In contrast to forskolin and 3-isobutyl-1-methylxanthine, amrinone stimulates the cardiac voltage-sensitive release mechanism without increasing calcium-induced calcium release.

The objective of this study was to determine whether the voltage-sensitive release mechanism (VSRM) can be stimulated independently from Ca(2+)-induced Ca(2+) release (CICR) by drugs that elevate intracellular cAMP. Contractions were measured in voltage-clamped guinea pig ventricular myocytes at 37 degrees C. Na(+) current was blocked. We compared effects of agents that elevate cAMP through activation of adenylyl cyclase (1 microM forskolin), nonspecific inhibition of phosphodiesterases (PDEs) [100 microM 3-isobutyl-1-methylxanthine (IBMX)], and selective inhibition of PDE III (100-500 microM amrinone) on contractions initiated by the VSRM and CICR. Forskolin and IBMX significantly increased peak Ca(2+) current and CICR. In addition, these agents also markedly increased contractions elicited by test steps from -65 to -40 mV, which activate the VSRM. However, because these steps also induced inward current in the presence of forskolin or IBMX, CICR could not be excluded. In contrast, amrinone caused a large, concentration-dependent increase in VSRM contractions but had no effect on CICR contractions or Ca(2+) current. Sarcoplasmic reticulum Ca(2+), assessed by rapid application of caffeine (10 mM), was increased only modestly by all three drugs. Normalization of contractions to caffeine contractures indicated that amrinone increased fractional release by the VSRM, but not CICR. Forskolin and IBMX increased fractional release elicited by steps to -40 mV. Increases in CICR induced by forskolin and IBMX were proportional to caffeine contractures. Thus, positive inotropic effects of cAMP on VSRM contractions may be compartmentalized separately from effects on Ca(2+) current and CICR.

Cardiac excitation-contraction coupling: role of membrane potential in regulation of contraction.

The steps that couple depolarization of the cardiac cell membrane to initiation of contraction remain controversial. Depolarization triggers a rise in intracellular free Ca(2+) which activates contractile myofilaments. Most of this Ca(2+) is released from the sarcoplasmic reticulum (SR). Two fundamentally different mechanisms have been proposed for SR Ca(2+) release: Ca(2+)-induced Ca(2+) release (CICR) and a voltage-sensitive release mechanism (VSRM). Both mechanisms operate in the same cell and may contribute to contraction. CICR couples the release of SR Ca(2+) closely to the magnitude of the L-type Ca(2+) current. In contrast, the VSRM is graded by membrane potential rather than Ca(2+) current. The electrophysiological and pharmacological characteristics of the VSRM are strikingly different from CICR. Furthermore, the VSRM is strongly modulated by phosphorylation and provides a new regulatory mechanism for cardiac contraction. The VSRM is depressed in heart failure and may play an important role in contractile dysfunction. This review explores the operation and characteristics of the VSRM and CICR and discusses the impact of the VSRM on our understanding of cardiac excitation-contraction coupling.

Diverse lamb genotypes. 3. Eating quality and the relationship between its objective measurement and sensory assessment.

The effect of genotype on eating quality was evaluated on m. Longissimus thoracis et lumborum (LTL) muscle of 60 lambs. The lambs were sired by a selection of Texel (T), Poll Dorset (PD), Border Leicester (BL) and Merino (M) rams, crossed with Border Leicester x Merino (BLM) and Merino (M) ewes giving six genotypes (TxBLM, PDxBLM, TxM, PDxM, BLxM and MxM). The relationships between sensory panel assessment of eating quality attributes and pH, cooking loss and shear force were also investigated. No significant differences were observed between genotypes for panel assessment of tenderness, juiciness, aroma liking, aroma strength, flavour liking, overall acceptability and rating. MxM lambs had a significantly (P<0.05) higher flavour strength than BLxM lambs. pH was a poor indicator of any eating quality attributes, except aroma strength (r=0.3, P<0.05). Warner Bratzler shear force value (WB) and tenderness showed a significant (P<0.001) negative correlation (-0.7). Tenderness, flavour and juiciness were the most important sensory attributes, explaining 86.5% of the total variation in overall acceptability.

Regulation of a voltage-sensitive release mechanism by Ca(2+)-calmodulin-dependent kinase in cardiac myocytes.

A role for Ca(2+)-calmodulin-dependent kinase (CamK) in regulation of the voltage-sensitive release mechanism (VSRM) was investigated in guinea pig ventricular myocytes. Voltage clamp was used to separate the VSRM from Ca(2+)-induced Ca(2+) release (CICR). VSRM contractions and Ca(2+) transients were absent in cells dialyzed with standard pipette solution but present when 2-5 microM calmodulin was included. Effects of calmodulin were blocked by KN-62 (CamK inhibitor), but not H-89, a protein kinase A (PKA) inhibitor. Ca(2+) current and caffeine contractures were not affected by calmodulin. Transient-voltage relations were bell-shaped without calmodulin, but they were sigmoidal and typical of the VSRM with calmodulin. Contractions with calmodulin exhibited inactivation typical of the VSRM. These contractions were inhibited by rapid application of 200 microM of tetracaine, but not 100 microM of Cd(2+), whereas CICR was inhibited by Cd(2+) but not tetracaine. In undialyzed myocytes (high-resistance microelectrodes), KN-62 or H-89 each reduced amplitudes of VSRM contractions by approximately 50%, but together they decreased VSRM contractions by 93%. Thus VSRM is facilitated by CamK or PKA, and both pathways regulate the VSRM in undialyzed cells.

Losartan improves recovery of contraction and inhibits transient inward current in a cellular model of cardiac ischemia and reperfusion.

Losartan, a selective angiotensin II (AII) type I receptor antagonist, may protect against myocardial stunning and arrhythmia in ischemia and reperfusion. To examine the cellular basis for these protective actions, we studied effects of losartan and AII on contractile and electrical activity of ventricular myocytes exposed to simulated ischemia and reperfusion. Ionic currents were measured with voltage-clamp techniques and contractions were measured with a video edge detector. After 10 min of superfusion with Tyrode's solution at 37 degrees C, cells were exposed to simulated ischemia (hypoxia, acidosis, hyperkalemia, hypercapnia, lactate accumulation, and substrate deprivation) for 30 min followed by 25 min of reperfusion with normal Tyrode's solution. During ischemia, drug-treated cells were exposed to either 0.1 microM AII, 10 microM losartan, or both simultaneously. In reperfusion, contractions were depressed to 42% of preischemic levels in untreated cells. Losartan treatment significantly improved contractile recovery to 84% (P <. 05) of preischemic levels. AII-treated cells showed contractile recovery similar to untreated cells (40%), whereas cells treated with losartan plus AII recovered to 101% of preischemic levels. Cells exposed to losartan or losartan plus AII also exhibited reduced incidence of transient inward current (I(TI)) (20%, P <.05; 36%) relative to untreated cells (60%). However, I(TI) incidence was not altered by treatment with AII alone (57%). Treatment with exogenous agonist did not potentiate contractile depression or I(TI) incidence, and losartan exerted protective effects in the presence and absence of AII. Thus, losartan may have effects that are independent of AII receptor blockade.

Regulation of contraction and relaxation by membrane potential in cardiac ventricular myocytes.

Control of contraction and relaxation by membrane potential was investigated in voltage-clamped guinea pig ventricular myocytes at 37 degrees C. Depolarization initiated phasic contractions, followed by sustained contractions that relaxed with repolarization. Corresponding Ca(2+) transients were observed with fura 2. Sustained responses were ryanodine sensitive and exhibited sigmoidal activation and deactivation relations, with half-maximal voltages near -46 mV, which is characteristic of the voltage-sensitive release mechanism (VSRM) for sarcoplasmic reticulum Ca(2+). Inactivation was not detected. Sustained responses were insensitive to inactivation or block of L-type Ca(2+) current (I(Ca-L)). The voltage dependence of sustained responses was not affected by changes in intracellular or extracellular Na(+) concentration. Furthermore, sustained responses were not inhibited by 2 mM Ni(2+). Thus it is improbable that I(Ca-L) or Na(+)/Ca(2+) exchange generated these sustained responses. However, rapid application of 200 microM tetracaine, which blocks the VSRM, strongly inhibited sustained contractions. Our study indicates that the VSRM includes both a phasic inactivating and a sustained noninactivating component. The sustained component contributes both to initiation and relaxation of contraction.

Increased expression of the gene for alpha-interferon-inducible protein in cardiomyopathic hamster heart.

Cardiomyopathic (CM) hamsters have a disruption in the delta-sarcoglycan gene which leads to progressive cardiac necrosis by 30 to 40 days of age, hypertrophy by 120 days, and heart failure by 250 days. We used differential display to detect other changes in mRNA levels in 30-, 60-, and 90-day-old wild-type and CM hamsters. We identified a 400-bp cDNA with sequence similarity to the human alpha-interferon-inducible protein (p27). This cDNA annealed with a 570-base mRNA whose steady-state levels were increased in 30-, 60-, and 90-day-old CM compared to wild-type heart. Increased expression of this hamster homolog of p27 (p27-h) was detected in CM hamster cardiac and skeletal muscle at 60 days of age but not in liver, kidney, or brain. Thus, an inherited defect in CM hamsters leads to increased expression of p27-h in advance of the development of hypertrophy and heart failure.

Role of voltage-sensitive release mechanism in depression of cardiac contraction in myopathic hamsters.

We investigated excitation-contraction (EC) coupling in isolated ventricular myocytes from prehypertrophic cardiomyopathic (CM) hamster hearts. Conventional and voltage-clamp recordings were made with high-resistance microelectrodes, and cell shortening was measured with a video-edge detector at 37 degrees C. Contractions were depressed in myocytes from CM hearts, whether they were initiated by action potentials or voltage-clamp steps. As in guinea pig and rat, contraction in hamster myocytes could be triggered by a voltage-sensitive release mechanism (VSRM) or Ca(2+)-induced Ca(2+) release (CICR). Selective activation of these mechanisms demonstrated that the defect in EC coupling was primarily caused by a defect in the VSRM. However, activation and inactivation properties of the VSRM were not altered. When the VSRM was inhibited, the remaining contractions induced by CICR exhibited identical bell-shaped contraction voltage relations in normal and CM myocytes. Inward Ca(2+) current was unchanged. Thus a defect in the VSRM component of EC coupling precedes the development of hypertrophy and failure in CM hamster heart.

Tetracaine can inhibit contractions initiated by a voltage-sensitive release mechanism in guinea-pig ventricular myocytes.

1. Effects of tetracaine on membrane currents and cell shortening were measured with high resistance electrodes, single-electrode voltage clamp (switch clamp) and a video edge detector at 37 C in cardiac ventricular myocytes. 2. Sequential voltage steps from -65 mV to -40 and 0 mV were used to activate two mechanisms of excitation-contraction (EC) coupling separately. The step to -40 mV activated the voltage-sensitive release mechanism (VSRM); the step to 0 mV1 activated Ca2+-induced Ca2+ release (CICR) coupled to inward Ca2+ current (IL). 3. Exposure to 100-300 microM tetracaine inhibited VSRM contractions but not CICR contractions. Inhibition of VSRM contractions was independent of INa blockade. In contrast, 100 microM Cd2+ blocked IL and CICR contractions, but not VSRM contractions. Simultaneous application of both agents blocked both mechanisms of EC coupling. 4. Contraction-voltage relationships were sigmoidal when the VSRM was available. However, when the VSRM was inhibited with 100-300 microM tetracaine, contraction-voltage relationships became bell-shaped. The tetracaine-insensitive contractions were abolished by 0.1 microM ryanodine, indicating that they were dependent on release of SR Ca2+. 5. At a higher concentration (1 mM) tetracaine also inhibited IL and contractions triggered by IL; however, the time course of effects on IL and associated contractions were different than for VSRM contractions. 6. With continuous application of tetracaine, the VSRM remained inhibited although SR Ca2+ stores increased 4-fold as assessed with caffeine. CICR contractions were not inhibited and maximum amplitude of contraction was not reduced. 7. Rapid application of tetracaine just before and during test steps also inhibited VSRM contractions, but without significantly affecting sarcoplasmic reticulum (SR) Ca2+ stores or CICR contractions. Maximum amplitude of contraction was reduced. 8. Rapid application of tetracaine (100-300 microM) allows preferential inhibition of the VSRM and provides a pharmacological method to assess the contribution of the VSRM to EC coupling.

Role of cAMP-dependent protein kinase A in activation of a voltage-sensitive release mechanism for cardiac contraction in guinea-pig myocytes.

1. Ionic currents and unloaded cell shortening were recorded from guinea-pig ventricular myocytes with single electrode voltage clamp techniques and video edge detection at 37 C. Patch pipettes (1-3 MOmega) were used to provide intracellular dialysis with pipette solutions. 2. Na+ currents were blocked with 200 microM lidocaine. Contractions initiated by the voltage-sensitive release mechanism (VSRM) and Ca2+-induced Ca2+ release (CICR) in response to L-type Ca2+ current (ICa,L) were separated with voltage clamp protocols. 3. Without 8-bromo cyclic adenosine 3',5'-monophosphate (8-Br-cAMP) in the pipette, small VSRM-induced contractions occurred transiently in only 13% of myocytes. In contrast, large ICa,L-induced contractions were demonstrable in 100% of cells. 4. Addition of 10 or 50 microM 8-Br-cAMP to the pipette increased the percentage of cells exhibiting VSRM contractions to 68 and 93%, respectively. With 50 microM 8-Br-cAMP, contractions initiated by the VSRM and ICa,L were not significantly different in amplitude. 5. 8-Br-cAMP-supported VSRM contractions had characteristics of the VSRM shown previously in undialysed myocytes. Cd2+ (100 microM) blocked ICa,L and ICa,L contractions but not VSRM contractions. 8-Br-cAMP-supported contractions exhibited steady-state inactivation with parameters characteristic of the VSRM, as well as sigmoidal contraction-voltage relations. 6. Without 8-Br-cAMP in the pipette, contraction-voltage relations determined with steps from a post-conditioning potential (Vpc) of either -40 or -65 mV were bell shaped, with a threshold near -35 mV. With 50 microM 8-Br-cAMP in the pipette, contraction-voltage relations from a Vpc of -65 mV were sigmoidal and the threshold shifted to near -55 mV. Contraction-voltage relations remained bell shaped in the presence of 8-Br-cAMP when the Vpc was -40 mV. 7. H-89, which inhibits cAMP-dependent protein kinase A (PKA), significantly reduced the amplitudes of VSRM contractions by approximately 84% with 50 microM 8-Br-cAMP in the pipette. H-89 also significantly reduced the amplitudes of peak ICa, L and ICa,L contractions, although to a lesser extent. 8. We conclude that intracellular dialysis with patch pipettes disrupts the adenylyl cyclase-PKA phosphorylation cascade, and that the VSRM requires intracellular phosphorylation to be available for activation. Intracellular dialysis with solutions that do not maintain phosphorylation levels inhibits a major mechanism in cardiac excitation- contraction coupling.

Ischemic preconditioning: antiarrhythmic effects and electrophysiological mechanisms in isolated ventricle.

The objective of this study was to identify cellular electrophysiological mechanisms by which ischemic preconditioning decreases arrhythmias in an isolated ventricular tissue model of ischemia and reperfusion. Electrical activity was recorded with microelectrodes from endocardium and epicardium of paced guinea pig right ventricular free walls. Control preparations were exposed for 15 min to Tyrode solution modified to simulate selected ischemic conditions and then were reperfused for 30 min with normal solution. Preconditioned tissues were exposed to a 2- or 5-min period of simulated ischemia before this same protocol. Neither preconditioning protocol affected incidence of ventricular tachycardia (VT) in ischemia; however, the 5-min protocol significantly decreased premature beats (PVB) and transmural conduction block. Preconditioning for 5 min, but not 2 min, significantly decreased reperfusion-induced VT and PVB. Ischemic preconditioning did not change effects of ischemia or reperfusion on action potential duration, effective refractory period, or endocardial conduction time. However, preconditioning markedly attenuated depression of transmural conduction by ischemia and early reperfusion and thereby prevented conduction delays necessary for transmural reentry.

Contribution of a voltage-sensitive calcium release mechanism to contraction in cardiac ventricular myocytes.

The contribution of a voltage-sensitive release mechanism (VSRM) for sarcoplasmic reticulum (SR) Ca2+ to contraction was investigated in voltage-clamped ventricular myocytes at 37 degrees C. Na+ current was blocked with lidocaine. The VSRM exhibited steady-state inactivation (half-inactivation voltage: -47.6 mV; slope factor: 4.37 mV). When the VSRM was inactivated, contraction-voltage relationships were proportional to L-type Ca2+ current (ICa-L). When the VSRM was available, the relationship was sigmoidal, with contractions independent of voltage positive to -20 mV. VSRM and ICa-L contractions could be separated by activation-inactivation properties. VSRM contractions were extremely sensitive to ryanodine, thapsigargin, and conditioning protocols to reduce SR Ca2+ load. ICa-L contractions were less sensitive. When both VSRM and ICa-L were available, sigmoidal contraction-voltage relationships became bell-shaped with protocols to reduce SR Ca2+ load. Myocytes demonstrated restitution of contraction that was slower than restitution of ICa-L. Restitution was a property of the VSRM. Thus activation and recovery of the VSRM are important in coupling cardiac contraction to membrane potential, SR Ca2+ load, and activation interval.

The 1996 Merck Frosst Award. The voltage-sensitive release mechanism: a new trigger for cardiac contraction.

Contraction in mammalian heart is initiated by a rapid rise in intracellular free calcium (Ca2+) triggered by excitation of the sarcolemma. Traditional views of cardiac excitation-contraction coupling have focused on the importance of Ca(2+)-induced Ca2+ release from the sarcoplasmic reticulum as a major source for this increase in Ca2+. Influx of Ca2+, primarily through L-type Ca2+ channels and the sodium-calcium (Na(+)-Ca2+) exchanger, is considered to be the main trigger for Ca(2+)-induced Ca2+ release. However, we recently have discovered a new trigger for excitation-contraction coupling in experiments on isolated ventricular myocytes under voltage clamp conditions. This trigger is a voltage-sensitive release mechanism that initiates release of Ca2+ from the sarcoplasmic reticulum. This article reviews the development of the concept of voltage-activated Ca2+ release in heart and discusses the importance of this discovery to the physiology, pathophysiology, and pharmacology of cardiac contraction.

"Voltage-activated Ca release" in rabbit, rat and guinea-pig cardiac myocytes, and modulation by internal cAMP.

It is widely believed that Ca release from the sarcoplasmic reticulum (SR) in heart muscle is due to "Ca-induced Ca-release" (CICR), triggered by transmembrane Ca entry. However, in intact guinea-pig cells or cells dialysed with cAMP there may be an additional mechanism - SR release may be activated directly by membrane depolarisation without Ca entry. The first objective of the present study was to investigate whether this "voltage-activated Ca release" (VACR) mechanism is present across species such as rabbit, rat and guinea-pig. The second objective was to characterise the dependence of a VACR mechanism on internal [cAMP]. Membrane current was measured with the whole-cell patch-clamp technique, intracellular [Ca] was monitored with Fura-2 (or a combination of Fluo-3/SNARF-1). Rapid changes of superfusate (within 100 ms) were made using a system which maintained cell temperature at 37 degrees C. We used a train of conditioning pulses to ensure a standard SR load before each test pulse. In rabbit myocytes dialysed with 100 microM cAMP, 89.6 +/- 7.0% of the control intracellular Ca (Cai) transient was still elicited by depolarisation during a switch to 5 mM Ni, which blocked pathways for Ca entry. This suggested that rabbit myocytes possess a VACR mechanism. The percentage of control Cai transient elicited by depolarisation in the presence of 5 mM Ni (i.e. magnitude of VACR) increased in a graded fashion with the pipette [cAMP] between zero and 100 microM. In rat myocytes dialysed with 50 microM cAMP, 64.4 +/- 6.2% of SR release was activated by depolarisation in the presence of 5 mM Ni, suggesting the presence of a VACR mechanism. The extent to which VACR triggered SR release increased with the pipette [cAMP] between zero and 50 microM. In guinea-pig myocytes dialysed with 100 microM cAMP, 74.6 +/- 3.6% of the control Cai transient was elicited by depolarisation in the presence of 5 mM Ni. The degree to which VACR triggered SR release was also graded with the pipette [cAMP] between zero and 100 microM. It therefore appears that each of the three species might possess a VACR mechanism which can be modulated by the internal [cAMP]. This may reflect an effect of cAMP to phosphorylate key proteins involved in excitation-contraction coupling. Under normal physiological conditions with a basal [cAMP] between 2 and 20 microM, VACR may play a role in triggering SR release. The role of VACR may increase under conditions which increase internal [cAMP].

Proarrhythmic actions of flecainide in an isolated tissue model of ischemia and reperfusion.

Flecainide may increase the incidence of cardiac arrhythmias in acute ischemia. The objective of this study was to determine the cellular actions underlying this effect in an isolated tissue model of acute ischemia and reperfusion. Transmembrane electrical activity was recorded with conventional microelectrode techniques from epi- and endocardial surfaces of right ventricular free walls from guinea pig hearts. Endocardium was stimulated. Tissues were equilibrated in Tyrode's solution for 60 min, then exposed to simulated ischemia (hypoxia, acidosis, lactate, hyperkalemia, no glucose) for 15 min and reperfused with normal Tyrode's solution for 30 min. In the absence of flecainide, sustained and nonsustained ventricular tachycardia occurred in 78% of hearts during ischemic conditions and 78% in early reperfusion (n = 14). Premature beats occurred in 14% of hearts in early reperfusion. Ventricular tachycardia was associated with abbreviation of endocardial effective refractory period and action potential duration, plus prolongation of transmural conduction time. Flecainide abolished premature beats at a concentration of 1 mumol/l or higher. However, an increase in the incidence of ventricular tachycardia occurred in both ischemia and reperfusion at all concentrations of flecainide (0.03-10.0 mumol/l). Proarrhythmic effects of flecainide were associated with selective prolongation of transmural conduction time in ischemia and early reperfusion. In epicardial slices flecainide lengthened conduction time transverse, but not parallel to fiber orientation. Our results suggest that proarrhythmic effects of flecainide in acute ischemia and reperfusion are mediated by potentiation of the arrhythmogenic effects of ischemia on anisotropic properties of the myocardium.

Transient inward current is conducted through two types of channels in cardiac Purkinje fibres.

Two-microelectrode voltage-clamp technique was applied and a number of experimental manoeuvres were used to determine the charge-carrying systems of the arrhythmogenic transient inward current (TI) in rabbit cardiac Purkinje fibres. Increasing [Ca2+]o to 30 mmol/l (in the presence of [Na+]o) induced TI with a clear-cut reversal potential around -23 mV. This observation suggests that the TI is conducted through an ionic channel. Nickel chloride (2.5 mmol/l) which blocks Na+/Ca2+ exchange current greatly decreased peak inward TI (57 +/- 3%) but significantly increased peak outward TI (86.4 +/- 9.6%), which suggests that Na+ Ca2+ exchange is not the charge-carrying system for TI. In experiments in which TI was induced when [Na+]o was replaced by either N-methyl-D-glucamine, choline, or sucrose (to eliminate Na+ Ca2+ exchange), Ni2+ still decreased inward TI and increased outward TI. There was no significant difference between the effects of Ni2+ in the presence and absence of [Na+]o. The effects of Ni2+ in the absence of [Na+]o confirm that Ni(2+)-induced attenuation of inward TI is not mediated by the Na+ Ca2+ exchange, but rather through an inhibition of TI channels. Acute exposure to Mn2+ (5 mmol/l) almost abolished inward TI at a time when outward TI just showed a slight decrease. A third divalent cation, Cd2+ (0.25-1.0 mmol/l), strongly suppressed both inward and outward TI at the same time. The opposite effects of Ni2+ on inward v outward TI and the preferential inhibition of inward TI by Mn2- suggest involvement of multiple ionic channels in conducting TI. 4.4'-diisothiocyanatostilbene-2.2' disulfonic acid (DIDS, 10 mumol/l) and 4-acetamido-4'-isothicyanatostilbene-2.2' disulfonic acid (SITS. 0.2 mmol/l), which block CI- conductance in cardiac tissues, dramatically suppressed outward TI (80 +/- 9%) but to a lesser extent inward TI (20 +/- 4%). Our results suggest that, in cardiac Purkinje fibres, high [Ca2+]o induced TI is conducted mainly through TI channels which fall into two different populations: cationic and anionic.

Losartan exerts antiarrhythmic activity independent of angiotensin II receptor blockade in simulated ventricular ischemia and reperfusion.

The purpose of this study was to determine whether specific angiotensin II (AII) type 1 receptor blockade with losartan would affect arrhythmia generation in an isolated guinea pig ventricular model of simulated ischemia and reperfusion. Effects of losartan were evaluated in the presence and absence of exogenous AII. Transmembrane potentials and an electrocardiogram were recorded during perfusion with normal Tyrode's solution, exposure to simulated ischemia for 15 min (hypoxia, acidosis, lactate, hyperkalemia, glucose-free) and reperfusion for 30 min. Under normal conditions, losartan did not affect endocardial or transmural conduction times, action potential duration at 90% repolarization or effective refractory period (ERP). However, losartan and AII each had significant effects on electrophysiological parameters during simulated ischemia and reperfusion. Further, losartan and AII, both independently and in combination, exerted antiarrhythmic effects in early reperfusion. Neither losartan nor AII affected action potential duration at 90% repolarization during simulated ischemia or reperfusion. However, AII exerted antiarrhythmic effects by preventing pronounced shortening of ERP in simulated ischemia and early reperfusion. Losartan by itself had no effect on ERP, but completely blocked the antiarrhythmic action of AII on ERP. Nevertheless, losartan preserved antiarrhythmic efficacy by attenuating prolongation of transmural conduction times in stimulated ischemia and early reperfusion. This antiarrhythmic action occurred in the absence or presence of AII. Our results indicate that losartan has antiarrhythmic efficacy which is independent of AII type 1 receptor blockade.

Saralasin suppresses arrhythmias in an isolated guinea pig ventricular free wall model of simulated ischemia and reperfusion.

The effects of saralasin on electrophysiological changes and arrhythmias induced by simulated ischemia and reperfusion were examined in an isolated tissue model. Segments of guinea pig right ventricles, stimulated regularly, were exposed to simulated ischemia for 15 min and then were reperfused with normal Tyrode's solution for 30 min. Transmembrane electrical activity and a high-gain electrogram were recorded. Arrhythmias and electrophysiological changes accompanying simulated ischemia and reperfusion in control preparations were compared to those in preparations treated with 0.1 or 1 microM saralasin. Simulated ischemia caused abbreviation of action potential duration measured at 90% repolarization, abbreviation of endocardial effective refractory period (ERP) and prolongation of transmural conduction time. Premature ventricular beats, ventricular tachycardia and conduction block were observed in approximately 35% of control preparations during simulated ischemia. Rapid sustained or nonsustained ventricular tachycardia occurred in approximately 60% of control preparations in early reperfusion. The overall incidence of arrhythmias and the incidence of ventricular tachycardia in early reperfusion were significantly decreased by 1 microM but not 0.1 microM saralasin. Saralasin (1 microM) prolonged the ERP in normoxic tissues, but it did not alter changes induced by ischemia or reperfusion in ERP or the action potential duration at 90% repolarization. Prolongation of transmural conduction time during ischemia and early reperfusion was significantly inhibited by both concentrations of saralasin. However, only 1 microM saralasin reduced the ratio of transmural conduction time to ERP enough to prevent arrhythmias. Our observations demonstrate that saralasin exerts antiarrhythmic effects in myocardial reperfusion by a mechanism independent of circulatory and central actions.

Effects of adenosine in simulated ischemia and reperfusion in guinea pig ventricular myocytes.

Effects of adenosine (ADN) on cardiac cellular electrical and contractile activity were determined during ischemia and reperfusion. Electrical activity was recorded with conventional and voltage-clamp techniques. Contractions were monitored with a video edge detector. Myocytes were exposed to simulated ischemia (20 min), in the presence or absence of ADN (1-50 microM), and reperfused with Tyrode solution. ADN had no effects under control conditions. However, action potential abbreviation during ischemia was greater in the presence of ADN than for control, and recovery was delayed. In ischemia, Ca2+ current declined equally, and contractions were abolished in control and ADN-treated myocytes. In early reperfusion, oscillatory afterpotentials (OAP), transient inward current (ITI) and aftercontractions appeared, and contractions increased above preischemic levels. ADN abolished contractile overshoot and reduced incidence of OAP, ITI, and aftercontractions from 78 to 37.5%. The effects of exogenous ADN were inhibited by ADN A1-receptor blockade. Inhibition of endogenous ADN by 8-phenyltheophylline only increased incidence of ITI. Thus exogenous ADN in ischemia may protect the myocardium in reperfusion via A1 receptors.

Contractions in guinea-pig ventricular myocytes triggered by a calcium-release mechanism separate from Na+ and L-currents.

1. Unloaded cell shortening and membrane currents were examined in isolated guinea-pig ventricular myocytes at 37 degrees C using video edge detection and single-electrode voltage clamp. 2. Inward Na+ currents were eliminated by lidocaine, tetrodotoxin, replacement of extracellular Na+ with choline chloride or sucrose, or by voltage inactivation of Na+ channels. In the absence of Na+ current, the threshold for contraction was approximately -50 or -55 mV. 3. Verapamil (5 microM) and nifedipine (2 microM) failed to inhibit contractions at negative membrane potentials when positive conditioning pulses were used to maintain intracellular Ca2+ stores via Na(+)-Ca2+ exchange. In contrast, 200 microM Ni2+ inhibited these contractions. 4. Contractions were abolished when the extracellular solution was nominally Ca2+ free. However, contractions were restored by as little as 50 microM extracellular Ca2+. 5. Ryanodine (30 nM) completely abolished contractions initiated by depolarizing steps from -65 to -40 mV, but had minimal effects on contractions initiated by depolarizing steps from -40 to +5 mV. Subtraction of contraction-voltage relations determined in the presence of ryanodine from control relations revealed a ryanodine-sensitive component of contraction. This component activated at -55 mV and reached a plateau near -25 mV. 6. The amplitudes of contractions initiated by depolarizing steps from -40 mV were directly proportional to the magnitude of Ca2+ current (ICa). In contrast, contractions initiated by steps from either -55 or -65 mV were not proportional to ICa. These contractions appeared at potentials negative to the threshold for L-type Ca2+ current, increased to a plateau at more positive potentials and did not decrease at potentials at which ICa decreased. 7. Subtraction of the contraction-voltage relationship determined from a membrane potential of -40 mV from that at -55 mV revealed a component of contraction with a negative activation threshold whose amplitude was not proportional to inward current. The shape of this relationship was virtually identical to that of the ryanodine-sensitive component of contraction. 8. This study identifies a component of contraction associated with Ca2+ release from sarcoplasmic reticulum (SR) which can be separated from other mechanisms of contraction on the basis of membrane potential. Our observations suggest that this voltage-dependent release mechanism is a true trigger mechanism which activates a portion of cardiac contraction which is attributable to SR Ca2+ release.