supplementary information - Nature Research · are S.E.M.). Uncropped images of blots are shown in...

s u p p l e m e n ta ry i n f o r m at i o n

www.nature.com/naturecellbiology 1

DOI: 10.1038/ncb2126

Figure S1 Calcineurin/NFAT-dependent transcriptional induction of miR-199b. (a) Dynamin-1 (Dnm1) expression, the host gene of miR-199b, remains unaltered in cardiac disease. Analysis of expression of Dnm1 transcripts by real time PCR in cardiac tissue of two experimental models of heart failure: transgenic mice engineered to overexpress a constitutively activated mutant of calcineurin in the postnatal myocardium (MHC-CnA) and mice subjected to pressure overload by transverse aortic constriction (TAC). (b) Luciferase reporters encompassing 1 kb upstream regions of the miR-199b gene related to the genomic location of miR-199b, located on the opposite strand of the

mouse dynamin 1 (Dnm1) gene on chromosome 2 in the murine genome. Reporters were tested for calcineurin/NFAT responsiveness in transient co-transfection experiments with vectors expressing an active calcineurin mutant and NFATc2. (c) Sequence alignment and evolutionary conservation between different species of three candidate NFAT sites located within -3.5 kb relative to the mmu-miR-199b gene. Sequences were compared to a minimal consensus NFAT binding motif (A/TGGAAA or TTTCCT/A) and site-directed nucleotide mutations used in luciferase reporters are shown in blue. Data are presented as mean (error bars are S.E.M.).

Supplemental Figure 1

a

0

0.4

0.8

1.2

nTg MHC-CnA TAC

Dnm

-1 tr

ansc

ript a

bund

ance

(fold

con

trol)

0

0.4

0.8

1.2

sham

b

1415

pre-miR-199b5P 3P

dnm1

luciferaseTATA

10 5 10

fold activation

-calcineurin/NFATc2

-0.5 0.5-1.5 Kb

XX

X

.

F4

F1

F2

F3

XXF2*

XXF2**

XF4*

*#

*

-2.5-3.5 ..

F9

F8

F6

F11

-5.5-7.5-9.5-11.5

c-0.5 0.5-1.5 Kb. -2.5-3.5

N3- 2698- 2708

5’ 3’Human

ChimpanzeeMacaque

MarmosetCowPigRat

Mouse

CAAGTTACCAACAATTTCCCAACAATTTCCCAACAATTTCCCAATACTTTTCCAGCTTCTTCCCAGCCATTTCTCAACAGTTTCCCAA

TTTCC-AT

CAGTAACCCAA

consensus

mutated

..N2a

5’ 3’GGTTGTCCACAGGTTGTCCACAGGTTGTCCAAA- - - - - - - - - - - - -GGTTATCCACGGGTTGTCCACGGATTATCCACAGATTGTCCACA

GATCATCCACA

TCATTTCCTGATCATTTCCTGATCATTTCCTGATCATTTTCCTGTCATTTCCTGATCATTTCCTGATCATTTCCTGATCATTTCCTGA

TCAGATCCTGA

5’ 3’

N2b- 937 - 927

- 581 - 571

TTTCC-AT TTTCC-AT

n = 3

n = 3 n = 3

© 2010 Macmillan Publishers Limited. All rights reserved.


2 www.nature.com/naturecellbiology

Figure S2 In vitro validation of miR-199b target mRNAs. (a) mRNA levels of predicted miR-199b target genes were assessed by standard RT-PCR. L7 served as a loading control. (b) Western blot validation of four putative miR-199b target genes in Dox inducible miR-199b clone. Dyrk1a, dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1a; Myl6b, myosin, light polypeptide 6B; GPCR5A, G protein-coupled receptor, family C, group 5, member A; AP1G1, adaptor protein complex AP-1, gamma 1 subunit. GAPDH, glyceraldehyde-3-phosphate dehydrogenase,

was used as a loading control. (c) Activity of luciferase reporter construct harboring the 3’UTR of Dyrk1a after transfection of indicated synthetic precursor microRNAs. (d) Activity assay of luciferase reporter constructs harboring an intact (pMIR-Dyrk1a) or mutated Dyrk1a 3’UTR (pMIR-Dyrk1a mut) after doxycylin-induced miR-199b-5p expression in clones from (b). *P < 0.05 vs corresponding control group (error bars are S.E.M.). Uncropped images of blots are shown in Supplementary Information, Fig.S6.


luciferase Dyrk1A 3’UTR

miR-199b

pMIR-Dyrk1a

c

Luci

fera

se a

ctiv

ity(%

con

trol

)

pMIR-Dyrk1a0

0.5

1.0

1.5

pre-miR-199b

+

-

-

+

-

+

-

+

-pre-miR-216a

+

-

+

-

+

-

**

*

map3k11

nfe2l1

extl3

myl6b

ttn

hspa5

ap1g1

m11s1

grpc5a

dyrk1a

l7

nTgMHCCnA

a

Dyrk1A

Myl6b

GPCR5a

AP1G1

GAPDH

: Dox- +b

Luci

fera

se a

ctiv

ity(%

con

trol

)

pMIR-Dyrk1a0

0.5

1.0

1.5

pMIR-Dyrk1a mut

DOX

+

-

-

+

-

+

-

+

-

-

+

+

x

d

luciferase Dyrk1a 3’UTR

miR-199b

pMIR-Dyrk1a

luciferase Dyrk1a 3’UTR pMIR-Dyrk1a mut

*

n = 3

n = 3




Figure S3 miR-199b overexpression sensitizes the myocardium to cardiac hypertrophy. (a) Gravimetric analysis of corrected heart weights in MHC-199b mice at 6 months of age. (b) Real time PCR analysis of transcript abundance for fetal marker genes and rcan1-4 in hearts from MHC-199b mice at 6 months of age. (c) Quantification of Rnu6-2 corrected Northern blot miR-199b-5p signals from non-transgenic (nTg), single transgenic (MHC-199b or MHC-CnA) and double transgenic mice (DTg). (d) Quantification of GAPDH corrected Western blot Dyrk1A signals from nTg, MHC-199b, MHC-CnA or DTg mice from Fig. 3d. (e) Quantification of

Rnu6-2 corrected Northern blot miR-199b-5p signals from non-transgenic (nTg) and MHC-199b transgenic mice subjected to sham or pressure overload by TAC (1 week). (f) Western blot analysis and quantification of Dyrk1a and GAPDH in non-transgenic (nTg) and MHC-199b transgenic mice subjected to sham or TAC. (g) Real time PCR analysis of transcript abundance for indicated fetal marker genes in hearts from nTg or MHC-199b mice, subjected to 1 week sham or TAC. *P < 0.05 vs corresponding control group; #P < 0.05 vs experimental group (error bars are S.E.M.). Uncropped images of blots are shown in Supplementary Information, Fig.S6.

Supplemental Figure 3.

0

1

2

3

4

5

miR-199

b/Rnu

6-2

(fold

con

trol

)

nTg

MH

C-1

99b

MH

C-C

nA

DTg

*

*

0

1

2

3

4

5

miR-199b

/Rnu6-2

(fold

con

trol

)

sham TAC

sham

nTgMHC199b nTg

MHC199b

TAC

Dyrk1a

GAPDH

c d

e

a b

0

1

2

3

4

HW

/BW

(m

g/g

)

nTg MHC199b

0

1

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3nppa nppb myh7 acta-1

nTg n = 3 MHC-199b n = 5

rela

tive

mR

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expr

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rcan1-4

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Dyr

k1A

(fold

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**

#

nTg

MH

C-1

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MH

C-C

nA

DTg

*

*

#

f

**

#

0

3

180

260

nppa nppb myh7 acta-1

nTg sham n = 3MHC-199b sham n = 5 nTg TAC n = 5 MHC-199b TAC n = 5

rela

tive

mR

NA

expr

essi

on

*

*

6

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100 **

*

*

*

*

320

g

#

#

#

Dyr

k1a

(fold

co

ntro

l)

0

0.5

1

Sham TAC

* ** nTg

MHC-199b

#nTgMHC-199b

n = 6

n = 5 5 5 3

n = 5 5 5 3

n = 3 5 5 5

n = 3 5 5 5




Figure S4 Dyrk1A haploinsufficiency sensitizes the myocardium to cardiac hypertrophy. (a) Western blot analysis and quantification of Dyrk1A and GAPDH in hearts from Dyrk1A+/+ and Dyrk1a haploinsufficient mice (Dyrk1A+/-) subjected to sham or TAC. (b) Real time PCR analysis of transcript abundance for indicated fetal marker genes in hearts from Dyrk1A+/+ and Dyrk1a+/- mice subjected to sham or TAC surgery for 1 week. (c) Gravimetric analysis of corrected heart weights in 3 week old Dyrk1a+/+, MHC-CnA/Dyrk1a+/+, Dyrk1a+/- and MHC-CnA/Dyrk1a+/- mice. (d) Western blot analysis and quantification of Dyrk1A and GAPDH in hearts from Dyrk1a+/+, MHC-CnA/

Dyrk1a+/+, Dyrk1a+/- and MHC-CnA/Dyrk1a+/- mice. (e) Representative image of whole hearts (top panels), H&E- (mid panels) or Sirius Red stained (lower panels) histological sections of hearts from Dyrk1a+/+, MHC-CnA/Dyrk1a+/+, Dyrk1a+/- and MHC-CnA/Dyrk1a+/- mice. (f) Real time PCR analysis of transcript abundance for fetal marker genes and rcan1-4 in hearts from Dyrk1a+/+, MHC-CnA/Dyrk1a+/+, Dyrk1a+/- and MHC-CnA/Dyrk1a+/- mice. Data are presented as mean ±S.E.M.; *P < 0.05 vs corresponding control group; #P < 0.05 vs experimental group (error bars are S.E.M.). Uncropped images of blots are shown in Supplementary Information, Fig.S6.

Dyrk1a

GAPDH

Dyr

k1a

(fold

co

ntro

l)

0

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dyrk1a+/-

TAC

dyrk1a+/-

Sham TAC

* **

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16

HW

/BW

(mg/

g)

dyrk

1a+/

+M

HC

-CnA

dyrk

1a+/

+dy

rk1a

+/-

MH

C-C

nAdy

rk1a

+/-

a

dyrk1a+/+ dyrk1a+/- MHC-CnAdyrk1a+/+

MHC-CnAdyrk1a+/-

H&

ES

irius

red

Dyrk1a

GAPDH

Dyr

k1a

(fold

co

ntro

l)

0

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c

d

dyrk1a+/+

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Dyrk1a+/+Dyrk1a+/-

e

#

* *

*

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75


rela

tive

mR

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50 * *

**

*

*

dyrk1a+/+ sham n = 3dyrk1a+/- sham n = 3 dyrk1a+/+ TAC n = 5 dyrk1a+/- TAC n = 5

*

*

b

#

##

**

#

dyrk

1a+/

+

MH

C-C

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rk1a

+/+

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-

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C-C

nAdy

rk1a

+/-

dyrk

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+M

HC

-CnA

dyrk

1a+/

+dy

rk1a

+/-

MH

C-C

nAdy

rk1a

+/-

50μm

5mm

50μm

0

5

10

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dyrk1a+/+ n = 3MHC-CnA/dyrk1a+/+ n = 3dyrk1a+/- n = 5

rela

tive

mR

NA

expr

essi

on

15

rcan1-4

0

4

8

16

12

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25

**

*

* **

*

*

f

#

##

#

*

*

MHC-CnA/dyrk1a+/- n = 5

n = 3 3 5 5

n = 5

n = 5





Figure S5 Antagomir-mediated miR-199b silencing prevents and reverses cardiac remodeling and dysfunction. (a) Northern blot analysis of miR-199a-5p expression (encoded by mmu-miR-199a-1 on murine chromosome 9 and mmu-miR-199a-2 on murine chromosome 1) in hearts from mice treated with vehicle or antagomir-199b as a control for specificity of antagomir-199b, Rnu6-2 expression was used as loading control. (b) Western blot quantification of Dyrk1A and GAPDH in hearts from nTg or MHC-CnA mice treated with antagomir-199b. (c) Northern blot and Western blot analysis of miR-199b and Dyrk1A, respectively, in hearts from wildtype mice subjected to sham or TAC for 6 weeks (prevention study, p) and treated with antagomir-199b, the small RNA molecule U6 small nuclear (Rnu6-2) and GAPDH were used as loading controls, respectively. (d) Quantification of Rnu6-2 corrected Northern blot miR-199b-5p signals from mice in

(c). (e) Quantification of GAPDH corrected Western blot Dyrk1A signals from mice in (c). (f) Real time PCR analysis of transcript abundance for fetal marker genes and rcan1-4 in hearts from mice in (c). (g) Northern blot and Western blot analysis of miR-199b and Dyrk1A, respectively, in hearts from wildtype mice subjected to sham or TACr for 6 weeks (r, reveral study). Rnu6-2 and GAPDH were used as loading controls, respectively. (h) Quantification of Rnu6-2 corrected Northern blot miR-199b-5p signals from mice in (g). (i) Quantification of GAPDH corrected Western blot Dyrk1A signals from mice in (g). (j) Real time PCR analysis of transcript abundance for fetal marker genes and rcan1-4 in hearts from (g). *P < 0.05 vs corresponding control group; #P < 0.05 vs experimental group (error bars are S.E.M.). Uncropped images of blots are shown in Supplementary Information, Fig.S6.


vehicle antagomir

nTg MHC-CnA nTg MHC-CnA

miR-199a

Rnu6-2

miR-199b

Rnu6-2

vehicle antagomir

sham sham TACpTACp

Dyrk1a

GAPDH

miR-199b

/Rnu6-2

(fold

co

ntro

l)

0

1

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4

5

sham TACp

Dyr

k1a

(fold

co

ntro

l)

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sham TACp 0

5

10

25


sham vehicle n = 3TACp vehicle n = 5sham antagomir n = 3 TACp antagomir n = 5

rela

tive

mR

NA

expr

essi

on

15

*

*

20

*

miR-199b

Rnu6-2

vehicle antagomir

sham sham TACrTACr

Dyrk1a

GAPDH

miR-199b

/Rnu6-2

(fold

co

ntro

l)

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sham TACr

6

7

Dyr

k1a

(fold

co

ntro

l)

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sham TACr

0.5

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sham vehicle n = 3TACr vehicle n = 5sham antagomir n = 3 TACr antagromir n = 5

rela

tive

mR

NA

expr

essi

on

15

20*

a c d

e f g

h i j

Dyr

k1a

(fold

co

ntro

l)

0

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vehicle antagomir nTg MHC-CnA

b

vehicle antagomir

#

*

#

**

*#

# *

*

#

#

#

**

#

#

*

*

#

vehicleantagomir

vehicleantagomir

vehicleantagomir

**

*

*

*

*

#

#

#

#

rcan1-4

0

1

2

4

3

5

rcan1-4

0

1

2

4

3

5

*#

*

#

*

n = 4 n = 4

n = 4

n = 4 n = 4




Figure S6 Uncropped scans of all western and northern blots depicted in the individual figures.

Figure 1a left Figure 1a right Figure 1b Figure 1d Fig ure 1f Figure 1i

miR-199b

U6

~21nt

~70nt

Figure 1j

NFAT n3 mut NFAT n3

n3

myoglobin

miR-199b

U6

Dyrk1A

GAPDH

13010070

35

Figure 2a Figure 2d Figure 2h

miR199b

U6

miR199b Dyrk1A

GAPDHU6

13010070

35

Figure 2j

miR199b

U6

Figure 3a

10070

35

GAPDH

Dyrk1A

Figure 3d (WB) Figure 3d (IP)

miR199b

U6

Figure 4g

35

10070

Dyrk1A

GAPDH

Figure 4j (WB) Figure 4j (IP)

10075

10075

Figure 5f

Dyrk1A Myl6b GPCR5a AP1G1 GAPDH

Sup. Figure 2b

3510070

Dyrk1A GAPDH

Sup. Figure 3f

10070

35

Dyrk1A

GAPDH

Sup. Figure 4h

10070

35

Dyrk1A

GAPDH

Sup. Figure 4k

miR199b

U6

Sup. Figure 5a

miR199b

U6

10070

35

Dyrk1A

GAPDH

Sup. Figure 5c

miR199b

U6

10070

35

Dyrk1A

GAPDH

Sup. Figure 5g


U6

miR-199b

U6

miR-199b

U6 U6

miR-199b miR-199b

miR-199b

U6

8529

39

91

37


Supplementary Table 1

No MicroRNA Signal (wild type) Signal (MHC-CnA) Fold change (log2)

1 mmu-miR-206 19.24 232.60 3.582 mmu-miR-466i 137.70 17.00 -3.063 mmu-miR-466f-3p 208.35 32.22 -2.864 mmu-miR-467f 193.84 29.20 -2.685 mmu-miR-1195 1,802.24 320.68 -2.496 mmu-let-7d* 1,463.11 286.37 -2.407 mmu-miR-691 108.47 23.26 -2.228 mmu-miR-208b 179.15 767.51 2.109 mmu-miR-669f 82.80 17.87 -2.06

10 mmu-miR-199b-5p 362.05 1,362.70 1.9111 mmu-miR-199b-3p 327.76 1,130.56 1.8012 mmu-miR-146b 583.25 1,704.25 1.6613 mmu-miR-199a-5p 440.10 1,348.73 1.5514 mmu-miR-709 55,668.48 19,682.48 -1.5015 mmu-miR-483 231.17 79.73 -1.4916 mmu-miR-1196 157.56 54.80 -1.5617 mmu-miR-181b 1,515.81 540.53 -1.4518 mmu-miR-1187 358.71 120.51 -1.4119 mmu-miR-126-5p 14,956.99 5,326.77 -1.4020 mmu-miR-181d 1,515.64 608.43 -1.3521 mmu-miR-186 77.16 190.75 1.3422 mmu-miR-154 58.79 147.90 1.2923 mmu-miR-21 13,641.76 32,647.08 1.2724 mmu-miR-149 791.98 328.86 -1.2325 mmu-miR-27b* 109.23 242.63 1.2126 mmu-miR-301a 81.25 169.73 1.1527 mmu-miR-224 179.74 349.90 1.1528 mmu-miR-148a 7,481.78 16,463.35 1.1529 mmu-miR-335-5p 6,390.13 3,117.85 -1.1430 mmu-miR-148b 633.94 1,414.46 1.1231 mmu-miR-151-3p 1,748.93 794.82 -1.1032 mmu-miR-497 240.05 506.56 1.0833 mmu-miR-221 1,231.80 2,572.45 1.0634 mmu-miR-145 14,501.63 29,389.36 1.0635 mmu-miR-29b 372.81 806.71 1.0536 mmu-miR-181a 4,150.74 2,112.45 -1.0337 mmu-miR-31 182.40 369.72 1.0138 mmu-miR-139-5p 5,305.03 2,655.26 -1.0039 mmu-miR-214 5,433.41 10,767.98 0.9940 mmu-miR-423-5p 2,604.47 1,398.66 -0.9641 mmu-miR-204 1,199.81 573.83 -0.9442 mmu-miR-328 1,186.93 664.42 -0.8543 mmu-miR-140* 869.31 1,541.94 0.8344 mmu-miR-222 911.98 1,643.78 0.8245 mmu-miR-199a-3p 13,806.96 24,337.26 0.8246 mmu-miR-152 4,684.11 7,924.26 0.7747 mmu-miR-101a 413.21 661.92 0.7548 mmu-miR-223 1,883.83 3,248.01 0.75

Supplemental Table 1



49 mmu-miR-25 11,288.05 6,784.27 -0.7550 mmu-miR-690 7,140.20 4,322.67 -0.7351 mmu-miR-106a 839.13 1,433.31 0.7252 mmu-miR-155 2,842.51 4,731.39 0.6953 mmu-miR-351 644.60 363.89 -0.6954 mmu-miR-30e* 5,911.83 3,706.66 -0.6755 mmu-miR-720 1,773.49 1,097.18 -0.6656 mmu-miR-574-5p 988.38 624.82 -0.6657 mmu-miR-574-3p 746.01 475.40 -0.6358 mmu-miR-17 1,865.78 2,894.91 0.6259 mmu-miR-99a 8,347.74 12,725.00 0.6260 mmu-miR-1 65,157.02 42,938.44 -0.6061 mmu-miR-22 5,019.44 7,875.21 0.6062 mmu-miR-24-2* 582.39 851.91 0.5963 mmu-miR-34a 424.60 630.78 0.5964 mmu-miR-455 534.91 785.87 0.5665 mmu-miR-365 1,419.24 968.97 -0.5566 mmu-miR-322* 948.41 661.19 -0.5567 mmu-miR-22* 1,333.93 1,883.47 0.5368 mmu-miR-28 2,555.01 3,702.72 0.5369 mmu-miR-128 3,614.74 2,467.83 -0.5270 mmu-miR-92b 4,982.50 3,464.35 -0.5171 mmu-miR-674* 805.97 562.32 -0.5172 mmu-miR-499 15,893.52 22,098.14 0.5073 mmu-miR-20b 900.85 1,257.85 0.4974 mmu-miR-320 5,301.00 3,808.19 -0.4875 mmu-miR-100 8,212.64 10,887.62 0.4776 mmu-miR-10a 3,596.88 2,527.65 -0.4677 mmu-miR-27b 21,347.44 29,269.07 0.4678 mmu-miR-379 816.13 1,115.13 0.4579 mmu-miR-15b 13,891.93 18,599.51 0.4480 mmu-miR-322 5,873.87 8,408.55 0.4481 mmu-miR-378* 1,155.63 853.96 -0.4482 mmu-miR-92a 9,586.38 7,102.89 -0.4383 mmu-miR-24 17,727.16 22,359.90 0.4184 mmu-miR-1224 1,738.54 2,342.29 0.4085 mmu-miR-30a* 5,205.21 4,013.62 -0.4086 mmu-miR-10b 4,777.28 3,525.96 -0.3787 mmu-miR-342-3p 2,753.39 3,381.47 0.3788 mmu-miR-27a 20,741.02 26,728.48 0.3689 mmu-miR-126-3p 39,802.67 29,224.49 -0.3690 mmu-miR-20a 2,792.33 3,509.46 0.3491 mmu-miR-486 20,021.98 15,820.51 -0.3492 mmu-miR-132 1,772.50 2,212.74 0.3393 mmu-miR-29a 14,709.71 18,648.94 0.3394 mmu-miR-15a 6,497.83 7,767.71 0.3295 mmu-miR-361 3,707.16 4,694.41 0.3196 mmu-miR-30d 17,829.49 14,979.14 -0.3197 mmu-let-7a 48,320.09 38,557.62 -0.3198 mmu-miR-705 5,044.32 4,158.39 -0.2999 mmu-miR-30e 11,966.19 10,007.72 -0.28



100 mmu-miR-143 15,148.69 18,053.83 0.28101 mmu-miR-30a 20,585.97 17,160.55 -0.28102 mmu-miR-30b 36,337.25 29,806.60 -0.27101 mmu-miR-133a 29,003.86 23,096.01 -0.27102 mmu-miR-133b 28,426.99 23,065.81 -0.26103 mmu-miR-195 19,710.14 23,690.51 0.25104 mmu-miR-151-5p 8,886.72 10,360.36 0.23105 mmu-let-7i 25,149.34 29,031.36 0.23106 mmu-miR-23b 31,276.02 35,246.85 0.22107 mmu-miR-16 23,993.87 27,704.11 0.21108 mmu-let-7e 36,091.31 31,122.40 -0.21109 mmu-miR-125a-5p 25,396.40 21,361.20 -0.21110 mmu-miR-30c 35,286.16 30,001.09 -0.20111 mmu-miR-26a 39,284.48 33,929.92 -0.20112 mmu-miR-125b-5p 27,872.79 30,385.55 0.20113 mmu-miR-150 12,376.15 10,953.77 -0.19114 mmu-let-7b 38,848.08 35,046.18 -0.19115 mmu-miR-451 17,057.99 18,840.44 0.17116 mmu-miR-26b 30,281.83 27,912.63 -0.15117 mmu-miR-378 8,320.01 9,590.22 0.15


Table 2. Prevention study: echocardiographical characteristics of wildtype mice following sham operation, transverse aortic constriction, and/or antagomir-199b treatment

Sham TAC

Vehicle Antagomir-199b Vehicle Antagomir-199b

0 3 wk 6 wk 0 3 wk 6 wk 0 3 wk 6 wk 0 3 wk 6 wk

n 5 7 7 5 7 7 5 7 7 5 7 7

IVSd (mm) 0.72±0.06 0.77±0.03 0.91±0.03 0.77±0.03 0.81±0.06 0.90±0.06 0.75±0.02 0.83±0.07 0.83±0.06 0.69±0.08 0.73±0.03 0.74±0.04

IVSs (mm) 1.28±0.02 1.33±0.02 1.47±0.04 1.32±0.04 1.35±0.04 1.37±0.03 1.29±0.04 1.36±0.03 1.41±0.03 1.29±0.06 1.31±0.03 1.28±0.03

LVIDd (mm) 2.65±0.04 2.77±0.04 2.84±0.07 2.56±0.05 2.79±0.08 2.86±0.09 2.57±0.15 3.52±0.16*# 3.92±0.07*# 2.49±0.11 2.56±0.12§ 2.80±0.10§

LVIDs (mm) 1.30±0.04 1.30±0.06 1.32±0.05 1.29±0.05 1.25±0.05 1.35±0.11 1.33±0.05 2.38±0.12*# 2.71±0.12*# 1.67±0.08 1.72±0.12§ 1.77±0.11§

LVPwd (mm) 0.75±0.03 0.89±0.04 0.94±0.03 0.77±0.06 0.87±0.06 0.89±0.03 0.78±0.02 0.79±0.04 0.69±0.04 0.88±0.04 0.92±0.03 0.90±0.05

LVPws (mm) 1.26±0.02 1.40±0.01 1.48±0.09 1.33±0.05 1.36±0.05 1.42±0.05 1.30±0.06 1.34±0.06 1.30±0.05 1.26±0.11 1.30±0.07 1.35±0.05

FS (%) 51±1 50±1 49±3 48±2 54±2 47±1 57±2 31±1*# 22±2*# 41±4 44±2§ 46±3§

LV mass (mg) 53±3 54±2 56±3 58±2 54±1 55±2 54±2 64±3*# 75±4*# 50±3 53±2§ 55±2§

VcF (circ/s) 5.6±1.1 5.4±1.6 5.1±0.9 5.4±1.8 5.2±0.9 5.8±0.7 5.8±1.4 4.1±0.2*# 3.9±0.6*# 5.6±0.8 5.6±0.8§ 5.7±0.6§

E/A (mm/s) 1.2±0.03 1.3±0.04 1.3±0.06 1.2±0.01 1.1±0.03 1.3±0.03 1.1±0.03 1.5±0.04 1.6±0.01*# 1.1±0.05 1.2±0.02 1.2±0.06

Data are expressed as means ± SEM. IVSd, interventricular septal thickness at end-diastole; IVSs, interventricular septal thickness at end-systole; LVIDd, left ventricular internal dimension at end-diastole; LVIDs, left ventricular internal dimension at end-systole; LVPwd, left ventricular posterior wall thickness at end-diastole; LVPws, left ventricular posterior wall thickness at end-systole; FS, fractional shortening; LV, left ventricular; VcF, circumferential fiber shortening; E/A, Doppler E/A ratio. * indicates P<0.05 vs corresponding sham group; # indicates P<0.05 vs t=0, same group; § indicates P<0.05 vs vehicle treated group, same time point.


Table 3. Reversal study: echocardiographical characteristics of wildtype mice following sham operation, transverse aortic constriction, and/or followed by antagomir-199b treatment Sham TAC

Vehicle Antagomir-199b Vehicle Antagomir-199b

0 3 wk 6 wk 6 wk 0 3 wk 6 wk 6 wk

n 5 7 7 7 5 7 7 7

IVSd (mm) 0.72±0.06 0.79±0.02 0.81±0.07 0.97±0.06 0.88±0.04 0.93±0.07 0.75±0.16 0.77±0.03

IVSs (mm) 1.24±0.04 1.28±0.04 1.36±0.06 1.36±0.09 1.49±0.11 1.46±0.03 1.45±0.04 1.32±0.07§

LVIDd (mm) 2.70±0.05 2.73±0.04 2.81±0.07 2.65±0.07 2.89±0.09 3.47±0.12*# 4.0±0.06*# 2.88±0.07§

LVIDs (mm) 1.30±0.03 1.33±0.03 1.34±0.06 1.27±0.06 1.22±0.07 2.27±0.09*# 2.81±0.42*# 1.51±0.04

LVPwd (mm) 0.76±0.03 0.83±0.06 0.90±0.05 0.88±0.03 0.87±0.02 0.68±0.14 0.74±0.03 0.98±0.04

LVPws (mm) 1.27±0.02 1.34±0.04 1.43±0.04 1.19±0,17 1.48±0.08 1.44±0.03 1.44±0.04 1.34±0.05§

FS (%) 56±3 54±1 55±3 56±2 47±1 30±1*# 23±2*# 42±1§

LV mass (mg) 52±1 53±3 53±2 53±1 54±1 67±3*# 84±10*# 59±4§

VcF (circ/s) 5.8±1.6 5.7±1.3 5.5±1.2 5.6±0.2 5.7±1.0 3.9±0.2*# 3.3±0.2*# 5.6±0.3§

E/A (mm/s) 1.0±0.05 1.1±0.02 1.1±0.07 1.2±0.04 1.3±0.05 1.6±0.07* 1.6±0.07*# 1.2±0.03§

Data are expressed as means ± SEM. IVSd, interventricular septal thickness at end-diastole; IVSs, interventricular septal thickness at end-systole; LVIDd, left ventricular internal dimension at end-diastole; LVIDs, left ventricular internal dimension at end-systole; LVPwd, left ventricular posterior wall thickness at end-diastole; LVPws, left ventricular posterior wall thickness at end-systole; FS, fractional shortening; LV, left ventricular; VcF, circumferential fiber shortening; E/A, Doppler E/A ratio. * indicates P<0.05 vs corresponding sham group; # indicates P<0.05 vs t=0, same group; § indicates P<0.05 vs vehicle treated group, same time point.


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