Smith Eric D, Jin Karen, Ferguson Brianna, Tsan Yao-Chang, DePalma Samuel J, Meisner Joshua, Renberg Aaron, Bedi Kenneth, Friedline Sabrina, Margulies Kenneth B, Baker Brendon M, Helms Adam S
Department of Internal Medicine, Division of Cardiovascular Medicine, University of Michigan, Ann Arbor, MI, USA.
Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA.
bioRxiv. 2025 Jun 3:2025.06.01.657304. doi: 10.1101/2025.06.01.657304.
Truncating variants in desmoplakin (tv), are a leading cause of arrhythmogenic cardiomyopathy (ACM), often presenting with early fibrosis and arrhythmias disproportionate to systolic dysfunction. DSP is critical for cardiac mechanical integrity, linking desmosomes to the cytoskeleton to withstand contractile forces. While loss-of-function is implicated, direct evidence, both for DSP haploinsufficiency in human hearts and for the impact of mechanical stress on cardiomyocyte adhesion, has been limited, leaving the pathogenic mechanism unclear.
We analyzed explanted human heart tissue from patients with tv (N=3), titin truncating variants (tv, N=5), and controls (N=5) using RNA-sequencing and mass spectrometry. We generated human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) harboring patient-derived or CRISPR-Cas9 engineered tv to model a range of DSP expression levels. Using a 2D cardiac muscle bundle (CMB) platform enabling live visualization of cell junctions, we developed an assay to assess cell-cell adhesion upon heightened contractile stress in response to the contractile agonist endothelin-1. CRISPR-interference (CRISPRi) was used to confirm the role of DSP loss, and CRISPR-activation (CRISPRa) was tested for therapeutic rescue.
Compared to both control and tv hearts, tv human hearts exhibited reduced at both the mRNA and protein level, as well as broadly disrupted desmosomal stoichiometry. Transcriptomic and proteomic analyses implicated cell adhesion, extracellular matrix, and inflammatory pathways. iPSC-CM models recapitulated DSP haploinsufficiency and desmosomal disruption. tv CMBs showed normal baseline contractile function. However, they displayed marked cell-cell adhesion failure with contractile stress (75% failure vs. 8% in controls, p<0.001). Adhesion failure was prevented by the myosin inhibitor, mavacamten. CRISPRi-mediated DSP knockdown replicated this susceptibility to adhesion failure. Conversely, CRISPRa robustly increased DSP expression and rescued cell-cell adhesion failure in tv CMBs (9% failure post-CRISPRa, p<0.001 vs. un-treated). Rescue occurred even when only the DSPII isoform was upregulated in a model with biallelic transcript 1 loss of function.
DSP haploinsufficiency is the major cause of DSP cardiomyopathy with a primary consequence of conferring vulnerability to cardiomyocyte cell-cell adhesion failure under heightened contractile stress. Transcriptional activation of reverses this defect in preclinical models, establishing proof-of-concept for a potential therapeutic strategy in DSP cardiomyopathy.
桥粒斑蛋白截短变异体（tv）是致心律失常性心肌病（ACM）的主要病因，常伴有早期纤维化和心律失常，且心律失常程度与收缩功能障碍不成比例。DSP对于心脏机械完整性至关重要，它将桥粒与细胞骨架相连以承受收缩力。虽然功能丧失与之相关，但关于人类心脏中DSP单倍体不足以及机械应力对心肌细胞黏附影响的直接证据一直有限，致病机制尚不清楚。
我们使用RNA测序和质谱分析了来自tv患者（N = 3）、肌联蛋白截短变异体（tv，N = 5）患者以及对照者（N = 5）的离体人心脏组织。我们生成了携带患者来源或经CRISPR - Cas9工程改造的tv的人诱导多能干细胞衍生心肌细胞（iPSC - CM），以模拟一系列DSP表达水平。使用二维心肌束（CMB）平台能够实时可视化细胞连接，我们开发了一种检测方法，以评估在收缩激动剂内皮素 - 1作用下收缩应激增强时的细胞间黏附。使用CRISPR干扰（CRISPRi）来确认DSP缺失的作用，并测试CRISPR激活（CRISPRa）用于治疗性挽救。
与对照心脏和tv心脏相比，tv人心脏在mRNA和蛋白质水平均表现出降低，以及桥粒化学计量广泛破坏。转录组学和蛋白质组学分析涉及细胞黏附、细胞外基质和炎症途径。iPSC - CM模型重现了DSP单倍体不足和桥粒破坏。tv CMB显示出正常的基线收缩功能。然而，它们在收缩应激下表现出明显的细胞间黏附失败（75%失败，而对照组为8%，p < 0.001）。肌球蛋白抑制剂mavacamten可预防黏附失败。CRISPRi介导的DSP敲低重现了这种对黏附失败的易感性。相反，CRISPRa强烈增加DSP表达并挽救了tv CMB中的细胞间黏附失败（CRISPRa后9%失败，与未处理相比p < 0.001）。即使在双等位基因转录本1功能丧失的模型中仅上调DSPII亚型时也出现了挽救。
DSP单倍体不足是DSP心肌病的主要原因，其主要后果是在收缩应激增强时使心肌细胞间黏附失败变得脆弱。在临床前模型中，DSP的转录激活可逆转这一缺陷，为DSP心肌病的潜在治疗策略建立了概念验证。
