Fig. 7

DNA binding activity of Abl1 on p53 targeted genes in the heart of doxorubicin-treated animals. Panels A: EMSA with nuclear protein extracts of cardiac tissue. We performed gel electrophoresis mobility shift assays with oligoprobes that harbour gene specific promoter binding sites for Abl1. Depicted are autoradiographs of heart nuclear protein complexes which were isolated from control and doxorubicin-treated animals. The 32P-labeled oligo probes are specific for Tcpn1, Pla2g2a and Sycp2, and competition assays were done with perfect matched (wt) and mutated probes (mut). Supershifts were carried out with two different antibodies which recognize Abl1 as detailed in the method section. In competition assays with 100-fold excess of the wt-probe the binding of the nuclear protein is significantly diminished whereas the mutated probes failed to shift the band. Furthermore, in super-shift assays, the antibody recognized Abl1 protein within the nuclear protein complex, and as detailed in the results section, variable amounts of the Abl1 protein were super-shifted. Panel B: Western blots of CDK1 and Abl1 protein. Immunoblots were performed with pooled nucler protein extracts (N = 3 animals) of cardiac tissue from control and doxorubicin-treated animals. We observed a clear dose-related decline in CDK1 protein after 8 h of treatment which returned to control values and slightly above after 24 and 72 h. The antibody for Abl1 recognized a 120 kDa and 140 kDa band. Initially, Abl1 expression was reduced and subsequently increased after 3 and 6 days. Note the dose-related differences in nuclear Abl1 protein expression. Panel C: Regulation of Abl1, p53 and p73 protein in the heart of control and doxorubicin-treated rats. We performed IHC with cardiac tissue of control and doxorubicin-treated animals (10 mg/kg for 6 days) and found Abl1 expression to be minimal to slight in cardiomyocytes of control animals. Conversely, its cytosolic expression in ventricular cardiomyocytes is marked (image 2), and we observed prominent nuclear as well as cytosolic expression of Abl1 in atrial cardiomyocytes (image 3). Cardiomyocytes of control animals do not express p53. In strong contrast, doxorubicin treatment elicited marked nuclear (image 2) and cytosolic expression of p53 in ventricular and atrial cardiomyocytes. Although p73 is not expressed in cardiomyocytes of control animals, it is strongly induced in atrial cardiomyocytes, in mast and endothelial cells (marked by the arrow, image 2). Strikingly, p73 expression is minimally in ventricular tissue and confined to a subpopulation of cardiomyocytes. Together, the strong induction of the proteins is highly suggestive for an activated Abl1-p53/p73 signaling axis in doxorubicin-dependent cardiomyopathies