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

基本信息

批准号：
10301250
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
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10301250
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/antagonist insight mdx mouse membrane model mitochondrial membrane mouse model new therapeutic target novel novel strategies premature prevent targeted treatment uptake

项目摘要

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.

摘要项目摘要肌肉营养不良（MD）是一个遗传疾病家族，其特征是进行性肌肉浪费，丧失肌肉功能和过早死亡。 MD病理学是由肌体不稳定性驱动的，导致膜破裂，坏死和肌纤维死亡。研究表明，MD肌纤维的细胞增加 Ca2+水平和线粒体膜通透性孔（MPTP）依赖肌纤维死亡贡献致MD。有证据表明，MD肌纤维线粒体的Ca2+水平升高，但孤立的MD线粒体 CA2+摄取率也降低了MCUB的表达，MCUB是Ca2+摄取的负调节剂。在心脏中限制线粒体Ca2+摄取可保护MPTP依赖性细胞死亡，而基因促进CA2+过载会导致MPTP激活和细胞死亡增加。因此，我们将测试 MD中的线粒体Ca2+稳态（AIM＃1）。我们将破坏主线粒体CA2+外排通过删除MD的MDX模型中的肌纤维线粒体Na+/Ca2+交换器（NCLX）来测试 Ca2+过载促进MPTP依赖性的肌纤维死亡和病理学的假设。我们也会通过删除线粒体Ca2+ Uniporter（MCU），破坏急性线粒体Ca2+摄取机制 MD的MDX模型中的MyOfbers测试抑制Ca2+摄取的假设将减少MPTP依赖性 MD中的肌纤维死亡病理学。我们将在MD的MDX模型中删除Myofiber MCUB来检验假设 MD肌纤维中MCUB表达的增加是MD疾病的保护性。更好地了解 MPTP在MD疾病中，我们将直接以遗传为目标MPTP。我们最近证明了腺嘌呤核苷酸转运剂（ANT）蛋白是构成MPTP的至少两种蛋白质之一。它最近已经观察到，分离的MD肌纤维线粒体对MPTP的敏感性提高激活以及ANT2表达的特异性增加。因此，我们将测试ANT依赖性的作用 MD中的MPTP激活（AIM＃2）。我们将在MD的SGCD - / - 模型中研究ANT1敲除小鼠，该小鼠缺乏主要的肌肉蚂蚁同工型，以检验依赖Ant依赖的MPTP（MPTPANT）的假设有助于MD 病理。我们将在MD的SGCD - / - 模型中删除Myofiber Ant2，以测试ANT2上升的假设在MD中，线粒体在MD中专门促进MPTP激活和病理。此外，我们将生成在MD的SGCD - / - 模型中缺少ANT的所有鼠同工型的小鼠以测量mptpant的总贡献致MD。由于我们已经证明了在总蚂蚁敲除的背景下抑制环蛋白D（CYPD）足以完全抑制MPTP，我们将治疗缺乏CYPD抑制剂所有蚂蚁同工型的SGCD - / - 小鼠 DeBio-025测试总肌纤维死亡和MD病理的比例取决于MPTP激活。这项研究将是线粒体Ca2+在MD病理生物学中的作用的首次测试，也是对生理系统中MPTP的蚂蚁模型。这项研究可能会发现治疗MD的新颖策略，因为目前尚无治愈或有效的治疗方法。