Novel cardioskeletal myopathy associated with MYL2

 描述(申请人提供)：先前在荷兰和意大利家庭中报道过同时发生严重心肌病的婴儿I型纤维营养不良，其基因与编码人类肌球蛋白调节轻链MLC2ventr/Slow的MYL2基因有关，该基因在脑室和慢抽动的骨骼肌中表达。出生后不久，患者经历了进行性缓慢抽动骨骼肌病，并最终在4至6个月大时死于心力衰竭。已知MYL2基因的显性突变可导致家族性肥厚型心肌病(FHC)，其病程、发病年龄和症状严重程度具有广泛的多样性。将使用我们的转基因(TG)小鼠来评估触发心脏病理重塑的分子事件的突变特异性失调，这些小鼠在MLC2ventr/low中表达恶性的：R58Q和D166V和良性的：K104E突变。除了心脏表型外，这项应用还将首次包括慢抽动骨骼肌和MYL2剪接位点IVS6-1突变的研究，这些突变被证明会导致人类严重的肌病和IVS6-1纯合子患者的过早死亡。目的1：确定MLC2ventR/Slow突变引起的心脏骨骼功能障碍的分子机制。我们假设，突变诱导的结构变化触发心脏和缓慢的骨骼肌的病理性重塑，导致收缩能力改变和心脏骨骼肌病。蛋白质组学研究将被用来确定与FHC突变相关的心脏骨骼功能障碍的信号通路。利用小角X射线衍射图，心脏中MLC2ventR/Slow突变的结构表型将与慢抽动骨骼肌中的各自表型相关联。组织病理学和电子显微镜(EM)将补充突变对心脏和比目鱼肌肌节结构重组的影响。对所有建议建立的FHC小鼠模型的皮肤乳头/比目鱼肌纤维的收缩力、力-PCA关系和肌球蛋白跨桥动力学的测量将完成MLC2ventr/慢特异性心脏骨骼肌病的表型特征。重要的是，我们还将研究IVS6-1突变与早产儿心源性死亡的关系。目的：在体内检测FHC诱导的心脏表型，并在表达组成型磷酸化P-MLC2的转基因小鼠中探索新的拯救机制。我们假设，通过改变依赖于钙离子的肌肉收缩调节，D166V和R58Q突变增加了受影响患者患恶性疾病表型的倾向。我们还假设其潜在机制与空间位阻抑制肌球蛋白轻链激酶依赖的MLC2的磷酸化有关。这些假说将使用我们最近开发的双突变TG-S15D-D166V救援小鼠来解决，旨在减轻恶性D166V突变与组成磷酸化的Ser-15(S15D)的影响。

 DESCRIPTION (provided by applicant): Infantile fiber-type I hypotrophy with simultaneously occurring severe onset of cardiomyopathy were previously reported in Dutch and Italian families and genetically linked to the MYL2 gene encoding for the human myosin regulatory light chain MLC2ventr/slow expressed in the ventricles and in slow-twitch skeletal muscles. Shortly after birth the patients experienced progressive slow-twitch skeletal myopathy and ultimately died of heart failure between 4 and 6 months of age. Dominant mutations in MYL2 have been known to cause familial hypertrophic cardiomyopathy (FHC) of extensive diversity in the course of the disease, age of onset and severity of symptoms. The mutation-specific dysregulation of the molecular events that trigger pathological remodeling of the heart, will be assessed using our transgenic (Tg) mice expressing the malignant: R58Q and D166V and benign: K104E mutations in MLC2ventr/slow. In addition to cardiac phenotypes, this application for the first time will include the slow-twitch skeletal muscle and the study of the splice site IVS6-1 mutation in MYL2 shown to cause severe myopathy in humans and premature death of IVS6-1-homozygous patients. AIM 1: Identify molecular mechanisms responsible for cardioskeletal dysfunction caused by MLC2ventr/slow mutations. We hypothesize that the mutation-induced structural changes trigger pathological remodeling of the heart and slow skeletal muscle leading to altered contractility and cardioskeletal myopathy. Proteomics study will be employed to identify the signaling pathways involved in cardioskeletal dysfunction associated FHC mutations. Structural phenotypes specific to MLC2ventr/slow mutations in the heart will be correlated with the respective phenotypes in the slow-twitch skeletal muscles using small angle X-ray diffraction patterns. Histopathology and electron microscopy (EM) will complement the effect of mutations on structural reorganization of the sarcomere in the heart and soleus muscle. Measurements of contractile force, force-pCa relationship and the myosin cross-bridge kinetics in skinned papillary/soleus muscle fibers from all proposed Tg mouse models of FHC will complete the phenotypic characterization of MLC2ventr/slow-specific cardioskeletal myopathy. Importantly, we will also study the IVS6-1 mutation associated with premature infantile cardiac death. AIM 2: Determine FHC induced cardiac phenotypes in vivo and explore novel rescue mechanisms in transgenic mice expressing constitutively phosphorylated P-MLC2. We hypothesize that by altering the Ca2+-dependent regulation of muscle contraction, D166V and R58Q mutations increase the propensity of affected patients toward malignant disease phenotypes. We also hypothesize that the underlying mechanisms relate to the steric inhibition of myosin light chain kinase dependent phosphorylation of MLC2. These hypotheses will be addressed using our recently developed double mutant Tg-S15D-D166V rescue mice, designed to mitigate the effects of the malignant D166V mutation with a constitutively phosphorylated Ser-15 (S15D).