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. 2008 Jul;295(1):R290-6.
doi: 10.1152/ajpregu.90374.2008. Epub 2008 May 14.

Chronically ischemic mouse skeletal muscle exhibits myopathy in association with mitochondrial dysfunction and oxidative damage

Iraklis I Pipinos  1 , Stanley A Swanson, Zhen Zhu, Aikaterini A Nella, Dustin J Weiss, Tanuja L Gutti, Rodney D McComb, B Timothy Baxter, Thomas G Lynch, George P Casale
Affiliations

Chronically ischemic mouse skeletal muscle exhibits myopathy in association with mitochondrial dysfunction and oxidative damage

Iraklis I Pipinos et al. Am J Physiol Regul Integr Comp Physiol. 2008 Jul.

Abstract

A myopathy characterized by mitochondrial pathology and oxidative stress is present in patients with peripheral arterial disease (PAD). Patients with PAD differ in disease severity, mode of presentation, and presence of comorbid conditions. In this study, we used a mouse model of hindlimb ischemia to isolate and directly investigate the effects of chronic inflow arterial occlusion on skeletal muscle microanatomy, mitochondrial function and expression, and oxidative stress. Hindlimb ischemia was induced by staged ligation/division of the common femoral and iliac arteries in C57BL/6 mice, and muscles were harvested 12 wk later. Muscle microanatomy was examined by bright-field microscopy, and mitochondrial content was determined as citrate synthase activity in muscle homogenates and ATP synthase expression by fluorescence microscopy. Electron transport chain (ETC) complexes I through IV were analyzed individually by respirometry. Oxidative stress was assessed as total protein carbonyls and 4-hydroxy-2-nonenal (HNE) adducts and altered expression and activity of manganese superoxide dismutase (MnSOD). Ischemic muscle exhibited histological features of myopathy and increased mitochondrial content compared with control muscle. Complex-dependent respiration was significantly reduced for ETC complexes I, III, and IV in ischemic muscle. Protein carbonyls, HNE adducts, and MnSOD expression were significantly increased in ischemic muscle. MnSOD activity was not significantly changed, suggesting MnSOD inactivation. Using a mouse model, we have demonstrated for the first time that inflow arterial occlusion alone, i.e., in the absence of other comorbid conditions, causes myopathy with mitochondrial dysfunction and increased oxidative stress, recapitulating the muscle pathology of PAD patients.

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Figures

Fig. 1.
Fig. 1.
Comparable histopathological changes in ischemic skeletal muscle of a chronically ligated mouse and a patient with arterial disease. Control and ischemic muscle from a sham-operated (A) and a chronically ligated (B) mouse, and a control patient (C) and an age- and sex-matched patient with arterial disease (D), were stained with hematoxylin and eosin and captured under bright-field conditions with a ×10 objective. Myofibers of control muscles (A and C) exhibit peripherally located nuclei, have a regular polygonal shape, and are fairly uniform in size. Myofibers of ischemic muscles (B and D) display more centrally located nuclei and decreased cross-sectional area. These features are consistent with myopathic alterations in the ischemic mouse muscle. The calibration bar at the bottom right of D represents 50 μm.
Fig. 2.
Fig. 2.
Representative images illustrating similar labeling patterns and increased expression of ATP synthase and manganese superoxide dismutase (MnSOD) in ischemic skeletal muscle of a chronically ligated mouse. Tibialis anterior muscle from sham-operated (A, C, and E) and chronically ligated (B, D, and F) mice was fixed in methacarn, embedded in paraffin, and sectioned at 4 μm. Control (A and C) and ischemic (B and D) muscles were labeled with specific monoclonal antibodies against ATP synthase. High magnification (×670) with a ×20 objective (A and B) revealed the classic tubular microanatomy of the mitochondrial system and intensely fluorescent nodules associated with mitochondrial labeling. At a lower magnification (×260) with a ×10 objective (C and D), labeling exhibits subsarcolemmal aggregates and a sarcoplasmic dotlike pattern typical of mitochondrial labeling of skeletal myofibers. ATP synthase expression is variable among myofibers of ischemic and control muscle, reflecting differences in mitochondrial content. Overall, ischemic muscle exhibits increased ATP synthase expression compared with control muscle. The same pattern of labeling was seen in adjacent serial sections of control (E) and ischemic (F) muscle labeled with a monoclonal antibody specific for MnSOD. Myofibers treated with isotype control exhibited no fluorescence labeling (not shown). Calibration bars at the bottom right of B and F represent 25 μm. Magnification was calculated by dividing the length of each bar in the image by 25 μm.
Fig. 3.
Fig. 3.
Increased 4-hydroxy-2-nonenal (HNE) content in ischemic calf muscle of chronically ligated mice. Myofibers in control (A) and ischemic (B) muscle were labeled with a mouse monoclonal antibody (gift of Dr. Koji Uchida) specific for HNE-protein adducts and Alexa Fluor-conjugated secondary antibody. Images were captured with a Leica fluorescence microscope (×10 objective). HNE signal is markedly increased in myofibers of the ischemic muscle. The bar at the bottom right of B represents 25 μm. C: homogenates prepared with ischemic (1–3) and control (5–7) muscle were analyzed by reverse-phase protein array analysis (RPPA). Homogenate preparations and dilutions (1/20, 1/40, 1/80, and 1/160) of stock HNE-BSA were arrayed in triplicate on nitrocellulose slides and labeled with the monoclonal anti-HNE antibody and IRDye 800 CW conjugated secondary antibody. Integrated intensity of the HNE signal is higher in ischemic muscle of the chronically ligated mice. For both RPPA and fluorescence microscopy, the isotype control produced no fluorescence signal (not shown).
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