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Alterations to cellular Ca2+ homeostatic mechanisms are
often implicated in the pathophysiology of heart disease. A
number of studies have reported abnormal myocyte Ca2+
handling during the heart failure that follows a prolonged
period of pressure overload. In studies using tissue from
either human (Gwathmey et al. 1987; Beuckelmann et al.
1992; Mattiello et al. 1998; Dipla et al. 1999) or animal
(Bing et al. 1991; Brooks et al. 1994; Gómez et al. 1997)
models, the impaired mechanical performance of hearts in
failure has been associated with various defects. These have
included remodelling of the ventricular chamber,
extracellular matrix hyperplasia, loss of myocytes by
apoptosis, and decreased myocyte contractile function.
Experimental models have employed a range of different
species as well as different myocardial preparations.
Results from these studies have generally found that, along
with the contractile dysfunction, there are corresponding
alterations in the Ca2+ transport mechanisms associated
with excitation–contraction coupling (for a review see
Movsesian & Schwinger, 1998).
The spontaneously hypertensive rat (SHR), introduced by
Okamoto & Aoki in 1963, is an animal model of genetic
systemic hypertension in which the development of
hypertension leads to heart failure. The progression of
disease in SHR follows a predictable pattern, with
hypertension being the primary stimulus for the
development of hypertrophy. End-stage heart failure
routinely develops in SHR between 18 and 24 months of
age (Perreault et al. 1990; Bing et al. 1991; Brooks et al.
1994), following a long period of stable hypertrophy,
during which contractile function is preserved or
enhanced (Conrad et al. 1991). Furthermore, unlike most
other animal models, the myocardial changes due to
senescence that usually accompany the human disease are
mimicked in SHR (Assayag et al. 1997; Fitzsimons et al.
1999). We therefore chose the SHR as our model of heart
failure, using Wistar-Kyoto rats of a similar age as
normotensive controls.
A major problem in studying isolated cardiac muscle is the
need to minimise metabolic demand in the preparation,
Reduced contraction strength with increased intracellular
[Ca2+] in left ventricular trabeculae from failing rat hearts
Marie-Louise Ward, Adèle J. Pope, Denis S. Loiselle and Mark B. Cannell
Department of Physiology, Faculty of Medicine and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand
Intracellular calcium ([Ca2+]i) and isometric force were measured in left ventricular (LV) trabeculae
from spontaneously hypertensive rats (SHR) with failing hearts and normotensive Wistar-Kyoto
(WKY) controls. At a physiological stimulation frequency (5 Hz), and at 37 °C, the peak stress of
SHR trabeculae was significantly (P ≤ 0.05) reduced compared to WKY (8 ± 1 mN mm_2 (n = 8) vs.
21 ± 5 mN mm_2 (n = 8), respectively). No differences between strains in either the time-to-peak
stress, or the time from peak to 50% relaxation were detected. Measurements using fura-2 showed
that in the SHR both the peak of the Ca2+ transient and the resting [Ca2+]i were increased compared
to WKY (peak: 0.69 ± 0.08 vs. 0.51 ± 0.08 mM (P ≤ 0.1) and resting: 0.19 ± 0.02 vs. 0.09 ± 0.02 mM
(P ≤ 0.05), SHR vs. WKY, respectively). The decay of the Ca2+ transient was prolonged in SHR, with
time constants of: 0.063 ± 0.002 vs. 0.052 ± 0.003 s (SHR vs. WKY, respectively). Similar results
were obtained at 1 Hz stimulation, and for [Ca2+]o between 0.5 and 5 mM. The decay of the caffeine-
evoked Ca2+ transient was slower in SHR (9.8 ± 0.7 s (n = 8) vs. 7.7 ± 0.2 s (n = 8) in WKY), but this
difference was removed by use of the SL Ca2+-ATPase inhibitor carboxyeosin. Histological
examination of transverse sections showed that the fractional content of perimysial collagen was
increased in SHR compared to WKY (18.0 ± 4.6% (n = 10) vs. 2.9 ± 0.9% (n = 11) SHR vs. WKY,
respectively). Our results show that differences in the amplitude and the time course of the Ca2+
transient between SHR and WKY do not explain the reduced contractile performance of SHR
myocardium per se. Rather, we suggest that, in this animal model of heart failure, contractile
function is compromised by increased collagen, and its three-dimensional organisation, and not by
reduced availability of intracellular Ca2+.
(Received 22 July 2002; accepted after revision 30 October 2002; first published online 29 November 2002)
Corresponding author M.-L. Ward: Department of Physiology, Faculty of Medicine and Health Sciences, University of
Auckland, Private Bag 92019, Auckland, New Zealand. Email: m.ward@auckland.ac.nz
J Physiol (2003), 546.2, pp. 537–550 DOI: 10.1113/jphysiol.2002.029132
© The Physiological Society 2002 www.jphysiol.org

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which is not generally perfused. For this reason,
experiments are commonly carried out at low
temperatures and low stimulation frequencies. It is
therefore possible that cellular processes observed under
these conditions may not be representative of those in vivo.
The need to reduce stimulation frequency and
temperature in order to avoid metabolic insufficiency can
be obviated by the use of cardiac trabeculae. These
preparations are structurally homologous to ventricular
wall tissue, being made up predominantly of
longitudinally arranged myocytes surrounded by a narrow
rim of endothelial cells (Hanley et al. 1999). Although
isolated myocytes provide minimal diffusion distances,
they were not used in this study, since they do not permit
quantitative force measurements at working sarcomere
lengths. The axial alignment of myocytes in trabeculae
make them ideal preparations for simultaneous
measurement of isometric force and [Ca2+]i. Using this
preparation we have found that although force is reduced,
resting [Ca2+]i and the amplitude of the Ca2+ transient are
increased, showing a dissociation between contractile
function and [Ca2+]i availability.
METHODS
Experimental animals and haemodynamic measurements
Spontaneously hypertensive rats (SHR) and normotensive
Wistar-Kyoto rats (WKY) of both sexes were obtained at 6 weeks
of age and were housed under control conditions with ad libitum
food and water. The SHR progresses to heart failure with
senescence, and after ~20 months of age signs of worsening
myocardial performance (such as respiratory distress, weight loss,
lethargy, poor grooming) could be detected. As soon as overt signs
of heart failure were apparent, SHR were used for
experimentation. Systolic blood pressure and heart rate were
measured with a tail cuff sphygmomanometer (Model 179, IITC
Inc., Life Science Instruments, Woodland Hills, CA, USA), and
animals were weighed, anaesthetised with halothane and killed by
decapitation. Experimental procedures were approved by the
Animal Ethics Committee of the University of Auckland. Healthy
WKY control animals of comparable age were selected, and
subjected to the same experimental protocols. The diagnosis of
heart failure in SHR was confirmed by post mortem examination.
In some cases, M-mode echocardiography was performed on
lightly anaesthetised (20 mg kg_1 I.P. of Teletamine HCl and
Zolazepam HCl; Vibrac Laboratories (NZ) Ltd) SHR to confirm
the signs of failure, and on matched WKY controls. Analysis of the
M-mode data confirmed ventricular dilatation (end diastolic
volume, SHR: 0.369 ± 0.036 ml (n = 3), and WKY: 0.174 ±
0.043 ml (n = 4), P ≤ 0.05) and reduced fractional shortening in
SHR compared to WKY (SHR: 0.34 ± 0.05 (n = 3), WKY:
0.56 ± 0.04 (n = 4), P ≤ 0.05).
Dissection and mounting of left ventricular trabeculae
Perfusion of the coronary circulation with oxygenated dissection
solution (see below) was maintained throughout dissection.
When present, an unbranched, cylindrical trabecula (average
length 1.9 ± 0.1 mm, cross-sectional area 0.038 ± 0.003 mm2,
n = 56) from the left ventricle was dissected free with a small block
of ventricular tissue at either end. The trabecula was then
transferred to a Perspex bath (volume 450 ml) on the stage of an
inverted microscope (Nikon Diaphot 300, Japan), as described
previously (Hanley & Loiselle, 1998). One end of the trabecula was
mounted in a wire cradle extending from the silicon beam of a
force transducer (model AE801, SensoNor, Horten, Norway)
while the other end of the trabecula was held in a monofilament
(30 mm diameter) nylon snare protruding from a stainless steel
tube attached to a micromanipulator. Trabeculae were
continuously superfused with oxygenated modified Krebs-
Henseleit solution (see below) at a flow rate of 7 ml min_1. Field
stimulation (5 ms pulse) was applied at 0.1 Hz by a Digitimer
D100 (Digitimer, Welwyn Garden City, Herts, UK), using
platinum electrodes. The central portion of the trabecula was
viewed using a w 40 objective (NA 0.55) and a charge-coupled
device camera connected to a video monitor to allow striations to
be observed. Trabeculae were adjusted to a sarcomere length of
2.1–2.2 mm. This sarcomere length has previously been shown to
be comparable to that maximally attained in vivo (Rodriguez et al.
1992) such as might occur at the end of diastolic filling.
Fura-2 AM (100 mg; Texas Fluorescent Laboratories, Austin, TX,
USA) was dissolved in 30 ml of freshly prepared anhydrous
dimethyl sulphoxide (DMSO; Aldrich) with 5% w/v pluronic
F127, and added to 10 ml of Krebs-Henseleit superfusate ([Ca2+]o
1 mM). Oxygenated loading solution was continuously circulated
for a period of 2 h, at room temperature (20–22 °C), whilst
stimulating the trabeculae at a frequency of 0.1 Hz. After loading
was complete, the superfusate was switched to Krebs-Henseleit
solution to remove any extracellular remnants of the loading
solution.
Chemicals and solutions
Trabeculae were superfused with a modified Krebs-Henseleit
solution containing (mM): 118 NaCl, 4.75 KCl, 1.18 MgSO4, 1.18
KH2PO4, 24.8 NaHCO3 and 10 D-glucose. The solutions were
continuously bubbled with 95% O2 and 5% CO2; pH was
maintained at 7.4. [Ca2+]o was adjusted, as required, by addition of
CaCl2 from a 1 M stock solution. The dissection solution
contained 0.25 mM Ca2+ plus 20 mM 2,3-butanedione monoxime
(BDM) to protect the myocardium from a high rate of energy
utilisation, by reducing cross-bridge cycling (Mulieri et al. 1989)
and SR content (Steele & Smith, 1993; Adams et al. 1998). On
removal of BDM from the superfusate, [Ca2+]o was increased
gradually to 1 mM. To maximise retention of indicator in the
cytoplasm at 37 °C, 1 mM probenecid was added to all solutions
following fura-2 loading (Di Virgilio et al. 1990). A modified
Tyrode solution was used for a number of trabeculae in which
caffeine contractures were carried out in both normal Na+
(143 mM) and Na+-free solutions. We used 5,6-carboxyeosin
diacetate (CE, Molecular Probes) to inhibit the sarcolemmal (SL)
Ca2+-ATPase (Choi & Eisner, 1999). Trabeculae were loaded for
30 min with 20 mM CE followed by > 10 min period for de-
esterification. The Tyrode solution was composed of the following
(mM): 141.8 NaCl, 6 KCl, 1.2 MgSO4.7H20, 1.2 Na2HPO4,
10 Hepes, 2 CaCl2 and 10 D-glucose, adjusted to pH 7.4 with
NaOH. A Na+-free and Ca2+-free Tyrode solution was obtained by
equimolar substitution of NaCl with LiCl, and the use of
K2HPO4.3H2O in the place of Na2HPO4. CaCl2 was replaced with
1 mM EGTA, and the pH was adjusted with KOH. Tyrode
solutions were continuously bubbled with 100% oxygen. All
chemicals used were purchased from Sigma (Sigma Aldrich,
Australia) unless otherwise stated.
Measurement of [Ca2+]i using fura-2
Trabeculae were illuminated using a 75 W xenon arc lamp, and a
spectrophotometric system (Cairn Research, Faversham, Kent,
M.-L. Ward, A. J. Pope, D. S. Loiselle and M. B. Cannell538 J Physiol 546.2