PT  - JOURNAL ARTICLE
AU  - Michael Haug
AU  - Barbara Reischl
AU  - Stefanie Nübler
AU  - Leonit Kiriaev
AU  - Davi A.G. Mázala
AU  - Peter J. Houweling
AU  - Kathryn N. North
AU  - Oliver Friedrich
AU  - Stewart I. Head
TI  - Absence of the Z-disc protein α-actinin-3 impairs the mechanical stability of <em>Actn3KO</em> mouse fast-twitch muscle fibres without altering their contractile properties or twitch kinetics
AID  - 10.1101/2021.11.09.467867
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.11.09.467867
4099  - http://biorxiv.org/content/early/2021/11/10/2021.11.09.467867.short
4100  - http://biorxiv.org/content/early/2021/11/10/2021.11.09.467867.full
AB  - Background A common polymorphism (R577X) in the ACTN3 gene results in 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 L0+ 20% stretch did not cause eccentric damage. In contrast, L0+ 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.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 α-actinin-3 does not affect the unloaded maximum speed of contraction, visco-elastic properties or myofibrillar force production. Eccentric contractions demonstrated that chemically skinned Actn3KO fibres are mechanically weaker being prone to breakage when eccentrically contracted. Furthermore, SHG images reveal disruptions in the myofibrillar alignment of Actn3KO fast-twitch fibres with an increase in Y-shaped myofibrillar lattice shifts.Conclusions 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 L0+ 30%, but not, L0+ 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 fibres breakages.Competing Interest StatementThe authors have declared no competing interest.CASCosine angle sumCSACross sectional areadLDistance shorteneddTTime shortenedEDLExtensor digitorum longusFabsMaximum absolute forceFRRestoration forceFrelaxForce at steady stateFsigEnd of fast phaseFDBFlexor digitorum brevisKapparentApparent dissociation constantKOKnock outL0Optimal lengthMyHCMyosin heavy chainpCaCalcium concentrationpCa50Calcium concentration at 50% of maximum forceSHGSecond harmonic generationτrelaxTime to reach steady stateVDVernier densityVfastFast velocityWTWild type