I(Kr) contributes to the altered ventricular repolarization that determines long-term cardiac memory.

OBJECTIVE: Cardiac memory (CM) is characterized by an altered T-wave morphology, which reflects altered repolarization gradients. We hypothesized that the delayed rectifier currents, I(Kr) and I(Ks), might contribute to these repolarization changes. METHODS: We studied conscious, chronically instrumented dogs paced from the postero-lateral left ventricular (LV) wall at rates 5-10% faster than sinus rate for 3 weeks. ECGs during sinus rhythm were recorded on days 0, 7, 14 and 21 of pacing. Within 3 weeks, CM achieved steady state, hearts were excised, and epicardial and endocardial tissues and myocytes were studied. RESULTS: In unpaced controls, action potential duration to 50% and 90% repolarization (APD) in epicardium was shorter than in endocardium (P < 0.05); in CM epicardial APD increased at CL > or = 500 ms, while endocardial APD was either unchanged or decreased such that the transmural gradient seen in controls diminished (P < 0.05). A transmural I(Kr) gradient occurred in controls (epicardium>endocardium, P < 0.05) and was reversed in CM. No I(Ks) transmural gradient was found in controls, while in CM endocardial I(Ks) was greater than epicardial at greater than +50 mV. Canine ERG (cERG) mRNA and protein in epicardium > endocardium in controls (P < 0.05), and this difference was lost in CM. Expression levels of KCNQ1 and KCNE1 protein were similar in all groups. CONCLUSIONS: A transcriptionally induced change in epicardial I(Kr) contributes to the altered ventricular repolarization that characterizes CM.

The cAMP response element binding protein modulates expression of the transient outward current: implications for cardiac memory.

OBJECTIVE: Long-term cardiac memory (LTCM), expressed as a specific pattern of T-wave change on ECG, is associated with 1) reduced transient outward potassium current (I(to)), 2) reduced mRNA for the pore-forming protein of I(to), Kv4.3, 3) reduced cAMP response element binding protein (CREB), and 4) diminished binding to its docking site on the DNA, the cAMP response element (CRE). We hypothesized a causal link between the decrease of the transcription factor CREB and down-regulation of I(to) and one of its channel subunits, KChIP2, in LTCM. METHODS: After three weeks of left ventricular pacing to induce LTCM (8 paced, 7 sham control dogs), epicardial KChIP2 mRNA and protein levels were assessed by real-time PCR and Western blotting. Mimicking the CREB down-regulation in LTCM, CREB was knocked down in situ in other dogs using adenoviral anti-sense. Effects on the action potential notch, reflecting I(to), were investigated in situ using monophasic action potential (MAP) recordings and at the cellular level by the whole-cell patch clamp technique. CREB binding in the KChIP2 promoter region was ascertained by electrophoretic mobility-shift assays. RESULTS: In LTCM, epicardial KChIP2 mRNA and protein were reduced by 62% and 76%, respectively, compared to shams (p < 0.05). CREB binding by the canine KChIP2 promoter region was demonstrated. CREB knockdown led to disappearance of the phase1 notch in MAP and ablation of I(to). CONCLUSIONS: These results strengthen the hypothesis that down-regulation of CREB-mediated transcription underlies the attenuation of epicardial I(to) in LTCM. They also emphasize that ventricular pacing exerts effects at a subcellular level contributing to memory and conceivably to other forms of cardiac remodeling.

Heterogeneous ventricular repolarization provides a substrate for arrhythmias in a German shepherd model of spontaneous arrhythmic death.

BACKGROUND: German shepherd dogs with inherited arrhythmias and sudden death appear to be a model for catecholamine-dependent ventricular tachycardias in human subjects. We tested the hypothesis that heterogeneity of left ventricular repolarization creates an arrhythmogenic substrate for pause-dependent ventricular tachycardia in these animals. METHODS AND RESULTS: We used microelectrode techniques to record action potentials (AP) from midmyocardial sections of anteroseptal, anterobasal, and posterobasal left ventricular (LV) wall of unafflicted and afflicted dogs. There were no differences in AP duration to 90% repolarization (APD) among LV regions in unafflicted dogs. In contrast, in afflicted dogs, there was significant heterogeneity, with the longest APD in anterobasal and shortest in anteroseptal regions. Isoproterenol did not affect repolarization in unafflicted dogs, whereas in afflicted dogs, it shortened APD anterobasally and prolonged APD anteroseptally. We studied the repolarizing currents, IKr and IKs, in single anteroseptal and anterobasal LV myocytes with the use of a whole-cell voltage clamp. There were no differences in IKr and IKs between anteroseptal and anterobasal regions in unafflicted dogs, whereas in afflicted dogs, IKr was smaller anterobasally (P<0.05). Isoproterenol produced a more prominent leftward shift in IKs voltage-dependent activation in anterobasal regions of afflicted than unafflicted dogs. CONCLUSIONS: Spatial heterogeneity in expression and catecholamine responsiveness of IKr and IKs results in heterogeneous LV repolarization in afflicted German shepherd dogs, contributing importantly to the arrhythmogenic substrate.

Mechanisms of alpha-adrenergic potentiation of ventricular arrhythmias in dogs with inherited arrhythmic sudden death.

OBJECTIVE: In German shepherd dogs having inherited arrhythmias and sudden death, pause-dependent arrhythmias are triggered by early afterdepolarizations (EADs) originating from left ventricular (LV) Purkinje fibers (PF). Heterogeneity of LV repolarization provides the arrhythmogenic substrate. To elucidate the mechanisms whereby alpha-adrenergic stimulation exacerbates these arrhythmias we tested the effects of phenylephrine on both arrhythmogenic trigger and substrate. METHODS AND RESULTS: We used microelectrode techniques to record action potentials from LV and right ventricular (RV) PF and from midmyocardial sections of anteroseptal, anterobasal and posterobasal LV wall of unafflicted and afflicted dogs. EADs occurred spontaneously in 8 of 12 LV PF and in no RV PF from afflicted dogs and in no PF from unafflicted dogs. In LV PF from afflicted dogs, phenylephrine (10(-9)-10(-5) M) concentration-dependently decreased membrane potential, induced abnormal automaticity at membrane potentials from -65 to -45 mV in 6 LV PF and potentiated EADs in another 6. To determine the mechanisms of membrane depolarization we studied phenylephrine effects on IK1 in voltage-clamped single LV and RV PF cells from afflicted dogs. In LV PF, phenylephrine (10(-5) M) reduced IK1 over the range of -120 to -40 mV and had no effects on RV PF. Regional heterogeneity of LV repolarization was observed in afflicted dogs only. Phenylephrine had no effects on repolarization in either group. CONCLUSION(S): Alpha-adrenergic stimulation exacerbates arrhythmias in afflicted dogs by increasing the arrhythmogenic trigger while leaving the substrate unchanged. Decrease in IK1 contributes importantly to alpha-adrenergic effects on LV PF.

Developmental changes in IKr and IKs contribute to age-related expression of dofetilide effects on repolarization and proarrhythmia.

OBJECTIVE: Clinical and experimental studies suggest that immature hearts are as or more sensitive than adult hearts to adverse effects of I(Kr) blocking drugs. We hypothesized that age-dependent changes in I(Kr) and I(Ks) contribute to the different repolarization reserves and proarrhythmic effects of I(Kr) blockers in the young and adult heart. METHODS: Dogs aged 1-150 days and adults were used to study (1) proarrhythmic effects in situ of the I(Kr) blocker dofetilide; (2) dofetilide effects on action potential duration (APD) recorded with microelectrodes from left ventricular (LV) slabs; (3) I(Kr) and I(Ks) in single LV myocytes using whole-cell voltage clamp. RESULTS: In situ, dofetilide-induced proarrhythmia occurred in 40% of adults, 86% of young (20-150 day) dogs and 0% of neonatal (1-19 day) dogs (P<0.05). Isolated tissue experiments showed no transmural gradient for repolarization from neonate through 3 months of age, after which the gradient increased through adulthood. In the presence of dofetilide, the greatest APD prolongation occurred in neonates. Yet, transmural dispersion did not increase in neonates but significantly increased in young and adults. Dofetilide-induced early after depolarization (EAD) incidence was 23% in adults, 59% in young and 8% in neonates (P<0.05). I(Kr) but not I(Ks) was expressed at <30 days, whereas both currents were present in adult myocardium. CONCLUSIONS: Our data suggest that a lack of I(Ks) results in a greater dependence on I(Kr) for repolarization in neonates and is associated with exaggerated effects of I(Kr)-blockade on APD. However, APD prolongation alone is insufficient for expression of proarrhythmia, which also requires transmural dispersion of repolarization and EADs. The extent to which APD prolongation, transmural dispersion and EADs are manifested at various ages in the absence and presence of I(Kr) blocking drugs appears to be the ultimate determinant of proarrhythmia.

Developmental evolution of the delayed rectifier current IKs in canine heart appears dependent on the beta subunit minK.

OBJECTIVES: We tested the hypothesis that the developmental changes occurring in I(Kr) and I(Ks) can be explained by changes in the expression of ERG encoding I(Kr), and KCNQ1, the beta subunit minK, and the recently reported subunit FHL2 encoding I(Ks). BACKGROUND: The delayed rectifier current contributes importantly to the developmental evolution of the canine myocardial action potential. Specifically, in left ventricular epicardial myocytes, I(Ks) is absent and I(Kr) is the major repolarizing current until age 4 weeks. With subsequent development, I(Ks) density increases and I(Kr) decreases, resulting in an altered voltage-time course of repolarization. METHODS: We used Western blotting and real-time polymerase chain reaction to compare the expression of ERG, KCNQ1, minK, and FHL2 in 1-week-old pups and adult dogs. RESULTS: ERG levels are high at 1 week and decrease significantly with age, consistent with developmental decrease in I(Kr). Whereas expression of KCNQ1 and FHL2 is unchanged between the two age groups, minK is minimally expressed at 1 week and increases in adults, consistent with developmental increase in I(Ks). CONCLUSIONS: A reduction in ERG explains the developmental decrease in I(Kr), whereas the accessory subunit minK appears to be the critical determinant of developmental evolution of I(Ks).