S100β Inhibits α1-Adrenergic Induction of the Hypertrophic Phenotype in Cardiac Myocytes
1997; Elsevier BV; Volume: 272; Issue: 50 Linguagem: Inglês
10.1074/jbc.272.50.31915
ISSN1083-351X
AutoresJames N. Tsoporis, Alexander Marks, Harriette J. Kahn, Jagdish Butany, Peter P. Liu, David O'Hanlon, Thomas G. Parker,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoIn an experimental rat model of myocardial infarction, surviving cardiac myocytes undergo hypertrophy in response to trophic effectors. This response involves gene reprogramming manifested by the re-expression of fetal genes, such as the previously reported isoform switch from adult α- to embryonic β-myosin heavy chain. We now report the transient re-expression of a second fetal gene, skeletal α-actin in rat myocardium at 7 days post-infarction, and its subsequent down-regulation coincident with the delayed induction of S100β, a protein normally expressed in brain. In cultured neonatal rat cardiac myocytes, co-transfection with an S100β-expression vector inhibits a pathway associated with hypertrophy, namely, α1-adrenergic induction of β-myosin heavy chain and skeletal α-actin promoters mediated by β-protein kinase C. The induction of β-myosin heavy chain by hypoxia was similarly blocked by forced expression of S100β. Our results suggest that S100β may be an intrinsic negative regulator of the hypertrophic response of surviving cardiac myocytes post-infarction. Such negative regulators may be important in limiting the adverse consequences of unchecked hypertrophy leading to ventricular remodeling and dysfunction. In an experimental rat model of myocardial infarction, surviving cardiac myocytes undergo hypertrophy in response to trophic effectors. This response involves gene reprogramming manifested by the re-expression of fetal genes, such as the previously reported isoform switch from adult α- to embryonic β-myosin heavy chain. We now report the transient re-expression of a second fetal gene, skeletal α-actin in rat myocardium at 7 days post-infarction, and its subsequent down-regulation coincident with the delayed induction of S100β, a protein normally expressed in brain. In cultured neonatal rat cardiac myocytes, co-transfection with an S100β-expression vector inhibits a pathway associated with hypertrophy, namely, α1-adrenergic induction of β-myosin heavy chain and skeletal α-actin promoters mediated by β-protein kinase C. The induction of β-myosin heavy chain by hypoxia was similarly blocked by forced expression of S100β. Our results suggest that S100β may be an intrinsic negative regulator of the hypertrophic response of surviving cardiac myocytes post-infarction. Such negative regulators may be important in limiting the adverse consequences of unchecked hypertrophy leading to ventricular remodeling and dysfunction. In a rat model of myocardial infarction, surviving terminally differentiated cardiac myocytes undergo a phenotypic transition post-infarction, involving cellular hypertrophy and re-expression of genes normally restricted to the fetus, including the embryonic β-myosin heavy chain (β-MHC), 1The abbreviations used are: β-MHC, β-myosin heavy chain; CAT, chloramphenicol acetyltransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NE, norepinephrine; PE, phenylephrine; PKC, protein kinase C; RT-PCR, reverse transcriptase polymerase chain reaction; skACT, skeletal α-actin; bp, base pair(s); RSV, Rous sarcoma virus; LUX, luciferase. the skeletal isoform of α-actin (skACT), atrial natriuretic factor, and a fetall-type calcium channel (1Orenstein T.L. Parker T.G. Butany J.W. Goodman J.M. Dawood F. Wen W.-H. Wee L. Martino T. McLaughlin P.R. Liu P.P. J. Clin. Invest. 1995; 96: 858-866Crossref PubMed Scopus (103) Google Scholar, 2Gidh-Jain M. Huang B. Jain P. Battula V. El-Sherif N. Biochem. Biophys. Res. Commun. 1995; 216: 892-897Crossref PubMed Scopus (59) Google Scholar, 3Hanatani A. Yoshiyama M. Kim S. Omura T. Toda I. Akioka K. Teragaki M. Takeuchi K. Iwao H. Takeda T. J. Mol. Cell. Cardiol. 1995; 27: 1905-1914Abstract Full Text PDF PubMed Scopus (88) Google Scholar, 4Kanda T. Nakano M. Arai M. Yamamoto A. Suzuki T. Murata K. Exp. Clin. Endocrinol. 1993; 101: 173-177Crossref PubMed Scopus (10) Google Scholar). This response is comparable to the hypertrophic response of the heart to acute pressure overload, during which the induction of several fetal genes including β-MHC and skACT has been observed (5Schwartz K. de la Bastie D. Bouveret P. Oliviéro P. Alonso S. Buckingham M. Circ. Res. 1986; 59: 551-555Crossref PubMed Scopus (219) Google Scholar, 6Izumo S.J. Lompré A.-M. Matsuoka R. Gideon K. Schwartz K. Nadal-Ginard B. Mahdavi V. J. Clin. Invest. 1987; 79: 970-977Crossref PubMed Scopus (372) Google Scholar, 7Nadal-Ginard B. Mahdavi V. Basic Res. Cardiol. 1993; 88: 65-79PubMed Google Scholar). The existence of feedback mechanisms limiting the hypertrophic response is suggested by the observation that the induction of β-MHC and skACT, in response to pressure overload, is down-regulated after 8–11 days in association with moderation of the increase in myocardial mass (5Schwartz K. de la Bastie D. Bouveret P. Oliviéro P. Alonso S. Buckingham M. Circ. Res. 1986; 59: 551-555Crossref PubMed Scopus (219) Google Scholar, 6Izumo S.J. Lompré A.-M. Matsuoka R. Gideon K. Schwartz K. Nadal-Ginard B. Mahdavi V. J. Clin. Invest. 1987; 79: 970-977Crossref PubMed Scopus (372) Google Scholar). Since the attenuation of the hypertrophic response post-infarction may be beneficial in tempering adverse ventricular remodeling (1Orenstein T.L. Parker T.G. Butany J.W. Goodman J.M. Dawood F. Wen W.-H. Wee L. Martino T. McLaughlin P.R. Liu P.P. J. Clin. Invest. 1995; 96: 858-866Crossref PubMed Scopus (103) Google Scholar), evidence for similar feedback mechanisms would be important to define the balance among opposing physiological interactions that come into play following ischemic injury. The signaling pathways activated by myocardial infarction are likely to be multifactorial and the relative contributions of hypoxia, ischemia, and activation of local and systemic trophic factors in the hypertrophy of surviving myocytes is unknown. Hypertrophy and its associated program of fetal gene re-expression can be reproduced in vitro in cultured neonatal rat cardiac myocytes by treatment with a number of trophic effectors including peptide growth factors and α1-adrenergic agonists such as norepinephrine (NE) and phenylephrine (PE) (8Simpson P.C. Circ. Res. 1985; 56: 884-894Crossref PubMed Scopus (405) Google Scholar, 9Bishopric N.H. Simpson P.C. Ordahl C.P. J. Clin. 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Martinson E.A. Van Bilsen M. Chien K.R. Brown J.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1305-1309Crossref PubMed Scopus (153) Google Scholar). Studies in this cell culture model have implicated several signaling mechanisms including activation of protein kinase C (PKC) in triggering this end-response (13Dummon P.M. Iwaki K. Henderson S.A. Sen A. Chien K.R. J. Mol. Cell. Cardiol. 1990; 22: 901-910Abstract Full Text PDF PubMed Scopus (125) Google Scholar, 15Chien K.R. Knowlton K.U. Zhu H. Chien S. FASEB J. 1991; 5: 3037-3046Crossref PubMed Scopus (696) Google Scholar, 17Shubeita H.E. Martinson E.A. Van Bilsen M. Chien K.R. Brown J.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1305-1309Crossref PubMed Scopus (153) Google Scholar, 18Kariya K. Karns L.R. Simpson P.C. J. Biol. Chem. 1991; 266: 10023-10026Abstract Full Text PDF PubMed Google Scholar). This has allowed the tracing of individual biochemical pathways that are associated with hypertrophy in myocytes, for example, the induction of β-MHC and skACT as a consequence of activation of β-PKC by α1-adrenergic agonists (9Bishopric N.H. Simpson P.C. Ordahl C.P. J. Clin. Invest. 1987; 80: 1194-1199Crossref PubMed Scopus (160) Google Scholar, 12Waspe L.E. Ordahl C.P. Simpson P.C. J. Clin. Invest. 1990; 85: 1206-1214Crossref PubMed Scopus (157) Google Scholar, 18Kariya K. Karns L.R. Simpson P.C. J. Biol. Chem. 1991; 266: 10023-10026Abstract Full Text PDF PubMed Google Scholar, 19Karns L.R. Kariya K. Simpson P.C. J. Biol. Chem. 1995; 270: 410-417Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The focus of the current research is to identify the intermediate components of these pathways that bring about the transcription of the end-response genes (19Karns L.R. Kariya K. Simpson P.C. J. Biol. Chem. 1995; 270: 410-417Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 20Kariya K. Farrance I.K.G. Simpson P.C. J. Biol. Chem. 1993; 268: 26658-26662Abstract Full Text PDF PubMed Google Scholar, 21Kariya K. Karns L.R. Simpson P.C. J. Biol. Chem. 1994; 269: 3775-3782Abstract Full Text PDF PubMed Google Scholar). To date, no intrinsic candidates for limiting the hypertrophic response have been put forward. S100 protein is a 20-kDa Ca2+-binding protein dimer composed of two subunits S100α and S100β, that have different tissue distributions (22Isobe T. Okuyama T. Eur. J. Biochem. 1978; 89: 379-388Crossref PubMed Scopus (268) Google Scholar, 23Isobe T. Okuyama T. Eur. J. Biochem. 1981; 116: 79-86Crossref PubMed Scopus (215) Google Scholar, 24Kato K. Kimura S. Biochim. Biophys. Acta. 1985; 842: 146-150Crossref PubMed Scopus (144) Google Scholar, 25Zimmer D.B. Van Eldik L.J. Am. J. Physiol. 1987; 252: C285-C289Crossref PubMed Google Scholar). While human myocardium normally expresses S100α (24Kato K. Kimura S. Biochim. Biophys. Acta. 1985; 842: 146-150Crossref PubMed Scopus (144) Google Scholar, 26Haimoto H. Kato K. Eur. J. Biochem. 1988; 171: 409-415Crossref PubMed Scopus (70) Google Scholar), we detected, unexpectedly, in post-mortem myocardium of human subjects deceased with chronic lung disease, the presence of S100β, a subunit usually expressed in brain, presumably induced in heart prior to death by elevated levels of endogenous or exogenously administered catecholamines (27Kahn H.J. Baumal R. Van Eldik L.J. Marks A. Dunn R.J. Modern Pathol. 1991; 4: 698-701PubMed Google Scholar). S100β has been shown to bind specifically in vitro to several substrates of PKC inhibiting their phosphorylation by the enzyme, with a preferential inhibition of phosphorylation of some substrates by recombinant β-PKC relative to the α- and γ-PKC subtypes (28Albert K.A. Wu W.C.-S. Nairn A.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3622-3625Crossref PubMed Scopus (118) Google Scholar, 29Baudier J. Mochly-Rosen D. Newton A. Lee S.-H. Koshland Jr., D.E. Cole R.D. Biochemistry. 1987; 26: 2886-2893Crossref PubMed Scopus (123) Google Scholar, 30Baudier J. Delphin C. Grunwald D. Khochbin S. Lawrence J.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11627-11631Crossref PubMed Scopus (302) Google Scholar, 31Sheu F.-S. Azmitia F.C. Marshak D.R. Parker P.J. Routtenberg A. Mol. Brain Res. 1994; 21: 62-66Crossref PubMed Scopus (59) Google Scholar). In the present article, report that, first, as in response to pressure overload, the induction of skACT in rat myocardium post-infarction is transient, and its down-regulation is temporally associated with the delayed induction of S100β. Second, transfection of an S100β expression vector into cultured neonatal rat cardiac myocytes inhibits the β-PKC mediated induction of β-MHC and skACT by NE and PE, respectively. Third, hypoxia induces β-MHC transcription in cultured myocytes, a response similarly blocked by forced expression of S100β. These results suggest that the appropriately timed induction of S100β is part of an intrinsic negative regulatory pathway that limits the hypertrophic response following myocardial infarction. The experimental model of lateral wall myocardial infarction by surgical left coronary artery ligation was established in male Sprague-Dawley rats aged 12–14 weeks as described previously (1Orenstein T.L. Parker T.G. Butany J.W. Goodman J.M. Dawood F. Wen W.-H. Wee L. Martino T. McLaughlin P.R. Liu P.P. J. Clin. Invest. 1995; 96: 858-866Crossref PubMed Scopus (103) Google Scholar). A DNA construct DO3.44 encoding the complete amino acid sequence of human S100β flanked by 16 and 650 nucleotides of 5′- and 3′-untranslated sequences, respectively, was assembled by joining the cDNA clone KN3 (32Allore R. O'Hanlon D. Price R. Neilson K. Willard H.F. Cox D.R. Marks A. Dunn R.J. Science. 1988; 239: 1311-1313Crossref PubMed Scopus (189) Google Scholar) with the genomicEcoRI-BamHI fragment DO2 that is contiguous with the 3′-end of KN3 cDNA at the EcoRI site and extends to the BamHI site near the 3′-end of exon 3 of S100β (33Allore R.J. Friend W.C. O'Hanlon D. Neilson K.M. Baumal R. Dunn R.J. Marks A. J. Biol. Chem. 1990; 265: 15537-15543Abstract Full Text PDF PubMed Google Scholar) (see Fig. 2). A control DNA construct DO3.5 was assembled by joining DO2 with KN3 in the reverse orientation. The two constructs were inserted into the pECE expression vector (34Ellis J. Clauser E. Morgan D.O. Edery M. Roth R.A. Rutter W.J. Cell. 1986; 45: 721-732Abstract Full Text PDF PubMed Scopus (696) Google Scholar) to derive plasmids pPDO3.44 (directing the expression of S100β mRNA) and pPDO3.5 (control). The reporter plasmids β-MHC-CAT, skACT-CAT, and α-MHC-CAT in which the respective rat β-MHC, skACT, and α-MHC promoters are joined to chloramphenicol acetyltransferase (CAT), and RSV-LUX, in which a luciferase (LUX) reporter gene is constitutively driven by the Rous sarcoma virus (RSV) long terminal repeat, as well as the expression plasmids SRα-PKCβ (specifying wild-type β-PKC) and SRα-ΔPKCβ (specifying a constitutively active β-PKC) (18Kariya K. Karns L.R. Simpson P.C. J. Biol. Chem. 1991; 266: 10023-10026Abstract Full Text PDF PubMed Google Scholar), were obtained from Dr. P. C. Simpson (18Kariya K. Karns L.R. Simpson P.C. J. Biol. Chem. 1991; 266: 10023-10026Abstract Full Text PDF PubMed Google Scholar, 19Karns L.R. Kariya K. Simpson P.C. J. Biol. Chem. 1995; 270: 410-417Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Neonatal cardiac myocytes were isolated from the ventricles of 2-day old Sprague-Dawley rats and established in primary culture as described (8Simpson P.C. Circ. Res. 1985; 56: 884-894Crossref PubMed Scopus (405) Google Scholar, 10Parker T.G. Packer S.E. Schneider M.D. J. Clin. Invest. 1990; 85: 507-514Crossref PubMed Scopus (309) Google Scholar). Transfection was carried out by CaPO4 precipitation (18Kariya K. Karns L.R. Simpson P.C. J. Biol. Chem. 1991; 266: 10023-10026Abstract Full Text PDF PubMed Google Scholar), using the specific quantities of the following plasmids: β-MHC-CAT or skACT-CAT, 2 pmol (9 μg); RSV-LUX, 0.02 pmol (0.1 μg); pECE, pPDO3.44 or pPDO3.5, 2 pmol (9 μg); SRα-PKCβ or SRα-ΔPKCβ, 0.2 pmol (1 μg), as indicated in legends. Myocyte cultures were maintained in medium supplemented with 5% fetal bovine serum for 18 h following transfection, prior to transfer to serum-free medium and treatment with NE (20 μm), PE (20 μm), or vehicle (100 μm ascorbic acid) for 48 h as specified in legends. The cell lysates were assayed for LUX and CAT activity using published techniques (35De Wet J.R. Wood K.V. De Luca M. Helinski D.R. Subramani S. Mol. Cell. Biol. 1987; 7: 725-737Crossref PubMed Scopus (2478) Google Scholar, 36Gorman C.M. Moffat L.F. Howard B.M. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5294) Google Scholar). Co-transfection with any of the plasmids or treatment of cultures as described above did not affect LUX activity. The differences of the respective LUX and CAT activities between duplicate dishes were <10% of their mean. CAT activity was normalized for transfection efficiency on the basis of LUX activity in the same dish. Treated/control ratios were tested for deviation from unity by calculation of confidence limits. Cardiac myocytes under normoxic conditions were maintained in a well humidified incubator at 37 °C in 1.5% CO2, 98.5% air. Hypoxic conditions were achieved by placing myocyte cultures in an incubator for 18 h, flushed with a gas mixture containing 5% CO2 and 95% N2. RNA was isolated from residual non-infarcted rat left ventricular myocardium outside the territory supplied by the ligated coronary artery by a one-step acid guanidinium phenol method (37Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63167) Google Scholar). RNase protection assays to determine steady-state levels of skACT mRNA, S100β mRNA, and glyceraldehyde-3 phosphate dehydrogenase (GAPDH) mRNA were performed by modifying published conditions (38Rokosh D.G. Bailey B.A. Stewart A.F.R. Karns L.R. Long C.S. Simpson P.C. Biochem. Biophys. Res. Commun. 1994; 200: 1177-1184Crossref PubMed Scopus (130) Google Scholar). Antisense riboprobes for the rat S100β (39Dunn R. Landry C. O'Hanlon D. Dunn J. Allore R. Brown I. Marks A. J. Biol. Chem. 1987; 262: 3562-3566Abstract Full Text PDF PubMed Google Scholar), skACT (a gift from Dr. L. K. Karns, Laboratory of Molecular Neuro-Oncology, University of Virginia Health Sciences, Charlottesville, VA), and GAPDH (Ambion, Austin, TX) were labeled with [α-32]UTP (800 Ci/mmol, Amersham, Oakville, Ontario) by in vitro transcription with SP6 RNA polymerase of appropriate RNA synthesis vectors. The S100β, skACT, and GAPDH probes (2 ng, 106 dpm) were hybridized with 25 μg (S100β) or 15 μg (skACT, GAPDH) of total RNA from rat heart or brain, or rat tRNA for 18 h at 45 °C and RNase-resistant hybrids were recovered using a commercial kit (Ambion), analyzed on 8m urea, 6% polyacrylamide sequencing gels and visualized by autoradiography. RNA was isolated from myocyte cultures (37Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63167) Google Scholar) and pretreated with RNase-free DNase I (Boehringer Mannheim, Laval, Quebec) prior to first strand cDNA synthesis with random hexamer primers. The primers used for PCR amplification were 5′-TGGACAATGATGGAGACGG-3′ and 5′-ATTAGCTACAACACGGCTGG-3′ located in the coding and 3′-untranslated regions, respectively, of exon 3 of the human S100β gene (indicated in Fig. 2). These primers direct the synthesis of a 210-bp fragment from human but not rat DNA. PCR was performed for 30 cycles, with denaturation at 94 °C for 1 min, annealing at 50 °C for 1 min, and extension at 72 °C for 1 min, with an extra 5-min extension for the last cycle. Aliquots of PCR products (10 μl) were separated by electrophoresis on 1.5% agarose gels in 40 mm Tris acetate, 1 mm EDTA, pH 8.0. Gels were visualized after ethidium bromide staining under UV transillumination. The steady-state levels of skACT and S100β mRNAs were measured in a rat model of myocardial infarction resulting from coronary artery ligation (1Orenstein T.L. Parker T.G. Butany J.W. Goodman J.M. Dawood F. Wen W.-H. Wee L. Martino T. McLaughlin P.R. Liu P.P. J. Clin. Invest. 1995; 96: 858-866Crossref PubMed Scopus (103) Google Scholar). The skACT mRNA, normally present in fetal and neonatal, but absent from adult rat myocardium, was re-expressed up to 7 days post-infarction and absent at 21 and 35 days, suggesting a down-regulation of the mRNA (Fig.1 A). S100β mRNA, absent from normal heart, was induced beginning at 7 days and increased up to day 35 post-infarction (Fig. 1 B). GAPDH steady state mRNA levels served as a control for the quality and loading of the mRNA. To investigate a possible functional role for S100β induction in cardiac myocytes surviving infarction, we took advantage of primary cultures of neonatal rat cardiac myocytes (8Simpson P.C. Circ. Res. 1985; 56: 884-894Crossref PubMed Scopus (405) Google Scholar). As a first step toward investigating if S100β could modulate pathways associated with hypertrophy in cell culture, we verified the expression of S100β mRNA in cardiac myocytes following transfection with the human S100β expression plasmid pPDO3.44 (Fig.2). Since 3–5% of cultured cardiac myocytes are transfected by exogenous DNA (18Kariya K. Karns L.R. Simpson P.C. J. Biol. Chem. 1991; 266: 10023-10026Abstract Full Text PDF PubMed Google Scholar), we used RT-PCR to demonstrate that pPDO3.44 directed the expression of human S100β mRNA following transfection (Fig.3). The uptake of co-transfected plasmids by the same competent cells in cardiac myocyte cultures allows the measurement of the modulation of reporter constructs by co-transfected signaling molecules in the setting of low transfection efficiency (18Kariya K. Karns L.R. Simpson P.C. J. Biol. Chem. 1991; 266: 10023-10026Abstract Full Text PDF PubMed Google Scholar, 19Karns L.R. Kariya K. Simpson P.C. J. Biol. Chem. 1995; 270: 410-417Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The use of reporter constructs in which cardiac-specific promoters are fused to CAT and expression vectors specifying putative signaling molecules in this system offers an effective strategy for dissecting the pathways associated with hypertrophy. While signal transduction pathways activated following myocardial infarction are likely to be multifactorial, α1-adrenergic stimulation of cardiac myocyte cultures results in a hypertrophic phenotype involving the reproducible induction of β-MHC and skACT (9Bishopric N.H. Simpson P.C. Ordahl C.P. J. Clin. Invest. 1987; 80: 1194-1199Crossref PubMed Scopus (160) Google Scholar, 11Parker T.G. Chow K.-L. Schwartz R.J. Schneider M.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7066-7070Crossref PubMed Scopus (77) Google Scholar, 12Waspe L.E. Ordahl C.P. Simpson P.C. J. Clin. Invest. 1990; 85: 1206-1214Crossref PubMed Scopus (157) Google Scholar, 19Karns L.R. Kariya K. Simpson P.C. J. Biol. Chem. 1995; 270: 410-417Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 21Kariya K. Karns L.R. Simpson P.C. J. Biol. Chem. 1994; 269: 3775-3782Abstract Full Text PDF PubMed Google Scholar), as described above for surviving myocardium following coronary artery ligation. To investigate whether S100β could modulate this specific pathway, we co-transfected cultured myocytes with β-MHC-CAT and pPDO3.44 prior to NE treatment. The approximately 3-fold induction of β-MHC-CAT by NE was inhibited by co-transfection with pPDO3.44, but not by pPDO3.5 or pECE (Fig. 4), suggesting that expression of S100β was responsible for the abrogation of the NE response. S100β had no effect on basal transcription from the β-MHC promoter. In parallel experiments, we showed that co-transfection of pPDO3.44 also inhibited the induction of skACT-CAT by PE (Fig. 5).Figure 5Induction of the skACT promoter by PE is inhibited by co-transfection with an S100β expression vector.Duplicate cultures of cardiac myocytes were co-transfected with plasmids skACT-CAT and RSV-LUX in combination with one of the following plasmids: pECE, pPD03.44, or pPDO3.5, and treated for 48 h with PE or vehicle alone (control). Bars denote mean ± S.E. of the ratio of CAT activities of PE-treated to control cultures (set at 1.0) or four independent experiments with two different preparation of S100β expression plasmids. Significant differences between PE-treated and control cultures are indicated by * (p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The induction of β-MHC and skACT by α1-adrenergic agonists in cultured cardiac myocytes can be reproduced experimentally by transfecting these cells with a constitutively active β-PKC (ΔPKCβ), suggesting the involvement of the PKC signaling pathway in this induction (18Kariya K. Karns L.R. Simpson P.C. J. Biol. Chem. 1991; 266: 10023-10026Abstract Full Text PDF PubMed Google Scholar, 19Karns L.R. Kariya K. Simpson P.C. J. Biol. Chem. 1995; 270: 410-417Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). To establish that S100β blocked the NE or PE activation of the β-MHC or skACT promoters, respectively, by interrupting the PKC signaling pathway, we examined the effect of co-transfection of a constitutively active mutant of β-PKC (ΔPKCβ) and S100β on β-MHC and skACT transcription. Transfection with ΔPKCβ (but not wild type β-PKC) induced the β-MHC and skCAT promoters approximately 2.5-fold (Figs.6 and 7), as described previously (18Kariya K. Karns L.R. Simpson P.C. J. Biol. Chem. 1991; 266: 10023-10026Abstract Full Text PDF PubMed Google Scholar, 19Karns L.R. Kariya K. Simpson P.C. J. Biol. Chem. 1995; 270: 410-417Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Co-transfection with S100β blocked the induction of β-MHC-CAT and skACT-CAT by ΔPKCβ when the cells were concomitantly treated with NE and PE, respectively, or by increasing the extracellular Ca2+ concentration from 2 to 3 mm (Figs. 6 and 7). Since these treatments promote release of Ca2+ from intracellular stores (40Eckel J. Gerlach-Eskuchen E. Reinauer H. J. Mol. Cell Cardiol. 1991; 23: 617-625Abstract Full Text PDF PubMed Scopus (27) Google Scholar), or increase extracellular Ca2+ influx, and by themselves (i.e. in the absence of co-transfection with S100β) do not block the induction of β-MHC or skACT by ΔPKCβ (data not shown), our results suggest that S100β interruption of the PKC signaling pathway is Ca2+ dependent. In additional experiments, we demonstrated that expression of S100β in cardiac myocytes did not block the induction of the α-MHC promoter by thyroid hormone, a non-PKC mediated signaling pathway (data not shown). This result confirmed the specificity of action of S100β on the PKC signaling pathway in cardiac myocytes and eliminated the possibility of a generalized inhibition of trans-activation.Figure 7Induction of the skACT promoter by a constitutively active β-PKC (ΔPKCβ) is inhibited by co-transfection with an S100β expression vector in the presence of PE or an increased extracellular Ca2+ concentration.Duplicate culture of cardiac myocytes were co-transfected with skACT-CAT and RSV-LUX together with expression plasmids SRα-PKCβ (specifying wild type β-PKC) or SRα-ΔPKCβ (specifying a constitutive active β-PKC) and, in addition, where shown, with pECE or pPDO3.44. As indicated, in some experiments, cultures co-transfected with pPDO3.44 were concomitantly treated with PE (20 μm) or exposed to a higher extracellular Ca2+ concentration (increased from 2 to 3 mm) for 48 h following transfection. Bars denote mean ± S.E. of a ratio of CAT activities of experimental to control cultures (co-transfected with the expression vector SRα, skACT-CAT, and RSV-LUX, and treated with vehicle) (set at 1.0) of four independent experiments. The significant differences between experimental and control cultures are indicated by * (p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To examine the potential role of hypoxia as an alternate mechanism for regulating the cardiac phenotype post-infarction, we examined the effect of hypoxia on β-MHC transcription and modulation by S100β in cultured myocytes. Hypoxia induced the β-MHC promoter approximately 2.5-fold with β-MHC transcription returning to baseline following 24 h of re-exposure to normal oxygen tension (Fig.8). S100β overexpression significantly attenuated the hypoxic induction of the β-MHC promoter to approximately 1.3-fold over baseline. Confirming no effect on basal transcription, S100β did not alter β-MHC promoter activity under either normoxic conditions or following hypoxia with subsequent re-oxygenation. Hypoxia did not result in cellular necrosis as myocyte numbers in culture remained unchanged during the time course of these experiments (data not shown). Acute myocardial infarction remains the leading cause of death in the developed world. Among survivors, subsequent morphological and biochemical alterations in spared cardiac muscle can lead to congestive heart failure, contributing to additional morbidity and mortality (41Pfeffer M.A. Braunwald E. Moyé L.A. Basta L. Brown Jr., E.J. Cuddy T.E. Davis B.R. Geltman E.M. Goldman S. Flaker G.C. Klein M. Lamar G.A. Packer M. Rouleau J. Rouleau J.L. Rutherford J. Wertheimer J.H. Hawkins C.-M. N. Eng. J. Med. 1992; 327: 669-677Crossref PubMed Scopus (5464) Google Scholar). 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