Wei Chuanyu, Chan Shing-Fai, Saguner Ardan M, Brunckhorst Corinna, Duru Firat, Marine Joseph E, James Cynthia A, Calkins Hugh, Judge Daniel P, Shou Weinian, Chen Huei-Sheng Vincent
bioRxiv. 2024 Sep 9:2024.09.09.612149. doi: 10.1101/2024.09.09.612149.
Pathological fibrosis is a major finding in cardiovascular diseases and can result in arrhythmia and heart failure. Desmosome gene mutations can lead to arrhythmogenic cardiomyopathy (ACM). Among ACM, pathogenic desmoplakin ( ) variants cause a distinctive cardiomyopathy with excessive cardiac fibrosis that could precede ventricular dysfunction. variants are also linked to other fibrotic diseases. Whether DSP plays any role in pathological fibrosis remain unknown.
Mesenchymal stromal cells (MSCs) are resident fibroblast-like cells that are responsible for fibrogenesis in most organs, including hearts. We first used unbiased genome-wide analyses to generate cardiac fibroblasts-like, induced pluripotent stem cell-derived MSCs from normal donors and ACM patients with mutations. We then studied the fibrogenic responses of cardiac MSCs to transforming growth factor beta-1 (TGF-β1) using Western/Co-IP, autophagy assay, gene knockdowns/over-expressions, genomic analyses, mouse DSP knockdown models, immunostaining, and qPCR.
TGFβ1 induced excessive accumulations of vimentin (VIM)/fibrillar collagens, and over-activated fibrotic genes in mutant MSCs when compared to normal MSCs. In normal MSCs, VIMs bind to wild-type DSP during normal fibrogenesis after TGFβ1. mutant MSCs exhibited a haplo-insufficient phenotype with increased DSP-unbound VIMs that sequestered beclin-1 (BECN1) from activating autophagy and caveolin-1 (CAV1)-mediated endocytosis. Decreased autophagy caused collagen accumulations and diminished CAV1 endocytosis resulted in abnormal CAV1 plaque formation that over-activated fibrotic genes [ and fibronectin ( )] via heightened p38 activities after TGFβ1. Genome-wide analysis and DSP knockdown in mouse fibroblasts confirmed this novel role of mutations in pathological fibrosis. Overexpression of VIM-binding domains of DSP could suppress pathological fibrosis by increasing collagen autophagic degradation and decreasing fibrotic gene expressions.
Our data reveal that DSP deficiency in MSCs/fibroblasts leads to exaggerated fibrogenesis in DSP-cardiomyopathy by decreasing BECN1 availability for autophagy and CAV1-endocytosis. Overexpression of VIM binding domains of DSP could be a new strategy to treat pathological fibrosis.
病理性纤维化是心血管疾病的主要表现，可导致心律失常和心力衰竭。桥粒基因突变可导致致心律失常性心肌病（ACM）。在ACM中，致病性桥粒斑蛋白（DSP）变异可引发一种独特的心肌病，伴有过度的心脏纤维化，且可能先于心室功能障碍出现。DSP变异还与其他纤维化疾病有关。DSP在病理性纤维化中是否发挥作用尚不清楚。
间充质基质细胞（MSC）是驻留的成纤维细胞样细胞，在包括心脏在内的大多数器官的纤维化形成中起作用。我们首先使用无偏倚的全基因组分析，从正常供体和携带DSP突变的ACM患者中生成心脏成纤维细胞样的诱导多能干细胞衍生的MSC。然后，我们使用蛋白质免疫印迹/免疫共沉淀、自噬检测、基因敲低/过表达、基因组分析、小鼠DSP敲低模型、免疫染色和定量聚合酶链反应，研究心脏MSC对转化生长因子β1（TGF-β1）的纤维化反应。
与正常MSC相比，TGFβ1诱导DSP突变的MSC中波形蛋白（VIM）/纤维状胶原蛋白过度积累，并过度激活纤维化基因。在正常MSC中，TGFβ1后正常纤维化过程中VIM与野生型DSP结合。DSP突变的MSC表现出单倍体不足的表型，未与DSP结合的VIM增加，这些VIM将自噬相关蛋白1（BECN1）隔离，使其无法激活自噬以及小窝蛋白1（CAV1）介导的内吞作用。自噬减少导致胶原蛋白积累，而CAV1内吞作用减弱导致异常的CAV1斑块形成，在TGFβ1后通过增强p38活性过度激活纤维化基因[Ⅰ型胶原蛋白（COL1A1）和纤连蛋白（FN1）]。小鼠成纤维细胞的全基因组分析和DSP敲低证实了DSP突变在病理性纤维化中的这一新作用。DSP的VIM结合域过表达可通过增加胶原蛋白的自噬降解和降低纤维化基因表达来抑制病理性纤维化。
我们的数据表明，MSC/成纤维细胞中DSP缺乏通过减少自噬和CAV1内吞作用中BECN1的可用性，导致DSP心肌病中纤维化形成加剧。DSP的VIM结合域过表达可能是治疗病理性纤维化的新策略。
