Alterations of the intercellular coupling protein, connexin-43, during ventricular fibrillation and sinus rhythm restoration demonstrated in male and female rat hearts: A pilot study

Ventricular fibrillation (VF) is the main cause of sudden cardiac death; therefore, understanding the mechanisms responsible for the onset and maintenance of VF is essential, along with investigating the factors that might affect defribrillation threshold and VF termination. It has been previously suggested that connexin-43 (Cx43) facilitates the development of malignant arrhythmias, and the hypothesis is that VF deteriorates Cx43 and, thus, hampers cardioversion into sinus rhythm. This study examined whether myocardial expression and phosphorylation status of Cx43 is altered due to VF and during sinus rhythm restoration in 10-month-old Wistar rats.

Abstract

Ventricular fibrillation (VF) is a life-threatening arrhythmia, whose occurrence precedes the development of myocardial arrhythmogenic substrate resulting from either chronic or acute pathophysiological conditions. The authors’ previous and current studies suggest that downregulated and/or heterogeneously distributed cell-to-cell coupling protein – connexin-43 (Cx43) – facilitates the development of malignant arrhythmias. It was hypothesized that VF itself deteriorates Cx43, and may hamper cardioversion into sinus rhythm. The purpose of the present study was to examine whether myocardial expression and the phosphorylated status of Cx43 is altered due to VF and during sinus rhythm restoration. Experiments were performed using 10-month-old male and female Wistar rats. Isolated Langendorff-mode-perfused rat hearts were subjected to the following events: basal condition, electrically induced VF lasting 2 min, electrically induced VF lasting 10 min, and sustained VF followed by spontaneous sinus rhythm restoration due to transient stop perfusion. The hearts were snap frozen at each event; ventricular tissue was sent for Cx43 immunoblotting using rabbit antiCx43 polyclonal antibody to detect phosphorylated (P-Cx43) as well as unphosphorylated (noP-Cx43) forms of Cx43, and mouse antiCx43 monoclonal antibody to detect noP-Cx43 only. Compared with basal conditions, total Cx43 expression did not change during experiments in either male or female rat hearts. However, P-Cx43 and the ratio of P-Cx43 to total Cx43 decreased significantly due to VF lasting 2 min and 10 min in male rat hearts only. In parallel, there was a significant increase in noP-Cx43 due to VF lasting 2 min and 10 min in male rat hearts only. Surprisingly, an enhancement of noP-Cx43 linked with suppression of P-Cx43 was detected during stop perfusion-induced termination of VF lasting 2 min, followed by sinus rhythm restoration in both male and female rat hearts. Sinus rhythm was not restored after 10 min of VF, which caused pronounced Cx43 dephosphorylation. In conclusion, there is a downregulation of Cx43 due to sustaining of VF, and it occurs earlier in male rat hearts compared with female rat hearts. It appears that transient no-flow-related inhibition of cell-to-cell coupling, as indicated by an increase in nonP-Cx43, can terminate VF followed by sinus rhythm restoration depending on the degree of previous Cx43 downregulation.

Experimental and clinical studies have shown that diseased or failing hearts are prone to develop ventricular fibrillation (VF), which is the main cause of sudden cardiac death (SCD) particularly during a heart attack. To prevent SCD due to VF in high-risk patients, an implantable cardioverter defibrillator (ICD) is used to revert VF into sinus rhythm by electrical shock. However, it should be noted that there are common problems, such as failure of electrical defibrillation and postshock reinitiation of VF, that consequently increase the number of shocks (1,2). Despite current therapies based on ICD, SCD due to VF remains a major health problem (3). Thus, understanding the mechanisms responsible for the onset and maintenance of VF in animal models and humans is essential to both identify individuals at risk and to develop specific therapies to mitigate that risk. Moreover, it is of interest to investigate the possible factors that might affect defibrillation threshold and VF termination.

The general classification of cardiac arrhythmias assumes that all disturbances of rhythm result from one of two primary abnormalities in electrical activity. The first is an abnormality in impulse initiation and, the second, an abnormality in impulse propagation; both may coexist (4,5). The former is associated particularly with triggered activity and/or abnormal automaticity, whereas the latter with conduction blockage and re-entry. It has been reported that intercellular electrical coupling and communication mediated by connexin (Cx) channels at the gap junctions may determine conduction velocity, and that alterations in Cx distribution and/or defective cell-to-cell coupling contribute to abnormal conduction facilitating the development of re-entrant arrhythmias such as VF (5,6). The major protein of cardiac gap junction channels is Cx43. Genetically Cx43-deficient mice indeed exhibit markedly slower myocardial conduction that facilitates re-entrant arrhythmias and sudden arrhythmic death (7,8). Heart diseases and aging are accompanied by the down-regulation of Cx43 and/or its mislocalization (remodelling), which contributes to the development of arrhythmogenic substrate and electrical instability (9–14). While acute dysfunction of cell-to-cell coupling, particularly due to alterations in Cx43 phosphorylation and Ca²⁺ overload, may account for triggering and maintaining malignant arrhythmias, including VF (7,10,15–20). Taking into account that Cx43 is an active signalling molecule for synchronous cardiac function, we hypothesized that VF itself impairs cell-to-cell coupling and may hamper cardioversion into sinus rhythm. The purpose of the present study was, therefore, to examine whether myocardial expression and phosphorylation status of Cx43 is altered due to VF and during sinus rhythm restoration.

METHODS

All animal experiments were performed in accordance with the rules issued by the State Veterinary Administration of the Slovak Republic, legislation No 289/2003, and with the regulations of the Animal Research and Care Committee of Institute for Heart Research. This study was conducted using 10-month-old male (n=20) and female (n=20) Wistar rats. The rats were anesthesized using ether, the chests were opened and the hearts were rapidly excised and perfused via cannulated aorta in Langendorff mode with oxygenated Krebs-Henseleit solution at a constant pressure of 80 mmHg and a temperature of 37°C. Left ventricular pressure (LVP) was measured isovolumetrically by a water-filled latex balloon, which was introduced into the left ventricle through the mitral orifice and connected to a pressure transducer. Bipolar electrocardiography was continuously monitored during the entire experiment. VF was induced using 1 s bursts of electrical rectangular pulses (100 pulses/s), 1 ms in duration at 35 mA (Electrostimulator ST-3“, Medicor, Hungary) delivered via stimulating electrodes attached to the epicardium of the right ventricle. When sustained VF lasting 2 min occurred, the ability of the heart to restore sinus rhythm was registered using stop perfusion-induced cardioversion.

Experimental protocol of isolated Langendorff-perfused heart and tissue sampling

Isolated perfused hearts were subjected to the following: basal condition, electrically induced VF lasting 2 min, electrically induced VF lasting 10 min, VF lasting 2 min and its termination by stop perfusion, followed by spontaneous sinus rhythm restoration. The heart was snap frozen in liquid nitrogen at each event, and ventricular tissues were taken for examination of Cx43 expression and its phosphorylated state.

Western blot analysis of Cx43

Frozen tissue from both ventricles was lyophilized and solubilized in SB20 solution (20% SDS, 10 mmol/L EDTA, 0.1 mol/L TRIS and pH 6.8) by sonicator UP 100H (Hielscher, Germany). An equal amount of total protein in each sample was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred electrophoretically to nitrocellulose membrane. Two different anti-Cx43 antibodies were used in the present study: A rabbit polyclonal antibody (Sigma-Aldrich, Germany) was generated against amino acids 363 to 382 of the Cx43 protein, and has been used extensively to exhibit high Cx43 specificity; and a mouse monoclonal antibody (designated 13-8300) generated against amino acids 360 to 376 of the Cx43 protein (Zymed Laboratories, USA). This antibody only recognizes Cx43 when the serine at residue 368 is unphosphorylated. To detect both phosphorylated (P-Cx43) and unphosphorylated (noP-Cx43) forms of Cx43, the membrane was incubated overnight with primary rabbit polyclonal antibodies (diluted 1:2000) at 4°C, followed by further incubation for 1 h at room temperature with the secondary donkey antibody (peroxidase-labelled antirabbit imunoglobulin, Amersham Biosciences, United Kingdom, 1:2000). The mouse antiCx43 monoclonal antibody (diluted 1:500) was used to detect the unphosphorylated form only. Bound antibodies were visualized by the enhanced chemiluminescence method. The optical density of individual bands was analyzed using PCBAS 2.08e software (Raytest Inc, Germany), and the optical density of each band was normalized relative to the optical density of GAPDH as an internal control.

Statistical analysis

The data are expressed as mean ± SD. One-way ANOVA followed by Duncan’s post hoc test was used to analyze statistical significance between means. Comparison between the two groups was performed using the unpaired Student’s t test. Values were considered to be statistically significant at P<0.05.

RESULTS

There was a sex-related difference in the susceptibility of the heart to electrically inducible VF. While one or two stimuli induced sustained VF in male rats, three to five stimuli were needed to induce sustained VF in female rats.

Compared with basal conditions, total Cx43 expression did not change during the experimental protocol either in male or female rat hearts (Figures 1B and 2B). However, the ratio of P-Cx43 to total Cx43 decreased significantly due to VF lasting 10 min in both male and female rat hearts. The decrease in this parameter did not reach significance when VF lasted 2 min in males, and did not change in females (Figures 1C and 2C). Furthermore, VF lasting 10 min resulted in a significant decrease in P-Cx43 and an increase in noP-Cx43 in both male and female rat hearts, and a decrease in the ratio of P-Cx43 to noP-Cx43 (Figures 3A, 3B and 3C, and 4A, 4B and 4C). These Cx43 alterations were less pronounced due to VF lasting 2 min, and they were only significant in males (Figures 3A, 3B, 3C and 4A, 4B, 4C). Surprisingly, an enhancement of noP-Cx43 that paralleled the suppression of P-Cx43 was detected in both male and female rat hearts at the moment of stop perfusion-induced termination of VF lasting 2 min, followed by spontaneous sinus rhythm restoration (Figures 3A, 3B, 3C and 4A, 4B, 4C). However, sinus rhythm was not restored by this manoeuvre when VF lasted 10 min, which caused pronounced Cx43 dephosphorylation.

Figure 1).

A Representative immunoblot showing unphosphorylated connexin 43 (noP-Cx43) and phosphorylated Cx43 (P-Cx43) in male Wistar rat hearts subjected to the following – 1: Basal conditions; 2: Electrically induced VF lasting 2 min; 3: Electrically induced VF lasting 10 min; 4: VF lasting 2 min and its termination by stop perfusion, followed by spontaneous sinus rhythm restoration. B Densitometric quantification of total Cx43 protein expression normalized to GAPDH. C Ratio of the phosphorylated forms of Cx43 to total Cx43 levels. Results are presented as mean ± SD. *P<0.01 versus 1

Figure 2).

A Representative immunoblot showing unphosphorylated connexin 43 (noP-Cx43) and phosphorylated Cx43 (P-Cx43) in female Wistar rat hearts subjected to the following – 1: Basal conditions; 2: Electrically induced VF lasting 2 min; 3: Electrically induced VF lasting 10 min; 4: VF lasting 2 min and its termination by stop perfusion, followed by spontaneous sinus rhythm restoration. B Densitometric quantification of total Cx43 protein expression normalized to GAPDH. C Ratio of the phosphorylated forms of Cx43 to total Cx43 levels. Results are presented as mean ± SD. *P<0.05 versus 1

Figure 3).

A Expression of phosphorylated forms of connexin 43 (Cx43) in male Wistar rat hearts subjected to the following – 1: Basal conditions; 2: Electrically induced VF lasting 2 min; 3: Electrically induced VF lasting 10 min; 4: VF lasting 2 min and its termination by stop perfusion, followed by spontaneous sinus rhythm restoration. *P<0.05 versus 1; ^†P<0.001 versus 1; ^‡P<0.01 versus 2. B Expression of unphosphorylated forms of Cx43 using specific antiCx43 monoclonal antibody, which recognizes Cx43 only when the serine at residue 368 is unphosphorylated. *P<0.05 versus 1; ^†P<0.01 versus 1; ^‡P<0.05 versus 2. C Ratio of the phosphorylated forms of Cx43 to the unphosphorylated forms. Results are presented as mean ± SD. *P<0.05 versus 1; ^†P<0.001 versus 1, ^‡P<0.05 versus 2

Figure 4).

A Expression of phosphorylated forms of connexin 43 (Cx43) in female Wistar rat hearts subjected to the following – 1: Basal conditions; 2: Electrically induced VF lasting 2 min; 3: Electrically induced VF lasting 10 min; 4: VF lasting 2 min and its termination by stop perfusion, followed by spontaneous sinus rhythm restoration. *P<0.05 versus 1; ^†P<0.05 versus 2; B Expression of unphosphorylated forms of Cx43 using specific antiCx43 monoclonal antibody, which recognizes Cx43 only when the serine at residue 368 is unphosphorylated. *P<0.001 versus 1; ^†P<0.01 versus 2. C Ratio of phosphorylated forms to unphosphorylated forms of Cx43. Results are presented as mean ± SD. *P<0.01 versus 1

DISCUSSION

Using an ex-vivo perfusion system of rat heart and electrically induced VF, we have found for the first time that fibrillation activity itself results in clear-cut alterations of myocardial Cx43 expression. Our results also suggest an inverse relationship between the phosphorylated state of Cx43 and sustained VF. Even when the total ventricular levels of Cx43 did not change due to VF lasting 2 min or 10 min, the phosphorylated state of this dominant gap junction channel protein was significantly altered. Time- and sex-dependent acute Cx43 alterations were demonstrated, further supporting the notion that sex differences in Cx43 expression can contribute to differences in arrhythmia susceptibility between males and females (21). Accordingly, VF lasting 2 min (considered in isolated heart models as sustained VF) caused reductions in P-Cx43, a decrease in the ratio of P-Cx43 to noP-Cx43, and an increase in noP-Cx43 that was significant in male rat hearts only. However, prolonged VF lasting 10 min resulted in significant changes in the phoshorylated status of Cx43 compared with a shorter VF period in males. Moreover, a significant downregulation of Cx43 was also found in females. Phosphorylation of gap junctional Cx43 appears to regulate channel function and the rates of channel assembly and turnover. Several protein kinases have been shown to influence the conduction and permeability of Cx43 channels (20,22). Phosphorylation of Cx43 has been described mainly on serine, but also at tyrosine residues (23). Using mouse monoclonal anti-Cx43 antibody, it was revealed that the serine residue at position 368 was markedly dephosphorylated due to VF, suggesting that this epitope may be implicated in myocardial cell-to-cell uncoupling. This fact seems to be very important because this unique phosphorylation site must be phosphorylated before phosphorylation of Cx43 at other sites (24). Moreover, it has been reported that phosphorylation of Cx43 at serine 368 by protein kinase C (which is involved in cardioprotective signalling) regulates intercellular gap junction channel communication (25). Taken together, the question arises whether preservation of phosphorylation of this specific site might protect from VF.

Our previous studies (10–12) have shown that chronic heart diseases are accompanied by either suppression of Cx43 phoshorylation or its hyperphosphorylation, whereby the former promotes VF unlike the latter. Downregulation of Cx43 most likely also contributes to a high risk of SCD in humans (14). In this context, it should be noted that dephosphorylation of Cx43 (associated with translocation of the protein from the gap junctions to the cytoplasm or redistribution to lateral cell surfaces) manifests as acute pathophysiological conditions such as ischemia, hypoxia, hypokalemia (7,10,16,18,19,26,27) and also VF, as we have shown in the present study. Nevertheless, these events result in an impairment of myocardial cell-to-cell coupling and uncoupling. The latter is most likely due to conformational changes of Cx43 protein induced by altered phosphorylated status and the presence of Ca²⁺ overload (15,17,19,23). Heterogeneously distributed myocardial cell-to-cell uncoupling has been shown to precede the occurrence of re-entrant arrhythmias, such as VF and AF (18,28), and most likely is involved in their persistance. Subsequent myocardial electrical heterogeneity hampers ventricular defibrillation and can also increase the defibrillation threshold (29,30). All of this should be taken into account, particularly in diseased hearts, when treating VF.

It is known that some animals exhibit transient VF (31) and that exposure of the Langendorff-heart preparation to short-lasting ischemia often induces transient VF, while prolonged ischemia results in sustained VF (32). During VF, the perfusion of the heart still persists as a consequence of constant perfusion pressure although the coronary flow is disturbed by fibrillating activity. In the present study, we have demonstrated for the first time that electrically induced sustained VF in an isolated heart preparation can be terminated and reverted to sinus rhythm by brief stop perfusion. The time interval from the stop perfusion until occurrence of sinus rhythm is variable and very likely reflects the ability of the heart to terminate VF. We hypothesized that the level of Cx43-mediated cell-to-cell coupling might affect this ability. Contrary to our expectation, we have found that during sinus rhythm restoration, there is an enhancement of noP-Cx43. It indicates that instead of improvement of Cx43 channel function, their global transient inhibition is probably involved in cardioversion. This notion is supported by the finding that demonstrated paradoxical improvement of impulse conduction in cardiac tissue by partial cellular uncoupling (33), and that showed a decrease in defibrillation threshold in isolated rabbit hearts by gap junction channel blockers (34). However, it seems that the severity of myocardial Cx43 alterations affects cardioversion, since it was hampered in the hearts subjected to 10 min VF, which caused much pronounced Cx43 downregulation. Nevertheless, future studies should elucidate whether sudden global inhibition of Cx43 channels resulting from stop perfusion may facilitate termination of VF and sinus rhythm restoration. It seems that Cx43-targeted approaches might be useful in clinics even in the era of sophisticated ICD.

CONCLUSION

There is a downregulation of Cx43 due to sustained VF, and it occurs earlier in male compared with female rat hearts. It appears that transient no-flow-related inhibition of cell-to-cell coupling, as indicated by an increase in nonP-Cx43, can terminate VF followed by sinus rhythm restoration depending on the degree of previous Cx43 downregulation.