TRPC1 Channels Are Critical for Hypertrophic Signaling in the Heart

TRPC1 Channels Are Critical for Hypertrophic Signaling inthe Heart

Malini Seth, Zhu-Shan Zhang, Lan Mao, Victoria Graham, Jarrett Burch, Jonathan Stiber,Leonidas Tsiokas, Michelle Winn, Joel Abramowitz, Howard A. Rockman,

Lutz Birnbaumer, Paul Rosenberg

Rationale: Cardiac muscle adapts to increase workload by altering cardiomyocyte size and function resulting incardiac hypertrophy. G protein–coupled receptor signaling is known to govern the hypertrophic responsethrough the regulation of ion channel activity and downstream signaling in failing cardiomyocytes.

Objective: Transient receptor potential canonical (TRPC) channels are G protein–coupled receptor operatedchannels previously implicated in cardiac hypertrophy. Our objective of this study is to better understand howTRPC channels influence cardiomyocyte calcium signaling.

Methods and Results: Here, we used whole cell patch clamp of adult cardiomyocytes to show upregulation of anonselective cation current reminiscent of TRPC channels subjected to pressure overload. This TRPC currentcorresponds to the increased TRPC channel expression noted in hearts of mice subjected to pressure overload.Importantly, we show that mice lacking TRPC1 channels are missing this putative TRPC current. Moreover,Trpc1�/� mice fail to manifest evidence of maladaptive cardiac hypertrophy and maintain preserved cardiacfunction when subjected to hemodynamic stress and neurohormonal excess. In addition, we provide amechanistic basis for the protection conferred to Trpc1�/� mice as mechanosensitive signaling throughcalcineurin/NFAT, mTOR and Akt is altered in Trpc1�/� mice.

Conclusions: From these studies, we suggest that TRPC1 channels are critical for the adaptation to biomechanicalstress and TRPC dysregulation leads to maladaptive cardiac hypertrophy and failure. (Circ Res. 2009;105:1023-1030.)

Key Words: transient receptor potential channels � G protein receptor signaling � cardiac hypertrophy

Cardiac myocytes respond to changing mechanical work-loads by altering the frequency and amplitude of their

calcium transients.1,2 Encoded in these calcium transients aresignals that alter not only the immediate contractile responsebut also initiate and maintain a remodeling response thatadjusts cellular mass, ionic currents, kinetic properties ofcontractile proteins, and metabolic capacity.3 It is likely thatpersistence of these signals modulate the calcium signalingevents resulting in a hypertrophic response and adverseremodeling. To identify the proximal signals that regulatecardiac hypertrophy, attention has begun to focus on ionchannels because they may link mechanical activity to cellsignaling.4 Recent work has raised the possibility that hyper-trophic agonists linked to G-protein coupled receptors acti-vate calcium entry through transient receptor potential canon-ical (TRPC) channels.5–10

TRPC channels encompass a large family of nonselectivecation channels found in many different cell types.11 TRPCchannels are activated downstream of G-protein receptor

through the phospholipase C signaling by inositol trisphos-phate (TRPC1/4/5) or by diacyl glycerol (TRPC3/6/7).5,12

More recently TRPC1/6 channels were found to be mechano-sensitive channels that mediate nonselective cation entry inresponse to increased membrane stretch.6,7 These findingsraise interesting possibilities about TRPC channels as mech-anosensitive channels that may be operative during cardio-myocyte stretch associated with pressure overload. In fact,several groups have linked increased TRPC channel activityto cardiac hypertrophy and failure.8–10,13 TRPC1/C3/C6 havebeen found to be upregulated in response to pressure overloadand a model of calcineurin-mediated cardiomyopathy. More-over, transgenic mice overexpressing either TRPC3 orTRPC6 channels in the heart manifest an exaggerated hyper-trophic response to pressure overload or die prematurely fromheart failure.9,13 In contrast, a TRPC3 specific small moleculeinhibitor prevented the development of cardiac hypertrophyin wild-type (WT) mice subjected to pressure overload.14

Despite some evidence suggesting a link between TRPC

Original received November 6, 2008; resubmission received August 5, 2009; revised resubmission received September 7, 2009; accepted September15, 2009.

From the Department of Medicine (M.S., Z.-S.Z., L.M., V.G., J.B., J.S., M.W., H.A.R., P.R.), Duke University School of Medicine, Durham, NC;Department of Cell Biology (L.T.), University of Oklahoma Health Sciences Center, Oklahoma City; and Laboratory of Neurobiology (J.A., L.B.),National Institute of Environmental Health Sciences, Research Triangle Park, NC.

Correspondence to Paul Rosenberg, Suite 200, 4321 Medical Park Dr, Durham, NC 27704. E-mail [email protected]© 2009 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.109.206581

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channels and cardiac hypertrophy, the molecular mechanismby which TRPC channels contribute to cardiac calciumsignaling is not known. We therefore tested the hypothesisTRPC1 channels contribute to biomechanical signaling follow-ing pressure overload in the cardiomyocytes and we providedirect evidence that TRPC1 is a key mediator of the cardiachypertrophic response. Our findings support a concept thattherapeutic strategies designed to block TRPC1 channels mayhave clinical benefit in the hypertrophic failing heart.

MethodsAn expanded Methods section is available in the Online DataSupplement at http://circres.ahajournals.org. All animal experimen-tal procedures were reviewed and approved by the InstitutionalAnimal Care and Use Committees of Duke University.

In brief, Trpc1�/� and WT mice were maintained in a 129background for more greater than seven generations. Paired micewere used in each study. Cardiac stress was induced using transverseaortic constriction (TAC) surgery or chronic angiotensin infusion.Adult cardiomyocytes were prepared using Langendorff perfusion todisaggregate single cells. Patch clamp using whole cell techniqueand calcium imaging with Fura-2 acetoxymethyl ester were used tomeasure TRPC1 currents and calcium entry. Cardiac lysates wereprepared for biochemical assays of relevant signaling pathways.Cryosectioning of hearts was performed for histological studies.

ResultsTrpc1�/� and Cardiac HypertrophyWe first designed studies to test the hypothesis that TRPC1mediated calcium entry in cardiomyocytes activates hyper-trophic signaling. Trpc1�/� mice and WT mice were sub-jected to TAC to induce cardiac hypertrophy. Histologicalsections of hearts from WT mice demonstrated a markedincrease in left ventricle (LV) size after 8 weeks of TACcompared to sham operated mice (Figure 1A, top row). Incontrast, the hearts of Trpc1�/� mice did not demonstratesignificant cardiac hypertrophy (Figure 1A, top row), nor was

there an increase in collagen deposition as determined bySirius red staining compared to the heart sections of WT mice(Figure 1A, bottom row).

Serial echocardiograms (performed at 0, 4, and 8 weeks)demonstrated a progressive decline in the percentage frac-tional shortening of WT mice, whereas Trpc1�/� micemaintained preserved percentage fractional shortening over awide range of systolic pressure gradients (0 to 150 mm Hg)(Figure 1B and Online Table I). A detailed statistical analysisfor fractional shortening and LV mass/body weight (BW)ratio has been shown in Online Table III and Online Figure II.Invasive LV hemodynamic parameters measured from WTand Trpc1�/� mice showed significant differences in the LV�dP/dtmax and LV �dP/dtmin only after the TAC operation.These data indicate that the loss of TRPC1 is associated withpreserved contractility despite a marked increase in pressureload (Online Table I).

Consistent with the observations made by echocardiogra-phy and hemodynamics, we found marked difference incardiac mass between hearts of Trpc1�/� mice subjected toTAC compared to WT mice. The increase in cardiac massfollowing TAC was significantly reduced in the Trpc1�/�

mice as was evident by the change in LV mass/BW ratio(3.1�0.23 to 4.5�0.175, before and after TAC, respectively)as compared to WT mice (3.3�0.14 to 6.8�0.42) (Figure 1Cand Online Table I). Thus, it is apparent from these data thatTrpc1�/� mice respond to pressure overload with a modestincrease in cardiac mass and preserved cardiac function,whereas the same level of pressure overload in WT miceproduced significant cardiac impairment. We also consideredwhether changes in TRPC2–7 channel expression occurred inthe hearts of the Trpc1�/� mice as mechanism for theprotection seen in the INSC. In fact, TRPC channels (mRNAor protein) at baseline and after pressure overload did notdiffer between WT and Trpc1�/� mice (Online Figure I, Athrough D).

TRPC channels are known to be receptor operated cationchannels activated downstream of G-protein coupled recep-tors, eg, angiotensin II (Ang II), raising the possibility thatAng II mediated hypertrophic signaling may influence TRPCsignaling9,15–17 WT and Trpc1�/� mice infused with Ang II(1000 ng/kg per minute) for 4 weeks (28 days) to inducecardiac hypertrophy (Figure 1D and Online Table II).

INSC in Adult CardiomyocytesNext, we designed whole cell voltage clamp studies tomeasure TRPC currents from isolated adult cardiomyocytestaken from WT or Trpc1�/� mice. Solutions were configuredso as to limit voltage gated Ca2� and K� currents mainly byincluding inhibitors of L-type calcium channels and Cs� toblock K� channels. We used an inverse ramp protocol from�100 mV to �100 mV to limit voltage gated Na� channelcurrents and holding potential of 0 mV (see expandedMethods section in the Online Data Supplement).18 Figure 2Adisplays current–time plots recorded from WT (brown traces)and Trpc1�/� (blue traces) cardiomyocytes 8 weeks after theTAC operation. WT cardiomyocytes displayed a greatercurrent recorded at both �80 mV and �80 mV membranepotentials compared to Trpc1�/� cardiomyocytes. The non-

Non-standard Abbreviations and Acronyms

ANF atrial natriuretic factor

Ang II angiotensin II

AT1R angiotensin type 1 receptor

BNP brain natriuretic peptide

BW body weight

GPCR G protein–coupled receptor

INSC nonselective current

Iswell nonselective current in response to swelling

LV left ventricle

NFAT nuclear factor of activated T cells

OAG 1-oleoyl-2-acetyl-sn-glycerol

SERCA sarcoplasmic/endoplasmic calcium ATPase

shTRPC1 transient receptor potential canonical-1 short hairpin RNA

siTRPC1 transient receptor potential canonical-1 small interferingRNA

TAC transverse aortic constriction

TRPC transient receptor potential canonical

WT wild type

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selective currents were inhibited by gadolinium (Gd3�), aknown TRPC channel blocker (Figure 2A). We noted that thecurrent–voltage relationship recorded from sham and TACoperated mice was linear in shape with a reversal potential of0 mV, features reminiscent of nonselective TRPC channels(Figure 2B). Interestingly nonselective currents recordedfrom cardiomyocytes taken from Trpc1�/� mice displayedsimilar current–voltage relations with zero reversal potential(Figure 2C). However, the current density of the nonselectivecurrents recorded from Trpc1�/� cardiomyocytes, both shamand TAC operated mice, was markedly reduced compared tothe WT cardiomyocytes (Figure 2D).

To characterize the INSC recorded from cardiomyocytes,we sought evidence for the contributions of TRPC fromcation selectivity, pharmacological profiling and gatingmechanism (Figure 2E). Replacing Na� in the externalsolution with N-methyl-D-glucamine dramatically reduced thenonselective current by greater than 50% from WT cardio-myocytes indicating the current is in part permeable to Na�.In addition, the INSC was rapidly blocked by the addition ofgadolinium (Gd3�, 10 �mol/L) to the perfusion bath. Triva-lent cation block of the INSC is a characteristic feature ofTRPC currents.19 We also found that the putative TRPC1current was equally permeable to calcium and barium whichwould further suggest this current is in part contributed byTRPC1 (Figure 2E). We found no difference in the Li3�

sensitive currents in cardiomyocytes isolated from WT andTrpc1�/� mice, indicating no change in NCX1 (Na�/Ca2�

exchange) currents (data not shown).20 In addition, we foundthat immunolabeled TRPC1 only partially overlapped with thatof NCX1 (Online Figure I, E). When considered in total theseresults indicate that TRPC1 is likely to contribute to thenonselective cation background current recorded in adultcardiomyocytes.

Given the central role of the neurohormone angiotensin-II(Ang II) in the stretch activated signaling in the cardiomyo-cyte,21 we tested whether Ang II application influenced thenonselective cation current attributed to TRPC1. Ang IIstimulation of WT cardiomyocytes resulted in two-fold in-crease in the current density of the nonselective current(Figure 2F). In contrast, Ang II failed to augment the TRPCcurrent in cardiomyocytes isolated from Trpc1�/� mice(Figure 2F). We also tested whether TRPC1 currents wereactivated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a stablecell permeable analog of DAG (Figure 2G). Here, perfusionof cardiomyocytes with OAG (10 �mol/L) activated a non-selective current similar to that seen with Ang II. We foundthat the current density of OAG activated currents fromTrpc1�/� and WT cardiomyocytes were not different (Figure2G). These results indicate that TRPC1 contributes to the AngII–induced nonselective current, whereas TRPC1 does notinfluence the DAG-induced current. TRPC1 has also been

Figure 1. Trpc1�/� mice are protected from pressure overload and lack a significanthypertrophic response. A, Photomicrographs of hearts from WT (sham and TAC) andTrpc1�/� (sham and TAC) mice at 16 weeks of age. Histological sections are stainedwith hematoxylin/eosin (middle) and Sirius red (bottom). B (top), Echocardiographicmeasurements of fractional shortening (%) as a function of pressure gradient in TAC-operated WT and Trpc1�/� mice. *P�0.05 (see Online Table II). B (bottom), Averagepercentage fractional shortening from WT sham (n�6) and TAC (n�30) and Trpc1�/�

sham (n�5) and TAC (n�29). C (top), LV mass/BW ratio in WT sham and TAC andTrpc1�/� sham and TAC mice plotted against systolic pressure gradients measuredat study termination. C (bottom), LV mass/BW ratio in WT and Trpc1�/� in sham vsTAC mice. *P�0.05 in all pairs, as analyzed by Tukey–Kramer test. D, WT andTrpc1�/� mice were infused with 1000 ng/kg Ang II per minute for 4 weeks and then

euthanized to measure their heart weight/BW ratio. The increase in heart weight/BW ratio in Trpc1�/� was significantly lower than thatof WT. *P�0.01 WT vs Trpc1�/� Ang II–infused mice.

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implicated in store operated calcium entry as a SOC channelin many cell types including cardiomyocytes. However, wedid not find a difference in the rate of Ca2� entry followingstore depletion in Trpc1�/� neonatal cardiomyocytes com-pared to WT cells (Online Figure III). These findings supportour model in which TRPC1 channels respond to G proteinsignaling that is involved in the pressure overload response.

TRPC1 and the Stretch ResponseWe next determined whether cardiomyocytes from Trpc1�/�

mice responded differently to mechanical stretch than WTcardiomyocytes. Isolated neonatal cardiomyocytes subjectedto a cyclic stretch at 15% strain at 1-Hz frequency for 8 to 24hours duration expressed augmented levels of brain natri-uretic factor (BNP) and atrial natriuretic factor (ANF) in atime-dependent manner (Figure 3A) peaking at 24 hours witha 7 fold increase in the BNP and ANF expression. Interest-ingly, treating WT cells with losartan to block angiotensintype 1 receptors (AT1Rs) prevented the induction of thestretch gene program. Moreover, Trpc1�/� cardiomyocytesshowed no change in the expression of BNP and ANF overany time point examined. Thus, neonatal cardiomyocyteslacking TRPC1 displayed a blunted response to stretch incomparison to WT cardiomyocytes. The blocking of stretchresponse by losartan treatment suggests that TRPC1 mediatedstretch signaling is downstream of the AT1R.

It has been recently shown that mechanical stretch acti-vates AT1R in an agonist independent manner that can result

in activation of a nonselective cation current in vascularsmooth muscle cells.22 These results may explain the recentfindings demonstrating TRPC1 and TRPC6 channels asputative stretch activated channels.7 We, therefore, testedwhether stretch influences the TRPC1 current in adult car-diomyocytes. Here, osmotic stress applied to the cardiomyo-cytes, by changing the osmolarity of the perfusion bath from305 to 205 mOsm, increased the nonselective current (Iswell)in WT cardiomyocytes. The current–voltage relationship ofthe Iswell from adult cardiomyocytes strongly resembled thatobserved above for pressure overload and Ang II. Osmoticstress did not activate the nonselective current in cardiomyo-cytes lacking TRPC1 (Figure 3B and 3C). We furthercharacterized the Iswell in WT cardiomyocytes to determinewhether Ang II signaling was involved in the osmoticactivated currents. Inclusion of the phospholipase C blockerU-73122 in the pipette solution decreased the nonselectivecurrent at baseline and following cell swelling induced byosmotic stress (Figure 3D and 3F). We also demonstrate thatthe Iswell in WT cardiomyocytes signals through the Ang IIreceptor as the current was significantly blocked by losartan(10 �mol/L) compared to vehicle treated WT cardiomyocytes(Figure 3E and 3F). These data indicate Ang II signaling isfundamental to the current activated by cell swelling. More-over, WT cardiomyocytes treated with tarantula toxinGsMTx-4, an inhibitor of stretch-activated channels alsoblocked the nonselective current induced by cell swelling,

Figure 2. Pressure overload induces nonselective whole cell currents (INSC) in adult cardiomyocytes that are attributed to TRPC1. A,Examples of membrane current recorded from isolated cardiomyocytes from WT TAC (brown) and Trpc1�/� TAC (blue). INSC was nor-malized by membrane capacitance. The currents were recorded at �80 mV and �80 mV. B and C, Representative current–voltagerelationship of membrane currents in cardiomyocytes from WT sham and TAC (B) and Trpc1�/� sham and TAC mice (C). D, Groupmean values of INSC at �80 and �80 mV in WT sham (red filled bar, n�25) and TAC (brown bar, n�33); Trpc1�/� sham (light blue bar,n�27) and TAC (dark blue filled bar, n�27) cardiomyocytes. *P�0.05, WT TAC vs Trpc1�/� TAC. E, Relative group means of changesof peak membrane currents at �80 mV in the presence of barium (2 mmol/L, n�11), N-methyl-D-glucamine (NMDG) (n�12) and Gd3�

(n�10) in the external solutions. Open bar represents control (100%). *P�0.05, **P�0.01 vs control. F, Relative group means ofchanges (%) of peak membrane current INSC at �80 mV caused by perfusion of Ang II (10 �mol/L) in WT (red) and Trpc1�/� (blue) car-diomyocytes. *P�0.05, unstimulated vs Ang II; #P�0.05, WT vs Trpc1�/� (after Ang II perfusion). G, Relative group mean changes (%)of INSC at �80 mV after perfusion of OAG (50 �mol/L) in WT and Trpc1�/� cardiomyocytes. P�0.05 WT vs Trpc1�/�.

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providing additional evidence that TRPC1 acts as a stretchactivated channel (data not shown).6,23 Collectively, thesestudies show that Trpc1�/� cardiomyocytes, in comparison toWT cells, fail to respond to different forms of stretch asindicated by the lack of stretch activated nonselective cationcurrent (swelling and positive pressure) or changes in ANFand BNP mRNA expression (radial stretch). These resultsprovide evidence in support of our model in which TRPC1channels reside downstream of stretch-activated G protein–coupled receptor (GPCR) signaling to confer stretch depen-dent signaling associated with cardiac hypertrophy.

Loss of TRPC1 Alters Hypertrophic SignalingTo this point, our studies indicated that mice lacking TRPC1are protected from the deleterious effects of pressure over-load. Because it is now well established that calcium entrythrough TRPC channels influences calcineurin/NFAT (nu-clear factor of activated T cells) signaling in many cell typesincluding cardiomyocytes, we examined the phosphorylationstate of NFATC3 in cardiac lysates prepared from WT andTrpc1�/� mice as a marker of calcineurin/NFAT signaling.NFATC3 is dephosphorylated by calcineurin in pressure-overloaded hearts as was detected in the WT mice in Figure

4A. We then examined whether the loss of TRPC1 influencedNFAT signaling. First, we found a significant increase inphospho-NFATC3 in the heart lysates prepared from shamoperated Trpc1�/� mice compared to WT mice indicatingthat more NFATC3 exists in the inactive state in the shamoperated Trpc1�/� mice (Figure 4A). No differences in totalNFAT were seen among WT and Trpc1�/� mice, but agreater fraction of NFATC3 existed in the phosphorylatedstate in TAC operated Trpc1�/� mice compared to TACoperated WT mice (Figure 4A). These data suggest thatcalcineurin/NFAT activity in the hearts of mice lackingTRPC1 is less active at baseline and after pressure overload(Figure 4B).

We next designed studies using a transgenic NFAT indi-cator mice24,25 to further examine the relationship betweenTRPC1 and calcineurin/NFAT signaling. Neonatal cardio-myocytes from NFAT indicator mice were infected withadenoviruses carrying either TRPC1 small interfering RNA(siTRPC1) or control small interfering RNA (scrambled)constructs (Figure 4C). Cardiomyocytes were stimulated withAng II to alter the frequency of spontaneous calcium oscil-lations and activate the hypertrophic program (Figure 4D). Incells with TRPC1 silencing, we found a marked attenuation

Figure 3. A, TRPC1 influences the stretch acti-vated program. ANF and BNP normalized toGAPDH in response to 8, 16, and 24 hours ofcyclic stretch (15% strain) in WT and Trpc1�/�

neonatal cardiomyocytes. *P�0.01 (WTstretched [8, 16, and 24 hours vs 0 time pointfor ANF and BNP]). Losartan (24�L)(10 mmol/L) incubated cardiomyocytes werestretch for 24 hours. B through E, Current–volt-age relationships of INSC recorded from WT (B)and Trpc1�/� (C) adult cardiomyocytes in iso-tonic and hypotonic solutions. Osmotic-induced currents were blocked in WT cardio-myocytes by phospholipase C (PLC) inhibitionby including 10 �mol/L U73122 in the pipettesolution or by AT1R blockade with 10 �mol/Llosartan. F, Group mean changes of INSC at�80 mV caused by osmotic stress in cardio-myocytes of WT (red bar, n�4) and Trpc1�/�

(blue bar, n�6) mice WT cardiomyocytes withphospholipase C blockade U73122 (purple bar,N�5) or AT1R with losartan (green bars N�5).*P�0.01, Trpc1�/� vs WT; #P�0.05, losartan-stimulated cells vs WT cells.

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of the calcium oscillations compared to control silenced cells(Figure 4D through 4F). This change in the oscillationfrequency in the siTRPC1 neonatal cardiomyocytes corre-sponded to a reduction in NFAT transactivation, as measuredby LacZ reporter gene compared to control neonatal cardio-myocytes. These results support our in vivo studies suggest-ing that TRPC1 channels provide calcium entry needed forhypertrophic signaling through the calcineurin/NFAT signal-ing pathway in cardiomyocytes (Figure 4G).

TRPC1 and Hypertrophic Gene ExpressionCardiac hypertrophy with diminished cardiac function haslong been associated with changes in profiles of gene expres-sion.26 We, therefore, measured the mRNA for BNP and ANFfrom the hearts of Trpc1�/� and WT mice (sham- andTAC-operated). These neurohormones are released by car-diomyocytes subjected to increased wall stress and areimportant biomarkers for the activation of the fetal geneprogram in response to cardiac stress.27 WT mice subjected topressure overload were found to have a nine fold increase inthe expression of BNP and 16-fold increase in ANF; whereasTrpc1�/� mice subjected to TAC showed no significantchange in BNP and ANF expression (Figure 5A). We alsofound that the expression of the calcium pump, sarcoplasmic/endoplasmic calcium ATPase (SERCA)2a, was decreased inthe TAC-WT mice, whereas there was no correspondingreduction in SERCA2a expression in Trpc1�/� mice sub-jected to TAC. SERCA2a is known to be diminished in heartfailure and may contribute to abnormal calcium signaling.28 Itis clear from these studies that the attenuated cardiac hyper-trophy and preserved cardiac function observed in micelacking TRPC1 accompanied no changes in the gene expres-sion profile associated with maladaptive cardiac hypertrophy.

To better understand the survival advantage offered toTrpc1�/� mice in response to pressure overload, we nexttested the hypothesis that Akt signaling pathways associatedwith cardiomyocyte survival would be enhanced in theTrpc1�/� mice. In fact, we were unable to discern differencesin the level of phospho-Akt between the sham and TACoperation in WT mice. In contrast, Akt phosphorylation wasnotably increased in heart lysates prepared from Trpc1�/�

mice following the TAC operation. Likewise, phospho-mTOR (mammalian target of rapamycin) was markedlyincreased in the heart lysates of Trpc1�/� TAC operated micecompared to WT mice. No differences were noted in the totalAkt or mTOR expression of WT or Trpc1�/� hearts (Figure5B and 5C). These results suggest that the preserved cardiacfunction and minimal changes to cardiac mass followingpressure overload seen in mice lacking TRPC1 may be due inpart to the preserved Akt and mTOR signaling which areassociated with cell survival.

DiscussionIn the present work, we reveal a highly specific role forTRPC1 as a nonselective cation channel in cardiomyocytesthat participates in cardiac hypertrophic signaling. In partic-ular, we show that mice lacking TRPC1 are protected fromthe deleterious effects of increased intracardiac pressuresimposed by various forms of cardiac stress (pressure overloador chronic Ang II stimulation). These findings provide thestrongest support to date that TRPC channels play a signifi-cant role in the pathophysiology of cardiac hypertrophy.

Recent reports have suggested that TRPC1 and TRPC6 arestretch activated cation channels.7,22 How the TRPC channelssense changes in stretch and what molecular mechanism leadsto TRPC1 channel gating is presently unknown. It has been

Figure 4. Calcineurin/NFAT signaling isless sensitized in Trpc1�/� mice follow-ing pressure overload. A, Immunoblot-ting for p-NFATC3, NFATC3, andGAPDH from cardiac lysates of WT andTrpc1�/� mice. B, Quantification ofp-NFAT/NFATC3 (total) in WT (shamand TAC) and Trpc1�/� (sham and TAC)mice. *P�0.03, WT TAC vs sham andWT sham vs Trpc1�/� sham. C, Silenc-ing TRPC1 expression in neonatal car-diomyocytes with adenovirus deliveredTRPC1 short hairpin RNA (shTRPC1)compared to noninfected control (NV)and sh-scrambled adenoviral infectedcells. D through E, Ang II increases thefrequency of Ca2� oscillations in WTcardiomyocytes (D) but not in cardio-myocytes infected with siTRPC1 adeno-virus (E). F, Average relative oscillationfrequency (MHz) from neonatal cardio-myocytes (control and siTRPC1-infected) stimulated with Ang II.*P�0.03, control virus–infected cells vsshTRPC1-infected cells stimulated withAng II. G, LacZ was measured from car-diomyocytes isolated from NFAT indica-tor mice. Cardiomyocytes infected withadenovirus for control or shTRPC1 andthen stimulated with vehicle or Ang II for

18 hours. *P�0.01, control virus–infected cells vs shTRPC1-infected cells stimulated with Ang II. CTL indicates control.

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proposed that TRPC channels either respond directly todeformation of the lipids in plasma membranes or as adownstream consequence of AT1R activation.22,29 We haverecently suggested that the scaffolding protein homer-1 linksTRPC1 with the cytoskeleton in skeletal muscle.30 In thepresent study, cardiomyocytes lacking TRPC1 are protectedfrom pressure overload which further supports our model.Under normal hemodynamic conditions TRPC1 plays a rolein adjusting cytoskeletal stiffness associated with loadingconditions. However, under pathological workloads TRPC1currents are augmented in response to more TRPC1 channelexpression resulting in the activation of adverse remodelingassociated with cardiac hypertrophy. It will be important toknow in future studies whether TRPC channels connected tothe homer-cytoskeleton are influenced by GPCR activation ashas been suggested for TRPC1, homer and mGluR receptorsin neurons.31,32

TRPC channels are nonselective cation channels and cationflux through TRPC channels have been implicated in othermammalian physiological processes including learning andmemory in the brain and glomerular slit diaphragm functionin kidney epithelial cells.33,34 The present work, along withother recent reports, establishes that TRPC channels arelocated at the sarcolemma where TRPC1 mediated cation fluxinfluences the transition from adaptive to maladaptive cardiachypertrophy. What is the common link between TRPCchannel activity in neurons, podocytes and cardiomyocytes?In neurons TRPC channels are important modulators of thecytoskeleton where growth cones turn and extend processesin response to local growth factor concentrations.34–38 In the

podocyte, activating mutations in TRPC6 disrupt the normalfunction of the foot process which is highly dependent on theintegrity of the actin cytoskeleton and GPCR signaling.39,40

According to our model, TRPC channels act downstream ofGPCR signaling that influences membrane–cytoskeletal in-teractions to accommodate specific cellular signaling events.Under conditions of pressure overload, TRPC1 calcium entryis likely to influence hypertrophic signaling resulting in theremodeling response that leads to heart failure. Future effortsto block these channels may offer novel strategies to treatcardiac hypertrophy and failure.

AcknowledgmentsWe thank Dr Sanjeev Ahuja and Dr Cary Ward.

Sources of FundingThis work was supported by NIH grant R01-HL093470 (to P.R.), theMandel Center at Duke (P.R.), and a Muscular Dystrophy Associa-tion research award (P.R.) and, in part, by the Intramural Program ofthe NIH, National Institute of Environmental Health Sciences (toJ.A. and L.B.).

DisclosuresNone.

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Figure 5. Hypertrophic signaling in WT andTrpc1�/� mice. A, Relative change in theexpression of ANF, BNP, SERCA2a, and�-myosin heavy chain (�MHC) (normalizedto GAPDH) in WT and Trpc1�/� mice sub-jected to sham or TAC operation. *P�0.03,WT TAC vs sham mice and Trpc1�/� TACvs sham mice. B, Immunoblotting for Aktand p-Akt in cardiac lysates prepared fromWT and Trpc1�/� mice. *P�0.03, WT TACvs WT sham and Trpc1�/� sham andTrpc1�/� TAC. C, Immunoblotting formTOR and p-mTOR in cardiac lysates pre-pared from Trpc1�/� (TAC vs sham mice)and WT (TAC vs sham mice) *P�0.03, WTTAC vs WT sham and Trpc1�/� sham vsTrpc1�/� TAC.

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SUPPLEMENT MATERIAL Detailed methods for Seth et al. TRPC1 KO mice: The TRPC1 knockout mice were generated and genotyped as previously described 1. The TRPC1 knockout mice were bred with 129sv WT mice for 9-10 generations to generate pure 129sv mice; for subsequent studies Trpc1+/- mice were bred to generate WT and Trpc1-/- mice. We have established TRPC6 null mice in both the C57/bl6 and 129sv backgrounds. We conducted studies (TAC and Ang-II) in small numbers of animals in both backgrounds but studies in this manuscript reflect those performed with 129 animals as they have similar background to the TRPC1 studies. WT animals in the revised studies are matched to littermate controls. In vivo pressure overload: 8-10 weeks old mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (2.5 mg/kg), and TAC was performed as described previously 2. Eight weeks after surgery, the trans-stenotic pressure gradient was assessed by recording simultaneous measurement of right carotid and left axillary arterial pressures. Histological analysis: Histological analysis was performed using standard techniques as described previously 3. Excised hearts were rinsed in PBS, fixed in 4% Para formaldehyde for 16h at 4°C and dehydrated in a series of ethanol washes. Samples were subsequently cleared in xylene and mounted in paraffin. Sections of 7µM in thickness were cut and stained with Hematoxylin and Eosin to analyze tissue morphology or Sirius Red to analyze collagen content and fibrosis. Whole-cell Patch Clamp Recordings: Patch clamp experiments were performed to record currents in the whole cell mode with pipettes filled with a low chloride pipette solution containing 3 mM cesium aspartate, 8 mM MgCl2, 10 mM HEPES, 5 mM EGTA, pH 7.2 (with CsOH), 305 mOsm (with d-Mannitol). The extracellular bath solution containing 130 mM sodium aspartate, 4mM CsCl, 6 mM NaCl, 2mM CaCl2, 1mM MgCl2, 10 mM HEPES, 10 mM glucose, pH 7.4 (with NaOH), 315 mOsm (with d-Mannitol). Potassium channel activity was blocked by replacing potassium with cesium in the solutions. L-type Ca2+ channel activity was blocked by verapamil 10μM in external solution. The voltage-dependent sodium channel was inactivated by the stimulation protocol, and chloride current was minimized by a reduced and equal chloride ion concentration in the external and internal solution. The osmolarity of each solution was verified with a freezing-point osmometer (Advanced Instruments). Currents were recorded using an Axonpatch-200A amplifier with Digidata 1200 interface and analyzed with pCLAMP software. Currents were induced by 200 ms voltage ramp protocols (1 mV/ms, from 100mV to –100mV), at a holding potential 0mV. Experiments were performed at room temperature with a sample rate of 4 kHz (filter 2 kHz). Cell Culture and stretching of cardiomyocytes: Neonatal ventricular cardiomyocytes were prepared from 1-2 day old mice as described previously 4. Stretching of cardiomyocytes was carried out by plating the cells at a density of 1×105 /cm2 on silicone rubber dishes. Cardiomyocytes were cyclically stretched to 15 % strain at a frequency of 1Hz using the FX-4000 Tension Plus cell stretcher for 8 - 24hours. Cells were harvested after 8, 16 and 24 h and RNA was isolated from the cells using the TRIZOL reagent (Invitrogen) according to the manufacturer’s protocol.  Ang­II infusion of mice: A 28-day infusion of Ang II (1000 ng/kg/min; Sigma-Aldrich) dissolved in sterile saline was performed using appropriately primed osmotic mini-pumps (Alzet model 2004; Alza Corp) implanted subcutaneously in the dorsal region, under isoflurane anesthesia (3%). Real-Time RT- PCR: Total RNA was isolated from the culture neonatal ventricular myocytes or adult mouse hearts using TRIZOL (Invitrogen) according to the manufacturer’s protocol. cDNA was prepared from the RNA using the iscript cDNA synthesis kit (Bio-Rad). Taqman primers and probes for mouse TRPC1, 3, 4, 6, BNP, ANF, GAPDH, β Myosin Heavy Chain, SERCA 2a and 18S were purchased from Applied Biosystems. Real-time quantitative PCR was carried out using the Applied biosystems 7000 Sequence Detection System. The threshold count values were normalized to either 18S or GAPDH to calculate the fold change. Immunostaining: For immunofluorescence studies, mouse left ventricular cardiomyocytes were isolated from 8 week old mice enzymatically using a protocol described previously 5 were isolated and stained for TRPC1 with IF-1 antibody or a C-terminal polyclonal antibody 3, 6 . TRPC3 polyclonal antibodies were generated against the C-terminal domain and are described at www.Larial.com. Secondary antibodies were Alexa-Fluor AF 488 anti-mouse and Alexa-Fluor AF555 anti-rabbit. The immunostaining was imaged on a laser scanning Zeiss 510 confocal microscope at 63X magnification. Activation of Akt , mTOR and NFATc4:  Activation of Akt , mTOR and NFATc4 was assessed from LV total extracts as the capacity to phosphorylate Akt and mTOR and detected by immunoblotting with antibodies specific for 

phosphorylated and total forms of Akt/mTOR /NFATc4. Activation/Inactivation of these pathways was calculated by the ratio of phosphorylated form to the total.  Echocardiography and hemodynamic measurements: Transthoracic 2-dimensional guided M-mode echocardiography was

performed in conscious mice using an HDI 5000 echocardiograph (Philips) as previously described 2. Cardiac catheterization of anesthesized mice was carried out as described in the cited reference 7. Data was recorded digitally at 1000Hz with PVAN analysis software (Millar Instruments). Trpc1 silencing and Angiotensin-II stimulation: Neonatal cardiomyocytes from rat or NRE-LACZ mice 8 were grown to confluency and serum starved for 48 hours before infecting with the adenovirus for scrambled and TRPC1silencing constructs3 and harvested after 72 hours or stained for LACZ. The cells were stimulated with (1µM, Ang-II) 48 h after infection for LACZ staining or for stimulation of hypertrophic oscillatory response. Store operated calcium entry: The neonatal cardiomyocytes were imaged as described in 3 . The cells were stimulated with CPA and ryanodine, CPA, ryanodine and caffeine, CPA and ryanodine in divalent free medium and then calcium was added back to measure store operated calcium entry. Statistics: Data are presented as means ± SEM. Unpaired Student’s t test was used for comparison between two groups and ANOVA with Tukey-Kramer’s test was used for comparison among multiple groups. Values of P<0.05 was considered significant.

Online Supplemental Results Pressure Overload. The LV+dP/dtmax measured from sham and TAC operated WT mice declined from 9206±1075 mmHg/s to 7402±912 mmHg/s consistent with deterioration in LV function often seen with chronic pressure overload. In contrast, LV+dP/dtmax measured from the hearts of Trpc1-/- mice significantly increased from 8876±970 mmHg/s in the sham operated mice to 10,778±315 mmHg/s in the TAC operated Trpc1-/- mice (supplemental table 1).

WT sham (8)

WT TAC (30)

Trpc1-/- sham (6)

Trpc1-/-TAC (29)

ECHO LVEDd (mm) 3.5±0.108 4.61±0.16 3.08±0.145 3.6 ± 0.08

LVESD ( mm)

1.3±0.089

3.01±0.20

1.06±0.074

1.6 ± 0.08

IVS (mm) 0.99±0.055 1.14±0.17 1.00±0.08 1.08 ± 0.03 PW (mm) 1.00±0.041 1.01±0.035 0.96±0.05 1.10 ± 0.03

FS(%) 63.6±2.1 37±2.3 62±3.1 56 ± 1.50 Vcfc(circ/s) 5.13±0.36 2.80±0.20 4.9±0.22 4.7 ± 0.23 HR (bpm) 592±29.9 600±16.8 713±59 604 ± 20.0

Organ morphometry

BW (g) 23.8±1.3 26.1±0.80 22.8±1.07 29.14±0.61 LV/BW (mg/g) 3.34±0.136 6.95 ±0.32 3.1±0.23 4.44 ±0.14 H/BW (mg/g) 4.84±0.084 10.5±0.60 4.5±0.26 6.4±0.24

Lung/BW (mg/g) 3.72±0.44 6.04±0.6 4.36±1.01 4.85±0.33

Hemodynamics WT sham

(N=4) WT TAC

(N=8) Trpc1-/- sham

(N=5) Trpc1-/- TAC

(N=7) LVESP 101.5±4.2 173±18.7 99.6±2.2 207±6 LVEDP 1.9±0.98 14±4.2 3.2±1.2 7±3.63

dP/dt max 9201±847 7402±912 8876±970 10738±315 dP/dt min -8069±896 -7034±911 -7580±915 -10276±350

HR 447±9.3 394±25 423±41 400±12.5 SP (prox) 180±14 199±5.4 SP (distal) 93±8 115±5.8

Pressure gradient 0 80±30.2 0 84±9.4 P<0.01 for WT TAC versus Trpc1-/- TAC for dp/dt max and dp/dt min Online Table 1

Angiotensin induced cardiac hypertrophy. Cardiac mass of WT mice increased significantly following Ang-II perfusion indicating the hearts of these mice were hypertrophic, whereas Trpc1-/- mice exposed to Ang-II for 4 weeks displayed a much smaller change in cardiac mass (Figure 1D). Ang-II chronic infusion resulted in a significant change in the LV hemodynamics of WT and Trpc1-/- mice where the change in contractility was similar between both groups (supplemental Table 2). These results, when considered along with the TAC model of cardiac hypertrophy, provide additional evidence that mice lacking TRPC1 are protected from the deleterious effects of chronic hemodynamic and neurohormonal excess (TAC procedure and Ang-II infusion).

WT (vehicle) (N=6)

WT (ANG-II) (N=10)

Trpc1-/- (Vehicle)

(N=5)

Trpc1-/-

(ANG-II) (N=7)

ECHO LVEDd (mm) 3.2±0.11 4.1±0.3 3.14±0.14 3.94±0.06

LVESD ( mm)

1.3±0.09

1.94±0.3 1.1±0.05

1.61±0.05

IVS (mm) 1.0±0.09 0.88±0.06 0.99±0.05 0.85±0.02 PW (mm) 0.96±0.05 0.91±0.03 1.00±0.041 0.91±0.01

FS(%) 63±4.6 55±4.3 64±2.5 60 ± 0.6 Vcfc(circ/s) 5.1±0.36 4.15±0.3 4.81±0.21 4.61±0.1 HR (bpm) 592±29.8 587±27.2 713±25.2 547 ± 20.3

Organ morphometry

BW (g) 21.4±1.3 25.08±0.30 22.8±1.07 26.8±0.55 H/BW (mg/g) 4.72±0.32 6.6±0.24 4.7±0.26 5.50±0.15

Hemodynamics

WT (vehicle)

(N=5)

WT (ANG-II)

(N=6)

Trpc1-/- (vehicle)

(N=6)

Trpc1-/- (ANG-II)

(N=6)

LVESP 98.6±4.3 106.4±3.2 100±2 113±3.5 LVEDP 1.6±0.32 1.91±0.82 3.2±1.1 2.53±0.73

dP/dt max 8489±823 10582±910 9119±829 10869±390 dP/dt min -7552±1152 -8934±692 -7595±749 -10116±560

HR 411±21 452±29 425±32 485±34 Fractional shortening: P<0.08 (not significant) (WT Ang-II and Trpc1-/- Ang-II) H/BW: P<0.001(WT Ang-II and Trpc1-/- Ang-II) Online table 2

Analysis of Variance by One-way Anova Percent Fractional Shortening

Source DF Sum of Squares

Mean Square

F ratio Probability>F

Type 3 5752.3 1917.50 22.67 <0.0001* Error 48 4059.1 84.57

C.total 51 9811.7 Means for one-way Anova

Level Number Mean Standard Error

Lower 95% Upper 95%

Trpc1-/- sham

6 62.8 3.75 55.3 70.4

Trpc1-/- TAC

29 56.8 2.05 52.8 61.0

WT sham 8 62.8 3.25 56.3 69.4 WT TAC 30 37.8 2.16 33.5 42.2

LV/BW (mg/g)

Source DF Sum of Squares

Mean Square

F ratio Probability>F

Type 3 186.8 62.3 35.8 <0.0001* Error 51 88.8 1.7

C.total 54 275.6 Means for one-way Anova

Level Number Mean Standard Error

Lower 95% Upper 95%

Trpc1-/- sham

6 3.1 0.54 1.99 4.15

Trpc1-/- TAC

29 4.5 0.29 3.94 5.13

WT sham 8 3.3 0.44 2.45 4.22 WT TAC 30 7.7 0.29 7.06 8.24

Standard error uses a pooled estimate of error variance Online table 3

Online Figure 1 Online figure legend 1Increased protein expression of TRPC1 in mouse model of cardiac hypertrophy. (A) Western blot for TRPC1 in WT and Trpc1-/- heart lysates to test the specificity of the antibody (1F-1 for TRPC1).(B)Western blot analysis for TRPC1 in the hearts from the WT sham and TAC operated mice after 4 and 8 weeks of banding. (C) Western blot analysis for TRPC3 and 6 in the hearts from the WT sham and TAC and Trpc1-/- sham and TAC after 8 weeks of TAC. (D) RNA was isolated from the hearts of WT and Trpc-/- and real time PCR was carried out using the cDNA for TRPC channels (1-7) (E)Adult cardiomyocytes from WT (A-C) and Trpc1-/- (D-F) mice stained with antibodies for NCX1 (A and D), TRPC1 (B and E) and merge (C and F).

Online Figure 2 Online figure legend 2 (A) Analysis of variance (ANOVA) of the change in percent fractional shortening in four groups of mice; Trpc1 -/- sham (light blue), mean fractional shortening is 63% (n=6), the lower 95% limit is 55.3 and the upper 95% is 70.4; TAC (dark blue), mean fractional shortening is 57% (n=29) and the upper 95% is 61 and lower 95% is 53. WT sham (light red), mean fractional shortening is 63% (n=8) and the upper 95% is 69 and the lower 95% is 56, TAC (dark red) average fractional shortening is 38% (n=30) and the upper 95% limit is 42 and the lower 95% limit is 33.4 in all pairs as analyzed by Tukey-Kramer’s test. (B) ANOVA of the change in LV/BW (mg/g) ratio in four groups of mice; Trpc1 -/- sham (light blue), mean LV/BW ratio is 3.1 (n=6), upper 95% is 4.1 and lower 95% is 2 ; TAC (dark blue), mean is 4.5, upper 95% is 5 and lower 95% is 3.9. WT sham (red) mean ratio is 3.2, upper 95% is 4.2 and lower 95% is 2.5, TAC (brown) mean ratio is 7.5 and the upper 95% limit is 8.1 whereas lower 95% limit is 6.9. P < 0.05 in all pairs as analyzed by Tukey-Kramer’s test.

Online Figure 3 Online figure legend 3 (A) Neonatal cardiomyocytes from WT and Trpc1-/- were treated with CPA in zero calcium solution and the store operated calcium entry was measured by adding back calcium. Red is for WT and blue is for Trpc1-/- . (B) Average rate of calcium entry in WT (N=50) and Trpc1-/-(N=38) neonatal cardiomyocytes. (C) Average peak amplitude of calcium reentry in the neonatal cardiomyocytes from WT and Trpc1-/- mice. Supplemental References 1. Dietrich A, Kalwa H, Storch U, Mederos YSM, Salanova B, Pinkenburg O, Dubrovska G, Essin K, Gollasch M,

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3. Stiber JA, Zhang ZS, Burch J, Eu JP, Zhang S, Truskey GA, Seth M, Yamaguchi N, Meissner G, Shah R, Worley PF, Williams RS, Rosenberg PB. Mice lacking Homer 1 exhibit a skeletal myopathy characterized by abnormal transient receptor potential channel activity. Molecular and cellular biology. 2008;28(8):2637-2647.

4. Seth M, Sumbilla C, Mullen SP, Lewis D, Klein MG, Hussain A, Soboloff J, Gill DL, Inesi G. Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) gene silencing and remodeling of the Ca2+ signaling mechanism in cardiac myocytes. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(47):16683-16688.

5. Zhou YY, Wang SQ, Zhu WZ, Chruscinski A, Kobilka BK, Ziman B, Wang S, Lakatta EG, Cheng H, Xiao RP. Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology. Am J Physiol Heart Circ Physiol. 2000;279(1):H429-436.

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7. Lima B, Lam GK, Xie L, Diesen DL, Villamizar N, Nienaber J, Messina E, Bowles D, Kontos CD, Hare JM, Stamler JS, Rockman HA. Endogenous S-nitrosothiols protect against myocardial injury. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(15):6297-6302.

8. Rosenberg P, Hawkins A, Stiber J, Shelton JM, Hutcheson K, Bassel-Duby R, Shin DM, Yan Z, Williams RS. TRPC3 channels confer cellular memory of recent neuromuscular activity. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(25):9387-9392.

and Paul RosenbergLeonidas Tsiokas, Michelle Winn, Joel Abramowitz, Howard A. Rockman, Lutz Birnbaumer

Malini Seth, Zhu-Shan Zhang, Lan Mao, Victoria Graham, Jarrett Burch, Jonathan Stiber,TRPC1 Channels Are Critical for Hypertrophic Signaling in the Heart

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 2009 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

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