Langendorff-perfused Guinea Pig Hearts Research Articles

Acetaminophen-mediated cardioprotection via inhibition of the mitochondrial permeability transition pore-induced apoptotic pathway

Our laboratory has previously reported that acetaminophen confers functional cardioprotection following cardiac insult, including ischemia/reperfusion, hypoxia/reoxygenation, and exogenous peroxynitrite administration. In the present study, we further examined the mechanism of acetaminophen-mediated cardioprotection following ischemia/reperfusion injury. Langendorff-perfused guinea pig hearts were exposed to acute treatment with acetaminophen (0.35 mM) or vehicle beginning at 15 min of a 30-min baseline stabilization period. Low-flow global myocardial ischemia was subsequently induced for 30 min followed by 60 min of reperfusion. At the completion of reperfusion, hearts were homogenized and separated into cytosolic and mitochondrial fractions. Mitochondrial swelling and mitochondrial cytochromec release were assessed and found to be significantly and completely reduced in acetaminophen- vs. vehicle-treated hearts following reperfusion. In a separate group of hearts, ventricular myocytes were isolated and subjected to fluorescence-activated cell sorting. Acetaminophen-treated hearts showed a significant decrease in late stage apoptotic myocytes compared with vehicle-treated hearts following injury (58 +/- 1 vs. 81 +/- 5%, respectively). These data, together with electron micrograph analysis, suggest that acetaminophen mediates cardioprotection, in part, via inhibition of the mitochondrial permeability transition pore and subsequent apoptotic pathway.

THE EFFECTS OF ISOSTEVIOL AGAINST MYOCARDIUM INJURY INDUCED BY ISCHAEMIA–REPERFUSION IN THE ISOLATED GUINEA PIG HEART

1. This study aimed to investigate the protective effects of isosteviol against myocardial ischaemia-reperfusion (IR) injury and its effects on mitochondrial adenosine triphosphate (ATP)-sensitive potassium channel (mitoK(ATP)) activity in vitro. 2. Groups of eight guinea pigs were treated as follows: constant perfusion control (PC), IR control, ischaemic preconditioning (IPC) + IR, isosteviol (50, 250 or 500 nmol) + IR, 5-hydroxydecanoate acid (5-HD) (5 micromol) + isosteviol (500 nmol) + IR. The guinea pig heart was isolated and perfused in Langendorff mode with modified Tyrode solution at a flow rate of 10 mL/min. Ischaemia was introduced for 30 min followed by reperfusion for 20 min. Cardiac function, coronary arterial flow rate, lactate dehydrogenase (LDH) and creatine kinase (CK) activities in the perfusate were measured prior to ischaemia and at the end of reperfusion. 3. There were no significant (P > 0.05) changes in cardiac function or markers of cell damage (i.e. activities of LDH and CK) in the PC group. In contrast, cardiac function was adversely affected in the IR group, with significant (P < 0.05) decreases in left ventricular developing pressure (LVDevP), dP/dt(max) and dP/dt(min) compared with baseline and the PC group. In addition, there were increases in activity of LDH (20%) and CK (67%) compared with baseline and the PC group. 4. Ischaemic preconditioning and pretreatment with isosteviol, at all dose levels, resulted in a significant (P < 0.05) attenuation of IR injury. Lactate dehydrogenase and CK activities were not significantly (P < 0.05) different compared with baseline. Isosteviol did not increase coronary flow, suggesting that the protective effect of isosteviol on the myocardium was not mediated by dilation of the coronary blood vessels. 5. Pretreatment with the mitoK(ATP) blocker 5-HD partially antagonized the effects of 500 nmol isosteviol, with a statistically significant attenuation of its protective effects on HR, LVDevP, dP/dt(max) and dP/dt(min) compared with isosteviol alone pretreatment. 6. The IR injury on the Langendorff perfused guinea pig heart was alleviated by isosteviol, which appears to mediate its effects through mitoK(ATP) channels. Future research might aim to investigate the interaction of isosteviol with mitoK(ATP) channels in order to clarify its mechanism of action.

QT prolongation in guinea pigs for preliminary screening of torsadogenicity of drugs and drug‐candidates. II

Experimental approaches on anaesthetised guinea pigs have been shown recently to be satisfactorily predictive of the torsadogenic risk of drugs. This work aimed at obtaining additional data, for a further understanding of the reliability and/or the limits of this model. Clonidine (non-torsadogenic in humans) induced a lengthening of the ECG parameter of RR in anaesthetised guinea pigs, without any corresponding increase of QT (corrected by the algorithms of Bazett and Fridericia). Thus, 'QT correct' prolonging effects produced by drugs torsadogenic in humans, on the guinea pig model are primarily due to inhibition of cardiac repolarisation. The corresponding RR prolongation is a consequence (not the cause) of this primary effect. Astemizole, haloperidol and terfenadine, torsadogenic in humans, produced in Langendorff perfused guinea pig hearts a prolongation of the QT interval. Chlorprotixene (non-torsadogenic) did not produce any significant effect on QT. These results are fully consistent with previous observations in anaesthetised guinea pigs. In Langendorff perfused hearts, pentobarbital does not affect cardiac repolarisation and does not potentiate the QT-prolonging effect of astemizole. Together with the findings reported by many authors, these data suggest that ECG recording in anaesthetised guinea pigs is a reliable model for cardiac safety studies evaluating the influence of drugs on the repolarisation process.

Histamine H 3-receptor signaling in cardiac sympathetic nerves: Identification of a novel MAPK-PLA 2-COX-PGE 2-EP 3R pathway

We hypothesized that the histamine H 3-receptor (H 3R)-mediated attenuation of norepinephrine (NE) exocytosis from cardiac sympathetic nerves results not only from a Gα i-mediated inhibition of the adenylyl cyclase-cAMP-PKA pathway, but also from a Gβγ i-mediated activation of the MAPK-PLA 2 cascade, culminating in the formation of an arachidonate metabolite with anti-exocytotic characteristics (e.g., PGE 2). We report that in Langendorff-perfused guinea-pig hearts and isolated sympathetic nerve endings (cardiac synaptosomes), H 3R-mediated attenuation of K +-induced NE exocytosis was prevented by MAPK and PLA 2 inhibitors, and by cyclooxygenase and EP 3-receptor (EP 3R) antagonists. Moreover, H 3R activation resulted in MAPK phosphorylation in H 3R-transfected SH-SY5Y neuroblastoma cells, and in PLA 2 activation and PGE 2 production in cardiac synaptosomes; H 3R-induced MAPK phosphorylation was prevented by an anti-βγ peptide. Synergism between H 3R and EP 3R agonists (i.e., imetit and sulprostone, respectively) suggested that PGE 2 may be a downstream effector of the anti-exocytotic effect of H 3R activation. Furthermore, the anti-exocytotic effect of imetit and sulprostone was potentiated by the N-type Ca 2+-channel antagonist ω-conotoxin GVIA, and prevented by an anti-Gβγ peptide. Our findings imply that an EP 3R Gβγ i-induced decrease in Ca 2+ influx through N-type Ca 2+-channels is involved in the PGE 2/EP 3R-mediated attenuation of NE exocytosis elicited by H 3R activation. Conceivably, activation of the Gβγ i subunit of H 3R and EP 3R may also inhibit Ca 2+ entry directly, independent of MAPK intervention. As heart failure, myocardial ischemia and arrhythmic dysfunction are associated with excessive local NE release, attenuation of NE release by H 3R activation is cardioprotective. Accordingly, this novel H 3R signaling pathway may ultimately bear therapeutic significance in hyper-adrenergic states.

Preconditioning protects endothelium by preventing ET-1-induced activation of NADPH oxidase and xanthine oxidase in post-ischemic heart

The hypothesis was tested that endothelin-1 (ET-1)-induced superoxide (O 2 −) generation mediates post-ischemic coronary endothelial injury, that ischemic preconditioning (IPC) affords endothelial protection by preventing post-ischemic ET-1, and thus O 2 −, generation, and that opening of the mitochondrial ATP-dependent potassium channel (mK ATP) triggers the mechanism of IPC. Furthermore, the study was aimed at identifying the source of O 2 − mediating the endothelial injury. Langendorff-perfused guinea-pig hearts were subjected either to 30 min ischemia/35 min reperfusion (IR) or were preconditioned prior to IR with three cycles of either 5 min ischemia/5 min reperfusion or 5 min infusion/5 min washout of mK ATP opener diazoxide (0.5 mM). Coronary flow responses to acetylcholine (ACh) served as a measure of endothelium-dependent vascular function. Myocardial outflow of ET-1 and O 2 − and functional recoveries were followed during reperfusion. NADPH oxidase and xanthine oxidase (XO) activities were measured in cardiac homogenates. IR augmented ET-1 and O 2 − outflow and impaired ACh response. All these effects were attenuated or prevented by IPC and diazoxide, and 5-hydroxydecanoate (a selective mK ATP blocker) abolished the effects of IPC and diazoxide. Superoxide dismutase and tezosentan (a mixed ET-1-receptor antagonist) mimicked the effects of IPC, although they had no effect on the ET-1 generation. IR augmented also the activity of NADPH oxidase and XO. Apocynin treatment, that resulted in NADPH oxidase inhibition, prevented XO activation and O 2 − generation in IR hearts. The inhibition of XO, either by allopurinol or feeding the animals with tungsten-enriched chow, prevented post-ischemic O 2 − generation, although these interventions had no effect on the NADPH activity. In addition, the post-ischemic activation of NADPH oxidase and XO, and O 2 − generation were prevented by IPC, tezosentan, thenoyltrifluoroacetone (mitochondrial complex II inhibitor), and tempol (cell-membrane permeable O 2 − scavenger). In guinea-pig heart: (i) ET-1-induced O 2 − generation mediates post-ischemic endothelial dysfunction; (ii) IPC and diazoxide afford endothelial protection by attenuating the ET-1, and thus O 2 − generation, and the mK ATP opening triggers the protection; (iii) the NADPH oxidase maintains the activity of XO, and the XO-derived O 2 − mediates the endothelial injury, and (iv) ET-1 and O 2 − (probably of mitochondrial origin) are upstream activators of the NADPH oxidase–XO cascade, and IPC prevents the cascade activation and the endothelial dysfunction by preventing the ET-1 generation.

Ventricular Fibrillation During No‐Flow Global Ischemia in Isolated Rabbit Hearts

The dominant frequency (DF) during ventricular fibrillation (VF) in Langendorff-perfused guinea pig hearts is higher in left ventricle (LV) than in right ventricle (RV). However, the onset of VF invariably leads to global ischemia. Whether or not a high DF source exists in LV during global ischemia is unknown. By using a two-camera optical mapping system, epicardial activation patterns of VF were studied in 12 isolated rabbit hearts during baseline, no-flow global ischemia, and reperfusion. Simultaneous endocardial electrode recording was performed in 4 of the 12 hearts. Optical mapping showed type 1 VF at baseline, with multiple wandering and short-lived wavelets. After the onset of global ischemia, VF showed progressively increased spatiotemporal periodicity. The majority (65%) of VF recorded after 7 minutes of global ischemia showed type 2 VF, containing a single epicardial site with stable (> or = 3.85 seconds in duration) repetitive activities. Among the 33 sites with these activities, 24 were located near the interventricular septum, and 27 showed an epicardial breakthrough pattern with centrifugal propagation and wavebreaks distant from the focal site. After 10 minutes of global ischemia, the DF was lower on LV epicardium (5.0 +/- 1.4 Hz) than on RV epicardium (8.6 +/- 2.5 Hz, P < 0.001). However, there was no DF gradient between RV and LV endocardium (9.7 +/- 1.0 vs 9.6 +/- 0.9 Hz). VF during prolonged global ischemia is consistent with type 2 VF with a single subepicardial source of rapid activation, mostly near the interventricular septum. The DF in LV is not higher than in RV.

A Comparison of Three Phosphodiesterase Type III Inhibitors on Mechanical and Metabolic Function in Guinea Pig Isolated Hearts

Little is known about of the comparative cardiac lusitropic and coronary vasoactive effects of type III phosphodiesterase inhibitors independent of their systemic circulatory effects. We hypothesized that phosphodiesterase inhibitors have dissimilar concentration-dependent effects on cardiac function and metabolism and that their coronary vasodilatory effects are solely dependent on flow autoregulation secondary to positive inotropic effects. Our aim was to compare the dose-response electrophysiologic, mechanical, vasodilatory, and metabolic properties of three clinically available phosphodiesterase inhibitors in isolated Langendorff perfused guinea pig hearts. We found that, over a range from 10(-7) to 10(-4) M, amrinone, enoximone, and milrinone each produced maximal concentration-dependent positive chronotropic (12%, 18%, 26%), inotropic (16%, 26%, 26%), and lusitropic (14%, 21%, 19%) effects. At clinical concentrations, all phosphodiesterase inhibitors increased heart rate, but only milrinone significantly enhanced contractility and relaxation (11%). Each phosphodiesterase inhibitor similarly increased contractility at its highest concentration; this was accompanied by an increase in oxygen consumption, which was matched by comparable increases in coronary flow and oxygen delivery. Coronary flow reserve was preserved at the highest concentration of each drug, indicating that an increased metabolic rate was responsible for the increase in coronary flow by each drug at each concentration. Over the concentrations examined, we conclude that each of the phosphodiesterase inhibitors does not directly promote coronary vasodilation and that milrinone has the most prominent effects on contractility and relaxation at clinically relevant concentrations.

Cardiac mast cell-derived renin promotes local angiotensin formation, norepinephrine release, and arrhythmias in ischemia/reperfusion

Having identified renin in cardiac mast cells, we assessed whether its release leads to cardiac dysfunction. In Langendorff-perfused guinea pig hearts, mast cell degranulation with compound 48/80 released Ang I-forming activity. This activity was blocked by the selective renin inhibitor BILA2157, indicating that renin was responsible for Ang I formation. Local generation of cardiac Ang II from mast cell-derived renin also elicited norepinephrine release from isolated sympathetic nerve terminals. This action was mediated by Ang II-type 1 (AT1) receptors. In 2 models of ischemia/reperfusion using Langendorff-perfused guinea pig and mouse hearts, a significant coronary spillover of renin and norepinephrine was observed. In both models, this was accompanied by ventricular fibrillation. Mast cell stabilization with cromolyn or lodoxamide markedly reduced active renin overflow and attenuated both norepinephrine release and arrhythmias. Similar cardioprotection was observed in guinea pig hearts treated with BILA2157 or the AT1 receptor antagonist EXP3174. Renin overflow and arrhythmias in ischemia/reperfusion were much less prominent in hearts of mast cell-deficient mice than in control hearts. Thus, mast cell-derived renin is pivotal for activating a cardiac renin-angiotensin system leading to excessive norepinephrine release in ischemia/reperfusion. Mast cell-derived renin may be a useful therapeutic target for hyperadrenergic dysfunctions, such as arrhythmias, sudden cardiac death, myocardial ischemia, and congestive heart failure.

Importance of spatiotemporal heterogeneity of cellular restitution in mechanism of arrhythmogenic discordant alternans

Spatially discordant cellular alternans form a substrate for development of unidirectional block and ventricular fibrillation. However, the mechanisms responsible for discordant alternans remain poorly understood. Previous work suggests electrical restitution is critical to the development of alternans in single cells. The purpose of this study was to investigate the hypothesis that spatial and temporal heterogeneities of restitution underlie the mechanism eliciting discordant alternans. Steady-state pacing was used to elicit concordant cellular alternans in nine Langendorff-perfused guinea pig hearts. A single extrastimulus (S2) was applied every 51st beat following either the even or the odd beat of alternans. The cellular response to S2 was determined using optical mapping to generate action potential duration (APD) restitution curves from 256 ventricular sites for both the even and the odd beats. Restitution kinetics were temporally heterogeneous during alternans, as restitution curves between the even and the odd beats differed significantly. Temporal heterogeneity was quantified by the average separation of restitution between the two curves, or Delta-restitution. Delta-Restitution was spatially heterogeneous and proportional to the amount of alternans at a given ventricular site. A computer simulation based on the experimental results showed the mechanism of discordant alternans was dependent on both spatial and temporal heterogeneities of restitution. Both temporal and spatial heterogeneities of restitution exist during cellular alternans in the intact heart. Temporal heterogeneities of restitution, quantified by Delta-restitution, are proportional to the magnitude of cellular alternans. The combination of spatial and temporal heterogeneities of restitution may underlie the genesis of discordant alternans.

Molecular mechanisms and global dynamics of fibrillation: an integrative approach to the underlying basis of vortex-like reentry

Art Winfree's Scientific legacy has been particularly important to our laboratory whose major goal is to understand the mechanisms of ventricular fibrillation (VF). Here, we take an integrative approach to review recent studies on the manner in which nonlinear electrical waves organize to result in VF. We describe the contribution of specific potassium channel proteins and of the myocardial fiber structure to such organization. The discussion centers on data derived from a model of stable VF in the Langendorff-perfused guinea pig heart that demonstrates distinct patterns of organization in the left (LV) and right (RV) ventricles. Analysis of optical mapping data reveals that VF excitation frequencies are distributed throughout the ventricles in clearly demarcated domains. The highest frequency domains are found on the anterior wall of the LV at a location where sustained reentrant activity is present. The optical data suggest that a high frequency rotor that remains stationary in the LV is the mechanism that sustains VF in this model. Computer simulations predict that the inward rectifying potassium current (IK1) is an essential determinant of rotor stability and frequency, and patch-clamp results strongly suggest that the outward component of IK1 of cells in the LV is significantly larger than in the RV. Additional computer simulations and analytical procedures predict that the filaments of the reentrant activity (scroll waves) adopt a non-random configuration depending on fiber organization within the ventricular wall. Using the minimal principle we have concluded that filaments align with the trajectory of least resistance (i.e. the geodesic) between their endpoints. Overall, the data discussed have opened new and potentially exciting avenues of research on the possible role played by inward rectifier channels in the mechanism of VF, as well as the organization of its reentrant sources in three-dimensional cardiac muscle. Such an integrative approach may lead us toward an understanding of the molecular and structural basis of VF and hopefully to new preventative approaches.

Pharmacological modulation of cardiac gap junctions to enhance cardiac conduction: evidence supporting a novel target for antiarrhythmic therapy.

Disease-induced alterations of cardiac gap junctions lead to intercellular uncoupling, which is an important mechanism of arrhythmogenesis. Therefore, drugs that selectively open gap junctions potentially offer a novel strategy for antiarrhythmic therapy. Because the peptide ZP123 was found to increase conductance between paired myocytes, we hypothesized that ZP123 would suppress acidosis-induced gap junction closure in the intact heart. High-resolution optical mapping was used to measure conduction velocity (CV) and action potential duration from ventricular epicardium of Langendorff-perfused guinea pig hearts at baseline (pH 7.4) and during 45 minutes of perfusion with acidotic (pH 6.0) Tyrode's solution with (n=8) and without (control, n=7) ZP123 (80 nmol/L). Acidosis produced conduction slowing transverse (29.1+/-0.1 to 16.8+/-0.2 cm/s, P<0.0001) and longitudinal (47.2+/-2.4 to 33.2+/-4.8 cm/s, P<0.0001) to cardiac fibers. Importantly, ZP123 inhibited conduction slowing during acidosis by approximately 60%. The peak effect of ZP123 was achieved after 16 minutes of acidosis, consistent with inhibition of uncoupling. ZP123 did not affect Na+ current in isolated myocytes, additionally affirming that preservation of CV was attributable to the compound's action on gap junctions. ZP123 had no effect on CV in the absence of acidosis, suggesting that drug activity targets gap junctions under metabolic stress. Action potential duration heterogeneity was significantly reduced by ZP123 (6.7+/-0.8 ms) compared with controls (9.7+/-3.1 ms, P<0.05), presumably by enhancing cell-to-cell coupling. These data suggest that ZP123 significantly attenuates gap junction closure during acidosis. Preservation of intercellular coupling diminished CV slowing and heterogeneous repolarization, eliminating arrhythmogenic substrates.

Effect of thymol on calcium handling in mammalian ventricular myocardium

Concentration-dependent effects of thymol on calcium handling were studied in canine and guinea pig cardiac preparations (Langendorff-perfused guinea pig hearts, canine ventricular trabeculae, canine sarcoplasmic reticular vesicles and single ryanodine receptors). Thymol induced a concentration-dependent negative inotropic action in both canine and guinea pig preparations (EC 50 = 297 ± 12 μM in dog). However, low concentrations of thymol reduced intracellular calcium transients in guinea pig hearts without decreasing contractility. At higher concentrations both calcium transients and contractions were suppressed. In canine sarcoplasmic reticular vesicles thymol induced rapid release of calcium (V max = 0.47 ± 0.04 nmol s −1, EC 50 = 258 ± 21 μM, Hill coefficient = 3.0 ± 0.54), and decreased the activity of the calcium pump (EC 50 = 253 ± 4.7 μM, Hill coefficient = 1.62 ± 0.05). Due to the less sharp concentration-dependence of the ATPase inhibition, this effect was significant from 50 μM, whereas the thymol-induced calcium release only from 100 μM. In single ryanodine receptors incorporated into artificial lipid bilayer thymol induced long lasting openings, having mean open times increased with 3 orders of magnitude, however, the specific conductance of the channel remained unaltered. This effect of thymol was not voltage-dependent and failed to prevent the binding of ryanodine. In conclusion, the negative inotropic action of thymol can be explained by reduction in calcium content of the sarcoplasmic reticulum due to the combination of the thymol-induced calcium release and inhibition of the calcium pump. The calcium-sensitizer effect, observed at lower thymol concentrations, indicates that thymol is likely to interact with the contractile machinery also.

Hysteresis Effect Implicates Calcium Cycling as a Mechanism of Repolarization Alternans

T-wave alternans is due to alternation of membrane repolarization at the cellular level and is a risk factor for sudden cardiac death. Recently, a hysteresis effect has been reported in patients whereby T-wave alternans, once induced by rapid heart rate, persists even when heart rate is subsequently slowed. We hypothesized that alternans hysteresis is an intrinsic property of cardiac myocytes, directly related to an underlying mechanism for repolarization alternans that involves intracellular calcium cycling. Stepwise pacing was used to induce alternans in Langendorff-perfused guinea pig hearts from which optical action potentials were recorded simultaneously at 256 ventricular sites with voltage-sensitive dyes and in whole-cell patch-clamped cardiac myocytes treated with or without BAPTA-AM (1,2-bis[2-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid tetrakis [acetoxymethyl ester]). Alternans hysteresis was observed in every isolated heart: threshold heart rate for alternans was 280+/-12 bpm, but during subsequent deceleration of pacing, alternans persisted to significantly slower heart rates (238+/-5 bpm, P<0.05). Optical mapping showed that this effect also applied to the threshold for spatially discordant alternans (313+/-2.2 bpm during acceleration versus 250+/-6.6 bpm during deceleration, P<0.05). Alternans hysteresis was also observed in isolated cardiac myocytes. Moreover, calcium chelation by BAPTA-AM raised the threshold for alternans and inhibited hysteresis in a dose-dependent manner with no effect on baseline action potential duration. Alternans hysteresis is an intrinsic property of cardiac myocytes that can lead to persistence of arrhythmogenic discordant alternans even after heart rate is slowed. These results also support an important underlying role of calcium cycling in the mechanism of alternans.

Intracellular calcium handling heterogeneities in intact guinea pig hearts.

Regional heterogeneities of ventricular repolarizing currents and their role in arrhythmogenesis have received much attention; however, relatively little is known regarding heterogeneities of intracellular calcium handling. Because repolarization properties and contractile function are heterogeneous from base to apex of the intact heart, we hypothesize that calcium handling is also heterogeneous from base to apex. To test this hypothesis, we developed a novel ratiometric optical mapping system capable of measuring calcium fluorescence of indo-1 at two separate wavelengths from 256 sites simultaneously. With the use of intact Langendorff-perfused guinea pig hearts, ratiometric calcium transients were recorded under normal conditions and during administration of known inotropic agents. Ratiometric calcium transients were insensitive to changes in excitation light intensity and fluorescence over time. Under control conditions, calcium transient amplitude near the apex was significantly larger (60%, P < 0.01) compared with the base. In contrast, calcium transient duration was significantly longer (7.5%, P < 0.03) near the base compared with the apex. During isoproterenol (0.05 microM) and verapamil (2.5 microM) administration, ratiometric calcium transients accurately reflected changes in contractile function, and, the direction of base-to-apex heterogeneities remained unchanged compared with control. Ratiometric optical mapping techniques can be used to accurately quantify heterogeneities of calcium handling in the intact heart. Significant heterogeneities of calcium release and sequestration exist from base to apex of the intact heart. These heterogeneities are consistent with base-to-apex heterogeneities of contraction observed in the intact heart and may play a role in arrhythmogenesis under abnormal conditions.

Differences in electrophysiological and contractile properties of mammalian cardiac tissues bathed in bicarbonate - and HEPES-buffered solutions.

The aim of this study was to compare the action potential configuration, contractility, intracellular Ca2+ and H+ concentrations in mammalian cardiac tissues bathed with Krebs and Tyrode solutions at 37 degrees C. In Langendorff-perfused guinea-pig hearts, loaded with the fluorescent Ca2+-indicator Fura-2, or H+-sensitive dye carboxy-SNARF, shifts from Krebs to Tyrode solution caused intra-cellular acidification, increased diastolic pressure and [Ca2+]i, decreased systolic pressure and [Ca2+]i, leading to a reduction in the amplitude of [Ca2+]i transients and pulse pressure. Contractility was also depressed in canine ventricular trabeculae when transferred from Krebs to Tyrode solution. Shifts from Krebs to Tyrode solution increased the duration of action potentials in multicellular cardiac preparations excised from canine and rabbit hearts but not in isolated cardiomyocytes. All these changes in action potential morphology, contractility, [Ca2+]i and [H+]i were readily reversible by addition of 26 mmol L(-1) bicarbonate to Tyrode solution. Effects of dofetilide and CsCl, both blockers of the delayed rectifier K current, on action potential duration were compared in Krebs and Tyrode solutions. Dofetilide lengthened rabbit ventricular action potentials in a significantly greater extent in Tyrode than in Krebs solution. Exposure of canine Purkinje fibres to CsCl evoked early after depolarizations within 40 min in all preparations incubated with Tyrode solution, but not in those bathed with Krebs solution. It is concluded that the marked differences in action potential morphology, [Ca2+]i, [H+]i and contractility observed between preparations bathed with Krebs and Tyrode solutions are more likely attributable to differences in the intracellular buffering capacities of the two media.

Toward an understanding of the molecular mechanisms of ventricular fibrillation.

A major goal of basic research in cardiac electrophysiology is to understand the mechanisms responsible for ventricular fibrillation (VF). Here we review recent experimental and numerical results, from the ion channel to the organ level, which might lead to a better understanding of the cellular and molecular mechanisms of VF. The discussion centers on data derived from a model of stable VF in the Langendorff-perfused guinea pig heart that demonstrate distinct patterns of organization in the left (LV) and right (RV) ventricles. Analysis of optical mapping data reveals that VF excitation frequencies are distributed throughout the ventricles in clearly demarcated domains. The highest frequency domains are usually found on the anterior wall of the LV, demonstrating that a high frequency reentrant source (a rotor) that remains stationary in the LV is the mechanism that sustains VF in this model. Computer simulations predict that the inward rectifying potassium current (IK1) is an essential determinant of rotor stability and rotation frequency, and patch-clamp results strongly suggest that the outward component of the background current (presumably IK1) of cells in the LV is significantly larger in the LV than in the RV. These data have opened a new and potentially exciting avenue of research on the possible role played by inward rectifier channels in the mechanism of VF and may lead us toward an understanding of its molecular basis and hopefully lead to new preventative approaches.

Endothelin in the mechanism of endothelial injury and neutrophil adhesion in the post-ischemic guinea-pig heart

This study addressed the hypothesis that endothelin promotes neutrophil accumulation in ischemic/reperfused myocardium, not only via its direct effect on neutrophils, but also because it mediates post-ischemic endothelial injury. Langendorff-perfused guinea-pig hearts were subjected to 30 min ischemia/35 min reperfusion, and infusion of neutrophils between 15 and 25 min of reperfusion. The infusion of the endothelin ET A/ET B receptor antagonist, tezosentan, the endothelin ET A receptor antagonist, BQ 123 [cyclo(- d-Trp- d-Asp-Pro- d-Val-Leu-], and superoxide dismutase was terminated at reperfusion, 5 min before the start of the neutrophil infusion, to avoid the contact of the drugs with neutrophils. Coronary flow responses to acetylcholine and nitroprusside were used as measures of endothelium-dependent and -independent vascular function, respectively. Neutrophil adhesion and endothelium glycocalyx ultrastructure were assessed in histological preparations. Ischemia/reperfusion resulted in a 54%-impaired acetylcholine response, endothelium glycocalyx disruption, and enhanced neutrophil adhesion (21.6% of microvessels contained neutrophils vs. 2.6% in sham group), the latter prevented by a selectin blocker, sulfatide, 20 μg/ml. These alterations were completely prevented by 0.5 and 5 nM, but not 0.05 nM, tezosentan, and were greatly attenuated by BQ 123, 1 and 10 nM. The glycocalyx-protective effect of these interventions preceded their effect on neutrophil adhesion. Superoxide dismutase, 150 IU/ml, reported before by us to protect post-ischemic endothelium glycocalyx, here prevented the post-ischemic endothelial dysfunction and neutrophil adhesion. The data imply that neutrophil adhesion in ischemic/reperfused guinea-pig heart is a selectin-dependent process, secondary to mostly endothelin ET A receptor- and free radical-mediated functional and/or structural changes in the coronary endothelium. Thus, endothelin ET A/ET B as well as ET A receptor antagonists may be useful in attenuation of the inflammatory response in ischemic/reperfused heart. The antagonists may be effective because of their direct effect on neutrophils, as demonstrated by others, and because they provide endothelial protection, as demonstrated here.

Rectification of the background potassium current: a determinant of rotor dynamics in ventricular fibrillation.

Ventricular fibrillation (VF) is the leading cause of sudden cardiac death. Yet, the mechanisms of VF remain elusive. Pixel-by-pixel spectral analysis of optical signals was carried out in video imaging experiments using a potentiometric dye in the Langendorff-perfused guinea pig heart. Dominant frequencies (peak with maximal power) were distributed throughout the ventricles in clearly demarcated domains. The fastest domain (25 to 32 Hz) was always on the anterior left ventricular (LV) wall and was shown to result from persistent rotor activity. Intermittent block and breakage of wavefronts at specific locations in the periphery of such rotors were responsible for the domain organization. Patch-clamping of ventricular myocytes from the LV and the right ventricle (RV) demonstrated an LV-to-RV drop in the amplitude of the outward component of the background rectifier current (I(B)). Computer simulations suggested that rotor stability in LV resulted from relatively small rectification of I(B) (presumably I(K1)), whereas instability, termination, and wavebreaks in RV were a consequence of strong rectification. This study provides new evidence in the isolated guinea pig heart that a persistent high-frequency rotor in the LV maintains VF, and that spatially distributed gradients in I(K1) density represent a robust ionic mechanism for rotor stabilization and wavefront fragmentation.

Changes in extracellular pH and myocardial ischaemia alter the cardiac effects of diadenosine tetraphosphate and pentaphosphate.

1. The structural conformation of diadenosine tetraphosphate (Ap(4)A) and pentaphosphate (Ap(5)A) has been reported to alter as pH is reduced. As such, it is possible that the cardiac effects of Ap(4)A and Ap(5)A vary during acidosis and myocardial ischaemia due to changes in ligand structure, receptor proteins or intracellular signalling. 2. We investigated whether the cardiac electrophysiological and coronary vasomotor effects of Ap(4)A and Ap(5)A are preserved under conditions of extracellular acidosis (pH 6.5) and alkalosis (pH 8.5) and whether Ap(4)A has any electrophysiological or antiarrhythmic effects during ischaemia. 3. Transmembrane right ventricular action potentials, refractory periods and coronary perfusion pressure were recorded from isolated, Langendorff-perfused guinea-pig hearts under constant flow conditions. The effects of 1 nM and 1 microM Ap(4)A and Ap(5)A were studied at pH 7.4, 6.5 and 8.5. The effects of 1 microM Ap(4)A were studied during global low-flow ischaemia and reperfusion. 4. At pH 7.4, Ap(4)A and Ap(5)A increased action potential duration (APD(95)) and refractory period (RP) and reduced coronary perfusion pressure. The electrophysiological effects were absent at pH 6.5 while the reductions in perfusion pressure were attenuated. At pH 8.5, Ap(4)A increased RP but the effects of Ap(4)A and Ap(5)A on perfusion pressure were attenuated. During ischaemia, Ap(4)A had no antiarrhythmic or electrophysiological effects. 5. These data demonstrate the importance of extracellular pH in influencing the effects of Ap(4)A and Ap(5)A on the heart and indicate that any potentially cardioprotective effects of these compounds during normal perfusion at physiological pH are absent during ischaemia.

Letter to the Editor

To the Editor: Analysis of the spatiotemporal organization of wave propagation during ventricular fibrillation (VF) can provide important clues about underlying mechanisms.1,2 Although VF may be the result of the breakup of multiple, randomly propagating wavelets,3 it may also result from rotor-generating wavefronts that break at fixed heterogeneities.4 These two apparently vastly different mechanisms are hypothesized to have different signatures in terms of VF dynamics, the latter showing, of course, more organization and stability in its patterns of wave propagation.5,6 In a recent article published in Circulation Research , Choi et al7 presented results of a study of the dynamics of VF in the Langendorff-perfused guinea pig heart. They used optical mapping and various methods of analysis, including spectral and velocity …