Mitochondrial Volume Density Research Articles

Consistent changes in muscle phenotype and mitochondrial abundance underlie dive performance across multiple lineages of diving ducks.

Diving animals must sustain high muscle activity with finite oxygen (O2) to forage underwater. Studies have shown that some diving mammals exhibit changes in the metabolic phenotype of locomotory muscles compared with non-divers, but the pervasiveness of such changes across diving animals is unclear, particularly among diving birds. Here, we examined whether changes in muscle phenotype and mitochondrial abundance are associated with dive capacity across 17 species of ducks from three distinct evolutionary clades (tribes) in the subfamily Anatinae: the longest diving sea ducks, the mid-tier diving pochards and the non-diving dabblers. In the gastrocnemius (the primary swimming and diving muscle), mitochondrial volume density in both oxidative and glycolytic fiber types was 70% and 30% higher in sea ducks compared with dabblers, respectively. These differences were associated with preferential proliferation of the subsarcolemmal subfraction, the mitochondria adjacent to the cell membrane and nearest to capillaries, relative to the intermyofibrillar subfraction. Capillary density and capillary-to-fiber ratio were positively correlated with mitochondrial volume density, with no variation in the density of oxidative fiber types across tribes. In the pectoralis, sea ducks had greater abundance of oxidative fiber types than dabblers, whereas pochards were intermediate between the two. These data suggest that skeletal muscles of sea ducks have a heightened capacity for aerobic metabolism and an enhanced ability to utilize O2 stores in the blood and muscle while diving.

Heterogeneous distribution of mitochondria and succinate dehydrogenase activity in human airway smooth muscle cells

AbstractSuccinate dehydrogenase (SDH) is a key mitochondrial enzyme involved in the tricarboxylic acid cycle, where it facilitates the oxidation of succinate to fumarate, and is coupled to the reduction of ubiquinone in the electron transport chain as Complex II. Previously, we developed a confocal‐based quantitative histochemical technique to determine the maximum velocity of the SDH reaction (SDHmax) in single cells and observed that SDHmax corresponds with mitochondrial volume density. In addition, mitochondrial volume and motility varied within different compartments of human airway smooth muscle (hASM) cells. Therefore, we hypothesize that the SDH activity varies relative to the intracellular mitochondrial volume within hASM cells. Using 3D confocal imaging of labeled mitochondria and a concentric shell method for analysis, we quantified mitochondrial volume density, mitochondrial complexity index, and SDHmax relative to the distance from the nuclear membrane. The mitochondria within individual hASM cells were more filamentous in the immediate perinuclear region and were more fragmented in the distal parts of the cell. Within each shell, SDHmax also corresponded to mitochondrial volume density, where both peaked in the perinuclear region and decreased in more distal parts of the cell. Additionally, when normalized to mitochondrial volume, SDHmax was lower in the perinuclear region when compared to the distal parts of the cell. In summary, our results demonstrate that SDHmax measures differences in SDH activity within different cellular compartments. Importantly, our data indicate that mitochondria within individual cells are morphologically heterogeneous, and their distribution varies substantially within different cellular compartments, with distinct functional properties.

C2 Spinal Cord Hemisection Reduces Mitochondrial Volume Density in Phrenic Motor Neurons

The diaphragm muscle (DIAm) is the major inspiratory pump for breathing. Neural control of the DIAm during breathing involves the recruitment of fatigue resistant motor units comprising smaller phrenic motor neurons (PhMNs), which innervate type I and IIa fibers. More fatigable DIAm motor units comprise larger PhMNs that innervate type IIx/IIb fibers, which generate greater forces but are more fatigable. The fatigable motor units are only infrequently recruited for expulsive behaviors of the DIAm. Thus, there are marked differences in the activation history of DIAm motor units. Consistent with their higher activity levels, previous studies from our lab have shown that smaller PhMNs and type I and IIa DIAm fibers have greater mitochondrial volume density (MVD). PhMNs located in the cervical spinal cord (C3-C5) receive direct excitatory input from medullary premotor neurons, which is primarily ipsilateral. Thus, C2 spinal cord hemisection (C2SH) disrupts the neural pathway essential for breathing and significantly reduces DIAm activity on the same side. Therefore, we hypothesized that C2SH significantly reduces MVD of smaller PhMNs. In Sprague Dawley rats, PhMNs on both sides were retrogradely labeled by intrapleural injection of cholera toxin B (CTB) 3 days prior to C2SH (right side). In addition, DIAm EMG activity was recorded on both sides by inserting bipolar electrodes. Mitochondria were labeled by intraspinal injection of MitoTracker Red and imaged in identified PhMNs using 3D confocal microscopy. A series of optical slices (0.5 mm) of PhMNs were deconvolved to improve contrast, then thresholded for MitoTracker labeling followed by 3D reconstruction, providing detailed images of mitochondrial morphometry and distribution within PhMNs. In sham rats, the MVD of smaller (lower somal surface area) PhMNs was higher compared to larger PhMNs. C2SH reduced DIAm EMG activity on the ipsilateral side across a 14-day period, while activity on the contralateral side increased. After 14 days, C2SH reduced MVD by ~25% in smaller PhMNs on the ipsilateral side. We conclude that the reduced activity of smaller PhMNs induced by C2SH induced mitochondrial morphological changes, most likely due to reduced mitochondrial biogenesis. These results indicate that reduced DIAm activity associated with mechanical ventilation may impose mitochondrial remodeling that reduces mitochondrial oxidative capacity in PhMNs. These adaptations may disrupt mitochondrial function and impair weaning from mechanical ventilation. Research support by NIH grants HL166204, HL146114 and AG44615. 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.

Knockdown of the SDHB Subunit Induces Mitochondrial Remodeling in Human Airway Smooth Muscle Cells

Succinate dehydrogenase (SDH) is a mitochondrial enzyme that serves a dual role in mitochondrial respiration: catalyzing the oxidation of succinate to fumarate in the tricarboxylic acid (TCA) cycle and transferring electrons from succinate to ubiquinone as complex II in the electron transport chain (ETC). SDH is composed of four subunits (SDHA-SDHD) and the loss of SDHB subunit, specifically, has been shown to increase oxidative stress. As a part of the catalytic core of the SDH complex, the SDHB subunit mediates electron transfer to ubiquinone. In human airway smooth muscle (hASM) cells with siRNA-mediated knockdown of SDHB, the maximum velocity of the SDH reaction (SDHmax) is decreased. Moreover, SDHB-knockdown leads to an increase in mitochondrial volume density, indicating a role of SDH in mitochondrial remodeling. However, the impact of SDHB-knockdown on mitochondrial dynamics has not been explored. Therefore, we hypothesize that SDHB-knockdown promotes mitochondrial fragmentation as part of mitochondrial remodeling in hASM cells. To investigate this hypothesis, bronchiolar tissue samples were collected in female and male patients undergoing lung surgery with no current history of smoking or respiratory diseases, the smooth muscle layer was dissected and hASM cells were dissociated. The targeted knockdown of SDHB was achieved by siRNA-mediated knockdown. Mitochondria in hASM cells were labeled with MitoTracker Green (200 nM) and imaged in 3D with a 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. Optical slices were deconvolved to enhance contrast and the MitoTracker labeling was thresholded to create binary images, which were then reconstructed in 3D. From these images, the mitochondrial complexity index (MCI) was calculated to quantify the extent of fragmentation. To identify the signaling pathway involved in mitochondrial fragmentation, the expression and phosphorylation of dynamin-related protein 1 (DRP1) was quantified by Western blot. Mitochondrial fragmentation is associated with increased phosphorylation of DRP1 at Ser616 (pDRP1s616). Furthermore, mitophagy was assessed by determining the mitochondrial expression of PINK1 and the phosphorylation of Parkin at Ser65 (pParkinS65). Mitochondrial biogenesis was also evaluated through the expression of PGC1α, NRF1/2 and TFAM. Our results show that SDHB-knockdown increased mitochondrial fragmentation (reduced MCI) in hASM cells, and increased pDRP1s616 phosphorylation and its translocation to mitochondria. SDHB-knockdown also increased mitochondrial expression of PINK1 and pParkins65 phosphorylation indicating activation of mitophagy. We also confirmed that SDHB-knockdown increased expression of PGC1α, NRF1/2 and TFAM consistent with mitochondrial biogenesis. These findings unveil the mechanisms underlying SDHB-knockdown-mediated mitochondrial remodeling. Supported by NIH grant R01-HL157984 (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.

Impact of Reduced Activity on Endurance of Rat Diaphragm Muscle

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.

Mitochondrial Fragmentation Increases with Aging in Rats

The diaphragm muscle (DIAm) is the major pump muscle for breathing. Neural control of the DIAm involves recruitment of phrenic motor neurons (PhMNs) comprising motor units that vary in their mechanical and fatigue properties. Smaller PhMNs innervating fatigue resistant DIAm fibers are highly active being recruited during breathing, while larger PhMNs are infrequently recruited during more forceful expulsive behaviors. Previously, we found that to support their higher activity levels, smaller PhMNs have higher mitochondrial volume density and are less fragmented compared to larger PhMNs. With aging, larger PhMNs are more vulnerable and ~30% are lost, and those surviving display greater mitochondrial fragmentation. We also found that older rats exhibit elevated levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFa), which in other tissue has been linked to mitochondrial fragmentation via activation of the IRE1a S724-mediated endoplasmic reticulum (ER) stress pathway. Accordingly, we hypothesized that in 24-month-old rats, increased TNFa levels activate the pIRE1aS724-mediated ER stress pathway, leading to XBP1s splicing, which transcriptionally increases expression of cyclin-dependent kinases (CDK1 and CDK5) that mediate phosphorylation of DRP1 at Serine 616 (pDRP1S616), and ultimately mitochondrial fragmentation. We found that serum TNFa levels increased ~3.5 fold in 24-month rats compared to 6-month rats. Consistent with elevated TNFa, we found increased pIRE1aS724 and XBP1s splicing in the cervical ventral horn of 24-month rats. In retrogradely labeled PhMNs, we found XBP1s was localized to the nucleus, consistent with transcriptional activation of gene expression. Bioinformatic analysis and ChIP assay confirmed XBP1s binding to the CDK1 and CDK5 promoter regions in rat cervical ventral horn. Both CDK1 and CDK5 mRNA and protein were elevated in the cervical ventral of 24-month rats. In aged rats, pDRP1S616 phosphorylation increased with a corresponding increase in mitochondrial fragmentation. Our results support our hypothesis that chronic inflammation (elevated TNFa levels) in older rats activate the pIRE1aS724-mediated ER stress pathway, leading to XBP1s splicing, which transcriptionally targets CDK1 and CDK5 expression mediating pDRP1S616 phosphorylation and mitochondrial fragmentation. Research Supported by NIH R01 grant AG44615. 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.

BDNF/TrkB Induces pCREBS133 Phosphorylation and Activates Mitochondrial Biogenesis in Rat Phrenic Motor Neurons

Neural control of the diaphragm muscle (DIAm) is essential to sustain breathing. During breathing, fatigue resistant DIAm motor units are recruited comprising smaller phrenic motor neurons (PhMNs) and type I and IIa muscle fibers. Larger PhMNs comprise more fatigable DIAm motor units that are infrequently active during expulsive behaviors. The difference in activation history is reflected in the higher mitochondrial volume density in smaller PhMNs. In PhMNs, brain-derived neurotrophic factor (BDNF) signals through its high-affnity receptor, tropomyosin receptor kinase B (TrkB.FL). Previously, we found that intrathecal BDNF treatment induces CREB phosphorylation at Serine 133 (pCREBs133). We hypothesized that pCREBs133 binds to the PGC1a promoter, transcriptionally targeting PGC1a expression, mediating downstream increased expression of NRF1, NRF2, and TFAM and mitochondrial biogenesis. To test this hypothesis, we used a chemosensitive TrkBF616 rat model with a knock-in allele sensitive to 1NMPP1 in that TrkB kinase is inhibited following 1NMPP1, a small molecule derivative of the protein kinase inhibitor protein phosphatase 1 that plays a significant role inhibiting the TrkB phosphorylation and activation. Systemic treatment of 1NMPP1 in TrkBF616 rats inhibits TrkB kinase activity. A bioinformatic analysis was performed to identify a putative binding site for pCREBs133 in the PGC1a promoter sequence in rats. Using a chromatin immunoprecipitation (ChIP) assay we found that pCREBS133 binds to the PGC1a promoter in rat cervical ventral horn. We showed that intrathecal BDNF treatment increased pCREBs133 phosphorylation, which was blocked by 1NMPP1 treatment in TrkBF616 rats. PGC1a, NRF1/2, and TFAM protein was compared in cervical ventral horn lysates from BDNF- and BDNF+1NMPP1-treated TrkBF616 rats by qRT-PCR and Western Blot. Relative mtDNA copy number (normalized to nuclear DNA) was quantified by qPCR to assess the mitochondrial biogenesis. All results were analyzed by paired t-test. Following 1NMPP1-induced inhibition of TrkB kinase, PGC1a, NRF1/2, and TFAM mRNA and protein expression was reduced. Consistent with these findings, mtDNA copy number was decreased following TrkB kinase inhibition by 1NMPP1. We conclude that inhibition of TrkB kinase by 1NMPP1 reduces PGC1a-mediated mitochondrial biogenesis in rat cervical ventral horn. Supported by NIH grants AG44615 and HL146114. 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.

Skeletal muscle mitochondria demonstrate similar respiration per cristae surface area independent of training status and sex in healthy humans.

The impact of training status and sex on intrinsic skeletal muscle mitochondrial respiratory capacity remains unclear. We examined this by analysing human skeletal muscle mitochondrial respiration relative to mitochondrial volume and cristae density across training statuses and sexes. Mitochondrial cristae density was estimated in skeletal muscle biopsies originating from previous independent studies. Participants included females (n=12) and males (n=41) across training statuses ranging from untrained (UT, n=8), recreationally active (RA, n=9), active-to-elite runners (RUN, n=27) and cross-country skiers (XC, n=9). The XC and RUN groups demonstrated higher mitochondrial volume density than the RA and UT groups while all active groups (RA, RUN and XC) displayed higher mass-specific capacity of oxidative phosphorylation (OXPHOS) and mitochondrial cristae density than UT. Differences in OXPHOS diminished between active groups and UT when normalising to mitochondrial volume density and were lost when normalising to muscle cristae surface area density. Moreover, active females (n=6-9) and males (n=15-18) did not differ in mitochondrial volume and cristae density, OXPHOS, or when normalising OXPHOS to mitochondrial volume density and muscle cristae surface area density. These findings demonstrate: (1) differences in OXPHOS between active and untrained individuals may be explained by both higher mitochondrial volume and cristae density in active individuals, with no difference in intrinsic mitochondrial respiratory capacity (OXPHOS per muscle cristae surface area density); and (2) no sex differences in mitochondrial volume and cristae density or mass-specific and normalised OXPHOS. This highlights the importance of normalising OXPHOS to muscle cristae surface area density when studying skeletal muscle mitochondrial biology. KEY POINTS: Oxidative phosphorylation is the mitochondrial process by which ATP is produced, governed by the electrochemical gradient across the inner mitochondrial membrane with infoldings named cristae. In human skeletal muscle, the mass-specific capacity of oxidative phosphorylation (OXPHOS) can change independently of shifts in mitochondrial volume density, which may be attributed to variations in cristae density. We demonstrate that differences in skeletal muscle OXPHOS between healthy females and males, ranging from untrained to elite endurance athletes, are matched by differences in cristae density. This suggests that higher OXPHOS in skeletal muscles of active individuals is attributable to an increase in the density of cristae. These findings broaden our understanding of the variability in human skeletal muscle OXPHOS and highlight the significance of cristae, specific to mitochondrial respiration.

PSXIV-26 Live Animal Longissimus Thoracis Mitochondrial Capacities at 10, 13, and 18 Months of Age Are Related to Meat Quality in Angus and Brahman Steers

Abstract Mitochondrial abundance and efficiency correlate with meat color, but relationships between live animal mitochondria and other markers of beef quality remain largely unknown. To test the hypothesis that live animal skeletal muscle mitochondrial markers would be correlated with post-harvest hot carcass weight, ribeye area, calculated yield grade, backfat thickness, and marbling, samples were collected from the longissimus thoracis of Brahman and Angus steers at 10 mo (25 Angus, 23 Brahman), 13 mo (35 Angus, 33 Brahman), and 18 mo (35 Angus, 33 Brahman) of age. Mitochondrial volume density and function were quantified by citrate synthase (CS) and cytochrome c oxidase activities, respectively. Mitochondrial oxidative phosphorylation (OxP) and electron transfer (E) capacities were determined via high-resolution respirometry. Relationships between mitochondrial variables and carcass quality were determined using PROC CORR in SAS (v9.4). Within Angus at 10 mo, integrative (per mg tissue) complex I-supported OxP (PCI), maximal OxP (PCI+II), maximal E (ECI+II), and E supported by complex II (ECII) were positively correlated with hot carcass weight (P ≤ 0.05). Also, within 10 mo Angus, integrative OxPCI and flux control ratio (FCR) for OxPCI were positively correlated with backfat thickness (P ≤ 0.03). Within 10 mo Brahman, integrative PCI was positively correlated with ribeye area (P = 0.04). Within 13 mo Brahman, integrative mitochondrial proton LEAK was positively correlated with hot carcass weight (P = 0.02) and intrinsic (relative to CS activity) LEAK, PCI, and ECI+II were positively correlated with marbling (P ≤ 0.05). Conversely, CS activity was negatively correlated with marbling in 13 mo Brahman (P = 0.05). Integrative, FCR, and intrinsic LEAK were negatively correlated with ribeye area (P ≤ 0.05) but positively correlated with calculated yield grade in 13 mo Angus (P ≤ 0.03). However, at 18 mo, integrative, FCR, and intrinsic LEAK were positively correlated with ribeye area (P ≤ 0.008) but negatively correlated with calculated yield grade (P ≤ 0.01) in Angus steers. Furthermore, integrative and intrinsic OxPCI+II, ECI+II, and ECII were positively correlated with marbling in 18 mo Angus (P ≤ 0.04). Within 18 mo Brahman, CS activity was positively correlated with ribeye area (P = 0.006). Cytochrome c oxidase activity was not correlated with any markers of meat quality. These results suggest that mitochondrial capacity for energy production in Angus and Brahman steers at different life and production stages may dictate potential for skeletal muscle growth and lipid metabolism leading to differences in beef quality at slaughter. This project was supported by USDA NIFA Award #2021-67015-33392.

Effects of Mifepristone Solid Lipid Nanoparticles on Cardiac Structure and Function in Rats Undergoing Induction of Labor at Full Term

This research was aimed to analyze the biological characteristics of mifepristone solid lipid nanoparticles (MFP/SLNs) and their effects on the cardiac function of rats undergoing induction of labor at full term (FTIL). MFP was loaded into SLNs to prepare MFP/SLNs. The morphology and particle size (PS) of MFP/SLNs were observed by transmission electron microscopy (TEM), and the PS distribution (PSD) and potential distribution of MFP/SLNs were analyzed by Zeta analyzer. The drug loading (DL) and encapsulation efficiency (EE) of MFP/SLNs were calculated, and the crystal form of the drug in the carrier was detected by differential scanning calorimetry (DSC). Fifteen pregnant rats were randomly rolled into a blank (BLK) group, an MFP group, and an MFP/SLNs group, with five rats in each. Those in the MFP/SLNs and the MFP groups were administered MFP/SLNs (10 mg) and MFP by gavage on the 20th day of pregnancy. The changes in myocardial tissue of rats in the MFP/SLNs and MFP groups were observed within 24 hours after delivery and analyzed by a multifunctional true-color pathological image analysis system. The results indicated that MFP/SLNs had a spherical shape and uniform PSD, with an average PS of about 153 nm. The drug EE of MFP/SLNs exceeded 88% when the drug dosage was 50 mg. The MFP group showed obvious cytoplasmic edema in myocardial cells, an increased average mitochondrial volume density (MVD), and glycogen granule deposition. The area of myocardial cells in the MFP group was obviously larger than that in the MFP/SLNs group (P < 0.05), and the nuclear-cytoplasmic ratio (NCR) was much smaller (P < 0.05). These findings suggested that MFP/SLNs were successfully prepared, and MFP can cause changes in the cardiac structure of rats undergoing FTIL, resulting in hypoxic injury. However, MFP/SLNs may protect the cardiac structure and function of rats.

Prenatal transportation stress does not impact resting skeletal muscle mitochondrial function or antioxidant activity in Brahman calves

In cattle, prenatal transportation stress has been associated with differential methylation of genes related to metabolism, but the effects of prenatal transportation stress on skeletal muscle mitochondria and oxidative stress have not been investigated. We tested the hypothesis that prenatally stressed calves would exhibit increased skeletal muscle mitochondrial function resulting in greater oxidative stress than calves from non-stressed dams. Serum and longissimus thoracis muscle samples were collected from yearling Brahman calves whose mothers were stressed by transportation at five time points during gestation [i.e., prenatally stressed (PNS); eight bulls and six heifers] and control calves (CON; four bulls and six heifers). Serum was evaluated for concentration of the stress hormone, cortisol and for a marker of muscle perturbation, creatine kinase activity. Muscle samples were analyzed for concentration of a by-product of lipid peroxidation, malondialdehyde, and activity of the antioxidants, superoxide dismutase and glutathione peroxidase. Additionally, muscle mitochondrial volume density and function were estimated by citrate synthase and cytochrome c oxidase activities, respectively. Data were analyzed using mixed linear models with sex, treatment, and the sex × treatment interaction as fixed effects. No investigated variable differed between CON and PNS calves (p ≥ 0.3). These data suggest that prenatal transportation stress does not have an impact on skeletal muscle mitochondrial metabolism or markers of stress or muscle damage in Brahman yearling calves at rest. However, previously reported negative impacts of prenatal stress on inflammatory responses suggest that PNS calves may be differentially equipped to handle an acute stressor. Future research should investigate the energetic and inflammatory implications of acute stressors in animals subjected to prenatal stress.

30 Prenatally Stressed Brahman Bull Calves had Increased Pro-Inflammatory Cytokines and Transportation-Induced Mitochondrial Impairments

Abstract Prenatal transportation stress has been reported to alter behavior and adrenal glucocorticoid secretion in calves; however, the impact on mitochondrial and inflammatory responses to an industry-relevant stressor have not been explored. Sixteen Brahman bull calves were used to test the hypothesis that calves previously stressed by transportation in utero (PNS; n = 8; mean ± SD, 654 ± 71 kg, 11.7 ± 0.4 months) would have impaired mitochondrial capacity and greater plasma concentrations of two pro-inflammatory cytokines [interleukin (IL)-6 and tumor necrosis factor (TNF)-a] following a 3-hr transportation stressor compared with calves that experienced no prenatal stress (CON; n = 8; 645 ± 90 kg, 11.8 ± 0.5 months). Longissimus thoracis muscle samples were collected from calves, pre-transportation, and 6- and 24-hour post-transportation and were analyzed for citrate synthase activity (CS) as a marker of mitochondrial volume density and for mitochondrial oxidative phosphorylation (P) and electron transport (E) capacities via high resolution respirometry. Plasma was collected pre-transportation and post-transportation at hour 0, 1, 6, 24, and 48 for analysis of cytokine concentrations using custom MesoScale Discovery U-Plex kits. Data were analyzed using PROC MIXED with repeated measure (time) in SAS v9.4 with time, treatment, and time × treatment as fixed effects. Integrative (relative to tissue wet weight) E with complex II only (ECII), intrinsic (relative to CS) ECII, the contribution of P with complex I (PCI) to total E (flux control ratio, FCRPCI), and FCR ECII decreased from pre- to 6 hour post-transport (P ≤ 0.04) and remained suppressed at 24 hour post-transport (P ≤ 0.02) in all animals regardless of prenatal treatment. Integrative PCI was also decreased 24-hour post-transportation compared with pre-transport (P = 0.02). Intrinsic ECII tended to be greater in CON than PNS bulls overall (P = 0.07). Intrinsic Leak, integrative Leak, and FCR Leak tended to be impacted by the interaction of treatment and time (P ≤ 0.10) whereby all 3 measures were greater in PNS than CON bulls at 6-hour post-transport (P ≤ 0.02) but were similar between treatments at 24-hour post-transport. Both plasma IL-6 and TNF-a were greater in PNS than CON bulls overall (P ≤ 0.008) but neither cytokine was affected by postnatal transport. Bull calves subjected to prenatal transportation stress had sustained increased plasma pro-inflammatory cytokine concentrations and an increase in mitochondrial leak with a decrease in complex II supported electron transfer following a single postnatal transportation stressor. Importantly, the postnatal stressor utilized in this study did not affect plasma cytokines. A more stressful postnatal stimulus may provide a clearer assessment of potential impacts of prenatal stress on the progeny’s physiological responses to stress.

30 Intrinsic skeletal muscle mitochondrial function decreases while mitochondrial volume density increases from 6 months to 3-years-old in Thoroughbreds

As the primary source of cellular energy, mitochondria are critical for athletic performance, and the intricacies of mitochondrial electron transfer may be related to Thoroughbred race performance. However, little is known about specific mitochondrial adaptations to growth in the equine athlete. To test the hypothesis that mitochondrial metrics would increase with age, gluteus medius samples were collected from the same Thoroughbred horses as weanlings (4 colts, 9 fillies; mean ± SD, 6 ± 1mo), yearlings (4 colts, 10 fillies; 20 ± 1mo), 2-year-olds (2yo; 3 geldings, 10 mares; 32 ± 1mo), and 3yo (2 geldings, 3 mares; 44 ± 1mo) from the same Kentucky farm over 4 years. Thoroughbreds were broke to ride in August of their yearling year then began race training. Mitochondrial oxidative phosphorylation (P) and electron transfer (E) capacities were determined via high-resolution respirometry. Citrate synthase (CS) and cytochrome c oxidase (CCO) activities were quantified as measures of mitochondrial volume density and function, respectively. Data were analyzed using PROC MIXED in SASv9.4 with age and sex as fixed effects, sire and dam as random effects, and age as a repeated effect with horse as the subject. Intrinsic (relative to CS activity) CCO activity, P supported by complex I (PCI), maximal P (PCI+II), and maximal E (ECI+II) weregreatest in weanlings compared with all other ages (P ≤ 0.003) and greater in yearlings than 2yo (P ≤ 0.05). Intrinsic E supported by complex II only (ECII) was greater in weanlings than 2 or 3yo (P ≤ 0.01) and greater in yearlings than 2yo (P ≤ 0.04). Weanlings also had a greater contribution of PCI+II to ECI+II than yearlings, 2yo, or 3yo (P ≤ 0.05). However, CS activity was greatest in 3yo (P ≤ 0.03) and lowest in weanlings (P ≤ 0.0001) with yearlings and 2yo intermediate, indicating expansion of the mitochondrial network with age and training. Additionally, 3yo had the greatest integrative (per mg tissue) PCI of all ages (P ≤ 0.005). Two and 3yo had greater integrative PCI+II, ECI+II, and ECII capacities than weanlings (P ≤ 0.05). Overall, weanlings had the greatest intrinsic mitochondrial capacities and function but 2 and 3yo Thoroughbreds had greater mitochondrial volume density resulting in greater integrative capacities. Skeletal muscle mitochondria of young Thoroughbreds appear to adapt to meet energetic demands of growth and race training by increasing volume density rather than intrinsic capacity, but further research is warranted to characterize the impacts of various training programs on mitochondrial metrics.

TNF alpha induces mitophagy in human airway smooth muscle cells

Mitophagy is a regulated multistep process of selective removal of dysfunctional/damaged mitochondria by autophagy and is critical for the regulation of mitochondrial quality control in many physiological processes. The best characterized pathway of mitophagy involves PTEN-induced kinase 1 (PINK1) and E3-ubiquitin ligase Parkin. Upon mitochondrial depolarization, PINK1 stabilizes on the outer mitochondrial membrane (OMM) and recruits Parkin. After recruitment, PINK1 phosphorylates Parkin at Ser65, thus activating Parkin which promotes the ubiquitination of OMM proteins resulting in the subsequent lysosomal degradation of the mitochondria. Previously, we reported that tumor necrosis factor α (TNFα), a proinflammatory cytokine produced during inflammatory airway diseases, increases force generation and ATP hydrolysis, and results in increased mitochondrial fragmentation in human airway smooth muscle (hASM) cells. We also showed that TNFα induces PGC1α signaling, thus promoting mitochondrial biogenesis and increased mitochondrial volume density. In the present study, we hypothesize that TNFα induces mitophagy to promote selective clearance of dysfunctional mitochondria, thus maintaining a healthy mitochondrial pool to meet the increased energy demand in hASM cells. Experiments were performed in hASM cells dissociated from bronchiolar tissue samples collected during bronchiolar surgery from female and male donors with no current history of smoking or respiratory diseases. The hASM cells were serum deprived for 48 h and the phenotype was confirmed prior to experimentation. hASM cells were separated into three 6-h treatment groups: 1) TNFα (20 ng/mL), 2) FCCP (5 μM; positive control), and 3) untreated controls. The mitochondria and lysosomes in hASM cells were labelled with MitoTracker green (200 nM) and LysoTracker red (100 nM), respectively and 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 3D images were used to determine the coefficient for colocalization of mitochondria with lysosomes, lysosomal volume density, mitochondrial volume density, and mitochondrial complexity index (MCI). Protein expression and phosphorylation status of PINK1, Parkin, phospho-Parkin, VDAC (voltage-dependent anion channel), and LAMP1 (Lysosomal-associated membrane protein 1) were quantified by western blot. We observed that TNFα and FCCP treatment increased lysosomal volume density and mitochondrial colocalization with lysosomes in hASM cells, indicating the formation of mitophagosomes, characteristic of mitophagy. Consistent with previous findings, TNFα treatment increased mitochondrial volume density and decreased MCI, indicative of increased mitochondrial fragmentation. In addition, TNFα treatment increased the expression and mitochondrial translocation of PINK1 and Parkin. The results suggest that TNFα activates PINK1/Parkin-mediated mitophagy in hASM cells. Supported by NIH grants HL157984 (GCS) and HL105355 (OARR) 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.

1 Repeated muscle collections induce few physiological changes in mature sedentary horses

Repeated skeletal muscle collections are often used to determine tissue-specific responses to interventions or stressors but little is known about the physiological impacts of the collections themselves. Twenty-six mature sedentary horses (8 geldings, 18 mares; mean ± SD 9.5 ± 3.5y) were used to test the hypothesis that repeated muscle collections would not impact mitochondrial function or markers of inflammation and muscle damage. Horses were stratified by age and sex and randomly assigned to have gluteus medius samples collected at: 1) 0 and 24 h (n = 7); 2) 0 and 6 h (n = 6); 3) 0, 6, and 12 h (n = 7); or 4) 0, 6, 12, and 24 h (n = 6). Blood was collected from all horses every 6 h for 72 h total, starting 24 h before the first muscle collection. Muscle mitochondrial oxidative phosphorylation and electron transfer (E) capacities were determined via high-resolution respirometry, and mitochondrial volume density and function by citrate synthase (CS) and cytochrome c oxidase activities. Muscle and plasma concentrations of interleukin (IL)- 4, IL-8, IL-10, and tumor necrosis factor (TNF)α were quantified, as well as serum amyloid A (SAA) and cortisol concentrations and creatine kinase (CK) activity. Data were analyzed using linear models in SAS with time as a repeated measure, horse as the subject, time as a fixed effect, and sex when significant. For blood parameters, additional fixed effects includedcollection group and the time × group interaction. Integrative (per mg tissue) E supported by complex II decreased from 0 to 6 h (P = 0.004) but returned to baseline by 12 h post-initial muscle collection. Intrinsic (per unit CS) Leak was greater at 6 than at 0 and 12 h (P < 0.03) but did not differ from 24 h. Activity of CS was greater at 0 than 12 and 24 h (P ≤ 0.02). Regardless of group, serum CK activity was similar from −24 through 0 h but increased in all horses at 6 h and remained elevated through 48 h, peaking at 24 and 30 h post-initial muscle collection (P < 0.05). Geldings had greater serum CK activity and plasma IL-8 concentrations than mares (P ≤ 0.006). Serum cortisol concentrations displayed a circadian pattern throughout the 72-h collection period (P < 0.0001) but were not impacted by tissue collection group. Overall, horses in group 2 (0 and 6 h) had greater SAA concentrations than all other groups (P < 0.006). No other variable was impacted by repeated tissue collections. In unstressed, mature horses, repeated gluteal muscle collections did not profoundly impact local and systemic markers of mitochondrial function, select cytokines, and stress indicators; however, serum CK should be interpreted with caution in studies employing repeated tissue collections.

Increased mitochondrial surface area and cristae density in the skeletal muscle of strength athletes.

Mitochondria are the cellular organelles responsible for resynthesising the majority of ATP. In skeletal muscle, there is an increased ATP turnover during resistance exercise to sustain the energetic demands of muscle contraction. Despite this, little is known regarding the mitochondrial characteristics of chronically strength-trained individuals and any potential pathways regulating the strength-specific mitochondrial remodelling. Here, we investigated the mitochondrial structural characteristics in skeletal muscle of strength athletes and age-matched untrained controls. The mitochondrial pool in strength athletes was characterised by increased mitochondrial cristae density, decreased mitochondrial size, and increased surface-to-volume ratio, despite similar mitochondrial volume density. We also provide a fibre-type and compartment-specific assessment of mitochondria morphology in human skeletal muscle, which reveals across groups a compartment-specific influence on mitochondrial morphology that is largely independent of fibre type. Furthermore, we show that resistance exercise leads to signs of mild mitochondrial stress, without an increase in the number of damaged mitochondria. Using publicly available transcriptomic data we show that acute resistance exercise increases the expression of markers of mitochondrial biogenesis, fission and mitochondrial unfolded protein responses (UPRmt ). Further, we observed an enrichment of the UPRmt in the basal transcriptome of strength-trained individuals. Together, these findings show that strength athletes possess a unique mitochondrial remodelling, which minimises the space required for mitochondria. We propose that the concurrent activation of markers of mitochondrial biogenesis and mitochondrial remodelling pathways (fission and UPRmt ) with resistance exercise may be partially responsible for the observed mitochondrial phenotype of strength athletes. KEY POINTS: Untrained individuals and strength athletes possess comparable skeletal muscle mitochondrial volume density. In contrast, strength athletes' mitochondria are characterised by increased cristae density, decreased size and increased surface-to-volume ratio. Type I fibres have an increased number of mitochondrial profiles with minor differences in the mitochondrial morphological characteristics compared with type II fibres. The mitochondrial morphology is distinct across the subcellular compartments in both groups, with subsarcolemmal mitochondria being bigger in size when compared with intermyofibrillar. Acute resistance exercise leads to signs of mild morphological mitochondrial stress accompanied by increased gene expression of markers of mitochondrial biogenesis, fission and mitochondrial unfolded protein response (UPRmt ).

Size-dependent differences in mitochondrial volume density in phrenic motor neurons.

Neuromotor control of diaphragm muscle (DIAm) motor units is dependent on an orderly size-dependent recruitment of phrenic motor neurons (PhMNs). Slow (type S) and fast, fatigue resistant (type FR) DIAm motor units, which are frequently recruited to sustain ventilation, comprise smaller PhMNs that innervate type I and IIa DIAm fibers. More fatigable fast (type FF) motor units, which are infrequently recruited for higher force, expulsive behaviors, comprise larger PhMNs that innervate more type IIx/IIb DIAm fibers. We hypothesize that due to the more frequent activation and thus higher energy demand of type S and FR motor units, the mitochondrial volume density (MVD) of smaller PhMNs is greater compared with larger PhMNs. In eight adult (6 mo old) Fischer 344 rats, PhMNs were identified via intrapleural injection of Alexa488-conjugated cholera toxin B (CTB). Following retrograde CTB labeling, mitochondria in PhMNs were labeled by transdural infusion of MitoTracker Red. PhMNs and mitochondria were imaged using multichannel confocal microscopy using a ×60 oil objective. Following optical sectioning and three-dimensional (3-D) rendering, PhMNs and mitochondria were analyzed volumetrically using Nikon Elements software. Analysis of MVD in somal and dendritic compartments was stratified by PhMN somal surface area. Smaller PhMNs (likely S and FR units) had greater somal MVDs compared with larger PhMNs (likely FF units). By contrast, proximal dendrites or larger PhMNs had higher MVD compared with dendrites of smaller PhMNs. We conclude that more active smaller PhMNs have a higher mitochondrial volume density to support their higher energy demand in sustaining ventilation.NEW & NOTEWORTHY Type S and FR motor units, comprising smaller phrenic motor neurons (PhMNs) are regularly activated to perform indefatigable ventilatory requirements. By contrast, type FF motor units, comprising larger PhMNs, are infrequently activated to perform expulsive straining and airway defense maneuvers. This difference in activation history is mirrored in the mitochondrial volume density (MVD), with smaller PhMNs having higher MVD than larger PhMNs. In proximal dendrites, this trend was reversed, with larger PhMNs having higher MVD than smaller PhMNs, likely due to the maintenance requirements for the larger dendritic arbor of FF PhMNs.

Molecular Mechanisms Underlying TNFα-Induced Mitochondrial Biogenesis in Human Airway Smooth Muscle.

Proinflammatory cytokines such as TNFα mediate airway inflammation. Previously, we showed that TNFα increases mitochondrial biogenesis in human ASM (hASM) cells, which is associated with increased PGC1α expression. We hypothesized that TNFα induces CREB and ATF1 phosphorylation (pCREBS133 and pATF1S63), which transcriptionally co-activate PGC1α expression. Primary hASM cells were dissociated from bronchiolar tissue obtained from patients undergoing lung resection, cultured (one-three passages), and then differentiated by serum deprivation (48 h). hASM cells from the same patient were divided into two groups: TNFα (20 ng/mL) treated for 6 h and untreated controls. Mitochondria were labeled using MitoTracker green and imaged using 3D confocal microscopy to determine mitochondrial volume density. Mitochondrial biogenesis was assessed based on relative mitochondrial DNA (mtDNA) copy number determined by quantitative real-time PCR (qPCR). Gene and/or protein expression of pCREBS133, pATF1S63, PCG1α, and downstream signaling molecules (NRFs, TFAM) that regulate transcription and replication of the mitochondrial genome, were determined by qPCR and/or Western blot. TNFα increased mitochondrial volume density and mitochondrial biogenesis in hASM cells, which was associated with an increase in pCREBS133, pATF1S63 and PCG1α expression, with downstream transcriptional activation of NRF1, NRF2, and TFAM. We conclude that TNFα increases mitochondrial volume density in hASM cells via a pCREBS133/pATF1S63/PCG1α-mediated pathway.

Structural and Functional State of Leukocytes During Long-term Adaptation of Experimental Animals to the Climatic and Geographical Conditions of High Mountains

This study presents the results of studies of the structural and functional state of leukocytes during long-term adaptation of experimental animals to high altitude conditions, obtained using the electron microscopic method of morphometry. It has been shown that under the influence of extreme factors of high altitude in the process of adaptation in experimental animals there is a statistically significant increase in the volume density of mitochondria and ribosomes in neutrophils, lymphocytes, eosinophils, monocytes, which can be regarded as an indicator of activation of the synthesis of nucleic acids and proteins, activation of the biosynthesis of mitochondrial structures and increase energy production in white blood cells. The volumetric value of specific granules also increased. The volume density of vacuoles in all white blood cells by the 30th day of stay at altitude was statistically significantly reduced, however, in neutrophils, eosinophils and lymphocytes, this indicator did not reach the values of animals in the control group. Thus, adaptation to high mountains has a certain training effect, the state of white blood cells under these conditions is a kind of marker of adaptive processes occurring in the body during the stay of humans and animals in high mountains.

Impact of Coenzyme Q10 Supplementation on Skeletal Muscle Respiration, Antioxidants, and the Muscle Proteome in Thoroughbred Horses.

Coenzyme Q10 (CoQ10) is an essential component of the mitochondrial electron transfer system and a potent antioxidant. The impact of CoQ10 supplementation on mitochondrial capacities and the muscle proteome is largely unknown. This study determined the effect of CoQ10 supplementation on muscle CoQ10 concentrations, antioxidant balance, the proteome, and mitochondrial respiratory capacities. In a randomized cross-over design, six Thoroughbred horses received 1600 mg/d CoQ10 or no supplement (control) for 30-d periods separated by a 60-d washout. Muscle samples were taken at the end of each period. Muscle CoQ10 and glutathione (GSH) concentrations were determined using mass spectrometry, antioxidant activities by fluorometry, mitochondrial enzyme activities and oxidative stress by colorimetry, and mitochondrial respiratory capacities by high-resolution respirometry. Data were analyzed using mixed linear models with period, supplementation, and period × supplementation as fixed effects and horse as a repeated effect. Proteomics was performed by tandem mass tag 11-plex analysis and permutation testing with FDR < 0.05. Concentrations of muscle CoQ10 (p = 0.07), GSH (p = 0.75), and malondialdehyde (p = 0.47), as well as activities of superoxide dismutase (p = 0.16) and catalase (p = 0.66), did not differ, whereas glutathione peroxidase activity (p = 0.003) was lower when horses received CoQ10 compared to no supplement. Intrinsic (relative to citrate synthase activity) electron transfer capacity with complex II (ECII) was greater, and the contribution of complex I to maximal electron transfer capacity (FCRPCI and FCRPCIG) was lower when horses received CoQ10 with no impact of CoQ10 on mitochondrial volume density. Decreased expression of subunits in complexes I, III, and IV, as well as tricarboxylic acid cycle (TCA) enzymes, was noted in proteomics when horses received CoQ10. We conclude that with CoQ10 supplementation, decreased expression of TCA cycle enzymes that produce NADH and complex I subunits, which utilize NADH together with enhanced electron transfer capacity via complex II, supports an enhanced reliance on substrates supplying complex II during mitochondrial respiration.