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Am J Physiol Heart Circ Physiol 289: H2167-H2175, 2005. First published July 22, 2005; doi:10.1152/ajpheart.00392.2005
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Cardioprotection induced by cardiac-specific overexpression of fibroblast growth factor-2 is mediated by the MAPK cascade

Stacey L. House,1 Kevin Branch,1 Gilbert Newman,1 Thomas Doetschman,2 and Jo El J. Schultz1

Departments of 1Pharmacology and Cell Biophysics and 2Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio

Submitted 21 April 2005 ; accepted in final form 7 July 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Our laboratory showed previously that cardiac-specific overexpression of FGF-2 [FGF-2 transgenic (Tg)] results in increased recovery of contractile function and decreased infarct size after ischemia-reperfusion injury. MAPK signaling is downstream of FGF-2 and has been implicated in other models of cardioprotection. Treatment of FGF-2 Tg and wild-type hearts with U-0126, a MEK-ERK pathway inhibitor, significantly reduced recovery of contractile function after global low-flow ischemia-reperfusion injury in FGF-2 Tg (86 ± 2% vehicle vs. 66 ± 4% U-0126; P < 0.05) but not wild-type (61 ± 7% vehicle vs. 67 ± 7% U-0126) hearts. Similarly, MEK-ERK inhibition significantly increased myocardial infarct size in FGF-2 Tg (12 ± 3% vehicle vs. 31 ± 2% U-0126; P < 0.05) but not wild-type (30 ± 4% vehicle vs. 36 ± 7% U-0126) hearts. In contrast, treatment of FGF-2 Tg and wild-type hearts with SB-203580, a p38 inhibitor, did not abrogate FGF-2-induced cardioprotection from postischemic contractile dysfunction. Instead, inhibition of p38 resulted in decreased infarct size in wild-type hearts (30 ± 4% vehicle vs. 11 ± 2% SB-203580; P < 0.05) but did not alter infarct size in FGF-2 Tg hearts (12 ± 3% vehicle vs. 14 ± 1% SB-203580). Western blot analysis of ERK and p38 activation revealed signaling alterations in FGF-2 Tg and wild-type hearts during early ischemia or reperfusion injury. In addition, MEK-independent ERK inhibition by p38 was observed during early ischemic injury. Together these data suggest that activation of ERK and inhibition of p38 by FGF-2 is cardioprotective during ischemia-reperfusion injury.

extracellular signal-regulated kinase; p38; ischemia-reperfusion injury; myocardial infarction; signaling cross talk; mitogen-activated protein kinase

THE ELUCIDATION OF MECHANISMS to protect the myocardium from ischemia-reperfusion injury is vital to the advancement of therapeutics for ischemic heart disease, which is a major cause of morbidity and mortality in the industrialized world. Studies have shown that several stimuli including brief ischemic insults, pharmacological agents, and endogenous molecules can elicit protection from cardiac ischemia-reperfusion injury (15, 34). Recently, growth factors including those of the FGF family have been suggested to be among the endogenous mediators of cardioprotection (25). A member of this protein family, basic FGF or FGF-2, was recently implicated as a potential mediator of cardioprotection in multiple pharmacological and in vitro studies (10).

Our laboratory showed recently (23) in an ex vivo mouse model of ischemia-reperfusion injury that genetically overexpressing FGF-2 in the heart decreases ischemic damage, as measured by the recovery of postischemic ventricular function and myocardial infarct size. Furthermore, this cardioprotection is not a result of increased vascularization of the heart but a more direct effect on the myocardium. Although this evidence suggests that FGF-2 is an important endogenous cardioprotective mediator, the mechanisms through which FGF-2 elicits this cardioprotection are yet to be understood.

FGF-2 has been shown in various tissues and developmental stages to signal through the MAPK pathway (37). ERK and p38 are two members of the MAPK protein family. p38 has also been termed a stress-activated protein kinase because of its activation by multiple cellular stresses including oxidative stress, ischemia-reperfusion injury, and osmotic stress (41). ERK has been shown to be pro-cell survival and growth, whereas p38 is known to be involved in apoptosis and inflammation (38). In addition, both ERK and p38 have been implicated in multiple models of cardioprotection from ischemia-reperfusion injury (1), but their necessity in FGF-2-induced cardioprotection is presently unknown.

Given the important cardioprotective role of the MAPK cascade and its known relationship with FGF-2 in many tissues, our laboratory set forth to determine the involvement of ERK and p38 signaling in cardioprotection elicited by cardiac-specific overexpression of FGF-2. Our results demonstrate that overexpression of FGF-2 causes ERK activation, leading to protection from postischemic contractile dysfunction and myocardial infarction, and that FGF-2 also inhibits p38 activation during ischemia and reperfusion, leading to decreased cell death after ischemia-reperfusion injury. These findings are of clinical importance as phase I and II clinical trials are currently under way studying the efficacy and safety of FGF-2 therapy for patients with ischemic heart disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice were housed in a pathogen-free facility and handled in accordance with standard use protocols, animal welfare regulations, and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All protocols were approved by the University of Cincinnati Institutional Animal Care and Use Committee. Wild-type (WT) mice and mice with a cardiac-specific overexpression of all four isoforms of human FGF-2 [FGF-2 transgenic (Tg) mice] were randomly assigned to 60-min ischemia, 120-min reperfusion studies (30 WT and 38 FGF-2 Tg), time course studies (36 WT and 64 FGF-2 Tg), and MAPK cross talk studies (56 WT and 66 FGF-2 Tg). Generation of FGF-2 Tg mice was described previously (23). Exclusion criteria were based on signs of aortic or pulmonary leaks in the work-performing heart preparation. Two independently derived FGF-2 Tg lines were used in all experiments to ensure that results were not due to random transgene insertion affecting other genetic loci. Because similar results were obtained in the two FGF-2 Tg lines, all figures depict combined data from both lines.

Isolated work-performing heart model of global low-flow ischemia. Age (10–12 wk)- and sex-matched WT and FGF-2 Tg mice were anesthetized with pentobarbital sodium (80 mg/kg ip). The isolated work-performing heart preparation and global low-flow ischemia protocol were completed as previously described (23), and two ischemia protocols were used: either 30 min of low-flow ischemia followed by 30 min of reperfusion or 60 min of low-flow ischemia followed by 120 min of reperfusion (Fig. 1). Briefly, the heart was removed from the thoracic cavity and perfused with oxygenated 37.7°C Krebs-Henseleit solution. Cannulas in the aorta, left atrium, and left ventricle recorded pressure development throughout the cardiac cycle. The hearts were allowed to equilibrate for 30 min with a venous return of 5 ml/min and an aortic pressure of 50 mmHg, resulting in a basal workload of 250 ml·min–1·mmHg–1. To induce ischemic damage, venous return to the heart was quickly reduced by 1-ml increments to a venous return of 1 ml/min for 30 or 60 min. After this global ischemic insult, the venous return was quickly increased in 1-ml increments to a venous return of 5 ml/min and the heart was reperfused for either 30 or 120 min. Pacing of the heart to its baseline heart rate was used to maintain the work demand on the myocardium throughout the experiment.



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  		Fig. 1. Schematic of low-flow ischemia protocols. A: time-course protocol for ERK and p38 activation during ischemia-reperfusion injury: 30-min equilibration followed by 30-min global low-flow ischemia and 30-min reperfusion. Arrows indicate time of ischemia or reperfusion when hearts were arrested and homogenized for Western blot analysis. B: drug treatment protocol for ERK and p38 inhibitor studies: 30-min equilibration followed by 60-min global low-flow ischemia and 120-min reperfusion. Pharmacological agents were administered for 15 min before and during the beginning of ischemia and 15 min before and during the beginning of reperfusion.

 
Pharmacological agents. 1,4-Diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U-0126; Calbiochem), an inhibitor of MEK1/2 [upstream kinases of ERK activation (11)], or 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole HCl (SB-203580; Calbiochem), a p38 inhibitor (8), was administered during the 60-min low-flow ischemia and 120-min reperfusion model of ischemia-reperfusion injury. Studies were performed with varying concentrations (1–10 µmol/l) of these compounds in the ischemia-reperfusion model. Concentrations of 2.5 µmol/l for U-0126 and 2 µmol/l for SB-203580 were chosen because these drug concentrations inhibited ERK and p38 activation, respectively, without the adverse effects (i.e., cardiac arrhythmias or increased left atrial pressure) that were observed at higher drug concentrations (data not shown). The MEK-ERK pathway inhibitor or the p38 inhibitor was administered for the last 15 min of baseline and the first 15 min of ischemia and again for the last 15 min of ischemia and the first 15 min of reperfusion (Fig. 1B) because alterations in ERK and p38 activation were observed during early ischemia and early reperfusion injury. In sham-treated hearts, administration of U-0126 resulted in a small but significant increase in cardiac contractility, whereas SB-203580 had no effect on any cardiac functional parameter (data not shown).

Measurement of infarct size. After the 60-min ischemia and 120-min reperfusion study, drug-treated or vehicle-treated WT and FGF-2 Tg hearts were perfused with 1% 2,3,5-triphenyltetrazolium chloride (Sigma-Aldrich) (12), and infarct size was determined as previously described (23).

Western blot analysis of MAPK phosphorylation. WT and FGF-2 Tg hearts subjected to various time points of ischemia-reperfusion injury were analyzed to determine the timing of alterations in ERK or p38 phosphorylation during ischemia-reperfusion injury. Time points studied included the following: sham treatment (30 min of equilibration), 5 min of ischemia, 15 min of ischemia, 30 min of ischemia, and 30 min of ischemia followed by 5, 15, or 30 min of reperfusion (Fig. 1A). Hearts were snap-frozen in liquid nitrogen and homogenized in buffer containing (in mmol/l) 25 HEPES, 150 NaCl, and 5 EDTA, with 1% Triton X-100, 1% glycerol, and various protease and phosphatase inhibitors (Roche complete mini EDTA-free protease inhibitor cocktail, PMSF, okadaic acid). Homogenates were centrifuged at 3000 g to remove cell debris, and 100 µg of each supernatant was subjected to SDS-PAGE. Western blot analysis was performed with phospho-specific ERK and p38 antibodies (1:1,000 dilution; Cell Signaling). Equal protein loading was assessed via Ponceau S (Sigma-Aldrich) staining of total protein content and Western blot analysis with antibodies to total ERK (1:1,000 dilution; BD Transduction Labs) and total p38 (1:1,000 dilution; Santa Cruz Biotechnology). Densitometry of protein bands was performed with a Fluorchem 8800 gel imager (Alpha Innotech).

MAPK cross-talk analysis. To identify cross talk within the MAPK cascade, U-0126 (2.5 µmol/l) or SB-203580 (2 µmol/l) was administered as described above, and drug- or vehicle-treated FGF-2 Tg or WT hearts were snap-frozen in liquid nitrogen after being subjected to 30-min equilibration followed by either 5-min ischemia or 60-min ischemia and 5-min reperfusion. In addition, FGF-2 Tg and WT hearts were subjected to 30-min equilibration and 5-min ischemia in the presence of both U-0126 and SB-203580. All hearts were homogenized and subjected to SDS-PAGE and Western blot analysis of phosphorylated ERK or p38 as described in Western blot analysis of MAPK phosphorylation.

Western blot analysis of heat shock protein-27 phosphorylation. To determine the efficacy of the p38 inhibitor SB-203580, Western blot analysis of the activation/phosphorylation state of a major downstream target of p38, heat shock protein (HSP)-27, was performed. WT and FGF-2 Tg hearts subjected to either 5-min ischemia or 60-min ischemia and 5-min reperfusion in the presence and absence of SB-203580 (2 µmol/l) were snap-frozen in liquid nitrogen and homogenized as described above (Western blot analysis of MAPK phosphorylation). One hundred micrograms of each heart homogenate was subjected to SDS-PAGE, and Western blot analysis was performed with a phospho-specific HSP-27 antibody (1:250 dilution; Santa Cruz Biotechnology). Equal protein loading was assessed via Ponceau S staining of total protein content and Western blot analysis with antibodies to total HSP-27 (1:1,000 dilution; BD Transduction Labs). Densitometry of protein bands was performed with a Fluorchem 8800 gel imager.

Statistical analysis. All values are expressed as means ± SE. Differences at various time points of percent functional recovery were compared with a two-way ANOVA for time and treatment with repeated measures followed by a Student's t-test. Myocardial infarct size was compared with a one-way ANOVA followed by a Student's t-test. Western immunoblotting data were compared with a Student's t-test. Statistical significance was determined by a P value <0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of cardiac-specific overexpression of FGF-2 on MAPK signaling. Our laboratory previously reported (23) that cardiac-specific overexpression of FGF-2 in two independent FGF-2 Tg mouse lines resulted in cardioprotection from contractile dysfunction and myocardial infarction after ischemia-reperfusion injury. These FGF-2 Tg mice exhibited a 25- to 35-fold overexpression of FGF-2 in the heart but had only a 2-fold increase in the level of circulating FGF-2 (23). As previously described, FGF-2 Tg mice had no known abnormalities in cardiac or vascular development (23).

To elucidate whether overexpression of FGF-2 primes known cardioprotective pathways such as the MAPK cascade, the activation/phosphorylation states of ERK and p38 were examined in nonischemic FGF-2 Tg and WT hearts. Cardiac-specific overexpression of FGF-2 had no effect on the level of phosphorylation of either ERK or p38 in nonischemic hearts (data not shown), suggesting that there was no priming effect of FGF-2 overexpression on ERK or p38 activation under basal conditions. Because alterations in MAPK signaling have been reported in response to ischemia-reperfusion injury in several models (38) and MAPK signaling is a primary pathway of many of the actions of FGF-2, the effect of overexpression of FGF-2 on MAPK activation in the context of ischemia-reperfusion injury was studied. FGF-2 Tg and WT hearts were subjected to 5, 15, or 30 min of global low-flow ischemia or 30 min of ischemia with 5, 15, or 30 min of reperfusion (Fig. 1A). FGF-2 Tg hearts showed significantly increased ERK phosphorylation compared with WT hearts after 5-min ischemia, 30-min ischemia, or 30-min ischemia followed by 5-min reperfusion (P < 0.05; Fig. 2A). In contrast, FGF-2 Tg hearts showed markedly decreased levels of p38 phosphorylation compared with WT hearts during the first 5 min of either ischemia or reperfusion (P < 0.05; Fig. 2B).



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  		Fig. 2. Time course of MAPK phosphorylation (i.e., activation) in FGF-2 transgenic (FGF-2 Tg) and wild-type (WT) hearts in response to ischemia-reperfusion injury. A: phosphorylation of ERK was significantly increased in FGF-2 Tg hearts at 5 min of ischemia (5'I), 30 min of ischemia (30'I), and 30-min ischemia, 5-min reperfusion (30'I,5'R) compared with WT hearts. 30'I,15'R, 30-min ischemia, 15-min reperfusion; 30'I,30'R, 30-min ischemia, 30-min reperfusion. B: phosphorylation of p38 was decreased significantly in FGF-2 Tg hearts during early ischemia (5'I) and early reperfusion (30'I,5'R) compared with WT hearts. n = 4–12 hearts/group. *P < 0.05 vs. WT at same time point.

 
MEK-ERK pathway mediates FGF-2-induced cardioprotection from global low-flow ischemia-reperfusion injury. Pharmacological inhibitors of the MAPK pathways were administered to FGF-2 Tg and WT hearts during the 60-min global low-flow ischemia and 120-min reperfusion model of ischemia-reperfusion injury to provide direct evidence of whether these signaling pathways are mediating FGF-2-induced cardioprotection (Fig. 1B). U-0126, an inhibitor of MEK1/2 (upstream kinases of ERK), significantly abrogated the recovery of postischemic contractile function after ischemia-reperfusion injury in FGF-2 Tg hearts (86 ± 2% vehicle treated vs. 66 ± 4% U-0126 treated; P < 0.05) without affecting contractile recovery in WT hearts (61 ± 7% vehicle treated vs. 67 ± 7% U-0126 treated; Fig. 3A). In addition, MEK-ERK pathway inhibition resulted in significantly larger myocardial infarcts in FGF-2 Tg hearts (31 ± 2%) compared with vehicle-treated FGF-2 Tg hearts (12 ± 3%, P < 0.05; Fig. 3B). Infarct size in WT hearts was not significantly affected by MEK-ERK inhibition (30 ± 4% vehicle treated vs. 36 ± 7% U-0126 treated). These results suggest that activation of the MEK-ERK signaling pathway is necessary to mediate FGF-2-induced cardioprotection from both postischemic contractile dysfunction and myocardial infarction.



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  		Fig. 3. Effect of MEK-ERK pathway inhibition on FGF-2-induced cardioprotection. A: cardiac-specific overexpression of FGF-2 significantly increased postischemic recovery of derivative of increase in pressure over time (+dP/dt), a measure of systolic function. Inhibition of MEK-ERK activation with U-0126 significantly abrogated FGF-2-mediated recovery of postischemic contractile function. +dP/dtmax, maximum +dP/dt. n = 7–16 hearts/group. B: cardiac-specific overexpression of FGF-2 significantly decreased myocardial infarct size after 60-min ischemia and 120-min reperfusion. Inhibition of ERK abolished FGF-2-induced cardioprotection from myocardial infarction. % Infarct Size, infarct size as a percentage of the whole heart. n = 5–9 hearts/group. *P < 0.05 vs. WT of the same treatment group; {dagger}P < 0.05 vs. vehicle-treated cohort.

 
Inhibition of p38 during ischemia-reperfusion injury protects against myocardial cell death. To assess the necessity of the p38 pathway in FGF-2-induced cardioprotection, an inhibitor of p38, SB-203580, was administered to FGF-2 Tg and WT mice subjected to ischemia-reperfusion injury. Administration of SB-203580 did not significantly affect postischemic contractile recovery in either FGF-2 Tg or WT hearts (Fig. 4A). Inhibition of p38 with SB-203580 also did not significantly affect myocardial infarct size in FGF-2 Tg hearts (12 ± 3% vehicle treated vs. 14 ± 1% SB-203580 treated; Fig. 4B). In contrast, inhibition of p38 did have an effect on infarct size in WT hearts. SB-203580 treatment resulted in significantly smaller myocardial infarcts (30 ± 4% vehicle treated vs. 11 ± 2% SB-203580 treated, P < 0.05; Fig. 4B). Together these results suggest a role for inhibition of p38 by FGF-2 in the determination of infarct size but not in postischemic functional recovery.



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  		Fig. 4. Effect of p38 inhibition on FGF-2-mediated cardioprotection. A: cardiac-specific overexpression of FGF-2 significantly increased postischemic recovery of +dP/dt, but inhibition of p38 with SB-203580 did not reduce this recovery. n = 7–16 hearts/group. B: cardiac-specific overexpression of FGF-2 significantly decreased myocardial infarct size after 60-min ischemia and 120-min reperfusion; however, p38 inhibition did not affect FGF-2's protection against myocardial infarction. p38 inhibition decreased infarct size in WT hearts to levels similar to FGF-2 Tg hearts. n = 4–11 hearts/group. *P < 0.05 vs. WT of the same treatment group; {dagger}P < 0.05 vs. vehicle-treated cohort.

 
Signaling cross talk between ERK and p38 during ischemia-reperfusion injury. Recent studies have demonstrated the existence of signaling cross talk between different protein kinase cascades including MAPK family members (7, 48, 53). Nonetheless, whether interplay between arms of MAPK signaling occurs to elicit cardioprotection, specifically as it relates to FGF-2-induced protection, is unknown. U-0126 or SB-203580 was administered to FGF-2 Tg and WT hearts subjected to ischemia-reperfusion injury. Hearts were arrested during early ischemia or early reperfusion injury because these were the times when the levels of ERK and p38 phosphorylation had been significantly different between FGF-2 Tg and WT hearts (see Fig. 2). ERK or p38 activation (phosphorylation) was determined in the presence of MEK1/2 inhibition, p38 inhibition, or both.

FGF-2 Tg hearts exhibited significantly decreased p38 phosphorylation compared with WT hearts during both early ischemia and early reperfusion injury (P < 0.05; Fig. 5), as seen in previous time-course experiments. Treatment with SB-203580 reduced p38 phosphorylation in WT hearts to FGF-2 Tg levels during both early ischemia and early reperfusion injury, suggesting that the concentration of SB-203580 used in these experiments did inhibit p38. To confirm that SB-203580 inhibited p38 activity, the phosphorylation state of HSP-27, a downstream target of the p38 pathway, was evaluated. Similar to p38, HSP-27 showed a significantly decreased level of phosphorylation in FGF-2 Tg hearts during early ischemia and early reperfusion injury (P < 0.05; Fig. 6). SB-203580 treatment significantly reduced HSP-27 phosphorylation in WT hearts to levels similar to those of FGF-2 Tg hearts (P < 0.05). MEK-ERK pathway inhibition with U-0126 did not significantly affect p38 phosphorylation at either time point, suggesting that p38 activation was not downstream of ERK activation in FGF-2-induced cardioprotection (Fig. 5).



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  		Fig. 5. p38 phosphorylation in response to MEK-ERK or p38 inhibition. A: p38 phosphorylation was significantly decreased in FGF-2 Tg hearts at 5 min of ischemia compared with WT hearts. Treatment with SB-203580 reduced p38 phosphorylation, but MEK-ERK pathway inhibition had no significant effect on p38 activation. Also, coadministration of SB-203580 and U-0126 resulted in significantly reduced p38 phosphorylation, but p38 phosphorylation was not different from that with SB-203580 treatment alone. B: p38 activation was significantly decreased in FGF-2 Tg hearts at 60-min ischemia, 5-min reperfusion. Treatment with SB-203580 reduced p38 phosphorylation in WT hearts similar to FGF-2 Tg levels. MEK-ERK pathway inhibition did not have a significant effect on p38 activation. n = 7–16 hearts/group. *P < 0.05 vs. WT; {dagger}P < 0.05 vs. vehicle treatment.

 


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  		Fig. 6. Phosphorylation of heat shock protein (HSP)-27 in response to p38 inhibition. FGF-2 Tg hearts have significantly reduced phosphorylation of HSP-27, a downstream target of the p38 pathway, at 5-min ischemia (A) and 60-min ischemia, 5-min reperfusion (B). Administration of SB-203580 significantly reduced HSP-27 phosphorylation to levels similar to those of FGF-2 Tg hearts. n = 4–6 hearts/group. *P < 0.05 vs. WT; {dagger}P < 0.05 vs. vehicle treatment.

 
Western blot analysis of FGF-2 Tg hearts showed significantly increased ERK phosphorylation compared with WT hearts during early ischemia and early reperfusion (P < 0.05; Fig. 7), as was seen in the previous time-course experiments. Treatment with U-0126 significantly reduced ERK phosphorylation in both WT and FGF-2 Tg hearts during early ischemia and early reperfusion, as expected. p38 inhibition with SB-203580 did not significantly affect ERK phosphorylation during early reperfusion (Fig. 7B) but significantly increased ERK phosphorylation in WT hearts during early ischemia (P < 0.05; Fig. 7A). These data suggest that p38 inhibits ERK phosphorylation during early ischemia.



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  		Fig. 7. ERK phosphorylation in response to MEK-ERK or p38 inhibition. A: level of ERK phosphorylation was significantly increased in FGF-2 Tg hearts at 5-min ischemia compared with WT hearts. Treatment with U-0126 significantly reduced ERK phosphorylation in both WT and FGF-2 Tg hearts. p38 inhibition with SB-203580 significantly increased ERK phosphorylation in WT hearts to FGF-2 Tg levels. Similarly, coadministration of U-0126 and SB-203580 resulted in significantly increased ERK phosphorylation in WT hearts to the same level as SB-203580 treatment alone. B: FGF-2 Tg hearts have increased ERK phosphorylation at 60-min ischemia, 5-min reperfusion compared with WT hearts. Treatment with U-0126 significantly reduced ERK phosphorylation in both WT and FGF-2 Tg hearts. p38 inhibition with SB-203580 did not significantly affect ERK phosphorylation during early reperfusion. n = 4–11 hearts/group. *P < 0.05 vs. WT; {dagger}P < 0.05 vs. vehicle treatment.

 
To ascertain whether this inhibitory effect of p38 was MEK1/2 dependent, FGF-2 Tg and WT hearts were subjected to 5-min ischemia in the presence of the MEK1/2 inhibitor U-0126 and the p38 inhibitor SB-203580. p38 phosphorylation was significantly reduced in these hearts, similar to results seen with treatment of SB-203580 alone (P < 0.05; Fig. 5A). The administration of both U-0126 and SB-203580 resulted in ERK phosphorylation in WT and FGF-2 Tg hearts that was not significantly different from that seen with SB-203580 treatment alone (Fig. 7A), suggesting that the effect of p38 inhibition on ERK phosphorylation was MEK1/2 independent. Together, these data indicate that during early ischemia, p38 inhibition resulted in increased ERK activation through a MEK-independent mechanism, whereas there did not appear to be cross talk from p38 to ERK during early reperfusion.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study has demonstrated that overexpression of FGF-2 in the heart results in cardioprotection from ischemia-reperfusion injury through alterations in MAPK signaling. Increased activation of ERK and decreased activation of p38 have been shown to occur in FGF-2 Tg hearts during early ischemia and early reperfusion injury (Fig. 2). These data are consistent with other reports showing ERK and p38 activation throughout both ischemia and reperfusion injury (35, 44, 50). There is evidence that FGF-2 regulates ERK and p38 activation in multiple tissues (9, 46). This is, however, the first report of activation of ERK and inhibition of p38 by FGF-2 in the heart in response to ischemia-reperfusion injury.

In the present study, pharmacological inhibitors of the MAPK cascade were used to elucidate the necessity of this pathway for FGF-2-induced cardioprotection. Administration of U-0126 (2.5 µmol/l), a noncompetitive inhibitor of MEK1/2 (kinases upstream of ERK), resulted in significantly reduced recovery of postischemic contractile function (Fig. 3A) and significantly increased myocardial infarct size (Fig. 3B) in FGF-2 Tg hearts. The specificity of U-0126 for inhibition of the MEK-ERK signaling cascade has been well established (11), and inhibition of ERK phosphorylation by U-0126 during ischemia-reperfusion injury was confirmed (Fig. 7). These data, together with the increased phosphorylation of ERK seen in FGF-2 Tg hearts subjected to ischemia-reperfusion injury (Fig. 2A), implicate activation of the MEK-ERK pathway by FGF-2 during ischemia-reperfusion injury in protection against contractile dysfunction and myocardial cell death. Consistent with our findings, other studies have demonstrated a role for ERK in reduction of ischemia-reperfusion damage both in vitro (52) and in vivo (42) and have shown that the ERK pathway mediates cardioprotection elicited through ischemic preconditioning (42) and several types of pharmacological preconditioning including opioid treatment (16). Until now, however, the necessity for MEK-ERK pathway activation for FGF-2-induced cardioprotection has been unknown.

Administration of SB-203580 (2 µmol/l) in our model of global low-flow ischemia-reperfusion injury was performed to assess the necessity of p38 signaling for FGF-2-induced cardioprotection. SB-203580 was originally reported to be a p38 inhibitor (8), but recent studies have shown that it can also inhibit JNK (6), which has also been implicated to be involved in cardioprotection (26). The concentration of SB-203580 used in our experiments, however, is lower than the smallest concentration of SB-203580 shown to inhibit JNK in these previous studies, suggesting that any inhibitory effect on JNK would be negligible compared with the p38 inhibition by SB-203580 in our model. Treatment of FGF-2 Tg and WT hearts with the p38 inhibitor SB-203580 did not significantly alter postischemic contractile function (Fig. 4A), suggesting that alterations in p38 signaling do not mediate FGF-2's protective effect against ventricular dysfunction. Similarly, p38 inhibition did not alter myocardial infarct size in FGF-2 Tg hearts compared with vehicle-treated FGF-2 Tg hearts. In contrast, WT hearts treated with SB-203580 showed significantly decreased infarct size compared with vehicle-treated WT hearts (Fig. 4B). These data suggest that p38 may actually be inhibited by FGF-2 overexpression, resulting in protection against myocardial cell death. Indeed, FGF-2 Tg hearts did have significantly decreased p38 phosphorylation during early ischemia and early reperfusion compared with WT hearts (Fig. 2B). The use of SB-203580 may have reduced p38 activity in WT hearts to basal levels similar to those seen in FGF-2 Tg hearts during ischemia-reperfusion injury, which would reduce myocardial infarct size in WT hearts to FGF-2 Tg levels. Consistent with this hypothesis, SB-203580 administration resulted in decreased p38 phosphorylation in WT hearts similar to the level of p38 phosphorylation in FGF-2 Tg hearts during early ischemia and early reperfusion (Fig. 5). SB-203580 has been shown to inhibit the catalytic activity of p38 by occupying the ATP-binding pocket without effecting MKK-dependent phosphorylation of p38 (20). Our data, which demonstrated a decreased level of p38 phosphorylation in WT hearts during ischemia-reperfusion injury in response to SB-203580 administration, suggest an autophosphorylation mechanism of p38. Recent studies have shown MKK-independent, SB-203580-sensitive phosphorylation of p38 resulting from intramolecular p38 autophosphorylation (17, 47). This type of autophosphorylation seems to be stimulus specific, but there is some evidence that it may occur during myocardial ischemic damage (33), as our data suggest. With the efficacy of SB-203580 in p38 pathway inhibition confirmed, HSP-27 phosphorylation was significantly reduced in WT hearts subjected to p38 inhibition to levels similar to those of FGF-2 Tg hearts (Fig. 6).

In the present study, a protective effect of p38 inhibition during early ischemia and early reperfusion injury downstream of FGF-2 was observed and resulted in the reduction of myocardial infarct size. However, with respect to the best studied form of cardioprotection, ischemic preconditioning, controversy exists as to the role of p38 in ischemia-reperfusion injury such that studies have identified either a cardioprotective effect or a deleterious effect of p38 (41). These conflicting reports are likely due to several factors, including the timing of the administration of p38 inhibitors and the known nonspecific inhibitory activity of the SB compounds at high concentrations, which could account for some of these discrepancies. In addition, there are four different isoforms of p38 of which p38{alpha} and p38{beta} have been shown to be expressed in cardiac tissue with a preponderance of p38{alpha} in murine (49) and human (27) hearts. These isoforms have been reported to have different biological functions, with p38{alpha} having proapoptotic actions and p38{beta} having a prosurvival and progrowth function (49). Differential activation of these opposing p38 isoforms may also contribute to the confusion over the role of p38 in the preconditioning literature. Some studies have shown that ischemia-reperfusion primarily activates p38{alpha} and inhibits p38{beta} (4, 39). The mechanism for p38 activation may also influence whether its effect is protective or deleterious. The autophosphorylation of p38, which likely occurs at least to some extent in our model, has been shown to result in increased myocardial cell death (47).

Consistent with our data, several studies have identified a protective effect of p38 inhibition in cardiac ischemia-reperfusion injury. In cardiomyocytes subjected to simulated ischemia, p38 inhibition reduced lactate dehydrogenase release and myocyte apoptosis, suggesting that p38 inhibition results in increased cardiomyocyte viability after ischemia (32). In addition, p38 inhibition has been shown to improve postischemic contractile function and reduce myocyte apoptosis and necrosis in isolated rat hearts (31, 40) and to reduce infarct size in pig myocardium in vivo (2). The present data show a relationship between p38 inhibition and reduction in infarct size but no effect of p38 inhibition on postischemic contractile recovery (Fig. 4). It was shown in another mouse model of low-flow ischemia that p38 inhibition does not abrogate myocardial stunning, which is consistent with our own findings (19). In addition, p38 inhibition has been shown in isolated cardiomyocytes to have a positive inotropic effect (28). SB-203580 treatment did not, however, alter baseline contractility in sham-treated hearts in our model (data not shown). Contractile status during early reperfusion injury has been suggested to affect postischemic myocardial cell death (43). In isolated rat hearts, p38 inhibition has been shown to result in both increased postischemic contractility and increased myocardial infarction. Reduction of this increased contractility during early reperfusion with a {beta}-adrenergic blocker or a contractile blocker (2,3-butanedione monoxime) resulted in reduced infarct size, suggesting that enhanced contractility during reperfusion may increase myocyte susceptibility to cell death. This proposed dichotomy between contractility and cell viability may also be an important factor in the discrepancies seen within the cardioprotection literature with respect to the role of p38 inhibition.

Our data have revealed that FGF-2 overexpression causes activation of ERK and inhibition of p38 during ischemia-reperfusion injury, leading to cardioprotection from postischemic contractile dysfunction and myocardial infarction. The substrates of ERK and p38 that mediate these effects are unknown, but there are several possibilities. On activation, both ERK and p38 are known to translocate to the nucleus and interact with several transcription factors to affect gene transcription. This mechanism is unlikely to be mediating the cardioprotection observed in FGF-2 Tg hearts because there was no difference in MAPK signaling in nonischemic FGF-2 Tg hearts compared with WT hearts (data not shown). Signaling alterations were only seen during ischemia-reperfusion injury (Fig. 2), suggesting that the effects of MAPK signaling in our model are more acute and are not due to differences in gene transcription. ERK and p38 are also known to cause posttranslational modifications of several proteins, which might play a role in FGF-2-induced cardioprotection. For example, activated ERK can phosphorylate and activate p90 ribosomal S6 kinase (14), which then phosphorylates and deactivates proapoptotic proteins such as BAD (5, 45). In addition, ERK can phosphorylate cytoplasmic phospholipase A2, resulting in increased arachidonic acid and lysophospholipid production and in the triggering of other cardioprotective signal transduction cascades (29). Both ERK and p38 are known to phosphorylate MAPK-associated protein 2, which in turn phosphorylates the cardioprotective molecule HSP-27 (13). Activated p38 has been shown to induce apoptosis by inducing cleavage of caspase-3 and Bid (54) and causing Bax translocation to mitochondria (18). Elucidation of the specific substrates acted on by ERK and p38 to elicit FGF-2-induced cardioprotection is beyond the scope of this manuscript but is the subject of future studies.

In vitro studies in HEK-293 cells, HeLa cells, and macrophages have implicated the existence of cross talk between several members of the MAPK cascade (51, 53). Many of these studies have shown reciprocal regulation of ERK and p38 in which inhibition of one of the signaling proteins enhances activation of the other. There is, however, some in vitro evidence that p38 activation may cause ERK activation (48). It was recently proposed that the discrepancies in the nature of the cross talk between ERK and p38 are likely due to agonist-specific differences in ERK and p38 cross talk (22). Our data showed no significant effect of MEK-ERK pathway inhibition on p38 activation during ischemia-reperfusion injury (Fig. 5). There was, however, an increase in ERK phosphorylation in SB-203580-treated WT hearts during early ischemia, which was MEK independent (Fig. 7A). It has been suggested that a direct interaction exists between phosphorylated p38 and ERK, resulting in ERK deactivation (53), and that this deactivation is mediated, in isolated cardiomyocytes, by protein phosphatase 2A (30). Inhibition of p38 autophosphorylation with SB-203580 in our model may disrupt this interaction, resulting in increased ERK phosphorylation in WT hearts. This increased ERK activation may also contribute to the reduction in myocardial infarct size seen in WT hearts subjected to p38 inhibition (Fig. 4).

The present study has demonstrated involvement of the MAPK cascade in FGF-2-induced cardioprotection from ischemia-reperfusion injury, but it is possible that other signaling cascades may also mediate this effect. FGF-2 has also been shown to signal through the PKC and nitric oxide synthase (NOS) pathways (3, 36). PKC and inducible NOS have also been shown to be involved in postischemic recovery of contractile function in hearts treated with recombinant FGF-2 (21, 24). These pathways may also mediate cardioprotection resulting from cardiac-specific overexpression of FGF-2, but this topic is beyond the scope of the current manuscript and will be the subject of future studies.

In summary, our findings have unequivocally demonstrated, for the first time, the necessity of the MAPK cascade in FGF-2-induced cardioprotection from ischemia-reperfusion injury. Cardiac-specific overexpression of FGF-2 has been shown to increase ERK phosphorylation at specific time points of ischemia-reperfusion injury, resulting in decreased postischemic contractile dysfunction and myocardial infarction (Fig. 8). FGF-2 also inhibited p38 activation, causing reduction in myocardial cell death. Furthermore, cross talk between p38 and ERK occurred during early ischemic injury that was MEK independent, which may contribute to the cardioprotective actions of FGF-2. In association with clinical trials of FGF-2 to assess the therapeutic potential of angiogenesis in patients with coronary artery disease, these data may aid in the development of FGF-2 as a cardioprotective therapy independent of angiogenic effects or lead to the elucidation of novel therapeutic strategies for patients with ischemic heart disease.



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  		Fig. 8. Schematic of roles of the MAPK cascade in FGF-2-induced cardioprotection. During ischemia-reperfusion injury, overexpression of FGF-2 results in activation of the MEK-ERK pathway, resulting in increased recovery of postischemic contractile function and decreased myocardial infarct size. At the same time, overexpression of FGF-2 in the heart reduces p38 activation, resulting in decreased myocardial infarction. During early ischemia, p38 negatively regulates ERK activation through a MEK1/2-independent mechanism.

 

   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the American Heart Association (230044N), the Pharmaceutical Research and Manufacturers of America Foundation (Research Starter Grant), and the National Heart, Lung, and Blood Institute (RO1-HL-075633-01A), awarded to J. J. Schultz.


   ACKNOWLEDGMENTS
 
The investigators acknowledge M. Bender and A. Whittaker for excellent animal husbandry work and J. O'Toole and D. Porter for mouse genotyping.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. J. Schultz, Dept. of Pharmacology and Cell Biophysics, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, ML 0575, Cincinnati, OH 45267 (e-mail: schuljo{at}email.uc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 DISCUSSION
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