Author + information
- Stefan Dhein, MD, PhDa,
- Ronald Wilders, PhDa,
- Habo J Jongsma, PhDa and
- Jaques-Antoine Haefliger, PhDa ()
In our study (1)we have shown changes in the expression and localization of the gap junction proteins connexin40 (Cx40) and connexin43 (Cx43) in patients suffering from chronic atrial fibrillation (AF). We found increased Cx40 and unchanged Cx43 expression. Moreover, we observed lateralizations of both connexins that were no longer confined to the cellular poles. In a rat model of pacing-induced AF, lateralization was also found along with a change in anisotropy.
Regarding the communication from van der Velden and colleagues, there is pobably some misunderstanding: It is stated clearly in our report that we produced a polyclonal antibody against a polypeptide comprising amino acids 231–330 of rat Cx40. There is no putative amino acid sequence homologous to Cx43.
This antibody did not show crossreactivity with other connexins. This was tested in Western blots from HeLa cells transfected with either Cx43 or Cx40. Immunocytochemistry demonstrated that Cx40 staining differs from Cx43 staining. In rat and mice, we did not see any Cx40 staining in the smooth muscle cells (SMC) of aortae, whereas Cx43 antibodies recognized Cx43 in SMC. The specificity of the antibody has also been shown in rat kidney (2). When freshly isolated ventricular cardiomyocytes from two-month-old rats were cultured, redifferentiation was found with differential time-dependent changes in connexin expression and phosphorylation and no crossreactivity between Cx40 and Cx43.
Regarding the immunostainings, there is autofluorescence from connective tissue. However, this is typical for this kind of human tissue. It is difficult to differentiate between connective tissue and specific staining (especially in the reproductions), but it is possible because the connective tissue shows typical thin, curly, fluorescent lines. Moreover, there is yellowish fluorescence from lipofuscine, which also disturbs. Thus, although we agree that it is difficult to evaluate, we have seen enhanced presence of specific immunofluorescence at the lateral sides of the cells in AF patients.
Regarding the functional meaning, we have hypothetized that this lateralization may result in a change in anisotropy, which was supported by our rat model. In that model a higher degree of lateralization was seen than observed in human chronic AF. However, it is difficult to extrapolate from the rat experiments to the human immunostaining results, for we cannot know how much of the protein positively staining for connexins resembles functional channels. In the rat model did we observe both lateralization and changes in anisotropy. However, we completely agree with van der Velden and colleagues that inhomogenicities in connexin distribution as described in their study in a goat model (3)should have a high impact on the biophysics of the tissue. In contrast to our findings in humans, they describe a decrease in Cx40, which from our point of view might either be due to species difference or to the fact that they investigated goat atria during the first 16 weeks of sustained pacing, while we investigated chronic AF of one-year duration in patients.
- American College of Cardiology Foundation
- Polontchouk L,
- Haefliger J.A,
- Ebelt B,
- et al.
- Van der Velden H.M.W,
- Ausma J,
- Rook M.B,
- et al.