Cardiac P2X purinergic receptors as a new pathway for increasing Na⁺ entry in cardiac myocytes.

P2X4 receptors (P2X4Rs) are ligand-gated ion channels capable of conducting cations such as Na(+). Endogenous cardiac P2X4R can mediate ATP-activated current in adult murine cardiomyocytes. In the present study, we tested the hypothesis that cardiac P2X receptors can induce Na(+) entry and modulate Na(+) handling. We further determined whether P2X receptor-induced stimulation of the Na(+)/Ca(2+) exchanger (NCX) has a role in modulating the cardiac contractile state. Changes in Na(+)-K(+)-ATPase current (Ip) and NCX current (INCX) after agonist stimulation were measured in ventricular myocytes of P2X4 transgenic mice using whole cell patch-clamp techniques. The agonist 2-methylthio-ATP (2-meSATP) increased peak Ip from a basal level of 0.52 ± 0.02 to 0.58 ± 0.03 pA/pF. 2-meSATP also increased the Ca(2+) entry mode of INCX (0.55 ± 0.09 pA/pF under control conditions vs. 0.82 ± 0.14 pA/pF with 2-meSATP) at a membrane potential of +50 mV. 2-meSATP shifted the reversal potential of INCX from -14 ± 2.3 to -25 ± 4.1 mV, causing an estimated intracellular Na(+) concentration increase of 1.28 ± 0.42 mM. These experimental results were closely mimicked by mathematical simulations based on previously established models. KB-R7943 or a structurally different agent preferentially opposing the Ca(2+) entry mode of NCX, YM-244769, could inhibit the 2-meSATP-induced increase in cell shortening in transgenic myocytes. Thus, the Ca(2+) entry mode of INCX participates in P2X agonist-stimulated contractions. In ventricular myocytes from wild-type mice, the P2X agonist could increase INCX, and KB-R7943 was able to inhibit the contractile effect of endogenous P2X4Rs, indicating a physiological role of these receptors in wild-type cells. The data demonstrate a novel Na(+) entry pathway through ligand-gated P2X4Rs in cardiomyocytes.

Treatment of heart failure by a methanocarba derivative of adenosine monophosphate: implication for a role of cardiac purinergic P2X receptors.

Evidence is accumulating to support a potentially important role for purinergic (P2X) receptors in heart failure (HF). We tested the hypothesis that a hydrolysis-resistant nucleotide analog with agonist activity at myocardial P2X receptors (P2XRs) improves the systolic HF phenotype in mouse and dog models. We developed a hydrolysis-resistant adenosine monophosphate derivative, (1'S,2R,3S,4'R,5'S)-4-(6-amino-2-chloro-9H-purin-9-yl)-1-[phosphoryloxymethyl] bicycle[3.1.0]hexane-2,3-diol) (MRS2339), with agonist activity at native cardiac P2XRs. Chronic MRS2339 infusion in postinfarct and calsequestrin (CSQ) mice with HF resulted in higher rates of pressure change (+dP/dt), left ventricle (LV)-developed pressure, and cardiac output in an in vitro working heart model. Heart function in vivo, as determined by echocardiography-derived fractional shortening, was also improved in MRS2339-infused mice. The beneficial effect of MRS2339 was dose-dependent and was identical to that produced by cardiac myocyte-specific overexpression of the P2X(4) receptor. The HF improvement was associated with the preservation of LV wall thickness in both systole and diastole in postinfarct and CSQ mice. In dogs with pacing-induced HF, MRS2339 infusion reduced left ventricular end-diastolic pressure, improved arterial oxygenation, and increased +dP/dt. MRS2339 treatment also decreased LV chamber size in mice and dogs with HF. In murine and canine models of systolic HF, in vivo administration of a P2X nucleotide agonist improved contractile function and cardiac performance. These actions were associated with preserved LV wall thickness and decreased LV remodeling. The data are consistent with a role of cardiac P2XRs in mediating the beneficial effect of this agonist.

An estrogen metabolite, 2-methoxyestradiol, disrupts cardiac microtubules and unmasks muscarinic inhibition of calcium current.

Microtubules provide a chemical signaling function as well as structural support for heart cells. Microtubules modulate autonomic signaling in the heart, and their disruption by colchicine unmasks muscarinic inhibition of Ca (ICa) current. In this study, we compare the actions of the estrogen metabolite, 2-methoxyestradiol (2-ME), with those of colchicine on microtubule stability and chemical signal function in guinea pig-isolated ventricular myocytes. Like colchicine, 2-ME binds to microtubules and disrupts the cytoskeleton of cardiac myocytes. Incubation with 2-ME increased the soluble fraction of tubulin and decreased the polymerized fraction at concentrations ranging from 10 to 100 microM. 2-ME was less potent than colchicine in causing microtubular disruption. Treatment with 2-ME for up to 4 h was accompanied by a progressive increase of I(Ca) amplitude. There was no change in the rates of ICa inactivation. Carbachol, which has no effect on ICa in untreated ventricular myocytes, inhibited this current in the presence of 2-ME. The extent of inhibition increased with incubation time in 2-ME such that carbachol completely removed the increment of ICa by the estrogen metabolite. The results illustrate the important role of microtubules in modulating cardiac autonomic signaling.

Extracellular ATP-stimulated current in wild-type and P2X4 receptor transgenic mouse ventricular myocytes: implications for a cardiac physiologic role of P2X4 receptors.

P2X receptors, activated by extracellular ATP, may be important in regulating cardiac function. The objective of the present study was to characterize the electrophysiologic actions of P2X4 receptors in cardiac myocytes and to determine whether they are involved in mediating the effect of extracellular ATP. Membrane currents under voltage clamp were determined in myocytes from both wild-type (WT) and P2X4 receptor-overexpressing transgenic (TG) mice. The P2X agonist 2-meSATP induced an inward current at -100 mV that was greater in magnitude (2-fold) in TG than in WT ventricular cells. In the presence of the P2X4 receptor-selective allosteric enhancer ivermectin (3 microM), the 2-meSATP-stimulated current increased significantly in both WT and TG ventricular cells, consistent with an important role of P2X4 receptors in mediating the ATP current not only in TG but also WT myocytes. That the current in both WT and TG cells showed similar voltage-dependence and reverse potential (approximately 0 mV) further suggests a role for this receptor in the normal electrophysiological action of ATP in WT murine cardiac myocytes. The P2X antagonist suramin was only able to block partially the 2-meSATP-stimulated current in WT cells, implying that both P2X4 receptor and another yet-to-be-identified P2X receptor mediate this current.

Reversal of cardiac myocyte dysfunction as a unique mechanism of rescue by P2X4 receptors in cardiomyopathy.

Binary cardiac transgenic (Tg) overexpression of P2X(4) receptors (P2X(4)R) improved the survival of the cardiomyopathic calsequestrin (CSQ) mice. Here we studied the mechanism of rescue using binary P2X(4)R/CSQ Tg and CSQ Tg mice as models. Cellular and intact heart properties were determined by simultaneous sarcomere shortening (SS) and Ca(2+) transients in vitro and echocardiography in vivo. Similar to a delay in death, binary mice exhibited a slowed heart failure progression with a greater left ventricular (LV) fractional shortening (FS) and thickness and a concomitant lesser degree of LV dilatation in both systole and diastole at 8 or 12 wk. By 16 wk, binary hearts showed similarly depressed FS and thinned out LV and equal enlargement of LV as did 12-wk-old CSQ hearts. Binary cardiac myocytes showed higher peak basal cell shortening (CS) and SS as well as greater basal rates of shortening and relaxation than did the CSQ myocytes at either 8 or 12 wk. Similar data were obtained in comparing the Ca(2+) transient. At 16 wk, binary myocytes were like the 12-wk-old CSQ myocytes with equally depressed CS, SS, and Ca(2+) transient. CSQ myocytes were longer than myocytes from wild-type and binary mice at 12 wk of age. At 16 wk, the binary myocyte length increased to that of the 12-wk-old CSQ myocyte, parallel to LV dilatation. The data suggest a unique mechanism, which involves a reversal of cardiac myocyte dysfunction and a delay in heart failure progression. It represents an example of targeting the abnormal failing myocyte in treating heart failure.

Role of P2X purinergic receptors in the rescue of ischemic heart failure.

Evidence is accumulating to support the presence of P2X purinergic receptors in the heart. However, the biological role of this receptor remains to be defined. The objectives here were to determine the role of cardiac P2X receptors in modulating the progression of post-myocardial infarction ischemic heart failure and to investigate the underlying mechanism. The P2X4 receptor (P2X4R) is an important subunit of native cardiac P2X receptors, and the cardiac-specific transgenic overexpression of P2X4R (Tg) was developed as a model. Left anterior descending artery ligation resulted in similar infarct size between Tg and wild-type (WT) mice (P > 0.1). However, Tg mice showed an enhanced cardiac contractile performance at 7 days, 1 mo, and 2 mo after infarction and an increased survival at 1 and 2 mo after infarction (P < 0.01). The enhanced intact heart function was manifested by a greater global left ventricular developed pressure and rate of contraction of left ventricular pressure in vitro and by a significantly increased fractional shortening and systolic thickening in the noninfarcted region in vivo (P < 0.05). The salutary effects on the ischemic heart failure phenotype were seen in both sexes and were not the result of any difference in infarct size in Tg versus WT hearts. An enhanced contractile function of the noninfarcted area in the Tg heart was likely an important rescuing mechanism. The cardiac P2X receptor is a novel target to treat post-myocardial infarction ischemic heart failure.

Characterization and mechanism of P2X receptor-mediated increase in cardiac myocyte contractility.

Cardiac P2X purinergic receptors can mediate an increase in myocyte contractility and a potentially important role in the heart. The P2X(4) receptor (P2X(4)R) is an important subunit of native cardiac P2X receptors. With transgenic mice with cardiac-specific overexpression of P2X(4)R (Tg) used as a model, the objectives here were to characterize the P2X receptor-mediated cellular contractile and Ca(2+) transient effects and to determine the mechanism underlying the receptor-induced increase in myocyte contractility. In response to the agonist 2-methylthioATP (2-meSATP), Tg myocytes showed an increased intracellular Ca(2+) transient, as defined by fura 2 fluorescence ratio, and an enhanced contraction shortening that were unaccompanied by cAMP accumulation or L-type Ca(2+) channel activation. The increased Ca(2+) transient was not associated with any alteration in action potential duration, resting membrane potential, or diastolic fluorescence ratio or rates of rise and decline of the Ca(2+) transient. Simultaneous Ca(2+) transient and contraction measurements did not show any agonist-mediated change in myofilament Ca(2+) sensitivity. However, activation of the overexpressed P2X(4) receptor caused an enhanced SR Ca(2+) loading, as evidenced by a 2-meSATP-evoked increase in the caffeine-induced inward current and Ca(2+) transient. Similar data were obtained in wild-type mouse ventricular myocytes. Thus an increased SR Ca(2+) content, occurring in the absence of cAMP accumulation or L-type Ca(2+) channel activation, is the principal mechanism by which cardiac P2X receptor mediates a stimulatory effect on cardiac myocyte contractility.

On the role of phosphatase in regulation of cardiac L-type calcium current by cyclic GMP.

Does cGMP, via protein kinase G, inhibit cAMP-stimulated Ca(2+) current (I(Ca(L))) in mammalian ventricular myocytes by phosphorylating the calcium channel at a site different from that acted on by cAMP or by dephosphorylating the calcium channel through phosphatase(s)? We tested these possibilities in guinea pig ventricular myocytes superfused with Tyrode's solution (35 degrees C) and dialyzed with adenosine 5'-O-(3-thiotriphosphate) ([ATPgammaS](pip)). ATPgammaS is a kinase substrate but thiophosphorylated proteins are not phosphatase substrates. With 5 mM [ATPgammaS](pip), I(Ca(L)) increased gradually over 20 to 25 min and then rapidly in the presence of 3-isobutyl-1-methylxanthine. 8-Bromo-cGMP (8-Br-cGMP; 1 mM) did not inhibit I(Ca(L)) significantly (-3 +/- 11.8%, n = 21) in contrast to results with ATP dialysis (). Similar results were obtained with 0.1 mM carbachol (CCh). I(Ca(L)) increased after longer dialysis (>/=40 min) with ATPgammaS; again, 8-Br-cGMP had no effect. Also, isoproterenol (ISO) did not stimulate and CCh, alone or in the presence of ISO, did not inhibit I(Ca(L)). Block of CCh effect by ATPgammaS, although consistent with cGMP action in muscarinic inhibition, could be explained by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) formation from ATPgammaS via nucleoside diphosphate kinase. GTPgammaS uncouples muscarinic and beta-adrenoceptors from intracellular effectors. Failure of 8-Br-cGMP to reduce I(Ca(L)) irreversibly excludes calcium channel phosphorylation as an inhibitory mechanism. We propose that cGMP inhibits I(Ca(L)) by activating phosphatase(s) in guinea pig ventricular myocytes.

Autonomic regulation of calcium and potassium channels is oppositely modulated by microtubules in cardiac myocytes.

We recently showed that colchicine treatment of rat ventricular myocytes increases the L-type Ca2+ current (I(Ca)) and intracellular Ca2+ concentration ([Ca2+](i)) transients and interferes with adrenergic signaling. These actions were ascribed to adenylyl cyclase (AC) stimulation after G(s) activation by alpha,beta-tubulin. Colchicine depolymerizes microtubules into alpha,beta-tubulin dimers. This study analyzed muscarinic signals in myocytes with intact or depolymerized microtubules. Myocytes were loaded with the Ca2+ indicator fluo 3 and were field stimulated at 1 Hz or voltage clamped. In untreated cells, carbachol (CCh; 1 microM) induced ACh-activated K(+) current [I(K(ACh))], which happens via betagamma-subunits from the activation of G(i). Carbachol also reduced [Ca2+](i) transients and contractions. Once G(i) is activated by muscarinic agonist, the alpha(i)-subunit is released from the betagamma-subunits, but it is silent, and its inhibition of the AC/cAMP cascade, manifested by I(Ca) reduction, is not seen unless AC has been previously activated. In colchicine-treated cells, CCh caused greater reductions of [Ca2+](i) transients and contractions than in untreated cells. The alpha(i)-subunit became effective in signaling through the AC/cAMP cascade and reduced I(Ca) without changing its voltage-dependence. Isoproterenol (Iso) regained its efficacy and reversed I(Ca) inhibition by CCh. Stimulation of I(Ca) by forskolin persisted in colchicine-treated cells when Iso was ineffective. The effect of CCh on I(K(ACh)) was occluded in colchicine-treated cells. Colchicine treatment, per se, may increase I(K(ACh)) by betagamma-subunits released from G(s) to mask this effect of CCh. Microtubules suppress I(Ca) regulation by alpha(i); their disruption releases restraints that unmask muscarinic inhibition of I(Ca). Summarily, colchicine treatment reverses regulation of ventricular excitation-contraction coupling by autonomic agents.