Journal of the American College of Cardiology
© 2000 by the American College of Cardiology
Published by Elsevier Science Inc.
Vol. 36, No. 1, 2000
ISSN 0735-1097/00/$20.00
PII S0735-1097(00)00703-8
Heart Failure
Is Myocardial Na⫹/Ca2⫹ Exchanger Transcription
a Marker for Different Stages of Myocardial
Dysfunction? Quantitative Polymerase
Chain Reaction of the Messenger RNA in
Endomyocardial Biopsies of Patients with Heart Failure
Cornelia Piper, MD,* Johannes Bilger, MD,† Eva-Maria Henrichs,† Heinz-Peter Schultheiss, MD, FESC,†
Dieter Horstkotte, MD, FESC,* Andrea Doerner, PHD†
Bad Oeynhausen and Berlin, Germany
This study was designed to determine the stage of myocardial dysfunction at which an
upregulation of the Na⫹/Ca2⫹exchanger (EXCH) transcription takes place.
BACKGROUND Because EXCH is an important regulator of intracellular calcium homeostasis, alterations in
EXCH expression may occur before the onset of end-stage heart failure (HF) to maintain
normal intracellular Ca2⫹ concentrations. We analyzed whether the EXCH transcription
level is correlated to the degree of myocardial dysfunction and whether it can be a suitable
molecular marker to define the transition to myocardial decompensation early on.
METHODS
By quantitative polymerase chain reaction technique, the level of EXCH transcription was
analyzed in myocardial biopsies from 40 patients with various degrees of myocardial
dysfunction due to valvular heart disease (VHD; n ⫽ 22) or dilated cardiomyopathy (DCM;
n ⫽ 18). Additionally, biopsies from 7 individuals with excluded heart disease and explanted
heart tissue from 13 patients with end-stage HF were investigated.
RESULTS
The level of EXCH transcription of controls (2.6 ⫾ 1.2 attomoles [amol]/ng total RNA) did
not differ from that of patients with DCM (2.3 ⫾ 1.5 amol/ng) or VHD (2.1 ⫾ 1.5 amol/ng).
No alteration in the EXCH transcription was found in VHD and DCM patients with respect
to the severity of myocardial dysfunction. However, patients with end-stage HF showed a
four-fold increase in EXCH transcription, amounting to 8.9 ⫾ 1.9 amol/ng (p ⬍ 0.05).
CONCLUSIONS The upregulation in EXCH transcription either occurs very late in human heart failure or is
a phenomenon of heart transplantation in end-stage HF. Consequently, myocardial EXCH
transcription cannot be used as a marker for early myocardial decompensation. (J Am Coll
Cardiol 2000;36:233– 41) © 2000 by the American College of Cardiology
OBJECTIVES
An upregulation of the sarcolemmal Na⫹/Ca2⫹ exchanger
(EXCH), an important regulator of the intracellular calcium
concentration in both mRNA and protein levels, has been
demonstrated in end-stage heart failure (HF) (1,2). The
electrogenic exchange of one Ca2⫹ for three Na⫹ ions across
the sarcolemmal membrane by the EXCH maintains low
diastolic cytosolic Ca2⫹ levels and enables diastolic relaxation to take place (3,4). In addition, the EXCH improves
cardiac contractility by increasing the systolic influx of Ca2⫹
ions into the cell (2,5,6). An upregulation of the EXCH
may help to compensate abnormally high intracellular Ca2⫹
concentrations that contribute to the systolic and diastolic
functional impairment in the failing heart (7–9).
As diastolic and systolic functions are disturbed long
before HF manifests itself, we investigated the stage of
From the *Department of Cardiology, Heart Center North Rhine-Westphalia,
University Hospital of the Ruhr University of Bochum, Bad Oeynhausen, Germany;
and †Department of Cardiology, Benjamin Franklin Hospital, Free University of
Berlin, Berlin, Germany.
This study was supported by a grant from the Franz-Loogen Foundation.
Manuscript received May 27, 1999; revised manuscript received December 30,
1999, accepted March 1, 2000.
myocardial dysfunction at which the EXCH transcription
alters, and whether this alteration in the EXCH is a suitable
molecular marker for defining the prognostically important
transition from adequate to inadequate myocardial adaptation due to chronic pressure and/or volume overload (10 –
14).
To this end, and for the first time ever, the EXCH
transcription level was determined in endomyocardial biopsies from patients with mild to severe myocardial dysfunction and was compared with that measured in hearts
explanted from patients with end-stage HF.
METHODS
Patients
Forty patients (age range 31– 85 years, mean 57 ⫾ 13 years;
27 men, 13 women) with chronic pressure overload (aortic
stenosis [AS], 11 patients), volume overload (aortic regurgitation [AR], 5 patients; primary mitral regurgitation
[MR], 6 patients) or dilated cardiomyopathy (18 patients)
were examined. All patients underwent diagnostic right and
left heart catheterization including coronary angiography.
234
Piper et al.
Myocardial Naⴙ/Ca2ⴙ Exchanger Transcription
Abbreviations and Acronyms
AR
⫽ aortic regurgitation
AS
⫽ aortic stenosis
CI
⫽ cardiac index
DCM
⫽ dilated cardiomyopathy
EF
⫽ ejection fraction
EXCH ⫽ Na⫹/Ca2⫹ exchanger
ICM
⫽ ischemic cardiomyopathy
MR
⫽ mitral regurgitation
NYHA ⫽ New York Heart Association
PLaorta ⫽ transaortic pressure loss
RFaorta ⫽ transaortic regurgitation
RFmitral ⫽ transmitral regurgitation
VAD
⫽ ventricular assist device
Significant (⬎50%) coronary artery disease was excluded in
all patients. During right heart catheterization, small biopsies were taken from the interventricular septum, immediately frozen in liquid nitrogen and stored at ⫺80°C. The
EXCH expression has been shown to be the same in the
interventricular septum and the right and left ventricles in
hearts explanted from patients with end-stage HF (15).
Biopsies were screened to exclude the presence of myocarditis according to the Dallas classification (16). The study
was approved by the ethics committee of the Free University
of Berlin, Germany.
The clinical and hemodynamic characteristics of the
patients enrolled in the study are listed in Tables 1 and 2.
The severity of aortic valve obstruction was graded according to the transvalvular pressure loss (PLaorta), the mean
transaortic pressure gradient (dp) and effective stroke volume coefficient, which was determined from the simultaneous records of the left ventricular (transseptal technique)
and aortic pressure curves. Effective left ventricular stroke
volume was determined by the thermodilution method.
Hemodynamically relevant AS was defined as PLaorta ⱖ
1 mm Hg per ml stroke volume (14). Aortic regurgitation
and MR were estimated using the transvalvular regurgitant
fraction (RF), which is the difference between total (measured angiographically) and effective left ventricular stroke
volume (measured by thermodilution technique). Regurgitant fractions of ⬎30% were considered hemodynamically
relevant (13) (Table 1).
In addition to valvular patients, endomyocardial samples
were taken from the right ventricular septum of 13 explanted hearts (Table 3) failing because of dilated cardiomyopathy (DCM; n ⫽ 2) or ischemic cardiomyopathy
(ICM; n ⫽ 11). Endomyocardial samples from two healthy
donors and from five individuals without myocardial dysfunction, who underwent cardiac catheterization and myocardial biopsies to exclude coronary artery disease and
myocarditis, served as controls.
Assessment of Ventricular Function
Left ventricular ejection fraction (EF) and total stroke
volume were determined by cineangiography using the
JACC Vol. 36, No. 1, 2000
July 2000:233–41
analytic calibration method (17,18). Cardiac index (CI) was
calculated as the average of five thermodilution measurements. Hemodynamic data are given in Tables 1 and 2. All
patients had transthoracic echocardiography including measurement of left ventricular end-diastolic (LVEDD) and
end-systolic (LVESD) dimensions, fractional shortening
(FS), end-diastolic left ventricular posterior wall thickness
(LVPW) and maximal systolic meridional wall stress (ws)
(g/cm2) (Tables 1 and 2). Wall stress was determined
according to the Laplace law. Left ventricular radius
(LVEDD/2), LVPW and left ventricular peak systolic
pressure (LVPRsyst), measured during catheterization, were
used to calculate left ventricular wall stress according to the
following equation (10):
ws ⫽
共LVEDD/2兲 ⫻ 共0.334 ⫻ LVPRsyst兲
g/cm2
LVPW
Quantitative Polymerase Chain Reaction
The competitive quantitative polymerase chain reaction
(q-PCR) was employed to determine the EXCH messenger
ribonucleic acid (mRNA) amount in the endomyocardial
specimens from the patients described. An internal RNA
standard was used, simultaneously reversely transcribed with
the target RNA and competitively amplified in the PCR.
Construction of the competitor. A bluescript vector,
containing 1.3 kb of the coding and 0.2 kb of the noncoding
sequence from guinea pig EXCH complementary deoxyribonucleic acid (cDNA), was used for the synthesis of
competitor complementary RNA. An 82-bp fragment was
released from the coding region by digestion with Bcll and
Hpal. After religation 10 ␮g plasmid was linearized with
Xhol according to the instructions of the manufacturer
(Boehringer Mannheim). Transcription was performed in a
reaction mixture containing 1 ␮g plasmid, 5 ␮l 5⫻ transcription puffer, 5 mmol/liter DTT, 10 U RNAsinInhibitor (Boehringer Mannheim) and 4 mmol/liter each of
rNTP and 100 U T3-polymerase (Stratagene) at 37°C for
1 h. The transcription mixture was subjected to a 1%
low-melting gel electrophoresis. cRNA was extracted with
phenol/chloroform and was precipitated with 0.1 vol%
sodium acetate pH 5.2 and 2.5 vol% ethanol at ⫺80°C
overnight. The RNA was stabilized with tRNA (100 ng
tRNA/␮l cRNA). The purity of the standard was proved by
PCR using nontranscribed standard RNA, which produced
no detectable signal.
Quantitative PCR. Total RNA was extracted from the
endomyocardial biopsies using the LiCl method (19). The
conditions of the reverse transcription and the PCR were
optimized, especially in view of the cycles, the amount of
isolated total RNA and standard cRNA (data not shown).
The following conditions were employed: equal amounts of
total RNA (7.5 ng) isolated from the endomyocardial
specimens were mixed with increasing amounts of competitor (3.5, 7, 14, 28, 56, 112, 224, and 448 attomoles [amol])
in a reverse transcription (RT) mixture of 12.5 ␮l, contain-
Angiocardiography
Echocardiography
AS
AS
AS
AS
AS
AS
AS
AS
AS
AS
AS
AR
AR
AR
AR
AR
MR
MR
MR
MR
MR
MR
m
f
m
f
f
f
f
f
m
m
f
m
m
m
m
m
m
f
m
f
f
m
85
79
75
75
68
65
65
64
63
52
60
58
57
77
66
43
54
58
62
72
76
34
1
3
2
3
3
3
3
2
1
1
4
3
3
3
1
1
1
3
3
3
4
3
2.9
2.9
0.36
1.35
0.74
1.38
0.95
0.71
0.42
0.36
1.05
0
0
0.25
0
0
0
0
0
0
0
0
0
0
0
0
0
22
19
18
11
0
0
30
25
46
33
38
0
0
13
0
11
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
39
40
55
43
22
30
40
50
77
63
34
50
50
63
61
66
57
38
48
49
55
52
66
66
45
53
58
66
25
27
56
45
39
25
51
61
46
44
38
37
43
33
51
61
54
48
23
26
34
61
2
2
4.4
2.4
3.6
2.4
3.7
3.8
3
3.3
3.2
1.8
3.7
2.7
4.5
3.9
3.5
3.9
2.2
2.1
2.4
3.3
5
11
14
16
20
11
14
23
13
12
23
30
19
14
18
12
6
17
9
5
7
4
218
186
216
216
164
140
197
218
157
164
150
136
148
186
154
132
190
110
106
106
130
100
50
50
53
52
48
62
53
58
47
58
60
72
64
61
63
67
61
55
67
51
55
65
34
28
25
36
32
44
33
36
31
38
35
52
46
35
42
45
36
34
47
32
47
42
32
44
53
31
33
28
38
42
36
33
42
28
27
42
34
34
41
38
30
38
14
35
15
12
11
15
14
12
16
15
13
14
12
11
11
14
13
9
9
10
10
9
11
12
1.7
2.1
2.2
1.9
1.8
2.6
1.8
2.1
1.6
2.1
2.5
3.3
2.9
2.2
2.4
3.7
3.4
2.8
3.3
2.8
2.5
2.7
121
129
174
125
94
121
110
140
94
113
125
149
144
135
125
164
215
101
119
100
109
90
AS ⫽ aortic stenosis; AR ⫽ aortic regurgitation; CI ⫽ cardiac index; EF ⫽ ejection fraction; FS ⫽ fractional shortening; LVEDD ⫽ left ventricular end-diastolic diameter; LVEDP ⫽ left ventricular end-diastolic pressure; LVESD
⫽ left ventricular end-systolic diameter; LVPRsyst ⫽ left ventricular peak systolic pressure; LVPW ⫽ left ventricular posterior wall thickness; MR ⫽ mitral regurgitation; PLaorta ⫽ transaortic pressure loss; RFaorta ⫽ transaortic
regurgitation; RFmitral ⫽ transmitral regurgitation; r/Th ⫽ ratio of left ventricular radius to left ventricular posterior wall thickness; SVleff ⫽ effective left ventricular stroke volume index; ws ⫽ max systolic meridional wall stress.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
RF
EF
SVI eff
Patient Diagnosis Gender Age NYHA
PL Aorta
RF
CI
LVEDP
LVPR
LVEDD LVESD FS LVPW r/Th
ws
(n)
(yr)
mm Hg/mlSV Aorta Mitral Angio (ml/min/m2) (l/min/m2) (mm Hg) (syst, mm Hg)
(mm)
(mm)
(%) (mm)
(g/cm2)
(%)
(%)
(%)
Clinical Data
Table 1. Clinical, Hemodynamic and Echocardiographic Characteristics of Patients With Valvular Heart Disease
JACC Vol. 36, No. 1, 2000
July 2000:233–41
Piper et al.
Myocardial Naⴙ/Ca2ⴙ Exchanger Transcription
235
DCM
DCM
DCM
DCM
DCM
DCM
DCM
DCM
DCM
DCM
DCM
DCM
DCM
DCM
DCM
DCM
DCM
DCM
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
m
m
m
m
m
m
m
m
m
m
m
m
f
m
m
f
f
m
Gender
47
57
33
40
57
42
39
36
63
53
59
44
57
58
31
56
62
59
Age
(yr)
2
3
1
4
4
3
2
2
3
2
2
3
3
4
1
1
3
3
NYHA
27
34
50
36
50
56
24
71
26
34
33
40
61
51
57
34
29
27
EF Angio
(%)
53
58
44
44
32
58
40
48
33
62
62
28
44
44
62
62
40
40
SVI eff
(ml/min/m2)
4.1
5
4.3
3.2
3.9
4.2
3.3
4.6
2.6
3.8
2.6
3.1
3.5
3.7
4.4
3.8
3.6
3.6
CI
(l/min/m2)
4
12
26
5
23
5
8
4
16
29
13
16
8
12
22
10
17
13
LVEDP
(mm Hg)
Angiocardiography
110
132
144
98
120
100
90
123
112
112
134
104
136
128
131
100
134
110
LVPR
(syst, mm Hg)
86
83
67
73
77
72
89
59
62
63
74
70
60
62
60
84
86
62
LVEDD
(mm)
78
68
45
61
65
59
73
38
52
53
61
64
41
53
48
68
47
49
LVESD
(mm)
11
17
33
16
15
18
18
36
15
22
18
9
31
13
20
20
30
19
FS
(%)
10
8
9
14
9
9
7
9
9
12
13
10
10
8
10
9
10
9
LVPW
(mm)
Echocardiography
4.3
5.2
3.7
2.6
4.3
4
6.4
3.3
3.4
2.6
2.8
3.5
3
3.9
3
4.7
4.3
3.4
r/Th
156
229
179
85
172
134
191
137
129
98
127
122
136
157
131
156
193
127
ws
(g/cm2)
CI ⫽ cardiac index; EF ⫽ ejection fraction; FS ⫽ fractional shortening; LVEDD ⫽ left ventricular end-diastolic diameter; LVEDP ⫽ left ventricular end-diastolic pressure; LVESD ⫽ left ventricular end-systolic diameter; LVPRsyst
⫽ left ventricular peak systolic pressure; LVPW ⫽ left ventricular posterior wall thickness; r/Th ⫽ ratio of left ventricular radius to left ventricular posterior wall thickness; SVleff ⫽ effective left ventricular stroke volume index; ws ⫽
max systolic meridional wall stress.
Diagnosis
Patient
(n)
Clinical Data
Table 2. Clinical, Hemodynamic and Echocardiographic Characteristics of Patients With Dilated Cardiomyopathy
236
Piper et al.
Myocardial Naⴙ/Ca2ⴙ Exchanger Transcription
JACC Vol. 36, No. 1, 2000
July 2000:233–41
Piper et al.
Myocardial Naⴙ/Ca2ⴙ Exchanger Transcription
JACC Vol. 36, No. 1, 2000
July 2000:233–41
Table 3. Clinical and Hemodynamic Characteristics of Patients
Undergoing Heart Transplantation Because of End-Stage HF
Caused by Ischemia (End-Stage ICM) or Dilated
Cardiomyopathy (End-Stage DCM)
Clinical Data
Angiocardiography
Patient
(n)
Diagnosis
Gender
Age
(yr)
EF
Angio
(%)
CI
(l/min/m2)
1
2
3
4
5
6
7
8
9
10
11
12
13
end-stage-ICM
end-stage-ICM
end-stage ICM
end-stage-ICM
end-stage-ICM
end-stage-ICM
end-stage-ICM
end-stage ICM
end-stage-ICM
end-stage-ICM
end-stage-ICM
end-stage-DCM
end-stage-DCM
m
m
m
m
m
m
m
m
m
m
m
m
m
61
62
61
52
52
68
66
59
68
63
50
47
55
20
22
13
15
13
11
11
16
15
22
14
13
20
1.4
1.6
1.7
1.9
2
1.3
1.6
2.1
1.3
2
1.4
1.3
1.7
EF ⫽ ejection fraction; CI ⫽ cardiac index.
ing 2.5 ␮l 5 ⫻ first strand buffer (GIBCO, Karlsruhe,
Germany), 5 U RNAse Inhibitor (Boehringer Mannheim),
0.4 mmol/liter of dNTP mix (Boehringer Mannheim), 8 ng
random primer, 8 mmol/liter DTT and 200 U MMLVReverse transcriptase (GIBCO).
Sense and antisense primers (5⬘: 5⬘-AAT GAG CTT
GGT TTC ACA-3⬘ and 3⬘: 5⬘-CCG CCG ATA CAG
CAG CAC-3⬘) were chosen to lie in homologous regions of
the human (20) and guinea pig (21) cDNA sequences.
Competitive PCRs were carried out in a 25-␮l reaction
mixture containing 10 mmol/liter Tris-HCl, pH 8.3; 50
mmol/liter KCl; 1.5 mmol/liter MgCl2; 200 ␮mol/liter of
each dNTPs; 0.4 ␮mol/liter of each primer; 5 ␮l RT
mixture and 0.63 U of Taq polymerase (Cetus Perkin
Elmer, Weiterstadt, Germany). The denaturation, annealing and extension conditions for the amplification were 45 s
at 94°C, 45 s at 60°C and 90 s at 72°C. Thirty amplification
cycles were performed with a thermal cycler (Biozyme).
Polymerase chain reaction products were subjected to agarose gel electrophoresis and stained with ethidium bromide.
Gels were scanned by a computer-based video system
(Polaroid, Offenbach, Germany). The intensity of the
ethidium– bromide-stained bands of the target and the
competitor RNA were evaluated by a computer-based
imaging system (NIH image). To correct for differences in
the size of the target (861 bp) and the competitor (778 bp)
PCR products, the band densities of the respective competitors were multiplied by the specific factor of 1.11. The
target/competitor ratio was plotted against the amount of
competitor in a log scale. The linear regression was calculated. The amount of competitor corresponding to the log
ratio value of 0 is equivalent to the amount of total
EXCH-specific RNA in the specimens. The level of EXCH
transcription was expressed as amol/ng of total RNA.
237
Statistical analysis. All data are given as mean ⫾ SD. For
statistical analysis nonparametric tests were used; for comparisons of more than two groups the Kruskal Wallis test
was applied for testing the global hypothesis. In case of a
significant result, the nonparametric Dunn’s test was then
used to assess the two-by-two differences. The test of Dunn
is based on the comparison in pairs of the mean ranks of the
groups. All analyses were performed using SPSS 8.0 software, except for the Dunn’s test (SAS 6.13, Cary, North
Carolina).
The p values are given in the following classes: ⬍0.01 and
⬍0.05. A p value of ⬍0.05 was considered significant.
RESULTS
This study was designed to clarify whether changes in
cardiac function are associated with an altered transcription
of the sarcolemmal EXCH in patients with valvular heart
disease or DCM. To determine the EXCH mRNA copy
number in small endomyocardial biopsy samples, the quantitative PCR technique was employed, using an internal
RNA standard as a competitor (Fig. 1).
The EXCH mRNA level amounted to an average of
2.6 ⫾ 1.2 amol/ng total RNA in the control group. A
fourfold increase (8.9 ⫾ 1.9 amol/ng) was found in hearts
explanted from patients with end-stage HF due to ischemic
or DCM, whereas no differences were seen in patients with
AS (1.8 ⫾ 1.4 amol/ng), AR/MR (2.0 ⫾ 1.5 amol/ng) or
DCM (2.3 ⫾ 1.5 amol/ng) (Fig. 2).
No significant changes in EXCH mRNA amount were
found in the group of patients with normal to mildly
impaired (EF ⬎ 50%) left ventricular EF, with an EF of
30% to 49% or with an EF ⬍ 30% (Fig. 3). Similar results
were obtained when EXCH amount was correlated to CI,
as patients with normal CI (CI ⬎ 3.5 liters/min/m2),
reduced CI (CI 2.4 –3.5 liters/min/m2) or very low CI
(CI ⬍ 2.4 liters/min/m2) had similar EXCH amounts of
2.0 ⫾ 1.6, 2.0 ⫾ 1.5 or 2.5 ⫾ 1.3 amol/ng total RNA.
There was no correlation to other invasively measured
hemodynamic parameters and echocardiographic data,
nor was there a correlation to the hemodynamic severity
of valvular stenosis or valvular regurgitations (Tables 1
and 2).
Not even patients with severe HF (New York Heart
Association [NYHA] grade IV) and a critical hemodynamic
condition showed an increase in EXCH transcription,
which was found only in tissue from explanted hearts. The
EXCH mRNA tended to be lower in patients with heart
valve disease and NYHA class I or II than in those with
heart valve disease and NYHA class III or IV which was not
seen in patients with dilated cardiomyopathy (Fig. 4).
Systolic wall stress, a parameter indicating the state of
myocardial adaptation to increased workload, did not induce
an increase in the EXCH mRNA level (Fig. 5).
238
Piper et al.
Myocardial Naⴙ/Ca2ⴙ Exchanger Transcription
JACC Vol. 36, No. 1, 2000
July 2000:233–41
Figure 1. Total EXCH mRNA quantification by competitive PCR. A constant amount of total RNA (7.5 ng), isolated from an endomyocardial biopsy
from a patient suffering from DCM, was mixed with a decreasing amount of competitor cRNA, reverse transcribed and amplified by PCR.
DISCUSSION
Transcription and translation of EXCH is known to be
upregulated in myocardium of failing hearts. This may be
related to changes in calcium homeostasis. The stage of
cardiac dysfunction at which upregulation occurs is unknown so far. We therefore examined the EXCH mRNA
level in endomyocardial biopsies from patients with mildly,
moderately and severely impaired left ventricular function,
employing the quantitative PCR technique.
EXCH and calcium homeostasis. Consistent with earlier
findings (1,2), we found a significant increase in the EXCH
mRNA amount in explanted human myocardium from
patients with end-stage HF. However, we did not see any
alterations of EXCH in endomyocardial biopsies from
patients with heart failure of varying severity. These findings
indicate that the EXCH mRNA level does not change until
the myocardium has been finally damaged.
In severe, but not end-stage, HF the intracellular calcium
homeostasis seems to be maintained without alteration of
the EXCH expression (22). It is likely that the upregulation
of the EXCH observed in end-stage HF is necessary to
compensate intracellular calcium overload by increasing the
calcium efflux across the sarcolemmal membrane (23).
Schillinger et al. (24) reported that a subset of patients with
Figure 2. Plots showing a significant difference (p ⬍ 0.05) between the
Na⫹/Ca2⫹ exchanger mRNA levels of explanted hearts (expl) from
patients with end-stage heart failure and those found in endomyocardial
tissues of controls (c), of patients with aortic stenosis (AS), aortic or mitral
regurgitation (AR/MR), and of dilated cardiomyopathy (DCM).
JACC Vol. 36, No. 1, 2000
July 2000:233–41
Figure 3. No significant changes in the Na⫹/Ca2⫹ exchanger mRNA
transcription level were obtained within patients with chronic heart failure
(CHF) irrespective of left ventricular ejection fractions (EFs): EF ⱖ 50%,
EF ⫽ 30%– 49% or EF ⬍ 30%. But a significant (p ⬍ 0.01) difference in
EXCH transcription was found between patients with severe CHF (EF ⬍
30%) and explanted hearts (expl), although EF was reduced similarly.
end-stage HF had higher levels of EXCH. This study
divided the patients into groups with either predominant
systolic or predominant diastolic alteration of the force
development in isolated muscle strips. Patients with systolic
dysfunction showed only slightly increased protein levels of
EXCH, whereas EXCH levels doubled in patients with
diastolic dysfunction. In the latter group, an inverse relationship between EXCH levels and the severity of diastolic
dysfunction was observed, pinpointing the importance of
EXCH for diastolic function.
EXCH upregulation. In our study, upregulation of the
myocardial EXCH was found to occur only after manifestation of end-stage HF. Whether this upregulation of
EXCH occurred shortly before or during heart transplantation is not known. Dipla et al. (25) observed, in favor for
EXCH upregulation before heart transplantation, alterations in calcium homeostasis just before heart transplanta-
Piper et al.
Myocardial Naⴙ/Ca2ⴙ Exchanger Transcription
239
Figure 5. No significant elevation in EXCH transcription level in CHF
according to systolic wall stress, a parameter indicating the state of
myocardial adaptation to increased workload, is demonstrated.
tion was performed. They showed that if patients were put
on a ventricular assist device (VAD) before transplantation,
contractility, relaxation and catecholamine responsiveness of
myocytes isolated from explanted hearts improved, because
VAD therapy resulted in higher Ca2⫹ transportation rates.
This indicates an interaction between central hemodynamics and calcium homeostasis. Whether alterations in the
EXCH expression were involved in the improvement of
calcium transportation rates and of myocardial function
remains speculative, although an upregulation of the EXCH
expression has been shown to be related to the severity of
diastolic dysfunction in failing human hearts (24,26).
Potential mechanisms of EXCH upregulation. The rise
in the EXCH amount may be due to an extremely decelerated relaxation, a reduced response to beta-adrenergic
stimulation, decreased force production (25,27) or biochemical changes (acidosis and ATP depletion in endomyocardial
Figure 4. Plots showing a significant difference (p ⬍ 0.01) in the EXCH transcription level, even between CHF in NYHA IV and expl, although the clinical
situation was similar. Otherwise, a nonsignificant tendency toward lower EXCH mRNA levels was found in NYHA I/II compared with NYHA III/IV
for VHD, not seen in DCM.
240
Piper et al.
Myocardial Naⴙ/Ca2ⴙ Exchanger Transcription
ischemia [28], increased levels of circulating cytokines [29]
or neurohumoral activation [28,30]) frequently observed in
HF.
Otherwise, upregulation of the EXCH mRNA amount is
likely to be a phenomenon induced by heart transplantation
or perioperative procedures/medications, especially in patients with severely damaged heart tissue (31,32). An
alteration in the EXCH expression was shown to occur
within 1 to 2 h of acute pressure overload (33), pharmacologically induced increase of Na⫹ influx (33) or acute
ischemia induced in animal models. For example, a significant upregulation of the EXCH expression was seen after
adrenergic stimulation with phenylephrine in ventricular
cardiocytes of rats, but not if the alpha-receptors had been
blocked with prazosin (34). These data demonstrate that, in
rats, alpha-adrenergic stimulation induces an increase in
EXCH mRNA within 1 h and an increase in protein levels
within 24 h. However, in patients with end-stage HF,
catecholamines do not seem to influence the upregulation of
EXCH transcripts, as elevated EXCH levels were documented even when all patients who had received catecholamines preoperatively were excluded (2).
Volatile anesthetics such as halothane are known to
influence EXCH by decreasing the activity of the exchanger, which in turn may induce an elevation of EXCH
transcription to compensate for the loss of EXCH activity
(35,36).
Myocardial adaptation. Our data show that alterations in
EXCH transcription do not parallel myocardial dysfunction, but occur only in conjunction with end-stage HF.
Therefore, EXCH expression is not an early marker for
inadequate myocardial adaptation to chronic pressure
and/or volume overload (37,38), and timing of surgical
intervention may not be directed by this parameter (39,40).
Study limitations. In this study, the EXCH mRNA
levels, but not the EXCH protein levels, were measured in
endomyocardial tissue from patients with various degrees of
HF. The limited biopsy material forced us to neglect this
parameter. However, this parameter is not essential for the
assessment of EXCH alterations in patients with myocardial dysfunction, as other groups have shown that an
increase in mRNA level is always accompanied by an
increase in EXCH protein level.
Conclusion. Our data indicate that the EXCH transcription level is upregulated in explanted human hearts with
end-stage HF, but not in myocardial biopsies from patients
with moderate to severe HF. Upregulation of EXCH
expression either occurs very late in the natural history of
HF as an indication of irreversible myocardial damage or is
a phenomenon of hemodynamic, autocrine, endocrine, ischemic and/or drug-induced changes during heart transplantation.
Acknowledgments
We would like to thank Mr. Helmut Drexler and Mr. Hans
Reinecke for their technical assistance, and Mr. Rito Berge-
JACC Vol. 36, No. 1, 2000
July 2000:233–41
mann from the Institute of Medical Outcome Research
(IMOR) for the statistical analyses.
Reprint requests and correspondence: Dr. Andrea Doerner,
Department of Cardiology, Benjamin Franklin Hospital, Free
University of Berlin, Hindenburgdamm 30, 12000 Berlin, Germany. Email: [email protected].
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