Cellular FLICE-Inhibitory Protein Protects Against Cardiac Remodeling Induced by Angiotensin II in Mice
The development of cardiac hypertrophy in response to increased hemodynamic load and neurohormonal stress is initially a compensatory response that may eventually lead to ventricular dilatation and heart failure. Cellular FLICE-inhibitory protein (cFLIP) is a homologue of caspase 8 without caspase activity that inhibits apoptosis initiated by death receptor signaling. Previous studies showed that cFLIP expression was markedly decreased in the ventricular myocardium of patients with end-stage heart failure. However, the critical role of cFLIP on cardiac remodeling remains unclear. To specifically determine the role of cFLIP in pathological cardiac remodeling, we used heterozygote cFLIP+/− mice and transgenic mice with cardiac-specific overexpression of the human cFLIPL gene. Our results demonstrated that the cFLIP+/− mice were susceptible to cardiac hypertrophy and fibrosis through inhibition of mitogen-activated protein kinase kinase-extracellular signal–regulated kinase 1/2 signaling, whereas the transgenic mice displayed the opposite phenotype in response to angiotensin II stimulation. These studies indicate that cFLIP protein is a crucial component of the signaling pathway involved in cardiac remodeling and heart failure.
Cardiac hypertrophy is a response of the myocardium to increased workload, characterized by increase of myocardial mass and accumulation of extracellular matrix.1–3 The initial cardiac hypertrophy acts likely an adaptive mechanism; however, prolonged and severe hypertrophy is a risk factor for arrhythmias, sudden death, and heart failure. Although much is known about the signal transduction pathways that promote hypertrophic responses, mechanisms that antagonize these pathways have not been as clearly defined. A better understanding of these antagonistic mechanisms in cardiac myocytes may lead to novel strategies for suppressing cardiac hypertrophy.
Cellular FLICE inhibitory protein (cFLIP) is a mammalian homolog of the viral FLICE-inhibitory protein and a natural modulator of tumor necrosis factor signaling.4 Multiple cFLIP splice variants have been found, and 2 main forms have been well characterized, cFLIP short form (cFLIPS: 26 kDa) and long form (cFLIPL: 55 to 60 kDa).4 Both splice variants have death effector domains, with which they bind to Fas-associated death domain at the death-inducing signaling complex and inhibit caspase 8 activation. It has been well documented that elevated cFLIP expression protects cells from death receptor-mediated apoptosis, whereas downregulation of cFLIP by chemicals or small interfering RNA sensitizes cells to death receptor-mediated apoptosis.4 A clear reduction in the expression of cFLIP has been demonstrated in the ventricular myocardium of patients with end-stage heart failure and rodents after myocardial infarction.5,6 cFLIP−/− mice also exhibit severe defects in cardiac trabecula formation, as well as a thinner myocardium.7 In addition, Huber et al8 found that T-cell–specific overexpression of cFLIPL diminished the severity of coxsackievirus B3-induced myocarditis. These data suggest that cFLIP plays a vital role in cardiac development and cardiomyocyte survival after stress. Nevertheless, the role of cFLIP in cardiac hypertrophy remains largely unclear. Recently, Giampietri et al9 reported that mouse cFLIPL mild overexpression reduced transverse aortic constriction–induced hypertrophy. However, it should be noted that the cFLIP transgenic (TG) mice in their study are generated under the control of the testis-specific Stra 8 promoter. More importantly, mice with cardiac-specific overexpression or deletion of cFLIPL gene are optimal animals to investigate the role of cFLIP in cardiac hypertrophy and the related mechanisms. In the present study, using cardiac-specific TG mice and cFLIP heterozygous knockout mice, we show for the first time that the cFLIP+/− (HET) mice exacerbate cardiac hypertrophy and fibrosis by blocking mitogen-activated protein kinase (MAPK) kinase (MEK)-extracellular signal-regulated kinase (ERK)1/2 signaling, whereas the TG mice with cardiac constitutive expression of human cFLIPL displayed the opposite phenotype in response to angiotensin II (Ang II) stimulation. Our study indicates that cFLIP is a pivotal inhibitor of cardiac remodeling and heart failure.
Methods and Materials
Antibodies for the MAPK and Smad pathways were purchased from Cell Signaling Technology. The antibody used to recognize GATA-4 was purchased from Upstate Biotechnology. The anti-cFLIPL (reactive with mouse or human) antibody was purchased from Abcam Inc. [3H]-Proline was purchased from Amersham. The details for other reagents and adenoviral vectors are given in the online Data Supplement at http://hyper.ahajournals.org.
Animals, Ang II Infusion Model, Blood Pressure, and Echocardiography
All protocols were approved by the Animal Care and Use Committee of University Health Network (Toronto, Ontario, Canada) and Renmin Hospital of Wuhan University (Wuhan, China). The details for mice information, Ang II infusion model, echocardiography, and cardiac catheterization are given in the online Data Supplement. Hearts and lungs of the euthanized mice were dissected and weighed to compare heart weight (HW)/body weight (BW; milligram per gram) and lung weight (LW)/BW (milligram per gram) ratios in different groups.
Several sections of heart (4 to 5 μm thick) were prepared and stained with hematoxylin-eosin for histopathology or Picrosirius red (PSR) 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.
Quantitative Real-Time RT-PCR, Western Blotting, and Electrophoretic Mobility Shift Assays
For quantitative real-time PCR, Western blot, and electrophoretic mobility shift assay analyses, please see the online Data Supplement. Nuclear proteins were isolated as described previously.10
Recombinant Adenoviral Vectors, Cultured Neonatal Rat Cardiac Myocytes, and Fibroblasts
Recombinant adenoviral vectors includes Ad-cFLIP, Ad-LacZ, Ad-shcFLIP, and Ad-shRNA. Primary cultures of cardiac myocytes and fibroblasts were prepared as described.10,11 For details, please see the online Data Supplement.
Cardiac Myocyte Area and Collagen Synthesis Assay
The surface areas of cardiac myocytes and [3H]-proline incorporation were measured as described previously.10 For details, please see the online Data Supplement.
Determination of Apoptosis and Myocardial Caspase 3, Caspase 8, and Caspase 9 Activity
Apoptosis was evaluated by TUNEL assay and caspase activities assay, as described previously.12,13
Data are expressed as mean±SEM. Differences among groups were tested 2-way ANOVA followed by post hoc Tukey test. Comparisons between 2 groups were performed by unpaired Student t test. A value of P<0.05 was considered to be significantly different.
Decreased cFLIP Level Exacerbated Cardiac Hypertrophy Induced by Ang II In Vivo
Because cFLIP−/− mice exhibit severe defects in cardiac formation and do not survive beyond day 12 of gestation, we used HET mice whose cFLIP mRNA and protein levels were significantly decreased in the heart (Figure S1A and S1B, available in the online Data Supplement) to assess the impact of deletion of 1 allele of cFLIP on cardiac hypertrophy. To investigate whether cFLIP expression is regulated by Ang II infusion or pressure overload, wild-type (WT) mice were subjected to Ang II infusion or aortic banding for different durations. As shown in Figure S1C, cFLIP protein levels were significantly decreased at week 4 after Ang II infusion. In addition, cFLIP expression increased by 2.4-fold over basal levels (n=6) at week 2 of aortic banding. However, cFLIP expression in the left ventricle (LV) was markedly decreased compared with basal levels after 8 weeks of aortic banding (Figure S1D). Thus, cFLIP expression is regulated during LV remodeling induced by chronic Ang II infusion or pressure overload.
To examine the potential effect of cFLIP on hypertrophy mediated by Ang II infusion, osmotic minipumps were implanted subcutaneously for a 4-week administration period, followed by cardiac functional assessment. The ratios of HW/BW and LW/BW cross-sectional area of cardiac myocytes were significantly increased in both WT and HET mice, and such increases were more pronounced in HET mice than in WT mice after 4 weeks of Ang II infusion (Figure 1A). We next assessed cardiac function in WT and HET mice by echocardiography. The cardiac functional parameters showed that decreased fractional shortening with dilated LV end-systolic diameter and LV end-diastolic diameter, as well as increased LV diastolic septum, and LV posterior wall thickness, were seen in WT mice. In HET mice, significantly higher LV end-systolic diameter and LV end-diastolic diameter, bigger increased LV diastolic septum, and LV posterior wall thickness, as well as lower fractional shortening, were observed compared with WT mice (Figure 1B), indicating that HET mice have a greater level of cardiac hypertrophy and dysfunction compared with WT mice in response to hypertrophic stimuli. Gross heart and wheat germ agglutinin and hematoxylin-eosin staining analysis further confirmed the exaggerated effect of inhibition of cFLIP on cardiac remodeling after Ang II stimulation (Figure 1C). Atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), myosin heavy polypeptide 7 cardiac muscle β (Myh7), and actin α1 skeletal muscle (Acta1) are markers for cardiac hypertrophy.14 To determine whether cFLIP affected the mRNA expression levels of these markers, we performed real-time PCR. Our results showed that the expression levels of ANP, BNP, Myh7, and Acta1 mRNA were higher in HET mice than those in WT mice (Figure S1E). These findings suggest that decreased cFLIP level promotes the development of cardiac hypertrophy induced by Ang II stimulation in vivo.
Effect of cFLIP on MEK-ERK1/2 Signaling Pathway
MAPK signal transduction pathways have been shown to be activated by a variety of hypertrophic stresses, including neurohormonal stimuli and hemodynamic overload.15,16 To explore the molecular mechanisms through which decreased cFLIP level enhances the hypertrophic response, we examined the state of activation of MAPK in HET and WT hearts in the Ang II–infused hypertrophic model. We found that the increased phosphorylated levels of MEK1/2 and ERK1/2 induced by Ang II were more pronounced in HET hearts (Figure 2A), whereas p38 and Jun kinase (JNK)1/2 were similarly activated in the 2 groups (data not shown). 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 HET mice (data not shown). Collectively, these data suggest that decreased expression of cFLIPL increases the activation of MEK-ERK1/2 signaling, although it has no effect on p38, JNK, or Akt activation in hearts subjected to Ang II stimulation.
To further examine the role of cFLIP on MEK-ERK1/2 signaling in the heart, we exposed cultured neonatal rat cardiomyocytes to 1 μmol/L of Ang II infected with Ad-cFLIP or Ad-shcFLIP (recombinant adenoviral vector cFLIP short hairpin RNA). At the beginning, we screened 3 shcFLIP and found that No. 2 shcFLIP markedly inhibited cFLIP expression in cardiac myocytes (Figure S2A). Therefore, we chose No. 2 shcFLIP for the following experiments. Further studies showed that Ang II induced a significant phosphorylation of MEK1/2 and ERK1/2 that was almost completely blocked and sustained for all of the tested time points by overexpression of cFLIP (Figure S2B and S2C). More importantly, decreased cFLIP levels by infection of Ad-shcFLIP resulted in pronounced activation of MEK1/2 and ERK1/2 in cardiac myocytes (Figure S2B and S2C). Our findings suggest that cFLIP inhibits MEK-ERK1/2 signaling in vitro and in vivo in response to hypertrophic stimuli. To examine whether MEK-ERK signaling has a causative role in cFLIP-mediated inhibition of cardiac hypertrophy, further in vitro experiments were performed. As expected, decreased cFLIP levels led to pronounced hypertrophy induced by Ang II as assessed by surface area measurements (Figure S2D). This response was strongly blunted by U0126, an MEK inhibitor that prevented ERK1/2 phosphorylation. These results suggest that cFLIPL inhibits cardiac hypertrophy through direct inhibition of MEK-ERK1/2 signaling in cardiac myocytes. The inhibition of MEK1/2 and ERK1/2 phosphorylation by cFLIPL prompted us to investigate whether cFLIPL directly interacts with MEK-ERK signaling. Inconsistent with recent findings that demonstrated the direct interaction of cFLIPL with mitogen-activated protein kinase kinase 7 (MKK7) to suppress JNK activation,17 our immunoprecipitation analysis showed that cFLIPL directly interacted with MEK1 (Figure S2E).
Gain- and loss-of-function analyses have indicated that the MEK1-ERK1/2 pathway activates critical transcription factors, including GATA-4, required for the activation of cardiac genes during hypertrophy.18–20 As expected, decreased cFLIP level enhanced GATA-4 phosphorylation and its DNA-binding activity induced by Ang II infusion (Figure 2B). In addition, we found that Ang II–induced GATA-4 phosphorylation and DNA-binding activity were almost completely inhibited by infection with dominant-negative ERK1/2 (Ad-dnERK) or treatment with U0126 but were augmented by infection with constitutive-active ERK1/2 (Ad-caERK1/2; Figure S2F). These data suggest that GATA-4 phosphorylation and activation require MEK-ERK1/2 signaling in cardiac myocytes.
Decreased cFLIP Level Exacerbated Cardiac Fibrosis Induced by Ang II In Vivo
Cardiac hypertrophy is associated with increased fibrosis in the myocardium, characterized by the overproduction of extracellular matrix proteins.1 To determine the extent of fibrosis in the heart, paraffin-embedded slides were stained with PSR. Marked perivascular fibrosis and interstitial fibrosis were present in the WT mice subjected to Ang II infusion by PSR staining but much more prominent in the HET mice (Figure 3A). Subsequent analysis of mRNA expression levels of known mediators of fibrosis, including transforming growth factor-β1 (Tgfβ1), procollagen type I α1 (Col1α1), procollagen type III α1(Col3α1), plasminogen activator inhibitor 1 (Pai-1), and connective tissue growth factor (Ctgf), demonstrated an increased response in HET mice (Figure S3).
Effect of cFLIP on Transforming Growth Factor-β1/Smad Signaling
Transforming growth factor (TGF)-β1 induces collagen synthesis via activation of a number of transcription factors, including Smads.21 To further elucidate the cellular mechanisms underlying the antifibrotic effects of cFLIP, we assessed the regulatory role of cFLIP on Smad cascade activation. Our results showed that HET mice induced higher levels of phosphorylation of Smad 2 and more nuclear translocation of Smad 2/3 compared with that of WT mice (Figure 3B). We then infected cardiac fibroblasts with Ad-cFLIP or Ad-shcFLIP and treated with TGF-β1 for specific time periods. Western blot analyses revealed significant phosphorylation of Smad 2 and translocation of Smad 2/3 without any significant alterations in Smad 2 after TGF-β1 treatment. Ad-cFLIP infection, however, almost completely suppressed Smad 2 phosphorylation, as well as Smad 2/3 nuclear translocation (Figure S4A and S4B). Importantly, Ad-shcFLIP infection enhanced the effects of TGF-β1 (Figure S4A and S4B). To further examine the mechanisms involved, we used confluent cardiac fibroblasts infected with Ad-GFP, Ad-caERK1/2, or Ad-dnERK1/2 or treated with U0126. Activation of ERK1/2 by infection with Ad-caERK1/2 revealed a significant increase, while blocking ERK1/2 activity by U0126 or Ad-dnERK1/2 infection almost completely abrogated collagen synthesis, as well as Smad 2 phosphorylation and Smad 2/3 nuclear translocation in response to TGF-β1 (Figure S4C and S4D). These findings suggest that cFLIP blocks collagen synthesis by disrupting MEK-ERK1/2–dependent TGF-β-Smad signaling.
Characterization of Human cFLIPL TG Mice
The above findings suggested that increased cFLIP levels in the heart will attenuate cardiac remodeling. To confirm this notion, we generated TG mice with full-length human cFLIPL cDNA under the control of the α-myosin heavy chain promoter (Figure S5A). Four lines of TG mice were confirmed by PCR. All of the experiments reported were performed with male mice that were 7 to 8 weeks old. We analyzed cFLIPL protein levels in various tissues by Western blot analysis using a human-specific anti-cFLIPL antibody. We found a robust expression of human cFLIPL protein in the heart but did not detect it in other organs (Figure S5B). Among 4 established lines of TG mice, the line that expressed the highest levels of the human cFLIPL protein in the heart was used for further experiments (Figure S5C). Western blot analysis further demonstrated that the expression level of the mouse cFLIPL protein was not modified by expression of the human cFLIPL gene (Figure S5D).
Human cFLIPL TG Mice Develop Decreased Cardiac Hypertrophy and Fibrosis
The Ang II–induced increase in the ratios of HW/BW, LW/BW, and cardiomyocyte cross-sectional area were attenuated in TG mice compared with WT mice (Figure 4A). Cardiac-specific overexpression of cFLIP abrogated Ang II–induced cardiac chamber dilatation and wall thickness and dysfunction, as evidenced by improvements in LV end-systolic diameter, LV end-diastolic diameter, LV posterior wall thickness, increased LV diastolic septum, and fractional shortening (Figure 4B). Histology analysis further confirmed the inhibitory effect of cFLIP on cardiac hypertrophy mediated by Ang II infusion (Figure 4C). Consistent with the functional data, the mRNA expression levels of hypertrophy markers ANP, BNP, Myh7, and Acta1 were severely blunted in TG mice (Figure S5E). Further studies showed that the cardiac overexpression of cFLIPL abolished MEK and ERK1/2 phosphorylation, as well as GATA-4 phosphorylation and its DNA-binding activity induced by Ang II infusion (Figure S5F and S5G). Moreover, the Ang II–induced interstitial fibrosis and perivascular fibrosis were present in WT mice but were remarkably reduced in the TG mice (Figure 4D). Ang II treatment also led to a lower increase of the mRNA expression of fibrotic markers Tgfβ1, Col1α1, Col3α1, Pai-1, and Ctgf in TG mice than in WT mice (Figure S5H). Further studies showed that the increased level of Smad 2 phosphorylation and Smad 2/3 nuclear translocation was attenuated in TG mice in response to Ang II infusion (Figure 4E). These results suggest that constitutive expression of cFLIPL in the heart leads to decreased cardiac hypertrophy and fibrosis in response to Ang II stimulation.
Effect of cFLIP on Apoptosis
It is well known that apoptosis is involved in pathological cardiac remodeling.2,6,12,13 Because cFLIP has been shown to protect cells from death receptor-mediated apoptosis, we further examined whether the cardioprotective role of cFLIP is related to its antiapoptotic effect in the Ang II–infused hypertrophic model. Myocardial tissue sections were stained with TUNEL staining to detect apoptosis. TUNEL-positive nuclei were present in the control mice subjected to Ang II infusion, whereas their number was markedly increased in HET mice and was significantly decreased in TG mice (Figure S6A and S6B). To further elucidate the mechanisms underlying the antiapoptotic effect of cFLIP, we assessed the regulatory role of cFLIP on caspases activation. Ang II stimulation increased caspase 3, caspase 8, and caspase 9 activity modestly in control hearts and massively in HET hearts; however, the activation of these caspases was rarely seen in TG hearts (Figure S6A and S6B). These results suggest that cFLIP inhibits cardiomyocyte apoptosis, at least in part, by blocking the activation of caspase 3, caspase 8, and caspase 9.
Previous studies have demonstrated that the expression of cFLIP was significantly decreased in the ventricular myocardium of patients with heart failure.5,6 However, its function during cardiac hypertrophy was unclear. In the present study, we examined the role of cFLIP in cardiac hypertrophy by using cardiac-specific cFLIPL HET mice and TG mice. The results demonstrated that decreased levels of cFLIP protein expression in HET mice profoundly exaggerated hypertrophy, chamber dilatation, and fibrosis via enhancement of MEK-ERK1/2 signaling after Ang II stimulation. In contrast, elevated cFLIP levels cause a blunt response of pathological cardiac remodeling. These findings suggest that cFLIP protein is a crucial component of the signaling pathway involved in cardiac remodeling and heart failure.
The mechanisms underlying the antihypertrophic effects of cFLIP remain largely unclear. It is well known that the MAPK signaling pathway plays a critical role in the pathogenesis of cardiac hypertrophy.22–24 The MAPK pathway consists of a sequence of successively acting kinases, including p38, JNKs, and ERKs, and is initiated in cardiac myocytes by stress stimuli.25,26 To examine the molecular mechanisms involved in cFLIP’s ability to suppress cardiac hypertrophy, we examined the activation status of the MAPK pathway in our hypertrophic models. Our data showed that MEK activation and ERK1/2 activation were enhanced further by decreased cFLIP levels, whereas MEK and ERK1/2 phosphorylation levels were almost completely blocked by cardiac expression of human cFLIPL in response to Ang II stimulation. However, the phosphorylation of p38, JNK1/2, and Akt was not affected by cFLIP. Therefore, MEK-ERK1/2 signaling seems to be a critical pathway through which cFLIP influences cardiomyocyte hypertrophic response. In accordance with our finding, Giampietri et al9 reported that cFLIP mild overexpression reduces cardiac hypertrophy in response to pressure overload. However, they demonstrated that the antihypertrophic effect of cFLIP is probably achieved through blocking Akt/GSK3β signaling. The discrepancy on mechanisms may be related to a different promoter controlling cFLIP overexpression, the cardiac-specific α-myosin heavy chain promoter in our study and the testis-specific Stra 8 promoter in their study. More importantly, we performed gain- and loss-of-function analyses both in vivo and in vitro, confirming the MEK-ERK1/2–related mechanism in the antihypertrophic effect of cFLIP. Our further study demonstrated that cFLIPL can bind directly to MEK1, disrupt its interactions, and finally block its activation. These findings are consistent with a recent study that showed that cFLIPL binds to MKK7 and MEK1 in a tumor necrosis factor–dependent manner and abrogates their interactions with MEKK1 and apoptosis signal-regulating kinase 1.17 Conversely, the results of the present study conflict with previous observations that FLIP can spontaneously engage the signaling pathway, leading to the activation of ERK1/2 through recruitment of Raf-1 in T cells.27 In addition, a study from Lüschen et al28 demonstrated that overexpression of cFLIP leads to enhanced and prolonged activation of ERK after tumor necrosis factor treatment in Hela and HEK293 cells. The reason for the discrepancy in the role of cFLIP in the activation of MEK-ERK1/2 signaling remains unclear. We postulate that it may relate to differences in experimental models, cell types, or differences in the strains of mice.
The downstream molecular mechanisms involved in MEK-ERK1/-mediated hypertrophy include transcription factors such as GATA-4. Recent studies revealed that ERK1/2 phosphorylates GATA-4 and increases its DNA-binding ability.29,30 In this study, we examined the potential role of cFLIP in regulating these mechanisms. We observed GATA-4 hyperphosphorylation and increased DNA-binding activity in response to hypertrophic stimuli, which is consistent with recent reports demonstrating that the hypertrophic stimuli trigger GATA-4 activation. 29 Importantly, in our hypertrophic models, forced expression of cFLIP abolished GATA-4 phosphorylation and DNA-binding activity, whereas decreased cFLIP levels resulted in augmentation. Furthermore, the inhibitory effects of cFLIP on cardiac hypertrophy, as well as on GATA-4 phosphorylation and activation, were through inhibition of MEK-ERK1/2 signaling. These findings suggest that cFLIP blunts cardiac hypertrophy by switching off MEK-ERK1/2–dependent GATA-4 activation.
Cardiac fibrosis, which is characterized by interstitial and perivascular fibrosis, is another classic feature of pathological cardiac hypertrophy, and approaches to limit collagen deposition in the heart have been limited to date.31,32 This study for the first time revealed that cFLIP blocks cardiac fibrosis in vivo and inhibits TGF-β1–induced collagen synthesis in vitro. In an attempt to elucidate the mechanisms underlying the antifibrotic effect of cFLIP, we analyzed key components of TGF-β1-Smad signaling, which is a crucial pathway in the regulation of fibrosis.10 Our results demonstrated that cFLIP abrogates Smad 2 phosphorylation and Smad 2/3 translocation in both cultured cardiac fibroblasts and hypertrophied hearts, thus inhibiting collagen synthesis and fibrosis. Recent studies suggest that the TGF-β1/Smad pathway can be regulated by the MEK-ERK1/2 pathway.33 We further investigated the effects of MEK-ERK1/2 activation on fibrosis and found that blocking MEK-ERK1/2 activation results in significant inhibition, whereas activation of MEK-ERK1/2 resulted in upregulation of collagen synthesis, as well as Smad 2 phosphorylation and Smad 2/3 translocation. The results indicate that cFLIP blocks cardiac fibrosis by inhibiting the MEK-ERK1/2 pathway.
In conclusion, this is the first study that defines the role of cFLIP in reducing cardiac remodeling in response to hypertrophic stimuli. The molecular mechanisms for the cardioprotective effects of cFLIP, at least in part, ascribe to inhibition of the MEK-ERK1/2 signaling pathway. We propose that targeting of the cFLIP signaling pathway may develop novel promising strategies for the treatment of cardiac hypertrophy and heart failure.
In the present study, we provide evidence for the protective role of cFLIP in cardiac remodeling through the use of Ang II–induced hypertrophic model in cardiac-specific cFLIP TG mice and cFLIP heterozygous knockout mice. Therapies designed to overexpress cFLIP in the heart might be beneficial in the prevention and treatment of cardiac hypertrophy and heart failure worthy of further validation and investigation.
Sources of Funding
This study was supported in part by grants from the Heart and Stroke Foundation of Ontario, and the Canadian Institutes of Health Research, and by the National Natural Science Foundation of China (grants 30900524 and 30972954) and supported by the Support Program for Disciplinary Leaders in Wuhan (200951830561), the Fundamental Research Funds for the Central Universities (3081013), and the National Basic Research Program of China grant 2011CB503902.
See Editorial Commentary, pp 1045-1046
- Received June 3, 2010.
- Revision received June 18, 2010.
- Accepted September 29, 2010.
Imanishi T, Murry CE, Reinecke H, Hano T, Nishio I, Liles WC, Hofsta L, Kim K, O'Brien KD, Schwartz SM, Han DK. Cellular FLIP is expressed in cardiomyocytes and down-regulated in TUNEL-positive grafted cardiac tissues. Cardiovasc Res. 2000; 48: 101–110.
Steenbergen C, Afshari CA, Petranka JG, Collins J, Martin K, Bennett L, Haugen A, Bushel P, Murphy E. Alterations in apoptotic signaling in human idiopathic cardiomyopathic hearts in failure. Am J Physiol Heart Circ Physiol. 2003; 284: H268–H276.
Bian ZY, Huang H, Jiang H, Shen DF, Yan L, Zhu LH, Wang L, Cao F, Liu C, Tang QZ, Li H. LIM and cysteine-rich domains 1 regulates cardiac hypertrophy by targeting calcineurin/nuclear factor of activated T cells signaling. Hypertension. 2010; 55: 257–263.
Sadoshima J, Izumo S. Molecular characterization of angiotensin II–induced hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts: critical role of the AT1 receptor subtype. Circ Res. 1993; 73: 413–423.
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.
Molkentin JD. Calcineurin-NFAT signaling regulates the cardiac hypertrophic response in coordination with the MAPKs. Cardiovasc Res. 2004; 63: 467–475.
Yin G, Haendeler J, Yan C, Berk BC. GIT1 functions as a scaffold for MEK1-tracellular signal-regulated kinase 1 and 2 activation by angiotensin II and epidermal growth factor. Mol Cell Biol. 2004; 24: 875–885.
Satoh M, Ogita H, Takeshita K, Mukai Y, Kwiatkowski DJ, Liao JK. Requirement of Rac1 in the development of cardiac hypertrophy. Proc Natl Acad Sci U S A. 2006; 103: 7432–7437.
Badorff C, Seeger FH, Zeiher AM, Dimmeler S. Glycogen synthase kinase 3β inhibits myocardin-dependent transcription and hypertrophy induction through site-specific phosphorylation. Circ Res. 2005; 97: 645–654.
Xu J, Kimball TR, Lorenz JN, Brown DA, Bauskin AR, Klevitsky R, Hewett TE, Breit SN, Molkentin JD. GDF15/MIC-1 functions as a protective and antihypertrophic factor released from the myocardium in association with SMAD protein activation. Circ Res. 2006; 98: 342–350.
Papanicolaou KN, Izumiya Y, Walsh K. Forkhead transcription factors and cardiovascular biology. Circ Res. 2008; 102: 16–31.
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: 718–723.
Liang Q, Wiese RJ, Bueno OF, Dai YS, Markham BE, Molkentin JD. The transcription factor GATA4 is activated by extracellular signal-regulated kinase 1- and 2-mediated phosphorylation of serine 105 in cardiomyocytes. Mol Cell Biol. 2001; 21: 7460–7469.
Morimoto T, Hasegawa K, Kaburagi S, Kakita T, Wada H, Yanazume T, Sasayama S. Phosphorylation of GATA-4 is involved in α1-adrenergic agonist-responsive transcription of the endothelin-1 gene in cardiac myocytes. J Biol Chem. 2000; 275: 13721–13726.
Epstein JA, Parmacek MS. Recent advances in cardiac development with therapeutic implications for adult cardiovascular disease. Circulation. 2005; 112: 592–597.
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.
Liu X, Sun SQ, Hassid A, Ostrom RS. cAMP inhibits transforming growth factor-β -stimulated collagen synthesis via inhibition of extracellular signal-regulated kinase 1/2 and Smad signaling in cardiac fibroblasts. Mol Pharmacol. 2006; 70: 1992–2003.