Could early ischemic arrhythmia triggered by purinergic activation of the transient receptor potential channels be prevented by creatine?

Abstract

Despite its degradation by ectonucleotidases, a low ATP concentration is present in the interstitial space; moreover, its level can markedly increase during various physiopathological conditions. ATP and uridine 5′-triphosphate (UTP) releases correlate with the occurrence of ventricular premature beats and ventricular tachycardia. ATP facilitates several voltage-dependent ionic currents including the L-type Ca²⁺ current. More recently, ATP and UTP were also shown to induce a poor voltage-dependent, long-lasting current carried by the heterotetrameric transient receptor potential (TRP) channels TRPC3/7. ATP effects result from its binding to metabotropic P2Y2 receptors that lead to diacylglycerol formation and activation of phospholipase Cβ and inositol-1,4,5-triphosphate production. ATP also favours TRPM4 activation by increasing Ca²⁺ release from the sarcoplasmic reticulum. Indeed, TRPM4 current properties match those of the Ca²⁺-activated, nonselective cationic current supporting the delayed afterdepolarizations observed under conditions of Ca²⁺ overload. In the present article, it was hypothesized that creatine, at a relatively high concentration, would serve as a buffer for the sudden release of ATP and UTP during the early phase of ischemia in association with previously described arrhythmic events. The potential preventive effect of creatine was tested by analyzing its ability to antagonize the arrhythmia that occurred on inducing a coronary ligature in rats that were or were not preinjected with creatine. Electrocardiogram recordings of creatine-injected rats clearly demonstrated that both ventricular premature beats and, particularly, ventricular tachycardia markedly decreased. The effect of creatine was even more striking in early deaths. However, an injection of beta-guanidinopropionate, a creatine analogue with 1000-fold lower kinetics, had no significant protective effect.

ATP, a high-energy phosphate donor, has been extensively studied since the role for extracellular purines was described by Drury and Szent-Györgyi in 1929 (1). Bolus venosus ATP injections have been successfully used for years in Europe for prompt termination of paroxysmal supraventricular tachycardia (2) despite the fact that ATP induces an initial tachycardia in approximately 50% of subjects (3). On the whole heart, extracellularly applied ATP slows the heart rate at low doses and induces atrioventricular and His bundle block accompanied by transient ectopic beats of atrioventricular junctional origin attributable to re-entry phenomena at higher doses (4). However, during various physiopathological conditions, such as ischemia, extracellular purines and pyrimidines are released so that ATP and UTP accumulate despite their brief biological half-life due to rapid degradation by ubiquitously distributed ectonucleotidases (5). Measurements of ATP in the effluent during reperfusion of an isolated rat heart showed a 79% disappearance of ATP infused on the atrial side, such that not ATP itself but its metabolite adenosine induces an increase in myocardial water content (6). Furthermore, it was recently demonstrated that phosphohydrolysis of ATP constitutes an important source of adenosine generation in cardioprotection by ischemic conditioning (7). The key enzyme appears to be CD39, an ectonucleoside-triphosphatase diphosphohydrolase, with apyrase providing pharmacological activity similar to that of CD39 while CD39 inhibitors increase infarct sizes. In control tissues, CD39 is expressed mainly on endothelia while ischemic preconditioning induces its expression on cardiomyocytes after 90 min.

Despite its degradation by ectonucleotidases, a low ATP concentration is present in the interstitial space; moreover, its level can markedly increase during various physiopathological conditions (4). Particularly, ATP is released during ischemia from various cell types, including cardiomyocytes (8), as previously shown using intrawall microdialysis (9). In the latter study (9), ATP release was correlated with the occurrence of ventricular premature beats and ventricular tachycardia. It has also been reported that uridine 5′-triphosphate (UTP) plasma levels estimated in the coronary sinus correlate with ventricular arrhythmia in pigs. Similarly, UTP is released in humans during cardiac infarction (10,11). Thus, during the first few minutes after an ischemic period, released ATP/UTP could accumulate in the vicinity of the cardiomyocytes before diffusing and being degraded, allowing for autocrine/paracrine purinergic stimulation. However, the mechanisms that lead to cardiac arrhythmia are unknown. This is of importance because the early phase of arrhythmia during an ischemic period in patients is highly deleterious and is not sensitive to presently known pharmacological agents.

Extracellular ATP activates the ionotropic (ligand-gated) receptors, the P2X_1–7 receptor family, and the metabotropic (G-protein coupled) receptors, P2Y_1–14 receptor families (4). Among the latter, P2Y_2,4,6 could also be activated by UTP to an extent (4,12). Of note, a single cardiac ventricular myocyte houses most of these P2X and P2Y purinoceptors (4). P2-purinergic stimulation has multiple effects on cardiac ionic currents: it increases the L-type Ca²⁺ current and most K⁺ currents and, in guinea pig atrial cells, activates a Cl⁻ current (4). Besides, on cells held at resting potential, a fast application of ATP at a micromolar concentration elicits a transient inward current, as initially reported in frog atrial cells (13), that requires extracellular Mg²⁺ (14–16). Furthermore, during ATP application in the presence of Mg²⁺ or not, a weak sustained inward current flows on cells held at −80 mV (15,17). The nature of the channel protein that carries this sustained current activated by ATP is unknown.

Transient receptor potential (TRP) channels constitute a family of ionic channels with low, if any, voltage dependency. The founding protein member was discovered in Drosophila melanogaster, in which it contributes to phototransduction by conducting calcium ions; however, a mutation induces a transitory response despite sustained lighting (18). The corresponding trp gene was cloned in 1989 (19) that led to identification of a cationic channel permeable to Ca²⁺ ions. Mammalian homologues encode channel proteins that have six transmembrane domains and assemble into heterotetramers (20–22). TRP channels are widely distributed in mammalian tissues and are involved in several cardiovascular functions and diseases (23,24). Similar to P2X purinoceptors, most TRP channels are nonselective to cations and act to shift the membrane potential to approximately 0 mV, thus depolarizing cells from their resting potential and allowing Ca²⁺ influx and cell automaticity. The TRPC subfamily is composed of seven members, TRPC1-7, with the TRPC3,6,7 subgroup being directly activated by diacylglycerol (25). TRPC7-expressing cells were first demonstrated to have both constitutively activated and ATP-enhanced inward currents that allow Ca²⁺ influx (26). Recently, TRPC6 and TRPC6/7 have been identified as essential parts of the α₁-adrenoceptor-activated cation currents in smooth muscle cells (27) while, in the heart, TRPC3 and TRPC6 proteins are essential for angiotensin II-induced hypertrophy (28,29) and TRPC3 is essential to the potentiated insulin-induced current (30). In the whole heart, the expression of several TRP channels (TRPC1,3–7; TRPV2,4; TRPM4,5,7 and TRPP2/1) has been demonstrated by reverse-transcription polymerase chain reaction or biochemical studies (31,32).

Mechanisms of ATP-induced arrhythmia in single cardiomyocytes

The mechanisms by which ATP could induce cell depolarization and trigger arrhythmia are multiple.

In isolated ventricular myocytes of the guinea pig, ATP alone does not exert significant electrophysiological effects; however, when it is applied with drugs known to increase intracellular Ca²⁺, ATP facilitates the induction of afterdepolarizations and triggered activity in approximately 60% of the cells (33). During heart failure, common features are an increased beta-adrenergic stimulation, which could reinforce the ATP-facilitated T- and L-type Ca²⁺ currents and the elevated sarcoplasmic reticulum Ca²⁺ release, which could evoke a reverse Na⁺/Ca²⁺-exchange current. In the presence of isoproterenol, ATP increases the amplitude of the transient inward current, delayed afterdepolarizations and L-type Ca²⁺ current (33). Of note, ATP alone induces significant increase in intracellular Ca²⁺ (34).

Activation of TRPM4:

Since the first measurements of single-channel openings in cardiomyocytes revealing a Ca²⁺-activated nonselective cation channel, the so-called CNRS channel (35), considerable effort has been devoted to identify its molecular candidate. Functional characterization of a Ca²⁺-activated nonselective cationic current in human atrial cardiomyocytes showed that the channel is equally permeable to Na⁺ and K⁺, but not permeable to Ca²⁺, and exhibits a 25 picosiemens (pS) conductance. These properties match those of TRPM4 (36) and make this channel protein a serious candidate supporting the delayed afterdepolarizations observed under conditions of Ca²⁺ overload. Various agonists, including ATP, could favour TRPM4 activation by increasing Ca²⁺ release from the sarcoplasmic reticulum following inositol-1,4,5-triphosphate production. Also, as proposed earlier, during ATP application, the transient early surge of nonselective cationic current could be consequent to activation by free Ca²⁺ following subsarcolemmal acidification induced by activation of the HCO₃⁻/Cl⁻ exchanger and Ca²⁺ release after its displacement by H⁺ from binding sites (15). In support of this assumption, both acidosis and transient current activation request the presence of Mg²⁺. Thus, TRPM4 could be a key player in the generation and/or perturbation of cardiac rhythm in any condition implying Ca²⁺ overload.

Activation of TRPC3/7 channels via P2Y2 receptors:

Besides the fast transient nonspecific cationic current that was reported earlier (15), a sustained current with lower amplitude is also activated on ATP application on single cardiomyocytes (37). However, while the activation of the fast transient current requires the presence of Mg²⁺, suggesting that MgATP is the probable agonist, the sustained current could be elicited by ATP and UTP, in their free forms – ATP^4– and UTP^4– (Figure 1). The ATP-induced current reversed near 0 mV exhibited a weak inward rectification and was inhibited by increasing external Ca²⁺ concentrations. It was verified that channel activity was negligible in control conditions. Changes in fluorescence induced by Ba²⁺ or Mn²⁺ influx in a Ca²⁺-free medium were observed only when ATP was applied in the 10 μM range. The nonselective cationic channel is a heterotetramer composed of TRPC3 and TRPC7 since the two proteins co-immunoprecipitate. The channel allows cationic flux only in the presence of agonists and exhibits two main conductance levels of 14 pS and 23 pS. The intracellular application of an anti-TRPC3 antibody markedly inhibits the current. Furthermore, it was shown that this sustained current results from the activation of the metabotropic P2Y2 receptor that leads to diacylglycerol formation and the activation of the phospholipase Cβ. Of note, transgenic mice deficient for the ligand-operated purinergic receptors P2X1, P2X4 or P2X1-P2X4 always exhibited one or the other of the ATP-induced transitory and sustained cationic currents recorded in controls (37).

Figure 1).

ATP-induced inward currents at resting potential. A Currents elicited by the application of 1 mM ATP on a rat cardiomyocyte clamped at a holding potential of −80 mV in the presence of Mg²⁺ or not. In the latter condition, the inward current was sustained for several minutes. B Dose-response curve of the sustained inward current elicited at −80 mV by various concentrations of ATP in a Mg²⁺-free, 300 μM Ca²⁺ solution. The apparent half-effective ATP concentration (EC_50ATP) was 558 μM, corresponding to a calculated EC_50ATP^4– of 58 μM. Redrawn with permission from reference 37. pA/pF Picoamperes per picofarad

Experimentally ATP-induced arrhythmia on cardiac tissues

The extracellular application of ATP on isolated cardiomyocytes is known to trigger various forms of cell electrical activity (4). However, our attempts to elicit some automatic responses by superfusing ATP (30 μM to 100 μM) on papillary muscles or ventricular strips generally failed despite some muscles showing a 5 mV decrease in resting potential (38). This was attributed to the fact that ATP could be rapidly degraded by ectonucleosidases. To avoid this effect, rat papillary muscles were bathed in a solution that contained caged ATP. Basal activity or the triggered contractions were unaffected. However, on ultraviolet flash photo release, ATP elicited one or more contractions (Figure 2).

Figure 2).

ATP and arrhythmia. Ultraviolet (UV) photo-released ATP triggered automatic contractile activity of a rat papillary muscle. Note that UV flashes were ineffective before caged ATP addition, while one or more contractions were elicited after each flash. The preparation was initially bathed in the control solution, with 0.3 mM Ba²⁺ added to reduce the depolarization threshold. Stimulations (Stim) were initially applied at 2 Hz. Caged ATP was added at 1 mM in the 2 mL experimental bath and photolyzed by flashes produced through a 300 nm cut-off filter using a xenon short-arc flash lamp; the flash duration was 1 ms and the total energy was 1 J

ATP/UTP-induced cell automaticity can be prevented by creatine transphosphorylation

Thirty years previously, creatine phosphate, under the trade name Neoton (Schiaparelli Farmaceutica, Italy), was commonly given orally to patients after cardiac surgery to significantly improve cardiac contractile function. Creatine phosphate (10 mM) has also been reported to markedly reduce the incidence of ventricular ectopic beats and tachycardia and fibrillation, which normally results from acute coronary ligation in the rat after creatine injection in the lumen (39). It also protects against reperfusion-induced arrhythmia in the rat heart; the electrophysiological changes, the instantaneous rate of voltage change over time (dV/dt), action potential duration and electrocardiogram (ECG) recordings are noted to be insufficient to explain the potent antiarrhythmic properties of creatine phosphate (40). Added to the cardioplegic crystalloid solution, 10 mM creatine phosphate improved the spontaneous restoration of cardiac rhythm and the spontaneous restoration of sinus rhythm in patients with initial atrial fibrillation without changes in ECG recordings (41). Although these studies suggest that creatine phosphate has biophysical, membrane-stabilizing effects, one should consider that due to the creatine kinase present in the interstitial space, most of the orally given creatine phosphate will have been dephosphorylated to increase circulating and interstitial creatine.

Due to the presence of interstitial creatine kinase, it could be hypothesized that as long as creatine is at a relatively high concentration, it serves as a buffer for the sudden release of ATP/UTP during the early phase of ischemia in association with the arrhythmic events as previously described (10,11,37). The potential preventive effect of creatine was tested by checking its ability to antagonize the arrhythmia that occurred on inducing a coronary ligature in rats that were or were not preinjected with creatine, taking advantage of the fact that creatine kinase is also released together with ATP/UTP during ischemic injury. ECG recordings in creatine-injected rats clearly demonstrated that both ventricular premature beats and particularly ventricular tachycardia markedly decreased, even if there was a very broad range of anomalous beats (a few to several hundred per hour) recorded in different animals (Figure 3). The creatine effect was even more striking in early deaths. Indeed no death was observed during the first 2 h following the coronary ligation in creatine-injected rats. Of note, beta-guanidinopropionate injection, a creatine analogue with 1000-fold lower kinetics (42), had no significant protective effect.

Figure 3).

Creatine transphophorylation prevents ATP-induced arrhythmia. The intraperitoneal injection of creatine at 0.075 g/kg 1 h before surgery prevented early death of the rat submitted to coronary ligature compared with the death recorded during the first 2 h following ligature in control conditions or after similar injection of beta-guanidinopropionate (GPA) (more than 30 rats in each condition). Also, analyzing electrocardiogram recordings during the first 30 min demonstrated that ventricular premature beats (VPB, including doublets and triplets) and, more specifically, ventricular tachycardia (VT, episodes of four beats as well as VT of up to 18 s) were reduced in creatine-injected animals compared with control and beta-GPA conditions (more than 25 rats in each condition). *P<0.05 versus control

CONCLUSION

The present article reveals a new, potentially deleterious role of TRPC channels. We report that following localized release of ATP and UTP during early ischemic events, ATP^4–/UTP^4– binding to P2Y₂ purinergic receptors activates TRPC3/7 channels, together with an early surge of current of unknown origin requiring Mg²⁺. Furthermore, ATP triggers the release of Ca²⁺, which could also activate TRPM4 channels. The consequent inward currents contribute to cell depolarization and Ca²⁺ overload such as to induce arrhythmic foci. Creatine, allowing for transphorylation-induced ATP/UTP control, markedly reduces arrhythmia occurring during the early ischemic phase. This sequence of events is summarized in Figure 4. Taking into consideration its weak noxious effects, interstitial creatine load should be a promising therapeutic approach for individuals at risk.

Figure 4).

Schematic representation of the cascade of events involved during an early ischemic period and leading to cell automaticity. The activation of the P2Y₂ receptors by the free forms of ATP and uridine 5′-triphosphate (UTP) (ATP^4– and UTP^4–) released from neighbouring cardiomyocytes leads to the opening of the TRPC3/7 channels via a G protein, phospholipase Cβ (PLCβ) and diacylglycerol (DAG) and inositol trisphosphate (IP₃) production. The consequent membrane depolarization triggers cell automaticity (shown as Ca²⁺ fluorescence recording on a Fura-2 loaded cardiomyocyte). In the presence of creatine, the creatine kinase (CK) allows the transphosphorylation of ATP and UTP to phosphocreatine (PCr)