Table S1. Features of patients with acute myocardial infarction or unstable angina and control subjects

parameters Healthy controls Unstable angina AMI P value

Serum Histamine (ng/ml) 2.2±0.1 2.3±0.13 6.5±1.26 0.000*

male 2.4±0.13 2.4±0.13 5.9±0.17  

female 2.0±0.14 2.2±0.24 8.8±3.86  

Gender (male/female) 0.92 (24/26) 2.33 (14/6) 2.88 (23/8) 0.042#

Age (years) 46±1.5 55±2.3 58±2.4 0.000$

male 46±2.2 52±1.9 56±2.3  female 46±2.1 63±5.4 66±5.9  

Use of β-blocker (%) 0 (0/50) 50 (10/20) 25.8 (8/31) 0.000#

Use of ACEI (%) 0 (0/50) 35 (7/20) 25.8 (8/31) 0.000#

AMI: acute myocardial infarction. * ANOVA test after the values were logarithm-transformed, # Chi-square test. ACEI: angiotensin convert enzyme inhibitors

Table S2. Sequences of primers used for RT-PCR

Name Sequences Product (bp) TM (℃)H2R (mouse) (F) 5’-GCTGTCACGGACCCACTGCG-3’ 763 59

  (R)5’-GGAGGCTCAGGCTCAGGAGACA-3’    β-actin (mouse) (F) 5’-TTCTACAATGAGCTGCGTGTGGC-3 456 58

  (R) 5’-CTCATAGCTTCTCCAGGGAGGA-3’    ANP (rat) (F) 5’-GGGCTCCTTCTCCATCACCA-3’ 420 56

  (R) 5’-GCACTGTATACGGGATTTGCTCC-3’    β-MHC (rat) (F) 5’-TCGTGGAGCGGCGCAACAAC-3’ 849 60

  (R) 5’-TCAAAGGCTCCAGGTCTCAGGGCT-3’    GADPH (rat) (F) 5’- ACCAACTGCTTAGCCCCCC-3’ 281 58

  (R) 5’-GCATGTCAGATCCACAACGG-3’    H1R (human) (F) 5’-GACTGTGTAGCCGTCAACCGGA-3’ 316 55

  (R) 5’-TGAAGGCTGCCATGATAAAACC-3’    H2R (human) (F) 5’-TCGTGTCCTTGGCTATCAC-3’ 330 51

  (R) 5’- CTTTGCTGGTCTCGTTCCT-3’    H3R (human) (F) 5’- CCTCCGCACCCAGAACAACTT-3’ 194 55

  (R) 5’- TGAGCACGATGTTGAAGGCAG-3’    TNFα (rat) (F) 5’- GGCTTTCGGAACTCACTGGA-3’ 420 56

(R) 5’- CCCGTAGGGCGATTACAGTC-3’

TNFα (human) (F) 5’- AGTGATCGGCCCCCAGAGGGA -3’ 198 59(R) 5’- CTCAGCTCCACGCCATTGGC -3’

GADPH (human) (F) 5’-CTTCTTTTGCGTCGCCAGCCGA-3’ 236 59  (R) 5’-CCCGTTCTCAGCCTTGACGGTG-3’    

1

Supplementary Materials

Front

Right side Left side

Rearleft

auricleright

auricleright

auricle

right auricle

left auricle

A B WT HO HE

1000 bp

298 bp

Normal state

Immediately after LCA ligation

45 mins after LCA ligation

Immediately after LCA reperfusion

24 hours after LCA reperfusion

1 hour after LCA reperfusion

Normal state

Immediately after LCA ligation

24 hours after LCA ligation

C

D

Figure S1. Genotyping and confirmation of myocardial infarction model in mice. (A) Genotyping of H2R knockout mice using PCR. (B) Whole view of heart for slice cutting after Evans blue injection. (C) Serial confirmation of ECG changes in response to ischemia for 45 min and reperfusion for 24 h. (D) Serial confirmation of ECG changes in response to ischemia for 24 h.

model Unstandard coefficients

Standard coefficients t value P value

Constant 0.939±0.175     0.000

Gender 0.008±0.047 0.016 0.174 0.862Age (years) -0.001±0.002 -0.069 -0.741 0.460Diagnosis -0.194±0.030 -0.662 -6.496 0.000Use of β-blocker -0.003±0.065 -0.004 -0.040 0.968Use of ACEI -0.118±0.067 -0.168 -1.767 0.081

Table S3. Correlations between logarithm of serum histamine and other factors

Female=1, male=2; AMI=1, unstable angina=2, healthy controls=3; use of β-blocker=1, non-use of β-blocker=0; use of ACEI=1, non-use of ACEI=0

2

0 0.01 0.1

1 10 100

Histamine (μM)

Figure S2. Representative pictures of Hoechst stained neonatal rat cardiomyocytes (panoptic cells were indicated by bright blue) exposed to different concentrations of histamine. Scale bar = 100 m.

3

Control AD His

p-E

RK

/ER

K

# #

p-ERK

ERK

Control AD His

Figure S3. Effects of H2R activation on activities of moesin and ERK as well as Bax translocation in neonatal rat cardiomyocytes. (A) Both amthamine dihydrobromide (AD, a selective H2R agonist) and histamine (His) increased mitochondrial translocation of Bax. (B) Both AD and His increased the phosphorylation of ERK. (C) MEK inhibitor U0126 exerted a persistent inhibitory effect on the phosphorylation of ERK (Cont: control without using U0126). (D) Histamine exerted no influence on the phosphorylation of moesin. # p < 0.01 vs. the control, n = 3-5 in each group. Doses of histamine and AD in panel A-C were 1 μM.

0 0.1 1 10Rat

io o

f p-

moe

sin/

moe

sin

Concentrations of histamine (μM)

p-moesin

moesin

β-actin

0 0.1 1 10

Concentrations of histamine (μM)D

Bax

Cox-Ⅳ

Control AD His

Control AD His

#

#

Bax

/CO

X-I

VA

B

His+10 μM U0126

His 0.5h 24hp-ERK

ERK

C

4

5

# # # # # #

# #

† † ††

† ††

††

Figure S4. Influence of histamine on endothelial permeability. (A) Effect of histamine receptor H1 antagonist on the dynamic changes of transmembrane resistance (TMR) in cultured HUVECs in response to stimulation of histamine (1 M) . # p<0.01 vs. control; † p<0.01 vs. histamine; n=20 in the control group and n=4 in other groups. (B). HUVECs were treated with famotidine (1 M) or diphenhydramine hydrochloride (1 M) to block the H2R and H1R, respectively. # p<0.01 vs. control; † p<0.01 vs. histamine or reoxygenation; n=20 in the control group and n=4 in other groups.

B

A

† † † #

# #

#

ReoxygenationHypoxia

Start of drug treatment

A B

ReceptorsGAPDH

H1 H2 H3 H4

C

p-m

oesi

n/m

oesi

n *

*

*

p-m

oesi

n/m

oesi

n

Control AD Fam Fam+AD

*

†

His+10μM U0126

His 0.5h 24hp-ERK

ERK

Control AD Fam Fam+AD p-moesin

moesinp-moesin

Control AD His

Control AGEs His

p-moesin

moesin

moesin

p-ERK

ERK

Control His

p-ERK

ERK

Control AD

p-E

RK

/ER

K *

*

D

E

Figure S5. Effects of H2R activation on activities of moesin and ERK in HUVECs. (A) mRNA expression of histamine receptors. (B) Both amthamine dihydrobromide (AD, a selective H2R agonist) and histamine (His) increased the phosphorylation of ERK. (C) MEK inhibitor U0126 exerted a persistent inhibitory effect on the phosphorylation of ERK. (D) AGEs (advanced glycation end products), His and AD increased the phosphorylation of moesin. (E) Famotidine (Fam) abrogated the AD-induced phosphorylation of moesin. * p < 0.05 vs. the control, †p < 0.05 vs. AD. n = 3-5 in each group. Doses of histamine, AD, famotidine and AGEs were 1 μM.

6

7

HE

Anti-M

PO

Isch

emia

24

hA

nti-caspase 3

Figure S6. Immunohistochemical detection of myocardial myeloperoxidase (MPO) and caspase 3. In response to 24 ischemia in a wildtype (WT) mouse, example views of HE, MPO and caspase 3 stain under different magnifications.

100 μm

Isch

emia

45m

in/R

eper

fusi

on 2

4 h

WT-Sham WT-IR WT-IR+AD

KO-IR WT-IR+Fam WT-IR+Fam+AD

Isch

emia

45m

in/R

eper

fusi

on 2

4 h

WT-Sham WT-IR WT-IR+AD

KO-IR WT-IR+Fam WT-IR+Fam+AD

Figure S7. Immunohistochemical Detection of Myocardial Myeloperoxidase (MPO) and Caspase 3 in Mice in response to 45 min of ischemia/24 h of reperfusion . (A) Representative MPO. (B) Representative caspae-3 staining . WT, wild-type mice; KO, H2R knockout mice; MI, myocardial infarction; AD, amthamine dihydrobromide; Fam, famotidine.

A

B

8