ABSTRACT
Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these changes do not always reflect what occurs in other tissues, such as skeletal muscle, these cells are an accessible and valuable source of viable mitochondria from human subjects. PBMCs are exposed to systemic signals that impact their bioenergetic state. Thus, expanding our tools to interrogate mitochondrial metabolism in this population will elucidate mechanisms related to disease progression. Functional assays of mitochondria are often limited to using respiratory outputs following maximal substrate, inhibitor, and uncoupler concentrations to determine the full range of respiratory capacity, which may not be achievable in vivo. The conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) by ATP-synthase results in a decrease in mitochondrial membrane potential (mMP) and an increase in oxygen consumption. To provide a more integrated analysis of mitochondrial dynamics, this article describes the use of high-resolution fluorespirometry to measure the simultaneous response of oxygen consumption and mitochondrial membrane potential (mMP) to physiologically relevant concentrations of ADP. This technique uses tetramethylrhodamine methylester (TMRM) to measure mMP polarization in response to ADP titrations following maximal hyperpolarization with complex I and II substrates. This technique can be used to quantify how changes in health status, such as aging and metabolic disease, affect the sensitivity of mitochondrial response to energy demand in PBMCs, T-cells, and monocytes from human subjects.
Subject(s)
Leukocytes, Mononuclear , Membrane Potential, Mitochondrial , Humans , Membrane Potential, Mitochondrial/physiology , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/cytology , Rhodamines/chemistry , Adenosine Diphosphate/metabolism , Adenosine Diphosphate/pharmacology , Oxygen Consumption/physiology , Mitochondria/metabolism , Fluorescent Dyes/chemistryABSTRACT
BackgroundCurrent studies suggest mitochondrial dysfunction is a major contributor to impaired physical performance and exercise intolerance in chronic kidney disease (CKD). We conducted a clinical trial of coenzyme Q10 (CoQ10) and nicotinamide riboside (NR) to determine their impact on exercise tolerance and metabolic profile in patients with CKD.MethodsWe conducted a randomized, placebo-controlled, double-blind, crossover trial comparing CoQ10, NR, and placebo in 25 patients with an estimated glomerular filtration rate (eGFR) of less than 60mL/min/1.73 m2. Participants received NR (1,000 mg/day), CoQ10 (1,200 mg/day), or placebo for 6 weeks each. The primary outcomes were aerobic capacity measured by peak rate of oxygen consumption (VO2 peak) and work efficiency measured using graded cycle ergometry testing. We performed semitargeted plasma metabolomics and lipidomics.ResultsParticipant mean age was 61.0 ± 11.6 years and mean eGFR was 36.9 ± 9.2 mL/min/1.73 m2. Compared with placebo, we found no differences in VO2 peak (P = 0.30, 0.17), total work (P = 0.47, 0.77), and total work efficiency (P = 0.46, 0.55) after NR or CoQ10 supplementation. NR decreased submaximal VO2 at 30 W (P = 0.03) and VO2 at 60 W (P = 0.07) compared with placebo. No changes in eGFR were observed after NR or CoQ10 treatment (P = 0.14, 0.88). CoQ10 increased free fatty acids and decreased complex medium- and long-chain triglycerides. NR supplementation significantly altered TCA cycle intermediates and glutamate that were involved in reactions that exclusively use NAD+ and NADP+ as cofactors. NR decreased a broad range of lipid groups including triglycerides and ceramides.ConclusionsSix weeks of treatment with NR or CoQ10 improved markers of systemic mitochondrial metabolism and lipid profiles but did not improve VO2 peak or total work efficiency.Trial registrationClinicalTrials.gov NCT03579693.FundingNational Institutes of Diabetes and Digestive and Kidney Diseases (grants R01 DK101509, R03 DK114502, R01 DK125794, and R01 DK101509).
Subject(s)
Renal Insufficiency, Chronic , Humans , Middle Aged , Aged , Cross-Over Studies , Renal Insufficiency, Chronic/drug therapy , TriglyceridesABSTRACT
The ability to respond to physical stress that disrupts normal physiological homeostasis at an older age embraces the concept of resilience to aging. A physical stressor could be used to induce physiological responses that are age-related, since resilience declines with increasing age. Increased fat and sugar intake is a nutritional stress with a high prevalence of obesity in older people. In order to determine the effect of this type of diet on resilience to aging, 18-month-old C57BL/6J male mice were fed a diet high in saturated fat (lard) and sucrose (HFS) for ten months. At the end of the 10-month study, mice fed the HFS diet showed increased cognitive impairment, decreased cardiac function, decreased strength and agility, and increased severity of renal pathology compared to mice fed a rodent chow diet low in saturated fat and sucrose (LFS). The degree of response aligned with decreased resilience to the long-term adverse effects of the diet with characteristics of accelerated aging. This observation suggests additional studies could be conducted to investigate the relationship between an accelerated decline in resilience to aging and enhanced resilience to aging under different dietary conditions.
ABSTRACT
Aging and poor diet are independent risk factors for heart disease, but the impact of high-sucrose (HS) consumption in the aging heart is understudied. Aging leads to impairments in mitochondrial function that result in muscle dysfunction (e.g., cardiac remodeling and sarcopenia). We tested whether HS diet (60%kcal sucrose) would accelerate muscle dysfunction in 24-month-old male CB6F1 mice. By week 1 on HS diet, mice developed significant cardiac hypertrophy compared to age-matched chow-fed controls. The increased weight of the heart persisted throughout the 4-week treatment, while body weight and strength declined more rapidly than controls. We then tested whether HS diet could worsen cardiac dysfunction in old mice and if the mitochondrial-targeted drug, elamipretide (ELAM), could prevent the diet-induced effect. Old and young mice were treated with either ELAM or saline as a control for 2 weeks, and provided with HS diet or chow on the last week. As demonstrated in the previous experiment, old mice had age-related cardiac hypertrophy that worsened after one week on HS and was prevented by ELAM treatment, while the HS diet had no detectable effect on hypertrophy in the young mice. As expected, mitochondrial respiration and reactive oxygen species (ROS) production were altered by age, but were not significantly affected by HS diet or ELAM. Our findings highlight the vulnerability of the aged heart to HS diet that can be prevented by systemic targeting of the mitochondria with ELAM.
Subject(s)
Heart Diseases , Sugars , Mice , Male , Animals , Cardiomegaly/etiology , Aging , Heart Diseases/complications , Sucrose , Dietary SugarsABSTRACT
Background: Disruption of metabolic and bioenergetic homeostasis related to mitochondrial dysfunction is a key driver of aging biology. Therefore, targeting mitochondrial function would be a rational approach to slowing aging. Elamipretide (Elam, a.k.a. SS-31) is a peptide known to target mitochondria and suppress mammalian signs of aging. The present study was designed to examine the phenotypic effects of long-term Elam treatment on aging in C57BL/6 mice starting at 18 months of age. Methods: Mice were fed regular chow (RC diet) or a diet high in fat and sugar (HF diet) and treated with 3 mg/kg of Elam or saline subcutaneously 5 days per week for 10 months. Physiological performance assessments were conducted at 28 months of age. Results: Elam improved the physical performance of males but not females, while in females Elam improved cognitive performance and enhanced the maintenance of body weight and fat mass. It also improved diastolic function in both males and females, but to a greater extent in males. The HF diet over 10 months had a negative effect on health span, as it increased body fat and decreased muscle strength and heart function, especially in females. Conclusions: Elam enhanced healthy aging and cardiac function in both male and female mice, although the specific effects on function differed between sexes. In females, the treatment led to better cognitive performance and maintenance of body composition, while in males, performance on a rotating rod was preserved. These overall observations have translational implications for considering additional studies using Elam in therapeutic or preventive approaches for aging and age-related diseases.
ABSTRACT
Purpose: The purpose of this study was to present our hypothesis that aging alters metabolic function in ocular tissues. We tested the hypothesis by measuring metabolism in aged murine tissues alongside retinal responses to light. Methods: Scotopic and photopic electroretinogram (ERG) responses in young (3-6 months) and aged (23-26 months) C57Bl/6J mice were recorded. Metabolic flux in retina and eyecup explants was quantified using U-13C-glucose or U-13C-glutamine with gas chromatography-mass spectrometry (GC-MS), O2 consumption rate (OCR) in a perifusion apparatus, and quantifying adenosine triphosphatase (ATP) with a bioluminescence assay. Results: Scotopic and photopic ERG responses were reduced in aged mice. Glucose metabolism, glutamine metabolism, OCR, and ATP pools in retinal explants were mostly unaffected in aged mice. In eyecups, glutamine usage in the Krebs Cycle decreased while glucose metabolism, OCR, and ATP pools remained stable. Conclusions: Our examination of metabolism showed negligible impact of age on retina and an impairment of glutamine anaplerosis in eyecups. The metabolic stability of these tissues ex vivo suggests age-related metabolic alterations may not be intrinsic. Future experiments should focus on determining whether external factors including nutrient supply, oxygen availability, or structural changes influence ocular metabolism in vivo.
Subject(s)
Aging/physiology , Retina/metabolism , Retinal Pigment Epithelium/metabolism , Adenosine Triphosphate/metabolism , Animals , Color Vision/physiology , Electroretinography , Flicker Fusion/physiology , Gas Chromatography-Mass Spectrometry , Glucose/metabolism , Glutamine/metabolism , Light , Male , Metabolomics , Mice , Mice, Inbred C57BL , Night Vision/physiology , Oxygen Consumption/physiology , Photic StimulationABSTRACT
Aging and poor nutrition are independent risk factors for the development of chronic disease. When young animals are given diets high in fat or sugar, they exhibit hallmarks of aging like mitochondrial dysfunction and inflammation, and also develop a greater risk for age-related disease. The same mitochondrial dysfunction and inflammation that progress with aging may also further predispose older individuals to dietary insults by fat and sugar. The purpose of this work is to review the most recent studies that address the impact of fat and sugar consumption on hallmarks of aging (mitochondrial dysfunction and inflammation). Findings from these studies show that obesogenic, high-fat diets can exacerbate age-related disease and hallmarks of aging in young animals, but high-fat diets that are non-obesogenic may play a beneficial role in old age. In contrast, high-sugar diets do not require an obesogenic effect to induce mitochondrial dysfunction or inflammation in young rodents. Currently, there is a lack of experimental studies addressing the impact of sugar in the context of aging, even though empirical evidence points to the detrimental effect of sugar in aging by contributing to a variety of age-related diseases. Fig. 1 Mitochondrial dysfunction and altered cellular communication (e.g. inflammation) progress with advancing age and increase the risk for age-related disease (ARD). Given the physiological changes that occur with age, the impact of high-fat (HFD) and high-sugar diets (HSD) may differ in later and earlier stages of life. HFD can promote the development of hallmarks of aging in young animals and can also exacerbate the risk for ARD when consumed at an old age. However, non-obesogenic high-fat diets may also reduce the risk for ARD in old age by acting on these hallmarks of aging. On the other hand, HSD promotes mitochondrial dysfunction and inflammation without necessarily inducing weight gain in young animals. Empirical evidence points to sugar as a major contributor to age-related disease and more experimental studies are needed to clarify whether aged individuals are more susceptible to its effects.
Subject(s)
Obesity , Sugars , Animals , Inflammation/etiologyABSTRACT
High intensity exercise is a popular mode of exercise to elicit similar or greater adaptive responses compared to traditional moderate intensity continuous exercise. However, the molecular mechanisms underlying these adaptive responses are still unclear. The purpose of this pilot study was to compare high and low intensity contractile stimulus on the Nrf2-mediated redox stress response in mouse skeletal muscle. An intra-animal design was used to control for variations in individual responses to muscle stimulation by comparing a stimulated limb (STIM) to the contralateral unstimulated control limb (CON). High Intensity (HI - 100Hz), Low Intensity (LI - 50Hz), and Naïve Control (NC - Mock stimulation vs CON) groups were used to compare these effects on Nrf2-ARE binding, Keap1 protein, and downstream gene and protein expression of Nrf2 target genes. Muscle stimulation significantly increased Nrf2-ARE binding in LI-STIM compared to LI-CON (p = 0.0098), while Nrf2-ARE binding was elevated in both HI-CON and HI-STIM compared to NC (p = 0.0007). The Nrf2-ARE results were mirrored in the downregulation of Keap1, where Keap1 expression in HI-CON and HI-STIM were both significantly lower than NC (p = 0.008) and decreased in LI-STIM compared to LI-CON (p = 0.015). In addition, stimulation increased NQO1 protein compared to contralateral control regardless of stimulation intensity (p = 0.019), and HO1 protein was significantly higher in high intensity compared to the Naïve control group (p = 0.002). Taken together, these data suggest a systemic redox signaling exerkine is activating Nrf2-ARE binding and is intensity gated, where Nrf2-ARE activation in contralateral control limbs were only seen in the HI group. Other research in exercise induced Nrf2 signaling support the general finding that Nrf2 is activated in peripheral tissues in response to exercise, however the specific exerkine responsible for the systemic signaling effects is not known. Future work should aim to delineate these redox sensitive systemic signaling mechanisms.
Subject(s)
Muscle, Skeletal , NF-E2-Related Factor 2 , Animals , Kelch-Like ECH-Associated Protein 1/genetics , Kelch-Like ECH-Associated Protein 1/metabolism , Mice , Muscle, Skeletal/metabolism , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Oxidation-Reduction , Pilot ProjectsABSTRACT
Endurance training (ET) is recommended for the elderly to improve metabolic health and aerobic capacity. However, ET-induced adaptations may be suboptimal due to oxidative stress and exaggerated inflammatory response to ET. The natural antioxidant and anti-inflammatory dietary supplement astaxanthin (AX) has been found to increase endurance performance among young athletes, but limited investigations have focused on the elderly. We tested a formulation of AX in combination with ET in healthy older adults (65-82 years) to determine if AX improves metabolic adaptations with ET, and if AX effects are sex-dependent. Forty-two subjects were randomized to either placebo (PL) or AX during 3 months of ET. Specific muscle endurance was measured in ankle dorsiflexors. Whole body exercise endurance and fat oxidation (FATox) was assessed with a graded exercise test (GXT) in conjunction with indirect calorimetry. Results: ET led to improved specific muscle endurance only in the AX group (Pre 353 ± 26 vs. Post 472 ± 41 contractions), and submaximal GXT duration improved in both groups (PL 40.8 ± 9.1% and AX 41.1 ± 6.3%). The increase in FATox at lower intensity after ET was greater in AX (PL 0.23 ± 0.15 g vs. AX 0.76 ± 0.18 g) and was associated with reduced carbohydrate oxidation and increased exercise efficiency in males but not in females.
Subject(s)
Antioxidants/pharmacology , Dietary Supplements , Exercise , Adaptation, Physiological/drug effects , Aged , Aged, 80 and over , Calorimetry, Indirect , Exercise/physiology , Exercise Test/drug effects , Female , Humans , Male , Physical Endurance/drug effects , Sex Factors , Xanthophylls/pharmacologyABSTRACT
Background: Eccentric contractions induce muscle damage (EIMD) that compromises muscle function. Poor recovery from EIMD has been suggested to be a contributor to the decline in muscle function evident in sarcopenia, but it is unclear which aspects of muscle function are more susceptible to disruption by EIMD in old versus young muscle. The purpose of this study was to determine the extent of impairment in contractile function (force, fatigue, tetanus and twitch kinetics) during the recovery from EIMD in very old (VO) mice compared to young adult (YA). Methods: Male CB6F1 were obtained from National Institure of Aging colony. VO mice were 29-31 months of age, and YA mice were 7-9 months of age. The plantarflexor muscles were subjected to 20 eccentric contractions in vivo to induce injury (EIMD). Changes in tetanic force and kinetics were assessed before EIMD, immediately after EIMD and 3 days after EIMD (3dEIMD). Force-frequency and rates of fatigue were assessed 3d-EIMD and compared with baseline. Histological analysis was conducted in injured and non-injured contralateral gastrocnemius muscle. Results: There was a greater loss in isometric tetanic force immediately following EIMD in VO compared with YA (-31.6% ± 10.4 vs. -21.7% ± 6.0, P < 0.05). At 3d-EIMD, the rate of contraction of tetanus began to recover in VO, but not in YA (20.8% vs. -6.8%, P < 0.05), whereas the extent of recovery of force tended to be greater in VO than YA (39.3% vs. 17.1%, P = 0.08) when compared with tetanic function immediately after injury. Compared with function pre-injury (baseline), VO and YA had similar deficits in tetanic force (-7.3% ± 5.3 vs. -9.2% ± 6.0, respectively) and kinetics at Day 3. Twitch kinetics (rate of relaxation) recovered faster in VO compared with YA. The rate of muscle fatigue was similar to baseline values, with VO continuing to be more fatigue resistant than YA 3d-EIMD. There were no detectable differences in muscle mass or myofibre cross-sectional area despite continued deficits in force following EIMD in either age group. Conclusions: Despite clear functional deficits and greater susceptibility to injury, aged sarcopenic muscle exhibited a similar ability to recover contractile function to younger muscle following EIMD. In addition, neither age group showed accelerated muscle fatigue in the recovery phase after EIMD; thus, sarcopenic mouse muscles do not appear to be more susceptible to long-term functional impairment than young healthy muscles.
ABSTRACT
KEY POINTS: Breast cancer 1 early onset gene codes for the DNA repair enzyme, breast cancer type 1 susceptibility protein (BRCA1). The gene is prone to mutations that cause a loss of protein function. BRCA1/Brca1 has recently been found to regulate several cellular pathways beyond DNA repair and is expressed in skeletal muscle. Skeletal muscle specific knockout of Brca1 in mice caused a loss of muscle quality, identifiable by reductions in muscle force production and mitochondrial respiratory capacity. Loss of muscle quality was associated with a shift in muscle phenotype and an accumulation of mitochondrial DNA mutations. These results demonstrate that BRCA1 is necessary for skeletal muscle function and that increased mitochondrial DNA mutations may represent a potential underlying mechanism. ABSTRACT: Recent evidence suggests that the breast cancer 1 early onset gene (BRCA1) influences numerous peripheral tissues, including skeletal muscle. The present study aimed to determine whether induced-loss of the breast cancer type 1 susceptibility protein (Brca1) alters skeletal muscle function. We induced genetic ablation of exon 11 in the Brca1 gene specifically in the skeletal muscle of adult mice to generate skeletal muscle-specific Brca1 homozygote knockout (Brca1KOsmi ) mice. Brca1KOsmi exhibited kyphosis and decreased maximal isometric force in limb muscles compared to age-matched wild-type mice. Brca1KOsmi skeletal muscle shifted toward an oxidative muscle fibre type and, in parallel, increased myofibre size and reduced capillary numbers. Unexpectedly, myofibre bundle mitochondrial respiration was reduced, whereas contraction-induced lactate production was elevated in Brca1KOsmi muscle. Brca1KOsmi mice accumulated mitochondrial DNA mutations and exhibited an altered mitochondrial morphology characterized by distorted and enlarged mitochondria, and these were more susceptible to swelling. In summary, skeletal muscle-specific loss of Brca1 leads to a myopathy and mitochondriopathy characterized by reductions in skeletal muscle quality and a consequent kyphosis. Given the substantial impact of BRCA1 mutations on cancer development risk in humans, a parallel loss of BRCA1 function in patient skeletal muscle cells would potentially result in implications for human health.
Subject(s)
BRCA1 Protein/genetics , Mitochondria, Muscle/pathology , Muscle Weakness/genetics , Muscle, Skeletal/pathology , Animals , DNA, Mitochondrial/genetics , Female , Male , Mice , Mice, Inbred C57BL , Mutation/geneticsABSTRACT
BACKGROUND: Massive rotator cuff tears (RCTs) begin as primary tendon injuries and cause a myriad of changes in the muscle, including atrophy, fatty infiltration (FI), and fibrosis. However, it is unclear which changes are most closely associated with muscle function. PURPOSE: To determine if FI of the supraspinatus muscle after acute RCT relates to short-term changes in muscle function. STUDY DESIGN: Controlled laboratory study. METHODS: Unilateral RCTs were induced in female rabbits via tenotomy of the supraspinatus and infraspinatus. Maximal isometric force and rate of fatigue were measured in the supraspinatus in vivo at 6 and 12 weeks after tenotomy. Computed tomography scanning was performed, followed by histologic analysis of myofiber size, FI, and fibrosis. RESULTS: Tenotomy resulted in supraspinatus weakness, reduced myofiber size, FI, and fibrosis, but no differences were evident between 6 and 12 weeks after tenotomy except for increased collagen content at 12 weeks. FI was a predictor of supraspinatus weakness and was strongly correlated to force, even after accounting for muscle cross-sectional area. While muscle atrophy accounted for the loss in force in tenotomized muscles with minimal FI, it did not account for the greater loss in force in tenotomized muscles with the most FI. Collagen content was not strongly correlated with maximal isometric force, even when normalized to muscle size. CONCLUSION: After RCT, muscle atrophy results in the loss of contractile force from the supraspinatus, but exacerbated weakness is observed with increased FI. Therefore, the level of FI can help predict contractile function of torn rotator cuff muscles. CLINICAL RELEVANCE: Markers to predict contractile function of RCTs will help determine the appropriate treatment to improve functional recovery after RCTs.
Subject(s)
Adipose Tissue/diagnostic imaging , Rotator Cuff Injuries/diagnosis , Rotator Cuff/physiopathology , Adipose Tissue/physiopathology , Animals , Female , Prognosis , RabbitsABSTRACT
The breast cancer type 1 susceptibility protein (Brca1) is a regulator of DNA repair in mammary gland cells; however, recent cell culture evidence suggests that Brca1 influences other processes, including those in nonmammary cells. In this study, we sought to determine whether Brca1 is necessary for metabolic regulation of skeletal muscle using a novel in vivo mouse model. We developed an inducible skeletal muscle-specific Brca1knockout (BRCA1KOsmi) model to test whether Brca1 expression is necessary for maintenance of metabolic function of skeletal muscle when exposed to a high-fat diet (HFD). Our data demonstrated that deletion of Brca1 prevented HFD-induced alterations in glucose and insulin tolerance. Irrespective of diet, BRCA1KOsmi mice exhibited significantly lower ADP-stimulated complex I mitochondrial respiration rates compared to age-matched wild-type (WT) mice. The data show that Brca1 has the ability to localize to the mitochondria in skeletal muscle and that BRCA1KOsmi mice exhibit higher whole-body CO2 production, respiratory exchange ratio, and energy expenditure, compared with the WT mice. Our results demonstrate that loss of Brca1 in skeletal muscle leads to dysregulated metabolic function, characterized by decreased mitochondrial respiration. Thus, any condition that results in loss of Brca1 function could induce metabolic imbalance in skeletal muscle.-Jackson, K. C., Tarpey, M. D., Valencia, A. P., Iñigo, M. R., Pratt, S. J., Patteson, D. J., McClung, J. M., Lovering, R. M., Thomson, D. M., Spangenburg, E. E. Induced Cre-mediated knockdown of Brca1 in skeletal muscle reduces mitochondrial respiration and prevents glucose intolerance in adult mice on a high-fat diet.
Subject(s)
Dietary Fats/adverse effects , Gene Knockdown Techniques , Glucose Intolerance/prevention & control , Integrases , Mitochondria, Muscle/metabolism , Muscle, Skeletal/metabolism , Oxygen Consumption , Tumor Suppressor Proteins/deficiency , Animals , BRCA1 Protein , Dietary Fats/pharmacology , Glucose Intolerance/chemically induced , Glucose Intolerance/genetics , Glucose Intolerance/metabolism , Mice , Mice, Knockout , Mitochondria, Muscle/genetics , Mitochondria, Muscle/pathology , Muscle, Skeletal/pathology , Tumor Suppressor Proteins/metabolismABSTRACT
BACKGROUND: Rotator cuff (RTC) tears are a common clinical problem resulting in adverse changes to the muscle, but there is limited information comparing histopathology to contractile function. This study assessed supraspinatus force and susceptibility to injury in the rat model of RTC tear, and compared these functional changes to histopathology of the muscle. METHODS: Unilateral RTC tears were induced in male rats via tenotomy of the supraspinatus and infraspinatus. Maximal tetanic force and susceptibility to injury of the supraspinatus muscle were measured in vivo at day 2 and day 15 after tenotomy. Supraspinatus muscles were weighed and harvested for histologic analysis of the neuromuscular junction (NMJ), intramuscular lipid, and collagen. RESULTS: Tenotomy resulted in eventual atrophy and weakness. Despite no loss in muscle mass at day 2 there was a 30% reduction in contractile force, and a decrease in NMJ continuity and size. Reduced force persisted at day 15, a time point when muscle atrophy was evident but NMJ morphology was restored. At day 15, torn muscles had decreased collagen-packing density and were also more susceptible to contraction-induced injury. CONCLUSION: Muscle size and histopathology are not direct indicators of overall RTC contractile health. Changes in NMJ morphology and collagen organization were associated with changes in contractile function and thus may play a role in response to injury. Although our findings are limited to the acute phase after a RTC tear, the most salient finding is that RTC tenotomy results in increased susceptibility to injury of the supraspinatus.
Subject(s)
Muscle Contraction , Rotator Cuff Injuries/physiopathology , Rotator Cuff/physiopathology , Adiposity , Animals , Biomarkers , Fibrosis , Male , Muscular Atrophy , Neuromuscular Junction/pathology , Random Allocation , Rats, Sprague-Dawley , Rotator Cuff/pathology , Rotator Cuff Injuries/pathologyABSTRACT
Duchenne muscular dystrophy (DMD) is a genetic disorder in which the absence of dystrophin leads to progressive muscle degeneration and weakness. Although the genetic basis is known, the pathophysiology of dystrophic skeletal muscle remains unclear. We examined nuclear movement in wild-type (WT) and muscular dystrophy mouse model for DMD (MDX) (dystrophin-null) mouse myofibers. We also examined expression of proteins in the linkers of nucleoskeleton and cytoskeleton (LINC) complex, as well as nuclear transcriptional activity via histone H3 acetylation and polyadenylate-binding nuclear protein-1. Because movement of nuclei is not only LINC dependent but also microtubule dependent, we analyzed microtubule density and organization in WT and MDX myofibers, including the application of a unique 3D tool to assess microtubule core structure. Nuclei in MDX myofibers were more mobile than in WT myofibers for both distance traveled and velocity. MDX muscle shows reduced expression and labeling intensity of nesprin-1, a LINC protein that attaches the nucleus to the microtubule and actin cytoskeleton. MDX nuclei also showed altered transcriptional activity. Previous studies established that microtubule structure at the cortex is disrupted in MDX myofibers; our analyses extend these findings by showing that microtubule structure in the core is also disrupted. In addition, we studied malformed MDX myofibers to better understand the role of altered myofiber morphology vs. microtubule architecture in the underlying susceptibility to injury seen in dystrophic muscles. We incorporated morphological and microtubule architectural concepts into a simplified finite element mathematical model of myofiber mechanics, which suggests a greater contribution of myofiber morphology than microtubule structure to muscle biomechanical performance.NEW & NOTEWORTHY Microtubules provide the means for nuclear movement but show altered organization in the muscular dystrophy mouse model (MDX) (dystrophin-null) muscle. Here, MDX myofibers show increased nuclear movement, altered transcriptional activity, and altered linkers of nucleoskeleton and cytoskeleton complex expression compared with healthy myofibers. Microtubule architecture was incorporated in finite element modeling of passive stretch, revealing a role of fiber malformation, commonly found in MDX muscle. The results suggest that alterations in microtubule architecture in MDX muscle affect nuclear movement, which is essential for muscle function.
Subject(s)
Cell Nucleus/metabolism , Cell Nucleus/pathology , Models, Biological , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/pathology , Muscular Dystrophies/pathology , Muscular Dystrophies/physiopathology , Animals , Cells, Cultured , Computer Simulation , Female , Finite Element Analysis , Male , Mice , Mice, Inbred C57BL , Mice, Inbred mdxABSTRACT
In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Animal models provide unmitigated access to histological samples, which provide a "direct measure" of damage. However, even with unconstrained access to tissue morphology and biochemistry assays, the findings typically do not account for loss of muscle function. Thus, the most comprehensive measure of the overall health of the muscle is assessment of its primary function, which is to produce contractile force. The majority of animal models testing contractile force have been limited to the muscle groups moving the ankle, with advantages and disadvantages depending on the equipment. Here, we describe in vivo methods to measure torque, to produce a reliable muscle injury, and to follow muscle function within the same animal over time. We also describe in vivo methods to measure tension in the leg and thigh muscles.
Subject(s)
Muscle Contraction , Muscle, Skeletal/physiology , Animals , Mechanical Phenomena , Mice , Muscle Strength , Muscle, Skeletal/injuries , Muscle, Skeletal/physiopathology , Muscular Diseases/physiopathology , Rabbits , RatsABSTRACT
BACKGROUND: For unknown reasons a woman's risk for developing Metabolic Syndrome (MetS) increases dramatically with age and/or loss of ovarian function. The MetS is characterized by hepatic insulin resistance (IR), which is strongly associated with intrahepatic lipid (IHL) accumulation, mitochondrial dysfunction, and oxidative stress. Although circumstantial evidence suggests that the endocrine function of the ovary can directly impact hepatic mitochondrial function, this hypothesis remains untested. Thus, the purpose of this study was to assess the influence of age and secretory function of the ovary on mechanisms that regulate hepatic mitochondrial function. METHODS: Adult (10 week-old) and aged (88 week-old) female C57BL/6 mice were separated into two groups to undergo bilateral ovariectomy (OVX) or control surgery (SHAM). Eight weeks after surgery hepatic tissue was removed for measurements of total IHL and fatty acid species within hepatic triglycerides, mitochondrial function, and reactive oxygen species (ROS) production. RESULTS: Hepatic IHL content was not affected by OVX, but was increased by age. OVX had no effect on mitochondrial respiration, however, hepatic mitochondria from aged mice had lower O2 consumption, lower complex IV and higher complex I content. Mitochondrial H2O2 production was highest in OVX groups and exacerbated by age, while mitochondrial lipid peroxidation was highest in the aged mice and exacerbated by OVX. Regardless of age, OVX resulted in lower mitochondrial content of antioxidant glutathione peroxidase 1 (Gpx1). Isolated liver tissue from a sub-set of animals were acutely treated with conditioned ovarian media which increased Gpx1 mRNA expression compared to vehicle treated liver tissue. CONCLUSION: Ovarian secretory function is necessary for the maintenance of hepatic ROS buffering capacity in the mitochondria, while age significantly influences mitochondrial respiration. These data suggest that when age is coupled with loss of ovarian function there is an increased risk for developing hepatic mitochondrial dysfunction, which may influence the onset of metabolic disease. Thus, in females there is critical organ cross-talk occurring between hepatic tissue and the ovary that impacts hepatic mitochondrial function.
Subject(s)
Aging/metabolism , Glutathione Peroxidase/physiology , Mitochondria, Liver/metabolism , Ovary/physiology , Oxidative Stress/physiology , Animals , Body Weight/physiology , Fatty Acids/metabolism , Female , Gene Expression Regulation/physiology , Glutathione Peroxidase/genetics , Hydrogen Peroxide/metabolism , Lipid Peroxidation/physiology , Liver/metabolism , Mice, Inbred C57BL , Ovariectomy , Oxygen Consumption/physiology , RNA, Messenger/genetics , Triglycerides/metabolism , Glutathione Peroxidase GPX1ABSTRACT
The rotator cuff (RTC) muscles not only generate movement but also provide important shoulder joint stability. RTC tears, particularly in the supraspinatus muscle, are a common clinical problem. Despite some biological healing after RTC repair, persistent problems include poor functional outcomes with high retear rates after surgical repair. Animal models allow further exploration of the sequela of RTC injury such as fibrosis, inflammation, and fatty infiltration, but there are few options regarding contractility for mouse, rat, and rabbit. Histological findings can provide a "direct measure" of damage, but the most comprehensive measure of the overall health of the muscle is contractile force. However, information regarding normal supraspinatus size and contractile function is scarce. Animal models provide the means to compare muscle histology, imaging, and contractility within individual muscles in various models of injury and disease, but to date, most testing of animal contractile force has been limited primarily to hindlimb muscles. Here, we describe an in vivo method to assess contractility of the supraspinatus muscle and describe differences in methods and representative outcomes for mouse, rat, and rabbit.
Subject(s)
Muscle Contraction/physiology , Rotator Cuff/physiology , Animals , Disease Models, Animal , Male , Mice , Mice, Inbred C57BL , Muscular Atrophy/physiopathology , Muscular Diseases/physiopathology , Rabbits , Rats , Rats, Sprague-DawleyABSTRACT
Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease in which weakness, increased susceptibility to muscle injury, and inadequate repair appear to underlie the pathology. While most attention has focused within the muscle fiber, we recently demonstrated in mdx mice (murine model for DMD) significant morphologic alterations at the motor endplate of the neuromuscular junction (NMJ) and corresponding NMJ transmission failure after injury. Here we extend these initial observations at the motor endplate to gain insight into the pre- vs. postsynaptic morphology, as well as the subsynaptic nuclei in healthy (WT) vs. mdx mice. We quantified the discontinuity and branching of the terminal nerve in adult mice. We report mdx- and age-dependent changes for discontinuity and an increase in branching when compared to WT. To examine mdx- and age-dependent changes in the relative localization of pre- and postsynaptic structures, we calculated NMJ occupancy, defined as the ratio of the footprint occupied by presynaptic vesicles vs. that of the underlying motor endplate. The normally congruent coupling between presynaptic and postsynaptic morphology was altered in mdx mice, independent of age. Finally we found an almost two-fold increase in the number of nuclei and an increase in density (nuclei/area) underlying the NMJ. These outcomes suggest substantial remodeling of the NMJ during dystrophic progression. This remodeling reflects plasticity in both pre- and postsynaptic contributors to NMJ structure, and thus perhaps also NM transmission and muscle function.
ABSTRACT
Breast cancer type 1 (BRCA1) susceptibility protein is expressed across multiple tissues including skeletal muscle. The overall objective of this investigation was to define a functional role for BRCA1 in skeletal muscle using a translational approach. For the first time in both mice and humans, we identified the presence of multiple isoforms of BRCA1 in skeletal muscle. In response to an acute bout of exercise, we found increases in the interaction between the native forms of BRCA1 and the phosphorylated form of acetyl-CoA carboxylase. Decreasing BRCA1 content using a shRNA approach in cultured primary human myotubes resulted in decreased oxygen consumption by the mitochondria and increased reactive oxygen species production. The decreased BRCA1 content also resulted in increased storage of intracellular lipid and reduced insulin signaling. These results indicate that BRCA1 plays a critical role in the regulation of metabolic function in skeletal muscle. Collectively, these data reveal BRCA1 as a novel target to consider in our understanding of metabolic function and risk for development of metabolic-based diseases.