Acute systemic administration of the β2-adreneroreceptor agonist, formoterol, induces widespread changes in gene expression in skeletal muscle
The entire data set is available via Gene Expression Omnibus (accession number GSE15793). Expression profiling was performed on 16 mice in total using 46K Illumina Sentrix BeadArray chips. Skeletal muscle samples from 16 independent animals were removed at 1 and 4 hours following a single i.p.
injection of the β2
-AR agonist, formoterol, or saline (vehicle control). Each timepoint consisted of eight animals with four saline and four formoterol treated animals. The tibialis anterior muscle was chosen for all analyses as it contains predominantly type II fibers, and (in rodents) is known to exhibit marked increases in protein content and lean mass (hypertrophy) in response to β-AR agonist administration [31
Using a p value cutoff of p < 0.05 (see methods for full statistical analysis) and a fold change cutoff of 1.85, at one hour following formoterol administration, 23 probes were significantly altered and 112 probes were significantly altered at four hours. Significant annotated genes from both timepoints are shown in table . Significant non-annotated genes (Riken cDNAs and hypothetical proteins) are included in Table .
Significant differentially expressed genes in skeletal muscle after acute systemic administration of β2-AR agonist
Significant differentially expressed non-annotated genes in skeletal muscle after acute systemic administration of β2-AR agonist
Functional categorization of genes differentially regulated by β2-AR activation
Genes presented in table were grouped according to their potential relevant function in skeletal muscle. The potential relevant function is based on the authors' opinion gained from a combination of Illumina Gene Ontology classifications, Ingenuity Pathway Analysis http://www.ingenuity.com
, Online Mendelian Inheritance in Man (OMIM; http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim
), and AceView http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/
searches. The Illumina BeadArray expression analysis revealed significant changes in the expression of genes in several functional categories at 1 and 4 hours following formoterol administration, including genes involved in skeletal muscle hypertrophy/growth, myoblast differentiation, metabolism, circadian rhythm, transcription, histones, oxidative stress, angiogenesis, solute carriers, apoptosis, cell cycle, cancer, DNA repair, and the ubiquitin-proteasome system.
Validation of differential gene expression by quantitative RT-PCR
The expression of 16 genes (Stat3, Idb1, Smad1, Smad3, Hk2, Pdk4, Sorbs1, Pgc1α, Lipin1α, FoxO1, Ucp3, Nfil3, Dbp, Nurr1, Crem, and Cebpb) that were identified as differentially expressed by Illumina beadarray analysis (Table ) and associated with the regulation of skeletal muscle mass, circadian rhythm and metabolism were validated and examined in greater detail following acute (1-24 h) and chronic (1-28 days) formoterol administration via quantitative RT-PCR (qRT-PCR; Figures , , , and ). All qRT-PCR analyses were performed on an independent/different set of formoterol treated mice (n = 5 per timepoint) than the group used in the Illumina BeadArray study. All 16 gene analyzed by qRT-PCR on independent animals closely mirrored the Illumina changes at both timepoints, highlighting the robust nature of the Illumina platform.
Figure 1 Acute systemic administration of formoterol alters the expression of genes associated with muscle growth and differentiation at multiple timepoints. Quantitative RT-PCR was used to assay the expression of A. Stat3, B. Idb1, C. Smad1, D. Acvr2b, E. Smad3 (more ...)
Figure 2 Chronic systemic administration of formoterol alters the expression of genes associated with skeletal muscle hypertrophy and myogenesis at multiple timepoints. Quantitative RT-PCR was used to assay the expression of A. Stat3, B. Idb1, C. Smad1, D. Acvr2b (more ...)
Figure 3 Acute systemic administration of formoterol alters the expression of genes associated with metabolism. Quantitative RT-PCR was used to assay the expression of A. Hk2, B. Pdk4, C. Sorbs1, D. Pgc1α, E.Lipin1α, F. FoxO1, and G.Ucp3 mRNAs (more ...)
Figure 4 Chronic systemic administration of formoterol alters the expression of genes associated with metabolism. Quantitative RT-PCR was used to assay the expression of A. Hk2, B. Pdk4, C. Sorbs1, D. Pgc1α, E. Lipin1α, F. FoxO1, and G.Ucp3 mRNAs (more ...)
Figure 5 Acute and chronic systemic administration of formoterol alters the expression of genes associated with circadian rhythm and transcriptional regulation. Quantitative RT-PCR was used to assay the expression of A. Nfil3, B. Dbp, C. Nurr1, D. Creb, and E (more ...)
Formoterol administration alters the expression of genes associated with skeletal muscle hypertrophy and differentiation: attenuation of myostatin signaling
Several differentially expressed genes associated with the regulation of muscle differentiation and mass (identified from the Illumina BeadArray) were examined over an acute and chronic time course of formoterol administration using qRT-PCR. In addition we also examined the expression of myostatin and the myostatin receptor, activin receptor IIB (Acvr2b) that are critical modulators of muscle mass. We examined these genes using qRT-PCR as Acvr2b was down-regulated at 4 hour in the Illumina BeadArray, however it did not pass statistical analysis (data not shown). Tibialis anterior muscle was isolated from groups (n = 5) of male mice, treated with either the specific β2-AR agonist treatment or saline (vehicle control) and assayed at 0, 1, 4, 8 and 24 h post treatment. Significant changes in expression at one or more timepoints were observed in the mRNAs encoding signal transducer and activator of transcription 3 (Stat3; Figure ), inhibitor of DNA binding 1 (Idb1; Figure ), small mothers against decapentaplegic homolog 1 (Smad1; Figure ), Acvr2b (Figure ), and small mothers against decapentaplegic homolog 3 (Smad3; Figure ). We did not observe any significant changes in myostatin expression, after acute β2-AR agonist treatment (Figure )
To examine chronic changes induced by formoterol administration, qRT-PCR was used to examine expression of genes from chronically treated mice (after 1, 7 and 28 days of agonist treatment). Similar to acute timepoints, tibialis anterior was isolated from groups (n = 5) of male mice, treated daily with either the specific β2-AR agonist formoterol or saline (vehicle control) and assayed at 0, 1, 7 and 28 days of treatment. Chronic formoterol administration was associated with a significant attenuation in the expression of the mRNAs encoding Idb1 (Figure ), Smad3 (Figure ) and myostatin (Figure ) after 7 or 28 days. No significant changes were observed following chronic formoterol administration in the expression of the mRNAs encoding Stat3 (Figure ), Smad1 (Figure ), and Acvr2b (Figure ), despite significant repression following acute formoterol administration.
To examine the effect of chronic β2-AR agonist treatment on critical regulators of the myostatin signaling pathway we examined the levels of the Myostatin precursor (pro-Myostatin), Smad3, phosphorylated Smad3 relative to Gapdh. We assayed levels by Western blotting analysis of tibialis anterior muscle (contralateral to muscle used for qRT-PCR analysis) following 28 days of formoterol/saline administration in four animals for each treatment (Figure ). Consistent with the qRT-PCR data, at the protein level, pro-Myostatin appears subtley (but consistently) suppressed following 28 days of formoterol administration. In concordance, the levels of Smad3 phosphorylation following the chronic formoterol administration are also reduced (in 3 out of 4 mice), while total Smad3 appears unchanged.
In summary, β2-adrenergic stimulation mediates changes in the expression of several genes associated with myostatin signaling, and the regulation of muscle mass (Figure ).
Formoterol administration alters the expression of genes associated with metabolism and circadian rhythm
Many genes that regulate and/or are directly involved in metabolism are regulated in a circadian manner. The Illumina BeadArray study identified differential expression of several genes involved in these pathways. Consequently, we utilized qRT-PCR to validate the differential expression of these genes after acute or chronic administration of fomoterol (vs vehicle) in tibialis anterior as detailed above. Significant expression changes at one or more timepoints were observed in several genes involved in metabolism, including, hexokinase 2 (Hk2
; Figure ), pyruvate dehydrogenase kinase 4 (Pdk4
; Figure ), sorbin and SH3 domain containing 1 (Sorbs1
; Figure ), PPARγ coactivator 1 alpha (Pgc1α
; Figure ), Lipin1α
(Figure ); forkhead box O1 (FoxO1
; Figure ), and uncoupling protein 3 (Ucp3
; Figure ). In the context of crosstalk between β-AR signaling and Nor-1
(NR4A3) signaling in skeletal muscle, we have previously identified and examined the induction of Pdk4
, Pgc1α, FoxO1
, and Lipin1α
over following acute β-AR activation [28
Chronic formoterol administration significantly altered the expression of FoxO1 (Figure ) and Ucp3 (Figure ) at 7 and 28 days respectively, while Hk2 (Figure ), Pdk4 (Figure ), Sorbs1 (Figure ), Pgc1α (Figure ), and Lipin1α (Figure ) were not significantly altered.
The expression of two peripheral tissue regulators of circadian rhythm, albumin D-box binding protein (Dbp), and nuclear factor interleukin 3 regulated (Nfil3) were significantly dysregulated by both acute and chronic formoterol administration (Figures and ).
In summary, formoterol administration mediated the significant modulates of several metabolic genes (for example Pgc1α, Lipin1α, Pdk4, FoxO1, Hk2, Ucp3, Sorbs1 etc) associated with the transient induction of oxidative metabolism, particularly following acute stimulation of β-AR's. Interestingly, the expression of these genes was normalized 24 h post treatment, and remained at control levels throughout the 28 days of formoterol administration. In addition, acute and chronic β2-AR agonist treatment significantly regulates the expression of two critical regulators of circadian cycling.
Altered transcriptional regulation following formoterol administration
We have previously demonstrated that β-AR agonists markedly increased the expression of the NR4A subgroup (Nur77
) of nuclear receptor transcription factors in skeletal muscle [27
]. From the Illumina BeadArray, ten transcription factors (not placed in other categories) were induced by formoterol 1 and 4 h post-administration (Table ). At one or more timepoints, acute administration of formoterol significantly induced nuclear receptor related 1 protein (Nurr1
; Figure ), cAMP responsive element modulator (Crem
; Figure ), and CCAAT/enhancer binding protein β (Cebpb
; Figure ). In contrast, Crem
was the only transcription factor to remain elevated throughout the 28 day formoterol administration period (Figure compared to Figures and ).