Subendocardial Purkinje Fibers Research Articles

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease.

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 µm) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 µm) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

A review of cardiac autonomics: from pathophysiology to therapy.

The effective management of cardiovascular diseases requires knowledge of intrinsic and extrinsic innervation of the heart and an understanding of how perturbations of said components affect cardiac function. The innate cardiac conduction system, which begins with cardiac pacemaker cells and terminates with subendocardial Purkinje fibers, is modulated by said systems. The intrinsic component of the cardiac autonomic nervous system, which remains incompletely elucidated, consists of intracardiac ganglia and interconnecting neurons that tightly regulate cardiac electrical activity. Extrinsic components of the autonomic nervous system, such as carotid baroreceptors and renin-angiotensin-aldosterone system, modulate sympathetic input to the heart through the stellate ganglion and parasympathetic input via the vagus nerve. There remains a need for additional therapies to treat conditions, such asadvanced heart failure and refractory arrhythmias, and a better understanding of autonomics may be key to their development.

The fine-scale architecture of defibrillation

See article by Sharifov et al. (pages 448–456) in this issue Ventricular fibrillation is fatal, unless it self-terminates or is terminated by an intervention. It probably is partially responsible for most non-violent deaths and is the immediate cause of death in most sudden cardiac deaths, which form up to a fifth of adult premature deaths in the developed world [1]. The only effective treatment is prompt defibrillation by a brief, large-amplitude electrical shock that produces a field of more than 5 V/cm in the myocardium. Since DC cardiac defibrillation/cardioversion was pioneered by Lown [2] in the 1960s, there have been substantial improvements in the engineering of defibrillators, leading to implantable devices for high-risk patients and public access automatic defibrillators in public areas [1,3]. However, the cellular and tissue electrophysiological mechanisms that underlie defibrillation remain poorly understood and controversial. Improvement in the spatial resolution of optical recording of the spatio-temporal pattern of activity across the ventricular wall produced by electrical shocks has helped to clarify these mechanisms. Fibrillation is produced by irregular, self-sustained propagation of activation wave fronts through the ventricle. The wave front will propagate into tissue that has recovered its excitability (the excitable gap), with a velocity that depends on both its local curvature and the relative refractoriness of the tissue (determined by the time since the previous activation, or the rate of activity). During normal sinus rhythm, the ventricular activation wave front forms a continuous surface, propagating from the sub-endocardial Purkinje fibers towards the epicardium. At a line break in the activation surface, produced by some heterogeneity, propagation may curl back on itself and … *Tel.: +44 113 343 4251; fax: +44 113 343 4230. Email address: arun{at}cbiol.leeds.ac.uk

Significant damage of the conduction system during cardioplegic arrest is due to necrosis not apoptosis.

Ventricular conduction disturbances following cardioplegic arrest remains a serious, yet unsolved problem. In the present study we examined whether myocardial conduction cells (MCC, Purkinje fibers) are more vulnerable to ischemia/reperfusion injury than working myocardial cells and whether the damage is due to necrosis or apoptosis. Mini-pigs were subjected to 60 min of crystalloid (St Thomas; n = 15 group I) or blood (Buckberg; n = 6 group II) cardioplegic arrest followed by 3 h of reperfusion. Animals not subjected to either procedures served as controls (n = 5). Ventricular myocardial specimens were investigated by hematoxylin and eosin (HE) and periodic acid Schiff (PAS) staining and immunohistochemical labeling of apoptosis-associated proteins (Bax, Bcl-2, Caspase-3). DNA-breaks were visualized by in situ end labeling (terminal deoxynucleotidyl transferase dUTP-biotin nick-end labeling, TUNEL). Electron microscopy confirmed apoptosis or necrosis. MCC of control hearts intrinsically expressed Bax, Bcl-2, and Caspase-3 without signs of either apoptotic or necrotic damage. Subendocardial Purkinje fibers of groups I and II hearts exhibited focal damage, with reduced labeling of apoptosis-associated proteins, glycogen loss, karyopycnosis and increased eosinophilia (15/21 hearts). The majority of damaged MCC displayed nuclear TUNEL-positivity (2.8+/-2.5% of MCC), whereas the average TUNEL-rate of the adjacent working myocardium was low (<0.1%). Electron microscopy demonstrated ischemic changes in MCC consistent with cellular necrosis. Ischemia/reperfusion injury due to cardioplegic arrest inflicts significant damage on subendocardial MCC, but not on working myocardium. Ultrastructural and light-microscopic findings are consistent with coagulation necrosis, rather than apoptosis.

Ca(2+) transients and Ca(2+) waves in purkinje cells : role in action potential initiation.

Purkinje cells contain sarcoplasmic reticulum (SR) directly under the surface membrane, are devoid of t-tubuli, and are packed with myofibrils surrounded by central SR. Several studies have reported that electrical excitation induces a biphasic Ca(2+) transient in Purkinje fiber bundles. We determined the nature of the biphasic Ca(2+) transient in aggregates of Purkinje cells. Aggregates (n=12) were dispersed from the subendocardial Purkinje fiber network of normal canine left ventricle, loaded with Fluo-3/AM, and studied in normal Tyrode's solution (24 degrees C). Membrane action potentials were recorded with fine-tipped microelectrodes, and spatial and temporal changes in [Ca(2+)](i) were obtained from fluorescent images with an epifluorescent microscope (x20; Nikon). Electrical stimulation elicited an action potential as well as a sudden increase in fluorescence (L(0)) compared with resting levels. This was followed by a further increase in fluorescence (L(1)) along the edges of the cells. Fluorescence then progressed toward the Purkinje cell core (velocity of propagation 180 to 313 microm/s). In 62% of the aggregates, initial fluorescent changes of L(0) were followed by focally arising Ca(2+) waves (L(2)), which propagated at 158+/-14 microm/s (n=13). Spontaneous Ca(2+) waves (L(2)*) propagated like L(2) (164+/-10 microm/s) occurred between stimuli and caused slow membrane depolarization; 28% of L(2)* elicited action potentials. Both spontaneous Ca(2+) wave propagation and resulting membrane depolarization were thapsigargin sensitive. Early afterdepolarizations were not accompanied by Ca(2+) waves. Action potentials in Purkinje aggregates induced a rapid rise of Ca(2+) through I(CaL) and release from a subsarcolemmal compartment (L(0)). Ca(2+) release during L(0) either induced further Ca(2+) release, which propagated toward the cell core (L(1)), or initiated Ca(2+) release from small regions and caused L(2) Ca(2+) waves, which propagated throughout the aggregate. Spontaneous Ca(2+) waves (L(2)*) induce action potentials.

Cellular basis for the normal T wave and the electrocardiographic manifestations of the long-QT syndrome.

This study probes the cellular basis for the T wave under baseline and long-QT (LQT) conditions using an arterially perfused canine left ventricular (LV) wedge preparation, which permits direct temporal correlation of cellular transmembrane and ECG events. Floating microelectrodes were used to record transmembrane action potentials (APs) simultaneously from epicardial, M-region, and endocardial sites or subendocardial Purkinje fibers. A transmural ECG was recorded concurrently. Under baseline and LQT conditions, repolarization of the epicardial action potential, the earliest to repolarize, coincided with the peak of the T wave; repolarization of the M cells, the last to repolarize, coincided with the end of the T wave. Thus, the action potential duration (APD) of the longest M cells determine the QT interval and the Tpeak-Tend interval serves as an index of transmural dispersion of repolarization. Repolarization of Purkinje fibers outlasted that of the M cell but failed to register on the ECG. The morphology of the T wave appeared to be due to currents flowing down voltage gradients on either side of the M region during phase 2 and phase 3 of the ventricular action potential. The interplay between these opposing forces determined the height of the T wave as well as the degree to which the ascending or descending limb of the T wave was interrupted, giving rise to bifurcated T waves and "apparent T-U complexes" under LQT conditions. Spontaneous and stimulation-induced polymorphic ventricular tachycardia with characteristics of torsade de pointes (TdP) developed in the presence of dl-sotalol. Our results provide the first direct evidence that opposing voltage gradients between epicardium and the M region and endocardium and the M region contribute prominently to the inscription of the ECG T wave under normal conditions and to the widened or bifurcated T wave and long-QT interval observed under LQT conditions. Our data suggest that the "pathophysiological U" wave observed in acquired or congenital LQTS is more likely to be a second component of an interrupted T wave, and argue for use of the term T2 in place of U to describe this event.

Zatebradine slows ectopic ventricular rhythms in canine heart 24 hours after coronary artery ligation.

Arrhythmias occur 24 h after occlusion of the left anterior descending (LAD) coronary artery in the canine heart and have been attributed to the abnormal spontaneous activity in subendocardial Purkinje fibers, which are markedly depolarized. The major current underlying normal automaticity in these fibers is i(f). Although the i(f) activation range is generally considered to be more negative than the diastolic membrane potential in these depolarized fibers in infarcts, this activation range has been shown to shift in a positive direction in response to hormonal influences. Thus i(f) could still mediate automaticity in these fibers in infarcts. Furthermore, recent reports indicate that a depolarizing diastolic current, probably i(f), also can be measured in ventricular muscle during abnormal experimental conditions, which may occur during ischemia. To test whether there is a role of i(f) currents in sustaining ventricular ectopy, we administered the selective i(f) channel blocker, zatebradine, 24 h after LAD ligation in canine hearts. We report that intravenous injections of zatebradine (0.25 or 1.0 mg/kg) significantly slow ventricular rhythms (with average reductions of 19 or 26%, respectively). Moreover, because zatebradine also slows sinus nodal rate, it can lead to an increased incidence of ectopic beats. However, during right atrial pacing, when sinus slowing has no effect on ventricular rhythms, capture of ventricular rhythms occurs at lower rates in the presence of zatebradine. The reduction of capture threshold is comparable to the reduction in the rate of the ectopic rhythm. Thus zatebradine eliminated the arrhythmia when the right atrium was paced at the original sinus rate.

Effects of chemical subendocardial ablation on activation rate gradient during ventricular fibrillation.

The mechanism by which an endocardial-epicardial activation rate gradient develops after 1 or 2 min of sustained ventricular fibrillation is unknown. We recorded from electrodes on the epicardium and from hook electrodes in the endocardium in three open-chest control dogs during prolonged ventricular fibrillation. The same recordings were also made in seven dogs after right ventricular subendocardial ablation with Lugol solution and in three dogs after substitution of air for the cavitary blood. The effects of these interventions, i.e., Lugol ablation (n = 2) and the exposure to air (n = 2), on the subendocardial Purkinje fiber transmembrane action potential properties were also evaluated in vitro using microelectrode recording techniques. The in vivo studies showed a significant endocardial-epicardial rate gradient in the control dogs and in dogs that had air substituted for the cavitary blood. In comparison, in dogs that underwent chemical subendocardial ablation, the activation cycle lengths for the endocardium and epicardium were not significantly different. The in vitro studies showed that subendocardial Purkinje fiber action potentials could still be recorded for up to 10 min of exposure to air. In comparison, in the tissues subjected to chemical ablation, no transmembrane action potentials could be recorded from either the Purkinje fibers or superficial ventricular muscle cells. We conclude that the development of an endocardial-epicardial activation rate gradient during prolonged ventricular fibrillation depends on the presence of intact subendocardial Purkinje fibers and ventricular myocytes. The retained cavitary blood is not responsible for the development of the rate gradient.

Enrichment of G protein α-subunit mRNAs in the AV-conducting system of the mammalian heart

We investigated the expression pattern of the heterotrimeric G proteins Gs alpha, Gi alpha-2 and Go alpha in rat and guinea-pig heart by in situ hybridization. Cryosections were hybridized with single-stranded 35S-cRNA probes complementary to subtype-specific sequences of the respective mRNAs. Hybridization signals were visualized by exposition to X-ray films and dipping autoradiography. The rank order of abundance was Gi alpha-2 approximately Gs alpha >> Go alpha. In general, G protein alpha-subunit mRNAs were evenly distributed in the heart including endo- and epicardium, large vessels and valves. Go alpha-mRNA levels were significantly higher in atria than in ventricles. In contrast to the rather uniform labeling of working myocardium, expression of all three G proteins was enriched in small intramural blood vessels and in subendocardial Purkinje fibers of septum and papillary muscles. A more marked enrichment of Gs alpha-, Gi alpha-2- and especially Go alpha-mRNA was seen in neuronal ganglionic cells in the atrial septum and posterior regions of the atrium. The main finding, however, was an enrichment of all three G protein mRNAs in the atrioventricular conductive tissue. The accumulation was strictly co-localized with acetylcholinesterase-positive regions identified as the atrioventricular node, the bundle of His and the right and left bundle branches and was seen similarly in rat and guinea-pig hearts. Quantitative in situ hybridization revealed Gs alpha-, Gi alpha-2- and Go alpha-mRNA levels in the bundle of His to be 206 +/- 0.13%. 191 +/- 0.15% and 165 +/- 0.06%, respectively, of that in the surrounding interventricular working myocardium. These findings indicate that heterotrimeric G proteins play an important role in modulation of electrical conductance in the heart.

Electrophysiologic effects of cocaine on subendocardial Purkinje fibers surviving 1 day of myocardial infarction.

Cocaine has been shown to have broad cardiovascular effects that could be life threatening. Most of the reported electrophysiologic effects of cocaine have been studied in normal but not infarcted myocardium. Using microelectrode techniques, we investigated the electrophysiologic effects of cocaine on endocardial canine Purkinje fibers that survived 1 day of myocardial infarction. In quiescent infarcted preparations, stimulated trains were followed by subthreshold delayed afterdepolarizations (DADs), in the presence of propranolol (1 microM). Cocaine (10 microM) decreased the amplitude of DADs from 6.1 +/- 1.8 mV to 3.0 +/- 1.3 mV (P < 0.05, n = 6). When stimulated preparations (n = 23) showing no triggered activity during control (+propranolol) were superfused with a low concentration of caffeine (1 mM) or high extracellular Ca2+ (8.1 mM), triggered activity was induced. Subsequent cocaine (10 microM) superfusion prevented the induction of caffeine- and high Ca(2+)-induced triggered activity. Cocaine's effects were reversible upon washout. In preparations that showed triggered activity during control conditions (+propranolol), the mean cycle length of triggered activity was 755 +/- 45 msec. Cocaine (10 microM) superfusion lengthened the cycle length to 1030 +/- 141 msec and terminated triggered activity with a subthreshold DAD (n = 12). In addition, cocaine and ryanodine (10 microM) suppressed triggered activity in a similar manner when tested in the same preparations (n = 4). During control conditions, cocaine did not cause any significant change on the rate of rise of action potential upstroke (from 55.6 +/- 24.3 to 54.5 +/- 28.6 V/sec, n = 8) and maximum diastolic potential (from -58.4 +/- 4.3 to -56.6 +/- 6.5 mV, n = 8). In the absence of propranolol, 50 microM but not 10 microM cocaine induced early afterdepolarizations in 62% of the preparations exhibiting triggered activity during control conditions. The results suggest that cocaine modulates DADs and triggered activity in infarcted endocardial fibers via direct inhibition of cyclic release of Ca2+ from sarcoplasmic reticulum (SR) independently from a local anesthetic or sympathomimetic effect. This SR inhibition could account for the myocardial depressant effect of cocaine. However, while cocaine suppressed DADs, its induction of EADs can precipitate malignant ventricular arrhythmias in the setting of cocaine overdose and infarction.

Electrophysiologic characteristics of M cells in the canine left ventricular free wall.

Recent studies have described the existence of M cells in the deep structures of the canine and human ventricle. The present study was designed to further characterize the M cell with respect to its distribution across the canine left ventricular free wall and the dependence of its action potential on [K+]o. We used standard microelectrode techniques to record transmembrane activity from deep subepicardial or transmural strips isolated from the canine left ventricular free wall near the base as well as subendocardial Purkinje fibers. M cells behavior (steep APD-rate relation) was observed at depths of 1 to 7 mm from the epicardial surface (deep subepicardium to mid-myocardium). M cells were found to be distributed uniformly in the deep subepicardium and did not appear in discrete bundles. We observed transitional behavior throughout the wall. The maximum rate of rise of the action potential upstroke, Vmax, increased sharply between epicardium and deep subepicardium (176 +/- 13 to 332 +/- 61 V/sec), remained high throughout the mid-myocardium and deep subendocardium, and returned to lower values only in the superficial layers of the endocardium (205 +/- 21 V/sec). The relationship between Vmax and takeoff potential in the M cell was fit by a Boltzmann equation with a V0.5 of -68.6 +/- 1.5 mV and k of 3.4 +/- 0.5. The relationship between resting membrane potential (RMP) and [K+]o in the M cell was exponential from 8 to 20 mmol/L (58 mV change in RMP per 10-fold change in [K+]o), deviating from K+ electrode behavior at [K+]o < 8 mmol/L. RMP in M cells continued to hyperpolarize at [K+]o < 2.5 mmol/L, reaching potentials of approximately -110 mV at [K+]o of 1 mmol/L. In contrast, subendocardial Purkinje fibers depolarized at these low levels of [K+]o. Unlike endocardium and epicardium, M cells developed early afterdepolarizations at low [K+]o and slow rates. Our data indicate that the M cells are widely distributed in the intramural layers of the canine left ventricular free wall. M cells and transitional cells occupy 30% to 40% of the left ventricular wall and an estimated 20% to 40% of the mass of the ventricles of the normal canine heart. They display characteristics common to both myocardial and specialized conducting cells. Like Purkinje fibers, M cells exhibit a relatively large Vmax and steep APD-rate relations that are modulated by [K+]o. Unlike Purkinje fibers, M cells do not appear in bundles, they do not depolarize at [K+]o < 2.5 mmol/L, nor do they exhibit phase 4 depolarization.

Transient outward currents in subendocardial Purkinje myocytes surviving in the infarcted heart.

Altered electrical activity of subendocardial Purkinje fibers contributes to arrhythmias in the 48-hour infarcted canine heart. Changes in the transmembrane action potentials of these fibers include marked action potential prolongation. The ionic basis for these changes is unknown. We used whole-cell voltage-clamp techniques to study 4-aminopyridine (4-AP)-sensitive voltage-dependent transient outward currents (Ito1) in Purkinje myocytes isolated from LV subendocardial (n = 14) and free-running (n = 15) bundles of the normal canine heart. Ito1 in these two groups of control cells (normal-zone Purkinje cells [NZPCS]) did not differ. NZPCS Ito1 was then compared with Ito1 of Purkinje myocytes dispersed from subendocardium of infarcted hearts 48 hours after total coronary artery occlusion (IZPC48, n = 14). Ito1 amplitude and current density were significantly reduced (P < .01) in IZPC48s (1650 +/- 389 pA, 9 +/- 2 pA/pF) compared with NZPCS (2917 +/- 267 pA, 20.2 +/- 2 pA/pF) at Vt = +55 mV, Vh = -60 mV, where Vt is test potential and Vh is holding potential. Decay of Ito1 was biexponential in all NZPCS but monoexponential in 71% of IZPC48s. Both NZPCS and IZPC48s have a sustained 4-AP-sensitive component (at 250 ms, Vt = +55 mV: 4 +/- 1 pA/pF, 3 +/- 1 pA/pF, respectively). Ito1 voltage dependence of inactivation did not differ between groups. In IZPC48s, recovery of Ito1 from inactivation was slowed significantly. Furthermore, significantly more Ito1 was seen with rapid pacing in NZPCS (cycle length [CL] 5000 ms = 100%, CL 1300 ms = 73%, CL 330 ms = 46%) than in IZPC48s (CL 5000 ms = 100%, CL 1300 ms = 58%, CL 330 ms = 31%). In three IZPC48s, no Ito1 was seen at CL 330 ms. Ito1 plays a major role in normal Purkinje myocyte electrophysiology, contributing both a large transient and a sustained component that are 4-AP-sensitive. In subendocardial Purkinje myocytes that survive in the 48-hour infarcted heart, density of Ito1 is markedly reduced and the remaining Ito1 showed specific changes in kinetics. The alterations observed in both Ito1 density and function could contribute to abnormally long transmembrane action potentials of these arrhythmogenic Purkinje myocytes of the infarcted heart.

Reversibility of Electrophysiological Abnormalities in Subacute Ischemia

Twenty-four hours after occlusion of the left anterior descending coronary artery in the dog, ventricular tachycardia is the predominant rhythm. At this time, records of transmembrane potentials from the subendocardial Purkinje fibers adjacent to the infarct show a low maximum diastolic potential, prominent phase 4 depolarization, and slow response action potentials. Exposure of the fibers to pinacidil, 25-100 microM, increases resting potential to the estimated value of EK, abolishes the phase 4 depolarization, and restores action potential amplitude and Vmax toward normal. Perfusion of the bed of the occluded coronary artery with Tyrode's solution prior to isolation of the subendocardial tissues results in similar normalization of transmembrane potentials. These findings indicate: (a) that the major cause of the abnormal transmembrane potentials of the subendocardial tissues is the loss of resting potential; and (b) that abnormalities of the transmembrane potentials are caused by some substance that can be washed out by perfusion and not by a direct effect of ischemia.

Reduced calcium currents in subendocardial Purkinje myocytes that survive in the 24- and 48-hour infarcted heart.

The abnormal transmembrane action potentials of subendocardial Purkinje fibers that survive 24 to 48 hours after coronary artery occlusion can be a source of the multiform ventricular tachycardias that occur during this time. A change in the density or function of either or both the T-type and L-type cardiac Ca2+ channels may contribute to the altered electrical activity of these Purkinje myocytes. The purpose of this study was to determine the function of the T- and L-type Ca2+ currents (iCat and iCaL, respectively) in Purkinje myocytes dispersed from the subendocardium of the left ventricle 24 and 48 hours after coronary artery occlusion (IZPC24 and IZPC48, respectively). To do this we compared whole-cell Ca2+ currents from Purkinje myocytes enzymatically dispersed from free-running fiber bundles (SPCs), from the subendocardium of the noninfarcted canine heart (NZPCs), and from IZPC24 and IZPC48. ICaL and iCat were recorded with Cs(+)- and EGTA-rich pipettes and in Na(+)-K(+)-free external solutions to eliminate overlapping currents. ICaL density was significantly reduced in IZPC48 compared with NZPC or IZPC24. This was not accompanied by a shift in the current-voltage relation or by a change in the time course of decay of iCaL. Replacement of Ca2+ with equimolar Ba2+ increased iCaL density in all cell types, but peak iBaL of IZPC48 remained reduced compared with control iBaL values. T-type Ca2+ currents were recorded in all SPCs and NZPCs. In IZPC24 and IZPC48 there was a reduction in peak iCat amplitudes and densities. This was not accompanied by a shift in the current-voltage relation or by a change in the time course of decay of peak iCat. However, there was a hyperpolarizing shift in the steady-state availability relations in both IZPC24 and IZPC48. In addition, the maximally available iCat in IZPC24 was not different from control, whereas it was significantly reduced in IZPC48. The L-type ICa density in subendocardial Purkinje myocytes that survive in the infarcted heart is significantly decreased by 48 hours after the time of coronary artery occlusion. The peak T-type ICa density is decreased in subendocardial Purkinje myocytes that survive in the infarcted heart at 24 hours, but further reduction occurs in these myocytes by 48 hours. This loss in Ca2+ channel function could contribute to the abnormal transmembrane potentials of these myocytes surviving in the infarcted heart.

Reversibility of electrophysiologic abnormalities of subendocardial Purkinje fibers induced by ischemia.

During the subacute phase of infarction in the canine heart, the subendocardial Purkinje fibers subtended by the infarct show depolarization greater than can be accounted for by the decrease in [K+]i, and generate abnormal action potentials and spontaneous rhythms due to abnormal automaticity. We have used pinacidil to hyperpolarize these fibers and evaluate the extent to which an increase in resting potential can normalize action potential generation. Twenty-four hours after two-stage ligation of the canine left anterior descending coronary artery, preparations of subendocardial Purkinje fibers were studied in vitro by recording transmembrane potentials through standard microelectrodes and exposing the preparation to pinacidil and increases in [K+]o. Pinacidil increased resting potential to the estimated value of EK, abolished the abnormal automaticity, and restored action potentials of normal amplitude with normal values of Vmax. This effect often persisted after washout of pinacidil. Elevation of [K+]o from 4.0 to 20.0 mM slightly increased maximum diastolic potential, suggesting that the excess (over the change in EK) depolarization was caused by a decrease in gK1. The ventricular arrhythmias seen during the subacute stage of infarction probably are caused by abnormal automaticity. Our findings support the conclusion that this abnormal automaticity arises in partially depolarized subendocardial Purkinje fibers. This loss of resting potential is due in large part to a decrease in gK1. Restoration of resting potential to the value of EK permits the Purkinje fibers to develop essentially normal action potentials. An agent capable of reversing the partial block of IK,1 thus might be an effective drug for some types of arrhythmias.

Drug‐Induced Afterdepolarizations and Triggered Activity Occur in a Discrete Subpopulation of Ventricular Muscle Cells (M Cells) in the Canine Heart:

Oscillations of membrane potential that attend or follow the cardiac action potential and depend on preceding transmembrane activity for their manifestation are known as afterdepolarizations. Early afterdepolarizations (EADs) interrupt or retard repolarization of the cardiac action potential, whereas delayed afterdepolarizations (DADs) arise after full repolarization. EADs and DADs can give rise to spontaneous action potentials or triggered activity believed to be responsible for a variety of cardiac arrhythmias. Recent studies from our laboratory have highlighted differences in the electrophysiology and pharmacology of three functionally distinct myocardial cell types found in the canine ventricle. Epicardial, M region, and endocardial tissues and cells show distinct, sometimes opposite, responses to a variety of drugs, including those capable of inducing EADs and DADs. In the present study, we used standard microelectrode techniques to examine the pharmacologic response of these cellular subtypes to therapeutic levels of quinidine and toxic levels of digitalis. Quinidine readily produced prominent EADs and EAD-induced triggered activity in tissue preparations from the M region (deep subepicardium), but not in those from epicardium, endocardium, or deep subendocardium of the canine ventricle. Acetylstrophanthidin produced prominent DADs in M cell preparations and subendocardial Purkinje fibers but only minute DADs, if any, in epicardium, endocardium, or deep subendocardium. DAD-induced triggered activity was observed to arise only in Purkinje and M cells and never in myocardial tissues from the epicardial, endocardial, or deep subendocardial regions of the ventricular wall. We conclude that EADs, DADs, and triggered activity caused by therapeutic levels of quinidine and toxic levels of digitalis are limited to or much more readily induced in a select population of cells in the deep subepicardial (M cell) region of the canine ventricle in addition to the Purkinje system of the heart.

Effects of subendocardial ablation on anodal supernormal excitation and ventricular vulnerability in open-chest dogs.

In Langendorff-perfused hearts and in hearts on cardiopulmonary bypass, chemical ablation of the subendocardium of both ventricles decreases ventricular vulnerability to fibrillation. It was hypothesized that the effects of ablation are a result of the elimination of the subendocardial Purkinje fiber network. This hypothesis has been supported by recent observations that the supernormal excitability that is demonstrable in the Purkinje fibers is associated with arrhythmogenesis. We tested this hypothesis on 10 open-chest dogs by evaluating the strength-interval curves of anodal and cathodal stimulation with the assistance of computerized mapping techniques. The ventricular fibrillation threshold was also determined. The same test was then performed after chemical ablation of the subendocardium of either the right ventricle (six dogs) or both ventricles (four dogs). Anodal supernormality was consistently demonstrated in all the dogs studied both before and after subendocardial ablation. The ventricular fibrillation thresholds were 23 +/- 5 mA both before and after right ventricular subendocardial ablation (p = NS). The ventricular fibrillation thresholds before and after biventricular subendocardial ablation were 25 +/- 3 and 22 +/- 10 mA, respectively (p = NS). We conclude that 1) subendocardial ablation does not decrease ventricular vulnerability when the heart is in situ and is not on cardiopulmonary bypass and 2) anodal supernormal excitability can be demonstrated in ventricles without a subendocardial Purkinje fiber network.

Patterns of Structural Deterioration Due to Ischemia in Purkinje Fibres and Different Layers of the Working Myocardium

Cellular and mitochondrial swelling are regarded as typical intra-ischemic alterations ("IIA"), contraction band lesions (CBL), in contrast, as products of post-ischemic reperfusion. The occurrence of both types of structural deterioration was investigated in Purkinje fibres and subendocardial and intramural working myocardium: initially after St. Thomas- or HTK cardioplegia, then during ensuing global ischemia up to the "practical limit of resuscitability", and following post-ischemic reperfusion. Generally, Purkinje fibres are not better preserved than neighbouring working myocardium. Comparing St. Thomas- and HTK cardioplegia, considerable quantitative, but not qualitative differences in the reaction patterns of different cell types or layers arise. Immediately after cardioplegia, CBL are completely lacking in both cell types. During ischemia, CBL occur occasionally in Purkinje fibres and seldom in subendocardial working myocardium, "IIA" predominate. During post-ischemic reperfusion "IIA" tend to reverse in all layers, whereas CBL are found to remain in the subendocardial cell types. In intramural layers, CBL occur only during reperfusion. Thus, we deduce that cardioplegia only modulates the severity of "IIA" and the frequency of CBL, but cannot abolish the particular sensitivity of subendocardial Purkinje fibres to global ischemia. Prerequisites for the development of irreversible CBL are on the one hand ischemic metabolic alterations and corresponding energy deficits, and, on the other hand, a supply of oxygen. The oxygen may be inadequately supplied via diffusion during ischemia or may be subsequently provided by reperfusion.

Ventricular tachycardia after in vivo DC shock ablation in dogs. Electrophysiologic and histologic correlation.

DC shock catheter ablation for the treatment of ventricular tachycardia (VT) may induce VT episodes that disappear within days. A 30-J cathodal shock was delivered to the endocardial left ventricular wall in 15 closed-chest dogs. All dogs had VT during the first day after ablation. Eleven of these dogs were studied on the first day. Extensive epicardial and endocardial activation mapping in vivo, in Langendorff-perfused hearts, and in tissue blocks in a tissue bath localized the site of origin of VT to subendocardial Purkinje fibers in a border zone surrounding the central necrotic ablation lesion. Intracellular recording showed that this zone consisted of a subendocardial superficial layer (SSL) of cells with abnormal characteristics, a resting membrane potential (RMP) of -58 +/- 11 mV (mean +/- SD), and an action potential amplitude (APA) of 61 +/- 20 mV. In addition, the steepness of phase 0 of the action potential was markedly reduced. In three dogs abnormal automaticity was found in a very small area. Immediately below the SSL, cells were normal with an RMP of -78 +/- 5 mV and an APA of 107 +/- 8 mV. Histology confirmed a thin SSL with edematous and necrotic cells, hemorrhage, and infiltration. The other four dogs were studied at 1 week after ablation when VT was absent. Microelectrode impalement in the SSL was either impossible or showed nearly normal action potential characteristics. Histological examination showed a markedly thickened fibrotic subendocardial layer at places where impalement was impossible. Normal subendocardium was found in other areas of the border zone. Our results indicate that VT after DC shock ablation originates from cells with abnormal automaticity in the superficial subendocardial border zone around the central ablation lesion. Within 1 week edematous and necrotic cells in this border zone are replaced by a fibrotic layer, and this transition is associated with the disappearance of VT.