Citations

Neuromotor control of diaphragm muscle is dependent on the orderly recruitment of phrenic motor neurons (PhMNs) in a size‐dependent manner. Type S and FR motor units comprise smaller, more easily recruited PhMNs that innervate fatigue resistant, lower force‐producing type I and type IIa muscle fibers. Type FInt and FF motor units, comprise larger, rarely recruited PhMNs innervating the more fatigable, higher force‐producing type IIx and/or IIb muscle fibers. The activity and energy demand upon PhMNs of type S and FR motor units is greater than that of PhMNs from type FInt and FF units, due to the high duty cycle (~35%) of ventilation. Accordingly, activity of type FInt and FF motor units is required for higher force behaviors that are performed intermittently over much shorter durations. These include the expulsive/straining maneuvers during coughing, sneezing, defecation and parturition. The energy demand in both PhMNs and diaphragm muscle fibers is met by mitochondria. To preserve homeostasis, mitochondrial functions are regulated by dynamic, continuous cycles of fusion and fragmentation. We hypothesized that mitochondrial density and mitochondrial fusion will be greater in smaller PhMNs, due to their more frequent activation during ventilation. In six adult (6‐month old) Fischer 344 rats, PhMNs were retrogradely labeled by intrapleural injection of Alexa488‐conjugated CTB. Three days later, cervical transdural infusion of MitoTracker Red was performed every 10 min for 1.5 h under anesthesia. Spinal cord tissue was collected following transcardial perfusion with 4% paraformaldehyde, sectioned longitudinally at 70 μm, and mounted onto slides. Two‐channel sequential confocal z‐stack (0.50 μm step size) mosaics of PhMNs and mitochondria were acquired using a 60x oil immersion objective and analyzed using Elements software (Nikon). The surface areas of PhMNs were measured and divided into tertiles. The lower tertile of PhMNs (comprising type S and FR motor units) had greater mitochondrial volume densities, greater mitochondrial aspect ratios and form factors and greater mean individual mitochondrial volumes than upper tertile PhMNs (comprising type FInt and FF motor units). We further characterized the differences in mRNA expression of mitochondrial dynamics associated proteins (Mfn2 and Drp1) in large vs. small PhMNs using fluorescent in‐situ hybridization techniques. Our results suggest that PhMNs of type S and FR motor units have increased mitochondrial density, supporting their high energy demand to sustain ventilation. Their increased mitochondrial fusion may underlie their resilience to PhMN loss in aging and disease. Support or Funding Information Supported by a National Institutes of Health grants R01‐AG044615

Congenital diaphragmatic hernia (CDH) occurs in approximately 1 in 2,500 live births; it is associated with high rates of neonatal mortality, especially in cases with severe pulmonary hypoplasia and pulmonary arterial hypertension. Treatments have focused on hernia repair and ameliorating lung hypoplasia, with the precise pathophysiological mechanisms of CDH remain unknown. We investigated whether or not diaphragm muscle (DIAm) weakness is apparent in CDH, evaluating DIAm force generation and ventilatory function in a Nitrofen rat model. 7 pregnant rats received 100 mg of Nitrofen in 2 mL of olive oil at 9 days gestational age and rat pups were collected at birth. Ventilatory function was assessed in isolated plethysmography chambers (Buxco). Following these functional assessments, neonatal pups were euthanized, and the DIAm with its rib attachments excised. The presence or absence of a CDH was confirmed and a DIAm strip was obtained either from control pups, or from the unaffected side in CDH pups. The isometric force generation of these DIAm strips and their fatigueability was assessed using field stimulation of muscle at various frequencies (5–100 Hz) and normalized to DIAm cross‐sectional area. Statistical significance was determined using a Student’s t‐test or Mann‐Whitney u test at the P <0.05 level. A marked reduction in both neonatal respiratory rate (−42%) and respiratory minute ventilation (−43%) was observed in CDH pups compared to controls ( P <0.01). We observed reduced DIAm twitch (−40%), maximum tetanic (−72%) and residual force following fatigue (−69%) in CDH pups compared to controls ( P <0.05). CDH neonatal rat pups exhibit DIAm weakness and ventilatory disturbances. Based on previous modelling of ventilatory function, it is possible that DIAm weakness contributes to the poor ventilation, as CDH DIAm forces following fatigue were less than control groups. This work has important considerations when assessing novel treatment outcomes in clinical CDH.

Pro‐inflammatory cytokines, like TNFα, mediate a cellular stress response to inflammation that may target mitochondria. In the present study, we hypothesize that in motor neuron‐like NSC‐34 cells, TNFα induces mitochondrial fragmentation and biogenesis in order to maintain overall O 2 consumption of these cells. NSC‐34 cells were differentiated by serum depletion (~24 h) until neurite structures (>50 μm) were observed. Cells were then exposed to TNFα (20 ng/ml) for 24 h. Mitochondria were labeled using MitoTracker Green and imaged using confocal microscopy. Mitochondrial morphometry was assessed by measuring aspect ratio (ratio of long to short axis) and form factor (perimeter 2 /4p·Area). Expression of mitofusin 2 (Mfn2 – fusion protein) and dynamin related protein 1 (Drp1 – fission) was determined by Western blot. From 3D reconstruction of optical slices of NSC‐34 cells, cell and mitochondrial volumes were determined and mitochondrial volume density calculated. Mitochondrial biogenesis was assessed by measuring mitochondrial DNA, and expression of PGC1α was measured. An Agilent Seahorse XF Analyzer was used to measure cellular O 2 consumption. A quantitative histochemical method was used to measure the maximum velocity of the succinate dehydrogenase (SDH max ) reaction within NSC‐34 cells to determine the functionality of the tri‐carboxylic acid (TCA) cycle and the electron transport chain (ETC). Finally, reactive oxygen species (ROS) formation was measured using MitoSox fluorescence. After exposure to TNFα, aspect ratio and form factor of mitochondria decreased by ~15% compared to untreated controls with consistent changes in Mfn2 and Drp1 expression. After TNFα exposure, mitochondrial DNA expression increased as did mitochondrial volume density, together indicating increased mitochondrial biogenesis. After TNFα exposure, O 2 consumption per mitochondrion (normalized for mitochondrial volume density) decreased. This was consistent with a TNFα‐induced decrease in SDH max . Despite the decrease in O 2 consumption and SDH max per mitochondrion after TNFα exposure, ROS formation increased reflecting mitochondrial dysfunction. We conclude that mitochondrial fragmentation, biogenesis and increased mitochondrial volume density are a homeostatic mechanism to decrease O 2 consumption per mitochondrion thereby damping ROS formation, while maintaining overall O 2 consumption to meet ATP demand. Support or Funding Information Supported by NIH grants AG44615 and HL105355.

There is an age‐associated loss of phrenic motor neurons (PhMNs) and withdrawal of pre‐synaptic terminals at neuromuscular junctions (NMJs) in 24‐month old Fischer 344 (F344) rats. Previously, we showed that age‐related PhMN loss is concomitant with neuromuscular transmission failure and diaphragm muscle (DIAm) sarcopenia (an age‐related atrophy of muscle fibers and a decrease in specific force). It is unknown if pre‐synaptic terminal withdrawal precedes PhMN loss, which would implicate a reduction in a trophic influence emanating from muscle fibers (myogenic). We hypothesized that age‐related PhMN loss results from pre‐synaptic terminal withdrawal and reduced myogenic influence, which occurs predominantly at type IIx and/or IIb DIAm fibers (innervated by larger PhMNs). F344 rats were obtained from the National Institutes of Aging. Experimental ages were chosen to represent 100% and 50% survival at 6 months and 24 months‐old, respectively. PhMNs were first retrogradely labeled by intrapleural injection of cholera toxin‐subunit B (CTB), which is dependent on binding to pre‐synaptic terminal gangliosides. Thus, with pre‐synaptic terminal withdrawal, PhMNs would not be labeled. Subsequently (3 days after CTB), PhMNs were retrogradely labeled by sectioning the phrenic nerve and dipping the proximal end into a Rhodamine dextran (Rho) solution – this labeling is not dependent on pre‐synaptic terminals. CTB and Rho labeled PhMNs were visualized by confocal microscopy. Only optical slices containing the nucleus of Rho‐ and/or CTB‐labeled PhMNs were counted. We analyzed the differences in labeling of PhMNs by CTB and Rho in both age groups. In labeled PhMNs, short and long axis measurements were used to determine somal surface areas by assuming a prolate spheroid shape. In 6‐month old rats, there was no significant difference in the number of PhMNs labeled by CTB or Rho. In 24‐month old rats, the total number of labeled PhMNs was reduced compared to the 6‐month old rats, which primarily affected larger PhMNs. Furthermore, at 24‐months, fewer PhMNs were labeled with CTB compared to Rho and those labeled with Rho had larger somal surface areas. These results support our hypothesis that the age‐related loss of larger PhMNs results from pre‐synaptic terminal withdrawal and reduced myogenic influence. Support or Funding Information Support: NIH R01‐AG044615 American Physiological Society

Inflammation triggers a homeostatic response that is mediated by pro‐inflammatory cytokines such as TNFα. We hypothesize that in a motor neuron‐like cell line, NSC‐34, TNFα decreases mitochondrial function, specifically succinate dehydrogenase (SDH) activity, which is a key enzyme in the tricarboxylic acid (TCA) cycle as well as complex II in the electron transport chain (ETC). Experiments were performed on cultured NSC‐34 cells that were differentiated by serum depletion until neurite structures (>50 μm) were observed. The differentiated cells were treated with TNFα (20 ng/ml) for 24 h. To measure the maximum velocity of the SDH reaction (SDH max ), cells were incubated in a solution containing 80 mM succinate and 1.5 mM nitro blue tetrazolium (NBT), the reaction indicator. As the SDH reaction proceeded, images were acquired every 15 s using a 40× objective on an Olympus IX71 inverted microscope. Linearity of the SDH reaction was confirmed across the 8‐min period, and SDH max was determined and expressed as mM fumarate/L tissue/min. To determine mitochondrial volume density, the cells were loaded with MitoTracker Green and imaged in 3D using a confocal microscope. The cell volume occupied by labeled mitochondria was determined from 3D reconstructions of 1 mm optical slices. In NSC‐34 cells exposed to TNFα, we found that mitochondrial volume density increased, and mitochondria were fragmented as compared to untreated controls. We also found that TNFα exposure decreased SDH max when normalized for mitochondrial volume density. In other cell types, we found that TNFα‐induces an increase in PGC1a and Drp1 expression and a decrease in Mfn2 expression, all of which affect mitochondrial structure. In related studies, we also found that TNFα exposure decreases O 2 consumption per mitochondrion in NSC‐34 cells. We conclude that by reducing the redox potential (SDH max ) of mitochondria, O 2 consumption per mitochondrion is reduced, thereby damping ROS formation. The impact of this homeostatic response is offset by mitochondrial biogenesis and an increase in mitochondrial volume density, thereby maintaining overall cellular O 2 consumption and ATP production but at reduced cost of ROS formation. Support or Funding Information Supported by NIH grants AG44615 and HL105355.

Neuroinflammation triggers a homeostatic response that is mediated by pro‐inflammatory cytokines such as TNFα. We hypothesize that in a motor neuron‐like cell line, NSC‐34, TNFα decreases mitochondrial O 2 consumption. NSC‐34 cells were differentiated by serum depletion (~24 h) until neurite structures (>50 μm) were observed. The cells were then exposed to TNFα (20 ng/ml) for 24 h. To determine mitochondrial volume density, the cells were loaded with MitoTracker Green and imaged in 3D using a Nikon A1R confocal microscope (60x oil immersion 1.4 NA). After treatment with TNFα, mitochondria appeared fragmented but mitochondrial volume density increased by ~50%, as compared to control untreated cells. A Agilent Seahorse XF Analyzer was used to determine the O 2 consumption of treated and untreated cells during a stress test that determines basal respiration, ATP production and maximum respiration. O 2 consumption was normalized to mitochondrial volume density in both treated and untreated groups. After TNFα exposure, basal respiration, ATP production and maximum respiration decreased per mitochondrion. However, overall O 2 consumption of the cells was unchanged after TNFα exposure due to an offsetting increase in mitochondrial volume density. Using MitoSox, we also measured reactive oxygen species (ROS) formation in treated and untreated NSC‐34 cells. ROS is a byproduct of mitochondrial O 2 consumption, and can cause cellular damage. After TNFα exposure, ROS formation increased compared to untreated cells. We conclude that mitochondrial biogenesis and an increase in mitochondrial volume density represent a homeostatic mechanism to reduce O 2 consumption the per mitochondrion, thereby damping ROS formation while maintaining overall cellular O 2 consumption and ATP production. Support or Funding Information Supported by NIH grant AG44615 and HL105355.

Nerve-muscle interaction is poorly understood in the context of early-onset spasticity and hypertonia. In an animal model of early-onset spasticity, spa mice, we found a marked impairment of tibialis anterior neuromuscular transmission. This impairment is associated with an increased innervation ratio (mean number of muscle fibers innervated by a single motor neuron). These disturbances may underlie weakness and gait disturbances observed in individual with developmental hypertonia and spasticity.

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.

In aged rats, diaphragm muscle (DIAm) reduced specific force and fiber cross-sectional area, sarcopenia, is selective for vulnerable type IIx and/or IIb DIAm fibers, with type I and IIa fibers being resilient. In humans, the oxidative capacity [as measured by maximum succinate dehydrogenase (SDH max ) activity] of fast-type muscle is reduced with aging, with slow-type muscle being unaffected. We hypothesized that in aged Fischer rat DIAm exhibiting sarcopenia, reduced SDH max activity would occur in type IIx and/or IIb fibers. Rats obtained from the NIA colony (6, 18, and 24 mo old) were euthanized, and ~2-mm-wide DIAm strips were obtained. For SDH max and fiber type assessments, DIAm strips were stretched (approximately optimal length), fresh frozen in isopentane, and sectioned on a cryostat at 6 μm. SDH max , quantified by intensity of nitroblue tetrazolium diformazan precipitation, was assessed in a fiber type-specific manner by comparing serial sections labeled with myosin heavy chain (MyHC) antibodies differentiating type I (MyHC Slow ), IIa (MyHC 2A ), and IIx and/or IIb fibers. Isometric DIAm force and fatigue were assessed in DIAm strips by muscle stimulation with supramaximal pulses at a variety of frequencies (5–100 Hz) delivered in 1-s trains. By 24 mo, DIAm sarcopenia was apparent and SDH max in type IIx and/or IIb fibers activity was reduced ~35% compared with 6-mo-old control DIAm. These results underscore the remarkable fiber type selectivity of type IIx and/or IIb fibers to age-associated perturbations and suggest that reduced mitochondrial oxidative capacity is associated with DIAm sarcopenia. NEW & NOTEWORTHY We examined the oxidative capacity as measured by maximum succinate dehydrogenase activity in older (18 or 24 mo old) Fischer 344 rat diaphragm muscle (DIAm) compared with young rats (6 mo old). In 24-mo-old rats, SDH activity was reduced in type IIx/b DIAm fibers. These SDH changes were concomitant with sarcopenia (reduced specific force and atrophy of type IIx/b DIAm fibers) at 24 mo old. At 18 mo old, there was no change in SDH activity and no evidence of sarcopenia.