Oxidative Response Networks in Chagasic Cardiomyopathy

Chagas disease (CD) caused by Trypanosoma cruzi (Tc) represents third greatest tropical disease burden as it affects >7 million people and causes >17,000 deaths and ~$8.0 billion in health care expenses and lost productivity every year. Ca2+-mitochondria disorders and oxidative/inflammatory stress are hallmarks of chagasic cardiomyopathy and heart failure. Our preliminary studies have identified a new dimension of CD pathogenesis. We found that alternative splicing (AS) of Ca2+ cycling genes specifically Atp2b1 (PMCA1) and Atp2a3 (SERC3) that signal nuclear Ca2+ and gene transcription, and alternative polyadenylation (APA) of mitochondria function related mRNAs (mf-mRNAs) were increased in CD. Rbfox2 (encodes an RNA binding protein of FOX family) switch from wild-type to exon1/2-skipping isoform was linked to AS events in CD, Rbfox2 knockdown led to similar AS pattern and delayed Ca2+ transients as were noted in infected cardiomyocytes, while PARP1 (polyADP-ribose polymerase) deletion improved the Atp2b1/Atp2a3 splicing pattern. Further, we identified that RBFOX2 binds to polyA tail nascent mRNAs, polyADP-ribosylation of CPSF73 and PAP that have essential function in polyA site selection and polyA synthesis was increased in CD, and Parp1-/- mice exhibited improved myocardial mf-mRNAs’ levels, and mitochondrial and LV function that were compromised in Tc-infected wild-type mice. Building on these and other preliminary data, we hypothesize that PARP1/RBFOX2 dictated RNA processing events create an environment for nCa2+-mitochondrial disequilibrium and resultant oxidative/inflammatory stress and LV dysfunction in CD. Using Parp1+/+, Parp1-/-, and Parp1CKO (newly generated by us) mice that exhibit none, whole body, and cardiomyocyte-specific PARP1 deletion, respectively, and advanced molecular tools, proposed studies will identify the mechanisms by which maturation, stability, and cellular function of mRNAs are dysregulated in CD. Studies in aim 1 will examine the time-course of Rbfox2 isoform switch, regulatory mechanisms involved in AS in CD and determine if enhancing RBFOX2/PARP1 balance can restore nCa2+/transcription equilibrium, and cardiomyocytes function in Tc infection. Studies in aim 2 will examine the novel mechanisms by which PARP1 disturbs site-specific function of CPSF73/PAP/RBFOX2 to influence the polyadenylation and stability of mf-mRNAs in CD. The proposed work is highly innovative as it will demonstrate for the first time the mechanisms by which pre-mRNAs processing and maturation are disturbed in infectious cardiomyopathies. The findings of global AS/APA networks in healthy and diseased heart will present promising and exciting fields for future research. The impact of the proposed studies is very high, as testing of PARP1 inhibitors, RBFOX2 enhancers and antisense oligonucleotides (ASOs) will offer broad-range new therapeutics targeting splicing and polyadenylation functions with relevance to preserving the nCa2+-mitochondrial homeostasis in diseased heart.