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Laboratorio de Investigación Cardiovascular, Servicio de Cardiología, Hospitals Vall d'Hebron, Barcelona, Spain
Submitted 2 May 2005 ; accepted in final form 14 September 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-glycyrrhetinic acid, and palmitoleic acid on intracellular Ca2+ concentration during simulated hypoxia (2 mM NaCN) in isolated cardiomyocytes. Next, we analyzed their effects on developed and diastolic tension and electrical impedance in 47 isolated rat hearts submitted to 40 min of hypoxia and reoxygenation. All treatments were applied only during the hypoxic period. Cell injury was determined by lactate dehydrogenase (LDH) release. Heptanol, but not 18-glycyrrhetinic acid nor palmitoleic acid, attenuated the increase in cytosolic Ca2+ concentration induced by simulated ischemia in cardiomyocytes and delayed rigor development (rigor onset at 7.31 ± 0.71 min in controls vs. 14.76 ± 1.44 in heptanol-treated hearts, P < 0.001) and the onset of the marked changes in electrical impedance (tissue resistivity: 4.02 ± 0.29 vs. 7.75 ± 1.84 min, P = 0.016) in hypoxic rat hearts. LDH release from hypoxic hearts was minimal and was not significantly modified by drugs. However, all gap junction uncouplers, given during hypoxia, attenuated LDH release during subsequent reoxygenation. Dose-response analysis showed that increasing heptanol concentration beyond the level associated with maximal effects on cell coupling resulted in further protection against hypoxic injury. In conclusion, gap junction uncoupling during hypoxia has a protective effect on cell death occurring upon subsequent reoxygenation, and heptanol has, in addition, a marked protective effect independent of its uncoupling actions.
hypoxia; electrical properties; calcium
-glycyrrhetinic acid and palmitoleic acid (19), an effect associated with an attenuation in the recovery of electrical tissue impedance during reperfusion. However, gap junction uncouplers have protective effects not only when given during reperfusion but also when they are administered after the onset of ischemia (14), or before it (22). The protective effect seen when given in these two last situations could be explained by residual protective effects on the posterior reperfusion, or by the fact that gap junction-mediated intercellular communication persists during ischemia and allows propagation of rigor contracture, and probably cell injury (21). The role of gap junction-mediated intercellular communication in the progression of ischemic injury is supported by observations indicating that heterozygous connexin 43 (Cx43)-deficient mice underexpressing Cx43 showed reduced infarct size after permanent coronary occlusion (9). The protective effects on reperfusion injury of gap junction uncouplers seem to be more powerful in the case of heptanol than with the other drugs tested, not only when analyzing infarct size but also when functional recovery was taken into account (14, 19). The higher efficacy of heptanol could be explained by other nonjunctional effects of this substance, including inhibition of Na+ and Ca2+ inward currents (15, 25). In addition, a recent study demonstrated that heptanol is able to reduce intracellular Ca2+ levels even at low concentrations (150 µM) in isolated rat mesenteric small arteries stimulated with norepinephrine (13). A reduction in intracellular Ca2+ overload has been demonstrated to be protective after ischemia-reperfusion in isolated rat hearts (7). Thus the aims of this work were to analyze the effects of heptanol and other gap junction uncouplers on intracellular Ca2+ concentration ([Ca2+]i), myocardial electrical impedance, and rigor development during myocardial hypoxia.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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HL-1 Cardiomyocytes and Freshly Isolated Rat Cardiomyocytes
HL-1 cardiomyocytes. HL-1 cardiac myocytes, an atrial-derived mouse cardiomyocyte cell line obtained from Dr. W. C. Claycomb, were cultured at 37°C, under a 5% CO2 atmosphere, in Claycomb medium supplemented with 10% FBS (JRH Biosciences), 4 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from GIBCO), and 100 µM norepinephrine (Sigma). Cells were seeded at a 20,000 cells/cm2 density in a culture flask precoated with 25 µg/ml fibronectin-0.02% gelatin solution until a 70–80% confluence was achieved, as previously described (3).
Freshly isolated rat cardiomyocytes. Because HL-1 cardiomyocytes are, in contrast to freshly isolated cardiomyocytes, electrically coupled, which may affect the response to gap junction uncouplers, we decided to repeat the experiments in freshly isolated rat cardiomyocytes. Ventricular heart muscle cells were isolated from adult male Sprague-Dawley rats (300 g) as previously described (20). Rat hearts were cannulated by the aorta and perfused in a Langendorff system with a modified Krebs buffer (containing in mM: 110 NaCl, 2.6 KCl, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, 11 glucose, and 0.045 CaCl2) continuously gassed with 95% O2-5% CO2. A 0.03% type II collagenase (Serva) was added to the buffer during the whole perfusion procedure. Cells from dissociated tissue were subjected to a progressive normalization of Ca2+ levels up to 1 mmol/l. The final proportion of rod-shaped cells was increased by means of 25 min gravity sedimentation in a 4% BSA gradient. The pellet of sedimented cells was resuspended in medium 199-HEPES (Sigma) and plated in laminin-pretreated glass-bottomed dishes. After being plated (1 h), nonattached cells were discarded by changing the medium.
Simulated hypoxia.
Cells were washed with normoxic buffer (in mM: 140 NaCl, 3.6 KCl, 2 CaCl2, 1.2 MgSO4, 20 HEPES, and 5 glucose, pH 7.4) and then subjected to 40 min of simulated ischemia (SI) in the same buffer lacking glucose and containing 2 mM NaCN and 20 mM 2-deoxyglucose in the absence (control cells) or in the presence of 10–3 M heptanol, 10–5 M 18-glycyrrhetinic acid, or 3 x 10–5 M palmitoleic acid.
Analysis of [Ca2+]i.
Analysis of [Ca2+]i was performed at 37°C (Digital Warm Stage Controller; Linkam) on the stage of an inverted microscope (Olympus IX70) at either x20 or x40 fluorite objectives. Changes in [Ca2+]i both in HL-1 and freshly isolated cardiac myocytes were analyzed throughout a simulated ischemic period in the presence or in the absence of 10–3 M heptanol, 10–5 M 18-glycyrrhetinic acid, or 3 x 10–5 M palmitoleic acid by a ratiofluorescence imaging system (QuantiCell2000; Visitech). Briefly, cells were loaded for 40 min at 37°C with 5 µM of the acetoxymethyl ester of fura 2 (Molecular Probes), washed two times, and postincubated in control medium to allow hydrolysis of the ester within the cells. Cells were then alternatively excited at 340 nm and 380 nm, with a bandwidth of 15 nm, by means of a fast-speed monochromator. Exposure time was set for each excitation wavelength at 100 ms. Emitted light (510 nm) was collected by an air-cooled intensified digital camera. Counts from clusters of 2 x 2 pixels within cell images were pooled to improve the signal-to-noise ratio. A 340/380 ratio value was calculated from individual fluorescence intensities obtained in each pixel cluster, and color-coded images were automatically generated using commercially available software (QuantiCell 2000; Visitech). Changes in the average ratio for previously defined regions of interest were analyzed throughout time every 10 s. In each cell, these changes were normalized with respect to the initial ratio value corresponding to control, normoxic conditions.
Isolated Rat Hearts
Experimental preparation. Forty-seven adult male Sprague-Dawley rats (250–350 g) were killed by an intraperitoneal overdose of pentobarbital sodium. The hearts were removed quickly and retrogradely perfused through the aorta with an oxygenated (95% O2-5% CO2) Krebs solution at 37°C (in mM: 118 NaCl, 4.7 KCl, 1.2 MgSO4, 2.5 CaCl2, 25 NaHCO3, 1.2 KH2PO4, and 11 glucose, pH 7.4). Both atria were removed, and the His bundle was crushed with a thin forceps to induce complete atrioventricular conduction block. After opening of the right ventricle and interventricular septum through a longitudinal incision from the cardiac base to the apex, hearts were pinned to a silicon membrane placed at the bottom of an organ bath, exposing the endocardial surface of the left ventricle (19). Cut epicardial arteries were ligated to prevent deficient perfusion of the remaining myocardium. A 2.0 silk-snare was placed in the septum of the hearts and connected to an isometric force transducer (FSG-01, SG-M DC bridge amplifier module; Experimentia, London, UK). Resting tension was 1 g. Preparations were paced from the cardiac base using rectangular pulses of 2.5 ms duration and 4 V of amplitude, at 400 ms basic cycle length.
Time of conduction blockade.
The time of conduction blockade was determined in transmembrane action potential recordings from the apical region of the heart, as previously described (19). Transmembrane action potentials were obtained using conventional glass microelectrodes (tip resistance 30–40 M
) filled with 3 M KCl and placed at 5–7 mm of distance of the stimulating electrode. A reference electrode was positioned in the bath near the recording microelectrode. Both the recording and the reference microelectrodes were connected via Ag-AgCl interfaces to high-input impedance amplifiers (VF-102 and IS-100; Biologic, Claix, France), and the signals were displayed on a digital storage oscilloscope (CS-8010; Kenwood), digitized at 10 kHz, and stored in a computer for further analysis. Conduction time was measured as the time between the stimulus artifact and the onset of the rapid depolarization of the action potential. Using this method, the estimated conduction velocity represents an averaged velocity between the stimulus site and the recording site, since the pathway of activation is not known. Conduction blockade was defined as complete loss of excitability at the pacing conditions used.
Myocardial electrical impedance.
Measurement of myocardial electrical impedance is an overall estimation of the passive electrical properties of the myocardium, which includes the intra- and extracellular resistance and the membrane capacitance. Myocardial electrical impedance is better characterized by measuring its two components: the in-phase component of voltage (V) with respect to current intensity (I) [i.e., tissue resistivity (R), calculated by direct application of Ohm's law, R = V/I] and the phase angle (
). Because biological tissues are not purely resistive, the capacitance of cell membranes must be considered. Phase angle is determined from the time delay between the voltage and intensity curves, by applying the formula = 360·
t·f, where t is the time delay and f the frequency of the injected current (Fig. 1). Its absolute value depends on membrane capacitance and is influenced by extracellular, intracellular, and membrane resistances. Myocardial impedance measurement was performed using a four-electrode probe placed in the septum, at 7 KHz, as previously described (19). The impedance probe consisted of a linear array of four platinum electrodes (length: 5 mm, diameter: 0.4 mm, interelectrode distance of 2.5 mm). An alternating current (10 µA) was applied through the outer pair of electrodes, and the in-phase components of voltage and phase angle were recorded every 10 s through the inner pair of electrodes (model 5110, high-input impedance lock-in amplifier; Princeton Applied Research).
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Experimental protocol.
After 30 min of equilibration, hearts were submitted to 40 min of hypoxia followed by 1 h of reoxygenation. Hypoxia was performed by changing the oxygenated Krebs solution perfused through the aorta by an hypoxic solution at pH 7.4 (in mM: 118 NaCl, 4.7 KCl, 1.2 MgSO4, 2.5 CaCl2, 25 NaHCO3, 1.2 KH2PO4, and 11 sucrose; bubbled with 95% N2-5% CO2). Sixteen of the hearts were used as controls, whereas in the other 19 hearts the effects of heptanol, given during the first 35 min of hypoxia, were analyzed at different concentrations, first at 10–3 M (n = 8) and thereafter from 10–4 M to 1.78 x 10–3 M (n = 2–3). In the remaining 12 hearts, the effects of other chemically unrelated gap junction uncouplers [18-glycyrrhetinic acid (10–5 M, n = 7) and palmitoleic acid (3 x 10–5 M, n = 5)], administered during the first 15 min of hypoxia, were analyzed and compared with those of control and heptanol-treated groups. In one-half of the control rat hearts, 0.15% DMSO was added to the hypoxic Krebs solution to exclude an effect of the vehicle. Because no difference was observed with respect to those controls untreated with DMSO, their data were pooled. The drugs and concentrations used have been previously demonstrated to be protective against reperfusion injury in isolated rat hearts and cause a reduction in normoxic conduction velocity of 
40, 85, and 26% for heptanol, 18-glycyrrhetinic acid, and palmitoleic acid, respectively (19). Palmitoleic acid and glycyrrhetinic acid were administered only during the initial 15 min of hypoxia because of toxic effects with more prolonged times (19). Changes in developed and diastolic tension, conduction velocity, and myocardial electrical resistivity and phase angle were continuously and simultaneously recorded during the whole experiments, and cell injury was evaluated by measurement of lactate dehydrogenase (LDH) release during reoxygenation.
Chemicals.
Heptanol (1-heptanol), 18-glycyrrhetinic acid (3
-hydroxy-11-oxo-18,20-olean-12-en-29-oic acid), and palmitoleic acid (cis-9-hexadecenoic acid) were obtained from Sigma Chemical. Palmitoleic acid and 18-glycyrrhetinic acid were initially dissolved in DMSO and then dissolved in Krebs solution to the desired concentration. Final DMSO concentration was 0.15%. Heptanol was directly dissolved in Krebs solution.
Statistical analysis. Statistical analysis was performed using commercially available software (SPSS for Windows 8.0). Data are expressed as means ± SE. ANOVA or repeated-measures ANOVA (MANOVA) and Dunnett's post hoc tests were used to assess differences between the different groups of treatment. A P value <0.05 was considered to be significant.
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RESULTS |
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Effects of heptanol on [Ca2+]i.
During SI (Figs. 2 and 3), control HL-1 cells and rat cardiomyocytes showed an increase in [Ca2+]i. Heptanol (10–3 M), but not 18-glycyrrhetinic acid nor palmitoleic acid, added at the onset of the SI period markedly delayed the onset of this Ca2+ overload and attenuated its maximal value (Figs. 2 and 3). In fact, 18-glycyrrhetincic acid-treated HL-1 cardiomyocytes showed a tendency toward a more pronounced Ca2+ overload compared with controls after 15–20 min of treatment. Rigor onset in isolated rat cardiomyocytes was significantly delayed by heptanol compared with control cells, but not by glycyrrhetinic acid nor palmitoleic acid (Fig. 3B).
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Effects of gap junction uncouplers on isolated rat hearts during hypoxia/reoxygenation. HYPOXIC RIGOR CONTRACTURE.
Developed tension decreased during hypoxia to reach a minimal value several minutes later. Rigor contracture, detected as a sudden increase in diastolic tension, occurred at 7.31 ± 0.71 min after the onset of hypoxia in control hearts (Fig. 4A). Heptanol, given during hypoxia at a concentration of 10–3 M, significantly attenuated rigor development (Fig. 5A) and delayed its onset (14.76 ± 1.44 min, P < 0.001; Fig. 4A). In contrast to heptanol effects, other two chemically unrelated gap junction uncouplers (18-glycyrrhetincic acid and palmitoleic acid, at 10–5 M and 3 x 10–5 M, respectively), when given during the first 15 min of hypoxia, did not modify the time course nor the onset of rigor development during oxygen deprivation (Figs. 4A and 5A).
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-glycyrrhetincic acid and palmitoleic acid-treated hearts did not show an improvement of function (0.82 ± 0.82 and 0.0 ± 0.0% of baseline tension in 18-glycyrrhetincic acid and palmitoleic acid-treated hearts, respectively).
CONDUCTION BLOCKADE.
Hypoxia caused a progressive reduction in conduction velocity in isolated rat hearts (Fig. 6). Conduction blockade was observed at 11.61 ± 0.9 min of hypoxia and was significantly delayed by treatment during hypoxia with heptanol (19.38 ± 1.45 min, P < 0.001; Fig. 4B). Palmitoleic acid did not modify the time of conduction blockade, whereas in hearts treated with 18-glycyrrhetincic acid it occurred slightly earlier (Fig. 4B).
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·cm, P < 0.001) and a marked negative shift in phase angle (from –1.83 ± 0.09 to –4.85 ± 0.51°, P < 0.001). In contrast, in heptanol-treated hearts, changes in myocardial electrical impedance were attenuated significantly (Fig. 5, B and C). Moreover, the onset of the marked changes in electrical impedance, which some authors have related to the onset of cell-to-cell electrical uncoupling based on an associated increase in intracellular but not extracellular resistance (18), was also significantly delayed when compared with control hearts (for tissue resistivity: 7.75 ± 1.84 min of hypoxia in heptanol-treated vs. 4.02 ± 0.29 min in control hearts, P = 0.016; for phase angle: 8.56 ± 1.81 vs. 4.71 ± 0.40 min, P = 0.015; Fig. 4, C and D). In contrast to heptanol effects, 18-glycyrrhetincic acid and palmitoleic acid, at 10–5 M and 3 x 10–5 M, respectively, given during the first 15 min of hypoxia did not modify the time course of the changes in myocardial electrical impedance during oxygen deprivation compared with control hearts (Fig. 5, B and C). Similarly, the onset of the marked changes in electrical impedance was not significantly modified by both drugs (Fig. 4, C and D). During reoxygenation, changes in myocardial electrical impedance showed a slow recovery toward baseline values in all groups. Higher concentrations of 18-glycyrrhetincic acid or palmitoleic acid, or more prolonged times of administration (n = 8), did not mimic the protective effects of heptanol against the increase in myocardial electrical impedance and the development of rigor contracture during hypoxia (data not shown).
REOXYGENATION-INDUCED LDH RELEASE.
LDH release during hypoxia was minimal and not significantly modified by any of the gap junction uncouplers tested (accumulated LDH release during the entire 40-min period was 24.08 ± 5.47, 10.75 ± 3.27, 19.54 ± 2.66, and 14.18 ± 4.22 U·g dry tissue–1·40 min–1, for control, heptanol, 18-glycyrrhetincic acid, and palmitoleic acid-treated hearts, respectively). In contrast, reoxygenation induced a marked release of LDH in control rat hearts that peaked during the first 2–4 min of oxygen restoration and was followed by a continuous decay (Fig. 7). LDH release was clearly attenuated by gap junction uncouplers (Fig. 7). The magnitude of the protective effect against LDH release was particularly prominent in the case of heptanol and palmitoleic acid (Fig. 7).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-glycyrrhetinic acid, and palmitoleic acid, when given during hypoxia, are protective against hypoxia/reoxygenation injury, thus providing further support to the hypothesis that gap junction-mediated spread of cell death significantly contributes to the final extent of necrosis induced by transient myocardial ischemia. However, the effects of heptanol on the changes in diastolic tension and myocardial electrical impedance induced by hypoxia were completely different from those observed with the other two gap junction uncouplers, suggesting an additional antihypoxic action of this alcohol, independent of its effects on gap junction channels. Results obtained in isolated cardiomyocytes confirm this antihypoxic action by demonstrating that heptanol attenuates and delays intracellular Ca2+ overload during SI. Protective Effects of Gap Junction Uncouplers During Hypoxia/reoxygenation
Previous studies have demonstrated that gap junction inhibitors are protective against myocardial reperfusion injury (4, 19). Heptanol, a gap junction uncoupler, prevented cell-to-cell progression of hypercontracture in cardiomyocytes and reduced LDH release and infarct size when given during reperfusion in isolated rat and in situ porcine hearts (4, 19). The protective effect of heptanol on reperfusion injury is shared by other chemically unrelated gap junction uncouplers, e.g., 18-glycyrrhetinic acid and palmitoleic acid (19). An action of these drugs on gap junction channels is supported by the fact that the protective effect is associated with an attenuation in the recovery of tissue electrical impedance during reperfusion (19). However, gap junction uncouplers have protective effects not only when given during reperfusion but also when they are administered after the onset of ischemia (14), or before it (22). Our present data also show protection when gap junction uncouplers are given during a hypoxic period. Although in the present work isolated cardiomyocytes and isolated rat hearts were submitted to global and theoretically uniform hypoxia, we believe that gap junctions can still play a role in propagation of injury in these conditions, since previous studies have demonstrated that isolated cardiomyocytes submitted to metabolic inhibition respond asynchronically regarding the time course of intracellular Ca2+ rise (21). Similarly, in globally ischemic isolated rabbit hearts, marked spatial heterogeneity in intracellular Ca2+ transients (17) and action potential duration, conduction time, and total repolarization time (10) have been also demonstrated. The protective effect seen when these drugs are given before or during the ischemic/hypoxic insult could be explained by a residual protective effect on the posterior reperfusion/reoxygenation. However, the absence of any effect of gap junction uncouplers administered during hypoxia on myocardial electrical impedance during initial reoxygenation, as it has been previously described when gap junction uncouplers are given during reperfusion (19), speaks against this possibility. It is also plausible that gap junction uncouplers exert their protective effect during the hypoxic period. In fact, previous studies have demonstrated that gap junction-mediated intercellular communication persists during energy deprivation and allows propagation of rigor contracture (21). The fact that heterozygous Cx43-deficient mice underexpressing Cx43 showed reduced infarct size after permanent coronary occlusion (9) supports the notion that gap junction-mediated intercellular communication may play a role in the progression of ischemic injury. However, Schwanke et al. (23) observed no differences between Cx43+/– and wild-type mice subjected to transient ischemia (23).
Gap Junction-independent Protective Effects of Heptanol in Isolated Rat Hearts
The present study demonstrates that heptanol exerts additional effects on the progression of the ischemic insult not shared by the other two gap junction uncouplers tested, since only this alcohol attenuated Ca2+ overload, rigor development, and changes in electrical impedance during hypoxia.
In addition to their well-described effects on gap junctional communication at the concentrations used in the present study (1, 6, 25), all three gap junction uncouplers tested have actions on other cell systems. Thus heptanol has been described to reduce several nonjunctional ionic currents, including Na+ and Ca2+ inward currents (15, 25). Similarly, 18-glycyrrhetinic acid and palmitoleic acid have been demonstrated to have other effects, such as inhibition of sarcolemmal Ca2+ currents and of mRNA synthesis (6, 24). These side actions of gap junction uncouplers could contribute to the observed protective effect of these drugs against hypoxia/reoxygenation injury. The marked protective effect of heptanol against cytosolic Ca2+ overload induced by energy deprivation in isolated cells suggests that this is likely to be true in the case of this drug.
In fact, a recent study has demonstrated that heptanol reduces intracellular Ca2+ levels even at low concentrations (150 µM) in isolated rat mesenteric small arteries stimulated with norepinephrine (13). A reduction in intracellular Ca2+ overload has been demonstrated to be protective during ischemia/reperfusion in isolated rat hearts (7). Thus the effect of heptanol on Ca2+ overload (among other intracellular changes) during hypoxia could contribute to its protective effect against cell death during hypoxia/reoxygenation. The results obtained in isolated cardiomyocytes submitted to SI, in which heptanol markedly attenuated Ca2+ overload, as assessed by analysis of changes in 340/380 fura 2 ratio fluorescence, indicate that this is likely true in the case of heptanol. These findings could explain the delay observed during hypoxia in the changes in electrical impedance and rigor development in isolated rat hearts treated with heptanol, but not with palmitoleic acid nor glycyrrhetinic acid, suggestive of an antihypoxic effect. The paradoxical lack of increase of myocardial electrical impedance during hypoxia in the presence of heptanol does not necessarily mean that heptanol is not causing some degree of gap junction closure but that it is counteracted by the lower cytosolic derangements occurring in the myocardium (lower Ca2+ overload), affecting not only gap junction closure but also other determinants of electrical impedance.
Implications in Preconditioning Studies
Recent studies have suggested a role for Cx43 in cardioprotection by ischemic preconditioning. Thus ischemic preconditioning preserves Cx43 phosphorylation during the posterior index ischemia (8, 14). Supporting a role for gap junctions during ischemic preconditioning were studies demonstrating that heptanol, given before the preconditioning cycles in isolated mouse hearts, abolished the protective effect of preconditioning (11) and that heterozygous Cx43-deficient mice (Cx43+/–), underexpressing Cx43, cannot be preconditioned (23). However, the gap junction-independent protective effect of heptanol shown in the present study raises the possibility that the absence of preconditioning in heptanol-pretreated hearts could in part be because of alterations of the preconditioning insult. According to this, gap junction uncouplers other than heptanol should be tested to prove the hypothesis of the role of gap junctions in ischemic preconditioning. In fact, the role of Cx43 in ischemic preconditioning could be, at least in part, gap junction independent. Not only heterozygous Cx43-deficient mice, but also isolated cardiomyocytes obtained from these hearts, cannot be preconditioned (12). Furthermore, experiments in in situ pig hearts and in isolated rat heats have demonstrated a reduction in infarct size with ischemic preconditioning that was independent of any effect on ischemia-induced changes in electrical impedance (16).
In conclusion, the present results further support a prominent role of gap junction-mediated cell-to-cell communication in the pathophysiology of myocardial cell death secondary to ischemia-reperfusion, stresses the potential value of gap junction blockade to prevent it, and points out the gap junction-independent effects of heptanol that limits its value as a research tool in this context.
Study Limitations
Measurement of myocardial electrical impedance is an overall estimation of the passive electrical properties of the tissue, composed by the extra- and intracellular resistances, including that of gap junctions, and the membrane capacitance. This complex nature makes the use of changes in electrical impedance as an index of changes in gap junction-mediated cell coupling difficult, particularly when measured at a single frequency. However, changes in electrical resistivity during hypoxia, measured at a single frequency, have been well characterized in isolated rabbit papillary muscles (18), a model that allows discrimination between extracellular and intracellular resistivities. In this model, after an initial small decrease in total resistivity, because of changes in the extracellular compartment, there is a marked increase in total myocardial resistance because of a rise in the resistivity of the intracellular compartment (gap junctional and/or cytoplasmic resistances). Based on the observed increase in intracellular but not extracellular resistance, Riegger et al. (18) related the onset of the marked increase in total resistivity to the onset of cell-to-cell electrical uncoupling. The fact that, in contrast to the isolated rabbit papillary muscle preparation, our model is characterized by a complex cellular structure and fiber orientation resulting in different anisotropy in the different myocardial layers from endocardium to epicardium may complicate the interpretation of our results. However, the pattern of the changes induced by hypoxia or ischemia in total resistance is very similar in both models. In the present study, a role for extracellular resistance in the marked increase in total resistivity can be discarded, since we kept the preparations under constant hypoxic perfusion, thus theoretically not modifying the extravascular compartment. Moreover, because in our system the onset of rigor contracture is posterior to that of the increase in total resistivity, cytoplasmic changes should not participate in the initial rise in total resistivity, and thus it is probably the result of changes in gap junctional communication.
Although measurement of myocardial electrical impedance at several frequencies gives additional information compared with measurements obtained at a single frequency, both methods share the limitations derived from simultaneous changes in several tissue compartments. We used a frequency of 7 kHz, since previous experiments of impedance spectroscopy demonstrated that this frequency maximizes the differences between normal and ischemic tissue (2).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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