Abstract:

1 The purpose of this study was to determine whether endocannabinoids can protect the heart against ischaemia and reperfusion. 2 Rat isolated hearts were exposed to low-flow ischaemia (0.5-0.6 ml min(-1)) and reperfusion. Functional recovery as well as CK and LDH overflow into the coronary effluent were monitored. Infarct size was determined at the end of the experiments. Phosphorylation levels of p38, ERK1/2, and JNK/SAPK kinases were measured by Western blots. 3 None of the untreated hearts recovered from ischaemia during the reperfusion period. Perfusion with either 300 nM palmitoylethanolamide (PEA) or 300 nM 2-arachidonoylglycerol (2-AG), but not anandamide (up to 1 micro M), 15 min before and throughout the ischaemic period, improved myocardial recovery and decreased the levels of coronary CK and LDH. PEA and 2-AG also reduced infarct size. 4 The CB(2)-receptor antagonist, SR144528, blocked completely the cardioprotective effect of both PEA and 2-AG, whereas the CB(1)-receptor antagonist, SR141716A, blocked partially the effect of 2-AG only. In contrast, both ACEA and JWH015, two selective agonists for CB(1)- and CB(2)- receptors, respectively, reduced infarct size at a concentration of 50 nM. 5 PEA enhanced the phosphorylation level of p38 MAP kinase during ischaemia. PEA perfusion doubled the baseline phosphorylation level of ERK1/2, and enhanced its increase upon reperfusion. The cardioprotective effect of PEA was completely blocked by the p38 MAP kinase inhibitor, SB203580, and significantly reduced by the ERK1/2 inhibitor, PD98059, and the PKC inhibitor, chelerythrine. 6 In conclusion, endocannabinoids exert a strong cardioprotective effect in a rat model of ischaemia-reperfusion that is mediated mainly through CB(2)-receptors, and involves p38, ERK1/2, as well as PKC activation.

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Endocannabinoids protect the rat isolated heart against ischaemia

^¹Faculté de Pharmacie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, QC, Canada H3C 3J7

^{Author for correspondence:}ac.laertnomu@engatnomal.leinad

^{Shared first authorship.}

^{Present address: Center for Neuronal Survival, Montreal Neurological Institute, 3801 University Street, Montreal, QC, Canada H3A 2B4}

Received 2003 Jan 28; Revised 2003 Feb 2; Accepted 2003 Apr 7.

Abstract

The purpose of this study was to determine whether endocannabinoids can protect the heart against ischaemia and reperfusion.
Rat isolated hearts were exposed to low-flow ischaemia (0.5–0.6 ml min^{) and reperfusion. Functional recovery as well as CK and LDH overflow into the coronary effluent were monitored. Infarct size was determined at the end of the experiments. Phosphorylation levels of p38, ERK1/2, and JNK/SAPK kinases were measured by Western blots.}
None of the untreated hearts recovered from ischaemia during the reperfusion period. Perfusion with either 300 nM palmitoylethanolamide (PEA) or 300 nM 2-arachidonoylglycerol (2-AG), but not anandamide (up to 1 μM), 15 min before and throughout the ischaemic period, improved myocardial recovery and decreased the levels of coronary CK and LDH. PEA and 2-AG also reduced infarct size.
The CB₂-receptor antagonist, SR144528, blocked completely the cardioprotective effect of both PEA and 2-AG, whereas the CB₁-receptor antagonist, SR141716A, blocked partially the effect of 2-AG only. In contrast, both ACEA and JWH015, two selective agonists for CB₁- and CB₂- receptors, respectively, reduced infarct size at a concentration of 50 nM.
PEA enhanced the phosphorylation level of p38 MAP kinase during ischaemia. PEA perfusion doubled the baseline phosphorylation level of ERK1/2, and enhanced its increase upon reperfusion. The cardioprotective effect of PEA was completely blocked by the p38 MAP kinase inhibitor, SB203580, and significantly reduced by the ERK1/2 inhibitor, PD98059, and the PKC inhibitor, chelerythrine.
In conclusion, endocannabinoids exert a strong cardioprotective effect in a rat model of ischaemia–reperfusion that is mediated mainly through CB₂-receptors, and involves p38, ERK1/2, as well as PKC activation.

Keywords: Cannabinoid, CB₂-receptors, ischaemia–reperfusion, myocardial infarction, protein kinase C, MAP kinases, rat isolated heart

Abstract

Acknowledgments

This project was supported by a grant from the Canadian Institutes of Health Research (CIHR MOP- 15047). PL holds studentship from the FRSQ. JFB held studentships from the FRSQ and from PMAC-HRF/MRC, and currently holds a Fellowship from the CIHR. CL received a Fellowship from the Ministère de l'éducation du Québec. The authors are grateful to S. Maltais and H.H. Dao for their advices regarding the Western blots.

Acknowledgments

Abbreviations

ACEA	arachidonyl-2′-chloroethylamide
2-AG	sn-2 arachidonoylglycerol
BCA	bicinchoninic acid
CCP	coronary perfusion pressure
CHO	Chinese hamster ovary
CK	creatine kinase
DMSO	dimethylsulphoxide
DTT	dithiothreitol
EDP	left ventricular end-diastolic pressure
EDTA	ethylenediaminetetraacetic acid
EGTA	ethyleneglycol bis(β-aminoethyl)-N,N,N′,N′-tetraacetic acid
ERK1/2	extracellurar regulated kinase 1/2
HSP27	heat-shock protein 27
JNK/SAPK	janus kinase/stress-activated protein kinase
JWH015	[2-methyl-1-propyl-1H-indol-3-yl]-1-naphthalenylmethanone
K–H	Krebs–Henseleit buffer
LDH	lactate dehydrogenase
MAP kinase	mitogen-activated protein kinase
MAPKAP2/3	MAP kinase-activated protein kinase 2/3
PD98059	2′-amino-3′-methoxyflavone
PEA	palmitoylethanolamide
PKC	protein kinase C
PMSF	phenylmethylsulphonyl fluoride
SB203580	4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridil)1H-imidazole
SDS	sodium dodecyl sulphate
s.e.m.	standard error of the mean
TBST	Tris-buffered saline containing 0.1% Tween 20

Abbreviations

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