Download Cardiac Contractility Modulation Electrical Signals Normalize Activity

Journal of Cardiac Failure Vol. 15 No. 1 2009
Basic Science and Experimental Studies
Cardiac Contractility Modulation Electrical Signals
Normalize Activity, Expression, and Phosphorylation
of the NaD-Ca2D Exchanger in Heart Failure
RAMESH C. GUPTA, PhD,1 SUDHISH MISHRA, PhD,1 MENGJUN WANG, MD,1 ALICE JIANG, MD,1 SHARAD RASTOGI, MD,1
BENNY ROUSSO, PhD,2 YUVAL MIKA, PhD,2 AND HANI N. SABBAH, PhD1
Detroit, Michigan; Orangeburg, New York
ABSTRACT
Background: Expression and phosphorylation of the cardiac Naþ-Ca2þ exchanger-1 (NCX-1) are upregulated in heart failure (HF). We examined the effects of chronic cardiac contractility modulation
(CCM) therapy on the expression and phosphorylation of NCX-1 and its regulators GATA-4 and FOG2 in HF dogs.
Methods and Results: Studies were performed in LV tissue from 7 CCM-treated HF dogs, 7 untreated
HF dogs, and 6 normal (NL) dogs. mRNA expression of NCX-1, GATA-4, and FOG-2 was measured using reverse transcriptase polymerase chain reaction, and protein level was determined by Western blotting.
Phosphorylated NCX-1 (P-NCX) was determined using a phosphoprotein enrichment kit. Compared with
NL dogs, NCX-1 mRNA and protein expression and GATA-4 mRNA and protein expression increased in
untreated HF dogs, whereas FOG-2 expression decreased. Compared with NL dogs, the level of P-NCX-1
normalized to total NCX-1 increased in untreated HF dogs (0.80 6 0.10 vs 0.37 6 0.04; P ! .05). CCM
therapy normalized NCX-1 expression, GATA-4, and FOG-2 expression, and the ratio of P-NCX-1 to total
NCX-1 (0.62 6 0.10).
Conclusion: Chronic monotherapy with CCM restores expression and phosphorylation of NCX-1. These
findings are consistent with previous observations of improved LV function and normalized sarcoplasmic
reticulum calcium cycling in the left ventricles of HF dogs treated with CCM therapy. (J Cardiac Fail
2009;15:48e56)
Key Words: Animal models, Congestive heart failure, Contractility, Gene expression, Protein expression,
Protein phosphorylation, Sodium-calcium exchanger, Ventricular performance.
Heart failure (HF) remains a leading cause of mortality
and morbidity in developed countries despite considerable
advances in therapy. Angiotensin-converting enzyme
(ACE) inhibitors, ß-adrenergic receptor blockers, and,
more recently, aldosterone receptor antagonists have markedly improved survival in patients with chronic HF.1e3 Despite these improvements, a large number of patients with
advanced HF are refractory to standard medical therapy
and follow a clinical course of progressive worsening of
the disease state manifested by multiple episodes of cardiac
decompensation that ultimately culminate in death. The use
of drugs that increase cardiac contractility in this patient
population, such as dobutamine and milrinone, that also increase myocardial oxygen consumption is often associated
with increased mortality.4e6 The need for further therapeutic interventions in this patient population has given rise to
a host of device-based therapies, such as cardiac resynchronization therapy. Resynchronization therapy has been
shown to improve left ventricular (LV) function in
patients with HF but is most likely effective only in those
patients with a wide QRS complex suggestive of intraventricular conduction disturbances.7e12 Electrical signals,
termed ‘‘cardiac contractility modulation (CCM)’’ signals,
that are delivered to the failing myocardium during the
From the 1Department of Medicine, Division of Cardiovascular Medicine,
Henry Ford Heart and Vascular Institute, Detroit, Michigan; and 2Impulse
Dynamics (USA) Inc., Orangeburg, New York.
Manuscript received December 21, 2007; revised manuscript received
August 18, 2008; revised manuscript accepted August 29, 2008.
Reprint requests: Hani N. Sabbah, PhD., Director, Cardiovascular Research, Henry Ford Health System, 2799 West Grand Boulevard, Detroit,
Michigan 48202.
1071-9164/$ - see front matter
Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.cardfail.2008.08.011
48
Cardiac Sodium-Calcium Exchanger in Heart Failure
absolute refractory period are another device-based therapy
targeting this population with advanced HF. Studies in patients with HF and animal models of experimentally induced HF have shown that CCM therapy is safe,
improves LV systolic function, and is associated with improved quality of life in patients.4,13e18 The improvement
in LV systolic function elicited by CCM therapy is not associated with an increase of myocardial oxygen consumption.13,17
Intracellular Ca2þ homeostasis is clearly abnormal in
constituent cardiomyocytes of the failing heart.19e22 In normal cardiomyocytes, excitation-contraction coupling is initiated by influx of Ca2þ through voltage-dependent Ca2þ
channels that, in turn, trigger the release of Ca2þ from
the sarcoplasmic reticulum (SR). Ca2þ influx into the cell
is balanced by Ca2þ efflux, the latter mediated by the cardiac-specific sarcolemmal Naþ-Ca2þ exchanger-1 (NCX1), which plays a major role for extruding Ca2þ out of
the cell and the adenosine triphosphate-dependent Ca2þ
pump. Several studies have reported that activity of NCX1, protein expression, and phosphorylation are increased
in experimental HF and human failing hearts.19,23e28
Recent studies have shown that the transcriptional factor
GATA-4 is activated in failing hearts and modulates expression of NCX-1.29,30 Other studies have shown that the
expression of GATA-4 is regulated by its cofactor FOG231e33 via interaction with the N-terminal zinc finger of
GATA-4.34 The present study explored the potential effects
of chronic CCM therapy on the activity, expression, and
phosphorylation of the cardiac-specific NCX-1 and on the
expression of its direct and indirect regulators GATA-4
and FOG-2 in LV myocardium of dogs with intracoronary
microembolization-induced HF.
Materials and Methods
Materials
Dogs were purchased from Marshal Farms (North Rose, NY).
Chemicals and supplies for electrophoresis and electrotransfer
were purchased from Bio-Rad (Hercules, CA). Biochemical supplies were obtained from Sigma Chemical (St Louis, MO). Gene
primers were synthesized by Operon Technologies, Inc (Alameda,
CA). Primary antibodies for NCX-1 and calsequestrin (CSQ) were
obtained from ABR, Inc (Golden, CO), and primary antibodies for
Fog-2 and GATA-4 were obtained from Santa Cruz Biotechnologies, Inc (Santa Cruz, CA). Secondary antibodies were obtained
from Amersham Biosciences (Piscataway, NJ).
Animal Model
The canine model of chronic HF used in the present study has
been described in detail.35 In the present study, HF was produced
by intracoronary microembolizations in 14 healthy mongrel dogs
weighing 19 and 30 kg. Microembolizations were performed during cardiac catheterizations under general anesthesia and sterile
conditions. Animals were induced with intravenous oxymorphone
hydrochloride (0.22 mg/kg) and diazepam (0.17 mg/kg), and
a plane of anesthesia was maintained with 1% to 2% isoflurane.
The study was approved by the Henry Ford Heath System
Gupta et al
49
Institutional Animal Care and Use Committee and conformed to
the National Institutes of Health ‘‘Guide and Care for Use of Laboratory Animals’’ and ‘‘Position of the American Heart Association on Research Animal Use.’’
Device Implantation and Protocol
Two weeks after the target LV ejection fraction (!30%) was
reached, all HF dogs were anesthetized as above. The right external jugular vein was exposed and used to position the CCM leads
as previously described.17 Briefly, 2 standard active fixation leads
were advanced into the right ventricle, positioned on the anterior
and posterior septal grooves, and used to sense ventricular activity
and deliver CCM signals (Figure 1). A third lead was positioned in
the right atrium for P-wave sensing. The leads were connected to
a CCM signal generator (Figure 1) (OPTIMIZER II, Impulse Dynamics USA, Inc, Orangeburg, NY) that was implanted in a subcutaneous pocket created on the right side of the neck. Two weeks
after OPTIMIZER implantation, HF dogs were randomized to
an active treatment group (n 5 7) or a sham-operated control
group (n 5 7). In the active treatment group, the OPTIMIZER
was activated to deliver CCM therapy. CCM therapy was administered for 5 hours per day based on a duty cycle of 1 hour ON
(CCM signal 6 7.73 volts) and 3 hours and 48 minutes OFF for
3 months. In sham-operated dogs, the OPTIMIZER was not activated and dogs were also followed for 3 months. At the end of 3
months of therapy, and while under general anesthesia, the dogs’
chests were opened and the hearts were rapidly harvested, and tissue from the LV free wall was obtained and prepared for biochemical evaluation. LV tissue from 6 NL dogs was obtained for
comparisons. All tissue was stored at 70 C until needed.
Isolation and Orientation of Isolated Sarcolemmal
Membranes
Sarcolemmal membrane fractions were isolated from LV tissue
as previously described.36 Sarcolemmal protein was measured by
Lowry’s method, and the protein yield was calculated and
expressed as milligrams of sarcolemmal protein per gram of LV
tissue. Sarcolemmal Mg2þ-adenosine triphosphatase (ATPase) activity, which was not altered in failing heart,19 was determined as
previously described19 and expressed as mmol Pi released/milligrams of protein per hour. To calculate leakiness and sidedness
of the isolated sarcolemmal preparations, specific endoenzyme,
Mg2þ-ATPase and exoenzyme, 5’nucleotidase was also measured
in isolated sarcolemmal vesicles in the absence and presence of
alamethicin as previously described.37 Isolated membrane was
treated with and without alamethicin at a ratio of 1/1.5’ nucleotidase activity inhibited by adenosine 5’-[a, ß-methylene] diphosphonate was measured as previously described and expressed as
micromoles of substrate liberated per hour per milligram of protein. On the basis of the activities of exo- and endoenzymes in
the absence and presence of alamethicin, percent of inside-out,
right-side out vesicles, and leakiness was calculated as previously
described.38
NCX-1 Activity Measurement
NCX-1 activity, measured as Naþ-dependent Ca2þ uptake, was
implemented as previously described previously19 with some
modifications. Briefly, 10 mL of sarcolemmal membrane (2 mg/
mL; 20 mg protein/tube) preloaded with NaCl-MOPS buffer at
37 C for 30 minutes were rapidly diluted 50 times with Ca2þ uptake medium containing 140 mmol/L KCl, 20 mmol/L MOPS, 0.4
50 Journal of Cardiac Failure Vol. 15 No. 1 February 2009
A
B
RA Lead
RA
LV
CCM
Leads
RV
Fig. 1. A, The Optimizer-II generator (Impulse Dynamics (USA) Inc., Orangeburg, NY) attached to 3 standard active fixation pacing leads. B,
Diagram illustrating the location of CCM leads. RA, right atrium; RV, right ventricle; LV, left ventricle; CCM, cardiac contractility modulation.
mM valinomycin, and 0.5 mCi 45Ca2þ, pH 7.4. After 20,’’ the
reaction was stopped by adding ice-cold 30 mL of the stopping
solution containing 140 mmol/L KCl, 1 mmol/L LaCl3, and
20 mmol/L MOPS, pH 7.4. Radioactivity was retained on glass
fiber filter, washed 3 times with 2 mL of stopping solution, and
counted with a Beckman LS scintillation counter. In parallel
with these samples, nonspecific Ca2þ-uptake was measured by
placing the Naþ-loaded sarcolemmal membrane in Ca2þ uptake
medium containing 140 mmol/L NaCl instead of KCl. Naþdependent Ca2þ uptake activity (NCX-1 activity) was calculated
by subtracting the nonspecific Ca2þ uptake value from the total
Ca2þ uptake activity and expressed as nmol45Ca2þ/mg protein/20.’’
mRNA Expression
Total RNA was isolated from frozen LV tissue in RNA Stat-60
(Tel-Test Inc, Friendswood, TX) using the guanidinium thiocyanate phenol-chloroform method according to the manufacturer’s
instructions. Concentration and quality of the isolated RNA in
each sample were determined spectrophotometrically. RNA with
an absorbance ratio (260 nm/280 nm) O 1.7 and that exhibited 3
major bands, namely, 28S, 18S, and 5.8S on 1.2% agarose with
28S being much stronger than 18S, was considered of good
quality. Approximately 5 mg RNA was reverse-transcribed into
cDNA in an assay volume of 100 mL using the High-capacity
cDNA Archive Kit (Applied Biosystems). The assay contained
(final concentration) 1 reverse transcriptase buffer, 1 deoxyribonucleoside triphosphate (deoxyadenosine triphosphate, deoxythymidine triphosphate, deoxyguanosine triphosphate, and
deoxycytidine triphosphate), 1 random primers, 250 units MultiScribe reverse transcriptase, 50 units RNasin (Invitrogen). Assay
tubes were incubated at 42 C for 60 minutes and then at 96 C for
10 minutes for denaturation. For each polymerase chain reaction,
2 mL first-strand cDNA was added to 18 mL of a reaction mixture
containing 20 pmol of each gene-specific forward and reverse
primer, 200 mM of each dNTP, 10 mmol/L Tris-HCl (pH 8.8),
50 mmol/L KCl, 0.1% Triton-X100, and 3.0 mmol/L MgCl2. After
heating the tube to 95 C for 5 minutes, 1-unit platinum Taq DNA
polymerase (Invitrogen, Carlsbad, CA) was added, and the polymerase chain reaction was allowed to proceed for 20 to 40 cycles.
Polymerase chain reaction products were analyzed by subjecting
20 mL of each reaction mixture to electrophoresis on 1% to
1.5% ethidium-bromide-agarose gels. Band size of the products
was compared with standard DNA size markers and confirmed
by sequencing. The forward (F) and reverse (R) primers for
NCX-1 ((F: 5’-GCCATGTCCGACAGCGAGAAG-3’; R: 5’eTC
CATGTTCCCCGTGACAGGTG-3’),GATA-4 (F: 5’-GTGTCA
AYTGTGGGGCYATGT -3’; R: 5’-ATTTATTCAGGTTCTT C3’), FOG-2: (F: 5’-AGGAGTGGAAGACAGCAAAAC -3’; R:
5’eCTTGAGT GAGA TGCGAGAACAG -3’), and 18s (a housekeeping gene): (F: 5’-TCAAGAACGAAA GT CGGAGG-3,’ R:
5’-GGACATCTAAGGGCATCAC-3’) were based on the gene sequences reported to GenBank. The amplified products exhibited
0.43 kb, 0.35 kb, 0.40 kb, and 0.528 kb product size for NCX-1,
GATA-4, FOG-2, and 18S, respectively. Band intensity was quantified in arbitrary densitometric units (du) using a Bio-Rad Gel
densitometer.
Protein Immunoblot Analysis
Protein levels of NCX-1, GATA-4, FOG-2, and CSQ, a cardiac
SR Ca2þ-binding protein reportedly unchanged in HF,39 were
measured in sodium dodecyl sulfate extract of LV tissue using
Western blotting as previously described.39,40,41 A primary monoclonal or polyclonal antibody specific to each protein was diluted
according to the supplier’s instructions. In all instances, the
Cardiac Sodium-Calcium Exchanger in Heart Failure
antibody was present in excess over the antigen and the density of
each protein band was in the linear scale. Band intensity was
quantified using a Bio-Rad gel densitometer and expressed in
densitometric units.
Gupta et al
51
Na+-dependent Ca2+-uptake in Sarcolemma
(nmol/mg protein/20”)
350
*
300
Measurement of Phosphorylated NCX-1
Phosphorylated NCX-1 in LV tissue was determined by 2 procedures: indirect (by in vitro phosphorylation) and direct (using
BD Bioscience [San Jose, CA] phosphoprotein enrichment kit).
Briefly, for the indirect method, NCX-1 was immunoprecipitated
from 200 mg protein extract of LV tissue as previously described.27
Half of the immunoprecipitated sample was used for the Western
blotting, and the rest was used for in vitro phosphorylation. In vitro phosphorylation was performed in 1 mg of the catalytic subunit
of cyclic adenosine monophosphate-dependent protein kinase
(Sigma) and 10 mCi of [g-32P] adenosine triphosphate at 37 C
for 15 minutes and the reaction was stopped as previously described.27 Both samples for Western blot and protein kinase A
phosphorylation were subjected to 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis followed by electrotransfer
of proteins from gel to nitrocellulose. Blot for Western blot was
developed as described above in the section of ‘‘Protein Immunoblot Analysis.’’ The blot for in vitro phosphorylation was
subjected to autoradiography, and the band’s intensity was quantified using a Bio-Rad gel densitometer.
Phosphorylated NCX-1 was also determined by direct method,
that is, Western blotting in phosphoprotein-enriched fraction prepared from LV tissue using BD Bioscience (San Jose, CA) phosphoprotein enrichment kit according to the supplier’s instructions
as previously described40 for protein phosphatase 1 inhibitor-2.
This kit uses a Phosphate Metal Affinity Chromatography Resin,
which binds proteins that carry a phosphate group on any amino
acid, including serine, tyrosine, or threonine. Non-phosphorylated
proteins are simply passed through the resin and enriched solutions of phosphorylated proteins are eluted from the column. In
both methods, phosphorylated NCX-1 was normalized to total
NCX-1 and represents partial normalization.
Data Analysis
Comparisons among study groups were carried out using 1-way
analysis of variance, with a set at 0.05. If significance was
achieved, pairwise comparisons were performed using the Student-Neuman-Keuls test. For this test, a probability value less
than .05 was considered significant. All data are expressed as
means 6 standard error of the mean.
Results
The hemodynamic results obtained in all dogs included
in this study have been reported.42 Briefly, in sham-operated HF control dogs, LV ejection fraction measured angiographically decreased from 27% 6 1% to 23% 6 1%
during the 3 months of follow-up (P 5 .001). In contrast,
LV ejection fraction increased from 27% 6 1% before initiating CCM therapy to 33% 6 1% after 3 months of CCM
therapy (P 5 .0001).20
250
200
**
150
100
50
0
NL
HF-Sham
HF+CCM
Fig. 2. Naþ-dependent Ca2þ uptake activity (NCX-1 activity) in
sarcolemmal membrane isolated from LV tissue of NL dogs, untreated HF dogs (HF-sham), and HF dogs treated with chronic
CCM monotherapy for 3 months (HF þ CCM). *P ! .05 vs NL
and **P ! .05 vs HF-sham. Values are average of the standard
error of the mean of 6 NL dogs, 7 untreated HF dogs, and 7
CCM-treated HF dogs. NL, normal; HF, heart failure; CCM, cardiac contractility modulation.
122.33 6 10* nmol 45Ca2þ uptake/mg protein.20’’1, *P
! .05) (Figure 2). This maladaptation was normalized in
HF dogs treated with chronic CCM monotherapy
(160.17 6 11** nmol 45Ca2þ uptake/mg protein.20’’1,
**P ! .05 vs untreated-HF dogs, Figure 2). The beneficial
effects of chronic CCM monotherapy on sarcolemmal
NCX-1 activity was not confounded by artifacts of the
sarcolemmal preparation because the sarcolemmal yield
(NL: 0.73 6 0.04, HF-sham: 0.72 6 0.07, HF þ CCM:
0.70 6 0.08 mg/g LV tissue) and Mg2þ-ATPase activity
(NL: 64.56 6 6, HF-sham: 66.01 6 5, HF þ CCM:
70.51 6 7 mmolPi released/mg protein. h1) were not affected by CCM monotherapy. In addition, orientation,
right-side out and inside-out vesicles and leakiness of the
isolated sarcolemmal membranes in all 3 groups are not
different (Table 1).
Expression of 18s, CSQ, and NCX-1
mRNA expression of 18s was unchanged among all 3
study groups. The band intensity of 18s in NL dogs, untreated HF controls, and CCM-treated dogs was 16.8 6 0.3
du, 17.1 6 0.1 du, and 17.2 6 0.3 du, respectively. NCX-1
mRNA expression increased significantly in control HF
Table 1. Orientation and Percentage of Leakiness of the
Isolated Sarcolemmal Vesicles
NL
HF-Sham
HF þ CCM
76.00 6 2.04
19.58 6 2.76
4.43 6 0.45
76.24 6 3.23
19.75 6 1.96
4.11 6 0.42
76.09 6 1.46
19.82 6 1.33
4.10 6 0.40
NCX-1 Activity
ROV (%)
IOV (%)
Leaky (%)
NCX-1 activity in sarcolemmal membrane was increased
in LV tissue of HF dogs compared with NL (278.33 6 22 vs
ROV, right-side out vesicle; IOV, inside-out vesicle; NL, normal; HF,
heart failure; CCM, cardiac contractility modulation.
52 Journal of Cardiac Failure Vol. 15 No. 1 February 2009
mRNA Expression of NCX-1
(Densitometric units)
140.0
Protein Expression of NCX-1
(Densitometric units)
8.0
A
120.0
7.0
*
C
*
6.0
100.0
5.0
**
80.0
4.0
60.0
**
3.0
40.0
2.0
20.0
1.0
0.0
NL
B
HF-Sham
HF+CCM
NCX-1
0.0
NL
HF-Sham
HF+CCM
D
NCX-1
18s
NL
HF-Sham
HF+CCM
CSQ
NL
HF-Sham
HF+CCM
Fig. 3. A, Band intensity in densitometric units for mRNA expression of NCX-1 in LV myocardium of 6 NL dogs, 7 untreated shamoperated heart failure (HF-sham) control dogs, and 7 HF CCM-treated dogs (CCM þ HF). B, Ethidium-bromide-agarose gel electrophoretic
bands for NCX-1 and 18s ribosomal RNA in LV myocardium of 2 NL dogs, 2 HF-sham dogs and 2 HF CCM-treated dogs. C, Band intensity in densitometric units for protein level of Naþ-Ca2þ-exchanger-1 (NCX-1) in LV myocardium of 6 NL dogs, 7 untreated HF-sham
control dogs, and 7 HF CCM-treated dogs. D, Western blot showing NCX-1 and CSQ in 2 NL dogs, 2 untreated HF-sham control dogs, and
2 HF CCM-treated dogs. *P ! .05 vs NL; **P ! .05 vs HF-sham. NL, normal; HF, heart failure; CCM, cardiac contractility modulation;
NCX-1, Naþ-Ca2þ exchanger-1; CSQ, calsequestrin.
dogs compared with NL dogs (98 6 14 vs 30 6 2 du, P !
.05). Three months of CCM therapy restored mRNA expression of NCX-1 to near-normal level (48 6 4 du) (Figure 3).
Protein level of CSQ was essentially unchanged among all
3 study groups. The band intensity of CSQ in NL dogs, untreated HF control dogs, and CCM-treated dogs was
43 6 3 du, 40 6 1 du, and 44 6 3 du, respectively. Total
NCX-1 protein level increased significantly in HF control
dogs compared with NL dogs (6.37 6 0.31 vs 4.29 6 0.10
du, P ! .05) but returned to a near-normal level in CCMtreated dogs (3.78 6 0.40 du) (Figure 3).
Phosphorylated NCX-1
Back-phosphorylation experiments showed that 32Pincorporation into NCX1 was reduced significantly in
control HF dogs compared with NL dogs (Figure 4A).
This finding suggests that phosphorylation of NCX-1 was
increased in failing hearts. Because the amount of phosphorylated NCX-1 depends on the total amount of NCX1, which was found to be increased in control HF dogs,
phosphorylated NCX-1 was normalized to total NCX-1
estimated under similar conditions as shown in
Figure 4A. A ratio of phosphorylated NCX-1 to total
NCX-1 was found to be reduced (0.65 6 0.10 du vs
2.67 6 0.40 du, P ! .05 vs NL) significantly in HF control
dogs compared with NL (Figure 4A). Three months of therapy with CCM restored (1.39 6 0.20 du, P ! .05 vs HF)
the ratio near to normal (Figure 4A). Similar results were
also seen when phosphorylated NCX-1 was measured using
phosphoprotein enriched fractions followed by Western
blotting (Figure 4B). In the direct method, phosphoprotein
enriched fractions. The ratio of phosphorylated NCX-1 to
total NCX-1 was also found to be increased significantly
in control HF dogs compared with NL dogs (0.80 6 0.10
du vs 0.37 6 0.04 du, P ! .05). CCM therapy significantly
reduced this ratio (0.62 6 0.10 du, P ! .05) (Figure 4B).
Expression of GATA-4 and FOG-2
Because GATA-4 and FOG-2 are suggested to be direct
and indirect regulators of NCX-1, their expression was
measured in LV myocardium of CCM-treated dogs, untreated-HF dogs, and NL dogs. Expression of GATA-4
mRNA was increased in HF control dogs compared with
NL dogs (204 6 19 du vs 103 6 31, du; P ! .05), whereas
treatment with CCM signals reduced the expression to nearnormal levels (106 6 28, du) (Figure 5). Similar results
were found with respect to protein expression of GATA-4.
Protein level of GATA-4 was increased in HF control
dogs compared with NL dogs (138 6 11 vs 96 6 5, du; P
! .05), whereas treatment with CCM signals reduced the
expression to near-normal levels (110 6 10, du) (Figure 5).
Consistent with these findings, FOG-2 expression was significantly lower in HF control dogs compared with NL dogs
(mRNA: 2.10 6 0.14 du vs 4.80 6 0.60 du; P ! .05;
protein: 3.53 6 0.20 du vs 6.13 6 0.11 du; P ! .05) but
remained elevated in CCM-treated HF dogs (mRNA:
3.4 6 0.3 du; protein: 4.71 6 0.27 du) (Figure 6).
Discussion
CCM electrical signals delivered to the myocardium during the absolute refractory period have been shown to
Cardiac Sodium-Calcium Exchanger in Heart Failure
32P-NCX-1
4.0
normalized to Total NCX-1
A
3.0
2.0
**
1.0
*
0.0
NL
HF-Sham
HF+CCM
32
P-NCX-1
T-NCX-1
Phosphorylated NCX-1 normalized to total NCX-1
1.0
B
*
0.8
**
0.6
0.4
0.2
0.0
NL
HF-Sham
HF+CCM
Fig. 4. A, 32P-incorporation into NCX-1 normalized to total NCX1. Band intensity of 32P-NCX-1 phosphorylated by cyclic adenosine monophosphate-dependent protein kinase in back-phosphorylation experiment was normalized to total NCX-1 in LV
myocardium of 6 NL dogs, 7 untreated heart failure shamoperated (HF-sham) control dogs, and 7 CCM-treated HF dogs
(HF þ CCM). *P ! .05 vs NL; **P ! .05 vs HF-sham. B,
Band intensity of phosphorylated NCX-1 normalized to total
NCX-1 in LV myocardium of 6 NL dogs, 7 untreated heart failure
sham-operated (HF-sham) control dogs, and 7 CCM-treated HF
dogs (HF þ CCM). *P ! .05 vs NL; **P ! .05 vs HF-sham.
HF-sham. NL, normal; HF, heart failure; CCM, cardiac contractility modulation; P-NCX-1, Phosphorylated Naþ-Ca2þ exchanger1; T-NCX-1, total Naþ-Ca2þ exchanger-1.
improve LV function both when delivered acutely and after
long-term delivery in dogs with experimentally induced
HF.4,14,40 In HF dogs, CCM therapy was associated with
improved SR calcium cycling, as evidenced by increased
activity and expression of calcium-ATPase and increased
phosphorylation of phospholamban.40 CCM therapy in patients with chronic HF (New York Heart Association class
III symptoms) was found to be safe and was associated
Gupta et al
53
with improvements in patients with New York Heart Association class III for quality of life assessed by the Minnesota Living with Heart Failure Questionnaire and LV ejection
fraction.13,15e17 The results of the present study clearly
show that long-term therapy with CCM signals in dogs
with chronic HF is associated with normalization of activity, expression, and phosphorylation of the cardiac specific
NCX-1. The expression was associated with normalization
of its direct and indirect modulators GATA-4 and FOG-2.
The normalization of NCX-1 activity, expression, and phosphorylation in the failing heart after chronic CCM signals is
in line with previous observations of normalization of cardiac SR calcium cycling and represents a potential contributing factor to the improved LV function in HF.
The NCX-1 is a transsarcolemmal protein that plays an
important role in the control of intracellular Ca2þ levels
during the cardiac cycle.25,43,44 The exchanger has been regarded as a Ca2þ extrusion system that contributes to the
low diastolic Ca2þ levels and diastolic relaxation.43 There
is experimental evidence, however, that suggests that the
NCX also serves as a source of Ca2þ influx into the
cell.43 Apparently, the exchanger is responsible for the extrusion of approximately 20% of the Ca2þ from the cytosol
during diastole, whereas the second Ca2þ extrusion system,
the SR Ca2þ ATPase, is responsible for approximately 80%
of Ca2þ sequestration from the cytosol into the SR.43 The
reverse mode exchange can be enhanced by prolongation
of the action potential, which occurs in HF,45 decreasing
the Naþ gradient or increasing the transsarcolemmal Ca2þ
gradient. Activity of the NCX-1 is regulated by the levels
of intracellular Naþ and Ca2þ concentrations that are altered in HF.43,46 When SR function is impaired, a greater
dependence on trans-sarcolemmal NCX-1 is expected. In
the present study, increased Naþ-dependent Ca2þ uptake
activity representing reverse mode of NCX-1 activity was
found to be increased significantly, and this abnormality
may contribute to an increased cytosolic Ca2þ during diastole in the failing heart. With minimal exceptions,47,48 similar findings were also shown by other investigators in
hearts of cardiomyopathic hamsters.28,49 Although we did
not measure forward mode of NCX-1 activity, abnormal diastolic Ca2þ handling in cardiomyocytes isolated from rabbits with experimentally induced HF was attributed to
decreased forward mode and enhanced reverse mode of
the NCX-1 secondary to increased intracellular Naþ concentration and prolongation of the action potential.50 In
LV midmyocardial myocytes isolated from NL and tachycardiac pacing-induced failing canine hearts, when SR
function was blocked by thapsigargin, both reverse-mode
and forward-mode NCX currents were increased more
than 2-fold in failing cells.24 These authors maintained
that, in HF, enhanced reverse and forward mode may
account for increased contribution of the NCX-1 to E-C
coupling.51 In normal cardiomyocytes with normal SR
function, this Ca2þ influx helps maintain and regulate SR
Ca2þ load, whereas in failing cardiomyocytes with poor
SR function, this Ca2þ influx directly contributes to
54 Journal of Cardiac Failure Vol. 15 No. 1 February 2009
Protein Expression of GATA-4
(densitometric units)
mRNA Expression of GATA-4
(densitometric units)
300
250
175
A
150
*
B
*
**
125
200
100
150
**
75
100
50
50
25
0
0
NL
HF-Sham
HF+CCM
NL
HF-Sham
HF+CCM
GATA-4
NL
HF-Sham
GATA-4
HF+CCM
NL
HF-Sham
HF+CCM
Fig. 5. A, Band intensity in densitometric units for mRNA expression of GATA-4 in LV myocardium of 6 NL dogs, 7 untreated heart failure
sham-operated (HF-sham) control dogs, and 7 HF CCM-treated dogs (HF þ CCM). Ethidium-bromide-agarose gel electrophoretic bands
for GATA-4 ribosomal RNA in LV myocardium of 2 NL dogs, 2 untreated HF-sham control dogs, and 2 HF CCM-treated dogs. B,
Band intensity in densitometric units for protein level of GATA-4 in LV myocardium of 6 NL dogs, 7 untreated HF-sham control dogs,
and 7 HF CCM-treated dogs. Western blot showing GATA-4 in 2 NL dogs, 2 untreated HF-sham control dogs, and 2 HF CCM-treated
dogs. *P ! .05 vs NL; **P ! .05 vs HF-sham. NL, normal; HF, heart failure; CCM, cardiac contractility modulation.
contraction.51 These authors concluded that the Ca2þ transient of the failing human ventricular myocytes has a higher
than normal dependence on Ca2þ influx via the reverse
mode of the NCX-1.51 Thus, on face value, enhanced activity of the reverse mode of NCX-1 in HF seems to counter
the existing abnormality in SR function and may improve
contractility in the short term. Studies in isolated failed human tissue suggested that BDF 9148, a Naþ channel agonist, can enhance NCX activity and lead to increased
systolic contraction.43 However, long-term sustained increase in reversed mode NCX could be detrimental in
that it contributes to ‘‘calcium overload’’ that is characteristic of the disease state.
In addition to Naþ and Ca2þ concentrations, activity of
the NCX-1 is also regulated by its expression level. Observations made in the present study indicate that NCX-1
mRNA and protein expression levels in LV tissues of dogs
with intracoronary microembolization-induced HF are significantly increased compared with NL dogs. These observations are consistent with those reported in failing human
hearts43,44 and in animal models of experimentally induced
HF.52 In failed human hearts with end-stage idiopathic
mRNA Expression of FOG-2
(densitometric units)
6.0
5.0
Protein Expression of FOG-2
(densitometric units)
7.0
A
6.0
4.0
B
**
5.0
**
*
4.0
3.0
*
3.0
2.0
2.0
1.0
1.0
0.0
0.0
NL
HF-Sham
HF+CCM
NL
HF-Sham
HF+CCM
FOG-2
FOG-2
NL
HF-Sham
HF+CCM
NL
HF-Sham
HF+CCM
Fig. 6. A, Band intensity in densitometric units for mRNA expression of FOG-2 in LV myocardium of 6 NL dogs, 7 untreated heart failure
sham-operated (HF-sham) control dogs, and 7 HF CCM-treated dogs (HF þ CCM). Ethidium-bromide-agarose gel electrophoretic bands
for FOG-2 ribosomal RNA in LV myocardium of 2 NL dogs, 2 untreated HF-sham control dogs, and 2 HF CCM-treated dogs. B Band
intensity in densitometric units for protein level of FOG-2 in LV myocardium of 6 NL dogs, 7 untreated HF-sham control dogs, and 7
HF CCM-treated dogs. Western blot showing FOG-2 in 2 NL dogs, 2 untreated HF-sham control dogs, and 2 HF CCM-treated dogs.
*P ! .05 vs NL; **P ! .05 vs HF-sham. NL, normal; HF, heart failure; CCM, cardiac contractility modulation.
Cardiac Sodium-Calcium Exchanger in Heart Failure
dilated cardiomyopathy, Studer et al.44 demonstrated that
LV mRNA and protein levels of NCX-1 are significantly increased. In dogs with pacing-induced HF, O’Rourke and colleagues52 reported an approximate 2-fold increase in the
expression of NCX compared with control NL dogs. In the
present study, long-term CCM therapy improved LV function and normalized the expression of NCX-1. In addition
to expression, phosphorylation level of NCX-1 has also
been found to be increased in failing heart.27,53 Our studies
clearly demonstrated increased phosphorylation of NCX-1
in LV tissue of HF dogs compared with NL dogs. Phosphorylation of NCX-1 might result in increased basal current,
and this abnormality results in reduced beta-adrenergic receptor responsiveness to catecholamines, a characteristic
feature of the failing heart. In another study, isoproterenol
and carbachol both regulated NCX current through phosphorylation of the exchanger in LV myocardium of normal
and atrial-paced pigs with HF.53 This study clearly demonstrated that atrial-paced HF dogs had increased phosphorylation of cardiac NCX because of reduced activity and
level of type-1 protein phosphatase, which is bound with
NCX macromolecule. In the present study, CCM therapy
was associated with reduced levels of phosphorylated
NCX-1, which can lead to improved responsiveness of failing myocardium to catecholamines.53 The former may occur
as a result of the improved cardiac function elicited by CCM
therapy, which, in turn, leads to lower levels of circulating
catecholamines and reduced NCX-1 phosphorylation.
CCM therapy has also been shown to be associated with normalized expression of SR Ca2þ ATPase and phosphorylated
phospholamban.42 These findings when viewed in concert
with observations of the present study suggest that CCM
therapy promotes normalization of overall SR Ca2þ cycling
in HF, a functional restoration that can clearly lead to overall
improvement in performance of the failing LV.
Recent studies showed that NCX-1 expression is regulated by GATA-4.29 In this study, we observed increased
GATA-4 expression (protein and mRNA) in LV tissue of
dogs with microembolization-induced HF compared with
NL dogs and chronic CCM therapy restored expression of
GATA-4 to near-normal levels. The transcriptional factor
GATA-4 has been reported to regulate a spectrum of cardiac-specific genes, including brain natriuretic peptide
and myosin heavy chain, in addition to NCX-1.34 Studies
in LV tissue obtained from human hearts failing as the result of idiopathic dilated cardiomyopathy also showed increased protein expression of GATA-4 compared with
non-failing human LV tissue.54 GATA-4 itself seems to be
regulated reciprocally by a cofactor FOG-2.33 In the present
study, we observed decreased FOG-2 mRNA expression in
the LV tissue of dogs with microembolizations-induced HF
compared with NL dogs, and chronic CCM therapy restored mRNA expression of FOG-2 to near-normal levels.
We are not aware of any studies that examined the expression of FOG-2 in failing LV myocardium. This study, therefore, is the first to report reduced mRNA and protein
expression of FOG-2 in experimental HF.
Gupta et al
55
Conclusions
The results of the present study indicate that long-term
therapy with CCM electric signals normalize the NCX-1,
GATA-4, and FOG-2 signaling pathway in HF dogs. Normalization of components of this signaling pathway is consistent with and provides further support to previous
observations of improved SR calcium cycling in HF dogs
treated long-term with CCM electrical signals. These improvements in NCX-1 also represent a potential contributing factor to the improved LV function in HF.
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