Tumor Suppressor A20 Protects Against Cardiac Hypertrophy and Fibrosis by Blocking Transforming Growth Factor-β–Activated Kinase 1–Dependent Signaling
A20 or tumor necrosis factor–induced protein 3 is a negative regulator of nuclear factor κB signaling. A20 has been shown previously to attenuate cardiac hypertrophy in vitro and postmyocardial infarction remodeling in vivo. In the present study, we tested the hypothesis that overexpression of A20 in the murine heart would protect against cardiac hypertrophy in vivo. The effects of constitutive human A20 expression on cardiac hypertrophy were investigated using in vitro and in vivo models. Cardiac hypertrophy was produced by aortic banding in A20 transgenic mice and control animals. The extent of cardiac hypertrophy was quantitated by echocardiography, as well as by pathological and molecular analyses of heart samples. Constitutive overexpression of human A20 in the murine heart attenuated the hypertrophic response and markedly reduced inflammation, apoptosis, and fibrosis. Cardiac function was also preserved in hearts with increased A20 levels in response to hypertrophic stimuli. Western blot experiments further showed A20 expression markedly blocked transforming growth factor-β–activated kinase 1–dependent c-Jun N-terminal kinase/p38 signaling cascade but with no difference in either extracellular signal-regulated kinase 1/2 or AKT activation in vivo and in vitro. In cultured neonatal rat cardiac myocytes, [3H]proline incorporation and Western blot assays revealed that A20 expression suppressed transforming growth factor-β–induced collagen synthesis and transforming growth factor-β–activated kinase 1–dependent Smad 2/3/4 activation. In conclusion, A20 improves cardiac functions and inhibits cardiac hypertrophy, inflammation, apoptosis, and fibrosis by blocking transforming growth factor-β–activated kinase 1–dependent signaling.
Heart failure is increasing in prevalence and is a debilitating disease with high rates of mortality and morbidity.1,2⇓ Cardiac hypertrophy is a common precursor to many forms of heart failure, of which molecular and cellular determinants remain largely unknown. After a period of compensatory adaptation, hypertrophy is associated with functional and histological deterioration of the myocardium, fibrosis, inflammation, and altered cardiac gene expression.3,4⇓ Accumulating evidence suggests that the nuclear factor κB (NF-κB) signaling system is a critical regulator of this process.5–7⇓⇓ Modulation of NF-κB signaling in the heart may provide a novel approach to attenuate the development of heart failure after cardiac hypertrophy.
A20 is a zinc finger protein originally identified as a tumor necrosis factor (TNF)–responsive gene in endothelial cells.8 It is an inducible and broadly expressed cytoplasmic protein that inhibits TNF-induced NF-κB activity. Recent studies showed that A20 expression protects various cell types from TNF-mediated apoptosis.9,10⇓ We also found that A20 expression protects against oxidized low density lipoprotein–induced macrophage apoptosis and inhibits the proliferation of vascular smooth muscle cells.11,12⇓ A20-deficient mice demonstrate spontaneous inflammation, cachexia, and premature death, and A20-deficient fibroblasts cannot properly terminate TNF-induced NF-κB activity.13 A20 is also an inducible ubiquitin-editing enzyme that restricts both toll-like receptor and TNF-induced responses by regulating the ubiquitination of key signaling proteins.14 Our data demonstrated that forced expression of A20 in the heart resulted in markedly improved functional recovery, decreased inflammation, reduced apoptosis, and diminished interstitial fibrosis after acute myocardial infarction.15 Cook et al16 reported that A20 is dynamically regulated during acute biomechanical stress in the heart and functions to attenuate cardiac hypertrophy in vitro. Despite the potentially significant roles of A20 in attenuating NF-κB–dependent apoptotic, inflammatory, and hypertrophic signaling, it has remained unclear whether A20 could regulate cardiac hypertrophy in vivo and whether targeted myocardial overexpression of A20 is cardioprotective. Thus, in the present study, our aim was to investigate the role of A20 in cardiac hypertrophy mediated by pressure overload and to clarify the related molecular mechanisms.
Methods and Materials
The antibodies against extracellular signal-regulated kinase (ERK) 1/2, p38, c-Jun N-terminal kinase (JNK), caspase 3/8/9, phospho-Smad2, transforming growth factor (TGF)-β–activated kinase 1 (TAK1), phospho-p65, inhibitor of κB (IκB) kinase (IKK)-α, IKKβ, phospho-IκBα, and IκBα were purchased from Cell Signaling Technology. [3H]-leucine and [3H]-proline were purchased from Amersham. The details for other reagents and adenoviral are given in the online Data Supplement, available at http://hyper.ahajournals.org.
Animals, Aortic Banding Surgery, Blood Pressure, and Echocardiography
The study protocol was approved by the animal care and use committee of our hospital. The details for mice information, aortic banding (AB) model, echocardiography, and cardiac catheterization are given in the online Data Supplement. Hearts and lungs of euthanized mice were dissected and weighed to compare heart weight/body weight (in milligrams per gram) and lung weight/body weight (in milligrams per gram) in transgenic (TG) and control mice.
Histological Analysis and Determination of Apoptosis
Several sections of heart (4 to 5 μm thick) were prepared and stained with hematoxylin-eosin for histopathology or Picrosirius red for collagen deposition, then visualized by light microscopy. For myocyte cross-sectional area, a single myocyte was measured with an image quantitative digital analysis system (National Institutes of Health Image 1.6). The outline of 100 to 200 myocytes was traced in each group. Apoptosis was evaluated by TUNEL assay and caspase activities assay.
Western Blot Analysis, Northern Blot, and Reporter Assays
For Northern blot and Western blot analyses, as well as atrial natriuretic peptide promoter activity, please see the online Data Supplement.
Electrophoretic Mobility Shift Assay, IKK Assay, and TAK1 Kinase Assay
Electrophoretic mobility shift assays were performed according to the manufacturer’s instructions (Gel Shift Assay System E3300, Promega). Nuclear proteins were isolated using our method described previously.6,7⇓ To determine the effect of A20 on IKK activation, the IKK assay was performed as described previously.6,7⇓ TAK1 immunoprecipitates were assayed using His-MKK6 as a substrate, as described previously.17
Cultured Neonatal Rat Cardiac Myocytes and Fibroblasts
[3H]-Leucine and [3H]-Proline Incorporation and Surface Area
All of the values are expressed as mean±SEM. Differences between 2 groups were determined by a Student t test. Comparisons between groups on Western blotting data were assessed by 1-way ANOVA followed by a Bonferroni correction. A value of P<0.05 was considered statistically significant.
Forced A20 Expression Attenuates Pathological Cardiac Hypertrophy
To investigate the role of A20 in biomechanical stress in the heart, we performed AB surgery on 8- to 10-week–old TG and wild-type (WT) mice. As shown in Table S1, heart weight:body weight and lung weight:body weight ratios were significantly decreased in TG mice compared with WT mice. Cardiac function was examined by echocardiography after 8 weeks of surgery. The increases in left ventricle chamber dimensions and wall thickness induced by pressure overload were also markedly reduced during both systole and diastole in TG mice compared with WT littermates (Table S1). Gross heart and hematoxylin-eosin staining further confirmed the inhibitory effect of A20 on cardiac remodeling in response to AB (Figure 1). We examined the expression of several cardiac hypertrophy markers in TG and WT mice after AB surgery by Northern blot analysis. Expression levels of atrial natriuretic peptide, brain natriuretic peptide, and β-myosin heavy chain were induced to a higher level in WT mice after AB, and such increases were markedly attenuated in TG mice (Figure S1). These results indicate that A20 overexpression in cardiomyocytes decreases the expression of cardiac hypertrophy markers atrial natriuretic peptide, brain natriuretic peptide, and β-myosin heavy chain and results in attenuated cardiac hypertrophy induced by pressure overload.
Forced A20 Expression Attenuates Mechanical Stress-Mediated p38/JNK1/2 Signaling
To examine the molecular mechanisms of A20 on cardiac hypertrophy, we investigated activation of the mitogen-activated protein kinase (MAPK) pathway in our hypertrophic models. We found that the phosphorylated levels of p38, JNK1/2, and ERK1/2 were significantly increased by AB in WT hearts. However, the phosphorylation of p38 and JNK1/2 was almost completely blocked in TG hearts, whereas ERK1/2 activation was similar in the 2 groups after AB (Figure 2). Although AKT signaling plays a crucial role in the regulation of cardiac remodeling and apoptosis, we did not observe any differences in AKT activation between WT and TG mice, as determined by immunoblotting for phosphorylation of AKT (Figure 2). Collectively, these data suggest that A20 overexpression suppresses the activation of p38 and JNK, although it has no effects on ERK1/2 or AKT activation in hearts subjected to AB. In vitro studies further demonstrated that p38 and JNK phosphorylation levels were enhanced after the reduction of A20 expression by RNA interference in response to hypertrophic stimuli. In contrast, p38 and JNK activations were almost completely blocked by increased A20 expression in cultured cardiac myocytes (Figure S2). These findings suggest that p38/JNK signaling was critical to the influence of A20 on cardiac hypertrophy.
Forced A20 Expression Impairs TAK1 Signaling Involved in Hypertrophy
Activation of TAK1, an upstream regulator of p38 and JNK, has been shown to participate in cardiac dysfunction after the development of hypertrophy. We, therefore, determined cardiac TAK1 activation by in vitro kinase activity assay. TAK1 activity was markedly increased in response to AB in WT mice (Figure 3). In the AB model, TAK1 activity was increased at 24 hours, peaked after 4 weeks, and then decreased, although the level remained higher than in the sham group. In contrast, the activity of TAK1 in response to AB was significantly abolished in TG hearts (Figure 3), suggesting that A20 overexpression may suppress TAK1 activation. The total protein level of TAK1 was not different among all of the tested groups. Consistent with our in vivo results, in vitro results showed that overexpression of A20 by infection of AdA20 blocked angiotensin (Ang) II–induced TAK1 activation, whereas downregulation of A20 expression by infection with AdsiA20 promoted angiotensin II–induced TAK1 activation (Figure S3). To further investigate the molecular mechanisms of the function of A20, we examined the effects of TAK1 activation on p38/JNK and cardiac hypertrophy. Blocking TAK1 activation by dominant-negative TAK1 (AddnTAK1) abrogated angiotensin II–mediated p38/JNK phosphorylation and cardiac hypertrophy, whereas activation of TAK1 by constitutively active TAK1 (AdcaTAK1) augmented these effects, as demonstrated by Western blot, ANF promoter activity, and [3H]-leucine incorporation (Figure S4 and S5). These results indicate that A20 attenuates cardiac hypertrophy by blocking TAK1-dependent JNK/p38 signaling pathways.
Forced A20 Expression Attenuates Fibrosis In Vivo
Heart sections were stained with Picrosirius red to detect fibrosis. In both groups, collagen continued to accumulate in the heart after 8 weeks of AB. As shown in Figure 4A, Picrosirius red staining and quantitative analysis showed that increased collagen deposition was significantly attenuated in TG mice. Reduced fibrosis in TG mice may represent increased collagen degradation or decreased collagen synthesis in response to tissue damage. We, therefore, examined the synthesis of collagen by examining the expression of mRNA and protein-encoding connective tissue growth factor, collagen I, collagen III, TGF-β1, and TGF-β3, known to be involved in the proliferation of cardiac fibroblasts and the biosynthesis of extracellular matrix proteins. The results showed that connective tissue growth factor, collagen I, collagen III, TGF-β1, and TGF-β3 mRNA, as well as protein expressions, were significantly lower in TG than in WT mice in response to hypertrophic stimuli (Figures 4B and S6). We then assessed the regulatory role of A20 in Smad cascade activation. TG animals showed suppressed Smad-2 phosphorylation, and almost complete inhibition of Smad-2/3/4 nuclear translocation but negligible effects on Smad-2/3/4 protein expression (Figure S7).
Forced A20 Expression Inhibits Collagen Synthesis Induced by TGF-β1 In Vitro
To confirm our in vivo fibrosis data, we examined the potential antifibrotic effect of A20 by [3H]-proline incorporation assay in cardiac fibroblasts. Cells were infected with AdA20 or AdsiA20 for 24 hours, then serum starved for 24 hours in 0.5% FCS, and subsequently treated with different concentrations of TGF-β1 for 48 hours or with 15 ng/mL of TGF-β1 for the indicated time. TGF-β1 stimulated [3H]-proline incorporation in a time- and dose-dependent manner (data not shown). More importantly, overexpression of A20 by infection of AdA20 inhibited TGF-β1–induced [3H]-proline incorporation, connective tissue growth factor, and collagen I/III protein expression. Conversely, downregulation of A20 expression by infection with AdsiA20 promoted these effects (Figure 5). To further investigate the molecular mechanisms of A20 on fibrosis, we examined the effects of A20 on Smad signaling in vitro. Western blot analysis revealed significant phosphorylation of Smad 2 and translocation of Smad 2/3/4 without any significant alterations in Smad 2/3/4 protein expression after TGF-β1 treatment in adenovirus containing green fluorescent protein (AdGFP) and Adsi control groups (Figure S8). AdA20 infection, however, almost completely suppressed Smad 2 phosphorylation, as well as Smad-2/3/4 nuclear translocation, whereas AdsiA20 enhanced these effects (Figure S8).
We then examined the effects of A20 on TGF-β1-induced TAK1 activity. Our further experiments demonstrated that forced expression of A20 significantly blocked TAK1 activity mediated by TGF-β1, whereas decreased A20 expression promoted TAK1 activity in cultured cardiac fibroblasts (Figure S9). Confluent cardiac fibroblasts were infected with AdGFP, AdcaTAK1, or AddnTAK1 and incubated with TGF-β1 for an indicated time. Activation of TAK1 induced a significant increase in collagen synthesis by TGF-β1, whereas blocking TAK1 activity by infection with AddnTAK1 almost completely abrogated the TGFβ1-induced responses (Figure S10). Furthermore, immunoblot analysis demonstrated that TGF-β1 incubation of cardiac fibroblasts infected with AdcaTAK1 resulted in markedly increased phosphorylation of Smad-2 and nuclear translocation of Smad-2/3/4 in response to TGF-β1. Conversely, infection with AddnTAK1 almost completely blocked these effects (Figure S11).
A20 Expression Inhibits Apoptosis and Inflammatory Response Induced by AB
We next examined the effects of A20 on apoptosis by TUNEL assays after 8 weeks of AB. Apoptotic cells were detected in TG and control mice, and the fraction of apoptotic versus total cells was significantly lower in TG mice than in WT mice (Figure 6A). To determine whether TG mice are resistant to apoptotic signals, we examined the cleavage of caspase 3, caspase 8, and caspase 9, as well as that of poly (ADP-ribose) polymerase (PARP). As expected, TG mice displayed a significant delay of cleavage of caspase 3, caspase 8, and caspase 9, as well as PARP degradation in response to AB (Figure 6B). To determine whether expression of A20 prevents the inflammatory responses in the hearts, cytokine induction was characterized by Western blot analyses. TG mice have significantly lower TNF-α, interleukin 6, and monocyte chemoattractant protein 1 protein levels in cardiac tissue after 8 weeks of surgery compared with WT mice (Figure S12). To determine the molecular mechanisms by which A20 attenuated cytokine induction in vivo, we analyzed NF-κB signaling pathways. We detected NF-κB activation, IKKβ, and IκBα phosphorylation, as well as IκBα degradation, clearly after 8 weeks of AB in WT mice. Interestingly, NF-κB activation, IKKβ and IκBα phosphorylation, and IκBα degradation were evidently blocked in TG mice (Figure S13).
In the present study, we demonstrate that the expression of A20 in the heart protects against cardiac hypertrophy. The cardioprotection of A20 is mediated by interruption of TAK1 activity–dependent signaling pathways (Figure 7). This results in the protection of the host from the combined deleterious effects of cardiac hypertrophy, apoptosis, inflammation, and fibrosis (Figure 7). The ability of A20 to prevent cardiac dysfunction and hypertrophy mediated by sustained pressure overload suggests that it may be an effective therapeutic candidate.
The MAPK signaling cascade is initiated in cardiac myocytes by activation of G protein–coupled receptors, receptor tyrosine kinases, and stress stimuli.19,20⇓ Once activated, downstream p38, JNKs, and ERKs each phosphorylate a wide array of intracellular targets, including numerous transcription factors, resulting in the reprogramming of cardiac gene expression. A significant finding of the present study is that the increase in JNK and p38 phosphorylation levels in response to hypertrophic stimuli was almost completely blocked in TG mice. The phosphorylations of ERK1/2 and AKT in myocytes were not affected by A20 expression. Further in vitro studies showed that inhibition of A20 expression significantly enhanced the activation of JNK and p38 but not that of ERK1/2 and AKT. Therefore, JNK/p38 signaling was the mediator of influences of A20 on cardiac myocyte growth. Suppression of the JNK/p38 signaling pathway by A20 in the heart attenuates cardiac remodeling. However, there is still some controversy on whether activation of the JNK and p38 MAPK pathway is protective or detrimental. Blocking JNK or p38 signaling by either genetic or pharmacological approaches has previously demonstrated cardioprotective effects.21,22⇓ In contrast, other studies suggest that JNK/p38 signaling may protect against apoptosis.23,24⇓ Another study found that dual JNK/p38 inhibition also leads to increased apoptosis in the heart25; however, this report also shows that the proapoptotic effects of the dual JNK/p38 inhibitor are possibly attributable to suppression of JNK, as opposed to p38 MAPK. These previous studies suggest that, although inhibition/activation of either p38 or JNK pathways produces the same cardiac phenotype, the temporal manifestation of the disease possibly depends on the overall extent of cellular signaling inhibition/activation and especially the upstream molecules of JNK/p38MAPK. This view is further supported by 2 recent studies on mixed-lineage kinase 7 and heat shock protein 20.26,27⇓ Mixed-lineage kinase 7 was reported to activate both JNK and p38 MAPK, and overexpression of mixed-lineage kinase 7 resulted in cardiac hypertrophy and promoted cell death in the heart, indicating that dual activation of JNK/p38 is more catastrophic than either alone. Another study showed that heat shock protein 20 overexpression blocks cardiac hypertrophy and fibrosis through inhibition of the ASK1-p38/JNK cascade.27 These findings suggest that inhibition of the upstream regulator of p38 and JNK may be beneficial in halting cardiac remodeling and the progression of heart failure.
To further investigate the molecular mechanisms by which A20 inhibits cardiac hypertrophy, we examined another protein upstream of p38/JNK, TAK1. TAK1 is an MAPK kinase kinase family member originally identified as a mediator in the TGF-β signaling pathway and can be activated in response to stress stimuli.28 Genetic and biochemical evidence has established TAK1 as a key kinase that mediates the activation of IKK, p38, and JNK by diverse cellular stimuli.29 Recent studies showed that TAK1 is critically important in the cardiac hypertrophic response.30 We found that A20 not only suppressed TAK1 activity in vivo in response to hypertrophic stimuli but also blocked TAK1 activity induced by angiotensin II in vitro. Our in vitro study also showed that a decreased A20 expression level effectively enhanced TAK1 activity resulting from angiotensin II. In addition, our data confirmed that inhibition of TAK1 activity abrogated the activation of JNK/p38, whereas activation of TAK1 activity augmented the phosphorylation of JNK/p38 in response to hypertrophic stimuli in vitro. These findings indicate that A20 attenuates cardiac hypertrophy by blocking TAK1-JNK/p38 signaling.
Cardiac fibrosis is another classic feature of pathological hypertrophy and is characterized by the expansion of the extracellular matrix attributed to the accumulation of collagen.31 Thus, it is important to understand the mechanisms that stimulate collagen deposition in the heart and define approaches to limit these processes. We found that A20 blocks cardiac fibrosis in vivo and inhibits collagen synthesis in vitro. Our study demonstrated, for the first time, that A20 blocks AB-induced fibrosis in vivo and TGF-β1–induced collagen synthesis in cardiac fibroblasts. In addition, our data suggest, for the first time, that A20 abrogates Smad 2 phosphorylation and Smad 2/3/4 translocation in both cardiac fibroblasts and hypertrophied hearts, thus inhibiting collagen synthesis and fibrosis. There is considerable evidence for synergy between the TAK-dependent and Smad-dependent TGF-β signaling pathways. TAK1 has been reported to interact with Smad 7 to inhibit TGF-β signaling by a negative feedback mechanism. More recently, TAK1 has been shown to interact with Smads and to inhibit BMP signaling.32 The relative contributions, however, of TAK-dependent and Smad-dependent pathways to cardiac fibrosis remain undetermined. We demonstrated that blocking TAK1 activation led to complete inhibition, whereas activation of TAK1 led to upregulation of collagen synthesis and Smad 2/3/4 activation in vitro. We also showed that TGF-β1–induced collagen synthesis depends on TAK1 signaling, indicating that the inhibitory effects of A20 on fibrosis and collagen synthesis are mediated by blocking TAK1-dependent signaling.
Cardiac myocyte apoptosis plays an important role in the transition of cardiac hypertrophy to heart failure.33 The experimental findings here show a correlation between an increase in the frequency of apoptosis and the extent of cardiac remodeling. Consistent with previous reports, A20 expression in the heart markedly decreased the number of apoptotic cells in response to long-term pressure overload. Furthermore, it has been demonstrated that A20 blocks apoptosis of various cell types, associated with inhibition of caspase-3, -8, and -9 activities.9,10⇓ Indeed, we found that overexpression of A20 attenuates myocardial apoptosis and is associated with abrogated cleavages of caspase-3 and -9, as well as that of PARP. The effects of A20 on these signaling molecules may explain the protection from apoptosis observed in TG hearts subjected to AB. In addition to apoptosis, there is evidence that proinflammatory cytokines play a role in pathological cardiac hypertrophy and heart failure.34,35⇓ We found a marked induction of cytokine expression in the heart in response to hypertrophic stimuli that was observably attenuated by cardiac forced expression of A20. One possible mechanism for such a protective effect is that A20 expression directly blocks NF-κB activation, attenuating the inflammatory response and subsequent myocardial hypertrophy. Our present data suggest that A20 abrogates NF-κB activation by disrupting DNA binding and phosphorylation of IκB. By blocking NF-κB signaling, A20 may inhibit the early steps of inflammation and modulate the amplification of multiple cytokine signaling cascades. In summary, the present work demonstrates that A20 protects against cardiac remodeling and heart failure in response to hypertrophic stimuli. The mechanism underlying the protective effects of A20 appears to involve the inhibition of the TAK1-JNK/p38 signaling pathway. The potential for A20 as a therapeutic target should be considered in future studies.
The current study provides a new insight into the role of A20 in the development of cardiac hypertrophy and fibrosis induced by pressure overload. Our findings suggest that A20 behaves as an endogenous and negative regulator of hypertrophic response, which may provide a novel therapeutic target for cardiac hypertrophy and fibrosis.
Sources of Funding
This research was supported by the National Natural Science Foundation of China (30900524, 30972954, and 30770733) and by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (2006-331).
H.H., Q.-Z.T., and A.-B.W. are joint first authors.
- Received January 6, 2010.
- Revision received January 26, 2010.
- Accepted June 6, 2010.
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- ↵Li HL, She ZG, Li TB, Wang AB, Yang Q, Wei YS, Wang YG, Liu DP. Overexpression of myofibrillogenesis regulator-1 aggravates cardiac hypertrophy induced by angiotensin II in mice. Hypertension. 2007; 49: 1399–1408.
- ↵Malynn BA, Ma A. A20 takes on tumors: tumor suppression by an ubiquitin-editing enzyme. J Exp Med. 2009; 206: 977–980.
- ↵Coornaert B, Carpentier I, Beyaert R. A20: central gatekeeper in inflammation and immunity. J Biol Chem. 2009; 284: 8217–8221.
- ↵Li HL, Wang AB, Zhang R, Wei YS, Huang Y, Liu DP, Liang CC. A20 inhibits oxidized low-density lipoprotein-induced apoptosis through negative Fas/Fas ligand-dependent activation of caspase-8 and mitochondrial pathways in murine RAW264.7 macrophages. J Cell Physiol. 2006; 208: 307–318.
- ↵Li HL, Zhuo ML, Wang D, Wang AB, Cai H, Sun LH, Yang Q, Huang Y, Wei YS, Liu PP, Liu DP, Liang CC. Targeted cardiac overexpression of A20 improves left ventricular performance and reduces compensatory hypertrophy after myocardial infarction. Circulation. 2007; 115: 1885–1894.
- ↵Cook SA, Novikov MS, Ahn Y, Matsui T, Rosenzweig A. A20 is dynamically regulated in the heart and inhibits the hypertrophic response. Circulation. 2003; 108: 664–667.
- ↵Muslin AJ. MAPK signalling in cardiovascular health and disease: molecular mechanisms and therapeutic targets. Clin Sci (Lond). 2008; 115: 203–218.
- ↵Molkentin JD. Calcineurin-NFAT signaling regulates the cardiac hypertrophic response in coordination with the MAPKs. Cardiovasc Res. 2004; 63: 467–475.
- ↵Wang Y. Mitogen-activated protein kinases in heart development and diseases. Circulation. 2007; 116: 1413–1423.
- ↵Kyoi S, Otani H, Matsuhisa S, Akita Y, Tatsumi K, Enoki C, Fujiwara H, Imamura H, Kamihata H, Iwasaka T. Opposing effect of p38 MAP kinase and JNK inhibitors on the development of heart failure in the cardiomyopathic hamster. Cardiovasc Res. 2006; 69: 888–898.
- ↵Fan GC, Yuan Q, Song G, Wang Y, Ashraf M, Kranias EG. Small heat-shock protein Hsp20 attenuates β-agonist-mediated cardiac remodeling through apoptosis signal-regulating kinase 1. Circ Res. 2006; 99: 1233–1242.
- ↵Rosenkranz S. TGF-β1 and angiotensin networking in cardiac remodeling. Cardiovasc Res. 2004; 63: 423–432.
- ↵Cai J, Yi FF, Yang L, Shen DF, Yang Q, Li A, Ghosh AK, Bian ZY, Yan L, Tang QZ, Li H, Yang XC. Targeted expression of receptor-associated late transducer inhibits maladaptive hypertrophy via blocking epidermal growth factor receptor signaling. Hypertension. 2009; 53: 539–548.
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- ↵Di Zhang A, Cat AN, Soukaseum C, Escoubet B, Cherfa A, Messaoudi S, Delcayre C, Samuel JL, Jaisser F. Cross-talk between mineralocorticoid and angiotensin II signaling for cardiac remodeling. Hypertension. 2008; 52: 1060–1067.