ABBS 2005,38(06): Effects of N-n-butyl Haloperidol Iodide on Myocardial Ischemia/reperfusion Injury and Egr-1 Expression in Rats

Abstract        We have previously shown that N-n-butyl
haloperidol iodide (F2)
derived from haloperidol reduces ischemia/reperfusion-induced myocardial injury
by blocking intracellular Ca2+ overload. This study tested the hypothesis
that cardio-protection with F2 is associated with an attenuation in the
expression of early growth response gene 1 (Egr-1). In an in vivo
rat model of 60 min coronary occlusion followed by 180 min of reperfusion,
treatment with F2 significantly reduced myocardial injury
evidenced by the reduction in release of plasma creatine kinase, myocardial
creatine kinase isoenzyme and lactate dehydrogenase. In cultured neonatal rat
cardiomyocytes of hypoxia for 3 h and reoxygenation for 1 h, F2 treatment attenuated necrotic and apoptotic cell death, as
demonstrated by electron microscopy. Concomitant with cardio-protection by F2, the increased expression
levels of Egr-1 mRNA and protein were significantly reduced in
myocardial tissue and cultured cardiomyocytes as detected by reverse
transcription-polymerase chain reaction, immunohistochemistry and
immunocytochemistry. In conclusion, these results suggest that the protective
effect of F2 on ischemia/reperfusion- or hypoxia/reoxygenation-induced
myocardial injury might be partly mediated by downregulating Egr-1
expression.

Key
words        N-n-butyl
haloperidol iodide; ischemia/reperfusion injury; early growth response gene-1;
myocardium; cardiomyocyte

Ischemia/reperfusion (I/R) in the heart initiates a series of rapid
inductions of pathological changes resulting in inflammatory and endothelial
cell-cell interactions, cardiac dysfunction and cardiomyocyte cell death [13].
Therapeutic strategies focusing on attenuation of I/R-mediated cellular events,
such as depletion of inflammatory cells from circulation, prevention of
abnormal cell-cell interactions and reduction of intracellular Ca2+ overload, have shown attenuation in
myocardial injury [4,5]. Recently, more attention has been placed on the
molecular mechanisms in myocardial I/R injury [6,7].

Early growth response gene 1 (Egr-1), a member of the zinc
finger family of transcription factors, is one of the immediate-early genes that
responds to stimulation, and the upregulation of its expression level has been
demonstrated in the heart, lung, gut and kidney after I/R [8]. With Egr-1-null
mice or antisense Egr-1 oligodeoxyribonucleotide , previous studies have
showed that Egr-1 might be a master switch in the pathogenesis of I/R
injury due to its coordinating upregulation of divergent gene families
underlying the pathophysiological
event of I/R [9,10]. In addition, other studies have reported that
overexpression of Egr-1 is triggered by elevated intracellular Ca2+ [11]. Therefore, it is reasonable to
speculate that an attenuation of intracellular Ca2+ overload has the potential
to reduce myocardial injury by reducing Egr-1 expression after I/R.

Haloperidol, an antipsychotic compound, has been shown to possess
vasodilatory and cardioprotective effects [12,13]. However, the extrapyramidal
side-effects of haloperidol have limited its potential clinical application. We
have synthesized a series of quaternary ammonium salt derivatives of haloperidol
and screened as N-n-butyl haloperidol iodide (F2) (Fig. 1), which was granted a
Chinese national invention patent (No. ZL96119098.1). As we reported
previously, F2 has vasodilatory effects, but without
haloperidol-induced side-effects [14]. In addition, F2 reduces myocardial I/R injury and preserves
ATPase activity, largely mediated by blocking intracellular Ca2+ overload [15]. However,
we do not know whether these protective effects elicited by F2 are also related to a modulation in molecular
changes, for example, Egr-1 expression after I/R. Therefore, this study
tested the hypothesis that F2 reduces tissue and cell
damage by modulating expression of Egr-1 mRNA and protein in an in
vivo rat model of I/R as well as in cultured neonatal rat cardiomyocytes of hypoxia
(H) and reoxygenation (Re). The hypothesis is based on previous reports of in
vitro and in vivo observations, which showed that intracellular Ca2+ overload-mediated Egr-1 expression
causes inflammation, vascular hyperpermeability and induction of myocardial
injury [4,9–11] and that F2 has the capability to reduce intracellular Ca2+ overload [15].

Materials and Methods

Surgical preparation of
animals and experimental protocol

Male adult Sprague-Dawley rats weighing 200–All rats were randomly assigned into four groups: sham (n=10),
the ligature was placed under the LAD without occlusion for a total of 4 h of
the experimental period; I/R control (n=10), heart was subjected to 60
min LAD occlusion followed by 180 min of reperfusion; F2 (n=10), F2 at a dose of 2 mg/kg dissolved in 100 ml of polyethylene glycol (PEG) 400 and 0.2 ml of saline was injected
through the sublingual vein 5 min before ischemia; and vehicle (n=10),
100 ml of PEG and 0.2 ml of saline without F2 was given through the sublingual vein 5 min
before ischemia. At the end of the experiment, blood was taken from the carotid
artery for analyzing activities of creatine kinase (CK), creatine kinase MB
isoenzyme (CK-MB) and lactate dehydrogenase (LDH). The area-at-risk
myocardium was excised for immunohistochemical examination, or stored at –70 ºC for determining Egr-1 mRNA.

Activities of plasma CK, CK-MB
and LDH

At the end of the experiment, the arterial blood sample (2 ml) was
centrifuged at  

Isolation of neonatal rat
cardiomyocytes and preparation of H/Re

Neonatal rat ventricular myocytes were isolated from 1–4-day-old Sprague-Dawley rats. The hearts were rapidly excised,
minced and dissociated with 0.1% trypsin. The dispersed cells were plated in
the medium with 15% fetal calf serum for 30 min to remove non-cardiomyocytes [16]. The isolated cardiomyocytes at a density of 2.5106 cells per well were then cultured in the
medium with  

Experimental protocols

After 5–7 d of cell culture in normoxic medium, the
cardiomyocytes were randomly divided into four groups: sham (n=5), the
cardiomyocytes were seeded on the plate for a total of 4 h of normoxic
incubation; H/Re control (n=5), the cardiomyocytes underwent 3 h of H
and 1 h of Re; F2 (n=5), F2 dissolved in PEG (110– 

Transmission electron
microscopy

The attached cardiomyocytes were digested with 0.25% trypsin and
0.2% EDTA (V/V, 1:1). After they were washed twice with
phosphate-buffered saline, the cell suspensions were centrifuged at  

Reverse
transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted from tissue or cultured cells using Trizol
reagent (Invitrogen,  

Immunohistochemistry and
immunocytochemistry

For immuohistochemical analysis, the tissue block was initially
immersed in 4% paraformaldehyde for 6 h then transferred to 30% sucrose
overnight. The tissue blocks were dehydrated in 30% sucrose, washed, embedded
and frozen in optimal cutting temperature compound. Cryosections of the tissue
blocks (10 mm) were cut using a Leica CM 100 cryostat
(Leica,  

Statistical analysis

Data are
shown as the mean±standard error of the mean. The significance of differences
was determined by using One-way anova,
followed by Student-Newman-Keuls test. p<0.05 was considered statistically significant.

Results

Changes in enzymatic
activities in plasma

A comparison in levels of plasma CK, CK-MB and LDH among all groups
at the end of the experiment is shown in Table 1. Compared with the sham
group, the plasma CK, CK-MB, and LDH activities in the I/R control group were
distinctly increased by 2.7-fold, 2.5-fold and 2.0-fold, respectively. A
similar pattern in changes of these parameters in the vehicle group was also
demonstrated (2.5-fold, 2.7-fold and 2.1-fold, respectively). Administration of
F2 distinctly attenuated changes in plasma CK,
CK-MB and LDH with an increase of 1.5-fold, 1.3-fold and 1.4-fold,
respectively, which were statistically different to those in the I/R control
group.

Changes in cardiomyocyte
ultrastructure

In the sham group, the shapes of organelles were normal and the
chromatin was distributed evenly in the nucleus of cardiomyocytes [Fig. 2(A)].
Relative to normal cardiomyocytes, H/Re caused significant morphological
changes with two distinct types of cell death: necrosis and apoptosis [Fig.
2(B)]. Necrosis was characterized by cell swelling, membrane structure
destruction and organelle breakdown, whereas apoptosis was distinguished as
chromatin condensation and margination. At the same time, degeneration characterized
by sparse cytoplasm and swollen or vacuolized mitochondria were observed in
many cardiomyocytes. Addition of PEG without F2 had no effect on changes in cardiomyocyte ultrastructure [Fig.
2(C)], which showed similar morphological changes as those in the I/R
control group. However, these changes in cardiomyocyte ultrastructure were
markedly inhibited when F2 was added to the cells
before H/Re. The necrotic and apoptotic cells were barely observed. Although
vacuolar mitochondria were also observed, the cytoplasm was rich in organelles
[Fig. 2(D)].

Levels of Egr-1 mRNA in
myocardial tissue and cultured cardiomyocytes

Levels of Egr-1 mRNA in the four groups are shown in Fig.
3. Relative to the sham group, levels of Egr-1 mRNA in myocardial tissue
and cultured cardiomyocytes in the I/R (H/Re) control group were significantly
increased at the end of the experiment. These changes were not altered by PEG,
but significantly reduced when F2 was given to the animals or
added to the cultured cells.

Detection of Egr-1 protein
expression in myocardial tissue and cultured cardiomyocytes

In the sham group, the weak immunostaining for Egr-1
expression was detected in myocardium (51 per sight, n=4), but not in
microvasculature [Fig. 4(A)]. In contrast, markedly enhanced Egr-1
expression was observed in both myocardium and microvasculature in the I/R
control group (293 per sight, n=4, P<0.05) [Fig. 4(B)]. These changes were not
altered by PEG (303 per sight, n=4, P<0.05) [Fig. 4(C)], but significantly downregulated
when F2 was administered before I/R (162 per sight, n=4, P<0.05) [Fig. 4(D)]. In addition, Egr-1 expression was also demonstrated in
cultured cardiomyocytes. Relative to the sham group (71 per sight, n=4)
[Fig. 4(E)], H/Re significantly increased Egr-1
expression (274 per sight, n=4, P<0.05), localized mainly in the nucleus of cardiomyocytes [Fig.
4(F)], which was not altered by PEG (283 per sight,
n=4, P<0.05) [Fig.
4(G)], but significantly inhibited by F2 (132 per sight, n=4, P<0.05) [Fig. 4(H)], consistent with a
down-regulation of Egr-1 expression in myocardial tissue.

Discussion

The present study demonstrates that F2 reduces myocardial I/R injury in an in vivo model as
evidenced by a reduction in leakage of myocardial enzymes such as CK, CK-MB and
LDH. In cultured neonatal cardiomyocytes, F2 also reduces H/Re-induced cell death. Consistent with attenuation in
myocardial and cell injury by F2, the expression levels of Egr-1 mRNA and protein are significantly
reduced.

It has been reported that the rapid activation of Egr-1 is
associated with I/R-induced tissue and cell injury [8,17–20]. Stimulation of Egr-1 expression causes release of
interleukin 1b, macrophage inflammatory protein 2,
intercellular adhesion molecule 1, tissue factor, plasminogen-activator
inhibitor 1, vascular endothelial growth factor and platelet-derived growth
factor A [9,10,20,21]. The expressions of these representative genes mediate
coagulation, inflammatory and vascular permeability, which are the main
pathological changes associated with I/R injury. Thus, Egr-1 has been
designated a central and unifying role in the pathogenesis of I/R injury [10].
Combining these data with our results, we suggest  that the alteration in the induction and activation of Egr-1
might play a significant role in myocardial and cellular injury associated with
I/R.

A previous study has demonstrated the time-dependent expression of EgrA number of gene products that participate in the pathophysiological
responses after I/R have been associated with enhanced expression of Egr-1
[9,10,21]. However, the signaling pathways in activating Egr-1
expression after I/R are not yet well defined. Recent studies have demonstrated the
potential roles of protein kinase C (PKCb), the Raf-MEK-Erk pathway and intracellular Ca2+ [11,22–24]. PKC-null mice or wild-type mice treated with PKCb inhibitor displayed increased survival compared with the control
animals after single-lung I/R. Protection was associated with significant
downregulation in Egr-1 expression [22]. In a rat model of liver
transplantation, the application of immunomodulator FTY720 has been shown to
reduce hepatic damage and increase survival rates by inhibiting Egr-1
expression-mediated pathways by the Raf-MEK-Erk pathway [23]. Mobilization of Ca2+ has also been implicated in the induction of Egr-1
expression with Ca2+-ionophore or EDTA [11,24]. In the present study, the inhibition in
overexpression of Egr-1 mRNA and protein as well as protection with F2 from in vivo and in vitro models
accorded with these results. We have previously shown in isolated
cardiomyocytes that F2 inhibits intracellular Ca2+ influx and overload, further maintains the
integrity of the cell membrane, and minimizes ATP depletion [15]. In in vivo
rat and rabbit models, F2 reduced I/R myocardial
injury [15,25]. Concomitant with the fact that Egr-1 expression can be
triggered by elevated intracellular Ca2+ overload [11, 24] and F2 reduces tissue and cell
damage by inhibiting Ca2+ overload [15], we conclude
that myocardial and cardiomyocyte protection by F2 might, in part, be a result of the inhibition of Ca2+ overload-mediated Egr-1 expression.
However, direct demonstration in roles of Egr-1 expression-induced
myocardial injury and Ca2+ overload-mediated Egr-1
expression after I/R requires further investigation.

In summary, these data provide evidence that F2 reduces myocardial injury in an in vivo
model of I/R, demonstrated by the attenuation in release of enzymes. In
cultured cardiomyocytes, F2 preserves cellular
ultrastructure, shown by the reduction in both necrosis and apoptosis after
H/Re. In addition, F2 decreases the expression
levels of Egr-1 mRNA and protein identified by RT-PCR,
immunohistochemistry and immunocytochemistry in myocardium and cardiomyocytes.
The data from present and previous studies in our laboratory [15,25] further
suggest that cardioprotection by F2 is associated with the
inhibition of Ca2+ overload and Egr-1
expression after I/R. Therefore, an understanding of the molecular events that
accompany myocardial I/R injury will enhance the development of therapeutic
strategies for treatment of cardiac injury.

Acknowledgments

This work was partly done in the Center for Molecular Biology,  

References

 1   Lopez-Neblina F,  2   Steinhoff G, Behrend M, Richter N, Schlitt
HJ, Cremer J, Haverich A. Distinct expression of cell–cell and cell–matrix adhesion molecules on endothelial
cells in human heart and lung transplants. J Heart Lung Transplant 1995, 14:
1145–1155

 3   Zhao ZQ, Morris CD, Budde JM, Wang NP, Muraki
S, Sun HY, Guyton RA. Inhibition of myocardial apoptosis reduces infarct size
and improves regional contractile dysfunction during reperfusion. Cardiovasc
Res 2003, 59: 132–142

 4   Hoffman JW Jr, Gilbert TB, Poston RS,
Silldorff EP. Myocardial reperfusion injury: Etiology, mechanisms, and
therapies. J Extra Corpor Technol 2004, 36: 391–411

 5    6   Mizukami Y, Iwamatsu A, Aki T, Kimura M,
Nakamura K, Nao T, Okusa T et al. ERK1/2 regulates intracellular ATP
levels through alpha-enolase expression in cardiomyocytes exposed to ischemic
hypoxia and reoxygenation. J Biol Chem 2004, 279: 50120–50131

 7   Stephanou A. Role of STAT-1 and STAT 8   Chen Y, Lui VC,  9   Okada M, Fujita T, Sakaguchi T, Olson KE,
Collins T, Stern DM, Yan SF et al. Extinguishing Egr-1-dependent
inflammatory and thrombotic cascades after lung transplantation. FASEB J 2001,
15: 2757–2759

10  Yan SF, Fujita T, Lu J, Okada K, Shan Zou Y, Mackman N, Pinsky DJ et
al. Egr-1, a master switch coordinating upregulation of divergent
gene families underlying ischemic stress. Nat Med 2000, 6: 1355–1361

11  Josefsen K, Sorensen LR, Buschard K, Birkenbach M. Glucose induces
early growth response gene (Egr-1) expression in pancreatic beta cells.
Diabetologia 1999, 42: 195–203

12  Shi GG, Xu SF. Effect of phencyclidine on contraction of porcine
coronary vessel strips. Zhong guo
13  Shi GG, Xu SF. Study on heart injury induced by phencyclidine in
rats. Shanghai Yi Ke Da Xue Xue Bao 1994, 21: 257–259

14 Shi GG, Zheng JH, Li CC, Chen JX, Zhuang XX, Chen SG, Liu XP. The
effect of quaternary ammonium salt derivation of haloperidol on coronary
artery. Zhong Guo 15  Huang ZQ, Shi GG, Zheng JH, Liu B. Effects of N-n-butyl
haloperidol iodide on rat myocardial ischemia and reperfusion injury and L-type
calcium current. Acta Pharmacol Sin 2003, 24: 757–763

16  Jonassen AK, Brar BK, Mjos OD, 17 Liang TB, Man K, Kin-Wah Lee T, Hong-Teng Tsui S, Lo CM, Xu X, Zheng
SS et al. Distinct intragraft response pattern in relation to graft size
in liver transplantation. Transplantation 2003, 75: 673–638

18  Okada M, Yan SF, Pinsky DJ. Peroxisome proliferator-activated
receptor-gamma (PPAR-gamma) activation suppresses ischemic induction of Egr-1
and its inflammatory gene targets. FASEB J 2002, 16: 1861–1868

19  Man K, Lee TK, Liang TB, Lo CM, Fung PC, Tsui SH, Li XL et al.
FK 409 ameliorates small-for-size liver graft injury by attenuation of portal
hypertension and down-regulation of Egr-1 pathway. Ann Surg 2004, 240:
159–168

20  Okada M, Wang CY, Hwang DW, Sakaguchi T, Olson KE, Yoshikawa Y,
Minamoto K et al. Transcriptional control of cardiac allograft
vasculopathy by early growth response gene-1 (Egr-1). Circ Res 2002, 91:
135–142

21  Yan SF, Zou YS, Gao Y, Zhai C, Mackman N, Lee SL, Milbrandt J et
al. Tissue factor transcription driven by Egr-1 is a critical
mechanism of murine pulmonary fibrin deposition in hypoxia. Proc Natl Acad Sci 22  Fujita T, Asai T, Andrassy M, Stern DM, Pinsky DJ, Zou YS, Okada M et
al. PKCbeta regulates ischemia/reperfusion injury in the lung. J Clin
Invest 2004, 113: 1615–1623

23  Man K, Ng KT, Lee TK, Lo CM, Sun CK, Li XL, Zhao Y et al.
FTY720 attenuates hepatic ischemia-reperfusion injury in normal and cirrhotic
livers. Am J Transplant 2005, 5: 40–49

24  Schaefer A, Magocsi M, Fandrich A, Marquardt H. Stimulation of the
Ca2+-mediated
egr-1 and c-fos expression in murine erythroleukaemia cells by
cyclosporin A. Biochem J 1998, 335: 505–511

25  Gao FF, Shi GG, Zheng JH, Liu B. Protective effects of N-n-butyl
haloperidol iodide on myocardial ischemia-reperfusion injury in rabbits. Chin J
Physiol 2004, 47: 61–66