Extracellular matrix remodeling mechanisms are understudied in cardiac development and congenital heart defects. We show that matrix-degrading metalloproteases ADAMTS1 and ADAMTS5, are extensively co-expressed during mouse cardiac development. The mouse mutants of each gene have mild cardiac anomalies, however, their combined genetic inactivation to elicit cooperative roles is precluded by tight gene linkage. Therefore, we coupled inactivation with pharmacologic ADAMTS5 blockade to uncover stage-specific cooperative roles and investigated their potential substrates in mouse cardiac development. ADAMTS5 blockade was achieved in null mouse embryos using an activity-blocking monoclonal antibody during distinct developmental windows spanning myocardial compaction or cardiac septation and outflow tract rotation. Synchrotron imaging, RNA in situ hybridization, immunofluorescence microscopy and electron microscopy were used to determine the impact on cardiac development and compared to and ADAMTS-cleavage resistant versican mutants. Mass spectrometry-based N-terminomics was used to seek relevant substrates. Combined inactivation of ADAMTS1 and ADAMTS5 prior to 12.5 days of gestation led to dramatic accumulation of versican-rich cardiac jelly and inhibited formation of compact and trabecular myocardium, which was also observed in mice with ADAMTS cleavage-resistant versican. Combined inactivation after 12.5 days impaired outflow tract development and ventricular septal closure, generating a tetralogy of Fallot-like defect. N-terminomics of combined ADAMTS knockout and control hearts identified a cleaved glypican-6 peptide only in the controls. ADAMTS1 and ADAMTS5 expression in cells was associated with specific glypican-6 cleavages. Paradoxically, combined ADAMTS1 and ADAMTS5 inactivation reduced cardiac glypican-6 and outflow tract transcription. Notably, hearts demonstrated similar rotational defects as combined ADAMTS inactivated hearts and both had reduced hedgehog signaling. Thus, versican proteolysis in cardiac jelly at the canonical Glu-Ala site is cooperatively mediated by ADAMTS1 and ADAMTS5 and required for proper ventricular cardiomyogenesis, whereas, reduced glypican-6 after combined ADAMTS inactivation impairs hedgehog signaling, leading to outflow tract malrotation.

中文翻译：

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在时间特异性窗口中紧密连接的 ADAMTS 蛋白酶的遗传-药物联合失活揭示了 versican 蛋白水解和 glypican-6 在心脏发育中的不同作用


在心脏发育和先天性心脏缺陷中，细胞外基质重塑机制的研究不足。我们表明，基质降解金属蛋白酶 ADAMTS1 和 ADAMTS5 在小鼠心脏发育过程中广泛共表达。每个基因的小鼠突变体都有轻微的心脏异常，然而，它们的联合基因失活以引发合作作用被紧密的基因连锁所排除。因此，我们将灭活与药物ADAMTS5阻断相结合，以揭示阶段特异性合作作用，并研究它们在小鼠心脏发育中的潜在底物。在跨越心肌压实或心脏间隔和流出道旋转的不同发育窗口期间，使用活性阻断单克隆抗体在无效小鼠胚胎中实现ADAMTS5阻断。同步加速器成像、RNA 原位杂交、免疫荧光显微镜和电子显微镜用于确定对心脏发育的影响，并与 ADAMTS 抗切割的 versican 突变体进行比较。使用基于质谱的 N 端组学寻找相关底物。妊娠 12.5 天前 ADAMTS1 和 ADAMTS5 的联合失活导致富含 Versican 的心脏胶质急剧积累并抑制致密和小梁心肌的形成，这在具有 ADAMTS 切割抗性 Versican 的小鼠中也观察到。12.5 天后联合失活损害了流出道发育和室间隔闭合，产生了法洛样缺损四联症。ADAMTS 敲除和对照心脏联合的 N 端组学仅在对照中鉴定出裂解的 glypican-6 肽。细胞中 ADAMTS1 和 ADAMTS5 的表达与特异性 Glypican-6 切割相关。 矛盾的是，ADAMTS1 和 ADAMTS5 联合灭活减少了心肌 glypican-6 和流出道转录。值得注意的是，心脏表现出与 ADAMTS 联合灭活心脏相似的旋转缺陷，并且两者都减少了刺猬信号传导。因此，经典 Glu-Ala 位点的心脏胶状体中的藤蛋白水解由 ADAMTS1 和 ADAMTS5 协同介导，并且是正常室性心肌生成所必需的，而联合 ADAMTS 失活后 glypican-6 的减少会损害刺猬信号传导，导致流出道旋转不良。