Supplementary Information Figures 1-19 Adora2b-elicited Per2 stabilization promotes

Supplementary InformationFigures 1-19

Adora2b-elicited Per2 stabilization promotes a HIF-dependent metabolic switch

critical for myocardial adaptation to ischemia

Tobias Eckle, Katherine Hartmann, Stephanie Bonney, Susan Reithel, Michel Mittelbronn, Lori A. Walker, Brian D. Lowes, Jun Han, Christoph H. Borchers, Peter M.

Buttrick, Douglas J. Kominsky, Sean P. Colgan and Holger K. Eltzschig

Figure S1. Experimental set-up for in situ ischemic preconditioning (IP) and ischemia exposure. (a) In studies of IP-elicited changes in gene expression, preconditioned myocardium was analyzed for transcript or protein levels following indicated time periods of reperfusion.

a

Suppl. Figure 1

Suppl. Figure 2

Figure S2. Canonical pathway analysis. Wild-type mice or gene-targeted mice for the Adora2b (Adora2b-/- ) were exposed to ischemic preconditioning (4 cycles consistent of 5 min ischemia followed by 5 min of reperfusion). Following two hours of reperfusion, cardiac tissue from preconditioned myocardium was compared to control tissue without preconditioning. Published online January 14th 2010, NCBI, Gene Expression Omnibus. (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE19875). We found a total of 30 differentially regulated genes during cardiac IP comparing WT and Adora2b-/- mice. Canonical pathway analysis was assessed by Ingenuity Software . Only pathways that reached the threshold according to Ingenuity Software analysis are displayed.

Suppl. Figure 3

Figure S3. First of two possible networks based on 30 differentially regulated genes during cardiac IP in wild-type vs. Adora2b-/- mice assessed by Ingenuity Software. Wild-type mice or gene-targeted mice for the Adora2b (Adora2b-/- ) were exposed to ischemic preconditioning (4 cycles consistent of 5 min ischemia followed by 5 min of reperfusion). Following two hours of reperfusion, cardiac tissue from preconditioned myocardium were compared to controls without preconditioning. (red=up regulated genes, green= down regulated genes based on the microarray analysis; comparison wild-type mice over Adora2b-/- mice; drawn connections indicate directly or indirectly interacting genes based on Ingenuity database. published online January 14th 2010, NCBI, Gene Expression Omnibus, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE19875).

Suppl. Figure 4

Figure S4. Second of two possible networks based on 30 differentially regulated genes during cardiac IP in wild-type vs. Adora2b-/- mice assessed by Ingenuity Software. Wild-type mice or gene-targeted mice for the Adora2b (Adora2b-/- ) were exposed to ischemic preconditioning (4 cycles consistent of 5min ischemia followed by 5 min of reperfusion). Following two hours of reperfusion, cardiac tissue from preconditioned myocardium were compared to controls without preconditioning. (red=up regulated genes, green= down regulated genes based on the microarray data; regulation means wild-type mice over Adora2b-/- mice; drawn connections indicate directly or indirectly interacting genes based on Ingenuity database published online January 14th 2010, NCBI, Gene Expression Omnibus, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE19875).

Clock

Actb

Cry1

Actb

Timeless

Actb

Prkaa1

Actb

a

b

c

d

Wildtype Adora2b-/-

Figure S5. Analysis of the circadian clock network in Adora2b-/- mice following cardiac IP. Adora2b-/- mice or littermate controls matched in age, weight and gender were subjected to in situ IP treatment consistent of 4 cycles of IP (5 minutes of ischemia, 5 minutes of reperfusion). Cardiac preconditioned tissue was shock-frozen and analyzed for protein levels. (a-d) Western blot of Clock (Clock), Cryptochrome 1 (Cry1), Timeless (Timeless) and AMP-activated protein kinase, alpha 1 (Prkaa1). Note: Microarray data revealed no changes for Clock, Cry1 or Prkaa1 in contrast to Western Blot analysis.

C IP CIP Fold change 0.8 Micro Array IP vs CFold change 1.1

Wildtype Adora2b-/-


Wildtype Adora2b-/-


Wildtype Adora2b-/-


Suppl. Figure 5

Figure S6. Experimental set-up for in situ ischemic preconditioning (IP) and ischemia exposure. (a) In vivo model of myocardial ischemic preconditioning and subsequent determination of myocardial injury. Following induction of anesthesia (A) and thoracotomy (T), mice were exposed to 60 minutes of myocardial ischemia with or without previous IP pretreatment. After 120 min of reperfusion, the area at risk was determined by retrograde injection of Evan’s blue, cardiac tissues were harvested and infarct staining was performed using 2,3,5-triphenyltetrazolium chloride (TTC). Myocardial injury was assessed by measuring infarct sizes determined as percentage of the infarcted area from the area at risk or by measurements of plasma troponin I levels.

a

Suppl. Figure 6

Suppl. Figure 7

WT -/-

Per1

0

20

40

60

80

Tro

pon

in (

ng

ml-1

)

b

c

C-Adora2b-/- IP-WT IP-Adora2b-/- C-WT

WT -/-

Per1

0

1

2

Gly

cog

en(

mol

gly

cosy

l res

idue

per

g t

issu

e)

d e

Per1IHC

Per1-/-

Hif1a

Actb

C IP

Figure S7. Per1 in myocardial ischemia. Adora2b−/− or littermate control mice matched in age, gender and weight were subjected to in situ preconditioning with 4 cycles of IP (5 minutes of ischemia, 5 minutes of reperfusion). (a,b) Per1 protein levels determined by Western blot (a) or immunohistochemistry (b) following IP-treatment without reperfusion. One representative image of three is displayed. * significant differential regulation based on microarray data (c) Per1-/- were exposed to 60 min of in situ myocardial ischemia followed by 2h of reperfusion, infarct sizes were measured by double staining with Evan’s blue and triphenyltetrazolium chloride or Troponin I serum levels (mean±SD; n=6, scale bare 50 m) (d) Glycogen content of Per1-/- hearts IP (mean±SD; n=6). (e) Western blot for Hif1a after cardiac IP treatment. One representative blot of three is displayed.

Wildtype Adora2b-/-

Per1

Actb

a

C IP CIP Fold change 2.0* Micro Array IP vs CFold change 0.8

WT -/-

Per1

0

20

40

60

80

Infa

rct

size

(%

are

a at

ris

k)

a

Figure S8. In vitro model of hypoxic preconditioning. For in vitro preconditioning with hypoxia, cardiomyocytes were exposed to 3 cycles of 45 minutes hypoxia (1%) followed by 20 minutes of normoxia (21%). Following 180 min of normoxia exposure, cells were harvested and analyzed for transcript levels by real-time RT-PCR or for protein levels by Western blot analysis, respectively.

Suppl. Figure 8

Figure S9. Per2 Western blot analysis obtained from cardiac biopsies of patients suffering from severe ischemic heart disease (IHD) or healthy controls (cardiac donations). For statistical analysis and overview, see Figure 1h; patient characteristics are given in Table S1.

Suppl. Figure 9

PER2

ACTB

Control IHD

1 2 3 4 1 2 3 4 Patient No.

PER2

ACTB

Control IHD

5 6 7 5 6 7 Patient No.

PER2

ACTB

Control IHD

8 9 10 8 9 10 Patient No.

a

b

c

1 1 0.8 1.7 3.2 2.26 3.56 1.56 Densitometry

1 1.9 2.1 3.57 2.8 3.4 Densitometry

1 0.9 1.2 3.6 2.4 2.2 Densitometry

ADORA1

ADORA2A

ADORA2B

ADORA30

2

4

6

8P < 0.05

P < 0.05

P < 0.05A

R t

ran

scrip

t le

vels

rel

ativ

e to

AD

OR

A2

A

Vehicl

e 20'

30'

60'

60'

0.0

0.5

1.0

1.5

BAY 60-6583 FSK (min)

P < 0.05

P < 0.05

P < 0.05

P < 0.05

cAM

P (

pm

ol m

l-1)

0' 10'15

'20

'30

' 0' 10'15

'20

'30

'0

1

2

3

HMEC SCHMEC ADORA2B KD

P < 0.05

P < 0.05

P < 0.05

P < 0.05

BAY 60-6583 treatment (min)

pC

RE

B n

g p

er 1

07 c

ells

PER2

ACTB

BAY 60-6583 [ZT]0 6 12 0 6 12

HMEC SC HMEC ADORA2B KD

b

d

a

c

Figure S10. Validation of human microvasuclar endothelial cells (HMEC-1) as in vitro model for PER2 regulation. (a) Transcript levels of adenosine receptors in HMEC-1 determined by real time RT PCR (mean±SD, n=3). (b) ELISA for cAMP in HMEC-1 after ADORA2B agonist (10 mM, BAY 60-6583) or forskolin (30 mM) treatment (mean±SD, n=3). (c) ELISA for Phospho-CREB in wildtype HMEC-1 (HMEC SC, treated with scrambled siRNA) or ADORA2B knockdown HMEC-1 (HMEC ADORA2B KD, treated with ADORA2B siRNA) after ADORA2B agonist (10 mM, BAY 60-6583) treatment (mean±SD, n=3). (d) Synchronized HMEC SC or HMEC ADORA2B KD cells were treated with vehicle, or ADORA2B agonist BAY 60-6583 and blotted after indicated time periods; one of three representative experiments is displayed.

Suppl. Figure 10

Chromosome 2239,152,679 239,197,749

PER2

b

aSuppl. Figure 11

Figure S11. PER2 promoter analysis. (a) Chromosomal localization of PER2. (b) PER2 promoter with putative CREB binding sites (yellow) and truncations (green/bold) used in promoter assays. (c) Nucleotide sequence alignments of conserved regions shared by human and mouse Per2 promoter. H and M represent human and mouse sequences, respectively. The bold , underlined letters represent a conserved CREB binding site between human and mouse, which was identified to be functional (Fig. 1i). Promoter analysis was performed using Genomatix Software (http://www.genomatix.de) and TESS (http://www.cbil.upenn.edu/cgi-bin/tess). Alignment was performed using BLAST (NCBI). (d) Schematic of plasmids expressing sequence corresponding to full length PER2 promoter (FLPER2) or indicated truncations: -88, -216, -357,-461 with putative CREB binding sites (red = CREB binding site that significantly increased luciferase activity over baseline activity upon ADORA2B activation, see Fig. 1j).

c H:-AGGCCCCGCCCC-TCATGTATGCAGATGAGACGG-AGTCGCGGCCAATGGCGGAGGC-C ||| ||||||| || ||||||| ||||| || | || || |||||||| | || | M:CAGGTTCCGCCCCGCCA-GTATGCAAATGAGGTGGCACTC-CGACCAATGGC-G-CGCGC H:GGGGGCGGGCGCGGCGCGCGCGGTCACGTTTTCCACTATGTGACAGCGG ||||||||| | |||||||||||||||||||||||||||||||||||| M:AGGGGCGGGCTCAGCGCGCGCGGTCACGTTTTCCACTATGTGACAGCGG

ATGGTACGCGCCACTCCGCGCTCCCCGAGCTGGCGGGCTTGAGGGCGTAGTGAATGGAAGGCGCCGACGCCGGAAGTGGATGAGACCACTAGGGAGGACGACGGGTAGCACGAACGCGCCGCGTCTCCATTGAGGAACCGACGAGGTGAACATGGAGTTCCATGTGCGTCTTATGTAAAAAGAGCGACGGGCGCGGCCACCAATGGGCGCGCGGCGTTCGTAGGCCCCGCCCCTCATGTATGCAGATGAGACGGAGTCGCGGCCAATGGCGGAGGCCGGGGGCGGGCGCGGCGCGCGCGGTCACGTTTTCCACTATGTGACAGCGGCGACTCGGCCGCGGCGGAGGCGGCGGCGCTGAGGGGATACGTGCAGCTGTGGGCGGCGGCGGCGGGCGCGGGGCCGGGCGGACAGAGCCGCGAGTCGGCGGAGGGACCGGCGGACGGGCTGACGCGGGCGCGGCCGGCGGTAAGTGGCGCGGCGCGGCCCCGCTGCGGCTTACGTAACCGCCGCCGGCGCGCGGGCCTCGGGCAGGTCGGGGTCCCAGCGCCGGCTCGGGCAGCGGAGGCGCCGCCGGAAGTTCCTTGGGCTGCTGGACTCCTCGGCTTGAAACGGCGCCGGCGTGGGGGCGTGTGCCCTTGGCCCTG

Per2 Promoter 239, 197,207 - 239,197,749

-461Luc

CREB -357Luc

CREBCREB -216Luc

CREBCREBCREB -88Luc

TSS-461-357-216-88ATG

CREBCREBCREBCREB FLLucd

0.0

0.5

1.0

1.5P < 0.05

Fol

d c

han

ge

in C

SN

5 t

ran

scrip

t

csiR

siCSN5

b

-

CSN5

ACTB

ZT60

Vehicle

BAY 60-6583

csiRsiCSN5+- +- -

- + - + -

+ - - - -

- +- +---

+

Figure S12. In vitro studies on ADORA2B dependent CUL deneddylation as molecular mechanism for PER2 stabilization. (a) Validation of actinomycin (ACT) and cycloheximide (CXM) treatment for studies on the influence of transcriptional, translational or post-translational mechanisms on Period 2 protein levels (Fig. 2b,c). (a,left) Inhibition of transcription using ACT. # significant inhibition of Per2 transcription; * significant increase of Per2 transcript due to pharmacological treatment (forskolin, FSK). (b, right) Inhibition of translation using CXM. (Fig. 2b,c).(b) Neddylated cullin enhances ubiquitin ligase (SCF) activity leading to proteasomal degradation. The COP9 signalosome or signaling via ADOAR2B leads to cullin deneddylation. (c,d) Synchronized HMEC-1 treated with vehicle or ADORA2B agonist BAY 60-6583 (10mM) were blotted after indicated time periods; one of three representative experiments is displayed, and quantified for neddylated CULLIN using a specific Nedd8 antibody (*p<0.05, n=3). (e, f) siRNA knockdown of the COP9 signalosome subunit CSN5. RT PCR or western blot for CSN5 from HMEC-1 after siRNA treatment. Note: siRNA knockdown revealed a 98 % reduction of CSN5 transcript or protein, respectively.

f

c

Suppl. Figure 12a

0 6 12 240.0

0.5

1.0

1.5 Vehicle

BAY 60-6583

ZT

*

Fol

d c

han

ge

in C

UL

NE

DD

8

CULNEDD8

ACTB

6 12 24 6 12 240

Vehicle BAY 60-6583

ZT

e

d

CFSK C

FSK0

1

2

3

4

Vehicle, 6hr

*

# #

Actinomycin 6 hr

Fol

d c

han

ge

PE

R2

tra

nsc

ript

ZT6

Ve

hic

le

FS

K

CX

M+

FS

K

CX

M

PER2

ACTB

S-Cul-Fubiquitin ligase

Nedd8

S-Cul-Fubiquitin ligase

Proteasomaldegradation

Proteasomaldegradation

ADORA2B/COP9

(CSN1-9)

WT HZ KO0.0

0.5

1.0

P < 0.05

P < 0.05

Per

2 t

ran

scrip

t (h

eart

)WT Per2-/-

0

20

40

60

80

100

Baseline

EF

(%

)

0

20

40

60

80

100

Baseline

LV

ED

V ( l

)Gys1

Baseline

Per2 -/-wildtype

Actb

Actb

Cpt1

Baseline

Per2-/-wildtype

Suppl. Figure 13

b

e

a

f

Figure S13. Baseline characteristics of Per2-/-mice. (a) Cardiac characterization of Per2-/- mice. Per2 transcript levels were measured in cardiac tissues by real-time RT-PCR relative to housekeeping gene β-actin. (WT: wildtype; HZ - heterozygote, KO - homozygous deletion of Per2, n=3). (b,c) Hearts from wildtype and Per2-/- mice were analyzed for glycogen (b) or long chain fatty acids (c) using an enzymatic ELISA KIT from Biovision (mean±SD; n=3). (d, e) Baseline protein levels of glycogen synthase 1(Gys1,d) or carnitine-palmitoyltransferase I (Cpt1,e). One representative blot of three is displayed. (f) Echocardiographic analysis of hearts at baseline comparing wild-type and Per2-/- mice (EF= ejection fraction [%], LVEDV=left ventricular end diastolic volume, mean±SD; n=3).

0

1

2

3

4P < 0.05

Baseline

Gly

cog

en[

mol

gly

cosy

l res

idue

per

g t

issu

e]

0.0

0.1

0.2

0.3

0.4P < 0.05

Baseline

Lon

g c

hai

n fa

tty

aci

ds

(n

mol

mg

-1 h

eart

tis

sue)

c d

0

2

4

6

C IP

P < 0.05

P < 0.05

Fol

d c

han

ge

in P

gk

1 t

ran

scrip

t

0

1

2

3

C IP

P < 0.05

P < 0.05

Fol

d c

han

ge

in P

dk

2 t

ran

scrip

t

0

5

10

15

C IP

P < 0.05

P < 0.05

Fol

d c

han

ge

in P

dk4

tra

nsc

ript

0

5

10

C IP

P < 0.05

P < 0.05

Fol

d c

han

ge

in H

k4 t

ran

scrip

t

0

2

4

6

8

C IP

P < 0.05

P < 0.05

P < 0.05

Fol

d c

han

ge

in G

apd

h t

ran

scrip

t

0

2

4

C IP

P < 0.05

P < 0.05

P < 0.05

Fol

d c

han

ge

in L

dh

b t

ran

scrip

t

Suppl. Figure 14WT Per2-/-

b fca d e

Figure S14. Consequences of ischemic preconditioning (IP) on glucose metabolism in wild-type or Per2-/- mice. Per2-/- mice or littermate controls matched in age, weight and gender were subjected to IP, ischemia alone or ischemia with IP (IP; 4 cycles of 5 min ischemia followed by 5 min of reperfusion). (a-j) Cardiac transcript levels of glycolytic enzymes determined by real-time RT-PCR relative to Actb and expressed as fold induction relative to sham-operated controls (mean±SD n=3), hexokinase 4 (Hk4), phosphofructokinase-M (Pfkm), glyceraldehyde 3-phosphate dehydrogenase (Gapdh), phosphoglycerate kinase 1 (Pgk1), pyruvate kinase (Pk), pyruvate dehydrogenase kinase isozyme 1, 2 and 4 (Pdk1, Pdk2, Pdk4), lactate dehydrogenase a and b (Ldha, Ldhb). (k, l) Pyruvate kinase and lactate dehydrogenase enzyme activity (mean±SD n=3).

0

2

4

6

C IP

P < 0.05

P < 0.05

Fol

d c

han

ge

in P

k tr

ansc

ript

0

2

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8

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P < 0.05

P < 0.05

Fol

d c

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dk1

tra

nsc

ript

0

2

4

6

8

C IP

P < 0.05

P < 0.05

P < 0.05F

old

ch

ang

e in

Pfk

m t

ran

scrip

t

0

2

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C IP

P < 0.05

P < 0.05

Pk

activ

ity (

U m

in-1

ml-1

)

0

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P < 0.05

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P < 0.05

Fol

d c

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in L

dh

a tr

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h lig j k

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C IP

P < 0.05

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P < 0.05

Ld

h a

ctiv

ity (

U m

in-1

ml-1

)

C IIP

+I IRIP

+IR C I

I+IP IR

IP+I

R0

2

4

6P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05P < 0.05

P < 0.05

P < 0.05

[C1

3C

6]

fru

ctos

e 1

,6 b

isp

hos

ph

ate

(fol

d c

han

ge)

d

0

1

2

3

P < 0.05

P < 0.05

P < 0.05P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05

TC

A-f

lux

[13

C2

] g

luta

mat

e/cr

eatin

e (f

old

ch

ang

e)

C IIP

+I IRIP

+IR C I

IP+I IR

IP+I

R0

2

4

6P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05P < 0.05

P < 0.05

P < 0.05

[C1

3C

3]

lact

ate

(fol

d c

han

ge)

0

2

4

6

P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05

Gly

cog

en(

mol

gly

cosy

l res

idue

per

g t

issu

e)

WT Per2-/- Suppl. Figure 15

Figure S15. Consequences of Period 2 deficiency on cardiac metabolism during myocardial ischemia or during reperfusion with or without IP pretreatment. (a-e) Per2-/- mice or littermate controls matched in age, weight and gender were exposed to 60 min of in situ myocardial ischemia and 60 minutes of reperfusion with or without ischemic preconditioning (IP; 4 cycles of 5 min ischemia followed by 5 min of reperfusion) prior to myocardial ischemia. C13-glucose for the studies during the ischemic period were administered 30 minutes prior to the onset of ischemia while for studies during the reperfusion phase, we applied the tracers at the onset of reperfusion. Both times the tracer was applied via intravascular injection into a catheter placed into the carotid artery. Isotopically labeled 13C-glucose was purchased from Cambridge Isotope Labs . All UPLC-MS data were acquired with a Waters Acquity UPLC system coupled to a Water Synapt HDMS quadrupole time-of-flight mass spectrometer. Metabolites were measured from the area at risk. (a) 13C-glucose (b) 13C-fructose-1,6-bisphosphate. (c) 13C-lactate. (d) Glucose oxidation (TCA cycle flux rates determined by the ratio of 13C3-glutamate and total creatine). (e) Glycogen content assessed by Glycogen Assay Kit. Per2-/- mice exhibited enhanced glycogen content at baseline, however did not recover during reperfusion (mean±SD; n=3).

b ca

0

2

4

6 P < 0.05

P < 0.05

P < 0.05P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05

[C1

3C

6]

glu

cose

(fo

ld c

han

ge)

e

0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 240

1

2

3 WT

Per2-/-

day course cardiac tissue[6 a.m. - 6 p.m.; ZT]

* *

n.s. changesover ZT0

Fol

d c

han

ge

in H

if1.2

tr

ansc

ript

0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 240

2

4

6

8WT

Per2-/-


*

*

*

# ## # #

Fol

d c

han

ge

in H

if1.1

tr

ansc

ript

0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 240

1

2

3

4WT

Per2-/-


* *


**

Fol

d c

han

ge

in L

dh

a t

ran

scrip

t

ba

c d

Figure S16. Hif1a regulation in Per2-/- mice. Analysis of cardiac transcript levels from wild-type or Per2-/- hearts during a 24 h period for Hif1a isoforms Hif 1.1 and 1.2, Pdk1 and Ldh1. (a) Hif 1.1 (b) Hif 1.2. (c) Pdk1 (pyruvate dehydrogenase kinase isozyme 1). (d) Ldha (lactate dehydrogenase a). Real-time RT-PCR relative to Actb and expressed as fold change of transcript relative to wild-type controls at ZT0 (mean±SD, n=3, #,*p<0.05 over control).

Suppl. Figure 16

0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 240

1

2

3 WT

Per2-/-

day course cardiac tissue[6 a.m. - 6 p.m. ; ZT]

* *


**

Fol

d c

han

ge

in P

dk1

tr

ansc

ript

Suppl. Figure 17

Cre+ DMOG

CardiacHif1a-/- DMOG

Cre+ control

Hif1a

Actb

Nx Hx Nx Hx

Cre+ myocytes

CardiacHif1a-/-

myocytes

Nx 21 % O2

Hx 1 % O2

b

a

Figure S17. Characterization of cardiac specific Hif1a-/- mice. (a) Comparison of immunoreactivity for Hif1a in untreated controls (Cre+) versus DMOG treated cardiac tissue from controls (Cre+) or from animals with a Hif1aloxp/loxp Myosin‐Cre+ background (cardiacHif1a-/- , magnification x 20, one of three representative images is displayed). Note: Hearts from animals with a HIF1aloxp/loxpMyosin‐Cre background (cardiacHif1a-/-) under TMX display absent to very low sarcoplasmatic Hif1a expression compared to strong sarcoplasmic and nuclear Hif1a expression seen in Cre+ animals with DMOG; scale bar 50 m. (b) Adult Myocytes were isolated from controls (Cre+) or from animals with a Hif1aloxp/loxp Myosin‐Cre+ background (cardiacHif1a-/- ) and exposed to ambient hypoxia [1%, 4h]. One representative blot of three is displayed.

[4h]

Suppl. Figure 18ba

Figure S18. Hif1a regulation in Adora2b-/-. Analysis of cardiac Hif1a isoforms Hif 1.1 (a) and Hif 1.2 (b), Pdk1 (pyruvate dehydrogenase kinase isozyme 1, c) and Ldha (lactate dehydrogenase a, d) transcript levels from wild-type or Adora2b-/- hearts during a 24 h time period. Real-time RT-PCR relative to Actb and expressed as fold change of transcript relative to wild-type controls at ZT0 (mean±SD, n=3, #, *p<0.05 over control).

dc

0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 240

1

2

3WT

Adora2b-/-


*


*

Fol

d c

han

ge

in H

if1.2

tra

nsc

ript

0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 240

1

2

3WT

Adora2b-/-


* *

### # #

Fol

d c

han

ge

in H

if1.1

tra

nsc

ript

0 3 6 9 12 15 182124 0 3 6 9 121518 21240

1

2

3

4WT

Adora2b-/-


*


**

Fol

d c

han

ge

in P

dk1

tra

nsc

ript

0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 240

1

2

3

4WT

Adora2b-/-

day course cardiac tissue(6 a.m. - 6 p.m.; ZT)

*


*

Fol

d c

han

ge

in L

dh

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ansc

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Suppl. Figure 19

Figure S19. Light-induced stabilization of cardiac Per2 provides potent protection from myocardial ischemia. (a) Naturally occurring stabilization of cardiac Per2 where ZT 0 is the start of the subjective day. (b) Per2-/- mice or littermate controls matched in age, gender and weight underwent light therapy as described above over indicated time periods, followed by exposure to in situ myocardial ischemia (60 min) followed by 2h of reperfusion. Myocardial injury was assessed by measurement of infarct size or troponin I plasma levels (Fig. 6i; n=6 mice per experimental group, mean±SD); scale bar 500 m (c) Wild-type mice were subjected to in situ myocardial ischemia (60 min) followed by 2h of reperfusion over a 24 h time period (mean±SD). Myocardial injury was assessed by measurement of infarct size or troponin I plasma levels (Fig. 6k; n=8 mice per experimental group, mean±SD).

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Supplementary Information Figures 1-19 Adora2b-elicited Per2 stabilization promotes