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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

Michael Haug, Barbara Reischl, Stefanie Nübler, Leonit Kiriaev, Davi A.G. Mázala, Peter J. Houweling, Kathryn N. North, Oliver Friedrich, Stewart I. Head
doi: https://doi.org/10.1101/2021.11.09.467867
Michael Haug
1Institute of Medical Biotechnology, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany
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Barbara Reischl
1Institute of Medical Biotechnology, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany
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Stefanie Nübler
1Institute of Medical Biotechnology, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany
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Leonit Kiriaev
2School of Medicine, Western Sydney University, Sydney, NSW, Australia
3School of Medical Science, University of New South Wales, Sydney, NSW, Australia
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Davi A.G. Mázala
4Department of Kinesiology, College of Health Professions, Towson University, Towson, MD, USA
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Peter J. Houweling
5Murdoch Children’s Research Institute, Melbourne, Victoria, Australia
6Department of Paediatrics, University of Melbourne, Melbourne, Victoria, Australia
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Kathryn N. North
5Murdoch Children’s Research Institute, Melbourne, Victoria, Australia
6Department of Paediatrics, University of Melbourne, Melbourne, Victoria, Australia
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Oliver Friedrich
1Institute of Medical Biotechnology, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany
2School of Medicine, Western Sydney University, Sydney, NSW, Australia
3School of Medical Science, University of New South Wales, Sydney, NSW, Australia
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Stewart I. Head
2School of Medicine, Western Sydney University, Sydney, NSW, Australia
3School of Medical Science, University of New South Wales, Sydney, NSW, Australia
5Murdoch Children’s Research Institute, Melbourne, Victoria, Australia
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  • For correspondence: s.head@westernsydney.edu.au
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ABSTRACT

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 Statement

The authors have declared no competing interest.

  • List of abbreviations

    CAS
    Cosine angle sum
    CSA
    Cross sectional area
    dL
    Distance shortened
    dT
    Time shortened
    EDL
    Extensor digitorum longus
    Fabs
    Maximum absolute force
    FR
    Restoration force
    Frelax
    Force at steady state
    Fsig
    End of fast phase
    FDB
    Flexor digitorum brevis
    Kapparent
    Apparent dissociation constant
    KO
    Knock out
    L0
    Optimal length
    MyHC
    Myosin heavy chain
    pCa
    Calcium concentration
    pCa50
    Calcium concentration at 50% of maximum force
    SHG
    Second harmonic generation
    τrelax
    Time to reach steady state
    VD
    Vernier density
    Vfast
    Fast velocity
    WT
    Wild type
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    Posted November 10, 2021.
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    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
    Michael Haug, Barbara Reischl, Stefanie Nübler, Leonit Kiriaev, Davi A.G. Mázala, Peter J. Houweling, Kathryn N. North, Oliver Friedrich, Stewart I. Head
    bioRxiv 2021.11.09.467867; doi: https://doi.org/10.1101/2021.11.09.467867
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    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
    Michael Haug, Barbara Reischl, Stefanie Nübler, Leonit Kiriaev, Davi A.G. Mázala, Peter J. Houweling, Kathryn N. North, Oliver Friedrich, Stewart I. Head
    bioRxiv 2021.11.09.467867; doi: https://doi.org/10.1101/2021.11.09.467867

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