Ginseng (Panax quinquefolius) Attenuates Leptin-Induced CardiacHypertrophy through Inhibition of p115Rho Guanine NucleotideExchange Factor-RhoA/Rho-Associated, Coiled-Coil ContainingProtein Kinase-Dependent Mitogen-Activated Protein KinasePathway Activation

Melissa Moey, Venkatesh Rajapurohitam, Asad Zeidan, and Morris KarmazynDepartment of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario,London, Ontario, Canada (M.M., V.R., M.K.); and Department of Anatomy, Cell Biology, and Physiological Sciences, AmericanUniversity of Beirut, Beirut, Lebanon (A.Z.)

Received April 1, 2011; accepted August 25, 2011

ABSTRACTLeptin is a 16-kDa peptide primarily derived from white adipocytesand is typically elevated in plasma of obese individuals. Althoughleptin plays a critical role in appetite regulation, leptin receptorshave been identified in numerous tissues including the heart andhave been shown to directly mediate cardiac hypertrophy throughRhoA/ROCK (Ras homolog gene family, member A/Rho-associ-ated, coiled-coil containing protein kinase)-dependent p38 mito-gen-activated protein kinase (MAPK) activation; however, the ba-sis for RhoA stimulation is unknown. Rho guanine nucleotideexchange factors (GEFs) catalyze the exchange of GDP for GTPresulting in Rho activation and may be the potential upstreamfactors mediating leptin-induced RhoA activation and therefore apotential target for inhibition. We investigated the effects of NorthAmerican ginseng (Panax quinquefolius), reported to reduce car-diac hypertrophy, on RhoA/ROCK and MAPK activation in ven-tricular cardiomyocytes exposed to leptin (50 ng/ml) and the pos-

sible role of p115RhoGEF and p63RhoGEF in these responses.Leptin produced a robust hypertrophic response that was asso-ciated with RhoA/ROCK activation resulting in a significant in-crease in cofilin-2 phosphorylation and actin polymerization, thelatter evidenced by a reduction in the G/F actin ratio. These effectswere prevented by ginseng (10 �g/ml). The stimulation of RhoA/ROCK by leptin was associated with significantly increasedp115RhoGEF gene and protein expression and exchange activity,all of which were completely prevented by ginseng. The ability ofginseng to prevent leptin-induced activation of RhoA/ROCK wasfurther associated with diminished p38 MAPK activation and nu-clear translocation. These results demonstrate a potent inhibitoryeffect of ginseng against leptin-induced cardiac hypertrophy, aneffect associated with prevention of p115RhoGEF-RhoA/ROCK-dependent p38 MAPK activation.

IntroductionThe prevalence of obesity in North America has significantly

increased over the past 5 years (Luo et al., 2007) along with thedetrimental accompanying risks for the development of cardio-vascular disease (Bui et al., 2011). The underlying mechanisms

for obesity-associated cardiovascular disease are not well un-derstood although emerging evidence implicates a potential roleof leptin, a member of the family of peptides known as adipo-kines, which is produced by the ob (obesity) gene (Zhang et al.,1994) and has been reported by a number of investigators toproduce a direct hypertrophic effect on the heart (reviewed byKarmazyn et al., 2008). The primary source of leptin is whiteadipose tissue, and plasma levels of the peptide have beenshown to be closely correlated to the degree of adiposity (Maffeiet al., 1995). We have previously shown that the rat heartproduces leptin within the cardiomyocyte and also expressesleptin receptors, suggesting that the heart is a target for leptin’s

This work was supported by the Canadian Institutes of Health Research[Grant MOP 62764]; and the Ontario Ginseng Innovation and Research Consor-tium. M.M. was supported by the Ontario Ginseng Innovation and ResearchConsortium. M.K. holds a Tier 1 Canada Research Chair in Experimental Cardi-ology.

Article, publication date, and citation information can be found athttp://jpet.aspetjournals.org.

doi:10.1124/jpet.111.182600.

ABBREVIATIONS: RhoA, Ras homolog gene family, member A; ROCK, Rho-associated, coiled-coil containing protein kinase; �-SA, �-skeletalactin; GEF, guanine nucleotide exchange factor; F actin, filamentous actin; G actin, globular actin; ERK1/2, extracellular signal-related kinase 1/2;MAPK, mitogen-activated protein kinase; Ob-Rb, leptin receptor isoform b; PCR, polymerase chain reaction; PBS, phosphate-buffered saline;BSA, bovine serum albumin.

0022-3565/11/3393-746–756$25.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 339, No. 3Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics 182600/3728325JPET 339:746–756, 2011 Printed in U.S.A.

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effects, potentially in a paracrine/autocrine manner (Purdhamet al., 2004). It is noteworthy that plasma levels of leptin areelevated in patients with heart failure independently of obesity(Schulze et al., 2003).

Several molecular signaling pathways up-regulated by leptinin cardiac hypertrophy have been identified, namely the RhoA/ROCK (Ras homolog gene family, member A/Rho-associated,coiled-coil containing protein kinase), p38, and extracellularsignal-regulated kinase 1/2 (ERK1/2) mitogen-activated proteinkinase (MAPK) pathways (Fruhbeck, 2006; Zeidan et al., 2006,2008), all of which can be activated by leptin. A particularimportance of the RhoA/ROCK pathway is that its activation,and subsequent changes in actin dynamics, is critical for selec-tive p38 MAPK translocation into nuclei, thus initiating thehypertrophic process (Zeidan et al., 2006, 2008). Although wehave previously demonstrated the potent stimulation of theRhoA/ROCK pathway by leptin, the specific mechanism of ac-tivation has not yet been defined. Regulators of small G pro-teins such as RhoA include GTPase-activating proteins thathydrolyze GTP to GDP, thereby deactivating RhoA, guaninenucleotide dissociation inhibitors that sequester GDP-boundsmall G proteins in the cytoplasm, and guanine nucleotideexchange factors (GEFs) that catalyze the exchange of GDP toGTP resulting in activation of RhoA (Schmidt and Hall, 2002;Rossman et al., 2005; Bos et al., 2007). RhoA GEFs (RhoGEFs),specifically p115RhoGEF and p63RhoGEF, have been shown tobe involved in the up-regulation of RhoA/ROCK in cardiac hy-pertrophy in response to G protein-coupled, receptor-linked hy-pertrophic agonists such as endothelin-1 (Porchia et al., 2008)and angiotensin II (Guilluy et al., 2010).

Ginseng (genus Panax) is a medicinal herb that has beenused widely in Asia for more than 2000 years (Goldstein,1975; Lu et al., 2009). Its principal bioactive components areginsenosides, which are triterpene saponins and are consid-ered the main constituents responsible for ginseng’s medici-nal effects (Attele et al., 1999). Emerging understanding ofthe chemistry of ginseng and its potential therapeutic usehas resulted in increasing interest in western countries forthe use of ginseng as a pharmacological agent for the treat-ment of a number of diseases. With respect to the cardiovas-cular system, ginsenosides have been shown to inhibit thedevelopment of atherosclerosis (Li et al., 2011), hypertension(Jeon et al., 2000), and cardiac hypertrophy, the latter effectbeing seen in a number of experimental models (Jiang et al.,2007; Qin et al., 2008; Deng et al., 2010; Guo et al., 2011).Whether ginseng affects leptin-induced hypertrophy has notbeen demonstrated. Accordingly, in this study, we deter-mined the effect of North American ginseng (Panax quinque-folius) on leptin-induced cardiomyocyte hypertrophy andstudied the potential underlying mechanisms for these ef-fects. Our study centered on the possible modulatory effect ofginseng on the RhoA/ROCK pathway after leptin addition

and the role of GEFs. In addition, we studied the relationshipbetween RhoA and MAPK pathway activation.

Materials and MethodsTreatment and Experimental Groups. Neonatal ventricular

cardiomyocytes were isolated and cultured from 1- to 3-day-oldSprague-Dawley rats as described previously (Rajapurohitam et al.,2006). Cells were grown in fetal bovine serum medium for up to 48 hafter 24-h serum starvation. For cell size and Western blotting time-course experiments, cardiomyocytes were pretreated with the alco-holic extract of North American ginseng (P. quinquefolius). Extractswere prepared by Naturex (South Hackensack, NJ) using ginsengroots supplied from five different farms in Ontario, Canada, asdescribed previously (Guo et al., 2011) and studied at concentrationsof 0.1, 1, 10, and 100 �g/ml for up to 24 h in the presence or absenceof 3.1 nM (50 ng/ml) leptin (Sigma-Aldrich, Oakville, Ontario, Can-ada), a concentration representative of plasma levels in obese indi-viduals (Maffei et al., 1995). For all subsequent experiments, cellswere pretreated with a ginseng concentration of 10 �g/ml for 1 hin the presence or absence of leptin for up to 24 h. Treatmentdurations reflected the period of peak activation of the parameterunder study. The protocols for the use of animals were approvedby the University of Western Ontario Animal Care and Use Com-mittee and conformed to guidelines in the Guide for the Care andUse of Laboratory Animals (Institute of Laboratory Animal Re-sources, 1996) and the Canadian Council of Animal Care (Ottawa,Ontario, Canada).

Cell-Surface Area Measurement. Cardiomyocyte images weretaken with a microscope (Leica, Wetzlar, Germany) equipped with anInfinity 1 camera at magnification 100�. The surface area of aminimum of 50 cells per treatment group was measured using Sig-maScan Pro 5 software (Systat Software, Inc., San Jose, CA) andaveraged.

RNA Isolation, Reverse Transcription, and Real-Time Poly-merase Chain Reaction. RNA was collected from cultured andtreated neonatal ventricular cardiomyocytes using TRIzol reagent(Invitrogen, Carlsbad, CA), according to the manufacturer’s instruc-tions, and reverse-transcribed to cDNA for real-time polymerasechain reaction (PCR) analysis of �-skeletal actin (�-SA), myosinheavy chain �, p115RhoGEF, and p63RhoGEF. In brief, cDNA wassynthesized from 4 �g of total RNA using random primers (Invitro-gen) and Moloney murine leukemia virus reverse transcriptase (In-vitrogen) following the manufacturer’s protocol. The reaction wasperformed with a SYBR Green Master Mix (Applied Biosystems,Foster City, CA), and the gene products were quantified with a DNAEngine Opticon 2 thermal cycler (MJ Research, Watertown, MA).Primer sequences (Invitrogen) for the genes of interest are listed inTable 1. PCR cycle conditions involved 40 cycles of denaturation at95°C for 30 s, followed by annealing at 60°C for 30 s, and finally byelongation at 72°C for 30 s. The housekeeping gene 18S was mea-sured and quantified to normalize cDNA levels.

[3H]Leucine Incorporation Measurement. Leucine incorpora-tion was performed as described previously (Zeidan et al., 2006) toanalyze protein synthesis under different experimental conditions.Cardiomyocytes were cultured in 24-well Primaria culture plates for48 h in serum medium, followed by 24-h serum starvation. Leptin

TABLE 1Gene-specific primer sequences of analyzed genes of interest

Gene Forward Primer Sequence Reverse Primer Sequence

�-Skeletal actin 5�-CACGGCATTATCACCAACTG-3� 5�-CCGGAGGCATAGAGAGACAG-3�Myosin heavy chain 5�-CATCACCGGAGAATCCGGAGC-3� 5�-CTATTGAGGCCACAGTCGTC-3�p115RhoGEF 5�-TAGAGGACTTCCGCTCCAAA-3� 5�-CAGTGACCACAGCAGCACTT-3�p63RhoGEF 5�-TATGTGGACGACTTGGGACA-3� 5�-TGATGAACAGCTGAGCCAAC-3�18S 5�-GTAACCCGTTGAACCCCATT-3� 5�-CCATCCAATCGGTAGTAGCG-3�

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was administered with or without ginseng pretreatment in the pres-ence of 2 �Ci of [3H]leucine for 24 h. Myocytes were washed thefollowing day with ice-cold PBS, and proteins were precipitated with5% trichloroacetic acid for 30 min on ice followed by two washes ofice-cold trichloroacetic acid (5%). Protein precipitates were resus-pended in 0.5 N NaOH and neutralized with 0.5 N HCl. Totalradioactivity was measured by liquid scintillation counting.

Isolation of Cytosolic-Enriched and Membrane Fractions.Cytosolic-enriched and membrane fractions from treated cell lysateswere prepared using differential centrifugation as described previ-ously (Zeidan et al., 2008). In brief, cell lysates were collected andhomogenized in an ice-cold buffer containing 20 mM Tris-HCl, 2 mMEDTA, 137 mM NaCl, 1 mM sodium orthovanadate, 2 mM sodiumpyrophosphate, 10% glycerol, 1 mM 4-(2-aminorthyl)-benzenesulfo-nyl fluoride, and 10 mg/ml leupeptin (buffer A). After clarification ofthe homogenate by centrifugation at 750g for 20 min at 4°C, thecollected lysate was further centrifuged at 10,000g for 20 min at 4°C,and the cytosolic-enriched fraction (supernatant) was obtained. Theremaining pellet was resuspended in a second ice-cold buffer B(buffer A with 2% SDS) and kept on ice to be used as the nuclear-containing membrane fraction.

Western Blotting. Total cellular lysates were collected using alysis buffer and protease cocktail inhibitor mixture as describedpreviously (Zeidan et al., 2006, 2008) for the measurement of pro-teins of interest. Proteins were loaded equally on 7.5, 10, or 15% SDSgels as appropriate after protein quantification via dye reagent (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’sinstructions. For time-course experiments, ventricular cardiomyo-cytes were treated for 5, 10, 15, 30, or 60 min with leptin (50 ng/ml)in the presence or absence of ginseng (10 �g/ml). For the quantifica-tion of cofilin-2 phosphorylation, cells were pretreated with ginsengfor 1 h followed by administration of leptin for 10 min. For alladditional protein measurements, cells were pretreated with ginsengfor 1 h in the presence or absence of leptin for 24 h. The primaryantibodies and respective dilutions used in this study include total(1:1000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA)and phosphorylated (Thr180/Tyr182) p38 forms (1:1000 dilution;Cell Signaling Technology, Danvers, MA), total (1:1000 dilution;Santa Cruz Biotechnology, Inc.) and phosphorylated ERK1/2(Thr202/Tyr204) forms (1:1000 dilution; Cell Signaling Technology),actin (1:1000 dilution; Cytoskeleton Inc., Denver, CO), p115RhoGEF(1:250 dilution; Santa Cruz Biotechnology, Inc.), p63RhoGEF (1:200dilution; Santa Cruz Biotechnology, Inc.), and phosphorylated (1:1000 dilution; Santa Cruz Biotechnology, Inc.) and total (1:1000dilution; Millipore Corporation, Billerica, MA) cofilin-2. Goat anti-rabbit IgG and goat anti-mouse IgG horseradish peroxidase conju-gate (Bio-Rad Laboratories) were used at 1:5000 dilution, and don-key anti-goat IgG horseradish peroxidase conjugate (Santa CruzBiotechnology, Inc.) was used at 1:10,000 dilution as appropriate.�-Actin (1:1000 dilution; Cytoskeleton Inc.) and proliferating cellnuclear antigen (1:1000 dilution) were used for cytosolic and nuclearloading controls, respectively. Spot densitometry using FluorChem(Alpha Innotech Corporation, Santa Clara, CA) software was per-formed to quantify protein.

GST-RhoG17A Bead Preparation. GST-RhoG17A beads wereprepared as described previously (García-Mata et al., 2006; Kakiash-vili et al., 2009). The nucleotide-free RhoG17A cDNA construct mu-tant was generously provided by Dr. Katalin Szaszi (St. Michael’sHospital, Toronto, ON, Canada) and Dr. Keith Burridge (Universityof North Carolina, Chapel Hill, NC).

Coimmunoprecipitation of p115RhoGEF. Active p115RhoGEFfrom cells treated with leptin in the presence or absence of leptin wereimmunoprecipitated with the nucleotide-free (does not bind to GTP orGDP) GST-RhoG17A-prepared beads, which has a high affinity for activeRhoGEFs, including p115RhoGEF, as described previously (García-Mataet al., 2006; Kakiashvili et al., 2009).

Immunofluorescence. Cells were prepared for immunofluores-cence on collagen-coated (3 �l of collagen/1 ml of PBS A) glass

coverslips and incubated at 37°C for a minimum of 30 min. Cellswere allowed to attach to prepared coverslips in serum medium for24 h followed by serum-free medium starvation for an additional 24 hbefore appropriate treatment. Immunofluorescence measurementsfrom cells administered leptin were performed for 3 h for total p38and total ERK1/2, for 10 min for p115RhoGEF and RhoA, and for24 h for globular (G)/filamentous (F) actin with or without ginsengpretreatment.

Total p38, Total ERK1/2, p115RhoGEF, and RhoA Immuno-fluorescence. Cells were fixed with 2:5 acetone/methanol for 1 h at4°C followed by permeabilization of cells for 15 min with 0.2% (v/v)Triton X-100 and blocking with blocking solution (1% BSA, 0.1%Triton X-100) for 1 h. Cells were incubated with the primary anti-body of interest (1:100 dilution) in 2% BSA in PBS A overnight at4°C. Cells were subsequently probed with the appropriate secondaryantibody, IgG anti-mouse Alex Fluor-488 (Invitrogen) or IgG anti-rabbit Alex Fluor-596 (Invitrogen) (1:250 dilution) in 2% BSA in PBSA for 1 h at room temperature under light-free conditions. Fordetection of the nucleus, cells were incubated with Hoechst dye for 30min before mounting on microscope slides (VWR, West Chester, PA)for image capture using a Carl Zeiss Inc. (Jena, Germany) invertedfluorescence microscope at magnification 630�.

G and F Actin Immunofluorescence. Cells were prepared asdescribed previously (Albinsson et al., 2004). Cells were fixed with3.7% (w/v) paraformaldehyde in PBS A for 1 h followed by a similarprotocol for permeabilization and blocking as indicated above. Todetect G and F actin, cells were incubated with 1 �g/ml phalloidin-fluorescein isothiocyanate, 10 �g/ml deoxyribonuclease I, and TexasRed conjugate in 2% BSA with 0.1% Triton X-100 in PBS A for 1 h atroom temperature under light-free conditions. Likewise, Hoechst dyewas used to detect the nucleus, and glass coverslips were subse-quently mounted onto microscope slides (VWR) for visualization ofG/F actin. All immunofluorescence images shown under Results arerepresentative of a minimum of three independent experiments.

Measurement of p115RhoGEF and RhoA Colocalization. Co-localization of p115RhoGEF and RhoA was measured from mergedimmunofluorescence images detecting p115RhoGEF and RhoA un-der different experimental conditions using a built-in colocalizationplug-in (Li et al., 2004) of ImageJ (National Institute of MentalHealth, Bethesda, MD), which is quantitatively represented by Pear-son’s correlation coefficient (Rr).

Measurement of RhoA Activity. RhoA activity, measured byRhoA-GTP levels, was quantified using the RhoA G-LISA ActivityBiochem Assay kit (Cytoskeleton Inc.) according to the manufac-turer’s protocol. Measurements were performed using a SpectraMaxM5 (Molecular Devices, Sunnyvale, CA) plate reader at an absor-bance of 490 nm.

p115RhoGEF Activity Assay. Measurement of immunopre-cipitated p115RhoGEF activity was measured according to themanufacturer’s protocol using the RhoGEF Biochem ExchangeAssay (Cytoskeleton Inc). Fluorescence was measured using aSpectraMax M5 (Molecular Devices) plate reader at an excitationof 360 nm and emission of 440 nm.

G/F Actin Measurement. G and F actin were isolated usingultracentrifugation as described previously (Albinsson et al., 2004).In brief, cell lysates were collected and homogenized at 37°C in alysis and F actin stabilizing buffer (50 mM PIPES, 50 mM NaCl, 5mM MgCl2, 5 mM EGTA, 1 mM ATP, 5% glycerol, 0.1% Nonidet-P40,0.1% Triton X-100, 0.1% Tween 20, 0.1% �-mercaptoethanol, 1:100protease inhibitor cocktail, and 0.0001% anti-foam). Cell lysateswere ultracentrifuged at 100,000g at 30°C for 1 h, and the superna-tant (soluble, G actin) was collected at diluted 1:2 in Laemmli buffer.The remaining pellet (F actin) was resuspended in ice-cold distilledwater with 1 �M cytochalasin D. The resuspended pellet was kept onice for 45 to 60 min to dissociate the F actin and was then diluted 1:4with Laemmli buffer. Protein concentrations were then quantified,equalized, and loaded onto 12.5% bis-acrylamide gels for Western

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blotting. Membranes were probed with anti-actin antibody (Cyto-skeleton Inc.) and quantified using spot densitometry.

Statistics. Data were analyzed with one-way analysis of variancefollowed by a post hoc Student’s t test. P values of �0.05 wereconsidered statistically significant.

ResultsGinseng Inhibits Leptin-Induced Cardiomyocyte

Hypertrophy. To first determine an appropriate concentra-tion for studying the effects of ginseng on leptin-inducedcardiac hypertrophy, cardiomyocytes were subjected to in-creasing concentrations of ginseng (0.1, 1, 10, and 100 �g/ml)for 1 h before the addition of leptin for a total incubation timeof 24 h (Fig. 1, top). As shown in Fig. 1 (middle), leptininduced a significant increase (p � 0.05) in cell size, whichwas attenuated by ginseng in a concentration-dependentmanner. A concentration of 10 �g/ml ginseng was used for allsubsequent experiments because this represented the lowestconcentration that completely abrogated the hypertrophicresponse to leptin (Fig. 1, middle).

An increase in cell-surface area in leptin-treated cells after24 h was additionally associated with a significant increase

(p � 0.05) in protein synthesis as indicated by increased[3H]leucine incorporation (Fig. 1, bottom left) and myosinheavy chain � (Fig. 1, bottom center) and �-SA (Fig. 1,bottom right) gene expression, as quantified by real-timePCR. As shown in Fig. 1, ginseng alone had no effect onany parameter.

Ginseng Inhibits Leptin-Induced RhoA Activation,Cofilin-2 Phosphorylation (Inactivation), and the De-crease in G/F Actin Ratio. RhoA activation was measuredafter 10 min of leptin administration in the presence orabsence of ginseng. As demonstrated in Fig. 2A, ginseng sig-nificantly inhibited (p � 0.05) leptin-induced RhoA activation,returning RhoA-GTP levels to control values, whereas ginsengalone had no effect on its own. Activation of the RhoA/ROCKpathway was additionally indirectly measured by quantifyingphosphorylated cofilin-2 (inactivated form) by Western blotting.Cofilin-2 is a ubiquitous enzyme responsible for depolymerizingF to G actin, thereby regulating cellular actin dynamics. Achange in this ratio favoring a higher F-to-G actin content as aresult of RhoA activation and subsequent cofilin-2 phosphory-lation (inactivation) has been previously demonstrated to rep-resent a key mechanism underlying the hypertrophic effects of

Fig. 1. Ginseng inhibits leptin-induced increase in cell-surface area, [3H]leucine incorporation, and expression of the gene markers of cardiachypertrophy, �-skeletal actin, and myosin heavy chain. Top, micrographs show representative images of neonatal ventricular cardiomyocytes with orwithout treatment with increasing ginseng concentrations (0.1, 1.0, 10, and 100 �g/ml, respectively) in the presence (top row) or absence (bottom row)of leptin (3.1 nM) pretreatment. Middle, surface area. Bottom, [3H]leucine incorporation (left) and expression of myosin heavy chain (MHC; center) and�-skeletal actin (right) with different treatments. Data represent means � S.E.M. n � 8 to 10 for surface area, n � 6 for leucine incorporation, andn � 6 to 8 for molecular markers of hypertrophy. �, p � 0.05 versus control; †, p � 0.05 versus leptin. Con, control; Lep, leptin; Gin, ginseng.

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leptin (Zeidan et al., 2006). The results, as shown in Fig. 2B,reveal a significant (25%) increase in p-cofilin-2 in cells admin-istered leptin (p � 0.05), which was attenuated and returned tocontrol levels in the presence of ginseng although ginseng alonehad no significant effect on p-cofilin-2 or RhoA-GTP levels.

To further characterize the effects of ginseng on leptin-induced RhoA/ROCK pathway activation, the G/F actin ratiowas assessed by Western blotting of isolated G and F actinfractions (Fig. 3A) and visualization using immunofluores-cence (Fig. 3C) after 24 h. In leptin-treated cells, the G/Factin ratio was decreased as measured by quantification of Gactin (supernatant) and F actin (pellet) using Western blot-ting (Fig. 3A), whereas pretreatment with ginseng restoredthis ratio to control values (Fig. 3B). This was similarlyobserved in the representative immunofluorescence images(Fig. 3C, Lep column) of leptin-treated cells as depicted by alighter red staining of G actin and intensified green stainingof F actin, which was returned to control conditions by pre-treatment with ginseng (Fig. 3C, LepGin column). Treat-ment with ginseng alone had no direct effect on the G/F actindynamics.

Ginseng Inhibits Leptin-Induced p38 and ERK1/2MAPK Phosphorylation. Leptin significantly induced bothp38 (Fig. 4A) and ERK1/2 (Fig. 5A) phosphorylation as earlyas 5 min after addition, with maximum activation seen at 15min followed by values returning to control by 30 min. Pre-treatment with ginseng inhibited leptin-induced p38 (Fig.4A) and ERK1/2 (Fig. 5A) activation at all time points al-though ginseng had no direct effect on its own.

Ginseng Inhibits Leptin-Induced p38 Nuclear Trans-location. Leptin induced a significant increase (p � 0.05) inp38 expression in the nuclear-containing membrane fraction(Fig. 4C), which was complemented by a significant decrease(p � 0.05) in cytosolic p38 levels, indicative of nuclear trans-location in p38 in leptin-treated cells (Fig. 4B). Nucleartranslocation of p38 was further visualized by immunofluo-rescence (Fig. 4D) where total p38, indicated by red fluores-cence, was much more centralized in the nuclear region ofleptin-treated cells. The ability of leptin to induce p38 trans-location was significantly inhibited by ginseng. As summa-rized in Fig. 5, B–D, leptin had no effect on ERK1/2 nucleartranslocation.

Fig. 2. Ginseng inhibits leptin-induced RhoA activation and phosphorylation (inactivation) of cofilin-2. A, RhoA-GTP (activated RhoA) levels. B,Western blots and densitometric values for phosphorylated cofilin-2. Data represent means � S.E.M. n � 6. �, p � 0.05 versus control; †, p � 0.05versus leptin. Con, control; Lep, leptin; Gin, ginseng.

Fig. 3. Ginseng inhibits leptin-induced decrease in G/F actin. A, Western blots for actin dynamics with respect to G actin in supernatant (S) and Factin in pellet fraction (P) with different treatments. B, densitometric values. C, representative fluorescence images of cardiomyocytes. For thesestudies, cells were fixed on collagen-coated glass coverslips, and G actin (first row) and F actin (second row) were visualized with deoxyribonucleaseI Texas Red conjugate and phalloidin-fluorescein isothiocyanate, respectively. Hoechst staining was used to detect nuclei, whereas the overlay depictsall three stains merged. In B, data represent means � S.E.M. n � 6 to 9. �, p � 0.05 versus control; †, p � 0.05 versus leptin. Con, control; Lep, leptin;Gin, ginseng.

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Ginseng Inhibits Leptin-Induced Increase inp115RhoGEF Protein and Gene Expression. The effectsof ginseng on leptin-induced RhoGEF activation was first de-termined by measurement of p115RhoGEF and p63RhoGEFgene expression through real-time PCR and protein expressionusing Western blotting (Fig. 6). A 6-fold increase (p � 0.05) inp115RhoGEF gene expression after 24 h was observed in leptin-treated cells, which was abolished in the presence of ginseng(Fig. 6A). Likewise, pretreatment with ginseng significantlyinhibited leptin-induced increase in p115RhoGEF protein ex-pression (Fig. 6B), whereas ginseng alone had no direct effect oneither parameter. Compared with p115RhoGEF, neither leptinnor ginseng exerted any effect on p63RhoGEF gene (Fig. 6C) orprotein (Fig. 6D) expression.

Ginseng Inhibits Leptin-Induced p115RhoGEF Mem-brane Translocation with RhoA Colocalization andp115RhoGEF Activity. Several studies have shown mem-brane translocation with RhoA colocalization as a component

of p115RhoGEF activation (Kozasa et al., 1998; Rossman etal., 2005; Aittaleb et al., 2010). Initial time-course experi-ments with leptin treatment revealed translocation andRhoA colocalization as early as 5 min after leptin addition(data not shown). Our previous studies have correspondinglyindicated activation of RhoA as early as 10 min (Zeidan et al.,2006, 2008). Consequently, cardiomyocytes were treated withginseng before the administration of leptin for 10 min andprepared for immunofluorescence. As depicted in Fig. 7A(row c), p115RhoGEF membrane translocation with RhoAcolocalization was observed in leptin-treated cells as indi-cated by evident yellow fluorescence at the cardiomyocyteborder. In addition, isolation of the colocalized pixels (Fig.7A, row d) revealed colocalization of p115RhoGEF and RhoAin leptin-treated cells, which was inhibited by pretreatmentwith ginseng. Treatment with ginseng alone was without anyeffect (Fig. 7A, Gin column).

Using the same treatment protocol, cell lysates were col-

Fig. 4. Ginseng inhibits leptin-induced phosphorylation and nuclear translocation of p38. A, Western blots and corresponding densitometric valuesfor phosphorylated p38. B and C, Western blots and corresponding densitometric values for p38 MAPK content in cytoplasmic-enriched (B) andnuclear-containing (C) membrane fractions. D, representative immunofluorescence images measuring total p38 indicated by red staining (IgG AlexFluor-596; left), Hoechst nuclear staining (center), and merged staining (right). Data in A–C represent means � S.E.M. n � 5 to 8. �, p � 0.05 versuscontrol; †, p � 0.05 versus leptin. Con, control; Lep, leptin; Gin, ginseng. Minutes indicate time after leptin addition.

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lected at 10 min after leptin administration and incubatedwith GST-RhoG17A, a nucleotide-free RhoA mutant that hasa high affinity for active RhoGEFs (García-Mata et al., 2006),to immunoprecipitate p115RhoGEF. Measurement of acti-vated p115RhoGEF from leptin-treated cells in the presenceor absence of ginseng to facilitate guanine nucleotide ex-change activity by purified small GTPase RhoA was thenperformed. p115RhoGEF activation was increased 2-fold inleptin-treated cells compared with control (Fig. 7B). Partic-ularly of interest, this observed increase in guanine nucleo-tide exchange activity was significantly abolished in cellspretreated with ginseng. p115RhoGEF activity was unaf-fected by ginseng alone.

DiscussionIncreasing evidence from a number of laboratories has

demonstrated that leptin exerts a direct hypertrophic effect

on cardiomyocytes (Rajapurohitam et al., 2003; Xu et al.,2004; Madani et al., 2006; Hou et al., 2010) and intact myo-cardium in vivo (Abe et al., 2007). In addition, blocking leptinreceptors attenuates remodeling and heart failure in thepostinfarcted rat heart (Purdham et al., 2008). Although theprecise mechanism of action of leptin accounting for its hy-pertrophic effect is not completely understood, we have pre-viously suggested that activation of the RhoA/ROCK path-way plays a critical role in mediating leptin-induced cardiachypertrophy, possibly through activation and subsequent nu-clear translocation of p38 MAPK, the latter effect dependenton alterations in actin dynamics (Zeidan et al., 2006, 2008).In the present study, we assessed the effects of North Amer-ican ginseng (P. quinquefolius) on leptin-induced cardiac hy-pertrophy based on emerging evidence that ginseng exertsanti-hypertrophic effects in a varied number of experimentalmodels (Jiang et al., 2007; Qin et al., 2008; Deng et al., 2010)

Fig. 5. Ginseng inhibits leptin-induced ERK1/2 phosphorylation with no effect on nuclear translocation. A, Western blots and correspondingdensitometric values for phosphorylated ERK1/2. B and C, Western blots and corresponding densitometric values for ERK1/2 MAPK content incytoplasmic-enriched (B) and nuclear-containing (C) membrane fractions. D, representative immunofluorescence images measuring total ERK1/2indicated by red staining (IgG Alex Fluor-596; left), Hoechst nuclear staining (center), and merged staining (right). Data in A–C represent means �S.E.M. n � 5 to 8. �, p � 0.05 versus control; †, p � 0.05 versus leptin. Con, control; Lep, leptin; Gin, ginseng. Minutes indicate time after leptinaddition.

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and also reduces the severity of heart failure in rats subjectedto chronic coronary artery ligation (Guo et al., 2011). Wehypothesized that ginseng would attenuate leptin-inducedcardiac hypertrophy by attenuating RhoA/ROCK activationafter leptin administration. Our study shows for the first timethat ginseng is a potent inhibitor of leptin-induced hypertrophy,and indeed this occurs through a mechanism associated withthe abrogation of RhoA/ROCK activation. Moreover, we identi-fied a potential key role of p115RhoGEF in facilitating RhoA/ROCK-dependent p38 and ERK1/2 MAPK pathway activationin leptin-induced cardiac hypertrophy and, critically, the abilityof ginseng to target p115RhoGEF as a mechanism for its abilityto prevent RhoA/ROCK activation, thus preventing cardiomyo-cyte hypertrophy.

Leptin-induced hypertrophy was manifested by an in-crease in cell-surface area, a 2-fold increase in [3H]leucineincorporation, and increased expression of two molecular hy-pertrophic gene markers, �-SA and myosin heavy chain, allof which were significantly attenuated by ginseng. Moreover,the hypertrophic effect of leptin was associated with an acti-vation of the RhoA/ROCK pathway as exhibited by an in-crease in RhoA-GTP levels, in support of our previous find-ings (Zeidan et al., 2006, 2008). RhoA/ROCK activation wasfurther demonstrated by increased phosphorylation (inacti-vation) of cofilin-2, a ubiquitous enzyme downstream of RhoAthat depolymerizes actin, resulting in a decrease in the G/Factin ratio. We previously reported that leptin-induced RhoA/ROCK activation was critical for p38 MAPK, but not ERK1/2MAPK nuclear translocation and the subsequent hypertro-phic response, a response probably dependent on the changesin actin dynamics (Zeidan et al., 2008). Together, the abilityof ginseng to completely prevent activation of the RhoA/ROCK pathway, p38 translocation, and the associated hyper-trophic response strongly suggests that inhibition of RhoA/

ROCK represents a key mechanism for the anti-hypertrophiceffect of ginseng as seen in our study.

We next assessed the potential target mediating the ability ofginseng to inhibit RhoA/ROCK activation. Our study centeredprimarily on the potential role of RhoGEFs, which are criticalfor downstream RhoA activation (Rossman et al., 2005). How-ever, the role of RhoGEFs in the cardiac hypertrophic programhas not been studied extensively. Although a number of Rho-GEFs have been identified, p115RhoGEF and p63RhoGEFwere considered of particular interest because their expressionhas been demonstrated in cardiovascular tissues including theheart (Souchet et al., 2002; Porchia et al., 2008; Wuertz et al.,2010) and their activation has been shown in response to vari-ous hypertrophic agonists including angiotensin II (Guilluy etal., 2010) and endothelin-1 (Porchia et al., 2008). Our studyshows for the first time that leptin induced a significant in-crease in p115RhoGEF gene and protein expression withoutaffecting p63RhoGEF. We next studied whether increased ex-pression of p115RhoGEF is also associated with increased GEFactivity. Activation of p115RhoGEF involves receptor-mediatedmembrane translocation and colocalization with RhoA (Kozasaet al., 1998; Rossman et al., 2005; Aittaleb et al., 2010). Ourresults revealed membrane translocation of p115RhoGEF withRhoA colocalization after 10 min of leptin stimulation and alsodemonstrated an increase in p115RhoGEF activity.

The ability of ginseng to inhibit p115RhoGEF activationsuggests this as a target for the ability of ginseng to inhibitleptin-induced cardiomyocyte hypertrophy, and it is reason-able to assume that p115RhoGEF activation by leptin andinhibition by ginseng represent the main regulatory sitesinfluencing subsequent p38 and ERK1/2 phosphorylation ortranslocation of the former. However, a limitation of our studyis that it does not establish a direct causal relationship betweenp115RhoGEF activation by leptin and stimulation of the RhoA

Fig. 6. Ginseng inhibits leptin-inducedup-regulation of p115RhoGEF but notp63RhoGEF protein and gene expres-sion. Gene expression (A and C) and pro-tein levels (B and D) for p115RhoGEFand p63RhoGEF in cardiomyocytestreated with leptin in the absence or pres-ence of ginseng are shown. Data repre-sent means � S.E.M. n � 8. �, p � 0.05versus control; †, p � 0.05 versus leptin.Con, control; Lep, leptin; Gin, ginseng.

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pathway. Additional studies are required to confirm this, par-ticularly by determining the effect of p115RhoGEF down-regu-lation on the ability of leptin to activate RhoA.

As reported previously (Zeidan et al., 2008), leptin inducesphosphorylation of both p38 and ERK1/2 although only theformer is translocated into nuclei, thus suggesting that phos-phorylation is not a precondition for nuclear transport. Al-though the precise mechanisms for selective p38 transloca-tion into nuclei are not known, the phenomenon is possiblymediated by changes in actin dynamics because the effect isprevented by latrunculin B, which prevents actin polymer-ization as a result of RhoA activation (Zeidan et al., 2008).The selective translocation of p38 into nuclei after leptinaddition also helps to explain our initial finding that phar-macological inhibition of p38, but not ERK1/2, prevents lep-tin-induced hypertrophy (Rajapurohitam et al., 2003). Thespecific mechanism of p115RhoGEF-RhoA/ROCK-dependentp38 and ERK1/2 MAPK inhibition by ginseng, however, stillremains unclear. The chemical structures of ginsenosides,which are triterpene saponins and are considered the pri-mary active constituents contributing to the medicinal effectsof ginseng (Attele et al., 1999), have been compared withsteroidal structures such as estrogen. Leung et al. (2007)

alluded to the competitive binding of the specific ginsenosideRb1 (the predominant ginsenoside of North American gin-seng), selective to the estrogen receptor �, where it wastheorized to be engulfed with the bound ginsenoside throughendocytosis into the cytoplasm in which it translocates intothe nucleus to bind to transcription factors eliciting its effectsas an antiangiogenic factor. Indeed, we previously haveshown that estrogen (as 17�-estradiol) exerts a pro-hypertro-phic effect on cultured ventricular myocytes at very low (1pM) concentrations but has antihypertrophic actions at nano-molar concentrations (Kilic et al., 2009). The observation ofthe ability of ginsenoside Rb1 to bind to estrogen receptorsis intriguing and raises the question of potential gender-specific effects of ginseng. Although it seems that estrogenreceptors are expressed in ventricular myocytes of bothmale and female rats, nonetheless, potential gender-dependent effects of ginseng are deserving of furtherstudy. In the present study, an alcoholic ginseng extractcontaining a large number of ginsenosides was used, and,consequently, the specific ginsenoside(s) responsible forthe observed inhibition of leptin-induced effects currentlycannot be identified.

Another potential mechanism for the observed inhibition of

Fig. 7. Ginseng inhibits leptin-inducedp115RhoGEF colocalization with RhoAand guanine nucleotide exchange activ-ity. A, representative immunofluores-cence images of p115RhoGEF transloca-tion and colocalization with RhoA afteradministration and incubation with lep-tin for 10 min in the presence or absenceof ginseng. Row a shows p115RhoGEF(IgG Alex Fluor-594) stained in red; row brepresents RhoA (IgG Alex Fluor-488)stained in green; row c represents mergedimages of p115RhoGEF and RhoA; row dshows isolated colocalized pixels ofp115RhoGEF and RhoA; and row e rep-resents isolated colocalized pixels ofmerged images of p115RhoGEF andRhoA. The Pearson’s correlation coeffi-cient (Rr) as calculated by the colocaliza-tion plug-in of ImageJ software is repre-sented at the bottom right of row d.B, p115RhoGEF activity determined byfluorescence assay at an excitation of 360nm and emission at 440 nm. The buffergroup depicts results using exchange buf-fer alone plus RhoA. Con, control; Lep,leptin; Gin, ginseng.

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these pathways by ginseng may occur extracellularly at thelevel of the leptin-receptor long isoform (Ob-Rb), consideredthe principal receptor mediating the biological effects of lep-tin (reviewed by Villanueva and Myers, 2008), potentially asa result of early binding and antagonism of Ob-Rb, conse-quently down-regulating further leptin signaling (Fig. 8).Although it was not an aim of the current study, competitivebinding studies between leptin and ginseng at the Ob-Rb aredeserving of further investigation.

In conclusion, our results show for the first time thatginseng markedly attenuates the direct hypertrophic effect ofleptin. Moreover, our results are strongly supportive of theconcept that this effect of ginseng against leptin-inducedhypertrophy occurs via inhibition of p115RhoGEF expressionand activity, thus abrogating RhoA/ROCK activation. Thelatter results in diminished p38 MAPK phosphorylation andtranslocation into nuclei, thus attenuating transcription andreducing the hypertrophic response. As noted previously,further work is necessary to demonstrate a precise causalrelationship between leptin-induced p115RhoGEF activationand subsequent activation of downstream pathways. In ad-dition, because ginseng exerts numerous and diverse effectson the heart (reviewed by Karmazyn et al., 2011), the contri-bution of other pathways as targets for the antihypertrophiceffects of ginseng cannot be excluded. Moreover, it is impor-tant to point out that the present study was carried out usingneonatal ventricular myocytes and therefore extrapolation ofthese results to the adult myocardium, particularly under invivo conditions, should be done cautiously. Although the roleof leptin in cardiac pathology still remains to be fully deter-mined, our overall results suggest that ginseng could be aneffective therapeutic approach aimed at mitigating potentialdeleterious cardiovascular complications associated with hy-perleptinemia, particularly those involving a cardiac hyper-trophic phenotype.

Acknowledgments

We thank Dr. Katalin Szaszi (St. Michael’s Hospital, University ofToronto, Toronto, Canada) for providing the RhoG17A construct andreference to appropriate protocols; Dr. Keith Burridge (University ofNorth Carolina, Chapel Hill, NC), for permission to use the con-struct; and the laboratory of Dr. Peter Chidiac (The University ofWestern Ontario) for providing the expertise, reagents, and equip-ment to perform these assays.

Authorship Contributions

Participated in research design: Moey, Rajapurohitam, Zeidan,and Karmazyn.

Conducted experiments: Moey, Rajapurohitam, and Zeidan.Contributed new reagents or analytic tools: Moey.Performed data analysis: Moey and Rajapurohitam.Wrote or contributed to the writing of the manuscript: Moey,

Zeidan, and Karmazyn.

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Address correspondence to: Dr. Morris Karmazyn, Department of Physiologyand Pharmacology, The University of Western Ontario, London, Ontario, CanadaN6A 5C1. E-mail: Morris.Karmazyn@schulich.uwo.ca

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