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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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. up-arrow , Time of sampling.

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 gamma -phosphate of [gamma -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 delta -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 delta -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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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

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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Table 2.   Effect of reperfusion on SR CaMK activity in the hypoxic and/or glucose-deprived rat hearts

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 delta -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 delta -CaMK in all groups. Figure 4 shows a significant reduction in the delta -CaMK protein content in hypoxic, glucose-deprived, reperfused hearts compared with controls.


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Fig. 4.   Autoradiograms depicting RyR, SERCA2a, PLB, and delta -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.

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).

                              
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Table 3.   Effect of I/R on cardiac performance, SR function, and SR CaMK activity in rat hearts


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 delta -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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 283(4):C1306-C1312
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