Transcriptome analysis of skeletal muscle tissue from Hras G12V mutant mice

Costello syndrome (CS) is a congenital disorder caused by heterozygous activating germline HRAS mutations in the canonical Ras/mitogen-activated protein kinase (Ras/MAPK) pathway. CS is one of the RASopathies, a large group of syndromes due to mutations within various components of the Ras/MAPK pathway. An important part of the phenotype that greatly impacts quality of life is hypotonia. To gain a better understanding of the mechanisms underlying hypotonia in CS, a mouse model with an activating HrasG12V allele was utilized. We identified a skeletal myopathy that was due in part to an inhibition of embryonic myogenesis and myofiber formation, resulting in a reduction of myofiber size and number that led to reduced muscle mass and strength. In addition to hyperactivation of the Ras/MAPK and PI3K/AKT pathways, there was a significant reduction of p38 signaling, as well as global transcriptional alterations consistent with the myopathic phenotype. Inhibition of Ras/MAPK pathway signaling using a MEK inhibitor rescued the HrasG12V myopathy phenotype both in vitro and in vivo, demonstrating that increased MAPK signaling is the main cause of the muscle phenotype in CS. The study consisted of 2 groups of mice with triplicate samples per group. The experimental group consisted of mice that were heterozygous (HrasG12V/+) for the HrasG35T (p.G12V) mutation. Control mice were homozygous (Hras+/+) for wild type Hras. Gastrocnemius muscle tissue was harvested from 21-day-old mice and snap-frozen for subsequent RNA extraction and transcriptome profiling with RNA-Sequencing (RNA-Seq) analysis.科斯特洛综合征（Costello syndrome, CS）是一类由经典Ras/丝裂原活化蛋白激酶（Ras/mitogen-activated protein kinase, Ras/MAPK）通路中杂合激活型种系HRAS突变所引发的先天性疾病。CS属于RASopathies（RAS通路相关疾病群）——一类因Ras/MAPK通路多种组分发生突变而导致的综合征大类。对患者生活质量造成显著影响的核心表型之一为肌张力低下。为深入解析CS患者肌张力低下的潜在发病机制，本研究采用了携带激活型HrasG12V等位基因的小鼠模型。研究人员在该模型中发现了一种骨骼肌病变，其部分成因在于胚胎肌发生与肌纤维形成过程受到抑制，进而导致肌纤维体积与数量减少，最终造成肌肉质量与力量下降。除Ras/MAPK及PI3K/AKT通路过度激活外，该模型还存在p38信号通路显著下调，以及与肌病表型相符的全基因组转录组改变。通过MEK抑制剂抑制Ras/MAPK通路信号，可在体外与体内环境中挽救HrasG12V介导的肌病表型，证实MAPK信号过度增强是CS肌肉表型的主要致病因素。本研究设置两组小鼠，每组设置三份生物学重复样本：实验组为携带HrasG35T（p.G12V）突变的杂合子小鼠（HrasG12V/+）；对照组为野生型Hras纯合子小鼠（Hras+/+）。研究人员采集21日龄小鼠的腓肠肌组织，经快速冷冻后用于后续RNA提取，以及通过RNA测序（RNA-Sequencing, RNA-Seq）开展转录组分析。