Abstract
The phospholipid l‐α‐lysophosphatidylinositol (LPI), an endogenous ligand for GPR55, is elevated in patients with acute coronary syndrome, and a GPR55 antagonist cannabidiol (CBD) reduces experimental ischemia/reperfusion (I/R) injury. While LPI activates multiple signaling pathways, little is known about which ones are important in cardiomyocytes. In this study we explored whether activation of the Rho kinase/ROCK/p38 MAPK pathway is responsible for LPI‐induced extension of I/R injury. Using a high‐throughput screening method (dynamic mass redistribution; DMR), mouse‐ and human‐induced pluripotent stem cell (iPSC) cardiomyocytes exposed to LPI were shown to exhibit a rapid, sustained, and concentration‐dependent (1 nmol L−1‐30 μmol L−1) cellular response. Y‐27632 (ROCK inhibitor; 10 & 50 μmol L−1) and CBD (1 μmol L−1) both abolished the DMR response to LPI (10 μmol L−1). In murine iPSC cardiomyocytes, LPI‐induced ROCK and p38 MAPK phosphorylation, both of which were prevented by Y‐27632 and CBD, but did not induce JNK activation or cleavage of caspase‐3. In hearts isolated from wild type (WT) mice subjected to 30 minutes global I/R, LPI (10 μmol L−1) administered via the coronary circulation increased infarct size when applied prior to ischemia onset, but not when given at the time of reperfusion. The exacerbation of tissue injury by LPI was not seen in hearts from GPR55−/− mice or in the presence of Y‐27632, confirming that injury is mediated via the GPR55/ROCK/p38 MAPK pathway. These findings suggest that raised levels of LPI in the vicinity of a developing infarct may worsen the outcome of AMI.
Highlights
The data presented from this study has shown for the first time that LPI, a platelet-derived lysophospholipid that is elevated in acute coronary syndrome, exacerbates the extent of myocardial injury and that, as in other cell types, LPI exerts its cellular responses in cardiomyocytes via activation of the GPR55 receptor and downstream activation of both Rho-assisted protein kinase (ROCK) and p38 mitogen-activated protein kinase (MAPK)
We have shown for the first time that exposure of miPSC cardiomyocytes to LPI induces both ROCK and p38 MAPK activation, and that this is dependent upon an action at GPR55
This study has focused on the ROCK/p38 MAPK pathway, LPI has been shown to activate additional signaling pathways on noncardiomyocyte cells, including c-Jun N-terminal kinase (JNK) and AKT/ERK
Summary
The underlying principle of determining changes in cellular DMR has previously been described in detail.[10,11,41] To measure DMR, cells are seeded on to a microplate containing a resonant waveguide grating biosensor, which measures changes in the local index of refraction upon mass redistribution within a living cell monolayer grown on the biosensor. When the ROCK inhibitor Y-27632 (10 and 50 μmol L−1; volume delivered 500 μL over 30 second) was applied to WT hearts via the aortic cannula 5 minutes prior to administration of either LPI or vehicle, the LPI-induced exacerbation of infarct size was abrogated by the higher concentration (Figure 6C, D and H), implicating ROCK in the signaling pathway downstream of GPR55. The data presented from this study has shown for the first time that LPI, a platelet-derived lysophospholipid that is elevated in acute coronary syndrome, exacerbates the extent of myocardial injury and that, as in other cell types, LPI exerts its cellular responses in cardiomyocytes via activation of the GPR55 receptor and downstream activation of both ROCK and p38 MAPK. P38 MAPK inhibition during I/R has been shown to prevent mitochondrial reactive oxygen species production and Ca2+ overload,[42] pointing to a
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