Fig 1.
(A) Survival rate, (B) parasitemia and (C to F) histological evaluation of cardiac tissue.
A-B: Mice were infected with 100 blood trypomastigotes of the Colombian strain of T. cruzi and the (A) survival rate and (B) parasite load were evaluated 15, 30 or 45 days after infection. Each point represents the group mean and the vertical bars represent the standard error (n = 12 animals per group). C-F: Representative images of histological characteristics of infected animals after (C and D) haematoxylin and (E and F) eosin or anti-T. cruzi staining. The arrows indicate (C; inflammatory infiltrate; blue arrow; 45 dpi; 2,5x), myocytes vacuolization, myocarditis and fiber necrosis (D; 45 dpi; black arrow; 40x) and amastigote nests (E and F; green arrows; 45 dpi; 10x and 20x).
Fig 2.
Electrocardiographic alterations in infected animals.
Mice were tranquilized with Diazepam (10 mg/Kg) and the transducers were carefully placed subcutaneously according to chosen preferential derivation (DII). The traces were recorded for 2 minutes. A: Representative ECG registers segments showing second degree atrioventricular block (30 dpi) and arrhythmia (45 dpi). B-E: Electrocardiographic parameters evaluated 13, 30 or 45 dpi. Results were expressed as medians and interquartile ranges (n = 12 animals per group). Groups were compared by a non-parametrical test (Mann-Whitney Rank Sum Test) with GraphPad Prism software (version 5.0.4). * p < 0,05. F: Percentage of afflicted animals at each time point.
Fig 3.
Characterization of microRNA expression profile.
A: Principal component analysis (PCA) plot of samples was performed using all probe sets, by using a median centering of the data set. B: Hierarchical clustering based on squared Euclidean distance measure and Ward's method for linkage analysis and Z score normalization. Each line represents one microRNA and each column represents one sample. The colour scale illustrates the microRNAs relative expression (ΔCt) after global normalization; red and blue represents upregulation and downregulation, respectively. Each square represents one sample. C: Venn diagram showing the number of differentially expressed microRNAs in each time point. D: Heat-map showing the fold change over control for the 17 miRNAs expressed in all time points post infection. The colour scale illustrates the fold change in microRNAs expression relative to uninfected controls; red and blue represents upregulation and downregulation respectively. Each square represents the group mean.
Fig 4.
Kinetics of microRNA expression during infection.
The line represents the fold change in microRNAs expression in each time point (15, 30 and 45 dpi) relative to uninfected controls (0 dpi).
Fig 5.
Correlations between microRNA expression and parasitemia and QTc interval alterations.
Each point represents one sample; green and orange represents correlation with QTc interval and parasitemia respectively. Lines represent the linear regression for Pearson correlation, p < 0, 05.
Table 1.
Correlations between microRNA expression and both parameters: Parasitemia and QTc interval.
Fig 6.
Network of miRNAs and molecules related to heart QT interval regulation.
In silico analysis done using the IPA software (Qiagen, USA) showing a biological network built with 4 miRNAs (miR-142-5p, miR-21-5p, miR-145-5p and miR-149-5p) from the 6 (miR-146b, miR-21-5p, miR-142-3p, miR-142-5p, miR-145-5p and miR-149) uploaded for the analysis. The resulted network shows how the 4 miRNAs and the multiple molecules that are regulated directly or indirectly by them are related to the regulation of heart QT interval. The miRNAs are represented in graduation of red and green based on their fold change in expression at 45 dpi compared to 0 dpi.
Fig 7.
Individual mature miRNAs and their respective putative gene targets: A) miR-149-5p and CACAN1C B) miR-21-5p, miR-145-5p and KCNA1 C) miR-145-5p and KCNA1, GJA5, RNF207 and D) miR-142-5p and SLC18A2 were measured by real time RT-PCR in each time point post infection (15, 30 and 45) in four animals per group.
The expression was calculated as the mean ± s.d. for each group as individual data points using the relative expression (fold change over CONT) by the 2-ΔΔct method, where Ct is the threshold cycle. Groups were compared by a non-parametrical test (Mann-Whitney Rank Sum Test) with GraphPad Prism software (version 5.0.4). Results were expressed as medians and interquartile ranges. * P-values were considered significant if <0.05.