Effects of L-Leucine Supplementation and Resistance Training on Adipokine Markers in Untrained Perimenopausal and Postmenopausal Women.

ABSTRACT: Chapman-Lopez, TJ, Funderburk, LK, Heileson, JL, Wilburn, DT, Koutakis, P, Gallucci, AR, and Forsse, JS. Effects of L-leucine supplementation and resistance training on adipokine markers in untrained perimenopausal and postmenopausal women. J Strength Cond Res 38(3): 526-532, 2024-This study examined the effects of supplementing 5 g of leucine compared with a placebo during a 10-week resistance training program on body composition parameters and adipokine concentrations in untrained, perimenopausal and postmenopausal women. Thirty-five women were randomly assigned to 2 groups-leucine (LEU, n = 17) and placebo (PLC, n = 18)-in a double-blind, placebo-controlled trial. Each group consumed the supplement or placebo every day and completed a resistance training program for 10 weeks. Using 3-day food records, a diet was assessed before the intervention and after its cessation. Body composition was assessed preintervention and postintervention using dual-energy x-ray absorptiometry. Moreover, the concentrations of adipokines, such as adiponectin, visfatin, leptin, and monocyte chemoattractant protein-1 (MCP-1), were assessed preintervention and postintervention. Both groups showed an increase in visceral adipose tissue (VAT) area ( p = 0.030) and fat-free mass (FFM; p = 0.023). There were significant group differences in concentrations of visfatin ( p = 0.020) and leptin ( p = 0.038) between the PLC and LEU groups. Visfatin displayed higher concentrations in the PLC group and leptin displayed higher concentrations in the LEU group. In addition, there were significant decreases in adiponectin concentrations for both groups (LEU: 652 ± 513 to 292 ± 447 pg·ml -1 ; PLC: 584 ± 572 to 245 ± 356 pg·ml -1 , p = 0.002) and MCP-1 only decreased in the PLC group (253 ± 119 to 206 ± 106 pg·ml -1 , p = 0.004). There were significant decreases in adiponectin concentrations in both groups and a decrease in MCP-1 concentrations in the PLC group. These decreases may be due to both adipokines possible relationship with VAT area. However, it is not known whether leucine has underlying properties that hinder changes in MCP-1 concentrations.

Aging alters gastrocnemius muscle hemoglobin oxygen saturation (StO2) characteristics in healthy individuals.

PURPOSE: Functional limitations during exercise from alterations in the balance of oxygen supply and demand-as reported by lower tissue oxygen saturation and longer recovery time-are well documented in clinical populations. We aimed to assess changes in skeletal muscle hemoglobin oxygen saturation (StO2) characteristics during exercise as a result of aging in otherwise healthy individuals. METHODS: We recruited healthy male and female participants (n = 101) from three age ranges-young (18-39 years), middle age (40-65 years), and older (> 65 years)-to complete exercise tests commonly used in clinical populations. Using near-infrared spectroscopy (NIRS) we assessed StO2 in the medial gastrocnemius during the Gardner Treadmill Protocol and 6 min walk test (6MWT). RESULTS: Minimum StO2 (%) during the treadmill test was significantly lower for both middle-age (36.1 ± 20.6) and older (27.3 ± 19.4) participants compared to young (46.8 ± 14.8) (p < 0.05 and p < 0.01 respectively), and recovery time (minutes) was significantly prolonged (young = 0.22 ± 0.34; middle age = 0.66 ± 0.52; older = 1.04 ± 1.00) (p < 0.001 for both middle age and older compared to young). Similar results were shown during the 6MWT, as minimum StO2 (%) was lower in middle-age (41.7 ± 17.2) and older (40.0 ± 25.9) participants compared to young (53.6 ± 14.5) (p < 0.05), and recovery times (minutes) were prolonged (young: 0.11 ± 0.17; middle age: 0.46 ± 0.42; older: 0.93 ± 0.43) (p < 0.001 for both middle age and older compared to young). Simple linear regression analyses demonstrated that age predicted treadmill recovery and 6MWT recovery. CONCLUSION: Our study provides evidence that aging, even in otherwise healthy individuals, negatively impacts muscle StO2 characteristics. In older individuals, working muscle tissue may reach lower oxygen saturation during exercise and take longer to return to baseline oxygen saturation post-exercise.

Expanding the Reactive Sulfur Metabolome: Intracellular and Efflux Measurements of Small Oxoacids of Sulfur (SOS) and H2S in Human Primary Vascular Cell Culture.

Hydrogen sulfide (H2S) is an endogenous signaling molecule which is important for cardiovascular health, but its mechanism of action remains poorly understood. Here, we report measurements of H2S as well as its oxidized metabolites, termed small oxoacids of sulfur (SOS = HSOH and HOSOH), in four human primary vascular cell lines: smooth muscle and endothelial cells derived from both human arterial and coronary tissues. We use a methodology that targets small molecular weight sulfur species; mass spectrometric analysis allows for species quantification to report cellular concentrations based on an H2S calibration curve. The production of H2S and SOS is orders of magnitude higher in smooth muscle (nanomolar) as compared to endothelial cell lines (picomolar). In all the primary lines measured, the distributions of these three species were HOSOH >H2S > HSOH, with much higher SOS than seen previously in non-vascular cell lines. H2S and SOS were effluxed from smooth muscle cells in higher concentrations than endothelial cells. Aortic smooth muscle cells were used to examine changes under hypoxic growth conditions. Hypoxia caused notable increases in HSOH and ROS, which we attribute to enhanced sulfide quinone oxidase activity that results in reverse electron transport.

Quantification of Daily Physical Activity and Sedentary Behavior of Claudicating Patients.

BACKGROUND: Claudication is the most common manifestation of peripheral artery disease (PAD), producing significant ambulatory compromise. Limited information exists on the routine physical activity of claudicating patients. Our objective was to record the intensity/time profiles of physical activity and the timing and duration of sedentary behavior of a sample of community-dwelling claudicating patients. METHODS: Forty-four claudicating patients referred to our vascular clinic were recruited. Physical activity was recorded using the ActiGraph GT1M activity monitor. The Actigraph monitor is a lightweight instrument designed to measure human movement through changes in acceleration, measured as counts over 1-minute time periods. Data from 7 consecutive days were used for the calculations. We processed the data using the ActiLife software program. RESULTS: The average daily activity of the claudicating patients shows a steady increase beginning approximately 05:30 AM until a peak plateau from approximately 10:00 AM to 01:30 PM followed by a steady decrease until approximately 09:30 PM, when a sustained period of inactivity begins. The average claudicating patient takes 3586 steps per day at an average intensity of 1.77 metabolic equivalents of task (METs, a physiological measure expressing the energy cost of physical activities). Average physical activity intensity and peak intensity fluctuate very little during the day, and they rarely exceed the level of light activity (light = <3 METs maximum effort, such as casual walking or light housework). During awake time, approximately 7 hours are spent in sedentary behaviors (<1.5 METs), and sedentary time is spread throughout the day mostly in short intervals between periods of low-energy activity. CONCLUSIONS: Our study objectively demonstrates the reduced physical activity of claudicating patients and documents physical activity/duration profiles throughout the day. The intensity of the physical activity of the average claudicating patient fluctuates very little during the day and rarely exceeds a light intensity level. Claudicating patients spend approximately half of their awake time in sedentary behavior and when they walk they do it in short bursts followed by several minutes of rest. We anticipate that changes in routine physical activity/duration profiles of patients with PAD will provide relevant, sensitive, and direct measures of the effectiveness of therapeutic interventions.

Resistance Training, Antioxidant Status, and Antioxidant Supplementation.

Resistance training is known to promote the generation of reactive oxygen species. Although this can likely upregulate the natural, endogenous antioxidant defense systems, high amounts of reactive oxygen species can cause skeletal muscle damage, fatigue, and impair recovery. To prevent these, antioxidant supplements are commonly consumed along with exercise. Recently, it has been shown that these reactive oxygen species are important for the cellular adaptation process, acting as redox signaling molecules. However, most of the research regarding antioxidant status and antioxidant supplementation with exercise has focused on endurance training. In this review, the authors discuss the evidence for resistance training modulating the antioxidant status. They also highlight the effects of combining antioxidant supplementation with resistance training on training-induced skeletal muscle adaptations.

Role of Transforming Growth Factor-ß in Skeletal Muscle Fibrosis: A Review.

Transforming growth factor-beta (TGF-ß) isoforms are cytokines involved in a variety of cellular processes, including myofiber repair and regulation of connective tissue formation. Activation of the TGF-ß pathway contributes to pathologic fibrosis in most organs. Here, we have focused on examining the evidence demonstrating the involvement of TGF-ß in the fibrosis of skeletal muscle particularly. The TGF-ß pathway plays a role in different skeletal muscle myopathies, and TGF-ß signaling is highly induced in these diseases. In this review, we discuss different molecular mechanisms of TGF-ß-mediated skeletal muscle fibrosis and highlight different TGF-ß-targeted treatments that target these relevant pathways.

Chloroquine improves the response to ischemic muscle injury and increases HMGB1 after arterial ligation.

OBJECTIVE: We have previously shown that exogenous administration of the nuclear protein high mobility group box 1 (HMGB1) improves angiogenesis after tissue ischemia. Antagonizing HMGB1 prolongs muscle necrosis and deters regeneration. In this study, we evaluated HMGB1 expression in peripheral arterial disease (PAD) and the mechanisms that promote its release in a murine model of hindlimb ischemia. Specifically, we investigated how chloroquine (CQ), a commonly employed disease-modifying antirheumatic drug, promotes HMGB1 release from muscle. We hypothesized that CQ could increase HMGB1 locally and systemically, allowing it to mediate recovery from ischemic injury. METHODS: Muscle biopsies were performed on patients undergoing lower extremity surgery for non-PAD-related disease as well as for claudication and critical limb ischemia. Clinical symptoms and ankle-brachial indices were recorded for each patient. HMGB1 was detected in muscle sections using immunohistochemical staining. Unilateral femoral artery ligation was performed on both wild-type and inducible HMGB1 knockout mice. Wild-type mice were administered intraperitoneal CQ 2 weeks before and after femoral artery ligation. Laser Doppler perfusion imaging was used to determine perfusion recovery. Serum and tissue levels of HMGB1 were measured at designated time points. In vitro, cultured C2C12 myoblasts were treated with increasing doses of CQ. HMGB1, autophagosome formation, p62/SQSTM1 accumulation, caspase-1 expression and activity, and lactate dehydrogenase levels were measured in supernatants and cell lysates. RESULTS: Nuclear expression of HMGB1 was prominent in patients with claudication and critical limb ischemia (P < .05) compared with controls. CQ-treated mice had elevated serum HMGB1 and diffuse HMGB1 staining in muscle (P < .01). In wild-type mice, CQ treatment resulted in higher laser Doppler perfusion imaging ratios in the ischemic limb at 7 days (P < .03) and less fat replacement after 2 weeks (P < .03). In cultured myoblasts, CQ induced autophagosome accumulation, inhibited p62/SQSTM-1 degradation, and activated caspase-1. CONCLUSIONS: HMGB1 is prominently expressed in PAD muscle but mostly confined to the nucleus. Our in vivo data suggest that HMGB1 mobilization into the sarcoplasm and serum can be increased with CQ, possibly through caspase-1-mediated pathways. Whereas HMGB1 can be released by many cell types, these studies suggest that the muscle may be an important additional source that is relevant in PAD.

Effects of Limb Revascularization Procedures on Oxidative Stress.

Revascularization procedures to treat patients with peripheral artery disease are among the most common operations performed by vascular surgeons. However, there are major limitations to revascularizations, readmission rates due to procedural complications are high, and greater risks of cardiovascular and limb adverse outcomes have been reported for patients with peripheral artery disease undergoing limb revascularization. Specifically, surgical revascularization may be associated with increased generation of reactive oxygen species based on the ischemia reperfusion injury theory, as restored blood flow and reoxygenation of ischemic areas may be accompanied by increased oxidative stress. In this review, we present the current evidence regarding the effects of revascularization procedures on oxidative stress. We also discuss potential therapeutic interventions to prevent ischemia reperfusion injury-mediated tissue damage.

An endovascular model of ischemic myopathy from peripheral arterial disease.

OBJECTIVE: Peripheral arterial disease (PAD) is a significant age-related medical condition with limited pharmacologic options. Severe PAD, termed critical limb ischemia, can lead to amputation. Skeletal muscle is the end organ most affected by PAD, leading to ischemic myopathy and debility of the patient. Currently, there are not any therapeutics to treat ischemic myopathy, and proposed biologic agents have not been optimized owing to a lack of preclinical models of PAD. Because a large animal model of ischemic myopathy may be useful in defining the optimal dosing and delivery regimens, the objective was to create and to characterize a swine model of ischemic myopathy that mimics patients with severe PAD. METHODS: Yorkshire swine (N = 8) underwent acute right hindlimb ischemia by endovascular occlusion of the external iliac artery. The effect of ischemia on limb function, perfusion, and degree of ischemic myopathy was quantified by weekly gait analysis, arteriography, hindlimb blood pressures, femoral artery duplex ultrasound scans, and histologic examination. Animals were terminated at 5 (n = 5) and 6 (n = 3) weeks postoperatively. Ossabaw swine (N = 8) fed a high-fat diet were used as a model of metabolic syndrome for comparison of arteriogenic recovery and validation of ischemic myopathy. RESULTS: There was persistent ischemia in the right hindlimb, and occlusion pressures were significantly depressed compared with the untreated left hindlimb out to 6 weeks (systolic blood pressure, 31 ± 21 vs 83 ± 15 mm Hg, respectively; P = .0007). The blood pressure reduction resulted in a significant increase of ischemic myopathy in the gastrocnemius muscle in the treated limb. Gait analysis revealed a functional deficit of the right hindlimb immediately after occlusion that improved rapidly during the first 2 weeks. Peak systolic velocity values in the right common femoral artery were severely diminished throughout the entire study (P < .001), and the hemodynamic environment after occlusion was characterized by low and oscillatory wall shear stress. Finally, the internal iliac artery on the side of the ischemic limb underwent significant arteriogenic remodeling (1.8× baseline) in the Yorkshire but not in the Ossabaw swine model. CONCLUSIONS: This model uses endovascular technology to produce the first durable large animal model of ischemic myopathy. Acutely (first 2 weeks), this model is associated with impaired gait but no tissue loss. Chronically (2-6 weeks), this model delivers persistent ischemia, resulting in ischemic myopathy similar to that seen in PAD patients. This model may be of use for testing novel therapeutics including biologic therapies for promoting neovascularization and arteriogenesis.

Transforming growth factor-beta 1 produced by vascular smooth muscle cells predicts fibrosis in the gastrocnemius of patients with peripheral artery disease.

BACKGROUND: Lower leg ischemia, myopathy, and limb dysfunction are distinguishing features of peripheral artery disease (PAD). The myopathy of PAD is characterized by myofiber degeneration in association with extracellular matrix expansion, and increased expression of transforming growth factor-beta 1 (TGF-ß1; a pro-fibrotic cytokine). In this study, we evaluated cellular expression of TGF-ß1 in gastrocnemius of control (CTRL) and PAD patients and its relationship to deposited collagen, fibroblast accumulation and limb hemodynamics. METHODS: Gastrocnemius biopsies were collected from PAD patients with claudication (PAD-II; N = 25) and tissue loss (PAD-IV; N = 20) and from CTRL patients (N = 20). TGF-ß1 in slide-mounted specimens was labeled with fluorescent antibodies and analyzed by quantitative wide-field, fluorescence microscopy. We evaluated co-localization of TGF-ß1 with vascular smooth muscle cells (SMC) (high molecular weight caldesmon), fibroblasts (TE-7 antigen), macrophages (CD163), T cells (CD3) and endothelial cells (CD31). Collagen was stained with Masson Trichrome and collagen density was determined by quantitative bright-field microscopy with multi-spectral imaging. RESULTS: Collagen density increased from CTRL to PAD-II to PAD-IV specimens (all differences p < 0.05) and was prominent around microvessels. TGF-ß1 expression increased with advancing disease (all differences p < 0.05), correlated with collagen density across all specimens (r = 0.864; p < 0.001), associated with fibroblast accumulation, and was observed exclusively in SMC. TGF-ß1 expression inversely correlated with ankle-brachial index across PAD patients (r = -0.698; p < 0.001). CONCLUSIONS: Our findings support a progressive fibrosis in the gastrocnemius of PAD patients that is caused by elevated TGF-ß1 production in the SMC of microvessels in response to tissue hypoxia.

Abnormal myofiber morphology and limb dysfunction in claudication.

BACKGROUND: Peripheral artery disease (PAD), which affects an estimated 27 million people in Europe and North America, is caused by atherosclerotic plaques that limit blood flow to the legs. Chronic, repeated ischemia in the lower leg muscles of PAD patients is associated with loss of normal myofiber morphology and myofiber degradation. In this study, we tested the hypothesis that myofiber morphometrics of PAD calf muscle are significantly different from normal calf muscle and correlate with reduced calf muscle strength and walking performance. METHODS: Gastrocnemius biopsies were collected from 154 PAD patients (Fontaine stage II) and 85 control subjects. Morphometric parameters of gastrocnemius fibers were determined and evaluated for associations with walking distances and calf muscle strength. RESULTS: Compared with control myofibers, PAD myofiber cross-sectional area, major and minor axes, equivalent diameter, perimeter, solidity, and density were significantly decreased (P < 0.005), whereas roundness was significantly increased (P < 0.005). Myofiber morphometric parameters correlated with walking distances and calf muscle strength. Multiple regression analyses demonstrated myofiber cross-sectional area, roundness, and solidity as the best predictors of calf muscle strength and 6-min walking distance, whereas cross-sectional area was the main predictor of maximum walking distance. CONCLUSIONS: Myofiber morphometrics of PAD gastrocnemius differ significantly from those of control muscle and predict calf muscle strength and walking distances of the PAD patients. Morphometric parameters of gastrocnemius myofibers may serve as objective criteria for diagnosis, staging, and treatment of PAD.

Reliability and validity of a wireless accelerometer for the assessment of postural sway.

Clinicians are in need of valid and objective measures of postural sway. Accelerometers have been shown to be suitable alternatives to expensive and stationary force plates. We evaluated the test-retest reliability and balance task discrimination capability of a new wireless triaxial accelerometer (YEI 3-Space Sensor). Four testing conditions (eyes open or closed, while on a firm or compliant surface) were used to progressively challenge the static balance of 20 healthy male (n = 8) and female (n = 12) older adults (mean age 81 ± 4.3 y). Subjects completed 2 blocks of three 30-second trials per condition. The accelerometer was positioned on the lower back to acquire mediolateral (M-L) and anterior-posterior (A-P) accelerations. Intraclass correlation coefficients were all good to excellent, with values ranging from .736 to .972 for trial-to-trial and from .760 to .954 for block-to- block. A significant stepwise increase in center of mass acceleration root mean square values was found across the 4 balance conditions (F[1.49, 28.26] = 39.54, P < .001). The new accelerometer exhibited good to excellent trial-to-trial and block-to-block reliability and was sensitive to differences in visual and surface conditions and acceleration axes.

Mitochondrial fatty acid beta-oxidation: a possible therapeutic target for skeletal muscle lipotoxicity in peripheral artery disease myopathy.

Peripheral artery disease (PAD) is an atherosclerotic disease impacting over 200 million individuals and the prevalence increases with age. PAD occurs when plaque builds up within the peripheral arteries, leading to reduced blood flow and oxygen supply to the outer extremities. Individuals who experience PAD suffer from ischemia, which is typically accompanied by significant damage to skeletal muscles. Additionally, this tissue damage affects mitochondria, causing them to become dysregulated and dysfunctional, resulting in decreased metabolic rates. As there is no known cure for PAD, researchers are exploring potential therapeutic targets by examining coexisting cardiovascular conditions and metabolic risk factors, such as the aging process. Among these comorbidities, type-two diabetes mellitus and obesity are particularly common in PAD cases. These conditions, along with aging itself, are associated with an elevated accumulation of ectopic lipids within skeletal muscles, similar to what is observed in PAD. Researchers have attempted to reduce excess lipid accumulation by increasing the rate of fatty acid beta oxidation. Manipulating acetyl coenzyme A carboxylase 2, a key regulatory protein of fatty acid beta oxidation, has been the primary focus of such research. When acetyl coenzyme A carboxylase 2 is inhibited, it interrupts the conversion of acetyl-CoA into malonyl-CoA, resulting in an increase in the rate of fatty acid beta oxidation. By utilizing samples from PAD patients and applying the pharmacological strategies developed for acetyl coenzyme A carboxylase 2 in diabetes and obesity to PAD, a potential new therapeutic avenue may emerge, offering hope for improved quality of life for individuals suffering from PAD.

Oxidative damage in the gastrocnemius predicts long-term survival in patients with peripheral artery disease.

Patients with peripheral artery disease (PAD) have increased mortality rates and a myopathy in their affected legs which is characterized by increased oxidative damage, reduced antioxidant enzymatic activity and defective mitochondrial bioenergetics. This study evaluated the hypothesis that increased levels of oxidative damage in gastrocnemius biopsies from patients with PAD predict long-term mortality rates. Oxidative damage was quantified as carbonyl adducts in myofibers of the gastrocnemius of PAD patients. The oxidative stress data were grouped into tertiles and the 5-year, all-cause mortality for each tertile was determined by Kaplan-Meier curves and compared by the Modified Peto test. A Cox-regression model was used to control the effects of clinical characteristics. Results were adjusted for age, sex, race, body mass index, ankle-brachial index, smoking, physical activity, and comorbidities. Of the 240 study participants, 99 died during a mean follow up of 37.8 months. Patients in the highest tertile of oxidative damage demonstrated the highest 5-year mortality rate. The mortality hazard ratios (HR) from the Cox analysis were statistically significant for oxidative damage (lowest vs middle tertile; HR = 6.33; p = 0.0001 and lowest vs highest; HR = 8.37; p < 0.0001). Survival analysis of a contemporaneous population of PAD patients identifies abundance of carbonyl adducts in myofibers of their gastrocnemius as a predictor of mortality rate independently of ankle-brachial index, disease stage and other clinical and myopathy-related covariates.

Effects of chronic alcohol intoxication on aerobic exercise-induced adaptations in female mice.

Chronic alcohol intoxication decreases muscle strength/function and causes mitochondrial dysfunction. Aerobic exercise training improves mitochondrial oxidative capacity and increases muscle mass and strength. Presently, the impact of chronic alcohol on aerobic exercise-induced adaptations was investigated. Female C57BL/6Hsd mice were randomly assigned to one of four groups: control sedentary (CON SED; n = 26), alcohol sedentary (ETOH SED; n = 27), control exercise (CON EX; n = 28), and alcohol exercise (ETOH EX; n = 25). Exercise mice had running wheel access for 2 h a day, 7 days a week. All mice were fed either control or an alcohol-containing liquid diet. Grip strength testing and EchoMRI were performed before and after the interventions. After 6 wk, hindlimb muscles were collected for molecular analyses. A subset of mice performed a treadmill run to fatigue (RTF), then abstained from alcohol for 2 wk and repeated the RTF. Alcohol decreased lean mass and forelimb grip strength compared with control-fed mice. Alcohol blunted the exercise-induced increase in muscle mass (plantaris and soleus), type IIa fiber percentage in the plantaris, and run time to fatigue. Mitochondrial markers (Citrate synthase activity and Complex I-IV, COXIV and Cytochrome C protein expression) were increased with exercise regardless of ETOH in the gastrocnemius but not tibialis anterior muscle. Two weeks of alcohol abstinence improved RTF time in ETOH EX but not in ETOH SED. These data suggest that alcohol impairs some exercise-induced adaptations in skeletal muscle, but not all were negatively affected, indicating that exercise may be a beneficial behavior even while consuming alcohol.NEW & NOTEWORTHY Alcohol consumption during an aerobic exercise training period prevented training-induced increases in run to fatigue time and grip strength. Cessation of alcohol allowed for recovery of endurance performance within 2 wk. The worsened exercise performance after alcohol was unrelated to impairments in markers of mitochondrial health. Therefore, some adaptations to exercise training are impaired with alcohol use (endurance performance, muscle growth, and strength), while others remain mostly unaffected (mitochondrial health).

Skeletal muscle desmin alterations following revascularization in peripheral artery disease claudicants.

Peripheral artery disease (PAD) is characterized by varying severity of arterial stenosis, exercise induced claudication, malperfused tissue precluding normal healing and skeletal muscle dysfunction. Revascularization interventions improve circulation, but post-reperfusion changes within the skeletal muscle are not well characterized. This study investigates if revascularization enhanced hemodynamics increases walking performance with concurrent improvement of mitochondrial function and reverses abnormal skeletal muscle morphological features that develop with PAD. Fifty-eight patients completed walking performance testing and muscle biopsy before and 6 months after revascularization procedures. Muscle fiber morphology, desmin structure, and mitochondria respiration assessments before and after the revascularization were evaluated. Revascularization improved limb hemodynamics, walking function, and muscle morphology. Qualitatively not all participants recovered normal structural architecture of desmin in the myopathic myofibers after revascularization. Heterogenous responses in the recovery of desmin structure following revascularization may be caused by other underlying factors not reversed with hemodynamic improvements. Revascularization interventions clinically improve patient walking ability and can reverse the multiple subcellular functional and structural abnormalities in muscle cells. Further study is needed to characterize desmin structural remodeling with improvements in skeletal muscle morphology and function.

Peripheral artery disease causes consistent gait irregularities regardless of the location of leg claudication pain.

BACKGROUND: The most common symptom of peripheral artery disease (PAD) is intermittent claudication that involves the calf, thigh, and/or buttock muscles. How the specific location of this leg pain is related to altered gait, however, is unknown. OBJECTIVES: We hypothesized that because the location of claudication symptoms uniquely affects different leg muscle groups in people with PAD, this would produce distinctive walking patterns. METHODS: A total of 105 participants with PAD and 35 age-matched older volunteers without PAD (CTRL) were recruited. Participants completed walking impairment questionnaires (WIQ), Gardner-Skinner progressive treadmill tests, the six-minute walk test, and we performed an advanced evaluation of the biomechanics of their overground walking. Participants with PAD were categorized into 4 groups according to their stated pain location(s): calf only (C, n = 43); thigh and calf (TC, n = 18); buttock and calf (BC, n = 15); or buttock, thigh, and calf (BTC, n = 29). Outcomes were compared between CTRL, C, TC, BC and BTC groups using a one-way ANOVA with post-hoc comparisons to identify and assess statistically significant differences. RESULTS: There were no significant differences between CTRL, C, TC, BC and BTC groups in distances walked or walking speed when either pain-free or experiencing claudication pain. Each participant with PAD had significantly dysfunctional biomechanical gait parameters, even when pain-free, when compared to CTRL (pain-free) walking data. During pain-free walking, out of the 18 gait parameters evaluated, we only identified significant differences in hip power generation during push-off (in C and TC groups) and in knee power absorption during weight acceptance (in TC and BC groups). There were no between-group differences in gait parameters while people with PAD were walking with claudication pain. CONCLUSIONS: Our data demonstrate that PAD affects the ischemic lower extremities in a diffuse manner irrespective of the location of claudication symptoms. DATABASE REGISTRATION: ClinicalTrials.gov NCT01970332.

Oxidative damage and myofiber degeneration in the gastrocnemius of patients with peripheral arterial disease.

Peripheral arterial disease (PAD), a manifestation of systemic atherosclerosis that produces blockages in arteries supplying the legs, affects an estimated 27 million people in Europe and North America. Increased production of reactive oxygen species by dysfunctional mitochondria in leg muscles of PAD patients is viewed as a key mechanism of initiation and progression of the disease. Previous studies demonstrated increased oxidative damage in homogenates of biopsy specimens from PAD gastrocnemius compared to controls, but did not address myofiber-specific damage. In this study, we investigated oxidative damage to myofibers as a possible cause of the myopathy of PAD. To achieve this, we developed and validated fluorescence microscopy procedures for quantitative analysis of carbonyl groups and 4-hydroxy-2-nonenal (HNE) adducts in myofibers of biopsy specimens from human gastrocnemius. PAD and control specimens were evaluated for differences in 1) myofiber content of these two forms of oxidative damage and 2) myofiber cross-sectional area. Furthermore, oxidative damage to PAD myofibers was tested for associations with clinical stage of disease, degree of ischemia in the affected leg, and myofiber cross-sectional area. Carbonyl groups and HNE adducts were increased 30% (p < 0.0001) and 40% (p < 0.0001), respectively, in the myofibers of PAD (N = 34) compared to control (N = 21) patients. Mean cross-sectional area of PAD myofibers was reduced 29.3% compared to controls (p < 0.0003). Both forms of oxidative damage increased with clinical stage of disease, blood flow limitation in the ischemic leg, and reduced myofiber cross-sectional area. The data establish oxidative damage to myofibers as a possible cause of PAD myopathy.

Morphometric analysis of gastrocnemius muscle biopsies from patients with peripheral arterial disease: objective grading of muscle degeneration.

Peripheral arterial disease (PAD), which affects ~10 million Americans, is characterized by atherosclerosis of the noncoronary arteries. PAD produces a progressive accumulation of ischemic injury to the legs, manifested as a gradual degradation of gastrocnemius histology. In this study, we evaluated the hypothesis that quantitative morphological parameters of gastrocnemius myofibers change in a consistent manner during the progression of PAD, provide an objective grading of muscle degeneration in the ischemic limb, and correlate to a clinical stage of PAD. Biopsies were collected with a Bergström needle from PAD patients with claudication (n = 18) and critical limb ischemia (CLI; n = 19) and control patients (n = 19). Myofiber sarcolemmas and myosin heavy chains were labeled for fluorescence detection and quantitative analysis of morphometric variables, including area, roundness, perimeter, equivalent diameter, major and minor axes, solidity, and fiber density. The muscle specimens were separated into training and validation data sets for development of a discriminant model for categorizing muscle samples on the basis of disease severity. The parameters for this model included standard deviation of roundness, standard deviation of solidity of myofibers, and fiber density. For the validation data set, the discriminant model accurately identified control (80.0% accuracy), claudicating (77.7% accuracy), and CLI (88.8% accuracy) patients, with an overall classification accuracy of 82.1%. Myofiber morphometry provided a discriminant model that establishes a correlation between PAD progression and advancing muscle degeneration. This model effectively separated PAD and control patients and provided a grading of muscle degeneration within clinical stages of PAD.

Gastrocnemius mitochondrial respiration: are there any differences between men and women?

INTRODUCTION: Work on human and mouse skeletal muscle by our group and others has demonstrated that aging and age-related degenerative diseases are associated with mitochondrial dysfunction, which may be more prevalent in males. There have been, however, no studies that specifically examine the influence of male or female sex on human skeletal muscle mitochondrial respiration. The purpose of this study was to compare mitochondrial respiration in the gastrocnemius of adult men and women. METHODS: Gastrocnemius muscle was obtained from male (n = 19) and female (n = 11) human subjects with healthy lower-extremity musculoskeletal and arterial systems and normal ambulatory function. All patients were undergoing operations for the treatment of varicose veins in their legs. Mitochondrial respiration was determined with a Clark electrode in an oxygraph cell containing saponin-skinned muscle bundles. Complex I-, II-, III-, and IV-dependent respiration was measured individually and normalized to muscle weight, total protein content, and citrate synthase (CS, index of mitochondrial content). RESULTS: Male and female patients had no evidence of musculoskeletal or arterial disease and did not differ with regard to age, race, body mass index, or other clinical characteristics. Complex I-, II-, III-, and IV-dependent respiration normalized to muscle weight, total protein content, and CS did not statistically differ for males compared with females. CONCLUSIONS: Our study evaluates, for the first time, gastrocnemius mitochondrial respiration of adult men and women who have healthy musculoskeletal and arterial systems and normal ambulatory function. Our data demonstrate there are no differences in the respiration of gastrocnemius mitochondria between men and women.