Supplemental Figure 1 A B C -...

Supplemental Figure 1

0

4000

8000

12000

FCSExog. cAMP

++

--

+-

*** ***

3 H-T

hym

idin

e in

corp

orat

ion

(AU

)

0

2

4

6

Col

1a2

mR

NA

leve

l

Iso/PEExog. cAMP

++

--

+-

0

2

4

6

Iso/PEExog. cAMP

++

--

+-

Col

3a1

mR

NA

leve

l** * ** *A CB

Supplemental Figure 1.Extracellular cAMP reduces the activation and proliferation of cardiac fibroblasts. (A and B) Quantitative PCR analysis of mRNAs encoding Col1a2 (A) and Col3a1 (B) in myocardial tissue from mice after 7 days of Iso/PE infusion (30 mg/kg/day each) in presence of absence of exogenous cAMP (30 mg/kg/day). n = 9-10 mice per group. (C) 3H-Thymidine incorporation by adult rat cardiac fibroblasts treated with 10% FCS and exogenous cAMP. Time of incubation: 48 h. n = 6 independent experiments. * p<0.05; ** p< 0.01;*** p <0.001 determined by one-way ANOVA with Bonferroni’s post hoc analysis.

0

0.005

0.010

0.015

0.020

0.025

Apo

ptot

ic c

ells

(%)

Iso/PE + + +- +Exog. cAMP + + +- -

- + -- -A1R Antag.- - +- -A2R Antag.

n.s.n.s.n.s.n.s.


+ A1R Antag. + A2R Antag.Control Iso/PE

Iso/PE + exog. cAMP

+ NaCl

A B

Supplemental Figure 2.Exogenous cAMP does not alter cardiomyocyte apoptosis in vivo. (A) Representative pictures of TUNEL staining in mouse hearts of animals after 7 days of infusion with Iso/PE (30 mg/kg/day each), exogenous cAMP (30 mg/kg/day), an A1 adeno-sine receptor antagonist (PSB-16P, 5 mg/kg/day) or an A2A adenosine receptor antagonist (MSX-3, 5 mg/kg/day). (B) Quantification of data from (A). n = 6-8 mice/group. NS: not significant, determined by one-way ANOVA with Holm-Sidak’s post hoc analysis.

A

ENTPD1

ENTPD2

ENTPD3

ENPP1

ENPP2

ENPP3ALP

LNT5E

3

4

5

6

7

8

pKi

C

0

5

10

cAM

P (p

mol

/ml)

TACSha

m

*

D

FE


0

5

10

15

20

Fibr

osis

(%)

**

Sham TAC TAC + ENPP1 inhib.

[SYL-001], M

% re

sidu

al a

ctiv

ity o

f EN

PP

1

B

10-11 10-10 10-9 10-8 10-7 10-6

020406080

1001 / [S]

1 / v

0 nM SYL-001

15 nM SYL-001

60 nM SYL-001

-0.10 -0.05 0.05 0.10-0.01

0.01

0.02

0.03

0.04

0.05

ENPP1-inhibitor(SYL-001)

0 h 24 h

Aorticbanding (TAC)

48 h

Collectpericardial fluid

Start of ENPP1-inhibitor infusion(SYL-001)

Day 1 Day 2

Aorticbanding (TAC)

Day 9

Harvesttissue

200

300

400

500

CM

are

a (µ

m2 )

** *

Sham TAC TAC + ENPP1 inhib.

WG

A /

Nuc

lei

TACENPP1-Inhib.

++

--

+-

TACENPP1-Inhib.

++

--

+-

Supplemental Figure 3.Inhibition of the cAMP-metabolizing enzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) exacerbates TAC-induced cardiac remodeling. (A) Concentration-inhibition curve for SYL-001 at human ENPP1 (Ki = 27 ± 0.8 nM, n = 3). (B) Lineweaver-Burk plot of ENPP1 inhibition by SYL-001. [S], substrate concentration of ATP (μM); v, velocity of enzyme (nmol/min/mg protein). (C) Potency of SYL-001 at various human enzymes: Ectonucleoside Triphosphate Diphosphohydrolases (ENTPD1-3), ENPP1-3, tissue-nonspecific alkaline phosphatase (ALPL) and ecto-5’-nucleotidase (NT5E). Data are given as means ± SEM of pKi values. The compound was inactive at ENTPD1-3 at concentrations up to 1 mM. (D) Quantification of endogenous cAMP in the pericardial fluid of mice after chronic cardiac pressure overload. 8 weeks old mice were subjected to transverse aorta constriction (TAC) and to sham surgeries. To prevent the degradation of cAMP, all mice received infusions with an inhibitor that blocks ENPP1 activity (SYL-001, 7.5 mg/kg/day) one day before TAC and sham surgeries. 24 h after surgery, mice were sacrificed and the pericardium was incised and washed with 350 μl PBS to collect pericardial fluid. 50 μl thereof were used for cAMP quantification. * p <0.05. (E-F) ENPP1 inhibition exacerbates TAC-in-duced cardiomyocyte hypertrophy and cardiac fibrosis. (E) (Top row) Wheat germ agglutinin staining of left ventricle tissue (scale bar: 50 μm) after the indicated treatments and (Bottom row) quantitative analysis. (F) Myocardial tissue sections after staining with Sirius Red and Fast Green counterstaining and quantification of myocardial fibrosis. Scale bar: 100 μm. n = 6-8 mice/group.

HW

/TL

(mg/

mm

)

** * ***

5

6

7

8

Iso/PE + +- +Exog. cAMP + +- -

- +- - ENPP1 Inhib.

0

2

4

6

Fibr

osis

(%)

** ** *


A B

Iso/PE + +- +Exog. cAMP + +- -

- +- - ENPP1 Inhib.

Supplemental Figure 4.Extracellular cAMP requires processing by ENPP1 to exert antihypertrophic effects. (A) Ratio of heart weight to tibia length (HW/TL) and (B) quantification of myocardial fibrosis of mice chronically infused with Iso/PE (30 mg/kg/day each), exogenous cAMP (30 mg/kg/day) and the ENPP1 antagonist (SYL-001, 7.5 mg/kg/day). After 7 days, mice were sacrificed for the assess-ment of cardiac hypertrophy. n = 5-8 mice per group. * p<0.05; ** p< 0.01; *** p <0.001; determined by one-way ANOVA with Bonferroni’s (A) and Holm-Sidak’s (B) post hoc analysis.

Cardiac

fibrob

lasts

0

2

4

6

8

Abc

c4 m

RN

A e

xpre

ssio

n

Cardiac

myo

cytes


0.5

1.0

1.5

0

5

10

15

0

Cardiomyocytes Cardiac fibroblasts

mR

NA

rela

tive

to G

apdh

A1 A2AA2B A3

Adenosine receptorsubtype

mR

NA

rela

tive

to G

apdh

Adenosine receptorsubtype

A B C

Supplemental Figure 5.Quantification of endogenous adenosine receptor subtype expression in isolated adult mouse cardiomyocytes (A) and cardiac fibroblasts (B). (C) Quantitative PCR analysis of Abcc4 mRNA by qPCR in RNA isolated from neonatal rat cardiac fibroblasts and cardiomyocytes. n=3-6 experiments in duplicate.

A1 A2AA2B A3

A B

Intra

cellu

lar c

AM

P(%

FR

ET

chan

ge)

Iso

Exog.

cAMP

5

10

15

20

cAM

P fo

rmat

ion

(% F

RE

T ch

ange

)

0

2

4

6

8

Exogenous cAMP (μM)100 100010

Ctrl.

A2R antagonist cAM

P fo

rmat

ion

(Rat

io C

FP/Y

FP, n

orm

.)

Time (sec)

Exog.cAMP

Iso

0 20 40 60 80 100

1.0

1.1

1.2

Sensor cardiac fibroblasts (Adrb1-/-/Adrb2-/- )


******

*

***

Cardiac fibroblasts

0

Supplemental Figure 6.Extracellular cAMP induces the formation of intracellular cAMP in cardiac fibroblasts. (A) Intracellular cAMP was detected in neonatal rat cardiac fibroblasts infected by an adenovirus to express the EPAC-FRET sensor. Intracellular cAMP formation occured as a concentration-dependent response to exogenously provided cAMP. An antagonist against adenosine receptor type 2A (ZM-241385, 100 nM) suppressed this response (n ≥ 17 cells per group). ** p< 0.01; *** p <0.001 determined by one-way ANOVA with holm-Sidak’s post hoc analysis. (B) Insensitivity of Adrb1/Adrb2-deficient sensor cardiac fibroblasts to isoproter-enol, but not to exogenous cAMP. (Left) Representative FRET measurement in sensor cardiac fibroblasts, isolated from adult Adrb1-/-/Adrb2-/- mice and stimulated with cAMP (100 µM) or with Iso (10 µM). Arrow indicates the addition of Iso or exogenous cAMP. (Right) Quantification of the changes in cAMP-FRET ratio. n=13-19 cells. *** p< 0.001 determined by Mann-Whitney test.

PBS IsoPBS Iso

Ext

race

llula

r cA

MP

(pm

ol/d

ish)

Ext

race

llula

r ATP

(pm

ol/d

ish)

PBS Iso

Ext

race

llula

r cG

MP

(pm

ol/d

ish)


0

10

20

30 ***

0

10

20

30

0

10

20

30

n.s.

n.s.

Supplemental Figure 7.Quantification of nucleotide efflux from neonatal rat cardiac myocytes (NRCM) after β-adrenoceptor stimulation in vitro. NRCM were cultured for 24 h and then stimulated for 5 min with Iso (10 µM) or mock-treated with PBS. To block degradation of cAMP or cGMP, IBMX was added (300 µM) together with Iso. As for the quantification of ATP, the E-NTPDase inhibitor POM-1 was added (30 µM) together with Iso. n = 4-5 experiments in duplicate. ** p< 0.01; NS, not significant vs. PBS determined by unpaired t test with Welch’s correction.

02

4

6

8

10

Iso

******

Iso

+ABCC Antag. +AR Antag.

Time (sec)0 50 100

1.00

1.04

1.08

1.12

Ctrl

Intra

cellu

lar c

AM

P(R

atio

CFP

/YFP

, nor

m.)

Intra

cellu

lar c

AM

P(%

FR

ET

chan

ge)

***Addition of supernatantfrom NRCM

Supplemental Figure 8.Conditioned medium from Iso-stimulated neonatal rat cardiomyocytes (NRCM) induces cAMP formation in sensor cardiac fibroblasts. The experiment was carried out analogous to Figure 3, except that no centrifugation of CM was applied, in order to prevent potential cell damage. Medium from cultured, Iso-treated NRCM was transferred onto Adrb1-/-/Adrb2-/- CF expressing the cAMP sensor. FRET recordings from individual cells (A) and statistical assessment of data from multiple cells (B) indicate that the Iso-conditioned medium increased cAMP formation in CF more than in ctrl-conditioned medium. Pretreatment of the CM with the general ABCC antagonist (MK571, 50 µM), or addition of the adenosine receptor antagonist to CF (DPSPX, 10 nM) completely abolished the Iso effect of conditioned medium. Quantitative FRET data were obtained from at least 7 cells per group * p<0.05; ** p< 0.01; vs Iso determined by one-way ANOVA with Holm-Sidak’s post hoc analysis.


A B

AR Antag.ABCC4 Antag.

---

+ ++

+

+-- -

-

HW

/TL

(mg/

mm

)

WTAbcc4-/-

- +Iso/PE - +4

6

8

10 * ***


0

2

4

6

8

10

- +Iso/PE - +

Fibr

osis

(%)

WTAbcc4-/-

C

A

D

B

100150200250300

-5

0

5

10

15

20

100150200250300

0 100 200 300 400

Abcc4-/- Iso

WT Iso

A1R Antag.

A1R Antag.

Time (sec)

Forc

e (m

g)

Cha

nge

in c

ontra

ctile

forc

e (m

g)

WT IsoAbcc4-/- Iso

*

Supplemental Figure 9.ABCC4 reduces cardiac remodeling and contractility. (A-B) ABCC4 is required to prevent catecholamine-induced cardiac remodeling. (A) Ratio of heart weight-to-tibia length (HW/TL) and (B) quantification of myocardial fibrosis of WT and Abcc4-/- mice (age of 8 months) after Iso/PE infusion (30 mg/kg/day each) for 7 days. (C-D) Contractility was analyzed on living myocar-dial slices from WT and Abcc4-/- mice in the presence of adenosine A1 receptor antagonist DPCPX (100 nM). (C) Representative twitch force recordings from WT (black) and Abcc4-/- (orange) slices after incubation with Iso (100 nM) and DPCPX (100 nM). (D) Quantification of data. n= 5-6 mice per group. * p< 0.05 determined by unpaired t test with Welch’s correction.


Supplemental Figure 10.Synthesis of the water–soluble phosphate prodrug PSB-16P of the A1-selective antagonist PSB-16. See the Methods section for a detailed description of the synthesis.

Supplemental Figure 1 A B C -...

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