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.
s u p p l e m e n ta ry i n f o r m at i o n
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.
Supplemental Figure 2
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
© 2010 Macmillan Publishers Limited. All rights reserved.
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 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
2
3nppa nppb myh7 acta-1
nTg n = 3 MHC-199b n = 5
rela
tive
mR
NA
expr
essi
on
rcan1-4
0
1
2
3
Dyr
k1A
(fold
co
ntro
l)
0
0.5
1
**
#
nTg
MH
C-1
99b
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
20
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
© 2010 Macmillan Publishers Limited. All rights reserved.
s u p p l e m e n ta ry i n f o r m at i o n
4 www.nature.com/naturecellbiology
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
0.5
1
sham
dyrk1a+/-
TAC
dyrk1a+/-
Sham TAC
* **
0
4
8
12
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
0.5
1
c
d
dyrk1a+/+
dyrk1a+/+
Dyrk1a+/+Dyrk1a+/-
e
#
* *
*
0
3
75
100nppa nppb myh7 acta-1
rela
tive
mR
NA
expr
essi
on
6
25
50 * *
**
*
*
dyrk1a+/+ sham n = 3dyrk1a+/- sham n = 3 dyrk1a+/+ TAC n = 5 dyrk1a+/- TAC n = 5
*
*
b
#
##
**
#
dyrk
1a+/
+
MH
C-C
nAdy
rk1a
+/+
dyrk
1a+/
-
MH
C-C
nAdy
rk1a
+/-
dyrk
1a+/
+M
HC
-CnA
dyrk
1a+/
+dy
rk1a
+/-
MH
C-C
nAdy
rk1a
+/-
50μm
5mm
50μm
0
5
10
20
30nppa nppb myh7 acta-1
dyrk1a+/+ n = 3MHC-CnA/dyrk1a+/+ n = 3dyrk1a+/- n = 5
rela
tive
mR
NA
expr
essi
on
15
rcan1-4
0
4
8
16
12
20
25
**
*
* **
*
*
f
#
##
#
*
*
MHC-CnA/dyrk1a+/- n = 5
n = 3 3 5 5
n = 5
n = 5
Supplemental Figure 4
© 2010 Macmillan Publishers Limited. All rights reserved.
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 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.
Supplemental Figure 5
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
2
3
4
5
sham TACp
Dyr
k1a
(fold
co
ntro
l)
0
1
2
3
sham TACp 0
5
10
25
30nppa nppb myh7 acta-1
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)
0
1
2
3
4
5
sham TACr
6
7
Dyr
k1a
(fold
co
ntro
l)
0
1
sham TACr
0.5
0
5
10
25
30nppa nppb myh7 acta-1
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
1
2
3
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
© 2010 Macmillan Publishers Limited. All rights reserved.
s u p p l e m e n ta ry i n f o r m at i o n
6 www.nature.com/naturecellbiology
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
Supplemental Figure 6
U6
miR-199b
U6
miR-199b
U6 U6
miR-199b miR-199b
miR-199b
U6
8529
39
91
37
© 2010 Macmillan Publishers Limited. All rights reserved.
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
© 2010 Macmillan Publishers Limited. All rights reserved.
No MicroRNA Signal (wild type) Signal (MHC-CnA) Fold change (log2)
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
© 2010 Macmillan Publishers Limited. All rights reserved.
No MicroRNA Signal (wild type) Signal (MHC-CnA) Fold change (log2)
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
© 2010 Macmillan Publishers Limited. All rights reserved.
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.
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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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