Alpha-Actinin-3 Deficiency Might Affect Recovery from Non-Contact Muscle Injuries: Preliminary Findings in a Top-Level Soccer Team.

There are recent data suggesting an association between the R577X polymorphism (rs1815739) in the gene encoding α-actinin-3 (ACTN3) and the risk of musculoskeletal injuries. The purpose of this study was to analyze the association of rs1815739 with risk of, and recovery time from non-contact soft-tissue muscle injuries in professional soccer players. Forty-six (22 male and 24 female) players from a top-level professional soccer team were assessed during five consecutive seasons: the genotype distribution was: RR, 41.3%; RX, 47.8%; and XX, 10.9%. There was a trend towards a higher risk of muscle injury associated with the XX genotype (p = 0.092, with no injury-free XX player during the 5-year study period) and a significant genotype effect for the time needed to return to play (p = 0.044, with the highest value shown for the XX genotype, i.e., 36 ± 26 days, vs. 20 ± 10 and 17 ± 12 days for RR and RX, respectively). In conclusion, the XX genotype might be associated not only with a higher risk of non-contact muscle injuries, but also of recovery time from these conditions. However, more research in larger cohorts is needed to confirm this preliminary hypothesis.

Parvins Are Required for Endothelial Cell-Cell Junctions and Cell Polarity During Embryonic Blood Vessel Formation.

OBJECTIVE: During vascular development, integrin-mediated signaling regulates the formation and stabilization of cell-cell junctions, which are required for endothelial cell (EC) apical-basal polarity and proper deposition of the vascular basement membrane. Parvins are actin-binding proteins that facilitate the interaction of integrins with the actin cytoskeleton. The endothelium expresses 2 parvin isoforms: α-pv (α-parvin) and ß-pv (ß-parvin). Recently, we have shown that α-pv is critical for vessel growth and vessel stability at late embryonic developmental stages. The role of parvins during early embryonic development is unknown. APPROACH AND RESULTS: To investigate the role of endothelial parvins in the developing vasculature, we generated mice with ECs lacking both parvin isoforms by deleting α-pv in ECs in global ß-pv-/- mice (α-pvΔEC;ß-pv-/- mice). Here, we show that α-pvΔEC;ß-pv-/- mice die around embryonic day 11.5 and exhibit hemorrhages, immature capillary beds, and severe vascular defects in the central nervous system, including reduced vessel branching, increased vessel diameter, and balloon-like hemorrhagic clusters of ECs. Vessels in α-pvΔEC;ß-pv-/- embryos display disorganized cell-cell junctions, impaired endothelial apical-basal polarity, and discontinuous basement membranes. These vascular defects are accompanied by defective pericyte-vessel interaction. CONCLUSIONS: Our results show that parvins are critical for the organization of endothelial cell-cell junctions, the establishment of endothelial apical-basal polarity, and the integrity of the basement membrane.

Exploring the relationship between α-actinin-3 deficiency and obesity in mice and humans.

Obesity is a worldwide health crisis, and the identification of genetic modifiers of weight gain is crucial in understanding this complex disorder. A common null polymorphism in the fast fiber-specific gene ACTN3 (R577X) is known to influence skeletal muscle function and metabolism. α-Actinin-3 deficiency occurs in an estimated 1.5 billion people worldwide, and results in reduced muscle strength and a shift towards a more efficient oxidative metabolism. The X-allele has undergone strong positive selection during recent human evolution, and in this study, we sought to determine whether ACTN3 genotype influences weight gain and obesity in mice and humans. An Actn3 KO mouse has been generated on two genetic backgrounds (129X1/SvJ and C57BL/6J) and fed a high-fat diet (HFD, 45% calories from fat). Anthropomorphic features (including body weight) were examined and show that Actn3 KO 129X1/SvJ mice gained less weight compared to WT. In addition, six independent human cohorts were genotyped for ACTN3 R577X (Rs1815739) and body mass index (BMI), waist-to-hip ratio-adjusted BMI (WHRadjBMI) and obesity-related traits were assessed. In humans, ACTN3 genotype alone does not contribute to alterations in BMI or obesity.

Evidence for ACTN3 as a genetic modifier of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is characterized by muscle degeneration and progressive weakness. There is considerable inter-patient variability in disease onset and progression, which can confound the results of clinical trials. Here we show that a common null polymorphism (R577X) in ACTN3 results in significantly reduced muscle strength and a longer 10 m walk test time in young, ambulant patients with DMD; both of which are primary outcome measures in clinical trials. We have developed a double knockout mouse model, which also shows reduced muscle strength, but is protected from stretch-induced eccentric damage with age. This suggests that α-actinin-3 deficiency reduces muscle performance at baseline, but ameliorates the progression of dystrophic pathology. Mechanistically, we show that α-actinin-3 deficiency triggers an increase in oxidative muscle metabolism through activation of calcineurin, which likely confers the protective effect. Our studies suggest that ACTN3 R577X genotype is a modifier of clinical phenotype in DMD patients.

How does α-actinin-3 deficiency alter muscle function? Mechanistic insights into ACTN3, the 'gene for speed'.

An estimated 1.5 billion people worldwide are deficient in the skeletal muscle protein α-actinin-3 due to homozygosity for the common ACTN3 R577X polymorphism. α-Actinin-3 deficiency influences muscle performance in elite athletes and the general population. The sarcomeric α-actinins were originally characterised as scaffold proteins at the muscle Z-line. Through studying the Actn3 knockout mouse and α-actinin-3 deficient humans, significant progress has been made in understanding how ACTN3 genotype alters muscle function, leading to an appreciation of the diverse roles that α-actinins play in muscle. The α-actinins interact with a number of partner proteins, which broadly fall into three biological pathways-structural, metabolic and signalling. Differences in functioning of these pathways have been identified in α-actinin-3 deficient muscle that together contributes to altered muscle performance in mice and humans. Here we discuss new insights that have been made in understanding the molecular mechanisms that underlie the consequences of α-actinin-3 deficiency.

Analysis of the ACTN3 heterozygous genotype suggests that α-actinin-3 controls sarcomeric composition and muscle function in a dose-dependent fashion.

A common null polymorphism (R577X) in ACTN3 causes α-actinin-3 deficiency in ∼ 18% of the global population. There is no associated disease phenotype, but α-actinin-3 deficiency is detrimental to sprint and power performance in both elite athletes and the general population. However, despite considerable investigation to date, the functional consequences of heterozygosity for ACTN3 are unclear. A subset of studies have shown an intermediate phenotype in 577RX individuals, suggesting dose-dependency of α-actinin-3, while others have shown no difference between 577RR and RX genotypes. Here, we investigate the effects of α-actinin-3 expression level by comparing the muscle phenotypes of Actn3(+/-) (HET) mice to Actn3(+/+) [wild-type (WT)] and Actn3(-/-) [knockout (KO)] littermates. We show reduction in α-actinin-3 mRNA and protein in HET muscle compared with WT, which is associated with dose-dependent up-regulation of α-actinin-2, z-band alternatively spliced PDZ-motif and myotilin at the Z-line, and an incremental shift towards oxidative metabolism. While there is no difference in force generation, HET mice have an intermediate endurance capacity compared with WT and KO. The R577X polymorphism is associated with changes in ACTN3 expression consistent with an additive model in the human genotype-tissue expression cohort, but does not influence any other muscle transcripts, including ACTN2. Overall, ACTN3 influences sarcomeric composition in a dose-dependent fashion in mouse skeletal muscle, which translates directly to function. Variance in fibre type between biopsies likely masks this phenomenon in human skeletal muscle, but we suggest that an additive model is the most appropriate for use in testing ACTN3 genotype associations.

Rodent models for resolving extremes of exercise and health.

The extremes of exercise capacity and health are considered a complex interplay between genes and the environment. In general, the study of animal models has proven critical for deep mechanistic exploration that provides guidance for focused and hypothesis-driven discovery in humans. Hypotheses underlying molecular mechanisms of disease and gene/tissue function can be tested in rodents to generate sufficient evidence to resolve and progress our understanding of human biology. Here we provide examples of three alternative uses of rodent models that have been applied successfully to advance knowledge that bridges our understanding of the connection between exercise capacity and health status. First we review the strong association between exercise capacity and all-cause morbidity and mortality in humans through artificial selection on low and high exercise performance in the rat and the consequent generation of the "energy transfer hypothesis." Second we review specific transgenic and knockout mouse models that replicate the human disease condition and performance. This includes human glycogen storage diseases (McArdle and Pompe) and α-actinin-3 deficiency. Together these rodent models provide an overview of the advancements of molecular knowledge required for clinical translation. Continued study of these models in conjunction with human association studies will be critical to resolving the complex gene-environment interplay linking exercise capacity, health, and disease.

Altered Ca2+ kinetics associated with α-actinin-3 deficiency may explain positive selection for ACTN3 null allele in human evolution.

Over 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. α-Actinin-3 deficiency is detrimental to sprint performance in elite athletes and beneficial to endurance activities. 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, appearing to provide a survival advantage where food resources are scarce and climate is cold. We have previously demonstrated that α-actinin-3 deficiency in the Actn3 KO mouse results in a shift in fast-twitch fibres towards oxidative metabolism, which would be more "energy efficient" in famine, and beneficial to endurance performance. Prolonged exposure to cold can also induce changes in skeletal muscle similar to those observed with endurance training, and changes in Ca2+ handling by the sarcoplasmic reticulum (SR) are a key factor underlying these adaptations. On this basis, we explored the effects of α-actinin-3 deficiency on Ca2+ kinetics in single flexor digitorum brevis muscle fibres from Actn3 KO mice, using the Ca2+-sensitive dye fura-2. Compared to wild-type, fibres of Actn3 KO mice showed: (i) an increased rate of decay of the 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 an additional explanation for the positive selection of the ACTN3 577X null allele in populations living in cold environments during recent evolution.

α-Actinin-3 deficiency alters muscle adaptation in response to denervation and immobilization.

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.

Global protein quantification of mouse heart tissue based on the SILAC mouse.

Metabolic labeling of living organisms with stable isotopes has become a powerful tool for global protein quantitation. The SILAC (stable isotope labeling with amino acids in cell culture) approach is based on the incorporation of nonradioactive-labeled isotopic forms of amino acids into cellular proteins. The effective SILAC labeling of immortalized cells and single-cell organisms (e.g., yeast and bacteria) was recently extended to more complex organisms, including worms, flies, and even rodents. The administration of a (13)C6-lysine (heavy) containing diet for one mouse generation leads to a complete exchange of the natural (light) isotope (12)C6-lysine. SILAC-labeled organisms are mainly used as a heavy "spike-in" standard into nonlabeled counterparts, and the combination with high-performance mass spectrometers allows for global proteomic screening. Here we used the fully labeled SILAC mice to identify proteins based on SILAC pairs from isolated cardiomyocytes, and we analyzed ß-parvin-deficient hearts. Our approach confirmed the absence ß-parvin and revealed simultaneously a clear up regulation of α-parvin in heart tissue. In this protocol, we describe the generation of a SILAC mouse colony and show two approaches to perform a proteome-wide analysis of heart tissue. Thus, the SILAC mouse spike-in approach is a readily available procedure and allows for a straightforward systematic analysis of disease models and knockout mice.

Alpha-actinin-3 deficiency does not significantly alter oxidative enzyme activity in fast human muscle fibres.

AIM: In Western European populations, about 18% of all individuals have a complete deficiency of the alpha-actinin-3 protein owing to homozygosity for a stop codon mutation (R577X) in the ACTN3 gene. Actn3(-/-) knock-out mice show increased activity of multiple enzymes in the aerobic metabolic pathway in fast muscle fibres. Whether this observation is also present in human XX genotype carriers compared to RR carriers has not been studied in a fibre-type-specific approach in humans. The purpose of this study was therefore to compare fibre-type-specific oxidative enzyme activity in humans with a different ACTN3 R577X genotype. METHODS: Vastus lateralis muscle biopsy samples of 17 XX and 16 RR subjects were used to measure markers of oxidative capacity [cytochrome c oxidase (CYTOX) and succinate dehydrogenase (SDH)] in a fibre-type-specific assay using enzyme histochemistry. RESULTS: Cytochrome c oxidase staining showed no significant genotype group differences in type I or type II muscle fibres. Also, we found no significant differences in SDH staining of fast fibres comparing XX and RR carriers. CONCLUSION: In conclusion, the increase in oxidative enzyme activity of fast muscle fibres, as reported in an Actn3(-/-) knock-out mouse, was not observed in our human samples. Known differences in metabolic characteristics of muscle fibres in rodents compared to humans may in part explain this discrepancy in findings.

α-Actinin-3 deficiency is associated with reduced bone mass in human and mouse.

Bone mineral density (BMD) is a complex trait that is the single best predictor of the risk of osteoporotic fractures. Candidate gene and genome-wide association studies have identified genetic variations in approximately 30 genetic loci associated with BMD variation in humans. α-Actinin-3 (ACTN3) is highly expressed in fast skeletal muscle fibres. There is a common null-polymorphism R577X in human ACTN3 that results in complete deficiency of the α-actinin-3 protein in approximately 20% of Eurasians. Absence of α-actinin-3 does not cause any disease phenotypes in muscle because of compensation by α-actinin-2. However, α-actinin-3 deficiency has been shown to be detrimental to athletic sprint/power performance. In this report we reveal additional functions for α-actinin-3 in bone. α-Actinin-3 but not α-actinin-2 is expressed in osteoblasts. The Actn3(-/-) mouse displays significantly reduced bone mass, with reduced cortical bone volume (-14%) and trabecular number (-61%) seen by microCT. Dynamic histomorphometry indicated this was due to a reduction in bone formation. In a cohort of postmenopausal Australian women, ACTN3 577XX genotype was associated with lower BMD in an additive genetic model, with the R577X genotype contributing 1.1% of the variance in BMD. Microarray analysis of cultured osteoprogenitors from Actn3(-/-) mice showed alterations in expression of several genes regulating bone mass and osteoblast/osteoclast activity, including Enpp1, Opg and Wnt7b. Our studies suggest that ACTN3 likely contributes to the regulation of bone mass through alterations in bone turnover. Given the high frequency of R577X in the general population, the potential role of ACTN3 R577X as a factor influencing variations in BMD in elderly humans warrants further study.

Alpha-actinin-4 and CLP36 protein deficiencies contribute to podocyte defects in multiple human glomerulopathies.

Genetic alterations of α-actinin-4 can cause podocyte injury through multiple mechanisms. Although a mechanism involving gain-of-α-actinin-4 function was well described and is responsible for a dominantly inherited form of human focal segmental glomerulosclerosis (FSGS), evidence supporting mechanisms involving loss-of-α-actinin-4 function in human glomerular diseases remains elusive. Here we show that α-actinin-4 deficiency occurs in multiple human primary glomerulopathies including sporadic FSGS, minimal change disease, and IgA nephropathy. Furthermore, we identify a close correlation between the levels of α-actinin-4 and CLP36, which form a complex in normal podocytes, in human glomerular diseases. siRNA-mediated depletion of α-actinin-4 in human podocytes resulted in a marked reduction of the CLP36 level. Additionally, two FSGS-associated α-actinin-4 mutations (R310Q and Q348R) inhibited the complex formation between α-actinin-4 and CLP36. Inhibition of the α-actinin-4-CLP36 complex, like loss of α-actinin-4, markedly reduced the level of CLP36 in podocytes. Finally, reduction of the CLP36 level or disruption of the α-actinin-4-CLP36 complex significantly inhibited RhoA activity and generation of traction force in podocytes. Our studies reveal a critical role of the α-actinin-4-CLP36 complex in podocytes and provide an explanation as to how α-actinin-4 deficiency or mutations found in human patients could contribute to podocyte defects and glomerular failure through a loss-of-function mechanism.

Properties of extensor digitorum longus muscle and skinned fibers from adult and aged male and female Actn3 knockout mice.

Absence of α-actinin-3, encoded by the ACTN3 "speed gene," is associated with poorer sprinting performance in athletes and a slowing of relaxation in fast-twitch muscles of Actn3 knockout (KO) mice. Our first aim was to investigate, at the individual-fiber level, possible mechanisms for this slowed relaxation. Our second aim was to characterize the contractile properties of whole extensor digitorum longus (EDL) muscles from KO mice by age and gender. We examined caffeine-induced Ca(2+) release in mechanically skinned EDL fibers from KO mice, and measured isolated whole EDL contractile properties. The sarcoplasmic reticulum of KO muscle fibers loaded Ca(2+) more slowly than that of wild-types (WTs). Whole KO EDL muscles had longer twitch and tetanus relaxation times than WTs, and reduced mass and cross-sectional area. These effects occurred in both male and female mice, but they diminished with age. These changes in KO muscles and fibers help to explain the effects of α-actinin-3 deficiency observed in athletes.

The effect of α-actinin-3 deficiency on muscle aging.

Deficiency of the fast-twitch muscle protein α-actinin-3 due to homozygosity for a nonsense polymorphism (R577X) in the ACTN3 gene is common in humans. α-Actinin-3 deficiency (XX) is associated with reduced muscle strength/power and enhanced endurance performance in elite athletes and in the general population. The association between R577X and loss in muscle mass and function (sarcopenia) has previously been investigated in a number of studies in elderly humans. The majority of studies report loss of ACTN3 genotype association with muscle traits in the elderly, however, there is some indication that the XX genotype may be associated with faster muscle function decline. To further explore these potential age-related effects and the underlying mechanisms, we examined the effect of α-actinin-3 deficiency in aging male and female Actn3 knockout (KO) mice (2, 6, 12, and 18 months). Our findings support previous reports of a diminished influence of ACTN3 genotype on muscle performance in the elderly: genotype differences in intrinsic exercise performance, fast muscle force generation and male muscle mass were lost in aged mice, but were maintained for other muscle function traits such as grip strength. The loss of genotype difference in exercise performance occurred despite the maintenance of some "slower" muscle characteristics in KO muscles, such as increased oxidative metabolism and greater force recovery after fatigue. Interestingly, muscle mass decline in aged 18 month old male KO mice was greater compared to wild-type controls (WT) (-12.2% in KO; -6.5% in WT). These results provide further support that α-actinin-3 deficient individuals may experience faster decline in muscle function with increasing age.

A gene for speed: the emerging role of alpha-actinin-3 in muscle metabolism.

A common polymorphism (R577X) in the ACTN3 gene results in complete deficiency of alpha-actinin-3 protein in approximately 16% of humans worldwide. The presence of alpha-actinin-3 protein is associated with improved sprint/power performance in athletes and the general population. Despite this, there is evidence that the null genotype XX has been acted on by recent positive selection, likely due to its emerging role in the regulation of muscle metabolism. alpha-Actinin-3 deficiency reduces the activity of glycogen phosphorylase and results in a fundamental shift toward more oxidative pathways of energy utilization.

Alpha-actinin-3 deficiency results in reduced glycogen phosphorylase activity and altered calcium handling in skeletal muscle.

Approximately one billion people worldwide are homozygous for a stop codon polymorphism in the ACTN3 gene (R577X) which results in complete deficiency of the fast fibre muscle protein alpha-actinin-3. ACTN3 genotype is associated with human athletic performance and alpha-actinin-3 deficient mice [Actn3 knockout (KO) mice] have a shift in the properties of fast muscle fibres towards slower fibre properties, with increased activity of multiple enzymes in the aerobic metabolic pathway and slower contractile properties. alpha-Actinins have been shown to interact with a number of muscle proteins including the key metabolic regulator glycogen phosphorylase (GPh). In this study, we demonstrated a link between alpha-actinin-3 and glycogen metabolism which may underlie the metabolic changes seen in the KO mouse. Actn3 KO mice have higher muscle glycogen content and a 50% reduction in the activity of GPh. The reduction in enzyme activity is accompanied by altered post-translational modification of GPh, suggesting that alpha-actinin-3 regulates GPh activity by altering its level of phosphorylation. We propose that the changes in glycogen metabolism underlie the downstream metabolic consequences of alpha-actinin-3 deficiency. Finally, as GPh has been shown to regulate calcium handling, we examined calcium handling in KO mouse primary mouse myoblasts and find changes that may explain the slower contractile properties previously observed in these mice. We propose that the alteration in GPh activity in the absence of alpha-actinin-3 is a fundamental mechanistic link in the association between ACTN3 genotype and human performance.

The evolution of skeletal muscle performance: gene duplication and divergence of human sarcomeric alpha-actinins.

In humans, there are two skeletal muscle alpha-actinins, encoded by ACTN2 and ACTN3, and the ACTN3 genotype is associated with human athletic performance. Remarkably, approximately 1 billion people worldwide are deficient in alpha-actinin-3 due to the common ACTN3 R577X polymorphism. The alpha-actinins are an ancient family of actin-binding proteins with structural, signalling and metabolic functions. The skeletal muscle alpha-actinins diverged approximately 250-300 million years ago, and ACTN3 has since developed restricted expression in fast muscle fibres. Despite ACTN2 and ACTN3 retaining considerable sequence similarity, it is likely that following duplication there was a divergence in function explaining why alpha-actinin-2 cannot completely compensate for the absence of alpha-actinin-3. This paper focuses on the role of skeletal muscle alpha-actinins, and how possible changes in functions between these duplicates fit in the context of gene duplication paradigms.

alpha-actinin-3 and performance.

The human sarcomeric alpha-actinins (ACTN2 and ACTN3) are major structural components of the Z line in skeletal muscle; they play a role in the maintenance of sarcomeric integrity and also interact with a wide variety of structural, signaling and metabolic proteins. ACTN2 is expressed in all muscle fibers, and expression of ACTN3 is restricted to the type 2 (fast glycolytic) fibers that are responsible for forceful contraction at high velocity. There is a common stop codon polymorphism R577X in the ACTN3 gene. Homozygosity for the R577X null-allele results in the absence of alpha-actinin-3 in fast muscle fibers with frequencies that vary from < 1% in Africans to approximately 18% in Caucasians. A number of association studies have demonstrated that the ACTN3 R577X genotype influences athletic performance in Caucasians; the frequency of the XX genotype is significantly lower than controls in sprint athletes, and it appears that alpha-actinin-3 deficiency is detrimental to sprint performance. In the general population, the ACTN3 genotype contributes to the normal variations in muscle strength and sprinting speed. In an Actn3 knockout mouse model, alpha-actinin-3 deficiency is associated with a shift in the characteristics of fast, glycolytic 2B muscle fibers towards a slow phenotype, with decreased muscle mass and fiber diameter, slower contractile properties, increased fatigue resistance, and an increase in oxidative enzyme activity. The shift towards a more efficient oxidative metabolism may underlie the selective advantage of the X-allele during evolution. In turn, the shift towards a 'slow' muscle phenotype in fast muscle fibers likely explains why loss of alpha-actinin-3 is detrimental to sprint performance.

Fast-twitch sarcomeric and glycolytic enzyme protein loss in inclusion body myositis.

Inclusion body myositis (IBM) is an inflammatory disease of skeletal muscle of unknown cause. To further understand the nature of the tissue injury in this disease, we developed methods for large-scale detection and quantitation of proteins in muscle biopsy samples and analyzed proteomic data produced by these methods together with histochemical, immunohistochemical, and microarray data. Twenty muscle biopsy samples from patients with inflammatory myopathies (n = 17) or elderly subjects without neuromuscular disease (n = 3) were profiled by proteomic studies using liquid chromatographic separation of peptides followed by mass spectrometry. Thirteen of the diseased samples additionally underwent microarray studies. Seventy muscle specimens from patients with a range of neuromuscular disorders were examined by ATPase histochemical methods. Smaller numbers of samples underwent immunohistochemical and immunoblot studies. Mass spectrometric studies identified and quantified approximately 300 total distinct proteins in each muscle sample. In IBM and to a lesser extent in polymyositis, proteomic studies confirmed by histochemical, immunohistochemical, and immunoblot studies showed loss of many fast-twitch specific structural proteins and glycolytic enzymes despite relative preservation of transcript levels. Increased abundance of a nuclear membrane protein, immunoglobulins, and two calpain-3 substrates were present. The atrophy present in IBM muscle is accompanied by preferential loss of fast-twitch structural proteins and glycolytic enzymes, particularly glycogen debranching enzyme, with relative preservation of the abundance of their respective transcripts. Although muscle atrophy has long been recognized in IBM, these studies are the first to report specific proteins which are reduced in quantity in IBM muscle.