Roel L. H. M. G. Spätjens

Probing the Contribution of IKs to Canine Ventricular Repolarization: Key Role for β-Adrenergic Receptor Stimulation

Background—In large mammals and humans, the contribution of IKs to ventricular repolarization is still incompletely understood. Methods and Results—In vivo and cellular electrophysiological experiments were conducted to study IKs in canine ventricular repolarization. In conscious dogs, administration of the selective IKs blocker HMR 1556 (3, 10, or 30 mg/kg PO) caused substantial dose-dependent QT prolongations with broad-based T waves. In isolated ventricular myocytes under baseline conditions, however, IKs block (chromanols HMR 1556 and 293B) did not significantly prolong action potential duration (APD) at fast or slow steady-state pacing rates. This was because of the limited activation of IKs in the voltage and time domains of the AP, although at seconds-long depolarizations, the current was substantial. Isoproterenol increased and accelerated IKs activation to promote APD95 shortening. This shortening was importantly reversed by HMR 1556 and 293B. Quantitatively similar effects were obtained in ventricular-tissue preparations. Finally, when cellular repolarization was impaired by IKr block, IKs block exaggerated repolarization instability with further prolongation of APD. Conclusions—Ventricular repolarization in conscious dogs is importantly dependent on IKs. IKs function becomes prominent during &bgr;-adrenergic receptor stimulation, when it promotes AP shortening by increased activation, and during IKr block, when it limits repolarization instability by time-dependent activation. Unstimulated IKs does not contribute to cellular APD at baseline. These data highlight the importance of the synergism between an intact basal IKs and the sympathetic nervous system in vivo.

Repolarizing K+ Currents ITO1 and IKs Are Larger in Right Than Left Canine Ventricular Midmyocardium

BACKGROUND The ventricular action potential exhibits regional heterogeneity in configuration and duration (APD). Across the left ventricular (LV) free wall, this is explained by differences in repolarizing K+ currents. However, the ionic basis of electrical nonuniformity in the right ventricle (RV) versus the LV is poorly investigated. We examined transient outward (ITO1), delayed (IKs and IKr), and inward rectifier K+ currents (IK1) in relation to action potential characteristics of RV and LV midmyocardial (M) cells of the same adult canine hearts. METHODS AND RESULTS Single RV and LV M cells were used for microelectrode recordings and whole-cell voltage clamping. Action potentials showed deeper notches, shorter APDs at 50% and 95% of repolarization, and less prolongation on slowing of the pacing rate in RV than LV. ITO1 density was significantly larger in RV than LV, whereas steady-state inactivation and rate of recovery were similar. IKs tail currents, measured at -25 mV and insensitive to almokalant (2 micromol/L), were considerably larger in RV than LV. IKr, measured as almokalant-sensitive tail currents at -50 mV, and IK1 were not different in the 2 ventricles. CONCLUSIONS Differences in K+ currents may well explain the interventricular heterogeneity of action potentials in M layers of the canine heart. These results contribute to a further phenotyping of the ventricular action potential under physiological conditions.

Accumulation of slowly activating delayed rectifier potassium current (IKs) in canine ventricular myocytes

In guinea‐pig ventricular myocytes, in which the deactivation of slowly activating delayed rectifier potassium current (IKs) is slow, IKs can be increased by rapid pacing as a result of incomplete deactivation and subsequent current accumulation. Whether accumulation of IKs occurs in dogs, in which the deactivation is much faster, is still unclear. In this study the conditions under which accumulation occurs in canine ventricular myocytes were studied with regard to its physiological relevance in controlling action potential duration (APD). At baseline, square pulse voltage clamp experiments revealed that the accumulation of canine IKs could occur, but only at rather short interpulse intervals (< 100 ms). With action potential (AP) clamp commands of constant duration (originally recorded at rate of 2 Hz), an accumulation was only found at interpulse intervals close to 0 ms. Transmembrane potential recordings with high‐resistance microelectrodes revealed, however, that at the fastest stimulation rates with normally captured APs (5 Hz) the interpulse interval exceeded 50 ms. This suggested that no IKs accumulation occurs, which was supported by the lack of effect of an IKs blocker, HMR 1556 (500 nM), on APD. In the presence of the β‐adrenergic receptor agonist isoproterenol (isoprenaline, 100 nM) the accumulation with AP clamp commands of constant duration was much more pronounced and a significant accumulating current was found at a relevant interpulse interval of 100 ms. HMR 1556 prolonged APD, but this lengthening was reverse rate dependent. AP clamp experiments in a physiologically relevant setting (short, high rate APs delivered at a corresponding rate) revealed a limited accumulation of IKs in the presence of isoproterenol. In conclusion, a physiologically relevant accumulation of IKs was only observed in the presence of isoproterenol. Block of IKs, however, led to a reverse rate‐dependent prolongation of APD indicating that IKs does not have a dominant role at short cycle lengths.

Dominant-Negative Control of cAMP-Dependent IKs Upregulation in Human Long-QT Syndrome Type 1

Rationale: The mutation A341V in the S6 transmembrane segment of KCNQ1, the &agr;-subunit of the slowly activating delayed-rectifier K+ (IKs) channel, predisposes to a severe long-QT1 syndrome with sympathetic-triggered ventricular tachyarrhythmias and sudden cardiac death. Objective: Several genetic risk modifiers have been identified in A341V patients, but the molecular mechanisms underlying the pronounced repolarization phenotype, particularly during &bgr;-adrenergic receptor stimulation, remain unclear. We aimed to elucidate these mechanisms and provide new insights into control of cAMP-dependent modulation of IKs. Methods and Results: We characterized the effects of A341V on the IKs macromolecular channel complex in transfected Chinese hamster ovary cells and found a dominant-negative suppression of cAMP-dependent Yotiao-mediated IKs upregulation on top of a dominant-negative reduction in basal current. Phosphomimetic substitution of the N-terminal position S27 with aspartic acid rescued this loss of upregulation. Western blot analysis showed reduced phosphorylation of KCNQ1 at S27, even for heterozygous A341V, suggesting that phosphorylation defects in some (mutant) KCNQ1 subunits can completely suppress IKs upregulation. Functional analyses of heterozygous KCNQ1 WT:G589D and heterozygous KCNQ1 WT:S27A, a phosphorylation-inert substitution, also showed such suppression. Immunoprecipitation of Yotiao with KCNQ1-A341V (in the presence of KCNE1) was not different from wild-type. Conclusions: Our results indicate the involvement of the KCNQ1-S6 region at/or around A341 in cAMP-dependent stimulation of IKs, a process that is under strong dominant-negative control, suggesting that tetrameric KCNQ1 phosphorylation is required. Specific long-QT1 mutations, including heterozygous A341V, disable this regulation.

End–diastolic myofiber stress and ejection strain increase with ventricular volume overload

AbstractBackgroundMyocardial stress and strain are considered primary mechanical stimuli for hypertrophic remodeling. Their values and signifi– cance in the intact beating heart during chronic overload remain poorly characterized.Methods and resultsLeft–ventricular (LV) dimensions (echocardiography) and pressure (invasive) were simultaneously recorded in anesthetized dogs at sinus rhythm (SR), acute and 1, 2, 6, 12 weeks of atrioventricular block (AVB), leading to structural, electrical and contractile remodeling. Mechanical load of the myocardium was quantified as myofiber stress (σf), being force along myofiber orientation per cross–sectional area, and natural myofiber strain (ef), being change in natural logarithm of myofiber length (l) divided by its reference length: ef = ln(l/lref). Time courses of σf and ef were calculated from LV pressure and dimensions, using a validated mathematical model of cardiac mechanics. End–diastolic σf increased from 2.0 ± 0.1 kPa at SR to 3.4 ± 0.3 kPa at acute AVB, remaining elevated for > 6 weeks. Systolic σf was not affected by AVB. Ejection strain rose instantly upon AVB, reaching a maximum at 2 weeks: 0.24 ± 0.02 vs. 0.10 ± 0.01 at SR. The increase of myofiber stroke work (σf–ef loop area) from 3.1 ± 0.3 at SR to 6.0 ± 0.5 kJ/m3/beat at 1 week AVB was attributed mainly to an increase of strain during ejection. Stroke work and ejection strain remained elevated up to 12 weeks. The rate of LV–mass increase was maximal (2.2 ± 0.4 g/day) at 1 week AVB.ConclusionsSerial mechanical phenotyping is feasible in the intact anesthetized dog with chronic ventricular overload. Our new approach yields values of mechanical load that are comparable to those found in isolated myocardium by others. In chronic AVB, both end–diastolic myofiber stress and ejection strain are increased. Early increases of both parameters coincide with peak hypertrophic growth, suggesting their important role for mechanotransduction. Peak systolic σf is likely not important for hypertrophy in this model, since it does not change throughout the experiment.