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BioRxiv : the Preprint Server For... Jun 2024Volumetric Muscle Loss (VML) injuries are characterized by significant loss of muscle mass, usually due to trauma or surgical resection, often with a residual open wound...
Volumetric Muscle Loss (VML) injuries are characterized by significant loss of muscle mass, usually due to trauma or surgical resection, often with a residual open wound in clinical settings and subsequent loss of limb function due to the replacement of the lost muscle mass with non-functional scar. Being able to regrow functional muscle in VML injuries is a complex control problem that needs to override robust, evolutionarily conserved healing processes aimed at rapidly closing the defect in lieu of restoration of function. We propose that discovering and implementing this complex control can be accomplished by the development of a Medical Digital Twin of VML. Digital Twins (DTs) are the subject of a recent report from the National Academies of Science, Engineering and Medicine (NASEM), which provides guidance as to the definition, capabilities and research challenges associated with the development and implementation of DTs. Specifically, DTs are defined as dynamic computational models that can be personalized to an individual real world "twin" and are connected to that twin via an ongoing data link. DTs can be used to provide control on the real-world twin that is, by the ongoing data connection, adaptive. We have developed an anatomic scale cell-level agent-based model of VML termed the Wound Environment Agent Based Model (WEABM) that can serve as the computational specification for a DT of VML. Simulations of the WEABM provided fundamental insights into the biology of VML, and we used the WEABM in our previously developed pipeline for simulation-based Deep Reinforcement Learning (DRL) to train an artificial intelligence (AI) to implement a robust generalizable control policy aimed at increasing the healing of VML with functional muscle. The insights into VML obtained include: 1) a competition between fibrosis and myogenesis due to spatial constraints on available edges of intact myofibrils to initiate the myoblast differentiation process, 2) the need to biologically "close" the wound from atmospheric/environmental exposure, which represents an ongoing inflammatory stimulus that promotes fibrosis and 3) that selective, multimodal and adaptive local mediator-level control can shift the trajectory of healing away from a highly evolutionarily beneficial imperative to close the wound via fibrosis. Control discovery with the WEABM identified the following design principles: 1) multimodal adaptive tissue-level mediator control to mitigate pro-inflammation as well as the pro-fibrotic aspects of compensatory anti-inflammation, 2) tissue-level mediator manipulation to promote myogenesis, 3) the use of an engineered extracellular matrix (ECM) to functionally close the wound and 4) the administration of an anti-fibrotic agent focused on the collagen-producing function of fibroblasts and myofibroblasts. The WEABM-trained DRL AI integrates these control modalities and provides design specifications for a potential device that can implement the required wound sensing and intervention delivery capabilities needed. The proposed cyber-physical system integrates the control AI with a physical sense-and-actuate device that meets the tenets of DTs put forth in the NASEM report and can serve as an example schema for the future development of Medical DTs.
PubMed: 38895374
DOI: 10.1101/2024.06.04.595972 -
Nutrients May 2024Fructose is a commonly consumed monosaccharide implicated in developing several metabolic diseases. Previously, elevated branched-chain amino acids (BCAA) have been...
UNLABELLED
Fructose is a commonly consumed monosaccharide implicated in developing several metabolic diseases. Previously, elevated branched-chain amino acids (BCAA) have been correlated with the severity of insulin resistance. Most recently, the effect of fructose consumption on the downregulation of BCAA catabolic enzymes was observed. Thus, this mechanistic study investigated the effects of physiologically attainable levels of fructose, both with and without concurrent insulin resistance, in a myotube model of skeletal muscle.
METHODS
C2C12 mouse myoblasts were treated with fructose at a concentration of 100 µM (which approximates physiologically attainable concentrations in peripheral circulation) both with and without hyperinsulinemic-mediated insulin resistance. Gene expression was assessed by qRT-PCR, and protein expression was assessed by Western blot. Oxygen consumption rate and extracellular acidification rate were used to assess mitochondrial oxidative and glycolytic metabolism, respectively. Liquid chromatography-mass spectrometry was utilized to analyze leucine, isoleucine and valine concentration values.
RESULTS
Fructose significantly reduced peak glycolytic and peak mitochondrial metabolism without altering related gene or protein expression. Similarly, no effect of fructose on BCAA catabolic enzymes was observed; however, fructose treatment resulted in elevated total extracellular BCAA in insulin-resistant cells.
DISCUSSION
Collectively, these observations demonstrate that fructose at physiologically attainable levels does not appear to alter insulin sensitivity or BCAA catabolic potential in cultured myotubes. However, fructose may depress peak cell metabolism and BCAA utilization during insulin resistance.
Topics: Animals; Insulin Resistance; Fructose; Amino Acids, Branched-Chain; Mice; Muscle Fibers, Skeletal; Cell Line; Mitochondria; Glycolysis; Myoblasts; Oxygen Consumption
PubMed: 38892515
DOI: 10.3390/nu16111582 -
Developmental Biology Jun 2024Anorectal malformation (ARM) is the most common congenital digestive tract anomaly in newborns, and children with ARM often have varying degrees of underdevelopment of...
Anorectal malformation (ARM) is the most common congenital digestive tract anomaly in newborns, and children with ARM often have varying degrees of underdevelopment of the pelvic floor muscles (PFMs). To explore the effects of RARα and Pitx2 on the development of rat PFMs, we constructed a rat ARM animal model using all-trans retinoic acid (ATRA), and verified the expression of RARα and Pitx2 in the PFMs of fetal rats. Additionally, we used rat myoblasts (L6 cells) to investigate the regulatory roles of RARα and Pitx2 in skeletal muscle myoblast differentiation and their interactions. The results indicated a significant decrease in the expression of RARα and Pitx2 in the PFMs of fetal rats with ARM. ATRA can also decrease the expression of RARα and Pitx2 in the L6 cells, while affecting the differentiation and fusion of L6 cells. Knocking down RARα in L6 cells reduced the expression of Pitx2, MYOD1, MYMK, and decreased myogenic activity in L6 cells. When RARα is activated, the decreased expression of Pitx2, MYOD1, and MYMK and myogenic differentiation can be restored to different extents. At the same time, increasing or inhibiting the expression of Pitx2 can counteract the effects of knocking down RARα and activating RARα respectively. These results indicate that Pitx2 may be downstream of the transcription factor RARα, mediating the effects of ATRA on the development of fetal rat PFMs.
PubMed: 38878992
DOI: 10.1016/j.ydbio.2024.06.006 -
Biochimica Et Biophysica Acta.... Jun 2024Recessive mutations in the Parkin gene (PRKN) are the most common cause of young-onset inherited parkinsonism. Parkin is a multifunctional E3 ubiquitin ligase that plays...
Recessive mutations in the Parkin gene (PRKN) are the most common cause of young-onset inherited parkinsonism. Parkin is a multifunctional E3 ubiquitin ligase that plays a variety of roles in the cell including the degradation of proteins and the maintenance of mitochondrial homeostasis, integrity, and biogenesis. In 2001, the R275W mutation in the PRKN gene was identified in two unrelated families with a multigenerational history of postural tremor, dystonia and parkinsonism. Drosophila models of Parkin R275W showed selective and progressive degeneration of dopaminergic neuronal clusters, mitochondrial abnormalities, and prominent climbing defects. In the Prkn mouse orthologue, the amino acid R274 corresponds to human R275. Here we described an age-related motor impairment and a muscle phenotype in R274W +/+ mice. In vitro, Parkin R274W mutation correlates with abnormal myoblast differentiation, mitochondrial defects, and alteration in mitochondrial mRNA and protein levels. Our data suggest that the Parkin R274W mutation may impact mitochondrial physiology and eventually myoblast proliferation and differentiation.
PubMed: 38878834
DOI: 10.1016/j.bbadis.2024.167302 -
Antioxidants & Redox Signaling Jun 2024Redox signaling plays a key role in skeletal muscle remodeling induced by exercise and prolonged inactivity, but it is unclear which oxidant triggers myofiber...
AIMS
Redox signaling plays a key role in skeletal muscle remodeling induced by exercise and prolonged inactivity, but it is unclear which oxidant triggers myofiber hypertrophy due to the lack of strategies to precisely regulate individual oxidants in vivo. In this study, we used tetrathiomolybdate (TM) to dissociate the link between superoxide and H2O2 and thereby to specifically explore the role of superoxide in muscle hypertrophy in C2C12 cells and mice.
RESULTS
TM can linearly regulate intracellular superoxide levels by inhibition of superoxide dismutase 1 (SOD1). A 70% increase in superoxide levels in C2C12 myoblast cells and mice is necessary and sufficient for triggering hypertrophy of differentiated myotubes, and can enhance exercise performance by more than 50% in mice. SOD1 knockout blocks TM-induced superoxide increments and thereby prevents hypertrophy, whereas SOD1 restoration rescues all these effects. Scavenging superoxide with antioxidants abolishes TM-induced hypertrophy and the enhancement of exercise performance, while the restoration of superoxide levels with a superoxide generator promotes muscle hypertrophy independent of SOD1 activity.
INNOVATION AND CONCLUSION
These findings suggest that superoxide is an endogenous initiator of myofiber hypertrophy, and that TM may be used to treat muscle wasting diseases. Our work not only suggests a novel druggable mechanism to increase muscle mass but also provides a tool for precisely regulating superoxide levels in vivo.
PubMed: 38877802
DOI: 10.1089/ars.2024.0595 -
Tissue & Cell May 2024Skeletal muscle function is highly dependent on the energy supply provided by mitochondria. Besides ATP production, mitochondria have several other roles, such as...
Skeletal muscle function is highly dependent on the energy supply provided by mitochondria. Besides ATP production, mitochondria have several other roles, such as calcium storage, heat production, cell death signaling, autophagy regulation and redox state modulation. Mitochondrial function is crucial for skeletal muscle fiber formation. Disorders that affect mitochondria have a major impact in muscle development and function. Here we studied the role of mitochondria during chick skeletal myogenesis. We analyzed the intracellular distribution of mitochondria in myoblasts, fibroblasts and myotubes using Mitotracker labeling. Mitochondrial respiration was investigated in chick muscle cells. Our results show that (i) myoblasts and myotubes have more mitochondria than muscle fibroblasts; (ii) mitochondria are organized in long lines within the whole cytoplasm and around the nuclei of myotubes, while in myoblasts they are dispersed in the cytoplasm; (iii) the area of mitochondria in myotubes increases during myogenesis, while in myoblasts and fibroblasts there is a slight decrease; (iv) mitochondrial length increases in the three cell types (myoblasts, fibroblasts and myotubes) during myogenesis; (v) the distance of mitochondria to the nucleus increases in myoblasts and myotubes during myogenesis; (vi) Rotenone inhibits muscle fiber formation, while FCCP increases the size of myotubes; (vii) N-acetyl cysteine (NAC), an inhibitor of ROS formation, rescues the effects of Rotenone on muscle fiber size; and (viii) Rotenone induces the production of ROS in chick myogenic cells. The collection of our results suggests a role of ROS signaling in mitochondrial function during chick myogenesis.
PubMed: 38875923
DOI: 10.1016/j.tice.2024.102423 -
BMC Methods 2024Functional evaluation of molecules that are predicted to promote stem cell mediated endogenous repair often requires in vivo transplant studies that are low throughput...
BACKGROUND
Functional evaluation of molecules that are predicted to promote stem cell mediated endogenous repair often requires in vivo transplant studies that are low throughput and hinder the rate of discovery. To offer greater throughput for functional validation studies, we miniaturized, simplified and expanded the functionality of a previously developed muscle endogenous repair (MEndR) in vitro assay that was shown to capture significant events of in vivo muscle endogenous repair.
METHODS
The mini-MEndR assay consists of miniaturized cellulose scaffolds designed to fit in 96-well plates, the pores of which are infiltrated with human myoblasts encapsulated in a fibrin-based hydrogel to form engineered skeletal muscle tissues. Pre-adsorbing thrombin to the cellulose scaffolds facilitates in situ tissue polymerization, a critical modification that enables new users to rapidly acquire assay expertise. Following the generation of the 3D myotube template, muscle stem cells (MuSCs), enriched from digested mouse skeletal muscle tissue using an improved magnetic-activated cell sorting protocol, are engrafted within the engineered template. Murine MuSCs are fluorescently labeled, enabling co-evaluation of human and mouse Pax7 cell responses to drug treatments. A regenerative milieu is introduced by injuring the muscle tissue with a myotoxin to initiate endogenous repair "in a dish". Phenotypic data is collected at endpoints with a high-content imaging system and is analyzed using ImageJ-based image analysis pipelines.
RESULTS
The miniaturized format and modified manufacturing protocol cuts reagent costs in half and hands-on seeding time ~ threefold, while the image analysis pipelines save 40 h of labour. By evaluating multiple commercially available human primary myoblast lines in 2D and 3D culture, we establish quality assurance metrics for cell line selection that standardizes myotube template quality. In vivo outcomes (enhanced muscle production and Pax7 cell expansion) to a known modulator of MuSC mediated repair (p38/β MAPK inhibition) are recapitulated in the miniaturized culture assay, but only in the presence of stem cells and the regenerative milieu.
DISCUSSION
The miniaturized predictive assay offers a simple, scaled platform to co-investigate human and mouse skeletal muscle endogenous repair molecular modulators, and thus is a promising strategy to accelerate the muscle endogenous repair discovery pipeline.
SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at 10.1186/s44330-024-00005-4.
PubMed: 38872952
DOI: 10.1186/s44330-024-00005-4 -
Skeletal Muscle Jun 2024Adult muscle-resident myogenic stem cells, satellite cells (SCs), that play non-redundant role in muscle regeneration, are intrinsically impaired in Duchenne muscular...
BACKGROUND
Adult muscle-resident myogenic stem cells, satellite cells (SCs), that play non-redundant role in muscle regeneration, are intrinsically impaired in Duchenne muscular dystrophy (DMD). Previously we revealed that dystrophic SCs express low level of anti-inflammatory and anti-oxidative heme oxygenase-1 (HO-1, HMOX1). Here we assess whether targeted induction of HMOX1 affect SC function and alleviates hallmark symptoms of DMD.
METHODS
We generated double-transgenic mouse model (mdx;HMOX1) that allows tamoxifen (TX)-inducible HMOX1 expression in Pax7 positive cells of dystrophic muscles. Mdx;HMOX1 and control mdx mice were subjected to 5-day TX injections (75 mg/kg b.w.) followed by acute exercise protocol with high-speed treadmill (12 m/min, 45 min) and downhill running to worsen skeletal muscle phenotype and reveal immediate effects of HO-1 on muscle pathology and SC function.
RESULTS
HMOX1 induction caused a drop in SC pool in mdx;HMOX1 mice (vs. mdx counterparts), while not exaggerating the effect of physical exercise. Upon physical exercise, the proliferation of SCs and activated CD34 SC subpopulation, was impaired in mdx mice, an effect that was reversed in mdx;HMOX1 mice, however, both in vehicle- and TX-treated animals. This corresponded to the pattern of HO-1 expression in skeletal muscles. At the tissue level, necrotic events of selective skeletal muscles of mdx mice and associated increase in circulating levels of muscle damage markers were blunted in HO-1 transgenic animals which showed also anti-inflammatory cytokine profile (vs. mdx).
CONCLUSIONS
Targeted expression of HMOX1 plays protective role in DMD and alleviates dystrophic muscle pathology.
Topics: Animals; Heme Oxygenase-1; Satellite Cells, Skeletal Muscle; Muscular Dystrophy, Duchenne; Mice, Inbred mdx; Mice, Transgenic; Muscle, Skeletal; Mice; PAX7 Transcription Factor; Male; Mice, Inbred C57BL; Physical Conditioning, Animal; Membrane Proteins
PubMed: 38867250
DOI: 10.1186/s13395-024-00346-2 -
FASEB Journal : Official Publication of... Jun 2024Mitochondrial disease is a devastating genetic disorder, with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and m.3243A>G...
Mitochondrial disease is a devastating genetic disorder, with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and m.3243A>G being the most common phenotype and genotype, respectively. The treatment for MELAS patients is still less effective. Here, we performed transcriptomic and proteomic analysis in muscle tissue of MELAS patients, and discovered that the expression of molecules involved in serine catabolism were significantly upregulated, and serine hydroxymethyltransferase 2 (SHMT2) increased significantly in both the mRNA and protein levels. The SHMT2 protein level was also increased in myoblasts with m.3243A>G mutation, which was transdifferentiated from patients derived fibroblasts, accompanying with the decreased nicotinamide adenine dinucleotide (NAD)/reduced NAD (NADH) ratio and cell viability. After treating with SHMT2 inhibitor (SHIN1), the NAD/NADH ratio and cell viability in MELAS myoblasts increased significantly. Taken together, our study indicates that enhanced serine catabolism plays an important role in the pathogenesis of MELAS and that SHIN1 can be a potential small molecule for the treatment of this disease.
Topics: Humans; MELAS Syndrome; Glycine Hydroxymethyltransferase; Serine; Myoblasts; NAD; Male; Proteomics; Female; Transcriptome; Multiomics
PubMed: 38865203
DOI: 10.1096/fj.202302286RRR -
Frontiers in Cell and Developmental... 2024Vertebrate body axis formation initiates during gastrulation and continues within the tail bud at the posterior end of the embryo. Major structures in the trunk are...
Vertebrate body axis formation initiates during gastrulation and continues within the tail bud at the posterior end of the embryo. Major structures in the trunk are paired somites, which generate the musculoskeletal system, the spinal cord-forming part of the central nervous system, and the notochord, with important patterning functions. The specification of these different cell lineages by key signalling pathways and transcription factors is essential, however, a global map of cell types and expressed genes in the avian trunk is missing. Here we use high-throughput sequencing approaches to generate a molecular map of the emerging trunk and tailbud in the chick embryo. Single cell RNA-sequencing (scRNA-seq) identifies discrete cell lineages including somites, neural tube, neural crest, lateral plate mesoderm, ectoderm, endothelial and blood progenitors. In addition, RNA-seq of sequential tissue sections (RNA-tomography) provides a spatially resolved, genome-wide expression dataset for the avian tailbud and emerging body, comparable to other model systems. Combining the single cell and RNA-tomography datasets, we identify spatially restricted genes, focusing on somites and early myoblasts. Thus, this high-resolution transcriptome map incorporating cell types in the embryonic trunk can expose molecular pathways involved in body axis development.
PubMed: 38863942
DOI: 10.3389/fcell.2024.1382960