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MUSCLE CELL BIOLOGY AND CELL MOTILITY

Aberrant cell-to-cell coupling in Ca²⁺-overloaded guinea pig ventricular muscles

Departments of ¹Pharmacology, ²Cardiology, ³Anatomy, and ⁴Cardiovascular Surgery, Juntendo University School of Medicine, Tokyo, Japan

Submitted 12 September 2007 ; accepted in final form 11 April 2008

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
To investigate how intercellular coupling can be changed during Ca²⁺ overloading of ventricular muscle, we studied Ca²⁺ signals in individual cells and the histochemistry of the major gap junction channel, connexin43 (Cx43), using multicellular preparations. Papillary muscles were obtained from guinea pig ventricles and loaded with rhod-2. Sequential Ca²⁺ images of surface cells were obtained with a confocal microscope. In intact muscles, all cells showed simultaneous Ca²⁺ transients in response to field stimulation over a field of view of 0.3 x 0.3 mm². In severely Ca²⁺-overloaded muscles, obtained by high-frequency stimulation in nonflowing Krebs solution, cells became less responsive to stimulation. Furthermore, nonsimultaneous but serial onsets of Ca²⁺ transients were often detected, suggesting a propagation delay of action potentials. The time lag of the onset between two aligned cells was sometimes as long as 100 ms. Similar lags were also observed in muscles with gap junction channels inhibited by heptanol. To investigate whether the phosphorylation state of Cx43 is affected in Ca²⁺-overloaded muscles, the distributions of phosphorylated and nonphosphorylated Cx43 were determined using specific antibodies. Most of the Cx43 was phosphorylated in the nonoverloaded muscles, whereas nonphosphorylated Cx43 was significantly elevated in severely Ca²⁺-overloaded muscles. Our results suggest that the propagation delay of action potential within a small area, a few square millimeters, can be a cause of abnormal conduction and a microreentry in Ca²⁺-overloaded heart. Inactivation of Na⁺ channels and inhibition of gap junctional communication may underlie the cell-to-cell propagation delay.

Ca²⁺ transient; connexin43; propagation delay; gap junction; arrhythmia

In general, there are two major mechanisms for ventricular arrhythmias, i.e., abnormal spontaneous excitation in nonpacemaker regions and abnormal conductivity between cells, which favors reentry. The most widely accepted mechanism for the abnormal excitability in Ca²⁺-overloaded muscles suggests that Ca²⁺ is spontaneously released from the overloaded SR via Ca²⁺ release channel/ryanodine receptor (RyR2) as Ca²⁺ waves. This Ca²⁺ release leads to aftercontraction and delayed afterdepolarization (DAD) by activating Na⁺-Ca²⁺ exchange activity (6, 11, 16, 47). The DAD can trigger extrasystolic action potentials if it exceeds the threshold for voltage-dependent Na⁺ channel opening (8, 28). Recently, this mechanism has received particular attention because mutations in RyR2, a Ca²⁺ release channel, or in calsequestrin (CASQ2), a Ca²⁺ binding protein in the SR, have been shown to enhance spontaneous Ca²⁺ release from SR and cause catecholamine-induced polymorphic ventricular tachycardia (12, 35, 36).

There is also evidence for abnormal conductivity in Ca²⁺-overloaded ventricular muscles. Increased cytoplasmic Ca²⁺ can suppress the conductivity of excitation between cells (4, 9, 17, 34), and may create reentry circuits (40, 42), thereby increasing the propensity for ventricular arrhythmias. The decreased conductivity can be explained by closure or redistribution of gap junction channels (13, 22, 45). Gap junction channels are formed by paired oligomeric membrane hemichannels (connexons), which are composed of six connexin proteins. In ventricular muscle, the major gap junction channels consist of connexin 43 (Cx43). Cx43 is highly phosphorylated at multiple carboxyterminal serine and threonine residues. Among them, the phosphorylated (P-Cx43) and nonphosphorylated (NP-Cx43) states of Ser368 can be determined by specific antibodies (5, 32, 33). It has been reported that a decrease in P-Cx43 along with an accumulation of NP-Cx43 correlates with loss of conductivity (5, 21, 24, 26, 41, 43).

To evaluate the mechanisms of ventricular arrhythmias in the Ca²⁺-overloaded heart as described above, it is necessary to study activities of individual cells in multicellular cardiac tissues and to define cell-to-cell communication. Ca²⁺ signals in individual cells in the whole Langendorff-perfused heart (1, 3, 18, 38) and isolated heart muscle preparations (19, 29, 48) have been described previously by several groups including us. Our subsequent studies reveal that abnormalities in action potential-induced Ca²⁺ transients frequently occur in muscles stimulated at high frequency in nonflowing Krebs solution. This preparation resembles the condition of ventricular tachycardia in which cardiac cells are hyperactive in terms of O₂ consumption but O₂ supply is compromised by cardiac dysfunction. We also determined the change of phosphorylation state of Cx43 in these muscles. On the basis of the results, we propose that suppressed cell-to-cell coupling may contribute to ventricular arrhythmias in Ca²⁺-overloaded heart, and that the propagation delays are probably due to functional changes in Cx43 in addition to reduced excitability of cardiac cells. A preliminary report of this work was presented to the Biophysical Society (20).

	MATERIALS AND METHODS

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Preparation. All experiments were carried out in accordance with the Juntendo University Ethics Committee guidelines and were approved by the Committee for Animal Experimentation of Juntendo University. Cardiac muscle preparations were obtained following the procedure of Kurebayashi et al. (19). Briefly, guinea pigs were anesthetized with an intraperitoneal injection of pentobarbital (100 mg/kg). The heart was quickly removed from the chest and then perfused via the aorta with a high-K⁺ Krebs solution (see below). Papillary muscle bundles (0.3 1.2 mm in diameter, 2 6 mm in length) were excised from the left and right ventricles in high-K⁺ solution. Usually, two to four preparations were obtained from one animal. The muscle bundles were then incubated with 5 µM rhod-2 AM in normal Krebs solution for 2 h at room temperature. After rhod-2 AM was washed out, the preparations were kept in Krebs solution and used within 4 h.

Confocal Ca²⁺ imaging. The muscle bundle was connected with silk thread to hooks in a chamber (8 mm in width, 30 mm in length, 2 mm in depth), stretched to its slack length, and gently pushed toward the bottom with a Plexiglas plate so that the lower surface of the bundle was 5–10 µm above the bottom. This gap allowed the cells at the lower surface access to the bathing solution. When the muscle surface was in direct contact with the bottom, the cells lost excitability within a few minutes at 0.5-Hz stimulation. The muscle was usually superfused with normal Krebs solution at a rate of 2 ml/min and conditioned by stimulation at 0.5 Hz with a pair of platinum wire electrodes (0.5 mm in diameter, 15 mm in length), with current pulses of 1.5 threshold voltage (2-ms duration). Experiments were carried out at 25°C.

Ca²⁺ imaging was carried out as described previously (19). The surface cells of the papillary muscle bundles were viewed with a confocal laser-scanning microscope system (Oz, Noran Instruments) equipped with an Argon Krypton Ion Laser System (488 and 568 nm excitation). Rhod-2 was excited at 568 nm, and fluorescence of >590 nm was detected. A x20 (0.75 numerical aperture, Plan Apo, Nikon) objective lens was used.

In typical experiments, each image (0.3 mm x 0.3 mm in size) was taken with 256 x 240 pixels every 8.3 ms, and eight images were averaged to obtain a single image. This produced an averaged image every 67 ms, and 200–500 averaged images were obtained continuously for 13 30 s for each data set. In experiments that needed higher time resolution, four images were averaged to give averaged images every 33 ms, with 200–300 averaged images (6.6 10 s) being obtained.

Procedure for Ca²⁺ overloading. In the present study, we prepared three types of Ca²⁺-overloaded muscles: moderately overloaded muscles, severely overloaded muscles, and fatally Ca²⁺-overloaded muscles (Fig. 1). The moderately overloaded muscles were produced by stimulation at 2 Hz for 3 5 min in flowing Krebs solution. The severely Ca²⁺-overloaded muscles were obtained by stimulation at 2 Hz for 10 20 min in nonflowing Krebs solution. Stopping the flow of the Krebs solution resulted in the gradual reduction in dissolved O₂ available to the bundle, especially at the gap between the bundle and Plexiglas bottom. The fatally overloaded muscles were obtained by stimulation at 0.5 Hz in the presence of a toxic dose of strophanthidin (1 µM) for 1 h.

View larger version (47K): [in this window] [in a new window]   Fig. 1. Ca2+ transients and Ca2+ waves in a cell in moderately (A), severely (B), or fatally overloaded ventricular muscle (C). Time-based scan images were obtained along the longitudinal axis of single cells in muscle preparations. Arrows indicate field stimulation at 2 Hz. A: cell showed constant amplitude of Ca2+ transients in response to stimulation, and Ca2+ waves at lengthening intervals after stimulation was stopped. Ba: cell showed normal Ca2+ transients in flowing normal Krebs solution, whereas after 20 min of stimulation (2 Hz) in nonflowing solution, it showed irregular amplitude of Ca2+ transients and frequent Ca2+ waves. The resting fluorescence level from rhod-2 was increased by 55% [fluorescence intensity (FI) was increased from 67 to 130, whereas photomultiplier gain was 25% higher in Bb than in Ba]. Note that the decay times of Ca2+ waves were longer in B than in A. C: cell did not respond to stimulation but showed periodic Ca2+ waves with prolonged decay time. The half-times of Ca2+ waves were 0.23 ± 0.06 s, n = 3 (A); 0.47 ± 0.06 s, n = 9 (B); and 0.90 ± 0.13 s, n = 8 (C). See MATERIALS AND METHODS for the detailed protocol used in these preparations.

Immunohistochemistry. After microscopic observation of Ca²⁺ signals, the muscles were immediately fixed with 4% formaldehyde in phosphate-buffered saline (PBS, pH 7.4), followed by treatment with 0.3% Triton X-100 in PBS. The muscles were incubated for 2 h with a mixture of primary antibodies, i.e., rabbit anti-phospho-Cx43 antibody (polyclonal, Cell Signaling Technology, Danvers, MA) for detection of the form phosphorylated at Ser368, and mouse anti-Cx43 (monoclonal, Zymed Laboratory, San Francisco, CA) for the nonphosphorylated form at Ser368 (27). After the muscles were washed, they were incubated for 1 h with a mixture of secondary antibodies, Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 594 goat anti-mouse IgG (Molecular Probes). The immunofluorescent-labeled samples were examined using a confocal-laser scanning microscope (Noran Instruments) with 512 x 480 pixels at 8 bits using a x20 (0.75 numerical aperture; Plan Apo) or x60 (1.20 numerical aperture, water immersion; Plan Apo) objective lens. The fluorescence intensity of each image was analyzed using ImageJ software (National Institutes of Health). Background fluorescence intensities were obtained from a specimen stained with the secondary antibodies alone.

Data analysis. Data are reported as means ± SE. The series of image data (obtained with the Oz system of Noran Instruments) were converted to stacks of TIFF images for analysis using ImageJ software. Time-based scan images, which correspond to line-scan images, were obtained along lines drawn on the stacks of images with the "Reslice" command. Statistical analysis was performed using Student's t-test and Kruskal-Wallis analysis of variance. P values <0.05 were considered to be significant.

	RESULTS

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When muscle fibers were stimulated by conditioning pulses, cells in healthy muscles showed global Ca²⁺ transients in response to the stimulation without showing Ca²⁺ waves between or after the stimuli. However, when muscles were overloaded with Ca²⁺ by high-frequency stimulation, incubation with high extracellular Ca²⁺, or treatment with a cardiac glycoside, muscle cells showed Ca²⁺ waves (19, 23). In the present study, we used three types of Ca²⁺-overloaded muscles: moderately overloaded muscles, severely overloaded muscles, and fatally Ca²⁺-overloaded muscles (Fig. 1). The moderately overloaded muscles, produced by stimulation at 2 Hz for 3 5 min in flowing Krebs solution, showed normal Ca²⁺ transients in response to electrical stimulation and temporary Ca²⁺ waves ceasing within a few minutes after the cessation of stimulation (Fig. 1A). The severely Ca²⁺-overloaded muscles, obtained by stimulating at 2 Hz for 10 20 min in nonperfusing Krebs solution, generated Ca²⁺ transients but with occasional failure (Fig. 1B; see also Figs. 3 and 4) and showed frequent Ca²⁺ waves lasting for more than 10 min after the cessation of stimulation. Although stopping the superfusion is not always necessary for the induction of Ca²⁺ waves, it does increase the occurrence of Ca²⁺ waves by three- to fivefold (19). The diastolic Ca²⁺ inferred from rhod-2 fluorescence was also increased by 86 ± 12% (n = 12). Similar increases in resting cytoplasmic Ca²⁺ concentration in ischemic and Ca²⁺-overloaded heart muscle have been described by other groups and us (19, 23, 30). The fatally overloaded muscles, obtained by stimulation at 0.5 Hz in the presence of a toxic dose of strophanthidin (1 µM), showed recurrent waves but no action potential-induced Ca²⁺ transients at 0.5 or 2 Hz [Fig. 1C; see also Fig. 6 of Kurebayashi et al. (19)]. This may correspond to muscles showing agonal waves reported by Kaneko et al. (18). Comparison of the half-times (t_1/2) of Ca²⁺ waves in these three conditions indicates that Ca²⁺ handling ability was decreased in the severely (t_1/2 = 0.47 ± 0.06 s, n = 9) and the fatally Ca²⁺-overloaded muscles (t_1/2 = 0.90 ± 0.13 s, n = 8) compared with the moderately overloaded muscles (t_1/2 = 0.23 ± 0.06 s, n = 3; see also Fig. 2B).

View larger version (50K): [in this window] [in a new window]   Fig. 3. Reduced responsiveness to field stimulation in cells in severely Ca2+-overloaded muscles. Muscles had been stimulated at 2 Hz for 20 min without superfusion of Krebs solution, and sequential Ca2+ images were obtained during the last part of the 2-Hz stimulation. A, left, and B: surface image of the papillary muscle cells. Aa–Ac: time-based scan images along lines a, b, and c in A and B. Note that individual cells sometimes failed to respond to stimulation and/or showed variable amplitudes of Ca2+ transients. Arrows indicate field stimulation. Horizontal bars are 100 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 4. Desynchronized action potential-induced Ca2+ transients in severely Ca2+-overloaded muscles. The same preparation shown in Fig. 3A was stimulated at 0.5 Hz after the 2-Hz stimulation. Aa: surface image of papillary muscle. Ab: a time-based scan image of Ca2+ transients obtained from two aligned cells (red line in a, same as line b in Fig. 3A). Ac and Ad: a time-based scan image of Ca2+ transients shown in Ab on an expanded time scale. Note that the onsets of the Ca2+ transients in two aligned cells (cells 1 and 2) were not simultaneous. Ba and Bb: sequential Ca2+ images taken every 67 ms (a) and their difference images (b) during one Ca2+ transients. Bc: overlay of difference images indicated with pseudo colors. C: normalized time courses of Ca2+ transients in cells 1, 2, and 3.

View larger version (81K): [in this window] [in a new window]   Fig. 6. Localization of phosphorylated (P-Cx43) and nonphosphorylated (NP-Cx43) states of connexin43 in control and Ca2+-overloaded muscles. A: images at similar magnification as Ca2+ imaging. P, P-Cx43 (green); NP, NP-Cx43 (red). Bar is 100 µm. Bottom: surface plot of NP-Cx43 images. B: high-magnification view of merged images. Control, control muscle; desync, a muscle that showed desynchronized Ca2+ transients; stroph, a muscle treated with a toxic dose (10–6 M) of strophanthidin for 60 min that showed recurrent Ca2+ waves but no Ca2+ transients. Bar is 20 µm.

View larger version (35K): [in this window] [in a new window]   Fig. 2. Simultaneous action potential-evoked Ca2+ transients in moderately Ca2+-overloaded ventricular muscle. The muscle was stimulated at 2 Hz for 5 min and then stimulation was stopped. Aa: image of the papillary muscle. Ab: time-based scan image during the last part of the stimulation (stim) and following resting period, obtained along a line drawn on two aligned cells in a. Ac and Ad: time-based scan images of Ca2+ transients (c) and Ca2+ waves (d) depicted in b on an expanded time scale. Arrows indicate field stimulation. B: normalized time courses of Ca2+ transients in cell 1 (), cell 2 (), and cell 3 ().

Examples of experiments using severely Ca²⁺-overloaded muscles are shown in Fig. 3. After 20 min of stimulation at 2 Hz without superfusion of Krebs solution, Ca²⁺ transients tended to alternate between cells and between pulses (Figs. 1B and 3A and supplemental video 2), which is similar to the observation reported by Aistrup et al. (1). Furthermore, overloaded cells often failed to respond to electrical stimulation at 2 Hz (Fig. 3Ab and Ac). In healthy muscles, this kind of alternation or failed response was not observed at 2 Hz at 25°C (Figs. 1 and 2) but was observed in 80% of severely Ca²⁺-overloaded preparations (20 muscles out of 26). These results indicate that cardiac cells become less responsive to electrical stimulation in severely Ca²⁺-overloaded muscles. It should be noted that when muscles were stimulated for a longer period, i.e., 30–60 min, without Krebs superfusion, the cells lost their responsiveness to electrical stimulation and often showed recurrent Ca²⁺ waves (see Fig. 6B of Ref. 19). Eventually the cells became inexcitable and exhibited no change in Ca²⁺ signal.

Although cells in the severely Ca²⁺-overloaded muscle often failed to be excited at 2 Hz, as shown in Fig. 3Ab, the same muscle cells were able to respond at lower frequency, i.e., 0.5 Hz, as shown in Fig. 4Ab. However, examination of the time-based scan image (Fig. 4Ac) indicates that the Ca²⁺ transients in cells were not perfectly synchronized. The time course of the Ca²⁺ transients in three cells are shown in Fig. 4C. The rising phases of the Ca²⁺ transients were not simultaneous (see also supplemental video 3). To determine the order of onset of Ca²⁺ transients, we obtained a stack of difference images between every two sequential images (Fig. 4Bb). From these difference images, we were able to detect the timing of the onset of Ca²⁺ transients because the difference in fluorescence intensity was largest during the initial period of Ca²⁺ transients. The difference images during one Ca²⁺ transient were colored with different colors and overlaid in Fig. 4Bc. Across the entire image, cells at the lower side showed earlier Ca²⁺ transients (red cells) than those at the upper side (blue or green cells), suggesting that the excitation propagates from the lower to upper side. When Ca²⁺ transients in two adjacent cells were compared, we sometimes found considerable time differences. For example, in Fig. 4, the onset of Ca²⁺ transient in cell 1 occurred >100 ms earlier than in cell 2. The time differences in the onsets were not constant from stimulation to stimulation, ranging between zero and 130 ms (59 ± 19 ms, n = 5), but less responsive cells always showed more or less delayed Ca²⁺ transients (see Fig. 4A). Time lags of 33 ms or more in the onset of Ca²⁺ transients in two adjacent cells were detected in 15 out of 20 preparations with the procedure we adopted here. Average value of maximal time lags in the onset of Ca²⁺ transients between two adjacent cells were 66 ± 8 ms (n = 15). The maximal time difference between fastest and slowest onsets of Ca²⁺ transients within a 0.3 x 0.3 mm² field of view was, on average, 89 ± 12 (n = 15, range 33–280 ms).

The results in Fig. 4 suggest that propagation of action potentials may be suppressed in severely Ca²⁺-overloaded muscles. The underlying mechanisms for this phenomenon involve the suppression of voltage-dependent Na⁺ channels and gap junction channels. The importance of Na⁺ channels is evident from the data presented in Fig. 3, where cell responsiveness to field stimulation was partially impaired. To investigate the influence of the gap junction channels, i.e., whether the inhibition of gap junctions can mimic the delay, we examined the effects of a gap junction channel inhibitor on propagation of Ca²⁺ transients. To assess the propagating ability of excitation, we induced spontaneous, synchronized Ca²⁺ transients in normal papillary muscles with 10^–7 M isoproterenol (25) and monitored the Ca²⁺ transients (Fig. 5). The onsets of Ca²⁺ transients were simultaneous within the field of view (Fig. 5Ab, 5C left), although we could not identify their origins. When 1 mM heptanol was applied to the muscle, time lags were noticed in the onsets of Ca²⁺ transients in some cells in the field of view. For example, the time courses of Ca²⁺ transients were not simultaneous in cells 1 and 2 shown in Fig. 5C, right (see supplemental video 4). The difference images during a Ca²⁺ transient were obtained every 67 ms (Fig. 5Ba) and overlaid with different colors (Fig. 5Bb). In this field of view, the last excited cell lagged 200 ms behind the first one (Fig. 5B, C right). Similar results were obtained in six experiments. These results indicate that inhibition of gap junction channels can mimic delayed onset of Ca²⁺ transients observed in severely Ca²⁺-overloaded muscles, and that the delay between two adjacent cells can be as long as 200 ms. The effect of heptanol was reversible; after withdrawal of the drug, cells in the field of view recovered synchronism (data not shown).

View larger version (37K): [in this window] [in a new window]   Fig. 5. Artificial inhibition of gap junction channels by heptanol. Aa: an image of a papillary muscle showing spontaneous activity in the presence of isoproterenol. Ab and Ac: time-based scan images obtained along a line drawn on two aligned cells in the absence (b) and presence (c) of 1 mM heptanol. B: difference images obtained from sequential images taken every 67 ms during one Ca2+ transient in the presence of heptanol (a) and overlay of the difference images indicated with pseudo colors (b). Horizontal bar is 100 µm. C: normalized time courses of Ca2+ transients in cell 1 (solid red square), cell 2 (open blue triangle), and cell 3 (solid green circle) in the presence (right) and absence (left) of heptanol.

In the experiments shown in Fig. 6, three papillary muscle preparations were obtained from the same individual animal, and each muscle was treated with one of three treatments. The control muscle was incubated in normal Krebs solution, observed with a confocal microscope to confirm normal Ca²⁺ response to conditioning pulses, and then fixed. The second preparation was kept stimulated at 2 Hz without superfusion of Krebs solution for 20 min until desynchronized onsets of Ca²⁺ transients appeared, and then immediately fixed. The third, which was used as a positive control for NP-Cx43 (41), was incubated with 10^–6 M strophanthidin at 0.5 Hz stimulation. After 1 h of incubation, the muscle showed frequent spontaneous Ca²⁺ waves but lost responsiveness to electrical stimulation (data not shown). The muscle was then fixed. These muscles were stained with primary antibody followed by secondary antibody in the same manner (see MATERIALS AND METHODS) and were observed with a laser-scanning confocal microscope with the same settings.

In the control, healthy muscle, the majority of Cx43 was in the phosphorylated state (Fig. 6A, left) as previously described by Matsushita et al. (27). NP-Cx43 was very faintly stained, and the level of staining was not different from the background level as stained with the secondary antibody alone. In a muscle treated with a toxic dose of strophanthidin, the level of P-Cx43 was decreased by half, whereas NP-Cx43 level was eight times higher compared with the control muscle (Fig. 6A, right). In the muscle that showed desynchronized occurrence of Ca²⁺ transients, P-Cx43 was still dominant. However, closer examination revealed that the level of NP-Cx43 was increased by three times when compared with that in control muscle (Fig. 6A, middle). The differences in NP-Cx43 levels under the three conditions were also shown as surface plots of NP-Cx43 images, which help to visualize low levels of staining intensity (bottom panels in Fig. 6A). It is noticeable that the staining of NP-Cx43 in the intracellular space was higher in severely Ca²⁺-overloaded and highest in fatally Ca²⁺-overloaded muscles. This was more clearly recognized at higher magnification (Fig. 6B).

In four sets of experiments using the same procedure as described in Fig. 6A, we obtained a total of 12 pairs of P-Cx43 and NP-Cx43 images for each group (control, desynchronized, and strophanthidin-treated). The total fluorescence intensity counts in each image, which included both the cell border and intracellular space, were determined using ImageJ software and are plotted in Fig. 7. On average, muscles treated with a toxic dose of strophanthidin showed a considerable decrease in P-Cx43 (from 1.53 ± 0.12 to 1.00 ± 0.08 x 10⁶ counts/mm², P < 0.05) and a massive increase in NP-Cx43 compared with control muscles (from 0.065 ± 0.008 to 0.855 ± 0.198 x 10⁶ counts/mm², P < 0.01). In muscle that showed desynchronized Ca²⁺ transients, significant increase in the level of NP-Cx43 (from 0.065 ± 0.008 to 0.233 ± 0.072 x 10⁶ counts/mm², P < 0.05) was also detected.

View larger version (13K): [in this window] [in a new window]   Fig. 7. Comparison of P-Cx43 and NP-Cx43 levels in muscles at various Ca2+-overloading. Fluorescence intensities of labeling of P-Cx43 and NP-Cx43 are plotted. See text for more detail. Values are means ± SE (n = 12). *P < 0.05, **P < 0.01 compared with control.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

Ca²⁺ overloading and other concomitant changes. Increases in cytoplasmic resting Ca²⁺ in ischemic or Ca²⁺-overloaded heart muscles were also described previously (19, 23, 30). In Ca²⁺-overloaded cardiac muscles, many changes other than elevated Ca²⁺ concentration are expected to occur. Elevation of intracellular Ca²⁺ accelerates consumption of ATP and causes lowered cytoplasmic pH, lowered ATP concentration, accumulation of lactic acid, and abnormal mitochondrial function. These factors then impair Ca²⁺ handling to further accelerate Ca²⁺ overloading (11, 14, 44). As a result, the above factors are both the cause and consequence of Ca²⁺ overload.

We prepared severely Ca²⁺-overloaded muscles by stimulation at 2 Hz in nonflowing Krebs solution. Stopping the superfusion, which results in lowered O₂ delivery and accumulation of lactate, increased the occurrence of Ca²⁺ waves by three- to fivefold (19) along with elevated diastolic Ca²⁺ (19, 23, 30) and also caused delayed Ca²⁺ transients. These results suggest that secondary changes, such as lowered intracellular pH, decrease in ATP, and accumulation of lactate, are involved in the delayed conductivity as well as being important factors for acceleration of Ca²⁺ overloading. The procedure may be a model of one typical Ca²⁺-overloaded state, ventricular tachycardia, in which an insufficient O₂ supply due to cardiac dysfunction is concomitant with increased cellular activity.

Decreased excitability in Ca²⁺-overloaded muscles. In severely Ca²⁺-overloaded muscles, cells often failed to show Ca²⁺ transients at 2-Hz stimulation (Fig. 3), whereas, in healthy muscles, all cells showed simultaneous Ca²⁺ transients (Fig. 2). The failure of the Ca²⁺ transient indicates that cardiac cells become less responsive to triggering stimulation, and this can be attributed to resting membrane potential depolarization. As inferred from the rhod-2 fluorescence, the diastolic intracellular Ca²⁺ in severely Ca²⁺-overloaded muscle was considerably higher than in normal muscles, and the decay time of Ca²⁺ transients was significantly prolonged (see Figs. 1, 2, and 4). Therefore the Ca²⁺ extrusion by Na⁺-Ca²⁺ exchanger would be enhanced and would lead to depolarization of the resting membrane potential (6, 7), which, in turn, would result in inactivation of Na⁺ channels. Since the action potential in ventricular muscle cells critically depends on the regenerative activation of Na⁺ channels, slowing the recovery of Na⁺ channels and increasing the threshold for the next action potential would cause failure of action potential generation by field stimulation in some cells.

Delayed onset of Ca²⁺ transients in Ca²⁺-overloaded muscle. In severely Ca²⁺-overloaded muscles, lag times of up to 100 ms in the Ca²⁺ transients were detected between two adjacent cells. Because the duration of the field stimulation pulse was 2 ms, Ca²⁺ transients occurring with such a delay after the stimulation could not have been induced directly by the stimulation. Instead, they must have resulted from propagated action potentials from adjacent cells. This phenomenon can be expected when, in some cells, 1) responsiveness to field stimulation is decreased but is still sufficient to generate action potentials, and 2) cell-to-cell electrical communication is limited but not completely blocked. For this to be the case, some particular cells in a multicellular system would not generate action potentials in response to field stimulation, whereas other surrounding healthy cells would. The depolarization of the surrounding cells during the action potential should gradually bring the less-responsive cells to their threshold and should finally result in action potential generation. This may explain why cell-to-cell propagation was delayed by several tens of milliseconds or more.

If the poor coupling region includes only two or a few cells, the poor intercellular coupling would be expected to prevent action potential spread from cell to cell and would not affect conduction of excitation because a small current source would not be able to activate the surrounding large current sink to the level required to produce a conducting action potential. But when the Ca²⁺-overloaded region has a larger number of cells with a mixture of cells with variable coupling ability, the overloaded region may then be expected to affect action potential spreading. In actuality, Ca²⁺ transients were variable from cell to cell and beat to beat (Fig. 3) under conditions of Ca²⁺ overloading. Therefore, with reduced coupling ability, cardiac cells in tissue may affect each other in action potential generation and spreading. Those regions may change the propagation routes of excitation, which would result in reentry and/or action potential dispersion (1), although we have not succeeded in capturing images of reentrant phenomenon in our preparation. Further studies are required to clarify the mechanisms of action potential spreading in Ca²⁺-overloaded regions.

Among the two plausible underlying conditions for delayed onsets, the decreased responsiveness is readily explained by inactivation of Na⁺ channels as described above. For the cell-to-cell propagation delay, there are two probable mechanisms. One likely mechanism also involves the inactivation of Na⁺ channels. If Na⁺ channels are partially inactivated, not only responsiveness to stimulation but also cell-to-cell propagation of electrical signals will be suppressed. The other explanation is the inhibition of gap junction channels. This idea is consistent with the fact that a similar lag in onset of Ca²⁺ transients can also be observed in the presence of a gap junction inhibitor, heptanol. Furthermore, NP-Cx43 was increased and redistributed into the intracellular space in muscles that showed desynchronized Ca²⁺ transients. This strongly suggests that decreased conductivity via gap junction had occurred. It is, therefore, reasonable to assume that both inactivation of Na⁺ channels and inhibition of gap channel activity underlie the propagation delay, although the quantitative contribution of each is not clear.

A major difference between the conditions in vivo and in our experiments was temperature. Our results were obtained at room temperature. Arutunyan et al. (2) have reported that propagation velocity was increased twofold from 25° to 37°C in two-dimensional cardiomyocyte monolayers. Our preliminary experiments suggested that the asynchrony became less obvious with an increase in temperature but was restored as the temperature returned to room temperature (data not shown). Therefore, delayed propagation of action potentials is also expected in Ca²⁺-overloaded cardiac muscles at 37°C, although the precise features of cell-to-cell propagation remain to be determined at this temperature.

Phosphorylation status of Cx43 in Ca²⁺-overloaded muscles. We examined the phosphorylation level of Cx43 after the observation of Ca²⁺ signals and detected a significant increase in NP-Cx43 in muscles that showed desynchronized Ca²⁺ transients (Figs. 6 and 7). The NP-Cx43 is known to be elevated at high intracellular Ca²⁺ and/or low intracellular pH, which enhance closure of gap junction channels (22, 46). Furthermore, NP-Cx43 tends to redistribute from intercalated discs to nondisk and intracellular region (5, 27, 31, 41, 43), with a resulting reduction in cell-to-cell coupling. Therefore the phosphorylation status and distribution of Cx43 are good reporters of cell-to-cell coupling in the heart. Our results suggest that the conductivity via Cx43 may be decreased in severely Ca²⁺-overloaded ventricular muscles.

Conclusions. One of the proposed mechanisms for ventricular arrhythmia in Ca²⁺-overloaded heart is that spontaneous Ca²⁺ release from the SR can cause waves, which, in turn, cause DAD and develop triggered action potentials (6, 11, 16, 47). We observed spontaneous Ca²⁺ transients in the presence of a β-agonist (Fig. 5), which leads to Ca²⁺ overload and enhanced excitability (25). In addition to this mechanism, we found indication of delayed propagation of action potentials. These can be a cause of beat-to-beat changes in spread of excitation. We suggest here that both spontaneous Ca²⁺ release-induced triggered activity and impaired conductivity can cause ventricular arrhythmias in the Ca²⁺-overloaded heart.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported in part by a Grant-in Aid (no. 13670096 and no. 18590241) and a High Technology Research Center Grant for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We also thank the Uehara Memorial Foundation for financial support.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. George B. McClellan for invaluable comments in editing the manuscript. We also thank Dr. Masato Konishi (Tokyo Medical University) for comments on the manuscript.

	FOOTNOTES

 

Address for reprint requests and other correspondence: N. Kurebayashi, Dept. of Pharmacology, Juntendo Univ. School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan (e-mail: nagomik{at}med.juntendo.ac.jp)

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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