Pflügers Arch – Eur J Physiol (1996) 431 : 652–657
© Springer-Verlag 1996
O R I G I NA L A RT I C L E
Witold Tuganowski
The effects of phosphocreatine introduced simultaneously into many cardiac cells
Received: 19 April 1995 / Received after revision: 25 August 1995 / Accepted: 8 September 1995
Abstract The aim of the present study was to ascertain whether or not phosphocreatine (PC) could produce electrophysiological and inotropic effects in isolated rabbit cardiac preparations. Exogenous PC (50 mmol/l) was introduced into many cells simultaneously by the “cut-end” and “saponinated-end” methods. PC that entered the cells (opened by cutting or chemical disruption of the sarcolemma) in the loading region, passed through the preparation intercellularly and evoked the following effects in the test region. PC enhanced the spontaneous rate and probably shifted the pacemaker in sinus node strips. On the other hand, PC elevated the action potential amplitude and duration and increased the isometric tension in atrial and ventricular strips. Furthermore, PC applied into ventricular cells partially prevented the effects of hypoxia. These findings suggest that PC may act in cardiac muscle as an intercellular energy carrier. The effects of PC introduced intracellularly resembled these evoked by O-benzyl-phosphocreatine – a permeant synthetic phosphagen – applied via superfusion. Key words Phosphocreatine · Cardiac muscle · Cut-end method · Saponinated-end method · Electrophysiology · Inotropy · Intercellular communication
Introduction Phosphocreatine (PC) injected into single ventricular cells increased the Ca2+ current, and thereby enhanced the action potential amplitude and duration [11, 16]. On the other hand, PC applied via superfusion did not
W. Tuganowski (*) Department of Physiology, Silesian School of Medicine, Medyków 18, PL-40 762 Katowice, Poland
influence either electrical activity or contractility in the multicellular cardiac specimens [18]. However, O-benzyl-phosphocreatine (BPC)–a permeant synthetic PC analogue–added to Tyrode solution evoked marked electrophysiological and inotropic effects in isolated cardiac preparations [18]. In the present experiments, PC was introduced into many cardiac cells simultaneously by the cutend method [4, 5, 20] and saponinated-end (sapend) method in order to find out whether or not this phosphagen could evoke electrophysiological and inotropic effects in isolated tissue. The results obtained verify the specificity of the effects of BPC and support the view that PC may carry metabolic energy intercellularly.
Materials and methods Adult rabbits of both sexes weighing 2.5–3.5 kg were anaesthetised with ethyl urethane (1 g / kg body wt.), and the hearts were quickly removed. Sinus node strips (0.7 mm wide, 0.7 mm thick), atrial trabeculae (less than 0.9 mm in diameter), right ventricle papillary muscles (less than 0.9 mm in diameter) and free-running ventricular trabeculae (less than 0.9 mm in diameter) were cut immediately from the hearts and mounted in a two-compartment Perspex chamber with interchangeable rubber partitions (0.2 mm thick). In each partition a hole was made to fit the individual cross-sectional area of the specimen. The partition divided the chamber into a test compartment (400 µl) perfused with warmed (30o C) Tyrode solution (NaCl 139, KCl 5.4, CaCl2 1.8, MgCl2 0.5, NaHCO3 12, glucose 5.5 mmol /l) saturated with carbogen (95 % O2 + 5 % CO2 gas mixture), and a loading compartment (200 µl). Each preparation was pulled partway through the hole and protruded both into the test compartment (2–2.5 mm) and into the loading compartment (1–1.5 mm). Both compartments were perfused continuously at a rate of 1 ml / min. In the cut-end experiments the loading compartment was perfused initially (30 min) with Tyrode solution. Then, an isotonic KCl solution containing 0.5 mmol /l ethylenebis (oxonitrilo) tetraacetate (EGTA) (pH adjusted to 7.4 by addition of NaHCO3) was applied for 10 min. Finally, this compartment was perfused with loading solution (PC dipotassium salt 50, KCl 75, EGTA 0.5 mmol / l, pH adjusted to 7.7 by addition of NaHCO3), and the loading region
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Fig. 1 Peak effects of phosphocreatine introduced by the sap-end method (A,D) and cut-end method (B,C) on action potentials and isometric tension. C Control, PC effects of phosphocreatine in the sinus node strip (A), atrial trabecula (B), papillary muscle (C) and ventricular trabecula (D) of the preparation was cut transversely at about 0.5 mm from the partition. In the sap-end experiments, saponin was applied in order to destroy the cell membrane in the loading region. Originally, saponin was used for cell membrane disruption in open-cell patch-clamp experiments [7]. In the present experiments the loading compartment was perfused initially with Tyrode solution for 30 min. Then, the Tyrode solution was replaced by an isotonic KCl solution containing 0.5 mmol/l EGT and 0.5% w /v saponin. The pH was 8.5. The saponin-containing solution was used for the next 10 min, and then was replaced by the loading solution. The drugs used were: PC dipotassium salt (Calbiochem, San Diego, Calif., USA), creatine, EGTA, saponin (Sigma, St. Louis, Mo., USA). The sinus node strips were spontaneously active, the atrial and ventricular specimens were driven with square-wave pulses (1 Hz, 2–4 ms duration, 1.5 threshold voltage) delivered from Ag /AgCl electrodes (one placed in the test compartment, the second in the loading compartment). Transmembrane potentials were recorded by means of glass capillary microelectrodes with an inner filament. The microelectrodes were impaled into distal cells of the test regions. The isometric tension was measured with an F 30 H Sachs transducer. The records were displayed and stored by a Tektronix 2221A oscilloscope, and then plotted by a Tektronix HC 100 plotter. Each record was the average of eight unitary transients.
Results The initial perfusion of the both compartments with Tyrode solution stabilised the control values in the test region within 10–25 min. Subsequent replacement of Tyrode fluid in the loading compartment by an isotonic KCl solution (with or without saponin) did not change the control values. This was used to establish that the compartments were sufficiently separated from each other; if they were not, a leakage of KCl or KCl plus saponin abolished any activity in the test region within 10–20 s. In five control experiments, the loading region of the papillary muscle was superfused with saponincontaining KCl solution for 60 min. This superfusion did not affect the transmembrane potentials and isometric tension in the test region. On the other hand, addition of saponin (0.1 % w / v) into the test compartment evoked irreversible cessation of any activity within 20–30 s. PC applied by the cut-end or sap-end method into sinus node strips increased the spontaneous rate to a maximum within 35–50 min and 25–40 min respectively (Fig. 1, Table 1). The peak effects lasted 15–25 min, and then there was a decay to the control values within a further 20–30 min.
654 Table 1 The peak effects of 50 mmol/l of phosphocreatine (PC) applied by the cut-end method and saponinated-end (sap-end) method into cardiac preparations. Each value is the mean ± SEM of 10 experiments. The percentage change is the mean ± SEM of differences between the control values and those in the presence of PC. (SF Spontaneous frequency, RP resting potential, AP action potential; APD75 75% of the action potential duration, IT isometric tension)
Preparation and conditions
Parameter SF (Hz)
Sinus node: Control PC (cut-end) Change (%) Control PC (sap-end) Change (%)
RP (mV)
2.4 ± 0.1 3.2 ± 0.3 27 ± 3.1** 2.5 ± 0.1 3.3 ± 0.1 33 ± 3.6**
AP (mV)
APD75 (ms)
IT (mg)
72 ± 1.3 76 ± 1.1 5 ± 1.3* 72 ± 1.7 73 ± 0.7 0.8 ± 1.1ns
Atrial trabecula: Control PC (cut-end) Change (%)
80 ± 0.6 81 ± 0.5 0.9 ± 0.5ns
106 ± 0.7 113 ± 0.8 6.2 ± 0.6**
83 ± 1.8 101 ± 2.2 22 ± 1.2**
51 ± 3.1 177 ± 46 244 ± 21**
Control PC (sap-end) Change (%)
80 ± 1.9 81 ± 1.2 1.7 ± 1.2ns
103 ± 1.3 111 ± 1.4 7.6 ± 1.1**
85 ± 2.6 108 ± 3.6 28 ± 3.0**
67 ± 5.8 321 ± 34 382 ± 35**
Ventricular tissue: Control PC (cut-end) Change (%) Control PC (sap-end) Change (%)
79 ± 0.6 80 ± 0.5 0.4 ± 0.9ns 80 ± 1.2 81 ± 1.9 1.4 ± 0.8ns
102 ± 1.4 110 ± 1.5 6.6 ± 0.8** 105 ± 2.4 113 ± 3.2 7.2 ± 0.5**
196 ± 4.5 237 ± 9.1 21 ± 3.1** 185 ± 9.3 228 ± 9.8 24 ± 3.1**
60 ± 7.3 171 ± 12 200 ± 25** 55 ± 11 318 ± 57 495 ± 23**
*P < 0.01, **P < 0.001 vs the control; ns, not significant (Student’s t-test for paired data)
PC introduced into atrial and ventricular cells by the cut-end method or sap-end method evoked a peak increase in the action potential amplitude and duration and the maximal positive inotropic response within 50–70 min (Fig. 1, Table 1). In the cut-end experiments the peak effects lasted for 10 –20 min, and then there was a decline to the control values within Fig. 2A, B The effects of hypoxia and phosphocreatine applied by the sap-end method on the transmembrane potentials and isometric tension in the ventricular specimens. A Hypoxia evoked before application of phosphocreatine. [C Control, H hypoxia (20th min), H + PC phosphocreatine added during hypoxia (70th min)]. Note the depolarisation and increase in the resting tension, B Hypoxia evoked after introduction of phosphocreatine. [C Control, PC effects of phosphocreatine (60th min), PC + H effects of phosphocreatine during hypoxia (120th min)]
a further 5–20 min. In the sap-end experiments the maximal effects lasted 60–70 min, and then there was a decrease to the control values within a further 40–50 min. In the other experimental group, PC was administered to ventricular tissue by the sap-end method during hypoxia induced in the test region. Hypoxia was evoked by gassing the Tyrode solution with 95% N2+5 % CO2 rather than carbogen. In five experiments, hypoxia was induced before (20 min) the addition of PC. Under these conditions the presence of PC in the loading compartment was entirely ineffective. Hypoxia abolished the contractility of the examined preparations within 20–30 min, and after a further 30–40 min it made the tissue inexcitable (Fig. 2).
655 Table 2 Effects of PC (50 mmol/l) applied by the sap-end method and of hypoxia (H) induced in ventricular preparations. Each value is the mean ± SEM of 5 experiments. The percentage change is the mean ± SEM of differences between effects in the presence of PC (60 min) and PC + H (120 min)
Conditions
Parameter RP (mV)
Control PC (60 min) PC + H (120 min) Change (%)
81 ± 1.3 83 ± 1.5 82 ± 1.4 [1.3 ± 0.8ns
APD75 (ms)
IT (mg)
105 ± 1.5 115 ± 1.9 113 ± 2.0 [1.8 ± 1.1ns
208 ± 8.7 270 ± 9.5 224 ± 8.9 [18 ± 3.8**
62 ± 9 205 ± 16 172 ± 10 [16 ± 4.5*
*P < 0.05, **P < 0.001
Fig. 3 Peak effects of creatine applied by the sap-end method (A) and added to the superfusion medium (B) on action potentials and isometric tension in the ventricular preparations. (C Control, Cr effects of creatine)
In five other experiments, PC was introduced into normoxic preparations. Then, after 60 min, the test region was superfused with hypoxic Tyrode solution for a further 60 min. Hypoxia did not significantly change the resting and action potential amplitudes. However, the action potential duration and isometric tension were reduced compared with the respective maximal effects of PC (Fig. 2, Table 2). In five sap-end experiments performed on the ventricular preparations, the composition of the loading solution was (in mmol/l): creatine 50, KCl 125, EGTA 0.5, pH adjusted to 7.7 by addition of NaHCO3. Creatine reduced the action potential amplitude and duration Table 3 Effects of creatine applied by the sap-end method (50 mmol/l) and added to the superfusion medium (5 mmol/l) in ventricular preparations. Each value is the mean ± SEM of 5 experiments. The percentage change is the mean ± SEM of differences between control values and those in the presence of creatine
AP (mV)
Conditions
and evoked a negative inotropic response. These effects reached their maximum within 35–45 min and lasted for 20–30 min (Fig. 3, Table 3). Then, there was a return to the control values within a further 10–15 min. In five other experiments, creatine (5 mmol/l) was added to Tyrode solution which superfused the ventricular preparation. The effects of creatine reached their maximum after 6–8 min of superfusion (Fig. 3, Table 3). Washing out the preparations with pure Tyrode solution allowed them to recover their control values within 3–5 min.
Discussion The present results reveal that some impermeant compounds, such as PC, can be introduced into many
Parameter RP (mV)
Sap-end: Control Creatine Change (%) Superfusion: Control Creatine Change (%)
82 ± 0.9 81 ± 1.0 [1.5 ± 1.3ns 83 ± 1.1 82 ± 1.5 [1.4 ± 1.2ns
*P < 0.01, **P < 0.001 vs the control
AP (mV) 107 ± 1.2 96 ± 1.9 [9.1 ± 0.6** 110 ± 1.4 94 ± 2.1 [14 ± 1.2**
APD75 (ms)
IT (mg)
191 ± 6.3 162 ± 9.0 [15 ± 3.2*
77 ± 11 21 ± 3.1 [71 ± 3.1**
220 ± 7.6 153 ± 8.8 [30 ± 4.1**
65 ± 14 21 ± 3.1 [67 ± 4.2**
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cardiac cells simultaneously by the cut-end as well as by the sap-end method. However, the maximal effects observed in the sap-end experiments were greater and lasted longer than in the cut-end ones. Probably, the saponin-induced chemical skinning enabled a direct diffusion into the cells because of the disrupted surface membranes. The present control experiments showed that when isotonic KCl solutions (without or with saponin) perfused the loading compartment, they did not affect the electrical activity and contractility of the test region. This fact indicated that any significant leakage or interstitial diffusion did not occur between the compartments. It was also noteworthy that continuous perfusion of either compartment reduced the effects of any leakage. These findings suggest that extracellular diffusion of PC into the test compartment is also negligible, the rather that the phosphagen does not cross the cell membrane [2, 3, 13, 14], and does not influence the examined parameters when added extracellularly [18]. On the other hand, various antiarrhythmic, cardioprotective and membrane-stabilising actions of PC have been reported. These effects, however, were observed only in anoxic and ischaemic hearts [14]. In these experiments PC applied into the loading compartment probably entered the cells, travelled intercellularly and evoked electrophysiological and inotropic effects in the test region. This view is corroborated by the present experiments in which hypoxia was used as an uncoupling agent [8, 21]. PC, applied before hypoxia, could, at least partially, prevent its effects, probably because the phosphagen supplied continuously was able to keep the gap junctions in the open state in spite of subsequent hypoxia. This effect of PC can be related to the direct action of ATP on connexons. ATP, in a dose-dependent manner, increased cell coupling in pairs of ventricular cells [15]. In this way, PC after transphosphorylation into ATP can prevent the uncoupling effects of hypoxia. On the other hand, PC did not counteract the effects of existing hypoxia. In this case, PC probably could not open the connexons that were already closed due to the decrease in the ATP content induced by hypoxia. The ATP concentration in ventricular tissue can be reduced to about 25% of the normoxic control value within 20 min of undergoing hypoxia [10]. Under these conditions intercellular communication was probably diminished. Further superfusion with hypoxic Tyrode solution, even in the presence of PC, could only intensify the uncoupling effects (see Fig. 2A). The ineffectiveness of PC applied during existing hypoxia suggests that the phosphagen cannot act directly on the connexons. Otherwise, PC introduced into the cardiac cells should be able to counteract the effects of hypoxia. One might assume that the electrophysiological and inotropic effects of PC were evoked by its metabo-
lite – creatine. However, the control experiments with creatine applied either intra- or extracellularly excluded such a possibility. PC and creatine exerted directly opposite effects. Similar results were obtained by other authors : creatine injected intracellularly reduced the action potential amplitude and duration in isolated ventricular cells [16]. Thus, the effects observed after the intracellular application of PC must in fact have been evoked by this phosphagen. One cannot exclude that PC exerts some effects by acting directly on the membrane channels. However, the involvement of ATP in the PC-induced effects is much more probable. This opinion is supported by the following facts : (1) PC can be transphosphorylated only into ATP; (2) there is no other known biochemical reaction involving PC [1, 6, 14, 19]; (3) intracellular ATP directly influences some classes of the Ca2+ and K+ channels [7, 11, 16, 17] : (4) BPC enhances the ATP content in the atrial preparations [18]. Therefore, the effects of PC can be attributed to the direct action of ATP on the Ca2+ and K+ channels. In light of the above-described findings, the effects of creatine also seem to be associated with the action of ATP. Creatine, whether applied extra- or intracellularly, evoked similar depressive effects on the action potential and contractility. These facts suggest that creatine can migrate through the cell membrane. Addition of creatine would increase its content in the cardiac cells and may shift the reaction: creatine + ATP ¢ PC+ADP to the right, so reducing ATP levels. Decreased ATP content can enhance the K+ conductance and reduce the Ca2+ current [7, 11, 16, 17]. Such effects can evoke a decrease in the action potential amplitude and duration, as well as the negative inotropic response. On the other hand, the introduction of PC would tend to shift to ATP synthesis, while the creatine excess could leave the cell. These proposals do not exclude some direct action of creatine in the cardiac cell. The electrophysiological and inotropic effects of PC applied intracellularly resembled, at least qualitatively, these obtained during superfusion of BPC except that this drug was capable of counteracting the effects of hypoxia [18]. BPC could pass through the surface membrane directly, whereas PC could only travel intercellularly. These facts support the idea that BPC acted specifically in the rabbit heart, i.e. BPC was probably converted into PC in the cardiac cells. Cell-to-cell diffusion is mainly dependent on the molecular weight of the examined substance, on the ratio of the gap junction area to the total membrane area and on the cell dimensions [4, 5, 20]. From theoretical calculations the permeability of the gap junction for PC (210 Da) is about 10[3 cm /s [5]. This indicates that the first molecules of PC may reach the most distal cells of the examined preparations within 3–4 min. However, the concentration of PC (and other migrating compounds) rises along the preparation
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length at a very slow rate [4, 5, 20]. Moreover, some of the PC can be stored and / or transphosphorylated throughout the course of its intercellular diffusion. These factors may explain the delay required to evoke the maximal effects of PC. Although the gap junctions between the small-sized sinus node cells occupied only 0.2 % of the cell border length [9], the peak effects of PC appeared after a considerably shorter time period than in the atrial and ventricular specimens. One may assume that PC shifted the pacemaker dominance towards the intercompartment partition where the phosphagen concentration was maximal. The gap junctions between the atrial and ventricular cells take up 1–3 % of the cell border length [9, 12], but the peak effects were visible after a long delay (about 1 h). It should be noted, however, that the peak effects were evoked by a maximal increase in the PC concentration in the whole test region of the atrial and ventricular specimens. Thus, the maximal possible rise in the PC content in the most distal cells may delimit the time period required to reach the peak inotropic and electrophysiological effects. Many experimental data suggest that PC is a temporal reservior of metabolic energy as well as a spatial energy buffer. The latter term signifies that PC is responsible for metabolic energy transport from the sites of ATP production to the sites of ATP utilisation: ATP is transphosphorylated into PC in the production sites, then PC migrates to the utilisation sites, and finally the phosphagen is transphosphorylated into ATP. Thus, PC acts as an energy carrier in an intracellular system called a “PC shuttle” or “PC circuit”. This system has been found in cardiac cells [1, 6, 19]. In summary, the present results indicate that PC can pass intercellularly in isolated cardiac preparations. Thus, one may assume that PC can also carry metabolic energy intercellularly. In this way, PC may equalise the energy levels between cardiac cells, and it can control ATP-operated Ca2+, and K+ channels [7, 11, 16, 17]. Such mechanisms may influence various processes dependent on the K+ and Ca2+ currents, e.g. the excitability of the cardiac cells and intercellular synchronisation of the pacemaker and inotropic responses.
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