(Received for publication, September 16, 1995; and in revised form, January 8, 1996)
From the
Measurements of free calcium ion concentration in the
sarcoplasmic reticulum ([Ca]
)
and an evaluation of its relationship to changes in cytosolic free
calcium and energy state of the cell, as well as heterogeneity of the
SR calcium pool, were performed using
F NMR in Langendorff
perfused rabbit hearts loaded with acetoxymethyl ester of
1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N`,N`-tetraacetic
acid. We report a base-line time-average
[Ca
]
value of 1.5 mM (n = 13) in the beating heart, similar to the
value measured at diastole. We further report that
[Ca
]
decreases by
30% at
the start of systole and that there is no evidence of spacial
heterogeneity in [Ca
]
during
the contraction cycle. However, there appears to be a heterogeneous
response to SR calcium channel release activator (caffeine) and SR
calcium-ATPase inhibitor (cyclopiazonic acid), consistent with studies
suggesting that there are subpopulations of SR. Raising cytosolic free
calcium by depolarizing the cell with 30 mM extracellular KCl,
resulted in an increase in
[Ca
]
; however, the calcium
gradient was unchanged. Lowering cell phosphorylation potential, which
would reduce the free energy available for the SR
Ca
-ATPase, leads to a decrease in the calcium
gradient across the SR, but this reduced gradient was primarily due to
an increase in cytosolic free calcium and not a net release of SR
calcium.
The sarcoplasmic reticulum (SR) ()plays an important
role in regulation of mammalian cardiac muscle contraction. It is
generally accepted that contraction is activated by Ca
influx through the sarcolemmal L-type channel, which
subsequently releases SR Ca
via the
Ca
-induced Ca
-release
mechanism(1) . During relaxation, Ca
is
resequestered into the SR by the SR Ca
-ATPase and
extruded by the sarcolemmal Na
-Ca
exchanger(2, 3) . The SR Ca
content available for release is an important determinant of
contractile state. In spite of the importance of SR
Ca
, little is known about the levels of free SR
Ca
concentration
([Ca
]
) and how it is altered
by physiologic or pathologic perturbations. Although previous studies (4, 5) using rapid cooling contractures have provided
a valuable index of SR Ca
load in cultured myocytes,
there are currently no direct measurements of
[Ca
]
. It is generally agreed
that the SR Ca
-ATPase maintains a calcium gradient
between the SR matrix and the cytosol which is close to the theoretical
limit based on the free energy available from ATP hydrolysis. However,
the exact degree of efficiency is debated due to the lack of precise
knowledge of [Ca
]
(see (6) ). Values for free SR calcium have been estimated (based on
binding to calsequestrin) to be in the range of 0.3-5
mM(6, 7, 8) . Furthermore, if the SR
Ca
-ATPase is operating near its theoretical limit, a
fall in phosphorylation potential, for example under conditions of
ischemia (9, 10) , could affect the Ca
gradient across the SR.
The present study provides direct
measurements of [Ca]
using
F NMR in Langendorff perfused rabbit hearts loaded with
acetoxymethyl ester of
1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N`,N`-tetraacetic
acid (TF-BAPTA). Our laboratory has recently developed TF-BAPTA and
applied it to measurements of cytosolic
[Ca
] in perfused rat
hearts(11, 12) . TF-BAPTA has a high K
for Ca
(65
µM) and combines both a large shift sensitivity with
fast-intermediate exchange kinetics at typical magnetic field
strengths(11, 12) . Such an indicator offers the
potential for simultaneous determinations of Ca
concentrations in different cellular compartments, contingent on
the degree of indicator loading into these compartments. For TF-BAPTA,
the chemical shift rather than the ratio of resonance intensities
provides information regarding
[Ca
](11, 12) . The
6-fluorine resonance in TF-BAPTA is insensitive to calcium binding and
serves as a shift reference, and the 5-fluorine resonance shifts
downfield on Ca
complexation. Using this approach we
directly measure an ionized calcium concentration in the SR of
1.5
mM, a value in good agreement with estimates obtained using
calsequestrin binding constants(7) . In addition we calculate
the free energy required for the Ca
gradient across
the SR under control conditions, under conditions of cardiac arrest
induced by high extracellular potassium concentration, and under
conditions of reduced phosphorylation potential.
Hearts were placed in a standard 30-mm
NMR tube. After 10 min of control perfusion, loading with 1000 ml of 5
µM acetoxymethyl ester of TF-BAPTA was started. With
typical flow rates of 30-50 ml/min, loading took about 30 min. To
monitor contractility, a latex balloon was inserted into the left
ventricle. The balloon was inflated to give an end diastolic pressure
of 5-10 cm HO. As observed in our previous study of
perfused rat hearts(12) , TF-BAPTA did not cause a significant
reduction in contractility.
To examine whether
[Ca]
varies during systole and
diastole, the NMR pulse was gated to the contraction cycle through a
homemade system, which provides a precise, adjustable trigger at the
desired point on the pressure wave. The gating system utilized the
dP/dt signal to generate a standard pulse signal which initiated the
NMR pulse. A delay relative to the standard pulse signal was included
in the pulse sequence so that the NMR pulse could be imposed at the
desired time during the cardiac cycle. At exactly the same time as the
NMR pulse, a triggering signal (TS) was sent out. We recorded
simultaneously left ventricular pressure (LVP), dP/dt, and TS to verify
the timing of the NMR acquisition. Once the NMR pulse was generated, a
free induction decay was acquired. Since the peak of the cytosolic
Ca
transient occurs near the beginning of the
upstroke of LVP wave(13, 14) , one NMR pulse was gated
at the time shown as TS 1 to measure systolic
[Ca
]
; and another NMR pulse
was delayed by 150 ms (TS 2) to measure diastolic
[Ca
]
. The time interval
between the scans depended on the heart rate. For a typical heart rate
of
150-200 beats/min, the interval was
300-400
ms. 1000-2000 consecutive gated scans were acquired to achieve an
acceptable signal-to-noise ratio. The other parameters were the same as
those used in the ungated study.
For the gating data to be valid,
the kinetics of calcium binding to the indicator should be fast and the
relaxation time should be short on the NMR time scale. These conditions
were met in these experiments. First, the lifetime of the
Ca-chelator complex is very short (
33
µs)(11) . Second, the NMR pulse is also negligibly short
(20 µs). Third, the apparent transverse relaxation time is
1.2
ms, so the acquisition time for the [Ca
]
measurements was only 2 ms. Thus the time resolution should be <3
ms, which is <1% of the typical cardiac cycle.
where K = 65
µM,
= 5.13,
+
= 13.41,
= 8.53,
= 14.83 (for
[Ca
] > 7 µM), or
= 11.44 (for [Ca
]
< 7 µM),
+
= 11.21, and
= 7.83. pH and
[Mg
] were taken as 7.1 and 1 mM,
respectively.
The following equation was used to
calculate G
, with
G
= -30.5 kJ/mol.
By thermodynamic convention values for G
and
G
are negative for exergonic
reactions, but after calculating
G
we refer
to the values in the text as absolute values. The
[ATP]
/[ADP]
ratio was
calculated by assuming that the creatine kinase reaction is at
equilibrium. The apparent equilibrium constant (K`
) was calculated according to (18) .
PCr and Cr contents were measured biochemically and converted to
concentration by assuming that these metabolites are entirely cytosolic
and that cytosolic volume equals 2.3 ml/g (dry weight).
[P] was obtained by comparing the NMR peak area
to that of the basal PCr peak, after correction for NMR saturation, and
assuming that the P
peak is entirely cytosolic.
Ionized free calcium in compartments that contain different
calcium concentrations can be measured from the F NMR
spectra of TF-BAPTA loaded hearts from the shift difference between the
Ca
-insensitive 6F resonance and the
Ca
-sensitive 5F resonance of TF-BAPTA. In rat heart
we observe one Ca
-sensitive peak (
5 ppm)
corresponding to a time-average cytosolic free Ca
concentration ([Ca
]
) of
600 nM(12) . However, in rabbit heart, as shown
in Fig. 1, the
F spectrum shows two
calcium-sensitive resonances: a cytosolic free calcium resonance at
5 ppm, and an additional resonance peak at
14 ppm that
corresponds to a [Ca
] of
1.5
mM. To assess if the
14 ppm peak represents SR
Ca
, hearts were treated with a SR
Ca
-ATPase inhibitor or a SR Ca
release channel activator or were arrested with 30 mM [K
]
to determine if these
manipulations would alter the measured [Ca
]
as would be expected if the peak reflected indicator in the SR.
Figure 1:
F NMR spectrum (addition
of spectra from four experiments), using 0.26-s intervals between scans
with a pulse of 40
(20 µs). The spectral width was
±7060 Hz, and 4000 data points were collected. The
free-induction decay was multiplied by an exponential function
corresponding to 100 Hz-line broadening.
If
the resonance at 14 ppm originates from the SR, then perfusion
with 50 µM CPA, a specific SR Ca
-ATPase
inhibitor(19) , should decrease
[Ca
]
. Consistent with our
expectation, after
10 min of perfusion with CPA, there was an
upfield shift of the resonance near 14 ppm, indicating a decrease in
[Ca
]
(Fig. 2a). As shown in the inset, the
shift and broadening observed with CPA addition is consistent with
heterogeneity of the SR Ca
pool. The spectrum at
20-25 min of CPA perfusion could be modeled, assuming that 66% of
the SR pool had a [Ca
] of 100 µM and 33% of the SR had a [Ca
] of 260
µM. The change in contractility observed on addition of
CPA is typical of SR Ca
-ATPase inhibition, i.e. a moderate decline in left ventricular developed pressure to 80
± 14 mm Hg compared to the initial level of 106 ± 16 mm
Hg, and a moderate increase in end diastolic pressure (see Fig. 2b).
Figure 2:
a, typical F NMR spectra from
a heart perfused with 50 µM CPA, a
Ca
-ATPase inhibitor. This is representative of three
experiments. During 5-10 min of CPA perfusion, the peak that was
initially located at 14.4 ppm is inhomogeneously shifted upfield to
13 ppm with a marked broadening, corresponding to a decline in
[Ca
]
from 1.5 mM to
300 µM. As shown in the inset, this could be
modeled assuming that there are two separate pools of calcium, as
discussed in the text. b, LVP of a perfused rabbit heart
before and during perfusion with 50 µM CPA.
We further tested whether SR
Ca could be depleted by the addition of caffeine,
which activates the SR Ca
-dependent Ca
release channel (20) . To minimize the caffeine-induced
contracture so that we could maintain tissue perfusion in the presence
of caffeine, 10 mM BDM was administered prior to
caffeine(21) . Subsequent combined perfusion with 10 mM caffeine + BDM resulted in a moderate increase in end
diastolic pressure, while left ventricular developed pressure was
reduced to 16.2 ± 3.1 mm Hg compared to the initial level of 101
± 6 mm Hg. As shown in Fig. 3, addition of caffeine
resulted in a loss of the resonance at 14.3 ppm, presumably due to
lowering of [Ca
]
and exchange
broadening of the peak. Also shown in Fig. 3, when caffeine and
BDM were washed out, the resonance at
14 ppm returned, as did
contractility, consistent with an SR location for the calcium pool at
14 ppm. This broadening of the
14 ppm resonance during
caffeine perfusion followed by the reappearance of a sharp resonance
during caffeine washout indicates that the
14 ppm resonance is not
due to extracellular indicator, and that the caffeine effect is not due
to loss of indicator from the SR.
Figure 3:
Typical F NMR spectra from a
heart perfused with 10 mM caffeine, a SR Ca
release channel activator, in the presence of 10 mM BDM. This
is representative of three experiments. The peak at 14.3 ppm becomes so
broad that it is virtually undetectable after 25 min of caffeine
perfusion. During the washout period without drugs, the peak returns to
very near the original position, corresponding to a
[Ca
]
of 1.2
mM.
We were also interested in testing
whether this resonance at 14 ppm could be shifted by perfusion
with high [K
]
(30 mM),
which depolarizes the myocytes, arrests the heart, and increases
diastolic [Ca
]
(22) . If
the peak at
14 ppm arises from SR Ca
, one might
expect a downfield shift on perfusion with high
[K
]
. As demonstrated in Fig. 4, the putative SR resonance shifts from 14.4 ppm to 14.7
ppm, corresponding to an increase in
[Ca
]
. The shift to 14.7 ppm is
very near saturation for the indicator, which makes it difficult to
quantitate accurately the change in
[Ca
]
. The calculated value for
[Ca
]
increased from the
control value of 1.5 mM to 5 mM during high
[K
]
perfusion.
Figure 4:
Typical F NMR spectra from a
heart perfused with 30 mM [K
]
in the perfusate. This
is representative of three experiments. The peak at 14.4 ppm is shifted
to 14.7 ppm, corresponding to an increase in
[Ca
]
from 1.5 mM to 5
mM.
After
confirming that the resonance at 14 ppm originates from SR, we
investigated the magnitude of the change in
[Ca
]
during the cardiac cycle,
which we thought might have a similar time course to the cytosolic
Ca
transient. This is done by gating the NMR pulse
and acquisition to the contraction cycle. As shown in Fig. 5a, [Ca
]
was measured at the start of systole (TS 1) and after the heart
had fully relaxed (TS 2). The corresponding spectra are shown in Fig. 5b. The chemical shift of the SR peak during
diastole is 14.4 ± 0.04 (n = 3), corresponding
to a [Ca
]
of 1.5 mM,
and the shift in early systole is 14.2 ± 0.03 (n = 3), corresponding to a
[Ca
]
of 1.0 mM. The
difference (0.5 mM) represents a decrease in
[Ca
]
at the start of systole
of
30%. We also gated at other points during systole (for example
at +10 ms compared to TS 1, near the middle of the upstroke). At
this point there is already partial recovery of
[Ca
]
, to a value of
1.2
mM. Thus the fall in [Ca
]
at the start of systole is very brief, which accounts for our
finding that the time-average value of
[Ca
]
(1.49 ± 0.06
mM, n = 13) is essentially the same as the
diastolic value.
Figure 5:
a, simultaneous recording of LVP, dP/dt,
and TS. Panel A shows the location of the NMR triggering
signal at the start of systole (TS 1) and panel B shows the
location of the NMR triggering signal during diastole (TS 2). b, typical F NMR spectra from a heart triggered
at systole (TS 1) and at diastole (TS 2). This is representative of
three experiments. The chemical shift of the SR Ca
resonance at diastole is
0.2 ppm upfield compared to the
chemical shift at systole, corresponding to a decrease in
[Ca
]
of
0.5 mM (
30%) at the start of systole.
The free energy required for the SR
Ca-ATPase can be calculated from the diastolic values
of [Ca
]
and
[Ca
]
. Using the
[Ca
]
value of 1.49 ±
0.06 mM and a diastolic [Ca
]
of 100 nM(22) , and assuming no membrane
potential across the sarcoplasmic reticulum(16) , the free
energy (
G) required for the SR
Ca
-ATPase is
49.5 kJ/mol. Using metabolites
measured in parallel perfused rabbit hearts (Table 1), we have
calculated the
G for ATP hydrolysis
(
G
) to be 59 kJ/mol (Table 2),
consistent with the reported values in the range of 55-60
kJ/mol(9, 23) . After 30 min of total ischemia, we
find that
G
falls to <48 kJ/mol, as
shown in Table 2. A
G
of <48
kJ/mol would not be sufficient to support the SR/cytosol calcium
gradient of 1.5
10
measured under basal conditions;
this might result in reversal of the Ca
-ATPase and
net calcium release from the SR during ischemia. To investigate how the
decrease in the free energy of ATP hydrolysis that occurs during
ischemia affects the SR calcium gradient, we subjected hearts to
ischemia and determined the effect on
[Ca
]
. As illustrated in Fig. 6a, even at 25-30 min of ischemia, the
chemical shift of the SR Ca
peak is unchanged. If we
assume that pH in the SR is the same as pH
, which declined
progressively (to be 6.08 ± 0.10 at the end of 30 min of
ischemia, n = 4), the calculated
[Ca
]
was not changed
significantly (Fig. 6b), despite a marked reduction in
G
, which we measured to be <48 kJ/mol.
However, there is a substantial decrease in the SR/cytosol calcium
gradient, due to the increase in [Ca
]
to a value of
3 µM (Fig. 6c),
which reduces the energy requirement (
G) for the
Ca
-ATPase during ischemia, to a value of 32 kJ/mol
after 30 min of ischemia. Thus with a decrease in the energy available
from ATP hydrolysis during ischemia, the SR/cytosol calcium gradient is
not maintained as [Ca
]
increases. This is in contrast to the effect of cardiac arrest
with high [K
]
perfusion, where
an increase in [Ca
]
(from
100 nM to
350 nM) is accompanied by an
increase in [Ca
]
(from
1.5 mM to
5 mM). The
G for
ATP hydrolysis does not decrease under these conditions, and therefore
the SR/cytosol calcium gradient can remain constant. These data suggest
that a rise in cytosolic free calcium would normally be associated with
an increase in [Ca
]
; however,
this does not occur during ischemia, possibly due to the decline in
G
. Although the free energy available to
pump calcium into the SR is reduced during ischemia, the
G
is adequate to prevent net release of SR
calcium during 30 min of total ischemia in the isolated rabbit heart.
Figure 6:
a, F NMR spectra (addition of
spectra from 4 hearts) during control perfusion, during global ischemia
and during the first 5 min of reperfusion. b and c,
time course of calculated [Ca
]
(b) and [Ca
]
(c) before and during ischemia. Asterisk indicates p<0.05 (tested by fully factorial
(M)ANOVA).
We report a value for time-average
[Ca]
of 1.5 mM measured in the beating perfused rabbit heart under basal
conditions. When the [Ca
]
measurement was gated to the contraction cycle, the diastolic
value was essentially the same as the time-average value. This value
agrees well with calculated values based on estimates of total calcium
and calsequestrin binding sites(7) . Calsequestrin, the major
calcium-binding protein in the SR has a K
for
Ca
in the range of 1
mM(24, 25, 26) . Thus, the
[Ca
]
values measured in this
study suggest that [Ca
]
is
being maintained near the K
of calsequestrin.
These data also allow calculation of the free energy requirement of the
SR Ca
-ATPase. The data in this report suggest a
calcium gradient across the SR of 1.5
10
at
diastole, and assuming no membrane potential, this would correspond to
a
G for the Ca
-ATPase of 49.5 kJ/mol, a
value that agrees well with that estimated
previously(6, 7, 8) . We further show that
high [K
]
perfusion, which we
have shown previously (22) to increase diastolic
[Ca
]
from
100 nM to 350 nM, increases the
[Ca
]
to
5 mM;
this corresponds to a
G for the
Ca
-ATPase of 49.3 kJ/mol. Thus, perfusion with 30
mM potassium elevates both cytosolic and SR
[Ca
], but the
G for the
Ca
-ATPase is unchanged. We also show that during
ischemia there is a parallel decline in the free energy of ATP
hydrolysis and the energy requirement of the SR
Ca
-ATPase. These data suggest that there is tight
coupling between the SR calcium gradient and the free energy of ATP
hydrolysis, and that the Ca
-ATPase works at near its
theoretical limit.
SR calcium appears to be well buffered during a
normal calcium transient. The difference in
[Ca]
between the start of
systole and diastole suggests that calcium release from the SR to
trigger contraction causes a transient decrease in
[Ca
]
of
30%. It has been
reported that the fractional SR calcium release varies with SR calcium
load and trigger calcium, but an estimate obtained under physiologic
conditions suggests that
35% of the SR calcium content may be
released with each twitch(27) . This is similar to an earlier
report that only about half of SR Ca
is released
during a twitch(5) . The relatively small and transient nature
of the changes in [Ca
]
during
the contraction cycle is also indicated by the sharpness of the
14
ppm resonance observed in the time-average measurement of
[Ca
]
in the beating heart,
which is similar to that observed in hearts arrested with high
[K
]
. The sharp resonance also
suggests that under basal conditions there is little spacial
heterogeneity in [Ca
]
.
The
modeling in the inset of Fig. 2shows that the spectra
observed after addition of CPA are consistent with a heterogeneous loss
of calcium from the SR network. One possible explanation for this
observation is that there are distinct types of SR, which may respond
differently to SR calcium-ATPase inhibitors. Another possibility is
that the signals were obtained from the whole heart, which displays a
great deal of heterogeneity in cellular structure and function. In
support of the first concept, Jorgensen et al.(28) showed that the total SR calcium measured by electron
probe microanalysis (EPMA) varied within subpopulations of SR. They
suggest that Ca is accumulated into the network SR,
which immunoelectron microscopic studies show have
Ca
-ATPase, which is apparently absent from the
junctional and corbular SR. They further suggest that Ca
accumulated by the network SR is then transferred and stored by
the junctional and corbular SR which contain large amounts of
calsequestrin. Further support for distinct populations of SR comes
from a study by Kijima et al.(29) , which reports
different intracellular localization of inositol 1,4,5-trisphosphate
and ryanodine receptors in cardiomyocytes. The results would also be
consistent with different leak rates from different parts of the SR
network. The data in this report suggest that different pools of SR
respond differently to CPA and caffeine.
One question that we have
not yet addressed is the possible contribution of mitochondrial
Ca under the conditions of our experiments. A number
of studies have demonstrated that [Ca
] in
mitochondria ([Ca
]
) is in the
same range as
[Ca
]
(30, 31) .
Thus, if TF-BAPTA loads into mitochondria, the mitochondrial peak might
not be distinguishable from the cytosolic peak and would be expected to
be well separated from the putative SR peak at
14 ppm. While
excitation-contraction coupling of normal rabbit myocytes under
physiologic conditions is heavily dependent on calcium fluxes across
the plasma membrane and the SR membrane, with relatively little
contribution from the mitochondria(3) , under conditions where
SR function is inhibited by caffeine, mitochondria can be involved in
calcium removal from the cytosol(3) . However, in our
experiments, we never observed a separate resonance or a shoulder on
the
5 ppm peak that could be attributed to the mitochondria. This
reflects either the lack of indicator loading into the mitochondria or
very tight coupling between [Ca
]
and [Ca
]
.
The total
calcium content of the SR and the percentage released during an action
potential, tetanus, or other interventions have been measured by EPMA
and estimated by caffeine release and rapid cooling contractures, using
fluorescent calcium indicators to measure the change in
[Ca]
. The EPMA studies of frog
skeletal muscle suggest that the resting content of total calcium in
the terminal cisternae (TC) is
120 mmol/kg (dry weight) of TC; a
1.2-s tetanus releases
70 mmol/kg (dry weight)(32) .
Similarly, addition of caffeine reduces TC calcium to
40 mmol/kg
(dry weight) (33) . Thus these studies show that greater than
half the total TC calcium is released during tetanus or addition of
caffeine in skeletal muscle. Using either caffeine or rapid cooling
contractures, estimates of the percentage of SR calcium release have
also been made in cardiac muscle. Baro et al.(34) report that caffeine released
71% of the total
calcium in cardiac muscle. The data presented here are generally in
agreement with this estimate of caffeine-releasable calcium. We find
that [Ca
]
decreases by more
than 80% upon addition of CPA or caffeine.
The EPMA data also
suggest that tetanus or caffeine addition results in a 50%
increase in total SR magnesium(16, 32, 33) .
The increase in free magnesium is likely to be much less than the
increase in total magnesium, due to buffering. However, assuming that
caffeine results in a 50% increase in free SR magnesium, this would
increase our calculated [Ca
]
by only 0.01 mM.
In summary, several conclusions can
be drawn from the data in this report. First, the time-average
[Ca]
in the beating rabbit
heart is
1.5 mM, which is not significantly different
than [Ca
]
measured at
diastole. Second, [Ca
]
decreases by
30% at the start of systole but this decrease
is very brief, with
50% recovery in 10 ms. Third, there is little
spacial heterogeneity in [Ca
]
in the beating heart. Fourth, there may be a heterogeneous
response to caffeine and CPA consistent with previous studies
suggesting that there are subpopulations of SR with different calcium
handling characteristics. Fifth, the data are consistent with tight
coupling between the SR Ca
gradient and the free
energy of ATP hydrolysis.