(A&B) The mtDNA level in PARP1+/- chagasic mice. Mice (WT and PARP1+/-) were infected with T. cruzi and monitored at 150 days’ post-infection. Representative gel images (A, n = 3 mice/group) show myocardial levels of 10 kb mtDNA and short 177-bp mtDNA and 96-bp nuDNA (GAPDH) fragments as controls. PCR amplification was performed for 28 cycles. Densitometry analysis was performed on PCR gels representing n? 6 mice/group, and density of the 10 kb mtDNA band, normalized against mtDNA and nuDNA fragments, is presented in B.a&b. (C-H) Effect of PARP1 inhibitor on cardiomyocytes infected with T. cruzi. Cardiac myocytes were infected with T. cruzi in presence or absence of PJ34 for 24 h. RT-qPCR was employed to evaluate the mRNA level for PARP1 and several components of the POLG replisome machinery, and data were normalized to GAPDH mRNA. (I-K) Cardiomyocytes were incubated for 24 with Tc in presence and absence of PJ34. ROS release was measured by an amplex red assay (I). MitoSOX red fluorescence detects mitochondrial O2•? level (J). Ratio of fluorescence intensity of J-monomers (green) to J-aggregates (red) indicates mitochondrial depolarization (K). Data in C-K were acquired by using three biological replicates (duplicate analysis per sample). Data in all bar graphs are plotted as mean value ± SEM, and statistical significance are marked as *WT.Tc vs. WT, and #WT.Tc vs. genetically-modified/infected or infected/PJ34-treated (#p<0.05, ***,###p<0.001).
C57/129SJ wild type (WT) and PARP1-/- mice were infected with Trypanosoma cruzi (10,000 Tc/mouse), and sacrificed at 150 days’ post-infection (pi) corresponding to chronic disease phase. (A) RT-qPCR evaluation of myocardial levels of PARP1 mRNA, normalized to GAPDH mRNA (n ? 5 mice/group, triplicate observations per mouse). (B-K) Myocardial tissue homogenates and fractionated organelles were prepared as detailed in Materials and Methods. Representative Western blot images (n = 3 mice/group) are shown for total heart homogenate levels of PARP1 and GAPDH (B), cytosolic levels of PARP1 and PAR with GAPDH as loading control (D&F), nuclear levels of PARP1 and PAR with Lamin B as loading control (D&G), and mitochondrial levels of PARP1 and PAR with COIV as loading control (D&H). Densitometry analysis was performed for all Western blot gels from n ? 6 mice/group, and data for PARP1 and PAR levels in total heart homogenates and cytosolic fractions (C,E,&I), nuclear fractions (E&J) and mitochondrial fractions (E&K) are presented. In bar graphs, data are plotted as mean value ± SEM. Statistical significance is marked as *WT.Tc vs. WT, &PARP1-/-.Tc vs. PARP1-/-, and #WT.Tc vs. PARP1-/-.Tc (**,&&,##p<0.01, ***,&&&,###p<0.001)..