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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

Downloaded from jpet.aspetjournals.org at ASPET Journals on August 4, 2015

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

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

Drugs targeting G protein-coupled receptors (GPCRs) are
used in a wide range of diseases. Indeed, modulation of
GPCR function is one of the most common approaches to drug
discovery, either with agonists to mimic endogenous receptor
activation or antagonists to block endogenous agonist bind-
ing. These strategies have successfully targeted dozens of
GPCRs in the discovery of new therapeutic agents (Hopkins
and Groom, 2002). However, the network of signaling events
downstream of a receptor is complex and usually regulates
multiple cellular and tissue responses, not all of which are
intended targets of classic agonists and antagonists. In ad-
All work was funded by Trevena Inc., a privately held drug discovery
company.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.173005.
□S The online version of this article (available at http://jpet.aspetjournals.org)
contains supplemental material.
effects have historically presented a major barrier to the
development of safe, effective therapeutics.
However, not all GPCR signaling derives from G protein
signaling; GPCRs can activate parallel and sometimes dis-
tinct signals. The most general of these are mediated by
␤-arrestins, which bind to activated receptors to desensitize
G protein signaling, promote receptor internalization, and
activate distinct signal transduction cascades that can be
independent of G protein coupling (Reiter and Lefkowitz,
2006; DeWire et al., 2007). Nearly all GPCRs will couple to at
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-
ated components (Schmid and Bohn, 2009).
Other work has shown that the experimental separation of
GPCR effects can also be achieved pharmacologically: certain
GPCR ligands can selectively activate subsets of receptor
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

2007; Tran et al., 2009; Zidar et al., 2009). These “biased
ligands” are agonists when assaying some receptor functions,
but they are antagonists or even inverse agonists when as-
saying other receptor functions (Violin and Lefkowitz, 2007;
Kenakin, 2009). One of the most studied GPCRs in this
regard is the angiotensin II type 1 receptor (AT1R). This
receptor engages all three classes of ␤-arrestin function: de-
sensitization (Violin et al., 2006), internalization (Kule et al.,
2004), and signaling (Barnes et al., 2005; DeWire et al., 2008;
Ahn et al., 2009). It was also one of the first receptors for
which a ␤-arrestin biased ligand was described: 1Sar, 4Ile,
8
Ile-angiotensin II (SII) induces ␤-arrestin recruitment, re-
ceptor internalization, and ␤-arrestin-mediated signals with-
out activating G protein coupling (Wei et al., 2003; Ahn et al.,
2004; Kim et al., 2009). Unfortunately, this peptide displays
low receptor binding affinity (Holloway et al., 2002), which
has made in vivo characterization of its effects challenging.
However, ex vivo studies revealed that SII promotes contrac-
tility of isolated cardiac myocytes via the AT1R and ␤-arres-
tin2 (Rajagopal et al., 2006) and mitogen-activated protein
kinase (MAPK) activation in perfused hearts (Aplin et al.,
2007), indicating that ␤-arrestin biased ligands may have
significant impact in a physiological setting. However, it
remains unclear whether AT1R signals can be separated by
biased ligands in vivo to allow precise targeting of desired
AT1R effects. More broadly, despite increasing speculation in
the research literature and numerous examples of biased
ligands eliciting unique pharmacology in vitro, it remains
unclear whether ligand bias can elicit unique, differentiated
pharmacology in vivo compared with unbiased ligands by
simultaneously activating one signal transduction pathway
while antagonizing another at the same receptor.
Thus we set out to discover new ␤-arrestin biased ligands
for the AT1R, seeking potent, selective compounds to enable
an in vivo comparison of biased and unbiased ligands to test
whether AT1R ligand bias translates from isolated cellular
systems to elicit unique integrated responses in vivo. Such a
ligand would also allow us to test whether biased ligands
truly uncover new opportunities for safer, more efficacious
therapeutics for cardiovascular diseases of impaired cardiac
performance.
Materials and Methods
Synthesis of Peptides. Peptides were synthesized by Genscript
USA Inc. (Piscataway, NJ) to more than 98% purity; quality control
was by high-performance liquid chromatography and mass spec-
trometry. The peptides described here are as follows: TRV120023,
Sar-Arg-Val-Tyr-Lys-His-Pro-Ala-OH; TRV120026, Sar-Arg-Val-
Tyr-Tyr-His-Pro-NH2; and TRV120027, Sar-Arg-Val-Tyr-Ile-His-
Pro-D-Ala-OH.
Cell Culture and Preparation. HEK-293 cells were stably
transfected to overexpress ␤-arrestin2 fused to a ␤-galactosidase
fragment and human AT1R or rat AT1aR fused to a complemen-
tary ␤-galactosidase fragment. The growth medium used was
MEM with 10% FBS, 1% penicillin/streptomycin, 150 ␮g/liter of
neomycin, and 150 ␮g/liter of hygromycin. ␤-Arrestin recruitment
and inositol monophosphate (IP1) accumulation were measured in
small-volume, 384-well plates; immunoblot experiments were in
12-well or 10-cm plates. U2-osteosarcoma (U2-OS cells) were sta-
bly transfected to overexpress rat AT1aR and cultured in MEM
with 10% FBS, 1% penicillin/streptomycin, and 150 ␮g/liter of
neomycin. Cells were plated in 6- or 12-well tissue culture plates
for immunoblot experiments.
AT1R ␤-Arrestin Bias Improves Cardiac Function 573
␤-Arrestin Recruitment. The PathHunter protein complemen-
tation assay (DiscoveRx Corporation, Fremont, CA) was performed
according to the manufacturer’s protocol and read for chemilumines-
cent signaling on a PheraStar reader (BMG Labtech, Durham, NC).
In brief, complementary halves of ␤-galactosidase were genetically
fused to the carboxyl termini of the AT1R and ␤-arrestin2. When
cotransfected, the two fusion proteins serve as a proximity sensor;
when ␤-arrestin2 translocates to active receptor, the ␤-galactosidase
fragments interact to form a functional enzyme, which is detected by
a chemoluminescent substrate.
Inositol Monophosphate Accumulation. IP1 was measured
with the IP-One Tb HTRF kit (Cisbio, Bedford, MA). Plates were
read on a PheraStar reader using a time-resolved fluorescence ratio
(665 nm/620 nm).
Immunoblotting. U2-OS or HEK-293 cells stably expressing the
rat AT1aR were grown in MEM supplemented with 10% FBS. After
overnight serum starvation, cells were stimulated with 1 ␮M angioten-
sin II, valsartan, TRV0100027, TRV0100023, or vehicle for 5 min.
Lysates were immunoblotted with phospho-Y416-Src, phospho-T202/
Y204-ERK, or phospho-S1177-eNOS antibodies per the manufacturer’s
Downloaded from jpet.aspetjournals.org at ASPET Journals on August 4, 2015
instructions (Cell Signaling Technologies, Danvers, MA). For RNA in-
terference experiments, siRNAs for ␤-arrestin2 were transfected as
described previously (DeWire et al., 2008), and cells were plated as
above at 48 h after siRNA transfection. The signal transduction protein
array assay was performed per the manufacturer’s protocol (Proteome
Profiler ARY003; R&D Systems, Minneapolis, MN). The activated en-
zymes detected in this array relied on antibodies to phospho-Y397 on
FAK and phospho-S63 on c-Jun. Immunoreactive bands or spots were
visualized and quantitated by using GeneSnap and GeneTool software
(Syngene, Frederick, MD).
Myocyte Contractility. Isolated murine cardiomyocytes were
isolated and contractility was measured by video edge detection as
described previously (Jaleel et al., 2008).
In Vivo Studies. All in vivo studies were performed at Qtest Labs
(Columbus OH) for cardiovascular assays or ChemPartner Co.
(Shanghai, China) for pharmacokinetics, under protocols reviewed
and approved by the respective Institutional Animal Care and Use
Committees.
Rat Hemodynamics during Angiotensin II Dose Escalation.
TRV120027, vehicle, and losartan were dosed to anesthetized (sodium
pentobarbital)/ventilated healthy male rats. Under anesthesia, the an-
imals were instrumented with ECG electrodes and micromanometers
into a femoral artery and the left ventricle. Mean arterial pressure
(MAP) and left ventricular hemodynamic and mechanical indices (end
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-
tion (QT/QTcF) were measured. Two dose levels of TRV120027, low (1
␮g/kg/min) and high (10 ␮g/kg/min), were assayed. The cardiovascular
responses of test article-treated animals to an eight-dose angiotensin II
challenge were evaluated and compared against those obtained in an-
imals receiving either losartan (3 mg/kg bolus injection) or vehicle
(infusion).
Rat Pressure-Volume Loop Analysis. Telmisartan and
TRV120027 were dosed to anesthetized (sodium pentobarbital)/ven-
tilated healthy male rats. Under anesthesia, the animals were in-
strumented with ECG electrodes, a micromanometer into a femoral
artery, a conductance/micromanometer into the left ventricle, and a
hydraulic occluder around the inferior vena cava (via a right thora-
cotomy). MAP and left ventricular mechanical (end systolic pressure,
end diastolic pressure, dP/dtmax, Tau, Vmax) and geometrical (end
diastolic volume, stroke volume, dV/dt) indices were obtained from
the ventricular pressure/conductance (volume) signals. The myocar-
dial inotropic [end systolic pressure-volume (PV) relationship
(ESPVR), preload recruitable stroke work (PRSW)], lusitropic (end
diastolic PV relationship), and energetic (PV area, stroke work) state
were examined by means of PV curves; PV families/relationships
were generated via preload reductions (inferior vena cava occlusion).
574 Violin et al.
ECG-derived indices of conduction (PQ, QRS) and repolarization
duration (QT/QTcF) were measured.
Statistics. Statistics were assessed by using Prism version 5
(GraphPad Software Inc., San Diego, CA). Schild analysis fitting the
Schild model of competitive antagonism to a series of dose-response
curves with varying amounts of antagonist was also performed with
Prism software.
Results
The AT1R Is Capable of Potent, Robust Efficacy by
␤-Arrestin Biased Ligands. To identify new, higher-
potency ␤-arrestin biased ligands, we initiated an iterative
evaluation of custom-synthesized peptides based on SII,
using assays of G protein coupling (inositol monophos-
phate accumulation) and ␤-arrestin recruitment to the
AT1R (enzyme complementation) to characterize the po-
tency, efficacy, and ␤-arrestin bias of new compounds at
overexpressed human AT1R. This search led to the iden-
tification of two ␤-arrestin biased ligands with improved
potency compared with SII: TRV120023 and TRV120027
selectively and potently engage ␤-arrestin2 recruitment
(EC50 ⫽ 44 and 17 nM, respectively at the human AT1R)
without any detectable activation of G protein coupling
(data for TRV120027 shown in Fig. 1). This contrasts with
the robust G protein and ␤-arrestin2 signals elicited by
angiotensin II (EC50 of 1.1 and 9.7 nM for IP1 accumula-
tion and ␤-arrestin2 recruitment, respectively) and the
complete lack of G protein or ␤-arrestin2 efficacy of angiotensin
receptor blockers (ARBs) such as valsartan. TRV120023 and
TRV120027 had very similar potency, efficacy, and bias at the
rat AT1aR, with EC50 for ␤-arrestin2 recruitment of 37 and 23
nM, respectively.
To verify ␤-arrestin efficacy, we also showed that angio-
tensin II, TRV120023, and TRV120027 evoked ␤-arrestin1
and ␤-arrestin2 recruitment, as measured by ␤-arrestin–
yellow fluorescent protein interaction with human AT1R–
cyan fluorescent protein (Rajagopal et al., 2006), whereas
ARBs did not, consistent with our enzyme complementa-
tion result (data not shown). In these same experiments,
we saw clear translocation of ␤-arrestin2–yellow fluores-
cent protein from cytosol to membrane compartments and
internalization of AT1R– cyan fluorescent protein for an-
giotensin II-, TRV120023-, and TRV120027-treated cells,
but no effect of the ARBs valsartan, losartan, or telmisar-
tan, consistent with ␤-arrestin2 recruitment and coupling
to endocytic machinery (Supplemental Fig. 1A). This was
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.
confirmed by whole-cell radioligand binding studies, which
showed that TRV120027, but not the ARB losartan, re-
duced angiotensin II binding sites in HEK cells overex-
pressing human AT1R (Supplemental Fig. 1B).
We chose TRV120027, the more potent ligand, for more
thorough characterization. Escalating concentrations of
TRV120027 shifted the EC50 of angiotensin II-evoked G
protein coupling without suppressing maximal efficacy,
consistent with a purely competitive mechanism of action
(Fig. 1D). Fitting the data to a Schild model of competitive
antagonism (Lew and Angus, 1995) supported this conclu-
sion (Schild slope ⫽ 1.04 ⫾ 0.06), with an estimated Kd of
19 nM. This is consistent with radioligand binding studies with
125
I-angiotensin II, which showed a Ki for TRV120027 of 16 nM
(Supplemental Table 1). These studies also showed that
TRV120027 has apparent first-order binding, with a kon of 3.9 ⫻
106 M⫺1 䡠 min⫺1, koff of 4.7 ⫻ 10⫺2 min⫺1, corresponding to a
residence half-time of 16 min, similar to that of losartan. The
Downloaded from jpet.aspetjournals.org at ASPET Journals on August 4, 2015
TRV120027 Kd, as calculated from on and off rates, was 12 nM,
consistent with the apparent Ki.
TRV120027 was also remarkably specific for the AT1R: at
58 different receptors and channels 10 ␮M TRV120027
showed less than 15% inhibition of reference radioligand
binding (Supplemental Methods). The only receptor other
than the AT1R for which TRV120027 had affinity was the
angiotensin II type 2 receptor (AT2R), with apparent Ki as
measured by radioligand binding of 7 nM.
The Signaling Profile of ␤-Arrestin Biased Ligands
Is a Subset of Angiotensin II-Mediated Signaling. Con-
sistent with ␤-arrestin recruitment, in U2-OS cells over-
expressing rat AT1aR, angiotensin II, TRV120023, and
TRV120027 activated the MAPK ERK1/2, contrasting with
valsartan (Fig. 2A) and a range of other ARBs that were
inactive (data not shown). Similar results were found in
HEK cells (data not shown), which have been widely used
to assess ␤-arrestin signaling (Ahn et al., 2004); in these
cells TRV120027 stimulated ERK1/2 with an EC50 of ap-
proximately 1 nM. It is noteworthy that of these com-
pounds only angiotensin II activated p38 MAPK (Fig. 2B),
highlighting that ␤-arrestin biased ligands, devoid of G
protein coupling, activate only a subset of the normal
complement of AT1R signals. We also found that angioten-
sin II, TRV120023, and TRV120027, but not valsartan,
stimulated activation of both Src (Fig. 2C) and eNOS (Fig.
2D). These data suggested that ␤-arrestin recruitment to
the AT1R could engage a pathway whereby Src phosphor-
 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,

Downloaded from jpet.aspetjournals.org at ASPET Journals on August 4, 2015

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
fact ␤-arrestin2-mediated.
We then searched more broadly for signals engaged by our
␤-arrestin biased ligands, using an antibody array for 55
different signal transduction proteins, largely selective for
phosphorylated “active” or “inactive” proteins. Of these, two
were strongly activated by the ␤-arrestin biased ligands but
not valsartan: phospho-c-Jun (Fig. 3A) and phospho-FAK
(Fig. 3B). Together, these results establish that ␤-arrestin
biased ligands display a pharmacological profile in vitro that
is distinct from classic antagonists and here represents a
subset of unbiased agonist signaling.
␤-Arrestin Biased Ligands Stimulate Isolated Car-
diomyocyte Contractility. Previous work showed that
SII stimulated contractility of isolated murine cardiomyo-
cytes via the AT1aR and ␤-arrestin2 (Rajagopal et al.,
2006), so we used this experimental system to test the
cellular effects of TRV120023 and TRV120027 related to
cardiovascular function. In agreement with the effects of
SII, we found that TRV120027 stimulated cardiomyocyte
contractility, but the unbiased antagonist valsartan did
not (Fig. 4A). Valsartan, which is highly selective for the
AT1R, including selectivity versus the AT2R (Criscione et
al., 1993), blocked TRV120027-stimulated cardiomyocyte
contractility, indicating that this effect is AT1R mediated.
Separate experiments showed similar results for TRV120023
(Fig. 4B) and a lack of effect for the ARBs losartan and
Fig. 3. FAK and c-Jun phosphorylation stimulated by ␤-arrestin biased
ligands, identified from a signal panning screen for changes in expression
or phosphorylation of 55 different signaling proteins. Comparison of
TRV120023 and TRV120027 to an unbiased agonist and an unbiased
antagonist in U2-OS cells overexpressing rat AT1aR identified c-Jun
phosphorylation (A) and FAK phosphorylation (B). Data are means ⫾
standard errors of three to seven independent experiments. ⴱ, p ⬍ 0.05
versus vehicle and valsartan and ⴱⴱ, p ⬍ 0.05 versus the same compound
with control siRNA, as measured by ANOVA with the Bonferroni multi-
ple comparison test.
telmisartan at receptor-saturating concentrations (data not
shown).
␤-Arrestin Biased Ligands Block the AT1R Pressor
Response While Stimulating Cardiac Performance.
In vivo, acute AT1R-mediated blood pressure effects are
regulated by G protein-stimulated calcium mobilization
(Harris et al., 2007; Wynne et al., 2009). Consistent with
this, and with the in vitro findings in Fig. 2, both
TRV120027 and the unbiased antagonist losartan compet-
itively antagonized the effect of escalating angiotensin II
on MAP in rats, as evidenced by an increase in the angio-
tensin II EC50 (Fig. 5A). For losartan, this competitive
antagonism was the only obvious effect. In contrast,
TRV120027 also reduced blood pressure independent of
angiotensin II and depressed the maximum angiotensin
II-evoked blood pressure increase.
In the same experiment, when we evaluated effects of
losartan and TRV120027 alone, before the addition of angio-
576 Violin et al.
Fig. 4. TRV120027 and TRV120023 increase mouse cardiomyocyte con-
tractility. A, angiotensin II (10 ␮M) and TRV120027 increased fractional
shortening, as measured by video edge detection microscopy. Valsartan (1
␮M) had no effect, but completely blocked the effect of TRV120027. B, a
separate experiment revealed similar properties for TRV120023 and also
compared responses with those elicited by 10 nM isoproterenol. Data are
presented as mean ⫾ standard error of the percentage increase in short-
ening of single myocytes after drug treatment. n ⫽ 3 independent exper-
iments, each using a different heart, with two myocytes tested for each
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-
roni multiple comparison test.
Fig. 5. TRV120027 competitively antagonizes angiotensin II blood pres-
sure increases while stimulating cardiac contractility in normal rats.
A, losartan and TRV120027 shift the angiotensin II dose-escalation blood
pressure response. B, in the same experiment, before angiotensin II
treatment, TRV120027, but not losartan, increased cardiomyocyte con-
tractility, as measured by the maximum rate of myocardial contractility
vehicle (Vmax). Data are means ⫾ standard errors of seven animals per
group. ⴱ, p ⬍ 0.05 versus vehicle, as measured by ANOVA with the
Bonferroni multiple comparison test.
tensin II, we noted that myocardial shortening velocity
(Vmax), a measure of cardiac contractility relatively insensi-
tive to changes in vascular or arterial pressure, was in-
creased by TRV120027 but not by losartan (Fig. 5B). This is
consistent with our cardiomyocyte contractility study (Fig.
4), a known ␤-arrestin2-mediated effect (Rajagopal 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
TRV120027 to eliminate potential nondrug effects; the
effects of telmisartan on cardiac function are shown in
Supplemental Table 2.
TRV120027 and telmisartan both produced dose-dependent
decreases in MAP (Fig. 6A). Neither compound increased heart
rate (Fig. 6B), but TRV120027, unlike telmisartan, increased
cardiac contractility: TRV120027 dose-dependently increased
the slope of ESPVR (Fig. 6C), a load-independent measure of
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-
versible: 5 to 7 min after ceasing infusion, MAP and ESPVR had
returned nearly to baseline values (Supplemental Fig. 3). This
rapid reversibility is consistent with the short half-life of
TRV120027 in vivo: after infusion into normal rats, TRV120027
had a half-life of 1.5 min (Supplemental Methods).
Similar to TRV120027, TRV120023 reduced MAP while
increasing ESPVR (Supplemental Fig. 4, A and B).
TRV120026, a third ␤-arrestin biased ligand with very sim-
ilar potency for ␤-arrestin recruitment (EC50 ⫽ 24.5 nM at
the human AT1R) but 80-fold higher relative specificity for
binding the AT1R over the AT2R, showed similar effects, at
the same doses, as TRV120027 and TRV120023 on MAP and
ESPVR (Supplemental Fig. 4, C and D). Indeed the biggest
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

Downloaded from jpet.aspetjournals.org at ASPET Journals on August 4, 2015

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
AT2R is consistent with an AT1R-mediated effect. The effects
of the affinity of TRV120027 for the AT2R, if any, remain
unclear; however, these findings, along with the blockade of
cardiomyocyte contractility in vitro by an AT1R-selective an-
tagonist, suggest that the enhancement of cardiac contractil-
ity of TRV120027 is AT1R-mediated.
Together, the findings presented here demonstrate that
TRV120027 depresses blood pressure while increasing car-
diac performance, consistent with its antagonist efficacy
against angiotensin II pressor activity (Fig. 5), its ␤-arrestin-
mediated stimulation of cardiac contractility in vitro (Fig. 4),
and Vmax and ESPVR in rats (Figs. 5 and 6). The stimulation
of contractility by TRV120027 is modest compared with the
classic inotrope dobutamine, which raises dP/dtmax and more
strongly increases ESPVR and also increases heart rate
(Supplemental Table 3). This modest efficacy of TRV120027,
in conjunction with its afterload reduction, may explain the
apparently beneficial energetics of reduced stroke work. This
profile suggests that TRV120027 and related ␤-arrestin bi-
ased AT1R agonists could have a beneficial impact on cardio-
vascular syndromes characterized by hypertension and in-
sufficient cardiac performance.
The results presented here describe the unique in vitro
and in vivo pharmacology of TRV120027, a novel, selective,
and potent ␤-arrestin biased ligand of the AT1R. This work
demonstrates that biased and unbiased ligands are phar-
macologically distinct in vivo. TRV120027 functions as an
antagonist, analogous to ARBs, when evaluated for G pro-
tein coupling, other cellular signals such as p38 MAPK
activation, and effects on blood pressure. However, when
assessed for efficacy in recruiting ␤-arrestins to the AT1R,
and for downstream effects including receptor internaliza-
tion, ␤-arrestin-mediated signals, and cardiomyocyte con-
tractility, TRV120027 is an agonist. This bias is not merely
an artifact of differentially amplified assay readouts: IP1
accumulation in response to angiotensin II is more sensi-
tive than ␤-arrestin2 recruitment, as measured by po-
tency, but undetectable despite strong ␤-arrestin re-
sponses for TRV120023 and TRV120027. This is a
hallmark of intrinsic ligand bias, reflecting a stabilization
of different receptor conformations than are stabilized by
angiotensin II or the unbiased ARBs (Kenakin, 2007). This
assay-dependent efficacy violates classic models of molec-
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.
related and instead requires newer more complicated mod-
els of drug action (Kenakin, 2009; Tran et al., 2009).
Because TRV120027 reduces MAP while increasing car-
diac contractility, it is intriguing to speculate what bene-
fits it might have in acute heart failure, a syndrome usu-
ally comprising low cardiac output, normal or elevated
blood pressure, and an elevated renin-angiotensin system.
This syndrome represents a vicious cycle in which insuffi-
cient cardiac output causes angiotensin II-mediated vaso-
constriction, further impairing cardiac output by raising
the work required to maintain end organ perfusion, thus
leading to further deterioration of cardiac performance.
TRV120027 could block the vasoconstrictive effects of re-
nin-angiotensin system activation while promoting cardiac
performance via ␤-arrestin signaling, potentially breaking
this vicious cycle and improving patient outcomes. This is
a unique profile, which in light of reduced afterload (Ea),
myocardial oxygen demand (PV area), and work (stroke
work) during TRV120027 treatment, suggests improved
energetic or mechanical function of the heart. This is re-
flected in improved ventriculo-arterial coupling (Ea/ES-
PVR slope) for TRV120027 not seen with telmisartan (Ta-
ble 1). Future studies will test the effects of TRV120027 in
disease models and elucidate the molecular mechanism
whereby these compounds increase cardiac contractility.
Nevertheless, this work clearly establishes TRV120027
as a biased ligand with unique physiological effects com-
pared with unbiased ligands. Our data strongly support
two major themes of contemporary GPCR research: the
importance of ␤-arrestin signaling as a distinct set of path-
ways operating parallel to classic G protein signaling and
the utility of biased ligands to selectively engage desired
signal transduction pathways from a single receptor to
elicit specific physiological effects. Because biased ligands
have been identified for numerous receptors (Violin and
Lefkowitz, 2007; Kenakin, 2009), ligand bias may be a
general strategy that can dissect the biology of GPCRs as
both research tools and investigational new drugs. This
has important implications for GPCR-targeted drug dis-
covery by suggesting that in many cases the optimal eval-
uation of GPCR ligands requires characterization across a
range of signal transduction pathways, linking the result-
ing profiles to the relevant pathophysiological setting.
More broadly, as the interplay of ␤-arrestin and G pro-
tein signaling networks continues to be elucidated, biased
ligands offer a general strategy for more precisely target-
ing GPCR functions. The findings presented here demon-
strate successful identification of biased ligands with
unique pharmacology that translates from in vitro to in
vivo studies. Such biased ligands allow an unprecedented
level of control of receptor functions, enabling both basic
research into GPCR signal transduction mechanisms and
potential development of safer, more efficacious therapeu-
tics.
Acknowledgments
We thank Steven Houser and Xiong Wen Chen for performing the
isolated cardiomyocyte contractility studies and Robert Lefkowitz,
Howard Rockman, and David Soergel for critically reviewing the
manuscript.
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AT1R ␤-Arrestin Bias Improves Cardiac Function 579
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Address correspondence to: Jonathan D. Violin, Trevena Inc., 1018 West
8th Avenue, Suite A, King of Prussia, PA 19406. E-mail: jviolin@
trevenainc.com
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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)