Citations

Type I and IIa diaphragm muscle (DIAm) fibers comprise fatigue resistant slow and fast motor units that are recruited to accomplish breathing. In contrast, type IIx/IIb fibers comprise more fatigable fast motor units that are infrequently recruited only during airway protective and straining behaviors. The activity and energetic demand for type I and IIa DIAm fibers is markedly increased compared to type IIx/IIb fibers. Cervical spinal cord hemisection at C 2 (C 2 SH) removes excitatory drive for inspiratory activity of the DIAm and thereby imposing prolonged inactivity of type I and IIa fibers. We hypothesize that mitochondrial structure and function in type I and IIa DIAm fibers adapt in response to imposed inactivity induced by spinal cord hemisection at C 2 (C 2 SH). After C 2 SH, inactivity was confirmed by the absence of DIAm EMG, and after 14 days, and the effect of inactivity on mitochondrial structure and function was assessed in type identified fibers. Mitochondria in DIAm fibers were labeled using MitoTracker Green and imaged in 3‐D by fluorescence confocal microscopy. Mitochondrial volume density and morphometry were analyzed using ImageJ. In alternate serial sections of the same DIAm fibers, the maximum velocity of the succinate dehydrogenase reaction (SDH max ) was determined using a quantitative histochemical technique. Overall, mitochondrial volume density was higher in type I and IIa DIAm fibers and mitochondria were more filamentous compared to type IIx/IIb fibers. Inactivity reduced mitochondrial volume density in type I and IIa DIAm fibers and induced mitochondrial fragmentation. The SDH max values normalized for fiber volume and for mitochondrial volume were both higher in type I and IIa DIAm fibers compared to type IIx/IIb fibers. After 14 days of inactivity induced by C 2 SH, SDH max (normalized for fiber volume and for mitochondrial volume) decreased in type I and IIa. These results are consistent with mitochondrial structural and functional adaptions to the marked reduction in the energetic demand of type I and IIa DIAm fibers imposed by C 2 SH.

The diaphragm (DIAm) and tibialis anterior (TA) muscles comprise both fatigue resistant (type I and IIa) and fatigable (type IIx/IIb) muscle fibers. Type I and IIa fibers in the DIAm are very frequently recruited for breathing, whereas in the TA, type I and IIa fibers are recruited for balance and locomotion. More fatigable type IIx/IIb fibers are only infrequently recruited for higher force behaviors in both the DIAm and TA. The repetitive activation of the DIAm during breathing is associated with a duty cycle of 40‐50%, making the DIAm one of, if not the most active muscle in the body ‐ much more active than the TA. These differences in activation history lead to differences in energetic demands between the DIAm and TA. We hypothesized that type I and IIa fibers in the DIAm exhibit a greater mitochondrial volume density and respiratory capacity (SDH max ) compared to type I and IIa fibers in the TA. Muscle segments from both DIAm and TA were dissected from adult female and male Sprague‐Dawley rats (~300 g), pinned to cork at optimal sarcomere length and rapidly frozen in melting isopentane. Subsequently, transverse serial sections of muscle fibers were cut at 10 µm thickness. In a series of transverse sections, muscle fiber types were determined by immunoreactivity to specific myosin heavy chain (MyHC) isoform antibodies. In an alternate series of sections of the same muscle fibers, mitochondria were labeled using MitoTracker Green. In a third series of alternate sections, the maximum velocity of the succinate dehydrogenase reaction (SDH max ) was measured using a quantitative histochemical assay in which accumulation of the reaction product, nitroblue tetrazolium diformazan (NBT dfz ), was measured as a change in optical density (OD) in muscle fibers every 15 s for a 10‐min period. Muscle fiber sections were imaged in 3D using an Olympus Fluoview confocal microscope (40X oil immersion lens; NA 1.4) with an optical slice of 0.5 µm. In the mitoTracker labeled sections, each optical slice was deconvolved to increase spatial resolution (0.125 µm) and after thresholding, binarized images were reconstructed in 3D and analyzed for mitochondrial volume and mitochondrial complexity index (MCI= SA 3 /16µ 2 V 2 ) using ImageJ and NIS elements. Mitochondrial volume density and morphometry were analyzed using ImageJ. The mitochondrial volume density and SDH max were greater in type I and IIa fibers of the DIAm compared to the TA. On the other hand, mitochondrial volume density and SDH max of type IIx/IIb fibers were comparable between the DIAm and TA. These results support mitochondrial structural and functional adaptations in type I and IIa DIAm fibers based on activation history and greater energetic demand.

The intrinsic and extrinsic tongue muscles manipulate the position and shape of the tongue and are activated during many oral and respiratory behaviors. In old age, we found that sarcopenia results in selective atrophy of type IIx/IIb diaphragm muscle (DIAm) fibers and a reduction in maximum specific force. We hypothesized that tongue muscles are vulnerable to sarcopenia depending on the relative contribution of type IIx/IIb fibers to total muscle mass. In 6 and 24‐month‐old Fischer 344 rats (n=8 per age), different intrinsic and extrinsic togue muscles were dissected, mounted in a tissue chamber at optimal length and stimulated directly using platinum plate electrodes. Maximum isometric force and fatigue properties were then determined. After mechanical measurements, muscle segments were frozen at optimal sarcomere length in melting isopentane. Transverse sections of muscle fibers were cut at 10 µm, and fiber type was determined based on immunoreactivity to specific myosin heavy chain (MyHC) isoform antibodies. Muscle fiber images were acquired using a confocal microscope (40x 1.3NA objective), and fiber cross‐sectional areas as well as interstitial space were measured. In superior and inferior longitudinal intrinsic muscles specific force was reduced by ~25% in older rats and type IIx/IIb fibers were ~20% smaller. In the genioglossus muscle, specific force was reduced by ~20% in older rats, and the cross‐sectional areas of type IIx/IIb fibers were ~20% smaller. There were no age‐related changes in specific force or fiber cross‐sectional areas in the transversalis muscle. The residual force following 2‐min of fatiguing contractions remained similar in young compared to old rats, consistent with the selective effect of sarcopenia on type IIx/b fibers. The results of this study support our hypothesis that tongue muscles are susceptible to age‐related sarcopenia. The presence of sarcopenia may contribute to the increased incidence of obstructive sleep apnea in older individuals.

Diaphragm muscle (DIAm) sarcopenia involves selective atrophy of type IIx/IIb DIAm fibers. In addition, there is an age‐related decrease in the maximum velocity of the succinate dehydrogenase reaction (SDH max ) in type IIx/IIb fibers. SDH is a key enzyme of the tricarboxylic acid (TCA) cycle as well as complex II of the electron transport chain (ETC). These results suggest that sarcopenia may reduce mitochondrial volume density in type IIx/IIb DIAm fibers and thereby reduce SDH max . Alternatively, sarcopenia may reduce the intrinsic respiratory capacity of mitochondria in type IIx/IIb DIAm fibers. We hypothesized that there is an age‐related selective reduction in mitochondrial volume density and SDH max per mitochondrial volume in type IIx/IIb DIAm fibers. In 24 Fischer 344 rats (6‐mo: n=6 female, n=6 male; 24‐mo: n=6 female, n=6 male), DIAm samples were dissected from the mid‐costal region and rapidly frozen at optimal sarcomere length. In 10 mm cross‐sections, DIAm fiber types were determined by immunoreactivity to specific myosin heavy chain (MyHC) isoform antibodies. Mitochondria were labelled using Mitotracker Green and imaged using 3D confocal microscopy (40X oil immersion lens; NA 1.4) with an optical slice of 0.5 µm. Each optical slice was deconvolved to increase spatial resolution (0.125 µm) and after thresholding, binarized images were reconstructed in 3D and analyzed for mitochondrial volume and mitochondrial complexity index (MCI=SA 3 /16µ 2 V 2 ) using ImageJ and NIS elements. In alternate 6 mm sections, SDH max was measured using a quantitative histochemical assay in which accumulation of nitro blue tetrazolium diformazan (NBT dfz ) was measured in DIAm fibers every 15 s for 10 min. Consistent with previous findings, type IIx/IIb DIAm fibers were ~36% smaller in 24‐mo compared to 6‐mo rats (P<0.0001), whereas there were no age‐related differences in cross‐sectional areas (CSA) of type I and IIa fibers. Mitochondrial volume density was lower in IIx/IIb fibers of 24‐mo compared to 6‐mo rats (P<0.0001), whereas there were no age‐related differences in type I and IIa fibers. The MCI in type IIx/IIb fibers was lower (greater fragmentation) in 24‐mo compared to 6‐mo rats (P<0.0001), whereas the MCIs of type I and IIa fibers were similar across ages. Finally, SDH max per fiber volume was lower in type IIx/IIb DIAm fibers in 24‐mo compared with 6‐mo rats (P<0.0001), whereas there were no age‐related differences in type I and IIa fibers. When SDH max was normalized for mitochondrial volume, there was no age‐related difference in the intrinsic mitochondrial respiratory capacity in type IIx/IIb fibers. Therefore, aging results in reduced mitochondrial volume density and increased fragmentation in type IIx/IIb DIAm fibers, but no change in the intrinsic respiratory capacity of mitochondria.

In rats, there is a strong association between the expansion of phrenic motor neuron (PhMN) size (from ~21‐28 days postnatal), the emergence of mature diaphragm muscle (DIAm) myosin heavy chain (MyHC 2X and MyHC 2B ) isoform expression, alongside the manifestation of mature morphological and functional properties. Despite mice models being de rigueur for developmental investigations, the postnatal development of mouse PhMN and DIAm properties have not been comprehensively characterized. Using wildtype C57 mice at ages P7, P14, P21, P28, and >3 months (maturity) we evaluated PhMN and DIAm physiologic properties. PhMNs were labelled via rhodamine phrenic nerve dip, processed for longitudinal cryosectioning (70 µm) and prepared for confocal imaging and PhMN assessment. Ex vivo DIAm strips were used for functional assessments of maximum specific force (P o ) and fatigue. DIAm strips were also fresh‐frozen, with 8 µm cross‐sections immuno‐labelled for MyHC slow , MyHC 2A , MyHC 2X and MyHC 2X and processed for fluorescent assessment. The number of PhMNs remained stable throughout postnatal life (~195), while PhMN surface area increased steadily, ~10% from P14 to P21, P21 to P28, and P28 to maturity, with overall ~30% increase from P14 to maturity. The biggest changes in number of primary dendrites occurred between P14 and P28 and between P28 and maturity, 25% decrease in primary dendrites between each group. DIAm maximum P o increased in two phases, 3.5‐fold from P7 to P14 and remained similar until P21. The second large increase was ~3‐fold from P21 to P28 and remained steady to maturity. The fatiguability of the DIAm increased with age in two phases, with fatigability being similar from P7 and P14 and increasing by ~20% at P21. Following, fatigability of the DIAm was similar from P21 to P28 and increased by ~35% at maturity. These major PhMN and DIAm changes coincide with the steady growth of PhMN and the emergence of MyHC 2X and MyHC 2B expression in DIAm from P14 to P21 onwards. The maturation of orderly PhMN recruitment likely depends on an interplay between PhMN size properties and network inputs to a refined dendritic tree. We show that DIAm muscle fiber maturation in mice hinges on the expression of MyHC 2X and MyHC 2B isoforms that is associated with an ~10 fold increase in DIAm force‐generation capacity.

For the individual tongue muscles, relatively little quantification of uniaxial force, fatigue, and fiber type-specific properties has been documented. Here, we assessed uniaxial-specific force generation, fatigability, and muscle fiber type-specific properties in the superior and inferior longitudinal muscles, the transversalis, and the genioglossus in Fischer 344 rats. The longitudinal muscles produced the greatest isometric tetanic-specific forces. The genioglossus was more fatigue resistant and comprised higher proportions of I and IIa fibers.

Mitochondrial dysfunction and fragmentation precede neuronal death in many neurodegenerative disorders, including those involving motor neurons. In other cell types, pro‐inflammatory agents such as TNF‐α cause marked mitochondrial fragmentation. These inflammatory insults may underlie the vulnerability of specific neurons during age‐associated conditions, such as sarcopenia. We hypothesize that motor neuron exposure to pro‐inflammatory cytokines induces mitochondrial fragmentation. Experiments were conducted using a mouse motor neuron‐like cell line, NSC‐34. These cells were differentiated by serum depletion until neurite structures (>50 um) were observed, ~24 h. The differentiated NSC‐34 cells were then treated with mouse TNF‐α at varying concentrations; control (0 ng), 10 ng/ml, and 20 ng/ml for 24 h. The cells were then loaded with MitoTracker Green to image mitochondria using a Nikon A1R confocal microscope (60x oil immersion 1.4 NA). Images were analyzed using MatLab macro to calculate form factor, aspect ratio and mitochondrial volume density. TNF‐α exposure caused mitochondrial fragmentation as reflected by decreased form factor (27% reduction) and aspect ratio (12 % reduction). These results are consistent with inflammatory cytokine exposure leading to mitochondrial fragmentation in motor neurons which may increase their vulnerability mitochondrial dysfunction and cell death. Support or Funding Information NIH: RO1AG044615 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

The diaphragm muscle (DIAm) comprises four muscle fiber/ motor unit types: Fatigue resistant type S and FR motor units with type I and IIa fibers, respectively that are recruited for ventilatory behaviors; and more fatigable type FInt and FF motor units with type IIx and/or IIb fibers that are only recruited during high force expulsive/straining behaviors of the DIAm. With advanced aging in Fischer 344 rats (F344), type IIx and/or type IIb DIAm fibers are vulnerable to sarcopenia (reductions in specific force and cross‐sectional area, CSA), while type I and IIa fibers are resilient. This is consistent with the fact that ventilatory behaviors are conserved with aging, while airway clearance behaviors are compromised. Throughout life, breathing imposes a high duty cycle (>33 %) of DIAm activity and thus, a high energy demand for type I and IIa fibers that is met by a high oxidative capacity. However, the effect of advanced age on DIAm fiber oxidative capacity has not been examined. Accordingly, we hypothesize that the oxidative capacity in type I and IIa DIAm fibers is not affected by age, whereas concomitant with sarcopenia, the oxidative capacity of type IIx and/or IIb fibers is reduced. To assess DIAm fiber oxidative capacity, we measured succinate dehydrogenase (SDH) activity in type‐identified DIAm fibers from 6, 18 and 24‐month old male and female F344 rats. Following euthanasia, the DIAm was excised and 2–3 mm strips from the mid‐costal region were dissected, stretched to optimal length and rapidly frozen in melting isopentane. Serial transverse sections (6 μm) were cut to assess fiber type (based on myosin heavy chain, MyHC isoform), cross‐sectional area (CSA) and SDH activity. For SDH, sections were incubated at 25°C for 8 min in solutions containing 0 or 80 mM succinate and 1.5 mM nitro blue tetrazolium (reaction indicator), 5 mM EDTA, 0.2 mM PMS and 0.1 mM azide in 0.1 M phosphate buffer. Images were acquired every 5 s across a 15‐min period using a 20x objective on an Olympus IX71 inverted microscope. Linearity of the SDH reaction (change in optical density‐ OD) was confirmed across the 15‐min period for each fiber type. The presence of DIAm sarcopenia was confirmed in 24‐month old rats by a 25 % decrease in specific force and a 20% decrease in type IIx and/or IIb CSA. At all ages, the SDH activity of type I and IIa fibers was higher than type IIx/IIb fibers. No age‐related difference was observed in the SDH activity of type I or IIa fibers, whereas SDH activity of type IIx and/or IIb DIAm fibers was reduced at 24 months compared to younger animals. These results underscore the remarkable vulnerability of IIx and/or IIb fibers to sarcopenia, with profound resilience in I and IIa fibers. We propose that mitochondrial abnormalities may underlie the selective vulnerability of IIx and/or IIb fibers possibly due to changes at the phrenic motor neuron level. Support or Funding Information Supported by NIH grant AG44615 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

The diaphragm muscle (DIAm) comprises a mixed population of motor units, with FInt and FF units (type IIx and/or IIb fibers) vulnerable to sarcopenia ‐ age‐associated reductions of specific force and cross‐sectional area (CSA). Fatigue‐resistant S and FR units (type I and IIa fibers) are relatively spared. There is controversy as to whether aging confers an enhanced resistance to DIAm fatigue, with interpretations relying on relative measurements of force. We assessed the loss of DIAm force in 6‐ and 24‐month old Fischer 344 rats at 10, 40 and 75 Hz stimulation in 330 ms duration trains repeated each s (33% duty cycle) across a 2 min period. Although the initial specific force of the DIAm was lower in older rats across all stimulation frequencies, the final residual DIAm specific force after 2 min of stimulation was the same (~5 N/cm 2 ) at all ages. This gave the misconception of an improved fatigue resistance, especially at 40 and 75 Hz. We also found that aging is associated with an increase in the relative contributions of type I and IIa fiber CSA to DIAm mass with decreased contributions of type IIx and/or IIb fibers. When these changing contributions are factored into a fiber type‐specific model of DIAm force generation, we found that ventilatory behaviors require activation of only type I and IIa fibers, regardless of age or fatigue. By contrast, the model predicts that aging impairs the ability of the DIAm to effectively perform expulsive manoeuvres, even in the non‐fatigued condition. In summary, relative fatigue measurements are not indicative of improved fatigue resistance in aging and are an artifact of reduced initial specific force contributions from FInt and FF units. These results are consistent with previous studies showing the relative vulnerability of FInt and FF units and conserved ventilatory behaviors in aging DIAm. Support or Funding Information R01‐AG044615 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Age related loss of larger phrenic motor neurons (PhMNs) may underlie diaphragm muscle sarcopenia. We found increased mitochondrial fragmentation in larger PhMNs, similar to that reported to be associated with motor neuron loss in amyotrophic lateral sclerosis (ALS). In rodents, we observed diminished brain‐derived neurotrophic factor (BDNF) signaling via its high affinity TrkB.FL receptor in older animals. TrkB.FL is expressed on the outer mitochondrial membrane, and in ALS models, BDNF/TrkB.FL signaling promotes motor neuron survival and mitochondrial fusion. In the present study, we hypothesized that enhancing BDNF/TrkB.FL signaling increases Mfn2 expression, thereby promoting mitochondrial fusion. Studies were conducted using a mouse motor neuron‐like cell line (NSC‐34), which were differentiated by serum depletion until neurite structures were observed. The differentiated NSC‐34 cells were treated with BDNF (12.5 ng/ml for 1 h) and then loaded with MitoTracker Green to image mitochondria using a Nikon A1R confocal microscope (60× oil immersion 1.4 NA). BDNF treatment increased both form factor and aspect ratio of mitochondria by ~ 20% and 15%, respectively, while also causing a threefold increase in expression of Mfn2, indicating mitochondrial fusion. These experiments were performed in six different trials with two replicates each. The observed relationship between BDNF/TrkB.FL signaling and mitochondrial fusion/fragmentation proteins supports our hypothesis. These results are consistent with diminished BDNF/TrkB.FL signaling underlying mitochondrial fragmentation and dysfunction in age‐related PhMN loss. Support or Funding Information Supported by NIH grant AG44615 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .