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. 2009 Mar;174(3):999-1008.
doi: 10.2353/ajpath.2009.080537. Epub 2009 Jan 29.

Valproic Acid Activates the PI3K/Akt/mTOR Pathway in Muscle and Ameliorates Pathology in a Mouse Model of Duchenne Muscular Dystrophy

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Free PMC article

Valproic Acid Activates the PI3K/Akt/mTOR Pathway in Muscle and Ameliorates Pathology in a Mouse Model of Duchenne Muscular Dystrophy

Praveen B Gurpur et al. Am J Pathol. .
Free PMC article

Abstract

Duchenne muscular dystrophy is a lethal neuromuscular disease that currently has no effective therapy. Transgenic overexpression of the alpha7 integrin in mdx/utrn(-/-) mice, a model of Duchenne muscular dystrophy ameliorates the disease. We have isolated and used alpha7(+/-) muscle cells expressing beta-galactosidase, driven by the endogenous alpha7 promoter, to identify compounds that increase alpha7 integrin levels. Valproic acid (VPA) was found to enhance alpha7 integrin levels, induce muscle hypertrophy, and inhibit apoptosis in myotubes by activating the Akt/mTOR/p70S6K pathway. This activation of the Akt pathway occurs within 1 hour of treatment and is mediated by phosphatidylinositol 3-OH kinase. To evaluate the potential use of VPA to treat muscular dystrophy, mdx/utrn(-/-) mice were injected with the drug. Treatment with VPA lowered collagen content and fibrosis, and decreased hind limb contractures. VPA-treated mice also had increased sarcolemmal integrity and decreased damage, decreased CD8-positive inflammatory cells, and higher levels of activated Akt in their muscles. Thus, VPA has important biological effects that may be applicable for the treatment of muscular dystrophy.

Figures

Figure 1
Figure 1
VPA increases α7 integrin levels in α7+/− myotubes. A: FDG-based β-galactosidase assay was done after exposing 2-day-old α7+/− myotubes to various concentrations of VPA for 24 hours. Data represent the mean ± SD of triplicate samples. B: Top: Western blotting of α7 integrin in extracts of α7+/− myotubes exposed for 48 or 72 hours to 2 mmol/L VPA in duplicate plates. Bottom: Quantification of α7 band intensities. Data represent mean ± SD of duplicate samples. *P < 0.05.
Figure 2
Figure 2
VPA promotes hypertrophy and inhibits apoptosis in α7+/− myotubes. Two mmol/L VPA was added to day 2 α7+/− differentiating cultures. A: Top: Phase-contrast micrographs of α7+/− myotubes exposed to VPA for 72 hours compared with untreated control cells. Bottom: Myotubes exposed to VPA for 72 hours and stained using anti-myosin heavy chain antibody. The graph shows the ratios of myotube area to the number of nuclei within myotubes in VPA-treated and control cultures. Data represent mean ± SD of the ratios in 10 random fields. *P < 0.05. B: Top: Phase-contrast micrographs of α7+/− myotubes treated with VPA for 6 days (starting on day 2 of differentiation) compared with untreated controls. Bottom: α7+/− myotubes in eight-well chamber slides were exposed to either 2 mmol/L VPA (four chambers) or no treatment (four chambers) for 6 days. TUNEL assays identify FITC-labeled apoptotic nuclei. DAPI staining shows all nuclei in the same fields. The total number of TUNEL-positive nuclear clusters in four chambers each for VPA-treated and control cultures was counted. Data represent mean ± SD of quadruplicate values. *P < 0.05.
Figure 3
Figure 3
VPA activates the Akt/mTOR/p70S6K pathway in α7+/− myotubes. A: Two mmol/L VPA was added to duplicate cultures of day 2 α7+/− myotubes for 72 or 96 hours, followed by Western blotting using antibodies against the signaling proteins. Higher levels of activated Akt, mTOR, and p70S6K and lower levels of activated ERK were detected in the VPA-treated myotubes. B: α7+/− myotubes in duplicate plates were treated with 2 mmol/L VPA and protein was extracted every 24 hours for up to 144 hours. Western blotting was done to quantify phospho and total Akt. The ratio of band intensities of phospho to total Akt every 24 hours shows sustained activation of Akt in the VPA-treated cells.
Figure 4
Figure 4
Akt activation by VPA is dependent on PI3K. A: α7+/− myotubes were treated for 72 hours with 2 mmol/L VPA with or without 100 nmol/L wortmannin. Fresh drugs were added every 24 hours. Phase-contrast micrographs show that the hypertrophic effect of VPA on myotubes is attenuated by wortmannin, indicating that VPA depends on PI3K for this effect. B: α7+/− myotubes treated as above were extracted and immunoblotted for phospho and total Akt. Ratios of phospho to total Akt band intensities were plotted. The Akt-activating effect of VPA was attenuated by wortmannin.
Figure 5
Figure 5
VPA activates Akt within 1 hour in a dose-dependent manner and this brief exposure promotes hypertrophy in α7+/− myotubes. A: α7+/− myotubes were exposed to various concentrations of VPA for 1 hour, followed by extraction and Western blotting for Akt. Band intensities from the blots were quantified and the ratios of activated to total Akt were plotted. VPA activates Akt in a dose-dependent manner, with highest activation seen at 30 mmol/L VPA. B: α7+/− myotubes were exposed to 30 mmol/L VPA with or without 100 nmol/L wortmannin for 1 hour, washed, and fresh medium was added. Microscopic observations 72 hours later show hypertrophy in myotubes exposed to VPA. This effect was attenuated by wortmannin, confirming the ability of VPA to activate Akt within 1 hour is dependent on PI3 kinase.
Figure 6
Figure 6
VPA inhibits muscle fibrosis in dystrophic muscle and ameliorates hind limb contractures in mdx/utrn−/− mice. A: On the 35th day of treatment, mdx/utrn−/− mice injected with saline (n = 9) or VPA (n = 9) were tested for contractures in their hind limbs by lifting of their tails. The presence of contractures prevents full extension of the hind limbs. The graph shows the proportion of mice with contractures in the saline-injected group (four of nine, 44.4%) was fourfold higher than the VPA-injected group (one of nine, 11.1%). B: Cryosections of quadriceps stained with Masson’s trichrome. Collagen content is higher in muscle from saline-injected mice (as seen by the higher intensity of blue), compared with VPA-injected mice. C: Sodium dodecyl sulfate extracts of quadriceps from saline (n = 9)- and VPA (n = 9)-injected mice were subjected to Western blotting for collagen type VI. Quantification of band intensities show greater than threefold more collagen type VI in the saline-injected mice compared with animals injected with VPA. Data represent mean ± SD. *P < 0.05.
Figure 7
Figure 7
VPA increases myofiber integrity and decreases inflammation in dystrophic muscle. A: Cryosections of quadriceps from saline (n = 9)- and VPA (n = 9)-injected mdx/utrn−/− mice that also had been injected with Evans blue dye (50 mg/kg). FITC-wheat germ agglutinin was used to delineate myofibers. The red fluorescence shows Evans blue dye-positive fibers. The percentage of Evan’s blue dye-positive fibers in three sections, 40 μm apart, were counted (∼5000 fibers total) for each mouse. Quantification shows an ∼2.5-fold decrease in proportion of Evans blue-positive fibers in VPA-injected mice. Data represent mean ± SD. *P < 0.05. B: Cryosections of quadriceps from saline- and VPA-injected mice were stained for CD8-positive cytotoxic T cells using a FITC-labeled anti-CD8a antibody. The sections were co-stained with DAPI to delineate nuclei. The number of CD8-positive cells and the total number of nuclei in the same field were counted in 10 random fields for each mouse. The ratio of the number of CD8-positive cells to the total number of nuclei were calculated, averaged, and plotted for the saline- and VPA-treated groups. Quantification shows a 2.1-fold decrease in number of CD8-positive cells in VPA-treated mice. Data represent mean ± SD. *P < 0.05.
Figure 8
Figure 8
VPA-treated mice have higher proportions of activated Akt in skeletal muscle. Extracts of quadriceps from saline- and VPA-injected mdx/utrn−/− mice were subjected to Western blotting for activated and total Akt. Quantification of band intensities and ratios of activated to total Akt show higher levels of activated Akt in the VPA-injected mice. Data represent mean ± SD. *P < 0.05.

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