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1.  Inhibition of a TREK-like K+ channel current by noradrenaline requires both β1- and β2-adrenoceptors in rat atrial myocytes 
Cardiovascular Research  2014;104(1):206-215.
Noradrenaline plays an important role in the modulation of atrial electrophysiology. However, the identity of the modulated channels, their mechanisms of modulation, and their role in the action potential remain unclear. This study aimed to investigate the noradrenergic modulation of an atrial steady-state outward current (IKss).
Methods and results
Rat atrial myocyte whole-cell currents were recorded at 36°C. Noradrenaline potently inhibited IKss (IC50 = 0.90 nM, 42.1 ± 4.3% at 1 µM, n = 7) and potentiated the L-type Ca2+ current (ICaL, EC50 = 136 nM, 205 ± 40% at 1 µM, n = 6). Noradrenaline-sensitive IKss was weakly voltage-dependent, time-independent, and potentiated by the arachidonic acid analogue, 5,8,11,14-eicosatetraynoic acid (EYTA; 10 µM), or by osmotically induced membrane stretch. Noise analysis revealed a unitary conductance of 8.4 ± 0.42 pS (n = 8). The biophysical/pharmacological properties of IKss indicate a TREK-like K+ channel. The effect of noradrenaline on IKss was abolished by combined β1-/β2-adrenoceptor antagonism (1 µM propranolol or 10 µM β1-selective atenolol and 100 nM β2-selective ICI-118,551 in combination), but not by β1- or β2-antagonist alone. The action of noradrenaline could be mimicked by β2-agonists (zinterol and fenoterol) in the presence of β1-antagonist. The action of noradrenaline on IKss, but not on ICaL, was abolished by pertussis toxin (PTX) treatment. The action of noradrenaline on ICaL was mediated by β1-adrenoceptors via a PTX-insensitive pathway. Noradrenaline prolonged APD30 by 52 ± 19% (n = 5; P < 0.05), and this effect was abolished by combined β1-/β2-antagonism, but not by atenolol alone.
Noradrenaline inhibits a rat atrial TREK-like K+ channel current via a PTX-sensitive mechanism involving co-operativity of β1-/β2-adrenoceptors that contributes to atrial APD prolongation.
PMCID: PMC4174890  PMID: 25205295
Background K+ current; Beta-adrenoceptor; K2P channel; Steady-state outward current; Osmotic stretch; TREK-1; Arachidonic acid
2.  Clinically-relevant consecutive treatment with isoproterenol and adenosine protects the failing heart against ischaemia and reperfusion 
Consecutive treatment of normal heart with a high dose of isoproterenol and adenosine (Iso/Ade treatment), confers strong protection against ischaemia/reperfusion injury. In preparation for translation of this cardioprotective strategy into clinical practice during heart surgery, we further optimised conditions for this intervention using a clinically-relevant dose of Iso and determined its cardioprotective efficacy in hearts isolated from a model of surgically-induced heart failure.
Isolated Langendorff-perfused rat hearts were treated sequentially with 5 nM Iso and 30 μM Ade followed by different durations of washout prior to 30 min global ischaemia and 2 hrs reperfusion. Reperfusion injury was assessed by measuring haemodynamic function, lactate dehydrogenase (LDH) release and infarct size. Protein kinase C (PKC) activity and glycogen content were measured in hearts after the treatment. In a separate group of hearts, Cyclosporine A (CsA), a mitochondria permeability transition pore (MPTP) inhibitor, was added with Iso/Ade. Failing hearts extracted after 16 weeks of ligation of left coronary artery in 2 months old rats were also subjected to Iso/Ade treatment followed by ischaemia/reperfusion.
Recovery of the rate pressure product (RPP) in Iso/Ade-treated hearts was significantly higher than in controls. Thus in Iso/Ade treated hearts with 5 nM Iso and no washout period, RPP recovery was 76.3 ± 6.9% of initial value vs. 28.5 ± 5.2% in controls. This was associated with a 3 fold reduction in LDH release irrespective to the duration of the washout period. Hearts with no washout of the drugs (Ade) had least infarct size, highest PKC activity and also showed reduced glycogen content. Cardioprotection with CsA was not additive to the effect of Iso/Ade treatment. Iso/Ade treatment conferred significant protection to failing hearts. Thus, RPP recovery in failing hearts subjected to the treatment was 69.0 ± 16.3% while in Control hearts 19.7 ± 4.0%. LDH release in these hearts was also 3 fold lower compared to Control.
Consecutive Iso/Ade treatment of normal heart can be effective at clinically-relevant doses and this effect appears to be mediated by glycogen depletion and inhibition of MPTP. This intervention protects clinically relevant failing heart model making it a promising candidate for clinical use.
PMCID: PMC4045901  PMID: 24885907
Ischaemia/reperfusion; Cardioprotection; Isoproterenol; Adenosine; Heart failure; Mitochondria permeability transition pore
3.  Stimulation of ICa by basal PKA activity is facilitated by caveolin-3 in cardiac ventricular myocytes☆ 
L-type Ca channels (LTCC), which play a key role in cardiac excitation–contraction coupling, are located predominantly at the transverse (t-) tubules in ventricular myocytes. Caveolae and the protein caveolin-3 (Cav-3) are also present at the t-tubules and have been implicated in localizing a number of signaling molecules, including protein kinase A (PKA) and β2-adrenoceptors. The present study investigated whether disruption of Cav-3 binding to its endogenous binding partners influenced LTCC activity. Ventricular myocytes were isolated from male Wistar rats and LTCC current (ICa) recorded using the whole-cell patch-clamp technique. Incubation of myocytes with a membrane-permeable peptide representing the scaffolding domain of Cav-3 (C3SD) reduced basal ICa amplitude in intact, but not detubulated, myocytes, and attenuated the stimulatory effects of the β2-adrenergic agonist zinterol on ICa. The PKA inhibitor H-89 also reduced basal ICa; however, the inhibitory effects of C3SD and H-89 on basal ICa amplitude were not summative. Under control conditions, myocytes stained with antibody against phosphorylated LTCC (pLTCC) displayed a striated pattern, presumably reflecting localization at the t-tubules. Both C3SD and H-89 reduced pLTCC staining at the z-lines but did not affect staining of total LTCC or Cav-3. These data are consistent with the idea that the effects of C3SD and H-89 share a common pathway, which involves PKA and is maximally inhibited by H-89, and suggest that Cav-3 plays an important role in mediating stimulation of ICa at the t-tubules via PKA-induced phosphorylation under basal conditions, and in response to β2-adrenoceptor stimulation.
Graphical abstract
•Basal L type calcium current was reduced by interfering with caveolin-3 binding.•L type calcium current is tonically regulated by PKA phosphorylation.•Interfering with caveolin-3 binding reduced beta2 adrenergic stimulation of ICa.
PMCID: PMC3980375  PMID: 24412535
t-tubules; Ca; Phosphorylation; β2-Adrenoceptors
4.  Nickel inhibits β-1 adrenoceptor mediated activation of cardiac CFTR chloride channels 
► We report the block of the β-adrenoceptor-activated cardiac CFTR Cl− current by Ni2+. ► Extracellular Ni2+ inhibits the current activated by β1-adrenoceptors in a concentration-dependent manner. ► The action of Ni2+ is insensitive to β2-blockade. ► Ni2+ does not affect the β-adrenoceptor-activated current from the intracellular side. ► The data are consistent with an action of Ni2+ at the β1-adrenoceptor from the external side.
Cardiac ventricular myocytes exhibit a protein kinase A-dependent Cl− current (ICl.PKA) mediated by the cystic fibrosis transmembrane conductance regulator (CFTR). There is conflicting evidence regarding the ability of the divalent cation nickel (Ni2+), which has been used widely in vitro in the study of other cardiac ionic conductances, to inhibit ICl.PKA. Here the action of Ni2+ on ICl.PKA activated by β-adrenergic stimulation has been elucidated. Whole-cell patch-clamp recordings were made from rabbit isolated ventricular myocytes. Externally applied Ni2+ blocked ICl.PKA activated by 1 μM isoprenaline with a log IC50 (M) of −4.107 ± 0.075 (IC50 = 78.1 μM) at +100 mV and −4.322 ± 0.107 (IC50 = 47.6 μM) at −100 mV. Thus, the block of ICl.PKA by Ni2+ was not strongly voltage dependent. Ni2+ applied internally via the patch-pipette was ineffective at inhibiting isoprenaline-activated ICl,PKA, but in the same experiments the current was suppressed by external Ni2+ application, indicative of an external site of Ni2+ action. In the presence of 1 μM atenolol isoprenaline was ineffective at activating ICl.PKA, but in the presence of the β2-adrenoceptor inhibitor ICI 118,551 isoprenaline still activated Ni2+-sensitive ICl.PKA. Collectively, these data demonstrate that Ni2+ ions produce marked inhibition of β1-adrenoceptor activated ventricular ICl.PKA at submillimolar [Ni2+]: an action that is likely to involve an interaction between Ni2+ and β1-adrenoceptors. The concentration-dependence for ICl.PKA inhibition seen here indicates the potential for confounding effects on ICl,PKA to occur even at comparatively low Ni2+ concentrations, when Ni2+ is used to study other cardiac ionic currents under conditions of β-adrenergic agonism.
PMCID: PMC3686155  PMID: 23376720
Rabbit cardiomyocytes; PKA-dependent Cl− current; CFTR; CFTR-inhibitor; Nickel; Ni2+
5.  Acute desensitization of acetylcholine and endothelin-1 activated inward rectifier K+ current in myocytes from the cardiac atrioventricular node 
► ACh and ET-1 activate a K+ current in cardiac atrioventricular nodal cells. ► Tertiapin-Q sensitive IKACh activated via M2 receptors shows bi-exponential ‘fade’. ► ET-1 activates a similar current that also fades. ► The fade reflects desensitization rather than altered K+ ion driving force. ► Acetylcholine is able to cross-desensitize the AVN cell response to endothelin-1.
The atrioventricular node (AVN) is a vital component of the pacemaker-conduction system of the heart, co-ordinating conduction of electrical excitation from cardiac atria to ventricles and acting as a secondary pacemaker. The electrical behaviour of the AVN is modulated by vagal activity via activation of muscarinic potassium current, IKACh. However, it is not yet known if this response exhibits ‘fade’ or desensitization in the AVN, as established for the heart’s primary pacemaker – the sinoatrial node. In this study, acute activation of IKACh in rabbit single AVN cells was investigated using whole-cell patch clamp at 37 °C. 0.1–1 μM acetylcholine (ACh) rapidly activated a robust IKACh in AVN myocytes during a descending voltage-ramp protocol. This response was inhibited by tertiapin-Q (TQ; 300 nM) and by the M2 muscarinic ACh receptor antagonist AFDX-116 (1 μM). During sustained ACh exposure the elicited IKACh exhibited bi-exponential fade (τf of 2.0 s and τs 76.9 s at −120 mV; 1 μM ACh). 10 nM ET-1 elicited a current similar to IKACh, which faded with a mono-exponential time-course (τ of 52.6 s at −120 mV). When ET-1 was applied following ACh, the ET-1 activated response was greatly attenuated, demonstrating that ACh could desensitize the response to ET-1. For neither ACh nor ET-1 was the rate of current fade dependent upon the initial response magnitude, which is inconsistent with K+ flux mediated changes in electrochemical driving force as the underlying mechanism. Collectively, these findings demonstrate that TQ sensitive inwardly rectifying K+ current in cardiac AVN cells, elicited by M2 muscarinic receptor or ET-1 receptor activation, exhibits fade due to rapid desensitization.
PMCID: PMC3400056  PMID: 22683635
Acetylcholine (ACh); Atrioventricular node; AV node; AVN; Endothelin-1 (ET-1); GIRK; IKACh; Inward rectifier; Muscarinic potassium current; Tertiapin-Q
6.  Modulation by Endothelin-1 of Spontaneous Activity and Membrane Currents of Atrioventricular Node Myocytes from the Rabbit Heart 
PLoS ONE  2012;7(3):e33448.
The atrioventricular node (AVN) is a key component of the cardiac pacemaker-conduction system. Although it is known that receptors for the peptide hormone endothelin-1 (ET-1) are expressed in the AVN, there is very little information available on the modulatory effects of ET-1 on AVN electrophysiology. This study characterises for the first time acute modulatory effects of ET-1 on AVN cellular electrophysiology.
Electrophysiological experiments were conducted in which recordings were made from rabbit isolated AVN cells at 35–37°C using the whole-cell patch clamp recording technique.
Application of ET-1 (10 nM) to spontaneously active AVN cells led rapidly (within ∼13 s) to membrane potential hyperpolarisation and cessation of spontaneous action potentials (APs). This effect was prevented by pre-application of the ETA receptor inhibitor BQ-123 (1 µM) and was not mimicked by the ETB receptor agonist IRL-1620 (300 nM). In whole-cell voltage-clamp experiments, ET-1 partially inhibited L-type calcium current (ICa,L) and rapid delayed rectifier K+ current (IKr), whilst it transiently activated the hyperpolarisation-activated current (If) at voltages negative to the pacemaking range, and activated an inwardly rectifying current that was inhibited by both tertiapin-Q (300 nM) and Ba2+ ions (2 mM); each of these effects was sensitive to ETA receptor inhibition. In cells exposed to tertiapin-Q, ET-1 application did not produce membrane potential hyperpolarisation or immediate cessation of spontaneous activity; instead, there was a progressive decline in AP amplitude and depolarisation of maximum diastolic potential.
Acutely applied ET-1 exerts a direct modulatory effect on AVN cell electrophysiology. The dominant effect of ET-1 in this study was activation of a tertiapin-Q sensitive inwardly rectifying K+ current via ETA receptors, which led rapidly to cell quiescence.
PMCID: PMC3315568  PMID: 22479400
7.  β-Adrenoceptor/PKA-stimulation, Na+–Ca2+ exchange and PKA-activated Cl− currents in rabbit cardiomyocytes: A conundrum 
Cell Calcium  2011;49(4):233-239.
Investigations into the functional modulation of the cardiac Na+–Ca2+ exchanger (NCX) by acute β-adrenoceptor/PKA stimulation have produced conflicting results. Here, we investigated (i) whether or not β-adrenoceptor activation/PKA stimulation activates current in rabbit cardiac myocytes under NCX-‘selective’ conditions and (ii) if so, whether a PKA-activated Cl−-current may contribute to the apparent modulation of NCX current (INCX). Whole-cell voltage-clamp experiments were conducted at 37 °C on rabbit ventricular and atrial myocytes. The β-adrenoceptor-activated currents both in NCX-‘selective’ and Cl−-selective recording conditions were found to be sensitive to 10 mM Ni2+. In contrast, the PKA-activated Cl− current was not sensitive to Ni2+, when it was activated downstream to the β-adrenoceptors using 10 μM forskolin (an adenylyl cyclase activator). When 10 μM forskolin was applied under NCX-selective recording conditions, the Ni2+-sensitive current did not differ between control and forskolin. These findings suggest that in rabbit myocytes: (a) a PKA-activated Cl− current contributes to the Ni2+-sensitive current activated via β-adrenoceptor stimulation under recording conditions previously considered selective for INCX; (b) downstream activation of PKA does not augment Ni2+-sensitive INCX, when this is measured under conditions where the Ni2+-sensitive PKA-activated Cl− current is not present.
PMCID: PMC3092849  PMID: 21439639
Cardiac myocyte; CFTR; NCX; Rabbit atrial myocyte; Rabbit ventricular myocyte; Whole-cell patch-clamp recording
8.  The sodium channel Nav1.5a is the predominant isoform expressed in adult mouse dorsal root ganglia and exhibits distinct inactivation properties from the full-length Nav1.5 channel 
Nav1.5 is the principal voltage-gated sodium channel expressed in heart, and is also expressed at lower abundance in embryonic dorsal root ganglia (DRG) with little or no expression reported postnatally. We report here the expression of Nav1.5 mRNA isoforms in adult mouse and rat DRG. The major isoform of mouse DRG is Nav1.5a, which encodes a protein with an IDII/III cytoplasmic loop reduced by 53 amino acids. Western blot analysis of adult mouse DRG membrane proteins confirmed the expression of Nav1.5 protein. The Na+ current produced by the Nav1.5a isoform has a voltage-dependent inactivation significantly shifted to more negative potentials (by ~5 mV) compared to the full-length Nav1.5 when expressed in the DRG neuroblastoma cell line ND7/23. These results imply that the alternatively spliced exon 18 of Nav1.5 plays a role in channel inactivation and that Nav1.5a is likely to make a significant contribution to adult DRG neuronal function.
PMCID: PMC2726334  PMID: 17433712

Results 1-8 (8)