- Email: [email protected]
Granulocyte colony-stimulating factor improves early remodeling in isoproterenol-induced cardiac injury in rats
Pharmacological Reports
Copyright © 2012 by Institute of Pharmacology Polish Academy of Sciences
2012, 64, 643649 ISSN 1734-1140
Granulocyte colony-stimulating factor improves early remodeling in isoproterenol-induced cardiac injury in rats Ludimila Forechi, Marcelo P. Baldo, Diana Meyerfreund, José G. Mill Department of Physiological Sciences, Federal University of Espirito Santo, Av. Marechal Campos 1468, Maruipe, 29042-755, Vitória, Espírito Santo, Brazil Correspondence: José G. Mill, e-mail:
[email protected]; [email protected]
Abstract: Background: Granulocyte colony-stimulating factor (G-CSF) has been used in some animal models and humans with wellestablished cardiovascular diseases. However, its effects in the initial stage of progressive non-ischemic heart failure are unknown. Methods: Wistar rats (260–300 g) were divided into three groups: control (without any intervention), ISO (150 mg/kg isoproterenol hydrochloride sc, once a day for two consecutive days), and ISO-GCSF (50 μg/kg/d G-CSF for 7 days beginning 24 h after the last administration of ISO). Echocardiography was performed at baseline and after 30 days of follow-up. Subsequently, animals were anesthetized for hemodynamic analysis. The left ventricle was removed for analysis of interstitial collagen deposition and cardiomyocyte hypertrophy. Results: Isoproterenol led to left ventricular dilation (control, 7.7 ± 0.14 mm; ISO, 8.7 ± 0.16 mm; ISO-GCSF 7.8 ± 0.09 mm; p < 0.05), myocardial fibrosis (control, 2.0 ± 0.18%; ISO, 9.1 ± 0.81%; ISO-GCSF 5.9 ± 0.58%; p < 0.05) and cardiomyocyte hypertrophy (control, 303 ± 10 μm ; ISO, 356 ± 18 μm ; ISO-GCSF 338 ± 11 μm ; p < 0.05). However, G-CSF partially prevented collagen deposition and left ventricular enlargement, with a slight effect on hypertrophy. Characterizing a compensated stage of disease, hemodynamic analysis did not change. Conclusion: G-CSF administered for 7 days was effective in preventing the onset of ventricular remodeling induced by high-dose isoproterenol with decreased collagen deposition and chamber preservation. Key words: isoproterenol, heart failure, G-CSF, collagen
Abbreviations: CVF – collagen volume fraction, dP/dt – maximal rate of pressure, FS – fractional shortening, G-CSF – granulocyte colony-stimulating factor, HR – heart rate, ISO – isoproterenol, LVEDD – left ventricular end-diastolic dimension, LVEDP – left ventricle end-diastolic pressure, LVESD – left ventricular peak systolic dimension, LVSP – left ventricle systolic pressure, MCSA – myocyte cross sectional area
Introduction Massive sympathetic discharge, as observed in extreme stress or with high activity of the central nervous system, has deleterious effects on the heart. In
these cases, high oxygen consumption by the myocardium leads to subendocardial ischemia and infarction followed by high levels of oxidative stress, ventricular dilation, fibrosis, progressive systolic dysfunction and heart failure [6, 17, 29]. Non-invasive animal models have been used to mimic the pathophysiological characteristics of this cardiomyopathy in humans [7]. Rats treated with high doses of the nonselective b-adrenergic receptor agonist isoproterenol are a widely used model to assess progressive left ventricular dysfunction. Although the mechanisms by which isoproterenol induces myocardial injury are not completely understood, b-adrenergic receptor overstimulation leads to intense coroPharmacological Reports, 2012, 64, 643649
643
nary vasoconstriction and the development of microinfarcts that more frequently affect the subendocardium or transmural region of the left ventricle wall [10, 27, 28]. Teerlink et al. [29] showed progressive left ventricular dilation and dysfunction associated with long-term mortality rates after high doses of isoproterenol. Recently, the therapeutic use of granulocyte colony-stimulating factor (G-CSF) has been reported in some tissues, such as heart, kidney and brain [4, 21, 31]. Studies have suggested that this cytokine can act in damaged hearts by recruiting bone marrow-derived cells and promoting angiogenesis, reducing apoptosis and modulating collagen content, thus improving overall cardiac function [1, 2, 12, 16, 19, 22, 23]. Most of these studies have been performed in different models of ischemic cardiomyopathy. Little is known about the potential cardioprotective effects of G-CSF in non-ischemic cardiomyopathies. Therefore, this study sought to evaluate the effects of G-CSF treatment on structural and functional cardiac parameters during cardiomyopathy induced by high-dose isoproterenol in rats.
Echocardiography
Transthoracic echocardiography was performed at baseline and 4 weeks after isoproterenol treatment as previously described [9]. Rats were intraperitoneally anesthetized with ketamine (50 mg/kg; Agener, Brazil) plus xylazine (10 mg/kg; Bayer, Brazil) and placed in the supine position, and electrodes were placed in the subcutaneous tissue of limbs for continuous electrocardiogram recordings. M-mode images were acquired with an echocardiograph (Aplio XG SSA-790, Toshiba, Japan) using a 7.5 MHz transducer transverse to the left ventricle at the level of papillary muscles. M-mode values of left ventricular enddiastolic dimension (LVEDD) and left ventricular peak systolic dimension (LVESD) were measured for each animal as the average of four consecutive cardiac cycles under regular rhythm. Heart rate (HR), fractional shortening {FS = ((LVEDD – LVESD)/ LVEDD) × 100)} and ejection fraction were obtained. The measures were performed by an investigator blinded to groups at both times. Hemodynamic analysis
Materials and Methods
Animals and groups
Male Wistar rats (260–300 g) were acquired from the animal facility at the Federal University of Espírito Santo and randomly assigned into three groups: a control group, without any intervention; the ISO group, which received isoproterenol hydrochloride (150 mg/kg, sc, Sigma-Aldrich Inc., USA) once a day for two consecutive days; and the ISO-GCSF group, which received G-CSF (Biosintetica, 50 μg/kg, sc) treatment for 7 days beginning 24 h after the last isoproterenol dose. Cardiac function in these animals was studied 3 weeks after the last G-CSF dose. Animals had free access to water and standard rat chow throughout the experimental period, and all of the procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85–23, revised 1996) and were approved by the institutional animal research committee (No. 012/2010). 644
Pharmacological Reports, 2012, 64, 643649
Four weeks after the first isoproterenol dose, animals were anesthetized with ketamine (90 mg/kg) and xylazine (10 mg/kg), and hemodynamic measurements were performed as previously reported [3]. Briefly, the right common carotid artery was dissected to insert a fluid-filled polyethylene catheter (P50) connected to a blood pressure transducer (TRI 21, Letica Scientific Instruments, Spain). The catheter was then advanced into the left ventricle to record peak systolic pressure (LVSP), end-diastolic pressure (LVEDP), and heart rate (HR) in cardiac cycles under regular cardiac beats. The maximal rate of pressure rise and fall (+dP/dt and –dP/dt) was obtained electronically (Powerlab/4SP ML750, AdInstruments, Australia) and used as left ventricular contractility and relaxation indexes, respectively. Determination of myocyte hypertrophy and fibrosis
After hemodynamic recordings, the heart was removed and rapidly washed with cold saline solution, and the ventricles were separated from the atria, blotted dry and weighed. The left ventricle was divided into three slices of approximately 2 mm, which were
G-CSF prevents early cardiomyopathy in rats Ludimila Forechi et al.
prepared for histology. Each slice was serially cut into 6 μm-thick transverse sections and stained with Sirius red to determine collagen volume fraction (CVF). Slices were also stained with hematoxylin-eosin to determine the myocyte cross sectional area (MCSA). The percentage of picrosirius red staining, which indicated CVF, was measured in images obtained with a video camera (Olympus, X-750, Indonesia) coupled to an optical microscope (Top Light B2, Bel Engineering, Italy) under 400× magnification. Nine areas of high-power fields were analyzed in the subendocardial layer, and nine were analyzed in the subepicardial layer. For MCSA evaluation, 40 to 60 myocytes positioned perpendicularly to the plane of the section with a visible nucleus and a clearly outlined and unbroken cell membrane were selected in each animal. Cell images viewed with a video camera were projected onto a monitor and traced. Images for CVF and MCSA evaluation were processed with Image J software (v. 1.43u, National Institutes of Health, USA). Statistical analysis
All of the data are expressed as the mean ± standard error of the mean (SEM). The goodness of fit for normal distribution was evaluated using the Kolmogorov-Smirnov test. Levene’s test was used to assess the equality of variances. One-way analysis of variance (ANOVA) was used to compare means between the experimental groups. Comparison among repeated
Tab. 1.
measurements of echocardiography was assessed by a two-way ANOVA, followed by Fischer’s post-hoc test for multiples comparisons. Statistical significance was set at p < 0.05.
Results General characteristics
The mortality during the 48 h of isoproterenol treatment was 18%, with the majority (67%) occurring after the first dose. Soon after each isoproterenol administration, signs of agitation and pelage erection were evident in treated animals. While body weight gain was observed in control rats, body weight loss was evident in both groups treated with isoproterenol (control: 12 ± 2.5 g; ISO: –12 ± 2.4 g; ISO + G-CSF: –17 ± 1.7 g; p < 0.05). However, the acute body weight loss observed in isoproterenol-treated rats was fully recovered during follow-up. As observed in Table 1, no significant difference was observed in relation to the morphometric parameters at the end of the follow-up period. Functional and structural assessment
Comparing the evolution of the echocardiographic parameters, an increased end-diastolic dimension was
Morphometric and hemodynamic parameters four weeks after isoproterenol treatment
Sample size (n) BW (g) LV/BW (mg/g) RV/BW (mg/g) HR (bpm) SBP (mmHg) DBP (mmHg) LVSP (mmHg) LVEDP (mmHg) dP/dt+ (mmHg/s) dP/dt– (mmHg/s)
Control
ISO
ISO-GCSF
12 401 ± 9 1.99 ± 0.04 0.51 ± 0.01 245 ± 6 114 ± 4.1 84 ± 4.1 127 ± 5 7 ± 0.7 4856 ± 188 3369 ± 196
19 411 ± 7 2.01 ± 0.03 0.49 ± 0.01 241 ± 6 113 ± 2.9 82 ± 2.9 126 ± 3 7 ± 0.6 4806 ± 133 3731 ± 173
16 396 ± 6 2.03 ± 0.04 0.50 ± 0.01 235 ± 6 113 ± 2.9 83 ± 2.4 126 ± 4 7 ± 0.9 4775 ± 181 3593 ± 177
BW body weight; LV left ventricle; RV right ventricle; HR heart rate; SBP systolic blood pressure; DBP diastolic blood pressure; LVSP left ventricular systolic pressure; LVEDP left ventricular end diastolic pressure; dP/dt maximum rate of pressure rise and fall. Data are depicted as the mean ± SEM Pharmacological Reports, 2012, 64, 643649
645
Tab. 2. Echocardiographic
parameters at baseline and after four weeks of follow-up
LVEDD (mm) Baseline 4 weeks Variation LVESD (mm) Baseline 4 weeks Variation EF (%) Baseline 4 weeks Variation FS (%) Baseline 4 weeks Variation
Control (n = 8)
ISO (n = 8)
ISO-GCSF (n = 7)
7.0 ± 0.24 7.7 ± 0.14 0.7 ± 0.25
7.1 ± 0.12 8.7 ± 0.16* 1.6 ± 0.12*
7.2 ± 0.11 7.8 ± 0.09# 0.6 ± 0.15#
3.5 ± 0.16 3.9 ± 0.21 0.4 ± 0.21
3.8 ± 0.13 4.8 ± 0.22* 1.0 ± 0.22*
3.9 ± 0.15 4.1 ± 0.10# 0.2 ± 0.09#
82.5 ± 2.55 84.9 ± 1.93 2.4 ± 1.53
81.8 ± 1.23 80.9 ± 1.82 -0.8 ± 2.51
79.0 ± 3.05 83.5 ± 1.55 4.6 ± 3.04
49.8 ± 1.00 49.2 ± 2.06 –0.6 ± 2.22
47.4 ± 1.18 45.6 ± 1.63 –1.8 ± 2.15
46.0 ± 1.58 47.9 ± 1.26 1.9 ± 0.85
LVEDD left ventricular end diastolic dimensions; LVESD left ventricular systolic dimensions; EF ejection fraction; FS shortening fraction. * p < 0.05 vs. CONTROL; # p < 0.05 vs. ISO
observed in all three groups (Tab. 2). In animals treated with isoproterenol, the increase was higher compared with other groups. The left ventricular end-systolic dimension was also increased in these animals. Treatment with G-CSF was effective in preventing left ventricular enlargement, as evaluated by the end-diastolic diameter. Fractional shortening and ejection fraction did not differ significantly between groups. Moreover, hemodynamic analysis did not change in any parameter evaluated (Tab. 1).
Morphological evaluation
When myocardial fibrosis was assessed in Sirius redstained sections, we found higher myocardium collagen content in rats receiving isoproterenol than in the control group. G-CSF treatment attenuated the increase in the collagen fractional volume induced by isoproterenol (control: 2.0 ± 0.18%; ISO: 9.1 ± 0.81%; ISO-GCSF: 5.9 ± 0.58%; p < 0.05). The transverse area of cardiomyocytes was higher after isoproterenol administration, which was partially reversed by G-CSF (control: 303 ± 10 μm2; ISO: 356 ± 18 μm2; ISO-GCSF: 338 ± 11 μm2; p < 0.05) (Fig. 1). 646
Pharmacological Reports, 2012, 64, 643649
Discussion
Our study showed that a high isoproterenol dose administered during two consecutive days induces ventricular remodeling and increases collagen in the left ventricle wall in rats. These changes can be partially prevented by G-CSF because animals treated with this cytokine 7 days after isoproterenol administration clearly showed less ventricular dilation and reduced collagen content in the left ventricular myocardium. Teerlink et al. [29] described the kinetics of cardiac structural and functional changes after different doses of isoproterenol in rats. They showed progressive left ventricular dilation at all tested doses (85, 170, and 340 mg/kg) up to 16 weeks of follow-up through passive pressure-volume curves but without affecting hemodynamic parameters. Similar results were found in our study, in which isoproterenol-treated rats (150 mg/kg) showed similar results regarding left ventricular dilation, as evaluated by echocardiographic analysis, but without hemodynamic repercussions. These data are in accordance with Teerlink et al. [29] because the animals presented an initial stage of cardiomyopathy development. Grimm et al. [11], in
G-CSF prevents early cardiomyopathy in rats Ludimila Forechi et al.
Fig. 1. Histological analysis of the left ventricle 4 weeks after isoproterenol. ( A) Representative images of hematoxylin-eosin- (top) and picrosirius red-stained (below) sections. (B) Myocyte cross-sectional area and (C) collagen volume fraction. Images were acquired at 400 ´ magnification. * p < 0.05 vs. control; # p < 0.05 vs. ISO
contrast, reported increased left ventricular enddiastolic pressure only after two weeks of a single dose of isoproterenol (150 mg/kg). These data, however, were not confirmed in our study. Several studies have shown that G-CSF improves cardiac function in animals submitted to ischemic injury by preventing enlargement of cardiac cavities and reducing mortality [14, 16, 20, 25]. The mecha-
nism by which G-CSF acts on the cardiovascular system remains under intense discussion. Several studies have shown that these beneficial effects may depend on the interaction of different mechanisms, such as interference in anti-apoptotic signaling [1, 12], myocyte regeneration [8], and modulation of synthesis and degradation of components in the extracellular matrix [19]. In this paper, we observed that G-CSF prevents Pharmacological Reports, 2012, 64, 643649
647
early dilation, as evaluated by left ventricular diastolic dimension four weeks after a high dose of isoproterenol. These results suggest that acute G-CSF can prevent the early structural changes leading to systolic dysfunction and heart failure development after catecholaminergic aggression. It was previously described that adrenergic overstimulation leads to myocyte apoptosis [26]. Moreover, apoptosis is directly involved in heart failure progression [24]. Studies have shown that G-CSF therapy seems to reduce heart failure progression through an antiapoptotic effect [1, 12, 13]. For example, G-CSF reduced FasL expression and cardiomyocyte apoptosis in rats with heart failure [13]. Thus, the reduction in left ventricular dilation observed in our work could be explained by a reduction in cardiomyocyte apoptosis by early G-CSF treatment. Cardiac hypertrophy is another effect associated with high-dose isoproterenol administration and the first step to cardiac decompensation. It was reported that high isoproterenol doses (85 and 150 mg/kg) led to left ventricular hypertrophy [5, 11]. We observed slight myocyte hypertrophy caused by isoproterenol, despite no significant changes in the left ventricular mass. It is likely that the increased volume of remaining myocytes compensated for the myocyte loss, mainly in the subendocardial layer, in which fibrosis is more intense. Similarly, Teerlink et al. [29] did not observe differences in left ventricular weight early after isoproterenol administration. G-CSF reduced myocyte hypertrophy, which can be explained by reduction of left ventricular dilation in G-CSF-treated animals. In the present study, isoproterenol increased collagen deposition in the myocardium. This finding is in accordance with a previous description in which a high dose of isoproterenol induced intense collagen deposition [5]. G-CSF partially prevented collagen deposition. Several studies have also shown that GCSF decreases myocardial fibrosis after cardiac damage [15, 19, 22]. This G-CSF effect does not seem to be restricted to hypoxic damage because Macambira et al. [17] recently observed that repeated injections of G-CSF decreased inflammation and fibrosis in a mouse model of Chagasic cardiomyopathy. We cannot rule out the possibility that the absence of hemodynamic impairment could be due the cardiodepressor effect of ketamine plus xylazine anesthesia. However, similar results were found by Teerlink et al. [29] using the same pharmacological model and a dif-
648
Pharmacological Reports, 2012, 64, 643649
ferent anesthetic agent. Another possibility lies in the difficulty in detecting small differences with a fluidfilled catheter. The tip catheter has important properties that improve the sensitivity of hemodynamic measurements. Conversely, the means from the three groups are closely similar and seem to be true and not due to the method used. In summary, our results showed that acute administration of G-CSF can modulate cardiac remodeling by preventing collagen deposition, myocyte hypertrophy and left ventricular dilation, contributing to avoiding hemodynamic deterioration and heart failure development. Further experiments need to be performed before these results can be used in clinical settings. However, it has been proven that G-CSF is safe for human use, and thus, could help as a possible adjuvant therapy for heart failure induced by sympathetic overload.
Acknowledgments:
This study was supported by grants from National Council for Scientific and Technological Development (CNPq) and Espírito Santo Research Foundation (FAPES/PRONEX).
References: 1. Baldo MP, Davel AP, Damas-Souza DM, NicolettiCarvalho JE, Bordin S, Carvalho HF, Rodrigues SL et al.: The antiapoptotic effect of granulocyte colonystimulating factor reduces infarct size and prevents heart failure development in rats. Cell Physiol Biochem, 2011, 28, 33–40. 2. Baldo MP, Davel AP, Nicoletti-Carvalho JE, Bordin S, Rossoni LV, Mill JG: Granulocyte colony-stimulating factor reduces mortality by suppressing ventricular arrhythmias in acute phase of myocardial infarction in rats. J Cardiovasc Pharmacol, 2008, 52, 375–380. 3. Baldo MP, Forechi L, Morra EAS, Zaniqueli D, Machado RC, Lunz W, Rodrigues SL, Mill JG: Longterm use of low dose spironolactone in spontaneously hypertensive rats. Effects on left ventricular hypertrophy and stiffness. Pharmacol Rep, 2011, 63, 975–982. 4. Baldo MP, Rodrigues SL, Mill JG: Granulocyte colonystimulating factor for ischemic heart failure: should we use it? Heart Fail Rev, 2010, 15, 613–623. 5. Brooks WW, Conrad CH: Isoproterenol-induced myocardial injury and diastolic dysfunction in mice: structural and functional correlates. Comp Med, 2009, 59, 339–343. 6. Broskova Z, Knezl V: Protective effect of novel pyridoindole derivatives on schemia/reperfusion injury of the isolated rat heart. Pharmacol Rep, 2011, 63, 967–974.
G-CSF prevents early cardiomyopathy in rats Ludimila Forechi et al.
7. Carll AP, Willis MS, Lust RM, Costa DL, Farraj AK: Merits of non-invasive rat models of left ventricular heart failure. Cardiovasc Toxicol, 2011, 11, 91–112. 8. Dawn B, Guo Y, Rezazadeh A, Huang Y, Stein AB, Hunt G, Tiwari S et al.: Postinfarct cytokine therapy regenerates cardiac tissue and improves left ventricular function. Circ Res, 2006, 98, 1098–1105. 9. dos Santos L, Mello AF, Antonio EL, Tucci PJ: Determination of myocardial infarction size in rats by echocardiography and tetrazolium staining: correlation, agreements, and simplifications. Braz J Med Biol Res, 2008, 41, 199–201. 10. Feng W, Li W: The study of ISO induced heart failure rat model. Exp Mol Pathol, 2010, 88, 299–304. 11. Grimm D, Elsner D, Schunkert H, Pfeifer M, Griese D, Bruckschlegel G, Muders F et al.: Development of heart failure following isoproterenol administration in the rat: role of the rennin-angiotensin system. Cardiovasc Res, 1998, 37, 91–100. 12. Harada M, Qin Y, Takano H, Minamino T, Zou Y, Toko H, Ohtsuka M et al.: G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes. Nat Med, 2005, 11, 305–311. 13. Hou XW, Son J, Wang Y, Ru YX, Lian Q, Majiti W, Amazouzi A et al.: Granulocyte colony-stimulating factor reduces cardiomyocyte apoptosis and improves cardiac function in adriamycin-induced cardiomyopathy in rats. Cardiovasc Drugs Ther, 2006, 20, 85–91. 14. Kanellakis P, Slater NJ, Du XJ, Bobik A, Curtis DJ: Granulocyte colony-stimulating factor and stem cell factor improve endogenous repair after myocardial infarction. Cardiovasc Res, 2006, 70, 117–125. 15. Li L, Takemura G, Li Y, Miyata S, Esaki M, Okada H, Kanamori H et al.: Granulocyte colony-stimulating factor improves left ventricular function of doxorubicininduced cardiomyopathy. Lab Invest, 2007, 87, 440–455. 16. Li Y, Takemura G, Okada H, Miyata S, Esaki M, Maruyama R, Kanamori H et al.: Treatment with granulocyte colony-stimulating factor ameliorates chronic heart failure. Lab Invest, 2006, 86, 32–44. 17. Macambira SG, Vasconcelos JF, Costa CR, Klein W, Lima RS, Guimarães P, Vidal DT et al.: Granulocyte colony-stimulating factor treatment in chronic Chagas disease: preservation and improvement of cardiac structure and function. FASEB J, 2009, 23, 3843–3850. 18. Mill JG, Stefanon I, dos Santos L, Baldo MP: Remodeling in the ischemic heart: the stepwise progression for heart failure. Braz J Med Biol Res, 2011, 44, 890–898. 19. Minatoguchi S, Takemura G, Chen XH, Wang N, Uno Y, Koda M, Arai M et al.: Acceleration of the healing process and myocardial regeneration may be important as a mechanism of improvement of cardiac function and remodeling by postinfarction granulocyte colony-stimulating factor treatment. Circulation, 2004, 109, 2572–2580.
20. Miyata S, Takemura G, Kawase Y, Li Y, Okada H, Maruyama R, Ushikoshi H et al.: Autophagic cardiomyocyte death in cardiomyopathic hamsters and its prevention by granulocyte colony-stimulating factor. Am J Pathol, 2006, 168, 386–397. 21. Nogueira BV, Palomino Z, Porto ML, Balarini CM, Pereira TM, Baldo MP, Casarini DE et al.: Granulocyte colony stimulating factor prevents kidney infarction and attenuates renovascular hypertension. Cell Physiol Biochem, 2012, 29, 143–152. 22. Okada H, Takemura G, Li Y, Ohno T, Li L, Maruyama R, Esaki M et al.: Effect of a long-term treatment with a low-dose granulocyte colony-stimulating factor on post-infarction process in the heart. J Cell Mol Med, 2008, 12, 1271–1283. 23. Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F, Nadal-Ginard B et al.: Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci USA, 2001, 98, 10344–10349. 24. Palojoki E, Saraste A, Eriksson A, Pulkki K, Kallajoki M, Voipio-Pulkki LM, Tikkanen I: Cardiomyocyte apoptosis and ventricular remodeling after myocardial infarction in rats. Am J Physiol Heart Circ Physiol, 2001, 280, H2726–2731. 25. Park C, Yoo JH, Jeon HW, Kang BT, Kim JH, Jung DI, Lim CY et al.: Therapeutic trial of granulocyte-colony stimulating factor for dilated cardiomyopathy in three dogs. J Vet Med Sci, 2007, 69, 951–955. 26. Prabhu SD, Wang G, Luo J, Gu Y, Ping P, Chandrasekar B: b-Adrenergic receptor blockade modulates Bcl-X5 expression and reduces apoptosis in failing myocardium. J Mol Cell Cardiol, 2003, 35, 483–493. 27. Ribeiro DA, Buttros JB, Oshima CT, Bergamaschi CT, Campos RR: Ascorbic acid prevents acute myocardial infarction induced by isoproterenol in rats: role of inducible nitric oxide synthase production. J Mol Hist, 2009, 40, 99–105. 28. Rona G: Catecholamine cardiotoxicity. J Mol Cell Cardiol, 1985, 17, 291–306. 29. Teerlink JR, Pfeffer JM, Pfeffer MA: Progressive ventricular remodeling in response to diffuse isoproterenolinduced myocardial necrosis in rats. Circ Res, 1994, 75, 105–113. 30. Wittstein IS, Thiemann DR, Lima JA, Baughman KL, Schulman SP, Gerstenblith G, Wu KC et al.: Neurohumoral features of myocardial stunning due to sudden emotional stress. N Engl J Med, 2005, 352, 539–548. 31. Yung MC, Hsu CC, Kang CY, Lin CL, Chang SL, Wang JJ, Lin MT et al.: A potential for granulocyte colony stimulating factor for use as a prophylactic agent for heatstroke in rats. Eur J Pharmacol, 2011, 661, 109–117.
Received: July 14, 2011; in the revised form: January 6, 2012; accepted: February 17, 2012.
Pharmacological Reports, 2012, 64, 643649
649
Copyright © 2012 by Institute of Pharmacology Polish Academy of Sciences
2012, 64, 643649 ISSN 1734-1140
Granulocyte colony-stimulating factor improves early remodeling in isoproterenol-induced cardiac injury in rats Ludimila Forechi, Marcelo P. Baldo, Diana Meyerfreund, José G. Mill Department of Physiological Sciences, Federal University of Espirito Santo, Av. Marechal Campos 1468, Maruipe, 29042-755, Vitória, Espírito Santo, Brazil Correspondence: José G. Mill, e-mail:
[email protected]; [email protected]
Abstract: Background: Granulocyte colony-stimulating factor (G-CSF) has been used in some animal models and humans with wellestablished cardiovascular diseases. However, its effects in the initial stage of progressive non-ischemic heart failure are unknown. Methods: Wistar rats (260–300 g) were divided into three groups: control (without any intervention), ISO (150 mg/kg isoproterenol hydrochloride sc, once a day for two consecutive days), and ISO-GCSF (50 μg/kg/d G-CSF for 7 days beginning 24 h after the last administration of ISO). Echocardiography was performed at baseline and after 30 days of follow-up. Subsequently, animals were anesthetized for hemodynamic analysis. The left ventricle was removed for analysis of interstitial collagen deposition and cardiomyocyte hypertrophy. Results: Isoproterenol led to left ventricular dilation (control, 7.7 ± 0.14 mm; ISO, 8.7 ± 0.16 mm; ISO-GCSF 7.8 ± 0.09 mm; p < 0.05), myocardial fibrosis (control, 2.0 ± 0.18%; ISO, 9.1 ± 0.81%; ISO-GCSF 5.9 ± 0.58%; p < 0.05) and cardiomyocyte hypertrophy (control, 303 ± 10 μm ; ISO, 356 ± 18 μm ; ISO-GCSF 338 ± 11 μm ; p < 0.05). However, G-CSF partially prevented collagen deposition and left ventricular enlargement, with a slight effect on hypertrophy. Characterizing a compensated stage of disease, hemodynamic analysis did not change. Conclusion: G-CSF administered for 7 days was effective in preventing the onset of ventricular remodeling induced by high-dose isoproterenol with decreased collagen deposition and chamber preservation. Key words: isoproterenol, heart failure, G-CSF, collagen
Abbreviations: CVF – collagen volume fraction, dP/dt – maximal rate of pressure, FS – fractional shortening, G-CSF – granulocyte colony-stimulating factor, HR – heart rate, ISO – isoproterenol, LVEDD – left ventricular end-diastolic dimension, LVEDP – left ventricle end-diastolic pressure, LVESD – left ventricular peak systolic dimension, LVSP – left ventricle systolic pressure, MCSA – myocyte cross sectional area
Introduction Massive sympathetic discharge, as observed in extreme stress or with high activity of the central nervous system, has deleterious effects on the heart. In
these cases, high oxygen consumption by the myocardium leads to subendocardial ischemia and infarction followed by high levels of oxidative stress, ventricular dilation, fibrosis, progressive systolic dysfunction and heart failure [6, 17, 29]. Non-invasive animal models have been used to mimic the pathophysiological characteristics of this cardiomyopathy in humans [7]. Rats treated with high doses of the nonselective b-adrenergic receptor agonist isoproterenol are a widely used model to assess progressive left ventricular dysfunction. Although the mechanisms by which isoproterenol induces myocardial injury are not completely understood, b-adrenergic receptor overstimulation leads to intense coroPharmacological Reports, 2012, 64, 643649
643
nary vasoconstriction and the development of microinfarcts that more frequently affect the subendocardium or transmural region of the left ventricle wall [10, 27, 28]. Teerlink et al. [29] showed progressive left ventricular dilation and dysfunction associated with long-term mortality rates after high doses of isoproterenol. Recently, the therapeutic use of granulocyte colony-stimulating factor (G-CSF) has been reported in some tissues, such as heart, kidney and brain [4, 21, 31]. Studies have suggested that this cytokine can act in damaged hearts by recruiting bone marrow-derived cells and promoting angiogenesis, reducing apoptosis and modulating collagen content, thus improving overall cardiac function [1, 2, 12, 16, 19, 22, 23]. Most of these studies have been performed in different models of ischemic cardiomyopathy. Little is known about the potential cardioprotective effects of G-CSF in non-ischemic cardiomyopathies. Therefore, this study sought to evaluate the effects of G-CSF treatment on structural and functional cardiac parameters during cardiomyopathy induced by high-dose isoproterenol in rats.
Echocardiography
Transthoracic echocardiography was performed at baseline and 4 weeks after isoproterenol treatment as previously described [9]. Rats were intraperitoneally anesthetized with ketamine (50 mg/kg; Agener, Brazil) plus xylazine (10 mg/kg; Bayer, Brazil) and placed in the supine position, and electrodes were placed in the subcutaneous tissue of limbs for continuous electrocardiogram recordings. M-mode images were acquired with an echocardiograph (Aplio XG SSA-790, Toshiba, Japan) using a 7.5 MHz transducer transverse to the left ventricle at the level of papillary muscles. M-mode values of left ventricular enddiastolic dimension (LVEDD) and left ventricular peak systolic dimension (LVESD) were measured for each animal as the average of four consecutive cardiac cycles under regular rhythm. Heart rate (HR), fractional shortening {FS = ((LVEDD – LVESD)/ LVEDD) × 100)} and ejection fraction were obtained. The measures were performed by an investigator blinded to groups at both times. Hemodynamic analysis
Materials and Methods
Animals and groups
Male Wistar rats (260–300 g) were acquired from the animal facility at the Federal University of Espírito Santo and randomly assigned into three groups: a control group, without any intervention; the ISO group, which received isoproterenol hydrochloride (150 mg/kg, sc, Sigma-Aldrich Inc., USA) once a day for two consecutive days; and the ISO-GCSF group, which received G-CSF (Biosintetica, 50 μg/kg, sc) treatment for 7 days beginning 24 h after the last isoproterenol dose. Cardiac function in these animals was studied 3 weeks after the last G-CSF dose. Animals had free access to water and standard rat chow throughout the experimental period, and all of the procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85–23, revised 1996) and were approved by the institutional animal research committee (No. 012/2010). 644
Pharmacological Reports, 2012, 64, 643649
Four weeks after the first isoproterenol dose, animals were anesthetized with ketamine (90 mg/kg) and xylazine (10 mg/kg), and hemodynamic measurements were performed as previously reported [3]. Briefly, the right common carotid artery was dissected to insert a fluid-filled polyethylene catheter (P50) connected to a blood pressure transducer (TRI 21, Letica Scientific Instruments, Spain). The catheter was then advanced into the left ventricle to record peak systolic pressure (LVSP), end-diastolic pressure (LVEDP), and heart rate (HR) in cardiac cycles under regular cardiac beats. The maximal rate of pressure rise and fall (+dP/dt and –dP/dt) was obtained electronically (Powerlab/4SP ML750, AdInstruments, Australia) and used as left ventricular contractility and relaxation indexes, respectively. Determination of myocyte hypertrophy and fibrosis
After hemodynamic recordings, the heart was removed and rapidly washed with cold saline solution, and the ventricles were separated from the atria, blotted dry and weighed. The left ventricle was divided into three slices of approximately 2 mm, which were
G-CSF prevents early cardiomyopathy in rats Ludimila Forechi et al.
prepared for histology. Each slice was serially cut into 6 μm-thick transverse sections and stained with Sirius red to determine collagen volume fraction (CVF). Slices were also stained with hematoxylin-eosin to determine the myocyte cross sectional area (MCSA). The percentage of picrosirius red staining, which indicated CVF, was measured in images obtained with a video camera (Olympus, X-750, Indonesia) coupled to an optical microscope (Top Light B2, Bel Engineering, Italy) under 400× magnification. Nine areas of high-power fields were analyzed in the subendocardial layer, and nine were analyzed in the subepicardial layer. For MCSA evaluation, 40 to 60 myocytes positioned perpendicularly to the plane of the section with a visible nucleus and a clearly outlined and unbroken cell membrane were selected in each animal. Cell images viewed with a video camera were projected onto a monitor and traced. Images for CVF and MCSA evaluation were processed with Image J software (v. 1.43u, National Institutes of Health, USA). Statistical analysis
All of the data are expressed as the mean ± standard error of the mean (SEM). The goodness of fit for normal distribution was evaluated using the Kolmogorov-Smirnov test. Levene’s test was used to assess the equality of variances. One-way analysis of variance (ANOVA) was used to compare means between the experimental groups. Comparison among repeated
Tab. 1.
measurements of echocardiography was assessed by a two-way ANOVA, followed by Fischer’s post-hoc test for multiples comparisons. Statistical significance was set at p < 0.05.
Results General characteristics
The mortality during the 48 h of isoproterenol treatment was 18%, with the majority (67%) occurring after the first dose. Soon after each isoproterenol administration, signs of agitation and pelage erection were evident in treated animals. While body weight gain was observed in control rats, body weight loss was evident in both groups treated with isoproterenol (control: 12 ± 2.5 g; ISO: –12 ± 2.4 g; ISO + G-CSF: –17 ± 1.7 g; p < 0.05). However, the acute body weight loss observed in isoproterenol-treated rats was fully recovered during follow-up. As observed in Table 1, no significant difference was observed in relation to the morphometric parameters at the end of the follow-up period. Functional and structural assessment
Comparing the evolution of the echocardiographic parameters, an increased end-diastolic dimension was
Morphometric and hemodynamic parameters four weeks after isoproterenol treatment
Sample size (n) BW (g) LV/BW (mg/g) RV/BW (mg/g) HR (bpm) SBP (mmHg) DBP (mmHg) LVSP (mmHg) LVEDP (mmHg) dP/dt+ (mmHg/s) dP/dt– (mmHg/s)
Control
ISO
ISO-GCSF
12 401 ± 9 1.99 ± 0.04 0.51 ± 0.01 245 ± 6 114 ± 4.1 84 ± 4.1 127 ± 5 7 ± 0.7 4856 ± 188 3369 ± 196
19 411 ± 7 2.01 ± 0.03 0.49 ± 0.01 241 ± 6 113 ± 2.9 82 ± 2.9 126 ± 3 7 ± 0.6 4806 ± 133 3731 ± 173
16 396 ± 6 2.03 ± 0.04 0.50 ± 0.01 235 ± 6 113 ± 2.9 83 ± 2.4 126 ± 4 7 ± 0.9 4775 ± 181 3593 ± 177
BW body weight; LV left ventricle; RV right ventricle; HR heart rate; SBP systolic blood pressure; DBP diastolic blood pressure; LVSP left ventricular systolic pressure; LVEDP left ventricular end diastolic pressure; dP/dt maximum rate of pressure rise and fall. Data are depicted as the mean ± SEM Pharmacological Reports, 2012, 64, 643649
645
Tab. 2. Echocardiographic
parameters at baseline and after four weeks of follow-up
LVEDD (mm) Baseline 4 weeks Variation LVESD (mm) Baseline 4 weeks Variation EF (%) Baseline 4 weeks Variation FS (%) Baseline 4 weeks Variation
Control (n = 8)
ISO (n = 8)
ISO-GCSF (n = 7)
7.0 ± 0.24 7.7 ± 0.14 0.7 ± 0.25
7.1 ± 0.12 8.7 ± 0.16* 1.6 ± 0.12*
7.2 ± 0.11 7.8 ± 0.09# 0.6 ± 0.15#
3.5 ± 0.16 3.9 ± 0.21 0.4 ± 0.21
3.8 ± 0.13 4.8 ± 0.22* 1.0 ± 0.22*
3.9 ± 0.15 4.1 ± 0.10# 0.2 ± 0.09#
82.5 ± 2.55 84.9 ± 1.93 2.4 ± 1.53
81.8 ± 1.23 80.9 ± 1.82 -0.8 ± 2.51
79.0 ± 3.05 83.5 ± 1.55 4.6 ± 3.04
49.8 ± 1.00 49.2 ± 2.06 –0.6 ± 2.22
47.4 ± 1.18 45.6 ± 1.63 –1.8 ± 2.15
46.0 ± 1.58 47.9 ± 1.26 1.9 ± 0.85
LVEDD left ventricular end diastolic dimensions; LVESD left ventricular systolic dimensions; EF ejection fraction; FS shortening fraction. * p < 0.05 vs. CONTROL; # p < 0.05 vs. ISO
observed in all three groups (Tab. 2). In animals treated with isoproterenol, the increase was higher compared with other groups. The left ventricular end-systolic dimension was also increased in these animals. Treatment with G-CSF was effective in preventing left ventricular enlargement, as evaluated by the end-diastolic diameter. Fractional shortening and ejection fraction did not differ significantly between groups. Moreover, hemodynamic analysis did not change in any parameter evaluated (Tab. 1).
Morphological evaluation
When myocardial fibrosis was assessed in Sirius redstained sections, we found higher myocardium collagen content in rats receiving isoproterenol than in the control group. G-CSF treatment attenuated the increase in the collagen fractional volume induced by isoproterenol (control: 2.0 ± 0.18%; ISO: 9.1 ± 0.81%; ISO-GCSF: 5.9 ± 0.58%; p < 0.05). The transverse area of cardiomyocytes was higher after isoproterenol administration, which was partially reversed by G-CSF (control: 303 ± 10 μm2; ISO: 356 ± 18 μm2; ISO-GCSF: 338 ± 11 μm2; p < 0.05) (Fig. 1). 646
Pharmacological Reports, 2012, 64, 643649
Discussion
Our study showed that a high isoproterenol dose administered during two consecutive days induces ventricular remodeling and increases collagen in the left ventricle wall in rats. These changes can be partially prevented by G-CSF because animals treated with this cytokine 7 days after isoproterenol administration clearly showed less ventricular dilation and reduced collagen content in the left ventricular myocardium. Teerlink et al. [29] described the kinetics of cardiac structural and functional changes after different doses of isoproterenol in rats. They showed progressive left ventricular dilation at all tested doses (85, 170, and 340 mg/kg) up to 16 weeks of follow-up through passive pressure-volume curves but without affecting hemodynamic parameters. Similar results were found in our study, in which isoproterenol-treated rats (150 mg/kg) showed similar results regarding left ventricular dilation, as evaluated by echocardiographic analysis, but without hemodynamic repercussions. These data are in accordance with Teerlink et al. [29] because the animals presented an initial stage of cardiomyopathy development. Grimm et al. [11], in
G-CSF prevents early cardiomyopathy in rats Ludimila Forechi et al.
Fig. 1. Histological analysis of the left ventricle 4 weeks after isoproterenol. ( A) Representative images of hematoxylin-eosin- (top) and picrosirius red-stained (below) sections. (B) Myocyte cross-sectional area and (C) collagen volume fraction. Images were acquired at 400 ´ magnification. * p < 0.05 vs. control; # p < 0.05 vs. ISO
contrast, reported increased left ventricular enddiastolic pressure only after two weeks of a single dose of isoproterenol (150 mg/kg). These data, however, were not confirmed in our study. Several studies have shown that G-CSF improves cardiac function in animals submitted to ischemic injury by preventing enlargement of cardiac cavities and reducing mortality [14, 16, 20, 25]. The mecha-
nism by which G-CSF acts on the cardiovascular system remains under intense discussion. Several studies have shown that these beneficial effects may depend on the interaction of different mechanisms, such as interference in anti-apoptotic signaling [1, 12], myocyte regeneration [8], and modulation of synthesis and degradation of components in the extracellular matrix [19]. In this paper, we observed that G-CSF prevents Pharmacological Reports, 2012, 64, 643649
647
early dilation, as evaluated by left ventricular diastolic dimension four weeks after a high dose of isoproterenol. These results suggest that acute G-CSF can prevent the early structural changes leading to systolic dysfunction and heart failure development after catecholaminergic aggression. It was previously described that adrenergic overstimulation leads to myocyte apoptosis [26]. Moreover, apoptosis is directly involved in heart failure progression [24]. Studies have shown that G-CSF therapy seems to reduce heart failure progression through an antiapoptotic effect [1, 12, 13]. For example, G-CSF reduced FasL expression and cardiomyocyte apoptosis in rats with heart failure [13]. Thus, the reduction in left ventricular dilation observed in our work could be explained by a reduction in cardiomyocyte apoptosis by early G-CSF treatment. Cardiac hypertrophy is another effect associated with high-dose isoproterenol administration and the first step to cardiac decompensation. It was reported that high isoproterenol doses (85 and 150 mg/kg) led to left ventricular hypertrophy [5, 11]. We observed slight myocyte hypertrophy caused by isoproterenol, despite no significant changes in the left ventricular mass. It is likely that the increased volume of remaining myocytes compensated for the myocyte loss, mainly in the subendocardial layer, in which fibrosis is more intense. Similarly, Teerlink et al. [29] did not observe differences in left ventricular weight early after isoproterenol administration. G-CSF reduced myocyte hypertrophy, which can be explained by reduction of left ventricular dilation in G-CSF-treated animals. In the present study, isoproterenol increased collagen deposition in the myocardium. This finding is in accordance with a previous description in which a high dose of isoproterenol induced intense collagen deposition [5]. G-CSF partially prevented collagen deposition. Several studies have also shown that GCSF decreases myocardial fibrosis after cardiac damage [15, 19, 22]. This G-CSF effect does not seem to be restricted to hypoxic damage because Macambira et al. [17] recently observed that repeated injections of G-CSF decreased inflammation and fibrosis in a mouse model of Chagasic cardiomyopathy. We cannot rule out the possibility that the absence of hemodynamic impairment could be due the cardiodepressor effect of ketamine plus xylazine anesthesia. However, similar results were found by Teerlink et al. [29] using the same pharmacological model and a dif-
648
Pharmacological Reports, 2012, 64, 643649
ferent anesthetic agent. Another possibility lies in the difficulty in detecting small differences with a fluidfilled catheter. The tip catheter has important properties that improve the sensitivity of hemodynamic measurements. Conversely, the means from the three groups are closely similar and seem to be true and not due to the method used. In summary, our results showed that acute administration of G-CSF can modulate cardiac remodeling by preventing collagen deposition, myocyte hypertrophy and left ventricular dilation, contributing to avoiding hemodynamic deterioration and heart failure development. Further experiments need to be performed before these results can be used in clinical settings. However, it has been proven that G-CSF is safe for human use, and thus, could help as a possible adjuvant therapy for heart failure induced by sympathetic overload.
Acknowledgments:
This study was supported by grants from National Council for Scientific and Technological Development (CNPq) and Espírito Santo Research Foundation (FAPES/PRONEX).
References: 1. Baldo MP, Davel AP, Damas-Souza DM, NicolettiCarvalho JE, Bordin S, Carvalho HF, Rodrigues SL et al.: The antiapoptotic effect of granulocyte colonystimulating factor reduces infarct size and prevents heart failure development in rats. Cell Physiol Biochem, 2011, 28, 33–40. 2. Baldo MP, Davel AP, Nicoletti-Carvalho JE, Bordin S, Rossoni LV, Mill JG: Granulocyte colony-stimulating factor reduces mortality by suppressing ventricular arrhythmias in acute phase of myocardial infarction in rats. J Cardiovasc Pharmacol, 2008, 52, 375–380. 3. Baldo MP, Forechi L, Morra EAS, Zaniqueli D, Machado RC, Lunz W, Rodrigues SL, Mill JG: Longterm use of low dose spironolactone in spontaneously hypertensive rats. Effects on left ventricular hypertrophy and stiffness. Pharmacol Rep, 2011, 63, 975–982. 4. Baldo MP, Rodrigues SL, Mill JG: Granulocyte colonystimulating factor for ischemic heart failure: should we use it? Heart Fail Rev, 2010, 15, 613–623. 5. Brooks WW, Conrad CH: Isoproterenol-induced myocardial injury and diastolic dysfunction in mice: structural and functional correlates. Comp Med, 2009, 59, 339–343. 6. Broskova Z, Knezl V: Protective effect of novel pyridoindole derivatives on schemia/reperfusion injury of the isolated rat heart. Pharmacol Rep, 2011, 63, 967–974.
G-CSF prevents early cardiomyopathy in rats Ludimila Forechi et al.
7. Carll AP, Willis MS, Lust RM, Costa DL, Farraj AK: Merits of non-invasive rat models of left ventricular heart failure. Cardiovasc Toxicol, 2011, 11, 91–112. 8. Dawn B, Guo Y, Rezazadeh A, Huang Y, Stein AB, Hunt G, Tiwari S et al.: Postinfarct cytokine therapy regenerates cardiac tissue and improves left ventricular function. Circ Res, 2006, 98, 1098–1105. 9. dos Santos L, Mello AF, Antonio EL, Tucci PJ: Determination of myocardial infarction size in rats by echocardiography and tetrazolium staining: correlation, agreements, and simplifications. Braz J Med Biol Res, 2008, 41, 199–201. 10. Feng W, Li W: The study of ISO induced heart failure rat model. Exp Mol Pathol, 2010, 88, 299–304. 11. Grimm D, Elsner D, Schunkert H, Pfeifer M, Griese D, Bruckschlegel G, Muders F et al.: Development of heart failure following isoproterenol administration in the rat: role of the rennin-angiotensin system. Cardiovasc Res, 1998, 37, 91–100. 12. Harada M, Qin Y, Takano H, Minamino T, Zou Y, Toko H, Ohtsuka M et al.: G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes. Nat Med, 2005, 11, 305–311. 13. Hou XW, Son J, Wang Y, Ru YX, Lian Q, Majiti W, Amazouzi A et al.: Granulocyte colony-stimulating factor reduces cardiomyocyte apoptosis and improves cardiac function in adriamycin-induced cardiomyopathy in rats. Cardiovasc Drugs Ther, 2006, 20, 85–91. 14. Kanellakis P, Slater NJ, Du XJ, Bobik A, Curtis DJ: Granulocyte colony-stimulating factor and stem cell factor improve endogenous repair after myocardial infarction. Cardiovasc Res, 2006, 70, 117–125. 15. Li L, Takemura G, Li Y, Miyata S, Esaki M, Okada H, Kanamori H et al.: Granulocyte colony-stimulating factor improves left ventricular function of doxorubicininduced cardiomyopathy. Lab Invest, 2007, 87, 440–455. 16. Li Y, Takemura G, Okada H, Miyata S, Esaki M, Maruyama R, Kanamori H et al.: Treatment with granulocyte colony-stimulating factor ameliorates chronic heart failure. Lab Invest, 2006, 86, 32–44. 17. Macambira SG, Vasconcelos JF, Costa CR, Klein W, Lima RS, Guimarães P, Vidal DT et al.: Granulocyte colony-stimulating factor treatment in chronic Chagas disease: preservation and improvement of cardiac structure and function. FASEB J, 2009, 23, 3843–3850. 18. Mill JG, Stefanon I, dos Santos L, Baldo MP: Remodeling in the ischemic heart: the stepwise progression for heart failure. Braz J Med Biol Res, 2011, 44, 890–898. 19. Minatoguchi S, Takemura G, Chen XH, Wang N, Uno Y, Koda M, Arai M et al.: Acceleration of the healing process and myocardial regeneration may be important as a mechanism of improvement of cardiac function and remodeling by postinfarction granulocyte colony-stimulating factor treatment. Circulation, 2004, 109, 2572–2580.
20. Miyata S, Takemura G, Kawase Y, Li Y, Okada H, Maruyama R, Ushikoshi H et al.: Autophagic cardiomyocyte death in cardiomyopathic hamsters and its prevention by granulocyte colony-stimulating factor. Am J Pathol, 2006, 168, 386–397. 21. Nogueira BV, Palomino Z, Porto ML, Balarini CM, Pereira TM, Baldo MP, Casarini DE et al.: Granulocyte colony stimulating factor prevents kidney infarction and attenuates renovascular hypertension. Cell Physiol Biochem, 2012, 29, 143–152. 22. Okada H, Takemura G, Li Y, Ohno T, Li L, Maruyama R, Esaki M et al.: Effect of a long-term treatment with a low-dose granulocyte colony-stimulating factor on post-infarction process in the heart. J Cell Mol Med, 2008, 12, 1271–1283. 23. Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F, Nadal-Ginard B et al.: Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci USA, 2001, 98, 10344–10349. 24. Palojoki E, Saraste A, Eriksson A, Pulkki K, Kallajoki M, Voipio-Pulkki LM, Tikkanen I: Cardiomyocyte apoptosis and ventricular remodeling after myocardial infarction in rats. Am J Physiol Heart Circ Physiol, 2001, 280, H2726–2731. 25. Park C, Yoo JH, Jeon HW, Kang BT, Kim JH, Jung DI, Lim CY et al.: Therapeutic trial of granulocyte-colony stimulating factor for dilated cardiomyopathy in three dogs. J Vet Med Sci, 2007, 69, 951–955. 26. Prabhu SD, Wang G, Luo J, Gu Y, Ping P, Chandrasekar B: b-Adrenergic receptor blockade modulates Bcl-X5 expression and reduces apoptosis in failing myocardium. J Mol Cell Cardiol, 2003, 35, 483–493. 27. Ribeiro DA, Buttros JB, Oshima CT, Bergamaschi CT, Campos RR: Ascorbic acid prevents acute myocardial infarction induced by isoproterenol in rats: role of inducible nitric oxide synthase production. J Mol Hist, 2009, 40, 99–105. 28. Rona G: Catecholamine cardiotoxicity. J Mol Cell Cardiol, 1985, 17, 291–306. 29. Teerlink JR, Pfeffer JM, Pfeffer MA: Progressive ventricular remodeling in response to diffuse isoproterenolinduced myocardial necrosis in rats. Circ Res, 1994, 75, 105–113. 30. Wittstein IS, Thiemann DR, Lima JA, Baughman KL, Schulman SP, Gerstenblith G, Wu KC et al.: Neurohumoral features of myocardial stunning due to sudden emotional stress. N Engl J Med, 2005, 352, 539–548. 31. Yung MC, Hsu CC, Kang CY, Lin CL, Chang SL, Wang JJ, Lin MT et al.: A potential for granulocyte colony stimulating factor for use as a prophylactic agent for heatstroke in rats. Eur J Pharmacol, 2011, 661, 109–117.
Received: July 14, 2011; in the revised form: January 6, 2012; accepted: February 17, 2012.
Pharmacological Reports, 2012, 64, 643649
649