Kim et al.
LKB1 in Atrial Fibrillation
LKB1 in Atrial Fibrillation
Kim et al
SUPPLEMENTAL MATERIALS
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Kim et al.
LKB1 in Atrial Fibrillation
Supplemental Table
Supplemental Table 1. List of genes and primers used in the transcript study experiments.
Gene
# Symbol
1
Atp1a2
2
Atp2a2
Gene Name, Synonyms
ATPase, NA+/+ transporting,
alpha 2
SERCA, variant 1 and 2
NCBI Ref
Seq
Forward primer sequence
Reverse primer sequence
NM_178405.3 GACCTATTTAAAGCTACCCTGT
GAGCTCCTTCTCTTTCTGTTT
NM_0011101
TACTGACCCTGTCCCTGACC
40
CACCACCACTCCCATAGCTT
NM_009781.3 AATGACACGATCTTCACCAA
GACATAGTCTGCATTGCCTA
NM_021415.4 CCATCCTCGTCAATACTCTG
AAAGATGTTGTAGGGGTTCC
NM_0011110
ACCACAGACTTCCCCAACTG
59
CGGATTCCAGAGCATTTGAT
NM_007742
CCTTCTGGATCAAGTGGTGAA
CACCACGATCGCCATTCT
NM_010217
AGCAGCTGGGAGAACTGTGT
GCTGCTTTGGAAGGACTCAC
10 Fn1
NM_010104
CGGAGCTGAGAATGGAGTGC
NM_0011463
GTACCCACAACAGGTCTCGC
50
NM_010233.1 ATGAGCGCCCTAAAGATTCC
CAGTCCATACGGTACGACGC
endoglin (Eng), transcript
variants 1&2
fibronectin 1, Fn, Fn-1
11 Gja1
gap junction protein, alpha 1,
connexin 43, Cx43
NM_010288.3 TACAGCGCAGAGCAAAATCG
GCTGTCGTCAGGGAAATCAAAC
12 Gja5
gap junction protein, alpha 5,
connexin 40, Cx40, Gja-5
NM_008121.2 CACCCACCGTTCCTCTAAAA
AGAAGAACCCGAGAAGCACA
13 Gjc1
gap junction protein, gamma 1,
NM_008122
connexin 45, Cx45
14 Itpr2
inositol 1,4,5-triphosphate
receptor 2, transcript variant 1
NM_019923.4 TACAGCAACGTTATCCAACT
GGCATCAGAACGACTTTATC
15 Kcna4
Kv1.4
NM_021275.4 CTCTGCAATACCCCCTAGCC
ACTTCACCATTCCCAGCAAGT
16 Kcna5
Kv1.5
NM_145983
17 Kcnb1
Kv2.1
NM_008420.4 GCTCCCTACCACAGAGGGTA
CACAGACTTGTCGTGGCTCT
18 Kcnd2
Kv4.2
NM_019697.3 TCGTGTCGAACTTCAGTCGG
TCAGTAGCCCATTCCGCTTG
19 Kcnd3
Kv4.3
NM_0010393 CACTGCTTAGAAAAGACCACTA
GTCTTCTTGCTACGACGGGA
47.1
ACC
20 Kcnh2
Kv11.1
Kir6.2, potassium inwardly
rectifying channel, subfamily J,
member 11 ; ATP-sensitive
inward rectifier potassium
channel 11
Kir2.4
Mus musculus potassium
inwardly-rectifying channel,
subfamily J, member 2 (Kcnj2),
Kir2.1 (Version 3)
Kir3.4
3
Cacna1c
4
Cacna1h
5
Cd34
6
Col1a1
7
Ctgf
Mus musculus calcium
channel, voltage-dependent, L
type, alpha 1C subunit
(Cacna1c), transcript variant 1;
Cav1.2
calcium channel, voltagedependent, T type, alpha 1H;
Cav3.2
CD34 antigen, transcript
variant 1
collagen, type I, alpha 1 ;
Cola1; Mov13; Cola-1; Mov-13;
Col1a-1
Ccn2; Hcs24; Fisp12; fisp-12
Edn1
endothelin 1
8
9
Eng
21 Kcnj11
22 Kcnj14
23 Kcnj2
24 Kcnj5
GAAACGGAAGAGGACCATGA
ACACTCTCTGACTCCGTCCC
AGTGCAATGGGATTTCCGGG
CCTTTTCCATTTCCCAGACA
AAAGCCCACCTCAAACACAG
AGGCAGCAGAAAAACCTCCT
NM_013569.2 CCGGGTCGACAGACAGGTG
GGATTCCCGCTCTGCTTAGTG
NM_010602.2 GTAGGGGACCTCCGAAAGAG
CAGGAAGATGCCGTTACCAC
NM_145963.2 CCCGAGACCCCTTTATGAGC
TTTCTCCACACACGGTCCAG
NM_010605
GCAAGCAGTGTCTTGGGAAT
TGCTGGTACAGGATCATGGG
CTCTCTGCGCTACAAGGGAAG
GTAGCCATCTTGGAGAGGCG
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LKB1 in Atrial Fibrillation
25 Kcnj8
Kir6.1
26 Kcnq1
KvLQT1; Kv7.1
ribosomal protein L32, rpL323A
Ryanodine receptor 2, cardiac
27 Rpl32
28 Ryr2
29 Scn5a
30 Stk11
sodium channel, voltagegated, type V, alpha, mH1,
Nav1.5, Nav1.5c, SkM1
serine/threonine kinase 11,
Lkb1, Par-4
NM_0012704
GTGCACTATGGATCGCACCT
22.1
NM_008434.2 AGTCTTCATTCACCGCCAGG
CGTCCTCCTAGAAGACTCGG
ATCTGCGTAGCTGCCAAACT
NM_172086.2 GCAAGTTCCTGGTCCACAAT
GACGGCAGGTTTTGTGATTT
NM_023868.2 AACTGATGATGAGGTGGTTC
TGAACTTCCACTTTTCCACA
NM_021544.4 CTGAAGACAATCGTGGGAGCC TCCGTGGTGCCATTCTTGAG
NM_011492.3 GAGAGGCCAACGTCAAGAAG
GTAGGTATTCCAGGCCGTCA
Supplemental Table Legends
Supplemental Table 1. List of genes and primers used in the transcript study experiments.
The list of genes and their primers used in the RT-qPCR experiments of this study are shown.
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Supplemental Figures
Supplemental Figure 1. Cardiac LKB1 expression and AMPK activation are decreased in
MHC-Cre LKB1fl/fl mice. LKB1 expression was measured at (A) transcript and (B) protein
level. Transcript levels were analyzed according to the ΔΔCT method, relative to the
housekeeping gene Rpl32, and then normalized to that of the age-matched control LKB1fl/fl atrial
group. *p<0.05, **p<0.01 vs. age-matched control group, n=5-6, two-tailed Student’s T-test. (C)
Representative immunoblots of the LKB1-downstream pathways, examining phosphorylated (p)
and total (t) levels of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase
(ACC).
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LKB1 in Atrial Fibrillation
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Supplemental Figure 2. No evidence of structural changes at embryonic day (ED) 15.5.
(A) Representative images of Picrosirius red stained ED15.5 heart left atrial (LA) or ventricular
(LV) regions, imaged under brightfield, are shown. Scale bars represent 100 μm. (B) Collagen
fibers were quantitated from micrographs viewed under circular polarized filter (not shown), and
shown as averaged values calculated from at least two views/animal (n=3-5 animals per group).
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Supplemental Figure 3. Apoptosis is selectively increased in 1-day-old ventricles of LKB1deleted hearts. Whole-heart sections of 1-day-old MHC-Cre LKB1fl/fl and littermate LKB1fl/fl
control mice were stained with TUNEL and DAPI. (A) Representative images of left atria and
left ventricles (upper and lower panels, respectively) at day 1 are shown. (B) The number of
TUNEL-positive cells, expressed per nuclei, is graphed. *p<0.01 vs. respective control group,
n=4-7, two-tailed Student’s T-test. (C) Representative images of 2-week-old atria and ventricles
(upper and lower panels, respectively) stained with TUNEL and DAPI. In all images, scale bars
represent 50 μm.
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Supplemental Figure 4. Cx40 and Cx43 are downregulated in LKB1-deficient hearts at
ED15.5. (A) Representative immunofluorescence images of ED15.5 atrial sections, stained for
Cx40 (above) or Cx43 (below), both labeled with green fluorescence.
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Supplemental Figure 5. Changes in protein expression correlate those at the transcript
level. (A) Representative immunoblots of tissue homogenates comparing protein expression
levels at week 2 for selected ion channels, including Cx40, Cx43, Cx45, Nav1.5, SERCA2,
phospho-S15/S16 phospholamban (PLN), total PLN and Na+/K+ ATPase. Averaged
densitometry values are shown as bar graphs to the right. **p<0.01, ***p<0.001 vs. control
group, n=3-6, two-tailed Student’s T-test.
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Supplemental Figure 6. Nav1.5 inactivation and myocyte capacitance are comparable
between LKB1fl/fl control and MHC-Cre LKB1fl/fl myocytes. (A) I/V plot of the inward
current measured at baseline and following 1 minute incubation with 10 μM TTX (*p<0.01, 2way ANOVA with Bonferroni’s multiple comparison test, n=3 cells). (B) A superimposed,
representative traces before and after addition of TTX, at -15mV. (C) Myocyte capacitance of
isolated atrial myocytes from MHC-Cre LKB1fl/fl LKB1fl/fl was not statistically different between
the two groups (n=14-17 cells from 6-8 animals). (D) Inactivation kinetics of inward sodium
current at -25mV, with current trace normalized to peak amplitude. Only cells with a voltagedependent INa were included in this analysis. Inset shows averaged tau values from exponential
decay fit curves (n=6-15 cells from 5-10 animals per group).
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Supplemental Figure 7. Changes in level of transcripts encoding calcium handling and
potassium channels. RT-qPCR experiments were performed to compare the transcript levels of
potassium and calcium channels, as described for Figure 3. (A) Calcium-handling and potassium
channels, as well as (B) potassium channels low in transcript level expression, compared to the
housekeeping gene Rpl32, or of unknown function in cardiomyocyte action potential are
included in this supplemental figure. *p<0.05, **p<0.01, ***p<0.001 vs. age-matched control
group, n=6, two-tailed Student’s T-test.
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Supplemental Figure 8. LKB1 deletion leads to premature death. (A) Survival of MHC-Cre
LKB1fl/fl and control mouse cohorts (n=18 per group) monitored from birth to 42 weeks of age.
*p<0.001; n=18 per group.
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Supplemental Figure 9. Prolonged AP duration and delayed inter-atrial conduction in
LKB1-deleted right atrium. (A) Action potential duration (APD) at 50% repolarization is
plotted against the pacing cycle length (PCL), from the same experiments as shown in Figure 5.
**p=0.001 against control, n=4-6, 2-way ANOVA with Sidak multiple comparison test. (B) A
typical bi-atrial preparation used for detailed inter-atrial optical AP mapping studies is shown.
Right and left atrial imaging were performed sequentially during RA pacing in all preparations.
Size and location of RA mapping field is indicated by the black square. Also shown in the
anatomical CCD image of the mapped RA field. APs were recorded simultaneously from
proximal [1] and distal [2] sites to the pacing electrode in preparations from LKB1fl/fl control (C)
and MHC-Cre LKB1fl/fl (D) hearts. In control preparations, rapid AP conduction was always
observed between proximal and distal sites during both slow (140 ms PCL, left) and fast (70 ms
PCL, right) pacing (C). In contrast, delayed AP conduction and a 2:1 pattern of conduction block
were observed in MHC-Cre LKB1fl/fl preparations during pacing at 140 and 70 ms PCLs,
respectively (D).
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Supplemental Figure 10. Normal electrical activity of the AMPK kinase-dead (KD) hearts.
(A) Representative surface ECG lead-II traces of WT and KD mice at week 2. All animals were
in sinus rhythm. (B) Graphs plotting average P wave duration, PR interval, QRS duration, and
heart rate (HR) in KD versus wild-type (WT) mice (n=10 per group). (C) Transcript levels of
channels and gap junction proteins in the atria and ventricles of AMPK KD mice at week 2.
*p<0.05, vs. control group, n=5-7, two-tailed Student’s T-test. (D) Level of acetyl-CoA
carboxylase (ACC) Ser79 phosphorylation in the KD vs. MHC-Cre LKB1fl/fl mice at week 2.
Values are expressed as normalized to LKB1fl/fl control. *, Δ p<0.001 vs. LKB1fl/fl or WT control
group, or #p<0.001 against MHC-Cre LKB1fl/fl, n=5-7, one-way ANOVA.
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Supplemental Figure 11. LKB1 deletion but not AMPK α2 inactivation leads to
cardiomyocyte hypertrophy. (A) Average diameters of atrial and ventricular myocytes.
*p<0.05, **p<0.01, vs. respective control, n=3 animals, with 5 images/animal, one-way
ANOVA.
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