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Fig 4. Mitochondrial respiration and ROS production in chronically infected WT and PARP1-/- mice.

WT and PARP1-/- mice were harvested at 150 days’ post-infection. (A-D) Cardiac myofiber bundles (~2 mg) were permeabilized, and mitochondrial respiratory function was measured using an Oxygraph-2k respirometer. After recording the state 4 respiration with addition of CI substrates (glutamate/pyruvate/malate), ADP was added, and state 3 respiration with electron input from complex CI was recorded (A). Rotenone (inhibits complex I) and CII substrate (succinate) were added to record CII-supported state 3 respiration (B). Competence of the outer mitochondrial membrane was assessed with addition of cytochrome c (C). The CII driven respiratory control ratio (RCR) is presented in D. (E-G) Isolated cardiac mitochondria were incubated in presence of CI (E) and CII (F) substrates, and the rate of H2O2 production was recorded. The total H2O2 levels in heart homogenates is shown in G. Data in all bar graphs are plotted as mean value ± SEM (n ? 5 mice/group, triplicate observations per sample). Statistical significance are marked as *WT.Tc vs. WT, &PARP1-/-.Tc vs. PARP1-/-, and #WT.Tc vs. PARP1-/-.Tc (*,&,#p<0.05, **,##p<0.01).


Mitochondrial respiratory dysfunction can result in increased release of electrons to molecular O2 and O2? formation. Next, we determined if PARP1 depletion arrested the mtROS generation in chagasic heart. Isolated cardiac mitochondria of WT.Tc (vs. WT) mice exhibited 55% and 114% increase in CI- and CII-dependent H2O2 release, respectively, and myocardial H2O2 level was increased by >7-fold in chagasic WT mice (Fig 4E–4G, all, p<0.05). The PARP1-/- mice exhibited non-significant changes in Tc-induced mtROS release, and only a modest increase in myocardial H2O2 level (Fig 4G). Together, the results presented in Fig 4, along with those presented in Fig 2 and Fig 3, suggest that a) PARP1 effects on mtDNA content contributed to a decline in mitochondrial OXPHOS capacity and an increase in mtROS production, and b) PARP1 depletion was beneficial in preserving the mitochondrial health in chagasic myocardium.

We confirmed the effects of PARP1/PAR on mtDNA content and antioxidant/oxidant balance by using a small molecule inhibitor of PARP1. Mice were infected with T. cruzi and then treated with a selective PARP1 inhibitor (PJ34, 12.5 mg/kg) as described in Materials and Methods. The PJ34 treatment abolished the >2-fold increase in PARP1 mRNA and protein levels observed in the myocardium of chagasic mice (Fig 5A–5C, p<0.001), as has also been observed previously in brain endothelial cells [25]. The Tc-induced increase in PARylation level, a measure of PARP1 activity, was also controlled by PJ34 in a dose dependent manner (Fig 5D & 5E). Further, PJ34 treatment resulted in >90% recovery of the mtDNA content (Fig 5F & 5G.a&b), 54–100% control of myocardial (Fig 5H–5K) and plasma (Fig 5L & 5M) levels of H2O2, 3-nitrotyrosine, protein carbonyls, and lipid hydroperoxides, and 21–64% recovery of the myocardial and plasma antioxidant capacity (Fig 5N & 5O) in chagasic mice. No effects of PJ34 were noted on the parasite load in chagasic/treated (vs. chagasic/untreated) mice (Fig 5P). Similar to the findings in mice, treatment of cardiac myocytes with PJ34 controlled the T. cruzi induced increase in the levels of PARP1 mRNA (S2C Fig) and cellular and mitochondrial oxidative stress (S2I and S2J Fig), and the decline in the expression of the components of the mtDNA replication machinery (S2D–S2H Fig) and mitochondrial health measured by the JC1 red/green ratio (S2K Fig). Together, these results suggest that PJ34-dependent control of PARP1/PAR was effective in preserving the mtDNA content, mitochondrial health, and antioxidant/oxidant balance that otherwise were profoundly disturbed in the cardiomyocytes and murine heart by T. cruzi infection.


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