- Email: [email protected]
MEF2 is upregulated during cardiac hypertrophy and is required for normal post-natal growth of the myocardium
Brief Communication
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MEF2 is upregulated during cardiac hypertrophy and is required for normal post-natal growth of the myocardium Steven M. Kolodziejczyk, Ling Wang, Kim Balazsi, Yves DeRepentigny, Rashmi Kothary and Lynn A. Megeney followed by an in vitro kinase reaction. Although this assay labeled a number of targets, one protein migrating at approximately 53 kDa displayed an enhanced phosphate incorporation, which increased in mdx:MyoD–/– lysates (Figure 1a). To identify the 53 kDa protein, lysates were subjected to a pre-immunoprecipitation with antibodies to known p38 targets (including NFκB, MEF-2 and AP-1), followed by the same immunoprecipitation/kinase assay described above. Only the pre-immunoprecipitation with a MEF2 antibody resulted in a specific reduction in the phosphate labeling of the 53 kDa protein, which was similar to the reported size of MEF2 isoforms (Figure 1b,c). In addition, mdx:MyoD–/– cardiac lysates subjected to MEF2 immunoprecipitation revealed an elevated association with p38 (as detected by western blotting), confirming the enhanced interaction of these proteins during hypertrophy (data not shown).
Figure 1
Correspondence: Lynn A. Megeney E-mail: [email protected]
kDa 66
–MEF2
46 *
(b)
0960-9822/99/$ – see front matter © 1999 Elsevier Science Ltd. All rights reserved.
Results and discussion The stress-activated kinase p38 undergoes enhanced activation concurrent to the development of cardiomyocyte hypertrophy in a murine model of dystrophy, the mdx:MyoD–/– mouse [17]. Moreover, p38 activation leads to cardiomyocyte hypertrophy in vitro, suggesting an important role for this kinase in regulating post-natal growth of the myocardium [18,19]. To discover the mechanism by which p38 induces cardiomyocyte hypertrophy, we identified protein targets of p38 during hypertrophic growth. Lysates from wild-type and mdx:MyoD–/– mice were subjected to co-immunoprecipitation with a p38 antibody
kDa 66 46
–/ –
IP: anti-p38 In vitro kinase assay
Published: 11 October 1999 Current Biology 1999, 9:1203–1206
*
W ild md type x:M yo D
Received: 23 April 1999 Revised: 4 August 1999 Accepted: 13 September 1999
200 µg
nt md rol x: Co Myo ntr D –/– md ol x 50 :Myo x c D– / 1 µ old o – la lig 3 µ nti-M o la E nti F2 -M EF 2
Address: Ottawa Hospital Research Institute, Ottawa Hospital General Campus, Centre for Molecular Medicine, Ottawa, Ontario, Canada K1H 8L6.
(c) 50 µg
Co
–/
–
(a)
W ild md type x: Pr Myo e md -I P D –/– x:M yo D
In mammals, growth of the fetal heart is regulated by proliferation of cardiac muscle cells. At later stages of pre-natal life, this proliferation diminishes profoundly [1,2] and the dramatic expansion in heart size during the transition to adulthood is due exclusively to hypertrophy of individual cardiomyocytes [3–5]. Cardiomyocyte hypertrophy also contributes to the pathology of most post-natal heart disease [6–10]. Within this context, numerous signal transduction pathways have been implicated as the link between the effector(s) and altered cardiac gene expression [11–16]. A common pathway has yet to be discovered, however. Here, we found that the activity of the stressactivated kinase p38 was enhanced in both types of cardiomyocyte hypertrophy. We also found that a target of the activated p38 kinase is the cardiac transcription factor MEF2. Transgenic mice expressing a dominant-negative form of MEF2C displayed attenuated post-natal growth of the myocardium. These results provide the first evidence for a single pathway regulating both normal and pathologic cardiomyocyte hypertrophy.
MEF2A MEF2D MEF2C –p38
Western: anti-MEF2 and anti-p38
Gel-shift assay Current Biology
MEF2 activity in heart lysates of wild-type and mdx:MyoD–/– mice. (a) The p38-directed phosphorylation of a 53 kDa protein, identified as MEF2 in a pre-immunoprecipitation (pre-IP) using an anti-MEF2 antibody. Asterisk, autophosphorylated form of p38. (b) Western blot with anti-p38 and anti-MEF2 antibodies. (c) Gel-shift assay using a labeled MEF2 consensus oligonucleotide. The total amount of protein used is indicated at the top. Specificity of the MEF2 complex (asterisk) was determined by competitive inhibition with unlabeled MEF2 consensus oligonucleotide (oligo) and supershifting of the complex with anti-MEF2 antibody (arrowhead). Similar results were obtained in three independent experiments.
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kDa 46 30
12 weeks
–p38
kDa 66 46
Ne w 2 w bor n 7 w eek s e 12 ek we s ek s
IP: anti-PTyr Western: anti-p38 (b)
Summary of α-MHC–MEF2C transgenic lines.
(d)
Ne 2 wwbo r 7 w eek n s e 12 ek we s ek s + An +A ti-M +Nnti-M E F2 Fκ E K B 7 oli go
(a)
Table 1
Ne w 2 w bor n 7 week s e 12 ek we s ek s
Figure 2
*
*
–MEF2
* IP: anti-p38 In vitro kinase assay
kDa 66 46
MEF2A MEF2D MEF2C –p38 Western: anti-MEF2 and anti-p38
1 5 15 17 22 24 25 28 30 31 37
Age at death (weeks)
Cardiac phenotype
2 (sudden) 8 (sacrificed) 8 (sacrificed) 4 (sudden) 7 (sacrificed) Still alive Still alive Still alive 7 (sacrificed) 4 (sudden) Still alive
Hypoplastic Dilated/hypoplastic Mild Hypoplastic Mild n.d. n.d. n.d. Dilated/hypoplastic Hypoplastic n.d.
n.d. = not determined
Ne w 2 w bor 7 w eek n 12 eek s we s ek s
(c)
Transgenic line
Gel-shift assay
Current Biology
MEF2 activity in heart lysates from newborn mice and mice aged 2, 7 and 12 weeks. (a) Immunoprecipitation (IP) with an antiphosphotyrosine (anti-PTyr) antibody, followed by western blotting with anti-p38 antibody. (b) The p38-directed phosphorylation of MEF2 (53 kDa). Asterisk, autophosphorylated p38. (c) Western blotting with anti-p38 and anti-MEF2 antibodies. (d) Gel-shift assay using a labeled MEF2 consensus oligonucleotide. Formation of a MEF2 complex (asterisk) remained unaffected in the presence of either a non-specific antibody (MEK7) or a non-specific transcription factor binding site (NFκB oligonucleotide). Similar results were obtained in three independent experiments. The arrowhead indicates a supershift of the complex with the anti-MEF2 antibody.
To examine MEF2 activity during cardiomyocyte hypertrophy, we performed gel mobility-shift assays using a MEF2-binding site as a probe. Compared with wild-type mice, heart lysates from mdx:MyoD–/– mice displayed a significant increase in MEF2 complex formation (Figure 1c), which is similar to the enhanced MEF2 binding reported for pressure overload hypertrophy [20]. Consequently, these observations suggest a role for the activated kinase in pathologic cardiomyocyte hypertrophy. It is interesting that members of the MEF2 family of transcription factors, specifically MEF2A and MEF2C, have been shown to be indispensible for regulating cardiac gene expression, and are highly expressed in both the developing and mature myocardium (Figure 1c; reviewed in [21]). As normal post-natal cardiomyocyte growth exhibits many features common to disease hypertrophy, there may be considerable overlap in the molecular cascades that regulate each process. Accordingly, we investigated the interaction between p38 kinase and MEF2 transcription factors in the post-natal developing heart. Tyrosine phosphorylation of p38 was elevated in lysates derived
from early-stage hearts and decreased in heart lysates of mature animals (Figure 2a). The heart lysates from the developmental time course were also subjected to p38 immunoprecipitation/kinase assays, which revealed a high level of MEF2 phosphate labeling (53 kDa protein) at early stages, declining with maturity (Figure 2b). In addition, gel mobility-shift assays demonstrated a high level of MEF2-binding activity for heart lysates derived from young mice (≤ 2 weeks of age), with a substantial reduction in activity as animals aged (Figure 2d). Therefore, upregulation of p38/MEF2 activity occurs at developmental stages when the heart is subject to the largest degree of cardiomyocyte hypertrophy. Interestingly, the elevated MEF2-binding activity was concurrent to an increased level of MEF2 proteins (Figure 2c), suggesting a further synergy for p38-mediated activation of this transcription factor. To determine whether increased p38/MEF2 activity was required for post-natal cardiomyocyte hypertrophy, we generated transgenic mice that expressed a truncated MEF2C protein (the carboxy-terminal transactivation domain was deleted) under the control of the α-myosin heavy chain promoter (α-MHC–MEF2C) [13,22]. We hypothesized that removal of this region of MEF2C, which includes the known p38-regulatable serine and threonine residues [23,24], would result in a loss of functional protein. Indeed, similar truncations in MEF2A have been shown to produce a dominant-negative protein in vitro [25]. Cardiac lysates from MEF2 transgenic mice revealed a significant reduction in MEF2–DNA complex formation (Figure 3e), which is similar to the dominant-negative effect that has been observed with overexpression of full-length MEF2C containing mutations in the p38-responsive residues [23]. Thus, our observations suggest that p38-mediated phosphorylation of MEF2 leads to an enhanced formation of MEF2–DNA complexes, which would directly affect MEF2-dependent transcription.
Brief Communication
Taken together, our data suggest that p38 activation of MEF2 is indispensible for precipitating the hypertrophic response in post-natal cardiomyocytes. Importantly, this interaction was evident in both normal and pathologic hypertrophy, which suggests a conserved molecular mechanism for cardiomyocyte adaptation. Finally, the data support the view that effective post-natal cardiac adaptation may be dependent on activating multiple cardiac transcription factors (that is, NFAT, GATA4 and MEF2), which recapitulates the paradigm observed for developmental cardiac morphogenesis [27].
Materials and methods Histological analysis Hearts were removed and fixed in 4% paraformaldehyde for 4–5 days, embedded in paraffin, sectioned at 4 µm and stained with hematoxylin–eosin [28].
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A simple interpretation of our observations suggests that p38 activation of MEF2 proteins in the hypertrophic heart may be specific to a p38–MEF2C interaction as, first, the p38 immunoprecipitation/kinase assays did not detect phosphorylation of a protein the size of MEF2A (~68–70 kDa) and, second, the competitive inhibition of the phosphorylated 53 kDa protein matched the known size of MEF2C (~53 kDa). Nevertheless, MEF2A protein, which is abundant in the adapting heart (Figure 2c), has been shown to be a responsive target for p38-directed activation [24,26]. Moreover, the cardiac phenotype of the MEF2C dominant-negative mouse does not exclude a role for MEF2A. Specifically, as MEF2A and MEF2C use a common consensus site, the underlying molecular basis for the dominant-negative MEF2C transgene would be equally effective in disrupting both MEF2C and MEF2A interactions with DNA.
Figure 3
W ild
Of the 11 independent founder mice that were obtained, three died early (≤ 4 weeks of age) with severe hypoplastic hearts (Figure 3b and Table 1). Histological analysis of the affected hearts revealed a dramatic reduction in cardiomyocyte size compared with hearts from age-matched controls (Figure 3c,d). Examination of hearts from surviving founder mice provided further support for a hypoplastic phenotype; two out of four mice had dilated ventricle chambers while maintaining outward cardiac dimensions similar to wild-type mice (data not shown). In addition, hearts from affected mice displayed a significant reduction (~2–3-fold) in the expression of genes associated with pathologic hypertrophy, such as Atrial natriuretic factor (ANF) (Figure 3f) and α-skeletal actin (data not shown). Thus, disruption of MEF2 activity leads to impaired cardiomyocyte hypertrophy.
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ANF northern
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Inhibited myocardial growth in mice expressing a truncated MEF2C protein, dmMEF2C. (a,c) Wild-type and (b,d) dmMEF2C-expressing mice. Hematoxylin–eosin stained sections of hearts (cut transversely at mid-ventricle) revealed a substantial reduction in the size of the myocardium in (b) 4 week old dmMEF2C mice compared with (a) age-matched wild-type mice. Histological comparison of hearts revealed a marked reduction in size of individual cardiomyocytes from (d) dmMEF2C mice compared with (c) wild-type mice of the same age (magnification 40 ×). (e) Reduced MEF2–DNA complex formation (asterisk) in dmMEF2C (mutant) heart lysates. (f) The decrease in ANF gene expression in dmMEF2C (mutant) hearts was related to the severity of the phenotype; the hypoplastic heart phenotype was visibly more severe in mutant B than in mutant A, with a corresponding reduction in ANF expression. The scale bars represent 1 mm in (a,b) and 50 µm in (c,d). duction Labs), 500 µg total protein was used with 5 µg PY20; for immunoprecipitations using anti-p38 (Santa Cruz) or anti-MEF-2 antibodies, 750 µg total protein was used with 2 µg of either anti-p38 or anti-MEF2 antibodies; all contained 25 µl protein G–Sepharose beads (Pharmacia). Immunoprecipitation was carried out for 16 h at 4°C. Precipitates were centrifuged, washed (3 ×) and subjected to SDS–PAGE followed by transfer to Immobilon-P membranes (Millipore). Detection of proteins by western blotting used the following dilutions for primary antibodies: p38 and MEF-2 1:500 (Santa Cruz); secondary 1:2000.
Immunoprecipitation and western blot analysis Immunoprecipitations and western blotting from the ventricle lysates were performed as described previously [17]. Briefly, for immunoprecipitations using the pan-tyrosine-phosphorylated antibody PY20 (Trans-
In vitro kinase assays Immunoprecipitations with anti-p38 antibody were performed as described above. For MEF2 inhibition experiments, a pre-immunoprecip-
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itation (3 h at 4°C) with 2 µg anti-MEF2 antibody was performed before the p38 immunoprecipitation. Following this procedure, the precipitates were washed twice with kinase buffer [17]. Reactions were then carried out with 10 µCi [γ-32P]ATP in a total kinase buffer volume of 30 µl. Reactions were allowed to proceed for 30 min at 30°C and then terminated with the addition of 2 × SDS loading buffer and subjected to SDS–PAGE. Gels were dried and exposed to film and/or subjected to phosphoimaging to quantitate the signal.
Gel mobility-shift assays Heart lysates were subjected to protein extraction as described above. Briefly, 200 µg total protein lysate per sample was incubated with a labeled ([γ-32P]ATP) MEF2 consensus oligonucleotide probe (Santa Cruz) in the presence of 2 µg poly [dI-dC], 2 µl buffer (250 mM HEPES, pH 7.6, 50 mM MgCl2, 340 mM KCl) in a final volume of 20 µl for 30 min at 37°C, followed by gel electrophoresis. Competitor oligonucleotides were added at 50-fold molar excess, while supershift experiments were performed with 2 µg specific or non-specific antibodies. Signal quantification was determined by phosphorimager analysis.
Northern analysis Total RNA extraction and northern blotting for ANF detection were performed as described previously [29].
Transgenic mice A MEF2C construct encoding amino acids 1–95 was produced by PCR amplification using primers corresponding to the required 5′ and 3′ regions. To direct post-natal cardiac-specific expression of the MEF2C construct, the sequence-verified PCR product was cloned into an expression vector containing the α-MHC promoter [22]. The fusion vector was then digested with BamHI, and the α-MHC–MEF2C transgene was gelpurified and eluted in oocyte injection buffer. Injection and derivation of transgenic mice were performed as described [30].
Acknowledgements We thank David Picketts, Michael Rudnicki and Ilona Skerjanc for helpful discussions, and J. Robbins and J. Molkentin for providing the alpha-MHC promoter. S.M.K. was supported by a postdoctoral fellowship from the Foundation for Cell and Gene Therapy. This work was supported by a grant from MDA (USA) to L.A.M.
References 1. Nag AC, Carey TR, Cheng M: Assessment of cardiomyocyte DNA synthesis in rat heart cells after injury and the regeneration of myocardia. Tissue Cell 1983, 15:597-613. 2. Soonpa MH, Field LJ: Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts. Am J Physiol 1997, 272:H220-H226. 3. Claycomb WC: Control of cardiac muscle cell division. Trends Cardiovasc Med 1992, 2:231-236. 4. Kim HD, Kim DJ, Lee IJ, Rah BJ, Sawa Y, Schaper J: Human fetal heart development after mid-term: morphometry and ultrastructural study. J Mol Cell Cardiol 1992, 24:949-965. 5. Li F, Wang X, Capasso JM, Gerdes AM: Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development. J Mol Cell Cardiol 1996, 28:1737-1746. 6. Schwartz K, Boheler KR, de la Bastie D, Lompre AM, Mercadier JJ: Switches in cardiac muscle gene expression as a result of pressure and volume overload. Am J Physiol 1992, 262:R364-R369. 7. Katz AM: The cardiomyopathy of overload: an unnatural growth response. Eur Heart J 1995, 16 Suppl. O:110-114. 8. Vikstrom KL, Leinwand LA: Contractile protein mutations and heart disease. Curr Opin Cell Biol 1996, 8:97-105. 9. Bonne G, Carrier L, Richard P, Hainque B, Schwartz K: Familial hypertrophy cardiomyopathy: from mutations to functional defects. Circ Res 1998, 83:580-593. 10. Hudlicka O, Brown MD: Postnatal growth of the heart and its blood vessels. J Vasc Res 1996, 33:266-287. 11. Chien KR, Zhu H, Knowlton KU, Miller-Hance W, van Bilsen M, Obrien TX, Evans SM: Transcriptional regulation during cardiac growth and development. Annu Rev Physiol 1993, 55:77-95.
12. Sadoshima J, Izumo S: The cellular and molecular response of cardiac myocytes to mechanical stress. Annu Rev Physiol 1997, 59:551-571. 13. Molkentin JD, Lu J-R, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, Olson EN: A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 1998, 93:215-228. 14. Sussman MA, Lim HW, Gude N, Taigen T, Olson EN, Robbins J, et al.: Prevention of cardiac hypertrophy in mice by calcineurin inhibition. Science 1998, 281:1690-1693. 15. Luo Z, Shyu KG, Gualberto A, Walsh K: Calcineurin inhibitors and cardiac hypertrophy. Nat Med 1998, 4:1092-1093. 16. Mende U, Kagen A, Cohen A, Aramburu J, Schoen FJ, Neer EJ: Transient cardiac expression of constitutively active Gaq leads to hypertrophy and dilated cardiomyopathy by calcineurindependent and independent pathways. Proc Natl Acad Sci USA 1998, 95:13893-13898. 17. Megeney LA, Kablar B, Perry RLS, Ying C, May L, Rudnicki MA: Severe cardiomyopathy in mice lacking dystrophin and MyoD. Proc Natl Acad Sci USA 1999, 96:220-225. 18. Zechner D, Thueraf DJ, Hanford DS, McDonough PM, Glembotski CC: A role for the p38 mitogen-activated protein kinase pathway in myocardial cell growth, sarcomeric organization, and cardiac-specific gene expression. J Cell Biol 1997, 139:115-127. 19. Nemoto S, Sheng Z, Lin A: Opposing effects of Jun kinase and p38 mitogen-activated protein kinases on cardiomyocyte hypertrophy. Mol Cell Biol 1998, 18:3518-3526. 20. Molkentin JD, Markham BE: Myocyte-specific enhancer-binding factor (MEF-2) regulates alpha-cardiac myosin heavy chain gene expression in vitro and in vivo. J Biol Chem 1993, 268:19512-19520. 21. Black BL, Olson EN: Transcriptional control of muscle development by myocyte enhancer factor-2 (MEF2) proteins. Annu Rev Cell Dev Biol 1998, 14:167-196. 22. Jones WK, Sanchez A, Robbins J: Murine pulmonary myocardium: developmental analysis of cardiac gene expression. Dev Dyn 1994, 200:117-128. 23. Han J, Jiang Y, Li Z, Kravcenko VV, Ulevitch, RJ: Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation. Nature 1997, 386:296-299. 24. Zhao M, New L, Kravcehnko VV, Kato Y, Gram H, di Padova F, et al.: Regulation of the MEF2 family of transcription factors by p38. Mol Cell Biol 1999, 19:21-30. 25. Ornatsky OI, Andreucci JJ, McDermott JC: A dominant-negative form of transcription factor MEF2 inhibits myogenesis. J Biol Chem 1997, 272:33271-33278. 26. Ornatsky OI, Cox DM, Tangirala P, Andreucci JJ, Quinn ZA, Wrana JL, et al.: Post-translational control of the MEF2A transcriptional regulatory protein. Nucleic Acids Res 1999, 27:2646-2654. 27. Olson EN, Srivastava D: Molecular pathways controlling heart development. Science 1996, 272:671-676. 28. Bancroft JD, Stevens A: Theory and Practice of Histological Techniques. Edinburgh: Churchill Livingston; 1990. 29. Jones WK, Grupp IL, Doetschman T, Grupp G, Osinska H, Hewett TE, et al.: Ablation of the murine α-myosin heavy chain gene leads to dosage effects and functional deficits in the heart. J Clin Invest 1996, 98:1906-1917. 30. Guy L-G, Kothary R, DeRepentigny Y, Delvoye N, Ellis J, Wall L: The β-globin locus control region enhances transcription of but does not confer position-independent expression onto the lacZ gene in transgenic mice. EMBO J 1996, 15:3713-3721.
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MEF2 is upregulated during cardiac hypertrophy and is required for normal post-natal growth of the myocardium Steven M. Kolodziejczyk, Ling Wang, Kim Balazsi, Yves DeRepentigny, Rashmi Kothary and Lynn A. Megeney followed by an in vitro kinase reaction. Although this assay labeled a number of targets, one protein migrating at approximately 53 kDa displayed an enhanced phosphate incorporation, which increased in mdx:MyoD–/– lysates (Figure 1a). To identify the 53 kDa protein, lysates were subjected to a pre-immunoprecipitation with antibodies to known p38 targets (including NFκB, MEF-2 and AP-1), followed by the same immunoprecipitation/kinase assay described above. Only the pre-immunoprecipitation with a MEF2 antibody resulted in a specific reduction in the phosphate labeling of the 53 kDa protein, which was similar to the reported size of MEF2 isoforms (Figure 1b,c). In addition, mdx:MyoD–/– cardiac lysates subjected to MEF2 immunoprecipitation revealed an elevated association with p38 (as detected by western blotting), confirming the enhanced interaction of these proteins during hypertrophy (data not shown).
Figure 1
Correspondence: Lynn A. Megeney E-mail: [email protected]
kDa 66
–MEF2
46 *
(b)
0960-9822/99/$ – see front matter © 1999 Elsevier Science Ltd. All rights reserved.
Results and discussion The stress-activated kinase p38 undergoes enhanced activation concurrent to the development of cardiomyocyte hypertrophy in a murine model of dystrophy, the mdx:MyoD–/– mouse [17]. Moreover, p38 activation leads to cardiomyocyte hypertrophy in vitro, suggesting an important role for this kinase in regulating post-natal growth of the myocardium [18,19]. To discover the mechanism by which p38 induces cardiomyocyte hypertrophy, we identified protein targets of p38 during hypertrophic growth. Lysates from wild-type and mdx:MyoD–/– mice were subjected to co-immunoprecipitation with a p38 antibody
kDa 66 46
–/ –
IP: anti-p38 In vitro kinase assay
Published: 11 October 1999 Current Biology 1999, 9:1203–1206
*
W ild md type x:M yo D
Received: 23 April 1999 Revised: 4 August 1999 Accepted: 13 September 1999
200 µg
nt md rol x: Co Myo ntr D –/– md ol x 50 :Myo x c D– / 1 µ old o – la lig 3 µ nti-M o la E nti F2 -M EF 2
Address: Ottawa Hospital Research Institute, Ottawa Hospital General Campus, Centre for Molecular Medicine, Ottawa, Ontario, Canada K1H 8L6.
(c) 50 µg
Co
–/
–
(a)
W ild md type x: Pr Myo e md -I P D –/– x:M yo D
In mammals, growth of the fetal heart is regulated by proliferation of cardiac muscle cells. At later stages of pre-natal life, this proliferation diminishes profoundly [1,2] and the dramatic expansion in heart size during the transition to adulthood is due exclusively to hypertrophy of individual cardiomyocytes [3–5]. Cardiomyocyte hypertrophy also contributes to the pathology of most post-natal heart disease [6–10]. Within this context, numerous signal transduction pathways have been implicated as the link between the effector(s) and altered cardiac gene expression [11–16]. A common pathway has yet to be discovered, however. Here, we found that the activity of the stressactivated kinase p38 was enhanced in both types of cardiomyocyte hypertrophy. We also found that a target of the activated p38 kinase is the cardiac transcription factor MEF2. Transgenic mice expressing a dominant-negative form of MEF2C displayed attenuated post-natal growth of the myocardium. These results provide the first evidence for a single pathway regulating both normal and pathologic cardiomyocyte hypertrophy.
MEF2A MEF2D MEF2C –p38
Western: anti-MEF2 and anti-p38
Gel-shift assay Current Biology
MEF2 activity in heart lysates of wild-type and mdx:MyoD–/– mice. (a) The p38-directed phosphorylation of a 53 kDa protein, identified as MEF2 in a pre-immunoprecipitation (pre-IP) using an anti-MEF2 antibody. Asterisk, autophosphorylated form of p38. (b) Western blot with anti-p38 and anti-MEF2 antibodies. (c) Gel-shift assay using a labeled MEF2 consensus oligonucleotide. The total amount of protein used is indicated at the top. Specificity of the MEF2 complex (asterisk) was determined by competitive inhibition with unlabeled MEF2 consensus oligonucleotide (oligo) and supershifting of the complex with anti-MEF2 antibody (arrowhead). Similar results were obtained in three independent experiments.
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kDa 46 30
12 weeks
–p38
kDa 66 46
Ne w 2 w bor n 7 w eek s e 12 ek we s ek s
IP: anti-PTyr Western: anti-p38 (b)
Summary of α-MHC–MEF2C transgenic lines.
(d)
Ne 2 wwbo r 7 w eek n s e 12 ek we s ek s + An +A ti-M +Nnti-M E F2 Fκ E K B 7 oli go
(a)
Table 1
Ne w 2 w bor n 7 week s e 12 ek we s ek s
Figure 2
*
*
–MEF2
* IP: anti-p38 In vitro kinase assay
kDa 66 46
MEF2A MEF2D MEF2C –p38 Western: anti-MEF2 and anti-p38
1 5 15 17 22 24 25 28 30 31 37
Age at death (weeks)
Cardiac phenotype
2 (sudden) 8 (sacrificed) 8 (sacrificed) 4 (sudden) 7 (sacrificed) Still alive Still alive Still alive 7 (sacrificed) 4 (sudden) Still alive
Hypoplastic Dilated/hypoplastic Mild Hypoplastic Mild n.d. n.d. n.d. Dilated/hypoplastic Hypoplastic n.d.
n.d. = not determined
Ne w 2 w bor 7 w eek n 12 eek s we s ek s
(c)
Transgenic line
Gel-shift assay
Current Biology
MEF2 activity in heart lysates from newborn mice and mice aged 2, 7 and 12 weeks. (a) Immunoprecipitation (IP) with an antiphosphotyrosine (anti-PTyr) antibody, followed by western blotting with anti-p38 antibody. (b) The p38-directed phosphorylation of MEF2 (53 kDa). Asterisk, autophosphorylated p38. (c) Western blotting with anti-p38 and anti-MEF2 antibodies. (d) Gel-shift assay using a labeled MEF2 consensus oligonucleotide. Formation of a MEF2 complex (asterisk) remained unaffected in the presence of either a non-specific antibody (MEK7) or a non-specific transcription factor binding site (NFκB oligonucleotide). Similar results were obtained in three independent experiments. The arrowhead indicates a supershift of the complex with the anti-MEF2 antibody.
To examine MEF2 activity during cardiomyocyte hypertrophy, we performed gel mobility-shift assays using a MEF2-binding site as a probe. Compared with wild-type mice, heart lysates from mdx:MyoD–/– mice displayed a significant increase in MEF2 complex formation (Figure 1c), which is similar to the enhanced MEF2 binding reported for pressure overload hypertrophy [20]. Consequently, these observations suggest a role for the activated kinase in pathologic cardiomyocyte hypertrophy. It is interesting that members of the MEF2 family of transcription factors, specifically MEF2A and MEF2C, have been shown to be indispensible for regulating cardiac gene expression, and are highly expressed in both the developing and mature myocardium (Figure 1c; reviewed in [21]). As normal post-natal cardiomyocyte growth exhibits many features common to disease hypertrophy, there may be considerable overlap in the molecular cascades that regulate each process. Accordingly, we investigated the interaction between p38 kinase and MEF2 transcription factors in the post-natal developing heart. Tyrosine phosphorylation of p38 was elevated in lysates derived
from early-stage hearts and decreased in heart lysates of mature animals (Figure 2a). The heart lysates from the developmental time course were also subjected to p38 immunoprecipitation/kinase assays, which revealed a high level of MEF2 phosphate labeling (53 kDa protein) at early stages, declining with maturity (Figure 2b). In addition, gel mobility-shift assays demonstrated a high level of MEF2-binding activity for heart lysates derived from young mice (≤ 2 weeks of age), with a substantial reduction in activity as animals aged (Figure 2d). Therefore, upregulation of p38/MEF2 activity occurs at developmental stages when the heart is subject to the largest degree of cardiomyocyte hypertrophy. Interestingly, the elevated MEF2-binding activity was concurrent to an increased level of MEF2 proteins (Figure 2c), suggesting a further synergy for p38-mediated activation of this transcription factor. To determine whether increased p38/MEF2 activity was required for post-natal cardiomyocyte hypertrophy, we generated transgenic mice that expressed a truncated MEF2C protein (the carboxy-terminal transactivation domain was deleted) under the control of the α-myosin heavy chain promoter (α-MHC–MEF2C) [13,22]. We hypothesized that removal of this region of MEF2C, which includes the known p38-regulatable serine and threonine residues [23,24], would result in a loss of functional protein. Indeed, similar truncations in MEF2A have been shown to produce a dominant-negative protein in vitro [25]. Cardiac lysates from MEF2 transgenic mice revealed a significant reduction in MEF2–DNA complex formation (Figure 3e), which is similar to the dominant-negative effect that has been observed with overexpression of full-length MEF2C containing mutations in the p38-responsive residues [23]. Thus, our observations suggest that p38-mediated phosphorylation of MEF2 leads to an enhanced formation of MEF2–DNA complexes, which would directly affect MEF2-dependent transcription.
Brief Communication
Taken together, our data suggest that p38 activation of MEF2 is indispensible for precipitating the hypertrophic response in post-natal cardiomyocytes. Importantly, this interaction was evident in both normal and pathologic hypertrophy, which suggests a conserved molecular mechanism for cardiomyocyte adaptation. Finally, the data support the view that effective post-natal cardiac adaptation may be dependent on activating multiple cardiac transcription factors (that is, NFAT, GATA4 and MEF2), which recapitulates the paradigm observed for developmental cardiac morphogenesis [27].
Materials and methods Histological analysis Hearts were removed and fixed in 4% paraformaldehyde for 4–5 days, embedded in paraffin, sectioned at 4 µm and stained with hematoxylin–eosin [28].
(c)
(d)
(e)
(f)
nt M rol ut a M nt A ut an tB
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A simple interpretation of our observations suggests that p38 activation of MEF2 proteins in the hypertrophic heart may be specific to a p38–MEF2C interaction as, first, the p38 immunoprecipitation/kinase assays did not detect phosphorylation of a protein the size of MEF2A (~68–70 kDa) and, second, the competitive inhibition of the phosphorylated 53 kDa protein matched the known size of MEF2C (~53 kDa). Nevertheless, MEF2A protein, which is abundant in the adapting heart (Figure 2c), has been shown to be a responsive target for p38-directed activation [24,26]. Moreover, the cardiac phenotype of the MEF2C dominant-negative mouse does not exclude a role for MEF2A. Specifically, as MEF2A and MEF2C use a common consensus site, the underlying molecular basis for the dominant-negative MEF2C transgene would be equally effective in disrupting both MEF2C and MEF2A interactions with DNA.
Figure 3
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Of the 11 independent founder mice that were obtained, three died early (≤ 4 weeks of age) with severe hypoplastic hearts (Figure 3b and Table 1). Histological analysis of the affected hearts revealed a dramatic reduction in cardiomyocyte size compared with hearts from age-matched controls (Figure 3c,d). Examination of hearts from surviving founder mice provided further support for a hypoplastic phenotype; two out of four mice had dilated ventricle chambers while maintaining outward cardiac dimensions similar to wild-type mice (data not shown). In addition, hearts from affected mice displayed a significant reduction (~2–3-fold) in the expression of genes associated with pathologic hypertrophy, such as Atrial natriuretic factor (ANF) (Figure 3f) and α-skeletal actin (data not shown). Thus, disruption of MEF2 activity leads to impaired cardiomyocyte hypertrophy.
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Inhibited myocardial growth in mice expressing a truncated MEF2C protein, dmMEF2C. (a,c) Wild-type and (b,d) dmMEF2C-expressing mice. Hematoxylin–eosin stained sections of hearts (cut transversely at mid-ventricle) revealed a substantial reduction in the size of the myocardium in (b) 4 week old dmMEF2C mice compared with (a) age-matched wild-type mice. Histological comparison of hearts revealed a marked reduction in size of individual cardiomyocytes from (d) dmMEF2C mice compared with (c) wild-type mice of the same age (magnification 40 ×). (e) Reduced MEF2–DNA complex formation (asterisk) in dmMEF2C (mutant) heart lysates. (f) The decrease in ANF gene expression in dmMEF2C (mutant) hearts was related to the severity of the phenotype; the hypoplastic heart phenotype was visibly more severe in mutant B than in mutant A, with a corresponding reduction in ANF expression. The scale bars represent 1 mm in (a,b) and 50 µm in (c,d). duction Labs), 500 µg total protein was used with 5 µg PY20; for immunoprecipitations using anti-p38 (Santa Cruz) or anti-MEF-2 antibodies, 750 µg total protein was used with 2 µg of either anti-p38 or anti-MEF2 antibodies; all contained 25 µl protein G–Sepharose beads (Pharmacia). Immunoprecipitation was carried out for 16 h at 4°C. Precipitates were centrifuged, washed (3 ×) and subjected to SDS–PAGE followed by transfer to Immobilon-P membranes (Millipore). Detection of proteins by western blotting used the following dilutions for primary antibodies: p38 and MEF-2 1:500 (Santa Cruz); secondary 1:2000.
Immunoprecipitation and western blot analysis Immunoprecipitations and western blotting from the ventricle lysates were performed as described previously [17]. Briefly, for immunoprecipitations using the pan-tyrosine-phosphorylated antibody PY20 (Trans-
In vitro kinase assays Immunoprecipitations with anti-p38 antibody were performed as described above. For MEF2 inhibition experiments, a pre-immunoprecip-
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itation (3 h at 4°C) with 2 µg anti-MEF2 antibody was performed before the p38 immunoprecipitation. Following this procedure, the precipitates were washed twice with kinase buffer [17]. Reactions were then carried out with 10 µCi [γ-32P]ATP in a total kinase buffer volume of 30 µl. Reactions were allowed to proceed for 30 min at 30°C and then terminated with the addition of 2 × SDS loading buffer and subjected to SDS–PAGE. Gels were dried and exposed to film and/or subjected to phosphoimaging to quantitate the signal.
Gel mobility-shift assays Heart lysates were subjected to protein extraction as described above. Briefly, 200 µg total protein lysate per sample was incubated with a labeled ([γ-32P]ATP) MEF2 consensus oligonucleotide probe (Santa Cruz) in the presence of 2 µg poly [dI-dC], 2 µl buffer (250 mM HEPES, pH 7.6, 50 mM MgCl2, 340 mM KCl) in a final volume of 20 µl for 30 min at 37°C, followed by gel electrophoresis. Competitor oligonucleotides were added at 50-fold molar excess, while supershift experiments were performed with 2 µg specific or non-specific antibodies. Signal quantification was determined by phosphorimager analysis.
Northern analysis Total RNA extraction and northern blotting for ANF detection were performed as described previously [29].
Transgenic mice A MEF2C construct encoding amino acids 1–95 was produced by PCR amplification using primers corresponding to the required 5′ and 3′ regions. To direct post-natal cardiac-specific expression of the MEF2C construct, the sequence-verified PCR product was cloned into an expression vector containing the α-MHC promoter [22]. The fusion vector was then digested with BamHI, and the α-MHC–MEF2C transgene was gelpurified and eluted in oocyte injection buffer. Injection and derivation of transgenic mice were performed as described [30].
Acknowledgements We thank David Picketts, Michael Rudnicki and Ilona Skerjanc for helpful discussions, and J. Robbins and J. Molkentin for providing the alpha-MHC promoter. S.M.K. was supported by a postdoctoral fellowship from the Foundation for Cell and Gene Therapy. This work was supported by a grant from MDA (USA) to L.A.M.
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