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Abstract
Mitogen-activated protein kinase kinase (MKK)4 is a critical member of the mitogen-activated protein kinase family. It is able to activate the c-Jun NH2-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase in response to environmental stresses. JNK and p38 are strongly implicated in pathological cardiac hypertrophy and heart failure; however, the regulatory mechanism whereby the upstream kinase MKK4 activates these signaling cascades in the heart is unknown. To elucidate the biological function of MKK4, we generated mice with a cardiac myocyte-specific deletion of mkk4 (MKK4cko mice). In response to pressure overload or chronic β-adrenergic stimulation, upregulated NFAT (nuclear factor of activated T-cell) transcriptional activity associated with exacerbated cardiac hypertrophy and the appearance of apoptotic cardiomyocytes were observed in MKK4cko mice. However, when subjected to swimming exercise, MKK4cko mice displayed a similar level of physiological cardiac hypertrophy compared to controls (MKK4f/f). In addition, we also discovered that MKK4 expression was significantly reduced in heart failure patients. In conclusion, this study demonstrates for the first time that MKK4 is a key mediator which prevents the transition from an adaptive response to maladaptive cardiac hypertrophy likely involving the regulation of the NFAT signaling pathway.
Pathological cardiac hypertrophy presents as a complex of cardiac remodeling, characterized by fetal gene reactivation, interstitial fibrosis, myocyte apoptosis, and contractile dysfunction. It occurs in patients with hypertension, ischemic infarction, or valvular heart diseases. Although pathological hypertrophy is initially likely to be an adaptive response, sustained stress inevitably leads to heart failure. In contrast to pathological hypertrophy, cardiac growth during normal postnatal development or the hypertrophy induced by exercise and by pregnancy is referred to as “physiological” and is characterized by more forceful ejection of blood.1,2 Although these 2 forms of hypertrophy have been well described, the regulation mechanisms determining the molecular differences between physiological and pathological hypertrophy, and the signaling pathways involved in the induction of pathological hypertrophy, are largely unknown and, in many cases, remain controversial.
c-Jun NH2-terminal protein kinase (JNK) and p38 belong to the stress-activated mitogen-activated protein kinase (MAPK) family.3,4 Because of their involvement in regulating cardiac hypertrophy and heart failure,5–7 the role of JNK and p38 MAPK in the heart has been the focus of much research. The analyses of transgenic and conventional gene-targeting knockout mouse models revealed that JNK, as well as p38, buffer the magnitude of pathological cardiac hypertrophy through inhibition of calcineurin-NFAT (nuclear factor of activated T-cell) signaling.8,9 However, a recent study of cardiac-specific p38α knockout mice demonstrated that p38 was required for cardiomyocyte survival but not for pathological cardiac hypertrophy.10 Additionally, p38 was also shown to regulate physiological hypertrophy by suppressing Ser/Thr protein kinase (PK)B (also known as AKT) activity.11 These observations and discrepancies reflect the complexity of the MAPK cascades and highlight the need for further study of the signaling regulation of JNK and p38 in the heart.
JNKs are phosphorylated and activated by MKK4 and MKK7 on tyrosine 185 and threonine 183, respectively.12,13 Differing from MKK7, MKK4 was reported to activate p38 in some cell types.14,15 The targeted deletion of the mkk4 or mkk7 gene leads to embryonic lethality, providing genetic evidence that MKK4 and MKK7 have nonredundant roles in vivo.12 This concept is underscored by the observation in mice with brain-specific ablation of mkk4 that decreased JNK activity causes a defect in neuronal migration and premature death.15
MKK4 is proposed to be involved in cardiac hypertrophy, however, previous studies provided conflicting results, and the role of MKK4 in the heart is still far from conclusive. For example, overexpression of dominant-negative MKK4 in either cultured cardiomyocytes or the adult rat heart attenuated stress-induced cardiac hypertrophy16,17; conversely, diminished JNK activation with enhanced hypertrophy was detected in mekk1−/− mice (MEKK1 is the upstream kinase of MKK4) subjected to pressure overload.18 Together, these controversial data prompted this study, in which we proposed to determine whether MKK4 is a nodal kinase activating JNK and p38 in the heart, and whether MKK4 regulates physiological or pathological cardiac hypertrophy. Analysis of cardiac-specific MKK4 knockout mice subjected to pressure overload or chronic β-adrenergic stimulation revealed that reduced JNK activity was associated with exacerbated cardiac hypertrophy and a significant increase in the NFAT transcription activity. Moreover, apoptotic cardiomyocytes were evident in hypertrophic MKK4cko mice. Following swimming exercise, the mutant mice displayed a similar level of cardiac hypertrophy as the control group. These data suggest that the MKK4/JNK signaling pathway is specifically required for pathological cardiac hypertrophy/remodeling, but not for physiological cardiac hypertrophy.
Materials and Methods
An expanded Materials and Methods section describing generation of MKK4 mutant mice, osmotic minipump infusion, in vivo physiology analysis, histological analysis, immunoblot analysis, quantitative real-time PCR, small interfering (si)RNA transfection, and luciferase reporter assay is available in the online data supplement at http://circres.ahajournals.org.
Transverse Aortic Constriction
Eight-week-old male MKK4f/f and MKK4cko mice were subjected to transverse aortic constriction (TAC) or a sham operation under isoflurane anesthesia, 0.1 mg/kg buprenorphine, and artificial ventilation as previously described.19 Hearts were analyzed for cardiac hypertrophy at different time points following TAC.
Swimming Exercise
Eight-week-old male MKK4f/f and MKK4cko mice were subjected to a swimming program. Mice swam for a period of up to 90 minutes twice a day in tanks containing water at 30°C to avoid thermal stress. Constant monitoring was given to ensure the safety of the mice. After the 28-day swimming program, the hearts were analyzed to evaluate hypertrophic growth.
Human Tissue Samples
Myocardial tissue samples from heart failure patients and normal subjects were obtained from Asterand (Herts, United Kingdom). Informed consent for the use of human tissue samples was obtained by Asterand and approved by the United Kingdom Human Tissue Authority.
Data Analysis
Data are expressed as means±SEM and were analyzed using the paired Student’s t test or 2-way ANOVA followed by post hoc multiple comparison test where appropriate, with a value of P<0.05 considered to be statistically significant.
Results
Characterization of MKK4cko Mice
To study the functional importance of MKK4 in the heart, we generated mutant mice with a cardiac-specific deletion of mkk4 (MKK4cko) using the Cre-LoxP system. MKK4cko mice were born at the expected Mendelian ratio; they developed to term and appeared normal. Immunoblot analysis of ventricular extracts from MKK4cko mice at 8 weeks of age showed an almost complete ablation of MKK4 protein (Figure 1A). This result was confirmed by immunostaining of isolated single cardiomyocytes (Figure 1B). It was evident that excision of MKK4 was specific to the heart because protein levels of MKK4 were equivalent in brain, liver, and skeletal muscle from both MKK4cko and MKK4f/f mice (Figure 1A). The absence of MKK4 in the heart did not cause any compensatory changes in the protein levels of MKK7, JNK, and p38 (Figure 1A). To determine whether MKK4 is able to activate JNK and p38 in vivo, ventricular tissues from 2 groups were collected 30, 60, and 90 minutes after isoproterenol treatment (10 mg/kg). Immunoblot analysis showed a rapid increase in JNK activity in controls 30 minutes after the treatment, whereas we barely detected any activation in the mutant hearts (Figure 1C). Consistent with this, MKK4 activity in controls was also increased 30 minutes after isoproterenol treatment (Figure I in the online data supplement). In contrast, the activation of p38 in both groups escalated to a similar level at 30 minutes after the treatment, sustained to 60 minutes, and then gradually declined (Figure 1C). This is confirmed by results obtained from in vitro kinase assays (supplemental Figure II). JNK reached only 25% of the level of activation seen in the controls following isoproterenol treatment, whereas p38 activity in both groups was comparable. These results suggest that MKK4 is the specific activator for JNK in the heart. The disruption of MKK4 in cardiomyocytes does not affect p38 activity.
Figure 1. The absence of MKK4 in cardiomyocytes severely reduces JNK activity. A, Western blot analysis to determine specificity of the deletion of MKK4 in the left ventricle (LV) compared to protein extracts from various tissues. Expression levels of MKK4, MKK7, JNK, and p38 MAPK in cardiomyocytes were determined; tubulin expression served as the protein loading control. B, Immunostaining of isolated single cardiomyocytes to verify efficiency of the excision of MKK4. Immune signals were captured with secondary antibodies conjugated to Alexa Fluoro 568 (red for MKK4), or to Alexa Fluoro 488 (green for α-actinin) (n=3). Scale bar=10 μm. C, Isoproterenol-induced activation of JNK or p38 was examined by immunoblot analysis at various time points, demonstrating that the loss of MKK4 specifically attenuates JNK activation in the heart. The ratios of p-JNK to total JNK expression, and p-p38 to total p38 expression are shown in bar graphs (n=3 for each group). #P<0.001, *no difference vs corresponding untreated group, respectively. Data are presented as means±SEM.
Pressure Overload Caused Exacerbated Cardiac Hypertrophic Remodeling in MKK4cko Mice
At baseline, there was no difference in the ratio of heart weight to tibia length (HW/TL) in MKK4cko mice compared to controls, and cardiac function, as measured by echocardiography, was not altered (supplemental Table I). To determine whether MKK4 is essential for pressure overload-induced cardiac hypertrophy, MKK4f/f and MKK4cko mice were subjected to TAC for 1 or 5 weeks.
After 1 week of TAC, MKK4cko mice showed an increased response to the hypertrophic stimulus demonstrated by enhanced HW/TL (58.3% [MKK4cko] versus 18.3% [MKK4f/f]) (Figure 2B) and greater cross-sectional area of cardiomyocytes (346.5±3.26 μm2 [MKK4cko] versus 252.4±6.88 μm2 [MKK4f/f]) (Figure 2A and 2C). Echocardiography showed a decline in contractility in the mutant hearts (supplemental Figure III); however, no apparent interstitial fibrosis and apoptosis was associated with the hypertrophy at this time point (data not shown). The transcripts of hypertrophic gene markers atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and regulator of calcineurin 1.4 (RCAN1.4) (a marker for calcineurin/NFAT activity) were significantly upregulated in MKK4cko-TAC mice compared to the controls (Figure 2D).
Figure 2. Enhanced hypertrophy in MKK4cko mice in response to TAC for 1 week. A (top), MKK4cko mice develop increased cardiac hypertrophy after 1 week of TAC (scale bar=2 mm). A (bottom), Hematoxylin/eosin staining of heart cross-sections (scale bar=20 μm). B, HW/TL ratios of MKK4f/f and MKK4cko mice were calculated following 1 week of TAC or sham operation (n=5). C, The measurement of mean cross-sectional area demonstrates significantly enlarged MKK4cko cardiomyocytes (n=5). D, Quantitative real-time PCR analyses of hypertrophic gene markers, ANP, BNP, and RCAN1.4. The data are derived from 3 independent experiments performed in triplicate and are normalized to the GAPDH content (n=3). Data are presented as means±SEM.
When subjected to prolonged TAC treatment (5 weeks), an adaptive hypertrophic response was seen in MKK4f/f mice characterized by preserved cardiac function (dP/dtmax: 5125±480 mm Hg/sec; dP/dtmin: −5642±552 mm Hg/sec; fractional shortening: 35.19±1.07%) (Figure 3E and 3F); however, the extent of hypertrophic remodeling in MKK4cko hearts was much more pronounced, which was evident by a conspicuous increase in HW/TL (103.92% [MKK4cko] versus 23.43% [MKK4f/f]) (Figure 3B), with substantially enlarged cross-sectional area of cardiomyocytes (446.7±8.48 μm2 [MKK4cko] versus 265.45±4.74 μm2 [MKK4f/f]) (Figure 3A and 3C) and extensive ventricular fibrosis (9.5±0.4% [MKK4cko] versus 2.9±0.79% [MKK4f/f]) (Figure 3D). These structural changes were accompanied by a detrimental change in cardiac function in MKK4cko mice (dP/dtmax: 3384±254 mm Hg/sec; dP/dtmin: −2868±187 mm Hg/sec; fractional shortening: 10.05±2.03%) (Figure 3E and 3F). As expected, the transcripts of hypertrophic gene markers ANP, BNP, and Myh7 (myosin heavy polypeptide 7 cardiac muscle β); the fibrosis maker genes procollagen type I, α2 (Col1α2), and procollagen type III, α1 (Col3α1); as well as RCAN1.4 were significantly upregulated in MKK4cko-TAC mice compared to the controls (supplemental Figure IV).
Figure 3. MKK4 is essential for preventing maladaptive hypertrophic remodeling. A (top), MKK4cko mice show exaggerated cardiac hypertrophy following 5 weeks of TAC (scale bar=2 mm). A (bottom), Hematoxylin/eosin staining of heart cross-sections (scale bar=20 μm). B, HW/TL ratios of MKK4f/f and MKK4cko mice were calculated following TAC or sham operation (n=7). C, The measurement of mean cross-sectional area demonstrates significantly enlarged MKK4cko cardiomyocytes (n=8). D, Masson’s trichrome staining of cross-sections shows substantial ventricular interstitial fibrosis in MKK4cko hearts (left image; arrows indicate areas of fibrosis; scale bar=50 μm). Quantification of the relative area of fibrosis is expressed as percentage of the fibrosis area in the microscope views (n=7). In vivo hemodynamic analysis (dP/dtmax: contractile response; dP/dtmin: lusitropic response) (E) and echocardiography assessment (fractional shortening percentage) (F) demonstrate detrimental cardiac function in MKK4cko-TAC mice (n=4 to 7 per group). Data are presented as means±SEM.
After 5 weeks of TAC, we observed pronounced apoptosis in MKK4cko cardiomyocytes (the number of TUNEL-positive nuclei was 6.3 times of that in controls), along with an increased level in activated caspase 3 (Figure 4A and 4B). In addition, MKK4cko-TAC mice showed an augmented protein level of Bax (proapoptotic Bcl-2 family member), with a higher ratio of Bax/Bcl-2 (1.81 times higher compared to controls), suggesting the Bcl family members are involved in hypertrophy-induced apoptosis in MKK4cko hearts (Figure 4B).
Figure 4. MKK4 is required for cardiomyocyte survival in response to hypertrophic stress. A, Increased apoptosis in MKK4cko ventricular myocardium after 5 weeks of TAC was detected by TUNEL assay. Triple staining was performed: TUNEL staining (green), DAPI marking nuclear chromatin (blue), α-actinin marking cardiomyocytes (red). Arrows indicate TUNEL-positive nuclei. Scale bar=20 μm. The bar graph summarizes the number of apoptotic nuclei in MKKcko hearts compared to that in control hearts after 5 weeks of TAC. B, Immunoblot analysis of protein levels of active caspase 3, the proapoptotic Bcl-2 family member Bax, and the antiapoptotic protein Bcl-2. Tubulin expression is the protein loading control. The increased ratios of active caspase 3 to tubulin and Bax/Bcl-2 in the mutant hearts are represented by the bar graphs (graphs). Data are means±SEM (n=4 per group).
To further investigate the mechanisms involved in this increased hypertrophic response, we examined the activation of both pro- and antihypertrophic pathways after 1 and 5 weeks of TAC. The activity of the prohypertrophic genes extracellular signal-regulated kinase (ERK)1/2, ERK5, and PKB in the 2 genotypes was comparable. GSK3β and JunD, which are known to prevent hypertrophy, also showed similar levels of activity in the 2 groups (supplemental Figures V and VI). In parallel with these data, we also detected that after TAC, the upregulation of JNK activity seen in MKK4f/f mice was not evident in MKK4cko hearts. The activation of p38 MAPK and MKK7 was comparable in both groups (supplemental Figure VII).
The Reduction of MKK4 Enhanced the NFAT Transcriptional Activity
Led by the result that the transcript level of RCAN1.4 was upregulated in MKK4cko-TAC hearts, we further examined whether the NFAT transcriptional activity was regulated by MKK4. Using an siRNA method, we specifically suppressed MKK4 expression by 66% in neonatal rat cardiomyocytes; this reduction of MKK4 led to attenuated JNK activation (Figure 5A and 5B), and a significant increase in the NFAT activity following isoproterenol treatment (Figure 5C). This finding suggests that the MKK4/JNK pathway regulates cardiac hypertrophy most likely through NFAT activity.
Figure 5. MKK4 is involved in regulating the NFAT activity in cardiomyocytes. A, Neonatal rat cardiomyocytes (NRCMs) were transfected with MKK4 siRNA (100 nmol/L) or negative control siRNA. Cells were cultured in serum-free medium for 72 hours before preparation of protein lysates for analysis of MKK4 protein level by immunoblotting. MKK7 protein level was also determined to examine the specificity of siRNA-mediated knockdown of MKK4. The ratio of MKK4 expression to MKK7 is presented as a bar graph. B, siRNA-transfected NRCMs were treated with isoproterenol (10 μmol/L) for 30 minutes before detecting JNK phosphorylation by immunoblotting. The ratio of phosphorylation to expression of JNK is presented as a bar graph. C, siRNA-transfected NRCMs were infected with Ad-NFAT-Luc (25 multiplicities of infection) for 24 hours, followed by stimulation with isoproterenol (10 μmol/L). The NFAT-dependent transcriptional activity was measured by the luciferase reporter assay system. Data are means±SEM (n=3 per group).
Chronic β-Adrenergic Stimulation Enhances the Cardiac Hypertrophic Response in MKK4cko Mice
We next investigated the requirement of MKK4 in isoproterenol-induced hypertrophy. After 7 days of administration of isoproterenol (10 mg/kg per day), MKK4cko mice developed advanced hypertrophy reflected by a 56.31% increase in HW/TL, compared to controls (32.54%). Consistently, enlarged cross-sectional area of cardiomyocytes and ventricular fibrosis were more notable in the mutant mice (supplemental Figures VIII and IX). However, cardiac function in MKK4cko mice remained relatively normal (supplemental Figure IX). Furthermore, the transcriptional levels of ANP, BNP, Myh7, Col1α2, Col3α1, and RCAN1.4 were found to be remarkably elevated in the MKK4cko hearts, compared to controls (supplemental Figure X).
MKK4cko and MKK4f/f Mice Exhibited a Similar Level of Physiological Cardiac Growth in Response to Swimming Exercise
Next, we explored whether MKK4 was necessary for physiological cardiac hypertrophy. Subjected to a 28-day swimming program, both groups displayed a comparable level of hypertrophic growth, with no significant difference in the HW/TL ratio and the cross-sectional area of cardiomyocytes (Figure 6A and 6B). Ventricular fibrosis was not detected by histological analysis, and echocardiography presented unperturbed cardiac function in both genotypes (data not shown). Because p38 and PKB have been implicated in the regulation of physiological hypertrophy,10,20 we examined the activation of JNK, p38, and PKB following swimming. We noted a similar rise in both p38 and PKB activity 30 minutes after swimming, whereas swimming exercise did not induce JNK or MKK4 activation within this time frame (supplemental Figures XI and I, B). Together, our data suggest that the MKK4/JNK signaling pathway is not required for the development of physiological hypertrophy.
Figure 6. MKK4 is not required for swimming-induced cardiac growth. A, MKK4f/f-swim and MKK4cko-swim mice exhibit a similar level of cardiac growth demonstrated by HW/TL ratios (n=11). B, Histological analysis of heart cross-sections by hematoxylin/eosin staining (left; scale bar=20 μm). Mean cross-sectional areas of cardiomyocytes in MKK4f/f and MKK4cko mice were measured (right; n=11). Data are presented as means±SEM.
Analysis of MKK4 Expression and Activity Following Hypertrophic Stress and in Human Failing Heart
To determine the response of MKK4 to hypertrophic stresses, we analyzed the protein level and activity of MKK4 following TAC, or swimming exercise in genetically normal myocardium (C57BL/6:129Sv mice). Intriguingly, both expression and activity of MKK4 were markedly upregulated after 5 weeks of TAC, but after 12 weeks of TAC, no detectable MKK4 activation was seen, and there was a trend toward a decrease in MKK4 protein level (86.5% of MKK4 expressed in sham groups, P=0.16) (Figure 7A). Following 28 days of swimming exercise, MKK4 expression and activity were unchanged (data not shown). We also examined the expression and phosphorylation of MKK4 in heart failure patients and in normal subjects. Interestingly, a significant reduction of expression (around 60%) associated with diminished activity of MKK4 was observed in heart failure patients (Figure 7B).
Figure 7. Immunoblot analyses of expression and activity of MKK4 in ventricular tissue from mice and heart failure patients. A, After 5 or 12 weeks of TAC, MKK4 expression and phosphorylation levels were detected. B, MKK4 protein level in the human failing hearts was significantly reduced compared to the normal subjects. The tubulin expression is the protein loading control. Data are means±SEM and presented in bar graphs as ratios (n=3 per group). *No differences between 2 groups.
Discussion
Data presented here depict a clear picture of the signaling regulation underlying 2 distinct forms of cardiac hypertrophy. The major finding of this study is that MKK4 acts as a modulator to protect the heart from pathological hypertrophy likely through the JNK-regulated NFAT activity, but it is not required for the development of physiological cardiac hypertrophy. Data obtained from heart failure patients showing a remarkable decrease in MKK4 expression strengthens such a notion and, for the first time, provides evidence for the functional importance of MKK4 in the human heart.
Pathological cardiac hypertrophy is a multifaceted process affecting myocardial growth, cardiomyocyte survival, cardiac pumping capacity, and fetal gene reactivation.21,22 The calcineurin-NFAT signaling pathway is believed to be a major character mediating pathological hypertrophy.23 It is reported that JNK antagonizes myocardial growth by directly phosphorylating NFAT and inhibiting its transcriptional activity.8 In line with this paradigm, our data showed that the transcript level of RCAN1.4 was significantly increased in TAC- or isoproterenol-treated MKK4cko hearts. Because the transcription of RCAN1.4 (also known as MCIP1.4) is tightly modulated by NFAT transcription factors,24,25 our observation implicates an increase in the NFAT activity in MKK4cko hearts. In addition, no change in the transcription of RCAN1.4 was found in swimming-activated MKK4cko hearts (data not shown), which is consistent with a previous study showing that the calcineurin-NFAT signaling pathway participated only in maladaptive hypertrophy and heart failure.26 Furthermore, in response to isoproterenol stimulation the NFAT activity was found to be elevated in rat cardiomyocytes in which MKK4 was exclusively knocked down, establishing the direct evidence that MKK4 is engaged in regulating the NFAT signaling. In contrast, De Windt et al reported that calcineurin-mediated hypertrophy was attenuated by overexpression of dominant negative MKK4 adenovirus in neonatal rat cardiomyocytes (NRCMs).27 Such a discrepancy can be explained by the use of different experimental models. Neonatal cardiomyocytes, which lack mature sarcomeric organization, are not regarded as an ideal model for studying pathological hypertrophic response in adults. Moreover, overexpression of dnMKK4 in NRCMs may cause some unwanted effects, thereby masking the true phenotypes; however, in our study, NRCMs with a specific knockdown of MKK4 closely resemble MKK4cko cardiomyocytes, making this a suitable experimental model for measuring the NFAT activity by luciferase assay.
In parallel, we also investigated the activation of a number of hypertrophic regulators following TAC stress, including ERK1/2,28 ERK5,29 and PKB,30 known as promoters for hypertrophy, and GSK3β31 and JunD,32 which have inhibitory functions on hypertrophic growth. However, we did not observe any difference in their activities between the 2 genotypes after either 1 or 5 weeks of TAC. This strongly suggests that the effect of MKK4 in preventing pathological hypertrophy is likely to be mediated via specific regulation of the NFAT activity and not through crosstalk with other hypertrophic regulators.
Apart from hypertrophic changes, we also detected a much higher prevalence of apoptotic cardiomyocytes with increased Bax protein level in TAC-treated mutant hearts, which clearly indicates that MKK4 protects against cardiomyocyte loss in response to hypertrophic stress by attenuating the mitochondrion-dependent apoptotic pathway. However, previous studies provided data to dispute the role of MKK4 in cardiomyocyte apoptosis, which may, in part, be attributable to the lack of a physiologically relevant experimental model.33,34 Therefore, the availability of MKK4cko mice will facilitate future studies regarding the precise contribution of MKK4 in cardiac apoptosis in response to distinct stimuli of various durations.
MKK4cko mice displayed detrimental cardiac contractility following 5 weeks of TAC, which was possibly related to enhanced apoptosis and massive ventricular fibrosis. Of note, in 1-week TAC-treated mutant hearts, the contractility was moderately blunted but no maladaptive remodeling was seen, implying that the loss of MKK4 may directly affect cardiac contractility. However, cardiac function in MKK4cko mice at baseline was normal, and there were no changes in the expression levels of Ca2+ handling proteins in MKK4cko hearts, such as SERCA2 (sarcoplasmic reticulum Ca2+-ATPase 2), Na+/Ca2+ exchanger, and phospholamban (data not shown). It is therefore uncertain whether MKK4 primarily regulates intracellular Ca2+ transients following hypertrophic stress.
Exercise-induced cardiac hypertrophy is considered a beneficial reaction in the heart. A wealth of evidence suggests that PKB is a critical participant for physiological hypertrophy, which acts by promoting protein synthesis via its downstream effectors.1 In addition, a recent study using cardiac-specific p38α-deficient mice and apoptosis signal-regulating kinase (ASK)1 total knockout mice has provided genetic evidence that the ASK1/p38 signaling pathway negatively regulates physiological hypertrophy by inhibiting PKB activity.10 ASK1 is a mitogen-activated protein kinase kinase kinase lying upstream of MKK4,35 which leads us to hypothesize whether the MKK4/JNK pathway has a dual function in regulating not only pathological hypertrophy/remodeling, but also physiological hypertrophy. Although our results do not support this hypothesis, this study provides valuable information clarifying the signaling determinant underlying physiological hypertrophy.
In this study, we also examined the expression and activity of MKK4 in wild-type mice. Interestingly, phosphorylation and expression of MKK4 were significantly upregulated after 5 weeks of TAC, whereas no increase in MKK4 expression was after 12 weeks of TAC. This result suggests that increased MKK4 may initially act as a brake to slow down hypertrophic growth; however, when sustained stress is applied, MKK4 recedes, thereby contributing to advanced hypertrophic growth (HW/TL: 38% [12 weeks of TAC] versus 23% [5 weeks of TAC]). These data are in line with our findings in MKK4cko mice and are further underpinned by the observation that MKK4 is markedly reduced in heart failure patients.
Previous studies showed that MKK4 was able to phosphorylate p38 MAPK in both in vitro and in vivo systems.14,15 However, in this study, under β-adrenergic stimulation or pressure overload, we consistently discerned that only JNK activation was abolished in MKK4-deficient myocardium, whereas p38 activity was unimpaired. This result is in agreement with a previous finding that overexpression of dominant-negative MKK4 blocked endothelin-1–induced hypertrophy in NRCMs via the inhibition of JNK activity, without affecting activation of p38 MAPK.16 Such discrepancies reflect the complexity of the MKK4-dependent signaling regulation in different cell types or tissues. It would be of great interest to further dissect the role of MKK4 in regulating JNK and/or p38 MAPK activation in various pathophysiological settings. Based on this study, MKK4 is the major, yet specific activator for JNK in hypertrophic remodeling. This further suggests that MKK7, another JNK upstream activator, may have distinct functions differing from those of MKK4 in the heart. This notion is supported by the analysis of a mouse model overexpressing dominant-active MKK7 in ventricular myocytes.36 It was revealed that MKK7 interacted with the transforming growth factor-β signaling pathway affecting extracellular matrix composition; also, MKK7 was involved in regulating ventricular electric conduction.36,37 It is known that MKK4 and MKK7 preferentially phosphorylate the Tyr and Thr residues within the T-X-Y motif of JNK15; however, whether it is the molecular determinant underlying the different effects of MKK4 and MKK7 in the heart requires further investigation. Meanwhile, to distinguish the distinct roles of MKK4 and MKK7 in myocardium, a cardiac-specific MKK7 knockout mouse model is certainly needed.
In conclusion, this study provides convincing evidence to clearly address the in vivo role of MKK4 in the heart. MKK4 modulates the progression of pathological hypertrophy likely by regulating the NFAT transcription factor. The recognition of the functional importance of MKK4 in preventing maladaptive cardiac hypertrophy and ventricular remodeling and the fact that it does not interfere with physiological hypertrophy may shed light on the development of new therapeutic targets modulating the heart failure process.
Acknowledgments
Sources of Funding
The work was supported by the British Heart Foundation and a Research Council UK Fellowship, together with the British Pharmacological Society’s Integrative Initiative Fund (to X.W.). Support for the National Institute for Health Research Manchester Biomedical Research Centre is also acknowledged.
Disclosures
None.
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