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.

项目摘要摘要 肌营养不良症(MD)是一种遗传性疾病，其特征是进行性肌肉萎缩、缺失 肌肉功能和过早死亡。MD的病理是由肌膜不稳定导致膜 破裂、坏死和肌纤维死亡。研究表明，MD肌纤维增加了细胞 钙离子水平与线粒体膜通透性孔(MPTP)依赖性肌纤维死亡的关系 致医学博士。有证据表明，MD肌纤维线粒体的钙离子水平升高，但MD线粒体是孤立的 也降低了钙摄取速率，增加了钙摄取的负调节因子MCUb的表达。 在心脏，限制线粒体钙摄取可以保护细胞免受MPTP依赖的细胞死亡，而从基因上讲 促进钙超载导致MPTP激活增加和细胞死亡。因此，我们将测试 MD患者线粒体钙稳态(目标1)。我们将扰乱线粒体主要的钙外流 去掉MDX模型中肌纤维线粒体Na~+/Ca~(2+)交换器(NCLx)的作用机制 钙超载促进MD依赖MPTP的肌纤维死亡和病理的假说。我们还将 通过缺失线粒体钙单一转运体(MCU)阻断急性线粒体钙摄取机制 在MD的MDX模型中检测抑制钙摄取将减少MPTP依赖的假说 MD的肌纤维死亡病理。我们将在MD的MDX模型中删除肌纤维Mcub以检验假设 增加MD肌纤维中MCUb的表达在MD疾病中具有保护作用。为了更好地理解 MPTP在MD疾病中的作用我们将从基因上直接靶向MPTP。我们最近证明了腺嘌呤 核苷酸转运子(ANT)蛋白代表组成MPTP的至少两种蛋白质物种之一。它 最近观察到，分离的MD肌纤维线粒体对MPTP- 激活以及ANT2表达的特异性增加。因此，我们将测试蚂蚁依赖的角色 MD的MPTP激活(目标2)。我们将在MD的SGCD-/-模型中研究ANT1基因敲除小鼠，该模型缺乏 主要肌肉ANT亚型，以检验蚂蚁依赖MPTP(MPTPANT)参与MD的假设 病理学。我们将删除MD SGCD-/-模型中的肌纤维Ant2，以验证ANT2上调的假设 在MD中，线粒体特异性地促进了MD中MPTP的激活和病理。此外，我们还将生成 在MD的Sgcd-/-模型中缺乏所有小鼠ANT亚型的小鼠以测量MPTPANT的总贡献 致医学博士。因为我们已经证明了在完全蚂蚁基因敲除背景下亲环素D(CypD)的抑制 足以完全抑制MPTP，我们将用CypD抑制剂治疗缺乏所有ANT亚型的SGCD-/-小鼠 DeBio-025来测试总肌纤维死亡和MD病理的比例取决于MPTP的激活。 这项研究将是第一次测试线粒体钙在MD病理生物学中的作用，也是第一次对线粒体钙在MD病理生物学中的作用进行研究 生理系统中MPTP的蚂蚁模型。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。