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：
10292934
负责人：
Julie Nouet
金额：
$ 4.02万
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 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10292934
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.

概括杜氏肌营养不良症 (DMD) 是一种严重的 X 连锁神经肌肉疾病，影响男性患者。罪魁祸首是肌营养不良蛋白的突变。心肌细胞肌膜处缺乏抗肌营养不良蛋白由于机械损伤，骨骼肌纤维导致细胞膜脆弱。一小部分 DMD 女性携带者也容易遭受肌营养不良蛋白损失。这些有症状的 DMD 女性携带者会出现一系列骨骼无力、心肌病和心电图异常。许多疗法用于正在 mdx 小鼠模型中研究雄性 DMD。然而，针对有 DMD 症状的女性的研究携带者滞后，部分原因是没有忠实的 DMD 症状携带者小鼠模型已测试。最近，我们创建了第一个有症状的 DMD 女性携带者小鼠模型。我们开发了通过将 mdx（鼠 DMD）胚胎干细胞（ESC）注射到野生型（WT）囊胚中来培育镶嵌小鼠（mdx/WT 嵌合体）。 mdx/WT 小鼠出现心肌和骨骼肌异常，模拟有症状的 DMD 女性携带者的特征。我们实验室的最新发现显示了重要作用连接蛋白 43 (Cx43)。在 DMD 患者和 mdx 小鼠中，Cx43 病理性上调和重塑远离 ID，可能是导致患者出现心肌病和心律失常的原因。在 DMD、Cx43 在丝氨酸残基三联体中表现出独特的磷酸化模式。我们已经启动研究使用携带 Cx43 磷酸模拟形式的突变敲入小鼠，其中三联体已突变为谷氨酸（Cx43S3E）。该突变被整合到 mdx 小鼠中，并且 Cx43 被保留到 ID 中。重要的是，所有心脏缺陷都得到了挽救。与心脏相反，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，我们发现营养不良的骨骼肌纤维之间的单核细胞中 Cx43 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变化。 mdx/WT DMD 女性携带者的骨骼肌。这可以防止心脏中的 Cx43 重塑。根据我们的结果，我们假设心脏和心脏中 Cx43 重塑和水平的调节骨骼肌改善 mdx/WT 女性携带者的 DMD 表现。我们首先要确定是否 Cx43 的修饰形式无法重塑，克服了 mdx/WT 嵌合体中心肌病的发展老鼠。我们将通过将 mdx ESC 注射到 WT:Cx43(S3E) 囊胚中来生成 mdx/WT:Cx43(S3E) 小鼠（目标1）。我们还将确定 Cx43 水平是否通过基因拷贝数减少而标准化克服了 mdx/WT 嵌合小鼠骨骼肌病理学的发展。 Cx43 不表达在骨骼纤维，但在纤维之间的单核细胞中表达，并且较高的 Cx43 蛋白水平在 DMD 骨骼肌中观察到。体内嵌合实验将通过共培养得到补充实验，其中成人 WT 和 mdx 骨骼肌纤维将在 mdx 存在的情况下进行培养使用或未使用 Cx43 半通道抑制剂 Gap19 (Aim2) 处理的巨噬细胞（或其分泌成分）。由于 Cx43 的肽模拟抑制剂正在临床试验中进行测试，并具有毒理学特征，这一培训机会将为解决心脏和骨骼肌病理学问题开辟新的治疗场所对有症状的 DMD 携带者进行了充分研究。