Early Trypanosoma cruzi Infection Triggers mTORC1-Mediated Respiration Increase and Mitochondrial Biogenesis in Human Primary Cardiomyocytes

FIGURE 1. Differential gene expression in response to Trypanosoma cruzi infection. (A) Comparative expression at times 0, 3, 6, and 12 hpi (fold change ≥ 2; p ≤ 0.01). (B) Hierarchical clustering of the differentially expressed genes. (C) Venn diagrams comparing (a) up-regulated and (b) down-regulated genes from infected vs. control cardiomyocytes at the different times post infection.

FIGURE 2. Pathway analysis and RT-qPCR. (A) Heat map of the most significantly altered pathways from the differentially expressed genes (p ≤ 0.01 Fc ≥ 2). C and I denotes control and infected cardiomyocytes respectively. (B) Quantification of selected genes by real-time PCR. The bars shows the relative fold changes of up-regulated genes (rps10, rpl37, eif3h, ndufb4, ldhb, mdh2, anxa1, hla-c) analyzed by qPCR at t0. Values are means of three biological replicates ± SEM. ∗p ≤ 0.05. The dotted line shows the value at which there is no difference in expression (1) between infected and control cells. (C) Kinetic expression analysis between 0–24 hpi in respect to each control by qPCR for selected genes. The dotted line shows the value at which there is no difference in expression (1) between infected and control cells. Values are means of two or three biological replicates ± SEM. ∗p ≤ 0.05.

FIGURE 3. Mitochondrial bioenergetics. (A) QO2 were evaluated in control (filled circles) and infected (open circles) cells at 6 or 24 hpi following the addition of oligomycin (2 μM), FCCP (1 μM) and AA-ROT (1 μM each). QO2 is expressed as pmol O2 consumed/min/104 cardiomyocytes and are the mean of three independent determinations. (B) Mitochondrial bioenergetics parameters were calculated after subtraction of non-mitochondrial respiration (QO2 after AA-ROT addition) to all data. Basal respiration (no drug added), maximal respiration (QO2 after FCCP addition) and spare respiratory capacity (calculated as the ratio between QO2FCCP and QO2BASAL) at the different time points analyzed (6, 17, and 24 hpi). Each data point represent the mean ± SEM n = 10. ∗∗p < 0.01, ∗∗∗p < 0.001.

FIGURE 4. Evaluation of glucose and fatty acid metabolism. (A) Kinetic of the PPR response of control and T. cruzi infected cardiomyocytes (24 hpi) to glucose (10 mM), oligomycin (2 μM) and 2-DG (100 mM). (B) Cardiomyocyte QO2 response of control and T. cruzi infected cardiomyocytes (24 hpi) to glucose (10 mM), oligomycin (2 μM) and 2-DG (100 mM). (C,D) Kinetic of QO2 response in the absence (E) or presence (F) of the CPT1 inhibitor etomoxir (100 μM), oligomycin (2 μM), FCCP (1 μM), and AA-ROT (1 μM each) in control and T. cruzi infected cardiomyocytes at 24 hpi. Culture media was the Human Cardiomyocytes Primary Cell Culture (Celprogen).

FIGURE 5. Mitochondrial biogenesis and ROS evaluation. (A) Quantification of the mtDNA relative to the nuclear DNA in infected cardiomyocytes, HeLa and primary Huvec cells versus the respective controls at 24 hpi determined by qPCR. Values are means of three biological replicates ± SEM. ∗∗∗p ≤ 0.001. (B) Confocal microscopy using anti-cytochrome c antibody (dilution 1/100) and DAPI for DNA staining after 24 hpi. White arrows denote the presence of intracellular amastigotes. (C) Measure of the means ± SD of the fluorescence intensity of infected and control cells. ∗∗∗p ≤ 0.001. (D) The production of ROS at 24 hpi was monitored by DCF fluorescence. Results are expressed graphically as percentages of the fluorescence intensity of the control cells. Values are means ± SEM of the results from three independent experiments performed in triplicate (C = uninfected and I = infected cells; ∗∗∗p < 0.002). (E) Relative quantification of pgc-1α by qPCR, showing the relative fold change of T. cruzi infected cells (I) vs. control cardiomyocytes (C) at 24 hpi. Values represents the mean of three independent biological replicates.

FIGURE 6. Analysis of AKT/mTORC1 pathway. (A) Western blot analysis of phosphorylated and non-phosphorylated AKT, mTOR, 4EBP1, p70S6k in control and T. cruzi infected cardiomyocytes at 24 hpi. GAPDH was used as loading control. (B) Densitometry analysis of western blots. Values represents the mean ± standard deviation of three or two independent biological replicates (∗p < 0.05).

FIGURE 7. Effect of rapamycin. (A) Western blot analysis of phosphorylated and non-phosphorylated p70S6k in control and infected cardiomyocytes at 24 hpi (C, I), vs. the same cells treated with rapamycin for 24 h before the infection (CR, IR). (B) Densitometric analysis of two independent experiments showing p-p70S6k protein expression normalized against the non-phosphorylated protein in control cells (∗p < 0.05). (C) Kinetic of QO2 response to oligomycin (2.0 μM), FCCP (1.0 μM) and AA-ROT (1.0 μM each) in control and T. cruzi infected cardiomyocytes cells 24 hpi with and without Rapamycin treatment (2.5 μM for 24 h before infection). Arrows indicates the injection of the different additions. Basal respiration (no drug added) and maximal respiration (QO2 after FCCP addition) with and without the rapamycin treatment were calculated. Each data point represent the mean ± SEM n = 5. ∗p < 0.05. (D) Relative fold changes of the mtDNA gene relative to the nuclear gene beta actin in infected cardiomyocytes (I), infected cardiomyocyte treated with rapamycin (2.5 μM) 24 h before infection (IR), and controls treated with rapamycin (2.5 μM) (CR) versus control cells at 24 hpi (C). Values are the mean…