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BRIEF RAPID COMMUNICATION
Effects of an Increase in Intracellular Free
[Mg2+] After Myocardial Stunning on
Sarcoplasmic Reticulum Ca2+ Transport
Stephen M. Krause, PhD, and Dennis Rozanski, BS
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Background. Myocardial stunning has been associated with a greater than twofold increase in
intracellular free [Mg+] from 0.6 to 1.5 mM. The effect of this increase in free [Mg+] on the
function of the sarcoplasmic reticulum (SR) Ca2+ pump was assessed in SR isolated from
Langendorif perfused, isovolumic rabbit hearts after 15 minutes of global ischemia.
Methods and Results. Our results indicate that myocardial stunning results in a shift in the Ca24
sensitivity of oxalate-supported, Ca>+ transport over the entire range of free [Ca2+] associated
with the cardiac cycle. Using 0.6 mM free Mg2+ as control, maximal rates of Ca2+ transport
occurred at 1 ,uM free Ca24 (control, 519±32; stunned, 337+37 nmol Ca21 min-1 * mg-t). At 0.56
,uM free Ca24, SR Ca24 transport was reduced from a control of 351±49 to 263±12 nmol
Ca2+ * min' * mg-t at 0.6 mM free [Mg2`]. Moreover, an increase in the free [Mg2`1 from 0.6 to
1.5 mM results in a greater shift in the Ca2+ activation curve with no change in the level of
maximal activation. Ca2+ transport at 0.56 ,M free Ca2+ was shifted in the stunned SR from
263±12 to 138+29 nmol Ca2+ mint * mg'1 at 0.6 and 1.5 mM free Mg2+, respectively.
Conclusions. These results indicate that an increase in free [Mg2`] after stunning in combination with the inherent defect in the SR Ca2+ ATPase may reduce the ability of the cell to regulate
Ca2+ to a greater extent than previously observed. This impairment in Ca2' regulatory function
may contribute directly to the increase in diastolic tone and indirectly to the reduced systolic
function characteristic of the stunned myocardium. (Circulation 1991;84:1378-1383)
.
yocardial stunning is associated with a prolonged but reversible mechanical dysfunction after a brief period of ischemia
insufficient to produce cell necrosis.12 The mechanism
by which this sustained depression in contractile dysfunction occurs, however, is not clear. Current theories appear to implicate a deficiency in the handling of
intracellular Ca2+3-6 rather than an alteration in the
supply of MgATP.2 Studies have shown not only that
Ca2+ transport by the sarcoplasmic reticulum (SR) is
altered,3,6 but have also implicated a decrease in the
Ca2+ sensitivity of the contractile proteins.47 A possible explanation for a change in Ca2+ sensitivity may
involve an increase in free [Mg2+] after reperfusion.
Recent studies have shown that the free [Mg2+] is
elevated after reperfusion.8$9 The effect this increase
would have on intracellular function is only beginning to be characterized. However, investigators8-11
have speculated that a rise in free [Mg2] may
M
From the Department of Physiology, Jefferson Medical College,
Philadelphia, Pa.
Supported in part by a grant from the Southeastern Pennsylvania Chapter of the American Heart Association.
Address for correspondence: Stephen M. Krause, PhD, Merck,
Sharp & Dohme Research Laboratories, WP 26-265, West Point,
PA 19486.
substantially alter intracellular enzyme function because Mg> is an important cofactor for many reactions and can compete with Ca> at various Ca>
binding sites. This is supported by studies using intact
cardiac cells from normal hearts that have shown that
increases in free [Mg`] will decrease SR Ca> loading12 and reduce Ca>2 sensitivity of the myofilaments,
resulting in a decrease in contractile activity.13 In
addition, Mg2+ is also able to modulate Ca' release
from the SR.14 Whether the increase in free [Mg2+]
that occurs with stunning will significantly affect Ca>2
loading of the SR, however, has not been examined.
Accordingly, we examined the effect an increase in
free [Mg2>] would have on the function of cardiac SR
after myocardial stunning.
The SR was examined because of its importance in
the regulation of the activation Ca>2 in the cardiac cell.
If the increase in free [Mg2+] after stunning was sufficient to shift the Ca-2 sensitivity of the SR ATPase,
then Ca2+ loading of the SR would be reduced. In
addition to the alteration in enzyme function previously
reported,3'6 this would decrease the amount of Ca>
available for release (and consequently the level of
tension development) and increase diastolic tone
caused by the slower rate of Ca> accumulation; both
hallmarks of myocardial stunning.
Krause et al Myocardial Stunning and Sarcoplasmic Reticulum Function
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Methods
Global myocardial stunning (15 minutes of ischemia) was induced in isolated, perfused New
Zealand White rabbit hearts as previously detailed.15
These animal studies conformed to the guiding principles of the American Physiological Society. Developed pressure, +dP/dt, coronary flow, and heart rate
were continuously monitored. After stabilization,
stunned hearts (n=6) were subjected to 15 minutes
of global ischemia (37°C) followed by 50 minutes of
reperfusion. Control hearts (n=7) were perfused for
70 minutes after stabilization.
The protocol followed kept the total perfusion
time for all hearts similar (70-80 minutes). Accordingly, the experimental time course for control hearts
was at least 15 minutes less than those subjected to
global stunning. In addition, stunned hearts were
perfused for an additional 10 minutes to assure that
the recovery of mechanical function after reperfusion
had stabilized. This additional 10 minutes of perfusion time is not associated with any deterioration of
function in control hearts (results not shown).
Subsequently, all hearts were rapidly cooled by
infusion of 25 ml ice-cold normal saline (0.9% NaCl),
removed from the perfusion apparatus, and placed in
ice-cold normal saline. The atria and right ventricle
were removed and the remaining left ventricular tissue
was weighed and finely minced with scissors in 10 mM
imidazole (pH 7.1) containing 25 ,uM phenylmethylsulfonyl fluoride (PMSF) according to the ratio of 3
ml/g wet weight. The minced tissue was gently homogenized on ice in a Thomas glass tissue grinder
equipped with a Teflon pestle rotating at 500-800
rpm. A portion of the homogenate was reserved for
characterization and the remainder processed for
isolation of a microsomal fraction enriched in SR
using techniques previously outlined.16
Steady-state Ca' transport by the SR was assessed
under maximally stimulated conditions (15 ,uM free
[Ca'], 3.4 mM MgATP) by a rapid filtration technique
in the presence of 10 mM K-oxalate, 200 ,uM 45CaC12 at
37°C as previously described.16 The Ca>2 sensitivity of
SR Ca21 transport, a more physiological representation
of intracellular function, was measured by varying the
total [45Ca21] at a constant [EGTA] of 0.5 mM to obtain
a free [Ca>2] ranging from 10 nM to 10 ,uM in the
presence of 5 mM K-oxalate, 5 mM NaN3, 20 mM
imidazole (pH 7.15) at a total ionic strength of 0.16 M
balanced with KCl using 0.1 mg/ml SR. The ionic
concentrations of each bath were calculated with the
computer program of Fabiato17 using the absolute
stability constants reported by Fabiato,18 which were
corrected for 37°C and pH 7.15.
To evaluate the effect of an increase in free [Mg2>]
on SR activity, the free [Mg>] was either 0.6 mM for
control SR or 1.5 mM for stunned SR. These free
[Mg2+] were chosen to represent the ionic conditions
in the control and stunned heart cells based on the
measurements of Murphy et a18 using 4F-APTRA.
The measurements using 4F-APTRA were used be-
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cause this compound has less affinity for Ca2' than
other analogues and is insensitive to physiological
changes in intracellular pH. Although other investigators have reported different free [Mg2`] after 15
minutes of ischemia,9 these have used an indirect
method of calculating the free [Mg2`] based on the ato-f3-ATP shift using 31P NMR and are not as
reliable as the direct method used by Murphy et al.8
For a discussion of the limitations of using 31P NMR
for measuring free [Mg2], see Murphy et al.8
Data Analysis
Comparisons between baseline control hemodynamic measurements at the 30-minute time point
and subsequent values were performed using the
Student's t test for paired values. Measurements of
the Ca' dependence of SR Ca' transport from
stunned and control groups were analyzed for statistical changes using a one-way ANOVA test followed
by a Dunnet and Newman-Keuls post hoc comparison. Ca2+ transport rates in homogenate and SR from
duplicate measurements from each heart were compared using the Student's t test. The results were
considered statistically significant at a confidence
level of p<0.05.
Results
Myocardial stunning is characterized by a prolonged depression in mechanical function after a
brief period of ischemia. In the rabbit heart, this is
exhibited as a sustained decline in developed pressure as well as an increase in end-diastolic pressure.
Figure 1 presents the mechanical function of both
control and stunned hearts during control periods
and after 50 minutes of reperfusion. After a marked
45% reduction in developed pressure (Figure 1A),
stunned hearts gradually regained systolic function to
stabilize at 85% of control values after 50 minutes of
reperfusion (p<0.05). End-diastolic pressure (Figure 1B) initially rose dramatically before stabilizing
at 12.3+2.1 mm Hg. This level was significantly
greater than the control (4.8± 1.3 mm Hg; p<0.03),
indicating a possible defect in the ability of the
cardiac cell to maintain intracellular Ca>2 homeostasis and to relax completely. Myocardial contractility
(+dP/dt; Figure IC) was significantly reduced in the
stunned hearts after 50 minutes of reperfusion
(p<O.Ol). The rate of relaxation, using -dP/dt as an
index (Figure 1D), was also reduced 18% in comparison with control hearts (p<0.02) after 50 minutes of
reperfusion.
Despite the reduction in mechanical function,
heart rate and coronary flow did not differ significantly between the two groups. During the initial
stabilization period, coronary flow was 76.2+6.2 and
83.3 +4.5 ml/min for control and stunned hearts,
respectively. At the end of the experimental protocol,
coronary flow was 70+±4.1 ml/min in the stunned
hearts compared with 72±+6.8 ml/min in controls.
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FIGURE 1. Plots show time course of developed pressure (panel A), diastolic pressure (panel B), contractility (+dP/dt; panel C),
and rate of relaxation (-dP/dt; panel D) for control hearts and hearts subjected to 15 minutes of ischemia and 50 minutes of
reperfusion (stunned). Each point represents the mean and each bar the standard error. *p<0.05 in comparison with preischemic
values at the 30-minute time point.
Upon reperfusion, however, stunned hearts did show
a hyperemic response with a 5% increase in coronary
flow during the initial 3 minutes (87.6+2.5 mUmin).
Heart rate at the end of the protocol averaged 144 +6
beats/min in controls compared with 148±5.8 beats/
min in the stunned hearts. This represented an 8%
decrease from the beginning of the protocol.
The reduction in -dP/dt would infer that the rate of
Ca>2 removal from the cell was reduced in comparison
with the control. This is borne out by assessing the
function of the SR. Biochemically, the SR from the
stunned hearts showed a statistically significant reduction in function when compared under identical conditions (15 ,uM free Ca>+, 0.6 mM free Mg'). Homogenate Ca2+ transport was reduced 20% in stunned hearts
from a control of 39.2±+1.9 to 31.3±+ 1.5 nmol
Ca> mg-1min-1 (p<0.01). These rates are significant in that no fractionation of the homogenized tissue
occurred. Thus, the results can be used to estimate the
rate at which the SR in the intact muscle can accumulate Ca2+.19 A similar 20% reduction in Ca2+ transport
was also observed in the isolated SR from a control of
495±29 to 397±+27 nmol Ca> * mg-1 * minm1 (p<0.01)
in the stunned heart. The Ca> ATPase activity, measured during Ca> transport, was reduced from 733 67
to 545±52 nmol Ca> mg-' * min' (p<0.04) for con±
trol and stunned SR, respectively. However, the coupling ratio (Ca' transport rate/Ca' ATPase rate) was
not significantly different between the two groups.
These data, obtained under supramaximal conditions
(15 /LM free Ca'), indicate that under identical conditions, there is an alteration in the enzyme function
similar to that observed in a regional model of stunning.6 These studies, however, do not provide any
information regarding the activity of the SR Ca' pump
under ionic conditions that represent a typical cardiac
cycle (0.1-1.0 ,cM free Ca').
To obtain this information, the Ca' sensitivity of
the SR Ca2' pump was assessed. Figure 2 shows the
result for both the control and stunned hearts. When
analyzed using a free [Mg>] of 0.6 mM to represent
the normal free [Mg2>], SR activity in the stunned
hearts was less than controls over the entire range of
free [Ca>]. This reduction ranged from 25 to 40% of
control between 0.18 and 1.0 ,uM free [Ca>'], which
was greater than observed at 15 itM free [Ca>].
More striking was the effect that an increase in the
free [Mg2>] would have on the function of the SR.
Although under maximal activation conditions (3.2
and 10 ,uM free [Ca>2]) there was no change in
activity, over the physiological range of free [Ca>]
Krause et al Myocardial Stunning and Sarcoplasmic Reticulum Function
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FIGURE 2. Plot shows effect of an increase in free
on Ca2' sensitivity of sarcoplasmic reticulum (SR) Ca' transport rates for SR isolated from
control and stunned hearts. Rates of Ca2+ transport
for control SR were assessed at 0.6 mM free Mg2+.
Rates of Ca21 transport for stunned SR were assessed at 0.6 mM and 1.5 mMfree Mg2+ to duplicate
in vivo conditions of the stunned cardiac cell. Each
point represents the mean and each bar the standard
error. *p<0.05, control vs. stunned; `p <0.02,
stunned 0.6 mM vs. 1.5 mMfree [Mg2+J.
[Mg2`]
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there was a shift in Ca2' sensitivity. With an increase
in the free [Mg2+] to 1.5 mM, there was a 70%
reduction in activity at 0.32 (p<0.03) and 0.56 ,iM
(p<0.02) free [Ca2"] compared with controls. Under
these conditions, a greater free [Ca2"] would be
required to achieve the same level of Ca2' pump
activity. Thus, when measured at a free [Mg2+]
similar to that observed in the stunned myocardium
(1.5 mM), SR Ca21 transport is reduced to a greater
degree than observed when assessed at either normal
free [Mg2+] (0.6 mM) or only under maximally stimulating conditions.
It should be noted that Ca2+ transport in normal
SR is also affected by an increase in the free [Mg2+].
The EC50 at 0.6 mM Mg2+ was 0.43 ,uM free Ca2 ,
whereas an increase in free Mg2+ to 1.5 mM shifted
the EC50 to 0.61 ,uM free Ca21 (data not shown). The
maximal activation, however, was not altered.
Discussion
The purpose of this study was to determine
whether the observed elevation in free [Mg2+] reported to occur with myocardial stunning has an
effect on the function of the cardiac cell that could be
related to the decline in mechanical function of the
stunned heart. Although it has been speculated that
the increase in free [Mg2+] could substantially alter
subcellular function,8-11 studies in support of this
theory are limited. Our study is among the first to
assess the effect an increase in free [Mg2+] will have
on subcellular function in the stunned myocardium.
The results indicate that the twofold increase in free
[Mg2e] can markedly affect the activity of one of the
principal organelles responsible for the regulation of
intracellular Ca2+: the SR. An increase in free [Mg2e]
comparable with that observed with myocardial stunning (1.5 mM) would be expected to decrease the
Ca2+ sensitivity of the SR Ca2+ pump. As a result, a
higher free [Ca2+] would be required to achieve the
same level of activation. In the absence of a compensating increase in intracellular free [Ca2+], Ca2+
accumulation during the relaxation phase of the
cardiac cycle will be less, leading to an increase in
diastolic [Ca2+] and elevated diastolic tone. In addition, the amount of Ca2' available for release from
the SR will be reduced, thus limiting the level of
myofilament activation.
Alterations in diastolic function that may be attributable in part to a defect in the SR are not limited to
models of global stunning. In a regional model of
stunning, Charlat et a120 observed that diastolic function was severely impaired and did not recover for 24
hours. Both the mean half-rate of end-diastolic thinning during early diastole and late diastole were
reduced, indicating a slower rate of LV relaxation
and an increase in diastolic tone. As a result, LV
filling would be reduced. Similar alterations in LV
relaxation rates have been reported in studies on
patients with coronary artery disease. de Bruyne et
a121 have shown a reduction in early LV diastolic
filling after balloon angioplasty. A reduction in early
diastolic filling has been attributable to a delayed or
incomplete relaxation whose rate is dependent primarily upon Ca2' removal from troponin C by the
SR.22,23 A persistent increase in diastolic thickness,
indicating incomplete relaxation, has also been
shown in patients with exercise-induced ischemia24
and after angioplasty.25 These diastolic defects may
be directly related in part to a decrease in the ability
of the SR to remove Ca2' at a rate sufficient to bring
about complete relaxation.
The effect of stunning on the function of the SR in
vivo may not be as severe as the in vitro studies
indicate. There is evidence to suggest that a partial
compensation may occur by an increase in intracellular [Ca>]. Although the mean cellular free [Ca2]
remains normal,26 Kusuoka et a17 observed an increase in the peak transient free [Ca>'] after reperfusion. Despite this increase, contractile function
remained depressed. This implies the increase in the
peak transient free [Ca2] may only partially reverse
the Mg2+-induced shift in Ca2+ sensitivity.
The results from our study are in line with other
studies that conclude that either the handling or
regulation of intracellular Ca>2 is a primary defect in
the stunned heart. Ito et a14 observed that stunned
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hearts could respond with normal contractile activity
if they were subjected to increased extracellular Ca>
infusion. Similar potentiation of contractile function
can be achieved with the use of positive inotropic
agents27,28 that increase intracellular [Ca>2]. Both of
these mechanisms could overcome a decrease in SR
Ca>2 loading, thus allowing a return to normal function. These studies suggest that stunning either involves a decrease in the level of intracellular free
Ca>2 available to the myofilaments (conclusions supported by our study) or a shift in the Ca>2 sensitivity
of the contractile proteins.7 This latter possibility will
require further study.
A decrease in the function of the SR with myocardial stunning is not a new observation. Previous
studies by Krause et a16 and Limbruno et a13 have
demonstrated that myocardial stunning is associated
with a decrease in the function of the SR. The
current study expands these observations in the isolated rabbit heart by showing a reduction in SR Ca>2
transport over the entire range of free [Ca>2] expected during the cardiac cycle. When this decline in
function is combined with the significant shift in the
Ca> sensitivity of the Ca' pump caused by the
increase in free [Mg2>], the result is a potentiation of
the loss in Ca>2 transport not taken into account
previously.
With a decline in SR Ca>2 loading, the amount of
Ca>2 available for release will be lower and will lead to
a decrease in contractile protein activation. This may
be the principal underlying mechanism relating to the
reduction in systolic function in the stunned heart.2,4,6
As a result, a portion of the activating Ca> for the
myofilaments may have to come from other sources
such as the trans-sarcolemmal flux. Recent studies by
Lewartowski et a129 have shown that the trans-sarcolemmal Ca>2 flux can activate the myofilaments
when SR Ca>2 fluxes are reduced. However, myofilament activation is less. These observations, along with
a probable decrease in myofilament Ca>2 sensitivity as
a result of an increase in free [Mg2+],13 may explain
why tension development is reduced in the stunned
myocardium whereas peak [Ca>2] reached during the
cardiac cycle appears to be elevated.7
As a result of the prominent role of Ca> in
reperfusion injury, attention to alterations in other
intracellular ion concentrations has not been as
actively pursued. It has been assumed that the ionic
concentration of viable postischemic cells was similar
to the normal cell. This is especially true for Mg2>.
Restricted by inaccurate means of measuring intracellular free [Mg2>] and uncertainty whether such
levels changed under pathological conditions, cytosolic free Mg2> was merely seen as a metabolic
cofactor. Previous measurements of free [Mg2>]
ranged from 2.4 to 3.5 mM,3031 indicating a limited
role for regulation of cellular function. Current refinements in measuring free [Mg2>] have provided
estimates between 0.4 and 0.8 mM.8,9,32 At this
concentration, Mg2+ can now be considered a regulatory ion.
With the ability to resolve free [Mg>] directly in
the beating heart,89 investigators now have the ability
to follow changes in free [Mg>] during ischemia and
reperfusion. Murphy et al,8 using a fluorinated Mg>
indicator 4F-APTRA, found that the free [Mg>]
increased from 0.6 mM to 2.1 mM after 15 minutes of
global ischemia. Similar increases have also been
observed by other investigators during 932 and 159
minutes of ischemia using 31P-NMR. After reperfusion, Murphy et a18 observed that the free [Mg2>] did
not return to control levels but remained elevated at
1.5 mM. Similar twofold increases in free [Mg>]
were also observed by Kirkels et a19; however, 9
minutes of ischemia was not associated with a sustained increase with reperfusion.32 It should be noted
that 9 minutes of ischemia would also not be expected to result in myocardial stunning.
For our studies, a free [Mg2>] of 1.5 mM was used,
based on the assumption that similar changes in free
[Mg2>] occur in the rabbit as in the rat heart.8
Unfortunately, studies on free [Mg2>] alterations
with ischemia have not yet been performed in other
species. However, one would expect a similar increase to occur since the source of the free Mg2>
appears to be the decrease in the adenine nucleotide
pool.8 Because the ATP levels after myocardial stunning are reduced to a similar level in the rat8 and
rabbit,28 one would expect that the free [Mg2>] would
increase in a proportional manner because the dissociation constants for Mg2> and the adenine nucleotides are not species specific.
To fully understand the contribution of Mg2> to
the process of myocardial stunning, more studies will
be required in other species. It will also be necessary
to follow the time course of the normalization of free
[Mg2>]. Current studies have only followed free Mg2>
changes for 30 minutes of reperfusion.8,9 It would be
interesting to see whether the return to normal free
[Mg2>] would follow a similar time course as the
repletion of the adenine nucleotide pool and normalization of rates of relaxation.20
We have shown that the increase in free [Mg2>]
that occurs after reperfusion of the stunned heart can
potentiate the inherent defect in SR function, leading to an accentuated depression in the ability to
regulate intracellular [Ca>2]. This impairment in
Ca>2 regulatory function may contribute to the elevated diastolic tone and reduced systolic function
characteristic of the stunned myocardium.
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KEY WORDS * reperfusion injury * myocardial ischemia
calcium regulation * Brief Rapid Communications
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Effects of an increase in intracellular free [Mg2+] after myocardial stunning on
sarcoplasmic reticulum Ca2+ transport.
S M Krause and D Rozanski
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Circulation. 1991;84:1378-1383
doi: 10.1161/01.CIR.84.3.1378
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