BACKGROUND β2-adrenergic receptors (β2ARs) are the target of catecholamines and play fundamental roles in cardiovascular, pulmonary, and skeletal muscle physiology. An important action of β2AR stimulation on skeletal muscle is anabolic growth, which has led to the use of agonists such as clenbuterol by athletes to enhance muscle performance. While previous work has demonstrated that β2ARs can engage distinct signaling and functional cascades mediated by either G proteins or the multifunctional adaptor protein, β-arrestin, the precise role of β-arrestin in skeletal muscle physiology is not known. Here, we tested the hypothesis that agonist activation of the β2AR by clenbuterol would engage β-arrestin as a key transducer of anabolic skeletal muscle growth. METHODS The contractile force of isolated extensor digitorum longus muscle (EDL) and calcium signaling in isolated flexor digitorum brevis (FDB) fibers were examined from the wild-type (WT) and β-arrestin 1 knockout mice (βarr1KO) followed by chronic administration of clenbuterol (1 mg/kg/d). Hypertrophic responses including fiber composition and fiber size were examined by immunohistochemical imaging. We performed a targeted phosphoproteomic analysis on clenbuterol stimulated primary cultured myoblasts from WT and βarr1KO mice. Statistical significance was determined by using a two-way analysis with Sidak's or Tukey's multiple comparison test and the Student's t test. RESULTS Chronic administration of clenbuterol to WT mice enhanced the contractile force of EDL muscle and calcium signaling in isolated FDB fibers. In contrast, when administered to βarr1KO mice, the effect of clenbuterol on contractile force and calcium influx was blunted. While clenbuterol-induced hypertrophic responses were observed in WT mice, this response was abrogated in mice lacking β-arrestin 1. In primary cultured myoblasts, clenbuterol-stimulated phosphorylation of multiple pro-hypertrophy proteins required the presence of β-arrestin 1. CONCLUSIONS We have identified a previously unappreciated role for β-arrestin 1 in mediating β2AR-stimulated skeletal muscle growth and strength. We propose these findings could have important implications in the design of future pharmacologic agents aimed at reversing pathological conditions associated with skeletal muscle wasting.

中文翻译：

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β-arrestin1调节β2-肾上腺素能受体介导的骨骼肌肥大和收缩。

背景技术β2-肾上腺素能受体（β2ARs）是儿茶酚胺的靶标，并且在心血管，肺和骨骼肌生理中起着基本作用。β2AR刺激对骨骼肌的重要作用是合成代谢的增长，这导致运动员使用了诸如盐酸克伦特罗这样的激动剂来增强肌肉性能。尽管先前的研究表明β2ARs可以参与由G蛋白或多功能衔接蛋白β-arrestin介导的不同信号传导和功能级联，但β-arrestin在骨骼肌生理中的确切作用尚不清楚。在这里，我们测试了一种假设，即克仑特罗对β2AR的激动剂激活将使β-arrestin参与合成代谢骨骼肌生长的关键转导。方法：从野生型（WT）和β-arrestin1基因敲除小鼠（βarr1KO）中，分别检测经分离的趾长伸肌（EDL）的收缩力和离体的短指屈短肌（FDB）纤维中的钙信号。盐酸克仑特罗（1 mg / kg / d）。通过免疫组织化学成像检查肥大性反应，包括纤维成分和纤维大小。我们对克伦特罗刺激的WT和βarr1KO小鼠原代培养的成肌细胞进行了靶向磷酸化蛋白质组学分析。统计显着性是通过使用Sidak或Tukey的多重比较检验和St​​udent t检验的双向分析来确定的。结果对野生型小鼠长期服用盐酸克伦特罗可增强离体FDB纤维中EDL肌肉的收缩力和钙信号传导。相反，当对βarr1KO小鼠给药时，盐酸克伦特罗对收缩力和钙流入的作用减弱。虽然在野生型小鼠中观察到了克仑特罗诱导的肥大性反应，但在缺乏β-arrestin1的小鼠中这种反应被消除了。在原代培养的成肌细胞中，克仑特罗刺激的多种肥大性肥大蛋白的磷酸化需要β-arrestin1的存在。已经确定了β-arrestin1在介导β2AR刺激的骨骼肌生长和力量中的前所未有的作用。我们认为这些发现可能对未来旨在逆转骨骼肌消瘦相关病理状况的药物的设计具有重要意义。虽然在野生型小鼠中观察到了克仑特罗诱导的肥大性反应，但在缺乏β-arrestin1的小鼠中这种反应被消除了。在原代培养的成肌细胞中，克仑特罗刺激的多种肥大性肥大蛋白的磷酸化需要β-arrestin1的存在。已经确定了β-arrestin1在介导β2AR刺激的骨骼肌生长和力量中的前所未有的作用。我们认为这些发现可能对未来旨在逆转骨骼肌消瘦相关病理状况的药物的设计具有重要意义。虽然在野生型小鼠中观察到了克仑特罗诱导的肥大性反应，但在缺乏β-arrestin1的小鼠中这种反应被消除了。在原代培养的成肌细胞中，克仑特罗刺激的多种肥大性肥大蛋白的磷酸化需要β-arrestin1的存在。已经确定了β-arrestin1在介导β2AR刺激的骨骼肌生长和力量中的前所未有的作用。我们认为这些发现可能对未来旨在逆转骨骼肌消瘦相关病理状况的药物的设计具有重要意义。克仑特罗刺激的多种肥大性前体肥大蛋白的磷酸化需要存在β-arrestin1。结论我们已经确定了β-arrestin1在介导β2AR刺激的骨骼肌生长和强度中的作用，这一作用以前未被认识。我们认为这些发现可能对未来旨在逆转骨骼肌消瘦相关病理状况的药物的设计具有重要意义。克仑特罗刺激的多种肥大性前体肥大蛋白的磷酸化需要存在β-arrestin1。结论我们已经确定了β-arrestin1在介导β2AR刺激的骨骼肌生长和强度中的作用，这一作用以前未被认识。我们认为这些发现可能对未来旨在逆转骨骼肌消瘦相关病理状况的药物的设计具有重要意义。