- Email: [email protected]
2-signaling
The International Journal of Biochemistry & Cell Biology 41 (2009) 2351–2355
Contents lists available at ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Medicine in focus
Cardiac hypertrophy: Targeting Raf/MEK/ERK1/2-signaling Kristina Lorenz a,∗ , Joachim P. Schmitt a , Marie Vidal a , Martin J. Lohse a,b a b
Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Straße 9, 97078 Würzburg, Germany Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, University of Würzburg, Versbacher Straße 9, 97078 Würzburg, Germany
a r t i c l e
i n f o
Article history: Received 23 May 2009 Received in revised form 26 July 2009 Accepted 4 August 2009 Available online 8 August 2009 Keywords: Cardiac hypertrophy ERK1/2 Signaling
a b s t r a c t Over the past two decades, basic research has revealed a complex network of regulatory mechanisms that control the ERK1/2-signaling cascade. ERK1/2 mediate cardiac hypertrophy, a major risk factor for the development of arrhythmias, heart failure and sudden death, but also beneficial effects, e.g. protection of the heart from cell death and ischemic injury. Selective targeting of these ambiguous ERK functions could provide a powerful tool in the treatment of cardiac disease. This short review will discuss new mechanistic insights into ERK1/2-dependent development of cardiac hypertrophy and the prospect to translate this knowledge into future therapeutic strategies. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction The primary response of the heart to increased workload is myocyte growth in an attempt to enhance cardiac contractile function (Hill and Olson, 2008). The increase of heart muscle mass reduces elevated ventricular wall stress and/or compensates increased hemodynamic demand. Therefore, also physiological conditions, e.g. physical exercise, induce hypertrophy of the heart. Unlike physiological hypertrophy, pathological stimuli, e.g. arterial hypertension or myocardial infarction, lead to interstitial fibrosis and expression of a fetal-gene program, ultimately to myocardial stiffness and declining cardiac output. This type of cardiac hypertrophy predisposes for sudden cardiac death, arrhythmia and the development of heart failure. A fundamental endeavour of heart failure research is to delineate adaptive from maladaptive mechanisms that cause cardiac hypertrophy. Since hypertrophy occurs in response to physiological or pathological stimuli, it is efficiently prevented by eliminating the underlying stimulus. For example, treatment of arterial hypertension, a prominent cause of cardiac hypertrophy, with anti-hypertensive drugs attenuates ventricular wall stress and myocardial growth and improves survival. However, often the cause of cardiac hypertrophy is not drugable or unknown. Studies using genetically engineered mice have suggested that direct targeting of the hypertrophic response itself can also be beneficial and, thus, may provide a suitable therapeutic option in such cases
∗ Corresponding author. Tel.: +49 931 201 48266; fax: +49 931 201 48539. E-mail addresses: [email protected] (K. Lorenz), [email protected] (J.P. Schmitt), [email protected] (M. Vidal), [email protected] (M.J. Lohse). 1357-2725/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2009.08.002
(Esposito et al., 2002; Frey et al., 2004; Hill and Olson, 2008). For example, mice with impaired G␣q -signaling (mice overexpressing an G␣q -inhibitory peptide) and mice lacking endogenous norepinephrine and epinephrine synthesis (dopamine -hydroxylase gene-targeted mice) showed less hypertrophy compared to wild type controls in response to pressure overload and little deterioration in cardiac function despite high left ventricular wall stress (Esposito et al., 2002). In line with these findings, regression of left ventricular hypertrophy in patients with high risk of vascular disease or diabetes reduced the risk of death, myocardial infarction, stroke and congestive heart failure (Mathew et al., 2001). These data indicate that the development of myocardial hypertrophy may not be mandatory for maintaining hemodynamic function under pathophysiological conditions and suggest blunting of the heart’s hypertrophic response as a promising therapeutic strategy.
2. Pathophysiological signaling Biomechanical stress and neurohumoral factors are the two main triggers of cardiac hypertrophy (Fig. 1). It is still illusive how biomechanical stress is sensed by the cardiomyocyte sarcomere and how it is transduced into intracellular signals (Debold et al., 2007; Heineke and Molkentin, 2006; Hill and Olson, 2008; Schmitt et al., 2006; Sheikh et al., 2008). Stretch-sensitive ion channels, intregrins, sarcomeric and cytoskeletal proteins appear to be entangled in a complex network to transduce biomechanical stress into a hypertrophic response. In contrast, neurohumoral factors and their downstream signaling pathways that mediate cardiac growth are well known and include endothelin-1, angiotensin II, IGF-1 and catecholamines (Heineke and Molkentin, 2006). Several neurohumoral factors mediate their hypertrophic
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K. Lorenz et al. / The International Journal of Biochemistry & Cell Biology 41 (2009) 2351–2355
Fig. 1. Myocyte signaling of hypertrophy. Biomechanical stress and neurohumoral factors are the main triggers of cardiac hypertrophy. While it is still unclear how mechanical stress is translated into a hypertrophic response, neurohumoral factors transmit hypertrophic signals to the cell via receptor tyrosine kinases (RTK) or by activating G-protein coupled receptors (GPCRs)—mainly those that couple to G␣q . The consequent release of calcium from intracellular stores plays a central role in the induction of hypertrophic signaling by the activation of calcium-dependent protein kinases, phosphatases or mitogen-activated protein kinases. These molecular events initiate hypertrophic signaling pathways such as the CamKII/HDAC-pathway, the calcineurin-NFAT-pathway and the extracellular-regulated kinases 1 and 2 (ERK1/2). Additional to its hypertrophic function, the ERK1/2 cascade is also involved in the control of cell survival. Akt, protein kinase B; ASK1, apoptosis signal-regulating kinase 1; BAD, Bcl2 antagonist of cell death; Bcl-2, B-cell lymphoma 2; CamK, calmodulin-dependent kinase; HDAC, histone deacetylases; MEK, mitogen-activated protein kinase kinase; NFAT, nuclear factor of activated T cells; PI3K, phosphatidylinositol 3-kinase.
effects via receptor tyrosine kinases (RTK), but the majority activates G-protein coupled receptors—mainly those that couple to G␣q (Fig. 1). The effector protein of these G␣q -proteins, phospholipase C, leads to the generation of inositol-1,4,5-trisphosphate, which in turn gives rise to the release of calcium from intracellular stores. A large body of signaling pathways has been implicated in cardiac hypertrophy, but there appears to be consensus that alterations in intracellular calcium represent a crucial initiating step leading to the activation of calcium-dependent protein kinases, phosphatases or mitogen-activated protein kinases. As a result, the calcineurin-NFAT-pathway, the CamKII/HDAC-pathway or the extracellular-regulated kinases 1 and 2 (ERK1/2) may be switched on (Gutkind and Offermanns, 2009; Heineke and Molkentin, 2006; Muslin, 2008) (Fig. 1). This short review will focus on the regulation of hypertrophic signaling via ERK1/2. 2.1. The relevance of ERK1/2 in the heart ERK1/2 are part of a mitogen-activated protein kinase (MAPK) cascade consisting of sequentially functioning kinases—Raf, MEK and ERK1/2 (Raman et al., 2007). Active ERK1/2 induce reprogramming of gene expression by phosphorylating various intracellular target proteins and transcription factors and thereby initiate cell growth, proliferation, differentiation and anti-apoptotic effects. The large variety of biological processes that involve ERK1/2 is mirrored by the diversity of affected organ systems in patients with Noonan and related syndromes such as the Costello, LEOPARD and CFC (cardio-facio-cutaneous) syndromes (Aoki et al., 2008; Kontaridis et al., 2008; Nakamura et al., 2007; Pandit et al., 2007). These autosomal dominant diseases are mostly caused by mutations in positive regulators of the Ras/MAP-kinase pathway or Raf-1 itself and can affect the heart, muscles, skeleton, blood and nerves as well as the gastro-intestinal, genito-urinary and immune system. Cardiac manifestations most frequently are pulmonary valve stenosis and hypertrophic cardiomyopathy (Kontaridis et al., 2008; Pandit et al., 2007). The hypertrophic stimuli depicted above, e.g. angiotensin II or biomechanical stress, activate ERK1/2 and thereby induce hypertrophic responses such as cardiomyocyte growth, increased fetal-gene expression and/or cytoskeletal reorganization in neonatal cardiomyocytes (Heineke and Molkentin, 2006; Muslin, 2008; Wang, 2007). The attenuation of hypertrophic responses by the use
of pharmacological inhibitors of MEK1/2, dominant-negative Raf1 or MEK1 and antisense oligonucleotides against ERK1/2 further substantiate the important role of the Raf–MEK–ERK1/2 cascade for cardiomyocyte hypertrophy (Heineke and Molkentin, 2006; Muslin, 2008; Wang, 2007). 2.2. Gain-of-function approaches in transgenic mice Since a detailed understanding of the physiological function of the cascade cannot be gained from cells isolated from their physiological environment, the in vivo role of the Raf/MEK/ERK1/2 pathway has been investigated using gain- or loss-of-function mouse mutants. Cardiac expression of a constitutively active Rasmutant (H-v12-Ras), the upstream activator of this kinase cascade, led to significant left ventricular hypertrophy, decreased contractility, diastolic dysfunction associated with interstitial fibrosis, induction of the fetal-gene program and sudden death (Mitchell et al., 2006; Zheng et al., 2004). Similarly, stable activation of MEK1 by overexpression of a MEK1-mutant, which simulates the Raf-1 phosphorylations, MEK1S217E,S221E , induced left ventricular hypertrophy (Bueno et al., 2000). The development of cardiac hypertrophy in these mice was independent of transgene-expression levels and hearts showed enhanced systolic but reduced diastolic function and increased expression of hypertrophic markers (ANF, BNP, ␣skeletal actin and -MHC). While both gain-of-function approaches, H-v12-Ras and MEK1S217E,S221E , result in an increased hypertrophic response, MEK activation – unlike Ras activation – did not exhibit cardiotoxic effects. The discrepancy regarding cardiac function between these two hypertrophic phenotypes, the pathological and the benign, may be explained by alternative (ERK-independent) Ras-signaling pathways. A second possible explanation could be that activated H-v12-Ras recruits modulatory proteins, e.g. G␥-subunits, which influence the function of the Raf/MEK/ERK1/2 cascade (Slupsky et al., 1999). A general activation of the entire cascade by such modulatory proteins could be circumvented by constitutive activation of downstream proteins, such as MEK1S217E,S221E . This would resemble a situation, where different ERK1/2 functions are selectively switched on, e.g. the protective, anti-apoptotic functions of ERK1/2 more than their hypertrophic functions. It would be interesting to know, whether the combination of upstream signals that contribute such modulatory proteins and MEK1S217E,S221E overexpression would influence the cardiac phenotype in these mice
K. Lorenz et al. / The International Journal of Biochemistry & Cell Biology 41 (2009) 2351–2355
differently. Furthermore, the constitutive activation of ERK1/2 in these transgenic models may be distinct from the carefully tuned transient activation pattern of endogenous MAP kinases (Luttrell and Luttrell, 2003; Wang, 2007). 2.3. Loss-of-function approaches Several loss-of-function approaches have been applied to further analyse the physiological function of ERK1/2. In contrast to the gain-of-function studies, mice with cardiac-specific overexpression of a dominant-negative Raf mutant (Raf-1K375H ) had no overt phenotype, but showed little hypertrophy and hypertrophic gene expression (ANF, -MHC) in response to cardiac pressure overload (transverse aortic constriction (TAC)) (Harris et al., 2004). However, cardiomyocyte apoptosis and animal mortality were significantly increased after TAC. ERK1/2 activity was markedly reduced in these mice, suggesting that Raf-1 signaling is mandatory for the development of cardiac hypertrophy and for cardiomyocyte survival in response to pressure overload. Since Raf-1K375H also inhibits other Raf-isoforms, a heartspecific Raf-1 knockout mouse (Raf-1−/− ) was generated (Hindley and Kolch, 2002; Mikula et al., 2001; Yamaguchi et al., 2004). Cardiac-specific Raf-1−/− mouse hearts showed systolic dysfunction, dilatation, an increased number of apoptotic cells, normal weight and cardiomyocyte size. ERK1/2-signaling was not disrupted in these hearts, most likely because absent Raf1/ERK function was compensated by other proteins, e.g. other Raf-isoforms. These findings suggested that in the context of normal ERK1/2 activity, Raf-1 may promote cardiomyocyte survival through a MEK/ERK-independent mechanism. The inhibition of MEK-function was studied in vivo using specific pharmacological inhibitors (PD98059, U0126). These inhibitors attenuated cardiac growth and ANF gene expression in a rat model of systemic hypertension induced by the NO synthase inhibitor L-NAME after endothelin-1 and isoprenaline application but enhanced myocyte apoptosis and pathological remodeling in a model of ischemia/reperfusion injury (Munzel et al., 2005; Sanada et al., 2003; Yue et al., 2000). These studies underlined the promi-
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nent role of the ERK1/2-signaling pathway in cardiac hypertrophy and its anti-apoptotic function. However, it cannot be ruled out that the pharmacological MEK-inhibitors used may have toxic side effects and possibly inhibit other kinases as well. Even though a large body of evidence supports a role for MEK and ERK1/2 in the development of cardiac hypertrophy, recent reports on ERK knockout mice substantially challenged this hypothesis because mice lacking ERK1 and one ERK2 allele (ERK1−/− ERK2+/− ) showed a normal hypertrophic response to pressure overload and to exercise (Purcell et al., 2007). These results suggested that ERK1/2 may not be mandatory for the growth of cardiomyocytes. In these mice, however, residual ERK activity due to the remaining ERK2 allele cannot be excluded. Further, due to the high embryonic lethality of ERK1−/− ERK2+/− mice, it is conceivable that the surviving mice express modifier genes that partially compensate for the loss of ERK1/2 function. In a second approach to block ERK1/2 activity, Purcell and coworkers generated an ERK1/2 phosphatase model by cardiac-specific and inducible expression of the dual specificity phosphatase 6 (DUSP6), an ERK1/2 specific phosphatase (Luttrell and Luttrell, 2003; Owens and Keyse, 2007; Purcell et al., 2007). TAC operation and phenylephrine infusion instantly induce ERK1/2 phosphorylation. Overexpression of DUSP6 completely inhibited this effect, while phosphorylation of other MAP kinases (p38, JNK or ERK5) was unchanged. As in ERK1−/− ERK2+/− mice, the degree of left ventricular hypertrophy after TAC, neuroendocrine agonist infusion and exercise was indistinguishable from non-transgenic mice. Fibrosis and apoptosis was increased in DUSP6 transgenic hearts in response to pressure overload indicating a protective role of ERK1/2 under stress conditions. Even though it cannot be excluded that DUSP6 has other targets than ERK1/2, which could at least partially cause this phenotype, this mouse model further supported the hypothesis that cardiac hypertrophy can develop independently of ERK1/2. In summary, these mouse models provide strong evidence for an important role of the Raf/MEK/ERK1/2 cascade in the heart: a protective anti-apoptotic function as well as a hypertrophic function under some instances—especially when ERK1/2 function was dominant-negatively inhibited under hypertrophic stress stimuli.
Fig. 2. ERK1/2-mediated cardiac hypertrophy is triggered by a new autophosphorylation of ERK1/2 at Thr 188. Activation of the ERK1/2-signaling cascade leads to the canonical phosphorylation of ERK1/2 at Thr183 and Tyr185. Subsequent dimerization of ERK1/2 and the release of G␥-subunits from G␣q leads to direct interaction of ERK1/2 with G␥ and thereby causes Thr188 autophosphorylation of ERK1/2. Thr188-phosphorylation of ERK1/2 specifically triggers ERK1/2-mediated cardiac hypertrophy without affecting other ERK1/2 functions in the cell.
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However, the question remains unsolved, whether cardiac hypertrophy can develop without concomitant ERK1/2 activation or whether ERK1/2 activation is indispensible for the transduction of a hypertrophic response. 2.4. Identification of a specific switch for ERK1/2-mediated cardiac hypertrophy? A recent study of the role of ERK1/2 in cardiac hypertrophy described a new regulatory mechanism of ERK1/2 function, which involves a previously unrecognized autophosphorylation site of ERK2 at Thr188 (Thr208 in ERK1) (Lorenz et al., 2009) (Fig. 2). This autophosphorylation occurs only in response to hypertrophic stimuli (angiotensin II, phenylephrine, neuregulin1 and TAC), and is not induced by non-hypertrophic stimuli such as carbachol. Thr188-phosphorylation requires the integration of upstream signals, particularly G␣q -coupled receptors, which trigger the release of G␥ subunits as well as the activation of the entire Raf\EK\ERK1/2 cascade, including canonical phosphorylation of ERK1/2 and ERK dimerization. Thr188-phosphorylation directs ERK to the nucleus, leading to enhanced phosphorylation of nuclear ERK1/2 targets such as Elk-1, c-Myc and MSK1. This new phosphorylation site was investigated in transgenic mice by overexpressing ERK2Thr188 -mutants that were either deficient in Thr188-phosphorylation (by exchanging threonine188 to alanine or serine, ERK2T188A or ERK2T188S , respectively) or simulated constitutive Thr188-phosphorylation (by exchanging threonine188 to aspartic acid, ERK2T188D ). Analysis of these mice revealed that TAC-induced hypertrophy is dependent on Thr188-phosphorylation. Hypertrophy was significantly increased in mutants simulating Thr188-phosphorylation, whereas phosphorylation deficient mutants attenuated the hypertrophic response. Furthermore, simulation of Thr188-phosphorylation was able to convert a non-hypertrophic stimulus (carbachol, an agonist on cardiac M2 muscarinic receptors) into a hypertrophic response, while a phosphorylation deficient mutant, ERK2T188S , attenuated angiotensin II induced cardiac hypertrophy. The extent of hypertrophy correlated well with the regression of hypertrophic marker genes and interstitial fibrosis. Interestingly, even though Thr188-phosphorylation was sufficient to induce or to attenuate ERK1/2-mediated hypertrophy, the overall activity of ERK1/2 was identical under basal conditions as well as after 6 weeks of TAC. These results indicate that Thr188-phosphorylation represents an important switch mechanism towards hypertrophic signaling. Since overall ERK1/2 activity remains unchanged, ERK1/2 functions other than hypertrophic signaling might be unaffected by this mechanism. 3. Future directions Cardiac hypertrophy is associated with an increased risk for heart failure, cardiac arrhythmia and sudden death. Efficient therapeutic strategies that directly interfere with the development of hypertrophy are still lacking. As pointed out in this review, targeting of the Raf–MEK–ERK1/2 cascade seems to provide a powerful tool against cardiac hypertrophy (Gutkind and Offermanns, 2009; Muslin, 2008; Wang, 2007). This concept would be most efficient if maladaptive signals that trigger hypertrophy could be selectively blocked without affecting the cardioprotective effects of ERK1/2 and particularly Raf-1 (Bueno et al., 2000; Harris et al., 2004; Zhai et al., 2007). Further, its complex role in various cellular functions during embryogenesis and adulthood, health and disease, prohibits a general inhibition of this signaling cascade. Therefore, only detailed knowledge about the differential molecular mechanisms of ERK1/2 activation will hold out improved therapeutic oppor-
tunities. The identification of Thr188-phosphorylation of ERK1/2 represents a first step in this direction. This phosphorylation can selectively activate hypertrophic ERK1/2 functions and, therefore, may help to target hypertrophy selectively. Mutant mice that lack this phosphorylation site showed a preserved cardiac structure and function, but attenuated hypertrophic response demonstrating that ERK1/2 functions indeed can be targeted selectively. Future efforts will have to focus on finding additional such mechanisms by further dissecting hypertrophic signaling cascades and by identifying downstream nodal endpoints of hypertrophic signaling. Such an analysis will help to identify the molecular events that discriminate between adaptive and pathological hypertrophic remodeling processes in the heart. The translation of this knowledge into clinical use will be both exciting and challenging. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft, the Leducq Foundation and the Fonds der Chemischen Industrie. We thank Dr. Ralf Schreck for critical review and helpful comments. References Aoki Y, Niihori T, Narumi Y, Kure S, Matsubara Y. The RAS/MAPK syndromes: novel roles of the RAS pathway in human genetic disorders. Hum Mutat 2008;29:992–1006. Bueno OF, De Windt LJ, Tymitz KM, Witt SA, Kimball TR, Klevitsky R, et al. The MEK1-ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice. EMBO J 2000;19:6341–50. Depold EP, Schmitt JP, Patlak JB, Beck SE, Moore JR, Seidman JG, et al. Hypertrophic and dilated cardiomyopathy mutations differentially affect the molecular force generation of mouse alpha-cardiac myosin in the laser trap assay. Am J Physiol Heart Circ Physiol 2007;293:H284–91. Esposito G, Rapacciuolo A, Naga Prasad SV, Takaoka H, Thomas SA, Koch WJ, et al. Genetic alterations that inhibit in vivo pressure-overload hypertrophy prevent cardiac dysfunction despite increased wall stress. Circulation 2002;105:85–92. Frey N, Katus HA, Olson EN, Hill JA. Hypertrophy of the heart: a new therapeutic target? Circulation 2004;109:1580–9. Gutkind JS, Offermanns S. A new Gq-initiated MAPK-signaling pathway in the heart. Dev Cell 2009;16:163–4. Harris IS, Zhang S, Treskov I, Kovacs A, Weinheimer C, Muslin AJ. Raf-1 kinase is required for cardiac hypertrophy and cardiomyocyte survival in response to pressure overload. Circulation 2004;110:718–23. Heineke J, Molkentin JD. Regulation of cardiac hypertrophy by intracellular signalling pathways. Nat Rev Mol Cell Biol 2006;7:589–600. Hill JA, Olson EN. Cardiac plasticity. N Engl J Med 2008;358:1370–80. Hindley A, Kolch W. Extracellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK)-independent functions of Raf kinases. J Cell Sci 2002;115:1575–81. Kontaridis MI, Yang W, Bence KK, Cullen D, Wang B, Bodyak N, et al. Deletion of Ptpn11 (Shp2) in cardiomyocytes causes dilated cardiomyopathy via effects on the extracellular signal-regulated kinase/mitogen-activated protein kinase and RhoA signaling pathways. Circulation 2008;117:1423–35. Lorenz K, Schmitt JP, Schmitteckert EM, Lohse MJ. A new type of ERK1/2 autophosphorylation causes cardiac hypertrophy. Nat Med 2009;15:75–83. Luttrell DK, Luttrell LM. Signaling in time and space: G protein-coupled receptors and mitogen-activated protein kinases. Assay Drug Dev Technol 2003;1:327–38. Mathew J, Sleight P, Lonn E, Johnstone D, Pogue J, Yi Q, et al. Reduction of cardiovascular risk by regression of electrocardiographic markers of left ventricular hypertrophy by the angiotensin-converting enzyme inhibitor ramipril. Circulation 2001;104:1615–21. Mikula M, Schreiber M, Husak Z, Kucerova L, Ruth J, Wieser R, et al. Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf-1 gene. EMBO J 2001;20:1952–62. Mitchell S, Ota A, Foster W, Zhang B, Fang Z, Patel S, et al. Distinct gene expression profiles in adult mouse heart following targeted MAP kinase activation. Physiol Genomics 2006;25:50–9. Munzel F, Muhlhauser U, Zimmermann WH, Didie M, Schneiderbanger K, Schubert P, et al. Endothelin-1 and isoprenaline co-stimulation causes contractile failure which is partially reversed by MEK inhibition. Cardiovasc Res 2005;68:464–74. Muslin AJ. MAPK signalling in cardiovascular health and disease: molecular mechanisms and therapeutic targets. Clin Sci (Lond) 2008;115:203–18. Nakamura T, Colbert M, Krenz M, Molkentin JD, Hahn HS, Dorn 2nd GW, et al. Mediating ERK 1/2 signaling rescues congenital heart defects in a mouse model of Noonan syndrome. J Clin Invest 2007;117:2123–32. Owens DM, Keyse SM. Differential regulation of MAP kinase signalling by dualspecificity protein phosphatases. Oncogene 2007;26:3203–13.
K. Lorenz et al. / The International Journal of Biochemistry & Cell Biology 41 (2009) 2351–2355 Pandit B, Sarkozy A, Pennacchio LA, Carta C, Oishi K, Martinelli S. Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy. Nat Genet 2007;39:1007–12. Purcell NH, Wilkins BJ, York A, Saba-El-Leil MK, Meloche S, Robbins J, et al. Genetic inhibition of cardiac ERK1/2 promotes stress-induced apoptosis and heart failure but has no effect on hypertrophy in vivo. Proc Natl Acad Sci USA 2007;104:14074–9. Raman M, Chen W, Cobb MH. Differential regulation and properties of MAPKs. Oncogene 2007;26:3100–12. Sanada S, Node K, Minamino T, Takashima S, Ogai A, Asanuma H, et al. Long-acting Ca2+ blockers prevent myocardial remodeling induced by chronic NO inhibition in rats. Hypertension 2003;41:963–7. Schmitt JP, Debold EP, Ahmad F, Armstrong A, Frederico A, Conner DA, et al. Cardiac myosin missense mutations cause dilated cardiomyopathy in mouse models and depress molecular motor function Proc Natl Acad Sci U S A 2006;103:14525–30. Sheikh F, Raskin A, Chu PH, Lange S, Domenighetti AA, Zheng M, et al. An FHL1-containing complex within the cardiomyocyte sarcomere mediates hypertrophic biomechanical stress responses in mice. J Clin Invest 2008;118: 3870–80.
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Slupsky JR, Quitterer U, Weber CK, Gierschik P, Lohse MJ, Rapp UR. Binding of Gbetagamma subunits to cRaf1 downregulates G-protein-coupled receptor signalling. Curr Biol 1999;9:971–4. Wang Y. Mitogen-activated protein kinases in heart development and diseases. Circulation 2007;116:1413–23. Yamaguchi O, Watanabe T, Nishida K, Kashiwase K, Higuchi Y, Takeda T, et al. Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis. J Clin Invest 2004;114:937–43. Yue TL, Wang C, Gu JL, Ma XL, Kumar S, Lee JC, et al. Inhibition of extracellular signal-regulated kinase enhances ischemia/reoxygenation-induced apoptosis in cultured cardiac myocytes and exaggerates reperfusion injury in isolated perfused heart. Circ Res 2000;86:692–9. Zheng M, Dilly K, Dos Santos Cruz J, Li M, Gu Y, Ursitti JA, et al. Sarcoplasmic reticulum calcium defect in Ras-induced hypertrophic cardiomyopathy heart. Am J Physiol Heart Circ Physiol 2004;286:H424–33. Zhai P, Gao S, Holle E, Yu X, Yatani A, Wagner T, et al. Glycogen synthase kinase-3␣ reduces cardiac growth and pressure overload-induced cardiac hypertrophy by inhibition of extracellular signal-regulated kinases. J Biol Chem 2007;282:33181–91.
Contents lists available at ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Medicine in focus
Cardiac hypertrophy: Targeting Raf/MEK/ERK1/2-signaling Kristina Lorenz a,∗ , Joachim P. Schmitt a , Marie Vidal a , Martin J. Lohse a,b a b
Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Straße 9, 97078 Würzburg, Germany Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, University of Würzburg, Versbacher Straße 9, 97078 Würzburg, Germany
a r t i c l e
i n f o
Article history: Received 23 May 2009 Received in revised form 26 July 2009 Accepted 4 August 2009 Available online 8 August 2009 Keywords: Cardiac hypertrophy ERK1/2 Signaling
a b s t r a c t Over the past two decades, basic research has revealed a complex network of regulatory mechanisms that control the ERK1/2-signaling cascade. ERK1/2 mediate cardiac hypertrophy, a major risk factor for the development of arrhythmias, heart failure and sudden death, but also beneficial effects, e.g. protection of the heart from cell death and ischemic injury. Selective targeting of these ambiguous ERK functions could provide a powerful tool in the treatment of cardiac disease. This short review will discuss new mechanistic insights into ERK1/2-dependent development of cardiac hypertrophy and the prospect to translate this knowledge into future therapeutic strategies. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction The primary response of the heart to increased workload is myocyte growth in an attempt to enhance cardiac contractile function (Hill and Olson, 2008). The increase of heart muscle mass reduces elevated ventricular wall stress and/or compensates increased hemodynamic demand. Therefore, also physiological conditions, e.g. physical exercise, induce hypertrophy of the heart. Unlike physiological hypertrophy, pathological stimuli, e.g. arterial hypertension or myocardial infarction, lead to interstitial fibrosis and expression of a fetal-gene program, ultimately to myocardial stiffness and declining cardiac output. This type of cardiac hypertrophy predisposes for sudden cardiac death, arrhythmia and the development of heart failure. A fundamental endeavour of heart failure research is to delineate adaptive from maladaptive mechanisms that cause cardiac hypertrophy. Since hypertrophy occurs in response to physiological or pathological stimuli, it is efficiently prevented by eliminating the underlying stimulus. For example, treatment of arterial hypertension, a prominent cause of cardiac hypertrophy, with anti-hypertensive drugs attenuates ventricular wall stress and myocardial growth and improves survival. However, often the cause of cardiac hypertrophy is not drugable or unknown. Studies using genetically engineered mice have suggested that direct targeting of the hypertrophic response itself can also be beneficial and, thus, may provide a suitable therapeutic option in such cases
∗ Corresponding author. Tel.: +49 931 201 48266; fax: +49 931 201 48539. E-mail addresses: [email protected] (K. Lorenz), [email protected] (J.P. Schmitt), [email protected] (M. Vidal), [email protected] (M.J. Lohse). 1357-2725/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2009.08.002
(Esposito et al., 2002; Frey et al., 2004; Hill and Olson, 2008). For example, mice with impaired G␣q -signaling (mice overexpressing an G␣q -inhibitory peptide) and mice lacking endogenous norepinephrine and epinephrine synthesis (dopamine -hydroxylase gene-targeted mice) showed less hypertrophy compared to wild type controls in response to pressure overload and little deterioration in cardiac function despite high left ventricular wall stress (Esposito et al., 2002). In line with these findings, regression of left ventricular hypertrophy in patients with high risk of vascular disease or diabetes reduced the risk of death, myocardial infarction, stroke and congestive heart failure (Mathew et al., 2001). These data indicate that the development of myocardial hypertrophy may not be mandatory for maintaining hemodynamic function under pathophysiological conditions and suggest blunting of the heart’s hypertrophic response as a promising therapeutic strategy.
2. Pathophysiological signaling Biomechanical stress and neurohumoral factors are the two main triggers of cardiac hypertrophy (Fig. 1). It is still illusive how biomechanical stress is sensed by the cardiomyocyte sarcomere and how it is transduced into intracellular signals (Debold et al., 2007; Heineke and Molkentin, 2006; Hill and Olson, 2008; Schmitt et al., 2006; Sheikh et al., 2008). Stretch-sensitive ion channels, intregrins, sarcomeric and cytoskeletal proteins appear to be entangled in a complex network to transduce biomechanical stress into a hypertrophic response. In contrast, neurohumoral factors and their downstream signaling pathways that mediate cardiac growth are well known and include endothelin-1, angiotensin II, IGF-1 and catecholamines (Heineke and Molkentin, 2006). Several neurohumoral factors mediate their hypertrophic
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Fig. 1. Myocyte signaling of hypertrophy. Biomechanical stress and neurohumoral factors are the main triggers of cardiac hypertrophy. While it is still unclear how mechanical stress is translated into a hypertrophic response, neurohumoral factors transmit hypertrophic signals to the cell via receptor tyrosine kinases (RTK) or by activating G-protein coupled receptors (GPCRs)—mainly those that couple to G␣q . The consequent release of calcium from intracellular stores plays a central role in the induction of hypertrophic signaling by the activation of calcium-dependent protein kinases, phosphatases or mitogen-activated protein kinases. These molecular events initiate hypertrophic signaling pathways such as the CamKII/HDAC-pathway, the calcineurin-NFAT-pathway and the extracellular-regulated kinases 1 and 2 (ERK1/2). Additional to its hypertrophic function, the ERK1/2 cascade is also involved in the control of cell survival. Akt, protein kinase B; ASK1, apoptosis signal-regulating kinase 1; BAD, Bcl2 antagonist of cell death; Bcl-2, B-cell lymphoma 2; CamK, calmodulin-dependent kinase; HDAC, histone deacetylases; MEK, mitogen-activated protein kinase kinase; NFAT, nuclear factor of activated T cells; PI3K, phosphatidylinositol 3-kinase.
effects via receptor tyrosine kinases (RTK), but the majority activates G-protein coupled receptors—mainly those that couple to G␣q (Fig. 1). The effector protein of these G␣q -proteins, phospholipase C, leads to the generation of inositol-1,4,5-trisphosphate, which in turn gives rise to the release of calcium from intracellular stores. A large body of signaling pathways has been implicated in cardiac hypertrophy, but there appears to be consensus that alterations in intracellular calcium represent a crucial initiating step leading to the activation of calcium-dependent protein kinases, phosphatases or mitogen-activated protein kinases. As a result, the calcineurin-NFAT-pathway, the CamKII/HDAC-pathway or the extracellular-regulated kinases 1 and 2 (ERK1/2) may be switched on (Gutkind and Offermanns, 2009; Heineke and Molkentin, 2006; Muslin, 2008) (Fig. 1). This short review will focus on the regulation of hypertrophic signaling via ERK1/2. 2.1. The relevance of ERK1/2 in the heart ERK1/2 are part of a mitogen-activated protein kinase (MAPK) cascade consisting of sequentially functioning kinases—Raf, MEK and ERK1/2 (Raman et al., 2007). Active ERK1/2 induce reprogramming of gene expression by phosphorylating various intracellular target proteins and transcription factors and thereby initiate cell growth, proliferation, differentiation and anti-apoptotic effects. The large variety of biological processes that involve ERK1/2 is mirrored by the diversity of affected organ systems in patients with Noonan and related syndromes such as the Costello, LEOPARD and CFC (cardio-facio-cutaneous) syndromes (Aoki et al., 2008; Kontaridis et al., 2008; Nakamura et al., 2007; Pandit et al., 2007). These autosomal dominant diseases are mostly caused by mutations in positive regulators of the Ras/MAP-kinase pathway or Raf-1 itself and can affect the heart, muscles, skeleton, blood and nerves as well as the gastro-intestinal, genito-urinary and immune system. Cardiac manifestations most frequently are pulmonary valve stenosis and hypertrophic cardiomyopathy (Kontaridis et al., 2008; Pandit et al., 2007). The hypertrophic stimuli depicted above, e.g. angiotensin II or biomechanical stress, activate ERK1/2 and thereby induce hypertrophic responses such as cardiomyocyte growth, increased fetal-gene expression and/or cytoskeletal reorganization in neonatal cardiomyocytes (Heineke and Molkentin, 2006; Muslin, 2008; Wang, 2007). The attenuation of hypertrophic responses by the use
of pharmacological inhibitors of MEK1/2, dominant-negative Raf1 or MEK1 and antisense oligonucleotides against ERK1/2 further substantiate the important role of the Raf–MEK–ERK1/2 cascade for cardiomyocyte hypertrophy (Heineke and Molkentin, 2006; Muslin, 2008; Wang, 2007). 2.2. Gain-of-function approaches in transgenic mice Since a detailed understanding of the physiological function of the cascade cannot be gained from cells isolated from their physiological environment, the in vivo role of the Raf/MEK/ERK1/2 pathway has been investigated using gain- or loss-of-function mouse mutants. Cardiac expression of a constitutively active Rasmutant (H-v12-Ras), the upstream activator of this kinase cascade, led to significant left ventricular hypertrophy, decreased contractility, diastolic dysfunction associated with interstitial fibrosis, induction of the fetal-gene program and sudden death (Mitchell et al., 2006; Zheng et al., 2004). Similarly, stable activation of MEK1 by overexpression of a MEK1-mutant, which simulates the Raf-1 phosphorylations, MEK1S217E,S221E , induced left ventricular hypertrophy (Bueno et al., 2000). The development of cardiac hypertrophy in these mice was independent of transgene-expression levels and hearts showed enhanced systolic but reduced diastolic function and increased expression of hypertrophic markers (ANF, BNP, ␣skeletal actin and -MHC). While both gain-of-function approaches, H-v12-Ras and MEK1S217E,S221E , result in an increased hypertrophic response, MEK activation – unlike Ras activation – did not exhibit cardiotoxic effects. The discrepancy regarding cardiac function between these two hypertrophic phenotypes, the pathological and the benign, may be explained by alternative (ERK-independent) Ras-signaling pathways. A second possible explanation could be that activated H-v12-Ras recruits modulatory proteins, e.g. G␥-subunits, which influence the function of the Raf/MEK/ERK1/2 cascade (Slupsky et al., 1999). A general activation of the entire cascade by such modulatory proteins could be circumvented by constitutive activation of downstream proteins, such as MEK1S217E,S221E . This would resemble a situation, where different ERK1/2 functions are selectively switched on, e.g. the protective, anti-apoptotic functions of ERK1/2 more than their hypertrophic functions. It would be interesting to know, whether the combination of upstream signals that contribute such modulatory proteins and MEK1S217E,S221E overexpression would influence the cardiac phenotype in these mice
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differently. Furthermore, the constitutive activation of ERK1/2 in these transgenic models may be distinct from the carefully tuned transient activation pattern of endogenous MAP kinases (Luttrell and Luttrell, 2003; Wang, 2007). 2.3. Loss-of-function approaches Several loss-of-function approaches have been applied to further analyse the physiological function of ERK1/2. In contrast to the gain-of-function studies, mice with cardiac-specific overexpression of a dominant-negative Raf mutant (Raf-1K375H ) had no overt phenotype, but showed little hypertrophy and hypertrophic gene expression (ANF, -MHC) in response to cardiac pressure overload (transverse aortic constriction (TAC)) (Harris et al., 2004). However, cardiomyocyte apoptosis and animal mortality were significantly increased after TAC. ERK1/2 activity was markedly reduced in these mice, suggesting that Raf-1 signaling is mandatory for the development of cardiac hypertrophy and for cardiomyocyte survival in response to pressure overload. Since Raf-1K375H also inhibits other Raf-isoforms, a heartspecific Raf-1 knockout mouse (Raf-1−/− ) was generated (Hindley and Kolch, 2002; Mikula et al., 2001; Yamaguchi et al., 2004). Cardiac-specific Raf-1−/− mouse hearts showed systolic dysfunction, dilatation, an increased number of apoptotic cells, normal weight and cardiomyocyte size. ERK1/2-signaling was not disrupted in these hearts, most likely because absent Raf1/ERK function was compensated by other proteins, e.g. other Raf-isoforms. These findings suggested that in the context of normal ERK1/2 activity, Raf-1 may promote cardiomyocyte survival through a MEK/ERK-independent mechanism. The inhibition of MEK-function was studied in vivo using specific pharmacological inhibitors (PD98059, U0126). These inhibitors attenuated cardiac growth and ANF gene expression in a rat model of systemic hypertension induced by the NO synthase inhibitor L-NAME after endothelin-1 and isoprenaline application but enhanced myocyte apoptosis and pathological remodeling in a model of ischemia/reperfusion injury (Munzel et al., 2005; Sanada et al., 2003; Yue et al., 2000). These studies underlined the promi-
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nent role of the ERK1/2-signaling pathway in cardiac hypertrophy and its anti-apoptotic function. However, it cannot be ruled out that the pharmacological MEK-inhibitors used may have toxic side effects and possibly inhibit other kinases as well. Even though a large body of evidence supports a role for MEK and ERK1/2 in the development of cardiac hypertrophy, recent reports on ERK knockout mice substantially challenged this hypothesis because mice lacking ERK1 and one ERK2 allele (ERK1−/− ERK2+/− ) showed a normal hypertrophic response to pressure overload and to exercise (Purcell et al., 2007). These results suggested that ERK1/2 may not be mandatory for the growth of cardiomyocytes. In these mice, however, residual ERK activity due to the remaining ERK2 allele cannot be excluded. Further, due to the high embryonic lethality of ERK1−/− ERK2+/− mice, it is conceivable that the surviving mice express modifier genes that partially compensate for the loss of ERK1/2 function. In a second approach to block ERK1/2 activity, Purcell and coworkers generated an ERK1/2 phosphatase model by cardiac-specific and inducible expression of the dual specificity phosphatase 6 (DUSP6), an ERK1/2 specific phosphatase (Luttrell and Luttrell, 2003; Owens and Keyse, 2007; Purcell et al., 2007). TAC operation and phenylephrine infusion instantly induce ERK1/2 phosphorylation. Overexpression of DUSP6 completely inhibited this effect, while phosphorylation of other MAP kinases (p38, JNK or ERK5) was unchanged. As in ERK1−/− ERK2+/− mice, the degree of left ventricular hypertrophy after TAC, neuroendocrine agonist infusion and exercise was indistinguishable from non-transgenic mice. Fibrosis and apoptosis was increased in DUSP6 transgenic hearts in response to pressure overload indicating a protective role of ERK1/2 under stress conditions. Even though it cannot be excluded that DUSP6 has other targets than ERK1/2, which could at least partially cause this phenotype, this mouse model further supported the hypothesis that cardiac hypertrophy can develop independently of ERK1/2. In summary, these mouse models provide strong evidence for an important role of the Raf/MEK/ERK1/2 cascade in the heart: a protective anti-apoptotic function as well as a hypertrophic function under some instances—especially when ERK1/2 function was dominant-negatively inhibited under hypertrophic stress stimuli.
Fig. 2. ERK1/2-mediated cardiac hypertrophy is triggered by a new autophosphorylation of ERK1/2 at Thr 188. Activation of the ERK1/2-signaling cascade leads to the canonical phosphorylation of ERK1/2 at Thr183 and Tyr185. Subsequent dimerization of ERK1/2 and the release of G␥-subunits from G␣q leads to direct interaction of ERK1/2 with G␥ and thereby causes Thr188 autophosphorylation of ERK1/2. Thr188-phosphorylation of ERK1/2 specifically triggers ERK1/2-mediated cardiac hypertrophy without affecting other ERK1/2 functions in the cell.
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However, the question remains unsolved, whether cardiac hypertrophy can develop without concomitant ERK1/2 activation or whether ERK1/2 activation is indispensible for the transduction of a hypertrophic response. 2.4. Identification of a specific switch for ERK1/2-mediated cardiac hypertrophy? A recent study of the role of ERK1/2 in cardiac hypertrophy described a new regulatory mechanism of ERK1/2 function, which involves a previously unrecognized autophosphorylation site of ERK2 at Thr188 (Thr208 in ERK1) (Lorenz et al., 2009) (Fig. 2). This autophosphorylation occurs only in response to hypertrophic stimuli (angiotensin II, phenylephrine, neuregulin1 and TAC), and is not induced by non-hypertrophic stimuli such as carbachol. Thr188-phosphorylation requires the integration of upstream signals, particularly G␣q -coupled receptors, which trigger the release of G␥ subunits as well as the activation of the entire Raf\EK\ERK1/2 cascade, including canonical phosphorylation of ERK1/2 and ERK dimerization. Thr188-phosphorylation directs ERK to the nucleus, leading to enhanced phosphorylation of nuclear ERK1/2 targets such as Elk-1, c-Myc and MSK1. This new phosphorylation site was investigated in transgenic mice by overexpressing ERK2Thr188 -mutants that were either deficient in Thr188-phosphorylation (by exchanging threonine188 to alanine or serine, ERK2T188A or ERK2T188S , respectively) or simulated constitutive Thr188-phosphorylation (by exchanging threonine188 to aspartic acid, ERK2T188D ). Analysis of these mice revealed that TAC-induced hypertrophy is dependent on Thr188-phosphorylation. Hypertrophy was significantly increased in mutants simulating Thr188-phosphorylation, whereas phosphorylation deficient mutants attenuated the hypertrophic response. Furthermore, simulation of Thr188-phosphorylation was able to convert a non-hypertrophic stimulus (carbachol, an agonist on cardiac M2 muscarinic receptors) into a hypertrophic response, while a phosphorylation deficient mutant, ERK2T188S , attenuated angiotensin II induced cardiac hypertrophy. The extent of hypertrophy correlated well with the regression of hypertrophic marker genes and interstitial fibrosis. Interestingly, even though Thr188-phosphorylation was sufficient to induce or to attenuate ERK1/2-mediated hypertrophy, the overall activity of ERK1/2 was identical under basal conditions as well as after 6 weeks of TAC. These results indicate that Thr188-phosphorylation represents an important switch mechanism towards hypertrophic signaling. Since overall ERK1/2 activity remains unchanged, ERK1/2 functions other than hypertrophic signaling might be unaffected by this mechanism. 3. Future directions Cardiac hypertrophy is associated with an increased risk for heart failure, cardiac arrhythmia and sudden death. Efficient therapeutic strategies that directly interfere with the development of hypertrophy are still lacking. As pointed out in this review, targeting of the Raf–MEK–ERK1/2 cascade seems to provide a powerful tool against cardiac hypertrophy (Gutkind and Offermanns, 2009; Muslin, 2008; Wang, 2007). This concept would be most efficient if maladaptive signals that trigger hypertrophy could be selectively blocked without affecting the cardioprotective effects of ERK1/2 and particularly Raf-1 (Bueno et al., 2000; Harris et al., 2004; Zhai et al., 2007). Further, its complex role in various cellular functions during embryogenesis and adulthood, health and disease, prohibits a general inhibition of this signaling cascade. Therefore, only detailed knowledge about the differential molecular mechanisms of ERK1/2 activation will hold out improved therapeutic oppor-
tunities. The identification of Thr188-phosphorylation of ERK1/2 represents a first step in this direction. This phosphorylation can selectively activate hypertrophic ERK1/2 functions and, therefore, may help to target hypertrophy selectively. Mutant mice that lack this phosphorylation site showed a preserved cardiac structure and function, but attenuated hypertrophic response demonstrating that ERK1/2 functions indeed can be targeted selectively. Future efforts will have to focus on finding additional such mechanisms by further dissecting hypertrophic signaling cascades and by identifying downstream nodal endpoints of hypertrophic signaling. Such an analysis will help to identify the molecular events that discriminate between adaptive and pathological hypertrophic remodeling processes in the heart. The translation of this knowledge into clinical use will be both exciting and challenging. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft, the Leducq Foundation and the Fonds der Chemischen Industrie. We thank Dr. Ralf Schreck for critical review and helpful comments. References Aoki Y, Niihori T, Narumi Y, Kure S, Matsubara Y. The RAS/MAPK syndromes: novel roles of the RAS pathway in human genetic disorders. Hum Mutat 2008;29:992–1006. Bueno OF, De Windt LJ, Tymitz KM, Witt SA, Kimball TR, Klevitsky R, et al. The MEK1-ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice. EMBO J 2000;19:6341–50. Depold EP, Schmitt JP, Patlak JB, Beck SE, Moore JR, Seidman JG, et al. Hypertrophic and dilated cardiomyopathy mutations differentially affect the molecular force generation of mouse alpha-cardiac myosin in the laser trap assay. Am J Physiol Heart Circ Physiol 2007;293:H284–91. Esposito G, Rapacciuolo A, Naga Prasad SV, Takaoka H, Thomas SA, Koch WJ, et al. Genetic alterations that inhibit in vivo pressure-overload hypertrophy prevent cardiac dysfunction despite increased wall stress. Circulation 2002;105:85–92. Frey N, Katus HA, Olson EN, Hill JA. Hypertrophy of the heart: a new therapeutic target? Circulation 2004;109:1580–9. Gutkind JS, Offermanns S. A new Gq-initiated MAPK-signaling pathway in the heart. Dev Cell 2009;16:163–4. Harris IS, Zhang S, Treskov I, Kovacs A, Weinheimer C, Muslin AJ. Raf-1 kinase is required for cardiac hypertrophy and cardiomyocyte survival in response to pressure overload. Circulation 2004;110:718–23. Heineke J, Molkentin JD. Regulation of cardiac hypertrophy by intracellular signalling pathways. Nat Rev Mol Cell Biol 2006;7:589–600. Hill JA, Olson EN. Cardiac plasticity. N Engl J Med 2008;358:1370–80. Hindley A, Kolch W. Extracellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK)-independent functions of Raf kinases. J Cell Sci 2002;115:1575–81. Kontaridis MI, Yang W, Bence KK, Cullen D, Wang B, Bodyak N, et al. Deletion of Ptpn11 (Shp2) in cardiomyocytes causes dilated cardiomyopathy via effects on the extracellular signal-regulated kinase/mitogen-activated protein kinase and RhoA signaling pathways. Circulation 2008;117:1423–35. Lorenz K, Schmitt JP, Schmitteckert EM, Lohse MJ. A new type of ERK1/2 autophosphorylation causes cardiac hypertrophy. Nat Med 2009;15:75–83. Luttrell DK, Luttrell LM. Signaling in time and space: G protein-coupled receptors and mitogen-activated protein kinases. Assay Drug Dev Technol 2003;1:327–38. Mathew J, Sleight P, Lonn E, Johnstone D, Pogue J, Yi Q, et al. Reduction of cardiovascular risk by regression of electrocardiographic markers of left ventricular hypertrophy by the angiotensin-converting enzyme inhibitor ramipril. Circulation 2001;104:1615–21. Mikula M, Schreiber M, Husak Z, Kucerova L, Ruth J, Wieser R, et al. Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf-1 gene. EMBO J 2001;20:1952–62. Mitchell S, Ota A, Foster W, Zhang B, Fang Z, Patel S, et al. Distinct gene expression profiles in adult mouse heart following targeted MAP kinase activation. Physiol Genomics 2006;25:50–9. Munzel F, Muhlhauser U, Zimmermann WH, Didie M, Schneiderbanger K, Schubert P, et al. Endothelin-1 and isoprenaline co-stimulation causes contractile failure which is partially reversed by MEK inhibition. Cardiovasc Res 2005;68:464–74. Muslin AJ. MAPK signalling in cardiovascular health and disease: molecular mechanisms and therapeutic targets. Clin Sci (Lond) 2008;115:203–18. Nakamura T, Colbert M, Krenz M, Molkentin JD, Hahn HS, Dorn 2nd GW, et al. Mediating ERK 1/2 signaling rescues congenital heart defects in a mouse model of Noonan syndrome. J Clin Invest 2007;117:2123–32. Owens DM, Keyse SM. Differential regulation of MAP kinase signalling by dualspecificity protein phosphatases. Oncogene 2007;26:3203–13.
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