Citations

In previous studies, we found that after cervical spinal cord hemisection at C2 (SH), neuromuscular transmission improved in the rat diaphragm muscle (DIAm). We also found that exogenous brain‐derived neurotrophic factor (BDNF) treatment enhanced neuromuscular transmission, whereas inhibiting activation of its receptor (TrkB) worsened neuromuscular transmission. We hypothesized that expression of BDNF and/or its receptor TrkB at phrenic motoneurons would increase following SH. In the present study, adult male rats underwent SH or sham surgery. Phrenic motoneurons were labeled by injection of cholera toxin B into the DIAm and retrogradely‐labeled phrenic motoneurons were sampled by laser capture microdissection (LCM). BDNF and TrkB receptor (truncated – T1 and full length – FL) mRNA were measured in LCM‐microdissected motoneurons using quantitative real‐time RT‐PCR and expressed as a ratio to ribosomal S16 RNA. We found that BDNF mRNA expression in phrenic motoneurons increased by 3 days after SH (~2‐fold). Similarly, both TrkB.T1 and TrkB.FL mRNA increased by 3 days after SH (~17‐ and ~11‐fold, respectively). It is possible that the relative expression of neurotrophins and their receptors (truncated or full‐length) may determine the time‐course of restoration of phrenic activity following SH. Supported by NIH grants AR51173 and HL37680 and the Mayo Foundation.

DIAm motor units are normally recruited in an orderly fashion to accomplish different ventilatory and non‐ventilatory behaviors. Following a cervical spinal cord hemisection at C2 (SH), there is restoration of phrenic activity over time. We hypothesized that an orderly recruitment of DIAm motor units is maintained with the restoration of rhythmic DIAm electromyographic (EMG) activity following SH. A pair of electrode wires was implanted into the midcostal regions of the DIAm of adult male rats for chronic EMG recording. Three days later, these animals underwent SH surgery. We examined changes in integrated DIAm EMG activity across different motor behaviors (i.e., eupnea, hypercapnia, tracheal occlusion and fictive sneezing) assuming that DIAm EMG activity during fictive sneezing represents the maximum recruitment of DIAm motor units. As expected in controls, DIAm EMG activity increased from eupnea (~27% of max) to hypercapnia (5% CO 2; ~45% of max) to sustained tracheal occlusion (~60% of max). By 14 days after SH with restored rhythmic DIAm activity, the amplitude of EMG activity on the ipsilateral side was reduced, and the changes in EMG activity induced by increased ventilatory and non‐ventilatory drive were blunted, although an orderly recruitment of motor units was still observed. These results support a generalized strengthening of contralateral presynaptic drive over time following spinal cord injury. Supported by NIH grants AR51173 and HL37680 and the Mayo Foundation.

Phrenic motor neuron (PhMN) development in early onset hypertonia is poorly understood. Yet, respiratory disorders are a common cause of morbidity and mortality. In spa mice, an animal model of early onset hypertonia, we found ~30% fewer PhMNs, compared with controls. This PhMN loss disproportionately affected larger PhMNs. Thus, the number and heterogeneity of the PhMN pool are decreased in spa mice, likely contributing to the hypertonia, impaired neuromotor control, and respiratory disorders.

Rewarming shock (acute cardiac failure) during resuscitation from profound hypothermia is partially ameliorated by low dose epinephrine (EPI). However, hypothermia‐induced cardiac Ca 2+ overload persists with rewarming. The reactive oxygen species (ROS) scavenger superoxide dismutase (SOD) protects isolated hearts from hypothermia‐induced Ca 2+ overload. In electrically stimulated rat cardiac myocytes loaded with Ca 2+ indicators fluo‐3 (cytosolic) and rhod‐2 (mitochondria) and imaged using confocal microscopy, we investigated the effects of EPI and SOD on cytosolic vs. mitochondrial Ca 2+ during rewarming. Myocytes were cooled from 37 to 15 °C over 1h, maintained at 15 °C for 30min, and rewarmed over 1h to 37 °C. EPI 0.125 μg/ml was administered from 27 °C upwards, and SOD 0.1 mg/ml from 15 °C upwards during rewarming. Control cells did not receive drugs. Other cells received EPI alone. Hypothermia resulted in elevated cytosolic and mitochondrial Ca 2+ . Compared to normothermia, mitochondrial Ca 2+ uptake and release was slowed by hypothermia, and not immediately reversed with rewarming. EPI prolonged mitochondrial Ca 2+ overload during rewarming. With SOD, mitochondrial as well as cytosolic Ca 2+ were comparable to controls during hypothermia and rewarming. These data suggest that during rewarming, mitochondrial Ca 2+ buffering remains impaired, and that ROS may play a role in Ca 2+ overload. Administration of EPI may exacerbate mitochondrial dysfunction, potentially worsening rewarming shock. Supported by Mayo Foundation Clinical Research grants, and NIH grants HL74309 and GM56686.

The mechanisms underlying rewarming shock (cardiac failure during rewarming from profound hypothermia) are not known. Increased myocardial Ca 2+ levels and altered mitochondrial ultrastructure have been reported. We hypothesized that mitochondrial buffering of cytosolic Ca 2+ contributes to cardiac dysfunction. Enzymatically‐dissociated rat cardiac myocytes loaded with Ca 2+ indicators fluo‐3 (cytosolic) and rhod‐2 (mitochondrial) were electrically stimulated (0.5 Hz) and visualized using real time confocal microscopy. Myocytes were cooled from 37 to 15 °C over 1h, maintained at 15 °C for 30min, and rewarmed over 1h back to 37 °C. Hypothermia increased baseline mitochondrial Ca 2+ (134% of normothermic control), which returned to control levels after rewarming. In contrast, rewarming elevated cytosolic Ca 2+ significantly compared to controls (126% vs. 94%). During hypothermia both cytosolic and mitochondrial peak Ca 2+ , time to peak, and time to 50% decay increased by 135%, 150% and 200%, respectively, but returned to control levels during rewarming. These data indicate that during hypothermia, mitochondria buffer cytosolic Ca 2+ , and during rewarming, mitochondrial Ca 2+ is redistributed to the cytosol, leading to continued elevation in cytosolic Ca 2+ . The temporary Ca 2+ overload in mitochondria may further affect subsequent energy production and myocyte function. Supported by Mayo Foundation Clinical Research grants, and NIH grants HL74309 and GM56686.

The aim of the study was to evaluate the effect of severe short‐term nutritional deprivation (ND) on the fractional synthesis rates (FSR) specific proteins and protein pools in the diaphragm muscle (DiaM). Male rats were divided into two groups: 20% of usual food intake for 4 days (ND) with water provided at libitum and free‐eating control (Ctl). [U‐13‐C] leucine was continuously infused through the jugular vein for 3–3.5 h after a priming dose of the isotope. Blood samples were collected from the carotid artery at baseline and every 30 min at precursor pool plateau. DiaM was excised at the end of the isotope infusion. Mixed muscle protein (MMP), myosin heavy chain (MHC), sarcoplasmic (Sarco) and mitochondrial (Mito) proteins were extracted from DiaM and the incorporation of the label into each protein(s) was determined using mass spectrometry. The FSR of MMP, MHC, Sarco and Mito in the rat DiaM were calculated using the precursor‐product relationship. ND resulted in the reduction of the FSRs of MMP (71%), MHC (69%), Sarco (44%) and Mito (55%) in DiaM. In summary, short‐term ND is associated with a global decrease in the synthetic rate of DiaM proteins, suggesting a compromise in the ability of the DiaM to synthesize functionally important proteins in the ND state. The underlying mechanisms for this depressed state of protein synthesis in DiaM related to ND remain to be elucidated.

Signaling via the tropomyosin-related kinase receptor subtype B (TrkB) regulates neuromuscular transmission, and inhibition of TrkB kinase activity by 1NMPP1 in TrkB F616A mice worsens neuromuscular transmission failure (NMTF). We hypothesized that acute inhibition of TrkB kinase activity will impair the ability of the diaphragm muscle to produce maximal transdiaphragmatic pressure (Pdi) without impacting the ability to generate forces associated with ventilation, consistent with the greater susceptibility to NMTF in motor units responsible for higher-force nonventilatory behaviors. Adult male and female TrkB F616A mice were injected with 1NMPP1 ( n = 8) or vehicle (DMSO; n = 8) 1 h before Pdi measurements during eupneic breathing, hypoxia/hypercapnia (10% O 2 /5% CO 2 ), tracheal occlusion, spontaneous deep breaths (“sighs”) and during maximal activation elicited by bilateral phrenic nerve stimulation. In the vehicle-treated group, Pdi increased from ~10 cmH 2 O during eupnea and hypoxia/hypercapnia, to ~35 cmH 2 O during sighs and tracheal occlusion, and to ~65 cm H 2 O during maximal stimulation. There was no effect of acute 1NMPP1 treatment on Pdi generated during most behaviors, except during maximal stimulation (~30% reduction; P < 0.05). This reduction in maximal Pdi is generally similar to the worsening of NMTF previously reported with TrkB kinase inhibition in rodents. Accordingly, impaired TrkB signaling limits the range of motor behaviors accomplished by the diaphragm muscle and may contribute to neuromuscular dysfunction, primarily by impacting fatigable, higher force-generating motor units. NEW & NOTEWORTHY TrkB signaling plays an important role in maintaining neuromuscular function in the diaphragm muscle and may be necessary to accomplish the various motor behaviors ranging from ventilation to expulsive, behaviors requiring near-maximal forces. This study shows that inhibition of TrkB kinase activity impairs maximal pressure generation by the diaphragm muscle, but the ability to generate the lower pressures required for ventilatory behaviors is not impacted.

Airway inflammation is a key aspect of diseases such as asthma. Proinflammatory cytokines such as TNFα mediate the inflammatory response. In various diseases, inflammation leads to endoplasmic reticulum (ER) stress, the accumulation of unfolded proteins, which triggers homeostatic responses to restore normal cellular function. We hypothesized that TNFα triggers ER stress through an increase in reactive oxygen species generation in human airway smooth muscle (hASM) with a downstream effect on mitofusin 2 (Mfn2). In hASM cells isolated from lung specimens incidental to patient surgery, dose- and time-dependent effects of TNFα exposure were assessed. Exposure of hASM to tunicamycin was used as a positive control. Tempol (500 μM) was used as superoxide scavenger. Activation of three ER stress pathways were evaluated by Western blotting: 1) autophosphorylation of inositol-requiring enzyme1 (IRE1α) leading to splicing of X-box binding protein 1 (XBP1); 2) autophosphorylation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) leading to phosphorylation of eukaryotic initiation factor 2α; and 3) translocation and cleavage of activating transcription factor 6 (ATF6). We found that exposure of hASM cells to tunicamycin activated all three ER stress pathways. In contrast, TNFα selectively activated the IRE1α/XBP1 pathway in a dose- and time-dependent fashion. Our results indicate that TNFα does not activate the PERK and ATF6 pathways. Exposure of hASM cells to TNFα also decreased Mfn2 protein expression. Concurrent exposure to TNFα and tempol reversed the effect of TNFα on IRE1α phosphorylation and Mfn2 protein expression. Selective activation of the IRE1α/XBP1 pathway in hASM cells after exposure to TNFα may reflect a unique homeostatic role of this pathway in the inflammatory response of hASM cells.