Chronic inhibition of fatty acid amide hydrolase by URB597 produces differential effects on cardiac performance in normotensive and hypertensive rats

Abstract

Background and Purpose

Fatty acid amide hydrolase (FAAH) inhibitors are postulated to possess anti‐hypertensive potential, because their acute injection decreases BP in spontaneously hypertensive rats (SHR), partly through normalization of cardiac contractile function. Here, we examined whether the potential hypotensive effect of chronic FAAH inhibition by URB597 in hypertensive rats correlated with changes in cardiac performance.

Experimental Approach

Experiments were performed using perfused hearts and left atria isolated from 8‐ to 10–week‐old SHR, age‐matched deoxycorticosterone acetate (DOCA)‐salt rats and normotensive controls chronically treated with URB597 (1 mg·kg⁻¹) or vehicle.

Key Results

URB597 decreased BP only in the DOCA‐salt rats, along with a reduction of ventricular hypertrophy and diastolic stiffness, determined in hypertension. We also observed normalization of the negative inotropic atrial response to the cannabinoid receptor agonist CP55940. In the SHR model, URB597 normalized (atria) and enhanced (hearts) the positive ino‐ and chronotropic effects of the β‐adrenoceptor agonist isoprenaline respectively. Ventricular CB₁ and CB₂ receptor expression was decreased only in the DOCA‐salt model, whereas FAAH expression was reduced in both models. URB597 caused translocation of CB₁ receptor immunoreactivity to the intercalated discs in the hearts of SHR. URB597 increased cardiac diastolic stiffness and modified the ino‐ and lusitropic effects of isoprenaline in normotensive rats.

Conclusion and Implications

Hypotensive effect of chronic FAAH inhibition depend on the model of hypertension and partly correlate with improved cardiac performance. In normotensive rats, chronic FAAH inhibition produced several side‐effects. Thus, the therapeutic potential of these agents should be interpreted cautiously.

Abbreviations

AEA

anandamide

AM3506

5‐(4‐hydroxyphenyl)pentanesulfonyl fluoride

CP55940

2‐[(1R,2R,5R)‐5‐hydroxy‐2‐(3‐hydroxypropyl)cyclohexyl]‐5‐(2‐methyloctan‐2‐yl)phenol

CPP

coronary perfusion pressure

DOCA

deoxycorticosterone acetate

FAAH

fatty acid amide hydrolase

HR

heart rate

LVP

left ventricular pressure

RPP

rate‐pressure product

SBP

systolic BP

SHR

spontaneously hypertensive rats

URB694

6‐hydroxy‐[1,1′‐biphenyl]‐3‐yl‐cyclohexyl‐[11C–carbonyl]carbamate

URB597

3‐(3‐carbamoylphenyl)phenyl N‐cyclohexylcarbamate

WKY

Wistar–Kyoto rats

Introduction

Hypertensive heart disease, which includes ventricular hypertrophy, contractile dysfunction and their clinical manifestations (arrhythmias and heart failure), is a leading cause of death associated with high BP (Drazner, 2011). The endocannabinoid system is suggested to buffer increases in BP in hypertension. In support of this, the plasma level of the best known endocannabinoid, anandamide (AEA), was higher in hypertensive patients (Engeli et al., 2012) and in spontaneously hypertensive rats (SHR; Li et al., 2009). Moreover, in acute experiments, (1) the CB₁ receptor‐mediated hypotension elicited by AEA was stronger in anaesthetized SHR than in normotensive controls (Lake et al., 1997; Bátkai et al., 2004); (2) in conscious SHR, AEA decreased BP, but increased it in normotensive controls (Lake et al., 1997); and (3) the systemic acute blockade of fatty acid amide hydrolase (FAAH; the key enzyme responsible for AEA degradation) by URB597 (Bátkai et al., 2004) and AM3506 (Godlewski et al., 2010) has been shown to normalize BP in SHR. Consequently, FAAH inhibitors are postulated to be potential antihypertensive agents (Bátkai et al., 2004; Godlewski et al., 2010). The chronic administration of URB597 to deoxycorticosterone acetate (DOCA)‐salt hypertensive rats reduced BP in older rats (in a manner partly dependent on vascular changes) and diminished cardiac hypertrophy in younger animals (Baranowska‐Kuczko et al., 2016; Toczek et al., 2016).

Cannabinoids have a negative inotropic effect in human (Bonz et al., 2003) and rat atrial muscle (Sterin‐Borda et al., 2005) via CB₁ receptors and a positive inotropic effect via CB₂ receptors (Sterin‐Borda et al., 2005). Coronary vasodilation is mediated by CB₁ (Wagner et al., 2005) or via non‐CB₁/CB₂ receptors (Ford et al., 2002). The reduction of increased cardiac contractility in hypertension is suggested to be one of the major components of the antihypertensive action of FAAH inhibitors after their acute injection (Bátkai et al., 2004; Godlewski et al., 2010). Chronic treatment with the FAAH inhibitor URB694 normalized resting heart rate (HR) and prevented the occurrence of arrhythmia in stressed animals (Carnevali et al., 2015a). In FAAH knockout mice, age‐related cardiac dysfunction (Bátkai et al., 2007) was decreased, but doxorubicin‐induced cardiotoxicity was enhanced (Mukhopadhyay et al., 2011). Thus, the first aim of our study was to examine whether the potential hypotensive effect of chronic FAAH inhibition by URB597 in hypertensive rats correlated with changes in cardiac performance.

In hypertension, an elevated sympathetic tone alters β‐adrenoceptor‐induced effects (Guyenet, 2006), which might additionally be modified by cannabinoids. There is evidence that cannabinoid agonists HU210 (Maslov et al., 2004), AEA (Gaskari et al., 2005) and WIN55212‐2 (Liao et al., 2013) can diminish the following responses to the non‐selective β‐adrenoceptor agonist isoprenaline: its positive ino‐ and/or chronotropic effects and/or stimulation of cAMP production in isolated hearts, ventricular papillary muscle and neonatal cardiomyocytes respectively. Moreover, acute administration of URB694 reduced the occurrence of isoprenaline‐induced ventricular tachyarrhythmias (Carnevali et al., 2015b). Therefore, the second aim of our study was to examine the influence of chronic URB597 administration on β‐adrenoceptor‐mediated effects in cardiac tissues isolated from normotensive and hypertensive rats.

Methods

Animals

All animal care, surgical procedures and experimental protocols were performed in accordance with the European Directive (2010/63/EU) and Polish legislation and were approved by the local Animal Ethics Committee in Białystok (Poland). Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015). The study was carried out in compliance with the replacement, refinement or reduction (the 3Rs). Male rats were obtained from the Centre for Experimental Medicine of the Medical University of Białystok (Poland). They were housed in collective plastic cages (two rats per cage) with sawdust on the bottom in a temperature‐controlled room at 22 ± 1°C under a 12:12 h light–dark cycle with free access to water and standard laboratory food unless otherwise noted. We used age‐matched rats with comparable primary (SHR; the most frequently studied genetic hypertensive model) and secondary (DOCA‐salt) hypertension in order to distinguish changes induced by hypertension from those related to any one particular hypertension model. We used the DOCA‐salt model because a salt‐rich diet is one of the main lifestyle factors leading to hypertension.

DOCA‐salt hypertension was induced in Wistar rats (6–7 weeks old; initially weighing 260–300 g) as described previously (Toczek et al., 2016). Animals were anaesthetized by i.p. injection of pentobarbital sodium (70 mg·kg⁻¹, i.e. ~300 μmol·kg⁻¹). The right kidney was removed in all rats via a right lateral abdominal incision. After 1 week of recovery, hypertension was induced by subcutaneous injections of 11‐DOCA (25 mg·kg⁻¹, i.e. ~67 μmol·kg⁻¹; 0.4 mL·kg⁻¹) twice weekly for 6 weeks and replacement of drinking water with 1% NaCl solution. Control sham‐operated rats (SHAM) received the vehicle for DOCA (N,N‐dimethylformamide) twice weekly and drank tap water.

Chronic treatment with URB597

We applied the same protocol and dose of URB597 as reported previously (Toczek et al., 2016) and age‐matched DOCA‐salt hypertensive rats, in which 2 weeks of URB597 administration was previously reported to decrease BP (Toczek et al., 2016). URB597 (1 mg·kg⁻¹, i.e. ~3 μmol·kg⁻¹; 1 mL·kg⁻¹) or its vehicle – a mixture of DMSO, Tween 80 and saline (1:2:7) – was injected i.p. for 14 days, twice daily for the following four groups of rats: hypertensive (1) DOCA‐salt (4 weeks after the onset of DOCA‐salt administration); (2) 8‐ to 10–week‐old male SHR (270–320 g) and their respective normotensive controls: (3) age‐matched SHAM (4 weeks after the onset of vehicle for DOCA‐salt administration); and (4) Wistar–Kyoto rats (WKY; 290–390 g). Rats were assigned randomly to the different experimental groups. Before the first dose of URB597 or its vehicle and 12 h after the final dose, systolic BP (SBP) was recorded in conscious rats by the non‐invasive tail‐cuff method using the Rat Tail Blood Pressure Monitor from Hugo Sachs Elektronik‐Harvard Apparatus (March–Hugstetten, Germany).

Isolated Langendorff heart preparation

Twelve hours after the final dose of URB597 (or its vehicle), rats were anaesthetized with 300 μmol·kg⁻¹ pentobarbital sodium i.p. and then injected with 500 IU heparin i.p. (Polfa, Warsaw, Poland). Hearts were rapidly excised, weighed and immediately mounted in a Langendorff system (Hugo Sachs Elektronik‐Harvard Apparatus GmbH, March‐Hugstetten, Germany). Hearts were perfused at a constant flow rate (12 mL·min⁻¹) with oxygenated (95% O₂ and 5% CO₂) Krebs–Henseleit solution of the following composition (mM): NaCl 118.0, KCl 4.8, CaCl₂ 1.8, NaHCO₃ 24.0, KH₂PO₄ 1.2, MgSO₄ 1.2, glucose 11.0, Na‐pyruvate 5.0, EDTA 0.03 (pH 7.4; 37°C). A distilled water filled latex balloon connected to a pressure transducer and inflated to increase the left ventricular end‐diastolic pressure to approximately 8–10 mmHg was inserted into the left ventricle through the mitral valve to measure the left ventricular pressure (LVP). Another pressure transducer located just above the aorta recorded coronary perfusion pressure (CPP). Hearts were allowed to beat spontaneously. After a 5 min stabilization period, increments in balloon volume (using a 50 mL Hamilton syringe connected to the fluid‐filled pressure transduction circuit) were applied to the heart at 0, 5, 10, 15, 20 and 30 mmHg, and left ventricular end‐diastolic pressure was recorded. Myocardial diastolic stiffness was calculated as the diastolic stiffness constant (κ, dimensionless), the slope of the linear relation between tangent elastic modulus (E, dyne·cm⁻²) and stress (σ, dyne/cm²) as previously described (Loch et al., 2007).

Heart parameters were continuously monitored using the ISOHEART^R software. Preparations were allowed to stabilize for 25 min. Two hearts (one from WKY URB597 and one from SHAM URB597 group) with persistent arrhythmias and poor contractility (LVP < 75 mmHg) were excluded from the study. After the stabilization period, increasing concentrations of isoprenaline (0.01 nM – 1 μM) were infused by a peristaltic pump (Ascor, Warsaw, Poland) at one hundredth of the current flow rate into the coronary arteries until a plateau was reached; concentrations referred to the final concentrations in the heart. Measurements recorded after the initial equilibration period were considered as the baseline values. The cardiac performance of the Langendorff‐perfused heart was evaluated from HR, LVP (which is an index of contractile activity) and the rate‐pressure product (RPP: HR × LVP, mmHg × beats·min⁻¹), which is an index of cardiac work (Angelone et al., 2008), the maximum rate of positive +(LVdP/dt)_max (inotropism) or negative −(LVdP/dt)_max (lusitropism) changes in LVP and CPP as an index of coronary dilation. All parameters are expressed as Δ changes from baseline before addition of the first concentration of isoprenaline. After experiments, the left atrium and the right and left ventricle with septum were weighed, and their weights were expressed as a ratio of tissue weight (mg) to total body weight (g).

Isolated left atria

The left atria were dissected before the isolated heart perfusions (see above). They were weighed and mounted in 10 mL organ baths containing Krebs solution of the following composition (mM): NaCl 118, KCl 4.8, MgSO₄ 1, NaHCO₃ 29, NaHPO₄x12H₂0 1, CaCl₂ 2.25, glucose 10, Na‐pyruvate 5, EDTA 0.04 (pH 7.4; 37°C). Atria were continuously stimulated electrically with square wave pulses (just over threshold, 5 ms duration, 2 Hz) using a pair of platinum electrodes. Contractions were recorded using an isometric force transducer (PIM 100RE, Bio‐Sys‐Tech, Białystok, Poland). Tissue was allowed to equilibrate for 60 min. When a stable amplitude of contractions had been reached, the cannabinoid receptor agonist CP55940 (1 nM – 30 μM) was cumulatively added to the atria. Atria were then washed several times and sensitized by a brief exposure to 5 × 10⁻⁶ M isoprenaline (Dincer et al., 2000). When steady basal values were obtained again, concentration–response curves for isoprenaline (0.01 nM – 3 μM) were constructed. The initial contractile forces (in mN) of atria in normotensive SHAM and WKY measured before the first concentration of CP55940 were as follows: 2.4 ± 0.1 (n = 11) and 2.5 ± 0.1 (n = 10), respectively, and before the first concentration of isoprenaline were the same 2.4 ± 0.1 for SHAM (n = 10) and WKY (n = 9). Hypertension and URB597 did not affect the basal values of this parameter (data not shown). Agonist responses were measured as the decrease or increase in the basal force (expressed as % of basal values) of the left atrium.

Western blots

Routine Western blotting procedure was used to examine protein expression, as has been described previously (Baranowska‐Kuczko et al., 2016). Briefly, samples from the left ventricles were homogenized in RIPA buffer containing a cocktail of protease and phosphatase inhibitors (Roche Diagnostics GmbH, Mannheim, Germany). In addition, protein concentration was measured using the bicinchoninic acid method with BSA as a standard. Following this, homogenates were reconstituted in Laemmli buffer, separated by 10% SDS‐PAGE and transferred onto nitrocellulose membranes. The membranes were incubated overnight at 4°C with the corresponding primary antibodies in appropriate dilutions: CB₁ (1:200, cat no. Ab23703; Abcam, UK), CB₂ (1:500, cat no. Ab3561; Abcam, UK), FAAH1 (1:500; cat no. Ab54615; Abcam, UK) and GAPDH (1:500, cat no. sc‐32 233; Santa Cruz Biotechnology, USA). Thereafter, nitrocellulose membranes were incubated with the appropriate secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology, USA). After adding a suitable substrate for horseradish peroxidase (Thermo Scientific, Rockford, IL, USA), protein bands were quantified densitometrically using a ChemiDoc visualization system EQ (Bio‐Rad, Warsaw, Poland). Equal protein loading in each line was confirmed by Ponceau S staining. The level of protein expression was standardized to GAPDH.

Immunohistochemistry

For immunohistochemistry, the EnVision method was used (Baranowska‐Kuczko et al., 2016) using antibodies against: CB₁ (1:2000 rabbit; Anti‐CB₁ cat no. Ab23703; Abcam, UK) and CB₂ (1:200 rabbit Anti‐CB₂ cat no. Ab3561; Abcam, UK). To test the specificity of the antibodies, a negative control was included, in which the antibodies were replaced by normal rabbit serum (Vector Laboratories, CA, USA) at the respective dilution (no staining), and a positive control, which was prepared from rat hippocampus for CB₁ and human tonsil for CB₂ (data not shown). The obtained results of immunohistochemical staining were viewed on an Olympus BX41 microscope with an Olympus DP12 camera under a magnification of 200× (20× lens and 10× eyepiece).

Data and statistical analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). The results are given as the mean ± SEM (n = number of animals). The exact group size (n) for each experimental group/condition is provided in Tables 1, 2, 3, 4 and ‘n’ refers to independent values, not replicates. Researchers were not blinded to the experimental conditions, but efforts were made to be close to the conditions of blinded assays, and our analysis did not include any subjective evaluation. Thus, (1) all the cardiac tissues were obtained via the same procedures; (2) they were treated in the same way; and (3) all data were obtained via direct recording of physiological parameters.

Table 1.

Influence of URB597 on physiological parameters of DOCA‐salt and SHR and their respective normotensive SHAM and WKY

		SHAM	SHAM URB597	DOCA‐salt	DOCA‐salt URB597	WKY	WKY URB597	SHR	SHR URB597
Parameters	n	10	10	10	11	10	9	10	10
SBP (mmHg)
day 0		131 ± 7	131 ± 8	217 ± 16*	201 ± 13	104 ± 8	105 ± 6	183 ± 7*	199 ± 12
day 14		137 ± 5	118 ± 10	217 ± 17*	159 ± 10†, #	105 ± 6	92 ± 4	196 ± 10*	182 ± 7
HR (beats·min−1)
day 0		362 ± 8	363 ± 10	360 ± 17	374 ± 11	343 ± 23	333 ± 9	383 ± 12	376 ± 10
day 14		357 ± 6	350 ± 14	370 ± 15	355 ± 11	280 ± 8#	317 ± 12†	357 ± 8*	349 ± 7#
Body weight (g)
day 0		302 ± 7	291 ± 10	264 ± 2*	267 ± 4	333 ± 10	340 ± 11	288 ± 6*	295 ± 6
day 14		310 ± 8	304 ± 10	271 ± 9*	277 ± 6	334 ± 10	343 ± 10	287 ± 5*	291 ± 5
Heart weight/body weight (mg·g−1)		4.0 ± 0.2	4.7 ± 0.3	5.9 ± 0.3*	5.5 ± 0.2	4.7 ± 0.2	4.8 ± 0.2	5.6 ± 0.1*	5.8 ± 0.1
LV + septum weight/body weight (mg·g−1)		2.26 ± 0.07	2.32 ± 0.11	3.60 ± 0.14*	3.15 ± 0.14†	2.53 ± 0.06	2.47 ± 0.04	3.33 ± 0.04*	3.54 ± 0.10
RV weight/body weight (mg·g−1)		0.61 ± 0.05	0.66 ± 0.04	0.87 ± 0.04*	0.74 ± 0.05	0.71 ± 0.03	0.67 ± 0.02	0.76 ± 0.02	0.78 ± 0.02
LA weight/body weight (mg·g−1)		0.073 ± 0.010	0.082 ± 0.004	0.155 ± 0.014*	0.129 ± 0.009	0.091 ± 0.005	0.088 ± 0.002	0.079 ± 0.002	0.076 ± 0.004
Diastolic stiffness constant (κ)		22.9 ± 0.7	26.3 ± 0.8†	32.4 ± 1.0*	26.2 ± 0.9†	21.6 ± 1.5	35.1 ± 1.1†	30.8 ± 0.9*	29.6 ± 2.8

Units are given in brackets. URB597 (1 mg·kg⁻¹) or its vehicle was injected i.p. every 12 h for 14 days. SBP and HR were recorded before (day 0) the first dose of URB597 or its vehicle and 14 days later. Heart and body weights were determined 12 h after the last dose of URB597 or its vehicle. Diastolic stiffness was measured at the beginning of Langendorff experiment. Weights of right and left ventricle (RV and LV), septum and left atrium (LA) were determined after the end of in vitro experiments. Data are given as the means ± SEM.

^* P < 0.05 DOCA‐salt and SHR versus SHAM and WKY respectively;

^† P < 0.05, significant effect of URB597; ANOVA with Bonferroni post hoc test

^# P < 0.05, significantly different from values before URB597 treatment (day 0 vs. day 14); Student's t‐test for paired data.

Table 2.

Influence of URB597 on basal parameters of hearts isolated from DOCA‐salt and SHR and their respective normotensive SHAM and WKY

		SHAM	SHAM URB597	DOCA‐salt	DOCA‐salt URB597	WKY	WKY URB597	SHR	SHR URB597
Parameters	n	10	10	10	11	10	9	10	10
HR (beats·min−1)		273 ± 7	293 ± 9	273 ± 9	269 ± 8	274 ± 14	254 ± 7	282 ± 10	260 ± 8
LVP (mmHg)		120 ± 8	131 ± 10	132 ± 10	131 ± 9	90 ± 5	90 ± 3	134 ± 10*	137 ± 8
+(LVdP/dt)max (mmHg·s−1)		3353 ± 217	3472 ± 274	3482 ± 240	3492 ± 268	2431 ± 222	2161 ± 631	3610 ± 204*	3307 ± 122
−(LVdP/dt)max (mmHg·s−1)		−2422 ± 174	−2846 ± 250	−2560 ± 240	−2798 ± 222	−1918 ± 125	−1924 ± 58	−2898 ± 234*	−3031 ± 165
RPP (mmHg·beats·min−1)		30 210 ± 2309	36 106 ± 3190	33 676 ± 2488	33 698 ± 2527	21 773 ± 1143	20 162 ± 620	34 243 ± 1587*	32 286 ± 1666
CPP (mmHg)		94 ± 8	92 ± 9	86 ± 8	85 ± 5	64 ± 4	66 ± 3	75 ± 3	76 ± 3

Units are given in brackets. URB597 (1 mg·kg⁻¹) or its vehicle was injected i.p. every 12 h for 14 days. Hearts were isolated 12 h after the final dose of URB597 or its vehicle. Data are given as the means ± SEM.

^* P < 0.05, SHR significantly different from WKY; ANOVA with Bonferroni post hoc test; LVP, the maximum rate of positive +(LVdP/dt)_max and negative −(LVdP/dt)_max changes in LVP.

Table 3.

Influence of URB597 on the isoprenaline (0.01 nM – 1 μM)‐induced changes in parameters of hearts isolated from DOCA‐salt and SHR and their respective normotensive SHAM and WKY

		SHAM		SHAM URB597		DOCA‐salt		DOCA‐salt URB597
	n	10		10		10		11
Parameters		pEC50	Emax	pEC50	Emax	pEC50	Emax	pEC50	Emax
HR (beats·min−1)		8.9 ± 0.2	107 ± 6	8.3 ± 0.2	98 ± 10	8.5 ± 0.2	89 ± 12	8.8 ± 0.3	94 ± 12
LVP (mmHg)		9.8 ± 0.7	55 ± 7	8.8 ± 0.4	30 ± 6*	8.7 ± 0.5	29 ± 6†	8.8 ± 0.3	39 ± 5
+(LVdP/dt)max (mmHg·s−1)		8.7 ± 0.4	4925 ± 522	8.5 ± 0.2	4124 ± 420	8.4 ± 0.2	2885 ± 397†	8.6 ± 0.2	3933 ± 512
−(LVdP/dt)max (mmHg·s−1)		9.3 ± 0.4	−2697 ± 303	8.8 ± 0.3	−2543 ± 335	8.5 ± 0.3	−2148 ± 325	8.7 ± 0.3	−2225 ± 350
RPP (mmHg·beats·min−1)		9.5 ± 0.5	30 487 ± 3894	8.7 ± 0.3	22 267 ± 2928	8.7 ± 0.4	20 097 ± 4442	8.7 ± 0.3	24 931 ± 4466
CPP (mmHg)		9.4 ± 0.6	−28 ± 6	9.3 ± 0.3	−32 ± 6	–	–	–	–

		WKY		WKY URB597		SHR		SHR URB597
	n	10		9		10		10
		pEC50	Emax	pEC50	Emax	pEC50	Emax	pEC50	Emax
HR (beats·min−1)		8.5 ± 0.3	95 ± 7	9.0 ± 0.1	105 ± 7	8.4 ± 0.3	83 ± 11	8.8 ± 0.2	116 ± 11*
LVP (mmHg)		9.7 ± 0.3	81 ± 9	8.7 ± 0.1	85 ± 8	9.0 ± 0.6	32 ± 10†	8.6 ± 0.4	40 ± 8
+(LVdP/dt)max (mmHg·s−1)		9.4 ± 0.3	5526 ± 684	8.4 ± 0.2*	5279 ± 615	8.4 ± 0.2†	4413 ± 585	8.3 ± 0.2	4410 ± 454
−(LVdP/dt)max (mmHg·s−1)		9.2 ± 0.4	−2970 ± 290	8.6 ± 0.2	−3166 ± 451	8.3 ± 0.2	−1847 ± 177†	8.2 ± 0.2	−1878 ± 222
RPP (mmHg·beats·min−1)		9.5 ± 0.3	29 409 ± 3123	8.6 ± 0.1*	37 038 ± 1066	8.7 ± 0.2†	22 539 ± 2833	8.7 ± 0.2	24 680 ± 680
CPP (mmHg)		9.7 ± 0.5	−26 ± 4	9.1 ± 0.2	−28 ± 3	–	–	–	–

Values are based on the concentration–response curves shown in Figures 1 and 2. Maximal effects (E_max) determined for isoprenaline (0.01 nM – 0.1 μM) represent maximum changes from baseline. Units for E_max are given in brackets. Data are given as the means ± SEM.

^* P < 0.05, significant effect of URB597.

^† P < 0.05 DOCA‐salt and SHR significantly different from SHAM and WKY respectively; ANOVA with Bonferroni post hoc test; LVP, the maximum rate of positive +(LVdP/dt)_max and negative −(LVdP/dt)_max changes in LVP.

Table 4.

Influence of URB597 on the isoprenaline (0.01 nM – 3 μM)‐induced positive inotropic and the CP55940 (1 nM – 30 μM)‐induced negative inotropic effects in left atria isolated from DOCA‐salt and SHR and their respective normotensive SHAM and WKY

Group	SHAM			SHAM URB597			DOCA‐salt			DOCA‐salt URB597
			n			n			n			n
	pEC50	Emax		pEC50	Emax		pEC50	Emax		pEC50	Emax
Isoprenaline	8.1 ± 0.2	74 ± 9	9	7.9 ± 0.2	81 ± 12	9	8.3 ± 0.2	65 ± 13	10	8.2 ± 0.2	81 ± 15	11
CP55940	6.1 ± 0.3	−38 ± 4	10	6.8 ± 0.4	−36 ± 4	10	5.0 ± 0.2#	−25 ± 2	8	6.1 ± 0.2†	−48 ± 6†	10

	WKY		n	WKY URB597		n	SHR		n	SHR URB597		n
	pEC50	Emax		pEC50	Emax		pEC50	Emax		pEC50	Emax
Isoprenaline	8.3 ± 0.1	156 ± 12	9	8.3 ± 0.1	230 ± 15†	10	8.3 ± 0.1	67 ± 6#	10	8.6 ± 0.1	125 ± 11†	8
CP55940a	7.3 ± 0.2	−54 ± 4	9	7.3 ± 0.1	−48 ± 4	10	6.6 ± 0.2	−34 ± 6#	9	7.3 ± 0.2†	−36 ± 5	10

Values are based on the concentration–response curves shown in Figure 3. Maximal effects (E_max) are expressed in % of basal values.

^a In the case of CP55940, E_max was determined for the concentration of 30 μM. Data are given as the means ± SEM.

^† P < 0.05, significant effect of URB597;

^# P < 0.05 DOCA‐salt and SHR significantly different from SHAM and WKY respectively. ANOVA with Bonferroni post hoc test.

Maximal effects of agonists (E_max) and their potencies (as pEC₅₀ values) were determined from the individual concentration–response curves. Values of E_max for isolated hearts and atria represent respective maximal changes from baseline and % of basal values, as their basal values in control normotensive SHAM and WKY were different in hearts (Table 2) and comparable in atria (see above). Due to dual cardiac effect of isoprenaline and the lack of the pronounced E_max for CP55940 in atria, E_max were determined for the following agonist concentrations: isoprenaline in the heart: 0.01 nM – 0.1 μM; and CP55940 in atria – 30 μM. Statistical analysis was performed using Graph Pad Prism 5 (GraphPad Software, La Jolla, CA, USA). Intergroup statistical comparisons were made by ANOVA followed by Bonferroni's multiple comparison test of the entire data set. Post hoc tests were only performed where the F‐ratio of the ANOVA highlighted a significant difference (P < 0.05). The Student's t‐test for paired and unpaired data was used as appropriate. Differences were considered significant at P < 0.05.

Materials

CP55940 (Tocris, Bristol, UK); heparin sodium (Polfa, Warsaw, Poland); 11‐DOCA, N,N‐dimethylformamide, (−)‐isoprenaline (±)‐bitartrate salt, Tween 80 (Sigma‐Aldrich, Munich, Germany); URB597; Cayman Chemical Company, Ann Arbor, MI, USA); pentobarbital sodium (Biowet, Puławy, Poland). Stock solutions of isoprenaline were prepared in distilled water and further diluted with Krebs solution. CP55940 was dissolved in DMSO; further dilutions were made with Krebs solution such that the final concentration of the DMSO in the organ bath was less than 0.01%.

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015a,b).

Results

General

Before application of the first dose of URB597, or its vehicle, SBP was about 70% higher in both hypertensive models. compared with normotensive controls (Table 1). URB597 reduced SBP in DOCA‐salt by about 20% but did not change SBP in SHR. HR or body weight were also unchanged in DOCA‐salt and SHR. Basal HR was reduced (by about 10%) in WKY and SHR both in the absence and the presence of URB597, 2 weeks after the experiment began.

Heart weight to body weight ratios were higher by about 50 and 20% in DOCA‐salt and SHR compared with SHAM and WKY respectively (Table 1). The cardiac hypertrophy in DOCA‐salt was connected with an increase in hypertrophy indices in all cardiac compartments. In contrast, in SHR, cardiac hypertrophy was associated with an increase in the ratio of left ventricular and septum weight to body weight only. URB597 reduced the elevated index of left ventricular and septum weight to body weight observed in DOCA‐salt.

Diastolic stiffness was increased by about 40% in hearts of both hypertensive models (Table 1). URB597 diminished the enhanced diastolic stiffness observed in hypertension by about 20% in DOCA‐salt, but not in SHR. Surprisingly, in normotensive animals, URB597 increased diastolic stiffness by about 15 and 50% in SHAM and WKY respectively.

Influence of hypertension and URB597 on basal parameters of isolated hearts

Values of all basal parameters of hearts isolated from DOCA‐salt and SHAM were comparable (Table 2). In SHR, indices of cardiac contractility (LVP), work (RPP), maximal rate of LV contraction and relaxation were higher by about 50% than in WKY. These values were comparable with the respective values in DOCA‐salt. URB597 did not modify any basal parameters of isolated hearts in normo‐ or hypertension.

Effects of hypertension and chronic URB597 administration on isoprenaline‐induced cardiostimulatory effects in isolated hearts

Isoprenaline (0.01 nM – 1 μM) caused concentration‐dependent increases of all cardiac parameters, which were comparable in hearts isolated from normotensive rats of both groups (for the respective pEC₅₀ and E_max values determined for isoprenaline 0.01 nM – 0.1 μM, see Table 3; Figures 1 and 2). One exception was a lower increase in LVP (for E_max P < 0.05; but not its pEC₅₀ value) in SHAM compared with WKY. Hypertension attenuated all cardiostimulatory effects of isoprenaline (mainly at the lower concentrations; Table 3; Figures 1 and 2). Maximal responses were reduced by about 50–60% for LVP (DOCA‐salt and SHR) and by about 40% for +(LVdP/dt)_max (DOCA‐salt) and for −(LVdP/dt)_max (SHR). Vasodilatory effects of isoprenaline on coronary arteries were completely prevented by hypertension. A weaker potency of isoprenaline was only observed for two parameters in SHR: +(LVdP/dt)_max and RPP.

Figure 1.

Influence of URB597 on the isoprenaline‐induced changes in LVP (A, A′), the maximum rate of positive +(LVdP/dt)_max (B, B′) and negative −(LVdP/dt)_max (C, C′) changes in LVP (ΔLVP) of hearts isolated from DOCA‐salt and SHR and their respective normotensive SHAM and WKY. URB597 (1 mg·kg⁻¹) or its vehicle was injected i.p. every 12 h for 14 days. Values shown are changes from baseline (Table 2). Data are given as the means ± SEM of 9–11 rats. *P < 0.05, significant effect of URB597, in normotensive rats only: ^# P < 0.05, DOCA‐salt and SHR significantly different from SHAM and WKY respectively; ANOVA with Bonferroni post hoc test. In few cases, SEM is smaller than or equal to the size of symbols.

Figure 2.

Influence of URB597 on the isoprenaline‐induced changes in HR (A, A′), RPP (B, B′) and CPP (C, C′) of hearts isolated from DOCA‐salt and SHR and their respective normotensive SHAM and WKY. URB597 (1 mg·kg⁻¹) or its vehicle was injected i.p. every 12 h for 14 days. Values shown (ΔHR etc) are changes from baseline (Table 2). Data are given as the means ± SEM of 9–11 rats. *P < 0.05, significant effect of URB597, in normotensive rats only with the exception of HR in SHR; ^# P < 0.05, DOCA‐salt and SHR significantly different from SHAM and WKY respectively; ANOVA with Bonferroni post hoc test. In few cases, SEM is smaller than or equal to the size of symbols.

URB597 tended to enhance, the reduced in hypertension increases in LVP, +(LVdP/dt)_max and RPP (but not other parameters), which were induced by higher (0.01 nM – 0.1 μM) isoprenaline concentrations in DOCA‐salt hypertensive rats; in the case of maximal responses by about 35, 40 and 25% respectively (for the respective pEC₅₀ and E_max values, see Table 3, Figures 1 and 2). In contrast, the only effect of URB597 in SHR was enhancement of the positive chronotropic effect of isoprenaline at higher (0.01 nM – 0.1 μM) concentrations (by approximately 40% for E_max). However, the potency of isoprenaline was not affected (Figure 2A′, Table 3).

In both normotensive groups, URB597 reduced the following cardiostimulatory effects of isoprenaline at lower concentrations: LVP, +(LVdP/dt)_max, −(LVdP/dt)_max and RPP. Moreover, in SHAM, URB597 decreased the increases in LVP and HR induced by higher and lower concentrations of isoprenaline respectively (Figures 1 and 2). In WKY, URB597 diminished the potency of isoprenaline for +(LVdP/dt)_max and RPP (Table 3). However, isoprenaline‐induced coronary vasodilatation in hypertensive and normotensive rats (Figure 2C, C′) was not altered by FAAH inhibition.

Effects of hypertension and chronic URB597 administration on isoprenaline‐ and CP55940‐induced changes in the contractile force of isolated left atria

Isoprenaline (0.01 nM – 3 μM) induced a concentration‐dependent increase in the contractile force of atria isolated from normo‐ and hypertensive rats. Hypertension and URB597 did not alter the positive inotropic effect of isoprenaline in DOCA‐salt and in SHAM (for the respective pEC₅₀ and E_max values, see Table 4, Figure 3A).

Figure 3.

Influence of URB597 on the isoprenaline‐induced (A, A′) increases and on the CP55940‐induced (B, B′) decreases in contractile force of left atria isolated from DOCA‐salt and SHR and their respective normotensive SHAM and WKY. URB597 (1 mg·kg⁻¹) or its vehicle was injected i.p. every 12 h for 14 days. Data are expressed in % of basal values (see Results). Data are given as the means ± SEM of 8–11 rats; *P < 0.05, significant effect of URB597; ^# P < 0.05, DOCA‐salt and SHR significantly different from SHAM and WKY respectively; ANOVA with Bonferroni post hoc test. In few cases, SEM is smaller than or equal to the size of symbols.

The inotropic effect of the higher concentration of isoprenaline in atria isolated from WKY was higher than in SHAM (in the case of the E_max value by approximately 110%; P < 0.05). The E_max value in SHR was lower by about 60% than in WKY. URB597 almost completely restored the positive inotropic effect of isoprenaline in SHR. Interestingly, it also strongly enhanced isoprenaline‐induced increase in contractile force observed in WKY. The maximal response was increased by about 50% (for the respective pEC₅₀ and E_max values, see Table 4, Figure 3A′). There was no alteration in the potency of isoprenaline (Table 4) in either model of hypertension or after administration of URB597.

The vehicle alone for CP55940 reduced atrial contractile force by about 5–10% in all experimental groups (Figure 3), in hypertension and in the presence of URB597 (data not shown). CP55940 (1 nM – 30 μM) caused concentration‐dependent decreases in the contractile force of left atria (a direct negative inotropic effect); stronger in WKY than in SHAM (P < 0.05 for pEC₅₀ and E_max values; Figure 3B, B′; for the respective pEC₅₀ and E_max values induced by CP55940 at 30 μM, see Table 4). The concentration–response curves for the negative inotropic effect of CP55940 in normotensive rats were shifted to the right, in atria isolated from DOCA‐salt and SHR. URB597 completely restored the negative inotropic effect of CP55940 in the DOCA‐salt and the potency of CP55940 in SHR. URB597 did not modify the negative inotropic effect of CP55940 observed in atria isolated from normotensive animals (Figure 3B, B′, Table 4).

Influence of hypertension and chronic administration of URB597 on cardiac expression of CB_1, CB₂ receptors and FAAH

Analysis of CB receptor and FAAH expression in left cardiac ventricles by Western blotting showed a single immunoreactive band of the molecular size expected for CB₁ receptors (60 kDa), CB₂ (40 kDa) and FAAH (67 kDa). The expression of CB_1, CB₂ receptors and FAAH decreased in tissues from hypertensive DOCA‐salt rats, whereas in SHR, only FAAH was down‐regulated. URB597 did not modify expression levels in normotensive animals. It just prevented the previously observed decrease in FAAH expression and tended to do so with respect to CB₁ and CB₂ receptor expression, in DOCA‐salt but not in SHR (Figure 4).

Figure 4.

Western blots of CB₁ (A, A′), CB₂ (B, B′) and FAAH (C, C′) protein in left ventricle isolated from DOCA‐salt and SHR and their respective normotensive SHAM and WKY. URB597 (1 mg·kg⁻¹) or its vehicle was injected i.p. every 12 h for 14 days. On the bottom of the panel, representative Western blots for CB₁, CB₂, FAAH and GADPH (which served as loading control) are given. Mean ± SEM of six rats; *P < 0.05, significant effect of URB597; ^# P < 0.05, DOCA‐salt and SHR significantly different from SHAM and WKY respectively; ANOVA with Bonferroni post hoc test.

Immunolocalization of CB₁ and CB₂ receptors (Figure 5) revealed their appearance in cardiomyocytes of the left cardiac ventricle, although the immunoreactivity for CB₁ receptors appeared more intense. Both CB₁ and CB₂ immunoreactivities were less intense in both models of hypertension (especially in DOCA‐salt) compared with their respective normotensive controls. URB597 increased labelling for CB₁ receptors in both hypertensive models and for CB₂ receptors in DOCA‐salt only. Interestingly, in both models of hypertension treated with URB597, a clearly visible signal for CB₁ receptors was noticed (stronger in SHR rather than DOCA‐salt rats), in the region of the intercalated discs of the heart.

Figure 5.

Representative micrographs of immunohistochemical staining of CB₁ and CB₂ receptors in cross sections of left ventricle isolated from DOCA‐salt and SHR and their respective normotensive SHAM and WKY treated i.p. every 12 h for 14 days with URB597 (1 mg·kg⁻¹) or its vehicle. Scale bar: 50 μm.

Discussion

Chronic URB597 administration produces age‐dependent hypotension in DOCA‐salt hypertensive rats (Toczek et al., 2016), which is partly dependent on vascular changes (Baranowska‐Kuczko et al., 2016). Here, we examined whether the potential hypotensive effect of chronic FAAH inhibition in hypertension was dependent on cardiac performance. Cardiac function was assessed in two in vitro models using tissue taken from the same animal: Langendorff hearts and paced left atria in which contractility is dependent or independent of contraction frequency respectively.

Changes related to hypertension

Hypertensive animals had higher SBP, cardiac hypertrophy and diastolic stiffness. Isoprenaline‐induced vasodilatation and its ino‐ (with the exception of atria from DOCA‐salt animals), chrono‐ and lusitropic effects in hearts and/or atria were attenuated in hypertension. This may result from a reduction of myocardial β‐adrenoceptor density in SHR (Böhm et al., 1988). In DOCA‐salt hypertension, no change or a decrease in the number of β‐adrenoceptors has been reported (Böhm et al., 1992).

The cannabinoid receptor agonist CP55940 induced a concentration‐dependent decrease in atrial contractile force, with a stronger effect in WKY compared with SHAM. The maximum negative inotropic effect of CP55940 was comparable with that induced by AEA in atrial muscle of humans (Bonz et al., 2003) and rats (Sterin‐Borda et al., 2005). This is the first study to demonstrate that the negative inotropic effects of CP55940 are diminished in hypertension. This was accompanied by decreased ventricular immunoreactivity and/or expression of FAAH and CB receptors (especially in DOCA‐salt), which can induce negative (CB₁) and positive (CB₂) inotropic responses in rat left atria (Sterin‐Borda et al., 2005). By contrast, Bátkai et al. (2004) reported an up‐regulation of cardiac CB₁ receptors and FAAH, associated with an AEA‐induced sustained decrease in cardiac contractility, in anaesthetized SHR. However, Bátkai et al. (2004) used older rats for their in vivo experiments (8–10 months vs. 8–10 weeks in our study), in which the negative inotropic effect of AEA was modified by a decreased HR. We examined atrial but not ventricular contractility. However, hypertension‐induced ventricular changes are accompanied by modifications of atrial function (Pluteanu et al., 2015). In contrast to the reduced effects and expression of cardiac CB₁ receptors in DOCA‐salt reported here, we have found in the same model up‐regulation of vascular CB₁ receptors and CB₁ receptor‐mediated vasodilatation (Baranowska‐Kuczko et al., 2016) and enhanced inhibition of sympathetic noradrenaline release (Toczek et al., 2015) in resistance vessels. This suggests that these receptors may play a protective role in hypertension.

Influence of chronic URB597 administration on hearts of hypertensive rats

FAAH inhibitors are postulated to possess anti‐hypertensive potential, because their acute systemic injection decreased BP in SHR partially through normalization of cardiac contractile performance, associated with up‐regulation of cardiac CB₁ receptors and FAAH (Bátkai et al., 2004; Pacher et al., 2008; Godlewski et al., 2010; O'Sullivan, 2015). We found that the effect of chronic FAAH inhibition was dependent on the hypertension model. More marked alterations were observed in secondary hypertension. This was probably due to the more dynamic progress of hypertension in DOCA‐salt compared with SHR (the comparable hypertension of about 200 mmHg before the first dose of URB597 was obtained after 4 weeks of DOCA‐salt procedure vs. 8–10 weeks of hypertension development in SHR) and the potential development of adaptive changes in SHR (Zicha and Kunes, 1999).

Chronic URB597 administration decreased BP only in DOCA‐salt (consistent with our previous studies; Baranowska‐Kuczko et al., 2016; Toczek et al., 2016) but not in SHR. We measured BP 12 h after the final dose of URB597 (1 mg·kg⁻¹) in conscious rats. Higher doses were not used due to potential side effects and neurotoxicity (1.5 and 3 mg·kg⁻¹; Su et al., 2015). In contrast, the strong hypotensive effects of acute administration of the FAAH inhibitors URB597 (used at the maximally effective dose of 10 mg·kg⁻¹; Bátkai et al., 2004) and AM3506 (Godlewski et al., 2010) were determined immediately after i.v. injection in older anaesthetized SHR.

The present results demonstrate that URB597 induced a fall in BP in DOCA‐salt hypertension, which was partly mediated by vascular (Baranowska‐Kuczko et al., 2016) and cardiac mechanisms. URB597 reduced hypertrophy of the left ventricle and septum and cardiac diastolic stiffness (observed in hypertension) and completely restored the negative inotropic effect of CP55940 in isolated atria. These changes were accompanied by enhanced ventricular expression of CB₁ and CB₂ receptors (immunohistochemistry) and FAAH (Western blot) that was reduced in hypertension. It is the first report demonstrating the up‐regulation of FAAH expression following chronic FAAH inhibition. So far, a decrease (Baranowska‐Kuczko et al., 2016; Su et al., 2016) or no change (Okine et al., 2012; Baranowska‐Kuczko et al., 2016) in FAAH expression has been demonstrated in other tissues, after chronic URB597 administration. We can only speculate that in our hands, inhibition of enzyme activity might cause a compensatory increase in enzyme expression (King et al., 1991). The possible involvement of cardiac mechanisms in the hypotensive effect of URB597 is partly confirmed by the observation that, in SHR, the same procedure did not decrease BP or normalize cardiac hypertrophy, diastolic stiffness, the negative inotropic effect of CP55940 or expression of CB receptors and FAAH. Our results on isolated atria from DOCA‐salt validated the findings from in vivo experiments (Bátkai et al., 2004) that changes in cannabinoid‐induced inotropic effects are related to a direct influence on the myocardium but not through inhibition of sympathetic tone. Moreover, the significance of CB₁ receptor mediated cardiac alternations underlined observations that chronic administration of the CB₁ receptor antagonist rimonabant improved cardiac function and remodelling after myocardial infarction, in obese SHR (Slavic et al., 2013).

The most visible effects of URB597 in SHR were the normalization (atria) and enhancement (hearts) of the positive ino‐ and chronotropic effects of isoprenaline respectively. It remains to be established whether these changes are related to redistribution of CB₁ receptors to the region of intercalated discs, clearly observed in SHR. We are the first to demonstrate this intriguing relationship between cardiac intercalated discs and CB₁ receptors. Intercalated discs are responsible for mechanical (desmosomes and adheren junctions) and electrical (gap junctions) communication between adjacent cardiomyocytes (Campbell et al., 2014). AEA can inhibit gap junctions in certain cell types, such as endothelial cells, but not myocytes (Oz, 2006). Improving cardiac gap junction communication, which is also modified by β‐adrenoceptor stimulation (Campbell et al., 2014), has been proposed as a new antiarrhythmic mechanism (Dhein et al., 2010). Thus, normalization of basal HR and prevention of arrhythmia by chronic URB694 administration to stressed animals (Carnevali et al., 2015b) could be associated with gap junction modification.

Pre‐incubation of ventricular papillary muscle (isolated from rats with cirrhotic cardiomyopathy) with the CB₁ receptor antagonist 1‐(2,4‐dichlorophenyl)‐5‐(4‐iodophenyl)‐4‐methyl‐N‐piperidin‐1‐ylpyrazole‐3‐carboxamide restored its blunted contractile response to isoprenaline (Gaskari et al., 2005). In our hands, chronic URB597 administration restored the positive inotropic effect of isoprenaline in atria, which was reduced in SHR and tended towards the same in isolated hearts from DOCA‐salt.

Influence of chronic URB597 administration on hearts of normotensive rats

To date, it is generally accepted that endocannabinoids are not involved in the regulation of cardiovascular parameters under physiological conditions, as genetic ablation or acute pharmacological blockade of CB receptors or FAAH does not modify basal haemodynamic parameters including left ventricle stiffness (Pacher et al., 2005; Malinowska et al., 2012; O'Sullivan, 2015) and parameters of isolated rat hearts (Wagner et al., 2005). Surprisingly, we found that chronic URB597 administration produced two detrimental effects in normotensive rats, namely, increased cardiac diastolic stiffness and modified cardiostimulatory effects of isoprenaline. Thus, it diminished the following effects of lower concentrations of isoprenaline on the heart: ino‐ and lusitropic actions, increases in RPP and the chronotropic effect (the latter in SHAM only). Moreover, the positive inotropic effect of higher concentrations of isoprenaline was enhanced in atria from WKY. These data indicate that the modification of β‐adrenoceptor function by cannabinoids, found in acute experiments (Maslov et al., 2004; Gaskari et al., 2005; Liao et al., 2013; Carnevali et al., 2015b; see Introduction), may also extend to chronic studies.

There is no clear explanation of the negative effect of URB597 in normotensive rats. In the light of pro‐fibrotic effects of CB₁ receptor activation in diabetic mice (Rajesh et al., 2012) or anti‐fibrotic influence of the CB₁ receptor antagonist rimonabant in experimental metabolic syndrome (Slavic et al., 2013), it could be not so surprising to see increased cardiac stiffness in response to URB597 increasing level of endocannabinoids acting via CB receptors. URB597 is generally regarded as a selective FAAH inhibitor (Piomelli et al., 2006). However, proteome‐wide studies identified the off‐target effects of URB597 (Ahn et al., 2007). Thus, side‐effects of URB597 might be connected with its off‐target response and with direct or indirect activation of alternative pathways of AEA metabolism. Importantly, other unexpected effects of chronic URB597 administration in SHAM animals have been observed previously in small resistance arteries: impaired acetylcholine‐induced vasodilatation, potentiated phenylephrine‐induced vasoconstriction (Baranowska‐Kuczko et al., 2016) and enhanced oxidative stress in liver (Biernacki et al., 2016). Similarly, cardiac injury and enhanced oxidative/nitrative stress were greater in FAAH^−/− compared with FAAH^+/+ mice, and AEA was reported to enhance cell death in human cardiomyocytes pretreated with an FAAH inhibitor (Mukhopadhyay et al., 2011).

In summary, the present findings show that hypotensive effect of chronic FAAH inhibition depends on the model of hypertension used and partly correlates with improved cardiac performance. The fall in BP in response to chronic URB597 administration was observed only in DOCA‐salt rats. This was associated with decreased left ventricular hypertrophy and cardiac diastolic stiffness and an enhanced negative inotropic atrial response to CP55940. The most visible influence of URB597 in SHR was an enhanced positive inotropic effect to isoprenaline in isolated hearts, accompanied by a redistribution of CB₁ receptors to the intercalated disc regions. However, chronic URB597 administration induced side‐effects in normotensive rats such as enhanced cardiac diastolic stiffness and modified cardiostimulatory effects to isoprenaline, stressing the importance of examination of bidirectional interactions of endocannabinoids with the cardiac effects of β‐adrenoceptors. Thus, the use of FAAH inhibitors as potential pharmacotherapy in normotensive patients, for example, for control of pain and inflammation (Fowler, 2015) or psychological‐cardiac comorbidity (Carnevali et al., 2016), should be approached cautiously, especially considering the side effects of FAAH inhibitors that have been reported in clinical studies (Mallet et al., 2016).

Author contributions

A.P.‐B. designed and conducted isolated heart experiments, analysed data and co‐wrote the manuscript; J.W. performed isolated atria experiments and analysed the data; M.T. performed the in vivo studies and helped in interpretation of data; M.B.K. contributed to the revised manuscript; I.K. designed and analysed immunohistochemical experiments; E.H.‐S. designed and analysed Western blot experiments; B.M. conceived, designed and supervised experiments and wrote the manuscript. All authors approved the final manuscript.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Pędzińska‐Betiuk, A. , Weresa, J. , Toczek, M. , Baranowska‐Kuczko, M. , Kasacka, I. , Harasim‐Symbor, E. , and Malinowska, B. (2017) Chronic inhibition of fatty acid amide hydrolase by URB597 produces differential effects on cardiac performance in normotensive and hypertensive rats. British Journal of Pharmacology, 174: 2114–2129. doi: 10.1111/bph.13830.