Identify a new non-canonical role of MEF2D⍺2 protein isoform in skeletal muscle metabolism

确定 MEF2D™2 蛋白亚型在骨骼肌代谢中的新非典型作用

• 批准号: 10732402
• 负责人: Ravi K. Singh
• 金额: $ 20.59万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-17 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10732402
• 关键词: ASCL1 gene; Adult; Affect; Age; Alternative Splicing; Amino Acids; Bioenergetics; Biological Assay; Biotin; Birth; Body Weight; CD36 gene; CRISPR/Cas technology; Cells; Consumption; Contralateral; Cytoplasm; Cytosol; Data; Development; Disease; Disease Outcome; Eko; Embryo; Evolution; Exercise; Exons; Family; Fatty Acids; Fiber; Fishes; Gastrocnemius Muscle; Genes; Genetic Transcription; Glucose; Goals; High Fat Diet; Homeostasis; Human; Immunoblot Analysis; Immunoprecipitation; In Vitro; Knock-out; Knockout Mice; Label; Life Style; Ligase; Ligation; Mass Spectrum Analysis; Mediating; Metabolic; Metabolic Diseases; Metabolism; Minor; Mitochondria; Modernization; Mus; Muscle; Muscle Development; Muscle Mitochondria; Muscle Proteins; Muscle function; Muscular Dystrophies; Myoblasts; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Nuclear Localization Signal; Obesity; Play; Process; Production; Protein Isoforms; Proteins; RNA Splicing; RNA-Binding Proteins; Research; Role; Running; Site; Skeletal Muscle; Streptavidin; Testing; Transactivation; Transcript; Wild Type Mouse; Work; aged; exercise training; experimental study; fatty acid metabolism; fatty acid oxidation; fetal; glucose metabolism; improved; in vivo; insight; mRNA Expression; member; muscle aging; muscle form; muscle metabolism; myocyte-specific enhancer-binding factor 2; novel; oxidation; paralogous gene; postnatal; postnatal period; response; skeletal muscle metabolism; transcription factor; transcriptome; transcriptome sequencing

Project Summary: Despite the widespread occurrence of alternative splicing in skeletal muscle, the role of very few muscle-specific protein isoforms produced by alternative splicing has been studied. In contrast, altered transcript splicing and splicing regulator expression is frequently found in muscle dystrophies and aging-associated decline in muscle function and metabolism. Skeletal muscle makes up to 40% of body weight in healthy human adults and plays a predominant role in regulating whole-body metabolism. Yet, the role of alternate protein products of alternative splicing in skeletal muscle function and metabolism is largely unknown. My recent work demonstrated that the Rbfox family of RNA-binding proteins is vital for regulating skeletal muscle homeostasis in adulthood. Inducible Rbfox knockout in adult mouse skeletal muscle caused ~50% reduction in muscle mass within four weeks, altered glucose metabolism, and splicing of >740 gene transcripts. Many RBFOX-regulated alternative exons are evolutionarily conserved, suggesting roles for the alternate protein isoforms in adult skeletal muscle function. RBFOX proteins regulate mutually exclusive ⍺1 and ⍺2 exons of the MEF2D transcription factor to produce the predominant adult skeletal muscle-specific isoform, MEF2D⍺2. The four MEF2 (MEF2A-D) members of the highly conserved family of transcription factors are important for embryonic muscle development, but their role in the adult skeletal muscle is not known. The ⍺2 exon inclusion increases to >75% after birth to produce the predominant MEF2D⍺2 isoform in adult skeletal muscle. To determine the role of MEF2D⍺2, I deleted the ⍺2-exon of Mef2d using CRISPR-Cas9 to generate, Mef2d⍺2 Eko mouse line. Compared to wild-type mice, Mef2d ⍺2 Eko mice displayed reduced running capacity and muscle fatty acid oxidation. Our preliminary data indicate minimal to no change in muscle transcriptome in muscles of Mef2d⍺2 Eko mice. We also found that most MEF2D is present in the cytosolic fraction of skeletal muscle and interacts with mitochondrial and muscle metabolic proteins. Given the reduced muscle fatty acid oxidation in skeletal muscles of Mef2d⍺2 Eko mice, we hypothesize that MEF2D⍺2 protein interacts with metabolic proteins in the cytosol to optimize fatty oxidation in adult skeletal muscle. In aim1, we will identify and validate proteins interacting with MEF2D⍺2 exclusively or preferentially in vivo. In aim2, we will determine the impact of the loss of MEF2D⍺2 on its interactors and muscle fatty acid oxidation and validate top MEF2D⍺2-protein interactions in human skeletal muscle tissues. A disruption in skeletal muscle glucose and fatty acid metabolism often manifests before the development of type II diabetes and obesity, one of the most prevalent lifestyle diseases of the modern world. Thus, our work will identify a new non-canonical role of MEF2D⍺2 in muscle metabolism, which we expect to be conserved across evolution as MEF2D⍺2 exon and splice sites are conserved from fish to humans.

项目摘要：尽管选择性剪接在骨骼肌中广泛存在，但其作用非常重要。 相反，很少有人研究过通过选择性剪接产生的肌肉特异性蛋白质亚型。 转录本剪接和剪接调节因子表达常见于肌肉营养不良和衰老相关疾病中 肌肉功能和新陈代谢下降，骨骼肌占健康人体重的 40%。 然而，替代蛋白质的作用在成人中起着主导作用。 骨骼肌功能和代谢中的选择性剪接产物在很大程度上是未知的。 我最近的工作表明，RNA 结合蛋白的 Rbfox 家族对于调节骨骼肌至关重要。 成年小鼠骨骼肌的诱导性 Rbfox 敲除导致约 50% 的肌肉稳态下降。 4 周内肌肉质量增加，葡萄糖代谢发生改变，以及超过 740 个基因转录本的剪接。 RBFOX 调节的替代外显子在进化上是保守的，表明替代蛋白的作用 成人骨骼肌功能中的亚型 RBFOX 蛋白调节相互排斥的 ⍺1 和 ⍺2 外显子。 MEF2D 转录因子，可产生主要的成人骨骼肌特异性亚型 MEF2D⍺2。 高度保守的转录因子家族的四个 MEF2 (MEF2A-D) 成员非常重要 对于胚胎肌肉发育，但它们在成年骨骼肌中的作用尚不清楚 ⍺2 外显子包含。 出生后增加到 >75%，在成人骨骼肌中产生主要的 MEF2D⍺2 亚型 确定。 为了发挥MEF2D⍺2的作用，我使用CRISPR-Cas9删除了Mef2d的⍺2外显子来生成Mef2d⍺2 Eko小鼠品系。 与野生型小鼠相比，Mef2d ⍺2 Eko 小鼠表现出跑步能力和肌肉脂肪酸降低 氧化。 我们的初步数据表明 Mef2d⍺2 Eko 肌肉中的肌肉转录组变化极小甚至没有变化 我们还发现大多数 MEF2D 存在于骨骼肌的胞质部分中并与骨骼肌相互作用。 鉴于骨骼肌中肌肉脂肪酸氧化减少。 Mef2d⍺2 Eko 小鼠，我们追踪 MEF2D⍺2 蛋白与细胞质中的代谢蛋白相互作用， 优化成人骨骼肌中的脂肪氧化 在目标 1 中，我们将识别并验证与之相互作用的蛋白质。 MEF2D⍺2仅在体内或优先在体内。在aim2中，我们将确定MEF2D⍺2的丢失对体内的影响。 其相互作用因子和肌肉脂肪酸氧化，并验证人类骨骼中的顶级 MEF2D⍺2 蛋白质相互作用 肌肉组织。 骨骼肌葡萄糖和脂肪酸代谢的破坏通常在发育之前就表现出来 II 型糖尿病和肥胖症是现代世界最普遍的生活方式疾病之一。 将确定 MEF2D⍺2 在肌肉代谢中的新的非规范作用，我们希望该作用得到保留 在整个进化过程中，MEF2D⍺2 外显子和剪接位点从鱼类到人类都是保守的。