Jan H. Ravesloot

Ionic Remodeling of Sinoatrial Node Cells by Heart Failure

Background—In animal models of heart failure (HF), heart rate decreases as the result of an increase in intrinsic cycle length of the sinoatrial node (SAN). In this study, we evaluate the HF-induced remodeling of membrane potentials and currents in SAN cells. Methods and Results—SAN cells were isolated from control rabbits and rabbits with volume and pressure overload–induced HF and patch-clamped to measure their electrophysiological properties. HF cells were not hypertrophied (capacitance, mean±SEM, 52±3 versus 50±4 pF in control). HF increased intrinsic cycle length by 15% and decreased diastolic depolarization rate by 30%, whereas other action potential parameters were unaltered. In HF, the hyperpolarization-activated “pacemaker” current (If) and slow component of the delayed rectifier current (IKs) were reduced by 40% and 20%, respectively, without changes in voltage dependence or kinetics. T-type and L-type calcium current, rapid and ultrarapid delayed rectifier current, transient outward currents, and sodium-calcium exchange current were unaltered. Conclusions—In single SAN cells of rabbits with HF, intrinsic cycle length is increased as the result of a decreased diastolic depolarization rate rather than a change in action potential duration. HF reduced both If and IKs density. Since IKs plays a limited role in pacemaker activity, the HF-induced decrease in heart rate is attributable to remodeling of If.

Ca2+‐activated Cl− current in rabbit sinoatrial node cells

The Ca2+‐activated Cl− current (ICl(Ca)) has been identified in atrial, Purkinje and ventricular cells, where it plays a substantial role in phase‐1 repolarization and delayed after‐depolarizations. In sinoatrial (SA) node cells, however, the presence and functional role of ICl(Ca) is unknown. In the present study we address this issue using perforated patch‐clamp methodology and computer simulations. Single SA node cells were enzymatically isolated from rabbit hearts. ICl(Ca) was measured, using the perforated patch‐clamp technique, as the current sensitive to the anion blocker 4,4′‐diisothiocyanostilbene‐2,2′‐disulphonic acid (DIDS). Voltage clamp experiments demonstrate the presence of ICl(Ca) in one third of the spontaneously active SA node cells. The current was transient outward with a bell‐shaped current‐voltage relationship. Adrenoceptor stimulation with 1 μm noradrenaline doubled the ICl(Ca) density. Action potential clamp measurements demonstrate that ICl(Ca) is activate late during the action potential upstroke. Current clamp experiments show, both in the absence and presence of 1 μm noradrenaline, that blockade of ICl(Ca) increases the action potential overshoot and duration, measured at 20 % repolarization. However, intrinsic interbeat interval, upstroke velocity, diastolic depolarization rate and the action potential duration measured at 50 and 90 % repolarization were not affected. Our experimental data are supported by computer simulations, which additionally demonstrate that ICl(Ca) has a limited role in pacemaker synchronization or action potential conduction. In conclusion, ICl(Ca) is present in one third of SA node cells and is activated during the pacemaker cycle. However, ICl(Ca) does not modulate intrinsic interbeat interval, pacemaker synchronization or action potential conduction.

Two types of action potential configuration in single cardiac Purkinje cells of sheep

Membrane potentials and currents of isolated sheep Purkinje and ventricular cells were compared using patch-clamp and microelectrode techniques. In ∼50% of Purkinje cells, we observed action potentials that showed a prominent phase 1 repolarization and relatively negative plateau (LP cells). Action potential configuration of the remaining Purkinje cells was characterized by little phase 1 repolarization and relatively positive plateau (HP cells). Microelectrode impalement of Purkinje strands also revealed these two types of action potential configuration. In LP cells, the density of L-type Ca2+ current ( I Ca,L) was lower, whereas the density of transient outward K+ current was higher, than in HP cells. Action potentials of HP cells strongly resembled those of ventricular cells. Densities of inward rectifier current and I Ca,L were significantly higher in ventricular cells compared with densities in both LP and HP Purkinje cells. Differences in current densities explain the striking differences in action potential configuration and the stimulus frequency dependency thereof that we observed in LP, HP, and ventricular cells. We conclude that LP Purkinje cells, HP Purkinje cells, and ventricular cells of sheep each have a unique action potential configuration.

Reduced swelling-activated Cl− current densities in hypertrophied ventricular myocytes of rabbits with heart failure

OBJECTIVE Hypertrophied myocytes of failing hearts have prolonged action potential durations. It is unknown how the swelling-activated Cl(-) current (I(Cl,swell)) affects the abnormal AP configuration. METHODS We studied I(Cl,swell) in ventricular myocytes isolated from failing and age-matched normal rabbit hearts. We applied whole-cell patch-clamp methodology and activated I(Cl,swell) by lowering tonicity of the superfusate. RESULTS Neither with ruptured-patch nor with amphotericin B perforated-patch, whole-cell clamp we found I(Cl,swell) active under isotonic conditions in either the normal or the hypertrophied failing heart (HFH) myocytes. I(Cl,swell) caused AP shortening and resting membrane potential (V(m)) depolarization in an osmotic gradient-dependent fashion. However, in the HFH myocytes swelling-induced AP changes were significantly smaller, even though the cells underwent the same relative change in planar cell surface area. Voltage-clamp experiments revealed that in HFH myocytes I(Cl,swell) current density was approximately 50% reduced. CONCLUSION Reduced I(Cl,swell) densities in HFH myocytes cause limited AP shortening and V(m) depolarization upon swelling of the cells.

Role of Ca2+‐Activated Cl− Current in Ventricular Action Potentials of Sheep During Adrenoceptor Stimulation

Adrenoceptor stimulation enhances repolarising and depolarising membrane currents to different extents in cardiac myocytes. We investigated the opposing effects of the repolarising Ca2+‐activated Cl− current (ICl(Ca)) and depolarising L‐type Ca2+ current (ICa,L) on the action potential configuration of sheep ventricular myocytes stimulated with noradrenaline. Whole‐cell current‐clamp recordings revealed that noradrenaline accelerated and prolonged phase‐1 repolarisation. We define the minimal potential at the end of phase‐1 repolarisation as ‘notch level’. Noradrenaline (1 μM) caused the notch level to fall from 14 ± 2.6 to 7.8 ± 2.8 mV (n= 24), but left action potential duration, resting membrane potential or action potential amplitude unaffected. Whole‐cell voltage‐clamp recordings showed that 1 μM noradrenaline increased both ICa,L and ICl(Ca), but it had no significant effect on the principal K+ currents. Blockage of ICl(Ca) by 0.5 mM 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid (DIDS) in both the absence and the presence of noradrenaline abolished phase‐1 repolarisation. In the presence of noradrenaline, DIDS caused elevation of the plateau phase amplitude and an increase in the action potential duration. In conclusion, elevation of the plateau phase amplitude and action potential prolongation associated with an increased ICa,L upon adrenoceptor stimulation is prevented by an increased ICl(Ca) in sheep ventricular myocytes.

Dietary omega-3 polyunsaturated fatty acids suppress NHE-1 upregulation in a rabbit model of volume- and pressure-overload

Background: Increased consumption of omega-3 polyunsaturated fatty acids (ω3-PUFAs) from fish oil (FO) may have cardioprotective effects during ischemia/reperfusion, hypertrophy, and heart failure (HF). The cardiac Na+/H+-exchanger (NHE-1) is a key mediator for these detrimental cardiac conditions. Consequently, chronic NHE-1 inhibition appears to be a promising pharmacological tool for prevention and treatment. Acute application of the FO ω3-PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) inhibit the NHE-1 in isolated cardiomyocytes. We studied the effects of a diet enriched with ω3-PUFAs on the NHE-1 activity in healthy rabbits and in a rabbit model of HF induced by volume- and pressure-overload. Methods: Rabbits were allocated to four groups. The first two groups consisted of healthy rabbits, which were fed either a diet containing 1.25% (w/w) FO (ω3-PUFAs), or 1.25% high-oleic sunflower oil (ω9-MUFAs) as control. The second two groups were also allocated to either a diet containing ω3-PUFAs or ω9-MUFAs, but underwent volume- and pressure-overload to induce HF. Ventricular myocytes were isolated by enzymatic dissociation and used for intracellular pH (pHi) and patch-clamp measurements. NHE-1 activity was measured in HEPES-buffered conditions as recovery rate from acidosis due to ammonium prepulses. Results: In healthy rabbits, NHE-1 activity in ω9-MUFAs and ω3-PUFAs myocytes was not significantly different. Volume- and pressure-overload in rabbits increased the NHE-1 activity in ω9-MUFAs myocytes, but not in ω3-PUFAs myocytes, resulting in a significantly lower NHE-1 activity in myocytes of ω3-PUFA fed HF rabbits. The susceptibility to induced delayed afterdepolarizations (DADs), a cellular mechanism of arrhythmias, was lower in myocytes of HF animals fed ω3-PUFAs compared to myocytes of HF animals fed ω9-MUFAs. In our rabbit HF model, the degree of hypertrophy was similar in the ω3-PUFAs group compared to the ω9-MUFAs group. Conclusion: Dietary ω3-PUFAs from FO suppress upregulation of the NHE-1 activity and lower the incidence of DADs in our rabbit model of volume- and pressure-overload.

Development of a test for evaluation of taste perception after tongue reduction

When performing a tongue reduction a frequently asked question is how operation will influence taste of the patient. Different kinds of taste tests are designed, most of these being non-specific ways to determine taste sensation in which high concentration of taste solutions are used to detect if a person is able to taste. To be able to judge the influence of tongue reduction on taste we wanted to develop a validated test that could be used in early childhood. No specific tasting areas were found. This test can be used to evaluate tongue reduction procedures.

Increased sarcolemmal Na+/H+ exchange activity in hypertrophied myocytes from dogs with chronic atrioventricular block

Dogs with compensated biventricular hypertrophy due to chronic atrioventricular block (cAVB), are more susceptible to develop drug-induced Torsade-de-Pointes arrhythmias and sudden cardiac death. It has been suggested that the increased Na+ influx in hypertrophied cAVB ventricular myocytes contribute to these lethal arrhythmias. The increased Na+ influx was not mediated by Na+ channels, in fact the Na+ current proved reduced in cAVB myocytes. Here we tested the hypothesis that increased activity of the Na+/H+ exchanger type 1 (NHE-1), commonly observed in hypertrophic hearts, causes the elevated Na+ influx. Cardiac acid-base transport was studied with a pH-sensitive fluorescent dye in ventricular myocytes isolated from control and hypertrophied cAVB hearts; the H+ equivalent flux through NHE-1, Na+-HCO−3 cotransport (NBC), Cl−/OH− exchange (CHE), and Cl−/HCO−3 exchange (AE) were determined and normalized per liter cell water and corrected for surface-to-volume ratio. In cAVB, sarcolemmal NHE-1 flux was increased by 65 ± 6.3% in the pHi interval 6.3–7.2 and NBC, AE, and CHE fluxes remained unchanged. Accordingly, at steady-state intracellular pH the total sarcolemmal Na+ influx by NHE-1 + NBC increased from 8.5 ± 1.5 amol/μm2/min in normal myocytes to 15 ± 2.4 amol/μm2/min in hypertrophied cAVB myocytes. We conclude that compensated cardiac hypertrophy in cAVB dogs is accompanied with an increased sarcolemmal NHE-1 activity. This in conjunction with unchanged activity of the other acid-base transporters will raise the intracellular Na+ in hypertrophied cAVB myocytes.