Andrew F. James

VEGF Activates Receptor-Operated Cation Channels in Human Microvascular Endothelial Cells

Objective—Vascular endothelial growth factor (VEGF) exerts many of its effects by stimulating endothelial calcium influx, but little is known about channels mediating VEGF-induced cation entry. The aim of this study was to measure and characterize for the first time the VEGF-activated cation current in human microvascular endothelial cells (HMVECs). Methods and Results—Whole-cell patch-clamp recordings were made from HMVECs. During applied voltage ramps, VEGF activated a current that reversed at 0 mV, was sensitive to gadolinium, and required extracellular cations. Noise analysis yielded a single-channel conductance of 27 pS. The current was not dependent on intracellular calcium stores, and was not blocked by inositol triphosphate (IP3) receptor or serine/threonine kinase inhibition but was partially inhibited by flufenamic acid. A similar current was activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analog of diacylglycerol (DAG). To determine whether VEGF could activate recombinant ion channels with similar properties, we investigated the effect of VEGF on Chinese hamster ovary cells cotransfected with VEGFR2 and the canonical transient receptor potential (TRPC) channels, TRPC3 or TRPC6. VEGF induced a similar current to that described above in VEGFR2-TRPC3 and VEGFR2-TRPC6 cells but not in cells transfected with either cDNA alone. Conclusions—VEGF activates a receptor-operated cation current in HMVECs and OAG can activate directly a similar current in these cells. VEGF is also able to activate heterologously expressed TRPC3/6 channels through VEGFR2.

Atrial Remodeling and the Substrate for Atrial Fibrillation in Rat Hearts with Elevated Afterload

Background— Although arterial hypertension and left ventricular hypertrophy are considered good epidemiological indicators of the risk of atrial fibrillation (AF) in patients, the link between elevated afterload and AF remains unclear. We investigated atrial remodeling and the substrate for arrhythmia in a surgical model of elevated afterload in rats. Methods and Results— Male Wistar rats (aged 3–4 weeks) were anesthetized and subjected to either partial stenosis of the ascending aorta (AoB) or sham operation (Sham). Experiments were performed on excised hearts 8, 14, and 20 weeks after surgery. Unipolar electrograms were recorded from the left atrial epicardial surface of perfused hearts using a 5×5 electrode array. Cryosections of left atrial tissue were retained for histological and immunocytochemical analyses. Compared to Sham, AoB hearts showed marked left atrial hypertrophy and fibrosis at 14 and 20 weeks postsurgery. The incidence and duration of pacing-induced AF was increased in hearts from AoB rats at 20 weeks postsurgery. The substrate for arrhythmia was associated with reduced vectorial conduction velocity and greater inhomogeneity in conduction but without changes in effective refractory period. Left atrial expression of the gap junction protein, connexin43, was markedly reduced in AoB compared with Sham hearts. Conclusions— Using a small-animal model, we demonstrate that elevated afterload in the absence of systemic hypertension results in increased inducibility of AF and left atrial remodeling involving fibrosis, altered atrial connexin43 expression, and marked conduction abnormalities.

Distribution of cAMP-Activated Chloride Current and CFTR mRNA in the Guinea Pig Heart

Guinea pig ventricular myocytes exhibit a Cl(-)-selective current regulated by the cAMP-dependent pathway. We have investigated the distribution of cAMP-activated Cl- channel current density and cystic fibrosis transmembrane-conductance regulator (CFTR) mRNA in three regions of the guinea pig heart: the atrium, and the epicardium and endocardium of the free wall of the left ventricle. The regional differences in the Cl- current density were investigated in enzymatically isolated myocytes using the whole-cell patch-clamp technique. Forskolin (1 mumol/L) activated Cl(-)-selective currents in all ventricular myocytes and 21% of atrial myocytes examined. The conductance density, estimated as the outward chord conductance normalized to cell capacitance, was greatest in epicardial myocytes (79.8 +/- 8.4 pS/pF, n = 21) and significantly lower in endocardial (59.8 +/- 9.5 pS/pF, n = 22) and atrial (10.9 +/- 5.0 pS/pF, n = 38) myocytes. The regional differences in CFTR mRNA expression levels were investigated by competitive reverse-transcribed polymerase chain reaction. The regional distribution of the mRNA levels was similar to that of the Cl- conductance density, ie, highest in the epicardium (23230 +/- 1840 molecules/microgram total RNA, n = 3), significantly lower in endocardium (10610 +/- 780 molecules/microgram total RNA, n = 3), and lowest in atrium (1450 +/- 290 molecules/microgram total RNA, n = 3). The data indicate that regional differences in CFTR mRNA expression in the guinea pig heart are responsible, at least in part, for the regional differences in cAMP-activated Cl- current density.

Gender-related differences in ventricular myocyte repolarization in the guinea pig

Abstract.It is well established that gender-differences exist in cardiac electrophysiology and these are thought to contribute to the increased risk of women, compared to men, for the potentially lethal ventricular arrhythmia, torsades de pointes. Data from animal models with abbreviated estrus cycles suggest that androgens may play a protective role in males. However, the role of female sex hormones in gender-differences in cardiac electrophysiology is less clear. This report describes gender differences in ventricular electrophysiology, investigated using the guinea pig heart. Ionic currents and action potentials were compared between ventricular myocytes isolated from male guinea pig hearts and those from females on the day of estrus (day 0) and 4 days post-estrus (day 4). The density of inward rectifier K+ current (IK1) at –120 mV was significantly greater in male myocytes than in female myocytes either at day 0 or day 4. The peak L-type Ca2+ current (ICa) at +10 mV was also significantly larger in male myocytes than in day 0 and day 4 female myocytes. Moreover, ICa differed significantly between day 0 and day 4 female myocytes, strongly suggesting that ICa density varies around the estrus cycle. Delayed rectifier (IK) tail currents were significantly different between male and female day 4 myocytes. Action potential duration (at 90% repolarization; APD90) was significantly shorter in male myocytes than in female myocytes at day 0, but not at day 4, broadly consistent with the combined differences in IK and ICa between the three groups. Taken together, our data are consistent with the contribution of multiple factors, rather than a single hormone, to gender differences in ventricular repolarization. Since female guinea pigs possess a conventional estrus cycle, our data suggest that this species may be well suited to elucidating the modulatory influence of ovarian steroids on ventricular repolarization and arrhythmic risk. Our findings suggest that further work examining the basis to gender differences in ventricular repolarization in the guinea pig is warranted.

Expression of voltage-gated K+ channels in human atrium.

Abstract. Voltage-gated K+ channels underlie repolarisation of the cardiac action potential and represent a potential therapeutic target in the treatment of cardiac dysrhythmias. However, very little is known about the relative expression of K+ channel subunits in the human myocardium. We used a semi-quantitative RT-PCR technique to examine the relative expression of mRNAs for the voltage-gated K+ channel subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv4.2, Kv4.3, KvLQT1, HERG and IsK in samples of human atrial appendage. Data were expressed as a percentage expression density relative to an 18S ribosomal RNA internal standard. The most abundant K+ channel mRNAs were Kv4.3 (80.7 ± 10.1 %), Kv1.5 (69.7 ± 11.2 %) and HERG (55.9 ± 21.5 %). Significant expression of KvLQT1 (33.5 ± 5.5 %,) and Kv1.4 (26.7 ± 9.6 %) was also detected. Levels of mRNAs for Kv1.2 and IsK were very low and neither Kv2.1 nor Kv4.2 mRNA were detected in any experiments. Whole-cell patch-clamp techniques were used to examine the outward currents of isolated human atrial myocytes at 37 °C. These recordings demonstrated the existence of transient (Ito1) and sustained (Iso) outward currents in isolated human atrial myocytes. Ito1, and not Iso, showed voltage-dependent inactivation during 100 ms pre-pulses. Both Ito1 and Iso were inhibited by high concentrations (2 mM) of the K+ channel blocker, 4-aminopyridine (4-AP). However, lower concentrations of 4-AP (10 μM) inhibited Iso selectively. Ito1 recovered from inactivation relatively rapidly (t ∼21 ms). These data, with published information regarding the properties of expressed K+ channels, suggest that Kv4.3 represents the predominant K+ channel subunit underlying Ito1 with little contribution of Kv1.4. The sensitivity of Iso to very low concentrations of 4-aminopyridine and the relatively low expression of mRNA for Kv1.2 and Kv2.1 is consistent with the major contribution of Kv1.5 to this current. The physiological significance of the expression of KvLQT1 and Kv1.4 mRNA in the human atrium warrants further investigation.

Inflammation, oxidative stress and postoperative atrial fibrillation in cardiac surgery

Postoperative atrial fibrillation (POAF) is a common complication of cardiac surgery that occurs in up to 60% of patients. POAF is associated with increased risk of cardiovascular mortality, stroke and other arrhythmias that can impact on early and long term clinical outcomes and health economics. Many factors such as disease-induced cardiac remodelling, operative trauma, changes in atrial pressure and chemical stimulation and reflex sympathetic/parasympathetic activation have been implicated in the development of POAF. There is mounting evidence to support a major role for inflammation and oxidative stress in the pathogenesis of POAF. Both are consequences of using cardiopulmonary bypass and reperfusion following ischaemic cardioplegic arrest. Subsequently, several anti-inflammatory and antioxidant drugs have been tested in an attempt to reduce the incidence of POAF. However, prevention remains suboptimal and thus far none of the tested drugs has provided sufficient efficacy to be widely introduced in clinical practice. A better understanding of the cellular and molecular mechanisms responsible for the onset and persistence of POAF is needed to develop more effective prediction and interventions.

Inhibition of the HERG K+ channel by the antifungal drug ketoconazole depends on channel gating and involves the S6 residue F656

The mechanism of human ether‐à‐go‐go‐related gene (HERG) K+ channel blockade by the antifungal agent ketoconazole was investigated using patch‐clamp recording from mammalian cell lines. Ketoconazole inhibited whole‐cell HERG current (I HERG) with a clinically relevant half‐maximal inhibitory drug concentration (IC50) value of 1.7 μM. The voltage‐ and time‐dependent characteristics of I HERG blockade by ketoconazole indicated dependence of block on channel gating, ruling out a significant role for closed‐state channel inhibition. The S6 HERG mutations Y652A and F656A produced ∼4‐fold and ∼21‐fold increases in IC50 for I HERG blockade, respectively. Thus, ketoconazole accesses the HERG channel pore‐cavity on channel gating, and the S6 residue F656 is an important determinant of ketoconazole binding.

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 approximately 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.

Altered distribution of ICa impairs Ca release at the t-tubules of ventricular myocytes from failing hearts

In mammalian cardiac ventricular myocytes, Ca influx and release occur predominantly at t-tubules, ensuring synchronous Ca release throughout the cell. Heart failure is associated with disrupted t-tubule structure, but its effect on t-tubule function is less clear. We therefore investigated Ca influx and release at the t-tubules of ventricular myocytes isolated from rat hearts ~ 18 weeks after coronary artery ligation (CAL) or corresponding Sham operation. L-type Ca current (ICa) was recorded using the whole-cell voltage-clamp technique in intact and detubulated myocytes; Ca release at t-tubules was monitored using confocal microscopy with voltage- and Ca-sensitive fluorophores. CAL was associated with cardiac and cellular hypertrophy, decreased ejection fraction, disruption of t-tubule structure and a smaller, slower Ca transient, but no change in ryanodine receptor distribution, L-type Ca channel expression, or ICa density. In Sham myocytes, ICa was located predominantly at the t-tubules, while in CAL myocytes, it was uniformly distributed between the t-tubule and surface membranes. Inhibition of protein kinase A with H-89 caused a greater decrease of t-tubular ICa in CAL than in Sham myocytes; in the presence of H-89, t-tubular ICa density was smaller in CAL than in Sham myocytes. The smaller t-tubular ICa in CAL myocytes was accompanied by increased latency and heterogeneity of SR Ca release at t-tubules, which could be mimicked by decreasing ICa using nifedipine. These data show that CAL decreases t-tubular ICa via a PKA-independent mechanism, thereby impairing Ca release at t-tubules and contributing to the altered excitation–contraction coupling observed in heart failure.

Inhibition of the cardiac L-type calcium channel current by the TRPM8 agonist, (-)-menthol

(-)-Menthol and icilin are agonists of the thermoreceptor non-selective cation channel, TRPM8, and are commonly used to investigate TRPM8 function without a full appreciation of their non-specific effects. To investigate the hypothesis that (-)-menthol and icilin inhibit cardiovascular-type L-type Ca(2+) channel currents (I(Ca,L)), the actions of the TRPM8 agonists on rabbit ventricular myocyte I(Ca,L) were examined at near-physiological temperature (≈35°C) using whole-cell recording. Icilin (3-100 μM) did not significantly inhibit I(Ca,L). (3) in contrast, (-)-menthol concentration-dependently inhibited peak I(Ca,L) (IC(50)=74.6 μM; log(10)IC(50)(M)=-4.13±0.14). (-)-Menthol blocked the late I(Ca,L) remaining at the end of depolarising pulses with greater efficacy (96.1±2.4% block at 1 mM) than peak I(Ca,L) (68.9±5.7% block at 1 mM, P<0.01), although there was no difference in potency of block of peak and late currents. Block by (-)-menthol showed no voltage-dependence. The actions of (-)-menthol were compared with those of nimodipine. Nimodipine was a more efficacious (97.3±1.5 % block at 30 μM, P<0.01) and potent (IC(50)=0.74 μM; log(10)IC(50)(M)=-6.13±0.08, P<0.0001) blocker of peak I(Ca,L) than (-)-menthol. In contrast to (-)-menthol, nimodipine showed greater potency (IC(50)=0.056 μM; log(10)IC(50)(M)=-7.25±0.17, P<0.0001), but not greater efficacy, in block of late compared with peak I(Ca,L). In summary, these data demonstrate that, at near-physiological temperature, (-) -menthol blocks cardiac I(Ca,L) at concentrations similar to those reportedly effective in TRPM8-agonism. The data suggest that the mechanism of L-type Ca(2+) channel block by (-)-menthol differs from that of nimodipine.