Electrophysiological and antiarrhythmic effects of the atrial selective 5-HT(4) receptor antagonist RS-100302 in experimental atrial flutter and fibrillation.

BACKGROUND: Stimulation of 5-HT(4) receptors increases atrial chronotropic and inotropic responses. Whether other electrophysiological effects are produced is unknown. In humans and swine, 5-HT(4) receptors are present only in atrium. Therefore, the effects of a novel 5-HT(4) receptor antagonist, RS-100302, and the partial agonist cisapride on atrial flutter and fibrillation induced in swine were studied to delineate the role of the 5-HT(4) receptor in modulating atrial electrophysiological properties and the antiarrhythmic potential of RS-100302. METHODS AND RESULTS: In 17 anesthetized, open-chest, juvenile pigs, atrial flutter or fibrillation was induced by rapid right atrial pacing with or without a right atrial free wall crush injury, respectively. Atrial effective refractory period (ERP), conduction velocity, wavelength, and dispersion of refractoriness were determined during programmed stimulation via a 56-electrode mapping plaque sutured to the right atrial free wall. Ventricular electrophysiological parameters were also measured. All electrophysiological parameters were measured at baseline and after infusion of RS-100302 and cisapride. In the atrium, RS-100302 prolonged mean ERP (115+/-8 versus 146+/-7 ms, P<0.01) and wavelength (8.3+/-0.9 versus 9.9+/-0.8 cm, P<0.01), reduced dispersion of ERP (15+/-5 versus 8+/-1 ms, P<0.01), and minimally slowed conduction velocity (72+/-4 versus 67+/-5 cm/s, P<0.01). These effects were all partially reversed by cisapride. RS-100302 produced no ventricular electrophysiological effects. RS-100302 terminated atrial flutter in 6 of 8 animals and atrial fibrillation in 8 of 9 animals and prevented reinduction of sustained tachycardia in all animals. CONCLUSIONS: The electrophysiological profile of RS-100302 suggests that it may have atrial antiarrhythmic potential without producing ventricular proarrhythmic effects.

Role of angiotensin in pressure overload-induced hypertrophy in rats: effects of angiotensin-converting enzyme inhibitors, an AT1 receptor antagonist, and surgical reversal.

The renin-angiotensin system (RAS) has been proposed to play a major role in causing the heart to hypertrophy during pressure overload. We examined whether blockade of this system by the angiotensin-converting enzyme (ACE) inhibitors enalapril (0.5 to 20 mg/kg p.o.) or ramipril (1.0 mg/kg p.o.) or the angiotensin receptor (AT1) antagonist losartan (3.0 mg/kg p.o.) could prevent pressure overload-induced hypertrophy. Pressure overload was produced by abdominal aortic constriction in rats. Cardiac hypertrophy was assessed by an increase in the ratio of left ventricular (LV) weight to body weight and total protein content of the left ventricle. Treatment with enalapril or ramipril, initiated 3 weeks after aortic banding and continued for 3 more weeks, failed to prevent the progression or cause regression of cardiac hypertrophy. Treatment for 6 weeks with ramipril initiated immediately after aortic banding also failed to prevent cardiac hypertrophy. Losartan treatment initiated 3 weeks after aortic banding and continued for 3 more weeks resulted in a slight but significant reduction in the extent of cardiac hypertrophy (45.6% hypertrophy in controls and 35.6% hypertrophy in losartan-treated animals, p < 0.05, n = 11 and 10, respectively). Surgical removal of bands 3 weeks after placement reduced cardiac hypertrophy to a greater extent than that observed in losartan-treated animals. These results suggest that angiotensin may not play a major role in causing pressure overload-induced hypertrophy or in maintaining such hypertrophy.

Ischaemia induced alternans of action potential duration in the intact-heart: dependence on coronary flow, preload and cycle length.

Clinical and experimental evidence relate action potential duration (APD) alternans to ischaemic heart disease and ventricular arrhythmias. The present investigation was performed to study the quantitative relationship between APD alternans and the degree of ischaemia, loading conditions and cycle length (CL) in an intact heart. Monophasic action potentials (MAP) were simultaneously recorded by contact electrodes from two left (LV) and one right ventricular (RV) sites in 20 Langendorff-perfused rabbit hearts. The preparations were subjected to global ischaemia at flow rates ranging from 40% of normal flow to complete cessation of flow. Pacing was performed at either constant or regularly changing CL. The magnitude of APD alternans was expressed as beat-to-beat differences in action potential duration of two consecutive MAPs. During normal perfusion, neither very fast pacing at a CL of 200 ms nor periodical rate switches resulted in persistent APD alternans. Pacing at a constant CL of 800 ms did not induce APD alternans at complete cessation of flow for 6 min. However, alternans developed progressively at a constant CL of 400 ms after 2.8 +/- 0.3 min of complete ischaemia at the pre-loaded LV, and after 4.6 +/- 0.4 min at the unloaded RV (P < 0.01). The reduction of preload at the LV from 15 to 5 mmHg end-diastolic pressure delayed development of APD alternans from 2.8 +/- 03 min to 4.3 +/- 0.4 min (P < 0.05) at 400 ms CL. Following graded underperfusion of 40%, 20% and 10% of initial flow, persistent APD alternans developed in relation to the degree of flow reduction and increased progressively with duration of ischaemia. APD alternans at the LV always preceded the onset of APD alternans at the RV. In experiments with identical flow rates the shortest CL of 200 ms resulted in the greatest and earliest initiation of APD alternans compared to the longer CL (P < 0.01, P < 0.001). An increase in CL from 400 to 800 ms immediately abolished APD alternans, generated by the shorter CL, at any time during the 6 min period of complete ischaemia. Similarly, increasing the cycle length from 200 or 400 to 600 ms eliminated APD alternans up to 6 min of ischaemia and significantly reduced its magnitude between 7 and 10 min within a few beats. We conclude that persistent APD alternans is a characteristic findings in the rabbit heart during global ischaemia4 It is a sensitive parameter of the severity of ischaemia and depends.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of ischemia and hypercarbic acidosis on myocyte calcium transients, contraction, and pHi in perfused rabbit hearts.

The time courses of changes in pHi and cytosolic calcium were compared in isolated perfused rabbit hearts with the use of the calcium-sensitive fluorescent indicator indo-1 and the pH indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Cell-permeant forms of these indicators were loaded into myocytes by arterial infusion or by direct infusion into the extravascular space. Indo-1 fluorescence was recorded from the epicardial surface of the left ventricle at an excitation wavelength of 360 nm and emission wavelengths of 400 and 550 nm. BCECF fluorescence was recorded at an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Calibration procedures were developed for each indicator that allowed [Ca2+]i and pHi to be quantified during ischemia. Global ischemia decreased contractility and caused a rapid increase in both the systolic and end-diastolic levels of the calcium transients. Ninety seconds of ischemia increased peak systolic [Ca2+]i from 609 +/- 29 to 1,341 +/- 159 nM, while end-diastolic [Ca2+]i increased from 315 +/- 25 to 553 +/- 52 nM. The observed increase in diastolic [Ca2+]i, was shown not to arise from indo-1-loaded endothelial cells. The initial increase in [Ca2+]i was followed by a gradual decline and then a secondary rise occurring between 5 and 15 minutes of ischemia. In contrast, ischemia caused a monotonic decrease in pHi from a baseline of 7.03 +/- 0.06 to 6.83 +/- 0.02 after 2 minutes, 6.32 +/- 0.1 after 10 minutes, and 6.11 +/- 0.04 after 15 minutes. Perfusion of hearts with acidified (hypercarbic) saline increased the systolic and diastolic levels of the calcium transients, but only when pHi fell below a threshold value, which was more acidic than values achieved during the first 2 minutes of ischemia (6.83 +/- 0.03). Lesser degrees of acidification caused a decrease in contractility but did not affect the calcium transients. Effects of pHi on the calcium transients were not due to altered calcium sensitivity of indo-1. These results suggest that cytosolic acidification may contribute to the increase in [Ca2+]i during the first 15 minutes of global ischemia, but the [Ca2+]i increase during the first 2 minutes is mediated by other factors.

Effects of verapamil and nifedipine on mechanisms of arrhythmia in an in vitro model of ischemia and reperfusion.

The effects of verapamil and nifedipine on cellular mechanisms of arrhythmia were examined in isolated canine Purkinje fiber-papillary muscles. Microelectrode recordings were made simultaneously from both tissues. Preparations were superfused with Tyrode's solution modified to mimic specific conditions of ischemia for 40 min with or without calcium channel blockers. Verapamil or nifedipine resulted in significantly greater depolarization of Purkinje tissue in response to ischemic conditions and increased the incidence of inexcitability or conduction block in Purkinje and muscle tissues. These calcium channel blockers caused only minor changes in ischemia-induced depolarization of muscle. In Purkinje tissue, return to nonischemic conditions in the absence of drugs caused, in sequence, oscillatory afterpotentials, temporary depolarization to inexcitability, and a phase of automaticity at low membrane potential. These events did not occur in muscle. Verapamil or nifedipine abolished oscillatory afterpotentials and low membrane potential automaticity in Purkinje tissue. However, reperfusion-induced depolarization and inexcitability of Purkinje tissue was delayed but not attenuated. This study demonstrates that verapamil or nifedipine exacerbate depolarization and depression of conduction in Purkinje tissue exposed to ischemic conditions. However, verapamil and nifedipine suppress some but not all potential mechanisms of arrhythmia induced by reperfusion.

Effect of platelet release products on cytosolic calcium in cardiac myocytes.

The effect of platelet release products on cytosolic calcium [( Ca++]i) was examined by monitoring the fluorescence of chick embryonic heart cells loaded with the fluorescent calcium indicator indo-1 AM. Cell free filtrate of platelet release products was obtained from rabbit platelets activated with thrombin or collagen. This filtrate caused a rapid increase in both systolic and diastolic [Ca++]i in a dose-dependent manner. The effect was not blocked by pretreating the platelets with aspirin or a thromboxane synthetase inhibitor. It was not mimicked by a thromboxane analog, or by several substances known to be released from platelets including ADP, serotonin, or platelet activating factor. Apyrase or ATP-gamma S had no effect on the activity. The responsible product was heat-sensitive, trypsin-sensitive, and partitioned into the aqueous phase of a chloroform suspension. It has a low molecular weight (less than 3kD) and is sensitive to 2-mercaptoethanol. Protease inhibitor appears to prolong the activity. These results suggest that trypsin-sensitive peptide(s) released from activated platelets can increase [Ca++]i in cardiac cells.

Effect of thrombin on calcium homeostasis in chick embryonic heart cells. Receptor-operated calcium entry with inositol trisphosphate and a pertussis toxin-sensitive G protein as second messengers.

UNLABELLED: Thrombin increases intracellular calcium ([Ca++]i) in several cell types and causes a positive inotropic effect in the heart. We examined the mechanism of the thrombin-induced [Ca++]i increase in chick embryonic heart cells loaded with the fluorescent calcium indicator, indo-1. Thrombin (1 U/ml) increased both systolic and diastolic [Ca++]i from 617 +/- 62 and 324 +/- 46 to 1041 +/- 93 and 587 +/- 38 nM, respectively. An initial rapid [Ca++]i increase was followed by a more sustained increase. There were associated increases in contraction strength, beat frequency, and action potential duration. The [Ca++]i increase was not blocked by tetrodotoxin or verapamil, but was blocked by pretreatment with pertussis toxin (100 ng/ml). The thrombin-induced [Ca++]i increase was partly due to intracellular calcium release, since it persisted after removal of external calcium. The [Ca++]i increase in zero calcium was more transitory than in normal calcium and was potentiated by 10 mM Li+. Thrombin also induced influx of calcium across the surface membrane, which could be monitored using Mn++ ions, which quench indo-1 fluorescence when they enter the cell. Thrombin-induced Mn++ entry was insensitive to verapamil, but was blocked by 2 mM Ni++. Thrombin increased inositol trisphosphates by 180% at 90 s and this effect was also blocked by pretreatment with pertussis toxin. CONCLUSION: thrombin promotes calcium entry and release in embryonic heart cells even when action potentials are inhibited. Both modes of [Ca++]i increase may be coupled to the receptor by pertussis toxin-sensitive G proteins.

Effects of ischemic conditions and reperfusion on depolarization-induced automaticity.

The inducibility of slow-response automaticity was assessed during ischemic conditions and reperfusion by application of extracellular current. Isolated canine Purkinje fibers were depolarized to membrane potentials less than -65 mV to elicit depolarization-induced automaticity (DIA). Ischemic conditions increased the cycle length of DIA and, in some tissues, prevented sustained DIA or completely abolished DIA. The magnitude of depolarization required to elicit DIA also increased. Inhibition of DIA occurred at a time when action potential plateaus were abbreviated. The effect of reperfusion on DIA was biphasic. Initial reappearance of DIA was followed by inhibition and reduction of the membrane potential range over which DIA could be elicited. Plateaus of action potentials initiated at high membrane potential were abbreviated at this time. DIA returned again as reperfusion effects dissipated. Phasic changes in the inducibility of DIA may represent changes in availability of the slow inward current and may regulate the timing and types of arrhythmic activity occurring with ischemia and reperfusion.

Effect of ischemia on calcium-dependent fluorescence transients in rabbit hearts containing indo 1. Correlation with monophasic action potentials and contraction.

The effects of acute global ischemia on cytosolic calcium transients were studied in perfused rabbit hearts loaded with the fluorescent calcium indicator indo 1. Indo 1-loaded hearts were illuminated at 360 nm, and fluorescence was recorded simultaneously at 400 and 550 nm from the epicardial surface of the left ventricle. The F400/F550 ratio was calculated by an analog circuit, which allowed cancellation of optical motion artifact. Resulting calcium transients demonstrated a rapid upstroke and slow decay similar to those recorded in isolated ventricular myocytes. Global ischemia rapidly suppressed contraction, but it produced a concurrent increase in the systolic and diastolic levels of the calcium transients, together with an increase in the duration of the peak. The effects of ischemia were reversed by reperfusion, inhibited by verapamil, and mimicked by perfusion of nonischemic hearts with acidified (CO2-rich) solution. In addition to elevation of the calcium transients, ischemia caused a pattern of intracellular calcium alternans that was discernible after 2-3 minutes. The pattern of alternans was stable at a given epicardial site, but it could be out of phase at different sites. Similar nonuniformities were observed in contraction strength and in the duration of monophasic action potentials recorded immediately adjacent to the fiber-optic probe. Abnormalities in intracellular calcium may be a causal factor in the loss of electrical and mechanical synchrony in the acutely ischemic heart.

Cytosolic calcium transients from the beating mammalian heart.

To elucidate the role of cytosolic calcium, [Ca2+]i, in the physiology of the normal and ischemic heart, we have developed a method for recording [Ca2+]i transients from the epicardial surface of the rabbit ventricle after arterial perfusion with the cell-permeant cytosolic calcium indicator indo-1 AM. Hearts were illuminated at 360 nm, and fluorescence was recorded simultaneously at 400 and 550 nm. The F400/F550 fluorescence ratio was calculated by an analog circuit that allowed cancelation of small movement artifacts that were present at single wavelengths. Clear [Ca2+]i transients were present in the F400/F550 signal and were remarkable for their slow decay. Slow decay of the transients was not due to buffering of [Ca2+]i by indo-1, since there was no associated impairment of contraction or relaxation. The peak amplitude of the [Ca2+]i transients was increased by ouabain, adrenaline, postextrasystolic potentiation, and acetylcholine. The extent to which the transients decayed diminished with shortening of the interbeat interval, but decay of the transients could be further diminished by acetylcholine or caffeine. A major advantage of the intact heart over isolated myocytes is the ability to measure changes in [Ca2+]i during ischemia. Ischemia produced a marked increase in both peak systolic and end-diastolic [Ca2+]i, which was most rapid during the first 30 sec, and approached a plateau value after 90 sec. This increase in [Ca2+]i was associated with a characteristic broadening of the peak of the transient. The increase in [Ca2+]i during ischemia is consistent with a proposed causative role of [Ca2+]i in mediating early electrophysiological abnormalities.