Background
The transient receptor potential vanilloid type 1 (TRPV1) is a member of the mammalian transient receptor potential family of ion channels [
1] and was cloned in 1997 [
2]. TRPV1 is a non-selective cation channel with a preference for calcium, which can be directly activated by noxious heat (> 43°C), extracellular acidification, as well as a large heterogeneous group of natural compounds such as dihydrocapsaicin (DHC) from chili pepper [
3,
4]. The TRPV1 receptor is widely expressed in the human body [
5,
6] particularly in "port-of-entry" tissues, the central nervous system (CNS), and in the peripheral nervous system in primary small to medium diameter sensory neurons such as dorsal root, trigeminal, and nodose ganglia that give rise to C-fibers and Aδ-fibers [
2,
7]. The TRPV1 receptor is involved in several biological systems including thermosensation and regulation [
8,
9] and the sensation of pain [
10‐
12]. Also, in the lung the TRPV1 receptors play an important role in the regulation of respiratory functions [
13]. Thus, via stimulation of lung TRPV1 receptors by inhaled irritants may elucidate airway reflexes including cough and bronchoconstriction [
13].
We have demonstrated the feasibility of using TRPV1 agonist for obtaining drug-induced mild therapeutic hypothermia in healthy animals (accompanying manuscript). Accordingly this pharmacological approach for obtaining therapeutic hypothermia may prove beneficial in patients resuscitated from a cardiac arrest. On the other hand, a transient receptor potential vanilloid type 1 agonist may differentially affect the cardiovascular system in health and in the compromised situation as following cardiac arrest and cardiopulmunary resuscitation. The heart is richly innervated by sensory and vagal nerve endings. These nerves transduce chemical and mechanical changes from the heart to the brain. Interestingly, sensory nerve endings supplying the heart express TRPV1 [
14]. However, the role of TRPV1 receptors in the cardiovascular system is currently not understood and the reported data are equivocal. Thus, it has been shown that administration of capsaicin caused bradycardia and hypotension in anaesthetized dogs or rabbits [
15,
16], whereas in studies in anaesthetized dogs using intravenous infusion of a chemically pure capsaicin a transient increase in heart rate and blood pressure was demonstrated [
17]. Moreover, in anaesthetized guinea pigs it has been observed that capsaicin evokes a biphasic change of heart rate with a prominent bradycardia as an initial component [
18] or a triphasic blood pressure response in anesthetized rat following intravenous administration of the TRPV1 agonist anandamide [
19]. Interestingly, in TRPV1 receptor knock-out mice injection of capsaicin caused no changes in arterial blood pressure in contrast to the clear response in control mice expressing TRPV1 [
20].
Activation of the peripheral termini of TRPV1 receptors causes the release of various pro-inflammatory neuropeptides such as substance P (SP), neurokinin A (NKA), and calcitonin gene-related peptide (CGRP) [
4,
21], which in turn affect the cardiovascular system. The primary pharmacological action of SP in the isolated spontaneously beating heart is to decrease heart rate and relax coronary resistance vessels [
22‐
24]. The effects of SP on the cardiac system can be blocked by atropine, a competitive antagonist of the muscarinic acetylcholine receptor, showing that it is mediated by the cholinergic nerves [
23,
25]. On the other hand, the effect of NKA, which is co-stored with SP in capsaicin-sensitive nerves also causes bradycardia, but via both cholinergic and non-cholinergic nerves [
18]. In contrast to SP and NKA, GCRP causes tachycardia in isolated guinea pig heart preparations via stimulation of the GCRP receptor [
26,
27].
In the present study, our goals were to investigate the effect of continuous intravenous infusion of the TRPV1 agonist DHC on the cardiovascular system in conscious rats, and in a model of a compromised cardiovascular system, i.e. the anesthetized rat following cardiac arrest and cardiopulmonary resuscitation. The TRPV1 agonist DHC and infusion paradigm was selected based on feasibility studies of the ability to induce mild therapeutic hypothermia (accompanying manuscript).
Discussion
On two occasions in the healthy rats we observed a transient cardiovascular episode of bradycardia and hypotension accompanied by a motor component during the infusion of DHC, a TRPV1 agonist. Saito and Yamamoto reported that oral administration of capsaicin to rats caused tremor, clonic convulsion, dyspnea and lateral or prone position before death, and speculated that the cause of death may be associated to hypotension [
31]. In anaesthetized artificially ventilated dogs administered intravenously with capsaicin elicited the Bezold-Jarisch reflex [
32], which is characterized by hypotension and bradycardia [
33]. Moreover, studies in TRPV1 receptor knock-out mice have emphasized the role of the TRPV1 receptors in the activation of the Bezold-Jarisch reflex [
20]. Here we report transient episodes of bradycardia and hypotension, which was accompanied by convulsions/spasmodic movements. Also in pilot dose finding experiments during infusion of very high levels of DHC to healthy rats, we observed convulsions or spasmodic movements followed by death in about 20% of the tested rats (data not shown). While we cannot exclude that the origin of the cardiovascular collapse observed might be ascribed to unidentified factors causing hypotension and bradycardia, collectively our observations are consistent with an activation of the Bezold-Jarish reflex by stimulation of the TRPV1 receptor with the agonist DHC.
This is the first study to characterize the cardiovascular profile of a TRPV1 agonist in a model of a compromised cardiac system - the resuscitated rats. The results indicate that the susceptibility of the resuscitated cardiac arrest rats towards TRPV1 agonist induced Bezold-Jarisch reflex is increased compared to the healthy situation. The duration of the episodes of bradycardia/hypotension was relatively short in the resuscitated rats compared to the healthy rats. On the other hand 100% of the resuscitated rats compared to 33% of the healthy rats displayed episodes of bradycardia/hypotension. Also, the episodes occurred at lower levels of DHC in the resuscitated cardiac arrest animals compared to the healthy rats. The reason for this difference is not known, but again may relate to the structural and functional differences of the myocardium in the two models. Cardiac arrest causes a decrease in myocardial contractility [
34] and persistent cardiovascular derangements following CPR is seen including decreased cardiac output, arrhythmias and morphological myocardial damage [
35]. The ischemia and associated myocardial damage leads to an increase of CO
2 and lactic acid in the interstitial compartment, which in turn cause cellular acidosis and a decrease in pH [
36]. Accordingly, buffer agents administered during CPR may ameliorate post-resuscitation myocardial dysfunction and thereby improve survival [
37]. Since the TRPV1 receptor is sensitized by extracellular protons [
38], the observed increased susceptibility of the resuscitated rats versus the healthy rats towards DHC-induced episodes of bradycardia and hypotension therefore may be from a difference in the myocardial pH in the two situations. However, we did not measure myocardial pH in our experiment, and therefore we cannot exclude that other factors such as the level of anesthesia or the rate of infusion may have impacted on the reported results.
Intravenous infusion of capsaicin caused a dose-dependent increase in heart rate and blood pressure in anaesthetized dogs [
17]. Here, using DHC, we confirm these results in healthy conscious rats and in resuscitated rats and show that arterial blood pressure and heart rate increased by intravenous infusion. Similarly we have observed tachycardia and hypertension in response to intravenously infused DHC in conscious calves (data not shown). This chronotropic effect of DHC may be related to the release of GCRP following activation of the TRPV1 receptor [
26,
27]. Using the design of a step-wise incremental dose of DHC by increasing the infusion rate we here demonstrate that the general tachycardia/hypertension precedes the episodes of bradycardia/hypotension in the healthy rats. Accordingly the episodes of bradycardia/hypotension, which in turn may be mediated by the release of NKA and SP (20, 27) may occur at higher level of TRPV1 activation: i.e., the biphasic or triphasic change of heart rate previously reported following administration of a TRPV1 agonist [
18,
19] may be a result of pharmacokinetic properties.
In the healthy rats, the blood pressure changes during the 2.0-mg/kg/hr infusion were not dose proportional. During this infusion period, body temperature reached its lowest level (approximately 4.5°C lower than vehicle treatment). Because central control of blood pressure is influenced by body temperature, the substantial decrease in body temperature observed during the 2.0-mg/kg/hr infusion may be responsible for the lack of dose proportionality noted in the blood pressure changes. Ejection time would normally be shorter in response to higher heart rate and longer during lower heart rates. In this experiment, heart rate was higher, but ejection time also increased. This could be a predictor of a loss of cardiac contractility.
Interestingly, the episodes of bradycardia and hypotension and third degree AV block elucidated by DHC in the resuscitated situation could be fully prevented by pre-treatment with the muscarinic acetylcholine receptor antagonist atropine. It has been previously reported that the cardiovascular effects of SP [
23,
25], but not of NKA [
18], could be prevented by atropine. Our data emphasizes the role of the cholinergic nerves in the mediation of the Bezold-Jarish reflex. It is not known whether the observed cardiac effects were a result of a direct stimulation of the vagus nerve, which do express TRPV1 [
14], or occurs indirectly with the vagus nerve transmitting a signal generated via stimulation of TRPV1 receptors in the CNS, and this may be subject for further experimentation.
Competing interests
The study was sponsored by Neurokey AS. At the time of the study K. Fosgerau, M Jayatissa, M. Axelsen1, JW Gotfredsen, UJ Weber and C Videbaek where employed at the company. JW Gotfredsen, UJ Weber, L Køber and C Torp-Pedersen were founders of the company.
Authors' contributions
All authors have read and approved the final manuscript. KF: have made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data, and writing of article. GR, MJ, CV: Have made contributions to conception and design, or acquisition of data in cardiac arrest rats. MA, JG, UW, LK, CT: have been involved in drafting the manuscript or revising it critically for important intellectual content.