Greising, Sarah M.

The diaphragm muscle is essential for breathing, and its dysfunctions can be fatal. Many disorders affect the diaphragm, including muscular dystrophies. Despite the clinical relevance of targeting the diaphragm, there have been few studies evaluating diaphragm function following a given experimental treatment, with most of these involving anti-inflammatory drugs or gene therapy. Cell-based therapeutic approaches have shown success promoting muscle regeneration in several mouse models of muscular dystrophy, but these have focused mainly on limb muscles. Here we show that transplantation of as few as 5000 satellite cells directly into the diaphragm results in consistent and robust myofiber engraftment in dystrophin- and fukutin-related protein-mutant dystrophic mice. Transplanted cells also seed the stem cell reservoir, as shown by the presence of donor-derived satellite cells. Force measurements showed enhanced diaphragm strength in engrafted muscles. These findings demonstrate the feasibility of cell transplantation to target the diseased diaphragm and improve its contractility.

The diaphragm muscle (DIAm) must be able to generate sufficient forces to sustain ventilatory and non‐ventilatory behaviors throughout the lifespan. Aging‐related decreases in DIAm fiber cross‐sectional area and specific force, i.e., sarcopenia, may impair the ability to generate transdiaphragmatic pressures (Pdi) necessary to accomplish expulsive, non‐ventilatory behaviors essential for airway clearance. We examined the effects of aging on DIAm function in mice at 6, 18, and 24 months of age. At each age, DIAm‐phrenic nerve preparations were used to determine maximal force by ex vivo contractility of the DIAm as well as neuromuscular transmission failure by repetitive nerve and muscle stimulation. By 18 months, animals had reduced Pdi during maximal forces (e.g., during tracheal occlusion) and increased DIAm neuromuscular transmission failure compared to 6 months, but no change in DIAm maximum specific force. By 24 months, Pdi remained reduced during occlusion, DIAm neuromuscular transmission failure remained increased compared to 6 months, and DIAm maximum specific force was decreased compared to both 6 and 18 months. Using this global measure of neuromuscular transmission, it is evident that impaired transmission precedes muscle weakness. Support or Funding Information Supported by R01‐AG044615.

Neurotrophic signaling is essential to the maintenance of diaphragm muscle function across the lifespan. With aging, there is an age‐related loss of neurotrophic signaling and a resultant increase in diaphragm muscle denervation and impairment in neuromuscular transmission. We hypothesized that enhancing neurotrophic signaling via the tropomyosin‐related kinase receptor subtype B (TrkB) using the small molecule TrkB agonist 7,8‐dihydroxyflavone (7,8‐DHF) would mitigate age‐related changes in diaphragm neuromuscular transmission and possibly sarcopenia (muscle atrophy and force loss). Adult male TrkB F616A mice (n=32) were randomized to receive 6‐month oral treatment with vehicle (control), 7,8‐DHF, or co‐treatment with 7,8‐DHF and 1NMPP1(a specific inhibitor of TrkB kinase activity in TrkB F616A mice) beginning at 18 and ending at 24 months of age. Mice were analyzed for neuromuscular transmission failure using diaphragm muscle‐phrenic nerve preparations, diaphragm muscle force, and morphologic characteristics of sarcopenia. Chronic 7,8‐DHF treatment did not improve diaphragm neuromuscular function or force (P≥0.25). Co‐treatment with 7,8‐DHF and 1NMPP1 decreased the ratio of the initial nerve stimulation‐derived to initial muscle stimulation‐derived force (~22%) compared to vehicle (p=0.01), consistent with a deleterious effect of inhibiting TrkB kinase activity on aging diaphragm muscle. With age, there was fiber type specific atrophy of type IIa and type IIx and/or IIb diaphragm muscle fibers. There was no evidence of a protective effect of 7,8‐DHF on muscle fiber cross‐sectional area. Taken together, these results support a role for BDNF/TrkB signaling in old age but do not support 7,8‐DHF as a therapeutic agent to mitigate age‐related effects on diaphragm muscle function. Support or Funding Information Supported by R01‐AG044615, T32‐HL105355, and the Mayo Clinic.

Neurotrophin brain-derived neurotrophic factor (BDNF) enhances neuromuscular transmission via activation of tropomyosin-related kinase receptor B (TrkB). Rat phrenic nerve-diaphragm muscle preparations were used to determine the rapid effect of BDNF on synaptic quantal release during repetitive stimulation. BDNF treatment significantly enhanced quantal release at all fiber types. BDNF increased synaptic vesicle cycling (measured using FM4-64 fluorescence uptake); conversely, inhibiting BDNF/TrkB signaling decreased FM4-64 uptake.

The force within a muscle may be assessed using intramuscular pressure measurements (IMP). However, a limiting factor in implementing this technology in the clinic is the observed variability of IMP measurements. The variability may be due in part to the flow of interstitial fluid. The objective of this study was to develop a technique for tracking interstitial fluid flow during muscle lengthening using fluorescent microspheres. Additionally, the effect of microsphere diameter on dispersion was evaluated. Fluorescent microspheres were injected into the rat tibialis anterior (TA) either before (right limb) or after (left limb) lengthening to 10% strain (n = 5). To help discriminate the effects of microsphere size, 10 µl of microspheres (0.25% by volume) were injected simultaneously: 0.1 µm in sky blue and 0.2 µm in yellow green. Once frozen, 20 µm muscle cross‐sections were stained and imaged using confocal microscopy to evaluate the microsphere's axial dispersion as a percent of muscle length. The microsphere dispersion was normalized to the muscle length. The fluorescent microspheres flowed with the interstitial fluid and did not penetrate the muscle fiber. There was significantly greater microsphere dispersion with passive strain (p‐value > 0.01) for lengthened samples (31±7%) compared to static samples (17±9%). Dispersion did not differ for the two bead sizes. In conclusion, this illustrates the microspheres track the interstitial fluid and passive lengthening results in additional dispersion. Supported by NIH under award number R01HD31476 and T32HL105355.

Sarcopenia is the age‐related decline of skeletal muscle function. Sarcopenia involves loss of motor neurons and the subsequent denervation of muscle fibers. Denervated muscle fibers can be re‐innervated through terminal axonal sprouting of neighboring axons. Given the predominant role of motor neurons in establishing muscle fiber type, we hypothesized that sarcopenia (and the associated partial denervatin effects) result in clustering of muscle fibers of the same type (classified by myosin heavy chain isoform composition). Fiber type specific immunofluorescence histology was used to quantify fiber clustering in murine diaphragm muscle (n=15) at 6 and 24 months of age. Clustering was analyzed using the distance between fibers of the same fiber type as well as quantification of the number of type‐identified fibers in a cluster. There was a 14% decrease to the closest fiber for type I and a 24% increase for type IIx and/or IIb fibers (P<0.001) in old vs. young mice. Additionally, a 34% increase to the 3 closest type IIx and/or IIb fibers was found (P<0.001). Changes in the interfiber distances for type‐identified fibers reveals important effects that consider changes in fiber size and proportion across disease conditions. This novel method of analyzing fiber type clustering may be useful in examining pathophysiological conditions of motor unit loss in neuromuscular disorders, myopathies, dystrophies, injuries, or amyotrophic lateral sclerosis. Supported by R01‐AG044615, T32‐HL105355, and the Mayo Clinic.

The diaphragm muscle (DIAm) must be able to generate sufficient forces to sustain ventilatory and non‐ventilatory behaviors throughout the lifespan. Age‐related decreases in DIAm fiber cross‐sectional area and specific force, i.e., sarcopenia, may impair the ability to generate transdiaphragmatic pressures (Pdi) necessary to accomplish expulsive, non‐ventilatory behaviors essential for airway clearance. We examined the effects of aging on DIAm function in both male and female mice at 6 and 24 months of age (n=79). Maximum DIAm specific force was 30% lower in old mice (P蠄0.001) and Pdi generated during non‐ventilatory behaviors was ~40% less in old mice (P蠄0.001). No differences in either DIAm force or Pdi were found between males and females. Collectively, these results indicate that DIAm sarcopenia is present in both sexes and significantly affects the ability to generate Pdi needed for airway clearance in old age. Supported by R01‐AG044615, T32‐HL105355, and the Mayo Clinic.

Aging is associated with respiratory complications and muscle dysfunction that may reduce the ability to perform expulsive, non‐ventilatory behaviors. We hypothesized that ventilatory function is impaired with aging and that the diaphragm muscle (DIAm) is susceptible to sarcopenia. Unrestrained‐awake ventilatory function, DIAm specific force, and fiber cross‐sectional area (CSA) were examined in a validated mouse model of advanced aging (expression of a hypomorphic allele ( H ) of BubR1 ), naturally aged (28 mo) and adult (5 mo) wild type ( wt ) mice (same genetic background). Minute ventilation and breathing frequency were decreased 17% in BubR1 H/H and 33% in aged wt mice vs. adult wt mice. Tidal volume was not different across groups. DIAm specific force was lower in both aged mice groups compared to the adult wt mice, being 62% lower in BubR1 H/H mice. DIAm fiber CSA from BubR1 H/H and aged wt mice was smaller than adult wt mice (23 and 10%, respectively). These results indicate that aging is associated with reduced ventilatory function and evidence of DIAm sarcopenia. In addition, aged mice recruit a greater percentage of DIAm fibers for eupneic breathing (greater fraction of maximum force). Together, these age‐related changes may limit the ability to accomplish expulsive, non‐ventilatory behaviors essential for airway clearance, and thus contribute to respiratory complications. Supported by Mayo Clinic.

The diaphragm muscle (DIAm) is responsible for breathing and must provide sufficient force to accomplish both ventilatory and non‐ventilatory behaviors. Age‐related changes to the DIAm, especially sarcopenia, may limit the ability to accomplish expulsive, non‐ventilatory behaviors essential for airway clearance, and thus contribute to respiratory complications. Age‐related changes in load (as a result of chest wall stiffening or lung compliance) may impact DIAm function. Adult male mice (6 and 24 months old) underwent measurements of respiratory system compliance and transdiaphragmatic pressure (Pdi) during: eupnea, hypoxia (10% O 2 ) –hypercapnia (5% CO 2 ), spontaneously occurring deep breaths, airway occlusion, and bilateral phrenic nerve stimulation. Overall respiratory system compliance decreased with age (increased load), while Pdi required for ventilatory behaviors did not change, consistent with the reserve capacity of the DIAm. The Pdi generated during deep breaths and airway occlusion were reduced 30–45% with age. These results indicate that DIAm dysfunction during aging is associated with reduced force to perform expulsive, non‐ventilatory behaviors. Reduced reserve capacity in old age may underlie increased susceptibility to lung infections and augment respiratory compromise associated with other age‐related diseases. Supported by T32‐HL‐105355 and the Mayo Clinic.

The age‐related mechanisms of diaphragm muscle (DIAm) sarcopenia are largely unknown. We hypothesize that sarcopenia may be related to neurotrophic signaling, specifically that of brain derived neurotrophic factor (BDNF) acting through the high‐affinity tropomyosin‐related kinase receptor subtype B (TrkB). In total 47, adult male TrkB F616A mice were examined at 6, 18, and 24 months of age (mo), representing survival rates of 100, 90, and 75%. At each age, ex vivo contractility of the DIAm was assessed to determine maximal force as well DIAm‐phrenic nerve neuromuscular transmission failure under the following preincubation conditions; control (vehicle) solution, BDNF, 1NMPP1 (inhibiting TrkB kinase activity), or co‐treatment of BDNF and 1NMPP1. Specific DIAm maximal twitch and isometric tetanic force, in addition to fatigue index were all negatively impacted at 24 mo (P蠄0.02). A significant interaction between age and neurotrophic treatment was found for DIAm‐phrenic nerve neuromuscular transmission failure (P蠄0.01). Specifically, at 6 and 18 mo BDNF decreased failure, while this was lost at 24 mo. Conversely, TrkB kinase inhibition increased failure at 6 mo, but this relationship was lost at both 18 and 24 mo. Taken together, neurotrophic signaling is key to the physiological function of the DIAm, however there is likely an age‐related loss of BDNF and TrkB signaling that detrimentally affects the DIAm. Grant Funding Source : Supported by R01‐AG044615, T32‐HL105355, and the Mayo Clinic.