Institute of Medical Biotechnology, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany.
School of Medicine, Western Sydney University, Sydney, NSW, 2560, Australia.
Skelet Muscle. 2022 Jun 23;12(1):14. doi: 10.1186/s13395-022-00295-8.
A common polymorphism (R577X) in the ACTN3 gene results in the complete absence of the Z-disc protein α-actinin-3 from fast-twitch muscle fibres in ~ 16% of the world's population. This single gene polymorphism has been subject to strong positive selection pressure during recent human evolution. Previously, using an Actn3KO mouse model, we have shown in fast-twitch muscles, eccentric contractions at L + 20% stretch did not cause eccentric damage. In contrast, L + 30% stretch produced a significant ~ 40% deficit in maximum force; here, we use isolated single fast-twitch skeletal muscle fibres from the Actn3KO mouse to investigate the mechanism underlying this.
Single fast-twitch fibres are separated from the intact muscle by a collagenase digest procedure. We use label-free second harmonic generation (SHG) imaging, ultra-fast video microscopy and skinned fibre measurements from our MyoRobot automated biomechatronics system to study the morphology, visco-elasticity, force production and mechanical strength of single fibres from the Actn3KO mouse. Data are presented as means ± SD and tested for significance using ANOVA.
We show that the absence of α-actinin-3 does not affect the visco-elastic properties or myofibrillar force production. Eccentric contractions demonstrated that chemically skinned Actn3KO fibres are mechanically weaker being prone to breakage when eccentrically stretched. Furthermore, SHG images reveal disruptions in the myofibrillar alignment of Actn3KO fast-twitch fibres with an increase in Y-shaped myofibrillar branching.
The absence of α-actinin-3 from the Z-disc in fast-twitch fibres disrupts the organisation of the myofibrillar proteins, leading to structural weakness. This provides a mechanistic explanation for our earlier findings that in vitro intact Actn3KO fast-twitch muscles are significantly damaged by L + 30%, but not L + 20%, eccentric contraction strains. Our study also provides a possible mechanistic explanation as to why α-actinin-3-deficient humans have been reported to have a faster decline in muscle function with increasing age, that is, as sarcopenia reduces muscle mass and force output, the eccentric stress on the remaining functional α-actinin-3 deficient fibres will be increased, resulting in fibre breakages.
ACTN3 基因的一个常见多态性（R577X）导致世界上约 16%的人的快肌纤维中完全缺失 Z 盘蛋白 α-辅肌动蛋白-3。这种单一基因多态性在人类进化的最近阶段受到了强烈的正向选择压力。以前，我们使用 Actn3KO 小鼠模型表明，在快肌中，20%拉伸的偏心收缩不会引起偏心损伤。相比之下，30%拉伸导致最大力显著下降约 40%；在这里，我们使用来自 Actn3KO 小鼠的分离的单个快肌纤维来研究这种现象的机制。
通过胶原酶消化程序将单个快肌纤维从完整肌肉中分离出来。我们使用无标记的二次谐波产生（SHG）成像、超快视频显微镜和我们的 MyoRobot 自动化生物机械系统的去皮纤维测量来研究来自 Actn3KO 小鼠的单个纤维的形态、粘弹性、力产生和机械强度。数据表示为平均值±标准差，并使用 ANOVA 进行显著性检验。
我们表明，缺乏 α-辅肌动蛋白-3不会影响粘弹性或肌球蛋白纤维的力产生。偏心收缩表明，化学去皮的 Actn3KO 纤维在偏心拉伸时更容易断裂，因此机械强度较弱。此外，SHG 图像显示 Actn3KO 快肌纤维的肌原纤维排列紊乱，Y 形肌原纤维分支增加。
快肌纤维 Z 盘缺乏 α-辅肌动蛋白-3会破坏肌原纤维蛋白的组织，导致结构脆弱。这为我们之前的发现提供了一种机制解释，即体外完整的 Actn3KO 快肌在受到 30%而不是 20%的偏心收缩应变时会受到显著损伤。我们的研究还为以下观点提供了一种可能的机制解释，即为什么缺乏 α-辅肌动蛋白-3 的人随着年龄的增长肌肉功能下降更快，即随着肌肉减少症减少肌肉质量和力量输出，剩余功能正常的 α-辅肌动蛋白-3 缺乏纤维上的偏心应力将会增加，导致纤维断裂。
