Tyrphostin AG-556 reduces myocardial infarct size and improves cardiac performance in the rat

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Experimental and Molecular Pathology 74 (2003) 314 –318

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Tyrphostin AG-556 reduces myocardial infarct size and improves cardiac performance in the rat Jacob George,a,* Simon Biner,a Pnina Keren,a Iris Barshack,b Iris Goldberg,b Jack Sherez,a Alexander Levitzki,c Gad Keren,a and Arie Rotha a

Department of Cardiology and the Cardiovascular Research Laboratory, Tel-Aviv Medical Center, Tel-Aviv, Israel b Institute of Pathology, Sheba Medical-Center, Tel-Aviv, Israel c Department of Biological Chemistry, The Hebrew University, Jerusalem, Israel Received 4 January 2003

Abstract TNF-␣ is a proinflammatory cytokine, abundantly expressed after myocardial infarction. It has been suggested that it exhibits myocardial suppressive and cytotoxic effects. AG-556 is a tyrosine kinase inhibitor synthesized based on its ability to reduce TNF-␣ production and cell toxicity, and to improve experimental models mediated by TNF-␣ (i.e., peritontitis and experimental autoimmune encephalomyelitis). Daily, for 7 days, rats were injected ip with either AG-556 dissolved in DMSO or with the control vehicle. Infarct size was determined in the hearts as well as in fibrous scar formation. Cardiac TNF-␣ expression was evaluated by ELISA and immunohistochemistry. Functional hemodynamic parameters were evaluated employing echocardiography prior to sacrifice. AG-556 treatment reduced MI size at 7 days with a parallel effect on fibrous tissue formation. TNF-␣ production by splenocytes was reduced upon AG-556 treatment, whereas no differences were evident between the groups with regard to myocardial cytokine expression. AG-556 attenuated the decrease in fractional shortening at the expense of preserving end systolic diameter. AG-556 has proven beneficial in reducing myocardial infarct size and attenuated consequent hemodynamic deterioration in the rat model. If reconfirmed, AG-556 may be of potential clinical use in post-MI patients. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Myocardial infarct; Rat; TNF-␣; Remodeling; Inflammation

Introduction Coronary ischemic events represent the major cause of congestive heart failure (CHF) in the Western world (Fuster et al., 1992). Through a series of adaptive and maladaptive processes, collectively termed cardiac remodeling, large myocardial infarctions (MI) can result in hemodynamic compromise bringing about overt heart failure. Complex and some yet unresolved processes are involved in reshaping the organization of the cellular and extracellular components within the myocardial tissue following MI (Fuster et al., 1992). One of the impediments to understanding of the remodeling processes is the complex interaction be-

* Corresponding author. The Department of Cardiology, Tel-Aviv Medical Center, 6 Weizmann St., Tel-Aviv, Israel. Fax: ⫹972-3-5469832. E-mail address: [email protected] (J. George).

tween neurohumoral and inflammatory mechanisms with regard to their effects on the cardiomyocyte and the extracellular matrix (St. John et al., 2000). Cytokines are important determinants of the compensatory myocardial response to MI. Experimental studies have demonstrated the presence of IL-1␤, IL-6, and TNF-␣ gene expression and protein products within the infarcted rat heart (Ono et al., 1998). These findings were supported by a subsequent study, showing the early expression of TNF-␣ and its receptors that was also evident in noninfarcted regional cardiomyoctes (Irwin et al., 1999). TNF-␣ is a pleiotropic cytokine found intracellularly within most cells and is thought to signify a nonspecific response to tissue stress (Vassalli, 1992). Several in vitro and in vivo studies support a role for TNF-␣ in mediating myocardial dysfunction (Feldman et al., 2000). (1) It has been shown to exhibit a direct negative inotropic effect

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J. George et al. / Experimental and Molecular Pathology 74 (2003) 314 –318

(Finkel et al., 1992) (2). It activates metalloproteinases and attenuates expression of metalloproteinase inhibitors, thus influencing exracellular remodeling (Li et al., 1999). (3) It induces a hypertrophic growth response and apoptosis in cardiac myocytes (Krown et al., 1996). Additionally, mice overexpressing TNF-␣ exhibit a phenotype that recapitulates congestive heart failure in humans (Bryant et al., 1997). These collective observations imply that TNF-␣ inhibition commenced early after myocardial infarction may have favorable effects on the functional status of the heart. Tyrphostins are inhibitors of tyrosine phophorylation that belong to the class of synthetic pharmacophores. AG556 is a representative tyrphostin identified and synthesized based on its effective inhibition of lipopolysacharide-induced TNF-␣ production (Novogrodsky et al., 1994; Vanichkin et al., 1996). It was also found to ameliorate TNF-␣ mediated cytotoxicity in vivo, thus providing a dual inhibitory mechanism on this target cytokine. In the present study, we evaluated the effect of AG-556 administered immediately following induction of MI on resultant cardiomyocyte loss and hemodynamics in a rat model. Materials and methods Experimental MI Male Wistar rats (300 –350 g) were used for the study, in which MI was induced by permanent left anterior descending artery ligation (Ono et al., 1998). AG-556 administration AG-556 was synthesized as previously described (Novogrodsky et al., 1994) and dissolved in dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO). Rats were injected daily either with 2 mg of AG-556 (n ⫽ 14) or DMSO (n ⫽ 15), following induction of MI and until sacrifice 7 days later. The chosen dose was determined based on its beneficial effects in previous experimental models and on the ability of such concentrations to suppress TNF-␣ production by spleen cells (Novogrodsky et al., 1994; Vanichkin et al., 1996).

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transducer (Acuson, USA) (Ono et al., 1998). LV enddiastolic diameter (EDD) and end-systolic diameter (ESD) were obtained from M-mode tracing. Fractional shortening (FS) was calculated by the formula LVFS共%兲 ⫽ 关共EDD ⫺ ESD兲/EDD兴 ⫻ 100. Cardiac myeloperoxidase (MPO) activity Heart samples were homogenized and supernatants were collected for determination of myocardial MPO activity (an index of leukocyte infiltration) as described previously (Lee et al., 1998). The results were expressed as the percentage of MPO detected in the control rats. Immunohistochemistry Frozen sections were studied employing the following primary antibodies: goat anti-Rat TNF-␣ (R&D systems) and rabbit anti-iNOS2, (Santa Cruz Biotechnology). Positively stained cells were counted at myocardial inflammatory areas at ⫻200 magnification. Stained myocytes were not included. An average number from five different 0.5 mm2 fields was calculated. Myocardial, serum, and conditioned medium TNF-␣ levels Homogenates of hearts from AG-556 or controls and their respective sera were evaluated for TNF-␣ levels by a Duoset Kit (R&D Systems). Conditioned medium obtained from splenocytes of AG-556 or control treated rats, upon priming with concavaline A for 24 h, was assessed for TNF-␣ in the same manner. Statistical analysis Comparison was done using the Student’s t test. P ⬍ 0.05 was considered statistically significant. Results are expressed as mean ⫾ SEM.

Evaluation of myocardial infarct size

Results

LV was sliced at three different plains, fixed in 10% formaldehyde, embedded in paraffin, and cut into 5 ␮m sections. Evaluation of MI size divided by total LV area was determined as described by Ono et al. (1998). To evaluate fibrous scar formation, parallel slides were stained by Masson’s trichrome.

A preliminary study was performed that demonstrated that daily DMSO injections for seven consecutive days did not alter MI size as compared with noninjected animals. Animals’ general health and weight were not altered in rats administered with AG-556 as compared with controls (data not shown). Treatment with AG-556 significantly reduced MI size (59.9%) as compared with DMSO-treated control rats (P ⫽ 0.015; Fig. 1A). None of the rats treated with AG-556 experienced rupture. To evaluate whether scar formation was compromised in AG-556 treated rats, slides were stained with Masson’s trichrome. Scar area was re-

Echocardiography Transthoracic echocardiography was performed prior to sacrifice, under mild anesthesia with a 7 mHz ultrasound

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Fig. 1. The effect of AG-556 on MI size and composition. (A) MI size as percentage of LV. (B) Scar formation (LV-fibrous occupancy) by Masson’s trichrome staining. (C) Quantitative cytokine expression in the hearts. (D) MPO activity. *P ⬍ 0.05

duced in AG-556 as compared to DMSO-treated animals, to a similar extent as the reduction in MI size (Fig. 1B). To explore the effect of AG-556 on the proinflammatory cytokines, TNF-␣ and IFN-␥ levels were assessed within the myocardium by ELISA. No significant differences were evident between AG-556 and DMSO treated rats with respect to TNF-␣ (42.03 ⫾ 7.8 pg/mg vs 48.37 ⫾ 10.8 pg/mg, respectively) or IFN-␥ (1230 ⫾ 245 pg/mg vs 901 ⫾ 400 pg/mg, respectively) concentrations. Conditioned medium obtained from Con-A primed splenocytes exhibited significant reduction of TNF-␣ production in AG-556 (169 ⫾ 7.5 ␮g/ml) as compared with DMSO (126 ⫾ 12.7 ␮g/ml; P ⬍ 0.05) treated rats. Serum TNF-␣ concentrations were below the detection threshold. Mean TNF-␣ positive inflammatory cells did not differ significantly in AG-556 and control rats (Fig. 1C), confirming the ELISA results. Inducible NOS positive cells were also similar in number between both groups (Fig. 1C). No differences were evident with regard to cardiac MPO activity between both experimental groups, signifying similar inflammatory cell infiltration (Fig. 1D). Baseline FS in all animals was 45 ⫾ 4%. Echocardiographic images taken prior to sacrifice showed that AG-556 treatment was associated with improved FS as compared with controls. The effect on FS was solely attributed to an attenuated ESD, whereas no effect was evident on EDD (Table 1, Fig. 2). In a separate preliminary experiment, it was shown that administration of AG-556 to noninfacted

rats did not influence cardiac performance by echocardiography precluding an intotropic effect. Discussion We have shown that the tyrphostin AG-556 reduces MI size induced by permanent ligation of the LAD. AG-556 is a tyrosine-kinase inhibitor synthesized based on a functional screening, confirming its ability to suppress TNF-␣ production and cellular toxicity (Novogrodsky et al., 1994; Vanichkin et al., 1996). AG-556 exhibited pronounced beneficial effects in experimental peritonitis and a multiplesclerosis model (Novogrodsky et al., 1994; Vanichkin et al., 1996; Sevransky et al., 1997). TNF-␣ has proven to be a key cytokine regulating myocardial cell and extracellular matrix functions (Feldman et al., 2000). These findings are supported by experimental studies demonstrating its expression, concomitant with its receptors, in infarcted hearts (Irwin et al., 1999). Cell death following cessation of coronary blood flow results from both necrotic and apoptotic processes (Itoh et al., 1995). TNF-␣ may well be one of the humoral mediators of cardiomyocyte death and thus a candidate for modulation. We failed to observe reduction in myocardial TNF-␣ by ELISA or by immunohistochemistry. These results were obtained despite a reduced TNF-␣ production by splenocytes from AG-556 treated rats. The inhibitory TNF-␣ activity of AG-556 was demonstrated previously on a myeloid

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Fig. 2. The effect of AG556 on hemodynamic parameters. M-mode image of short axis views from a representative AG-556 (A) and DMSO (B) treatedrat.

cell line (Novogrodsky et al., 1994). It is thus possible that nonmyeloid cells within the heart could have “overcompensated” for the decreased TNF-␣ production by mononuclear cells in the typhostin-treated animals. As AG-556 was also found potent in reducing TNF-␣ mediated cell cytotoxicity

Table 1 Hemodynamic parameters in AG-556 treated rats following MI

ESD (mm) EDD (mm) LV-FS (%) PWT (mm)

AG-556

DMSO

P value

0.45 ⫾ 0.02 0.75 ⫾ 0.03 39.2 ⫾ 2.5 0.18 ⫾ 0.01

0.57 ⫾ 0.02 0.79 ⫾ 0.02 28.7 ⫾ 1.7 0.18 ⫾ 0.01

0.015 0.3 0.008 1.0

Note. ESD, end systolic diameter; EDD, end diastolic diameter; FS, fractional shortening; LV, left ventricle; PWT, posterior-wall thickness.

(Vanichkin et al., 1996), we presume that the favorable effect of the tyrphostin was the result of myocardial cellular protection, rather than a mere attenuation of its local expression. We have shown here that the reduction in fractional shortening induced by MI induction was attenuated by AG556. This effect contributed to the preservation of the mean ESD, with no alteration of EDD, or LV thickness (Table 1). It is thus the effect of TNF-␣ inhibition by AG-556 on global myocardial contractility, that is likely to be responsible for the relatively preserved FS, rather than an attenuated remodeling process. It cannot be ruled out that a beneficial effect on remodeling could be evident in a longer term treatment. We have also addressed the potential influence of TNF-␣ inhibition on scar formation. These data are of prime im-

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portance as inflammatory cell recruitment could also stabilize the infarcted zone and prevent subsequent rupture. We have shown that scar formation as exhibited by the Masson’s trichrome staining, was not compromised and mirrored the reduction in infarct size. Furthermore, none of the rats exhibited signs of cardiac rupture (evident by sudden death). In this respect, it is worthwhile to mention that TNF-␣ inhibition by the soluble TNF-␣ receptor Etanercept, has proven safe in patients with CHF (Deswal et al., 1999). In conclusion, we have demonstrated the efficacy of the tyrphostin AG-556 in reducing myocardial infarct size and ameliorating the consequent reduced contractility.

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