PRECLINICAL STUDY
Activation of p38 Mitogen-Activated Protein Kinase Contributes to the Early Cardiodepressant Action of Tumor Necrosis Factor
Mohamed Bellahcene, PhD*,
Sebastien Jacquet, PhD*,
Xue B. Cao, MD*,
Masaya Tanno, MD, PhD*,
Robert S. Haworth, PhD*,
Joanne Layland, PhD ,
Alamgir M. Kabir, MB, BS,
Matthias Gaestel, PhD ,
Roger J. Davis, PhD ,
Richard A. Flavell, PhD||,
Ajay M. Shah, MD, FESC*,
Metin Avkiran, PhD, DSc* and
Michael S. Marber, PhD, FACC*,*
* Cardiovascular Division, King’s College London, The Rayne Institute, St. Thomas’ Hospital, London, United Kingdom
Cardiovascular Division, King’s College London, King’s College Hospital, London, United Kingdom
Institute of Biochemistry, Medical School Hannover, Hannover, Germany
Howard Hughes Medical Institute, University of Massachusetts, Worcester, Massachusetts
|| Section of Immunobiology, Howard Hughes Medical Institute and Yale University School of Medicine, New Haven, Connecticut.
Manuscript received September 2, 2005;
revised manuscript received December 21, 2005,
accepted February 7, 2006.
* Reprint requests and correspondence: Prof. Michael S. Marber, Cardiovascular Division, King’s College London, The Rayne Institute, St. Thomas’ Hospital, London SE1 7EH, United Kingdom. (Email: mike.marber{at}kcl.ac.uk).
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Abstract
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OBJECTIVES: The purpose of this study was to determine whether p38 mitogen-activated protein kinase (p38-MAPK) contributes to tumor necrosis factor-alpha (TNF )-induced contractile depression.
BACKGROUND: Tumor necrosis factor has both beneficial and detrimental consequences that may result from the activation of different downstream pathways. Tumor necrosis factor activates p38-MAPK, a stress-responsive kinase implicated in contractile depression and cardiac injury.
METHODS: In isolated hearts from mice lacking the p38-MAPK activator, MAPK kinase 3 (MKK3), perfused at constant coronary pressure or flow, we measured the left ventricular developed pressure (LVDP) and the relationship between end-diastolic volume and LVDP in the presence and absence of 10 ng/ml TNF.
RESULTS: Within 15 min at constant pressure, TNF significantly reduced LVDP and coronary flow in outbred and mkk3+/+ mice. This early negative inotropic effect was associated with a marked phosphorylation of both p38-MAPK and its indirect substrate, HSP27. In hearts lacking MKK3, TNF failed to activate p38-MAPK or to cause significant contractile dysfunction. The actions of TNF were similarly attenuated in MAPK-activated protein kinase 2 (MK2)-deficient hearts, which have a marked reduction in myocardial p38-MAPK protein content, and by the p38-MAPK catalytic site inhibitor SB203580 (1 µmol/l). Under conditions of constant coronary flow, the p38-MAPK activation and contractile depression induced by TNF, though attenuated, remained sensitive to the absence of MKK3 or the presence of SB203580. The role of p38-MAPK in TNF-induced contractile depression was confirmed in isolated murine cardiac myocytes exposed to SB203580 or lacking MKK3.
CONCLUSIONS: Tumor necrosis factor activates p38-MAPK in the intact heart and in isolated cardiac myocytes through MKK3. This activation likely contributes to the early cardiodepressant action of TNF.
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Abbreviations and Acronyms
| | LVDP = left ventricular developed pressure | | LVEDP = left ventricular end-diastolic pressure | | MK2 = mitogen-activated protein kinase-activated protein kinase 2 | | MKK3 = mitogen-activated protein kinase-kinase 3 | | p38-MAPK = p38 mitogen-activated protein kinase | | TNF = tumor necrosis factor-alpha |
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Tumor necrosis factor-alpha (TNF) is a proinflammatory cytokine elevated in conditions of depressed cardiac contractility such as cardiac allograft rejection (1), myocardial infarction (2), and congestive heart failure (3). A causative, rather than associative, role is indicated in vivo by the contractile depression and remodeling that accompany systemic increases in TNF (4–6). Similarly, exposure of isolated hearts (7) and cardiac myocytes (8) to TNF causes almost immediate contractile depression in most studies (9). On the basis of these and other observations, 3 clinical trials have targeted TNF in patients with heart failure. Surprisingly the results of these trials suggest that anti-TNF therapy has no benefit (10) or may even be harmful (11). One possible explanation for the dichotomy between the preclinical, early clinical, and multicenter clinical trial data is that TNF exposure triggers beneficial (12,13) as well as detrimental changes within the heart (14). It is likely that the mechanisms responsible for the benefits of TNF differ from those that cause harm (5,14). If this is the case, it may be possible to specifically target a downstream detrimental signal while leaving others intact. Among the myocardial signaling pathways activated by TNF is p38 mitogen-activated protein kinase (p38-MAPK) (12,15). Previously we have found that although this pathway is activated by TNF it does not mediate the protection against myocardial infarction that follows exposure (12,15). Under other circumstances, not involving TNF, the activation of p38-MAPK can cause contractile dysfunction (16,17). We therefore hypothesized that p38-MAPK activation may play a role in TNF-induced contractile dysfunction.
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Methods
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Treatment of animals conformed to the rules of the United Kingdom Home Office Guidance on the Operation of Animals (Scientific Procedures) Act 1986, published by Her Majesty’s Stationary Office, London.
MKK3- and MK2-deficient mice.
Mitogen-activated protein kinase kinase 3 (MKK3) and mitogen-activated protein kinase–activated protein kinase 2 (MK2) are kinases that lie immediately upstream and downstream of p38-MAPK, respectively. Targeting strategies that disrupt MKK3 and MK2 are described previously (18,19). Colonies of mkk3+/– and mk2+/– mice were derived from these sources by crossing mkk3–/– and mk2–/– mice with outbred c57BL/6 mice (Harlan, Sussex, United Kingdom). To reduce background genetic variability (20), we used male mkk3–/– and mkk3+/+, as well as male mk2–/– and mk2+/+ progeny of heterozygote matings, which whenever possible were litter mates. In addition, outbred male c57BL/6 mice were used to ensure that findings were not the result of within-colony idiosyncrasies (20). All gene-targeted lines were bred and maintained in the same facility under identical conditions.
Murine heart perfusion.
Male mice (25 to 30 g) were killed with pentobarbital (300 mg/kg with 150 IU heparin, IP). After midline sternotomy, hearts were rapidly excised and perfused as previously described (12,15,21) at a constant pressure of 80 mm Hg with a modified Krebs-Henseleit buffer 1 containing (in mmol/l) NaCl 118.5, NaHCO3 25.0, KCl 4.75, KH2PO4 1.18, MgSO4 1.19, D-glucose 11.0, and CaCl2 1.41 at pH 7.4, saturated with 95% O2 and 5% CO2.
Left ventricular (LV) pressure was measured by a fluid-filled balloon while hearts were paced at 580 beats/min (bpm). Additionally, some hearts were perfused under constant flow conditions. Constant flow was set at the value that generated a perfusion pressure of 80 mm Hg during the last 10 min of stabilization using a servo-controlled peristaltic pump (Gilson, Luton, United Kingdom). The average set coronary flow was 3.4 ± 0.4 ml/min.
In some experiments, the Frank-Starling (or end-diastolic volume versus left ventricular developed pressure [LVDP]) relationship was determined beyond 15 min of TNF exposure by emptying the intraventricular balloon and then slowly refilling in 4-µl increments every 30 s.
Experimental protocols.
After cannulation, hearts were stabilized for 40 min. All hearts had to fulfill the following inclusion criteria: coronary flow between 1.5 and 4.5 ml/min, heart rate >300 beats/min (unpaced), LVDP >60 mm Hg, time from thoracotomy to aortic cannulation <3 min, and no persistent dysrhythmia during stabilization. Hearts were exposed to 10 ng/ml TNF (murine TNF-; Sigma cat. no. T7539) for 15 min without recirculation. Some hearts were pretreated with SB203580 (1 µmol/l; Sigma) starting 5 min before and continuing during TNF exposure. The SB203580 was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of <0.01%. At the end of the protocols, hearts were freeze-clamped and stored at –80°C for protein analysis, or the end-diastolic volume versus LVDP relationship was determined during continued TNF exposure.
Murine cardiomyocyte isolation.
Male outbred c57BL/6, mkk3+/+, and mkk3–/– mice (25 to 30 g) were killed with pentobarbital (300 mg/kg with 150 IU heparin, IP) and the hearts rapidly excised. Ventricular myocytes were isolated as previously described (22). Finally, myocytes were stored at room temperature in a HEPES buffer at pH 7.4 containing (in mmol/l) NaCl 130, KCl 5.4, NaH2PO4 0.4, MgCl21.4, glucose 10, D-glucose 10.0, creatine 10, taurine 20, HEPES 10, ascorbic acid 0.03, EGTA 0.01, and CaCl2 1.0 and used within 4 to 5 h.
Western blot analysis.
Samples were obtained from murine cardiomyocytes treated for 30 min with increasing concentrations of TNF (0, 50, 100 ng/ml). Some cells were exposed to coincident 1 µmol/l SB203580 starting 5 min before TNF exposure. Cells were then washed 3 times in ice-cold phosphate-buffered solution and harvested in electrophoresis sample (ES) buffer (250 mmol/l Tris-HCl, 4% sodium dodecyl sulfate, 10% glycerol, and 2% ß-mercaptoethanol, pH 6.8). Frozen heart samples were similarly homogenized in ES buffer. Proteins, separated by electrophoresis and transferred to nitrocellulose membranes, were exposed to the following primary antibodies: anti-dual phospho-p38-MAPK (Thr180 and Tyr182) (Sigma) at 1:1000, anti-p38 at 1:1000, anti-HSP27 at 1:1000, anti-MKK3/6 at 1:1000, anti–phospho-MKK3/6 (Ser189/Ser207) at 1:500 (the latter 4 from Santa Cruz, Wembley, United Kingdom), and anti–phospho-HSP27 (Ser83) at 1:400 (Amersham, Little Chalfont, United Kingdom), before appropriate second antibody and ECL detection. Autophotographic images of Western blots were scanned and quantified using NIH image analysis software, and results are expressed as mean ± SEM.
Murine cardiomyocyte contraction.
Freshly isolated murine cardiomyocytes were randomly assigned to 1 of the following groups: control, TNF (10 ng/ml for 30 min); SB (1 µmol/l SB203580 for 35 min), and SB + TNF. To assess the reversibility of TNF-induced effects, myocytes were washed 3 times with Tyrode’s solution (see the following text) and allowed to recover for 45 min. All treatments were performed at room temperature and away from light before transferring cells to a superfusion chamber with a glass cover slip base resting on the stage of an inverted microscope (Nikon, Tokyo, Japan). Cells were superfused at 1 to 2 ml/min at 37°C, with the HEPES-based buffer supplemented with 10 ng/ml TNF, 1 µmol/l SB203580, or both according to the preincubation group. Myocytes were observed using a 40x Fluor objective (Nikon), and the image was relayed via a video camera and frame grabber to a PC running IonWizard software (IonOptix, Milton, Massachusetts). Myocytes were selected according to previous criteria (8) and sarcomere length recorded during field stimulation at 0.5 Hz (MyoPacer; IonOptix) by video analysis (IonOptix).
Statistical analysis.
Baseline characteristics, as well as sarcomere shortening data, were compared using 1-way analysis of variance (ANOVA) followed by Newman-Keuls post hoc analysis. Hemodynamic variables were compared by 2-way repeated measures ANOVA with time and group as factors. Results are shown as mean ± SEM, except where otherwise indicated, and p < 0.05 was considered significant.
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Results
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The baseline characteristics of the mice and hearts are shown in Table 1. There was no difference between outbred c57BL/6, mkk3+/+, mkk3–/–, mk2+/+, and mk2–/– mice.
TNF in mkk3–/– hearts.
Under constant pressure perfusion, TNF (10 ng/ml) induced a rapid decrease of LVDP in outbred c57BL/6 and mkk3+/+ hearts that reached  50% after 15 min (Fig. 1A). However, a similar decrement was not seen in mkk3–/– hearts, with contractile performance remaining similar to time-matched outbred mkk3+/+ and mkk3–/– control mice. The TNF-induced contractile depression was further confirmed by the end-diastolic volume versus LVDP relationship (Fig. 1B). Tumor necrosis factor (10 ng/ml) significantly depressed contraction, with severely blunted contractile recruitment at balloon volumes greater than 12 to 16 µl, corresponding to end-diastolic pressure of 20 to 30 mm Hg, in both c57BL/6 outbred and mkk3+/+ but not mkk3–/– hearts (Fig. 1B). To further explore the role of p38-MAPK, we measured its dual phosphorylation, as well as HSP27 phosphorylation, after 15 min exposure to TNF (10 ng/ml) (Fig. 1D). The TNF-induced negative inotropy in mkk3+/+ hearts was associated with a significant phophorylation of both p38-MAPK and HSP27 (24-fold and 8-fold, respectively) under constant perfusion pressure conditions. However, in mkk3–/– hearts, resistant to the negative inotropic effect of TNF, there was no detectable p38-MAPK activation. Interestingly, our coronary flow data (Fig. 1C) highlighted the acute vasoconstrictive action of TNF reported elsewhere: (9,23) TNF induced a 40% reduction in coronary flow in all treated hearts, which, although present, tended to be less marked in mkk3–/– hearts. We have recently shown (21) that reductions in coronary flow of a similar magnitude cause myocardial p38-MAPK activation after 60 or 120 min. Although we did not measure p38-MAPK activation after just 15 min of flow reduction, the possibility remains that, under constant pressure conditions, the observed p38-MAPK activation is the result of TNF-induced coronary vasoconstriction rather than a direct action on the myocardium. To explore this possibility, we measured p38-MAPK activation and contraction during TNF exposure with constant coronary flow (Fig. 2). The cardiodepressant action of TNF was less marked than under constant pressure (compare Fig. 2A with Fig. 1A) but remained significant and was especially evident when contractile reserve was examined by incremental increases in LV balloon volume (Fig. 2B). Furthermore, in keeping with findings in Figure 1, TNF failed to reduce contractility in mkk3–/–hearts (Figs. 2A and 2B). Under constant flow conditions, TNF-induced vasoconstriction manifested as a rise in coronary perfusion pressure (Fig. 2C), which may have partially offset the cardiodepressant action of TNF though the physiologically antagonistic action of the Gregg effect (24). Under constant flow, p38-MAPK activation was less pronounced than under constant pressure (compare Fig. 2D with Fig. 1D). Taken together, these results suggest that TNF-induced contractile dysfunction may be partially dependent on coronary flow. However TNF also has a direct cardiodepressant action independent of changes in coronary flow. Importantly, these direct and indirect effects act synergistically and are associated with p38-MAPK activation. This association was further explored using an independent mouse line.
TNF in mk2–/– hearts.
The disruption of the mk2 allele is associated with a marked reduction in myocardial total p38-MAPK (25). Therefore, to further confirm the role of p38-MAPK in TNF-induced negative inotropy we used mk2–/– and mk2+/+ mouse hearts perfused with a constant pressure. As shown in Figure 3A, TNF reduced contractility by 30% in mk2+/+ but only by 15% in mk2–/–. Accordingly, mk2–/– hearts contained less than half the p38-MAPK of mk2+/+ hearts (Fig. 3C) (25). The reductions in coronary flow (Fig. 3B) were similar to those seen in outbred c57BL/6 and mkk3+/+ hearts (Fig. 1B). Similar levels of MKK3 and MKK6 protein were found in mk2–/– and mk2+/+ hearts (Fig. 3C). Nonetheless, it seems that MKK3 is preferentially activated over MKK6 by TNF (Fig. 3C), a finding in keeping with Figure 1D and previous reports in other cell types (26). Consequently, TNF induces p38-MAPK dual phosphorylation in both mk2–/– and mk2+/+ hearts, but the reduced levels of p38-MAPK in the knockout hearts result in less dual phosphorylation. Once again, a mouse line with defective p38-MAPK activation in response to TNF is relatively resistant to TNF-induced cardiac depression.
Effects of SB203580.
To complement the experiments in targeted mouse lines, we investigated the effect of the p38-MAPK catalytic site inhibitor SB203580 (1 µmol/l), on TNF-induced cardiac dysfunction in outbred c57BL/6 mice. With constant pressure perfusion, SB203580 attenuated TNF-induced dysfunction (Fig. 4A). Neither SB203580 alone nor DMSO (<0.01%) had any effect on contractility (Figs. 4A and 4C). However SB203580, but not DMSO, attenuated the contractile depression caused by TNF. Surprisingly, SB203580, in contrast to the absence of MKK3, also reversed the vasoconstrictive action of TNF (Fig. 4B), consistent with the observation that SB203580 relaxes smooth muscle cell contraction through inhibition of p38-MAPK (27). By inference, it is likely that MKK3 is not responsible for p38-MAPK activation in this case. Under constant flow conditions, pretreatment with SB203580 also abolished the TNF effect on contractility (Fig. 4C) and inhibited the rise in perfusion pressure (Fig. 4D). As shown in Figure 4E, SB203580 blunted p38-MAPK dual phosphorylation as well as HSP27 phosphorylation. This reduction in dual phosphorylation may reflect the component arising from coronary vasoconstriction (Figs. 1D and 2D), in keeping with our previous findings (15,21). Taken together these results suggest that SB203580 reduces both direct and indirect effects of TNF on contractility, and that this seems to occur through inhibition of TNF-induced p38-MAPK activity.
Characterization of TNF effects in murine cardiomyocytes.
To clarify the effect of TNF without the confounding effect on coronary flow/pressure, we measured murine cardiomyocyte shortening in response to TNF. In cardiomyocytes from adult c57BL/6 mice we noted a substantial basal p38-MAPK dual phosphorylation (Fig. 5A). In these cells, 10 ng/ml TNF increased p38-MAPK dual phosphorylation 3-fold, and higher concentrations of TNF did not produce further p38-MAPK activation. As shown in Figure 5B, the p38-MAPK activation status in whole heart displays a greater dynamic range than in cardiomyocytes, owing to a combination of lower basal and greater TNF-stimulated activation.
TNF effects on mkk3–/– cardiomyocytes.
Cardiomyocytes from adult outbred c57BL/6, mkk3+/+, and mkk3–/– mice were examined. The effects of TNF (10 ng/ml) on contractility were assessed by measuring sarcomere shortening in the presence and absence of SB203580 (1 µmol/l). Figures 6A and 6B both show that TNF induced a 50% reduction in contraction amplitude in outbred C57BL/6 and mkk3+/+ myocytes. This effect was at least partially attenuated in mkk3–/– myocytes. Tumor necrosis factor caused a marked decrease of the maximal rates of sarcomere shortening and relengthening (Fig. 6B). In addition, these effects were reversible on TNF wash-out. Surprisingly, SB203580 completely inhibited TNF-induced contractile depression in outbred c57BL/6, mkk3+/+, as well as mkk3–/– myocytes. Figure 6C shows that TNF (10 ng/ml) exposure induced p38-MAPK activation in mkk3+/+ but not in mkk3–/– myocytes. Pretreatment with SB203580 inhibited TNF-induced p38-MAPK activity in mkk3+/+ cells, based on HSP27 phosphorylation, without any effect on mkk3–/– cells. Taken together, these results confirmed a direct negative inotropic effect of TNF mediated, at least in part, by p38-MAPK. Pretreatment of cardiomyocytes with SB203580 totally reverses TNF-induced negative inotropy.
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Figure 6 Effects of tumor necrosis factor-alpha (TNF) (10 ng/ml) and SB203580 (SB, 1 µmol/l) on mkk3–/– cardiomyocytes. Freshly isolated cardiomyocytes from outbred C57BL/6, mkk3+/+, and mkk3–/– mice underwent the following exposures: TNF (10 ng/ml; n = 24, 29, and 32, respectively) for 30 min; SB (1 µmol/L; n = 25, 29, and 34, respectively); and SB for 35 min starting 5 min before TNF exposure (n = 20, 28, and 32, respectively). After TNF and SB treatments, some cells were allowed to recover for 45 min in Tyrode’s buffer to assess the reversibility of the contractile deficit (n = 16, 13, and 24, respectively). Values are mean ± SEM. (A) Experimental traces showing sarcomere contraction in outbred C57BL/6, mkk3+/+, and mkk3–/– mouse cardiomyocytes preincubated for 30 min with TNF (10 ng/ml). (B) Normalized sarcomere contraction amplitude, maximal rate of sarcomere contraction, and maximal rate of sarcomere relengthening. Continued on next page.(C) Characterization of SB effect on TNF-induced p38-MAPK activation in murine cardiomyocytes. Upper part shows representative Western blots of total and phosphorylated p38-MAPK and HSP27 in mkk3+/+ and mkk3–/– murine cardiomyocytes exposed to TNF (10 ng/ml) for 15 min, SB (1 µmol/l) for 35 min, and SB for 35 min given 5 min before TNF treatment. Lower part shows quantitative data expressed as fold increase of phosphorylation relative to control (mean ± SEM; n = 3). Importantly, TNF treatment induced p38-MAPK activation in mkk3+/+ but not in mkk3–/– myocytes.
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Discussion
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In the present study, we show that the early contractile dysfunction induced by TNF is dependent on myocardial p38-MAPK activation. This conclusion, based on 2 different targeted mouse lines and a pharmacologic inhibitor, is consistent with other recent studies implicating myocardial p38-MAPK activation in contractile dysfunction (16,17,28). Furthermore, at least 1 of the early benefits of TNF is independent of p38-MAPK activation (12). Taken together, these findings suggest that inhibition of p38-MAPK activation downstream of TNF may provide greater specificity than interventions that target TNF.
TNF and p38-induced cardiac dysfunction.
The activation of p38-MAPK by genetic or other means has been shown to cause contractile dysfunction (16,17). Although the exact underlying mechanism is not known, it does not seem to involve alterations in the calcium transient (16,17), a direct phosphorylation of troponin I, or a fall in pH (16) but rather a decrease in myofilament calcium sensitivity, and/or force production, by another mechanism (16,17). The mechanism by which TNF causes early contractile depression in vitro, despite being more thoroughly studied, remains similarly uncertain, with evidence supporting both calcium and myofilament-dependent and -independent effects (9). However, greater consensus exists regarding the proximal signaling pathway responsible for TNF-induced contractile depression, which likely includes TNF receptor 1-dependent activation of sphingomyelinases to form ceramide (9). This pathway also mediates the cardioprotection that follows TNF exposure (29) and leads to p38-MAPK activation in multiple cell types. Our data suggest that the p38-MAPK activation downstream of this pathway results in contractile depression, whereas other data suggest it does not lead to cardioprotection (12). Therefore, inhibition of p38-MAPK is likely to achieve greater physiologic specificity than inhibition of sphingomyelinases. Furthermore, there is accumulating evidence that inhibiting p38-MAPK is a valid strategy.
Previous studies have examined the immediate effect of pharmacologic inhibition of p38-MAPK on basal contraction or contractile dysfunction induced by arsenite (16,17). These findings, together with our own, which employed complementary nonpharmacologic approaches in the whole heart, suggest that p38-MAPK is a valid and realistic target in heart failure and that inhibition could improve short-term contractility and perhaps disease progression (30).
Findings in models of longer-term contractile dysfunction have also generally shown improvements with p38-MAPK inhibition. However interpreting results of these studies is complex. p38-MAPK inhibition occurred early in the disease process and caused beneficial remodeling (31,32), making it is difficult to dissect the contribution of improved contraction from that caused by more favorable left ventricular morphology. Furthermore, the elegant recent study by Li et al. (32) demonstrates that p38-MAPK is critical to an autocrine loop because it lies both upstream and downstream of TNF released from cardiac myocytes. This finding further complicates interpretation of underlying mechanisms but reinforces potential clinical utility.
The mechanisms of p38-MAPK activation in response to TNF.
In this and our previous study (15), the activation of p38-MAPK seen in response to TNF is MKK3 dependent. This observation is in keeping with findings in other cell types (26). The mechanism by which TNF activates MKK3 is not known for certain but may involve oxidation-sensitive modulation of the upstream kinase apoptosis signal-regulating kinase 1 (ASK1) by thioredoxin (33).
Because TNF has been shown to induce coronary constriction (24), we had to verify whether p38-MAPK–mediated effects of TNF on contractility were due to a direct effect of TNF on cardiomyocytes or whether instead TNF exerts this effect indirectly through flow-contraction matching. Using constant pressure versus constant flow models of heart perfusion, we showed that direct and indirect effects of TNF on cardiomyocytes act in concert. In addition, it is likely that TNF-induced coronary vasoconstriction also contributes to p38-MAPK activation, because dual phosphorylation is greater under constant coronary pressure than in constant coronary flow conditions (compare Figs. 1 and 2). Furthermore, the reductions in coronary flow seen with TNF may have been sufficient to synergistically dual phosphorylate/activate p38-MAPK through an independent mechanism that may involve autophosphorylation and therefore result in partial sensitivity to inhibition of p38-MAPK catalytic activity with SB203580 (15). Alternatively, the reduction in p38-MAPK dual phosphorylation may simply result from the lack of coronary vasoconstriction and consequent normalization of flow in the presence of TNF with SB203580. The ability of SB203580 to prevent coronary vasconstriction may be a reflection of TNF-induced p38-MAPK activation within vascular smooth muscle which is known to cause contraction (27). However, this agent also inhibits a number of other kinases and proteins (34), which may explain why it improved contractility in mkk3–/– cardiomyocytes in the absence of detectable TNF-induced p38-MAPK dual phosphorylation (Figs. 6B and 6C). If the abolition of coronary vasoconstriction is the result of p38-MAPK inhibition within the vasculature, this implies that the mechanisms of activation differ from those within the myocardium, because they are not abrogated by the absence of MKK3 or MK2.
In this study, we show that p38-MAPK activation is at least in part responsible for the early cardiodepressant action of TNF. Although TNF is implicated in the pathogenesis of heart failure, clinical trials targeting this cytokine have not shown a benefit (10,11), possibly because TNF is involved in adaptive signaling (12,13). Our results, together with other findings (12,32), imply that p38-MAPK may represent a more favorable, but related, target that is already under investigation in a number of other disease areas (35).
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Footnotes
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Supported by grants from the Wellcome Trust (0645447 and 074653) and British Heart Foundation (02/105/14432). The authors thank Semjidmaa Dashnyam, who was funded through a Medical Research Council Co-operative Group Core Grant (G0001112), for her help in myocyte isolation.
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