Davis, Leah A.

Abstract The mammalian neuromuscular junction (NMJ) comprises a presynaptic terminal, a postsynaptic receptor region on the muscle fiber (endplate), and the perisynaptic (terminal) Schwann cell. As with any synapse, the purpose of the NMJ is to transmit signals from the nervous system to muscle fibers. This neural control of muscle fibers is organized as motor units, which display distinct structural and functional phenotypes including differences in pre‐ and postsynaptic elements of NMJs. Motor units vary considerably in the frequency of their activation (both motor neuron discharge rate and duration/duty cycle), force generation, and susceptibility to fatigue. For earlier and more frequently recruited motor units, the structure and function of the activated NMJs must have high fidelity to ensure consistent activation and continued contractile response to sustain vital motor behaviors (e.g., breathing and postural balance). Similarly, for higher force less frequent behaviors (e.g., coughing and jumping), the structure and function of recruited NMJs must ensure short‐term reliable activation but not activation sustained for a prolonged period in which fatigue may occur. The NMJ is highly plastic, changing structurally and functionally throughout the life span from embryonic development to old age. The NMJ also changes under pathological conditions including acute and chronic disease. Such neuroplasticity often varies across motor unit types. © 2022 American Physiological Society. Compr Physiol 12: 3731–3766, 2022.

Sarcopenia is defined as age-related atrophy of muscle fibers as well as a decrease in specific force (force per cross-sectional area). In the rat diaphragm muscle (DIAm), sarcopenia primarily affects type IIx/IIb fibers that generate greater specific force, have the fastest maximum shortening velocities, but display greater fatigue with repeated activation. We hypothesized that as a result of sarcopenia, maximum shortening velocity and power output of the DIAm decrease with age. DIAm strips ~2-3 mm wide were excised from 6-month and 24-month female (n=4 per age group) and male (n=4 per age group) rats. DIAm strips were suspended in a tissue chamber containing Rees–Simpson solution maintained at 26oC, with the costal margin clamped and the central tendon attached to a Cambridge Dual-Mode force/length transducer. To determine maximum isometric specific force, DIAm strips were stimulated using platinum plate electrodes with supramaximal current (~250 mA) pulses (1 ms) at 75 Hz delivered in 1-s duration train. The muscle strips were then allowed to shorten over a 30-ms period against different percentages of maximum isometric load. Based on the load/velocity relationship, the maximum shortening velocity (Vmax) and peak power output were determined. Subsequently, the muscle strip was repetitively stimulated and allowed to shorten at 30% maximum load (approximating peak power output). In this protocol, the muscle strip was stimulated at 75 Hz an generated isometric force during the initial 225 ms of the 330 ms duration train the muscle. Thereafter, the load was reduced to 30% maximum and the muscle strip was allowed to shorten for the subsequent 105 ms. Finally, the DIAm strip was stretched to ~150% its resting length (corresponding to optimal sarcomere length) and rapidly frozen in melting isopentane. Transverse serial sections of DIAm fibers were cut, and fiber types identified by immunoreactivity to specific myosin heavy chain (MyHC) isoforms. As previously observed, the cross-sectional areas of type IIx/IIb DIAm fibers and maximum isometric specific force were reduced in 24-month compared to 6-month rats confirming sarcopenia. The maximum shortening velocity of the DIAm in 24-month rats was slower than in the 6-month rats and peak power output of the DIAm was reduced in 24-month rats. The duration during which peak power output could be maintained (i.e., endurance) of the DIAm was prolonged in 24-month compared to 6-month rats (120 s vs 60 s, respectively). These results support our hypothesis that aging affects maximum shortening velocity and peak power output of the DIAm. It is important to note that endurance of the DIAm is preserved, consistent with resilience of breathing motor function in old age. Research supported by NIH grant AG44615 This is the full abstract presented at the American Physiology Summit 2023 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.

Two weeks of diaphragm (DIAm) inactivity imposed by C2SH caused reduced mitochondrial volume density, mitochondrial fragmentation, and a concomitant reduction of SDH max in type I and IIa DIAm fibers on the lesioned side. Type I and IIa DIAm fibers were far more sensitive to inactivation than type IIx/IIb fibers, which exhibited little pathology. Our results indicate that mitochondria in DIAm fibers are plastic in response to varying levels of activity.

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 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.

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.