Characterization of a newly identified myosin, Myo18Aγ, in cardiac sarcomeres

心脏肌节中新鉴定的肌球蛋白 Myo18Aγ 的表征

• 批准号: 512106520
• 负责人: Professor Dr. Peter Hanley
• 金额: --
• 依托单位: HMU Research, Development und Innovation gGmbH (HMU RDI)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/512106520?language=en
• 关键词: Characterization; newly; identified; myosin; Myo18A

Using various Myo18a knockout mouse models, microscopy and RNA-sequence analysis, we unexpectedly discovered Myo18Aγ as a new component of cardiac sarcomeres (published preliminary data). Myo18Aγ localizes to the sarcomeric A-band and may be a missing piece of the puzzle towards understanding sarcomere assembly and function. In unpublished preliminary data, we confirmed that the γ-isoform of Myo18A is indeed specific for striated muscle. We also found that the expression of Myo18Aγ but not Myo18Aα increases during cardiac differentiation and decreases during cardiac de-differentiation, consistent with a role for Myo18Aγ in sarcomerogenesis (myofibrillogenesis). In the current project, we plan to precisely locate Myo18Aγ on the sarcomere landscape using electron microscopy and single-molecule localization microscopy (SMLM). Preliminary data using adenoviral vectors indicate that the N-terminus of Myo18Aγ is sufficient for localization to the A-band. We also plan to knockdown Myo18Aγ (and Myo18Aα) in conditionally immortalized rat atrial myocytes and neonatal rat ventricular myocytes to explore the roles of Myo18Aγ (and Myo18Aα) in sarcomere assembly and maintenance, respectively. Western blot analyses confirmed that the rat Myo18Aγ-specific shRNAs (delivered by adenoviruses) are effective. In addition, using Myo18aexon1 deletion and inducible Myo18a knockout mouse models, we will determine whether the α-isoform of Myo18A is redundant for cardiac development and sarcomere function and whether loss of Myo18A expression causes cardiomyopathy. Finally, we will map the Myo18Aγ protein interactome using co-immunoprecipitation and BioID and validate putative interaction proteins. We anticipate that knockdown/knockout studies, together with precise localization of Myo18Aγ in the sarcomeric A-band and identification/confirmation of interaction partners, should significantly advance our understanding of the roles of class 18 myosins in sarcomere assembly and cardiac health.

利用各种Myo18A基因敲除的小鼠模型，显微镜和序列分析，我们意外地发现了Myo18Aγ作为心脏肌节的一个新成分(已公布初步数据)。Myo18Aγ定位于肌节A带，可能是理解肌节组装和功能的一个缺失片段。在未发表的初步数据中，我们证实了Myo18A的γ-亚型确实是横纹肌所特有的。我们还发现，Myo18Aγ的表达在心脏分化过程中增加，而不是Myo18Aα的表达增加，而在心脏去分化过程中表达下降，这与Myo18Aγ在肌瘤发生(肌纤维形成)中的作用一致。在目前的项目中，我们计划使用电子显微镜和单分子定位显微镜在肌节景观上精确定位Myo18Aγ。使用腺病毒载体的初步数据表明，Myo18Aγ的N末端足以定位到A带。我们还计划在条件永生化的大鼠心房肌细胞和新生大鼠的心室肌细胞中敲除Myo18Aγ(和Myo18Aα)，以分别探讨Myo18Aγ(和Myo18Aα)在肌节组装和维持中的作用。Western印迹分析证实，大鼠Myo18Aγ特异性shRNA(由腺病毒递送)是有效的。此外，利用Myo18aexon 1缺失和可诱导的Myo18a基因敲除小鼠模型，我们将确定Myo18A的α异构体是否对心脏发育和肌节功能是多余的，以及Myo18A表达缺失是否会导致心肌病。最后，我们将使用免疫共沉淀和生物ID来定位Myo18Aγ蛋白的相互作用组，并验证可能的相互作用蛋白。我们预计，基因敲除/基因敲除研究，加上肌节A带上Myo18Aγ的精确定位和相互作用伙伴的识别/确认，应该会显著促进我们对18类肌球蛋白在肌节组装和心脏健康中的作用的理解。