Effect of chronic contractile activity on mRNA stability in skeletal muscle

Abstract

Repeated bouts of exercise promote the biogenesis of mitochondria by multiple steps in the gene expression patterning. The role of mRNA stability in controlling the expression of mitochondrial proteins is relatively unexplored. To induce mitochondrial biogenesis, we chronically stimulated (10 Hz; 3 or 6 h/day) rat muscle for 7 days. Chronic contractile activity (CCA) increased the protein expression of PGC-1alpha, c-myc, and mitochondrial transcription factor A (Tfam) by 1.6-, 1.7- and 2.0-fold, respectively. To determine mRNA stability, we incubated total RNA with cytosolic extracts using an in vitro cell-free system. We found that the intrinsic mRNA half-lives (t(1/2)) were variable within control muscle. Peroxisome proliferator-activated receptor-gamma, coactivator-1alpha (PGC-1alpha) and Tfam mRNAs decayed more rapidly (t(1/2) = 22.7 and 31.4 min) than c-myc mRNA (t(1/2) = 99.7 min). Furthermore, CCA resulted in a differential response in degradation kinetics. After CCA, PGC-1alpha and Tfam mRNA half-lives decreased by 48% and 44%, respectively, whereas c-myc mRNA half-life was unchanged. CCA induced an elevation of both the cytosolic RNA-stabilizing human antigen R (HuR) and destabilizing AUF1 (total) by 2.4- and 1.8-fold, respectively. Increases in the p37(AUF1), p40(AUF1), and p45(AUF1) isoforms were most evident. Thus these data indicate that CCA results in accelerated turnover rates of mRNAs encoding important mitochondrial biogenesis regulators in skeletal muscle. This adaptation is likely beneficial in permitting more rapid phenotypic plasticity in response to subsequent contractile activity.

Fig. 1.

Effect of chronic contractile activity (CCA) on peroxisome proliferator-activated receptor- coactivator-1α (PGC-1α) protein and mRNA levels. Animals were subjected to CCA for 7 days. A: cytochrome c oxidase (COX) enzyme activity of whole muscle. The EDL muscles of animals were subjected to CCA for 3 or 6 h/day for 7 days [n = 18; *P < 0.05, control (CTRL) vs. stimulation (STIM)]. B: PGC-1α protein, along with a loading control (aciculin), were measured in extracts from CTRL or STIM EDL muscles (n = 6; *P < 0.05, CTRL vs. STIM). C: PGC-1α and S12 mRNA transcripts were measured by RT-PCR from total RNA isolated from CTRL or STIM EDL muscles. A representative ethidium bromide (EtBr)-stained agarose gel is illustrated along with a graphical representation of the data (n = 10).

Fig. 2.

Effect of CCA on PGC-1α mRNA stability in skeletal muscle. A: total RNA (12 μg) from the tibualis anterior (TA) muscle was incubated with RNase-free isolation buffer alone for 0, 10, 20, and 40 min. PGC-1α and S12 mRNA transcripts were examined by RT-PCR and EtBr-stained agarose gel electrophoresis. B: total RNA (30 μg) was incubated with 20 μg of cytosolic proteins for 0, 5, 10, 20, and 30 min in the presence or absence of a ribonuclease inhibitor. Reactions in the absence of RNA or cDNA are also shown as negative controls. C: total RNA isolated from TA muscle was incubated with cytosolic proteins from CTRL or STIM EDL muscles of animals subjected to CCA for 7 days. After each time point, total RNA was reisolated and PGC-1α (n = 5; *P < 0.05, CTRL vs. STIM) along with an internal control S12 (n = 10) were examined by RT-PCR. A representative EtBr gel is illustrated along with a graphical representation of the data. Reactions in the absence of cytosolic proteins, as well as the absence of RNA or cDNA, are shown as positive and negative controls, respectively.

Fig. 3.

Effect of CCA on c-myc steady-state protein, mRNA levels, and mRNA stability. Animals were subjected to CCA for 7 days. A: c-myc protein, along with a loading control (aciculin), were measured in extracts from CTRL or STIM EDL muscles (n = 8; *P < 0.05, CTRL vs. STIM). B: c-myc and S12 mRNA transcripts were measured by RT-PCR from total RNA isolated from CTRL or STIM EDL muscles. A representative EtBr-stained agarose gel is illustrated along with a graphical representation of the data (n = 10). C: total RNA isolated from TA muscle was incubated with cytosolic proteins from CTRL or STIM EDL muscles of animals subjected to CCA for 7 days. After each time point, total RNA was reisolated, and c-myc (n = 4) along with an internal control S12 (n = 10) were examined by RT-PCR. A representative EtBr gel is illustrated along with a graphical representation of the data. Reactions in the absence of cytosolic proteins, as well as the absence of RNA or cDNA, are shown as positive and negative controls, respectively.

Fig. 4.

Effect of CCA on mitochondrial transcription factor A (Tfam) steady-state protein and mRNA levels and mRNA stability. Animals were subjected to CCA for 7 days. A: Tfam protein, along with a loading control (GAPDH), were measured in extracts from CTRL or STIM EDL muscles (n = 8; *P < 0.05, CTRL vs. STIM). B: Tfam and S12 mRNA transcripts were measured by RT-PCR from total RNA isolated from CTRL or STIM EDL muscles. A representative EtBr-stained agarose gel is illustrated along with a graphical representation of the data (n = 8). C: total RNA isolated from TA muscle was incubated with cytosolic proteins from CTRL or STIM EDL muscles of animals subjected to CCA for 7 days. After each time point, total RNA was reisolated and Tfam (n = 7, *P < 0.05 CTRL vs. STIM) along with an internal control S12 (n = 10) were examined by RT-PCR. A representative EtBr gel is illustrated along with a graphical representation of the data. Reactions in the absence of cytosolic proteins as well as the absence of RNA or cDNA are shown as positive and negative controls, respectively.

Fig. 5.

Effect of CCA on RNA-binding proteins, human antigen R (HuR), and ARE-RNA binding factor 1 (AUF1). Animals were subjected to CCA for 7 days. A: HuR, along with a loading control aciculin, were measured in extracts from CTRL or STIM EDL muscles (n = 8; *P < 0.05, CTRL vs. STIM). B: analyses and graphical representations of protein expression of p37^AUF1, p40^AUF1, p42^AUF1, p45^AUF1, and aciculin, the loading control are shown (n = 6, *P < 0.05, CTRL vs. STIM). C: distribution of various isoforms are shown. (n = 6; *P < 0.05 CTRL vs. STIM).

Fig. 6.

Effect of CCA on mRNA stability in skeletal muscle. Total RNA isolated from TA muscle was incubated with cytosolic proteins from CTRL or STIM EDL muscles of animals subjected to CCA for 7 days. After each time point, total RNA was reisolated and PGC-1α, c-myc, and Tfam along with an internal control S12 (n = 4–10) were examined by RT-PCR. * and †, Statistical significance from respective CTRLs and a main effect for all CTRL mRNAs, respectively.

Fig. 7.

PGC-1α 3′ downstream sequences contain AU-rich elements. A: schematic representation of the aligned sequence beginning from the stop codon (TAA) up to the consensus poly(A) signal (bold) and immediate downstream residues. For some species, this stretch of sequence is an amalgamation of the end and the beginning of two adjacent contigs, where bases may be missing. B: tabular result of PGC-1α sequence between the stop codon and the poly(A) signal comparing human (column A) against six mammals (column B) using the software ClustalW2. A pairwise score was calculated as the number of identities in the best alignment divided by the number of residues compared. Gap positions were excluded (30). C: sequence alignments of one of several putative HuR and AUF1 binding sites designated ARE (bold) within the PGC-1α 3′ downstream sequences suggest that this region is evolutionarily conserved.