Direct evidence of bradycardic effect of omega-3 fatty acids acting on nucleus ambiguus

Abstract

Docosahexaenoic acid (DHA) an omega-3 polyunsaturated fatty acid, is an agonist of FFA1 receptor. DHA administration reduces the heart rate via unclear mechanisms. We examined the effect of DHA on neurons of nucleus ambiguus that provide the parasympathetic control of heart rate. DHA produced a dose-dependent increase in cytosolic Ca²⁺ concentration in cardiac-projecting nucleus ambiguus neurons; the effect was prevented by GW1100, a FFA1 receptor antagonist. DHA depolarized cultured nucleus ambiguus neurons via FFA1 activation. Bilateral microinjection of DHA into nucleus ambiguus produced bradycardia in conscious rats. Our results indicate that DHA decreases heart rate by activation of FFA1 receptor on cardiac-projecting nucleus ambiguus neurons.

1. Introduction

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are found in fatty fish, seafood, and dietary supplements, and not synthesized de novo in mammals [48]. n-3 PUFAs have been reported to provide therapeutic benefits in pain [22], aging [44], dementia, Alzheimer’s disease [12], Parkinson’s disease [6], cystic fibrosis [37] and cardiovascular diseases [3].

n-3 PUFAs activate a family of GPCR, namely FFA1, FFA2, FFA3 and FFA4 [17, 46]. Long-chain fatty acids, such as DHA and EPA activate FFA1 receptor, previously known as GPR40 [10] and FFA4 receptor, previously known as GPR120 [25], while short-chain fatty acids activate FFA2 and FFA3 receptors [11]. FFA1 receptor is mainly expressed in the pancreas and brain [10].

Epidemiological data support an inverse relationship between fatty acids consumption and cardiovascular morbidity and mortality [1, 18, 19, 31]. In addition to the improvement of lipid panel and the anti-inflammatory vascular effect, n-3 PUFAs lower the heart rate via incompletely characterized mechanisms [4, 26, 33, 36, 45].

The parasympathetic control of heart rate emerges from cardiac-projecting brainstem neurons of nucleus ambiguus [34]. FFA1 receptor expression was identified in medulla oblongata [10, 30], but its role at this level has not been investigated. We examined the effect of DHA on nucleus ambiguus neurons in vitro and in vivo on heart rate.

2. Materials and Methods

2.1. Ethical approval

Animal protocols were approved by the Institutional Animal Care and Use Committee from each institution. All animal experiments comply with the ARRIVE guidelines.

2.2. Animals

Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA), neonates and adults, were used in this study. Neonatal rats were used for retrograde labeling of nucleus ambiguus and neuronal culture for in vitro studies, and adult male rats were used for in vivo studies.

2.3. Chemicals

Docosahexaenoic acid (DHA) was from Tocris Bioscience (Bio-Techne, Minneapolis, MN) and GW1100, FFA1 receptor antagonist, was from Cayman Chemical (Ann Arbor, MI)

2.4. Retrograde labeling and neuronal culture

Cardiac vagal neurons of nucleus ambiguus were retrogradely labeled by intrapericardial injection of rhodamine [X-rhodamine-5-(and-6)-isothiocyanate; 5(6)-XRITC] (Invitrogen, ThermoFisher Scientific, Grand Island, NY), as previously reported [8]. 24 h later, rats were euthanized by decapitation, and the brain dissected in ice-cold Hanks’ balanced salt solution (HBSS; Mediatech, Manassas, VA). Nucleus ambiguus neurons were dissociated by enzymatic and mechanical dissociation and cultured on poly-lysine-coated glass coverslips in Neurobasal-A medium containing GlutaMax (1%), antibiotic-antimycotic (2%), and fetal bovine serum (10%). Cultures were maintained at 37 °C, in a humidified atmosphere with 5% CO₂.

2.5. Calcium imaging

The intracellular Ca²⁺ concentration, [Ca²⁺]_i, was assessed using Fura-2 AM methods, as previously described [7, 8]. Neurons were incubated with Fura-2 AM (5 μM in HBSS, 45 min), followed by incubation in dye-free HBSS. Coverslips were mounted in an open bath chamber on the stage of Nikon Eclipse TiE microscope equipped with a a Photometrics CoolSnap HQ2 CCD camera (Photometrics, Tucson, AZ). Fura-2 AM fluorescence (excitation - 340 and 380 nm, emission 510 nm) was acquired and analyzed using NIS-Elements AR software (Nikon Melville, NY), and was converted to Ca²⁺ concentrations.

2.6. Measurement of membrane potential

The membrane potential was assessed in neurons loaded with bis-(1,3-dibutylbarbituric acid)-trimethine-oxonol, DiBAC₄(3) a slow-response voltage-sensitive dye, as reported [8]. Neurons were incubated with DiBAC₄(3) (0.5 mM in HBSS, 30 min) and the fluorescence (excitation/emission 480nm/540nm) was monitored.

2.7. Surgical procedures and telemetric heart rate monitoring

Adult male rats (250–300 g) were anesthetized with ketamine hydrochloride (100–150 mg/kg) and acepromazine maleate (0.2 mg/kg) [8]. A guide C315G cannula was bilaterally inserted into nucleus ambiguus, which was identified based on stereotaxic coordinates (12.24 mm posterior to bregma, 2.1 mm from midline and 8.2 mm ventral to the dura mater). A calibrated transmitter (E-mitters, series 4000; Mini Mitter, Sunriver, OR) was inserted in the intraperitoneal space [8]. Series 4000 receivers and VitalView™ software (Mini Mitter) were used to collect the signal from transmitters, as reported [8].

2.8. Microinjection into nucleus ambiguus

Bilateral microinjections into the nucleus ambiguus were performed one week after surgery, without animal handling. The functional identification of nucleus ambiguus was based on stereotaxic coordinates [42] and the bradycardia induced by microinjection of L-glutamate (L-Glu) at this site [8].

2.9. Statistical analysis

Data were expressed as mean ± standard error of mean and compared for statistically significant differences using one-way ANOVA followed by post hoc Bonferroni test; P < 0.05 was considered statistically significant.

3. Results

3.1. DHA increases cytosolic Ca²⁺ concentration in cardiac preganglionic nucleus ambiguus neurons

In rhodamine-labeled cardiac vagal neurons of nucleus ambiguus, DHA (5–30 μM) increased F340/380 fluorescence ratio and cytosolic Ca²⁺ concentration, [Ca²⁺]_i in a dose-dependent manner (n = 8–10 for each concentration) (Fig. 1). In neurons pretreated with GW1100 (20 μM), a FFA1 receptor antagonist [9], the response to DHA (20 μM) was markedly decreased (n = 8) (Fig. 1 A–C).

Fig. 1. DHA increases [Ca²⁺]_i in cardiac vagal neurons of nucleus ambiguus via FFA1 receptor activation.

A, F340/380 ratio in rhodamine-labeled cardiac-projecting neurons of nucleus ambiguus; the response was prevented by pretreatment with GW1100, a FFA1 receptor antagonist. B, Example of DHA-induced increase in [Ca²⁺]_i; the response was abolished by GW1100. C, DHA (5–30 μM) produced a dose-dependent increase in [Ca²⁺]_i; P<0.05 as compared to basal (*) or to the effect of DHA (20 μM) (#).

3.2. DHA produces depolarization of cardiac preganglionic nucleus ambiguus neurons

Voltage imaging studies indicate that DHA (20 μM) depolarized cardiac vagal neurons of nucleus ambiguus (n = 11); the response was prevented by GW1100 (20 μM) (n = 8) (Fig. 2).

Fig. 2. DHA depolarizes cardiac vagal neurons of nucleus ambiguus via FFA1 receptor activation.

A, Example of DHA (20μM)-induced depolarization; the effect was prevented by GW1100. B, Comparison of the amplitude of the depolarization. P<0.05 as compared to basal (*) or to the effect of DHA (20 μM) (#).

3.3. DHA microinjection into nucleus ambiguus elicits bradycardia in conscious rats

In conscious rat, bilateral microinjection of DHA (20 μM, 50 nL) into nucleus ambiguus reduced the heart rate; the effect was prevented by GW1100 (20 μM, 50 nL) (n = 6) (Fig. 3). Microinjection of saline and L-Glu (5 mM, 50 nL) was used as negative and positive control, respectively. Microinjection sites in nucleus ambiguus are indicated on diagrams of coronal brainstem sections (Fig. 3C, dark circles).

Fig. 3. Microinjection of DHA into nucleus ambiguus elicits bradycardia in conscious rats.

A, Telemetric heart rate recordings illustrating the bradycardia produced by bilateral microinjection of DHA (20 μM, 50 nL) into nucleus ambiguus; the effect was abolished by GW1100 (20 μM, 50 nL). Microinjection of saline and L-Glu (5 mM, 50 nL) was used as negative and positive control, respectively. B, Comparison of the amplitude of the decrease in heart rate produced by microinjection into nucleus ambiguus of DHA, and DHA + GW1100. (*) P<0.05 as compared to the effect of DHA. C, Illustration of microinjection sites (dark circles) in coronal medullary sections. Abbreviations: Amb, nucleus ambiguus; NTS, nucleus tractus solitarius; 4V, fourth ventricle.

4. Discussion

Consumption of omega-3 polyunsaturated fatty acids (n-3 PUFAs) such as DHA and EPA has been associated with lower risk of cardiovascular disease and reduced mortality [3, 18, 19, 31]. Clinical and experimental studies indicate that ingestion or intravenous administration of n-3 PUFAs reduces the heart rate and increases the heart rate variability [4, 14–16, 50]. A meta-analysis of 51 randomized controlled trials with approximately 3000 participants indicates that DHA but not EPA reduced the heart rate [24]. Notably, DHA supplementation during pregnancy increased the fetal heart rate variability [23]. Moreover, n-3 PUFAs have been shown to attenuate the heart rate response to exercise in animal and human studies [4, 5, 35].

FFA1 receptor expression was identified in medulla oblongata [9] and DHA can cross the blood-brain barrier [13, 32, 38]; this prompted us to examine the effect of DHA on cardiac vagal neurons of nucleus ambiguus that provide the parasympathetic control of heart rate [20, 34].

We found that DHA increased [Ca²⁺]_i and produced depolarization of cardiac vagal neurons of nucleus ambiguus; the effect was prevented by the antagonist of FFA1 receptor. The concentrations of DHA used in our study are similar to those reported by other studies in vitro [2, 28, 29, 49] and to the DHA concentrations reported in the rat brain [43, 47]. FFA1 receptor couples with G_q/11 proteins leading to Ca²⁺ mobilization G [10, 27]. Since activation of cardiac-projecting nucleus ambiguus neurons produces release of acetylcholine in the cardiac ganglia and consequent bradycardia, we examined the effect of microinjection of DHA in nucleus ambiguus on heart rate. DHA microinjected into nucleus ambiguus produced bradycardia in conscious rats. Our results support a heart rate-lowering effect of DHA by activation of FFA1 receptor in cardiac-projecting neurons of nucleus ambiguus (Fig. 4).

Fig. 4. Diagram summarizing the mechanism proposed.

DHA, acting on FFA1 receptor (FFA1R) in nucleus ambiguus neurons, increased [Ca²⁺]_i and produced depolarization leading to bradycardia. The effect of DHA was prevented by GW1100, a FFA1 receptor antagonist.

Resting heart rate, reflective of sympathetic-parasympathetic balance, is considered an independent predictor of cardiovascular mortality in the general population and in those with cardiovascular diseases [21, 39]. An elevated resting heart rate is associated with a greater risk for sudden cardiac death [4, 39]. The reduction in heart rate produced by DHA supports its use as nutraceutical with potential beneficial effects in cardiovascular disease. Very recently, the increase in heart variability produced by n-3 PUFAs was proposed to be used as a monitoring parameter in patients with sepsis treated with n-3 PUFAs [40].

5. Conclusions

Our results support a vagal-mediated effect of DHA on lowering the heart rate via FFA1 receptor activation in nucleus ambiguus.

Highlights.

• Omega 3 fatty acids reduce heart rate via unclear mechanisms

• Docosahexaenoic acid (DHA) increases cytosolic Ca²⁺and depolarizes nucleus ambiguus

• Microinjection of DHA into ambiguus produces bradycardia in conscious rats

• DHA modulates cardiac vagal tone by activating FFA1 receptor in nucleus ambiguus

Abbreviations

[Ca²⁺]_i

cytosolic Ca²⁺ concentration

DHA

docosahexaenoic acid

EPA

eicosapentaenoic acid

HBSS

Hank’s balanced salt solution

n-3 PUFAs

omega-3 polyunsaturated fatty acids