Lack of both oxygen and glucose contributes to I/R-induced
changes in cardiac SR function
Rana M.
Temsah,
Thomas
Netticadan,
Ken-Ichi
Kawabata, and
Naranjan S.
Dhalla
Institute of Cardiovascular Sciences, St. Boniface General Hospital
Research Centre, and Department of Physiology, Faculty of Medicine,
University of Manitoba, Winnipeg, Manitoba R2H 2A6, Canada
 |
ABSTRACT |
Although ischemia-reperfusion
(I/R) has been shown to depress cardiac performance and sarcoplasmic
reticulum (SR) function, the mechanisms underlying these alterations
are poorly understood. Because lack of oxygen and substrate deprivation
are known to occur during the ischemic phase, we examined the
effects of reperfusion on cardiac performance and SR function in hearts
subjected to hypoxia and substrate lack. For this purpose, isolated rat
hearts were perfused with hypoxic and/or glucose-free medium for 30 min and then reperfused with normal medium for 1 h; the SR vesicles were isolated for studying the Ca2+-transport activities.
Reperfusion with normal medium of hearts deprived of oxygen or glucose
showed no changes in cardiac performance and SR function. However,
reperfusion of hearts perfused with hypoxic glucose-free medium showed
~45% decrease in cardiac contractile activities as well as 23 and
64% reduction in SR Ca2+-uptake and
Ca2+-release activities, respectively, without any change
in the level of SR Ca2+-cycling proteins. Depressed SR
function in these hearts was associated with a reduction in
Ca2+/calmodulin-dependent protein kinase (CaMK)
phosphorylation of the SR Ca2+-cycling proteins and 34%
decrease in SR CaMK activity. These changes in cardiac performance, SR
function, and SR CaMK activity in the hypoxic, glucose-deprived,
reperfused hearts were similar to those observed in hearts subjected to
30 min of global ischemia and 60 min of reperfusion. The
results therefore suggest that the lack of both oxygen and substrate
during the ischemic phase may contribute to the I/R-induced
alterations in cardiac performance and SR function. Furthermore, these
abnormalities were associated with reduced SR CaMK activity.
sarcoplasmic reticulum; hypoxia-reoxygenation; ischemia-reperfusion; calcium/calmodulin-dependent protein kinase
 |
INTRODUCTION |
ISCHEMIA-REPERFUSION
(I/R) injury is known to occur during clinical procedures such as
coronary bypass surgery, angioplasty, thrombolytic therapy, and cardiac
transplantation (2, 4). Earlier it has been shown that I/R
caused abnormalities in cardiac performance and sarcoplasmic reticulum
(SR) function as well as defects in the phosphorylation of the SR
proteins by Ca2+/calmodulin-dependent protein kinase (CaMK)
and cAMP-dependent protein kinase (PKA) (19, 22, 28, 29).
However, it is not clear whether these changes occur due to alterations
induced in the ischemic phase or reperfusion phase. Some
studies have indicated that oxidative stress (29) and
activation of the
-adrenergic system (28), which are
known to occur during the reperfusion phase, may be partially
responsible for the occurrence of cardiac and SR changes in the I/R
hearts. On the other hand, no such information regarding the influence
of ischemic phase on the I/R-induced changes in the heart is
available in the literature.
Under conditions of coronary occlusion, there is a limited supply of
oxygen and exogenous substrate to the myocardium, which leads to
ischemia and subsequent infarction (10). Although
hypoxia and substrate depletion have been shown to depress SR
Ca2+ transport and heart function (5, 13, 17),
the contribution of these factors in the I/R-induced changes in the
heart has not been demonstrated in any experimental model. This study
was therefore undertaken to examine if reperfusion for 60 min with
normal medium exerts similar effects in isolated rat hearts subjected
to 30 min of hypoxia, substrate lack, or global ischemia. The
data of this study indicate that a combination of both hypoxia and
glucose deprivation, which occur during the ischemic phase, may
be one of the underlying mechanisms responsible for the observed
changes in the I/R hearts.
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MATERIALS AND METHODS |
Heart perfusion and experimental protocol.
Male Sprague-Dawley rats weighing 250-350 g were anesthetized with
a mixture of ketamine (60 mg/kg) and xylazine (10 mg/kg). The hearts
were rapidly excised, cannulated to the Langendorff apparatus, and
perfused with Krebs-Henseleit (KH) medium (37°C), gassed with a
mixture of 95% O2-5% CO2, pH 7.4, containing
(in mM) 120 NaCl, 25 NaHCO3, 11 glucose, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, and 1.25 CaCl2. The hearts were electrically stimulated at a rate of
300 beats/min (Phipps and Bird, Richmond, VA), and the perfusion rate
was maintained at 10 ml/min. A water-filled latex balloon was inserted
in the left ventricle (LV) and connected to a pressure transducer
(model 1050BP; Biopac System, Goleta, CA) for the LV developed pressure
(LVDP); the LV end-diastolic pressure (LVEDP) was adjusted at 10 mmHg.
The LV pressure was differentiated to estimate the rate of ventricular
pressure development (+dP/dt) and the rate of ventricular
pressure decline (
dP/dt) using the Acknowledge 3.03 software for Windows (Biopac System). To study the effects of hypoxia
and glucose deprivation/reperfusion, one set of hearts were stabilized
for a period of 30 min before use and was randomly distributed into
several groups: 1) control hearts, perfused for 90 min with
normal KH medium gassed with 95% O2-5% CO2,
pH 7.4; 2) hypoxic-reperfused hearts, perfused for 30 min
with KH medium gassed with 95% N2-5% CO2, pH
7.4, followed by 60 min of perfusion with normal KH medium;
3) glucose-deprived/reperfused hearts, perfused for 30 min
with glucose-free KH medium gassed with 95% O2-5%
CO2, where glucose was replaced with Tris · HCl to
maintain osmolarity, pH 7.4, followed by 60 min of perfusion with
normal KH medium; and 4) hypoxic and
glucose-deprived/reperfused hearts, perfused for 30 min with
glucose-free KH medium gassed with 95% N2-5%
CO2, pH 7.4, followed by 60 min of perfusion with normal KH
medium. The perfusion procedures have been adapted from previous
studies (6, 27). Under the same experimental conditions, another set of hearts was subjected to global I/R; all hearts were
stabilized for a period of 30 min before use and were randomly distributed into two groups 1) control hearts, perfused for
90 min with normal KH medium gassed with 95% O2-5%
CO2, pH 7.4; and 2) I/R hearts, which underwent
30 min of global ischemia followed by 60 min of perfusion with
normal KH medium. The experimental protocol for each group is given in
Fig. 1. It is pointed out that separate
controls were used for both sets (hypoxia and/or glucose
deprivation/reperfusion set and the I/R set).

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Fig. 1.
Schematic representation of the experimental design for the hypoxia
and/or glucose deprivation-reperfusion model (A) and
ischemia-reperfusion model (B). See materials and
methods for details. , Time of sampling.
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Isolation of SR vesicles and measurement of
Ca2+-uptake and -release activities.
SR membranes were prepared by a method used previously
(29). Ca2+-uptake activity of the SR vesicles
was determined by the procedure of Netticadan et al. (18),
whereas Ca2+-release activity of the SR vesicles was
measured by the procedure used by Temsah et al. (29). The
Ca2+-induced Ca2+ release was completely
prevented (95-97%) by the treatment of SR preparations with 20 µM ryanodine.
Measurement of protein phosphorylation by CaMK and PKA.
For the phosphorylation experiments, the SR preparation was isolated in
the presence of a phosphatase inhibitor to prevent any
dephosphorylation occurring during the isolation procedure. Both the
homogenization buffer and the phosphorylation assay medium contained 1 mM sodium pyrophosphate. SR protein phosphorylation by CaMK was
determined by the procedure described by Netticadan et al.
(19). The initial concentration of free Ca2+
determined by the computer program of Fabiato (7) was 3.7 µM. The assay for phosphorylation by PKA was carried out according to
the procedure described elsewhere (19, 22). The intensity of each phosphorylated band was scanned by an Imaging Densitometer with
the aid of Molecular Analyst software version 1.3 (Bio-Rad, Hercules, CA).
Measurement of CaMK activity.
The SR CaMK activity was measured by using Upstate Biotechnology assay
kits as described earlier (18, 19). For the measurement of
CaMK activity, the SR fraction was isolated in the presence of a
phosphatase inhibitor. The assay kit for CaMK activity is based on the
phosphorylation of a specific substrate peptide (KKALRRQETVDAL) by the
transfer of
-phosphate of [
-32P]ATP by CaMK II.
Western blot analysis.
Protein contents of the SR proteins ryanodine receptor (RyR),
sarcoendoplasmic reticulum Ca2+-pump ATPase
(SERCA2a), phospholamban (PLB), and
-CaMK were determined according to the method described earlier (29). Protein
samples (20-25 µg of total protein/lane) were separated by
SDS-PAGE on 5% (for RyR), 10% (for SERCA2a), 12% (for
-CaMK), and
15% (for PLB) gels, transferred to polyvinylidene difluoride
membranes, probed with appropriate antibodies, and detected by the
enhanced chemiluminesence (ECL) kit (Amersham Life Science, Oakville,
ON, Canada).
Statistical analysis.
Results are expressed as means ± SE and statistically evaluated
by one-way ANOVA test followed by Student's unpaired t-test for multiple comparisons. A level of P < 0.05 was
considered the threshold for statistical significance between the
control and experimental groups.
 |
RESULTS |
Hypoxic and/or glucose-deprived/reperfused hearts.
Hypoxic or glucose-deprived hearts showed a significant decrease in
LVDP, +dP/dt, and
dP/dt and a marked increase
in LVEDP (Table 1). On reperfusion for 60 min with normal perfusion medium, hypoxic hearts recovered completely,
whereas the glucose-deprived hearts showed a near complete recovery.
Hearts perfused with hypoxic glucose-free medium for 30 min exhibited a
further decrease (in comparison to hypoxic or glucose-deprived hearts)
in LVDP, +dP/dt, and
dP/dt, as well as a
further elevation in LVEDP. These alterations persisted after 60 min of
perfusion with normal medium although there was a tendency toward
recovery. Hypoxia or glucose deprivation followed by reperfusion had no
effect on the SR function, whereas hypoxic, glucose-deprived,
reperfused hearts showed a significant decrease in the
Ca2+-uptake and -release activities compared with controls
(Table 1). Because the objective of the study was to examine the role of hypoxia and glucose deprivation in the I/R-induced changes, the
samples were not collected after the deprivation period, and therefore
the SR Ca2+-uptake and -release activities were not
determined at the end of that phase. Furthermore, the presence of
myocardial necrosis in this model was not examined.
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Table 1.
Effect of reperfusion of hypoxic and/or glucose-deprived rat hearts on
cardiac performance and SR function
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Because protein phosphorylation plays an important role in regulating
SR function, the in vitro CaMK- and PKA-mediated phosphorylations of
the SR Ca2+-cycling proteins were examined. Figure
2 shows that CaMK phosphorylation of RyR,
SERCA2a, and PLB, unlike that observed in the hypoxic or
glucose-deprived hearts, was significantly decreased in the hypoxic,
glucose-deprived, reperfused hearts compared with controls. On the
other hand, the PKA phosphorylation of RyR or PLB was not different
among all experimental groups (Fig. 3).
Because reduced CaMK phosphorylation of SR proteins may be due to
alterations in the kinase activity, the SR CaMK activity was assessed.
To examine whether the changes in CaMK activity were restricted to the
SR, the cytosolic CaMK activity was also measured. Table
2 shows that the endogenous SR CaMK
activity was significantly depressed in the hypoxic,
glucose-deprived, reperfused hearts but unaffected in the other groups
compared with the control.

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Fig. 2.
Ca2+/calmodulin (CM)-dependent protein kinase (CaMK)
phosphorylation of ryanodine receptor (RyR), sarcoendoplasmic reticulum
Ca2+-pump ATPase (SERCA2a), high-molecular-weight
phospholamban [PLB-(H)], and low-molecular-weight PLB [PLB-(L)]:
autoradiogram (A) and analysis of data (B) on
phosphorylation in control (C), hypoxia-reperfusion (H/R), glucose
deprivation-reperfusion (G/R), and hypoxia-glucose
deprivation-reperfusion (H/G/R) groups. PLB phosphorylation was the sum
of PLB-(H) and PLB-(L) phosphorylation. Controls of each set
(containing 4 groups: C, H/R, G/R, and H/G/R) were considered as 100%.
When other groups were compared with controls, the difference was
calculated as % of control. The results are means ± SE of 4 hearts/group. * P < 0.05 vs. control.
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Fig. 3.
cAMP-dependent protein kinase (PKA) phosphorylation of PLB-(H) and
PLB-(L): autoradiogram (A) and analysis of data
(B) on phosphorylation in C, H/R, G/R, and H/G/R groups. PLB
phosphorylation was the sum of PLB-(H) and PLB-(L) phosphorylation.
Controls of each set (containing 4 groups: C, H/R, G/R, and H/G/R) were
considered as 100%. When other groups were compared with controls, the
difference was calculated as % of control. The results are means ± SE of 4 hearts/group.
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Alterations in the CaMK-mediated phosphorylation of RyR, SERCA2a, and
PLB may also be due to changes in the protein content of these
substrates. Figure 4 shows that the
levels of the SR proteins were not different among all experimental
groups except that there was a significant increase in the SERCA2a
protein content in the glucose-deprived hearts. It should be noted that
the protein levels of RyR and
-CaMK also appeared to increase in
glucose-deprived hearts (Fig. 4); however, this increase was not
statistically significant. Because the reduction in CaMK-mediated
protein phosphorylation and SR CaMK activity may be due to a decrease
in its protein content, we examined the levels of SR
-CaMK in all
groups. Figure 4 shows a significant reduction in the
-CaMK protein
content in hypoxic, glucose-deprived, reperfused hearts compared with
controls.

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Fig. 4.
Autoradiograms depicting RyR, SERCA2a, PLB, and -CaMK
protein bands (A) and analysis of data (B) in C,
H/R, G/R, and H/G/R groups. Controls of each set (containing 4 groups:
C, H/R, G/R, and H/G/R) were considered as 100%. When other groups
were compared with controls, the difference was calculated as % of
control. The results are means ± SE of 4 hearts/group.
* P < 0.05 vs. control.
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I/R hearts.
For the purpose of comparison, the cardiac function as well as SR
Ca2+-uptake and -release activities were assessed in the
control and I/R groups. A marked depression in LVDP, +dP/dt,
and
dP/dt, as well as an increase in LVEDP, was observed
in hearts subjected to 30 min of global ischemia followed by 60 min of reperfusion (Table 3). The SR
Ca2+-uptake and Ca2+-release activities were
also depressed in these I/R hearts compared with controls (Table 3).
The SR CaMK activity was significantly reduced in the I/R group
compared with control hearts (Table 3).
 |
DISCUSSION |
Ischemic injury has been attributed to several factors
such as depressed levels of ATP, acidosis, alterations in the osmotic control, membrane disruption, glucose-deprivation, and hypoxia (4, 9, 24). In an attempt to understand the contribution of ischemic factors in I/R injury, we investigated the effect of hypoxia and glucose deprivation on cardiac performance, SR function,
and its regulation by CaMK and PKA phosphorylation. Previous reports
(5, 13) have indicated depressions in cardiac performance
and SR Ca2+ uptake in hearts deprived of oxygen and/or
glucose. The omission of glucose from the hypoxic medium was also
reported to accelerate the deleterious effect of hypoxia on cardiac
performance and SR Ca2+ uptake (13). These
defects were attributed to insufficiency in energy generation
(5), taking into consideration that no changes in membrane
lipid composition were observed in the hypoxic and substrate-deprived
hearts. In the present study reperfusion with normal perfusion medium
after hypoxia or glucose deprivation resulted in a complete or partial
functional recovery, respectively, as well as a complete recovery of SR
Ca2+-transport activities. On the other hand, a combination
of hypoxia and glucose deprivation was essential to induce
abnormalities similar to those observed in I/R hearts, i.e., cardiac
dysfunction as well as depressed SR Ca2+-uptake and
-release activities (28, 29). It can be argued that the
defects observed in these reperfused hearts may be attributed to other
factors such as acidosis or alterations in osmotic control. However, it
is important to mention that this may not be applicable under our
experimental conditions because the accumulation of metabolic end
products and associated acidosis (25) were prevented by
maintaining the coronary flow during the hypoxic and/or
glucose-deprivation period. Nonetheless, further studies are required
to rule out the effect of these factors as well as those of alterations
in ATP generation and utilization during the ischemic phase.
There is increasing evidence to suggest a major role of CaMK- and
PKA-mediated protein phosphorylation in regulating SR function (1, 3, 8, 12). Phosphorylation of PLB by CaMK and PKA has
been reported to diminish the inhibitory effect of PLB on SERCA2a and
increase the affinity of SERCA2a for Ca2+ as well as
Vmax for Ca2+ transport (1,
11, 12, 26). Although phosphorylation of SERCA2a by CaMK and its
functional consequences were questioned (21, 23), Xu and
colleagues (32) have shown that SERCA2a is a substrate for
the endogenous CaMK in isolated SR membranes. Furthermore, serine
phosphorylation of SERCA2a has been demonstrated in the intact beating
heart (34). The CaMK phosphorylation site on SERCA2a has
also been identified (30). In addition, Xu and Narayanan
(33) have shown an increase in Vmax
of Ca2+ uptake on selective phosphorylation of SERCA2a in
the absence of PLB phosphorylation. Earlier data from our laboratory
(18, 19, 22) support the fundamental observations of Xu
and colleagues (8, 32, 33, 34). SR
Ca2+-release is also subjected to regulation by CaMK and
PKA phosphorylation of the RyR. PKA phosphorylation of RyR has been
reported to promote SR Ca2+ release (16).
Although there have been conflicting results from bilayer measurements
examining the functional consequences of RyR phosphorylation by CaMK
(15, 31), KN-93, a specific inhibitor of CaMK, was
reported to inhibit SR Ca2+ release in intact
cardiomyocytes, suggesting a positive role for CaMK (14).
Ryanodine (low µM), a specific activator of SR Ca2+
release, and ruthenium red, a specific blocker of SR Ca2+
release, caused activation and inhibition, respectively, of CaMK phosphorylation of RyR in native cardiac SR vesicles, supporting a
positive role for this phosphorylation in SR Ca2+ release
(20). Thus it appears that CaMK phosphorylation promotes SR Ca2+ release.
The results from this study suggest that depressed SR
Ca2+-uptake and -release activities of the hypoxic,
substrate-deprived, reperfused hearts may be related to a reduction in
the in vitro CaMK phosphorylation of RyR, SERCA2a, and PLB. These
changes in CaMK-mediated phosphorylation may be of specific nature
because the in vitro PKA phosphorylation of SR Ca2+-cycling
proteins was unaffected in the hypoxic, substrate-deprived, reperfused
hearts. Because the contents of RyR, SERCA2a, and PLB were unaltered in
the hypoxic substrate deprived-reperfused hearts, the decrease in the
endogenous CaMK phosphorylation seems to be due to reduced SR CaMK
activity. Furthermore, this reduction in activity could be directly
linked to a significant decrease in the SR
-CaMK protein content.
To test whether I/R produces changes (under the same experimental
conditions) similar to those observed due to hypoxia plus glucose
deprivation-reperfusion, some salient characteristics exhibited by the
hypoxic, glucose-deprived, reperfused hearts were examined in the I/R
hearts. Our results showing depressed cardiac performance, SR function,
CaMK phosphorylation of SR Ca2+-cycling proteins, and CaMK
activity in I/R hearts are consistent with similar alterations in the
hypoxic, glucose-deprived, reperfused hearts. Furthermore, the
alterations observed in the I/R hearts in this study are similar to
those reported by us earlier (19, 22, 28, 29). Thus our
study is the first to demonstrate that a combination of hypoxia and
substrate deprivation, unlike hypoxia or substrate lack alone, is
required for the occurrence of I/R injury with respect to changes in
cardiac performance and SR function. Furthermore, we demonstrate a
depression in CaMK phosphorylation of the SR Ca2+-cycling
proteins and CaMK activity in hearts perfused with hypoxic, glucose-deprived medium. These data seem to indicate the role of the
ischemic phase and explain the underlying mechanisms involved in cardiac dysfunction under acute conditions of I/R injury and therefore advance our understanding of the ischemic heart disease.
 |
ACKNOWLEDGEMENTS |
The work reported in this study was supported by a grant from the
Canadian Institutes of Health Research (CIHR) Group in Experimental Cardiology. N. S. Dhalla holds the CIHR/Pharmaceutical Research and Development Chair in Cardiovascular Research supported by Merck
Frosst (Canada). R. M. Temsah was a predoctoral fellow of the
Heart and Stroke Foundation of Canada.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: N. S. Dhalla, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Ave., Winnipeg, MB R2H 2A6, Canada
(E-mail: cvso{at}sbrc.ca).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
June 20, 2002;10.1152/ajpcell.00138.2002
Received 26 March 2002; accepted in final form 11 June 2002.
 |
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