Xu Chen, Zhang Ge, Wang Xinjian, Huang Xiaozhi, Zhang Jiayin, Han Shuxian, Wang Jinxi, Hall Duane D, Xu Ruoqing, He Feng, Chang Xing, Wang Fudi, Xie Wenjun, Wu Zhichao, Song Long-Sheng, Han Peidong
Center for Genetic Medicine, The Fourth Affiliated Hospital (C.X., G.Z., X.W., X.H., J.Z., S.H., R.X., F.H., P.H.), Zhejiang University School of Medicine, Hangzhou, China.
Institute of Genetics, Zhejiang University International School of Medicine, Hangzhou, China (C.X., G.Z., X.W., X.H., J.Z., S.H., R.X., F.H., P.H.).
Circulation. 2024 Apr 23;149(17):1375-1390. doi: 10.1161/CIRCULATIONAHA.123.065767. Epub 2024 Jan 12.
Cardiac transverse tubules (T-tubules) are anchored to sarcomeric Z-discs by costameres to establish a regular spaced pattern. One of the major components of costameres is the dystrophin-glycoprotein complex (DGC). Nevertheless, how the assembly of the DGC coordinates with the formation and maintenance of T-tubules under physiological and pathological conditions remains unclear.
Given the known role of Ptpn23 (protein tyrosine phosphatase, nonreceptor type 23) in regulating membrane deformation, its expression in patients with dilated cardiomyopathy was determined. Taking advantage of Cre/Loxp, CRISPR/Cas9, and adeno-associated virus 9 (AAV9)-mediated in vivo gene editing, we generated cardiomyocyte-specific and (α-actinin-2, a major component of Z-discs) knockout mice. We also perturbed the DGC by using dystrophin global knockout mice (). MM 4-64 and Di-8-ANEPPS staining, Cav3 immunofluorescence, and transmission electron microscopy were performed to determine T-tubule structure in isolated cells and intact hearts. In addition, the assembly of the DGC with and dystrophin loss of function was determined by glycerol-gradient fractionation and SDS-PAGE analysis.
The expression level of Ptpn23 was reduced in failing hearts from dilated cardiomyopathy patients and mice. Genetic deletion of resulted in disorganized T-tubules with enlarged diameters and progressive dilated cardiomyopathy without affecting sarcomere organization. AAV9-mediated mosaic somatic mutagenesis further indicated a cell-autonomous role of in regulating T-tubule formation. Genetic and biochemical analyses showed that Ptpn23 was essential for the integrity of costameres, which anchor the T-tubule membrane to Z-discs, through interactions with α-actinin and dystrophin. Deletion of α-actinin altered the subcellular localization of Ptpn23 and DGCs. In addition, genetic inactivation of dystrophin caused similar T-tubule defects to loss-of-function without affecting Ptpn23 localization at Z-discs. Last, inducible knockout at 1 month of age showed Ptpn23 is also required for the maintenance of T-tubules in adult cardiomyocytes.
Ptpn23 is essential for cardiac T-tubule formation and maintenance along Z-discs. During postnatal heart development, Ptpn23 interacts with sarcomeric α-actinin and coordinates the assembly of the DGC at costameres to sculpt T-tubule spatial patterning and morphology.
心脏横管（T 管）通过肌小节外周肌动蛋白重复序列（costameres）锚定在肌节 Z 盘上，以建立规则的间隔模式。肌小节外周肌动蛋白重复序列的主要成分之一是肌营养不良蛋白-糖蛋白复合物（DGC）。然而，在生理和病理条件下，DGC 的组装如何与 T 管的形成和维持相协调仍不清楚。
鉴于蛋白酪氨酸磷酸酶非受体型 23（Ptpn23）在调节膜变形中的已知作用，测定了其在扩张型心肌病患者中的表达。利用 Cre/Loxp、CRISPR/Cas9 和腺相关病毒 9（AAV9）介导的体内基因编辑，我们构建了心肌细胞特异性的α-辅肌动蛋白-2（Z 盘的主要成分）敲除小鼠。我们还使用肌营养不良蛋白全身敲除小鼠来干扰 DGC。进行 MM 4-64 和 Di-8-ANEPPS 染色、Cav3 免疫荧光和透射电子显微镜检查，以确定分离细胞和完整心脏中的 T 管结构。此外，通过甘油梯度分级分离和 SDS-PAGE 分析确定 DGC 与α-辅肌动蛋白的组装以及肌营养不良蛋白功能丧失情况。
扩张型心肌病患者和小鼠衰竭心脏中 Ptpn23 的表达水平降低。α-辅肌动蛋白-2 的基因缺失导致 T 管紊乱，直径增大，并进展为扩张型心肌病，而不影响肌节组织。AAV9 介导的镶嵌体细胞诱变进一步表明α-辅肌动蛋白-2 在调节 T 管形成中具有细胞自主作用。遗传和生化分析表明，Ptpn23 通过与α-辅肌动蛋白和肌营养不良蛋白相互作用，对于将 T 管膜锚定到 Z 盘的肌小节外周肌动蛋白重复序列的完整性至关重要。α-辅肌动蛋白的缺失改变了 Ptpn23 和 DGC 的亚细胞定位。此外，肌营养不良蛋白的基因失活导致与α-辅肌动蛋白-2 功能丧失相似的 T 管缺陷，但不影响 Ptpn23 在 Z 盘的定位。最后，1 月龄时可诱导的α-辅肌动蛋白-2 敲除表明，Ptpn23 对于成年心肌细胞中 T 管的维持也是必需的。
Ptpn23 对于沿 Z 盘的心脏 T 管形成和维持至关重要。在出生后心脏发育过程中，Ptpn23 与肌节α-辅肌动蛋白相互作用，并协调 DGC 在肌小节外周肌动蛋白重复序列处的组装，以塑造 T 管的空间模式和形态。
