Ravil Z. Gainullin

Impact of Sex and Gonadal Steroids on Prolongation of Ventricular Repolarization and Arrhythmias Induced by IK-Blocking Drugs

BackgroundMechanisms for longer rate-corrected QT intervals and higher incidences of drug-induced torsade de pointes in women than in men are incompletely defined, although gonadal steroids are assumed to be important determinants of these differences. Methods and ResultsWe used microelectrode techniques to study isolated rabbit right ventricular endocardium from control male and female and castrated male (ORCH) and female (OVX) rabbits. Action potential duration to 30% repolarization (APD30) was significantly shorter in male than female and in ORCH than OVX at a cycle length of 500 ms. The IKs blocker chromanol 293B had no effect on APD in males or females. The IKr blocker dofetilide prolonged APD in female and ORCH more than in male and OVX. At 10−6 mol/L dofetilide (cycle length=1 second), the incidence of early afterdepolarizations was: female, 67%; ORCH, 56%; male, 40%; and OVX, 28%. Serum 17&bgr;-estradiol levels were unrelated to the effects of dofetilide, but as testosterone levels increased, the dofetilide effect to increase APD diminished, as did early afterdepolarization incidence. ConclusionsSex-related differences in basal right ventricular endocardial AP configuration persist in castrated rabbits, suggesting that extragonadal factors contribute to the differences in ventricular repolarization. In this model, drugs that block IKr but not IKs prolong repolarization in a way that suggests that protection from excess prolongation in males is attributable to testosterone, whereas the risk of excess prolongation of repolarization in females is related to sex-determined factors in addition to estrogen.

Cellular electrophysiologic properties of old canine atria provide a substrate for arrhythmogenesis

OBJECTIVE The incidence of atrial fibrillation increases with age. We hypothesized that aging-associated changes in the atrial action potential (AP) and conduction velocity provide a substrate for abnormal conduction and arrhythmogenesis. METHODS We used microelectrode techniques to record AP from the endocardium of the right atrial wall of dogs aged 1-5 (adult) and >8 years (old). Conduction velocity was measured between two microelectrodes 3-10 mm apart. Histological study was carried out to assess fibrosis. RESULTS Whereas resting potential, AP amplitude and V(max) did not differ with age, the plateau was more negative and AP duration was longer in old tissue. The L-type calcium current (I(Ca,L)) agonist Bay K8644 (10(-8)-10(-6) mol/l) elevated the plateau and shortened APD more in old than in adult, such that AP contour in old atria approached that of adult. In contrast, the I(Ca,L) blocker nisoldipine (10(-8)-10(-5) mol/l) depressed the plateau in adult and had no effect in old. There was no difference between the two groups in conduction velocity of normal beats, whereas for early premature impulses, reduced conduction velocity and a wider time window manifesting slow conduction were detected in old in comparison to adult tissue. A twofold increase in the amount of fibrous tissue was detected in old atria. CONCLUSIONS Our data show significant differences in contour of AP in adult and old atria. The responses to Bay K8644 and nisoldipine suggest a decreased I(Ca,L) in old atrial tissue. The alterations in AP contour and increased fibrosis may be responsible for slower conduction of early premature beats in old atria. The age-related changes in conduction of premature beats are consistent with those observed in patients with paroxysmal atrial fibrillation and may contribute to the greater propensity to atrial fibrillation in the aged.

Role of L-Type Calcium Channels in Pacing-Induced Short-Term and Long-Term Cardiac Memory in Canine Heart

Background—We tested the hypothesis that ICa,L is important to the development of cardiac memory. Methods and Results—The effects of L-type Ca2+ channel blockade and &bgr;-blockade were tested on acutely anesthetized and on chronically instrumented, conscious dogs. Short-term memory (STM) was induced by 2 hours of ventricular pacing and long-term memory (LTM) by ventricular pacing for 21 days. STM dogs received placebo, nifedipine, or propranolol, and LTM dogs received placebo, atenolol, or amlodipine. AT1 receptor blockade (candesartan) and ACE inhibition (trandolapril) were also tested in LTM. Microelectrodes were used to record transmembrane potentials from isolated epicardial and endocardial slabs using a protocol simulating STM in intact animals. Left ventricular epicardial myocytes from LTM or sham control dogs were dissociated, and ICa,L was recorded (whole-cell patch-clamp technique). Evolution of STM and LTM was attenuated by ICa,L blockers but not &bgr;-blockers. Neither AT1 receptor blockade nor ACE inhibition suppressed LTM. In microelectrode experiments, pacing induced an epicardial-endocardial gradient change mimicking STM that was suppressed by nifedipine. In patch-clamp experiments, peak ICa,L density in LTM and control were equivalent, but activation was more positive and time constants of inactivation longer in LTM (P <0.05). Conclusions—ICa,L blockade but not &bgr;-adrenergic blockade suppresses cardiac memory. LTM evolution is unaffected by angiotensin II blockade and is associated with altered ICa,L kinetics.

Cardiac Memory Is Associated With Decreased Levels of the Transcriptional Factor CREB Modulated by Angiotensin II and Calcium

Abstract— Cardiac memory (CM) has short- (STCM) and long-term (LTCM) components modulated by calcium and angiotensin II. LTCM is associated with reduced Ito and Kv4.3 mRNA levels. Because the cAMP response element binding protein, CREB, contributes to CNS memory transcription, we hypothesized that it might be a transcriptional factor in CM, influenced by calcium and angiotensin II. We studied STCM in dogs that were AV sequentially paced (AVP) for 2 hours or sham-operated. STCM was evaluated with ECG and vectorcardiogram (VCG), and subepicardial biopsies were taken at 5 to 120 minutes and investigated for CREB. LTCM was studied in dogs paced for 3 weeks and in sham controls. At 3 weeks the heart was excised, biopsies obtained, and CRE binding tested. STCM induction occurred in AVP dogs but not in sham or AVP dogs treated with saralasin or nifedipine. Nuclear CREB was significantly decreased at 2 hours in the AVP no-drug group only. LTCM dogs manifested reduced binding of nuclear proteins to CRE, and CRE binding activity in the promoter region of Kv4.3. In conclusion, there is an association between STCM induction and decreased nuclear CREB that is angiotensin-modulated and calcium-dependent. Moreover, the decreased CRE binding after 3 weeks of AVP combined with CRE binding activity in the Kv4.3 promoter can explain the Kv4.3 mRNA and Ito downregulation that characterize LTCM.

Cardiac Memory Evolves With Age in Association With Development of the Transient Outward Current

Background—Calcium-insensitive transient outward current (Ito) is important to the development of cardiac memory (CM), which itself reflects the capacity of the heart to remodel electrophysiologically. We used cardiac pacing to test the hypothesis that CM evolution can be explained by developmental maturation of Ito. Methods and Results—Acutely anesthetized dogs from 1 day old to adult were paced from the left ventricle (VP, n=29) or left atrial appendage (AP, n=12) to induce CM. T-wave vector displacement (TVD) obtained during VP was greater than with AP (adults, 0.39±0.06 mV; neonates, 0.04±0.01 mV; P<0.05). TVD began to increase at ≈40 days of age, reaching adult levels by ≈200 days. Microelectrode studies performed in 18 dogs (ages 3 to 94 days) after completing the CM protocol and 20 additional dogs (1 day old to adult) revealed that the epicardial action potential notch was absent in neonates, became apparent in the young, and was deepest in adults. The relationship between TVD and epicardial notch was such that as notch magnitude increased, TVD increased (r=−0.65, P<0.05). KChIP2 and Kv4.3 mRNA (measured via reverse transcription–polymerase chain reaction) also increased with age. Conclusions—The inducibility of CM gradually increases with age in association with evolution of the epicardial action potential notch and mRNA expression for KChIP2 and Kv4.3. This suggests that the capacity of the heart to remodel electrophysiologically and to manifest memory during development depends in part on evolution of the determinants of Ito.

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.

Interactions between antiarrhythmic drugs and cardiac memory.

OBJECTIVE Ventricular pacing or arrhythmias can induce cardiac memory (CM). We hypothesized that clinically administered antiarrhythmic drugs alter the expression of CM, and that the repolarization changes characteristic of CM can modulate the effects of antiarrhythmic drugs. METHODS We studied conscious, chronically-instrumented dogs paced for two 1-h periods to study the effects of drugs on the evolution of memory (protocol 1) or for 21 days (protocol 2) to observe the effects of steady-state memory on drug actions. Dogs were treated in both settings with quinidine, lidocaine or E4031, in random order, and within therapeutic serum concentration ranges. RESULTS Pacing, alone, for 2 h significantly prolonged ERP only near the left ventricular pacing site, whereas pacing alone for 21 days prolonged ERP at all sites (P<0.05). Quinidine and E4031, but not lidocaine, prolonged repolarization and ERP and suppressed evolution of CM in protocol 1. However, quinidines effect in prolonging repolarization was diminished in both protocols, while its effect in prolonging ERP was diminished in the 21-day protocol only. In contrast, the effects of E4031 were additive to those of CM, prolonging repolarization and ERP in both protocols, while lidocaine showed no changes in effect at all. CONCLUSIONS Pacing to induce CM significantly affects ventricular repolarization and refractoriness, and there are interactions between CM, quinidine and E4031. Depending on the specific drug, these interactions have the potential to be anti- or proarrhythmic, and may impact importantly on the clinical efficacy of drugs as well as on electrophysiologic testing of drug actions.

β1 and β2-adrenergic receptor subtype effects in German shepherd dogs with inherited lethal ventricular arrhythmias

Objective: Delayed afterdepolarization-induced triggered activity originating in ventricular myocardium is a mechanism for some age-dependent, inherited ventricular tachycardias in a colony of German shepherd dogs. Methods: We used standard microelectrode techniques to study β-adrenergic receptor subtype modulation of the triggered activity in anteroseptal left ventricular myocardium from eleven of these dogs and seven unafflicted, age-matched German shepherd controls. Results : During sustained stimulation at cycle lengths of 300–4000 ms, 10−9–10−7 M isoproterenol concentration-dependently shortened action potential duration (APD) to 90% repolarization more in myocardium from afflicted than from unafflicted dogs. This shortening was prevented by a β1-blocker CGP20712A (10−7 M) while a β2-blocker ICI118551 (10−7 M) did not modify the effect of isoproterenol in either group. The β2-agonist zinterol 10−8–10−6 M had no effect on APD. Stimulation at a cycle length of 250 ms in the presence of 10−7 M isoproterenol induced more triggered AP in myocardium from afflicted than unafflicted dogs. β1-Blockade completely eliminated, while β2-blockade facilitated, and the β2-agonist zinterol did not induce triggered activity in the two groups. Conclusion : Isoproterenol effects on APD and triggered activity in the myocardium of dogs with inherited arrhythmias are due primarily to an abnormality of β1-adrenoceptor mediated signaling that is subject to β2-adrenergic modulation.

Effects of mibefradil, a T-type calcium current antagonist, on electrophysiology of Purkinje fibers that survived in the infarcted canine heart.

Mibefradils Effects on Canine Purkinje Myocytes. Introduction: We studied the effects of mibefradil (MIB), a nondihydropyridine T‐type Ca2+ channel antagonist, on T‐ and L‐type Ca2+ (ICaT, ICaL) currents in Purkinje myocytes dispersed from the subendocardium of the left ventricle of normal (NZPC) and 48‐hour infarcted (IZPC) hearts.

Long-term electrophysiological effects of regional cardiac sympathetic denervation of the neonatal dog

OBJECTIVE In many cardiac arrhythmias, both a triggering factor and a favorable myocardial substrate are required. Whereas the sympathetic nervous system may trigger tachyarrhythmias, its function as a long-term modulator of the myocardial substrate is less well understood. Therefore, we tested the hypothesis that regional sympathetic denervation at birth would produce an abnormal myocardial substrate. The comparator was the substrate associated with inherited, lethal tachyarrhythmias at 5 months of age in German shepherd dogs with incomplete sympathetic innervation. METHODS Mongrel dogs underwent right cardiac stellectomy (RSX) within the first day of life and were terminally studied with control littermates at 5 months of age. RESULTS On days 1-21 of life, RSX animals manifested significant QT prolongation on ECG and sudden, asystolic death. Beyond this age, QT intervals normalized and deaths did not occur. At 5 months, action potentials (AP) were recorded from Purkinje fibers (PF) and midmyocardial preparations in anteroseptal (AS) and posterobasal (PB) left ventricle. Early afterdepolarizations occurred only in left ventricular PF from RSX dogs. Isoproterenol prolonged AP duration in AS and shortened it in PB of RSX but not control dogs. The incidence of isoproterenol-initiated triggered activity and the amplitude of delayed afterdepolarizations were greater in RSX than control dogs. CONCLUSION Five months after RSX heterogeneous alterations of LV electrophysiological properties were similar to those previously observed in animals having inherited deficits in sympathetic innervation and sudden death. This implicates the sympathetic nerves as long-term modulators of an arrhythmogenic substrate. That 5-month-old RSX dogs did not experience tachyarrhythmias or sudden death indicates that further anomalies--beyond those explicable by the substrate change--must exist to induce sudden death.