MD disease. To better understand the role of MPTP in MD disease we will directly target the MPTP genetically. We have recently demonstrated that adenine nucleotide translocator (ANT) proteins represent one of at least two protein species that comprise the MPTP. It has recently been observed that isolated MD myofiber mitochondria have increased sensitivity to MPTP- activation as well as a specific increase in ANT2 expression. We will therefore test the role of ANT-dependent MPTP activation in MD (Aim #2). We will study Ant1 knockout mice in the Sgcd-/- model of MD, which lack the main muscle ANT isoform, to test the hypothesis that ANT-dependent MPTP (MPTPANT) contributes to MD pathology. We will delete myofiber Ant2 in the Sgcd-/- model of MD to test the hypothesis that ANT2 upreglation in MD mitochondria specifically promotes MPTP activation and pathology in MD. Additionally, we will generate mice lacking all murine isoforms of ANT in the Sgcd-/- model of MD to measure the total contribution of MPTPANT to MD. Since we have demonstrated that inhibition of cyclophilin D (CypD) in the context of total ANT knockout is sufficient to completely inhibit MPTP, we will treat Sgcd-/- mice lacking all ANT isoforms with the CypD inhibitor Debio-025 to test what proportion of total myofiber death and MD pathology is dependent on MPTP-activation. This research will be the first test of the role of mitochondrial Ca2+ in MD pathobiology and the first study of the ANT model of MPTP in a physiological system. This research may uncover novel strategies to treat MD, for which there is currently no cure or effective treatment.

项目摘要 肌营养不良症（MD）是一个家族的遗传性疾病，其特征在于进行性肌肉萎缩、肌肉萎缩、肌肉萎缩和肌肉萎缩。 肌肉功能和过早死亡MD病理由肌膜不稳定性驱动，导致膜 破裂、坏死和肌纤维死亡。研究表明，MD肌纤维增加了细胞 Ca 2+水平和线粒体膜通透性孔（MPTP）依赖性肌纤维死亡有助于 到MD。有证据表明MD肌纤维线粒体具有升高的Ca 2+水平，但分离的MD线粒体 还具有降低的Ca 2+摄取速率和增加的MCUb表达，MCUb是Ca 2+摄取的负调节剂。 在心脏中，限制线粒体Ca 2+摄取可防止MPTP依赖性细胞死亡，而在遗传学上， 促进Ca 2+超负荷导致MPTP活化和细胞死亡增加。因此，我们将测试 MD中线粒体Ca 2+稳态（目标#1）。我们将破坏主要的线粒体Ca 2+外流 通过在MD的Mdx模型中删除肌纤维线粒体Na+/Ca 2+交换器（Nclx）来测试 假设钙超载促进MPTP依赖性肌纤维死亡和病理在MD。我们还将 通过删除线粒体Ca 2+单向转运体（Mcu），破坏急性线粒体Ca 2+摄取机制， 在MD的Mdx模型中使用myofbers，以检验抑制Ca 2+摄取将减少MPTP依赖性 肌纤维死亡病理学。我们将在MD的Mdx模型中删除肌纤维Mcub来检验这一假设 MD肌纤维中MCUb表达的增加在MD疾病中具有保护作用。为了更好地理解 在MD疾病中，我们将直接靶向MPTP基因。我们最近证明腺嘌呤 核苷酸转运子（ANT）蛋白代表构成MPTP的至少两种蛋白质种类之一。它 最近观察到，分离的MD肌纤维线粒体对MPTP的敏感性增加， 激活以及ANT 2表达的特异性增加。因此，我们将测试ANT依赖的作用， MD中的MPTP激活（目标#2）。我们将在MD的Sgcd-/-模型中研究Ant 1敲除小鼠，该模型缺乏 主要肌肉ANT亚型，以检验ANT依赖性MPTP（MPTPANT）有助于MD的假设 病理我们将在MD的Sgcd-/-模型中删除肌纤维Ant 2，以检验Ant 2上调 在MD中，线粒体特异性促进MD中MPTP的激活和病理学。此外，我们将生成 在MD的Sgcd-/-模型中缺乏ANT的所有鼠同种型的小鼠，以测量MPTPANT的总贡献 到MD。由于我们已经证明，在ANT基因完全敲除的情况下， 足以完全抑制MPTP，我们将用CypD抑制剂治疗缺乏所有ANT同种型的Sgcd-/-小鼠 Debio-025来测试总肌纤维死亡和MD病理学的比例依赖于MPTP激活。 这项研究将是线粒体Ca 2+在MD病理生物学中作用的第一个测试，也是第一个研究线粒体Ca 2+在MD病理生物学中的作用的研究。 生理系统中MPTP的ANT模型。这项研究可能会发现治疗MD的新策略， 目前还没有治愈或有效的治疗方法。