Passive electrical properties, mechanical activity, and extracellular potassium in arterially perfused and ischemic rabbit ventricular muscle. Effects of calcium entry blockade or hypocalcemia

The relation among passive electrical resistive properties, longitudinal conduction velocity, extracellular potassium concentration, [K+]o, and mechanical activity was investigated in the isolated rabbit papillary muscle during normal arterial perfusion and no-flow ischemia in the presence and absence of verapamil, or a reduced extracellular Ca2+ concentration [Ca2+]o. During normal arterial perfusion, verapamil (0.5 microM, free [Ca2+]o = 1.0 mM) and hypocalcemic blood perfusate (free [Ca2+]o = 0.4 mM) reduced the maximal isometric twitch tension by 48% and 78%, depolarized the resting membrane by +3 and +7 mV, decreased the extracellular longitudinal resistance (ro) by 15% and 26%, and increased conduction velocity by 4% and 6%, respectively. The changes in conduction velocity during these interventions were consistent with those predicted by linear cable theory (+3% and +9%) for the observed changes in ro. In contrast, verapamil shortened whereas a reduced [Ca2+]o lengthened action potential duration. Comparison of simultaneously measured longitudinal whole tissue resistance (rt), intracellular longitudinal resistance (ri), [K+]o, and resting tension during ischemia showed a close association between abrupt cell-to-cell electrical uncoupling, development of ischemic contracture, and the secondary rise of [K+]o, which all started to develop after approximately 15 minutes of ischemia. Electrical cell-to-cell uncoupling was completed within 15 minutes. In the presence of verapamil, the relation among the onset of electrical cell-to-cell uncoupling, secondary rise of [K+]o, and onset of ischemic contracture in ischemia was qualitatively the same as in its absence; however, these events were postponed by approximately 10 minutes, and the rates of contracture development and uncoupling were diminished. Conduction velocity decreased after 12 minutes of ischemia from 54 to 36 cm/sec in the absence of and from 61 to 46 cm/sec in the presence of verapamil. This slowing effect on impulse conduction could not be attributed to changes of electrical cell-to-cell coupling because at this time an increase in ri had not yet taken place. In the presence of a reduced [Ca2+]o, the resting tension and ri increased almost immediately after the onset of ischemia. Although the resting tension rose progressively throughout the course of ischemia, the ri showed a biphasic increase characterized by an early transient increase that reached a peak at 8 minutes (+87%) and a second, irreversible increase beginning at approximately 12 minutes. This final onset of electrical cell-to-cell uncoupling and the secondary rise of [K+]o were not different from the findings with a normal [Ca2+]o.(ABSTRACT TRUNCATED AT 400 WORDS)     

Blinded validation of the isolated arterially perfused rabbit ventricular wedge in preclinical assessment of drug-induced proarrhythmias

BACKGROUND: The development of preclinical models with high predictive value for the identification of drugs with a proclivity to induce Torsade de Pointes (TdP) in the clinic has long been a pressing goal of academia, industry and regulatory agencies alike. The present study provides a blinded appraisal of drugs, in an isolated arterially-perfused rabbit ventricular wedge preparation, with and without the potential to produce TdP. METHODS AND RESULTS: Thirteen compounds were tested for their potential for TdP using the rabbit left ventricular wedges. All investigators were blinded to the names, concentrations and molecular weights of the drugs. The compounds were prepared by the study sponsor and sent to the investigator as 4 sets of 13 stock solutions with the order within each set being assigned by a random number generator. Each compound was scored semi-quantitatively for its relative potential for TdP based on its effect on ventricular repolarization measured as QT interval, dispersion of repolarization measured as T(p-e)/QT ratio and early afterdepolarizations. Disclosure of the names and concentrations after completion of the study revealed that all compounds known to be free of TdP risk received a score of less or equal to 0.25, whereas those with known TdP risk received a score ranging from 1.00 to 7.25 at concentrations less than 100X their free therapeutic plasma C(max). CONCLUSIONS: Our study provides a blinded evaluation of the isolated arterially-perfused rabbit wedge preparation demonstrating both a high sensitivity and specificity in the assessment of 13 agents with varying propensity for causing TdP.     

Cardiac muscle: Excitability and passive electrical properties

The field of cellular cardiac electrophysiology has made excellent progress in the last decade in its effort to understand the electrical properties of the heart. It has profited from progress in membrane electrophysiology that has increased our understanding of the basic ionic mechanisms. It has developed quantitative methods for study of these mechanisms, in spite of the geometrical complexity of the heart. Indeed this complexity has added a richness and challenge to this area.²⁰ We have shared with skeletal muscle the problem of electromechanical coupling.³⁷This review has discussed the present state of our knowledge of basic membrane mechanisms of importance in understanding normal and pathological function of heart muscle. It is intended to lay the groundwork for the following articles dealing with special aspects of cardiac electrophysiology. There is a continual interchange of ideas and concepts between applied and basic electrophysiology, and, as a result of this interchange, we can expect both areas to grow greatly in the coming years.     

Electrical uncoupling and increase of extracellular resistance after induction of ischemia in isolated, arterially perfused rabbit papillary muscle

Extracellular and intracellular longitudinal resistances (ro and ri), transmembrane potentials, and conduction velocity were determined in arterially blood-perfused rabbit papillary muscles. Cable analysis was made possible by placing the muscle in a H2O-saturated gaseous environment, which acted as an electrical insulator. Ischemia was produced by exchanging the O2 in the atmosphere by N2 (94% N2-6% CO2) in addition to arresting coronary flow. The first 10-15 minutes of ischemia were characterized by an increase in ro, while ri remained constant. The early part of the increase in ro coincided with the drop in perfusion pressure and was probably due to the diminution of intravascular volume. Rapid electrical uncoupling, reflected by an increase in ri (threefold within 5 minutes), occurred thereafter. The dissociation between the early increase in ro and the delayed increase in ri produced an initial increase in the ratio ro:ri, which subsequently decreased. The decrease in conduction velocity was less than observed in intact hearts with ischemia. This difference is explained by the relatively small changes in resting membrane potential and action potential amplitude in the preparation used. Our results suggest that in the early, reversible phase of ischemia, the increase in ro contributes to a small but significant extent to the slowing of conduction. After 15-20 minutes, the rapid cellular uncoupling, which was most likely coincident with breakdown of cellular homeostasis, may contribute to the occurrence of arrhythmias during this phase of ischemia. Moreover, the early change in the ratio ro:ri will influence the amplitude of the extracellular electrograms following coronary occlusion (TQ-segment and ST-segment shifts).     

Influence of oxygen on perfused capillary density and capillary red cell velocity in rabbit skeletal muscle

The pattern of capillary blood flow changes was investigated in rabbit tenuissimus muscle as a function of oxygen tension in the solution suffusing the muscle. The density of perfused capillaries decreased from 269 ± 22 (mean ± SD) to 6 ± 10 cap/mm² when ambient pO₂ was elevated progressively from 5 to 100 mm Hg. In the same pO₂ range capillary red cell velocity decreased from 0.29 ± 0.14 (mean ± SD) to 0.18 ± 0.06 mm/sec in the capillaries which were perfused. During exposure of the muscle to atmospheric oxygen tension (pO₂ = 150 mm Hg) there were no capillaries perfused and hence flow velocity was zero in all capillaries. The results demonstrate that oxygen influences capillary blood flow over an extensive range of oxygen tensions. The anatomical structures responsible for the alterations in both capillary density and capillary flow velocity are the arterioles. This site of control determines actively the total blood flow through the capillary bed, whereas passive factors seem to determine the distribution of the flow between specific capillaries. The results do not support the existence of precapillary sphincters controlling perfused capillary density.     

Effect of glibenclamide on extracellular potassium accumulation and the electrophysiological changes during myocardial ischaemia in the arterially perfused interventricular septum of rabbit

STUDY OBJECTIVE--The aim was to study the effects of glibenclamide on the rate of rise of extracellular potassium concentration ([K+]o) and the electrophysiological changes that occur during myocardial ischaemia. DESIGN--The study was performed in isolated, arterially perfused interventricular septa from the rabbit. Six septa were treated with glibenclamide 10(-6) mol.litre-1 and there were six untreated controls (vehicle only). [K+]o and electrophysiological variables were compared before and during a 30 min period of global zero flow ischaemia. MEASUREMENTS AND MAIN RESULTS--Prior to ischaemia, the extracellular potassium concentrations measured using potassium sensitive valinomycin electrodes were similar in the control and glibenclamide groups being 4.0 (SEM 0.1) and 4.0 (0.1) mmol.litre-1 respectively. [K+]o rose during ischaemia in both groups, and at 30 min was 13.3 (0.7) mmol.litre-1 in the control group. The increase in the glibenclamide group was less marked, reaching 9.2 (0.5) mmol.litre-1 (p less than 0.0005; unpaired t test). Glibenclamide had no electrophysiological effects prior to ischaemia. However, during ischaemia the decrease in action potential amplitude, action potential duration (APD), maximum upstroke velocity of the action potentials (dV/dtmax), and the extent of resting membrane potential (Em) depolarisation were less in the glibenclamide group than in the controls. The effective refractory period (ERP) progressively shortened over the 30 min of ischaemia in both groups, to a similar extent. When taken in conjunction with the relative changes in action potential duration the degree of post-repolarisation refractoriness (ERP-APD) that developed was less in the glibenclamide group than in the controls. CONCLUSIONS--Glibenclamide attenuated the ischaemic rise in [K+]o, with preservation of both membrane potential and action potential amplitude, duration, and upstroke velocity together with less post-repolarisation refractoriness. These effects could be potentially antiarrhythmic in acute myocardial ischaemia.     

Changes in passive electrical properties of guinea-pig ventricular muscle exposed to low K+ and high Ca2+ conditions

Changes in passive electrical properties of guinea-pig papillary muscle exposed to low K+, high Ca2+ conditions were examined using a single sucrose gap technique. While quiescent preparations exposed for 5 mins did not develop delayed afterdepolarizations, those placed in the test solution for 30 mins with or without stimulation developed afterdepolarizations. Changes occurring during a short exposure to low K+, high Ca2+ solution were increases in membrane resistance, membrane time constant and space constant by 47%, 83% and 17% compared with the control, respectively. There were no significant changes in internal longitudinal resistance and membrane capacity. During long exposure to the test solution (30 mins), delayed afterdepolarizations developed. There were similar increases in membrane resistance and in time constant as found during the short exposures. Internal longitudinal resistance was calculated to have increased by 24% during the long exposure. A 19% increase in membrane capacity was also found during the test condition. High Ca2+ or low K+ alone did not cause a significant increase in internal longitudinal resistance. The conduction velocity in the longitudinal direction decreased from 107 +/- 23 cm/s during the control to 80 +/- 7 cm/s during the test period for 30 mins. These results suggest that, in addition to the abnormal impulse formation based on afterdepolarizations, low K+, high Ca2+ solution changes the passive electrical properties of the fibers, resulting in a lower rate of impulse conduction.     

Influence of thyroid hormone levels on the electrical and mechanical properties of rabbit papillary muscle

We have examined the influence of chronic in vivo alterations of thyroxine levels on the electrophysiological and mechanical properties of rabbit ventricular papillary muscles measured in vitro. Marked changes in the repolarization phase of the action potential and the time to peak tension of isometric twitches were observed when thyroid hormone levels were increased above or decreased below normal. The time to peak tension was consistently shorter than normal in hyperthyroid and longer than normal in hypothyroid preparations. In hypothyroid preparations the action potential duration was greater than that of controls at all stimulation frequencies tested (0.1 to 1.0 Hz). In hyperthyroid preparations, the repolarization phase consisted of an initial phase of fast repolarization to membrane potential values between -20 and -40 mV followed by a plateau. The early phase of repolarization persisted at all stimulation frequencies tested (0.1 to 1.0 Hz) but the plateau component increased markedly as the stimulation frequency was decreased. The early phase of repolarization was markedly reduced in the presence of 4-aminopyridine. These results suggest that thyroxine levels may modulate the kinetics of a transient outward current which in rabbit papillary muscle normally is responsible for the frequency dependence of action potential duration. Action potential amplitude, maximum rate of rise, resting membrane potential, and peak isometric twitch tension were not markedly different between the three classes of preparations. A second depolarizing response occurred in all hyperthyroid preparations at low stimulation frequencies (0.1 to 0.4 Hz) but not in control or hypothyroid preparations. This second depolarizing response, which was eliminated by D-600 treatment, was similar to the calcium-dependent slow action potentials recorded in other cardiac preparations. These two component action potentials could represent either intrinsic single cell activity, or a re-entry wave of depolarization which results from nonhomogeneous excitation.     

Effect of acid perfusion on passive electrophysiological properties of rabbit esophagus in vivo

In the present paper we studied early acid-induced changes in the passive electrical properties of the rabbit esophageal epithelium in vivo by measurements of the transluminal potential difference (PD) during acid perfusion and by estimating the transmucosal electrical resistance (R _m) using cable analysis. Perfusion with acid (pH 1) for 45 min produced a rapid (<1 min) negative shift in the lumen-negative PD followed by a slow lumen-negative drift. The acid-induced change in PD was dependent on the accompanying anion, the largest anion (sulfate) producing the largest change. The acid-induced changes in PD were parallelled by reductions in R _m, these reductions also being dependent on the accompanying anion. Interpretation of resistance and net current (estimated by Ohms law) time curves suggest that the initial acid-induced changes of the PD reflect properties of the naive mucosa whereas the later drift will reflect a diffusion driven increase in transmucosal proton permeability. Further, coapplication of the protective drug sucrose octasulfate attenuated the hydrochloric acid-induced changes of all measured and estimated electrophysiological parameters. The electrophysiological results were to some extent corroborated by light microscopic findings, although no large acid-induced change in mucosal appereance was observed.     

Effect of alpha-adrenergic receptor stimulation on ventricular electrical properties in the normal canine heart

We examined the effects of alpha-adrenergic stimulation on ventricular excitability, refractoriness, and vulnerability to fibrillation. Methoxamine or phenylephrine was infused in five dogs each before and after aortic arch and carotid sinus baroreceptor denervation, in doses which increased mean arterial blood pressure by 20 to 30 mm Hg. Methoxamine or phenylephrine caused an increase in the ventricular fibrillation threshold (VFT) (from 27% to 41%) and in the repetitive extrasystole threshold (RET) (from 28% to 39%). This effect was abolished by baroreceptor denervation. Neither drug altered mid-diastolic threshold or effective refractory period duration either before or after denervation. We conclude that alpha-receptor activation exerts no direct effect on ventricular excitability or refractoriness in the normal intact heart.     

Different effects of reoxygenation on the electrical activity of ventricular muscle

Response of a hypoxic and acidotic (HA, with exogenous lactate) ventricular muscle tissue to subsequent reoxygenation in the absence of substrate (0 mM dextrose) was different from that of a Purkinje fiber. The K+ concentration in this solution (4.6 mM) was slightly higher than that in Tyrode solution (2.7 mM). The observed effects of reoxygenation of such a ventricular tissue were also variable. The ventricular muscle tissue exhibited the following different responses on reoxygenation after hypoxia and acidosis: (1) arrhythmias, without much depolarization of the membrane potential, (2) oscillatory after-potentials (OAPs) during the late diastole, which lessened in amplitude as the time of reoxygenation increased, but no arrhythmias, or (3) a pronounced slowed phase of repolarization (hump), but no arrhythmias. These different effects of reoxygenation did not occur if concentration of K+ in HA was very much higher than 4.6 mM. Common to these three different responses was the prolongation of the action potential durations during reoxygenation at the 50% and 90% levels of repolarization (APD50 and APD90) and a slight increase in the resting tension after 30-40 minutes of reoxygenation. Some of the observed responses of ventricular muscles were well mimicked by increasing extracellular calcium, but the different and variable effects of arrhythmias, OAPs, and prolonged APD require further analysis.     

溶血磷脂酸对兔心室肌电稳定性的影响

目的:评价溶血磷脂酸(lysophosphatidic acid,LPA)对兔心室肌电稳定性的影响.方法:20只新西兰大白兔随机分为LPA高浓度组和低浓度组,制备兔左室楔形心肌块灌注模型,分别灌流含LPA 10 μmol/L和 1 μmol/L的台式液.利用浮置玻璃微电极法同步记录楔形心肌块内、外膜心肌细胞动作电位(Endo-APD90、Epi-APD90)和跨室壁心电图.分别观察2组Endo-APD90、Epi-APD90,跨室壁复极离散度(TDR),QT间期以及S1S2刺激下的室性心动过速诱发率在给药前后的变化.结果:高浓度组给药后与给药前比较:QT间期、Endo-APD90、TDR均增大(均P<0.01),S1S2刺激的室性心动过速诱发率也明显增高(P<0.05),但Epi-APD90无明显变化(P＞0.05). 低浓度组给药后与给药前比较,其电生理参数变化差异无统计学意义(P＞0.05).结论:高于生理浓度的LPA使兔心室肌的电不稳定性增加,有致心律失常作用.     

Differential effects of L-propionylcarnitine on the electrical and mechanical properties of guinea pig ventricular muscle in normal and acidic conditions

We studied the effects of L-propionylcarnitine (PC) on transmembrane action potentials and isometric contractile tension in isolated guinea pig ventricular papillary muscles. The effects of 5 concentrations of PC (10(-5), 10(-4), 10(-3), 10(-2) and 3 X 10(-2)M) were examined in both normal (pH 7.4) and acidic (pH 6.9) conditions. The concentrations of 10(-5) to 10(-2)M had no significant effect on action potential amplitude, maximum upstroke velocity of phase 0 and resting potential, in either condition. At pH 7.4, action potential duration (ADP) was significantly (P less than 0.05) or insignificantly shortened by the drug depending upon the concentration used. At pH 6.9, however, the APD was prolonged by moderate PC concentrations (10(-3) and 10(-2)M), in which the effective refractory period (ERP) was also lengthened, associated with an increased ERP/APD ratio. In both pH conditions, the highest concentration (3 X 10(-2)M) significantly (P less than 0.05) decreased all these action potential parameters. PC had a biphasic effect on the developed tension. In both pH conditions, low PC concentrations (10(-5) to 10(-3)M) produced an initial augmentation of the contraction, followed by subsequent reduction. The initial augmentation disappeared by pretreatment with reserpine or propranolol, suggesting the involvement of beta-adrenoceptors. In the steady state, all PC concentrations produced a negative inotropic effect at pH 7.4, while at pH 6.9 only high concentrations (10(-2)M and 3 X 10(-2)M) had this effect. These results suggest that the effects of PC in an acidic condition differ considerably from those in a normal pH condition and that limited concentrations of PC (10(-3) to 10(-2)M) may prevent re-entrant arrhythmias from developing under acidic conditions via lengthening of the ERP, without deleterious effects on the contractile force.     

肥胖对家兔心房肌组织结构和电生理特性的影响

目的:探讨肥胖对家兔心房肌组织结构和电生理特性的影响,为进一步了解肥胖影响房颤(AF)易感性的相关机制和AF的早期干预提供实验依据。方法:将30只家兔随机分为正常组、肥胖组和肥胖+他汀药物组,雌雄各半,每组10只。肥胖组给予高脂饲料,造模成功后,采用Langendorff离体灌注法对3组家兔分别进行离体心脏灌注。采用心脏电生理刺激仪监测心房肌的电生理特性参数:肥胖组家兔房内传导时间(HRA-HIS)、心房有效不应期(AERP)、窦房结恢复时间(SNRT)、校正后窦房结恢复时间(CSNRT)。剪取部分心耳组织,经MASSON染色观察心房肌纤维化的程度。采用TUNEL法测定心房肌细胞的凋亡率。结果:与正常组相比,肥胖组家兔HRAHIS、AERP、SNRT和CSNRT等电生理参数均出现不同程度的延长,并导致AERP频率自适应性降低。与肥胖组比较,肥胖+他汀药物组的心肌基质胶原纤维显著减少(P0.05),心肌胶原容积分数(CVF)显著减低(P0.01),心肌细胞凋亡率(%)明显降低(P0.01)。结论:肥胖可引起家兔心房肌组织结构与电生理基质改变,利于AF的发生与维持,而应用他汀类药物干预后,这些改变有所改善。     

Relaxant effect of oxygen free radicals on rabbit tracheal smooth muscle

We investigated the effect of exogenously generated superoxide anions (O(2)(-)), hydrogen peroxide (H(2)O(2)) and hydroxyl radicals (.OH) on isolated rabbit tracheal smooth muscle suspended in Krebs-Ringer solution. The ability of oxygen free radicals (OFRs) to affect acetyicholine (Ach)-induced contraction in these muscles was also investigated. OFRs, in general, produced a concentration-dependent relaxation of the tracheal smooth muscle in the doses used. However, in large concentrations, O(2)(-) and H(2)O(2) produced effects which were smaller than those obtained with lower concentrations. The relaxant effects of these oxyradicals were progressive and lasted throughout the 20min observation period. At all concentrations used, the OFRs tended to abolish or reduce Ach-induced contraction in a concentration-dependent manner. O(2)(-) was more potent than H(2)O(2) or DHF in relaxing the Ach-precontracted muscle and in inhibiting the response of the muscle to Ach. OFR-induced relaxation of the Ach-contracted muscle was not due to inactivation of the Ach by OFRs. Relaxation produced by OFRs was greater in preparations with intact epithelium than in those denuded of epithelium. The relaxant effects were blocked by indomethacin, a cyclooxygenase inhibitor. OFRs in the presence of indomethacin produced contraction only in the preparations with intact epithelium, suggesting a release of contractile factor(s) from epithelium. These results suggest that OFRs relax rabbit tracheal smooth muscle. The relaxation appears to be mediated through the synthesis and release of prostaglandins from the epithelium and smooth muscles.     

Isotonic muscle and sarcomere shortening in rabbit right ventricular preparations

Summary Cardiac muscle fibers are suspended within and attached to an elaborate connective tissue matrix that includes numerous compliant interconnections. Myocardial muscle fibers are not branched, but connect at small angles to each other to form a branched array. Therefore, fiber shortening occurs as a vector within a connective tissue framework and individual fiber work may exceed external muscle work. To evaluate the latter we measured isotonic muscle shortening simultaneous with sarcomere shortening. The hearts were obtained from rabbits (n=4) anesthetized with intravenous pentobarbital sodium. We isolated right ventricular trabeculae or free wall papillary muscles in Krebs-Ringer's solution (2.5 mM Ca²⁺, 28°C). Cross-sectional area was 0.038±0.003 mm² (±SE throughout) and resting sarcomere length was 2.33±0.12 m. Sarcomere length was measured with laser diffraction (He–Ne, =632.8 nm) during force clamps in single- and paired-stimulation twitches. Relative sarcomere shortening (¢SL) was isotonic sarcomere shortening divided by sarcomere length at the onset of isotonic shortening. Relative muscle shortening (ML) was isotonic muscle shortening divided by muscle length at zero load; the latter was estimated from the stress-strain relation of elastic recoil at the onset of load clamps. Average SL/ML at peak shortening was 3.38±0.16 and was independent of stimulus pattern, isotonic load, amount of shortening, time during a twitch or laser beam position along a muscle. Therefore, the ratio >1 was neither a function of activation nor heterogeneous sarcomere length change. To account for (SL/ML) >1 we developed a geometrical model based on the fact that muscle fibers, interconnect at small acute angles and are suspended in and interconnected by a connective tissue matrix. Developed force of individual angled muscle fibers must be greater than external muscle force. As a result, the inter-fiber connective tissue matrix will be strained and there will be proportionately more shortening of muscle fibers and their sarcomeres than overall muscle shortening. Also, fiber angle decrease during shortening may accommodate some excess fiber shortening. In this model fiber and sarcomere work during shortening will be greater than work measured at the muscle's ends.