Number Of Myofibers Research Articles

Spiny mice are primed but fail to regenerate volumetric skeletal muscle loss injuries

BackgroundIn recent years, the African spiny mouse Acomys cahirinus has been shown to regenerate a remarkable array of severe internal and external injuries in the absence of a fibrotic response, including the ability to regenerate full-thickness skin excisions, ear punches, severe kidney injuries, and complete transection of the spinal cord. While skeletal muscle is highly regenerative in adult mammals, Acomys displays superior muscle regeneration properties compared with standard laboratory mice following several injuries, including serial cardiotoxin injections of skeletal muscle and volumetric muscle loss (VML) of the panniculus carnosus muscle following full-thickness excision injuries. VML is an extreme muscle injury defined as the irrecoverable ablation of muscle mass, most commonly resulting from combat injuries or surgical debridement. Barriers to the treatment of VML injury include early and prolonged inflammatory responses that promote fibrotic repair and the loss of structural and mechanical cues that promote muscle regeneration. While the regeneration of the panniculus carnosus in Acomys is impressive, its direct relevance to the study of VML in patients is less clear as this muscle has largely been lost in humans, and, while striated, is not a true skeletal muscle. We therefore sought to test the ability of Acomys to regenerate a skeletal muscle more commonly used in VML injury models.MethodsWe performed two different VML injuries of the Acomys tibialis anterior muscle and compared the regenerative response to a standard laboratory mouse strain, Mus C57BL6/J.ResultsNeither Acomys nor Mus recovered lost muscle mass or myofiber number within three months following VML injury, and Acomys also failed to recover force production better than Mus. In contrast, Acomys continued to express eMHC within the injured area even three months following injury, whereas Mus ceased expressing eMHC less than one-month post-injury, suggesting that Acomys muscle was primed, but failed, to regenerate.ConclusionsWhile the panniculus carnosus muscle in Acomys regenerates following VML injury in the context of full-thickness skin excision, this regenerative ability does not translate to regenerative repair of a skeletal muscle.

Testosterone hormone levels and breast muscle performance of Pelung chickens after zinc sulfate and synthetic testosterone supplementation

Background and Aim: Pelung chicken Gallus gallus gallus (Linnaeus, 1758) is a chicken endemic to Cianjur, West Java, Indonesia. This study aimed to determine the effects of zinc sulfate (ZnSO4) and synthetic testosterone supplementations for 56 days on testosterone hormone levels and breast muscle performance in Pelung chickens. Materials and Methods: This study used 12 Pelung chickens with three treatment groups (G) and four replications, namely, control (G0), ZnSO4 0.9 mg/kg (G1), and synthetic testosterone 3 mg/day (G2). Chickens were acclimatized for 7 days and then supplemented for 56 days. Drinking water and commercial standard feed were provided ad libitum. Blood was collected through the brachial vein for the analysis of testosterone levels using the enzyme-linked immunosorbent assay. The samples were collected every 14 days; on days 0, 14, 28, 42, and 56. Breast muscles were collected for texture analysis, and breast muscle preparations were made with hematoxylin-eosin staining to measure the fascicle area (FA), number of myofibers in one fascicle (NMOF), and myofiber area (MA). The collected data were analyzed using analysis of variance at a 95% confidence level with the help of Statistical Package for the Social Sciences v.29.0. Furthermore, principal component analysis (PCA) was performed with the help of Minitab v. 19. Results: Statistical analysis results on 56 days of testosterone level parameters showed that G2 was significantly different from all treatments (p < 0.05). The results of statistical analysis on Pelung chicken breast muscle performance, especially hardness, chewiness, FA, NMOF, and MA, were significantly different (p < 0.05) compared with the other treatments. The results of PCA showed that the testosterone level parameters were positively correlated with FA, NMOF, MA, hardness, and chewiness, whereas the fracture parameters were negatively correlated with all parameters except the springiness index and were significantly different between the G2 group and the other groups (p < 0.05). Conclusion: It can be concluded that supplementing synthetic testosterone 3 mg/day body weight for 56 days can improve testosterone levels and breast muscle performance, especially hardness, chewiness, FA, NMOF, and MA in Pelung chickens. Keywords: muscle, Pelung chicken, performance, testosterone, texture.

Effects of Short-Term Treatment of Rabbit Extraocular Muscle With Ciliary Neurotrophic Factor.

Little is known about the effect of ciliary neurotrophic factor (CNTF) on extraocular muscles, but microarray studies suggested CNTF might play a role in the development and/or maintenance of strabismus. The effect of short-term treatment of adult rabbit extraocular muscle with injected CNTF was examined for its ability to alter muscle characteristics. Eight adult New Zealand white rabbits received an injection into one superior rectus muscle of 2 µg/100 µL CNTF on 3 consecutive days. One week after the first injection, the rabbits were euthanized, and the treated and contralateral superior rectus muscles were assessed for force generation capacity and contraction characteristics using an in vitro stimulation protocol and compared to naïve control superior rectus muscles. All muscles were analyzed to determine mean cross-sectional areas and expression of slow twitch myosin heavy chain isoform. Short-term treatment of rabbit superior rectus muscles with CNTF resulted in a significant decrease in muscle force generation, but only at the higher stimulation frequencies. Significantly decreased myofiber cross-sectional areas of the treated muscles correlated with the decreased generated force. In addition, there were significant changes to contractile properties of the treated muscles, as well as a decrease in the number of myofibers expressing slow twitch myosin heavy chain. We show that short-term treatment of a single rabbit superior rectus muscle results in decreased myofiber size, decreased force, and altered contractile characteristics. Further studies are needed to determine if it can play a role in improving alignment in animal models of strabismus.

Development of a local controlled release system for therapeutic proteins in the treatment of skeletal muscle injuries and diseases

The present study aims to develop and characterize a controlled-release delivery system for protein therapeutics in skeletal muscle regeneration following an acute injury. The therapeutic protein, a membrane-GPI anchored protein called Cripto, was immobilized in an injectable hydrogel delivery vehicle for local administration and sustained release. The hydrogel was made of poly(ethylene glycol)-fibrinogen (PEG-Fibrinogen, PF), in the form of injectable microspheres. The PF microspheres exhibited a spherical morphology with an average diameter of approximately 100 micrometers, and the Cripto protein was uniformly entrapped within them. The release rate of Cripto from the PF microspheres was controlled by tuning the crosslinking density of the hydrogel, which was varied by changing the concentration of poly(ethylene glycol) diacrylate (PEG-DA) crosslinker. In vitro experiments confirmed a sustained-release profile of Cripto from the PF microspheres for up to 27 days. The released Cripto was biologically active and promoted the in vitro proliferation of mouse myoblasts. The therapeutic effect of PF-mediated delivery of Cripto in vivo was tested in a cardiotoxin (CTX)-induced muscle injury model in mice. The Cripto caused an increase in the in vivo expression of the myogenic markers Pax7, the differentiation makers eMHC and Desmin, higher numbers of centro-nucleated myofibers and greater areas of regenerated muscle tissue. Collectively, these results establish the PF microspheres as a potential delivery system for the localized, sustained release of therapeutic proteins toward the accelerated repair of damaged muscle tissue following acute injuries.

Whole-body replacement of larval myofibers generates permanent adult myofibers in zebrafish

Drastic increases in myofiber number and size are essential to support vertebrate post-embryonic growth. However, the collective cellular behaviors that enable these increases have remained elusive. Here, we created the palmuscle myofiber tagging and tracking system for in toto monitoring of the growth and fates of ~5000 fast myofibers in developing zebrafish larvae. Through live tracking of individual myofibers within the same individuals over extended periods, we found that many larval myofibers readily dissolved during development, enabling the on-site addition of new and more myofibers. Remarkably, whole-body surveillance of multicolor-barcoded myofibers further unveiled a gradual yet extensive elimination of larval myofiber populations, resulting in near-total replacement by late juvenile stages. The subsequently emerging adult myofibers are not only long-lasting, but also morphologically and functionally distinct from the larval populations. Furthermore, we determined that the elimination-replacement process is dependent on and driven by the autophagy pathway. Altogether, we propose that the whole-body replacement of larval myofibers is an inherent yet previously unnoticed process driving organismic muscle growth during vertebrate post-embryonic development.

Skeletal myoblast proliferation and differentiation are reduced in growth restricted fetal sheep

Fetal growth restriction (FGR) increases the risk of type 2 diabetes due in part to deficits in fetal skeletal muscle growth that are not fully compensated after birth. During prenatal development, muscle progenitor cells proliferate, differentiate, and fuse with existing fibers to support myofiber hypertrophy. The total number of myofibers is set in utero, and reductions in myofiber number, size, and myonuclear accretion suggest that myogenesis is impaired in FGR. Previous studies identify similar rates of proliferation and differentiation in FGR and control (CON) myoblasts exposed to enriched growth media in vitro, despite deficits in myoblast proliferation/differentiation in whole muscle of FGR fetuses. Whether muscle progenitor cells of FGR fetuses exhibit impaired myogenic capacity in vivo remains unknown. Using a novel flow cytometry approach to quantify stages of myogenesis in vivo, we aimed to identify the cellular origins of dysregulated skeletal muscle development in FGR due to placental insuffciency. Pregnant sheep were exposed to elevated temperatures (n=15) from early to mid-gestation to induce placental insuffciency and subsequent FGR, defined as birth weight 2 SD below the mean for gestational age. At 90% gestation, fetuses were weighed, and muscle progenitor cells were isolated from biceps femoris of FGR (n=8) and CON (n=5) fetuses for flow cytometry. Muscle cells were identified by anti-CD56, anti-Pax7, and live/dead dye. DAPI was used to quantify resting (G0/1) versus replicating (S/G2) myoblasts. Myogenic regulatory factors, anti-MyoD and anti-myogenin (MyoG), distinguished non-committed (MyoD-/MyoG-) and activated (MyoD+/MyoG-) myoblasts from those beginning (MyoD+/MyoG+) and completing (MyoD-/MyoG+) differentiation. Statistical significance was determined by Student’s t-tests with α=0.05. Percentages are relative to total myoblasts (live, singlets expressing Pax7/CD56). Total myoblasts per live singlets were not different between FGR and CON muscle. FGR fetuses had more muscle progenitors in G0/1 (97% vs 95%, p<0.0001) and fewer replicating myoblasts in S/G2 compared to CON (3% vs 5%, p<0.0001). Myofactor analysis found decreased activated myoblasts (MyoD+/MyoG-) in FGR versus CON (2.5% vs 5%, p<0.005), though the fraction of non-committed progenitors (MyoD-/MyoG-) was similar between groups (14% vs 15%). Despite reduced myogenic induction in FGR, there were no differences between FGR and CON myoblasts expressing MyoG in early (MyoD+, 38% vs 47%) or terminal differentiation (MyoD-, 47% vs 34%). Correlation analyses of all fetuses (n=15) revealed positive relationships between fetal weight and MyoD+/MyoG- (r=0.57) as well as MyoD+/MyoG+ myoblasts (r=0.56), while MyoD-/MyoG+ cells correlated negatively with fetal weight (r=-0.60). FGR fetuses have decreased myoblast proliferation in vivo. Myogenic lineage commitment is reduced in FGR progenitor cells, and positive correlations between fetal weight and myogenic induction suggest that deficits in muscle size and body mass may result from impaired initiation of myogenesis in utero. The elevated percentage of terminally differentiated myocytes in low birthweight fetuses may be a consequence of impaired fusion, which likely contributes to reductions in myonuclei within FGR myofibers. Since the capacity for growth is conserved in FGR skeletal muscle, indicated by retention of myoblast number and proliferation/differentiation capacity in vitro, therapies that stimulate myogenic induction and/or myoblast fusion during fetal development may be necessary/suffcient to restore muscle growth in FGR. NIH R01HD079404; Evonuk Graduate Fellowship; UO VPRI funds. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Integrating miRNA and full-length transcriptome profiling to elucidate the mechanism of muscle growth in Muscovy ducks reveals key roles for miR-301a-3p/ANKRD1

BackgroundThe popularity of Muscovy ducks is attributed not only to their conformation traits but also to their slightly higher content of breast and leg meat, as well as their stronger-tasting meat compared to that of typical domestic ducks. However, there is a lack of comprehensive systematic research on the development of breast muscle in Muscovy ducks. In addition, since the number of skeletal muscle myofibers is established during the embryonic period, this study conducted a full-length transcriptome sequencing and microRNA sequencing of the breast muscle. Muscovy ducks at four developmental stages, namely Embryonic Day 21 (E21), Embryonic Day 27 (E27), Hatching Day (D0), and Post-hatching Day 7 (D7), were used to isolate total RNA for analysis.ResultsA total of 68,161 genes and 472 mature microRNAs were identified. In order to uncover deeper insights into the regulation of mRNA by miRNAs, we conducted an integration of the differentially expressed miRNAs (known as DEMs) with the differentially expressed genes (referred to as DEGs) across various developmental stages. This integration allowed us to make predictions regarding the interactions between miRNAs and mRNA. Through this analysis, we identified a total of 274 DEGs that may serve as potential targets for the 68 DEMs. In the predicted miRNA‒mRNA interaction networks, let-7b, miR-133a-3p, miR-301a-3p, and miR-338-3p were the hub miRNAs. In addition, multiple DEMs also showed predicted target relationships with the DEGs associated with skeletal system development. These identified DEGs and DEMs as well as their predicted interaction networks involved in the regulation of energy homeostasis and muscle development were most likely to play critical roles in facilitating the embryo-to-hatchling transition. A candidate miRNA, miR-301a-3p, exhibited increased expression during the differentiation of satellite cells and was downregulated in the breast muscle tissues of Muscovy ducks at E21 compared to E27. A dual-luciferase reporter assay suggested that the ANKRD1 gene, which encodes a transcription factor, is a direct target of miR-301a-3p.ConclusionsmiR-301a-3p suppressed the posttranscriptional activity of ANKRD1, which is an activator of satellite cell proliferation, as determined with gain- and loss-of-function experiments. miR-301a-3p functions as an inducer of myogenesis by targeting the ANKRD1 gene in Muscovy ducks. These results provide novel insights into the early developmental process of black Muscovy breast muscles and will improve understanding of the underlying molecular mechanisms.

In-ovo feeding with creatine monohydrate: implications for chicken energy reserves and breast muscle development during the pre-post hatching period.

The most dynamic period throughout the lifespan of broiler chickens is the pre-post-hatching period, entailing profound effects on their energy status, survival rate, body weight, and muscle growth. Given the significance of this pivotal period, we evaluated the effect of in-ovo feeding (IOF) with creatine monohydrate on late-term embryos' and hatchlings' energy reserves and post-hatch breast muscle development. The results demonstrate that IOF with creatine elevates the levels of high-energy-value molecules (creatine and glycogen) in the liver, breast muscle and yolk sac tissues 48h post IOF, on embryonic day 19 (p < 0.03). Despite this evidence, using a novel automated image analysis tool on day 14 post-hatch, we found a significantly higher number of myofibers with lower diameter and area in the IOF creatine group compared to the control and IOF NaCl groups (p < 0.004). Gene expression analysis, at hatch, revealed that IOF creatine group had significantly higher expression levels of myogenin (MYOG) and insulin-like growth factor 1 (IGF1), related to differentiation of myogenic cells (p < 0.01), and lower expression of myogenic differentiation protein 1 (MyoD), related to their proliferation (p < 0.04). These results imply a possible effect of IOF with creatine on breast muscle development through differential expression of genes involved in myogenic proliferation and differentiation. The findings provide valuable insights into the potential of pre-hatch enrichment with creatine in modulating post-hatch muscle growth and development.

Research Note: Muscle hypertrophy is associated with reversed sexual dimorphism in body size of quail

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-week-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.

PSV-5 The Effect of a Prenatal Immune Challenge on Offspring Skeletal Muscle Characteristics in the Longissimus Muscle of Weaned Pigs

Abstract An insult to the health status of an animal is associated with decreased skeletal muscle growth. Furthermore, an immune challenge to a gestating dam may inhibit pre- and postnatal muscle growth of the offspring. The objective of this study was to determine if the administration of low-dose lipopolysaccharide (LPS) to gestating sows would alter muscle characteristics of the longissimus muscle (LM) in weaned pigs exposed to LPS. Pregnant Camborough sows were randomly assigned to receive LPS (LPS; n = 7) at a dose of 2.5 μg/kg BW or saline (CON; n= 7) on 78 ± 1.8 d of gestation. At weaning (21 ± 1.3 d of age), barrows (CON n = 17; LPS n = 17) from each sow received a secondary LPS challenge. Following the postnatal challenge, barrows were euthanized (31 ± 1.3 d of age), and both LM were collected. The left LM was weighed, measured for length, and measured for circumference at the cranial, middle, and caudal portion of the muscle. Two samples from the right LM were preserved for histological analyses. Morphometric, fiber type count, primary myofiber clustering, and satellite cell counts were analyzed using the MIXED procedure of SAS 9.4 with a main effect of prenatal treatment and covariate of body weight. There were no differences (P &amp;gt; 0.248) between any morphometric measures. The average diameter for myosin heavy chain (MHC) type I and IIB/X fibers was increased (P &amp;lt; 0.048) 8.6 and 5.9% respectively in LPS offspring compared with CON offspring. Average cross-sectional area (CSA) was increased 10.6% (P = 0.030) in MHC IIB/X fibers and tended to be increased 14.9% (P = 0.080) in MHC 1 fibers of LPS offspring. Number of myofibers within the LM was increased 16.1% (P = 0.032) in CON compared with LPS offspring. Diameter, CSA, and total CSA of oxidative myofibers (MHC type I and IIA) clusters was increased (P &amp;lt; 0.001) 13.8, 27.6, and 22.3% respectively in LPS compared with CON offspring. There were no differences (P &amp;gt; 0.186) between treatment groups for total nuclei or nuclei positive for MYF5, PAX7, or MYF5 and PAX7 nuclei. Based on the timing of the maternal challenge, it can be theorized that LPS hinders hyperplasia during secondary myogenesis following an immune challenge to the dam, with a rescue response of increased hypertrophy of MHC I and IIB/X myofibers post-weaning and glycolytic myofiber profile. However, due to the reduction in total myofibers, the impact of LPS on parameters such as average daily gain, feed efficiency, and lean skeletal muscle accretion should be studied further.

Naringenin improves muscle endurance via activation of the Sp1-ERRγ transcriptional axis

Skeletal muscle function declines in the aging process or disease; however, until now, skeletal muscle has remained one of the organs most undertreated with medication. In this study, naringenin (NAR) was found to build muscle endurance in wild-type mice of different ages by increasing oxidative myofiber numbers and aerobic metabolism, and it ameliorates muscle dysfunction in mdx mice. The transcription factor Sp1 was identified as a direct target of NAR and was shown to mediate the function of NAR on muscle. Moreover, the binding site of NAR on Sp1 was further validated as GLN-110. NAR enhances the binding of Sp1 to the CCCTGCCCTC sequence of the Esrrg promoter by promoting Sp1 phosphorylation, thus upregulating Esrrg expression. The identification of the Sp1-ERRγ transcriptional axis is of great significance in basic muscle research, and this function of NAR has potential implications for the improvement of muscle function and the prevention of muscle atrophy.

Transcriptomic Analysis on Pectoral Muscle of European Meat Pigeons and Shiqi Pigeons during Embryonic Development.

In avian muscle development, embryonic muscle development determines the number of myofibers after birth. Therefore, in this study, we investigated the phenotypic differences and the molecular mechanism of pectoral muscle development of the European meat pigeon Mimas strain (later called European meat pigeon) and Shiqi pigeon on embryonic day 6 (E6), day 10 (E10), day 14 (E14) and day 1 after birth (P1). The results showed that the myofiber density of the Shiqi pigeon was significantly higher than that of the European meat pigeon on E6, and myofibers with a diameter in the range of 50~100 μm of the Shiqi pigeon on P1 were significantly higher than those of European meat pigeon. A total of 204 differential expressed genes (DEGs) were obtained from RNA-seq analysis in comparison between pigeon breeds at the same stage. DEGs related to muscle development were found to significantly enrich the cellular amino acid catabolism, carboxylic acid catabolism, extracellular matrix receptor interaction, REDOX enzyme activity, calcium signaling pathway, ECM receptor interaction, PPAR signaling pathway and other pathways. Using Cytoscape software to create mutual mapping, we identified 33 candidate genes. RT-qPCR was performed to verify the 8 DEGs selected-DES, MYOD, MYF6, PTGS1, MYF5, MYH1, MSTN and PPARG-and the results were consistent with RNA-seq. This study provides basic data for revealing the distinct embryonic development mechanism of pectoral muscle between European meat pigeons and Shiqi pigeons.

Greater numbers and sizes of muscle bundles in the breast and leg muscles of broilers compared to layer chickens.

Meat-type (broiler) and egg-type (layer) chickens were bred by intensive selection over the years, resulting in more numbers and larger sizes of myofibers. Although the characteristics are important parameters in muscle growth and meat quality, muscle bundle characteristics have not been studied in poultry. Therefore, this study aimed to compare the histological characteristics of myofibers and muscle bundles in muscles between male broiler (Ross broiler breed) chickens and layer (Hy-Line) chickens. Chicken muscles, pectoralis major (PM) and gastrocnemius (GM), were sampled at the age of 49 days and stained to analyze histological characteristics. Expectedly, body weights (BWs) and weights of PM and GM muscles in 49-day-old broilers were significantly heavier than those in layers. Within PM, broilers exhibited greater number and cross-sectional area (CSA) of myofibers than layers (3.3- and 3.3-fold, respectively). The total number and CSA of PM muscle bundles were approximately 1.5 and 6.6 times greater, respectively, in broilers than layers. Moreover, broilers exhibited 2 times greater number of myofibers per bundle of PM muscle than layers. Within GM, myofiber number and CSA were 2.3- and 2.4-fold greater, respectively, in broilers than layers. In addition, the total number of muscle bundles and bundle CSA were 2.5- and 2.1-fold greater, respectively, in broilers than in the layers. The novel findings of the current study provide evidence that greater muscle mass of broilers occurs by both hyperplasia and hypertrophy of muscle bundles and myofibers.

Nystagmus Associated With the Absence of MYOD Expression Across the Lifespan in Extraocular and Limb Muscles.

The extraocular muscles (EOMs) undergo significant levels of continuous myonuclear turnover and myofiber remodeling throughout life, in contrast to limb skeletal muscles. Activation of the myogenic pathway in muscle precursor cells is controlled by myogenic transcription factors, such as MYOD. Limb muscles from MyoD-/- mice develop normally but have a regeneration defect, and these mice develop nystagmus. We examined MyoD-/- mice to determine if they have an aging phenotype. Eye movements of aging MyoD-/- mice and littermate controls (wild type) were examined using optokinetic nystagmus (OKN). We assessed limb muscle function, changes to myofiber number, mean cross-sectional area, and abundance of the PAX7 and PITX2 populations of myogenic precursor cells. Aging did not significantly affect limb muscle function despite decreased mean cross-sectional areas at 18+months. Aging wild type mice had normal OKN responses; all aging MyoD-/- mice had nystagmus. With OKN stimulus present, the MyoD-/- mice at all ages had shorter slow phase durations compared to wild type age matched controls. In the dark, the MyoD-/- mice had a shorter slow phase duration with age. This correlated with significantly decreased fiber numbers and cross-sectional areas. The EOM in MyoD-/- mice had increased numbers of PAX7-positive satellite cells and significantly decreased PITX2-positive myonuclei. The absence of MYOD expression in aging mice causes a decrease in on-going myofiber remodeling, EOM fiber size, and number, and is associated with the development of spontaneous nystagmus. These results suggest that muscle-specific mutations can result in nystagmus, with increasing aging-related changes in the MyoD-/- EOM.

Reduced Expression of Septin7 Hinders Skeletal Muscle Regeneration.

Septins are considered the fourth component of the cytoskeleton with the septin7 isoform playing a critical role in the formation of diffusion barriers in phospholipid bilayers and intra- and extracellular scaffolds. While its importance has already been confirmed in different intracellular processes, very little is known about its role in skeletal muscle. Muscle regeneration was studied in a Sept7 conditional knock-down mouse model to prove the possible role of septin7 in this process. Sterile inflammation in skeletal muscle was induced which was followed by regeneration resulting in the upregulation of septin7 expression. Partial knock-down of Sept7 resulted in an increased number of inflammatory cells and myofibers containing central nuclei. Taken together, our data suggest that partial knock-down of Sept7 hinders the kinetics of muscle regeneration, indicating its crucial role in skeletal muscle functions.

Emergence and Progression of Behavioral Motor Deficits and Skeletal Muscle Atrophy across the Adult Lifespan of the Rat.

The facultative loss of muscle mass and function during aging (sarcopenia) poses a serious threat to our independence and health. When activities of daily living are impaired (clinical phase), it appears that the processes leading to sarcopenia have been ongoing in humans for decades (preclinical phase). Here, we examined the natural history of sarcopenia in male outbred rats to compare the occurrence of motor behavioral deficits with the degree of muscle wasting and to explore the muscle-associated processes of the preclinical and clinical phases, respectively. Selected metrics were validated in female rats. We used the soleus muscle because of its long duty cycles and its importance in postural control. Results show that gait and coordination remain intact through middle age (40-60% of median lifespan) when muscle mass is largely preserved relative to body weight. However, the muscle shows numerous signs of remodeling with a shift in myofiber-type composition toward type I. As fiber-type prevalence shifted, fiber-type clustering also increased. The number of hybrid fibers, myofibers with central nuclei, and fibers expressing embryonic myosin increased from being barely detectable to a significant number (5-10%) at late middle age. In parallel, TGFβ1, Smad3, FBXO32, and MuRF1 mRNAs increased. In early (25-month-old) and advanced (30-month-old) aging, gait and coordination deteriorate with the progressive loss of muscle mass. In late middle age and early aging due to type II atrophy (>50%) followed by type I atrophy (>50%), the number of myofibers did not correlate with this process. In advanced age, atrophy is accompanied by a decrease in SCs and βCatenin mRNA, whereas several previously upregulated transcripts were downregulated. The re-expression of embryonic myosin in myofibers and the upregulation of mRNAs encoding the γ-subunit of the nicotinic acetylcholine receptor, the neuronal cell adhesion molecule, and myogenin that begins in late middle age suggest that one mechanism driving sarcopenia is the disruption of neuromuscular connectivity. We conclude that sarcopenia in rats, as in humans, has a long preclinical phase in which muscle undergoes extensive remodeling to maintain muscle mass and function. At later time points, these adaptive mechanisms fail, and sarcopenia becomes clinically manifest.

Chronic repetitive cooling and caffeine-induced intracellular Ca2+ elevation differentially impact adaptations in slow- and fast-twitch rat skeletal muscles.

Intracellular Ca2+ concentration ([Ca2+]i) is considered important in the regulation of skeletal muscle mass. This study tested the hypothesis that chronic repeated cooling and/or caffeine ingestion would acutely increase [Ca2+]i and hypertrophy muscles potentially in a fiber-type-dependent manner. Control rats and those fed caffeine were subjected to repeated bidiurnal treatments of percutaneous icing, under anesthesia, to reduce the muscle temperature below ∼5°C. The predominantly fast-twitch tibialis anterior (TA) and slow-twitch soleus (SOL) muscles were evaluated after 28 days of intervention. The [Ca2+]i elevating response to icing was enhanced by caffeine loading only in the SOL muscle, with the response present across a significantly higher temperature range than in the TA muscle under caffeine-loading conditions. In both the TA and SOL muscles, myofiber cross-sectional area (CSA) was decreased by chronic caffeine treatment (mean reductions of 10.5% and 20.4%, respectively). However, in the TA, but not the SOL, CSA was restored by icing (+15.4 ± 4.3% vs. noniced, P < 0.01). In the SOL, but not TA, icing + caffeine increased myofiber number (20.5 ± 6.7%, P < 0.05) and satellite cell density (2.5 ± 0.3-fold) in cross sections. These contrasting muscle responses to cooling and caffeine may reflect fiber-type-specific [Ca2+]i responses and/or differential responses to elevated [Ca2+]i.

Exploring skeletal muscle tolerance and whole-body metabolic effects of FDA-approved drugs in a volumetric muscle loss model.

Volumetric muscle loss (VML) is associated with persistent functional impairment due to a lack of de novo muscle regeneration. As mechanisms driving the lack of regeneration continue to be established, adjunctive pharmaceuticals to address the pathophysiology of the remaining muscle may offer partial remediation. Studies were designed to evaluate the tolerance and efficacy of two FDA-approved pharmaceutical modalities to address the pathophysiology of the remaining muscle tissue after VML injury: (1) nintedanib (an anti-fibrotic) and (2) combined formoterol and leucine (myogenic promoters). Tolerance was first established by testing low- and high-dosage effects on uninjured skeletal muscle mass and myofiber cross-sectional area in adult male C57BL/6J mice. Next, tolerated doses of the two pharmaceutical modalities were tested in VML-injured adult male C57BL/6J mice after an 8-week treatment period for their ability to modulate muscle strength and whole-body metabolism. The most salient findings indicate that formoterol plus leucine mitigated the loss in muscle mass, myofiber number, whole-body lipid oxidation, and muscle strength, and resulted in a higher whole-body metabolic rate (p ≤ 0.016); nintedanib did not exacerbate or correct aspects of the muscle pathophysiology after VML. This supports ongoing optimization efforts, including scale-up evaluations of formoterol treatment in large animal models of VML.

Advanced Methodology for Rapid Isolation of Single Myofibers from Flexor Digitorum Brevis Muscle.

Isolated individual myofibers are valuable experimental models that can be used in various conditions to understand skeletal muscle physiology and pathophysiology at the tissue and cellular level. This report details a time- and cost-effective method for isolation of single myofibers from the flexor digitorum brevis (FDB) muscle in both young and aged mice. The FDB muscle was chosen for its documented history in single myofiber experiments. By modifying published methods for FDB myofiber isolation, we have optimized the protocol by first separating FDB muscle into individual bundles before the digestion, followed by optimizing the subsequent digestion medium conditions to ensure reproducibility. Morphological and functional assessments demonstrate a high yield of isolated FDB myofibers with sarcolemma integrity achieved in a shorter time frame than previous published procedures. This method could be also adapted to other types of skeletal muscle. Additionally, this highly reproducible method can greatly reduce the number of animals needed to yield adequate numbers of myofibers for experiments. Thus, this advanced method for myofiber isolation has the potential to accelerate research in skeletal muscle physiology and screening potential therapeutics "ex vivo" for muscle diseases and regeneration.

In ovo probiotic supplementation promotes muscle growth and development in broiler embryos

In chickens, muscle development during embryonic growth is predominantly by myofiber hyperplasia. Following hatch, muscle growth primarily occurs via hypertrophy of the existing myofibers. Since myofiber number is set at hatch, production of more muscle fibers during embryonic growth would provide a greater myofiber number at hatch and potential for posthatch muscle growth by hypertrophy. Therefore, to improve performance in broilers, this study investigated the effect of in ovo spray application of probiotics on overall morphometry and muscle development in broiler embryos. For the study, fertile Ross 308 eggs were sprayed with different probiotics; Lactobacillus paracasei DUP 13076 (LP) and L. rhamnosus NRRL B 442 (LR) prior to and during incubation. The embryos were sacrificed on d 7, 10, 14, and 18 for embryo morphometry and pectoralis major muscle (PMM) sampling. Muscle sections were stained and imaged to quantify muscle fiber density (MFD), myofiber cross-sectional area (CSA), and nuclei density. Additionally, gene expression assays were performed to elucidate the effect of probiotics on myogenic genes. In ovo probiotic supplementation was found to significantly improve embryo weight, breast weight, and leg weight (P < 0.05). Further, histological analysis of PMM revealed a significant increase in MFD and nuclei number in the probiotic-treated embryos when compared to the control (P < 0.05). In 18-day-old broiler embryos, myofibers in the treatment group had a significantly smaller CSA (LP: 95.27 ± 3.28 μm2, LR: 178.84 ± 15.1 μm2) when compared to the control (211.41 ± 15.67 μm2). This decrease in CSA was found to be associated with a concomitant increase in MFD (fibers/mm2) in the LP (13,647 ± 482.15) and LR (13,957 ± 463.13) group when compared to the control (7,680 ± 406.78). Additionally, this increase in myofibrillar hyperplasia in the treatment groups was associated with upregulation in the expression of key genes regulating muscle growth including MYF5, MYOD, MYOG, and IGF-1. In summary, in ovo spray application of probiotics promoted overall embryo growth and muscle development in broilers.