Inhibition of ER Stress by 2-Aminopurine Treatment Modulates Cardiomyopathy in a Murine Chronic Chagas Disease Model

Figures

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  Fig. 1.. Treatment with 2AP during indeterminate stage improved the morphology of the heart during murine chronic CD at 100 DPI (n=5/group). (A) A bar graph representing the ultrasound analysis of the hearts at 100 DPI. Ultrasound analysis of the hearts both at diastole (d) and systole (s) condition showed a significant decrease in the left ventricle internal diameter (LVID) and significant increase in the right ventricle internal diameter (RVID) in the infected mice compared to uninfected mice at 100 DPI. However, the infected mice treated with 2AP displayed significantly modified LVID (s) and RVID (both d and s) compared to infected untreated mice at 100 DPI. (B) Representative cardiac ultrasound images of mice (uninfected, infected, uninfected+2AP treated and infected 2AP treated). Statistical significance compared to uninfected untreated mice and infected untreated mice are represented by **p≤0.01, ***p≤0.001, and #p≤0.05 or ##p≤0.01, respectively.

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  Fig. 2.. 2AP inhibits cardiac ER stress in chronic CD mice (n=10/group). (A) Immunoblot analysis demonstrated a significant decrease in the levels of ER stress markers BIP, p-elF2α and CHOP in the hearts of infected mice treated with 2AP compared to infected untreated mice at 120 DPI. (B) Fold changes in the protein levels of BIP, pELF2α and CHOP were normalized to guanosine nucleotide dissociation inhibitor (GDI) expression and represented as the bar graph. (C) qPCR analysis demonstrated a significant decrease in the mRNA levels of ER stress response genes such as BIP, PERK, Erol-α, ATF4 and CHOP in the hearts of infected mice treated with 2AP compared to infected untreated mice at 120 DPI. Statistical significance compared to uninfected untreated mice and infected untreated mice are represented by *p≤0.05, **p≤0.001, and #p≤0.05 or ##p≤0.01, respectively.

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  Fig. 3.. Treatment with 2AP during indeterminate stage decreased apoptotic signaling in chronic T. cruzi infected mice (n=10/group). qPCR analysis demonstrated a significant increase in the cardiac mRNA levels of anti-apoptotic BCL-Xl, and significant decrease in mRNA levels of pro-apoptotic markers TNF-R1 and BAK in the hearts of infected mice treated with 2AP compared to infected untreated mice at 120 DPI. Statistical significance compared to uninfected untreated mice and infected untreated mice are represented by **p≤0.01, ***p≤0.001, and #p≤0.05 or ##p≤0.01, respectively.

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  Fig. 4.. Inhibition of cardiac ER stress by 2AP modified mitochondrial function by upregulating anti-oxidant genes during chronic T. cruzi infection (n=10). (A) qPCR analysis demonstrated a significant increase in the cardiac mRNA levels of genes involved in mitochondrial functions such as COX3, CYTB, ATP6 and ND1in infected mice treated with 2AP compared to infected untreated mice at 120 DPI. (B) 2AP treatment significantly upregulates mRNA levels of anti-oxidant genes such as MNSOD, CAT, GPX1, GPX2, PGC1α and GSK3 in the hearts of infected mice treated with 2AP compared to infected untreated mice at 120 DPI as demonstrated by qPCR analysis. Statistical significance compared to uninfected untreated mice and infected untreated mice are represented by *p≤0.05, ***p≤0.001, and #p≤0.05 or ##p≤0.01, respectively.

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  Fig. 5.. Treatment with 2AP during the indeterminate stage reduced cardiac inflammation in chronic T. cruzi infected mice (n=10). (A) Immunoblot analysis demonstrated a significant decrease in the level of TNFα in the hearts of infected mice treated with 2AP compared to infected untreated mice at 120 DPI. (B) Fold changes in the protein levels of TNFα were normalized to GDI expression and represented as the bar graph. (C) qPCR analysis demonstrates a significant decrease in the mRNA levels of TNF-A in the hearts of infected mice treated with 2AP compared to infected untreated mice at 120 DPI. Statistical significance compared to uninfected untreated mice and infected untreated mice are represented by *p≤0.05, ***p≤0.001, and #p≤0.05 or ##p≤0.01, respectively.

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  Fig. 6.. Amelioration of cardiac pathology (inflammation, fibrosis, degenerating cardiac muscle fibre and accumulated lipid droplets) by 2AP in mice during chronic infection at 120 DPI (n=8, minimum five images/section were analyzed). (A) H&E staining displayed significantly more damage (inflammation – long black arrow, fibrosis – red arrow head, degenerating cardiac muscle fibre – black arrow head (See Supplementary Fig. 3) and presence of adipocytes or lipid granules – red long arrow (See Supplementary Fig. 3)) in infected mice hearts compared to the hearts of uninfected mice. However, infected 2AP treated mice displayed significantly reduced damage (p≤0.01) compared to infected untreated mice (bar −100 um). Additional images are presented as Supplementary Fig. 3. (B) The photomicrographs of trichrome Masson stained hearts sections demonstrated significant (p≤0.01) increase in cardiac fibrosis and collagen deposition in infected mice compared to uninfected mice. Infected 2AP treated mice showed significantly reduced damage (p≤0.01) compared to infected untreated mice (bar −100 um). (C) qPCR analysis demonstrated a significant increase in the cardiac mRNA levels of collagen I and III,…