Exogenous Lysophosphatidylinositol Exacerbates Myocardial Tissue Injury Via A GPR55/ROCK Dependent Mechanism

O J Robertson-Gray1, S K Walsh1, A C Jönsson-Rylander2, C L Wainwright1. 1Robert Gordon University, Aberdeen, UK, 2AstraZeneca, Mölndal, Sweden

Studies exploring the role of G protein coupled receptor 55 (GPR55) have suggested that it may represent a new target in cardiac dysfunction.[1] However, whilst it has been demonstrated that GPR55 mRNA is expressed in the heart,[2] the role of GPR55 in this organ remains unknown. With regard to myocardial ischaemia/reperfusion injury, it is now widely accepted that [Ca2+]i overload mediated by this condition promotes cardiomyocyte death and myocardial tissue injury.[3] Interestingly, lysophosphatidylinositol (LPI), the proposed endogenous ligand of GPR55, has been shown to increase [Ca2+]i in neonatal ventricular cardiomyocytes.[1] It is possible that an LPI/GPR55-induced increase in [Ca2+]i in cardiomyocytes may contribute to tissue injury in the setting of myocardial ischaemia/reperfusion thus the present study was carried out to investigate this.

Wild-type (WT) and homozygous GPR55-/- knockout mice (male/female; 9-12 week old; 18-32g) were anaesthetized with ketamine and xylazine (120mg kg-1 and 16mg kg-1, respectively, i.p.). Hearts were mounted onto Langendorff apparatus and retrogradely perfused with Kreb’s Henseleit buffer (pH7.4; 37oC; 2-2.5ml min-1). A 15 minute stabilisation period preceded a 30 minute no-flow global ischaemia (GI)/30 minute reperfusion period. Hearts were treated by bolus dose with vehicle (0.1% DMSO) or LPI (10µM) pre-GI or during early reperfusion (post-GI). LPI/GPR55 signalling studies were subsequently carried out where the ROCK inhibitor, Y-27632 dihydrochloride (10µM or 50µM) was administered alone or pre-LPI administration pre-GI. Hearts were subsequently frozen, sectioned and stained using 1% 2,3,5-triphenyltetrazolium chloride and infarct size measured via computerised planimetry (ImageJ). Data are expressed as mean ± S.E.M. and were compared using a one-way ANOVA test followed by a ‘Bonferroni’ post-hoc test or ‘Student’s unpaired t-test’.

Preliminary studiesshowed that infarct size did not significantly differ between WT (n=14) and GPR55-/- (n=5) mice (33.9±2.9 vs. 37.0±3.2%). However, LPI administration pre-GI to WT hearts, increased infarct size (48.0±4.7 vs. 33.9±2.9%; P<0.05), an effect which did not occur if LPI was administered post-GI (n=14 and n=9, respectively; 38.1±3.3 vs. 33.9±2.9%). Additionally, pre-GI LPI administration to GPR55-/- (n=7) hearts did not alter infarct size compared to controls (43.8±4.6%). When investigating the signalling mechanisms by which the LPI/GPR55 system exacerbates myocardial tissue injury, the lower concentration (10µM; n=6) of the ROCK inhibitor, Y-27632 dihydrochloride, ameliorated the deleterious effects of LPI (33.2±3.9%; P<0.05) in WT hearts. However, the higher concentration (50µM; n=7) did not. Interestingly, when 50µM Y-27632 dihydrochloride was administered alone (n=7) to WT hearts, it increased myocardial tissue injury (55.3±7.1 vs. 33.9±2.9%; P<0.05) suggesting that the signalling mechanism(s) involved are complex and require further investigation. To conclude, exogenous LPI exacerbates myocardial tissue injury via a GPR55/ROCK dependent mechanism.

(1) Yu et al., (2013) J Biol Chem. 288:22481-92

(2) Henstridge et al., (2011) Mol Endocrinol. 25:1835-48

(3) Mozaffari et al., (2013) Am J Cardiovasc Dis. 3:180-96