Energy-Linked Calcium Transport
in Subcellular Fractions of the
Failing Rat Heart
By John R. Muir, M.R.C.P., Naranjon S. Dholla, Ph.D.,
Josefina M. Ortexa, M.D., and Robert E. Olson, M.D.
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ABSTRACT
The isolated rat heart perfused without substrate provides a good model in
which to study biochemical changes in the failing myocardium. In this
preparation, there is a decline in the ability of both the mitochondria and the
sarcoplasmic reticulum to accumulate calcium in the absence of oxalate after 30
minutes of perfusion. Calcium binding by mitochondria fell from control levels
of 47 ± 8 m^imoles/mg protein to 17.5 ± 4.9 m/nmoles/mg protein and that by
microsomes from 29.8 ± 5.1 m^unoles/mg protein to 15.2 ± 4.8 rmxmoles/mg
protein. This drop coincided with die start of the decline in myocardial
contractility. The calcium uptake by the sarcoplasmic reticulum in the presence
of oxalate decreased from control levels of 534.1 ± 32.0 to 160.5 ± 27.2
ffuzmoles/mg protein after 2 hours of perfusion, at which time the myocardial
contractility had dropped to below 10% of control levels. This change in the
calcium uptake by the sarcoplasmic reticulum is associated with an increase in
its ATPase activity from 1.91 ± 0.21 to 3.00 ± 0.31 /imoles P,/mg
protein/min over 2 hours of perfusion without substrate. This suggests tJiat
there is an uncoupling of the microsomal ATP-dependent calcium pump in
these late stages of heart failure due to substrate lack. The change in calcium
binding to reticulum occurred in association with the onset of contractile
failure, whereas changes in calcium uptake in the presence of oxalate were
delayed and probably represent irreversible disorganization of the intracellular
membranes.
ADDITIONAL KEY WORDS
sarcoplasmic reticulum
calcium binding
heart failure due to substrate lack
mitochondria
calcium uptake
• The regulation of intracellular calcium
concentration occupies a central position in
current concepts of excitation-contraction
coupling in skeletal muscle (1-3). The instantaneous concentration of calcium appears to
be regulated by opposing forces related to the
extent of depolarization of the T-system on
the one hand to an energy-linked calcium
From the Department of Biochemistry, Saint Louis
University School of Medicine, Saint Louis, Missouri
63104.
This investigation was supported in part by U. S.
Public Health Service grants-in-aid (1 F05 TW 145801, FR 5388-07, 5 T01 HE5672-04, and HE0977204). Dr. Muir was a U. S. Public Health Service
Postdoctoral Research Fellow. His present address is
National Heart Hospital, London, W.I., England.
Received May 19, 1969. Accepted for publication
February 4, 1970.
CircmUtUm Rtsfrcb,
Vol. XXVI, April 1970
ATPase activity
oligomycin
transport system located in the vesicles of the
longitudinal system on the other (1, 4-7). It is
possible that similar events occur in the
myocardium because cardiac contractile proteins, like those of skeletal muscle, are
sensitive to changes in the ionic strength of
calcium (8, 9). Some workers have found a
disappointingly low calcium-binding capacity
in cardiac sarcoplasmic reticulum (8), although more recently higher activities have
been reported (2, 10). It has therefore been
suggested that the mitochondria may play an
important role in regulating calcium concentrations in the cardiac cells, since they are
capable
of
accumulating
considerable
amounts of calcium (11-13).
Recently, Gertz et al. (14) reported a
429
430
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reduced level of calcium uptake and ATPase
activity of sarcoplasmic reticulum isolated
from spontaneously failing heart lung preparations, and Lee et al. (15) have also shown a
decrease in both the rate and the total calcium
uptake by the sarcoplasmic reticulum of
ischemic dog heart muscle. In view of these
findings, we decided to study the effect of
heart failure induced by perfusion with
substrate-free medium on the calcium transport and ATPase activity of the myocardial
subcellular fragments of the isolated rat heart,
a preparation demonstrated in this laboratory
to be an excellent model in which biochemical
and physiological events can be studied
during the course of heart failure (16-18).
The degree of heart failure can be measured
directly in terms of the myocardial contractility. It appears that the primary defect
underlying cardiac failure in this system is a
reduced rate of ATP formation, which is
diminished by a lack of substrate rather than
oxygen. A study in our laboratory of the
changes in adenine nucleotides during the
onset of cardiac failure in the rat heart
perfused without substrate has demonstrated
that it imitates the effects of hypoxia on a
longer time scale.
In the present study, the term "calcium
binding" is applied to the ability of microsomes or mitochondria to take up calcium in
the absence of oxalate. It is understood that
this convention employs an arbitrary meaning
for binding, since it is likely that both binding
and a small amount of accumulation occur
under these conditions. On the other hand, for
purposes of clarity, the term "calcium uptake"
is employed in this paper to signify calcium
accumulation by microsomes in the presence
of 5 mM potassium oxalate.
Materials and Methods
Albino male rats weighing 250 to 325 g were
decapitated, and the hearts were quickly removed
and chilled in ice-cold oxygenated perfusion
medium. The fat and connective tissue were
trimmed away and the hearts were arranged for
coronary perfusion by the Langendorff technique.
The perfusion medium was a modified KrebsHenseleit solution containing half of the original
amount of Ca2 + , i.e., 1.25 mM. It was equili-
MUIR, DHALLA, ORTEZA, OLSON
brated with a gas mixture of 95% O2 and 5% CO2
and maintained at 37°C. In some of the
experiments, the perfusion medium also contained
8 mM glucose. The contractile force and heart
rate were monitored on a Sanborn recorder by a
force displacement transducer.
In each experiment, two hearts were pooled
after perfusion for the desired time and were
immediately immersed in ice-cold homogenizing
solution. The tissue was cut into small pieces and
homogenized with 5 volumes of a solution
containing 5 HIM histidine, 5 mM potassium
oxalate, 100 mM potassium chloride and 320 mM
sucrose at pH 7.4. The oxalate was omitted from
the homogenizing solution when hearts were
being prepared for studies of calcium binding.
The homogenate was centrifuged at 800 X g for
20 minutes to remove the cell debris. The
supernatant fluid was centrifuged at 8,000 X g
for 30 minutes to obtain the mitochondrial pellet
and then for an additional hour at 37,000 X g to
obtain the sarcoplasmic reticulum pellet. The
pellets were resuspended in 2 ml of the
homogenizing solution for determination of calcium uptake in the presence of oxalate and in 1 ml
of homogenizing solution for estimation of
calcium binding.
The calcium uptake by the sarcoplasmic
reticulum in the presence of oxalate was
measured by using 0.2 ml of the resuspended
reticulum in an incubation medium containing 2
mM histidine, 5 mM potassium oxalate, 150 mM
potassium chloride, 5 mM magnesium chloride, 5
mM ATP, and 0.125 mM calcium chloride labeled
with Ca4li. The pH of the medium was 7.4 and
the total volume 4.0 ml. The final concentration
of Ca4B in the incubation mixture was 0.2 /xc/ml.
The reaction was started by adding ATP and
was carried out for 20 minutes at 25°C with
constant stirring. Aliquots removed at various
times after the start of the reaction were filtered
through a Swinney filter holder containing a 13
mm Millipore filter (type HA, size 0.45) using a
syringe. The filtration was usually completed in 2
to 3 seconds. Filtrate in the amount of 0.05 ml
was added to 15 ml of Bray's solution (19) and
counted in a Packard Tri-Carb liquid scintillation
spectrometer. Using a control, in which tissue was
absent, the amount of calcium retained by the
reticulum was calculated from the total free
calcium in the reaction mixture and the percent
isotopic Ca45 retained. This is, of course, a
minimum value, since the total free calcium pool
was not measured chemically in these experiments. In most experiments, these data are
presented as myu, moles Ca 2+ taken up/mg
protein/unit time.
Calcium binding was studied in a similar
manner, except that potassium oxalate was
Circulation Restdrcb, Vol. XXVI, April 1970
431
CALCIUM TRANSPORT IN HEART FAILURE
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excluded from all solutions. The time course of
the incubation was shorter than that for the
measurement of calcium uptake, aliquots being
taken from the incubation flask at 15, 30, and 45
seconds and 1 and 2 minutes after the reaction
was started by adding ATP. In these experiments,
uptakes were generally complete between 15 and
30 seconds, but the values are expressed per 2
minutes, the total time of the experiment. The
subcellular fractions were characterized by studying the influence of oligomycin (2.5 fig/ml) on
their ability to bind calcium. Oligomycin inhibits
ATP-driven calcium binding by mitochondria
(12) but has little or no effect on the calcium
binding of fresh sarcoplasmic reticulum (20).
The protein concentrations of die sample were
determined by either the Biuret reaction or by
Lowry's method (21) using bovine serum
albumin as a standard.
The ATPase activity of the reticulum was
measured by two methods. In the first, the
increase in inorganic phosphate content of the
filtrates from the calcium uptake studies was
measured by the Fiske-Subbarow method (22).
In addition, the initial rate of ATP breakdown by
a suspension of sarcoplasmic reticulum was
measured by following the decline in DPNH in a
system containing phosphoenol-pyruvate, pyruvate kinase and lactic dehydrogenase. In this
system, the ADP formed by hydrolysis of ATP is
rephosphorylated by phosphoenol-pyruvate. The
resulting pyruvate is dien reduced to lactate by
lactic dehydrogenase and the decline in DPNH
was followed in an Eppendorf fluorometer at an
excitation wave length of 340 mfi. The final
composition of the reaction medium within the
cuvette was as follows: 61.5 ITIM Tris buffer pH
7.5; 30.8 mu
KC1; 7.7 ITIM MgCl2; 9.2 HIM
phosphoenol-pyruvate; 46 ITIM DPNH; lactic
dehydrogenase, 8 /ig/ml; pyruvate kinase, 8
ug/ml; and 3 mM ATP. The reaction was started
by adding 2 to 4 fig of sarcoplasmic reticulum.
Recent studies on the subcellular distribution of
adenine mononucleotide phosphatase (AMPase)
activity have indicated that this enzyme is
confined to the sarcoplasmic reticulum in the rat
myocardium (23). The AMPase activity of the
subcellular fractions was therefore used as a
marker for sarcoplasmic reticulum. It was estimated by measuring the release of inorganic
phosphate from AMP by the subcellular fractions
in the presence of 0.05M glycine-sodium hydroxide buffer (pH 8.5), 5 HIM magnesium and 5 mM
AMP. The reaction was started by adding AMP
and stopped after 10 minutes with 10% trichloracetic acid. The inorganic phosphate content was
measured by the Taussky-Schorr method (24).
Zero time levels were obtained by adding the
trichloracetic acid before the AMP. The AMPase
activity was finally expressed in terms of the
release of inorganic phosphate per minute per
milligram of protein.
For electron microscopic examination, the
pellet was fixed for 2 hours with osmium tetroxide
buffer at pH 7.4 (25) and was then dehydrated
with increasing concentrations of ethanol and
embedded in Epon 812. The sections were cut
with a glass knife using the Porter-Blum
ultramicrotome (MT-1 model), stained widi
uranyl acetate and lead citrate (26, 27) and
examined with a Phillips 200 electron microscope.
Results
Yield of Subcellular Fractions.—The yield of
both the mitochondrial and the reticulum
pellets for pairs of hearts perfused without
TABLE 1
Fresh Weight and Yields of Miiochondrial and Reticulum for Ral Hearts Perfused for Ten Minutes
and Two Hours Without Substrate
Wet weight
Perfusion time
10 min
(c)
3.5
(12)
±0.1
2 hours (10)
3.4
-0.2
Paired r*t hearti
Mitochondrial
yield
(mg)
(mg)
97
*24
81
25
*3
28
±12
±3
Values are means ± SE for pairs of rat hearts; number of experiments is in parentheses. The
wet weight of hearts perfused 2 hours has been corrected for the increased hydration over 2-hour
perfusion without substrate. The yield of fresh particles has been calculated by multiplying the
protein content by 5. The overall yields of mitochondria and reticulum are 10 ± 2% and 15 ±
2%, respectively, of that initially present in the heart. There is no significant difference between
the mitochondrial or reticulum yields after 10 minutes and after 2 hours of perfusion (P > 0.05
and P > 0.05).
Circulaion Rtst^cb,
Vol. XXVI. April
1970
MUIR, DHALLA, ORTEZA, OLSON
432
100
80
60
Contract! la Forc»
161
Mltochrondrii d l d u n Binding
(mumol d + + Imj at ProMn / 2 r a i n )
(61
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Rrfculum C J W U O Binding
(mprool C i * * / m g of ProMn / 2 m l n )
(6)
0
10
30
60
90
120
PERFUSIOHTIMEOalnl
FIGURE 1
Changes in heart rate, myocardial contractility, and
calcium binding by the mitochondria and the sarcoplasmic reticulum during perfusion without substrate
of the isolated rat heart. Mean and SE are gioen;
number of experiments is in parentheses. The drop in
calcium binding by both the mitochondria and the
reticulum is unlikely to have arisen by chance (0.01 >
P > 0.005; 0.05 > P > 0.025).
substrate for 10 minutes and 2 hours are
shown in Table 1. The wet weights of the
hearts perfused for 2 hours have been
corrected for the increase in hydration that
occurs during perfusion (18). The dry-to-wet
weight ratio of rat hearts declines to 8535 of the
control value during this time. The fresh
weight of mitochondria initially present in rat
heart was calculated from values for cytochrome C and ubiquinone in whole rat heart
(28) and in mitochondria (29); the ratios
obtained indicate that rat heart contains 29 to
3235 mitochondria by weight. The electron
microscopic estimations of reticulum in muscle
(30) were used to arrive at a figure of 5% fresh
weight for cardiac reticulum. There was no
significant difference in the yield of either
mitochondria or reticulum from hearts perfused for 10 minutes and for 2 hours (P > 0.05
and P>0.05), although the overall yields
were low.
Myocardial Contractility, Heart Rate, and
Calcium Binding by the Mitochondria and
Sarcoplasmic Reticulum.—Isolated hearts
were perfused for varying times with substratefree medium. The myocardial contractile
force, heart rate, and calcium binding by the
mitochondria and by the sarcoplasmic reticulum are shown in Figure 1. The decline in
contractile force after 30-minute perfusion and
the negligible change in heart rate over the 2hour period confirm results already reported
from this laboratory (18). The calcium
binding is expressed in terms of the accumulation of calcium per milligram of protein
after 2 minutes of incubation. These conditions were uniformly followed because we
have found that the calcium binding reaches a
plateau 15 seconds after the addition of ATP.
TABLE 2
Effect of Restoring Glucose to Failing Substrate-Deprived Heart on Contractility and Ca** Binding
of Reticulum
Condition
No.
Time
(min)
Contractility
(% control)
C* 3i binding
(m/iM/raf protein)
No substrate
No substrate
No substrate
Glucose added
at 30 min
8
8
8
10
30
60
100 ± 5
95 •*> 10
54 ± 8
29 =*= 5
4
60
90 ± 10
31 ± 5
13 ± 4
16 ± 3
Values are means **= BE. The difference in contractility and calcium binding induced by addition of glucose at 30 minutes is highly significant (P < 0.02).
Circulation Resttrcb, Vol. XXVI, April
1970
433
CALCIUM TRANSPORT IN HEART FAILURE
1000
TABLE 3
CaWum Uptake
Ircpmol Ca++/rogo( protein/ 20 mln.1
161
Initial ATPase Activity of Sarcoplasmic Reticulum
from Control Rat Hearts and from Hearts Perfused for
Two Hours without Substrate
Activity
Perfusioa t4m»
0
(7)
2 hours (9)
ATPase Activity
(pniot Pi /rag of protein/ 20 rain.)
15.X
12.00
IV
(9)
16)
10
30
~
(jaoolea Pi/mg protem/mm)
1.9
3.0
0.2
0.3
Rates were measured in the Eppendorf fluorimeter
(see text for details). Values are means =*= SB; number
of experiments is in parentheses. The difference between the two values was unlikely to have arisen by
chance (P < 0.025).
9.00
6.00
3.X
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0
60
90
120
PER FUSION TIME (min)
FIGURE Z
Calcium uptake and ATPase activity of the sarcoplasmic Teticulum during perfusion without substrate of
the isolated rat heart. The ATPase activity is expressed
in terms of the release of inorganic phosphate from
ATP over the 20-minute incubation period. The drop
in the calcium uptake by the reticulum after 2 hourperfusion is unlikely to have arisen by chance (P <
0.0O5).
Katz and Repke (9) have reported similar
findings. The calcium-binding capacity of
both the mitochondria and the sarcoplasmic
reticulum fell after 30-minute perfusion without substrate, and these changes were unlikely
to have arisen by chance (0.01 > P > 0.005;
0.05>P>0.025). When glucose was added
to substrate-deprived hearts at 30 minutes,
both contractility and the ability of the
reticulum to bind calcium were restored, as
shown in Table 2.
Calcium Uptake and ATPase Activity of the
Sarcoplasmic Reticulum.—The ability of the
sarcoplasmic reticulum to accumulate calcium
in the presence of oxalate was not altered over
the first 60 minutes of perfusion without
substrate. However, after 2 hours of perfusion,
the calcium uptake of the sarcoplasmic
reticulum was reduced to a level approximately 30* of that found in control hearts (Fig. 2)
(P<0.005).
The ATPase activity of the sarcoplasmic
reticulum, measured as the average increase in
Circulation Rtstarcb, Vol. XXVI, April
1970
inorganic phosphate in the filtrate in which
calcium uptake was observed over the 20minute period, was unchanged by 2-hour
perfusion without substrate (Fig. 2). However, when the true initial rate of ATPase
activity of the sarcoplasmic reticulum was
measured by ADP formation from ATP in the
fluorimeter, there was a significant increase in
the activity following the 2-hour perfusion
period (P<0.025) Table 3.
Influence of Glucose on the Calcium
Uptake of Sarcoplasmic Reticulum.—When
isolated rat hearts are perfused with medium
containing 8 mM glucose, the contractile force
does not change over a 2-hour period of
perfusion (18). The calcium uptake of the
sarcoplasmic reticulum was therefore compared in two groups of hearts perfused with
and without addition of 8 mM glucose to the
perfusate. The results are shown in Figure 3.
It is apparent that the ability of the sarcoplasmic reticulum to accumulate calcium in
the presence of oxalate is preserved when
substrate is present in the perfusion medium
over a 2-hour period.
Influence of Oligomycin on Calcium Accumulation.—As the various subcellular fractions separated by differential centrifugation
are by no means pure (31), the effect of
oligomycin, a known inhibitor of ATP-driven
mitochondrial calcium transport (12), on the
calcium uptake and binding by the different
fractions was studied. Both the calcium
uptake and binding by reticulum were unaffected by oligomycin, indicating that contaminating mitochondria were not contribut-
MUIR, DHALLA, ORTEZA, OLSON
434
Sarcoplasmic Reticulum:
750
Calcium Uptake
(mumol Ca ++ /mg of protein)
(10)
(6)
(6)
500
(10)
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lOmin 2 hr
lOmin 2 hr
perfused
without
glucose
perfused
with
glucose
protein/min and for ten pairs of hearts
perfused for 2 hours without substrate 0.167
( S E ± 0.018) ^imoles Pi/mg protein/min.
There was no significant difference between
the two mean levels (P > 0.10).
Electron Microscopic Examination of Subcellular Fractions of the Perfused Heart.—The
pellets of both the sarcoplasmic reticulum and
the mitochondrial fractions of the hearts
perfused for 10 minutes and 2 hours with
substrate-free medium were fixed with
osmium tetroxide, dehydrated with alcohol,
and examined under the electron microscope.
When compared with hearts perfused for 10
minutes, the sarcoplasmic reticulum from the
2-hour perfused hearts showed insignificant
changes, possibly a slight increase in swelling.
FIGURE 3
Comparison of the calcium uptake by rat heart sarcoplasmic reticulum perfused for 10 minutes and 2 hourt
with and without substrate. Mean and SE are shown;
number of experiments is in parentheses. Addition of
substrate to the perfusion medium maintained the
calcium uptake at control levels over the 2-hour
perfusion.
ing to the calcium accumulation. On the other
hand, as shown in Figure 4, the calcium
binding by the mitochondrial fraction was
inhibited approximately 853> by oligomycin,
suggesting that the calcium binding in this
fraction was, in fact, due to mitochondria.
Oligomycin had similar effects on the calcium
binding by the subcellular fractions isolated
from hearts perfused for 2 hours without
substrate.
AAlPase Activity of 8,000 to 37,000 g
Subcellular Fraction.—The AMPase activity
of the sarcoplasmic reticulum fraction (8,000
to 37,000 g) was used as an enzymatic marker
for this fraction. The mean activity for this
fraction from ten pairs of control nonperfused
hearts was 0.143 ( S E ± 0.016) /imoles P,/mg
75
Calcium Binding
(mumol Ca + + /mg of protein)
(6)
50
25
(6)
A
B
Mitochondria
Reticulum
FIGURE 4
Influence of oligomycin (2.5 ng/ml) on calcium binding
by the mitochondrial and reticular fractions of the
rat heart; A, without oligomycin; B, with oligomycin.
Calcium binding by the mitochondrial fraction was
reduced by 85% in the presence of oligomycin, whereat
the activity of the sarcoplasmic reticulum was unaffected.
R,st*rcb, Vol. XXVI. April 1970
435
CALCIUM TRANSPORT IN HEART FAILURE
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FIGURE 5
Electron micrographs of the mitochondrUd pellets from rat hearts perfused for 10 minutes (top)
and 2 hours (bottom). The mitochondria are swollen and the cristae disrupted after 2-hour
perfusion without substrate.
In contrast to the sarcoplasmic reticulum, the
changes in the ultrastructure of mitochondria
of the perfused heart were pronounced (Fig.
OrcuUsitm Rtsurcb, Vol. XXVI, April 1970
5). Mitochondria from hearts perfused without substrate were considerably swollen, and
their cristae were disrupted.
MUIR, DHALLA, ORTEZA, OLSON
436
Discussion
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It has aheady been shown in this laboratory
that the dechne in myocardial contractility in
the isolated rat heart perfused without substrate is associated with a drop in the ATP and
a rise in the AMP content of the heart muscle
(18). These data suggested that the failing
contractility in this preparation reflected a
failing rate of oxidative phosphorylation. If
the control of intracellular calcium concentration by sarcoplasmic reticulum and possibly
mitochondria is important in cardiac excitation-contraction coupling, a change in the
ability of the various subcellular fractions to
accumulate calcium might also occur under
these conditions and contribute to the pathogenesis of failure. The results of the present
study show that in the isolated heart failing
from substrate lack, the calcium-binding
capacity of both the sarcoplasmic reticulum
and the mitochondria declines after 30
minutes. This change coincides with the onset
of the decline in both the myocardial contractility and the ATP-AMP ratio in the heart
(18). The calcium uptake in the presence of
oxalate by the reticulum is also reduced, but
the change occurs much later, when myocardial contractility is reduced to a negligible
force after 2 hours of perfusion. The delayed
onset of the defect in calcium uptake in the
presence of oxalate is not unexpected, since
this measure of calcium transport is less
sensitive than calcium binding. Since the fall
in contractility and calcium binding are
coincident, it is possible that the fall in
calcium binding contributes to the onset of
cardiac failure in this preparation.
The decline in myocardial contractility in
the isolated rat heart following perfusion
without substrate can be reversed if glucose is
added to the perfusate after 30 minutes to 1
hour (18). A similar reversibility in calcium
binding by the reticulum has also been
demonstrated. However, after 90 minutes, the
changes become irreversible. It was suggested
that the phase of irreversible myocardial
failure is associated with irreversible alterations in intracellular membranes. The observation that the dechne in reticular calcium
uptake with oxalate coincides with irreversible
myocardial failure supports this concept, since
calcium uptake is dependent on the integrity
of the reticular membrane (32, 33). It was
also demonstrated that both myocardial contractility and calcium uptake can be maintained at normal levels in the isolated heart
for up to 2 hours if glucose is added initially to
the perfusion medium. The changes in the
ultrastructure of the subcellular fragments
following perfusion with substrate-free medium are also compatible with such a hypothesis.
Although the dechne in the ability of the
sarcoplasmic reticulum to accumulate calcium
was not accompanied by a fall in the ATPase
activity of the reticulum, measured in terms of
the total inorganic phosphate released over a
20-minute period, this may reflect the insensitivity of this method of measuring initial
ATPase rates. On the contrary, the initial rate
of reticular ATPase activity was significantly
elevated following 2 hours of perfusion
without substrate, indicating an uncoupling of
reticular ATPase activity from calcium uptake.
Other investigators have also reported a
similar uncoupling with trypsin and sodium
oleate (34, 35), and during muscle maturation
(36). The ability of trypsin (34) to uncouple
ATPase activity from calcium uptake by the
reticulum suggests that the reticular membrane must be intact if the energy released by
the breakdown of ATP is to be utilized
efficiently by the calcium transport system.
Therefore, the uncoupling of the ATPasecalcium uptake system in the sarcoplasmic
reticulum following 2-hour perfusion without
substrate may again be due to disorganization
of the intracellular membranes.
It is not likely that the observed dechne in
calcium accumulation by the subcellular fractions over the 2-hour period of perfusion was
an artifact of particle isolation because (1)
the yields of microsomal material from the
hearts perfused for 10 minutes and 2 hours
were the same, (2) the AMPase activity of the
sarcoplasmic reticulum was unchanged over
the perfusion time, and (3) addition of
CircrUtio* Rtsurcb, Vol. XXVI, April 1970
437
CALCIUM TRANSPORT IN HEART FAILURE
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glucose prevented the change in calcium
uptake.
The results presented here show that in
myocardial failure due to substrate lack there
is a decrease in calcium binding by mitochondria and sarcoplasmic reticulum after 30
minutes that correlates with the decline in
myocardial contractility and in the ATP-AMP
ratio in the heart muscle. When the failure
becomes irreversible and not responsive to
restoration of substrate, there is a decrease in
calcium uptake by the sarcoplasmic reticulum
and a rise in the microsomal ATPase. It is
suggested that these latter changes in the final
stage of failure are associated with irreversible
alterations of the intracellular membranes.
Acknowledgment
10. INESL
1. WEBER, A., AND WINICUR, S.: Role of calcium in
the superprecipitation of actomyosin. J Biol
Chem 236: 3198, 1961.
2. WEBER, A.: Energized calcium transport and
relaxing factors. Curr Top Bioenerg 1: 203,
1966.
R.
J.,
AND CONSTANTTN,
L.
L.:
Regulation by calcium of the contraction and
relaxation of muscle fibres. Fed Proc 23: 933,
1964.
4. EBASHI, S.: Calcium binding activity of vesicular
relaxing factor. J Biochem 50: 236, 1961.
5. EBASHI,
S.,
AND LIPMANN,
S.,
AND WATANABE,
S.:
11. SLATER, E. C , AND CLELAND, K. W.: Effect of
calcium on the respiratory and phosphorylative activities of heart-muscle sarcosomes.
Biochem J 55: 566, 1953.
12. BRJERLEY, G. P., MURER, E., AND BACHMANN,
E.: Studies on ion transport: III. Accumulation
of calcium and inorganic phosphate by heart
mitochondria. Arch Biochem 105: 89, 1964.
13. PATRIARCA, P., AND CARAFOU, E.: A study of
intracellular transport of calcium in rat heart. J
Cell Physiol 72: 29, 1968.
14. GERTZ, E. W., HESS, M. L., LAIN, R. F., AND
BRICGS, F. N.: Activity of the vesicular
calcium pump in the spontaneously failing
heart-lung preparation. Circ Res 20: 477,
1967.
Decreased Ca 2 + uptake by sarcoplasmic reticulum after coronary artery occlusion for 60 and
90 minutes. Circ Res 21: 439, 1967.
16. DHALLA, N. S., AND OLSON, R. E.: Correlation of
the endogenous fuel supply and contractile
force in the isolated perfused rat heart (abstr).
Fed Proc 26: 352, 1967.
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Energy-Linked Calcium Transport in Subcellular Fractions of the Failing Rat Heart
JOHN R. MUIR, NARANJAN S. DHALLA, JOSEFINA M. ORTEZA and ROBERT E. OLSON
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Circ Res. 1970;26:429-438
doi: 10.1161/01.RES.26.4.429
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