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Role of Connexin43 in the heart and skeletal muscle in a model for Duchenne muscular dystrophy symptomatic carriers

Connexin43 在杜氏肌营养不良症状携带者模型中心脏和骨骼肌中的作用

基本信息

批准号：
10062690
负责人：
Julie Nouet
金额：
$ 3.97万
依托单位：
RBHS-NEW JERSEY MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10062690
关键词：
Address Adult Affect Arrhythmia Cardiac Cardiac Myocytes Cardiomyopathies Cell membrane Cells Characteristics Chimera organism Clinical Trials Coculture Techniques Complement Connexin 43 Connexins Development Duchenne muscular dystrophy Dystrophin Ensure Exhibits Female Fiber Gene Dosage Giant Cells Glutamic Acid Goals Heart Heart Abnormalities Heart failure Intercalated disc Intervention Knock-in Mouse Link Longevity Mechanics Modeling Mononuclear Mosaicism Mus Muscle Fibers Mutate Mutation Myocardium Neuromuscular Diseases Pathologic Pathology Patients Pattern Phosphorylation Play Prevention Proteins Regulation Research Role Sarcolemma Serine Signal Transduction Skeletal Development Skeletal Muscle Testing Therapeutic Time Toxicology Triplet Multiple Birth Wild Type Mouse base blastocyst embryonic stem cell experience experimental study improved in vivo inhibitor/antagonist insight macrophage male mdx mouse mimetics mouse model mutant novel therapeutics peptidomimetics respiratory skeletal stem cells training opportunity

项目摘要

SUMMARY Duchenne Muscular Dystrophy (DMD) is a severe x-linked neuromuscular disorder that affects male patients. The culprit is a mutation in dystrophin. Absence of dystrophin at the sarcolemma of cardiomyocytes and skeletal muscle fibers leads to fragility of the cell membrane due to mechanical-induced damage. A fraction of DMD female carriers are also vulnerable to dystrophin loss. These symptomatic DMD female carriers develop a range of skeletal weakness, cardiomyopathy, and electrocardiographic abnormalities. Many therapeutics for male DMD are being investigated in the mdx mouse model. However, research on DMD symptomatic female carriers lags behind, partly because there was no faithful mouse model of DMD symptomatic carriers to be tested. Recently, we created the first symptomatic mouse model of DMD female carriers. We developed mosaic mice by injecting mdx (murine DMD) embryonic stem cells (ESCs) into wild-type (WT) blastocysts (mdx/WT chimera). mdx/WT mice develop cardiac and skeletal muscle abnormalities that model the characteristics of symptomatic DMD female carriers. Recent discoveries in our lab showed an important role for connexin-43 (Cx43). In DMD patients and mdx mice, Cx43 is pathologically upregulated and remodeled away from the ID and is likely responsible for the cardiomyopathy and arrhythmias patients experience. In DMD, Cx43 exhibits a distinct phosphorylation pattern in a triplet of serine residues. We have initiated studies using mutant knock-in mice harboring a phospho-mimetic form of Cx43 where the triplet has been mutated to glutamic acids (Cx43S3E). The mutation was incorporated into mdx mice, and Cx43 was retained to the ID. Importantly, all the cardiac defects were rescued. In contrast to the heart, Cx43 is not expressed in the skeletal muscle fibers. This is because the fibers form a syncytium, and thus, do not need connectors. However, we found exacerbated Cx43 expression in mononuclear cells between the dystrophic skeletal muscle fibers. Our preliminary results indicate that reduction of Cx43 copy number eliminates pathology in the heart and the skeletal muscle of mdx/WT DMD female carriers. This leads to prevention of Cx43 remodeling in the heart. Based on our results, we hypothesize that regulation of Cx43 remodeling and levels in the heart and in the skeletal muscle improves DMD manifestation in mdx/WT female carriers. We will first determine whether a modified form of Cx43, unable to remodel, overcomes development of cardiomyopathy in mdx/WT chimeric mice. We will generate mdx/WT:Cx43(S3E) mice by injecting mdx ESCs into WT:Cx43(S3E) blastocysts (Aim1). We will also determine whether normalization of Cx43 levels by gene-copy number reduction overcomes development of skeletal muscle pathology in mdx/WT chimeric mice. Cx43 is not expressed in the skeletal fibers but expressed in the mononuclear cells between fibers, and higher Cx43 protein levels are observed in DMD skeletal muscle. Chimeric experiments in-vivo will be complemented by co-culture experiments, where adult WT and mdx skeletal muscle fibers will be cultured in the presence of mdx macrophages (or their secreted components) treated or not with Cx43 hemichannel inhibitor Gap19 (Aim2). Because peptide mimetic inhibitors of Cx43 are being tested in clinical trials, with toxicological profiles in place, this training opportunity will open new therapeutic venues to address cardiac and skeletal muscle pathology in understudied symptomatic DMD carriers.

摘要杜氏肌营养不良症(DMD)是一种严重的x连锁神经肌肉疾病，影响男性患者。罪魁祸首是营养不良蛋白的突变。肌萎缩蛋白在心肌细胞和心肌细胞的肌膜上缺失由于机械损伤，骨骼肌纤维导致细胞膜的脆性。一小部分 DMD女性携带者也容易发生肌营养不良蛋白丢失。这些有症状的DMD女性携带者会一系列骨骼无力、心肌病和心电图异常。多种疗法治疗男性DMD正在MDX小鼠模型中进行研究。然而，对DMD症状女性的研究携带者落后，部分原因是没有忠实的DMD症状携带者的小鼠模型测试过。最近，我们建立了第一个有症状的DMD雌性携带者小鼠模型。我们开发了将MDX(小鼠DMD)胚胎干细胞(ESCs)注入野生型(WT)囊胚的嵌合体小鼠 (MDX/WT嵌合体)。MDX/WT小鼠出现心肌和骨骼肌异常，症状性DMD女性携带者的特征。我们实验室最近的发现显示了一个重要的作用连接蛋白-43(Cx43)。在DMD患者和MDX小鼠中，Cx43在病理上上调和重塑远离ID，可能是患者经历心肌病和心律失常的原因。在……里面 DMD，Cx43在三联体丝氨酸残基中显示出明显的磷酸化模式。我们已经开始了研究使用携带模拟磷酸形式的Cx43的突变敲入小鼠，其中三联体已突变为谷氨酸(Cx43S3E)。该突变被整合到MDX小鼠中，而Cx43保留为ID。重要的是，所有的心脏缺陷都被挽救了。与心脏不同，Cx43在骨骼中不表达肌肉纤维。这是因为纤维形成合胞体，因此不需要连接器。然而，我们发现在营养不良的骨骼肌纤维之间的单个核细胞中Cx43的表达加剧。我们的初步结果表明，Cx43拷贝数的减少消除了心脏和心脏的病理 MDX/WT DMD女性携带者的骨骼肌。这可以防止Cx43在心脏中的重塑。根据我们的结果，我们假设Cx43在心脏和心脏中的重塑和水平的调节骨骼肌改善了MDX/WT女性携带者的DMD表现。我们将首先确定一个修饰形式的Cx43不能重塑，克服了MDX/WT嵌合体心肌病的发展老鼠。我们将通过将MDX ESCs注射到WT：Cx43(S3E)囊胚中来产生MDX/WT：Cx43(S3E)小鼠 (目标1)。我们还将确定是否通过减少基因拷贝数来使Cx43水平正常化克服MDX/WT嵌合小鼠骨骼肌病理的发展。Cx43不表达在但骨骼纤维表达于单个核细胞之间的纤维，且Cx43蛋白水平较高在DMD骨骼肌中观察到。体内嵌合实验将得到共培养的补充成年WT和MDX骨骼肌纤维在MDX存在下培养的实验巨噬细胞(或其分泌成分)是否经Cx43半通道抑制剂Gap19(AIM2)处理。由于Cx43的多肽模拟抑制剂正在进行临床试验，毒理学特征已经到位，这一培训机会将打开新的治疗场所，以解决心肌和骨骼肌病理对症状性DMD携带者研究不足。