Cardiac hypertrophy induced by prostaglandin may be mediated by

Document Sample
Cardiac hypertrophy induced by prostaglandin may be mediated by Powered By Docstoc
					742                                                                                Acta Physiologica Sinica, December 25, 2005, 57 (6): 742-748

Research Paper

Cardiac hypertrophy induced by prostaglandin F2α may be mediated by
calcineurin signal transduction pathway in rats
JIANG Qing-Song1, 2 , HUANG Xie-Nan1,*, YANG Gui-Zhong1, 2, DAI Zhi-Kai1, ZHOU Qi-Xin2 , SHI Jing-Shan1,
WU Qin1
    Department of Pharmacology, Zunyi Medical College, Zunyi 563003, China; 2 Department of Pharmacology, Chongqing Medical
University, Chongqing 400016, China

Abstract: In this paper, we studied the relationship between the prostaglandin F2α (PGF2α)-induced cardiac hypertrophy and calcineurin
(CaN) signal transduction pathway in vivo and in vitro. Male Sprague-Dawley rats were given a single i.p. injection with monocrotaline
(MCT) (60 mg/kg) and then given orally with celecoxib (20 mg/kg) or vehicle once a day for 14 d before (from d 1 to d 14) or after (from
d 15 to d 28) right ventricular hypertrophy (RVH) was formed. Body weight (BW), right ventricular weight (RV), left ventricular with
septum weight (LV), as well as lung weight were determined. RVH index (RVHI=RV/LV), RV/BW, and lung weight/BW were calculated
and histological changes were observed with transmission electron microscope. PGF2α level, atrial natriuretic peptide (ANP) and CaN
mRNA expressions, expression of CaN and its downstream effectors, NFAT 3 and GATA4 protein were assayed by EIA kit, RT-PCR,
and Western blotting, respectively. The cardiomyocyte hypertrophy in primary culture induced by PGF2α (0.1 µmol/L) was evaluated
by measuring the cell diameter, protein content, and ANP mRNA as well as CaN mRNA expressions. It was found that 14 d or 28 d after
MCT was given, the RVHI, RV/BW, and lung weight/BW were significantly increased by 47%, 53% and 118%, and by 64%, 94% and
156%, respectively; at the same time PGF2α levels in RV tissue were increased by 44% and by 51% with increasing RVHI, and elevated
expressions of ANP and CaN mRNA, as well as CaN, NFAT 3 and GATA4 proteins in a positive correlation manner. Furthermore, some
histological injuries were found in RV tissue. Celecoxib, a cyclooxygenase inhibitor, obviously blunted the elevation of RVHI, RV/BW,
and lung weight/BW no matter it was given before or after RVH. In vitro experiments showed that 0.1 µmol/L PGF2α significantly
increased the cardiomyocyte diameter and protein content, and promoted ANP and CaN mRNA expressions, which was blocked by
cyclosporin A, a CaN inhibitor. Our results indicate that PGF2α may be involved in cardiac hypertrophy induced by MCT in rats
through CaN signal transduction pathway.

Key words: prostaglandin F2α; cardiac hypertrophy; calcineurin; monocrotaline

                                                                                  F 2α
                  1,2             1,*                  1,2             1            2              1            1

1                                                            2
                                               563003                                          400016

                                (monocrotaline         MCT)                                                                     F2α (prostaglandin
F2α           PGF2α )                                            (calcineurin    CaN)                                     Sprague-Dawley
                MCT (60 mg/kg)          i.p.                                       (20 mg/kg)          /            2
                                                   EIA                          PGF2α       RT-PCR                      (atrial natriuretic peptide
ANP)             CaN mRNA                                          CaN                   NFAT 3        GATA4
                    ANP mRNA                       0.1 µmol/L PGF2α                                        CaN mRNA
                        CaN                    A   PGF2α                         CaN mRNA                                 MCT          2       (M2W
     )                        (RVHI)               /               /                     47%      53%        118%          4    (M4W       )

      Received 2005-03-30          Accepted 2005-08-15
      This work was supported by the Science Foundation of Guizhou Province (No.2004-3057).
             Corresponding author. Tel : +86-852-8205416; E-mail:
JIANG Qing-Song et al: Cardiac Hypertrophy Induced by PGF2α May be Mediated by CaN                                             743

64%    94%     156%                                                            PGF2α        M2W      M4W                  44%
51%       RVHI     ANP       CaN    mRNA              CaN/NFAT 3 /GATA4
                             MCT                                                  PGF2α (0.1 µmol/L)
                 ANP     CaN mRNA                        CaN               A           PGF2α                          CaN mRNA
                                       PGF2α          MCT                          CaN

               Q257; R542.2

It has been well known that cardiac hypertrophy, which is           lates several downstream effectors such as calcineurin
caused by chronic increase in cardiac workload, may be              (CaN) and Ca 2+/calmodulin-dependent protein kinases
beneficial for body to maintain normal cardiac function.            (CaMKs). CaN is a key mediator of cardiac hypertrophy [10, 11].
On the other hand, hypertrophy definitely leads to dys-             It dephosphorylates nuclear factor of activated T cells
function of heart, even congestive heart failure, arrhyth-          (NFAT), which then translocates to the nucleus where it
mia and sudden death[1]. Therefore, it is very important to         acts combinatorially with other transcription factors (e.g.
understand the precise mechanisms and mediators of car-             GATA4) for activation of downstream targets to induce
diac hypertrophy.                                                   cardiac hypertrophy [12,13]. However, whether PGF2α-in-
   It has been reported that many vasoactive substances             duced cardiac hypertrophy is mediated by CaN-signal path-
were implicated in cardiac and vascular remodeling[2], but          way has not been known yet.
only a few studies focused on prostaglandin F2α (PGF2α)                In this study, we took right ventricular hypertrophy
which caused cultured rat cardiomyocytes over hypertro-             (RVH) model of rat induced by monocrotaline (MCT)[14]
phy and over expression of atrial natriuretic peptide (ANP)[3].     and cultured neonatal rat cardiomyocytes to investigate the
Some data showed that experimental infarction, ischemia,            possible relationship between CaN-signaling pathway and
and pressure overload increased the formation of prostag-           PGF2α in cardiac hypertrophy.
landins (PGs) in the myocardium[3-5]. These findings indi-
cate that PGF2α is a potential candidate to mediate the car-        1 MATERIALS AND METHODS
diac hypertrophy.                                                   1.1 Animals
   In general, PGF2α exerts its biological effects through FP       Male Sprague-Dawley rats (200~250 g, Grade II, Certifi-
prostanoid receptor, which is coupled with activation of            cate No. SCXK Chongqing 20020003, China) were housed
phospholipase C via Gq protein, leading to inositol triphos-        in a standard environment with a 12 h light /12 h dark
phate-induced elevation of intracellular calcium and                cycle, where they were freely access to food and water.
diacylglycerol activation of protein kinase C (PKC)[6].             One week after being fed adaptively, the rats were injected
Stimulation of Gq protein-linked receptors can also lead to         (i.p.) with MCT solution (Sigma Chemical Co., USA, 60
activation of members of the mitogen-activated protein ki-          mg/kg, n=40) or equal volumes of vehicle (n=21). After
nase (MAPK) family, including extracellular signal-regu-            that, the animals were divided into 7 groups: (1) vehicle-
lated kinase (ERK), p38 mitogen-activated protein kinase            treated rats (3 groups): after vehicle was given, the rats
(p38), and c-Jun NH2-terminal kinase (JNK), which are               were given i.g. with vehicle once a day for 0, 2 or 4 weeks
thought to play a critical role in cell proliferation and differ-   (N0W, N2W, and N4W groups) (n=7), respectively; (2)
entiation as well as cardiac hypertrophy[7]. It was said that       MCT-treated rats (2 groups): after MCT-treatment, the rats
PGF2α-induced cardiomyocyte hypertrophy and ANP ex-                 were given i.g. with vehicle once a day for 2 or 4 weeks
pression is independent of PKC, p38, or ERK pathways[8],            (M2W or M4W groups) (n=10); (3) MCT plus celecoxib-
therefore, the possible signal transduction pathway is quite        treated rats (2 groups): after MCT-treatment, the rats were
different from that of known activation of Gq.                      given i.g. with celecoxib (G.D. Searle & Co., USA) 20
   Ca2+ signaling has been reported to play a critical role in      mg/kg·d-1 for 14 d from d 1 to d 14 or from d 15 to d 28
the generation of cardiac hypertrophy induced by various            (PC2W and TC2W groups) (n=10).
hypertrophic stimuli, including angiotensin II, endothelin-            MCT was prepared by dissolving in 1 mol/L HCl. The
I, α-adrenergic agents and mechanical stress[9]. The in-            pH was neutralized with 0.5 mol/L NaOH, and final con-
creased intracellular Ca2+ binds to calmodulin and regu-            centration was adjusted to 2% with phosphate-buffered
744                                                              Acta Physiologica Sinica, December 25, 2005, 57 (6): 742-748

saline (PBS, pH 7.4).                                          AAG CTG TTG CAG CCT A -3’; (2) CaN: sense 5’- ACT
1.2 Measurement of hemodynamics                                GGC ATG CTC CCC AGC GGA -3’, antisense 5’- GTG
The animals were weighed and anesthetized with chloral         CCG TTA GTC TCT GAG GCG -3’; (3) β-actin: sense
hydrate solution (0.4 g/kg, i.p.), and then the carotid ar-    5’- GAC TAC CTC ATG AAG ATC CTG ACC -3’, antisense
tery was dissociated so as to insert the artery cannulae to    5’- TGA TCT TCA TGG TGC TAG GAG CC -3’; the
measure systolic pressure/diastolic pressure (SP/DP), mean     predicted product size were 202 bp, 244 bp, and 423 bp,
arterial pressure (AP) and heart rate (HR).                    respectively. These primers were synthesized by Beijing
1.3 Determination of heart weights                             Dingguo Shengwu Jishu Fazhan Ltd. Co. (China). RT-PCR
After measurement of hemodynamics, the rats were killed        was performed according to the RT-PCR Kit protocols
by decapitation, and then the lung and heart were removed      (Promega, USA) in a DNA thermal cycler (PCR instrument,
and weighed quickly. The heart was separated into the          Bio-Rad, USA). Cycling conditions were: (1) 1 cycle of
right ventricle (RV) and the left ventricle with septum (LV)   48 °C for 45 min, 94 °C for 2 min; (2) 35 cycles of 94 °C
and weighed separately. Finally, the lung weight/body          for 30 s, 60 °C for 30 s, and 72 °C for 1 min; (3) 72 °C for
weight (BW), RV hypertrophic index (RVHI=RV/LV) and            7 min. The products were electrophoresed on a 1% agar-
RV/BW were calculated. After weighing, the RV tissue was       ose gel and visualized with ethidium bromide. The optical
quickly frozen and kept at –70 °C until PGF2α, protein or      density values (OD) for each band of ANP, CaN and β-
total RNA were isolated.                                       actin on the gel were assayed by BI2000 Imaging Analysis
1.4 Histological observation                                   System (Chengdu Taimeng Sci-Tec Ltd. Co., China). β-
After RV walls (1 mm3) were fixed by immersion in cold         actin was used as an internal control for semi-quantitative
2.5% glutaraldehyde solution (pH 7.4), they were rinsed        assay.
and postfixed with 1% osmium tetroxide in 0.1 mol/L PBS        1.7 Western blotting for RV tissue proteins
for 2 h at room temperature, and then dehydrated through       Forty microgrammes of protein from the supernatant of
a graded series of ethanol to propylene oxide and embed-       homogenized rat RV tissues centrifuged at 12 000 g was
ded in epoxy resin. 60 nm section in thick were made and       separated by 10% SDS-PAGE and transferred on
then observed with transmission electron microscope.           polyvinylidene difluoride nylon membranes. Protein con-
1.5 Myocyte PGF2α isolation and measurements                   centrations were determined by the Bradford assay[18] us-
Three hearts per group were collected for myocyte isola-       ing bovine γ-globulin as standard. The blots were probed
tion using the methods described by Zhang [15] and PGF2α       with mouse anti-CaNA-α (1:200 dilution) or anti-NFAT3
contents were measured with EIA kit (Cayman Chemical           (1:100 dilution) or anti-GATA4 antibodies (1:100 dilution)
Co., USA). In brief, frozen tissues (30 mg) were homog-        (Santa Cruz Biotechnology, USA) followed by horseradish
enized immediately in 1.0 ml absolute ethanol and normal       peroxidase-conjugated goat anti-mouse IgG (1:2 500
saline (2:8) using a motor-driven glass homogenizer. After     dilution) antibodies. Immunodetection was carried out us-
added with HCl 50 µl at 0.1 mol/L and ethyl acetate 5.0 ml,    ing BI2000 Imaging Analysis System.
the homongenate was centrifuged at 2 500 g for 20 min,         1.8 Cell culture
then the supernatant was lyophilized with nitrogen and         Neonatal rat ventricular myocytes from 1- to 3-day-old
stored at –70 °C. For the measurement of PGF2α, the lyo-       Sprague-Dawley rats were prepared and cultured for 48 h
philized residues were dissolved in 0.5 ml HEPES and its       in Dulbecco’s modified Eagle’s medium (DMEM) con-
absorption were assayed at 405 nm. The PGF2α content of        taining 20% fetal bovine serum and bromodeoxyuridine
each sample was calculated by the standard curve.              (0.1 mmol/L) as described previously[19]. The media was
1.6 RNA extraction and RT-PCR                                  replaced by serum-free DMEM for further 24 h before
Total RNA extracted from right ventricles according to the     treatments. PGF2α (0.1 µmol/L) was used to stimulate neo-
RNeasy Mini Handbook protocol (Qiagen Company,                 natal rat cardiomyocytes, and cyclosporin A, a CaN
Germany) was redissolved in 30 to 50 µl of DEPC water,         inhibitor, was used to investigate the relation between the
and stored at –70 °C. RNA was spectrophotometrically           myocyte hypertrophy induced by PGF2α and CaN signal
quantified by measuring the optical density of samples at      pathway. Cell diameter was measured by BI2000 Imaging
260/280 nm.                                                    Analysis System. Protein concentration and ANP mRNA
   The nucleotide sequence of the primers used in this ex-     as well as CaN mRNA expressions were determined by
periment were as follows[16, 17]: (1) ANP: sense 5’- GCC       the methods described above.
CTG AGC GAG CAG ACC GA -3’, antisense 5’- CGG                  1.9 Statistical analysis
JIANG Qing-Song et al: Cardiac Hypertrophy Induced by PGF2α May be Mediated by CaN                                                    745

All of the data were expressed as mean ± SD. The data                  2.3 Myocardial morphology
were analyzed statistically using the SPSS 11.5 for Win-               Pathomorphology of the RV was observed with electron
dows statistical program by one-way ANOVA or Student’s                 microscopy. It was shown that the hypertrophied RV tis-
t-test. Correlation analyses were used for observing the               sues from MCT-treated animals showed some injuries: dis-
correlation between PGF2α level with cardiac hypertrophy               ruption of the intercalated disk, irregular pattern of cross-
or with CaN signaling pathway. Differences were consid-                striation, and aggregation of swollen and misshapen mito-
ered statistically significant when P value was less than              chondria (Fig. 1).
0.05.                                                                  2.4 Levels of PGF2α
                                                                       The calculating 50% B/B0 was 41 pg/ml and the detection
2 RESULTS                                                              limit (80% B/B0) was 9 pg/ml using standard curve. The
                                                                       PGF2α content was (6.30 ± 0.50) pg/mg tissue in N0W.
2.1 Hemodynamics
                                                                       No changes were found in N2W and N4W [(6.03 ± 0.59),
The HR, SP/DP and AP were (351 ± 28) bpm and (136 ±
                                                                       (6.53 ± 0.29) pg/mg tissue, respectively]. But in MCT-
12), (108 ± 8), (117 ± 10) mmHg, respectively, in N0W
                                                                       treated rats, the PGF2α contents significantly increased by
group. No changes were found in other groups, including
                                                                       44% and 51% in M2W and M4W, respectively (P<0.05,
N2W, N4W, M2W, M4W, PC2W and TC2W groups (data
                                                                       n=6) (Fig.2), which was related to RVHI with correlation
not shown).
                                                                       coefficient of 0.75 (P<0.001).
2.2 RV tissue weights
                                                                       2.5 Expressions of ANP and CaN mRNA
Table 1 showed that the BW in vehicle-treated group (N0W,
N2W and N4W) increased in time-dependent manner, but                   Expressions of the cardiac ANP and CaN mRNA from RV
in M2W and M4W decreased by 16% and 21%,                               tissues were shown in Table 2. It was obvious that there
respectively, compared with that in N2W and N4W. The                   were no significant changes among N0W, N2W, and N4W
RVHI, RV/BW, and lung weight/BW increased by 47%,                      groups. However, in M2W and M4W groups, ANP mRNA
53%, and 118% in M2W and 64%, 94%, and 156% in                         and CaN mRNA expressions distinctly increased in time-
M4W, in comparison with corresponding control. Pretreat-               dependent manner and were related to PGF2α levels with
ment with celecoxib could significantly blunt the decrease             correlation coefficients of 0.92 and 0.86 (P<0.001),
of BW and the increase of RVHI, RV/BW and lung/BW                      respectively.
(PC2W vs M2W). Although the BW in TC2W group had                       2.6 CaN, NFAT3, GATA4 protein levels
no change, its RVHI, RV/BW and lung weight/BW were                     Figure 3 showed that the CaN, NFAT3, and GATA4 protein
obviously decreased by 9%, 18%, and 17%, respectively,                 levels were increased by 28%, 24%, and 15% in M2W
when compared with that in M4W.                                        (P<0.05), 44%, 28% and 21% in M4W (P<0.05),

 Table 1. General pathomorphological changes of heart and lung in rats treated with vehicle or monocrotaline or celecoxib + monocrotaline

   Groups            BW (g)                RVHI (g/g)                   RV/BW (      10-4, g/g)         Lung weight/BW (     10-4, g/g)

   N0W              214 ± 13             0.2768 ± 0.0196                    5.41 ± 0.41                     51.98 ± 5.33
   N2W              249 ± 21             0.2807 ± 0.0323                    5.79 ± 0.52                     52.39 ± 5.86
   N4W              271 ± 22 ***#        0.2831 ± 0.0238                    5.77 ± 0.65                     53.30 ± 12.28
   M2W              210 ± 23 ##          0.4113 ± 0.0470 ***###             8.86 ± 1.22 ***###             114.15 ± 16.36 ***###
   M4W              215 ± 16 +++         0.4638 ± 0.0537 ***+++            11.17 ± 1.42 ***+++             136.42 ± 21.44***+++
                               *                           **##                           ***###
   PC2W             231 ± 16             0.3485 ± 0.0501                    7.14 ± 0.60                      89.91 ± 10.17***###
   TC2W             219 ± 21+++          0.4209 ± 0.0195 ***+++             9.18 ± 0.83 ***+++             112.76 ± 17.84***+++

mean SD, ncontrol=7, nMCT=10. Groups: male Sprague-Dawley rats were injected (i.p.) with monocrotaline (60 mg/kg) and then given
orally with vehicle for 2 weeks (M2W) or 4 weeks (M4W), or with celecoxib (20 mg/kg) from d 1 to d 14 (PC2W) or from d 15 to d 28
(TC2W) after monocrotaline was given. Control groups were injected (i.p.) with vehicle and then given orally with vehicle for 0, 2, or 4
weeks (N0W, N2W, N4W). BW, body weight; RVHI, right ventricular hypertrophy index; RV, right ventricular weight. *P<0.05, **P<0.5,
    P<0.001 vs N0W; #P<0.05, ##P<0.01, ###P<0.001 vs N2W; +++P<0.001 vs N4W; P<0.05                 P<0.01,        P<0.001 vs M2W;
   P<0.05,    P<0.01 vs M4W.
746                                                                            Acta Physiologica Sinica, December 25, 2005, 57 (6): 742-748

Fig. 1. Electron microscopic findings of the right ventricular wall of the rats treated with vehicle (A) or with monocrotaline and then fed for 2
weeks (B). A did not show any abnormal changes, but B showed some myocardial injuries such as disruption of the intercalated disk (arrow),
an irregular pattern of cross-striation, and an aggregation of swollen and misshapen mitochondria (M). Scale bar, 2.2 µm.

                                                                            Table 2. Expressions of ANP or CaN mRNA in right ventricle of rats
                                                                            treated with vehicle or monocrotalline

                                                                               Groups          ANP mRNA (OD)             CaN mRNA (OD)

                                                                                N0W             0.010 ± 0.002             0.226 ± 0.023
                                                                                N2W             0.007 ± 0.003             0.227 ± 0.013
                                                                                N4W             0.008 ± 0.001             0.227 ± 0.020
                                                                                M2W             0.025 ± 0.002             0.394 ± 0.016**
                                                                                M4W             0.026 ± 0.003             0.424 ± 0.033##
Fig. 2. PGF2α levels in right ventricle of rats treated with vehicle or     mean ± SD, n =3. ANP, atrial natriuretic peptide; CaN, calcineurin.
monocrotaline. Groups: male Sprague-Dawley rats were injected               Groups: male Sprague-Dawley rats were injected (i.p.) with
(i.p.) with monocrotaline (60 mg/kg) and then given orally with ve-         monocrotaline (60 mg/kg) and then given orally with vehicle for 2
hicle for 2 weeks (M2W) or 4 weeks (M4W). Control groups were               weeks (M2W) or 4 weeks (M4W). Control groups were injected
injected (i.p.) with vehicle and then given orally with vehicle for 0, 2,   (i.p.) with vehicle and then given orally with vehicle for 0, 2, or 4
or 4 weeks (N0W, N2W, N4W). *P<0.05 vs corresponding control.               weeks (N0W, N2W, N4W). **P<0.01, ***P<0.001 vs N2W; ##P<
                                                                            0.01, ###P<0.001 vs N4W.

                                                                            respectively, compared with corresponding controls. The
                                                                            correlation analysis of them with PGF2α levels showed that
                                                                            the correlation coefficients were 0.58, 0.57 and 0.63
                                                                            (P<0.05), respectively.
                                                                            2.7 Effect of cyclosporin A on cardiomyocyte hyper-
                                                                            trophy induced by PGF2α
                                                                            Table 3 showed that the treatment of PGF2α (0.1 µmol/L)
Fig. 3. Expression of CaN, NFAT3 or GATA4 in right ventricle of             significantly increased the diameter, protein content and
rats treated with vehicle (N0W) or monocrotalline (60 mg/kg) i.p. and       ANP mRNA expressin of the primary cultured
then fed for 2 weeks (M2W) or 4 weeks (M4W) by Western blotting             cardiomyocytes and cyclosporin A (1 µmol/L) significantly
(OD). *P<0.05, **P<0.01 vs N0W.                                             decreased the elevation (P<0.01). Similar result was found
JIANG Qing-Song et al: Cardiac Hypertrophy Induced by PGF2α May be Mediated by CaN                                                    747

Table 3. Effects of cyclosporin A on the increasing cardiaomyocyte diamter, protein content, and ANP mRNA and CaN mRNA
expressions induced by PGF2α (mean ± SD)

      Groups (µmol/L)                  Cardiomyocyte           Protein content          ANP mRNA                    CaN mRNA
                                     diameter (µm) (n=3)        (pg/cell)(n=6)      expression (OD) (n=3)       expression (OD) (n=3)

 Control                                   36 ± 11             419.2 ± 41.6             0.010 ± 0.004              0.225 ± 0.004
                                                     ***                      ***                       ***
 PGF2α (0.1)                              115 ± 23             548.5 ± 59.2             0.026 ± 0.004              0.343 ± 0.030***
                                                     ###                      ##                        ##
 Cyclosporin A (1)+PGF2α (0.1)             67 ± 15             420.5 ± 69.7             0.019 ± 0.001              0.252 ± 0.033##

ANP, atrial natriuretic peptide; CaN, calcineurin. ***P<0.001 vs control; ##P<0.01, ###P<0.001 vs PGF2α (0.1 µmol/L ) treatment.

in the CaN mRNA expression of cardiomyocytes treated                 phy of cultured cardiac myocytes, it was considered that
by PGF2α and interfered by cyclosporin A.                            PGF2α might participate in the process of RVH caused by
                                                                     MCT. Similar report from Lai et al.[3] supports our opinion.
3 DISCUSSION                                                            Many molecular mechanisms are involved in the pro-
                                                                     cess of cardiac hypertrophy, including the factors and their
Current data for identification of factors that induce car-
                                                                     interactions in the MAPK and PKC pathways[7]. Recently,
diac hypertrophy overwhelmingly derived from animal LV
                                                                     the Ca2+/calmodulin- activated CaN signaling pathway was
hypertrophy models, such as spontaneously hypertensive
                                                                     also described as an important pathway in Gq-coupled re-
rats and aortic banding in normotensive Sprague-Dawley
                                                                     ceptor induced hypertrophy[23, 24]. In MCT-induced hyper-
rats, or from cultured cardiac myocyte in vitro. In the
                                                                     trophic model, we observed that there existed significant
present study, we used MCT-induced RVH rats to probe
                                                                     parallel elevations about CaN mRNA expression, CaN,
the possible role of PGF2α in the pathogenesis of RVH and
                                                                     NFAT3, GATA4 protein expressions, and PGF2α contents
its signal transduction pathway because it has been well
                                                                     in RV tissue. However, in order to determine whether this
known that MCT is converted in the liver to MCT-pyrrole              hypertrophic model is related to CaN signal pathway and
which causes injury of the lung vasculature leading to pul-          PGF2α, it is necessary to observe if the CaN inhibitor de-
monary hypertension and subsequently to RVH[20]. In our              presses the cardiac hypertrophy and CaN mRNA expre-
study, the significant increase lung weight/BW ratio, RVHI,          ssion. Because current CaN inhibitors have so many con-
and RV/BW ratio without changes of blood pressure and                founding effects that are associated with systemic admin-
HR strongly suggest that there exists the formation of pul-          istration of the drugs and make results confused, it is sel-
monary hypertension and RVH without LV hypertrophy                   dom to use them as tools for testing the CaN-pathway in
(LVH) in MCT-treated rats, which meant that the estab-               pressure-overload hypertrophy in vivo[10]. For this reason,
lishment of RVH model was successful. Furthermore, the               we took the myocyte hypertrophy model caused by PGF2α
observation that the changes of myocardial pathomorphol-             in vitro and found cyclosporin A, a CaN inhibitor, obvi-
ogy were similar to the description of RVH with increasing           ously blocked the myocyte hypertrophy process and CaN
expression of ANP mRNA in MCT-treated rats provided                  mRNA expression. Our results from in vivo and in vitro
another support[21].                                                 experiments indicate that RVH induced by MCT in rat may
    Great deals of investigations suggest that vasoactive            be related to PGF2α and at least partly, mediated by CaN
substances are concerned with cardiac hypertrophy[22]. For           signal transduction pathways.
example, angiotensin II, endothelin-1, and phenylephrine
are known to interact with Gq-coupled receptors and cause                                    REFERENCES
changes in morphology and gene expression in cardiac                 1   Hunter JJ, Chien KR. Signaling pathways for cardiac hypertro-
myocytes[7]. Our results showed that in MCT-induced RVH                  phy and failure. N Engl J Med 1999; 341 (17): 1276-1283.
model, the increase in RVHI and ANP mRNA expression                  2   Swynghedauw B. Molecular mechanisms of myocardial
were in positive correlation with the elevation of PGF2α                 remodeling. Physiol Rev 1999; 79 (1): 215-262.
level from myocardium, and celecoxib, a cyclooxygenase               3   Lai J, Jin H, Yang R, Winer J, Li W, Yen R, King KL, Zeigler F,
inhibitor, which could blunt the hypertrophic response in-               Ko A, Cheng J, Bunting S, Paoni NF. Prostaglandin F2α induces
duced by MCT no matter it was given before or after for-                 cardiac myocyte hypertrophy in vitro and cardiac growth in vivo.
mation of RVH. Together with the results from in vitro                   Am J Physiol 1996; 271 (6 Pt 2): H2197-H2208.
experiment in which, extraneous PGF2α induced hypertro-              4   Karmazyn M. Synthesis and relevance of cardiac eicosanoids
748                                                                          Acta Physiologica Sinica, December 25, 2005, 57 (6): 742-748

      with particular emphasis on ischemia and reperfusion. Can J            pulmonary vascular injury by the Na+-H + exchange inhibitor
      Physiol Pharmacol 1989; 67 (8): 912-921.                               cariporide. J Pharmacol Exp Ther 2001; 298(2): 469-476.
5     Mentz P, Pawelski KE, Giessler C, Mest HJ, Mannes F, Rotzoll        15 Zhang JT (         ). Phamarcologic Experimental Methods in
      S. Myocardial biosynthesis of prostacyclin and the influence of        Progress. 1st ed. Beijing: Chinese Academy of Medical Sciences
      cardiac loading and drugs. Biomed Biochim Acta 1988; 47 (10-           and Peking Union Medical College Press, 1998, 414-429
      11): S244-S247.                                                        (Chinese).
6     Fujino H, Srinivasan D, Pierce KL, Regan JW. Differential regu-     16 Taigen T, De Windt LJ, Molkentin JD. Targeted inhibition of
      lation of prostaglandin F2α receptor isoforms by protein kinase        calcineurin prevents agonist-induced cardiomyocyte
      C. Mol Pharmacol 2000; 57 (2): 353-358.                                hypertrophy. Proc Natl Acad Sci USA 2000; 97(3): 1196-1201.
7     Chien KR. Molecular Basis of Cardiovascular Disease. 1st ed.        17 Abassi Z, Brodsky S, Gealekman O, Rubinstein I, Hoffman A,
      Beijing: People’s Medical Publishing House, 2001, 211-                 Winaver J. Intrarenal expression and distribution of
      250.                                                                   cyclooxygenase isoforms in rats with experimental heart failure.
8     Adams JW, Sah VP, Henderson SA, Brown HJ. Tyrosine kinase              Am J Physiol Renal Physiol 2001; 280 (1): F43-F53.
      and c-jun NH2-terminal kinase mediate hypertrophic response to      18 Bradford MM. A rapid and sensitive method for the quantitation
      prostaglandin F2α in cultured neonatal rat ventricular myocytes.       of microgram quantities of protein utilizing the principle of pro-
      Circ Res 1998; 83(2): 167-178.                                         tein-dye binding. Anal Biochem 1976; 72: 248-254.
9     Frey N, McKinsey TA, Olson EN. Decoding calcium signals             19 Xu SY (         ), Bian RL, Chen X. Methodologies of Pharma-
      involved in cardiac growth and function. Nat Med 2000; 6 (11):         cological Experiments. 3rd ed. Beijing: People’s Medical Publish-
      1221-1227.                                                             ing House, 2002, 567-569 (Chinese).
10 Zhang W. Old and new tools to dissect calcineurin’s role in            20 Boor PJ, Gotlieb AI, Joseph EC, Kerns WD, Roth RA,
      pressure-overload cardiac hypertrophy. Cardiovasc Res 2002;            Tomaszewski KE. Chemical-induced vasculature injury. Toxicol
      53 (2): 294-303.                                                       Appl Pharmacol 1995; 132(2): 177-195.
11 De Windt LJ, Lim HW, Haq S, Force T, Molkentin JD.                     21 Ahn BH, Park HK, Cho HG, Lee HA, Lee YM, Yang EK, Lee
      Calcineurin promotes protein kinase C and c-jun NH2-terminal           WJ. Estrogen and enalapril attenuate the development of right
      kinase activation in the heart, cross-talk between cardiac hyper-      ventricular hypertrophy induced by monocrotaline in ovariecto-
      trophic signaling pathways. J Biol Chem 2000; 275(18): 13571-          mized rats. J Korean Med Sci 2003; 18 (5): 641-648.
      13579.                                                              22 Yamazaki T, Yazaki Y. Molecular basis of cardiac hypertrophy.
12 Hogan PG, Chen L, Nardone J, Rao H. Transcriptional regula-               Z Kardiol 2000; 89(1): 1-6.
      tion by calcium, calcineurin, and NFAT. Genes Dev 2003; 17          23 Wettschureck N, Rutten H, Zywietz A, Gehring D, Wilkie TM,
      (18): 2205-2232.                                                       Chen J, Chien KR, Offermanns S. Absence of pressure overload
13 Vega RB, Bassel-Duby R, Olson EN. Control of cardiac growth               induced myocardial hypertrophy after conditional inactivation
      and function by calcineurin signaling. J Biol Chem 2003; 278           of Galphaq/Galpha11 in cardiomyocytes. Nat Med 2001; 7(11):
      (39): 36981-36984.                                                     1236-1240.
14 Chen L, Gan XT, Haist JV, Feng Q, Lu X, Chakrabarti S,                 24 Molkentin JD, Dorn II GW. Cytoplasmic signaling pathways
      Karmazyn M. Attenuation of compensatory right ventricular              that regulate cardiac hypertrophy. Annu Rev Physiol 2001; 63:
      hypertrophy and heart failure following monocrotaline-induced          391-426.