Propagation of Cardiomyocyte Hypercontracture by Passage of Na⁺ Through Gap Junctions

Cardiomyocyte Isolation Procedure

Animals were handled according to the Declaration of Helsinki, and the experimental procedures were approved by the Research Commission of the Hospital Vall d’Hebron. Ventricular heart muscle cells were isolated from adult male Sprague-Dawley rats (300 g) as previously described.¹⁶ Briefly, whole hearts were retrogradely perfused in a Langendorff system with a modified Krebs buffer (in mmol/L, NaCl 110, KCl 2.6, KH₂PO₄ 1.2, MgSO₄ 1.2, NaHCO₃ 25, and glucose 11) containing 45 μmol/L CaCl₂ and 0.03% collagenase (type II, Serva). Cells from dissociated tissue were subjected to a progressive normalization of Ca²⁺ levels to a final concentration of 1 mmol/L. Rod-shaped cells were selected by gravity sedimentation in a 4% BSA gradient (fraction V, Boehringer Mannheim) and plated in glass-bottomed dishes with medium 199/HEPES (Sigma) and 4% FCS (Gibco). Two hours after plating, broken nonattached cells were discarded by changing the medium. Five to ten end-to-end–connected myocytes were found in each culture dish, and propagation of hypercontracture between adjacent cells, GJ permeability, and changes in intracellular Na⁺ and Ca²⁺ concentrations were studied in these cell pairs.

Microinjection and Experimental Conditions

One of the cells of each pair was microinjected with extracellular medium to simulate sarcolemmal disruption occurring during reoxygenation-induced hypercontracture. A hydraulic micromanipulator with digital control (digital micromanipulator model 5171 and transjector model 5246, Eppendorf) was used. Microinjection was performed through a 0.2- to 0.5-μm–tip sterile micropipette (Sterile Femtotips, Eppendorf) by a pulse pressure of 400 hPa of 0.1-second duration. The axial and depth limits were set to allow penetration of the micropipette 3 to 5 μm below cell surface at an angle of 45°. Previous studies using the same micropipettes and similar solutions showed that these microinjection parameters allow effective injection without detectable mechanical injury.¹⁷ In control experiments, the extracellular medium was a buffer containing (in mmol/L) NaCl 140, KCl 3.6, MgSO₄ 1.2, CaCl₂ 1, and HEPES 20. The modifications of the extracellular buffer included the addition of (1) 10 μmol/L ryanodine, (2) 10 μmol/L nifedipine, or (3) 15 μmol/L and 20 μmol/L KB-R7943, or the replacement of Ca²⁺ by 2 mmol/L EGTA. The microinjected solution was modified in some experiments by replacing Ca²⁺ by 2 mmol/L EGTA. To assess GJ permeability, 2% Lucifer Yellow was added to the microinjection solution in a series of experiments. All experiments were performed at 37°C (Digital Warm Stage Controller, Linkam) on the stage of an inverted microscope (Olympus IMT-2). Cell images were continuously videorecorded at ×400 magnifications. Relative changes in cell length were measured on the videorecorded images. Measurements were performed in both cells every 2 seconds during the first 30 seconds after microinjection, except when indicated otherwise. Transfer dye through GJs was monitored under fluorescent microscopy using a 420-nm excitation light with a bandwidth of 15 nm.

Measurement of [Ca²⁺]_i and Na⁺ Concentrations

Ratiofluorescence studies were performed in a separate series of experiments under the previously described conditions. Changes in [Ca²⁺]_i and Na⁺ concentrations were analyzed in myocytes loaded with fura-2 or SBFI (Molecular Probes), respectively. For loading, cells were incubated for 30 minutes at 37°C in medium 199 with the acetoxymethyl ester of fura-2 (5 μmol/L) or SBFI (10 μmol/L). Myocytes were then washed twice and postincubated for 20 minutes in medium 199 to allow hydrolysis of the acetoxymethyl esters within the cells. Petri dishes with fura-2 or SBFI-loaded myocytes were positioned on the temperature-controlled stage of an inverted fluorescent microscope (Olympus IX70) with a fluorite objective (UplaFL ×200 or ×400, Olympus). Ratiofluorescence measurements were performed by a commercially available imaging system (QuantiCell 900, Applied Imaging). Cells were alternatively excited at 340 and 380 nm, with a bandwidth of 15 nm, by means of a fast-speed monochromator. Exposure time was set for each excitation wavelength according to fluorescence intensity and was typically 40 to 80 ms. Emitted light was collected by an air-cooled intensified digital camera with a resolution of 640×640 pixels. Counts from clusters of 2×2 or 4×4 pixels were pooled to use shorter exposure times and to improve signal/noise ratio. Ratios of 340/380 were calculated for each pixel cluster from signal intensities in that cluster in pairs of images consecutively obtained at the 2 wavelengths, and color-coded 340/380 ratio images were generated. The average ratio was calculated for regions of interest defined in these images, and the changes in these average ratio values through time were analyzed.

Field Stimulation of Cardiomyocytes

To assess the potential effects of KB-R7943 on cytosolic Ca²⁺ transients and contractility, changes in cell length and ratiofluorescence were analyzed in cardiomyocytes submitted to field stimulation at 1 Hz, as previously described.¹⁸ Biphasic electrical pacing composed of 2 equal but opposed rectangular 40-V stimuli of 0.5-ms duration were applied between 2 silver electrodes immersed in the fluid. Measurements were performed before addition of any drug to the extracellular fluid and after addition of KB-R7943 at 15 μmol/L.

Role of [Ca²⁺]_o in the Propagation of Hypercontracture

In control conditions (extracellular and microinjected solutions composed of HEPES buffer with ionic composition mimicking physiological extracellular media), microinjection of extracellular buffer consistently induced hypercontracture of the injected myocyte, and the rapid rise in cytosolic Ca²⁺ concentration in the microinjected cell was followed within a few seconds by a similar rise in the adjacent cell and by its hypercontracture (Figure 1⇓). However, when Ca²⁺ was replaced in the extracellular medium by the Ca²⁺ chelator EGTA, but not in the injected buffer, the rise in Ca²⁺ concentration and hypercontracture observed in the injected myocyte were not propagated to the adjacent cell in 16 of 18 cell pairs (Figure 2⇓). Failure of propagation of hypercontracture in the absence of [Ca²⁺]_o was not due to reduced GJ permeability, because transfer of the GJ-permeant dye Lucifer Yellow from the microinjected to the adjacent cell was not modified in the absence of [Ca²⁺]_o (Figure 2⇓).

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Figure 1.

Propagation of hypercontracture induced by microinjection of extracellular medium in pairs of isolated cardiomyocytes. A, Sequential 340/380 ratiofluorescence images of a fura-2–loaded cell pair obtained after microinjection (arrow). *Position of intercalated disc between the 2 cells, as identified by direct observation at ×400. Only the first 1 of every 6 images consecutively obtained are shown. The first image (top left) and the last image (bottom right) are separated by 30 seconds. Note that microinjection is followed by a change in the color in both cells, reflecting Ca²⁺ overload. B, Average changes in cell length in 12 cases induced by microinjection in the injected myocyte (○) and the adjacent one (•). In <30 seconds, both cells were completely hypercontracted. C, Average changes in fura-2 ratio in 4 experiments, demonstrating the rapid increase in [Ca²⁺]_i induced by microinjection in both cells. Change in ratio (▵) at a given time is defined as the value measured at that time divided by the initial value measured before microinjection. Data are mean±SEM.

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Figure 2.

A, Lack of propagation of hypercontracture in [Ca²⁺]_o-free conditions is illustrated in the following sequence of 3 images. A1, Image was obtained immediately after injection of control buffer in one of the cells of a pair. A2, 30 seconds after microinjection, injected cell was completely hypercontracted, but hypercontracture did not progress to the adjacent cell. A3, Fluorescent image of the same cell pair illustrates the passage of Lucifer Yellow from microinjected to adjacent cell despite the absence of propagation of hypercontracture, which demonstrates that GJs were permeable. *Position of the intercalated disc; arrow indicates micropipette. B, Average changes in cell length in 16 experiments performed under [Ca²⁺]_o-free conditions. C, With normal [Ca²⁺]_o, addition of 10 μmol/L nifedipine did not prevent or modify propagation of hypercontracture. D, Blockade of Ca²⁺ release from the SR with 10 μmol/L ryanodine had no effect on propagation of hypercontracture from the microinjected to the adjacent cell. In all cases, ○ indicates microinjected myocyte, and •, adjacent cell. Data are mean±SEM.

On the basis of the observed role of [Ca²⁺]_o in propagation of hypercontracture, we investigated L-type Ca²⁺ channels as a likely route of Ca²⁺ entry into the adjacent cell. However, this possibility was ruled out by repeating the microinjection in the presence of 10 μmol/L of the channel blocker nifedipine. Blockade of L-type Ca²⁺ channels did not modify either Ca²⁺ rise in the adjacent cell or propagation of hypercontracture (Figure 2⇑).

Ca²⁺ release from the sarcoplasmic reticulum (SR) had no effect on cell propagation of hypercontracture. Addition of ryanodine to the extracellular medium (10 μmol/L for 15 minutes) did not prevent Ca²⁺ rise or hypercontracture in the adjacent cell (Figure 2⇑).

Passage of Na⁺ and Role of Reverse Na⁺/Ca²⁺ Exchange

We used ratiofluorescence imaging of cells loaded with the Na⁺ indicator SBFI to demonstrate a rapid and almost simultaneous increase in cytosolic Na⁺ concentration in the microinjected and the adjacent cell (Figure 3⇓). To determine the role of reverse Na⁺/Ca²⁺ exchange in the increase of [Ca²⁺]_i concentration and hypercontracture of the adjacent cell, microinjection of extracellular medium was performed in the presence of the novel isothiourea derivative KB-R7943, a highly selective blocker of Na⁺/Ca²⁺ exchange in its reverse mode.^{19 20} Addition of 20 μmol/L of KB-R7943 to the extracellular medium prevented propagation of hypercontracture in 12 of 15 cell pairs and markedly delayed it in the remaining 3 (Figure 3⇓). A similar effect was observed at 15 μmol/L (propagation was observed only in 1 of 5 cell pairs). However, at 10 μmol/L of KB-R7943, propagation was observed in 12 of 13 cell pairs, although it was significantly delayed (after 30 seconds of microinjection, hypercontracture was propagated in only 6 of 13 cell pairs). KB-R7943 at 15 μmol/L had no influence on either cell shortening (10.3±0.4% and 10.0±0.5%, respectively, in the presence and the absence of the drug) or Ca²⁺ transients induced by field stimulation (Figure 4⇓). Despite the absence of propagation of hypercontracture, KB-R7943 at 20 μmol/L did not alter GJ permeability, as assessed by cell-to-cell diffusion of Lucifer Yellow (Figure 3⇓).

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Figure 3.

Role of passage of Na⁺ through GJs in propagation of hypercontracture. A, Sequential SBFI ratiofluorescence images from a pair of myocytes obtained after microinjection of one of them (arrow), demonstrating a rapid increase in intracellular Na⁺ concentration in both cells. The last image (bottom right) is separated from the first (top left) by 60 seconds. *Position of intercalated disc; arrow indicates micropipette. The end of a third cell, very close to the injected cell but without physical contact with it, can also be observed (broken line). B, Average changes in SBFI ratio in the microinjected cell (○) and the adjacent one (•) in 6 experiments performed under control conditions. Change in ratio (▵) at a given time is defined as the value measured at that time divided by the initial value before microinjection. C, Effect of blockade of reverse Na⁺/Ca²⁺ exchange on propagation of hypercontracture in the presence of KB-R7943 in the extracellular medium, immediately before (left) and 240 seconds after microinjection (right). *Position of the intercalated disc. Note that despite chemical coupling (passage of Lucifer Yellow from microinjected to adjacent cell), blockade of reverse Na⁺/Ca²⁺ exchange prevented propagation of hypercontracture. D, Average changes in cell length in 15 experiments performed in the presence of KB-R7943. In 12 of 15 cases, blockade of reverse Na⁺/Ca²⁺ exchange prevented propagation of hypercontracture. In the other 3 cell pairs, hypercontracture was propagated, although with some delay. E, Microinjection of Ca²⁺-free buffer containing 2 mmol/L EGTA with normal [Ca²⁺]_o concentration (1 mmol/L). Images of a cell pair obtained before microinjection (left) and 60 seconds after microinjection (right) demonstrate hypercontracture of the adjacent cell but not of the microinjected one. F, Average changes in cell length in 5 cell pairs microinjected with extracellular buffer in which Ca²⁺ had been replaced by EGTA. In all cases, ○ indicates injected myocyte; •, adjacent cell. Data are mean±SEM.

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Figure 4.

Representative traces of changes in cell length (left) and 340/380 fura ratio (right) in cells submitted to field stimulation at 1 Hz in the absence (control) and in the presence of KB-R7943 at 15 μmol/L. Ratios of 340/380 represent the average values from individual pixels within the cell image (64×60 pixels) obtained every 17 ms. Cell length was derived from high-resolution images (256×240 pixels) obtained under visible light (400 nm) every 10 ms. The lower panels show the time-averaged traces. KB-R7943 at 15 μmol/L had no detectable influence on either cell shortening or Ca²⁺ transients induced by electrical stimulation.

The role of passage of Na⁺ through GJ and subsequent exchange with [Ca²⁺]_o in cell-to-cell propagation of hypercontracture was further tested in a series of experiments in which Ca²⁺ was replaced by 2 mmol/L EGTA in the microinjection buffer. This consistently resulted in hypercontracture of the adjacent cell despite the absence of any change in length in the microinjected cell (Figure 3⇑) and clearly ruled out the mechanical distortion of GJs as the main cause of propagation of hypercontracture. The fact that the hypercontracture in the adjacent cell is delayed in these experiments may indicate that passage of Ca²⁺ from the primarily injured cell can contribute to hypercontracture of the adjacent cell.