Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCA185

Poster Communications

Calcium transients in single muscle fibres isolated from the flexor digitorium brevis of α-actinin-3-deficient mice

S. Head1, P. Houweling2, K. North2

1. Physiology, University of New South Wales Australia, Sydney, New South Wales, Australia. 2. Murdoch Childrens Research Institute, Melbourne, Victoria, Australia.

Worldwide around 1.5 billion people lack the skeletal muscle fast-twitch fibre protein α-actinin-3 due to homozygosity for a common null polymorphism (R577X) in the ACTN3 gene. In the human genome, it is very difficult to find single-gene loss-of-function variants that bear signatures of positive selection, yet intriguingly, the ACTN3 null variant has undergone strong positive selection during recent evolution in humans. We have previously demonstrated Head et al. (2015) that α-actinin-3 deficiency in the Actn3 KO mouse results in (i) an increased rate of decay of the Ca2+ twitch transient; (ii) a fourfold increase in the rate of SR Ca2+ leak; (iii) a threefold increase in the rate of SR Ca2+ pumping; and (iv) enhanced maintenance of tetanic Ca2+ during fatigue. The SR Ca2+ pump, SERCA1, and the Ca2+-binding proteins, calsequestrin and sarcalumenin, showed markedly increased expression in muscles of KO mice. Together, these changes in Ca2+ handling in the absence of α-actinin-3 are consistent with cold acclimatisation and thermogenesis and offer one possible explanation for the positive selection of the ACTN3 577X null allele in humans. Here we investigate amplitude of Ca2+ transients, the relationship of Ca2+ release to stimulation frequency and releasable SR Ca2+ content in single FDB Fast-twitch fibres from ACTN3 KO mice. An intracellular microelectrode was used to ionophorese the free acid form of fura-2 or low affinity fura-ff. Fibres were electrically stimulated. In some cases fibres were immobilized with BTS. We utilised a published deconvolution formula to correct peak values of individual twitch Ca 2+ transients for the high affinity of fura-2 Bakker et al. (1997). Ca2+-frequency curves were obtained using stimulation frequencies of 2 to 100 Hz. SR Ca2+ content was assessed using the method described in Loy et al. (2011). The SR was completely emptied of Ca2+ using a mixture comprised of 10 μM ionomycin, 30 μM cyclopiazonic acid and 100 μM EGTA. In FDB fibres of a-actinin-3-deficient KO mice twitch Ca2+ transients displayed a lower peak of Ca2+ compared to fibres from WT. Peak Ca2+ was 865nM ± 22.57, n=31 fibres in KO and 1580nM ± 34.77, n=36 fibres in WT (P<0.0001). The rate of SR Ca2+ release was higher in KO (1.82 ± 0.19 ratio units/s in KO, n=6 fibres), versus 1.01 ± 0.19 ratio units/s in WT, n=6 fibres). However there does not appear to be a difference in releasable Ca2+ (445 ± 101 ratio units in KO, n=6 fibres), versus 357 ± 110 ratio units in WT, n=6 fibres). Ca2+-frequency curves showed no significant differences between fibres from KO and WT mice. Results to date suggests that while ACTN3 polymorphism may alter SR Ca2+ content it does not significantly change SR Ca2+ release parameters.

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