This study aimed to investigate the effects of dietary crude protein (CP) and lysine levels on growth performance, slaughter performance, meat quality, and myofiber characteristics of slow-growing chicken. A 3 × 3 factorial experiment was arranged, and the chickens were fed with 3 levels of dietary CP (16.0%, 17.0%, 18.0%) and 3 levels of dietary lysine (0.69%, 0.84%, 0.99%). A total of 540 8-week-old Beijing-You Chicken (BYC) female growing chickens were randomly allocated to 9 groups, 5 replicates per group, and 12 chickens per replicate. The birds were randomly allocated to one of the 9 experimental diets. Growth performance, slaughter performance, meat quality, and myofiber characteristics were determined at 16 weeks of age. The results showed that dietary CP level and the interaction of dietary CP and lysine levels affected average feed intake (AFI) (p < 0.05). The AFI in the 16.0% CP and 17.0% CP groups was higher than in the 18.0% CP group (p < 0.05). Dietary CP levels significantly affected body weight gain (BWG) (p < 0.05) at 9 to 16 weeks. The 18.0% CP group had the highest BWG (93.99 g). Dietary CP levels affected the percentage of leg muscle yield, and the percentage of leg muscle yield of the 16.0% CP group was significantly lower than that in the other two groups (p < 0.05). Dietary CP and lysine levels alone and their interactions did not affect pH24h, drip loss, and cooking loss of breast muscle (p > 0.05). The shear force of the 18.0% CP group (29.55 N) was higher than that in the other two groups (p < 0.01). Dietary CP level affected myofiber characteristic (p < 0.01), with the lowest myofiber density (846.35 p·mm-2) and the largest myofiber diameter (30.92 μm) at 18.0% CP level. Dietary lysine level affected myofiber diameter, endomysium thickness, perimysium thickness (p < 0.01), with the largest myofiber diameter (29.29 μm) obtained at 0.84% lysine level, the largest endomysium thickness (4.58 μm) at 0.69% lysine level, and the largest perimysium thickness (9.26 μm) at 0.99% lysine level. Myofiber density was negatively correlated with myofiber diameter and endomysium thickness (R = -0.883, R = -0.523, p < 0.01); perimysium thickness had a significant negative correlation with shear force (R = -0.682, p < 0.05). Therefore, reducing dietary CP level and adding appropriate lysine can reduce myofiber diameter and increase perimysium thickness, reducing shear force and improving meat tenderness. A high lysine level (0.99%) in the low-CP (16.0%) diet can improve meat tenderness by regulating the myofiber characteristic without affecting production performance.

The development and characteristics of muscle fibers in broilers are critical determinants that influence their growth performance, as well as serve as essential prerequisites for the production of high-quality chicken meat. Guanidinoacetic acid (GAA) is a crucial endogenous substance in animal creatine synthesis, and its utilization as a feed additive has been demonstrated the capabilities to enhance animal performance, optimize muscle yield, and augment carcass quality. The objective of this study was to investigate the regulation and molecular mechanism underlying muscle development in broilers at different levels of GAA via multiple omics analysis. The 90 Cobb broilers, aged 1 day, were randomly allocated into three treatments consisting of five replicates of six chickens each. The control group was provided with a basal diet, while the Normal GAA and High GAA groups received a basal diet supplemented with 1.2 g/kg and 3.6 g/kg of GAA, respectively. After a feeding period of 42 days, the pectoralis muscles were collected for histomorphological observation, transcriptome and metabolomic analysis. The results demonstrated that the addition of 1.2 g/kg GAA in the diet led to an augmentation in muscle fiber diameter and up-regulation of IGF1, IHH, ASB2, and ANKRD2 gene expression. However, a high dose of 3.6 g/kg GAA in the diet potentially reversed the beneficial effects on chicken breast development by excessively activating the TGF-β signaling pathway and reducing nucleotide metabolite content. These findings would provide a theoretical foundation for enhancing the performance and meat quality of broilers by incorporating GAA as a feed additive.

BACKGROUND: Duchenne muscular dystrophy (DMD) is associated with impaired muscle regeneration, progressive muscle weakness, damage, and wasting. While the cause of DMD is an X-linked loss of function mutation in the gene encoding dystrophin, the exact mechanisms that perpetuate the disease progression are unknown. Our laboratory has demonstrated that pannexin 1 (Panx1 in rodents; PANX1 in humans) is critical for the development, strength, and regeneration of male skeletal muscle. In normal skeletal muscle, Panx1 is part of a multiprotein complex with dystrophin. We and others have previously shown that Panx1 levels and channel activity are dysregulated in various mouse models of DMD.
METHODS: We utilized myoblast cell lines derived from DMD patients to assess PANX1 expression and function. To investigate how Panx1 dysregulation contributes to DMD, we generated a dystrophic (mdx) mouse model that lacks Panx1 (Panx1-/-/mdx). In depth characterization of this model included histological analysis, as well as locomotor, and physiological tests such as muscle force and grip strength assessments.
RESULTS: Here, we demonstrate that PANX1 levels and channel function are reduced in patient-derived DMD myoblast cell lines. Panx1-/-/mdx mice have a significantly reduced lifespan, and decreased body weight due to lean mass loss. Their tibialis anterior were more affected than their soleus muscles and displayed reduced mass, myofiber loss, increased centrally nucleated myofibers, and a lower number of muscle stem cells compared to that of Panx1+/+/mdx mice. These detrimental effects were associated with muscle and locomotor functional impairments. In vitro, PANX1 overexpression in patient-derived DMD myoblasts improved their differentiation and fusion.
CONCLUSIONS: Collectively, our findings suggest that PANX1/Panx1 dysregulation in DMD exacerbates several aspects of the disease. Moreover, our results suggest a potential therapeutic benefit to increasing PANX1 levels in dystrophic muscles.

RNA fluorescence in situ hybridization (FISH) is a powerful method to determine the abundance and localization of mRNA molecules in cells. While modern RNA FISH techniques allow quantification at single molecule resolution, most methods are optimized for mammalian cell culture and are not easily applied to in vivo tissue settings. Single-molecule RNA detection in skeletal muscle cells has been particularly challenging due to the thickness and high autofluorescence of adult muscle tissue and a lack of in vitro models for mature muscle cells (myofibers). Here, we present a method for isolation of adult myofibers from mouse skeletal muscle and detection of single mRNA molecules and proteins using multiplexed RNA FISH and immunofluorescence.

Camellia seed oil (CO) has high nutritional value and multiple bioactivities. However, the specific anti-fatigue characteristics and the implied mechanism of CO have not yet been fully elucidated. Throughout this investigation, male C57BL/6J mice, aged 8 weeks, underwent exhaustive exercise with or without CO pretreatment (2, 4, and 6 mL/kg BW) for 28 days. CO could extend the rota-rod and running time, reduce blood urea nitrogen levels and serum lactic acid, and increase muscle and hepatic glycogen, adenosine triphosphate, and anti-oxidative indicators. Additionally, CO could upregulate the mRNA and Nrf2 protein expression levels, as well as enhance the levels of its downstream antioxidant enzymes and induce the myofiber-type transformation from fast to slow and attenuate the gut mechanical barrier. Moreover, CO could ameliorate gut dysbiosis by reducing Firmicutes to Bacteroidetes ratio at the phylum level, increasing the percentage of Alistipes, Alloprevotella, Lactobacillus, and Muribaculaceae, and decreasing the proportion of Dubosiella at the genus level. In addition, specific bacterial taxa, which were altered by CO, showed a significant correlation with partial fatigue-related parameters. These findings suggest that CO may alleviate fatigue by regulating antioxidant capacity, muscle fiber transformation, gut mechanical barrier, and gut microbial composition in mice. PRACTICAL APPLICATION: Our study revealed that camellia seed oil (CO) could ameliorate exercise-induced fatigue in mice by modulating antioxidant capacity, muscle fiber, and gut microbial composition in mice. Our results promote the application of CO as an anti-fatigue functional food that targets oxidative stress, myofiber-type transformation, and microbial community.

Aging is accompanied by a progressive loss of skeletal muscle mass and strength. The mechanisms underlying this phenomenon are certainly multifactorial and still remain to be fully elucidated. Changes in the cell nucleus structure and function have been considered among the possible contributing causes. This review offers an overview of the current knowledge on skeletal muscle nuclei in aging, focusing on the impairment of nuclear pathways potentially involved in age-related muscle decline. In skeletal muscle two types of cells are present: fiber cells, constituting the contractile muscle mass and containing hundreds of myonuclei, and the satellite cells, i.e., the myogenic mononuclear stem cells occurring at the periphery of the fibers and responsible for muscle growth and repair. Research conducted on different experimental models and with different methodological approaches demonstrated that both the myonuclei and satellite cell nuclei of aged skeletal muscles undergo several structural and molecular alterations, affecting chromatin organization, gene expression, and transcriptional and post-transcriptional activities. These alterations play a key role in the impairment of muscle fiber homeostasis and regeneration, thus contributing to the age-related decrease in skeletal muscle mass and function.

Cardiomyocytes are viscoelastic and key determinants of right ventricle (RV) mechanics. Intracellularly, microtubules are found to impact the viscoelasticity of isolated cardiomyocytes or trabeculae; whether they contribute to the tissue-level viscoelasticity is unknown. Our goal was to reveal the role of the microtubule network in the passive anisotropic viscoelasticity of the healthy RV. Equibiaxial stress relaxation tests were conducted in healthy RV free wall (RVFW) under early (6%) and end (15%) diastolic strain levels, and at sub- and physiological stretch rates. The viscoelasticity was assessed at baseline and after the removal of microtubule network. Furthermore, a quasi-linear viscoelastic (QLV) model was applied to delineate the contribution of microtubules to the relaxation behavior of RVFW. After removing the microtubule network, RVFW elasticity and viscosity were reduced at the early diastolic strain level and in both directions. The reduction in elasticity was stronger in the longitudinal direction, whereas the degree of changes in viscosity were equivalent between directions. There was insignificant change in RVFW viscoelasticity at late diastolic strain level. Finally, the modeling showed that the tissue\'s relaxation strength was reduced by the removal of the microtubule network, but the change was present only at a later time scale. These new findings suggest a critical role of cytoskeleton filaments in RVFW passive mechanics in physiological conditions.

Cardiomyocytes are viscoelastic and contribute significantly to right ventricle (RV) mechanics. Microtubule, a cytoskeletal protein, has been shown to regulate cardiomyocyte viscoelasticity. Additionally, hypertrophied cardiomyocytes from failing myocardium have increased microtubules and cell stiffness. How the microtubules contribute to the tissue-level viscoelastic behavior in RV failure remains unknown. Our aim was to investigate the role of the microtubules in the passive anisotropic viscoelasticity of the RV free wall (RVFW) during pulmonary hypertension (PH) progression. Equibiaxial stress relaxation tests were conducted in the RVFW from healthy and PH rats under early (6%) and end (15%) diastolic strains, and at sub- (1Hz) and physiological (5Hz) stretch-rates. The RVFW viscoelasticity was also measured before and after the depolymerization of microtubules at 5Hz. In intact tissues, PH increased RV viscosity and elasticity at both stretch rates and strain levels, and the increase was stronger in the circumferential than longitudinal direction. At 6% of strain, the removal of microtubules reduced elasticity, viscosity, and the ratio of viscosity to elasticity in both directions and for both healthy and diseased RVs. However, at 15% of strain, the effect of microtubules was different between groups - both viscosity and elasticity were reduced in healthy RVs, but in the diseased RVs only the circumferential viscosity and the ratio of viscosity to elasticity were reduced. These data suggest that, at a large strain with collagen recruitment, microtubules play more significant roles in healthy RV tissue elasticity and diseased RV tissue viscosity. Our findings suggest cardiomyocyte cytoskeletons are critical to RV passive viscoelasticity under pressure overload. STATEMENT OF SIGNIFICANCE: This study investigated the impact of microtubules on the passive anisotropic viscoelasticity of the right ventricular (RV) free wall at healthy and pressure-overloaded states. We originally found that the microtubules contribute significantly to healthy and diseased RV viscoelasticity in both (longitudinal and circumferential) directions at early diastolic strains. At end diastolic strains (with the engagement of collagen fibers), microtubules contribute more to the tissue elasticity of healthy RVs and tissue viscosity of diseased RVs. Our findings reveal the critical role of microtubules in the anisotropic viscoelasticity of the RV tissue, and the altered contribution from healthy to diseased state suggests that therapies targeting microtubules may have potentials for RV failure patients.

This perspective article is concerned with the question of how proteomics, which is a core technique of systems biology that is deeply embedded in the multi-omics field of modern bioresearch, can help us better understand the molecular pathogenesis of complex diseases. As an illustrative example of a monogenetic disorder that primarily affects the neuromuscular system but is characterized by a plethora of multi-system pathophysiological alterations, the muscle-wasting disease Duchenne muscular dystrophy was examined. Recent achievements in the field of dystrophinopathy research are described with special reference to the proteome-wide complexity of neuromuscular changes and body-wide alterations/adaptations. Based on a description of the current applications of top-down versus bottom-up proteomic approaches and their technical challenges, future systems biological approaches are outlined. The envisaged holistic and integromic bioanalysis would encompass the integration of diverse omics-type studies including inter- and intra-proteomics as the core disciplines for systematic protein evaluations, with sophisticated biomolecular analyses, including physiology, molecular biology, biochemistry and histochemistry. Integrated proteomic findings promise to be instrumental in improving our detailed knowledge of pathogenic mechanisms and multi-system dysfunction, widening the available biomarker signature of dystrophinopathy for improved diagnostic/prognostic procedures, and advancing the identification of novel therapeutic targets to treat Duchenne muscular dystrophy.

Sexual dimorphism is phenotypic differences between males and females in the same species. In general, males in most animals are larger than females at the same age, however, in quail, females have a larger body size with greater muscle mass than males. To understand what characteristics in muscle growth play roles in reversed sexual dimorphism in quail, the weights and the characteristics of the pectoralis major and gastrocnemius muscles (PM and GM, respectively) of male and female quail were compared in the current study. The data showed that 15-wk-old female quail have significantly heavier bodies, and PM and GM weights compared to male quail (1.27-folds, 1.29-folds, and 1.16-folds, respectively). To compare muscle characteristics such as hypertrophy (increased size) and hyperplasia (increased cell number), the PM and GM were stained using hematoxylin and eosin, and then histological characteristics such as total cross-sectional area (CSA), number and size of myofibers, and muscle bundle of the muscles were measured and analyzed. In both PM and GM, there were no differences in total numbers of myofibers and muscle bundles as well as the average numbers of myofibers per bundle between sexes. However, the sizes of myofiber and the bundle were significantly increased in female compared to male (1.33-folds and 1.28-folds in PM, and both 1.23-folds in GM, respectively). The findings of the current study suggest that muscle hypertrophy in female quail, not hyperplasia, can be attributed to the sexual dimorphism in quail size.