Morphometry, ultrastructure, myosin isoforms, and metabolic capacities of the “mini muscles” favoured by selection for high activity in house mice

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Abstract

Prolonged selective breeding of mice (Mus musculus) for high levels of voluntary wheel running has favoured an unusual phenotype (“mini muscles”), apparently caused by a single Mendelian recessive allele, in which most hind-limb muscles are markedly reduced in mass, but have increased mass-specific activities of mitochondrial enzymes. We examined whether these changes reflect changes in fibre size, number or ultrastructure in normal and “mini-muscle” mice within the two (of four) selectively bred lines (lab designations L3 and L6) that exhibit the phenotype at generations 26 and 27. In both lines, the gastrocnemius and plantaris muscles are smaller in mass (by > 50% and 20%, respectively) in affected individuals. The mass-specific activities of mitochondrial enzymes in the gastrocnemius and plantaris muscles were increased in the mini phenotype in both lines, with stronger effects in the gastrocnemius muscle. In the gastrocnemius, the % myosin heavy chain (MHC) IIb was reduced by 50% in L3 and by 30% in L6, whereas the % MHC IIa and I were higher, particularly in L3. Fibre number in the plantaris muscle did not significantly differ between mini and normal muscles, although muscle mass was a significant positive correlate of fibre number. Small fibres were more abundant in mini than normal muscles in L3. Mitochondrial volume density was significantly higher in mini than normal muscle fibres in L3, but not in L6. Microscopy revealed a surprising attribute of the mini muscles: an abundance of small, minimally differentiated, myofibril-containing cells positioned in a disorderly fashion, particularly in the surface layer. We hypothesise that these unusual cells may be satellite cells or type IIb fibres that did not complete their differentiation. Together, these observations suggest that mice with the mini phenotype have reduced numbers of type IIb fibres in many of their hind-limb muscles, leading to a decrease in mass and an increase in mass-specific aerobic capacity in muscles that typically have a high proportion of type IIb fibres. Moreover, the several statistically significant interactions between muscle phenotype and line indicate that the effect of the underlying allele is altered by genetic background.

Introduction

Adaptive phenotypic evolution in response to natural selection is a central and undisputed paradigm underlying contemporary biology, but its direct study is difficult. Experimental evolution protocols provide a means of evaluating adaptive mechanisms under more-or-less defined and controlled selective regimes (Bennett, 2003, Garland, 2003, Folk and Bradley, 2005, Swallow and Garland, 2005). For example, selective breeding of house mice for high voluntary wheel running was used to create four replicate lines (Swallow et al., 1998a) that run approximately 170% more revolutions/day as compared with four randomly bred control lines (e.g., see Garland et al., 2002, Garland, 2003, Belter et al., 2004, Rezende et al., 2006b). As a group, the four selected lines exhibit a number of characteristics that appear to represent adaptations for high levels of sustained, aerobically supported wheel running. These include higher whole-animal maximal oxygen consumption (VO2max, especially in males: Swallow et al., 1998b, Rezende et al., 2006a, Rezende et al., 2006b), increased insulin-stimulated glucose uptake in extensor digitorum longus muscle (Dumke et al., 2001), and more symmetrical hind-limb bones and larger femoral condyles (Garland and Freeman, 2005, Kelly et al., in press).

In addition to traits that differ consistently between the four selected and four control lines, some distinct responses have occurred among the replicate selected lines. Such differences are expected because random genetic processes (founder effects, genetic drift, mutation) will necessarily alter the gene pool within each line (isolated population) at the same time that any adaptive responses to selection are occurring. The most dramatic example of among-line variation in the wheel-running selection experiment is that two selected lines have evolved a high frequency of a “mighty mini-muscle” phenotype, whose primary characteristic is an approximate halving of hind-limb muscle mass with a coincident doubling of mass-specific activities of mitochondrial enzymes (Houle-Leroy et al., 2003). The phenotype appears attributable to an autosomal recessive allele that was present in the base (starting) population at a frequency of about 7% (Garland et al., 2002). The fact that two of the selected lines have never shown the phenotype is presumably attributable to random genetic drift, i.e., as a rare allele, it was lost by chance before selection could increase its frequency (see Garland et al., 2002). Interestingly, although the mini phenotype has been favoured by selection, the absolute running performance (revolutions/day) of these mice usually does not differ statistically from that of selected mice with normally sized muscles (Garland et al., 2002, Houle-Leroy et al., 2003, Swallow et al., 2005). However, individuals with the mini phenotype often run significantly faster on wheels as compared with selected individuals with normally sized muscles (Kelly et al., in press; see also Syme et al., 2005), and they have higher VO2max during forced exercise tests in hypoxia (Rezende et al., 2006a). Therefore, whatever the characteristics that led it to be favoured by selection for high wheel running, the mini-muscle phenotype represents what may be considered an alternate solution to the physiological challenge imposed by the selection regime, not necessarily a better one as compared with normal-sized hind-limb muscles (Garland, 2003).

Recently, we reported contractile characteristics of soleus and medial gastrocnemius muscles from selected lines L3 (all mini individuals) and L6 (both normal and mini) (Syme et al., 2005). We found that soleus of mini individuals was actually larger than for normal mice. In spite of the increased mass, contractile characteristics of the soleus did not significantly differ between mini and normal mice. However, medial gastrocnemius muscles of mini mice exhibited slower twitches, a more curved force–velocity relationship, produced about half the mass-specific isotonic power, 20–50% of the mass-specific cyclic work and power, and fatigued at about half the rate of normal muscles (i.e., had greater endurance). Gastrocnemius muscles of mini individuals also exhibit increased glycogen concentration as compared with the normal phenotype (Gomes et al., 2004).

The primary goal of the present study was to evaluate mechanisms that may underlie the altered size, aerobic capacity, and contractile properties of mini muscles. For example, the reduced mass could reflect a reduction in fibre number and/or fibre size. The increased aerobic capacity of mini muscles could reflect a greater mitochondrial volume density in the fibres. We evaluated these hypotheses using a combination of biochemical, morphometric, and ultrastructural techniques. Previous biochemical studies of the mini muscles examined the “triceps surae” complex (gastrocnemius, soleus, plantaris: Belter et al., 2004) or mixed hind-limb muscle as a pool (Houle-Leroy et al., 2000, Houle-Leroy et al., 2003; but see Gomes et al., 2004). Thus, to identify the structural and physiological changes associated with the expression of the mini-muscle allele, we compared specific individual muscles in mice with mini muscles with those of their line-mates that had normally sized muscles. Our first step was to compare the mass of the gastrocnemius, plantaris, and soleus muscles. Next we compared the enzymatic profiles of the muscles that were smaller in the mini phenotype. Given the small size, cylindrical shape, and homogeneity of fibre distribution in the plantaris muscle, we chose it rather than the larger gastrocnemius to compare fibre morphometry and ultrastructure of mini- and normal-sized muscles (see also Discussion). We quantified the number of muscle cells in cross-sections, compared the frequency distributions of fibre sizes, and evaluated whether such ultrastructural characteristics as mitochondrial and myofibrillar volume density and mitochondrial cristae density differ between mini- and normally sized muscles. As a first step towards evaluating whether differences in fibre type proportions might underlie the mass reduction, we characterised the myosin heavy and light chain (MHC and MLC) composition in the gastrocnemius. By carrying out these measurements in both selected lines that expressed the mini phenotype, we also evaluated whether mini muscles show the same characteristics in both lines or whether the effect of the allele depends on genetic background.

Section snippets

Animals and the selection experiment

Swallow et al. (1998a) provide full details of the selection experiment, which involves four lines of mice (Mus musculus) selectively bred for high voluntary wheel running and four additional lines maintained as controls. Mice used for our metabolic, morphometric, and ultrastructural determinations were 55 males aged between 152–169 days and sampled from generation 26 of the artificial selection experiment for increased voluntary activity on running wheels. For the characterisation of myosin

Body and muscle masses

In generation 26, of the 55 experimental male mice (33 from L3, 22 from L6), 18 individuals (12 from L3, 6 from L6) showed the mini-muscle phenotype (Fig. 1; open symbols). The body mass of these individuals was significantly lower (P < 0.0001) than that of mice with normally sized muscles from these selected lines, and the difference was greater in L3 (Table 1, interaction P = 0.039). Results were different for the females of generation 27, as body mass did not differ between mice with the mini

Discussion

The central finding of this study is that multi-generation selective breeding for high levels of voluntary running has favoured a phenotype in which hind-limb muscles that are usually rich in type IIb fibres decrease in mass and show an abundance of unusual “mini” cells. These cells contained myofibrils and mitochondria, but were vastly smaller than typical muscle fibres. In the plantaris muscle, these “mini” cells primarily occurred in the outer fibre layers. Various lines of evidence suggest

Acknowledgements

We thank A. M. Bronikowski for assistance with dissections. This research was supported by funds from NSERC to H. Guderley and from NSF to T. Garland (IBN-0212567).

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