Effects of acetylcholine on time-dependent currents in sheep cardiac Purkinje fibers

Voltage clamp experiments were carried out on sheep Purkinje fibers to determine the effect of Ach on the time-dependent currents.On the pacemaker current (i _{K 2}) Ach 10^–6 mol·l^–1 had the following effects: shift of the activation curve by a few mV in the depolarizing direction, without change in the rectifier ratio. The potential dependence of the time constants for activation and deactivation was influenced in a similar way as the activation curve.Ach had no effect on the positive dynamic current (i _qr ) or the late plateau outward current (i _x ).The slow inward current (i _si ) as well as the transient inward current (T.I.) were reduced in amplitude and slowed in time course by Ach.The changes in pacemaker current are important in explaining the increased rate of diastolic depolarization in the presence of Ach. The decrease of slow inward current by Ach cannot be made responsible for the plateau shift or the prolongation of the action potential.Supported by F.G.W.O. Belgium 3.0087.74     

Effect of acetylcholine on time-independent currents in sheep cardiac Purkinje fibers

Voltage-clamp experiments were carried out in sheep Purkinje fibers in order to find an explanation for the prolongation of the action potential, the positive shift of the plateau, the hyperpolarization of the maximum diastolic potential and the increase in rate of diastolic depolarization, occurring in the presence of acetylcholine (Ach).In the presence of Ach the instantaneous current-voltage relation is shifted in the inward direction for potentials positive to –75 mV, while the opposite shift is obtained for more negative potentials; the results suggest a decrease in background conductance.The contribution of K, Cl, Na and Ca to the Ach sensitive current was studied by varying K₀ concentration or adding 20 mmol·l^–1 Cs, by omitting Cl or Na, and by changing the Ca concentration.In 20 mmol·l^–1 Cs the apparent reversal potential of the Ach sensitive current is –50 mV, as compared to –75 mV in normal Tyrode. The component of the Ach sensitive current, which is suppressed by Cs, shows inward going rectification. In different K₀ concentrations the reversal potential of the Ach sensitive current is changed; the shift obeys the theoretical change in equilibrium potential of K. The results are consistent with a decrease in K background current by Ach (inward and outward rectifier).In Cl free media the Ach sensitive current is not decreased excluding a major contribution of Cl ions. The Ach effect also persists in Na free media; the reversal potential of the Ach sensitive current is slightly shifted in the hyperpolarizing direction. These results indicate that active electrogenic pumps (Na or Na–Ca) do not play an important role; they are in accord with a reduction in inward Na background current by Ach. The shift of the current-voltage relation by Ach was greater the lower the Ca_o concentration; the mechanism is not clear.The inward shift of the current at –40 mV was dependent on the Ach concentration. Half-maximum effect was obtained at 3·10^–7 mol·l^–1 Ach; the Hill coefficient was 1.12.It is concluded that Ach interacts in a one to one reaction with a muscarinic receptor and reduces the background current mainly carried by K (inward and outward rectifier), and less by Na (and probably Ca).Supported by F.G.W.O. Belgium 3.0087.74     

Existence of two transient outward currents in sheep cardiac Purkinje fibers

Voltage clamp analysis of the transient outward (positive dynamic) current was performed in sheep Purkinje fibers at a pulse frequency of 1/min. 4-aminopyridine (4-AP, 1 mM) suppressed most of the transient outward current, thus revealing the slow inward current,i _si, and an associated brief outward current,i _bo. The long lasting component of the current suppressed by 4-AP was labelledi _lo. In the presence of 4-AP,i _bo was suppressed either by caffeine 10 mM or when Sr was substituted for Ca, both conditions makingi _si clearly detectable. Mn ions suppressed bothi _si andi _bo. Current decay was a monoexponential process fori _bo (=12 ms) and a two exponential process fori _lo (₁=80–100 ms, ₂=250–400 ms). The peak amplitude-E _m relationships were different for the two currents. It was shown that the reversal potential ofi _lo was not measureble by the usual method probably because of the too fast activation-deactivation kinetics of the current.It is concluded that not one but two transient outward currents with different electrophysiological and pharmacological characteristics exist in the sheep Purkinje fiber. The reason of the caffeine-sensitivity ofi _bo is discussed.Supported by F.G.W.O. Belgium 3.0087.74.     

Alinidine modifies the pacemaker current in sheep Purkinje fibers

(1) The specific bradycardic agent alinidine reduces the slope of the diastolic depolarization in sinoatrial tissue and Purkinje fibers. In short Purkinje fibers of sheep, alinidine (28 M) decreased the pacemaker current by a dual action. The voltage dependence ofi _f activation was shifted in the hyperpolarizing direction by 7.8±0.6 mV (n=18,p<0.001) and the conductance of the fully activated current was reduced to 73±2% (n=18,p<0.001) of its control value. These effects were reversible and dose-dependent. (2) Ionophoretic injections of alinidine caused reversible reductions of the diastolic depolarization rate and simultaneous transient hyperpolarizing shifts of thei _f activation range. (3) Some prolongation of the action potential duration was observed at 28 M and more pronounced at higher concentration. This was presumably the consequence of a reduction by alinidine of outward repolarizing current carried by the background inward rectifier and plateau currenti _x. (4) The action of alinidine oni _f resulted in a slower activation of a reduced fraction of the pacemaker current at the maximal diastolic potential level. This explains the decrease of the diastolic depolarization rate observed in Purkinje fibers.     

Electrophysiological characterization of histamine receptor subtypes in sheep cardiac Purkinje fibers

The histamine-receptor-subtype-mediated effects on action potentials of electrically driven and spontaneously active isolated sheep cardiac Purkinje fibers were investigated using H₁-and H₂-selective agonists and antagonists.In electrically stimulated Purkinje fibers, histamine (3 mol/l) increased the action potential plateau height, decreased the action potential duration measured at a repolarization level of –60 mV and enhanced the pacemaker activity. These effects were abolished by the H₂-selective antagonist cimetidine (30 mol/l), but were not impaired by the H₁-selective antagonist dimetindene (0.3 mol/l).In spontaneously active Purkinje fibers, histamine (10 mol/l) increased the spontaneous rate by 24%, the slope of diastolic depolarization by 45% and shortened the duration of the diastole by 32% of the respective control measurements. These effects were blocked by 30 mol/l cimetidine, but remained unchanged in the presence of 0.3 mol/l dimetindene.Concentration-response curves of histamine were shifted to the right by approximately 2 logarithmic units in the presence of 30 mol/l cimetidine, but were not influenced in the presence of 0.3 mol/l dimetindene. The H₂-selective agonist impromidine (0.001–0.3 mol/l) had similar actions as histamine on spontaneously active Purkinje fibers, while the H₁-selective agonist 2-(2-pyridyl-)ethylamine was ineffective. It is concluded that the pronounced stimulatory action of histamine on spontaneous activity in sheep cardiac Purkinje fibers is exclusively mediated by H₂ receptors.Dedicated to Prof. Dr. E. Mutschler on the occasion of his 60th birthday.Supported by Ministerium für Wissenschaft und Forschung, Nordrhein-Westfalen, Projekt-Nr. 40008786.     

Luciani periods arising by the action of acetylcholine and eserine on automatic activity of Purkinje fibers

Experiments on isolated rabbit hearts with complete atrioventricular block showed that Luciani periods arise under the influence of acetylcholine (1·10^–7–2·10^–6 g/ml) or eserine (1·10^–7–2·10^–6 g/ml). These periods disappear as a result of the action of atropine (1·10^–6 g/ml). Microelectrode recordings showed that Luciani periods produced under the influence of acetylcholine or eserine are due to periodic inhibition of pacemaker activity of the Purkinje fibers. This supperssion of pacemaker activity is regarded as the result of relative insufficiency of the process of active ion transport.Laboratory of General and Clinical Physiology of the Heart, Institute of Normal and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 79, No. 4, pp. 33–36, April, 1975.     

Overdrive excitation: Onset of activity following fast drive in cardiac Purkinje fibers exposed to norepinephrine

Summary Purkinje fibers perfused in vitro in 5.4 mM K Tyrode remained quiescent in the presence of 8.8×10^–7 M norepinephrine. When these fibers were driven at 30/min and then overdriven at 120/min for 2 min, the cessation of drive was followed by the onset of spontaneous activity (overdrive excitation). It was found that overdrive excitation: 1. required both overdrive and norepinephrine to occur; 2. occurred when during the overdrive there was an increase in maximum diastolic potential (E _max) but not ifE _max decreased; 3. was caused by a steepening of diastolic depolarization following overdrive; 4. was not inhibited by a burst of fast drive; and 5. subsided through a progressive decrease ofE _max and of the slope of diastolic depolarization.It is proposed that overdrive excitation is due to a faster fall of the slowly changing potassium current to a smaller value. These changes, in turn, are caused by the combined action of: 1. a hyperpolarization due to both overdrive and norepinephrine; and 2. a shift in a depolarizing direction of the relationship between voltage and steady state slow potassium conductance due to norepinephrine.Supported by a grant from the New York Heart Association.Dr. Carpentier is an N. I. H. International Fellow from the University of Chile.     

Effects of antikaliuretic agents on cardiac electrophysiology—Measurements in papillary heart muscle and in purkinje fibers

Summary Electrophysiological parameters of myocardial excitability of heart muscle in vitro were studied under the influence of antikaliuretic diuretics. Potassium canrenoate, amiloride and triamterene were added in final concentrations of 5–440 µg/ml incubation medium. Resting and action potentials as well as refractory periods of single muscle cells and Purkinje fibers were measured by the microelectrode technique. — The antikaliuretic substances potassium canrenoate, amiloride and triamterene led to a significant concentration-dependent prolongation of action potential duration and, correspondingly, to a lengthening of the refractory period. Glycoside-induced shortening of the refractory period could be demonstrated to be antagonized by triamterene. — Results suggest that aldosterone antagonists, amiloride and triamterene may exhibit antiarrhythmic properties in the ventricular myocardium as well as in the conducting tissue. Drugs are equally effective when compared in molar concentrations. The application of antikaliuretic diuretics seems to be useful in the therapy of congestive heart failure with respect to their extrarenal cardiac effects, too.Presented in part at the 7th European Congress of Cardiology Amsterdam 1976     

Activity of Purkinje cells, parallel fibers, and climbing fibers in the developing rabbit cerebellum

A study has been made of the activity of Purkinje cells, parallel fibers, and climbing fibers in 120 rabbits aged from a few hours to 20 days. Spontaneous simple spike activity due to activation by the mossy fiber-granular cell-Purkinje cell system has been found, and also a complex spike related to direct activation by the climbing fibers from the first few hours of life. Statistical analysis of unit discharges shows that there is a general linear increase in their frequency with age. The presence of a propagated spike in the parallel fibers and of complex discharges in the Purkinje cells in response to stimulation of afferent climbing fibers confirms that a large part of the cerebellar synaptic system is operative from the time of birth.     

The effects of extracellular potassium,ouabain, and prostaglandins on intracellular potassium activity in sheep cardiac Purkinje fibers

(1) Intracellular K activity (a _K ⁱ ) of sheep heart Purkinje fibers was measured using K-selective microelectrodes (liquid ion exchanger).a _K ⁱ in the resting state with an extracellular K of 4 mmol·l^–1 was 112.9±6.1 mmol·l^–1 (n=47) for a membrane potential (V _M) of –73.3±0.9 mV.V _M deviated from the calculated potassium equilibrium potential (E _K=–93 mV). (2) When extracellular K was decreased to 2 mmol·l^–1 or increased to 6 and 10 mmol·l^–1 E _K changed from –114 to –84 and –73 mV, with little change ina _K ⁱ . (3)a _K ⁱ andV _M significantly decreased after administration of 10^–6 mol·l^–1 ouabain. (4) Prostaglandins (PGI₂ 10–100 g·l^–1 and PGE₂ 0.01–1 g·l^–1) decreaseda _K ⁱ without greatly changingV _M. The differences betweenV _M andE _K became smaller. These effects indicate an increase in K permeability and may explain the antiarrhythmic action of prostaglandins.This study was supported by the Deutsche Forschungsgemeinschaft (grant Wi 328). In preliminary form part of the data has been presented (Pflügers Arch. 384: R 13, 1980, and Proc. XXVIII. Int Congr Physiol Sci, Vol XIV: 279, 1980)     

Norepinephrine and potassium fluxes in cardiac Purkinje fibers

Summary Dog ventricular Purkinje fibers, loaded with⁴²K, were mounted in a tissue bath located over a beta-detector and washed in Tyrode solution. Tissue radioactivity was measured at intervals and transmembrane potential recorded by means of microelectrodes. Relative potassium influx was estimated by re-exposing the fiber to radioactive solution for fixed periods and measuring the tissue radio-activity increase at the end of the uptake period. Potassium efflux was estimated by measuring tissue and effluent radioactivities. After control recordings, administration of norepinephrine caused the following changes during the period it was applied: 1. increase in potassium influx; 2. increase in potassium efflux in nonstimulated fibers; 3. a smaller increase in potassium efflux in fibers stimulated electrically at a constant rate; 4. increase in potassium content in fibers loaded to equilibrium with⁴²K; 5. a smaller increase in K uptake in fibers driven at a high rate; and 6. enhancement of potassium uptake even in the presence of high [K]₀. It is concluded that norepinephrine increases both influx and efflux in Purkinje fibers. The larger enhancement of the influx with respect to the efflux and the increase in potassium content suggests that norepinephrine stimulates active transport of potassium. This stimulatory action can be dissociated from the stimulation of automaticity.This investigation was supported by a grant from N. I. H. (Nr. HE-10097).     

Effect of isoprenaline on active Na transport in sheep cardiac Purkinje fibres

The effect of isoprenaline (ISO; 5·10^–8 to 10^–6 M) on active Na transport was studied in depolarized sheep cardiac Purkinje fibres. Membrane current (I) and intracellular Na activity were measured simultaneously during enhanced Na pumping in voltage clamped preparations. ISO stimulated enhanced active Na transport but did not affect membrane current or intracellular Na concentration (c _na ⁱ ) in the steady state under the chosen experimental conditions. The stimulatory effect of ISO was mediated by ₁-adrenoceptors via a cAMP dependent pathway. The effect depended on the extracellular K concentration (c _k ⁰ ) and was inhibited by external Ba ions. Complementary experiments on isolated sheep Purkinje cells revealed no ISO induced alteration of the Na pump current.The mechanism of the ISO induced stimulation of enhanced Na pumping in sheep Purkinje fibres probably involves an augmented K efflux. A direct effect on the pump molecule seems unlikely.This work was supported by the Deutsche Forschungsgemeinschaft (Forschergruppe Konzell)On leave from the Institute of Experimental Biology, Armenian Academy of Sciences, Yerevan, USSR     

Mechanism of acetylcholine action on pacemaker current (i f) in canine Purkinje fibers

We have recently reported in canine Purkinje fibers that acetylcholine (ACh) can reverse the positive voltage shift of the pacemaker current (i _f) induced by -adrenergic stimulation while having no direct action of its own [3]. We have now investigated this effect of ACh on the cyclic adenosine monophosphate (cAMP) cascade in more detail. We find that addition of a membrane permeable analogue of cAMP (8-chlorophenylthio cAMP), 0.5–1 mM, increased the amplitude of i _f .This action was not reversed by 1 M ACh, implying that ACh acts at a step prior to cAMP action. We then looked at the steps controlling intracellular concentration of cAMP. Inhibiting the phosphodiesterase with 100 M isobutyl-1-methylxanthine (IBMX) increased i _f. This action, however, was reversed by ACh. Finally we investigated whether the action of forskolin, a direct activator of adenylyl cyclase, could be reversed by ACh. Forskolin (10–20 M) increased i _f, and ACh at 1 M partially reversed this action of forskolin. These results suggest that, in canine Purkinje fibers, ACh reverses the positive action of -adrenergic agents on i _f via a decrease in cAMP production.     

Electrophysiological characterization of histamine receptor subtypes in sheep cardiac Purkinje fibres

The histamine-receptor subtype mediated effects on action potentials of electrically driven and spontaneously active isolated sheep cardiac Purkinje fibres were investigated using H₁-and H₂-selective agonists and antagonists.In electrically stimulated Purkinje fibres histamine (3 mol/l) increased action potential plateau height, decreased action potential duration at –60mV, and enhanced pacemaker activity. These effects were abolished by 30 mol/l cimetidine (H₂-selective antagonist), but not impaired by 0.3 mol/l dimethindene (H₁-selective antagonist).     

Electrophysiological characterization of histamine receptor subtypes in sheep cardiac Purkinje fibres

The histamine-receptor subtype mediated effects on action potentials of electrically driven and spontaneously active isolated sheep cardiac Purkinje fibres were investigated using H₁-and H₂-selective agonists and antagonists.

In electrically stimulated Purkinje fibres histamine (3 μmol/l) increased action potential plateau height, decreased action potential duration at −60mV, and enhanced pacemaker activity. These effects were abolished by 30 μmol/l cimetidine (H₂-selective antagonist), but not impaired by 0.3 μmol/l dimethindene (H₁-selective antagonist).

In spontaneously active Purkinje fibres, histamine (10 μmol/l) increased the spontaneous rate by 24%, the slope of diastolic depolarization by 45% and shortened the duration of the diastole at −60mV by 32% of the respective control measurements. These effects were blocked by 30 μmol/l cimetidine, but not by 0.3 μmol/l dimethindene. The concentration-response relationship of histamine was shifted to higher histamine concentrations by approximately 2 logarithmic units in the presence of 30 μmol/l cimetidine, but no displacement was found in the presence of 0.3 μmol/l dimetindene. The H₂-selective agonist impromidine (0.001–0.3 μmol/l) has similar actions to histamine on spontaneously active Purkinje fibres, while the H₁-selective agonist 2-(2-pyridyl-)ethylamine was ineffective. It is concluded that the pronounced stimulatory action of histamine on spontaneous activity in sheep cardiac Purkinje fibres is exclusively mediated by H₂-receptors.

     

Existence of a calcium-dependent potassium channel in the membrane of cow cardiac Purkinje cells

Single-channel currents were recorded in the membrane of cow cardiac Purkinje cells using the patch-clamp technique. Recordings from cell-attached and cell-free patches demonstrated large outward single-channel currents associated with depolarizing voltage-clamp pulses. The time course of the reconstructed mean current showed a rapid activation phase followed by a slower inactivation following a single exponential time course with a time-constant in the range 30 ms to 100 ms. The current-voltage relation of the channel was linear in the voltage range between + 10 mV and + 110 mV with a slope conductance of 120 pS in 10.8 mM external K. The results indicated that the channel is selective for K ions. In inside-out patches, when the internal Ca activity was raised from 0.01 M to 1 M, the frequency of opening of the K channel during a depolarizing pulse was markedly increased, indicating Ca-dependence of these channels. The relation between this ion channel and the previously described transient outward current in cow Purkinje fibres is discussed. In sheep Purkinje cells a channel, carrying a transient outward current, with different properties was found.     

Spring-like fibers for cardiac tissue engineering

Recapitulation of the cellular microenvironment of the heart, which promotes cell contraction, remains a key challenge in cardiac tissue engineering. We report here on our work, where for the first time, a 3-dimensional (3D) spring-like fiber scaffold was fabricated, successfully mimicking the coiled perimysial fibers of the heart. We hypothesized that since in vivo straightening and re-coiling of these fibers allow stretching and contraction of the myocardium in the direction of the cardiomyocytes, such a scaffold can support the assembly of a functional cardiac tissue capable of generating a strong contraction force. In this study, the mechanical properties of both spring-like single fibers and 3D scaffolds composed of them were investigated. The measurements showed that they have increased elasticity and extensibility compared to corresponding straight fibers and straight fiber scaffolds. We have also shown that cardiac cells cultivated on single spring-like fibers formed cell–fiber interactions that induced fiber stretching in the direction of contraction. Moreover, cardiac cells engineered within 3D thick spring-like fiber scaffolds formed a functional tissue exhibiting significantly improved function, including stronger contraction force (p = 0.002), higher beating rate (p < 0.0001) and lower excitation threshold (p = 0.02), compared to straight fiber scaffolds. Collectively, our results suggest that spring-like fibers can play a key role in contributing to the ex vivo formation of a contracting cardiac muscle tissue. We envision that cardiac tissues engineered within these spring-like fiber scaffolds can be used to improve heart function after infarction.     

The cardioplegic solution HTK: effects on membrane potential,intracellular K+ and Na+ activities in sheep cardiac Purkinje fibres

The effects of the cardioplegic solution HTK on membrane potential (E_M) and intracellular K and Na activities (a _K ⁱ , a _Na ⁱ ) were studied in sheep cardiac Purkinje fibres by means of conventional and ion-selective microelectrodes. HTK contains (mM): Na 15, K 10, Ca 0, Mg 4, histidine 180, (1) In control conditions E_M was –74.3±3.3 mV (n=25), a _K ⁱ was 116.4±4.1 mM (n=7) and a _Na ⁱ was 8.2±1.4 mM (n=15). (2) Exposure to HTK led to a depolarization to –59.7±3.6 mV (n=25) which exceeded by about 5–7 mV that induced in a Tyrode solution of 10 mM K and in a modified HTK solution supplemented by 2 mM Ca (n=6). (3) Addition of 0.5 mM barium eliminated the difference in the steady-state depolarization. (4) HTK superfusion increased a _K ⁱ to 120.1±4.4 mM (n=7) and decreased a _Na ⁱ to 3.9±0.9 mM (n=15). (5) The decrease in a _Na ⁱ was insensitive to amiloride (1 mM) and to external alkalization but was slightly increased by addition of 2 mM calcium. (6) When the calcium in Tyrode solution was lowered from 2.0 mM to 0.05 mM, a _Na ⁱ hardly decreased during subsequent exposure to unmodified HTK and it increased in the presence of 0.1 mM dihydroouabain. We propose the hypothesis (1) that the difference in membrane depolarization between HTK and a 10 mM K-Tyrode is caused by a decrease in K conductance by the HTK solution and (2) that the a _Na ⁱ decline mainly results from a coupled Ca influx via Na-Ca exchange due to a delayed washout of external calcium.This work was supported by the Deutsche Forschungsgemeinschaft, SFB 330 — Organprotektion