rad gtpase deficiency leads to cardiac hypertrophy

Rad GTPase Deficiency Leads to Cardiac HypertrophyLin Chang, MD, PhD*; Jifeng Zhang, MS*; Yu-Hua Tseng, PhD; Chang-Qing Xie, MD, PhD;

Jacob Ilany, MD; Jens C. Brüning, PhD; Zhongcui Sun, MD; Xiaojun Zhu, MD;Taixing Cui, MD, PhD; Keith A. Youker, PhD; Qinglin Yang, MD, PhD;Sharlene M. Day, MD; C. Ronald Kahn, MD; Y. Eugene Chen, MD, PhD

Background—Rad (Ras associated with diabetes) GTPase is the prototypic member of a subfamily of Ras-related smallG proteins. The aim of the present study was to define whether Rad plays an important role in mediating cardiachypertrophy.

Methods and Results—We document for the first time that levels of Rad mRNA and protein were decreased significantlyin human failing hearts (n�10) compared with normal hearts (n�3; P�0.01). Similarly, Rad expression was decreasedsignificantly in cardiac hypertrophy induced by pressure overload and in cultured cardiomyocytes with hypertrophyinduced by 10 �mol/L phenylephrine. Gain and loss of Rad function in cardiomyocytes significantly inhibited andincreased phenylephrine-induced hypertrophy, respectively. In addition, activation of calcium-calmodulin–dependentkinase II (CaMKII), a strong inducer of cardiac hypertrophy, was significantly inhibited by Rad overexpression.Conversely, downregulation of CaMKII� by RNA interference technology attenuated the phenylephrine-inducedhypertrophic response in cardiomyocytes in which Rad was also knocked down. To further elucidate the potential roleof Rad in vivo, we generated Rad-deficient mice and demonstrated that they were more susceptible to cardiachypertrophy associated with increased CaMKII phosphorylation than wild-type littermate controls.

Conclusions—The present data document for the first time that Rad is a novel mediator that inhibits cardiac hypertrophythrough the CaMKII pathway. The present study will have significant implications for understanding the mechanismsof cardiac hypertrophy and setting the basis for the development of new strategies for treatment of cardiac hypertrophy.(Circulation. 2007;116:2976-2983.)

Key Words: cardiomyopathy � genes � heart diseases � hypertrophy � natriuretic peptides

Cardiac hypertrophy is an important adaptive growthresponse to facilitate an increase in myocardial contrac-

tility. Although sustained hypertrophy is an initial compen-satory mechanism to preserve cardiac function, it is anindependent major risk factor for cardiac morbidity andmortality.1 Emerging data suggest that pathological structuralchanges in the heart are induced in part by small G proteins(also called GTPases).2 In the cardiovascular system, small Gproteins are implicated in regulation of endothelial function3;vascular smooth muscle cell (VSMC) contraction, prolifera-tion, and migration; and cardiac hypertrophy.4

Clinical Perspective p 2983

Rad (Ras associated with diabetes) GTPase, a 33- to35-kDa protein, was originally identified from skeletal mus-

cle of patients with type 2 diabetes mellitus.5,6 Rad and itsclosely related GTPases (Gem, Kir, Rem, and Rem2) form anRGK subfamily in the Ras family of small GTPases.2 To date,it has been reported that Rad can inhibit insulin-stimulatedglucose uptake in myocyte and adipocyte cell lines.7 Inaddition, RGK GTPases are able to function as potentinhibitors of voltage-dependent calcium channels by directlybinding to their �-subunit.8 Moreover, Rad and Gem canmodulate cytoskeleton remodeling through the Rho/Rho-kinase pathway.9 Thus, Rad is a key GTPase that has manyimportant biological functions.

In recent studies designed to explore the role of Rad in thecardiovascular system at large, we demonstrated that Radexpression was upregulated in VSMCs during vascular lesionformation. Overexpression of Rad significantly inhibited the

Received April 2, 2007; accepted September 25, 2007.From the Cardiovascular Center (L.C., J.Z., C.-Q.X., T.C., S.M.D., Y.E.C.), Department of Internal Medicine, University of Michigan Medical Center,

Ann Arbor, Mich; Joslin Diabetes Center (Y.-H.T., J.I., J.C.B., C.R.K.), Harvard Medical School, Boston, Mass; Institute of Molecular Medicine (Z.S.,X.Z.), Peking University, Beijing, People’s Republic of China; Department of Cardiology (K.A.Y.), The Methodist Hospital Research Institute, Houston,Tex; and Cardiovascular Research Institute (Q.Y.), Morehouse School of Medicine, Atlanta, Ga.

*The first 2 authors contributed equally to this work.The online-only Data Supplement, consisting of an expanded Methods section and figures, is available with this article at http://circ.ahajournals.org/

cgi/content/full/CIRCULATIONAHA.107.707257/DC1.Correspondence to Jifeng Zhang or Dr Y. Eugene Chen, Cardiovascular Center, Department of Internal Medicine, University of Michigan Medical

Center, Ann Arbor, MI 48109. E-mail [email protected] or [email protected]© 2007 American Heart Association, Inc.

Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.107.707257

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attachment and migration of VSMCs and suppressed neoin-timal formation after balloon injury.10 Interestingly, we andothers11 recently found that Rad is most abundantly expressedin the heart, which indicates that Rad may be involved incardiac physiology and/or pathophysiological processes. Inthe present study, using gain-of-function and loss-of-functionstrategies both in vivo and in vitro, we have documented forthe first time that Rad is a novel mediator that inhibits cardiachypertrophy through the calmodulin-dependent kinase II(CaMKII) pathway.

MethodsCardiomyocyte CulturesPrimary cardiomyocytes were prepared from 2-day-old Sprague-Dawley rats as described previously.12 Cardiomyocytes were platedat a density of 2.5�105 cells/well in 12-well plates and cultured for16 to 20 hours in DMEM that contained 10% fetal bovine serum.Overexpression of Rad was achieved by infecting the cardiomyo-cytes with 10 plaque-forming units (pfu) per cell of the recombinantadenovirus containing the human Rad cDNA (Ad-Rad) for 48 hours.The adenovirus carrying green fluorescent protein (Ad-GFP) wasused as the negative control for Ad-Rad, as described previously.10

Knockdown of Rad was achieved by infecting the cardiomyocyteswith 10 pfu/cell of adenovirus carrying Rad short hairpin RNA(shRNA) oligonucleotides (Ad-Rad-RNAi), which we generatedusing the Knockout Adenoviral RNAi System 1 from Clontech(Mountain View, Calif). The Rad RNAi oligonucleotide sequencefor the knockdown is 5�-CGAGACCTTCAGGC GGCGCTA-3�.The adenovirus carrying scrambled Rad shRNA oligonucleotides(Ad-Rad-RNAi-Control) was used as the negative control for Ad-Rad-RNAi. CaMKII� Stealth small interfering RNA oligonucleo-tides (5�-AUAUUCUGCCACUUCCCAUCACGGC-3� and 5�-GCCGUGAUGGGAAGUGGCAGAAUAU-3�) were purchasedfrom Invitrogen (Carlsbad, Calif). Knockdown of CaMKII� incardiomyocytes was achieved by transfection of 20 nmol/LCaMKII� small interfering RNA oligonucleotides (CaMKIIi) withLipofectamine 2000 (Invitrogen) for 24 hours.

Induction and Characterization of CardiomyocyteHypertrophy In VitroCardiomyocyte hypertrophy was induced by treatment of quiescentcardiomyocytes (serum-free DMEM, 24 hours) with 10 �mol/Lphenylephrine (PE) for the required times. The effects of PE wereconfirmed by determining the mRNA levels of cardiomyocyte hyper-trophic markers by use of quantitative real-time polymerase chainreaction (qRT-PCR) normalized to 18S rRNA. Cardiomyocyteprotein synthesis was determined by 3H-leucine incorporation. Inbrief, neonatal rat cardiomyocytes infected with Ad-Rad, Ad-GFP,Ad-Rad-RNAi, or Ad-Rad-RNAi-Control were kept in serum-freeDMEM for 24 hours. Next, the cells were treated with 10 �mol/L PEfor 24 hours, and [3H]Leu (1 �Ci/mL) was added to the wells 18hours before the cells were harvested. To precipitate proteins, cellswere washed 3 times with ice-cold PBS, and subsequently, ice-cold10% trichloroacetic acid was added to the wells for 2 hours. Thetrichloroacetic acid was then removed, and wells were washed with95% ethanol. After they were dried at room temperature, 200 �L of0.5 mol/L NaOH was added to the wells. Samples were transferredto scintillation vials to measure 3H-Leu incorporation.

Thoracic Transverse Aortic Constriction–InducedCardiac HypertrophyRad knockout mice (Rad-KO; Data Supplement) and their wild-typelittermate control mice (10 weeks old) were anesthetized by intra-peritoneal injection of xylazine (5 mg/kg) and ketamine (100mg/kg).13 A heating pad was used to maintain body temperature. Theuse of a horizontal incision at the level of the suprasternal notchallows direct visualization of the transverse aorta without entering

the pleural space and thus obviates the need for mechanical venti-lation. We banded the transverse aorta between the right innominateand left carotid arteries to the diameter of a 27-gauge needle (bodyweight 25 to �27 g) using a 7-0 silk suture. Sham operations on sex-and age-matched mice only omitted the actual aortic banding andserved as a control for all experimental groups. Fourteen days aftersurgery, hearts were harvested. Cardiac hypertrophy was determinedby heart weight–to–body weight ratio, myocardial cross sections,and expression levels of 2 cardiac hypertrophy markers, atrialnatriuretic factor (ANF) and brain natriuretic peptide (BNP).

Western BlottingWestern blot analyses were performed as described previously.10 Arabbit anti-Rad polyclonal antibody (1:2000 dilution) reported pre-viously6 was used in the present study. Rabbit anti-phospho-CaMKII(1:1000 dilution) polyclonal antibodies and rabbit anti-CaMKII(1:1000 dilution) polyclonal antibodies were purchased from UpstateInc (Charlottesville, Va).

CaMKII Activity AssayLeft ventricles isolated from mice or neonatal rat cardiomyocyteswere washed 3 times with ice-cold PBS and then lysed with modifiedRIPA buffer (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1%Nonidet-P40, 1% sodium deoxycholate, 1 mmol/L sodium vanadate,10 mmol/L sodium pyrophosphate, 10 mmol/L NaF, 1% TritonX-100, 0.5% SDS, 0.1% EDTA, 10 �g/mL leupeptin, 10 �g/mLaprotinin, and 1 mmol/L PMSF). Lysates were cleared by centrifu-gation, and the total protein concentration was determined by theBio-Rad protein assay reagent (catalog #500-0006, Hercules, Calif).CaMKII in the cell lysates (200 �g of proteins) was immunopre-cipitated by incubation with anti-CaMKII antibody and protein Abeads at 4°C overnight. CaMKII kinase activities were examined byan assay kit from Upstate (catalog #17-135) according to themanufacturer’s instructions.

Histological AnalysisHearts were harvested from anesthetized mice after fixation viatranscardial perfusion with 4% formaldehyde (pH 8.0) and fixedovernight. After dehydration, samples were embedded in paraffinwax according to standard laboratory procedures. Sections of 5 �mwere stained with hematoxylin and eosin for routine histopatholog-ical examination with light microscopy. For determination of myo-cyte cross-sectional areas, 70 individual cells per slide were deter-mined by digitization of the images and computerized pixelcounting. Only nucleated cardiac myocytes from areas of trans-versely cut muscle fibers were included in the analysis.14

Patient SamplesLeft ventricular myocardium was obtained from patients undergoingheart transplantation owing to end-stage heart failure (n�10). Meanage was 49�18 years, and all patients were in New York HeartAssociation class IV with ejection fractions �20%. All patients weretaking ACE inhibitors and �-blockers. For comparison, we obtainedleft ventricular tissue samples from donor hearts of accident victimswithout known cardiac trauma that could not be transplanted fortechnical reasons (n�3). Mean age of the normal heart donors was47�11 years. The study was approved by the Institutional ReviewBoard for human subject research at the Baylor College of Medicineand the University of Michigan.

Statistical AnalysisResults are reported as mean�SD. Differences between mean valueswere evaluated by Student t test or ANOVA, with values of P�0.05indicating a significant difference. Two independent samples fromFigure 1C and 1D were used for a 2-tailed, unpaired Student t test.Data taken at different points in time, as shown in Figure 2A and 2B,were analyzed by repeated-measures ANOVA and 1-way ANOVAfollowed by Newman-Keuls multiple comparison procedure toaccount for multiple testing. Two-way ANOVA was used to analyzethe data in Figures 3 and 4, with small samples in each comparison

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group. One-way ANOVA followed by Newman-Keuls multiplecomparison was used in Figure 5B, 5C, and 5D. Figure 6B and 6Cwere analyzed by repeated-measures ANOVA and 1-way ANOVAfollowed by Newman-Keuls multiple comparison procedure toaccount for multiple testing.

The authors had full access to the data and take full responsibilityfor its integrity. All authors have read and agree to the manuscript aswritten.

ResultsRad Expression Is Significantly Decreased inHuman Failing HeartsRad is highly expressed in the human heart and in skeletalmuscle (Data Supplement Figure I), in agreement with arecent report.11 The abundant expression of Rad in the heartsuggests that Rad may play an important role in cardiacdiseases. As the first step in exploring the potential role ofRad in cardiac hypertrophy, we collected left ventricularmyocardium samples from patients undergoing heart trans-plantations because of end-stage heart failure. These failinghearts displayed severe hypertrophic morphology, as shownin Figure 1A. Using qRT-PCR, we demonstrated that 2markers of cardiac hypertrophy, ANF and BNP, were in-creased significantly by 11-fold and 6.8-fold, respectively, inthese human failing hearts (Figure 1B; P�0.01 versus normalhearts). Intriguingly, Western blotting showed that Rad pro-tein levels were decreased significantly in all human failing

hearts (n�5) compared with normal hearts (n�3; Figure 1C).Similar data were also observed in samples from 5 otherhuman failing hearts (data not shown). In addition, relativeRad mRNA levels were decreased in human failing heartscompared with normal hearts according to qRT-PCR (Figure1D; 0.42�0.21 versus 1.12�0.38, P�0.01). Thus, the pres-ent data strengthen the notion that Rad plays an importantrole in the mediation of cardiac remodeling.

Rad Expression Is Decreased in ExperimentalHypertrophic CardiomyocytesIn Vivo and In VitroTo explore the potential function of Rad in the heart, we usedthe well-established cardiac hypertrophy model induced bythoracic transverse aortic constriction (TAC). We docu-mented that Rad protein levels were decreased significantlyin a time-dependent manner from 24 hours to day 14 afterTAC during the progression of cardiac hypertrophy (Figure2A). Furthermore, we established a PE-induced cardiomyo-cyte hypertrophy model in vitro using primary cultures ofneonatal rat cardiomyocytes. In this model, we found thatlevels of Rad protein were downregulated significantly 4hours after PE treatment (10 �mol/L) and remained low for atleast 24 hours after PE treatment (Figure 2B). Taken together,these results demonstrate for the first time that decreased Radexpression is associated with cardiac hypertrophy, whichsuggests a potentially important role of Rad in this process.

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Figure 1. Expression levels of Rad weredownregulated in human failing hearts.A, Representative hematoxylin and eosinstain of human heart cross sections ofnormal and failing human hearts (scalebar�50 �m). B, qRT-PCR was used tomeasure expression levels of 2 cardiachypertrophy markers, ANF and BNP. Val-ues for the specific mRNA levels normal-ized by 18S rRNA levels are expressed asmean�SD (n�3 for normal human heartsand n�10 for failing human hearts,**P�0.01 versus normal human hearts).C and D, Expression levels of Rad weredownregulated in human failing hearts asjudged by Western blot analysis (C) andqRT-PCR (D). �-Tubulin and 18S rRNAwere used as internal controls, respec-tively. Data are shown as mean�SD(**P�0.01 versus normal human hearts).

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Figure 2. Rad is downregulated in cardio-myocyte hypertrophy models in vivo andin vitro. Representative Western blotsshow expression levels of Rad protein.A, Samples from TAC-induced cardiachypertrophy. B, Samples from PE (10�mol/L)-induced neonatal rat cardiomyo-cyte hypertrophy. Average values normal-ized by �-tubulin from 3 independentexperiments are shown in the bottompanel (*P�0.05 and **P�0.01 comparedwith 0 hours).

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Rad Inhibits 3H-Leu Incorporation inCardiomyocytes Induced by PETo explore whether Rad has a protective role in cardiacremodeling, we performed gain-of-function and loss-of-function studies in cultured cardiomyocytes using adenoviruscontaining human Rad cDNA10 and Rad shRNA, as describedin Methods. Ad-Rad infection (10 pfu/cell) led to a substan-tial increase in the level of Rad protein of �4-fold, andAd-Rad-RNAi (10 pfu/cell) effectively decreased Rad proteinexpression by �90% in neonatal rat cardiomyocytes, respec-tively (Data Supplement Figure IIA). Using 3H-Leu incorpo-ration, we found that Rad overexpression significantly re-duced PE-induced protein synthesis (2031�506 versus3186�273 cpm, P�0.01), whereas Rad knockdown dramat-ically increased protein synthesis by �2-fold compared withthe corresponding Ad-Rad-RNAi control (6122�836 versus

3154�694 cpm, P�0.01; Figure 3A). In addition, increasedRad levels inhibited expression of the PE-induced hypertro-phy marker ANF by 47% in cardiomyocytes treated withAd-Rad (1.9�0.3 versus 2.8�0.5, P�0.01), whereas Radknockdown led to a significant increase of ANF expression(4.3�0.3 versus 2.5�0.2, P�0.01; Figure 3B). Taken to-gether, these findings suggest that Rad may play an importantprotective role during cardiac remodeling through a mecha-nism that involves inhibition of cardiomyocyte hypertrophy.

Rad-KO Mice Are More Susceptible toTAC-Induced Cardiac HypertrophyTo uncover the potential role of Rad in vivo, we generatedRad-deficient mice (Rad-KO) for the present study (DataSupplement Figure IIIA). Rad expression was absent in theheart of Rad-KO mice (Data Supplement Figure IIIB and

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Figure 3. Rad inhibits cardiomyocyte hypertrophy. Rat neonatal cardiomyocytes were infected by 10 pfu/cell of Ad-Rad or Ad-Rad-RNAi (Ad-Rad i) and used in 10 �mol/L PE-induced 3H-Leu incorporation assay (A) and to determine expression of ANF, a hypertrophymarker in cardiomyocytes, by qRT-PCR analyses (B). Ad-GFP and Ad-Rad RNAi-Control (Ad-Rad ic) were used as controls for Ad-Radand Ad-Rad-RNAi, respectively. Control was cardiomyocytes without adenoviral infection. Values are expressed as mean�SD (n�6 ineach group). Vehicle: PBS. ANF mRNA levels were normalized by 18S rRNA levels.

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Figure 4. Rad-deficient mice are moresusceptible to pressure overload–inducedcardiac hypertrophy. A, Heart weight tobody weight ratio (HW/BW). Hearts wereharvested at day 14 after TAC procedure.n�6. B, Representative images of hema-toxylin-and-eosin–stained left ventricularcross sections (bars�20 �m) of wild-typesham, Rad-KO sham, wild-type TAC, andRad-KO TAC mice. C, Cross-sectionalcell area in wild-type sham, Rad-KOsham, wild-type TAC, and Rad-KO TACmice. *P�0.05, Rad-KO TAC vs wild-typeTAC. D, qRT-PCR was used to measureexpression levels of 2 cardiac hypertro-phy markers, ANF and BNP, in thesemice at 14 days after TAC. Values formRNA levels normalized by 18S rRNAlevels are expressed as mean�SD (n�6;**P�0.01 versus wild-type TAC).



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IIIC). Rad-KO mice showed normal heart weight, bodyweight, and heart-weight–to–body-weight ratio comparedwith wild-type littermate controls in the present study (Figure4A). The morphology of myocardial cross sections was alsosimilar, as judged by hematoxylin-and-eosin staining (Figure4B, Sham). Hemodynamic analysis by in vivo cardiac cath-eterization revealed that basal left ventricular contractility(assessed by maximal �dP/dt) and diastolic function (as-

sessed by maximal dP/dt) were similar in Rad-KO andcontrol mice (Data Supplement Figure IV). Dobutaminestimulation increased left ventricular contractility anddiastolic function in a dose-dependent manner in bothRad-KO and control mice; however, Rad-KO mice showedweaker left ventricular contractility than control mice athigher doses of dobutamine (2.5 and 5.0 �g/kg body weight).Taken together, these results suggest that Rad deficiency

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Figure 5. Rad inhibits CaMKII phosphorylation and autonomous CaMKII activity. A, Representative Western blots showing phosphory-lated CaMKII and total CaMKII levels in rat neonatal cardiomyocytes infected with 10 pfu/cell of Ad-GFP, Ad-Rad, Ad-Rad-RNAi (Ad-Rad i), or Ad-Rad-RNAi-Control (Ad-Rad ic). B, Cell lysates of neonatal rat cardiomyocytes infected with 10 pfu/cell Ad-GFP, Ad-Rad,Ad-Rad I, or Ad-Rad ic were used to measure autonomous CaMKII activity as described in Methods. C and D, 3H-Leu incorporation(C) and ANF mRNA (D) levels in the presence of 10 �mol/L of PE-treated rat neonatal cardiomyocytes were determined for each group.Ad-Rad ic was used as the control for Ad-Rad-RNAi. Scrambled CaMKII� RNAi oligonucleotide (CaMK II ic) was used as the control forCaMKII� RNAi (CaMK II i). Control was rat neonatal cardiomyocytes without adenoviral infection. Values are expressed as mean�SD(n�6 in each group). ANF mRNA levels were normalized by 18S rRNA levels.

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Figure 6. Rad deficiency increasesCaMKII phosphorylation in the heart. A,Representative images of Western blotsshowing increased CaMKII phosphoryla-tion in Rad-KO mice compared with wild-type littermate controls after TAC. B andC, CaMKII activity in left ventricles afterTAC. Extracts from left ventricles of TACmice were assayed in vitro for Ca2�-dependent CaMKII activity (B) in thepresence of Ca2�/CaM or for autono-mous CaMKII activity (C) in the presenceof 5 mmol/L EGTA. Values are expressedas mean�SD (n�6, **P�0.01 versuswild-type littermate controls).



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affects cardiac systolic function in response to increasedcardiac stress.

To further define the role of endogenous Rad in cardiacremodeling, we performed TAC in Rad-KO and wild-typelittermate control mice. We documented that heart-weight–to-body-weight ratio (Figure 4A) and myocardial cross sec-tions (Figure 4B and 4C; Data Supplement Figure V) wereincreased significantly in Rad-KO mice compared with wild-type littermates 14 days after TAC. In addition, we examinedthe expression of several cardiac hypertrophy markers inRad-KO mice and wild-type littermate controls after TACusing qRT-PCR analyses. At 10 weeks of age, Rad-KO andcontrol mice had similar basal expression levels of the cardiachypertrophy markers ANF and BNP. After the TAC proce-dure, both ANF and BNP expression levels were upregulatedsignificantly by 2.4-fold (3.77�0.46 versus 1.56�0.16,P�0.01) and 1.9-fold (2.76�0.2 versus 1.44�0.32, P�0.01),respectively, in Rad-KO mice compared with wild-typelittermate controls (Figure 4D). Thus, these data provideevidence that downregulation of Rad in vivo can promote theprogression of cardiac hypertrophy.

Rad Interacts With the Ca2�-Calmodulin-CaMKIIPathway in CardiomyocytesIt has been well documented that activated (phosphorylated)CaMKII is a strong inducer of cardiac hypertrophy.15 Ourprevious studies demonstrated that Rad can interact withcalmodulin (CaM) and CaMKII in skeletal muscle cells.16 Todetermine whether the Ca2�-CaM-CaMKII pathway mediatesthe protective effects of Rad in cardiac hypertrophy, weperformed a coimmunoprecipitation assay in cultured neona-tal rat cardiomyocytes. Using this method, we found thatthere is a strong interaction between Rad and CaM, as well asbetween Rad and CaMKII, in cardiomyocytes (Data Supple-ment Figure VI), which is similar to our previous findings inskeletal muscle cells.16 Furthermore, overexpression of Radsignificantly inhibited CaMKII phosphorylation, whereasRad knockdown increased CaMKII phosphorylation in car-diomyocytes (Figure 5A). We also demonstrated thatCaMKII activity was decreased in Rad-overexpressing cellsbut increased in Rad knockdown cells (Figure 5B). To date,emerging data suggest that CaMKII� plays a critical role incardiac hypertrophy.17 To further address whether CaMKII�is the critical mediator of Rad-inhibited cardiac hypertrophy,we reduced CaMKII� expression by RNAi technology inneonatal rat cardiomyocytes in combination with Rad knock-down. Interestingly, double knockdown of CaMKII� and Radin cardiomyocytes significantly decreased PE-induced 3H-Leu incorporation (Figure 5C; 3957�492 versus 6622�786cpm, P�0.01) and ANF mRNA expression (Figure 5D;3.1�0.2 versus 4.5�0.4, P�0.01) compared with only Radknockdown. In agreement with this observation, we furtherdocumented that in the present in vivo model, TAC-inducedCaMKII phosphorylation in the heart was significantly in-creased in Rad-KO mice compared with wild-type littermatecontrols (Figure 6A).

To further define the effects of Rad on CaMKII activity inthe heart, we measured the Ca2�-dependent CaMK activity ofventricular extracts from wild-type and Rad-KO mouse hearts

using a specific peptide (KKALRRQETVDAL) as the sub-strate provided with the CaMKII kinase assay kit fromUpstate Inc. As shown in Figure 6B, basal levels of Ca2�-dependent CaMK activity in the hearts of Rad-KO mice andwild-type control littermates are similar when a reactionsystem with 1 mmol/L CaCl2 is used. Although Ca2�-dependent CaMK activity is significantly increased in bothwild-type and Rad-KO mice at 7 and 14 days after the TACprocedure, the Rad-KO mice showed significantly higherCa2�-dependent CaMK activity (32% increase, n�6, P�0.01).Because CaMKII becomes independent of Ca2�/CaM whenactivated, and its Ca2�-independent kinase activity can beassayed in the presence of EGTA,18 we measured Ca2�-independent CaMKII activity (autonomous activity ofCaMKII). Rad-KO hearts showed significantly greater auton-omous CaMKII activity (33% increase, n�6, P�0.01) thanwild-type littermate controls at 7 and 14 days after the TACprocedure, although the basal levels (day 0) of autonomousCaMK activity were similar (Figure 6C). Taken together, thepresent data imply that Rad is a novel cardioprotectivemediator that prevents cardiac hypertrophy through the inhi-bition of CaMKII activity.

DiscussionCardiac hypertrophy is an important adaptive growth re-sponse that facilitates an increase in myocardial contractility.It is postulated that pathological structural changes in theheart are induced in part by small G proteins that govern awide spectrum of functions, including regulation of cellproliferation, migration, apoptosis, and cytoskeletal rear-rangement.2,4 Rad was originally identified by subtractioncloning and found to be overexpressed in skeletal muscle ofpatients with type 2 diabetes mellitus.6 To date, the functionof Rad remains largely unknown in the cardiovascular sys-tem. In the present study, we have shown that although Radis highly expressed in the normal myocardium, it is dramat-ically decreased in human failing hearts. In addition, thepresent data demonstrated for the first time that Rad is a noveland critical mediator that prevents and inhibits cardiac hy-pertrophy through inhibition of the CaMKII pathway.

To date, �100 identified small GTPases constitute asuperfamily that comprises at least 6 subfamilies: Ras, Rho(Rho, Rac, and Cdc42), Arf (ADP ribosylation factor), Rab,Ran, and RGK (Rad/Gem/Kir).2,19 In the cardiovascularsystem, small GTPases are implicated in regulation of endo-thelial function3; VSMC contraction, proliferation, and mi-gration10; and cardiac hypertrophy.4,20–22 We have reportedthat inhibition of the Rho/ROK (Rho-kinase) signaling path-way reduces VSMC migration and decreases neointimalformation.10 The inhibition of Ras attenuates atherosclerosisin apolipoprotein E–deficient mice and neointimal formationin porcine coronary balloon angioplasty.23 Furthermore, inhi-bition of Rac1 induces regression of coronary atherosclerosisin a pig model treated with interleukin-1�.24 Activation ofRhoA is a critical component of hypertension, whereasoxyhemoglobin-induced Rho/ROK activation is a majorcausative component of cerebral vasospasm.25,26 Thus, thetargeting of small GTPases and their downstream signalingcould provide novel therapeutic approaches for the treatment



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of cardiovascular disorders. Actually, the importance of smallGTPases as targets for therapeutic purposes in the cardiovas-cular system is underscored by experimental and clinicalstudies with statins that protect against cardiac hypertrophyand atherosclerosis by targeting GTPases beyond their cho-lesterol-lowering effects.27,28

Rad, a 33- to 35-kDa protein (with 61% amino acidsequence identity to Gem and Kir)5,29 is the prototypicmember of a new subfamily of Ras-related GTPases thatlacks the typical prenylation motifs at the C terminus and wasoriginally identified from skeletal muscle of patients withtype 2 diabetes mellitus.6 Genetic studies linking a trinucle-otide-repeat polymorphism in Rad and type 2 diabetes mel-litus are controversial, with 2 studies in favor30,31 and 1 largerpopulation study against.32 Although an association betweenRad upregulation and type 2 diabetes mellitus could not beconfirmed, a correlation with obesity was reported.33 It wasalso reported that Rad inhibits insulin-stimulated glucoseuptake in myocyte and adipocyte cell lines.7 A recent reportrevealed that Rad is highly expressed in a breast cancer cellline with high tumorigenic and metastatic potential, promot-ing growth and accelerating cell cycle transition.34 However,during our previous studies of Rad in VSMCs, we found thatoverexpression of Rad in VSMCs had no effect on cellproliferation,10 which suggests that its role is cell-specific andcontextual. Interestingly, a previous study demonstrated thatGem and Rad can negatively regulate the function of theRho/ROK signaling pathway through association with theRho effector, ROK, in neuroblastoma.35 Indeed, our pub-lished data showed that Rad also physically interacts withROK but not with RhoA in VSMCs.10 Compared with othertissues, Rad is highly expressed in the heart; however, thefunctions of Rad in heart remain largely unknown. Intrigu-ingly, a recent study demonstrated that overexpression of aRad dominant-negative mutant in cardiomyocytes leads toQT prolongation and causes ventricular arrhythmias viamodulation of L-type Ca2� channels in the heart.36

Multifunctional CaMKs are transducers of Ca2� signals.Several transgenic mouse models have confirmed thatCaMKs play an important role in the development of cardiachypertrophy.37 CaMKII is a proarrhythmic signaling mole-cule in cardiac hypertrophy in vivo.38 CaMKII proteins areencoded by 4 genes, �, �, �, and �. Whereas the �- and�-genes are neuron specific, the �- and �- genes are expressedin most somatic cells.39 To date, emerging data suggest thatCaMKII� plays a critical role in cardiac hypertrophy.17

Indeed, it was reported that genetic mouse model of cardiacCaMKII inhibition, which genetically targeted a conservedregion of the CaMKII regulatory domain with cDNA encod-ing an inhibitory peptide, substantially prevented maladaptiveremodeling from excessive �-adrenergic receptor stimulationand myocardial infarction.40 In the present study, we demon-strated a direct physical interaction between Rad and CaMKIIin cardiomyocytes by coimmunoprecipitation. Also, we doc-umented that overexpression of Rad in cardiomyocytes in-hibits CaMKII phosphorylation and autonomous activity,whereas CaMKII phosphorylation and autonomous activityare dramatically increased in cardiomyocytes with Radknockdown. In the present in vitro model, cardiomyocytes

with Rad knockdown were more susceptible to PE-inducedcardiomyocyte hypertrophy, as inferred from 3H-Leu incor-poration and ANF expression; however, cells with bothCaMKII� and Rad knockdown were resistant to PE-inducedcardiomyocyte hypertrophy. Using TAC, a procedure thatmimics the pressure overload–induced cardiac hypertrophymodel, we further documented that Rad expression wasdecreased significantly in hypertrophic hearts and that Raddeficiency led to significantly exacerbated cardiac hypertro-phy in association with increased CaMKII phosphorylationand activation in the heart. Thus, the present results stronglysuggest that Rad is a novel mediator of cardiac hypertrophyvia inhibition of CaMKII activity.

In summary, the present data document for the first timethat Rad, through its ability to strongly decrease CaMKIIphosphorylation and activation, is an endogenous inhibitor ofcardiac hypertrophy. Our studies provide new insights intounderstanding the mechanisms of cardiac hypertrophy andmay have new, significant implications for the developmentof novel strategies for treatment of cardiac hypertrophythrough specific targeting of the Rad signaling pathwaywithin this family of small GTPases.

Sources of FundingDr Chen was supported by National Institutes of Health grantsHL068878 and HL075397. Drs Chang and Xie were supported by anAmerican Heart Association Postdoctoral Fellowship from theGreater Midwest Affiliate (0625705Z) and a Southeast Affiliate(0525510B) grant, respectively. Dr Zhu was supported by theNatural Science Foundation of China (30671027).

DisclosuresNone.

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cellular signalling pathways. Nat Rev. 2006;7:589–600.2. Kelly K. The RGK family: a regulatory tail of small GTP-binding

proteins. Trends Cell Biol. 2005;15:640–643.3. Fryer BH, Field J. Rho, Rac, Pak and angiogenesis: old roles and newly

identified responsibilities in endothelial cells. Cancer Lett. 2005;229:13–23.

4. Satoh M, Ogita H, Takeshita K, Mukai Y, Kwiatkowski DJ, Liao JK.Requirement of Rac1 in the development of cardiac hypertrophy. ProcNatl Acad Sci U S A. 2006;103:7432–7437.

5. Maguire J, Santoro T, Jensen P, Siebenlist U, Yewdell J, Kelly K. Gem:an induced, immediate early protein belonging to the Ras family. Science.1994;265:241–244.

6. Reynet C, Kahn CR. Rad: a member of the Ras family overexpressed inmuscle of type II diabetic humans. Science. 1993;262:1441–1444.

7. Moyers JS, Bilan PJ, Reynet C, Kahn CR. Overexpression of Rad inhibitsglucose uptake in cultured muscle and fat cells. J Biol Chem. 1996;271:23111–23116.

8. Finlin BS, Crump SM, Satin J, Andres DA. Regulation of voltage-gatedcalcium channel activity by the Rem and Rad GTPases. Proc Natl AcadSci U S A. 2003;100:14469–14474.

9. Ward Y, Kelly K. Gem protein signaling and regulation. MethodsEnzymol. 2005;407:468–483.

10. Fu M, Zhang J, Tseng YH, Cui T, Zhu X, Xiao Y, Mou Y, De Leon H,Chang MM, Hamamori Y, Kahn CR, Chen YE. Rad GTPase attenuatesvascular lesion formation by inhibition of vascular smooth muscle cellmigration. Circulation. 2005;111:1071–1077.

11. Hawke TJ, Kanatous SB, Martin CM, Goetsch SC, Garry DJ. Rad istemporally regulated within myogenic progenitor cells during skeletalmuscle regeneration. Am J Physiol Cell Physiol. 2006;290:C379–C387.

12. Kaburagi S, Hasegawa K, Morimoto T, Araki M, Sawamura T, Masaki T,Sasayama S. The role of endothelin-converting enzyme-1 in the devel-



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13. Peng X, Kraus MS, Wei H, Shen TL, Pariaut R, Alcaraz A, Ji G, ChengL, Yang Q, Kotlikoff MI, Chen J, Chien K, Gu H, Guan JL. Inactivationof focal adhesion kinase in cardiomyocytes promotes eccentric cardiachypertrophy and fibrosis in mice. J Clin Invest. 2006;116:217–227.

14. Buitrago M, Lorenz K, Maass AH, Oberdorf-Maass S, Keller U,Schmitteckert EM, Ivashchenko Y, Lohse MJ, Engelhardt S. Thetranscriptional repressor Nab1 is a specific regulator of pathologicalcardiac hypertrophy. Nat Med. 2005;11:837– 844.

15. Zhang T, Maier LS, Dalton ND, Miyamoto S, Ross J Jr, Bers DM, BrownJH. The deltaC isoform of CaMKII is activated in cardiac hypertrophyand induces dilated cardiomyopathy and heart failure. Circ Res. 2003;92:912–919.

16. Moyers JS, Bilan PJ, Zhu J, Kahn CR. Rad and Rad-related GTPasesinteract with calmodulin and calmodulin-dependent protein kinase II.J Biol Chem. 1997;272:11832–11839.

17. Zhang T, Brown JH. Role of Ca2�/calmodulin-dependent protein kinase IIin cardiac hypertrophy and heart failure. Cardio Res. 2004;63:476–486.

18. Hanson PI, Kapiloff MS, Lou LL, Rosenfeld MG, Schulman H.Expression of a multifunctional Ca2�/calmodulin-dependent proteinkinase and mutational analysis of its autoregulation. Neuron. 1989;3:59–70.

19. Meller N, Merlot S, Guda C. CZH proteins: a new family of Rho-GEFs.J Cell Sci. 2005;118:4937–4946.

20. Brown JH, Del Re DP, Sussman MA. The Rac and Rho hall of fame: adecade of hypertrophic signaling hits. Circ Res. 2006;98:730–742.

21. Grounds HR, Ng DC, Bogoyevitch MA. Small G-protein Rho is involvedin the maintenance of cardiac myocyte morphology. J Biol Chem. 2005;95:529–542.

22. Higuchi Y, Otsu K, Nishida K, Hirotani S, Nakayama H, Yamaguchi O,Hikoso S, Kashiwase K, Takeda T, Watanabe T, Mano T, Matsumura Y,Ueno H, Hori M. The small GTP-binding protein Rac1 induces cardiacmyocyte hypertrophy through the activation of apoptosis signal-regulating kinase 1 and nuclear factor-kappa B. J Biol Chem. 2003;278:20770–20777.

23. Work LM, McPhaden AR, Pyne NJ, Pyne S, Wadsworth RM, WainwrightCL. Short-term local delivery of an inhibitor of Ras farnesyltransferaseprevents neointima formation in vivo after porcine coronary balloon angio-plasty. Circulation. 2001;104:1538–1543.

24. Morishige K, Shimokawa H, Eto Y, Kandabashi T, Miyata K, MatsumotoY, Hoshijima M, Kaibuchi K, Takeshita A. Adenovirus-mediated transferof dominant-negative rho-kinase induces a regression of coronary arte-riosclerosis in pigs in vivo. Arterioscler Thromb Vasc Biol. 2001;21:548–554.

25. Seko T, Ito M, Kureishi Y, Okamoto R, Moriki N, Onishi K, Isaka N,Hartshorne DJ, Nakano T. Activation of RhoA and inhibition of myosinphosphatase as important components in hypertension in vascular smoothmuscle. Circ Res. 2003;92:411–418.

26. Wickman G, Lan C, Vollrath B. Functional roles of the rho/rho kinasepathway and protein kinase C in the regulation of cerebrovascular con-

striction mediated by hemoglobin: relevance to subarachnoid hemorrhageand vasospasm. Circ Res. 2003;92:809–816.

27. Rikitake Y, Liao JK. Rho GTPases, statins, and nitric oxide. Circ Res.2005;97:1232–1235.

28. Takemoto M, Node K, Nakagami H, Liao Y, Grimm M, Takemoto Y,Kitakaze M, Liao JK. Statins as antioxidant therapy for preventingcardiac myocyte hypertrophy. J Clin Invest. 2001;108:1429–1437.

29. Dorin D, Cohen L, Del Villar K, Poullet P, Mohr R, Whiteway M, WitteO, Tamanoi F. Kir, a novel Ras-family G-protein, induces invasivepseudohyphal growth in Saccharomyces cerevisiae. Oncogene. 1995;11:2267–2271.

30. Wang GY, Niu TH, Chen CZ, Li QF, Xu XP. A novel Rad genepolymorphism combined with obesity increases risk for type 2 diabetesmellitus. Chin Med J (Engl). 2004;117:770–771.

31. Yuan X, Yamada K, Ishiyama-Shigemoto S, Koyama W, Nonaka K.Analysis of trinucleotide-repeat combination polymorphism at the radgene in patients with type 2 diabetes mellitus. Metabolism. 1999;48:173–175.

32. Orho M, Carlsson M, Kanninen T, Groop LC. Polymorphism at the radgene is not associated with NIDDM in Finns. Diabetes. 1996;45:429–433.

33. Paulik MA, Hamacher LL, Yarnall DP, Simmons CJ, Maianu L, PratleyRE, Garvey WT, Burns DK, Lenhard JM. Identification of Rad’s effector-binding domain, intracellular localization, and analysis of expression inPima Indians. J Cell Biochem. 1997;65:527–541.

34. Tseng YH, Vicent D, Zhu J, Niu Y, Adeyinka A, Moyers JS, Watson PH,Kahn CR. Regulation of growth and tumorigenicity of breast cancer cellsby the low molecular weight GTPase Rad and nm23. Cancer Res. 2001;61:2071–2079.

35. Ward Y, Yap SF, Ravichandran V, Matsumura F, Ito M, Spinelli B, KellyK. The GTP binding proteins Gem and Rad are negative regulators of theRho-Rho kinase pathway. J Cell Biol. 2002;157:291–302.

36. Yada H, Murata M, Shimoda K, Yuasa S, Kawaguchi H, Ieda M, AdachiT, Murata M, Ogawa S, Fukuda K. Dominant negative suppression ofRad leads to QT prolongation and causes ventricular arrhythmias viamodulation of L-type Ca2� channels in the heart. Circ Res. 2007;101:69–77.

37. Passier R, Zeng H, Frey N, Naya FJ, Nicol RL, McKinsey TA, OverbeekP, Richardson JA, Grant SR, Olson EN. CaM kinase signaling inducescardiac hypertrophy and activates the MEF2 transcription factor in vivo.J Clin Invest. 2000;105:1395–1406.

38. Anderson ME. Calmodulin kinase and L-type calcium channels: a recipefor arrhythmias? Trends Cardiovasc Med. 2004;14:152–161.

39. Tobimatsu T, Fujisawa H. Tissue-specific expression of four types of ratcalmodulin-dependent protein kinase II mRNAs. J Biol Chem. 1989;264:17907–17912.

40. Zhang R, Khoo MS, Wu Y, Yang Y, Grueter CE, Ni G, Price EE Jr, ThielW, Guatimosim S, Song LS, Madu EC, Shah AN, Vishnivetskaya TA,Atkinson JB, Gurevich VV, Salama G, Lederer WJ, Colbran RJ,Anderson ME. Calmodulin kinase II inhibition protects against structuralheart disease. Nat Med. 2005;11:409–417.

CLINICAL PERSPECTIVEThis study identifies a novel role for the small G protein-Rad GTPase in the process of cardiac hypertrophy induction,which is notable because it appears to be relevant to human cardiovascular disease. Recently, Rad GTPase has emergedas an important protein in cardiac function since, as it is most abundantly expressed in the heart. The data shown in thepresent study strongly support the idea that Rad GTPase serves as a cardiac hypertrophy inhibitor. First, human samplesdisplaying clinical heart failure showed decreased Rad GTPase protein and mRNA expression levels. Secondly, a murinemodel of Rad GTPase deficiency was more susceptible to cardiac hypertrophy than were wild-type littermates. Finally, anassociation between Rad GTPase and inhibition of calmoudulin kinase II phosphorylation and activity was documented,thereby inhibiting the progression toward cardiac hypertrophy. Viewed this way, therapeutic activation of Rad GTPasesignaling offers a theoretical but testable strategy for counteracting hypertrophic stimuli at the cardiomyocyte level.



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Ronald Kahn and Y. Eugene ChenZhongcui Sun, Xiaojun Zhu, Taixing Cui, Keith A. Youker, Qinglin Yang, Sharlene M. Day, C.

Lin Chang, Jifeng Zhang, Yu-Hua Tseng, Chang-Qing Xie, Jacob Ilany, Jens C. Brüning,Rad GTPase Deficiency Leads to Cardiac Hypertrophy

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 2007 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation doi: 10.1161/CIRCULATIONAHA.107.707257

2007;116:2976-2983; originally published online December 3, 2007;Circulation.

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Chang et al., The Role of Rad in Cardiac Hypertrophy

Supplemental Text

Generation of Rad knock out (Rad-KO) mice

A genomic clone that included the murine Rad gene was isolated from a

129/SvJ1 mouse library. A targeting vector was designed to replace Exon 2 of the Rad

gene by a neomycin cassette and generation of mice was performed according to

standard procedures (Brninget al, 1998). Briefly, electroporation of the linearized vector

into 129/SvJ1 embryonic stem cells and the selection of G418-resistant transformants

were performed as described (Brninget al, 1998). G418-resistant colonies were first

analyzed for homologous recombination by PCR method and further confirmed by

Southern blot analysis after Hind III digestion with a 5' genomic probe that contained

sequences that reside outside the targeting vector. Correctly targeted clones were

injected into C57BL/6 blastocysts to produce chimeras, which were crossed with

C57BL/6 female mice to generate heterozygous Rad+/- mice. The Rad-KO and the

littermate control mice used in the current study were back crossed with C57BL/6

female mice for 4 generations. The resulting offspring were genotyped by PCR with the

following primers: Rad-forward 5’agtctgaacaggggtctacgagtg3’; Rad-reverse

5’tctggccctgtgtccgagttc3’; Rad-KO-forward 5’gagcacgtactcggatggaagc3’; Rad-KO-

reverse 5’gcgatagaaggcgatgcgctgc3’. The expected sizes of the wild-type allele and

deleted allele were 507bp and 347bp, respectively. Antibodies able to recognize the C-

terminus of Rad failed to detect any Rad expression (data not shown) in the heart of

these animals indicating that the strategy used resulted in complete depletion of the

Rad protein. Mice were housed in a specific pathogen-free facility. All animal

experiments were performed according to National Institutes of Health guidelines and

Chang et al., The Role of Rad in Cardiac Hypertrophy MS ID#: 707257-R3

were approved by the University Committee on Use and Care of Animals at the

University of Michigan.

Cardiac function analysis by cardiac catheterization

Cardiac catheterizations were performed as described previously2. In brief, sex-

matched, 10-week-old mice were anesthetized with avertin (375 mg/kg body weight,

i.p.). Mice were laid naturally on a warm cushion (37°C) and fixed with adhesive tape. A

1.4-F Millar Mikro-Tip catheter transducers (Model SPR-671, Millar Instruments Inc.,

Houston, TX,) was inserted through the right carotid artery into left ventricle, where

pressure and volume were recorded. We monitored the position of the tip by the length

of transducers advanced and observation of the pressure wave form. Hemodynamic

measurements were recorded at baseline and following injection of dobutamine (0 to 5

μg/kg body weight). Analog inputs from the pressure transducer were amplified using an

ARIA (Millar Instruments Inc.) amplifier and digitized with a data-acquisition system

(Harvard Apparatus) at a rate of 1,000 Hz. All parameters were calculated from the

average of 30 consecutive beats at the stabilized stage.

References:

1. Bruning JC, Michael MD, Winnay JN, Hayashi T, Horsch D, Accili D, Goodyear LJ,

Kahn CR. A muscle-specific insulin receptor knockout exhibits features of the

metabolic syndrome of NIDDM without altering glucose tolerance. Mol Cell. 1998;

Nov;2(5):559-69.

2


2. Peng X, Kraus MS, Wei H, Shen TL, Pariaut R, Alcaraz A, Ji G, Cheng L, Yang Q,

Kotlikoff MI, Chen J, Chien K, Gu H, Guan JL. Inactivation of focal adhesion

kinase in cardiomyocytes promotes eccentric cardiac hypertrophy and fibrosis in

mice. J Clin Invest. 2006;116:217-27.

Supplemental Figure Legend

Supplemental Figure S1: Rad mRNA expression levels in normal human tissues

Total RNA from various human tissues were purchased from Clontech (Mountain

View, CA). Northern blot was performed with a standard protocol. In brief, 10 mg of total

RNA from different human tissues was subjected to electrophoresis through 1%

formaldehyde-agarose gels. After transferring to nylon membranes, the RNA was cross-

linked to the membrane with a UV cross-linker (Stratagene, La Jolla, CA). A 32P-labeled

human Rad cDNA probe was generated using the random primer labeling system

(Invitrogen). The RNA image by ethidium bromide-staining was served as the loading

control.

Supplemental Figure S2: Characterization of Rad and CaMK IIδ RNAi.

A: Representative Western blots showing increased and decreased levels of Rad

expression in cardiomyocytes by Ad-Rad and Ad-Rad-RNAi (Ad-Rad i), respectively.

Neonatal rat cardiomyocytes were infected with 10 pfu/cell of Ad-GFP, Ad-Rad, Ad-Rad

I, or Ad-Rad-RNAi-Control (Ad-Rad ic) for 48 h in 10% FBS of DMEM medium. Control

3


is rat neonatal cardiomyocytes without adenoviral infections. β-tubulin was used as the

loading control. B: Representative Western blots showing a decreased level of CaMK II

expression in cardiomyocytes transfected with CaMK IIδ RNAi. Neonatal rat

cardiomyocytes were transfected with 20 nmol/L of CaMK IIδ RNAi oligonucleotides

(CaMK II i) or the scrambled CaMK IIδ RNAi oligonucleotides (CaMK II ic) using

Lipofectamine 2000 (Invitrogen) for 24h. Control is rat neonatal cardiomyocytes without

RNAi oligonucleotide transfections. β-tubulin was used as the loading control.

Supplemental Figure S3: Generation of Rad-deficient mice.

A: Schematic map of the Rad gene targeting strategy. The position of the exons

of mouse Rad gene is indicated as open boxes. H: restriction sites of Hind III; PGK-Neo:

neomycin resistance gene cassette driven by the phosphoglycerate kinase (PGK)

promoter; DTA: diphtheria toxin-A chain gene. The 5' probe and PCR primers shown in

the figure were used for the confirmation of homologous recombination in ES cells. B:

Representative image of genotyping results showing the confirmation of wild-type,

heterozygous, and homozygous Rad-KO mice. C: Representative Western bolts

showing no Rad expression in the heart of Rad-KO mice (Rad-/-,) and a significant

decrease of Rad expression in the heart of heterozygous mice (Rad+/-) compared to the

wild-type littermate control mice (Rad+/+)

Supplemental Figure S4: Rad deficiency affects cardiac systolic function in

response to the increased cardiac stress.

4


Cardiac catheterizations were performed in sex-matched, 10-week-old mice. A

1.4-F Millar Mikro-Tip catheter transducers (Model SPR-671, Millar Instruments Inc.

Houston, TX,) was inserted through the right carotid artery into left ventricle.

Hemodynamic measurements were recorded at baseline and following injection of

dobutamine (0 to 5 μg/kg body weight). All parameters were calculated from the

average of 30 consecutive beats at the stabilized stage (**p<0.01 vs 0 ng/g/min

dobutamine treatment).

Supplemental Figure S5: Representative images showing intact hearts from Rad-

KO mice and wild-type littermate controls at day 14 after TAC.

A: The harvested mouse hearts were washed with PBS for 3 times and then

fixed in 4% formalin for 24 h before being photographed with a Nikon digital camera. B:

Representative images showing the histological evaluation of longitudinal sections of

the whole hearts (Scale bar=1.25 mm).

Supplemental Figure S6: Rad physically interacts with CaM and CaMK II.

Neonatal rat cardiomyocyte lysates (200 μg) were subjected to

immunoprecipitation with anti-CaM or anti-CaMK II antibodies from Upstate

(Charlottesville, VA). Bound fractions were then used for Western blot analysis with the

Rad antibody. IP: immunoprecipitation; NC: negative control of immunoprecipitation;

Input: 10% of input of cell lysates; and IB: immunoblot.

5

Chang et al., Supplemental Fig. S1 A

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2H

17 kb

5’probe

Wild-type allele

Targeting construct

DTAH

PGK-Neo

H

Targeted allele

HH8.1 kb 10 kb

PCR primer pair

1 543H

1 543PGK-Neo

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507 bp347 bp

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Rad

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Rad: +/+ +/- -/-

Chang et al., Supplemental Fig. S4

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IP: input CaM CaMK II NC

IB: Rad

Chang et al., Supplemental Fig. S6Page 11

rad gtpase deficiency leads to cardiac hypertrophy

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