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1 Cardiovascular Division and 4 Angiogenesis Research Center, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215; 2 Department of Cardiology, Medical University, D-30625 Hannover, Germany; and 3 Department of Surgery University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Basic fibroblast growth factor
(FGF-2) may protect the heart from ischemia-reperfusion injury
(stunning) by stimulating nitric oxide (NO) production. To test this
hypothesis, we pretreated coronary-perfused mouse hearts with 1 µg/ml
FGF-2 or vehicle control before the onset of ischemia. Intracellular
calcium (Cai2+) was estimated by aequorin, and NO
release was measured with an NO-selective electrode. Hearts perfused
with FGF-2 maintained significantly better left ventricular (LV)
function during ischemia than hearts perfused with vehicle. FGF-2
significantly delayed the onset of ischemic contracture and improved LV
recovery during reperfusion. Cai2+ was similar in both
groups at baseline during ischemia and reperfusion. L-N6-(1-iminoethyl)lysine, a
selective inhibitor of inducible NO synthase (NOS2), obliterated the
protective effects of FGF-2. In transgenic hearts deficient in the
expression of NOS2 (NOS2
/), FGF-2 did not attenuate
ischemia-induced LV dysfunction. Measurements of NO release
demonstrated that FGF-2 perfusion significantly increased NO in
wild-type but not in NOS2/ hearts. We conclude that basic FGF
attenuates myocardial stunning independent of alterations in
Cai2+ by stimulating NO production via an
NOS2-dependent pathway.
ischemia; nitric oxide; intracellular calcium; myocardial function
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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BASIC FIBROBLAST GROWTH FACTOR (FGF-2) has been shown in vivo to promote angiogenesis in the ischemic myocardium (see Ref. 43 for review). In animals treated with FGF-2, resting collateral flow values significantly exceeded those of controls, resulting in improved myocardial perfusion and performance (14, 25, 27, 38, 42). A number of studies, however, have suggested that the action of FGF-2 and other members of the FGF family of heparin-binding growth factors extend beyond stimulation of growth and proliferation of different cell types. These nonmitogenic actions of FGF-2 include induction of hypotension (8), alterations in intracellular ion homeostasis (20, 30, 31), and negative inotropy (20). Moreover, FGF-2 and FGF-1 administered to the heart have been shown to improve cardiac resistance to ischemia-reperfusion injury (17, 18, 34, 44). Although the mechanisms of these observations remain largely unknown, current data suggest that FGF-2 has physiological effects that are mediated, in part, through activation of nitric oxide synthase (NOS) and the subsequent generation of nitric oxide (NO) (5, 19, 23, 37). Because NO has been shown to play a significant role in mitigating ischemic stress (2-4, 40), we hypothesized that FGF-2 may provide cardioprotection via production of NO.
To determine whether nonmitogenic effects of FGF-2 could be beneficial
to the heart during acute myocardial ischemia and reperfusion, FGF-2
was administered in a recently described murine model of myocardial
stunning (12). The advantages of this mouse model are
well-defined markers of ischemia-reperfusion injury, including ischemic
contracture, alteration in calcium homeostasis, and prolonged ventricular dysfunction, occurring within a time window too short to
activate the mitogenic properties of FGF-2. Transgenic mouse hearts
deficient in the expression of the inducible isoform of NOS (NOS2/)
(24) were used to further investigate the coupling of
FGF-2 and NO during acute myocardial ischemia and reperfusion.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Isolated Heart Preparation
The method for perfusion of the isovolumic mouse heart was described previously (12). Briefly, hearts were excised from adult C57/BL6 mice of either sex that had been anesthetized and heparinized (500 U/100 g body wt). The aorta was slipped over a 20-gauge blunt-tipped stainless steel needle through which oxygenated (95% O2-5% CO2) Krebs- Henseleit (KH) buffer (in mM: 118.0 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.5 CaCl2, 1.2 MgCl2, 23.0 NaHCO3, 10.0 dextrose, and 0.3 EDTA, pH 7.4) was pumped at a rate of ~3 ml/min. An intraventricular balloon catheter system specially designed for the mouse heart (12) was passed through the mitral annulus into the left ventricle, and the distal end of the balloon catheter was connected to a Statham P23b (Gould, Cleveland, OH) transducer to record intraventricular pressure. Left ventricular (LV) pressure recordings were analyzed with regard to LV developed pressure (LVP), LV end-diastolic pressure, peak rate of pressure development (dP/dtmax), time to 90% pressure decline, and peak rate of pressure decline (dP/dtmin).Measurement of Intracellular Ca2+
In hearts in which intracellular Ca2+ (Cai2+) was estimated, aequorin was injected into the apex of the heart as previously described (12, 13). Briefly, after the perfusate was modified to contain 0.5 mM CaCl2, 0.6 mM MgCl2, and 20 mM dextrose, 1-3 µl of aequorin were injected with a glass micropipette into a localized region of 2 mm2 at the apex of the heart. The heart was positioned in an organ bath such that the aequorin-loaded region was ~2 mm from the bottom of the bath. The Ca2+ and Mg2+ concentrations of the perfusate were increased to 2.5 mM Ca2+ and 1.2 mM Mg2+ in a stepwise fashion over a period of 40 min. The entire isolated heart preparation was positioned in a light-tight box for collection of the aequorin light signal as previously described (12). Aequorin luminescence was detected by a photomultiplier tube and recorded as anodal current. For estimation of Cai2+, Triton X-100 was injected into the coronary perfusate to quickly permeabilize the myocardial cell membranes and expose the remaining active aequorin to saturating Ca2+. This resulted in a burst of light, the integral of which approximated the maximum light (Lmax) against which light signals of interest (L) provided the fractional luminescence (L/Lmax). L/Lmax was referred to a calibration equation to estimate Cai2+ (12, 22).Experimental Protocol
Drugs. Recombinant bovine FGF-2 (rFGF-2) was obtained from Chiron (Sunnyvale, CA). NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, was obtained from RBI (Natick, MA). L-N6-(1-iminoethyl)lysine (L-NIL), a selective inhibitor of NOS2, was obtained from Sigma (St. Louis, MO). All studies were conducted at 30°C, and hearts were paced at 6 Hz to minimize consumption of aequorin. After a 15-min equilibrium period, baseline conditions were recorded. Subsequently, hearts were divided into the following perfusion groups: perfusion with KH for 40 min (control, n = 10), perfusion with KH for 20 min followed by perfusion with KH plus 1 µg/ml rFGF-2 for 20 min (rFGF-2, n = 10), perfusion with KH plus 400 µM L-NAME for 20 min followed by perfusion with KH plus 400 µM L-NAME plus 1 µg/ml rFGF-2 for 20 min (L-NAME + rFGF-2, n = 6), and perfusion with KH plus 400 µM L-NIL for 20 min followed by perfusion with KH plus 400 µM L-NIL plus 1 µg/ml rFGF-2 for 20 min (L-NIL + rFGF-2, n = 5). To test the effect of perfusion with the NOS inhibitors in the absence of rFGF-2, the following two additional perfusion groups were studied: perfusion with KH for 20 min followed by perfusion with KH plus 400 µM L-NAME for 20 min (L-NAME, n = 5), and perfusion with KH for 20 min followed by perfusion with KH plus 400 µM L-NIL for 20 min (L-NIL, n = 5).
Ischemia and reperfusion. The hearts were subjected to no-flow ischemia for 15 min. The organ bath was evacuated of its oxygenated solution and refilled with nitrogen-saturated perfusate. Pacing was maintained during ischemia. LV pressure was monitored throughout ischemia and reperfusion. All hearts ceased to contract within 3 min. The time for LVP to fall to 10% of baseline (TLVP10) was measured to quantify differences in LV function during early ischemia. Mean ischemic Cai2+ was calculated as the mean Cai2+ recorded from the 2nd through the 14th minute of ischemia. Contracture was defined as an abrupt and sustained rise in intraventricular pressure above 4 mmHg. Contracture time was measured as the time from the onset of ischemia to the onset of contracture. At the end of 15 min of ischemia, the nitrogen-saturated bath was replaced by the original bath maintained at 30°C. Flow was recommenced. Mean Cai2+ during early reflow was calculated as the mean of the peaks of Cai2+ recorded during the 1st minute of reperfusion. After 20 min of reperfusion, Cai2+ and functional parameters were again measured.
NOS2/ mice hearts.
Additional hearts were excised from NOS2/ mice (24)
and age-matched homozygous littermate controls (NOS2+/+). These hearts were instrumented and perfused as described in Isolated Heart Preparation and divided into two perfusion groups:
perfusion with KH for 40 min (n = 7), and perfusion
with KH for 20 min followed by perfusion with KH plus 1 µg/ml rFGF-2
for 20 min (n = 7) before 15 min of ischemia and 20 min
of reperfusion. Aequorin was injected into control and NOS2/ hearts
for estimation of Cai2+.
Measurement of NO
Additional NOS2+/+ (n = 5) and NOS2/ hearts
(n = 5) were used to measure NO concentration in the
coronary effluent using an amperometric sensor (ISO-NO, World Precision
Instrument, Sarasota, FL), according to a technique previously
described (29). Briefly, after 20 min of perfusion with
either vehicle or 1 µg/ml rFGF-2, the electrode was positioned in the
effluent to measure the amount of NO released from the coronary sinus.
Electrode calibration was performed before each experiment with NO
generated from the reaction of
S-nitroso-N-acetylpenicillamine (Sigma) with
cupric sulfate (Sigma) and acidic solution.
Quantification of NOS Gene Expression
To determine NOS2 and NOS3 mRNA levels in FGF-2-treated compared with control hearts, we performed 30 cycles of RT-PCR on equal amounts of total RNA from six control and six rFGF-2-treated hearts using primers corresponding to human NOS3 and NOS2 sequences. For NOS3, primers were as follows: 5' (sense), 5'-CAGTGTCCAACATGCTGCTGGAAATTG-3' (bases 1,050-1,076); antisense, 5'-TAAAGGTCTTCTTGGTGATGCC-3' (bases 1,511-1,535). For NOS2, primers were as follows: 5' (sense), 5'-GCCTCGCTCTGGAAAGA-3' (bases 1,425-1,441); antisense, 5'-TCCATGCAGACAACCTT-3' (bases 1,908-1,924). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was amplified from the same amount of RNA at the same time to correct for variation between different samples. The PCR products, separated on 1% agarose gels, were scanned and quantitated using Image-Quant software (Molecular Dynamics).For Northern analysis of NOS1 and NOS3 mRNA levels in hearts of
NOS2/ and wild-type mice, total RNA was prepared from freshly excised hearts as previously described (26), subjected to
electrophoresis on 1% paraformaldehyde-agarose gel, transferred to the
GeneScreen Plus membrane (Dupont), and probed with random-primed mouse
NOS1 and NOS3 cDNA probes. GAPDH cDNA probe was used to control for loading. Quantification was achieved using Image-Quant software.
Statistical Analysis
Observations made before and after drug administration were compared using Student's two-tailed paired t-test. Observations made before and after the ischemia-reperfusion protocol within a group were compared using Student's two-tailed paired t-test. Between-group comparisons were made using analysis of variance. When an overall significance was observed, multiple comparisons were performed using the Bonferroni-modified t-test. A value of P < 0.05 was considered significant. Data are expressed as means ± SE.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Baseline Conditions and Effects of Ischemia
Baseline parameters of cardiac function including myocardial Cai2+ were similar at baseline in all groups (Table 1) and were not affected by administration of L-NAME, L-NIL (not shown), or rFGF-2. Interruption of coronary flow led to an abrupt fall in LV pressure in all hearts. This fall in LV pressure during early ischemia was significantly attenuated in hearts pretreated with rFGF-2 compared with control hearts (Fig. 1). Pretreatment with rFGF-2 prolonged TLVP10 (124 ± 9 vs. 74 ± 5 s, rFGF-2 vs. control, P < 0.05) and significantly delayed the onset of contracture (893 ± 7 vs. 819 ± 36 s, rFGF-2 vs. control, P < 0.01) (Table 2).
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To explore the role of NO in mediation of this cardioprotective effect of FGF-2, we used L-NAME to inhibit all isoforms of NOS in the heart. Pretreatment with L-NAME completely blocked the cardioprotective effects of rFGF-2 during ischemia, significantly reducing TLVP10 (79 ± 2 vs. 124 ± 9 s, L-NAME + rFGF-2 vs. rFGF-2, P < 0.05) and accelerating the onset of ischemic contracture (674 ± 24 vs. 893 ± 7 s, L-NAME + rFGF-2 vs. rFGF-2, P < 0.05). However, perfusion with L-NAME alone (in the absence of rFGF-2) did not affect either TLVP10 [69 ± 3 vs. 74 ± 5 s, L-NAME vs. control, P = not significant (NS)] or the onset of ischemic contracture (820 ± 24 vs. 819 ± 36 s, L-NAME vs. control, P = NS).
To further define the type of NOS enzyme involved in this FGF-2 response, we utilized a NOS2-selective inhibitor, L-NIL. Similarly to L-NAME, L-NIL fully inhibited the cardioprotective effects of rFGF-2, significantly reducing TLVP10 (62 ± 3 vs. 124 ± 9 s, L-NIL + rFGF-2 vs. rFGF-2, P < 0.05) and accelerating the onset of ischemic contracture (652 ± 16 vs. 893 ± 7 s, L-NIL + rFGF-2 vs. rFGF-2, P < 0.05). Similarly to perfusion with L-NAME, perfusion with L-NIL alone, in the absence of rFGF-2, did not affect either TLVP10 (67 ± 6 vs. 74 ± 5 s, L-NIL vs. control, P = NS) or the onset of ischemic contracture (740 ± 39 vs. 819 ± 36 s, L-NIL vs. control, P = NS).
Stunning
Fifteen minutes of global ischemia followed by twenty minutes of reperfusion resulted in prolonged ventricular dysfunction characterized by reduced levels of LVP generation as well as significant decreases in dP/dtmax and dP/dtmin. Pretreatment with rFGF-2 significantly improved the extent of recovery of LVP compared with control (untreated) hearts (83 ± 5 vs. 61 ± 6%) and equally significant preservation of dP/dtmax and dP/dtmin (86 ± 3 vs. 65 ± 6% and 85 ± 5 vs. 60 ± 5%, respectively; Fig. 2). Stunning in hearts perfused with either NOS inhibitor by itself was not different from that in control hearts. Functional recovery of LVP in untreated control hearts (61 ± 6%) was not significantly different from that in hearts perfused with either L-NAME alone (59 ± 9%) or L-NIL alone (57 ± 6%). Depression of dP/dtmax and dP/dtmin (65 ± 6 and 60 ± 5%, respectively) in untreated hearts was similar to that in hearts perfused with L-NAME alone (60 ± 9 and 55 ± 8%, respectively) and hearts perfused with L-NIL alone (57 ± 9 and 67 ± 4%, respectively).
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Unlike initial pretreatment with rFGF-2, addition of the growth factor
to the coronary perfusate after the onset of ischemia, immediately
before reperfusion, did not improve LV function 20 min after
reperfusion (LVP 60 ± 4%, dP/dtmax
62 ± 4%, and dP/dtmin 58 ± 4%, all
P = NS vs. control). As in the case of acute ischemic changes, pretreatment with either L-NAME or
L-NIL led to a complete inhibition of rFGF-2
effects (Figs. 2 and 3).
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Role of NOS2
The preceding studies suggested that the NOS2 isoform was the primary NOS isoform responsible for FGF-2-induced preservation of myocardial function in this model. To further corroborate these results, we repeated the same studies in hearts from NOS2/ mice using their NOS2+/+ littermates as controls. Continuous recordings of
LV pressure at baseline and during ischemia and reperfusion in NOS2+/+
and NOS2/ hearts pretreated with rFGF-2 or control buffer are
illustrated in Fig. 3. As in the case of previous studies, ischemia in
both NOS2+/+ and NOS2/ hearts was characterized by an abrupt fall
in LV pressure, a gradual onset of ischemic contracture, and prolonged
ventricular dysfunction throughout 20 min of reperfusion. rFGF-2
pretreatment prolonged TLVP10, reduced the onset
of contracture, and improved LV recovery throughout reperfusion
(Table 2 and Fig. 2). However, in NOS2/ hearts, rFGF-2 failed to
provide any protective effects against global ischemia and stunning
as measured by changes in LVP, dP/dtmax, and
dP/dtmin after 20 min of reperfusion (Table 2
and Fig. 2).
Myocardial Calcium Homeostasis
Changes in myocardial Cai2+ are thought to play an important role in ischemia-induced myocardial dysfunction. Therefore, additional experiments were carried out to assess the effect of rFGF-2 administration on myocardial ionized calcium levels. Myocardial Cai2+ measured at baseline was not different between NOS2+/+ and NOS2/hearts, and pretreatment with rFGF-2 had no effect
on these levels (Table 2). Interruption of coronary flow produced
abrupt alterations in Cai2+ in all hearts, with a
gradual rise in diastolic and peak Cai2+ as ischemia
progressed. Mean ischemic Cai2+, Cai2+
averaged from the 2nd through the 14th minute of ischemia, was not
affected by rFGF-2 pretreatment and was the same in NOS2+/+ and
NOS2/ hearts (Table 2). Restoration of coronary flow was followed
by a marked increase in myocardial Cai2+. Neither the
extent of this increase nor peak Cai2+ levels were
affected by rFGF-2 administration in NOS2+/+ or NOS2/ hearts (Table
2).
Release of NO and FGF-2 Effects on NOS Gene Expression
To directly demonstrate the role of rFGF-2-induced NO release, we measured the concentration of NO in coronary effluent before and after rFGF-2 administration. NO concentration increased significantly after perfusion with rFGF-2 compared with measurements after perfusion with vehicle (236 ± 24 vs. 190 ± 25 nM/g, P < 0.05) in wild-type hearts. In contrast, perfusion with rFGF-2 did not increase NO concentration in NOS2/ hearts
compared with NO values measured after perfusion with vehicle (170 ± 24 vs. 154 ± 46 nM/g, P = NS) (Fig.
4A). To assess whether rFGF-2
increased NO production by stimulating NOS enzyme or increasing its
gene expression, we carried out RT-PCR analysis of NOS2 and NOS3 mRNA
levels before and after 40 min of exposure to rFGF-2. No differences in
either NOS2 or NOS3 levels were detected (Fig. 4B).
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The "knockout" of the NOS2 gene may have affected expression of
NOS1 or NOS3 genes in these mice. To evaluate this possibility, we
performed Northern analysis of NOS1 and NOS3 gene expression in hearts
from C57/BL6 NOS2+/+ and NOS2/ mice. No significant changes in
expression of either gene compared with that in control mice were
detected (Fig. 4, C and D).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Myocardial stunning is the phenomenon described by Braunwald and Kloner (6) whereby an ischemic insult interferes with normal cardiac function, cellular processes, and ultrastructure for prolonged periods. Numerous mechanisms of myocardial stunning have been proposed, the most probable of which include generation of oxygen-derived free radicals, metabolic impairment, and calcium overload (see Ref. 3 for review). Recently, a number of pharmacological agents and physiological manipulations have been shown to induce early or late ischemic preconditioning, a state characterized by reduced susceptibility to postischemic decline in myocardial function. In particular, FGF-2 has been demonstrated to improve myocardial function in the setting of acute myocardial ischemia both in vivo (17, 44) and in isolated rat heart studies (34). The well-known angiogenic effects of FGF-2, however, occur too gradually to be relevant in such settings (43). The purpose of this study, therefore, was to study the potential role of NO release in FGF-2-mediated cardioprotection and to define the NOS isoform responsible for FGF-2-induced NO release.
We chose a recently described murine model of myocardial stunning, characterized by a rise in Cai2+, myocardial contracture during ischemia, Cai2+ overload during early reperfusion, and prolonged ventricular dysfunction (12). In this model, pretreatment with FGF-2 before the onset of ischemia significantly delayed the fall of LV pressure during ischemia, reduced ischemic contracture, and improved functional recovery following reperfusion. Interestingly, administration of FGF-2 at the time of reperfusion did not improve myocardial recovery. This would suggest that FGF-2 activates a signaling pathway that is protective before the onset of ischemia, akin to other instances of ischemic preconditioning. Perfusion with FGF-2 did not alter LV function or Cai2+ at baseline (Tables 1 and 2). The inotropic state of the heart before the induction of ischemia, therefore, could not account for the differences in recovery.
A number of experimental observations suggest that NO generation before ischemia may ameliorate ischemic stress (9, 33, 34). In particular, enhanced NO release appears to be an important mechanism in development of late preconditioning against myocardial stunning (4, 36). Although the specific isoform of NOS involved in this response has not been identified, a recent study implicated NOS2 as a potential key enzyme (40). The levels of NO production that we report in isolated mouse hearts are similar to those reported in isolated rat hearts (29). Moreover, previous studies (10) showed that augmenting endogenous NO production before ischemia and reperfusion significantly improved functional recovery. However, others (28) showed that NO augmentation may increase postischemic injury and that NOS antagonists may improve recovery from ischemia and reperfusion. Moreover, increased NO production by cardiomyocytes may contribute to cytokine-induced contractile dysfunction characteristic of systemic sepsis and some cardiomyopathies (1).
The antagonism of FGF-2 benefits by pretreatment with L-NAME in the current study would suggest that NO generation was a factor in resistance to ischemic injury in our model. To test whether the NOS2 isoform is a primary mediator of FGF-2-induced ischemic preconditioning, we examined the effects of the selective NOS2 inhibitor L-NIL in a concentration shown to be effective in selectively blocking NOS2 (7, 11). Whereas pretreatment with L-NIL by itself did not alter any of the baseline parameters or change the extent of recovery, it completely abrogated the cardioprotective effects of FGF-2. To confirm this conclusion, we employed a mouse strain with deletions of both copies of the NOS2 gene. In these hearts, unlike those of NOS2+/+ littermate controls, pretreatment with FGF-2 was completely ineffective in either increasing NO production before ischemia (Fig. 4) or reducing ischemia-induced myocardial stunning (Figs. 2 and 3). Taken together, these results demonstrate that the cardioprotective effects of FGF-2 are mediated by NO and implicate NOS2 as the primary activator.
Given the relatively short time interval from FGF-2 treatment to the observed effect, it appears more likely that the growth factor stimulated the activity of the NOS2 enzyme rather than increasing its levels of expression. RT-PCR analysis of NOS2 mRNA 40 min after FGF-2 administration failed to demonstrate any change in NOS2 message level, suggesting that changes in enzyme activity, and not expression, are responsible for the observed effect.
The activity of the NOS2 enzyme is thought to be determined by the level of the NOS2 protein, by the availability of a cofactor, tetrahydrobiopterin (BH4), and by cellular levels of glutathione (39). In particular, BH4 levels, primarily determined by the activity of GTP cyclohydrolase I, play a critical role in regulation of inducible NOS activity. Expression of human NOS2 gene in rat smooth muscle cells that do not produce BH4 under normal conditions is ineffective in stimulating NOS2 activity. However, cotransfection of NOS2 with GTP cyclohydrolase I in the same cells resulted in significant increase of NOS2 activity (41). Similarly, glutathione presence is essential for maximal NOS2 enzyme activity (15). Reduction of glutathione levels, which would occur in the setting of ischemia, would lead to a significant reduction in NOS2 activity. Thus rFGF-2 could potentially induce NOS2 activity by increasing cellular levels of BH4 or preventing ischemia-induced reduction in glutathione concentration.
There may be multiple mechanisms by which NO provides protection from
ischemia-reperfusion injury. NO may reduce reperfusion injury by acting
as a scavenger of oxygen free radicals (29), whereas
inhibition of NO synthesis may worsen myocardial stunning due to a
mismatch between oxygen demand and supply (16).
Furthermore, endogenous NO has an energy-sparing effect that reduces
the amount of stunning after reperfusion (33). NO may also
play a significant role in transmembrane signaling in the ischemic
heart (21, 39), including the activation of
protein kinase C-
(35). Finally, the role of NO in
mitigating stunning may be related to NO stimulation of guanylyl
cyclase within ventricular myocytes (2). Furthermore, reduction of cGMP by NO (32) and consequent reduction in
Ca2+ influx may attenuate the harmful increases in
Cai2+ during ischemia and reperfusion.
Our Cai2+ data, however, suggest that FGF-2 may play
only a minor role in modulating levels of Cai2+ at
baseline, during ischemia, and during early reperfusion. These observations contradict a recent report that FGF-2 has a negative inotropic effect on adult rat cardiac myocytes that may have resulted from alterations in Cai2+ homeostasis
(20). No differences in Cai2+ overload
during early reperfusion are apparent between NOS2/ hearts perfused
with or without rFGF-2. The energy-sparing effects provided by
FGF-2-induced NO production, therefore, may be a predominant mechanism
for cardioprotection during ischemia, because FGF-2 prolongs
TLVP10 during ischemia and delays the onset of
ischemic contracture in wild-type hearts. No such effects were observed in NOS2/ hearts perfused with FGF-2. We speculate, therefore, that
FGF-2 activation of NO may attenuate the effects of depletion of energy
substrates during acute myocardial ischemia and reperfusion, thereby
mitigating ischemic stress and reducing the extent of stunning
after reperfusion.
In summary, FGF-2 attenuates myocardial stunning, independent of alterations in Cai2+, by increasing NO production via NOS2-dependent pathways. Pretreatment with FGF-2 may be an effective way of modulating NO levels and limiting ischemia-induced alterations in myocardial function.
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ACKNOWLEDGEMENTS |
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We express appreciation to Diego Yepes for expert assistance in preparation of this manuscript.
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FOOTNOTES |
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This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-53793 and an American Heart Association (AHA) Established Investigator Award (to M. Simons), AHA-Massachusetts Affiliate Grant 13-455-967 (to T. G. Hampton), and a research grant from Chiron Corporation. I. Amende received support from Förderkreis zur Verbesserung des Gesundheitswesens eV. M. Simons is an Established Investigator of the AHA.
This work was presented in part at the 71st Scientific Sessions of the AHA, Dallas, TX, November 1998.
Address for reprint requests and other correspondence: M. Simons, Angiogenesis Research Center, RW-453, Beth Israel Deaconess Medical Center, RW453, 330 Brookline Ave., Boston, MA 02215 (E-mail: msimons{at}caregroup.harvard.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. §1734 solely to indicate this fact.
Received 4 October 1999; accepted in final form 12 January 2000.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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