Activation of Cannabinoid Receptors Attenuates Endothelin-1-Induced Mitochondrial Dysfunction in Rat Ventricular Myocytes.

Evidence suggests that the activation of the endocannabinoid system offers cardioprotection. Aberrant energy production by impaired mitochondria purportedly contributes to various aspects of cardiovascular disease. We investigated whether cannabinoid (CB) receptor activation would attenuate mitochondrial dysfunction induced by endothelin-1 (ET1). Acute exposure to ET1 (4 hours) in the presence of palmitate as primary energy substrate induced mitochondrial membrane depolarization and decreased mitochondrial bioenergetics and expression of genes related to fatty acid oxidation (ie, peroxisome proliferator-activated receptor-gamma coactivator-1α, a driver of mitochondrial biogenesis, and carnitine palmitoyltransferase-1β, facilitator of fatty acid uptake). A CB1/CB2 dual agonist with limited brain penetration, CB-13, corrected these parameters. AMP-activated protein kinase (AMPK), an important regulator of energy homeostasis, mediated the ability of CB-13 to rescue mitochondrial function. In fact, the ability of CB-13 to rescue fatty acid oxidation-related bioenergetics, as well as expression of proliferator-activated receptor-gamma coactivator-1α and carnitine palmitoyltransferase-1β, was abolished by pharmacological inhibition of AMPK using compound C and shRNA knockdown of AMPKα1/α2, respectively. Interventions that target CB/AMPK signaling might represent a novel therapeutic approach to address the multifactorial problem of cardiovascular disease.

INTRODUCTION

Endocannabinoids are endogenous polyunsaturated fatty acids that bind to cannabinoid receptors to elicit physiological effects. These G-protein–coupled receptors include CB1 and CB2 receptor subtypes. Both CB1 and CB2 receptors are present in the cardiovascular system (ie, heart[1,2] and blood vessels[3,4]), although CB1 and CB2 receptors are also profusely expressed in the brain[5] and immune cells.[6]

Aside from CB receptors, other constituents of the endocannabinoid signaling system are present in the heart. These include endocannabinoid ligands, such as arachidonoylethanolamide (AEA or anandamide)[7] and 2-arachidonoylglycerol (2-AG),[8] and the enzyme fatty acid amide hydrolase,[9,10] which hydrolyzes anandamide and 2-AG.[10,11]

Manipulating the endocannabinoid system may be a salutary approach worth pursuing, as endocannabinoid signaling plays a diverse role in modulating central and peripheral physiology.[12] In fact, extant evidence suggests that cannabinoids are protective in the ischemic heart. For example, infarct size is reduced by endogenous and synthetic cannabinoid agonists,[13,14] reportedly via CB2,[15] and this was associated with recovery of ventricular function.[13,14] We also previously reported that cannabinoid receptor signaling prevents cardiac myocyte hypertrophy.[16] Here, we extended these findings by determining the effects of CB receptor activation on mitochondrial function.

The heart requires a large amount of adenosine triphosphate (ATP), for which mitochondria are the major source. In the healthy heart, cardiac myocytes use predominantly fatty acids as energy substrate, and this accounts for 50%–70% of total ATP production.[17,18] The electron transport chain (ETC) embedded within the mitochondrial inner membrane is responsible for oxidative phosphorylation, thereby yielding approximately 95% of the total ATP.[19] Accordingly, mitochondria constitute a significant 30% of cardiac myocyte volume.[20] Given this key role, mitochondrial dysfunction has been linked to ischemia reperfusion injury,[21] cardiomyopathy,[22] left ventricular hypertrophy,[23] and heart failure.[24]

Here, we determined the early effects of endothelin-1 (ET1) on mitochondrial membrane permeability, membrane polarization, and bioenergetics in cardiac myocytes, in the presence of fatty acids as primary energy substrate. Furthermore, we tested the hypothesis that CB receptor signaling would rescue mitochondrial function and probed the signaling pathway(s) involved.

MATERIALS AND METHODS

Materials

Endothelin-1 (ET1), compound C, β-actin antibody, l-carnitine hydrochloride, oligomycin, carbonylcyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), rotenone, antimycin A, and etomoxir were from Sigma Aldrich (St Louis, MO). CB-13 and the JC-1 mitochondrial membrane potential assay kit were from Cayman Chemical (Ann Arbor, MI). Calcein-AM (Molecular Probes) and carnitine palmitoyltransferase (CPT)-1β primers were from Life Technologies (Carlsbad, CA). p-AMPKα (Thr172) and AMPKα antibodies were from Cell Signaling (2535S and 2603S, respectively; Whitby, Canada). CB1 and CB2 antibodies were from Abcam (ab23703 and ab45942, respectively; Toronto, Canada). Proliferator-activated receptor-gamma coactivator (PGC)-1α antibody was from EMD Millipore (ST1202; Temecula, CA). XF24 FluxPaks were from Agilent (Santa Clara, CA).

Neonatal Rat Ventricular Myocytes

This study was approved by the University of Manitoba Animal Care Committee and follows Canadian Council of Animal Care guidelines. Ventricular myocytes were isolated from 1-day-old neonatal Sprague Dawley rats by digestion with several cycles of 0.1% trypsin and mechanical disruption, as previously described.[25] Cells were cultured on gelatin-coated plates in Dulbecco's Modified Eagle Medium containing 10% cosmic calf serum (Hyclone) for 18–24 hours before experimentation.

Treatments

As applicable, myocytes were subjected to lentiviral infection. Myocytes were rendered quiescent by serum deprivation for 24 hours and then exposed to ET1 (0.1 µM; 4 hours) in the presence or absence of vehicle, CB-13 (1 μM), and/or a chemical inhibitor of AMPK (compound C; 1 μM; 1 hour). The concentration of ET1 (0.1 µM) is predicated on our extensive use of neonatal rat cardiac myocytes treated with ET1 as our experimental paradigm of cardiac myocyte hypertrophy.[26-28] Accordingly, we used this experimental paradigm to generate our finding that ligand activation of cannabinoid receptors attenuates hypertrophy of neonatal rat cardiomyocytes.[16] Here, this concentration of ET1 increased hypertrophic parameters (myocyte cell size and fetal gene activity) in an AMPK-dependent manner.[16] The 4-hour incubation time is based first, on the literature precedent in which Sun et al[29] reported disruption of mitochondrial function in pulmonary arterial endothelial cells after 4 hours, and second, time course experiments, in which we found that CB-13 significantly activated AMPK at 4 hours.[16] Ligands remained in the culture media for the remainder of the experiment. CB-13 is a nonselective CB1/CB2 agonist with limited brain penetration (Ki: CB1 = 6.1 nM vs. CB2 = 27.9 nM).[30,31] The concentration of CB-13 was selected based on, first, our finding that it attenuates myocyte hypertrophy,[16] and second, that micromolar plasma concentrations are achievable following oral administration of CB-13 (3 mg/kg).[30] Levels of CB1 and CB2 receptor expression are unaffected by CB-13 in the presence or absence of ET1 (see Figure S6, Supplemental Digital Content 1, http://links.lww.com/JCVP/A425).

shRNA Knockdown of AMPKα

Lentiviral vectors expressing shRNA against AMPK α1 and α2 were prepared, as previously described.[16] Myocytes were infected for 24 hours, and then cultured for a further 72 hours to allow knockdown before further experimentation. Degree of knockdown was confirmed by Western blotting (see Figure S5, Supplemental Digital Content 1, http://links.lww.com/JCVP/A425).

Measurement of Mitochondrial Respiration

An XF24 Analyzer (Agilent) was used to measure mitochondrial bioenergetics. The XF24 creates a reversible 7-μL enclosure above cells; this facilitates real-time monitoring of oxygen consumption rate (OCR) before reequilibration with the unenclosed incubation medium.[32,33] Fatty acid–dependent OCRs, driven by palmitate/bovine serum albumin (BSA) conjugate, were measured under basal conditions. Briefly, myocytes were seeded at 100,000 cells per well and exposed to assay media 1 hour before the assay. Independent experiments were conducted using distinct neonatal myocyte preparations to generate an n-value ≥3 (5 replicates per n-value). Krebs–Henseleit buffer containing l-carnitine hydrochloride (0.4 mM), glucose (2.5 mM), and palmitate/BSA conjugate (200 μM) was used to measure fatty acid–dependent respiration. Following the measurement of basal OCR, oligomycin (1 μg/mL), FCCP (palmitate assays—10 µM), and rotenone + antimycin A (1 µM each) were sequentially injected. Oligomycin is an ATP synthase inhibitor and eliminates OCR associated with ATP synthesis (ATP-linked OCR). Remaining OCR represents oxygen consumption attributable to proton leak. Mitochondrial coupling efficiency is the ratio of ATP-linked OCR to basal OCR. FCCP is a protonophore that uncouples the ETC and allows protons to flow back into the mitochondrial matrix to reduce oxygen. Thus, OCR in the presence of FCCP reflects maximal respiratory capacity, and the difference between maximal OCR and basal OCR reflects spare respiratory capacity. Finally, rotenone + antimycin A abolishes electron flow through complexes I–III, preventing oxygen consumption by cytochrome c oxidase. The remaining OCR in this case is the result of nonmitochondrial respiration.[33] Nonmitochondrial respiration was subtracted during the calculation of all bioenergetic parameters, as previously described.[32,33] The CPT1 inhibitor, etomoxir (40 µM), was used to verify that fatty acid–dependent respiration was the result of oxidation of exogenous palmitate, and BSA served as negative control. OCR is represented as picomoles per minute per 10-µg protein and is expressed as % untreated control. The aforementioned experimental conditions are predicated on optimization experiments (see Figures S1–S4, Supplemental Digital Content 1, http://links.lww.com/JCVP/A425), showing that OCR peaked at a seeding density of 75,000–100,000 cells per well, as previously reported,[34] that reduction in OCR by oligomycin is concentration dependent, and that the maximal effect of FCCP on fatty acid oxidation (FAO) was achieved at 10 μM. Also, etomoxir (40 µM) dramatically inhibited palmitate-related OCR, and BSA only generated negligible OCR, thereby confirming that oxygen consumption was the result of oxidation of exogenous palmitate.

Measurement of Mitochondrial Membrane Permeability Transition

Calcein-AM and CoCl2 dual staining is a well-established method used to assess the extent of membrane permeability transition (mPT) in intact cells.[35] Calcein-AM is a membrane-permeant dye that, upon entry into the cell, is converted to green fluorescent calcein by intracellular esterases. CoCl2 selectively quenches cytosolic calcein fluorescence, thereby improving detection of bright fluorescent calcein puncta within mitochondria. Mitotracker-Red (0.1 μM) was also coloaded, and overlap between calcein and Mitotracker-Red verified mitochondrial localization of calcein puncta (images not shown). When mPT pores (pPTPs) open, calcein leaks from mitochondria into the cytosol, thereby decreasing the fluorescence contrast between mitochondria and cytosol.

Myocytes were cultured in 48-well plates (0.25 × 106 cells/well) and pretreated with CB-13 (1 μM; 1 hour) or vehicle in Krebs–Henseleit buffer supplemented with palmitate/BSA substrates (200 μM), l-carnitine hydrochloride (0.4 mM), and glucose (2.5 mM). Myocytes were then coloaded with calcein-AM (2 μM), CoCl2 (2 mM), and MitoTracker-Red (0.1 μM) for 15 minutes, followed by a 5-minute wash with phosphate-buffered saline. The excitation/emission wavelengths for calcein and MitoTracker-Red are 494/517 nm and 579/599 nm, respectively. Images were acquired using an Olympus inverted fluorescence microscope before and after treatment (ie, 0 and 15 minutes) with ET1 (0.1 μM) or vehicle. Fluorescence contrast was determined as the difference in fluorescent calcein intensity between mitochondria (eg, puncta) and cytosol using Image J. For each replicate, images were captured for 5 myocytes, and within each myocyte, fluorescent calcein intensity was quantified in 3 separate fields within mitochondria. Results are presented as percent values of posttreatment:pretreatment fluorescence contrast. Cells were incubated with ionomycin (2 μM) for 5 minutes at the end of the experiment; this causes mPT and served as positive control.

Mitochondrial Membrane Potential (Δψm) Imaging

The lipophilic fluorescent probe JC-1 was used to investigate changes in Δψm, as per the manufacturer's protocol. In healthy mitochondria with relatively high Δψm, JC-1 concentrates as J-aggregates and emits red fluorescence. In contrast, in mitochondria with reduced Δψm, JC-1 presents mainly as monomeric form because of decreased concentration and emits green fluorescence. The ratio of aggregate to monomer fluorescence serves as an indicator of changes in Δψm.

Myocytes were loaded with JC-1 for 60 minutes at 37°C in Krebs–Henseleit buffer containing palmitate/BSA substrates (200 μM), l-carnitine hydrochloride (0.4 mM), and glucose (2.5 mM) to assess fatty acid–dependent Δψm. Myocytes were then washed with phosphate-buffered saline for 5 minutes, and images were acquired using an Olympus inverted fluorescence microscope. Samples were excited at 485 nm for monomer fluorescence and at 560 nm for JC-1 aggregate fluorescence. Emission fluorescence images were recorded at 535 nm for JC-1 monomer and 595 nm for JC-1 aggregates. Fluorescence intensity was also quantified using a SpectraMax Gemini XS fluorescence microplate reader. Also, as JC-1 may respond to plasma membrane depolarization, FCCP (1 μM) was added at the end of each experiment to achieve maximal dissipation of Δψm and served as positive control.

Western Blotting

Myocytes were cultured in 6-well plates (2 × 106 cells/well). Following treatments, cell lysates were prepared in radioimmune precipitation assay buffer and clarified by centrifugation. Antibodies against p-AMPK (1:1000), AMPK (1:1000), and PGC-1α (1:1000) were used for the detection by conventional Western blotting. Membranes were stripped and reprobed with β-actin antibody to account for loading variations among lanes.

RNA Extraction and Real-Time Polymerase Chain Reaction

Myocytes were cultured in 12-well plates (1 × 106 cells/well). Following treatments, total RNA was extracted from myocytes using the RNeasy mini kit (QIAGEN, Hilden, Germany). Real-time polymerase chain reaction (PCR) was performed using the iScript One-Step RT-PCR SYBR Green kit (Bio-Rad, ON, Canada) in the presence of CPT-1β primers (forward: 5′-CTTCTCAGTATGGTTCATCTTCTC-3′; reverse: 5′-CGAACATCCACCCATGATAG-3′). Glyceraldehyde 3-phosphate dehydrogenase was employed as the internal control (forward: 5′-CTCATGACCACAGTCCATGC-3′; reverse: 5′-TTCAGCTCTGGGATGACCT-3′).

Statistics

Data are presented as mean ± SEM. As applicable, 1-way analysis of variance, followed by a Newman–Keuls or Dunn Multiple Comparison test, was used to detect between-group differences. P value of < 0.05 was considered significant.

RESULTS

CB-13 attenuates ET1-induced aberrations of FAO-related mitochondrial bioenergetics. As shown in Figure 1, ET1 reduced a number of bioenergetic parameters pertaining to FAO using palmitate, including (vs. control) basal OCR (82% ± 5%; P < 0.05), coupling efficiency (86% ± 6%; P < 0.05), maximal (78% ± 4%; P < 0.01) and spare (72% ± 5%; P < 0.01) respiratory capacity, and respiratory control ratio (81% ± 5%; P < 0.01). Basal OCR consists of both ATP-linked and proton leak–linked OCR; Figures 1C, D suggest that reduction in basal OCR was solely attributable to a decrease in ATP-linked OCR (74% ± 7%; P < 0.05 vs. control). CB-13 pretreatment partially attenuated the depression of basal OCR (95% ± 3%, not significant (ns) vs. control nor ET1) and coupling efficiency (97% ± 2%, ns vs. control nor ET1), and significantly restored maximal (97% ± 5%, P < 0.05 vs. ET1) and spare respiratory capacity (97% ± 4%, P < 0.01 vs. ET1), as well as respiratory control ratio (94% ± 2%, P < 0.05 vs. ET1). Proton leak–related OCR was unaffected by either ET1 or CB-13.

FIGURE 1.

CB-13 attenuates ET1-induced depression of FAO-related respiration. Serum-deprived myocytes were pretreated with CB-13 (1 μM; 2 hours) followed by the addition of ET1 (0.1 μM; 4 hours) and provided palmitate/BSA conjugates (200 μM) as energy substrate. A, Representative plots. Left panel B–G, quantitative data demonstrate that ET1 reduced (B) basal OCR, (C) ATP-linked OCR, (E) coupling efficiency, (F) maximal, and (G) spare respiratory capacity, as well as (H) respiratory control ratio. CB-13 attenuated ET1 effects. D, Proton leak–linked OCR was unaffected by ET1 or CB-13. Right panel B–G, quantitative data demonstrate that the ability of CB-13 to attenuate ET1-induced reductions in (B) basal OCR, (C) ATP-linked OCR, (E) coupling efficiency, (F) maximal, and (G) spare respiratory capacity were attenuated, at least in part, by compound C. D, Proton leak–linked OCR and (H) respiratory control ratio were unaffected. n = 4–7 (5 replicates/n); *P < 0.05 and **P < 0.01 versus control (open bars); ns = not significant; †P < 0.05 and ‡P < 0.01 versus ET1. Mean ± SEM.

AMPK contributes to CB-13–dependent correction of FAO-related mitochondrial bioenergetics in hypertrophied myocytes. AMPK maintains or promotes ATP production by improving FAO.[36,37] Thus, we queried whether AMPK mediates preservation of FAO by CB-13. CB-13 effects on FAO-dependent bioenergetics in ET1-treated myocytes were abolished by a chemical inhibitor of AMPK, compound C. We first determined that compound C treatment alone (1 µM) did not affect bioenergetic parameters (data not shown). However, in the presence of compound C, CB-13 failed to rescue (vs. control) basal OCR (66% ± 6%; P < 0.01), ATP-linked OCR (64% ± 9%; P < 0.01), and maximal (67% ± 4%; P < 0.01) and spare (65% ± 6%; P < 0.01) respiratory capacity (Fig. 1) in ET1-treated myocytes. Interestingly, fatty acid–related respiration was also impaired in the CB-13 + compound C group (vs. control), as shown by reduced basal OCR (81% ± 3%; P < 0.05), ATP-linked OCR (77% ± 4%; P < 0.05), coupling efficiency (92% ± 2%; P < 0.05), maximal (78% ± 4%; P < 0.01) and spare (71% ± 7%; P < 0.01) respiratory capacity, and respiratory control ratio (88% ± 2%; P < 0.01) (Fig. 1).

ET1-Induced mPT is Prevented by CB-13

Myocytes were first pretreated with CB-13 or its vehicle, dimethyl sulfoxide, followed by loading of calcein-AM and CoCl2. Images were acquired before (t = 0 minutes) and after treatment (t = 15 minutes) with ET1 or H2O. Fluorescence contrast between mitochondria and cytosol was measured to reflect the status of mPTPs. Lower fluorescence contrast indicates greater calcein leak from mitochondria to cytosol, and it is evidence of higher levels of mPT. As shown in Figure 2, ET1 induced a significant reduction in fluorescence contrast compared with H2O (ET1: 31% ± 6% vs. H2O: 84% ± 9%; P < 0.01), suggesting an increase in mPT. In contrast, myocytes pretreated with CB-13 exhibited similar fluorescence contrast after treatment with H2O or ET1 (ET1: 64% ± 2% vs. H2O: 75% ± 3%, not significant), suggesting that CB-13 prevented ET1-dependent mPT. At the end of the experiment, myocytes from all groups were treated with ionomycin (2 μM; 5 minutes). Ionomycin causes Ca2+ overload and induces mPT and thus served as positive control. Mitochondrial fluorescence puncta were dissipated by ionomycin in all groups (data not shown).

FIGURE 2.

CB-13 ameliorates ET1-induced mPT. Serum-deprived myocytes were pretreated with CB-13 (1 μM, 1 hour) or DMSO (vehicle for CB-13), followed by the addition of ET1 (0.1 μM, 15 minutes) or H2O in media containing palmitate/BSA (200 μM) as substrate. Calcein fluorescence contrast between mitochondria and cytosol, an indicator that negatively correlates with mPT, was measured within randomly selected myocytes before (t = 0 minutes) and after (t = 15 minutes) ET1/H2O treatments. A, Results are presented as percent of posttreatment:pretreatment fluorescent contrast. Addition of ET1 (0.1 μM) to myocytes for 15 minutes significantly dissipated fluorescence calcein contrast between mitochondria and cytosol compared with H2O-treatment, suggesting increased mPT. In the presence of CB-13 (1 μM), ET1 failed to reduce fluorescence calcein contrast between mitochondria and cytosol, suggesting preserved mPT. B, Representative fluorescent images, arrows indicate regions of calcein fluorescence contrast between mitochondria and cytosol. n = 3.15 mitochondrial regions from 5 myocytes were analyzed per replicate. **P < 0.01 versus H2O treatment with DMSO (open bar); ‡P < 0.01 versus ET1 treatment with DMSO. Mean ± SEM. DMSO, dimethyl sulfoxide.

CB-13 Prevents ET1-Induced Mitochondrial Membrane Depolarization in an AMPK-Independent Manner

The ratio of red J-aggregates to green monomer declined in ET1-treated cells (80% ± 3%; P < 0.05 vs. control), reflecting membrane depolarization (Fig. 3A). Depolarization was attenuated by CB-13 pretreatment (106% ± 10%; P < 0.05 vs. ET1). The ability of CB-13 to prevent mitochondrial depolarization was unaffected by the AMPK chemical inhibitor, compound C (Fig. 3B).

FIGURE 3.

In the presence of palmitate as energy substrate, CB-13 prevents ET1-induced mitochondrial membrane depolarization in an AMPK-independent manner. Serum-deprived myocytes were pretreated with CB-13 (1 μM, 2 hours) in the presence or absence of compound C (AMPK inhibitor, 1 μM; 1 hour), followed by the addition of ET1 (0.1 μM; 4 hours) in media containing palmitate/BSA (200 μM) as substrate. Results are presented as representative fluorescent images and percent of normalized red/green fluorescence ratio versus control (open bar). A, The ability of ET1 to induce mitochondrial membrane depolarization, indicated by decreased ratio of JC-1 aggregated red signal to monomeric green signal, was attenuated by pretreatment with CB-13. B, Rescue of mitochondrial membrane potential by CB-13 was unaffected by compound C (data not shown). n = 7–8 (≥3 replicates per n-value). *P < 0.05 and **P < 0.01 versus control (open bars); †P < 0.05 versus ET1. Mean ± SEM.

CB-13 Attenuates ET1-Reduced Expression of PGC-1α and CPT-1β Through AMPK

CB-13 treatment (4 hours) increased phosphorylation of AMPKα at Thr172 (303% ± 60%; P < 0.01 vs. control) (Fig. 4A), which is an indicator of AMPK activation status.[38,39] PGC-1α, a central regulator of mitochondrial metabolism,[40] was reduced by ET1 by 41% ± 7% (P < 0.01 vs. control), and this was prevented by CB-13 (96% ± 2%; P < 0.05 vs. ET1). CB-13 alone increased PGC-1α (140% ± 27%; P < 0.01 vs. control). We next performed knockdown of AMPKα1/2 to ascertain its contribution to CB-13 effects. Infection of cardiomyocytes with lentiviral constructs expressing shRNA against AMPKα1 and AMPKα2 produced significant and simultaneous reductions in AMPKα1 and AMPKα2 (see Figure S5, Supplemental Digital Content 1, http://links.lww.com/JCVP/A425). shRNA knockdown of AMPKα1 and AMPKα2 abrogated the ability of CB-13 to increase PGC-1α (Fig. 4B); in fact, CB-13 then reduced PGC-1α expression, whether in the presence (32% ± 7%; P < 0.01 vs. control) or absence (59% ± 10%; P < 0.01 vs. control) of ET1 (Fig. 4B).

FIGURE 4.

ET1-induced downregulation of PGC-1α, and CPT-1β is attenuated by CB-13 in an AMPK-dependent manner. A, Exposure to CB-13 (1 µM) significantly increased phosphorylation of AMPKα at Thr172, which is an indicator of AMPK activation status. AMPK phosphorylation in the presence of CB-13, and ET1 is comparable to AMPK phosphorylation levels in the presence of CB-13 alone. n = 3–9. **P < 0.01 versus control (open bar). B, PGC-1α was reduced by ET1 (0.1 μM; 4 hours), and this was prevented by CB-13. Upregulation of PGC-1α by CB-13 in untreated myocytes was abolished by AMPKα1/α2 knockdown. Likewise, the ability of CB-13 to restore PGC-1α expression in ET1-treated myocytes was blocked by AMPKα1/α2 knockdown. n = 3–12. *P < 0.05 and **P < 0.01 versus control (open bars); †P < 0.05 versus ET1. C, ET1 decreased CPT-1β RNA expression, and this was partially attenuated by CB-13. AMPKα1/α2 knockdown blocked CB-13 effects. n = 5–8 (≥3 replicates per n value). *P < 0.05 versus control (open bars); ns = not significant versus control (open bars). Mean ± SEM.

We next examined CPT-1, which is a rate-limiting enzyme that facilitates the transport of fatty acids into the mitochondria for use as energy substrates.[41] CPT-1β, the predominant isoform of CPT-1 in the heart,[42] was assessed by real-time PCR. ET1 reduced CPT-1β expression to 82% ± 4% (P < 0.05 vs. control), and this was attenuated by CB-13 (91% ± 6%, ns from control or ET1) (Fig. 4C). However, simultaneous knockdown of AMPKα1 and AMPKα2 abrogated the ability of CB-13 to rescue CPT-1β (73% ± 8%; P < 0.05 vs. control) (Fig. 4C).

DISCUSSION

Mitochondrial aberrations have been linked to numerous aspects of cardiovascular disease, such as ischemia reperfusion injury,[21] cardiomyopathy,[22] and heart failure.[24] In fact, mitochondrial dysfunction purportedly contributes to the development of cardiac hypertrophy vis-à-vis, for example, altered mitochondrial biogenesis, decreased energy production/state, worsened redox status, and impaired Ca2+ homeostasis. Accordingly, improvement in mitochondrial function has been proposed as a therapeutic target toward the treatment of cardiac hypertrophy.[43] The present study shows that the activation of the endocannabinoid system attenuates mitochondrial aberrations in cardiac myocytes subjected to an acute, prehypertrophic exposure of ET1. CB-13, a dual CB1/CB2 receptor agonist,[30,31] attenuated ET1-induced depression of fatty acid–dependent respiration (a surrogate marker of FAO), mitochondrial mPT, and mitochondrial inner membrane depolarization These protective effects of CB-13 were largely dependent on AMPK and were associated with rescued expression of 2 key regulators of mitochondrial function: PGC-1α and CPT-1β.

Acute Exposure to ET1 Impairs FAO-dependent Mitochondrial Bioenergetics

Our data suggest that an early response to ET1 is depressed FAO. ET1 reduced palmitate-dependent basal OCR (Fig. 1). This was attributable solely to a decrease in ATP-linked OCR and translated into compromised coupling efficiency (ie, efficiency of ATP production).

In ET1-treated cardiac myocytes, the net result of depressed FAO is a decrease in oxidative phosphorylation, yet hypertrophic growth, for example, is an energy-consuming process. From a bioenergetic perspective, this may explain, at least in part, the contribution of prolonged hypertrophy to the development of heart failure and functional decompensation.[44-46] We previously reported that ET1 impaired contractile function of cardiac myocytes by reducing shortening and relengthening velocities,[16] and a link exists between decreased conduction velocities, mitochondrial dysfunction, and inefficient cellular ATP utilization.[47,48] Moreover, maximal and spare respiration capacities, which reflect the ability of mitochondria to respond to higher energy demand, were also reduced by ET1. This predicts a limited capacity to adjust to conditions such as hemodynamic overload that require extra ATP, rendering myocytes vulnerable to secondary stress.[32]

Mitochondrial Membrane Integrity in ET1-Treated Cardiac Myocytes in the Presence of Palmitate

Mitochondrial Δψm results from the electrochemical gradient across the inner membrane, which is established as protons are pumped from the mitochondrial matrix to the intermembrane space by ETC complexes I, III, and IV (extrusion). In contrast, Δψm might be dissipated via proton leak and the ATP synthase pore (reentry). Thus, Δψm is influenced by the rates of proton extrusion and reentry.

Our data suggest that ET1-induced mitochondrial depolarization was attributable to reductions in proton extrusion (Fig. 3). In the presence of palmitate, ET1 reduced net proton reentry (as evidenced by reduced ATP-linked OCR and unaffected proton leak; Fig. 1). Nevertheless, mitochondrial Δψm was reduced, indicating that the decline in proton extrusion exceeded the decline in proton reentry. Thus, dissipation of Δψm was because of the reduction in active proton extrusion.

CB-13 restored Δψm in ET1-treated myocytes, and this appeared to be AMPK independent (Fig. 3). In the latter, the failure of compound C to abolish protective CB-13 effects on Δψm does not seem to reconcile with ablation of CB-13 effects on ATP-linked OCR (ie, proton reentry; Fig. 1C). This disparity might be explained by a concomitant further decrease in proton extrusion, where treatment with ET1 + compound C + CB-13 reduced not only proton re-entry via ATP synthase (Fig. 1C) but also proton extrusion. This would yield a normalized mitochondrial Δψm. This might be achieved by a blunting of ETC complexes I, III, and IV expression and/or activity in the absence of AMPK signaling. Decreased AMPK activity and ETC complex expression were observed in pulmonary artery endothelial cells from fetal lambs with persistent pulmonary hypertension.[49] Decreased AMPK activity[50,51] and reduced gene expression of complex I and ATP synthase were also detected in rat ventricles,[52] and in the spontaneously hypertensive rat, hypertrophy is linked to suppressed activities of complex I and AMPK.[53] Another mechanism by which loss of AMPK might impair proton extrusion is via reduced CPT-1β levels, as shown in response to ET1 + CB-13 + AMPK knockdown treatment (Fig. 4C). Suppression of CPT-1β would reduce mitochondrial fatty acid uptake and entry into the tricarboxylic acid cycle, electron donor concentrations (NADH/FADH2), and therefore ETC-dependent proton extrusion. Thus, our findings suggest that during FAO, disrupting AMPK abolishes the ability of CB-13 to maintain both proton extrusion rate and proton reentry, yielding a net noneffect on mitochondrial Δψm. Thus, rather than suggesting noninvolvement of AMPK, the inability of compound C to reverse CB-13 rescue of Δψm reflects a parallel loss of CB-13/AMPK effects on proton extrusion and reentry.

Effects of liganded CB receptor activation on mitochondrial signaling cascades is explained in Supplemental Digital Content 1 (schematic representation, see Figure S7, http://links.lww.com/JCVP/A425).

We identified PGC-1α as a candidate mediator of CB-13 actions. PGC-1α is a key transcriptional coactivator that regulates mitochondrial function. ET1, angiotensin II, and phenylephrine in myocytes[54] or pressure overload in vivo[55] downregulate PGC-1α. Activators of AMPK increase PGC-1α expression, and when PGC-1α is absent, the expression of several target mitochondrial genes of AMPK is ablated.[56] AMPK might also stimulate PGC-1α by increasing NAD+:NADH, thereby activating sirtuin-1 (SIRT1), an NAD+-dependent deacetylase. SIRT-1 activates PGC-1α by deacetylating lysine sites,[57,58] and AMPK and SIRT1 reciprocally upregulate each other.[59,60] PGC-1α regulates mitochondrial biogenesis, ATP synthesis, and ROS defense mechanisms,[61,62] and PGC-1α overexpression rescues cardiac mitochondrial function.[43] ET1 reduced PGC-1α and, consistent with reports that deactivation of the PPARγ/PGC-1α complex leads to downregulation of FAO genes,[57,63,64] CPT-1β expression (Fig. 4) as well. Upregulation of CPT-1 involves AMPK,[65] so while CB-13 rescued PGC-1α and CPT-1β expression, AMPKα1/2 knockdown abrogated CB-13 effects.

When we disrupted AMPK signaling, by shRNA knockdown or using compound C, CB-13 treatment alone reduced PGC-1α (Fig. 4B) and, as would then be expected, FAO-related mitochondrial respiration (Fig. 1). We speculate that CB-13 activates AMPK via CB2 receptors, whereas CB1 receptors invoke other signaling. JWH-133, a CB2-selective agonist, activates AMPK (data not shown), and a CB2 agonist is sufficient to stimulate PGC-1α.[66] This suggests that without AMPK signaling, CB-13 might be stimulating other signaling cascades to reduce PGC-1α, thus depressing FAO-related mitochondrial bioenergetics. CB-13 is a dual agonist of CB1 and CB2 receptors; our previous findings showed that anandamide, which is equally potent at CB1 and CB2 receptors,[67] stimulates PGC-1α expression in the Sprague Dawley rat heart, and moreover, JWH-133 stimulates AMPK activity (data not shown). In addition, Zheng et al[66] reported that a CB2 agonist activated PGC-1α. Therefore, we speculate that CB1-induced signaling emerges to exert opposing effects when CB2/AMPK signaling is inhibited. Indeed, Tedesco et al[68] observed decreased AMPK activity and endothelial nitric oxide synthase expression, as well as depressed mitochondrial biogenesis in liver, muscle, and white adipose tissues in mice treated with a CB1-selective agonist. Perwitz et al[69] also showed that blockage of CB1 receptors enhanced mitochondrial respiration and increased AMPK activity and PGC-1α expression in adipocytes. Other studies also reported the opposite effects of CB1 and CB2 receptors, where CB1 is detrimental and CB2 is beneficial.[70-72] Therefore, an explanation for our finding that CB-13 impairs FAO when AMPK signaling is inhibited might be that CB1 (deleterious) and CB2 (salutary) act in opposition, at least in cardiac myocyte mitochondria, and that CB2 receptor–stimulated AMPK pathways dominate over CB1 receptor signaling in cardiac myocytes to achieve regulation of mitochondrial function by CB-13. This remains to be tested.

CONCLUSIONS

We previously reported that manipulation of the endocannabinoid system represents a viable strategy to prevent cardiac hypertrophy.[16] Here, activation of CB receptors exerted early protective effects on mitochondrial function in cardiac myocytes exposed to a prohypertrophic agonist. Dual agonism of CB1 and CB2 receptors restored mitochondrial Δψm and prevented depression of FAO-related mitochondrial bioenergetics. AMPK played a central role, at least in part by upregulating PGC-1α and CPT-1β, which are key regulators of FAO. Given that fatty acids are the primary energy source in the heart, the ability of CB-13 to restore FAO strengthens its cardioprotective potential. Thus, activation of peripheral CB1/CB2 receptors may be a new therapeutic approach to address mitochondrial dysfunction in the context of cardiac disease.