Sieck, Dylan C.

Histamine appears to be intimately involved with skeletal muscle responses during and following exercise. Blocking histamine’s actions during endurance exercise training by taking common over-the-counter antihistamines resulted in diminished gains in fitness. These results indicate that exercise-induced histamine release is important in generating many of the positive adaptations to exercise training.

In skeletal muscles, force gradation depends on the level of motor unit (MU) activation. MU recruitment generally follows a specific order, with MUs comprising type I or type IIa fibers more active than type IIx and/or IIb. Direct evidence of MU recruitment across motor behaviors is limited to samples of relatively few MUs within a pool. The purpose of this study was to determine the extent of diaphragm MU recruitment across different motor behaviors. We hypothesized that in vivo labeling of individual neuromuscular junctions (NMJs) by FM4‐64 reflects the activation of MUs . Adult rats were exposed to eupneic (E), hypoxic/hypercarbic (H) conditions or supramaximal phrenic nerve stimulation (S). Individual diaphragm NMJs were type‐identified by myosin heavy chain expression. Average FM4‐64 labeling for NMJs at type I or IIa fibers vs. type IIx and/or IIb fibers increased progressively from E to H to S. The proportion of labeled NMJs of each type also increased significantly. At type I or IIa fibers, 40%, 59% and 90% of NMJs were labeled during E, H and S, respectively. At type IIx and/or IIb fibers, 21%, 32% and 82% of NMJs were labeled during E, H and S, respectively. Thus, using an in vivo labeling method there is direct evidence of progressive MU recruitment across different ventilatory and non‐ventilatory behaviors in the rat diaphragm. Supported by NIH grants HL37680 and AR51173, 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.

Neuregulin (NRG) is important for expression of acetylcholine (ACh) receptors during development of the neuromuscular junction (NMJ). NRG treatment significantly increases ACh‐induced currents in hippocampal neurons, but it is not known if NRG exerts a similar effect at NMJs. Diaphragm muscle (DIAm)‐phrenic nerve preparations from 7‐ and 14‐day old rats were used to determine the effects of NRG (heregulin β177–244) on neuromuscular transmission and DIAm contractile properties. Neuromuscular transmission failure (NMTF) was assessed by the difference in force elicited by repetitive phrenic nerve stimulation (0.2 ms pulses in 40 Hz, 330 ms trains) and superimposed, intermittent direct DIAm stimulation (1 ms pulses in 40 Hz, 330 ms trains applied every 15 seconds). NMTF was higher in 7‐day old compared to 14‐day old rats (76% vs. 65%; p<0.05). Exogenous NRG treatment significantly improved neuromuscular transmission in 7‐day old rats (NMTF: 52%; p<0.05), but not in 14‐day old rats (65%). Isometric DIAm contractile properties were not affected by NRG treatment in either age group. These results indicate that NRG has important synaptic effects during early postnatal development of the DIAm. Whether NRG effects on neuromuscular transmission result from acute changes in AChR expression or alternative mechanisms remains to be explored. Supported by NIH grant AR51173 and a Career Development Award from Mayo Clinic.

Activation of the tropomyosin-related kinase receptor B (TrkB) by brain-derived neurotrophic factor acutely regulates synaptic transmission at adult neuromuscular junctions (NMJs). The role of TrkB kinase activity in the maintenance of NMJ function and structure at diaphragm muscle NMJs was explored using a chemical-genetic approach that permits reversible inactivation of TrkB kinase activity in TrkB F616A mice by 1NMPP1. Inhibiting TrkB kinase activity for 7 days resulted in significant, yet reversible, impairments in neuromuscular transmission at diaphragm NMJs. Neuromuscular transmission failure following 2 min of repetitive phrenic nerve stimulation increased from 42% in control to 59% in 1NMPP1-treated TrkB F616A mice ( P = 0.010). Recovery of TrkB kinase activity following withdrawal of 1NMPP1 treatment improved neuromuscular transmission ( P = 0.006). Electrophysiological measurements at individual diaphragm NMJs documented lack of differences in quantal content in control and 1NMPP1-treated mice ( P = 0.845). Morphological changes at diaphragm NMJs were modest following inhibition and recovery of TrkB kinase activity. Three-dimensional reconstructions of diaphragm NMJs revealed no differences in volume at motor end plates (labeled by α-bungarotoxin; P = 0.982) or presynaptic terminals (labeled by synaptophysin; P = 0.515). Inhibition of TrkB kinase activity by 1NMPP1 resulted in more compact NMJs, with increased apposition of presynaptic terminals and motor end plates ( P = 0.017) and reduced fragmentation of motor end plates ( P = 0.005). Recovery of TrkB kinase activity following withdrawal of 1NMPP1 treatment resulted in postsynaptic remodeling likely reflecting increased gutter depth ( P = 0.007), without significant presynaptic changes. These results support an essential role for TrkB kinase activity in maintaining synaptic function and structural integrity at NMJs in the adult mouse diaphragm muscle.