Data_Sheet_1_Phosphorylation at Serines 157 and 161 Is Necessary for Preserving Cardiac Expression Level and Functions of Sarcomeric Z-Disc Protein Telethonin.pdf

Aims: In cardiac myocytes, the sarcomeric Z-disc protein telethonin is constitutively bis-phosphorylated at C-terminal residues S157 and S161; however, the functional significance of this phosphorylation is not known. We sought to assess the significance of telethonin phosphorylation in vivo, using a novel knock-in (KI) mouse model generated to express non-phosphorylatable telethonin (TcapS157/161A).Methods and Results:TcapS157/161A and wild-type (WT) littermates were characterized by echocardiography at baseline and after sustained β-adrenergic stimulation via isoprenaline infusion. Heart tissues were collected for gravimetric, biochemical, and histological analyses. At baseline, TcapS157/161A mice did not show any variances in cardiac structure or function compared with WT littermates and mutant telethonin remained localized to the Z-disc. Ablation of telethonin phosphorylation sites resulted in a gene-dosage dependent decrease in the cardiac telethonin protein expression level in mice carrying the S157/161A alleles, without any alteration in telethonin mRNA levels. The proteasome inhibitor MG132 significantly increased the expression level of S157/161A telethonin protein in myocytes from TcapS157/161A mice, but not telethonin protein in myocytes from WT mice, indicating a role for the ubiquitin–proteasome system in the regulation of telethonin protein expression level. TcapS157/161A mice challenged with sustained β-adrenergic stimulation via isoprenaline infusion developed cardiac hypertrophy accompanied by mild systolic dysfunction. Furthermore, the telethonin protein expression level was significantly increased in WT mice following isoprenaline stimulation but this response was blunted in TcapS157/161A mice.Conclusion: Overall, these data reveal that telethonin protein turnover in vivo is regulated in a novel phosphorylation-dependent manner and suggest that C-terminal phosphorylation may protect telethonin against proteasomal degradation and preserve cardiac function during hemodynamic stress. Given that human telethonin C-terminal mutations have been associated with cardiac and skeletal myopathies, further research on their potential impact on phosphorylation-dependent regulation of telethonin protein expression could provide valuable mechanistic insight into those myopathies.

目的：在心肌细胞中，肌节Z盘蛋白telethonin在C端残基S157和S161处持续进行双磷酸化；然而，这种磷酸化的功能意义尚不清楚。本研究旨在通过构建表达非磷酸化telethonin（TcapS157/161A）的新型敲入（KI）小鼠模型，在体内评估telethonin磷酸化的意义。方法与结果：对TcapS157/161A和野生型（WT）同窝小鼠进行基线及异丙肾上腺素持续β-肾上腺素能刺激后的超声心动图检查。收集心脏组织进行质量测量、生化分析和组织学分析。在基线状态下，TcapS157/161A小鼠与WT同窝小鼠相比，在心脏结构和功能上未显示出任何差异，突变型telethonin仍定位于Z盘。消除telethonin磷酸化位点导致携带S157/161A等位基因的小鼠心脏telethonin蛋白表达水平基因剂量依赖性降低，而telethonin mRNA水平未发生任何改变。蛋白酶体抑制剂MG132显著提高了TcapS157/161A小鼠心肌细胞中S157/161A telethonin蛋白的表达水平，但未影响WT小鼠心肌细胞中的telethonin蛋白，这表明泛素-蛋白酶体系统在调节telethonin蛋白表达水平中发挥作用。通过异丙肾上腺素持续β-肾上腺素能刺激的TcapS157/161A小鼠出现心脏肥大伴随轻度收缩功能障碍。此外，在异丙肾上腺素刺激后，WT小鼠的telethonin蛋白表达水平显著增加，但在TcapS157/161A小鼠中这种反应被抑制。结论：总体而言，这些数据揭示了telethonin蛋白在体内的周转以新颖的磷酸化依赖性方式调节，并表明C端磷酸化可能保护telethonin免受蛋白酶体降解，并在血流动力学应激期间维持心脏功能。鉴于人类telethonin C端突变与心脏和骨骼肌病相关，进一步研究其潜在影响磷酸化依赖性调节telethonin蛋白表达，可能为这些肌病的机制研究提供宝贵的见解。