Differential Activation of Cultured Neonatal Cardiomyocytes by Plasmalemmal Versus Intracellular G Protein-coupled Receptor 55*

Background: The LPI-sensitive receptor GPR55 signals through Ca2+. Results: Activation of sarcolemmal versus intracellular GPR55 mobilizes Ca2+ from distinct pools and associates with cardiomyocyte depolarization and hyperpolarization, respectively. Conclusion: GPR55 location critically affects LPI-induced modulation of cardiomyocyte function. Significance: We identify GPR55 as a new receptor regulating cardiac function at two cellular sites. The L-α-lysophosphatidylinositol (LPI)-sensitive receptor GPR55 is coupled to Ca2+ signaling. Low levels of GPR55 expression in the heart have been reported. Similar to other G protein-coupled receptors involved in cardiac function, GPR55 may be expressed both at the sarcolemma and intracellularly. Thus, to explore the role of GPR55 in cardiomyocytes, we used calcium and voltage imaging and extracellular administration or intracellular microinjection of GPR55 ligands. We provide the first evidence that, in cultured neonatal ventricular myocytes, LPI triggers distinct signaling pathways via GPR55, depending on receptor localization. GPR55 activation at the sarcolemma elicits, on one hand, Ca2+ entry via L-type Ca2+ channels and, on the other, inositol 1,4,5-trisphosphate-dependent Ca2+ release. The latter signal is further amplified by Ca2+-induced Ca2+ release via ryanodine receptors. Conversely, activation of GPR55 at the membrane of intracellular organelles promotes Ca2+ release from acidic-like Ca2+ stores via the endolysosomal NAADP-sensitive two-pore channels. This response is similarly enhanced by Ca2+-induced Ca2+ release via ryanodine receptors. Extracellularly applied LPI produces Ca2+-independent membrane depolarization, whereas the Ca2+ signal induced by intracellular microinjection of LPI converges to hyperpolarization of the sarcolemma. Collectively, our findings point to GPR55 as a novel G protein-coupled receptor regulating cardiac function at two cellular sites. This work may serve as a platform for future studies exploring the potential of GPR55 as a therapeutic target in cardiac disorders.

within the cell. Thus, we used Ca 2ϩ and voltage imaging and both extracellular and intracellular administration (microinjection) of GPR55 ligands to test the involvement of this receptor in the regulation of cardiomyocyte signaling.
Real-time PCR-Total RNA was isolated from isolated rat neonatal cardiomyocytes, cortex, and olfactory bulb using an RNeasy Midi kit or RNeasy Fibrous Tissue Midi kit with proteinase K and DNase I digestion (Qiagen). cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems), and real-time PCR was performed with TaqMan gene expression master mix (Applied Biosystems) using the StepOne Plus real-time PCR system (Applied Biosystems). The following primer were used: Rn03037213-s1 (GPR55) and Rn01775763-g1 (GAPDH for normalization). They were obtained from Applied Biosystems. All samples were run in triplicate, and data were analyzed using Applied Biosystems Comparative CT Method (⌬⌬CT).
Immunocytochemistry and Confocal Imaging Studies-Cultured neonatal ventricular cardiomyocytes transiently transfected with GFP-tagged rat GPR55 receptor and Rab7-red fluorescent protein (RFP) (Addgene, Cambridge MA) 48 h earlier were fixed with paraformaldehyde 4%, washed in PBS, and mounted with DAPI Fluoromont G (Southern Biotech, Birmingham, AL). Cells were imaged using a Carl Zeiss 710 twophoton confocal microscope with a ϫ63 oil immersion objective using ϫ1 digital zoom with excitations set for DAPI, GFP, and DsRed at 405 nm, 488 nm, and 561 nm, respectively. Images were analyzed using Zen 2010 (Zeiss) as reported previously (26).
Calcium Imaging-Measurements of [Ca 2ϩ ] i were performed as described previously (27)(28)(29). Cells were incubated with 5 M Fura-2 AM (Invitrogen) in Hanks' balanced salt solu-tion at room temperature for 45 min, in the dark, washed three times with dye-free Hanks' balanced salt solution, and then incubated for another 45 min to allow for complete de-esterification of the dye. Coverslips (25-mm diameter) were subsequently mounted in an open bath chamber (RP-40LP, Warner Instruments, Hamden, CT) on the stage of an inverted microscope (Nikon Eclipse TiE, Nikon Inc., Melville, NY). The microscope is equipped with a Perfect Focus System and a CoolSnap HQ2 CCD camera (Photometrics, Tucson, AZ). During the experiments, the Perfect Focus System was activated. Fura-2 AM fluorescence (emission ϭ 510 nm), following alternate excitation at 340 and 380 nm, was acquired at a frequency of 0.25 Hz. Images were acquired and analyzed using NIS-Elements AR 3.1 software (Nikon Inc.). The ratio of the fluorescence signals (340/380 nm) was converted to Ca 2ϩ concentrations (30). In Ca 2ϩ -free experiments, CaCl 2 was omitted. In experiments using KCl, 50 mM NaCl was replaced by 50 mM KCl.
Intracellular Microinjection-Injections were performed using the Femtotips II, InjectMan NI2, and FemtoJet systems (Eppendorf) as reported previously (24,28,29). Pipettes were backfilled with an intracellular solution containing 110 mM KCl, 10 mM NaCl, 20 mM HEPES (pH 7.2), and dimethyl sulfoxide 0.004% (31) or the compounds to be tested. The injection time was 0.4 s at 60 hectopascals with a compensation pressure of 20 hectopascals to maintain the microinjected volume to less than 1% of cell volume, as measured by microinjection of a fluorescent compound (Fura-2 free acid) (31). The intracellular concentration of chemicals was determined on the basis of the concentration in the pipette and the volume of injection.
Measurement of Membrane Potential-The relative changes in membrane potential of single cardiomyocytes were evaluated using bis-(1,3-dibutylbarbituric acid) trimethine oxonol, bis-(1,3-dibutylbarbituric acid) trimethine oxonol, a slow-response, voltage-sensitive dye, as described previously (32)(33)(34). Upon membrane hyperpolarization, the dye concentrates in the cell membrane, leading to a decrease in fluorescence intensity, whereas depolarization induces the sequestration of the dye into the cytosol, resulting in an increase of the fluorescence intensity (35). Cultured ventricular cardiomyocytes were incubated for 30 min in Hanks' balanced salt solution containing 0.5 mM bis-(1,3-dibutylbarbituric acid) trimethine oxonol, and fluorescence was monitored at 0.17 Hz (excitation/emission 480 nm/540 nm). Calibration of bis-(1,3-dibutylbarbituric acid) trimethine oxonol fluorescence following background subtraction was performed using the Na ϩ -K ϩ ionophore gramicidin in Na ϩ -free physiological solution and various concentrations of K ϩ (to alter membrane potential) and N-methylglucamine (to maintain osmolarity) (35). Under these conditions, the membrane potential is approximately equal to the K ϩ equilibrium potential determined by the Nernst equation. The intracellular K ϩ and Na ϩ concentration were assumed to be 130 mM and 10 mM, respectively.
Electrophysiology-Briefly, pipettes were pulled from tubing with an outer diameter of 1.5 mm (Garner Glass, Claremont, CA). After fire polishing and backfilling, pipette resistances were ϳ3-5 M⍀. Membrane potentials from rat neonatal myocytes were recorded using current-clamp configuration with Statistics-Data were expressed as mean Ϯ S.E. One-way analysis of variance, followed by post hoc Bonferroni and Tukey tests, were used to assess significant differences between groups. Mann-Whitney U test was used for comparison of the traces shown in Fig. 4A. p Ͻ 0.05 was considered statistically significant.

Extracellular Administration of LPI Elicits GPR55-dependent
Cytosolic Ca 2ϩ Elevation in Ventricular Cardiomyocytes, Which Is Partially Contingent on Ca 2ϩ Entry-To initially determine whether GPR55 activation can elevate cytosolic Ca 2ϩ in cultured neonatal ventricular myocytes, the cells were stimulated with increasing concentrations of LPI in the absence and presence of a GPR55 antagonist. The cardiomyocytes responded to a bath application of LPI (0.1, 1 and 10 M) with a concentration-dependent increase in [Ca 2ϩ ] i of 12 Ϯ 2.6 nM (n ϭ 17 cells), 94 Ϯ 3.8 nM (n ϭ 23), and 727 Ϯ 8.4 nM (n ϭ 19). The effects reached statistical significance (p Ͻ 0.05) for concentrations of 1 and 10 M (Fig. 1A). LPI induced a sustained Ca 2ϩ response that was relatively slow in onset, with 1-to 2-min latency, and reached a maximum within 3-4 min (Fig. 1, B and C). Pretreatment with the GPR55 antagonist ML-193 (30 M, 10 min) (36) significantly attenuated the effect of LPI (10 M), supporting GPR55 dependence of the response (⌬[Ca 2ϩ ] i was 21 Ϯ 6.2 nM; n ϭ 26; Fig. 1, B and C). In the presence of nifedipine (NIF), the effect of extracellular LPI (10 M) was reduced to 348 Ϯ 7.4 nM, indicating partial involvement of L-type Ca 2ϩ channels (LTCC) (Fig. 1, A and B). Expression of GPR55 in rat neonatal cardiomyocytes was confirmed relative to positive control brain tissue samples via real-time PCR, revealing a substantially lower gene expression level than that found in either cortex or olfactory bulb tissue (Fig. 2), consist-ent with another report showing high levels of GPR55 expression in various brain regions and low GPR55 mRNA levels in the whole heart (8).
Ryanodine inhibits Ca 2ϩ mobilization either by blocking Ca 2ϩ release channels or by promoting the leakage of Ca 2ϩ from the SR (38). To distinguish between these possibilities, we treated neonatal cardiomyocytes with thapsigargin (1 M in Ca 2ϩ -free saline), an inhibitor of the sarco/endoplasmic reticulum Ca 2ϩ ATPase, conditions which deplete both ryanodineand IP 3 -sensitive Ca 2ϩ pools (39). As shown in Fig. 4, A-D, application of thapsigargin (1 M) increased the fluorescence ratio (340/380 nm) of Fura-2 AM-loaded neonatal cardiomyo-  Relative expression (log 10 ) of GPR55 mRNA in rat neonatal cortex (black bar) and rat neonatal olfactory bulb (gray bar) tissues with a known high expression of GPR55 in comparison to rat neonatal cardiomyocytes (white bar). Data are expressed as the mean relative quantitation (RQ) value Ϯ RQ min and RQ max from each tissue performed in triplicate.
cytes with similar amplitudes and kinetics in the absence and presence of ryanodine (10 M, 30 min). Should ryanodine promote the basal Ca 2ϩ leak from the SR, this would result in a reduced SR Ca 2ϩ store level, further manifested as a significantly lower Ca 2ϩ response to thapsigargin in ryanodinetreated versus untreated cells. Moreover, in Ca 2ϩ -free saline, neonatal cardiomyocytes pretreated with ryanodine (10 M, 30 min) did not respond to caffeine (10 mM) with an increase in [Ca 2ϩ ] i (Fig. 4, A and B). Caffeine is known to release intracellular Ca 2ϩ by targeting RyRs in various cell types, including neonatal cardiomyocytes (40). In view of these findings, we con-clude that the effects of ryanodine (10 M, 30 min) observed in our study are due to inhibition of ryanodine receptors.
Several control experiments (n ϭ 6 cells/experiment) were performed to ensure that inhibition of LPI-induced Ca 2ϩ responses was due to ML-193 antagonism at GPR55 rather than off-target effects at Ca 2ϩ entry or Ca 2ϩ release channels. High concentrations of KCl produce depolarization-induced activation of LTCCs in cardiomyocytes (41). As shown in Fig. 5,A and B, the Ca 2ϩ response of cultured neonatal ventricular cardiomyocytes to 50 mM KCl was largely identical in shape and amplitude in the absence (the ⌬[Ca 2ϩ ] i was 509 Ϯ 6.7 nM) and   Fig. 6A). LPI produced a fast, robust, and transitory increase in [Ca 2ϩ ] i (Fig. 6, B and D), sensitive to intracellular blockade of GPR55 with ML-193 (3 M; the ⌬[Ca 2ϩ ] i was 57 Ϯ 11 nM; n ϭ 6; Fig. 6, A, B, and E). To exclude any possible involvement of plasmalemmal GPR55 in LPI-induced responses, ML-193 (30 M, 1-min incubation) was also present in the extracellular solution. Blockade of intracellular GPR55 was a mandatory step to prevent the effect of microinjected LPI because the extent to which ML-193 crosses the sarcolemma even after 10 min of incubation is not sufficient to completely abolish the Ca 2ϩ response of cultured neonatal ventricular myocytes to intracellular LPI. 10-min incubation with ML-193 reduces the LPI-induced effect on [Ca 2ϩ ] i from 492 Ϯ 7 nM to 213 Ϯ 44 nM, n ϭ 6 cells (Fig. 6, F and G). Conversely, a 10-min incubation with ML-193 was sufficient to block the effect of extracellular LPI (Fig. 1).
The  (Fig. 7, n ϭ 6 cells/experiment). These findings suggest that NAADP-dependent lysosomal Ca 2ϩ stores are critically involved in LPI signaling upon activation of intracellularly located GPR55. This signal is further amplified by CICR via RyR.
Intracellular GPR55 Is Located at Endolysosomes in Neonatal Rat Cardiomyocytes-Extensive colocalization was observed when GFP-tagged GPR55 was coexpressed with RFP-tagged Rab7, a small GTPase associated with both endosomes and lysosomes (46). When cells where incubated with bafilomycin A1, a V-type ATPase that prevents lysosomal acidification (47), the GPR55 and Rab7 signal was abolished, confirming endolysosomal location of intracellular GPR55 (Fig. 8).

Antipodal Changes in Resting Membrane Potential in Response to Intra-versus Extracellular LPI Administration to Cultured Neonatal Ventricular Myocytes and Relation to LPIinduced Changes in [Ca 2ϩ
] i -Cultured cardiomyocytes had a mean resting membrane potential of Ϫ72.7 Ϯ 0.05 mV. An extracellular application of LPI (10 M) produced a relatively long-lasting membrane depolarization with a mean amplitude of 12.6 Ϯ 0.7 mV (n ϭ 17) that was abolished by pretreatment with ML-193 (30 M, ⌬V m ϭ 0.9 Ϯ 0.3 mV, Fig. 9, A and B) (n ϭ 11). Conversely, intracellular microinjection of LPI (1 M) to ventricular myocytes resulted in a rapid, short-lived hyperpolarization with a mean amplitude of Ϫ7.3 Ϯ 0.5 mV (Fig. 9, A  and B) (n ϭ 6). Similar to the plasma membrane-initiated response, the effect of microinjected LPI was sensitive to GPR55 blockade with ML-193 (3 M). In the presence of the GPR55 antagonist, microinjection of LPI (1 M) elicited an insignificant response (⌬V m ϭ Ϫ1.2 Ϯ 0.3 mV, n ϭ 6) largely similar to that of control buffer microinjection (⌬V m ϭ Ϫ0.9 Ϯ 0.2 mV, n ϭ 6, Fig. 9, A and B). To evaluate how the GPR55-dependent Ca 2ϩ signaling correlates with the voltage changes observed in this study, cells were loaded with BAPTA-AM (20 M, 20 min), a potent Ca 2ϩ chelator. As expected, BAPTA-AM-treated cells did not elevate their [Ca 2ϩ ] i in response to extracellular or intracellular administration of LPI. As shown in Fig. 9, C and D, incubation with BAPTA-AM markedly reduced the cardiomyocyte Ca 2ϩ response to extracellularly applied LPI from 727 Ϯ 8.4 nM to 14 Ϯ 2.1 nM (n ϭ 19) and that of micro- injected LPI from 492 Ϯ 7 nM (n ϭ 6) to 11 Ϯ 2.7 (n ϭ 6 cells). However, even in the presence of BAPTA-AM, a bath application of LPI (10 M) resulted in a depolarization of 12.79 Ϯ 0.8 mV (n ϭ 12) similar to that observed in the absence of BAPTA-AM pretreatment (Fig. 9, A and B). Conversely, LPI (1 M) microinjection to BAPTA-AM-incubated neonatal cardiomyocytes did not significantly modify sarcolemmal potential (the ⌬V m was Ϫ1.1 Ϯ 0.3 mV; n ϭ 9 cells; Fig. 9, A and B). In patch-clamp experiments, a bath application of LPI (10 M) did not trigger action potential but produced a 10.2 Ϯ 3.2 mV (n ϭ 6 cells) depolarization of cardiomyocyte membrane similar to that observed using voltage-sensitive dyes (Fig. 9, E and F).

DISCUSSION
GPCR signaling in the heart has important physiological and pathological implications (48). Cardiac cell processes such as contractility, response to ischemia, proliferation, apoptosis, and gene expression are GPCR-regulated, making GPCRs an important therapeutic target in a variety of heart diseases (49,50). Importantly, in addition to the "classical" GPCRs involved in heart function, such as those for angiotensin II, endothelin 1, or catecholamines (48, 49), a wide variety of recently deorpha-nized GPCRs are expressed in cardiac cells (50). Defining the signaling pathways coupled with these newly recognized cardiac GPCRs may yield critical insights into cardiac cell function and is imperative if new strategies are to be employed in the treatment of heart disorders (48,50).
The expression of the LPI/cannabinoid-sensitive receptor GPR55 has been detected in the whole heart by others (8) and in rat neonatal cardiomyocytes in this study, suggesting that GPR55 may play a role in cardiac cell function. Given that GPR55 is a Gq-coupled receptor that signals through Ca 2ϩ (17,51) and that Ca 2ϩ -dependent signaling is critical in cardiomyocytes (15,52), in a first series of experiments we tested whether GPR55 activation at the sarcolemma elicits [Ca 2ϩ ] i elevation. Indeed, extracellular application of LPI increased [Ca 2ϩ ] i in cultured neonatal ventricular myocytes, a response completely sensitive to GPR55 antagonism.
In cardiomyocytes, Ca 2ϩ signaling regulates the coupling of excitation with contraction and transcription (15,52). In excitation-contraction coupling, membrane depolarization triggers Ca 2ϩ entry through plasma membrane LTCC, which further induces CICR via RyRs and a global rise in [Ca 2ϩ ] i that activates myofilaments to produce cardiac contraction. An increase in  [Ca 2ϩ ] i may also induce gene expression and transcription, eliciting hypertrophic responses, a process occurring on a longer time scale and known as excitation-transcription coupling (15). In both processes, a major role is played by Ca 2ϩ release from internal stores (52), although it was shown recently that the latter is also triggered by Ca 2ϩ influx (53). Thus, identification of the Ca 2ϩ pools mobilized by LPI is critical for the characterization of the GPR55-initiated signaling pathways in ventricular cardiomyocytes.
We noticed that LTCC blockade reduced to half the effect of bath-applied LPI on [Ca 2ϩ ] i . Moreover, the response was rather brief and no longer showed a plateau phase. Likewise, in the absence of extracellular Ca 2ϩ , LPI application induced a transient [Ca 2ϩ ] i rise with a decreased amplitude. Thus, both Ca 2ϩ entry and Ca 2ϩ release occur downstream of sarcolemmal GPR55 activation in cultured neonatal ventricular cardiomyocytes. In addition to the endo/sarcoplasmic reticulum, which expresses Ca 2ϩ release channels such as IP 3 R and RyR (54), the endolysosomal system also functions as a Ca 2ϩ store involved in Ca 2ϩ mobilization (45,55).
Using a pharmacological approach, we demonstrate that Ca 2ϩ release occurs primarily via IP 3 Rs and is further amplified by a RyR-dependent CICR mechanism, whereas the endolysosomal NAADP-dependent Ca 2ϩ stores are not involved. The IP 3 dependence of the response is not surprising because GPR55 has been shown to couple with Gq (51). Compared with RyR, IP 3 R expression is scarce in ventricular myocytes, indicating a rather minor physiological role. While IP 3 R activation may be beneficial in that it serves as an inotropic support for ventricles, its predominant effect is to promote arrhythmias (56,57). To explain the increased arrhythmogenicity of IP 3 R stimulation, it has been suggested that Ca 2ϩ release via IP 3 Rs may activate additional currents such as the store-operated Ca 2ϩ currents or the Na ϩ /Ca 2ϩ exchange current (56). These currents may be partially responsible for the plateau phase of the Ca 2ϩ response elicited by extracellular application of LPI to cultured neonatal ventricular myocytes incubated with Ca 2ϩcontaining saline. On a different note, studies on adult and neonatal cardiomyocytes correlated both Ca 2ϩ influx via LTCC (53) and the IP 3 -dependent Ca 2ϩ release with cardiac hypertrophy and heart failure (52,(57)(58)(59), suggesting that sarcolemmal GPR55 activation may produce hypertrophic signals.
Because cardiovascular cell function is also regulated by intracellularly expressed GPCRs (60,61) and preliminary data from our laboratory suggested functionality of intracellular GPR55 (23), we evaluated the effect of LPI microinjection in cultured neonatal ventricular myocytes. Similar to the response elicited at the plasma membrane, the [Ca 2ϩ ] i elevation triggered by intracellular injection of LPI was GPR55-dependent. However, the Ca 2ϩ response pattern was strikingly different in the two paradigms. Extracellular administration of LPI resulted in a relatively slow response that reached a peak within 1-2 min and continued with a plateau phase, whereas LPI microinjection induced a fast and transitory effect similar to that reported upon activation of other intracellular GPCRs, such as those for angiotensin II (29,33,62) or endothelin 1 (28). We have noticed likewise discrepancies in the plasmalemmal-initiated versus intracellularly initiated Ca 2ϩ responses by the G protein-coupled estrogen receptor GPER/GPR30 (34,63), which is reportedly expressed at both sites (64,65). LPI is a lipid messenger, expected to get partitioned in the lipid bilayer and reach the other side of the sarcolemma. However, LPI cannot passively diffuse across the plasma membrane. Rather, after its intracellular synthesis by the phospholipase A2, LPI is pumped out of the cell via the multidrug resistance-associated protein 1 (Mrp1/ABCC1) (7). Mrp1 is also expressed in the sarcolemma of rodent heart (66,67).
Furthermore, we found that the [Ca 2ϩ ] i rise produced by intracellular administration of LPI is completely contingent on Ca 2ϩ mobilization from internal stores. Importantly, the Ca 2ϩ is released from the endolysosomes via the NAADP-sensitive TPCs (45,55), a signal further amplified by RyR-dependent CICR from the sarcoplasmic reticulum. NAADP-dependent Ca 2ϩ release from acidic stores has been shown to elevate the sarcoplasmic reticulum Ca 2ϩ load, thus enhancing cardiomyocyte contraction (68). Moreover, ␤-adrenoreceptors in ventricular myocytes can signal through NAADP (43,68). In line with these observations, intracellular GPR55 may play a role in the regulation of cardiomyocyte contraction.
The accumulating evidence pointing to endolysosomes as intracellular locations where GPCRs initiate signaling (19,24,28,69) prompted us to probe for colocalization of GFP-tagged GPR55 and RFP-tagged Rab7, an endolysosomal-associated small GTPase (46). We noticed extensive colocalization of GPR55 and Rab7. In addition, lysosomal disruption with bafilomycin A1 markedly reduced the fluorescence intensity of both GPR55 and Rab7, suggesting that GPR55 localizes intracellularly at the endolysosomes. GPR55 localization at this site correlates well with its ability to trigger NAADP-dependent Ca 2ϩ responses because NAADP-generating enzymes (70,71) have been also reported to localize at the membrane of acidic organelles (72).
An important discrepancy in the responses initiated by sarcolemmal versus intracellular GPR55 stimulation is that the former depolarized, whereas the latter hyperpolarized the membrane of neonatal ventricular cardiomyocytes. The depolarizing effect of bath-applied LPI was independent of Ca 2ϩ signaling, whereas LPI induced GPR55-dependent Ca 2ϩ influx via LTCC. A direct effect of LPI on LTCC was considered less likely because it has been reported that, similar to other phospholipids, LPI slightly suppresses LTCC activity (73). The LPIinduced depolarization was not large enough to generate action potentials and, thus, activate LTCC. Rather, the explanation of the LPI-induced effects sits in the fact that cardiac LTCC can be directly activated by Gq (74) because GPR55 is Gq-coupled (17). Conversely, hyperpolarization in response to intracellular GPR55 activation was short-lived and Ca 2ϩ -dependent. Ca 2ϩ extrusion is a fast process, explaining why the hyperpolarization is transient. Similarly, intracellular microinjection of an acknowledged intracrine, angiotensin II, hyperpolarizes ventricular myocytes via intracellular AT 1 receptors (75). Although hyperpolarization may be beneficial in that it may prevent arrhythmias, it could also trigger I f /I h . I f /I h is typically a Na ϩ /K ϩ inward current associated with hyperpolarization-activated cyclic nucleotide-gated channels, playing a pivotal role in cardiac pacemaking but associated with cardiac hypertrophy and failure when expressed in the ventricles (76). LPI has been shown to have GPR55-independent hyperpolarizing effects on the endothelial cell membrane (77). However, in the presence FIGURE 10. Proposed mechanisms initiated by LPI at the sarcolemma or within the neonatal ventricular cardiomyocyte via GPR55. LPI activates GPR55 located on the membrane of endolysosomes (Endo-Lys) to promote NAADP-dependent Ca 2ϩ release from these organelles, which further induces CICR via RyRs located at the SR and converging to membrane hyperpolarization (top, blue). GPR55 stimulation at the sarcoplasmic membrane results, on one hand, in Ca 2ϩ entry through LTCCs and, on the other, in IP 3dependent Ca 2ϩ release from the intracellular stores, a signal further amplified by CICR via RyRs. Independently of Ca 2ϩ signaling, GPR55 activation results in membrane depolarization (bottom, red). of the GPR55 antagonist , LPI administration to neonatal ventricular cardiomyocytes produced non-significant responses, supporting GPR55-dependence of the LPI effect in our paradigm.
In animal models of ischemia-reperfusion injury, deletion of the main LPI-synthesizing enzyme cPLA2␣ has been shown to be either cardioprotective (13) or deleterious to the heart (14).
In light of our current findings, the discrepancy between these results may be correlated with the ability of LPI to activate GPR55 either at the sarcoplasmic membrane or within the cardiomyocyte and trigger distinct mechanisms with opposite outcomes.
Collectively, our study shows for the first time that the LPIresponsive receptor GPR55 is involved in the regulation of cardiomyocyte Ca 2ϩ signaling. Furthermore, functional GPR55 can elicit signaling both at the sarcolemma and at the endolysosomal compartment, and, as shown in Fig. 10, the downstream cascades are distinct. Activation of plasmalemmal GPR55 triggers Ca 2ϩ influx through LTCC and IP 3 -dependent Ca 2ϩ release that is amplified by CICR via RyR. Independently of this Ca 2ϩ signaling cascade, plasmalemmal GPR55 couples to depolarization of the cardiomyocyte membrane. Intracellular GPR55 activation associates with NAADP-dependent Ca 2ϩ mobilization from acidic-like Ca 2ϩ stores, which is amplified by CICR via RyR, converging to membrane hyperpolarization. This work may serve as a platform for future studies exploring the potential of GPR55 as a therapeutic target in cardiac disorders.