Am J Physiol Heart Circ Physiol 285: H907-H914, 2003.
First published March 27, 2003; doi:10.1152/ajpheart.00856.2002
0363-6135/03 $5.00
INNOVATIVE METHODOLOGY
24-h Langendorff-perfused neonatal rat heart used to study the impact of adenoviral gene transfer
S. Wiechert,1
A. El-Armouche,2
T. Rau,2
W. -H. Zimmermann,2 and
T. Eschenhagen2
1Institute of Experimental and Clinical
Pharmacology, University Erlangen-Nürnberg, 91054 Erlangen, Germany; and
2Institute of Experimental and Clinical Pharmacology,
University Hospital Hamburg-Eppendorf, 20246 Hamburg, Germany
Submitted 5 November 2002
; accepted in final form 27 February 2003
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ABSTRACT
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The human genome project has increased the demand for simple experimental
systems that allow the impact of gene manipulations to be studied under
controlled ex vivo conditions. We hypothesized that, in contrast to adult
hearts, neonatal hearts allow long-term perfusion and efficient gene transfer
ex vivo. A Langendorff perfusion system was modified to allow perfusion for
>24 h with particular emphasis on uncompromised contractile activity,
sterility, online measurement of force of contraction, inotropic response to
-adrenergic stimulation, and efficient gene transfer. The hearts were
perfused with serum-free medium (DMEM + medium 199, 4 + 1) supplemented with
hydrocortisone, triiodothyronine, ascorbic acid, insulin, pyruvate,
L-carnitine, creatine, taurine, L-glutamine, mannitol,
and antibiotics recirculating (500 ml/2 hearts) at 1 ml/min. Hearts from 2
day-old rats beat constantly at 135–155 beats/min and developed active
force of 1–2 mN. During 24 h of perfusion, twitch tension increased to
165% of initial values (P < 0.05), whereas the inotropic
response to isoprenaline remained constant. A decrease in total protein
content of 10% and histological examination indicated moderate edema, but
actin and calsequestrin concentration remained unchanged and perfusion
pressure remained constant at 7–11 mmHg. Perfusion with a LacZ-encoding
adenovirus at 3 x 108 active virus particles yielded
homogeneous transfection of 80% throughout the heart and did not affect
heart rate, force of contraction, or response to isoprenaline compared with
uninfected controls (n = 7 each). Taken together, the 24-h
Langendorff-perfused neonatal rat heart is a relatively simple, inexpensive,
and robust new heart model that appears feasible as a test bed for functional
genomics.
functional genomics; heart failure; molecular biology
WITH SUCCESSFUL TERMINATION of the human genome project, the
scientific community is faced with about 30,000 genes and an estimated
threefold higher number of gene products whose functions in the cell are far
from being understood in the vast majority of cases
(3,
13,
19). Simple, efficient, and
valid assays are crucial to study gene-function relationships. Whereas
principal gene functions can easily be studied in vitro or in simple
experimental systems such as Xenopus oocytes or mammalian tumor cell
lines, the contribution of a single gene or changes in its expression levels
to the overall function of an organ or an organism remains difficult to
elucidate.
Tremendous progress has been made by transgenic techniques, i.e., the
generation of animals overexpressing foreign or modified endogenous genes or
carrying an inactivated gene locus
(15,
22). Transgenic animals are
undisputably the gold standard to study gene-function relationships under
(patho)physiological conditions, but limitations remain. First, generation of
transgenic animals still requires considerable time and resources that
preclude its application as a screening method. Second, despite important
progress in conditional transgene expression in terms of timing, organ
specificity, and quantity, transgenic techniques are prone to confounders such
as developmental and physiological compensations and genetic background.
Finally, ethic considerations exist.
In cardiovascular research attempts have been made to create easier and
more efficient experimental models that are less prone to compensational
responses but yet provide the necessary complex cardiac context. Examples are
cultured neonatal rat cardiac myocytes
(11), freshly isolated adult
cardiac myocytes (5,
6,
16), isolated trabeculae of
adenovirus-perfused rabbit hearts
(17), injection of adenovirus
into neonatal rat hearts in vivo
(4), or adenovirus infusion
into adult rat hearts in vivo
(12). Cells in culture are
easily transfected but are either immature or prone to cell isolation and
dedifferentiation artifacts. In addition, measurement of force of contraction
is indirect at best. More complex systems likely better reflect the
physiological situation and allow force measurement, but they require high
virus titers, i.e., are expensive and time consuming, require higher technical
demands, and have not yet been shown to be applicable as a routine method.
Recently, we have introduced the method of engineered heart tissue that may
serve as an intermediate between cell culture and animal model by allowing
efficient adenoviral gene transfer and measurement of isometric force of
contraction (9,
23). However, adenoviral gene
toxicity appears to limit its usefulness as a routine method
(7). Thus none of the existing
systems appears to be a perfect test bed for adenoviral gene transfer. The
classic Langendorff heart may be ideally suited for this purpose because it
allows measurement of complex cardiac function under relatively physiological
conditions. Yet, in hearts from adult animals of various species development
of interstitial edema limits perfusion time to maximally 6–8 h, and
attempts to circumvent this problem by modifying the perfusion conditions have
failed (21). A recent review
describes that hypothermic arrest periods cause tissue preservation up to 24 h
and longer (20), but no
attempts have been made to use this model for genetic testing. We hypothesized
that neonatal rat hearts are less sensitive to the artificial conditions of in
vitro perfusion with crystalloidal solutions, partly because they are very
hypoxia/ischemia resistant (1,
10) and have a less developed
vascular system and therefore may serve as a suitable, improved model to study
effects of adenoviral gene transfer.
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METHODS
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All procedures were approved by the local animal protection authority
(TS-00/20 Pharma) and conformed to the Guide for the Care and Use of
Laboratory Animals published by the National Institutes of Health (NIH
Publication 85-23, Revised 1985).
Animals, Langendorff perfusion setup, and medium. A total number
of 710 Wistar pups (days 0–7) was used. The mean weight was 7.9
g. Animals received an intraperitoneal injection of heparin (500 units)
10–20 min before being euthanized. After cervical dislocation, hearts
were rapidly excised (mean time to perfusion 2.5 min) and mounted on a
Langendorff perfusion system (LDH 4103, Föhr Medical Instruments)
equipped with a 23-gauge (0.6 mm) needle
(Fig. 1, A and
B). The system consists mainly of different
carbogen-gassed, water-jacketed reservoirs for the medium, a pump, sterile
one-way filters, special glass equipment to warm the medium, and a bubble trap
directly above the needle holding the heart. The perfusion medium was composed
of DMEM + medium 199 4+1, 100 U/ml penicillin (Pen), 100 µg/ml streptomycin
(Strep), 2 mmol/l L-glutamine (Biochrom), 5 µg/ml
hydrocortisone, 5 mmol/l creatine, 1 nmol/l triiodothyronine, 1.87 µg/ml
amphotericin B, 80 mmol/l mannitol (Sigma), 5 mmol/l pyruvate, 2 mmol/l
L-carnitine (Fluka), 100 µg/ml ascorbic acid (Roth), 20 IU/l
insulin (Aventis), and 5 mmol/l taurine (Applichem). Temperature was
maintained at 37°C, and the pH was initially 7.59 ± 0.02 and 7.63
± 0.02 after 24 h without significant change (n = 8). Force of
contraction was measured with a self-made transducer consisting of a beam in
bending equipped with four strain gauges (no. 3/350DY11, Hottinger Baldwin
Messtechnik). Twitch tension (TT), resting tension, contraction duration (time
from 10% to peak force development), and relaxation duration (time to 90%
relaxation) were evaluated by BMON software (Ingenieurbüro Jäckel;
Hanau, Germany).
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Fig. 1. Experimental setup for 24-h Langendorff perfusion of neonatal rat hearts.
A: schematic diagram of the setup. 1 and 2,
carbogen-gassed, water-jacketed medium reservoirs; 3, one-way sterile
filter; 4, pump; 5, water-jacketed organ bath; P, perfusion pressure
transducer; SG, force transducer with strain gauges; PC personal computer.
B: photograph of a detail of the perfusion setup. BT, bubble trap;
LH, Langendorff-perfused heart; SW, steal wire connecting the heart with the
force transducer; SG, force transducer with strain gauges.
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Measures to ensure and to determine sterility. Given that
sterility was a major requirement for success, several measures were taken to
minimize contamination and growth of germs. At the end of a week, the complete
system was thoroughly rinsed with water to remove the medium, cleaned with
citric acid (0.1 mol/l) and SDS (10 mmol/l) at 60°C, and subsequently
thoroughly rinsed with water. Before the next experiment, the complete device
was autoclaved, and new sterile filters were build in. The latter were changed
daily. The instruments were stored in 70% ethanol. Handling was performed with
gloves to minimize contamination. The organ baths were partly closed with a
lid, and the perfusion medium contained Pen-Strep at standard and amphotericin
B at one-third of the standard concentration (1.87 µg/ml). After
establishment of standard conditions, sterility was tested for 1 wk after each
24-h run. Samples of the perfusion medium were taken directly above the
23-gauge needle holding the heart. Samples were subjected to a standard
quantitative bacteriological examination kindly provided by the Institute of
Microbiology, University of Erlangen-Nuremberg. This evaluation showed no sign
of bacterial or fungal growth.
Heart wet-to-dry weight and protein determination. To determine
heart-to-body weight ratios, animals were weighed, and hearts including atria
were excised and gently, but thoroughly dabbed between two layers of paper
towels either before or after the 24-h perfusion, weighed, and then
freeze-dried (24 h) in preweighed Eppendorf cups. To determine protein content
using the Bradford method (Bio-Rad), hearts either native or after the 24-h
perfusion were homogenized in lysis buffer (Tris, pH 7.5, 25 mmol/l, SDS 5%),
further diluted with water, and measured with IgG as standard.
Adenovirus production and titering. The adenovirus coding for
bacterial -galactosidase with a nuclear localization signal (Ad5LacZ)
was a kind gift from Y. Fromes, Paris, France. It was propagated in HEK 293
cells and titered on neonatal rat cardiac myocytes as described previously
(23). Importantly, the number
of biologically active virus (bav) was defined as the virus number necessary
to positively transfect (stain blue) a given number of neonatal rat cardiac
myocytes in culture. For example, 1 x 106 bav was just
sufficient to transfect 1 x 106 cardiac myocytes or 50% of 2
x 106 cardiac myocytes.
Adenoviral gene transfer: standard experiment. The hearts were
infected with Ad5LacZ 3 h after the start of perfusion. To increase gene
transfer efficiency (7,
16), the hearts were perfused
with medium containing histamine and serotonin (10 µmol/l each) that was
drained off after passage through the hearts. After 10 min, the perfusion was
stopped and the virus was injected directly into the tube above the heart.
Perfusion was started for 1 min, stopped for another 2 min, and periods of
continuous and halted perfusion were repeated twice as described by others
(7,
16). The medium containing the
virus was collected in a 50-ml reservoir and recirculated until the end.
Inotropic response to isoprenaline. In the series of experiments
designed to evaluate the influence of a 24-h perfusion on force of contraction
and inotropic response to -adrenergic agonists, isoprenaline was infused
for 15 min 3 h after the start of perfusion, the perfusate was drained,
isoprenaline was washed out for 20 min, and recirculation was reestablished.
This procedure was repeated after 21 h of perfusion. In the series of
experiments designed to test the influence of virus infection on
-adrenergic responses, isoprenaline was applied only 21 h after
infection. To enlarge the inotropic response, the perfusion medium in this
series of experiments was exchanged to a low-calcium (1 mmol/l) Tyrode
solution before application of isoprenaline. This reduced force of contraction
by 80% (see Fig. 6) but
yielded enhanced inotropic response to isoprenaline.
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Fig. 6. Histological analysis of Ad5LacZ-infected 24-h Langendorff-perfused
neonatal rat hearts. Photomicrograph of a paraffin section (4 µm) of a
-galactosidase-stained, formaldehyde-fixed (4%, overnight), PBS-rinsed
(overnight) heart. Section was counterstained with hematoxylin and eosin. Note
that with this technique, histological preservation was superior, but
transfection efficiency was underestimated. Bar = 30 µm.
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Histological procedures. Hearts were fixed in 4% formaldehyde in
PBS overnight at 4°C, either directly after excision or after the 24-h
perfusion, and subjected to standard paraffin embedding. Slices (4 µm) were
cut and stained with hematoxylin and eosin (H&E) according to standard
protocols. To determine transfection efficiency after adenoviral gene transfer
with Ad5LacZ, hearts were not fixed but immediately frozen in liquid nitrogen
and embedded in TissueTek, and 10-µm slices were cut. Sections were washed
with PBS, fixed with 70% ethanol for 1 min, washed with PBS twice, and
incubated with LacZ assay solution {20 mmol/l
K3[Fe(CN)6], 20 mmol/l
K4[Fe(CN)6], 2 mmol/l
MgCl2· 6H2O, 0.02% Igepal CA-630,
0.01% sodium deoxycholate, and 1 mg/ml X-Gal} for 10 min. Slices were rinsed
with PBS, postfixed in 4% formaldehyde in PBS, and counterstained with
H&E.
Statistical analysis. Data were calculated as arithmetic means
± SE and analyzed using an unpaired t-test. A P value
of <0.05 was considered significant. n indicates the number of
independent experiments.
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RESULTS
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Characterization of the 24-h Langendorff perfusion model
sterility. An inherent problem of 24-h perfusions of biological specimens
excised under semisterile conditions in a half-open system is bacterial and
fungal infection. Indeed, initial experiments with medium containing only
Pen-Strep at standard concentrations showed severe contamination in both the
medium and the heart. Inclusion of four sterile one-way filters
(Fig. 1), exclusive use of
silicone tubing and glass connectors, autoclaving the entire unit once a week,
i.e., for perfusion of eight hearts, and inclusion of amphotericin B at
one-third of the standard concentration eliminated bacterial or fungal growth
(10 samples taken during several weeks, data not shown).
Online measurement of beating rate, force of contraction, and perfusion
pressure. Force of contraction was measured by connecting a hook in the
apex of the perfused heart with a self-made force transducer
(Fig. 1). Online recording was
interrupted during the night by applying a weight of 4 mN to the apex (removed
the next morning) because it was shown to increase the reproducibility of
force measurements after 24 h (n = 4, data not shown). Under these
conditions (n = 23), the hearts beat at a mean frequency of 145
beats/min 3 h after the start of perfusion and 137 beats/min 24 h after the
start of perfusion (Fig.
2A). Isoprenaline (0.1 µmol/l) increased beating rate
to 179 beats/min 3 h after the start of perfusion and 168 beats/min 24 h after
the start of perfusion (Fig.
2A). Interestingly, force of contraction increased
between 3 h and 24 h after the start of perfusion from 1.8 to 2.9 mN (+61%;
Fig. 2B). Isoprenaline
(0.1 µmol/l) increased force of contraction to 3.7 mN (+106%) 3 h after the
start of perfusion and to 4.7 mN (+62%) 24 h after start of perfusion
(Fig. 2B). In absolute
terms, the increase in force of contraction was similar (1 mN). The perfusion
pressure was measured online for 24 h and, after a short equilibration period
with slightly higher pressures, remained constant at 1 ml/min
(Fig. 2C). Perfusion
at 2 ml/min raised pressures to 50 mmHg (declining over time to 30 mmHg) but
did not lead to better contractile function after 24 h (n = 7–8
per group; not shown). Because sterile filters obstructed, we found the 1
ml/min rate more suitable.
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Fig. 2. Functional performance of Langendorff-perfused rat hearts (n = 23)
over 24 h. A: spontaneous and isoprenaline-stimulated beating rate 3
h and 24 h after the start of perfusion. bpm, Beats/min. B:
spontaneous and isoprenaline-stimulated twitch tension (TT) 3 h and 24 h after
the start of perfusion. C: perfusion pressure over the entire 24-h
period. *P < 0.05 vs. 3 h.
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Conditions of perfusion. The medium was designed according to
standard cell culture techniques, published protocols
(14), and our experience. Not
all of the supplements were systematically tested. First, amphotericin B at
standard concentration reduced force of contraction below 1 mN or hampered
positive inotropic effect of isoprenaline (exclusion criteria) in five of
seven experiments. Second, force of contraction and the positive inotropic
effect of isoprenaline 24 h after the start of perfusion were 16% and 25%
higher in hearts perfused with 1 nM triiodothyronine than with 0.1 nM
(Fig. 3). Third, at a later
stage of the experiments, a series of experiments showed that omitting insulin
from the medium did not affect basal force of contraction but doubled the
inotropic response to isoprenaline (n = 6 per group; not shown).
Finally, addition of fetal calf serum or albumin was not compatible with the
continuous bubbling of the medium.
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Fig. 3. Effect of medium composition on contractile function. Spontaneous and
isoprenaline-stimulated TT 24 h after start of perfusion in the presence of
0.1 nmol/l (n = 7 hearts) or 1.0 nmol/l (n = 10 hearts)
triiodothyronine (T3).
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Heart wet weight and dry weight, protein concentration, and
histology. Given that interstitial edema represents a limiting factor of
Langendorff-perfused adult hearts, several parameters were studied to quantify
edema in 24-h perfused neonatal rat hearts. As shown in
Fig. 4, the dry weight-to-wet
weight ratio showed a 10% decrease compared with native, unperfused hearts
from animals of similar age (Fig.
4A). Protein concentration decreased from 9.3 to 8.4%
(–10%; Fig. 4B).
Standard H&E histology showed a less compact tissue structure in the 24-h
perfused hearts, an increase in interstitial space, and a decrease in myocyte
diameter and eosinophilic staining (Fig. 4,
C and D). However, both actin and calsequestrin,
two markers of cardiac myocytes, remained unchanged when assayed as such or
normalized to GAPDH (Fig.
4E). This indicates that the changes, if present, are
minor.
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Fig. 4. Tissue properties of 24-h Langendorff perfusion of neonatal rat hearts.
A: dry weight-to-wet weight ratio of native and 24-h perfused hearts.
B: protein concentration in percentage of wet weight in native and
24-h perfused hearts. n, Number of hearts studied. C:
photomicrograph of an hematoxylin and eosin-stained paraffin section of a
native neonatal rat heart (day 2). D: 24-h perfused heart of
same age; bar = 50 µm. E: representative Western blot and
statistical analysis of actin, GAPDH, and calsequestrin (CSQ) in native and
24-h perfused neonatal rat hearts (n = 8 each). P < 0.05
vs. native.
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Adenoviral gene transfer into 24-h Langendorff-perfused neonatal rat
heart. Hearts were perfused with Ad5LacZ in a volume of about 5 ml under
three times 2-min stop-flow conditions in the presence of histamine and
serotonin. The virus was then recirculated for the rest of the time in a 50-ml
volume. These conditions were chosen after initial experiments with different
application modes and increasing concentrations of virus and were found to
yield a high transfection efficiency throughout the hearts partly excluding
the atria (Fig. 5).
Importantly, the transfection efficiency of 80% was stable in seven
consecutive experiments. Histologically, -galactosidase staining showed
nuclear concentration, but the myoplasma was stained blue as well
(Fig. 6). Virus infection did
not affect beating rate (Fig.
7A) or force of contraction under basal or
isoprenaline-stimulated conditions compared with uninfected controls studied
in parallel (Fig.
7B).
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Fig. 7. Effect of Ad5LacZ on contractile function of 24-h Langendorff-perfused
neonatal rat hearts (n = 7). A: beating rate and contraction
kinetics of hearts 24 h after the start of perfusion in the absence or
presence of Ad5LacZ. dF/dt+, maximal increase in force over time
(mN/s); dF/dt–, maximal decrease in force over time (mN/s);
T1, time from 10% to peak force development;
T2, time to 90% relaxation. B: TT and response to
isoprenaline 24 h after the start of perfusion in the absence or presence of
Ad5LacZ. Force of contraction was determined in normal perfusion medium
(medium), 45 min after change of perfusion to low Ca2+
Tyrode solution (tyrode), and 15 min after application of isoprenaline (0.1
µmol/l).
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DISCUSSION
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We developed a new experimental model, the 24-h Langendorff-perfused
neonatal rat heart, and showed that it is stable for >24 h and allows
highly efficient adenoviral gene transfer at relatively low virus
concentration without apparent adverse effect on contractile behavior. It is
relatively simple, inexpensive [complete setup 11,000 Euro (circa
$10,330), each run 22 Euro (circa $21)], and robust, yet relatively
complex and physiological and may therefore be suitable as a screening model
for studying gene-function relationships ex vivo.
During the establishing phase we found several parameters to be critical.
First, air bubbles were avoided by scrupulous control of the tubing. In
addition, bubble traps directly above the heart are essential. Second,
continuous filtering of the perfusate is necessary when using a recirculating
mode and can easily be achieved with one-way filters. Third, sterility is
difficult to ensure because the heart is manipulated and is beating in an open
reservoir. For logistical reasons we did not perform manipulations under the
sterile hood. Thus germs were present from the beginning of the experiment and
likely entered the perfusate continuously during the 24 h in a normal cell
culture laboratory. We solved this problem by autoclaving the entire perfusion
system once a week and by inclusion of Pen-Strep and amphotericin B. The
latter was used at one-third of the recommended concentration for cell culture
to avoid toxicity. In addition, sterile filters in the circulation were
changed for each experiment. Fourth, medium composition is certainly critical,
but we did not extensively test different compositions. Instead, we modified
an existing protocol (14)
according to our cell culture experience. Two statements can be made:
increasing triiodothyronine to 1 nmol/l improved contractile preservation, and
interestingly, omitting insulin improved the inotropic response to
isoprenaline (note that all experiments depicted here have been performed in
the presence of insulin). Initial experiments with fetal calf serum did not
indicate improvement and added the problem of foaming in the continuously
gassed reservoir. Fifth, an important parameter of success is mechanical
loading of the heart. We found the hearts to perform better when a constant
weight was applied to the apex during most of the perfusion time and if the
heart can perform work. Even though we did not systematically study
contraction mechanics, it appears obvious that the heart performs
"auxotonic" work when lifting a weight with each heart beat. This
is more physiological than either an isometric or isotonic mode of
contraction. The weight of 4 mN (400 mg) is about 10 times the weight of a
neonatal rat heart. Finally, the conditions of virus infection were chosen
according to recent studies in adult heart models
(8,
18) showing improvement of
transfection efficiency with vasodilating receptor agonists such as histamine,
serotonin, bradykinin, and vascular endothelial growth factor, as well as by
stop flow. We did not systematically study the impact of these parameters on
transfection efficiency in our model. One may argue that the stop-flow method
induced preconditioning. However, the similar values for force of contraction
in the virus-perfused (stop flow) and nonperfused (continued flow; compare
Figs. 2 and
3 with
7B) and the fact that
21 h separated stop flow from force measurements argue against a significant
impact of this intervention.
Even though most of the parameters studied demonstrated good performance of
the model, limitations exist. An obvious, inherent problem of the model is
that the neonatal rat heart is immature, e.g., lacks a well-developed
sarcoplasmic reticulum/T-tubular system
(2). Thus it may not be optimal
for studying the impact of sarcoplasmic reticulum-related genes. Moreover, a
relatively low heart rate and short action potential differ significantly from
the mature rat heart. Second, 24 h of perfusion induced edema, thinning of
myocytes, and a reduction in heart weight. This indicates some damage. Yet,
with about 10% this effect was relatively modest and apparently did not affect
perfusion pressure, contractile function, or beating rate. Actin and
calsequestrin concentration also remained constant, arguing against
significant myofilament degradation. Accordingly, force of contraction
significantly increased. We believe that the latter does not indicate real
improvement of contractile force but rather some geometric adaptation to the
direction of strain on the heart. This relates to another principal
shortcoming of the model, namely that contractile function is not measured by
a balloon but by a hook in the apex and a force transducer. The latter was
constructed not to measure force of contraction in an isometric manner, but
rather auxotonic. Thus it behaves like a spring and allows the heart to work
(see above). We are aware that this setup is not optimal in terms of muscle
physiology, but installation of a balloon in the left ventricular cavity of a
neonatal rat heart, one-third of the size of a mouse heart, is technically
demanding and not suitable for screening purposes (at present 4 hearts are run
in parallel). Given the well-developed inotropic response to isoprenaline
(Fig. 6B) we feel that
the present setup is a good alternative.
When compared with existing experimental ex vivo systems, the 24-h perfused
neonatal rat heart has certain advantages and disadvantages that should be
taken into consideration when designing an experiment. Whereas it is clearly
more laborious than culturing and infecting neonatal or adult cardiac
myocytes, it undoubtedly allows better functional evaluation of contractile
parameters. This new method is easier to perform and control (in a
reproducible manner) than the model of direct virus injections in the living
heart or the cultured trabeculae system in which sufficient transfection
efficiencies are only obtained with >100 times more virus
(17). We feel that
particularly the ease of virus infection, the reproducibility of high
transfection rates at low titers, and the absence of signs of virus toxicity
are important advantages. With one virus preparation from 40 dishes (145 mm)
of HEK cells, we routinely get 1 x 1011 bavs. Thus 100
hearts can be studied with one virus preparation. The latter can be thoroughly
titered, which is critical for quantitative statements and comparison of
different experiments. Another advantage is the efficiency in terms of animal
usage. Existing cell isolation methods yield about 2 x 106
cells/neonatal rat heart, the number of cells necessary to plate one 60-mm
cell culture dish at high density. A principal advantage of the intact heart
is that cardiac myocyte function can be studied in the intact
three-dimensional context, devoid of the confounding factors of cell
isolation. Finally, it integrates the function of the entire cardiac cell
population, whereas isolated cells necessarily represent a selection of cells
that survive the harsh conditions of enzymatic cell isolation.
In conclusion, the new model should allow studies on the function of genes
in a cardiac context and the impact of quantitative changes in gene
expression. As such, it may be a useful complementation of existing
experimental models.
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DISCLOSURES
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This study was supported by the German Ministry for Education and Research
to T. Eschenhagen (collaborative project with Medigene, Martinsried, Germany)
and by the German Research Foundation (DFG GRK 750).
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ACKNOWLEDGMENTS
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We thank Thomas Müller and Johann Hügel for excellent technical
support and ideas for the construction of the setup device.The results are
part of the doctoral thesis of S. Wiechert and have been published in abstract
form (Eur Heart J Suppl 23: 675, 2002).
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FOOTNOTES
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Address for reprint requests and other correspondence: T. Eschenhagen,
Martinistr. 52, 20246 Hamburg, Germany (E-mail:
t.eschenhagen{at}uke.uni-hamburg.de).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
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REFERENCES
|
|---|
- Bert P.
Lecons sur la Physiologie Comparee de la Respiration.
Paris: Baillere, 1870.
- Bishop SP,
Anderson PG, and Tucker DC. Morphological development of the rat heart
growing in oculo in the absence of hemodynamic work load. Circ
Res 66:
84–102, 1990.[Abstract/Free Full Text]
- Chiche JD,
Cariou A, and Mira JP. Bench-to-bedside review: fulfilling promises of the
Human Genome Project. Crit Care
6: 212–215,
2002.[ISI][Medline]
- Christensen G,
Minamisawa S, Gruber PJ, Wang Y, and Chien KR. High-efficiency, long-term
cardiac expression of foreign genes in living mouse embryos and neonates.
Circulation 101:
178–184, 2000.[Abstract/Free Full Text]
- Davia K, Hajjar
RJ, Terracciano CM, Kent NS, Ranu HK, O'Gara P, Rosenzweig A, and Harding
SE. Functional alterations in adult rat myocytes after overexpression of
phospholamban with use of adenovirus. Physiol Genomics
1: 41–50,
1999.[Abstract/Free Full Text]
- del Monte F,
Harding SE, Dec GW, Gwathmey JK, and Hajjar RJ. Targeting phospholamban by
gene transfer in human heart failure. Circulation
105: 904–907,
2002.[Abstract/Free Full Text]
- Ditz D,
Pamminger T, El-Armouche A, and Eschenhagen T. Effect of green fluorescent
protein encoding adenoviral vectors on contractility in cardiac myocytes.
Naunyn Schmiedebergs Arch Pharmacol
365: R1–R164,
2002.
- Donahue JK,
Kikkawa K, Thomas AD, Marban E, and Lawrence JH. Acceleration of
widespread adenoviral gene transfer to intact rabbit hearts by coronary
perfusion with low calcium and serotonin. Gene Ther
5: 630–634,
1998.[ISI][Medline]
- Eschenhagen T,
Fink C, Remmers U, Scholz H, Wattchow J, Weil J, Zimmermann W, Dohmen HH,
Schafer H, Bishopric N, Wakatsuki T, and Elson EL. Three-dimensional
reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart
muscle model system. FASEB J
11: 683–694,
1997.[Abstract]
- Fazekas JF,
Alexander FAD, and Himwich HE. Tolerance of the newborn to anoxia.
Am J Physiol 134:
281–287, 1941.[Free Full Text]
- Giordano FJ, He
H, McDonough P, Meyer M, Sayen MR, and Dillmann WH. Adenovirus-mediated
gene transfer reconstitutes depressed sarcoplasmic reticulum
Ca2+-ATPase levels and shortens prolonged cardiac
myocyte Ca2+ transients.
Circulation 96:
400–403, 1997.[Abstract/Free Full Text]
- Hajjar RJ,
Schmidt U, Matsui T, Guerrero JL, Lee KH, Gwathmey JK, Dec GW, Semigran MJ,
and Rosenzweig A. Modulation of ventricular function through gene transfer
in vivo. Proc Natl Acad Sci USA
95: 5251–5256,
1998.[Abstract/Free Full Text]
- Hwang JJ, Dzau
VJ, and Liew CC. Genomics and the pathophysiology of heart failure.
Curr Cardiol Rep 3:
198–207, 2001.[Medline]
- Janssen PM,
Lehnart SE, Prestle J, Lynker JC, Salfeld P, Just H, and Hasenfuss G. The
trabecula culture system: a novel technique to study contractile parameters
over a multiday time period. Am J Physiol Heart Circ
Physiol 274:
H1481–H1488, 1998.[Abstract/Free Full Text]
- Kimura S and
Gonzalez FJ. Applications of genetically manipulated mice in
pharmacogenetics and pharmacogenomics. Pharmacology
61: 147–153,
2000.[ISI][Medline]
- Kirshenbaum LA,
MacLellan WR, Mazur W, French BA, and Schneider MD. Highly efficient gene
transfer into adult ventricular myocytes by recombinant adenovirus.
J Clin Invest 92:
381–387, 1993.[ISI][Medline]
- Lehnart SE,
Janssen PM, Franz WM, Donahue JK, Lawrence JH, Marban E, Prestle J, and
Hasenfuss G. Preservation of myocardial function after adenoviral gene
transfer in isolated myocardium. Am J Physiol Heart Circ
Physiol 279:
H986–H991, 2000.[Abstract/Free Full Text]
- Logeart D,
Hatem SN, Rucker-Martin C, Chossat N, Nevo N, Haddada H, Heimburger M,
Perricaudet M, and Mercadier JJ. Highly efficient adenovirus-mediated gene
transfer to cardiac myocytes after single-pass coronary delivery.
Hum Gene Ther 11:
1015–1022, 2000.[ISI][Medline]
- O'Shaughnessy KM. The genetics of essential hypertension.
Br J Clin Pharmacol 51:
5–11, 2001.[ISI][Medline]
- Sutherland FJ and Hearse DJ. The isolated blood and perfusion fluid perfused heart.
Pharmacol Res 41:
613–627, 2000.[ISI][Medline]
- Woo-Ming M, Wu
NM, Lester JL, III, and Malinin TI. Water content of hearts perfused with
crystalloid and nutrient solutions. J Mol Cell Cardiol
12: 371–386,
1980.[ISI][Medline]
- Yamamoto A, Hen
R, and Dauer WT. The ons and offs of inducible transgenic technology: a
review. Neurobiol Dis 8:
923–932, 2001.[ISI][Medline]
- Zimmermann WH,
Fink C, Kralisch D, Remmers U, Weil J, and Eschenhagen T.
Three-dimensional engineered heart tissue from neonatal rat cardiac myocytes.
Biotechnol Bioeng 68:
106–114, 2000.[ISI][Medline]
Copyright © 2003 by the American Physiological Society.
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