| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Departments of
* Pharmacology and Toxicology, Biocenter Oulu;
Pathology;
Physiology and
Anatomy and Cell Biology, University of Oulu, Oulu, Finland
1Correspondence: Department of Pharmacology and Toxicology, Faculty of Medicine, University of Oulu, P.O. Box 5000, University of Oulu, Oulu FIN-90014, Finland. E-mail: heikki.ruskoaho{at}oulu.fi
SPECIFIC AIMS
Adverse left ventricular (LV) remodeling after myocardial infarction (MI) is a major cause of heart failure. Despite optimal treatment with existing drugs, the prognosis of heart failure remains poor, and thus adjunctive strategies that are designed to specifically prevent or attenuate postinfarction LV remodeling may play an important role in the clinical treatment of heart failure. Mitogen-activated protein kinase (MAPK) cascade, consisting of three terminal MAP kinases, p38 kinase, extracellular signal-regulated protein kinase (ERK), and c-jun N-terminal protein kinase (JNK), is one of the most conserved signal transduction systems in the heart and a critical regulator of cell growth, survival, and death. However, neither the detailed changes in MAPK activities nor their functional importance during postinfarction remodeling is known, and the role of p38 kinase in other cardiovascular disease models is under much debate. The present study was aimed to determine the role of p38 kinase in postinfarction remodeling and as a potential target for heart failure gene therapy.
PRINCIPAL FINDINGS
1. LV function, morphology, and gene expression after MI
To study the role of MAPKs in postinfarction remodeling, the left anterior descending coronary artery (LAD) was ligated in Sprague-Dawley rats. MI progressively decreased LV ejection fraction (EF) and fractional shortening (FS) and caused LV dilatation as assessed by echocardiography during the 4-wk follow-up period. The LV dilatation, thinning of the anterior wall, and hypertrophy of the posterior wall were also observed in hematoxylin-eosin stained myocardial sections. The functional and morphological changes were accompanied by an increase in A-type and B-type natriuretic peptide gene expression in the left ventricle.
2. Distinct inactivation of p38 kinase after MI
Consistent with ischemia-reperfusion models, a rapid activation (1.5-fold, P<0.05 vs. sham) of p38 kinase was observed at 10 min after ligation of LAD. Thereafter, p38 kinase was sustainedly down-regulated in the infarcted hearts. At day 1, the ratio of phosphorylated p38 to total p38 was decreased by 76%, and a 54% decrease was still observed 2 wk after MI. The changes in p38 kinase activity were confirmed with an ATF-2 based kinase assay showing a corresponding inactivation at day 1 and at 2 wk. To assess the localization of p38 kinase inactivation in the heart, Western blots were performed from protein extracted from the infarcted anterior wall and noninfarcted posterior wall. Phospho-p38 kinase was down-regulated in both regions at day 1 after MI.
The activity of p38 kinase isoforms is mainly regulated by dual phosphorylation by two specific upstream kinases in the p38 cascade, MAP kinase kinase 3b (MKK3b), and MAP kinase kinase 6b (MKK6b). To characterize the role of these upstream mechanisms in the reduction of p38 kinase activity, the levels of MKK3b were assessed by Western blotting. Similarly to p38 kinase, also MKK3b was down-regulated at day 1 after MI.
In contrast to p38 kinase, ERK 1/2 activity was not altered 1 day after MI but increased 1.5-fold at 2 wk. The kinase assays using ELK-1 as a substrate confirmed the activation of ERK 1/2. A 3.1-fold increase was observed in phospho-JNK vs. total JNK-ratio at day 1, although this was likely due to a relative decrease in total JNK levels.
3. Adenovirus-mediated overexpression of MKK3bE and wild-type p38
increases p38 activity in normal hearts and rescues p38 kinase activity after MI
To study the importance of reduced p38 kinase activity in LV remodeling after MI, we established a protocol to locally increase p38 MAPK activity. We first tested the effects of the direct adenovirus-mediated gene transfer of constitutively active MKK3b (MKK3bE) and MKK6b (MKK6bE), specific upstream activators of p38 kinase, and wild-type (WT) p38 and p38ßbeta; in the normal heart in several combinations. The virus constructs were injected directly into the LV free wall, and LV MAPK levels were assessed by Western blotting 3 days after gene transfer. This protocol has previously been demonstrated as an efficient cardiac-specific gene delivery approach. Control animals were injected with adenovirus expressing the Escherichia coli ßbeta;-galactosidase (LacZ) gene at an appropriate concentration, and the efficiency and localization of the adenoviral gene transfer were confirmed by X-gal staining. The strongest up-regulation of p38 kinase activity was observed when MKK3bE at 6 x 108 infectious units was coinjected WT p38 at 2 x 108 infectious units. Gene transfer of MKK3bE and WT p38 did not change the levels of phospho-ERK 1/2 or phospho-JNK, indicating that this combination is a specific approach to activate p38 kinase.
Since coadministration of MKK3bE and WTp38 produced the most effective increase in p38 kinase activity in the normal heart, the combination was used to reverse decreased p38 levels in the infarcted hearts. A solution containing either LacZ or MKK3bE together with WT p38 recombinant adenoviruses was injected into the LV free wall, and LAD was ligated immediately thereafter. At 3 days after MI, the levels of phospho-p38 were similar in the sham-operated animals and animals with MI treated with MKK3bE+WTp38, indicating that the gene transfer completely normalized the inactivation of p38 kinase.
4. Rescue of p38 kinase activity by MKK3bE and WTp38 improves cardiac function during postinfarction remodeling
Next, we studied the functional consequences of normalized p38 kinase activity by echocardiography. Despite a comparable decrease in systolic function 3 days after MI, infarcted hearts treated with MKK3bE+WTp38 gene transfer showed a significant functional improvement after 2 wk. Normalization of p38 kinase activity increased FS and EF by 1.5-fold compared with the LacZ-injected group and significantly attenuated LV dilatation. LV diameter, EF, and LV wt/body wt ratio (LV/BW) of MKK3bE+WTp38-treated animals without MI did not significantly differ from sham-operated or LacZ-treated animals (Fig. 1
).
|
5. Decrease in infarct size by normalization of LV p38 kinase activity
The late LV remodeling is characterized by persistent thinning of the ventricular wall at the site of infarction, hypertrophy of surviving cardiomyocytes, and progressive enlargement of the infarcted area. To study whether the functional improvement by p38 kinase activity normalization is related to the reduction of infarct size, histologic sections from sham-operated, LacZ-treated, and MKK3bE+WTp38-treated hearts were stained with Masson’s trichrome or Sirius red. Consistent with the improved cardiac function, the size of the infarcted area assessed from the LV circumference was significantly reduced by the MKK3bE+WTp38 gene transfer (26±3% vs. 47±4%, P<0.05 vs. LacZ).
6. Rescue of p38 kinase activity attenuates fibrosis and apoptosis but does not affect c-kit positive cardiac stem-like cells
Pathological fibrosis, apoptotic cell death, and cardiac stem cell recruitment have been proposed to contribute to the late LV remodeling. The fibrotic area of the LV was assessed from Masson’s trichrome or Sirius red stained sections at 2 wk after MI. Fibrosis was significantly attenuated by MKK3bE+WTp38 gene transfer (16±3% vs. 34±8%, P<0.05). Furthermore, a significant decrease in the rate of apoptosis was observed at day 3 in MKK3bE+WTp38-treated hearts as assessed by TUNEL (35±7 vs. 69±13 cells, P<0.01). In contrast to fibrosis and apoptosis, no difference in the number of c-kit positive cardiac stem-like cells between LacZ and MKK3bE+WTp38-treated groups was noted.
7. Angiogenesis in the peri-infarct region of MKK3bE+WTp38-treated hearts
In addition to stem cell recruitment, apoptosis, and fibrosis, angiogenesis in the ischemic border zone of the infarct may affect the remodeling process. Therefore, we studied the effects of the normalization of p38 kinase activity on the microvessels of the peri-infarct area. Two weeks after MI and gene transfer, the histological sections were stained with lectin-GSL, a specific marker for endothelium. MKK3bE+WTp38-treated hearts showed a significant increase in the capillary density and a marked dilatation of the microvessel network in the border zone of the infarction compared with those treated with LacZ virus (Fig. 2
), as assessed from 5 high-power fields of lectin-stained sections. To characterize further the angiogenic mechanisms of p38 kinase overexpression, we examined the gene expression of selected angiogenic growth factors after MKK3bE+WTp38 gene transfer in the normal hearts. At day 3 after gene transfer, a significant increase in basic fibroblast growth factor (bFGF) expression (2.3-fold vs. LacZ-treated hearts, P<0.01) and platelet-derived growth factor (PDGF-A) mRNA levels (1.7-fold vs. LacZ, P<0.05) was noted.
|
CONCLUSIONS AND SIGNIFICANCE
A key finding of the present study is that p38 kinase is sustainedly down-regulated after MI, and reduced p38 MAPK activity is one of the mechanisms in the process that leads from MI to a failing heart. Normalization of p38 kinase activity with MKK3bE+WTp38 gene transfer prevented the vicious circle of postinfarction LV remodeling and heart failure both at physiological and morphological level, since the infarcted hearts treated with p38 kinase gene transfer showed both significantly improved cardiac function and reduced infarct size. Lectin-stained histological sections from peri-infarct area demonstrated a significant increase in capillary density and microvessel size in the MKK3bE+p38 injected hearts. This angiogenic response may improve the oxygen supply in the ischemic border zone, resulting in increased myocyte survival and reduction of infarct size. Moreover, we observed a significant decrease in the number of apoptotic cells, likely also contributing to the beneficial structural and functional effects of p38 kinase gene transfer.
Growing evidence shows that the reversal of structural remodeling represents a key therapeutic target in postinfarction management and treatment of established heart failure. MAPKs are central signaling molecules regulating myocyte stress response, but the functional effects attributed to MAPK signaling in postinfarction LV remodeling and heart failure have not been known. The present study reveals reduced p38 kinase activity as a causative factor in postinfarction remodeling and identifies it as the first conserved intracellular signaling target for early postinfarction heart failure gene therapy. Normalization of p38 kinase activity may represent a new preventive approach against the progression of heart failure, since in addition to limiting structural remodeling of the left ventricle, it has the potential for functional improvement through a distinct angiogenic and antiapoptotic mechanism.
|
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5618fje
This article has been cited by other articles:
|
|
 |
|
  D. Westermann, A. Riad, O. Lettau, A. Roks, K. Savvatis, P. M. Becher, F. Escher, A.H. Jan Danser, H.-P. Schultheiss, and C. Tschope Renin Inhibition Improves Cardiac Function and Remodeling After Myocardial Infarction Independent of Blood Pressure Hypertension, December 1, 2008; 52(6): 1068 - 1075. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Sato, T. Horinouchi, D. S. Hutchinson, B. A. Evans, and R. J. Summers Ligand-Directed Signaling at the beta3-Adrenoceptor Produced by 3-(2-Ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate (SR59230A) Relative to Receptor Agonists Mol. Pharmacol., November 1, 2007; 72(5): 1359 - 1368. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
