Duration: 10/01/2025 to 09/30/2030

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

This project describes a fundamental research approach to an important agricultural issue, providing the world a high-quality source of protein. The current membership has grown substantially and is highly qualified for making immediate and unprecedented progress towards the goals outlined in this revised project proposal.

Statement of Issues and Justification

The overall goal of this cooperative, multi-state, multidisciplinary, basic research project is to increase the efficiency and sustainability of meat production in the US and around the globe. Based on most predictions, world-wide meat production must increase nearly 50% by 2050 to meet the demands of the burgeoning world population. Moreover, meat animal production and allied industries contribute some $1.3 trillion to the US economy in 2019 and support 1.8M jobs to the US workforce. As such, meat production is not only necessary to support the health and well-being of a growing global citizenry but is also a vibrant driver of the economy. To increase the efficiency and sustainability of lean muscle accretion, a more comprehensive understanding of those basic biological mechanisms undergirding skeletal muscle tissue is necessary.  Moreover, greater information is needed so data-driven decision-making abilities can be used to optimize further animal growth efficiency.  While we have made great progress towards this goal under the current NC1184 project, participation in the project continues to increase. In fact, over 50 scientists claim membership in NC1184 from over 25 states across the country, many outside the NC region. Regardless, this heightened level of interest coupled with rejuvenated, new membership has spawned a real enthusiasm for understanding the nuances of animal growth and meat production. This revitalization of investigators and the continued emergence of cutting-edged technologies will lead to increased knowledge of high-quality meat production. The subgoal of this multi-state, multidisciplinary, basic research project is to utilize these tools to elucidate the molecular and cellular processes that control skeletal muscle growth and function; thereby, providing opportunities to increase the efficiency of high-quality lean meat production in animals.

This renewal describes the efforts from different Agricultural Experiment Stations that will characterize various aspects of the molecular and cellular mechanisms that regulate skeletal muscle growth and function.  Salient points that support continuation of this important, fundamental research project are as follows.

1. Meat is a significant source of protein for human consumption. Meeting the needs of a growing population will require significant growth in meat production world-wide. Though some growth will be realized by increasing animal units, greater outputs must be realized per animal unit. This will require a more thorough understanding of those mechanisms responsible for the efficiency of protein accretion.
2. In 2024, the totality of the meat animal industry was responsible for nearly 6% of the GDP and over 10% of the total employment in the US. Ensuring the viability of this industry in times of elevated energy costs and concerns for the environment will require a better understanding of those inputs, and outputs, required by optimal meat animal production.
3. The project continues to relate directly to the national agricultural research priorities set forth in the NIFA Strategic Plan (2022 – 2026). Strategic Goal 2: Ensure America’s Agricultural System is Equitable, Resilient and Prosperous.
4. One of the NIFA strategic goals is foundational, and applied research ensures that strategies to manage national and global challenges in food, nutrition and agriculture are rooted in robust peer-reviewed evidence and theories, areas strongly supported by NC1184.
5. The NC1184 Committee continues to be highly productive. Many papers emanating from this group of scientists are published in top tier journals often reserved for reporting fundamental knowledge. This reflects well on the aptitude of the membership and the basic nature of their approach in creation of new knowledge.
6. The NC1184 project is both a multi-state and a multidisciplinary project, involving the effort of investigators from over 25 different State Agricultural Experiment Stations. The Principal Investigators represent a variety of basic science disciplines that complement each other and provide the expertise necessary to complete the objectives.
7. The project continues to enjoy a strong collaborative environment. During the current project, there have been exchanges of data, scientists, students, samples, cell lines, reagents, etc. In addition, there has been vast knowledge sharing of techniques and on occasion, use of equipment only available at some stations. The fruits of these collaborations are best illustrated by the number publications jointly co-authored.
8. Members of the NC-1184 committee have been and will continue to be highly successful in obtaining outside support to fund this research. Funding from the USDA NRICGP (AFRI) Program, NIH, NSF, health-related granting agencies, and industry sources are used to maintain a high level of productivity.

 

 

Related, Current and Previous Work

While there were several active multistate projects that ‘show-up’ when using the key words muscle, muscle growth, and muscle growth and development, all focus on other disciplines or on the end-product of muscle, meat. Some have overlap with the current project but that is expected as muscle represents some 40% of the mass of most animals and forms the basis for meat production. Moreover, muscle tissue has the capacity to regenerate and as such, contains stem or stem-like cells which makes it an ideal tissue for studying global cellular programing. Finally, muscle competes with adipose tissue for nutrients, so it is often considered when studying adipose tissue growth and development. In fact, some current members of the NC1184 project are also members of the NCC210 multistate entitled Regulation of Adipose Tissue Accretion in Meat-Producing Animals, though the future of that project is currently unknown.  Regardless, cross fertilization and multiplicity of projects is highly valuable and helps foster interdisciplinary thinking and problem-solving.

Below are thumbnail sketches of scientific areas of inquiry that may hold potential for increasing meat animal production worldwide.

MYOGENESIS: Muscle fibers are multi-nucleated muscle cells that are created by the fusion of muscle precursors or myoblasts that represent the earliest stages of skeletal muscle development (Chal and Pourquié, 2017). Postnatal muscle growth is largely predicated on the amount of muscle tissue developed during this process.  Embryonic myogenesis is regulated by a variety of factors including maternal nutrition and other physiological conditions.  Maternal over-nutrition and obesity impair myogenesis exerting long-term effects on skeletal muscle properties in offspring.  Understanding the molecular cues responsible for controlling this entire process may be exploited as a means of increasing animal productivity and meat production. 

 

EXTRACELLULAR MATRIX: Connective tissue in muscle is known as extracellular matrix (ECM) and is an active participant in the control of muscle growth and development and plays a significant role in the overall palatability of this tissue when it is transformed into meat (Velleman, 2000). Anomalies in ECM are thought to be partially responsible for wooden breast syndrome that results in widespread loss of muscle fibers in chicken breast muscle that ultimately is replaced by connective tissue. This costly meat quality defect plaguing the global commercial poultry industry manifests in the fastest-growing, heaviest-muscled broiler chickens and costs the commercial US broiler producers at least $200 million per year. Identifying key features of how the collective ECM facilitates, maintains and contributes to the overall eating satisfaction of meat products will dramatically affect overall productivity of production agriculture. 

GROWTH FACTORS AND HORMONES: Myogenesis and the subsequent hypertrophy of muscle are regulated by growth factors (Hernandez-Hernandez et al., 2017). A number of these growth factors modulate various steps in myogenesis. Moreover, growth factors modulate animal growth when steroid-based implants are used in the feedlot cattle.  These implants have revolutionized beef production in the US and are ultimately responsible for much of our recent gains in animal growth rate and increased feed efficiencies.  Further delineation of this biology will result in greater augmentation of animal growth that will increase meat production. In addition, long-chain fatty acids also play a role in skeletal muscle plasticity and repair but mechanisms responsible for this growth promotion are largely unknown but may hold value to augmenting muscle growth.

SATELLITE AND STEM CELL BIOLOGY:  Satellite cells (SC) are undifferentiated stem-like cells held in a quiescent state until needed by adjacent muscle cells for regeneration or growth (Moss and Leblond, 1971). Mechanisms controlling their quiescence, activation and subsequent replication and fusion into growing muscle fibers (cells) are important to animal agriculture, as incorporation of these cells are considered a rate-limiting step to support muscle hypertrophy. Broiler chickens affected with WB may also have abnormalities in their muscle satellite/stem cell populations. Identifying mechanisms that increase satellite cell incorporation may lead to increased lean tissue accretion.  Satellite cells are also a mixture of multiple subtypes with each likely playing distinct roles. To facilitate muscle growth, an improved understanding of how these cells differ from one another, how their actions are initiated following metabolic challenges and how nutrition can regulate the timing of their activation is required. 

Cellular agriculture aims to provide a source of animal-free meat products, which would decrease worldwide nutritional dependency on animal husbandry and reduce the impact of the industry on Earth's climate. However, while some studies have produced lab-based meat on a small scale, scalability of this approach requires significant optimization of methodologies to ensure its viability on an industrial scale. One of the methodological promises of in vitro meat production is the application of cell suspension-based bioreactors. Hence, complex transcriptomic comparison of adherent undifferentiated, differentiated and suspension-cultured myosatellite cells may allow for more efficient cellular agriculture development.

CELLULAR ENERGY SENSORS: Muscle cells, like all cells must be able to monitor nutrient availability in their respective niche. Understanding how cells sense these changes may lead to development of different management strategies for optimizing animal growth efficiency or possibly how to overcome more effectively adverse stresses to the homeostatic controls of the tissue.  One such ‘fuel gauge’ in skeletal muscle is AMP kinase (AMPK). Once activated by cellular levels of AMP, this kinase activates mitochondrial biogenesis in response to a decrease in energy availability. At the same time, however, this kinase blunts energy using pathways to conserve energy (Steinberg and Hardie, 2023).  One such anabolic pathway curtailed is protein synthesis, the foundation for muscle growth.  Defining the exact role of how muscle cells respond to changes in energy availability will aid development of more effective feeding regimes. 

GENETICS: Genomic information is now routinely used to predict the complex phenotype of an individual with a specific genotype. As many advances in biology have been driven by genome sequence information, animal genomics is on the cusp of a new era in predictive biology (Rexroad et al., 2019). An excellent, highly contiguous porcine genome assembly has recently been completed, and as costs continue to decrease, availability of genotype information is no longer the bottleneck. The next challenge is to be able to read the complexity of these instructions across animals and to predict the resulting phenotypes. Identifying these genes and regulatory elements in skeletal muscle will increase our ability to annotate functional regions related to growth and other important phenotypes.

MITOCHONDRIAL FUNCTION: The mitochondria form the basis for nutrient utilization in skeletal muscle cells.  Mitochondria are not just oblong, individual organelles located in the subsarcolemmal space but rather these ATP generating powerhouses are intricately connected throughout the innermost workings of the muscle fiber and intimately associated with the sarcoplasmic reticulum.  As such, these organelles likely impact calcium homeostasis muscle.  Moreover, these electrostatic compartments create and dispense of reactive oxygen species, the balance of which can have a profound impact on mitochondria function. When mitochondria are damaged, they are cleared through autophagy (Leduc-Gaudet et al., 2021). Developing a better understanding of mitochondrial function is important for improving efficiency of meat production. Further, free radical injury has been linked to problems in meat production; however, pre-harvest mitochondria markers animals have yet to be examined as a predictor of meat quality at harvest.

Mitochondrial respiration via oxidative phosphorylation (OXPHOS) is a primary mechanism for generating ATP. Disruptions or reductions in OXPHOS can slow metabolism and impede non-essential activities such as growth (Leduc-Gaudet et al., 2021). Previous work has demonstrated that poor maternal diet during gestation, both increased and reduced intake, can impact mitochondrial number and/or function. Thus, mitochondria appear to be sensitive to gestational insults, which may impact pre- and post-natal growth.

HEAT STRESS – Thermic stresses continue to jeopardize animal production, though underlying mechanisms are poorly understood (Gonzalez-Rivas et al., 2019).  Etiological interventions are needed to counter lost production efficiency, but development is limited by our poor understanding of the metabolic modifications caused by heat stress (HS) and is further compounded by a poor appreciation of the role of sex on these outcomes. Given the sheer mass of skeletal muscle, this tissue is of additional interest as it may significantly impact systemic and metabolic pathophysiological responses to HS. Bos indicus cattle breeds, such as Brahman, excel in tropical and subtropical environments but are discounted compared to other types of beef cattle. Understanding the mechanisms responsible for differences in heat tolerance and animal type may lead to improvements in cattle growth efficiency in adverse regions of the world. Moreover, the full spectrum of cell signaling pathways and regulatory mechanisms involved in successful adaptation to environmental stress are not completely understood and specific mechanisms by which muscle cells regulate protein synthesis in response to environmental stress are not fully understood. Identifying the mechanisms and factors involved in this selectivity can provide insights into how the synthesis of specific proteins is prioritized under environmental stress conditions.

COMPENSATORY OR ‘CATCH-UP’ GROWTH: Compensatory growth, or catch-up growth, refers to the accelerated growth observed in animals following a period of growth retardation caused by factors such as nutritional restriction, impaired homeostasis, or metabolic changes (Hornick et al., 2000). However, the practical application of compensatory growth is challenging due to its complexity and the underlying molecular mechanisms driving compensatory growth remain poorly understood. Modeling this phenomenon in pigs provides a useful model for studying the mechanisms responsible for increased growth efficiencies during periods of catch-up growth.

Differences in offspring growth and development depend on maternal parity (i.e., offspring birth order), offspring sex, litter size, and maternal environment. However, there is no clear consensus for the effects of maternal parity on growth outcomes. Primiparous animals tend to have smaller offspring than subsequent pregnancies or multiparous animals (Hyatt et al., 2009). Interestingly, offspring of primiparous ewes demonstrate “catch-up” growth and are of similar size to offspring of multiparous dams within several months of age. Catch-up growth generally results in greater adiposity and it is likely that poor maternal diet during gestation can further exacerbate the effects of parity. However, because multigenerational studies commonly use primiparous F1 dams to produce the F2 generation, it is unknown whether the first born F2 offspring are more or less affected by the diet of the parental generation than subsequent offspring. Thus, it is critical to determine the independent, but potentially additive, effects of parity and maternal nutrition on offspring growth and development.

Mammalian target of rapamycin (mTOR) and other associated kinases are commonly activated to stimulate protein accretion and muscle growth (Liu and Sabatini, 2020). At the same time, protein degradation is equally complex.  Targeting individual proteins within this highly organized and filamentous structure is first accomplished by the calpain system or caspases followed by degradation by the ubiquitin proteasome pathway using a family of ubiquitin-based ligases, or by the autophagy-lysosomal pathway, which involves the creation of autophagosomes and fusion to lysosomes.  The entirety of the aforementioned is a highly dynamic and ever-changing field of inquiry. The potential ramifications of better understanding the control of protein accretion for animal agriculture are monumental.

Micronutrients, such as vitamins, are a critical component of an animal’s diet, but are often overlooked in production livestock animal nutrition but are essential for growth (Messersmith et al., 2021). The role that vitamins play in skeletal muscle growth has not been thoroughly explored.

ADIPOSE TISSUE: While advances in animal growth research have improved skeletal muscle growth and beef cattle production efficiency, producing high-quality beef remains a paramount to consumer demand. A recent national survey of beef producers revealed that only 3.8% of carcasses were graded prime, with less than 4% achieving a marbling score of slightly abundant or above. Marbling, resulting from intramuscular adipose tissue (IMAT) accumulation is essential for the desired meat beef. Differentiation of adipoblasts is responsible for the formation of intramuscular adipose tissue in mice. These progenitor cells are composed of individual cells with the ability to undergo adipogenesis and generate adipocytes in the skeletal muscle (Dodson et al., 2015). Adipocyte progenitor cells also have fibrogenic capacity and are able to differentiate into fibroblasts/myofibroblasts. However, the presence of collagenous intramuscular connective tissue (IMCT), which contributes to background toughness, can be detrimental to beef palatability.  This underscores the urgent need for research into the mechanisms controlling the development of IMAT and IMCT to develop strategies that optimize marbling deposition while limiting IMCT. Current methods to increase IMAT largely depend on prolonged high-concentrate feeding, which often leads to the excessive accumulation of subcutaneous and visceral adipose tissues. This not only reduces feed efficiency and yield but also diminishes profit margins. IMAT is the last depot to develop, making it particularly challenging to promote its accumulation selectively. Understanding the molecular control of progenitor cell differentiation may lead to our understanding of the differences in highly marbled and tender meat.

RESIDUAL FEED INTAKE:  Feed costs account for 60-70% of total production expenses in most swine production operations (Patience et al., 2015). Therefore, the molecular pathways governing protein synthesis and degradation in skeletal muscle have emerged as crucial determinants of feed efficiency, explaining approximately 37% of the variation in Residual Feed Intake (RFI). Notably, mitochondrial function significantly influences these molecular pathways and subsequent feed efficiency outcomes. Since its introduction, RFI has become a fundamental metric for assessing production efficiency. Studies have provided significant insights into muscle fiber composition and nutrient utilization pathways, particularly highlighting RFI's inverse correlation with molecular pathways controlling lean tissue accretion and positive correlation with pathways regulating fat deposition. Understanding mitochondrial function as related to energy production and substrate utilization represents a key mechanistic link between cellular metabolism and feed efficiency.

MEAT QUALITY: In response to increasing consumer demand for high-quality meat, it is necessary to understand the factors controlling the development of meat quality attributes. Meat quality is largely determined through changes in the biochemical processes occurring during muscle’s transformation into meat (England et al., 2013). Because mitochondrial respiration is impeded by the lack of oxygen in postmortem muscle, these organelles are usually considered irrelevant to the development of meat quality. However, recent research demonstrates that mitochondria impact postmortem metabolism and meat quality during storage. Delineating the role of mitochondria in modulating the rate of postmortem pH decline and meat color stability during storage could improve the quality of fresh meat.

Objectives

1. Characterize the molecular mechanisms controlling skeletal muscle tissue growth, development and composition.
   
2. Characterize the cellular mechanisms that regulate skeletal muscle metabolism.
   
3. Characterize mechanisms of protein synthesis and degradation in skeletal muscle.
   

Methods

Objective 1: Characterize the molecular mechanisms controlling skeletal muscle tissue growth, development and composition.

The Louisiana and Washington stations will collaborate and take muscle biopsies from Wagyu and Brahman cattle at different growth stages. Single-nucleus multiomic (RNA-seq + ATAC-seq) will be performed to profile the gene expression and chromatin accessibility of different cell types at the single-nucleus level. Differential gene expression and chromatin accessibility between the two breeds in individual cell types. Key enhancers and transcription factors responsible for the gene expression and chromatin accessibility differences will be identified through joint analysis of the two modalities, which will construct a GRN with improved accuracy. Identified enhancers and transcription factors will be validated in vitro, with a focus on those regulating adipogenesis and fibrogenesis-related genes.

Similarly, the Connecticut station will study the impact of parity on offspring muscle development using ewes fed control (100%), over (140%), or restricted (60%) diets from day 30 of gestation through parturition. To determine the basal inflammatory status of the offspring in response to poor maternal nutrition, blood will be collected from F2 lambs at 3 mo of age for gene expression analysis of inflammatory markers. Whole blood will be added to a Tempus blood RNA tube to precipitate and stabilize RNA from white blood cells. RNA will be isolated from blood and reverse transcribed to generate cDNA. Expression of differentially expressed genes from F1 and F2 offspring will be analyzed by real-time PCR.  To understand the effects of poor maternal nutrition during gestation on offspring mitochondrial function, fetal liver and muscle samples collected from offspring of control-, over-, and restricted-fed ewes, and concentrations of key mitochondrial metabolites, substrates and co-factors will be evaluated. Real-time PCR will be performed to quantify expression of genes related to mitochondrial biogenesis and mitochondrial function.

The Utah station will isolate primary bovine satellite cells and grow them in culture and study their myogenic properties in response to different stimuli (trace minerals, hormones found in anabolic implants, amino acid derivatives, cells from cattle of different breed types, etc.).  They will also assess how these stimuli impact growth of skeletal muscle by assessing proliferation, differentiation, and protein synthesis. In addition, they will also measure abundance of both mRNA and protein that relate to skeletal muscle growth and breakdown, immune response, oxidative stress, and other factors that are related to skeletal muscle growth. Finally, this group will analyze the transcriptome, proteome, and metabolome of skeletal muscle collected from beef cattle that differ in growth rates during the feedlot phase of production to understand the molecular mechanisms through which skeletal muscle grows.

Using in vivo and in vitro methods, the Georgia station will study the effects the silent mating type information regulation 2 homolog 1 (SIRT-1) pathway and mitochondria biogenesis on primary and secondary muscle fiber development and growth. They will also conduct post-hatch studies to determine the effects these embryonic manipulations have on growth performance and meat quality of different muscles in poultry carcass.

Using molecular, cellular, and biochemical tools, the Virginia station will study rapid-fusing SC progenitors and self-renewing SC precursors.  Dietary supplements will be used to alter skeletal muscle composition, and the impact of glycolytic and oxidative metabolism will be examined on SC bioactivity. In addition, systemic and local factors that affect SC biology including steroids, growth factors, and gut-derived factors will be examined in the regulation of skeletal muscle hypertrophy.  

The Michigan, South Dakota, and Wisconsin stations will utilize in vitro primary cell cultures, establishing lines from 80 animals with diverse genetic backgrounds and performing screening via flow cytometry for biological replication. They will test various scenarios involving thermal conditions and different animal growth stages. Findings at the cellular level are being and will be further confirmed by animal trials in a thermoregulated physiological room. In both in vitro and in vivo approaches, analysis includes transcriptomics, gene and protein abundance, and immunohistochemistry.

The Alabama station will use a combination of in vivo and in vitro approaches to investigate the functionality of these essential stem cells in WB-affected broiler chickens. Specifically, they will assess the extent to which satellite cell function is impaired in WB-affected birds and determine whether the aberrant function of the satellite cell populations is due to an issue with the SC themselves and/or their environment.  In addition, investigators will assess the extent to which hatching egg incubation conditions are negatively impacting the development of chicks such that they develop the costly WB myopathy. They will use a combination of experimental approaches including cryohistology, immunofluorescence staining, and digital image analysis as well as functional proteomics via quantitative protein sequencing to investigate the functionality of these essential stem cells in broiler chickens and determine if improper incubation temperatures during both early and late stage hatching egg incubation are contributing to the development of the WB meat quality defect. 

The Hawaii station will study the relationship between vitamin A, E, and B12 in proliferating ovine satellite cells.  Weaned wethers will be used for separation of satellite cells. The effects of vitamin A, E and B12 on muscle cell differentiation will be determined.

The Kansas station will study mitochondria as a regulator of porcine myogenesis by isolating satellite cells from young pigs to knockout genes in the electron transport chain using Crispr-Cas9. Knockout cells will then be evaluated for proliferative and differentiation capacity. To investigate the mitochondrial signaling mediating porcine myogenesis, they will assess ATP-independent functions of mitochondria such as metabolite utilization, calcium handling, mitochondrial dynamics, and amino acid metabolism.

Finally, the North Carolina station will use in vitro techniques to gain a better understanding of myogenesis to produce meat in bioreactors. They will use a series of studies regarding developing novel stem cell lines for cultured meat production, a better understanding of in vitro metabolism and novel scaffolding to improve in vitro meat production.  Modern next-generation sequencing (RNA-seq) will be used to determine the levels of transcripts in the cultures' cell samples. Then, differential expression and pathway analyses will be performed using bioinformatical methods. Moreover, telomerase activity is highly correlated to the proliferation capacity and immortality of cells. To evaluate the possibility of continuous culture, myoblasts will be isolated from the pectoralis thoracicus muscle of newborn turkeys and maintained in 2D (adherence based) and suspension cultures. Telomerase activity will be evaluated in all types of obtained cultures. Overall results will show whether satellite cells and myoblasts are able to grow in suspension without losing their myogenic properties.

Objective 2: Characterize the cellular mechanisms that regulate skeletal muscle metabolism.

The Texas station will collaborate with the Virginia, Iowa and Nebraska stations to use functional mitochondrial analyses to understand changes in muscle tissue energy metabolism using the Oroboros O2k. Recent addition of fluorescent modules to many stations will allow more in-depth analyses of ATP production potential, ROS production, membrane potential, and the roles of calcium and magnesium in these areas of muscle physiology. We analyze antioxidant and inflammatory mediators, linking thermal environment and physiological stress with bioenergetics, which impact production and performance, and therefore, sustainability of multiple animal systems. Finally, they will use metabolomics and proteomics to better understand the role of metabolism in production and performance.

The Utah station will use both in vitro and in vivo techniques to understand how the hormones found in anabolic implants alter metabolism of skeletal muscle by using and integrating several different -omics technologies (proteomics, transcriptomics, and metabolomics).

The Kansas station will study target genes in regulating nutrient utilization in skeletal muscle will be determined by generating knockout and overexpressing myotubes in vitro. Myotubes will be assessed for changes in the abundance and activity of enzymes involved in glycolysis and mitochondrial metabolism as well as altered nutrient preference through stable isotope tracing experiments.

The Indiana station will study the role of arachidonic acid release on muscle injury using mice as a model.  Briefly, 1.2% tibialis anterior (TA) muscle injury will be induced by BaCl₂.  Bright field (H & E and picrosirius red) and fluorescent images will be captured and evaluated for total muscle fiber number, myofiber cross-sectional area, muscle fiber type, and regenerating (centrally nucleated) myofiber counts will be analyzed.  Finally, isolated satellite cells will be FACS sorted and induced to differentiate into myotubes. Fluorescent images will be captured and morphological analysis of proliferation/viability (total DAPI⁺ nuclei per field of view), the differentiation index (% DAPI⁺ nuclei within MHC⁺ cytoplasm), fusion index (% nuclei within MHC⁺ multinucleated cells), and mean myotube diameter will be examined.

The New Jersey station will delineate circadian rhythms in the skeletal muscle metabolome during dietary amino acid insufficiency. This study will employ the use of time restricted feeding on mice that are either genetically intact (wild type) or carrying a global deletion in the nutrient sensing kinase called GCN2. Metabolomics on serum and tissues will be conducted every 4 h over 24 h in mice fed diets that are sufficient or insufficient in an essential amino acid. The group will also examine the impact of branched chain amino acid (BCAA) deficiency on muscle metabolism. In this project, they will use stable isotopes to measure metabolism in skeletal muscle and other tissues of intact mice and mice lacking the gene Bckdk that are consuming normal versus low protein diets. Animals with defective or deleted Bckdk have reduced BCAA concentrations in blood and skeletal muscle and show reduced muscle growth and impaired performance. Whole body metabolism and physical activity will be assessed using a Comprehensive Lab Animal Monitoring System.  Biochemical endpoint measurements will be complemented by histochemical approaches and electron microscopy to visualize subcellular processes.

The Utah station will also determine the role of mitochondria in the transformation of muscle to meat and the ultimate quality of meat.  Market-weight steers will be harvested, and the longissimus and psoas major muscles will be collected 24 h postmortem. Each muscle will be fabricated into eight steaks. Steaks will be randomly injected with either mitoTEMPO (mitochondrial-targeted ROS scavenger) or saline (control). One steak from each treatment will be used immediately, and the remaining steaks will be packaged and placed in a display case maintained at 4 °C for 24 h, 168 h, or 336 h. At the end of each display period, pH, color, myoglobin redox forms, mitochondrial efficiency, ROS level, mPTP opening, cytochrome c level, and lipid oxidation will be evaluated.  Further, differences in metabolomic profiles of porcine skeletal muscles known to vary in mitochondrial content and rates of pH decline (masseter, longissimus, and semimembranosus) at 0, 60, 120, 480, and 1440 min postmortem will be assessed. Finally, they will examine differences in metabolomic profiles of 3 different bovine muscles known to vary in mitochondrial content and color stability (psoas major, longissimus, and semimembranosus) at 0, 1, 7, or 14 d.

Objective 3: Characterize mechanisms of protein assembly and degradation in skeletal muscle.

The Wisconsin station will explore the molecular mechanisms underlying compensatory growth in pigs. Studies will use an economically efficient pig model that undergoes compensatory growth after a period of attenuated growth induced by consumption of diets containing 2% ammonium chloride. Efforts will focus specifically on the role of RNA binding motif protein 20 (RBM20) and its regulation of titin isoform switching, which plays a critical role in muscle hypertrophy during recovery phases. Proteins of the muscle splicesome will be characterized and potential changes in stoichiometry during growth and development will be examined using Western blotting, mass spectrometry, and immunofluorescence methods. Primary emphasis will be placed on the SR class of splicing factors.

Similarly, the Virginia station will investigate molecular mechanisms controlling porcine muscle growth and feed efficiency using surgical catheterization techniques with sophisticated amino acid analysis methods. Building on established substrate oxidation protocols and validated RFI calculation models, the research design will incorporate proven dietary intervention strategies within a carefully controlled experimental framework. This integrated approach examines the molecular signaling pathways regulating muscle development and metabolism, with particular focus on mitochondrial function and its impact on nutrient utilization efficiency, while enabling systematic evaluation of various nutritional interventions, ultimately providing robust insights into the fundamental mechanisms controlling skeletal muscle growth, differentiation, and their impact on feed efficiency in commercial swine production.

The Utah station will use both in vivo and in vitro methods to assess how different conditions (trace minerals, hormones found in anabolic implants, amino acid derivatives, cells from cattle of different breed types, etc.) impact growth of skeletal muscle by assessing the balance between protein synthesis and degradation. In addition, they will also measure abundance of mRNA, protein, and metabolites to understand the molecular pathways responsible for observed differences in skeletal muscle growth. Similarly, the Alabama station will use in vitro and in vivo models, including a unique bromodeoxyuridine proliferating cell labeling method to understand the impact of various nutritional factors (vitamins, minerals, amino acids, fatty acids, etc.) and management (including egg incubation) strategies on skeletal muscle development and growth in poultry and pigs.

The Texas, Oklahoma and Utah stations will characterize how specific genetic regulatory and signaling mechanisms affect meat quality endpoints. Their overall hypothesis is that physiological response to environmental conditions and stressors is mediated in part by molecular genetic regulators whose function is affected by individual genetic variation. They will also continue to identify candidate genomic, transcriptomic, and proteomic factors that influence overall meat quality phenotypes (tenderness, marbling, and color) and focus on transcriptomic variants that affect meat quality, including messenger RNAs as well as non-coding regulatory transcripts. They intend to identify candidate molecular genetic regulators of the physiological processes that determine skeletal muscle phenotypes, and test hypotheses experimentally through in vitro methods as well as direct sequencing and animal evaluation.

The Kansas station will leverage insulin genetic models that alter sarcomere numbers with protein labeling techniques to visualize nascent protein addition during muscle growth and Transcriptomic approaches will also be used to identify insulin targets that promote sarcomere addition remodeling.

The New Jersey station will study circadian rhythms in the skeletal muscle transcriptome and translatome during dietary amino acid insufficiency. They will employ Ribo-tag mice which are genetically engineered to carry a ribosomal protein gene with a floxed C-terminal exon followed by an identical exon tagged with hemagglutinin (HA). Using antibodies against the HA epitope tag allows for the immunoprecipitation of ribosome bound mRNAs. Subsequent analysis of mRNA abundances by qRT-PCR or RNA-sequencing allows for assessment of both global and gene-specific mRNA translation in skeletal muscle and other relevant tissues. Relevant nutrient sensing and signaling pathways such as the mechanistic target of rapamycin and the integrated stress response will also be analyzed using immunoblotting. The group will also examine the impact of temperature stress on gene-specific translation in muscle of pigs and rodents. To reveal translational control mechanisms in the skeletal muscle of animals during temperature stress, they will conduct sucrose density gradient centrifugation on tissues collected from animals maintained at room temperature or subjected to acute cold or chronic exposure to heat. The optical density is used to infer the translational status of the tissue in a general sense. To analyze the translatome more specifically, total RNA is isolated from all sucrose fractions under the various experimental conditions and transcript abundances are analyzed using RNA sequencing or RT-qPCR.  Biochemical measurements of mRNA and protein levels will be complemented by histochemical approaches and electron microscopy to visualize subcellular processes as necessary. Experiments focused on heat stress mechanisms in porcine skeletal muscle will be conducted in collaboration with the Iowa station.

Measurement of Progress and Results

Outputs

• Research supported by this project will result in education of new researchers equipped to use molecular and cell biology methods to understand the proliferation and differentiation of muscle cells.
• Understand the role of extracellular matrix in controlling lean tissue accretion.
• Discover how growth factors and hormones modulate animal growth.
• Define the role of vitamins and minerals in improving growth efficiencies and subsequently impact animal performance.
• Increased understanding of the role of specific proteases in protein degradation in skeletal muscle.
• Greater understanding of how muscle increased DNA accumulation during growth.
• Appreciate connective tissue cell contributions to high quality meat development.

Outcomes or Projected Impacts

• Publications will be the primary tangible result expected from the project because it focuses on basic research. It is anticipated that utilization of these published results will lead to improved efficiency of lean meat production in domestic livestock and poultry.
• The Committee will sponsor a Symposium on Molecular Mechanisms Regulating Skeletal Muscle Growth and Differentiation. This symposium will provide a mechanism to convey new research information and will increase the visibility of the project.
• Increased understanding of the role of growth factors and cell signaling pathways in regulating proliferation and differentiation of muscle cells may lead to molecular- and cellular biology-based strategies for more efficient production of lean meat which will benefit both producers and consumers. For example, it may be possible to alter fiber type distribution in muscle to increase muscle mass and/or meat quality. Additionally, it may be possible to increase or prolong the proliferative activity of muscle satellite cells so that they provide more DNA to support postnatal muscle fiber growth.
• Elucidation of mechanisms responsible for muscle protein degradation may provide information necessary to utilize molecular- or cell biology-based methods to reduce protein degradation in growing muscle. This will allow more rapid muscle growth without increasing energy requirements.
• Gene expression studies may lead to identification of genes that may be used to produce transgenic animals that more efficiently produce muscle. Additionally, because this project combines the skills of muscle biologists and molecular biologists, collaboration between committee members should result in elucidation of the function in muscle growth and differentiation of proteins produced by identified genes.

Milestones

(2026):Sponsor a symposium at ASAS/ADSA National Meeting on Cellular Mechanisms Regulating Skeletal Muscle Growth and the Impact on Sustainable Agriculture. This will allow us to convey research findings to interested individuals in these organizations.

Projected Participation

View Appendix E: Participation

Outreach Plan

Dissemination of the results of the research will be done via the timely publication of refereed articles in established journals. Because this a basic research project, publications will be the primary tangible result expected from the project. It is anticipated that utilization of these published results will lead to improved efficiency of lean meat production in domestic livestock and poultry. Additionally, the committee will organize a symposium on Cellular Mechanisms Regulating Skeletal Muscle Growth and the Impact on Sustainable Agriculture at the ASAS/ADSA National Meeting that will convey recent research findings to the members of these organizations. 

Organization/Governance

The Technical Committee will consist of at least one representative from each participating unit, appointed as described in the Manual for Cooperative Regional Research, with one representative from each unit designated as the voting member. The North Central Regional Association of Directors will appoint one Director to serve as Administrative Advisor and a representative of the USDA/CSREES will also serve as an Advisor; both will be ex officio member of this committee. The Technical Committee will elect a Chair and a Secretary to serve for a period of one year, and these officers will continue to be voting members. In succeeding years, the Secretary will become Chair, and only a new Secretary will be elected. The Chair, the Secretary, the immediate Past Chair and the Administrative Advisor will serve as the Executive Committee, and they will have the authority to act on behalf of the Technical Committee during the periods between meetings. Because this is a proposal for renewal of the NC-1184 project, the current Chair will serve as Past Chair for the first year of the revised project and the current Secretary will become Chair if the renewal is approved.

The Chair, with approval of the Administrative Advisor, will call yearly meetings of the Technical Committee and interim meetings of the Executive Committee if needed. A report of research results from each unit will be presented orally and in writing at the Technical Committee meetings. The written report should include a list of publications resulting from research related to the project for the year. Reports will be critically reviewed by the Technical Committee and recommendations will be made for future research and coordination of research between units to maximize attainment of the Objectives. The Secretary will prepare minutes and an Annual Report for distribution to Technical Committee members, Directors and Department Chairs of participating State Agricultural Experiment Stations and USDA Laboratories, and the Regional Research Office, CSREES. Because submission of a yearly progress report is essential to assess progress, ensure participation, and prepare the Annual Report, any unit that does not submit a written progress report for two consecutive years will be considered not to be participating and will be dropped from the project.

A station, agency, or institution not currently listed as a participant in the project can petition to join by submitting an addendum to the Methods section of this proposal. This addendum should describe the proposed work and how it contributes to the project. The Administrative Advisor will circulate the addendum to the voting members of the Technical Committee for their consideration and approval. If approved by a majority of the voting members of the Technical Committee, the addendum will be added to the official project outline. 

Literature Cited

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Attachments

Land Grant Participating States/Institutions

AL, AR, GA, HI, IA, IN, KS, MS, NC, NJ, TX, UT, VA, WA, WI

Non Land Grant Participating States/Institutions