Amiodarone inhibits Trypanosoma cruzi infection and promotes cardiac cell recovery with gap junction and cytoskeleton reassembly in vitro

Abstract

We present the results of the first detailed study of the antiproliferative and ultrastructural effects of amiodarone on Trypanosoma cruzi, the causative agent of Chagas' disease. Moreover, we report the effects of this compound on the recovery of F-actin fibrils, connexin43, and contractility in T. cruzi-infected cardiac myocytes. Amiodarone is the most prescribed class III antiarrhythmic agent and is frequently used for the symptomatic treatment of Chagas' disease patients with cardiac compromise. In addition, recent studies identified its antifungal and antiprotozoal activities, which take place through Ca(2+) homeostasis disruption and ergosterol biosynthesis blockade. We tested different concentrations of amiodarone (2.5 to 10 μM) on infected primary cultures of heart muscle cells and observed a dose- and time-dependent effect on growth of the clinically relevant intracellular amastigote form of T. cruzi. Ultrastructural analyses revealed that amiodarone had a profound effect on intracellular amastigotes, including mitochondrial swelling and disorganization of reservosomes and the kinetoplast and a blockade of amastigote-trypomastigote differentiation. Amiodarone showed no toxic effects on host cells, which recovered their F-actin fibrillar organization, connexin43 distribution, and spontaneous contractility concomitant with the drug-induced eradication of the intracellular parasites. Amiodarone is, therefore, a promising compound for the development of new drugs against T. cruzi.

FIG. 1.

Time and concentration dependence of the effects of amiodarone on infection of HMCs by T. cruzi (protocol i). Amiodarone (2.5 to 10 μM) was added to the cultures after 2 h of infection. (A, B) We observed significant effects of the drug on the percentage of infected cells (A) and the number of intracellular parasites (B), which were followed as a function of time. (C) The concentrations of the drug used in these studies were not toxic to the cardiac myocytes. Asterisks indicate statistical differences in relation to control cultures. The graphs show the means and standard deviations from triplicates of one representative experiment of four independent experiments. Cont, control.

FIG. 2.

Time and concentration dependence of the effects of amiodarone on infection of HMCs by T. cruzi (protocol ii). (A to C) Amiodarone (5 to 10 μM) was added to the cultures after 48 h of infection, and the percentage of infected cells (A), number of intracellular parasites per host cell (B), and number of released trypomastigotes in the supernatant (C) were followed as a function of time. (D) We did not observe significant cytotoxic effects of amiodarone with the concentrations used throughout the experiments. Asterisks indicate statistical differences in relation to control cultures. The graphs show the means and standard deviations from triplicates of one representative experiment of four independent experiments.

FIG. 3.

Effects of amiodarone on the intracellular cycle of T. cruzi. Primary cardiac myocytes were obtained and infected with the Y strain of T. cruzi. After 2 h of infection, cultures were treated with 5 or 10 μM amiodarone. At 96 h postinfection (hpi), untreated cultures (A) displayed cells with trypomastigote forms of the parasite already evading host cells. Cultures treated with 5 μM (B) and 10 μM (C) of amiodarone displayed intracellular parasites with severe morphological alterations (arrows). (C) Interestingly, an amiodarone concentration of 10 μM induced a drastic reduction of parasitism. After 144 h of infection (corresponding to 142 h of treatment), we observed that in nontreated cultures (D), 70 to 80% of cardiomyocytes were infected. Treatment with 5 μM amiodarone (E) drastically reduced the number of parasites, whereas the use of 10 μM amiodarone (F) almost eliminated the parasitism of the cultures. Insets show details of intracellular parasites in higher magnification. P, parasites; N, nucleus.

FIG. 4.

(A) Effects of amiodarone on the ultrastructure of intracellular T. cruzi (Y strain) amastigotes. Untreated HMCs after 192 h of infection with T. cruzi, displaying severe intracellular damage and intact amastigote forms (P) and presenting a bar-shaped kinetoplast (K), nucleus (N), and flagellum (F). (B) Infected cultures treated with 5 μM amiodarone for 144 h showed parasites with membrane blebs (arrows), loss of intracellular material (stars), and kinetoplast (K) alterations. (C) Released parasites from untreated cultures displayed acidocalcisomes (AC) and a bar-shaped kinetoplast (K), reservosome (R), and flagellum (F), as expected. (D) Parasites obtained from cultures treated with 5 μM amiodarone (144 h) showed important alterations in the kinetoplast and Golgi apparatus (arrow).

FIG. 5.

Amiodarone induced the recovery of host cell homeostasis. After eradicating the infection, we observed the recovery of morphological and functional aspects of cardiac myocytes cultures, as assessed by immunofluorescence and measuring of the spontaneous electrocontractility of the cells. (A to C) Uninfected cultured myocytes displayed abundant connexin43 immunoreactivity (C) as well as striated patterns of F-actin staining (B). (D to F) Highly infected cultures, at 144 h of infection, presented destruction of the F-actin cytoskeleton (E) and loss of Cx43 plaques (F). (G to I) Treatment with 5 μM amiodarone for 142 h decreased parasitism and induced recovery of host cells, as evidenced by F-actin and Cx43 recovery, which included striations (H) and the presence of gap junction plaques (I). (J) The spontaneous electrocontractility of the myocytes culture was evaluated by counting the number of contractions per 10 s. We observed that T. cruzi-infected cultures had a progressive decrease in electrocontractility, whereas treatment with 5 and 10 μM amiodarone restored cultures to normal levels of electrocontractility. *, P < 0.05; **, P < 0.01 (ANOVA).