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Departments of Human Anatomy and Cell Sciences and Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada R2H 2A6
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ABSTRACT |
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 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
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We examined the effect of
fibroblast growth factor (FGF)-2 on myocardial resistance to injury
when administered after the onset of ischemia, in
vivo and ex vivo, and the role of FGF-2 receptors and protein kinase C
(PKC). FGF-2 was injected into the left ventricle of rats undergoing
permanent surgical coronary occlusion leading to myocardial infarction
(MI). After 24 h, FGF-2-treated hearts displayed significantly
reduced injury, determined by histological staining and troponin T
release, and improved developed pressure compared with untreated
controls. An FGF-2 mutant with diminished affinity for the tyrosine
kinase FGF-2 receptor 1 (FGFR1) was not cardioprotective. FGF-2-treated
hearts retained improved function and decreased damage at 6 wk after
MI. In the ex vivo heart, FGF-2 administration during reperfusion after
30-min ischemia improved functional recovery and increased
relative levels of PKC subtypes 
, 
, and 
in the particulate
fraction, in a chelerythrine-preventable mode; it also decreased loss
of energy metabolites. We conclude that intramyocardial FGF-2
administration shortly after the onset of ischemia confers
protection from acute and chronic cardiac dysfunction and damage; FGF-2
delivered during reperfusion protects from ischemia-reperfusion
injury; and protection by FGF-2 requires intact binding to FGFR1 and is
likely mediated by PKC.
cardioprotection; growth factors; reperfusion injury; signal transduction; protein therapy
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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FIBROBLAST GROWTH FACTOR (FGF)-2, a prototypical member of the larger family of heparin-binding growth factors, is a multifunctional protein that stimulates cell growth and angiogenesis, affects gene expression and differentiation, and protects cells from apoptosis and/or necrosis (21). There is strong evidence that, as an angiogenic agent, FGF-2 may represent a promising new long-term treatment during remodeling after myocardial infarction (MI) (48). FGF-2, given intra-arterially or by pericardiac implantation over a 4- to 6-wk period, has been shown to stimulate formation of collateral vessels in the chronic post-MI heart and thus improve perfusion (25, 47). Early clinical trials confirmed that FGF-2 is beneficial as a long-term treatment, although side effects were also noted (44).
In addition to its long-term effects on the circulatory system, FGF-2 affects adult cardiac myocytes directly; these cells express functional FGF-2 receptor 1 (FGFR1) (26) and respond to FGF-2 by changes in contractility (31) and hypertrophy (40). Furthermore, FGF-2 administered to the isolated rat or mouse heart before ischemia is clearly protective against subsequent ischemia and reperfusion injury (31, 32, 42). The acute protective effects of FGF pretreatment are proposed to engage a preconditioning-like mechanism (17, 31), and thus one theoretical therapeutic possibility for FGF-2 would be as an agent of primary injury prevention.
We now report on a third possibility for FGF-2 to be used as acute, local therapeutic treatment. We have examined the potential of FGF-2 to act as an agent of secondary injury prevention when administered locally after the onset of ischemia, in the presence or absence of reperfusion, and the role of FGFR1 and protein kinase C (PKC) in this context.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals. Male Sprague-Dawley rats (200-250 g) were used in all experiments and were obtained from the Central Animal Care Facility at the University of Manitoba. The investigations followed the guidelines of the Canadian Council on Animal Care and were approved by the local Animal Care Committee of the National Research Council of Canada.
Materials. Recombinant rat wild-type (wt) or mutant (mt) FGF-2 produced in Escherichia coli bacteria and purified according to previously published procedures was used (22, 31). Water-soluble chelerythrine chloride was purchased from Research Biochemicals International (Natick, MA). Monoclonal antibodies to cardiac troponin T (TnT; no. CT3), developed by Jim Jung-Ching Lin (University of Iowa, Iowa City, IA), were obtained as a culture supernatant from the Developmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development, and maintained by the Department of Biological Sciences, University of Iowa. The supernatant was used at 1:200 dilution for Western blotting.
Myocardial infarction. MI was produced by permanent ligation of the left coronary artery as described previously (7, 19). Briefly, rats were anesthetized with 2-2.5% isofluorane inhalation. The chest was opened by a left-side thoracotomy at the level of the fourth rib, the pericardium was incised, and the left coronary artery was ligated with a silk suture. Vehicle (saline), wtFGF-2 (0.2 and 2 µg), or E104A mtFGF-2 (2 µg) was then injected (in a total volume of 100 µl) into three sites at the lower half of the (ischemic, akinetic) left front ventricular wall, within 10 min of coronary ligation. The chest was then evacuated and closed; animals were placed in an incubated chamber and allowed to recover for 24-48 h (as required). Coronary ligation resulted in 30% mortality, similar in all groups. At various time points after ligation (4-24 h, 1 wk, or 6 wk), animals were anesthetized with a ketamine-xylazine cocktail (90 mg/kg-10 mg/kg ip). Blood samples were collected from the carotid artery and centrifuged to obtain plasma. Rats were euthanized by decapitation, and hearts were harvested for determination of hemodynamic function or infarct size (n = 6-8).
Determination of relative plasma TnT levels. Concentration of plasma TnT was determined by Western blotting with monoclonal anti-cardiac TnT antibodies. Five microliters of plasma was diluted with one hundred microliters of SDS-PAGE (24) loading buffer; 15 µl of the diluted sample was loaded per gel (15% acrylamide) lane. After transfer, the polyvinylidene difluoride membrane was blocked with 10% milk-Tris-buffered saline with 0.05% Tween 20 (TBST) for 1 h, incubated with anti-TnT antibody (1:200) for another hour, washed with 1% milk-TBST for 6 × 5 min and incubated with anti-mouse horseradish peroxidase (1:10,000, Bio-Rad) for 1 h, rinsed with 1× TBST for 6 × 5 min, and finally processed for chemiluminescence (enhanced chemiluminescence). Standard TnT was purchased from Sigma.
Determination of infarct size. Extent of MI was estimated as described previously (3). Briefly, the heart was cut into horizontal slices (2-mm vertical length) and incubated with 1% 2,3,5-triphenyltetrazolium chloride (TTC) at 37°C for 10 min. Infarct tissue appears pale because of the absence of critical tissue factors necessary to interact with TTC to form the dye. Viable tissue stains red because of formation of formazan dye in the cell. The extent of infarction was estimated as the ratio of the area that remained unstained by TTC over the total ventricular area, in all slices, with Sigma ScanPro photographic analysis software. Because rats do not possess cardiac collateral circulation, the infarcted area is expected to be very similar to area at risk.
Immunofluorescence. Hearts were processed for cryosectioning and simultaneous immunolocalization of FGF-2 and vinculin with polyclonal anti-FGF-2 and monoclonal anti-vinculin antibodies, exactly as described by us previously (30). Staining without the primary antibodies was used to control for nonspecific fluorescence.
Perfusion of isolated hearts. After removal from the animals and arrest in cold buffer, hearts were perfused in the Langendorff mode at 37°C as described previously (31). Briefly, a water-filled compliant balloon, connected to a pressure transducer (Stratham P23 ID) was inserted into the left ventricle via the mitral valve. Mechanical function was assessed as developed pressure (DP), end-diastolic pressure (EDP), and rate of contraction/relaxation. Functional data were acquired and analyzed with a PC-based system (Harvard Apparatus, Saint Laurent, PQ, Canada). Hearts were perfused at a constant pressure of 80 mmHg with a modified Krebs-Henseleit (K-H) buffer as described previously (31). For the ex vivo ischemia-reperfusion experiments, left ventricular EDP was adjusted to 5-10 mmHg by inflating the balloon. Global ischemia was induced by interrupting the flow under normothermic conditions. Reperfusion was achieved by reestablishing flow. At the beginning of reperfusion, FGF-2-supplemented (10 µg in 12 ml K-H buffer) or standard K-H buffer was infused directly into the perfusion buffer with a peristaltic pump at the point of entry to the heart. Four groups (n = 6-8/group) were used. All groups (1-4) were equilibrated with K-H buffer for 30 min. Group 1 was perfused with K-H buffer for another 5 min and then subjected to 30-min global ischemia and 60-min reperfusion in K-H buffer. Group 2 was subjected to 5 min of perfusion in K-H buffer-chelerythrine (5 µM) followed by 30-min global ischemia and 60-min reperfusion in K-H buffer-chelerythrine. Group 3 was perfused with K-H buffer for 5 min and subjected to 30-min ischemia, 12-min reperfusion in K-H buffer-FGF-2, and 48-min reperfusion in K-H buffer. Group 4 was subjected to 5 min of perfusion in K-H buffer-chelerythrine, followed by 30-min ischemia, 12-min reperfusion in K-H buffer-FGF-2-chelerythrine, and 48-min reperfusion in K-H buffer-chelerythrine. For assessment of contractile function after permanent coronary ligation in vivo, rats were killed at 4-24 h and 1-6 wk after MI and hearts were placed in a Langendorff apparatus. Hearts were maintained at constant pressure of 80 mmHg. ATP and creatine phosphate (CP) concentrations were determined as described previously (28).
Subcellular distribution of PKC subtypes , , and .
Cytosolic (Cyt) and Triton X-100-soluble membrane (M) fractions were
obtained from hearts (n = 3) subjected to 30-min
ischemia and 30-min reperfusion in the presence or absence of
FGF-2 exactly as described previously (45). The remaining
Triton X-100-insoluble (pellet, P) fractions were solubilized directly
in SDS-PAGE buffer (24). Cyt and total particulate
fractions were also obtained from hearts treated with FGF-2 in the
presence or absence of chelerythrine (n = 3). The
different fractions (20 µg/lane) were analyzed by SDS-PAGE and
Western blotting, probing for PKC subtypes , , and , exactly
as we described previously (31).
FGF-2(S) mutagenesis. The plasmid FGF-2(S).pET19b, containing the AUG-initiated 18-kDa form of rat FGF-2, was used as the template for PCR site-directed mutagenesis. The oligonucleotide primers synthesized by Bio.Synthesis corresponding to sequences within FGF-2 but bearing single-base pair mismatches were used for amplification of mutated subfragments in separate reactions. PCR mutagenesis was carried out in 2 steps. 1) The primer 5'-GTAGTTATTGGACTCCAGGCGTcCAAAGAAGAAACAC-3'(Glu104-Ala antisense strand primer) was used in combination with the FGF-2(S) sense strand primer 5'-GGATTGAGACTCCATATGGCTGCCGGCAGCATCCTTCG-3' to generate the upstream half of FGF-2 fragments termed GluM1. The primer 5'-GTGTTTCTTCTTTGcACGCCTGGAGTCCAATAAC-3' (Glu104-Ala sense primer) was used in combination with a T7 terminator primer 5'-GCTAGTTATTGCTCAGCGG-3' (corresponding to vector sequences) to generate the downstream half mutated FGF-2 fragment termed GluM2. After amplification, the PCR products were analyzed in 1% agarose gels and isolated for subsequent PCR. 2). To generate the full-length mtFGF-2 cDNA, the subfragments GluM1 and GluM2 were mixed and followed by PCR amplification through two cycles at 95°C for 1 min, 60°C for 1 min, 72°C for 10 min in a 30-µl reaction with 10 mM Tris, pH 8.3, 2.5 mM MgCl2, 50 mM KCl, 0.2 µg/µl gelatin, 83 µM dNTP, and 1 U of Taq polymerase. After two cycles, 20 µl of fresh solution containing the above-described buffer was added and further PCR reactions were carried out in 39 cycles consisting of denaturation at 95°C for 1 min, annealing at 60°C for 45 s, and extension at 72°C for 1 min. Full-length FGF-2 mutant fragment was ligated into TA(pCR2.1) cloning vector (Invitrogen) to generate pFGFm. The mtFGF-2 cDNA was released from pFGFm by NdeI-XhoI, gel isolated, and used to replace the full-length wtFGF-2 cDNA in the pET19 expression vector. The mutant's sequence was confirmed by the dideoxy method (µ-mol sequencing kit; Promega, Madison, WI).
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Effect of wt- and mtFGF-2 on injury and contractile dysfunction
after MI.
wtFGF-2, the (Glu104-Ala) FGF-2 mutant (mtFGF-2), or saline
was injected directly into the ischemic left ventricle within
10 min of permanent coronary ligation. At 4 and 24 h after MI,
animals were killed, hearts were removed, and the degree of myocardial damage was assessed with the tetrazolium method for staining followed by morphometric analysis. Results are shown in Fig.
1. Injection of wtFGF-2 (2 µg) resulted
in a significantly smaller infarct area compared with saline-injected
controls at both time points. A reduced wtFGF-2 dose (0.2 µg) also
produced a smaller infarct area compared with saline, although the
effect was smaller than with the higher wtFGF-2 dose. The extent of
infarction in the group injected with mtFGF-2 (2 µg) was similar to
that in the saline-treated group, signifying the lack of protection.
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Effect of FGF-2 administered during reperfusion on recovery:
Involvement of PKC.
The in vivo study examined the effect of FGF-2 on ischemic,
nonreperfused myocardium. Current strategies for effective management of an acute ischemic episode require prompt tissue reperfusion. Although restoration of blood flow is considered essential for salvaging myocardial function, it has been associated with a degree of
exacerbation of myocardial injury (36). To investigate
whether FGF-2 would be protective when administered during reperfusion, we used the ex vivo heart model of 30 min of global ischemia
followed by 60 min of reperfusion as previously described (31,
32). In this model, FGF-2 is infused into the reperfusion medium
during the first 12 min of reperfusion. A schematic representation of the different groups used for these studies is shown in Fig.
5.
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, , and , we examined their relative levels in the
Cyt, M, and P fractions. The M and P fractions represent further
fractionation of the "particulate" fraction remaining after removal
of cytosolic proteins and are enriched in membrane and cytoskeletal
intercalated disk proteins, respectively. As shown in Fig.
7, FGF-2 significantly decreased the
relative levels of all PKC subtypes tested in the cytosol. This was
accompanied by significant increases in the relative levels of PKC-
in the M fraction and those of PKC- and - in the P fraction. We
also examined whether chelerythrine affected relative levels of PKC subtypes in FGF-2-treated hearts (Fig. 7). Relative levels of all PKCs
in the cytosol of chelerythrine-FGF-2-treated hearts were significantly
higher than those in FGF-2-only-treated samples. Conversely, relative
levels of all PKCs in the particulate fraction (combined M and P
fractions) of chelerythrine-FGF-2-treated hearts was significantly
lower than in the FGF-2-treated samples (Fig. 7B). This is
in agreement with previous studies that showed chelerythrine to prevent
the translocation of PKC in cardiomyocytes (11). Our data
show that, in hearts reperfused after ischemia, FGF-2 induced
redistribution of PKCs from cytosolic to particulate fractions in a
chelerythrine-preventable mode.
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DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The need for effective treatments that reduce the extent of an evolving MI cannot be understated. No clinically useful agents capable of protecting the ischemic myocardium, in the presence or absence of reestablishment of flow, are currently available (49). We therefore examined whether FGF-2 has a protective effect on the ischemic myocardium in two different experimental models. In vivo, FGF-2 was injected directly into the ischemic ventricle during irreversible surgical coronary ligation to examine its ability to salvage ischemic tissue from damage. In the Langendorff-perfused heart, FGF-2 was administered during the reperfusion phase to examine its effects on dysfunction and loss of energy metabolites subsequent to ischemia and reperfusion. In addition, we examined the participation of specific elements of the FGF-2 signal transduction pathway in relation to cardioprotection. We found that 1) protection against myocardial injury and contractile dysfunction is achieved through single-dose intramyocardial administration of FGF-2 into the ischemic rat myocardium in a model of permanent coronary occlusion and is effective acutely (4-24 h) as well as at 1-6 wk after MI; 2) FGF-2, given during reperfusion, protects against ischemia-reperfusion injury of the ex vivo heart; 3) the protective effects of FGF-2 on ischemic myocytes require intact binding to FGFR1; and 4) these effects are likely mediated by PKC.
To our knowledge, FGF-2 is the only agent identified to date that exerts protection when administered locally to the ischemic heart and also during reperfusion. Our findings extend and are in broad agreement with previous reports of cardioprotection by systemic administration of FGF-1 (a factor belonging to the same family of heparin-binding growth factors as FGF-2) or FGF-2 in vivo (4, 5). Others, using intracoronary infusion of FGF-2 in a canine model, reported that FGF-2 significantly limited myocardial necrosis after acute coronary occlusion (15). A significant difference in our approach is that in the in vivo model we used local intramyocardial delivery to avoid overall systemic involvement, including potential side effects but also any uncertainty as to the direct involvement of heart muscle itself. In addition, we addressed the duration of the beneficial effects by assessing the hearts up to several weeks from treatment.
Injected FGF-2 induced protection of cardiomyocytes in vivo as indicated by infarct size estimates as well as relative TnT plasma levels (Fig. 1). FGF-2 was clearly retained by the myocardium and localized around myocytes, indicating its potential to directly influence these cells. We previously showed (26) that FGF-2 injected into the myocardium triggers tyrosine phosphorylation in the cardiac myocyte, a finding consistent with in situ activation of its tyrosine kinase (TK) FGFR1.
The biological effects of extracellular FGF-2 are mediated by binding to plasma membrane TK receptors (FGFR1-4) and HSPG "low"-affinity sites (27). The HSPG sites are considered to allow and/or facilitate binding to the TK receptors; they may also mediate independent signaling events (38). To examine whether the cardioprotective effects of FGF-2 required intact binding to FGFR1 (the only FGF-2 receptor expressed in adult rodent myocardium; Refs. 10, 18, 26, 33), we used mtFGF-2. Rat mtFGF contains a substitution of glutamine-104 with an alanine residue, equivalent to the glutamine-96 mutation on human FGF-2. This mutation produces FGF-2 that binds to the low-affinity HSPG sites with unchanged affinity but has diminished affinity for FGFR1 (50). Our own characterization confirmed that, although mtFGF-2 was retained by the heart (presumably by binding to HSPGs of the basement membrane and the extracellular matrix) and localized around cardiomyocytes (Fig. 2) in a manner identical to wtFGF-2, it was unable to displace 125I-labeled wtFGF-2 from the high-affinity plasma membrane sites (unpublished observations). mtFGF-2 had no cardioprotective properties. Thus it would appear that the effect of FGF-2 in the heart is dependent on its ability to bind to FGFR1 and that binding to HSPG sites is not sufficient for cardioprotection. Although FGFR1 is known to mediate cardiac growth in development, its role in the adult myocardium has been less clear. We propose that FGFR1 can mediate FGF-2 cardioprotection of the ischemic myocardium in vivo.
FGF-2 was injected in the middle of the lower half (nearer to the apex)
of the left ventricle, an area that was rendered ischemic as
judged by its changed color and development of akinesis after occlusion. Exogenous FGF-2 localized in association with viable myocardium surrounding the infarcted region, as well as with
irreversibly injured myocytes (Fig. 2). It is therefore presumed that
FGF-2 exerted its protection on cells suffering intermediate levels of
ischemic damage, at or near the infarct border. FGF-2
protection is likely to have included immediate effects, as is
discerned clearly in the ex vivo model, as well as effects on gene
expression, possibly causing a response similar to that of delayed
preconditioning. FGF-2 stimulates expression of nitric oxide synthase
(29) as well as expression of the transcription factor
nuclear factor-
B (16); both intermediates, as well as
PKC- (also stimulated by FGF-2; Ref. 31), are strongly
implicated in the induction of late preconditioning (1).
FGF-2 protection was discernible at 1-6 wk after MI. Although this
is likely a consequence of acute preservation of myocardium at the
early time points, we cannot exclude the possibility (not examined
here) that formation of new blood vessels may also have occurred, as
has been reported by others (41) who used comparable FGF-2
treatment. An angiogenic response would contribute to improved cardiac
function at the later (but not the early) time points.
Unlike FGF-1, which has been reported to prevent ischemia-reperfusion-induced apoptosis (6), we do not think it likely that FGF-2 protection in our in vivo system involved effects on apoptosis. Examination of several sections per heart revealed a negligible number of terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL)-positive nuclei in muscle or nonmuscle cells (unpublished observations), in agreement with reports linking apoptosis predominantly to reperfusion events (20).
In our previous studies (32) in which FGF-2 was administered to isolated rat hearts before ischemia, we noted that FGF-2 was retained by the myocardium even after 30 min of ischemia followed by 1 h of reperfusion. As a result, FGF-2 might be expected to continue exerting effects on myocytes during the reperfusion phase. Indeed, as presented here, FGF-2 administration during the reperfusion stage elicited a significant improvement in recovery of function. The extent of this recovery was similar to that induced by FGF-2 given before ischemia. Both preischemic (31, 32) and postischemic (Fig. 2) administration of FGF-2 resulted in hearts displaying a 1.6-fold increase in DP over that of untreated controls, after 30-min ischemia and 60-min reperfusion. Thus it would appear that, irrespective of whether it induces a preconditioning response in nonischemic cells, FGF-2 is cardioprotective when administered during reperfusion on ischemic myocytes.
Increased contractile recovery accompanied by increased levels of energy metabolites in the FGF-2-treated ex vivo ischemic hearts is consistent with decreased myocyte injury. Preservation against contractile dysfunction or "stunning" (14) or direct effects on energy metabolism may also have contributed to improved functional recovery and merit further investigation.
To address the mechanism of FGF-2 cardioprotection during reperfusion,
we examined the role of PKC. PKC, in particular the -isoform,
mediates the early and late preconditioning responses and
cardioprotection (35, 37, 43). FGF-2-triggered signal transduction in cardiomyocytes includes FGFR1-mediated activation of
phospholipase C and PKC- in cardiomyocytes (9,
31). In the present investigation, involvement of PKC
signaling was supported by the FGF-2-induced redistribution of PKC to
particulate compartments (Fig. 7). A recent paper showed that PKC
activity/redistribution during reperfusion serves to preserve myocytes
from reperfusion injury (45). PKC activation by FGF-2
during reperfusion therefore would be expected to enhance cardiac
resistance to injury, exactly as we found (Fig. 6). The inhibition of
FGF-2-induced cardioprotection (Fig. 6) and PKC translocation (Fig.
7B) by chelerythrine supports the notion that PKC activation
during reperfusion is required for the cardioprotective effects of
FGF-2.
FGF-2 affected all three PKC subtypes examined, representing all three
classes of PKC. Because there is evidence that PKC- mediates
protection during reperfusion (45), it is likely that at
least this subtype contributed to the protective effect of FGF-2. It is
of interest that FGF-2 caused translocation of PKC- to the P
fraction, containing the majority of at least one of its established
targets and interacting proteins, the gap junction protein connexin43
(2, 9, 34). We showed previously (8, 9) that
FGF-2 stimulates PKC--dependent phosphorylation of the gap junction
protein connexin43, leading to decreased coupling between
cardiomyocytes. Myocyte uncoupling caused by other agents such as
anesthetics is considered to be protective by preventing the spreading
of hypercontracture via gap junctions (12, 39). We
therefore speculate that FGF-2 cardioprotection during reperfusion may
include PKC-mediated effects on cardiac gap junctions.
In conclusion, FGF-2 is an excellent candidate for secondary prevention of evolving ischemic myocardial damage, at least in our animal model. The safety and efficacy of intramyocardial and/or local delivery of therapeutic agents is the subject of intense interest (23, 44). Recent studies using pig models indicated that fluoroscopy-guided intramyocardial injection is a feasible and safe procedure (13). It is thus reasonable to suggest that acute "protein therapy" with FGF-2 formulations may not be technically forbidding. Assuming that safety issues have been fully addressed, FGF-2 might be delivered intramyocardially, by catheterization, through the coronary artery, during performance of angiograms, and last, but not least, as an additional treatment during reestablishment of blood flow.
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ACKNOWLEDGEMENTS |
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This work was supported by the Canadian Institutes for Health Research (CIHR; Group in Experimental Cardiology) (E. Kardami) and a joint grant to E. Kardami and I. M. C. Dixon by the St. Boniface General Hospital Research Foundation. E. Kardami was supported by a CIHR Group scientist award. I. M. C. Dixon has a scholarship from CIHR. P. A. Cattini was a CIHR scientist. Z.-S. Jiang has a postdoctoral fellowship from the Manitoba Health Research Council.
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FOOTNOTES |
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* Z.-S. Jiang and R. R. Padua contributed equally to this work.
Present addresses: B. W. Doble, Ontario Cancer Institute, Division of Experimental Therapeutics, Toronto, ON, Canada M5G 2M9; H. Xu, Cardiovascular Pharmacology, GlaxoSmithKline, King of Prussia, PA 19406.
Address for reprint requests and other correspondence: E. Kardami, Inst. of Cardiovascular Sciences, SBGH Res. Cntr 3008, 351 Tache Ave., Winnipeg, MB, Canada R2H 2A6 (E-mail: ekardami{at}sbrc.ca).
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.
10.1152/ajpheart.00290.2001
Received 9 April 2001; accepted in final form 6 November 2001.
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REFERENCES |
|---|
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Bolli, R.
The late phase of preconditioning.
Circ Res
87:
972-983,
2000.
2.
Bowling, N,
Huang X,
Sandusky GE,
Fouts RL,
Mintze K,
Esterman M,
Allen PD,
Maddi R,
McCall E,
and
Vlahos CJ.
Protein kinase C- and - modulate connexin-43 phosphorylation in human heart.
J Mol Cell Cardiol
33:
789-798,
2001.
3.
Chopra, K,
Singh M,
Kaul N,
Andrabi KI,
and
Ganguly NK.
Decrease of myocardial infarct size with desferrioxamine: possible role of oxygen free radicals in its ameliorative effect.
Mol Cell Biochem
113:
71-76,
1992.
4.
Cuevas, P,
Carceller F,
Lozano RM,
Crespo A,
Zazo M,
and
Gimenez-Gallego G.
Protection of rat myocardium by mitogenic and non-mitogenic fibroblast growth factor during postischemic reperfusion.
Growth Factors
15:
29-40,
1997.
5.
Cuevas, P,
Carceller F,
Martinez-Coso V,
Cuevas B,
Fernandez-Ayerdi A,
Reimers D,
Asin-Cardiel E,
and
Gimenez-Gallego G.
Cardioprotection from ischemia by fibroblast growth factor: role of inducible nitric oxide synthase.
Eur J Med Res
4:
517-524,
1999.
6.
Cuevas, P,
Reimers D,
Carceller F,
Martinez-Coso V,
Redondo-Horcajo M,
Saenz de Tejada I,
and
Gimenez-Gallego G.
Fibroblast growth factor-1 prevents myocardial apoptosis triggered by ischemia reperfusion injury.
Eur J Med Res
2:
465-468,
1997.
7.
Dixon, IM,
Lee SL,
and
Dhalla NS.
Nitrendipine binding in congestive heart failure due to myocardial infarction.
Circ Res
66:
782-788,
1990.
8.
Doble, BW,
Chen Y,
Bosc DG,
Litchfield DW,
and
Kardami E.
Fibroblast growth factor-2 decreases metabolic coupling and stimulates phosphorylation as well as masking of connexin43 epitopes in cardiac myocytes.
Circ Res
79:
647-658,
1996.
9.
Doble, BW,
Ping P,
and
Kardami E.
The subtype of protein kinase C is required for cardiomyocyte connexin-43 phosphorylation.
Circ Res
86:
293-301,
2000.
10.
Engelmann, GL,
Dionne CA,
and
Jaye MC.
Acidic fibroblast growth factor and heart development. Role in myocyte proliferation and capillary angiogenesis.
Circ Res
72:
7-19,
1993.
11.
Fryer, RM,
Wang Y,
Hsu AK,
and
Gross GJ.
Essential activation of PKC-
in opioid-initiated cardioprotection.
Am J Physiol Heart Circ Physiol
280:
H1346-H1353,
2001.
12.
Garcia-Dorado, D,
Inserte J,
Ruiz-Meana M,
Gonzalez MA,
Solares J,
Julia M,
Barrabes JA,
and
Soler-Soler J.
Gap junction uncoupler heptanol prevents cell-to-cell progression of hypercontracture and limits necrosis during myocardial reperfusion.
Circulation
96:
3579-3586,
1997.
13.
Gwon, HC,
Jeong JO,
Kim HJ,
Park SW,
Lee SH,
Park SJ,
Huh JE,
Lee Y,
Kim S,
and
Kim DK.
The feasibility and safety of fluoroscopy-guided percutaneous intramyocardial gene injection in porcine heart.
Int J Cardiol
79:
77-88,
2001.
14.
Hampton, TG,
Amende I,
Fong J,
Laubach VE,
Li J,
Metais C,
and
Simons M.
Basic FGF reduces stunning via a NOS 2-dependent pathway in coronary-perfused mouse hearts.
Am J Physiol Heart Circ Physiol
279:
H260-H268,
2000.
15.
Horrigan, MC,
Malycky JL,
Ellis SG,
Topol EJ,
and
Nicolini FA.
Reduction in myocardial infarct size by basic fibroblast growth factor following coronary occlusion in a canine model.
Int J Cardiol
68, Suppl1:
S85-S91,
1999.
16.
Hoshi, S,
Goto M,
Koyama N,
Nomoto K,
and
Tanaka H.
Regulation of vascular smooth muscle cell proliferation by nuclear factor-B and its inhibitor, I-B.
J Biol Chem
275:
883-889,
2000.
17.
Htun, P,
Ito WD,
Hoefer IE,
Schaper J,
and
Schaper W.
Intramyocardial infusion of FGF-1 mimics ischemic preconditioning in pig myocardium.
J Mol Cell Cardiol
30:
867-877,
1998.
18.
Jin, Y,
Pasumarthi KB,
Bock ME,
Lytras A,
Kardami E,
and
Cattini PA.
Cloning and expression of fibroblast growth factor receptor-1 isoforms in the mouse heart: evidence for isoform switching during heart development.
J Mol Cell Cardiol
26:
1449-1459,
1994.
19.
Ju, H,
Zhao S,
Tappia PS,
Panagia V,
and
Dixon IM.
Expression of Gq and PLC-
in scar and border tissue in heart failure due to myocardial infarction.
Circulation
97:
892-899,
1998.
20.
Kajstura, J,
Cheng W,
Reiss K,
Clark WA,
Sonnenblick EH,
Krajewski S,
Reed JC,
Olivetti G,
and
Anversa P.
Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats.
Lab Invest
74:
86-107,
1996.
21.
Kazama, H,
and
Yonehara S.
Oncogenic K-Ras and basic fibroblast growth factor prevent Fas-mediated apoptosis in fibroblasts through activation of mitogen-activated protein kinase.
J Cell Biol
148:
557-566,
2000.
22.
Ke, LD,
Karaganis AG,
and
Shain SA.
A rapid, two-step method for high-yield purification of recombinant rat acidic and basic fibroblast growth factors.
Protein Expr Purif
3:
497-507,
1992.
23.
Kornowski, R,
Fuchs S,
Leon MB,
and
Epstein SE.
Delivery strategies to achieve therapeutic myocardial angiogenesis.
Circulation
101:
454-458,
2000.
24.
Laemmli, UK.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970.
25.
Lazarous, DF,
Scheinowitz M,
Shou M,
Hodge E,
Rajanayagam S,
Hunsberger S,
Robison WG, Jr,
Stiber JA,
Correa R,
Epstein SE,
and
Unger EF.
Effects of chronic systemic administration of basic fibroblast growth factor on collateral development in the canine heart.
Circulation
91:
145-153,
1995.
26.
Liu, L,
Pasumarthi KB,
Padua RR,
Massaeli H,
Fandrich RR,
Pierce GN,
Cattini PA,
and
Kardami E.
Adult cardiomyocytes express functional high-affinity receptors for basic fibroblast growth factor.
Am J Physiol Heart Circ Physiol
268:
H1927-H1938,
1995.
27.
McIntosh, I,
Bellus GA,
and
Jab EW.
The pleiotropic effects of fibroblast growth factor receptors in mammalian development.
Cell Struct Funct
25:
85-96,
2000.
28.
McPherson, CD,
Pierce GN,
and
Cole WC.
Ischemic cardioprotection by ATP-sensitive K+ channels involves high-energy phosphate preservation.
Am J Physiol Heart Circ Physiol
265:
H1809-H1818,
1993.
29.
Murphy, PR,
Limoges M,
Dodd F,
Boudreau RT,
and
Too CK.
Fibroblast growth factor-2 stimulates endothelial nitric oxide synthase expression and inhibits apoptosis by a nitric oxide-dependent pathway in Nb2 lymphoma cells.
Endocrinology
142:
81-88,
2001.
30.
Padua, RR,
and
Kardami E.
Increased basic fibroblast growth factor (bFGF) accumulation and distinct patterns of localization in isoproterenol-induced cardiomyocyte injury.
Growth Factors
8:
291-306,
1993.
31.
Padua, RR,
Merle PL,
Doble BW,
Yu CH,
Zahradka P,
Pierce GN,
Panagia V,
and
Kardami E.
FGF-2-induced negative inotropism and cardioprotection are inhibited by chelerythrine: involvement of sarcolemmal calcium-independent protein kinase C.
J Mol Cell Cardiol
30:
2695-2709,
1998.
32.
Padua, RR,
Sethi R,
Dhalla NS,
and
Kardami E.
Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury.
Mol Cell Biochem
143:
129-135,
1995.
33.
Partanen, J,
Makela TP,
Eerola E,
Korhonen J,
Hirvonen H,
Claesson-Welsh L,
and
Alitalo K.
FGFR-4, a novel acidic fibroblast growth factor receptor with a distinct expression pattern.
EMBO J
10:
1347-1354,
1991.
34.
Ping, P,
Zhang J,
Pierce WM, Jr,
and
Bolli R.
Functional proteomic analysis of protein kinase C epsilon signaling complexes in the normal heart and during cardioprotection.
Circ Res
88:
59-62,
2001.
35.
Ping, P,
Zhang J,
Qiu Y,
Tang XL,
Manchikalapudi S,
Cao X,
and
Bolli R.
Ischemic preconditioning induces selective translocation of protein kinase C isoforms and 
in the heart of conscious rabbits without subcellular redistribution of total protein kinase C activity.
Circ Res
81:
404-414,
1997.
36.
Piper, HM,
Garcia-Dorado D,
and
Ovize M.
A fresh look at reperfusion injury.
Cardiovasc Res
38:
291-300,
1998.
37.
Qiu, Y,
Ping P,
Tang XL,
Manchikalapudi S,
Rizvi A,
Zhang J,
Takano H,
Wu WJ,
Teschner S,
and
Bolli R.
Direct evidence that protein kinase C plays an essential role in the development of late preconditioning against myocardial stunning in conscious rabbits and that is the isoform involved.
J Clin Invest
101:
2182-2198,
1998.
38.
Quarto, N,
and
Amalric F.
Heparan sulfate proteoglycans as transducers of FGF-2 signaling.
J Cell Sci
107:
3201-3212,
1994.
39.
Ruiz-Meana, M,
Garcia-Dorado D,
Hofstaetter B,
Piper HM,
and
Soler-Soler J.
Propagation of cardiomyocyte hypercontracture by passage of Na+ through gap junctions.
Circ Res
85:
280-287,
1999.
40.
Schultz, JE,
Witt SA,
Nieman ML,
Reiser PJ,
Engle SJ,
Zhou M,
Pawlowski SA,
Lorenz JN,
Kimball TR,
and
Doetschman T.
Fibroblast growth factor-2 mediates pressure-induced hypertrophic response.
J Clin Invest
104:
709-719,
1999.
41.
Schumacher, B,
Pecher P,
von Specht BU,
and
Stegmann T.
Induction of neoangiogenesis in ischemic myocardium by human growth factors: first clinical results of a new treatment of coronary heart disease.
Circulation
97:
645-650,
1998.
42.
Sheikh, F,
Sontag DP,
Fandrich RR,
Kardami E,
and
Cattini PA.
Overexpression of FGF-2 increases cardiac myocyte viability after injury in isolated mouse hearts.
Am J Physiol Heart Circ Physiol
280:
H1039-H1050,
2001.
43.
Simkhovich, BZ,
Przyklenk K,
and
Kloner RA.
Role of protein kinase C as a cellular mediator of ischemic preconditioning: a critical review.
Cardiovasc Res
40:
9-22,
1998.
44.
Simons, M,
Bonow RO,
Chronos NA,
Cohen DJ,
Giordano FJ,
Hammond HK,
Laham RJ,
Li W,
Pike M,
Sellke FW,
Stegmann TJ,
Udelson JE,
and
Rosengart TK.
Clinical trials in coronary angiogenesis: issues, problems, consensus: an expert panel summary.
Circulation
102:
E73-E86,
2000.
45.
Stamm, C,
Friehs I,
Cowan DB,
Cao-Danh H,
Noria S,
Munakata M,
McGowan FX, Jr,
and
del Nido PJ.
Post-ischemic PKC inhibition impairs myocardial calcium handling and increases contractile protein calcium sensitivity.
Cardiovasc Res
51:
108-121,
2001.
46.
Steenbergen, C,
Hill ML,
and
Jennings RB.
Cytoskeletal damage during myocardial ischemia: changes in vinculin immunofluorescence staining during total in vitro ischemia in canine heart.
Circ Res
60:
478-486,
1987.
47.
Unger, EF,
Banai S,
Shou M,
Lazarous DF,
Jaklitsch MT,
Scheinowitz M,
Correa R,
Klingbeil C,
and
Epstein SE.
Basic fibroblast growth factor enhances myocardial collateral flow in a canine model.
Am J Physiol Heart Circ Physiol
266:
H1588-H1595,
1994.
48.
Ware, JA,
and
Simons M.
Angiogenesis in ischemic heart disease.
Nat Med
3:
158-164,
1997.
49.
Yellon, DM,
and
Baxter GF.
Protecting the ischaemic and reperfused myocardium in acute myocardial infarction: distant dream or near reality?
Heart
83:
381-387,
2000.
50.
Zhu, H,
Ramnarayan K,
Anchin J,
Miao WY,
Sereno A,
Millman L,
Zheng J,
Balaji VN,
and
Wolff ME.
Glu-96 of basic fibroblast growth factor is essential for high affinity receptor binding. Identification by structure-based site-directed mutagenesis.
J Biol Chem
270:
21869-21874,
1995.
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), in the
presence of FGF-2 (
), or in the presence of
chelerythrine (
) on % recovery in DP or EDP,
respectively. Values are expressed as means ± SE
(n = 6). Statistical analysis was done using ANOVA with
post hoc testing, where P < 0.05 was considered
significant. *Statistically significant differences between
FGF-2-treated and control hearts. B and D:
effects of reperfusion in the presence of chelerythrine-FGF-2
(
) on recovery of DP and EDP, respectively. Values
corresponding to control or FGF-2-treated hearts and shown in
A and C are included in B and
D to facilitate comparisons. Values are expressed as
means ± SE (n = 5-6). Data were analyzed by
ANOVA with post hoc testing where P < 0.05 was considered
significant. *Statistically significant differences between the
chelerythrine-FGF-2-treated group and the FGF-2-treated group. Absolute
values for DP and EDP in control hearts after stabilization
for 30 min and before ischemia were 103.3 ± 5.9 and 7.6 ± 0.5 mmHg, respectively. Corresponding values (before
ischemia) in: FGF-2-treated hearts were 97.2 ± 8.2 and
7.7 ± 0.6 mmHg; chelerythrine-treated hearts were at 86.2 ± 9.4 and 8.1 ± 0.6 mmHg; chelerythrine-FGF-2-treated hearts were
at 93.6 ± 8.8 and 8.1 ± 0.6 mmHg.
