Evlampia Papoutsi

Parallel Changes in Neuronal AT1R and GRK5 Expression Following Exercise Training in Heart Failure

Although exercise training (ExT) is an important therapeutic strategy for improving quality of life in patients with chronic heart failure (CHF), the central mechanisms by which ExT is beneficial are not well understood. The angiotensin II type 1 receptor (AT1R) plays a pivotal role in the development of CHF and is upregulated in a number of tissues owing, in part, to transcription factor nuclear factor kappa B (NF-&kgr;B). In addition, AT1R is marked for internalization and recycling via G-protein–coupled receptor kinase (GRK) phosphorylation. Because previous studies have shown that the beneficial effects of ExT in CHF rely on a reduction in angiotensin II, we hypothesized ExT would decrease AT1R, GRK5, and NF-&kgr;B protein expression in the paraventricular nucleus and rostral ventrolateral medulla of CHF rats. Following infarction by coronary artery ligation, animals were exercised 4 weeks postsurgery on a treadmill at a final speed of 25 miles per minute for 60 minutes, 5 days per week for 6 weeks. Western blot analysis of paraventricular nucleus and rostral ventrolateral medulla micropunches revealed an upregulation of AT1R, GRK5, and NF-&kgr;B in the infarcted group that was reversed by ExT. Furthermore, the relative expression of phosphorylated AT1R and AT1R/GRK5 physical association was increased in the CHF sedentary group and reversed by ExT. Overexpression of GRK5 in cultured CATH.a neurons blunted angiotensin II-mediated upregulation of AT1R and NF-&kgr;B; conversely, silencing of GRK5 exacerbated angiotensin II-mediated AT1R and NF-&kgr;B upregulation. Taken together, increased GRK5 may regulate AT1R expression in CHF, and ExT mitigates AT1R and its pathway components.

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.

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.

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.

Oxidative Stress and Arterial Dysfunction in Peripheral Artery Disease

Peripheral artery disease (PAD) is an atherosclerotic disease characterized by a narrowing of the arteries in the lower extremities. Disease manifestations are the result of more than just reduced blood flow, and include endothelial dysfunction, arterial stiffness, and inflammation. Growing evidence suggests that these factors lead to functional impairment and decline in PAD patients. Oxidative stress also plays an important role in the disease, and a growing amount of data suggest a link between arterial dysfunction and oxidative stress. In this review, we present the current evidence for the involvement of endothelial dysfunction, arterial stiffness, and inflammation in the pathophysiology of PAD. We also discuss the links between these factors and oxidative stress, with a focus on nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2)-derived reactive oxygen species (ROS) and decreased nitric oxide (NO) bioavailability. Finally, the potential therapeutic role of NOX2 antioxidants for improving arterial function and functional status in PAD patients is explored.