Yong Gao Wang

Functional coupling between glycolysis and excitation-contraction coupling underlies alternans in cat heart cells.

1 Electromechanical alternans was characterized in single cat atrial and ventricular myocytes by simultaneous measurements of action potentials, membrane current, cell shortening and changes in intracellular Ca2+ concentration ([Ca2+]i). 2 Using laser scanning confocal fluorescence microscopy, alternans of electrically evoked [Ca2+]i transients revealed marked differences between atrial and ventricular myocytes. In ventricular myocytes, electrically evoked [Ca2+]i transients during alternans were spatially homogeneous. In atrial cells Ca2+ release started at subsarcolemmal peripheral regions and subsequently spread toward the centre of the myocyte. In contrast to ventricular myocytes, in atrial cells propagation of Ca2+ release from the sarcoplasmic reticulum (SR) during the small‐amplitude [Ca2+]i transient was incomplete, leading to failures of excitation‐contraction (EC) coupling in central regions of the cell. 3 The mechanism underlying alternans was explored by evaluating the trigger signal for SR Ca2+ release (voltage‐gated L‐type Ca2+ current, ICa, L) and SR Ca2+ load during alternans. Voltage‐clamp experiments revealed that peak ICa, L was not affected during alternans when measured simultaneously with changes of cell shortening. The SR Ca2+ content, evaluated by application of caffeine pulses, was identical following the small‐amplitude and the large‐amplitude [Ca2+]i transient. These results suggest that the primary mechanism responsible for cardiac alternans does not reside in the trigger signal for Ca2+ release and SR Ca2+ load. 4 β‐Adrenergic stimulation with isoproterenol (isoprenaline) reversed electromechanical alternans, suggesting that under conditions of positive cardiac inotropy and enhanced efficiency of EC coupling alternans is less likely to occur. 5 The occurrence of electromechanical alternans could be elicited by impairment of glycolysis. Inhibition of glycolytic flux by application of pyruvate, iodoacetate or β‐hydroxybutyrate induced electromechanical and [Ca2+]i transient alternans in both atrial and ventricular myocytes. 6 The data support the conclusion that in cardiac myocytes alternans is the result of periodic alterations in the gain of EC coupling, i.e. the efficacy of a given trigger signal to release Ca2+ from the SR. It is suggested that the efficiency of EC coupling is locally controlled in the microenvironment of the SR Ca2+ release sites by mechanisms utilizing ATP, produced by glycolytic enzymes closely associated with the release channel.

Acetylcholine Elicits a Rebound Stimulation of Ca2+ Current Mediated by Pertussis Toxin–Sensitive G Protein and cAMP-Dependent Protein Kinase A in Atrial Myocytes

Cholinergic inhibition of atrial contraction is typically followed by a rebound positive inotropic response. In the present study, we used a nystatin-perforated patch whole-cell recording method to determine whether acetylcholine (ACh) elicits a rebound stimulation of L-type Ca2+ current (ICa,L) in cat atrial myocytes. ACh (1 mumol/L) decreased basal ICa,L (-19 +/- 2%). Within approximately 30 s of returning to ACh-free solution, basal ICa,L exhibited a rebound increase above the control level (+61 +/- 7%) that returned to the control level within 4 to 5 minutes. ACh elicited concomitant changes in cell shortening, ie, a decrease followed by a rebound increase. The EC50 and maximal response of ACh-induced inhibition and rebound stimulation of ICa,L were 1.9 x 10(-9) mol/L and -30%, respectively, and 2.9 x 10(-8) mol/L and +64%, respectively. All effects of ACh on ICa,L were blocked by prior exposure to 1 mumol/L atropine or 100 mumol/L AFDX116 and unaffected by 0.2 mumol/L pirenzepine or 1 mumol/L propranolol. In the presence of ACh, exposure to atropine elicited stimulation of ICa,L.ACh-induced inhibition and rebound stimulation of current were independent of external Ca2+. Rebound stimulation of ICa,L was associated with a negative shift in the voltage dependence of ICa,L activation. Inhibition of protein kinase A by 50 mumol/L Rp-cAMPs decreased basal ICa,L by 36 +/- 1% and abolished the rebound stimulation of ICa,L. Forskolin (0.01 mumol/L) or isoproterenol (0.01 mumol/L) had no effect on basal ICa,L, but each accentuated the rebound increase in ICa,L. When adenylate cyclase was maximally stimulated with 1 mumol/L isoproterenol plus 2 mumol/L forskolin, ACh decreased ICa,L but failed to elicit rebound stimulation of ICa,L. Milrinone (10 mumol/L) increased basal ICa,L by 70 +/- 7% and significantly attenuated the rebound stimulation of ICa,L. Exposure to 1 mmol/L 8-bromo-cGMP elicited a small decrease in basal ICa,L, attenuated ACh-induced inhibition, and enhanced the rebound stimulation of ICa,L. Incubation in pertussis toxin prevented all ACh-induced changes in ICa,L. Inhibition of nitric oxide synthase by 100 mumol/L NG-monomethyl-L-arginine (L-NMMA) decreased basal ICa,L by -20 +/- 5%, prevented ACh-induced inhibition, and markedly attenuated the rebound stimulation of ICa,L. We conclude that in cat atrial myocytes ACh acts via M2 muscarinic receptors and pertussis toxin-sensitive G protein to inhibit basal ICa,L and that on withdrawal ACh elicits a rebound stimulation of ICa,L. Rebound stimulation of ICa,L is mediated via cAMP-dependent protein kinase A enhanced by ACh-induced inhibition of phosphodiesterase.(ABSTRACT TRUNCATED AT 400 WORDS)

Laminin acts via β1 integrin signalling to alter cholinergic regulation of L‐type Ca2+ current in cat atrial myocytes

1 A perforated patch recording method was used to determine how plating cells on laminin (20 μg ml−1; >2 h) alters cholinergic regulation of L‐type Ca2+ current (ICa,L) in atrial myocytes. 2 Acetylcholine (ACh; 1 μm)‐induced inhibition of basal ICa,L was not different between cells on glass and laminin. However, stimulation of ICa,L elicited by ACh withdrawal was significantly smaller in cells on laminin (10 ± 2 %) than on glass (48 ± 5 %) (P= 0.001). 3 Stimulation of ICa,L induced by either spermine‐NO (200 μm), milrinone (10 μm), IBMX (100 μm) or forskolin (1 μm) was significantly smaller in cells plated on laminin than on glass. However, stimulation of ICa,L by 100 μm 8‐CPT‐cAMP or intracellular dialysis with 50 μM cAMP was not different between cells plated on laminin or glass. 4 Basal, forskolin‐ and IBMX‐stimulated cAMP content was significantly smaller in cells plated on laminin than on glass. 5 Stimulation of ICa,L by ACh withdrawal was significantly smaller in cells plated on an αβ1‐integrin antibody (10 ± 4 %) than on glass (38 ± 6 %; P= 0.001). In cells on laminin, prior exposure to 100 μg ml−1 YIGSR, a laminin receptor‐binding peptide, restored ACh‐induced stimulation of ICa,L (58 ± 14 %)laminin alone (7 ± 2 %; P= 0.05). 6 Addition of 20 μm cytochalasin D or 1 μM latrunculin A, agents that prevent actin polymerization, to cells on laminin restored ACh‐induced stimulation of ICa,L. 7 We conclude that laminin binding to β1 integrins acts in association with the actin‐based cytoskeleton to attenuate adenylate cyclase activity. As a result, laminin inhibits NO‐mediated stimulation of ICa,L elicited by ACh withdrawal. Laminin‐integrin signalling may be relevant to changes in autonomic regulation that occur during cardiac development and/or disease.

Withdrawal of Acetylcholine Elicits Ca2+-Induced Delayed Afterdepolarizations in Cat Atrial Myocytes

BACKGROUND Recent experiments in atrial myocytes indicate that withdrawal of cholinergic agonist can directly increase Ca2+ influx via L-type Ca2+ current and stimulate Ca2+ uptake into the sarcoplasmic reticulum (SR), thereby increasing intracellular Ca2+. Overload of cellular Ca2+ within the SR can initiate various types of atrial dysrhythmias. The present study was designed to determine whether withdrawal of acetylcholine (ACh) can elicit Ca2+-induced delayed afterdepolarizations (DADs) in atrial myocytes. METHODS AND RESULTS A nystatin perforated-patch whole-cell method and fluorescence microscopy (indo 1) were used to measure electrical activities and intracellular free Ca2+ ([Ca2+]i), respectively. Withdrawal of ACh (1 micromol/L) increased action potential duration, shifted plateau voltage toward positive, and generated DADs that initiated spontaneous action potentials. Voltage-clamp analysis revealed that withdrawal of ACh elicited a rebound stimulation of L-type Ca2+ current (I(Ca,L)) (+45%) and Na/Ca exchange current (I(NaCa)) (+16%) and the appearance of transient inward current (I(ti)) and spontaneous [Ca2+]i transients. Each of these changes induced by withdrawal of ACh was abolished by Rp-cAMPs (50 to 100 micromol/L) or H-89 (2 micromol/L), inhibitors of cAMP-dependent protein kinase A. Ryanodine (1 micromol/L) abolished I(NaCa) and the appearance of I(ti) without decreasing the rebound stimulation of I(Ca,L) elicited by withdrawal of ACh. CONCLUSIONS Withdrawal of ACh can elicit cAMP-mediated stimulation of Ca2+ influx via I(Ca,L) and uptake of SR Ca2+. As a result, cellular Ca2+ overload causes enhanced SR Ca2+ release and the initiation of DADs. These mechanisms may generate triggered and/or spontaneous atrial depolarizations elicited by withdrawal of vagal nerve activity.

Laminin binding to β1‐integrins selectively alters β1‐ and β2‐adrenoceptor signalling in cat atrial myocytes

1 Perforated patch recordings were used to determine how plating atrial cells on laminin alters β‐adrenergic receptor (β‐AR) regulation of L‐type Ca2+ current (ICa,L). 2 Isoproterenol (isoprenaline; ISO; 0.01 μM), a non‐selective β‐AR agonist, elicited a greater stimulation of ICa,L in cells plated on laminin (+79 ± 16 %; n= 17) than on glass (+33 ± 5 %; n= 23). Also, desensitization to ISO was greater in cells on laminin (−16 ± 2 %) than on glass (−3 ± 1 %). Atenolol (0.1 μM), a selective β1‐AR antagonist, inhibited the effects of ISO in cells on glass but not laminin. Conversely, 0.1 μM ICI 118,551, a selective β2‐AR antagonist, inhibited the effects of ISO in cells on laminin but not glass. With β2‐ARs blocked, ISO‐induced stimulation of ICa,L was greater in cells on glass than laminin. 3 Zinterol (0.01–0.1 μM), a selective β2‐AR agonist, elicited a greater stimulation of ICa,L in cells on laminin than on glass. The effects of zinterol were blocked by ICI 118,551. 4 ISO‐induced stimulation of ICa,L was greater in cells plated on an αβ1‐integrin antibody than on glass. Also, addition of 20 μM cytochalasin D to cells on laminin prevented the enhanced effects of ISO typically elicited in cells on laminin alone. 5 We conclude that laminin binding to αβ1‐integrins, in conjunction with the actin cytoskeleton, reduces β1‐AR and enhances β2‐AR signalling which regulates ICa,L. This novel mechanism may contribute to remodelling of β‐AR signalling in the failing heart.

Signaling Mechanisms That Mediate Nitric Oxide Production Induced by Acetylcholine Exposure and Withdrawal in Cat Atrial Myocytes

Abstract— Fluorescence microscopy and the NO-sensitive indicator 4,5-diaminofluorescein were used to determine the effects of acetylcholine (ACh) on intracellular NO (NOi) in cat atrial myocytes. Field stimulation (1 Hz) of cells or exposure of quiescent cells to ACh (1 to 10 &mgr;mol/L) had no effect on NOi. However, in field-stimulated cells, ACh exposure increased NOi, and ACh withdrawal elicited an additional, prominent increase in NOi production. During ACh exposure, addition of 1 &mgr;mol/L atropine increased NOi production similar to ACh withdrawal. ACh-induced increases in NOi were reduced by prior exposure to 1 mmol/L extracellular Ca2+ ([Ca2+]o) and prevented by 0.5 mmol/L [Ca2+]o, 1 &mgr;mol/L verapamil, 1 &mgr;mol/L atropine, 10 &mgr;mol/L L-N5-(1-iminoethyl)ornithine, 10 &mgr;mol/L W-7, or incubating cells in pertussis toxin or 10 &mgr;mol/L LY294002 (inhibits phosphatidylinositol 3-kinase). Switching to 0.5 mmol/L [Ca2+]o during ACh withdrawal prevented the additional increase in NOi. ACh exposure increased phosphorylation (Ser473) of protein kinase B (Akt), and this effect was blocked by LY294002 and unaffected in low (0.5 mmol/L) [Ca2+]o. Confocal microscopy revealed that ACh exposure increased NOi at local subsarcolemmal sites, and ACh withdrawal additionally increased NOi by recruiting additional subsarcolemmal release sites. Disruption of caveolae by 2 mmol/L methyl-&bgr;-cyclodextrin abolished ACh-induced NOi production. We conclude that in cat atrial myocytes, ACh stimulates NOi release from local subsarcolemmal sites. ACh-induced increases in NOi requires both muscarinic receptor–mediated Gi protein/phosphatidylinositol 3-kinase/Akt signaling and voltage-activated Ca2+ influx for stimulation of calmodulin-dependent endothelial NO synthase activity. Increases in NOi elicited by ACh withdrawal result from the recovery of Ca2+ influx after ACh inhibition. NO signaling elicited by ACh withdrawal stimulates rapid recovery from cholinergic atrial inhibition.

Phenylephrine acts via IP3‐dependent intracellular NO release to stimulate L‐type Ca2+ current in cat atrial myocytes

This study determined the effects of α1‐adrenergic receptor (α1‐AR) stimulation by phenylephrine (PE) on L‐type Ca2+ current (ICa,L) in cat atrial myocytes. PE (10 μm) reversibly increased ICa,L (51.3%; n= 40) and shifted peak ICa,L activation voltage by −10 mV. PE‐induced stimulation of ICa,L was blocked by each of 1 μm prazocin, 10 μml‐NIO, 10 μm W‐7, 10 μm ODQ, 2 μm H‐89 or 10 μm LY294002, and was unaffected by 10 μm chelerythrine or incubating cells in pertussis toxin (PTX). PE‐induced stimulation of ICa,L also was inhibited by each of 10 μm ryanodine or 5 μm thapsigargin, by blocking IP3 receptors with 2 μm 2‐APB or 10 μm xestospongin C or by intracellular dialysis of heparin. In field‐stimulated cells, PE increased intracellular NO (NOi) production. PE‐induced NOi release was inhibited by each of 1 μm prazocin, 10 μml‐NIO, 10 μm W‐7, 10 μm LY294002, 2 μm H‐89, 10 μm ryanodine, 5 μm thapsigargin, 2 μm 2‐APB or 10 μm xestospongin C, and unchanged by PTX. PE (10 μm) increased phosphorylation of Akt, which was inhibited by LY294002. Confocal microscopy showed that PE stimulated NOi release from subsarcolemmal sites and this was prevented by 2 mm methyl‐β‐cyclodextrin, an agent that disrupts caveolae formation. PE also increased local, subsarcolemmal SR Ca2+ release via IP3‐dependent signalling. Electron micrographs of atrial myocytes show peripheral SR cisternae in close proximity to clusters of caveolae. We conclude that in cat atrial myocytes PE acts via α1‐ARs coupled to PTX‐insensitive G‐protein to release NOi, which in turn stimulates ICa,L. PE‐induced NOi release requires stimulation of both PI‐3K/Akt and IP3‐dependent Ca2+ signalling. NO stimulates ICa,L via cGMP‐mediated cAMP‐dependent PKA signalling. IP3‐dependent Ca2+ signalling may enhance local SR Ca2+ release required to activate Ca2+‐dependent eNOS/NOi production from subsarcolemmal caveolae sites.

Nitric oxide signalling by selective β2‐adrenoceptor stimulation prevents ach‐induced inhibition of β2‐stimulated Ca2+ current in cat atrial myocytes

The present study determined the effects of acetylcholine (ACh) on the L‐type Ca2+ current (ICa,l) stimulated by β1‐ or β2‐adrenergic receptor (AR) agonists in cat atrial myocytes. When isoproterenol (ISO; 0.1 μm) plus the β2‐AR antagonist ICI 118,551 (ISO‐β1‐AR stimulation) or 0.1 μm fenoterol, a β2‐AR agonist (FEN‐β2‐AR stimulation) increased ICa,l, ACh (1 μm) inhibited ICa,l by –60 ± 4 and –63 ± 6 %, respectively. When ISO plus the β1‐AR antagonist atenolol (ISO‐β2‐AR stimulation) or 1 μm zinterol (ZIN‐β2‐AR stimulation) increased ICa,l, ACh‐induced inhibition of ICa,l was significantly smaller, at –21 ± 3 and −24 ± 3 %, respectively. l‐N5‐(1‐iminoethyl)ornithine (l‐NIO, 10 μm), an inhibitor of nitric oxide (NO) synthase, enhanced ACh‐induced inhibition of ICa,l when stimulated by ZIN‐β2‐ARs, but not when stimulated by ISO‐β1‐ARs or FEN‐β2‐ARs. Haemoglobin (50 μm), a NO scavenger, also enhanced ACh‐induced inhibition when ICa,l was stimulated by ZIN‐β2‐ARs, but not when stimulated by FEN‐β2‐ARs. ACh‐induced inhibition of ICa,l stimulated by ZIN‐β2‐ARs was not affected by 10 μm 1H‐[1,2,4] oxadiazolo[4,3‐a] quinoxaline‐1‐one (ODQ) a guanylate cyclase inhibitor, but was significantly enhanced by 500 μm reduced glutathione or 100 μm dithiothreitol, agents that act as sinks for S‐nitrosylation. ACh‐induced inhibition was smaller when ICa,l was stimulated by spermine/NO, a NO donor, than by milrinone, a phosphodiesterase type III inhibitor. ISO (ISO‐β1/β2‐AR stimulation) increased ICa,l and even though ISO releases NO, ACh prominently inhibited ICa,l. This inhibitory effect of ACh was enhanced by l‐NIO. Stimulation of ZIN‐β2‐ARs increased intracellular NO, whereas ISO‐β1‐ARs or FEN‐β2‐ARs failed to increase intracellular NO. These results indicate that in atrial myocytes, NO released by selective β2‐AR stimulation prevents ACh‐induced inhibition of ICa,l stimulated by β2‐ARs. NO acts via a cGMP‐independent, S‐nitrosylation mechanism. Although FEN acts via β2‐ARs, it fails to stimulate Gi‐/NO signalling and preferentially stimulates Gs‐/adenylate cyclase signalling, similar to β1‐ARs. These findings indicate that NO signalling modulates muscarinic receptor inhibition of atrial function stimulated by β2‐ARs.

β-Adrenergic Stimulation Induces Acetylcholine to Activate ATP-Sensitive K+ Current in Cat Atrial Myocytes

Our previous work on atrial myocytes suggested that the effect of acetylcholine (ACh) to increase K+ conductance can be potentiated by prior loading of the sarcoplasmic reticulum (SR) with Ca2+. The present study, therefore, sought to determine whether prior exposure to isoproterenol (ISO) potentiates ACh-induced increases in K+ conductance and the underlying mechanisms. A nystatin-perforated patch whole-cell configuration was used to record from cat atrial myocytes. Voltage-clamp ramps (40 mV/s) were used to assess total membrane conductance. The experimental protocol consisted of two consecutive 30-second ACh exposures (ACh1 and ACh2) separated by a 6-minute recovery period in ACh-free solution. In general, experimental interventions, such as exposure to ISO, were imposed during the period between ACh1 and ACh2 to determine their effects on the response to ACh2 in relation to ACh1. Under control conditions, K+ conductances induced by ACh1 and ACh2 were not different from one another with or without activation of L-type Ca2+ current (ICa,L) during the recovery period. When 1 mumol/L ISO plus ICa,L activation was imposed during the recovery period, ACh2 induced a significantly larger increase in K+ conductance than ACh1. The ACh2-induced K+ current potentiated by ISO was time independent and selectively blocked by 10 mumol/L glibenclamide and therefore identified as ATP-sensitive K+ current (IK,ATP). The effect of ISO to induce ACh2-activated IK,ATP was mimicked by 1 mumol/L forskolin or 200 mumol/L 8-(4-chlorophenylthio)-cAMP, but not by 0.5 mumol/L BAY K 8644, and was selectively abolished by (1) 5 mumol/L thapsigargin or 1 mumol/L ryanodine, agents that prevent accumulation of SR Ca2+, (2) inhibition of L-type Ca2+ current (ICa,L) by 1 mumol/L nisoldipine or zero external Ca2+, (3) 50 mumol/L Rp-cAMPs, an inhibitor of cAMP-dependent protein kinase A, or (4) 2 mumol/L propranolol. Atropine (1 mumol/L) abolished all ACh-induced currents. Moreover, ACh2-activated IK,ATP was selectively blocked by 0.2 mumol/L pirenzepine, an M1 muscarinic receptor antagonist, or 0.1 mumol/L calphostin C, a selective inhibitor of protein kinase C. AFDX116 (100 mumol/L), an M2 muscarinic receptor antagonist, blocked the conventional ACh-activated K+ current and revealed ACh2-activated IK,ATP. These results indicate that prior exposure to ISO potentiates ACh-induced increases in K+ current via ACh-activated IK,ATP.(ABSTRACT TRUNCATED AT 400 WORDS)

A Cellular Mechanism Contributing to Postvagal Tachycardia Studied in Isolated Pacemaker Cells From Cat Right Atrium

Vagal nerve-induced inhibition of the heartbeat is followed by a postvagal increase in heart rate above control levels, postvagal tachycardia. In the present study, we used a perforated-patch/whole-cell recording method to determine the role of L-type Ca2+ current (ICa,L) and the hyperpolarization-activated inward current (I(f)) in the positive chronotropic response elicited by withdrawal of acetylcholine (ACh). Experiments were performed on sinoatrial node (SAN) and latent atrial pacemaker (LAP) cells isolated from cat right atrium. Withdrawal of a 2-minute exposure to 1 mumol/L ACh elicited a rebound stimulation of ICa,L in both SAN (33 +/- 4%) and LAP (50 +/- 6%) cells above control. Similarly, withdrawal of ACh (1 mumol/L) elicited a rebound stimulation of I(f) in both SAN (21 +/- 4%) and LAP (20 +/- 6%) cells. During the rebound stimulation of ICa,L, peak amplitude was increased throughout the voltage range, and the voltage dependence of activation was shifted to more negative voltages. Action potential recordings from both SAN and LAP cells showed that following ACh-induced inhibition, withdrawal of ACh elicited a concomitant rebound increase in action potential amplitude ( + 21 +/- 2% and + 21 +/- 3%, respectively) and decrease in pacemaker cycle length (30 +/- 5% and 44 +/- 5%, respectively) compared with control. H-89 (2 mumol/L), an inhibitor of cAMP-dependent protein kinase A, abolished the rebound increase of ICa,L, I(f), action potential amplitude, and decrease in pacemaker cycle length elicited by withdrawal of ACh. In the presence of 2 mmol/L cesium, a blocker of I(f), the rebound decrease in pacemaker cycle length elicited by withdrawal of ACh was unchanged. We conclude that in SAN and LAP cells, withdrawal of ACh elicits a positive chronotropic response primarily through a cAMP-mediated rebound stimulation of ICa,L. These findings are the first demonstration of an intrinsic cellular mechanism that may contribute directly to the nonadrenergic component of postvagal tachycardia.