Panagiotis Koutakis

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.

Abnormal joint powers before and after the onset of claudication symptoms.

OBJECTIVE Claudication is the most common manifestation of peripheral arterial disease, producing significant ambulatory compromise. Our study evaluated patients with bilateral lower limb claudication and characterized their gait abnormality based on advanced biomechanical analysis using joint torques and powers. METHODS Twenty patients with bilateral claudication (10 with isolated aortoiliac disease and 10 with combined aortoiliac and femoropopliteal disease) and 16 matched controls ambulated on a walkway while 3-dimensional biomechanical data were collected. Patients walked before and after onset of claudication pain. Joint torques and powers at early, mid, and late stance for the hip, knee, and ankle joints were calculated for claudicating patients before and after the onset of claudication pain and were compared to controls. RESULTS Claudicating patients exhibited significantly reduced hip and knee power at early stance (weight-acceptance phase) due to decreased torques produced by the hip and knee extensors. In mid stance (single-limb support phase), patients had significantly reduced knee and hip power due to the decreased torques produced by the knee extensors and the hip flexors. In late stance (propulsion phase), reduced propulsion was noted with significant reduction in ankle plantar flexor torques and power. These differences were present before and after the onset of pain, with certain parameters worsening in association with pain. CONCLUSIONS The gait of claudication is characterized by failure of specific and identifiable muscle groups needed to perform normal walking (weight acceptance, single-limb support, and propulsion). Parameters of gait are abnormal with the first steps taken, in the absence of pain, and certain of these parameters worsen after the onset of claudication pain.

Joint torques and powers are reduced during ambulation for both limbs in patients with unilateral claudication

OBJECTIVES Symptomatic peripheral arterial disease (PAD) results in significant gait impairment. In an attempt to fully delineate and quantify these gait alterations, we analyzed joint kinematics, torques (rotational forces), and powers (rotational forces times angular velocity) in patients with PAD with unilateral claudication for both the affected and nonaffected legs. METHODS Twelve patients with unilateral PAD (age, 61.69 +/- 10.53 years, ankle-brachial index [ABI]: affected limb 0.59 +/- 0.25; nonaffected limb 0.93 +/- 0.12) and 10 healthy controls (age, 67.23 +/- 12.67 years, ABI >1.0 all subjects) walked over a force platform to acquire gait kinetics, while joint kinematics were recorded simultaneously. Data were collected for the affected and nonaffected limbs during pain free (PAD-PF) and pain induced (PAD-P) trials. Kinetics and kinematics were combined to quantify torque and powers during the stance period from the hip, knee, and ankle joints. RESULTS The affected limb demonstrated significantly (P <.05) reduced ankle plantar flexion torque compared to controls during late stance in both PAD-PF and PAD-P trials. There were significant reductions in ankle plantar flexion power generation during late stance for both the affected (P <.05) and nonaffected limbs (P <.05) compared to control during PAD-PF and PAD-P trials. No significant differences were noted in torque comparing the nonaffected limbs in PAD-PF and PAD-P conditions to control for knee and hip joints throughout the stance phase. Significant reductions were found in knee power absorption in early stance and knee power generation during mid stance for both limbs of the patients with PAD as compared to control (P <.05). CONCLUSION Patients with PAD with unilateral claudication demonstrate significant gait impairments in both limbs that are present even before they experience any claudication symptoms. Overall, our data demonstrate significantly reduced ankle plantar flexion torque and power during late stance with reduced knee power during early and mid stance for the affected limb. Further studies are needed to determine if these findings are dependent on the location and the severity of lower extremity ischemia and whether the changes in the nonaffected limb are the result of underlying PAD or compensatory changes from the affected limb dysfunction.

Oxidative damage in the gastrocnemius of patients with peripheral artery disease is myofiber type selective

Background Peripheral artery disease (PAD), a manifestation of systemic atherosclerosis that produces blockages in the arteries supplying the legs, affects approximately 5% of Americans. We have previously, demonstrated that a myopathy characterized by myofiber oxidative damage and degeneration is central to PAD pathophysiology. Objectives In this study, we hypothesized that increased oxidative damage in the myofibers of the gastrocnemius of PAD patients is myofiber-type selective and correlates with reduced myofiber size. Methods Needle biopsies were taken from the gastrocnemius of 53 PAD patients (28 with early PAD and 25 with advanced PAD) and 25 controls. Carbonyl groups (marker of oxidative damage), were quantified in myofibers of slide-mounted tissue, by quantitative fluorescence microscopy. Myofiber cross-sectional area was determined from sarcolemma labeled with wheat germ agglutinin. The tissues were also labeled for myosin I and II, permitting quantification of oxidative damage to and relative frequency of the different myofiber Types (Type I, Type II and mixed Type I/II myofibers). We compared PAD patients in early (N=28) vs. advanced (N=25) disease stage for selective, myofiber oxidative damage and altered morphometrics. Results The carbonyl content of gastrocnemius myofibers was higher in PAD patients compared to control subjects, for all three myofiber types (p<0.05). In PAD patients carbonyl content was higher (p<0.05) in Type II and I/II fibers compared to Type I fibers. Furthermore, the relative frequency and cross-sectional area of Type II fibers were lower, while the relative frequencies and cross-sectional area of Type I and Type I/II fibers were higher, in PAD compared to control gastrocnemius (p<0.05). Lastly, the type II-selective oxidative damage increased and myofiber size decreased as the disease progressed from the early to advanced stage. Conclusions Our data confirm increased myofiber oxidative damage and reduced myofiber size in PAD gastrocnemius and demonstrate that the damage is selective for type II myofibers and is worse in the most advanced stage of PAD.

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.

Protein Concentration and Mitochondrial Content in the Gastrocnemius Predicts Mortality Rates in Patients With Peripheral Arterial Disease

OBJECTIVE This study evaluated the hypothesis that protein concentration and mitochondrial content in gastrocnemius biopsies from patients with peripheral arterial disease (PAD) predict mortality rates. BACKGROUND PAD patients experience advancing myopathy characterized by mitochondrial dysfunction, myofiber degradation, and fibrosis in their ischemic legs, along with increased mortality rates. METHODS Samples from the gastrocnemius of PAD patients were used for all analyses. Protein concentration was normalized to muscle wet weight, and citrate synthase activity (standard measure of mitochondrial content in cells) was normalized to muscle wet weight and protein concentration. Protein and citrate synthase data were grouped into tertiles and 5-year, all-cause mortality for each tertile was determined with Kaplan-Meier curves and compared by the modified Peto-Peto test. A Cox-regression model for each variable controlled for the effects of clinical characteristics. RESULTS Of the 187 study participants, 46 died during a mean follow-up of 23.0 months. Five-year mortality rate was highest for patients in the lowest tertile of protein concentration. Mortality was lowest for patients in the middle tertile of citrate synthase activity when normalized to either muscle wet weight or protein concentration. The mortality hazard ratios (HRs) from the Cox analysis were statistically significant for protein concentration normalized to muscle wet weight (lowest vs middle tertile; HR = 2.93; P = 0.008) and citrate synthase normalized to protein concentration (lowest vs middle tertile; HR = 4.68; P = 0.003; and lowest vs highest tertile; HR = 2.36; P = 0.027). CONCLUSIONS Survival analysis of a contemporaneous population of PAD patients identifies protein and mitochondrial content of their gastrocnemius as predictors of mortality rate.

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.

Abnormal Accumulation of Desmin in Gastrocnemius Myofibers of Patients with Peripheral Artery Disease Associations with Altered Myofiber Morphology and Density, Mitochondrial Dysfunction and Impaired Limb Function

Patients with peripheral artery disease (PAD) develop a myopathy in their ischemic lower extremities, which is characterized by myofiber degeneration, mitochondrial dysfunction and impaired limb function. Desmin, a protein of the cytoskeleton, is central to maintenance of the structure, shape and function of the myofiber and its organelles, especially the mitochondria, and to translation of sarcomere contraction into muscle contraction. In this study, we investigated the hypothesis that disruption of the desmin network occurs in gastrocnemius myofibers of PAD patients and correlates with altered myofiber morphology, mitochondrial dysfunction, and impaired limb function. Using fluorescence microscopy, we evaluated desmin organization and quantified myofiber content in the gastrocnemius of PAD and control patients. Desmin was highly disorganized in PAD but not control muscles and myofiber content was increased significantly in PAD compared to control muscles. By qPCR, we found that desmin gene transcripts were increased in the gastrocnemius of PAD patients as compared with control patients. Increased desmin and desmin gene transcripts in PAD muscles correlated with altered myofiber morphology, decreased mitochondrial respiration, reduced calf muscle strength and decreased walking performance. In conclusion, our studies identified disruption of the desmin system in gastrocnemius myofibers as an index of the myopathy and limitation of muscle function in patients with PAD.

Patients with peripheral arterial disease exhibit reduced joint powers compared to velocity-matched controls

Previous studies have shown major deficits in gait for individuals with peripheral arterial disease before and after the onset of pain. However, these studies did not have subjects ambulate at similar velocities and potential exists that the differences in joint powers may have been due to differences in walking velocity. The purpose of this study was to examine the joint moments and powers of peripheral arterial disease limbs for subjects walking at similar self-selected walking velocities as healthy controls prior to onset of any symptoms. Results revealed peripheral arterial disease patients have reduced peak hip power absorption in midstance (p=0.017), reduced peak knee power absorption in early and late stance (p=0.037 and p=0.020 respectively), and reduced peak ankle power generation in late stance (p=0.021). This study reveals that the gait of patients with peripheral arterial disease walking prior to the onset of any leg symptoms is characterized by failure of specific and identifiable muscle groups needed to perform normal walking and that these gait deficits are independent of reduced gait velocity.

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.