α-actinin-3 deficiency does not affect the unloaded shortening biomechanics of single isolated fast-twitch FDB fibres from Actn3 KO mice

S. Wagner,³ O. Friedrich,³ S. Chan,¹ P.J. Houweling,² K.R. Quinlan,² R.M. Murphy,⁴ K.N. North² and S.I. Head,¹ ¹School of Medical Sciences, University of New South Wales, NSW 2052, Australia, ²Murdoch Children’s Research Institute, Melbourne, VIC 3052, Australia, ³Institute of Medical Biotechnology, Friedrich-Alexander-University Erlangen-Nuremberg, 90152, Germany and ⁴Department of Zoology, La Trobe University, VIC 3086, Australia.

Introduction. We have identified a common null polymorphism in ACTN3 (R577X) that has undergone strong positive selection during recent human evolution. Homozygosity for this allele (577XX) results in absence of α-actinin-3 in the fast-twitch (IIX human) muscle fibres of ∼18% of the world’s population. The polymorphism does not have any known disease phenotype. However, it is associated with a shift in the metabolic profile of the α-actinin null fast fibers from a predominantly anaerobic production of ATP to an aerobic production of ATP (MacArthur et al., 2007). There is a common misconception that this metabolic shift means that in Actn3 KO fast-twitch fibres have been switched to a slow-twitch contractile phenotype. This is not correct as has been demonstrated in a series of experiments carried out on muscles form the Actn3 KO mice, these include skinned fibre experiments and histochemical fiber typing along with isometric contractile measurements made from isolated extensor digitorum longus (EDL) fast-twitch muscles (Chan et al., 2011). These studies showed there is no change in the expression of fast myosin isoforms, Force-pCa curve or rise times of the twitch.

Aim. In the current study we measure the unloaded maximum speed of shortening in single intact enzymatically isolated fast twitch muscle fibers to find out whether it is altered by the absence of α-actinin-3.

Methods and results. Littermate control and Actn3 KO mice were killed by an overdose of the inhalation anaesthetic, halothane or isoflurane. The flexor digitorum brevis muscle (FDB) was dissected out and single fibres were obtained by collagenase dissociation. Single intact fibres were plated out on glass coverslips and viewed via a Nikon inverted microscope. Fibres were stimulated to contract by field stimulation from two insulated platinum wires. The stimulation protocol was; single pulses, 10 Hz, 20 Hz, 30 Hz and 100 Hz in order to record single twitch, unfused and fused tetanic contractile responses. Fibre shortening was recorded using a CMOS high-speed camera (hs1200, PCO, Kehlheim, Germany) connected to the side port of the inverted microscope. Adjusting the field of image acquisition on the chip allowed frame rates up to 4,500 frames per second. For analysis, a custom-made program was written consisting of the following steps: after loading the first image of an experiment XYT series, the fibre borders could be manually enclosed with a freehand ROI in order to exclude eventual debris or artifacts within the fibre area. The ROI mask was applied to all images within the series and converted to a binary mask on which border detection was performed. The longest distance between detected borders were taken as fibre length and followed up within subsequent images. A criterion was set to allow for maximum length change in successive images of 2.5 to 5% of initial fibre length. This resulted in a significant reduction in false-positive border detections from areas outside the fibre or floating particles in the solution. The length values were stored in a vector matrix upon which time derivatives were performed for speed of shortening assessment. Each experiment was plotted in an automated results file showing the l(t) and v(t) curves, both in relative (normalized to L₀) as well as absolute values (converting to μm after a grid calibration). Minimum shortening length and maximum speed of shortening did not significantly differ between wild-type (n=18) and Actn3 KO fibres (n=22), neither for the variable of genotype nor for stimulation frequency. For instance, both littermate controls and Actn3 KO recorded unloading shortening speeds of around 5.5 mm/s for single twitches and around 6.5 mm/s at 100Hz stimulations.

Conclusion. This is the first report of unchanged unloaded speed of shortening kinetics in fast-twitch fibres from Actn3 KO mice upon either twitch- or tetanic field stimulation. These findings support previous reports that the absence of α-actinin-3 does not change the heavy myosin chain compositions or isometric twitch rise times. We conclude that the Actn3 polymorphism results in fast-twitch fibres with normal contraction kinetics which have a metabolic shift towards slow-twitch fibre aerobic ATP production pathways.

Chan S, Seto JT, Houweling PJ, Yang N, North KN, and Head SI. (2011) Muscle and Nerve 43: 37-48.

MacArthur D, Seto JT, Raftery JM, Quinlan KG, Huttley GA, Hook JW, Lemckert FA, Kee AJ, Edwards MR, Berman Y, Hardeman EC, Gunning PW, Easteal S, Yang N, and North KN. (2007) Nature Genetics 39: 1261-1265.