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Ectopic catalase expression in mitochondria by adeno-associated virus enhances exercise performance in mice.

Li D, Lai Y, Yue Y, Rabinovitch PS, Hakim C, Duan D - PLoS ONE (2009)

Bottom Line: However, administration of generic antioxidants has failed to convincingly improve performance during exhaustive exercise.Our result provides a framework for further elucidating the underlying mechanism.It also raises the hope of applying similar strategies to remove excessive, pathogenic free radicals in certain muscle diseases (such as Duchenne muscular dystrophy) and ameliorate muscle disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO, USA.

ABSTRACT
Oxidative stress is thought to compromise muscle contractility. However, administration of generic antioxidants has failed to convincingly improve performance during exhaustive exercise. One possible explanation may relate to the inability of the supplemented antioxidants to effectively eliminate excessive free radicals at the site of generation. Here, we tested whether delivering catalase to the mitochondria, a site of free radical production in contracting muscle, could improve treadmill performance in C57Bl/6 mice. Recombinant adeno-associated virus serotype-9 (AV.RSV.MCAT) was generated to express a mitochondria-targeted catalase gene. AV.RSV.MCAT was delivered to newborn C57Bl/6 mouse circulation at the dose of 10(12) vector genome particles per mouse. Three months later, we observed a approximately 2 to 10-fold increase of catalase protein and activity in skeletal muscle and the heart. Subcellular fractionation western blot and double immunofluorescence staining confirmed ectopic catalase expression in the mitochondria. Compared with untreated control mice, absolute running distance and body weight normalized running distance were significantly improved in AV.RSV.MCAT infected mice during exhaustive treadmill running. Interestingly, ex vivo contractility of the extensor digitorum longus muscle was not altered. Taken together, we have demonstrated that forced catalase expression in the mitochondria enhances exercise performance. Our result provides a framework for further elucidating the underlying mechanism. It also raises the hope of applying similar strategies to remove excessive, pathogenic free radicals in certain muscle diseases (such as Duchenne muscular dystrophy) and ameliorate muscle disease.

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Systemic AV.RSV.MCAT infection leads to mosaic catalase expression in the mitochondria in striated muscles.A, Representative catalase immunofluorescence staining photomicrographs of AV.RSV.MCAT infected and uninfected skeletal muscle (top panels) and heart (bottom panels). Arrow, AAV transduced skeletal muscle myofiber. B, Representative double immunofluorescence staining photomicrographs of AV.RSV.MCAT infected heart. Bottom panels are high magnification images of the boxed region in respective top panels. Cytochrome C marks mitochondria (red color). Catalase is in green color. Yellow color in merged images reveals mitochondrial catalase expression.
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pone-0006673-g002: Systemic AV.RSV.MCAT infection leads to mosaic catalase expression in the mitochondria in striated muscles.A, Representative catalase immunofluorescence staining photomicrographs of AV.RSV.MCAT infected and uninfected skeletal muscle (top panels) and heart (bottom panels). Arrow, AAV transduced skeletal muscle myofiber. B, Representative double immunofluorescence staining photomicrographs of AV.RSV.MCAT infected heart. Bottom panels are high magnification images of the boxed region in respective top panels. Cytochrome C marks mitochondria (red color). Catalase is in green color. Yellow color in merged images reveals mitochondrial catalase expression.

Mentions: Three months after AV.RSV.MCAT infection, we examined catalase expression in skeletal muscle and the heart (Figures 1 and 2). On whole muscle lysate western blot, the intensity of the catalase band was substantially stronger in AV.RSV.MCAT infected mice (Figure 1B). Catalase overexpression was confirmed by the zymograph assay (Figure 1B). Interestingly, for reason(s) yet unknown, MCAT (Figure 1B arrowhead) appeared to migrate faster than endogenous PCAT in the zymography gel (Figure 1B arrows). Next, we quantified catalase activity in whole muscle lysate. Compared with uninfected mice, we observed an approximately 10-fold catalase activity increase in the heart of AV.RSV.MCAT infected mice. In skeletal muscle, the catalase activity was increased by 3 to 5-fold except for the soleus muscle which only showed a less than 2-fold increase (Figure 1C). To confirm mitochondrial catalase expression, we performed western blot using isolated skeletal muscle mitochondria preparation (Figure 1D) [24]. Prohibitin was used as the mitochondria marker. As expected, we observed the catalase band in the mitochondria isolated from AV.RSV.MCAT infected muscle but not from uninfected muscle (Figure 1D).


Ectopic catalase expression in mitochondria by adeno-associated virus enhances exercise performance in mice.

Li D, Lai Y, Yue Y, Rabinovitch PS, Hakim C, Duan D - PLoS ONE (2009)

Systemic AV.RSV.MCAT infection leads to mosaic catalase expression in the mitochondria in striated muscles.A, Representative catalase immunofluorescence staining photomicrographs of AV.RSV.MCAT infected and uninfected skeletal muscle (top panels) and heart (bottom panels). Arrow, AAV transduced skeletal muscle myofiber. B, Representative double immunofluorescence staining photomicrographs of AV.RSV.MCAT infected heart. Bottom panels are high magnification images of the boxed region in respective top panels. Cytochrome C marks mitochondria (red color). Catalase is in green color. Yellow color in merged images reveals mitochondrial catalase expression.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2723912&req=5

pone-0006673-g002: Systemic AV.RSV.MCAT infection leads to mosaic catalase expression in the mitochondria in striated muscles.A, Representative catalase immunofluorescence staining photomicrographs of AV.RSV.MCAT infected and uninfected skeletal muscle (top panels) and heart (bottom panels). Arrow, AAV transduced skeletal muscle myofiber. B, Representative double immunofluorescence staining photomicrographs of AV.RSV.MCAT infected heart. Bottom panels are high magnification images of the boxed region in respective top panels. Cytochrome C marks mitochondria (red color). Catalase is in green color. Yellow color in merged images reveals mitochondrial catalase expression.
Mentions: Three months after AV.RSV.MCAT infection, we examined catalase expression in skeletal muscle and the heart (Figures 1 and 2). On whole muscle lysate western blot, the intensity of the catalase band was substantially stronger in AV.RSV.MCAT infected mice (Figure 1B). Catalase overexpression was confirmed by the zymograph assay (Figure 1B). Interestingly, for reason(s) yet unknown, MCAT (Figure 1B arrowhead) appeared to migrate faster than endogenous PCAT in the zymography gel (Figure 1B arrows). Next, we quantified catalase activity in whole muscle lysate. Compared with uninfected mice, we observed an approximately 10-fold catalase activity increase in the heart of AV.RSV.MCAT infected mice. In skeletal muscle, the catalase activity was increased by 3 to 5-fold except for the soleus muscle which only showed a less than 2-fold increase (Figure 1C). To confirm mitochondrial catalase expression, we performed western blot using isolated skeletal muscle mitochondria preparation (Figure 1D) [24]. Prohibitin was used as the mitochondria marker. As expected, we observed the catalase band in the mitochondria isolated from AV.RSV.MCAT infected muscle but not from uninfected muscle (Figure 1D).

Bottom Line: However, administration of generic antioxidants has failed to convincingly improve performance during exhaustive exercise.Our result provides a framework for further elucidating the underlying mechanism.It also raises the hope of applying similar strategies to remove excessive, pathogenic free radicals in certain muscle diseases (such as Duchenne muscular dystrophy) and ameliorate muscle disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO, USA.

ABSTRACT
Oxidative stress is thought to compromise muscle contractility. However, administration of generic antioxidants has failed to convincingly improve performance during exhaustive exercise. One possible explanation may relate to the inability of the supplemented antioxidants to effectively eliminate excessive free radicals at the site of generation. Here, we tested whether delivering catalase to the mitochondria, a site of free radical production in contracting muscle, could improve treadmill performance in C57Bl/6 mice. Recombinant adeno-associated virus serotype-9 (AV.RSV.MCAT) was generated to express a mitochondria-targeted catalase gene. AV.RSV.MCAT was delivered to newborn C57Bl/6 mouse circulation at the dose of 10(12) vector genome particles per mouse. Three months later, we observed a approximately 2 to 10-fold increase of catalase protein and activity in skeletal muscle and the heart. Subcellular fractionation western blot and double immunofluorescence staining confirmed ectopic catalase expression in the mitochondria. Compared with untreated control mice, absolute running distance and body weight normalized running distance were significantly improved in AV.RSV.MCAT infected mice during exhaustive treadmill running. Interestingly, ex vivo contractility of the extensor digitorum longus muscle was not altered. Taken together, we have demonstrated that forced catalase expression in the mitochondria enhances exercise performance. Our result provides a framework for further elucidating the underlying mechanism. It also raises the hope of applying similar strategies to remove excessive, pathogenic free radicals in certain muscle diseases (such as Duchenne muscular dystrophy) and ameliorate muscle disease.

Show MeSH
Related in: MedlinePlus