Increased Connective Tissue Growth Factor Relative to ...hyper.ahajournals.org/content/hypertensionaha/49/5/1120.full.pdf · in AC rats, RNA isolation and Northern blot analysis,

Increased Connective Tissue Growth Factor Relative toBrain Natriuretic Peptide as a Determinant of

Myocardial FibrosisNorimichi Koitabashi, Masashi Arai, Shinya Kogure, Kazuo Niwano, Atai Watanabe, Yasuhiro Aoki,

Toshitaka Maeno, Takashi Nishida, Satoshi Kubota, Masaharu Takigawa, Masahiko Kurabayashi

Abstract—Excessive fibrosis contributes to an increase in left ventricular stiffness. The goal of the present study was toinvestigate the role of connective tissue growth factor (CCN2/CTGF), a profibrotic cytokine of the CCN (Cyr61, CTGF,and Nov) family, and its functional interactions with brain natriuretic peptide (BNP), an antifibrotic peptide, in thedevelopment of myocardial fibrosis and diastolic heart failure. Histological examination on endomyocardial biopsysamples from patients without systolic dysfunction revealed that the abundance of CTGF-immunopositive cardiacmyocytes was correlated with the excessive interstitial fibrosis and a clinical history of acute pulmonary congestion. Ina rat pressure overload cardiac hypertrophy model, CTGF mRNA levels and BNP mRNA were increased in proportionto one another in the myocardium. Interestingly, relative abundance of mRNA for CTGF compared with BNP waspositively correlated with diastolic dysfunction, myocardial fibrosis area, and procollagen type 1 mRNA expression.Investigation with conditioned medium and subsequent neutralization experiments using primary cultured cellsdemonstrated that CTGF secreted by cardiac myocytes induced collagen production in cardiac fibroblasts. Further, Gprotein–coupled receptor ligands induced expression of the CTGF and BNP genes in cardiac myocytes, whereasaldosterone and transforming growth factor-� preferentially induced expression of the CTGF gene. Finally, exogenousBNP prevented the production of CTGF in cardiac myocytes. These data suggest that a disproportionate increase inCTGF relative to BNP in cardiac myocytes plays a central role in the induction of excessive myocardial fibrosis anddiastolic heart failure. (Hypertension. 2007;49:1120-1127.)

Key Words: extracellular matrix � hypertrophy � cardiac function � connective tissue growth factor� natriuretic peptide

Epidemiological studies have established that 40% to 50%of patients with heart failure have normal or minimally

impaired left ventricular (LV) ejection fraction, a clinicalsyndrome that is commonly referred to as diastolic heartfailure (DHF). These patients typically have cardiac hyper-trophy that is induced by long-standing hypertension or byprimary hypertrophic cardiomyopathy, as well as increasedpassive LV stiffness.1 Among various molecular mechanismsthat regulate LV stiffness,2 abnormalities in the transcrip-tional or posttranscriptional regulation of the collagen genecan result in the disproportionate accumulation of fibroustissue and elevation of stiffness in the hypertrophied heart.2,3

Recent studies have shown that, in addition to mechanicalload, autocrine, paracrine, and endocrine factors, such asangiotensin II, aldosterone (Aldo), endothelin-1 (ET1), natri-uretic peptides, osteopontin, and transforming growthfactor-�1 (TGF-�), play important roles in the development

of myocardial hypertrophy and fibrosis.4,5 However, the precisemolecular mechanisms that initiate and promote myocardialfibrosis and increases in ventricular stiffness remain largelyunknown.

Connective tissue growth factor (CCN2/CTGF) belongs tothe CCN (Cyr61, CTGF, and Nov) family of immediate earlygenes, which are highly conserved among species.6 Thiscysteine-rich secreted protein may contribute to progressivefibrosis and excessive scarring in various systemic and localfibrotic diseases.6 Further, CTGF expression is increased inthe hypertrophied and failing myocardium of experimentalanimal models.7,8 CTGF is also an essential mediator for thebiological actions of TGF-�6 and its downstream signal trans-duction elements.9 However, a recent in vitro study dem-onstrated that CTGF is 1 of the earliest growth factorstranscriptionally induced by hypertrophic stimuli in cardiacmyocytes (CMs).10

Received July 24, 2006; first decision August 13, 2006; revision accepted February 25, 2007.From the Department of Medicine and Biological Science (N.K., M.A., S.K., K.N., A.W., Y.A., T.M., M.K.), Gunma University Graduate School of

Medicine, Gunma, Japan; and the Department of Biochemistry and Molecular Dentistry (T.N., S.K., M.T.), Okayama University Graduate School ofMedicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.

Correspondence to Masashi Arai, Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-22Showa-machi, Maebashi, Gunma 371-8511, Japan. E-mail [email protected]

© 2007 American Heart Association, Inc.

Hypertension is available at http://www.hypertensionaha.org DOI: 10.1161/HYPERTENSIONAHA.106.077537

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In this study, to confirm the involvement of CTGF in themyocardial fibrosis, we first investigated CTGF protein produc-tion in myocardial biopsy samples of patients with DHF.Secondly, by using the pressure overload rat model with asuprarenal abdominal aortic constriction (AC), which mimics amodel of DHF,5 we determined and compared the temporalchanges of CTGF, TGF-�, and an antifibrotic peptide, brainnatriuretic peptide (BNP).11 Because the collagen accumulationlevel is reflected by the balance of profibrotic factors andantifibrotic factors,12 we investigated their functional interac-tions, especially between CTGF and BNP, in the development ofmyocardial fibrosis and DHF.

MethodsAn expanded Methods section is available online at http://hyper.ahajournals.org.

Forty-six consecutive patients with normal or minimally impairedLV ejection fraction (�40%), estimated by echocardiography, whounderwent endomyocardial biopsy of the LV-free wall in GunmaUniversity Hospital were enrolled in this study (Table S1). All of thepatients were clinically stable when the biopsy was performed. Ofthese patients, 31 patients who had a previous history of overt heartfailure within the preceding year in the absence of impaired systolicfunction as estimated by echocardiography were designated as theDHF group. Another 15 patients without a previous history of heartfailure were designated as the nonfailing (NF) group. The clinicaldiagnosis and the exclusion criteria are described in the expandedMethods section.

AC was established with a 21G silver clip13 in male Wistar rats(Charles River, Japan) weighing 250 to 300 g. Cell culture, histo-chemical analysis and immunostaining, hemodynamic measurementsin AC rats, RNA isolation and Northern blot analysis, Westernblotting, and statistical analysis are described in the expanded Methodssection online.

ResultsElevated Levels of CTGF Protein in CMCorrelates With Myocardial Interstitial Fibrosis inPatients With Preserved Ejection FactorClinical characteristics of the NF and DHF groups aresummarized in Table S1. There were no significant differ-ences in age, sex, clinical diagnosis, and frequency ofcomplicated disease, except for atrial fibrillation, when com-paring the 2 groups. Sixty-one percent of DHF patients hadbeen given previous medication, including angiotensin-converting enzyme inhibitors and/or �-adrenoceptor block-ers, because of their previous history of congestive heartfailure. Pulmonary artery wedge pressure and LV end-dia-stolic pressure were not different when comparing the 2groups. Furthermore, there were no significant differences inechocardiographic parameters when comparing the 2 groupsexcept for left atrial dimension. Plasma BNP concentrationwas significantly elevated in the DHF group. RepresentativeLV biopsy samples taken from a patient from the NF groupwith hypertension and a patient from the DHF group are

Figure 1. Myocardial fibrosis and CTGFprotein in endomyocardial biopsy sam-ples. A through D, Main panels show high-power fields (�400). Left small panelsshow low-power fields (�100). All scalebars are 50 �m. A and C, Masson’strichrome staining; B and D, immunostain-ing for CTGF; A and B, NF group: A77-year–old man with mild LV hypertrophyand chronic hypertension, without previ-ous history of pulmonary congestion. HisLV ejection fraction was 45%. PlasmaBNP concentration was 76.2 pg/mL. Cand D, DHF group: A 74-year–old manwith mild LV hypertrophy, chronic atrialfibrillation, and hypertension. His LV ejec-tion fraction was 75%. Plasma BNP con-centration was 135 pg/mL. E, Comparisonbetween NF (n�15) and DHF (n�31) inMFA estimated by Masson’s trichromestaining in NF (n�15) and DHF (n�31). F,Comparison between NF (n�15) and DHF(n�31) in positive-stained area with CTGFantibody. G, Correlation between MFA andCTGF-stained area among all of thepatients enrolled (n�46). �, NF; ●, DHF.

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illustrated in Figure 1. Biopsies from the NF patient showedmild hypertrophic myocytes but no interstitial fibrosis byMasson’s trichrome staining (Figure 1A). CTGF immuno-staining of serial sections showed a small amount of CTGFprotein in the myocytes (Figure 1B). By contrast, biopsiesfrom the DHF patient showed interstitial fibrosis (Figure 1C)and an abundance of CTGF protein in CM (Figure 1D).Quantitative analysis revealed that myocardial fibrosis area(MFA) and CTGF-stained area were significantly elevated inDHF patients (Figure 1E and 1F). Interestingly, the CTGF-stained area correlated with MFA (r�0.638; P�0.001;Figure 1G).

CTGF and BNP Gene Expression AreCoordinately Induced Early in the Development ofCardiac Hypertrophy and FibrosisTo investigate the role of CTGF for the development ofcardiac fibrosis, we created a rat pressure overload cardiachypertrophy model by constricting the abdominal aorta. Inaccordance with the increase of systolic blood pressure, on

day 4 after AC operation and until day 28, LV weight/bodyweight ratio, a parameter of LV hypertrophy, significantlyincreased (Table S2). Furthermore, MFA was significantlyincreased on day 14 after AC.

Quantitative Northern blot analysis revealed that CTGFmRNA levels peaked on day 1, whereas TGF-� mRNA levelsincreased gradually and peaked on day 7 (Figure 2A).Furthermore, procollagen type 1�1 (COL1A1) mRNA levelswere significantly increased on day 7 and continued toincrease until day 28. Interestingly, the temporal course ofchanges in BNP mRNA was similar to that of CTGF mRNA,particularly from day 1 to day 7 (N�22; r�0.836; P�0.001;Figure 2B and 2D).

High CTGF/BNP Expression Ratio Is AssociatedWith Myocardial Fibrosis and VentricularStiffness at a Later Stage of Cardiac HypertrophyAlthough a correlation between CTGF and BNP mRNAexpression was observed during the entire experimentalperiod (N�69; r�0.804; P�0.001; Figure not shown), the

Figure 2. Gene expressions in hypertro-phied rat hearts. A, Temporal expressionpatterns of mRNAs normalized to 18SrRNA. S-0, rat euthanized immediatelyafter sham operation; AC-1, 4, 7, 14, and28, rats euthanized on designated dayafter AC or sham operation; *P�0.05 vssham-operated rats at same postoperativeperiods. B and C, Correlation betweenCTGF and BNP mRNA levels in AC rats ondays 1, 4, and 7 (B) and on days 14 and28 (C). D and E, Representative Northernblot analysis of CTGF, BNP, TGF-�, andCOL1A1 (procollagen type 1�1) mRNAsand 28S and 18S rRNAs on day 4 (D) andday 28 (E).

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correlation was weaker between day 14 and day 28 (N�19;r�0.645; P�0.001; Figure 2C and 2E) when compared withthe early stage of cardiac hypertrophy (day 1 to day 7;r�0.836; Figure 2B). As shown in Figure 2C, some ratsexpressed disproportionately abundant CTGF mRNA. Fur-thermore, those rats with high CTGF mRNA levels relative toBNP mRNA levels also showed marked upregulation ofCOL1A1 mRNA (Figure 2E, lanes 4 and 6). By contrast, ratswith proportional increases in both CTGF and BNP mRNAlevels showed only mild upregulation of COL1A1 mRNA(Figure 2E, lanes 3 and 5). Finally, rats with low CTGFmRNA levels relative to BNP mRNA levels showed lowlevels of COL1A1 mRNA (Figure 2E, lane 7).

Hemodynamic analysis was performed in rats on day 28.LV contractility indices, calculated using the pressure-volumerelationship, were comparable when comparing sham-operated and AC rats (Table S3). Diastolic indexes, that is,time constant of relaxation (�) and the slope of end-diastolicpressure-volume relationship (EDPVR slope), were signifi-cantly higher in the AC rats than in the sham-operated rats.

Representative hemodynamic data and histochemicalstaining of CTGF in a sham-operated and 2 AC rats withcomparable or disproportionate mRNA levels for CTGF andBNP are illustrated in Figure 3A and Figure S1. A rat withhigh CTGF levels related to BNP mRNA levels showed asteeper slope of EDPVR (Figure 3A), a high E/A ratio inDoppler echocardiography (Figure S1A), severe interstitialfibrosis, and positive immunostaining against CTGF in CM(Figure S1B) when compared with a sham-operated rat and arat with comparable mRNA levels of CTGF and BNP.

To further characterize hearts with high CTGF mRNAlevels relative to BNP mRNA levels, AC rats were classified

according to the upper or lower 50th percentile groups of theCTGF/BNP expression ratio on day 28. The mean ratio of theCTGF/BNP mRNA level was 1.2 (Figure 3B). AC rats witha higher CTGF/BNP mRNA ratio (n�7) showed elevatedEDPVR slope, higher E/A ratio, and increased MFA (Figure3B) relative to AC rats with the lower CTGF/BNP ratio(n�7). By contrast, LV relaxation (�), contractility (ejectionfactor), or LV hypertrophy (LV weight/body weight ratio)was similar when comparing the 2 groups (Figure 3B). Theprotein content of sarcomeric �-actin was also similar whencomparing the 2 groups (Figure 3B). Interestingly, the CTGF/BNP expression ratio correlated with EDPVR slope (r�0.720;P�0.001; Figure S2) and with COL1A1 mRNA expression(r�0.458; P�0.001; Figure S2). The ratio also significantlycorrelated with the E/A ratio, expression levels of procollagentype 3�1 (COL3A1), and MFA (Table S4). On the other hand,LV contractility indexes, �, and mRNA expressions of TGF�and sarcoplasmic reticulum Ca2� ATPase (SERCA) 2a were notcorrelated with the ratio (Table S4). Finally, plasma concentra-tion of Aldo was significantly elevated in AC rats with a higherCTGF/BNP ratio (Figure S3), whereas plasma TGF-� and ET-1concentration was not significantly different when comparingthe 2 groups.

CTGF Is Secreted From CMThe molecular basis of the production of CTGF in the heartand the functional interaction with other neurohumoral fac-tors was investigated using rat neonatal primary CM andcardiac fibroblasts (CFBs). Immunofluorescent study withanti-CTGF antibody revealed production of CTGF in culturedCMs (Figure 4A) and CFBs (vimentin-positive cells; FigureS4). Administration of recombinant CTGF resulted in a

Figure 3. LV diastolic function and myo-cardial fibrosis in rats with different ratiosof CTGF and BNP mRNAs. Representativepressure–volume loops (A) and echocardio-grams (B). Left, central and right panelsshow sham, an AC rat with comparablemRNA levels of CTGF and BNP, and an ACrat with disproportionate increase of CTGFagainst BNP, respectively. A, End-diastolicrelationships are depicted in broken lines.B, Differences in cardiac parametersbetween upper and lower 50th percentilegroups of CTGF/BNP expression ratio inday 28 AC rats. In each subset, � repre-sents the lower group (n�7), and f repre-sents the higher group (n�7). EDPVR,slope of end-diastolic pressure volumerelationship; �, monoexponential time con-stant of relaxation; LVW/BW, ratio of LVweight to body weight at sacrifice; Sarco-meric �-actin, the amount of sarcomeric�-actin in the heart extracts estimated byWestern blot.



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dose-dependent increase in COL1A1 mRNA levels in cul-tured CFB (Figure 4B). Profibrotic stimulation with TGF-�,ET1, and Aldo resulted in increased CTGF production andrelease into the culture medium from the myocytes (Figure 4C).Furthermore, conditioned medium from these CMs enhancedCOL1A1 mRNA levels in CFBs (Figure 4C), suggesting thatCMs may regulate collagen production in CFBs. When theTGF-�–treated medium was preincubated with anti-CTGF an-tibody, COL1A1 mRNA induction was abolished in CFBs(Figure 4D). Pretreatment of the CM-cultured medium with bothanti-CTGF and anti–TGF-� antibodies further suppressed theCOL1A1 mRNA, suggesting that the induction of the COL1A1gene by TGF-� and even the basal expression level of theCOL1A1 gene in CFBs are mediated through TGF-�–dependentand CTGF-dependent pathways.

Common and Uncommon Stimuli TriggeringCTGF and BNP Gene Transcription in CMsTo further characterize the correlation between CTGF andBNP mRNA induction in AC rats, the role of mechanohmoral

and neurohumoral stimuli on CTGF and BNP induction wasinvestigated. Cyclic stretch induced a rapid increase in CTGFand BNP mRNA levels (Figure 5A). Furthermore, G protein–coupled receptor ligands, such as ET1 (Figure 5B), norepi-nephrine, and angiotensin II (Figure S5A), increased CTGFand BNP levels in a dose-dependent manner. By contrast,Aldo and TGF-� stimulation resulted in increases in CTGFmRNA levels and decreases or no effect on BNP mRNAlevels (Figure 5B). The differential effect of TGF-� and Aldoon CTGF and BNP mRNA levels was also confirmed bycomparing the temporal induction pattern of these genes byTGF-� and Aldo with that induced by ET1 (Figure 5C).

BNP Suppresses CTGF Expression in CMsBecause BNP and TGF-� have opposing biological effects,14

the effect of BNP on CTGF expression was investigated.CTGF mRNA levels decreased 2 hours after administrationof synthetic BNP (Figure S5B). Synthetic BNP-mediatedinhibition of CTGF expression was completely blocked bythe protein kinase G inhibitor KT5823, suggesting that the

Figure 4. CTGF expression in cultured cardiac myocytes (CM) and its paracrine effect on COL1A1 expression in cardiac fibroblasts(CFB). A, Immunofluorescent imaging of CTGF (detected by Cy3) and actinin (detected by FITC) protein. Actinin is a sarcomeric protein,which indicates CM. Bar, 20 �m. B, COL1A1 mRNA levels in cultured cardiac fibroblasts (CFB) treated with recombinant human CTFGin designated concentrations for 24 h. The result was confirmed by triplicate experiments. C, COL1A1 mRNA levels in CFB 24 h afterthe addition of conditioned medium from CM cultured in the presence or absence of TGF-� (10 ng/mL), ET1 (0.1 �mol/L), or Aldo (1�mol/L) for 24 h. Upper and middle panels: Western blot showing the amount of CTGF protein in cell lysates and in the culture mediaof CM, respectively. Lower panel: Northern blot showing COL1A1 mRNA levels in CFB simulated by conditioned medium. Experimentswere performed in triplicate. D, The effect of an anti-CTGF neutralizing antibody on COL1A1 mRNA levels in CFB. Experimental condi-tions were identical to those in panel C. Veh-medium, medium with the solvent of TGF-�; �CTGF, anti-CTGF neutralizing antibody (�g/mL); �TGF�, anti-TGF-�neutralizing antibody (�m/mL); IgG; normal goat IgG, used as a control for the anti-CTGF antibody. The bargraphs show mean mRNA levels based on 4 independent experiments. *P�0.05 versus Veh-medium with normal IgG; †P�0.05 versusTGF�-treated-medium with normal IgG.



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BNP–cGMP–protein kinase G pathway plays a critical role inregulating CTGF expression in CM (Figure S5C). Further-more, the effect of BNP was also evident in the context ofenhanced production of the CTGF protein in response toprofibrotic stimuli, such as TGF-�, ET1, and Aldo (Figure 5D).

DiscussionDHF, Fibrosis, and CTGFIn the present study, patients with DHF had greater amountsof interstitial fibrosis when compared with patients without aprevious history of congestive heart failure. Excessive colla-gen deposition contributes to abnormal passive diastolicventricular stiffness3 and leads to pulmonary edema.1 Impor-tantly, MFA, the degree of the interstitial fibrosis, signifi-cantly correlated with the abundance of CTGF-positive CMs(Figure 1G). By contrast, neither MFA nor the percentage ofCTGF-positive CMs was correlated with LV ejection frac-tion, an index of systolic function, in our study subjects (datanot shown). Endomyocardial biopsy can merely disclosehistological changes of a limited portion of whole heart, andthis immunohistochemical analysis is not a quantitative mea-surement of CTGF protein. The amount of biopsy sampleswas not enough to isolate protein for Western blotting.However, multiple samplings from different portions of thesame heart and the staining of serial section with normal IgGas a reference of nonspecific staining minimized samplingand technical variations. Our study suggests that excess

fibrosis, through an increase of CTGF, significantly contrib-utes to the development of DHF. Based on the stainingpattern with CTGF antibody, strong staining was mainlyobserved in CMs rather than in the interstitium (Figure 1D),suggesting that CMs are largely responsible for the produc-tion of CTGF in the DHF heart.

CMs Produce CTGFCTGF is overexpressed in numerous fibrotic diseases, and thedegree of overexpression correlates with the severity ofdisease.6 Collagen is mainly produced by fibroblasts in manyorgans. However, various other cells, including fibroblasts,secrete humoral factors to initiate collagen production infibroblasts.4,5 Although previous reports mainly focused onCFBs as CTGF-producing cells in the heart,15 the presentstudy demonstrated that a significant amount of CTGF isproduced by CMs in the hypertrophied rat heart and in thehearts of patients with DHF.

Interestingly, cultured CFB had higher basal levels ofCTGF mRNA than CM (Figure S4A). However, unlike thesignificant induction in CM, CTGF mRNA levels in CFBswere only minimally affected by extrinsic stimuli (CFBs,Figure S4B; CMs, Figure 5B), which is consistent withobservations by Kemp et al.10 The inducibility of CTGF inCMs, along with the fact that BNP, an antifibrotic factor, isalso produced by CMs, raises the possibility that CTGFproduced in CMs regulates collagen production in CFBs.

Figure 5. Effects of various hypertrophy-associated stimuli on the CTGF and BNPexpression in CMs. A and B, Northern blotshowing the effect of cyclic cell stretching(A) and various humoral factors (4 hours;B) on CTGF and BNP mRNA levels inCMs. Experiments were performed at leastin triplicate. C, Temporal patterns of CTGFand BNP mRNA levels in response toTGF-� (10 ng/mL), ET1 (0.1 �mol/L), orAldo (1 �mol/L) stimulation. Each dot indi-cates the mean intensity of 4 independentexperiments relative to the value in thevehicle-treated sample in each time point.D, Western blot showing the effect ofTGF-�, ET1, and Aldo and the inhibitoryeffect of BNP on CTGF protein levels. Syn-thetic BNP (sBNP) was added with TGF-�,ET1, or Aldo, and cells were incubated for24 hours. Bar graphs show mean CTGFprotein levels based on 4 independentexperiments. *P�0.01 vs vehicle, †P�0.05vs maximal level of CTGF protein inducedby these stimuli.



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Indeed, the present study demonstrated that CTGF wassecreted into the cultured medium of CMs and that thisconditioned medium induced increases in COL1A1 mRNAlevels in CFBs (Figure 4C). In addition, neutralization ofconditioned medium with CTGF antibody sufficientlyblunted the fibrotic signal from CMs to CFBs (Figure 4D).These data suggest that there is molecular communication ina paracrine manner between CMs and CFBs and that thisprocess regulates production of collagen.

CTGF/BNP Balance Regulates Cardiac FibrosisBecause we could not obtain a well-working antibody forBNP immunostaining, we were unable to estimate the BNPprotein level in myocardial biopsy samples. However, thepercentage of CTGF-positive staining cells in biopsy samplescorrelated with the plasma BNP level (r�0.41; P�0.05;Figure not shown). A close correlation between CTGF andBNP mRNA induction was also seen in the pressure overloadrat heart and in cultured CMs under cell stretch or stimulationwith G protein–coupled receptor ligands. Although the pre-cise mechanisms for this induction were not investigated inthis study, preliminary studies demonstrated that the ET1-induced increase in CTGF and BNP mRNA levels wasblocked by inhibitors of mitogen-activating protein kinases,protein kinase C, and protein kinase A (data not shown).These data suggest that coordinated expression of the CTGFand BNP genes may be mediated by these signalingpathways.

Most importantly, the CTGF/BNP ratio in CM significantlycorrelated with indices of fibrosis and diastolic function, suchas the slope of EDPVR, E/A ratio, COL1A1 mRNA levels,and MFA (Figure 3 and Table S4). Furthermore, AC rats withcomparable levels of CTGF mRNA and BNP mRNA expres-sion showed mild production of CTGF protein and sparsefibrosis in the myocardium. Sarcomeric �-actin content wasnot different between the high CTGF/BNP ratio group andthe comparable ratio group, which suggests that myocyte lossis not responsible for the change of the ratio (Figure 3B).SERCA2a is a principal protein responsible for the initiationof the diastolic phase through its ability to remove cytoplas-mic Ca2�.2 However, SERCA2a mRNA level was not corre-lated with the CTGF/BNP ratio (Table S4). These datasuggest that the CTGF/BNP ratio does not associate with LVdiastolic function, which is related to Ca2� removal fromcytoplasm.

The identity of upstream factors responsible for the dispro-portionate expression of CTGF and BNP in CMs remainsunclear. AC causes severe hypertension and increases in thelevels of various neurohumoral factors, such as renin andangiotensin II.16 Interestingly, rats with a higher CTGF/BNPratio had a higher plasma Aldo concentration and a tendencytoward higher plasma TGF-� and ET1 concentrations thanthe rats with a lower CTGF/BNP ratio (Figure S3). Inaddition, in vitro study demonstrated that Aldo and TGF-�induced increases in CTGF mRNA but not in BNP mRNA incontrast to the response to cell stretch or G protein–coupledreceptor ligands (Figure 5A through 5C). Therefore, at leastin the present model, Aldo may be an upstream factorresponsible for disproportionate CTGF expression.

In addition to the effect on body fluid homeostasis andblood pressure control, BNP can exert antihypertrophic andantifibrotic effects in the stressed myocardium.11 The presentstudy demonstrated that BNP suppressed basal CTGF expres-sion level in CMs via its effects on protein kinase G (FigureS4C). The effect of BNP on CTGF expression was alsoobserved under various profibrotic stimuli, such as ET1,Aldo, and TGF-� (Figure 5D). Thus, the increase of CTGFand/or decrease of BNP in CMs may play a central role in theinduction of excessive myocardial fibrosis and abnormaldiastolic function (Figure 6).

To dissect the role of CTGF in the development of DHF,we used a rat pressure-overloaded model as a preservedsystolic but impaired diastolic function model. Given thatcollagen accumulation is regulated by a balance of its synthesisand degradation, the pressure-overloaded model may becharacterized as a “synthesis”-dominant model.12 On theother hand, myocardial infarction is a “accelerated synthesisand accelerated degradation” model with respect to collagenturnover.17 Myocardial infarction is another leading cause toprovoke cardiac fibrosis. Therefore, our hypothesis should bealso tested in the ischemic heart model, as well as thepressure-overloaded cardiac hypertrophy model.

PerspectivesCTGF is a secreted protein, and plasma CTGF concentrationcorrelates with the severity of several systemic fibroticdisorders.18 Measurement of plasma CTGF concentrations iseasier and less invasive than assessment of CTGF levels inbiopsy samples. Furthermore, the present data suggest thatplasma concentration of CTGF or the ratio of plasma con-centration of CTGF:BNP may be a diagnostic marker formyocardial fibrosis. In addition, our data showing the induc-ibility of CTGF in CMs and the myocardial responsiveness to

Figure 6. A scheme illustrating that an abundance of CTGF rela-tive to BNP in CMs promotes pathogenic collagen production inCFBs.



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exogenously administered CTGF suggest that CTGF plays anactive role in cardiac progressive fibrosis and, thus, becomesa good candidate molecule as a target of antifibrotic therapy.

ConclusionsThe present study demonstrated the following: (1) productionof CTGF from CMs is associated with the myocardialinterstitial fibrosis and DHF; (2) increased CTGF expressionrelative to BNP expression triggers excessive cardiac fibrosisvia BNP-mediated suppression of CTGF expression; and (3)Aldo and TGF-� induce a disproportionate induction ofCTGF and BNP expression, whereas a mechanical stretch ofCM and G protein–coupled receptor ligands induces propor-tionate CTGF and BNP expression. These data suggest thatCTGF is a key molecule in the process of cardiac fibrosis andthat it may serve as a diagnostic marker and therapeutic targetfor cardiac fibrosis and DHF.

AcknowledgmentsWe are grateful to Miki Yamazaki for her technical assistance withthe cardiac myocytes culture and Yoshiko Nonaka for her excellentpreparation of histological samples.

Sources of FundingThis work was supported in part by a Grant-in-Aid for ScientificResearch (KAKENHI B-17390224 and S-15109010) from the JapanSociety for the Promotion of Science.

DisclosuresNone.

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13. Takizawa T, Arai M, Yoguchi A, Tomaru K, Kurabayashi M, Nagai R.Transcription of the SERCA2 gene is decreased in pressure-overloadedhearts: A study using in vivo direct gene transfer into living myocardium.J Mol Cell Cardiol. 1999;31:2167–2174.

14. Kapoun AM, Liang F, O’Young G, Damm DL, Quon D, White RT,Munson K, Lam A, Schreiner GF, Protter AA. B-type natriuretic peptideexerts broad functional opposition to transforming growth factor-beta inprimary human cardiac fibroblasts: fibrosis, myofibroblast conversion,proliferation, and inflammation. Circ Res. 2004;94:453–461.

15. Ahmed MS, Oie E, Vinge LE, Yndestad A, Oystein Andersen G,Andersson Y, Attramadal T, Attramadal H. Connective tissue growthfactor–a novel mediator of angiotensin II-stimulated cardiac fibroblastactivation in heart failure in rats. J Mol Cell Cardiol. 2004;36:393–404.

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KurabayashiAoki, Toshitaka Maeno, Takashi Nishida, Satoshi Kubota, Masaharu Takigawa and Masahiko

Norimichi Koitabashi, Masashi Arai, Shinya Kogure, Kazuo Niwano, Atai Watanabe, YasuhiroDeterminant of Myocardial Fibrosis

Increased Connective Tissue Growth Factor Relative to Brain Natriuretic Peptide as a

Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright © 2007 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Hypertension doi: 10.1161/HYPERTENSIONAHA.106.0775372007;49:1120-1127; originally published online March 19, 2007;Hypertension.

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Increased connective tissue growth factor relative to brain natriuretic peptide

as a determinant of myocardial fibrosis

Norimichi Koitabashi, Masashi Arai, Shinya Kogure, Kazuo Niwano, Atai Watanabe,

Yasuhiro Aoki, Toshitaka Maeno, Takashi Nishida, Satoshi Kubota, Masaharu Takigawa,

Masahiko Kurabayashi

ONLINE DATA SUPPLEMENT

Address correspondence to:

Masashi Arai MD, PhD

Department of Medicine and Biological Science

Gunma University Graduate School of Medicine

3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan

E-mail: [email protected]

TEL: (+81) 27-220-8142 FAX: (+81) 27-220-8158

Koitabashi et al. CTGF vs BNP, and myocardial fibrosis HYPERTENSION/2006/077537/R4

Materials and Methods

Patients

The study was approved by the local ethics committee and conforms to the ethical

guidelines of the 1975 Declaration of Helsinki. Written informed consent was obtained

from all patients.

Forty-six consecutive patients with normal or minimally impaired left ventricular (LV)

ejection fraction (>40%) estimated by echocardiography who underwent endomyocardial

biopsy of the LV free wall in Gunma University Hospital were enrolled in this study.

Clinical diagnosis of these patients included hypertrophic cardiomyopathy (n=13),

hypertensive heart disease (n=15), dilated cardiomyopathy (n=7), alcoholic

cardiomyopathy (n=3), sick sinus syndrome (n=1), hyperthyroidism (n=1), idiopathic

ventricular tachycardia (n=1), and other diseases (n=5). Of these patients, 31 patients who

had previous history of overt heart failure within the preceding year (i.e. dyspnea and rales

due to pulmonary congestion, as confirmed by chest radiography) in the absence of

impaired systolic function as estimated by echocardiography were designated as the

diastolic heart failure (DHF) group. Heart failure was clinically diagnosed according to the

criteria used in the Framingham Heart Study project 1 and the elevation of plasma BNP

2


concentration was confirmed. All patients in the DHF group showed any of

echocardiographic criteria of DHF, i.e. impaired relaxation, pseudonormal, and restrictive

patterns. Another 15 patients without a previous history of heart failure were designated as

the non-failing (NF) group. Patients with significant coronary stenosis in angiography,

moderate or severe valvular disease, secondary hypertension, renal failure (serum creatinine

concentration >2.0mg/dl), myocarditis, epicarditis, or uncontrolled decompensated

congestive heart failure were excluded. Patients with cardiac sarcoidosis pathologically

diagnosed by their endomyocardial biopsy (i.e., lymphocytic infiltration) were also

excluded from the present analysis. At least two endomyocardial samples were obtained

from the LV free wall in each patient, and hemodynamic parameters were measured with an

LV and Swan-Ganz catheters. Two-dimensional, M-mode, and Doppler ultrasound

recordings were performed in each patient using transthoracic echocardiography, and left

ventricular ejection fraction and mass index was calculated from the echocardiogram.

Peripheral blood samples were obtained within a week before or after cardiac

catheterization for determination of plasma brain natriuretic peptide (BNP) concentration.

BNP levels were measured using immunoradiometric assay.

Histochemical analysis and immunostaining

3


Endomyocardial biopsy samples were immediately fixed in 10% buffered formalin,

embedded in paraffin. Masson’s trichrome staining was performed for detection of collagen,

and five high-power field (X200) color images were randomly selected in each sample.

Myocardial fibrosis area (MFA) was determined by blue staining and quantified by an

automated image analysis system (MacScope)2.

Immunostaining with a human connective tissue growth factor (CTGF) antibody (Santa

Cruz Biotechnology, Inc.) and with a normal goat IgG1 (R&D systems) as a negative

control for non-specific staining was performed in the same serial sections as that used in

the MFA study. Variation of control IgG1 staining among samples was minimized, and

sections that demonstrated significantly higher staining intensity with CTGF antibody than

with control IgG1 were selected for densitometry 3. Average data of the percentage of

positively stained area relative to the sample area in 5 different positions in each sample

was used to determine the “CTGF-stained area”.

Animal models

Constriction of the suprarenal abdominal aorta was established with a 21G silver clip 4 in

male Wister rats (Charles River, Japan) weighing 250-300 g after intraperitoneal (IP)

injection of pentobarbital. After hemodynamic measurement on the experimental day, the

4


heart was excised and weighed. The LV was divided into three pieces for histological

analysis, RNA isolation and protein extraction. All animal experiments were performed

according to the Guide for the Care and Use of Laboratory Animals published by the US

National Institutes of Health and were approved by the Animal Research Committee of the

Gunma University Graduate School of Medicine.

Hemodynamic measurements in rats

Before constriction of the suprarenal abdominal aorta, blood pressure was measured by the

tail-cuff method.

On Day 28, hemodynamic parameters were measured using a pressure-volume (PV)

catheter. Rats were anesthetized with 2% isoflurane, and tracheostomy was performed to

allow mechanical ventilation. The LV apex was exposed under sternotomy, and a microtip

PV catheter (SPR-838, Millar Instruments) was advanced through the apex along the

longitudinal axis. Absolute volume was calibrated, and PV data were measured at a steady

state and during transient reduction of venous return, as described previously 5. Blood

pressure was measured in rats before sacrifice on Days 1, 4, 7 and 14 using a fluid-filled

manometer via the carotid artery under isoflurane anesthesia 4

To assess ventricular function and hypertrophy, transthoracic echocardiography was

5


performed with a 10-MHz transducer (EUB-6000, HITACHI) in all rats on Days 0, 1, 4, 7,

14 and 28. Rats were sedated with ketamine (40 mg/kg IP) and xylazine (10 mg/kg IP) to

maintain blood pressures equivalent to the awake condition. M-mode tracings and

transmitral pulse wave Doppler spectra were measured as described previously 6.

RNA isolation and Northern blot analysis

Total cellular RNA was isolated using the ISOGEN reagent (Nippongene) in accordance

with the manufacturer’s instruction. Probes for Northern blots were as follows: 1) rat CTGF

(nucleotide +1201~1795 bp; Acc. No. NM_022266) 7 isolated using RT-PCR; 2) rat

procollagen type 1α1 (COL1A1) (nucleotide +5096~5669 bp; Acc. No. Z78279) isolated

using RT-PCR; 3) rat procollagen type 3α1 (COL3A1) (nucleotide +2046~2350 bp; Acc.

No. XM_216813) isolated using RT-PCR; 4) a 628-bp fragment of the rat BNP cDNA 8;

and 5) a 490-bp fragment of the rat transforming growth factor (TGF) β1 cDNA 9; 6) rat

sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) (nucleotide +3557-3865 bp; Acc. No.

J04023) isolated usinf RT-PCR. Messenger RNA levels were quantified using scanning

autoradiographs and computerized optical densitometry and were normalized with 28S

rRNA.

6


Plasma analysis in rats

Blood sample was obtained via the carotid artery before sacrifice. Plasma TGF-β

concentration was examined by enzyme-linked immunosorbent assay. Plasma endothelin

(ET)1 and aldosterone (Aldo) concentration were examined by radioimmunoassay.

Cell culture

Neonatal rat cardiac myocytes were isolated from 1- to 3-day-old Wistar rats, as previously

described 10, and were seeded on gelatin-coated tissue culture plates or FlexWell plates

(Flexcell International) 10. Cardiac myocytes were cultured for 24 h in Dulbecco’s modified

Eagle’s medium (DMEM) containing 10% fetal bovine serum and 0.1 mmol/L

bromodeoxyuridine and then switched to DMEM containing 0.1%

insulin/transferring/selenium (Gibco) before being stimulated with various agents 24 h later.

For cell stretch experiments, cardiac myocytes were stretched biaxially (15%, 0.5 Hz) using

a FlexCell Strain Unit (FX-4000; FlexCell International). Control myocytes were cultured

on FlexWell plates without mechanical stretch.

Neonatal rat cardiac fibroblasts were prepared as described previously 10. After the

second passage, cells were plated (3×105 cells) in 60 mm-culture dishes and grown in

DMEM containing 10% FBS. Just before reaching confluence, the medium was replaced

7


with serum-free DMEM or with conditioned medium from cardiac myocytes culture.

Cardiac myocyte-conditioned medium was prepared as a supernatant of cultured media

after 24 h stimulation of cardiac myocytes by TGF-β, ET-1 or Aldo. The medium for

neonatal cardiac fibroblasts was replaced with the cardiac myocyte-conditioned medium,

and fibroblasts were harvested 24 hrs after the replacement. In neutralizing antibody

experiments, antibodies for CTGF and TGF-β (R&D systems) and normal IgG (R&D

systems) were supplemented at the time of medium replacement.

Immunofluorescent microscopic analysis

Immunofluorescent microscopic analysis was performed with CTGF antibody and

Cy3-conjugated anti-goat IgG antibody (Sigma) in methanol-fixed cultured cells. Mouse

monoclonal sarcomeric actinin antibody (Sigma) and FITC-conjugated anti-mouse IgG

antibody (Sigma) were used for detection of cardiac myocytes. Mouse monoclonal

vimentin antibody (Sigma) was used for detection of cardiac fibroblasts.

Western blotting

The protein extracts of in vivo experiments were homogenized with buffer containing 10

mmol/L imidazole, 300 mmol/L sucrose, and protease inhibitors. Cultured cells were lysed

8


by adding ice-cold radioimmunoprecipitation buffer. Protein concentration was determined

by the Bradford dye-binding method (BioRad). The cell lysates or culture media were

subjected to electrophoresis on a SDS-13% polyacrylamide gel and transferred to

nitrocellulose membranes. Membranes were then blocked in TBS (10 mmol/L Tris, pH 7.6,

and 150 mmol/L NaCl) containing 5% skim milk, followed by overnight incubation with

anti-CTGF antibody (Santa Cruz). Chemiluminescent detection was performed with the

enhanced chemiluminescence protocol (ECL; Amersham Bioscience). After CTGF

detection, the membranes were stripped and reprobed with anti sarcomeric α-actin (Sigma)

as an internal control.

Reagents

Synthetic rat BNP and recombinant human ET-1 were obtained from the Peptide Institute.

Norepinephrine, Ang II, Aldo, and human recombinant TGF-β were obtained from Sigma,

Bachem, Acros Organics, and Roche, respectively. Recombinant human CTGF was purified

as previously described 11. KT5823 was obtained from Calbiochem.

Statistical analysis

Data are expressed as means±SD. Overall differences within groups were determined by

9


one-way analysis of variance. When this test indicated that differences existed, individual

experimental groups were compared by Bonferroni’s test. Categorical variables were

analyzed by the χ2 test or Fisher’s exact probability test when necessary. Bivariate

correlations between variables were assessed by simple least-squares linear regression

analysis. A probability value <0.05 was considered statistically significant.

10


References for Supplemental Methods

1. McKee PA, Castelli WP, McNamara PM, Kannel WB. The natural history of

congestive heart failure: the Framingham study. N Engl J Med.

1971;285:1441-1446.

2. Querejeta R, Varo N, Lopez B, Larman M, Artinano E, Etayo JC, Martinez Ubago

JL, Gutierrez-Stampa M, Emparanza JI, Gil MJ, Monreal I, Mindan JP, Diez J.

Serum carboxy-terminal propeptide of procollagen type I is a marker of myocardial

fibrosis in hypertensive heart disease. Circulation. 2000;101:1729-1735.

3. Wallace CK, Stetson SJ, Kucuker SA, Becker KA, Farmer JA, McRee SC, Koerner

MM, Noon GP, Torre-Amione G. Simvastatin decreases myocardial tumor necrosis

factor alpha content in heart transplant recipients. J Heart Lung Transplant.

2005;24:46-51.

4. Takizawa T, Arai M, Yoguchi A, Tomaru K, Kurabayashi M, Nagai R. Transcription

of the SERCA2 gene is decreased in pressure-overloaded hearts: A study using in

vivo direct gene transfer into living myocardium. J Mol Cell Cardiol.

1999;31:2167-2174.

11


5. Pacher P, Mabley JG, Liaudet L, Evgenov OV, Marton A, Hasko G, Kollai M, Szabo

C. Left ventricular pressure-volume relationship in a rat model of advanced

aging-associated heart failure. Am J Physiol Heart Circ Physiol.

2004;287:H2132-2137.

6. Masuyama T, Yamamoto K, Sakata Y, Doi R, Nishikawa N, Kondo H, Ono K,

Kuzuya T, Sugawara M, Hori M. Evolving changes in Doppler mitral flow velocity

pattern in rats with hypertensive hypertrophy. J Am Coll Cardiol.

2000;36:2333-2338.

7. Yokoi H, Mukoyama M, Sugawara A, Mori K, Nagae T, Makino H, Suganami T,

Yahata K, Fujinaga Y, Tanaka I, Nakao K. Role of connective tissue growth factor in

fibronectin expression and tubulointerstitial fibrosis. Am J Physiol Renal Physiol.

2002;282:F933-942.

8. Kojima M, Minamino N, Kangawa K, Matsuo H. Cloning and sequence analysis of

cDNA encoding a precursor for rat brain natriuretic peptide. Biochem Biophys Res

Commun. 1989;159:1420-1426.

9. Tsuji T, Okada F, Yamaguchi K, Nakamura T. Molecular cloning of the large subunit

of transforming growth factor type beta masking protein and expression of the

mRNA in various rat tissues. Proc Natl Acad Sci U S A. 1990;87:8835-8839.

12


10. Yokoyama T, Sekiguchi K, Tanaka T, Tomaru K, Arai M, Suzuki T, Nagai R.

Angiotensin II and mechanical stretch induce production of tumor necrosis factor in

cardiac fibroblasts. Am J Physiol. 1999;276:H1968-1976.

11. Nishida T, Nakanishi T, Shimo T, Asano M, Hattori T, Tamatani T, Tezuka K,

Takigawa M. Demonstration of receptors specific for connective tissue growth

factor on a human chondrocytic cell line (HCS-2/8). Biochem Biophys Res Commun.

1998;247:905-909.

13


Figure legends for supplemental figures

Supplemental Figure I

LV diastolic function and myocardial fibrosis in rats with different ratios of CTGF and BNP

mRNAs. Representative echocardiograms (A), and histological analysis (B); In each row,

left, central and right panels show sham, a AC rat with comparable mRNA levels of CTGF

and BNP, and a AC rat with disproportionate increase of CTGF against BNP, respectively;

(A) M-mode echocardiography (left) and transmitral Doppler flow pattern (right). Mean

E/A ratios of consecutive five beats are shown below the panels. (B) Histological analysis

of LVs; upper panel, Masson’s trichrome staining; lower panel, immunohistologic staining

with an anti-CTGF antibody. All scale bars are 50 μm. Arrows indicate CTGF-positive

cardiac myocytes (CM). Asterisks indicate vascular structure.

Supplemental Figure II

(A) Correlation between CTGF/BNP expression ratio and EDPVR in Day 28 sham (n=5)

and AC (n=14) rats.

(B) Correlation between CTGF/BNP expression ratio and COL1A1 mRNA expression level

estimated by quantitative Northern blot in Day 28 sham (n=5) and AC (n=14) rats.

14


Supplemental Figure III

Difference in plasma concentration of TGFβ, ET1 and Aldo between higher (filled columns,

n=7) and lower (open columns, n=7) groups of CTGF/BNP expression ratio in Day 28 AC

rats.

Supplemental Figure IV

Immunofluorescent imaging of CTGF (detected by Cy3) and vimentin (detected by FITC)

protein in cultured cardiac cells. Vimentin is an intermediated filament, which has been

shown to be abundant in fibroblasts. A right cell in these panels shows a cardiac fibroblast.

Supplemental Figure V

(A) Northern blot showing the effect of norepinephrine (NE) and angiotensin II (AngII) on

CTGF and BNP mRNA levels in cardiac myocytes.

(B) Northern blot showing the temporal changes of CTGF mRNA levels in CM in

response to synthetic BNP (sBNP: 0.1 μmol/L).

(C) Effect of the protein kinase G inhibitor, KT5823 (1 μmol/L), on the

BNP-mediated suppression of CTGF mRNA levels in CM. Four hours after

15


incubation with synthetic BNP in the presence or absence of KT5823, CTGF

mRNA expression was examined by Northern blot analysis. Experiments were

performed in triplicate.

Supplemental Figure VI

(A) Northern blot showing basal expression of CTGF in cultured neonatal rat cardiac

myocytes (CM) and cardiac fibroblasts (CFB). Cultured rat cardiac fibroblasts were used

after the second passage. Bar graphs show mean values of four independent experiments

relative to the mean level of CTGF mRNA in cardiac myocytes. * P<0.05 vs. cardiac

myocytes.

(B) Northern blot showing the effect of various humoral factors (4 hours) on CTGF mRNA

level in cultured cardiac fibroblasts. Bar graphs show mean values of four independent

experiments. Values in the vehicle-stimulated group are defined as 1. *P<0.05 vs.

vehicle-treated group.

16

Age

Sex (Male/Female)

HCM/HHD/other

Prior Medication

Hypertension(N)

Atrial Fibrillation(N)

Renal Failure(N)

Diabetes(N)

Prior PCI/CABG (N)

PAWP (mmHg)

LVEDP (mmHg)

LVEF (%)

LVEDD (mm)

LAD

LVM (g)

E/A

DcT (msec)

BNP (pg/mL)

58±15

9/6

7/3/5

2

3

0

0

3

1

11.8±3.0

15.0±8.8

62.5±9.6

48.4±8.8

37.5±4.6

153.6±134.5

0.89 ±0.55

226.7±24.6

68.2±73.5

57±16

20/11

6/12/13

19

12

14

2

3

0

10.5±6.8

18.0±8.3

54.1±12.8

52.3±11.3

43.6±6.3

189.4±131.6

0.79±0.72

195.0±31.9

263.5±214.2

VariableNF

(N=15)DHF

(N=31)

NS

NS*

NS*

<0.05†

NS†

<0.01†

NS†

NS†

NS†

NS

NS

NS

NS

<0.05

NS

NS

NS

<0.05

P

Supplemental Table I. Clinical characteristics in patients

Prior Medication: angiotensin converting enzyme inhibitor/angiotensin II receptor blocker/aldosterone

blocker/beta adrenoceptor blocker; HHD, hypertensive heart disease; HCM, hypertrophic

cardiomyopathy; PCI, percutaneous coronary intervention; CABG, coronary artery bypass graft; PAWP,

mean pulmonary artery wedge pressure; LVEDP, left ventricular end-diastolic pressure; MFA, myocardial

fibrosis area; LVEF, left ventricular ejection fraction; LVEDD, left ventricular end-diastolic diameter; LAD,

left atrial diameter, LVM, left ventricular mass; DcT, deceleration time; P, ANOVA with the exceptions

indicated as follows:* χ2 test; † Fisher’s exact probability test

17

Shamn=5

ACn=12

Shamn=5

ACn=14

Day 7 Day 28

BW, g

HR

SBP, mmHg

LVEDD, mm

FS, %

LVM, mg

E/A

LVW/BW, mg/g

MFA, %

257±1

312±11

103 ±10

7.15 ±0.23

32.4 ±2.6

526 ±29

2.17 ±0.3

2.03 ±0.11

1.10 ±0.07

250±14

320 ±18

155 ±25*

7.72 ±0.08

33.0 ±3.3

637 ±18

2.13 ±0.04

2.45 ±0.14*

1.38 ±0.15

Shamn=4

ACn=5

Day 14

Shamn=4

ACn=6

Day 4

281±7

300 ±19

98 ±9

7.42 ±0.78

33.0 ±0.8

540 ±29

1.92 ±0.11

2.00 ±0.06

1.06 ±0.10

261±7

341 ±18

149 ±27*

7.70 ±0.17

31.2 ±1.4

788 ±26*

2.55 ±0.25

2.80 ±0.10*

2.93 ±0.71

318±12

333 ±26

100 ±5.9

7.90 ±0.21

33.5 ±2.5

696 ±32

2.09 ±0.26

2.06 ±0.07

0.76 ±0.05

318±6

316 ±19

162 ±26*

7.70 ±0.24

33.9 ±1.6

958 ±64*

1.89 ±0.23

2.78 ±0.07*

2.66 ±0.19*

385±12

350 ±11

113±9

8.26 ±0.18

34.6 ±1.1

706 ±27

2.00 ±0.11

1.86 ±0.04

1.00 ±0.24

363±11

341 ±18

141 ±20*

8.03 ±0.17

32.3 ±1.6

991 ±84*

1.88 ±0.23

2.61 ±0.08*

2.85 ±0.64*

* P<0.05 vs. Sham

BW, body weight, HR, heart rate, LVEDD, left ventricular end-diastolic diameter, FS, fractional shortening, LVM, left ventricular mass, LVW/BW, left

ventricular weight to body weight ratio, MFA, myocardial fibrosis area

258±8

342±19

98 ±10

7.25 ±0.25

35.0 ±3.0

466 ±18

1.75±0.10

2.06 ±0.19

0.89 ±0.11

280±20

340 ±20

160 ±25*

7.70 ±0.03

32.2 ±1.3

457 ±60

2.01 ±0.17

2.19 ±0.17

1.01 ±0.37

Shamn=3

ACn=4

Day １

Supplemental Table II. Time-dependent changes in cardiac morphologic and functional parameters after aortic constriction

18

Supplemental Table III. Hemodynamic parameters in sham- and AC-operated rats measured by the Millar pressure-volume conductance catheter system

ShamDay 28

N=5

ACDay 28N=14

LVESP (mmHg)

LVEDP (mmHg)

EF (%)

dP/dtmax (mmHg/s)

dP/dtmin (mmHg/s)

PRSW (mmHg)

Emax (mmHg/μL)

EDPVR (mmHg/μL)

τ (msec)

κ

108.7±5.7

7.0±1.6

50.3±4.9

9229±723

-7971±536

123.0±25.3

2.02±0.77

0.010±0.004

12.1±0.9

0.002±0.001

141.0±10.8*

12.0±3.3

41.5±2.2

9178±958

-7810±725

112.4±17.0

1.27±0.27

0.052±0.013*

17.3±0.9*

0.004±0.003

* P<0.05 vs. Sham

LVESP, left ventricular end-systolic pressure; LVEDP, left ventricular end-diastolic

pressure; LVESV, left ventricular end-systolic volume; LVEDV, left ventricular end-

diastolic volume; EF, ejection fraction; dP/dtmax, maximal rate of pressure

development; dP/dtmin, maximal rate of pressure decline; PRSW, preload-recruitable

stroke work; Emax, maximal elastance; EDPVR, end-diastolic pressure-volume

relationship; τ, monoexponential time constant of relaxation; κ, constant of chamber

stiffness.

19

Variable

VariableCorrelationCoefficient P value

Hemodynamic parametersLVESPLVEDPdP/dtmax

dP/dtmin

PRSWEmaxτEDPVRFS*E/A*

Gene expressionsCOL1A1COL3A1TGFβSERCA2a

Histomorphological parameterMFA

0.2460.3490.310-0.1350.3300.2040.2700.7200.1610.315

0.4580.2700.0500.179

0.525

0.2740.1130.1630.5430.1590.3930.337<0.0010.1590.009

<0.0010.0060.6220.183

<0.001

Supplemental Table IV. Correlation between CTGF/BNP ratio and hemodynamic or genetic parameters

LVESP, left ventricular end-systolic pressure; LVEDP, left ventricular end-

diastolic pressure; dP/dtmax, maximal rate of pressure development; dP/dtmin,

maximal rate of pressure decline; PRSW, preload-recruitable stroke work;

Emax, maximal elastance; τ, monoexponential time constant of relaxation;

EDPVR, end-diastolic pressure-volume relationship; FS, fractional shortening;

COL1A1, procollagen type 1α1 mRNA, COL3A1; procollagen type 3 α1 mRNA;

TGFβ, transforming growth factor β1 mRNA; SERCA2a, sarcoplasmic reticulum

Ca2+ ATPase 2a mRNA; MFA, myocardial fibrosis area. * Parameters

estimated by echocardiography

20

Sham day 28 AC day 28CTGF=BNP

AC day 28CTGF>BNPA

E/A=1.6 E/A=1.3 E/A=5.2

Sham Day 28 AC Day 28CTGF>BNP

AC Day 28CTGF=BNP

B

*

*

*

*

*

*

*

*

EEAA

Supplemental Figure I

21

A B

r=0.720P<0.001

0

0.05

0.1

0.15

0.2

0 1 2 3CTGF/BNP ratio

EDPV

R (m

mH

g/μL

)

r=0.458 P<0.001C

OL1

A1

mR

NA

Fold

Incr

ease

0 1 2 3CTGF/BNP ratio

5

4

3

2

1

0

Supplemental Figure II

22

Plasma TGFβ

0

10

20

30

40

ng/m

L

Plasma Endothelin 1

0123456

pg/m

L

Plasma Aldosterone

*

0200400600800

10001200

pg/m

L

CTGF/BNP<1.2 CTGF/BNP>1.2

Supplemental Figure III

23

Supplemental Figure IV

CTGFVimentin Merged

24

NE (μmol/L)0 1010 101

AngII (μmol/L)

Supplemental Figure V

1 2 4

sBNPhrs0

CTGF

28S

0 0.5sBNP(μmol/L) 0.1 0 0.50.1

KT5823Vehicle

CTGF

28S

B C

A

28S

BNP

CTGF

25

CM CFB

CTGF

28S

0

1

2

3

Rel

ativ

e In

tens

ityA *

Veh 1 10 0.1 1 10 0.01 0.1 0.1 1

TGFβ(ng/mL)

AngII(μmol/L)

NE(μmol/L)

ET1(μmol/L)

Aldo(μmol/L)

CTGF

28S

0

0.5

1

1.5 *

Rel

ativ

e In

tens

ityB

Supplemental Figure VI

26

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Increased Connective Tissue Growth Factor Relative to ...hyper.ahajournals.org/content/hypertensionaha/49/5/1120.full.pdf · in AC rats, RNA isolation and Northern blot analysis,