Permeabilized Fiber Bundles Research Articles

Diaphragm Force and Mitochondrial Function Following Enhanced Nitric Oxide Availability During Mechanical Ventilation

During mechanical ventilation (MV), force developed by the diaphragm decreases over time much faster than locomotor muscles, known as ventilator-induced diaphragm dysfunction (VIDD). VIDD is accompanied by an increase in intramyofiber protein S-nitrosylation, a modification of cysteines by nitric oxide (NO). During the treatment of acute respiratory distress syndrome (ARDS), inhaled NO is commonly used to improve gas exchange, but little is known about the effects of increased NO availability to the diaphragm during MV and the intracellular mechanisms to protect against excessive S-nitrosylation. We hypothesize that the enzyme S-nitrosoglutathione reductase (GSNOR) protects against excessive protein S-nitrosylation in diaphragm myofibers in conditions where NO availability is enhanced during MV, thereby preserving contractile function. We tested this hypothesis by enhancing NO availability and blocking GSNOR activity in mice during MV. Mice were treated with GSNOR inhibitor (SPL-334; GSNORi) or both GSNORi and NO donor isosorbide dinitrate (GSNORi-ISDN) together, and mechanically ventilated for 2h. E x vivo diaphragm force and mitochondrial respiration were measured. Male (C57BL/6J) 3-4 month old mice (n=27) were anesthetized and subjected to MV for 0 (non-MV), 2, 4, or 6h (150 breaths/min, 10 cmH2O PIP, 3.5 cmH2O PEEP. 8 mL/kg VT). Alternatively, mice were treated with PBS/10% DMSO (DMSO, n=6) or 25 μg SPL-334 in PBS/DMSO (GSNORi, n=6) or 25 μg SPL-334 + 1.7 mg ISDN in PBS/DMSO (GSNORi-ISDN; n=6), and then subjected to MV for 2 h. After MV, mice were euthanized, and diaphragm strips were used for force, and permeabilized fiber bundles for mitochondrial oxidative phosphorylation and H2O2 generation measurements. Peak tetanic force was decreased by MV ~23% at 4 h and ~40% at 6 h (P=0.0183 and P=0.0062, respectively, one-way ANOVA, Tukey post-test) vs 0 h. However, peak force was not different between DMSO, GSNORi and GSNORi-ISDN at 2 h MV. Coupled-phosphorylating mitochondrial respiration (Kruskal-Wallis H=2.2, p=0.35) was not different but H2O2 flux was highest in GSNORi-ISDN vs DMSO and GSNORi (Kruskal-Wallis H=6.6, p=0.03). GSNORi and ISDN treatment during MV did not produce any changes to VIDD and to mitochondrial respiration, but increased ROS production. If exogenous NO is not provided, inhibiting GSNOR in vivo alone does not affect diaphragm function ex vivo during short MV. Support: TRDRP (T32IR5221 and T29KT0397CA, to L.N.) and SDSU 2023 SEED Grant (to L.N.). 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.

Role of Cardiorespiratory Fitness and Mitochondrial Oxidative Capacity in Reduced Walk Speed of Older Adults With Diabetes.

Cardiorespiratory fitness and mitochondrial oxidative capacity are associated with reduced walking speed in older adults. The impact of cardiorespiratory fitness and mitochondrial oxidative capacity on walking speed in older adults with diabetes has not been clearly defined. We examined differences in cardiorespiratory fitness and skeletal muscle mitochondrial oxidative capacity between older adults with and without diabetes as well as determine their relative contribution to slower walking speed in older adults with diabetes. Participants with diabetes (n=159) had lower cardiorespiratory fitness and mitochondrial respiration in permeabilized fiber bundles when compared to those without diabetes (n=717), following adjustments for covariates including BMI, chronic comorbid health conditions, and physical activity. 4-m and 400-m walking speeds were slower in those with diabetes. Mitochondrial oxidative capacity alone or combined with cardiorespiratory fitness mediated ∼20-70% of the difference in walk speed between older adults with and without diabetes. Additional adjustments with BMI and co-morbidities further explained the group differences in walk speed. Cardiorespiratory fitness and skeletal muscle mitochondrial oxidative capacity contribute to slower walking speeds in older adults with diabetes.

High-dose atorvastatin therapy progressively decreases skeletal muscle mitochondrial respiratory capacity in humans.

BACKGROUNDWhile the benefits of statin therapy on atherosclerotic cardiovascular disease are clear, patients often experience mild to moderate skeletal myopathic symptoms, the mechanism for which is unknown. This study investigated the potential effect of high-dose atorvastatin therapy on skeletal muscle mitochondrial function and whole-body aerobic capacity in humans.METHODSEight overweight (BMI, 31.9 ± 2.0) but otherwise healthy sedentary adults (4 females, 4 males) were studied before (day 0) and 14, 28, and 56 days after initiating atorvastatin (80 mg/d) therapy.RESULTSMaximal ADP-stimulated respiration, measured in permeabilized fiber bundles from muscle biopsies taken at each time point, declined gradually over the course of atorvastatin treatment, resulting in > 30% loss of skeletal muscle mitochondrial oxidative phosphorylation capacity by day 56. Indices of in vivo muscle oxidative capacity (via near-infrared spectroscopy) decreased by 23% to 45%. In whole muscle homogenates from day 0 biopsies, atorvastatin inhibited complex III activity at midmicromolar concentrations, whereas complex IV activity was inhibited at low nanomolar concentrations.CONCLUSIONThese findings demonstrate that high-dose atorvastatin treatment elicits a striking progressive decline in skeletal muscle mitochondrial respiratory capacity, highlighting the need for longer-term dose-response studies in different patient populations to thoroughly define the effect of statin therapy on skeletal muscle health.FUNDINGNIH R01 AR071263.

SKELETAL MUSCLE ENERGETICS ARE ASSOCIATED WITH PERFORMANCE FATIGABILITY

Abstract Performance fatigability manifests as insufficient energy to complete daily physical tasks and worsens with aging, exacerbating vulnerability to disability. Skeletal muscle energetics also declines with aging. Thus, we hypothesized muscle energetics may be an important contributor to performance fatigability. In the Study of Muscle, Mobility and Aging (SOMMA), participants completed a usual-paced 400m walk while wearing a wrist-worn ActiGraph, from which raw data were used to derive the Pittsburgh Performance Fatigability Index (PPFI, higher=more severe fatigability) that quantifies percent decline in the entire individual cadence-versus-time trajectory. Maximal oxidative phosphorylation (maxOXPHOS) in skeletal muscle mitochondria was quantified in vitro using high-resolution respirometry in permeabilized fiber bundles from vastus lateralis muscle biopsies. Maximal adenosine triphosphate production (ATPmax) was assessed in vivo by 31P magnetic resonance spectroscopy. We conducted separate tobit regressions to examine associations of maxOXPHOS and ATPmax with PPFI, adjusting for technician/site, age, sex, race, height, weight, and mins/day moderate-to-vigorous physical activity measured by ActiGraph in free-living, in N=795 participants with complete PPFI scores and >1 energetics measure (70-94 yrs, 58% women). Median PPFI scores were 1.4% [IQR: 0-2.9%]. After adjustment, each SD (18.4 pmol/(s*mg)) lower maxOXPHOS was associated with 0.55% (95% CI: 0.26, 0.85) higher PPFI scores, while each SD (0.2 mM/sec) lower ATPmax was associated with 0.54% (95% CI: 0.27, 0.81) higher PPFI scores. Our results indicate that lower skeletal muscle energetics were associated with more severe performance fatigability. This suggests that therapeutics targeting muscle energetics may thereby potentially mitigate fatigability and lessen susceptibility to disability among older adults.

MITOCHONDRIAL ENERGETICS AND PERCEIVED PHYSICAL FATIGABILITY SEVERITY IN OLDER ADULTS

Abstract It has been hypothesized that lower skeletal muscle energetics may contribute to onset of age-related perceived fatigability. Consistent with this idea, previous research found that maximal ATP production (ATPmax) was associated with higher perceived exertion (i.e., fatigability) measured in older adults with the Borg Scale at the end of a slow walking task. We extend this work to SOMMA examining skeletal muscle energetics (ATPmax and maximal complex I&II supported oxidative phosphorylation [maxOXPHOS]) with perceived physical fatigability using the Pittsburgh Fatigability Scale (PFS, range 0-50, higher=greater). PFS captures fatigue perception anchored to activities of varying intensity and duration. We assessed ATPmax in vivo by 31P magnetic resonance spectroscopy following an acute knee extensor exercise bout. maxOXPHOS was quantified ex vivo in permeabilized fiber bundles by high-resolution spectroscopy. Participants (N=721 with PFS and ≥1 energetics measure) were aged 76.4±5.0 years, 56.7% women, 86.7% White. Lower ATPmax (r=-0.21) and maxOXPHOS (r=-0.29) correlated with higher PFS scores (both p<0.001). Linear regressions adjusted for age, sex, technician, and total daily accelerometry-derived physical activity revealed that each one SD lower ATPmax (0.15 mM/sec) was associated with 1.15 (95% CI: 0.50, 1.79) higher PFS scores; each one SD lower maxOXPHOS (18.5 pmol/(sec*mg)) was associated with 1.79 (95% CI: 1.14, 2.45) higher PFS scores. Impaired skeletal muscle energetics may be implicated in the etiologic pathway of age-related fatigability. Next steps will examine the longitudinal association between changes in skeletal muscle energetics and age-related fatigability.

DECONSTRUCTING MULTIMORBIDITY AND MUSCLE MITOCHONDRIAL RESPIRATION AND ENERGETICS IN OLDER ADULTS

Abstract Lower cardiorespiratory fitness among older adults is associated with greater multimorbidity burden, including cardiovascular disease, cancer, and dementia. Those with poorer muscle mitochondrial energetics also have lower cardiorespiratory fitness. We have previously shown lower mitochondrial respiration and energetics (OXPHOS and ATPmax) have higher morbidity burden using a summary measure, but the associations among specific conditions have not been examined. We leverage data from the SOMMA cohort (N=856, 76.4yrs, 59% women, 85.5% white); participants self-reported physician diagnosis of 11 age-related conditions (i.e., cancer, cardiac arrhythmia, chronic kidney disease (CKD), chronic obstructive pulmonary disease, coronary heart disease (CHD), depression, diabetes). Mitochondrial respiration was assessed by high-resolution respirometry of vastus lateralis using permeabilized fiber bundles. 31P magnetic resonance spectroscopy of the quadriceps measured ATPmax. In adjusted logistic models with beta-coefficients reported per SD decrement in each mitochondrial measure, fatty-acid-supported Leak respiration was associated with CKD (OR: 2.3(95%CI=1.2,4.3)), fatty acid and complex I&II supported oxidative phosphorylation (OXPHOS) with depression (OR: 1.8(95%CI=1.1,2.7)). Many respirometry measures and ATPmax associated with diabetes, with maximal electron transport system (ETS) capacity having the highest odds (OR: 1.5(95%CI=1.1,2.0)). No other associations were statistically significant for conditions listed above. These results indicate that mitochondrial respiration and energetics are more impaired in depression, kidney disease, and diabetes. This raises new questions about the role of mitochondrial respiration and energetics as cause or consequences of these conditions. Overall, our analyses suggest compromised mitochondrial function may have implications for specific aging-related conditions, and global measures of morbidity burden may not reflect these distinctions.

Associations between skeletal muscle energetics and accelerometry-based performance fatigability: Study of Muscle, Mobility and Aging.

Performance fatigability is typically experienced as insufficient energy to complete daily physical tasks, particularly with advancing age, often progressing toward dependency. Thus, understanding the etiology of performance fatigability, especially cellular-level biological mechanisms, may help to delay the onset of mobility disability. We hypothesized that skeletal muscle energetics may be important contributors to performance fatigability. Participants in the Study of Muscle, Mobility and Aging completed a usual-paced 400-m walk wearing a wrist-worn ActiGraph GT9X to derive the Pittsburgh Performance Fatigability Index (PPFI, higher scores = more severe fatigability) that quantifies percent decline in individual cadence-versus-time trajectory from their maximal cadence. Complex I&II-supported maximal oxidative phosphorylation (max OXPHOS) and complex I&II-supported electron transfer system (max ETS) were quantified exvivo using high-resolution respirometry in permeabilized fiber bundles from vastus lateralis muscle biopsies. Maximal adenosine triphosphate production (ATPmax) was assessed invivo by 31P magnetic resonance spectroscopy. We conducted tobit regressions to examine associations of max OXPHOS, max ETS, and ATPmax with PPFI, adjusting for technician/site, demographic characteristics, and total activity count over 7-day free-living among older adults (N = 795, 70-94 years, 58% women) with complete PPFI scores and ≥1 energetics measure. Median PPFI score was 1.4% [25th-75th percentile: 0%-2.9%]. After full adjustment, each 1 standard deviation lower max OXPHOS, max ETS, and ATPmax were associated with 0.55 (95% CI: 0.26-0.84), 0.39 (95% CI: 0.09-0.70), and 0.54 (95% CI: 0.27-0.81) higher PPFI score, respectively. Our findings suggested that therapeutics targeting muscle energetics may potentially mitigate fatigability and lessen susceptibility to disability among older adults.

Exercise and ageing impact the kynurenine/tryptophan pathway and acylcarnitine metabolite pools in skeletal muscle of older adults.

Exercise-induced perturbation of skeletal muscle metabolites is a probable mediator of long-term health benefits in older adults. Although specific metabolites have been identified to be impacted by age, physical activity and exercise, the depth of coverage of the muscle metabolome is still limited. Here, we investigated resting and exercise-induced metabolite distribution in muscle from well-phenotyped older adults who were active or sedentary, and a group of active young adults. Percutaneous biopsies of the vastus lateralis were obtained before, immediately after and 3h following a bout of endurance cycling. Metabolite profile in muscle biopsies was determined by tandem mass spectrometry. Mitochondrial energetics in permeabilized fibre bundles was assessed by high resolution respirometry and fibre type proportion was assessed by immunohistology. We found that metabolites of the kynurenine/tryptophan pathway were impacted by age and activity. Specifically, kynurenine was elevated in muscle from older adults, whereas downstream metabolites of kynurenine (kynurenic acid and NAD+ ) were elevated in muscle from active adults and associated with cardiorespiratory fitness and muscle oxidative capacity. Acylcarnitines, a potential marker of impaired metabolic health, were elevated in muscle from physically active participants. Surprisingly, despite baseline group difference, acute exercise-induced alterations in whole-body substrate utilization, as well as muscle acylcarnitines and ketone bodies, were remarkably similar between groups. Our data identified novel muscle metabolite signatures that associate with the healthy ageing phenotype provoked by physical activity and reveal that the metabolic responsiveness of muscle to acute endurance exercise is retained [NB]:AUTHOR: Please ensure that the appropriate material has been provide for Table S2, as well as for Figures S1 to S7, as also cited in the text with age regardless of activity levels. KEY POINTS: Kynurenine/tryptophan pathway metabolites were impacted by age and physical activity in human muscle, with kynurenine elevated in older muscle, whereas downstream products kynurenic acid and NAD+ were elevated in exercise-trained muscle regardless of age. Acylcarnitines, a marker of impaired metabolic health when heightened in circulation, were elevated in exercise-trained muscle of young and older adults, suggesting that muscle act as a metabolic sink to reduce the circulating acylcarnitines observed with unhealthy ageing. Despite the phenotypic differences, the exercise-induced response of various muscle metabolite pools, including acylcarnitine and ketone bodies, was similar amongst the groups, suggesting that older adults can achieve the metabolic benefits of exercise seen in young counterparts.

AGE, PHYSICAL ACTIVITY AND MITOCHONDRIAL FUNCTION IN THE STUDY OF MUSCLE MOBILITY AND AGING (SOMMA)

Abstract We studied whether lower physical activity (PA) in older adults may partly explain the age-related decline in muscle mitochondrial function in the SOMMA cohort of 879 men and women, aged 70+. PA was measured both subjectively (CHAMPS questionnaire) and objectively (wrist-worn ActiGraph Link) classifying PA levels as sedentary, light, or moderate to vigorous (MVPA, ≥3 METS). Muscle mitochondrial function was assessed ex vivo via respirometry in permeabilized fiber bundles and as in vivo by 31Phosphorus magnetic resonance spectroscopy (31P-MRS) of the maximal rate of muscle ATP regeneration (ATPMAX in the quadriceps after contraction. Of 809 with respirometry or 31P-MRS, mean age was 76.4, 58.3% were women, 12.7% Black, 85.2% White and 2.1% other race/ethnicity. Mean OXPHOS and ATPMAX values were 1.91 pmol/(s*mg) and 0.016 mM/sec lower per 5-year age increment. Men self-reported more MVPA than women, but women recorded more MVPA with actigraphy. For both self-reported and recorded activity, associations with maximal tissue oxidative phosphorylation (OXPHOS) as well as ATPMAX were stronger for MVPA than for sedentary or light activity. After adjustment for age, sex, and clinic site, OXPHOS was lower for each standard deviation) fewer minutes/week of MVPA [reported, SD=408 min/wk: -3.66 pmol/(s*mg) (95% CI -4.93, -2.39); recorded, SD=600 min/wk: -1.93 pmol/(s*mg) (95% CI -3.31, -0.55)]. Similar associations were noted for ATPMAX. MVPA attenuated the association of age with mitochondrial function by 31–50% for both OXPHOS and ATPMAX, suggesting that MVPA could offset the lower mitochondrial function with age or that lower mitochondrial function impedes MVPA at older ages.

SEX DIFFERENCES IN SKELETAL MUSCLE ENERGETICS AMONG OLDER ADULTS

Abstract Mobility impairment is the most common disability among older adults, with an earlier presentation, higher prevalence, and greater severity in women compared to men. A decline in skeletal muscle metabolism contributes to the loss of mobility with age. However, it is unknown if sex-specific differences in muscle energetics can explain the disparity in mobility impairment among men and women. In the Study of Muscle, Mobility, and Aging (SOMMA), muscle energetics was characterized using in vivo Phosphorus Magnetic Resonance Spectroscopy and High-Resolution Respirometry of permeabilized fiber bundles from muscle biopsies. In this analysis of 773 participants aged 70–94 years, 519 were women, of which 16% were deemed lower extremity mobility impaired based on a Short Physical Performance Battery (SPPB) score

Mitochondrial Energetics in Skeletal Muscle Are Associated With Leg Power and Cardiorespiratory Fitness in the Study of Muscle, Mobility and Aging.

Mitochondrial energetics are an important property of aging muscle, as generation of energy is pivotal to the execution of muscle contraction. However, its association with functional outcomes, including leg power and cardiorespiratory fitness, is largely understudied. In the Study of Muscle, Mobility, and Aging, we collected vastus lateralis biopsies from older adults (n = 879, 70-94 years, 59.2% women). Maximal State 3 respiration (Max OXPHOS) was assessed in permeabilized fiber bundles by high-resolution respirometry. Capacity for maximal adenosine triphosphate production (ATPmax) was measured in vivo by 31P magnetic resonance spectroscopy. Leg extension power was measured with a Keiser press system, and VO2 peak was determined using a standardized cardiopulmonary exercise test. Gender-stratified multivariate linear regression models were adjusted for age, race, technician/site, adiposity, and physical activity with beta coefficients expressed per 1-SD increment in the independent variable. Max OXPHOS was associated with leg power for both women (β = 0.12 Watts/kg, p < .001) and men (β = 0.11 Watts/kg, p < .050). ATPmax was associated with leg power for men (β = 0.09 Watts/kg, p < .05) but was not significant for women (β = 0.03 Watts/kg, p = .11). Max OXPHOS and ATPmax were associated with VO2 peak in women and men (Max OXPHOS, β women = 1.03 mL/kg/min, β men = 1.32 mL/kg/min; ATPmax β women = 0.87 mL/kg/min, β men = 1.50 mL/kg/min; all p < .001). Higher muscle mitochondrial energetics measures were associated with both better cardiorespiratory fitness and greater leg power in older adults. Muscle mitochondrial energetics explained a greater degree of variance in VO2 peak compared to leg power.

Acl Injury Reduces Quadriceps Mitochondrial Oxidative Capacity That Does Not Recover With Rehabilitation

Protracted quadriceps weakness and localized muscle fatigue after ACL reconstruction result in poor knee mechanics. The biological underpinnings of poor functional recovery are unknown, limiting the development of evidence-based therapies. Mitochondrial dysfunction contributes to muscle atrophy and fatigue, but the effect of ACL injury and reconstruction on quadriceps mitochondrial capacity are unknown. PURPOSE: To define quadriceps oxidative capacity in human participants following ACL reconstruction and at the conclusion of rehabilitation. METHODS: Muscle biopsies (vastus lateralis) were obtained from ACL-injured and Healthy limbs of 11 participants (6 M,5F; 20 ± 3 yr) prior to surgical reconstruction and following the completion of post-surgical rehabilitation (4-6 months following surgery). Integrative (relative to tissue wet weight) mitochondrial oxidative phosphorylation capacity with complex I substrates (PCI), PCI plus complex II substrate (PCI + II), uncoupled electron transfer system capacity (ECI + II), and electron transfer system capacity with functional complex II only (ECII) was determined in permeabilized fiber bundles using high-resolution respirometry within 24 hr of biopsy collection. Oxidative phosphorylation and electron transport system capacity were compared using a mixed model. RESULTS: Following ACL injury, PCI + II is 19% lower in the injured limb (Healthy: 124 ± 8; ACL-injured: 97 ± 8 pmol O2 per second per mg tissue, p < 0.05) and did not recover following reconstruction and rehabilitation (Post-Rehab: 109 ± 12 pmol O2 per second per mg tissue, p > 0.05). Additionally, ECI + II was 18% lower in the ACL-injured quadriceps (Healthy: 140 ± 9; ACL-injured: 112 ± 12 pmol O2 per second per mg tissue, p < 0.05) and did not recover following reconstruction and rehabilitation (Post-Rehab: 123 ± 16 pmol O2 per second per mg tissue, p > 0.05). CONCLUSIONS: ACL injury and reconstruction reduce quadriceps oxidative capacity, and these deficits are obstinate to current standard of care rehabilitation. Quadriceps fatigue resistance is critical for stability of the healing knee, and there is a need for therapies targeting deficits in mitochondrial capacity to enhance muscular endurance. Supported by NIH R01AR072061 and R01AR071398

Residual force enhancement is reduced in permeabilized fiber bundles from mdm muscles.

Residual force enhancement (RFE) is the increase in steady-state force after active stretch relative to the force during isometric contraction at the same final length. The muscular dystrophy with myositis (mdm) mutation in mice, characterized by a small deletion in N2A titin, has been proposed to prevent N2A titin-actin interactions so that active mdm muscles are more compliant than wild type (WT). This decrease in active muscle stiffness is associated with reduced RFE. We investigated RFE in permeabilized soleus (SOL) and extensor digitorum longus (EDL) fiber bundles from WT and mdm mice. On each fiber bundle, we performed active and passive stretches from an average sarcomere length of 2.6-3.0 µm at a slow rate of 0.04 µms-1, as well as isometric contractions at the initial and final lengths. One-way ANOVA showed that SOL and EDL fiber bundles from mdm mice exhibited significantly lower RFE than WT mice (P<0.0001). This result is consistent with previous observations in single myofibrils and intact muscles. However, it contradicts the results from a previous study that appeared to show that compensatory mechanisms could restore titin force enhancement in single fibers from mdm psoas. We suggest that RFE measured previously in mdm single fibers was an artifact of the high variability in passive tension found in degenerating fibers, which begins after ∼24 days of age. The results are consistent with the hypothesis that RFE is reduced in mdm skeletal muscles owing to impaired Ca2+-dependent titin-actin interactions resulting from the small deletion in N2A titin.

Residual force enhancement is reduced in permeabilized fiber bundles from mdm muscles

Residual Force enhancement (RFE) is defined as the increase in steady state force after active stretch compared with force after isometric contraction at the same final length. Recent studies have demonstrated a pivotal role for titin in RFE. In myofibrils stretched beyond overlap of thick and thin filaments, Ca2+ activated myofibrils had a higher titin-based force and a steeper increase with force with stretch than passively stretched myofibrils at the same average sarcomere length. These results suggest higher titin-based stiffness in active myofibrils, perhaps due to N2A titin-actin interactions in active wild type (WT) muscle. Mdm mice carry a small deletion in titin that includes 53 amino acids in Ig83 in the N2A region. It has been proposed that the mdm mutation prevents N2A titin-actin interactions so that active mdm muscles are more compliant than WT muscles. This decrease in active muscle stiffness should be associated with a reduction in RFE. Based on this rationale, we investigated RFE in permeabilized soleus (SOL) and extensor digitorum longus (EDL) fiber bundles from 20-30 day old mdm mice (n=5), harvested at a young age to diminish the effects of degeneration and regeneration, and 30-60 day old WT mice (n=5). Contractile stress and RFE of EDL (mdm n=19 and WT n=20) and SOL fibers (WT n=19 and mdm n=16) was analyzed using JMP and Graph pad (PRISM) software. Statistics included Shapiro-Wilk (normality), Levene's and Bartlett (homogeneity of variances), Welch's one-way ANOVA (unequal variances), Dunnett's T3 (multiple comparisons), and T-tests with Welch's correction (unpaired). To investigate differences in RFE between genotypes, we performed isometric contractions, as well as active and passive stretch from an average sarcomere length of 2.6 - 3.0 µm on each fiber. This stretch amplitude, within the physiological range, resulted in no observable breakage or slipping of fibers during experiments. We predicted that mdm fiber bundles would show reduced RFE compared to WT fibers. Contractile stress when activated at 2.6 µm was significantly greater in WT EDL (Welch's one-way ANOVA; P<0.05) bundle and SOL (Welch's one-way ANOVA; P<0.05) compared to fiber bundles. Our results showed that mdm muscles exhibit significantly lower RFE than WT for both SOL (Welch's one-way ANOVA; P<0.0001) and EDL (Welch's one-way ANOVA; P<0.0001). This result is consistent with previous observations in single myofibrils and intact muscles. The data support the hypothesis that titin contributes to RFE. We suggest that RFE is reduced in mdm muscles due to impaired Ca2+ dependent titin-actin interactions, resulting from the small deletion in titin that includes 53 amino acids in Ig83 in the N2A region.

Mitochondrial respiration and H2O2 emission in saponin-permeabilized murine diaphragm fibers: optimization of fiber separation and comparison to limb muscle.

Diaphragm abnormalities in aging or chronic diseases include impaired mitochondrial respiration and H2O2 emission, which can be measured using saponin-permeabilized muscle fibers. Mouse diaphragm presents a challenge for isolation of fibers due to relatively high abundance of connective tissue in healthy muscle that is exacerbated in disease states. We tested a new approach to process mouse diaphragm for assessment of intact mitochondria respiration and ROS emission in saponin-permeabilized fibers. We used the red gastrocnemius (RG) as "standard" limb muscle. Markers of mitochondrial content were two- to fourfold higher in diaphragm (Dia) than in RG (P < 0.05). Maximal O2 consumption (JO2: pmol·s-1·mg-1) in Dia was higher with glutamate, malate, and succinate (Dia 399 ± 127, RG 148 ± 60; P < 0.05) and palmitoyl-CoA + carnitine (Dia 15 ± 5, RG 7 ± 1; P < 0.05) than in RG, but not different between muscles when JO2 was normalized to citrate synthase activity. Absolute JO2 for Dia was two- to fourfold higher than reported in previous studies. Mitochondrial JH2O2 was higher in Dia than in RG (P < 0.05), but lower in Dia than in RG when JH2O2 was normalized to citrate synthase activity. Our findings are consistent with an optimized diaphragm preparation for assessment of intact mitochondria in permeabilized fiber bundles. The data also suggest that higher mitochondrial content potentially makes the diaphragm more susceptible to "mitochondrial onset" myopathy. Overall, the new approach will facilitate testing and understanding of diaphragm mitochondrial function in mouse models that are used to advance biomedical research and human health.

Characterization of the passive mechanical properties of spine muscles across species

Passive mechanical properties differ between muscle groups within a species. Altered functional demands can also shift the passive force-length relationship. The extent that passive mechanical properties differ within a muscle group (e.g. spine extensors) or between homologous muscles of different species is unknown. It was hypothesized that multifidus, believed to specialize in spine stabilization, would generate greater passive tensile stresses under isometric conditions than erector spinae, which have more generalized functions of moving and stabilizing the spine; observing greater multifidus moduli in different species would strengthen this hypothesis. Permeabilized fibre bundles (n = 337) from the multifidus and erector spinae of mice, rats, and rabbits were mechanically tested. A novel logistic function was fit to the experimental data to fully characterize passive stress and modulus. Species had the greatest effect on passive muscle parameters with mice having the largest moduli at all lengths. Rats generated less passive stress than rabbits due to a shift of the passive force-length relationship towards longer muscle lengths. Rat multifidus generated slightly greater stresses than erector spinae, but no differences were observed between mouse muscles. The secondary objective was to determine the parameters required to simulate the passive force-length relationship. Experimental data were compared to the passive muscle model in OpenSim. The default OpenSim model, optimized for hindlimb muscles, did not fit any of the spine muscles tested; however, the model could accurately simulate experimental data after adjusting the input parameters. The optimal parameters for modelling the passive force-length relationships of spine muscles in OpenSim are presented.

Mitochondrial Respiration and H2O2 Emission in Saponin‐permeabilized Murine Diaphragm Fibers: Optimization of Fiber Separation and Comparison to Limb Muscle

Chronic diseases such as cancer, COPD, and heart failure impair diaphragm metabolism and mitochondrial function. Mice are used extensively to replicate disease conditions and offer the advantage of studying genetically modified animals. Mouse diaphragm contains a high abundance of connective tissue, with relatively thin (15–25 μm diameter) and fragile fibers. In preliminary studies, we discovered that standard fiber separation approaches utilized for limb muscles are unsuitable for the diaphragm. Thus, the purpose of this study was to optimize a protocol for diaphragm (Dia) fiber bundle preparation to evaluate the mitochondrial respiration and reactive oxygen species emission. We used the red gastrocnemius (Gast) muscle as a ‘standard’ for comparison.Michaelis‐Menten kinetics of ADP‐stimulated O2 consumption with complex I substrates (JO2 in pmol/s/mg wet weight) showed 3‐fold higher Vmax in Dia than Gast (Gast 69 ± 18, Dia 211 ± 20; p &lt; 0.05, n = 9 mice), whereas Km was not significantly different. Maximal JO2 in Dia was 3‐fold higher with complex I + II substrates (Gast 148 ± 25, Dia 387 ± 45; p &lt; 0.05), and 2‐fold higher with palmitoyl‐CoA + carnitine (Gast 7 ± 0, Dia 15 ± 2, p &lt; 0.05) compared to Gast. Baseline JH2O2 (pmol/min/mg wet weight) was higher in Dia (Gast 0.8 ± 0.2, Dia 1.7 ± 0.3; p &lt; 0.05), but succinate‐induced JH2O2 was not different between muscles. Citrate synthase activity was 3‐fold higher in Dia (μmol/min/mg protein: Gast 43 ± 3, Dia 118 ± 4; p &lt; 0.05). The protein abundance of electron transport chain complexes I‐V were 2–4 fold higher in the Dia than Gast (p &lt; 0.05). When we normalized maximal JO2 by citrate synthase activity, there were no differences between Dia and Gast for complex I and complex I + II substrates. JH2O2 normalized to citrate synthase activity was higher for Gast at baseline (pmol/min/mg wet weight/U CS: Gast 2.1 ± 0.3, Dia 0.3 ± 0.1; p &lt; 0.05), but there was no difference in succinate‐induced JH2O2 normalized to citrate synthase activity.The values for maximal diaphragm mitochondria JO2 in our study are 2–3 fold higher than reported in the literature using the standard ‘limb muscle’ approach for the diaphragm fiber preparation. The similar maximal JO2 normalized to citrate synthase activity between diaphragm and red gastrocnemius muscles suggest that our new approach is valid for the assessment of intact diaphragm mitochondrial function in permeabilized fiber bundles.Support or Funding InformationFunding source: NIH 1R01HL130318‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Direct real-time quantification of mitochondrial oxidative phosphorylation efficiency in permeabilized skeletal muscle myofibers.

Oxidative phosphorylation (OXPHOS) efficiency, defined as the ATP-to-O ratio, is a critical feature of mitochondrial function that has been implicated in health, aging, and disease. To date, however, the methods to measure ATP/O have primarily relied on indirect approaches or entail parallel rather than simultaneous determination of ATP synthesis and O2 consumption rates. The purpose of this project was to develop and validate an approach to determine the ATP/O ratio in permeabilized fiber bundles (PmFBs) from simultaneous measures of ATP synthesis (JATP) and O2 consumption (JO2 ) rates in real time using a custom-designed apparatus. JO2 was measured via a polarigraphic oxygen sensor and JATP via fluorescence using an enzyme-linked assay system (hexokinase II, glucose-6-phosphate dehydrogenase) linked to NADPH production. Within the dynamic linear range of the assay system, ADP-stimulated increases in steady-state JATP mirrored increases in steady-state JO2 (r(2) = 0.91, P < 0.0001, n = 57 data points). ATP/O ratio was less than one under low rates of respiration (15 μM ADP) but increased to more than two at moderate (200 μM ADP) and maximal (2,000 μM ADP) rates of respiration with an interassay coefficient of variation of 24.03, 16.72, and 11.99%, respectively. Absolute and relative (to mechanistic) ATP/O ratios were lower in PmFBs (2.09 ± 0.251, 84%) compared with isolated mitochondria (2.44 ± 0.124, 98%). ATP/O ratios in PmFBs were not affected by the activity of adenylate kinase or creatine kinase. These findings validate an enzyme-linked respiratory clamp system for measuring OXPHOS efficiency in PmFBs and provide evidence that OXPHOS efficiency increases as energy demand increases.

Chronological Age Does not Influence Ex-vivo Mitochondrial Respiration and Quality Control in Skeletal Muscle.

Considerable debate continues to surround the concept of mitochondrial dysfunction in aging muscle. We tested the overall hypothesis that age per se does not influence mitochondrial function and markers of mitochondria quality control, that is, expression of fusion, fission, and autophagy proteins. We also investigated the influence of cardiorespiratory fitness (VO2max) and adiposity (body mass index) on these associations. Percutaneous biopsies of the vastus lateralis were obtained from sedentary young (n = 14, 24±3 years), middle-aged (n = 24, 41±9 years) and older adults (n = 20, 78±5 years). A physically active group of young adults (n = 10, 27±5 years) was studied as a control. Mitochondrial respiration was determined in saponin permeabilized fiber bundles. Fusion, fission and autophagy protein expression was determined by Western blot. Cardiorespiratory fitness was determined by a graded exercise test. Mitochondrial respiratory capacity and expression of fusion (OPA1 and MFN2) and fission (FIS1) proteins were not different among sedentary groups despite a wide age range (21 to 88 years). Mitochondrial respiratory capacity and fusion and fission proteins were, however, negatively associated with body mass index, and mitochondrial respiratory capacity was positively associated with cardiorespiratory fitness. The young active group had higher respiration, complex I and II respiratory control ratios, and expression of fusion and fission proteins. Finally, the expression of fusion, fission, and autophagy proteins were linked with mitochondrial respiration. Mitochondrial respiration and markers of mitochondrial dynamics (fusion and fission) are not associated with chronological age per se, but rather are more strongly associated with body mass index and cardiorespiratory fitness.

Locomotor and Diaphragm Muscle Mitochondrial Oxidative Capacity in Lung‐Targeted VEGF Gene Deleted Mice Exposed to Cigarette Smoke

Locomotor skeletal muscle function is impaired in patients with chronic obstructive pulmonary disease (COPD). The mechanisms responsible for these skeletal muscle abnormalities are not well understood. We hypothesized that the severity of lung damage following chronic exposure to cigarette smoke (CS) would determine the mitochondrial oxidative capacity of locomotor and respiratory skeletal muscle.Adult VEGFLoxP mice with or without lung‐targeted VEGF gene deletion (5 months of age) were used as a model of progressively compromised static lung mechanics following exposure to 2 × 30 min of daily, nose‐only CS for an additional 4 months. Deletion of the VEGF gene was accomplished by instilling half the mice with AAV‐Cre one month before initiating CS exposure. The remaining mice received a control AAV (LacZ). Further, both groups (Cre and LacZ) were assigned either to CS exposure or room air control (RA). Mitochondrial respiration in permeabilized fiber bundles from the diaphragm and medial gastrocnemius was measured using high‐resolution respirometry.Gastrocnemius ADP‐stimulated O2 consumption with malate and glutamate as substrates was 78±6%, 66±7% and 60±11% lower in CS‐LacZ, RA‐Cre, and CS‐Cre, respectively, than RA‐LacZ (p&lt;0.05). Gastrocnemius electron transport system capacity was 68±5% (p&lt;0.05), 53±11% (p&lt;0.05) and 48±21% (NS) lower in the CS‐LacZ, RA‐Cre, and CS‐Cre, respectively, than RA‐LacZ. Diaphragm ADP‐stimulated O−2 consumption was 56±9% lower in CS‐LacZ fibers than RA‐LacZ (p&lt;0.05). Diaphragm fiber electron transport system capacity was not different between the groups.Mice with a COPD‐like phenotype exhibited impaired locomotor muscle oxidative capacity irrespective of whether the lung damage was caused by cigarette smoke exposure or VEGF gene deletion. In contrast, diaphragm mitochondrial respiration only showed a deficit in ADP‐stimulated O2 consumption with CS exposure. Thus, mitochondrial respiration in locomotor skeletal muscle appears to be less well‐preserved than diaphragm in this model of COPD.Support or Funding InformationDepartment of Veterans Affairs