A Gene with Multiple Transcriptsb

Gene 353 (2005) 31 – 40 www.elsevier.com/locate/gene

Characterization and functional significance of myotrophin: A gene with multiple transcriptsB

Gautam Adhikary1,2, Sudhiranjan Gupta1, Parames Sil3, Yasser Saad, Subha Sen*

Department of Molecular Cardiology, NB 50, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, United States

Received 1 December 2004; received in revised form 7 March 2005; accepted 22 March 2005

Received by J.A. Engler

Abstract

The underlying mechanism for the development of cardiac hypertrophy that advances to heart failure is not known. Many factors have been implied to play a role in this process. Among others, we have isolated and identified myotrophin, a factor that stimulates myocytes growth, from spontaneously hypertensive rat (SHR) heart and patients with dilated cardiomyopathy. The gene encoding myotrophin has been cloned and expressed in E. coli. Recently, myotrophin gene has been mapped and shown to be a novel gene localized in human chromosome 7q-33. To define the characteristics of each transcript and its pathophysiological significance, we examined transcripts of myotrophin in SHR heart during progression of hypertrophy. Northern blot analysis of myotrophin mRNA showed multiple transcripts. We isolated and characterized various myotrophin cDNA clones corresponding to the multiple transcripts by 5V ‘‘stretch plus’’ rat heart cDNA library screening. Sequence analysis of these cDNA clones indicates that each clone has a unique 5V UTR and multiple 3V UTR with varying lengths, repeated ATTTA motifs and many polyadenylation signals. In vitro transcripts generated from all these myotrophin-specific cDNA clones translate in vitro to a 12-kD protein. Among pathophysiological significance, we determined mRNA expression in 9 days old, 3 weeks old and 31 weeks old and observed a linear increased during the progression of hypertrophy. In WKY, this mRNA level remained the same throughout the growth and development of hypertrophy. Our data strongly suggest that myotrophin appears to be a candidate gene for cardiac hypertrophy and heart failure. D 2005 Elsevier B.V. All rights reserved.

Abbreviations: SHR, spontaneously hypertensive rat; WKY, Wisytar Kyto; cDNA, DNA complimentary to RNA; UTR, untranslated region; kD, kilo dalton; mRNA, messenger RNA; DCM, dilated cardiomyopathy; ANF, atrial natriuretic factor; h-MHC, h-myosin heavy chain; PKC, protein kinase C; DOCA-salt, deoxycorticosterone acetate salt hypertensive rat; NF-nB, nuclear factor kB; InBa, inhibitory molecule of NF-nB; EDTA, ethylene diamine tetra acetic acid; SDS, sodium dodicyl sulphate; SSC, standard sodium citrate; SSPE, standard sodium phosphate EDTA; kb, kilo base; kbp, kilo base pair; ORF, open reading frame; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; MTPN, myotropihin; NCBI, national center for biotechnology information; SNPs, single nucleotide polymorphism (s); MAS, myotrophin antisense primer; LL, lambda left primer; CPSF, cleavage and polyadenylation specific factor; ARE, adenylate and uridylate-rich element; AUF1, adenylate/uridylate (AU) rich RNA-binding factor 1; HUR, Hu antigen R; AUH, AU binding homolog of enoyl-CoA hydrase. i The nucleotide sequence(s) reported in this paper has been submitted to the GenBank Data Bank with accession number(s) AY951952. * Corresponding author. Tel.: +1 216 444 2056; fax: +1 216 444 3110. E-mail address: [email protected] (S. Sen). 1 Both authors participated equally in generating the data. 2 Current address: Case Western Reserve University, Cleveland, OH, United States. 3 Current address: Department of Chemistry, Bose Institute, Calcutta, India.

1. Introduction evidence that myotrophin plays an integral role in initiating cardiac hypertrophy. Cardiac hypertrophy associated with hypertension and The gene coding for myotrophin has been cloned and subsequent heart failure is the most leading cause of death in expressed in E. coli (Sivasubramanian et al., 1996). The developed countries with diverse clinical and pathological recombinant myotrophin showed the same biological manifestations (Maron et al., 1995; Maron, 1997). Unfortu- activity as native myotrophin. Sequence analysis of the nately, the underlying mechanism for the molecular changes cDNA clone revealed that myotrophin consists of 118 that take place during this progression process is not known. amino acids and contains two full and two half-ankyrin Studies from our laboratory have demonstrated that mech- repeats (Sivasubramanian et al., 1996). Interestingly, one of anism involved in the initiation or regression of hypertrophy the ankyrin repeat showed structural homology with InBa, in spontaneously hypertensive rats (SHR) cannot be fully an inhibitory molecule of NF-nB. In addition, structural explained as response to blood pressure control alone. We analysis also revealed the putative phosphorylation sites for hypothesized that the development of hypertrophy is PKC and casein kinase II (Sivasubramanian et al., 1996). initiated by a humoral or mechanical signal to the Myotrophin has multiple transcripts in the rat heart. Among myocardium, which in turn produces a soluble factor that these transcripts, the 4.3-kb transcripts are present in triggers protein synthesis and initiates myocardial growth. abundance, but the levels of the other myotrophin transcripts In exploring the factor that initiates cardiac hypertrophy, our in mRNA are relatively low. The complete profile of the laboratory has identified a factor, myotrophin, from SHR transcripts corresponding to each clone and also the and DCM human hearts (Sen et al., 1990; Sil et al., 1993) structural organization of myotrophin gene has not been that stimulates myocytes growth. Myotrophin has been established. To define the significance of each myotrophin shown to stimulate (a) incorporation of [3H] leucine as well transcript, we describe (1) the isolation and characterization as [14C] phenylalanine into myocyte protein in vitro, (b) cell of different myotrophin cDNA clones corresponding to their growth and (c) the increase in cell surface area in a dose- multiple transcripts from the rat heart 5V ‘‘stretch-plus’’ dependent fashion compared with controls (Sen et al., cDNA library (2) the organization of the myotrophin gene, 1990). Neonatal myocytes treated with myotrophin dis- and (3) the identification of different myotrophin mRNAs in played an accelerated myofibrillar growth and an organ- SHR heart by northern hybridization. ization into sarcomeres (Sen et al., 1990). Myotrophin has also been shown to be specific for myocytes only, because it has no effect on fibroblasts, endothelial cells, or smooth 2. Materials and methods muscle cells. It was observed that myotrophin selectively increased the transcript level of the proto-oncogenes c-myc, 2.1. Materials and methods c-fos and c-jun, along with connexin (gap junction protein), atrial natriuretic factor (ANF) and h-myosin heavy chain (h- All male SHRs were obtained from Taconic Farms, MHC) gene in cultured myocytes (Mukherjee et al., 1993). Germantown, NY. Timed pregnant rats were obtained from Furthermore, increased level of myotrophin has also been Hilton Farm, Scottsdale, PA. All animals were kept in correlated with the onset of hypertrophy in SHR and in accordance with NIH and Institutional guidelines. The rats humans (Sil et al., 1995). Myotrophin gene was mapped, for were housed three per cage and maintained under the first time, to human chromosome 7q33 (Mitra et al., controlled conditions of light, temperature and humidity. 2001). Moreover, data from our laboratory also showed that All rats had free access to water and food. Hearts were protein synthesis stimulatory activity of myotrophin action removed after ether anesthesia. Atria and blood vessels was mediated through PKC signaling pathway (Sil et al., were removed. Hearts were then blotted dry and immedi- 1998). Most recently, we examined myotrophin gene in ately frozen to À70 -C. three different hypertensive models, e.g. renal hypertension, DOCA-salt and aortic coarctation where myotrophin levels 2.2. Northern analysis of myotrophin mRNAs were elevated (Sil et al., 2004). To further evaluate the physiological relevance of myotrophin in vivo, we used a- Total RNA was isolated from 9-day-old SHR hearts myosin heavy chain promoter to generate transgenic mice in following the phenol chloroform extraction method which myotrophin is overexpressed in the heart (Sarkar et (Chomczynski and Sacchi, 1987). Poly (A) enriched RNA al., 2004). Overexpression of myotrophin specifically in the was isolated using the oligo(dT)-cellulose type-III (Collab- myocardium resulted in severe cardiac hypertrophy that orative Research, Bedford, MA) column at high-salt advanced to heart failure. These mice exhibited left conditions (10 mM Tris–HCl, pH 7.5, 1 mM EDTA, 0.5 ventricular hypertrophy, atrial dilation, myocyte necrosis, M NaCl). After washing the column with high-salt buffer, multiple focal fibrosis, pleural effusion and compromised poly (A) RNA was eluted with no-salt (10 mM Tris–HCl, cardiac function associated with significant reduction in pH 7.5, 1 mM EDTA) buffer. The poly (A) RNA eluted by ejection fraction and fractional shortening (Sarkar et al., this method was fractionated on 1% agarose formaldehyde 2004). All our findings provided a wealth of convincing gels, transferred to a zeta probe membrane (BIORAD, G. Adhikary et al. / Gene 353 (2005) 31–40 33

Hercules, CA) by the downward neutral (20ÂSSC) were 95 -C for 1 min, 55 -C for 1 min, and 72 -C for 3 min. capillary transfer method (Cicogna et al., 1997). The PCR products were purified through the QIAGEN (Santa random primer labeling method was used to generate a Clarita, CA) cartridges. The purified DNA was subcloned in radiolabeled myotrophin cDNA probe using the pCRII Myo the ‘‘TA’’ cloning vector pCRII (Invitrogen) between the SP6 #8 cDNA clone (Mukherjee et al., 1993; Sivasubramanian et and T7 promoters. The ligated products were transformed to al., 1996). Utilizing the same clone, a single-stranded the competent E. coli cells; DH5a and Plasmid DNA were myotrophin-specific radiolabeled antisense RNA probe isolated by the maxi kit method (QIAGEN, Santa Clarita, was made using the T7 RNA polymerase-directed in vitro CA). The orientation of the sense direction of each clone, transcription reaction. Two micrograms of ‘‘low salt-wash whether it was under the control of the SP6 or the T7 RNA’’ (Lane a in Fig. 1) and 5 Ag of pure poly (A) RNA promoter, was determined by PCR, using myotrophin (Lane b in Fig. 1) from 9-day-old SHR hearts were internal primer MS (5V GGAGACTTGGAT-GAGGT- fractionated on the agarose gel. The hybridization experi- GAAGGAC 3V), SP6 (5V ATTTAGGTGACACT-ATAG 3V), ment was done using very high stringency conditions for and MS, T7 (5V GTAATACGACTCACTATAG 3V) primers. cDNA probe (42 -C, 5ÂSSPE, 10ÂDenhardt’s, 50% Five cDNA clones of myotrophin corresponding to the 3.5-, formamide, 2% SDS, and 100 Ag/ml salmon sperm DNA) 2.4-, 1.8-, 1.0-, and 0.7-kb transcripts were sequenced. All and the antisense RNA probe (50 -C, 1.5ÂSSPE, 1% SDS, these clones contained the same 5V ends and the same open 0.5% BLOTTO, 50% formamide, 0.2 mg/ml tRNA, and 0.5 reading frame (ORF), but the 3V ends of the cDNA varied in mg/ml salmon sperm DNA). In both cases, membranes were length. washed vigorously (2ÂSSC, 15 min; 2ÂSSC 1% SDS, 15 min; 0.5ÂSSC 1% SDS, 15 min, all at room temperature. 2.4. 5V End amplification of different myotrophin transcripts Dried membranes were subjected to autoradiography at À70 -C with an intensifying screen. DNA was isolated from the 5V ‘‘stretch-plus’’ cDNA library (Clontech, Palo Alto, CA) by QIAGEN (Santa 2.3. Isolation and cloning of myotrophin cDNA clones Clarita, CA) cartridges. This library was made by extensive denaturation of mRNA by methyl mercuric hydroxide. The Initially, the rat heart Egt11 cDNA library was screened 5V ‘‘stretch-plus’’ cDNA library was made by using unique using a myotrophin cDNA probe (pCRII Myo #8), and two oligo (dT)25 d(A/C/G) primers, which helps the positioning of types of myotrophin cDNA clones were obtained with the primers during the first strand synthesis at the junction myotrophin insert size 1 kb and 0.7 kb, but no cDNA clones of the poly (A) tail and encoded transcripts. Therefore, only were obtained corresponding to higher molecular weight a relatively small fraction of the poly (A) tail needs to be transcripts of myotrophin. Therefore the 5V ‘‘stretch plus’’ reverse transcribed to get to the transcript and, consequently, Egt11 cDNA library of rat heart (Clontech, Palo Alto, CA) more mRNAs are fully reverse transcribed. The 5V ends of was screened with the myotrophin cDNA probe. Individual myotrophin cDNA clones were amplified by PCR using this positive plaques were screened several times to gel pure DNA (50 ng) as a template with myotrophin ORF-specific single cDNA clones. In the purified cDNA clones, myo- antisense primer MAS (5V AGGCTTCCTCCCA-CCCTC- trophin cDNA inserts were present between lambda left and TAGTGT 3V, 111–88 nt). The PCR conditions were 1 min lambda right arms. Myotrophin inserts were amplified by the denaturation at 95 -C, 1 min annealing at 50 -C, and 2 min PCR method using ampli Taq DNA polymerase (Perkin extension at 72 -C for 25 cycles. Elmer, Branchburg, NJ) with Egt11 leftward (LL) (5V ATCTGCT-GCACGCGGAAGAAGGCA 3V) and rightward 2.5. Southern hybridization (LR) (5V ATGGTAGCG-ACCGGCGCTCAGC-3V) primers as sense and antisense, respectively; these primers were Genomic DNA was isolated from SHR hearts follow- designed very close to the cDNA ends. The PCR conditions ing the QIAamp tissue kit method (QIAGEN, Santa

Fig. 1. Organization of human myotrophin gene (MTPN). MTPN genomic organization was obtained from the National Center for Biotechnology (NCBI, web- site address www.ncbi.nlm.nih.gov) using Locus Link ID 136319; accession number NT_007933. SNP, single nucleotide polymorphism. 34 G. Adhikary et al. / Gene 353 (2005) 31–40

Clarita, CA). The isolated genomic DNA (20 Ag) was 2.8. Ontogeny of myotrophin gene expression in SHR and digested separately with the restriction enzymes EcoRI, WKY hearts HindIII, and PstI for 24 h at 37 -C. Digested DNA was fractionated in 1% agarose gel, transferred to a zeta 9-day-, 3-week- and 31-week-old SHR and WKY rat probe nylon membrane (BIORAD, Hercules, CA) and hearts were used to study the ontogeny of myotrophin gene hybridized with a myotrophin-specific cDNA probe expression in SHR and WKY. Total RNA was isolated from (1Â106 cpm/ml) generated by the random primer label- all rat hearts following phenol chloroform extraction ing method. The membrane was de-probed by washing at methods (Chomczynski and Sacchi, 1987) and Northern 95 -C for 30 min twice in 0.2ÂSSC, 0.5% SDS solution blot analysis was performed as described previously and then hybridized with the myotrophin coding region, (Mukherjee et al., 1993). The transcript levels of myotro- 5V end and 3V end of the myotrophin cDNA clone as a phin were quantified by using a video densitometer and probe separately, generated by the random primer label- image analyzer from the autoradiograph. ing method using the PstI-digested fragment of the myotrophin cDNA clone as the DNA template (data not shown). 3. Results

2.6. In vitro transcription and translation of myotrophin 3.1. Structural organization of myotrophin gene cDNA clones We have shown that myotrophin (MTPN) was localized The T7 TNT coupled wheat germ extract system in the human chromosome 7q33 (Mitra et al., 2001). MTPN (Promega, Madison, WI) was used for a single-tube in genomic organization was obtained from the National vitro transcription and translation of various myotrophin Center for Biotechnology (NCBI, web-site address cDNA clones isolated from the 5V ‘‘stretch-plus’’ cDNA www.ncbi.nlm.nih.gov) using LocusLink ID 136319 (acces- library (Egt11) of rat heart. The clones isolated were sion number NT_007933) to define the intron–exon subcloned in the pCRII TA-cloning vector (Invitrogen) boundaries, number of exons and also the promoter region. between the SP6 and T7 promoters. The linear myotro- The human myotrophin gene (MTPN) is located on phin inserts were amplified from different myotrophin chromosome 7q33 between chromosomal locations clones and also from the 5V UTR-a-actin-myotrophin 135119319 to 135068764 bp in the 5V to 3V orientation. construct by the PCR method using the SP6 and T7 Chromosomal location was determined by adding 74410376 primers. The PCR products were purified using the to the NT_007933 contig position. MTPN consists of a 5V- QIAGEN (Santa Clarita, CA) PCR purification kit. The untranslated region (UTR), 4 exons, 3 introns, and a 3VUTR. linear DNA templates of the myotrophin inserts were used The gene codes for a 118-amino acid protein (U21661). for the coupled in vitro transcription and translation Public databases (NCBI) show a total of 114 single reaction (Promega, Madison, WI) using TNT wheat nucleotide polymorphisms (SNPs) for MTPN scattered extract, TNT reaction buffer, amino acid mixture minus throughout the gene, but mainly in intronic regions. For cysteine (0.02 mM), 35S-cysteine (40 ACi), RNasin exact chromosomal locations of these SNPs, access Locus- (ribonuclease inhibitor 0.004 U), DNA template (1 Ag), Link ID 136319. Included are the putative degradation and and TNT RNA polymerase (either SP6 or T7 RNA GT-rich motifs that may help explain the various transcript polymerase, depending on the orientation of the coding variants that are observed for this gene. region), in a total volume of 50 Al. The reaction mixture was incubated at 30 -C for 90 min. The control reaction 3.2. Myotrophin gene expression in SHR heart was done using luciferase DNA as the template. In vitro translation products were acetone precipitated two times to Expression of the myotrophin gene in SHR heart was remove unincorporated 35S-cysteine. Pellets were sus- determined by Northern blot analyses. From the coding pended in the protein sample buffer and the translated region of myotrophin cDNA clones, both a double-stranded products were fractionated on 10% Tris–tricine SDS– cDNA probe (Fig. 2) and a single-stranded antisense RNA PAGE. The gel was dried and the autoradiograph of the probe were used in different Northern blot experiments (Fig. gel was developed. Luciferase enzyme used as control 2, lane a). Very high stringency hybridization and wash was detected by the photographic method (data not conditions were used for these experiments. Initially, only shown). 4.3-kb and 3.5-kb transcripts were detected when pure poly (A) RNA was used (Fig. 2, lane b). These transcripts did not 2.7. Western blot analysis correspond to the myotrophin cDNA clones that we had isolated. We isolated full-length myotrophin cDNA clones Western blot analysis of in vitro translated product was corresponding to 0.7-kb and 1.0-kb transcripts. Since low- performed by using myotrophin antibody as a probe (Gupta molecular-weight myotrophin transcript signals were not and Sen, 2002). detected significantly in the poly(A) RNA, we included a G. Adhikary et al. / Gene 353 (2005) 31–40 35

Fig. 3. Southern hybridization of myotrophin inserts present in various Fig. 2. Northern blot analyses of myotrophin transcripts in 9-day-old SHR myotrophin cDNA clones, isolated from the 5V ‘‘stretch-plus’’ cDNA library hearts. Lane b represents the no-salt buffer-eluted poly (A) RNA (5 Ag), of rat hearts. Myotrophin inserts detected in various myotrophin cDNA probed with a myotrophin cDNA probe. Lane a represents RNA (2 Ag) clones were amplified by PCR. The PCR products were fractionated on a precipitated from the ‘‘low-salt-wash’’ fraction during the poly (A) RNA 1% agarose gel and transferred to a positively charged nylon membrane by isolation procedure and probed with the myotrophin-specific antisense the Southern transfer method. The membrane was then probed with a single-stranded RNA probe made by the in vitro transcription reaction. myotrophin cDNA probe. Lanes c and d represent re-probing of lanes a and b, respectively, with a h- MHC probe. specific antisense primer MAS and lambda primer LR, generated only one 300-bp PCR product (Fig. 4), but the ‘‘low-salt-wash’’ step in our oligo(dT)-cellulose purification other PCR with MAS and lambda left primer (LL) did not procedure (10 mM Tris–HCl, pH 7.5, 1 mM EDTA, 0.1 M produce any product. Sequence analysis showed that a NaCl) before eluting the poly(A) RNA with no-salt buffer. single ORF was present in all myotrophin cDNA clones. The RNA from this ‘‘low-salt-wash’’ fraction was ethanol The 3V ends were also the same in sequences up to the end precipitated and analyzed for myotrophin-specific tran- points for smaller clones, but the 3V ends were different in scripts. Interestingly, the majority of the myotrophin- length (Fig. 5) (GenBank Accession No. AY951952). The specific low-molecular-weight transcripts (2.4, 1.0, 0.7, observation of multiple types of cDNA clones with different and 0.4 kb) along with 4.3-kb and 3.5-kb transcripts were 3V UTRs suggested a mechanism for the observation of observed in the ‘‘low-salt-wash’’ RNA rather than the pure multiple transcripts in the Northern blot experiments. (poly A) RNA (Fig. 2, lane a). The integrity of the RNA Southern hybridization analysis of rat genomic DNA (Fig. samples was checked by re-probing the same blot with h- 6) suggests that the myotrophin gene is a single copy gene. MHC as the cDNA probe (Fig. 2, lanes c and d). h-MHC is A 6.4-kb EcoRI genomic DNA fragment was hybridized to expressed in heart as a single species of transcript size 6.2 the full-length myotrophin cDNA probe (Myo# 3.5) with a kb. The signals for 1.0-kb and 0.7-kb transcripts were strong signal and 3.0-kb fragment with weaker signal. In the stronger than other transcripts in the ‘‘low-salt-wash’’ RNA. HindIII digested lane, two DNA fragments were detected It is possible that the poly (A) tracts in these transcripts may with full-length myotrophin cDNA probes of 5.5 kb and 4.5 be quite short and thus eluted in the ‘‘low-salt-wash’’ kb. When the same blot was de-probed and re-probed fraction. separately with a 5V UTR and a 3V UTR specific myotrophin

3.3. Characteristics of myotrophin cDNA clones

Five myotrophin cDNA clones corresponding to transcripts were isolated from the 5V stretch-plus cDNA library. We isolated one myotrophin cDNA clone corresponding to a 1.8-kb transcript. In Northern hybridization, this transcript was not detected (Fig. 2), probably because of very low levels of this transcript in rat heart. Different myotrophin inserts corresponding to various transcripts were fractionated in 1% agarose gel and transferred to nylon membrane. Southern hybridization with a myotrophin cDNA probe confirmed the authenticity of different myotrophin clones (Fig. 3). One isolated 3.7-kb clone (Fig. 3), which was hybridized with the myotrophin probe, is not characterized Fig. 4. Agarose gel fractionation of the 5V ends of specific PCR products of different myotrophin cDNA clones. Using the 5V ‘‘stretch-plus’’ rat heart yet. Different myotrophin cDNA clones were sequenced. cDNA library as a template, PCR was done with the myotrophin-specific Amplification of the 5V ends by PCR, using 5V stretch-plus antisense primer MAS (111-88 nt) and LL, lambda left primer (lane 2), and rat heart library DNA as a template with the myotrophin- MAS; LR, lambda right primer (lane 3). 36 G. Adhikary et al. / Gene 353 (2005) 31–40

Fig. 5. (A) Structure of different myotrophin cDNA clones. The nucleotide numeration starts with +1 at the A of the translation initiation codon ATG, and TGA is the stop codon. (B) Nucleotide and deduced amino acid sequences of the rat myotrophin cDNA clone corresponding to the 3.5-kb transcripts. Nucleotide and deduced amino acid sequences corresponding to the 2.4-kbp, 1.8-kbp, 1.0-kbp, and 0.7-kbp myotrophin cDNA clones are superimposed on the 3.5-kbp myotrophin cDNA clone sequences. The deduced amino acid sequences are shown by the three-letter code below the nucleotide sequences (*** indicates the stop codon). Coding regions are indicated in upper case, 3V UTR and 5V UTR in lower case. Amino acid numbers are shown at the right of the nucleotide sequence and nucleotide numbers are in parentheses. Arrows (,) marked on the nucleotide indicates the end nucleotide of particular myotrophin cDNA clone. Nucleotide Fg_ (+546), Ft_ (+837), Fa_ (+1634) and Fa_ (+2013) positions are the end points of 0.7-kbp, 1.0-kbp, 1.8-kbp, and 2.4-kbp myotrophin cDNA clones, respectively. Poly (A) recognition signals (attaaa); (aataaa), (ataaa) and also mRNA destabilizing sequences (attta) are underlined. The -GT- rich nucleotide sequences at the end of 2.4-kbp and also 3.5-kbp myotrophin cDNA clones are underlined. (GenBank Accession No. AY951952). probe, the 5.5-kb fragment was detected by the 5V UTR must arise from a single copy myotrophin gene. Similar specific myotrophin probe and the 4.5-kb fragment by the 3V types of multiple transcripts have been observed for other UTR specific probe (data not shown). Myotrophin gene size genes, such as opsin, in mouse, rat, human, and frog (al- is approximately 10 kb. Therefore, the multiple transcripts Ubaidi et al., 1990), vimentin (Zehner and Paterson, 1983) G. Adhikary et al. / Gene 353 (2005) 31–40 37

(Fig. 7A). The a-actin 5V UTR-myo construct produced four times more 12-kD translated myotrophin protein than we would have expected from the natural myotrophin cDNA clones. For further clarification, we performed Western blot analysis using same in vitro translated product and myotrophin antibody. Our data show a single 12-kD protein band in Western analysis (Fig. 7B). Altogether, it suggests that the product we got after in vitro translation is the right product for myotrophin.

3.5. Ontogeny of myotrophin gene expression in SHR and WKY hearts

Expression of the myotrophin gene in SHR and WKY rat heart during development of hypertrophy was determined by Northern blot analysis. The strongest signal was obtained from the transcript levels of the 4.3-kb and 3.5-kb forms, which were quantified by video densitometry. The transcript levels from each group are summarized in Fig. 8. In 9-day-, 3-week-old and 31-week-old SHR, the amount of the 4.3-kb transcript was significantly increased compared with levels in WKY (Fig. 8). In WKY, the transcript level remained the Fig. 6. Rat genomic DNA (20 Ag) was digested with restriction enzymes same throughout the growth period. When the 3.5-kb EcoRI, HindIII, and PstI separately for 24 h. The same restriction digestion transcript level was quantified, a similar increase in was repeated another two times for complete restriction digestion. Digested DNA was ethanol precipitated, fractionated in a 1% agarose gel, and hybridized with a myotrophin-specific full-length cDNA probe (Myo # 3.5). Sizes of the digested fragments were EcoRI (6.4 kb) and (3.0 kb), HindIII (5.5, 4.5 kb), PstI (4.0, 2.9, 2.5, 1.9 and 1.7 kb). adenosine deaminase gene (Yeung et al., 1983)and dihydrofolate reductase gene (Yoder et al., 1983).

3.4. In vitro transcription and translation of myotrophin clones

The myotrophin coding regions in different myotrophin cDNA clones were amplified by PCR and the purified DNA was used for in vitro transcription and translation in a single tube with either SP6 or T7 RNA polymerase, depending on the orientation of the coding region of the clone in the pCRII vector. The translation efficiency of the normal myotrophin gene (pCRII Myo # 8) was predicted to be low based on Kozak sequences. The sequences around V Fig. 7. In vitro transcription and translation of various myotrophin cDNA the start codon of natural myotrophin are 5 CAGTGATGT clones. (A) Different myotrophin inserts, including the a-actin 5V UTR- 3V. The first three amino acids of myotrophin and a-actin myotrophin construct, were amplified from cDNA clones by PCR using are identical (Met-Cys-Asp). The Kozak context around the SP6 and T7 primers. The linear DNA templates (1 Ag) of myotrophin start codon of a-actin is 5 VCCACCATGT3V, and its cDNA clones [a-actin 5V UTR-myo (1), 0.7 kbp (2), 1.0 kbp (3), 1.8 kbp expression levels are higher. Therefore, another construct (4), 2.4 kbp (5), and 3.5 kbp (6)] were used for coupled in vitro transcription and translation (Promega, Madison, WI) using TNT wheat was made in which the myotrophin-coding region was germ extract, TNT reaction buffer, amino acid mixture minus cysteine (0.02 under the control of the 5V untranslated region (UTR) of mM), 35S-cysteine (40 ACi), RNasin (0.004 U), TNT RNA polymerase skeletal a-actin, and that construct was sub-cloned in the (either SP6 or T7 RNA polymerase, depending on the orientation of the pcDNA3 vector. The 12-kD in vitro translated, radiolabeled coding region) in a reaction volume of 50 Al. No template DNA was used in myotrophin protein was detected in the autoradiograph. The the second lane. The translated products were acetone precipitated; the V pellets were suspended in protein sample buffer and fractionated in 10% translated product from the construct, skeletal a-actin 5 Tris–tricine SDS–PAGE. (B) a-actin 5V UTR-myo was transferred onto UTR-myo, was four times more abundant than what we PVDF membrane and Western blot was performed using Myotrophin would have expected from normal myotrophin clones antibody as a probe. 38 G. Adhikary et al. / Gene 353 (2005) 31–40

for all myotrophin cDNA clones that code for the 12-kD protein. The 5V ends were identical in sequence. The 3V ends of smaller transcripts were exactly the same up to the end point compared with the 3.5-kb myotrophin cDNA clone (Fig. 4). In the 3.5-kb clone there were several poly (A) recognition signals and a stretch of 85 nucleotides of -GT- in the 3V UTR region. In situ hybridization using different 3V UTRs of myotrophin cDNA clone as probes would give the functional significance of multiple transcripts of myotrophin. Sequence analysis showed that in the 3V UTR of 1.8-kbp, 2.4-kbp, and 3.5-kbp clones, two stretches of ATTTA sequences in the UTR were thought to play a role in selective mRNA degradation. Several lines of evidence Fig. 8. Ontogeny of myotrophin gene expression in SHR and WKY. Northern blot analysis was performed using conditions described in the suggest that trans acting factors such as AUH, AUF-1 and text. Note: A significant increase in transcript level of myotrophin mRNA HUR showed their binding activity towards adenylate- and was observed in 9-day-, 3-week- and 31-week-old SHR, compared to age- uridylate-rich element (AREs) in 3V UTR of mRNAs; and and sex-matched WKY. binding to AREs frequently serve as a signal to target the mRNA for rapid degradation. The smaller myotrophin transcript levels at 9 days, 3 weeks, and 31 weeks was found transcripts (0.7 kbp and 1.0 kbp) might be generated by (data not shown). using different poly (A) recognition signals depending on the physiological needs. Birkenmeier et al. (1993) showed that transcripts of erythroid ankyrin-protein vary in the 3V UTRs. 4. Discussion Myotrophin protein contains ankyrin repeats and also multiple-transcript variation in the 3V UTR. The 3V UTRs In this study, we have shown that the myotrophin gene is of myotrophin transcripts may regulate transcript stability a single copy gene, consisting 4 exons separated by 3 (Jackson and Standart, 1990). Sundell and Singer (1991) introns. We have detected six transcripts in SHR heart. All showed the distinct sub-cellular localization of actin mes- myotrophin transcripts contain the same coding region and senger RNA. The 3V UTRs of myotrophin transcripts may the same 5V ends, but the 3V ends vary in length. The regulate the sub-cellular localization of myotrophin tran- significant finding presented in this study is characterization scripts. Moreover, AT-rich elements are known to be and importance of the multiple transcripts of myotrophin recognition sequences of several RNA-binding proteins. gene. All transcripts contain the similar length of open Together, our data suggest that the presence of ATTTA in 3V reading frame (ORF) and 5V UTR but varying in the 3V UTR. UTR of myotrophin gene may serve as a target for mRNA In addition, we observed a high level of translational instability or degradation. Furthermore, from the sequence product of myotrophin when the Cozak sequence was analysis, it was also observed that the 0.7-kbp cDNA clones replaced with a-actin 5VUTR as the first three amino acids of contained an ATTAAA motif 25 nucleotides upstream from myotrophin ORF showed a similarity with a-actin gene, the polyadenylation site. In the 1010-kbp myotrophin cDNA which normally expressed in higher level in animal. clone, this signal was not observed near the 3V ends. Our study showed that all myotrophin transcripts are Sequence analysis also showed that the 3V end of the 2.4- translatable in vitro, although the translation efficiency for kbp clones contained a stretch of 35 -GT- nucleotides. At the all cDNA clones is low because the Kozak context is poor. same position the 3.5-kb myotrophin clone has 85 -GT- To increase the translation efficiency of the natural stretch nucleotides. Generally, -GT- stretch nucleotides are myotrophin gene, we made another construct in which the present in the 3V UTR of pre-mRNA and help in the cleavage myotrophin coding region was under the control of the 5V and poly adenylation of mRNA by CPSF (cleavage and poly UTR sequence of the a-actin gene. The first three amino adenylation specific factor) (Keller, 1995). Both 3.5-kb and acids of myotrophin and a-actin are identical and a-actin is 2.4-kb myotrophin transcripts contain -GT- repeats in the 3V expressed at higher levels in animals. The a-actin 5V UTR- UTR region, a very unusual feature. It is possible that the myo construct gave a higher (at least 4-fold) level of 3.5-kbp and the 2.4-kbp myotrophin transcripts may act as myotrophin expression than the natural myotrophin cDNA storage forms of mRNA. Myotrophin transcripts are also clones (lane 1 in Fig. 6). Our observation of several detected in other organs (Sivasubramanian et al., 1996), but myotrophin-specific transcripts in the ‘‘low-salt-wash’’ the functional significance and the regulation of each RNA fraction suggests that these are translationally silenced transcript in each organ are currently unknown. It is thus mRNAs ready to be translated upon receiving the physio- speculate from this study that alternative polyadenylation logical signals (Huarte and Stutz, 1992). signals and an ARE sequence provide a novel mechanism for From sequence analyses of different myotrophin cDNA the regulation of the rat myotrophin gene expression. clones, it was observed that the coding region was the same Furthermore, it may also predict from the varying 3V UTR G. Adhikary et al. / Gene 353 (2005) 31–40 39 of myotrophin gene the predominance or abundance of hypertrophy in several studies (Vanderburgh, 1992). Ham- higher molecular weight transcript. mond et al. (1982) first showed the existence of a soluble To define the pathophysiological significance of various factor that stimulated protein synthesis in the heart. It is transcript levels of myotrophin, we determined all the known that some growth factors can affect the level of messages in both 9-day-old SHR and WKY rat heart and expression of other growth factor genes, as reported by compared the level of each transcript in the hearts of both Schneider and Parker (1991). Sadoshima et al. (1992) also the strains. The levels of all the transcripts (0.7, 1.0, 2.4, 3.5 reported that a 6-h treatment with angiotensin II increases and 4.3 kb) are significantly elevated in SHR hearts the level of expression of transforming growth factor-h1as compared with levels of the same transcript in WKY rat well as angiotensinogen genes in cardiac myocytes. We hearts. This result suggests that regardless of the transcript have identified a factor, myotrophin, from SHR and size, the changes of the levels are in the same direction. We cardiomyopathy human hearts appears to a good candidate quantified the message levels of 4.3 kb (most abundant for such complex process that translates mechanical load to transcript) at various age groups during development of a biochemical event (e.g. protein synthesis) and thereby hypertrophy in SHR and compared that to age- and sex- facilitates an increase in cardiac mass. It is evident that matched WKYs, as this transcript was the strongest and cardiac hypertrophy and heart failure are complex process. accurately quantifiable. A significant increase in the tran- So, it is not unexpected that the initiation and progression of script level was observed in the SHR during the progression this disease are a result of combined effects of many factors. of hypertrophy. This parallels an increase in heart size in We, therefore, predict that this protein may be a common SHRs, 13% by 3 weeks of age and 28% by 31 weeks of age link initiating different type of cardiac hypertrophy. Further (Sen et al., 1974). All these data showed that the myotrophin studies will reveal much more about myotrophin during the transcript and proteins both change with the development of progression of chronic heart failure, which may help in hypertrophy and hypertension in SHRs. designing therapeutic drug for patients with hypertensive The importance of myotrophin in various cardiac heart disease. disease states was substantiated by O’Brien et al. (2003), who has detected a significant high level of myotrophin in the plasma of heart failure patients. Recently, we demon- Acknowledgement strated that cardiac specific overexpression of myotrophin in the transgenic mice resulted in severe cardiac hyper- This study was supported in part by National Institutes of trophy that transits to heart failure (Sarkar et al., 2004). Health grant HL-47794 (S. Sen). We thank David Young for Myotrophin has been suggested to be an NF-nB binding preparation of recombinant myotrophin. We acknowledge protein (Knuefermann et al., 2002; Sivasubramanian et al., Chris Colazei for secretarial support and Christine Kassuba 1996). We recently demonstrated that probably myotrophin for editorial assistance. exerts its action by activating NF-nB signaling pathway (Gupta et al., 2002). Activation of NF-nB system is widely documented in heart failure and may be an important References phenomenon in the progression of heart failure (Maulik et al., 1998; Morgan et al., 1999; Ritchie, 1998; Valen et al., al-Ubaidi, M.R., Pittler, S.J., Champagne, M.S., Triantafyllos, J.T., 2001; Wong et al., 1998; Xuan et al., 1999). In addition, McGinnis, J.F., Baehr, W., 1990. Mouse opsin. Gene structure and molecular basis of multiple transcripts. J. Biol. 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