Klber G. snap frozen at each event; ventricular tissue was sent for Cx43 immunoblotting Rabbit Polyclonal to PMS1 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 BN82002 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 BN82002 previous Cx43 downregulation. 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 BN82002 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 ?and2B).2B). However, the ratio of P-Cx43 to total Cx43 decreased significantly due BN82002 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 ?and2C).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. Open in a BN82002 separate window Figure 1) A B C B C ??B ??C ???B ?C em Ratio of phosphorylated forms to unphosphorylated forms of Cx43. Results are presented as mean SD. *P 0.01 versus 1 /em 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.