igures with p,0.05 considered statistically significant. Results In the Myocardium, TrkA is Expressed Preferentially in CardioAGI 5198 site myocytes and TrkC in Cardiac Fibroblasts Early work by others demonstrated Trk receptors in cardiomyocytes. We used real-time PCR to determine basal levels of Trk receptors in primary cultures of adult and neonatal mouse CFs. We find that fibroblasts express TrkA and TrkC transcripts, TrkA predominating over TrkC, at levels comparable to those in cardiomyocytes. Adult and neonatal cardiac cells exhibited similar patterns of Trk expression. Visualization of TrkA and TrkC by immunofluorescence in cardiac fibroblasts is consistent with the mRNA findings. In light of this Trk expression we next determined transcript levels of the Trk ligands NGF, BDNF, and NT-3. NGF was most highly expressed in both adult and neonatal cardiac fibroblasts and myocytes, however BDNF transcripts were more prevalent in neonatal cells while NT-3 transcripts increased in adult mice. Dissociation curves and agarose gel analysis of qPCR products demonstrated the homogeneity 19239230 of amplicons. To determine TrkA and TrkC protein levels in the myocardium, we reacted heart tissue sections with antibodies against receptors TrkA or TrkC and against markers 9726632 of CF and cardiomyocytes, followed by fluorescent-labeled secondary antibodies. Then, we quantified specific fluorescence pixels in the receptors and markers dots, while viable cardiomyocytes are stained blue by the vital dye Hoechst 33342 and no PI staining. At least 200 cells/ well were counted per point. T cruzi Spurs Cardiac Fibroblast-Myocyte Crosstalk using NIH ImageJ software, revealing TrkA in both myocardial fibroblasts and myocytes but more prominently in the myocytes, and TrkC predominating in CFs. In validation of this expression pattern, visualization of stained cardiac tissue sections shows that, although TrkA merges with both MHC and vimentin, it prevails with the cardiomyocyte marker, quite the contrary of TrkC, which merges primarily with the fibroblast marker vimentin . T cruzi Infection of Cardiac Fibroblasts, but not Cardiomyocytes, Triggers a Robust Expression of Bioactive NGF Based on real-time PCR, basal level of NGF transcript in primary neonatal CFs is,3-fold higher than BDNF and,120fold higher than NT-3 transcript, analogous to primary cardiomyocyte expression except for NT-3, which is not detectable in the myocytes. As T cruzi interacts with both TrkA and TrkC, we tested whether NT expression in CFs and/or cardiomyocytes is altered in response to T cruzi infection. We find that T cruzi-infected CFs, but not cardiomyocytes, exhibit a dramatic increase in NGF expression. For example, NGF transcript in T cruzi-infected CFs increases by more than 9fold at 24-h post-infection whereas NGF protein in the culture supernatants is boosted by more than 80-fold at 72-h PI . This response is in sharp distinction to the one produced by T cruzi infection of primary cardiomyocytes and of the cardiomyocyte cell line H9c2, which upregulate NGF only slightly 
and not at all, respectively. Early microarray studies showed that T cruzi infection of primary cardiomyocytes upregulate NGF mRNA. Because BDNF and NT-3 transcripts, whether in CFs or myocytes, did not significantly change in response to T cruzi infection, they are not studied further here. Indirect immunofluorescence of cardiac fibroblasts/myocytes co-cultures validates the robust and cell-selective NGF upregulation. Co-staining for NGF
