Stewart I. Head

An Actn3 knockout mouse provides mechanistic insights into the association between α-actinin-3 deficiency and human athletic performance

A common nonsense polymorphism (R577X) in the ACTN3 gene results in complete deficiency of the fast skeletal muscle fiber protein alpha-actinin-3 in an estimated one billion humans worldwide. The XX null genotype is under-represented in elite sprint athletes, associated with reduced muscle strength and sprint performance in non-athletes, and is over-represented in endurance athletes, suggesting that alpha-actinin-3 deficiency increases muscle endurance at the cost of power generation. Here we report that muscle from Actn3 knockout mice displays reduced force generation, consistent with results from human association studies. Detailed analysis of knockout mouse muscle reveals reduced fast fiber diameter, increased activity of multiple enzymes in the aerobic metabolic pathway, altered contractile properties, and enhanced recovery from fatigue, suggesting a shift in the properties of fast fibers towards those characteristic of slow fibers. These findings provide the first mechanistic explanation for the reported associations between R577X and human athletic performance and muscle function.

Abnormalities in structure and function of limb skeletal muscle fibres of dystrophic mdx mice

In this study we have shown that the skeletal muscle fibres from adult (older than 26 weeks) mdx mice have gross structural deformities. W e have characterized the onset and age dependence of this feature in mdx mice. The three dimensional structure of these deformities has been visualized in isolated fibres and the orientation of these deform ities was determ ined w ithin the muscle by confocal laser scanning microscopy. We have also shown that the occurrence of morphologically abnormal fibres is greater in muscles w ith longer fibres (extensor digitorum longus (edl) and soleus, 6—7.3 mm long), than in muscles with shorter fibres (flexor digitorum brevis (f d b ), 0.3—0.4 m m long). A population of post-degenerative fibres, with both central and peripheral nuclei coexistent along the length of the fibre, has also been identified in the muscles studied. We showed that a mild protocol of lengthening (eccentric) contractions (the muscle was stretched by 12% during a tetanic contraction) caused a major reduction in the maximal tetanic force subsequently produced by mdx edl muscle. In contrast, maximal tetanic force production in normal soleus, normal edl and mdx soleus muscles was not altered by this protocol. We suggest that the deform ed fast glycolytic fibres which are found in adult mdx edl but not in adult mdx soleus muscles are the population of fibres dam aged by the lengthening protocol.

Membrane potential, resting calcium and calcium transients in isolated muscle fibres from normal and dystrophic mice.

1. Single skeletal muscle fibres were enzymatically isolated from the flexor digitorum brevis muscles (FDB) of dystrophic mdx and control C57BL/10 mice aged 3‐9 weeks. In this age range the majority (> 95%) of the mdx fibres were morphologically normal. 2. There was no significant difference between the resting membrane potential (RMP) of mdx and control mice, ‐71.2 +/‐ 1.21 (n = 26) and ‐70.6 +/‐ 1.15 mV (n = 42), respectively. 3. At RMP more negative than ‐60 mV the resting calcium (recorded with fura‐2, free acid ionophoresed into cell) in the dystrophic mdx cells was not significantly different from the normal animals, 45.7 +/‐ 4.1 (n = 10) and 46.2 +/‐ 3.9 nM (n = 9), respectively. 4. The resting cytosolic calcium concentration was measured simultaneously with the RMP. At RMP between ‐60 to ‐17 mV there was an increase in the resting calcium concentration in both mdx and control ranging from 79.3 to 252 nM. This increase was most probably due to the activation of the slow calcium current. 5. Fura‐2 calcium transients were produced via single action potential stimulation using an intracellular microelectrode both to stimulate the cell and record potential changes. There was no significant difference between the rise time (Tp) or half‐decay time (T1/2) at 22 degrees C of the calcium transient in response to a single action potential in mdx compared to normal animals, 5.9 +/‐ 0.34 (n = 8) and 5.4 +/‐ 0.36 ms (n = 7); 39.5 +/‐ 2.9 (n = 8) and 40.75 +/‐ 3.7 ms (n = 7), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+ levels in myotubes grown from the skeletal muscle of dystrophic (mdx) and normal mice.

1. Myotubes were grown in culture from normal (C57BL/ScSn) and mdx mice and the cytosolic [Ca2+] was monitored through development (5‐21 days in culture) using fura‐2 loaded via ionophoresis. Simultaneous measurements of the membrane potential and cytosolic [Ca2+] were made in normal and mdx myotubes before, during and after stimulation by action potentials elicited following anode break excitation. All experiments were undertaken at 22 degrees C. All data are expressed as means +/‐ S.E.M. 2. A new method was developed which enabled accurate determination of the fluorescence characteristics of fura‐2 in murine skeletal muscle fibres. In the under in vitro conditions by 14.60 +/‐ 0.05, 9.40 +/‐ 0.15 and 6.90 +/‐ 0.43% respectively. 3. The resting cytosolic [Ca2+] in the mdx myotubes was consistently higher than in the normal myotubes throughout the developmental period measured. Overall, the resting cytosolic [Ca2+] in mdx myotubes (134 +/‐ 9 nM, n = 22) was twofold higher than in normal myotubes (66 +/‐ 6 nM, n = 26). After stimulation (one to three action potentials) the cytosolic [Ca2+] of both mdx and normal myotubes remained elevated. The mdx myotubes (236 +/‐ 55 nM, n = 5) again had approximately double the cytosolic [Ca2+] of normal myotubes (109 +/‐ 19 nM, n = 9). 4. The time course and amplitude of the Ca2+ responses measured in the mdx and normal myotubes after action potential stimulation were variable. Two categories of Ca2+ response were observed in mdx and normal myotubes, the first consisted of a small, slow rise in [Ca2+] that remained elevated and the second consisted of a rapid (time to peak 7.4 +/‐ 1.5 ms) (n = 8) rise in [Ca2+] with amplitudes in the range 61‐773 nM and a [Ca2+] decay rate constant of 4.35 +/‐ 1.57 s‐1 (n = 8) (range 0.96‐15 s‐1). 5. In conclusion, the elevated cytosolic [Ca2+] reported here through development of cultured mdx myotubes suggests that this genetic disorder results in a defect which compromises the ability of the myotubes to strictly regulate cytosolic [Ca2+]. The results are consistent with the presence of functionally abnormal Ca2+ channels recently reported in mdx myotubes.

A gene for speed: contractile properties of isolated whole EDL muscle from an α-actinin-3 knockout mouse

The actin-binding protein alpha-actinin-3 is one of the two isoforms of alpha-actinin that are found in the Z-discs of skeletal muscle. alpha-Actinin-3 is exclusively expressed in fast glycolytic muscle fibers. Homozygosity for a common polymorphism in the ACTN3 gene results in complete deficiency of alpha-actinin-3 in about 1 billion individuals worldwide. Recent genetic studies suggest that the absence of alpha-actinin-3 is detrimental to sprint and power performance in elite athletes and in the general population. In contrast, alpha-actinin-3 deficiency appears to be beneficial for endurance athletes. To determine the effect of alpha-actinin-3 deficiency on the contractile properties of skeletal muscle, we studied isolated extensor digitorum longus (fast-twitch) muscles from a specially developed alpha-actinin-3 knockout (KO) mouse. alpha-Actinin-3-deficient muscles showed similar levels of damage to wild-type (WT) muscles following lengthening contractions of 20% strain, suggesting that the presence or absence of alpha-actinin-3 does not significantly influence the mechanical stability of the sarcomere in the mouse. alpha-Actinin-3 deficiency does not result in any change in myosin heavy chain expression. However, compared with alpha-actinin-3-positive muscles, alpha-actinin-3-deficient muscles displayed longer twitch half-relaxation times, better recovery from fatigue, smaller cross-sectional areas, and lower twitch-to-tetanus ratios. We conclude that alpha-actinin-3 deficiency results in fast-twitch, glycolytic fibers developing slower-twitch, more oxidative properties. These changes in the contractile properties of fast-twitch skeletal muscle from alpha-actinin-3-deficient individuals would be detrimental to optimal sprint and power performance, but beneficial for endurance performance.

Gadolinium reduces short‐term stretch‐induced muscle damage in isolated mdx mouse muscle fibres

Duchenne muscular dystrophy is a lethal muscle disease caused by absence of the protein dystrophin which is part of a glycoprotein complex located on the intracellular surface of the surface membrane. The precise function of dystrophin and the reason why its absence causes severe muscle damage are unclear. Stretch‐induced muscle damage is well recognised in normal muscle and is more severe in muscles from animals lacking dystrophin (mdx mice). It has been proposed that stretch‐induced damage underlies the progression of damage in muscular dystrophy. In the present study we confirm that single fibres from mdx muscle are more susceptible to stretch‐induced damage and show that there is an associated rise in intracellular sodium concentration ([Na+]i) which is greater than in wild‐type mice. We show that this rise in [Na+]i can be prevented by Gd3+, which is an established blocker of stretch‐activated channels. mdx fibres have a higher than normal resting [Na+]i and this is also reduced by Gd3+. If Gd3+ is applied over the period in which [Na+]i rises following stretched contraction, it prevents one component of the reduced force. The other component of reduced force is caused by inhomogeneity of sarcomeres and can be minimised by stretching the muscle to its new optimum length. These experiments show that part of the short‐term damage caused by stretch in mdx fibres can be prevented by blocking stretch‐activated channels.

Deficiency of α-actinin-3 is associated with increased susceptibility to contraction-induced damage and skeletal muscle remodeling

Sarcomeric α-actinins (α-actinin-2 and -3) are a major component of the Z-disk in skeletal muscle, where they crosslink actin and other structural proteins to maintain an ordered myofibrillar array. Homozygosity for the common null polymorphism (R577X) in ACTN3 results in the absence of fast fiber-specific α-actinin-3 in ∼20% of the general population. α-Actinin-3 deficiency is associated with decreased force generation and is detrimental to sprint and power performance in elite athletes, suggesting that α-actinin-3 is necessary for optimal forceful repetitive muscle contractions. Since Z-disks are the structures most vulnerable to eccentric damage, we sought to examine the effects of α-actinin-3 deficiency on sarcomeric integrity. Actn3 knockout mouse muscle showed significantly increased force deficits following eccentric contraction at 30% stretch, suggesting that α-actinin-3 deficiency results in an increased susceptibility to muscle damage at the extremes of muscle performance. Microarray analyses demonstrated an increase in muscle remodeling genes, which we confirmed at the protein level. The loss of α-actinin-3 and up-regulation of α-actinin-2 resulted in no significant changes to the total pool of sarcomeric α-actinins, suggesting that alterations in fast fiber Z-disk properties may be related to differences in functional protein interactions between α-actinin-2 and α-actinin-3. In support of this, we demonstrated that the Z-disk proteins, ZASP, titin and vinculin preferentially bind to α-actinin-2. Thus, the loss of α-actinin-3 changes the overall protein composition of fast fiber Z-disks and alters their elastic properties, providing a mechanistic explanation for the loss of force generation and increased susceptibility to eccentric damage in α-actinin-3-deficient individuals.

Branched fibres in old dystrophic mdx muscle are associated with mechanical weakening of the sarcolemma, abnormal Ca2+ transients and a breakdown of Ca2+ homeostasis during fatigue

In the dystrophinopathies, skeletal muscle fibres undergo cycles of degeneration and regeneration, with regenerated fibres displaying a branched morphology. This study tests the hypothesis that regenerated branched fibres are mechanically weakened by the presence of branches and are damaged by contractions which do not affect unbranched dystrophin‐negative fibres. Experiments were carried out on single fast‐twitch fibres and whole muscle from the dystrophin‐negative mdx mouse. Fura‐2 was ionophoresed into fibres to measure intracellular Ca2+ concentration ([Ca2+]i). Single branched mdx fibres have abnormal Ca2+ kinetics, with the [Ca2+]i transient at the peak of the twitch depressed, are damaged by fatiguing activation, resulting in a breakdown of Ca2+ homeostasis, and break at branch points when submaximally activated in skinned fibre experiments. When old intact isolated mdx muscles, with >90% branched fibres, are eccentrically activated with a moderate eccentric protocol there is a 40 ± 8% reduction in maximal force. Isolated single fibres from these muscles show areas of damage at fibre branch points. This same eccentric protocol causes no force loss in either littermate control muscles or mdx muscles with <10% branched fibres. I present a two‐stage hypothesis for muscle damage in the dystrophinopathies, as follows: stage 1, the absence of dystrophin disrupts ion channel function, causing an activation of necrotizing Ca2+‐activated proteases, which results in regenerated branched fibres; and stage 2, branched fibres are mechanically damaged during contraction. These results may have implications when considering therapies for boys with Duchenne muscular dystrophy. In particular, any therapy aimed at rescuing the defective gene will presumably have to do so before the number of branched fibres has increased to a level where the muscle is mechanically compromised.

GABAA RECEPTOR EXPRESSION AND INHIBITORY POST‐SYNAPTIC CURRENTS IN CEREBELLAR PURKINJE CELLS IN DYSTROPHIN‐DEFICIENT mdx MICE

1 Duchenne muscular dystrophy (DMD) is the second most common fatal genetic disease and arises as a consequence of an absence or disruption of the protein dystrophin. In addition to wasting of the skeletal musculature, boys with DMD have a significant degree of cognitive impairment. 2 We show here that there is no difference between littermate control and mdx mice (a murine model of DMD) in the overall expression of the GABAA receptor a1‐subunit, supporting the suggestion that it is the clustering at the synapse that is affected and not the expression of the GABAA receptor protein. 3 We report a significant reduction in both the frequency and amplitude of spontaneous inhibitory post‐synaptic currents in cerebellar Purkinje cells of mdx mice compared with littermate controls, consistent with the reported reduction in the number and size of GABAA receptor clusters immunoreactive for a1‐ and a2‐subunits at the post‐synaptic densities. 4 These results may explain some of the behavioural problems and cognitive impairment reported in DMD.

Long-term depression is reduced in cerebellar Purkinje cells of dystrophin-deficient mdx mice.

The mdx (muscular dystrophy X-linked) mouse is a model for human Duchenne muscular dystrophy (DMD) and is characterized by the absence of the cytoskeletal protein dystrophin. Using a cerebellar slice preparation, we show that postsynaptically mediated long-term depression (LTD) is significantly reduced in mdx Purkinje cells, while presynaptically mediated paired-pulse facilitation (PPF) is normal. This disruption of LTD could contribute to the cognitive deficit in boys with Duchenne muscular dystrophy.