Investigation into the intracellular mechanisms whereby long-chain fatty acids protect the heart in ischaemia/reperfusion
Date
2005-03
Authors
Engelbrecht, Anna-Mart
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: Although there is evidence for a protective role of long-chain polyunsaturated
fatty acids (PUFAs) in cardiovascular disease, their mechanism of action as
well as their participation in intracellular signalling processes remain to be
elucidated. Therefore the aims of this study were twofold: (i) to characterize
the roles of the mitogen-activated protein kinases (MAPKs) and protein kinase
B (PKB/Akt) in ischaemia/reperfusion-induced apoptosis of neonatal
cardiomyocytes and (ii) to establish whether long-chain PUFAs protect the
heart via manipulation of these kinases.
Rat neonatal ventricular myocytes exposed to simulated ischaemia and
reperfusion (Sl/R) were used to characterize the role(s) of extracellular signalregulated
kinase (ERK), p38 and c-Jun NH2-terminal protein kinase (JNK), as
well as PKB/Akt in apoptosis. The effects of an omega-3 fatty acid
(eicosapentaenoic acid - EPA) and an omega-6 fatty acid (arachidonic acid -
ARA) on the response of neonatal rat cardiomyocytes to Sl/R with regard to
the above parameters were determined.
Exposure of the myocytes to SI (energy depletion induced by KCN and 2-
deoxy-D-glucose) reduced cell viability, as measured by the 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, and
stimulated apoptosis (increased caspase-3 activation and poly(ADP-ribose)
polymerase (PARP) cleavage). However, morphological evidence of
increased apoptosis (Hoechst 33342 staining) occurred only after reperfusion.
A rapid activation of p38 and PKB/Akt Ser473 occurred during SI, while
significant activation of ERK and JNK was observed during reperfusion only.
Myocytes pre-treated with SB203580, a p38-inhibitor, displayed a significant
increase in cell viability and attenuation of apoptosis during Sl/R, while
SP600125, a specific JNK inhibitor, significantly increased both caspase-3
activation and the apoptotic index. However, PD98059, an ERK inhibitor, was
without effect. Wortmannin, a PI3-kinase inhibitor, reduced PKB/Akt Thr308 but
not Ser473 phosphorylation during Sl/R and caused a significant increase in Although there is evidence for a protective role of long-chain polyunsaturated
fatty acids (PUFAs) in cardiovascular disease, their mechanism of action as
well as their participation in intracellular signalling processes remain to be
elucidated. Therefore the aims of this study were twofold: (i) to characterize
the roles of the mitogen-activated protein kinases (MAPKs) and protein kinase
B (PKB/Akt) in ischaemia/reperfusion-induced apoptosis of neonatal
cardiomyocytes and (ii) to establish whether long-chain PUFAs protect the
heart via manipulation of these kinases.
Rat neonatal ventricular myocytes exposed to simulated ischaemia and
reperfusion (Sl/R) were used to characterize the role(s) of extracellular signalregulated
kinase (ERK), p38 and c-Jun NH2-terminal protein kinase (JNK), as
well as PKB/Akt in apoptosis. The effects of an omega-3 fatty acid
(eicosapentaenoic acid - EPA) and an omega-6 fatty acid (arachidonic acid -
ARA) on the response of neonatal rat cardiomyocytes to Sl/R with regard to
the above parameters were determined.
Exposure of the myocytes to SI (energy depletion induced by KCN and 2-
deoxy-D-glucose) reduced cell viability, as measured by the 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, and
stimulated apoptosis (increased caspase-3 activation and poly(ADP-ribose)
polymerase (PARP) cleavage). However, morphological evidence of
increased apoptosis (Hoechst 33342 staining) occurred only after reperfusion.
A rapid activation of p38 and PKB/Akt Ser473 occurred during SI, while
significant activation of ERK and JNK was observed during reperfusion only.
Myocytes pre-treated with SB203580, a p38-inhibitor, displayed a significant
increase in cell viability and attenuation of apoptosis during Sl/R, while
SP600125, a specific JNK inhibitor, significantly increased both caspase-3
activation and the apoptotic index. However, PD98059, an ERK inhibitor, was
without effect. Wortmannin, a PI3-kinase inhibitor, reduced PKB/Akt Thr308 but
not Ser473 phosphorylation during Sl/R and caused a significant increase in Although there is evidence for a protective role of long-chain polyunsaturated
fatty acids (PUFAs) in cardiovascular disease, their mechanism of action as
well as their participation in intracellular signalling processes remain to be
elucidated. Therefore the aims of this study were twofold: (i) to characterize
the roles of the mitogen-activated protein kinases (MAPKs) and protein kinase
B (PKB/Akt) in ischaemia/reperfusion-induced apoptosis of neonatal
cardiomyocytes and (ii) to establish whether long-chain PUFAs protect the
heart via manipulation of these kinases.
Rat neonatal ventricular myocytes exposed to simulated ischaemia and
reperfusion (Sl/R) were used to characterize the role(s) of extracellular signalregulated
kinase (ERK), p38 and c-Jun NH2-terminal protein kinase (JNK), as
well as PKB/Akt in apoptosis. The effects of an omega-3 fatty acid
(eicosapentaenoic acid - EPA) and an omega-6 fatty acid (arachidonic acid -
ARA) on the response of neonatal rat cardiomyocytes to Sl/R with regard to
the above parameters were determined.
Exposure of the myocytes to SI (energy depletion induced by KCN and 2-
deoxy-D-glucose) reduced cell viability, as measured by the 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, and
stimulated apoptosis (increased caspase-3 activation and poly(ADP-ribose)
polymerase (PARP) cleavage). However, morphological evidence of
increased apoptosis (Hoechst 33342 staining) occurred only after reperfusion.
A rapid activation of p38 and PKB/Akt Ser473 occurred during SI, while
significant activation of ERK and JNK was observed during reperfusion only.
Myocytes pre-treated with SB203580, a p38-inhibitor, displayed a significant
increase in cell viability and attenuation of apoptosis during Sl/R, while
SP600125, a specific JNK inhibitor, significantly increased both caspase-3
activation and the apoptotic index. However, PD98059, an ERK inhibitor, was
without effect. Wortmannin, a PI3-kinase inhibitor, reduced PKB/Akt Thr308 but
not Ser473 phosphorylation during Sl/R and caused a significant increase in PARP cleavage during reperfusion, but had no effect on caspase-3 activation
or the apoptotic index.
EPA and ARA (20 jiM, present before and after SI) significantly reduced
caspase-3 activation, PARP-cleavage and the apoptotic index during
reperfusion. This was associated with increased ERK- and decreased p38
phosphorylation. Vanadate (a tyrosine phosphatase inhibitor), but not okadaic
acid (a serine-threonine phosphatase inhibitor), significantly reduced ARAinduced
inhibition of p38 phosphorylation, suggesting involvement of tyrosine
phosphatases during Sl/R. MKP-1, a dual-specificity phosphatase, was
targeted and a significant induction of MKP-1 by ARA and EPA was observed.
An in vitro dephosphorylation assay confirmed that this phosphatase might be
responsible for the inhibition of p38 activation. It was also demonstrated that
the protective actions of ARA are PI3-K dependent.
The results suggest that p38 has a pro-apoptotic role while JNK
phosphorylation is protective and that these kinases act via caspase-3 to
prevent or promote cell survival in response to SI/R-induced injury. It was
demonstrated for the first time that EPA and ARA protect neonatal cardiac
myocytes from ischaemia/reperfusion-induced apoptosis through induction of
a dual-specific phosphatase, MKP-1, causing dephosphorylation of the proapoptotic
kinase, p38. These beneficial effects of ARA and EPA were also
reflected by improvement in functional recovery during ischaemia/reperfusion
of the isolated perfused rat heart model.
AFRIKAANSE OPSOMMING: Dit word algemeen aanvaar dat lang-ketting poli-onversadigde vetsure teen kardiovaskulere siektes beskerm, maar hul meganisme van aksie sowel as hul invloed op intrasellulere seinoordragpaaie is egter onbekend. Die doelwitte van hierdie studie is dus tweevoudig: (i) om die belang van mitogeen-geaktiveerde proteien kinases (MAPKs) en protein kinase B (PKB/Akt) in isgemie/herperfusie-geinduseerde apoptose vas te stel en (ii) om te bepaal of lang-ketting poli-onversadigde vetsure die hart, deur manipulering van hierdie kinases, beskerm. Rot neonatale ventrikulere miosiete, blootgestel aan gesimuleerde isgemie en herperfusie (Sl/H), is gebruik om die aktivering van ekstrasellulere seingereguleerde kinase (ERK), p38, c-Jun NH2-terminale protein kinase (JNK) asook PKB/Akt tydens apoptose, te karakteriseer. Die effek van ‘n omega-3 vetsuur (eikosapentaenoSsuur - EPA) en ‘n omega-6 vetsuur (aragidoonsuur - ARA) op die respons van bogenoemde kinases in neonatale kardiomiosiete tydens Sl/H, is ondersoek. Blootstelling van miosiete aan SI (energie-uitputting gemduseer deur kaliumsianied en 2-deoksi-D-glukose) het ‘n afname in die vermoe van die sel om te oorleef, soos gemeet deur die MTT (3-[4,5-dimetieltiazol-2-yl]-2,5- difeniel tetrazolium bromied) bepaling, tot gevolg gehad. ‘n Toename in apoptose (kaspase-3 aktivering en poli(ADP-ribose) polimerase (PARP) kliewing) is ook waargeneem. Morfologiese bewyse van apoptose (Hoechst 33342 kleuring) was egter eers tydens herperfusie sigbaar. SI is gekenmerk deur vinnige aktivering van p38 en PKB/Akt Ser473, terwyl ERK en JNK fosforilering slegs tydens herperfusie waargeneem is. Vooraf-behandeling met SB203580, ‘n p38 inhibitor, het ‘n beduidende toename in sellewensvatbaarheid asook ‘n afname in die apoptotiese indeks tydens Sl/H teweeggebring, terwyl SP600125, ‘n spesifieke JNK inhibitor, apoptose bevorder het. PD98059, ‘n ERK inhibitor, het geen invloed op apoptose tydens Sl/H gehad nie. Wortmannin, ‘n PI3-kinase inhibitor, het Thr308 (nie Ser473) fosforilering onderdruk, gepaargaande met ‘n toename in PARP kliewing, maar dit het geen invloed op kaspase-3 aktivering of die apoptotiese indeks gehad nie. EPA en ARA (20 (iM, teenwoordig voor en na SI) het kaspase-3 aktivering en PARP kliewing asook die apoptotiese indeks tydens herperfusie beduidend verminder. Beide vetsure het ook ‘n beduidende toename in ERK en afname in p38 fosforilering veroorsaak. Vanadaat (‘n serien-threonien fosfatase inhibitor), maar nie “okadaic” suur (‘n tirosien fosfatase inhibitor), kon die ARA-gemduseerde inhibisie van p38 ophef nie. Induksie van MKP-1, ‘n tweeledige-spesifieke fosfatase, is beduidend deur ARA en EPA tydens herperfusie verhoog. 'n In vitro defosforileringbepaling het bevestig dat hierdie fosfatase wel betrokke by die inhibisie van p38 kan wees. Daarbenewens is gevind dat die beskermende aksie van ARA PI3-K afhanklik is. Hierdie resultate wys dat fosforilering van p38 pro-apoptoties is, terwyl JNK beskermend is en dat hierdie kinases via kaspase-3 seldood of oorlewing tydens SI/H-geinduseerde beskadiging bemiddel. In hierdie model is daar vir die eerste keer getoon dat EPA en ARA neonatale kardiale miosiete teen isgemie/herperfusie-geinduseerde apoptose beskerm deur induksie van MKP- 1, wat defosforilering van die pro-apoptotiese kinase, p38 teweegbring. Hierdie voordelige effekte van EPA en ARA is ook sigbaar in die funksionele herstel tydens isgemie/herperfusie van die geisoleerde rothart model.
AFRIKAANSE OPSOMMING: Dit word algemeen aanvaar dat lang-ketting poli-onversadigde vetsure teen kardiovaskulere siektes beskerm, maar hul meganisme van aksie sowel as hul invloed op intrasellulere seinoordragpaaie is egter onbekend. Die doelwitte van hierdie studie is dus tweevoudig: (i) om die belang van mitogeen-geaktiveerde proteien kinases (MAPKs) en protein kinase B (PKB/Akt) in isgemie/herperfusie-geinduseerde apoptose vas te stel en (ii) om te bepaal of lang-ketting poli-onversadigde vetsure die hart, deur manipulering van hierdie kinases, beskerm. Rot neonatale ventrikulere miosiete, blootgestel aan gesimuleerde isgemie en herperfusie (Sl/H), is gebruik om die aktivering van ekstrasellulere seingereguleerde kinase (ERK), p38, c-Jun NH2-terminale protein kinase (JNK) asook PKB/Akt tydens apoptose, te karakteriseer. Die effek van ‘n omega-3 vetsuur (eikosapentaenoSsuur - EPA) en ‘n omega-6 vetsuur (aragidoonsuur - ARA) op die respons van bogenoemde kinases in neonatale kardiomiosiete tydens Sl/H, is ondersoek. Blootstelling van miosiete aan SI (energie-uitputting gemduseer deur kaliumsianied en 2-deoksi-D-glukose) het ‘n afname in die vermoe van die sel om te oorleef, soos gemeet deur die MTT (3-[4,5-dimetieltiazol-2-yl]-2,5- difeniel tetrazolium bromied) bepaling, tot gevolg gehad. ‘n Toename in apoptose (kaspase-3 aktivering en poli(ADP-ribose) polimerase (PARP) kliewing) is ook waargeneem. Morfologiese bewyse van apoptose (Hoechst 33342 kleuring) was egter eers tydens herperfusie sigbaar. SI is gekenmerk deur vinnige aktivering van p38 en PKB/Akt Ser473, terwyl ERK en JNK fosforilering slegs tydens herperfusie waargeneem is. Vooraf-behandeling met SB203580, ‘n p38 inhibitor, het ‘n beduidende toename in sellewensvatbaarheid asook ‘n afname in die apoptotiese indeks tydens Sl/H teweeggebring, terwyl SP600125, ‘n spesifieke JNK inhibitor, apoptose bevorder het. PD98059, ‘n ERK inhibitor, het geen invloed op apoptose tydens Sl/H gehad nie. Wortmannin, ‘n PI3-kinase inhibitor, het Thr308 (nie Ser473) fosforilering onderdruk, gepaargaande met ‘n toename in PARP kliewing, maar dit het geen invloed op kaspase-3 aktivering of die apoptotiese indeks gehad nie. EPA en ARA (20 (iM, teenwoordig voor en na SI) het kaspase-3 aktivering en PARP kliewing asook die apoptotiese indeks tydens herperfusie beduidend verminder. Beide vetsure het ook ‘n beduidende toename in ERK en afname in p38 fosforilering veroorsaak. Vanadaat (‘n serien-threonien fosfatase inhibitor), maar nie “okadaic” suur (‘n tirosien fosfatase inhibitor), kon die ARA-gemduseerde inhibisie van p38 ophef nie. Induksie van MKP-1, ‘n tweeledige-spesifieke fosfatase, is beduidend deur ARA en EPA tydens herperfusie verhoog. 'n In vitro defosforileringbepaling het bevestig dat hierdie fosfatase wel betrokke by die inhibisie van p38 kan wees. Daarbenewens is gevind dat die beskermende aksie van ARA PI3-K afhanklik is. Hierdie resultate wys dat fosforilering van p38 pro-apoptoties is, terwyl JNK beskermend is en dat hierdie kinases via kaspase-3 seldood of oorlewing tydens SI/H-geinduseerde beskadiging bemiddel. In hierdie model is daar vir die eerste keer getoon dat EPA en ARA neonatale kardiale miosiete teen isgemie/herperfusie-geinduseerde apoptose beskerm deur induksie van MKP- 1, wat defosforilering van die pro-apoptotiese kinase, p38 teweegbring. Hierdie voordelige effekte van EPA en ARA is ook sigbaar in die funksionele herstel tydens isgemie/herperfusie van die geisoleerde rothart model.
Description
Thessis (PhD)--Stellenbosch University, 2005.
Keywords
Ischemia, Unsaturated fatty acids, Reperfusion injury, Cardiovascular system -- Diseases, Dissertations -- Medicine