Download Distinct Cellular and Molecular Mechanisms Underlie Functional

Distinct Cellular and Molecular Mechanisms Underlie
Functional Remodeling of Repolarizing Kⴙ Currents With
Left Ventricular Hypertrophy
Céline Marionneau,* Sylvain Brunet,* Thomas P. Flagg, Thomas K. Pilgram,
Sophie Demolombe, Jeanne M. Nerbonne
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Abstract—Left ventricular hypertrophy (LVH) is associated with electric remodeling and increased arrhythmia risk,
although the underlying mechanisms are poorly understood. In the experiments here, functional voltage-gated (Kv) and
inwardly rectifying (Kir) K⫹ channel remodeling was examined in a mouse model of pressure overload–induced LVH,
produced by transverse aortic constriction (TAC). Action potential durations (APDs) at 90% repolarization in TAC LV
myocytes and QTc intervals in TAC mice were prolonged. Mean whole-cell membrane capacitance (Cm) was higher, and
Ito,f, IK,slow, Iss, and IK1 densities were lower in TAC, than in sham, LV myocytes. Although the primary determinant of
the reduced current densities is the increase in Cm, IK,slow amplitudes were decreased and Iss amplitudes were increased
in TAC LV cells. Further experiments revealed regional differences in the effects of LVH. Cellular hypertrophy and
increased Iss amplitudes were more pronounced in TAC endocardial LV cells, whereas IK,slow amplitudes were selectively
reduced in TAC epicardial LV cells. Consistent with the similarities in Ito,f and IK1 amplitudes, Kv4.2, Kv4.3, and
KChIP2 (Ito,f), as well as Kir2.1 and Kir2.2 (IK1), transcript and protein expression levels were similar in TAC and sham
LV. Unexpectedly, expression of IK,slow channel subunits Kv1.5 and Kv2.1 was increased in TAC LV. Biochemical
experiments also demonstrated that, although total protein was unaltered, cell surface expression of TASK1 was
increased in TAC LV. Functional changes in repolarizing K⫹ currents with LVH, therefore, result from distinct cellular
(cardiomyocyte enlargement) and molecular (alterations in the numbers of functional channels) mechanisms. (Circ Res.
2008;102:0-0.)
Key Words: hypertrophy 䡲 arrhythmia 䡲 heart failure
L
eft ventricular hypertrophy (LVH) is an adaptive response of the myocardium to an increase in load.1 LVH
is seen in various disease states including hypertension and
myocardial infarction, as well as in valvular and congenital
heart diseases.1 LVH is also observed in physiological states
following rigorous, prolonged exercise.2 Although physiological LVH does not confer increased morbidity and mortality,
pathological LVH is consistently associated with prolongation of ventricular action potentials and alterations in the
dispersion of repolarization, both of which result in electric
instability and increase the propensity to develop lifethreatening arrhythmias.3 Several lines of evidence suggest
that these electric changes reflect, at least in part, alterations
in the functioning of the K⫹ channels that underlie ventricular
action potential repolarization.3,4
Various experimental models of LVH,5–7 including pressure overload–induced LVH,8,9 have been developed to explore the mechanisms underlying K⫹ current remodeling.
Although several studies have examined regional differences
in remodeling,8 –11 few have probed the underlying molecular
and cellular mechanisms. In the studies here, a mouse model
of pressure overload–induced LVH, produced by transverse
aortic constriction (TAC), was exploited to quantify the
effects of LVH on repolarizing K⫹ currents in LV myocytes
and to delineate the mechanisms underlying K⫹ current
remodeling. These experiments revealed that APDs were
prolonged in TAC LV myocytes and that QTc intervals were
increased in TAC mice. Mean whole-cell membrane capacitance (Cm) was increased significantly, and the densities of
voltage-gated K⫹ (Kv) and inwardly rectifying K⫹ (Kir)
currents were reduced in TAC LV myocytes. Further electrophysiological, molecular, and biochemical analyses revealed marked regional differences in the effects of LVH
and that distinct cellular and molecular mechanisms contribute to the functional remodeling of repolarizing Kv and
Kir currents.
Original received August 13, 2007; resubmission received December 17, 2007; revised resubmission received April 17, 2008; accepted April 23, 2008.
From the Departments of Molecular Biology and Pharmacology (C.M., S.B., J.M.N.), Cell Biology and Physiology (T.P.F.), and Radiology (T.K.P.),
Washington University Medical School, St Louis, Mo; and INSERM U533, L’Institut du Thorax and Université de Nantes (S.D.), Faculté de Médecine,
France. Present address for S.B.: Department of Pharmacology, University of Washington, Seattle.
*Both authors contributed equally to this work.
Correspondence to Jeanne Nerbonne, Washington University School of Medicine, Molecular Biology and Pharmacology, 660 Euclid Ave, Box 8103,
St Louis, MO 63110. E-mail [email protected]
© 2008 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.107.170050
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June 6, 2008
Figure 1. Detection of LVH 7 days after TAC. A,
Representative echocardiographic M-mode images
of sham and TAC LV. Scale bars are 1 mm and
100 ms. AW and PW designate anterior and posterior wall thicknesses, respectively. B, LVM/BW in
sham (n⫽18) and TAC (n⫽39) mice; individual values and means⫾SEM are plotted. *P⬍0.001. C,
Mean⫾SEM mRNA expression levels (arbitrary
units) of the hypertrophy markers ANF and ␤-MHC
are significantly (*P⬍0.001; §P⬍0.05) higher in
TAC (n⫽6) than in sham (n⫽6) LV.
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Materials and Methods
Animals were handled in accordance with the Guide for the Care and
Use of Laboratory Animals (NIH). Detailed methods are provided in
the online data supplement at http://circres.ahajournals.org.
Induction of LVH
Pressure overload–induced LVH was produced in adult mice by
TAC.12 Sham-operated animals underwent surgery but without aortic
constriction. Seven days after surgery, echocardiographic images
were acquired and analyzed using described methods.12,13
Electrophysiological Recordings
Electrocardiographic (ECG) recordings were obtained from anesthetized mice.14 Myocytes were isolated from the LV (and RV), as well
as from the epicardial (EPI) and endocardial (ENDO) LV surfaces,
of sham and TAC hearts by enzymatic dissociation and mechanical
dispersion using described methods.15 Whole-cell membrane currents and action potentials were measured as previously described.15
Quantitative RT-PCR
Total RNA from RV, LV, EPI, and ENDO was isolated and DNase
treated using described methods.16 RNA concentrations were determined by optical density measurements. TaqMan low-density arrays
(Applied Biosystems) were used in a 2-step RT-PCR process as
described previously.16 The 96 genes selected for quantification
(supplemental Table I) encode 68 ion channel ␣, ␤, and regulatory
subunits; 11 Ca2⫹ homeostasis regulators; 6 transcription factors; 7
markers of vessels, neurons, fibroblasts, inflammation, and hypertrophy; and 4 controls.17 Data were analyzed using the threshold
cycle relative quantification method, with hypoxanthine guanine
phosphoribosyl transferase (HPRT) as the endogenous control.
Because hypertrophy is associated with a generalized increase in
transcript and protein content without an increase in cell numbers,18
transcript expression data determined in each sham and TAC (LV,
EPI and ENDO) sample were multiplied by the LV mass to body
weight ratio (LVM/BW) determined in the same animal.
The expression of genes encoding atrial natriuretic factor (ANF),
␤-myosin heavy chain (␤-MHC), and the 2-pore domain K⫹ (K2P)
channel subunits TASK1, TASK2, and TREK1 was determined by
SYBR green quantitative RT-PCR using sequence specific primers.
Expression data were normalized to HPRT and to the measured
LVM/BW.
Biochemical Analyses
Protein lysates were prepared from sham and TAC LV, EPI, and
ENDO using described methods.19 Biotinylation of isolated LV
myocytes was used to examine cell surface protein expression.
Protein quantification was performed with the BCA Protein Assay
Kit (Pierce). Proteins were loaded on SDS-PAGE gels in amounts
proportional to the relative LVM/BW in TAC and sham mice.
Statistics
Results are expressed as means⫾SEM. Statistical analyses were performed using the Student’s t test. Multiple regression analysis was used
to determine the significance of regional (EPI/ENDO) differences.
Results
Pressure Overload–Induced LVH
Preliminary echocardiographic experiments revealed that the
extent/severity of LVH did not vary appreciably in mice
examined 7 days to 3 months after TAC (not shown).
Experiments here, therefore, were completed 7 days following surgery. Reduced chamber volume and increased anterior
and posterior wall thicknesses were observed in echocardiographic 2D short axis cine loops of the LV in TAC mice
(Figure 1A). M-mode based LV mass measurements revealed
that mean⫾SEM LVM/BW was significantly (P⬍0.001)
higher in TAC (4.07⫾0.1 mg/g, n⫽39), than in sham
(2.8⫾0.06 mg/g, n⫽18), mice (Figure 1B and supplemental
Table II). In addition, the mean⫾SEM E/A wave ratio was
significantly (P⬍0.05) larger in TAC animals (supplemental
Table II), consistent with diastolic dysfunction.13 Fractional
shortening, however, was similar (supplemental Table II),
and there was no evidence of altered systolic function or heart
failure in TAC animals.
SYBR green quantitative RT-PCR revealed that expression
of the hypertrophy markers2 ANF (P⬍0.001) and ␤-MHC
(P⬍0.05) was increased significantly in TAC, compared with
sham, LV (Figure 1C), consistent with the presence of
pathological LVH.
Marionneau et al
Mechanisms Underlying Kⴙ Current Remodeling in LVH
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Table. Kv and Kir Currents in Sham and TAC LV, RV, EPI, and ENDO Myocytes
Cells
Cm
Ipeak
Ito,f
IK,slow
Iss
IK1
Sham
LV
132⫾3
␶d
84⫾3
1281⫾23
pA
6849⫾306
3533⫾219
2408⫾96
714⫾20
⫺1474⫾48
pA/pF
52.5⫾2.2
27.1⫾1.6
18.4⫾0.7
5.5⫾0.1
⫺11.2⫾0.2
n
RV
73
60
130⫾5
␶d
74⫾3
1155⫾28
pA
8909⫾607
5087⫾417
2702⫾157
690⫾33
⫺1618⫾75
pA/pF
68.3⫾3.8
38.9⫾2.8
20.7⫾0.9
5.3⫾0.1
⫺12.6⫾0.5
n
EPI
35
26
138⫾7
␶d
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87⫾5
1191⫾40
pA
8393⫾519
4105⫾308
3031⫾193
787⫾49
⫺1427⫾105
pA/pF
61.6⫾3.2
30.2⫾2.2
22.3⫾1.3
5.7⫾0.2
⫺10.4⫾0.5
n
ENDO
13
13
131⫾10
␶d
89⫾13
1434⫾51
pA
5131⫾469
2335⫾203
2010⫾214
686⫾62
⫺1579⫾110
pA/pF
39.8⫾2.3
18.3⫾1.3
15.3⫾0.9
5.3⫾0.3
⫺12.4⫾0.6
n
16
16
TAC
LV
172⫾5*
␶d
82⫾2
1553⫾39
pA
5744⫾314‡
3149⫾235
1747⫾83*
807⫾26†
pA/pF
34.1⫾1.8*
18.8⫾1.4*
10.4⫾0.4*
4.7⫾0.1*
n
RV
70
⫺1459⫾50
⫺8.6⫾0.3*
57
122⫾5
␶d
66.4⫾2
1286⫾39
pA
7123⫾570
4406⫾386
1858⫾150
662⫾38
⫺1398⫾155
pA/pF
58.4⫾4.1
36.1⫾2.7
15.2⫾1.1
5.4⫾0.2
⫺11.4⫾1
n
EPI
21
12
177⫾9†
␶d
56⫾2
1596⫾53
pA
5754⫾471*b
3684⫾354
1167⫾107*a
pA/pF
32.9⫾2.4*b
21.1⫾1.9†
6.7⫾0.6*a
n
ENDO
4.6⫾0.3†
23
⫺1310⫾88
⫺7.1⫾0.4*
21
215⫾11*a
␶d
70⫾3
1626⫾74
pA
5642⫾584
2719⫾369
1856⫾182
pA/pF
26.8⫾2.5*
13.2⫾1.7‡
8.7⫾0.8*
n
802⫾57
18
1039⫾73*a
4.8⫾0.2
⫺1720⫾99
⫺7.9⫾0.4*
19
All values are means⫾SEM. Cm, ␶ decay (␶d), amplitude, and density values are expressed in pF, ms, pA, and pA/pF, respectively.
Kv and Kir densities were determined at ⫹40 and ⫺120 mV (HP⫽⫺70 mV), respectively. Values were compared in TAC and sham
LV myocytes, in TAC and sham EPI myocytes, or in TAC and sham ENDO myocytes, and *P⬍0.001, †P⬍0.01, ‡P⬍0.05 values are
indicated. Impact of LVH is significantly (aP⬍0.05, bP⬍0.01) different in EPI and ENDO myocytes.
Kⴙ Current Remodeling in TAC LV Myocytes
Whole-cell recordings revealed that mean⫾SEM Cm was
significantly (P⬍0.001) higher in TAC, than in sham, LV
myocytes (Table). Consistent with the increased Cm, peak Kv
current (Figure 2A and 2C) and IK1 (Figure 2B and 2G)
densities were significantly (P⬍0.001) lower in TAC LV
cells (Table). The decay phases of the Kv currents in adult
wild-type mouse ventricular myocytes are best described by
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Figure 2. Alterations in repolarizing K⫹ currents in TAC LV myocytes. Representative whole-cell Kv currents (A), evoked during 4.5second voltage steps to potentials between ⫺40 and ⫹40 mV from a holding potential (HP) of ⫺70 mV, and Kir currents (B), evoked
during 350-ms voltage steps to potentials between ⫺40 and ⫺120 mV (HP⫽⫺70 mV) in sham and TAC LV cells. Mean⫾SEM Ipeak (C),
Ito,f (D), IK,slow (E), Iss (F), and IK1 (G) densities in sham and TAC LV myocytes are plotted as a function of test potential. Relative changes
in Cm (H), current amplitudes (I), and densities (J) in TAC compared with sham LV myocytes. Values in TAC and sham LV myocytes are
significantly (*P⬍0.001, #P⬍0.01) different.
the sum of 2 exponentials, reflecting the inactivating currents
Ito,f and IK,slow and a noninactivating current, Iss.15 Kinetic
analyses of the currents revealed that mean⫾SEM Ito,f (Figure
2D), IK,slow (Figure 2E), and Iss (Figure 2F) densities were
significantly (P⬍0.001) lower in TAC, than in sham, LV
myocytes (Table). Mean⫾SEM Ito,f and IK1 amplitudes in
TAC and sham LV myocytes, however, were indistinguishable (Figure 2I), suggesting that the reductions in Ito,f and IK1
densities (Figure 2J) reflect only the increase in Cm (Figure
2H). In contrast, mean⫾SEM IK,slow amplitude was significantly (P⬍0.001) smaller and mean⫾SEM Iss amplitude was
significantly (P⬍0.01) larger in TAC, than in sham, LV
myocytes (Figure 2I). The increase in Iss amplitude, however,
was not enough to offset the increase in Cm, and Iss density
was decreased (Figure 2J). There were no differences in the
kinetics or voltage dependences of the Kv currents in TAC
and sham LV myocytes, and no significant differences in Cm
or current amplitudes/densities in TAC and sham RV myocytes were observed (Table).
APDs at 90% repolarization (APD90) were significantly
(P⬍0.01) longer in TAC, than in sham, LV myocytes,
whereas resting membrane potentials and action potential
Marionneau et al
Mechanisms Underlying Kⴙ Current Remodeling in LVH
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Figure 3. Action potential and surface ECG abnormalities with TAC. A, Mean⫾SEM resting membrane potentials (Vm), action potential
amplitudes (APA), and durations at 50% (APD50) and 90% (APD90) repolarization in sham and TAC RV and LV myocytes. APD90 values
are significantly (#P⬍0.01) longer in TAC LV cells (supplemental Table III). B, Representative lead II ECGs from anesthetized sham and
TAC mice before and after surgery. QTc interval durations (C) and J wave amplitudes (D) in sham (n⫽8) and TAC (n⫽8) mice before and
after surgery; individual values and means⫾SEM are plotted (§P⬍0.05).
amplitudes were not significantly different (Figure 3A and
supplemental Table III). To assess the functional consequences of reduced K⫹ current densities and action potential
prolongation, ECGs were obtained from anesthetized sham
and TAC mice before and after surgery. Analysis of ECG
recordings revealed significantly (P⬍0.05) longer QT and
corrected QT (QTc) intervals in TAC, compared with sham,
mice (Figure 3B and 3C and supplemental Table IV). In
addition, the J wave, corresponding to the early repolarization
phase of murine ventricular action potentials,20 was significantly (P⬍0.05) flattened or inverted (Figure 3B and 3D and
supplemental Table IV), suggesting that the dispersion of
ventricular repolarization is altered in TAC LV.
Regional Differences in the Effects of
TAC-Induced LVH on Kⴙ Currents
Regional differences in the remodeling of LV K⫹ currents
would be expected to alter the dispersion of ventricular
repolarization.8 –11 Subsequent experiments focused, therefore, on investigating the effects of LVH on repolarizing K⫹
currents in cells isolated from the EPI and ENDO surfaces of
the LV wall. As previously reported in wild-type cells,15
mean⫾SEM Ipeak (Figure 4A and 4C), Ito,f (Figure 4D), and
IK,slow (Figure 4E) densities were significantly (P⬍0.001)
higher in sham EPI, than ENDO, LV myocytes (Table).
Regional differences in current densities15 have been suggested to contribute to the native transmural repolarization
gradient.21
There were also marked regional differences in the cellular
effects of LVH. The reduction in Ipeak densities was more
pronounced in TAC EPI, than ENDO, LV myocytes in spite
of the fact that mean⫾SEM Cm was higher in TAC ENDO
cells (Figure 4A through 4C and the Table). In addition,
mean⫾SEM Ipeak and IK,slow amplitudes were reduced significantly (P⬍0.001) in TAC EPI (Figure 4G), but not in TAC
ENDO (Figure 4H), LV myocytes. Mean⫾SEM Iss amplitude, in contrast, was increased significantly (P⬍0.001) in
TAC ENDO (Figure 4H), but not EPI (Figure 4G), LV
myocytes, and, as a result, Iss density was decreased in TAC
EPI (Figure 4I), but not ENDO (Figure 4J), cells. Similar to
results obtained in myocytes dispersed from whole LV, the
reduced Ito,f and IK1 densities in TAC EPI and ENDO
myocytes (Figure 4I, 4J) appear to reflect only cellular
hypertrophy (increased Cm), because no changes in Ito,f or IK1
amplitudes were observed in EPI or ENDO cells (Figure 4G
and 4H).
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Figure 4. Regional (EPI/ENDO) differences in the effects of LVH on repolarizing K⫹ currents and Cm. A, Representative Kv currents recorded, as described in the legend to Figure 2, from sham and TAC EPI and ENDO LV myocytes. B, Cm in sham and TAC EPI and
ENDO cells. Mean⫾SEM Ipeak (C), Ito,f (D), IK,slow (E), and Iss (F) densities in sham and TAC EPI and ENDO LV myocytes are plotted as a
function of test potential. Relative changes in current amplitudes (G and H) and densities (I and J) in TAC, compared with sham, EPI (G
and I) and ENDO (H and J) LV myocytes. Indicated values in TAC and sham myocytes are significantly (*P⬍0.001, #P⬍0.01, §P⬍0.05)
different.
Molecular Basis of Kⴙ Current Remodeling in
TAC LV
Subsequent experiments examined the impact of TACinduced LVH on the transcript and protein expression levels
of several channel pore-forming (␣) and accessory (␤) subunits encoding murine myocardial K⫹ channels.14,19,22–24 Because accumulating evidence suggests that cardiac ion channels function as components of macromolecular complexes,25
TaqMan low-density arrays16 were exploited to allow quantitative determinations of multiple transcripts simultaneously.
The amount of total RNA isolated from TAC LV was
significantly (P⬍0.01) higher (1.7-fold on average) than from
sham LV (Figure 5A). This observation is consistent with
previous findings demonstrating that RNA synthesis/content
in the LV is increased with hypertrophy, reflecting increased
myocyte size, without an increase in myocyte number.18
Transcript expression levels determined by quantitative RTPCR on sham and TAC LV, and on EPI and ENDO LV,
samples were, therefore, normalized to the LVM/BW determined in each animal. Consistent with the increase in total
RNA, expression of several endogenous control genes (18S
RNA, GAPDH, HPRT, RNA polymerase II [RNA Pol II])17
was increased in TAC, compared with sham, LV (Figure 5B).
In contrast with the global increases in transcript expression with hypertrophy, transcript expression of the Ito,f channel ␣ subunits Kv4.2 (KCND2) and Kv4.3 (KCND3)19,24 was
not significantly different in TAC and sham LV, and expression of the Ito,f channel accessory subunit KChIP2 (KCNIP2)24
was actually slightly lower in TAC LV (Figure 5C). The
expression of transcripts encoding IK1 channel ␣ subunits
Kir2.1 (KCNJ2) and Kir2.2 (KCNJ12),23 and of the K2P
channel subunit TASK1 (KCNK3), which has been suggested
to underlie Iss in rat cardiomyocytes,26 as well as TASK2
(KCNK5), was unaffected in TAC LV (Figure 5C). In
contrast, expression of the transcripts encoding IK,slow1 and
IK,slow2 channels Kv1.5 (KCNA5) and Kv2.1 (KCNB1),14,22 as
well as of the K2P channel subunit TREK1 (KCNK2), was
increased in TAC LV (Figure 5C). The expression levels of
several other Kv ␣ subunits Kv1.4 (KCNA4), Kv4.1
(KCND1), and KvLQT1 (KCNQ1), as well as a number of K⫹
channel regulatory proteins including Kv␤1 (KCNAB1),
Kv␤2 (KCNAB2), minK (KCNE1), KChAP (Pias3), PSD-95
(post-synaptic density 95 protein), and filamin C, were also
increased in TAC LV (Figure 5C).
Analyses of transcript expression in EPI and ENDO LV
samples from sham and TAC animals revealed no regional
differences in remodeling. As in wild-type LV,15 KCND2
expression was significantly (P⬍0.001) higher in sham EPI,
than ENDO, LV (Figure 5D). This gradient was maintained
in TAC LV, and expression of KCND3 and KCNIP2, as well
as KCNJ2 and KCNJ12, transcripts was also similar in TAC
EPI and ENDO (Figure 5D), consistent with the similarities
Marionneau et al
Mechanisms Underlying Kⴙ Current Remodeling in LVH
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Figure 5. Transcript expression profiling in TAC and sham LV. A, Total RNA content in sham (n⫽6) and TAC (n⫽8) LV; individual values
and means⫾SEM are plotted (#P⬍0.01). Mean⫾SEM RNA expression levels (arbitrary units) of control (B) and K⫹ channel subunit and
regulatory (C) genes in sham and TAC LV and of K⫹ channel subunit and regulatory genes in sham and TAC EPI and ENDO LV (D)
(n⫽6 mice in each group). Indicated values are significantly (*P⬍0.001, #P⬍0.01, §P⬍0.05) different in TAC and sham LV and in EPI
versus ENDO (†P⬍0.001) samples.
in Ito,f and IK1 amplitudes in TAC and sham EPI and ENDO
LV myocytes (Figure 4). The transcript expression levels of
Kv1.5, Kv2.1, and TREK1, as well as SAP-97, which has
been postulated to play a role in Kv1.5 trafficking,27 were
increased similarly in TAC EPI and ENDO LV (Figure 5D).
Western blot experiments were performed to determine the
(total and/or cell surface) expression levels of several Kv
channel subunit and regulatory proteins. Similar to the RNA data
and in accordance with previous reports,18 total LV protein
content was significantly (P⬍0.01) higher (1.2-fold on average)
in TAC, compared with sham, LV (Figure 6A). To compare
protein expression in sham and TAC LV and in sham and TAC
EPI and ENDO LV samples, therefore, protein loading on
SDS-PAGE gels was normalized to reflect the LVM/BW.
Consistent with the global increase in protein content (Figure
6A), expression of the endogenous (transferrin receptor17) control protein, as well as the structural proteins filamin C and
␤-actin, was increased significantly (P⬍0.05) in TAC, compared with sham, LV (Figure 6B and 6C). Consistent with the
transcript data (Figure 5), however, no significant differences in
the expression levels of the Ito,f (Kv4.2, KChIP2) or IK1 (Kir2.1,
Kir2.2) channel subunit proteins were observed in TAC and
sham LV (Figure 6B and 6C). Further experiments revealed no
regional differences in the impact of LVH: Kv2.1 and ␤-actin
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Figure 6. Protein expression in TAC and sham LV. A, Total protein content in sham (n⫽3) and TAC (n⫽4) LV; individual values and
means⫾SEM are plotted (#P⬍0.01). Representative Western blots of total protein expression of K⫹ channel subunits, as well as of the
control proteins, transferrin receptor (TransR), ␤-actin, and filamin C, in sham and TAC LV (B), EPI (D), and ENDO (E). Quantification of
relative differences in total protein expression in sham and TAC total LV (C) and in sham and TAC EPI and ENDO LV (F). Data are
means⫾SEM (n⫽3 to 6), and indicated values are significantly (§P⬍0.05) different in TAC and sham LV or TAC and sham EPI or ENDO
LV. As in wild-type (WT) LV,15 Kv4.2 expression is higher (†P⬍0.01) in (sham and TAC) EPI than ENDO LV.
expression were increased in both TAC EPI and ENDO LV,
whereas Kv4.2, TASK1, and TASK2 expression were unaffected (Figure 6D through 6F). Cell surface Kv2.1 expression
was also increased in TAC LV (Figure 7). Interestingly, however, although total TASK1 protein expression was unchanged
(Figure 6), cell surface TASK1 expression was markedly increased in TAC, compared with sham, LV myocytes (Figure 7),
in parallel with the increase in Iss amplitude (Figure 2).
Discussion
LVH in TAC Mice
The experiments here revealed that pathological LVH is
clearly evident in TAC mice 7 days following surgery. In
parallel with the increase in LVM/BW, mean LV myocyte
size, as determined by Cm measurements, was increased with
TAC, in agreement with previous reports.8,28 In contrast to
Figure 7. Cell surface protein expression in TAC
and sham LV. A, Representative Western blots of
cell surface expression of K⫹ channel subunit and
the control (TransR) proteins in sham and TAC LV.
B, Quantification of relative differences in cell surface protein expression in sham and TAC LV. Data
are means⫾SEM (n⫽3 to 6), and expression levels
are significantly (#P⬍0.01, §P⬍0.05) different in
TAC and sham LV.
Marionneau et al
Mechanisms Underlying Kⴙ Current Remodeling in LVH
these earlier reports, however, there was no evidence that
mean LVM/BW or myocyte size continued to increase at
longer times (up to 3 months) after TAC. Also, in contrast to
the effects on LV cells, there were no differences in mean Cm
in TAC and sham RV cells, demonstrating that the cellular
hypertrophy is specific for the LV.
Alterations in Repolarizing Kv and Kir Currents
in TAC LV Myocytes
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
Kv current and IK1 densities were markedly lower, and action
potentials were prolonged in TAC, compared with sham, LV
myocytes. In addition, QTc intervals were increased in TAC
mice. Reduced Kv current densities and action potential
prolongation are consistent findings in experimental LVH
models3–9,11 and in failing human hearts.10,29 The analyses
completed here, however, revealed that cellular hypertrophy
(increased Cm) is the main factor in determining reductions in
Ito,f and IK1 densities, because the mean amplitudes of these
currents in TAC and sham LV cells were not significantly
different. In contrast, mean IK,slow and Iss amplitudes were
altered in TAC LV myocytes, suggesting that functional IK,slow
and Iss channel expression is modulated with LVH.
Regional Differences in the Effects of
TAC-Induced LVH
As described in failing human hearts,10 as well as in various
LVH models,8,9,11 the experiments here revealed marked
regional differences in the effects of LVH. Specifically, Ipeak
and IK,slow densities were attenuated more in TAC EPI, than
TAC ENDO, LV myocytes. The larger reduction in (Ipeak/
IK,slow) densities in TAC EPI cells reflects the decrease in IK,slow
amplitudes, which were not affected in TAC ENDO cells.
In contrast to previous reports,8 –11,28 however, the experiments here also revealed that the cellular responses to pressure
overload are heterogeneous. Specifically, cellular hypertrophy
was greater in TAC ENDO, than TAC EPI, LV myocytes. This
regional difference may reflect the higher wall tension applied to
the ENDO, as compared with the EPI, LV surface.30 No changes
in mean Ito,f, IK,slow, and IK1 amplitudes were detected in TAC
ENDO myocytes; the reductions in the densities of these
currents in TAC ENDO cells appear to reflect only the increased
cell size. Interestingly, however, mean Iss amplitude was significantly increased in TAC ENDO myocytes, partially compensating for the increase in cell size. Because this increase occurs
only in TAC ENDO LV myocytes, where the increase in cell
size is greater, it is tempting to speculate that ENDO Iss channels
undergo remodeling to compensate (although inefficiently) for
cellular hypertrophy/stretch.
The marked reduction in the functional expression of EPI
IK,slow channels, together with the increased expression of
ENDO Iss channels, collapses the transmural Kv current
gradient in TAC LV and is reflected in the alteration in the
polarity of the J wave on ECG recordings.
Molecular Remodeling of Kⴙ Channels in
TAC-Induced LVH
Reductions in the densities of specific K⫹ currents in the
diseased myocardium have previously been reported to be
associated with alterations in the expression of the subunits
9
underlying these currents.3,4 In the molecular/biochemical
analyses completed here, however, no significant changes in
the expression levels of the Ito,f (Kv4.2, Kv4.3, KChIP2) or IK1
(Kir2.1, Kir2.2) channel subunits in TAC (EPI and/or ENDO)
LV were observed, consistent with the observed similarities
in Ito,f and IK1 amplitudes in TAC EPI and ENDO LV
myocytes. As previously demonstrated,18 the hypertrophic
growth of cardiomyocytes with LVH is characterized by an
increase in cell size without significant changes in cell
number. In agreement with these previous reports,18 TACinduced LVH was associated with global increases in LV
RNA and protein expression. Because cardiomyocytes contribute more than 90% to the total mass of the myocardium,1
the increase in LVM, as well as in RNA and protein content
with LVH, predominantly reflects cardiomyocyte enlargement. Taken together, therefore, the results here suggest that
the reductions in Ito,f and IK1 densities in TAC LV reflect the
fact that the expression of the subunits encoding these
channels is not increasing as the cells are enlarging.
In contrast to Ito,f and IK1, mean IK,slow amplitude was
markedly reduced in TAC EPI myocytes, suggesting downregulation of functional IK,slow channels. Unexpectedly, however, Kv1.5 and Kv2.1 transcripts were increased with TAC.
Although the lack of an anti-Kv1.5 antibody that can detect
endogenous Kv1.5 in cardiomyocytes reliably precluded
analyses of Kv1.5 protein expression, biochemical data presented here demonstrate increased total and cell surface
Kv2.1 protein expression in TAC EPI and ENDO LV. If
Kv1.5 protein expression increases in parallel with KCNA5,
the loss of functional IK,slow channels must reflect altered
expression of yet to be identified IK,slow accessory and/or
regulatory subunits and/or altered posttranslational processing of the Kv1.5 and/or Kv2.1 proteins.
The experiments here also revealed that Iss amplitude was
increased in TAC ENDO LV myocytes. Whereas the transcript and total protein expression levels were unchanged in
TAC EPI and ENDO, the cell surface expression of TASK1
was markedly increased in TAC LV myocytes. These results
are consistent with recent findings demonstrating a role for
TASK1 in the generation of Iss in rat cardiomyocytes.26
Relationship to Previous Studies
The results presented here demonstrate that distinct mechanisms
underlie functional K⫹ channel remodeling with LVH, with the
main factor being cellular hypertrophy. Several previous studies
have described reductions in K⫹ current densities with LVH or
heart failure.5–11,29 Most of these studies reported 30% to 50%
decreases in K⫹ current densities, with cell membrane capacitances increased by approximately the same percentage. Although not reported, it seems likely that K⫹ current amplitudes
were also largely unaffected in these previous studies and that
increased cell size was the primary determinant of the reductions
in K⫹ current densities. Some of these previous studies also
reported decreased expression of K⫹ channel subunits with
LVH. It appears, however, that these RNA/protein expression
data were not normalized to reflect the global RNA/protein
increases associated with cellular hypertrophy. Had the cellular
hypertrophy been taken into account in these previous LVH
studies, it seems certain that the expression levels of K⫹ channel
10
Circulation Research
June 6, 2008
subunits would, similar to the present findings, be shown to be
largely unchanged.
The recognition of the important role of cardiomyocyte
enlargement on functional K⫹ channel expression with LVH
will impact the way future investigations into K⫹ (particularly
Ito,f and IK1) channel remodeling with LVH are approached.
Indeed, the results presented here suggest that alterations in
the functional expression of Ito,f and IK1 channels with LVH do
not reflect transcriptional or translational downregulation of
the subunits encoding these channels, as has been previously
suggested.3,4,29 Rather, the results here suggest that although
global transcriptional and translational machineries are activated with LVH,18 the expression of channel subunits do not
increase corresponding to the increase in cell size. Therapeutic strategies aimed at reducing cellular hypertrophy or
increasing the functional expression of repolarizing K⫹ channels would, therefore, appear to be promising approaches in
the treatment of LVH-associated ventricular arrhythmias.
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
Acknowledgments
We thank the Mouse Cardiovascular Phenotyping Core for surgical
expertise and Drs Flavien Charpentier and Attila Kovacs for helpful
discussion concerning interpretation of ECGs and echocardiograms.
Sources of Funding
This work was supported by National Heart, Lung, and Blood
Institute grant HL-066388, the Heartland Affiliate of the American
Heart Association (Postdoctoral Fellowship), and Agence Nationale
de la Recherche grant ANR-05-PCOD-037-01.
Disclosures
None.
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Distinct Cellular and Molecular Mechanisms Underlie Functional Remodeling of
Repolarizing K + Currents With Left Ventricular Hypertrophy
Céline Marionneau, Sylvain Brunet, Thomas P. Flagg, Thomas K. Pilgram, Sophie Demolombe
and Jeanne M. Nerbonne
Circ Res. published online May 1, 2008;
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2008 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
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http://circres.ahajournals.org/content/early/2008/05/01/CIRCRESAHA.107.170050.citation
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2008/05/01/CIRCRESAHA.107.170050.DC1
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ONLINE SUPPLEMENT
Marionneau et al.
MATERIALS AND METHODS
Animals used in the present study were handled in accordance with the guidelines
published in the Guide for the Care and Use of Laboratory Animals (US National Institutes of
Health); all protocols were approved by the Washington University Animal Studies Committee.
Induction of Left Ventricular Hypertrophy
Pressure overload-induced Left Ventricular Hypertrophy (LVH) was produced in adult
(8-9 week) male C57BL6 mice by transverse aortic constriction (TAC), a procedure that results
in the development of concentric LVH within days1,2. For surgery, animals were anesthetized
with xylazine (16 mg/kg, i.p.) and ketamine (80 mg/kg, i.p.). Once deep anesthesia was
confirmed, the chest was opened and, following blunt dissection through the intercostal muscles,
the thoracic aorta was identified. A 7-0 silk suture was placed around the transverse aorta and
tied around a 26-gauge needle, which was subsequently removed. This procedure was performed
on a total of 86 animals. Of these, 21 animals died during the surgery or within the following 24
hours; the remaining 65 animals survived and were used for experiments. Sham-operated
animals (n=35) underwent the same surgical procedure, except that the aortic constriction was
not put in place and, in contrast to the TAC animals, all of the sham animals survived.
Echocardiography
Seven days after TAC, animals were examined by non-invasive transthoracic
echocardiography using described methods1,3. Under anesthesia (avertin, 0.25 mg/g, i.p.),
animals were examined using an Acuson Sequoia 256 echocardiography system (Acuson,
1
Stockton, CA, USA) equipped with a 15-MHz transducer. Digitally acquired and stored twodimensional short axis cine-loops of the LV of TAC and sham mice were viewed and displayed
next to images obtained from age-matched wild-type (WT) unoperated controls. Comparisons
were then made between the appearance of the LV (i.e., wall thickness and chamber size) in
TAC, sham and WT animals. Echocardiograms obtained from sham animals were not
significantly different from those recorded from WT animals. To validate this rapid semiquantitative selection process for the detection of LVH, the assignments were compared with the
results obtained with quantitative M-mode based LV mass measurements1,3 in a blinded fashion.
Results are expressed as means ± SEM from 18 sham and 39 TAC mice. Statistical differences
between TAC and sham animals were assessed using the Student’s t-test.
LV diastolic function was evaluated by the use of Doppler tissue imaging as described
previously4. Trans-mitral flow velocity was obtained from a modified apical 4-chamber view
with the pulse-wave Doppler sample volume positioned at the ventricular side of the mitral valve
leaflets.
Electrocardiograms
Surface electrocardiographic (ECG) recordings were obtained from anesthetized (avertin,
0.25 mg/g, i.p.) mice, before and after (TAC) surgery. For signal detection, needle electrodes
were inserted through the skin following a standard three-lead scheme (left foreleg, right foreleg
and left rear leg). ECG signals were amplified with a four channel differential amplifier (model
1700, A-M Systems, San Diego, CA, USA) and data were collected using a Digidata 1200
analog/digital converter using Axoscope 8 software (Axon Instrument). ECG data were analyzed
using both commercial (Clampfit 8.0, Molecular Devices, Sunnyvale, CA, USA) and custom5
softwares. RR, PR, QRS and QT interval durations, as well as J wave amplitudes, were measured
2
as previously described6,7. QT intervals were corrected (QTc) for heart rate using the formula,
QTc=QT/(RR/100)1/2, with QT and RR intervals measured in ms8. Results are expressed as
means ± SEM from 8 sham and 8 TAC mice. Statistical differences between TAC and sham
animals were assessed using the Student’s t-test.
Electrophysiological Recordings
Myocytes were isolated from the left (LV) and right (RV) ventricles of TAC and sham
mouse hearts by enzymatic dissociation and mechanical dispersion using previously described
methods9,10. In initial experiments, whole-cell voltage-clamp recordings were obtained from
(randomly dispersed) LV wall and RV wall myocytes isolated from TAC and sham animals at 7
and 9-11 days, respectively, following surgery. In experiments aimed at exploring the regional
effects of LVH, the epicardial (EPI) and endocardial (ENDO) surfaces of the LV were separated
using iridectomy scissors9; the resulting tissue pieces were mechanically dispersed and plated
separately. Electrophysiological experiments were conducted within 24 hours of cell isolation at
room temperature (22-24°C). Recording pipettes contained (in mmol/L): KCl 135, EGTA 10,
HEPES 10, and glucose 5 (pH 7.4, 295-310 mOsm). The bath solution contained (in mmol/L):
NaCl 136, KCl 4, MgCl2 2, CaCl2 1, CoCl2 5, tetrodotoxin 0.02, HEPES 10, and glucose 10 (pH
7.4, 295-300 mOsm). For whole-cell current-clamp recordings, tetrodotoxin and CoCl2 were
omitted from the bath solution. Experiments were performed using an Axopatch 1D amplifier
(Molecular Devices, Sunnyvale, CA, USA) connected to a Gateway 350-MHz Pentium computer
interfaced to the recording equipment with a Digidata 1200 analog/digital interface and the
pClamp 7 software package (Axon Instrument). Data were filtered at 5 KHz before storage.
Recording electrodes were fabricated from soda lime glass (Gerresheimer Glass Inc., Vineland,
NJ, USA), coated with Sylgard (Dow Corning, Midland, MI, USA) and fire-polished prior to
3
use. Whole-cell series resistances were routinely compensated electronically (≈ 85%); voltage
errors resulting from the uncompensated series resistance were always ≤ 8 mV and were not
corrected. Ca2+-independent depolarization-activated outward K+ (Kv) currents were routinely
recorded in response to 4.5 s voltage steps to test potentials between -40 and +40 mV from a
holding potential (HP) of -70 mV. Inwardly rectifying K+ currents (IK1) were recorded in
response to 350 ms voltage steps to test potentials between -40 and -120 mV from the same HP.
Whole-cell current-clamp recordings were used to obtain resting membrane potentials (Vm) and
to record action potentials, evoked in response to brief (1 ms) depolarizing current injections at a
frequency of 1 Hz.
Voltage-clamp data were compiled and analyzed using Clampfit 8.0 (Molecular Devices,
Sunnyvale, CA, USA) and Excel (Microsoft). For each cell, the spatial control of the membrane
voltage was assessed by analyzing the decay of the capacitative transient evoked during brief (25
ms) subthreshold ± 10 mV voltage steps from the HP (-70 mV); only cells with capacitative
transients well described by single exponentials were analyzed further. In each cell, integration
of the capacitative transient recorded during these brief ± 10 mV voltage steps provided the
whole-cell membrane capacitance (Cm), an estimate of cell size. Leak currents were always less
then 100 pA and were not corrected. Peak Kv current at each test potential was defined as the
maximum value of the outward Kv current during each 4.5 s voltage step. IK1 amplitude at each
test potential was measured at the end of each 350 ms voltage steps. The time constants of
inactivation (τ) of the Kv current components (Ito,f and IK,slow) and the amplitudes (A) of the
individual current components (Ito,f, IK,slow and Iss) were determined from exponential fits of the
decay phases of the currents using the following equation: At=A1 exp(-t/τ1)+A2 exp(-t/τ2)+Ass,
where At represents the amplitude of the current at time t, A1 and τ1 and A2 and τ2, represent the
4
amplitudes (A) and the time constants (τ) of the fast (Ito,f) and the slow (IK,slow) components of
current decay; Ass is the amplitude of the non-inactivating, steady-state component (Iss) of the
total outward Kv current. Current densities (in pA/pF) were obtained by normalizing the current
amplitudes (in pA) by the Cm (in pF). Action potential amplitudes (APA) and action potential
durations at 50% (APD50) and 90% (APD90) repolarization were measured using Clampfit.
All voltage- and current-clamp data are expressed as means ± SEM. The statistical
significance of observed differences between groups of cells was evaluated using the Student’s ttest. In addition, to determine the statistical significance of apparent regional (EPI/ENDO)
differences in the impact of TAC, multiple regression analysis was performed.
RNA Preparation
For the preparation of RNA, animals were sacrificed by cervical dislocation and the
hearts (from TAC and sham mice) were rapidly removed. RV and LV from each heart, or EPI
and ENDO from each LV, were dissected (n=6-8 in each group) and flash-frozen in liquid
nitrogen for further RNA isolation. Total RNA was isolated and DNase treated using the RNeasy
Fibrous Tissue Mini Kit (Qiagen, Valencia, CA, USA). The quality of total RNA was assessed
by gel electrophoresis, and RNA concentration was determined by optical density measurement
at 260 nm. Genomic DNA contamination was assessed by PCR amplification of total RNA
samples without prior cDNA synthesis; no genomic DNA was detected.
TaqMan Quantitative RT-PCR
TaqMan Low Density Arrays (TLDA, Applied Biosystems, Foster City, CA, USA) were
completed in a two-step RT-PCR process as described previously11. Briefly, first strand cDNA
was synthesized from 2 μg of total RNA (from individual sham and TAC samples) using the
5
High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). PCR reactions
were carried out in TLDAs using the ABI PRISM 7900HT Sequence Detection System (Applied
Biosystems, Foster City, CA, USA). The 384 wells of each array were pre-loaded with 96 x 4
pre-designed FAM-labeled fluorogenic TaqMan probes and primers. The probes were labeled
with the fluorescent reporter dye 6-carboxyfluorescein (FAM, Applera Corporation, Norwalk,
CT, USA) on the 5’ end and with a non-fluorescent quencher on the 3’ end. The genes selected
for analysis (see Online Table I) were ones encoding 68 cardiac ion channel pore-forming (α)
and accessory (β) subunit proteins as well as proteins involved in ion channel regulation, 11
proteins involved in Ca2+ homeostasis, 6 transcription factors, 7 specific markers of vessels,
neurons, fibroblasts, inflammation and hypertrophy, and 4 endogenous controls. Two ng of
cDNA from each sample was combined with 1X TaqMan Universal Master Mix (Applied
Biosystems, Foster City, CA, USA) and loaded into each well. The TLDAs were thermal-cycled
at 50°C for 2 min and 94.5°C for 10 min, followed by 40 cycles of 97°C for 30 s and 59.7°C for
1 min. Data were collected with instrument spectral compensations using the Applied
Biosystems SDS 2.2.2 software and analyzed using the threshold cycle (CT) relative
quantification (2-∆Ct) method12. Genes with CT values >32 were not analyzed further.
The hypoxanthine guanine phosphoribosyl transferase I (HPRT) gene was used as an
endogenous control to normalize the experimental data. As described previously, cardiac
hypertrophy results in global increases in the total RNA (Figure 5A) and protein (Figure 6A)
content of the LV, as well as increased LV mass and LV mass to body weight (LVM/BW) ratio
(Figure 1, Online Table II), without any increases in cell numbers13-15. To quantify transcript
expression levels on a per cell basis, therefore, the TLDA (HPRT-normalized) expression data
from each LV, EPI and ENDO sample were multiplied by the LVM/BW ratios determined in the
6
same (sham or TAC) animals. Results are expressed as means ± SEM from 6 mice in each group,
and statistical differences between samples from TAC and sham animals were assessed using the
Student’s t-test.
SYBR Green Quantitative RT-PCR
First strand cDNA was synthesized from 2 μg of total RNA using the High-Capacity
cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). The expression levels of genes
encoding the cardiac hypertrophy markers ANF (Atrial Natriuretic Factor) and β MHC (β
Myosin Heavy Chain), as well as the two-pore domain K+ channels TASK1 (KCNK3), TASK2
(KCNK5) and TREK1 (KCNK2), were determined by quantitative PCR using sequence specific
primer pairs and 1X SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA,
USA). PCR reactions were performed on 10 ng of cDNA in the ABI PRISM 7900HT Sequence
Detection System (Applied Biosystems, Foster City, CA, USA). The cycling conditions included
a hot start at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Primer
sequences
were
the
following:
ANF:
5’-CACTGTGGCGTGGTGAACA
and
5’-
TCGTGATAGATGAAGGCAGGAA; β MHC: 5’-TGAATGAGCACCGGAGCAA and 5’CTGGCTGGTGAGGTCATTGA;
5’-GCTTCCGCAACGTCTATGC
TASK1:
and
5’-
GGGATGGAGTACTGCAGCTTCT; TASK2: 5’-TCAGATCACGTGTACAGCCATCT and 5’CATTCTTCTGTCACCATGAACACA; TREK1: 5’-CCTTTTGTGCCCAGACTGTTTC and
5’-GCAAAGCATTCAGATTCATTCATAG; and HPRT: 5’-TGAATCACGTTTGTGTCATTA
GTGA and 5’-TTCAACTTGCGCTCATCTTAGG. PCR primer pairs were tested using mouse
cDNA, and primers giving 90-100% efficacy, as well as single amplicons of the appropriate
melting temperature or size, were selected. Negative control experiments using total RNA
samples incubated without reverse transcriptase during cDNA synthesis showed no
7
amplification. Data were analyzed according to the 2-∆Ct method12. As described above for the
TaqMan analysis, transcript expression data in each sham and TAC LV, EPI and ENDO sample
were first normalized using the endogenous control gene HPRT, and subsequently multiplied by
the LVM/BW ratios measured in the same (sham or TAC) animals. Results are expressed as
means ± SEM from 6 mice in each group, and statistical differences between TAC and sham
animals were assessed using the Student’s t-test.
To validate these analytical methods described above (i.e., multiplication of 2-∆Ct values
by the LVM/BW ratios), SYBR Green quantitative RT-PCR experiments on few channel
subunits, as well as several endogenous control genes, were also conducted starting with
normalized (by LVM/BW ratios) quantities of total RNA. As expected, the results of these
analyses were indistinguishable from those obtained when the normalization was done on the
analyzed (TaqMan or SYBR Green) RT-PCR data.
Western Blot Analyses
Using previously described methods16, western blot analyses were performed on total
protein extracts prepared from LV, RV, LV EPI and LV ENDO of sham and TAC mice (n=3-4
in each group). Tissues were homogenized in Dounce homogenizer in 10 volumes of ice-cold
lysis buffer (in mmol/L: Tris-HCl 50 (pH 7.5), EDTA 1, NaCl 150) containing protease inhibitor
cocktail tablet (Roche, USA), Pefabloc (1 mmol/L), pepstatin A (1 ug/ml) and Triton X-100
(1%). After 15-min incubation with slow rotation at 4°C, the insoluble fraction was removed by
centrifugation at 3000 rpm for 10 min at 4°C. Quantification of total proteins in each sample was
obtained by using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA). For Western blots,
the total amount of (sham or TAC) sample protein loaded on to each lane of the SDS-PAGE gels
was normalized to the LVM/BW ratio determined in sham and TAC animals to allow direct
8
comparisons of protein expression levels in comparable numbers of cells. Following
fractionation, proteins were transferred to PVDF membranes.
The following primary antibodies were used: rabbit polyclonal anti-Kv4.2, anti-Kir2.1,
anti-Kir2.2 (Chemicon, Temecula, CA, USA); rabbit polyclonal anti-TASK1, anti-TASK2 and
anti-TREK1 (Alomone labs, Jerusalem, Israel); mouse monoclonal anti-Kv2.1 and anti-KChIP2
(NeuroMab, Davis, CA, USA); rabbit polyclonal anti-Filamin C (Kinasource, Scotland, UK);
mouse monoclonal anti-transferrin receptor (Invitrogen Corporation, Carlsbad, CA, USA); and
mouse monoclonal anti-β-actin (Sigma, Saint Louis, MO, USA). The specificities of the antiKv4.2, anti-KChIP2, anti-Kv2.1 and anti-TREK1 antibodies were tested using total protein
extracts from mice in which the genes encoding these subunits have been eliminated by
homologous recombination16,17. In each case, no signal corresponding to the targeted channel
subunit was detected. Following blocking, PVDF membranes were incubated with one of the
anti-channel subunit specific antibodies, and subsequently with one of the anti-endogenous
control protein antibodies. After washing, the membranes were incubated with a goat anti-rabbit
or anti-mouse horseradish peroxidase-conjugated secondary antibody (Pierce, Rockford, IL,
USA). Membranes were incubated with the SuperSignal West Dura Extended Duration substrate
(Pierce, Rockford, IL, USA), and signal was exposed to x-ray film (Kodak, Rochester, NY,
USA). Films were scanned, and the densities of specific bands were determined. Results are
expressed as means ± SEM from 3-4 different sham or TAC mice. Statistical differences
between TAC and sham animals were assessed using the Student’s t-test.
Cell Surface Expression of K+ Channel Subunits in Mouse Ventricular Myocytes
Surface biotinylation of ventricular myocytes were completed as previously described18.
Briefly, freshly isolated LV myocytes from sham and TAC mice were incubated in Ringer’s
9
solution (in mmol/L: Hepes (pH 7.4) 10, NaCl 154, KCl 7.2, CaCl2 1.8) at 4oC to inhibit
membrane protein internalization, followed by 1 mg/ml Sulfo-NHS-SS-Biotin (Pierce, Rockford,
IL, USA) for 45 min with gentle agitation. The biotinylation reaction was quenched with Trissaline solution (in mmol/L: Tris (pH 7.4) 10, NaCl 120), the cells were extensively washed with
Tris-saline solution and collected. Detergent-soluble lysates were prepared, and biotinylated cell
surface proteins were affinity-purified using NeutrAvidin-conjugated agarose beads (Pierce,
Rockford, IL, USA). Purified cell surface proteins were then analyzed by western blots as
described above. Results are expressed as means ± SEM from 3-6 different sham or TAC mice.
Statistical differences between TAC and sham animals were assessed using the Student’s t-test.
10
REFERENCES
1.
Zhang S, Weinheimer C, Courtois M, Kovacs A, Zhang CE, Cheng AM, Wang Y,
Muslin AJ. The role of the Grb2-p38 MAPK signaling pathway in cardiac hypertrophy
and fibrosis. J Clin Invest. 2003;111:833-41.
2.
Rockman HA, Ross RS, Harris AN, Knowlton KU, Steinhelper ME, Field LJ, Ross J, Jr.,
Chien KR. Segregation of atrial-specific and inducible expression of an atrial natriuretic
factor transgene in an in vivo murine model of cardiac hypertrophy. Proc Natl Acad Sci
U S A. 1991;88:8277-81.
3.
Tanaka N, Dalton N, Mao L, Rockman HA, Peterson KL, Gottshall KR, Hunter JJ, Chien
KR, Ross J, Jr. Transthoracic echocardiography in models of cardiac disease in the
mouse. Circulation. 1996;94:1109-17.
4.
Chiu HC, Kovacs A, Blanton RM, Han X, Courtois M, Weinheimer CJ, Yamada KA,
Brunet S, Xu H, Nerbonne JM, Welch MJ, Fettig NM, Sharp TL, Sambandam N, Olson
KM, Ory DS, Schaffer JE. Transgenic expression of fatty acid transport protein 1 in the
heart causes lipotoxic cardiomyopathy. Circ Res. 2005;96:225-33.
5.
McLerie M, Lopatin AN. Dominant-negative suppression of I(K1) in the mouse heart
leads to altered cardiac excitability. J Mol Cell Cardiol. 2003;35:367-78.
6.
London B, Guo W, Pan X, Lee JS, Shusterman V, Rocco CJ, Logothetis DA, Nerbonne
JM, Hill JA. Targeted replacement of KV1.5 in the mouse leads to loss of the 4aminopyridine-sensitive component of I(K,slow) and resistance to drug-induced qt
prolongation. Circ Res. 2001;88:940-6.
11
7.
Liu G, Iden JB, Kovithavongs K, Gulamhusein R, Duff HJ, Kavanagh KM. In vivo
temporal and spatial distribution of depolarization and repolarization and the illusive
murine T wave. J Physiol. 2004;555:267-79.
8.
Mitchell GF, Jeron A, Koren G. Measurement of heart rate and Q-T interval in the
conscious mouse. Am J Physiol. 1998;274:H747-51.
9.
Brunet S, Aimond F, Li H, Guo W, Eldstrom J, Fedida D, Yamada KA, Nerbonne JM.
Heterogeneous expression of repolarizing, voltage-gated K+ currents in adult mouse
ventricles. J Physiol. 2004;559:103-20.
10.
Xu H, Guo W, Nerbonne JM. Four kinetically distinct depolarization-activated K+
currents in adult mouse ventricular myocytes. J Gen Physiol. 1999;113:661-78.
11.
Marionneau C, Couette B, Liu J, Li H, Mangoni ME, Nargeot J, Lei M, Escande D,
Demolombe S. Specific pattern of ionic channel gene expression associated with
pacemaker activity in the mouse heart. J Physiol. 2005;562:223-34.
12.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time
quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402-8.
13.
Bonnin CM, Sparrow MP, Taylor RR. Increased protein synthesis and degradation in the
dog heart during thyroxine administration. J Mol Cell Cardiol. 1983;15:245-50.
14.
Hannan RD, Jenkins A, Jenkins AK, Brandenburger Y. Cardiac hypertrophy: a matter of
translation. Clin Exp Pharmacol Physiol. 2003;30:517-27.
15.
Morgan HE, Gordon EE, Kira Y, Chua HL, Russo LA, Peterson CJ, McDermott PJ,
Watson PA. Biochemical mechanisms of cardiac hypertrophy. Annu Rev Physiol.
1987;49:533-43.
12
16.
Guo W, Jung WE, Marionneau C, Aimond F, Xu H, Yamada KA, Schwarz TL,
Demolombe S, Nerbonne JM. Targeted deletion of Kv4.2 eliminates I(to,f) and results in
electrical and molecular remodeling, with no evidence of ventricular hypertrophy or
myocardial dysfunction. Circ Res. 2005;97:1342-50.
17.
Kuo HC, Cheng CF, Clark RB, Lin JJ, Lin JL, Hoshijima M, Nguyen-Tran VT, Gu Y,
Ikeda Y, Chu PH, Ross J, Giles WR, Chien KR. A defect in the Kv channel-interacting
protein 2 (KChIP2) gene leads to a complete loss of I(to) and confers susceptibility to
ventricular tachycardia. Cell. 2001;107:801-13.
18.
Eder P, Probst D, Rosker C, Poteser M, Wolinski H, Kohlwein SD, Romanin C,
Groschner K. Phospholipase C-dependent control of cardiac calcium homeostasis
involves a TRPC3-NCX1 signaling complex. Cardiovasc Res. 2007;73:111-9.
13
FIGURE LEGENDS
Online Figure I. Transcript expression profiling in TAC and sham LV. Mean ± SEM transcript
expression (arbitrary units) of genes encoding (A) α and β subunits of Na+ and Ca2+ channels,
connexins (Cx), (B) Ca2+ homeostasis regulators, and (C) cardiac hypertrophy markers in sham
and TAC LV. Data are means ± SEM from 6 mice in each group. *P<0.001, #P<0.01, §P<0.05 in
TAC, compared with sham, LV.
14
300
0
*
200
100
#
#
Online Figure I. Marionneau et al.
TAC LV
10
*
*
*
Sham LV
*
0
B
Sham LV
400
TAC LV
200
*
*
Cx43
20
mRNA Expression (AU)
Sham LV
TAC LV
SERCA2a
#
Cx45
30
Ryr2
*
Cx40
*
PLB
*
Cavβ2
Cavα2δ1
*
NCX1
60
IP3R-2
Casq2
*
Cav3.1
Cav1.2
Navβ1
Nav2.3
2
Camk2d
40
Calm1
20
Calcineurin 2B
β1 Na/K ATPase
Nav1.5
Nav1.4
mRNA Expression (AU)
4
Gata 4
700
α1 skeletal actin
C
α2 Na/K ATPase
600
β MHC
0
α1 Na/K ATPase
mRNA Expression (AU)
0
BNP
mRNA Expression (AU)
A
60
§
40
20
#
Online Table I. Genes Analyzed by TaqMan Low Density Arrays (TLDA)
Gene
Protein
18S RNA
Gene/Protein Name
NCBI Gene Reference
Assay ID
Eukaryotic 18s rRNA
X03205
4342379-18S
Abcc8
SUR1
ATP-binding cassette, sub-family C (CFTR/MRP), member 8
NM_011510
Mm00803450_m1
Abcc9
ATP-binding cassette, sub-family C (CFTR/MRP), member 9
NM_011511
Mm00441638_m1
Acta1
SUR2
α1 skeletal actin
actin, alpha 1, skeletal muscle
NM_009606
Mm00808218_g1
Actn2
α2 actinin
actinin alpha 2
NM_033268
Mm00473657_m1
Ank2
AnkB
ankyrin 2, brain
NM_178655
Mm00618325_m1
Ank3
AnkG
α1 Na/K ATPase
ankyrin 3, epithelial
NM_146005
Mm00464776_m1
Atp1a1
ATPase, Na+/K+ transporting, alpha 1 polypeptide
NM_144900
Mm00523255_m1
Atp1a2
α2 Na/K ATPase
ATPase, Na+/K+ transporting, alpha 2 polypeptide
NM_178405
Mm00617899_m1
Atp1b1
β1 Na/K ATPase
ATPase, Na+/K+ transporting, beta 1 polypeptide
NM_009721
Mm00437612_m1
Atp2a2
SERCA2a
ATPase, Ca++ transporting, cardiac muscle, slow twitch 2
NM_009722
Mm00437634_m1
Cacna1c
Cav1.2
calcium channel, voltage-dependent, L type, alpha 1C subunit
NM_009781
Mm00437917_m1
Cacna1g
calcium channel, voltage-dependent, T type, alpha 1G subunit
NM_009783
Mm00486549_m1
Cacna2d1
Cav3.1
Cavα2δ1
calcium channel, voltage-dependent, alpha2/delta subunit 1
NM_009784
Mm00486607_m1
Cacnb2
Cavβ2
calcium channel, voltage-dependent, beta 2 subunit
NM_023116
Mm00659092_m1
Calm1
calmodulin 1
NM_009790
Mm00486655_m1
Camk2d
Calm1
CamkIIδ
calcium/calmodulin-dependent protein kinase II, delta
NM_023813
Mm00499266_m1
Casq2
Casq2
calsequestrin 2
NM_009814
Mm00486742_m1
Cav3
Caveolin 3
caveolin 3
NM_007617
Mm00725536_s1
Cdh2
Cadherin 2
cadherin 2
NM_007664
Mm00483213_m1
Cnn1
calponin 1
NM_009922
Mm00487032_m1
Col1a1
Calponin 1
α1 procollagen 1
procollagen, type I, alpha 1
NM_007742
Mm00801666_g1
Dlgh1
SAP-97
discs, large homolog 1 (Drosophila)
NM_007862
Mm00492174_m1
Dlgh4
PSD-95
discs, large homolog 4 (Drosophila)
NM_007864
Mm00492193_m1
Dmd
Dystrophin
dystrophin, muscular dystrophy
NM_007868
Mm00464475_m1
Dpp4
Dpp4
dipeptidylpeptidase 4
NM_010074
Mm00494548_m1
Dpp6
Dpp6
dipeptidylpeptidase 6
NM_010075
Mm00456605_m1
Dpp7
Dpp7
dipeptidylpeptidase 7
NM_031843
Mm00473420_m1
Dpp8
Dpp8
dipeptidylpeptidase 8
NM_028906
Mm00547049_m1
Dpp9
dipeptidylpeptidase 9
NM_172624
Mm00841122_m1
Dtna
Dpp9
α dystrobrevin
dystrobrevin alpha
NM_207650
Mm00494555_m1
Flnc
Filamin C, γ
filamin C, gamma (actin binding protein 280)
AF119148
Mm00471824_m1
Freq
Frequenin
frequenin homolog (Drosophila)
NM_019681
Mm00490552_m1
Gapdh
Gapdh
glyceraldehyde-3-phosphate dehydrogenase
NM_001001303
Mm99999915_g1
Gata4
Gata4
GATA binding protein 4
NM_008092
Mm00484689_m1
Gja1
Cx43
gap junction membrane channel protein alpha 1
NM_010288
Mm00439105_m1
Gja5
Cx40
gap junction membrane channel protein alpha 5
NM_008121
Mm00433619_s1
Gja7
Cx45
gap junction membrane channel protein alpha 7
NM_008122
Mm00433624_m1
Hcn2
Hcn2
hyperpolarization-activated, cyclic nucleotide-gated K+ 2
NM_008226
Mm00468538_m1
Hprt1
Hprt1
hypoxanthine guanine phosphoribosyl transferase 1
NM_013556
Mm00446968_m1
Hspa1l
Hsp70
heat shock protein 1-like
NM_013558
Mm00442854_m1
Hspca
Hsp90
heat shock protein 1, alpha
NM_010480
Mm00658568_gH
Il6
IL6
interleukin 6
NM_031168
Mm00446190_m1
Irx5
Irx5
Iroquois related homeobox 5 (Drosophila)
NM_018826
Mm00502107_m1
Itpr2
IP3R-2
inositol 1,4,5-triphosphate receptor 2
NM_010586
Mm00444937_m1
Kcna1
Kv1.1
potassium voltage-gated channel, shaker-related subfamily, member 1
NM_010595
Mm00439977_s1
Kcna2
Kv1.2
potassium voltage-gated channel, shaker-related subfamily, member 2
NM_008417
Mm00434584_s1
Kcna4
Kv1.4
potassium voltage-gated channel, shaker-related subfamily, member 4
NM_021275
Mm00445241_s1
Kcna5
Kv1.5
potassium voltage-gated channel, shaker-related subfamily, member 5
NM_145983
Mm00524346_s1
Kcna6
potassium voltage-gated channel, shaker-related, subfamily, member 6
NM_013568
Mm00496625_s1
Kcnab1
Kv1.6
Kvβ1
potassium voltage-gated channel, shaker-related subfamily, beta member 1
NM_010597
Mm00440018_m1
Kcnab2
Kvβ2
potassium voltage-gated channel, shaker-related subfamily, beta member 2
NM_010598
Mm00440022_m1
Kcnb1
Kv2.1
potassium voltage gated channel, Shab-related subfamily, member 1
NM_008420
Mm00492791_m1
Kcnd1
Kv4.1
potassium voltage-gated channel, Shal-related family, member 1
NM_008423
Mm00492796_m1
Kcnd2
Kv4.2
potassium voltage-gated channel, Shal-related family, member 2
NM_019697
Mm00498065_m1
Kcnd3
Kv4.3
potassium voltage-gated channel, Shal-related family, member 3
NM_019931
Mm00498260_m1
Kcne1
MinK
potassium voltage-gated channel, Isk-related subfamily, member 1
X60457
Mm00434615_m1
Kcnh2
Merg
potassium voltage-gated channel, subfamily H (eag-related), member 2
NM_013569
Mm00465370_m1
Kcnip1
KChIP1
Kv channel-interacting protein 1
NM_027398
Mm00471928_m1
Kcnip2
KChIP2
Kv channel-interacting protein 2
NM_030716
Mm00518914_m1
Kcnj11
Kir6.2
potassium inwardly rectifying channel, subfamily J, member 11
NM_010602
Mm00440050_s1
Kcnj12
Kir2.2
potassium inwardly-rectifying channel, subfamily J, member 12
NM_010603
Mm00440058_s1
Kcnj2
Kir2.1
potassium inwardly-rectifying channel, subfamily J, member 2
NM_008425
Mm00434616_m1
Kcnj3
Kir3.1
potassium inwardly-rectifying channel, subfamily J, member 3
NM_008426
Mm00434618_m1
Kcnj8
Kir6.1
potassium inwardly-rectifying channel, subfamily J, member 8
NM_008428
Mm00434620_m1
Kcnq1
potassium voltage-gated channel, subfamily Q, member 1
NM_008434
Mm00434641_m1
Myh7
KvLQT1
β MHC
myosin, heavy polypeptide 7, cardiac muscle, beta
NM_080728
Mm00600555_m1
Nedd4l
Nedd4-like
neural precursor cell expressed, developmentally down-regulated gene 4-like
NM_031881
Mm00459584_m1
Mm00452375_m1
Nfatc4
Nfatc4
nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 4
NM_023699
Nos1
Nos1
nitric oxide synthase 1, neuronal
NM_008712
Mm00435175_m1
Nos2
Nos2
nitric oxide synthase 2, inducible, macrophage
NM_010927
Mm00440485_m1
Nos3
Nos3
nitric oxide synthase 3, endothelial cell
NM_008713
Mm00435204_m1
Nppb
BNP
natriuretic peptide precursor type B
NM_008726
Mm00435304_g1
Pias3
KChAP
protein inhibitor of activated STAT 3
NM_146135
Mm00450739_m1
Pln
PLB
phospholamban
NM_023129
Mm00452263_m1
Polr2a
RNA Polymerase II
polymerase (RNA) II (DNA directed) polypeptide A
NM_009089
Mm00839493_m1
Ppap2b
phosphatidic acid phosphatase type 2B
NM_080555
Mm00504516_m1
Prkaca
Calcineurin 2B
PKA, catalytic, α
protein kinase, cAMP dependent, catalytic, alpha
NM_008854
Mm00660092_m1
Prkca
PKC, α
protein kinase C, alpha
NM_011101
Mm00440855_m1
Prkcb1
PKC, β1
protein kinase C, beta 1
NM_008855
Mm00435749_m1
Prkcd
PKC, δ
protein kinase C, delta
NM_011103
Mm00440891_m1
Prkce
PKC, ε
protein kinase C, epsilon
NM_011104
Mm00440894_m1
Ryr2
ryanodine receptor 2, cardiac
NM_023868
Mm00465877_m1
Scn1b
Ryr2
Navβ1
sodium channel, voltage-gated, type I, beta
NM_011322
Mm00441210_m1
Scn4a
Nav1.4
sodium channel, voltage-gated, type IV, alpha polypeptide
NM_133199
Mm00500103_m1
Scn5a
Nav1.5
sodium channel, voltage-gated, type V, alpha
NM_021544
Mm00451971_m1
Scn7a
Nav2.3
sodium channel, voltage-gated, type VII, alpha
NM_009135
Mm00801952_m1
Slc8a1
NCX1
solute carrier family 8 (sodium/calcium exchanger), member 1
NM_011406
Mm00441524_m1
Smyd1
m-Bop
SET and MYND domain containing 1
NM_009762
Mm00477663_m1
Snta1
Syntrophin a1
syntrophin, acidic 1
NM_009228
Mm00486270_m1
Sntb1
Syntrophin b1
syntrophin, basic 1
NM_016667
Mm00489473_m1
Sntb2
Syntrophin b2
syntrophin, basic 2
NM_009229
Mm00486275_m1
Sod3
Sod3
superoxide dismutase 3, extracellular
NM_011435
Mm00448831_s1
Stx1a
Syntaxin 1A
syntaxin 1A (brain)
NM_016801
Mm00444008_m1
Uchl1
UCHL1
ubiquitin carboxy-terminal hydrolase L1
NM_011670
Mm00495900_m1
Zfpm2
Fog2
zinc finger protein, multitype 2
NM_011766
Mm00496074_m1
Online Table II. Echocardiographic Parameters of Sham and TAC Mice 7 Days After TAC.
Sham
n
TAC
n
P value
BW
LVPWd
IVSd
LVIDd
LVPWs
IVSs
LVIDs
FS
LVM
LVM/BW
E/A
Dop Vel
(g)
(mm)
(mm)
(mm)
(mm)
(mm)
(mm)
(%)
(mg)
(mg/g)
(RU)
(m/s)
24.2 ± 0.5
0.63 ± 0.01
0.67 ± 0.01
3.42 ± 0.05
1.35 ± 0.03
1.47 ± 0.03
1.46 ± 0.05
57.5 ± 0.9
67.9 ± 1.8
2.8 ± 0.06
1.16 ± 0.05
1±0
18
18
18
18
18
18
18
18
18
18
10
18
22.5 ± 0.3
0.81 ± 0.02
0.89 ± 0.01
3.26 ± 0.04
1.46 ± 0.03
1.60 ± 0.03
1.46 ± 0.06
55.6 ± 1.5
91.4 ± 2.2
4.07 ± 0.1
1.61 ± 0.15
4.8 ± 0.09
39
39
39
39
39
39
39
39
39
39
9
39
<0.01
<0.001
<0.001
<0.05
<0.05
<0.001
NS
NS
<0.001
<0.001
<0.05
<0.001
All values are means ± SEM. Abbreviations: BW, body weight; LVPWd, LV posterior wall thickness at end-diastole; IVSd, interventricular septum thickness at end-diastole; LVIDd, LV internal dimension at enddiastole; LVPWs, LV posterior wall thickness at end-systole; IVSs, interventricular septum thickness at end-systole; LVIDs, LV internal dimension at end-systole; FS, fractional shortening; LVM, LV mass;
LVM/BW, LV mass to body weight ratio; E/A, E/A wave ratio; RU, relative units; Dop Vel, peak flow velocity across TAC; NS, not significant.
Online Table III. Resting and Active Membrane Properties of Sham and TAC LV and RV Myocytes.
Sham
TAC
Cm
Vm
APD90
(mV)
APA
(mV)
APD50
(pF)
(ms)
(ms)
LV
127 ± 9
73 ± 1
56 ± 0.3
3 ± 0.2
25 ± 3
12
RV
107 ± 7
71 ± 1
58 ± 1.3
2 ± 0.1
10 ± 1
9
LV
170 ± 8#
69 ± 1
60 ± 0.5
5 ± 0.7
36 ± 3#
19
RV
126 ± 13
70 ± 2
58 ± 1.1
2 ± 0.2
11 ± 1
7
n
All values are means ± SEM; Abbreviations: Cm, whole-cell membrane capacitance; Vm, resting membrane potential, APA, action
potential amplitude; APD50 and APD90, action potential durations at 50% and 90% repolarization. #P<0.01 in TAC, compared with
sham, LV myocytes.
Online Table IV. ECG Parameters in Anesthetized Sham and TAC Mice Before and After Surgery.
Sham
QTc
RR
(ms)
PR
(ms)
QRS
(ms)
QT
(ms)
(ms)
J
(mV)
Before
124 ± 4
45 ± 1
8 ± 0.3
58 ± 2
52 ± 2
0.07 ± 0.01
After
147 ± 5
55 ± 4
9 ± 0.4
61 ± 4
50 ± 4
0.06 ± 0.02
Before
147 ± 8
49 ± 2
9 ± 0.3
62 ± 4
51 ± 4
0.05 ± 0.02
After
163 ± 9
52 ± 1
9 ± 0.5
85 ± 8§
67 ± 6§
-0.01 ± 0.01§
n
8
TAC
8
All values are means ± SEM; Abbreviations: RR, PR, QRS, QT and QTc: RR-, PR-, QRS-, QT- and QTc-interval durations; J, J-wave amplitude.
§P<0.05 in TAC, compared with sham, mice.