Multiparameters dependance of tissue shape maintenance in myoblasts multicellular aggregates: the role of intermediate filaments.

Liquid and elastic behavior of tissues drives their morphology and their response to the environment. They appear as the first insight on tissue mechanics. We explore the role of individual cell properties on spheroids of mouse muscle precursor cells by developing a fully automated surface tension and Young's modulus measurement system. Flattening multicellular aggregates under magnetic constraint, we show that rigidity and surface tension act as highly sensitive macroscopic reporters closely related to microscopic local tension and effective adhesion. Shedding light on the major contributions of acto-myosin contractility, actin organization and intercellular adhesions, we reveal the role of desmin organization on the macroscopic mechanics of this tissue model.

β-Hydroxy-β-Methylbutyrate Supplementation Promotes Antitumor Immunity in an Obesity Responsive Mouse Model of Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the United States, and effective therapies for PDAC are currently lacking. Moreover, PDAC is promoted and exacerbated by obesity, while cachexia and sarcopenia are exceptionally common comorbidities that predict both poor survival and treatment response. Managing PDAC with immunotherapies has thus far proven ineffective, partly due to the metabolically hostile tumor microenvironment. β-hydroxy-β-methylbutyrate (HMB), a metabolite of leucine commonly used as a dietary supplement to boost muscle growth and immune function, may be an attractive candidate to augment PDAC therapy. We therefore sought to test the hypothesis that HMB would enhance antitumor immunity while protecting mouse muscle mass. Control and diet-induced obese C57BL/6 male mice bearing subcutaneously injected Panc02 tumors were supplemented with 1% HMB and treated with or without 50 mg/kg gemcitabine (n = 15/group). HMB was associated with reduced muscle inflammation and increased muscle fiber size. HMB also reduced tumor growth and promoted antitumor immunity in obese, but not lean, mice, independent of the gemcitabine treatment. Separately, in lean tumor-bearing mice, HMB supplementation promoted an anti-PD1 immunotherapy response (n = 15/group). Digital cytometry implicated the decreased abundance of M2-like macrophages in PDAC tumors, an effect that was enhanced by anti-PD1 immunotherapy. We confirmed that HMB augments M1-like macrophage (antitumor) polarization. These preclinical findings suggest that HMB has muscle-sparing and antitumor activities against PDAC in the context of obesity, and that it may sensitize otherwise nonresponsive PDAC to immunotherapy.

Exercise increases phosphorylation of the putative mTORC2 activity readout NDRG1 in human skeletal muscle

In mice, exercise is suggested to activate the mechanistic target of rapamycin complex 2 (mTORC2) in skeletal muscle, and mTORC2 is required for normal muscle glucose uptake during exercise. Whether this translates to human skeletal muscle and what signaling pathways facilitate the exercise-induced mTORC2 activation is unknown but important to determine given the important role of mTORC2 in metabolism. We herein tested the hypothesis that exercise increases mTORC2 activity in human skeletal muscle and investigated if β2-adrenergic receptor (AR) activation mediates exercise-induced mTORC2 activation. We examined several mTORC2 activity readouts (p-NDRG1 Thr346, p-Akt Ser473, p-mTOR S2481, and p-Akt Thr450) in human skeletal muscle biopsies after uphill walking or cycling exercise. In mouse muscles, we assessed mTORC2 activity readouts following acute activation of muscle β2-adrenergic or Gs signaling and during in vivo and ex vivo muscle contractions. Exercise increased phosphorylation of NDRG1 Thr346 in human soleus, gastrocnemius, and vastus lateralis muscle, without changing p-Akt Ser473, p-Akt Thr450, and p-mTOR Ser2481. In mouse muscle, stimulation of β2-adrenergic or Gs signaling and ex vivo contractions failed to increase p-NDRG1 Thr346, while in vivo contractions were sufficient to induce p-NDRG1 Thr346. In conclusion, the mTORC2 activity readout p-NDRG1 Thr346 is a novel exercise-responsive signaling protein in human skeletal muscle. Notably, contraction-induced p-NDRG1 Thr346 appears to require a systemic factor. Unlike exercise, and in contrast to published data obtained in cultured muscles cells, stimulation of β2-adrenergic signaling is not sufficient to trigger NDRG1 phosphorylation in mature mouse skeletal muscle.

Probenecid affects sarcoplasmic reticulum Ca2+ release and depresses contractile activation in mouse skeletal muscle

Pannexins are plasma membrane heptameric channels mediating ATP release from the cytosol to the extracellular space. Skeletal muscle activity is associated with Pannexin 1 (Panx1) channels activation, ATP release out to the extracellular space and subsequent activation of purinergic signaling pathways. In agreement, recent evidence has shown molecular and functional interactions between Panx1 and the excitation–contraction (EC) coupling machinery of skeletal muscle. In this framework, we tested whether pharmacological effectors of Panx1 affect EC coupling in differentiated muscle fibers. Using confocal detection of cytosolic Ca2+ in voltage-clamped mouse muscle fibers, we found that the Panx1 blocker probenecid (1 mM) affects intracellular Ca2+ handling and EC coupling: acute application of probenecid generates a rise in resting Ca2+ that also occurs in nominally Ca2+-free extracellular medium. This effect is associated with a reduction of Ca2+ release through the sarcoplasmic reticulum (SR) Ca2+ channel RYR1. The effect of probenecid persists with time, with muscle fibers incubated for 30 min in the presence of the drug exhibiting a 40% reduction in peak SR Ca2+ release. Under the same conditions, the other Panx1 blocker carbenoxolone (50 µM) produced a 70% reduction in peak SR Ca2+ release. Application of probenecid on electrically stimulated whole mouse muscle induced a slight rise in resting tension and a >50% reduction of tetanic force after 30 min of incubation. Our results provide further support for the strong links between Panx1 function and EC coupling. Because probenecid is used both in the clinic for several types of therapeutic benefits and as a hiding agent for doping in sport, our results question whether potential adverse muscular effects may have, so far, been overlooked.

Hindlimb Muscle Dissection, Permeabilization, and Respirometry v1

The use of permeabilized muscle fibers (PMF) has emerged as a gold standard for assessing skeletal muscle mitochondrial function. PMF provide an intermediate approach between in vivo strategies and isolated mitochondria that allows the mitochondria to be maintained in close to their native morphology in the myofiber while allowing greater control of substrate and inhibitor concentrations. However, like mitochondrial isolation, the primary drawback to PMF is disruption of the cellular environment during the muscle biopsy and preparation. Despite all the benefits of permeabilized muscle fibers in evaluating mitochondrial respiration and dynamics one of the major drawbacks is increased variability introduced during a muscle biopsy as well as intrinsic variation that exists due to sex and age. This study was designed to evaluate how age, sex, and biopsy preparations affect mitochondrial respiration in extensor digitorum longus, soleus, and gastrocnemius muscle of mice. Here we detail a modified approach to skeletal muscle biopsy of the gastrocnemius muscle of mice focused on maintenance of intact fibers that results in greater overall respiration compared to cut fibers. The improved respiration of intact fibers is sex specific as are some of the changes in mitochondrial respiration with age. This study shows the need for standard practices when measuring mitochondrial respiration in permeabilized muscle and provides a protocol to control for variation introduced during a typical mouse muscle biopsy.

Confocal Endomicroscopy of Neuromuscular Junctions Stained with Physiologically Inert Protein Fragments of Tetanus Toxin

Live imaging of neuromuscular junctions (NMJs) in situ has been constrained by the suitability of ligands for inert vital staining of motor nerve terminals. Here, we constructed several truncated derivatives of the tetanus toxin C-fragment (TetC) fused with Emerald Fluorescent Protein (emGFP). Four constructs, namely full length emGFP-TetC (emGFP-865:TetC) or truncations comprising amino acids 1066–1315 (emGFP-1066:TetC), 1093–1315 (emGFP-1093:TetC) and 1109–1315 (emGFP-1109:TetC), produced selective, high-contrast staining of motor nerve terminals in rodent or human muscle explants. Isometric tension and intracellular recordings of endplate potentials from mouse muscles indicated that neither full-length nor truncated emGFP-TetC constructs significantly impaired NMJ function or transmission. Motor nerve terminals stained with emGFP-TetC constructs were readily visualised in situ or in isolated preparations using fibre-optic confocal endomicroscopy (CEM). emGFP-TetC derivatives and CEM also visualised regenerated NMJs. Dual-waveband CEM imaging of preparations co-stained with fluorescent emGFP-TetC constructs and Alexa647-α-bungarotoxin resolved innervated from denervated NMJs in axotomized WldS mouse muscle and degenerating NMJs in transgenic SOD1G93A mouse muscle. Our findings highlight the region of the TetC fragment required for selective binding and visualisation of motor nerve terminals and show that fluorescent derivatives of TetC are suitable for in situ morphological and physiological characterisation of healthy, injured and diseased NMJs.

Neuromuscular connectomes across development reveal synaptic ordering rules

The connections between motor neurons and muscle fibers are dramatically reorganized in early postnatal life. This work attempts to better understand this synaptic rewiring by using a connectomic approach, i.e., tracing out all the connections between motor neurons and muscle fibers, at successive ages in a small mouse muscle. We reconstructed 31 partial-complete neuromuscular connectomes, using serial section scanning electron microscopy in a neonatal mouse and Brainbow-based and XFP-based fluorescent reconstructions in older animals. Our data included a total of more than 6000 neuromuscular junctions (NMJs), including complete connectomes from one newborn, seven developmental ages (P6-P9), and two adults. Analysis confirmed the massive rewiring that takes place as axons prune their motor units but add more synaptic areas at the NMJs with which they remain in contact. Interestingly, we found synaptic ordering rules that likely underlie this circuit maturation and yield the resulting adult neuromuscular pattern, as manifest in Henneman's size principle. In particular, by analyzing both the identities of axons sharing NMJs at developing ages and muscle fibers with multiple endplates, we found evidence suggesting an activity-based linear ranking of motor neurons such that neurons co-innervated the same endplates and same muscle fibers (if there were more than one endplate) when the axons were similar in activity and hence rank. In addition, this ranking provided a means for understanding action at a distance in which the activity at one neuromuscular junction can impact the fate of the axons at another junction at a different site on the same muscle fiber. These activity-dependent mechanisms provide insight into the means by which timing of activity among different axons innervating the same population of cells, that start out with nearly all-to-all connectivity, can produce a well-organized system of axons, a system that is necessary for the recruitment order of neurons during a graded behavior like muscle contraction.

In silico Identification of Key Factors Driving the Response of Muscle Sensory Neurons to Noxious Stimuli

Nociceptive nerve endings embedded in muscle tissue transduce peripheral noxious stimuli into an electrical signal [i.e., an action potential (AP)] to initiate pain sensations. A major contributor to nociception from the muscles is mechanosensation. However, due to the heterogeneity in the expression of proteins, such as ion channels, pumps, and exchangers, on muscle nociceptors, we currently do not know the relative contributions of different proteins and signaling molecules to the neuronal response due to mechanical stimuli. In this study, we employed an integrated approach combining a customized experimental study in mice with a computational model to identify key proteins that regulate mechanical nociception in muscles. First, using newly collected data from somatosensory recordings in mouse hindpaw muscles, we developed and then validated a computational model of a mechanosensitive mouse muscle nociceptor. Next, by performing global sensitivity analyses that simulated thousands of nociceptors, we identified three ion channels (among the 17 modeled transmembrane proteins and four endoplasmic reticulum proteins) as potential regulators of the nociceptor response to mechanical forces in both the innocuous and noxious range. Moreover, we found that simulating single knockouts of any of the three ion channels, delayed rectifier voltage-gated K+ channel (Kv1.1) or mechanosensitive channels Piezo2 or TRPA1, considerably altered the excitability of the nociceptor (i.e., each knockout increased or decreased the number of triggered APs compared to when all channels were present). These results suggest that altering expression of the gene encoding Kv1.1, Piezo2, or TRPA1 might regulate the response of mechanosensitive muscle nociceptors.