Sieck, Gary C.

Developmental motor control dysfunctions, including spastic cerebral palsy (sCP) often have respiratory components. Spa mutant mice exhibit a spastic phenotype closely resembling sCP symptoms. Using the spa mouse model of spastic cerebral palsy (sCP), we quantified transdiaphragmatic pressure deficits, diaphragm muscle weakness, and fiber type-specific atrophy, improving our understanding of respiratory dysfunctions in sCP.

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.

Cellular mitochondrial function can be assessed using high resolution respirometry that measures O 2 consumption rate during various conditions that systematically alter the tricarboxylic acid (TCA) cycle or the electron transport chain (ETC). However, current high resolution respirometry does not measure O 2 consumption rate at the single cell level, but actually measures average mitochondrial function across a number of cells (either isolated or in tissues). Thus, respirometry assumes physiological homogeneity across cells. However, in many tissues, mitochondrial function varies across cells and this heterogeneity is physiologically important. Therefore, a direct measurement of cellular mitochondrial function will provide valuable novel information and physiological insight. In the present study, we used a quantitative histochemical technique to measure the activity of succinate dehydrogenase (SDH), a key enzyme located in the inner mitochondrial membrane, and the only enzyme to participate in both the TCA cycle and the ETC as Complex II. SDH mediates the oxidation of succinate to fumarate in the TCA cycle, which is coupled to the reduction of ubiquinone to ubiquinol in the ETC. In this study we determined the maximum velocity of the SDH reaction (SDH max ) in isolated human airway smooth muscle (hASM) cells using 1‐methoxyphenazine methosulphate (mPMS), as an exogenous electron carrier, and azide to inhibit cytochrome oxidase. To measure SDH max , the cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium (NBT) as the reaction indicator. hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using a Nikon Eclipse A1 laser scanning confocal system with a ×60/1.4 NA oil‐immersion lens. In the quantitative histochemical procedure, changes in cell optical density (OD) due to the progressive reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) were measured every 15 s over a 10 min period. Linearity of the SDH reaction was confirmed across the 10 min period, and SDH max was expressed as mM fumarate/liter of tissue/min. Validation of this technique included specific ETC inhibitors including oligomycin (ATP synthase inhibitor), FCCP (proton ionophore), antimycin A (Complex III inhibitor) and rotenone (Complex I inhibitor), similar to those used in high resolution respirometry. We observed that FCCP‐mediated disruption of the mitochondrial proton gradient does not affect SDH max , while SDH max is decreased by rotenone and antimycin A. In addition, we used MitoTracker Green to label and image mitochondria in hASM cells and determined mitochondrial volume density. The SDH max was then normalized to mitochondrial content. Our results confirm that this quantitative technique is rigorous and reproducible, and that measurements of cellular SDH max can serve as a reliable surrogate for the measurement of maximum mitochondrial respiration in single cells.

The polymerization of globular to filamentous actin in smooth muscle occurs upon activation and is associated with cytoskeletal remodeling. Remodeling of the cortical cytoskeleton in this manner allows for tethering of contractile proteins and force transmission to the extracellular matrix. Muscle contraction involves ATP hydrolysis and cross‐bridge cycling, which are both sensitive to internal load. During isometric activation of smooth muscle, ATP hydrolysis rate peaks initially but decreases with time, reaching a steady‐state, while maintaining maximum force. Thus, tension cost, i.e., the rate of ATP hydrolysis per unit of force, decreases over time, reflecting increased efficiency of smooth muscle contraction. We hypothesized that the time‐dependent decrease in tension cost and ATP hydrolysis are associated with actin polymerization and a subsequent increase in internal loading on cross‐bridges. In permeabilized porcine airway smooth muscle (ASM) strips, ATP hydrolysis rate and isometric force were measured simultaneously after activation with maximum Ca 2+ (pCa 4.0). Two strips were tested from each animal. One strip was treated with Cytochalasin‐D (Cyto‐D; 1 μM for 30 min), an inhibitor of actin polymerization, and one was an untreated, time‐matched control. We found that peak and steady‐state isometric force decreased by ~40%, and peak and steady‐state ATP hydrolysis rate increased by ~30% and ~20%, respectively, following Cyto‐D treatment. Thus, tension cost increased by ~100% and ~60% at peak and steady‐state, respectively. These results support our hypothesis that the time‐dependent decrease in ATP hydrolysis rate relates to an increase in internal load on cross‐bridges via actin polymerization. Inhibition of actin polymerization induced by Cyto‐D in smooth muscle impairs contractile protein tethering and therefore reduces internal loading, resulting in increases in tension cost, ATP hydrolysis rate and faster cross‐bridge cycling.

Previously, we reported that airway inflammation mediated by pro‐inflammatory cytokines such as TNFα induces hypercontractility and increased ATP consumption in human airway smooth muscle (hASM). We also found that TNFα induces an increase in maximum O 2 consumption rate as well as mitochondrial biogenesis and an increase in mitochondrial volume density. However, when normalized to mitochondrial volume, maximum O 2 consumption rate per mitochondrion is reduced in hASM after TNFα exposure. Unfortunately, standard respirometry (e.g., Seahorse) measures only the averaged maximum O 2 consumption rate of a large number of cells. In the present study, we used a quantitative histochemical technique to determine the maximum velocity of the succinate dehydrogenase reaction (SDH max ) together with mitochondrial volume density in individual hASM cells. SDH is a key enzyme in the tricarboxylic acid (TCA) cycle as well as complex II in the electron transport chain (ETC), and SDH max reflects the maximum respiratory capacity of individual mitochondrion. We hypothesized that TNFα decreases SDH max per mitochondrion in hASM cells. hASM cells were dissociated from bronchial biopsies obtained during thoracic surgeries in patients with no history of asthma or chronic obstructive pulmonary disease. The hASM cells were treated with TNFα (20 ng/ml) or vehicle (DMSO) for 24 h, and SDH max was measured using a solution containing 80 mM succinate (maximum substrate concentration) and 1.5 mM nitro blue tetrazolium (NBT) as the reaction indicator. As the SDH reaction proceeded, the hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using an oil‐immersion ×60/1.4 NA objective on a Nikon Eclipse A1 laser scanning confocal system. The change in optical density (OD), reflecting the accumulation of NBT diformazan reaction product in the hASM cell, was measured every 15 s over a 10 min period. Based on the Beer‐Lambert equation, the slope of the change in OD was used to determine SDH max expressed as mM fumarate/L tissue/min. To determine mitochondrial volume density, the same hASM cells were also loaded with MitoTracker Green (500 nM) and imaged in 3D. We found that mitochondria were more fragmented after TNFα exposure although mitochondrial volume density increased. When SDH max was normalized to mitochondrial volume, we found that SDH max per mitochondrion decreased in hASM cells, consistent with the reduction in maximum O 2 consumption per mitochondrion that we previously observed. Thus, the increased energetic demand induced by inflammation (TNFα) are met by mitochondrial fragmentation leading to biogenesis and an increase in mitochondrial volume density rather than an increase in mitochondrial respiratory capacity.

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.

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.