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

Abstract

BackgroundPeripheral 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.ObjectivesIn 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.MethodsNeedle 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.ResultsThe 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.ConclusionsOur 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.

Abnormal myofiber morphology and limb dysfunction in claudication

“…Our group has quantified, precisely, parameters of myofiber morphology in PAD and control gastrocnemius samples obtained with minimally invasive needle biopsy. We found consistent and quantifiable changes in gastrocnemius myofiber morphology, which can separate PAD patients from controls and PAD patients with stage II disease from patients with critical limb ischemia [18,20]. In the present study, we evaluated myofiber morphometrics as predictors of objective measures of the limb function (calf muscle strength and walking distances) of PAD patients.…”

“…1 and 2). PAD muscle displays different patterns of myofiber degeneration with alteration of the size and shape of the myofibers [2,15,18,19], internal nuclei, target lesions, vacuolization, shift of myofiber phenotype toward type I fibers, myofiber necrosis, fatty infiltration and endomysial, perimysial and epimysial fibrosis [15–19]. In addition, the myopathy of PAD muscle has been observed by computerized tomographic scanning of the calf muscles of claudicating patients, demonstrating smaller muscle volume, higher fat content, and lower muscle density [27].…”

“…The frames corresponding to each fluorophore were montaged in one large image to represent the given (whole) specimen. Image segmentation was done with a custom-made Matlab algorithm (R2012a; MathWorks Inc, Natick, MA), using thresholding, edge detection, regionprops, and a set of heuristics [18,20]. Labeled sarcolemma acquired at 668 nm provided a binary image that served as an outline for each myofiber.…”

“…For each specimen, we extracted measurements of myofiber size, shape, and density. The methods describing how these parameters were measured have been described in detail [18,20]. Briefly, we calculated the following parameters of myofiber size: 1) myofiber cross-sectional area (μ 2 ), from the number of pixels enclosed within a segmented myofiber, 2) major and minor axes (μ), of an ellipse fitted in the myofiber, 3) equivalent diameter (μ), as the diameter of a circle that has the same area as the segmented myofiber, and 4) myofiber perimeter (μ), from the number of pixels on the boundary of the myofiber.…”

Disruption of mitochondrial quality control in peripheral artery disease: New therapeutic opportunities

“…Excessive 4-HNE adducts formation has been previously reported in ischemic hearts from both humans and rodents (100) and was associated with contractility dysfunction and impaired mitochondrial bioenergetics (33, 97, 98). Increased 4-HNE-modified proteins have also been reported in gastrocnemius muscle biopsies from individual with PAD and rodents subjected to hindlimb ischemia (28, 101, 102). Therefore, accumulation of 4-HNE appears to be a hallmark of ischemia-reperfusion injury in both cardiac and skeletal muscles.…”