Mantilla, Carlos B.

This study found that inhibiting TrkB kinase activity decreases the extent of neuromuscular transmission failure during repetitive stimulation at low frequency while increasing failure with high-frequency stimulation. The frequency-dependent effect of 1NMPP1 treatment suggests that the role of TrkB signaling in maintaining neuromuscular transmission during repetitive stimulation may differ between motor unit types (slow-twitch, fatigue-resistant vs. fast-twitch, fatigable) and likely reflect effects at the presynaptic terminal without major effects on the muscle itself.

Introduction The nerve-derived growth factor neuregulin (NRG) plays a role in the regulation of skeletal muscle mass through Akt and mTOR signaling transduction pathways that regulate both protein synthesis and degradation. We previously reported that NRG increases muscle protein synthesis (~20%) in a PI3 kinase (PI3K)/Akt-dependent manner. However, the effects of NRG on protein degradation are still poorly understood and are needed to elucidate the role of NRG in the maintenance of skeletal muscle protein balance. Methods Neonatal diaphragm muscle ex vivo preparations were pharmacologically treated with NRG and pharmacological inhibitors of PI3K (LY294002, 50 μM), MEK (PD98059, 50 μM) or mTOR (rapamycin, 100 nM). Tyrosine release from muscle was used as a surrogate measure of protein degradation. Results We report that basal protein degradation in the neonatal rat diaphragm muscle is significantly reduced by NRG treatment (19%). Basal protein degradation was increased following treatment with inhibitors of PI3K, MEK or mTOR, with inhibition of each pathway sufficient to increase basal protein degradation greater than 30%. Importantly, NRG treatment in the presence of each of these inhibitors blunts the increase in protein degradation induced by inhibition of PI3K, MEK or mTOR. NRG effects were significantly blunted by rapamycin (p < 0.05 compared to NRG alone), but not by LY294002 or PD98059. Discussion We suggest that both the PI3K/Akt and MAP kinase pathways are important for NRG effects on protein degradation, but that mTOR may be a critical modulator of these effects and thus of protein balance in skeletal muscle.

Aging selectively impacts the function of type IIx/IIb fibers in the diaphragm muscle. We show that sarcopenia reduces maximum specific force, maximum shortening velocity, and peak power output, whereas endurance during repetitive contractions is preserved. These findings explain why higher force airway clearance behaviors decline with age, whereas fatigue-resistant breathing behavior remains resilient.

Neuromotor control of sighs consists of two phases: Phase I, which resembles a preceding eupneic breath, and Phase II, an extra inspiration superimposed on Phase I. The diaphragm muscle (DIAm)—the primary inspiratory muscle in mammals—is activated during multiple behaviors, including sighs, and its activity can be quantified using electromyography (EMG). Although the biphasic structure of sighs has been described using airflow and pressure signals, how these phases are represented in DIAm EMG activity—and the underlying dynamics of DIAm motor unit (MU) recruitment—remains unclear. Previously we described that eupnea consists of a period of MU recruitment followed by a period of sustained MU activity before derecruitment. We hypothesize that during sighs Phase I follows a similar trajectory of MU recruitment as eupnea while Phase II reflects superimposed additional MU recruitment. DIAm EMG was recorded using fine wire electrodes in awake Sprague-Dawley rats (n=6) of both sexes. To determine the period of DIAm MU recruitment during both Phase I and Phase II, a stationarity analysis was performed. The stationarity analysis uses a reverse arrangement test applied to sequential, non-overlapping 10-ms bins across a longer analysis window. Because DIAm MU discharge rates rarely exceed 100 Hz, it is unlikely that a single MU fires more than once within each 10-ms window; thus, non-stationarity reflects MU recruitment rather than changes in discharge rate. An average of 17 ± 4 sighs per animal were identified (range: 10-20), with a frequency of 2.8 ± 0.1 min-1. Sighs had a burst duration of 1300 ± 405 ms, much longer than the burst duration of eupneic breaths (~598 ± 111 ms). During sighs there were two distinct periods of MU recruitment reflected by non-stationarity of the EMG signal. The MU recruitment duration of Phase I was significantly shorter than Phase II (100 ± 28 ms vs. 179 ± 53 ms, p = 0.041). The onset-to-peak duration during sighs averaged 489 ± 135 ms, with the duration of Phase I lasting longer than Phase II (298 ± 119 ms vs 179 ± 56 ms). It is important to note that Phase II is characterized by rapid, continuous recruitment throughout its duration. In contrast, Phase I had a longer overall duration, although the period of MU recruitment was relatively brief—similar to eupneic breathing. Using EMG stationarity in awake animals allowed us to characterize these underlying MU patterns without altering ventilation or disrupting the intact of the respiratory system. These results indicate that sighs reflect a distinct pattern generator superimposed on that generating eupneic breathing. It remains unclear whether sighs are triggered (e.g., reflexes) or spontaneously generated. Funding: NIH NHLBI 5R01HL146114 and NIA 5R01AG044615 This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Brain-derived neurotrophic factor (BDNF) signals through its high-affinity full length tropomyosin receptor kinase-B (TrkB.FL). One key downstream target of BDNF/TrkB.FL signaling is phosphorylation of the transcription factor cyclic AMP response element-binding (CREB) protein at serine 133 (pCREBS133). We hypothesized that a positive feedback loop exists in the spinal cord in which BDNF/TrkB.FL induces pCREBS133 which then transcriptionally targets pro-BDNF expression. To test this hypothesis, we employed a loss of function approach using a novel chemogenetic rat model (TrkBF616A) with a knock-in allele sensitive to the selective kinase inhibitor 1NMPP1 to inhibit TrkB.FL kinase activity. In female and male TrkBF616A rats, we then examined the effects of 1NMPP1 (orally through drinking water) induced TrkB.FL kinase inhibition on BDNF levels in the cervical (CSC) and lumbar (LSC) spinal cord. The spinal cord was collected at two time points at 0700 and 010 reflecting the trough and peak timepoints of endogenous BDNF expression. BDNF levels were quantified using ELISA. We found in the CSC, BDNF levels did not vary across timepoints in either 1NMPP1 or vehicle treated groups. However, 1NMPP1-induced TrkB.FL inhibition significantly reduced BDNF levels at both timepoints (P=0.004). This reduction suggests that cervical circuits rely on continuous CREB-dependent support of BDNF expression and that inhibiting TrkB.FL disrupts this loop throughout the day. In the LSC, control rats displayed time of day-dependent variations in BDNF with lower levels at 0700 compared to 0100 (P=0.02). In contrast, 1NMPP1-induced inhibition of TrkB.FL reduced BDNF levels at 0100—when BDNF normally peaks in the LSC (P=0.01). This nighttime specific deficit supports the idea that activity-driven pCREBS133 activation is necessary to elevate BDNF during the active phase and that blocking TrkB.FL signaling disrupts this pCREBS133-mediated positive feedback. Overall, these results support a positive feedback that is region- and time-specific and involves BDNF/TrkB.FL signaling inducing pCREBS133 phosphorylation which then targets pro-BDNF expression in the spinal cord. Supported by NIH grants HL146114 and AG44615. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Neuromotor control of diaphragm muscle (DIAm) involves size-dependent recruitment of phrenic motor neurons (PhMNs). To accomplish breathing, only smaller PhMNs innervating slow (type S) and fast, fatigue resistant (type FR) DIAm motor units are recruited. Thus, smaller PhMNs and type S and FR DIAm motor units are very active in sustaining ventilation throughout life. Larger PhMNs innervating more fatigable fast (type FF) motor units are very infrequently recruited for higher force, expulsive behaviors. Previously, we reported that smaller PhMNs have higher mitochondrial volume densities (MVD) reflecting their more frequent activation. Mitochondria are essential for maintaining energetic support and cellular homeostasis. We hypothesized that to sustain higher MVD, mitochondrial biogenesis is higher in smaller compared to larger PhMNs. In female and male Sprague Dawley rats (~3 months of age) PhMNs were retrogradely labeled by intrapleural injection of cholera toxin B (CTB). After 3 days, mitochondria in the cervical spinal cord were labeled by subdural injection of MitoTracker Red. The cervical spinal cord was collected and labeled PhMNs and mitochondria were imaged by high-resolution confocal microscopy and reconstructed in 3D. As previously reported, we found a significant negative correlation between MVD and PhMN somal surface area. By immunohistochemistry, we found an increased nuclear to cytosolic ratio of pCREBS1333 in smaller PhMNs compared to larger PhMNs. PGC1α and TFAM expression was also higher in smaller PhMNs. These findings support size-dependent differences in pCREBS133–PGC1α–TFAM pathway promoting mitochondrial biogenesis. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Mitochondria are organelles essential for maintaining energetic support and cellular homeostasis. In phrenic motor neurons (PhMNs), mitochondria support the high level of activity necessary to sustain neuromotor control of the diaphragm muscle during breathing. Previously, we observed that small PhMNs recruited during breathing have higher mitochondrial volume density (MVD) compared to large PhMNs that are recruited infrequently during higher force airway clearance behaviors. In support, we also found that the maximum velocity of the succinate dehydrogenase activity (SDHmax) was higher in small PhMNs compared to large PhMNs. However, total mitochondrial volume was comparable across PhMNs of varying size. This suggests that the total ATP demands of PhMNs do not vary with size. The distribution of mitochondria likely reflects the demands of ATP consumption. In airway smooth muscle cells, mitochondria are not homogeneously distributed, with a higher density in the perinuclear region. In addition, mitochondria appear to be localized in close proximity to the endoplasmic reticulum (ER) most likely to support cytosolic and mitochondrial Ca 2+ regulation. The ER is also clustered in the perinuclear compartment of cells. Perinuclear clustering of mitochondria most likely reflects the higher ATP consumption close to the nucleus. We hypothesized that MVD is higher in the perinuclear region of PhMNs compared to more distal compartments. PhMNs of young (6 month), female and male Fisher 344 rats were retrogradely labeled by intrapleural injection of Alexa 488-conjugated cholera toxin B (CTB). Three days later, mitochondria were labeled by subdural injection of MitoTracker Red CMXRos. Spinal cord tissue was collected following transcranial perfusion with 4% paraformaldehyde, sectioned longitudinally at 70 µm, and mounted onto slides. Mosaics of labeled PhMNs and mitochondria were acquired in 3D (Z optical slices of 0.5 μm) using a 60x Plan Apo oil-immersion objective (NA 1.40). Using a concentric (10 mm) shell method for analysis, MVD was quantified relative to the distance relative to the nuclear membrane of PhMNs. Our results show a disproportionately higher MVD within the perinuclear region compared to more distal regions of PhMNs. This perinuclear distribution of mitochondria was found across PhMNs of varying size. These results suggest that energetic requirements of nucleus and ER are major drivers of mitochondrial biogenesis. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Brain derived neurotrophic factor (BDNF) signals through its high-affinity full-length tropomyosin receptor kinase-B (TrkB.FL) and plays role in maintaining the survival and function of phrenic motor neurons (PhMNs), neuromuscular transmission and synaptic activity. Disruption of this pathway is a hallmark of several neurodegenerative diseases associated with dysfunction and death of PhMNs with impaired mitochondrial function. BDNF/TrkB.FL signaling phosphorylates CREB at serine 133 (pCREBS133) and promotes its nuclear localization in cervical ventral horn of rats, however its downstream molecular pathway has not yet been established. Recently, we demonstrated that in human airway smooth muscle cells, pCREBS133, as a transcription factor, binds to the CRE sequence in the PGC1a promoter and activates its expression. PGC1a co-transcriptionally activates mitochondrial transcription factor A (TFAM) expression promoting mitochondrial DNA (mtDNA) replication and mitochondrial biogenesis. In the present study, we hypothesized that BDNF/TrkB.FL signaling promotes pCREBS133 phosphorylation and transcriptional activation of PGC1a and downstream expression of TFAM, leading to mitochondrial biogenesis. In this study, we employed a loss of function approach using a novel chemogenetic rat model (TrkBF616A) with a knock-in allele sensitive to the selective kinase inhibitor 1NMPP1 to inhibit TrkB.FL kinase activity. One group of female and male rats were treated intrathecally at C4 with either artificial cerebrospinal fluid (aCSF) or BDNF (450 ng/ 300 g b.w.; diluted in aCSF) for 40 mins, followed by collection of C3-C6 cervical ventral horn. In a second group, female and male rats were treated with 1NMPP1 (25 uM; i.p.) or the vehicle DMSO for 2 h. After 2 h, both groups were treated intrathecally with BDNF for 40 min and the C3-C6 cervical ventral horn collected. Intrathecal BDNF increased the pTrkB.FLY817 autophosphorylation and downstream phosphorylation of pCREBS133. Bioinformatic analysis identified a putative binding site of pCREBS133 on PGC1a promoter, which was confirmed by Chromatin immunoprecipitation (ChIP) assay. Consistent with this, BDNF treatment increased the expression of PGC1a and downstream TFAM, with an increase in mtDNA copy number. 1NMPP1 inhibition of TrkB.FL kinase (blunted BDNF-induced pTrkB.FLY817 phosphorylation) inhibited downstream activation of the pCREBS133/PGC1a/TFAM signaling cascade. Additionally, 1NMPP1 treatment blunted the BDNF-induced increase in mtDNA copy number. We conclude that BDNF/TrKB.FL signaling mediates mitochondrial biogenesis in the cervical ventral horn suggesting that interventions targeting this pathway could be a therapeutic strategy for neurodegenerative diseases. This research was funded by NIH Grants HL146114 & AG44615. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

The present study provides novel physiological insights into aging by measuring airway compliance and residual volume via forced oscillation perturbations using a programmable mechanical ventilator (flexiVent—currently considered a gold standard for in vivo lung function measurements). In addition, dynamic measurements of lung inflation reported herein may serve as early indicators of altered elastic properties. Taken together, the findings offer a comprehensive assessment of age-related changes in lung structure and function.