Download Print - Circulation Research

Loss of PI3K␥ Enhances cAMP-Dependent MMP
Remodeling of the Myocardial N-Cadherin Adhesion
Complexes and Extracellular Matrix in Response to Early
Biomechanical Stress
Danny Guo, Zamaneh Kassiri, Ratnadeep Basu, Fung L. Chow, Vijay Kandalam, Federico Damilano,
Wenbin Liang, Seigo Izumo, Emilio Hirsch, Josef M. Penninger, Peter H. Backx, Gavin Y. Oudit
Rationale: Mechanotransduction and the response to biomechanical stress is a fundamental response in heart
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
disease. Loss of phosphoinositide 3-kinase (PI3K)␥, the isoform linked to G protein– coupled receptor signaling,
results in increased myocardial contractility, but the response to pressure overload is controversial.
Objective: To characterize molecular and cellular responses of the PI3K␥ knockout (KO) mice to biomechanical
stress.
Methods and Results: In response to pressure overload, PI3K␥KO mice deteriorated at an accelerated rate
compared with wild-type mice despite increased basal myocardial contractility. These functional responses were
associated with compromised phosphorylation of Akt and GSK-3␣. In contrast, isolated single cardiomyocytes
from banded PI3K␥KO mice maintained their hypercontractility, suggesting compromised interaction with the
extracellular matrix as the primary defect in the banded PI3K␥KO mice. ␤-Adrenergic stimulation increased
cAMP levels with increased phosphorylation of CREB, leading to increased expression of cAMP-responsive
matrix metalloproteinases (MMPs), MMP2, MT1-MMP, and MMP13 in cardiomyocytes and cardiofibroblasts.
Loss of PI3K␥ resulted in increased cAMP levels with increased expression of MMP2, MT1-MMP, and MMP13
and increased MMP2 activation and collagenase activity in response to biomechanical stress. Selective loss of
N-cadherin from the adhesion complexes in the PI3K␥KO mice resulted in reduced cell adhesion. The ␤-blocker
propranolol prevented the upregulation of MMPs, whereas MMP inhibition prevented the adverse remodeling
with both therapies, preventing the functional deterioration in banded PI3K␥KO mice. In banded wild-type
mice, long-term propranolol prevented the adverse remodeling and systolic dysfunction with preservation of the
N-cadherin levels.
Conclusions: The enhanced propensity to develop heart failure in the PI3K␥KO mice is attributable to a
cAMP-dependent upregulation of MMP expression and activity and disorganization of the N-cadherin/␤-catenin
cell adhesion complex. ␤-Blocker therapy prevents these changes thereby providing a novel mechanism of action
for these drugs. (Circ Res. 2010;107:00-00.)
Key Words: myocardial contractility 䡲 cardiomyopathy 䡲 signaling pathways 䡲 heart failure 䡲 hypertrophy
T
cell adhesion complexes which are modulated by both class
IA and IB PI3Ks.1,7 Although PI3Ks and lipid phosphatases
can modulate cytoskeletal interactions, stretch can in turn
activate Akt/PKB and GSK-3␤ activity in both cardiomyocytes and Langendorff-perfused hearts.8 In cardiac muscle,
cell adhesion and the cardiomyocyte stretch sensor machinery
play key roles in the complex mechanism leading to human
DCM and associated heart failures.9 Indeed, dilated cardio-
he phosphoinositide 3-kinase (PI3K) system has a fundamental role in cell signaling and is involved in cell
survival and growth and modulates myocardial contractility.1,2
In the heart, both PI3K␣3–5 and PI3K␥3,6 controls distinct
aspects of cardiac structure and function. Mechanotransduction plays a fundamental role in cardiac (and vascular)
function and it appears to involve interactions between
extracellular matrix and intracellular cytoskeletal proteins via
Original received November 30, 2009; resubmission received July 26, 2010; revised resubmission received September 2, 2010; accepted September 8, 2010.
In August 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.2 days.
From the Division of Cardiology, Department of Medicine (D.G., F.L.C., G.Y.O.); Mazankowski Alberta Heart Institute (D.G., Z.K., R.B., F.L.C.,
V.K., G.Y.O.); and Department of Physiology (Z.K., R.B., V.K.), University of Alberta, Edmonton, Canada; University of Torino (F.D., E.H.), Italy;
Department of Physiology (W.L., P.H.B.), University of Toronto, Canada; Beth Israel Deaconess Medical Center (S.I., J.M.P.), Harvard Medical School,
Boston Mass; and Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna.
Correspondence to Gavin Y. Oudit, MD, PhD, FRCP(C), Division of Cardiology, Department of Medicine, Mazankowski Alberta Heart Institute,
University of Alberta, Edmonton, Alberta, T6G 2S2, Canada. E-mail [email protected]
© 2010 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.110.229054
1
2
Circulation Research
November 12, 2010
Non-standard Abbreviations and Acronyms
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
AB
ANF
BNP
DIDA
ECM
ERK
GSK
KO
LV
MHC
MMP
phospho
PI3K
PI3K␣DN
PI3K␥KD
PI3K␥KO
PLN
TIMP
WT
aortic banding
atrial natriuretic factor
B-type natriuretic peptide
2⬘,5⬘-dideoxyadenosine
extracellular matrix
extracellular signal-regulated kinase
glycogen synthase kinase
knockout
left ventricular
myosin heavy chain
matrix metalloproteinase
phosphorylated
phosphoinositide 3-kinase
phosphoinositide 3-kinase ␣ dominant negative
phosphoinositide 3-kinase ␥ kinase dead
phosphoinositide 3-kinase ␥ knockout
phospholamban
tissue inhibitor of matrix metalloproteinases
wild type
myopathy in humans is associated with marked remodeling of
extracellular matrix and cell adhesion complexes.
Loss of p110␥, the catalytic subunit of the PI3K␥ complex,
leads to enhanced cAMP generation resulting from a loss of
phosphodiesterase activity, resulting in enhanced Ca2⫹ cycling and attributable primarily to increased phosphorylation
(and inhibition) of phospholamban (PLN) and increased
sarcoplasmic reticulum Ca2⫹ stores.3,6,10 Using the aortic
banding (AB) model, we showed that despite these biochemical changes, PI3K␥KO mice show enhanced susceptibility to
early biomechanical stress. Our results provide a crucial link
between PI3K␥ and the cellular responses to biomechanical
stress, with a loss of p110␥ resulting in elevated matrix
metalloproteinase (MMP) expression and activity, and correlates with a selective loss of N-cadherin from cell adhesion
complexes. Importantly, these adverse cellular changes are
not observed in the PI3K␣DN and in PI3K␥ kinase dead
(kDa) mutant mice in response to pressure overload. Inhibition of cAMP levels with propranolol and MMP inhibition
provided significant rescue of the dilated cardiomyopathy in
banded PI3K␥KO mice. Chronic ␤-blocker therapy also
resulted in prevention of adverse myocardial remodeling in
wild-type (WT) mice. These results illustrate the importance
of cell adhesion and extracellular matrix in the response to
biomechanical stress even in the setting of enhanced Ca2⫹
cycling and increased myocardial contractility.
Methods
An expanded Methods section is available in the Online Data
Supplement at http://circres.ahajournals.org.
Experimental Animals
PI3K␥KO, PI3K␣DN, and PI3K␥KD mice in C57Bl/6 background
have been described previously.3,6,11 All experiments were performed in accordance with institutional guidelines and Canadian
Council on Animal Care. Only male mice of 8 to 9 weeks of age
and littermate C57Bl/6 WT controls were used. For the propranolol experiments, mice were treated with propranolol in their
drinking water (0.2 g/L) to deliver 15 mg/kg per day; propranolol
was withdrawn 1 day before the echocardiographic and hemodynamic measurements. The broad-spectrum MMP inhibitor
PD166793 (Pfizer Inc) was administered orally by daily gavage as
described previously.12 Because of the rapid onset of ventricular
dilation in PI3K␥KO mice, PD166793 treatment (30 mg/kg per
day) began 3 days before AB and continued until mice were
euthanized.
Aortic Banding
The AB protocol was used as a means of pressure overload and
biomechanical stress as described previously.12,13 Briefly, the descending aortic arch was accessed via a left thoracotomy and the
descending thoracic aorta was surgically constricted to generate a
transstenotic pressure gradient of 50 to 60 mm Hg.
Histology, Hydroxyproline, and TUNEL Assays
For heart morphometry, hearts were arrested with KCl, fixed with
10% buffered formalin, and embedded in paraffin. Myocardial
interstitial fibrosis was determined as collagen volume fraction using
confocal microscopy on picrosirius red stained 10 ␮m thick sections
pretreated with PMA. Collagen content was also determined using
the hydroxyproline assay as previously reported.14 In situ DNA
fragmentation was labeled using the TUNEL assay (ApopTag Plus
kit; Oncor, Gaithersburg, Md).
Echocardiography and
Hemodynamic Measurements
Echocardiographic assessments and invasive hemodynamic measurements were carried out as described previously.3,12,13
Isolated Cardiomyocyte Contractility
Left ventricular (LV) cardiomyocyte contractility was carried out as
described previously.3
Isolation and Culture of Adult Cardiomyocytes
and Fibroblasts
The protocol used for isolation and culture of adult mouse cardiomyocytes and fibroblasts was modified from O’Connell et al (see
Online Methods).15
Measurement of cAMP Levels
The cAMP levels in isolated adult ventricular cardiomyocytes
and LV myocardial tissue were measured using the cAMP
competitive enzyme immunoassay system (GE Healthcare Amersham Biosciences).
Cell Adhesion Assay
The ECM-based cell adhesion assay was carried out using a
colorimetric-based assay (CytoSelect 48-Well Cell Adhesion Assay;
Cell Biolabs Inc).
TaqMan Real-Time PCR, Western Blot Analyses,
and Membrane Fractionation, Gelatin
Zymography, and Collagenase Activity Assay
RNA expression levels were quantified with TaqMan RT-PCR using
ABI Prism 7700 sequence detection system as described previously
(see Online Table I for primers and probes).16,17 Western blot
analyses, gelatin zymography, and collagenase activity were performed as described previously (see Online Methods).16,17 For the in
vitro analysis of N-cadherin cleavage, 100 ng of human recombinant
N-cadherin Fc Chimera protein (R&D) was incubated with 0, 1, 10,
100 nmol/L MMP2 (R&D) or MT1-MMP (R&D).
Role of PI3K␥ in Biomechanical Stress
Guo et al
A
B
WT
3 weeks AB
D
PI3Kγ-KO
3 weeks AB
WT
3 weeks AB
PI3Kγ-KO
3 weeks AB
WT
PI3Kγ-KO
10
*
PSR
CVF (%)
8
6
4
2
0
apoptotic myocytes (%)
PI3Kγ-KO
3 weeks AB
G
3 weeks AB
F
TUNEL Staining
WT
3 weeks AB
WT
PI3Kγ-KO
4
3
2
1
0
1 week AB
H
BNP/18S (AU)
2400
2000
1600
*
1200
800
3000
2000
1000
400
0
Sham
1 Week AB
3 Weeks AB
0
Figure 1. Development of early-onset
dilated cardiomyopathy in response to
biomechanical stress in PI3K␥KO mice.
A and B, Four-chamber trichrome-stained
heart sections showing concentric hypertrophy in WT mice (A) with ventricular dilation and eccentric remodeling in PI3K␥KO
mice (B) in response to after AB. C
through F, Increased interstitial fibrosis
shown using picrosirius red staining (C,
left) and quantification of the collagen volume fraction (CVF) (D) with lack of an
increase in apoptosis based on TUNEL
staining (E, left) and quantified as percentage of apoptotic nuclei (F, right)
(P⫽0.341; n⫽5 per group). G through I,
Expression profile of hypertrophy disease
markers showing early and markedly
increased expression of ANF (G), BNP (H),
and ␤MHC (I) in banded PI3K␥KO mice
compared with WT mice. n⫽5 for sham
groups and n⫽7 for AB groups. AU indicates arbitrary unit. *P⬍0.05 compared
with the WT group.
*
8
6
4
2
0
3 weeks AB
4
3
2
1
0
3 weeks AB
I
*
4000
WT
PI3Kγ-KO *
100
βMHC/18S (AU)
2800
ANF/18S (AU)
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
E
Hydroxyproline (ug/mg)
PI3Kγ-KO
1 week AB
apoptotic myocytes (%)
WT
1 week AB
C
3
Sham
1 Week AB
*
80
60
40
*
20
3 Weeks AB
0
Confocal Fluorescence Microscopy
Formalin-fixed sections were permeabilized with ice-cold methanol
for 10 minutes, blocked with goat serum and incubated with primary
antibodies (anti–N-cadherin [rabbit] and anti–␤-catenin [mouse],
1:500, Abcam) overnight at 4°C in a humidified chamber. After
several washes, Texas red– conjugated (for cadherin) or FITCconjugated (for catenin) secondary antibodies were incubated for
30-minute at room temperature. The sections were evaluated with a
Zeiss LSM510 confocal microscope and associated software.
Statistical Analysis
One-way ANOVA was used to test for overall significance, followed
by the Student–Newman–Keuls test for multiple comparison testing
between the various groups. All statistical analyses were performed
with the SPSS software (version 10.1). Data are presented as
means⫾SEM; n refers to the sample size.
Results
Accelerated Development of Ventricular Dilation
and Pathological Hypertrophy in PI3K␥KO Mice
in Response to Biomechanical Stress
In response to pressure overload, WT mice developed concentric remodeling with increased wall thickness and reduction in chamber size at 1 and 3 weeks following AB (Figure
1A). In contrast, LV size dilated rapidly in PI3K␥KO mice
characteristic of eccentric remodeling at 1 and 3 weeks
Sham
1 Week AB
3 Weeks AB
following AB as shown by trichrome-stained four-chamber
views (Figure 1B). The early development of dilated cardiomyopathy in the banded PI3K␥KO mice was associated with
increased interstitial myocardial fibrosis at 3 weeks after AB
as illustrated by picrosirius red staining (Figure 1C) and
quantified as collagen volume fraction (Figure 1D). Increased
myocardial fibrosis in banded PI3K␥KO mice was confirmed
by the biochemical assessment of hydroxyproline content
(Figure 1D) and occurred without a measurable increase in
apoptosis (Figure 1E and 1F). In banded PI3K␥KO mice,
mRNA expression profile showed greater myocardial expression of the disease markers, atrial natriuretic factor (ANF)
(Figure 1G), BNP (Figure 1H), and ␤MHC (Figure 1I) with
morphometric features of hypertrophy (Online Table I).
Activation of several signaling pathways is a key mediator of
the response to biomechanical stress.18,19 The increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2
(Figure 2A) and Akt (Figure 2B) were reduced in banded
PI3K␥KO mice compared with banded WT mice after 1 week
of pressure overload. However, following 3 weeks of pressure overload, the degree of ERK1/2 phosphorylation was
equivalent in WT and PI3K␥KO mice (Figure 2A). The
GSK-3 system is an important downstream mediator of
PI3K/Akt signaling1,2,20 and consistent with a loss of Akt
4
Circulation Research
November 12, 2010
1 wk AB
Sham
A
p-ERK 1/2
ERK 1/2
B
WT
AB
AB
Sh 1 wk 3 wk
PI3Kγ-KO
AB
AB
Sh 1 wk 3 wk
p-Akt (S473)
Akt
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
C
WT
PI3Kγ-KO
AB
AB
Sh 1 wk 3 wk
AB
AB
Sh 1 wk 3 wk
p-GSK-3α
GSK-3α
D
WT
PI3Kγ-KO
AB
AB
Sh 1 wk 3 wk
AB
AB
Sh 1 wk 3 wk
p-GSK-3β
GSK-3β
E
WT
PI3Kγ-KO
AB
AB
Sh 1 wk 3 wk
AB
AB
Sh 1 wk 3 wk
p-FAK
FAK
1.2
1.2
1.0
1.0
0.8
*
*
0.6
0.4
0.4
0.2
0.2
0.0
0.0
1.2
*
*
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
2.0
2.0
*
1.5
1.5
1.0
1.0
0.5
0.5
0.0
0.0
0.6
0.6
0.5
*
0.4
0.3
0.3
0.2
0.2
0.1
0.1
0.0
0.0
1.0
*
*
#
0.5
0.4
1.2
*
0.8
0.6
1.0
3 wk AB
PI3Kγ-KO
1.2
p-Akt (S473) / Akt (AU)
AB
AB
Sh 1 wk 3 wk
p-GSK-3α / GSK-3α (AU)
AB
AB
Sh 1 wk 3 wk
p-GSK-3β / GSK-3β (AU)
PI3Kγ-KO
p-FAK / FAK (AU)
WT
p-ERK 1/2 / ERK 1/2 (AU)
WT
*
1.2
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
*
*
Figure 2. Altered signaling in response to biomechanical stress in PI3K␥KO mice. A and B, Western blot analysis (left) and quantification (right) showing increased phosphorylation of the mitogen-activated protein kinase ERK1/2 (A) with loss of serine 473 Akt
phosphorylation (B) in the banded PI3K␥KO mice. C through E, Western blot analysis (left) and quantification (right) showing preservation of glycogen synthase kinase 3␣ (GSK-3␣) (C) and GSK-3␤ (D) phosphorylation in banded WT mice but with a loss of phosphorylation in the banded PI3K␥KO mice, whereas earlier phosphorylation of focal adhesion kinase (FAK) occurs in the banded PI3K␥KO
mice (E). n⫽5 per group. AU indicates arbitrary unit. *P⬍0.05 compared with the corresponding sham group; #P⬍0.05 compared with
WT sham.
signaling, phosphorylated (phospho)–GSK-3␣ (Figure 2C)
and phospho–GSK-3␤ (Figure 2D) were not affected initially
at 1 week and then showed a drastic reduction at 3 weeks after
AB in PI3K␥KO mice. Interestingly, baseline level of
phospho–GSK-3␤ was significantly greater in PI3K␥KO
compared with WT mice (Figure 2D). The phosphorylation of
focal adhesion kinase (FAK) is widely accepted as a fundamental response in myocardial mechanical stretch and hypertrophy.21 In the banded PI3K␥KO mice, increased phosphoFAK occurred at an earlier stage compared with WT mice
consistent with an aberrant response to biomechanical stress
(Figure 2E). These results show that PI3K␥KO mice devel-
Guo et al
oped a rapid onset of adverse myocardial remodeling and
pathological hypertrophy in response to biomechanical stress
with resultant alteration in the activation of mechanosensitive
and Akt-dependent signaling cascades.
Uncoupling Between In Vivo Myocardial
Contractility and Single-Cardiomyocyte
Contractility in Pressure-Overloaded
PI3K␥KO Mice
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
To examine whether these differences in myocardial remodeling in WT and PI3K␥KO mice result in functional alteration, we used transthoracic echocardiography to assess heart
function. LV size rapidly dilated with a marked and progressive decrement in systolic function at 1 and 3 weeks in
PI3K␥KO mice (Figure 3A through 3E; Online Table II). In
marked contrast, banded WT mice developed increased wall
thickness with reduced ventricular size and preserved systolic
function (Figure 3A through 3D; Online Table II). We used
invasive hemodynamic measurements to further characterize
these functional alterations which showed greater elevation in
LV end diastolic pressure (Online Table II) and reduced
myocardial contractility as assessed by ⫹dP/dtmax (Figure
3D; Online Table II) and ⫺dP/dtmin (Figure 3E; Online Table
II) in banded PI3K␥KO mice. The early and marked decompensation in banded PI3K␥KO mice despite enhanced Ca2⫹
cycling and increased basal myocardial contractility (Figure
3; Online Table II)3,6,10 suggests that myocardial cell adhesion
is compromised.
As such, we hypothesize that isolated cardiomyocyte
contractility would remain elevated in banded PI3K␥KO
mice. Increased phosphorylation of PLN is a critical target of
the elevated cAMP levels in PI3K␥KO mice.3,10,22 Western
blot analyses confirmed increased serine-16 phosphorylation
of PLN in PI3K␥KO compared with WT mice, which was
preserved in response to pressure overload (Figure 3F).
Analysis of cAMP levels in the LV from PI3K␥KO mice
confirmed elevated basal levels of cAMP which were maintained in response to 1 week of after AB (Figure 3G). Indeed,
isolated cardiomyocytes from banded PI3K␥KO mice
showed increased cell shortening (Figure 3H) and rate of
contractility (Figure 3I) compared with cardiomyocytes obtained from banded WT mice. Whereas ECM-based cell
adhesion was intact in the PI3K␥KO cardiomyocytes under
baseline conditions (sham), there was a drastic loss of
adhesive properties to collagen IV and laminin in response
to pressure overload (Figure 3J). In contrast, cardiomyocyte adhesion from WT mice remained intact following
pressure overload (Figure 3J). These results show that
isolated cardiomyocytes maintain an increased contractility despite the early deterioration in whole heart systolic
function providing strong evidence that the primary defect
in banded PI3K␥KO mice is a disorganized extracellular
matrix and/or cell adhesion.
Selective Upregulation of MMP2 and MT1-MMP
and MMP Inhibition Mediated Rescue of
Pressure-Overloaded PI3K␥KO Mice
Adverse remodeling of the extracellular matrix by MMPs is a
key determinant of the response to biomechanical stress.23
Role of PI3K␥ in Biomechanical Stress
5
MMP2 (gelatinase A) and MMP13 (collagenase-3) genes
contain promoter regions encoding a cAMP-response element
(CRE), which binds CRE binding protein (CREB)24 and
mediates cAMP-dependent increase in the synthesis of
MMP225 and MMP13,26 whereas MT1-MMP expression is
also positively regulated by cAMP.27 We hypothesize that
elevated cAMP levels in the setting of increased biomechanical stress synergistically increase MMP expression and/or
activity in banded PI3K␥KO mice. Indeed, both MMP2
(Figure 4A) and MT1-MMP (Figure 4B) myocardial mRNA
expression increased within 1 week of after AB and persisted
at 3 weeks in PI3K␥KO compared with WT mice, whereas
MMP13 expression was drastically increased in PI3K␥KO
mice at 3 weeks after AB (Figure 4C). In contrast, non–
cAMP-responsive MMPs such as MMP9 (gelatinase B)
(Figure 4D) and MMP8 (collagenase-2) (data not shown)
showed no differential change in expression in response to
after AB. To provide a more definitive connection between
cAMP and MMP expression/activity, we also examined the
expression of these MMPs in banded PI3K␣DN mice which
also develop an early dilated cardiomyopathy4 and in banded
PI3K␥KD mice, which lack PI3K␥ signaling without elevation in cAMP levels.6 Consistent with our hypothesis, myocardial expression levels of MMP2 (Figure 4A), MT1-MMP
(Figure 4B), and MMP13 (Figure 4C) were not increased in
PI3K␣DN and PI3K␥KD mice at 1 and 3 weeks after AB.
Next, we examined the changes in MMP expression in
cultured adult ventricular cardiomyocyte and cardiofibroblast
fractions in response to the activation of cAMP signaling
using the ␤-adrenergic agonist, isoproterenol. The levels of
cAMP in cardiomyocytes increase significantly in response to
isoproterenol stimulation preventable by ␤-adrenergic blockade using propranolol (25 ␮mol/L) and specific adenylate
cyclase inhibition using 2⬘,5⬘-dideoxyadenosine (DIDA)
(30 ␮mol/L; Figure 4E). Western blot analysis showed an
early and marked increase in serine-133 CREB phosphorylation in cultured cardiomyocytes and cardiofibroblasts (Figure
4F) and shown quantitatively (Figure 4G) in response to
␤-adrenergic receptor stimulation. Consistent with activation
of CREB, mRNA levels of cAMP-responsive MMPs following 24 hours of stimulation with 100 nmol/L isoproterenol
showed that MMP2 (Figure 4H), MT1-MMP (Figure 4I), and
MMP13 (Figure 4J) all showed a significant rise in mRNA
expression in cultured adult cardiomyocytes. Although the
basal expressions of MMP2 and MT1-MMP were greater in
cultured adult cardiofibroblasts, MMPs also showed a similar
increase in response to ␤-adrenergic stimulation in these cells
(Figure 4H through 4J). Importantly, the corresponding increase in mRNA expression of MMP2, MT1-MMP, and
MMP13 in both cardiomyocytes and cardiofibroblasts were
suppressed by both propranolol and DIDA (Figure 4H
through 4J).
We next assessed for direct biochemical evidence for
increased MMP activity in the PI3K␥KO mice in response to
pressure overload. Myocardial collagenase activity showed a
significant increase at 1 and 3 weeks after AB in the
PI3K␥KO mice compared with banded WT mice, which was
suppressible by the MMP inhibitor PD166793 (Figure 5A),
whereas gelatin zymography showed selective activation of
6
Circulation Research
November 12, 2010
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Figure 3. Uncoupling between in vivo myocardial contractility and isolated single cardiomyocyte contractility in PI3K␥KO mice
in response to early biomechanical stress. A through E, M-mode echocardiograms (A) and in vivo quantitative assessment of heart
function showing baseline hypercontractility followed by rapid and marked ventricular dilation (B) and reduction in systolic function
based on fractional shortening (C) and invasive hemodynamic measurement of ⫹dP/dtmax (D) and ⫺dP/dtmin (E) at 1 week after AB.
LVEDD indicates LV end-diastolic diameter. n⫽8 for sham groups; n⫽12 for AB groups. F through G, Western blot analysis (left) and
quantification (right) using myocardial membrane fractions showing increased phospho-PLN at baseline and in banded PI3K␥KO mice
(F) with elevated myocardial cAMP levels in PI3K␥KO hearts at baseline and at 1 week after AB (G). n⫽5 for all groups. H and I, Single
cardiomyocyte contractility measurements showing basal hypercontractility based on percentage (H) and rate of change (⫹dL/dt) (I) of
cell shortening in PI3K␥KO mice, which persists in response to 1 week of pressure overload. n⫽8 for sham groups; n⫽12 for AB
groups. J, ECM-based cell adhesion of isolated adult LV cardiomyocytes following 1 week of after AB showing an increase in fibronectin adhesion in WT cardiomyocytes and a marked decrease in adhesion to collagen IV and laminin in PI3K␥KO cardiomyocytes. AU
indicates arbitrary unit; Col I, collagen I; Col IV, collagen IV; FN, fibronectin; LN, laminin. n⫽4 for WT and PI3K␥KO. *P⬍0.05 compared
with corresponding WT group.
Role of PI3K␥ in Biomechanical Stress
Guo et al
WT
140
PI3Kα-DN
120
PI3Kγ-KO
PI3Kγ-KD
100
80
B
*
*
60
4
MT1-MMP/18S (AU)
MMP2/18S (AU)
A
40
7
*
3
*
2
1
20
0
C
Sham
1 Week AB
D
16
MMP9/18S (AU)
12
10
8
6
1 Week AB
3 Weeks AB
Sham
1 Week AB
3 Weeks AB
3
2
1
2
Sham
1 Week AB
E
Cardiomyocyte
F
40
*
30
Control
ISO
ISO + Prop
ISO + ACi
0
3 Weeks AB
Control
G
ISO 30 mins ISO 1 hour
p-CREB
CREB
Cardiofibroblast
20
Control
10
ISO 30 mins ISO 1 hour
p-CREB
0
I
10
5
0
*
Cardiomyocyte Cardiofibroblast
*
#
1.5
1.0
0.5
Cardiomyocyte Cardiofibroblast
40
30
20
10
0
10
MMP 13/18S (AU)
15
50
MT1-MMP/18S (AU)
MMP 2/18S (AU)
20
#
J
25
*
2.0
0.0
CREB
H
Control
ISO 30 mins
ISO 1 hour
#
#
2.5
p-CREB/CREB (AU)
0
cAMP (pmol/mg)
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
4
Sham
4
*
14
MMP13/18S (AU)
0
3 Weeks AB
*
Cardiomyocyte Cardiofibroblast
8
6
*
Control
ISO
ISO + Prop
ISO + ACi
4
2
0
*
Cardiomyocyte Cardiofibroblast
Figure 4. Upregulation of myocardial matrix metalloproteinase expression in banded PI3K␥KO mice with ␤-adrenergic receptor
stimulation of MMPs expression in isolated adult cardiomyocytes and cardiofibroblasts. A through D, Expression profiling of
MMPs revealed selective upregulation of myocardial MMP2 (A), MT1-MMP (B), and MMP13 (C) expression without a change in MMP9
(D) expression in the banded PI3K␥KO mice compared with banded WT, PI3K␣DN, and PI3K␥-kDa mice. *P⬍0.05 compared with all
other groups. E, Normalization of elevated cAMP levels in cultured adult cardiomyocytes in response to ␤-adrenergic receptor stimulation using isoproterenol (100 nmol/L) by ␤-adrenergic receptor blockade using propranolol (25 ␮mol/L) and specific adenylate cyclase
inhibition using DIDA (30 ␮mol/L); n⫽5 for each group. F and G, Western blot analyses showing early phosphorylation of cAMPresponse element (CRE) binding protein (CREB) in response to ␤-adrenergic receptor stimulation using isoproterenol (100 nmol/L) in
cultured adult cardiomyocytes and cardiofibroblasts (F) and shown quantitatively (G). n⫽5 for each group. #P⬍0.05 compared with the
control group. H through J, Expression analysis showing that long-term ␤-adrenergic receptor stimulation using isoproterenol (100
nmol/L) leads to an upregulation of mRNA expression of MMP2 (H), MT1-MMP (I), and MMP13 (J) in cultured adult cardiomyocytes
and cardiofibroblasts, which was prevented by the ␤-adrenergic receptor blocker propranolol (25 ␮mol/L) and specific adenylate cyclase inhibition using DIDA (30 ␮mol/L). AU indicates arbitrary unit. n⫽5 for each group. *P⬍0.05 compared with all other groups.
MMP2 in banded PI3K␥KO compared with WT mice (Figure
5B). MMP9 protein levels did not change in banded WT
hearts, whereas at 3 weeks, post-AB PI3K␥KO hearts showed
an increase (Online Figure III) despite minimal increase in
mRNA levels (Figure 4D), suggesting a posttranscriptional
mechanism for the increased MMP9 levels. Given the lack of
an increased MMP expression in banded PI3K␥KD mice,
myocardial collagenase activity was not elevated in these
November 12, 2010
WT
A
2.0
Collagenase activity (AU)
B
WT
PI3Kγ-KO
PI3Kγ-KD
PI3Kγ-KO
sh 1wk 3wks sh 1wk 3wks
72kDa MMP2
64kDa MMP2
*
*
1.5
1.0
WT
0.5
PI3Kγ-KO
sh 1wk 3wks sh 1wk 3wks
0.0
Sham
1wk AB
72kDa MMP2
64kDa MMP2
3 wks AB 1wk AB
+ MMPi
α-tubulin
MT1-MMP
Ponceau Red
E
*#
6
*
4
2
0
WT
5
*
4
3
*
2
1
0
WT
PI3Kγ-KO
4
*
*
3
*
2
1
0
WT
PI3Kγ-KO
D
PI3Kγ-KO
16
14
12
10
8
6
4
2
0
PI3Kγ-KO + AB + MMPi
TIMP3/18S (AU)
PI3Kγ-KO + AB + MMPi
*#
5
Sham
1 week AB
3 weeks AB
2.0
#
TIMP2/18S (AU)
sh 1wk 3wks sh 1wk 3wks
8
1.5
#
1.0
#
#
0.5
Sham
0
1 Week AB 3 Weeks AB
*
3
2
1
0
Sham
1 Week AB 3 Weeks AB
WT
PI3Kγ-KO
4
4
TIMP4/18S (AU)
PI3Kγ-KO
MT1-MMP/Ponceau Red (AU)
WT
TIMP1/18S (AU)
C
Sham
1 Week AB 3 Weeks AB
3
*
2
1
0
Sham
1 Week AB 3 Weeks AB
F
4
2
0
MMPi (-)
(-)
(+)
WT PI3Kγ-KO
10
8
6
4
4
3
2
3
10
1
0
WT PI3Kγ-KO
MMPi (-)
(-)
(+)
WT PI3Kγ-KO
30
20
0
*#
40
2
1
(+)
50
4
0
(-)
*
5
2
MMPi (-)
60
6
5
FS (%)
BNP/18S (AU)
6
*
6
*
12
LVESD (mm)
14
*
LVEDD (mm)
8
ANF/18S (AU)
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Sham
1 week AB
3 weeks AB
MMP2 (72kDa)/α-tubulin (AU)
Circulation Research
MMP2 (64kDa)/α-tubulin (AU)
8
*
0
MMPi (-)
(-)
(+)
WT PI3Kγ-KO
MMPi (-)
(-)
(+)
WT PI3Kγ-KO
Figure 5. Increased collagenase activity, MMP2 activation, and MT1-MMP levels with prevention of the dilated cardiomyopathy
by broad-spectrum MMP inhibition in banded PI3K␥KO mice. A, Increased myocardial collagenase activity in banded PI3K␥KO
mice suppressible by the MMP inhibitor PD166793 (30 mg/kg per day). B and C, Gelatin zymography showed increased pro-MMP2
and active MMP2 levels in the banded PI3K␥KO mice and Western blot analysis showing increased expression of active MMP2 (64
kDa) and pro-MMP2 (72 kDa) (B) and membrane-fractionated MT1-MMP (C) in banded PI3K␥KO at 1 and 3 weeks after AB. n⫽4 for
sham group and AB groups. *P⬍0.05 compared with the sham group. D, Expression profile of tissue inhibitors of metalloproteinases
(TIMP) showing equivalent upregulation in TIMP1 without alteration in TIMP2, whereas TIMP3 and TIMP4 levels were increased in
banded PI3K␥KO mice. n⫽6 for sham group and n⫽8 for AB groups. #P⬍0.05 compared with the sham group; *P⬍0.05 compared
with the WT group. E and F, Treatment with the broad-spectrum MMP inhibitor PD166793 (30 mg/kg per day) prevented the dilated
cardiomyopathy and disruption of the extracellular collagen network shown as picrosirius red staining (E) while reversing the upregulation of disease markers, ANF and BNP, ventricular dilation at end-diastole and end-systole, and reduction in systolic performance (F) in
banded PI3K␥KO mice. FS indicates fractional shortening; LVEDD, LV end diastolic diameter; LVESD, LV end systolic diameter. n⫽8
for each group. *P⬍0.05 compared with all other groups; #P⬍0.05 compared with WT group. AU indicates arbitrary unit.
Guo et al
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
mice (Figure 5A). Consistent with gelatin zymography, Western blot analysis and quantification (Figure 5B) revealed
increased myocardial expression of active MMP2 (64 kDa)
and pro-MMP2 (72 kDa) in banded PI3K␥KO mice at 1 and
3 weeks after AB. Similarly, membrane fractionation and
analysis of MT1-MMP levels showed an earlier and greater
increase in MT1-MMP levels in banded PI3K␥KO mice
(Figure 5C). The activity of MMPs are inhibited by tissue
inhibitor of matrix metalloproteinases (TIMPs), TIMP1,
TIMP2, TIMP3, and TIMP4 with TIMP3 playing a key role
in the myocardial response to biomechanical stress.12,28 Interestingly, TIMP1 levels increased similarly, whereas the
expression of TIMP2 did not change in banded WT and
PI3K␥KO mice (Figure 5D). By contrast, increase in TIMP3
and TIMP4 expression occurs at 3-weeks after AB in
PI3K␥KO mice possibly attributable to a negative-feedback
response to the increased MMP expression and activities
(Figure 5D). Our data provide a crucial link between the
adverse myocardial remodeling in banded PI3K␥KO mice
and the increase in activation and activity of MMPs. Human
recombinant MMP2 rather than MT1-MMP cleaves human
recombinant N-cadherin Fc chimera and was inhibited by the
MMP inhibitor, PD166793 (Online Figure II). We hypothesized that MMP inhibition will lead to a marked protection in
banded PI3K␥KO mice. Daily treatment of banded PI3K␥KO
mice with the broad-spectrum MMP inhibitor PD166793 (30
mg/kg per day)12 prevented the dilated cardiomyopathy,
fibrosis, and disruption of the extracellular collagen network
(Figure 5E). This also reversed the upregulation of disease
markers, ANF and BNP, and ventricular dilation at end-diastole and end-systole, resulting in improved systolic performance in banded PI3K␥KO mice (Figure 5F). These results
illustrate a key role of cAMP-mediated upregulation of
MMP2 and MT1-MMP expressions in mediating the adverse
myocardial remodeling in pressure-overloaded PI3K␥KO
mice.
Specific Loss of N-Cadherin From Adhesion
Complexes While ␤-Adrenergic Blocker Prevents
Upregulation of MMP and Loss of N-Cadherin in
Banded PI3K␥KO Mice
In addition to the degradation of the ECM, N-cadherin/␤catenin cell adhesion complexes are also important targets of
an activated MMP system.29,30 Western blot analysis of the
myocardial membrane fraction in banded WT and PI3K␣DN
mice showed a modest increase in N-cadherin levels (Figure
6A and 6B), whereas in banded PI3K␥KO mice, there was a
75% loss of N-cadherin levels (Figure 6A and 6B). In
contrast, in the banded PI3K␥KO (and PI3K␣DN) mice,
levels of ␤-catenin in the heart were significantly increased
compared with banded WT mice (Figure 6C). The relative
preservation of N-cadherin levels in the PI3K␥KD hearts is
consistent with a critical role of cAMP in mediating the loss
of N-cadherin from adhesion complexes independent of the
PI3K␥ lipid kinase activity per se. The quality of Western
blot analysis was confirmed by absence of the membranespecific protein (toll-like receptor 4) in the cytosolic fraction
and absence of the cytosolic-specific protein (caspase-3) in
the membrane fraction (Online Figure I). Immunofluores-
Role of PI3K␥ in Biomechanical Stress
9
cence microscopy confirmed colocalization of N-cadherin
and ␤-catenin in the end-to-end and side-to-side connections
between cardiomyocytes in banded WT hearts (Figure 6D).
Consistent with our Western blot analysis, there was a near
complete loss of N-cadherin from end-to-end and side-to-side
connections from pressure-overloaded PI3K␥KO myocardium (Figure 6E). Consistent with the ability of PD166793 to
prevent MMP2-mediated cleavage of N-cadherin (Online
Figure II) and rescue the dilated cardiomyopathy in banded
PI3K␥KO mice (Figure 5), Western blot analysis confirmed
reduced loss of membrane-associated N-cadherin (Figure 6F
and 6G), resulting in more pronounced staining of N-cadherin
in the myocardial cell adhesion junctions (Figure 6H) in
banded PI3K␥KO mice treated with PD166793. Given the
key role of integrin complexes in mediating cell adhesion in
the heart,31,32 we also examined changes in integrin levels in
response to pressure overload. Western blot analysis of
membrane fractionated ␤1D and ␣7B integrins showed increased levels in WT hearts at 1 and 3 weeks after AB with
a delayed increase in the PI3K␥KO hearts with a significant
increase only at 3 weeks after AB (Online Figure IV).
We then tested the hypothesis that elevated cAMP plays a
key in vivo role in the adverse remodeling in banded
PI3K␥KO mice by using the nonspecific ␤-adrenergic
blocker propranolol at a dose that has been previously shown
to normalize elevated cAMP levels.6 Propranolol treatment
normalizes the mRNA expression of MMP2, MT1-MMP, and
MMP13 (Figure 7A) with similar changes seen in the
expression of disease markers, ANF, ␤MHC, and BNP
(Figure 7B). Consistent with a reduction in MMP expression,
elevated collagenase activity in the banded PI3K␥KO mice
was normalized in response to propranolol (Figure 7C) with
relative preservation of N-cadherin levels in the myocardial
membrane fraction (Figure 7D). These biochemical and
cellular changes implies that cardiac function was rescued in
banded PI3K␥KO mice treated with propranolol. Indeed,
echocardiographic assessment showed a near normalization
of the increased LV dilation and improved fractional shortening with invasive hemodynamic parameters showing
marked increase in ⫹dP/dtmax and ⫺dP/dtmin in the banded
PI3K␥KO mice treated with propranolol (Figure 7E). These
results highlight a key reversible defect in the N-cadherin
system in the PI3K␥KO mice in response to biomechanical
stress caused by cAMP-dependent upregulation of MMP
activity.
␤-Blocker Therapy Prevents the Adverse
Remodeling in Response to Chronic
Pressure Overload
The beneficial effects of ␤-blocker therapy in the banded
PI3K␥KO mice suggest that ␤blockade may have a similar
protective role in a long-term (9 weeks) AB model of WT
mice. Aortic banding for 9 weeks resulted in marked increase
in expression of hypertrophy markers, ␣-skeletal actin,
␤MHC and BNP (Figure 8A) in association with LV dilation
and reduced systolic function (Figure 8B; Online Figure V).
Propranolol treatment prevented the adverse remodeling and
systolic dysfunction in response to chronic biomechanical
stress (Figure 8A and 8B; Online Figure V). Quantitative
10
Circulation Research
November 12, 2010
A
B
C
2.0
2.0
*
AB
Sh
AB
N-cadherin
β-catenin
Coomassie Blue
1.5
1.5
*
0.5
*
F
PI
3K
γKD
PI
3K
γKO
T
αDN
W
PI
3K
N-cadherin
G
PI3Kγ-KO AB
β-catenin
Co-localization
H
N-cadherin
Co-localization
PI3Kγ-KO AB + MMPi
PI3Kγ-KO
WT
AB
N-cadherin
Coomassie Blue
AB
AB + MMPi
N-Cadherin (AU)
1.2
β-catenin
1.0
N-cadherin
Co-localization
0.8
0.6
0.4
*
0.2
0.0
T
AB
PI
3
Kγ
-K
O
AB
MMPi (-) (-) (+)
W
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
β-catenin
0.5
0.0
E
WT AB
*
1.0
1.0
0.0
D
Sham
AB
αDN
PI
3K
γKD
PI
3K
γKO
Sh
T
AB
*
W
Sh
PI3Kγ-KO
PI
3K
AB
PI3Kγ-KD
β-Catenin (AU)
Sh
PI3Kα-DN
N-Cadherin (AU)
WT
Figure 6. Selective loss of N-cadherin from myocardial membrane fraction and the cell adhesion complexes in the myocardium
from banded PI3K␥KO mice. A through C, Western blot analysis of the myocardial membrane fraction (A) and quantification (B)
showing a selective loss of N-cadherin levels without alteration in ␤-catenin levels (C) in banded PI3K␥KO mice, whereas the level of
N-cadherin increases in banded WT and PI3K␣DN mice. *P⬍0.05 compared with the corresponding sham group. D and E, Immunofluorescence microscopy of the myocardial ␤-catenin and N-cadherin proteins showing end-to-end connections (top) and side-to-side
connections (bottom) in banded WT (D) and PI3K␥KO (E) mice with a distinct loss of N-cadherin in the banded PI3K␥KO mice. F
through H, Western blot analysis of the myocardial membrane fraction (F) and quantification (G) and immunofluorescence microscopy
of myocardial ␤-catenin and N-cadherin proteins (H) showing MMP inhibition prevents loss of N-cadherin in banded PI3K␥KO mice. AU
indicates arbitrary unit. n⫽5 for all groups. *P⬍0.05 compared with all other groups.
assessment of cardiac systolic function using echocardiography showed a marked improvement in fractional shortening
and reduction in LV end-diastolic dimension (Online Figure
V). Similarly, invasive hemodynamic measurement revealed
restoration of an elevated LV end-diastolic pressure and
normalization of ⫹dP/dtmax and – dP/dtmin in response to
chronic ␤-blocker therapy (Figure 8C). These functional
changes correlated with a reduction of MMP2 and MT1-
Guo et al
Role of PI3K␥ in Biomechanical Stress
11
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Figure 7. The nonspecific ␤-receptor blocker propranolol (15 mg/kg per day) prevents the molecular, biochemical, and functional
deterioration in response to biomechanical stress in PI3K␥KO mice. A and B, Expression profiling showing that treatment with propranolol prevents the increased expression of MMPs including MMP2, MT1-MMP, and MMP13 (A) and disease markers such as ANF, ␤MHC,
and BNP (B) in banded PI3K␥KO mice. *P⬍0.05 compared with all other groups. C and D, Suppression of the increased myocardial collagenase activity (C) with Western blot analysis and quantification (D) showing restoration of the membrane N-cadherin levels in banded
PI3K␥KO mice. *P⬍0.05 compared with all other groups. E, Reduction in LV dilation and preservation of systolic function in response to
treatment with propranolol in banded PI3K␥KO mice based on echocardiographic parameters, LV end diastolic diameter (LVEDD) and fractional shortening (FS), and hemodynamic assessment, ⫹dP/dtmax and ⫺dP/dtmin. AU indicates arbitrary unit. n⫽8 for all groups. *P⬍0.05
compared with all other groups.
12
Circulation Research
November 12, 2010
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Figure 8. Chronic ␤-blocker prevents pressure overload–induced adverse remodeling and preserves systolic function in WT mice. A,
Expression profile of hypertrophy disease markers showing markedly increased expression of ␣-skeletal actin, ␤MHC, and BNP in banded
WT mice at 9 weeks, which was dramatically reduced by treatment with propranolol. n⫽5 per group. B, Echocardiographic assessment of
systolic function with M-mode echocardiograms showing a marked reduction in LV dilation and increase in fractional shortening in response
to therapy with propranolol. C, Invasive hemodynamic assessment showing marked reduction in LVEDP and prevention of the deterioration in
myocardial contractility based on ⫹dP/dtmax and ⫺dP/dtmin in response to chronic ␤-blocker therapy. n⫽6 for WT sham and n⫽8 for WT AB
groups. D, Expression of MMPs showing a marked increase in MMP2 and MT1-MMP expression without a differential effect on MMP-13 levels and a normalization in response to chronic ␤-blocker therapy (n⫽5 per group). E, Western blot analysis revealed a greater increase in
myocardial N-cadherin levels with equivalent increase in ␤-catenin levels, leading to a relative preservation of the N-cadherin/␤-catenin ratio
in response to chronic ␤-blocker therapy. AU indicates arbitrary unit. n⫽5 per group. *P⬍0.05 compared with all other groups; #P⬍0.05
compared with WT⫹AB 9 weeks; ‡P⬍0.05 compared with WT sham group.
Guo et al
MMP expression in the absence of a differential effect on
MMP13 expression (Figure 8D). Western blot analysis
showed a higher level of membrane-associated N-cadherin
with an equivalent increase in ␤-catenin levels resulting in
preservation of the myocardial N-cadherin/␤-catenin ratio
(Figure 8E) in response to chronic ␤-blocker therapy. Our
results highlight a novel mechanism of chronic ␤-blocker
therapy in preventing the adverse myocardial remodeling in
pressure overload–induced heart failure.
Discussion
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
The response to biomechanical stress is a fundamental response in heart disease and plays a key adaptive role in
response to a pressure overload state characteristic of hypertension and valvular stenosis. Mechanotransduction plays a
fundamental role in cardiac structure and function and involves a concerted interaction between extracellular matrix,
intracellular cytoskeletal proteins, and cell adhesion complexes.7,33 Advanced heart failure in response to myocardial
injury including biomechanical stress leads to impaired Ca2⫹
cycling and strategies aimed at enhancing Ca2⫹ cycling are
currently being developed as therapies for heart failure.34,35
Loss of PI3K␥ increases basal cAMP levels, enhances
SERCA2 function and Ca2⫹ cycling, and increases basal
myocardial contractility.3,6,10 In the heart, loss of PI3K␥
prevents phosphorylation of Akt in response to G protein–
coupled receptor agonists,6,11,36 and PI3K␥KO mice are
resistant to the pathological effects of ␤-adrenergic stimulation.11 Despite these biochemical changes, PI3K␥KO mice
show enhanced susceptibility to biomechanical stress,6,37 and
we provide direct evidence that the primary defect is a
compromised cell-adhesion/extracellular matrix system
linked to elevated cAMP levels. In contrast, in PI3K␥KD
mice, which have normal myocardial cAMP levels,6 there is
no upregulation of MMPs with preservation of N-cadherin
levels consistent with the ability of these mice to maintain
normal systolic function in response to early biomechanical
stress.6
The PI3K/PTEN system may also have a more direct role
in the cardiac response to biomechanical stress.2,7,8,13
Whereas PI3K and lipid phosphatases can modulate cytoskeletal interactions, stretch can in turn activate Akt/PKB and
GSK-3␤ activity.8 Indeed, loss of PTEN prevents pressure
overload–induced heart failure,13 whereas loss of Akt1/PKB␣
leads to a rapid onset of ventricular dilation and systolic
dysfunction in response to pressure overload.38 Consistent
with a critical role of PI3K␥ in pathological G protein–
coupled receptor signaling,6,11,36 phosphorylation of ERK1/2
is impaired in response to early biomechanical stress in the
PI3K␥KO mice.6 However, this differential response is loss
at 3 weeks after AB, which is likely driven by severe
disruption of the ECM and/or cell adhesion leading to G
protein– coupled receptor–independent activation of ERK1/2
in the PI3K␥KO mice. Despite equivalent basal phospho-Akt
levels in WT and PI3K␥KO hearts, basal phospho–GSK-3␤
was increased in the PI3K␥KO mice, likely mediated by
PKA39 given the chronic elevation of myocardial cAMP
levels in these mice. The diverse downstream effects of
Role of PI3K␥ in Biomechanical Stress
13
elevated basal phospho–GSK-3␤40 may have contributed to
the adverse remodeling in the PI3K␥KO mice.
Elevated cAMP levels in response to increased biomechanical stress leads to increased MMP expression and
increased active (cleaved) MMP2 and collagenase activity,
leading to adverse myocardial remodeling. Activation of
pro-MMP2 occurs by proteolytic cleavage of the N-terminal
propeptide and requires two MT1-MMP molecules in association with TIMP2.41,42 In addition to degrading various
components of the ECM, increased MMP activity can also
adversely modify cell– cell adhesion complexes. Cardiomyocyte cell adhesion complexes provide an important mechanism by which cardiomyocytes (and cardiofibroblasts) are
anchored to the extracellular matrix while allowing force
transmission to the intracellular cytoskeletal network.31,33,43
In particular, we have shown that increased MMP expression
and activity can regulate N-cadherin function through proteolytic degradation.29,30,44 Cardiac-specific loss of N-cadherin
in the heart leads to a dilated cardiomyopathy attributable to
loss of the integrity of cell adhesion junctions.45 Because of
their homophilic binding and adhesive specificities, N-cadherin/catenin complex is required for cadherin-mediated cell
adhesion and linkage to the actin cytoskeleton. The delayed
increase in membrane ␤1D and ␣7B integrins in the banded
PI3K␥KO hearts may have also contributed to the early onset
of dilated cardiomyopathy.31,32 In addition, MT1-MMP is a
potent collagenase that also targets other ECM components
such as fibronectin,46 laminin,46 and integrins47,48 while
activating MMP13 (collagenase-3), thereby amplifying the
collagen-degradation process.16,49 Our results are consistent
with the conclusion that elevated cAMP (and its downstream
effects) is the primary driver of the adverse remodeling in
banded PI3K␥KO mice rather than loss of PI3K␥ signaling
per se. Our findings may help to explain the cardiomyopathy
in experimental models with enhanced ␤-adrenergic signaling
(and cAMP levels)50,51 and lack of a protection against
pressure overload and tumor necrosis factor–induced heart
failure despite enhanced Ca2⫹ cycling in the PLN knockout
mice.52,53 Indeed, whereas Ca2⫹ transients in cardiomyocytes
were normalized in tumor necrosis factor–induced cardiomyopathy in a PLN-null background, global systolic function
remained depressed and unchanged.53 We propose that enhancing cell– cell adhesion and cell–ECM interaction will
promote the salutary effects of enhanced intracellular Ca2⫹
cycling on whole heart function and booster the therapeutic
potential of normalizing intracellular Ca2⫹ cycling in patients
with heart failure. Increased sympathetic nervous system
activity and ␤-adrenergic receptor signaling are key aspects
of the pathophysiology of heart failure54 and in catecholamine-mediated cardiomyopathies.55,56 ␤-Adrenergic receptor blockers improve clinical outcomes in patients with
chronic heart failure.54 In contrast, agents that increase
myocardial cAMP such as PDE3 inhibitors are associated
with adverse outcomes and increased mortality in patients
with heart failure.57 Our data support a novel role of
␤-adrenergic blocker in reducing MMP expression and/or
activity and preservation of cell adhesion, thereby curtailing
adverse myocardial remodeling.
14
Circulation Research
November 12, 2010
Sources of Funding
This work was supported by the Heart and Stroke Foundation of
Canada (to G.Y.O.), Canadian Institute for Health Research (grant
86602 to G.Y.O. and grant 84279 to Z.K.), and the Alberta
Innovates–Health Solutions (to G.Y.O. and Z.K.).
Disclosures
None.
References
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
1. Oudit GY, Sun H, Kerfant BG, Crackower MA, Penninger JM, Backx
PH. The role of phosphoinositide-3 kinase and PTEN in cardiovascular
physiology and disease. J Mol Cell Cardiol. 2004;37:449 – 471.
2. Oudit GY, Penninger JM. Cardiac regulation by phosphoinositide
3-kinases and PTEN. Cardiovasc Res. 2009;82:250 –260.
3. Crackower MA, Oudit GY, Kozieradzki I, Sarao R, Sun H, Sasaki T,
Hirsch E, Suzuki A, Shioi T, Irie-Sasaki J, Sah R, Cheng HY, Rybin VO,
Lembo G, Fratta L, Oliveira-dos-Santos AJ, Benovic JL, Kahn CR, Izumo
S, Steinberg SF, Wymann MP, Backx PH, Penninger JM. Regulation of
myocardial contractility and cell size by distinct PI3K-PTEN signaling
pathways. Cell. 2002;110:737–749.
4. McMullen JR, Shioi T, Zhang L, Tarnavski O, Sherwood MC, Kang PM,
Izumo S. Phosphoinositide 3-kinase(p110alpha) plays a critical role for
the induction of physiological, but not pathological, cardiac hypertrophy.
Proc Natl Acad Sci U S A. 2003;100:12355–12360.
5. Inuzuka Y, Okuda J, Kawashima T, Kato T, Niizuma S, Tamaki Y,
Iwanaga Y, Yoshida Y, Kosugi R, Watanabe-Maeda K, Machida Y, Tsuji
S, Aburatani H, Izumi T, Kita T, Shioi T. Suppression of phosphoinositide 3-kinase prevents cardiac aging in mice. Circulation. 2009;120:
1695–1703.
6. Patrucco E, Notte A, Barberis L, Selvetella G, Maffei A, Brancaccio M,
Marengo S, Russo G, Azzolino O, Rybalkin SD, Silengo L, Altruda F,
Wetzker R, Wymann MP, Lembo G, Hirsch E. PI3Kgamma modulates
the cardiac response to chronic pressure overload by distinct kinasedependent and -independent effects. Cell. 2004;118:375–387.
7. Sugden PH. Ras, Akt, and mechanotransduction in the cardiac myocyte.
Circ Res. 2003;93:1179 –1192.
8. Baba HA, Stypmann J, Grabellus F, Kirchhof P, Sokoll A, Schafers M,
Takeda A, Wilhelm MJ, Scheld HH, Takeda N, Breithardt G, Levkau B.
Dynamic regulation of MEK/Erks and Akt/GSK-3beta in human
end-stage heart failure after left ventricular mechanical support: myocardial mechanotransduction-sensitivity as a possible molecular
mechanism. Cardiovasc Res. 2003;59:390 –399.
9. Knoll R, Hoshijima M, Hoffman HM, Person V, Lorenzen-Schmidt I,
Bang ML, Hayashi T, Shiga N, Yasukawa H, Schaper W, McKenna W,
Yokoyama M, Schork NJ, Omens JH, McCulloch AD, Kimura A,
Gregorio CC, Poller W, Schaper J, Schultheiss HP, Chien KR. The
cardiac mechanical stretch sensor machinery involves a Z disc complex
that is defective in a subset of human dilated cardiomyopathy. Cell.
2002;111:943–955.
10. Kerfant BG, Gidrewicz D, Sun H, Oudit GY, Penninger JM, Backx PH.
Cardiac sarcoplasmic reticulum calcium release and load are enhanced by
subcellular cAMP elevations in PI3Kgamma-deficient mice. Circ Res.
2005;96:1079 –1086.
11. Oudit GY, Crackower MA, Eriksson U, Sarao R, Kozieradzki I, Sasaki T,
Irie-Sasaki J, Gidrewicz D, Rybin VO, Wada T, Steinberg SF, Backx PH,
Penninger JM. Phosphoinositide 3-kinase gamma-deficient mice are protected from isoproterenol-induced heart failure. Circulation. 2003;108:
2147–2152.
12. Kassiri Z, Oudit GY, Sanchez O, Dawood F, Mohammed FF, Nuttall RK,
Edwards DR, Liu PP, Backx PH, Khokha R. Combination of tumor
necrosis factor-alpha ablation and matrix metalloproteinase inhibition
prevents heart failure after pressure overload in tissue inhibitor of
metalloproteinase-3 knock-out mice. Circ Res. 2005;97:380 –390.
13. Oudit GY, Kassiri Z, Zhou J, Liu QC, Liu PP, Backx PH, Dawood F,
Crackower MA, Scholey JW, Penninger JM. Loss of PTEN attenuates the
development of pathological hypertrophy and heart failure in response to
biomechanical stress. Cardiovasc Res. 2008;78:505–514.
14. Bradshaw AD, Baicu CF, Rentz TJ, Van Laer AO, Boggs J, Lacy JM,
Zile MR. Pressure overload-induced alterations in fibrillar collagen
content and myocardial diastolic function: role of secreted protein acidic
and rich in cysteine (SPARC) in post-synthetic procollagen processing.
Circulation. 2009;119:269 –280.
15. O’Connell TD, Rodrigo MC, Simpson PC. Isolation and culture of adult
mouse cardiac myocytes. Methods Mol Biol. 2007;357:271–296.
16. Kandalam V, Basu R, Abraham T, Wang X, Soloway PD, Jaworski DM,
Oudit GY, Kassiri Z. TIMP2 deficiency accelerates adverse postmyocardial infarction remodeling because of enhanced MT1-MMP
activity despite lack of MMP2 activation. Circ Res. 2010;106:796 – 808.
17. Zhong JC, Basu R, Guo D, Chow FL, Byrns S, Shuster M, Loibner H,
Wang X, Penninger JM, Kassiri Z, Oudit GY. Angiotensin converting
enzyme 2 suppresses pathological hypertrophy, myocardial fibrosis and
cardiac dysfunction. Circulation. 2010;122:717–728.
18. Sussman MA, McCulloch A, Borg TK. Dance band on the Titanic:
biomechanical signaling in cardiac hypertrophy. Circ Res. 2002;91:
888 – 898.
19. Heineke J, Molkentin JD. Regulation of cardiac hypertrophy by intracellular signalling pathways. Nat Rev Mol Cell Biol. 2006;7:589 – 600.
20. Matsuda T, Zhai P, Maejima Y, Hong C, Gao S, Tian B, Goto K, Takagi
H, Tamamori-Adachi M, Kitajima S, Sadoshima J. Distinct roles of
GSK-3alpha and GSK-3beta phosphorylation in the heart under pressure
overload. Proc Natl Acad Sci U S A. 2008;105:20900 –20905.
21. Romer LH, Birukov KG, Garcia JG. Focal adhesions: paradigm for a
signaling nexus. Circ Res. 2006;98:606 – 616.
22. Oudit GY, Kassiri Z. Role of PI3 kinase gamma in excitation-contraction
coupling and heart disease. Cardiovasc Hematol Disord Drug Targets.
2007;7:295–304.
23. Spinale FG. Myocardial matrix remodeling and the matrix metalloproteinases: influence on cardiac form and function. Physiol Rev. 2007;87:
1285–1342.
24. Sands WA, Palmer TM. Regulating gene transcription in response to
cyclic AMP elevation. Cell Signal. 2008;20:460 – 466.
25. Melnikova VO, Mourad-Zeidan AA, Lev DC, Bar-Eli M. Plateletactivating factor mediates MMP-2 expression and activation via phosphorylation of cAMP-response element-binding protein and contributes
to melanoma metastasis. J Biol Chem. 2006;281:2911–2922.
26. Quinn CO, Rajakumar RA, Agapova OA. Parathyroid hormone induces
rat interstitial collagenase mRNA through Ets-1 facilitated by cyclic AMP
response element-binding protein and Ca(2⫹)/calmodulin-dependent
protein kinase II in osteoblastic cells. J Mol Endocrinol. 2000;25:73– 84.
27. Shankavaram UT, Lai WC, Netzel-Arnett S, Mangan PR, Ardans JA,
Caterina N, Stetler-Stevenson WG, Birkedal-Hansen H, Wahl LM.
Monocyte membrane type 1-matrix metalloproteinase. Prostaglandindependent regulation and role in metalloproteinase-2 activation. J Biol
Chem. 2001;276:19027–19032.
28. Kassiri Z, Khokha R. Myocardial extra-cellular matrix and its regulation
by metalloproteinases and their inhibitors. Thromb Haemost. 2005;93:
212–219.
29. Ho AT, Voura EB, Soloway PD, Watson KL, Khokha R. MMP inhibitors
augment fibroblast adhesion through stabilization of focal adhesion
contacts and up-regulation of cadherin function. J Biol Chem. 2001;276:
40215– 40224.
30. Covington MD, Burghardt RC, Parrish AR. Ischemia-induced cleavage of
cadherins in NRK cells requires MT1-MMP (MMP-14). Am J Physiol
Renal Physiol. 2006;290:F43–F51.
31. Ross RS, Borg TK. Integrins and the myocardium. Circ Res. 2001;88:
1112–1119.
32. Babbitt CJ, Shai SY, Harpf AE, Pham CG, Ross RS. Modulation of
integrins and integrin signaling molecules in the pressure-loaded murine
ventricle. Histochem Cell Biol. 2002;118:431– 439.
33. Jamora C, Fuchs E. Intercellular adhesion, signalling and the
cytoskeleton. Nat Cell Biol. 2002;4:E101–E108.
34. Chien KR, Ross J Jr, Hoshijima M. Calcium and heart failure: the cycle
game. Nat Med. 2003;9:508 –509.
35. Jaski BE, Jessup ML, Mancini DM, Cappola TP, Pauly DF, Greenberg B,
Borow K, Dittrich H, Zsebo KM, Hajjar RJ. Calcium upregulation by
percutaneous administration of gene therapy in cardiac disease (CUPID
Trial), a first-in-human phase 1/2 clinical trial. J Card Fail. 2009;15:
171–181.
36. Naga Prasad SV, Esposito G, Mao L, Koch WJ, Rockman HA.
Gbetagamma-dependent phosphoinositide 3-kinase activation in hearts
with in vivo pressure overload hypertrophy. J Biol Chem. 2000;275:
4693– 4698.
37. Oudit GY, Kassiri Z, Crackower MA, Penninger JM, Backx PH. Loss of
PI3 kinase gamma leads to an accelerated progression to dilated cardiomyopathy in response to pressure overload. Circulation. 2004;
108(Suppl):IV-238.
Guo et al
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
38. DeBosch B, Treskov I, Lupu TS, Weinheimer C, Kovacs A, Courtois M,
Muslin AJ. Akt1 is required for physiological cardiac growth. Circulation. 2006;113:2097–2104.
39. Tanji C, Yamamoto H, Yorioka N, Kohno N, Kikuchi K, Kikuchi A.
A-kinase anchoring protein AKAP220 binds to glycogen synthase
kinase-3beta (GSK-3beta) and mediates protein kinase A-dependent inhibition of GSK-3beta. J Biol Chem. 2002;277:36955–36961.
40. Hardt SE, Sadoshima J. Glycogen synthase kinase-3beta: a novel regulator of cardiac hypertrophy and development. Circ Res. 2002;90:
1055–1063.
41. English JL, Kassiri Z, Koskivirta I, Atkinson SJ, Di Grappa M, Soloway
PD, Nagase H, Vuorio E, Murphy G, Khokha R. Individual Timp deficiencies differentially impact pro-MMP-2 activation. J Biol Chem. 2006;
281:10337–10346.
42. Page-McCaw A, Ewald AJ, Werb Z. Matrix metalloproteinases and the
regulation of tissue remodelling. Nat Rev Mol Cell Biol. 2007;8:221–233.
43. Stokes DL. Desmosomes from a structural perspective. Curr Opin Cell
Biol. 2007;19:565–571.
44. Pon YL, Auersperg N, Wong AS. Gonadotropins regulate N-cadherinmediated human ovarian surface epithelial cell survival at both posttranslational and transcriptional levels through a cyclic AMP/protein
kinase A pathway. J Biol Chem. 2005;280:15438 –15448.
45. Kostetskii I, Li J, Xiong Y, Zhou R, Ferrari VA, Patel VV, Molkentin JD,
Radice GL. Induced deletion of the N-cadherin gene in the heart leads to
dissolution of the intercalated disc structure. Circ Res. 2005;96:346 –354.
46. Ohtake Y, Tojo H, Seiki M. Multifunctional roles of MT1-MMP in
myofiber formation and morphostatic maintenance of skeletal muscle.
J Cell Sci. 2006;119:3822–3832.
47. Deryugina EI, Ratnikov BI, Postnova TI, Rozanov DV, Strongin AY.
Processing of integrin alpha (v) subunit by membrane type 1 matrix
metalloproteinase stimulates migration of breast carcinoma cells on vitronectin and enhances tyrosine phosphorylation of focal adhesion kinase.
J Biol Chem. 2002;277:9749 –9756.
Role of PI3K␥ in Biomechanical Stress
15
48. Ratnikov BI, Rozanov DV, Postnova TI, Baciu PG, Zhang H, DiScipio
RG, Chestukhina GG, Smith JW, Deryugina EI, Strongin AY. An alternative processing of integrin alpha(v) subunit in tumor cells by membrane
type-1 matrix metalloproteinase. J Biol Chem. 2002;277:7377–7385.
49. Itoh Y, Seiki M. MT1-MMP: a potent modifier of pericellular microenvironment. J Cell Physiol. 2006;206:1– 8.
50. Engelhardt S, Hein L, Wiesmann F, Lohse MJ. Progressive hypertrophy
and heart failure in beta1-adrenergic receptor transgenic mice. Proc Natl
Acad Sci U S A. 1999;96:7059 –7064.
51. Liggett SB, Tepe NM, Lorenz JN, Canning AM, Jantz TD, Mitarai S,
Yatani A, Dorn GW II. Early and delayed consequences of beta(2)adrenergic receptor overexpression in mouse hearts: critical role for
expression level. Circulation. 2000;101:1707–1714.
52. Kiriazis H, Sato Y, Kadambi VJ, Schmidt AG, Gerst MJ, Hoit BD,
Kranias EG. Hypertrophy and functional alterations in hyperdynamic
phospholamban-knockout mouse hearts under chronic aortic stenosis.
Cardiovasc Res. 2002;53:372–381.
53. Janczewski AM, Zahid M, Lemster BH, Frye CS, Gibson G, Higuchi Y,
Kranias EG, Feldman AM, McTiernan CF. Phospholamban gene ablation
improves calcium transients but not cardiac function in a heart failure
model. Cardiovasc Res. 2004;62:468 – 480.
54. Triposkiadis F, Karayannis G, Giamouzis G, Skoularigis J, Louridas G,
Butler J. The sympathetic nervous system in heart failure physiology,
pathophysiology, and clinical implications. J Am Coll Cardiol. 2009;54:
1747–1762.
55. Kloner RA, Hale S, Alker K, Rezkalla S. The effects of acute and chronic
cocaine use on the heart. Circulation. 1992;85:407– 419.
56. Gianni M, Dentali F, Grandi AM, Sumner G, Hiralal R, Lonn E. Apical
ballooning syndrome or Takotsubo cardiomyopathy: a systematic review.
Eur Heart J. 2006;27:1523–1529.
57. Nony P, Boissel JP, Lievre M, Leizorovicz A, Haugh MC, Fareh S, de
Breyne B. Evaluation of the effect of phosphodiesterase inhibitors on
mortality in chronic heart failure patients. A meta-analysis. Eur J Clin
Pharmacol. 1994;46:191–196.
Novelty and Significance
What Is Known?
●
●
Phosphoinositide 3-kinase (PI3K)␥ couples G protein– coupled receptors to downstream signaling pathways.
Loss of PI3K␥ enhances cAMP levels and enhances basal myocardial
contractility.
What New Information Does This Article Contribute?
●
●
●
Loss of PI3K␥ enhances susceptibility to biomechanical stress and
early-onset heart failure.
Activation of matrix metalloproteinase (MMP) and loss of N-cadherin–
mediated cell adhesion are critical mediators of this adverse
remodeling.
␤-Blocker suppresses cAMP-mediated MMP upregulation and preserves N-cadherin levels with minimization of cardiac systolic
dysfunction.
Altered PI3K signaling plays a fundamental role in heart disease.
Although loss of PI3K␥ enhances myocardial contractility, there
is a paradoxical rapid development of heart failure in response
to biomechanical stress. The uncoupling between basal hypercontractility and the response to pathological stimulus was
driven by adverse remodeling of the extracellular matrix and
N-cadherin mediated cell-adhesion. In particular, there was
upregulation of the myocardial MMP2 and MT1-MMP axis.
Inhibition of MMP prevented the accelerated development of
dilated cardiomyopathy in pressure-overloaded PI3K␥KO mice.
The molecular trigger of this adverse remodeling is excess
cAMP, and ␤-blocker therapy normalizes cAMP levels, suppresses MMP expression, and rescues the early onset of dilated
cardiomyopathy in the mutant mice. These findings provide
valuable insight into the phenotype of other gene-targeted
animal models with elevated Ca2⫹ cycling but an inability to
rescue heart failure. Our findings highlight the potential negative
impact chronic elevations in cAMP can have on the heart in the
setting of heart failure while highlighting a novel mechanism of
chronic ␤-blocker therapy in preventing the adverse myocardial
remodeling in pressure overload–induced heart failure.
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Loss of PI3Kγ Enhances cAMP-Dependent MMP Remodeling of the Myocardial
N-Cadherin Adhesion Complexes and Extracellular Matrix in Response to Early
Biomechanical Stress
Danny Guo, Zamaneh Kassiri, Ratnadeep Basu, Fung L. Chow, Vijay Kandalam, Federico
Damilano, Wenbin Liang, Seigo Izumo, Emilio Hirsch, Josef M. Penninger, Peter H. Backx and
Gavin Y. Oudit
Circ Res. published online September 16, 2010;
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2010 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/early/2010/09/16/CIRCRESAHA.110.229054.citation
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2010/09/16/CIRCRESAHA.110.229054.DC1
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further information
about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/
Guo D et al.
Role of PI3Kγ in Biomechanical Stress
ONLINE METHODS and DATA SUPPLEMENT
Loss of PI3Kγ enhances cAMP-dependent MMP remodeling of the myocardial N-cadherin
adhesion complexes and extracellular matrix in response to early biomechanical stress
by
Danny Guo BSc, Zamaneh Kassiri PhD, Ratnadeep Basu MD, Fung L. Chow MSc, Vijay
Kandalam BSc, Federico Damilano MD, Wenbin Liang MSc, MD, Seigo Izumo MD,
Emilio Hirsch MD, Josef M. Penninger MD, Peter H. Backx DVM, PhD
and Gavin Y. Oudit MD, PhD
ONLINE METHODS
Experimental Animals. PI3Kgamma knockout (PI3KγKO), PI3Kalpha dominant negative
(PI3KαDN), PI3Kgamma kinase dead (PI3KγKD) mice have been previously described.1-3 All
experiments were performed in accordance to institutional guidelines and Canadian Council on
Animal Care. Mice were backcrossed into a pure C57Bl/6 background for at least 10 generations and
only littermate male mice of 8-9 weeks of age were used. For the propranolol experiments, mice
were treated with propranolol in their drinking water (0.2 g/L) in order to deliver 15 mg/kg/day;
propranolol was withdrawn one day prior to the echocardiographic and hemodynamic measurements.
The broad spectrum MMP inhibitor, PD166793 (Pfizer Inc.), was administered orally by daily
gavage as previously described.4 Due to the rapid onset of ventricular dilation in PI3KγKO mice
following pressure-overload, PD166793 treatment (30 mg/kg/day) began 3 days prior to aorticbanding and continued until mice were sacrificed.
Aortic Banding. 8-9 week old male C57Bl/6 WT, PI3KαDN, PI3KγKO and PI3KγKD mice
weighing (20–25 g) were used in these experiments. Mice were anesthetized with 1.5 ketamine (100
mg/kg) and xylaxine (10 mg/kg). A topical depilatory agent was applied to the neck and chest area to
1
Guo D et al.
Role of PI3Kγ in Biomechanical Stress
remove fur at and around the area of incision. The skin was cleaned with Germex and Betadine. One
dose of penicillin (10 mg/kg, 0.1 mL i.p.) was administered prior to start of surgery. Mice were
placed supine and body temperature was maintained at 37oC with a heating pad. A horizontal skin
incision of 1 cm in length was made at the level of second intercostal space, once the animal was in
surgical plane of anesthesia (lack of reflex or response to toe-pinching). A 6-0 silk suture was passed
under the aortic arch. A bent 26-gauge needle was then placed next to the aortic arch and the suture
was snugly tied around the needle and aorta between the left carotid artery and the brachiocephalic
trunk. The needle was quickly removed allowing the suture to constrict the aorta. The incision was
closed in layers and the mice were allowed to recover on a warming pad until they were fully awake.
Immediately after the surgery, mice received one dose of buprenorphine and 5for the first 24 hours.
The sham animals underwent the same procedure without the aortic banding. The heart tissues were
snap frozen in liquid nitrogen and were kept in -80oC until being used for experiments.
Echocardiographic Imaging. Transthoracic M-mode and Doppler echocardiographic examination
at 1 wk and 3 wks post-aortic banding were performed using an Acuson® Sequoia C256 system
equipped with a 15-MHz linear transducer (15L8) (Version 4.0, Acuson Corporation, Mountain
View, California) as previously described.1,
4, 5
Data were stored on the hard drive and 230 MB
optical disk for image processing. Mice were placed on a heating pad and a nose cone with 0.75-1%
isoflurane in 100% oxygen was applied. The temperature was maintained at 36.5 to 37.5°C.
Ultrasound gel was placed on the chest of the anesthetized mouse. The ultrasound probe was placed
in contact with the ultrasound gel and scanning was performed over 30 min. The temperature, heart
rate (HR) and blood pressure (BP) were constantly monitored during the scanning. M-mode images
were obtained for measurements of left ventricular (LV) wall thickness (LVWT), LV end-diastolic
2
Guo D et al.
Role of PI3Kγ in Biomechanical Stress
diameter (LVEDD), and LV end-systolic diameter (LVESD). M-mode images were used to measure
LV chamber sizes and wall thicknesses. Fractional shortening and velocity of circumferential
shortening were calculated. For the propanolol experiments in the WT mice, the echocardiographic
measurements were made using the Vevo 770 high-resolution imaging system equipped with a 30MHz transducer (RMV-707B; VisualSonics, Toronto, Canada) as we have previously described.6
Isolated Cardiomyocyte Contractility. Ventricular cardiomyocytes were placed in a Plexiglas
chamber and continuously perfused with oxygenated Tyrodes buffer (137 mM NaCl, 5.4 mM KCl,
10 mM glucose, 10 mM HEPES, 0.5 mM NaH2PO4, 1 mM MgCl2, 1.2 mM CaCl2, and pH 7.4) at
2.5 mL/min at 36oC. Cardiomyocytes were stimulated at 1 Hz with a Grass S44 stimulator (pulse
duration 3 ms; 15-20 V) and a video edge detector (Crescent Electronics) was used to track myocyte
contractions. Steady state contractions were recorded at 1 kHz following a 4 min equilibrium period
using a Phillips 800 camera system (240 Hz) and Felix acquisition software (Photon Technologies
Inc.). Cardiomyocyte length, percent fractional shortening, shortening rate (+dL/dt) and relaxation
rate (-dL/dt) were determined at baseline.
Isolation and culture of adult cardiomyocytes and fibroblasts. Adult murine left ventricular
cardiomyocytes and cardiofibroblasts were isolated and cultured as previously described.7 Briefly, 11week old mice were injected with 0.05 mL of 1000USP/mL heparin for 15 min and then anesthetized
using 2% isoflurane (1 L/min oxygen flow rate) provided through a nose cone. After opening the chest
cavity, the heart was quickly excised and perfused using a Langendorff system within 45 s. Following
3 min perfusion, the heart was then digested with 2.4 mg/mL collagenase type 2 (Worthington) for 7-8
min. After sufficient digestion, the ventricles were removed, dissociated using forceps and transfer
3
Guo D et al.
Role of PI3Kγ in Biomechanical Stress
pipettes, and resuspended in stopping buffer (10% FBS perfusion buffer). The isolated
cardiomyocytes were then exposed to increasing calcium concentrations (100 µM, 400 µM, and 900
µM) for 15 min each before being plated onto laminin coated culture dishes in plating buffer (Eagle’s
MEM with 10% FBS, Sigma) and placed at 37oC in a sterile 2% CO2 incubator. The discarded
stopping buffer was set aside for cardiofibroblasts collection. One hour after plating, the plating buffer
was gently aspirated and replaced with culture buffer (serum free Eagle’s MEM with 0.1% BSA) and
then placed into the incubator for 18 h before treatment. The discarded stopping buffer is centrifuged
at 20 g for 3 min and the resulting supernatant was collected in a 15 mL conical tube. This was then
centrifuged at 1500 rpm for 5 min and the pellet was collected and washed in 10% FBS DMEM
(GIBCO). The solution is once again centrifuged at 1500 rpm for 5 min and the pellet was collected
and plated onto a 10 cm culture dish in 10% FBS DMEM. The cardiofibroblasts were then passaged 2
times and put into 24 h serum free DMEM prior to treatment.
TaqMan Real-time PCR. RNA expression levels of various genes were determined by TaqMan
Real-time PCR as previously described.4,
8
Total RNA was extracted from flash-frozen tissue or
cardiofibroblasts using TRIzol extraction protocol, and cDNA was synthesized from 1 μg RNA by
using random hexamers. For each gene, a standard curve was generated using known concentrations
of cDNA (0.625, 1.25, 2.5, 5, 10 and 20 µg) as a function of cycle threshold (CT). Expression analysis
of the reported genes was performed by TaqMan Real-time PCR using ABI 7900 Sequence Detection
System. The SDS2.2 software (integral to ABI7900 real-time machine) fits the CT values for the
experimental samples and generates values for cDNA levels. All samples were run in triplicates in 384
well plates. 18S rRNA was used as an endogenous control. The primers and probes for mRNA
expression analysis by Taqman Real-time PCR are listed in Supplementary Table 1 (see below).
4
Guo D et al.
Role of PI3Kγ in Biomechanical Stress
Western Blot Analysis and Membrane Fractionation. The phosphorylated and/or total proteins
from cultured cells and heart tissues of mice were measured by Western blot analysis as previously
described.8, 9 Protein was extracted in 50 mM Tris, 120 mM NaCl, 1 mM EDTA and 1% Triton X-100
(final pH 7.4). After quantification using the BCA Protein Array Kit (Pierce, Rockford, IL, USA),
protein samples were separated by 8%-12% SDS-PAGE gel electrophoresis and then transferred to
nitrocellulose membrane (Millipore). The membrane was blocked with 5% milk in Tris-Buffered
Saline Tween-20 (TBST) for 2 h and then incubated overnight at 4oC with primary antibody against
phosphorylated and total ERK1 (44kDA), ERK2 (42kDa), AKT (60kDA), GSK-3α (51kDa), GSK-3β
(46kDa) (Cell Signaling), FAK (125kDa) (Santa Cruz and BD Biosciences), PLN (25kDa) (Santa
Cruz), and CREB (43kDa) (Cell Signaling). Primary antibodies against N-Cadherin (130kDa), βCatenin (92kDa) (BD Biosciences), MMP2 (72 and 64 kDa) (Millipore), MT1-MMP (60 kDa)
(Millipore), MMP9 (92 kDa) (Millipore) and β1D-integrin (116 kDa) (Millipore) antibodies were also
used. The anti-α7B-integrin (26-34 kDa) was kindly provided by Dr. Stephen J. Kaufman, University
of Illinois, Urbana, Illinois and used as previously described.10 After primary antibody was removed,
the membrane was washed 3 times with TBST for 15 min each. The membrane was then incubated
with an appropriate horseradish peroxidase (HRP) coupled secondary antibody at a 1:5000 dilution in
TBST for 2 h at room temperature, then washed 3 times with TBST for 15 min each. Proteins were
detected by enhanced chemiluminescence (GE) using X-ray film (Fuji) and analyzed by the ImageJ
software (U.S. National Institutes of Health, Bethesda, MD). For the in vitro analysis of N-cadherin
cleavage, 100ng of N-Cadherin Fc Chimera protein (R&D) was incubated with 0, 1, 10, 100 nM of
MMP2 (R&D) or MT1-MMP (R&D) (see Online Supp. Fig. II). An additional group of 100 ng of NCadherin Fc Chimera and 100 nM of MMP2 or MT1-MMP was incubated with 10 μM of the MMP
5
Guo D et al.
Role of PI3Kγ in Biomechanical Stress
inhibitor PD166793. All components were dissolved in 50 mM Tris/HCl pH 7.8, 50 mL CaCl2 and 0.5
M NaCl. The mixture was incubated in 37oC for 4 h and then the proteolysis was stopped with 5X
SDS sample buffer. The samples were then immediately separated in a 12% SDS-PAGE gel and
blotted with an N-Cadherin specific antibody (Santa Cruz).
Membrane fraction was extracted from LV myocardium by homogenized frozen tissue in
RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA) with DTT (1 mM), PMSF (1
mM), protease inhibitor (Calbiochem, San Diego, USA) and phosphatase inhibitor cocktails (SigmaAldrich, Oakville, Canada), centrifugation at 2,900g for 20 min. The supernatant containing the
cytosolic and membrane proteins was centrifuged at 29,000g for 45 min. This high speed
centrifugation separated the cytosolic protein (supernatant) from the membrane protein (pellet). The
pellet was resuspended in 50 μL RIPA buffer (containing DTT, PMSF, protease and phosphatase
inhibitors, with 0.25% sodium deoxycholate and 1% NP40 and then centrifuged at 15,000 g for 20
min. This supernatant was stored at -80oC as the membrane fraction. Western blot analysis for Ncadherin, β-catenin and MT1-MMP were performed on the membrane fraction using a 10% SDSPAGE gel. The nitrocellulose membrane was then stripped with a mild stripping buffer (62.5 mM
Tris-HCl, pH 6.8, 69.4 mM SDS, and 0.7% β-mercaptoethanol; 30 min at 55oC), blocked with 10%
skim milk and blotted for Toll-like receptor 4 (TLR4) (1:500 dilution, Santa Cruz, Santa Cruz, USA),
and subsequently for caspase 3 (1:3000 dilution, Cell Signaling). The purity of the membrane fraction
was confirmed by the presence of the membrane specific protein, TLR4, and the absence of the
cytoplasmic protein, caspase 3.
Cell Adhesion Assay. The cell adhesion assay was performed in accordance with the protocol
provided by CELL BIOLABS, INC. CytoSelect 48-Well Cell Adhesion Assay (ECM Array,
6
Guo D et al.
Role of PI3Kγ in Biomechanical Stress
Colorimetric Format) as previously described.11 Adult murine cardiomyocytes from sham and 1 wk
post-AB hearts were freshly isolated and a hemocytometer was used to determine cell concentration
per mL. The concentration was adjusted to 100,000 cells/mL and the cells were plated into each well
at 20,000 cells/well. The cells were then placed into a 2% CO2 sterile 37oC incubator for 60 min for
cells to adhere. The adherent cells were washed with PBS before 200 µL of the Cell Stain solution was
added for 10 min. The cells were then washed 5 times with PBS to remove the staining solution and
left to air dry for 10 min. 200 µL of extraction solution was then added to each well and the plate was
incubated for 10 min on a rocker. Once the color of the solution stabilized, the 48-well plate was
placed in a plate reader and optical density (OD) was recorded at 560 nm. The reported OD values are
the raw OD values of samples minus the OD value of a blank.
Measurement of cAMP levels. The cAMP assay was performed following the protocol provided with
the GE Healthcare Amersham cAMP Biotrak Enzyme immunoassay System. Isolated ventricular cells
and LV myocardial tissue were homogenized with the provided lysis reagent and centrifuged to
remove debris. The homogenate was placed into the appropriate wells in the 96-well pre-made plate
and incubated with antiserum for 2 h at 4oC. After the incubation, cAMP-peroxidase conjugate was
pipetted into all the wells except blanks and the plate was then incubated at 4oC for 1 h before the
wells were aspirated, washed and dried. Enzyme substrate was then added to each well for 30 min at
room temperature before collecting the OD values at 630 nm. The OD values were converted to
concentrations using a standard curve prepared from provided standards. The effects of β-adrenergic
blockade using propranolol (25 µM) (Sigma) and specific adenylate cyclase inhibition using 2′,5′dideoxyadenosine (DIDA; 30 µM) (Sigma)12 on isoproterenol-induced increase in cAMP were also
examined.
7
Guo D et al.
Role of PI3Kγ in Biomechanical Stress
Confocal Fluorescence Microscopy. Formalin-fixed sections were permeablized with ice-cold
methanol for 10 min, blocked with goat serum and incubated with primary antibodies (N-cadherin
(rabbit) and β-catenin (mouse), 1:500, Abcam®) overnight at 4oC in a humidified chamber. After
several washes, Texas Red-conjugated (for N-cadherin) or FITC-conjugated (for β-catenin) secondary
antibodies were added and the sections were incubated for 30 min at room temperature. The sections
were evaluated with a Zeiss LSM510 confocal microscope and associated software.
References
1.
Crackower MA, Oudit GY, Kozieradzki I, Sarao R, Sun H, Sasaki T, Hirsch E, Suzuki A,
Shioi T, Irie-Sasaki J, Sah R, Cheng HY, Rybin VO, Lembo G, Fratta L, Oliveira-dosSantos AJ, Benovic JL, Kahn CR, Izumo S, Steinberg SF, Wymann MP, Backx PH,
Penninger JM. Regulation of myocardial contractility and cell size by distinct PI3KPTEN signaling pathways. Cell. 2002;110:737-749.
2.
Oudit GY, Crackower MA, Eriksson U, Sarao R, Kozieradzki I, Sasaki T, Irie-Sasaki J,
Gidrewicz D, Rybin VO, Wada T, Steinberg SF, Backx PH, Penninger JM.
Phosphoinositide 3-kinase gamma-deficient mice are protected from isoproterenolinduced heart failure. Circulation. 2003;108:2147-2152.
3.
Patrucco E, Notte A, Barberis L, Selvetella G, Maffei A, Brancaccio M, Marengo S,
Russo G, Azzolino O, Rybalkin SD, Silengo L, Altruda F, Wetzker R, Wymann MP,
Lembo G, Hirsch E. PI3Kgamma modulates the cardiac response to chronic pressure
overload by distinct kinase-dependent and -independent effects. Cell. 2004;118:375-387.
4.
Kassiri Z, Oudit GY, Sanchez O, Dawood F, Mohammed FF, Nuttall RK, Edwards DR,
Liu PP, Backx PH, Khokha R. Combination of tumor necrosis factor-alpha ablation and
matrix metalloproteinase inhibition prevents heart failure after pressure overload in tissue
inhibitor of metalloproteinase-3 knock-out mice. Circ Res. 2005;97:380-390.
5.
Oudit GY, Kassiri Z, Zhou J, Liu QC, Liu PP, Backx PH, Dawood F, Crackower MA,
Scholey JW, Penninger JM. Loss of PTEN attenuates the development of pathological
8
Guo D et al.
Role of PI3Kγ in Biomechanical Stress
hypertrophy and heart failure in response to biomechanical stress. Cardiovasc Res.
2008;78:505-514.
6.
Kandalam V, Basu R, Abraham T, Wang X, Soloway PD, Jaworski DM, Oudit GY,
Kassiri Z. TIMP2 deficiency accelerates adverse post-myocardial infarction remodeling
because of enhanced MT1-MMP activity despite lack of MMP2 activation. Circ Res.
2010;106:796-808.
7.
O'Connell TD, Rodrigo MC, Simpson PC. Isolation and culture of adult mouse cardiac
myocytes. Methods Mol Biol. 2007;357:271-296.
8.
Kassiri Z, Zhong J, Guo D, Basu R, Wang X, Liu PP, Scholey JW, Penninger JM, Oudit
GY. Loss of angiotensin-converting enzyme 2 accelerates maladaptive left ventricular
remodeling in response to myocardial infarction. Circ Heart Fail. 2009;2:446-455.
9.
Zhong JC, Yu XY, Lin QX, Li XH, Huang XZ, Xiao DZ, Lin SG. Enhanced angiotensin
converting enzyme 2 regulates the insulin/Akt signalling pathway by blockade of
macrophage migration inhibitory factor expression. Br J Pharmacol. 2008;153:66-74.
10.
Babbitt CJ, Shai SY, Harpf AE, Pham CG, Ross RS. Modulation of integrins and integrin
signaling molecules in the pressure-loaded murine ventricle. Histochem Cell Biol.
2002;118:431-439.
11.
Cervera AM, Apostolova N, Crespo FL, Mata M, McCreath KJ. Cells silenced for SDHB
expression display characteristic features of the tumor phenotype. Cancer Res.
2008;68:4058-4067.
12.
Desaubry L, Shoshani I, Johnson RA. 2',5'-Dideoxyadenosine 3'-polyphosphates are
potent inhibitors of adenylyl cyclases. J Biol Chem. 1996;271:2380-2382.
9
Guo D et al.
Role of PI3Kγ in Biomechanical Stress
SUPPLEMENTAL TABLES
Supplemental Table I. Taqman primers and probes
TIMP 1
TIMP 2
TIMP3
TIMP 4
MMP-9
MMP-2
MMP13
Forward Primer:
Reverse Primer:
Probe:
Forward Primer:
Reverse Primer:
Probe:
Forward Primer
(294):
Reverse Primer
(427):
Probe (376):
Reverse primer2
(423):
Forward Primer:
Reverse Primer:
Probe:
Forward Primer:
Reverse Primer:
Probe:
Forward Primer:
Reverse Primer:
Probe:
Forward Primer:
Reverse Primer:
Probe:
MMP14
MT1MMP
Forward Primer:
Reverse Primer:
Probe:
ANF
Forward Primer:
Reverse Primer:
Probe:
Forward Primer:
Reverse Primer:
Probe:
Forward Primer:
Reverse Primer:
Probe:
BNP
ß-MHC
5'-CATGGAAAGCCTCTGTGGATATG-3'
5'-AAGCTGCAGGCACTGATGTG-3'
5'-FAM-CTCATCACGGGCCGCCTAAGGAAC-TAMRA-3'
5'-CCAGAAGAAGAGCCTGAACCA-3'
5'-GTCCATCCAGAGGCACTCATC-3'
5'-FAM-ACTCGCTGTCCCATGATCCCTTGC-TAMRA-3'
5'- CCG CAG CGG ACC ACA AC-3'
5'- CCG GAT CAC GAT GTC GGA G-3'
5'- FAM-CTA CCA TGA CTC CCT GGC TT-TAMRA-3'
5'- GGA TCA CGA TGT CGG AGT TGC-3'
5'-TGCAGAGGGAGAGCCTGAA-3'
5'-GGTACATGGCACTGCATAGCA-3'
5'-FAM-CCACCAGAACTGTGGCTGCCAAATC-TAMRA3'
5’-CGAACTTCGACACTGACAAGAAGT -3’
5’- GCACGCTGGAATGATCTAAGC-3’
5’-TCTGTCCAGACCAAGGGTACAGCCTGTTC -3’
5’-AACTACGATGATGACCGGAAGTG -3’
5’-TGGCATGGCCGAACTCA -3’
5’-TCTGTCCTGACCAAGGATATAGCCTATTCCTCG -3’
5’-GGGCTCTGAATGGTTATGACATTC -3’
5’-AGCGCTCAGTCTCTTCACCTCTT -3’
5’-AAGGTTATCCCAGAAAAATATCTGACCTGGGATTC
-3’
5’- AGGAGACAGAGGTGATCATCATTG -3’
5’- GTCCCATGGCGTCTGAAGA -3’
5’-FAM- CCTGCCGGTACTACTGCTGCTCCTG-TAMRA 3’
5'-GGA GGA GAA GAT GCC GGT AGA-3'
5'-GCT TCC TCA GTC TGC TCA CTC A-3'
5'-TGA GGT CAT GCC CCC GCA GG-3'
5'-CTG CTG GAG CTG ATA AGA GA-3'
5'-TGC CCA AAG CAG CTT GAG AT-3'
5'-FAM-CTC AAG GCA GCA CCC TCC GGG-TAMRA-3'
5'-GTGCCAAGGGCCTGAATGAG-3'
5'-GCAAAGGCTCCAGGTCTGA-3'
5'-ATCTTGTGCTACCCAGCTCTAA-3'
10
Guo D et al.
Role of PI3Kγ in Biomechanical Stress
Supplemental Table II. Echocardiographic, hemodynamic and morphometric
parameters following aortic banding
N
HR (bpm)
PWT (mm)
LVEDD (mm)
LVESD (mm)
FS (%)
VCFc (circ/s)
+dP/dtmax
(mmHg/s)
-dP/dtmin
(mmHg/s)
LVEDP (mmHg)
LVW/BW (mg/g)
HW/BW (mg/g)
WT+
SHAM
PI3KγKO+
SHAM
8
554±14
0.68±0.06
3.81±0.11
1.71±0.09
55.1±1.8
10.8±0.32
10131±301
8
549±12
0.67±0.05
3.79±0.12
1.52±0.1*
59.9±1.9*
12.94±0.4*
12893±276*
WT+AB
1wk post-AB
10
561±14
0.74±0.09
3.78±0.14
1.69±0.16
55.3±2.5
10.9±0.38
9935±378
PI3KγKO+AB
1 wk post-AB
10
548±15
0.73±0.11
4.41±0.2*
2.63±0.18*
40.3±2.8*
7.48±0.31*
7138±330*
WT+AB
3 wks post-AB
10
539±16
0.86±0.12#
3.65±0.17
1.69±0.21
53.8±3.1
10.9±0.45
9984±423
PI3KγKO+AB
3 wks post-AB
10
568±17
0.84±0.14#
5.53±0.23*
4.58±0.19*
17.2±4.2*
4.52±0361*
5012±359*
9945±252
12671±311
9471±283
6902±363*
6145±369
4875±408*
5.23±1.96
3.75±0.13
4.9±0.15
6.56±2.7
3.71±0.17
4.88±0.15
7.71±2.8
4.17±0.21
5.21±0.23
12.8±2.2*
4.22±0.24
5.28±0.27
9.23±4.1
5.63±0.22#
6.27±0.41#
19.3±3.9*
5.69±0.29#
6.31±0.38#
AB, aortic banded; SHAM, sham-operated; HR, heart rate; PWT, posterior left ventricular wall thickness;
LVEDD, left ventricular end diastolic diameter; LVESD, left ventricular end systolic diameter; FS, fractional
shortening; VCFc, velocity of circumferential shortening corrected for heart rate; +dP/dtmax, maximum first
derivative of the change in left ventricular pressure; and -dP/dtmin, minimum first derivative of the change in
left ventricular pressure; LVEDP, left ventricular end-diastolic pressure. *p<0.05 compared with the
corresponding WT+AB group; #p<0.05 compared with corresponding SHAM group.
11
Guo D et al.
Role of PI3Kγ in Biomechanical Stress
SUPPLEMENTAL FIGURES
Online Figure I
Online Supplemental Figure I. The purity of protein fractionation was confirmed
using western blot for caspace-3 (a membrane bound protein) and toll-like receptor 4
(a cytosolic protein). Caspace-3 protein was only found in the cytosolic fraction and
not the membrane fraction whereas toll-like receptor 4 protein was present in the
membrane fraction but not in the cytosolic fraction.
12
Guo D et al.
Role of PI3Kγ in Biomechanical Stress
Online Figure II
Online Supplemental Figure II.
II MMP2,
MMP2 but not MT1-MMP,
MT1 MMP cleaves human
recombinant N-cadherin Fc Chimera. N-cadherin Fc Chimera (NCAD, 100 ng) was
incubated with 3 different concentrations of human recombinant MMP2 or MT1-MMP
and the MMP inhibitor (MMPi, PD166793 (10 μM)) for 4 hours at 37oC. Western
blotting detected bands at 65, 60, and 40kDa representing fragments of the N-cadherin
Fc Chimera.
13
Guo D et al.
Role of PI3Kγ in Biomechanical Stress
Online Figure III
Online Supplemental Figure III. Western blot analysis of MMP9 protein levels
showing an increase in the PI3KγKO hearts at 3 weeks after AB but not at 1 week
after AB whereas WT hearts showed no changes in MMP9 protein levels. n=4 for all
groups. *p<0.05 compared with sham and 1 week AB groups.
14
Guo D et al.
Role of PI3Kγ in Biomechanical Stress
Online Figure IV
Online Supplemental Figure IV. Western blot analysis of membrane β1D and α7B
integrin protein showing increased levels in WT hearts at 1 and 3 weeks after AB.
Both integrins increased in the PI3Kγ-KO at 3 weeks after AB. n=4 for all groups.
*p<0.05 compared with corresponding sham group; #p<0.05 compared with the
PI3Kγ-KO group.
15
Guo D et al.
Role of PI3Kγ in Biomechanical Stress
Online Figure V
Online Supplemental Figure V.
V Echocardiography shows that 9 weeks of AB in WT
mice induces LV systolic dysfunction characterized by a reduction in percentage of
fractional shortening (A) and an increase in left ventricular end diastolic diameter
(LVEDD) (B). These changes are reversed with a daily intake of the beta-blocker,
propranolol (15 mg/kg/day) (A-B). *p<0.05 compared with all other groups.
16