Mouse hearts subjected to repeated transplant surgery and ischemia/reperfusion injury develop substantial interstitial and perivascular fibrosis that was spatially associated with dysfunctional activation of fetal SMA gene expression in graft ventricular cardiomyocytes. Compared to cardiac fibroblasts where nuclear levels of the Sp1 and Smad 2/3 transcriptional activating proteins increased markedly after transplant injury, the most abundant SMA gene activating protein in cardiomyocyte nuclei was serum response factor (SRF). Additionally, cardiac intercalated discs in heart grafts contained substantial deposits of Pur, a mRNA-binding protein and known negative modulator of SRF-activated SMA gene transcription. Activation of fetal SMA gene expression in perfusion-isolated adult cardiomyocytes was linked to elevated binding of a novel protein complex consisting of SRF and Pur to a purine-rich DNA element in the SMA promoter called SPUR previously shown to be required for induction of SMA gene transcription in injury-activated myofibroblasts. Increased SRF binding to SPUR DNA plus one of two nearby CARG box consensus elements was observed in SMA-positive cardiomyocytes in parallel with enhanced Pur:SPUR protein:protein interaction. The data suggest that de novo activation of the normally silent SMA gene in reprogrammed adult cardiomyocytes is linked to elevated interaction of SRF with fetal-specific CARG and injury-activated SPUR elements in the SMA promoter as well as the appearance of novel Pur protein complexes in both the nuclear and cytosolic compartments of these cells.