Genotype Score for Iron Status Is Associated with Muscle Fiber Composition in Women

Human muscle fiber composition is heterogeneous and mainly determined by genetic factors. A previous study reported that experimentally induced iron deficiency in rats increases the proportion of fast-twitch muscle fibers. Iron status has been reported to be affected by genetic factors. As the TMPRSS6 rs855791 T/C and HFE rs1799945 C/G polymorphisms are strongly associated with iron status in humans, we hypothesized that the genotype score (GS) based on these polymorphisms could be associated with the muscle fiber composition in humans. Herein, we examined 214 Japanese individuals, comprising of 107 men and 107 women, for possible associations of the GS for iron status with the proportion of myosin heavy chain (MHC) isoforms (I, IIa, and IIx) as markers of muscle fiber composition. No statistically significant correlations were found between the GS for iron status and the proportion of MHC isoforms in all participants. When the participants were stratified based on sex, women showed positive and negative correlations of the GS with MHC-IIa (age-adjusted p = 0.020) and MHC-IIx (age-adjusted p = 0.011), respectively. In contrast, no correlation was found in men. In women, a 1-point increase in the GS was associated with 2.42% higher MHC-IIa level and 2.72% lower MHC-IIx level. Our results suggest that the GS based on the TMPRSS6 rs855791 T/C and HFE rs1799945 C/G polymorphisms for iron status is associated with muscle fiber composition in women.

Lifespan Analysis of Dystrophic mdx Fast-Twitch Muscle Morphology and Its Impact on Contractile Function

Duchenne muscular dystrophy is caused by the absence of the protein dystrophin from skeletal muscle and is characterized by progressive cycles of necrosis/regeneration. Using the dystrophin deficient mdx mouse model, we studied the morphological and contractile chronology of dystrophic skeletal muscle pathology in fast-twitch Extensor Digitorum Longus muscles from animals 4–22 months of age containing 100% regenerated muscle fibers. Catastrophically, the older age groups lost ∼80% of their maximum force after one eccentric contraction (EC) of 20% strain with the greatest loss of ∼92% recorded in senescent 22-month-old mdx mice. In old age groups, there was minimal force recovery ∼24% after 120 min, correlated with a dramatic increase in the number and complexity of branched fibers. This data supports our two-phase model where a “tipping point” is reached when branched fibers rupture irrevocably on EC. These findings have important implications for pre-clinical drug studies and genetic rescue strategies.

SR Ca2+ handling in unbranched, immediately post-necrotic fast-twitch mdx fibres is similar to wt littermates

There is a lack of consensus in the literature regarding the effects of dystrophin deficiency on the Ca2+ handling properties of the sarcoplasmic reticulum (SR) in mdx mice, an animal model of Duchenne muscular dystrophy. One possible reason for this is that only a few studies control for the presence of branched fibres. Fibre branching, a consequence of degenerative-regenerative processes such as muscular dystrophy, has in itself a significant influence on the function of the SR. In our present study we attempt to detect early effects of dystrophin deficiency on SR Ca2+ handling by using unbranched fibres from the immediate post-necrotic stage in mdx mice (just regenerated following massive necrosis). Using kinetically-corrected Fura-2 fluorescence signals measured during twitch and tetanus, we analysed the amplitude, rise time and decay time of Δ[Ca2+]i in unfatigued and fatigued fibres. Decay was also resolved into SR pump and SR leak components. Fibres from mdx mice were similar in all respects to fibres from wt littermates apart from: (i) a longer rise time and slower rate of rise of [Ca2+]i during a tetanus; and (ii) a mitigation of the fall in Δ[Ca2+]i amplitude during the course of fatigue. Our findings suggest that the early effects of a loss of dystrophin on SR Ca2+ handling are only slight, and differ from the widely held view that there is significant Ca2+ pathology in mdx mice. It may be that Ca2+pathology is magnified by progressive branching and degeneration.

Sarcoplasmic Reticulum from Horse Gluteal Muscle Is Poised for Enhanced Calcium Transport

We have analyzed the enzymatic activity of the sarcoplasmic reticulum (SR) Ca2+-transporting ATPase (SERCA) from the horse gluteal muscle. Horses are bred for peak athletic performance yet exhibit a high incidence of exertional rhabdomyolysis, with elevated levels of cytosolic Ca2+ proposed as a correlative linkage. We recently reported an improved protocol for isolating SR vesicles from horse muscle; these horse SR vesicles contain an abundant level of SERCA and only trace-levels of sarcolipin (SLN), the inhibitory peptide subunit of SERCA in mammalian fast-twitch skeletal muscle. Here, we report that the in vitro Ca2+ transport rate of horse SR vesicles is 2.3 ± 0.7-fold greater than rabbit SR vesicles, which express close to equimolar levels of SERCA and SLN. This suggests that horse myofibers exhibit an enhanced SR Ca2+ transport rate and increased luminal Ca2+ stores in vivo. Using the densitometry of Coomassie-stained SDS-PAGE gels, we determined that horse SR vesicles express an abundant level of the luminal SR Ca2+ storage protein calsequestrin (CASQ), with a CASQ-to-SERCA ratio about double that in rabbit SR vesicles. Thus, we propose that SR Ca2+ cycling in horse myofibers is enhanced by a reduced SLN inhibition of SERCA and by an abundant expression of CASQ. Together, these results suggest that horse muscle contractility and susceptibility to exertional rhabdomyolysis are promoted by enhanced SR Ca2+ uptake and luminal Ca2+ storage.

Six1 promotes skeletal muscle thyroid hormone response through regulation of the MCT10 transporter

Background The Six1 transcription factor is implicated in controlling the development of several tissue types, notably skeletal muscle. Six1 also contributes to muscle metabolism and its activity is associated with the fast-twitch, glycolytic phenotype. Six1 regulates the expression of certain genes of the fast muscle program by directly stimulating their transcription or indirectly acting through a long non-coding RNA. We hypothesized that additional mechanisms of action of Six1 might be at play. Methods A combined analysis of gene expression profiling and genome-wide location analysis data was performed. Results were validated using in vivo RNA interference loss-of-function assays followed by measurement of gene expression by RT-PCR and transcriptional reporter assays. Results The Slc16a10 gene, encoding the thyroid hormone transmembrane transporter MCT10, was identified as a gene with a transcriptional enhancer directly bound by Six1 and requiring Six1 activity for full expression in adult mouse tibialis anterior, a predominantly fast-twitch muscle. Of the various thyroid hormone transporters, MCT10 mRNA was found to be the most abundant in skeletal muscle, and to have a stronger expression in fast-twitch compared to slow-twitch muscle groups. Loss-of-function of MCT10 in the tibialis anterior recapitulated the effect of Six1 on the expression of fast-twitch muscle genes and led to lower activity of a thyroid hormone receptor-dependent reporter gene. Conclusions These results shed light on the molecular mechanisms controlling the tissue expression profile of MCT10 and identify modulation of the thyroid hormone signaling pathway as an additional mechanism by which Six1 influences skeletal muscle metabolism.

Genomic predictors of testosterone levels are associated with muscle fiber size and strength

Purpose Circulating testosterone levels are a heritable trait with anabolic properties in various tissues, including skeletal muscle. So far, hundreds of single nucleotide polymorphisms (SNPs) associated with testosterone levels have been identified in nonathletic populations. The aim of the present study was to test the association of 822 testosterone-increasing SNPs with muscle-related traits (muscle fiber size, fat-free mass and handgrip strength) and to validate the identified SNPs in independent cohorts of strength and power athletes. Methods One hundred and forty-eight physically active individuals (47 females, 101 males) were assessed for cross-sectional area (CSA) of fast-twitch muscle fibers. Significant SNPs were further assessed for fat-free mass and handgrip strength in > 354,000 participants from the UK Biobank cohort. The validation cohorts included Russian elite athletes. Results From an initial panel of 822 SNPs, we identified five testosterone-increasing alleles (DOCK3 rs77031559 G, ESR1 rs190930099 G, GLIS3 rs34706136 TG, GRAMD1B rs850294 T, TRAIP rs62260729 C) nominally associated (P < 0.05) with CSA of fast-twitch muscle fibers, fat-free mass and handgrip strength. Based on these five SNPs, the number of testosterone-increasing alleles was positively associated with testosterone levels in male athletes (P = 0.048) and greater strength performance in weightlifters (P = 0.017). Moreover, the proportion of participants with ≥ 2 testosterone-increasing alleles was higher in power athletes compared to controls (68.9 vs. 55.6%; P = 0.012). Conclusion Testosterone-related SNPs are associated with muscle fiber size, fat-free mass and strength, which combined can partially contribute to a greater predisposition to strength/power sports.

Absence of the Z-disc Protein α-actinin-3 Impairs the Mechanical Stability of Actn3KO Mouse Fast- twitch Muscle Fibres without Altering their Contractile Properties or Twitch Kinetics

Background: A common polymorphism (R577X) in the ACTN3 gene results in complete absence of the Z-disc protein α-actinin-3 from fast-twitch muscle fibres in ~16% of the world’s population. This single gene polymorphism has been subject to strong positive selection pressure during recent human evolution. Previously, using an Actn3KO mouse model, we have shown in fast-twitch muscles, eccentric contractions at L0+ 20% stretch did not cause eccentric damage. In contrast, L0+ 30% stretch produced a significant ~40% deficit in maximum force; here we use isolated single fast-twitch skeletal muscle fibres from the Actn3KO mouse to investigate the mechanism underlying this.Methods: Single fast-twitch fibres are separated from the intact muscle by a collagenase digest procedure. We use label-free second harmonic generation (SHG) imaging, ultra-fast video microscopy and skinned fibre measurements from our MyoRobot automated biomechatronics system to study the morphology, visco-elasticity, force production and mechanical strength of single fibres from the Actn3KO mouse. Data are presented as means ± SD and tested for significance using ANOVA.Results: We show that the absence of α-actinin-3 does not affect the unloaded maximum speed of contraction, visco-elastic properties or myofibrillar force production. Eccentric contractions demonstrated that chemically skinned Actn3KO fibres are mechanically weaker being prone to breakage when eccentrically contracted. Furthermore, SHG images reveal disruptions in the myofibrillar alignment of Actn3KO fast-twitch fibres with an increase in Y-shaped myofibrillar lattice shifts. Conclusions: Absence of α-actinin-3 from the Z-disc in fast-twitch fibres disrupts the organisation of the myofibrillar proteins, leading to structural weakness. This provides a mechanistic explanation for our earlier findings that, in vitro intact Actn3KO fast-twitch muscles are significantly damaged by L0+ 30%, but not, L0+ 20%, eccentric contraction strains. Our study also provides a possible mechanistic explanation as to why α-actinin-3 deficient humans have been reported to have a faster decline in muscle function with increasing age, that is; as sarcopenia reduces muscle mass and force output, the eccentric stress on the remaining functional α-actinin-3 deficient fibres will be increased, resulting in fibres breakages.

Ryanodine receptors mediate high intracellular Ca2+ and some myocyte damage following eccentric contractions in rat fast twitch skeletal muscle

Eccentric contractions (ECC) facilitate cytosolic calcium ion (Ca2+) release from the sarcoplasmic reticulum (SR) and Ca2+ influx from extracellular space. Ca2+ is a vital signaling messenger that regulates multiple cellular processes via its spatial and temporal concentration ([Ca2+]i) dynamics. We hypothesized that: 1) a specific pattern of spatial/temporal intramyocyte Ca2+ dynamics portends muscle damage following ECC, and 2) these dynamics would be regulated by the ryanodine receptor (RyR). [Ca2+]i in the tibialis anterior muscles of anesthetized adult Wistar rats was measured by ratiometric (i.e. ratio, R, 340/380 nm excitation) in vivo bioimaging with Fura-2 pre-ECC and at 5 and 24 hours post-ECC (5 x 40 contractions). Rats received RyR inhibitor dantrolene (DAN; 10 mg/kg i.p.) immediately post-ECC (+DAN). Muscle damage was evaluated by histological analysis on hematoxylin-eosin stained muscle sections. Compared to control (CONT, no ECC), [Ca2+]i distribution was heterogeneous with increased % total area of high [Ca2+]i sites (operationally defined as R > 1.39 i.e., > 1 SD of mean control) 5 hours post-ECC (CONT, 14.0 ± 8.0; ECC5h: 52.0 ± 7.4%, p < 0.01). DAN substantially reduced the high [Ca2+]i area 5 hours post-ECC (ECC5h+DAN: 6.4 ± 3.1%, p < 0.01) and myocyte damage (ECC24h, 63.2 ± 1.0%; ECC24h+DAN, 29.1 ± 2.2%, p < 0.01). Temporal and spatially-amplified [Ca2+]i fluctuations occurred regardless of DAN (ECC vs ECC+DAN, p > 0.05). These results suggest that the RyR-mediated local high [Ca2+]i itself is related to the magnitude of muscle damage while the [Ca2+]i fluctuation is an RyR-independent phenomenon.

Absence of the Z-disc protein α-actinin-3 impairs the mechanical stability of Actn3KO mouse fast-twitch muscle fibres without altering their contractile properties or twitch kinetics

Background: A common polymorphism (R577X) in the ACTN3 gene results in complete absence of the Z-disc protein α-actinin-3 from fast-twitch muscle fibres in ~16% of the worlds population. This single gene polymorphism has been subject to strong positive selection pressure during recent human evolution. Previously, using an Actn3KO mouse model, we have shown in fast-twitch muscles, eccentric contractions at L0+ 20% stretch did not cause eccentric damage. In contrast, L0+ 30% stretch produced a significant ~40% deficit in maximum force; here we use isolated single fast-twitch skeletal muscle fibres from the Actn3KO mouse to investigate the mechanism underlying this. Methods: Single fast-twitch fibres are separated from the intact muscle by a collagenase digest procedure. We use label-free second harmonic generation (SHG) imaging, ultra-fast video microscopy and skinned fibre measurements from our MyoRobot automated biomechatronics system to study the morphology, visco-elasticity, force production and mechanical strength of single fibres from the Actn3KO mouse. Data are presented as means ± SD and tested for significance using ANOVA. Results: We show that the absence of α-actinin-3 does not affect the unloaded maximum speed of contraction, visco-elastic properties or myofibrillar force production. Eccentric contractions demonstrated that chemically skinned Actn3KO fibres are mechanically weaker being prone to breakage when eccentrically contracted. Furthermore, SHG images reveal disruptions in the myofibrillar alignment of Actn3KO fast-twitch fibres with an increase in Y-shaped myofibrillar lattice shifts. Conclusions: Absence of α-actinin-3 from the Z-disc in fast-twitch fibres disrupts the organisation of the myofibrillar proteins, leading to structural weakness. This provides a mechanistic explanation for our earlier findings that, in vitro intact Actn3KO fast-twitch muscles are significantly damaged by L0+ 30%, but not, L0+ 20%, eccentric contraction strains. Our study also provides a possible mechanistic explanation as to why α-actinin-3 deficient humans have been reported to have a faster decline in muscle function with increasing age, that is; as sarcopenia reduces muscle mass and force output, the eccentric stress on the remaining functional α-actinin-3 deficient fibres will be increased, resulting in fibres breakages.