Online Supplemental Data
Animal preparations
Aged and young male New Zealand White rabbits (54 vs 6 months; n=24, 24
respectively) were used for the studies. Prior to the studies, rabbits underwent
echocardiographic examination under sedation with ketamine hydrochloride (38-45
mg/kg IM). The echo exam was usually lasts 10-15 minutes in duration, after which the
rabbit was closely monitored until recovery from ketamine sedation. Left ventricular (LV)
and left atrial (LA) dimensions of rabbit hearts were assessed using 2-D
echocardiography as described previously.1,
2
Specifically, LV & LA end-diastolic
dimension (LVEDD & LAEDD), LV & LA end-systolic dimension (LVESD & LAESD) and
fractional shortening were analyzed.1, 2
Rabbits were euthanized by intravenous injection (I.V.) of high dose pentobarbital
sodium 50mg/kg. Body weights, heart weights, and lung weights were obtained. LA
tissues were dissected from 6 young and 8 aged rabbit hearts. Part of the LA from each
heart was fixed in 10% formalin solution for immunohistological studies, and the
remaining LA tissue was flash-frozen in liquid nitrogen for biochemical studies. From
immunoblotting results we found that aged LA tissue exhibited markedly increased
levels of aging markers3 p15INK and p19ARF (50% and 215% respectively vs young
controls, p<0.05; Online Suppl_Figures 1A, 1B). Three young rabbits were used for
isolation of LA myocytes as previously described.1, 2 Freshly isolated LA myocytes were
then flash frozen for immunoblotting studies. Nine young and 10 aged rabbit hearts
were perfused via cannulated aorta with cold cardioplegic solution (in mmol/L, NaCl
1
110, KCl 16, MgCl2 16, CaCl2 1.2, and NaHCO3 10) followed by Tyrode’s buffer
perfusion for optical mapping studies.
Atrial arrhythmia induction
To activate JNK in vivo, young rabbits (n=4) were injected with anisomycin (a JNK
activator) at a dose of 15 mg/kg (I.V.) for a total of four injections over nine days.
Following the treatment, these rabbits were subjected to an in vivo atrial arrhythmia
induction procedure. Rabbits were anesthetized (2-4% isoflurane in 100% oxygen,
inhalation) and maintained in a surgical plane of anesthesia for an open-chest AT/AF
induction procedure. The same in vivo AF induction procedure was also performed in
aged and young rabbit without anisomycin challenge. A bipolar pacing electrode was
placed on the right atrium (RA). Four unipolar electrodes were sutured at four sites
(2mm apart) on the LA posterior free wall for electrical signal recording through a multichannel data acquisition system based on the method by Berul et al4 with modification.
First, atrial effective refractory period (AERP) was measured by giving a train of 10
basic stimuli (S1, 2x diastolic threshold, 2ms pulse duration) at a cycle length (CL) of
200ms, followed by a premature stimulus (S2). The S2 was delivered at 2x diastolic
threshold in 10ms decrements and followed in 5ms decrements until a response was
not evoked. The AERP was defined as the longest S1–S2 interval which failed to
produce a propagated response, and the procedure was repeated twice for the AERP
measurement. The RA was paced using a bipolar electrode and LA electrical signals
were recorded from four unipolar electrodes located on the LA. Atrial arrhythmias
(AT/AF) were induced using burst pacing starting at a pacing CL of 100 msec and then
reduced to 50 msec in 10 msec decrements for 2 sec duration at 3x diastolic threshold
2
(TH). The same pacing protocol was then repeated at 6x TH strength. Finally, 30 sec
rapid pacing (CL = ± 5msec of AERP) at 6x TH was attempted. The induced rhythm was
defined as AT/AF when the atrial bipolar electrogram showed fast (>8kz) and regular or
irregular cycles that lasted for at least 1sec. If the arrhythmia lasted for more than 30sec,
it was terminated by electrical shock. The duration of AT/AF was analyzed as the mean
value of all episodes in each rabbit. After the AT/AF induction procedure was completed,
tissue was dissected and flash-frozen for immunoblotting assay.
Optical mapping
Excised rabbit hearts were cannulated through the aorta and perfused with Tyrode’s
solution (in mmol/L, NaCl 128.5, Glucose 10, KCl 4.7, MgCl2 0.7, NaH2PO4 0.5, CaCl2
1.5, and NaHCO3 14) at 37ºC. The perfusion pressure was maintained at 40~50 mmHg
via adjusted flow rates. The LA posterior wall was positioned over a transparent imaging
window. A bipolar pacing electrode was located at the top edge of the LA. An
electromechanical uncoupling agent, 2, 3-butanedione monoxime (BDM) (15-20
mmol/L, Sigma), was used to suppress the heart’s contractions followed by staining with
a voltage sensitive dye RH-237 (Invitrogen). Excitation light from a 200-W Hg/Xe lamp
(Opti Quip, NY) was filtered (550 ± 32 nm), deflected by a dichroic mirror with
transmission at >595 nm, and focused on the LA by a photographic lens (Nikon, Japan).
The emitted fluorescence was collected by the same lens and filtered at >650 nm.
Fluorescence changes were measured using a 16x16 photodiode array (C4675-102,
Hamamatsu Corp., Japan) at a sampling rate of 1 kHz/channel as previously described.
14
Diastolic pacing threshold was measured as the smallest current that could result in
3
1:1 capture of the heart. Optical signals were recorded during pacing at various cycle
lengths (CL = 250, 200, 150, 100 msec) at 2x diastolic threshold. Atrial effective
refractory period (AERP) was determined as described above. Arrhythmias were
induced by burst pacing (50Hz for 2sec at 3x, and 6x diastolic threshold) on the LA
posterior wall. The induced rhythm was defined as AT/AF when the atrial bipolar
electrogram showed a fast (>8Hz) rate along with a uniform or nonuniform morphology
that lasted for at least 1sec. If an arrhythmia lasted for more than 30sec, it was
terminated by electrical shocks delivered via two mesh electrodes attached to opposite
walls on the perfusion chamber. If no AT/AF was induced or if the pacing-induced
AT/AF was self-terminated, up to 3 consecutive burst pacing trials in each heart were
attempted to induce sustained AT/AF.
Optical mapping data analysis
Optical fluorescence signals were analyzed using custom programs. The action
potential (AP) amplitude was defined as the difference between the maximal signal at
the peak of upstroke and the baseline fluorescence signal. Activation time was assigned
as the time when upstroke rose to 50% of AP amplitude. Action potential duration (APD)
was defined as the time interval from activation time to the 60% level of repolarization
(APD60). Upstroke slope was calculated as the maximal dV/dt of AP rise.5, 6 Conduction
velocities (CV) at pacing CLs of 250, 200, 150, and 100msec were calculated via vector
field analysis based on methods described by others6, 7 with modification. A CV vector
was calculated from activation times measured at 5 x 5 neighboring pixels. Pixels with
local CV larger than 100 cm/sec were excluded from the analyses. Conduction paths
4
were assigned according to the group of vectors spreading from the central stimulation
site for every 10-degrees (0-360◦). The pathway containing the largest population of
vectors was considered as the major CV pathway. For each animal, 3 beats were
analyzed at each CL. The average value of the major-pathway CV from the three beats
of each LA was used for statistical analysis.
To analyze the heterogeneity of optically mapped CV, maximal local activation
time differences were calculated as previously described.8-10 Activation time differences
between the center site and each of its 8 immediate neighbor pixels were adjusted by
dividing the linear distance between the two sites. The maximum adjusted activation
time delay was assigned as the local activation time differences. The absolute degree of
heterogeneity was defined as the width of the distribution (P5-P95; 5th and 95th
percentiles of the activation time differences). The heterogeneity index was calculated
as the absolute degree of heterogeneity divided by the median activation time difference
(P5-95/P50).
Western blot analysis
Western blotting was performed as previously described.1,
2
Specific antibodies were
used to assess p15INK (Abcam), p19ARF (Abcam), Cx43 (Zymed), Cx40 (Zymed), Ncadherin (Zymed), GAPDH (Chemicon), SCN5A (Sigma), JNK type1 and JNK type 2
isoforms (JNK1, JNK2; Cell Signaling), phosphor-JNK (JNK-P, Cell Signaling),
phosphor-mitogen-activated protein kinase kinases type 7 (MKK7-P) and type 4 (MKK4P) isoforms (Cell Signaling). Immunoblotting images were obtained and quantitatively
analyzed using a gel imaging system and Quantity-One software (Bio-Rad).
5
Masson’s Trichrome staining
The collagen deposition of LA tissues was stained using Masson’s Trichrome staining
protocol.11 Paraffin embedded tissue sections were treated with the following steps: 1)
de-waxed with xylene, and then hydrated in distilled water; 2) mordanted in Bouin’s
solution for 1 hour at 56ºC; 3) incubated with Weigert’s hematoxylin for 10 minutes; 4)
immersed Biebrich scarlet-acid fuchsin for 2 min; 5) incubated in phosphomolybdicphosphotungstic acid solution for 10-15 min; 6) then immersed in aniline blue solution
for 5 min; and 7) placed in 1% acetic acid solution for 3-5 min and then dehydrated with
95% absolute alcohol, cleared with two or three changes of xylene and mounted with
synthetic resin. After each step, slides were rinsed with distilled water. Following the
staining procedure the blue-stained collagen fibers were quantified as follows. For each
Masson’s Trichrome stained tissue section, 15 to 20 images were taken from a Zeiss
light microscope using a 40x objective oil-immersion lens. A MATLAB-based custom
program was used to quantify the number of pixels that exhibited blue staining specific
for collagen deposition.
Cultured HL-1 atrial cell line
We further employed a well-characterized cultured atrial cell line (HL-1, obtained from
Dr. William Claycomb, Louisiana State University, USA), which expresses cardiac
genes and proteins, including cardiac ion channels (i.e. L-type calcium channels)12 and
mature isoforms of sarcomeric contractile proteins13,
14
normally found in adult atrial
myocytes. The HL-1 atrial cells were cultured in a specially formulated growth medium
6
(Claycomb medium, Sigma) supplemented with 0.1mM norepinephrine, 2mM Lglutamine (Invitrogen) and 10% FBS (Sigma) as previously described. 13, 14 We found
that expression levels of Cx40, Cx43, Cx45, and SCN5A proteins in cultured HL-1 cells
were comparable to that in isolated young rabbit LA myocytes (Online Suppl_Figure
2A). Confocal immunostaining showed similar distribution patterns of Cx43 (green) and
Cx40 (red) between cultured HL-1 atrial cells and rabbit atrial tissue (Online
Suppl_Figure 2B). These results suggest that the content of expressed connexins in
HL-1 atrial cells is similar to that in rabbit atrial myocytes.
On the third day of cell culture, cells were treated with anisomycin (a JNK
activator)15-17 for 16 or 24hr. To confirm that specificity of anisomycin-induced Cx43
reduction was JNK-dependent, we pre-treated HL-1 atrial cells with SP600125
(2µmol/L, Sigma; a specific JNK inhibitor)18 for 8hr prior to anisomycin treatment, and
then continuously incubated cells with SP600125 along with anisomycin for 16hr. Cells
treated with 0.02% DMSO served as sham-controls. When HL-1 cells became confluent
on the fourth-fifth day (without spontaneous rhythm), the atrial myocyte monolayers
were washed either with ice cold PBS for biochemical studies or with 37°C Hank’s
solution (Sigma) for optical mapping studies. For biochemical studies, cells were lysed
in a RIPA buffer containing protease cocktails (Sigma) and followed by immunoblotting
assays. Comparable results of anisomycin-induced Cx43 suppression were found in
both anisomycin-treated freshly isolated young rabbit LA myocytes and cultured HL-1
atrial cells. Online Suppl_Figure 3 shows a 47% reduction in Cx43 in anisomycintreated (24hr) isolated young rabbit LA myocytes vs sham-controls (n=3,3). To rescue
JNK-induced Cx43 suppression, anisomycin pre-treated (24hr) HL-1 atrial cells were
7
infected with Adcx43WT for one hour in a serum-reduced culture medium, then cultured
in adenovirus-free medium for up to 24hr as described previously.19 Cultured HL-1 atrial
cells infected with AdLacZ only were used as a negative control, while cells pre-treated
with anisomycin for 24hr followed by AdCx43DN infection (an inactive form of dominant
negative Cx43) served as a positive control.
For optical mapping studies, cells were loaded with 20 mmol/L RH273 voltage
sensitive dye (Invitrogen) for 5 min at 37°C, then washed 3 times with Hank’s solution
for subsequent biochemical and optical mapping measurements as described.20
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Paul M, Bohm M. Contractile systolic and diastolic dysfunction in renin-induced
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Functional expression of l- and t-type ca2+ channels in murine hl-1 cells. J Mol
Cell Cardiol. 2004;36:111-119
White SM, Constantin PE, Claycomb WC. Cardiac physiology at the cellular
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structure and function. Am J Physiol Heart Circ Physiol. 2004;286:H823-829
Claycomb WC, Lanson NA, Jr., Stallworth BS, Egeland DB, Delcarpio JB,
Bahinski A, Izzo NJ, Jr. Hl-1 cells: A cardiac muscle cell line that contracts and
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9
Figure legends
Online Suppl_Figure 1.
Enhanced aging markers in aged rabbit LA. A. Immunoblotting images of aging
markers p15INK, p19RAF in young and aged rabbit LA. B. Summarized data normalized to
GAPDH (*p<0.05 vs young; n=6,8).
Online Suppl_Figure 2.
A. Immunoblots of Cxs and SCN5A proteins from HL-1 atrial myocytes and freshly
isolated rabbit LA myocytes (n=3, 3). B. Confocal images of triple immunostaining with
Cx43 (green), Cx40 (red), N-cadherin (N-cad; blue) antibodies in HL-1 myocytes.
Arrows indicate punctate patterns of Cx43 and Cx43 with N-cadherin proteins located at
the intercalated disc of the membrane between two myocytes.
Online Suppl_Figure 3.
Double immunostaining images with Cx43 (red) and Cx40 (green) antibodies and
overlapped Cx43 and Cx40 (yellow) in young and aged rabbit LA.
Online Suppl_Figure 4
A. Immunoblotting images show reduced Cx43 and activated JNK in anisomycin-treated
(24hr) isolated young rabbit LA myocytes, while Cx40 and SCN5A remain unchanged
(n=3, 3).
Online Suppl_Figure 5
Representative fluorescent signals of optically mapped action potentials from young (A)
and aged (B) rabbit LA.
10
Online Suppl_Figure 6
Summarized immunoblotting data (A) and conduction velocity (B, Fig.5F in the main
text) demonstrate JNK-induced Cx43 protein suppression and slow conduction in 16hr
and 24hr anisomycin pre-treated monolayers were rescued by JNK inhibition using a
specific JNK inhibitor SP600125. Although the recovery of the Cx43 protein in JNK
inhibited monolayers with 24hr anisomycin pre-treatment was 76% compared to sham
controls (**P < 0.01 or ***p <0.001 as indicated; n = 3, 4, 3, 3, 3; A), the increased Cx43
protein was adequate to improve conduction up to 82% (B) of that in sham-controls (**P
< 0.01 or ***p <0.001 as indicated; n = 9, 12, 7). A_16h = 16hr anisomycin treatment;
A_24h = 24hr anisomycin treatment; A_16h+SP610025 = SP610025 JNK inhibition
along with 16hr anisomycin treatment; A_24h+SP610025 = SP610025 JNK inhibition
along with 24hr anisomycin treatment.
Online Suppl_Movie 1
An example of a uniformly propagated AP in a sham-control HL-1 monolayer (the same
monolayer as used in Fig.5I).
Online Suppl_Movie 2
An example of a discontinuous AP wave in anisomycin-treated (24hr) monolayers (the
same monolayer as used in Fig.5J).
Online Suppl_Movie 3
An example of a uniformly propagated AP in an anisomycin pre-treated monolayer with
subsequent overexpressed exogenous wild-type Cx43 (AdCx43WT infection). It is the
same monolayer as used in Fig.5K.
11
Online Suppl_Movie 4
An example of a reentrant AP propagation in an anisomycin pre-treated monolayer with
subsequent overexpressed exogenous dominant-negative Cx43 (AdCx43DN infection).
It is the same monolayer as used in Fig.5L.
12
Table.1 Rabbit information
Young Male Aged Male
n=24
n=24
Age (month)
6.10±0.09
53.69±8.81
HW/BW(g/kg)
2.76±0.52
2.73±0.66
LW/BW(g/kg)
3.46±0.94
3.27±1.06
LVEDD
1.68±0.16
1.74±0.16
LVESD
0.96±0.21
0.98±0.22
LVSF
43.27±7.35
43.86±9.45
LAED Area
1.11±0.47
0.93±0.26
LAED Parameter
3.54±0.34
3.67±0.32
LAED Diameter
1.05±0.08
0.99±0.16
LAES Area
0.52±0.19
0.43±0.13
LAES Parameter
2.48±0.32
2.49±0.42
LAES Diameter
0.69±0.0
0.67±0.01
LAFS
54.79±8.77
53.29±10.29
HW=heart weight; LW=lung weight; BW=body weight;
LVEDD=left ventricular end diastolic dimension;
LVESD=left ventricular end systolic dimension;
LAED=left atria end diastolic; LAES=left atria end systolic;
LAFS=left atria fraction shortening; All p=NS;
13