Christopher R. Pitzer

Rehabilitative activity can improve injury repair, but it risks additional damage and reduces the functional recovery of regenerating muscle. This study tested the hypothesis that moderate electrically evoked contractions would slow restoration of neuromuscular function after cardiotoxin‐induced injury; however exogenous mitochondrial transplantation (MT) would enhance recovery of contractile function after injury. Cardiotoxin was injected into the tibialis anterior of C57BL/6 mice (10–12 weeks of age) to induce muscle necrosis. Exogenous mitochondria or phosphate‐buffered saline (PBS) were injected into the mouse tail vein after cardiotoxin injury. Injured muscles were either rested or given 40 Hz submaximal electrically evoked contractions to cardiotoxin‐injured muscles during the recovery period. Relative to intra‐animal non‐damaged control muscles restoration of peak tetanic torque after both rested and evoked contractions during recovery and twitch torque was greater, and the difference between control and injured muscle twitch one‐half relaxation time was lower in injured muscles that were rested for 10 days after injury and received MT compared to PBS‐treated muscles. Neuromuscular junction efficiency in cardiotoxin‐injured muscles was ∼70% of control undamaged muscles, but MT improved the recovery of neuromuscular junction efficiency to produce torque by 14 days after cardiotoxin injury in muscles that received additional damage induced by evoked contractions during the recovery period. These data suggest that MT enhances the recovery of neuromuscular function when the muscle is rested after injury, but it provides limited improvement in muscle function when the muscle is challenged with electrically evoked contractions in the recovery period after injury. image Key points: Mitochondrial transplantation by systemically infusing healthy donor mitochondria into injured mice improved the recovery of maximal torque production of injured muscles when evoked contractions were provided to the regenerating muscle during the recovery period after injury. Mitochondrial transplantation improved the restoration of neuromuscular junction efficiency after muscle injury. The recovery of maximal torque capabilities function following cardiotoxin‐induced tibialis anterior muscle injury was attenuated by electrically evoked muscle contractions conducted every other day during the recovery period in young adult mice.

Current evidence from the literature suggests a role for circulating extracellular vesicles (EVs) in the pathogenesis of multiple diseases as well as a potential therapeutic role via delivery of vesicular cargo. Type 2 Diabetes is among the most prevalent and costly metabolic diseases in the world. Lifestyle intervention including exercise can cause Type 2 Diabetes to enter effective remission but remain ineffective due to poor adherence. Exercise stimulates the release of EVs from the skeletal muscle (and other tissues), and these vesicles are thought to confer a portion of the metabolic benefits of exercise. However, the direct effects of EV’s to regulate muscle metabolic disruption or how exercise-modified EVs regulate skeletal muscle gene signaling in Type 2 Diabetes is not well understood. The purpose of this project was to study EVs on skeletal muscle cells to identify the mechanisms through which circulating extracellular vesicles contribute to the phenotype of Type 2 Diabetes; and to evaluate the effects of exercise associated EVs in a mouse model of Type 2 Diabetes. We addressed this problem in part via a novel model for investigation of EVs. In our model, we utilized exosome free cell culture media to replace depleted exosomes with those from control or diabetic mice. Using this approach, we demonstrate that exosomes from diabetic mice significantly impair insulin stimulated uptake of 2-NBDG (a fluorescent glucose analog). We also report that this occurs without alteration in phosphorylation of AKT. We also observed marked deposition on neutral lipids in myotubes after exposure with no changes in expression of a subset of genes important for lipid synthesis or import. Next, we employed 3’ end RNA seq to identify differentially regulated genes in response to exosomes associated with both aerobic exercise and type 2 diabetes. We found that exosomes associated with aerobic exercise robustly downregulated the expression of MAP2K6 whereas diabetes associated exosomes upregulated the expression of PRKD1. This is important because both genes may uniquely contribute to insulin sensitivity and overall muscle health. Finally, we investigated the therapeutic utility of intravenous administration of exercise associated exosomes for diabetic mice. While the exercise exosomes improved run to fatigue time and decreased intraperitoneal glucose tolerance test area under the curve, there was no improvement in muscle mass or force production, HOMA-IR index, or hemoglobin A1C in diabetic animals. We therefore must conclude that aerobic exercise associated exosomes confer functional and metabolic benefits associated with exercise but

Neuromuscular diseases and damage affect many people of all ages and are responsible for an exorbitant medical cost, more than $200 million annually. Finding an appropriate model to investigate potential curative interventions is necessary. One currently used involves the application of toxic agents on skeletal muscle followed by treatment interventions. A question regarding this model is whether such toxins impact not only muscle tissue, but also the neuromuscular junctions (NMJs) responsible for exciting the muscle tissue. This question was addressed here by forming 4 experimental groups: 1) Controls whose muscles had not been injured or treated, 2) muscles taken from mice that were injured and then treated with mitochondrial supplement, 3) muscles that had not been injured but still treated with mitochondria, and 4) muscles that were injured and received no mitochondrial treatment. Several pre- and post-synaptic features of NMJs received immunofluorescent staining before having morphological features assessed with confocal microscopy. Results revealed that only pre-synaptic acetylcholine (ACh) receptors showed any significant (p0.05) among treatment groups and structural features were not different between groups with the exception of dispersion of post-synaptic receptors. Overall, these results suggest that skeletal muscles damaged with toxin accurately mimic what occurs during toxin-induced damage and post-injury recovery.

Although many of the structures and organelles of vegetative cells are comparable to those of animal tissues, significant differences between the two kingdoms require modifications in histological techniques for both tissue processing steps and histochemical staining techniques. The authors investigated the challenges of working with plant tissues by collecting various flora to represent the four main plant organs: leaf, stem, root, and flower/fruit. Triplicate samples for each specimen were placed into formalin for paraffin embedding, placed into formalin for later frozen sections, and used fresh to undergo immediate frozen sectioning. Frozen sections of plant tissues were more difficult to obtain than formalin-fixed paraffin-embedded (FFPE) sections, exhibited tissue loss during staining, and were inferior morphologically to FFPE sections. Although, historically, plant tissue fixation and processing has employed several different reagents compared with those used in animal tissue processing and took significantly longer times, the current investigation determined reagents and protocols from a modern histology laboratory which processes mammalian tissues can be applied to plant tissue processing with only slight modifications in respect to reagent timing. Additionally, staining techniques were compared and while it is well known that plant cell walls stain well with safranin O, the current investigation determined the uptake of safranin O can be accelerated by incubating at 60°C.

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

The application of most chemical fixatives, such as formalin, in the anatomic pathology laboratory requires safety training and hazardous chemical monitoring due to the toxicity and health risks associated with their use. Consequently, the use of formalin has been banned in most applications in Europe; the primary exception is its use in the histology laboratory in lieu of a suitable and safer alternative. Glyoxal based solutions, several of which are available commercially, are the most promising alternative fixatives, because they are based on a mechanism of fixation similar to that of formalin. Unlike formalin, however, glyoxal based solutions do not dissociate from water and therefore do not require ventilation measures such as a fume hood. A primary barrier to the adoption of commercially available glyoxal based solutions is their low pH, which can produce undesirable morphological and antigenic tissue alterations; however, a recently available neutral pH glyoxal product (glyoxal acid free) (GAF) has been developed to mitigate the challenges of low pH. We compared the morphology and histochemistry among tissues fixed in 10% neutral buffered formalin, a commercially available acidic glyoxal product (Prefer), and GAF. Tissues fixed in formalin and Prefer exhibited similar morphology and staining properties; tissues fixed with 2% GAF exhibited deleterious effects.

Despite claims of safety or harm reduction for electronic cigarettes (E-cig) use (also known as vaping), emerging evidence indicates that E-cigs are not likely safe, or necessarily safer than traditional cigarettes, when considering the user’s risk of developing vascular dysfunction/disease. E-cigs are different from regular cigarettes in that E-cig devices are highly customizable, and users can change the e-liquid composition (such as the base solution, flavors, and nicotine level). Since the effects of E-cigs on the microvascular responses in skeletal muscle are poorly understood, we used intravital microscopy with an acute (one-time 10 puff) exposure paradigm to evaluate the individual components of e-liquid on vascular tone and endothelial function in the arterioles of the gluteus maximus muscle of anesthetized C57Bl/6 mice. Consistent with the molecular responses seen with endothelial cells, we found that the peripheral vasoconstriction response was similar between mice exposed to E-cig aerosol or cigarette smoke (i.e., 3R4F reference cigarette); this response was not nicotine dependent, and endothelial cell-mediated vasodilation was not altered within this acute exposure paradigm. We also report that, regardless of the base solution component [i.e., vegetable glycerin (VG)-only or propylene glycol (PG)-only], the vasoconstriction responses were the same in mice with inhalation exposure to 3R4F cigarette smoke or E-cig aerosol. Key findings from this work reveal that some component other than nicotine, in inhaled smoke or aerosol, is responsible for triggering peripheral vasoconstriction in skeletal muscle, and that regardless of one’s preference for an E-cig base solution composition (i.e., ratio of VG-to-PG), the acute physiological response to blood vessels appears to be the same. The data suggest that vaping is not likely to be ‘safer’ than smoking towards blood vessels and can be expected to produce and/or result in the same adverse vascular health outcomes associated with smoking cigarettes.

Musculoskeletal health is directly linked to independence and longevity, but disease and aging impairs muscle mass and health. Complete repair after a pathological or physiological muscle injury is critical for maintaining muscle function, yet muscle repair is compromised after disuse, or in conditions such as metabolic diseases, cancer, and aging. Regeneration of damaged tissue is critically dependent upon achieving the optimal function of satellite cells (muscle stem cells, MSCs). MSC remodeling in muscle repair is highly dependent upon its microenvironment, and metabolic health of MSCs, which is dependent on the functional capacity of their mitochondria. Muscle repair is energy demanding and mitochondria provide the primary source for energy production during regeneration. However, disease and aging induce mitochondrial dysfunction, which limits energy production during muscle regeneration. Nevertheless, the role of mitochondria in muscle repair likely extends beyond the production of ATP and mitochondria could provide potentially important regulatory signaling to MSCs during repair from injury. The scope of current research in muscle regeneration extends from molecules to exosomes, largely with the goal of understanding ways to improve MSC function. This review focuses on the role of mitochondria in skeletal muscle myogenesis/regeneration and repair. A therapeutic strategy for improving muscle mitochondrial number and health will be discussed as a means for enhancing muscle regeneration. Highlights: (a). Mitochondrial dysfunction limits muscle regeneration; (b). Muscle stem cell (MSC) function can be modulated by mitochondria; (c). Enhancing mitochondria in MSCs may provide a strategy for improving muscle regeneration after an injury.

Cancer cachexia is defined as unintentional weight loss secondary to neoplasia and is associated with poor prognosis and outcomes. Cancer cachexia associated weight loss affects both lean tissue (i.e., skeletal muscle) and adipose tissue. Exosomes are extracellular vesicles that originate from multivesicular bodies that contain intentionally loaded biomolecular cargo. Exosome cargo includes proteins, lipids, mitochondrial components, and nucleic acids. The cargo carried in exosomes is thought to alter cell signaling when it enters into recipient cells. Virtually every cell type secretes exosomes and exosomes are known to be present in nearly every biofluid. Exosomes alter muscle and adipose tissue metabolism and biological processes, including macrophage polarization and apoptosis which contribute to the development of the cachexia phenotype. This has led to an interest in the role of tumor cell derived exosomes and their potential role as biomarkers of cancer cell development as well as their contribution to cachexia and disease progression. In this review, we highlight published findings that have studied the effects of tumor derived exosomes (and extracellular vesicles) and their cargo on the progression of cancer cachexia. We will focus on the direct effects of tumor derived exosomes and their cellular cross talk on skeletal muscle and adipose tissue, the primary sites of weight loss due to cancer cachexia.

Sarcopenia is a debilitating skeletal muscle disease that accelerates in the last decades of life and is characterized by marked deficits in muscle strength, mass, quality, and metabolic health. The multifactorial causes of sarcopenia have proven difficult to treat and involve a complex interplay between environmental factors and intrinsic age-associated changes. It is generally accepted that sarcopenia results in a progressive loss of skeletal muscle function that exceeds the loss of mass, indicating that while loss of muscle mass is important, loss of muscle quality is the primary defect with advanced age. Furthermore, preclinical models have suggested that aged skeletal muscle exhibits defects in cellular quality control such as the degradation of damaged mitochondria. Recent evidence suggests that a dysregulation of proteostasis, an important regulator of cellular quality control, is a significant contributor to the aging-associated declines in muscle quality, function, and mass. Although skeletal muscle mammalian target of rapamycin complex 1 (mTORC1) plays a critical role in cellular control, including skeletal muscle hypertrophy, paradoxically, sustained activation of mTORC1 recapitulates several characteristics of sarcopenia. Pharmaceutical inhibition of mTORC1 as well as caloric restriction significantly improves muscle quality in aged animals, however, the mechanisms controlling cellular proteostasis are not fully known. This information is important for developing effective therapeutic strategies that mitigate or prevent sarcopenia and associated disability. This review identifies recent and historical understanding of the molecular mechanisms of proteostasis driving age-associated muscle loss and suggests potential therapeutic interventions to slow or prevent sarcopenia.