Holman Et Al., Acute Stress, Endocannabinoids, and the Heart: Supplemental Digital Content 1

Holman et al., Acute stress, endocannabinoids, and the heart: Supplemental Digital Content 1 Pg. 1

Supplemental Digital Content 1

Measures

Reagents and Chemicals. 2,5-dihydro-2,4,5-trimethylthiazoline (TMT) was purchased from

Contech Enterprises (Delta, B.C., Canada). Butyric acid (BA) was purchased from Sigma-

Aldrich (Saint Louis, MO). ELISA Kits for rat brain natriuretic peptide (BNP) and interleukin-6 (IL-

6) were obtained from Novatein Biosciences (Cambridge, MA). Troponin-I was measured using the Rat Cardiac Tn-I ELISA kit from Life Diagnostics, Inc. (West Chester, PA). EIA Kit for

Angiotensin-II was purchased from RayBiotech, Inc. (Norcross, GA). QuantiChrom™ thiobarbituric acid reactive substances (TBARS) Assay Kit was from BioAssay Systems

(Hayward, CA). The BNP ELISA Kit has a detection range of 31.2 pg/ml-1000pg/ml, a sensitivity of 5.0 pg/ml and an overall variation coefficient of precision between assays and precision within an assay < 10%. The IL-6 ELISA kit has a detection range of 6.25 pg/ml-200 pg/ml, a sensitivity of 1.0 pg/ml and an overall variation coefficient of precision between assays and precision within an assay < 10%. The EIA Angiotensin-II kit has a detection range of 0.1-1,000 pg/ml, a sensitivity of 2.62 pg/ml, and an intra-assay coefficient of variation < 10% and inter-assay coefficient of variation < 15%. The fluorimetric assay of TBARS Assay Kit has a lower limit of detection of 0.1 µM MDA; the intra-assay variation is below 5%. BCA protein assay was from

Pierce (Rockford, IL). Infinity Triacylglycerols colorimetric kit was from Thermo Fisher Scientific,

2 2 2 Inc. (Rockford, IL). [ H4]-OEA, [ H4]-PEA, [ H4]-anandamide and 1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-heptadecenoyl were prepared as described previously using the

2 appropriate fatty acid chlorides purchased from Nu-Chek Prep, Inc. (Elysian, MN). [ H8]-2-AG was from Cayman Chemical (Ann Arbor, MI). C17:0 heptadecanoic acid, C17:1 heptadecenoic acid, C17:1 diheptadecenoinglycerol and C17:1 monoheptadecenoinglycerol, were from Nu-

Check Prep, Inc. (Elysian, MN). C12 ceramide (d18:1/12:0) and 10-cis- heptadecenoylethanolamide were from Avanti® Polar Lipids, Inc. (Alabaster, AL). Holman et al., Acute stress, endocannabinoids, and the heart: Supplemental Digital Content 1 Pg. 2

Anandamide[ethanolamine-3H] was purchased from American Radiolabeled Chemicals (Saint

Louis, MO).

Hemodynamic parameters. Systolic (SBP), diastolic (DBP) and mean blood pressure (MBP) were measured with a tail cuff method by volume pressure recording technology using the Non-

Invasive Blood Pressure System CODA-6 (Kent Scientific Corporation, CODA, Torrington, CT).

Elevated plus maze (EPM). The EPM was used to evaluate anxiety-like behavior. The EPM has two open arms, 50 x 10 cm, and two enclosed arms, 50 x 10 x 40cm that intersect to form a plus sign; it is elevated to 60 cm. White lighting provided 35-45 lux in the open arms and 3-9 lux in the closed arms. Rats were placed individually in the center facing an open arm; behavior was scored for 5 minutes. A video camera was mounted vertically above the EPM and connected to a monitor located in an adjacent room. A trained observer counted open and closed arm entries and measured time spent in open and closed arms. Arm entry was defined as having all four feet in one arm (1). Three indices were calculated from these observations:

Ratio time is the amount of time spent in the open arms divided by the total time spent in the open and closed arms; ratio entry is the number of entries into the open arms divided by the total number of entries into the open and closed arms. The third is the “anxiety index” which is calculated from measurements of exploratory behavior as:

Higher scores indicate increased anxiety-like behavior (2). Total distance moved was measured using the EthoVision 3.0 software (Leesburg, VA). After each session the EPM was wiped with 10% ethanol and dried thoroughly.

Lipid extraction and liquid chromatography/mass spectrometry (LC/MS). Triacylglycerols

(TAG), free fatty acids (FFA), OEA, PEA, anandamide, 2-AG, 2-palmitoylglycerol (2-PG), 2- oleoylglycerol (2-OG) and ceramides were extracted from tissues and serum. Frozen tissues

2 were weighed and homogenized in methanol (1 ml/100 mg of tissue) containing [ H4]-OEA, Holman et al., Acute stress, endocannabinoids, and the heart: Supplemental Digital Content 1 Pg. 3

2 2 2 [ H4]-PEA, [ H4]-anandamide, [ H8]-2-AG, C12 ceramide (d18:1/12:0) and heptadecanoic acid as internal standards. Lipids were extracted using 2 volumes of chloroform and washed with 1 volume of water. After centrifugation at 2000gx at 4˚C, organic phases were collected and dried under nitrogen. The extracts were dissolved in 2 ml chloroform and fractionated onto small glass columns packed with Silica Gel G (60-Å 230-400 Mesh ASTM; Whatman, Clifton, NJ) (3). The first eluate was dried under nitrogen and reconstituted in 60 µl of Tert-butanol:Triton X-

100:Methanol (3:1:1) to determine the TAG content using a colorimetric kit (4). Lipids were eluted with 2 ml chloroform/methanol (9:1, vol/vol), reconstituted in 60 µl of methanol and quantified by liquid chromatography/mass spectrometry (LC/MS) (5,6).

2 2 To extract lipids from serum, 1 ml of ice-cold acetone containing [ H4]-OEA, [ H4]-PEA,

2 2 [ H4]-anandamide, [ H8]-2-AG, heptadecanoic acid and C12 ceramide (d18:1/12:0) as internal standards was added to 1 ml of serum. Proteins were precipitated by centrifugation at 1000xg for 20 min at 4C. The supernatant was collected and excess acetone was dried under nitrogen.

Tissue levels of OEA, PEA, anandamide, 2-AG, 2-PG, 2-OG and FFA were quantified by

LC/MS (7-9). A 1100-LC system coupled to a 1946A-MS detector (Agilent Technologies, Inc.,

Palo Alto, CA) equipped with an electrospray ionization interface was used. Fatty acid ethanolamides and monoacylglycerols were eluted on a XDB Eclipse C18 column (50 x 4.6 mm, inner diameter 1.8 µm, Zorbax, Agilent Technologies, Inc.) using a linear gradient of methanol in water (from 85% to 90% methanol in 2.5 min) at a flow rate of 1 ml/min. Column temperature was kept at 40C. MS detection was in positive ionization mode for OEA, PEA, anandamide, 2-

AG, 2-PG and 2-OG, capillary voltage was set at 3000 V, and fragmentor voltage was varied from 120 to 140 V. Nitrogen was used as drying gas at a flow rate of 13 L/min and a temperature of 350C. Nebulizer pressure was set at 60 psig. For quantification purposes we

+ + 2 monitored, in the selective ion-monitoring mode (SIM), the Na adducts ([M+Na ]) of [ H4]-OEA

2 (mass-to-charge ratio, m/z = 352), OEA (m/z = 348), [ H4]-anandamide (m/z = 374), Holman et al., Acute stress, endocannabinoids, and the heart: Supplemental Digital Content 1 Pg. 4

2 2 anandamide (m/z = 370), [ H4]-PEA (m/z = 326), PEA (m/z = 322), [ H8]-2-AG (m/z =409) and

2-AG (m/z = 401). FFA were eluted on a XDB Eclipse C18 column (50 x 4.6–mm inner diameter, 1.8 µm particle size, 80 Å of porous diameter, Zorbax, Agilent Technologies, Inc.).

Mobile phase A consisted of methanol containing 0.25% acetic acid and 5 mM ammonium acetate; mobile phase B consisted of water containing 0.25% acetic acid and 5 mM ammonium acetate. Lipids were eluted using a linear gradient from 90% A to 100% B in 2.5 min at a flow rate of 1.5 ml/min with column temperature at 40C. ESI was in the negative mode, capillary voltage was set at -4 kV and fragmentor voltage was 120 V. Nitrogen was used as drying gas at a flow rate of 13 L/min and a temperature of 350C. Nebulizer pressure was set at 60 psig. FFA were analyzed monitoring the m/z of the deprotonated molecular ions [M-H]- in SIM mode.

Detection and analysis were controlled by Agilent Chemstation. The following FFA were monitored: palmitic acid (C16:0, m/z = 255), heptadecanoic acid (C17:0, m/z = 269), stearic acid

(C18:0, m/z = 283), arachidic acid (C20:0, m/z = 311), docosanoic acid (C22:0, m/z = 339), tetracosanoic (C24:0, m/z = 367), hexacosanoic (C26:0, m/z = 395), palmitoleic acid (C16:1∆9, m/z = 253), oleic acid (C18:1∆9, m/z = 281), eicosenoic acid (C20:1∆9, m/z = 309), erucic acid

(C22:1∆9, m/z = 337), nervonic acid (C24:1∆9, m/z = 365), linoleic acid (C18:2∆9,12, m/z = 279), linolenic acid (C18:3∆9,12,15, m/z = 277), eicosatrienoic acid (20:3∆8,11,14, m/z = 305), eicosatrienoic acid (20:3∆5,8,11, m/z = 305), arachidonic acid (C20:4∆5,8,11,14, m/z = 303), eicosapentaenoic acid (C20:5∆5,8,11,14,17, m/z = 301), docosatetraenoic acid (22:4∆7,10,13,16, m/z =

331), docosapentaenoic acid (C22:5∆7,10,13,16,19, m/z = 329), docosapentaenoic acid

(C22:5∆4,7,10,13,16, m/z = 329), docosahexaenoic acid (C22:6∆4,7,10,13,16,19, m/z = 327).

Total FFA were calculated by summing the concentration of all FFA measured. Total saturated FFA, monounsaturated FFA (MUFA) and polyunsaturated FFA (PUFA) were calculated by summing the concentrations of individual FFA if detectable. Estimated desaturase and elongase activities and de novo lipogenesis were calculated by product/substrate ratios as Holman et al., Acute stress, endocannabinoids, and the heart: Supplemental Digital Content 1 Pg. 5 follows: Steaoryl delta-9 desaturase activities were calculated by the ratios of palmitoleic acid and palmitic acid or oleic and stearic acid respectively (16:1ω3/16:0, SCD-16; 18:1ω9/18:0,

SCD-18). Delta-5 (D5D) and delta-6 (D6D) desaturase activities were estimated by the ratios

20:4ω6/20:3ω6 and 18:3ω6/18:2ω6, respectively (10). The ratio of palmitic and linoleic acid

(16:0/18:2ω6) was used for estimation of de novo lipogenesis (11). Finally, elongase activity was estimated by the ratio of stearic to palmitic acid (18:0/16:0) and erucic acid to nervonic acid

(22:1ω9/24:1ω9) (12).

Tissue and serum ceramides were analyzed by tandem mass spectrometry (13), using an Agilent 1100 liquid chromatograph coupled to an ESI-ion-trap XCT mass detector.

Ceramides were eluted on a Poroshell 300 SB C18 column (2.1 x 75 mm, inner diameter 5 µm;

Agilent Technologies, Inc.) maintained at 30°C. A linear gradient of methanol in water containing

5 mM ammonium acetate and 0.25% acetic acid (from 80% to 100% of methanol in 3 min) was used at a flow rate of 1 ml/min. Detection was in the positive mode, capillary voltage was 4.5 kV, skim1 −40 V, and capillary exit −151 V. Nitrogen was used as drying gas at a flow rate of 12

L/min, temperature set at 350°C, and nebulizer pressure set at 80 psig. Helium was used as collision gas. Ceramide species were identified by comparison of its MSn fragmentation pattern and LC retention time with that of authentic standards. Extracted ion chromatograms were used to quantify the following ceramide species: d18:0/14:0 [M+H]+ (m/z = 510.5>492.5>264.3), d18:1/16:0 [M+H]+ (m/z = 538.5>520.5>264.3), d18:1/18:0 [M+H]+ (m/z = 566.5>548.5>264.3), d18:1/24:1 [M+H]+ (m/z = 648.6>630.8>264.3), d18:1/24:0 [M+H]+ (m/z = 650.5>632.5>264.3), d18:1/26:0 [M+H]+ (m/z = 678.5>660.5>264.3), using d18:1/12:0 [M+H]+ (m/z =

482.5>464.5>264.3) as an internal standard.

To determine serum levels of OEA, PEA, AEA, 2-AG, 2-PG and 2-OG lipids were eluted on a HP ODS Hypersil column (100 x 4.6 mm inner diameter, 5 µm particle size, Thermo

Scientific, West Palm Beach, FL) using a gradient of methanol in water (from 75% to 100% Holman et al., Acute stress, endocannabinoids, and the heart: Supplemental Digital Content 1 Pg. 6 methanol in 35 min) at a flow rate of 0.5 ml/min. Column temperature was kept at 20C. MS detection was in the positive ionization mode, capillary voltage was set at 3000 V, and fragmentor voltage was varied from 120 to 140 V. Nitrogen was used as drying gas at a flow rate of 13 L/min and a temperature of 350C. Nebulizer pressure was set at 30 psig. FFAs were eluted and monitored as described above.

Enzyme activities

Heart tissue was homogenized on ice-cold Tris-HCl (20 mM, pH 7.4, 10 vol) containing

0.32 M sucrose. Homogenates were centrifuged at 1,000xg for 15 min. Supernatants were collected and protein concentration determined using a bicinchoninic acid (BCA) assay kit

(Pierce, Rockford, IL).

N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) activity.

NAPE-PLD activity was measured at 37˚C for 30 min in Tris-HCl buffer (50 mM, pH 7.4) containing 0.1% Triton X-100, phenylmethylsulphonylfluoride (PMSF, 1 mM), protein (100 µg) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-heptadecenoyl (100 µM) as substrate. The reactions were stopped by adding chloroform/methanol (1:1, vol/vol) containing

2 [ H4]oleoylethanolamide (100 pmol/reaction). After centrifugation at 1,500xg at 4˚C for 15 min, the organic layers were collected and dried under nitrogen. The residues were suspended in 50

µl of methanol and analyzed by LC/MS. Fatty acid ethanolamides were eluted on a XDB Eclipse

C18 column (50 x 4.6 mm, inner diameter 1.8 µm, Zorbax, Agilent Technologies, Inc.) using a linear gradient of methanol in water (from 85% to 90% methanol in 2.5 min) at a flow rate of 1.5 ml/min. Column temperature was kept at 40C. MS detection was in positive ionization mode, capillary voltage was set at 4000 V, and fragmentor voltage was 150 V. Nitrogen was used as drying gas at a flow rate of 13 L/min and a temperature of 350C. Nebulizer pressure was set at

+ 2 60 psig. For quantification purposes, we monitored the [M+Na] ions of [ H4]-oleoylethanolamide

(m/z = 352) and N-heptadecenoylethanolamide (m/z = 334) (9,14). Holman et al., Acute stress, endocannabinoids, and the heart: Supplemental Digital Content 1 Pg. 7

Fatty acid amide hydrolase (FAAH) activity. Reactions were conducted at 37°C for 30 min in Tris-HCl buffer (50 mM, pH 7.5, 0.5 ml) containing fatty acid-free bovine serum albumin

(0.05%), protein (100 µg) and anandamide[ethanolamine-3H] (10,000 dpm, specific activity 20

Ci/mmol). After stopping the reactions with chloroform/methanol (1:1, vol/vol, 1 ml) and centrifugation at 1,500xg at 4˚C for 15 min, radioactivity was measured in the aqueous layers by liquid scintillation counting (15,16).

Monoacylglycerol lipase (MGL) activity. MGL activity was determined at 37°C for 10 min in Tris-HCL buffer (50 mM pH 8.0, 0.5 ml) containing fatty acid-free bovine serum albumin

(0.05%), protein (10 µg/reaction) and C17:1 monoheptadecenoinglycerol (10 µM/reaction) as substrate. The reactions were stopped by adding 1.5 ml of cold chloroform/methanol (2:1, vol/vol) containing C17:0 heptadecanoic acid 5 µM/reaction). After centrifugation at 1,500xg at

4˚C for 5 min, the organic layers were collected and dried under nitrogen. The residues were suspended in 100 µl of methanol and analyzed by LC/MS. FFA were eluted on a XDB Eclipse

C18 column (50 x 4.6–mm inner diameter, 1.8 µm particle size, 80 Å of porous diameter,

Zorbax, Agilent Technologies, Inc.). Mobile phase A consisted of methanol containing 0.25% acetic acid and 5 mM ammonium acetate; mobile phase B consisted of water containing 0.25% acetic acid and 5 mM ammonium acetate. Lipids were eluted using a linear gradient from 90% A to 100% B in 2.5 min at a flow rate of 1.5 ml/min with column temperature at 40C. ESI was in the negative mode, capillary voltage was set at -4 kV and fragmentor voltage was 120V.

Nitrogen was used as drying gas at a flow rate of 13 L/min and a temperature of 350C.

Nebulizer pressure was set at 60 psig. FFA were analyzed monitoring the m/z of the deprotonated molecular ions [M-H]- in SIM mode. For quantification purposes, we monitored the

267 (m/z) for the reaction product, C17:1 heptadecenoic acid, and 269 (m/z) for C17:0 heptadecanoic acid (17). Holman et al., Acute stress, endocannabinoids, and the heart: Supplemental Digital Content 1 Pg. 8

Diacylglycerol lipase (DGL) activity. Reactions were conducted at 37°C for 30 min in

Tris-HCL buffer (50 mM, pH 7.0, 0.2 ml) containing Triton X-100 (0.1%), protein (10 µg/reaction) and C17:1 diheptadecenoinglycerol (50 µM/reaction) as substrate. The reactions were stopped

2 by adding 0.6 ml of cold chloroform/methanol (2:1, vol/vol) containing [ H8]-2-AG as internal standard (25 pmol/reaction). After centrifugation at 1,500xg at 4˚C for 5 min, the organic layers were collected and dried under nitrogen. The residues were suspended in 60 µl of methanol and analyzed by LC/MS. Lipids were eluted on a XDB Eclipse C18 column (50 x 4.6 mm, inner diameter 1.8 µm, Zorbax, Agilent Technologies, Inc.) using a linear gradient of methanol in water (from 85% to 90% methanol in 2.5 min) at a flow rate of 1.5 ml/min. Column temperature was kept at 40C. MS detection was in positive ionization mode, capillary voltage was set at

4000 V, and fragmentor voltage was 150 V. Nitrogen was used as drying gas at a flow rate of

13 L/min and a temperature of 350C. Nebulizer pressure was set at 60 psig. The quantification was done by monitoring the [M+Na]+ ions for the reaction product monoheptadecanoinglycerol

2 (m/z = 365) and [ H8]-2-AG (m/z = 409) (18).

N-acylethanolamine-hydrolyzing acid amidase (NAAA) activity. Heart tissue was homogenized on ice and transferred into ice-cold Tris-HCl buffer (20 mM, pH 7.4) containing

0.32 M sucrose (final volume-to-weight ratio, 9:1). Samples were centrifuged at 1000xg for 10 min at 4°C, and the supernatants were ultra-centrifuged at 12,000xg for 30 min at 4°C. The pellets were suspended in ice-cold PBS (pH 7.4), subjected to one freeze/thaw cycle at -80°C, sonicated (5 seconds, amplitude 50%) in a digital sonifier (Branson Ultrasonics, Danbury, CT) and frozen again. Suspensions were centrifuged at 105,000xg for 1-h at 4°C and the supernatants were kept. Protein concentration was measured and samples stored at -80°C until use. Proteins (50 µg/reaction) were incubated in NAAA assay buffer [0.1 M NaH2PO4, 0.1 M sodium citrate, 0.1% NP-40, 3 mM dithiothreitol (DTT), pH 4.0] for 30 min at 37°C in the presence of substrate (10-cis-heptadecenoylethanolamide, 50 µM) at 37°C for 30 min. Holman et al., Acute stress, endocannabinoids, and the heart: Supplemental Digital Content 1 Pg. 9

Reactions were terminated by the addition of 0.6 ml ice-cold methanol/chloroform (1:2) containing 100 pmol heptadecanoic acid as internal standard. Organic phases were collected, dried under nitrogen and suspended in 100 µl of methanol/chloroform (3:1). Heptadecenoic

(17:1) and heptadecanoic (17:0) acids were eluted on an Eclipse XDB-C18 column (Agilent

Technologies, Santa Clara, CA) isocratically at 2.2 ml/min for 1 min with a solvent mixture of

95% methanol and 5% water, both containing 0.25% acetic acid and 5mM ammonium acetate.

The column temperature kept at was 50°C. Electrospray ionization was in the negative mode, capillary voltage was 4 kV, and fragmentor voltage was 100 V. N2 was used as drying gas at a flow rate of 13 L/min and a temperature of 350°C. Nebulizer pressure was set at 60 psig. We monitored [M-H]- in the selected-ion monitoring mode using heptadecanoic acid as internal standard. Calibration curves were generated using commercial 17:0 and 17:1 acids (m/z = 269 and m/z = 267, respectively) (14).

RNA extraction and gene expression analysis. Total RNA was extracted from heart with Trizol® (Life Technologies, Grand Island, NY) and purified by using the RNeasy® Plus Mini

Kit (Quiagen, Valencia, CA). RNA (2 µg) was then translated into cDNA using the High Capacity cDNA Reverse Transciption Kit (Life Technologies, Grand Island, NY). mRNA expression of genes of interest was determined by quantitative RT-PCR with Taqman® PCR Master Mix

(Applied Biosystems-Roche, Carlsbad, CA) in a Mx3000P plate thermocycler (Stratagene, La

Jolla, CA) and normalized to β-actin expression. TaqMan gene expression assays for β-actin, sterol regulatory element-binding protein-1 (SREBP-1, Rn01495769_m1), carnitine palmitoyltransferase-1β (CPT-1β, Rn00682395_m1) caspase-3 (Rn00563902_m1), interleukin-

1β (IL-1β, Rn00580432_m1) and interleukin-6 (IL-6, Rn01410330_m1) were from Applied

Biosystems-Roche (Carlsbad, CA). Primers and probes for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), peroxisome proliferator-activated receptor gamma (PPAR-γ), cluster of differentiation 36 (CD36), lipoprotein lipase (LPL), fatty acid synthase (FAS), NAPE-PLD1, N- Holman et al., Acute stress, endocannabinoids, and the heart: Supplemental Digital Content 1 Pg. 10 acylethanolamine-hydrolyzing acid amidase (NAAA), fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MGL), diacylglycerol lipase-β (DGL-β) and tumor necrosis factor-α

(TNF-α) were from TIB Molbiol (Adelphia, NJ).

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