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Journal of the American College of Cardiology Vol. 51, No. 13, 2008© 2008 by the American College of Cardiology Foundation ISSN 0735-1097/08/$34.00P

PRECLINICAL RESEARCH

Effect of ATP-Sensitive Potassium ChannelAgonists on Ventricular Remodeling in Healed Rat Infarcts

Tsung-Ming Lee, MD, FESC,* Mei-Shu Lin, PHD,† Nen-Chung Chang, MD, PHD, FACC‡

Tainan and Taipei, Taiwan

Objectives The purpose of this study was to determine whether ATP-sensitive potassium (KATP) channel agonists exert abeneficial effect on the structural, functional, and molecular features of the remodeling heart in infarcted rats.

Background Myocardial KATP channels have been implicated in the ventricular remodeling after myocardial infarction by inhi-bition of 70-kDa S6 (p70S6) kinase.

Methods Male Wistar rats after induction of myocardial infarction were randomized to either vehicle, agonists of KATP

channels nicorandil and pinacidil, an antagonist of KATP channels glibenclamide, or a combination of nicorandiland glibenclamide or pinacidil and glibenclamide for 4 weeks. To verify the role of p70S6 kinase in ventricularremodeling, rapamycin was also assessed.

Results Significant ventricular hypertrophy was detected by increased myocyte size at the border zone isolated by enzy-matic dissociation after infarction. Increased synthesis of p70S6 kinase messenger ribonucleic acid after infarc-tion in vehicle-treated rats was confirmed by reverse transcription-polymerase chain reaction, consistent with theresults of immunohistochemistry and Western blot for phosphorylated p70S6 kinase. Rats in the nicorandil- andpinacidil-treated groups significantly attenuated cardiomyocyte hypertrophy and phosphorylated p70S6 kinaseexpression with similar potency, as compared with vehicle. The beneficial effects of nicorandil and pinacidil wereabolished by administering either glibenclamide or 5-hydroxydecanoate. Addition of rapamycin attenuated ven-tricular remodeling and did not have additional beneficial effects compared with those seen in rats treated witheither nicorandil or pinacidil alone.

Conclusions Activation of KATP channels by either nicorandil or pinacidil can attenuate ventricular remodeling, probablythrough a p70S6 kinase-dependent pathway after infarction. (J Am Coll Cardiol 2008;51:1309–18) © 2008 bythe American College of Cardiology Foundation

ublished by Elsevier Inc. doi:10.1016/j.jacc.2007.11.067

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ardiac remodeling is an unfavorable evolution associatedith myocardial hypertrophy after myocardial infarction

MI) (1). Cardiac remodeling is a complex process involvingumerous signaling pathways. Pharmacological therapies,uch as angiotensin-converting enzyme inhibitors and beta-lockers through different molecular targets, have been usedo improve cardiac remodeling (2). Previous studies havehown that left ventricular (LV) remodeling is improvedfter nicorandil treatment (3), implying myocardial ATP-ensitive potassium (KATP) channels may play a role inentricular remodeling after infarction. We have shown airect relation between the activation of myocardial KATP

rom the *Cardiology Section, Department of Medicine, Taipei Medical Universitynd Chi-Mei Medical Center, Tainan, Taiwan; †Department of Pharmacy, Nationalaiwan University and Hospital, Taipei, Taiwan; and the ‡Cardiology Section,epartment of Medicine, Taipei Medical University and Hospital, Taipei, Taiwan.his work was supported by a grant from Chi-Mei Medical Center (CMFHT 9501,MFHR9502, CMFHR9506, and CM-TMU9501) and a grant from Nationalcience Council, Republic of China (NSC 95-2314-B-384-009).

1Manuscript received June 7, 2007; revised manuscript received October 29, 2007,

ccepted November 19, 2007.

hannels and ventricular remodeling by administering KATP

hannel antagonists in hyperlipidemic rabbits (4). Cabo andoyden (5) have shown a marked reduction of the currentensity of potassium current at the border zone cellsompared with that in the remote sites. Activation of KATP

hannels has been shown to attenuate cardiac hypertrophyy inhibition of 70-kDa S6 (p70S6) kinase (6), a key triggerf protein synthesis for hypertrophic changes. The activityf p70S6 kinase is controlled by multiple phosphorylation.hosphorylation of Thr389 most closely correlates with70S6 kinase activity in vivo (7). Although we have dem-nstrated a cardioprotection against infarct of any size andyocardial ischemia by activation of KATP channels in

nimals (8) and in humans (9) in acute settings, thebserved beneficial effects do not provide information onhether similar effects would be present on ventricular

emodeling with long-term administration of KATP channelgonists.

Cardiac myocytes contain 2 distinct KATP channels with
subtype located in the sarcolemma and the other in the

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1310 Lee et al. JACC Vol. 51, No. 13, 2008KATP Channels and Ventricular Remodeling April 1, 2008:1309–18

inner membrane of the mito-chondria (10). MitochondrialKATP channels share some phar-macological properties with sar-colemmal KATP channels, whilepossessing a distinct pharmaco-logic response. KATP channelsare activated by numerous com-pounds with distinct chemicalstructures (11). These agents,collectively termed as KATPchannel agonists, cause variousbiological effects through modu-lation of the sarcolemmal andmitochondrial KATP channels(11). Pinacidil has been shown toactivate both sarcolemmal andmitochondrial KATP channels,whereas nicorandil activates onlymitochondrial KATP channels(11). The purpose of this study

as: 1) to investigate how 2 KATP channel agonists, nic-randil and pinacidil, affect ventricular remodeling; 2) tossess the role of p70S6 kinase systems; and 3) toetermine which subtype of KATP channels plays a role inentricular remodeling in a rat MI model by the use of-hydroxydecanoate (5-HD), a specific mitochondrialATP channel blocker.

ethods

nimals. Male Wistar rats (250 to 300 g) were subjected toigation of the anterior descending artery, resulting innfarction of the LV free wall. Rats were randomly assignednto 9 groups so as to have approximately the same numberf survivors in each group: 1) vehicle group; 2) nicorandil0.1 mg/kg/day, Chugai Pharmaceutical Co., Tokyo, Ja-an), a specific mitochondrial KATP channel agonist; 3)inacidil (0.1 mg/kg/day, Sigma Chemical Co., St. Louis,issouri), a nonspecific KATP channel agonist; 4) gliben-

lamide (1.4 mg/kg/day), a nonspecific KATP channellocker; 5) a combination of nicorandil and glibenclamide;) a combination of pinacidil and glibenclamide; 7) rapa-ycin (0.5 mg/kg/day), a p70S6 kinase inhibitor; 8) a

ombination of nicorandil and rapamycin; and 9) a combi-ation of pinacidil and rapamycin. The doses of nicorandil12), pinacidil (13), and glibenclamide (14) used in thistudy have been shown to specifically modulate KATPhannels without the interference of hemodynamics. Toerify the role of p70S6 kinase in KATP channel inhibition-elated ventricular hypertrophy, we further assessed rapamy-in in the role of ventricular remodeling. The oral dose ofapamycin was according to previous studies (6), showingffective attenuation of cardiac remodeling induced byong-term inhibition of nitric oxide synthesis. The drugs

Abbreviationsand Acronyms

GAPDH � glyceraldehyde-3-phosphate-dehydrogenase

HD � hydroxydecanoate

KATP � ATP-sensitivepotassium

LV � leftventricle/ventricular

LVEDD � left ventricularend-diastolic diameterdimension

LVESD � left ventricularend-systolic diameterdimension

MI � myocardial infarction

p70S6 � 70-kDa S6

RT-PCR � reversetranscription-polymerasechain reaction

ere given orally by gastric gavage once a day. The drugs A

ere started 24 h after MI, during which drugs canaximize benefits at this timing window (15) and minimize

he possibility of a direct effect on infarct size. The studyuration was designed to be 4 weeks because the majority ofhe myocardial remodeling process in the rat (70% to 80%)s complete within 3 weeks (16). To prevent hypoglyce-

ic attacks during the administration of glibenclamide,.5% (weight/volume) sucrose in filtered tap water wasupplied and glucose examinations were performed onceer week by the 1-touch method. Sham operation serveds controls to exclude the possibility of drugs themselvesirectly to alter cardiomyocyte hypertrophy.Although results of the above study showed that KATP

hannel antagonist-induced ventricular remodeling was al-ered at 4 weeks after infarction (see Results section),libenclamide was used as an antagonist of KATP channels,hich can block both mitochondrial and sarcolemmal KATP

hannels and have multiple actions independent of KATPhannels. To further confirm the role of KATP channelgonist-induced ventricular remodeling and assess whichubtype of KATP channels plays a role in attenuating p70S6inase expression, we used 5-HD, a specific mitochondrialATP channel blocker, in an in vitro model. Four weeks

fter induction of MI by coronary ligation, infarcted ratearts were isolated and subjected to no treatment (vehicle),icorandil (50 �M), pinacidil (50 �M), 5-HD (100 �M),icorandil � 5-HD, or pinacidil � 5-HD. Each heart waserfused with a noncirculating modified Tyrode’s solutionontaining (in mM): NaCl 117.0, NaHCO3 23.0, KCl 4.6,aH2PO4 0.8, MgCl2 1.0, CaCl2 2.0, and glucose 5.5,

quilibrated at 37°C and oxygenated with a 95% O2 to 5%O2 gas mixture. The perfusion medium was maintained atconstant temperature of 37°C with a constant flow at 4l/min as previously described (17). Drugs were infused for

0 min. The doses of nicorandil (18), pinacidil (19), and-HD (20) used in this study have been shown to modulateATP channels in an isolated heart. At the end of the study,

ll hearts (n � 10 in each group) were used for performingestern blot from samples at the border zone. The animal

xperiment was approved and conducted in accordance withocal institutional guidelines for the care and use of labora-ory animals in the Chi-Mei Medical Center.xperimental MI. After anesthesia with ketamine (90g/kg) intraperitoneally, rats were intubated and the ante-

ior descending artery was ligated using a 6-0 silk asreviously described (17). Sham rats underwent the samerocedure except the suture was passed under the coronaryrtery and then removed. Mortality in the animals with MIas �50% within the first 24 h. None of the sham-operated

nimals died.chocardiogram. At 28 days after operation, rats were

ightly anesthetized with intraperitoneal injection of keta-ine HCl (25 mg/kg). Echocardiographic measurementsere done with an HP Sonos 5500 system with a 15-6L (6

o 15 MHz, SONOS 5500, Agilent Technologies, Palo
lto, California) probe. M-mode tracing of the LV was

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1311JACC Vol. 51, No. 13, 2008 Lee et al.April 1, 2008:1309–18 KATP Channels and Ventricular Remodeling

btained from the parasternal long-axis view to measure leftentricular end-diastolic diameter dimension (LVEDD)nd left ventricular end-systolic diameter dimensionLVESD), and fractional shortening (%) was calculated.fter this, the hearts quickly underwent hemodynamiceasurement after systemic heparinization.emodynamics and infarct size measurements. Using a

-F micromanometer-tipped catheter (Model SPR-407,illar Instruments, Houston, Texas) inserted through the

ight carotid artery as in our previous description (17), weeasured the maximal rate of LV pressure rise (�dP/dt)

nd decrease (�dP/dt). Next, the heart was rapidly excisednd divided into right and left atria, right ventricles,V, and the scarred area. Each tissue was then weighed

ndividually. Infarct size (%) was expressed as the ratio ofhe sum of external and internal perimeters of LV asescribed by Pfeffer and Braunwald (16). Samples of theV from the border zone (0 to 2 mm outside the infarct)ere cut transmurally, frozen rapidly in liquid nitrogen,

nd stored at �80°C until use. It has been shown thatypertrophy of the residual myocardium progresses afterI if infarct size is larger than 30% of the LV (16). Thus,

ith respect to clinical importance, only rats with infarc-ion larger than 30% of the LV were selected for analysis.everse transcription-polymerase chain reaction (RT-CR) analysis of p70S6 kinase. To further confirm theownstream activation of KATP channels, messenger ribo-ucleic acid (mRNA) levels of p70S6 kinase were measuredy real-time quantitative RT-PCR from samples obtainedrom the border zone with the TaqMan system (Prism700 Sequence Detection System, Applied Biosystems,ourtaboeuf, France) as in our previous description

17). For p70S6 kinase the primers were sense, 5=-AAGATTTATTGGTAGCCCACGAA-3=; antisense, 5=-CACCTCGTCCCCAGAAA-3=. For glyceraldehyde-3-

hosphate-dehydrogenase (GAPDH), the primers were-CTTCACCACCATGGAGAAGGC-3= and 5=-GCATGGACTGTGGTCATGAG-3=. For quantifica-

ion, p70S6 kinase expression was normalized to the ex-ressed housekeeping gene GAPDH. Reaction conditionsere programmed on a computer linked to the detector for0 cycles of the amplification step.

estern blot analysis of p70S6 kinase. Samples from theorder zone were homogenized, and the supernatant proteinoncentration was determined with the BCA protein assayeagent kit (Pierce, Rockford, Illinois); 20 �g protein waseparated by 8% SDS-PAGE and electrotransferred onto aitrocellulose membrane. After incubation with anti–hospho-p70S6 kinase antibodies (Thr389, #9205L, Cellignalling, Hitchin, Kent, United Kingdom) and anti–otal-p70S6 kinase antibodies (#9202, Cell Signalling,

itchin), antigen-antibody complexes were detected with-bromo-4-chloro-3-indolyl-phosphate and nitroblueetrazolium chloride (Sigma). Films were volume-
ntegrated within the linear range of the exposure using a c
canning densitometer. Experiments were replicated 3imes and results expressed as the mean value.mmunohistochemical analysis of p70S6 kinase. To con-rm the downstream pathways of KATP channel, immuno-istochemical staining of phospho-p70S6 kinase was per-ormed on LV muscle from the border zone. Cryosectionsere performed at a thickness of 5 �m and incubated withrabbit polyclonal anti–phospho-p70S6 kinase antibody

Santa Cruz Biotechnology, Santa Cruz, California) atilution 1:10. Immunostaining with p70S6 kinase antibod-es was performed using a standard immunoperoxidaseechnique (N-Histofine Simple Stain Rat MAX PO kit,ichirei Co., Tokyo, Japan). The antibody used had been

ested for specificity in the rat. Isotype-identical directlyonjugated antibodies served as a negative control.ell isolation. Because cardiac hypertrophy is a combina-

ion of reactive fibrosis and myocyte hypertrophy, weeasured cardiomyocyte sizes at the border zone besides

sing myocardial weight to avoid the interference of non-yocytes on post-infarction hypertrophy. Myocytes were

nzymatically isolated with collagenase (type II, Sigma) androtease (type XIV, Sigma) according to previously de-cribed techniques (17). Random high-power fields of theod-like relaxed myocytes with clear striations were selectedo eliminate selection bias. In the sham-operated group, cellidth and length were measured from the LV free wall for

omparisons. At least 100 cells from each section wereelected for measurement of cell length, width, and area,nd the mean value was used as the individual value for eachection.

orphometric determination of cardiac fibrosis. Heartections from the remote regions were stained with Siriused stain to distinguish areas of connective tissue as previ-usly described (21). The percentage of red staining, indic-tive of fibrosis, was measured in 10 fields randomly selectedn each section (Image Pro Plus, Media Cybernetics, Silverpring, Maryland). The value was expressed as the ratio ofirius red-stained fibrosis area to total infarct area. Allections were evaluated under blind conditions withoutrior knowledge as to which section belonged to which rat.aboratory measurements. To determine the confound-

ng roles of glucose and insulin in ventricular remodeling,lood samples from the aorta were assayed at the end of thetudy. Plasma insulin was measured by ultrasensitive ratnzyme immunoassay (Mercodia, Uppsala, Sweden).tatistical analysis. Results were presented as mean �tandard deviation. Statistical analysis was performed usinghe SPSS statistical package (version 10.0, SPSS Inc.,hicago, Illinois). A 1-way analysis of variance was used to

earch for possible effects of nicorandil, pinacidil, andlibenclamide on the measurements of hemodynamics andyocyte sizes. In case of a significant effect, the measure-ents between the groups were compared, with a value of� 0.007 (Bonferroni’s correction) as significant. Proba-

ility values were 2-tailed, and a value of p � 0.05 was
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ifferences in mortality between vehicle (n � 3, 25%) andreated groups (n � 20, 20%) were not found throughouthe study. No sham-operated rats had evidence of cardiacamage. Blood pressure, heart rate, infarct size, and glucose

evels did not significantly differ among the infarcted groupsTable 1). Insulin concentrations were significantly in-reased in rats that were administered glibenclamide.

orphometric studies. In the sham-operated rats, treat-ent with either nicorandil, pinacidil, or glibenclamide had

ittle effect on cardiac gross morphology (data not shown),hereas there were significant effects on the cardiac mor-hology after MI (Table 1). Four weeks after MI, thenfarcted area of the LV was very thin and was totallyeplaced by fully differentiated scar tissue. The vehicle-,libenclamide-, and a combination of nicorandil andlibenclamide- or pinacidil- and glibenclamide-treatedroups had an increase in right ventricular weight/bodyeight ratio, and lung weight/body weight ratio comparedith the sham-operated rats.To characterize the cardiac hypertrophy on a cellular

evel, we isolated cardiomyocytes from different treatedroups (Table 2). The cell areas isolated from the borderone in the vehicle significantly increased by 63% comparedith those from the same area of sham-operated hearts

4,664 � 187 �m2 in the vehicle vs. 2,863 � 92 �m2, p �.0001). Nicorandil and pinacidil reduced cell areas 20%nd 19% compared with vehicle (both p � 0.0001, respec-ively). Conversely, the rats to which glibenclamide wasdministered developed cardiomyocyte hypertrophy com-ared with the nicorandil- and pinacidil-treated groupslone. Besides, treatment of rapamycin significantly atten-ated cardiomyocyte hypertrophy and blunted the increasen cell area by about 15% compared with those treatedith vehicle at the border zone, a figure similar to that

n either the nicorandil- or pinacidil-treated group. How-ver, addition of rapamycin did not further attenuate ven-ricular hypertrophy in either the nicorandil- or pinacidil-reated group.

Fibrosis of the LV from the remote zone was examined inissue sections after Sirius red staining as shown in Figure 1.nfarcted rats treated with vehicle had significantly largerreas of intense focal fibrosis compared with sham-operatedats (0.09 � 0.04% vs. 0.02 � 0.02%, p � 0.0001).ompared with vehicle, treatment with either nicorandil orinacidil attenuated fibrosis. Collagen formation in the LVas significantly increased in rats treated with a combina-

ion of KATP channel agonists and glibenclamide comparedith KATP channel agonists-treated rats alone.chocardiographic data. Compared with sham-operatedearts, MI hearts showed structural changes such as in-reased LV diastolic and systolic diameters (Figs. 2 and 3),onsistent with LV remodeling. Both LVEDD andVESD in rats with MI were significantly reduced by
icorandil or pinacidil treatment (p � 0.0001). Left ven- Ca T N B H L L L R L � � In G In
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Characteristics of Isolated Cardiomyocytes

Table 2 Characteristics of Isolated Cardiomyocytes

Parameters Sham Vehicle Nic Rap Rap � Nic Rap � Pin

Number of animals 5 4 5 5 5 5

Border zone

Myocyte length, �m 132 � 5 179 � 11* 158 � 8*† 161 � 11*§ 156 � 14*§ 153 � 9*§

Myocyte width, �m 21 � 3 24 � 3* 21 � 2† 21 � 3§ 22 � 2§ 21 � 3§

Measured myocyte areas, �m2 2,863 � 92 4,664 � 187* 3,721 � 153*† ,963 � 186*§ 3,894 � 132*§ 3,595 � 126*§

Remote zone

Myocyte length, �m 135 � 6 143 � 13� 141 � 10� 145 � 11 138 � 11� 143 � 11

Myocyte width, �m 20 � 2 19 � 2� 19 � 3 19 � 2 20 � 3 19 � 2

Measured myocyte areas, �m2 2,861 � 97 2,959 � 89� 2,985 � 102� ,008 � 114� 2,983 � 86� 3,197 � 105

Values are mean � standard deviation. *p � 0.005 compared with the sham-operated group; †p � 0.007 co ted groups treated with vehicle and Pin � Glib; §p � 0.05 comparedwith vehicle; �p � 0.05 compared with respective data from border zones within the same group.

Abbreviations as in Table 1.

1313JACC

Vol.51,No.13,2008Lee

etal.

April1,2008:1309–18K

ATP

Channelsand

VentricularRem

odeling

Infarction Treated With

Pin Glib Nic � Glib Pin � Glib

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151 � 12*‡ 172 � 12* 177 � 11* 182 � 10*

22 � 2‡ 27 � 3* 26 � 2* 25 � 3*

3,760 � 85*‡ 4,782 � 162* 4,920 � 143* 5,090 � 86* 3

136 � 11� 146 � 13� 143 � 9� 144 � 12�

20 � 2 18 � 2� 19 � 2� 20 � 2�

3,104 � 98� 2,851 � 96� 2,911 � 88� 3,092 � 75� 3

mpared with infarcted groups treated with vehicle and Nic � Glib; ‡p � 0.007 compared with infarc

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ricular fractional shortening was significantly higher in theicorandil- or pinacidil-treated group compared with theehicle. Conversely, the rats to which glibenclamide wasdministered developed impaired LV systolic function androgressive LV dilation more than that in the nicorandil-nd pinacidil-treated groups alone. These data were corrob-rated by the results that �dP/dt and �dP/dt were signif-cantly improved in the nicorandil- and pinacidil-treated

Figure 2 Echocardiogram in Infarcted Rats

Representative M-mode image reveals a hypokinetic-to-akinetic anterior walland left ventricular (LV) dilation in the infarcted hearts (B to J) in contrast tonormal anterior wall motion in sham-operated heart (A). There are markedlydilated LV end-diastolic diameter and LV end-systolic diameter in groupstreated with vehicle (B), Glib (E), Nic � Glib (F), and Pin � Glib (G) comparedwith those in groups treated with Nic (C) and Pin (D). Besides, infarcted ratstreated with Rap (H), Rap � Nic (I), and Rap � Pin (J) showed a significantdecrease of LV end-diastolic diameter and LV end-systolic diameter comparedwith vehicle. IVS � interventricular septum thickness; other abbreviations as inFigure 1.

roups compared with the combined groups.

mmunohistochemical analyses, Western blot, and real-ime PCR of p70S6 kinase. Immunohistochemical analy-is of the infarcted myocardium revealed the presence ofhospho-p70S6 kinase immunoreactivity in the myocardialissue (Fig. 4). Stronger phospho-p70S6 kinase signals athe border zone of vehicle-treated rats were observed than inhe same region of sham rats. The intensity of the immu-

Figure 3 Comparison of LV Remodelingand Function by 2D Echocardiography Study

Values are mean � standard deviation. �p � 0.005 compared with the shamgroup; †p � 0.007 compared with infarcted groups treated with vehicle andNic � Glib; ‡p � 0.007 compared with infarcted groups treated with vehicleand Pin � Glib; §p � 0.007 compared with vehicle. LV � left ventricular; 2D �

2-dimensional; other abbreviations as in Figure 1.

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oreaction was reduced in the nicorandil- and pinacidil-reated groups compared with that in the vehicle.icorandil- and pinacidil-induced beneficial effects were

eversed by the addition of glibenclamide, implicating KATP

hannels as the relevant target.Western blot shows that the levels of LV phospho-p70S6

inase were significantly more up-regulated 1.9-fold at theorder zone in the vehicle than in sham-operated rats (p �.0001), whereas total p70S6 kinase remained constantFig. 5). Compared with vehicle in nicorandil- andinacidil-treated rats, LV phospho-p70S6 kinase levels wereignificantly lower at the border zone. The rats to whichlibenclamide was administered showed significantly higherhospho-p70S6 kinase levels than those in the nicorandil-

Figure 4 Immunohistochemical Microscopy ofp70S6 Kinase at the BZ in Different Treated Rats

Positive staining for phospho-p70S6 kinase (p70S6 kinase) (magnification200�) (brown color) in myocytes is significantly higher in groups treated withvehicle (B), glibenclamide (E), and a combination of nicorandil � gliben-clamide (F) and pinacidil � glibenclamide (G) than those in sham (A), nic-orandil- (C), pinacidil- (D), rapamycin- (H), rapamycin � nicorandil- (I), andrapamycin � pinacidil–treated rats (J). The line length corresponds to 200�m. BZ � border zone.

nd pinacidil-treated groups alone. To elucidate the role of

itochondrial KATP channels in modulating p70S6 kinase,-HD was assessed in an in vitro model. Figure 6 showshat 5-HD significantly inhibited attenuated expression ofhospho-p70S6 kinase compared with either nicorandil orinacidil alone, confirming the role of mitochondrial KATPhannels in mediating p70S6 kinase.

The mRNA levels of p70S6 kinase showed a 5.4 �.2-fold up-regulation at the border zone in the vehicleompared with the sham-operated rats (p � 0.0001)Fig. 7). Thus, the mRNA levels of p70S6 kinase changedn parallel to the tissue peptide levels, implying that theroduction of p70S6 kinase mRNA is a critical regulationtep for its local activation.

iscussion

his study demonstrates for the first time that chronicreatment for 4 weeks with KATP channel agonists leads toavorable ventricular remodeling, probably through attenu-ted activity of phospho-p70S6 kinase. These results wereoncordant for beneficial effects of KATP channel agonists,s documented structurally by reduction in myocyte sizesnd cardiac fibrosis, molecularly, by myocardial p70S6inase protein and mRNA levels, and functionally, by

Figure 5 Western Blot of Phospo-p70S6 Kinaseand Total p70S6 Kinase in an In Vivo Study

(Top) Western blot analysis of p70S6 kinase showing the effect of KATP chan-nels on immunorecognition of p70S6 kinase in homogenates of the LV fromthe border zone. p70S6 kinase activity was measured with phospho-specificantibody against phosphorylated p70S6 kinase (top band) and a total p70S6kinase antibody (bottom band). Ventricular remodeling after myocardial infarc-tion was associated with marked increase of phospho-p70S6 kinase. A signifi-cantly reduced phospho-p70S6 kinase had taken place in the groups treatedwith either nicorandil or pinacidil administration compared with that seen invehicle. (Bottom) Densitometric quantification of phosphorylation levels wasexpressed as the ratio of the density of phosphorylated band over total p70S6kinase. Each point is an average of 3 separate experiments. �p � 0.007 com-pared with vehicle-, Glib-, Nic � Glib-, and Pin � Glib–treated groups. Abbrevia-tions as in Figure 1.

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mprovement of echocardiography-derived systolic function.hese new observations strengthen the concept that KATP

hannels play a central role in the remodeling process anday improve our understanding of the beneficial effect of

arly administration of KATP channel agonists in post-nfarction remodeling.

The beneficial effect of KATP channels on LV remodelingas demonstrated as evidenced by 3 observations.irst, KATP channel-related LV remodeling was noted after

nfarction. Administration of KATP channel agonists atten-ates the dysfunction of chronically infarcted hearts. Car-iomyocyte sizes, cardiac fibrosis, and echocardiographicallyerived LVESD and LVEDD were significantly smaller inither the pinacidil- or nicorandil-treated group than thosen the vehicle. Our results were consistent with the findingsf Coromilas et al. (22), showing that KATP channels at theorder zone remain functional and can be activated by KATPhannel agonists. The involvement of KATP channels isurther supported by the antagonizing action of gliben-lamide. Second, either pinacidil or nicorandil administra-ion can attenuate cardiomyocyte hypertrophy with a similarotency at the border zone. A similar potency to attenuateypertrophy by pinacidil and nicorandil may suggest that

Figure 6 Western Blot of Phospo-p70S6 Kinaseand Total p70S6 Kinase in an In Vitro Study

(Top) Western blot analysis of p70S6 kinase to furthermore confirm the mito-chondrial KATP channels on p70S6 kinase activity in homogenates of the leftventricle from the border zone in a rat isolated heart model. P70S6 kinaseactivity was measured with phospho-specific antibody against phosphorylatedp70S6 kinase (top band) and a total p70S6 kinase antibody (bottomband). A significantly increased phospho-p70S6 kinase is noted in thegroups treated with a combination of KATP channel agonists and 5-hydroxy-decanoate (5-HD) compared with that seen in the groups treated with KATP

channel agonists alone. (Bottom) Densitometric quantification of phosphor-ylation levels was expressed as the ratio of the density of phosphorylatedband over total p70S6 kinase. Each point is an average of 3 separateexperiments. �p � 0.007 compared with vehicle-, 5-HD-, Nic � 5-HD-, andPin � 5-HD–treated groups. Abbreviations as in Figure 1.

itochondrial KATP channels play a role in regulating

ardiomyocyte hypertrophy. Although dissimilar structures,inacidil and nicorandil appear to share a common media-or, p70S6 kinase, in which transcription levels of p70S6inase may play a role in the signal transduction pathway.hird, the involvement of p70S6 kinase in mitochondrialATP channel inhibition-related ventricular remodeling af-

er infarction was further confirmed by administering aitochondrial KATP antagonist (5-HD). The pharmaco-

ogic selectivity of the used mitochondrial KATP channellocker, at the conventional inhibitory concentrations (20)sed in the present study may indicate the important rolef the mitochondrial KATP channel in modulating p70S6inase activity, leading to attenuate cardiac hypertrophy.urthermore, addition of rapamycin attenuated ventric-lar remodeling and did not have additional beneficialffects compared with rats treated with either nicorandilr pinacidil alone, confirming the critical role of p70S6inase in ventricular remodeling. Indeed, our results wereonsistent with the observation that cell hypertrophy isegulated by mitochondrial KATP channel-dependent70S6 kinase (23,24).It appears from our study that the attenuated ventricular

emodeling is related to an activation of KATP channels.he mechanisms by which KATP channel agonists attenuate

entricular remodeling remain to be defined. However,everal factors can be excluded: 1) hemodynamics: neitherinacidil nor nicorandil exerted any hemodynamic effects athe dose used in this study. Nicorandil has been shown toeduce hypertrophy in animals, which has been assumed toe due to the drug’s blood-pressure-lowering effect (25).he observation was not consistent with our stable hemo-ynamics throughout the study in nicorandil-treated rats.ndeed, in rats, Xu et al. (26) have shown that oral dose oficorandil (6 mg/kg/day), a dose much higher than that

Figure 7 Western Blot of p70S6 Kinase in an In Vivo Study

Each mRNA was corrected for an mRNA level of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). Each column and bar represents mean � standarddeviation. �p � 0.007 compared with vehicle-, Glib-, Nic � Glib-, and Pin �

Glib–treated groups. LV � left ventricular; other abbreviations as in Figure 1.

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sed in our study (0.1 mg/kg/day), did not lower bloodressure. 2) Differences in MI size: MI size is an importanteterminant of LV remodeling (27). However, there is aimilar MI size among the infarcted groups.

ther mechanisms. Although the present study suggestshat the mechanisms of KATP channel agonist-inducedttenuated ventricular remodeling may be related to inhibi-ion of p70S6 kinase, there are possible other candidatesodulating the antihypertrophic effects of KATP channel

gonists, such as endothelin-1 and angiotensin. First, block-de of endothelin-1 alleviated the development of cardiacypertrophy (28). Activation of KATP channels has beenhown to attenuate cardiac hypertrophy by inhibition ofndothelin-1 (29). Thus, KATP channel agonists may atten-ate cardiac hypertrophy by attenuated production ofndothelin-1. Second, blunting by KATP channel agonists ofntihypertropic effect may also result from attenuated effectsf angiotensin; KATP channel agonists have been shown tonhibit angiotensin II-induced increase in cell protein con-ent (30). Complex interactions between endothelin-1, an-iotensin II, and KATP channels exist that could affectardiac hypertrophy. Therefore, a variety of upstream reg-lators could interfere with hypertrophic signaling path-ays. The development of cardiac hypertrophy is a multi-enic, integrative response involving signal integration ofultiple pathways. The failure to completely abrogate the

ypertrophic process was not surprising in view of thenderlying complex of hypertrophic process, which is un-ikely to be amendable to one therapeutic intervention.

linical implications. After acute MI, patients remain atigh risk for recurrent cardiovascular events and mortality31). Previous studies have demonstrated that regionalyocyte hypertrophy parallels regional myocardial dysfunc-

ion during post-infarct remodeling (32). Attenuation ofentricular remodeling prevents the transition for compen-atory dilation to ventricular dysfunction and failure. Thus,he KATP channel agonists may have important biologicalffects that prevent occurrence of post-infarcted heart fail-re. Our results explained, at least in part, the clinicalndings of the IONA (Impact Of Nicorandil in Angina)tudy group (33), showing that treatment with nicorandilmproves outcome in terms of reducing events related tocute coronary disease and the associated requirement fordmission to hospital.

onclusions

hese findings are consistent with a pathogenetic role ofegional KATP channels in cardiac remodeling after MI.arly intervention with KATP channel agonists after MI can

ttenuate ventricular remodeling probably through inhibi-ion of phospho-p70S6 kinase pathway. The pharmacologicrofile of KATP channel agonists gives new perspectives in
he early treatment of acute MI.
eprint requests and correspondence: Prof. Nen-Chung Chang,ardiology Section, Department of Medicine, Taipei Medicalniversity and Hospital, 252, Wu-Hsing Street, Taipei, 110,aiwan. E-mail: [email protected].

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