已经观察到Slo1基因敲除小鼠的运动功能降低,某些Slo1突变的人有运动问题,但是没有答案是否运动障碍是由神经系统Slo1的丧失引起的,或者骨骼肌,或者两者兼而有之。这里,确定Slo1在哪些组织中起调节运动功能的作用,并在治疗相关运动障碍方面提供更深入的见解,我们产生了骨骼肌特异性Slo1基因敲除小鼠,研究Slo1缺乏骨骼肌的功能变化,并探讨其潜在机制。
我们使用骨骼肌特异性Slo1敲除小鼠(Myf5-Cre;Slo1flox/flox小鼠,称为CKO)作为体内模型,以检查Slo1在肌肉生长和肌肉再生中的作用。前肢握力测试用于评估骨骼肌功能,跑步机疲劳测试用于测试全身耐力。使用源自CKO(成肌细胞/CKO)小鼠的小鼠原代成肌细胞将发现扩展到对成肌细胞分化和融合的体外作用。实时定量PCR,使用蛋白质印迹和免疫荧光方法分析Slo1在成肌细胞分化和肌肉再生过程中的表达。探讨基因参与Slo1缺失诱导的肌肉功能障碍的调节,在原代成肌细胞中进行RNA-seq分析。使用免疫沉淀和质谱鉴定与Slo1相互作用的蛋白质。使用双荧光素酶报告基因测定来鉴定Slo1缺失是否影响NFAT活性。
我们发现,CKO小鼠的体重和大小与Slo1flox/flox小鼠(称为WT)的体重和大小没有显着差异。肌肉中Slo1的缺乏导致耐力降低(约减少30%,P<0.05)和强度(~30%降低,P<0.001)。尽管肌肉的一般形态没有差异,电子显微镜显示比目鱼肌中线粒体的含量显着减少(约40%减少,P<0.01)。我们发现Slo1主要在细胞膜上表达,在慢抽搐纤维中表达较高。Slo1蛋白表达在肌肉出生后发育和损伤后再生过程中逐渐降低,并且在成肌细胞分化期间表达强烈降低。Slo1缺失损害了成肌细胞分化和慢抽搐纤维形成。机械上,RNA-seq分析表明,Slo1影响与肌源性分化和慢抽搐纤维形成相关基因的表达。Slo1与FAK相互作用以影响成肌分化,Slo1缺失会降低NFAT活性。
我们的数据显示Slo1缺乏损害骨骼肌再生和慢抽搐纤维形成。
It has been observed that
Slo1 knockout mice have reduced motor function, and people with certain
Slo1 mutations have movement problems, but there is no answer whether the movement disorder is caused by the loss of Slo1 in the nervous system, or skeletal muscle, or both. Here, to ascertain in which tissues Slo1 functions to regulate motor function and offer deeper insight in treating related movement disorder, we generated skeletal muscle-specific Slo1 knockout mice, studied the functional changes in
Slo1-deficient skeletal muscle and explored the underlying mechanism.
We used skeletal muscle-specific Slo1 knockout mice (Myf5-Cre; Slo1flox/flox mice, called CKO) as in vivo models to examine the role of
Slo1 in muscle growth and muscle regeneration. The forelimb grip strength test was used to assess skeletal muscle function and treadmill exhaustion test was used to test whole-body endurance. Mouse primary myoblasts derived from CKO (myoblast/CKO) mice were used to extend the findings to in vitro effects on myoblast differentiation and fusion. Quantitative real-time PCR, western blot and immunofluorescence approaches were used to analyse Slo1 expression during myoblast differentiation and muscle regeneration. To investigate the involvement of genes in the regulation of muscle dysfunction induced by Slo1 deletion, RNA-seq analysis was performed in primary myoblasts. Immunoprecipitation and mass spectrometry were used to identify the protein interacting with Slo1. A dual-luciferase reporter assay was used to identify whether Slo1 deletion affects NFAT activity.
We found that the body weight and size of CKO mice were not significantly different from those of Slo1flox/flox mice (called WT). Deficiency of Slo1 in muscles leads to reduced endurance (~30% reduction, P < 0.05) and strength (~30% reduction, P < 0.001). Although there was no difference in the general morphology of the muscles, electron microscopy revealed a considerable reduction in the content of mitochondria in the soleus muscle (~40% reduction, P < 0.01). We found that Slo1 was expressed mainly on the cell membrane and showed higher expression in slow-twitch fibres. Slo1 protein expression is progressively reduced during muscle postnatal development and regeneration after injury, and the expression is strongly reduced during myoblast differentiation.
Slo1 deletion impaired myoblast differentiation and slow-twitch fibre formation. Mechanistically, RNA-seq analysis showed that Slo1 influences the expression of genes related to myogenic differentiation and slow-twitch fibre formation.
Slo1 interacts with FAK to influence myogenic differentiation, and
Slo1 deletion diminishes NFAT activity.
Our data reveal that Slo1 deficiency impaired skeletal muscle regeneration and slow-twitch fibre formation.