l‐α‐Lysophosphatidylinositol (LPI) aggravates myocardial ischemia/reperfusion injury via a GPR55/ROCK‐dependent pathway

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.

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.

K E Y W O R D S
cell signaling, GPR55, ischemia/reperfusion injury, lysophosphatidylinositol, rho kinase

| INTRODUC TI ON
Reperfusion of an occluded artery is vital to the salvage of the myocardium affected by ischemia, however the reintroduction of blood and oxygen paradoxically causes further injury and cardiomyocyte death known as ischemia/reperfusion (I/R) injury. 12 Immediate "lethal" I/R injury occurs within the first few minutes of reperfusion with the critical early events being an increase in oxidative stress, [Ca 2+ ] i overload, 13 opening of the mitochondrial permeability transition pore (MPTP 15 ) and the activation of proapoptotic pathways. 14,26 It is well established that the release of bioactive lipids from platelets at the site of clot formation during acute myocardial infarction (AMI) contributes to the eventual extent of myocardial I/R injury, with some lipids (eg sphingosine-1-phosphate; S-1-P) exerting cardioprotective effects (reviewed in 21) and others exacerbating (eg platelet activating factor: 22,32) I/R injury. Platelets also release the lysophospholipid lαlysophosphatidylinositol (LPI) and are thought to be the source of the elevated plasma levels of LPI seen in patients undergoing coronary angioplasty for acute coronary syndrome (ACS 27 ). LPI acts as an endogenous ligand at the G-protein coupled receptor GPR55 36 which is expressed in various rodent 40 and human 18 tissues including the cardiovascular system, where it is expressed on vascular smooth muscle and endothelial cells 6,7 and ventricular cardiomyocytes. 45,52 Although the functional importance of the GPR55/LPI system in cardiovascular physiology and pathophysiology is poorly understood, we have previously shown that mice lacking GPR55 exhibit moderate systolic dysfunction with age and have a decreased contractile reserve in response to adrenoceptor stimulation, 45 suggesting a physiological role in maintaining cardiac function. In contrast, observations that GPR55 tissue expression and circulating levels of LPI are raised in conditions where there is heightened cardiovascular risk (such as obesity and metabolic syndrome 33 ); that cardiac LPI levels rise during asphyxia-induced myocardial ischemia, 24,25 and that the GPR55 antagonist CBD reduces myocardial infarct size in rats in vivo 9,46 all point to a potential detrimental role for LPI/GPR55 in cardiovascular pathologies such as AMI.
Studies in GPR55-expressing HEK cells have linked GPR55mediated LPI responses to activation of several signaling pathways, including activation of RhoA and ROCK resulting in an increase in intracellular calcium ([Ca 2+ ] i ) and activation of G α13.

17
Similarly, LPI induces a rapid and transient increase in [Ca 2+ ] i in cardiomyocytes through an action at GPR55 receptors located on both the sarcolemma and the membranes of intracellular organelles, 52 although any involvement of RhoA/ROCK signaling in the response to LPI in cardiomyocytes remains to be investigated.
[Ca 2+ ] i overload is a major factor in cardiomyocyte death following myocardial I/R 5,28 and activation of the RhoA/ROCK pathway plays an important role in the setting of I/R injury as it is activated at the time of reperfusion to suppress the RISK pathway and extend reperfusion injury. 14 Thus, our underlying hypothesis was that activation of the GPR55/RhoA/ROCK pathway by LPI in the vicinity of the ischemic myocardium, may contribute to exacerbation of I/R injury. To explore this, we initially confirmed in murine and human iPSC cardiomyocytes that activation in response to LPI is mediated by GPR55, utilizing a well-established label-free, highthroughput screening system (Corning ® Epic ® ) that measures the dynamic mass redistribution (DMR) of cells and enables real-time detection of integrated cellular responses in living cells. 41 This allowed us to make a rapid determination of the optimum concentration and exposure time to LPI for subsequent studies in murine iPSC cardiomyocytes and isolated Langendorff-perfused hearts, which were undertaken to confirm that stimulation of GPR55 by LPI leads to activation of the RhoA/ROCK/p38 MAPK pathway and to an exacerbation of myocardial injury in cultured cardiomyocytes and isolated hearts, respectively.

| Dynamic mass redistribution (DMR) in response to LPI
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 a receptor is stimulated by a ligand, the change in DMR in the cells is manifested as a shift in the wavelength of light that is reflected from the sensor. The magnitude of this wavelength shift is proportional to the total change in the biomass proximal to the sensor surface (amount of DMR) and thus GPCR activation is translated into signaling pathway-specific optical signatures. This technique has been previously used to demonstrate activation of GPR55 in other cell types, 19 but not in cardiomyocytes, and has been identified as the preferred assay technique to study GPR55 pharmacology and function since it provides a much more accu-  the microplates incubated at 26°C for 1 hour. Baseline readings were then taken every minute for a 3-minute period prior to addition of LPI alone (1 nmol L −1 -30 μmol L −1 ), or LPI (10 μmol L −1 ) in the presence of the GPR55 antagonist cannabidiol (CBD; 1 μmol L −1 ), or the ROCK inhibitor Y-27632 (10 and 50 μmol L −1 ), using a Biomek ® NM P laboratory automated workstation (Beckman Coulter, Sweden); 100 μmol L −1 ATP was used as a positive control. The microplate was then reinserted into the Corning ® Epic ® System and the DMR measurements in each well were recorded at 1-minute intervals over a 90-minute time period. To confirm that any effects of Y-27632 and CBD on LPI responses were not due to a cytotoxic effect, cell viability of miPSC cardiomyocytes exposed to CBD (1 μmol L −1 ) or Y-27632 (10 and 50 μmol L −1 ) for 90 minutes was determined using an MTT (3-(4,5-d imethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Briefly, miPSC cardiomyocytes were seeded at a density of 20,000 cells/ well in a 96-well plate and cultured in Cor.At ® media (supplied by the manufacturer) for 7 days at 37°C (in 5% CO 2 ). Following treatment with pharmacological agents, media was removed, replaced with MTT (1 mg/mL) and plates incubated for 4 hour at 37°C. The formazan product was then dissolved by adding DMSO to the wells and absorbance measured at 560 nm using a plate reader (Bio-Tek).

| Activation of downstream signaling pathways by LPI in miPSC cardiomyocytes
Activation of ROCK, p38 MAPK, JNK, and caspase-3 cleavage in response to LPI stimulation was determined in miPSC cardiomyocytes cultured as described in Section 2.1. Experiments were performed with cells grown at a confluency of 70%-80% and then serum starved overnight (16 hours) prior to experimentation.
Following challenge with LPI, with or without the ROCK inhibitor Y-27632 or the GPR55 antagonist CBD, the cells were lysed and the resulting lysates subjected to SDS-PAGE and immunoblotting as previously described. 19 Briefly, following cell treatment, the cells  ground) mice were bred in-house and were routinely genotyped as previously described. 49 All animals were housed in the University of Aberdeen Medical Research Facility until experimentation at Robert Gordon University. All mice were grouped according to genotype, gender and age and housed in temperature (21 ± 2°C) and humidity (55 ± 10%) controlled rooms with a 12-hour light/ dark cycle (7 am/7 pm). Additionally, mice were housed (according to husbandry guidelines set by the UK Home Office) in groups not exceeding eight, with ad libitum access to water and food pellets and environmental enrichment. All animals (males and females) were aged between 9-12 weeks (body weights 18-32 g) at the time of use and were randomly allocated to experimental groups using random number generator software (Stat Trek, UK).

| Isolated heart studies
Mice were anesthetized with ketamine (120 mg/kg) and xylazine (16 mg/kg) via intraperitoneal (ip) injection and the heart rapidly excised, the aorta cannulated and the heart mounted onto a

| Statistical analysis
For all experiments, data are expressed as mean ± SEM of n observations. For the ROCK2 phosphorylation assay, n refers to the number of experiments performed using separate cell preparations and data were expressed as a ratio of phospho-ROCK2 to ROCK2. For the Epic studies, the time courses of the responses were obtained by plotting the DMR activity (arbitrary units) measured at 1-minute intervals and peak DMR activity was determined from the time course plots and measured at the point where the response reached a maximum before either reaching a plateau or declining; n refers to the number of plates tested for each intervention. For isolated heart studies, n refers to the number of hearts or tissue samples used for the study. Power calculations performed on data from previous studies indicated that to detect a 30% difference in infarct size with 85% power, the minimum group size is 5. Therefore, all experiments employed hearts from at least five animals for each intervention.
However, since the studies were performed over several months, contemporaneous control experiments in wild type mice were included throughout to avoid any influence of seasonal variation and therefore the data were pooled, resulting in larger group sizes.
Infarct size is expressed as a percentage of total ventricular area of the tissue slice. For both cell studies and isolated heart studies, comparisons were made using either Student's unpaired t-test (twogroup comparison) or one-way ANOVA followed by a Bonferroni post-hoc test (multiple comparisons). All statistical analyses were carried out using GraphPad Prism ® 4 software (GraphPad Software, Inc. USA). In all cases a value of P < 0.05 was taken to indicate statistical significance.

| Effect of LPI on cultured cardiomyocytesidentification of ROCK as a GPR55 signaling pathway
In order to explore whether LPI, acting through GPR55 and ROCK, could have an adverse effect on the outcome of AMI, it was important to initially characterize the responses to LPI in cardiomyocytes.   Figure 3A and B) and was similarly GPR55 and ROCK-mediated as shown by inhibition by CBD ( Figure 3C) and Y-27632 ( Figure 3D), respectively.

| LPI induces ROCK and p38 MAPK phosphorylation in murine iPSC cardiomyocytes
To confirm that the changes in DMR in response to LPI were indeed associated with ROCK and p38 MAPK phosphorylation, murine iPSC cardiomyocytes were stimulated with LPI alone (1 & 10 μmol ; and inverse agonism at CB 2 receptors, 37 ), the role of GPR55 in mediating the responses to LPI was further confirmed in these experiments by using the selective GPR55 antagonist CID16020046 (10 μmol L −1 ). ROCK phosphorylation by LPI was still clearly detectable after 40 minutes ( Figure 5A and B) and, as with CBD, CID16020046 completely abrogated the response providing further confirmation that this was mediated through GPR55 activation.

| LPI exacerbates I/R injury via activation of GPR55 and ROCK phosphorylation
To confirm that LPI was acting through GPR55, hearts from both WT and GPR55 −/− mice were used. Challenging WT hearts with LPI prior to the onset of global ischemia significantly increased infarct size compared to vehicle controls (Figure 6A, B and G), whereas this was not seen in seen GPR55 −/− hearts, confirming an action of LPI at GPR55 (Figure 6E

| D ISCUSS I ON AND CON CLUS I ON S
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.  CBD and Y-27632, in both murine and human-induced pluripotent stem cell cardiomyocytes, establishing that the same signaling pathway is important in both species. In HEK293 cells, GPR55 activation leads to the recruitment of several nuclear transcription factors, all of which activate RhoA leading to phosphorylation of ROCK. 17,19 In these present studies, 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.

| LPI signaling in cardiomyocytes
In cardiomyocytes, one consequence of p38 MAPK activation is the induction of apoptosis, 39 however we did not see any activation of JNK or caspase-3 (both markers of apoptosis), suggesting that the increased injury seen in the isolated hearts challenged with LPI prior to I/R was not due to induction of apoptotic cell death but to some other mechanism, such as calcium overload. It has been previously shown that LPI directly increases cardiomyocyte [Ca 2+ ] i through activation of both sarcolemmal GPR55 receptors, to stimulate Ca 2+ entry via L-type Ca 2+ channels (LTCC) and IP 3 -mediated Ca 2+ release from internal stores, and intracellular GPR55 receptors that promote internal Ca 2+ release via endolysosomal NAADP-sensitive two-pore channels. 52 There is generally scant information on any association  17 suggesting that in some tissues these pathways converge. Indeed, LPI causes a biphasic increase in [Ca 2+ ] i , in endothelial cells, the first phase of which is PLC-IP 3 -dependent and the second phase, ROCK-dependent. 1 Therefore, it is feasible that ROCK activation in cardiomyocytes is similarly linked to changes in intracellular calcium.

between ROCK activation and increases in [Ca
Although this study has focused on the ROCK/p38 MAPK pathway, LPI has been shown to activate additional signaling pathways on noncardiomyocyte cells, including JNK and AKT/ERK. However, as described above, we did not see any notable JNK activation, and we have found previously in rat neonatal cardiomyocytes that the activation of ERK in response to LPI is very transient (5 minutes) and would therefore be unlikely to account for the sustained response to LPI seen in the DMR studies ( Figure S1; unpublished observations).

| Mechanisms of LPI-induced cardiomyocyte injury
Total levels of LPI in the coronary circulation during AMI are significantly elevated (mean 2.95 ± 1.88 μmol L −1 ; range 1-12 μmol L −1 ), compared to levels seen in either stable angina or no coronary artery disease (mean 1.54 ± 0.99 μmol L −1 ; range 1-5 μmol L −1 27 ) and it is believed that the LPI is released from platelets activated at the site of plaque rupture and clot formation, rather than the myocardium per se. Platelet-derived substances released during AMI have ready access (via the coronary circulation) to the cardiac tissue in the vicinity of their release and therefore could influence the injury process within cardiac tissue. By using an isolated perfused heart model, we have demonstrated that application of LPI via the F I G U R E 3 The DMR response to LPI in human iPSC cardiomyocytes is identical to that seen in murine iPSC cardiomyocytes and is similarly inhibited by the GPR55 antagonist CBD and the ROCK inhibitor Y-27632. LPI (1 nmol L −1 -30 μmol L −1 ) produced a concentrationdependent increase in peak DMR response in human iPSC cardiomyocytes (Panels A & B). Both CBD (1 μmol L −1 ; Panel C) and Y-27632 (10 & 50 μmol L −1 ; Panel D) markedly reduced the peak DMR response to LPI (10 μmol L −1 ). ATP (100 μmol L −1 ) was employed as a positive control in all experiments. Values are shown as mean ± SEM; n = 4 experiments performed using separate cell preparations for all groups. *P < 0.001 vs vehicle; # P < 0.001 vs LPI alone. AU = Arbitrary Units pressor rather than a constrictor response. 1 It is noteworthy that we did not see any difference in the extent of injury between vehicletreated WT and GPR55 −/− hearts, supporting the notion that there is no endogenous release of LPI from the heart during I/R and that the source of elevated LPI seen during acute coronary events is extracardiac. Indeed, in a separate study (manuscript in preparation), we have found that total LPI levels in WT hearts subjected to I/R are in fact significantly lower than in sham hearts. Our studies also show that administration of LPI at the time of reperfusion does not exacerbate the extent of injury, demonstrating that the LPI-induced increase in myocardial injury occurs during ischemia.
We have also confirmed that the increase in infarct size following LPI application to isolated hearts is via activation of GPR55 receptors, since infarct size in hearts from GPR55 −/− mice was not increased by LPI. Utilizing hearts lacking GPR55, as opposed to the GPR55 antagonist CBD, was a more appropriate way of demonstrating a role for the receptor since, although we know that CBD acts relatively selectively at GPR55 in cell systems, when used in whole tissue or in vivo studies it displays a more complex pharmacology. 47 Moreover, CBD itself reduces infarct size 9,46 through mechanisms that are not exclusively GPR55-mediated, and which could have confounded the present studies. LPI also promotes Ca 2+ entry (via LTCCs 52 ) into cardiomyocytes, which in the setting of I/R would induce cellular injury through the induction of cardiomyocyte Ca 2+ overload, activation of mitochondrial K ATP channels and subsequent opening of the mitochondrial permeability transition pore (mPTP 13 ). Although an association between LPIinduced Ca 2+ entry via LTCC's and activation of ROCK has not been investigated in cardiomyocytes, Ca 2+ entry via LTCC's has been linked to ROCK activation in vascular smooth muscle cells. 31 Moreover, in GPR55-expressing HEK cells LPI activates phospholipase C (PLC) via a ROCK-dependent pathway, 19 with the subsequent production of IP 3 and stimulated Ca 2+ release from the sarcoplasmic reticulum, which again would cause Ca 2+ overload. ROCK has also been shown to work in concert with low voltage-activated T-type Ca 2+ channels (Cav3.2 channel), which have also been implicated in Ca 2+ overload during myocardial ischemia. 34 Clearly these are potential mechanisms of LPIinduced cardiomyocyte injury that require further investigation.
Although an investigation of the effect of ROCK inhibition per se on infarct size was not a primary aim of the present study, it is interesting that, in contrast to other studies showing an infarct-sparing effect of ROCK inhibition, we did not see an infarct-reducing effect of Y-27632 but rather we observed a slight (not statistically significant) trend toward an increase in infarct size. There are several plausible explanations for this. First, ROCK inhibition has been shown to decrease infarct size and preserve postinfarction cardiac systolic function through various mechanisms including increased collateral blood flow to the myocardium, 51 improved metabolic status of the ischemic tissue, 16 reduced myocardial fibrosis, 29 reduced apoptosis and inflammation 4 and preservation of the reperfusion injury salvage kinase (RISK) pathway. 30 Importantly, all of these studies used in vivo models and determined end points (inflammation and apoptosis) that occur over extended periods of time (hours to days) after reperfusion or implemented ROCK inhibition at the time of reperfusion. 14 In contrast, our studies were performed in blood-free perfused hearts in vitro, the hearts were administered Y-27632 prior to ischemia and relatively short (30 minutes) periods of I/R (when the main mechanisms contributing to cell death are calcium overload and oxidative stress) were used. Consistent with our findings, ROCK activation has been shown to promote cardiomyocyte survival via a PKD pathway 50 and more recent studies have shown that the two ROCK isoforms (ROCK-1 and ROCK-2) play opposing roles in the heart, with ROCK-1 protecting against cardiac dysfunction and oxidative stress and ROCK-2 promoting these. 43 Since Y-27632 is a nonselective inhibitor that has similar affinities for both ROCK isoforms, 2 this could explain why in vehicle-treated hearts, where I/R-induced ROCK-1 activation predominates and serves a pro-survival role, Y-27632 had a slightly deleterious effect, while in LPI-stimulated hearts where ROCK-2 is activated (as shown from our cardiomyocytes studies), it prevented the ROCK-2 mediated extension of injury.
Off-target actions of Y-27632 for ROCK could also potentially account for our findings since, when screened against a panel of kinases it has been shown to inhibit protein kinase-2 (PRK2 8 ; PKC epsilon (PKCε) and protein kinase N1 (PKN). 2 The expression of PRK2 is very low in the heart, 38 making it an unlikely target, but PKN is a pro-survival kinase expressed in response to I/R injury 44 while PKCε is a known trigger of delayed preconditioning. 20,48 Since our model F I G U R E 7 Proposed signaling pathway for LPI in cardiomyocytes that contributes to exacerbation of I/R injury. LPI is known to increase intracellular calcium in cardiomyocytes via direct entry through LTCC's and through PLC/IP 3 -mediated release from the sarcoplasmic reticulum (black arrows). Here we have shown that LPI results in both ROCK and p38 MAPK activation (blue arrows); while p38 MAPK activation has been linked to a pro-apoptotic effect during ischemia (pink), lack of evidence of activation of pro-apoptotic pathways suggests this is not the cause of LPI-induced cardiomyocyte cell injury. The proposed pathways (red arrows) are p38 MAPK-induced mitochondrial release of Ca2+ and ROS production In summary, we present the novel finding that LPI activates murine and human-induced pluripotent stem cell cardiomyocytes via a GPR55/RhoA/ROCK/p38 MAPK-dependent pathway, and that this same pathway is responsible for the exacerbation of injury seen in hearts challenged with LPI prior to an ischemia/reper-

D I SCLOS U R E S
None.