Ca and Na selectivity of the active membrane of rabbit AV nodal cells

The atrioventricular (AV) node is thought to have a slow ionic channel. These experiments were designed to measure the relative contributions of Na and Ca ions to inward currents in the AV nodal cells of rabbit heart superfused with Tyrode solution. The effects of tetrodoxin (TTX), Mn2+, and verapamil observed in this study were in agreement with reports by others. The overshoot of AV nodal (N) cells was related to external Ca, with a slope of 12 mV/decade, unchanged by addition of TTX. Similar dependence of overshoot on external Na was seen, with a slope of 20 mV/decade. The slope did not change on addition of TTX. Total removal of either Na or Ca from the solution abolished excitability. Using a constant field equation, we estimated relative permeability (P) of the membrane at the time of maximal overshoot to be PCa/PNa congruent to 60 similar to or approximately 100 and PK/PNa congruent to 1. Relative contributions of these ions to the currents were estimated as ICa congruent to 17%, INa congruent to 33% (inward currents), and IK congruent to 50% (outward current). In conclusion, AV nodal cells have "slow inward-current channels" that are selective for Ca over Na ions.     

The effect of polarization on the action potentials of the rabbit AV nodal cells

The resting potential of various parts of the AV nodal regions of an excised rabbit heart was changed by applying constant electrical current with a suction electrode. The membrane potential was recorded with an intracellular microelectrode and the maximum rate of rise of depolarization was computed. The action potential recorded from the AV nodal cell responded to constant current in a manner qualitatively similar to other myocardiums. The relationship between resting potential and amplitude of action potential was expressed by a straight line, while a sigmoidal relationship was observed between the resting potential and the maximum rate of rise of depolarization. Most of the initial current system in alpha region was inactivated at the control membrane potential. The maximum value of the maximum rate of rise of depolarization of alpha region was 30 V/sec which was lower than that of other parts of the nodal region.     

Altered nicotinic sensitivity of AV node in surgically denervated canine hearts

The responses of normal and cardiac-denervated (DNV) dogs to acetylcholine (ACh) and nicotine (NIC) were examined to determine if the intrinsic cardiac nerves (ICN) that modulate electrical conduction display denervation supersensitivity. Control (n = 18) and DNV (n = 18) animals were placed on cardiopulmonary bypass. Recording of intra-atrial (P-A), intraventricular (H-V), and atrioventricular (AV) nodal (A-H) conduction times were made from the region of the His bundle. ACh (0.1-10 micrograms) was used to produce muscarinic stimulation, whereas NIC (0.1-400 micrograms) was employed to stimulate the ICN. All drugs were administered intracoronary. No supersensitivity to either ACh or NIC was seen in the data from the P-A or H-V intervals of the His electrogram in the DNV animals. However, this group displayed approximately a 10-fold increase in the negative dromotropic effect of NIC on the AV node compared with control. No significant change in muscarinic sensitivity of the AV node was observed in the DNV animals. We conclude that 1) no denervation supersensitivity of the ICN mediating effects on intra-atrial and intraventricular conduction occurs; 2) the AV node itself does not show muscarinic supersensitivity following extrinsic denervation; 3) the ICN do display denervation supersensitivity as shown by a 10-fold increase in the effects of NIC on AV nodal conduction time.     

Left-right lineage analysis of AV cushion tissue in normal and laterality defective Xenopus hearts

The majority of complex congenital heart defects occur in individuals who are afflicted by laterality disease. We hypothesize that the prevalence of valvuloseptal defects in this population is due to defective left-right patterning of the embryonic atrioventricular (AV) canal cushions, which are the progenitor tissue for valve and septal structures in the mature heart. Using embryos of the frog Xenopus laevis, this hypothesis was tested by performing left-right lineage analysis of myocytes and cushion mesenchyme cells of the superior and inferior cushion regions of the AV canal. Lineage analyses were conducted in both wild-type and laterality mutant embryos experimentally induced by misexpression of ALK4, a type I TGF-beta receptor previously shown to modulate left-right axis determination in Xenopus. We find that abnormalities in overall amount and left-right cell lineage composition are present in a majority of ALK4-induced laterality mutant embryos and that much variation in the nature of these abnormalities exists in embryos that exhibit the same overall body situs. We propose that these two parameters of cushion tissue formation-amount and left-right lineage origin-are important for normal processes of valvuloseptal morphogenesis and that defective allocation of cells in the AV canal might be causatively linked to the high incidence of valvuloseptal defects associated with laterality disease.     

Histopathologic time course of myocardial infarct in rabbit hearts

Introduction: The histopathologic evolution of myocardial infarct and of remote zones in rabbit hearts was studied. Methods: The left coronary artery of 55 rabbits was ligated and rabbits were sacrificed at 2, 4, 6, 8, 12, 14, 16, 18, 26, 35 and 56 days post-ligature (n=5 per group). Two rabbits were used as control and four were sham-operated. The hearts were excised, cut in slices and stained with hematoxylin-eosin, Masson's trichrome and picrosirius red. The histological evaluation was semiquantitative (scale: 0 to ++). Results: At day 2, the presence of neutrophils was ++, decreasing suddenly at day 4 and disappearing completely at day 6. The proliferation of cells with features of fibroblasts increased from days 4 to 14 post-occlusion. Coagulation necrosis in mid-myocardium during the first week was ++. Subendocardial myocytolysis was evident from day 2 up to day 56 post-infarction. During the second week, proliferation of lymphocytes and macrophages (++), granulation tissue formation (++) and incipient traces of fibrosis that peaked at day 35 were observed. Scarring was complete at day 56 (++). In remote zones (right ventricle and septum), the proliferation of cells+ on Vimentin was observed at day 2, and perivascular, interstitial and endocardial fibrosis started to increase at day 6 and peaked at day 16. Conclusion: Although myocardial infarction in rabbits maintains the essence of the infarct chronology, some differences as the early presence of cells+ on Vimentin and subendocardial fibrosis in infarcted areas, and also the rapid increase and early disappearance of neutrophils appear when other species are considered. An interesting finding was the early proliferation of cells with features of fibroblasts in remote zones.     

Infarct size-limiting effect of calcium preconditioning in rabbit hearts

Recent studies demonstrated that brief period of Ca2+ depletion and repletion (Ca2+ preconditioning, CPC) has strong protective effects against ischemia in a rat heart. CPC and classic preconditioning (IPC) were compared in relation with infarct size and protein kinase C (PKC) isozymes. Isolated Langendorff-perfused rabbit hearts were subjected to 45-min ischemia (Isc) followed by 120-min reperfusion (R) with or without IPC, induced by 5-min Isc and 10-min R. In the CPC hearts, 5-min Ca2+ depletion and 10-min repletion (CPC) were given before 45-min Isc, with or without concurrent PKC inhibition (calphostin C, 200 nmol/L). IPC enhanced recovery of LV function, while CPC did not. Infarct size was significantly reduced by both CPC and IPC (p < 0.05 vs. ischemic control). Membrane PKC was significantly increased from 2.53 +/- 0.07 (baseline, nmol/g tissue) to 3.11+/-0.07, 3.34 +/- 0.11, 3.15 +/- 0.09, and 3.06 +/- 0.08 by IPC, IPC and 45-min Isc, CPC and 45-min Isc, respectively (p < 0.01). Immunoblots of membrane PKC were increased by IPC, IPC and 45-min Isc, and CPC. These effects were abolished by PKC inhibition. Thus, activation of PKC may have trigger role in the mechanism of cardioprotective effect by CPC.     

Sodium-calcium exchange and sarcolemmal enzymes in ischemic rabbit hearts

We have investigated alterations in sarcolemmal function that occur during myocardial ischemia. Rabbit ventricles were incubated at 37 degrees C for time periods ranging from 5 min to 2 h. The ischemic tissue was homogenized, and activities of the sarcolemmal enzymes Na+-K+-ATPase, K+-p-nitrophenylphosphatase (K+-pNPPase), and adenylate cyclase were measured in the crude homogenate. Na+-K+-ATPase and K+-pNPPase were substantially inhibited after only 10 min of ischemia, and activities for all three enzymes declined progressively up to 1 h of ischemia, when activities were 37-59% of control. Highly purified sarcolemmal membranes prepared from control tissue and myocardium that had been made ischemic for 1 h showed similar purification of sarcolemmal enzymes, passive Ca2+ binding, and passive permeability to Ca2+. However, the velocity of Na+-Ca2+ exchange in ischemic sarcolemmal vesicles was reduced approximately 50% due to a reduction in Vmax. Although the parallel decline in activities of several sarcolemmal functions might suggest a change in membrane structure, phospholipid and cholesterol contents in ischemic sarcolemma were the same as control.     

Prostacyclin-dependent coronary vasodilation in rabbit and guinea pig hearts

Isolated hearts from rabbits or guinea pigs were perfused according to Langendorff and the coronary flow was recorded continuously. In addition, the rabbit heart transmyocardial effluent's content of platelet anti-aggregatory (prostacyclin-like, PCLA) activity was assayed biologically at regular intervals. Perfusion was performed with a solution gassed with 95% O₂ in CO₂, switching at intervals to a solution gassed with 12% O₂ and 5% CO₂ in N₂. Perfusion with a hypoxic solution elicited reproducible increases in coronary flow. After pretreatment with indomethacin (5times 10^-5M), this increase was completely abolished and in several cases it was reversed to a marked reduction in coronary flow. The transmyocardial effluent contained, during perfusion with normoxic solution, no detectable or only negligible amounts of PCLA. During hypoxia the efflux of PCLA into the transmyocardial effluent increased markedly. This increase was completely abolished wheri indomethacin (5 × 10^-5M) was added to the solution perfusing the heart. The results strongly suggest that increased coronary vascular formation of prostacyclin plays a key role in the coronary vasodilation induced by hypoxia in rabbit and guinea pig hearts.     

Effects of activation rate on contractile properties of rabbit masseter motor units

Force-frequency curves of rabbit masseter motor units ( n=20) were determined, in order to study the capacity of these motor units for rate gradation and to establish the relationship between twitch contraction time (TCT) and the shape of the curves. Motor unit force responses were elicited by stimulating motoneurons in the trigeminal motor nucleus extracellularly. A sequence of pulse trains with increasing frequency rates was followed by trains with decreasing frequency rates. All motor units were classified as fast (F) units. The ascending force-frequency curves showed a distinct sigmoid appearance; the descending curves were shifted toward lower stimulation rates. The position and shape of the force-frequency curves related significantly to the TCT. The curves of slower units were located at lower frequencies and had a larger inclination. In addition, slower units had a lower fusion frequency and a larger twitch-tetanus ratio. Hence, slower units started to fuse and reached maximum force at lower stimulation rates than faster units and needed a smaller change in simulation frequency to achieve the same relative force. It can be concluded that the capacity for rate gradation differs between rabbit masseter motor units and that the TCT is a determinant for the position and shape of the force-frequency curves.     

Prostacyclin-dependent coronary vasodilation in rabbit and guinea pig hearts.

Isolated hearts from rabbits or guinea pigs were perfused according to Langendorff and the coronary flow was recorded continuously. In addition, the rabbit heart transmyocardial effluent's content of platelet anti-aggregatory (prostacyclin-like, PCLA) activity was assayed biologically at regular intervals. Perfusion was performed with a solution gassed with 95% O2 in CO2, switching at intervals to a solution gassed with 12% O2 and 5% CO2 in N2. Perfusion with a hypoxic solution elicited reproducible increased in coronary flow. After pretreatment with indomethacin (5 x 10-5M), this increase was completely abolished and in several cases it was reversed to a marked reduction in cornonary flow. The transmyocardial effluent contained, during perfusion with normoxic solution, no detectable or only negligible amounts of PCLA. During hypoxia the efflux of PCAL into the transmyocardial effluent increased markedly. This increase was completely abolished when indomethacin (5 x 10-5M) was added to the solution perfusing the heart. The results strongly suggest that increased coronary vascular formation of prostacyclin plays a key role in the coronary vasodilation induced by hypoxia in rabbit and guinea pig hearts.     

心麻痹液有助于缺血预处理幼兔的心肌保护作用

缺血预处理(ischemic preconditioning,IPC)是一个在许多种动物上都观察到的内源性心肌保护现象。一些研究认为ATP敏感性钾通道(KATP channel)开放可能与IPC产生心肌保护的机制有关。鉴于高钾浓度的心麻痹液是临床上最为常用的心肌保护措施之一,但细胞外高浓度钾是否会影响到KATP通道开放?因此,本研究利用Langendorff离体心脏灌注模型研究IPC单独应用和与心麻痹液(St．Thomas Ⅱ号停搏液)联合应用对兔未成熟心脏全心缺血再灌注的影响,以了解心麻痹液在IPC心肌保护效应中的作用。     

Dipole moment ofin vivo and isolated perfused rabbit hearts

We have measured magnitude and location of heart dipole moment during QRS in 46 New Zealand white rabbits. The spatial magnitude curve had one to three peaks. Mean values were M₁=80±10 μA-cm (N=5) pointing to right anterior and caudal, M₂=260±15 μA-cm (N=42) directed slightly to left of due anterior and caudal, and M₃=236±9 μA-cm (N=43) pointing towards left posterior and cephalad. The mean thorax resistivity was 250 ohm-cm. For 23 rabbits, M₂/M₃>1 and for 16 rabbits, M₂/M₃<1. Mean times of occurrence of the three peaks were 5.8, 11.2, and 19.6 ms, respectively. Spatial magnitude curves for hearts perfused at the center of a sphere showed usually one major peak at about 19 ms. Locuscardiograms ofin vivo hearts were also measured. By comparing M values forin vivo and isolated hearts, we found that M₁ values agreed closely but mean M₂ measured from the heartsin vivo was 2.5 times that for the isolated hearts, and M₃ forin vivo hearts was about two-thirds that for isolated hearts. We relate these differences to the effects of intracardiac blood and lungs on the measured dipole moment. A preliminary report of this work was given inAdvances in Electrocardiology, Proceedings XIth International Congress on Electrocardiology, P. d'Alche, ed. Caen, France, 1985, pp. 39–41 This project was supported by Grant No. HL 19486 (BCH) and by Research Career Award HL K6 1932 (CVN) from the National Heart, Lung and Blood Institute, U.S. Public, Health Service     

离体家兔心脏房压增加所致的心房肌电生理特性变化及机制

目的：探讨心脏房压增加引起的心脏电生理变化及链霉素（牵张激活性离子通道阻断剂）和维拉帕米对其的影响。方法：采用结扎腔静脉和肺静脉、贯通房间隔、调节肺动脉灌注压（5 cmH₂O到25 cmH₂O）的离体Langendorff心脏模型，观察房压增加前后心肌有效不应期（ERP）、单相动作电位时程MAPD₉₀和房颤阈（AFT）的变化，并比较链霉素和维拉帕米对这些电生理参数变化的影响。结果：房压上升（20 cmH₂O）引起ERP和MAPD₉₀缩短，AFT下降（P<0.01）；链霉素可有效抑制这些电生理变化；而维拉帕米对此没有明显影响。结论：牵张激活性离子通道的活化可能参与房压增加引起的心脏电生理变化过程。     

Oxygen consumption in rabbit Langendorff hearts perfused with a saline medium

Isolated rabbit hearts were perfused according to Langendorff at a temperature of 38°C and a pressure of 5.9 kPa with gassed Tyrode solution. Gas mixtures containing 5 % CO₂ and 15, 20, 30, 60, or 95% O₂ in N₂ were used to saturate the perfusion medium. In some cases lactate (50 or 500 μM) was present in the medium perfusing the heart. Coronary flow (CF), oxygen pressure in the perfusion medium and in the cardiac effluent and lactate in the effluent were analysed in all experiments. The oxygen uptake in the hearts perfused with a medium equilibrated at atmospheric pressure with 95% O₂ and 5% CO₂ (oxygen pressure?87 kPa, oxygen content 19 ml × 1^-1), averaged 3 mix 100 g w.w. ^-1× min^-1. Reduction of the oxygen pressure in the perfusion medium resulted in an increase in CF and in the fractional extraction of oxygen from the medium, making it possible to maintain the heart's oxygen uptake (V?o₂) down to an oxygen pressure in the perfusion medium of about 24 kPa (oxygen content?5 ml × 1^-1, the perfusion medium equilibrated with 20% O₂ and 5%CO₂ in N₂). Myocardial lactate production was low during perfusion at pO₂? 87 kPa but increased rapidly when the oxygen pressure was lowered. The addition of lactate (500 /°M) to the perfusion medium at pO₂? 87 kP induced a fractional uptake of about 20%. It is concluded that the V?o₂ observed during perfusion at pO₂? 87 kPa mainly reflects aerobic myocardial metabolism in this preparation. This assumption is based on the facts that coronary flow and fractional oxygen extraction are submaximal and that a considerable uptake of lactate occurs concurrently with a very limited production. However, even moderate reduction of the oxygen pressure in the perfusion medium (to ? 61 kPa) is followed by a significant increase in lactate production, indicating that myocardial oxygenation is inefficient.     

Hypoxia elicits liberation of anti-aggregatory substances from isolated rabbit hearts

The hypothesis was investigated that myocardial hypoxia stimulates the production of platelet anti-aggregatory substances in the heart. Rabbit hearts were perfused under normoxic or hypoxic conditions and the coronary and interstitial effluents from the hearts were separated. The occurrence of anti-aggregatory activity (AAA) in the interstitial effluent was detected in vitro from its capacity to inhibit ADP-induced platelet aggregation. The AAA in the effluent was deemed to be prostacyclin (PGI2) if its release was abolished by administration of indomethacin (5 X 10(-5) M) to the heart, and to be adenosine if it was abolished by incubation of the effluent with adenosine deaminase. During normoxic perfusion, only a minor efflux of AAA appeared from the heart; neither was the efflux appreciable during mild hypoxia (30 or 60% O2). Severe hypoxia (venous pO2 below 5 kPa), on the other hand, was associated with a marked release of AAA. Incubation of hypoxic effluent with adenosine deaminase resulted in a small loss of activity, indicating that the major part of the AAA was not ascribable to adenosine. After indomethacin treatment, significant amounts of AAA still appeared in the effluent during hypoxia. However, unlike the case before indomethacin, this AAA was completely destroyed by adenosine deaminase. From these data, we conclude that myocardial hypoxia can mobilize either of two independent mechanisms for protection against platelet aggregation: an activation of the synthesis and release of prostacyclin, and a more complete breakdown of ATP, leading to an increased formation and efflux of adenosine.     

Arrhythmogenic properties of dismantling cadherin-mediated adhesion in murine hearts

Objective

To evaluate the arrhythmogenic effects of dismantling cadherin-mediated adhesion by recombinant mouse aminopeptidase N (rmAPN) in murine hearts.

Methods

rmAPN was incubated with cultured neonatal rat cardiomyocytes as well as being infused in adult mice. The cell-cell connections were immunolabelled and observed by laser confocal microscopy. Disruption of the N-terminal of N-cadherin (N-cad) was detected by western blot and quantitative immunofluorescence. The risk of inducible ventricular tachyarrhythmia was evaluated in mice by an electrophysiological study.

Results

Disrupted cell-cell contact was observed in cultured neonatal rat cardiomyocytes in response to 30-40 ng/µL rmAPN. Loss of the N-terminal in N-cad and altered distribution of connexin 43 (Cx43) were observed in hearts from rmAPN-infused mice. In addition, a reduction of phosphorylated Cx43 was also detected concomitant with redistribution of Cx43. Electrophysiological studies of rmAPN-infused mice showed prolonged QRS duration and increased inducibility of ventricular tachycardias.

Conclusion

Disruption of N-cad by rmAPN contributes to gap junction remodeling and may elicit arrhythmogenic effects. The disorder of adherent junctions by proteolytic enzymes may play an important role in arrhythmogenic mechanisms in correlated diseases.