Absence of the Z-disc protein α-actinin-3 impairs the mechanical stability of Actn3KO mouse fast-twitch muscle fibres without altering their contractile properties or twitch kinetics

Background: A common polymorphism (R577X) in the ACTN3 gene results in the complete absence of the Z-disc protein alpha-actinin-3 from fast-twitch muscle fibres in similar to 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-0 + 20% stretch did not cause eccentric damage. In contrast, L-0 + 30% stretch produced a significant similar to 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. Methods 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. Results: We show that the absence of alpha-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. Conclusions: The absence of alpha-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-0 + 30%, but not L-0 + 20%, eccentric contraction strains. Our study also provides a possible mechanistic explanation as to why alpha-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 alpha-actinin-3 deficient fibres will be increased, resulting in fibre breakages.

Automated summary

The polymorphism in the ACTN3 gene affects the organization and mechanical strength of fast-twitch muscle fibers, leading to structural weakness and a rapid decline in muscle function with increasing age.