Abstract

Homozygosity for a common null polymorphism (R577X) in the ACTN3 gene results in the absence of the fast fibre-specific protein, α-actinin-3 in ∼16% of humans worldwide. α-Actinin-3 deficiency is detrimental to optimal sprint performance and benefits endurance performance in elite athletes. In the general population, α-actinin-3 deficiency is associated with reduced muscle mass, strength and fast muscle fibre area, and poorer muscle function with age. The Actn3 knock-out (KO) mouse model mimics the human phenotype, with fast fibres showing a shift towards slow/oxidative metabolism without a change in myosin heavy chain (MyHC) isoform. We have recently shown that these changes are attributable to increased activity of the calcineurin-dependent signalling pathway in α-actinin-3 deficient muscle, resulting in enhanced response to exercise training. This led us to hypothesize that the Actn3 genotype influences muscle adaptation to disuse, irrespective of neural innervation. Separate cohorts of KO and wild-type mice underwent 2 weeks immobilization and 2 and 8 weeks of denervation. Absence of α-actinin-3 resulted in reduced atrophic response and altered adaptation to disuse, as measured by a change in MyHC isoform. KO mice had a lower threshold to switch from the predominantly fast to a slower muscle phenotype (in response to immobilization) and a higher threshold to switch to a faster muscle phenotype (in response to denervation). We propose that this change is mediated through baseline alterations in the calcineurin signalling pathway of Actn3 KO muscle. Our findings have important implications for understanding individual responses to muscle disuse/disease and training in the general population.

INTRODUCTION

Muscle atrophy (loss of muscle mass) is strongly associated with a decrease in muscle strength and power, as well as increased morbidity and reduced quality of life in humans (1). Atrophy is a well-documented and common comorbidity in patients with a wide spectrum of disorders such as inherited and inflammatory myopathies and neuropathies, spinal cord injury, cancer, diabetes, AIDS, kidney disease and aging (2–8). These disorders are associated with reduced protein synthesis and increased proteolysis, causing a reduction in both muscle mass and muscle fibre size, and alterations in muscle contractility, fatigability and metabolism. Recent research has focused on the mechanisms underlying the adaptive response of muscle to changing physical demands in order to develop targeted therapeutics to increase muscle mass (9–11).

The sarcomeric α-actinins (ACTN2 and ACTN3) are major constituents of the muscle Z-disk, where they crosslink actin and bind to a wide range of structural, metabolic and signalling proteins in the muscle sarcomere (reviewed in 11). α-Actinin-2 is expressed in all muscle fibres, whereas α-actinin-3 has a highly specialized pattern of expression in only fast, glycolytic muscle fibres (12). A loss-of-function variant in the ACTN3 gene (ACTN3 R577X allele) is common in the human population. An estimated 1.5 billion people worldwide are homozygous for the ACTN3 577X allele (XX genotype) resulting in complete deficiency of α-actinin-3 (13).

α-Actinin-3 deficiency does not cause overt muscle disease. However, multiple association studies in human athlete and non-athlete cohorts have demonstrated that ACTN3 genotype influences human skeletal muscle performance. The frequency of the ACTN3 577XX genotype is significantly reduced in elite power and sprint athletes (14–20), suggesting that α-actinin-3 deficiency is detrimental to optimal function of fast muscle fibres. Some studies have demonstrated over-representation of the ACTN3 577XX (14,21) and 577RX (22) genotypes in elite endurance athletes; however, this association is not consistently seen across all studies (23). In non-athletes, α-actinin-3 deficiency has been associated with significantly slower 40 m sprint times (24), lower isometric maximal voluntary muscle contractions (25–27), reduced muscle mass (27–29) and fast fibre area (26), and increased response to resistance training (25). It has been estimated that the ACTN3 genotype contributes between 1 and 2.5% of the variance in human muscle strength and performance (24–27).

A number of studies have examined the effect of α-actinin-3 deficiency in response to aging in skeletal muscle (7,23,27–35) (summarized in Table 1). Two longitudinal studies demonstrated an association between α-actinin-3 deficiency and decline in muscle function with age; ACTN3 577XX men showed a greater decline in 400 m walk speed, while ACTN3 577XX women had a 35% greater risk for persistent difficulty climbing stairs or walking, and a 33% increased risk of falling (7,32). A meta-analysis of cohorts with a wide age range (52–82 years) found an association between reduced grip strength and α-actinin-3 deficiency in males, but no effect of the ACTN3 genotype on baseline performance in chair-raise, balance or 6 min timed walk test (23).

Table 1.

ACTN3 genotype in older populations

Population N Age/gender No effect of ACTN3 genotype Effect of ACTN3 genotype References 
Spanish 41 90–97 M&F 1RM leg press, handgrip strength, walking and stair climbing ability – (30
Caucasian 2568 70–79 M&F Baseline grip strength, knee extensor torque, mid-thigh muscle volume, muscle quality, 400 m walk times XX men had greater decline in 400 m walk speed, XX women had 35% greater risk in for incident persistent lower extremity limitation (28
Caucasian 157 56–74 M&F Knee extensor concentric peak power at baseline and after 10 weeks of unilateral knee extensor resistance training XX women had higher relative and absolute knee extensor concentric peak power (PP) compared with RR/RX genotypes. RR women had improved relative peak power vs. XX (7
Caucasian 100 60–70 M&F Lean mass, muscle strength (isokinetic knee extension) and twitch response of quadriceps Lower knee extensor shortening and lengthening peak torque in XX women (31
Caucasian 848 22–90 M&F Lean mass and muscle torque in males XX associated with lower peak torque (shortening and lengthening) in knee extensor muscles, lower lean mass compared with RX/RR (27
Japanese 109 50–78 F No effect physical activity XX had lower mid-thigh cross-sectional area compared with RX/RR (29
Caucasian 4163 >50 F – RX and XX women had a 33% increased risk of falling (32
Spanish 81 71–93 M&F Thigh muscle cross-sectional area (MRI) grip-strength, 30-s chair stand test, functional ability during activities of daily living (Barthel index) and bone mineral density (femur)  (33
Brazilian 246 61–80 F Quadriceps strength (isokinetic) at baseline or after a 24-week resistance program X-allele had higher relative fat free mass at baseline compared with RR (34
Spanish 23 60–80 F 1RM leg press, sit stand-test and one mile walk test  (35
Caucasian 
 NSHD 2900 53 No effect on female grip strength, male or female timed get-up and go, balance, chair raises, balance Trend for reduced grip strength in XX men (P = 0.09) (23
 ELSA 6231 >52 
 Eureka 1198 11–18 
 HAS 717 63–73 
 HCS 2997 59–73 
 BOrr 728 64–82 
 CaPS 2512 65–83 
 LBC 514 77–80 
Population N Age/gender No effect of ACTN3 genotype Effect of ACTN3 genotype References 
Spanish 41 90–97 M&F 1RM leg press, handgrip strength, walking and stair climbing ability – (30
Caucasian 2568 70–79 M&F Baseline grip strength, knee extensor torque, mid-thigh muscle volume, muscle quality, 400 m walk times XX men had greater decline in 400 m walk speed, XX women had 35% greater risk in for incident persistent lower extremity limitation (28
Caucasian 157 56–74 M&F Knee extensor concentric peak power at baseline and after 10 weeks of unilateral knee extensor resistance training XX women had higher relative and absolute knee extensor concentric peak power (PP) compared with RR/RX genotypes. RR women had improved relative peak power vs. XX (7
Caucasian 100 60–70 M&F Lean mass, muscle strength (isokinetic knee extension) and twitch response of quadriceps Lower knee extensor shortening and lengthening peak torque in XX women (31
Caucasian 848 22–90 M&F Lean mass and muscle torque in males XX associated with lower peak torque (shortening and lengthening) in knee extensor muscles, lower lean mass compared with RX/RR (27
Japanese 109 50–78 F No effect physical activity XX had lower mid-thigh cross-sectional area compared with RX/RR (29
Caucasian 4163 >50 F – RX and XX women had a 33% increased risk of falling (32
Spanish 81 71–93 M&F Thigh muscle cross-sectional area (MRI) grip-strength, 30-s chair stand test, functional ability during activities of daily living (Barthel index) and bone mineral density (femur)  (33
Brazilian 246 61–80 F Quadriceps strength (isokinetic) at baseline or after a 24-week resistance program X-allele had higher relative fat free mass at baseline compared with RR (34
Spanish 23 60–80 F 1RM leg press, sit stand-test and one mile walk test  (35
Caucasian 
 NSHD 2900 53 No effect on female grip strength, male or female timed get-up and go, balance, chair raises, balance Trend for reduced grip strength in XX men (P = 0.09) (23
 ELSA 6231 >52 
 Eureka 1198 11–18 
 HAS 717 63–73 
 HCS 2997 59–73 
 BOrr 728 64–82 
 CaPS 2512 65–83 
 LBC 514 77–80 

M, male; F, female; RM, repetition maximum; MRI, magnetic resonance imaging; NSHD, Medical Research Council National Survey of Health and Development; ELSA, English Longitudinal Study of Ageing; Eureka, Greek rural adolescent study; HAS, Hertfordshire Ageing Study; HCS, Hertfordshire Cohort Study; BOrr, Boyd Orr cohort; CaPS, Caerphilly Prospective Study; LBC, Lothian Birth Cohort 1921 Study.

Attempts to examine the effect of the ACTN3 genotype on muscle disease severity and progression have been hampered by small sample sizes and mixed populations (gender, age and ethnicity) (6,36–39) (summarized Table 2). However, α-actinin-3 deficiency was associated with enhanced exercise performance in patients with glycogen phosphorylase deficiency (McArdle's disease) (38) and with altered fibre-type proportions in paraplegics following spinal cord injury (6).

Table 2.

ACTN3 genotype in muscle disease populations

Population description N Age/Gender Study results References 
Merosin congenital muscular dystrophy (12) 54 M&F No correlation was found with degree of muscle degeneration or clinical course (36
Severe childhood autosomal recessive MD (12) 
Mild limb girdle (14) 
Human muscle glycogen phosphorylase deficiency (MPL/McArdle's disease) 99 8–81 M&F No significant relationships identified between clinical severity and ACTN3 genotype (37
40 34–41 M&F XX & RX individuals had a higher VO2 peak than RR. No differences were found in male patients (38
Inflammatory myopathies; dermatomyositis (DM); polymyositis (PM) DM 27
PM 10 
24–54 M&F Enhanced proportion XX genotypes in affected group (P < 0.001)
ACTN3 genotype not related to severity of expression of the phenotype or altered enzyme levels (CPK, LDH, AST, and ALT) 
(39
Paraplegics 28–48 M XX = 1, RX = 1, RR = 1, XX had no 2X fibres, RX had largest fibre diameter, R allele had highest unloaded shortening velocity, XX had less stiff type 2A/2X fibres, no difference in calcium sensitivity (6
Population description N Age/Gender Study results References 
Merosin congenital muscular dystrophy (12) 54 M&F No correlation was found with degree of muscle degeneration or clinical course (36
Severe childhood autosomal recessive MD (12) 
Mild limb girdle (14) 
Human muscle glycogen phosphorylase deficiency (MPL/McArdle's disease) 99 8–81 M&F No significant relationships identified between clinical severity and ACTN3 genotype (37
40 34–41 M&F XX & RX individuals had a higher VO2 peak than RR. No differences were found in male patients (38
Inflammatory myopathies; dermatomyositis (DM); polymyositis (PM) DM 27
PM 10 
24–54 M&F Enhanced proportion XX genotypes in affected group (P < 0.001)
ACTN3 genotype not related to severity of expression of the phenotype or altered enzyme levels (CPK, LDH, AST, and ALT) 
(39
Paraplegics 28–48 M XX = 1, RX = 1, RR = 1, XX had no 2X fibres, RX had largest fibre diameter, R allele had highest unloaded shortening velocity, XX had less stiff type 2A/2X fibres, no difference in calcium sensitivity (6

M, male; F, female; CPK, creatine phosphokinase; LDH, lactic dehydrogenase; AST, aspartate aminotransferase; ALT, alanine aminotransferase.

The Actn3 knock-out (KO) mouse mimics the phenotype of human α-actinin-3 deficiency. Compared with wild-type (WT) littermates, Actn3 KO mice have reduced body weight due to lower muscle mass, reduced grip strength (∼7%) and lower absolute muscle force (∼10%) (40–42). α-Actinin-3 deficiency is associated with enhanced endurance performance and response to endurance training; Actn3 KO mice can run 33% further, and have enhanced response to endurance training (1.3×), compared to their WT littermates (41,43). Isolated Actn3 KO muscles have longer twitch relaxation times and enhanced recovery from fatigue compared with WT (41,42). The Actn3 KO mouse 2B fibres (equivalent to 2X in humans) have significantly smaller cross-sectional diameter (∼34% less than WT) and stain more intensely for mitochondrial markers succinate dehydrogenase (SDH) and NADH (41,44). In whole muscle homogenates, Actn3 KO mice have significantly decreased lactate dehydrogenase activity in the anaerobic pathway and increased activity of mitochondrial enzymes in the aerobic pathway, with a 50% increase in glycogen content (41,44,45). At baseline, there is no change in the proportion of 2B fibres compared with WT; however, following endurance training Actn3 KO mice demonstrate an enhanced adaptive response to endurance training, associated with a lower threshold to switch muscle fibre type [as defined by myosin heavy chain (MyHC) isoform] from fast-twitch glycolytic (2B) towards fast-twitch oxidative fibres (2A) (43). These data demonstrate that α-actinin-3 deficiency in mice is associated with a shift in fast 2B fibre properties towards a more oxidative phenotype that would, in turn, be detrimental to optimal sprint and power performance.

We have also examined the effects of α-actinin-3 deficiency across the mouse lifespan (2–18 months) (46). Compared with WT, Actn3 KO mice continue to have reduced grip strength, decreased type 2B fibre size, greater force recovery following fatigue, increased aerobic enzyme activity and mitochondrial protein expression throughout life. The persistent relative decrease in muscle strength and muscle mass associated with α-actinin-3 deficiency in mice may explain why elderly ACTN3 577XX humans have a trend for reduced grip strength (23), have a 33% increased risk of falls (32) and have a greater decline in walk speed (28).

Recently, we have demonstrated that α-actinin-3 deficiency in mouse and humans is associated with increased activity of the calcineurin pathway (43)—a signalling pathway which has been shown to promote the switch from fast-to-slow fibre type. The increase in calcineurin activity provides a mechanistic explanation for the effects of the ACTN3 genotype on skeletal muscle performance in elite athletes and altered adaptation to changing physical demands in the general population.

The impact of α-actinin-3 deficiency on muscle disuse has not yet been investigated. Mouse models of immobilization and denervation display similar fibre atrophy responses to those found in human disuse conditions such as immobilization (47), injury (48,49) and aging (50). Mouse muscle is inherently faster than human muscle, and changes in fibre-type adaptation can differ between human and mouse muscle depending on the underlying disease or atrophic stimulus (51). Nevertheless, mouse models provide a useful platform to explore the effects of a specific genotype on the adaptive shifts in the muscle MyHC profile, under controlled conditions. In mice, gene expression profiling has shown that immobilization of the fast hindlimb muscles typically results in a shift towards a fast, glycolytic 2B MyHC profile (49,50); while long-term denervation results in a shift towards a slower type 1 MyHC profile (30,31,51).

On this basis, we explored the local impact of α-actinin-3 deficiency on muscle atrophy and muscle adaptation by studying the effect on Actn3 KO mice on two physiological forms of disuse—immobilization and denervation. These models result in atrophy and opposing adaptation shifts in MyHC isoforms. We hypothesized that the presence or absence of α-actinin-3 would have a local effect on the ability of muscle to adapt to disuse irrespective of neural innervation. The molecular regulation of this adaptive response is of great interest due to its potential applicability to human muscle disease and disuse.

RESULTS

Reduced muscle atrophy response in immobilized and denervated Actn3 KO mice

WT and Actn3 KO mice were immobilized (for 2 weeks) or denervated (for 2 and 8 weeks) to assess the effect of α-actinin-3 deficiency on muscle atrophy. Tibialis anterior (TA) mass was examined to determine the extent of atrophy. As previously reported, the TA muscles in Actn3 KO mice are on average 10% smaller than WT controls. With immobilization, there was less atrophy of the TA muscles in Actn3 KO mice (−38.2%) compared with WTs (−43.7%), so that there was no difference in TA muscle mass between WT and Actn3 KO post-immobilization (Fig. 1A). A similar response was found following denervation. Post 2 weeks denervation, KO TA mass was still significantly smaller than WT TA mass; however, with longer-term (eight weeks) denervation, Actn3 KO mouse muscle atrophied less than WT (−50.4 versus −61.6%) (Fig. 1B).

Figure 1.

Reduced atrophy of muscle in Actn3 KO TA in response to immobilization and denervation. (A) At baseline, TA muscle mass is significantly reduced in Actn3 KO mice compared with WT. Following 2 weeks immobilization, there is a significant reduction in TA muscle mass in both WT and KO mice—however, the atrophied Actn3 KO immobilized muscles no longer weigh significantly less than WT immobilized. (B) TA mass is significantly reduced in both WT and Actn3 KO mice in response to 2 weeks of denervation. With a longer period of denervation (8 weeks), the atrophied Actn3 KO denervated muscles no longer weigh significantly less than denervated WT muscles.

Figure 1.

Reduced atrophy of muscle in Actn3 KO TA in response to immobilization and denervation. (A) At baseline, TA muscle mass is significantly reduced in Actn3 KO mice compared with WT. Following 2 weeks immobilization, there is a significant reduction in TA muscle mass in both WT and KO mice—however, the atrophied Actn3 KO immobilized muscles no longer weigh significantly less than WT immobilized. (B) TA mass is significantly reduced in both WT and Actn3 KO mice in response to 2 weeks of denervation. With a longer period of denervation (8 weeks), the atrophied Actn3 KO denervated muscles no longer weigh significantly less than denervated WT muscles.

We also analysed the immobilized hindlimbs using DEXA (Supplementary Material, Fig. S1) as well as the mass of other muscles affected by immobilization and denervation (Supplementary Material, Tables S1 and S2). In all muscles assessed, there was a reduced muscle atrophy response in the Actn3 KO mouse muscle compared with WT.

Type 2B fibres in Actn3 KO mouse muscle atrophy less in response to immobilization and denervation

α-Actinin-3 is expressed predominantly in type 2B (fast, glycolytic) fibres in mouse muscle (12). To further investigate the observed reduced atrophy response in the Actn3 KO mouse, we examined the MyHC 2B fibre size distribution of the TA muscle of both immobilized and denervated WT and Actn3 KO mice (Fig. 2A). Following both immobilization and denervation, WT muscle showed a clear shift to the left in diameter, indicating a greater number of smaller fibres. In comparison, the shift in Actn3 KO fibre size in response to both immobilization and denervation was less pronounced. Mean 2B fibre size as a percentage of controls was higher in Actn3 KO muscles compared with WT, indicating that Actn3 KO mice maintained their 2B fibre size better than WT mice—following immobilization (KO 55% versus WT 40%; P = 0.0823) and denervation (KO 75% versus WTs 48%; P = 0.0079) (Fig. 2B).

Figure 2.

2B fibre size in the TA muscle of immobilized and denervated WT and Actn3 KO mice. (A) Fibre size histograms; 2B fibre size distribution curve of WT mice is shifted to the left in response to both immobilization and denervation. The Actn3 KO 2B fibre size curve does not shift to the same extent. (B) 2B fibre size expressed as a percentage (%) of fibre size in control WT and Actn3 KO mice. Following immobilization and 2 weeks denervation. Actn3 KO fast fibres undergo less atrophy compared to WT. In each group n = 5–7. **P < 0.01.

Figure 2.

2B fibre size in the TA muscle of immobilized and denervated WT and Actn3 KO mice. (A) Fibre size histograms; 2B fibre size distribution curve of WT mice is shifted to the left in response to both immobilization and denervation. The Actn3 KO 2B fibre size curve does not shift to the same extent. (B) 2B fibre size expressed as a percentage (%) of fibre size in control WT and Actn3 KO mice. Following immobilization and 2 weeks denervation. Actn3 KO fast fibres undergo less atrophy compared to WT. In each group n = 5–7. **P < 0.01.

Actn3 KO mouse muscle shows altered adaptation in response to immobilization

In addition to 2B fibre size, we assessed the overall adaptive response of the TA muscle post-immobilization using single section, fibre typing by immunohistochemistry (Fig. 3A and B). 2B, 2X, 2A and type 1 size, number and area were assessed in both WT and Actn3 KO mice (Fig. 3C–E).

Figure 3.

Muscle fibre analysis in response to immobilization shows altered adaptation in Actn3 KO mouse muscle. (A) Whole TA muscle cross-sections from WT and Actn3 KO controls were immunostained with dystrophin (to demonstrate the sarcolemma) and MyHC (fibre type) for quantitation. Images are representative cross-section images—dystrophin is stained green, type 2B fibres (red), 2A (green) and type 1 (blue). (B) Whole TA muscle cross-sections from immobilized WT and Actn3 KO mice were similarly stained, measured and counted. (C) Fibre size in controls and immobilized groups from left to right (2B, 2X, 2A). (D) Fibre number. (E) Fibre area. Significant results using the Mann–Whitney-U t-test to compare WT vs. KO groups have been marked; *P < 0.05, **P < 0.001.

Figure 3.

Muscle fibre analysis in response to immobilization shows altered adaptation in Actn3 KO mouse muscle. (A) Whole TA muscle cross-sections from WT and Actn3 KO controls were immunostained with dystrophin (to demonstrate the sarcolemma) and MyHC (fibre type) for quantitation. Images are representative cross-section images—dystrophin is stained green, type 2B fibres (red), 2A (green) and type 1 (blue). (B) Whole TA muscle cross-sections from immobilized WT and Actn3 KO mice were similarly stained, measured and counted. (C) Fibre size in controls and immobilized groups from left to right (2B, 2X, 2A). (D) Fibre number. (E) Fibre area. Significant results using the Mann–Whitney-U t-test to compare WT vs. KO groups have been marked; *P < 0.05, **P < 0.001.

In response to immobilization, all fibres were significantly atrophied in both WT and Actn3 KO mice compared with their genotype control groups (Fig. 3C, Supplementary Material, Table S3). Actn3 KO 2X and 2A fibres atrophied less, and after immobilization were 51 and 43% larger than the immobilized WT 2X and 2A fibres, respectively. Immobilization resulted in a decrease in 2A fibre number (Fig. 3D); however, Actn3 KO mice retained a greater number of 2A fibres and immobilized Actn3 KO mice had more than twice the number of 2A fibres than WT mice (138 ± 17 versus 302 ± 41, P = 0.004). Total fibre numbers (Supplementary Material, Table S3) were the same between control and immobilization groups.

The total 2B, 2X and 2A fibre surface areas in the overall muscle cross-section were compared between WT and Actn3 KO, control and immobilization groups (Fig. 3E, Supplementary Material, Table S4). In controls, WT mice had a 32% larger 2B surface area (309153 μm2; P = 0.03), while areas covered by 2X and 2A fibres were no different between WT and Actn3 KO mice. In response to immobilization, all fibre surface areas were significantly smaller. Reduced atrophy in Actn3 KO 2B fibres meant that there was no difference in 2B fibre size between WT and Actn3 KO after immobilization (Fig. 3E). Less 2X fibre size atrophy, but fewer 2X fibres post-immobilization in Actn3 KOs meant that there was no difference in surface area compared with WT. Actn3 KO 2A fibres also atrophied less but had greater numbers of fibres which meant overall surface area was greater. Actn3 KO muscle had a 30% larger 2A area post-immobilization compared with WT (81683 μm2; P = 0. 0.0043). Thus immobilization of the TA is associated with atrophy in both WT and Actn3 KO mice; however, WT mice also shift towards a fast MyHC profile which is not detected in KO mice.

Actn3 KO mouse muscle has a lower threshold to switch to slow MyHC fibre type in response to denervation

Both 2 and 8 weeks of denervation resulted in significant atrophy of muscle fibres compared with controls (Fig. 4; Supplementary Material, Table S5). As described above, the smaller (α-actinin-3 deficient) 2B fibres in Actn3 KO mice atrophied less with denervation compared with WT mice (Fig. 2B). 2X and 2A fibres responded equally in WT and Actn3 KO mice at each denervation time point (Supplementary Material, Table S5).

Figure 4.

Muscle fibre analysis in response to denervation shows altered adaptation in Actn3 KO mouse. (A) WT and Actn3 KO TA muscle cross-sections are mostly made up of fast fibres (in order of prevalence) 2B (red), 2X (unstained), 2A (green) with no detectable type 1 fibres (blue). Sarcolemma is stained with dystrophin (green). (B) In response to 2 weeks denervation, the TA muscle cross-sectional area and fibre size are smaller compared with controls but overall fast fibres predominance is maintained. (C) With 8 weeks denervation, the muscle cross-sectional area is smaller, fibres have atrophied and there is significant changes in fibre-type proportion, with greatest frequency of 2A fibres (labelled blue), followed by 2X (unstained) and type 1 (green). (D) Total cross-sectional area of each fibre type (2B, 2X, 2A) in control and denervated groups. (E) Percentage cross-sectional area of each fibre type (2B, 2X, 2A) in control and denervated groups. (F) Surface area of each fibre type (2B, 2X, 2A) in control and denervated groups.

Figure 4.

Muscle fibre analysis in response to denervation shows altered adaptation in Actn3 KO mouse. (A) WT and Actn3 KO TA muscle cross-sections are mostly made up of fast fibres (in order of prevalence) 2B (red), 2X (unstained), 2A (green) with no detectable type 1 fibres (blue). Sarcolemma is stained with dystrophin (green). (B) In response to 2 weeks denervation, the TA muscle cross-sectional area and fibre size are smaller compared with controls but overall fast fibres predominance is maintained. (C) With 8 weeks denervation, the muscle cross-sectional area is smaller, fibres have atrophied and there is significant changes in fibre-type proportion, with greatest frequency of 2A fibres (labelled blue), followed by 2X (unstained) and type 1 (green). (D) Total cross-sectional area of each fibre type (2B, 2X, 2A) in control and denervated groups. (E) Percentage cross-sectional area of each fibre type (2B, 2X, 2A) in control and denervated groups. (F) Surface area of each fibre type (2B, 2X, 2A) in control and denervated groups.

Total fibre number remained similar in denervated groups and was no different between WT and Actn3 KO mice. With 8 weeks of denervation, there was a large fibre-type shift towards a slower MyHC profile (2X and 2A) in the TA muscle—2A (fast oxidative) fibre numbers were significantly increased at 8 weeks in both WT and Actn3 KO mice, while 2X fibre numbers stayed relatively unchanged (Supplementary Material, Table S5). Interestingly, there was a significantly greater reduction in 2B (fast, glycolytic) fibre number in Actn3 KO mice, and at 8 weeks Actn3 KO mice had ∼50% of the mean 2B fibre numbers compared with WT mice (P = 0.03) without detectable difference in 2A or 2X fibre numbers (Supplementary Material, Table S5).

Denervation-induced fibre atrophy and MyHC fibre-type shifts resulted in a dramatic change in fibre-type proportions in both WT and Actn3 KO mice—with muscle switching to a slower MyHC profile (Fig. 4, Supplementary Material, Table S6). 2B and 2X fibre areas significantly decreased in both WT and Actn3 KO and the percentage of 2A fibre area was significantly increased. After 8 weeks of denervation, Actn3 KO mice had a 70% smaller 2B area than WT (P = 0.0317) (Fig. 4E). These results suggest an enhanced shift to a slower MyHC isoform profile in Actn3 KO mice due to a greater reduction in fast 2B fibres.

Actn3 KO mice resist the shift to a fast fibre phenotype and have an enhanced shift toward the slow fibre phenotype during muscle adaptive response to immobilization and denervation

To give an overall understanding of the genotype effect on a muscle's ability to adapt to both atrophy stimuli, we further examined 2B, 2X and 2A fibre-type cross-sectional area (Fig. 5). As previously reported, at baseline, Actn3 KO mice have a smaller 2B fibre area, and a slower metabolic profile, without change in the MyHC profile. In C57BL6 mice, mean fibre-type percentage cross-sectional area in WT control TA muscle was 67.0% 2B, 17.0% 2X and 16.0% 2A, while Actn3 KO control muscle was 49.9% 2B, 26.7% 2X and 22.4% 2A.

Figure 5.

Summary of muscle adaptation response to immobilization and denervation. Muscle fibre-type CSA of MyHC 2B, 2X and 2A (%) is shown for immobilized, controls and denervated groups of WT and Actn3 KO mice. Immobilization results in an adaptive shift in fibre type towards a fast MyHC profile in WT mice and this shift is less pronounced in Actn3 KO mice. Denervation results in an adaptive shift in fibre type towards a slow MyHC profile and this shift is more pronounced in Actn3 KO mice.

Figure 5.

Summary of muscle adaptation response to immobilization and denervation. Muscle fibre-type CSA of MyHC 2B, 2X and 2A (%) is shown for immobilized, controls and denervated groups of WT and Actn3 KO mice. Immobilization results in an adaptive shift in fibre type towards a fast MyHC profile in WT mice and this shift is less pronounced in Actn3 KO mice. Denervation results in an adaptive shift in fibre type towards a slow MyHC profile and this shift is more pronounced in Actn3 KO mice.

Immobilization induced significant changes in fibre size and number which altered the surface area covered by each fibre type. WT mice shifted to a faster MyHC profile with 70.0% 2B, 23.4% 2X and 6.6% 2A. Actn3 KO mice, however, resisted the shift to a fast MyHC, with the Actn3 KO fibre-type proportions remaining similar to controls with 53.0% 2B, 28.0% 2X and 19.0% 2A. Comparing the surface areas in WT and Actn3 KO muscles, post-immobilization demonstrated a greater difference in 2B surface area (−17%; P = 0.0043) and a 12% larger 2A surface area (P = 0.0043) in Actn3 KO relative to WT.

Eight weeks denervation also resulted in large changes in the muscle MyHC profile, shifting towards a slower MyHC profile. The WT mice showed reduced 2B and increased 2A and type 1 fibre-type percentage cross-sectional area (18% 2B, 16% 2X, 65% 2A and 5% type 1). The Actn3 KO fibre-type profile also switched towards a slower MyHC profile (7% 2B, 17% 2X, 73% 2A and 4% type 1). Actn3 KO mouse muscle demonstrated a trend towards a reduction in 2B surface area (10%; P = 0.06) and 2X surface area (10%; P = 0.06) compared with no change in WT mouse muscle, but these changes did not reach statistical significance.

Calcineurin pathway activity is altered in response to immobilization and denervation consistent with shifts in MyHC isoform profiles

We have previously demonstrated that α-actinin-3 deficiency results in greater calcineurin activity, with higher levels of a regulator of calcineurin, RCAN1-4 in Actn3 KO mouse at baseline and in ACTN3 577XX humans (43). We postulate that increased calcineurin activity provides an explanation for the shift towards an oxidative metabolic phenotype in Actn3 KO mouse muscle, and that Actn3 KO mouse muscle has a lower threshold to switch towards a slower MyHC isoform profile in response to changing physical demands.

We sought to determine whether these baseline differences in the calcineurin pathway were altered in response to muscle atrophy. Immobilization, which induced a switch to fast MyHC isoforms in WT muscle, caused no change in RCAN1-4 expression (Fig. 6A). In Actn3 KO muscle, there were higher RCAN1-4 levels at baseline. With immobilization, there was a trend towards reduced RCAN1-4 levels in the Actn3 KO muscle towards WT levels (P = 0.057) so that they were no longer significantly different.

Figure 6.

Regulator of calcineurin (RCAN1) response to immobilization and denervation in WT and Actn3 KO mice. (A) Western blot analysis (top) and densitometry quantification of the endogenous calcineurin-responsive 28 kDa RCAN1-4 isoform that reflects calcineurin activity. At baseline, RCAN1-4 is increased in Actn3 KO mice compared with WT. Following immobilization, there is a reduction in RCAN1-4 in Actn3 KO mouse muscle with no significant difference compared with WT mouse muscle. (B) Western blot analysis and quantification of RCAN1-4 at baseline and following denervation. Denervated mouse muscle shows a marked increase in the levels of RCAN1-4 after 2 weeks denervation in both WT and Actn3 KO mice.

Figure 6.

Regulator of calcineurin (RCAN1) response to immobilization and denervation in WT and Actn3 KO mice. (A) Western blot analysis (top) and densitometry quantification of the endogenous calcineurin-responsive 28 kDa RCAN1-4 isoform that reflects calcineurin activity. At baseline, RCAN1-4 is increased in Actn3 KO mice compared with WT. Following immobilization, there is a reduction in RCAN1-4 in Actn3 KO mouse muscle with no significant difference compared with WT mouse muscle. (B) Western blot analysis and quantification of RCAN1-4 at baseline and following denervation. Denervated mouse muscle shows a marked increase in the levels of RCAN1-4 after 2 weeks denervation in both WT and Actn3 KO mice.

Denervation, which resulted in a fibre-type switch towards a slower MyHC isoform profile, resulted in RCAN1-4 expression being significantly increased in both WT and Actn3 KO mouse muscle at both 2 and 8 weeks (Fig. 6B). In both WT and Actn3 KO mice, these changes preceded the slow fibre-type shift detected at 8 weeks. After denervation, the levels of RCAN1-4 expression were not measurably different between WT and Actn3 KO mice. Our results confirm baseline signalling calcineurin differences between WT and Actn3 KO mice, and the large post-atrophy RCAN1-4 increase corresponds to the significant ‘fast-to-slow’ fibre-type shift in both WT and KO muscle.

Structural protein changes in WT and Actn3 KO mice following atrophy

Previously, we have demonstrated up-regulation of a number of structural proteins in the Actn3 KO mouse (40,41,52). α-Actinin-3 deficiency is associated with compensatory up-regulation of the closely related isoform, α-actinin-2 in type 2B fibres (40). The structural remodelling proteins, myotilin, desmin and ZASP, interact with the α-actinins at the Z-line, and are also up-regulated in α-actinin-3 deficiency (52,53). As muscle atrophy results in significant muscle fibre remodelling over time, we compared WT and Actn3 KO mice after immobilization and denervation.

In response to 2 weeks of immobilization, the level of α-actinin-3 and α-actinin-2 did not change in WT mice (Fig. 7A). In Actn3 KO mice, α-actinin-2 expression did not change after immobilization, and remained higher (2-fold) compared with WT. Structural Z-line proteins (desmin and myotilin) were decreased following immobilization, and their levels were no longer higher than WT (Fig. 7A). We examined the integrity of the muscle after atrophy with a haematoxylin and eosin stain. Smaller fibres and greater numbers of nuclei were apparent with immobilization. However, there were no differences between WT and Actn3 KO (Supplementary Material, Fig. S4C).

Figure 7.

Structural and metabolic response to atrophy in WT and KO mice. (A) Western blot analysis (top) and densitometry quantification of proteins in WT and Actn3 KO muscle following immobilization. As previously shown, at baseline, α-actinin-2, the structural proteins desmin and myotilin, and the mitochondrial proteins cytochrome oxidase and porin are increased in Actn3 KO mice compared with WT. Following immobilization, α-actinin-2 remains significantly increased compared with WT, but the structural and mitochondrial proteins are decreased in the Actn3 KO and are not significantly different to WT. (B) Western blot analysis and densitometry quantification of proteins in WT and Actn3 KO muscle following 8 weeks denervation. α-Actinin-2, the structural proteins desmin and myotilin, and the mitochondrial proteins, cytochrome oxidase and porin are all up-regulated well above baseline values in both WT and Actn3 KO mice, with no significant differences between genotype. (C) In response to 8 weeks denervation, there were significant protein changes in compared with genotype controls. Reduced fold changes were detected in Actn3 KO muscle in α-actinin-2, desmin, myotilin and αB-crystallin compared with WT mice.

Figure 7.

Structural and metabolic response to atrophy in WT and KO mice. (A) Western blot analysis (top) and densitometry quantification of proteins in WT and Actn3 KO muscle following immobilization. As previously shown, at baseline, α-actinin-2, the structural proteins desmin and myotilin, and the mitochondrial proteins cytochrome oxidase and porin are increased in Actn3 KO mice compared with WT. Following immobilization, α-actinin-2 remains significantly increased compared with WT, but the structural and mitochondrial proteins are decreased in the Actn3 KO and are not significantly different to WT. (B) Western blot analysis and densitometry quantification of proteins in WT and Actn3 KO muscle following 8 weeks denervation. α-Actinin-2, the structural proteins desmin and myotilin, and the mitochondrial proteins, cytochrome oxidase and porin are all up-regulated well above baseline values in both WT and Actn3 KO mice, with no significant differences between genotype. (C) In response to 8 weeks denervation, there were significant protein changes in compared with genotype controls. Reduced fold changes were detected in Actn3 KO muscle in α-actinin-2, desmin, myotilin and αB-crystallin compared with WT mice.

Denervation resulted in marked atrophy at 2 weeks, and at 8 weeks this was accompanied by a significant shift to a slower MyHC profile. Structural protein levels were examined at both denervation time points. Similar to 2 weeks immobilization, there was no detectable change in α-actinin-2 levels in the denervated WT or Actn3 KO mouse muscle after 2 weeks denervation compared with controls (Supplementary Material, Fig. S2). Myotilin and desmin were both increased by 2 weeks, and there was no longer a detectable difference between WT and Actn3 KO mice (Supplementary Material, Fig. S2). At 8 weeks, there were large increases in the concentration of structural (desmin and myotilin) proteins in both WT and Actn3 KO mice (Fig. 7B). α-Actinin-3 was decreased by ∼50% in WT mice (P = 0.029), along with an increase in α-actinin-2 in WT (2.3-fold) and Actn3 KO (1.4-fold) mice. Eight-week denervated WT muscles showed increased myotilin (5-fold), desmin (20-fold), ZASP (7-fold) and αB-crystallin (6-fold) compared with WT controls (Fig. 7C). Relative to the WT response, denervated Actn3 KO muscles showed similar fold changes in ZASP (8.7-fold) but lower fold changes in myotilin (2.4-fold), desmin (8-fold) and αB-crystallin (3.4-fold) compared with Actn3 KO controls. In summary, α-actinin-2 and the structural remodelling proteins, myotilin, desmin and αB-crystallin are increased at baseline in Actn3 KO muscle, and have an altered adaptive response to denervation, compared with WT muscle.

Metabolic protein and oxidative enzyme changes in WT and Actn3 KO mice following atrophy

α-Actinin-3 deficiency is associated with an up-regulation of enzymes associated with the oxidative metabolic pathway (40). Disuse atrophy results in significant altered metabolic demands, and thus we also compared adaptation response of WT and Actn3 KO mouse muscle with both immobilization and denervation.

With 2 weeks immobilization, Actn3 KO mouse muscle atrophied but resisted the shift to a fast MyHC profile seen in WT mice. Consistent with maintaining a slower metabolic profile, the oxidative enzyme stains, SDH (mitochondria-specific) and NADH-TR (sarcoplasmic reticulum and mitochondrial specific) remained increased in Actn3 KO mice compared with WT (Supplementary Material, Fig. S4). When controlled for actin, the overall COX IV and porin protein levels in the Actn3 KO were decreased to a similar level in WT (Fig. 7A).

In response to denervation, there was a gradual shift to a slower muscle phenotype. At 2 weeks, there was an increase in both SDH and NADH staining in both WT and Actn3 KO mice which preceded any detectable change in MyHC profile (Supplementary Material, Fig. S3). After 8 weeks denervation, there was an increase in oxidative staining in both WT and Actn3 KO mouse muscle, so they were a similar intensity (Supplementary Material, Fig. S3). Significant metabolic protein changes were also detected, with large increases in the concentration of the oxidative COX IV protein in both WT and Actn3 KO mice (2.7-fold and 4.5-fold), compared with genotype controls (Fig. 7B). The muscle's metabolic adjustment to either immobilization atrophy (shift to fast) or denervation (shift to slow) shows a similar directional response in both genotypes, suggesting that the baseline metabolic differences between WT and Actn3 KO mice make a major contribution to the timeline of MyHC adaptation.

DISCUSSION

The Actn3 KO mouse has been specifically generated to mimic human α-actinin-3 deficiency. Loss of α-actinin-3 results in a fast-to-slow switch in muscle metabolic and physiological properties, without a change in MyHC isoform expression at baseline. The phenotypic changes seen in Actn3 KO mice include reduced muscle mass, increased oxidative capacity and an enhanced adaptation to endurance training. Using this mouse model, we have now addressed potential differences in muscle adaptation to atrophic stimuli. Using two experimental conditions—immobilization and denervation—we have demonstrated that Actn3 KO mice have reduced atrophy and altered adaptation to disuse, as measured by a change in MyHC isoform.

Immobilization results in atrophy through physical shortening and muscle disuse, while sciatic denervation causes disuse via neural inactivation (10,54). The loss of muscle mass, reduction in fibre size and shift in MyHC isoforms seen in both WT and Actn3 KO mouse muscle is a typical skeletal muscle adaptive response to immobilization (55,56) and denervation (54,57,58). We have previously demonstrated at baseline that Actn3 KO mice have significantly lower lean mass, muscle mass and smaller type 2B muscle fibres compared with WT (41). We hypothesized that the presence or absence of α-actinin-3 would have a local effect in response to muscle atrophy, irrespective of the muscles' innervation status. Consistent with this, Actn3 KO muscle responded in the same manner, regardless of the type of atrophy stimulus.

Actn3 KO mice showed significantly less reduction in hindlimb muscle mass and lean mass following immobilization. We then examined muscle fibre size, and demonstrated that the differential effect was most pronounced in type 2B fibres, where α-actinin-3 is normally expressed; deficiency of α-actinin-3 in 2B fibres reduced the rate of atrophy.

After 2 weeks of denervation, Actn3 KO 2B fibres atrophied 27% less than WT fibres. Slow fibres tend to atrophy less than fast fibres (59), consistent with the changes we observed in WT muscles (2A atrophied less than 2X and 2B). Actn3 KO fast glycolytic (2B) fibres show a response to atrophic stimuli that is more reminiscent of “slower” fibres (2X or 2A). An alternate explanation is that the decreased size of 2B muscle fibres at baseline in Actn3 KO mice may contribute to their reduced atrophy rate. It has been shown that the starting properties and size of the muscle influences the rate of muscle atrophy/degradation, and that smaller muscles have a decreased rate of muscle atrophy (60); however, the mechanistic basis of this is not yet known (61).

MyHC staining of the TA muscle provided more detailed analysis of changes in muscle fibre size as well as fibre number. Immobilization of the TA in WT mice resulted in atrophy and a shift to a faster MyHC profile, similar to that previously published (62,63). Actn3 KO mice had a higher threshold for this shift towards the fast fibre type and showed no evidence of fibre-type conversion with immobilization, consistent with altered muscle adaptation to disuse.

In response to long-term denervation (8 weeks), we observed atrophy and marked shifts in the MyHC profile towards slower isoforms. In WT mice, fast muscles typically shift to a slower, more oxidative profile, replacing their atrophied 2B and 2X myosin contractile machinery with MyHC 2A (54,57,58), as shown in Figure 3. After 8 weeks denervation, compared with WT, Actn3 KO mouse muscle demonstrated a greater increase in the number of fibres expressing the 2A (fast, oxidative) myosin isoform, with a consequential decrease in 2B (fast, glycolytic) fibres, consistent with a lower threshold to shift towards the slower more oxidative MyHC isoform profile.

Muscle fibre-type adaptation (as defined by changes in MyHC expression) is a complex process that can vary depending on the muscles starting properties and the type of disuse. Since mouse muscle has a different baseline MyHC isoform profile compared with human muscle, our results cannot be directly extrapolated. For example, in humans, denervation secondary to spinal cord injury results in a slow-to-fast (2X) fibre transition (48,51), whereas denervation of the mouse hindlimb results in a fast-to-slow fibre-type shift. Thus while we have demonstrated that Actn3 genotype influences muscle adaptation in response to disuse in mice, further studies in human cohorts are required to define the effect of the ACTN3 genotype on muscle disease and disuse.

We have previously reported that the loss of α-actinin-3 in fast fibres not only results in a smaller 2B fibre, but also a shift towards oxidative muscle metabolism and a slower contractile speed (41,42). This shift in metabolic properties has not yet been thoroughly assessed in α-actinin-3 deficient human muscles—one study found higher glycogen content in XX compared with RX individuals (45), while another study found no differences in succinate dehydrogenase staining or cytochrome oxidase enzyme activity (64). More recently, we have identified activation of the calcineurin pathway as the molecular mechanism responsible for this shift in muscle properties at baseline (43). In α-actinin-3 deficient muscle, there is up-regulation of α-actinin-2, which has increased binding affinity to the fast fibre protein, calsarcin-2, an inhibitor of calcineurin activation (43). α-Actinin-2 replaces α-actinin-3 at the Z-line of 2B fibres in Actn3 KO muscle, resulting in decreased availability of calsarcin-2 to bind to and inhibit calcineurin, and therefore activation of the calcineurin pathway.

On this basis, we examined the levels of RCAN1-4, a downstream regulator of calcineurin, which is specifically increased upon activation of calcineurin, at baseline and in response to atrophy. Overall, the calcineurin pathway response in immobilization and denervation reflected the adaptive response of the muscle and was consistent in both WT and Actn3 KO mice. We hypothesize that the increase in calcineurin activity and the resulting slower, more oxidative, muscle metabolic phenotype at baseline results in the altered MyHC adaptation to atrophic stimuli in the Actn3 KO mouse. Increased calcineurin activity at baseline would alter the propensity of a fibre to replace the contractile machinery (via ordered transcriptional and translation changes), and result in a delayed switch-to-fast and lower threshold for switch-to-slow MyHC profile in Actn3 KO mouse muscle compared with WT. Our data suggest that the starting properties of the muscle play a key role in determining muscle adaptive response (65).

Acting in parallel with the calcineurin pathway are signalling systems involved in the complex, highly ordered atrophy of the contractile elements of skeletal muscle. Denervation and immobilization caused significant muscle degradation in WT and Actn3 KO mice; however, the 2B fibres of Actn3 KO mice show a reduced rate of atrophy, suggesting they are less responsive to degradation. Coordinating players involved in muscle atrophy include the calpains, lysosomal proteases (cathepsins) and the ubiquitin-proteasome pathway. Calpains arrive early, cleaving myofibrillar proteins such as titin, vinculin, C-protein, nebulin as well as α-actinin. α-Actinin was thought to be a poor substrate of calpain (66)—however more recent findings show a catalyst is needed in the reaction (67,68). The lipid signaler phosphoinositide (PtdIns3,4,5,P3) binds to α-actinin and alters its structural confirmation to reveal a highly sensitive calpain cleavage site in the actin-binding domain. This results in substantial proteolysis with stable 80 and 65 kDa breakdown products (67,68). Studies to date have not focused on differentiating between the two sarcomeric isoforms, α-actinin-2 and α-actinin-3. We have recently demonstrated that structural and signalling skeletal muscle proteins (ZASP, titin, vinculin and calsarcin-2) preferentially bind to α-actinin-2 compared with α-actinin-3 (43,52). Thus protein degradation of α-actinin-2 in the 2B fibre environment could be slowed by reduced substrate specificity and/or reduced amounts of the phosphoinositide catalyst, which may alter downstream myofibre degradation. Our results suggest that in times of stress, α-actinin-2 at the Z-line resists proteolysis, resulting in the decreased atrophy response seen in α-actinin-3 deficient muscle.

In conclusion, the ACTN3 R577X loss-of-function variant is extremely common in the human population, and contributes to genetic variability in strength and muscle mass in both mice and humans. Our study provides novel data to demonstrate that the ACTN3 genotype is also a genetic modifier of the adaptive response to muscle disuse. Genetic association studies in human populations have already provided some evidence for this, by demonstrating that the ACTN3 genotype alters response to training (14), aging (28,32), progression of, and susceptibility to muscle disease (38,39) and more recently in spinal cord injury (6). Improved understanding of the contribution of the ACTN3 genotype to human muscle function will significantly improve our knowledge of human muscle adaptation to changes in activity, environment and as a modifier of muscle disease.

MATERIALS AND METHODS

This study was approved by CMRI/Children's Hospital Westmead Animal Care and Ethics Committee. Immobilization tests and analysis were performed on WT and Actn3 KO mice on a R129 genetic background, as described previously (40). Denervation studies were performed in WT and Actn3 KO mice on a C57BL/6 background. All mice were fed meat free mouse pellets (Specialty foods, Glen forest, Western Australia) and water ad libitum, and maintained in a 12:12 h cycle of light and dark.

Hindlimb immobilization and denervation

Separate cohorts of WT and Actn3 KO mice underwent 2 weeks of immobilization and either 2 or 8 weeks of denervation and were compared with age-matched controls. Immobilization was carried out as described in ref. (69). Briefly R129 mice were anaesthetized using a ketamine/xylazine injection (100 mg/20 mg/kg body weight) before being immobilized. The soleus (Sol) and gastrocnemius (GST) were held in a stretched position and the extensor digitorum longus and TA in a shortened position by bandaging the hind foot twice against the leg in a moderate dorsiflexed position. The leading edge of the bandage was continued up and wrapped across the back of the mouse to secure the bandage in place. This was repeated on the contralateral foot. Bandages were inspected daily to ensure complete immobilization of hindlimb. Age-matched mice that were not immobilized were used as controls. A pilot study was carried out in 6 WT mice to assess the optimal immobilization time point (1, 2 or 4 weeks) to see a loss in muscle mass, change in fibre size and proportion (data not shown).

For denervation experiments, C57BL/6 mice were anesthetized using 3.5% isoflurane in oxygen from a precision vaporizer. One group of female mice (age 20 weeks) had one leg denervated for 8 weeks (each group with 7 Actn3 KO and 5 WT). A second group of female mice (aged 13 weeks) had one leg denervated for 2 weeks. A sham control group were age matched at cull. The sciatic nerve was transected at the level of the femur, 5 mm of the nerve was removed to prevent reinnervation while in the sham control the nerve remained intact. Buprenorphine was administered as an analgesic (0.01 mg/kg) and mice were monitored regularly for signs of distress during the test period. Mice were confirmed as being denervated with complete disuse/paralysis of their hindlimb/s. Mice were monitored daily for the first 2 weeks after surgery, after which they were monitored every 2 days. Tissues were excised after mice were sacrificed via cervical dislocation. The GST, Sol and TA, muscles innervated by the sciatic nerve, were collected and used for analysis.

Tissue collection

Mice were euthanized by cervical dislocation immediately prior to tissue collection. Muscles were removed, weighed and immediately snap frozen in partially thawed isopentane for protein and muscle fibre analyses. Tissues were stored in liquid nitrogen until use.

MyHC isoforms staining and measurements

Staining of MyHC isoforms and measurements was performed as previously described in ref. (70). A transverse 8 μm section was cut from the mid-section of the frozen mouse TA muscle and blocked for 1 h at room temperature to prevent cross-reaction with endogenous mouse antibodies using AffiniPure Fab fragment goat anti-mouse IgG (1:25 dilution; Jackson ImmunoResearch). Detection of MyHC type 2B was performed using a monoclonal antibody raised from hybridoma culture (BF-F3; developmental study hybridoma bank). The section was also co-stained with dystrophin (dys6-10; kindly supplied by L. Kunkel) to identify the sarcolemma and define muscle fibre borders. Secondary incubation was performed at room temperature using Alexa Fluor 555 goat anti-mouse IgM and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes). To detect MyHC type 2A and type 1 fibres within the same section, we used the Zenon mouse IgG labelling as per kit instructions (Molecular Probes). MyHC 2A antibody (SC71) was pre-labelled with Zenon Alexa Fluor 488, while the MyHC type 1 antibody (MAB1628, Chemicon) was pre-labelled with the Zenon Alexa Fluor 350. Non-staining fibres are assumed to be type 2X fibres. All images were captured using the Olympus BX50 microscope attached to a Jenoptik ProgRes digital camera and ProgRes software (SciTech). Fibre analysis was performed using METAMORPH® software (Molecular Devices) (Denervation) and Image-Pro Plus 2.0 software (Media Cybernetics, Silver Spring, MD, USA) (Immobilization). Measurement files were exported to Microsoft Excel (2000). The whole TA muscle section from each mouse was measured to assess fibre-type area and proportions while being blinded to genotype. Fibre-type proportions were determined as the percentage of dominantly positive stained fibres vs. the total fibres in the TA muscle. Cross-sectional surface area (CSA) was estimated using fibre area multiplied by fibre number, and the value was expressed as proportion of the total CSA.

Immunoblotting

Sample preparation for western blots was performed as described by Seto et al. (52). Western blots were performed on TA samples. 40 × 8 μm cryosections were homogenized and sonicated in Laemmli buffer containing 4% SDS (62.5 mm Tris, pH 6.8, 4% sodium dodecyl sulphate). Sample protein was quantified using a BCA assay kit (BCA assay kit) and prepared at concentrations of 1 or 2 ug/µl with the addition of 0.1% bromophenol blue, 10% glycerol, 50 mm dithiothreitol and protease inhibitor (1:500; Sigma Aldrich). Samples, adjusted for loading were separated by SDS–PAGE on pre-cast mini-gels, transferred to polyvinylidene fluoride membranes (Millipore), probed with antibodies and developed with ECL chemiluminescent reagents (Amersham Biosciences). Antibodies used included; α-actinin-3 (1:20000 5B3 kindly provided by A. Beggs), α-actinin-2 (1:800000 4B3 kindly provided by A. Beggs), porin (20B12; 1:5000; Molecular Probes), cytochrome c oxidase (COX IV) (20E8; 1:2000; Molecular Probes), desmin (1:800, NCL-DER11; Leica), myotilin (1:800 NCL-MYOTILIN; Leica), ZASP (1:200000 LDB3 Proteintech), αβ-crystallin (1:2000, NCL-ABCrys-512; Leica) and RCAN1 (D6694; Sigma-Aldrich). α-Sarcomeric actin (5C5 1:2000 Sigma-Aldrich) was used as a loading control and densitometry was performed using ImageJ (71).

Histochemistry

NADH-tetrazolium reductase (NADH-TR) and SDH staining were carried out on separate 8 μm TA muscle via methods in MacArthur et al. (40). Both NADH-TR and SDH muscle sections were incubated for 60 min at 37°C. Following incubation, sections were removed from the reaction mixture, washed in distilled water and mounted for viewing.

Statistics

All comparisons reported in this study involved small sample sizes to which standard tests for normality could not be applied. As such, all comparisons of paired groups were performed using the non-parametric Mann–Whitney U-test. All histograms show mean values ± SEM unless otherwise stated. P < 0.05 was considered significantly different with values obtained using GraphPad Prism 6.0 software.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.

FUNDING

This work was funded in part by a grant from the National Health and Medical Research Council of Australia (1002033). J.T.S. and K.G.R.Q. are supported by Early Career Fellowships from the NHMRC of Australia (1036656, 0511981). F.C.G. is supported by an Australian Postgraduate Award.

ACKNOWLEDGEMENTS

We gratefully acknowledge the technical immobilization and denervation assistance provided by Dr Anthony Kee and Dr Josephine Joya from University of NSW. Author contributions; F.C.G and J.T.S. immobilized the mice, F.C.G. denervated the mice, F.C.G. performed the analysis for fibre diameter and fibre-type proportions, western blots, immunohistochemistry and histochemistry; J.T.S., N.Y. and P.J.H. maintained the mouse line; F.C.G., J.T.S., P.J.H. and N.Y. collected muscle weight data and harvested muscles for analysis; K.G.R.Q and F.C.G. performed the calcineurin assay; N.Y., J.T.S, K.N.N. and F.C.G. contributed to the experimental design; F.C.G., K.N.N, P.J.H., K.G.R.Q. and J.T.S. wrote the and edited the manuscript.

Conflict of Interest statement. None declared.

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