Sieck, Gary C.

Following unilateral C2 spinal hemisection (SH), descending excitatory drive to phrenic motoneurons is disrupted causing ipsilateral diaphragm muscle paralysis. After SH, there is gradual recovery of phrenic nerve activity reflecting neuroplasticity. We hypothesized that this neuroplasticity is dependent on BDNF signaling via its TrkB receptor and kinase activity. In this study we used TrkB(F616A) knockin mice, that permit rapid and selective inhibition of TrkB kinase activity by 1NMPP1. Adult TrkB(F616A) mice were treated with 1NMPP1 (25 μM) or vehicle in the drinking water starting 3 days post‐SH. At the terminal experiment, bilateral diaphragm EMG was monitored to determine the proportion of animals displaying recovery of ipsilateral phrenic activity. At 3 days post‐SH, no animals displayed ipsilateral diaphragm EMG activity (0/10). At 14 days post‐SH, 40% (6/15) of TrkB(F616A) mice displayed ipsilateral phrenic activity, although EMG amplitude remained substantially below contralateral levels. Following 1NMPP1 treatment, no animals displayed ipsilateral diaphragm EMG activity (0/10; p<0.05). In control, wild‐type mice, 1NMPP1 treatment (for 11 days) had no evident effect on bilateral diaphragm EMG activity (n=7). These results support an essential role for TrkB kinase activity in the spontaneous recovery of rhythmic phrenic activity post spinal cord injury. Supported by NIH grant HL096750.

Airway inflammation is a key feature of asthma, with inflammatory cytokines (e.g., TNFα and IL‐13) triggering hyperreactivity (enhance contractile state) and proliferation and remodeling (enhanced synthetic state) in airway smooth muscle (ASM). We hypothesize that TNFα and IL‐13 induce sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) stress, thereby altering expression of mitochondrial fission/fusion proteins (dynamin‐related protein ‐ Drp1 and mitofusin ‐ Mfn2) with downstream impact on ASM Ca 2+ regulation and contractility as well as proliferation. Human ASM cells were isolated from lung specimens obtained incidental to patient surgery. We found that after 24 h exposing ASM cells to TNFα and IL‐13: 1) expression of SR/ER stress protein markers (PERK, calnexin and XBP1) increased; 2) Drp1 expression increased Mfn2 expression decreased; 3) mitochondrial fragmentation increased; 4) [Ca 2+ ] cyt and [Ca 2+ ] mito responses to ACh stimulation were uncoupled; 5) net [Ca 2+ ] cyt increased; 6) contractility increased; and 7) cell proliferation increased. Together these results suggest that inflammation induces SR/ER stress that leads to changes in Mfn2 and Drp1 expression, which alter mitochondrial dynamics and Ca 2+ regulation resulting in ASM hyperreactivity and remodeling. Grant Funding Source : Supported by NIH grant HL74309 (GCS)

Cervical spinal hemisection at C2 (SH) paralyzes the diaphragm muscle by interrupting descending bulbospinal pathways to phrenic motoneurons. Signaling via the neurotrophin receptor TrkB in phrenic motoneurons enhances functional recovery after SH. However, it is not clear if the delay in gene expression resulting from adeno‐associated virus (AAV)‐mediated delivery would blunt the therapeutic efficacy of AAV delivered after SH. The goal of the present study was to examine the efficacy of intrapleurally‐delivered AAV7 encoding the full‐length TrkB receptor at 3 days post‐SH. Chronic diaphragm EMG recordings were conducted in adult male Sprague‐Dawley rats confirming absence of ipsilateral activity 3 days after SH. The proportion of animals displaying recovery of ipsilateral diaphragm EMG activity increased over time post‐SH and was greater in those treated with AAV‐TrkB (by 14 days post‐SH: AAV‐TrkB: 10/15 vs. AAV‐GFP: 2/11; p=0.01). Furthermore, peak root‐mean‐square amplitude of the diaphragm EMG activity increased progressively over time post‐SH. During eupnea, animals displaying recovery of ipsilateral diaphragm EMG activity that were treated with AAV‐TrkB achieved 40% of pre‐injury values (vs. 10% in those treated with AAV‐GFP). Targeting phrenic motoneurons via intrapleural delivery of AAV7‐TrkB promotes recovery of diaphragm activity when administered 3 days post‐injury. Grant Funding Source : Supported by R01 HL96750, T32 HL105355 and Mayo Clinic Center for Regenerative Medicine

Respiratory failure is one of the most important complications of cervical spinal cord injury. The level of injury critically determines the severity of the complications. We hypothesized that higher cervical contusion injuries will compromise the motor neuron pool and the descending bulbospinal projections, causing greater functional compromise than contusions at lower levels. Unilateral contusion was performed on adult rats at either the C3 or C5 levels vs. laminectomy. No deficit was seen in ventilatory and non‐ventilatory behaviors as assessed by whole body plethysmography or chronic diaphragm electromyography up to 2 weeks. Contusion injuries at either level resulted in similarly sized cavity formation. Phrenic motor neuron loss differed, with C3 contusion causing a 50% loss of phrenic motor neurons, and C5 contusion only 20% loss compared to the contralateral side. The percent of NMJs that were denervated followed a segmental pattern. Following C3 contusion, ventral regions of the diaphragm displayed denervation of ~30% of NMJs compared to 1‐3% in the C5 contusion and laminectomy groups. In contrast, following C5 contusion, denervation was evident only in dorsal regions (~10%). These results show minimal ventilatory impairment following contusion injuries to the mid‐cervical spinal cord that result in phrenic motor neuron loss and evidence of diaphragm muscle denervation. Grant Funding Source : Supported by NIH grant R01 HL096750 and the Mayo Clinic

Ventilatory behaviors across a range of species including humans require less than 50% of maximal force‐generating capacity by the diaphragm muscle, the main inspiratory muscle in mammals. Near maximal forces are only generated during expulsive behaviors usually associated with airway clearance. There is ongoing controversy regarding the impact of unilateral lesions (e.g., spinal cord injury, denervation) on ventilatory outcomes. We hypothesized that unilateral denervation of the diaphragm muscle in rats and mice would minimally impact ventilation. Transdiaphragmatic pressure (Pdi) measurements were used to estimate diaphragm muscle force in adult male rats and mice. Following acute, unilateral phrenic nerve transection in the neck, there was no change in Pdi during eupnea or hypoxia‐hypercapnia in rats or mice. Reduced Pdi was evident during deep breaths and tracheal occlusion (behaviors associated with ~70% of maximal Pdi elicited by bilateral phrenic nerve stimulation) in both species. These results are also consistent with the lack of changes in tidal volume or respiratory pattern during eupnea measured by whole body plethysmography in unrestrained, spontaneously breathing rats after mid‐cervical contusion or hemisection injuries. These results highlight the large reserve capacity for force generation by the diaphragm muscle in rodents and the importance of measuring multiple motor behaviors. Grant Funding Source : Supported by HL096750, AG044615, HL105355 and Mayo Center for Regenerative Medicine

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.

Upper cervical spinal cord injury (SCI) disrupts descending inspiratory‐related drive to phrenic motoneurons paralyzing the diaphragm muscle. Over time following unilateral C2 hemisection (SH), there is gradual recovery of rhythmic diaphragm activity ipsilaterally, consistent with neuroplasticity. We recently reported that functional recovery after SH is enhanced by chronic intrathecal delivery of BDNF to the region of the phrenic motor neuron pool. BDNF also exerts acute effects on synaptic transmission that may enhance functional recovery. Accordingly, we hypothesized that intrathecal delivery of BDNF to the phrenic motor neuron pool acutely enhances functional recovery post‐SH. Rats were implanted with diaphragm electrodes for chronic EMG recordings post‐SH, verifying absence of ipsilateral eupneic diaphragm EMG activity by 3 and 28 days after SH. In spontaneously breathing, anesthetized animals, BDNF was administered intrathecally (30 μl) at C4 and ipsilateral rhythmic diaphragm EMG activity was restored (~120% of pre‐injury diaphragm root‐mean‐square [RMS] EMG amplitude) within ~30 min of administration. Diaphragm RMS EMG increased further following exposure to hypoxia (10% O 2 )‐hypercapnia (5% CO 2 ) and intranasal capsaicin. These results highlight the acute role of BDNF signaling in the phrenic motor neuron pool in chronically‐injured rats displaying no spontaneous recovery. Grant Funding Source : Supported by NIH grant R01 HL096750.

Recovery from accidental hypothermia followed by rewarming (H/R) is often unsuccessful due to heart failure. Although the causes for H/R‐induced heart failure remain elusive, studies at the cellular level suggest alterations in both cytoplasmic and mitochondrial Ca 2+ regulation. We hypothesize that H/R is associated with mitochondrial Ca 2+ overload, which leads to: 1) excessive reactive oxygen species (ROS) generation, 2) mitochondrial membrane depolarization, and 3) mitochondrial cytochrome c release. To test this hypothesis, isolated cardiomyocytes from adult male Sprague‐Dawley rats were subjected to an in vitro H/R protocol (1 h cooling to 15 o C held for 30 min followed by 1 h rewarming to 37 o C). Cytoplasmic (CM‐H 2 DCFDA fluorescence) and mitochondrial (MitoSOX fluorescence) ROS generation increased during cooling and remained elevated throughout the H/R protocol. Mitochondrial membrane potential (TMRM fluorescence) depolarized during rewarming suggesting mitochondrial permeability transition pore opening. After rewarming, cytochrome c content (Western blot) in cytoplasmic to mitochondrial fractions increased indicating release. These results suggest that Ca 2+ overload associated with H/R induces ROS generation leading to mitochondrial disruption. Grant Funding Source : Supported by NIH Grant 5T32HL105355‐02 (GCS)

We recently determined the presence of sarcopenia in the diaphragm muscle (DIAm), specifically an age‐related reduction in force and muscle fiber cross‐sectional area, the latter being more pronounced at type IIx and/or IIb DIAm fibers. In limb muscles, age‐related changes at neuromuscular junctions (NMJs) include retraction of presynaptic terminals, disaggregation and fragmentation of the motor end‐plate. However, it is presently not clear whether aging impacts the morphology of DIAm NMJs. Adult male mice (n=14) at 6 and 24 months of age, representing survival rates of 100 and 75%, respectively, were used to examine DIAm NMJ structure, pre‐ and post‐synaptic morphology and overlap as measured in 3D by confocal microscopy (643 NMJs). No age related differences were evident in 2D planar area (mean: 328 µm 2 ) or complexity (i.e., fragmentation) of motor end‐plates. Pre‐ and post‐synaptic volumes were also similar across age groups. Evidence of partial or full denervation was present in ~6% of NMJs in both age groups. Thus, these results indicate that by 24 months of age, DIAm sarcopenia is not associated with evidence of ongoing denervation. Examination of earlier age groups will be necessary to rule out the contribution of NMJ denervation to DIAm sarcopenia given the loss of type IIx and/or IIb fibers by 24 months of age. Grant Funding Source : Supported by R01‐AG044615, T32‐HL105355, and the Mayo Clinic.

Regulation of gap junctional intercellular communication plays a very important role in many physiological and pathophysiological processes. Our hypothesis is that tumor necrosis factor (TNF) will decrease gap junction dependent cell‐to‐cell communication of vascular connexin by disturbing connexin‐cytoskeleton interactions. Transformed HeLa cells expressing vascular connexin 37 were used in these experiments. After 1 hour, TNF (20 ng/ml) treatment resulted in near total loss of dye‐coupling (Alexa Fluor‐480) in HeLaCx37 (p<0.02, n=16) and remained constant up to 2 hours. siRNA‐mediated knockdown of ZO‐1 restored dye coupling. TNF caused a significant increase in detergent solubility of Cx37. ZO‐1 was co‐IP with Cx37 only after TNF treatment, suggesting that TNF induces a ZO‐1 and Cx37 interaction. Actin was co‐IP with Cx37 but TNF did not affect this association. N‐cadherin was not co‐IP with Cx37. Immunofluorescence double labeling for Cx37/ZO‐1 and Cx37/actin confirm the co‐IP experiments. TNF reduces gap junction coupling of Cx37 when expressed alone in epithelial cells. TNF mediates Cx37 interaction with ZO‐1 but not actin. N‐cadherin does not interact with Cx37. TNF may affect Cx37/ZO‐1 interaction resulting in reduced dye coupling.