ABSTRACT
Zebrafish (Danio rerio) has emerged as a pivotal model organism in vertebrate development research over several decades. Beyond its contributions to developmental biology, zebrafish have increasingly played a crucial role in the field of lipidomics. Lipidomics, a comprehensive analysis of lipids within biological systems, offers profound insights into lipid metabolism and signaling pathways. This chapter explores the zebrafish's unique attributes that make it an ideal candidate for lipidomics studies. With a genome sharing numerous genetic similarities with humans, zebrafish serve as a powerful model for dissecting lipid metabolism and unraveling the complexities of lipid mediator-related diseases. In this chapter, we delve into specific protocols tailored for utilizing zebrafish in lipidomics research and similar investigations. Through a comprehensive exploration of zebrafish as a model organism, this chapter aims to provide researchers with valuable insights and methodologies for advancing lipidomics studies using zebrafish.
Subject(s)
Lipid Metabolism , Lipidomics , Zebrafish , Zebrafish/metabolism , Animals , Lipidomics/methods , Lipids/analysis , Models, Animal , HumansABSTRACT
This chapter conducts an in-depth exploration of the impact of musculoskeletal (MSK) disorders and injuries, with a specific emphasis on their consequences within the older population demographic. It underscores the escalating demand for innovative interventions in MSK tissue engineering. The chapter also highlights the fundamental role played by lipid signaling mediators (LSMs) in tissue regeneration, with relevance to bone and muscle recovery. Remarkably, Prostaglandin E2 (PGE2) emerges as a central orchestrator in these regenerative processes. Furthermore, the chapter investigates the complex interplay between bone and muscle tissues, explaining the important influence exerted by LSMs on their growth and differentiation. The targeted modulation of LSM pathways holds substantial promise as a beneficial way for addressing muscle disorders. In addition to these conceptual understandings, the chapter provides a comprehensive overview of methodologies employed in the identification of LSMs, with a specific focus on the Liquid Chromatography-Mass Spectrometry (LC-MS). Furthermore, it introduces a detailed LC MS/MS-based protocol tailored for the detection of PGE2, serving as an invaluable resource for researchers immersed in this dynamic field of study.
Subject(s)
Dinoprostone , Lipidomics , Tandem Mass Spectrometry , Humans , Lipidomics/methods , Dinoprostone/metabolism , Dinoprostone/analysis , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Musculoskeletal Diseases/diagnosis , Lipid Metabolism , Lipids/analysisABSTRACT
Intracellular Ca2+ can be conveniently monitored by sensitive Ca2+ fluorescent dyes in live cells. The Gαq involved lipid signaling pathways and, thus, can be studied by intracellular Ca2+ imaging. Here we describe the protocols to measure intracellular Ca2+ for studying PEG2-EP1 activity in esophageal smooth muscle cells. The ratiometric Fura-2 imaging provides quantitative data, and the Fluo-4 confocal microscopic imaging has high-spatial resolution.
Subject(s)
Calcium , Receptors, G-Protein-Coupled , Calcium/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Microscopy, Confocal/methods , Signal Transduction , Myocytes, Smooth Muscle/metabolism , Calcium Signaling , Humans , Xanthenes/metabolism , Fura-2/metabolism , Lipid Metabolism , Esophagus/metabolism , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Aniline CompoundsABSTRACT
Laparotomy (EL) is one of the most common procedures performed among surgical specialties. Previous research demonstrates that surgery is associated with an increased inflammatory response. Low psoas muscle mass and quality markers are associated with increased mortality rates after emergency laparotomy. Analysis of lipid mediators in serum and muscle by using liquid chromatography-mass spectrometry (LC-MS)-based lipidomics has proven to be a sensitive and precise technique. In this chapter, we describe an LC-MS/MS protocol for the profiling and quantification of signaling lipids formed from Eicosapentaenoic Acid (EPA) and Eicosatetranoic acid (ETA) by 5, 12, or 15 lipoxynases. This protocol has been developed for and validated in serum and muscle samples in a mouse model of surgical stress caused by laparotomy.
Subject(s)
Aging , Laparotomy , Lipidomics , Tandem Mass Spectrometry , Animals , Mice , Lipidomics/methods , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Aging/metabolism , Stress, Physiological , Disease Models, Animal , Lipids/analysis , Lipids/blood , Lipid MetabolismABSTRACT
Developing robust analytical techniques is a vital phase to facilitate understanding the roles and impacts of various omic profilings in cellular functions. The comprehensive analysis of various biological molecules within a biological system requires a precise sample preparation technique. Solid-Phase Extraction (SPE) has proven to be an indispensable method in lipidomic analysis, providing an uncomplicated and user-friendly technique for extraction and purification of lipid components from complex biological matrices. Of all the factors influencing the reliability and success of SPE, column or adsorbent materials, flow rate, and storage conditions are paramount in terms of their significance. In this chapter, we will discuss in detail the SPE steps for lipidomic analysis in biofluid samples (serum and plasma) and muscle tissues.
Subject(s)
Lipidomics , Lipids , Solid Phase Extraction , Solid Phase Extraction/methods , Lipidomics/methods , Lipids/isolation & purification , Lipids/chemistry , Humans , AnimalsABSTRACT
Bioactive lipids have been identified as dynamic signaling lipid mediators (LMs). These fats have the ability to activate responses and control bodily functions either directly or indirectly. Linoleic Acid (LA) and Alpha Linoleic Acid (ALA) are types of omega 3 fatty acids that possess inflammatory properties and promote resolution of inflammation either through their own actions or through their metabolites known as oxylipins. In this chapter, we provide an explanation of a method that combines chromatography with tandem mass spectroscopy (LC MS/MS) to identify and measure all the metabolites derived from LA and ALA. Additionally, we employed the described methodology to analyze human serum samples obtained before and after whole-body vibration exercise training. The results indicated an increase in some of the LA and ALA LMs that have beneficial effects in regulating the cardiovascular system.
Subject(s)
Linoleic Acid , Lipidomics , Tandem Mass Spectrometry , Vibration , Humans , Linoleic Acid/metabolism , Lipidomics/methods , Tandem Mass Spectrometry/methods , Chromatography, Liquid/methods , Exercise/physiology , Oxylipins/metabolism , Oxylipins/blood , Lipid MetabolismABSTRACT
Skeletal muscle injury affects the quality of life in many pathologies, including volumetric muscle loss, contusion injury, and aging. We hypothesized that the nicotinamide phosphoribosyltransferase (Nampt) activator P7C3 improves muscle repair following injury. In the present study, we tested the effect of P7C3 (1-anilino-3-(3,6-dibromocarbazol-9-yl) propan-2-ol) on chemically induced muscle injury. Muscle injury was induced by injecting 50 µL 1.2% barium chloride (BaCl2) into the tibialis anterior (TA) muscle in C57Bl/6J wild-type male mice. Mice were then treated with either 10 mg/kg body weight of P7C3 or Vehicle intraperitoneally for 7 days and assessed for histological, biochemical, and molecular changes. In the present study, we show that the acute BaCl2-induced TA muscle injury was robust and the P7C3-treated mice displayed a significant increase in the total number of myonuclei and blood vessels, and decreased serum CK activity compared with vehicle-treated mice. The specificity of P7C3 was evaluated using Nampt+/- mice, which did not display any significant difference in muscle repair capacity among treated groups. RNA-sequencing analysis of the injured TA muscles displayed 368 and 212 genes to be exclusively expressed in P7C3 and Veh-treated mice, respectively. There was an increase in the expression of genes involved in cellular processes, inflammatory response, angiogenesis, and muscle development in P7C3 versus Veh-treated mice. Conversely, there is a decrease in muscle structure and function, myeloid cell differentiation, glutathione, and oxidation-reduction, drug metabolism, and circadian rhythm signaling pathways. Chromatin immunoprecipitation-quantitative polymerase chain reaction (qPCR) and reverse transcription-qPCR analyses identified increased Pax7, Myf5, MyoD, and Myogenin expression in P7C3-treated mice. Increased histone lysine (H3K) methylation and acetylation were observed in P7C3-treated mice, with significant upregulation in inflammatory markers. Moreover, P7C3 treatment significantly increased the myotube fusion index in the BaCl2-injured human skeletal muscle in vitro. P7C3 also inhibited the lipopolysaccharide-induced inflammatory response and mitochondrial membrane potential of RAW 264.7 macrophage cells. Overall, we demonstrate that P7C3 activates muscle stem cells and enhances muscle injury repair with increased angiogenesis.
ABSTRACT
The intricate interplay between the muscle and bone tissues is a fundamental aspect of musculoskeletal physiology. Over the past decades, emerging research has highlighted the pivotal role of lipid signaling in mediating communication between these tissues. This chapter delves into the multifaceted mechanisms through which lipids, particularly phospholipids, sphingolipids, and eicosanoids, participate in orchestrating cellular responses and metabolic pathways in both muscle and bone. Additionally, we examine the clinical implications of disrupted lipid signaling in musculoskeletal disorders, offering insights into potential therapeutic avenues. This chapter aims to shed light on the complex lipid-driven interactions between the muscle and bone tissues, paving the way for a deeper understanding of musculoskeletal health and disease.
Subject(s)
Lipid Metabolism , Musculoskeletal Diseases , Signal Transduction , Animals , Humans , Bone and Bones/metabolism , Eicosanoids/metabolism , Muscle, Skeletal/metabolism , Musculoskeletal Diseases/metabolism , Phospholipids/metabolism , Sphingolipids/metabolismABSTRACT
This chapter provides an overview of the diverse range of applications associated with nanoparticles. The application of nanoparticles in the medical field has garnered considerable attention due to their unique properties and versatile compositions. They have shown promise in the treatment of cancer, fungal and viral infections, and pain management. These systems provide numerous benefits, such as increased drug stability, improved bioavailability, and targeted delivery to specific tissues or cells. The objective of this chapter is to provide a brief analysis of the differences between nanoparticles and lipid particles, focusing particularly on the importance of nanoparticle size and composition in their interactions with lipids. Additionally, the applications of nanoparticles in lipid signaling will be discussed, considering the vital roles lipids play in cellular signaling pathways. Nanoparticles have shown immense potential in the regulation and control of medical pathways. In this case, we will focus on the manufacture of liposomes, a type of nanoparticle composed of lipids. The reason behind the extensive investigation into liposomes as drug delivery vehicles is their remarkable biocompatibility and adaptability. This section will provide insights into the methods and techniques employed for liposome formulation.
Subject(s)
Lipids , Liposomes , Nanoparticles , Signal Transduction , Nanoparticles/chemistry , Humans , Liposomes/chemistry , Lipids/chemistry , Animals , Drug Delivery Systems/methods , Lipid MetabolismABSTRACT
With exercise, muscle and bone produce factors with beneficial effects on brain, fat, and other organs. Exercise in mice increased fibroblast growth factor 23 (FGF23), urine phosphate, and the muscle metabolite L-ß-aminoisobutyric acid (L-BAIBA), suggesting that L-BAIBA may play a role in phosphate metabolism. Here, we show that L-BAIBA increases in serum with exercise and elevates Fgf23 in osteocytes. The D enantiomer, described to be elevated with exercise in humans, can also induce Fgf23 but through a delayed, indirect process via sclerostin. The two enantiomers both signal through the same receptor, Mas-related G-protein-coupled receptor type D, but activate distinct signaling pathways; L-BAIBA increases Fgf23 through Gαs/cAMP/PKA/CBP/ß-catenin and Gαq/PKC/CREB, whereas D-BAIBA increases Fgf23 indirectly through sclerostin via Gαi/NF-κB. In vivo, both enantiomers increased Fgf23 in bone in parallel with elevated urinary phosphate excretion. Thus, exercise-induced increases in BAIBA and FGF23 work together to maintain phosphate homeostasis.
Subject(s)
Aminoisobutyric Acids , Fibroblast Growth Factor-23 , Fibroblast Growth Factors , Osteocytes , Signal Transduction , Animals , Signal Transduction/drug effects , Fibroblast Growth Factors/metabolism , Aminoisobutyric Acids/pharmacology , Mice , Osteocytes/metabolism , Osteocytes/drug effects , Stereoisomerism , Receptors, G-Protein-Coupled/metabolism , Male , Mice, Inbred C57BL , Humans , Adaptor Proteins, Signal Transducing/metabolism , Physical Conditioning, AnimalABSTRACT
Here, we investigated the mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development, including asymmetric cell division, cell-type specification, and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb-like (Numbl) in mouse myofibers caused weakness, disorganization of sarcomeres, and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb, Numbl knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb that Septin 7 is a potential Numb-binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets, and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb-binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function.
Subject(s)
Intracellular Signaling Peptides and Proteins , Membrane Proteins , Mice, Knockout , Muscle Contraction , Nerve Tissue Proteins , Sarcomeres , Septins , Animals , Septins/metabolism , Septins/genetics , Sarcomeres/metabolism , Mice , Muscle Contraction/physiology , Membrane Proteins/metabolism , Membrane Proteins/genetics , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Protein Binding , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/physiologyABSTRACT
PURPOSE: Mitofusin 2 (Mfn2) is one of two mitofusins involved in regulating mitochondrial size, shape and function, including mitophagy, an important cellular mechanism to limit oxidative stress. Reduced expression of Mfn2 has been associated with impaired osteoblast differentiation and function and a reduction in the number of viable osteocytes in bone. We hypothesized that the genetic absence of Mfn2 in these cells would increase their susceptibility to aging-associated metabolic stress, leading to a progressive impairment in skeletal homeostasis over time. METHODS: Mfn2 was selectively deleted in vivo at three different stages of osteoblast lineage commitment by crossing mice in which the Mfn2 gene was floxed with transgenic mice expressing Cre under the control of the promoter for Osterix (OSX), collagen1a1, or DMP1 (Dentin Matrix Acidic Phosphoprotein 1). RESULTS: Mice in which Mfn2 was deleted using DMP1-cre demonstrated a progressive and dramatic decline in bone mineral density (BMD) beginning at 10 weeks of age (n = 5 for each sex and each genotype from age 10 to 20 weeks). By 15 weeks, there was evidence for a functional decline in muscle performance as assessed using a rotarod apparatus (n = 3; 2 males/ 1 female for each genotype), accompanied by a decline in lean body mass. A marked reduction in trabecular bone mass was evident on bone histomorphometry, and biomechanical testing at 25 weeks (k/o: 2 male/1 female, control 2 male/2 female) revealed severely impaired femur strength. Extensive regional myofiber atrophy and degeneration was observed on skeletal muscle histology. Electron microscopy showed progressive disruption of cellular architecture, with disorganized sarcomeres and a bloated mitochondrial reticulum. There was also evidence of neurodegeneration within the ventral horn and roots of the lumbar spinal cord, which was accompanied by myelin loss and myofiber atrophy. Deletion of Mfn2 using OSX-cre or Col1a1-cre did not result in a musculoskeletal phenotype. Where possible, male and female animals were analyzed separately, but small numbers of animals in each group limited statistical power. For other outcomes, where sex was not considered, small sample sizes might still limit the strength of the observation. CONCLUSION: Despite known functional overlap of Mfn1 and Mfn2 in some tissues, and their co-expression in bone, muscle and spinal cord, deletion of Mfn2 using the 8 kB DMP1 promoter uncovered an important non-redundant role for Mfn2 in maintaining the neuromuscular/bone axis.
Subject(s)
Bone Density , GTP Phosphohydrolases , Animals , Female , GTP Phosphohydrolases/metabolism , GTP Phosphohydrolases/genetics , Male , Mice , Bone Density/genetics , Bone Density/physiology , Mice, Transgenic , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Bone and Bones/pathology , Bone and Bones/metabolism , Neuromuscular Junction/metabolism , Neuromuscular Junction/pathology , Osteoblasts/metabolism , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/geneticsABSTRACT
Healing in compromised and complicated bone defects is often prolonged and delayed due to the lack of bioactivity of the fixation device, secondary infections, and associated oxidative stress. Here, we propose amorphous silicon oxynitride (SiONx) as a coating for the fixation devices to improve both bioactivity and bacteriostatic activity and reduce oxidative stress. We aimed to study the effect of increasing the N/O ratio in the SiONx to fine-tune the cellular activity and the antioxidant effect via the NRF2 pathway under oxidative stress conditions. The in vitro studies involved using human mesenchymal stem cells (MSCs) to examine the effect of SiONx coatings on osteogenesis with and without toxic oxidative stress. Additionally, bacterial growth on SiONx surfaces was studied using methicillin-resistant Staphylococcus aureus (MRSA) colonies. NRF2 siRNA transfection was performed on the hMSCs (NRF2-KD) to study the antioxidant response to silicon ions. The SiONx implant surfaces showed a >4-fold decrease in bacterial growth vs. bare titanium as a control. Increasing the N/O ratio in the SiONx implants increased the alkaline phosphatase activity >1.5 times, and the other osteogenic markers (osteocalcin, RUNX2, and Osterix) were increased >2-fold under normal conditions. Increasing the N/O ratio in SiONx enhanced the protective effects and improved cell viability against toxic oxidative stress conditions. There was a significant increase in osteocalcin activity compared to the uncoated group, along with increased antioxidant activity under oxidative stress conditions. In NRF2-KD cells, there was a stunted effect on the upregulation of antioxidant markers by silicon ions, indicating a role for NRF2. In conclusion, the SiONx coatings studied here displayed bacteriostatic properties. These materials promoted osteogenic markers under toxic oxidative stress conditions while also enhancing antioxidant NRF2 activity. These results indicate the potential of SiONx coatings to induce in vivo bone regeneration in a challenging oxidative stress environment.
ABSTRACT
As both L- and D-BAIBA are increased with exercise, we sought to determine if circulating levels would be associated with physical performance. Serum levels of L- and D-BAIBA were quantified in 120 individuals (50% female) aged 20-85 years and categorized as either a "low" (LP), "average" (AP) or "high" performing (HP). Association analysis was performed using Spearman (S) and Pearson (P) correlation. Using Spearman correlation, L-BAIBA positively associated with (1) body mass index BMI (0.23) and total fat mass (0.19) in the 120 participants, (2) total fat mass in the 60 males (0.26), and (3) bone mineral density, BMD, (0.28) in addition to BMI (0.26) in the 60 females. In HP females, L-BAIBA positively associated with BMD (0.50) and lean mass (0.47). D-BAIBA was positively associated with (1) age (P 0.20) in the 120 participants, (2) age (P 0.49) in the LP females and (3) with gait speed (S 0.20) in the 120 participants. However, in HP males, this enantiomer had a negative association with appendicular lean/height (S - 0.52) and in the AP males a negative correlation with BMD (S - 0.47). No associations were observed in HP or AP females, whereas, in LP females, a positive association was observed with grip strength (S 0.45), but a negative with BMD (P - 0.52, S - 0.63) and chair stands (P - 0.47, S - 0.51). L-BAIBA may play a role in BMI and BMD in females, not males, whereas D-BAIBA may be a marker for aging and physical performance. The association of L-BAIBA with BMI and fat mass may reveal novel, not previously described functions for this enantiomer.
Subject(s)
Bone Density , Physical Functional Performance , Male , Humans , Female , Body Mass Index , Body Composition , Absorptiometry, PhotonABSTRACT
Declining physical performance with age and disease is an important indicator of declining health. Biomarkers that identify declining physical performance would be useful in predicting treatment outcomes and identifying potential therapeutics. γ-aminobutyric acid (GABA), a muscle autocrine factor, is a potent inhibitor of muscle function and works as a muscle relaxant. L-α-aminobutyric acid (L-AABA) is a biomarker for malnutrition, liver damage, and depression. We sought to determine if GABA and L-AABA may be useful for predicting physical performance. Serum levels of GABA and L-AABA were quantified in 120 individuals divided by age, sex, and physical capacity into low, average, and high performer groups. Analyses explored correlations between serum levels and physical performance. Both GABA and the ratio of GABA/AABA (G/A), but not AABA, were highly positively associated with age (Pearson correlations r = 0.35, p = 0.0001 for GABA, r = 0.31, p = 0.0007 for G/A, n = 120). GABA showed negative associations in the whole cohort with physical performance [fast gait speed, 6 min walk test (6MWT), PROMIS score, and SF36PFS raw score] and with subtotal and femoral neck bone mineral density. L-AABA was positively associated with usual gait speed, 6MWT, total SPPB score, and SF36PFS raw score in the total cohort of 120 human subjects, also with 6MWT and SF36PFS raw score in the 60 male subjects, but no associations were observed in the 60 females. As both GABA and L-AABA appear to be indicative of physical performance, but in opposite directions, we examined the G/A ratio. Unlike GABA, the G/A ratio showed a more distinct association with mobility tests such as total SPPB score, usual and fast gait speed, 6MWT, and SF36PFS raw score in the males, regardless of age and metabolic status. Serum G/A ratio could be potentially linked to physical performance in the male population. Our findings strongly suggest that GABA, L-AABA, and the G/A ratio in human serum may be useful markers for both age and physical function. These new biomarkers may significantly enhance the goal of identifying universal biomarkers to accurately predict physical performance and the beneficial effects of exercise training for older adults.
Subject(s)
Aminobutyrates , gamma-Aminobutyric Acid , Female , Humans , Male , Aged , Physical Functional Performance , Aging , BiomarkersABSTRACT
While mitochondria in different tissues have distinct preferences for energy sources, they are flexible in utilizing competing substrates for metabolism according to physiological and nutritional circumstances. However, the regulatory mechanisms and significance of metabolic flexibility are not completely understood. Here, we report that the deletion of Ptpmt1, a mitochondria-based phosphatase, critically alters mitochondrial fuel selection - the utilization of pyruvate, a key mitochondrial substrate derived from glucose (the major simple carbohydrate), is inhibited, whereas the fatty acid utilization is enhanced. Ptpmt1 knockout does not impact the development of the skeletal muscle or heart. However, the metabolic inflexibility ultimately leads to muscular atrophy, heart failure, and sudden death. Mechanistic analyses reveal that the prolonged substrate shift from carbohydrates to lipids causes oxidative stress and mitochondrial destruction, which in turn results in marked accumulation of lipids and profound damage in the knockout muscle cells and cardiomyocytes. Interestingly, Ptpmt1 deletion from the liver or adipose tissue does not generate any local or systemic defects. These findings suggest that Ptpmt1 plays an important role in maintaining mitochondrial flexibility and that their balanced utilization of carbohydrates and lipids is essential for both the skeletal muscle and the heart despite the two tissues having different preferred energy sources.
Cells are powered by mitochondria, a group of organelles that produce chemical energy in the form of molecules called ATP. This energy is derived from the breakdown of carbohydrates, fats, and proteins. The number of mitochondria in a cell and the energy source they use to produce ATP varies depending on the type of cell. Mitochondria can also switch the molecules they use to produce energy when the cell is responding to stress or disease. The heart and the skeletal muscles which allow movement are two tissues that require large amounts of energy, but it remained unknown whether disrupting mitochondrial fuel selection affects how these tissues work. To answer these questions, Zheng, Li, Li et al. investigated the role of an enzyme found in mitochondria called Ptpmt1. Genetically deleting Ptpmt1 in the heart and skeletal muscle of mice showed that while the development of these organs was not affected, mitochondria in these cells switched from using carbohydrates to using fats as an energy source. Over time, this shift damaged both the mitochondria and the tissues, leading to muscle wasting, heart failure, and sudden death in the mice. This suggests that balanced use of carbohydrates and fats is essential for the muscles and heart. These findings imply that long-term use of medications that alter the fuel that mitochondria use may be detrimental to patients' health and could cause heart dysfunction. This may be important for future drug development, as well as informing decisions about medication taken in the clinic.
Subject(s)
Heart Failure , Animals , Mice , Fatty Acids , Glucose , Heart Failure/genetics , Mice, Knockout , Mitochondria , Muscular AtrophyABSTRACT
Here, we investigated mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development including asymmetric cell division, cell-type specification and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb-like (NumbL) in mouse myofibers caused weakness, disorganization of sarcomeres and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb, NumbL knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb, that Septin 7 is a potential Numb binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function.
ABSTRACT
The L-enantiomer of ß-aminoisobutyric acid (BAIBA) is secreted by contracted muscle in mice, and exercise increases serum levels in humans. In mice, L-BAIBA reduces bone loss with unloading, but whether it can have a positive effect with loading is unknown. Since synergism can be more easily observed with sub-optimal amounts of factors/stimulation, we sought to determine whether L-BAIBA could potentiate the effects of sub-optimal loading to enhance bone formation. L-BAIBA was provided in drinking water to C57Bl/6 male mice subjected to either 7 N or 8.25 N of sub-optimal unilateral tibial loading for 2 weeks. The combination of 8.25 N and L-BAIBA significantly increased the periosteal mineral apposition rate and bone formation rate compared to loading alone or BAIBA alone. Though L-BAIBA alone had no effect on bone formation, grip strength was increased, suggesting a positive effect on muscle function. Gene expression analysis of the osteocyte-enriched bone showed that the combination of L-BAIBA and 8.25 N induced the expression of loading-responsive genes such as Wnt1, Wnt10b, and the TGFb and BMP signaling pathways. One dramatic change was the downregulation of histone genes in response to sub-optimal loading and/or L-BAIBA. To determine early gene expression, the osteocyte fraction was harvested within 24 hours of loading. A dramatic effect was observed with L-BAIBA and 8.25 N loading as genes were enriched for pathways regulating the extracellular matrix (Chad, Acan, Col9a2), ion channel activity (Scn4b, Scn7a, Cacna1i), and lipid metabolism (Plin1, Plin4, Cidec). Few changes in gene expression were observed with sub-optimal loading or L-BAIBA alone after 24 hours. These results suggest that these signaling pathways are responsible for the synergistic effects between L-BAIBA and sub-optimal loading. Showing that a small muscle factor can enhance the effects of sub-optimal loading of bone may be of relevance for individuals unable to benefit from optimal exercise. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
ABSTRACT
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.
Subject(s)
Muscle Fibers, Skeletal , Muscle, Skeletal , Mice , Animals , Reproducibility of ResultsABSTRACT
Skeletal muscle undergoes rapid and extensive atrophy following nerve transection though the underlying mechanisms remain incompletely understood. We previously showed transiently elevated Notch 1 signaling in denervated skeletal muscle that was abrogated by administration of nandrolone (an anabolic steroid) combined with replacement doses of testosterone. Numb is an adaptor molecule present in myogenic precursors and skeletal muscle fibers that is vital for normal tissue repair after muscle injury and for skeletal muscle contractile function. It is unclear whether the increase in Notch signaling observed in denervated muscle contributes to denervation and whether expression of Numb in myofibers slows denervation atrophy. To address these questions, the degree of denervation atrophy, Notch signaling, and Numb expression was studied over time after denervation in C57B6J mice treated with nandrolone, nandrolone plus testosterone or vehicle. Nandrolone increased Numb expression and reduced Notch signaling. Neither nandrolone alone nor nandrolone plus testosterone changed the rate of denervation atrophy. We next compared rates of denervation atrophy between mice with conditional, tamoxifen-inducible knockout of Numb in myofibers and genetically identical mice treated with vehicle. Numb cKO had no effect on denervation atrophy in this model. Taken together, the data indicate that loss of Numb in myofibers does not alter the course of denervation atrophy and that upregulation of Numb and blunting of the denervation-atrophy induced activation of Notch do not change the course of denervation atrophy.