Hernandez-Vizcarrondo, Genesis A.

Abstract Age‐associated muscle weakness and atrophy of limb muscles, termed sarcopenia, is a major factor in the morbidity of the elderly. It is becoming increasingly recognized that a substantial contribution to the sarcopenic phenotype arises from motor neuron (MN) death in old age and subsequent muscle denervation. In human and rodent models, mitochondrial dysfunction is a leading culprit in both muscle and MN deterioration with age. In other motor pools within the Fischer 344 (F344) aging rat model, we showed that sarcopenia is selective to type IIx/b muscle fibers. We also showed a loss of larger MNs and denervation of the IIx/b fibers, which together comprise more fatigable fast (type FF) motor units. This selective vulnerability of larger MNs and type IIx/b muscle fibers to sarcopenia is further reflected by reductions in oxidative capacity of IIx/b fibers, as assayed by determining the maximum velocity of the succinate dehydrogenase reaction (SDHmax). Here, we hypothesize similar changes will occur in F344 tibialis anterior (TA) muscle from young (6‐months) and old (24‐months) groups. We also predict lumbar MN death in young and old F344 rats. We found selective atrophy of type IIx/b fibers in the TA from old rats. In old age, SDHmax was reduced but only in IIx/b TA fibers. These muscle observations were concomitant with the death of larger lumbar MNs in old age. This study indicates the remarkable selectivity of type FF TA motor units to age‐associated perturbations and sarcopenia.

Phrenic motor neurons (PhMNs) innervate the diaphragm muscle (DIAm), the primary pump muscle involved in breathing. It has been shown that brain-derived neurotrophic factor (BDNF) signals through its high-affinity, full-length tropomyosin receptor kinase B (TrkB.FL) to promote PhMN survival in embryonic and early postnatal development. Reduced BDNF/TrkB.FL signaling also appears to be involved in the age-related loss of PhMNs. Previously, we developed a novel chemogenetic rat model ( TrkB F616 rat) in which insertion of a knock-in allele sensitive to 1NMPP1 (a selective kinase inhibitor) allows rapid inhibition of TrkB.FL kinase activity. Using this TrkB F616 rat, we found that in younger animals, a 14-day period of 1NMPP1-induced TrkB.FL kinase inhibition resulted in an ~20% loss of larger PhMNs similar to that found with aging. Previous studies from our laboratory demonstrated that BDNF/TrkB.FL signaling induces pCREB S133 phosphorylation at serine residue 133 in PhMNs. 1NMPP1 induced inhibition of TrkB.FL kinase activity in TrkB F616 rats acutely blunts pCREB S133 phosphorylation induced by intrathecal BDNF treatment. In another cell type, we showed that pCREB S133 transcriptionally targets PGC1α expression with downstream upregulation of expression of mitochondrial transcription factor A (TFAM) and mitochondrial biogenesis. Based on a bioinformatic analysis, we identified a putative binding site for pCREB S133 in the rat PGC1α promoter. In the present study, we hypothesized that BDNF/TrkB.FL signaling mediates pCREB S133 phosphorylation and downstream activation of PGC1α thereby promoting mitochondrial biogenesis. Adult female and male TrkB F616 rats (~ 3 months of age; 300 g) were anesthetized (ketamine/xylazine) and either 25 μM 1NMPP1 or DMSO (vehicle control) was injected ip. After 2 h, the rats were treated intrathecally (infused at C 4 ) with 100 ng/ml BDNF for 40 min. Thereafter, the animals were euthanized and the ventral cervical spinal cord containing PhMNs was dissected, and lysates were prepared for subsequent analyses. Western blot was performed to quantify pCREB S133 phosphorylation, and PGC1α mRNA and protein expression were measured by qPCR and Western blot, respectively. A chromatin immunoprecipitation (ChIP) assay confirmed that pCREB S133 binds to the PGC1α promoter and that compared to vehicle treated controls, 1NMPP1induced TrkB.FL kinase inhibition reduced the binding affinity of pCREB S133 . Consistent with these results, we found that pCREB S133 phosphorylation and PGC1α mRNA and protein expression were reduced following 1NMPP1-induced TrkB.FL kinase inhibition. We conclude that in the rat cervical ventral horn, BDNF/TrkB.FL signaling induces pCREB S133 phosphorylation which transcriptionally activates PGC1α expression and downstream mitochondrial biogenesis. The BDNF/TrkB.FL signaling pathway acting in concert with activity dependent signaling may converge on pCREB S133 phosphorylation to maintain the survival of PhMNs by promoting mitochondrial biogenesis. The loss of the contribution of BDNF/TrkB.FL signaling in old age and with neurodegeneration may lead to PhMN death. Supported by NIH grants AG44615 and HL146114 This abstract was presented at the American Physiology Summit 2025 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.

In the elderly, age-associated muscle weakness and atrophy (sarcopenia) is prevalent in both limb and respiratory muscles. In the tongue muscles, dyscoordination and sarcopenia predisposes elderly individuals to dysphagia, dysphonia and a greater risk for aspiration pneumonias and obstructive sleep apnea. In humans and in the Fischer 344 rat model of aging, motor neuron (MN) death occurs in old age, and the selective vulnerability of fast-fatigable motor units to loss is consistent with behavioral deficits in airway defense (e.g., cough). However, whether the timing of MN loss is gradual across the lifespan or precipitous from late-middle-age is unknown. Recently, the concept of an age -related increase of systemic inflammatory cytokines has been implicated in the development of sarcopenia, although the mechanisms by which this occurs remain obscure. In other cell types, inflammation triggers an unfolded protein response and mitochondrial fragmentation – processes implicated in neuronal death and neurodegeneration. In this study of female and male Fischer 344 rats at 6-, 18- and 24-months-old, we evaluated serum inflammatory cytokines (TNFα, IL1β & IL6) via ELISA, medullary unfolded protein response (pIRE1a S724 & CDK5) and mitochondrial fragmentation (pDRP1 S616 ) via Western blot, hypoglossal MN mitochondrial volume density and mitochondrial fragmentation via serial blockface electron microscopy (SBFEM), hypoglossal MN survival via Nissl histology and stereological counting and tongue muscle (longitudinal and genioglossus) sarcopenia via uniaxial specific force assessment and cross-sectional muscle fiber areas. All data were analyzed by 1-way ANOVAs or Kruskal-Wallis nonparametric tests, depending on data distributions. Here, we show that a systemic inflammation-induced unfolded protein response in the medulla and hypoglossal MNs mitochondrial fragmentation is present at 18-months-old. This mitochondrial fragmentation is consistent with increased medullary expression of pDRP1 S616 . At this age, we did not observe any evidence of tongue muscle weakness or atrophy, nor any evidence of hypoglossal MN death. At 24-months old, hypoglossal MN mitochondrial deficits remained readily observable using SBFEM, with hypoglossal MN death (specific to larger MNs) and tongue muscle sarcopenia (selective to fast-fatigable type IIx fibers) present at this age. Overall, we conclude that denervation-related sarcopenia is precipitous from late-middle-age to old age in Fischer 344 rats. This MN death is triggered (or at least preceded), by an unfolded protein response and mitochondrial fragmentation. Interventions ameliorating inflammation, the unfolded protein response and/or mitochondrial breakdown may preserve MNs and prevent or reduce age-associated sarcopenia. This work was supported by NIH grants: HL166204 (MJF), AG086136 (MJF), HL157984 (GCS) & AG44615 (GCS) This abstract was presented at the American Physiology Summit 2025 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 slow (type S) and fast, fatigue resistant (type FR) DIAm motor units are recruited. Thus, type s and FR DIAm motor units are very active in sustaining ventilation throughout life. Importantly, type S and FR DIAm motor units comprise small PhMNs that innervate type I and IIa DIAm fibers, respectively. More fatigable fast (type FF) motor units are very infrequently recruited for higher force, expulsive behaviors. Type FF DIAm motor units comprise large PhMNs that innervate type IIx/IIb DIAm fibers. Previously, we reported that compared to large PhMNs, small PhMNs have higher mitochondrial volume densities reflecting their more frequent activation. Using a quantitative histochemical technique to measure the maximum velocity of the succinate dehydrogenase reaction (SDH max ), we showed that type I and IIa DIAm fibers have higher oxidative capacity compared to type IIx/IIb fibers, which correlates with mitochondrial volume density and the more frequent activation of these muscle fibers. Accordingly, we hypothesize that the SDH max of small PhMNs is greater compared to large PhMNs. PhMNs in 6 month old, female and male Sprague Dawley rats were retrogradely identified by intrapleural injection of Alexa 647-conjugated cholera toxin B (CTB). Following retrograde CTB labeling, rats were perfused intracardially with a 0.1 M PBS, euthanized, and the C1–C6 segment of the spinal cord was dissected. Spinal cord sections were cut (40 mm) in longitudinal horizontal sections and mounted in 2-well Ibidi μ-slide plates. To measure SDH max , the sections were exposed to a solution containing 80 mM succinate (maximum substrate concentration) and 1.5 mM nitro blue tetrazolium (NBT; reaction indicator). As the SDH reaction proceeded, CTB-labeled PhMNs were imaged in 3D (Z optical slice of 0.5 μm) using an oil-immersion x60/1.4 NA objective on a Nikon Eclipse A1 laser scanning confocal system. The change in optical density (OD) was measured every 15 s over a 10-min period to measure the rate of accumulation of NBT diformazan (NBT dfz ; peak absorbance of 570 nm) in PhMNs. Using the Beer-Lambert equation, the change in OD was used to determine SDH max expressed as mmol fumarate/L cell/min. In separate experiments, following retrograde CTB labeling, mitochondria in PhMNs were labeled by transdural infusion of MitoTracker Red and imaged in 3D to determine mitochondrial volume density. Our results support a size-dependent heterogeneity in PhMN mitochondrial volume density and SDH max . We conclude that more active small PhMNs have a higher mitochondrial volume density and corresponding SDH max compared to large PhMNs. NIH grant AG44615 This abstract was presented at the American Physiology Summit 2025 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 diaphragm muscle (DIAm) as the major inspiratory pump is highly active. During breathing, fatigue resistant DIAm motor units are recruited that comprise type I and IIa fibers. Accordingly, these fibers have substantially higher mitochondrial volume density (MVD) and mitochondrial respiratory capacity (SDHmax) compared to more fatigable type IIx/IIb fibers. Cervical spinal cord injury disrupts descending inspiratory drive and markedly reduces eupneic activity of the DIAm. With C2 spinal cord hemisection (C2SH) DIAm activity on the ipsilateral side is reduced. Following a two-week period of C2SH, we found reduced MVD and SDHmax in type I and IIa DIAm fibers. As a result, we hypothesize that endurance of the DIAm is reduced. Female and male Sprague Dawley rats were used. EMG electrodes were placed in the left and right DIAm 3 days prior to C2SH surgery, and baseline recordings of eupneic DIAm activity were obtained in awake animals. The right C2 spinal cord was hemisected, and the extent of injury was confirmed by the absence of eupneic DIAm activity on the ipsilateral side while animals were anesthetized. Subsequently, DIAm EMG activity was recorded at 3, 7 and 14 days post C2SH. After two weeks, rats were anesthetized and the DIAm was excised. DIAm strips were cut and suspended in a tissue chamber containing Rees-Simpson solution maintained at 26oC, with thecostal margin clamped and the central tendon attached to a Cambridge Dual-Mode force/length transducer. DIAm strips were stimulated using platinum plate electrodes with supramaximal current (~250 mA) pulses (1 ms), and maximum specific force was determined at 75 Hz. The force-velocity relationship was then determined by systematically changing load, and maximum velocity (Vmax) was estimated based on curve fitting with the Hill-equation. Based on the force-velocity measurements peak power of the DIAm was calculated. Subsequently, the DIAm was repetitively stimulated and allowed to shorten at 30% maximum velocity (isovelocity approximating peak power output). Endurance of the DIAm was then determined as the duration that peak power output was sustained. Finally, the DIAm was rapidly frozen, and cross-sections were cut for fiber type and cross-sectional area (CSA) measurements. C2SH resulted in a marked decrease in eupneic DIAm EMG activity across the two-week period, which disproportionately affected type I and IIa DIAm fibers. However, there was no impact of C2SH on DIAm fiber type proportions or CSA. Following two-weeks C2SH, maximum specific force, Vmax, and peak power output of the DIAm were comparable between intact and injured sides. However, endurance of the DIAm was reduced on the injured side compared to the intact side. These results support our hypothesis that reduced activity of DIAm, reduces oxidative capacity of type I and IIa fibers and thereby reduces endurance. In clinical conditions such as mechanical ventilation DIAm activity is reduced and if maintained for longer periods of time this may affect DIAm endurance post weaning. Research supported by NIH grant: HL146114 (GCS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The diaphragm muscle (DIAm) shortens and produces force to generate negative intra‐thoracic pressure for airflow into the lungs and positive intra‐abdominal pressure for airway clearance and expulsive behaviors. Breathing is a low force repetitive behavior that requires recruitment of fatigue resistant type I and IIa DIAm fibers. In contrast, short duration airway clearance behaviors are infrequent and associated with greater DIAm force that require the additional recruitment of more fatigable type IIx/IIb fibers. Aging is characterized by selective atrophy of type IIx/IIb DIAm fibers and decreased specific force, i.e., sarcopenia. Older individuals are able to breathe without difficulty but have trouble with some higher force airway clearance behaviors of the DIAm such as coughing and sneezing. In 6‐ and 24‐month Fischer 344 rats (n=6 per group), DIAm strips were dissected from the mid‐costal region, mounted in a tissue chamber filled with Rees‐Simpson solution, and stimulated directly to measure maximum isometric specific force (Po) and maximum shortening velocity (V max ) using a Cambridge dual‐mode length‐force servo controller (model 300B). In addition, power fatigue was assessed during repetitive isovelocity (30%V max ) contractions evoked by 75 Hz in 400‐ms‐duration stimulus trains repeated for a 2‐min period. Additional muscle segments were rapidly frozen at optimal sarcomere length in melting isopentane, and transverse muscle fiber sections were cut at 10 µm thickness for determination of muscle fiber type (based on myosin heavy chain immunoreactivity) and fiber cross‐sectional areas. The relationship between force (load) and shortening velocity was determined using the Hill equation. Aging significantly reduced maximum isometric force, maximum shortening velocity, and maximum power output of the DIAm in older rats. The reduction in power during repetitive isovelocity shortening was reduced in older rats, but the residual power output after 2 min stimulation was comparable between young and old rats. These changes in mechanical and fatigue properties were consistent with the selective atrophy of type IIx/IIb DIAm fibers and reflected the sustained output of type I and IIa DIAm fibers, which are recruited for essential breathing behaviors.