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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 335, No. 3
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 173005/3638461
JPET 335:572–579, 2010 Printed in U.S.A.

Selectively Engaging ␤-Arrestins at the Angiotensin II Type 1


Receptor Reduces Blood Pressure and Increases Cardiac
Performance□S

Jonathan D. Violin, Scott M. DeWire, Dennis Yamashita, David H. Rominger, Lisa Nguyen,
Kevin Schiller, Erin J. Whalen, Maxine Gowen, and Michael W. Lark
Trevena Inc., King of Prussia, Pennsylvania
Received July 15, 2010; accepted August 25, 2010

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ABSTRACT
Biased G protein-coupled receptor ligands engage subsets of the thase phosphorylation via ␤-arrestin coupling. Consistent with
receptor signals normally stimulated by unbiased agonists. How- ␤-arrestin efficacy, and unlike unbiased antagonists, TRV120027
ever, it is unclear whether ligand bias can elicit differentiated increased cardiomyocyte contractility in vitro. In rats, TRV120027
pharmacology in vivo. Here, we describe the discovery of a po- reduced mean arterial pressure, as did the unbiased antagonists
tent, selective ␤-arrestin biased ligand of the angiotensin II losartan and telmisartan. However, unlike the unbiased antago-
type 1 receptor. TRV120027 (Sar-Arg-Val-Tyr-Ile-His-Pro-D- nists, which decreased cardiac performance, TRV120027 in-
Ala-OH) competitively antagonizes angiotensin II-stimulated creased cardiac performance and preserved cardiac stroke vol-
G protein signaling, but stimulates ␤-arrestin recruitment and ume. These striking differences in vivo between unbiased and
activates several kinase pathways, including p42/44 mitogen- ␤-arrestin biased ligands validate the use of biased ligands to
activated protein kinase, Src, and endothelial nitric-oxide syn- selectively target specific receptor functions in drug discovery.

Introduction dition to off-target side effects, these “on-target” adverse


effects have historically presented a major barrier to the
Drugs targeting G protein-coupled receptors (GPCRs) are
development of safe, effective therapeutics.
used in a wide range of diseases. Indeed, modulation of
However, not all GPCR signaling derives from G protein
GPCR function is one of the most common approaches to drug
signaling; GPCRs can activate parallel and sometimes dis-
discovery, either with agonists to mimic endogenous receptor
tinct signals. The most general of these are mediated by
activation or antagonists to block endogenous agonist bind-
ing. These strategies have successfully targeted dozens of ␤-arrestins, which bind to activated receptors to desensitize
GPCRs in the discovery of new therapeutic agents (Hopkins G protein signaling, promote receptor internalization, and
and Groom, 2002). However, the network of signaling events activate distinct signal transduction cascades that can be
downstream of a receptor is complex and usually regulates independent of G protein coupling (Reiter and Lefkowitz,
multiple cellular and tissue responses, not all of which are 2006; DeWire et al., 2007). Nearly all GPCRs will couple to at
intended targets of classic agonists and antagonists. In ad- least one G protein and at least one ␤-arrestin, and in many
cases the physiological effects of a single agonist activating a
GPCR can be separated into ␤-arrestin- and G protein-medi-
All work was funded by Trevena Inc., a privately held drug discovery ated components (Schmid and Bohn, 2009).
company.
Article, publication date, and citation information can be found at Other work has shown that the experimental separation of
http://jpet.aspetjournals.org. GPCR effects can also be achieved pharmacologically: certain
doi:10.1124/jpet.110.173005.
□S The online version of this article (available at http://jpet.aspetjournals.org)
GPCR ligands can selectively activate subsets of receptor
contains supplemental material. signals (Wei et al., 2003; Gesty-Palmer et al., 2006; Mailman,

ABBREVIATIONS: GPCR, G protein-coupled receptor; AngII, angiotensin II; AT1R, angiotensin II type 1 receptor; AT2R, angiotensin II type 2
receptor; SII, 1Sar, 4Ile, 8Ile-angiotensin II; ARB, angiotensin receptor blocker; eNOS, endothelial nitric-oxide synthase; MAPK, mitogen-activated
protein kinase; FAK, focal adhesion kinase; MAP, mean arterial pressure; PRSW, preload recruitable stroke work; PV, pressure volume; ESPVR,
end systolic pressure volume relationship; ANOVA, analysis of variance; HEK, human embryonic kidney; MEM, modified Eagle’s medium; FBS,
fetal bovine serum; IP1, inositol monophosphate; RVU, relative volume unit; ERK, extracellular signal-regulated kinase; U2-OS, U2-osteosarcoma;
siRNA, small interfering RNA; TRV120023, Sar-Arg-Val-Tyr-Lys-His-Pro-Ala-OH; TRV120026, Sar-Arg-Val-Tyr-Tyr-His-Pro-NH2; TRV120027,
Sar-Arg-Val-Tyr-Ile-His-Pro-D-Ala-OH.

572
AT1R ␤-Arrestin Bias Improves Cardiac Function 573
2007; Tran et al., 2009; Zidar et al., 2009). These “biased ␤-Arrestin Recruitment. The PathHunter protein complemen-
ligands” are agonists when assaying some receptor functions, tation assay (DiscoveRx Corporation, Fremont, CA) was performed
but they are antagonists or even inverse agonists when as- according to the manufacturer’s protocol and read for chemilumines-
saying other receptor functions (Violin and Lefkowitz, 2007; cent signaling on a PheraStar reader (BMG Labtech, Durham, NC).
Kenakin, 2009). One of the most studied GPCRs in this In brief, complementary halves of ␤-galactosidase were genetically
fused to the carboxyl termini of the AT1R and ␤-arrestin2. When
regard is the angiotensin II type 1 receptor (AT1R). This
cotransfected, the two fusion proteins serve as a proximity sensor;
receptor engages all three classes of ␤-arrestin function: de- when ␤-arrestin2 translocates to active receptor, the ␤-galactosidase
sensitization (Violin et al., 2006), internalization (Kule et al., fragments interact to form a functional enzyme, which is detected by
2004), and signaling (Barnes et al., 2005; DeWire et al., 2008; a chemoluminescent substrate.
Ahn et al., 2009). It was also one of the first receptors for Inositol Monophosphate Accumulation. IP1 was measured
which a ␤-arrestin biased ligand was described: 1Sar, 4Ile, with the IP-One Tb HTRF kit (Cisbio, Bedford, MA). Plates were
8
Ile-angiotensin II (SII) induces ␤-arrestin recruitment, re- read on a PheraStar reader using a time-resolved fluorescence ratio
ceptor internalization, and ␤-arrestin-mediated signals with- (665 nm/620 nm).
out activating G protein coupling (Wei et al., 2003; Ahn et al., Immunoblotting. U2-OS or HEK-293 cells stably expressing the
2004; Kim et al., 2009). Unfortunately, this peptide displays rat AT1aR were grown in MEM supplemented with 10% FBS. After
overnight serum starvation, cells were stimulated with 1 ␮M angioten-
low receptor binding affinity (Holloway et al., 2002), which
sin II, valsartan, TRV0100027, TRV0100023, or vehicle for 5 min.
has made in vivo characterization of its effects challenging. Lysates were immunoblotted with phospho-Y416-Src, phospho-T202/
However, ex vivo studies revealed that SII promotes contrac- Y204-ERK, or phospho-S1177-eNOS antibodies per the manufacturer’s

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tility of isolated cardiac myocytes via the AT1R and ␤-arres- instructions (Cell Signaling Technologies, Danvers, MA). For RNA in-
tin2 (Rajagopal et al., 2006) and mitogen-activated protein terference experiments, siRNAs for ␤-arrestin2 were transfected as
kinase (MAPK) activation in perfused hearts (Aplin et al., described previously (DeWire et al., 2008), and cells were plated as
2007), indicating that ␤-arrestin biased ligands may have above at 48 h after siRNA transfection. The signal transduction protein
significant impact in a physiological setting. However, it array assay was performed per the manufacturer’s protocol (Proteome
remains unclear whether AT1R signals can be separated by Profiler ARY003; R&D Systems, Minneapolis, MN). The activated en-
biased ligands in vivo to allow precise targeting of desired zymes detected in this array relied on antibodies to phospho-Y397 on
FAK and phospho-S63 on c-Jun. Immunoreactive bands or spots were
AT1R effects. More broadly, despite increasing speculation in
visualized and quantitated by using GeneSnap and GeneTool software
the research literature and numerous examples of biased
(Syngene, Frederick, MD).
ligands eliciting unique pharmacology in vitro, it remains Myocyte Contractility. Isolated murine cardiomyocytes were
unclear whether ligand bias can elicit unique, differentiated isolated and contractility was measured by video edge detection as
pharmacology in vivo compared with unbiased ligands by described previously (Jaleel et al., 2008).
simultaneously activating one signal transduction pathway In Vivo Studies. All in vivo studies were performed at Qtest Labs
while antagonizing another at the same receptor. (Columbus OH) for cardiovascular assays or ChemPartner Co.
Thus we set out to discover new ␤-arrestin biased ligands (Shanghai, China) for pharmacokinetics, under protocols reviewed
for the AT1R, seeking potent, selective compounds to enable and approved by the respective Institutional Animal Care and Use
an in vivo comparison of biased and unbiased ligands to test Committees.
Rat Hemodynamics during Angiotensin II Dose Escalation.
whether AT1R ligand bias translates from isolated cellular
TRV120027, vehicle, and losartan were dosed to anesthetized (sodium
systems to elicit unique integrated responses in vivo. Such a
pentobarbital)/ventilated healthy male rats. Under anesthesia, the an-
ligand would also allow us to test whether biased ligands imals were instrumented with ECG electrodes and micromanometers
truly uncover new opportunities for safer, more efficacious into a femoral artery and the left ventricle. Mean arterial pressure
therapeutics for cardiovascular diseases of impaired cardiac (MAP) and left ventricular hemodynamic and mechanical indices (end
performance. systolic pressure, end diastolic pressure, dP/dtmax, Tau, Vmax) were
obtained from the arterial/ventricular pressure signals. In addition,
ECG-derived indices of conduction (PQ, QRS) and repolarization dura-
Materials and Methods
tion (QT/QTcF) were measured. Two dose levels of TRV120027, low (1
Synthesis of Peptides. Peptides were synthesized by Genscript ␮g/kg/min) and high (10 ␮g/kg/min), were assayed. The cardiovascular
USA Inc. (Piscataway, NJ) to more than 98% purity; quality control responses of test article-treated animals to an eight-dose angiotensin II
was by high-performance liquid chromatography and mass spec- challenge were evaluated and compared against those obtained in an-
trometry. The peptides described here are as follows: TRV120023, imals receiving either losartan (3 mg/kg bolus injection) or vehicle
Sar-Arg-Val-Tyr-Lys-His-Pro-Ala-OH; TRV120026, Sar-Arg-Val- (infusion).
Tyr-Tyr-His-Pro-NH2; and TRV120027, Sar-Arg-Val-Tyr-Ile-His- Rat Pressure-Volume Loop Analysis. Telmisartan and
Pro-D-Ala-OH. TRV120027 were dosed to anesthetized (sodium pentobarbital)/ven-
Cell Culture and Preparation. HEK-293 cells were stably tilated healthy male rats. Under anesthesia, the animals were in-
transfected to overexpress ␤-arrestin2 fused to a ␤-galactosidase strumented with ECG electrodes, a micromanometer into a femoral
fragment and human AT1R or rat AT1aR fused to a complemen- artery, a conductance/micromanometer into the left ventricle, and a
tary ␤-galactosidase fragment. The growth medium used was hydraulic occluder around the inferior vena cava (via a right thora-
MEM with 10% FBS, 1% penicillin/streptomycin, 150 ␮g/liter of cotomy). MAP and left ventricular mechanical (end systolic pressure,
neomycin, and 150 ␮g/liter of hygromycin. ␤-Arrestin recruitment end diastolic pressure, dP/dtmax, Tau, Vmax) and geometrical (end
and inositol monophosphate (IP1) accumulation were measured in diastolic volume, stroke volume, dV/dt) indices were obtained from
small-volume, 384-well plates; immunoblot experiments were in the ventricular pressure/conductance (volume) signals. The myocar-
12-well or 10-cm plates. U2-osteosarcoma (U2-OS cells) were sta- dial inotropic [end systolic pressure-volume (PV) relationship
bly transfected to overexpress rat AT1aR and cultured in MEM (ESPVR), preload recruitable stroke work (PRSW)], lusitropic (end
with 10% FBS, 1% penicillin/streptomycin, and 150 ␮g/liter of diastolic PV relationship), and energetic (PV area, stroke work) state
neomycin. Cells were plated in 6- or 12-well tissue culture plates were examined by means of PV curves; PV families/relationships
for immunoblot experiments. were generated via preload reductions (inferior vena cava occlusion).
574 Violin et al.

ECG-derived indices of conduction (PQ, QRS) and repolarization confirmed by whole-cell radioligand binding studies, which
duration (QT/QTcF) were measured. showed that TRV120027, but not the ARB losartan, re-
Statistics. Statistics were assessed by using Prism version 5 duced angiotensin II binding sites in HEK cells overex-
(GraphPad Software Inc., San Diego, CA). Schild analysis fitting the pressing human AT1R (Supplemental Fig. 1B).
Schild model of competitive antagonism to a series of dose-response
We chose TRV120027, the more potent ligand, for more
curves with varying amounts of antagonist was also performed with
thorough characterization. Escalating concentrations of
Prism software.
TRV120027 shifted the EC50 of angiotensin II-evoked G
protein coupling without suppressing maximal efficacy,
Results consistent with a purely competitive mechanism of action
The AT1R Is Capable of Potent, Robust Efficacy by (Fig. 1D). Fitting the data to a Schild model of competitive
␤-Arrestin Biased Ligands. To identify new, higher- antagonism (Lew and Angus, 1995) supported this conclu-
potency ␤-arrestin biased ligands, we initiated an iterative sion (Schild slope ⫽ 1.04 ⫾ 0.06), with an estimated Kd of
evaluation of custom-synthesized peptides based on SII, 19 nM. This is consistent with radioligand binding studies with
125
using assays of G protein coupling (inositol monophos- I-angiotensin II, which showed a Ki for TRV120027 of 16 nM
phate accumulation) and ␤-arrestin recruitment to the (Supplemental Table 1). These studies also showed that
AT1R (enzyme complementation) to characterize the po- TRV120027 has apparent first-order binding, with a kon of 3.9 ⫻
tency, efficacy, and ␤-arrestin bias of new compounds at 106 M⫺1 䡠 min⫺1, koff of 4.7 ⫻ 10⫺2 min⫺1, corresponding to a
overexpressed human AT1R. This search led to the iden- residence half-time of 16 min, similar to that of losartan. The

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tification of two ␤-arrestin biased ligands with improved TRV120027 Kd, as calculated from on and off rates, was 12 nM,
potency compared with SII: TRV120023 and TRV120027 consistent with the apparent Ki.
selectively and potently engage ␤-arrestin2 recruitment TRV120027 was also remarkably specific for the AT1R: at
(EC50 ⫽ 44 and 17 nM, respectively at the human AT1R) 58 different receptors and channels 10 ␮M TRV120027
without any detectable activation of G protein coupling showed less than 15% inhibition of reference radioligand
(data for TRV120027 shown in Fig. 1). This contrasts with binding (Supplemental Methods). The only receptor other
the robust G protein and ␤-arrestin2 signals elicited by than the AT1R for which TRV120027 had affinity was the
angiotensin II (EC50 of 1.1 and 9.7 nM for IP1 accumula- angiotensin II type 2 receptor (AT2R), with apparent Ki as
tion and ␤-arrestin2 recruitment, respectively) and the measured by radioligand binding of 7 nM.
complete lack of G protein or ␤-arrestin2 efficacy of angiotensin The Signaling Profile of ␤-Arrestin Biased Ligands
receptor blockers (ARBs) such as valsartan. TRV120023 and Is a Subset of Angiotensin II-Mediated Signaling. Con-
TRV120027 had very similar potency, efficacy, and bias at the sistent with ␤-arrestin recruitment, in U2-OS cells over-
rat AT1aR, with EC50 for ␤-arrestin2 recruitment of 37 and 23 expressing rat AT1aR, angiotensin II, TRV120023, and
nM, respectively. TRV120027 activated the MAPK ERK1/2, contrasting with
To verify ␤-arrestin efficacy, we also showed that angio- valsartan (Fig. 2A) and a range of other ARBs that were
tensin II, TRV120023, and TRV120027 evoked ␤-arrestin1 inactive (data not shown). Similar results were found in
and ␤-arrestin2 recruitment, as measured by ␤-arrestin– HEK cells (data not shown), which have been widely used
yellow fluorescent protein interaction with human AT1R– to assess ␤-arrestin signaling (Ahn et al., 2004); in these
cyan fluorescent protein (Rajagopal et al., 2006), whereas cells TRV120027 stimulated ERK1/2 with an EC50 of ap-
ARBs did not, consistent with our enzyme complementa- proximately 1 nM. It is noteworthy that of these com-
tion result (data not shown). In these same experiments, pounds only angiotensin II activated p38 MAPK (Fig. 2B),
we saw clear translocation of ␤-arrestin2–yellow fluores- highlighting that ␤-arrestin biased ligands, devoid of G
cent protein from cytosol to membrane compartments and protein coupling, activate only a subset of the normal
internalization of AT1R– cyan fluorescent protein for an- complement of AT1R signals. We also found that angioten-
giotensin II-, TRV120023-, and TRV120027-treated cells, sin II, TRV120023, and TRV120027, but not valsartan,
but no effect of the ARBs valsartan, losartan, or telmisar- stimulated activation of both Src (Fig. 2C) and eNOS (Fig.
tan, consistent with ␤-arrestin2 recruitment and coupling 2D). These data suggested that ␤-arrestin recruitment to
to endocytic machinery (Supplemental Fig. 1A). This was the AT1R could engage a pathway whereby Src phosphor-

Fig. 1. TRV120027 is a highly potent ␤-arrestin biased ligand at the AT1R. A–C, ␤-arrestin2 recruitment (black circles) was measured by
chemiluminescent ␤-galactosidase activity, and G protein coupling (red squares) was measured by IP1 accumulation, in HEK cells expressing human
AT1R for angiotensin II (A), the ARB valsartan (B), and TRV120027 (C). D, TRV120027 preincubated at escalating concentrations shifts the
angiotensin II dose-response curve for IP1 accumulation, consistent with competitive antagonism. Data are means ⫾ standard error for three
independent experiments.
AT1R ␤-Arrestin Bias Improves Cardiac Function 575

Fig. 2. ␤-Arrestin biased ligands stimulate selective signaling in comparison with an unbiased agonist and an unbiased antagonist in U2-OS cells
overexpressing rat AT1aR. A–D, activation by phosphorylation was measured by immunoblot with phospho-specific antibodies for ERK1/2 (A),
p38 (B), Src (C), and eNOS (D) activation. E, ␤-arrestin2 siRNA prevents biased ligand-stimulated eNOS phosphorylation compared with a
control siRNA. Data are means ⫾ standard errors of three to seven independent experiments. ⴱ, p ⬍ 0.05 versus vehicle and valsartan and ⴱⴱ,

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p ⬍ 0.05 versus the same compound with control (CTL) siRNA, as measured by ANOVA with the Bonferroni multiple comparison test.

ylates and activates Akt, which in turn phosphorylates


eNOS (Haynes et al., 2003; Suzuki et al., 2006). eNOS
activation by phosphorylation may provide a link between
AT1R ␤-arrestin function and cardiovascular tone via reg-
ulation of nitric oxide. Consistent with ␤-arrestin bias,
eNOS activation by both TRV120023 and TRV120027 is
eliminated by siRNA silencing of ␤-arrestin2 (Fig. 2E). In
contrast, ␤-arrestin2 silencing reduced Ang II-stimulated
eNOS phosphorylation by approximately 50%, suggesting
that AngII activates eNOS by both ␤-arrestin2-dependent
and -independent pathways (data not shown); this may
also explain why the ␤-arrestin biased ligands do not elicit
as much eNOS phosphorylation as AngII (Fig. 2D). A sec-
ond siRNA sequence targeting ␤-arrestin2 yielded similar
results (data not shown). These results demonstrate that
this signaling effect of the ␤-arrestin biased ligands is in Fig. 3. FAK and c-Jun phosphorylation stimulated by ␤-arrestin biased
ligands, identified from a signal panning screen for changes in expression
fact ␤-arrestin2-mediated. or phosphorylation of 55 different signaling proteins. Comparison of
We then searched more broadly for signals engaged by our TRV120023 and TRV120027 to an unbiased agonist and an unbiased
␤-arrestin biased ligands, using an antibody array for 55 antagonist in U2-OS cells overexpressing rat AT1aR identified c-Jun
phosphorylation (A) and FAK phosphorylation (B). Data are means ⫾
different signal transduction proteins, largely selective for standard errors of three to seven independent experiments. ⴱ, p ⬍ 0.05
phosphorylated “active” or “inactive” proteins. Of these, two versus vehicle and valsartan and ⴱⴱ, p ⬍ 0.05 versus the same compound
were strongly activated by the ␤-arrestin biased ligands but with control siRNA, as measured by ANOVA with the Bonferroni multi-
not valsartan: phospho-c-Jun (Fig. 3A) and phospho-FAK ple comparison test.
(Fig. 3B). Together, these results establish that ␤-arrestin
biased ligands display a pharmacological profile in vitro that telmisartan at receptor-saturating concentrations (data not
is distinct from classic antagonists and here represents a shown).
subset of unbiased agonist signaling. ␤-Arrestin Biased Ligands Block the AT1R Pressor
␤-Arrestin Biased Ligands Stimulate Isolated Car- Response While Stimulating Cardiac Performance.
diomyocyte Contractility. Previous work showed that In vivo, acute AT1R-mediated blood pressure effects are
SII stimulated contractility of isolated murine cardiomyo- regulated by G protein-stimulated calcium mobilization
cytes via the AT1aR and ␤-arrestin2 (Rajagopal et al., (Harris et al., 2007; Wynne et al., 2009). Consistent with
2006), so we used this experimental system to test the this, and with the in vitro findings in Fig. 2, both
cellular effects of TRV120023 and TRV120027 related to TRV120027 and the unbiased antagonist losartan compet-
cardiovascular function. In agreement with the effects of itively antagonized the effect of escalating angiotensin II
SII, we found that TRV120027 stimulated cardiomyocyte on MAP in rats, as evidenced by an increase in the angio-
contractility, but the unbiased antagonist valsartan did tensin II EC50 (Fig. 5A). For losartan, this competitive
not (Fig. 4A). Valsartan, which is highly selective for the antagonism was the only obvious effect. In contrast,
AT1R, including selectivity versus the AT2R (Criscione et TRV120027 also reduced blood pressure independent of
al., 1993), blocked TRV120027-stimulated cardiomyocyte angiotensin II and depressed the maximum angiotensin
contractility, indicating that this effect is AT1R mediated. II-evoked blood pressure increase.
Separate experiments showed similar results for TRV120023 In the same experiment, when we evaluated effects of
(Fig. 4B) and a lack of effect for the ARBs losartan and losartan and TRV120027 alone, before the addition of angio-
576 Violin et al.

2006), indicating that the ␤-arrestin agonist efficacy of


TRV120027 modestly stimulates cardiac contractility, a
property not shared by ARBs. TRV120027 did not cause any
chronotropic effects and did not affect PQ, QRS, or QT inter-
vals, indicating no effect on conduction and instead suggest-
ing that any effect on contractility is probably a result of
favorable effects on the mechanics or energetics of myocyte
contractility.
To more directly assess how TRV120027 affects cardiac
performance, we compared TRV120027 to an ARB in rats
by using left ventricular PV loop analysis (PV loops). This
technique measures pressure and conductance (corre-
sponding to blood volume) of the left ventricle during in-
creasing occlusion of the inferior vena cava to reduce car-
diac-filling pressure. The family of PV loops, with each
loop representing the cycle of pressure and volume changes
during a single heart beat, allows direct assessment of
cardiac performance independent of preload (filling pressure)

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and afterload (resistance from aortic pressure) (Kohout et al.,
2001). The effects of TRV120027 on systolic, diastolic, and
cardiac conductance functions are shown in Table 1. For
this study we tested the ARB telmisartan because its high
solubility allowed for infusion with a volume matched to
Fig. 4. TRV120027 and TRV120023 increase mouse cardiomyocyte con-
tractility. A, angiotensin II (10 ␮M) and TRV120027 increased fractional
TRV120027 to eliminate potential nondrug effects; the
shortening, as measured by video edge detection microscopy. Valsartan (1 effects of telmisartan on cardiac function are shown in
␮M) had no effect, but completely blocked the effect of TRV120027. B, a Supplemental Table 2.
separate experiment revealed similar properties for TRV120023 and also TRV120027 and telmisartan both produced dose-dependent
compared responses with those elicited by 10 nM isoproterenol. Data are
presented as mean ⫾ standard error of the percentage increase in short- decreases in MAP (Fig. 6A). Neither compound increased heart
ening of single myocytes after drug treatment. n ⫽ 3 independent exper- rate (Fig. 6B), but TRV120027, unlike telmisartan, increased
iments, each using a different heart, with two myocytes tested for each cardiac contractility: TRV120027 dose-dependently increased
drug from each heart. ⴱ, p ⬍ 0.05 versus vehicle and valsartan and ⴱⴱ, p ⬍
0.05 versus TRV120027 alone, as measured by ANOVA with the Bonfer- the slope of ESPVR (Fig. 6C), a load-independent measure of
roni multiple comparison test. left ventricular contractility (Little, 1985; Georgakopoulos et al.,
1998). Consistent with these effects, TRV120027 also preserved
stroke volume (Fig. 6D) and increased normalized PRSW
(Table 1). TRV120027 reduced dP/dtmax, a commonly used
measure of contractility, but because this parameter corre-
lates with preload it probably reflects the actions of
TRV120027 on preload (Schmidt and Hoppe, 1978). In con-
trast to these effects, telmisartan reduced cardiac perfor-
mance, causing reduced ESPVR slope and stroke volume.
These responses are consistent with the failure of other ARBs
to increase cardiac performance (Chan et al., 1992). Repre-
sentative PV loop families for vehicle control, telmisartan,
and TRV120027 are shown in Supplemental Fig. 2. It is
noteworthy that TRV120027-enhanced cardiac performance co-
incided with reduced stroke work, suggesting improved myo-
cardial efficiency. Furthermore, TRV120027 did not cause any
electrocardiographic changes, and its effects were rapidly re-
Fig. 5. TRV120027 competitively antagonizes angiotensin II blood pres- versible: 5 to 7 min after ceasing infusion, MAP and ESPVR had
sure increases while stimulating cardiac contractility in normal rats. returned nearly to baseline values (Supplemental Fig. 3). This
A, losartan and TRV120027 shift the angiotensin II dose-escalation blood
pressure response. B, in the same experiment, before angiotensin II rapid reversibility is consistent with the short half-life of
treatment, TRV120027, but not losartan, increased cardiomyocyte con- TRV120027 in vivo: after infusion into normal rats, TRV120027
tractility, as measured by the maximum rate of myocardial contractility had a half-life of 1.5 min (Supplemental Methods).
vehicle (Vmax). Data are means ⫾ standard errors of seven animals per
group. ⴱ, p ⬍ 0.05 versus vehicle, as measured by ANOVA with the
Similar to TRV120027, TRV120023 reduced MAP while
Bonferroni multiple comparison test. increasing ESPVR (Supplemental Fig. 4, A and B).
TRV120026, a third ␤-arrestin biased ligand with very sim-
tensin II, we noted that myocardial shortening velocity ilar potency for ␤-arrestin recruitment (EC50 ⫽ 24.5 nM at
(Vmax), a measure of cardiac contractility relatively insensi- the human AT1R) but 80-fold higher relative specificity for
tive to changes in vascular or arterial pressure, was in- binding the AT1R over the AT2R, showed similar effects, at
creased by TRV120027 but not by losartan (Fig. 5B). This is the same doses, as TRV120027 and TRV120023 on MAP and
consistent with our cardiomyocyte contractility study (Fig. ESPVR (Supplemental Fig. 4, C and D). Indeed the biggest
4), a known ␤-arrestin2-mediated effect (Rajagopal et al., difference between TRV120026 and either TRV120027 or
AT1R ␤-Arrestin Bias Improves Cardiac Function 577
TABLE 1
Effect of TRV120027 on cardiovascular parameters
Data are mean ⫾ SEM of three animals.

Baseline 0.1 ␮g/kg/min 1 ␮g/kg/min 10 ␮g/kg/min Washout

Hemodynamics
Heart rate (bmp) 371 ⫾ 6 356 ⫾ 5 351 ⫾ 8 359 ⫾ 5 369 ⫾ 4
Mean arterial pressure (mm Hg) 103 ⫾ 3 92 ⫾ 3* 84 ⫾ 4* 72 ⫾ 5* 88 ⫾ 3
Stroke volume (␮l) 26.3 ⫾ 3.1 25.2 ⫾ 4 27.1⫾4 26.2 ⫾ 5.1 29.6 ⫾ 4
End diastolic volume (RVU) 15.6 ⫾ 1.6 15.5 ⫾ 1.6 15.3 ⫾ 1.5 15.4 ⫾ 1.7 15.5 ⫾ 1.7
End systolic pressure (mm Hg) 118 ⫾ 4 112 ⫾ 7 99 ⫾ 8 87 ⫾ 9* 104 ⫾ 8
End diastolic pressure (mm Hg) 6⫾2 6⫾2 6⫾3 5⫾2 7⫾2
Systolic function
dP/dtmax (mm Hg/s) 6060 ⫾ 198 5494 ⫾ 325 4697 ⫾ 365* 3755 ⫾ 248* 5304 ⫾ 316
Ees, ESPVR slope (mm Hg/RVU) 45 ⫾ 1 51 ⫾ 1 54 ⫾ 2* 60 ⫾ 4* 48 ⫾ 1
PRSW (SWU/RVU) 67 ⫾ 6 60 ⫾ 2 59 ⫾ 1 58 ⫾ 4 58 ⫾ 4
Stroke work (mm Hg ⫻ RVU) 110 ⫾ 3 104 ⫾ 5 93 ⫾ 13 66 ⫾ 13* 87 ⫾ 13
Normalized PRSW (1/RVU) 0.62 ⫾ 0.1 0.59 ⫾ 0.03 0.67 ⫾ 0.09 0.93 ⫾ 0.1 0.69 ⫾ 0.08
Diastolic function
dP/dtmin (mm Hg/s) ⫺6333 ⫾ 73 ⫺5602 ⫾ 167 ⫺4921 ⫾ 288* ⫺4036 ⫾ 295* ⫺5699 ⫾ 429
Tau (s) 12 ⫾ 1 12 ⫾ 1 12 ⫾ 0 12 ⫾ 1 11 ⫾ 0
Cardiac conduction
PQ (ms) 41 ⫾ 4 41 ⫾ 4 41 ⫾ 4 42 ⫾ 5 42 ⫾ 4

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QRS (ms) 24 ⫾ 2 24 ⫾ 1 23 ⫾ 2 23 ⫾ 2 23 ⫾ 2
QT (ms) 61 ⫾ 2 61 ⫾ 2 62 ⫾ 1 61 ⫾ 1 62 ⫾ 1
QTcF (ms) 113 ⫾ 2 111 ⫾ 3 112 ⫾ 1 112 ⫾ 2 113 ⫾ 3
RVU, relative volume unit; PRSW, stroke work vs. end diastolic pressure slope; SWU, stroke work units (mm Hg ⫻ volume unit).
* P ⬍ 0.05 by ANOVA with Dunnett’s multiple comparison test.

TRV120023 is an enhanced effect on ESPVR by TRV120026, Discussion


which in light of this compound’s reduced affinity for the
The results presented here describe the unique in vitro
AT2R is consistent with an AT1R-mediated effect. The effects
and in vivo pharmacology of TRV120027, a novel, selective,
of the affinity of TRV120027 for the AT2R, if any, remain
and potent ␤-arrestin biased ligand of the AT1R. This work
unclear; however, these findings, along with the blockade of
demonstrates that biased and unbiased ligands are phar-
cardiomyocyte contractility in vitro by an AT1R-selective an-
macologically distinct in vivo. TRV120027 functions as an
tagonist, suggest that the enhancement of cardiac contractil-
antagonist, analogous to ARBs, when evaluated for G pro-
ity of TRV120027 is AT1R-mediated.
tein coupling, other cellular signals such as p38 MAPK
Together, the findings presented here demonstrate that
activation, and effects on blood pressure. However, when
TRV120027 depresses blood pressure while increasing car-
assessed for efficacy in recruiting ␤-arrestins to the AT1R,
diac performance, consistent with its antagonist efficacy
and for downstream effects including receptor internaliza-
against angiotensin II pressor activity (Fig. 5), its ␤-arrestin-
tion, ␤-arrestin-mediated signals, and cardiomyocyte con-
mediated stimulation of cardiac contractility in vitro (Fig. 4),
tractility, TRV120027 is an agonist. This bias is not merely
and Vmax and ESPVR in rats (Figs. 5 and 6). The stimulation
of contractility by TRV120027 is modest compared with the an artifact of differentially amplified assay readouts: IP1
classic inotrope dobutamine, which raises dP/dtmax and more accumulation in response to angiotensin II is more sensi-
strongly increases ESPVR and also increases heart rate tive than ␤-arrestin2 recruitment, as measured by po-
(Supplemental Table 3). This modest efficacy of TRV120027, tency, but undetectable despite strong ␤-arrestin re-
in conjunction with its afterload reduction, may explain the sponses for TRV120023 and TRV120027. This is a
apparently beneficial energetics of reduced stroke work. This hallmark of intrinsic ligand bias, reflecting a stabilization
profile suggests that TRV120027 and related ␤-arrestin bi- of different receptor conformations than are stabilized by
ased AT1R agonists could have a beneficial impact on cardio- angiotensin II or the unbiased ARBs (Kenakin, 2007). This
vascular syndromes characterized by hypertension and in- assay-dependent efficacy violates classic models of molec-
sufficient cardiac performance. ular pharmacology that assume that all efficacies are cor-

Fig. 6. TRV120027 reduces blood pressure while increasing cardiac performance in normal rats. A, PV loop analysis in normal rats infused with either
TRV120027 or telmisartan revealed similar effects on MAP. B, neither compound substantially changes heart rate. C, TRV120027 increased, whereas
telmisartan decreased, load-independent cardiac performance, as measured by the slope of ESPVR. D, telmisartan, but not TRV120027, significantly
reduced stroke volume. Data shown are mean ⫾ standard errors from three independent experiments with three to four animals per group. ⴱ, p ⬍ 0.05
versus baseline, as measured by ANOVA with the Bonferroni multiple comparison test.
578 Violin et al.

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Acknowledgments Schmidt HD and Hoppe H (1978) Preload dependence of dP/dtmax, VCEmax and
calculated Vmax compared to the inotropic sensitivity of these indices of cardiac
We thank Steven Houser and Xiong Wen Chen for performing the contractility. Basic Res Cardiol 73:380 –393.
isolated cardiomyocyte contractility studies and Robert Lefkowitz, Suzuki H, Eguchi K, Ohtsu H, Higuchi S, Dhobale S, Frank GD, Motley ED, and
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manuscript. Tran JA, Chang A, Matsui M, and Ehlert FJ (2009) Estimation of relative micro-
AT1R ␤-Arrestin Bias Improves Cardiac Function 579
scopic affinity constants of agonists for the active state of the receptor in functional Wynne BM, Chiao CW, and Webb RC (2009) Vascular smooth muscle cell signaling
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Violin JD, Dewire SM, Barnes WG, and Lefkowitz RJ (2006) G protein-coupled G protein coupled receptor kinases (GRKs) encodes distinct functions of biased
receptor kinase and ␤-arrestin-mediated desensitization of the angiotensin II type ligands. Proc Natl Acad Sci USA 106:9649 –9654.
1A receptor elucidated by diacylglycerol dynamics. J Biol Chem 281:36411–36419.
Wei H, Ahn S, Shenoy SK, Karnik SS, Hunyady L, Luttrell LM, and Lefkowitz RJ Address correspondence to: Jonathan D. Violin, Trevena Inc., 1018 West
(2003) Independent ␤-arrestin 2 and G protein-mediated pathways for angiotensin 8th Avenue, Suite A, King of Prussia, PA 19406. E-mail: jviolin@
II activation of extracellular signal-regulated kinases 1 and 2. Proc Natl Acad Sci trevenainc.com
USA 100:10782–10787.

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Selectively engaging β-arrestins at the AT1R reduces blood pressure and increases cardiac performance
Jonathan D. Violin Ph.D., Scott M. DeWire Ph.D., Dennis Yamashita Ph.D., David H. Rominger, Lisa Nguyen, Kevin Schiller, Erin J. Whalen Ph.D., Maxine Gowen Ph.D., and Michael W. Lark Ph.D
The Journal of Pharmacology and Experimental Therapeutics

A
Vehicle Angiotensin II Valsartan Losartan

Telmisartan TRV120023 TRV120027

Supplementary Figure 1. TRV120027 stimulates receptor internalization. (A) rat AT1aR fused
to cyan fluorescent protein and expressed in HEK-293 cells exposed to vehicle, angiotensin II, ARBs
(valsartan, losartan, or telmisartan) or β-arrestin biased ligands (TRV120023 or TRV120027). (B)
Downregulation of cell-surface human AT1R binding sites in response TRV120027 or losartan at 37°C. Data
are the mean total [125I]-angiotensin II binding ± SEM from 2-3 independent experiments.
Selectively engaging β-arrestins at the AT1R reduces blood pressure and increases cardiac performance
Jonathan D. Violin Ph.D., Scott M. DeWire Ph.D., Dennis Yamashita Ph.D., David H. Rominger, Lisa Nguyen, Kevin Schiller, Erin J. Whalen Ph.D., Maxine Gowen Ph.D., and Michael W. Lark Ph.D
The Journal of Pharmacology and Experimental Therapeutics

A B

C D

E F

Supplementary Figure 2. Representative left ventricular pressure-volume


loops before (A) and after (B) vehicle infusion; before (C) and after(D)
telmisartan infusion; and before (E) and after (F) TRV120027 fusion. Each
family of loops represents a series of cardiac cycles during increasing occlusion
of the vena cava to reduce preload. Linear regression of the highlighed data
points (black circles, representing the end of left ventricular ejection and closing
of the aortic valve) yields a load-independent measure of contractility (Ees, the
slope of the end systolic pressure-volume relationship).
Selectively engaging β-arrestins at the AT1R reduces blood pressure and increases cardiac performance
Jonathan D. Violin Ph.D., Scott M. DeWire Ph.D., Dennis Yamashita Ph.D., David H. Rominger, Lisa Nguyen, Kevin Schiller, Erin J. Whalen Ph.D., Maxine Gowen Ph.D., and Michael W. Lark Ph.D
The Journal of Pharmacology and Experimental Therapeutics

A B

Supplementary Figure 3. Rapid reversibility of TRV120027 effects. Dose escalation of infused


TRV120027 every 10 minutes, followed by a post‐infusion reading 5 minutes after terminating
infusion. (A) mean arterial pressure. (B) cardiac performance, as measured by the slope of the
end systolic pressure‐volume relationship Ees. Data are mean +/‐ SEM for 3 animals.
Selectively engaging β-arrestins at the AT1R reduces blood pressure and increases cardiac performance
Jonathan D. Violin Ph.D., Scott M. DeWire Ph.D., Dennis Yamashita Ph.D., David H. Rominger, Lisa Nguyen, Kevin Schiller, Erin J. Whalen Ph.D., Maxine Gowen Ph.D., and Michael W. Lark Ph.D
The Journal of Pharmacology and Experimental Therapeutics

A B

C D

Supplementary Figure 4. Effects of the β‐arrestin biased ligands TRV120023 and


TRV120026 on mean arterial pressure (MAP) and cardiac performance, as measured
by the slope of the end systolic pressure‐volume relationship Ees. (A) Effect of
TRV120023 on MAP. (B) Effect of TRV120023 on Ees. (C) Effect of TRV120026 on
MAP. (D) Effect of TRV120026 on Ees.
Selectively engaging β-arrestins at the AT1R reduces blood pressure and increases cardiac performance
Jonathan D. Violin Ph.D., Scott M. DeWire Ph.D., Dennis Yamashita Ph.D., David H. Rominger, Lisa
Nguyen, Kevin Schiller, Erin J. Whalen Ph.D., Maxine Gowen Ph.D., and Michael W. Lark Ph.D
The Journal of Pharmacology and Experimental Therapeutics

Supplementary Methods

Selectivity Profiling

The following receptors and ion channels were tested for TRV120027 binding by radioligand
displacement - ExpresS Profile (Cerep, Potiers, France): (human) Adenosine A1, A2A and A3, Adrenergic
β1 and β2, Angiotensin-II AT1, AT2, Bradykinin B2, Cannabinoid CB1, Cholecystokinin CCK1 (CCKA),
Dopamine D1, and D2Short, Endothelin ETA, GABA, Galanin GAL2, Chemokines CXCR2, CCR1,
Histamine H1 and H2, Melanocortin MC4, Melatonin MT1 (ML1A), Muscarinic M1, M2 and M3,
Neurokinin NK2 and NK3, Neuropeptide Y1 and Y2, Neurotensin NTS1 (NT1), Opioid δ2DOP), Opioid
µ(MOP), Opioid NOP (ORL1), Prostanoid TP (TXA2/PGH2), Serotonin 5-HT1A, 5-HT2A, 5-HT2B , 5-HT3
, 5-HT5a , 5-HT6 and 5-HT7, Vasoactive intestinal peptide VPAC1 (VIP1) and V1a, Norepinephrine
transporter, Dopamine transporter, Serotonin 5-HT transporter, APJ (apelin), Angiotensin-converting
enzyme ACE; (rat) Adrenergic α1 and α2, Benzodiazepine BZD, Serotonin 5-HT1B, Opioid κ (KOP),
Ca2+ channel (L verapamil site), K+ channels KV and SKCa Na+ channel (site 2), Cl- channel (GABA-
gated); (murine) Somatostatin sst (non-selective).

Rat pharmacokinetics
During a 2-hour IV infusion, 10 ug/kg/min TRV120027 rapidly reached a steady-state concentration of
246 ng/mL (266 nM), and upon ending infusion revealed a half-life of 1.5 minutes with low volume of
distribution of 208 mL/kg in rats. The high clearance indicates that infusion is the optimal route of
administration to elicit pharmacological effects, and given the high affinity of TRV120027 for the AT1R,
predicts a pharmacologically effective dose of 0.1-10 ug/kg/min. The rapid clearance and volume of
distribution also suggest a rapidly reversible effect limited to the vascular space.

Rat hemodynamic studies

Rats were intraperitoneally (IP) anesthetized to effect with sodium pentobarbital (78.8 ± 1.5 mg/kg; 63.4
– 90.0 mg/kg). Following induction, the animals were prepared for surgery, endotracheally intubated (via
a tracheotomy), and mechanically ventilated (~90 breaths/min, ~2.5ml tidal volume with a 95% O2/ 5%
CO2 mixture) via an adjustable small animal ventilator (model 680; Harvard Apparatus). Anesthesia was
sustained with routine sodium pentobarbital injections (total, 28.0 ± 1.8 mg/kg IP) until completion of the
study. Temperature was continuously monitored throughout the duration of the surgical/experimental
procedures via a rectal probe, and was maintained within the physiological range via a heated
temperature-controlled (closed-loop) small animal surgical table/control unit (model 03-01; Vestavia
Scientific). Subsequently, transthoracic needle electrodes forming a single-lead ECG (lead II) were
placed. The right carotid artery was isolated, dissected free from the surrounding tissue, and cannulated
with a 2F high-fidelity micromanometer catheter (SPR-838; Millar Instruments). In order to determine
left-ventricular pressure this catheter was advanced retrogradely across the aortic valve, and into the LV.
Similarly, in order to record arterial pressures, a 2F high-fidelity micromanometer catheter (SPR-320;
Millar Instruments) was inserted into the right femoral artery, and advanced into the abdominal aorta.
Finally, indwelling catheters were placed in the left femoral and jugular veins for the administration of the
vehicle/control/test articles and of angiotensin-II (respectively). The animals were kept/placed in dorsal
recumbence until the end of the experiment.

After instrumentation and hemodynamic stabilization for approximately 10 min, baseline data were
collected (i.e., pre-dosing data, PRE). Subsequently, the animals were treated with the test/vehicle/control
articles and following hemodynamic stabilization, as well as data collection the peak cardiovascular
responses to eight single-dose AT-II injections (IV bolus) were evaluated. Throughout the experiments,
Selectively engaging β-arrestins at the AT1R reduces blood pressure and increases cardiac performance
Jonathan D. Violin Ph.D., Scott M. DeWire Ph.D., Dennis Yamashita Ph.D., David H. Rominger, Lisa
Nguyen, Kevin Schiller, Erin J. Whalen Ph.D., Maxine Gowen Ph.D., and Michael W. Lark Ph.D
The Journal of Pharmacology and Experimental Therapeutics

analog signals were digitally sampled (1000Hz) and recorded continuously with a data acquisition system
(IOX; EMKA Technologies). Heart rate (HR), and systemic/left-ventricular hemodynamic/mechanical
indices were measured both before/after dosing, as well as during the AT-II dose-response generation.
Mean (MAP) arterial pressure and left-ventricular hemodynamic/mechanical parameters (ESP, EDP,
dP/dt max/min, time-constant of relaxation – tau, contractility-index – Vmax) were measured from the
aortic/left-ventricular pressure signals (respectively). Meanwhile, the following ECG-derived indices/
parameters were measured both before and after vehicle/control/test-article administration only: HR, RR,
PQ, QRS, QT, and Fridericia’s rate-corrected QT (QTcF) 1. Data were acquired digitally (IOX; EMKA
Technologies), and analyzed offline (IOX/ECG Auto; EMKA Technologies).

Rat Pressure-Volume Loop studies

Healthy, acclimatized, male Sprague-Dawley rats (Harlan Laboratories, IN; 283 – 392g) were
intraperitoneally (IP) anesthetized to effect with sodium pentobarbital (~60mg/kg). Following induction,
the animals were prepared for surgery, endotracheally intubated (via a tracheotomy), and mechanically
ventilated (~90 breaths/min, ~2.5ml tidal volume with a 95% O2/ 5% CO2 mixture) via an adjustable
small animal ventilator (model 680; Harvard Apparatus). In-dwelling catheters were placed in a femoral
and a jugular vein for the administration of either anesthetic agents or vehicle/control/test articles
(respectively). Anesthesia was sustained with a continuous sodium pentobarbital infusion (~ 10 mg/kg/h
IV) until completion of the study. Once a proper anesthetic regime was established and verified (e.g.,
nonresponsive to toe pinch), the animals were paralyzed with pancuronium (1 mg/kg/h IV bolus).
Temperature was continuously monitored throughout the duration of the surgical/experimental procedures
via a rectal probe, and was maintained within the physiological range via a heated temperature-controlled
(closed-loop) small animal surgical table/control unit (model 03-01; Vestavia Scientific).

Subsequently, transthoracic needle electrodes forming a single-lead ECG (lead II) were placed. The right
carotid artery was isolated, dissected free from the surrounding tissue, and cannulated with a 2F high-
fidelity conductance/micromanometer catheter (SPR- 838; Millar Instruments). In order to simultaneously
determine left-ventricular pressure and volume (via conductivity; MPCU-200, Millar Instruments), this
catheter was advanced retrogradely across the aortic valve, and into the LV. The thoracic cavity was
opened via a right-thoracotomy, and a non-constricting hydraulic vessel-occluder (3mm; In Vivo Metrics)
filled with distilled water was securely placed around the inferior vena cava. In order to record arterial
pressures, a 2F high-fidelity micromanometer catheter (SPR-320; Millar Instruments) was inserted into a
femoral artery. Finally, the animals were placed in dorsal recumbency until the end of the experiment.

After instrumentation and hemodynamic stabilization, baseline data was collected. Subsequently,
escalating-dose infusions of the test/control article(s) were started, and measurements were taken once a
steady state was reached at each concentration (~10min). Finally, an additional set of measurements was
obtained during the washout period (~5-7min after the end of infusion). Meanwhile, in the vehicle group,
measurements were at baseline, just before the end of a timematched PBS (TRV-vehicle) infusion (i.e.,
~40-45min after infusion onset), and immediately after an acute inotropic challenge with dobutamine
(VEH+DOB). Left-Ventricular Mechanics: Once a steady hemodynamic state was reached at a given
time-point, left-ventricular pre-load was acutely reduced by means of brief (~8-10 beats) vena cava
occlusions (via transient inflation of the vessel occluder) in order to generate a family of pressure-volume
curves/loops; approximately three occlusions were performed at each time-point, allowing for
hemodynamic recovery between tests. The resulting left-ventricular pressure and volume data were
analyzed both on- and off-line (IOX/ECG Auto; EMKA Technologies) in order to generate relationships
representing the contractile and energetic state of the myocardium. At the end of each experiment,
conductance signals (in relative volume units, RVU) were calibrated in-vitro with warm blood, as recently
Selectively engaging β-arrestins at the AT1R reduces blood pressure and increases cardiac performance
Jonathan D. Violin Ph.D., Scott M. DeWire Ph.D., Dennis Yamashita Ph.D., David H. Rominger, Lisa
Nguyen, Kevin Schiller, Erin J. Whalen Ph.D., Maxine Gowen Ph.D., and Michael W. Lark Ph.D
The Journal of Pharmacology and Experimental Therapeutics

reviewed/summarized by Pacher et al. 2, in order to derive absolute left-ventricular volumes (in μL). In
short, blood was freshly collected, heparinized, and used to fill cylindrical wells of known
volume/dimensions (calibration cuvette model 910-1048; Millar Instruments); three wells with outer
diameters (OD) of 2, 3, and 4mm were used. Subsequently, average conductance values were obtained
from each blood-filled cylinder. In order to derive a calibration function (slope/intersect), the resulting
conductivity values were linearly regressed against the “known” volume of the wells; a unique function
(correction) was obtained for each animal. Throughout the experiments, analog signals were digitally
sampled (1000Hz) and recorded continuously with a data acquisition system (IOX; EMKA
Technologies). Digitally acquired signals were analyzed on/offline (IOX/ECG Auto; EMKA
Technologies). The following ECG-derived indices/parameters were measured offline with the aid of
pattern-recognition software (ECG Auto; EMKA Technologies): HR, RR, PQ, QRS, QT, and Fridericia’s
rate-corrected QT (QTcF) 1. Mean arterial (MAP) and endsystolic / end-diastolic left-ventricular (ESP,
EDP) pressures were measured. Meanwhile, left-ventricular mechanical/geometrical indices were
obtained from the pressure (dP/dt max/min, time-constant of relaxation – tau, contractility-index – Vmax)
and volume (ESV, EDV, SV, dV/dt) signals. In addition, the following measurements were derived from
left-ventricular pressure-volume data (PV loops) generated during brief periods of preload reduction:
Pressure volume area (PVA), and Stroke Work (SW), End-systolic (ESPVR) and end-diastolic (EDPVR)
pressure volume relationships. From the end-systolic PV-relationship, estimates of the left-ventricular
unstressed volume (volume-axis intercept, Vo) and elastance (slope, Ees), End systolic pressure and end-
diastolic volume relationship (Arterial Elastance,Ea), Slope of the stroke work (SW) and end-diastolic
volume (linear) relationship, also called preload recruitable stroke work (PRSW).

Visualization of mCFP receptors and mYFP β-arrestin2

HEK cells stable expressing rat AT1AR-mCFP and rat b-arrestin2-mYFP were produced under selection
with G418 (500ug/ml) and Zeocin (Invitrogen, 150ug/ml). Cells were plated on glass the night before the
experiment, and transferred to a phenol-red free culture media prior to the experiment. Individual dishes
were stimulated with 1uM compound for 30 minutes at 37C, and post-stimulation images were taken
using an epifluorescence microscope. Slidebook (Intelligent Imaging Innovations) software suite was
used for image analysis.

Radioligand binding membrane preparation

HEK-293 cells with stable expression of the human AT1R were harvested by centrifugation at 400xg for
30min at 4°C, washed once with a balanced salt solution, re-pelleted, and the pellet flash frozen in liquid
nitrogen. The cell pellets were stored at -80°C until processed for membranes. Pellets were resuspended
in buffer (50 mM HEPES, 2 mM EDTA pH 7.4 containing fresh protease inhibitors - Complete Brand
protease tablets from Roche Diagnostics (Indianapolis, IN) and subjected to nitrogen cavitation with a
Parr Cell Disruption Bomb (Parr Instrument Co., Moline, IL) at 1000 psi for 20 min on ice. Ruptured
cells were sedimented at 500g for 10 min at 4°C and the supernatant containing cellular membranes was
washed twice at 48,000g for 15 min. cell pellets were re-suspended at 4oC in 10 volumes of ice-cold
buffer A and cavitation, placed on ice. To remove large particles, a low speed centrifugation (500xg for
30 min at 4oC) was performed, followed by high-speed centrifugation (48,000xg for 45 min at 4oC),
resuspension in buffer plus protease inhibitor cocktail, and a final high speed centrifugation at (48,000g
for 45 min at 4oC). A dounce homogenizer was used to resuspend the final pellet using ice-cold buffer.
The membrane suspension was passed through a 23G needle, aliquoted, and stored at -80oC. Total
protein concentration of the membrane preparation was determined with a Coomassie Plus Reagent Kit
from Pierce Biotechnology (Rockford, IL) using bovine serum albumin as the standard.
Selectively engaging β-arrestins at the AT1R reduces blood pressure and increases cardiac performance
Jonathan D. Violin Ph.D., Scott M. DeWire Ph.D., Dennis Yamashita Ph.D., David H. Rominger, Lisa
Nguyen, Kevin Schiller, Erin J. Whalen Ph.D., Maxine Gowen Ph.D., and Michael W. Lark Ph.D
The Journal of Pharmacology and Experimental Therapeutics

[125I]Angiotensin II receptor competition and kinetic membrane radioligand binding assays

Membranes were diluted in assay buffer (50 mM Hepes, 150 mM NaCl, 5mM MgCl2 pH 7.2 at 23oC) to a
concentration of 10-20 µg protein/well. Assays were initiated by the addition of 97 µl of membrane
suspension to 200 µl of [125I]-Angiotensin II ([125I]-ANGII, specific activity 2200 Ci/mmol; PerkinElmer
Life and Analytical Sciences, Boston, MA), at 0.5-1 times Kd and various concentrations of inhibitors in
buffer plus a cocktail of protease inhibitors and 0.02% BSA to reduce non-specific binding. Compounds
were diluted in DMSO and tested at a final concentration of 1% DMSO (determined to be non-
detrimental to the assay). Binding assays were performed in duplicate in polypropylene 96 well plates
(Costar Corp., Cambridge, MA). Nonspecific binding was defined in the presence of 1µM saralasin.
Competition assays were performed at 23°C for 3-4 hours to allow adequate time for compounds and
radioligand to reach equilibrium for binding. The separation of bound from free radioligand was
accomplished by rapid vacuum filtration of the incubation mixture over GF/B uni-filter
(polyethylenamine-treated) plates (Perkin Elmer, Waltham, MA) using a Brandel cell harvester (Brandel,
Gaithersburg, MD). Filters were washed 2 times with 0.3 ml of ice-cold phosphate buffered saline pH 7.0
containing 0.01% Triton X100. Radioactivity on the filters was quantified using a MicroBeta TriLux
Liquid Scintillation Counter (PerkinElmer Life and Analytical Sciences, Waltham, MA). In radioligand
time course experiments, designed to determine unlabeled compound kinetic (association and
dissociation) rate constants, radioligand, membranes and unlabeled test compound were added to the
wells at various time points (0-4 hr) and the assay wells harvested simultaneously.

[125I]Angiotensin II receptor whole cell radioligand binding assay

AT1R downregulation was measured as loss of cell surface angiotensin II binding sites following
methods previously described 3,4. Briefly, HEK-293 cells, with stable expression of human AT1R(2x 105
cells per well) in 24-well plates (Poly-D-Lysine treated) were stimulated with either Opti-MEM (vehicle),
angiotensin II, TRV0120027 or losartan for 30 minutes at 37°C. Surface-bound ligands were removed by
a gentle acid wash (50 mM glycine-150 mM NaCl, pH 3) for 10 min at 4°C, which did not affect
subsequent receptor binding. After rinsing several times, a whole cell radioligand binding assay was
performed (3-5 hours at 4°C) to quantify receptors remaining cell surface receptors. [125I]-ANGII at a
concentration of 0.1 to 0.2 nM was added in buffer containing 150 mM NaCl, 5 mM MnCl2, 0.1%
DMSO and 0.02% bovine serum albumin. Total binding was measured with the addition of assay buffer
and nonspecific binding was defined in the presence of 1 µM saralasin. After 3-5 h incubation at 4°C,
cells were solubilized with 0.5 M NaOH-0.05% SDS, and total radioligand bound was quantified by
addition of scintillation fluid using a Microbeta TriLux scintillation counter. Downregulation was
examined bycomparing binding in the presence of TRV027 or losartan to control.

Data Analysis – radioligand binding

Apparent binding affinities, Ki = IC50/ (1+ [Ligand]/Kd) were performed using the nonlinear iterative
curve-fitting computer program GraphPad PRISM (San Diego, CA). The association and dissociation rate
constants of unlabeled ligands were determined using a previously described method 5,6 in which
association of a radiolabeled ligand is measured in the presence of a fixed concentration(s) of unlabeled
test ligand. The model assumes that the ligands bind in a competitive manner according to simple
bimolecular reactions.
Selectively engaging β-arrestins at the AT1R reduces blood pressure and increases cardiac performance
Jonathan D. Violin Ph.D., Scott M. DeWire Ph.D., Dennis Yamashita Ph.D., David H. Rominger, Lisa
Nguyen, Kevin Schiller, Erin J. Whalen Ph.D., Maxine Gowen Ph.D., and Michael W. Lark Ph.D
The Journal of Pharmacology and Experimental Therapeutics

References

1. Fridericia LS (1920) Die systolendauer im elektrokardiogramm bei normalen menschen und bei
herzkranken. Acta Med Scand 53:469-486
2. Pacher P, Nagayama T, Mukhopadhyay P, Bátkai S, Kass DA (2008) Measurement of cardiac
function using pressure-volume conductance catheter technique in mice and rats. Nat Protoc
3(9):1422-34
3. Modrall JG, Nanamori M, Sadoshima J, Barnhart DC, Stanley JC, Neubig RR. (2001) ANG II
type 1 receptor downregulation does not require receptor endocytosis or G protein coupling. Am J
Physiol Cell Physiol 281:C801-C809
4. Feng YH, Ding Y, Ren S, Zhou L, Xu C, and Karnik SS (2005) Unconventional homologous
internalization of the angiotensin II Type-1 receptor induced by G-Protein–independent signals.
Hypertension 46:419-425
5. Motulsky H and Mahan LC (1984) The kinetics of competitive radioligand binding predicted by
the law of mass action. Mol Pharmacol 25:1–9
6. Hoare SRJ and UsdinTB (2000) Tuberoinfundibular peptide (7-39) [TIP(7-39)], a novel,
selective, high-affinity antagonist for the parathyroid hormone-1 receptor with no detectable
agonist activity. J Pharmacol Exp Ther 295:761–770
Selectively engaging β-arrestins at the AT1R reduces blood pressure and increases cardiac performance
Jonathan D. Violin Ph.D., Scott M. DeWire Ph.D., Dennis Yamashita Ph.D., David H. Rominger, Lisa Nguyen, Kevin Schiller, Erin J. Whalen Ph.D., Maxine Gowen Ph.D., and Michael W. Lark Ph.D
The Journal of Pharmacology and Experimental Therapeutics

Supplementary Table 1. Kinetic parameters of TRV120027 and Losartan binding to the human AT1Rin
HEK293 cell membranes. Time course of [125I]-ANGII association binding with the hAT1 receptor in
HEK membranes to determine unlabeled compound kinetics was measured as described under
Supplementary Methods. Association time course data in the presence of unlabeled ligand were fitted
according to data analysis to obtain apparent values for kon and koff, respectively the association and
dissociation rate constants of the unlabeled ligand. The following parameters determined independently
for [125I]-ANG II were held constant in the analysis: [L], k1 8.3x 10 7mol/L-1 ⋅ min-1, k2 0.013 min-1 along
with [I] tested. Values presented are mean ± SEM from two independent experiments.

Ligand kon koff (t ½) koff / kon Competition


binding Ki
x 10 6mol/L-1 ⋅ min-1 min-1 nmol/L nmol/L
TRV120027 3.9 ± 0.15 0.047 ± 0.01 12 ± 4 16
(16 min)
Losartan 13.9 ± 0.85 0.1 ± 0.03 7.5 ± 3 7
(7 min)
Selectively engaging β-arrestins at the AT1R reduces blood pressure and increases cardiac performance
Jonathan D. Violin Ph.D., Scott M. DeWire Ph.D., Dennis Yamashita Ph.D., David H. Rominger, Lisa Nguyen, Kevin Schiller, Erin J. Whalen Ph.D., Maxine Gowen Ph.D., and Michael W. Lark Ph.D
The Journal of Pharmacology and Experimental Therapeutics

Supplementary Table 2. Effect of telmisartan on cardiovascular parameters. Data are


mean +/- SEM of 3 animals.

Hemodynamics Baseline 0.1 ug/kg/min 1 ug/kg/min 10 ug/kg/min Washout


Heart Rate (bmp) 352 ± 13 346 ± 24 348 ± 29 341 ± 36 316 ± 18
Mean Arterial Pressure (mmHg) 111 ± 6 83 ± 1* 80 ± 2* 70 ± 3* 70 ± 4*
Stroke Volume (uL) 29 ± 1.4 24.7 ± 1.1 22.4 ± 1.8* 20.5 ± *1.7 21.8 ± 2*
End Diastolic Volume (RVU) 17.1 ± 0.4 17.3 ± 0.6 17.3 ± 0.7 17.6 ± 0.7 17.3 ± 0.6
End Systolic Pressure (mmHg) 114 ± 9 86 ± 1* 82 ± 1* 74 ± 4* 78 ± 5*
End Diastolic Pressure (mmHg) 6± 1 5± 1 4± 1 4± 1 4± 2

Systolic Function
dP/dt max (mmHg/s) 5,487 ± 940 3,736 ± 141* 3,515 ± 186* 3,127 ± 236* 3,224 ± 223*
Ees, ESPVR slope (mmHg/RVU) 30 ± 2 25 ± 2 22 ± 4 20 ± 4* 20 ± 4*
PRSW (SWU/RVU) 65 ± 3 53 ± 8 38 ± 10 43 ± 3 52 ± 8
Stroke Work (mmHg * RVU) 176 ± 23 112 ± 2* 94 ± 4* 107 ± 7* 123 ± 4*
normalized PRSW (1/RVU) 0.38 ± 0.04 0.48 ± 0.08 0.39 ± 0.08 0.41 ± 0.06 0.42 ± 0.07

Diastolic Function
dP/dt min (mmHg/s) 6,333 ± 816 -4,357 ± 141* -4,020 ± 215* -3,468 ± 345* -3,687 ± 390*
Tau (s) 13 ± 1 14 ± 1 14 ± 2 15 ± 2 15 ± 1

Cardiac Conduction
PQ (ms) 44 ± 2 43 ± 2 43 ± 2 43 ± 2 44 ± 1
QRS (ms) 16 ± 1 16 ± 1 16 ± 1 15 ± 0 16 ± 1
QT (ms) 77 ± 4 76 ± 4 78 ± 5 76 ± 4 78 ± 3
QTcF (ms) 139 ± 5 136 ± 4 138 ± 4 135 ± 2 136 ± 3

*P < 0.05 by ANOVA with Dunnett's multiple comparison test


RVU = relative volume unit
ESPVR = end-systolic pressure volume relationship
PRSW = preload recruitable stroke work; SW vs EDV slope
SWU = stroke work units (mmHg * volume unit)
Selectively engaging β-arrestins at the AT1R reduces blood pressure and increases cardiac performance
Jonathan D. Violin Ph.D., Scott M. DeWire Ph.D., Dennis Yamashita Ph.D., David H. Rominger, Lisa Nguyen, Kevin Schiller, Erin J. Whalen Ph.D., Maxine Gowen Ph.D., and Michael W. Lark Ph.D
The Journal of Pharmacology and Experimental Therapeutics

Supplemental Table 3. Effect of dobutamine on cardiovascular


parameters. Data are mean +/- SEM of 3 animals

Hemodynamics Vehicle Dobutamine


Heart Rate (bmp) 369 ± 21 450 ± 11*
Mean Arterial Pressure (mmHg) 112 ± 9 103 ± 10
Stroke Volume (uL) 57.2 ± 4.3 68 ± 7.7
End Diastolic Volume (RVU) 17.5 ± 0.3 16.5 ± 0.6
End Systolic Pressure (mmHg) 123 ± 7 136 ± 11
End Diastolic Pressure ((mmHg)
g) 7±1 7±1

Systolic Function
dP/dt max (mmHg/s) 6,099 ± 372 10,608 ± 1345*
Ees, ESPVR slope (mmHg/RVU) 30 ± 8 42 ± 6
PRSW (SWU/RVU) 103 ± 10 120 ± 15
Stroke Work (mmHg * RVU) 279 ± 43 294 ± 43
normalized PRSW (1/RVU) 0.39 ± 0.1 0.43 ± 0.05

Diastolic Function
dP/dt min (mmHg/s) -7,262 ± 508 -8,917 ± 755
Tau (s) 13 ± 0 10 ± 0

Cardiac Conduction
PQ (ms) 42 ± 2 40 ± 1
QRS (ms) 16 ± 1 15 ± 1
QT (ms) 64 ± 3 65 ± 2
QTcF (ms) 117 ± 6 126 ± 5

*P < 0.05 by ANOVA with Dunnett's multiple comparison test


RVU = relative volume unit
ESPVR = end-systolic pressure volume relationship
PRSW = preload recruitable stroke work; SW vs EDV slope
SWU = stroke work units (mmHg * volume unit)