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Transcriptional Regulation of Phospholamban Gene and Translational Regulation of SERCA2 Gene Produces Coordinate Expression of These Two Sarcoplasmic Reticulum Proteins during Skeletal Muscle Phenotype Switching

1995; Elsevier BV; Volume: 270; Issue: 19 Linguagem: Inglês

10.1074/jbc.270.19.11619

1083-351X

Ping Hu, Chang Yin, Ke-Min Zhang, Leon D. Wright, Todd E. Nixon, Andrew S. Wechsler, John A. Spratt, F. Norman Briggs,

Neurobiology and Insect Physiology Research

Chronic 1 Hz stimulation of the canine latissimus dorsi muscle produced a time-dependent switch from the fast-twitch to the slow-twitch phenotype. This included changes in the proteins of the sarcoplasmic reticulum. After 3 days of muscle stimulation, there was down-regulation of fast-twitch Ca-ATPase (SERCA1a) mRNA and induction of slow-twitch Ca-ATPase (SERCA2a) mRNA; most changes in both mRNAs were nearly complete after 14 days of stimulation. Although the induction of phospholamban mRNA began after 3 days of muscle stimulation, its up-regulation was not completed until the muscle had been stimulated for 42 days. The time course of expression of SERCA2a protein was very different from that of SERCA2a mRNA, suggesting that SERCA2 gene expression is regulated at the translational as well as the transcriptional level. The time course of expression of phospholamban protein closely followed that of phospholamban mRNA, suggesting that this gene is under transcriptional control. Thus coordinated expression of SERCA2a and phospholamban proteins is achieved via translational control of the SERCA2 gene and transcriptional control of the phospholamban gene. Chronic 1 Hz stimulation of the canine latissimus dorsi muscle produced a time-dependent switch from the fast-twitch to the slow-twitch phenotype. This included changes in the proteins of the sarcoplasmic reticulum. After 3 days of muscle stimulation, there was down-regulation of fast-twitch Ca-ATPase (SERCA1a) mRNA and induction of slow-twitch Ca-ATPase (SERCA2a) mRNA; most changes in both mRNAs were nearly complete after 14 days of stimulation. Although the induction of phospholamban mRNA began after 3 days of muscle stimulation, its up-regulation was not completed until the muscle had been stimulated for 42 days. The time course of expression of SERCA2a protein was very different from that of SERCA2a mRNA, suggesting that SERCA2 gene expression is regulated at the translational as well as the transcriptional level. The time course of expression of phospholamban protein closely followed that of phospholamban mRNA, suggesting that this gene is under transcriptional control. Thus coordinated expression of SERCA2a and phospholamban proteins is achieved via translational control of the SERCA2 gene and transcriptional control of the phospholamban gene. Chronic low frequency electrostimulation of fast-twitch muscle induces a switch to the slow-twitch phenotype (1Salmons S. Sreter F.A. Nature. 1976; 263: 30-34Crossref PubMed Scopus (472) Google Scholar). During the phenotype switch the expression of the fast-twitch SERCA1a, 1The abbreviations used are: SERCA1a, expressed by the post-fetal gene of fast-twitch skeletal muscle; SERCA, for any isoform: can designate the gene, mRNA, or protein; SERCA2a expressed by the post-fetal gene of slow-twitch/cardiac muscle; PAGE, Polyacrylamide gel electrophoresis; kb, kilobase(s). isoform of the calcium pump protein is down-regulated, and the slow-twitch SERCA2a isoform (2Briggs F.N. Lee K.F. Feher J.J. Wechsler A.S. Ohlendieck K. Camphell K. FEBS Lett. 1990; 259: 269-272Crossref PubMed Scopus (56) Google Scholar) and phospholamban (3Briggs F.N. Lee K.F. Wechsler A.S. Jones L.R. J. Biol. Chem. 1992; 267: 26056-26061Abstract Full Text PDF PubMed Google Scholar) are induced. Although the effect of chronic low frequency stimulation of the canine latissimus dorsi appeared to be complete after 42 days of stimulation (2Briggs F.N. Lee K.F. Feher J.J. Wechsler A.S. Ohlendieck K. Camphell K. FEBS Lett. 1990; 259: 269-272Crossref PubMed Scopus (56) Google Scholar, 3Briggs F.N. Lee K.F. Wechsler A.S. Jones L.R. J. Biol. Chem. 1992; 267: 26056-26061Abstract Full Text PDF PubMed Google Scholar, 4Ohlendieck K. Briggs F.N. Lee K.F. Wechsler A.S. Camphell K.P. Eur. J. Biochem. 1991; 202: 739-747Crossref PubMed Scopus (46) Google Scholar), its time course was not investigated either at the level of transcript or protein. The first step in the induction of the SERCA2a and phospholamban genes is transcription. The time course of the up-regulation of these transcripts during discontinuous stimulation in rabbits have been described by Leberer et al. (5Leberer E. Hartner K. Brandl C.J. Fujii J. Tada M. MacLennan D.H. Pette D. Eur. J. Biochem. 1989; 185: 51-54Crossref PubMed Scopus (69) Google Scholar). The mRNA must be translated in to complete the expression of these genes. Leberer et al. (5Leberer E. Hartner K. Brandl C.J. Fujii J. Tada M. MacLennan D.H. Pette D. Eur. J. Biochem. 1989; 185: 51-54Crossref PubMed Scopus (69) Google Scholar) did not measure the protein products of these genes, so the time of onset of translation of these transcripts is unknown. Although eukaryote gene expression is usually regulated primarily at the transcriptional level, there are a number of exceptions (6Kozak M. J. Cell Biol. 1991; 115: 887-903Crossref PubMed Scopus (1451) Google Scholar). Here we report changes in SERCA and phospholamban mRNAs and proteins during muscle transformation and consider the control of these genes during the process. The latissimus dorsi muscles were stimulated indirectly at 1 Hz via the three major branches of the thoracodorsal nerve for 3–70 days as described (4Ohlendieck K. Briggs F.N. Lee K.F. Wechsler A.S. Camphell K.P. Eur. J. Biochem. 1991; 202: 739-747Crossref PubMed Scopus (46) Google Scholar). At the end of the stimulation period the animals were sacrificed by an overdose of sodium pentobarbital. The latissimus dorsi, cardiac, and vastus intermedius muscles were rapidly removed, washed, chilled in ice-cold saline, and cleaned of superficial connective tissue and fat. Samples were then removed, frozen in liquid nitrogen, and stored at –80 °C. The left and right muscles were the experimental and control, respectively. Conditioning of the left muscle had no effect on the expression of SERCA or phospholamban genes of right muscle (data not shown). The number of animals studied at each time period ranged from 4 to 9. A microsomal fraction containing vesicles derived from longitudinal and junctional sarcoplasmic reticulum, transverse tubules, and sarcolemma was isolated as described by Ohlendieck et al. (4Ohlendieck K. Briggs F.N. Lee K.F. Wechsler A.S. Camphell K.P. Eur. J. Biochem. 1991; 202: 739-747Crossref PubMed Scopus (46) Google Scholar) and stored at –80 °C. Total RNA was isolated from 1.0 g of fresh chilled or frozen muscle by the guanidinium thiocyanate method (7Chomcyzynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159PubMed Google Scholar). SERCA1a and SERCA2a mRNAs were detected by Northern blots as described in Sambrook et al. (8Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Thirty μg of total RNA was size fractionated on 1.2% agarose gels containing 0.66 M formaldehyde, blotted onto nitrocellulose membrane, and hybridized with a SERCA probe randomly labeled with [α-32P]dCTP. Slot-blot and Northern blot membranes were washed with 2 × SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0 solution) and 0.1% sodium dodecyl sulfate at 55 and 42 °C, respectively, for 30 min. The different sizes of the SERCA1a and SERCA2a mRNAs (9Arai M. Otsu K. MacLennan D.H. Periasamy M. Am. J. Physiol. 1992; 262: C614-C620Crossref PubMed Google Scholar) allow separation and quantification on Northern blots. Total SERCA mRNA was measured on slot-blots using the same probe. Phospholamban mRNA was measured by slot-blot analysis. The mRNA levels were measured densitometrically from x-ray film using a Pharmacia UltraScan XL densitometer. The film was exposed for periods of time that gave linear responses to the amount of radioactivity on the blots. The phospholamban cDNA, obtained from a cardiac λgt 10 library (10Palmer C.J. Scott B.T. Jones L.R. J. Biol. Chem. 1991; 266: 11126-11130Abstract Full Text PDF PubMed Google Scholar), was a gift from Larry R. Jones at the University of Indiana. It is a 604-base pair fragment that starts 191 base pairs 5′ to the coding region, includes the coding region and extends into the 3′ region. The SERCA probe, a 1.7-kb SERCA cDNA, nucleotides 1,798–3,594 (11MacLennan D.H. Brandl C.J. Korczak B. Green N.M. Nature. 1985; 316: 696-700Crossref PubMed Scopus (805) Google Scholar), was a gift from Muthu Periasamy at the University of Cincinnati. It cross-hybridizes with SERCA1 and SERCA2 mRNAs (9Arai M. Otsu K. MacLennan D.H. Periasamy M. Am. J. Physiol. 1992; 262: C614-C620Crossref PubMed Google Scholar). The proteins of the membrane fraction were separated by SDS-PAGE on linear 7–15 % Polyacrylamide gels with 4% stacking gels as described (12Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). The electrophoresed proteins were stained with Coomassie Brilliant Blue G and scanned with a Pharmacia UltroScan XL densitometer. The SERCA1 monoclonal antibody IIH11, and SERCA2 monoclonal antibody IID8, were gifts from Dr. Kevin P. Camphell at the University of Iowa and are described (13Jorgensen A.O. Arnold W. Pepper D.R. Kahl S.D. Mandel F. Camphell K.P. Cell Motil. Cytoskeleton. 1988; 9: 164-174Crossref PubMed Scopus (79) Google Scholar). The phospholamban monoclonal antibody 2D12 was a gift from Dr. Larry Jones at the University of Indiana and is described in detail 114). Western blots for SERCA1a and SERCA2a were carried out as described (2Briggs F.N. Lee K.F. Feher J.J. Wechsler A.S. Ohlendieck K. Camphell K. FEBS Lett. 1990; 259: 269-272Crossref PubMed Scopus (56) Google Scholar). and the phospholamban immunoblots were as described in (3Briggs F.N. Lee K.F. Wechsler A.S. Jones L.R. J. Biol. Chem. 1992; 267: 26056-26061Abstract Full Text PDF PubMed Google Scholar). Two modifications of the methods were made: 1) gelatin was used as a blocking protein, and 2) the immunoblots were visualized using 12r'I-antimouse IgG as secondary antibody. Radioautographs were made from transblots and scanned with a Pharmacia UltroScan XL densitometer. Proteins were measured by the method of Lowry et al. (15Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using bovine serum albumin as standard. Multiple comparisons were evaluated statistically with one way analysis of variance (ANOVAI or ranked ANOVA, depending on the data. Dunnet's test was used for normally distributed data and Dunn's test for ranked data. Differences were accepted at the ρ < 0.05 level. Pearson product-moment correlation was used to measure the strength of association between pairs of variables; e.g. changes in protein versus changes in mRNA. The value, r, is a measure of the strength of the correlation; Ρ is the probability of the correlation arising by chance. Pette (16Pette D. Med. Sci. Sports Exer. 1984; 16: 517-528Crossref PubMed Scopus (144) Google Scholar) reported that 14 days of stimulation of rabbit fast-twitch muscle induced a 3-fold rise in total RNA, mostly due to an increase in ribosomal RNA. With continued stimulation the total RNA level fell to twice that of the control muscle. Pette did not report mRNAs changes. In the canine latissimus dorsi muscle the level of SERCA (SERCA1a + SERCA2a) mRNA did not change during the 70 days of stimulation, Fig. 1, whether it was measured as a ratio to a-actin or to the control muscle. To switch from the fast to the slow-twitch phenotype requires down-regulation of the SERCA1 gene and induction of the SERCA2 gene. Typical Northern blots showing changes in the expression of SERCA1a and SERCA2a mRNAs are shown in Fig. 2. The changes in SERCA1a mRNA and SERCA2a mRNAs for all muscles are shown in FIG. 3, FIG. 4. The pattern that emerges is change in both mRNAs starting after 3 days of muscle stimulation. By 14 days of muscle stimulation the down-regulation of SERCA1a was 70% complete (Fig. 3) and the up-regulation of SERCA2a 75% complete (Fig. 4).FIG. 3Effect of chronic conditioning on muscle SERCA1a mRNA and membrane SERCA1a protein. A, Western blots of SERCA1a membrane protein. l = left latissimus dorsi, r = right latissimus dorsi, e = stimulated muscle, c = control muscle. Period of stimulation 0, 7, 21, 42, and 70 days. B, SERCA1a mRNA and protein. Mean ± S.E. for the ratios of stimulated muscles to control muscles in four to nine animals at each time point. ●. SERCA1a mRNA; A SERCA1a protein. *, p < 0.05 that the difference between experimental and control is due to chance. When error bars are not shown they are smaller than the symbol used to identify values.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIG. 4Effect of chronic conditioning on muscle SERCA2a mRNA and membrane SERCA2a protein. A, Western blots of SERCA2a membrane protein. Period of stimulation, 0, 14, 21, 42, and 70 days. The abbreviations are as in Fig. 3. B, SERCA2a mRNA and protein. Mean i S.E. for the ratios of stimulated muscles to control muscles in four to nine animals at each time point. A. SERCA2a membrane protein; ●, SERCA2a mRNA. *. see legend to Fig. 3. Pearson product-moment correlation gave r = 0.727 and ρ = 0.064.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The change in SERCA1a protein in response to chronic muscle stimulation is compared with the change in SERCA1a mRNA in Fig. 3. The decline in SERCA1a mRNA starts after 3 days of stimulation, as does the decline in SERCA1a protein. Although the down-regulation of mRNA and protein start simultaneously and show identical profiles, the magnitude of the decline in protein was not as great as that in mRNA until the muscle had been stimulated for 42 days. The parallel decline in mRNA and protein suggests that the expression of the SERCA1 gene is under transcriptional control during the change to slow-twitch phenotype. The changes in SERCA2a mRNA and SERCA2a protein in response to chronic muscle stimulation are recorded in Fig. 4. The increase in SERCA2a mRNA was 60% complete by 7 days and 75% complete by 14 days of stimulation. SERCA2a protein increased continuously between 7 and 42 days of stimulation, while the level of SERCA2a mRNA remained unchanged, suggesting that SERCA2a protein expression is under translational control. The rises in SERCA2a protein and mRNA were not significantly correlated. Briggs et al. (3Briggs F.N. Lee K.F. Wechsler A.S. Jones L.R. J. Biol. Chem. 1992; 267: 26056-26061Abstract Full Text PDF PubMed Google Scholar) showed that chronic electrostimulation of the canine latissimus dorsi induces the expression of phospholamban protein, a sarcoplasmic reticulum protein found in some slow-twitch but not fast-twitch muscles (3Briggs F.N. Lee K.F. Wechsler A.S. Jones L.R. J. Biol. Chem. 1992; 267: 26056-26061Abstract Full Text PDF PubMed Google Scholar, 17Jorgensen A.O. Jones L.R. J. Biol. Chem. 1986; 261: 3775-3781Abstract Full Text PDF PubMed Google Scholar). The stimulation induced expression of phospholamban mRNA and protein in the canine latissimus dorsi is shown in Fig. 5. The phospholamban mRNA began to rise with 3 days and phospholamban protein with 7 days of stimulation. The correlation between the rises in protein and in mRNA was highly significant. Although the up-regulation of SERCA2a and phospholamban mRNAs have distinctly different patterns, the patterns of expression of the two proteins are very similar, Fig. 6. It is clear that the two proteins are coexpressed during transformation of fast-twitch to slow-twitch muscle. The Pearson product-moment correlation gave a coefficient of 0.970 and a ρ value < 0.001. In the transforming canine skeletal muscle SERCA2a and phospholamban proteins were expressed simultaneously (Fig. 6). The mRNAs of these two genes were not coordinately expressed during the transformation, Fig. 4 versus 5. The induction and up-regulation of SERCA2a mRNA was completed before that of phospholamban mRNA. The delay between expression of SERCA2a mRNA and expression of SERCA2a protein, Fig. 4, requires translational control. The parallel increase in phospholamban mRNA and protein suggests that this gene is regulated at the transcriptional level. Thus, to achieve the coordinate expression of SERCA2a protein and phospholamban during transformation of skeletal muscle phenotype the expression of these genes were regulated at different levels; the SERCA2a gene at the translational and the phospholamban gene at the transcriptional level. To get such precise regulation of gene expression using two levels of control must require some mechanism to coordinate the expression of SERCA2a and phospholamban proteins. The data recorded in Fig. 6, showing that phospholamban expression precedes that of SERCA2a, may mean that phospholamban stimulates the translation of the SERCA2a transcript. A number of physiological interventions induce switches in muscle fiber type. In the unweighted rat soleus muscle (18Schulte L.M. Navarro J. Kandarian S.C. Am. J. Physiol. 1993; 264: C1308-C1315Crossref PubMed Google Scholar), increases in SERCA1a mRNA and protein proceeded with similar time courses, suggesting that up-regulation of the SERCA1a gene is transcriptionally regulated. Denervation of the rat extensor digitorum longus produces a rapid down-regulation in SERCA1a mRNA and a much slower and less marked down-regulation of SERCA1a protein. This slow decline in SERCA1a protein was attributed to the stability of the SERCA1a protein (19Kirchberger M.A. Tada M. J. Biol. Chem. 1976; 251: 725-729Abstract Full Text PDF PubMed Google Scholar), and not to translational control, an alternative interpretation. In the stimulated canine latissimus dorsi the fall in SERCA1a mRNA was paralleled by a fall in SERCA1a protein, suggesting that in this model SERCA1a protein expression is under transcriptional control. The down-regulation of SERCA1 gene expression appears to be under transcriptional control. Twenty-eight days after unloading the rat soleus muscle (18Schulte L.M. Navarro J. Kandarian S.C. Am. J. Physiol. 1993; 264: C1308-C1315Crossref PubMed Google Scholar), SERCA2a mRNA increased and SERCA2a protein decreased, indicating that under this condition SERCA2 gene expression is under translational control. This response is similar to that observed in the canine latissimus dorsi, which also showed that SERCA2a mRNA and SERCA2a protein levels do not change in close concert and suggests that for these two types of muscle stimulation the expression of SERCA2a protein is under translational control. The expression of SERCA2a and phospholamban can be differentially regulated. For example, SERCA2a is expressed before phospholamban during skeletal muscle development (9Arai M. Otsu K. MacLennan D.H. Periasamy M. Am. J. Physiol. 1992; 262: C614-C620Crossref PubMed Google Scholar). Phospholamban is expressed in the slow-twitch muscles of the rabbit (9Arai M. Otsu K. MacLennan D.H. Periasamy M. Am. J. Physiol. 1992; 262: C614-C620Crossref PubMed Google Scholar, 19Kirchberger M.A. Tada M. J. Biol. Chem. 1976; 251: 725-729Abstract Full Text PDF PubMed Google Scholar) and dog (3Briggs F.N. Lee K.F. Wechsler A.S. Jones L.R. J. Biol. Chem. 1992; 267: 26056-26061Abstract Full Text PDF PubMed Google Scholar, 17Jorgensen A.O. Jones L.R. J. Biol. Chem. 1986; 261: 3775-3781Abstract Full Text PDF PubMed Google Scholar), but not rat (20Kandarian S.C. Peters D.G. Taylor J.A. Williams J.H. Am. J. Physiol. 1994; 266: C1190-C1197Crossref PubMed Google Scholar). In animals treated with excess thyroid hormone (21Nagai R.N. Zarain-Herzberg A. Brandl C.J. Fuji J. Tada M. MacLennan D.H. Alpert N.R. Periasamy M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2966-2970Crossref PubMed Scopus (214) Google Scholar), there is an increase in cardiac muscle SERCA2a mRNA and a decrease in phospholamban mRNA, indicating that thyroid hormone produces opposite effects on cardiac SERCA2a and phospholamban gene expression. In contrast to the studies in the dog reported here, where SERCA2a and phospholamban mRNA rose with different time courses, Leberer et al. (5Leberer E. Hartner K. Brandl C.J. Fujii J. Tada M. MacLennan D.H. Pette D. Eur. J. Biochem. 1989; 185: 51-54Crossref PubMed Scopus (69) Google Scholar) have reported (for the one animal showing the greatest transformation at each time point) that SERCA2a and phospholamban mRNA increased with similar time courses, indicating that in the rabbit undergoing discontinuous stimulation the transcription of these two genes may be under a common control mechanism. The studies cited above indicate that SERCA2a and phospholamban can undergo independent regulation. On the other hand under some circumstances the transcription of these two genes can be coordinated.