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

Abstract

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.

Keywords: Cytoskeleton; desmin; intermittent claudication; muscle disease; myofiber.

Figure 1.

Schematic representation of the desmin intermediate filament network and its association with myofibrils, sarcolemma, mitochondria and the nucleus, in a skeletal myofiber. Desmin is a key protein of the intermediate filaments of the cytoskeleton in myofibers of skeletal muscle. Desmin’s main function is transmission of the forces produced by myofibrils. Desmin IFs link myofibrils into bundles through the Z-discs (dark blue) and connect them to specialized sites of adhesion (costameres) (green) in the sarcolemma. Myofibrillar forces are then transmitted from the costameres to the extracellular matrix and then to the tendons. In addition, Desmin provides a three-dimensional organization of the mitochondrial system (pink), connects the contractile apparatus to the myonuclei (purple), and may contribute to mechano-chemical signaling between the various compartments of the myofiber.

Figure 2.

Slide-mounted gastrocnemius sections from a control subject (A–C, G–I) and a Peripheral Artery Disease (PAD) subject (D–F, J–L) were labeled for fluorescence microscopy and captured with 20×, 40× and 100× objectives. In longitudinally sectioned control myofibers (A–C), desmin (green) exhibited a normal distribution along the Z-disks, seen as regular, horizontal striations of bright green labeling and representing a well-organized cytoskeletal network. In PAD myofibers (D–F), desmin was disorganized and seen as a more dense deposition along the Z-disks in some areas (arrowheads), where abnormal cross-links between adjacent Z-disks are evident, and dense accumulation along the walls of large vacuoles (filled arrows). In cross-sections of control myofibers (G–I), desmin exhibited a normal honeycomb appearance throughout the myofiber (100×), representing numerous myofibrils in cross-section, and a high concentration in the subsarcolemma region (20×, 40× and 100×) (filled arrows). In cross-sections of PAD myofibers (J–L), desmin exhibited an irregular honeycomb appearance seen as intense and uneven labeling along the boundaries of the myofibrils (100×) (line arrows) and extensive, irregular cytoplasmic aggregates (arrowheads).

Figure 3.

(A) Representative western blot showing increased desmin content in gastrocnemius tissue from Peripheral Artery Disease (PAD) subjects as compared with that of control subjects. A desmin band of higher mobility in PAD muscle suggests post-translational modification, possibly oxidative damage, and/or protein degradation. (B) Desmin mRNA content normalized to myosin mRNA was higher in PAD gastrocnemius specimens. (C) Mean desmin mRNA normalized to myosin mRNA was 126 times higher in PAD (n=30) as compared with control (n=30) gastrocnemius, suggesting inefficient translation of the gene transcript and/or high protein degradation.

Figure 4.

Abnormal desmin accumu-lation in the myofibers of Peripheral Artery Disease (PAD) gastrocnemius (A) and associated changes in myofiber morphology and extracellular matrix (B). Sections from the gastrocnemius of a PAD subject with moderate to severe myopathy were captured under fluorescence (A) and bright-field (B) conditions with a 10× objective. The specimen (A) was labeled with desmin antibody (green), wheat germ agglutinin (membranes and connective tissue; red) and DAPI nuclear stain (blue). The neighboring section (B) was stained with Masson’s Trichrome (sarcoplasm, red; connective tissue, blue; nuclei, black). The PAD myofibers display dense, irregular accumulations of desmin in the sarcoplasm (A). The myofibers are irregular in shape and size and include many large, rounded and swollen fibers and many small, irregularly shaped fibers (A and B). Several of the large myofibers show evidence of target lesions (filled arrows), vacuoles (arrow heads) and internal nuclei (line arrows) (B), findings consistent with myofiber degeneration. There is obvious expan-sion of the extracellular matrix. Scale, 100 µm.

Figure 5.

Representative fluorescence images of desmin (green) and ATP-Synthase (red) in slide-mounted sections of the gastrocnemius of control (row A) and Peripheral Artery Disease (PAD) subjects (row B). There is an irregular and patchy distribution of mitochondria in PAD myofibers, with an absence of mitochondrial labeling in the areas where desmin is abundant and highly aggregated. The mitochondria exhibit an uneven distribution throughout the remainder of the sarcoplasm and in the sub-sarcolemma region.

Figure 6.

Desmin content in Peripheral Artery Disease (PAD; n=30) gastrocnemius myofibers is inversely correlated with limb function. (A) Muscle strength exerted from the ankle plantarflexors during maximum isometric contraction. (B) Peak walking distance results. (C) Six-minute walking distance results.