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(Circulation. 1997;96:2501-2504.)
© 1997 American Heart Association, Inc.

Articles

Negative Inotropic Effect of Basic Fibroblast Growth Factor on Adult Rat Cardiac Myocyte

Yuji Ishibashi, MD; Yoshitoshi Urabe, MD; Hiroyuki Tsutsui, MD; Shintaro Kinugawa, MD; Masaru Sugimachi, MD; Masaru Takahashi, MD; Shimako Yamamoto, MD; Hirofumi Tagawa, MD; Kenji Sunagawa, MD; ; Akira Takeshita, MD

From the Research Institute of Angiocardiology and Cardiovascular Clinic, Kyushu University School of Medicine (Y.I., Y.U., H. Tsutsui, S.K., M.T., S.Y., H. Tagawa, A.T.), Fukuoka, and Department of Cardiovascular Dynamics, National Cardiovascular Center (M.S., K.S.), Suita, Osaka, Japan.

Correspondence to Hiroyuki Tsutsui, MD, PhD, Research Institute of Angiocardiology and Cardiovascular Clinic, Kyushu University School of Medicine, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-82, Japan.

	Abstract

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Background Basic fibroblast growth factor (bFGF) is highly expressed in the myocardium in some cardiac disorders, such as ischemia-reperfusion and cardiac allograft rejection. However, whether bFGF has any effects on myocardial contraction is unknown.

Methods and Results We examined the effects of bFGF on myocardial contractility using isolated adult rat cardiac myocyte preparations. bFGF exerted a direct negative inotropic effect that was concentration and time dependent. The pretreatment of myocytes with a neutralizing anti-bFGF antibody (100 ng/mL) abolished the negative inotropic effects of bFGF (100 ng/mL). Platelet-derived growth factor (12.5 ng/mL) and transforming growth factor-ß (1 ng/mL) did not exert such effects, which indicated that bFGF-induced negative inotropism was considered to be specific for this growth factor. bFGF decreased the peak intracellular Ca²⁺ transient by 46% during systole. The enhanced production of nitric oxide was unlikely to be responsible for the bFGF-induced negative inotropic effect.

Conclusions bFGF, primarily a potent growth promoter, produced acute negative inotropic effects in the adult cardiac myocyte that could have resulted from alterations in intracellular Ca²⁺ homeostasis. The negative inotropic effect of bFGF may contribute to myocardial dysfunction associated with ischemia-reperfusion injury and heart transplant rejection.

Key Words: growth substances • cells • muscles • contractility • calcium

	Introduction

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Basic fibroblast growth factor (also known as FGF-2) is a multifunctional regulator protein of growth promotion and differentiation in skeletal muscle¹ as well as in cardiac myocytes.² bFGF also has been shown to function in some way to promote survival in adult cardiac myocytes.³ bFGF and its receptor have been demonstrated to be present in fully differentiated adult myocytes.⁴ Recently, bFGF has been shown to have a sparing effect on myocardial infarction. Yanagisawa-Miwa and coworkers⁵ demonstrated that bFGF reduced infarct size and that this reduction was associated with an increase in capillary density. Recently, Horrigan et al⁶ demonstrated that bFGF could reduce myocardial infarct size after temporary coronary occlusion without exerting any hemodynamic effects on preexisting collateral flow or inducing neovascularization. Moreover, bFGF is highly expressed in myocardium suffering from ischemia-reperfusion injury,⁷ which is accompanied by transient depression of myocardial contractility. bFGF is also elevated in the myocardium during cardiac allograft rejection,⁸ wherein LV contractile dysfunction is frequently observed even though there are only minor histological changes in the myocardium. However, the role of bFGF in the pathogenesis of myocardial contractile defects in these pathological conditions is not known. It is conceivable that highly expressed bFGF participates in the development of contractile dysfunction. bFGF may exert an effect on myocardial contractility similar to IL-1ß and IL-6 by inducing NO because it shares structural similarities with the interleukins. However, to the best of our knowledge, no study has examined whether bFGF modulates myocardial contractility.

The purpose of the present study was to examine the direct functional effects of bFGF on myocardial contractility and [Ca²⁺]i handling at the cellular level. We also investigated whether the effect of bFGF on myocardial contractility was mediated by generation of NO in myocytes. We used isolated myocyte preparations in the present study because LV contractile function in vivo could be affected by the vasodilator effects of bFGF.⁹

	Methods

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Effects of bFGF on Myocyte Viability
Cardiac myocytes were isolated from the rat heart according to methods described previously,¹⁰ with some modifications. To exclude the possibility that bFGF (Takeda Chemical Industries) was acutely cytotoxic to the myocytes, freshly isolated cells were treated for 30 minutes with 100 ng/mL bFGF, and viability was assessed by use of the index of the percentage of rod-shaped cells in 10 randomly chosen 1x1-mm fields and the percentage of rod-shaped cells in 10 randomly chosen 1x1-mm fields excluding 0.4% trypan blue dye.

Effects of bFGF on Myocyte Morphology
To examine the effects of bFGF on myocyte morphology, photomicrographs of isolated myocytes were obtained during treatment with 100 ng/mL bFGF for 60 minutes, and their two-dimensional surface area was determined by digitizing the lateral edges of myocytes (final magnification x650). Myocyte length and width were determined from these cell images as the maximum values for each of these two parameters.

Effects of bFGF on Myocyte Contractility
To determine whether bFGF had direct effects on the contractility of isolated myocytes, we evaluated contractile function by analyzing sarcomere motion during electrical stimulation (0.25 Hz) using laser diffraction techniques as described previously.¹⁰

After baseline contraction was recorded, bFGF (0.01 to 100 ng/mL) was added to the superfusate and applied to the myocytes, and the contraction was sequentially recorded on the same cells to determine the effects of bFGF on myocyte contractile function. To determine the time course of the bFGF-induced effect on sarcomere shortening, bFGF was added to the bathing medium, and sequential samples of contraction were recorded from a single myocyte after treatment. To determine whether an increase in myocyte NO was responsible for mediating the negative inotropic effects of bFGF, myocytes were pretreated with the NO synthase inhibitor L-NAME (0.1 mmol/L) for 15 minutes according to the methods described previously,¹⁰ and the contractile function was examined sequentially on the same cells. To further determine whether the bFGF-induced effect was specific for this growth factor, we examined the effects of PDGF (12.5 ng/mL) and TGF-ß (1 ng/mL) on myocyte contractile function.

Measurement of [Ca²⁺]i Transient
To determine whether the bFGF-induced effect was the result of changes in Ca²⁺ activation of the cells, we measured the [Ca²⁺]i transient in the isolated myocyte preparations using the calcium-selective fluorescent dye indo 1 (Molecular Probes Inc). Cardiac myocytes, isolated as described above, were loaded with 5 µmol/L indo 1-AM in the buffer supplemented with fatty acid–free 0.5% BSA and 0.03% Pluronic F-127 at room temperature. The cells were subsequently washed with buffer with 1 mmol/L Ca²⁺ for 30 minutes before being used for experiments. Indo 1–loaded myocytes were placed in a chamber on the stage of an epifluorescence microscope (Olympus) and stimulated to contract by a pair of platinum wire electrodes. A single myocyte was then excited at 350 nm by epi-illumination, and indo 1 fluorescent emission light, split by a 455-nm dichroic mirror and selected by use of rectangular band-pass interference filters in the wavelength ranges of 380 to 430 nm (405-nm channel) and 455 to 505 nm (480-nm channel), was directed to a pair of photomultiplier tubes. The photocurrent from each tube was integrated at a 1-ms interval, and the ratio of indo 1 emission at the two wavelengths was calculated as an index of [Ca²⁺]i by a computer. The results were expressed as a fluorescence ratio rather than as absolute [Ca²⁺]i values because of the difficulties in obtaining quantitative calibration owing to significant variation in the degree of compartmentalization of this indicator from cell to cell. Analysis of [Ca²⁺]i transients was performed by averaging four to six successive recordings to improve the signal-to-noise ratio. We examined the effects of bFGF on [Ca²⁺]i by recording the sequential [Ca²⁺]i transients in the same cells after treatment of myocytes with 100 ng/mL bFGF. The methods used to apply bFGF to myocytes were identical to those used for myocyte contraction studies.

Statistical Analysis
Data are expressed as mean±SEM. For multiple comparisons, one-way ANOVA was used to evaluate mean differences in conjunction with post hoc t test with Scheffé's correction. The Student's paired t test was used to evaluate the effects of bFGF on [Ca²⁺]i transient parameters. All tests were considered statistically significant at P<.05.

	Results

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There was no significant decline in the percentage of viable rod-shaped cells (72±4% versus 71±5%; P=NS) after exposure to 100 ng/mL bFGF, and virtually all rod-shaped myocytes treated with bFGF excluded trypan blue dye, indicating that they were functionally intact during the period of study. bFGF did not affect the cellular size of isolated cardiac myocytes (data not shown). bFGF reduced the extent and maximum velocity of sarcomere shortening in a time-dependent manner (Fig 1A). It exerted a definite negative inotropic effect after 20 minutes of exposure, and no further decline was noted after 30 to 60 minutes of exposure. Fig 1B shows the dose-dependent effect of bFGF on the sarcomere shortening velocity, wherein its effect was evident at a dose of 1 ng/mL and peaked at 10 ng/mL. Diastolic sarcomere length did not change at any concentration of bFGF (data not shown). It took >20 minutes to determine the maximum response to bFGF, and therefore the effects of graded doses of bFGF could not be examined in the same cell. The dose-response relation was constructed from the data obtained in different myocytes; one myocyte was used for only one concentration of bFGF. The number of myocytes used for each concentration of bFGF was five to seven. The negative inotropic response persisted even 30 minutes after removal of bFGF from the superfusion medium. Neither PDGF nor TGF-ß exerted a negative inotropic effect on cardiac myocytes (Fig 1B). The specificity of the bFGF-induced negative inotropic effects was confirmed by use of anti-human bFGF antibody to neutralize the effects of bFGF. The anti-bFGF antibody alone (100 ng/mL) had no effects on the sarcomere-shortening mechanics, whereas pretreatment with this antibody completely abolished the negative inotropic effects induced by bFGF (Fig 1C). Pretreatment of myocytes with L-NAME itself did not significantly alter myocyte contractility and did not attenuate the bFGF-induced negative inotropic effects (Fig 1C).

View larger version (25K): [in this window] [in a new window]   Figure 1. A, Sequential samples from a single cardiac myocyte treated with 100 ng/mL of bFGF where for each time point, sarcomere length (SL) versus time is given in the upper panel and the rate of length change versus time is given in the lower panel. bFGF exerted a time-dependent decrease in sarcomere shortening and its velocity. B, Effects of varying concentrations of bFGF (0.01 to 100 ng/mL), PDGF (12.5 ng/mL), and TGF-ß (1 ng/mL) on cardiac myocyte contractile function. Compared with time-control myocytes treated with vehicle, the treatment of myocytes with bFGF resulted in a concentration-dependent decrease in the sarcomere shortening velocity (SSV). Neither PDGF nor TGF-ß affected SSV. Values are indicated as the percent of baseline values. C, Effects of a neutralizing anti-bFGF antibody (Ab) and L-NAME on the bFGF-induced negative inotropic effects. Anti-bFGF antibody alone had no effects on the SSV, whereas pretreatment of myocytes with this antibody completely abolished the effects by bFGF. L-NAME alone had no effects on myocyte contractility and did not attenuate the bFGF-induced negative inotropic effects. All data are expressed as mean±SEM. n=4 to 10 cells for each treatment. *P<.05 and **P<.01 compared with control values.

Fig 2A shows an example of [Ca²⁺]i fluorescence transient obtained from the myocyte before and 20 minutes after treatment with bFGF. bFGF decreased the peak fluorescence ratio without altering the resting fluorescence ratio (Fig 2B and 2C). The amplitude of [Ca²⁺]i was decreased to 54% of the baseline value. Furthermore, bFGF did not alter the time course of the [Ca²⁺]i transient (data not shown). The time-dependent decrease in the amplitude of the [Ca²⁺]i transient paralleled the changes in sarcomere shortening. L-NAME did not attenuate the reduction of the peak [Ca²⁺]i transient induced by bFGF (data not shown). Thus, the negative inotropic effects induced by bFGF likely resulted from the alteration in [Ca²⁺]i homeostasis.

View larger version (18K): [in this window] [in a new window]   Figure 2. A, An example of the bFGF-induced effects on [Ca2+]i transients. Recordings were obtained sequentially from a single cardiac myocyte before and after 20 minutes' exposure to bFGF (100 ng/mL). B and C, Summary data for the effects of bFGF on diastolic fluorescence ratio and transient amplitude (peak minus diastolic fluorescence ratio). bFGF decreased the peak indo 1 fluorescence ratio but did not alter the diastolic fluorescence ratio. bFGF also did not affect the time course of the [Ca2+]i transient.

	Discussion

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The results of the present study demonstrate for the first time that bFGF exerts a direct negative inotropic effect on fully differentiated adult myocytes that is concentration and time dependent. The pretreatment of myocytes with a neutralizing anti-bFGF antibody completely abolished the negative inotropic effects of bFGF. These effects were not observed in other growth factors and are considered to be a specific action of bFGF. Because the effect of bFGF on the contractile function was studied with the use of isolated myocyte preparations, we could obviate the potential problems of diffusion limitation and the presence of cells other than myocytes that might bind and respond to bFGF or metabolize this growth factor in intact tissue preparations. Thus, bFGF-induced effects were not mediated by release of soluble factors from nonmyocytes.

The rapidity of the bFGF-induced negative inotropic effects raises the possibility that bFGF may directly modulate excitation-contraction coupling. To determine whether bFGF-induced negative inotropic effects resulted from alteration of Ca²⁺ activation in the cells, we measured the [Ca²⁺]i transient in the same myocyte preparations using calcium-selective fluorescent dye and showed that bFGF decreased the amplitude of [Ca²⁺]i to 54% of the baseline values. Therefore, the negative inotropic effects of bFGF could be the direct result of alterations in [Ca²⁺]i homeostasis. However, contradictory results have been reported concerning the bFGF-induced effects on Ca²⁺ homeostasis in myoblasts,¹¹ neonatal rat myocytes,¹² and fibroblasts.¹³ Although the precise reasons for the discrepant findings between the present study and others are not apparent, the disparity may be related to the differences between skeletal and cardiac muscles or to the developmental differences between neonatal and adult myocyte preparations. The exact intracellular mechanism for the alterations in Ca²⁺ homeostasis is not clear from the present study, and further studies focusing on the effects of bFGF on sarcolemmal Ca²⁺ current using a patch-clamp technique¹³ and the Ca²⁺ uptake/release function of the sarcoplasmic reticulum are needed.

Recent reports have shown that inflammatory cytokines such as IL-6 and TNF- have negative inotropic effects that might be mediated, at least in part, by the enhanced production of NO in cardiac myocytes. In addition, bFGF could induce vasodilation, which has been shown to be mediated by ATP-sensitive potassium channels as well as by NO.⁹ However, the present results indicate that myocyte NO is unlikely to be responsible for the bFGF-induced contractile abnormalities. Furthermore, bFGF-induced cytotoxicity is unlikely to be responsible for the effect of bFGF on cellular function because there was no significant decline in the percentage of viable rod-shaped cells after exposure.

Compared with the effective doses of bFGF reported in other studies (1 to 10 ng/mL),¹⁴ the concentration of bFGF that produced significant effects on myocyte contractility in the present study (10 to 100 ng/mL) seems to be relatively high. However, the effects of bFGF obtained in cultured cells were difficult to interpolate to our experimental conditions. Moreover, it is uncertain whether the concentrations of bFGF used in the present study are comparable to those occurring in vivo under pathophysiological conditions. It may be possible that the accumulation of bFGF in an autocrine/paracrine manner might reach much higher local concentrations. The major advantage of assessing the myocardial contractility by use of isolated myocyte preparations is that it permits the direct examination of contraction in the absence of any confounding cell-to-cell or cell-to-interstitium interactions. Even though myocyte contractile performance obtained under externally unloaded conditions could not be simply equated with the multicellular tissue or ventricular level, the usefulness of the isolated cardiac myocyte model has been well validated.¹⁰ We therefore believe that the results obtained from isolated myocytes in the present study could be applied to cardiac function at the LV level.

The present study provides compelling evidence that bFGF exerts direct negative inotropic effects in terminally differentiated adult cardiac myocytes. bFGF has been shown to be highly expressed in ischemia-reperfusion⁷ and cardiac allograft rejection,⁸ in which LV ejection performance is often depressed. Although direct correlation between the short-term in vitro effect at the myocyte level and the long-term in vivo effects at the organ level may not be appropriate, the present study at least provides a potential cellular mechanism for the myocardial contractile dysfunction in pathological conditions such as ischemia-reperfusion and cardiac allograft rejection. bFGF might be cardioprotective against ischemia-induced myocardial damage by suppressing the energy requirement for contraction, which has been suggested by recent studies of ischemia- reperfusion.^{6 7}

	Selected Abbreviations and Acronyms

 

[Ca2+]i = intracellular Ca2+ bFGF = basic fibroblast growth factor IL = interleukin L-NAME = N-nitro-l-arginine methyl ester LV = left ventricular NO = nitric oxide PDGF = platelet-derived growth factor TGF = transforming growth factor

	Acknowledgments

 
This study was supported in part by grants from the Ministry of Education, Science, and Culture (No. 06670726, 06274223, 07266220, 07670789, and 08258221) and the CASIO Scientific Promotion Foundation. We thank Sayaka Shimizu and Erina Tajima for their technical assistance and Dr Nobumasa Ishide in the First Department of Internal Medicine, Tohoku University, for valuable suggestions.

	Footnotes

 
Dr Urabe's present address is Department of Cardiovascular Medicine, Kitakyushu Municipal Medical Center, 2-1-1 Bashaku, Kitakyushu, Fukuoka 802, Japan.

Previously published in abstract form (Circulation. 1995;92(suppl I):I-182).

Received June 19, 1997; revision received August 4, 1997; accepted August 7, 1997.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Kudla AJ, John ML, Bowen-Pope DF, Rainish B, Olwin BB. A requirement for fibroblast growth factor in regulation of skeletal muscle growth and differentiation cannot be replaced by activation of platelet-derived growth factor signaling pathways. Mol Cell Biol.. 1995;15:3238-3246.[Abstract]

2. Peters KG, Werner S, Chen G, Williams LT. Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse. Development.. 1992;114:233-243.[Abstract]

3. Speir E, Tanner V, Gonzalez AM, Farris J, Baird A, Casscells W. Acidic and basic fibroblast growth factors in adult rat heart myocytes: localization, regulation in culture, and effects on DNA synthesis. Circ Res.. 1992;71:251-259.[Abstract/Free Full Text]

4. Liu L, Pasumarthi KBS, Padua RR, Massaeli H, Fandrich RR, Pierce GN, Cattini PA, Kardami E. Adult cardiomyocytes express functional high-affinity receptors for basic fibroblast growth factor. Am J Physiol.. 1995;268:H1927–H1938.[Abstract/Free Full Text]

5. Yanagisawa-Miwa A, Uchida Y, Nakamura F, Tomaru T, Kido H, Kamijo T, Sugimoto T, Kaji K, Utsuyama M, Kurashima C, Ito H. Salvage of infarcted myocardium by angiogenic action of basic fibroblast growth factor. Science.. 1992;257:1401-1403.[Abstract/Free Full Text]

6. Horrigan MCG, MacIsaac AI, Nicolini FA, Vince DG, Lee P, Ellis SG, Topol EJ. Reduction in myocardial infarct size by basic fibroblast growth factor after temporary coronary occlusion in a canine model. Circulation.. 1996;94:1927-1933.[Abstract/Free Full Text]

7. Padua RR, Sethi R, Dhalla NS, Kardami E. Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury. Mol Cell Biochem.. 1995;143:129-135.[Medline] [Order article via Infotrieve]

8. Ationu A, Carter N. Ventricular expression of basic fibroblast growth factor gene after orthotopic cardiac transplantation. Transplantation.. 1994;57:1364-1366.[Medline] [Order article via Infotrieve]

9. Cuevas P, Carceller F, Ortega S, Zazo M, Nieto I, Gimenez-Gallego G. Hypotensive activity of fibroblast growth factor. Science.. 1991;254:1208-1210.[Abstract/Free Full Text]

10. Yamamoto S, Tsutsui H, Tagawa H, Saito K, Takahashi M, Tada H, Yamamoto M, Katoh M, Egashira K, Takeshita A. Role of myocyte nitric oxide in ß-adrenergic hyporesponsiveness in heart failure. Circulation.. 1997;95:1111-1114.[Abstract/Free Full Text]

11. Peters KG, Marie E, Wilson E, Ives HE, Escobedo J, Del Rosario M, Mirda D, Williams LT. Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca²⁺ flux but not mitogenesis. Nature.. 1992;358:678-681.[Medline] [Order article via Infotrieve]

12. Merle P-L, Feige J-J, Verdetti J. Basic fibroblast growth factor activates calcium channels in neonatal rat cardiomyocytes. J Biol Chem.. 1995;270:17361-17367.[Abstract/Free Full Text]

13. Munaron L, Distasi C, Carabelli V, Baccino FM, Bonelli G, Lovisolo D. Sustained calcium influx activated by basic fibroblast growth factor in Balb-c 3T3 fibroblasts. J Physiol.. 1995;484:557-566.[Abstract/Free Full Text]

14. Parker TG, Packer SE, Schneider MD. Peptide growth factors can provoke `fetal' contractile protein gene expression in rat cardiac myocytes. J Clin Invest.. 1990;85:507-514.

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