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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
ABSTRACT
As a leading cause of heart failure, postinfarction left ventricular remodeling represents an important target for therapeutic interventions. Mitogen-activated protein kinases regulate critical cellular processes including stress response and survival, but their role in left ventricular remodeling is unknown. In the present study, rats were subjected to myocardial infarction by ligating the left anterior descending coronary artery. Western blot and kinase assay analysis revealed an inactivation of p38 kinase after myocardial infarction. Local adenovirus-mediated cotransfection of wild-type (WT) p38 kinase and constitutively active MKK3b reduced infarct size (26±3% vs. 47±4%, P<0.05 vs. LacZ-treated control) associated with improved ejection fraction (66.9±5.5% vs. 44.4±4.0%, P<0.001), fractional shortening (30.2±2.1% vs. 19.7±2.2%, P<0.001), and decreased left ventricular diastolic diameter (8.5±0.4 mm vs. 9.5±0.2 mm, P<0.01). p38 kinase gene transfer increased capillary density (2423±107/mm2 vs. 1934±86/mm2, P<0.001) and resulted in microvessel enlargement in the ischemic border zone. Apoptosis (35±7 vs. 69±13 cells, P<0.01) and fibrosis (16±3% vs. 34±8%, P<0.05) were reduced, while the number of c-kit positive cardiac stem-like cells remained unchanged. These results indicate that reduced p38 signaling predisposes to adverse postinfarction remodeling. The rescue of failing myocardium with p38 kinase may be a potential new therapy for heart failure after myocardial infarction. —Tenhunen, O., Soini, Y., Ilves, M., Rysä, J., Tuukkanen, J., Serpi, R., Pennanen, H., Ruskoaho, H., Leskinen, H. p38 Kinase rescues failing myocardium after myocardial infarction: evidence for angiogenic and anti-apoptotic mechanisms.
Key Words: remodeling • signal transduction • gene therapy
ADVERSE LEFT VENTRICULAR (LV) remodeling after myocardial infarction (MI) is a major cause of heart failure and death (1)
. The loss of functional cardiac myocytes following acute myocardial infarction (AMI) initially triggers a cascade of events resulting in the expansion of the infarction area, which may cause early LV rupture or aneurysm formation (2)
. This early phase of cardiac remodeling is followed by late remodeling characterized by enlargement and scar formation of the infarction area, progressive fibrous replacement of myocardium, as well as hypertrophic growth of the noninfarcted region. These structural changes ultimately lead to a progressive functional decline of the left ventricle and clinical symptoms of congestive heart failure (1
, 2)
. Despite optimal treatment with existing drugs, the prognosis of heart failure remains poor (1
, 2)
, 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.
Postinfarction remodeling involves alterations in specific signaling molecules and their respective downstream pathways. Among the most conserved signal transduction systems in the heart is the mitogen-activated protein kinase (MAPK) cascade, which consists of sequentially acting protein kinases resulting in the activation of three terminal MAP kinases, p38 kinase, extracellular signal-regulated protein kinase (ERK), and c-jun N-terminal protein kinase (JNK) (3
, 4)
. MAPKs are serine/threonine kinases that transduce signals from the cell membrane to the nucleus in response to growth factors and G protein-coupled receptor agonists, as well as mechanical stress (3
, 4)
. Members of the MAPK cascade are known to regulate a wide array of critical cellular processes, such as cell growth, cell cycle, and differentiation (3
, 4)
. Due to the inability of cardiomyocytes to divide, the function of MAPKs in cardiac cells is related to stress response, cell survival, and apoptosis, which are all fundamental mechanisms of postinfarction remodeling (4)
. However, neither the activity nor the functional importance of MAP kinases in postinfarction remodeling and heart failure is known.
The aim of the present study was to determine the role of MAP kinases in LV remodeling after myocardial infarction and as potential targets for heart failure gene therapy. To address these questions, we subjected adult Sprague-Dawley rats to AMI by ligating the left anterior descending artery (LAD). MAPK activities were studied by Western blotting and kinase assays during the LV remodeling period. Since these experiments revealed that MI resulted in a sustained inactivation of p38 MAP kinase during ventricular remodeling process, we established a protocol to locally increase p38 kinase activity by using adenovirus-mediated gene transfer into the left ventricle simultaneously with MI. Our present study provides the first evidence that normalization of p38 kinase activity rescues failing heart after MI.
MATERIALS AND METHODS
Recombinant adenovirus vectors
The recombinant adenoviruses containing the coding regions of the consititutively active mitogen-activated protein kinase kinases (MKK), MKK3b (RAdMKK3bE) and MKK6b (RAdMKK6bE), and wild-type (WT) p38
(RAdp38) and WT p38ß (RAdp38ß) genes driven by the cytomegalovirus immediate early promoter were generated as described previously (5)
. Recombinant replication-deficient adenovirus RAdlacZ, which contains the Escherichia coli ß-galactosidase (LacZ) gene, was used as a control. The recombinant adenoviruses were generously supplied by Dr. Veli-Matti Kähäri from the University of Turku, Finland.
Myocardial infarction and cardiac gene transfer in vivo
MI was produced by ligation of the LAD (6)
. Male Sprague-Dawley rats weighing 250–300 g (n=141) were anesthetized with medetomidine hydrochloride (Domitor, 250 µg/kg ip) and ketamine hydrochloride (Ketamine, 50 mg/kg, ip). Rats were connected to the respirator through a tracheotomy. A left thoracotomy and pericardial incision were performed, and the LAD was ligated. After the operation, anesthesia was partially antagonized with atipamezole hydrochloride (Antisedan, 1.5 mg/kg ip) and rats were hydrated with 5 ml physiological saline solution given subcutaneously. For postoperative analgesia, buprenorphine hydrochloride (Temgesic, 0.05–0.2 mg/kg sc) was administered. The sham-operated rats underwent the same surgical procedure without the ligation of LAD. The experimental design was approved by the Animal Use and Care Committee of the University of Oulu. Adenovirus-mediated gene transfer into the LV free wall was done by the method recently described (7)
. Recombinant adenovirus (2–8x108 pfu) in a 100 µl volume was injected using a Hamilton precision syringe directly into the anterior wall of the left ventricle before the ligation of LAD. This approach has been shown to be a powerful method for local gene delivery into the heart, and it targets high expression of the gene to the left ventricle without affecting other organs or other regions of the heart (7)
. The adenoviral gene transfer to the normal hearts was performed using the same technique without the ligation of LAD.
Echocardiographic measurements
Transthoracic echocardiography was performed using the Acuson Ultrasound System (Sequoia 512) and a 15-MHz linear transducer (15L8) (Acuson, MountainView, CA). Before examination, rats were sedated with ketamine (50 mg/kg) and xylazine (10 mg/kg). With the use of two-dimensional imaging, a short axis view of the left ventricle at the level of the papillary muscles was obtained, and a two dimensionally guided M-mode recording through the anterior and posterior walls of the left ventricle was obtained. LV end-systolic (LVESD) and end-diastolic (LVEDD) dimensions as well as the thickness of the interventricular septum (IVS) and posterior wall (PW) were measured from the M-mode tracings. The LV fractional shortening (FS) and ejection fraction (EF) were calculated from the M-mode LV dimensions using the following equations: LVFS (%) = {(LVEDD-LVESD) / LVEDD} x 100, LVEF (%) = {(LVEDD)3 – (LVESD)3 / LVEDD3} x 100. An average of three measurements of each variable was used. After echocardiography, the animals were killed, the hearts were removed, and the cardiac chambers were separated. LV and right ventricular tissue samples were weighed, immersed in liquid nitrogen, and stored at –70°C for later analysis.
Histology and image analysis
For histological analysis, the hearts were fixed in 10% buffered formalin solution. Transversal sections of the left ventricle were embedded in paraffin, and 5 µm-thick sections were cut and stained with hematoxylin and eosin, Masson’s trichrome, or Sirius red. Biotinylated lectin GSL-1 (B-1205, Vector Laboratories, Burlingame, CA) was used to stain endothelial cells. Paraffin-embedded sections were deparaffinized in xylene and dehydrated in graded ethanol. After incubation with HRP-streptavidin (SA-5004, Vector Laboratories), the peroxidase label was developed by a peroxidase conjugated EnVision Detection Kit system (DakoCytomation) and the samples were counterstained with hematoxylin. To detect apoptotic cells, in situ labeling of the 3'-ends of the DNA fragments generated by apoptosis-associated endonucleases was performed using the ApopTag in situ apoptosis detection kit (Oncor, Gaithersburg, MD) as described previously (8)
. As a positive control, tissue sections from hyperplastic lymph nodes were used. The apoptotic cells and bodies were counted in five high-power fields from the peri-infarct regions choosing hot spot areas in each sample to make the results comparable. The area covered by microvessels was measured from lectin-stained sections using a digital image analysis system (MCID/M4 with software version 3.0, Imaging Research, St. Catharines, Canada). Five representative high power fields (x40) from the peri-infarct area of each section were measured, and the number of capillaries was calculated from the same fields. The fibrotic area of the left ventricle and infarct size were determined from the Masson’s trichrome stained histological sections using the same software.
Immunohistochemistry
Paraffin-embedded sections were deparaffinized in xylene and dehydrated in graded ethanol. A C-kit antibody (Ab; Santa Cruz Biotechnology, Santa Cruz, CA) was used to stain stem-like cells. The peroxidase label was developed by a peroxidase conjugated EnVision Detection Kit system (DakoCytomation), and the samples were counterstained with hematoxylin. The number of c-kit positive stem-like cells in the anterior wall of the left ventricle was counted. For detection of caspase-3 positive cells, a specific Ab for cleaved caspase-3 (Cell Signaling Technology, Beverly, MA) was used.
X-gal staining
Hearts were rinsed in PBS and fixed in PBS containing 4% paraformaldehyde for 10 min at room temperature, washed twice in 0.15 M sodium phosphate buffer (pH 7.2) for 15 min, and incubated with 1 mg/ml X-gal (5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) in reaction buffer [5 mM K3Fe(CN)6, 5 mM K4Fe(CN)63H2O, 2 mM MgCl2, 1x PBS] at 37°C for 3 h. Hearts were photographed. For frozen sections, cardiac samples were embedded in OCT compound (Tissue-Tek, Sakura, The Netherlands), frozen, and sectioned. Sections were counterstained with hematoxylin-eosin (HE) and analyzed with a light microscope.
Extraction of cytoplasmic protein
The tissue was broken in liquid nitrogen and homogenized in lysis buffer consisting of 20 mmol/l Tris (pH 7.5), 10 mmol/l NaCl, 0.1 mmol/l EDTA, 0.1 mmol/l EGTA, 1 mmol/l ß-glycerophosphate, 1 mmol/l Na3VO4, 2 mmol/l benzamidine, 1 mmol/l phenylmethylsulfoxide (PMSF), 50 mmol/l NaF, 1 mmol/l dithiothreitol (DTT), and 10 µg/ml each of leupeptin, pepstatin and aprotinin. After centrifugation, the supernatant was separated and protein samples were stored in –70°C until assayed. Protein concentrations were determined by the Bio-Rad Laboratories protein assay.
Western blotting
Western blots were performed as described previously (9)
. Briefly, 30 µg of protein were loaded onto an SDS-PAGE gel and transferred to nitrocellulose filters. The membranes were blocked in 5% nonfat milk and incubated with primary antibodies overnight. After incubation with horseradish peroxidase-linked secondary Ab, the protein amount was detected by enhanced chemiluminesence using hyperfilm MP (Amersham, Bucks, UK). For a second Western blot, the membranes were stripped for 30 min at + 60°C in stripping buffer containing 62.5 mmol/l Tris (pH 6.8), 2% SDS, and 100 mmol/l mercaptoethanol. The following primary antibodies were used: anti-Akt, antiphospho-p38, antiphospho-p44/42, antiphospho-JNK, antiphospho-MKK3, antip38, antip44/42, anti-JNK, antiphospho-ATF-2, antiphospho-c-jun, and antiphospho-ELK-1. Antibodies were obtained from Cell Signaling Technology (Beverly, MA), Chemicon International (Temecula, CA), and Santa Cruz Biotechnology.
Kinase assays
The p38 MAP kinase assay kit was obtained from Cell Signaling Technology and perfomed according to the manufacturer’s instructions. Two hundred micrograms of protein extract were immunoprecipitated with immobilized phospho-p38 monoclonal antibody (mAb). For the determination of p38 kinase activity, 2 µg of activating transcription factor 2 (ATF-2) fusion protein and 200 µM ATP were added to the kinase reaction, and phospho-ATF-2 production was determined by Western blotting. The assay for measuring ERK activity (Cell Signaling Technology) was similar to the p38 kinase assay, except that the protein was immunoprecipitated by immobilized phospho-p44/42 mAb, ELK-1 fusion protein was used as a substrate, and the samples were analyzed by Western blotting for phospho-ELK-1.
Isolation and analysis of RNA
The RNA extraction and Northern blot analysis were performed as described previously (9)
. The RNA was extracted from the left ventricles by using the guanidine-thiocyanate-CsCl method. For the Northern blot analyses, 20 µg samples of RNA were separated by electrophoresis and transferred to nylon membranes (Osmonics, Westborough, MA). The cDNA probes complementary to rat A-type [atrial natriuretic peptide (ANP)] and B-type natriuretic peptide (BNP) or ribosomal 18S RNA were random primer-labeled with [32P]dCTP, and the membranes were hybridized and washed 3 x 20 min at + 62°C. The membranes were exposed with PhosphorImager screens (Amersham Biosciences), which were scanned with a Molecular Imager FX Pro Plus, and quantitated using Quantity One software (Bio-Rad). The hybridization signals of ANP and BNP mRNAs were normalized to that of 18S RNA in each sample. Basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-A), and vascular endothelial growth factor (VEGF) mRNA levels were measured by quantitative reverse transcription-polymerase chain reaction (PCR) analysis as described previously (10)
. The sequences of the forward (F) and reverse (R) primers and probes (P) for RNA detection were as follows: bFGF (F) 5'-CCCGGCCACTTCAAGGAT-3', (R) 5'- GATGCGCAGGAAGAAGCC – 3', (P) 5'-Fam-CCAAGCGGCTCTACTGCAAGAACGG-Tamra-3'; PDGF-A (F) 5'-CGAGCGACTGGCTCGAA-3', (R)5'-GAGTCTATCTCCAAGAGTCGCTGG-3', (P) 5'-Fam-TCAGATCCACAGCATCCGGGACC-Tamra-3'; and VEGF (F) 5'-GATCCGCAGACGTGTAAATGTTC-3', (R) 5'-TTAACTCAAGCTGCCTCGCC-3', (P) 5'-Fam-TGCAAAAACACAGACTCGCGTTGCA-Tamra-3'.
Statistics
Results are expressed as mean ± SE. The data were analyzed with one way ANOVA followed by a least significant difference (LSD) post hoc test. For comparisons between two groups, Student’s t test was used. A P value of <0.05 was considered statistically significant.
RESULTS
LV function, morphology, and gene expression after MI
To study the role of MAP kinases in postinfarction remodeling, the LAD was ligated in rats. MI progressively decreased LV ejection fraction and fractional shortening and caused LV dilatation as assessed by echocardiography during the 4 wk follow-up period (Supplementary Table 1). The LV dilatation, thinning of the anterior wall, and hypertrophy of the posterior wall were seen also in hematoxylin-eosin stained myocardial sections (Supplementary Fig. 1A). The functional and morphological changes were accompanied by an increase in A-type and B-type natriuretic peptide gene expression in the left ventricle (Supplementary Fig. 1B). The levels of ANP and BNP mRNA were also significantly increased in the right ventricles (data not shown).
Distinct inactivation of p38 kinase after MI
The MAPK signaling pathways serve as pivotal transducers of diverse biological functions including cell growth, differentiation, proliferation, and apoptosis (3
, 4)
. 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 (4
, 11)
. 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 marked inactivation was still observed 2 wk after MI (Fig. 1
A). 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 (Fig. 1A
). 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 (Fig. 1B
). 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) (3
, 4)
. 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 (Fig. 1B
.
|
In contrast to p38 kinase, ERK 1/2 activity was not altered 1 day after MI but increased 1.5-fold at 2 wk (Fig. 1C
). 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, while at 2 wk phospho-JNK vs. total JNK-ratio was significantly decreased (Fig. 1D
). The total protein levels of Akt kinase were not altered (Fig. 1B
).
Adenovirus-mediated overexpression of MKK3bE and WT p38 increases p38 activity in normal hearts and rescues p38 kinase activity after MI
The p38 branch of MAP kinase cascade includes four separate isoforms, p38, p38ß, p38
, and p38
. The major isoform of p38 kinase expressed in the heart is p38, while p38ß is undetectable in murine heart (12
, 13)
. To study the importance of reduced p38 kinase activity in LV remodeling after MI, we established a protocol to locally increase p38 kinase activity. We first tested the effects of the direct adenovirus-mediated gene transfer of constitutively active MKK3b and MKK6b, specific upstream activators of p38 kinase, and WTp38 and p38ß in the normal heart. Control animals were injected with adenovirus expressing the Escherichia coli ß-galactosidase (LacZ) gene, and the efficiency and localization of the adenoviral gene transfer were confirmed by X-gal staining (Fig. 2
); 6 x 108 infectious units of recombinant adenovirus (RAdMKK3bE, RAdMKK6bE, RAdp38, RAdp38ß) in a 100 µl vol was injected directly into the LV free wall, and the animals were killed 3 days after the procedure. Western blot analysis revealed a significant increase in the levels of phospho-p38 and total p38 by adenovirus-mediated overexpression of WTp38 and p38ß, whereas no significant effect on the levels of phospho-p38 was observed by constitutively active MKK3b or MKK6b alone (Fig. 3
). Since coinfection of fibroblasts with MKK3bE and WTp38 can potentiate the activation of p38 phosphorylation in vitro (14)
, we next coinjected MKK3bE at 6 x 108 infectious units and WT p38 at 2 x 108 infectious units into the LV wall. The combined administration of MKK3bE and WT p38 resulted in the strongest up-regulation of phospho-p38 kinase levels in normal hearts (Fig. 3
and Fig. 4
A). Similarly, the administration of MKK3bE potentiated the up-regulation of p38 kinase by WT p38 in cultured cardiomyocytes (L. Kaikkonen, E. Koivisto, R. Kerkelä, H. Tokola, H. Ruskoaho, unpublished observation). Gene transfer of MKK3bE and WTp38 did not change the levels of phospho-ERK 1/2 or phospho-JNK (Fig. 4B and C
), indicating that this combination is a specific approach to activate p38 kinase. The expression profile of the transgene was transient: at 2 wk, the protein levels of phospho-p38 or total p38 did not differ between the MKK3bE+WTp38- and LacZ-treated hearts (phospho-p38: 0.8-fold vs. LacZ, P=NS, n=6; total p38: 0.86-fold vs. LacZ, P=NS, n=6). No significant differences in LVEF, FS, or LV diameter were observed 3 days after injection between the LacZ vs. MKK3bE+WTp38 injected groups as assessed by echocardiography, showing that overexpression of p38 kinase does not alter the LV function of normal rat hearts at this time point (data not shown).
|
|
|
Since coadministration of MKK3bE and WTp38 produced the strongest increase in p38 kinase activity in the normal heart, p38 is the predominant kinase isoform in the rodent heart (12
, 13)
, and MKK3 primarily phosphorylates p38 (4)
, this 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 d 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 (Fig. 4D
).
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 d after MI (Table 1
), infarcted hearts treated with MKK3bE+WTp38 gene transfer showed a significant functional improvement after 2 wk (Fig. 5
). Normalization of p38 kinase activity increased FS and LVEF by 1.5-fold compared with the LacZ-injected group (Fig. 5C-D
) and significantly attenuated LV dilatation (Fig. 5B
). LV diameter, FS, 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, except that there was a slight difference in FS between MKK3bE+WTp38-treated and sham-operated groups (Fig. 5A-D
).
|
|
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 (2)
. To study whether the functional improvement by p38 kinase activity normalization is related to the reduction of infarct size, histological 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 (Fig. 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 (2
, 15)
. The fibrotic area of the left ventricle 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 (Fig. 6)
. Furthermore, a significant decrease in the rate of apoptosis was observed at day 3 (Fig. 7
A) but not at 2 wk (data not shown) in MKK3bE+WTp38-treated hearts as assessed by TUNEL. The results of the TUNEL analysis were supported by immunostaining for activated caspase-3, which showed a similar decrease in the number of apoptotic cells (Fig. 7B
). To study whether the attenuation of LV remodeling is due to increased stem cell recruitment, immunohistochemistry using c-kit-specific Ab was performed and the number of c-kit positive cells in the anterior wall of the left ventricle was counted. 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 (5±1 vs. 4±1 cells/section, P=NS, n=5–6) was noted.
|
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 (16)
. 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-1, 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 to those treated with LacZ virus (Fig. 8
), as assessed from five high-power fields of lectin-stained sections. Also normal hearts treated with MKK3bE+WTp38 gene transfer showed a tendency for increased capillary density; however, this increase was not statistically significant (Fig. 8)
.
|
To characterize further the angiogenic mechanisms of p38 kinase overexpression, we examined the gene expression of selected angiogenic growth factors after MKK3bE+p38WT gene transfer in the normal hearts. At day 3 after gene transfer, a significant increase in bFGF expression (2.3-fold vs. LacZ-treated hearts, P<0.01, n=8–10) and platelet-derived growth factor A (PDGF-A_mRNA levels (1.7-fold vs. LacZ, P<0.05, n=8–10) was noted, while VEGF mRNA levels (1.0-fold vs. LacZ, P=NS, n=8–10) remained unchanged.
DISCUSSION
Growing evidence shows that the reversal of structural remodeling represents a key therapeutic target in postinfarction management and treatment of established heart failure (1
, 2)
. MAPKs are central signaling molecules regulating myocyte stress response, but the functional effects attributed to MAPK signaling in postinfarction LV remodeling and heart failure are not known (3
, 4)
. The present study reveals reduced p38 MAP kinase activity as a causative factor in postinfarction remodeling and identifies it as the first conserved intracellular signaling target for early postinfaction heart failure gene therapy. Furthermore, we demonstrated here that rescue of p38 kinase activity by adenovirus-mediated gene transfer during postinfarction LV remodeling results in significant reduction of infarct size and functional improvement with angiogenesis and reduced rate of apoptosis.
Given the central role of p38 kinase in the regulation of cellular stress response mechanisms, modulation of p38 kinase activity represents an attractive therapeutic approach in the treatment of several diseases. Indeed, small molecule p38 inhibitors have been suggested to have potentially beneficial effects in pulmonary disease, septic shock, and joint disease (17)
. The concept of p38 inhibition as a treatment for cardiovascular disease is based on the assumption that p38 kinase is activated in these diseases. Therefore, it is surprising to note that the detailed changes in p38 kinase activity during postinfarction remodeling have not been previously described. Our present results indicate that postinfarction LV remodeling and heart failure are characterized by a sustained down-regulation of p38 kinase activity. On the other hand, reversible myocardial ischemia as well as pressure overload has been reported to transiently activate p38 kinase (4
, 11
, 18)
. Thus, it appears that p38 signaling in postinfarction heart failure differs from other cardiovascular diseases, and instead of activation the functional and structural effects of p38 kinase are related to a sustained down-regulation of the kinase activity. Consistent with this, p38 kinase has been reported to be down-regulated in failing human hearts (13)
. Our present findings also indicate that the members of the MAP kinase family are distinctly regulated in postinfarction remodeling and heart failure. In contrast to p38 kinase, ERK 1/2 activity was significantly upregulated at 2 wk. JNK activity also appeared to increase at day 1 after MI, although the activation was likely due to the relative decrease in total JNK levels.
A key finding of our study is that reduced p38 kinase activity is one of the mechanisms in the process that leads from acute 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 levels, since the infarcted hearts treated with p38 kinase gene transfer showed both significantly improved cardiac function and reduced infarct size. On the other hand, a tendency to more dilatation and reduced function as well as an increase in LVW/BW was observed in MKK3bE+WTp38-treated animals at 3 days after MI (Table 1)
. Therefore, it is likely that the late improvement in cardiac function is a consequence of the reduced infarct size and scar formation rather than a direct effect of p38 kinase on cardiac contractility. Although most ischemia-reperfusion studies have suggested a damaging role for p38 kinase both in vitro and in vivo (4
, 11
, 18)
, supporting evidence in different experimental models exists for its protective role. Complete disruption of p38 is lethal, suggesting that maintenance of normal levels of p38 activity is necessary for the normal embryonic development (19)
. Dominant negative p38, MKK3 and MKK6 transgenic mice show enhanced cardiomyopathy (20)
, and cardiac-specific p38 knockout mice develop cardiac dysfunction and heart dilatation in response to pressure overload (21)
. p38 kinase has been suggested as a mediator of ischemic preconditioning in vitro (4)
, and transgenic mice overexpressing MKK6 were reported to show less myocardial damage and enhanced functional recovery from reversible ischemia-reperfusion injury (22)
. Of note, the majority of the transgenic studies had used myocyte-specific transgene expression, while in our experimental model the transgene is likely expressed by both cardiomyocytes and noncardiomyocytes, in agreement with the findings that adenoviruses infect a variety of cell types (12
, 20
21
22
23)
.
Remarkably, rescue of p38 kinase activity with local MKK3bE+WTp38 gene transfer attenuated remodeling through a distinct angiogenic mechanism. LV remodeling has been suggested to be explained by the impairment of oxygen and nutrient supply of otherwise viable myocytes in the border zone of the infarct scar (16)
. It is also known that neovascularization, although not in extent large enough to rescue the failing myocardium, occurs in the infarcted area and border zone. Moreover, increased neovascularization of ischemic myocardium by human bone marrow-derived angioblasts antagonizes pathological LV remodeling and improves cardiac function after experimental MI (16)
, and adenovirus-mediated overexpression of angiogenic growth factors has been suggested to prevent postinfarction heart failure (24)
. Interestingly, lectin-stained histological sections from the peri-infarct area showed a significant increase in capillary density and microvessel size in the MKK3bE+p38 injected hearts. This may improve the oxygen supply in the ischemic border zone, resulting in increased myocyte survival and reduction of infarct size. In support of our findings, activation of p38 kinase is known to be involved in endothelial cell responses including migration and cell survival (25)
. Furthermore, small molecule inhibitors of p38 kinase have been documented to prevent angiogenesis in an inflammatory angiogenesis model (26)
. VEGF and angiopoietin-1 are known to activate p38 kinase (25
, 27)
, and a similar microvessel enlargement as in our model has been observed in response to overexpression of VEGF-D in porcine heart (28)
. Interestingly, MKK3bE+WTp38 gene transfer significantly increased the gene expression of bFGF and PDGF-A in the normal heart, in agreement with the angiogenic effect of p38 kinase.
p38 has been linked to cardiomyocyte apoptosis (5
, 22)
, and in vivo studies have suggested that p38 kinase may protect cardiomyocytes from cell death (21)
. Importantly, we observed a significant decrease in the rate of apoptosis in response to normalization of p38 kinase activity at an early postinfarction time point, likely also contributing to the beneficial structural and functional effects of p38 kinase gene transfer. Of note, angioblast-induced postinfarction angiogenesis is also associated with reduced apoptosis (16)
. On the other hand, the number of c-kit positive cardiac stem cells did not differ between the hearts treated with MKK3bE+WTp38 and those treated with LacZ control virus, suggesting that increased stem cell recruitment is not the main mechanism underlying p38 kinase mediated cardioprotection. Histological data also revealed that the functional benefit of MKK3bE+WTp38 gene transfer was associated with the attenuation of pathological fibrosis, likely as a secondary consequence of angiogenesis and reduced apoptosis. However, p38 overexpression may as well have a direct effect on LV fibrosis, since it has been shown that p38 dominant negative mice develop massive cardiac fibrosis in response to pressure overload (21)
.
Our finding that early rescue of p38 kinase activity by local gene transfer attenuates postinfarction LV remodeling is not in contrast with the potential benefits of pharmacological p38 inhibitors at later time points. As a converge point of myocyte signaling, p38 kinase is known to affect multiple cellular processes, part of which may be harmful during chronic phases of heart failure. Indeed, improvements in cardiac performance and remodeling process have been reported with chronic in vivo administration of p38 inhibitors when initiation of treatment is delayed 1–2 wk after MI (29
, 30)
. Thus, during early remodeling process, p38 kinase seems to be protective, but becomes after this stage detrimental, suggesting a therapeutic potential for the use of either p38 gene therapy or synthetic small molecule p38 inhibitors. On the other hand, due to the complexity of intracellular signaling pathways and the factors activating them, it is difficult to separate the contributions of individual mechanisms of inhibitors of selected signaling molecules to their pharmacological actions. Therefore, the beneficial effects of pharmacological p38 kinase inhibitors in postinfarction heart failure may be related to their systemic effects rather than the inhibition of myocardial p38 kinase activity. For example, in contrast to down-regulated myocardial p38 activity observed in the present study and in end-stage failing human myocardium (13)
, vascular p38 MAP kinase is activated in postinfarction heart failure, and chronic inhibition of p38 kinase attenuates endothelial vasomotor dysfunction in heart failure (31)
.
Since the size of the infarcted area and the extent of postinfarction LV remodeling are two major factors that determine prognosis of patients after MI, development of therapies that affect these two processes is critical. Based on our results, normalization of down-regulated p38 kinase activity represents such 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. Previously, adenovirus-mediated gene transfer of hepatocyte growth factor (HGF) and VEGF has been reported to attenuate adverse remodeling after MI, but our study is the first to identify a conserved intracellular signaling pathway as a potential target for gene therapy in postinfarction heart failure (24
,32)
. Moreover, our present results reveal reduced p38 MAP kinase activity as a causative factor in the development of ischemic heart failure.
ACKNOWLEDGMENTS
We thank Sirpa Rutanen, Pirjo Korpi, Kaisa Penttilä, and Tuulikki Kärnä for expert technical assistance. This work was financially supported by grants from the Academy of Finland (H. Ruskoaho and R. Serpi), the Sigrid Juselius Foundation (H. Ruskoaho), the Finnish Foundation for Cardiovascular Research (O. Tenhunen, H. Ruskoaho, and H. Leskinen), the National Technology Foundation TEKES (H. Ruskoaho.), the Aarne Koskelo Foundation (O. Tenhunen), and the Finnish Medical Foundation (O. Tenhunen).
Received for publication January 3, 2006. Accepted for publication April 17, 2006.
REFERENCES
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