Cardiac purinergic signalling in health and disease

Abstract

This review is a historical account about purinergic signalling in the heart, for readers to see how ideas and understanding have changed as new experimental results were published. Initially, the focus is on the nervous control of the heart by ATP as a cotransmitter in sympathetic, parasympathetic, and sensory nerves, as well as in intracardiac neurons. Control of the heart by centers in the brain and vagal cardiovascular reflexes involving purines are also discussed. The actions of adenine nucleotides and nucleosides on cardiomyocytes, atrioventricular and sinoatrial nodes, cardiac fibroblasts, and coronary blood vessels are described. Cardiac release and degradation of ATP are also described. Finally, the involvement of purinergic signalling and its therapeutic potential in cardiac pathophysiology is reviewed, including acute and chronic heart failure, ischemia, infarction, arrhythmias, cardiomyopathy, syncope, hypertrophy, coronary artery disease, angina, diabetic cardiomyopathy, as well as heart transplantation and coronary bypass grafts.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9436-1) contains supplementary material, which is available to authorized users.

Synopsis

ᅟ

Introduction

ᅟ

Early history

ᅟ

Cardiac innervation

ᅟ

Sympathetic nerves

Parasympathetic non-adrenergic, non-cholinergic transmission

Sensory nerves

Intracardiac neurons

Central nervous system control of the heart

Vagal cardiovascular reflexes

Actions of adenine nucleosides and nucleotides

ᅟ

Cardiomyocytes 1979-1999

P1 receptors

P2 receptors

Cardiomyocytes 2000-2014

P1 receptors

P2 receptors

Atrioventricular and sinoatrial nodes

AV node

SA node

Atria

His-Purkinje fibres

Papillary muscle

Endocardium

Release and degradation of ATP

ᅟ

Release of ATP

Degradation of ATP

Pathophysiology and therapeutic potential

Acute and chronic heart failure

Ischaemia

Myocardial infarction

Arrhythmias

Paroxysmal supraventricular tachycardia

Ventricular arrhythmias

Atrial fibrillation

Cardiomyopathy

Syncope

Cardiac hypertrophy

Sick sinus syndrome

Coronary artery disease

Heart transplants and coronary bypass grafts

Heart transplantation

Coronary bypass grafts

Diabetic cardiomyopathy

Angina

Pulmonary hypertension

Introduction

This review is a historical account of purinergic signalling research, which has led to our current understanding of its roles in the complex cardiac physiology and pathophysiology (comparable to retrospective art exhibitions). In 1978, Burnstock proposed that there were two families of receptors for purines, which he named P1 receptors (R) (receptors activated by adenosine) and P2R (receptors activated by adenosine 5′-triphosphate (ATP) and adenosine 5′-diphosphate (ADP)). Methylxanthines antagonised effects mediated by P1R, but not P2R [1]. When the actions of ATP were equipotent or less potent than that of adenosine, this suggested that ATP was acting via P1R after its enzymatic breakdown to adenosine. This could then be confirmed by antagonism with methylxanthines, such as theophylline or caffeine, non-selective P1R antagonists. The distribution of P1R and P2R in the guinea pig and frog hearts was described on the basis of pharmacology [2]. Current knowledge of receptor subtypes, based on cloning, pharmacological characterisation and second messenger systems published in the 1990s, is as follows: P1R are G protein-coupled receptors (A1, A2A, A2B and A3 subtypes), P2XR are cationic channels (P2X1–7) and P2Y are G protein-coupled receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14) (see [3, 4]). The development of antibodies for P1R and P2R subtypes led to their localisation using immunohistochemistry and in situ hybridisation techniques, which led to subsequent advances in the field. The effects of adenosine and ATP acting on P1R and P2R on cardiomyocytes are summarised below. Thus, by definition, purinergic signalling means the actions of extracellular purine compounds mediated by cell surface receptors (i.e. P1R and P2R); moreover, the critical roles of intracellular purines in cellular energetics and metabolism are not discussed in this review.

Thousands of papers dealing with purines and the cardiovascular system in general and the heart in particular have been published since the first report on the effects of adenine compounds in the heart in 1929 [5], so it is inevitable that the coverage of the multiple areas of research in this field is limited and the citation of relevant papers is selective. Thus, we apologise if our selection does not include papers that others in the field feel should have been cited. The selection of papers published in the last decade about purinergic signalling in the heart is focused on pathophysiology. The vast majority of studies in this field deal with the two major purines: ATP and the product of its enzymatic degradation, adenosine.

Many reviews on various aspects of purinergic signalling in cardiac physiology and pathophysiology have been published over the years, including the following:

• Physiological roles of cardiac P2X and P2Y purinoceptors [6–16];

• Roles of adenosine in health and disease [17–35];

• Effects of ATP and adenosine on coronary myocytes [12, 36];

• Purine degradation pathways in the myocardium [18, 37];

• Myocardial nucleotide transport [38];

• Non-adrenergic, non-cholinergic (NANC) neural control of the atrial myocardium [39];

• Vagal cardiovascular reflexes [40];

• Genetic modulation of adenosine receptor function [41].

Pathophysiology

• Heart failure [16, 42];

• Coronary artery disease (CAD) [43];

• Congestive heart failure [44];

• Cardiac arrhythmia [45–47];

• Cardioprotection [48–54];

• Ischaemia [55, 56];

• Myocardial transplantation [57];

• Adenosine and kidney function in heart failure [58, 59];

• Paroxysmal supraventricular tachycardia (PSVT) and fibrillation [60–63].

  Broad reviews about purinergic signalling have been published that include a section about the heart [64–78].

Early history

The seminal paper by Drury and Szent-Györgyi [5] reported that extracellular purine compounds, in particular adenosine 5′-monophosphate (AMP), act on the coronary arteries of the guinea pig, cat, rabbit and dog. Later, it was shown in the perfused rabbit heart that adenosine is a powerful dilator of the coronary vessels [79]. The effects of adenosine on the human heart were also examined early on [80]. Honey et al. [81] concluded that adenosine was not useful for the treatment of heart disease. Intravenous administration of adenosine in patients led to paroxysmal tachycardia. A review summarising these early studies was published by Drury in 1936 [82]. He noted, in particular, unpublished observations that “ATP produces heart block in the guinea pig and appears to be more active than adenosine”. Heart block by ATP in the rabbit was also reported [83], and Gaddum and Holtz [84] found that ATP was more than three times more potent than adenosine in this regard. An important book entitled Biological Actions of the Adenine Nucleotides was published in 1950 by Green and Stoner [85], which described seminal studies of the effect of ATP on the heart.

ATP injections were first used for the treatment of angina pectoris associated with coronary disease in the 1940s and AMP was also employed for the treatment of angina [86]. ATP was used early on for the treatment of patients with coronary insufficiency ([87–90]; and see references from an article published by RONA LABORATORIES Ltd. (1955) The influence of adenosine triphosphoric acid on coronary circulation and heart muscle, pp. 1–16). Senile myocardial fibrosis was treated with adenylic derivatives [91].

Since then until the early 1960s, relatively few publications dealt with the actions of extracellular nucleosides and nucleotides in the cardiovascular system. In 1963, Berne [92], and independently Gerlach [93], proposed that adenosine was the physiological regulator of reactive hyperaemia in the heart, what became known as Berne-Gerlach’s Adenosine Hypothesis. In 1972, Burnstock’s proposal of purinergic neurotransmission led to increased interest in this field [94]. He reported that ATP exerts negative inotropic and chronotropic effects on the mammalian heart and speculated that ATP could be released from vagal nerve terminals. At that time, it was shown in the isolated rat and guinea pig hearts that dipyridamole inhibits the uptake of adenosine [95]. Adenosine analogues were shown to be potent coronary dilators [96]. ATP and ADP were the only consistent releasers of prostaglandins from the isolated perfused rabbit heart, an action abolished by indomethacin [97]. ATP, ADP, AMP and adenosine injected into the left atrium of the guinea pig produced a period of heart block identical in both latency and duration, which raised the possibility that ecto-enzymes degrade extracellular nucleotides to adenosine, the active compound [98]. This was supported later by seminal studies by Schrader and Gerlach [99].

Adenosine produced cyclic AMP (cAMP) accumulation in guinea pig ventricular myocardium [100]. Another landmark paper showed the release of ATP from isolated adult heart cells in response to hypoxia [101]. Inosine, in addition to having cardiostimulatory effects, was reported to reverse β-adrenoceptor blockade [102]. AMP, as well as adenosine, was claimed to act via a receptor on cultured muscle cells and coronary myocytes [103]. This group also identified the important additional action of adenosine, i.e. the inhibition of the myocardial effect of catecholamines, what became known as the indirect anti-β-adrenergic action of adenosine [104].

The Adenosine Hypothesis was contested by Burnstock [105], who claimed that ATP, released during hypoxia from endothelial cells leading to the production of nitric oxide (NO), was the compound initially responsible for reactive hyperaemia. This role of ATP is supported by subsequent studies indicating that extracellular ATP can serve as a substrate for the extracellular production of adenosine (mainly by cell surface-localised enzymes CD39 and CD73) in addition to its role as a primary signalling molecule (see [106]).

Cardiac innervation

There are intrinsic cardiac neurons as well as sympathetic, parasympathetic, and sensory innervation of the heart.

Sympathetic nerves

Sympathetic nerve stimulation led to release of ATP and adenosine from the perfused rabbit heart [107]. Adenosine was shown to modulate sympathetic nerve stimulation-induced release of noradrenaline (NA) in the isolated rabbit heart [108]. Rat sympathetic neurons grown singly on small islands of cardiac myocytes were shown to release NA and acetylcholine (ACh) as well as an active compound that hyperpolarised and inhibited contraction of the cells; experimental evidence indicated that this compound was adenosine, the source of which could have been ATP [109]. ATP and NA were implicated as cotransmitters in sympathetic nerves supplying the sinus venosus of the toad [110]. Direct contact between sympathetic nerves and rat cardiac myocytes in vitro increased expression of functional calcium channels [111], although it was not recognised in this paper that ATP was a cotransmitter. Von Kügelgen et al. [112] presented, for the first time, evidence that prejunctional inhibition of NA release from sympathetic nerves involved P2R as well as adenosine and P1R [112]. ATP was shown to be released by isoprenaline from sympathetic nerves in the guinea pig atrium [113]. P2XR on sympathetic nerve terminals in the guinea pig right atrium regulate release of NA [114]. Depressed cardiac contractility occurred during postnatal development in rats after clinical sympathectomy [115], perhaps due to ATP released as a cotransmitter from sympathetic nerves. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) and ecto-5′-nucleotidase were shown to be present on nerve fibres only in the rat left ventricle, suggesting a different status of sympathetic nerves in left vs. right ventricles [116].

Parasympathetic non-adrenergic, non-cholinergic transmission

Non-adrenergic, non-cholinergic (NANC) neurotransmission has been identified in the mammalian heart. The transmitters involved were not identified initially, although calcitonin gene-related peptide appeared to be at least one of them [39, 117].

Sensory nerves

Inhibitory adenosine A1R were identified on cardiac sensory nerves and it was suggested that they may have a modulatory action on cardiac NANC neurotransmission [118, 119]. It was concluded that both A1R and A2R were present on ventricular epicardial sensory nerve endings of dorsal root ganglion (DRG) neurons [120]. Using in situ hybridisation, it was shown that P2X3R are localised on sensory nerves in the heart [121]. Using a canine model in vivo, it was found that P2X2/3R are localised on vagal sensory nerve terminals in the infero-posterior wall of the left ventricle [122].

Intracardiac neurons

The presence of quinacrine-positive intramural nerve cell bodies and nerve fibres in guinea pig atria suggested that a sub-population of intracardiac nerves could be purinergic (see Fig. 1) [6, 123]. Allen and Burnstock [124, 125] studied the actions of ATP on intracardiac neurons of two main types, AH and M cells in ganglia in the atria and in interatrial septum of newborn guinea pig heart. Three different responses to ATP were observed; initially, rapid transient depolarisations of about 40 % of both AH and M cells with agonist potencies of ATP>ADP, but with AMP and adenosine ineffective. In a further 30 % of AH cells, ATP evoked an initial depolarisation, followed by hyperpolarisation and a slow prolonged depolarisation. Finally, in about 2 % of AH cells, ATP evoked a slow depolarisation [124, 125].

Fig. 1.

Quinacrine fluorescent histochemistry showing quinacrine-positive intramural nerve cell bodies and varicose fibres in the guinea pig atrium. a A fine plexus of quinacrine-positive nerve fibres. b A similar preparation to a after treatment with 6-hydroxydopamine (one intraperitoneal injection, 250 mg/kg, 24 h prior to sacrifice). c A cardiac ganglion showing 20–30 quinacrine-positive nerve cell bodies; d Another ganglion, containing a smaller proportion of quinacrine-positive cell bodies. All bars represent 100 μm (reproduced from [6], with permission)

In a later study by another group, the actions of ATP on dissociated neurons from rat cardiac ganglia supported and extended these findings [126]. Huang et al. studied purinergic modulation of adult guinea pig cardiomyocytes in long-term cultures and co-cultures with extracardiac and intrinsic cardiac neurons [127, 128]. They found that cardiac neurons expressing P2R could greatly enhance cardiac myocyte contractile rate when activated by ATP and also that some intracardiac neurons expressed adenosine receptors [127, 128]. Thus, intracardiac neurons are not all parasympathetic neurons controlled by nicotinic neurotransmission, but they may also contain sensory neurons playing a role in local reflex pathways [129]. Indeed, Armour and co-workers have shown that in situ canine nodose ganglion afferent neurons can be activated by adenosine and ATP [127, 130]. A later study showed that in rat intracardiac neurons, ATP activated P2Y2R to transiently raise [Ca²⁺]_i and activate an inward current [131]. The ecto-nucleotidase CD39 localised in intrinsic neurons of human and porcine heart was shown to modulate ATP-induced NA exocytosis [132].

Central nervous system control of the heart

The increase in heart rate caused by activation of A1R appears to be mediated by its action on the central nervous system (CNS), while the decrease in blood pressure by activation of A2AR appears to be mediated in the periphery [133].

Vagal cardiovascular reflexes

There was an early hint about possible effects of purines in the heart mediated by a central vagal reflex [5]. In anaesthetised dogs, ATP, but not adenosine, was shown to trigger a vagal reflex, which appeared to mediate, in part, the transient negative chronotropic and dromotropic effects on the sinoatrial (SA) and atrioventricular (AV) nodes, respectively [134, 135]. The right vagus was identified to play a dominant role in carrying cardiopulmonary vagal afferent traffic [136] and subsequently the dominant role of the right vagus in the ATP-triggered vagal reflex in dogs was shown [137–139]. This reflex is the result of the activation of P2X2/3R localised on vagal sensory nerve terminals in the infero-posterior wall of the left ventricle (see Fig. 2) [122]. Thus, the actions of extracellular ATP in the heart are mediated by adenosine, the product of its rapid degradation by ecto-enzymes as well as a central vagal reflex. This explains the differential potency of ATP vs. adenosine for the suppression of sinus node automaticity and AV nodal conduction [15].

Fig. 2.

Central vagal cardio-cardiac reflex triggered by ATP (modified and reproduced from [15])

In view of the vagal component of the bradycardic action of ATP in the heart, Flammang et al. [140] hypothesised that patients with neurally mediated syncope might be hyper-sensitive to an intravenous bolus injection of ATP. They found that there was a cohort of patients with neurally mediated syncope or syncope of unknown origin that manifested an exaggerated response to ATP and that these patients could benefit from pacemaker therapy [141]. The negative chronotropic and dromotropic vagal effect of ATP in syncopal patients is reproducible [142] and thus, ATP is now an accepted diagnostic tool in this setting [143].

Evidence has been presented to suggest that ATP attenuates reflex increases in renal sympathetic nerve activity by stimulating left ventricular chemoreceptors with cardiac vagal afferents [144]. Adenosine is an endogenous modulator of cardiac excitatory afferent nerves and this has been suggested to play a mechanistic role in vasovagal syncope [145]. There is functional and immunohistochemical evidence that the cardiopulmonary chemoreflex pathways in the caudal nucleus of the solitary tract in the brainstem are directly inhibited by A1R activation and indirectly inhibited by A2AR via γ-aminobutyric acid release [146].

Actions of adenine nucleosides and nucleotides

Cardiomyocytes 1979–1999

P1 receptors

There is evidence that all four subtypes of P1R are expressed in cardiomyocytes [34]. A1R mediate the direct negative chronotropic and dromotropic actions of adenosine as well as indirect anti-β adrenergic actions [28, 147, 148]. In addition, there is substantial evidence that the activation of all four adenosine receptors is cardioprotective [149].

A1R were found in the guinea pig myocardium and adenylate cyclase was shown to be coupled to these receptors in the ventricular membranes [150]. A1R are also found in the guinea pig atrium [151] and rat ventricular myocytes [152]. It was suggested that cardiac A1R are critically dependent on temperature [153]. Evoniuk et al. [154] confirmed that A1R mediated the negative chronotropic effect of adenosine, while A2R mediated its vasodilatory-hypotensive effect.

A1R mediate the indirect anti-adrenergic action of adenosine, which is manifested in the attenuation of the electrophysiologic, metabolic and inotropic effects of catecholamines. For example, adenosine antagonised catecholamine-elicited glycogenolysis [155]. In addition, adenosine antagonised the positive inotropic action mediated by β-, but not α-adrenoceptors in rabbit papillary muscle [156, 157]. Furthermore, adenosine abolished early after-depolarisations and triggered activity induced by isoproterenol in isolated guinea pig ventricular myocytes [158]. Under specific experimental settings, the actions of adenosine and catecholamines could be additive; e.g. stimulation of cAMP production produced by adenosine and isoproterenol combined was essentially the sum of the two individual responses [159]. Chronic dietary theophylline was shown to up-regulate cardiac A1R, without changing the anti-adrenergic or inhibitory inotropic and chronotropic actions of adenosine receptor agonists [160]. In addition to A1R-mediated inhibitory effects, activation of A2AR resulted in stimulation of contractility of cardiomyocytes [161] and attenuation of the anti-adrenergic actions of A1R activation [162]. Interestingly, a differential A1R reserve for the direct and indirect actions of adenosine in the guinea pig atrial myocytes has been documented [163].

Cardiac sympathetic neurotransmission was shown to be inhibited by adenosine [164–166]. Sympathetic nerve stimulation led to the formation and release of adenosine from the rabbit heart [167]. Presynaptic inhibitory action of adenosine on release of NA from sympathetic nerves supplying the guinea pig heart was reported [168].

Adenosine is transported into rat cardiac myocytes via a saturable process [169]. Potentiation of the effects of adenosine by diazepam as well as dipyridamole on cardiac muscle was suggested to be due to inhibition of adenosine uptake [170]. Coformycin, an inhibitor of adenosine deaminase (ADA), potentiated the concentration-dependent decrease in the action potential duration and contractile force of atrial preparations caused by adenosine [171].

Certain actions of adenosine manifest an age-dependency; e.g. young guinea pigs were sensitive to the positive inotropic effect of the A1R antagonist NPC 205. The sensitivity was lower in older animals and basal heart rate was also significantly lower [172]. A2R activation was shown to inhibit neutrophil adhesion and injury in isolated cardiac myocytes [173]. A decrease in A1R-mediated responses was reported in ageing rat heart [174, 175].

No evidence was found for the presence of A3R in the atrium [176–178]. However, induction of apoptosis in cardiac myocytes from newborn rats by an A3R agonist was reported [179].

A1R stimulation activated δ-protein kinase (PK) C in rat ventricular myocytes [180]. Adenosine was shown to stimulate NO synthesis in rat cardiac myocytes [181]. Adenosine stimulated atrial natriuretic peptide (ANP) expression in cultured ventricular cardiomyocytes [182]. A1R over-expression can reverse the inotropic, but not the chronotropic, effects of adenosine in mouse heart [183]. A2AR activation enhances cardiomyocyte shortening [184].

P2 receptors

ATP elicited a triple response of the frog ventricle: an initial increase in contractility, followed by a period when the twitch amplitude fell, sometimes below the control level and thirdly, a slowly developing and longer-lasting increase in contractile force [185]. ATP was shown to facilitate the discharge of calcium from the sarcoplasmic reticulum in frog heart cells [186], in retrospect via P2YR. Evidence was presented to suggest that the inhibitory responses produced by ATP in the rat ventricle were mediated by P2R [187].

ATP, ADP and β,γ-methylene ATP had negative chronotropic and inotropic effects on guinea pig atrium, while α,β-methylene ATP (α,β-meATP) (which acts on P2XR) induced a stimulatory response [188]. ATP directly affected junctional conductance between paired ventricular myocytes from the heart of guinea pigs which could be explained by a specific ligand-receptor interaction between ATP and gap junctional channel proteins [189].

ATP is released from rat ventricular myocytes in response to hypoxia and acidosis [190, 191] or adrenaline in the perfused heart [192]. ATP increased [Ca²⁺]_i and contractility of ventricular myocytes of rat [193–195] and atrial myocytes of guinea pig and rabbit [196]. Ca²⁺ mobilisation by ATP was claimed to be regulated by PKC and PKA [197]. ATP was shown to increase mechanical activity and inositol trisphosphate (InsP₃) production in rat heart [198, 199], in retrospect mediated by P2YR. When inhibitory adenosine receptors were blocked, ATP produced a positive inotropic effect, probably mediated by P2YR [200]. Purinergic stimulation of rat cardiomyocytes induced tyrosine phosphorylation, a major mechanism for InsP₃ generation [201].

P2X1R were identified on cardiac myocytes [202], and both P2X3 and P2X4R mRNA were detected in the human heart [121, 203]. In addition, P2X1, P2X3, P2X4 and P2Y2, P2Y4 and P2Y6R were identified in the human foetal heart [204]. Furthermore, P2X1R were found in low density on myocytes of the rat heart, with occasional high density patches near nerve varicosities, while P2X2 and P2X3R were confined to nerve fibres [205]. Photoaffinity labelling and functional assays of ATP receptors expressed by cardiac myocytes were first described by Giannattasio et al. [206, 207].

The mechanism underlying the positive inotropy induced by ATP in rat papillary muscle in vitro was determined to be mediated, in part, by increased Ca²⁺ inward current [208]. While in bovine atrial cells, dual control of inwardly rectifying K⁺ channels by ATP and ACh was reported [209]. Activation of chloride currents by purinergic stimulation of guinea pig atrial myocytes was described by Matsuura and Ehara [210] and later in rat and mouse ventricular myocytes [211, 212]. ATP-induced increase in [Ca²⁺]_i was attenuated in cardiomyocytes from vitamin B₆-deficient rats [213]. The activation of Cl⁻ channels in mouse atrial and ventricular myocytes is due to activation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channels through a novel intracellular signalling pathway involving purinergic activation of PKC and PKA [214]. L-type calcium channel activity was modulated by ATP and adenosine-5′-(γ-thio)-triphosphate in ferret and rat ventricular cells [215, 216] and sinoatrial nodal cells [217]. Increase in L-type calcium current amplitude induced by ATP was shown to be mediated by P2YR [218]. A later study confirmed that P2R as well as P1R contribute to the ATP-induced inhibition of L-type Ca²⁺ current in rabbit atrial myocytes [219, 220].

Experiments with electrically driven rat atria suggested that P2YR, as well as P1R (since 2-methylthio ATP (2-MeSATP) was effective) mediated rapid decrease in contractility, while P2XR (since α,β-meATP was effective) mediated the increase in contractility [221], in retrospect probably by P2X1 and/or P2X3R.

Molecular cloning and functional expression of a novel rat heart P2XR was reported [203]. Both P2XR and P2YR were cloned and characterised from the human foetal heart, but their role in developmental processes and physiological activation of the foetal heart remains to be determined [204]. Activation of P2R in guinea pig atrial cells was shown to increase the delayed rectifier K⁺ current through intracellular mechanisms independent of PKA, PKC or intracellular free Ca²⁺ [222]. Subsequently, it was claimed that P2YR were involved in this action [223]. Diadenosine polyphosphates activated guinea pig left atrium via P1R and P2R [224].

Activation of rat ventricular myocytes by ATP triggered oscillatory contractions and potentiated the amplitude of electrically triggered contractions [225]. ATP activated the muscarinic K⁺ channel via pertussis toxin-sensitive G proteins in guinea pig [226, 227] and dog atrial myocytes [228]. Uridine 5′-triphosphate (UTP) also activated muscarinic K⁺ channels, suggesting mediation by P2U (P2Y2 and/or P2Y4) receptors [229].

Cardiomyocytes 2000–2014

P1 receptors

A1R stimulation inhibited α₁-adrenergic activation of the cardiac sarcolemmal Na⁺/H⁺ exchanger [230]; it was also associated with increased production of NO and cyclic GMP [231], which prevents mitochondrial oxidant damage in rat cardiomyocytes [232]. Chronic caffeine treatment increased heart rate and resting blood pressure, which was concurrent with changes in P1R function [233]. A1R over-expression reverses, at least in part, the interaction of β-adrenergic and A1R stimulation suggesting that the receptor/effector coupling is dependent on receptor density at least in this experimental model [234]. Adenosine protects against apoptosis induced by angiotensin II in rat cardiomyocyte cultures [235].

A2AR activation increased contractility of isolated perfused hearts [236]. A2BR-mediated NO release in the mouse heart was blunted by knockout of the A2AR gene [237].

Diadenosine polyphosphates activated coronary vessels via P1R and P2R [238]. Diadenosine pentaphosphate (Ap₅A) was shown to produce A1R-mediated pro-arrhythmic effects in rabbit atrial myocardium [239]. Diadenosine monophosphate exerts indirect and direct negative inotropic effects in isolated human cardiac atrial preparations through A1R [240]. Adenosine 5′-tetraphosphate (Ap₄A) has been identified and characterised in human myocardial tissue [241] as well as Ap₅A and diadenosine hexaphosphate (Ap₆A) [242]. In human and guinea pig hearts, Ap₅A had positive inotropic and sustained anti-β-adrenergic effects, acting via P1R [243]. Ap₅A was identified as a potent activator of the sheep cardiac ryanodine receptors [244].

Free radicals potentiated the negative dromotropic effect of adenosine in guinea pig heart [245]. p38 Mitogen-activated protein kinase (MAPK) plays a mechanistic role in A1R-mediated anti-adrenergic action of adenosine in rat ventricular myocytes [246], while ERK1/2 signalling pathway activation by adenosine in cardiomyocytes resulted from an additive stimulation of A1R, A2AR and A3R, which involved G_i/o proteins, PKC, and tyrosine kinase for A1R and A3R, and G_s and PKA for A2AR; moreover, the A3R response also involved a cAMP/PKA pathway via PKC activation [247].

A3R stimulation reversed myocardial stunning of isolated atrial and papillary muscles [248]. An age-related reduction in expression of both A1R and A2R was found in the rat heart [249] in agreement with previous studies [174, 175]. The negative inotropic and chronotropic effects of fluoxetine, a selective serotonin re-uptake inhibitor antidepressant, on isolated guinea pig atria were suggested to be mediated by inhibition of re-uptake of adenosine or by the activation of A1R [250]. While the A1R is involved in the regulation of heart rate in the mouse, the magnitude of its involvement is more pronounced in males vs. females [251]. Inhibition of ADA enhanced the inotropic response mediated by A1R in hyperthyroid guinea pig atrium [252].

Several studies using genetic models of A1R knockout confirmed findings of previous studies, that the negative chronotropic action of adenosine is mediated by A1R. Thus, using A1R and A2AR knockout male mice, it was found that heart rate was higher in A1R knockout mice, but lower in A2AR knockout mice [253]. Also, the negative chronotropic effect of bolus injections of adenosine in vivo were abolished in A1R knockout mice [254].

It was reported that multivalent dendrimeric and monomeric adenosine receptor agonists attenuated cell death in HL-1 mouse cardiomyocytes expressing A3R [255]. Also, adenosine attenuated cardiomyocyte hypertrophy; adenosine kinase is an important mediator of this effect [256].

Differential effects of A2AR and A2BR on cardiac contractility were described [257]. A2BR mediated direct contractile effects without altering β-adrenergic or A1R-mediated anti-adrenergic effects, while A2AR mediated increase in cardiac contractility indirectly by modulating the A1R-mediated anti-adrenergic effect. Caffeine disrupted embryonic cardiac function and its response to hypoxia through blockade of A1R that raises concern regarding caffeine exposure during embryogenesis, particularly in pregnancies with increased risk of embryonic hypoxia [258]. NA release and stress-induced heart rate increase was selectively attenuated by partial A1R agonists, most likely due to a presynaptic attenuation of NA release [259].

There is reduced adenosine release from the heart of aged mammals, probably due to reduced mitochondrial purine synthesis [260]. A2BR mediate the release of interleukin (IL)-6 and IL-8 and vascular endothelial growth factor from cardiac stromal cells [261]. CD73, nucleoside transporters and inosine provoke arrhythmia mediated by A1R and A2AR in the developing heart [262]. The elevated plasma concentration of adenosine was probably the cause of bradycardia during experimental breath-hold diving [263]. A1R antagonists prevented the electrophysiological effects of amitriptyline, a tricyclic antidepressant, on atrial action potentials; it was suggested therefore, that A1R activation could mediate the cardiovascular toxic effects produced by amitriptyline [264]. Caffeine was shown to act via A1R in mouse embryos to alter adult cardiac function and DNA methylation [265].

P2 receptors

In concert with previous studies in animal models, it was found that ATP and adenosine also shorten action potential duration in atrial but not ventricular myocytes [266]. Regulation of muscarinic K⁺ channels, which mediate action potential shortening by ATP in guinea pig atrial myocytes was found to be mediated by the activation of phospholipase C (PLC) and subsequent cell membrane depletion of phosphatidylinositol 4,5-bisphosphate [267]. It was reported that P2X4R and P2X7R are expressed in the t-tubular network of rat ventricular cells [268]. Longitudinal stretch of rat atrial myocytes induced the activation of non-selective cation channels [269], which, in retrospect, may be due to release of ATP acting on P2XR. The involvement of death receptor signalling in mechanical stretch-induced cardiomyocyte apoptosis [270] might be mediated by P2X7R after release of ATP. P2XR activation enhanced cardiac contractility in isolated rat and mouse hearts [271]. Indeed, using transgenic mice with cardiac-specific over-expression of the human P2X4R [272], it was found that P2X4R mediate the increase in myocyte contractility in response to ATP [273]. In another study, an increase in ATP-induced inward current was observed in mouse cardiac myocytes over-expressing P2X4R [274]. Activation of cardiac P2XR was shown to augment the Ca²⁺ content of sarcoplasmic reticulum independent of cAMP and therefore likely to contribute to P2XR-mediated myocyte contractility [275]. In mouse cardiomyocytes, the positive inotropic effects of ATP were reported to be mediated by P2Y11R [276]. It was tentatively suggested that the positive inotropic effects of ATP might be mediated by P2X4-like receptors [277]. Interactions between purinergic and adrenergic receptors in the regulation of rat myocardial contractility in postnatal development was reported [278]. P2Y2/4R were involved in the regulation of myocardial contractility in growing rats [279].

It was suggested that ATP and UTP had opposite effects on the regulation of ANP secretion; ATP via adenosine and P1R increased secretion, while UTP via P2YR decreased secretion [280]. In a later paper, stimulation of ANP secretion was shown to be mediated by A3R [281].

P2YR were shown to mediate the regulation of CFTR chloride channels in mouse cardiac myocytes [282]. Regulation of UTP-activated Cl⁻ current involves P2YR, PLC-PKC signalling and ATP hydrolysis in mouse ventricular myocytes [283]. Outwardly rectifying chloride channel activity was up-regulated by intracellular ATP, but inhibited by extracellular nucleotides [284]. A study using guinea pig ventricular myocytes has indicated that extracellular ATP modulates the activity of K_ATP channels via P2YR coupled to phosphatidylinositol 4,5-bisphosphate [285]. P2X1R in human myocardium were found to be densely localised in gap junctions at intercalated discs between myocytes, closely associated with connexin 43 in some regions of some gap junctions, but spatially separate in others regions (Fig. 3) [286].

Fig. 3.

Immunohistochemical staining of P2X1 receptors in human left ventricle and co-localisation with connexin43. a P2X1 staining is shown (green, FITC) with arrows indicating the intercalated discs. Scale bar = 30 μm. b After pre-incubation of the antibody with epitope peptide, positive P2X1 staining at intercalated discs preferentially disappears, although some green and yellow auto-fluorescence remains (probably due to the highly fluorescent protein lipofuscin). Scale bar as in a. c Double labelling of P2X1 receptor and connexin43 in human left ventricle. Sections were double-labelled with anti-P2X1 (green, FITC) and anti-connexin43 (red, Cy3). The images were obtained by confocal microscopy. Both P2X1 and the gap junction protein connexin43 are localised in the intercalated discs. Note the variable degree of double labelling (yellow) in different discs. Scale bar 25 μm. d and e are higher magnification micrographs showing the variable amount of double labelling of P2X1 receptors and connexin43 in two different gap junctions. Scale bar in d and e 2 μm (reproduced from [286], with permission)

P2R-mediated signalling appears to be involved in the intercellular synchronisation of intracellular Ca²⁺ oscillations in cultured cardiac myocytes [287]. It was shown that maxi-anion channels expressed by neonatal cardiomyocytes were involved in ATP release [288].

Valvular myofibroblasts, together with endothelial cells, cardiac myocytes and smooth muscle from the cardiac valves were shown to be activated by P2Y2R-mediated Ca²⁺ release [289]. ATP and UTP activation of P2Y2R via a G protein and stimulation of PLCβ induces the opening of heteromeric TRPC3/7 channels, leading to a sustained, non-specific cationic current [290]. P2Y1R, P2Y2R, P2Y4R and P2Y6R as well as P2Y11-like receptors were co-expressed and induced function through G_q/11 protein coupling in neonatal rat cardiac myofibroblasts (see Fig. 4) [291].

Fig. 4.

Expression of P2Y receptor mRNA obtained from neonatal rat cardiac myofibroblasts. Total RNA was prepared and reverse transcription-polymerase chain reaction was carried out; 1.5 % agarose gel electrophoresis represent mRNA coding for β-actin (lanes 1–2), P2Y₁ (lanes 3–4), P2Y₂ (lanes 5–6), P2Y₄ (lanes 7–8), P2Y₆ (lanes 9–10), P2Y₁₂ (lanes 11–12), P2Y₁₃ (lanes 13–14) and P2Y₁₄ (lanes 15–16). Lanes 2, 4, 6, 8, 10, 12, 14, 16 correspond to the primer control without cDNA and lane L to the ladder. Gel images presented are from one experiment and representative of seven independent experiments (reproduced from [291], with permission from The British Pharmacological Society)

Increased P2X7R expression was shown in atrial cardiomyocytes of caveolin-1-deficient mice [292]. Stretch of atrial myocytes stimulates recruitment of macrophages via ATP released through gap-junction channels [293].

Atrioventricular and sinoatrial nodes

AV node

Both adenosine and ATP suppress atrioventricular (AV) nodal conduction. In 1949, Wayne et al. [294] showed that the depressant effects of ATP and adenosine were dominant on the AV node. AV nodal conduction block associated with myocardial ischaemia is mediated by adenosine [295, 296]. A subsequent study showed that endogenous adenosine, via A1R and a pertussis toxin-sensitive G protein, mediates hypoxia-induced AV nodal conduction block in guinea pig heart in vivo [297]. In the isolated guinea pig heart, the action of ATP was shown to be mediated by adenosine [295]. The suppression of AV nodal conduction by adenosine, manifested in the prolongation of the AH interval, is mediated by A1R [298]. The potency of the bradycardic effects of adenosine manifests species variability among guinea pig, rat and rabbit [299]. Chronic administration of R-phenyl isopropyl adenosine to guinea pigs desensitised the AV node to the negative dromotropic effect of adenosine in a homologous but not a heterologous manner and desensitisation of the AV node response to adenosine was associated with down-regulation of A1R, a decrease in the fraction of A1R in the high-affinity state and a decrease in the contents of G_i and G_o proteins [300].

At all concentrations tested, the negative chronotropic and dromotropic effects of ATP on canine AV and sinoatrial (SA) nodes in vivo were more pronounced than those of adenosine [301]. This differential potency was due to a vagal component in the chronotropic and dromotropic actions of ATP but not adenosine [301]. In human patients, intravenously administered adenosine and ATP were equally effective in producing AV block that was antagonised by aminophylline but not by atropine [302]. However, other studies in human subjects have shown a vagal component in the suppression of AV nodal conduction by ATP (see [303]).

In the isolated perfused rat heart, ATP induced arrhythmias, prolonged the PR interval and suppressed SA nodal activity and induced partial block of AV conduction [8]. AV node (and SA node) electrophysiological responses to ATP appeared to be different [304]. Freely moving mice with over-expression of A1R exhibited AV (and SA) nodal dysfunction and supraventricular arrhythmias [305].

SA node

Both adenosine and ATP suppress pacemaker activity of the sinus node, as well as junctional, His and Purkinje fibres automaticity [166, 306]. ATP (probably via adenosine) produced hyperpolarisation of cells in frog sinus venosus [307] and adenosine suppressed rabbit SA node automaticity [308]. The action of adenosine on the SA node was found to be mediated by the activation of a K⁺ outward current [309]. In the isolated perfused guinea pig heart, adenosine potentiated ventricular overdrive suppression probably by direct activation of K⁺ outward currents [310]. ATP increased sinus cycle length and SA conduction time in isolated blood-perfused dog atrium [311], and prolonged sinus cycle length in the canine heart in vivo [301].

mRNAs for P2X1R, P2X4R and P2Y1,2,4,6,12 and 14 receptors were expressed in human SA node, with P2Y14R manifesting the highest level (see Fig. 5) [312]. However, one study concluded that the electrophysiological effects of ATP on rabbit SA node pacemaker cells were via P1R, since no functional P2X1R or P2Y2R were found [313]. ATP is required to support the basal SA cell firing rate via the Ca²⁺, adenylate cyclise, and protein kinase pathways [314].

Fig. 5.

Relative abundance, as measured by quantitative PCR, of P2X and P2Y transcripts in the human right atrium and sinoatrial node. Means ± SEM (n = 4) shown (reproduced from [312], with permission from Springer)

Atria

Adenosine is also a modulator of L-type calcium channels in the atrial myocardium of guinea pigs [315]. In anaesthetised, open-chest cats, adenosine infusion significantly decreased atrial fibrillation (AF) threshold; this effect was associated with reduced contractility [316]. A similar action of adenosine in the canine atria was shown to be mediated by pertussis toxin-sensitive guanine nucleotide binding proteins (G proteins) coupled to adenosine receptors [228].

His-Purkinje fibres

Purkinje fibres are modified heart muscle fibres travelling from the AV node forward into the septum between the ventricles where they divide into right and left bundles. The initial portion of the bundle below the AV node is called the His bundle. The Purkinje fibres transmit the wave of depolarisation originating under physiological conditions in the sinus node and travelling from the atria, via the AV node to the ventricles. Under pathophysiological conditions, characterised by lack of depolarisation propagating from the AV junction, the His and Purkinje fibres can act as pacemakers. Both adenosine and ATP suppress the pacemaker activity of His and Purkinje fibres [166, 317, 318]. In guinea pig and dog, ventricular pacemakers were shown to be more sensitive to adenosine than the sinus node [166, 306]. Adenosine and ATP modulate the electrophysiological effects of catecholamines in Purkinje and ventricular tissue [319, 320]. The effects of ATP and adenosine on action potentials evoked in sheep Purkinje fibres under normal and simulated ischaemic conditions have been described [321]. Rosen et al. [318] have shown that attenuation by adenosine of the effects of epinephrine on canine Purkinje fibre automaticity was membrane potential-dependent. However, a later study has found that adenosine did not alter the NA-induced effects on automaticity of sheep Purkinje fibres under physiological or ischaemic-acidotic conditions in vitro (i.e. modified medium) [321]. But, both adenosine and ATP attenuated the reduction in the rate of rise of the upstroke and the amplitude of the action potential caused by ischaemic-acidotic conditions [321].

Papillary muscle

Papillary muscles are located in the lumen of the ventricles; they are attached to the cusps of the AV valves (i.e. the mitral and tricuspid valves). UTP prolonged the action potential duration in guinea pig papillary muscles via P2Y2R [322].

Endocardium

ATP, ADP, AMP and adenosine hyperpolarised in guinea pig endocardial endothelium-like cells studied either as small tissue preparation or freshly isolated cells [323].

In summary, adenosine and ATP exert negative chronotropic and dromotropic effects on cardiac pacemakers and AV nodal conduction, respectively. In addition, adenosine exerts a negative inotropic effect, while ATP may exert a positive inotropic effect on cardiac myocyte contractility. Both adenosine and ATP interact with the autonomic neural control of the heart via localised actions as well as central reflexes.

Both P1R and P2R are localised in the heart. mRNA and protein for all P2XR subtypes have been identified on cardiac myocytes [14, 205, 324]. mRNA for P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11 receptors is also expressed in cardiac muscle [14, 324–326].

Release and degradation of ATP

Release of ATP

Adenine nucleotides are present in variable amounts in the extracellular space of the heart; ATP is released from endothelial cells [327–329], from red blood cells (RBC) and activated platelets [14, 330]. The level of ATP in the coronary effluent of saline perfused hearts was in the range of 1 nM [331]. However, this low value reflects the rapid degradation of ATP by ecto-enzymes and indeed high quantities of adenosine have been detected in the perfusates. With the use of microdialysis, interstitial levels of ATP were established to be in the range of 40 nM [332]. The levels increased markedly during hypoxia and ischaemia [92, 101, 190, 192, 333]. ATP is released into the extracellular space during increased blood flow [334, 335], probably due to release of ATP from endothelial cells in response to shear stress (see [78]). ATP is also released as a cotransmitter from perivascular sympathetic nerves [336, 337]. ATP was also shown to be released from rat and mouse cardiac fibroblasts by hypotonic (mechanical) stimulation via connexin hemichannels [338]. A major source of extracellular ATP are RBC, which release ATP when tissue oxygen demand exceeds supply and/or under deformation and thereby, participate in vascular signalling and control the systemic circulation [339]. Other sources of ATP in the heart appear to be ischaemic myocytes, activated platelets, inflammatory cells and smooth muscle cells (see [14]). The mechanisms of ATP release involve vesicular exocytosis from both nerve terminals and vascular endothelial cells (see [328]) and there is more recent evidence for mediation of ATP release via connexin and pannexin hemichannels (see [340]). RBC are a major source of ATP, which participates in vascular signalling and control of the systemic circulation [330].

Degradation of ATP

The breakdown of ATP and ADP to adenosine and hypoxanthine was first reported by Jorgensen in 1956 [341]. Most of the ATP perfused into the heart is dephosphorylated during a single passage through the coronary vasculature [295, 333, 342]. The catabolism is likely to be due mostly to endothelial ecto-nucleotidase. 5′-Nucleotidase activity was also described in isolated mature rat cardiac myocytes [343]. The relative activities of 5′-nucleotidase and ADA in atrial and ventricular myocardium were determined by Choong and Armiger [344]. ADA degrades adenosine to inosine [345, 346]. ADA was localised by immunocytochemistry to the extracellular surface of endothelial cells of small coronary arteries [347]. ADA was also localised in mid-myocardium of all chambers of the rabbit heart [348].

Characterisation of a Ca²⁺/Mg²⁺ ecto-ATPase from rat heart sarcolemma was reported by Tuana and Dhalla [349]. Using immunogold methodology, 5′-nucleotidase was found in the cytoplasm of cardiac myocytes and coronary endothelium [350]. Heavy exercise training was shown to increase the activity of 5′-nucleotidase and ADA in the left ventricle of the rat heart [351]. In addition, 5′-nucleotidase of neonatal rat ventricular myocytes was shown to be stimulated by thyroid hormone [352].

Regulation of adenosine production by ecto-nucleotidases on adult rat ventricular myocytes was such that at the cell surface the level of nucleotides (especially ATP and ADP) was low, and the level of adenosine was high during periods of extracellular nucleotides supply [353]. Ectonucleoside triphosphate diphosphohydrolases (E-NTPDase or apyrase), ecto-nucleotidase pyrophosphatase (E-NPPs), ecto-5′-nucleotidase (CD73) and alkaline phosphatases are likely to be involved in the degradation of ecto-nucleotides (see [354]). ATP-diphosphohydrolase (apyrase) activity was reported in rat heart tissue [355–357]. Ecto-ATPases were localised histochemically on the plasma membranes of cardiac myocytes, capillary endothelial cells and nerve fibres in rat heart [358]. E-NTPDase1 was identified on cardiac sympathetic nerve endings modulating ATP-mediated feedback of NA release [359].

Cardiac fibroblasts

Adenosine, acting through A2BR, inhibits collagen and protein synthesis in cardiac fibroblasts; accordingly, it was suggested that A2BR agonists may protect against cardiac fibrosis [360]. In a later paper from this group, it was suggested that A2BR on proliferating cardiac fibroblasts play a role in regulating cardiac remodelling associated with myocardial infarction (MI) and ischaemic injury [361]. Over-expression of A2BR led to a decrease in basal levels of collagen and protein synthesis, while under-expression of A2BR yielded an increase in protein and collagen synthesis [362]. Elevated glucose increased the expression of A1R and A2AR, decreased expression of A3R and had no effect on A2BR, while insulin suppressed the expression of A1R and A2BR, but had no effect on A2AR and A3R expression [363]. Studies using RT-PCR revealed that mRNA for all four P1R subtypes, A1, A2A, A2B and A3, were expressed in rat cardiac fibroblasts, with A2R dominant, acting via cAMP second messenger [364]. In primary cultures of adult rat cardiac fibroblasts, adenosine activated the A2R-G_s-adenylyl cyclase pathway; the resultant cAMP reduced collagen synthesis via a PKA-independent, Epac-dependent pathway that feeds through PI3K [365]. In a recent abstract, it was shown that A2AR and A2BR mediate differential modulation of signal transduction and collagen production in murine cardiac fibroblasts; both A2AR and A2BR stimulation increased ERK phosphorylation, but only A2BR modulated collagen production [366].

P2YR mediate activation of c-fos gene expression and inhibition of DNA synthesis in cultured rat cardiac fibroblasts [367]. UTP, acting via P2Y2R, induced pro-fibrotic responses in rat and mouse cardiac fibroblasts and it was suggested that P2Y2R antagonists may provide a means to reduce cardiac fibrosis [368]. Thickening and scaring of connective tissue occurs, most often as a consequence of inflammation or injury. Purinergic signalling via P2Y6R in cardiomyocytes triggers pressure overload-induced cardiac fibrosis [369]. ATP was shown to up-regulate proliferation and migration of human cardiac fibroblasts, probably via P2Y2R, but also via P2X4R and/or P2X7R [370]. Recently, Lu and Insel [371] suggested that the fibrotic response in rat cardiac fibroblasts involves the integration of purinergic signalling that is pro-fibrotic by ATP, and anti-fibrotic by adenosine, the product of ATP degradation by ENTPDs.

Coronary blood vessels

AMP has been known to be a potent dilator of coronary vessels since 1929 [5] and later, adenosine, ATP and ADP were also shown to dilate coronary vessels [85, 92, 372].

The hypothesis by Berne [92] and independently by Gerlach [93] that adenosine was the physiological regulator of blood flow during reactive hyperaemia (what became known as the Adenosine Hypothesis) dominated the field for the next decade, even though conclusive supporting evidence was lacking (see [373, 374]; and see later section on “Ischaemia”).

ATP is released from coronary endothelial and RBC in response to shear stress, resulting from changes in blood flow and hypoxia (see [78, 330]). Release of ATP from cardiac endothelial cells has also been shown in response to ACh, bradykinin, 5-hydroxytryptamine and ADP [375]. ATP released from aggregating platelets and RBC also caused endothelium-dependent relaxation of canine coronary arteries [376, 377]. Intracoronary ATP [378], and also adenosine [379], produced maximal coronary vasodilatation in humans.

Large increases in myocardial blood flow were associated with ATP infusion in rat heart [380]. Intracoronary adenosine, ATP and ADP dilate these vessels largely via the endothelium [345], implying that P2R are expressed by coronary endothelial cells. ATP and adenosine hyperpolarised guinea pig cultured coronary endothelial cells; the adenosine-induced hyperpolarisation, but not that by ATP, was antagonised by theophylline [381], indicating that both P1R and P2R are present on these cells. A later study presented evidence for the presence of P2YR on endothelial cells mediating hyperpolarisation, and vascular relaxation via both P1R and P2R localised on smooth muscle cells, in guinea pig and rabbit coronary arteries [382]. It was suggested that ATP may be a more significant relaxant of canine large coronary arteries than adenosine, but that adenosine may be a more significant relaxant than ATP in small coronary arteries [383]. ATP also evoked endothelium-dependent vasodilation in human isolated coronary arteries [378, 384]. Direct smooth muscle (endothelium-independent) relaxation by ATP and UTP of human epicardial coronary arteries has also been reported [385], as well as hyperpolarisation of smooth muscle cells of the guinea pig coronary artery by ATP [386].

Vasodilatation by ATP in perfused guinea pig heart involves NO [387, 388], although in another study it was concluded that ATP-induced vasodilation of guinea pig heart did not depend on NO production, but may have been partly dependent on the production of prostaglandins [389]. Adenosine contributed little to the coronary vasodilation in guinea pig hearts resulting from bolus injection of ATP and ADP [389, 390]. The receptors involved in purinergic endothelium-dependent vasodilation were identified as P2Y1R and P1R subtypes in the rat [391, 392] and dog [393] heart microvessels. P2Y1R and P2Y2R were described in cultures of rat cardiac microvascular endothelial cells [394]. UTP-sensitive P2UR (P2Y2 and/or P2Y4), as well as P2Y1R, on human, canine and guinea pig cardiac endothelial cells have also been reported [388, 395, 396]. ATP and adenosine exerted opposing effects (stabilising and disrupting, respectively) on the barrier function (i.e. macromolecule permeability of microvascular endothelial cells and microvessels) of the rat coronary microvasculature [397].

In addition to vasorelaxation, predominantly via endothelial P2YR and P1R, P2XR and P2YR that mediate vasoconstriction are expressed on the coronary artery smooth muscle cells of human, porcine, rabbit and rat hearts [398–402]. Human coronary artery smooth muscle cells express P2YR and P2UR (P2Y2R and/or P2Y4R) leading to increases in [Ca²⁺]_i by 2-MeSATP and UTP, respectively [403]. RT-PCR studies of P2R in human coronary arteries showed pronounced expression of P2X1R and P2Y2R mRNA, while weaker expression of P2Y1, P2Y4 and P2Y6R mRNA [401]. This study also showed that contractile responses of human coronary arteries to α,β-meATP and the stable pyrimidine analogue, uridine 5′-O-3-thiotriphosphate, were consistent with activation of P2X1R and P2Y2R, respectively. UTP-evoked contractions of porcine coronary artery smooth muscle cells also appear to be mediated predominantly by P2Y2R [402]. Coexpression of mRNA for P2X1, P2X2 and P2X4R was found in smooth muscle cells of rat coronary arteries [404]. UTP elicited depolarisation of rat coronary artery smooth muscle [405]. Ap₅A and Ap₆A dinucleoside polyphosphates dilate or constrict rat coronary vessels via P2Y1R and P2XR on endothelial cells and smooth muscle, respectively [406]; adenosine 5′-tetraphosphate also dilated rat coronary vessels [241]. ATP can constrict human epicardial coronary veins [385]. Uridine adenosine-tetraphosphate is a novel vasodilator of the coronary microcirculation of swine hearts, acting via P1R [407]. ATP acting via P2YR seems to mediate NANC inhibitory transmission in lamb coronary small arteries [408]. One of the factors controlling coronary blood flow during exercise appears to be ATP [409–411].

Proliferation of porcine cultured coronary artery smooth muscle cells was promoted by ATP, via P2YR, and insulin acting synergistically [412]. A recent study indicates that P2X1R-mediated inhibition of the proliferation of human coronary smooth muscle cells involved the transcription factor NR4A₁ [413].

Nicotinamide adenine dinucleotide was reported to be a coronary vasodilator in 1971 [414] and adenosine is a potent coronary dilator in all mammalian species studied, including humans [415–417]. The vasodilator actions of adenosine are mediated by A2R [36, 418–420] since A2R-mediated coronary vasodilation was antagonised by methylxanthines [421], located on endothelial cells [422–426]. The adenosine may be released directly from cardiomyocytes and endothelial cells after intra- and extracellular breakdown of ATP, respectively [427].

Adenosine receptor-mediated hyperpolarisation of bovine and porcine coronary artery smooth muscle was reported [428, 429]. Adenosine was found also to hyperpolarise cultured guinea pig coronary endothelial cells; this finding supports the hypothesis that the hyperpolarisation of the endothelium induced by adenosine released into the perivascular space of the capillaries may be conducted electrotonically to the terminal arterioles and may cause vasodilation via current flow through myoendothelial gap junctions [430].

It was proposed that there was another P1R subtype, in addition to A2R, located on smooth muscle [431] since N⁶-cyclopentyladenosine, an A1R-selective agonist, had vasorelaxant activity in porcine and canine coronary arteries [432, 433] and A1R are expressed by smooth muscle cells isolated from the porcine coronary artery [434], which also express A2R [435]. Adenosine-mediated relaxation in human small coronary arteries via A2BR, which was independent of the endothelium and NO [436]. However, in a later paper, it was claimed that in human coronary arteriolar smooth muscle, the A2AR mediated vasodilation [437]. A2AR and A2BR mediate production of NO in cultured porcine coronary artery endothelial cells [438] and it was suggested that while A2AR are predominant, A2BR may also play a role in adenosine-induced vasodilation, possibly through the p38 MAPK pathway [439]. A later study showed that the A2AR mediated relaxation via the smooth muscle [440]. Endothelium-dependent coronary vasodilation in the guinea pig heart was shown to be due to multiple P1R subtypes; A1R mediated the release of both NO and prostaglandin I₂, and A2AR and A3R acted mainly via NO [441]. The presence of A3R in the rat coronary circulation has also been claimed [442]. A2AR and A2BR also mediate coronary vasodilation in mice [443, 444] and up-regulation of A2BR in A2AR knockout mouse coronary artery has been reported [237, 445]; an involvement of K_ATP channels in this action was also demonstrated [445].

Inosine transiently decreased coronary flow in the rat, but potentiated vasodilation by adenosine [446]. Porcine coronary vasodilation was produced by Ap₄A, probably via P1R [447]. Diadenosine polyphosphates are potent vasoconstrictors of human coronary artery, radial artery and saphenous vein bypass grafts and it was suggested that they may play a role in post-operative contraction of these grafts [448]. The role of adenosine in the dilation of human coronary vessels was reviewed by Heusch [449]. The P2Y12R antagonist, ticagrelor, enhanced adenosine-induced coronary vasodilatory responses in humans [450].

A period of maturation of the rabbit heart after birth was required before an adult level of coronary responsiveness to exogenous adenosine was demonstrated [451]. It was proposed that different adenosine receptor subtypes mediate coronary vasodilation in mature rats, but that there is a reduction in the response to adenosine with age that may be due to changes in the high-affinity receptor site [452], by a reduction in adenosine receptor transduction [453] and/or by a reduction of A3R-mediated activity [442, 454]. In vasculogenesis, long-term signalling by adenosine in cardiac microvascular cells has been described [455].

In male endurance athletes, myocardial blood flow during adenosine-mediated hyperaemia was reduced compared to untrained men and the fitter the athlete, the lower was the adenosine-induced myocardial blood flow, although A2AR density was unchanged [456]. Sexual dimorphism in the permeability response of coronary microvessels to adenosine was described [457].

More recently, a selective A2AR agonist has been introduced into the clinical setting to induce coronary vasodilation as a pharmacological stress agent in conjunction with radionuclide myocardial perfusion imaging in patients unable to undergo adequate exercise stress test, commonly used to diagnose coronary artery stenosis [458].

Pathophysiology and therapeutic potential

Acute and chronic heart failure

About one half of patients with chronic heart failure (CHF) and about two thirds of patients with acute heart failure have concomitant renal dysfunction, termed the cardiorenal syndrome.

There is endogenous adenosine accumulation in patients with CHF [459]. It was suggested that selective A1R blockade may be a useful adjunctive diuretic in heart failure. Long-term oral administration of dipyridamole, leading to high extracellular levels of adenosine, improved cardiac status of patients with mild to moderate heart failure [460]. A1R up-regulation accompanied decreasing myocardial adenosine levels in mice with left ventricular dysfunction [461]. Adenosine therapy was considered for its cardioprotective effect for CHF [462] mediated by A1R and A3R [463]. In contrast, impairment of adenosine action contributed to the pathophysiology of CHF [464] (see also the contradictory findings by [465]). A1R antagonists improved glomerular filtration, but simultaneously promoted natriuresis and diuresis in patients with heart failure [466–468]. A1R antagonists have been recommended for the treatment of cardiorenal syndrome [469]. Therefore, A1R antagonists are not recommended as a treatment for acute heart failure with renal dysfunction [470, 471], although rolofylline, a selective A1R antagonist, had some beneficial effects for high risk acute decompensated heart failure [472].

A2AR expression was decreased by chronic renal failure with or without left ventricular failure; this decrease was reduced in haemodialysed patients [473]. Intravenous infusion of adenosine improved left ventricular function in dogs with advanced heart failure [474]. A2BR mRNA was over-expressed in the left ventricle of minipigs with heart failure [475]. Adenosine release in CHF was found in the periphery and not in the myocardium [476]. In patients with CHF, following exercise the plasma levels of adenosine increased, while those of ammonia decreased and it was suggested that the enhanced cardioprotective actions of adenosine after exercise may be an adaptive response in patients with CHF [477]. The accumulation of adenosine in CHF was suggested to be due to the reduction of ADA gene expression [478].

Alterations in the expression of P2X1R in failing human atria were found [479]. P2X6R mRNA was reported to be up-regulated in left myocardium of patients with CHF [480]. P2XR activation protected the heart in heart failure models [481, 482]. A beneficial effect of MRS2339, a P2XR agonist in heart failure was demonstrated; it was identical to that produced by cardiac myocyte-specific over-expression of the P2X4R [483]. The improvement was associated with the preservation of left ventricular wall thickness in both systole and diastole in post-infarct mice and calsequestrin (CSQ) over-expression mice with cardiac-specific P2X4R over-expression and decreased left ventricular chamber size in CSQ mice with heart failure and dogs with pacing-induced heart failure [483]. Increases in ecto-5′-nucleotidase in the plasma and myocardium in patients with CHF were thought likely to contribute to the increased levels of adenosine [484]. In the rat coronary artery ligation-induced heart failure model, P2X4R mRNA was up-regulated by 93 % in cells of the SA node and in right atrial and left ventricular myocytes [312]. P2X7R are pro-thrombotic and knockout of the P2X7R gene was protective in a mouse model of coronary artery thrombosis [485].

Accumulation of blood within the heart, which is the result of back pressure in the veins, leads to congestive heart failure. There is congestion in the lungs and/or liver in heart failure, which may be associated with accumulation of fluid in tissues (oedema). Volume-overload congestive heart failure in dogs is associated with reduced myocardial inotropic responsiveness to the administration of β-adrenoceptor agonists. It was suggested that the elevated adenosine release in the failing myocardium contributed to these effects [486]. An A1R antagonist appeared to be beneficial for patients with congestive heart failure, but some side effects were noted [487].

An increase in cardiac P2X1R and P2Y2R mRNA was noted in congestive heart failure (Fig. 6) [488]. Enhanced P2YR-mediated dilatation by endothelium-derived hyperpolarising factor was also described in congestive heart failure [489]. However, in congestive heart failure, down-regulation of P2X1R was induced in peripheral resistance arteries [490]. In a rat model of ischaemic congestive heart failure, there was a selective down-regulation of P2XR-mediated pressor effects, while the hypotensive effects mediated by endothelial P2YR were unaffected and the adenosine-mediated inhibitory effects on heart rate were attenuated [491].

Fig. 6.

Up-regulation of P2X1 and P2Y₂ receptor mRNA levels in hearts from congestive heart failure (CHF) rats. P2X1 and P2Y₂ receptor mRNA molecule numbers/μg total RNA in the myocardium from sham operated and CHF rats. Values are means + s.e.m., n = 7–8. For each, receptor measurements were performed with the same concentration total RNA together with five increasing concentrations of competitor standard. The upper band represents the wild-type product and the lower band represents the competitor product. The left set represents a sham operated rat and the right the CHF rat. There is a clear shift in the equivalence point (arrow) in the CHF rat towards the right (higher concentration) for both the P2X1 and P2Y₂ receptor (reproduced from [488], with permission from Elsevier)

Ischaemia

Ischaemia leads to injury of the heart. Purine and pyrimidine nucleotides, released at the site of cell damage, generally contribute to injury, but adenosine is generally protective (see [492]).

As mentioned above, Berne [92] and Gerlach [93] put forward the adenosine hypothesis, according to which adenosine was the physiological regulator of blood flow during reactive hyperaemia following hypoxia. Although this hypothesis was supported by early papers [493, 494], data obtained in subsequent studies challenged this hypothesis. For example, theophylline, a non-selective adenosine receptor antagonist, blocked coronary vasodilation by perfused adenosine, but it did not block reactive hyperaemia [495–499]. Similarly, adenosine and ATP increased coronary blood flow, but after occlusion, reactive hyperaemia in the dog heart was not blocked by aminophylline, a non-selective adenosine receptor antagonist [500–503]. It was also shown that while reactive hyperaemia occurred about 10 s after resumption of blood flow, adenosine did not appear in the perfusate until about 90 s later. ATP, however, appeared early on in the coronary sinus effluent of the isolated working rat hearts in response to hypoxia [504]. Thus, Burnstock hypothesised [105] that the initial phase of vasodilation following hypoxia was due to ATP, released from endothelial cells [505] to cause vasodilation via NO [506], while adenosine, a breakdown product of ATP by ecto-enzymes, contributed only to the later stages of reactive hyperaemia by acting on P1R on the smooth muscle cells. The delay in appearance of adenosine in the perfusate was explained by the fact that ADP (derived from the rapid breakdown of ATP) inhibits 5′-nucleotidase, the enzyme that metabolises AMP to adenosine [507]. Ecto-enzymes involved in the breakdown of ATP by coronary microvascular endothelial cells from rat heart have been described [508].

An early study has found that a combination of suppression of contractility of right ventricular trabeculae and preservation of electrical stability could serve as a cardioprotective action by adenosine in ischaemia [509]. Adenosine decreased ischaemic damage and enhanced cardiac function during severe hypothermia [510]. Activation of adenosine receptors mimics the cardioprotective effect of pre-conditioning; intracoronary adenosine protects against reperfusion injury after coronary occlusion [511–514], an action mediated by A1R [515]. A reduced sensitivity to adenosine has been found in the ischaemic or hypoxic heart [374, 516]. A2R binding in the heart was shown to be modified by ischaemia [517] and ischaemia-reperfusion selectively attenuated coronary vasodilatation mediated by A2R, but not A1R agonists [432]. Attenuation of responsiveness would tend to counteract the cardioprotective effects of adenosine, but the importance of this is not yet clear.

Glycolysis inhibition and enhanced mechanical function of working rat hearts resulted from A1R stimulation during reperfusion following ischaemia [518]. Activation of adenosine receptors has been shown to reduce ischaemia-reperfusion injury in the heart [519, 520]. Transgenic A1R over-expression increased myocardial resistance to ischaemia [521]. A later study showed that the cardioprotective mechanism of A1R over-expression involved altered gene expression [522]. It was suggested that ischaemic stress-induced pre-conditioning is dependent on the concomitant stimulation of both adenosine and NA receptors and that P1R-mediated cardioprotection occured only if α₁-adrenoceptor activation was intact [523]. Evidence that activation of both A1R and A2R during hypoxia can attenuate myocardial injury was presented [524–526]. There is an age-dependent decrease in the cardioprotection provided by adenosine in reperfusion injury in rats [527]. However, a stronger anti-adrenergic effect of adenosine was reported in the ageing ischaemic rat myocardium, possibly due to cross-talk between A1R and A2AR [528]. Selective A3R activation is cardioprotective in wild-type hearts and hearts over-expressing A1R, although A3R gene deletion generates an ischaemia-tolerant phenotype [529]. In a later study, it was shown that improved resistance of the heart to ischaemic damage can be achieved by increasing the expression of A3R, without detrimental side effects on heart rate or systolic function [530]. Reduced A3R transcription may contribute to improved ischaemia tolerance in aged hearts [531]. Reduction in post-ischaemic inflammation and infarct size was achieved by perfusion of the canine myocardium with a selective A2AR agonist [532]. One study concluded that cardioprotection by adenosine was dependent on NO and was blunted by the ganglion blocker hexamethonium indicating that it was mediated mainly by the activation of a neurogenic pathway [533]. Apyrase (CD39), which catalyses the hydrolysis of ATP to AMP, provided myocardial protection against cardiac ischaemia-reperfusion injury [534]. UTP was claimed to reduce infarct size and improve mouse heart function after myocardial infarct via P2Y2R [535].

The mechanism underlying cardioprotection by adenosine is not fully understood. Activation by adenosine of the reperfusion injury salvage kinase pathway, involving phosphorylation of Akt and/or ERK1/2, which leads to inhibition of mitochondrial permeability transition pore formation [536–538], may be involved. Genetic deletion of A1R limits myocardial ischaemic tolerance [539]. The cardioprotective effect of ischaemic pre-conditioning was shown to be dependent on activation of adenosine A1R in the first few minutes of reperfusion [540]. In contrast, the infarct size-limiting effect of myocardial ischaemic post-conditioning was shown to be mediated by the activation of adenosine A2AR at the time of reperfusion [541]. Indeed, targeted deletion of A2AR attenuated the protective effects of myocardial post-conditioning [541]. Low dose adenosine infusion reduced the ischaemic burden and improved left ventricular regional systolic function in the ischaemic walls of patients with exercise-induced myocardial ischaemia [542]. The infarct-sparing effect of A2AR activation has been suggested to be primarily due to inhibition of CD4⁺ T cell accumulation and activation in the reperfused heart [543]. Another study using H9c2 cardiomyoblasts showed that the cardioprotection afforded by adenosine was microtubules-dependent and involved the stimulation by adenosine of cytosolic PKCε translocation to the nucleus and dephosphorylation at Ser729 [544].

It was first suggested that reflex responses mediated by cardiac sympathetic afferent nerves during myocardial ischaemia were caused by adenosine, released from the ischaemic myocardium, mediated by A1R [545], but later it was suggested that it was ATP that activated sympathetic afferents [546]. A brief intravenous infusion of ATL-146e, a selective adenosine A2AR agonist (added 30 min before reperfusion), reduced myocardial infarct size at 48 h after ischaemia-reperfusion perturbation [547]. Endogenous adenosine is an important mediator of ischaemic preconditioning and post-conditioning (see [548]). It has been claimed that A2AR and A2BR act in concert to induce strong protection against reperfusion injury in rat hearts [549] and co-operative activation of A1R and A2AR to produce cardioprotection in ischaemia-reperfused mouse heart has also been reported [550]. Indeed, endogenous adenosine makes a significant contribution to A1R agonist-mediated prevention of necrosis in a cardiac cell model of ischaemia by co-operative interactions with both A2AR and A2BR, but does not play a role in A3R agonist-mediated protection [551, 552]. Figure 7 shows a simplified depiction of the impact of adenosine receptors in protecting against myocardial injury following ischaemia or hypoxia.

Fig. 7.

Simplified depiction of the impact of adenosine receptors (AR) in ameliorating injury and promoting adaptation during and following myocardial insult. Responses may be modified in disease states. Receptor involvement (identified in either animal or human studies) is shown. *Support from human tissue studies; †supported from animal models; ‡speculative/debated (reproduced from [33], with permission)

It has been reported that A1R and A3R agonists reduce hypoxic injury through the involvement of p38 MAPK [553]. In situ ischaemic pre-conditioning conferred cardioprotection in A1R, A2AR and A3R, but not A2BR knockout mice, or in wild-type mice after inhibition of A2BR, and thus, it was suggested that 5′-nucleotidase and A2BR agonists might be considered as therapeutic agents for myocardial ischaemia [554, 555]. Activation of A3R protected against myocardial ischaemia-reperfusion injury in mice, an effect which disappeared in A3R knockout mice [529, 556, 557]. In male patients with stable angina, capadenoson, an oral A1R agonist, lowers heart rate during exercise, which was associated with prolongation of time to ischaemia [558]. CD73-derived adenosine promoted cardiac remodelling and recovery of ventricular performance after ischaemia-reperfusion, most likely by acting on T cells [559]. Adenosine was shown to stimulate the recruitment of endothelial progenitor cells to the ischaemic heart resulting in enhanced revascularisation [560]. Studies focusing on the attenuation of myocardial ischaemia by targeting A2BR have been reviewed recently [561]. Previous reviews discussed the role of adenosine in pre-conditioning and ischaemia-reperfusion injury [65, 562–570].

Although early on the focus was on the role of adenosine in ischaemic and reperfusion injuries, there is now increasing interest in the role of ATP in these settings. A possible role for nucleotides in cardiac ischaemia was first raised in 1948, with the emphasis on degradation of nucleotides which appears to take place within muscle cells during ischaemia [571]. Accordingly, local infusion of ATP was used to successfully delay the onset of irreversible ischaemic injury and the role of high energy phosphates in preservation of ischaemic myocardium was highlighted [572]. Delayed resynthesis of ATP following its depletion during myocardial ischaemia was proposed [573] and thus, ATP-MgCl₂ was used for the treatment of ischaemia (see [574–576]). Extracellular ATP and adenosine appear to play complementary, protective roles in ischaemic pre-conditioning through P2YR and P1R, respectively [577]. Pre-administration of UTP, by as much as 48 h before an ischaemic episode, had a cardioprotective effect [326, 578].

Ischaemia is accompanied by increased release of ATP from cardiac myocytes and sympathetic nerves [579–581]. Before its degradation to adenosine, ATP was shown to induce positive inotropy, but it may also depolarise cells and trigger arrhythmias [582]. Release of ATP from cardiac myocytes in response to ischaemia was suggested to be via connexin hemichannels [581]. In a later study, it was proposed that ATP released in this way acts as a paracrine agent to cause fibroblast activation leading to the development of fibrosis in the heart following MI [583]. Blocking P2X7R or pannexin-1 channels prevented the protective effect of both ischaemic pre- and post-conditioning [584, 585].

It has been suggested that release of ATP from cardiomyocytes is strictly regulated during ischaemia by a negative feedback mechanism consisting of maxi-anion channel-derived ATP-induced suppression of ATP release via hemichannels in cardiomyocytes [586]. In a more recent study, it was shown that ischaemic/hypoxic stress induced rapid ATP release from cultured cardiomyocytes and that distinct P2R, perhaps P2X7R and P2Y2R, regulated cardiomyocyte death [587]. Ischaemia-induced accumulation of ATP in the extracellular space was suggested to trigger enhancement of [Cl⁻]_i in ventricular muscle during ischaemic conditions [579].

Tissue transglutaminase 2 protects cardiomyocytes against ischaemia-reperfusion injury by regulating ATP synthesis [588]. There is up-regulation of P2X7R in rat superior cervical ganglia (SCG) after myocardial ischaemic injury [589]. Sensory-sympathetic coupling in SCG after myocardial ischaemic injury facilitates sympathoexcitatory action via up-regulated P2X7R [590]. The P2X7R antagonist brilliant blue G acting on stellate ganglia prevented the increased sympathoexcitatory reflex via sensory-sympathetic coupling induced by myocardial ischaemic injury [591]. It has been claimed that ATP post-conditioning provides a powerful protective effect in a rat model of ischaemia-reperfusion due partly to antioxidation and partly to inhibition of inflammation [592]. It has been claimed that puerarin, an active ingredient in the traditional Chinese medicine Ge-gen, blocked signalling transmission mediated by P2X3R in stellate ganglia and DRG and reduced myocardial ischaemic damage [593].

Extracellular purine nucleotides have been shown to be cardioprotective in various experimental models. Extracellular ATP protects against reperfusion-induced failure of the endothelial barrier in rat hearts [594]. Extracellular ATP also protects human cultured myocardial endothelial cells from apoptotic cell death during hypoxia, by activating P2Y2R-mediated MEK/ERK- and P13K/Akt-signalling pathways [595]. ATP administration, before or just after cardiac ischaemia, inhibited the development of ventricular muscle damage [584].

UTP, released during cardiac ischaemia, was claimed to act via P2Y2R and/or P2Y4R to play a substantial role in mediating cardioprotection from hypoxic damage [325, 326, 596, 597]. In isolated ischaemic rat hearts, ATP-loaded liposomes and ATP-loaded immunoliposomes effectively protected the myocardium from ischaemia-reperfusion damage as determined by systolic and diastolic function [598]. Moreover, in the rabbit heart in vivo with induced localised myocardial ischaemia, liposomal encapsulation of ATP significantly diminished the proportion of ventricular muscle at risk that was irreversibly damaged during reperfusion [598]. Thus, it was proposed that ATP-loaded liposomes could be developed as a treatment for myocardial ischaemia [598]. It has been suggested that P2Y6R and P2Y11R are involved in pyridoxal-5′-phosphate-induced cardiac pre-conditioning in rat hearts [599]. ADP acting on endothelial P2Y1R plays a major role in coronary flow during post-ischaemic hyperaemia [600]. ADP, acting via P2Y1R, mediates the release of tissue plasminogen activator during ischaemia and post-ischaemic hyperaemia. Tissue plasminogen activator is involved in maintaining the endothelial wall of blood vessels free of thrombi formation and it was suggested that this action of ADP may counteract some of the platelet activating effects of ADP [601]. It has been suggested that the P2Y4R could be a therapeutic target to regulate cardiac remodelling and post-ischaemic revascularisation [602]. P2Y2R activation protects cardiomyocytes from hypoxia in vitro and reduces post-ischaemic myocardial damage in vivo [603].

Extracellular ATP augments cardiac contractility by elevating intracellular calcium in cardiac myocytes. Impairment of extracellular ATP-induced Ca²⁺ mobilisation in rat hearts after ischaemia-reperfusion has been described [604]. Cardiac-specific over-expression of human P2X4R confers a beneficial effect in the left anterior descending artery ligation model of ischaemic cardiomyopathy [605]. Transgenic over-expression of P2X4R in mouse heart led to enhanced cardiac contractile performance after ischaemic infarction and increased survival at 1 and 2 months after infarction; this suggested that enhanced contractile function via P2X4R of the non-infarcted areas was likely to be a rescuing mechanism [606]. A recent review discusses P2X4R as targets for cardiac ischaemia [607].

Myocardial ischaemic nociceptive signalling was shown to be mediated by P2X3R in rat stellate ganglion neurons [608]. P2X3R were up-regulated in rat stellate and nodose ganglia after myocardial ischaemic injury and may underlie ischaemic pain [609–611]. Myocardial ischaemic injury induced an increase in expression of P2X3R in SCG and DRG neurons, which led to aggravated sympathoexcitatory reflexes [612]. The authors also claimed that oxymatrine, a Chinese herbal remedy for ulcers and tumours, may decrease the expression of P2X3R and depress the aggravated sympathoexcitatory reflex induced by ischaemic injury.

AZD6140, a reversible oral P2Y12R antagonist, inhibits adenosine uptake into erythrocytes and enhances coronary blood flow after local ischaemia or intracoronary adenosine infusion [613]. After ischaemia-reperfusion injury, the coronary vasodilator effect of Ap₄A was shown to be impaired, probably due to reduced responsiveness of P2YR to Ap₄A [614]. Extracellular tri- and di-phosphates were claimed to be responsible for cardioprotection against hypoxic damage, probably by preventing free radical formation [615]. Diadenosine triphosphate, a vasoactive mediator stored in platelet granules, induces coronary vasodilation, which is attenuated by ischaemia-reperfusion due to the functional impairment of P2YR [616]

A protective role of E-NTPDase 1/CD39 in ischaemia-reperfusion injury has also been shown. Specifically, targeted deletion of E-NTPDase 1/CD39 led to enhanced myocardial ischaemia-reperfusion injury, which was corrected by infusion of AMP or apyrase (which degrades nucleotides) [534]. More recently, transgenic expression of human E-NTPDase 1/CD39 in pigs protected against myocardial ischaemia-reperfusion injury [617]. Interestingly, robust and selective induction of E-NTPDase 1/CD39 was shown after pre-conditioning in the mouse heart [534]. Ecto-nucleotidase on sympathetic nerve endings attenuates ATP and NA exocytosis in myocardial ischaemia by reducing ATP levels and consequently the prejunctional facilitatory effects of ATP [580]. Conversely, over-expression of E-NTPDase 1/CD39 resulted in enhanced removal of exogenous ATP [618] and protection against murine myocardial ischaemic injury [619]. Since ATP availability greatly increases in myocardial ischaemia, it has been suggested that recombinant E-NTPDase 1/CD39 may offer a novel therapeutic approach to the damage caused by ischaemia by reducing sympathetic activity [618]. E-NTPDase 1/CD39 deletion led to an attenuation of the activity of sympathetic pre- and post-synaptic P2XR (attributed to P2XR desensitisation) perhaps due to prolonged exposure to ATP that accompanies E-NTPDase 1/CD39 deletion, and it was suggested that E-NTPDase 1/CD39 can potentially prevent the transition from myocardial ischaemia to infarction [620]. A recent study showed that decreased ecto-nucleotidase activity produced by ischaemia in rat heart may exacerbate subsequent reperfusion injury [621]. These observations are in congruence with an earlier finding that ischaemic pre-conditioning increased 5′-nucleotidase activity and adenosine release during myocardial ischaemia and reperfusion in dogs [622].

In summary, ATP is released from ischaemic cells and adenosine, the product of its degradation by ecto-enzymes, plays a major role in cardioprotection in the setting of myocardial ischaemia-reperfusion.

Myocardial infarction

Myocardial infarction (MI) is localised death of heart muscle in the setting of myocardial ischaemia (imbalance between oxygen supply and demand) caused by impaired coronary blood flow.

Protection against MI was mediated by A1R and also probably A3R in the rabbit heart [515, 623]. Activation of A2AR at reperfusion was shown to reduce infarct size in dog heart [519]. Inhibition of NO synthesis reduced infarct size via release of adenosine [624]. Magnesium reduced infarct size also via enhancement of the action of adenosine in rabbits [625]. Over-expression of A1R protected mice against MI [626]. The use of intravenous adenosine after primary coronary stenting reduced the infarct size [627]. Clinical outcomes in patients with acute MI undergoing reperfusion therapy (thrombolysis) were not significantly improved with adenosine infusion (3-h infusion of 50 or 70 μg/kg/min) [628]. However, it was reported later that continuous infusion of adenosine for 3 h improved left ventricular function and reduced infarct size in patients with ST-segment elevation MI [629]. The role of adenosine as an adjunct therapy in the prevention and treatment of no-reflow phenomenon in MI with ST-segment elevation was recently reviewed [630].

Acute Myocardial Infarction Study of Adenosine (AMISTAD) clinical trials showed that infusion of adenosine for 3 h resulted in a striking reduction in infarct size [631]. However, long-term stimulation of A2BR commenced after MI prevented cardiac remodelling in rats [632] and contributed to post-infarction heart failure [633]. In a more recent study, it was found that the selective blockade of A2BR reduced cardiac remodelling following acute MI in mice [634]. It was suggested that stimulation of A2BR blocks apoptosis of cardiac myocytes and that this may explain the anti-remodelling effect of A2BR stimulation after MI [635]. It was shown that A2BR signalling is linked to up-regulation of pro-angiogenic factors in cardiac Sca-1⁺CD31⁻ stromal cells, which is essential for overall improvement of cardiac recovery seen after their transplantation to the injured heart (post MI) [636]. Intracoronary adenosine improved myocardial microvascular perfusion in late reperfused MI [637].

Infarct size was reduced after a brief intravenous infusion of a selective A2AR agonist, ATL-146e [547]. In a clinical trial, a high dose of intracoronary adenosine given during percutaneous coronary intervention (PCI) did not reduce infarct size [638]. However, a later study provided evidence for contrasting beneficial effects of adenosine under similar conditions [639]. Intracoronary administration of adenosine, but not of nitroprusside, resulted in a significant improvement following acute MI [640].

Imidapril, an angiotensin-converting enzyme inhibitor, improved the attenuated inotropic responses to ATP in heart failure due to MI [641]. UTP reduced infarct size and improve heart function in rats after MI [578] and also in mice [535]. Positive inotropic effects in humans were elicited by UTP and uridine diphosphate via P2Y2R and P2Y6R, respectively [642]. Release of UTP from cardiomyocytes during MI was also claimed in this study. P2XR-mediated pressor reflex responses were augmented in animals with MI compared with healthy control animals, and congestive heart failure induced by MI was shown to up-regulate P2XR in the DRG [643]. There is increased risk of acute MI in carriers of the Thr-87 polymorphic variant of the P2Y11R; since extracellular ATP acting on the P2Y11R regulates inflammatory cells, the increased risk was hypothesised to involve an inflammatory mechanism [644]. Intravenous administration of high doses of ATP during primary PCI reduced left ventricular improved wall motion within an area at risk for MI suggesting that ATP can prevent reperfusion injury [645]. In addition, intravenous ATP administration significantly extended ST-segment resolution, reduced the no-reflow ratio, preserved left ventricular systolic function and prevented left ventricular remodelling [646]. An association of the P2Y2R gene with MI in Japanese men has been reported [647]. Clopidogrel and ticlopidine, P2Y12R antagonists used for thrombosis, are also used for prevention and treatment of MI, acting via L-type calcium currents on cardiomyocytes [648].

ATP, released from astrocytes within the rostral ventro-lateral medulla presympathetic circuits, induced increased sympathetic outflow contributing to the progression of heart failure following MI in rats [649]. These findings support the view that sympathetic overactivity in heart failure is detrimental, contributing to the progression of the disease. It was suggested that ATP metabolism is a target for protection against MI [650].

In summary, although there are conflicting reports regarding the actions of purine nucleosides and nucleotides during MI, it is clear that adenosine is effective in reducing MI size and is probably involved in post-infarct remodelling. Adenosine inhibits proliferation of cardiac fibroblasts and hypertrophy of cardiomyocytes, both of which play a role in cardiac remodelling after MI.

Arrhythmias

Paroxysmal supraventricular tachycardia

ATP was first used for the acute therapy of paroxysmal supraventricular tachycardia (PSVT) in the late 1940s [651] (and see reference to earlier studies therein) and later by others [652–658]. ATP was shown to evoke transient tachycardia at low doses and to induce AV block at high doses [659, 660]. Later, adenosine was also found to be effective in the acute termination of PSVT [661, 662] and was used effectively in children [663–666]. The mechanism of terminating the arrhythmias by either adenosine or ATP is the induction of transient complete AV nodal conduction block. Dipyridamole, which inhibits the uptake of adenosine and thereby increasing its extracellular levels, was found to reduce the amount of adenosine required for the termination of PSVT involving the AV node [667]. In contrast, it has been suggested that caffeine (a non-selective adenosine receptor antagonist) ingestion reduces adenosine efficacy in the treatment of PSVT [668], although, the methodology of the latter study was subsequently criticised [669].

Tachycardia can be induced by A2AR agonists and is mediated by direct stimulation of the sympathoexcitation system in awake rats [670]. Caffeine can also induce PSVT [669]. Slow infusion of calcium channel blockers are claimed to be a safe alternative to adenosine for the emergency treatment of PSVT [671]. The relative efficacy of adenosine and verapamil for the treatment of PSVT were found to be similar; adenosine had a higher rate of minor adverse effects, while verapamil had a higher rate of causing hypotension [672].

A comprehensive study of the clinical and electrophysiologic effects of ATP and verapamil on paroxysmal reciprocating junctional tachycardia was undertaken [673]. It was concluded that ATP was more effective and more rapid than verapamil, but with a higher incidence of cardiac and non-cardiac side effects. ATP was shown to be a safe and effective drug at low dose for re-entrant tachycardias, involving the AV node [674]. ATP and adenosine were found to be equally effective for the diagnosis and treatment of PSVT and the incidence and severity of side effects were similar [675]. However, other studies have shown that ATP is more potent than adenosine, probably due to the central cardio-cardiac vagal reflex triggered by the nucleotide (see [15]).

ATP was effective against wide QRS tachycardia [676], and it can be used as a diagnostic tool in the non-invasive diagnosis of dual AV nodal pathway as the substrate for PSVT [677–679]. The effect of ATP on PSVT seems likely to be due to its degradation by ecto-nucleotidases to adenosine to act via P1R [680]. However, continuous perfusion of ATP in animal models where adenosine receptors were blocked showed both negative and positive chronotropic and inotropic effects of ATP, which were dose dependent [122, 681].

Several reviews discuss the treatment of PSVT by ATP and adenosine [15, 61, 682–687]. Adenosine was recommended for the diagnosis of wide complex tachycardia [688].

Ventricular arrhythmias

Evidence was presented to suggest that adenosine in low concentrations contributed to ventricular dysrhythmia, while ATP and ADP and high concentrations of adenosine have anti-arrhythmic effects [689]. Adenosine was recommended as a safe and effective agent in the evaluation and treatment of infants and children with arrhythmias [46, 690–692]. However, several rare cases of life-threatening ventricular arrhythmias induced by adenosine have been reported [693, 694]. When the activity of intrinsic cardiac neurons was modified by locally applied adenosine, ventricular arrhythmias were induced [695]. An A2BR antagonist, GS-6201, reduced left ventricular dysfunction and ventricular arrhythmias 1 week after MI in a rat model [696].

It has been shown that acute adenosine administration in the rat activates cardiac vagal nerve terminals and it was suggested that this could make an important contribution to the anti-arrhythmic action of adenosine [697]. However, in cat, dog and man, the actions of adenosine (given as a rapid intravenous bolus injection) on SA and AV nodes do not involve the vagus (for adenosine-vagus interaction, see [303, 698]). Valuable articles discussing the role of adenosine in atrial arrhythmias and fibrillation have been published [699, 700].

It was proposed that the release of ATP in pathological conditions could induce a depolarisation, which would generate ventricular arrhythmias [701]. In isolated guinea pig ventricular myocytes, ATP alone did not induce after-depolarisations nor did it significantly alter the resting membrane potentials and action potentials. However, when it was applied with drugs known to increase intracellular Ca²⁺, ATP facilitated the induction of after-depolarisations and triggered activity in approximately 60 % of the cells [702]. ATP triggers arrhythmias in electrically stimulated rat cardiac myocytes [703]. ATP prevents the run-down of gap junctional communication between ventricular myocytes in newborn rats by promoting protein phosphorylation [704]. In a later paper, P2X1R were shown to be closely associated with connexin 43 in gap junctions in the human ventricular myocardium [286] and the possibility that decreased expression of P2X1R is related to arrhythmias in the heart in Chagas disease and diabetes was considered [705]. In the perfused mouse heart, the addition of ATP to the perfusate induced ventricular tachycardia; this effect was mediated by P2R and increased diastolic level of [Ca²⁺]_i [706].

Atrial fibrillation

Fibrillation is characterised by rapid unsynchronised beating of cardiac myocytes resulting in non-effective contractions of the atria or the ventricles. Transient episodes of atrial fibrillation (AF) occur in some patients after the administration of adenosine or ATP for the termination of PSVT; this is probably due to the shortening of action potential duration of atrial myocytes [228, 707, 708].

Abnormal calcium handling in AF was linked to up-regulation of A2AR [709]. High endogenous adenosine plasma concentrations were associated with AF during cardiac surgery with cardiopulmonary bypass [710]. However, the use of adenosine, instead of hyperkalemia, in cold crystalloid cardioplegia for the induction of cardiac arrest during coronary artery bypass grafting surgery, was found to reduce the incidence of post-operative AF [711].

Adenosine and ATP have been used in conjunction with various AF ablation procedures to expose dormant conductions between an arrhythmogenic superior vena cava (SVC) and pulmonary vein (PV) and the right atrium (i.e. acute adenosine/ATP-induced pulmonary vein reconnection may lead to a greater likelihood of recurrence of AF) [712–718].

Cardiomyopathy

Cardiomyopathy, or heart muscle disease, is a type of progressive chronic heart disease characterised by abnormally enlarged, thickened and/or stiffened myocardium. These pathological changes result in inadequate blood pumping and flow, causing heart failure and the backup of blood in the lungs or the rest of the body. Cardiomyopathy can also be associated with cardiac arrhythmias. It may be inherited, but can also be caused by other factors, including viral infections, alcoholism, vitamin B deficiency or amyloidosis.

In hereditary dystrophic cardiomyopathy in hamsters, adenosine reduced the force of contraction during β-adrenergic stimulation, but impaired adenosine feedback control of the heart did not play a role in the pathogenesis of this hereditary cardiomyopathy [719]. Pacing-induced regional differences in P1R mRNA expression were described in a swine model of dilated cardiomyopathy [720].

An increase in P2X1R expression in the atria of patients with dilated cardiomyopathy was reported [721]. A beneficial role for activation of cardiac P2X4R was described in a CSQ over-expression model of cardiomyopathy [722]. P2XR-mediated action on cardiac myocytes was beneficial in the CSQ model of cardiomyopathy, reducing hypertrophy and increasing life span [723].

Syncope

Syncope or transient loss of consciousness can be due to many factors involving the cardiovascular system and its neural control. Syncope of cardiac origin can be due to sinus node malfunctioning, paroxysmal AV nodal conduction block, PSVT or self terminating ventricular tachycardia. Patients with syncope of unknown origin or suspected neurally mediated syncope, were more susceptible to intravenous injection of ATP than those without syncope [140, 724]; patients with hyper-sensitivity to ATP can benefit from pacemaker therapy [140, 141, 725].

Adenosine and ATP have been used in conjunction with the head-up tilt table test (HUT) to provoke vasovagal reaction in susceptible patients during the test [726, 727]. Higher plasma levels of adenosine and increased expression of A2AR was found in syncopal patients with a positive HUT [728]. Since syncopes occur suddenly and unexpectedly, it is difficult to see a mechanistic role for chronic changes of A2AR expression and adenosine level in the acute syncopal events. A study of highly symptomatic vasovagal patients using an implantable loop recorder found that the heart rhythm during a spontaneous syncope was identical to the recurrent syncope but not that during HUT or ATP-test [729]. However, the method used by this group to quantify the effects of ATP during the test and the interchangeable use of adenosine and ATP as the provocateur agent has been criticised [141].

Cardiac hypertrophy

Activation of A1R attenuates cardiac hypertrophy and prevents heart failure in a mouse left ventricular pressure-overload model [730]. Phenylephrine-induced cardiomyocyte hypertrophy in rats was inhibited by activation of multiple adenosine receptor subtypes [731]. In addition, hypertrophy induced by phenylephrine in cultured rat ventricular myocytes was associated with compensatory up-regulation of the adenosine system manifested in the significant increase in gene and protein expression of adenosine A1R, A2AR and A3R [732], furthermore, phenylephrine-induced cardiomyocyte hypertrophy and calcification were shown to be regulated by the interaction of CD73 and alkaline phosphatase and inhibited by adenosine receptor activation [733].

Extracellular ATP induces immediate-early gene expression but not cellular hypertrophy [734], and inhibited adrenergic agonist-induced cardiac hypertrophy in cultured neonatal cardiac myocytes [735]. UTP, but not ATP, caused hypertrophic growth in neonatal cardiomyocytes [736]. Over-expression of human P2X4R in ventricular myocytes of a transgenic mouse increased basal cardiac contractility without cardiac hypertrophy or failure [274] and activation with a P2X4R agonist had beneficial effects [483]. Deletion of the P2Y4R gene in mice lowered exercise capacity and reduced myocardial hypertrophy [737].

Sick sinus syndrome

Sick sinus syndrome (SSS) is defined as malfunction of the sinus node. Intravenous injection of adenosine or ATP can be used to assess sinus node dysfunction and thereby diagnose SSS [738–745].

Coronary artery disease

Atherosclerosis of coronary vessels is known as coronary artery disease (CAD). The pharmacologic stress test is a common diagnostic procedure aimed at determining the extent of CAD and the affected coronaries in patients unable to perform conventional exercise stress. In this test, a vasodilating agent is administered followed by a radio-isotope (what is known as cardiac nuclear perfusion imaging). Affected coronaries fail to dilate properly resulting in a coronary steal phenomenon, manifested as a non-homogenous increase in coronary blood flow, which is reflected in the non-homogeneous distribution of the radio-isotope that can be mapped using a gamma-camera. Adenosine and ATP, and more recently a selective A2AR agonist (Lexiscan [458]), have been used as pharmacologic stressors in this setting [746–754]. The use of adenosine-cardiac magnetic resonance imaging for the detection of CAD has been recommended recently [755–757].

Administration of ATP into the left coronary artery has been used to treat patients with CAD in preference to papaverine [758]. ATP acts on endothelial P2YR to cause release of NO and subsequent vasodilation. Patients with CAD and patients with risk factors for the disease were shown to have reduced lymphocyte CD39 (NTPDase 1) ecto-nucleotidase activity compared to healthy controls [759].

Platelet adhesion and aggregation is a key step in the development of inflammation, thrombosis and atherosclerosis. Thus, platelet aggregation inhibitors, such as clopidogrel, prasugrel or cangrelor, antagonists of ADP-sensitive P2Y12R, have been used to prevent and treat CAD [760–763]. Several reviews discuss the use of anti-platelet drugs for the treatment and prevention of CAD [762, 764, 765]. It has been suggested that ADP-mediated migration of host smooth muscle-like cells and CD45⁺ leukocytes, via P2Y12R, may play a role in the development of transplant arteriosclerosis, partly by monocyte chemoattractant protein-1 [766]. Clinical trials with clopidogrel and ticlopidine in patients with CAD have shown significant benefit compared with aspirin. An association between the T744C polymorphism of the P2Y12R gene and the anti-platelet effect of clopidogrel was found in patients with CAD after coronary stenting [767]. ADP receptor antagonist discontinuation prior to coronary artery bypass surgery has been recommended [768, 769].

Continuous infusion and low-dose bolus injection of adenosine were early approaches to determine coronary vasodilator reserve in patients with CAD [770]. The safety of adenosine infusion during cardiac MRI was confirmed [771]. Coronary sinus adenosine was proposed to provide an index of myocardial ischaemia in patients with CAD [772]. Intravenous adenosine resulted in variable changes in systemic blood pressure, which can lead to alterations in fractional flow reserve lesion classification [773].

It was claimed that ADA played a key role in CAD [774]. Ischaemic pre-conditioning effects of adenosine were identified in patients with severe CAD that preserved coronary flow reserve [775]. It was reported that a high intracoronary bolus dose of adenosine was equal to or more effective than an intravenous infusion in assessing fractional flow reserve in patients with coronary heart disease [776].

Heart transplants and coronary bypass grafts

Heart transplantation

The responses to adenosine of the denervated donor sinus and AV nodes were of greater magnitude and duration than those of the innervated recipient and normal control hearts, suggesting supersensitivity to adenosine in the denervated human heart [777]. Cardioplegia is the technique used to stop the heart, either by injecting it with a salt solution, by hypothermia or by an electrical stimulus. This is used to enable complex cardiac surgery and transplantation to be performed safely. Adenosine injection prior to cardioplegia is used to protect against ischaemia-reperfusion injury during hypothermia [778–780]. ATP administered to pig hearts in cardioplegia and reperfusion had beneficial effects both on the myocardium and on peripheral vessels [781]. The introduction of ATP synthesis promoters did not improve the beneficial effects of adenosine in cardioplegia [782].

Selective up-regulation of P2X6R was observed in the myocardium of patients undergoing heart transplantation because of CHF [480]. The potential significance of this was studied in primary cardiac fibroblasts freshly isolated from young pigs. Tumour necrosis factor-α (TNFα), a proinflammatory cytokine implicated in CHF progression, increased cell death and similar effects were produced by ATP in a murine cardiomyocytic cell line. In cardiac fibroblasts, TNFα inhibited the down-regulation of P2X6R mRNA and may provide a basis for P2X6R up-regulation in CHF, which may contribute to CHF pathogenesis. There was significant reduction of ATP levels in 1-day-old blood compared to fresh blood, suggesting that fresh blood should be used for assessing T cell immune function in transplant patients [783]. A recent study found that P2X7R are specifically up-regulated in graft-infiltrating lymphocytes in cardiac-transplanted humans and mice [784]. Furthermore, a short-term treatment with a P2XR antagonist prolonged cardiac transplant survival [784].

Coronary bypass grafts

The purinoceptor distribution on endothelial cells of blood vessels used for coronary artery bypass grafts was examined [785]. The level of P2Y2R was much the same in human internal mammary arteries (IMA) and radial arteries (RA) and on saphenous veins (SV), whereas levels of P2X4R were low in IMA and RA, but high in SV. The authors suggested that the high levels of P2X4R in SV may be the principal reason why these vessels are found to be more susceptible to atherosclerosis and exhibit a reduction in graft patency longevity. Since P2X4R appears to play a role in arteriosclerosis and restenosis, it is suggested that IMA and RA are preferable for coronary bypass grafts. In another study, it was shown that P2X1R and P2Y6R mediate more prominent contractions in the SV than in the IMA and they suggested that P2X1R and P2Y6R antagonists could be used to prevent vasospasm and restenosis in the SV during and after revascularisation surgery [786]. The robust vasoconstriction produced by diadenosine polyphosphates observed in human RA was compared to the low incidence of vasoconstriction in the IMA [448]. It was concluded that the selective activity of diadenosine polyphosphates in RA would implicate them as potential mediators of post-operative contraction of the graft, confirming again the preference for the use of IMA for coronary bypass grafts.

Diabetic cardiomyopathy

The coronary dilator action of adenosine was shown to be reduced in diabetes [787, 788]. Activation of P1R was required for myocardial insulin responsiveness in vivo [789]. Atria from middle-aged diabetic rat models exhibited supersensitivity to the inotropic effect of adenosine [790]. Atria from 6-week streptozotocin (STZ)-induced diabetes in rats exhibited supersensitivity to the negative inotropic and chronotropic effects of adenosine [791]. A3R mRNA and protein levels were increased in whole heart from rats with STZ-induced diabetes [792]. The development of diabetes resulted in an increased uptake of adenosine by rat cardiac fibroblasts and reduction of their ability to release adenosine due to altered expression of nucleoside transporters [793]. The heart rate response to adenosine infusion was diminished in patients with diabetes mellitus, probably due to cardiovascular autonomic neuropathy [794]. Protective effects of adenosine on the diabetic myocardium against ischaemic-reperfusion injury were reported [795]. A predictor of adverse cardiac outcome in asymptomatic patients with type 2 diabetes is the heart rate response to adenosine [796].

Insulin improved the ATP decrease in the subendocardium of the left ventricle of STZ-induced diabetic, but not non-diabetic hearts [797]. The maximum response of [Ca²⁺]_i to exogenous ATP was increased in diabetic cardiomyocytes [798]. Modulation of purinergic neurotransmission via P2YR in isolated atria of STZ-induced diabetic rats has been reported [799].

Angina

Angina pectoris, pain in the centre of the chest, occurs when the demand for blood by the heart exceeds the supply by the coronary arteries that usually results from coronary artery atheroma. It can be treated with NO donors and β-blockers, but may need coronary angioplasty or coronary bypass grafts.

Adenosine was claimed to be a messenger between myocardial ischaemia and angina pectoris in a patient with a transplanted heart [800]. Intracoronary adenosine administration provoked anginal pain and aminophylline, a P1R antagonist, reduced both adenosine and exercise-induced chest pain [801]. Further, the severity of anginal pain produced by intravenous adenosine was milder in patients with silent ischaemia than in those with painful ischaemia. Adenosine infusion into both left and right coronary arteries also produced anginal pain [802]. It was shown later that adenosine, released during ischaemia, provoked anginal pain that was controlled by previous administration of theophylline, a non-selective P1R antagonist [803]. Another study found that intracoronary administration of adenosine produced anginal pain by a mechanism other than myocardial ischaemia [804]. Dipyridamole infusion, which inhibits adenosine uptake, increased anginal pain [805]. β-Endorphin counteracted adenosine-provoked anginal pain [806]. However, it was shown later that adenosine infusion provoked oscillation of chest pain without involvement of opioids [807]. Yet, A1R agonists were recommended for the treatment of neuropathic pain as well as cardioprotective agents [808].

Myocardial release of the breakdown products of ATP, inosine, hypoxanthine and lactate, during pacing-induced angina in humans with CAD was reported [809]. ATP was recommended as a sensitive and reliable method of detecting coronary lesions in patients with unstable angina pectoris [810].

Pulmonary hypertension

Several studies in animal models of pulmonary hypertension demonstrated the beneficial effects of ATP infusion in this setting [811–815]. Pulmonary hypertension is a serious problem in children with congenital heart defects. ATP-MgCl₂ (0.05, 0.1, and 0.2 mg of ATP/kg/min) was a safe and effective pulmonary vasodilator in children with pulmonary hypertension, secondary to congenital heart defects [816].

Concluding comments

What is clear from the voluminous literature about purinergic signalling in the heart is that:

1. Nucleoside and nucleotide receptors, i.e. P1R and P2R, respectively, play multiple roles in cardiac physiology and pathophysiology.

2. There are still important gaps in our knowledge and many questions still remain unanswered regarding purinergic signalling in the heart.

3. Many of the therapeutic strategies for a number of heart disorders based on the manipulation of purinergic signalling are not well defined. The side effects of some of the treatments need to be considered and strategies to overcome them defined.

The newly developed stable, small molecules that act as selective agonists and antagonists at purinergic receptors, which can be taken orally, as well as novel nucleotidase inhibitors and ATP transport blockers could be a major step forward towards resolving these problems. Studies with inhibitors of ATP release may also enhance our understanding of the relevant mechanisms including the genetic variations in response to purinergic compounds. In this context, immunological factors should also be taken into account.