Molecular Remodeling of Cardiac Sinus Node Associated with Acute Chagas Disease Myocarditis

Figures

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  Figure 1. Heart rate kinetics in mice. (A) Initial heart rate in beats per min (bpm) in control (black symbols) and infected (red symbols) mice. (B) The Δ heart rate, defined as the differential of the heart rate at point 0 and 5 min post-ivabradine single-dose treatment, in control (black symbols) and infected (red symbols) groups. * The p-value was estimated at 0.0425 by Student’s t-test. (C) Linear regression between initial heart rate and the Δ heart rate in control (black symbols) and infected (red symbols) groups. Slopes were considered significantly different with a p-value of 0.0036. (D) Control mice were treated (brown symbols) or not treated (black symbols) with a single bolus of 5 mg/kg of ivabradine five minutes before stimulation with 1.1 mg/kg isoproterenol; infected mice were treated (green symbols) or not treated (red symbols) with ivabradine, like the control mice.

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  Figure 2. Quantitative/qualitative analysis of ECG parameters after a single-dose treatment with ivabradine (Ivb) in mice at the EA phase of infection. (A). ECG intervals (QRS, P duration, and QTc) and amplitudes (T, S, and P) were determined for control and infected mice before (Pre-Ivb) and after (Post-Ivb) treatment with ivabradine. An unpaired Student’s t-test was performed for statistical comparison (* p < 0.05), and a one-way ANOVA post-test for trends indicated in arrows (* p ≤ 0.05 and *** p ≤ 0.001). (B) Representative ECG recordings pre- and post-Ivb treatment. Registers a–d were taken from different infected mice, where a indicates a junctional ectopic beat (arrow), b the QRS enlargement with S blockage, and c–d illustrate atrial ectopic beats.

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  Figure 3. Quantitative/qualitative analysis of ECG parameters after continuous treatment with ivabradine (Ivb) in mice at the EA phase of infection. (A) Differences in wave voltages and amplitudes between treated (Post-Ivb) and untreated (Pre-Ivb) mice. Statistical significance was analyzed by one-way ANOVA (** p < 0.01 and *** p < 0.001). (B) Bimodal P-wave morphology, as seen in the mean P-wave trace. (C) Supraventricular ectopic beats were registered after continuous ivabradine treatment in chagasic mice.

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  Figure 4. Histological sections of auricles stained with Van Gieson’s stain. The left panel shows the normal morphology of cells compatible with pyramidal P cells (***) and peripheral fusiform fibroblast-like cells (arrow) located externally to the nodal area. Structural relationships are well-defined and conserved. The right panel shows the right auricle of the infected tissue with loss of structure and depopulation of pyramidal cells. A moderate mononuclear infiltrate can be appreciated. Fusiform fibroblast-like cells preserve morphology and location (arrow).

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  Figure 5. HCN4 expression in heart tissue. (A) Confocal immunofluorescence staining of auricles from control and infected mice at the EA phase using anti-Cx43 and anti-HCN4 antibodies, along with DAPI for nuclear visualization and to delimitate structural relationships in the nodal region of the right auricle. (B) HCN4 mean fluorescence was determined in control and infected samples. (C) Western blot analysis of ventricles was performed to estimate HCN4 expression in control and infected mice at the EA phase. The membrane was stained with Ponceau red before incubating with antibodies as a protein load control. (D) Hcn4 mRNA expression in hearts of control and infected mice at the EA phase. Fold change in each infected group was calculated compared to control (“0” value in log 10 scale). Statistical analysis was undertaken using an unpaired Student’s t-test of the infected and the control groups in B and D (* p < 0.05; ** p < 0.01).