Heavy Chain Type Research Articles

Automated Microfluidics-Assisted Hydrogel-Based Wet-Spinning for the Biofabrication of Biomimetic Engineered Myotendinous Junction.

The muscle-tendon junction (MTJ) plays a pivotal role in efficiently converting the muscular contraction into a controlled skeletal movement through the tendon. Given its complex biomechanical intricacy, the biofabrication of such tissue interface represents a significant challenge in the field of musculoskeletal tissue engineering. Herein, a novel method to produce MTJ-like hydrogel yarns using a microfluidics-assisted 3D rotary wet-spinning strategy is developed. Optimization of flow rates, rotational speed, and delivery time of bioinks enables the production of highly compartmentalized scaffolds that recapitulate the muscle, tendon, and the transient MTJ-like region. Additionally, such biofabrication parameters are validated in terms of cellular response by promoting an optimal uniaxial alignment for both muscle and tendon precursor cells. By sequentially wet-spinning C2C12 myoblasts and NIH 3T3 fibroblasts, a gradient-patterned cellular arrangement mirroring the intrinsic biological heterogeneity of the MTJ is successfully obtained. The immunofluorescence assessment further reveals the localized expression of tissue-specific markers, including myosin heavy chain and collagen type I/III, which demonstrate muscle and tenogenic tissue maturation, respectively. Remarkably, the muscle-tendon transition zone exhibits finger-like projection of the multinucleated myotubes in the tenogenic compartment, epitomizing the MTJ signature architecture.

Analysis of immunoglobulin organization and complexity in mink (Neovison vison)

Mink are susceptible to viruses such as SARS-CoV-2, H1N1 and H9N2, so they are considered a potential animal model for studying human viral infections. Therefore, it is important to study the immune system of mink. Immunoglobulin (Ig) is an important component of humoral immunity and plays an important role in the body's immune defense. In this study, we described the gene loci structure of mink Ig germline by genome comparison, and analysed the mechanism of expression diversity of mink antibody library by 5′RACE and next-generation sequencing (NGS). The results were as follows: the IgH, Igκ and Igλ loci of mink were located on chromosome 13, chromosome 8 and chromosome 3, respectively, and they had 25, 36 and 7 V genes, 3, 5 and 7 J genes and 10 DH genes, respectively. Mink Ig heavy chain preferred the IGHV1, IGHD2 and IGHJ4 subgroups, κ chain mainly use the IGKV1, IGKJ1 and IGHL4 subgroups, and λ chain mainly use the IGLV3 and IGLJ3 subgroups. Linkage diversity analysis revealed that N nucleotide insertion was the main factor affecting the linkage diversity of mink Igs. On the mutation types of mink Ig Somatic Hypermutation (SHM), the high mutation types of heavy chain were mainly G > A, C > T, T > C, A > G, C > A, G > T, A > C, and T > G; the high mutation types of κ chain were G > A and T > C; and the high mutation types of λ chain were G > A and A > G. The objective of this study was to analyse the loci structure and expression diversity of Ig in mink. The results contribute to our comprehension of Ig expression patterns in mink and were valuable for advancing knowledge in mink immunogenetics, exploring the evolution of adaptive immune systems across different species, and conducting comparative genomics research.

Preparation of a monoclonal antibody against recombinant LSDV034 protein and its application in detecting lumpy skin disease virus through a competitive enzyme-linked immunosorbent assay (cELISA)

Lumpy skin disease (LSD) is a highly contagious disease caused by lumpy skin disease virus (LSDV) in bovines. Rapid and accurate diagnosis is very important to controll it. However, current commercial detection kits need to be improved in terms of sensitivity or specificity. This study aimed to develop a novel diagnostic competitive enzyme-linked immunosorbent assay (cELISA) based on the newly identified antigen gene LSDV034. The rLSDV034 protein was identified as a potential diagnostic antigen, and it was expressed, purified, and used to immunize BALB/c mice. Using laboratory-prepared indirect ELISA (iELISA), the positive cell lines were screened, and their blocking activity was further verified by competitive ELISA (cELISA). The cell line, 1H7, was chosen to produce mouse ascites, which were purified for a monoclonal antibody (mAb, 5.395 mg/mL). The heavy chain type of the 1H7 mAb was identified as IgG1a, and its light chain subtype was identified as κ. Furthermore, cELISA was developed to detect bovine serum antibodies, with rLSDV034 (4 μg/mL) as the coating antigen and HRP-1H7 mAb (1:300) as the competitive antibody. The cutoff value of cELISA was 55%, based on 32 negative bovine serum samples. The analytical sensitivity was 1:8, and no cross-reaction was detected with bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), Pasteurella multocida (P. multocida), or Mycoplasma bovis (M. bovis) from the serum samples. The diagnostic sensitivity and specificity of cELISA were 98.46% (95% confidence interval, CI: 91.7–100) and 100% (95% CI: 89.1–100), respectively, based on the analysis of 30 LSDV-infected bovine serum samples, 35 GTPV-vaccinated samples, and 32 negative samples. The overall coincidence of the cELISA with the virus neutralization test (VNT) reached 98.97% (95% CI: 94.4–100). Furthermore, we used cELISA to analyze 230 clinical bovine serum samples (including 59 infected and 171 vaccinated samples) and found that the serum positivity rates of the immunized samples (on d 60 postimmunization) and infected samples were 77.78% (95% CI: 70.8–83.8%) and 71.19% (95% CI: 57.9–82.2), respectively. These results indicate that the developed cELISA is promising for detecting serum antibodies in naturally infected or vaccinated cattle.

Transcriptomics reveals transient and dynamic muscle fibrosis and atrophy differences following spinal cord injury in rats.

The rate and magnitude of skeletal muscle wasting after severe spinal cord injury (SCI) exceeds most other disuse conditions. Assessing the time course of molecular changes can provide insight into the progression of muscle wasting post-SCI. The goals of this study were (1) to identify potential targets that may prevent the pathologic features of SCI in soleus muscles and (2) to establish therapeutic windows for treating these pathologic changes. Four-month-old Sprague-Dawley male rats received T9 laminectomy (SHAM surgery) or severe contusion SCI. Hindlimb locomotor function was assessed weekly, with soleus muscles obtained 1week, 2weeks, 1month and 3months post-surgery (n=6-7 per group per timepoint). RNA was extracted from muscles for bulk RNA-sequencing analysis (n=3-5 per group per timepoint). Differentially expressed genes (DEGs) were evaluated between age-matched SHAM and SCI animals. Myofiber size, muscle fibre type and fibrosis were assessed on contralateral muscles. SCI produced immediate and persistent hindlimb paralysis, with Basso-Beattie-Bresnahan locomotor scores remaining below 7 throughout the study, contributing to a progressive 25-50% lower soleus mass and myofiber atrophy versus SHAM (P<0.05 at all timepoints). Transcriptional comparisons of SCI versus SHAM resulted in 184 DEGs (1week), 436 DEGs (2weeks), 133 DEGs (1month) and 1200 DEGs (3months). Upregulated atrophy-related genes included those associated with cell senescence, nuclear factor kappa B, ubiquitin proteasome and unfolded protein response pathways, along with upregulated genes that negatively influence muscle growth through the transforming growth factor beta pathway and inhibition of insulin-like growth factor-I/Akt/mechanistic target of rapamycin and p38/mitogen-activated protein kinase signalling. Genes associated with extracellular matrix (ECM), including collagens, collagen crosslinkers, proteoglycans and those regulating ECM integrity, were enriched within upregulated DEGs at 1week but subsequently downregulated at 2weeks and 3months and were accompanied by >50% higher ECM areas and hydroxyproline levels in SCI muscles (P<0.05). Myofiber remodelling genes were enriched in upregulated DEGs at 2weeks and 1month and were downregulated at 3months. Genes that regulate neuromuscular junction remodelling were evident in muscles post-SCI, along with slow-to-fast fibre-type shifts: 1 week and 2 weeks SCI muscles were composed of 90% myosin heavy chain (MHC) type I fibres, which decreased to only 16% at 3months and were accompanied by 50% fibres containing MHC IIX (P<0.05). Metabolism genes were enriched in upregulated DEGs at 1month and were further enriched at 3months. Our results substantiate many known pathologic features of SCI-induced wasting in rat skeletal muscle and identify a progressive and dynamic transcriptional landscape within the post-SCI soleus. Future studies are warranted to consider these therapeutic treatment windows when countering SCI muscle pathology.

Muscular and neuronal control of voice production – forgotten findings, current concepts, and new developments

Voice production has been an area of interest in science since ancient times, and although advancing research has improved our understanding of the anatomy and function of the larynx, there is still little general consensus on these two topics. This review aims to outline the main developments in this field and highlight the areas where further research is needed. The most important hypotheses are presented and discussed highlighting the four main lines of research in the anatomy of the human larynx and their most important findings: (1) the arrangement of the muscle fibers of the thyroarytenoid muscle is not parallel to the vocal folds in the internal part (vocalis muscle), leading to altered properties during contraction; (2) the histological structure of the human vocal cords differs from other striated muscles; (3) there is a specialized type of heavy myosin chains in the larynx; and (4) the neuromuscular system of the larynx has specific structures that form the basis of an intrinsic laryngeal nervous system. These approaches are discussed in the context of current physiological models of vocal fold vibration, and new avenues of investigation are proposed.

Peripheral neural interfaces: Skeletal muscles are hyper-reinnervated according to the axonal capacity of the surgically rewired nerves.

Advances in robotics have outpaced the capabilities of man-machine interfaces to decipher and transfer neural information to and from prosthetic devices. We emulated clinical scenarios where high- (facial) or low-neural capacity (ulnar) donor nerves were surgically rewired to the sternomastoid muscle, which is controlled by a very small number of motor axons. Using retrograde tracing and electrophysiological assessments, we observed a nearly 15-fold functional hyper-reinnervation of the muscle after high-capacity nerve transfer, demonstrating its capability of generating a multifold of neuromuscular junctions. Moreover, the surgically redirected axons influenced the muscle's physiological characteristics, by altering the expression of myosin heavy-chain types in alignment with the donor nerve. These findings highlight the remarkable capacity of skeletal muscles to act as biological amplifiers of neural information from the spinal cord for governing bionic prostheses, with the potential of expressing high-dimensional neural function for high-information transfer interfaces.

Extracellular vesicles from obese and diabetic mouse plasma alter C2C12 myotube glucose uptake and gene expression.

Recent studies have indicated a role for circulating extracellular vesicles (EVs) in the pathogenesis of multiple diseases. However, most invitro studies have used variable and arbitrary doses of EVs rather than interpreting EVs as an existing component of standard skeletal muscle cell culture media. The current study provides an initial investigation into the effects of circulating EVs on the metabolic phenotype of C2C12 myotubes by replacing EVs from fetal bovine serum with circulating EVs from control mice or mice with obesity and type 2 diabetes (OT2D). We report that EVs associated with OT2D decrease 2-NBDG uptake (a proxy measure of glucose uptake) in the insulin-stimulated state compared to controls. OT2D associated EV treatment also significantly decreased myosin heavy chain type 1 (MHCI) mRNA abundance in myotubes but had no effect on mRNA expression of any other myosin heavy chain isoforms. OT2D-associated circulating EVs also significantly increased lipid accumulation within myotubes without altering the expression of a selection of genes important for lipid entry, synthesis, or catabolism. The data indicate that, in a severely diabetic state, circulating EVs may contribute to insulin resistance and alter gene expression in myotubes in a manner consistent with the skeletal muscle phenotype observed in OT2D.

MYH13, a superfast myosin expressed in extraocular, laryngeal and syringeal muscles.

MYH13 is a unique type of sarcomeric myosin heavy chain (MYH) first detected in mammalian extraocular (EO) muscles and later also in vocal muscles, including laryngeal muscles of some mammals and syringeal muscles of songbirds. All these muscles are specialized in generating very fast contractions while producing relatively low force, a design appropriate for muscles acting against a much lower load than most skeletal muscles inserting into the skeleton. The definition of the physiological properties of muscle fibres containing MYH13 has been complicated by the mixed fibre type composition of EO muscles and the coexistence of different MYH types within the same fibre. A major advance in this area came from studies on isolated recombinant myosin motors and the demonstration that the affinity of actin-bound human MYH13 for ADP is much weaker than those of fast-type MYH1 (type 2X) and MYH2 (type 2A). This property is consistent with a very fast detachment of myosin from actin, a major determinant of shortening velocity. The MYH13 gene arose early during vertebrate evolution but was characterized only in mammals and birds and appears to have been lost in some teleost fish. The MYH13 gene is located at the 3' end of the mammalian fast/developmental gene cluster and in a similar position to the orthologous cluster in syntenic regions of the songbird genome. MYH13 gene regulation is controlled by a super-enhancer in the mammalian locus and deletion of the neighbouring fast MYH1 and MYH4 genes leads to abnormal MYH13 expression in mouse leg muscles.

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.

Effects of exercise prehabilitation on muscle atrophy and contractile properties in hindlimb-unloaded rats.

Effective strategies for rapid recovery after surgery are needed. Therefore, we investigated the effects of exercise prehabilitation (EP) and hindlimb unloading (HU) on muscle loss and contractility. Twenty-two Sprague-Dawley rats (12 wk old) were divided into normal control (NCON, n = 5), hindlimb unloading control (HCON, n = 10), and exercise prehabilitation followed by hindlimb unloading (Ex-preH, n = 7) groups. Ex-PreH performed exercise training for 14 days before hindlimb unloading for 14 days. Body composition was evaluated, along with muscle strength and function. The soleus (SOL) and extensor digitorum longus (EDL) muscle contractile properties were analyzed at the whole-muscle level. The titin concentration and myosin heavy chain (MHC) type composition were analyzed. There were no effects of Ex-preH on total mass, lean mass, or muscle weight. Physical function was significantly higher in the Ex-preH group than in the HCON group (39.5° vs. 35.7°). The SOL twitch force (19.6 vs. 7.1 mN/m2 ) and specific force (107.3 vs. 61.2 mN/m2 ) were greater in Ex-preH group than in HCON group. EDL shortening velocity was higher in Ex-preH group than in HCON group (13.2 vs. 5.0 FL/s). The SOL full-length titin level was higher in Ex-preH group than in HCON group. Exercise prehabilitation did not prevent muscle mass loss followed by muscle wasting, although it minimized the reduction of physical function. Therefore, exercise prehabilitation should be considered for rapid functional recovery after disuse due to surgery and injuries.

Abstract 6073: Distinct subtype of multiple myeloma revealed by whole genome and transcriptome sequencing

Abstract Background: Advanced sequencing technologies have contributed to identifying genomic and transcriptomic features of various types of tumors. Despite several sequencing data from multiple myeloma (MM) being generated, understanding various properties of MM remained insufficient. Methods: The patients confirmed with MM by bone marrow examination were analyzed in the study. Myeloma cells were enriched from the bone marrow aspirates of the patients using CD138. Whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) were performed for isolated myeloma cells. Mutational signatures were analyzed using somatic mutations detected by WGS. Results: A total of 37 MM patients (median age 67) were enrolled in this study. WGS revealed somatic mutations of an average of 8872 single nucleotide variants (SNVs) and 934 insertions and deletions (Indels) from the patients, respectively. By the analysis of structural variants, 7 chromothripsis and 4 chromoplexy patients were identified. Mutational signature analysis showed that single base substitution (SBS) 9 signature largely varied between patients. SBS9 signature high patients were mostly diagnosed with non-IgG/non-IgA heavy chain and lambda light chain type myeloma. Notably, IgD myeloma patients were solely detected in the SBS9 signature high group compared to the low group (p = 0.001). Gene expression patterns obtained from WTS were well-divided into SBS9 signature high and low groups, and analysis of differentially expressed genes (DEG) between SBS9 signature high and low groups implied IgD myeloma features including high expression of IGHD. As a validation test, we tried mutational signature analysis on whole exome sequencing (WES) data of 784 MM patients from the MMRF database. Of the 38 patients with more than 500 exonic mutations, only the SBS9 signature high group had IgD myeloma candidates, whereas the SBS9 signature low group had no IgD myeloma candidate (p = 0.014). Conclusions: DNA and RNA sequencing of myeloma patients could classify specific subtypes of disease categories. Further study for SBS 9 signature high and IgD myeloma is needed to discover the underlying mechanism of distinct features and to find out their clinical implication. Citation Format: Sheehyun Kim, Hyundong Yoon, Youngil Koh, Sung-Soo Yoon. Distinct subtype of multiple myeloma revealed by whole genome and transcriptome sequencing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6073.

Effects of Probiotics Supplementation on the Intestinal Metabolites, Muscle Fiber Properties, and Meat Quality of Sunit Lamb.

The development of animal husbandry is closely related to the meat quality of small ruminants. Intestinal metabolites and the muscle fiber types of lambs are important factors that affect their meat quality, but few studies have examined the regulation of the "intestinal muscle axis" by probiotics. In this study, 12 Sunit lambs were divided into a control group (C) and a probiotics group (P). The gene expressions of the myosin heavy chain, metabolic enzyme activity, and short-chain fatty acids in the intestines were analyzed using gas chromatography-mass spectrometry (GC-MS) and quantitative real-time PCR. The results showed that levels of propionic acid and butyric acid in the intestines of group P were significantly higher than in group C (p < 0.05). In addition, probiotics increased the number and area ratio of type I muscle fibers. They also increased the mRNA expression of MyHC IIA and the activity of malate dehydrogenase (MDH) and succinate dehydrogenase (SDH). Propionic acid was negatively correlated with the number ratio of type IIB muscle fibers. Butyric acid was found to be significantly positively correlated with the number ratio of type IIA muscle fibers. Cooking loss, pH24h, and shear force decreased significantly in group P. In conclusion, intestinal metabolites (SCFAs) altered the activity of oxidative-myofibril-metabolizing enzymes and the expression of myosin heavy-chain type IIA, reduced the meat shear values, and improved meat tenderness. This study provides a new basis for improving the production and meat quality of small ruminants.

Resistance training restores skeletal muscle atrophy and satellite cell content in an animal model of Alzheimer’s disease

Alzheimer’s disease (AD) is the most common neurodegenerative disease, and numerous recent findings suggest that several pathologic signs, including loss of muscle strength and mass, are also detected in these patients. In the present study, we evaluated muscle cross-sectional area (CSA), myonuclear number, satellite cell (SC) content, and myosin heavy chain (MyHC) types in an animal model of AD and examined the possible role of resistance training in controlling skeletal muscle size in this disease. Fifty-eight male rats were randomly divided into four groups: healthy-control (H-C), healthy-exercise (H-Ex), Alzheimer-control (A-C), and Alzheimer-exercise (A-Ex). AD was induced by the single injection of 1–42 amyloid into the CA1 region of the hippocampus (1 μl/site). The rats in H-Ex and A-Ex groups performed a 5-week resistance training period (17 sessions). The results indicated that AD induces significant skeletal muscle atrophy and reduces the myonuclear number and SC content in gastrocnemius muscle in both whole muscle cross-sections and isolated myofibers. Interestingly, we did not find any significant differences in the different MyHC distributions of AD animals compared with controls, while resistance training significantly increased the CSA of MyHC IIb fibers in both AD and healthy animals. Altogether, these observations suggest that the skeletal muscle of AD animals are more prone to atrophy and loss of myonuclear number and satellite cell content, while resistance training successfully restores these impairments.

Serological proteome analysis identifies crustacean myosin heavy chain type 1 protein and house dust mite Der p 14 as cross-reacting allergens.

Allergies to house dust mite (HDM) and to crustaceans are clinically and pathogenically linked. Several homologous allergenic proteins have been identified, among which tropomyosin is the prototype, expressing epitopes endowed with variable levels of immunoglobulin E (IgE) cross-reactivity. Component-resolved diagnosis (CRD) does not allow a thorough characterization of all relevant IgE reactivities to these allergen sources. We studied 1 patient allergic to shrimp with positive skin prick test to HDM and negative scores for IgE to HDM allergen components routinely used in CRD (group 1 and 2 allergens, Der p 23 and tropomyosin). In order to identify the allergen(s) involved in IgE reactivity, we used serological proteome analysis (SERPA), which utilizes two-dimensional gel electrophoresis (2DE), immunoblotting and mass spectrometry (MS). The identified allergenic proteins were tested with sera from 20 crustacean-allergic patients and 19 grass-allergic patients serving as controls. Der p 14 and myosin heavy chain type 1 (MHC1) were identified as the components recognized by patient's IgE in the proteome of Dermatophagoides pteronyssinus and Penaeus monodon, respectively. The MHC1 protein shows about 30% sequence identity with Der p 14 in specific domains, and cross-reactivity against epitopes shared by the 2 proteins was demonstrated by reduced reactivity to shrimp extract following pre-incubation with Der p 14. Serum IgE from 5 out of 20 patients allergic to crustaceans reacted with MHC1, compared to none among 19 controls (p < 0.05). We identified MHC1 as a relevant allergic component in the proteome of Penaeus monodon, the prototypic allergen source used in diagnosis of allergy to crustaceans. Our data demonstrate MHC1 cross-reactivity between MHC1 and Der p 14 from Dermatophagoides pteronyssinus.

Semi-automated technique for bovine skeletal muscle fiber cross-sectional area and myosin heavy chain determination.

Myosin heavy chain (MyHC) type and muscle fiber size are informative but time-consuming variables of interest for livestock growth, muscle biology, and meat science. The objective of this study was to validate a semi-automated protocol for determining MyHC type and size of muscle fibers. Muscle fibers obtained from the longissimus and semitendinosus of fed beef carcasses were embedded and frozen within 45 min of harvest. Immunohistochemistry was used to distinguish MyHC type I, IIA, and IIX proteins, dystrophin, and nuclei in transverse sections of frozen muscle samples. Stained muscle cross sections were imaged and analyzed using two workflows: 1) Nikon workflow which used Nikon Eclipse inverted microscope and NIS Elements software and 2) Cytation5 workflow consisting of Agilent BioTek Cytation5 imaging reader and Gen5 software. With the Cytation5 workflow, approximately six times more muscle fibers were evaluated compared to the Nikon workflow within both the longissimus (P < 0.01; 768 vs. 129 fibers evaluated) and semitendinosus (P < 0.01; 593 vs. 96 fibers evaluated). Combined imaging and analysis took approximately 1 h per sample with the Nikon workflow and 10 min with the Cytation5 workflow. When muscle fibers were evaluated by the objective thresholds of the Cytation5 workflow, a greater proportion of fibers were classified as glycolytic MyHC types, regardless of muscle (P < 0.01). Overall mean myofiber cross-sectional area was 14% smaller (P < 0.01; 3,248 vs. 3,780) when determined by Cytation5 workflow than when determined by Nikon workflow. Regardless, Pearson correlation of mean muscle fiber cross-sectional areas determined by Nikon and Cytation5 workflows was 0.73 (P < 0.01). In both workflows cross-sectional area of MyHC type I fibers was the smallest and area of MyHC type IIX fibers was the largest. These results validated the Cytation5 workflow as an efficient and biologically relevant tool to expedite data capture of muscle fiber characteristics while using objective thresholds for muscle fiber classification.

Effects of the F94L myostatin gene mutation in beef × dairy crossed cattle on muscle fiber type, live performance, carcass characteristics, and boxed beef and retail cut yields.

Producer live performance data and carcasses from steers (n = 116) resulting from the mating of four Limousin/Angus sires heterozygous for the F94L myostatin mutation to Jersey/Holstein dams were utilized to evaluate the effects of one copy of the F94L allele on live performance, carcass traits and USDA grades, and boxed beef and retail yields. Slaughter data were collected at time of harvest and carcass data were collected 48 hours postmortem. One side from each of the 58 carcasses was fabricated into boxed beef and retail cuts by experienced lab personnel 5-8 d postmortem. One copy of the F94L allele did not affect gestation length, birth weight, percentage of unassisted births, feedlot average daily gain, live weight at harvest, hot carcass weight, or dressing percentage (P > 0.05). Muscle fiber analysis indicated that the increase in muscularity by the F94L allele in the semitendinosus and longissimus was likely due to hyperplasia as there was a 19% increase in the quantity of myosin heavy chain type IIA and IIX fibers in the semitendinosus (P < 0.05) with no effect on muscle fiber size (P > 0.05). Carcasses from steers with one F94L allele had larger ribeye areas (99.2 vs. 92.3 sq.cm.), greater ribeye width:length ratios (0.498 vs. 0.479), lower USDA yield grades (2.21 vs. 2.66), and lower marbling scores (438 vs. 480) (P < 0.05). Additionally, for boxed beef yields, one F94L allele, vs. zero F94L alleles, increased (P < 0.05) 85/15 trimmings (+0.59%), top round (+0.28%), strip loin (+0.12%), eye round (+0.11%), tenderloin (+0.07%), boneless foreshank (+0.07%), cap/wedge (+0.06%), and tri-tip (+0.04%). Overall, carcasses from steers with one F94L allele had a greater boxed beef yield (+1.06%), boxed beef plus 85/15 trimmings yield (+1.65%), and total retail cuts plus ground beef 85/15 yield (+1.78%) than carcasses from steers with zero F94L alleles (P < 0.05). One copy of the F94L allele utilized in beef-on-dairy breeding system had no significant impact on live performance traits but resulted in lower marbling scores and increased muscularity as evidenced through larger, more beef-shaped ribeyes, lower USDA yield grades, and greater carcass cutout yields (both boxed beef and retail yields).

Chronic Fetal Leucine Infusion Increases Rate of Leucine Oxidation but Not of Protein Synthesis in Late Gestation Fetal Sheep

BackgroundLeucine increases protein synthesis rates in postnatal animals and adults. Whether supplemental leucine has similar effects in the fetus has not been determined. ObjectiveTo determine the effect of a chronic leucine infusion on whole-body leucine oxidation and protein metabolic rates, muscle mass, and regulators of muscle protein synthesis in late gestation fetal sheep. MethodsCatheterized fetal sheep at ∼126 d of gestation (term = 147 d) received infusions of saline (CON, n = 11) or leucine (LEU; n = 9) adjusted to increase fetal plasma leucine concentrations by 50%–100% for 9 d. Umbilical substrate net uptake rates and protein metabolic rates were determined using a 1-13C leucine tracer. Myofiber myosin heavy chain (MHC) type and area, expression of amino acid transporters, and abundance of protein synthesis regulators were measured in fetal skeletal muscle. Groups were compared using unpaired t tests. ResultsPlasma leucine concentrations were 75% higher in LEU fetuses compared with CON by the end of the infusion period (P < 0.0001). Umbilical blood flow and uptake rates of most amino acids, lactate, and oxygen were similar between groups. Fetal whole-body leucine oxidation was 90% higher in LEU (P < 0.0005) but protein synthesis and breakdown rates were similar. Fetal and muscle weights and myofiber areas were similar between groups, however, there were fewer MHC type IIa fibers (P < 0.05), greater mRNA expression levels of amino acid transporters (P < 0.01), and a higher abundance of signaling proteins that regulate protein synthesis (P < 0.05) in muscle from LEU fetuses. ConclusionsA direct leucine infusion for 9 d in late gestation fetal sheep does not increase protein synthesis rates but results in higher leucine oxidation rates and fewer glycolytic myofibers. Increasing leucine concentrations in the fetus stimulates its own oxidation but also increases amino acid transporter expression and primes protein synthetic pathways in skeletal muscle.

Slow twitch paraspinal muscle dysregulation in adolescent idiopathic scoliosis exhibiting HIF-2α misexpression.

Adolescent idiopathic scoliosis (AIS) refers to a three-dimensional spinal deformity which has a typical onset during adolescence. In most cases, the cause of the deformity cannot be clearly identified. Unbalanced paraspinal muscle activity in AIS patients was reported and hypoxia was implicated to regulate myogenesis. This study aims to investigate the association between myogenesis/muscle toning and HIF-αs activity in the pathogenesis of AIS. HIF-αs expression was examined by enzyme-linked immunosorbent assay and western blot in paraspinal myoblasts isolated from 18 subjects who underwent deformity correction surgery. QPCR was conducted to measure the gene expression levels of perinatal muscle fiber markers MYH3, MYH8; slow twitch muscle fiber markers MHY7; fast twitch muscle fiber markers MYH4; and myogenic regulatory factors MYF5 and MYOG. Slow and fast twitch muscle fiber composition in concave/convex paraspinal musculature of AIS subjects was evaluated by immunostaining of myosin heavy chain type I (MyHC I) and myosin heavy chain type II (MyHC II). Reduced HIF-2α induction under hypoxia was found in paraspinal myoblast culture of 33% AIS subjects. We detected a suppression of perinatal and slow twitch muscle fiber associated genes, but not fast twitch muscle fiber-associated genes and myogenic regulatory factors in HIF-2α misexpressed AIS myoblasts. Distinct reduction of slow twitch muscle fiber was evidenced in convex paraspinal musculature, suggesting an asymmetric expression of slow twitch muscle fiber in HIF-2α misexpressed AIS patients. This study indicates an association of abnormal HIF-2α expression in paraspinal myoblasts and a disproportionate slow twitch muscle fiber content in the convexity of the curvature in a subset of AIS subjects, suggesting HIF-2α dysregulation as a possible risk factor for AIS. The role of HIF-2α in paraspinal muscle function during spinal growth and its relevance in AIS prognosis warrants further investigation.

Autonomic Nerve Fibers Aberrantly Reinnervate Denervated Facial Muscles and Alter Muscle Fiber Population.

The surgical redirection of efferent neural input to a denervated muscle via a nerve transfer can reestablish neuromuscular control after nerve injuries. The role of autonomic nerve fibers during the process of muscular reinnervation remains largely unknown. Here, we investigated the neurobiological mechanisms behind the spontaneous functional recovery of denervated facial muscles in male rodents. Recovered facial muscles demonstrated an abundance of cholinergic axonal endings establishing functional neuromuscular junctions. The parasympathetic source of the neuronal input was confirmed to be in the pterygopalatine ganglion. Furthermore, the autonomically reinnervated facial muscles underwent a muscle fiber change to a purely intermediate muscle fiber population myosin heavy chain type IIa. Finally, electrophysiological tests revealed that the postganglionic parasympathetic fibers travel to the facial muscles via the sensory infraorbital nerve. Our findings demonstrated expanded neuromuscular plasticity of denervated striated muscles enabling functional recovery via alien autonomic fibers. These findings may further explain the underlying mechanisms of sensory protection implemented to prevent atrophy of a denervated muscle.SIGNIFICANCE STATEMENT Nerve injuries represent significant morbidity and disability for patients. Rewiring motor nerve fibers to other target muscles has shown to be a successful approach in the restoration of motor function. This demonstrates the remarkable capacity of the CNS to adapt to the needs of the neuromuscular system. Yet, the capability of skeletal muscles being reinnervated by nonmotor axons remains largely unknown. Here, we show that under deprivation of original efferent input, the neuromuscular system can undergo functional and morphologic remodeling via autonomic nerve fibers. This may explain neurobiological mechanisms of the sensory protection phenomenon, which is because of parasympathetic reinnervation.

A Novel Missense Variant in Actin Binding Domain of MYH7 Is Associated With Left Ventricular Noncompaction.

Cardiomyopathies are a group of common heart disorders that affect numerous people worldwide. Left ventricular non-compaction (LVNC) is a structural disorder of the ventricular wall, categorized as a type of cardiomyopathy that mostly caused by genetic disorders. Genetic variations are underlying causes of developmental deformation of the heart wall and the resultant contractile insufficiency. Here, we investigated a family with several affected members exhibiting LVNC phenotype. By whole-exome sequencing (WES) of three affected members, we identified a novel heterozygous missense variant (c.1963C>A:p.Leu655Met) in the gene encoding myosin heavy chain 7 (MYH7). This gene is evolutionary conserved among different organisms. We identified MYH7 as a highly enriched myosin, compared to other types of myosin heavy chains, in skeletal and cardiac muscles. Furthermore, MYH7 was among a few classes of MYH in mouse heart that highly expresses from early embryonic to adult stages. In silico predictions showed an altered actin-myosin binding, resulting in weaker binding energy that can cause LVNC. Moreover, CRISPR/Cas9 mediated MYH7 knockout in zebrafish caused impaired cardiovascular development. Altogether, these findings provide the first evidence for involvement of p.Leu655Met missense variant in the incidence of LVNC, most probably through actin-myosin binding defects during ventricular wall morphogenesis.