Ginseng Inhibits Cardiomyocyte Hypertrophy and Heart … · Ginseng Inhibits Cardiomyocyte...

Ginseng Inhibits Cardiomyocyte Hypertrophy and HeartFailure via NHE-1 Inhibition and Attenuation of

Calcineurin ActivationJuan Guo, PhD; Xiaohong Tracey Gan, MSc; James V Haist, BSc; Venkatesh Rajapurohitam, PhD;

Asad Zeidan, PhD; Nazo Said Faruq; Morris Karmazyn, PhD

Background—Ginseng is a medicinal plant used widely in Asia that has gained popularity in the West during the pastdecade. Increasing evidence suggests a therapeutic role for ginseng in the cardiovascular system. The pharmacologicalproperties of ginseng are mainly attributed to ginsenosides, the principal bioactive constituents in ginseng. The presentstudy was carried out to determine whether ginseng exerts a direct antihypertrophic effect in cultured cardiomyocytesand whether it modifies the heart failure process in vivo. Moreover, we determined the potential underlying mechanismsfor these actions.

Methods and Results—Experiments were performed on cultured neonatal rat ventricular myocytes as well as adult ratssubjected to coronary artery ligation (CAL). Treatment of cardiomyocytes with the �1 adrenoceptor agonistphenylephrine (PE) for 24 hours produced a marked hypertrophic effect as evidenced by significantly increased cellsurface area and ANP gene expression. These effects were attenuated by ginseng in a concentration-dependent mannerwith a complete inhibition of hypertrophy at a concentration of 10 �g/mL. Phenylephrine-induced hypertrophy wasassociated with increased gene and protein expression of the Na�-H� exchanger 1 (NHE-1), increased NHE-1 activity,increased intracellular concentrations of Na� and Ca2�, enhanced calcineurin activity, increased translocation ofNFAT3 into nuclei, and GATA-4 activation, all of which were significantly inhibited by ginseng. Upregulation of thesesystems was also evident in rats subjected to 4 weeks of CAL. However, animals treated with ginseng demonstratedmarkedly reduced hemodynamic and hypertrophic responses, which were accompanied by attenuation of upregulationof NHE-1 and calcineurin activity.

Conclusions—Taken together, our results demonstrate a robust antihypertrophic and antiremodeling effect of ginseng,which is mediated by inhibition of NHE-1–dependent calcineurin activation. (Circ Heart Fail. 2011;4:79-88.)

Key Words: ginsenosides � phenylephrine � NHE-1 � calcineurin � heart failure

Ginseng is a popular herbal medicine that has been used inAsia for centuries, although, in recent years, its potential

therapeutic effects have become more widely recognized.Ginsenosides are the principal bioactive constituents of gin-seng, and �40 ginsenosides have been isolated to date.1

Ginseng exerts numerous pharmacological properties in mul-tiple species, including humans.2 The cardiovascular benefi-cial effect of ginseng has also been demonstrated for thetreatment of angina pectoris3 and for the reduction of adria-mycin-induced heart failure in rats.4 Moreover, ginseng hasbeen demonstrated to attenuate right and left ventricularhypertrophy in a number of experimental models.5–7 Becausethe underlying basis for the antihypertrophic effect of ginsengis poorly understood, we studied the mechanisms for theantihypertrophic effect of ginseng using cultured ventricular

myocytes and an in vivo model of heart failure secondary tochronic ischemia. The study centered primarily on theNa�-H� exchanger-1 isoform (NHE-1), which has beenextensively shown to contribute to hypertrophy and heartfailure.8,9 Accordingly, we determined the effect of ginsengon NHE-1 activity and its expression and subsequent

Clinical Perspective on p 88effects on key signaling mechanisms underlying the hyper-trophic program. For example, NHE-1 has been shown tocontribute to intracellular Ca2� overloading, resulting in theactivation of Ca2�-dependent prohypertrophic processes me-diated by the protein phosphatase calcineurin and subsequentactivation of prohypertrophic transcriptional factors.10,11

Here, we determined the role of this pathway in mediating theantihypertrophic effect of ginseng in cultured ventricular

Received June 7, 2010; accepted October 6, 2010.From the Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario

N6A 5C1, Canada. Dr Guo’s permanent address is the Department of Pharmacology, Shanghai University of Traditional Chinese Medicine, Shanghai201203, China.

Correspondence to Morris Karmazyn, PhD, Department of Physiology and Pharmacology, University of Western Ontario, Schulich School of Medicineand Dentistry, London, Ontario N6A 5C1, Canada. E-mail [email protected]

© 2011 American Heart Association, Inc.

Circ Heart Fail is available at http://circheartfailure.ahajournals.org DOI: 10.1161/CIRCHEARTFAILURE.110.957969

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myocytes subjected to hypertrophic stimuli and applied thesefindings to an in vivo model of heart failure in rats.

Methods

Neonatal Cardiac Myocytes Culture andTreatment ProtocolThe studies have been approved by the Animal Use Subcommittee ofthe University of Western Ontario and procedures conform to theguidelines of the Canadian Council on Animal Care (Ottawa,Ontario, Canada). Myocytes were prepared from hearts of 1- to4-day-old Sprague-Dawley rats as described previously12 and cul-tured for 24 hours in serum containing medium followed by 24 hoursin serum-free medium. To initiate hypertrophy, myocytes were thentreated with 10 �m/L �1 adrenoceptor agonist phenylephrine (PE)for 24 hours in the absence or presence of ginsenosides (0.1, 1, or 10�g/mL). For some experiments (see Results), cells were subjected toPE treatment for shorter durations.

Ginsenoside Extraction ProcedureFour-year-old North American ginseng (Panax quinquefolius) rootswere collected from 5 farms in Ontario, Canada and shipped toNaturex (South Hackensack, NJ) for ginsenoside extraction with useof a hydroalcoholic process. In brief, ground ginseng roots weresoaked 3 times over 5 hours in an ethanol/water (75:25 v/v) solutionat 40°C. The extract was filtered and excess solvent removed undervacuum at 45°C. The extract was concentrated again until the totalsolids on a dry basis were �60%. These concentrates were thenlyophilized at the Ontario Ginseng Innovation and Research Con-sortium central laboratory (University of Western Ontario) to pro-duce a powdered alcoholic ginseng extract, which was then subjectedto analysis by high-pressure liquid chromatography (HPLC) todetermine the presence of major (Rb1 and Re) and minor (Rg1, Rb2,Rd, and Rc) ginsenosides.

Measurement of Cell Surface AreaMyocytes were visualized using a Leica DMIL inverted microscope(Leica, Wetzlar, Germany) equipped with an Infinity 1 camera. Atleast 10 random photographs were taken from each dish, and the cellsurface area of a minimum of 30 cells from each treatment wasmeasured using SigmaScan Software (Systat, Richmond, Calif).

Measurement of Intracellular Na� andCa2� ConcentrationsMyocytes were incubated with CoraNa Red (excitation: 554 nm;emission: 578 nm) or Furo-2 (excitation: 338 nm; emission: 510 nm)for 30 minutes at 37°C, to measure Na� or Ca2� concentrations,respectively. Myocytes were washed twice with phosphate-bufferedsaline (PBS), and fluorescence intensity was measured using aspectra Max M5 plate reader. Fluorescence intensity was normalizedagainst control (CoroNa-Red-loaded cells or Furo-2-loaded cells)after subtraction of baseline (CoroNa-Red or Furo-2 without cells).

Measurement of Intracellular pH (pHi)The pHi was measured using the pH-sensitive dye 2�,7�-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein acetoxymethyl ester (In-vitrogen, Carlsbad, Calif). In brief, myocytes were loaded with thedye at 37°C for 30 minutes and placed on the stage of an invertedZeiss Axiovert 35 microscope. Myocytes were continuously per-fused at 1 mL/min with HCO3-free HEPES buffer solution. The pHi

in individual cardiomyocytes was recorded by photometry at 502.5and 440 nm for excitation and 528 nm for emission using amonochromatic Deltascan-4000 system (Photon Technology Inter-national, Birmingham, NJ). The NH4Cl prepulse technique was usedto determine activity of NHE-1 and the effect of treatments onNHE-1 activity in cardiomyocytes.13

Calcineurin Phosphatase Activity AssayCalcineuruin activity was determined using commercially availablekits according to the manufacturer’s instructions (Enzo Life Sci-ences, Plymouth Meeting, Pa).

Determination of NFAT3 TranslocationMyocytes were fixed in an acetone and methanol (20:80) mixture asdescribed previously.14

After permeabilization and blocking, cells were incubated withNFAT3 antibody (1:100 dilutions) overnight at 4°C followed byincubation with Alexa Fluor 594 goat anti-rabbit IgG (1:250 dilu-tions) for 1 hour at room temperature in darkness. The cells weremounted on the glass slide using DakoCytomation fluorescentmounting medium and visualized using a Zeiss Axio Observer D1fluorescence microscope (Zeiss, Gottingen, Germany).

Electrophoretic-Mobility Shift Assay (EMSA)EMSAs were performed using the Panomics EMSA Gel-Shift Kit(Panomics, Inc., Fremont, Calif) according to the manufacturer’sprotocol.

RNA Isolation, Reverse Transcription andReal-Time PCR AnalysisRNA was extracted using Trizol (Invitrogen, Carlsbad, Calif) ac-cording to the manufacturer’s instructions. RNA (2 �g) was used tosynthesize the first strand of cDNA using M-MLV reverse transcrip-tase according to the manufacturer’s protocol and was used as atemplate in the following PCR reactions. The expression of ANP,

Figure 1. Effect of different concentrations of ginsenosides (0.1to 10 �g /mL) on cell surface area and ANP gene expression inmyocytes treated with phenylephrine (10 �mol/L) for 24 hours.A, Representative micrographs of cardiomyocytes. B, Cell sur-face area. C, Expression of ANP gene. Data are shown asmean�SEM, *P�0.05 versus control; #P�0.05 versus PE; n�6 to7. Ctl indicates control; Gins, ginsenosides; PE, phenylephrine.

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NHE-1, MCIP1, and 18S rRNA (loading control) genes was determinedin 10-�L reaction volumes using SYBR green Jumpstart TagReadyMix DNA polymerase and fluorescence was measured andquantified using DNA Engine Opticon 2 System. The followingprimer sequences were used: 5�-CTGCTAGACCACCTGAGGA-3�(forward) and 5�-AAGCTGTTGCAGCCTAGTCC-3� (backward)for ANP; 5�-ATGTGGCTGGGAAACAAGAC-3� (forward) and5�-GACAGTCCCTCCCGTGTAAA-3� (backward) for NHE-1; 5�-GCC-CAATCCAGACAAACAGT-3� (forward) and 5�-TGATTTTTGGC-TTGGGTCTC-3� (backward) for MCIP1; and 5�-GTAACC-CTTGAACCCCATT-3� (forward) and 5�-CCATCCAATCGGTAGTAGCG-3� (backward) for 18S rRNA. PCR conditionsand cell cycle number were optimized for each set of primers.Melting curve analysis showed a single PCR product for each geneamplification. PCR conditions to amplify all 3 genes were 30seconds at 94°C followed by annealing at 60°C for 25 seconds forANP, MCIP1, and NHE1 and 54°C for 20 seconds for 18S rRNAfollowed by elongation at 72°C for 30 s. All genes were amplified for40 cycles except 18S rRNA, which was amplified for 35 cycles.

Western Blotting for GATA-4 and NHE-1After appropriate treatments, myocytes were washed with PBS andlysed with 150 �L of lysis buffer. Cell lysates were transferred to1.5-mL Eppendorf tubes, homogenized, and centrifuged at 10 000 gfor 5 minutes at 4°C. The supernatant was transferred to a fresh tubeand the protein concentration determined by the Bradford proteinassay method (Bio-Rad, Hercules, Calif). Thirty micrograms ofprotein were resolved on a 10% SDS-polyacrylamide gel andtransferred to nitrocellulose membranes. The membranes wereblocked in 5% milk for 1 hour and incubated with primary antibodyfor GATA-4 or NHE-1 for 1 hour, followed by a secondary antibodyfor 1 hour, then detected by enhanced chemiluminescence reagent(Amersham Biosciences Inc., Piscataway, NJ). The blots werestripped and reprobed with actin antibodies.

Coronary Artery LigationMale Sprague-Dawley rats (275 to 300 g) were randomly assigned tothe following 4 treatment groups: sham and coronary artery ligation(CAL) with or without ginsenosides (100 mg/kg) treatment started24 hours after surgery. Surgery was performed as previously de-scribed.15 All animals received 0.03 mg/kg buprenorphine immedi-ately after completion of surgery for pain management.

EchocardiographyFour weeks after CAL, rats were anesthetized with 2% isofluraneand placed in a supine position on a heated platform. The chest andabdomen were shaved and the extremities were fixed to electrodes onthe platform surface by use of tape and a highly conductive electrodegel. Echocardiography evaluations were performed using a Vevo 770high-resolution in vivo microimaging system equipped with areal-time microvisualization scan head of 17.5 MHz (VisualSonics,Toronto, Ontario, Canada). M-mode 2-dimensional echocardiogra-phy images were obtained from the parasternal short axis. Imageswere analyzed using the Vevo 770 Protocol-Based Measurementssoftware and calculations for the dimensions of the left ventricle(LV) diameter. Doppler measurements were taken to determine peakearly diastolic filling velocity (E wave), peak late diastolic fillingvelocity (A wave), and E/A ratios.

Hemodynamic MeasurementsThe rats were anesthetized with pentobarbital sodium (50 mg/kg, ip).An anterior thoracotomy was performed, and the LV was catheter-

Figure 2. Effect of 10 �g/mL ginsenosides on cell surface area(A) and ANP gene expression (B) in myocytes treated with 100nmol/L angiotensin II or 10 nmol/L endothelin-1. Data are shownas mean�SEM. *P�0.05 versus control; #P�0.05 versusrespective agonist alone. n�5. Ctl indicates control; Gins,ginsenosides. Figure 3. Effect of different concentrations of ginsenosides (0.1

to 10 �g/mL) on NHE-1 gene and protein expression and 10�g/mL Gins on NHE-1 activity in the cells treated with phenyl-ephrine (10 �mol/L) for 24 hours. A, Quantification of NHE-1gene expression. B, Representative Western blot and quantifica-tion of NHE-1 protein expression. C, pH recovery after intracel-lular acidosis. Data are shown as mean�SEM. *P�0.05 versuscontrol; #P�0.05 versus PE; n�5 to 9. Ctl, control; Gins, gin-senosides; PE, phenylephrine.

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ized retrogradely via the right carotid artery using a 2.0F P-VMikro-Tip catheter (Millar Instruments, Houston, Tex) as previouslydescribed.15 Data were recorded and analyzed by hemodynamic dataanalysis software (Notocord, Croissy-sur-Seine, France), digitizedwith a sampling rate of 1000 Hz, and recorded on a personalcomputer using Notocord-hem 4.2 software.

Statistical AnalysisResults are presented as means�SEM. The data were analyzed with1-way ANOVA and group differences were detected using aStudent-Newman-Keuls post hoc test when initial ANOVA analysisrevealed statistically significant differences. P values of �0.05 wereconsidered significant.

ResultsEffect of Ginseng on PE-InducedCardiomyocyte HypertrophyMyocytes treated with PE for 24 hours demonstrated asignificant increase in cell surface area from 850�14 �m2 to1090�20 �m2 (Figure 1A and 1B; P�0.05); whereas in thepresence of 10 �g/mL ginsenosides cell surface area in thepresence of PE was reduced to 942�44 �m2 (Figure 1B;P�0.05 PE alone). PE increased gene expression of ANP by�2-fold (Figure 1C; P�0.05) compared with controls;whereas this was almost completely prevented by 10 �g/mLginsenosides. Ginsenosides had no direct effect on eitherparameter in the absence of PE. Ginsenosides exertedsimilar effects against other prohypertrophic stimuli in-cluding either 100 nmol/L angiotensin II or 10 nmol/Lendothelin-1 (Figure 2).

Effect of Ginseng on PE-Induced Changes inNHE-1 Protein, Gene Expression and ActivityFigure 3 shows that PE induced a 1.56�0.16-fold upregula-tion of NHE-1 gene (Figure 3A) and a 1.38�0.09-foldincrease in protein abundance (Figure 3B) after 24 hours oftreatment, which was associated with increased NHE-1 ac-tivity (Figure 3C). The upregulation of NHE-1 gene andprotein expression was abrogated by the 2 highest concentra-

tions of ginsenosides with increases for gene and proteinlevels in the presence of 10 �g/mL ginsenosides reduced to1.18�0.04-fold and 1.07�0.10-fold, respectively (Figure 3Aand 3B). Moreover, stimulation of NHE-1 activity wasreduced to values not significantly different from control by10 �g/mL ginsenosides (Figure 3C).

Effect of Ginseng on PE-Induced Changes inIntracellular Na� and Ca2� ConcentrationsPE induced a rapid elevation in intracellular concentrations ofboth Na� and Ca2� that was evident 15 minutes after PEaddition (Figure 4). No effect of ginseng (10 �g/mL ginsen-osides) was observed up to 6 hours after its addition, althougha significant reduction in the intracellular concentrations ofboth Na� and Ca2� was evident 12 and 24 hours afteradministering ginsenosides with values not significantly dif-ferent from control after 24 hours.

Effect of Ginseng on PE-Induced Changes inCalcineurin ActivityAs shown in Figure 5A calcineurin activity was rapidly(within 15 minutes) increased after PE administration withactivity steadily declining in the presence of ginsenosidesafter 6 hours, although still significantly greater than control.Ginsenosides alone had no effect on calcineurin activity(Figure 5A).

Effect of Ginseng on PE-Induced Changes inNFAT3 Nuclear Import and GATA-4 ActivationLocalization of NFAT3 in control myocytes was primarilyrestricted to the cytosol (Figure 5B), although substantial trans-location to nuclei was evident after 24 hours of PE treatment.Ginsenosides clearly reduced PE-induced translocation resultingin substantial cytosolic localization of NFAT3 similar to thatseen under control conditions (Figure 5B).

PE significantly increased GATA-4 phosphorylation by 1.3-fold (Figure 5C; P�0.05) and increased GATA-4–DNA bind-

Figure 4. Effect of phenylephrine(10 �mol/L) treatment for indicated timepoints on intracellular Na� and Ca2�

concentrations in presence or absenceof 10 �g/mL ginsenosides. The cellswere treated with PE in the presence orabsence of ginsenosides for 15 minutes,3 hours, 6 hours, 12 hours, and 24hours. The quantification of intracellularNa� (A) and Ca2� (B) levels were mea-sured by using CoroNa-Red dye andFuro-2 dye, respectively. Data are shownas means�SEM. *P�0.05 versus control;#P�0.05 versus PE; n�8. Gins indicatesginsenosides; PE, phenylephrine.




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ing activity as determined by EMSA (Figure 5D). Both re-sponses were prevented by ginsenosides (Figure 5C and 5D).

Effect of Ginseng on CAL-Induced LeftVentricular DysfunctionWe next determined whether the direct antihypertrophiceffect of ginsenosides seen in cultured myocytes can betranslated to protection in vivo in rats subjected to 4 weeks ofsustained CAL. As shown in the Table, CAL producedmarked systolic and diastolic abnormalities, which wereattenuated by ginsenoside treatment. Moreover, ginsenosidetreatment significantly reduced the increase in left ventricularinner diameters in rats subjected to sustained CAL (Figure 6Aand 6B). In addition, CAL increased the E/A ratio obtainedfrom Doppler echocardiographic analysis, which was normal-ized in animals treated with ginseng indicative of improveddiastolic function (Figure 6C and 6D).

Effect of Ginseng on CAL-InducedCardiac HypertrophyAnimals subjected to CAL had significantly reduced bodyweights at the end of the 4-week ligation period, although this

was unaffected by ginsenosides (Figure 7A). Animals sub-jected to CAL exhibited significantly increased left ventricleweights as well as ANP expression (Figure 7D), indicatingdevelopment of left ventricular hypertrophy (Figure 7B to7D). These responses were completely prevented byginsenosides.

Effect of Ginseng on CAL-Induced NHE-1 andExpression and Calcineurin ActivationAs shown in Figure 8A, rats subjected to CAL had signifi-cantly increased NHE-1 expression (1.98�0.16-fold) al-though this was partially but significantly reduced by ginsen-oside treatment (1.41�0.11-fold) (Figure 8A). Twoindicators of calcineurin activity, namely modulatory cal-cineurin interacting protein 1(MCIP1) expression (Figure 8B)and calcineurin phosphatase activity (Figure 8C) were signif-icantly increased in hearts subjected to CAL, although theseresponses were completely abrogated by ginsenosides (Figure8B and 8C).

DiscussionAlthough ginseng has been used as a pharmacotherapeuticagent in Asian society for centuries, its potential cardiac

Figure 5. Effect of phenylephrine (10 �mol/L) treat-ment on calcineurin activity, NFAT3 translocation,and the DNA-binding activity of GATA-4 in thepresence or absence of 10 �g/mL ginsenosides.A, Quantification of calcineurin activity in the cellstreated with PE in the presence or absence of gin-senosides for 15 minutes, 3 hours, 6 hours, 12hours, and 24 hours. B, Immunofluorescentimages of NFAT3 translocation into nucleus in thecells treated with PE in the presence or absenceof ginsenosides for 24 hours. C, RepresentativeWestern blots and quantification of GATA-4 phos-phorylation. D, GATA-4/DNA-binding activity in thecells treated with PE in the presence or absenceof ginsenosides. Data are shown as means�SEM.*P�0.05 versus control; #P�0.05 versus PE, n�3to 5. Ctl, control; Gins, ginsenosides; PE,phenylephrine.




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therapeutic properties have not been extensively studied andare poorly understood. Ginseng is also among the mostcommon of the alternate medicines used by the Americanpopulation, although not necessarily for cardiovascular dis-orders.16 Whether the use of natural compounds such asginseng holds promise for the treatment of cardiovascular

disorders is not known, possibly because of a paucity of datademonstrating their effects in well-established experimentalmodels of cardiovascular disease and the lack of informationon the mechanism of action of these compounds. Here, wedetermined the potential antihypertrophic effect of the bio-logically active components of ginseng, the ginsenosides,

Table. Hemodynamic Data

Sham (n�8) Sham�Gins (n�6) CAL (n�8) CAL�Gins (n�10)

LVESP (mm Hg) 120.81�5.9 127.81�4.3 96.92�2.2* 113.75�3.3†

LVEDP (mm Hg) 3.64�0.30 4.01�0.53 11.73�0.46* 7.76�0.40†

�dP/dt (mm Hg/s) 9711�408 8519�342 4770�682* 6550�164.2†

�dP/dt (mm Hg/s) �8247�413 �7933�421 3755�860* �6568�195.8†

HR (beats/min) 395.68�8.2 385.98�7.4 397.68�6.1 393.62�9.0

LVESV (�L) 85.46�5.3 89.17�2.6 181.47�10* 108.45�6.0†

LVEDV (�L) 207.53�5.8 209.87�7.1 279.60�10.3* 215.27�8.6†

SV (�L) 124.70�4.6 127.56�3.8 98.17�4.7* 113.76�4.0†

EF (%) 59.64�2.3 60.29�1.1 38.43�2.3* 48.97�1.2†

CO (mL/min per kg) 122.95�4.2 119.79�3.6 100.26�3.3* 113.68�4.2†

All results are shown as means�SEM. CAL, coronary artery ligation; Gins, ginsenosides (100 mg/kg); LVESP, leftventricular end-systolic pressure; LVEDP, left ventricular end-diastolic pressure; �dP/dt and �dP/dt, rate of leftventricular pressure development and relaxation, respectively; HR, heart rate; ESV, end-systolic volume; EDV,end-diastolic volume; SV, stroke volume; EF, ejection fraction; CO, cardiac output. *P�0.05 versus sham; †P�0.05versus CAL.

Figure 6. Effect of ginsenosides on CAL-induced changes in left ventricular internal diameters and transmitral velocity. Animals weresubjected to either CAL or sham operation, with or without daily ginsenoside treatment (100 mg/kg) for 4 weeks. A, Short-axis biven-tricular M-mode images. B, Quantification of end-diastolic and end-systolic LVID. C, representative images of long-axis transmitralvelocity. D, Quantification the E/A ratio. Data are shown as mean�SEM. *P�0.05 versus respective sham; #P�0.05 versus CAL, n�6to 10. CAL indicates coronary artery ligation; LVID, left ventricular internal diameter; Gins, ginsenosides; LVIDd, end-diastolic LVID;LIVDs, end-systolic LVID.




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extracted from North American ginseng, on the hypertrophicresponse of myocytes exposed to the �1 adrenoceptor agonistPE. We show that ginsenosides provide a robust antihyper-trophic influence in this model of hypertrophy, although ourstudy also suggests that the antihypertrophic effect of ginsengis likely not restricted to PE particularly because it would beunlikely that the robust salutary effects seen in vivo weremediated solely by an effect restricted to inhibition of �1

adrenoceptor-mediated hypertrophy. Moreover, our studyshows that ginsenosides also markedly attenuate the directhypertrophic of both angiotensin II and endothelin-1 onmyocytes.

Overall, our study strongly suggests that the ability ofginsenosides to attenuate hypertrophy is related to preventingthe activation/upregulation of NHE-1, which has been exten-sively implicated in the hypertrophic and heart failure pro-cess.17,18 This probably occurs subsequent to receptor-dependent NHE-1 activation. NHE-1 activation results in anumber of intracellular alterations that can contribute to thehypertrophic program,10,19,20 although a particularly impor-tant consequence of NHE-1 activation is the elevation inintracellular Na� concentrations, which is followed by in-creases in intracellular Ca2� concentrations via reverse modeNa-Ca exchange activity.21 As discussed below, this in turnwould induce hypertrophy by activating key factors in thehypertrophic program, especially the phosphatase calcineurinthat results in transcriptional changes due to NFAT3 dephos-phorylation and its translocation into nuclei. The ability ofginsenosides to attenuate the hypertrophic effects of bothangiotensin II and endothelin-1, 2 NHE-1 activators,22 furthersupports NHE-1 as a target for their salutary effects. None-theless, the possibility that ginsenosides are acting throughother or additional nonspecific mechanisms cannot be ruledout and requires further studies.

In the present study we used an alcoholic extract ofginsenosides to demonstrate a potent antihypertrophic effectin vitro, as well as a highly effective ability to reducehypertrophy and heart failure in vivo, through what appears tobe identical mechanisms. The ability of ginsenosides to blockthe hypertrophic response to the �1 adrenoceptor agonist PEat the highest concentration equaled the antihypertrophiceffect observed with the NHE-1 inhibitor cariporide (data notshown). The antihypertrophic effect of ginseng in vitro is inpartial agreement with a previous study demonstrating thatginsenosides inhibit prostaglandin F2�-induced hypertrophythrough a mechanism involving attenuation in the increasedexpression levels of calcineurin and various transcriptionalfactors.23 We were unable to observe any changes in abun-dance (either gene or protein) of calcineurin (data not shown),but rather, activation of calcineurin was the primary responseto hypertrophic stimuli. Ginseng prevented the upregulationof NHE-1 gene and protein abundance and depressed NHE-1activity 24 hours after PE treatment. Addition of PE produceda rapid elevation of intracellular Na� and Ca2� concentra-tions and calcineurin activation. Interestingly, the early up-regulation of these factors was unaffected by ginseng up to 6hours after PE administration, except for a significant inhi-bition in intracellular Na� concentrations at 6 hours, whereasall factors were significantly inhibited 12 and 24 hours afterPE administration. Because NFAT3 translocation into nucleiand activation of the transcriptional factor GATA-4 24 hoursafter PE administration were markedly inhibited by ginseng,the results suggest that late inhibition of calcineurin activa-tion via Na�- and Ca2�-dependent mechanisms is importantin attenuating the hypertrophic response to PE, at least withrespect to the antihypertrophic effect of ginsenosides.

Using a rat CAL model we also show that oral admin-istration of ginseng reduces hypertrophy and hemodynam-

Figure 7. Body weights (A) and indicesof cardiac hypertrophy in animals sub-jected to either CAL or sham operation,with or without daily ginsenoside treat-ment (100 mg/kg) for 4 weeks. Gravimet-ric data for heart weight and left ventric-ular weight and quantification of ANPgene expression levels are shown in B,C, and D, respectively. Data are shownas mean�SEM, n�6 to 10. *P�0.05 ver-sus respective sham; #P�0.05 versusCAL. CAL indicates coronary artery liga-tion; Gins, ginsenosides.




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ic abnormalities in this well-established model of heartfailure. We used a ginseng dose of 100 mg/kg, whichproduced an optimal salutary effect in this model. Com-parison of this dose with the clinical use of ginseng isdifficult because of the lack of standardization in terms ofdosing or the paucity of well-controlled clinical trials.Moreover, different ginseng varieties exhibit varied bio-logical profiles. Nonetheless, the dose in our study wouldroughly approximate the mid high end of the dosingspectrum in humans, which can range between 0.5 and 15g/d. The salutary effect of ginseng was evident in terms ofsubstantial attenuation and, in some cases, the near nor-malization of hemodynamic dysfunction as assessed bothby invasive catheter-based determinations of hemodynam-ic parameters and by echocardiography. Moreover, theimproved hemodynamic function was associated with di-minished hypertrophy determined by gravimetric analysisand molecular markers. Although mechanistic insights aremore difficult to establish using in vivo approaches, thebeneficial effect of ginseng in this model demonstratedsubstantial concordance with the effect seen in culturedmyocytes vis-a-vis NHE-1 and calcineurin in that the

improved cardiovascular properties were associated withdiminished expression of NHE-1 and prevention of cal-cineurin activation, the latter determined by measurementof phosphatase activity and expression levels of MCIP1, anindex of calcineurin activation.24

Based on current knowledge, the ability of ginseng toinhibit NHE-1 activity may represent its pivotal effect interms of attenuating the hypertrophic response. As previouslynoted, from a general perspective, there is substantial evi-dence implicating NHE-1 as a key contributor to myocardialhypertrophy, remodeling, and heart failure, because the anti-porter is upregulated in heart failure and its inhibition reducesthe severity of the hypertrophic response and the develop-ment of cardiovascular dysfunction.10,15,25 The nature ofNHE-1 involvement in the hypertrophic response is notcompletely understood, although NHE-1 activation couldresult in a number of intracellular changes resulting instimulation of the hypertrophic program.10,19,20 Ca2�-mediated signaling has gained substantial attention amongmany molecular mechanisms that are known to coordinatedevelopment of pathological hypertrophy.26 In particular,Ca2�-dependent activation of calcineurin leads to dephos-phorylation of NFAT3 which subsequently translocates to thenucleus where it acts with other transcription factors includ-ing GATA-4 to initiate gene transcription. The ability ofginseng to attenuate calcineurin activation, NFAT3 translo-cation as well as GATA-4 phosphorylation and DNA-bindingactivity suggest that this represents the target for its antihy-pertrophic effect. Among these responses is the elevation inintracellular Ca2� concentrations, secondary to elevations inintracellular Na� concentrations, resulting in reverse-modeNa�-Ca2� exchange activation that results in the activeimport of Ca2� into the cell.27–31 There is emerging strongevidence in the literature that Ca2�-dependent initiation of thehypertrophic program is NHE-1 dependent. For example, theantihypertrophic effect of NHE-1 inhibition in spontaneouslyhypertensive rats has been shown to be associated withnormalization of the calcineurin pathway in the hearts ofthese animals.32 Moreover, a recent study demonstrated thatcardiac NHE-1 overexpression in transgenic mice activatesthe Ca2�-dependent hypertrophic program as manifested byincreased intracellular Ca2� concentrations, calcineurin acti-vation, and establishment of cardiac hypertrophy and heartfailure, in the absence of insult.10 Importantly, the activationof the Ca2�-dependent hypertrophic cell signaling processand the hypertrophy itself can be abrogated by the NHE-1specific inhibitor cariporide.10,33

In conclusion, we have used an integrative approach todemonstrate a robust antihypertrophic effect of ginseng incultured neonatal myocytes exposed to hypertrophic stim-uli as well as an in vivo model of heart failure secondaryto sustained CAL. (The effects of ginseng in vivo translateto improved hemodynamic status 4 weeks after CAL.) Ourresults further suggest a common underlying mechanisminvolving NHE-1 inhibition resulting in the attenuation ofcalcineurin activation. A present limitation of our study isour inability to identify the nature of the specific ginsen-oside that may account for the salutary effects on hyper-trophy and heart failure. As alluded to previously, �40

Figure 8. Attenuation of CAL-induced elevation in NHE-1,MCIP1 gene expression and calcineurin activation by ginsen-osides. Animals were subjected to either CAL or sham proce-dure, with or without daily ginsenoside treatment (100 mg/kg)for 4 weeks. A, Quantification of NHE-1 gene expression levels.B, Quantification of MCIP1 gene expression levels. C, Cal-cineurin phosphatase activity. Data are shown as mean�SEM,n�6 to 10. *P�0.05 versus respective sham; #P�0.05 versusCAL. CAL indicates coronary artery ligation; Gins, ginsenosides.




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ginsenosides have thus far been identified.1 Using HPLCanalysis, the primary ginsenosides identified in the extractused in the present study were Rb1 and Re (each �45% oftotal ginsenosides) whereas the content of minor ginsen-osides (Rg1, Rb2, Rd, and Rc) was between 1% and 7%(Dr EMK Lui, University of Western Ontario and Dr JTArnason, University of Ottawa, personal communication).From a quantitative perspective, it is attractive to speculatethat Rb1 and Re represent the principal ginsenosidesaccounting for the antihypertrophic/antiremodeling ef-fects, although this needs to be determined with furtherstudies. It is also possible that the beneficial effectsrepresent the combined actions of a number of thesecompounds present in the ginseng extract. This limitationnotwithstanding, our study suggests that administration ofginseng may represent an effective adjunct therapy for thelimitation of myocardial hypertrophic response and for thetreatment of heart failure. Indeed, interaction betweenginseng and therapeutic agents used for the treatment ofheart failure would be valuable for future studies toexplore.

Sources of FundingThis study was supported by the Institute of Circulatory andRespiratory Health of the Canadian Institutes of Health Research andthe Ontario Ginseng Innovation and Research Consortium (OGIRC)(Ontario Ministry of Research and Innovation Grant RE02-049). DrJ. Guo was supported by OGIRC. Dr M. Karmazyn holds a CanadaResearch Chair in Experimental Cardiology.

DisclosuresNone.

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CLINICAL PERSPECTIVEHeart failure is a major cause of hospitalization and is associated with high financial costs and societal burden. Cardiachypertrophy and myocardial infarction are major risk factors for the development of heart failure. In recent years, there hasbeen growing interest in botanicals as alternative medicines for cardiovascular disease. Ginseng is a popular traditionalherbal medicine and has been widely used to treat cardiovascular diseases for thousands of years in Asia. Despite increasinginterest in ginseng in Western societies, its efficacy and mechanism of action are poorly understood. We sought tounderstand the effect of ginsenosides on cardiomyocyte hypertrophy and myocardial infarction-induced heart failure. In invitro studies, we showed that ginsenosides were able to attenuate cardiomyocyte hypertrophy. Further studies revealed thatthe antihypertrophic effect of ginsenosides was mediated by inhibiting NHE-1 activity and blocking calcium-mediatedsignaling, which is evidenced by decreased intracellular calcium levels, calcineurin activity, and NFAT3 translocation. Insupport of in vitro findings, our in vivo studies demonstrated that oral administration of ginsenosides prevented theprogression of heart failure by reducing postinfarction myocardial hypertrophy and improving hemodynamics. Althoughresults from animal studies must be interpreted cautiously, our findings support the development of ginsenosides aspotential therapeutics for the treatment of cardiac diseases and demonstrate the cellular bases for these actions.




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Said Faruq and Morris KarmazynJuan Guo, Xiaohong Tracey Gan, James V Haist, Venkatesh Rajapurohitam, Asad Zeidan, Nazo

and Attenuation of Calcineurin ActivationGinseng Inhibits Cardiomyocyte Hypertrophy and Heart Failure via NHE-1 Inhibition

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