Muscle-specific microRNA1 (miR1) targets heat shock protein 70 (HSP70) during dexamethasone-mediated atrophy

Abstract

High doses of dexamethasone (Dex) or myostatin (Mstn) induce severe atrophy of skeletal muscle. Here we show a novel microRNA1 (miR1)-mediated mechanism through which Dex promotes skeletal muscle atrophy. Using both C2C12 myotubes and mouse models of Dex-induced atrophy we show that Dex induces miR1 expression through glucocorticoid receptor (GR). We further show that Mstn treatment facilitates GR nuclear translocation and thereby induces miR1 expression. Inhibition of miR1 in C2C12 myotubes attenuated the Dex-induced increase in atrophy-related proteins confirming a role for miR1 in atrophy. Analysis of miR1 targets revealed that HSP70 is regulated by miR1 during atrophy. Our results demonstrate that increased miR1 during atrophy reduced HSP70 levels, which resulted in decreased phosphorylation of AKT, as HSP70 binds to and protects phosphorylation of AKT. We further show that loss of pAKT leads to decreased phosphorylation, and thus, enhanced activation of FOXO3, up-regulation of MuRF1 and Atrogin-1, and progression of skeletal muscle atrophy. Based on these results, we propose a model whereby Dex- and Mstn-mediated atrophic signals are integrated through miR1, which then either directly or indirectly, inhibits the proteins involved in providing protection against atrophy.

FIGURE 1.

Dex up-regulates miR1 in C2C12 myotubes and mouse skeletal muscle. A, real time qPCR analysis of miR1–1 primary transcript expression in control (Ethanol) and Dex-treated C2C12 myotubes at 6- and 24-h time points. Relative gene expression was normalized to U6 RNA expression. The graphs depict fold-differences relative to controls at each time point. Values are mean ± S.E. (n = 3); **, p < 0.01. B, Northern blot analysis of miR1 in control (Ethanol) and Dex-treated C2C12 myotubes at 6- and 24-h time points. The expression of U6 was measured as a loading control. C, real time qPCR analysis of miR1–1 primary transcript expression in BF, quadriceps, and tibialis anterior muscle from saline- and Dex-injected wild type mice. Relative gene expression analysis was performed using the ΔΔC_t method and was normalized to U6 RNA expression. Values are mean ± S.E. (n = 3) and are expressed as the fold-difference relative to saline control; **, p < 0.01.

FIGURE 2.

miR1–1 is transcriptionally regulated by Mstn and GR during Dex-mediated muscle atrophy. A, Western blot analysis of Mstn protein levels in control (Ethanol) and Dex-treated C2C12 myotubes. The level of GAPDH was measured as a loading control. B, real time qPCR analysis of miR1–1 primary transcript expression in conditioned CHO media (CCM) and conditioned Mstn media (CMM)-treated C2C12 myotubes at 6- and 24-h time points. Relative gene expression analysis was performed using the ΔΔC_T method and was normalized to U6 RNA expression. The graphs depict fold-differences relative to controls at each time point. Values are mean ± S.E. (n = 3); **, p < 0.01. C, Northern blot analysis of miR1 expression in BF muscle from WT (lanes 1 and 2) and Mstn^−/− mice (lanes 3 and 4) following either saline or Dex injection. The expression of U6 was measured as the loading control. D, corresponding densitometric analysis of miR1 levels in WT and Mstn^−/− muscle following injection of either saline or Dex. Error bars represent mean ± S.E. (n = 3, **, p < 0.01). E, representative graph showing real time qPCR analysis of miR1–1 primary transcript expression in C2C12 myotubes treated with ethanol, Dex, or Dex + Ant1. Values are mean ± S.E. (n = 3) and are expressed as fold-differences relative to ethanol-treated controls; **, p < 0.01. F, Western blot analysis of GR levels in nuclear extracts from control (Ethanol) and Dex-treated C2C12 myotubes. Ponceau S staining was performed to ensure equal loading. G, schematic representation of the miR1–1 promoter showing the location and sequence of the GRE in the miR1–1 upstream enhancer sequence (top). The middle and bottom schematics represent the miR1–1/pGL3b promoter-reporter constructs with and without GRE, respectively. H, assessment of miR1–1 promoter-reporter activity during differentiation of C2C12 myoblasts into myotubes transfected with PC1 or empty vector control (pGL3b). miR1–1 promoter-reporter activity was normalized to Renilla luciferase and expressed as relative luminescence units (RLU). Each bar represents the mean ± S.E. (n = 3); **, p < 0.01. I, assessment of miR1–1 promoter-reporter activity in 72-h differentiated C2C12 myoblasts co-transfected with GR expression vector or empty vector and PC1, PC2, or empty vector control (pGL3b). miR1–1 promoter-reporter activity was normalized to Renilla luciferase and expressed as relative luminescence. Each bar represents the mean ± S.E. (n = 3); ***, p < 0.001. J, electrophoretic mobility shift assay (EMSA) with nuclear extracts from C2C12 myotubes treated with Dex for 6 h using oligonucleotides specific for the GRE within the mouse miR1–1 promoter as a probe. A band shift was noted after the addition of the nuclear extract and was enhanced in the presence of Dex. Lane 1 is the free oligo control and lanes 2 and 3 reveal the shifted band in control (Ethanol) and Dex-treated myotubes, respectively. K, EMSA with nuclear extracts from C2C12 myotubes treated with Dex for 6 h using oligonucleotides specific for the GRE within the mouse miR1–1 promoter as a probe together with competition oligos (unlabeled primers). A band shift was noted after the addition of the nuclear extract and was decreased in a dose-dependent manner upon addition of competition oligos (lanes 2–4). L, EMSA with nuclear extracts from C2C12 myotubes treated with Dex for 6 h using oligonucleotides specific for the GRE within the mouse miR1–1 promoter as a probe together with anti-GR antibody. The supershift upon the addition of anti-GR antibody confirmed the interaction of GR with the GRE in the mouse miR1–1 promoter upon Dex treatment (lanes 2 and 3). M, image of agarose gel depicting the interaction of endogenous GR with endogenous miR1–1 promoter in C2C12 myotubes when treated with 100 μm Dex as analyzed through ChIP. N, real time qPCR analysis of miR1–1 primary transcript expression in ethanol, Dex, and Dex + RU486-treated C2C12 myotubes. The graph displays the fold-change in miR1–1 expression relative to ethanol treated controls. Values are mean ± S.E. (n = 3); **, p < 0.01. O, Western blot analysis of GR levels in nuclear extracts from conditioned CHO media (CCM)- and CMM-treated C2C12 myotubes.

FIGURE 3.

miR1 targets HSP70 during Dex-mediated muscle atrophy. A, representative images of C2C12 myoblast differentiated for 72 h and transfected with AntagomiR1 or NS miR followed by 100 μm Dex or ethanol treatments for 24 h. Myotube cultures were fixed and stained with Gill's hematoxylin and eosin. Scale bar represents 100 μm. B, the graph showing frequency distribution of myotube area (μm²) for AntagomiR1 and NS miR transfected C2C12 myotubes from ethanol-treated controls (n = 3). C and D, the graphs show frequency distribution of myotube area (μm²) of ethanol- or Dex-treated NS miR and AntagomiR1-transfected C2C12 myotubes (n = 3). E, the graph represents the average myotube area (μm²) of NS miR or AntagomiR1 transfected and Dex- or ethanol-treated C2C12 myotubes quantified from 10 random images in triplicate per treatment. Values represent mean ± S.E. (n = 3); **, p < 0.01. F, real time quantitative PCR analysis of miR1 expression in mRNA extracted from NS miR- and AntagomiR1-transfected C2C12 myotubes. Values are mean ± S.E. (n = 3) expressed as fold-difference relative to NS miR-transfected controls; **, p < 0.01. G, Western blot analysis of HSP70, pFOXO3, FOXO3, pAKT, AKT, MuRF1, and Atrogin-1 protein levels in NS miR- and AntagomiR1-transfected C2C12 myotubes treated with ethanol or Dex. The level of GAPDH was measured as a loading control. H and I, corresponding densitometric analysis of the levels of phosphorylated FOXO3 and AKT, expressed as a percentage of total FOXO3 and AKT, respectively, in NS miR- and AntagomiR1-transfected C2C12 myotubes following treatment with ethanol or Dex. J, corresponding densitometric analysis of the levels of HSP70, MuRF1, and Atrogin-1 in NS miR- and AntagomiR1-transfected C2C12 myotubes, following treatment with ethanol or Dex. Values represent mean ± S.E. (n = 3); *, p < 0.05. K, bioinformatics analysis, using RNAhybrid version 2.0, showing the complementary miR1 sequence in the murine Hsp70 gene. The matched base pairs are in bold and connected by a vertical line, and the G:U/U:G wobble is indicated by bold letters connected by dots. L, Western blot analysis of HSP70 levels in C2C12 myotubes treated for 24 h with Dex. GAPDH was used as the loading control. M, densitometric analysis of HSP70 protein levels in Dex-treated C2C12 myotubes; **, p < 0.01. N, Western blot analysis of HSP70 levels in C2C12 myotubes transfected with miR1 expression vector, empty vector, and miR1 expression vector with AntagomiR1. GAPDH was used as the loading control. O, densitometric analysis of HSP70 protein levels in 72-h differentiated myoblasts transfected with miR1 expression vector, empty vector, and miR1 expression vector with AntagomiR1; **, p < 0.01. P, assessment of pMIR reporter activity in C2C12 myotubes following overexpression of miR1. C2C12 myoblasts were co-transfected with a miR1 expression vector or empty vector and the pMIR reporter construct containing either a wild type (WT) or mutated (MT) miR1 seed sequence from the murine Hsp70 gene. pMIR reporter activity was normalized to Renilla luciferase and expressed as relative luminescence units (RLU). Each bar represents the mean ± S.E. (n = 3); *, p < 0.05.

FIGURE 4.

Inhibition of HSP70 results in decreased AKT and FOXO3 phosphorylation and enhanced myotube atrophy. A, Western blot analysis of pFOXO3, FOXO3, pAKT, and AKT protein levels in C2C12 myotubes treated without (dimethyl sulfoxide; DMSO) or with increasing concentrations (10 and 20 μm) of HSP70 antagonist (VER155008). The level of GAPDH was measured as a loading control. B and C, corresponding densitometric analysis of the levels of phosphorylated FOXO3 and AKT, expressed as a percentage of total FOXO3 and AKT, respectively, in control (DMSO) and VER155008-treated C2C12 myotubes. Values represent mean ± S.E. (n = 3); *, p < 0.05, and **, p < 0.01. D, Western blot analysis of MuRF1 and Atrogin-1 protein levels in control (DMSO) or VER155008-treated C2C12 myotubes. The level of GAPDH was measured as a loading control. E and F, corresponding densitometric analysis of the levels of Atrogin-1 and MuRF1 in control (DMSO) or VER155008-treated C2C12 myotubes. Values represent mean ± S.E. (n = 3); *, p < 0.05 with respect to DMSO-treated controls. G, representative images of C2C12 myotubes treated with DMSO or 10 μm VER155008, fixed, and stained with Gill's hematoxylin and eosin. Scale bar represents 100 μm. H and I, the graphs show frequency distribution of the myotube area (μm²) (H) and average myotube area (μm²) (I) of DMSO- or VER155008-treated C2C12 myotubes quantified from 10 random images in triplicate per treatment (n = 3). Values represent mean ± S.E. (n = 3); **, p < 0.01. J, representative images of C2C12 myotubes treated with ethanol or Dex, fixed, and stained with Gill's hematoxylin and eosin. The scale bar represents 100 μm. K, the graph shows frequency distribution of myotube area (μm²) of C2C12 myotubes treated with ethanol or Dex. L, the graph represents average myotube area (μm²) of ethanol- or Dex-treated C2C12 myotubes quantified from 10 random images in triplicate per treatment. Values represent mean ± S.E. (n = 3); **, p < 0.01. M, Western blot analysis of GR protein levels in nuclear extracts from C2C12 myotubes treated without (DMSO) or with increasing concentrations (10 and 20 μm) of VER155008. N, corresponding densitometric analysis of the levels of GR in C2C12 myotubes treated without (DMSO) or with increasing concentrations (10 and 20 μm) of VER155008. Values represent mean ± S.E. (n = 3); *, p < 0.05 and **, p < 0.01 when compared with DMSO control. O, real time quantitative PCR analysis of miR1 expression in control (DMSO) and VER155008-treated (10 and 20 μm) C2C12 myotubes. Values are fold-change mean ± S.E. (n = 3) when compared with DMSO-treated controls, **, p < 0.01. P, pAKT is associated with HSP70 in skeletal muscle. Western blot analysis of HSP70 (top) and pAKT (middle) co-immunoprecipitated with pAKT in protein lysates from BF muscle of saline and Dex-injected wild type mice. The level of α-tubulin was measured in input fraction (bottom) to ensure an equal amount of protein was used for co-immunoprecipitation. Q, graph showing densitometric analysis of HSP70 protein co-immunoprecipitated with pAKT antibody in BF muscle of saline- and Dex-treated WT mice. HSP70 densitometry values were normalized to α-tubulin and pAKT values and are depicted as a percent decrease after Dex treatment. Data represent mean ± S.E. (n = 3); *, p < 0.05.

FIGURE 5.

Overexpression of HSP70 during Dex-induced atrophy resulted in rescue of enhanced levels of atrophy-related proteins. A, real time qPCR analysis of miR1 expression in mRNA extracted from empty vector or HSP70 expression plasmid-transfected C2C12 myotubes after ethanol or Dex treatments. Values are mean ± S.E. (n = 3) expressed as fold-difference relative to empty vector-transfected controls, *, p < 0.05. B, Western blot analysis of pFOXO3, FOXO3, pAKT, AKT, MuRF1, and Atrogin-1 protein levels in empty vector and HSP70 expression plasmid-transfected C2C12 myotubes treated with ethanol or Dex. The level of GAPDH was measured as a loading control. C and D, corresponding densitometric analysis of the levels of phosphorylated FOXO3 and AKT, expressed as a percentage of total FOXO3 and AKT, respectively, in empty vector or HSP70 expression plasmid-transfected C2C12 myotubes following treatment with ethanol or Dex. E, corresponding densitometric analysis of the levels of MuRF1 and Atrogin-1 in empty vector or HSP70 expression plasmid-transfected C2C12 myotubes, following treatment with ethanol or Dex. Values represent mean ± S.E. (n = 3); *, p < 0.05.

FIGURE 6.

Mstn regulates miR1 during Dex-mediated muscle atrophy. A, Western blot analysis of pFOXO3, FOXO3, HSP70, pAKT, AKT, MuRF1, and Atrogin-1 levels in whole cell lysates from C2C12 myotubes treated with Dex (lane 2) and Dex + Ant1 (lane 3), as compared with ethanol-treated controls (lane 1). The level of GAPDH was measured as a loading control. B and C, corresponding densitometric analysis of the levels of phosphorylated FOXO3 and AKT, expressed as a percentage of total FOXO3 and AKT, respectively. Error bars represent mean ± S.E. (n = 3); **, p < 0.01, and *, p < 0.05. D, representative graph showing densitometric analysis of HSP70, MuRF1, and Atrogin-1 protein levels (normalized to GAPDH) in ethanol-, Dex-, and Dex + Ant1-treated C2C12 myotubes. Error bars represent mean ± S.E. (n = 3); **, p < 0.01. E, Western blot analysis of pFOXO3, FOXO3, HSP70, pAKT, and AKT protein levels in BF muscle collected from saline- or Dex-injected WT (lanes 1 and 2) and Mstn^−/− (lanes 3 and 4) mice. The level of GAPDH was measured as a loading control. F and G, corresponding densitometric analysis of the levels of phosphorylated FOXO3 and AKT, expressed as a percentage of total FOXO3 and AKT, respectively, in WT and Mstn^−/− mice after injection with saline or Dex. H, densitometric analysis of HSP70 protein levels in WT and Mstn^−/− muscle from saline or Dex-injected mice; *, p < 0.05 and **, p < 0.01. Error bars represent mean ± S.E. (n = 3).

FIGURE 7.

Proposed mechanism behind Dex-induced miR1-mediated skeletal muscle atrophy. Dex activates GR, which translocates to the nucleus and up-regulates miR1. Dex also up-regulates Mstn, which increases nuclear translocation of GR and miR1 expression. miR-1-mediated loss of HSP70 and enhanced activation of GR leads to further up-regulation of miR1 expression. Thus, increased miR1 expression may feedforward to further enhance its own expression. The reduced levels of HSP70 observed following Dex and Mstn treatment would further exacerbate skeletal muscle atrophy by decreasing phosphorylation of AKT, which results in activation of downstream proteosomal signaling components, such as FOXO3, MuRF1, and Atrogin-1. Arrows represent stimulation and blunt-ended lines represent inhibition.