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The sarcomeric α-actinins perform a dynamic balancing act at the muscle Z-line

F.C. Garton,1,2 M.W. Hogarth,2 P.J. Houweling,1,2 X.F. Zheng,2 K.G. Quinlan,2 N. Yang,2 T. Tukiainen,3 D.G. MacArthur,3 P. Gregorevic4 and K.N. North,1,2 1Murdoch Childrens Research Institute, Royal Children's Hospital, Flemington Road, Parkville, VIC 3052 Australia, 2The Institute for Neuroscience and Muscle Research, The Children's Hospital at Westmead, Westmead, NSW 2145, Australia, 3Analytic and Translational Genetics Unit, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114, USA and 4Baker IDI Heart and Diabetes Institute, 75 Commercial Road, Melbourne, VIC 3004, Australia.

α-Actinin-3 is primarily expressed in fast (type 2) fibres in human skeletal muscle. A loss-of-function (LoF) polymorphism identified in the ACTN3 gene (R577X) results in a shift in muscle function and performance. Complete α-actinin-3 deficiency (577XX) occurs in ∼16% of humans worldwide and studies in elite, healthy and aged populations show this causes significant detriment to fast fibre function. We have developed an Actn3 knockout (KO) mouse which mimics the human phenotype. Despite the total pool of Z-line α-actinins being maintained with an up regulation of α-actinin-2, KO mice display reduced grip strength, a shift towards slow/oxidative metabolism in fast fibres and increased calcineurin signalling. Over 50% of the human population have one copy of the ACTN3 X allele, however heterozygosity (577RX) and dosage of α-actinin-3 has not been investigated. We assessed the relative impact of one copy of the ACTN3 X allele by phenotyping Actn3 heterozygous (HET) mouse. We hypothesized that HET mice would display an intermediate phenotype indicating a dosage effect associated with Actn3 genotype. This included an intramuscular rescue and over-expression experiment with α-actinin-2 and -3 to test reciprocal regulation and the dose-response limits of the sarcomere pool. For the results analysis, all tissues from mouse experiments were excised after cervical dislocation. During the intramuscular rAAV experiments, mice were anaesthetized using 3.5% isoflurane in oxygen from a precision vaporizer and Buprenorphine was administered as an analgesic (0.01mg/kg) before the injection.

We can report that heterozygous mice are biologically intermediate compared to their WT and Actn3 KO littermates. An additive gene-model can explain altered muscle mass, fast 2B fibre size, protein level of α-actinin-3, α-actinin-2 and downstream myofibrillar and metabolic proteins. Additionally we find post-natal delivery of α-actinin-3 into the Actn3 KO mouse improves contractile force and alters recovery from fatigue. These contractile changes are accompanied with a dose-responsive reduction in α-actinin-2 and other Z-line proteins to demonstrate a delicate balance and endogenous reciprocal regulation of the sarcomeric α-actinins which directly influences function. Independent of the α-actinin isoform, overexpression disrupted the sarcomeric pool, resulting in muscle degeneration/regeneration to indicate a physiological threshold.

Our results reveal that the total pool of α-actinin proteins is tightly regulated and the reciprocal balance of α-actinin-2 and -3 in skeletal muscle can regulate metabolic, myofibrillar, and signalling proteins in a dose dependant manner. These findings provide insight into local muscle sarcomere α-actinin balance relevant to ACTN3 genotype as well as individual responses to muscle disuse/disease and training in the human population.

Research Funding: National Health and Medical Research Council of Australia and Australian Research Council.