Depressed intracellular calcium transients and contraction
in myocytes from hypertrophied and failing guinea pig hearts
FRANCIS M. SIRI, JOHN KRUEGER, CHARLES NORDIN,
ZHEN MING, AND RONALD S. ARONSON
Cardiology Division, Department of Medicine, Albert Einstein College of Medicine,
Bronx, New York 10461
SIRI, FRANCIS M., JOHN KRUEGER, CHARLES NORDIN, ZHEN
MING, AND RONALD S. ARONSON. Depressed intraceZlular cal-
cium transients and contraction in myocytes from hypertrophied
and failing guinea pig hearts. Am. J. Physiol. 261 (Heart Circ.
Physiol. 30): H514-H530, 1991.-We investigated the basis for
impaired left ventricular function of hearts in which hypertro-
phy was produced by gradual pressure overload. We measured
myoplasmic free calcium concentration ([ Ca”+];) with fura-
and sarcomere shortening in single myocytes isolated from
control hearts and hypertrophied failing hearts. Diastolic
[ Ca2+]i was normal, but [Ca”+]; at the peak of contraction was
depressed in myocytes from failing hypertrophied hearts. In-
creasing drive rate from 0.20 Hz to 5.00 Hz increased both
diastolic and peak [Ca2+];. Norepinephrine (3 X lo-" M) in-
creased diastolic [Ca2+]; in all cells and tended to normalize
peak [ Ca”+]; in myocytes from hypertrophied failing hearts
during 5.00 Hz drive. Depressed peak [Ca2+]i in the hypertro-
phied cells was paralleled by significant decreases in both the
velocity and percent of sarcomere shortening, which were meas-
ured in cells not loaded with fura-2. Sarcomere length was
correlated with estimates of [Ca2+]i in intact cells and with
controlled levels of [Ca”‘] in chemically “skinned” myocytes. A
plot of sarcomere length against [Ca”‘] gave a single continuous
relationship that spanned resting and peak values at all drive
rates in both the control and hypertrophied myocytes. Thus
heart failure in this model is reflected in impaired myocyte
contraction, which is closely related to reduced levels of
[Ca”‘]i during systole rather than to depressed myofilament
sensitivity to Ca2+.
fura-2; myoplasmic free calcium; heart enlargement; heart fail-
ure; sarcomere dynamics; contraction; norepinephrine
CARDIAC HYPERTROPHY is an adaptive mechanism of the
heart to pressure and volume overloads (7, 28, 44). For
reasons that are still unclear, the adaptive phase of
cardiac hypertrophy can eventually deteriorate into a
phase of heart failure. Despite a number of previous
studies in which various aspects of excitation and con-
traction have been investigated, the basis for impaired
contractile function of the failing myocardium has not
been established.
In many models of hypertrophy, myosin adenosinetri-
phosphatase activity is depressed (52). Although this
depression has been correlated with various indexes of
contractility, it does not result in decreased tension
development in skinned muscle (25, 43) or in isolated
papillary muscle (10) and is not necessarily associated
with either reduced pressure-generating ability or dimin-
ished pump function of the intact heart (54). Further-
more, there is presently no evidence linking the impaired
function of the hypertrophied, failing left ventricular
myocardium to either reduced myofibrillar sensitivity to
[Ca2+]; or to a decrease in maximum Ca”+-activated force
(25, 37, 43, 49).
In virtually all models of hypertrophy induced by pres-
sure overload, the action potential is prolonged (2, 16,
18,23, 24,33,47,51,59). The prolonged action potential,
if due to increased Ca2+ current ( loa), might enhance
contraction by increasing activator Ca”+. In one study in
single myocytes isolated from rats with left ventricular
hypertrophy induced by renal hypertension, Ica increased
markedly and the total free myoplasmic calcium ([Ca”‘];)
estimated by calculation increased substantially in hy-
pertrophied cells over control levels (30). A more recent
study in myocytes isolated from rats with left ventricular
hypertrophy induced by abdominal aortic constriction
found that
I
cay
normalized for membrane capacitance,
was not significantly different in normal and hypertro-
phied myocytes, nor was there any difference in the time
course of decay (51). In single myocytes isolated from
cats with right ventricular hypertrophy, 1oa appeared to
be slightly reduced or the same as in normal myocytes
and the time course of inactivation of Ica was prolonged
(33). The cells used in all the voltage-clamp studies were
isolated from hypertrophied hearts without evidence of
heart failure.
The time course of activation and decay of Ica can
influence both the release from and loading of Ca2’ into
the sarcoplasmic reticulum (SR) (12, 13). The effect of
Ica on the [ Ca2+]i transient in intact cells is complex (5)
and may be altered in diseased cells. In addition, 1ca is
only one of many factors that can influence [Ca2+];. For
example, the prolonged duration of the action potential
characteristic of hypertrophied cells (2) could increase
[Ca2+]; via an electrogenic Na+-Ca2+ exchange (32, 41,
45) independent of 1ca.
Two previous studies have described alterations in the
time course, but not the peak value, of the [Ca2+]; tran-
sient as measured by the fractional luminescence of the
photoprotein aequorin in hypertrophied and failing mus-
cles from ferrets and humans (19, 20). Although frac-
tional luminescence was not converted into [Ca2+];, the
authors concluded that in the ferrets with experimental
pressure-overload hypertrophy, “the prolonged time
H514
0363-6135/91 $1.50 Copyright 0 1991 the American Physiological Society

MYOCYTE FREE CALCIUM ANL) CONTRACTION IN HEART FAILURE H515
course of tension development, but not the diminished
peak isometric tension response, may be related to
changes in intracellular calcium handling” (20). Prepa-
rations from patients with end-stage heart failure had
abnormally prolonged [ Ca”+] i transients and impaired
ability to restore diastolic [ Ca2+]i to normal low levels
(19). There was, however, no evidence of a consistent
decrease in the peak of the [Ca2+]i transient in failing
muscles.
Several studies have shown that SR function is altered
in heart failure. For example, the velocity of Ca2’ trans-
port into isolated SR vesicles is depressed by as much as
50% in some models of heart failure (27, 57). The rela-
tionship between this change and depressed contractile
function is not clear.
Thus the available experimental evidence does not
provide any convincing explanation for the reduced con-
tractile performance of hypertrophied muscle that has
failed. Therefore, we investigated the cellular basis for
depressed contractile performance in a model in which
chronic heart failure occurs after a gradually developing
left ventricular overload in guinea pigs. We have previ-
ously characterized the hemodynamic and contractile
abnormalities in the intact heart in this model (54). We
have also shown that single left ventricular myocytes
isolated from hypertrophied hearts are larger than con-
trol cells and maintain the prolongation of action poten-
tial duration characteristic of the hypertrophy process
(47). In this study, we directly measured intracellular
[ Ca2+]i transients in single cells with the Ca2+-sensitive
dye fura-2. The physiological relevance of our measure-
ments of [Ca’+]; are further supported by our observa-
tions that [Ca2+]; measured with fura- correlates
strongly with contraction. Our results show that the peak
of the [ Ca2+]; transient is markedly reduced in cells
isolated from failing hearts and thus provides an expla-
nation for the depressed contractile performance of cells
isolated from the failing myocardium. Preliminary re-
ports of this work have been presented as abstracts (37,
55) .
METHODS
Experimental animals and aortic banding procedure.
Male guinea pigs of the Hartley strain, initially weighing
225-275 g (2-3 wk of age), were used. Under methohex-
ital sodium anesthesia (30 mg/kg), half of the animals
underwent a lateral thoracotomy with placement of a
small Tygon band on the ascending aorta. This band was
of a size that produced very little left ventricular overload
at first but led to a progressive rise in systolic left
ventricular pressure as the banded animals grew. Each
banded animal was age and weight matched with an
unoperated control. With growth to -750 g body wt,
most aortic-banded animals had substantial left ventric-
ular hypertrophy, and a high percentage (50-60%) also
showed clinical dyspnea and cyanosis by 40 days after
banding. We have previously described the details of this
model and demonstrated that clinical dyspnea and cy-
anosis in the aortic-banded animals correlated with path-
ophysiological alterations consistent with heart failure:
high lung weight, right ventricular hypertrophy, and
depressed capacity of the intact left ventricle to generate
pressure (54). Thus we compared data from three groups:
control guinea pigs, aortic-band.ed guinea pigs without
evidence of congestive heart failure (AC), and aortic-
banded guinea pigs with evidence (dyspnea and mild
cyanosis) of congestive heart failure (ACF).
Because this study extended longer than 40 days, a
higher percentage of banded animals progressed to overt
heart failure (ACF), leaving a relatively low number of
animals in the AC group. This limited our statistical
analysis of the AC group, particularly with respect to the
subgroups used for testing norepinephrine’s effects and
for examining contractile parameters. In these cases only
the control and ACF groups are compared statistically.
We nevertheless present data from myocytes in the AC
group to illustrate the continuum of results. The major
differences occurred between the control and ACF
groups, and that is the principal focus of this study.
Isolation of cells and loading with fura-2. Single myo-
cytes were isolated from the left ventricles of guinea pig
hearts by enzymatic dispersion by a method described
previously (3, 41, 47). The isolated cells were suspended
in 10 ml of a solution of the following composition (in
mM): 118 NaCl, 4.8 KCl, 1.2 NaH2P04, 1.2 MgSOJ, 1.0
CaC12, 11 glucose, and 25 Na-N-Z-hydroxyethylpipera-
zine-2V’-2ethanesulfonic acid. One-half milliliter of the
cell suspension was added to a small tube containing I.5
ml of normal Tyrode solution of the following composi-
tion (in mM): 137.5 NaCl, 12.0 NaHC03, 1.8 NaH2P04,
4.0 KCl, 1.2 CaC12, 0.5 MgC12, and 5.5 glucose. Five
microliters of a 1 mM solution of the acetoxymethyl ester
derivative of fura- (fura-2/AM) in dimethyl sulfoxide
was added, after which the tube was capped and rotated
at 10 revolutions/min at room temperature (23°C) for
15-20 min.
Evaluation of distribution of cellular fluorescence. Cells
were selected for photomultiplier measurements based
on their rod-shaped morphology and good striations. It
is also important to verify that the fura- dye is uniformly
distributed throughout the cell, since under some condi-
tions it can become trapped in intracellular organelles
(62) and lead to artifacts in the estimation of [ Ca”+];.
Such compartmentalization can be minimized by loading
the cells with fura- at room temperature, then gradually
raising the perfusate temperature to the level at which
they are to be studied (42). To evaluate uniformity of
dye distribution in this study, we obtained fluorescent
video images of representative unstimulated myocytes
using a SIT camera (Dage-MTI, SIT66). Each image was
digitized rapidly (l/30 s) at 640 X 480 X 8 bit resolution
with a microcomputer (Macintosh II equipped with a
video-frame grabber, Data Translation, DT 2255). Noise
in individual video frames was reduced by averaging 16-
81 frames for each image obtained at each wavelength.
The signal-averaged image was stored on the hard disk
for retrospective image processing.
Figure 1, A and B, shows representative fluorescent
video images of a resting myocyte isolated from an ACF
heart. These images show a fairly uniform distribution
of fluorescence, and the lower intensity of the image due
to 340 nm excitation (compared with the 380-nm image)
is typical of an unstimulated myocyte with low [ Ca2+];.