Involvement of microRNAs in the regulation of muscle wasting during catabolic conditions

Ricardo José Soares, Stefano Cagnin, Francesco Chemello, Matteo Silvestrin, Antonio Musaro, Cristiano De Pitta, Gerolamo Lanfranchi, Marco Sandri, Ricardo José Soares, Stefano Cagnin, Francesco Chemello, Matteo Silvestrin, Antonio Musaro, Cristiano De Pitta, Gerolamo Lanfranchi, Marco Sandri

Abstract

Loss of muscle proteins and the consequent weakness has important clinical consequences in diseases such as cancer, diabetes, chronic heart failure, and in aging. In fact, excessive proteolysis causes cachexia, accelerates disease progression, and worsens life expectancy. Muscle atrophy involves a common pattern of transcriptional changes in a small subset of genes named atrophy-related genes or atrogenes. Whether microRNAs play a role in the atrophy program and muscle loss is debated. To understand the involvement of miRNAs in atrophy we performed miRNA expression profiling of mouse muscles under wasting conditions such as fasting, denervation, diabetes, and cancer cachexia. We found that the miRNA signature is peculiar of each catabolic condition. We then focused on denervation and we revealed that changes in transcripts and microRNAs expression did not occur simultaneously but were shifted. Indeed, whereas transcriptional control of the atrophy-related genes peaks at 3 days, changes of miRNA expression maximized at 7 days after denervation. Among the different miRNAs, microRNA-206 and -21 were the most induced in denervated muscles. We characterized their pattern of expression and defined their role in muscle homeostasis. Indeed, in vivo gain and loss of function experiments revealed that miRNA-206 and miRNA-21 were sufficient and required for atrophy program. In silico and in vivo approaches identified transcription factor YY1 and the translational initiator factor eIF4E3 as downstream targets of these miRNAs. Thus miRNAs are important for fine-tuning the atrophy program and their modulation can be a novel potential therapeutic approach to counteract muscle loss and weakness in catabolic conditions.

Keywords: Gene Expression; MicroRNA (miRNA); Muscle; Muscle Atrophy; Pathology; Protein Degradation; Signal Transduction; Skeletal Muscle; Transcription.

© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Figures

FIGURE 1.
FIGURE 1.
miRNA expression is deregulated in different models of skeletal muscle atrophy, miRNA-21 and miRNA-206 being the two most induced miRNAs in denervation.A, weight of gastrocnemius muscle in each atrophic condition studied. Data are shown as mean ± S.E.; n > 3 muscles per condition. *, p < 0.05. B, cluster analysis of atrophy conditions. Euclidean distance was used in the clustering. Denervated mice cluster together, separately from starvation, diabetes, and cancer cachexia conditions. Den3, denervation 3 days; Den7, denervation 7 days; Den14, denervation 14 days; STV24, starvation 24 h; STV48, starvation 48 h; STZ, diabetes induced by streptozotocin (STZ). C, heat map of miRNA differentially expressed between the different times of denervation. The indicated miRNAs functionally characterized are in bold. Color code is indicated as log2 (atrophy/control). Gray box represents data not available because under limit of detection. D, miRNA-206 expression levels are increased at different time points of denervation. qRT-PCR of miRNA expression levels in gastrocnemius of control and denervated animals at different time points. Data are shown as mean ± S.E.; n > 3 muscles per condition. *, p < 0.05. E, miRNA-21 expression levels are increased at different time points of denervation. qRT-PCR of miRNA expression levels in gastrocnemius of control and denervated animals at different time points. Data are shown as mean ± S.E.; n > 3 muscles per condition. *, p < 0,05. F, 48 h of fasting increases the miRNA-206 expression level. qRT-PCR of miRNA expression levels in gastrocnemius of fed and starved animals for 48 h. Data are shown as mean ± S.E.; n > 3 muscles per condition. *, p < 0.05. G, 48 h of fasting decreases the miRNA-21 expression level. qRT-PCR of miRNA expression levels in gastrocnemius of fed and starved animals for 48 h. Data are shown as mean ± S.E.; n > 3 muscles per condition. **, p < 0.01. H and I, diabetes does not alter the expression levels of miRNA-206 and miRNA-21. qRT-PCR of miRNA expression levels in gastrocnemius of vehicle or streptozotocin-injected animals (7 days after injection). Data are shown as mean ± S.E.; n > 3 muscles per condition. J, miRNA-206 and -21 luciferase sensors were inhibited in denervated muscles. Data are shown as mean ± S.E. n > 4 muscles per condition; *, p < 0.05.
FIGURE 2.
FIGURE 2.
miRNA-206 and -21 are selectively induced in atrophying muscles.A, miRNA-206, and B, miRNA-21 expression levels are increased after 15 days of denervation in both fast and slow muscles. Quantitative RT-PCR analysis on miRNA expression levels in TA and soleus muscles of control and denervated animals. Data are shown as mean ± S.E. n > 3 independent experiments; *, p < 0.05; **, p < 0.01. C, expression of miRNA-133b, the co-cistronic partner of miRNA-206, is not up-regulated during denervation. Quantitative RT-PCR analysis on miRNA expression levels in gastrocnemius of control and denervated animals at different time points. Data are shown as mean ± S.E., n = 3 independent experiments; *, p < 0.05. D, expression level of Tmem49, the host gene of miRNA-21, during 24 h fasting and 7 days of denervation. Quantitative RT-PCR analysis on mRNA expression levels in gastrocnemius muscles. Data are shown as mean ± S.E., n = 3 independent experiments.
FIGURE 3.
FIGURE 3.
Over-expression of miRNA-206 and miRNA-21 in vivo alters adult muscle fiber size.A and B, in vivo transfection of co-cistronic vectors expressing (A) miRNA-206 or (B) miRNA-21 together and GFP. Adult TA were transfected and collected 10 days after transfection. Cryosections were stained with anti-dystrophin, to identify the plasma membrane of the myofibers, and counterstained with Hoechst. Images were merged to demonstrate that the GFP signal lay under the sarcolemma and not in interstitial cells. C and D, qRT-PCR of miRNA-206 and miRNA-21 expression levels on transfected TA muscles, respectively. Data are shown as mean ± S.E., n > 3 muscles per condition, *, p < 0.05. E and F, qRT-PCR of miRNA-206 and miRNA-21 expression levels on C2C12 myoblasts transfected for 48 h with miRNA overexpressing vectors. Data are shown as mean ± S.E., n = 3 independent experiments; *, p < 0.05; **, p < 0.01. G and H, in vivo transfection of miRNA-206 or miRNA-21 expressing vectors produces functional mature miRNAs. Adult tibialis anterior muscles were co-transfected with miRNA-expressing vector and with the miRNA luciferase sensor for each miRNA. A Renilla luciferase vector was co-transfected to normalize for transfection efficiency. 7 days after transfection, muscles were collected and the luciferase/Renilla ratio was determined. Data are shown as mean ± S.E. n > 3 muscles per condition; *, p < 0.05. I, in vivo transfection of miRNA-206 induces skeletal muscle atrophy. Adult TA muscles were transfected and collected 10 days after transfection. Cross-sectional area of transfected fibers, identified by the GFP, was measured. Data are shown as mean ± S.E., n > 800 per each muscle, at least 3 muscles per condition were examined; **, p < 0.01. J, in vivo transfection of miRNA-21 does not affect skeletal muscle fiber size. Transfection and analyses were performed as described in I. K, over-expression of miRNA-206 in denervated muscles aggravates the atrophic phenotype. Adult tibialis anterior muscles were transfected with DNA vector over-expressing the mature miRNA-206 and simultaneously denervated. Muscles were collected 10 days after transfection. Analyses were performed as described in I, **, p < 0.01. L, over-expression of miRNA-21 in denervated muscles aggravates the atrophic phenotype. Adult tibialis anterior muscles were transfected with DNA vector over-expressing the mature miRNA-21. Muscles were collected 10 days after transfection. Analyses were performed as described in I; *, p < 0.05.
FIGURE 4.
FIGURE 4.
Inhibition of miRNA-206 in vivo induces skeletal muscle hypertrophy.A and B, transfection of C2C12 cells with constructs expressing miRNA sponges efficiently inhibits miRNA-206 or miRNA-21. C2C12 myoblasts were transfected with miRZIP and simultaneously with the luciferase sensor for the respective miRNA. 24 h after transfection luciferase levels were measured. Data are shown as mean ± S.E., n = 4 independent experiments, **, p < 0.01. C, in vivo inhibition of miRNA-206 induces skeletal muscle hypertrophy. Adult TA muscles were transfected with miRZIP-206. 10 days after electroporation muscles were collected and cryosectioned. Cross-sectional area of transfected fibers, identified by the presence of GFP, was measured. Data are shown as mean ± S.E.; n > 700 for each muscle, at least 3 muscles per condition were analyzed; **, p < 0.01. D, in vivo inhibition of miRNA-21 does not affect skeletal muscle fiber size. Adult TA muscles were transfected with miRZIP-21. 10 days after electroporation muscles were collected and cryosectioned. Muscles were analyzed as described in C. E, inhibition of miRNA-206 in denervated muscles partially protects from atrophy. Adult TA muscles were transfected miRZIP-206 and simultaneously denervated and collected 10 days after electroporation. Cross-sectional area were quantified as described in C. **, p < 0.01. F, inhibition of miRNA-21 in denervated muscles partially protects from atrophy. Adult TA muscles were transfected with miRZIP-21 and simultaneously denervated. Muscles were collected after 10 days and the cross-sectional area was quantified as described in C. **, p < 0.01.
FIGURE 5.
FIGURE 5.
Approach used to identify biologically relevant targets of miRNA-206 and miRNA-21.A, procedure of computational prediction of miRNA targets. miRNA and gene expression profiles were defined in atrophic muscles of the same mice. Target prediction was performed by the PITA algorithm, then expression data were integrated to improve the detection of functional correlation between miRNA and mRNA expression profiling. B, Venn diagram shows the number of down-regulated genes (blue circle) in the denervated condition (after 7 and 14 days) that are targets of miR-21 (green circle) and miR-206 (yellow circle). C, denervation down-regulates the expression levels of YY1, eIF4E3, and Pdcd10. qRT-PCR confirmed the down-regulation of YY1, eIF4E3, and Pdcd transcripts after denervation. Data are shown as mean ± S.E. n = 3 muscles; *, p < 0.05; **, p < 0.01. D, Western blot for YY1 and Ago2 in TA muscles of control and denervated condition at 7 and 14 days, and E, densitometric quantification. The YY1-V5 construct was transfected in C2C12 cells and the lysate was used as positive control for the Western blot. F–H, adult tibialis anterior muscles were electroporated with DNA vectors encoding for the luciferase sensor of YY1–3′UTR, eIF4E3–3′UTR, or PDCD10–3′UTR together with Renilla luciferase vector to normalize for electroporation efficiency. Simultaneously, the left hindlimb was denervated. 15 days after electroporation/denervation, muscles were collected and luciferase/Renilla ratio was determined. Data are shown as mean ± S.E. n > 3 independent experiments; *, p < 0.05; **, p < 0.01.
FIGURE 6.
FIGURE 6.
In vivo luciferase assays confirm the miRNA-mediated regulation of YY1, eIF4E3, and Pdcd10.A, YY1 is a target of miRNA-21. Adult TA muscles were co-transfected with expression plasmid for miRNA-206 or miRNA-21 and the construct encoding for the luciferase sensor of YY1–3′UTR. A Renilla luciferase vector was co-transfected to normalize for electroporation efficiency. Muscles were collected 7 days after electroporation and the luciferase/Renilla ratio determined. Data are shown as mean ± S.E., n > 3 muscles per condition; **, p < 0.01. B, eIF4E3 is a target of both miRNA-206 and miRNA-21. Adult TA muscles were transfected and processed as described in A; **, p < 0.01. C, Pdcd10 is a target of both miRNA-206 and miRNA-21. Adult TA muscles were transfected and processed as described in A; **, p < 0.01. D, mutation of the miRNA-206 binding site on the 3′UTR of eIF4E3 partially prevents miRNA-dependent inhibition. C2C12 myoblasts were co-transfected with control or mutated eIF4E3–3′UTR together with scramble or miRNA-206 expressing vector. Luciferase/Renilla ratio was determined 24 h after transfection. Data are shown as mean ± S.E., n = 3 independent experiments; **, p < 0.01; #, p < 0.05. E, mutation of the miRNA-21 binding sites on the 3′UTR of YY1 and eIF4E3 partially prevent miRNA-dependent inhibition. C2C12 myoblasts were co-transfected with the control or mutated version of the 3′UTR of YY1 or eIF4E3 together with scramble or miRNA-21 expressing vector. Luciferase/Renilla ratio was determined 24 h after transfection. Data are shown as mean ± S.E., n = 3 independent experiments; *, p < 0.05; **, p < 0.01; #, p < 0.05; ##, p < 0.01.
FIGURE 7.
FIGURE 7.
A, over-expression of miRNA-206 or miRNA-21 directly regulates the mRNA of YY1 and eIF4E3. C2C12 myoblasts were transfected with miRNA-206 or miRNA-21 expressing vector. RNA was extracted 48 h after transfection and the expression levels of YY1 and eIF4E3 were analyzed by qRT-PCR. Data are shown as mean ± S.E., n = 3 independent experiments; *, p < 0.05; **, p < 0.01. B, densitometric quantification of the Western blot for YY1 in C2C12 myoblasts over-expressing negative control or miRNA-21. Data are shown as mean ± S.E., n = 3 independent experiments.

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