Trypanosoma cruzi induces changes in cardiac connexin43 expression

Abstract

Gap junction proteins (connexins) are required for myocardial function, since they allow intercellular transmission of current carrying ions and signaling molecules. Previous studies demonstrated that rat cardiac myocytes infected with Trypanosoma cruzi lost gap junctional communication and decreased automaticity. We infected mouse cardiac myocytes with trypomastigotes of the Y strain of T. cruzi and observed alterations in connexin43 (Cx43) distribution. One hour post infection Cx43 levels were significantly increased. However, at longer time points post infection there was a significant loss of Cx43 staining in membranes of infected cardiac myocytes. Interestingly, there was also a significant reduction in myocardial Cx43 protein levels during acute infection. These data indicate that T. cruzi infection alters Cx43 expression both in vitro and in vivo. Disruptions in Cx43 may contribute to the pathogenesis of cardiac electrical alterations observed in T. cruzi infection.

Figure 1. Initiation of infection induced a transient increase in Cx43 in cardiac myocytes

A. Immunoblots for Cx43 of mouse cardiac myocytes had two upper phosphorylated bands (P1+P2) and one lower non-phosphorylated band (P0). B. Immunoblot analysis of non-infected (C) or T. cruzi-infected (Tc) cultured myocytes. GAPDH was used as loading control for all the experiments. C. Whole protein quantification of Cx43 showed that at one hour PI there was a 68% increase in Cx43 expression and at two and six hours, there was a 13.4% and 7.9% decrease respectively. D. Differential quantification for protein phosphorylation analyses of P1+P2 (phosphorylated) and P0 (non-phosphorylated) showed that both forms of Cx43 were transiently increased during the first hour of infection (60% and 20%, respectively). After this period, all three bands returned to levels near baseline (*: p<0.05, ANOVA).

Figure 2. T. cruzi alters cardiac myocyte Cx43 distribution at initial times of infection

Mouse cardiac myocytes infected after 72 hours of cultivation were fixed at one hour post infection and stained with anti-connexin43 (Sigma antibody), Phalloidin-FITC for F-actin filaments and DAPI for DNA. A. Merged image of a non-infected culture showing that cardiac myocytes were connected by Cx43 and well differentiated. B. Cx43 was localized at cell-cell contacts and also displayed large vesicular structures (arrowheads). C. Phalloidin staining showed that cells were differentiated into myocytes with well defined polarity and presence of myofibrils (MF). D. and E: After one hour of infection Cx43 lost its plaque distribution compared with uninfected cells and was evident in stress fibers. In addition, there was more perinuclear staining indicating intense synthesis of Cx43. F. Phalloidin staining remained similar to observed in controls (C). D and D.1 show in detail DAPI staining corresponding to host cell and trypomastigotes’ DNA (arrows). Bars= 50.0μm.

Figure 3. Cx43 levels were reduced during parasite proliferation

In vitro infection of mouse cardac myocytes was analyzed from 24 to 72hr. A and B. Immunoblots showed that Cx43 was increased after 24 hours PI (+32%±0.24) and was reduced at 48 (-21% ± 0.36) and 72 hours PI (-61% ± 0.14). C. Differential quantification of Cx43 phospho-bands revealed that 72 hours PI, P1+P2 bands had intensity higher then non-phosphorylated P0 band. (*: p<0.05, ANOVA). D. Biphasic effect of infection on mouse cardiac myocytes Cx43. There was a transient increase in Cx43 levels (68%) after one hour PI but it returned to baseline levels between 2hpi until 48hpi. Thereafter, Cx43 expression was reduced 61% at 72 hours PI (*: p<0,05, ANOVA).

Figure 4. Immunostaining of mouse embryo cardiac myocyte gap junction protein Cx43

A and C show the merged image of DIC and anti-Cx43 antibody. Non-infected (A and B) cultures presented abundant gap junction plaques (arrows) between coupled cardiac myocytes. After 72 hours of infection (C and D), heavily infected cells (*) lost Cx43 staining and non-infected cells in the same field displayed strong junction plaques, similar to controls (arrows). Note in C, amastigote forms of the parasite revealed by DIC microscopy. (Scale bars = 50.0μm).

Figure 5. Infection reduced Cx43 expression in the heart

Mice infected with 10⁴ trypomastigotes and reached the peak of parasitemia at day 9 PI (A). At day 11 PI hearts were collected and atria and ventricles were analyzed separately. B and C: Western blot analysis demonstrated a significant reduction in the expression of Cx43 in both atria (28%) and ventricles (24%), (* p<0.05, ANOVA).