Sieck, Gary C.

Motor units of the diaphragm muscle (DIAm) are recruited in an orderly fashion across ventilatory and non‐ventilatory tasks. Spinal hemisection at C2 (C2HS) blocks descending ipsilateral input to phrenic motoneurons and causes DIAm paralysis. With time after C2HS, there is spontaneous recovery of phrenic nerve activity ipsilateral to the injury. We hypothesized that recruitment order of DIAm motor units is maintained following recovery of DIAm electromyographic (EMG) activity post‐C2HS. DIAm EMG activity in adult male rats was recorded chronically using a pair of electrode wires implanted into the midcostal region bilaterally. DIAm EMG activity was strongly correlated to transdiaphragmatic pressure (Pdi) measured using esophageal and gastric balloons across different motor behaviors (Eupnea, hypoxia‐hypercapnia (10%O 2 and 5%CO 2 ), tracheal occlusion, fictive sneezing and bilateral supramaximal phrenic nerve stimulation). Fictive sneezing produced forces comparable to those elicited by phrenic nerve stimulation, and thus DIAm EMG activity during fictive sneezing was considered maximal. Absence of DIAm EMG activity was verified at the time of C2HS and 3 days (3D) post‐C2HS. Over time, a subset of animals displayed functional recovery of rhythmic DIAm EMG activity ipsilateral to C2HS, although DIAm EMG activity was reduced when compared to pre‐injury levels. EMG activity increased across ventilatory and non‐ventilatory tasks associated with increasing drive in C2HS animals displaying functional recovery, supporting a generalized strengthening of contralateral presynaptic drive over time following spinal cord injury.

Upper cervical spinal cord injury disrupts descending inspiratory‐related drive to phrenic motoneurons (PMN) paralyzing the diaphragm muscle (DIAm). There is gradual recovery of rhythmic DIAm activity ipsilateral to injury suggesting strengthened spared synaptic inputs to PMN. Neurotrophins play an important role in regulating synaptic function. Thus, we hypothesized that intrathecal brain‐derived neurotrophic factor (BDNF) enhances recovery of rhythmic phrenic activity after C2 spinal cord hemisection (C2HS), whereas quenching neurotrophins with TrkB‐IgG impairs functional recovery. An intrathecal catheter was placed at C4 to chronically infuse artificial CSF, BDNF or TrkB‐Fc using an osmotic pump. DIAm EMG electrodes were implanted bilaterally to verify complete hemisection at the time of C2HS and 3D post‐C2HS. In the artificial CSF group, functional recovery was evident in 33% of C2HS rats at 7D and 56% of rats at 14D post‐injury. Intrathecal BDNF treatment enhanced functional recovery, with 88% of rats at 7D and 100% at 14D post‐C2HS displaying rhythmic phrenic activity. In contrast, intrathecal TrkB‐Fc treatment completely prevented functional recovery up to 14D post‐C2HS. Our results demonstrate that BDNF/TrkB signaling at the level of the PMN pool is both sufficient and necessary for functional recovery of rhythmic phrenic activity after C2HS. Supported by NIH grant AR51173.

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.

Regulation of intracellular Ca2+ in human airway smooth muscle (ASM) involves sarcoplasmic reticulum (SR) Ca2+ release/reuptake and plasma membrane (PM) Ca2+ influx, including store‐operated Ca2+ entry (SOCE) following SR Ca2+ depletion. Studies in human ASM have shown that siRNA suppression of stromal interaction molecule 1 (STIM1) and Orai1 decrease SOCE; however, their exact role in regulating Ca2+ are still under investigation. In human ASM, STIM1 and STIM2 were found exclusively in SR membrane, and Orai1 only in PM (especially caveolae). GFP‐tagged STIM1 transfection showed intracellular/SR localization at baseline, with increased peri‐PM aggregation following store depletion with 10ƒÝM cyclopiazonic acid (CPA). Such aggregation was prevented by microtubule inhibition (nocodazole), but not actin inhibition (phalloidin). FRET analysis revealed co‐localization of STIM1 with TRPC3 and Orai1 in CPA‐stimulated cells. In fura‐2 loaded ASM cells, SOCE was significantly reduced by STIM1 and Orai1 siRNA (less so by STIM2 siRNA). However, basal Ca2+ and agonist (ACh, histamine)‐induced Ca2+ responses were substantially modulated by STIM2 suppression. These results indicate that in human ASM, STIM1 mediates SR‐PM interactions in regulating SOCE, while STIM2 modulates sub‐maximal agonist‐induced changes in Ca2+. Supported by NIH grant HL74309 (GCS), HL090595 (CMP) and HL088029 (YSP).

Enhanced airway contractility by inflammatory cytokines involves increased intracellular Ca 2+ levels in airway smooth muscle (ASM). In this study, we examined the role of the bidirectional plasma membrane Na + /Ca 2+ exchanger (NCX) in human ASM Ca 2+ regulation following exposure to the pro‐inflammatory cytokines tumor necrosis factor alpha (TNFα) or interleukin‐13 (IL‐13). Both cytokines significantly increased NCX1 expression, with these effects inhibited by dexamethasone (NFκB inhibitor) or PD98059 (MAPK inhibitor). In human ASM cells loaded with the Ca 2+ indicator fura‐2, or the intracellular Na + indicator SBFI, NCX‐mediated Ca 2+ influx (with simultaneous Na + efflux) was enhanced by both cytokines. NCX‐mediated Ca 2+ efflux (with simultaneous Na + influx) was also enhanced, but not to the same extent. The NCX inhibitor KBR7943 as well as NCX1 siRNA inhibited cytokine enhancement of both modes NCX and of Ca 2+ responses to bronchoconstrictor agonists. These data indicate that a) NCX normally operates in human ASM; b) cytokines increase NCX expression, leading to greater NCX‐mediated Ca 2+ influx, thus contributing to enhanced Ca 2+ in inflamed ASM. Supported by NIH grants HL74309 (GCS), HL090595‐S2 (VS) and HL090595 (CMP).

The balance between protein synthesis and degradation determines if there is a net loss (atrophy) or net gain (hypertrophy) in muscle mass. Recent studies suggest that PI3K‐dependent activation of Akt is an important regulator of both protein synthesis (via Erk and mTOR) and degradation (via the transcription factor FoxO1). Unilateral denervation (DNV) of the diaphragm muscle (DIAm) increases protein synthesis at 3d and degradation by 5d post‐DNV such that protein breakdown becomes evident by 5d after DNV. We hypothesized that DIAm DNV results in time‐dependent changes in Akt activation, leading to initial mTOR and subsequent FoxO1 activation. Akt, Erk, and mTOR phosphorylation and FoxO1 nuclear translocation were measured by Western blot analyses. At 1h and 6h post‐DNV, pAkt/Akt and pErk/Erk decreased. pErk/Erk increased at 1d and 3d, and pAkt/Akt increased at 5d post‐DNV. Although mTOR phosphorylation and expression increased 5d post‐DNV, the ratio of phospho‐mTOR/mTOR did not change at any time point post‐DNV. Nuclear translocation of FoxO1 increased 1d post‐DNV. Taken together, these results indicate that concomitant changes in PI3K/Akt, Erk, mTOR, and FoxO1 activity all contribute to, but do not completely account for, the time‐dependent changes in protein balance observed after DNV. Regulation of other signaling pathways, e.g., AMPK, and/or trophic influences, e.g., neuregulin, must be considered.

Skeletal muscle growth is achieved by a net increase in muscle protein synthesis, decreased protein degradation or both. The Akt and mTOR signaling transduction pathways regulate both protein synthesis and degradation. We previously reported that the nerve‐derived growth factor neuregulin (NRG) increases muscle protein synthesis (~20%) in a PI3 kinase (PI3K)/Akt‐dependent manner. In the present study, we report that basal protein degradation in the diaphragm muscle is also significantly reduced by NRG treatment (23%). The NRG‐induced reduction of basal protein degradation is abrogated by pretreatment with inhibitors of PI3K (LY294002, 50 μM), mTOR (rapamycin, 100 nM) or MEK (PD98059, 50 μM). In fact, inhibition of any of these pathways alone is sufficient to increase basal protein degradation by greater than 30%. Importantly, NRG treatment nullifies the increase in protein degradation induced by PI3K or MEK inhibition, but not that induced by mTOR inhibition. Thus, unlike NRG effects on protein synthesis, the NRG‐induced decrease in protein degradation is abrogated not only by inhibition of the PI3K/Akt pathway, but also by inhibition of the MAP kinase. Based on these results, we suggest that both the PI3K/Akt and MAP kinase pathways are important for NRG effects on protein degradation, and that mTOR is a critical regulator of NRG effects on protein balance in skeletal muscle. Supported by NIH grant AR51173.

Autophagy maintains cellular homeostasis by the elimination of damaged proteins or organelles. Our overarching hypothesis is that autophagy contributes to aging‐related neuromuscular dysfunction. The mechanisms regulating autophagy flux are promising therapeutic targets to mitigate the effects of neuromuscular dysfunction across conditions such as aging. In previous studies, we found that autophagy flux is reduced in early old age (18‐month old mice; 90% survival), with progressive autophagosome accumulation in old age evident by increased LC3 and p62 protein expression in cervical motor neurons (24‐month old mice; 75% survival). Neurotrophins acting through the tropomyosin‐related kinase receptor subtype B (TrkB) are critical to the maintenance of neuromuscular function including neuromuscular transmission in the diaphragm muscle and generation of maximal transdiaphragmatic pressure (Pdi), both in young and aged mice. Specifically, inhibition of TrkB signaling impairs maximal Pdi generation whereas activation of TrkB signaling blunts failure in neuromuscular transmission. We hypothesize that inhibiting TrkB kinase activity reduces autophagy flux resulting in increased LC3 and p62 expression, like the aging effects at phrenic motor neurons. TrkB F616A mice at 18‐ and 24‐ months of age, harboring a mutation that induces susceptibility to 1NMPP1‐mediated TrkB kinase inhibition, were randomized to receive 1‐week treatment with vehicle or 1NMPP1 (n=6 per age and treatment group). Protein expression of the autophagy markers LC3 and p62 in cervical spinal cord (CSC) segments C2‐C6 was analyzed by Western blotting. There was a significant effect on LC3 and p62 expression of age, but not of 1NMPP1 treatment, and no age*treatment interaction. LC3B expression increased by ~50% and p62 expression increased by ~30% in 24 month‐old compared to 18 month‐old mice. These results are consistent with previous reports that there is an aging‐related impairment of autophagy and accumulation of autophagosomes; however, inhibiting TrkB kinase activity does not modulate autophagy in these age groups. Understanding the role of TrkB signaling in mediating changes in autophagy and age‐related neuromuscular dysfunction will be important in advancing potential therapeutic applications of TrkB agonists.

Glycine (Gly) is a key inhibitory neurotransmitter at phrenic motor neurons (PhMN). In development, Gly is excitatory, transitioning to inhibitory action in the postnatal period. In adult conditions such as amyotrophic lateral sclerosis (ALS), excessive excitatory signaling onto motor neurons results in excitotoxic motor neuron death. However, it is not known how an imbalance of neurotransmission early in development impacts PhMNs at maturity. This is a knowledge gap, as some congenital hypertonia conditions exhibit an imbalance in neurotransmitter signaling, with these individuals suffering significant morbidity and mortality from respiratory disorders. Using an animal model of congenital hypertonia ( spa mouse [B6.Cg‐ Glrb spa /J] with a Gly receptor mutation), we evaluated PhMN and motor unit physiology. Similar to previous work in other Gly mutants, we hypothesized that spa mice will have excessive developmental PhMN loss with preserved PhMNs having a smaller somal size compared to wildtype (WT) mice. We also hypothesized that fewer PhMNs in spa mice will result in an increased diaphragm (DIAm) innervation ratio (number of DIAm fibers innervated by a PhMN) and increased DIAm neuromuscular transmission failure (NMTF). S pa and WT mice from our colony, ages 6 to 16 weeks, underwent unilateral retrograde labeling of PhMNs via rhodamine phrenic nerve dip. After three days, mice were euthanized, perfused with 4% paraformaldehyde, cervical spinal cord excised and processed for longitudinal cryosectioning (70 µm) and prepared for confocal imaging. Absolute PhMN counts were obtained using mosaic images of adjacent serial sections. Somal size measurements (i.e. surface area) were obtained using ImageJ. DIAm force was determined from DIAm strip preparations stretched to optimal length, and force was evoked by phrenic nerve stimulation at 40 Hz in 330 ms duration trains repeated each s (33% duty cycle) for 120 s. To assess NMTF, force evoked by phrenic nerve stimulation was compared to force evoked by direct DIAm stimulation superimposed every 15 s. Total DIAm fiber number was estimated in H&E stained cross‐sections. S pa mice had 30% fewer PhMNs compared to WT ( P =0.005) with somal size being 22% smaller in spa mice ( P =0.019). Spa mice had 20% NMTF during the initial stimulus train compared to 8% in WT mice with spa mice having 82% NMTF and WT having 40% NMTF at end of stimulation. Spa mice had a 39% greater innervation ratio of DIAm motor units compared to WT. Developmental PhMN loss and somal size is significantly disrupted in congenital hypertonia due to impaired Gly signaling and may impact performance of high‐force expulsive DIAm activities. In addition, the increased innervation ratio of DIAm motor units in spa mice, likely contributes to the NMTF due to branch point failure. Overall, this results in less effective airway defense and straining behaviors in congenital hypertonia. Our next step is to identify crucial developmental milestones when excessive PhMN loss occurs and the impact on large versus small PhMNs.

Vitamin D deficiency and vitamin D receptor (VDR) polymorphisms are associated with adverse pulmonary and diaphragm muscle (DIAm)-associated respiratory outcomes. We used a skeletal muscle-specific tamoxifen-inducible VDR knockout to investigate DIAm dysfunction following reduced VDR signaling. Marked DIAm weakness and atrophy of type IIx and/or IIb fibers are present in muscle-specific tamoxifen-induced VDR knockout mice compared with controls. These results show that the VDR has a significant biological effect on DIAm function independent of systemic effects on mineral metabolism.