Supplementary Figures

W T B e rk -S S

2 0

3 0

4 0

5 0

6 0

IVR

T [

ms

ec

]

P = 0 .0 6

W T B e rk -S S

0

1

2

3

4

MV

E/A

n s

Figure S1. Diastolic function in sickle mice by tissue Doppler imaging (TDI). Six to

nine month old age- and gender-matched sickle (Berk-SS) and wild type (WT) mice

underwent cardiac echocardiography with TDI. Panels depict (A) Isovolumic relaxation

time (IVRT) and (B) Ratio of mitral valve early to late filling (MV E/A). Data shown as

mean ± SEM. n= 8-14 mice in WT and 5-10 mice in Berk-SS, Mann-Whitney test, ns not

significant (P >0.05).

Figure S1

A B

Figure S2. Sickle cell mice have abnormal contractile and relaxation parameters

as evident by invasive assessment of the left ventricular function. (A) Trends in

maximal dP/dt max at baseline and with increasing beta-adrenergic stimulation show

normal contractility in sickle cell mice at baseline and with modest beta-adrenergic

stimulation, but significant contractile deficits at higher dobutamine doses. (B) The

time constant of relaxation, τ, is elevated in sickle cell mice compared to wild type. n

= Berk-SS (“MUT”), n = 3, 34-43 weeks old. ¢ = wild type (“NTG”), n = 6, 43 weeks

old. Valid statistical analyses cannot be performed for these experiments due to small

number of Berk-SS mice that survived to completion of the experiment.

Figure S2

0

2

4

6

8

10

12

τ(m

s)

WT

Berk-SS

0

5000

10000

15000

20000

25000

30000

Max d

P/d

t (m

mH

g/s)

WT

Berk-SS

**

*

*

* * *

Figure S3

Figure S3. Survival curves of Berk-SS (SS) mice as compared to survival of

wild-type (WT) and iron deficiency anemia (IDA) mice. Berk-SS mice used in

multiple experiments described are plotted, including those used for ECG studies and

comparisons with iron deficiency anemia mice. All mice with induced iron deficiency

anemia and corresponding WT controls are shown. The shaded rectangle represents

the WT and Berk-SS mice used in the ECG experiment. Statistical comparisons of the

survival curves is depicted [ns=not significant; **=P<0.01] for n= 25, n=36 and n=313

for WT, IDA and Berk-SS mice, respectively.

0 2 0 4 0 6 0 8 0

0

5 0

1 0 0

S u r v i v a l p r o p o r t i o n s : S u r v i v a l o f W e e k s O l d

W e e k s

Pe

rc

en

t s

ur

viv

al

W T

I D A

S S

n s

* ** *

-1 .0

-0 .5

0 .0

0 .5

1 .0

L

AD

[m

m]

***

B e rk -S S

ID A

3 M o n th s o f A n e m ia

Figure S4. Change in LAD is significantly higher in the sickle mice (Berk-SS)

when compared to their iron deficiency anemia controls (IDA) after 3 months of

anemia. LAD: left atrial anterior-posterior dimension measured by 2D and M-mode

transthoracic echocardiography. Data shown as mean ± SEM, n=10 mice in IDA and

8 mice in Berk-SS, Mann-Whitney test, ***P ≤ 0.001.

Figure S4

W T B e rk -S S

1 5

3 0

4 5

6 0

7 5

EC

V (

%)

* ***

D o n o r

H o s t W T

Figure S5. ECV%, a marker of fibrosis, is significantly higher in transplanted

sickle mice (Berk-SS) as compared to their age- and gender-matched

transplanted wild type controls (WT). Data shown as mean ± SEM. n=17 mice in

WT and 8 mice in Berk-SS, Mann-Whitney test, ****P ≤ 0.0001.

Figure S5

A

C

B

D

Figure S6. Hydroxyproline assay of the ventricles/interventricular septum (IVS) and

atria of 7-9 month old Berk-SS and WT mice hearts is shown as the total

hydroxyproline content in (A) and (B) and the concentration in (C) and (D). Data

shown as mean ± SEM. Mann-Whitney test, ns not significant (P >0.05), ** P ≤ 0.01.

Figure S6

W T B e rk -S S

3 0

3 5

4 0

4 5

5 0

5 5

6 0

To

tal

Hy

dro

xy

pro

lin

e

Co

nte

nt

[g

]

* *

V e n tr ic le s /IV S

W T B e rk -S S

0

1 0

2 0

3 0

4 0

To

tal

Hy

dro

xy

pro

lin

e

Co

nte

nt

[g

]

* *

A tr ia

W T B e rk -S S

0 .5

1 .0

1 .5

2 .0

2 .5

Hy

dro

xy

pro

lin

e [

g]

We

t T

iss

ue

W

eig

ht

[mg

] n s

A tr ia

W T B e rk -S S

0 .2

0 .3

0 .4

Hy

dro

xy

pro

lin

e [

g]

We

t T

iss

ue

W

eig

ht

[mg

]

**

V e n tr ic le s /IV S

Figure S7. Sickle cell anemia mice do not exhibit common classical causes of

restrictive cardiomyopathy. Prussian Blue (a, b) and Congo Red (c, d) staining of

the myocardium of 8 month old sickle (Berk-SS) and wild type (WT) mice. All sickle

and wild type mice heart sections were negative for iron (a,b) and amyloid deposition

(c,d).

Figure S7

a b

c d

10x

10x 10x

10x

WT Berk-SS

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

3 0 0 0

Sa

rco

me

re L

en

gth

[

m]

W T B e rk -S S B e rk -S SW T

2 m o n th 8 m o n th

* * * * * * * *

Figure S8. Sarcomere length is significantly shorter in the sickle mice (Berk-SS)

when compared to their age- and gender-matched wild type controls (WT) as

early as 2 months of age and becomes more apparent at 8 months of age. Data

shown as mean ± SEM, n=403 sarcomere in WT 2 mo, 556 sarcomere in Berk-SS 2

mo, 186 sarcomere in WT 8 mo and 150 sarcomere in Berk-SS 8 mo, 1 mouse per

arm, Mann-Whitney test, ****P ≤ 0.0001.

Figure S8

A

WT Berk-SS

-2 0 +2

WT Berk-SS

B

Figure S9. RNAseq analysis profile in sickle myocardium reveals downregulation of genes

involved in ion and voltage transport and regulation. A. Heat map of differentially expressed genes

between WT and Berk-SS mouse myocardium. B. Heat map and decreased fold expression of 31 ion

channel genes between WT and Berk-SS mouse myocardium (n=3 WT and 3 in Berk-SS).

Figure S9

Supplementary Tables

Table S1. Echocardiographic Features of Iron Deficiency Anemia Mice

Following 6 months of IDA

Measurement [Unit] WT IDA P value

IVSDd [mm] 0.95 ± 0.02 0.90 ± 0.05 0.14

LVPWd [mm] 0.85 ± 0.03 0.81 ± 0.03 0.19

LVM [mg] 109 ± 4 116 ± 8 0.35

LVIDd [mm] 3.96 ± 0.10 4.29 ± 0.06 0.006

LVSF [%] 32 ± 3 27 ± 1 0.10

LAD [mm] 2.01 ± 0.15 1.98 ± 0.08 0.35

IDA mice (n=7; 8 mo old, 6 months of IDA) compared to age- and gender-matched

wild type (WT) controls (n=6). Data presented as mean ± SEM. Mann-Whitney test.

IVSDd = end-diastolic interventricular septum thickness. LVPWd = end-diastolic left

ventricular posterior wall thickness. LVM = left ventricular mass. LVIDd = left

ventricular end-diastolic internal dimension. LVSF = left ventricular shortening

fraction. LAD= left atrial anterior-posterior dimension measured by 2D and M-mode

transthoracic echocardiography.

Table S1

Hematologic values of mice fully chimeric transplanted sickle and age- and gender-

matched WT bone marrow, n= 8 in each group, animals were 12-15 months of age. Data

presented as mean ± SEM. Mann-Whitney test. P < 0.005, t test. WBC: white blood cell

count, RBC: red blood cell count, Hgb: hemoglobin, Hct: hematocrit, RDW: rate

distribution width.

Table S2: Hematologic Parameters of Fully Chimeric Transplanted Wild Type and

Sickle Mice

WBC

[K/μL]

RBC

[M/μL]

Hgb

[g/dL]

Hct

[%]

RDW

[%]

Reticulocytes

[%]

WT 12.6 ± 0.9 9.7 ± 0.3 14.0 ± 0.2 53.6 ± 1.3 18.8 ± 0.8 3.7 ± 0.4

Sickle 25.8 ± 2.4 6.6 ± 0.1 7.9 ± 0.1 34.3 ± 0.5 35.2 ± 0.4 32.9 ± 0.9

Table S2

Table S3: Cardiac MRI Features of Transplanted Sickle Cell Anemia (SCA) Mice

Measurement [Unit] Wild Type Berk-SS P value

LVEDV [µL] 40 ± 2 63 ± 5 < 0.0001

RVEDV [µL] 19 ± 7 29 ± 10 0.007

LVM [mg] 78 ± 3 104 ± 3 < 0.0001

SV [µL] 24 ± 1 40 ± 3 < 0.0001

LVEF [%] 61 ± 3 65 ± 3 0.28

Transplanted Berk-SS SCA mice (n=9; 12-15 mo) compared to age- and gender-matched

transplanted wild type (WT) controls (n=18). Data presented as mean ± SEM. Mann-

Whitney test. LVEDV = left ventricular end-diastolic volume. RVEDV = right ventricular

end-diastolic volume. LVM = Left ventricular mass. SV = Stroke volume. LVEF= Left

ventricular ejection fraction.

Table S3

Table S4. Echocardiographic Features of Transplanted Sickle Cell Anemia (SCA)

Mice

Measurement [Unit] Wild Type Berk-SS P value

IVSDd [mm] 0.88 ± 0.03 0.99 ± 0.05 0.04

LVPWd [mm] 0.79 ± 0.02 0.84 ± 0.03 0.14

LVM [mg] 80 ± 3 133 ± 6 <0.0001

LVIDd [mm] 3.51 ± 0.10 4.41 ± 0.10 0.0001

LVSF [%] 35 ± 3 29 ± 2 0.03

LAD [mm] 1.88 ± 0.07 2.53 ± 0.16 0.0004

Transplanted Berk-SS SCA mice (n=6; 8 mo) compared to age- and gender-matched

transplanted wild type (WT) controls (n=12). Data presented as mean ± SEM. Mann-

Whitney test. IVSDd = end-diastolic interventricular septum thickness. LVPWd = end-

diastolic left ventricular posterior wall thickness. LVM = left ventricular mass. LVIDd = left

ventricular end-diastolic internal dimension. LVSF = left ventricular shortening fraction.

LAD = M-mode measured left atrial anterior-posterior dimension.

Table S4

Table S5: Detailed GO listing of the upgregulated (A.) and downregulated (B.) genes in

the sickle myocardial transcriptome as determined by RNAseq analysis.

A. Upregulated Genes (334 genes)

Category Name p-value

q-value

FDR

B&H

Hit Count

in Query

List Hit in Query List

GO: Biological

Process

cellular response to

transforming growth factor

beta stimulus 2.22E-03

4.51E-

02 11

PDGFB,GCNT2,PPP1R15A,CCL2,

SFRP1,ID1,TFDP2,ANKRD1,ENG,

SMAD6,SMAD7

GO: Biological

Process

extracellular matrix

organization 1.65E-05

1.75E-

03 20

RAMP2,PDGFB,ADAM15,SPOCK2

,SCX,TTR,MFAP2,FBLN2,AGT,KA

ZALD1,FOXC1,SFRP2,CIB1,VWF,

COMP,WT1,CTGF,ELN,SNCA,EN

G

GO: Biological

Process cell-matrix adhesion 3.40E-02

1.60E-

01 7

ACER2,ADAM15,AGT,SFRP1,CIB

1,APOD,CTGF

GO: Biological

Process vasculature development 2.44E-11

3.49E-

08 37

RAMP2,PDGFB,ADAM15,MCAM,S

OX18,GPR56,HMOX1,ADM,RASIP

1,GJA4,AGT,CCL2,FOXC1,TBXA2

R,NPPB,SFRP1,ATPIF1,SFRP2,CI

B1,THSD7A,GPX1,SOX17,WT1,C

XCL13,ID1,APOD,ECM1,PLCD3,H

EY1,ROM1,CTGF,EGFL7,TNFRSF

1A,LYL1,ENG,SMAD6,SMAD7

GO: Biological

Process Angiogenesis 2.16E-10

1.83E-

07 29

RAMP2,ADAM15,MCAM,SOX18,G

PR56,HMOX1,ADM,RASIP1,AGT,

CCL2,TBXA2R,NPPB,SFRP1,ATPI

F1,SFRP2,CIB1,THSD7A,GPX1,S

OX17,CXCL13,ID1,APOD,ECM1,P

LCD3,HEY1,CTGF,EGFL7,TNFRS

F1A,ENG

GO: Biological

Process response to lipid 4.80E-07

1.46E-

04 33

ACER2,RAMP2,RAMP3,PDGFB,P

DK3,HMOX1,PTGDS,ADH1C,ADM

,S100A9,BMP6,PTPRN,MIF,NQO1

,CCL2,CA2,TBXA2R,FMO1,SFRP1

,KRT19,GPX1,PTPRU,NR1D1,HE

Y1,ANKRD1,TREM2,CD24,CTGF,

TNFRSF1A,SNCA,ENG,IL4R,SMA

D6

GO: Molecular

Function extracellular matrix binding 2.53E-05

2.19E-

02 7

ACHE,GPR56,SPOCK2,TINAGL1,

FBLN2,ECM1,ELN

Interaction CSNK1D interactions 5.86E-04

1.61E-

01 5

DBNDD2,PPP1R14A,PER3,PER2,

SNCA

Interaction PER2 interactions 7.02E-04

1.61E-

01 3 PER3,PER2,CRY2

Mouse

Phenotype abnormal circadian period 8.55E-04

3.45E-

01 5 DBP,PER3,PER2,NR1D1,CRY2

Pubmed

TGF-beta signaling is

dynamically regulated during

the alveolarization of rodent

and human lungs. 6.46E-06

6.18E-

03 4 CTGF,ENG,SMAD6,SMAD7

Pubmed

TGFbeta superfamily signals

are required for

morphogenesis of the

kidney mesenchyme

progenitor population. 1.59E-02

2.95E-

02 3 BMP6,WT1,ENG

Table S5

B. Downregulated Genes (147 genes)

Category Name p-value

q-value

FDR

B&H

Hit Count in

Query List Hit in Query List

Coexpression

Atlas

geo_heart_top-relative-

expression-ranked_1000 6.06E-14 1.79E-10 33

DPT,MASP1,COL15A1,MAOB,CES1,

ZNF366,HLA-DQA1,PDK4,HLA-

DQB1,SH3BGR,ECM2,SMCO1,HLA-

DRB5,CASQ1,HTRA3,DDO,AQP1,LD

HD,KY,PHKG1,HMCN2,KCNJ3,SCAR

A5,SCN4B,HRC,KIAA1755,PLA2G5,C

D74,TESC,CLEC10A,HBA1,RPL3L,A

LOX5

GO:

Molecular

Function ion channel activity 6.70E-05 1.37E-02 12

CACNA1S,KCNV2,ANO10,AQP1,CLC

N1,PKD2L2,KCNA7,KCND2,KCNJ3,S

CN4A,SCN4B,SLC26A6

Mouse

Phenotype

abnormal circulating free

fatty acids level 9.18E-06 8.17E-03 9

CES1,PDK4,CDH23,PPARA,CPT1A,S

TAT5A,ADRA1B,ANGPTL3,CTF1

Mouse

Phenotype

abnormal free fatty acids

level 1.17E-05 8.17E-03 9

CES1,PDK4,CDH23,PPARA,CPT1A,S

TAT5A,ADRA1B,ANGPTL3,CTF1

Mouse

Phenotype loose skin 2.40E-05 1.26E-02 4 CACNA1S,DPT,LUM,HR

Mouse

Phenotype abnormal fatty acid level 8.89E-05 3.11E-02 9

CES1,PDK4,CDH23,PPARA,CPT1A,S

TAT5A,ADRA1B,ANGPTL3,CTF1

Pathway glycerolipid metabolic 1.62E-03 7.40E-02 3 CPT1A,CPT2,PLA2G5

Pathway Serotonergic synapse 1.97E-03 8.27E-02 5

CACNA1S,MAOB,KCND2,KCNJ3,AL

OX5

Transcription

Factor

Binding Site V$MEF2_03 5.36E-04 2.37E-01 8

CASQ1,AQP1,OGN,PPP1R3D,ARNT

L,CLCN1,GAL3ST3,SLC26A6

Transcription

Factor

Binding Site V$FOXO3_01 1.87E-03 4.13E-01 7

CDKN1C,PDK4,ENPP2,ZNF521,GFR

A1,CTLA4,HR

Table S5

Table S6. Electrocardiographic Parameters of Sickle Cell Anemia (SCA)

Mice

Measurement [Unit] Wild Type Berk-SS P value

P duration [msec] 14.7 ± 0.4 14.6 ± 0.3 0.8

PR Interval [msec] 33.7 ± 1.0 32.6 ± 0.5 0.3

RR Interval [msec] 89 ± 6 81 ± 4 0.003

Heart Rate [bpm] 683 ± 15 745 ± 12 0.004

QRS Duration [msec] 12.4 ± 0.3 15.2 ± 0.9 0.02

QTc [msec] 80 ± 6 102 ± 3 0.002

Berk-SS SCA mice (n=12; 5 weeks) compared to age- and gender-matched wild type

(WT) controls (n=8). Data presented as mean ± SEM. Multiple t-tests, Holm-Sidak

correction.

Table S6