Jian-Bing Shen

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 μM), 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 (∼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.—Shen, J.‐B., Pappano, A. J., Liang, B. T. Extracellular ATP‐stimulated current in wild‐type and P2X4 receptor transgenic mouse ventricular myocytes: implications for a cardiac physiologic role of P2X4 receptors. FASEB J. 20, 277–284 (2006)

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 P2X4 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.

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.

Cesium Abolishes the Barium‐Induced Pacemaker Potential and Current in Guinea Pig Ventricular Myocytes

Cesium Abolishes Barium‐Induced PM Current. Introduction: The ability of cesium to block barium‐induced diastolic depolarization (“Ba‐DD”) and pacemaker current was tested in isolated ventricular myocytes. Because Ba‐DD is due to decreasing k conductance and there is no If at the resting potential, this approach permits verification of whether Cs+ is a specific blocker of I, or if it instead also blocks a K+ pacemaker current.

Novel Protective Role of Endogenous Cardiac Myocyte P2X4 Receptors in Heart Failure

Background—Heart failure (HF), despite continuing progress, remains a leading cause of mortality and morbidity. P2X4 receptors (P2X4R) have emerged as potentially important molecules in regulating cardiac function and as potential targets for HF therapy. Transgenic P2X4R overexpression can protect against HF, but this does not explain the role of native cardiac P2X4R. Our goal is to define the physiological role of endogenous cardiac myocyte P2X4R under basal conditions and during HF induced by myocardial infarction or pressure overload. Methods and Results—Mice established with conditional cardiac-specific P2X4R knockout were subjected to left anterior descending coronary artery ligation–induced postinfarct or transverse aorta constriction–induced pressure overload HF. Knockout cardiac myocytes did not show P2X4R by immunoblotting or by any response to the P2X4R-specific allosteric enhancer ivermectin. Knockout hearts showed normal basal cardiac function but depressed contractile performance in postinfarct and pressure overload models of HF by in vivo echocardiography and ex vivo isolated working heart parameters. P2X4R coimmunoprecipitated and colocalized with nitric oxide synthase 3 (eNOS) in wild-type cardiac myocytes. Mice with cardiac-specific P2X4R overexpression had increased S-nitrosylation, cyclic GMP, NO formation, and were protected from postinfarct and pressure overload HF. Inhibitor of eNOS, L-N5-(1-iminoethyl)ornithine hydrochloride, blocked the salutary effect of cardiac P2X4R overexpression in postinfarct and pressure overload HF as did eNOS knockout. Conclusions—This study establishes a new protective role for endogenous cardiac myocyte P2X4R in HF and is the first to demonstrate a physical interaction between the myocyte receptor and eNOS, a mediator of HF protection.

CD13 is essential for inflammatory trafficking and infarct healing following permanent coronary artery occlusion in mice

Abstract Aims To determine the role of CD13 as an adhesion molecule in trafficking of inflammatory cells to the site of injury in vivo and its function in wound healing following myocardial infarction induced by permanent coronary artery occlusion. Methods and results Seven days post-permanent ligation, hearts from CD13 knockout (CD13KO) mice showed significant reductions in cardiac function, suggesting impaired healing in the absence of CD13. Mechanistically, CD13KO infarcts showed an increase in small, endothelial-lined luminal structures, but no increase in perfusion, arguing against an angiogenic defect in the absence of CD13. Cardiac myocytes of CD13KO mice showed normal basal contractile function, eliminating myocyte dysfunction as a mechanism of adverse remodelling. Conversely, immunohistochemical and flow cytometric analysis of CD13KO infarcts demonstrated a dramatic 65% reduction in infiltrating haematopoietic cells, including monocytes, macrophages, dendritic, and T cells, suggesting a critical role for CD13 adhesion in inflammatory trafficking. Accordingly, CD13KO infarcts also contained fewer myofibroblasts, consistent with attenuation of fibroblast differentiation resulting from the reduced inflammation, leading to adverse remodelling. Conclusion In the ischaemic heart, while compensatory mechanisms apparently relieve potential angiogenic defects, CD13 is essential for proper trafficking of the inflammatory cells necessary to prime and sustain the reparative response, thus promoting optimal post-infarction healing.

Inositol-1,4,5-trisphosphate increases contractions but not L-type calcium current in guinea pig ventricular myocytes

OBJECTIVE We studied the effects of intracellularly applied inositol-1,4,5-trisphosphate (InsP3) to test the hypothesis that InsP3 is a messenger for stimulation of L-type calcium current (ICa(L)) and contractions by muscarinic agonists. METHODS Voltage clamp pulses elicited ICa(L) that evoked contractions recorded with an edge detector in single guinea pig ventricular myocytes superfused with Tyrodes solution (36 degrees C). InsP3 or cyclic AMP (cAMP) was dialyzed into the cell at selected times via the patch electrode. RESULTS InsP3 (1-10 microM) transiently increased isotonic contractions when applied for 4-5 min; higher concentrations (50-300 microM) caused a sustained decrease in contractions. InsP3 had no effect on ICa(L) at any concentration tested. Caffeine (10 mM)-induced contractures were increased and decreased, respectively, at 3 and 100 microM InsP3. Pentosan polysulfate (50 micrograms/ml), an InsP3 receptor antagonist, opposed the increased contractions by InsP3. Intrapipette cyclic AMP (10-300 microM) caused sustained increases of ICa(L) and contractions. Cyclic AMP, but not InsP3, also increased ICa(L) when intrapipette Cs+ suppressed K+ currents. CONCLUSIONS Increased myocyte shortening at low InsP3 concentrations accords with receptor-initiated sarcoplasmic reticulum Ca2+ release. The transient stimulation of contractions at low concentrations and the sustained reduction of contractions at high concentrations are not consistent with a role for InsP in the persistent increase of contractions by muscarinic agonist in ventricular muscle and myocytes. The failure of InsP3 to change ICa(L) when contractions were increased or decreased militates against the L-type calcium channel being an effector of InsP3.

Barium-induced diastolic depolarization and controlling mechanisms in guinea pig ventricular muscle

Barium-induced diastolic depolarization (Ba(2+)-DD) and its modulation were studied in guinea pig papillary muscle and single ventricular myocytes. In papillary muscles, Cs+ (4 mM) abolished the DD induced by Ba2+ (0.05-0.2 mM) by abolishing the undershoot at the end of the action potential (AP; consistent with a block of an outward current). Acetylcholine (ACh 1 microM) had little effect on Ba(2+)-DD, whereas norepinephrine (NE 1 microM) enhanced it by increasing the undershoot and by inducing an oscillatory potential. Low [Ca2+]o (0.54 mM) decreased the resting potential and increased Ba(2+)-DD amplitude. High [Ca2+]o (8.1 mM) had opposite effects. Cs+ also reduced Ba(2+)-DD in low [Ca2+]o. In isolated myocytes, Ba(2+)-DD and the pacemaker current induced by Ba2+ (Ba(2+)-IKdd) increased on depolarization and reversed on hyperpolarization. Although not significantly, high [Ca2+]o slightly decreased and low [Ca2+]o slightly increased Ba(2+)-IKdd. Cd2+ markedly reduced the slow inward current ICa and the AP duration (APD), but did not affect Ba(2+)-IKdd. We conclude that Ba(2+)-DD (a) is entirely due to a voltage- and time-dependent decrease in gK1, since it is abolished by Cs+ (no contribution of a nonblocked decaying IK) by eliminating the undershoot (no If), (b) is potentiated by NE through an increased undershoot and an oscillatory potential, (c) is modified by high and low [Ca2+]o mostly through changes in the resting potential, and (d) is not affected by the block of the slow channel by Cd2+.

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.

On the Mechanism of Cesium-Induced Voltage and Current Tails in Single Ventricular Myocytes

The mechanisms by which different concentrations of cesium modify membrane potentials and currents were investigated in guinea pig single ventricular myocytes. In a dose-dependent manner, cesium reversibly decreases the resting potential and action potential amplitude and duration, and induces a diastolic decaying voltage tail (Vex), which increases at more negative and reverses at less negative potentials. In voltage-clamped myocytes, Cs+ increases the holding current, increases the outward current at plateau levels while decreasing it at potentials closer to resting potential, induces an inward tail current (Iex) on return to resting potential and causes a negative shift of the threshold for the inward current. During depolarizing ramps, Cs+ decreases the outward current negative to inward rectification range, whereas it increases the current past that range. During repolarizing ramps, Cs+ shifts the threshold for removal of inward rectification negative slope to less negative values. Cs+-induced voltage and current tails are increased by repetitive activity, caffeine (5 mM) and high [Ca2+]O (8.1 mM), and are reduced by low Ca2+ (0.45 mM), Cd2+ (0.2 mM) and Ni2+ (2 mM). Ni2+ also abolishes the tail current that follows steps more positive than ECa. We conclude that Cs+ (1) decreases the resting potential by decreasing the outward current at more negative potentials, (2) shortens the action potential by increasing the outward current at potentials positive to the negative slope of inward rectification, and (3) induces diastolic tails through a Ca2+-dependent mechanism, which apparently is an enhanced electrogenic Na-Ca exchange.