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Ligand Activation of Cannabinoid Receptors Attenuates Hypertrophy of Neonatal Rat Cardiomyocytes

Lu, Yan BSc*; Akinwumi, Bolanle C. MSc*; Shao, Zongjun PhD; Anderson, Hope D. PhD*,†

Journal of Cardiovascular Pharmacology: November 2014 - Volume 64 - Issue 5 - p 420–430
doi: 10.1097/FJC.0000000000000134
Original Article
Open

Abstract: Endocannabinoids are bioactive amides, esters, and ethers of long-chain polyunsaturated fatty acids. Evidence suggests that activation of the endocannabinoid pathway offers cardioprotection against myocardial ischemia, arrhythmias, and endothelial dysfunction of coronary arteries. As cardiac hypertrophy is a convergence point of risk factors for heart failure, we determined a role for endocannabinoids in attenuating endothelin-1–induced hypertrophy and probed the signaling pathways involved. The cannabinoid receptor ligand anandamide and its metabolically stable analog, R-methanandamide, suppressed hypertrophic indicators including cardiomyocyte enlargement and fetal gene activation (ie, the brain natriuretic peptide gene) elicited by endothelin-1 in isolated neonatal rat ventricular myocytes. The ability of R-methanandamide to suppress myocyte enlargement and fetal gene activation was mediated by CB2 and CB1 receptors, respectively. Accordingly, a CB2-selective agonist, JWH-133, prevented only myocyte enlargement but not brain natriuretic peptide gene activation. A CB1/CB2 dual agonist with limited brain penetration, CB-13, inhibited both hypertrophic indicators. CB-13 activated AMP-activated protein kinase (AMPK) and, in an AMPK-dependent manner, endothelial nitric oxide synthase (eNOS). Disruption of AMPK signaling, using compound C or short hairpin

RNA knockdown, and eNOS inhibition using L-NIO abolished the antihypertrophic actions of CB-13. In conclusion, CB-13 inhibits cardiomyocyte hypertrophy through AMPK-eNOS signaling and may represent a novel therapeutic approach to cardioprotection.

*Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada; and

Canadian Centre for Agri-Food Research in Health and Medicine, St Boniface General Hospital Research Centre, Winnipeg, Manitoba, Canada.

Reprints: Hope D. Anderson, PhD, Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba R3E 0T5, Canada (e-mail: handerson@sbrc.ca).

Supported by Heart and Stroke Foundation of Canada (Manitoba) and the Canadian Institutes of Health Research.

The authors report no conflicts of interest.

This is an open access article distributed under the terms of the Creative Commons Attribution-Noncommercial-No Derivatives 3.0 License, where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially.

Received February 06, 2014

Accepted June 02, 2014

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INTRODUCTION

Endocannabinoids are bioactive amides, esters, and ethers of long-chain polyunsaturated fatty acids. Two G protein-coupled receptors for endocannabinoids have been identified: CB1 receptors, which are expressed abundantly in the brain,1 but are also detectable in the heart2 and vasculature,3,4 and CB2 receptors, which are abundant in immune5 and hematopoietic cells,6 and are detected in heart at levels comparable with CB1 receptors.7 Endocannabinoid effects are terminated by uptake through a membrane transporter,8–11 followed by hydrolysis by enzymes such as fatty acid amide hydrolase (FAAH)12,13 and monoacylglycerol lipase.14–16 Endogenous CB receptor agonists, together with the proteins that transport and metabolize them, constitute the endocannabinoid system.17 This system has been implicated in multiple central and peripheral functions, leading to interest in therapeutic development associated with manipulating its activity.18

Cardiac hypertrophy is the increased myocardial mass provoked by hemodynamic stress or myocardial injury,19 and is a convergence point for risk factors of heart failure. Prolonged hypertrophy leads to functional decompensation,20–22 so mitigation of this process is considered a promising therapeutic target to prevent heart failure.23

Although components of the endocannabinoid system are detectable in the heart, including the cannabinoid receptor ligand anandamide,24 CB12,25 and CB2 receptors,7 and metabolizing enzymes,26 the effect of endocannabinoids on cardiac hypertrophy remains unclear. There are however reports of cannabinoids being cardioprotective, especially in the context of ischemic insult. CB2 receptors contribute to the ability of lipopolysaccharide to reduce infarct size in ischemic rat myocardium,27 and similar effects such as reduction of infarct size and improved postischemic ventricular recovery have been reported for a number of endogenous and synthetic cannabinoid ligands.28,29 Based on these considerations, we tested the hypothesis that activation of the endocannabinoid system attenuates cardiomyocyte hypertrophy using endothelin-1 (ET1)-treated neonatal rat ventricular myocytes as our experimental paradigm.

We also probed possible signaling mechanisms with particular focus on AMP-activated protein kinase (AMPK). AMPK is a serine/threonine kinase that senses the energy status of the cell and, in response to energy deprivation, coordinates a metabolic response to conserve adenosine triphosphate. Specifically in the heart, AMPK is an important regulator of cardiomyocyte energy homeostasis by mechanisms, which may include (1) increasing fatty acid uptake and oxidation, (2) accelerating glucose uptake, (3) stimulating glycolysis, and (4) shut down of energy-consuming pathways such as protein synthesis.30 There is evidence to suggest that activated AMPK is antihypertrophic. In vitro, AMPK activation blocked cardiomyocyte enlargement, protein synthesis, and hypertrophic gene expression in response to phenylephrine31,32 and angiotensin II,33 as well as signaling through prohypertrophic mediators such as NFAT, NF-κB, and MAPK.34 In vivo, AICAR, a chemical activator of AMPK, attenuated pressure-overload hypertrophy in rats,34 and pressure-overload hypertrophy was exaggerated in AMPKα2 gene knockout mice.35 Also, the ability of resveratrol36 and calorie restriction37 to impede hypertrophy has been attributed to AMPK signaling.

Notably, there is evidence to suggest that endocannabinoids activate AMPK, albeit derived mostly from the brain. Cannabinoid receptor ligands such as 2-AG,38 Δ9-tetrahydrocannabinol,38 and HU21039 increase AMPK phosphorylation and enzymatic activity in rat hippocampus through CB1 and CB2 receptors.39,40 There is less evidence from the heart, although 2-AG38 does activate AMPK. There is likewise evidence of crosstalk between endocannabinoids and NO in the nervous system,41 immune cells,42 adipocytes,43 and vasculature.44 NO contributes to the ability of CB receptors to reduce myocardial infarct size.29,45 Neuronal nitric oxide synthase, endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) are expressed in myocytes,46 and low levels of NO derived from eNOS protect from cardiac hypertrophy.47–51 This is in contrast to the deleterious cardiac effects of iNOS activation in the cytosol, whether due to high NO levels or superoxide/peroxynitrite formation.52–55 As activation of AMPK promotes phosphorylation and activation of eNOS at Ser1177,56,57 we tested the hypothesis that cannabinoid receptor signaling stimulates an AMPK-eNOS signaling axis that contributes to the attenuation of hypertrophy.

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METHODS

Materials

Anandamide, R-methanandamide, JWH-133, ET1, compound C, sarcomeric α-actinin antibody, and β-actin antibody were from Sigma–Aldrich (St. Louis, MO). AM251, AM281, AM630, and L-NIO were from Tocris Cookson (Minneapolis, MN). CB-13 was from Cayman Chemical (MI). Lipofectin, calcein AM (Molecular Probes), and propidium iodide (PI; Molecular Probes) were from Invitrogen (Carlsbad, CA). Texas Red-conjugated horse anti-mouse antibody and VECTASHIELD mounting medium containing DAPI were from Vector Laboratories (Carlsbad, CA). The -1595 human brain natriuretic peptide (BNP)-luciferase reporter construct was kindly provided by Dr. David Gardner (University of California, San Francisco, CA). Antibodies against phosphorylated AMPK (p-AMPK), AMPK, and phosphorylated eNOS (p-eNOS) were from Cell Signaling (Whitby, Canada).

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Animals

This study was conducted according to recommendations from the Animal Care Committee of the University of Manitoba and the Canadian Council of Animal Care. Neonatal rat pups were produced from a larger in house Sprague–Dawley rat breeding colony. The pups were born in an aspen bedding-enriched polycarbonate rat cage suspended in a racking system that forms the cage lid. Pups were subsequently housed alone with the mother. Animals were maintained at 22–25°C, 55%–60% humidity, and a 12-hour light–dark cycle, and allowed free access to water and food (PMI RMH-3000 feed).

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Neonatal Rat Ventricular Myocytes

Ventricular cardiomyocytes were isolated from 1-day-old neonatal Sprague–Dawley rats by digestion of ventricles with several cycles of 0.1% trypsin and mechanical disruption as previously described.58 Cells were cultured on gelatin-coated plates in DMEM containing 10% cosmic calf serum (Hyclone) for 18–24 hours before experimentation.

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Adult Rat Ventricular Myocytes

Adult myocytes were used for analyses of contractile function. Adult male Sprague–Dawley rats (200–250 g) were anesthetized with 3% isoflurane and injected with heparin into the saphenous vein (1000 U/mL at 1 mL/kg body weight). The heart was immediately removed and placed into a perfusion chamber and cannulated through the aorta. The heart was washed of blood with calcium-free buffer (mM: NaCl 90, KCl 10, KH2PO4 1.2, MgSO4 7H2O 5.0, NaHCO3 15, taurine 30, glucose 20, pH 7.4) for 5 minutes. The heart was then perfused for 20 minutes (at 37°C) with calcium-free buffer containing 179 U/mL collagenase II. After perfusion, ventricles were removed, minced, and incubated for 5 minutes at 37°C with recirculated collagenase buffer for further digestion. Isolated cardiomyocytes were then plated on plates precoated with laminin (10 μg/mL) and maintained for 2 hours at 37°C and 5% CO2 in a medium consisting of medium 199 containing 5% fetal bovine serum, 5% horse serum, and 1% penicillin/streptomycin. After 2 hours, the medium was replaced with medium 199 supplemented with 5 mM taurine, 2 mM L-carnitine, 1 mM creatine, 2 μM insulin, and 100 IU/mL penicillin/streptomycin.

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Treatments

As applicable, myocytes were subjected to transfection. Myocytes were then rendered quiescent by serum deprivation (0.5% serum) for 24 hours and pretreated for 1 hour with vehicle, anandamide (0.001–1 μM), R-methanandamide (1 μM), JWH-133 (0.001–1 μM), or CB-13 (0.001–1 μM) in the presence or absence of cannabinoid receptor antagonists (CB1 inhibitors - AM251 [0.1 μM] and AM281 [2 μM] or CB2 inhibitor AM630 [0.1 μM]). After the 1-hour pretreatment, ligands remained in the culture media for the remainder of the experiment. Hypertrophy was stimulated by addition of ET1 (0.1 μM); the length of exposure to ET1 was uniformly 48 hours, except for BNP polymerase chain reaction experiments, where ET1 treatment was 24 hours (which is sufficiently long to elicit a stable hypertrophic response).59 The concentrations of CB antagonists used in these experiments are predicated on reports that sub- or low micromolar concentrations ablate previously described cardiac or vascular effects of endocannabinoids.28,60,61

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Hypertrophic Indicators

Hypertrophy was assessed as previously described.59,62 Briefly, myocyte size was assessed by immunofluorescence, fluorescence microscopy, and computer-assisted planimetry. The -1395 hBNP promoter-luciferase activity and BNP messenger RNA levels determined by real-time polymerase chain reaction were used as markers of fetal gene activation.

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Lentiviral Preparation and Infection

Lentiviral vectors expressing short hairpin (shRNA) against AMPKα1 and α2 were obtained from the University of Manitoba OpenBiosystems library (AMPKα1: TRCN0000000860, TRCN0000024003; AMPKα2: V2LMM_73,754, V2LMM_71,195). Scrambled sequences served as non-silencing controls. Lentivirus vector plasmids were co-transfected with psPAX2 (packaging) and pMD2.G (enveloping) vectors using FuGENE6 Reagent (Roche; Indianapolis, IN). High-titer lentiviral stock was produced in HEK-293T cells 48 hours after transfection. Myocytes were infected for 24 hours by application of the lentivirus to the culture medium, and then cultured for a further 72 hours (to achieve knockdown) prior to treatments and further experimentation. Knockdown was confirmed by western blotting.

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Western Blotting

Cell lysates were prepared in RIPA buffer, clarified by centrifugation, and p-AMPK, AMPK, and p-eNOS were detected by conventional western blotting. Membranes were stripped and reprobed with β-actin antibody to account for loading variations among lanes.

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Measurement of Cardiomyocyte Viability

Cardiomyocyte viability was assessed by double staining with calcein AM and PI. Calcein AM is a membrane-permeant dye that is converted to a green-fluorescent calcein by intracellular esterases in viable cells. PI is a membrane-impermeant intercalating agent. Although excluded from viable cells with intact membranes, PI enters dead cells and fluoresces on binding to nucleic acids. After treatments and removal of media, 400 μL of a mixture of 3 μM calcein AM and 2.5 μM PI in warm phosphate-buffered saline was added to each well (24-well plate, 350,000 cells per well). The following were used as controls: background—no cells + calcein AM/PI; live controls—vehicle-treated cells; dead cell controls—cells treated with 0.2% Triton X-100 (15 minutes). Following dark incubation (37°C, 30 minutes), fluorescence was measured using a plate reader using excitation/emission wavelengths 485 nm/535 nm for calcein and 530 nm/620 nm for PI.

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Measurement of Ventricular Myocyte Contractile Function

Contractile properties of adult rat cardiomyocytes were assessed using a video-based edge-detection system (Ionoptix HyperSwitch Myocyte System). Cardiomyocytes were cultured on coverslips (0.3 × 106 cells per coverslip) and rendered quiescent. After treatments (as described in figures), coverslips were placed on a chamber mounted on the stage of an inverted microscope and perfused with a buffer containing (in mM): 131 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, at pH 7.4 and maintained at 37°C. Cells were stimulated to contract using the IonOptix Myopacer at a frequency of 0.5 Hz. Cardiomyocytes were displayed on a monitor display using an IonOptix Myocam camera. SoftEdge software (IonOptix) was used to compare changes in cell length during shortening (contraction) and relengthening (relaxation). Indices used to evaluate cell contractility included maximal velocity of shortening (+dL/dt) and maximal velocity of relengthening (−dL/dt). These are representations of systolic contraction and diastolic relaxation, respectively. Contractility was also measured as peak shortening.

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Statistics

Data are presented as mean ± SEM. All data were subjected to 1-way analysis of variance followed by a Newman–Keuls Multiple Comparison test to detect between-group differences. P < 0.05 was considered significant.

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RESULTS

Anandamide Suppresses ET1-dependent Induction of Markers of Hypertrophy

At the cardiomyocyte level, hypertrophy is characterized by increases in cell size, protein synthesis, sarcomeric reassembly, and changes in gene expression.63 As reinduction of fetal genes such as BNP is one of the most consistent markers of hypertrophy, BNP gene expression and BNP promoter–reporter constructs are used as experimental indicators of hypertrophy.64 ET1 treatment (0.1 μM; 24 hours) elicited cardiomyocyte hypertrophy, as evidenced by significant enlargement of myocytes (Fig. 1A) and activation of the BNP promoter (Fig. 1B). These ET1-induced indicators of hypertrophy were attenuated by anandamide.

FIGURE 1

FIGURE 1

We also considered the possibility that these effects might be related to uptake and metabolism of anandamide through the arachidonic acid cascade. However, R-methanandamide, which is a non-hydrolyzable, metabolically stable analog of anandamide,65 also suppressed ET1-dependent myocyte enlargement (Fig. 1C) and BNP promoter activity (Fig. 1D). These results suggest that ligand activation of CB receptors suppresses cardiomyocyte hypertrophy.

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CB1 and CB2 Receptors Mediate Distinct Aspects of the Antihypertrophic Actions of R-methanandamide

To determine which receptor mediates the antihypertrophic effects of R-methanandamide, we used selective pharmacological antagonists of CB1 and CB2 receptors. AM251 is a selective CB1 receptor antagonist.18 Ki values for AM251 are CB1 = 7.49 nM versus CB2 = 2290 nM.66 In contrast, AM630 is selective for CB2 receptors; Ki values are CB1 = 5152 nM versus CB2 = 31.2 nM.18,67 The ability of R-methanandamide to suppress myocyte enlargement was abolished by AM630 (Fig. 2A), whereas suppression of BNP promoter activation was abolished by AM251 (Fig. 2B). Given reports that AM251 might also act as a GPR55 agonist, we verified the involvement of CB1 receptors using AM281. In fact, the effects of AM281 agree with those of AM251. As with AM251, AM281 failed to affect the ability of R-methanandamide to suppress myocyte enlargement (data not shown). In contrast, AM281 abolished suppression of BNP gene activation by R-methanandamide (ie, in the presence of AM281, ET1: 284% ± 70%; ET1 + R-methanandamide: 365% ± 70%, both P < 0.05 vs. control). These results show dissociation of the trophic effect from the gene expression effect of ET1 in cardiomyocytes, and that agonism of both CB receptor subtypes is necessary to attenuate both hypertrophic events.

FIGURE 2

FIGURE 2

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Effects of a CB2-selective Ligand and a Peripherally Restricted CB1/CB2 Dual Agonist on Cardiomyocyte Hypertrophy

Clinically relevant cannabinoid-based therapeutic development has been hindered by the central psychoactive effects of CB1 receptors.68–70 Alternate strategies like using CB2-selective agonists or CB1/CB2 dual agonists with limited brain penetration have been proposed.68–71 Our data (Fig. 3) suggest that selective activation of CB2 receptors might not be sufficient to prevent all aspects of cardiac hypertrophy. Indeed, JWH-133, a selective CB2 receptor agonist (Ki: CB1 = 677 nM vs. CB2 = 3.4 nM),72 attenuated ET1-induced myocyte enlargement (Fig. 3A), but not ET1-induced BNP promoter activation (Fig. 3B). We therefore assessed the effects of a peripherally restricted dual CB1/CB2 agonist. CB-13 is a nonselective CB1/CB2 agonist with limited brain penetration (Ki: CB1 = 6.1 nM vs. CB2 = 27.9 nM).73,74 Peripheral administration of CB-13 yields extremely low brain-to-plasma concentration ratios, and is associated with analgesic effects that are not central nervous system (CNS)-mediated, but rather identified as peripheral site-of-action.73 As shown in Figure 4, CB-13 attenuated both indicators of hypertrophy (ie, cell enlargement and BNP gene activation).

FIGURE 3

FIGURE 3

FIGURE 4

FIGURE 4

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Effects of CB-13 on Cardiomyocyte Viability and Contractile Function

The use of micromolar concentrations of CB receptor ligands in our experiments is predicated on previous reports that their protective effects against ischemia in rat hearts occur within the low micromolar concentration range.28,29 Indeed, our dose response data confirm that the antihypertrophic actions of anandamide (Fig. 1A), JWH-133 and CB-13 (Figs. 3A, 4A) are exhibited at low micromolar concentrations. We found that micromolar CB-13 had no adverse effects on myocyte viability (Fig. 5). Moreover, although ET1 treatment prolonged shortening and relengthening velocities, CB-13 had no adverse effects on contractile function either in untreated or ET1-treated cardiomyocytes (Table 1).

FIGURE 5

FIGURE 5

TABLE 1

TABLE 1

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AMPK-eNOS Signaling Contributes to CB-13 Effects

CB-13 significantly increased phosphorylation of AMPKα at Thr172 (Fig. 6A), which is an indicator of AMPK activation status.75,76 Consistent with reports that activated AMPK promotes phosphorylation and activation of eNOS at Ser1177,56,57 CB-13 also activated eNOS (Fig. 6B). We next performed knockdown of AMPKα1/2 to ascertain its role in eNOS phosphorylation by CB-13. Infection of cardiomyocytes with lentiviral constructs expressing shRNA against AMPKα1 and AMPKα2 produced significant, simultaneous reductions to 17% ± 6% and 28% ± 14%, respectively (n = 3, P < 0.05). The ability of CB-13 to induce eNOS phosphorylation was abolished by AMPK knockdown (Figs. 6C, D). These findings suggest that CB-13 stimulates AMPK-eNOS signaling.

FIGURE 6

FIGURE 6

Moreover, the ability of CB-13 to attenuate cardiomyocyte hypertrophy was abolished by disruption of AMPK signaling using a chemical inhibitor (compound C/dorsomorphin; Ki = 109 nM; 1 μM) or by shRNA knockdown of AMPKα1/2 when normalized to their respective controls, (Fig. 7). The selective eNOS inhibitor N5-(1-iminoethyl)-L-ornithine (L-NIO; IC50 = 500 nM; 10× and 5× less potent at neuronal nitric oxide synthase and iNOS, respectively; 1 μM)77 also ablated the antihypertrophic effects of CB-13 (Fig. 7). It bears mentioning that shRNA knockdown of AMPKα1/2 and L-NIO increased baseline cardiomyocyte size to 161% ± 20% (n = 3, P < 0.05) and 159% ± 17% (n = 5, P < 0.01) versus control, respectively, suggesting basal antigrowth activities of AMPK and eNOS in cardiomyocytes. Collectively, these findings indicate that CB-13 attenuates cardiomyocyte hypertrophy through AMPK-eNOS signaling.

FIGURE 7

FIGURE 7

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DISCUSSION

To the best of our knowledge, this study shows for the first time that ligand activation of cannabinoid receptors attenuates hypertrophy of isolated cardiomyocytes through AMPK-eNOS signaling. Anandamide, a naturally occurring endocannabinoid,78 and 3 synthetic CB receptor ligands (R-methanandamide, JWH-133, and CB-13) prevented ET1-induced cardiomyocyte enlargement. As cardiac hypertrophy is a major risk factor for heart failure, we further investigated here the signaling mechanisms that underlie the antihypertrophic effects of CB receptors.

We first investigated the role of CB1 and CB2 receptors and found that distinct CB receptor subtypes mediate the antihypertrophic actions of R-methanandamide. In fact, selective antagonism of CB receptor subtypes uncoupled the inhibitory effects on ET1-induced myocyte growth from effects on hypertrophic gene expression. In particular, R-methanandamide prevents myocyte enlargement through CB2 receptors and was sensitive to AM630. In contrast, inhibition of fetal gene activation trafficked through CB1 receptors and was sensitive to AM251. We also considered the possibility that the ability of AM251 to block R-methanandamide effects might be due to agonism of GPR55 rather than antagonism of CB1.79 However, it is likely that AM251 was functioning here solely as a CB1 receptor antagonist. First, the concentration of AM251 that blocked R-methanandamide effects on BNP gene activation was 0.1 μM in accordance with its Ki at CB1 receptors (7.5 nM)80. In contrast, Kapur et al79 demonstrated GPR55-mediated effects of AM251 at 30 μM, with an EC50 of ∼10 μM, whereas at 0.1 μM, and even a logarithmic increment higher at 1.0 μM, AM251 failed to invoke GPR55 signaling. This information strongly suggests that the inhibitory effects of AM251 are attributable to antagonism of CB1 receptors. In support of this notion, we verified that AM281, a CB1 receptor antagonist lacking effects on GPR55, also abolished the ability of R-methanandamide to suppress BNP gene activation.

There exist literature precedents in which morphological changes are uncoupled from the hypertrophic gene program. For example, Thorburn et al81 reported that in isolated myocytes, ERK signaling mediates phenylephrine-dependent ANP promoter activation but not organization of contractile proteins such as actin. Furthermore, AP-1, and in particular c-Fos, is a key mediator of hypertrophic gene expression but not myocyte growth82. In vivo, activated GSK-3β can dissociate the expression of hypertrophic genes from cardiac growth overexpression; transgenic mice that overexpress calcineurin exhibit cardiac growth and BNP expression, whereas coexpression of activated GSK-3β inhibits cardiac growth but not BNP expression.83 Interestingly, there is evidence that endocannabinoids might interfere with c-Fos signaling84 through CB1 receptors85. We therefore speculate that activated CB1 receptors may attenuate BNP expression by suppressing ERK-AP-1 signaling, though this remains to be determined.

Selective agonism of CB2 receptors with JWH-133 suppressed myocyte enlargement but failed to prevent fetal gene activation. In contrast, we found that the antihypertrophic actions of a dual CB1/CB2 agonist, CB-13, extended beyond those of JWH-133, such that both myocyte enlargement and BNP gene expression were inhibited. These findings are in agreement with other reports of cardioprotective attributes that pertain predominantly to activation of CB2 receptors such as protection from ischemic insult,27–29,86 antiarrhythmic effects,87 and prevention of endothelial dysfunction of coronary arteries.7,88 However, although CB2 signaling may be cardioprotective, there is also evidence that activation of CB1 receptors is associated with dysfunction of vascular endothelium,89 exerts proatherosclerotic actions,90–92 and promotes oxidative stress, inflammation and/or cell death in vitro93 and in pathological conditions such as diabetic cardiomyopathy94 and doxorubicin-induced cardiomyopathy.95,96 Our data agree with the notion that sole activation of CB1 receptors is cardio-deleterious; in the context of cardiomyocyte hypertrophy, myocyte enlargement was suppressed by CB2 receptors, whereas only fetal gene activation was prevented by CB1 receptors. Thus, in the presence of sole activation of CB1 receptors and the absence of CB2 signaling, myocyte hypertrophy would persist and potentially give rise to adverse endpoints such as ischemia (vis-à-vis reduced capillary density), myocyte misalignment, and cardiac stiffening. Activation of CB2 receptors is therefore a necessary component of cannabinoid-based antihypertrophic therapy, which we speculate may ameliorate adverse effects of unopposed activation of CB1 receptors alone.

CB-13 stimulated phosphorylation of AMPKα (Thr172) and eNOS (Ser1177) at known activation sites (Figs. 6A, B),56,57,75,76 and we determined that eNOS phosphorylation is downstream of AMPK since it was inhibited by AMPKα knockdown (Figs. 6C, D). Inhibition of AMPK or eNOS signaling abolished the antihypertrophic effects of CB-13 (Fig. 7). Taken together, our findings suggest that signaling through AMPK-eNOS crosstalk might play a role in the antihypertrophic effects of CB receptors. This is consistent with reports by others, in which AMPK-eNOS crosstalk underlies the antigrowth effects of non-cannabinoid interventions such as metformin,97 calorie restriction,37 and resveratrol.98 Interestingly, phosphorylative activation of AMPK and eNOS peaked by 4 hours, and returned to baseline by 24 hours. This suggests that AMPK-eNOS crosstalk might occur at a pivotal, early point with downstream effects that will collectively prevent the hypertrophic response. Attenuation of prohypertrophic RhoA/RhoA kinase (ROCK) signaling might be one such downstream event. These members of the Rho family GTPases are well-established mediators of cardiac hypertrophy,99–101 and Hunter et al102 reported that NO inhibits ET1-induced cardiac myocyte hypertrophy by blocking the RhoA/ROCK cascade. Moreover, CB receptor activation inhibits RhoA signaling.103–105 We therefore speculate that CB-13, by transiently activating AMPK/eNOS crosstalk, elicits NO-dependent blockade of RhoA/ROCK, although the effects of CB-13 on RhoA/ROCK signaling remain to be determined.

We tested the antihypertrophic potential of a wide range of concentrations of anandamide (Fig. 1A), JWH-133 (Fig. 3A), and CB-13 (Fig. 4A). Clearly, low micromolar concentrations are required; CB-13 partially attenuated or completely abolished ET1-induced myocyte enlargement at 0.1 and 1 μM, respectively. Although seemingly high, these concentrations are achievable in blood as CB-13 exhibits good oral bioavailability. Dziadulewicz et al73 reported that following oral administration of CB-13 (3 mg/kg), a Cmax of 1.13 μM was observed at 1 hour postdose, whereas a maximal brain concentration of only 0.24 μmol/kg was reached 4 hours postdose. Moreover, CB-13 exerted biological effects (antihyperalgesia) in a rat model of neuropathic pain, but produced no CNS effects at this dose.73 Importantly, our results suggest that this concentration does not adversely affect cardiomyocyte viability (Fig. 4). Also, although ET1 reduces conduction velocity in isolated myocytes,106 and this is associated with increased expression and density of L-type Ca2+ channels,107 CB-13 did not worsen contractile function (Table 1). Therefore, our experimental concentration of CB-13 (1 μM) should be physiologically attainable and relevant.

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CONCLUSIONS

There is significant interest in manipulation of the endocannabinoid system as a therapeutic approach to treat disorders such as metabolic syndrome, inflammatory and neuropathic pain, and multiple sclerosis.68–70 Unfortunately, therapeutic use of cannabinoids is impeded by psychotropic side effects including dysphoria, memory impairment, reduced concentration, disorientation, motor incoordination, and possibly addiction.68,69 This undesirable psychoactivity is mediated by CNS CB1 receptors,68,69,108,109 so alternate strategies like using CB2-selective agonists and/or peripherally restricted CB1/CB2 dual agonists have been proposed.68–71 Activation of the endocannabinoid system remains a viable strategy to prevent cardiac hypertrophy, a major risk factor for heart failure. However, our findings suggest that to achieve cannabinoid-based cardioprotection devoid of undesirable central CB1-mediated side effects, the best approach warranting further study would be a CB1/CB2 receptor dual agonist with negligible brain penetration.

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ACKNOWLEDGMENTS

The authors thank Lam Dang and Ping Lu for technical assistance.

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Keywords:

cardiac hypertrophy; cardiac myocyte; endocannabinoid; anandamide; AMPK

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