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Cardiovascular Research 2002 55(4):768-777; doi:10.1016/S0008-6363(02)00494-7
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Copyright © 2002, European Society of Cardiology

Angiogenesis-independent cardioprotection in FGF-1 transgenic mice

Alexandra Buehlera, Alessandra Martirea,1, Claudia Strohma,1, Swen Wolframa, Borja Fernandeza, Meindert Palmenb, Xander H.T Wehrensb, Pieter A Doevendansb, Wolfgang M Franzd, Wolfgang Schapera and René Zimmermannc,*

aDepartment of Experimental Cardiology, Max-Planck-Institute, D-61231 Bad Nauheim, Germany
bCardiovascular Research Institute Maastricht, 6202 AZ Maastricht, The Netherlands
cDepartment of Vascular Genomics, Kerckhoff Clinic, Benekestr. 2–8, D-61231 Bad Nauheim, Germany
dMedizinische Universitätsklinik, D-23538 Lübeck, Germany

* Corresponding author. Tel.: +49-6032-996-2801; fax: +49-6032-996-2809 r.zimmer{at}vascular-genomics.de

Received 17 October 2001; accepted 13 May 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: This study was performed to evaluate the cardioprotective role of acidic fibroblast growth factor-1 (FGF-1) in transgenic mice with cardiac-specific overexpression of human FGF-1. Methods: Mice were subjected to coronary artery occlusion for 15–75 min with a continuously recorded 3-lead electrocardiogram (ECG). Infarct size was measured and ERK-1 and -2 activity was assessed by Western blot analysis. Creatine kinase and lactate dehydrogenase activity as marker for cell viability were measured in isolated ventricular myocytes subjected to simulated ischemia. Results: Infarct development was markedly delayed in transgenics with first signs of myocardial infarction visible at 45 min after coronary artery occlusion compared to 15 min in wildtype. Maximal infarct size (60% of risk area) did not differ, but transgenics reached maximal infarction after 75 min compared to 45 min in wildtype animals. ECG revealed delayed Q-wave development and delayed ST-segment elevation in transgenics. Creatine kinase and lactate dehydrogenase release was significantly attenuated from isolated transgenic myocytes at 4 and 8 h after simulated ischemia. The delay in infarct development is partially due to a constitutive higher expression of the extracellular signal-regulated kinases ERK-1 and -2 in the myocardium of transgenics. Additionally, injection of the ERK-1/2 inhibitor UO126 decreased the cardioprotective effect of FGF-1. Conclusion: Cardiac specific overexpression of FGF-1 provides cardioprotection at the level of the cardiac myocyte, independent from angiogenesis, and at least partially mediated via activation of the mitogen activated protein kinase (MAP) ERK-1 and -2.

KEYWORDS Ventricular function; Hemodynamics; Signal transduction; Ischemia; ECG


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The term ischemic preconditioning refers to the limitation or the delay of myocardial cell death following coronary occlusions that precede an occlusion sufficiently long to produce near total necrosis of the area at risk. Ischemic preconditioning is considered as an endogenous protection against myocardial infarction [1]. Several agonists or inhibitors were shown to mediate cardioprotection via different pathways [1–3], mimicking ischemic preconditioning. It was reported that local and systemic infusion of FGF-1 exerts an anti-ischemic effect that is comparable to the protection obtained by ischemic preconditioning [4,5]. Renaud et al. reported that in contrast to exogenous FGF-1, endogenous FGF-1 is not a mitogen but a survival factor [6].

The best known members of the FGF family are acidic FGF [7,8] and basic FGF (reviewed in Ref. [9]). FGFs play a role in numerous aspects of embryogenesis, angiogenesis, growth, and cell survival [10–13].

Previously, we reported the generation of transgenic mice (TG) with cardiac specific overexpression of the human FGF-1 gene. These mice showed significantly more arterioles and more branchings of the main coronary arteries, had a normal lifespan and fertility and lacked cardiac malformations [14]. Transgenic animals were subjected to in vivo regional myocardial ischemia and isolated ventricular cardiac myocytes to in vitro simulated ischemia in order to determine if permanent overexpression of FGF-1 increases cardiac tolerance towards ischemia by either an angiogenic/arteriogenic or a non-vascular mechanism. On one hand, the increased density of arterioles might result in a higher coronary collateral blood flow which could lead to a decrease in maximal infarct size. On the other hand, earlier experiments in pigs showed that intramyocardial infusion of FGF-1 and FGF-2 mimicked the effect of ischemic preconditioning in the myocardium [5]. FGFs, other peptide growth factors and Gq protein-coupled receptor agonists stimulate activation of ERKs in ischemic myocardium [15]. Therefore, we hypothesized a cardioprotective role of the ERK cascade in our model and studied the effect of administration of the MEK inhibitor UO126 on cardioprotection.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
All animal studies were approved by the Bioethical Committee of the District of Darmstadt, Germany. Animals were handled in accordance with the American Physiological Society Guidelines for Animal Welfare and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

The transgenic mice (TG) have been characterized and described recently [14]. For all experiments adult male FGF-1 TG (12–14 weeks) of lines L1 and L2 were used. Wildtype (WT) mice (12–14 weeks) from the same litter were used as controls. Numbers of animals for each experiment are indicated in the Results section.

Mice were anesthetised with an intraperitoneal injection of 0.1 mg/g bodyweight ketaminhydrochloride (Ketamin® 10%) and 0.02 mg/g bodyweight xylazinhydrochloride (Xylazin® 2%) (both from Medistar, Holzwickede, Germany) and artificially ventilated. Anesthesia was maintained via supplemental doses of this combination as needed. A 3-lead electrocardiogram was recorded continuously.

To measure blood pressure a 1.4F Millar Tip-Catheter was inserted into the aorta via the right carotid artery. Between the insertion of the tip catheter and the operation, the mouse was allowed to recover for 20 min. Blood pressure was recorded from the beginning until 30 min after coronary artery occlusion as described previously (Table 1) [16]. After an ischemic period of 15, 30, 45, 60 or 75 min the ligature was removed followed by reperfusion for 20 min. Finally, the coronary artery was reoccluded.


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  		Table 1 Mean arterial blood pressure (MABP) and heart rate from all groups starting at the beginning of the operation (baseline) until 30 min of ischemia

 
To determine infarct size we deviated from the classical triphenylterazolium chloride measurement and used propidium iodide (PI) instead because long reperfusions which are necessary with triphenylterazolium chloride can be avoided with propidium iodide, a nuclear stain, which can enter the cell only when the sarcolemma is irreversibly damaged. The superiority of this method was demonstrated in two recent papers by other authors [17,18].

Infarct area (IA) was assessed by injection of 0.125 ml PI 0.05% (m/v) (Sigma, Munich, Germany) at the onset of reperfusion. A 20-min period of reperfusion is long enough for propidium iodide to label irreversibly damaged cells. To delineate the perfused area of the left ventricle (LV) and, by exclusion, the area at risk (RA) (ischemic but not necessarily infarcted myocardium) 0.125 ml thioflavinS 5% (m/v) (Sigma) were injected after reocclusion (Fig. 1A) [19,20]. The heart was excised, the right ventricle removed and the LV frozen in liquid nitrogen. Cryosections of 16 µm were cut and propidium iodide/thioflavinS stainings were analyzed with a fluorescence microscope (Leica DM-RB). The IA, RA, and LV were measured by planimetry using the NIH Image 1.62 program.


Figure 1
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  		Fig. 1 Protocol of the in vivo myocardial ischemia experiments. (A) After occlusion of the left coronary artery (15–75 min) the artery was reperfused for 20 min and reoccluded afterwards. To determine the RA and the IA, propidium iodide and thioflavinS were injected directly after reperfusion or reocclusion, respectively. (B) In the second set of experiments UO126 in KHB/DMSO or KHB/DMSO alone was injected 30 min prior an ischemic period of 45 min. (C) Myocardial biopsies for Western blot analysis were taken after 10 min of ischemia only. (D) UO126 in KHB/DMSO or KHB/DMSO alone was injected 30 min prior to an ischemic insult. Myocardial biopsies for Western blot analysis were taken after 10 min of ischemia.

 
In order to evaluate the cardioprotective effect of FGF-1 without the possible influence of the vasculature, in vitro experiments with isolated ventricular myocytes were performed. Ventricular cardiac myocytes of transgenic and control mice were isolated as previously described for rats [21] with minor changes: cardiac myocytes were plated in 35-mm culture dishes (Falcon, Heidelberg, Germany), coated with 5 µg/ml laminin (Sigma) and 10 µg/ml of fibronectin (PromoCell, Heidelberg, Germany). No fetal calf serum was added to the culture medium. Ischemia of adult mouse ventricular cardiac myocytes was simulated by modifying experimental protocols described for different species [22–25]. In brief, dishes were washed three times with a glucose-free modified tyrode solution (110 mM NaCl, 2.6 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1 mM CaCl2, 10 mM HEPES, pH 7.4), placed in gas tight chambers and incubated at 37 °C in an environment of 95% N2 and 5% CO2 for 4, 8, and 12 h. Reoxygenation was achieved by switching to cell culture medium and incubation with 21% O2, 74% N2, and 5% CO2 for 4 h. Time matched control myocytes were incubated in an environment of 21% O2, 74% N2, and 5% CO2. The release of lactate dehydrogenase and creatine kinase was used as an indicator for loss of cell membrane integrity and as a sign of cell death. The activities of these enzymes in the supernatant were determined at normoxia and after simulated ischemia, reoxygenation and lysis of the cells with 1% Triton X-100. The release of enzymes was calculated as the percentage of activity after ischemia/reoxygenation versus total activity (simulated ischemia+reoxygenation+Triton X-100 extracts).

To study the role of ERK-1/2 in the cardioprotection by FGF-1, 500 µl of the MEK inhibitor UO126 (Promega, Heidelberg, Germany; 0.25 mg/kg body weight in Krebs-Henseleit-buffer/DMSO) or of Krebs-Henseleit-buffer/DMSO (KHB/DMSO) (0.5%) were injected in the ventral tail artery of TG and WT, groupwise 30 min before coronary artery occlusion. The ischemic period was either 10 min without reperfusion for Western blot analysis of ERK activity (Fig. 1C,D; see below) or 45 min, followed by 20 min of reperfusion, for measurement of infarct size (Fig. 1B).

From mice not undergoing IA determination (animals treated with UO126 or KHB/DMSO alone or animals without any treatment) myocardial biopsies were taken after 10 min of ischemia from the ischemic and the non-ischemic part of the LV or, in the case of non-occluded hearts, from the LV (Fig. 1C,D). The soluble fractions were prepared as reported previously [26]. They were subjected to Western blot analysis using a polyclonal anti-phospho-p44/42-MAPK antibody (New England BioLabs, Schwalbach/Taunus, Germany) and a polyclonal anti-ERK-2 (C-14) antibody (Santa Cruz Biotechnology, Heidelberg, Germany). The secondary antibody was a peroxidase labelled anti-rabbit immunoglobulin (Amersham, Freiburg, Germany). Bound antibodies were detected by the enhanced chemiluminescense Western blot detection method (Amersham, Freiburg, Germany).

2.1 Statistical analyses
All data are represented as mean±standard error of the mean (S.E.M.). Bonferroni-adjustments and pairwise mean differences (ANOVA) or Kruskal–Wallis test and Dunn's method were used to analyse the differences between experimental groups. A P-value smaller than 0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Myocardial ischemia
No differences in the ratio RA/LV could be detected between all tested groups. Despite similar-sized RA, WT hearts showed a significantly larger IA/RA-ratio after coronary occlusion compared with the TG for each time point studied (Fig. 2A,B). No differences could be observed among the transgenic lines. In WT infarctions were already histologically visible after 15 min of ischemia (IA/RA 9.06±1.41; n = 5) (Fig. 3A) whereas in TG similar values were observed after 45 min of ischemia (10.08±1.93 and 10.57±1.74 for L1 (n = 5) and L2 (n = 3), respectively). In WT maximal IA/RA was reached after 45 min of ischemia (57.76±1.78; n = 5), whereas TG reached comparable maximum values after 75 min (63.12±3.81, n = 5; 66.67±3.34, n = 3). Longer duration of ischemia in WT (60 min) showed no further increase in IA/RA ratio (IA/RA 63.73±1.47; n = 3) (Fig. 3A), designating the infarct size at these timepoints as maximal infarct size.


Figure 2
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  		Fig. 2 Heart sections of a wildtype and an FGF-1 transgenic mouse. LV, RA and IA are delineated by thioflavinS (TS) and propidium iodide (PI) staining in a representative midventricular heart section of a WT mouse after 45 min of ischemia (A) compared to a heart from a transgenic animal (B). Areas that stain blue (TS) are not at risk. Areas that appear red (PI) are infarcted. Areas that remain black are not infarcted but are within the risk area.

 

Figure 3
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  		Fig. 3 FGF-1 transgenic animals show enhanced cardioprotection. (A) IA versus RA in WT and TG mice (L1, L2). Data are represented in %. Transgenics reach maximum IA related to RA, but delayed when compared to WT mice. No significant differences could be observed between the different transgenic mouse lines. (B) IA versus RA in WT and TG mice (L1), either receiving UO126 or not. Data are represented in %. Injection of UO126 in TG leads to a significant reduction of cardioprotection after 45 min of ischemia. IA/RA remains significant smaller in TG mice compared to WT, both with or without UO treatment.

 
Injection of UO126 or KHB/DMSO in WT or TG of L1 (four animals each) did not alter the ratio RA/LV. Furthermore, in WT treatment with UO126 did not significantly affect the ratio IA/RA at 45 min of coronary artery occlusion (WT/UO126: 65.55±4.87; WT/–: 57.76±1.78). However, a significant increase in IA/RA could be seen at the same timepoint in TG following application of UO126 (TG/UO126: 25.15±4.4) compared to untreated TG (TG/–: 10.08±1.93; Fig. 3B). Application of KHB/DMSO without UO126 had no influence on RA/LV or IA/LV in TG (data not shown).

3.2 Simulated ischemia
In vitro experiments of simulated ischemia showed that creatine kinase release of transgenic myocytes was significantly decreased at 4 and 8 h of simulated ischemia when compared to wildtype myocytes (10.86±1.38 vs. 33.14±2.26% and 42.33±1.99 vs. 60.48±2.95%, respectively; Fig. 4A). Similarly, lactate dehydrogenase release of transgenic myocytes was also decreased at 4 and 8 h of simulated ischemia when compared to wildtype myocytes (33.59±2.51 vs. 58.64±3.02% and 73.94±3.18 vs. 86.87±3.46%, respectively; Fig. 4B). At 12 h of simulated ischemia the release of both enzymes from transgenic myocytes was not significantly different when compared to enzyme release from wildtype myocytes. Under normoxic conditions the release of creatine kinase and lactate dehydrogenase did not change significantly during the 16-h experimental protocol.


Figure 4
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  		Fig. 4 Cardioprotection in isolated myocytes. Release of creatine kinase (A) and lactate dehydrogenase (B) from transgenic and wildtype myocytes after 4, 8 and 12 h of simulated ischemia followed by a reoxygenation period of 4 h. Data are means±S.E.M. of six independent culture preparations. *P<0.05 versus wildtype myocytes; **P<0.001 versus wildtype myocytes.

 
3.3 Hemodynamics
Mean arterial blood pressure was measured in seven WT, six L1 and four L2 mice (Table 1).

Baseline mean arterial blood pressure (represented in mmHg) was measured after an equilibration period of 20 min. At this timepoint significant differences (P<0.05) between the different lines could be observed (121.17±5.51, 90.36±3.41 and 90.56±5.96 for WT, L1 and L2 mice, respectively). Furthermore, significant differences within the different lines were observed between the timepoints ‘baseline’ and ‘before coronary artery occlusion’. The timepoint ‘before coronary artery occlusion’ was recorded after the ligature had been put in place but not tied.

During coronary artery occlusion L1 and L2 mice showed lower mean arterial blood pressure than WT mice. Although statistically significant differences could be observed between lines L1 or L2 and WT at two time points, this could not be related to infarct size development. This is also true for the heart rate.

3.4 Electrocardiograms
A surface ECG (corresponding to lead II) was recorded continuously throughout the whole experiment. The most prominent ECG feature of coronary occlusion was a 2- to 3-fold increase in R-wave amplitude occurring within seconds after coronary artery occlusion. This pattern was comparable for WT and TG (Fig. 5A), although the R-wave amplitude remained increased for a longer period of time in TG (P<0.05).


Figure 5
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  		Fig. 5 Changes in mean R-wave amplitude (A) and ST-segment (B) during ischemia. (A) After coronary artery occlusion a 2- to 3-fold increase in R-wave amplitude height occurred within seconds. The R-wave height remained increased for a longer period of time in transgenic animals (*P<0.05). {dagger}P<0.05 versus coronary occlusion; {ddagger}P<0.05 versus baseline. (B) FGF-1 transgenic animals showed a delayed and prolonged ST-segment elevation compared to WT animals (*P<0.05). {dagger}P<0.05 versus coronary occlusion; {ddagger}P<0.05 versus baseline.

 
In addition, TG showed a delayed ST-segment elevation compared to WT animals (Fig. 5B). In WT mice, ST-segment elevation was already seen after 1 min of coronary artery occlusion while in TG the ST-segments were typically decreased or iso-electric after 1 and 5 min, followed by ST-segment elevation starting from 10–15 min of coronary artery occlusion. Whereas partial ST-segment normalization was observed in WT after 45 min of coronary artery occlusion, prolonged ST-segment elevation was observed in TG. In addition, the development of Q-waves was also delayed in the FGF-1 transgenic mice, occurring typically after 15 min in WT compared to 30 min in TG. Finally, there were no significant differences in the magnitude and time-course of ECG changes during coronary artery occlusion between the different transgenic lines L1 and L2.

3.5 Effect of FGF-1 on the activity of ERK-1/2
To evaluate the effect of FGF-1 on the activity of the MAP kinases ERK-1/2 myocardial biopsies were taken from TG of line L1 (n = 8) and WT (n = 8) after 10 min of ischemia and from TG (n = 8) and WT (n = 6) not subjected to coronary artery occlusion. Western blot analysis with phospho-ERK specific (Thr202/Tyr204) antibodies showed a higher amount of P-ERK-1 and P-ERK-2 (2.3- and 2.9-fold, respectively) in the LV of L1 compared to WT. Similar results were found in both transgenic lines (data not shown). Following 10 min of coronary artery occlusion levels of P-ERK-1 and -2 were significantly increased in WT and TG compared to untreated controls (P-ERK-1: 1.5-fold (WT) and 3.6-fold (TG); P-ERK-2: 2.6-fold (WT) and 4.4-fold (TG); Fig. 6).


Figure 6
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  		Fig. 6 Activity of MAP-Kinases ERK-1 and ERK-2. (A) Western blot assay of TG (L1) and WT myocardium of control and ischemic tissue (all experiments without drug- and solvent-infusions). The non-phosphorylated form of ERK-1/2 indicates equal loading of the gel. (B,C) Quantitative Western blot analysis (mean±S.E.M.; *P<0.05) of protein extracts from hearts of WT and TG with or without ischemia, using the phospho-specific ERK-1 (B) and ERK-2 (C) antibody. Data are expressed as percentage of the corresponding WT tissue without ischemia. 1=LV WT: control tissue of LV from a wildtype animal; 2=RA WT+Isch: tissue of the risk-area of a wildtype animal after 10 min of ischemia; 3=LV trans: tissue of the LV of transgenic animals; 4=RA trans+Isch: tissue of RA from FGF transgenic myocardium after 10 min of ischemia. The arrows on the right indicate the position of P-ERK-1 and -2.

 
In a second set of experiments we tested the effect of the MAP kinase inhibitor UO126 on ERK-1/2 activity in these animals. TG and WT (n = 4 for each group) were injected with either KHB/DMSO (KD) alone or with UO126 (0.25 mg/kg body weight in KHB/DMSO) and subjected to 10 min of ischemia as described in Methods. Animals without ischemia served as control. From the results it became obvious that treatment with UO126 abrogates nearly completely the increase of P-ERK-1 and -2 seen in TG following ischemia and KHB/DMSO treatment (P-ERK-1: 3.8-fold (TG+KD/ischemia) and 1.9-fold (TG+UO/ischemia); P-ERK-2: 4.7-fold (TG+KD/ischemia) and 2.6-fold (TG+UO/ischemia), both compared to WT without ischemia+KD; Fig. 7).


Figure 7
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  		Fig. 7 Influence of ischemia and UO126 on phosphorylation of MAP-Kinases ERK-1 and ERK-2. (A) Representative blot showing the effect of the application of UO126 (UO) and KHB/DMSO (KD) on ERK-1/2 phosphorylation in the heart of L1 transgenics (TG) and wildtype mice (WT). 1=LV WT+KD: control tissue of LV from WT after KHB/DMSO infusion; 2=LV trans+KD: tissue of the left ventricle of TG after KHB/DMSO infusion; 3=RA WT+KD+Isch: tissue of the risk-area of WT after 10 min of ischemia and KHB/DMSO infusion; 4=RA trans+KD+Isch: tissue of RA from FGF transgenic myocardium after 10 min of ischemia and KHB/DMSO infusion; 5=RA WT+UO126+Isch: tissue of the risk-area of WT after 10 min of ischemia and UO126 infusion; 6=RA trans+UO126+Isch: tissue of the risk-area of TG after 10 min of ischemia and UO126 infusion; 7=LV WT+UO126: tissue of left ventricle from WT after UO126 infusion; 8=LV trans+UO126: tissue of left ventricle from TG after UO126 infusion. The arrows on the right indicate the position of P-ERK-1 and -2. The non-phosphorylated form of ERK-1/2 indicates equal loading of the gel. (B,C) Effect of the application of UO126 (UO) and KHB/DMSO (KD) on ERK-1 (B) and ERK-2 (C) phosphorylation in the heart of TG and WT. Quantitative Western blot analysis was performed by using Phosphorimager SF (Molecular Dynamics). The data (mean±S.E.M.; *P<0.05) were obtained from Western blot assays performed after UO126 (0.25 mg/kg) or KHB/DMSO infusions with or without ischemia, using the phospho-specific ERK-1/2 antibody. They are expressed as percentage of the corresponding WT tissue after KHB/DMSO infusion.

 
In both experimental settings significant changes in the protein levels of unphosphorylated ERK-1/2 were not found.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Overexpression of FGF-1 in the heart by generation of transgenic mice expressing the FGF-1 cDNA under the heart-specific MLC2v promoter resulted in increased tolerance against ischemia, delayed infarct expansion and in constitutive activation of the ERK-1/2 pathway. Whereas maximal infarct size following coronary artery occlusion and reperfusion was reached after 45 min in WT, 75 min were necessary to obtain the same effect in TG. Eventually, the same infarct size was reached in both, WT and TG, despite the fact that TG had a denser arteriolar network [14].

FGF-1 is a multifunctional peptide with angiogenic and tissue protective effects [11–13,27–29]. FGF-1 is constitutively expressed in the normal heart at low levels and its expression is upregulated in chronic ischemia following cardiac microembolization in the pig [30]. Htun et al. were the first to show a cardioprotective effect of exogenous FGF-1 in vivo that was receptor mediated in contrast to the uptake into the nucleus which was not receptor dependent and was not associated with protection [5]. In view of the fact that endogenous FGF-1 does not have a leader sequence enabling secretion and that previous experiments had utilized only exogenous FGF-1 that binds to a receptor on the outside of the cell membrane, it appeared of interest to study overexpressed intracellular FGF-1. The generation and phenotype of FGF-1 transgenic mice was described recently by our group [14]. Transgenic hearts showed unchanged capillary but increased arteriolar density [14]. This increase represents more collateral connections and branches which were expected to exert a limiting effect on infarct size.

The results of the present study indeed show a significant delay in infarct development in both TG lines analysed, compared to controls. Moreover, changes in the ECG also suggest a delayed development of myocardial ischemia. ECG signs of myocardial ischemia were visible 5 min after coronary artery occlusion in WT compared to 10–15 min in TG. The increase in R-wave amplitude has been observed in patients with coronary ischemia [31] and in experimental models [31,32] and reflects myocardial ischemia. Characteristic for the mouse electrocardiogram is that no clear ST-segment can be distinguished because the T-wave merges with the final part of the QRS-complex [33,34]. However, in pathological conditions like QT-prolongation or myocardial ischemia the duration and magnitude of the ST-segment become more pronounced [35,36]. Therefore, the delayed ST-segment elevation in the TG mice is in line with a delay in myocardial necrosis in TG.

In fact, the first morphological evidence of myocyte cell death was observed after 15 min of coronary artery occlusion in WT and 45 min in TG. These results indicate that myocardial overexpression of FGF-1 is able to significantly delay infarct development. Increased levels of endogenous FGF-1 may also contribute to this effect. However, the finding that the maximal infarct size is not reduced in TG when compared to WT indicates that the anti-ischemic effect of FGF-1 is most probably not due to maintained perfusion by increased arterial density. Because a linear relationship exists between final infarct size and collateral blood flow [37] we suggest that the unchanged final infarct size in the FGF-1 TG reflects the absence of an influence on collateral flow. Since the infarct region in a mouse heart is only 2 mg of tissue the measurement of collateral flow remains a challenge.

To prove our hypothesis of the non-angiogenic cardioprotective effect of FGF-1 [5], we conducted experiments on isolated ventricular cardiac myocytes subjected to simulated ischemia. Overexpression of FGF-1 markedly increased myocyte viability at 4 and 8 h of simulated ischemia. However, after 12 h of simulated ischemia no significant difference in enzyme release from transgenic and wildtype myocytes could be observed, indicating a similar cell death. Thus, overexpression of FGF-1 markedly enhances cell viability of isolated ventricular cardiac myocytes subjected to simulated ischemia. Therefore, the delayed myocardial infarct development in TG is due to an increased cardiac myocyte viability caused by overexpression of FGF-1.

The molecular mechanisms of this protective effect is among others mediated by way of the tyrosine kinase receptors. FGF-1 and -2 share the same tyrosine kinase receptors (FGFR-1 to -4) [38], they bind to FGFR-1 and FGFR-2 equally and crossphosphorylation between the heterologous FGFs has been described [39]. Tyrosine kinase coupled receptors are discussed as mediators for ischemic preconditioning because inhibition of these receptors was found to block myocardial protection [40]. Moreover, tyrosine kinase inhibitors prevented the cardioprotective effect of FGF-1 infusion in the pig heart [5].

FGF-ligand-binding induces initiation of downstream signaling via the MAPKs ERK-1 and ERK-2, ending in the activation of transcription factors. Barancik et al. showed that brief ischemia and FGF-1 upregulate ERK-1 and -2, especially during reperfusion [41]. MAPKs are important mediators of signal transduction from the cell surface to the nucleus, being involved not only in the regulation of cell hypertrophy but also in the response to cellular stresses such as hypoxia or ischemia. We have recently shown that ERK-1 and -2 are important checkpoints in the survival pathway of ischemically stressed myocardium. Blockade with the ERK-specific inhibitors PD98059 and UO126 inhibits ischemic preconditioning and infarcts following long index ischemia become larger than non-conditioned controls which shows that ischemic preconditioning is at least in part mediated via transcription factors [15]. This is also true in the FGF-1 transgenic mouse where the cardioprotective effect of FGF-1 is partially attenuated after injection of the MEK-inhibitor UO126. Injection of the buffer and DMSO alone showed no differences in IA/RA ratio implying that these substances are not responsible for the differences we saw in infarct size development. The partial but not total loss of cardioprotection could be explained by the complex and multifactorial protection cascade of FGF-1. Palmen et al. showed in their experiments that in the same animals cardioprotection is also mediated via a cascade involving FGFR-1, tyrosine kinases and PKC [42]. Other molecules, not yet identified, may also be involved. In summary, it is an interesting finding that transgenic overexpression of FGF-1 leads to a constitutive activation of the ERK pathway and we believe that this is one of the molecular pathways of protection against ischemia because its inhibition partially abrogates the protective effect.

Patients suffering from acute MI die mainly within the first hours after infarction. Tissue ischemia is tolerated only for minutes or hours whereas arteriogenesis and angiogenesis require at least 1 day to become functionally relevant. Our main goal was to find treatments that can bridge the gap between the onset of ischemia and the increase in collateral dependent blood flow. In this regard, our findings of delayed infarct development and enhanced cell viability due to FGF-1 overexpression are indeed very encouraging. The ideal experimental approach for enhancing the viability of ischemic tissues is to increase the short-term protection and at the same time accelerate vessel growth to the ischemic tissues by the same growth factor or by combined treatments. Increasing the tolerance towards ischemia and restoring blood flow as fast as possible could reduce or even prevent tissue necrosis and potentially save many lives.

We conclude that FGF-1 overexpression in the heart delayed infarct size development by constitutively activating the ERK-1/2 branch of the MAP kinases. The finding that the maximal infarct size is delayed but not reduced in transgenic animals and that the cell viability is enhanced in isolated transgenic cardiomyocytes subjected to ischemia indicates that the anti-ischemic effect of FGF-1 is most probably not due to maintained perfusion by increased arteriolar density.

Time for primary review 27 days.


   Acknowledgements
 
The authors want to thank Professor Dr Jutta Schaper for her help with microscopy, Professor Dr Jos F.M. Smits for his help in the surgical procedure, Ronald J.P. Bronsaer and Claudia Ullmann for their help with the studies, Sigfried Langsdorf for the construction of the gas tight chambers and Gerd Staemmler for the statistical analysis.


   Notes
 
1 Both authors contributed equally to this study. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Carroll R., Yellon D.M. Myocardial adaptation to ischaemia—the preconditioning phenomenon. Int J Cardiol (1999) 68(Suppl_1):S93–S101.[CrossRef][Web of Science][Medline]
  2. Baines C.P., Wang L., Cohen M.V., Downey J.M. Myocardial protection by insulin is dependent on phospatidylinositol 3-kinase but not protein kinase C or KATP channels in the isolated rabbit heart. Basic Res Cardiol (1999) 94:188–198.[CrossRef][Web of Science][Medline]
  3. Kevelaitis E., Peynet J., Mouas C., Launay J.M., Menasche P. Opening of potassium channels: the common cardioprotective link between preconditioning and natural hibernation? Circulation (1999) 99:3079–3085.[Abstract/Free Full Text]
  4. Cuevas P., Carceller F., Lozano R.M., Crespo A., Zazo M., Gimenez-Gallego G. Protection of rat myocardium by mitogenic and non-mitogenic fibroblast growth factor during post-ischemic reperfusion. Growth Factors (1997) 15:29–40.[Web of Science][Medline]
  5. Htun P., Ito W.D., Hoefer I.E., Schaper J., Schaper W. Intramyocardial infusion of FGF-1 mimics ischemic preconditioning in pig myocardium. J Mol Cell Cardiol (1998) 30:867–877.[CrossRef][Web of Science][Medline]
  6. Renaud F., Oliver L., Desset S., et al. Up-regulation of aFGF expression in quiescent cells is related to cell survival. J Cell Physiol (1994) 158:435–443.[CrossRef][Web of Science][Medline]
  7. Burgess W.H., Mehlman T., Marshak D.R., Fraser B.A., Maciag T. Structural evidence that endothelial cell growth factor beta is the precursor of both endothelial cell growth factor alpha and acidic fibroblast growth factor. Proc Natl Acad Sci USA (1986) 83:7216–7220.[Abstract/Free Full Text]
  8. Jaye M., Howk R., Burgess W., et al. Human endothelial cell growth factor: cloning, nucleotide sequence, and chromosome localization. Science (1986) 233:541–545.[Abstract/Free Full Text]
  9. Nugent M.A., Iozzo R.V. Fibroblast growth factor-2. Int J Biochem Cell Biol (2000) 32:115–120.[CrossRef][Web of Science][Medline]
  10. Gospodarowicz D., Neufeld G., Schweigerer L. Molecular and biological characterization of fibroblast growth factor, an angiogenic factor which also controls the proliferation and differentiation of mesoderm and neuroectoderm derived cells. Cell Differ (1986) 19:1–17.[CrossRef][Web of Science][Medline]
  11. Clegg C.H., Linkhart T.A., Olwin B.B., Hauschka S.D. Growth factor control of skeletal muscle differentiation: commitment to terminal differentiation occurs in G1 phase and is repressed by fibroblast growth factor. J Cell Biol (1987) 105:949–956.[Abstract/Free Full Text]
  12. Folkman J., Klagsbrun M. Angiogenic factors. Science (1987) 235:442–447.[Abstract/Free Full Text]
  13. Klein S., Roghani M., Rifkin D.B. Fibroblast growth factors as angiogenesis factors: new insights into their mechanism of action. EXS (1997) 79:159–192.[Medline]
  14. Fernandez B., Buehler A., Wolfram S., et al. Transgenic myocardial overexpression of fibroblast growth factor-1 increases coronary artery density and branching. Circ Res (2000) 87:207–213.[Abstract/Free Full Text]
  15. Strohm C., Barancik T., Bruhl M.L., Kilian S.A., Schaper W. Inhibition of the ER-kinase cascade by PD98059 and UO126 counteracts ischemic preconditioning in pig myocardium. J Cardiovasc Pharmacol (2000) 36:218–229.[CrossRef][Web of Science][Medline]
  16. Michael L.H., Entman M.L., Hartley C.J., et al. Myocardial ischemia and reperfusion: a murine model. Am J Physiol (1995) 269:H2147–H2154.[Web of Science][Medline]
  17. Ito W.D., Schaarschmidt S., Klask R., et al. Infarct size measurement by triphenyltetrazolium chloride staining versus in vivo injection of propidium iodide. J Mol Cell Cardiol (1997) 29:2169–2175.[CrossRef][Web of Science][Medline]
  18. Wolff R.A., Chien G.L., van Winkle D.M. Propidium iodide compares favorably with histology and triphenyl tetrazolium chloride in the assessment of experimentally-induced infarct size. J Mol Cell Cardiol (2000) 32:225–232.[CrossRef][Web of Science][Medline]
  19. Holland R.P., Brooks H. Precordial and epicardial surface potentials during myocardial ischemia in the pig. A theoretical and experimental analysis of the TQ and ST segments. Circ Res (1975) 37:471–480.[Abstract/Free Full Text]
  20. Kloner R.A., Ganote C.E., Jennings R.B. The ‘no-reflow’ phenomenon after temporary coronary occlusion in the dog. J Clin Invest (1974) 54:1496–1508.[Web of Science][Medline]
  21. Kostin S., Hein S., Bauer E.P., Schaper J. Spatiotemporal development and distribution of intercellular junctions in adult rat cardiomyocytes in culture. Circ Res (1999) 85:154–167.[Abstract/Free Full Text]
  22. Gray M.O., Karliner J.S., Mochly-Rosen D. A selective epsilon-protein kinase C antagonist inhibits protection of cardiac myocytes from hypoxia-induced cell death. J Biol Chem (1997) 272:30945–30951.[Abstract/Free Full Text]
  23. Arstall M.A., Zhao Y.-Z., Hornberger L., et al. Human ventricular myocytes in vitro exhibit both early and delayed preconditioning responses to simulated ischemia. Cardiovasc Res (1998) 30:1019–1025.
  24. Taimor G., Lorenz H., Hofstaetter B., Schluter K.-D., Piper H.M. Induction of necrosis but not apoptosis after anoxia and reoxygenation in isolated adult cardiomyocytes of rat. Cardiovasc Res (1999) 41:147–156.[Abstract/Free Full Text]
  25. Ping P., Zhang J., Cao X., et al. PKC-dependent activation of p44/p42 MAPKs during myocardial ischemia-reperfusion in conscious rabbits. Am J Physiol (1999) 276:H1468–H1481.[Web of Science][Medline]
  26. Barancik M., Htun P., Schaper W. Okadaic acid and anisomycin are protective and stimulate the SAPK/JNK pathway. J Cardiovasc Pharmacol (1999) 34:182–190.[CrossRef][Web of Science][Medline]
  27. Gospodarowicz D., Massoglia S., Cheng J., Fujii D.K. Effect of fibroblast growth factor and lipoproteins on the proliferation of endothelial cells derived from bovine adrenal cortex, brain cortex, and corpus luteum capillaries. J Cell Physiol (1986) 127:121–136.[CrossRef][Web of Science][Medline]
  28. Padua R.R., Sethi R., Dhalla N.S., Kardami E. Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury. Mol Cell Biochem (1995) 143:129–135.[CrossRef][Web of Science][Medline]
  29. Slack J.M., Darlington B.G., Heath J.K., Godsave S.F. Mesoderm induction in early Xenopus embryos by heparin-binding growth factors. Nature (1987) 326:197–200.[CrossRef][Medline]
  30. Quinkler W., Maasberg M., Bernotat-Danielowski S., Luthe N., Sharma H.S., Schaper W. Isolation of heparin-binding growth factors from bovine, porcine and canine hearts. Eur J Biochem (1989) 181:67–73.[Web of Science][Medline]
  31. Birnbaum Y., Hale S.L., Kloner R.A. Changes in R wave amplitude: ECG differentiation between episodes of reocclusion and reperfusion associated with ST-segment elevation. J Electrocardiol (1997) 30:211–216.[CrossRef][Web of Science][Medline]
  32. Ferrara N., Abete P., Leosco D., et al. The hypothetical role of potassium conductance on the genesis of R-wave amplitude increase during ischemia in the isolated rat heart. Can J Physiol Pharmacol (1991) 69:994–1000.[Web of Science][Medline]
  33. Goldbarg A.N., Hellerstein H.K., Bruell J.H., Daroczy A.F. Electrocardiogram of the normal mouse, Mus musculus: general considerations and genetic aspects. Cardiovasc Res (1968) 2:93–99.[Abstract/Free Full Text]
  34. Berul C.I., Aronovitz M.J., Wang P.J., Mendelsohn M.E. In vivo cardiac electrophysiology studies in the mouse. Circulation (1996) 94:2641–2648.[Abstract/Free Full Text]
  35. Doevendans P.A., Daemen M.J., de Muinck E.D., Smits J.F. Cardiovascular phenotyping in mice. Cardiovasc Res (1998) 39:34–49.[Abstract/Free Full Text]
  36. Wehrens X.H., Kirchhoff S., Doevendans P.A.F.M. Mouse electrocardiography: an interval of 30 years. Cardiovasc Res (2000) 45:231–237.[Abstract/Free Full Text]
  37. Reimer K.A., Jennings R.B. Effects of calcium-channel blockers on myocardial preservation during experimental acute myocardial infarction. Am J Cardiol (1985) 55:107B–115B.[CrossRef][Medline]
  38. Neufeld G., Gospodarowicz D. Basic and acidic fibroblast growth factors interact with the same cell surface receptors. J Biol Chem (1986) 261:5631–5637.[Abstract/Free Full Text]
  39. Bellot F., Crumley G., Kaplow J.M., Schlessinger J., Jaye M., Dionne C.A. Ligand-induced transphosphorylation between different FGF receptors. EMBO J (1991) 10:2849–2854.[Web of Science][Medline]
  40. Baines C.P., Wang L., Cohen M.V., Downey J.M. Protein tyrosine kinase is downstream of protein kinase C for ischemic preconditioning's anti-infarct effect in the rabbit heart. J Mol Cell Cardiol (1998) 30:383–392.[CrossRef][Web of Science][Medline]
  41. Barancik M., Htun P., Maeno Y., Zimmermann R., Schaper W. Differential regulation of distinct protein kinase cascades by ischemia and ischemia/reperfusion in porcine myocardium. Circulation (1997) 96(Suppl I):1397. abstract.[Web of Science]
  42. Palmen M., Daemen M.J., Willemsen P., et al. FGF-1 protects the myocardium during ischemia and reperfusion through a tyrosine kinase and protein kinase C dependent pathway. Circulation (2000) 102:II–156. abstract.

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