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    PARP1/PAR role in mtDNA integrity (± T. cruzi)

    Because PARP1 is a DNA repair enzyme and mtDNA encodes essential components of the respiratory chain, we determined if mitochondrial translocation of PARP1/PAR preserved the mtDNA in chagasic myocardium. Long qPCR amplification of 10 kb mtDNA fragment showed the basal level of mtDNA content was not changed in WT, PARP1+/-, and PARP1-/- mice. The long mtDNA (vs. short mtDNA fragment) in chagasic WT, PARP1+/-, and PARP1-/- mice (vs. matched controls) was decreased by 70%, 46%, and 32%, respectively (Fig 2A, Fig 2B.a, and S2 Fig panels A & B.a, p<0.001). When compared to nuDNA fragment, the long mtDNA content was decreased by 85% and 30%, respectively, in the myocardium of chagasic WT and PARP1+/- mice (p<0.001) and no change was noted in PARP1-/- mice (Fig 2B.b, S2 Fig panel B.b). The protection of mtDNA content was associated with preservation of mtDNA-encoded genes’ expression at mRNA (ND4, CYTB, COI, ATP6, and ATP8, Fig 2C–2G) and protein (COI, Fig 2H & 2I) levels in chronically infected PARP1-/- (vs. control) mice. In comparison, WT.Tc (vs. WT) mice exhibited 21–51% decline in mtDNA-encoded gene expression and >80% decline in COI protein level (Fig 2C–2I, p<0.05). Mitochondrial (and total) levels of AMPK-like protein were equally increased in chagasic WT and PARP1-/- mice, and no changes in COIV (nuDNA encoded complex IV subunit) were noted in WT and PARP1-/- mice (Fig 2H, 2J & 2K). Our results suggest that a) Tc-induced PARP1/PAR were detrimental to mtDNA integrity and mtDNA-encoded gene expression, and b) depletion of PARP1/PAR prevented the loss in mtDNA content in chronic Chagas disease. Though a potential contamination with cytoplasmic material may explain the finding of AMPK-like protein in mitochondrial fractions, we believe that this observation indicates a novel role for AMPK in mitochondrial biogenesis.