1. Disregulation of Akt Associated with Enhanced Cardiotoxicity in ß2-Adrenergic Receptor (AR) Knockout Mice: Possible Mechanism of Crosstalk Between ß-Receptors and Her2 in Anthracycline Cardiotoxicity Daniel Bernstein †, Mingming Zhao †, Jennifer Powers †, Brian K. Kobilka*, Giovanni Fajardo † Departments of Pediatrics †, Molecular and Cellular Physiology* and Howard Hughes Medical Institute*, Stanford University, Stanford, CA Recent data suggest that ß-AR subtypes couple differentially to signaling pathways regulating cardiac function (chronotropy, inotropy) and remodeling (hypertrophy, apoptosis). We have previously shown that ß2-AR-/- mice have markedly enhanced cardiotoxicity to the anthracycline doxorubicin (DOX), an effect that is rescued by the additional deletion of the ß1-AR. Enhanced cardiotoxicity is also associated with administration of anti- Her2 (erbB2) antibodies (Herceptin). To examine potential crosstalk between the ß2-AR and Her2 pathways, we examined Her2 signaling in ß2-/- and WT mice at baseline and after administration of doxorubicin (DOX) 15 mg/kg i.v. Within 30 min. of DOX, 100% of ß2-/- mice died compared with 0% of WT. In ß2-/- mice, baseline expression of Her2 mRNA (quantitative RT-PCR) and activation (phospho- protein by Western blot) were unchanged compared to WT. Baseline activation of Akt (IP kinase assay with phospho- GSK3ß as substrate) was also not significantly altered. However, 30 min. after DOX, Akt activity was reduced by 75% in ß2-/- mice compared with no change in WT. ERK1 activity increased 2-fold in ß2-/- after DOX compared with a 50% decrease in WT. ERK2 activity also increased 2-fold compared with no change in WT. Thus, although Her2 receptor expression and activation is not altered in ß2-/- mice, there is disregulation of Akt, a point of convergence between these two pathways. Decreased Akt activity may predispose cardiomyocytes to apoptosis in response to cardiotoxic stimuli. ABSTRACT RESULTS CONCLUSIONS FIGURE 4.  2-/- mice show markedly enhanced doxorubicin cardiotoxicity. Additional deletion of the  1-receptor (  2-/-) fully rescues this toxicity Mortality (%) SYBR-RT-PCR FIGURE 5. Baseline Her-2 expression is not altered in ß2-/- mice. FIGURE 7. Similar to WT, Akt activity in ß1-/- and ß1/ß2-/- mice does not change with doxorubicin. Phospho-GSK3 (OD x mm 2 ) Agonist: Neuregulin-1ßAntagonist: Anti-ergB2 INTRODUCTION Patients receiving both doxorubicin (Adriamycin) and trastuzumab (Herceptin) are at increased risk for cardiotoxicity Herceptin is a monoclonal antibody directed against the Her-2 (erbB2) receptor tyrosine kinase erbB2 is a member of the epidermal growth factor receptor family (Figure 1)erbB2 is a member of the epidermal growth factor receptor family (Figure 1) erbB2 plays a role in cardiac development and myofillament organizationerbB2 plays a role in cardiac development and myofillament organization stimulation of erbB2 has anti-apoptotic effectsstimulation of erbB2 has anti-apoptotic effects erbB2-/- mice develop cardiomyopathyerbB2-/- mice develop cardiomyopathy erbB2 modulates doxorubicin toxicity in cultured rat myocytes (Sawyer et al. Circ. 2002, Figure 2).erbB2 modulates doxorubicin toxicity in cultured rat myocytes (Sawyer et al. Circ. 2002, Figure 2). FIGURE 1. Her-2 (erbB2) is a member of the epidermal growth factor receptor family. FIGURE 2. Her-2 agonists decrease (C) and antagonists increase (D) doxorubicin toxicity in cultured rat myocytes (Sawyer et al. Circ. 2002). FIGURE 3. Possible crosstalk between ß2-ARs and erbB2 METHODS Abnormalities in Her-2 or distal signaling pathways (e.g. Akt) are present in ß2-/- mice and may be responsible for their increased doxorubicin cardiotoxicity. HYPOTHESIS  3 mo. old male mice (WT, ß1-/-, ß2-/- and ß1/ß2-/-) treated with doxorubicin (15 mg/kg) i.v. and sacrificed at 30 min.  Her-2 expression measured using quantitative SYBR Green RT- PCR  Baseline and post-doxorubicin Akt activity measured using immunoprecipitation assay with GSK-3ß as substrate  MAPK (Phospho-ERK1 and 2, p38) measured by Western blot  Myocytes isolated from WT and knockout mice treated with 1 µM doxorubicin and TUNEL assay performed at 24 h. FIGURE 6. Baseline activity of Akt is not changed in ß2-/- mice (not shown). However, after receiving doxorubicin,  2-/- mice show a 75% decrease in Akt activity. In contrast, in WT mice Akt activity does not change. * * P < 0.005 Phospho-GSK3 (OD x mm 2 ) * FIGURE 8. p38 MAPK activity is increased 20-fold in ß2-/- mice after doxorubicin *p < 0.001 ß2-/- mice manifest a dramatic increase in cardiotoxicity after doxorubicin. This cardiotoxicity is fully rescued by the additional deletion of the ß1-AR. Her2 receptor expression is not altered in ß2-/- mice ß2-/- mice show disregulation of Akt activation, a point of convergence between the Her2 and ß-AR pathways. This disregulation tracks with the toxicity phenotype, i.e. it is not present in ß1-/- or ß1/ß2-/- mice. MAPK activity (p38,ERK1/2) is increased in ß2-/- mice after doxorubicin. Myocytes isolated from ß2-/- mice show increased apoptosis after doxorubicin. Decreased Akt activity in response to stress may leave ß2-/- myocytes vulnerable to cardiotoxic stimuli. Figure 9. ERK1 and ERK2 activities are also increased in ß2-/- mice after doxorubicin, although not as dramatically. * *p < 0.001 * ERK2 (P44 MAPK)ERK1 (P42 MAPK) Figure 10. Myocytes isolated from ß2-/- mice show increased TUNEL staining after doxorubicin vs. WT. In contrast, ß1/ß2-/- myocytes show decreased TUNEL staining after doxorubicin. WT, Dox 1µM  2-/-, Dox 1  M ß1/ß2-/-, Dox 1µM ß2-/- DOX-ß2-/- DOX+WT DOX-WT DOX+