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(Circulation. 1997;96:3579-3586.)
© 1997 American Heart Association, Inc.

Articles

Gap Junction Uncoupler Heptanol Prevents Cell-to-Cell Progression of Hypercontracture and Limits Necrosis During Myocardial Reperfusion

David Garcia-Dorado, MD; Javier Inserte, ScD; Marisol Ruiz-Meana, DVM; Miguel A. González, PharmD; Julia Solares, MD; Margarita Juliá, ScD; José A. Barrabés, MD; ; J. Soler-Soler, MD

From Servicio de Cardiología, Hospital General Universitari Vall d'Hebron, Paseo Vall d'Hebron, Barcelona, Spain, and Servicio de Anatomía Patológica (J.S.), Hospital San Pedro de Alcantara, Cáceres, Spain.

Correspondence to David Garcia-Dorado, Servicio de Cardiología. Hospital General Universitari Vall D'Hebron 119-129, Barcelona 08035, Spain. E-mail dgdorado{at}ar.vhebron.es

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background The objective of this study was to test the hypothesis that chemical interaction through gap junctions may result in cell-to-cell progression of hypercontracture and that this phenomenon contributes to the final extent of reperfused infarcts.

Methods and Results Cell-to-cell transmission of hypercontracture was studied in pairs of freshly isolated adult rat cardiomyocytes. Hypercontracture induced by microinjection of a solution containing 1 mmol/L Ca²⁺ and 2% lucifer yellow (LY) was transmitted to the adjacent cell (11 of 11 pairs), and the gap junction uncoupler heptanol (2 mmol/L) prevented transmission in 6 of 8 pairs (P=.003), with a perfect association between passage of the LY and transmission of hypercontracture. In the isolated, perfused rat heart submitted to 30 minutes of hypoxia, addition of heptanol to the perfusion media during the first 15 minutes of reoxygenation had a dose-related protective effect against the oxygen paradox, as demonstrated by a reduction of diastolic pressure and marked recovery of developed pressure (P<.001), as well as less lactate dehydrogenase release during reoxygenation (P<.001) and less contraction band necrosis (P<.001) than controls. In the in situ pig heart submitted to 48 minutes of coronary occlusion, the intracoronary infusion of heptanol during the first 15 minutes of reperfusion at a final concentration of 1 mmol/L limited myocardial shrinkage, reflecting hypercontracture (P<.05), reduced infarct size after 5 hours of reperfusion by 54% (P=.04), and modified infarct geometry with a characteristic fragmentation of the area of necrosis. Heptanol at 1 mmol/L had no significant effect on contractility of nonischemic myocardium.

Conclusions These results demonstrate that hypercontracture may be transmitted to adjacent myocytes through gap junctions and that heptanol may interfere with this transmission and reduce the final extent of myocardial necrosis during reoxygenation or reperfusion. These findings are consistent with the hypothesis tested and open a new approach to limitation of infarct size by pharmacological control of gap junction conductance.

Key Words: myocardial infarction • reperfusion • ischemia • occlusion • hypoxia

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Myocytes still viable at the time of reperfusion may develop hypercontracture, sarcolemmal disruption, and death upon restoration of coronary blood flow.^{1 2 3} Cytosolic Ca²⁺ concentration increases in myocytes after prolonged ischemia, and reperfusion-induced hypercontracture is mainly due to reenergization in the presence of elevated cytosolic Ca²⁺. However, other mechanisms appear to contribute to hypercontracture during in situ myocardial reperfusion. Several observations indicate that reoxygenation-induced hypercontracture and sarcolemmal disruption are influenced by cell-to-cell interactions. In reperfused infarcts, the areas of contraction band necrosis are composed of hypercontracted myocytes^{3 4} connected to each other to form a continuum, whose often complex geometry cannot be explained by gradients in collateral flow or microvascular distribution.⁵ Computer simulation studies indicate that some kind of cell-to-cell interaction must be taken into account to explain these histological features and that in its absence, hypercontracted myocytes should be scattered across the area at risk.⁵ It has been suggested that this cell-to-cell interaction could be mechanical, the exchange of forces imposed by intercellular junctions tearing apart the sarcolemma of adjacent cells,⁶ but it also could be chemical. Ca²⁺ and other messengers may diffuse through gap junctions,^{7 8 9} and previous studies have demonstrated cell-to-cell transmission of Ca²⁺ waves through gap junctions in various cell types.^{7 10 11}

This study tested the hypothesis that chemical signaling through gap junctions may result in cell-to-cell progression of hypercontracture and contribute to myocardial necrosis caused by ischemia-reperfusion. First, transmission of hypercontracture, its relation to gap junction permeability, and the effect of the gap junction uncoupler heptanol^{11 12} on this transmission were investigated in pairs of isolated cardiomyocytes. The effect of heptanol was then investigated in the reoxygenated rat heart and in the reperfused pig heart.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Animals were handled in accordance with the position of the American Heart Association rules on Research Animal Use, and all experimental procedures were approved by the Research Commission of the Hospital General Vall d'Hebron.

Studies in Pairs of Isolated Myocytes
Ventricular cardiomyocytes were isolated from adult Sprague-Dawley rat hearts, as previously described.¹³ Whole hearts were retrogradely perfused for 20 minutes with a buffer containing (in mmol/L) NaCl 110, KCl 2.6, KH₂PO₄ 1.2, MgSO₄ 1.2, NaHCO₃ 25, glucose 11 (pH 7.4), and collagenase 0.03% (type II, Boehringer Ingelheim). To increase the final number of end-to-end connected pairs of myocytes, 45 µmol/L of Ca²⁺ was added to the dissociation buffer.¹⁴ Dissociated tissue was filtered and centrifuged, and the pellet was subjected to a progressive normalization of Ca²⁺ levels to 1 mmol/L. Rod-shaped cells were selected by gravity sedimentation and plated in medium 199 (Sigma) with 4% fetal calf serum in preincubated Falcon dishes.¹⁵ Five to 10 end-to-end connected rod-shaped myocytes were found in each culture dish, and cell-to-cell transmission of hypercontracture and gap junction permeability were studied in these pairs of cells.

Study Protocols and Solutions
Hypercontracture was induced by microinjection of extracellular medium in one of the cells of each pair (Digital Micromanipulator 5171, Transjector 5246, Eppendorf) by a pulse pressure of 400 HPa of 0.1-second of duration through a 0.5-µm tip micropipette (Sterile Femtotips, Eppendorf). The injected solution had the same composition as the extracellular buffer except for the addition of 2% lucifer yellow (LY). In 20 pairs the extracellular medium was control buffer (in mmol/L: NaCl 24.2, KCl 3.6, MgSO₄ 1.2, CaCl₂ 1, and HEPES 20), and in 18 pairs Ca²⁺ was replaced by 0.2 mmol/L EGTA (Ca²⁺-free buffer). In 8 of the 20 experiments performed in control buffer and in 15 of the 18 experiments in Ca²⁺-free buffer, heptanol 2 mmol/L was added to the buffer. In additional experiments, heptanol did not prevent hypercontracture of myocytes reoxygenated after 1 hour of energy deprivation, either at 1 or 2 mmol/L (92±2% and 87±2% of cells hypercontracted respectively versus 95±1% without heptanol, P=.22).

The possibility of transmission of hypercontracture induced by reoxygenation was investigated in 9 end-to-end connected cell pairs submitted to reoxygenation after 40 minutes of metabolic inhibition. The time of onset of hypercontracture was determined in these cells and in 9 pairs formed by 2 separated cells selected at random from the same field.

Study Monitoring
Experiments were performed at 37°C on the stage of an inverted microscope (Olympus IMT-2; Digital Warm Stage Controller) with a Hoffman optical system (Hoffman Modulation Contrast). Cell images were continuously recorded at x400 magnification (Videocassette Recorder SVO-9500MDP, Sony). Cell length was measured every 2 seconds during the first 30 seconds after microinjection. Gap junction permeability was analyzed by the dye transfer method. Passage of LY to the adjacent cell was monitored under fluorescence microscopy.

Studies in the Isolated, Perfused Rat Heart
Whole hearts from male Sprague-Dawley rats (weight, 300 to 350 g) anesthetized with thiopental sodium (150 mg/kg), were excised, arrested in ice-cold saline, and retrogradely perfused (10 mL/min, 37°C) with oxygenated buffer (in mmol/L: NaCl 140, NaHCO₃ 24, KCl 2.7, KH₂PO₄ 0.4, MgSO₄ 1, CaCl₂ 1.8, and glucose 5, pH 7.4, equilibrated with 95% O₂–5% CO₂). Hypoxic perfusion was performed with the same buffer, equilibrated with 95% Ar–5% CO₂, and without glucose. Perfusion buffers were filtered through a 5-µm filter.

Study Protocols
Normoxic studies. The effect of heptanol on contractility and cell integrity was investigated in 15 hearts exposed, after 30 minutes of equilibration, to 15 minutes of normoxia with heptanol at 0, 0.4, 1, 2, and 6 mmol/L, and 75 minutes of normoxia without heptanol.

Hypoxia-reoxygenation studies. The effect of heptanol on reoxygenation injury was investigated in 45 hearts. After 30 minutes of equilibration, the hearts were switched to 40 minutes of hypoxic perfusion followed by 90 minutes of reoxygenation, and heptanol was added during the first 15 minutes of reoxygenation. The hearts were allocated to 7 treatment groups with different heptanol concentrations: 0 (control group), 0.2, 0.4, 1, 2, 4, and 6 mmol/L. In 2 additional groups the perfusion with 2 mmol/L heptanol was, respectively, extended to the first 30 minutes or reduced to the first 5 minutes of reoxygenation.

Study Monitoring
Left ventricular (LV) pressure was monitored by means of a fluid-filled latex balloon in the tip of a Cordis 5F catheter (Baxter 43600F pressure transducer). LV end-diastolic pressure (LVEDP) was set between 8 and 12 mm Hg. The signal was digitized (100 Hz) and continuously recorded on hard disk. In 14 animals the ECG was continuously recorded (Siemmens-Elema Mingolog-7) by means of two silver wire electrodes in the LV apex and the aortic cannula. High-speed recordings (200 mm/s) were obtained every minute.

Enzyme release. Lactate dehydrogenase (LDH) activity was spectrophotometrically measured (SLT Spectra Vision) in samples from the coronary effluent every 10 minutes before reoxygenation and at 1, 2, 3, 4, 6, 8, 10, 12, 15, 20 and then every 10 minutes during reoxygenation and expressed as units of activity released per gram of dry weight.

Histological analysis. A 3-mm-thick, cross-sectional, midventricular slice was embedded in paraffin, and 4-µm sections were obtained (Leika RM2145 microtome) and stained with phosphotungstic hematoxiline (PTAH) and Masson's trichrome. Myocardial necrosis was identified by positive PTAH staining, and contraction band necrosis as detected by Masson's trichrome was quantified as previously described.² The sections were divided in 4 low-magnification fields (x40), and a set of photographs of these fields was obtained. The necrosed fibers were identified by examination of the sections at x200 magnification and marked on color prints with a sharp-tipped marker. The color prints were digitized into 768x576 pixel images (Matrox IP8, Matrox Electronic Systems), and the patches of necrosis were planimetered digitally.

Studies in the In Situ Pig Heart
Farm pigs (weight, 20 to 30 kg) were premedicated with 10 mg/kg azaperone IM, anesthetized with thiopental 30 mg/kg IV, intubated, and mechanically ventilated with room air. Anesthesia was maintained with a continuous infusion of thiopental. A superficial abdominal vein and a femoral artery were catheterized, a midline sternotomy was performed, and the pericardium was opened. The left anterior descending coronary artery (LAD) was dissected free at its mid length, and a transit time flow probe (T-106, Transonic Systems) was placed at the dissected segment. A pressure transducer catheter (Mikro-tip, Millar) was advanced into the left ventricle. Two pairs of piezoelectric crystals (1-mm diameter) were inserted into the inner third of the LV wall in the LAD and circumflex territory, respectively, as previously described,² and were stimulated by a System 6 microsonometer (Triton). Before coronary occlusion, a 2.5F catheter for intracoronary infusion was advanced through a Judkins 8F guiding catheter into the LAD until its tip was placed immediately proximal to the dissection site.²

Study Protocols
Studies in control myocardium. The effects of heptanol on myocardial function and coronary blood flow were investigated in 5 pigs. In these animals, the same normoxic buffer used in the rat heart studies containing heptanol at 3 mmol/L was infused for 2 minutes into the LAD. The infusion rate was continuously adjusted to 33.3% of blood flow measured at the infusion site to obtain a final concentration of heptanol of 1 mmol/L. A control infusion of buffer not containing heptanol was performed according to the same protocol. Both infusions were performed in random order and separated by 15 minutes.

Ischemia-reperfusion studies. Ten pigs were submitted to 48 minutes of coronary occlusion of the LAD followed by 5 hours of reperfusion. Before occlusion, the animals were allocated to one of two groups receiving, during the first 15 minutes of reperfusion, an intracoronary infusion of the normoxic buffer used in the rat heart studies containing or not containing, respectively, heptanol at 3 mmol/L. In both groups, the infusion rate was continuously adjusted to obtain a final concentration of 1 mmol/L in the heptanol group.

Study Monitoring
Arterial pH, PaO₂, and PaCO₂ were monitored to adjust the ventilatory parameters. Lead II of the ECG, aortic pressure (Micron Instruments), the first derivative of LV pressure, and coronary blood flow signals were digitized at a sampling rate of 100 Hz (Tecfen ISC-16E/CR, RC Electronics), stored on a hard disk, and continuously recorded in a digital recorder (MT9500, Astro-Med).

Segment length measurements. Measurements were performed on the digitized signals (Enhanced Graphics Acquisition and Analysis, RC Electronics), as previously described.² Systolic shortening was calculated as the difference between end-diastolic length (EDL) and end-systolic length, divided by EDL, x100. All measurements were expressed as a percentage of values immediately before coronary occlusion.

Infarct size and histology. After 5 hours of reperfusion, the LAD was reoccluded and 5 mL of fluorescein was injected into the left atrium. The heart was excised, cooled at 4°C, and cut into five 7-mm slices that were imaged under UV light to outline the area at risk. The images were digitized on-line. The slices were then incubated (37°C, pH 7.4) in 1% triphenyltetrazolium chloride for 5 to 10 minutes to outline the area of necrosis and imaged again under white light with a reference scale. The zone at risk and the area of necrosis were measured in the digital images (Image Pro-Plus, Media Cybernetics). The mass of myocardium at risk and infarct size were calculated from these measurements and from the weight of slices.² The third slice was processed for histology, and 6-µm sections including both ventricles were mounted on 10x14-cm pieces of glass and stained with Masson's trichrome.

Statistical Analysis
Statistical analysis was performed by using commercially available software (SPSS PC+ 4.0). Comparisons involving more than two groups were performed by ANOVA after assessment of data for normal distribution. Individual comparisons were performed by means of the least significant difference test. Comparisons between two groups were performed by means of t tests for independent samples. Changes along time were assessed by multiple ANOVA analysis. A critical value of P=.05 was used for all tests. All values are expressed as mean±SEM.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Studies in Pairs of Isolated Myocytes
All cells microinjected with control buffer immediately developed hypercontracture, starting at the site of microinjection and progressing along the longitudinal axis of the cell. When hypercontracture reached the intercalated disk, it started in the adjacent cell, and in all instances both cells developed full hypercontracture within 30 seconds after micropuncture (Fig 1). Passage of the LY was assessed in five cases, and in all instances transmission of hypercontracture was associated to a marked fluorescence of the second cell, with a similar intensity to that observed in the injected cell 30 seconds after injection. In contrast, when 2 mmol/L of heptanol was added to the control buffer, transmission of hypercontracture was observed in only two of eight pairs (Fig 1). There was perfect concordance between passage of LY and transmission of hypercontracture.

View larger version (19K): [in this window] [in a new window]   Figure 1. Changes induced by microinjection of extracellular medium in the length of the injected cell (closed circles) and the adjacent cell (open circles) in end-to-end connected cell pairs. In the absence of heptanol (top), both cells hypercontracted within the first 30 seconds after microinjection. However, in the presence of heptanol (bottom), the second cell developed hypercontracture in two out of eight cases (dotted line) but not in the remaining six (broken line).

Microinjection in Ca²⁺-free conditions did not result in hypercontracture of the injected cell nor the adjacent cell. In the absence of heptanol, both cells were markedly fluorescent 30 seconds after injection (3 of 3, whereas in its presence the second cell was fluorescent in only 3 of 15 pairs (P<.001) (Fig 2).

View larger version (99K): [in this window] [in a new window]   Figure 2. Passage of lucifer yellow from the microinjected cell to the adjacent one. When Ca2+ was present both in the injection solution and in the extracellular medium, the dye passed to the second cell, and both cells hypercontracted (A). Addition of heptanol (2 mmol/L) prevented passage of the dye and transmission of hypercontracture (B). When both the injection solution and the extracellular medium were Ca2+ free, neither the microinjected cell nor the adjacent one hypercontracted, and passage of the dye to the adjacent cell was observed in the absence of heptanol (C) but not in its presence (D). Fluorescence microscopy images obtained 30 seconds after injection. Original magnification x400.

In cells submitted to 40 minutes of metabolic inhibition, hypercontracture started between 2 and 130 seconds after reoxygenation, but the difference between the times of hypercontracture of the two cells forming end-to-end connected pairs was 2.9±0.5 seconds, much less than the difference of 40±10.2 seconds observed between nonconnected cells from the same field (P=.002). There was an excellent correlation between the times of hypercontracture of pairs of connected cells (r=.992) but not in pairs of separated cells (r=.197).

Studies in the Isolated Perfused Heart
Normoxic Perfusion Studies
At the end of the equilibration period, heart rate averaged 296±4 bpm and LV developed pressure (LVdevP=peak systolic pressure-LVEDP) 91±5 mm Hg. After 120 minutes of normoxia, heart rate was 292±8 bpm and LVdevP 73±7 mm Hg. Addition of heptanol at concentrations 1 mmol/L rapidly induced a moderate reduction in heart rate (259±10 and 240±14 bpm, respectively, at 0.4 and 1 mmol/L) that was rapidly reversible upon its discontinuation. These changes were accompanied by a marked increase of the PR interval (from 56.1±1.1 to 72.0±2.6 ms at 1 mmol/L), without significant changes in the duration of QRS (from 27.9±1.0 to 28.3±1.2 ms). At concentrations 2 mmol/L, heptanol abolished all electrical activity. Heptanol induced dose-dependent reduction in LVdevP that was completely and rapidly reversible at concentrations 4 mmol/L and irreversible at 6 mmol/L (Fig 3). No measurable LDH release was observed during the 120 minutes of normoxic perfusion in hearts not receiving heptanol nor in those receiving it at 0.4, 1, or 2 mmol/L. Significant LDH release (69.3±8.6 IU/15 min per gram dry wt) was observed at 6 mmol/L.

View larger version (22K): [in this window] [in a new window]   Figure 3. Effect of heptanol at various concentrations on contractile function of the isolated, perfused rat heart under normoxic conditions. Heptanol induced a dose-related inhibition of contractility that was complete at 2 mmol/L and irreversible at 6 mmol/L. LVdevP indicates left ventricular developed pressure.

Hypoxia-Reoxygenation Studies
LV pressure. LVdevP was 88±7 mm Hg at the end of the 30-minute equilibration period, without intergroup differences. During anoxia LVdevP fell to 0, and LVEDP increased steeply (Fig 4). No functional recovery was observed during reoxygenation in the absence of heptanol. In contrast, a marked contractile recovery was observed in hearts receiving heptanol during the first 15 minutes of reoxygenation. During the heptanol perfusion the hearts were in electrical arrest or ventricular fibrillation, and recovery started immediately after cessation of heptanol perfusion (Fig 4). The magnitude of the recovery was dose dependent. It increased up to 2 mmol/L; beyond this point the increase in concentration was associated to a progressive reduction of contractile recovery (Fig 5). Heptanol also had a marked and dose-related influence on LVEDP (Fig 5). The effect of heptanol at 2 mmol/L on contractile recovery did not increase when the duration of perfusion was extended from the first 15 to the first 30 minutes of reoxygenation (69±6% and 60±10% of values before hypoxia, respectively, P=NS). In contrast, when the duration of the heptanol perfusion was reduced to 5 minutes, LVdevP recovery at the end of the reoxygenation period was reduced to 25±2% (P<.01).

View larger version (22K): [in this window] [in a new window]   Figure 4. Effect of addition of heptanol at 2 mmol/L concentration in the reoxygenation buffer during the first 15 minutes of reoxygenation in the isolated rat heart exposed to 30 minutes of hypoxia. A sustained recovery of left ventricular developed pressure (LVdevP, top) and a reduction in left ventricular end-diastolic pressure (LVEDP, bottom) were observed in hearts receiving heptanol, in sharp contrast to control hearts.

View larger version (19K): [in this window] [in a new window]   Figure 5. Effect of different concentrations of heptanol during the first 15 minutes of reoxygenation on left ventricular developed pressure (LVdevP, top) and left ventricular end-diastolic pressure (LVEDP, bottom) 75 minutes after cessation of heptanol infusion. Heptanol improved contractile recovery and reduced LVEDP in a dose-related way, the maximal benefit being observed at 2 mmol/L concentration.

LDH release. No LDH release was observed during equilibration or hypoxic perfusion. Reoxygenation-induced LDH release peaked 2 minutes after its onset. The addition of heptanol during the first 15 minutes of reoxygenation did not modify the time course of LDH release but markedly reduced its magnitude (Fig 6).

View larger version (16K): [in this window] [in a new window]   Figure 6. Effect of the presence of heptanol in the reoxygenation buffer on reoxygenation-induced lactate dehydrogenase (LDH) release during the first 15 minutes (filled bars) and the remaining 75 minutes of reoxygenation (open bars) in the isolated rat heart submitted to 30 minutes of hypoxia. A dose-related protective effect against early enzyme release was observed except at the highest concentration, which induced a massive enzyme release. No effects were observed on LDH release after the first 15 minutes of reoxygenation. *P<.05 in respect to no heptanol.

Histology
Control hearts had a compact area of contraction band necrosis involving 50.2±3.1% of the cross-sectional area of the LV wall. In contrast, hearts reoxygenated in the presence of 2 mmol/L heptanol had only small patches of contraction band necrosis involving only 2.4±0.6% of the cross-sectional myocardial surface (P<.01). All hearts had small areas of necrosis without contraction bands mainly distributed across the subendocardial myocardial layer, without group differences.

Studies in the In Situ Pig Heart
Infusion of Heptanol in Control Myocardium
The intracoronary infusion of heptanol at a final concentration of 1 mmol/L was associated with nonsignificant changes in heart rate (from 88±10 to 96±12 bpm), aortic pressure (from 77±6 to 80±5 mm Hg), and systolic shortening in the LAD segment (from 18.5±2.2% to 18.2±2.5%). The intracoronary infusion of normoxic buffer with or without heptanol induced a rapid increase in coronary blood flow (from 16±3 to 24±3 mL/min and from 17±3 to 24±5 mL/min, respectively).

Transient Coronary Occlusion and Reperfusion
Hemodynamic data and coronary blood flow. Heart rate and aortic pressure remained stable and within the normal range in all animals, and no intergroup differences were observed except for reactive hyperemia, which was significantly more pronounced in the heptanol group (Table 1). One animal in the control group and three in the heptanol group presented ventricular fibrillation during reperfusion (P=NS).

View this table: [in this window] [in a new window]   Table 1. Hemodynamic Data During Coronary Occlusion and Reperfusion in the In Situ Pig Heart

Regional wall function. During coronary occlusion, no differences in EDL or systolic shortening were observed between the heptanol and control groups (Table 2). After reperfusion, an important and abrupt reduction in EDL was observed in control hearts but not in heptanol-treated pigs. A small although transient recovery of systolic shortening was observed in the heptanol group but not in controls.

View this table: [in this window] [in a new window]   Table 2. Regional Wall Function During Coronary Occlusion and Reperfusion in the In Situ Pig Heart

Infarct size and histology. There were no differences in the area at risk between control and heptanol groups (Fig 7), but infarct size in the heptanol group was 50% smaller than in controls (27.7±2.7% versus 59.8±14.1% of the area at risk, respectively, P=.042). Macroscopic examination of infarcts demonstrated a more fragmented appearance of the areas of necrosis in the heptanol group (Fig 8). Histological analysis in both groups revealed infarcts composed almost exclusively of contraction band necrosis. The total extension of the areas of necrosis in each heart was clearly smaller in heptanol-treated animals than in controls. In hearts from the heptanol group, small patches of contraction band necrosis scattered within salvaged myocardium were often seen.

View larger version (18K): [in this window] [in a new window]   Figure 7. Effect of the intracoronary infusion of heptanol at a final concentration of 1 mmol/L on infarct size. The area at risk was similar in animals receiving heptanol and in controls, but infarct size was significantly smaller in the heptanol group.

View larger version (35K): [in this window] [in a new window]   Figure 8. Myocardial necrosis (black areas) in the third slice from the 10 pigs included in the study, five from the control group (A), and 5 from the heptanol group (B), as disclosed by computer-assisted analysis of tryphenyltetrazolium staining images. Hearts from the heptanol-treated animals showed significantly smaller and more fragmented areas of necrosis.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This study demonstrates for the first time that hypercontracture of a cardiomyocyte may induce hypercontracture of adjacent cells by chemical signaling through gap junctions. The gap junction uncoupler heptanol interferes with this transmission and markedly reduces the number of myocytes developing hypercontracture and the final extent of necrosis during myocardial reoxygenation or reperfusion. This protective effect is observed at concentrations that do not significantly alter contractility in vivo during normoxia. These results are consistent with the hypothesis that chemical cell-to-cell interaction through gap junctions may result in amplification of myocardial necrosis during reperfusion and open a new approach to infarct size limitation after transient ischemia.

Cell-to-Cell Progression of Hypercontracture
Gap junctions are permeable to second messengers such as cAMP, inositol trisphosphate, Ca²⁺, and other ions and have been involved in the cell-to-cell signal propagation in many cell types, including cardiomyocytes.^{7 8 10 11 16 17} High cytosolic Ca²⁺, intracellular acidosis, and ATP depletion result in gap junction closure in ischemic myocytes.^{18 19 20 21} However, the rapid reenergization, normalization of pH, and cytosolic Ca²⁺ occurring after reperfusion result in reopening of gap junctions in surviving cells.²¹

The hypothesis of cell-to-cell progression of reperfusion-induced hypercontracture through chemical interaction implies that gap junctions may be permeable in cells that have not regained full control of Ca²⁺ homeostasis, that cells able to survive are exposed to adjacent hypercontracting cells, and that the increased Ca²⁺ concentration causing hypercontracture does not cause uncoupling simultaneously. The beneficial effect of heptanol suggests that gap junctions may open before the cells have regained complete Ca²⁺ homeostasis. Reperfusion-induced hypercontracture may not occur immediately upon reflow,^{22 23} and sarcoplasmic reticulum–dependent Ca²⁺ oscillations may lead to hypercontracture after an initial recovery from intracellular acidosis and Ca²⁺ overload has taken place.²⁴ The presence of cell-to-cell communication between reoxygenated cells that have not reached stable Ca²⁺ concentration is further supported by experiments showing that reoxygenation-induced hypercontracture occurred virtually simultaneously in pairs of end-to-end connected cells but not in two single cells selected at random within the same optical field. The differences in the efficiency of individual cells to regain Ca²⁺ control during reperfusion is demonstrated by the coexistence of surviving cells with cells developing hypercontracture and necrosis. Finally, the transfer of LY immediately after intracellular injection of Ca²⁺ indicates that there is a time interval between Ca²⁺ rise and gap junction closure. This is in agreement with observations in the rabbit papillary muscle under different situations of energy deprivation.²¹ Cell-to-cell propagation of hypercontracture and necrosis in reperfused myocardium within the area at risk may be favored by mechanical fragility^{13 25} and increased susceptibility to Ca²⁺²⁶ associated to previous energy deprivation.

Effect of Heptanol
Heptanol was protective against reoxygenation or reperfusion injury at lower concentrations (1 mmol/L) than those required to prevent LY transfer and transmission of hypercontracture in normoxic isolated cell pairs or normal impulse propagation in the normoxic rat heart. This may reflect an increased susceptibility of gap junctions to heptanol in cells recovering from the derangements that had led to uncoupling during previous ischemia. Although heptanol at 1 mmol/L did not induce ECG changes in the normoxic isolated rat heart (apart from prolongation of the PR interval), it caused electrical arrest or fibrillation when applied during early reoxygenation after 30 minutes of hypoxia. This enhanced effect of heptanol during early reoxygenation was further confirmed in additional experiments involving alternative addition and withdrawal of the drug during the first 20 minutes of reoxygenation.

In this study heptanol had an inhibitory effect on contractility. At 1 mmol/L, this effect was moderate in the isolated rat heart and undetectable in in situ pig myocardium. Although heptanol influences Na⁺ current,^{27 28} the mechanism of this inhibitory effect, also observed in neonatal rat myocytes at concentrations 2 mmol/L but not at 1.5 mmol/L,¹¹ is not known. The negative inotropic action of heptanol, rather than its effect on gap junctions, could explain its protective effect during reperfusion. Nevertheless, in contrast to BDM, which required complete abolition of contractility to be effective,^{2 3} the beneficial effect of heptanol was observed at concentrations without effects on regional wall function. Moreover, 1 mmol/L heptanol did not prevent reoxygenation hypercontracture in isolated myocytes after 40 minutes of metabolic inhibition.

A trend toward a higher incidence of reperfusion-induced ventricular fibrillation was observed in pigs receiving intracoronary heptanol, and it cannot be ruled out that the uncoupling action of heptanol could have favored these arrhythmias. Nevertheless, Callans et al²⁹ did not observe spontaneous arrhythmias in the dog model during intracoronary infusion of heptanol at a final concentration of 1 mmol/L despite a 15% increase in activation time and an increased susceptibility to programmed electrical stimulation.

Previous Studies
Johnston et al³⁰ first described the uncoupling properties of heptanol and octanol in crayfish nerve cells. Heptanol at 1 to 2 mmol/L produces a rapid, marked, and reversible reduction in gap junction conductance and permeability in many cell types including cardiomyocytes.^{11 12 29} Although its exact mechanism of action is not known, it seems related to a decreased fluidity of membranous cholesterol reach domains resulting in a reduced open probability of the gap junction channels.^{12 28}

To our knowledge, no previous study has analyzed the effect of heptanol during myocardial reoxygenation or reperfusion. Diederichs³¹ described a protective effect of heptanol against the Ca²⁺ paradox in the isolated rat heart. Addition of 2 mmol/L heptanol to the Ca²⁺-containing media after a free preperfusion period markedly reduced enzyme release. However, enzyme release was abruptly initiated when heptanol was removed from the media after 10 minutes of reperfusion. This author interpreted that Ca²⁺ deprivation generated persistent leaks in gap junctions that allowed Ca²⁺ influx during Ca²⁺ reposition and that heptanol isolated the cytosol from the gap junction space. In our study, addition of heptanol during the first 15 minutes of reperfusion afforded permanent protection against reperfusion and reoxygenation injury, indicating that the stimulus for transmission of reoxygenation-induced hypercontracture lasts for a limited period of time.

Implications
Considering that myocytes are connected to multiple cells through gap junctions³² and that contraction band necrosis represents the largest fraction of reperfused infarcts, even a small probability of cell-to-cell progression of necrosis should have a major impact on infarct size. The possibility of interfering with this transmission pharmacologically without interfering with the function of normoxic myocardium thus has a large therapeutic potential.

	Acknowledgments

 
The authors acknowledge the excellent technical work of Yolanda Puigfel and Elisabeth Amon and the contribution of Dr Juan Cinca in reviewing the manuscript.

This work was partially supported by BIOMED Concerted Action BMH1-PL95/1254 from the European Union, and by grants FIS 97/0948 and CRHG-01-94-67.

Received April 8, 1997; revision received July 7, 1997; accepted July 15, 1997.

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