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

总结 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。在心肌细胞的肌膜上缺乏肌营养不良蛋白， 骨骼肌纤维由于机械引起的损伤而导致细胞膜的脆性。的一小部分 DMD女性携带者也容易出现肌营养不良蛋白丢失。这些有症状的DMD女性携带者 一系列的骨骼衰弱心肌病和心电图异常许多治疗方法， 在mdx小鼠模型中研究雄性DMD。然而，对DMD症状女性的研究 携带者落后，部分原因是没有可靠的DMD症状携带者小鼠模型可供研究。 测试.最近，我们创建了第一个DMD女性携带者的症状小鼠模型。我们开发 通过将mdx（鼠DMD）胚胎干细胞（ESC）注射到野生型（WT）胚泡中， (mdx/WT嵌合体）。mdx/WT小鼠出现心脏和骨骼肌异常， 有症状的DMD女性携带者的特征。我们实验室最近的发现表明 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，可能导致心肌病和心律失常患者经历。在 DMD，Cx43在丝氨酸残基三联体中表现出独特的磷酸化模式。我们已经开始研究 使用携带Cx43的磷酸模拟形式的突变敲入小鼠，其中三联体已经突变为 谷氨酸（Cx43 S3 E）。将突变掺入mdx小鼠中，并将Cx43保留至ID。 重要的是，所有的心脏缺陷都得到了挽救。与心脏相反，Cx43在骨骼肌中不表达。 肌肉纤维这是因为纤维形成合胞体，因此不需要连接器。但我们 发现在营养不良的骨骼肌纤维之间的单核细胞中Cx43的表达加剧。我们 初步结果表明，Cx43拷贝数的减少消除了心脏的病理， mdx/WT DMD女性携带者的骨骼肌。这导致预防心脏中的Cx43重塑。 基于我们的研究结果，我们假设调节心脏和心肌中的Cx43重塑和水平， 骨骼肌改善mdx/WT女性携带者DMD表现首先，我们将确定A Cx43的修饰形式，不能重塑，克服mdx/WT嵌合体中心肌病的发展 小鼠我们将通过将mdx ESC注射到WT：Cx43（S3 E）胚泡中来产生mdx/WT：Cx43（S3 E）小鼠 （目标1）。我们还将确定是否通过基因拷贝数减少使Cx43水平正常化， 克服了mdx/WT嵌合小鼠中骨骼肌病理学的发展。Cx43不表达于 但在纤维之间的单核细胞中表达，并且Cx43蛋白水平较高， 在DMD骨骼肌中观察到。体内嵌合实验将通过共培养来补充 实验，其中成年WT和mdx骨骼肌纤维将在mdx存在下培养 用Cx43半通道抑制剂Gap 19（Aim 2）处理或未处理的巨噬细胞（或其分泌组分）。 由于Cx43的肽模拟物抑制剂正在临床试验中进行测试，毒理学特征已经到位， 这一培训机会将开辟新的治疗场所，以解决心脏和骨骼肌病理学， 未被充分研究的症状性DMD携带者。

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.