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Negative Inotropy of the Gastric Proton Pump Inhibitor
Pantoprazole in Myocardium From Humans and Rabbits
Evaluation of Mechanisms
Wolfgang Schillinger, MD*; Nils Teucher, MD*; Samuel Sossalla, MS; Sarah Kettlewell, PhD;
Carola Werner, PhD; Dirk Raddatz, MD; Andreas Elgner, MS; Gero Tenderich, MD;
Burkert Pieske, MD; Giuliano Ramadori, MD; Friedrich A. Schöndube, MD;
Harald Kögler, MD; Jens Kockskämper, PhD; Lars S. Maier, MD; Harald Schwörer, MD;
Godfrey L. Smith, PhD; Gerd Hasenfuss, MD
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Background—Proton pump inhibitors are used extensively for acid-related gastrointestinal diseases. Their effect on
cardiac contractility has not been assessed directly.
Methods and Results—Under physiological conditions (37°C, pH 7.35, 1.25 mmol/L Ca2⫹), there was a dose-dependent
decrease in contractile force in ventricular trabeculae isolated from end-stage failing human hearts superfused with
pantoprazole. The concentration leading to 50% maximal response was 17.3⫾1.3 ␮g/mL. Similar observations were
made in trabeculae from human atria, normal rabbit ventricles, and isolated rabbit ventricular myocytes. Real-time
polymerase chain reaction demonstrated the expression of gastric H⫹/K⫹–adenosine triphosphatase in human and rabbit
myocardium. However, measurements with BCECF-loaded rabbit trabeculae did not reveal any significant
pantoprazole-dependent changes of pHi. Ca2⫹ transients recorded from field-stimulated fluo 3–loaded myocytes (F/F0)
were significantly depressed by 10.4⫾2.1% at 40 ␮g/mL. Intracellular Ca2⫹ fluxes were assessed in fura 2–loaded,
voltage-clamped rabbit ventricular myocytes. Pantoprazole (40 ␮g/mL) caused an increase in diastolic [Ca2⫹]i by
33⫾12%, but peak systolic [Ca2⫹]i was unchanged, resulting in a decreased Ca2⫹ transient amplitude by 25⫾8%. The
amplitude of the L-type Ca2⫹ current (ICa,L) was reduced by 35⫾5%, and sarcoplasmic reticulum Ca2⫹ content was
reduced by 18⫾6%. Measurements of oxalate-supported sarcoplasmic reticulum Ca2⫹ uptake in permeabilized
cardiomyocytes indicated that pantoprazole decreased Ca2⫹ sensitivity (Kd) of sarcoplasmic reticulum Ca2⫹ adenosine
triphosphatase: control, Kd⫽358⫾15 nmol/L; 40 ␮g/mL pantoprazole, Kd⫽395⫾12 nmol/L (P⬍0.05). Pantoprazole
also acted on cardiac myofilaments to reduced Ca2⫹-activated force.
Conclusions—Pantoprazole depresses cardiac contractility in vitro by depression of Ca2⫹ signaling and myofilament
activity. In view of the extensive use of this agent, the effects should be evaluated in vivo. (Circulation. 2007;116:00000000.)
Key Words: calcium 䡲 contractility 䡲 heart failure 䡲 inotropic agents 䡲 pharmacology
P
roton pump inhibitors (PPIs) like pantoprazole, omeprazole, esomeprazole, lansoprazole, and rabeprazole are the
most effective pharmacological means of reducing gastric
acid secretion by blocking the final step of proton secretion,
ie, the gastric acid pump, H⫹/K⫹–adenosine triphosphatase
(ATPase). The efficacy of this class of drugs has been
demonstrated in the treatment of a number of acid-related
gastrointestinal diseases, in particular, gastroesophageal reflux and ulcer disease.1 Hence, there is extensive use of these
Clinical Perspective p 0000
agents for patients in a variety of settings. In a US Medicaid
population, PPIs accounted for 5.6% of the net pharmacy
expenditures and ranked first in expenditures among all drug
therapy classes during the 12 months before the implementation of the PPI prior-authorization policy.2 Drug shortage
bulletins have been issued repeatedly for intravenous PPIs in
the United States.3
Received September 25, 2006; accepted May 1, 2007.
From the Herzzentrum, Kardiologie und Pneumologie, Universitaet Goettingen, Goettingen, Germany (W.S., S.S., A.E., B.P., H.K., J.K., L.S.M.,
G.H.); Herzzentrum, Thorax-, Herz-, und Gefaesschirurgie, Universitaet Goettingen, Goettingen, Germany (N.T., F.A.S.); Institute of Biomedical and Life
Sciences, University of Glasgow, Glasgow, UK (S.K., G.L.S.); Medizinische Statistik, Universitaet Goettingen, Goettingen, Germany (C.W.);
Gastroenterologie und Endokrinologie, Universitaet Goettingen, Goettingen, Germany (D.R., G.R., H.S.); and Herz- und Diabeteszentrum NordrheinWestfalen, Klinik fuer Thorax- und Kardiovaskularchirurgie, Bad Oeynhausen, Germany (G.T.).
*The first 2 authors contributed equally to this work.
Correspondence to Wolfgang Schillinger, MD, Herzzentrum, Kardiologie und Pneumologie, Georg-August Universitaet Goettingen, Robert-Koch
Strasse 40, 37099 Goettingen, Germany. E-mail [email protected]
© 2007 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/CIRCULATIONAHA.106.666008
1
2
Circulation
July 3, 2007
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It has been shown recently that the expression of H⫹/K⫹ATPase is not limited strictly to gastric tissue. It has also been
identified in renal4 and colonic epithelial cells,5 vascular
smooth muscle cells,6 and other tissues. In myocardium from
rats, the expression of H⫹/K⫹-ATPase has been demonstrated
at the transcriptional and protein levels.7 Biochemical evidence and physiological evidence for a myocardial H⫹/K⫹ATPase have also been found.8 Furthermore, Beisvag and
coworkers7 showed a contribution from the H⫹/K⫹-ATPase of
⬇25% of total 86Rb⫹ uptake in rat hearts and suggested that
the enzyme could contribute significantly to the regulation of
myocardial K⫹ and H⫹ homeostasis. Inhibition of H⫹/K⫹ATPase might therefore induce cellular acidosis, which is
known to depress myocardial contractility mainly at the level
of myofilament responsiveness to [Ca]i.9
Orally applied PPIs are considered safe1 and have been
found advantageous in regard to cardiovascular side effects
compared with histamine type 2 (H2) receptor antagonists
because of lack of chronotropic and inotropic effects. In
contrast to famotidine, omeprazole did not show any changes
in cardiac performance in healthy volunteers as measured by
impedance cardiography and mechanocardiography after
1-week oral treatment with therapeutic doses.10 Pantoprazole,
lansoprazole, and esomeprazole are currently available as
intravenous formulations in the United States. The rationale
for use has come primarily with the suggested efficacy in
reducing rebleeding after endoscopic hemostasis of bleeding
peptic ulcers (eg, References 11 and 12). The target goal for
gastric pH in these patients has been suggested to be ⬎6 in
order to promote hemostasis and minimize clot lysis, in
contradistinction to the target pH ⬎4 for treating patients to
prevent stress ulcer or heal ulcers or reflux esophagitis.1,11,12
As such, the dosing amounts of intravenous PPI have been
higher than that of oral PPI. Recently, omeprazole and pantoprazole have been dosed at 80 mg followed by 8 mg/h for 72
hours.11,12 However, information regarding the cardiac effects of
high doses is lacking. Moreover, few data are available regarding the direct effect of such agents on the myocardium.
Hence, because of the abundant use of this drug class and
because of the presence of a H⫹/K⫹-ATPase in myocardium,
we sought to investigate the effects of PPI on contractility of
isolated human myocardium using pantoprazole, which is the
most commonly used intravenous formulation. In addition,
the mode of action in cardiac tissue was further analyzed in
isolated cardiac tissue from rabbits.
Methods
Myocardial Tissue
Experiments were performed in left ventricular muscle strips from 8
end-stage failing human hearts obtained from patients undergoing
cardiac transplantation and right atrial trabeculae obtained from 16
patients who underwent cardiac surgery. Additional right ventricular
trabeculae were obtained from adult female Chinchilla Bastard
rabbits (weight, 2.0 to 2.5 kg; Charles River Deutschland, Kisslegg,
Germany). Myocardial trabeculae13 and adult rabbit ventricular
cardiac myocytes14 were isolated and forces of electrically stimulated preparations were investigated as described previously. Experimental procedures with human tissue were reviewed and approved
by the ethical committee of the University Clinics of Goettingen, and
the subjects gave informed consent. Procedures with rabbits were
performed in accordance with institutional guidelines for the care
and use of laboratory animals.
pHi Measurements
Intracellular pH (pHi) was measured in isolated trabeculae from
rabbit hearts as described previously.13 Briefly, trabeculae were
mounted in a cylindrical glass cuvette, connected to an isometric
force transducer, and loaded with 2’,7’-bis(carboxyethyl)-5(6)carboxyfluorescein (BCECF)-AM (15 ␮mol/L) in Tyrode’s solution
by 45-minute incubation at room temperature. Excitation light from
a mercury lamp was passed alternately through 2 bandpass filters
(450 nm/495 nm) and focused on the muscle strip. Fluorescence
emission was collected by a photomultiplier (Scientific Instruments)
after passage through a bandpass filter (535⫾5 nm). Values of pHi
were estimated from the ratio of the BCECF fluorescence signals
(F495/F450) after subtraction of background fluorescence. At the end of
each experiment, the BCECF fluorescence ratio was calibrated in vivo
by means of the high K⫹-nigericin method as previously reported.15
Voltage Clamp and Intracellular [Ca2ⴙ]
Measurements in Rabbit Cardiomyocytes
The cardiomyocytes were superfused with a solution consisting of
(mmol/L) 144.0 NaCl, 5.4 KCl, 0.3 NaH2PO4, 1.0 MgCl2, 5.0
HEPES, 11.1 glucose, 1.8 CaCl2, 0.1 niflumic acid, 5.0 4-AP (pH
7.4) at room temperature in a chamber mounted on the stage of an
inverted microscope. Voltage clamp was achieved with the use of
whole-cell patch-clamp technique with an Axoclamp 2A amplifier
(Axon Instruments, Foster City, Calif) in switch clamp (discontinuous) mode. Pipettes were filled with an intracellular solution of the
following composition (mmol/L): 20.0 KCl, 100.0 K aspartate (DL),
20.0 TEA Cl, 10.0 HEPES, 4.5 MgCl2, 4.0 Na2ATP, 1.0 Na2CrP, 2.5
EGTA (pH 7.25 with KOH) and were of resistance 3 to 6 mol/L⍀.
Intracellular [Ca2⫹] was measured from fura 2 fluorescence signals
by a dual-wavelength spectrophotometer method as previously
described.16 Cytosolic loading of fura 2 was achieved by incubating
cardiomyocytes with 5 ␮mol/L fura 2-AM at room temperature for
12 minutes.
Electrophysiological Protocols
Isolated rabbit cardiomyocytes were held at ⫺80 mV, and the
voltage was stepped to ⫺40 mV (50 ms) to inactivate the inward Na⫹
current before stepping to 0 mV (150 ms). Tetrodotoxin 3⫻10⫺5
mol/L was also used to block INa. This protocol was repeated 40
times at a rate of 1 Hz to achieve steady state Ca2⫹ transients.
Sarcoplasmic reticulum (SR) Ca2⫹ content and Na⫹/Ca2⫹ exchanger
(NCX) activity were then estimated by rapidly switching to
10 mmol/L caffeine to cause SR Ca2⫹ release. In the continued
presence of caffeine (20 seconds), the SR is unable to reaccumulate
Ca2⫹, and therefore Ca2⫹ removal is mainly via NCX. The time
course of the decay of [Ca2⫹] and the NCX-mediated inward current
(INCX) represent rates of extrusion of Ca2⫹ from the cell predominantly by NCX.17 These signals were fitted to exponential decays
over ⬎80% of their amplitude. The magnitude of non-NCX Ca2⫹removal mechanisms was estimated from the Ca2⫹ decay obtained by
rapidly switching to 10 mmol/L caffeine in the presence of
10 mmol/L NiCl2 (which blocks NCX), in which the absence of a
current indicated that the current obtained without NiCl2 was solely
due to NCX.
Simultaneous Measurements of Myocyte
Shortening and Intracellular [Ca2ⴙ]
Shortening and [Ca2⫹]i were measured simultaneously as reported
previously.18 Cells were loaded with 10 ␮mol/L fluo 3-AM (Molecular Probes, Carlsbad, Calif) for 15 minutes. Fluo 3 was excited at
480 nm, and fluorescence was measured at 535 nm. The fieldstimulation frequency was 1 Hz (37°C, pH 7.35). Normalized
amplitude of calcium transients (F/F0) was calculated by dividing
fluorescence F by the baseline fluorescence F0 after subtraction of
the background fluorescence (IonWizard, IonOptix Corp). Cells
were treated with 0/10/40 ␮g/mL pantoprazole followed by a
Schillinger et al
TABLE 1.
Negative Inotropy of Pantoprazole
3
Primer Pairs Used in Real-Time PCR
Gene
Human
H⫹/K⫹-ATPase
Sequence
Gene Bank Accession No.
Sense: 5⬘-CCACATCCACACAGCTGACAC-3⬘
M63962
Antisense: 5⬘-TCAAACGTCTGCCCTGACTG-3⬘
Rabbit H⫹/K⫹-ATPase
Sense: 5⬘-ACTCTGCACCGACATTTTCCC-3⬘
X64694
Antisense: 5⬘-TCAGCCTTCTCATACGCCAAG-3⬘
␤-Actin
Sense: 5⬘-CTGGCACCCAGCACAATG-3⬘
M10277
Antisense: 5⬘-CCGATCCACACGGAGTACTTG-3⬘
Transcription of Hⴙ/Kⴙ-ATPase in
Human Myocardium
washout. Fluorescence and shortening were analyzed at steady state
conditions.
RNA was isolated from myocardial tissue and gastric corpus mucosa
from humans and rabbits with the use of RNeasy Kits (Qiagen).
Quantitative reverse transcription polymerase chain reaction (RTPCR) was performed by real-time PCR as described earlier.19 Primer
pairs for H⫹/K⫹-ATPase and ␤-actin are summarized in the Table.
Each sample was analyzed in duplicate. The investigated number of
cDNA molecules was related to ␤-actin cDNA molecules detected in
the same sample to normalize for the quantity of RNA extracted and
the efficiency of cDNA synthesis. The relative expression was then
calculated as described.19
Measurements of SR Ca2ⴙ Uptake Characteristics
Statistical Analysis
Data are expressed as mean⫾SEM. Student paired t test (Figures 3
and 5) or repeated-measures ANOVA (Figures 1, 4, 6, and 7) were
performed to test for statistically significant differences between
different interventions. In general, a value of P⬍0.05 was accepted
as statistically significant; for post hoc analysis, probability values
were adjusted according to Bonferroni (Figures 1 and 4), or the
Tukey test was used (Figure 6).
The authors had full access to and take full responsibility for the
integrity of the data. All authors have read and agree to the
manuscript as written.
Skinned Fibers
Fibers dissected from rabbit right ventricles were skinned by
incubation with Triton (1%) for 24 hours at 4°C. Paired measurements of calcium sensitivity and force development of the myofilaments were performed with the use of 1 relaxation and 10 activation
solutions containing pantoprazole (10/40 ␮g/mL) or NaCl as control
with increasing Ca2⫹ concentrations (from 1.66⫻10⫺7 mol/L to
5.15⫻10⫺5 mol/L Ca2⫹). Active tension was measured via force
transducer and analyzed at steady state conditions. To exclude that
changes in the active tension were due to rundown of the preparation,
each exposure to pantoprazole was preceded and followed by 1
control step with NaCl 0.9%.
A
8
C on t r ol
(n = 15/12)
B
Dose-Response Relationship of PPIs in Isolated
Trabeculae From Human and Rabbit Myocardium
Figure 1A and 1B demonstrates a dose-dependent reduction
of isometric twitch force of electrically stimulated trabeculae
6
C o n t r ol
(n = 12/5)
4
P an t opr az o l e
(n = 16/12)
4
*
3
*
Pantoprazole
(n = 11/5)
2
*
1
2
Human atrium
0
0 0.625 1.25 3.125 6.25 12.5 25
C
Results
5
6
Developed Tension (mN/mm²)
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Measurements of SR Ca2⫹ uptake were performed as described
previously.14 Ventricular myocytes freshly dissociated from adult
rabbit hearts were permeabilized with 0.1 mg/mL ␤-escin and kept in
mock intracellular solution of the following composition (mmol/L):
100 K⫹, 40 Na⫹, 25 HEPES, 100 Cl⫺, 0.05 EGTA, 5 ATP, 10 CrP
(pH 7.0). To test the influence of pantoprazole on SR Ca2⫹ uptake,
myocytes were divided into 3 groups, and 0 (control), 10 ␮g/mL, and
40 ␮g/mL pantoprazole were added into the cuvette. Oxalate
(10 mmol/L) was included to maintain low and constant intra-SR
[Ca2⫹], and ruthenium red (2.7 ␮mol/L) was included to block SR
Ca2⫹ efflux. Myocyte suspensions were stirred and [Ca2⫹] was
monitored with the use of fura 2 (10 ␮mol/L). The decline of Ca2⫹
signal was used to calculate SR Ca2⫹ uptake characteristics.
50 Wash-out
30
0
D
Co n t r o l
( n = 11/11)
25
Human ventricle
0.625 1.25 3.125 6.25 12.5
25
50
Wash-out
6
5
20
**
Pantoprazole
15
10
4
3
C o n t r ol
(n = 14/ 12)
(n = 11/11)
2
5
0
E some pr azole
(n = 5/5)
Rabbit ventricle
0
0.625 1.25
2.5
1
5
10
20
40
Wash-out
Human atrium
0 0.625 1.25 3.125 6.25 12.5 25
Concentration (µg/mL)
50 Wash-out
Figure 1. Dose dependence of isometric
twitch force from pantoprazole (A to C)
and esomeprazole (D) in different types of
myocardium as indicated and partial
reversibility after washout of the drug. The
number of trabeculae and hearts used for
each experiment is indicated in parentheses. *P⬍0.025 (only concentrations with
potential clinical relevance have been
tested).
4
Circulation
July 3, 2007
Pantoprazole (µg/mL):
0
11
10
9
8
7
6
5
4
3
2
1
0
0
500
11
10
9
8
7
6
5
4
3
2
1
0
1000 0
10
500
11
10
9
8
7
6
5
4
3
2
1
0
1000 0
20
500
11
10
9
8
7
6
5
4
3
2
1
0
1000 0
40
500
11
10
9
8
7
6
5
4
3
2
1
0
1000 0
B
160
500
Developed Tension (mN/mm2)
Force (mN/mm²)
A
1000
6
4
2
1
6
6
6
6
6
5
5
5
5
5
4
4
4
4
4
3
3
3
3
3
2
2
2
2
2
1
1
1
1
1
0
0
500
0
1000 0
500
0
1000 0
500
0
1000 0
Time (ms)
500
0
1000 0
D
500
1000
Developed Tension (mN/mm2)
Force (mN/mm²)
C
10
100
Pantoprazole [µg/mL]
Time (ms)
6
4
2
0
1
10
100
Pantoprazole [µg/mL]
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Figure 2. Determination of EC50 for contractile depression of pantoprazole in human myocardium. Single twitches of original recordings
of typical experiments in human atrial (A) and ventricular (C) trabeculae are shown. Mean force values in atrial (B) (n⫽8 trabeculae from
4 hearts) and ventricular (D) (n⫽6/3) trabeculae are shown. Curves have been fitted to yield EC50 values as detailed in Results.
from nonfailing human atrial and failing ventricular myocardium under the influence of pantoprazole. Negative inotropy
was at least partially reversible after washout of the drug. The
pantoprazole-dependent negative inotropy was also present in
ventricular myocardium from healthy adult rabbits (Figure
1C). Moreover, a similar dose-dependent effect was found
with esomeprazole in human atrial myocardium (Figure 1D).
The effect was highly reproducible, and the maximum effect
usually occurred within a few minutes after exposure to
pantoprazole. To yield EC50 values of the negative inotropic
effect, additional dose-response experiments with maximum
pantoprazole concentrations of 160 ␮g/mL have been performed that induced nearly complete suppression of contractile force. Data points have been fitted with the use of logistic
curve fit with OriginPro 7 scientific software (OriginLab
Corp). The EC50 was 30.6⫾1.8 ␮g/mL in atrial human
myocardium (Figure 2B) and 17.3⫾1.3 ␮g/mL in ventricular
human myocardium (Figure 2D). Compared with control, at
doses of 6.25 and 12.5 ␮g/mL of pantoprazole, the contractile
forces in human ventricular myocardium were 3.0⫾0.4 versus 4.2⫾0.7 mN/mm2 and 2.4⫾0.3 versus 4.0⫾0.6 mN/mm2
(P⬍0.025, each). This corresponds to a depression of contractile forces by 27⫾9% and 42⫾8%, respectively. In
human atrial myocardium, forces in the presence of 12.5
␮g/mL pantoprazole were 5.1⫾0.8 versus 5.7⫾0.4 mN/mm2
compared with control (P⬍0.025, depression by 12⫾14%).
Preincubation of trabeculae with ouabain (0.2 ␮mol/L) induced an increase in contractile force by 31.1%. After
exposure to pantoprazole (40 ␮g/mL), force decreased by
65.2% compared with ouabain treatment and by 54.4%
compared with baseline values (P⬍0.05 each).
Expression of Hⴙ/Kⴙ-ATPase in Myocardium and
Gastric Mucosa From Humans and Rabbits
H⫹/K⫹-ATPase mRNA expression was detectable in both
gastric corpus mucosa and ventricular myocardium from
humans and rabbits. However, the expression was very low in
ventricular myocardium from both species. The relative
expression of H⫹/K⫹-ATPase in corpus mucosa compared
with ventricular myocardium was 1.2⫻106 times higher in
humans (1.3⫾0.8 versus 1.1⫻10⫺6⫾0.7⫻10⫺6; n⫽3) and
2.2⫻106 times higher in rabbits (1262⫾673 versus
580⫻10⫺6⫾217⫻10⫺6; n⫽3), respectively.
Pantoprazole Does Not Affect pHi
pHi is an important modulator of contractility. Therefore, we
tested whether pantoprazole induced changes in pHi that
might explain the observed negative inotropic effect. Figure
Figure 3. pHi measurements in ventricular trabeculae from rabbits. A, pHi
changes after application of pantoprazole (40 ␮g/mL) in an individual rabbit
ventricular muscle strip. B, Average
changes of pHi (left) and developed force
(right) induced by 20-minute treatment
with 40 ␮g/mL pantoprazole. Data were
obtained from 5 ventricular trabeculae
isolated from 5 rabbit hearts. **P⬍0.01
vs initial control.
Schillinger et al
A
10 µg/ mL
Negative Inotropy of Pantoprazole
5
40 µ g / m L W a s h - o ut
Length (µm)
1.90
1.85
1.80
1.75
0
100
20 0
300
Time (s)
B
Length (µm)
1.90
1.85
1.80
100
0
20 0
3 00
Time (s)
C
10
40
[Ca²+]i; 400 rel.
Fluorescence Units
PP (µg/mL): 0
1s
D
E
1.6
*
*
1
Control
2
[Ca2+]i (F/F0)
3
0
1.4
1.2
Control
*
Pantoprazole
4
Pantoprazole
Fractional Shortening (%)
Downloaded from http://circ.ahajournals.org/ by guest on June 15, 2017
Figure 4. Dependence of myocyte shortening and [Ca2⫹]i from pantoprazole in rabbit
myocytes. Original chart files of myocyte
shortening with (A) and without (B) addition
of pantoprazole are shown. C, Original recordings of Ca2⫹ transients in an isotonically contracting myocyte. D, Mean values
of fractional shortening. E, Ca2⫹ transient
measurements (n⫽16 myocytes from 10
animals for pantoprazole, and n⫽15/8 for
control). *P⬍0.025 vs control.
1.75
1.0
0
10
40
Wash-out
Concentration (µg/mL)
0
10
40
Wash-out
Concentration (µg/mL)
3A shows pHi changes of a BCECF-loaded rabbit ventricular
muscle strip before and in the presence of pantoprazole (40
␮g/mL). In this example, pHi was increased slightly by ⬇0.1
pH units. Simultaneously, developed force declined from
12.0 to 5.6 mN/mm2 or by 53% within the 20-minute
recording period (not shown). Average results from a total of
5 muscle strips (Figure 3B) revealed that pantoprazole did not
induce any significant alterations in pHi (⌬pHi⫽0.07⫾0.10;
P⫽NS), whereas developed force was reduced by 55⫾4%
(P⬍0.01). Thus, the negative inotropic effect of pantoprazole
was not accompanied by changes in pHi.
Ca2ⴙ Homeostasis in Isolated Rabbit Myocytes
Similar to the findings in isolated trabeculae, pantoprazole
induced a dose-dependent reduction of isotonic shortening of
isolated rabbit myocytes by 32.3⫾5.8% at 10 ␮g/mL and
65.4⫾3.4% at 40 ␮g/mL. This was paralleled by a depression
of Ca2⫹ transient amplitude measured by fluorescence of fluo
3–loaded myocytes (F/F0) by 9.0⫾2.6% and 10.4⫾2.1% at 10
and 40 ␮g/mL, respectively (Figure 4). Effects of pantoprazole on intracellular Ca2⫹ cycling were further investigated in
voltage-clamped and fura 2–loaded rabbit myocytes (Figure
5A and 5B). On addition of 40 ␮g/mL pantoprazole, diastolic
6
Circulation
July 3, 2007
A
B
Em (mV)
[Ca ] (nM)
800
2+
0
-40
-80
(ii) Pantoprazole
Ave rag e ch an ges
% change (+/-SEM)
Control
(i)
600
**
2+
Diastolic [Ca ]i
40
2+
Systolic [Ca ]i
Transient amplitude
20
0
-20
**
400
% change (+/-SEM)
400ms
1
0
-1
0
-1
-20
**
ICa,L amplitude
**
-40
2+
Ca influx via ICa,L
50ms
C
D
Con trol
(i)
(ii) Pantoprazole
Caffeine (10mM)
Ave rage cha nge s
Caffeine (10mM)
% change (+/-SEM)
Im (nA) [Ca2+] (nM)
i
1000
500
0
0.0
INCX.time integral
2+
20
Ca transient amplitude
2+
Rate of Ca transient decay
0
-20
-0.1
*
*
2s
Figure 5. Depolarization-induced Ca2⫹ transients recorded from rabbit ventricular cardiomyocytes. A, Records of membrane voltage
(Em), membrane current, and [Ca2⫹]i from single cardiomyocytes (average of 4 sequential signals) during control conditions (i) and after
perfusion with pantoprazole (40 ␮g/mL) (ii). B, Mean⫾SEM of the percent change in diastolic [Ca2⫹]I, systolic [Ca2⫹]i, and Ca2⫹ transient
amplitude (**P⬍0.01). C, Recordings of [Ca2⫹]i and membrane current recorded on rapid application of 10 mmol/L caffeine (as indicated
above the records) during control conditions (i) and after perfusion with pantoprazole (40 ␮g/mL) (ii). D, Mean⫾SEM of the percent
change in INCX · time integral diastolic [Ca2⫹]I, Ca2⫹ transient amplitude, and rate constant for the decay of the Ca2⫹ transient. *P⬍0.05.
[Ca2⫹]i was increased by 33⫾12% (P⬍0.05; n⫽13), with no
significant change in peak systolic [Ca2⫹]i (4⫾7%; n⫽14). As
a result of these changes, Ca2⫹ transient amplitude was
reduced by 25⫾8% (n⫽14; P⬍0.05) compared with control
cells. These changes were paralleled by a reduction in ICa,L
amplitude (by 35⫾5%; P⬍0.05; n⫽14). To measure SR and
Kd of SR Ca2+ uptake
Vmax of SR Ca2+ uptake
Control
Low Panto
High Panto
0.4
Contr ol
l ow P a n t o
High Panto
45 0
*
Kd (nmol/L)
Vmax (fmol/s/106 cells)
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Current (nA)
Current (nA)
0
0.3
0.2
0.1
A
*
40 0
35 0
B
Figure 6. Characteristics of SR Ca2⫹-ATPase activity as determined by use of a cuvette assay. Vmax (A) and Kd (B) of Ca2⫹ transport
activity in the presence of either 0 (control), 10 (low), or 40 (high) ␮g/mL of pantoprazole (Panto) are shown. *P⬍0.05 vs control.
Schillinger et al
7
[Ca
2+
]o (mol/L)
4 0 µ g/ m L
Pantoprazole
0
Figure 7. Measurements in skinned fibers. Dose
dependence of active tension at increasing Ca2⫹
concentrations in skinned fiber preparations at 10
␮g/mL (A) and 40 ␮g/mL (B) of pantoprazole is
shown. Maximal active tension at saturating Ca2⫹
concentration and Ca2⫹ sensitivity was significantly
lower at 40 ␮g/mL. *P⬍0.05.
10
-4
10
-4
10
-5
10
-6
10
-7
0
10
10
-5
1 0 µ g /m L
Pantoprazole
*
Control
10
-6
10
20
10
-7
Control
30
10
-8
20
Active Tension (mN/mm²)
B
30
10
-8
Active Tension (mN/mm²)
A
Negative Inotropy of Pantoprazole
[Ca 2+ ]o (mol/L)
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NCX function, the intracellular Ca2⫹ signals and the associated INCX were analyzed on rapid application of 10 mmol/L
caffeine (Figure 5C and 5D). A reduction in both the
amplitude of the caffeine induced Ca2⫹ release (by 18⫾6%;
n⫽10; P⬍0.05) and the associated time integral of INCX (by
21⫾6%; n⫽10; P⬍0.05) on exposure to pantoprazole (40
␮g/mL) indicated that pantoprazole decreased SR Ca2⫹ content. No changes in the rate of decay of the caffeine induced
Ca2⫹ transient (1⫾8%; n⫽10) and INCX (2⫾7%; n⫽10) were
observed after pantoprazole administration, indicating that
the sarcolemmal extrusion processes (particularly NCX) were
unaffected by the drug.
SR Ca2ⴙ Uptake Data
Figure 6 demonstrates the effects of pantoprazole on oxalatesupported Ca2⫹ uptake in permeabilized rabbit ventricular cardiomyocytes. In the presence of either carrier solution or low (10
␮g/mL) or high (40 ␮g/mL) concentrations of pantoprazole, the
[Ca2⫹] at which half maximal Ca2⫹ uptake occurred (Kd) and the
value of the maximum rate of Ca2⫹ uptake (Vmax) were estimated
with a fura 2– based SR Ca2⫹ uptake assay. Neither low nor high
concentration of pantoprazole influenced the Vmax value. However, the Kd values of SR Ca2⫹ uptake were increased significantly at both concentrations of pantoprazole (nmol/L: control,
358⫾15; low pantoprazole, 406⫾15; high pantoprazole,
395⫾12; P⬍0.05).
Effects of Pantoprazole in Skinned
Fiber Preparations
The effect of pantoprazole in skinned fibers is shown in
Figure 7. There was a reduction of the maximum active
tension at saturating Ca2⫹ concentration by 7.8⫾0.8% at 10
␮g/mL (P⫽NS) and 26.5⫾2.8% at 40 ␮g/mL (P⬍0.05). In
addition, a small but significant rightward shift of the Ca2⫹
response curve indicating a decrease of Ca2⫹ sensitivity was
found. The EC50 was 1.44 mol/L Ca2⫹ at 0 ␮g/mL and 1.59
mol/L Ca2⫹ at 40 ␮g/mL pantoprazole (P⬍0.05).
Discussion
The present study shows a negative inotropic effect of
pantoprazole in isolated myocardium. This was dose dependent, induced nearly complete inhibition of twitch force at
very high doses, and was partially reversible. Negative
inotropy of pantoprazole was present in myocardium from
different species (human and rabbit) and in myocardium from
different origins (atrial and ventricular), and it was found in
different myocardial preparations (multicellular and single
cells). The EC50 for contractile force depression was
30.6⫾1.8 ␮g/mL in nonfailing human atrial and 17.3⫾1.3
␮g/mL in failing human ventricular myocardium, respectively. Moreover, similar results could be obtained with
esomeprazole, which is suggestive of a class effect of PPIs.
Furthermore, we could reveal 2 underlying mechanisms for
the pantoprazole-dependent inhibition of contractile force: (1)
reduction in the amplitude of Ca2⫹ transients as a consequence of impaired SR Ca2⫹ uptake and reduced Ca2⫹ influx
via ICa,L and (2) reduced Ca2⫹ responsiveness of the myofilaments as a result of a reduced maximal active tension and a
slightly lower Ca2⫹ sensitivity. In contrast, despite the expression of the H⫹/K⫹-ATPase at the transcriptional level in
human and rabbit myocardium, no significant changes in pHi
could be detected in the presence of pantoprazole.
Note that the mechanisms underlying the effects of pantoprazole in myocardium are completely different from the mechanisms of the drug in gastric parietal cells and probably do not
involve inhibition of H⫹/K⫹-ATPase. In regard to gastric proton
pump inhibition, all PPIs are prodrugs and require acid to
become protonated and converted into the active form.1 After
intravenous administration or intestinal absorption, when orally
administered the lipophilic unprotonated form readily penetrates
cell membranes, including that of gastric parietal cells and
myocytes. In parietal cells, as it transverses the cell it is exposed
to the acidic environment in the secretory canaliculus of the
gastric site and becomes protonated, converting it to a hydrophilic drug that can no longer permeate cell membranes. The
drug becomes trapped in the canaliculus of the parietal cell. For
this reason, in parietal cells PPIs exhibit a substantial accumulation versus plasma at low pH, eg, 1000-fold for omeprazole
and 10000-fold for rabeprazole at a pH of 1. Moreover, protonation of the drug initiates a series of chemical reactions that
culminates in covalent binding of the drug with selected cysteine
residues of the H⫹/K⫹-ATPase.1
The present study is the first one reporting on the expression of H⫹/K⫹-ATPase in human and rabbit ventricular
myocardium. Recently, it has been suggested that H⫹/K⫹ATPase may contribute to pH homeostasis in rat myocardium.7 We therefore investigated the hypothesis that cellular
acidosis subsequent to proton pump inhibition might explain
the negative inotropy of PPIs. However, our measurements
with the H⫹-sensitive fluorescence dye BCECF did not reveal
8
Circulation
July 3, 2007
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any significant influence of pantoprazole on pHi. Moreover,
the absence of acidic compartments in cardiac tissue with pH
⬍1 precludes significant accumulation of pantoprazole in the
myocardium. In moderately acidic compartments like lysosomes, slow activation of pantoprazole theoretically may
occur with an activation half-life of 4.7 hours.20 However,
negative inotropy in our experiments usually occurred
quickly within several minutes. Moreover, the effect was at
least partially reversible after washout of the drug, whereas
the recovery of the gastric proton pump from pantoprazole
has a half-life of ⬇46 hours21 and was suggested to depend
mainly on the synthesis of new pump protein.1,22 In addition,
the expression of H⫹/K⫹-ATPase in myocardium was very
low compared with gastric mucosa. Therefore, it is unlikely
that the negative inotropy of pantoprazole in myocardium is
the consequence of H⫹/K⫹-ATPase inhibition.
We investigated the effects of pantoprazole on intracellular
Ca2⫹ homeostasis and myofilament Ca2⫹ responsiveness,
which are the 2 principal physiological mechanisms of the
myocardium to alter contractility. We found a reduction in the
Ca2⫹ transient amplitude in field-stimulated ventricular myocytes. Interpretation of this result in terms of Ca2⫹ signaling is
complicated by possible effects of pantoprazole on the action
potential. With fixed depolarizing pulses in voltage-clamped
myocytes, pantoprazole caused a rise in diastolic [Ca2⫹]i and
minimal changes in systolic [Ca2⫹]i, the net effect being a
reduced Ca2⫹ transient amplitude. Independent evidence for
pantoprazole-dependent inhibition of SERCA as an underlying mechanism was obtained with a fura 2– based SR calcium
uptake assay in permeabilized cardiomyocytes. Moreover,
Ca2⫹ influx via ICa,L was also depressed by pantoprazole. The
combination of reduced SR calcium uptake and reduced Ca2⫹
influx would explain the reduction in SR Ca2⫹ content
observed and raised diastolic [Ca2⫹]i.
We also investigated the influence of pantoprazole in
skinned fibers. These preparations allow for the investigation
of drug effects directly at the myofilament level under
well-defined in vitro conditions. Hence, it can be excluded
that the observed effects are mediated by changes in [Ca2⫹]i or
pHi. We were able to show a significant reduction in maximal
active tension of the contractile proteins at saturating Ca2⫹
concentrations by 26.5⫾2.8% at 40 ␮g/mL. Moreover, at 40
␮g/mL there was a slight but significant reduction in myofilament Ca2⫹ sensitivity. However, when the marked negative inotropy in multicellular trabeculae is considered, these
effects were too faint to explain entirely the mode of action of
pantoprazole in myocardium. Therefore, the negative inotropy of pantoprazole observed at 10 ␮g/mL mainly results
from decreased intracellular [Ca2⫹]. At the higher dose (40
␮g/mL), depression of myofilament responsiveness appears
to contribute to the negative inotropic effect. We also suggest
that the effects of pantoprazole in myocardium depend on the
native unprotonated form of the drug and do not require
activation by low pH because all effects occurred at pH 7.3 to
7.4.
In contrast to our data, Yenisehirli and Onur23 found a positive
inotropic effect of 3 different PPIs in rat atria that was potentiated by pretreatment with the Na⫹/K⫹-ATPase inhibitor ouabain.
Moreover, lansoprazole induced a prolongation of the action
potential. The authors speculated that this could be mediated by
inhibition of H⫹/K⫹-ATPase promoting altered intracellular
Ca2⫹ handling. In our study with human and rabbit myocardium,
pantoprazole decreased tension and reduced shortening and Ca2⫹
transient amplitude in “unclamped” trabeculae and single cells.
When single rabbit myocytes were clamped with a fixed voltage
clamp duration, pantoprazole decreased the Ca2⫹ transient amplitude to a degree similar to that seen in “unclamped” preparations. This suggests that in humans and rabbits, action potential
duration changes are not instrumental in the decreased Ca2⫹
transient and subsequent mechanical response caused by pantoprazole application. Moreover, a significant contribution of
H⫹/K⫹-ATPase to the action potential is unlikely because of the
low degree of expression. The different responses to PPIs may
therefore be related to species. Compared with human and rabbit
myocardium, in rats the action potential is short and intracellular
sodium is high. In particular, the latter condition favors Ca2⫹
entry by reverse-mode Na⫹-Ca2⫹ exchange, which contributes to
SR Ca2⫹ load and contractility when action potential duration
increases.9 In contrast to the study by Yenisehirli and Onur, the
negative inotropic effect of pantoprazole in rabbit myocardium
observed by us was not influenced by blockade of Na⫹/K⫹ATPase, and the magnitude of the effect was similar to that in
experiments without ouabain.
Finally, although this was beyond the scope of the present
study, we would like to speculate whether our findings might be
of clinical relevance. Because the negative inotropic effect was
partially reversible after washout of the drug and significant
accumulation in the myocardium is unlikely, the effect of
pantoprazole in vivo probably depends on plasma concentrations. Maximal pantoprazole plasma concentrations of 4.6 ␮g/
mL24 and 10.4 ␮g/mL (Altana Pharma AG, written communication, July 23, 2004) have been found after administration of
common oral (40 mg) and intravenous (80 mg) doses. In the
present work, similar concentrations induced a reduction of
contractile force of isolated trabeculae by 27⫾9% (6.25 ␮g/mL)
and 42⫾8% (12.5 ␮g/mL), which might indicate a potential
clinical relevance of our findings. However, the duration of
cardiac side effects is temporally limited because all PPIs are
quickly eliminated from blood with plasma elimination half-life
periods of ⬇1 to 2 hours.20,24 Moreover, cardiac side effects may
be attenuated in vivo because the activity of the active free
compound may be substantially lower because of high plasma
protein binding.20 On the other hand, particular conditions may
be associated with increased duration and intensity of side
effects. Patients with heart failure must be investigated for PPI
tolerance. These patients are much more susceptible to negative
inotropic drugs because of blunted contractile reserve subsequent to decreased sympathetic sensitivity25 or negative forcefrequency relationship.26 In addition, the dependence of H⫹
elimination from H⫹/K⫹-ATPase may be increased in heart
failure because of the impaired function of the Na⫹/H⫹ exchange
subsequent to increased [Na⫹]i.27 Moreover, all PPIs undergo
extensive hepatic biotransformation before elimination. In
CYP2C19-poor metabolizers that represent ⬇3% to 5% of
whites, a similar percentage of blacks, and 12% to 25% of
different Asian populations, much higher plasma concentrations
and longer elimination half-life periods have been found.1 The
same holds true for patients with severe liver impairment.28
Schillinger et al
Recent American29 and Canadian30 studies have shown that
appropriate use of intravenous PPIs was seen in less than half
of the patients. In view of our data, PPIs should be prescribed
carefully. We have recently initiated a clinical study for the
investigation of cardiac side effects of pantoprazole in
healthy volunteers. Moreover, we encourage clinical studies
to identify individuals with increased intrinsic risk for cardiac
side effects of PPIs.
Acknowledgments
We gratefully acknowledge the expert assistance of Hanna Schotola,
Astrid Steen, Aileen Rankin, Gudrun Muller, Elke Neumann, and
Michael Kothe.
Sources of Funding
This work was supported by grants from the Wellcome Trust (to Dr
Kettlewell), the British Heart Foundation (to Dr Smith), and the
Deutsche Forschungsgemeinschaft (to Dr Maier).
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Disclosures
None.
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10
Circulation
July 3, 2007
CLINICAL PERSPECTIVE
The proton pump inhibitor pantoprazole was evaluated for its effects on cardiac contractility in isolated myocardium from
humans and rabbits. We found a dose-dependent negative inotropic effect mainly resulting from alterations in intracellular
Ca2⫹ handling. At higher concentrations of the drug, depression of myofilament Ca2⫹ responsiveness was also observed.
However, despite the expression of the gastric proton pump in human and rabbit hearts, no relevant changes in pH
homeostasis could be detected. Moreover, expression of the pump was very low in myocardium compared with gastric
tissue. A similar effect was observed with esomeprazole. Thus, proton pump inhibitors affect cardiac contractility by
intracellular mechanisms that are distinct from their effects in gastric parietal cells. The effects in isolated myocardium
were observed at concentrations that might be of potential clinical relevance. Thus, cardiac effects of proton pump
inhibitors should be evaluated in vivo because of their extensive use for acid-related gastrointestinal diseases.
Downloaded from http://circ.ahajournals.org/ by guest on June 15, 2017
Negative Inotropy of the Gastric Proton Pump Inhibitor Pantoprazole in Myocardium
From Humans and Rabbits. Evaluation of Mechanisms
Wolfgang Schillinger, Nils Teucher, Samuel Sossalla, Sarah Kettlewell, Carola Werner, Dirk
Raddatz, Andreas Elgner, Gero Tenderich, Burkert Pieske, Giuliano Ramadori, Friedrich A.
Schöndube, Harald Kögler, Jens Kockskämper, Lars S. Maier, Harald Schwörer, Godfrey L.
Smith and Gerd Hasenfuss
Downloaded from http://circ.ahajournals.org/ by guest on June 15, 2017
Circulation. published online June 18, 2007;
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