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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 17, 2001 as doi:10.1096/fj.00-0870fje. |
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Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada
2Correspondence: Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Taché Ave., Winnipeg, Manitoba, Canada R2H 2A6. E-mail: cvso{at}sbrc.umanitoba.ca
SPECIFIC AIMS
We tested the hypothesis that hypoxia and/or glucose deprivation that occur during the ischemic phase and intracellular Ca2+ overload that occurs during the reperfusion phase and reoxygenation contribute to alterations in the sarcoplasmic reticulum (SR) gene expression of Ca2+ cycling proteins due to ischemia-reperfusion (IR). Isolated rat hearts deprived of oxygen and/or glucose with and without reperfusion with normal perfusion medium and hearts subjected to Ca2+ paradox (CP) upon Ca2+ depletion/repletion were used to study changes in SR gene expression.
PRINCIPAL FINDINGS
1. Hypoxia and/or glucose deprivation increased mRNA levels of the cardiac SR genes for SR proteins such as ryanodine receptor (RyR), Ca2+ pump ATPase (SERCA2a), phospholamban (PLB), and calsequestrin (CQS)
Hearts exposed to 30 min of hypoxia showed an increase in mRNA levels for RyR (by 12%) and SERCA2a (by 15%), whereas glucose lack for 30 min caused an increase in the transcript levels of all genes studied (RyR by 34%, SERCA2a by 42%, PLB by 28%, and CQS by 19%) (Fig. 1
A, B). A combination of hypoxia and glucose lack for 30 min enhanced expression levels for RyR, SERCA2a, and PLB by 11%, 24%, and 22%, respectively, without any change in CQS mRNA levels.
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2. Perfusion of the hypoxic or glucose-deprived hearts with control medium normalized the SR gene expression whereas perfusing hypoxic and glucose-deprived hearts with control medium decreased SR gene expression
When the hypoxic or glucose-deprived hearts were reperfused for 60 min with normal medium, the SR genes were expressed at the control level (Fig. 1A, B
). On the other hand, hearts deprived of both glucose and oxygen and reperfused with normal perfusion medium showed a significant decrease in the mRNA levels for RyR (by 49%), SERCA2a (by 35%), PLB (by 31%), and CQS (by 29%). These changes in SR gene expression were time dependent, as 10 min of deprivation of oxygen and glucose had no effect on the expression of SR genes but longer periods (20 to 60 min) of hypoxia and glucose lack were accompanied by a progressive decrease in the steady-state levels of RyR mRNA (by 30–78%), SERCA2a mRNA (by 11–50%), PLB mRNA (by 25–54%), and CQS mRNA (by 38–49%).
3. Intracellular Ca2+ overload may be a possible mechanism mediating changes in SR gene expression due to IR or hypoxia-reoxygenation
In view of the occurrence of intracellular Ca2+ overload in IR hearts, we used the CP hearts to study the effect of intracellular Ca2+ overload on SR gene expression. Ca2+ depletion for 5 min had no effect on the SR gene levels whereas Ca2+ repletion in Ca2+-depleted hearts decreased the RyR, SERCA2a, PLB, and CQS mRNA by 65, 85, 60, and 75%, respectively (Fig. 2
A, B).
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CONCLUSIONS AND SIGNIFICANCE
In this study, we have shown that the change induced by hypoxia and/or glucose deprivation per se has no deleterious effect on mRNA levels of SR proteins in the ischemic heart (Fig. 1)
. On the contrary, these factors were found to enhance mRNA levels; such a change may represent an attempt to rescue the heart against these stresses. These findings also suggest that SR genes may be sensitive to hypoxic and glucose-dependent regulatory elements or be up-regulated by other transcription factors that are activated by hypoxia and/or glucose deprivation. Since our data represent the steady-state levels of the SR genes, the enhanced expression can be attributed to either an increased rate of transcription and/or increased mRNA stability. Nevertheless, alterations in mRNA levels in the stunned myocardium as well as during differentiation and cardiac myogenesis have been implied to be due primarily to changes in the transcription rate. It can be argued that the depressed SR gene expression in the ischemic heart may be attributed to factors such as acidosis or accumulation of metabolites; however, these two factors may not play any role in the changes observed in this experimental model because the accumulation of metabolic end products and associated acidosis were prevented by maintaining the coronary flow during the hypoxic and glucose-free perfusion periods. In view of the important role of ATP in the maintenance of protein synthesis and Ca2+ gradient as well as protection against oxidative stress conditions in the heart, extensive work is necessary to determine the energy status of the heart under different conditions and its effect on the alterations in SR gene expression.
Perfusion of the hypoxic or glucose-deprived hearts with normal oxygenated medium indicates that the enhanced transcript levels of SR genes observed during the hypoxic or glucose deprivation phases were reversible (Fig. 1)
. When hearts were perfused after exposure to both hypoxia and glucose deprivation, there was a significant decrease in the expression of SR genes. In view of similar changes in SR gene expression observed in the IR hearts, this observation indicates that glucose and oxygen lack during the ischemic phase are both important for reperfusion-induced injury to occur. This hypothesis was confirmed by the time course study, where a progressive decrease in the expression of SR genes was observed when prolonging the perfusion period in the absence of both oxygen and glucose. These results are consistent with the view that there is a correlation between changes in the levels of SR gene expression and the severity of cardiac injury.
The depressed mRNA levels of SR genes in IR hearts can be explained by two mechanisms: the occurrence of intracellular Ca2+ overload and the development of oxidative stress. Ca2+ overload has been reported to occur in IR and hypoxia-reoxygenated hearts (deprived of glucose). Our study shows that Ca2+ depletion has no effect on the mRNA levels whereas Ca2+ repletion, which is known to cause a massive increase in intracellular Ca2+ concentration, induces a dramatic decrease in the expression of RyR, SERCA2a, PLB, and CQS (Fig. 2)
. In a previous study, we showed that treating hearts with superoxide dismutase and catalase (a system known to scavenge oxyradicals) protected the changes in SR gene expression due to IR. Furthermore, hearts perfused with H2O2 or xanthine + xanthine oxidase (a source of oxyradical generation) showed changes in SR gene expression similar to those observed in the IR hearts. Since isolated adult cardiomyocytes exposed to hypoxia and glucose deprivation were reported to show significant increase in intracellular Ca2+ content and a marked elevation in reactive oxygen species levels only after reoxygenation, it is possible that Ca2+ overload and oxidative stress may both be involved in producing depression in the SR gene expression. However, it is difficult to determine the cause-effect relationship between intracellular Ca2+ overload and oxidative stress or their direct modulatory effect on SR gene expression in the IR hearts.
The data presented in this study indicate that the lack of both oxygen and glucose may modulate the genetic machinery for the occurrence of reperfusion-induced injury (Fig. 3
). The alterations observed in SR gene expression in hearts subjected to both hypoxia and glucose lack may be a consequence of Ca2+ overload and/or oxidative stress that may occur during the reperfusion phase. Changes in SR gene expression observed within 30 min of exposure of the heart to both hypoxia and glucose lack as well as during the 60 min of reperfusion phase of the IR hearts may not be sufficient to cause changes in SR proteins. Thus, changes in SR function and subsequent cardiac performance under acute conditions of IR may not be due to alterations in SR gene expression. On the other hand, the depression in IR-induced SR gene expression may result in delayed recovery of SR function, which may explain the chronic effects of IR.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0870fje; to cite this article, use FASEB J. (September 17, 2001) 10.1096/fj.00-0870fje 
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