Shock-induced arrhythmogenesis is enhanced by 2,3-butanedione monoxime compared with cytochalasin D

Investigation of the mechanisms of arrhythmia genesis and maintenance has benefited from the use of optical mapping techniques that employ excitation-contraction uncouplers. We investigated the effects of the excitation-contraction uncouplers 2,3-butanedione monoxime (BDM) and cytochalasin D (Cyto D) on the induction and maintenance of arrhythmia by electric shocks. Electrical activity was optically mapped from anterior epicardium of rabbit hearts (n = 9) during shocks (-100 V, 8 ms) applied from a ventricular lead at various phases of action potential duration (APD). Restitution curves were obtained using S1-S2 protocol and measurement of APD values at 70% of repolarization. Compared with Cyto D, BDM significantly shortened APD at 90% of repolarization, although no significant difference in dispersion of repolarization was observed. Wavelength was also shortened with BDM. In general, shock-induced arrhythmias with BDM and Cyto D were ventricular tachycardic in nature. With respect to shock-induced sustained arrhythmias, the vulnerable window was wider and the incidence was higher with BDM than with Cyto D. There was also a difference in the morphology of ventricular tachycardia (VT) between the two agents. The arrhythmias with BDM usually resembled monomorphic VT, especially those that lasted >30 s. In contrast, arrhythmias with Cyto D more resembled polymorphic VT. However, the average number of phase singularities increased under Cyto D vs. BDM, whereas no significant difference in the dominant frequency of shock-induced sustained arrhythmia was observed. BDM reduced the slope of the restitution curve compared with Cyto D, but duration of arrhythmia under BDM was significantly increased compared with Cyto D. In conclusion, BDM increased arrhythmia genesis and maintenance relative to Cyto D.     

Mechanisms of shock-induced arrhythmogenesis during acute global ischemia

Little is known about the mechanisms of vulnerability and defibrillation under ischemic conditions. We investigated these mechanisms in 18 Langendorff-perfused rabbit hearts during 75% reduced-flow ischemia. Electrical activity was optically mapped from the anterior epicardium during right ventricular shocks applied at various phases of the cardiac cycle while the excitation-contraction decoupler 2,3-butanedione monoxime (BDM; 15 mM) was used to suppress motion artifacts caused by contraction of the heart. During ischemia, vulnerable window width increased [from 30-90% of the action potential duration (APD) in the control to -10 to 100% of the APD in ischemia]. Moreover, arrhythmia severity increased along with the reduction of APD (176 +/- 9 ms in control and 129 +/- 26 ms in ischemia, P < 0.01) and increased dispersion of repolarization (45 +/- 17 ms in control and 73 +/- 28 ms in ischemia, P < 0.01). Shock-induced virtual electrode polarization was preserved. Depolarizing (contrary to hyperpolarizing) response time constants increased. Virtual electrode-induced wavefronts of excitation had much more tortuous pathways leading to wavefront fractionation. Defibrillation failure at all shock strengths was observed in four hearts. Optical mapping revealed that the shock extinguished the arrhythmia; however, the arrhythmia self-originated after an isoelectric window of 339 +/- 189 ms. In conclusion, in most cases, virtual electrode-induced phase singularity (VEIPS) was responsible for shock-induced arrhythmogenesis during acute global ischemia. Enhancement of arrhythmogenesis was associated with an increased dispersion of repolarization and altered deexcitation. In four hearts, arrhythmogenesis could not be explained by VEIPS.     

Regional increase of extracellular potassium leads to electrical instability and reentry occurrence through the spatial heterogeneity of APD restitution

The heterogeneities of electrophysiological properties of cardiac tissue are the main factors that control both arrhythmia induction and maintenance. Although the local increase of extracellular potassium ([K(+)](o)) due to coronary occlusion is a well-established metabolic response to acute ischemia, the role of local [K(+)](o) heterogeneity in phase 1a arrhythmias has yet to be determined. In this work, we created local [K(+)](o) heterogeneity and investigated its role in fast pacing response and arrhythmia induction. The left marginal vein of a Langendorff-perfused rabbit heart was cannulated and perfused separately with solutions containing 4, 6, 8, 10, and 12 mM of K(+). The fluorescence dye was utilized to map the voltage distribution. We tested stimulation rates, starting from 400 ms down to 120 ms, with steps of 5-50 ms. We found that local [K(+)](o) heterogeneity causes action potential (AP) alternans, 2:1 conduction block, and wave breaks. The effect of [K(+)](o) heterogeneity on electrical stability and vulnerability to arrhythmia induction was largest during regional perfusion with 10 mM of K(+). We detected three concurrent dynamics: normally propagating activation when excitation waves spread over tissue perfused with normal K(+), alternating 2:2 rhythm near the border of [K(+)](o) heterogeneity, and 2:1 aperiodicity when propagation was within the high [K(+)](o) area. [K(+)](o) elevation changed the AP duration (APD) restitution and shifted the restitution curve toward longer diastolic intervals and shorter APD. We conclude that spatial heterogeneity of the APD restitution, created with regional elevation of [K(+)](o), can lead to AP instability, 2:1 block, and reentry induction.     

Nonlinearity between action potential alternans and restitution, which both predict ventricular arrhythmic properties in Scn5a+/- and wild-type murine hearts

Electrocardiographic QT- and T-wave alternans, presaging ventricular arrhythmia, reflects compromised adaptation of action potential (AP) duration (APD) to altered heart rate, classically attributed to incomplete Na(v)1.5 channel recovery prior to subsequent stimulation. The restitution hypothesis suggests a function whose slope directly relates to APD alternans magnitude, predicting a critical instability condition, potentially generating arrhythmia. The present experiments directly test for such correlations among arrhythmia, APD alternans and restitution. Mice haploinsufficient in the Scn5a, cardiac Na(+) channel gene (Scn5a(+/-)), previously used to replicate Brugada syndrome, were used, owing to their established arrhythmic properties increased by flecainide and decreased by quinidine, particularly in right ventricular (RV) epicardium. Monophasic APs, obtained during pacing with progressively decrementing cycle lengths, were systematically compared at RV and left ventricular epicardial and endocardial recording sites in Langendorff-perfused Scn5a(+/-) and wild-type hearts before and following flecainide (10 μM) or quinidine (5 μM) application. The extent of alternans was assessed using a novel algorithm. Scn5a(+/-) hearts showed greater frequencies of arrhythmic endpoints with increased incidences of ventricular tachycardia, diminished by quinidine, and earlier onsets of ventricular fibrillation, particularly following flecainide challenge. These features correlated directly with increased refractory periods, specifically in the RV, and abnormal restitution and alternans properties in the RV epicardium. The latter variables were related by a unique, continuous higher-order function, rather than a linear relationship with an unstable threshold. These findings demonstrate a specific relationship between alternans and restitution, as well as confirming their capacity to predict arrhythmia, but implicate mechanisms additional to the voltage feedback suggested in the restitution hypothesis.     

Effects of diacetyl monoxime and cytochalasin D on ventricular fibrillation in swine right ventricles

Whether or not the excitation-contraction (E-C) uncoupler diacetyl monoxime (DAM) and cytochalacin D (Cyto D) alter the ventricular fibrillation (VF) activation patterns is unclear. We recorded single cell action potentials and performed optical mapping in isolated perfused swine right ventricles (RV) at different concentrations of DAM and Cyto D. Increasing the concentration of DAM results in progressively shortened action potential duration (APD) measured to 90% repolarization, reduced the slope of the APD restitition curve, decreased Kolmogorov-Sinai entropy, and reduced the number of VF wave fronts. In all RVs, 15-20 mmol/l DAM converted VF to ventricular tachycardia (VT). The VF could be reinduced after the DAM was washed out. In comparison, Cyto D (10-40 micromol/l) has no effects on APD restitution curve or the dynamics of VF. The effects of DAM on VF are associated with a reduced number of wave fronts and dynamic complexities in VF. These results are compatible with the restitution hypothesis of VF and suggest that DAM may be unsuitable as an E-C uncoupler for optical mapping studies of VF in the swine RVs.     

Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression

The characterization of cellular phenotypes of heart disorders can be achieved by isolating cardiac myocytes from mouse models or genetically modifying wild-type cells in culture. However, adult mouse cardiac myocytes show extremely low tolerance to isolation and primary culture conditions. Previous studies indicate that 2,3-butanedione monoximine (BDM), a nonspecific excitation-contraction coupling inhibitor, can improve the viability of isolated adult mouse cardiac myocytes. The mechanisms of the beneficial and unwanted nonspecific actions of BDM on cardiac myocytes are not understood. To understand what contributes to murine adult cardiac myocyte stability in primary culture and improve this model system for experimental use, the specific myosin II inhibitor blebbistatin was explored as a media supplement to inhibit mouse myocyte contraction. Enzymatically isolated adult mouse cardiac myocytes were cultured with blebbistatin or BDM as a media supplement. Micromolar concentrations of blebbistatin significantly increased the viability, membrane integrity, and morphology of adult cardiac myocytes compared with cells treated with previously described 10 mM BDM. Cells treated with blebbistatin also showed efficient adenovirus gene transfer and stable transgene expression, and unlike BDM, blebbistatin does not appear to interfere with cell adhesion. Higher concentrations of BDM actually worsened myocyte membrane integrity and transgene expression. Therefore, the specific inhibition of myosin II activity by blebbistatin has significant beneficial effects on the long-term viability of adult mouse cardiac myocytes. Furthermore, the unwanted effects of BDM on adult mouse cardiac myocytes, perhaps due to its nonspecific activities or action as a chemical phosphatase, can be avoided by using blebbistatin.     

Remodeling of atrial ATP-sensitive K⁺ channels in a model of salt-induced elevated blood pressure

Hypertension is associated with the development of atrial fibrillation; however, the electrophysiological consequences of this condition remain poorly understood. ATP-sensitive K(+) (K(ATP)) channels, which contribute to ventricular arrhythmias, are also expressed in the atria. We hypothesized that salt-induced elevated blood pressure (BP) leads to atrial K(ATP) channel activation and increased arrhythmia inducibility. Elevated BP was induced in mice with a high-salt diet (HS) for 4 wk. High-resolution optical mapping was used to measure atrial arrhythmia inducibility, effective refractory period (ERP), and action potential duration at 90% repolarization (APD(90)). Excised patch clamping was performed to quantify K(ATP) channel properties and density. K(ATP) channel protein expression was also evaluated. Atrial arrhythmia inducibility was 22% higher in HS hearts compared with control hearts. ERP and APD(90) were significantly shorter in the right atrial appendage and left atrial appendage of HS hearts compared with control hearts. Perfusion with 1 μM glibenclamide or 300 μM tolbutamide significantly decreased arrhythmia inducibility and prolonged APD(90) in HS hearts compared with untreated HS hearts. K(ATP) channel density was 156% higher in myocytes isolated from HS animals compared with control animals. Sulfonylurea receptor 1 protein expression was increased in the left atrial appendage and right atrial appendage of HS animals (415% and 372% of NS animals, respectively). In conclusion, K(ATP) channel activation provides a mechanistic link between salt-induced elevated BP and increased atrial arrhythmia inducibility. The findings of this study have important implications for the treatment and prevention of atrial arrhythmias in the setting of hypertensive heart disease and may lead to new therapeutic approaches.     

Effects of Na(+) channel and cell coupling abnormalities on vulnerability to reentry: a simulation study

The role of dynamic instabilities in the initiation of reentry in diseased (remodeled) hearts remains poorly explored. Using computer simulations, we studied the effects of altered Na(+) channel and cell coupling properties on the vulnerable window (VW) for reentry in simulated two-dimensional cardiac tissue with and without dynamic instabilities. We related the VW for reentry to effects on conduction velocity, action potential duration (APD), effective refractory period dispersion and restitution, and concordant and discordant APD alternans. We found the following: 1). reduced Na(+) current density and slowed recovery promoted postrepolarization refractoriness and enhanced concordant and discordant APD alternans, which increased the VW for reentry; 2). uniformly reduced cell coupling had little effect on cellular electrophysiological properties and the VW for reentry. However, randomly reduced cell coupling combined with decoupling promoted APD dispersion and alternans, which also increased the VW for reentry; 3). the combination of decreased Na(+) channel conductance, slowed Na(+) channel recovery, and cellular uncoupling synergistically increased the VW for reentry; and 4) the VW for reentry was greater when APD restitution slope was steep than when it was flat. In summary, altered Na(+) channel and cellular coupling properties increase vulnerability to reentrant arrhythmias. In remodeled hearts with altered Na(+) channel properties and cellular uncoupling, dynamic instabilities arising from electrical restitution exert important influences on the VW for reentry.     

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches¹. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible¹. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization¹. The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart². It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling³. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart^4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy^8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view^10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia^14-16.     

Eliminating contraction during culture maintains global and local Ca2+ dynamics in cultured rabbit pacemaker cells

Pacemaker cells residing in the sinoatrial node generate the regular heartbeat. Ca²⁺ signaling controls the heartbeat rate—directly, through the effect on membrane molecules (NCX exchange, K⁺ channel), and indirectly, through activation of calmodulin-AC-cAMP-PKA signaling. Thus, the physiological role of signaling in pacemaker cells can only be assessed if the Ca²⁺ dynamics are in the physiological range. Cultured cells that can be genetically manipulated and/or virally infected with probes are required for this purpose. Because rabbit pacemaker cells in culture experience a decrease in their spontaneous action potential (AP) firing rate below the physiological range, Ca²⁺ dynamics are expected to be affected. However, Ca²⁺ dynamics in cultured pacemaker cells have not been reported before. We aim to a develop a modified culture method that sustains the global and local Ca²⁺ kinetics along with the AP firing rate of rabbit pacemaker cells.We used experimental and computational tools to test the viability of rabbit pacemaker cells in culture under various conditions. We tested the effect of culture dish coating, pH, phosphorylation, and energy balance on cultured rabbit pacemaker cells function. The cells were maintained in culture for 48 h in two types of culture media: one without the addition of a contraction uncoupler and one enriched with either 10 mM BDM (2,3-Butanedione 2-monoxime) or 25 μM blebbistatin. The uncoupler was washed out from the medium prior to the experiments. Cells were successfully infected with a GFP adenovirus cultured with either BDM or blebbistatin. Using either uncoupler during culture led to the cell surface area being maintained at the same level as fresh cells. Moreover, the phospholamban and ryanodine receptor densities and their phosphorylation level remained intact in culture when either blebbistatin or BDM were present. Spontaneous AP firing rate, spontaneous Ca²⁺ kinetics, and spontaneous local Ca²⁺ release parameters were similar in the cultured cells with blebbistatin as in fresh cells. However, BDM affects these parameters. Using experimental and a computational model, we showed that by eliminating contraction, phosphorylation activity is preserved and energy is reduced. However, the side-effects of BDM render it less effective than blebbistatin.     

Differences in distribution of fibrosis in the ventricles underlie dominant arrhythmia vulnerability of the right ventricle in senescent mice

Mutations that are supposed to affect right (RV) and left ventricular (LV) electrophysiology equally, often reveal dominant conduction slowing and arrhythmia vulnerability in RV. In this study we investigated the mechanism of dominant arrhythmia vulnerability of RV in senescent mice. We performed epicardial ventricular activation mapping on adult and senescent Langendorff perfused hearts. Longitudinal and transversal conduction velocity, as well as arrhythmia inducibility were determined. Subsequently, hearts were processed for immunohisto-chemistry and Picro Sirius Red staining. Senescent mice revealed decreased conduction velocity, increased aniso-tropic ratio and reduced excitation wavelength in RV, but not in LV. Arrhythmias were mainly induced in RV of senescent hearts. No arrhythmias were induced in adult hearts. Immunohistochemistry revealed that the amount of Connexin 43 and cardiac sodium channel Nav1 .5 were equally decreased, and that collagen content was equally increased in senescent RV and LV. However, patches of replacement fibrosis were found throughout the RV wall, but only in the sub-endocardium and mid-myocardium of LV. The study shows that the dominant arrhythmia vulnerability in RV of senescent mice is caused by the distribution of replacement fibrosis which involves the entire RV but only part of the LV. (Neth Heart J 2008; 16:356-8.)     

Steeper restitution slopes across right ventricular endocardium in patients with cardiomyopathy at high risk of ventricular arrhythmias

Steep action potential duration (APD) restitution slopes (>1) and spatial APD restitution heterogeneity provide the substrate for ventricular fibrillation in computational models and experimental studies. Their relationship to ventricular arrhythmia vulnerability in human cardiomyopathy has not been defined. Patients with cardiomyopathy [left ventricular (LV) ejection fraction <40%] and no history of ventricular arrhythmias underwent risk stratification with programmed electrical stimulation or T wave alternans (TWA). Low-risk patients (n = 10) had no inducible ventricular tachycardia (VT) or negative TWA, while high-risk patients (n = 8) had inducible VT or positive TWA. Activation recovery interval (ARI) restitution slopes were measured simultaneously from 10 right ventricular (RV) endocardial sites during an S1-S2 pacing protocol. ARI restitution slope heterogeneity was defined as the coefficient of variation of slopes. Mean ARI restitution slope was significantly steeper in the high-risk group compared with the low-risk group [1.16 (SD 0.31) vs. 0.59 (SD 0.19), P = 0.0002]. The proportion of endocardial recording sites with a slope >1 was significantly larger in the high-risk patients [47% (SD 35) vs. 13% (SD 21), P = 0.022]. Spatial heterogeneity of ARI restitution slopes was similar between the two groups [29% (SD 16) vs. 39% (SD 34), P = 0.48]. There was an inverse linear relationship between the ARI restitution slope and the minimum diastolic interval (P < 0.001). In cardiomyopathic patients at high risk of ventricular arrhythmias, ARI restitution slopes along the RV endocardium are steeper, but restitution slope heterogeneity is similar compared with those at low risk. Steeper ARI restitution slopes may increase the propensity for ventricular arrhythmias in patients with impaired left ventricular function.     

2,3-butanedione monoxime unmasks Ca(2+)-induced NADH formation and inhibits electron transport in rat hearts

We used 2,3-butanedione monoxime (BDM) to suppress work by the perfused rat heart and to investigate the effects of calcium on NADH production and tissue energetics. Hearts were perfused with buffer containing BDM and elevated perfusate calcium to maintain the rates of cardiac work and oxygen consumption at levels similar to those of control perfused hearts. BDM plus calcium hearts displayed higher levels of NADH surface fluorescence, indicating calcium activation of mitochondrial dehydrogenases. These hearts, however, displayed 20% lower phosphocreatine levels. BDM suppressed the rates of state 3 respiration of isolated mitochondria. Uncoupled respiration was suppressed to a lesser degree, and the state 4 respiration rates were not affected. Double-inhibitor experiments with liver mitochondria using BDM and carboxyatractyloside (CAT) were used to identify the site of inhibition. BDM at low levels (0-5 mM) suppressed respiration. In the presence of CAT at levels that inhibit respiration by 60%, low levels of BDM were without effect. Because these effects were not additive, BDM does not inhibit adenine nucleotide transport. This was supported by an assay of adenine nucleotide transport in liver mitochondria. BDM did not inhibit ATP hydrolysis by submitochondrial particles but strongly suppressed reversed electron transport from succinate to NAD(+). Oxidation of NADH by submitochondrial particles was inhibited by BDM but oxidation of succinate was not. We conclude that BDM inhibits electron transport at site 1.     

Dynamic origin of spatially discordant alternans in cardiac tissue

Alternans, a condition in which there is a beat-to-beat alternation in the electromechanical response of a periodically stimulated cardiac cell, has been linked to the genesis of life-threatening ventricular arrhythmias. Optical mapping of membrane voltage (V_m) and intracellular calcium (Ca_i) on the surface of animal hearts reveals complex spatial patterns of alternans. In particular, spatially discordant alternans has been observed in which regions with a large-small-large action potential duration (APD) alternate out-of-phase adjacent to regions of small-large-small APD. However, the underlying mechanisms that lead to the initiation of discordant alternans and govern its spatiotemporal properties are not well understood. Using mathematical modeling, we show that dynamic changes in the spatial distribution of discordant alternans can be used to pinpoint the underlying mechanisms. Optical mapping of V_m and Ca_i in paced rabbit hearts revealed that spatially discordant alternans induced by rapid pacing exhibits properties consistent with a purely dynamical mechanism as shown in theoretical studies. Our results support the viewpoint that spatially discordant alternans in the heart can be formed via a dynamical pattern formation process which does not require tissue heterogeneity.     

Spatial heterogeneity of action potential alternans during global ischemia in the rabbit heart

Cardiac ischemia causes beat-to-beat fluctuation in action potential duration (APD) alternans, which leads to T wave alternans and arrhythmias. Occurrence of APD alternans that is out of phase at two sites is especially important, but most APD alternans studies have involved rapid pacing of normal myocardium rather than ischemia. To determine the spatial features of APD alternans during ischemia, blood-perfused rabbit hearts were stained with 4-[beta-[2(di-n-butylamino)-6-napthyl]vinyl]pyridinium (di-4-ANEPPS) and imaged with a high-resolution camera. Hearts were perfused with oxygenated Tyrode solution at 37 degrees C for staining and then switched to a 50:50% blood/Tyrode mixture. Hearts were paced from the right ventricle at 3/s, and made ischemic by stopping flow for 6 min. Images of 10,000 pixels were obtained at 300 frames/s. Motion artifact was controlled by immobilization and by manual selection of undistorted single-pixel records. Upstroke propagation and conduction isochrones were displayed by computerized image processing. APD alternans was demonstrated in six of seven hearts, and was out of phase in different regions of the image in three hearts. The largest spatial variation in the onset of depolarization to 50% repolarization (APD50) was 155%. This caused beat-to-beat reversal of repolarization. An alternans map could be constructed for well-immobilized portions of the image. There were discrete regions of APD alternans separated by a boundary, as occurs with intracellular Ca2+ concentration alternans. Pixels as close together as 1.1 mm showed an APD alternans that was out of phase. The out-of-phase APD alternans was not due to conduction alternans, as shown by upstroke intervals and conduction isochrones. This contrasts with rapid pacing, where a causal relationship appears to exist. These new observations suggest distinct mechanisms for the genesis of arrhythmias during ischemia.     

Mechanisms of enhanced shock-induced arrhythmogenesis in the rabbit heart with healed myocardial infarction

Shock-induced vulnerability and defibrillation have been mostly studied in structurally normal hearts. However, defibrillation therapy is normally applied to patients with diseased hearts, frequently those with prior myocardial infarction (MI). Shock-induced vulnerability and defibrillation have not been well studied under this condition. We sought to examine the mechanisms of shock-induced arrhythmogenesis and arrhythmia maintenance in a rabbit model of healed MI (4 wk or more postinfarction). Ligation of the lateral division or posterolateral division of the left coronary artery at a level of 40-70% from the apex was performed 53 +/- 21 days before acute experiments. Shock-induced vulnerability was assessed in infarcted (n = 8) and structurally normal (n = 8) hearts by delivering internal monophasic shocks at different shock strengths and delivery phases. Electrical activities from the anterior epicardium during shock application and during shock-induced arrhythmias were optically recorded and quantitatively analyzed. Ligation resulted in a transmural left ventricular free wall infarction mainly located at the apical region with a consistent endocardial border zone (BZ) as confirmed by histological studies. There were significant increases in the incidence, severity, and duration of shock-induced arrhythmias in the infarcted hearts versus controls due to 1) postshock break-excitation wavefronts that frequently originated near the infarction BZ and 2) the existence of an infarction BZ that created an anatomic reentry pathway and facilitated arrhythmia maintenance. In conclusion, the infarction BZ contributes to both increased shock-induced arrhythmogenesis and arrhythmia maintenance in the rabbit model of healed MI.     

Action potential characterization in intact mouse heart: steady-state cycle length dependence and electrical restitution

Transgenic mice have been increasingly utilized to investigate the molecular mechanisms of cardiac arrhythmias, yet the rate dependence of the murine action potential duration and the electrical restitution curve (ERC) remain undefined. In the present study, 21 isolated, Langendorff-perfused, and atrioventricular node-ablated mouse hearts were studied. Left ventricular and left atrial action potentials were recorded using a validated miniaturized monophasic action potential probe. Murine action potentials (AP) were measured at 30, 50, 70, and 90% repolarization (APD(30)-APD(90)) during steady-state pacing and varied coupling intervals to determine ERCs. Murine APD showed rate adaptation as well as restitution properties. The ERC time course differed dramatically between early and late repolarization: APD(30) shortened with increasing S1-S2 intervals, whereas APD(90) was prolonged. When fitted with a monoexponential function, APD(30) reached plateau values significantly faster than APD(90) (tau = 29 +/- 2 vs. 78 +/- 6 ms, P < 0.01, n = 12). The slope of early APD(90) restitution was significantly <1 (0.16 +/- 0.02). Atrial myocardium had shorter final repolarization and significantly faster ERCs that were shifted leftward compared with ventricular myocardium. Recovery kinetics of intracellular Ca(2+) transients recorded from isolated ventricular myocytes at 37 degrees C (tau = 93 +/- 4 ms, n = 18) resembled the APD(90) ERC kinetics. We conclude that mouse myocardium shows AP cycle length dependence and electrical restitution properties that are surprisingly similar to those of larger mammals and humans.     

Cardiac alternans in embryonic mouse ventricles

T-wave alternans, an important arrhythmogenic factor, has recently been described in human fetuses. Here we sought to determine whether alternans can be induced in the embryonic mouse hearts, despite its underdeveloped sarcoplasmic reticulum (SR) and, if so, to analyze the response to pharmacological and autonomic interventions. Immunohistochemistry confirmed minimal sarcoplasmic-endoplasmic reticulum Ca-ATPase 2a expression in embryonic mouse hearts at embryonic day (E) 10.5 to E12.5, compared with neonatal or adult mouse hearts. We optically mapped voltage and/or intracellular Ca (Ca(i)) in 99 embryonic mouse hearts (dual mapping in 64 hearts) at these ages. Under control conditions, ventricular action potential duration (APD) and Ca(i) transient alternans occurred during rapid pacing at an average cycle length of 212 +/- 34 ms in 57% (n = 15/26) of E10.5-E12.5 hearts. Maximum APD restitution slope was steeper in hearts developing alternans than those that did not (2.2 +/- 0.6 vs. 0.8 +/- 0.4; P < 0.001). Disabling SR Ca(i) cycling with thapsigargin plus ryanodine did not significantly reduce alternans incidence (44%, n = 8/18, P = 0.5), whereas isoproterenol (n = 14) increased the incidence to 100% (P < 0.05), coincident with steepening APD restitution slope. Verapamil abolished Ca(i) transients (n = 9). Thapsigargin plus ryanodine had no major effects on Ca(i)-transient amplitude or its half time of recovery in E10.5 hearts, but significantly depressed Ca(i)-transient amplitude (by 47 +/- 8%) and prolonged its half time of recovery (by 18 +/- 3%) in E11.5 and older hearts. Embryonic mouse ventricles can develop cardiac alternans, which generally is well correlated with APD restitution slope and does not depend on fully functional SR Ca(i) cycling.     

The Transfer Functions of Cardiac Tissue during Stochastic Pacing

The restitution properties of cardiac action potential duration (APD) and conduction velocity (CV) are important factors in arrhythmogenesis. They determine alternans, wavebreak, and the patterns of reentrant arrhythmias. We developed a novel approach to characterize restitution using transfer functions. Transfer functions relate an input and an output quantity in terms of gain and phase shift in the complex frequency domain. We derived an analytical expression for the transfer function of interbeat intervals (IBIs) during conduction from one site (input) to another site downstream (output). Transfer functions can be efficiently obtained using a stochastic pacing protocol. Using simulations of conduction and extracellular mapping of strands of neonatal rat ventricular myocytes, we show that transfer functions permit the quantification of APD and CV restitution slopes when it is difficult to measure APD directly. We find that the normally positive CV restitution slope attenuates IBI variations. In contrast, a negative CV restitution slope (induced by decreasing extracellular [K⁺]) amplifies IBI variations with a maximum at the frequency of alternans. Hence, it potentiates alternans and renders conduction unstable, even in the absence of APD restitution. Thus, stochastic pacing and transfer function analysis represent a powerful strategy to evaluate restitution and the stability of conduction.     

Mechanisms of the acute ischemia-induced arrhythmogenesis--a simulation study

The underlying ionic mechanisms of ischemic-induced arrhythmia were studied by the computer simulation method. To approximate the real situation, ischemic cells were simulated by considering the three major component conditions of acute ischemia (elevated extracellular K(+) concentration, acidosis and anoxia) at the level of ionic currents and ionic concentrations, and a round ischemic zone was introduced into a homogeneous healthy sheet to avoid sharp angle of the ischemic tissue. The constructed models were solved using the operator splitting and adaptive time step methods, and the perturbation finite difference (PFD) scheme was first used to integrate the partial differential equations (PDEs) in the model. The numerical experiments showed that the action potential durations (APDs) of ischemic cells did not exhibited rate adaptation characteristic, resulting in flattening of the APD restitution curve. With reduction of sodium channel availability and long recovery of excitability, refractory period of the ischemic tissue was significantly prolonged, and could no longer be considered as same as APD. Slope of the conduction velocity (CV) restitution curve increased both in normal and ischemic region when pacing cycle length (PCL) was short, and refractory period dispersion increased with shortening of PCL as well. Therefore, dynamic changes of CV and dispersion of refractory period rather than APD were suggested to be the fundamental mechanisms of arrhythmia in regional ischemic myocardium.