Endogenous slow and fast myosin dynamics in myofibers isolated from mice expressing GFP-Myh7 and Kusabira Orange-Myh1
Endogenous slow and fast myosin dynamics in myofibers isolated from mice expressing GFP-Myh7 and Kusabira Orange-Myh1
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DOI：
10.1152/ajpcell.00415.2021
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发表时间：
2022-08-01
影响因子：
5.5
通讯作者：
Nishimura，Takanori
中科院分区：
文献类型：
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作者：
Ojima，Koichi;Kigaki，Masahiro;Nishimura，Takanori
骨骼肌由表达不同肌球蛋白亚型的慢肌纤维和快肌纤维组成。大约300个肌球蛋白在肌原纤维中形成单根粗丝，其中肌球蛋白不断交换。然而，内源性慢和快肌球蛋白动力学尚未完全了解。为了阐明这些动力学，在这里，我们产生了小鼠表达绿色荧光蛋白标记的慢肌球蛋白重链（GFP-Myh 7）和Kusabira橙子荧光蛋白标记的快肌球蛋白重链（KuO-Myh 1）。首先，这些小鼠使我们能够在荧光显微镜下区分GFP-和KuO-肌纤维：GFP-Myh 7和KuO-Myh 1分别只在慢肌纤维和快肌纤维中表达。接下来，为了监测内源性肌球蛋白动力学，进行光漂白后荧光恢复（FRAP）。GFP-Myh 7和KuO-Myh 1的移动的分数（Mf）几乎是恒定值，与肌纤维的区域和肌纤维分离的肌肉部分无关。有趣的是，蛋白酶体抑制剂治疗显着降低了Mf在GFP-Myh 7，但不是在KuO-Myh 1肌纤维，表明蛋白质周转的干扰的反应依赖于肌纤维类型。两者合计，目前的结果表明，我们产生的小鼠是有前途的工具，不仅用于区分GFP-和KuO-肌纤维，但也用于研究动态的内源性肌球蛋白异构体的活细胞荧光成像。
Skeletal muscle consists of slow and fast myofibers in which different myosin isoforms are expressed. Approximately 300 myosins form a single-thick filament in the myofibrils, where myosin is continuously exchanged. However, endogenous slow and fast myosin dynamics have not been fully understood. To elucidate those dynamics, here we generated mice expressing green fluorescence protein-tagged slow myosin heavy chain (GFP-Myh7) and Kusabira Orange fluorescence protein-tagged fast myosin heavy chain (KuO-Myh1). First, these mice enabled us to distinguish between GFP- and KuO-myofibers under fluorescence microscopy: GFP-Myh7 and KuO-Myh1 were exclusively expressed in slow myofibers and fast myofibers, respectively. Next, to monitor endogenous myosin dynamics, fluorescence recovery after photobleaching (FRAP) was conducted. The mobile fraction (Mf) of GFP-Myh7 and that of KuO-Myh1 were almost constant values independent of the regions of the myofibers and the muscle portions where the myofibers were isolated. Intriguingly, proteasome inhibitor treatment significantly decreased the Mf in GFP-Myh7 but not in KuO-Myh1 myofibers, indicating that the response to a disturbance in protein turnover depended on muscle fiber type. Taken together, the present results indicated that the mice we generated are promising tools not only for distinguishing between GFP- and KuO-myofibers but also for studying the dynamics of endogenous myosin isoforms by live-cell fluorescence imaging.