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Cardiovascular Research 2007 74(3):466-470; doi:10.1016/j.cardiores.2007.02.027
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Copyright © 2007, European Society of Cardiology

Post-infarction remodeling is independent of mitogen-activated protein kinase kinase 3 (MKK3)

James E. Clarka, Richard A. Flavellb, Matthew E. Fairclotha, Roger J. Davisc, Richard J. Headsa and Michael S. Marbera,*

aThe Cardiovascular Division, Kings College London, The Rayne Institute, St Thomas' Hospital, London, UK
bSection of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA
cHoward Hughes Medical Institute, University of Massachusetts, Worcester, MA, USA

* Corresponding author. Tel.: +44 20 7188 1008; fax: +44 20 7188 0970. Email address: mike.marber{at}kcl.ac.uk

Received 4 January 2007; revised 15 February 2007; accepted 19 February 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results and discussion
 References
 
Objectives:: Our aim was to examine the role of mitogen-activated protein kinase kinase 3 (MKK3) in the development of left ventricular (LV) remodeling following myocardial infarction (MI).

Methods:: MKK3-null mice were subjected to permanent coronary artery ligation. Twenty-eight days after MI, haemodynamics in male mkk3+/+(WT) and mkk3–/–(KO) littermates were assessed using a pressure-conductance catheter. MI groups were compared to un-operated time-matched WT and KO controls.

Results:: MI caused significant LV contractile dysfunction and dilatation which did not differ by genotype. Detailed morphometric analysis of excised hearts confirmed these similar global indices of remodeling and also demonstrated that pathological changes within remote myocardium and scar did not differ between KO and WT hearts.

Conclusions:: Despite numerous lines of evidence suggesting MKK3 is the relevant kinase upstream of p38 mitogen-activated protein kinase in LV remodeling these processes can continue in its absence.

KEYWORDS Infarction; Remodeling; MKK3; p38-MAPK; Haemodynamics


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results and discussion
 References
 
Many investigators have found that activation (by dual phosphorylation) of p38-mitogen-activated protein kinase (p38-MAPK) leads to myocyte contractile dysfunction, apoptosis, and cell death [1–4]. These findings are reflected in vivo where cardiac-specific activation of the {alpha} isoform of p38-MAPK causes, whilst specific pharmacological inhibition prevents, myocardial dysfunction and death [5–8]. Unfortunately, pharmacological inhibition of p38-MAPK, with different small molecules, in separate early clinical trials, has led to a common toxicity profile. This has prompted investigators to search for alternative therapeutic targets in the p38-MAPK pathway.

Activation of the p38-MAPK system is predominantly through the upstream mitogen-activated protein kinase kinases (MKKs), MKK6 and MKK3 [9]. Targeted disruption in mice has demonstrated that MKK3 is predominantly responsible for the activation of the pathophysiologically relevant isoform, p38{alpha} [10,11]. This is a finding recently verified by transient co-expression in vivo [12]. Moreover, Liao et al. have shown that active MKK3, but not MKK6, expressed within the cardiomyocyte compartment leads to a thin hypocontractile ventricle, with an increased LV end-systolic volume, pathognomic of the failing heart [2,7]. Whilst we have demonstrated that the short-term pathological effects of TNF-{alpha} on myocardial contractility are by MKK3-mediated p38-MAPK activation [13].

In summary, pharmacological inhibition suggests that p38-MAPK activation aggravates heart failure and predominantly involves the {alpha}-isoform which is preferentially activated by MKK3. Furthermore, MKK3 overexpression recapitulates the phenotype of the failing heart whilst the absence of MKK3 prevents TNF{alpha}-induced cardiac dysfunction. Considering this evidence, a potentially central role for MKK3 in the development and progression of heart failure is apparent. This study is the first to investigate the role of endogenous MKK3 in post-infarction left ventricular remodelling.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results and discussion
 References
 
All animal experiments were carried out in accordance with Home Office regulations as detailed in the Home Office Guidance on the Operation of Animals (Scientific Procedures) Act 1986, HMSO (London) which mirrors those found in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The strategy used to generate the mkk3–/– mice has been described previously [10]. For this study we used male mkk3–/– (n=12) and mkk3+/+ (n=12) mice that were always the offspring of heterozygote matings (mkk3+/–xmkk3+/–). In addition, when ever possible, we ensured that litters contributed equally to each genotype. Mice were anaesthetised by isofluorane inhalation, and ventilated by tracheal intubation. A left thoracotomy was performed and the left anterior descending (LAD) coronary artery was tied by the edge of the left atrium. Twenty-eight days after LAD ligation, mice were re-anaesthetised and intubated as described above. The right internal carotid artery (ICA) was exposed, and a miniature pressure–volume catheter (SPR-839, Millar Instruments) was inserted into the LV via the aortic valve. Pressure–volume loops were acquired as previously described [14,15]. To ascertain the pressure–volume relationship under different preload conditions, the inferior vena cava (IVC) was transiently occluded. Hearts were then excised and placed in 10% paraformaldehyde with a LV chamber pressure of 10 mm Hg, mounted in 5% agarose and sectioned (700 µm) from apex to base prior to computer-aided image analysis (Sigma Scan v5.0, SPSS).

In a parallel study, hearts were excised from animals just before, and 1 h, 1 day and 1 week following permanent occlusion of the LAD and rapidly frozen. Activation of MKK3/6, p38-MAPK and HSP27 (as a readout of p38 activity) was measured by immunoblotting using phospho-specific antibodies for p38-MAPK, MKK3/6 and HSP27 as previously described [16].

Results are expressed as mean±SD. All data sets were analysed by one-way analysis of variance followed by Tukey Multiple Comparison. A value of P<0.05 was considered statistically significant.


   3. Results and discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results and discussion
References
 
Four weeks following coronary artery ligation, there were no observed differences in the heart rate (HR), stroke volume (SV) and, consequently, cardiac output (CO) between MIs and controls (Table 1). A significant increase in end-systolic LV volume (ESV) (representative sections from hearts of each genotype and intervention are shown in Fig. 1A) significantly depressed the ejection fraction (EF) in infarcted hearts. Preload recruitable stroke work (PRSW) was markedly decreased and accompanied by a decline in the end systolic pressure–volume relationship (ESPVR), indicating a detrimental change in load-independent systolic function. In addition to these measures of systolic dysfunction, relaxation was abnormally prolonged as indicated by an increase in the time constant of isovolumic relaxation, {tau}. Ex vivo analyses verified these changes since heart weight, LV cavity size and LV muscle volume, were all significantly increased in infarcted hearts. Despite clear pathological remodeling, indices did not differ significantly between mkk3–/– and mkk3+/+ genotypes.


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  		Table 1 Morphometric and haemodynamic analysis of mkk3–/– and mkk3+/+ mice 28 days after LAD ligation or in time-matched controls

 

Figure 1
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  		Fig. 1 (A) Representative slices of hearts (n=6) from mice at baseline and in age- and weight-matched mkk3–/– and mkk3+/+ animals 28 days after LAD ligation (MI). (B) Representative immunoblots of hearts from age- and weight-matched mkk3–/– and mkk3+/+ animals at baseline, 1 h, 1 day, and 1 week after LAD ligation. The total MKK6/3 antibody used detects two specific proteins, the upper (heavier) band is MKK6 and the lower band is MKK3.

 
In view of the haemodynamic and morphometric data, we determined p38-MAPK activity by measuring its dual phosphorylation and the phosphorylation of the indirect downstream substrate HSP27 (Figs. 1B and 2Go). There is robust activation of p38-MAPK 1 h after ligation whereas at 1 day and 1 week the activity diminished. This effect was apparent in mice of both mkk3–/– and mkk3+/+ genotype despite there being no obvious phospho-MKK3/6 signal in the KO hearts. Serum TNF-{alpha} levels were elevated in both mkk3–/– and 1 week in mkk3+/+ mice post infarction (data not shown). In previous in vitro studies, we demonstrated that p38-MAPK activation by TNF-{alpha} is predominantly through MKK3 and that there is no short-term compensation for the absence of MKK3 in null hearts [13,17].


Figure 2
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  		Fig. 2 Quantification of immunoblots (n=3) for activated, phosphorylated, p38 (A), HSP27 (B) and MKK3 (C) in hearts from age- and weight-matched mkk3+/+ (open bars) and mkk3–/– (filled bars) animals at baseline, 1 h, 1 day, and 1 week after LAD ligation. MKK3 was undetectable (N/D) in hearts from mkk3–/– mice. Data are shown as a ratio of phosphorylated:total protein as mean±SEM of 3 independent experiments. *P<0.05 vs. control, {dagger} P<0.05 vs. 1 h of same genotype, **P<0.01 vs. control.

 
In this study we have, however, clearly shown that MKK3-mediated p38-MAPK activation is not necessary for pathological remodeling in the mouse heart following MI. Moreover there is no discernable activation of MKK6 in this model. A possible explanation for these observations is that p38-MAPK is not activated through the upstream MKKs in this circumstance. TGF-β-activated protein kinase-1 binding protein 1 (TAB1) is known to interact with p38-MAPK and induce its autophosphorylation [18], resulting in MKK-independent activation. In support of this, we and others have suggested that ischemic activation of p38-MAPK is through a TAB1-mediated pathway [17,19,20].

Despite the marked LV cavity dilation, the contractile function, as measured by dP/dtmax, stroke volume and end systolic pressure did not differ between the MI and control groups. Furthermore, following MI, the mice gained weight normally and seemed active without obvious tachypnoea or peripheral oedema. Thus our model is probably not one of an overt heart failure syndrome but rather of severe LV dysfunction. Furthermore from a physiological viewpoint heart failure was present since normalisation of load-dependent values of contractility was achieved at the expense of a significant increase in LVEDP. Despite the absence of clinical features of heart failure the changes we observed are of direct clinical relevance since LV end-systolic dimension is the most powerful predictor of mortality after infarction and the vast majority of patients requiring mortality-reducing treatment after MI do not have stage D heart failure [21,22].

The clinical development of pharmacological inhibitors of p38-MAPK has been hampered by toxicity resulting in a search for more specific targets within the same pathway. Based on our findings, despite numerous lines of indirect evidence, it seems unlikely that MKK3 constitutes such a target after myocardial infarction.


   Acknowledgement
 
This work was supported by the British Heart Foundation and Wellcome Trust.


   Notes
 
Time for primary review 11 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results and discussion
 References
 

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