Depletion of Vasohibin 1 Speeds Contraction and Relaxation in Failing Human Cardiomyocytes
Depletion of Vasohibin 1 Speeds Contraction and Relaxation in Failing Human Cardiomyocytes
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DOI：
10.1161/circresaha.119.315947
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发表时间：
2020-07-03
影响因子：
20.1
通讯作者：
Prosser, Benjamin L.
中科院分区：
文献类型：
--
作者：
Chen, Christina Yingxian;Salomon, Alexander K.;Prosser, Benjamin L.
理由：心肌松弛受损是心衰（HF）的一个顽固性特征。在人HF中，去酪氨酸微管使心肌细胞变硬并损害松弛。然而，去酪氨酸酶的身份仍然不明确，阻碍了机制研究和治疗发展。目的：我们旨在确定最近发现的VASH1/2（血管hibin 1/2）和SVBP（小血管hibin结合蛋白）复合物在心肌细胞中是否是一种活性的去酪氨酸酶，以及VASH-SVBP的遗传抑制是否足以降低心衰患者的僵硬性和改善收缩性。方法和结果：转录谱分析显示，在人类心脏中，vash1转录本的丰度是vash2的10倍。利用短发夹rna （shRNAs）对抗VASH1、VASH2和svbp，我们发现VASH1-和VASH2- svbp复合物在心肌细胞中都具有微管蛋白羧肽酶的功能，其中VASH1起主导作用。我们还生成了催化死版本的酪氨酸化酶TTL (TTL- e331q)，以分离TTL的微管解聚作用与其酶活性。微管稳定性测试显示TTL和TTL- e331q都能解聚微管，而VASH1和SVBP的耗尽则能独立于解聚而减少去酪氨酸。我们接下来探讨了对人类心肌细胞收缩性的影响。心衰患者的收缩动力学减慢，射血分数保留的心衰患者心肌细胞松弛明显减慢。VASH1的敲低使非衰竭心肌细胞的动力学得到了微妙的改善，同时显著改善了衰竭心肌细胞的动力学。此外，TTL，而不是TTL- e331q，稳健地加速弛豫。钙瞬态和收缩性的同时测量表明，VASH1耗竭加速了与钙循环变化无关的动力学。最后，原子力显微镜证实，VASH1的耗尽降低了衰竭的人心肌细胞的硬度。结论：VASH-SVBP复合物是心肌细胞中的活性微管蛋白羧肽酶。抑制VASH1或激活TTL足以降低心衰患者心肌细胞的僵硬和加速松弛，支持进一步追求去酪氨酸作为舒张功能障碍的治疗靶点。
Rationale: Impaired myocardial relaxation is an intractable feature of several heart failure (HF) causes. In human HF, detyrosinated microtubules stiffen cardiomyocytes and impair relaxation. Yet the identity of detyrosinating enzymes have remained ambiguous, hindering mechanistic study and therapeutic development. Objective: We aimed to determine if the recently identified complex of VASH1/2 (vasohibin 1/2) and SVBP (small vasohibin binding protein) is an active detyrosinase in cardiomyocytes and if genetic inhibition of VASH-SVBP is sufficient to lower stiffness and improve contractility in HF. Methods and Results: Transcriptional profiling revealed thatVASH1transcript is >10-fold more abundant thanVASH2in human hearts. Using short hairpin RNAs (shRNAs) againstVASH1,VASH2, andSVBP, we showed that both VASH1- and VASH2-SVBP complexes function as tubulin carboxypeptidases in cardiomyocytes, with a predominant role for VASH1. We also generated a catalytically dead version of the tyrosinating enzyme TTL (TTL-E331Q) to separate the microtubule depolymerizing effects of TTL from its enzymatic activity. Assays of microtubule stability revealed that both TTL and TTL-E331Q depolymerize microtubules, while VASH1 and SVBP depletion reduce detyrosination independent of depolymerization. We next probed effects on human cardiomyocyte contractility. Contractile kinetics were slowed in HF, with dramatically slowed relaxation in cardiomyocytes from patients with HF with preserved ejection fraction. Knockdown of VASH1 conferred subtle kinetic improvements in nonfailing cardiomyocytes, while markedly improving kinetics in failing cardiomyocytes. Further, TTL, but not TTL-E331Q, robustly sped relaxation. Simultaneous measurements of calcium transients and contractility demonstrated that VASH1 depletion speeds kinetics independent from alterations to calcium cycling. Finally, atomic force microscopy confirmed that VASH1 depletion reduces the stiffness of failing human cardiomyocytes. Conclusions: VASH-SVBP complexes are active tubulin carboxypeptidases in cardiomyocytes. Inhibition of VASH1 or activation of TTL is sufficient to lower stiffness and speed relaxation in cardiomyocytes from patients with HF, supporting further pursuit of detyrosination as a therapeutic target for diastolic dysfunction.