Daniel Bernstein † , Mingming Zhao † , Jennifer Powers † , Brian K. Kobilka*, Giovanni Fajardo †

1. ß2-/- DOX- ß2-/- DOX+ WT DOX- WT DOX+ * P < 0.005 Phospho-GSK3 (OD x mm2) * FIGURE 7. Similar to WT, Akt activity in ß1-/- and ß1/ß2-/- mice does not change with doxorubicin. Mortality (%) WT, Dox 1µM b2-/-, Dox 1 mM Agonist: Neuregulin-1ß Antagonist: Anti-ergB2 ß1/ß2-/-, Dox 1µM 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 HYPOTHESIS ABSTRACT FIGURE 1. Her-2 (erbB2) is a member of the epidermal growth factor receptor family. Figure 9. ERK1 and ERK2 activities are also increased in ß2-/- mice after doxorubicin, although not as dramatically. FIGURE 6. Baseline activity of Akt is not changed in ß2-/- mice (not shown). However, after receiving doxorubicin, b2-/- mice show a 75% decrease in Akt activity. In contrast, in WT mice Akt activity does not change. 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. 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. *p < 0.001 * *p < 0.001 * METHODS • 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. RESULTS ERK2 (P44 MAPK) ERK1 (P42 MAPK) FIGURE 4. b2-/- mice show markedly enhanced doxorubicin cardiotoxicity. Additional deletion of the b1-receptor (b1b2-/-) fully rescues this toxicity 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. FIGURE 2. Her-2 agonists decrease (C) and antagonists increase (D) doxorubicin toxicity in cultured rat myocytes (Sawyer et al. Circ. 2002). Phospho-GSK3 (OD x mm2) 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 plays a role in cardiac development and myofillament organization • stimulation of erbB2 has anti-apoptotic effects • erbB2-/- mice develop cardiomyopathy • erbB2 modulates doxorubicin toxicity in cultured rat myocytes (Sawyer et al. Circ. 2002, Figure 2). FIGURE 3. Possible crosstalk between ß2-ARs and erbB2 FIGURE 8. p38 MAPK activity is increased 20-fold in ß2-/- mice after doxorubicin FIGURE 5. Baseline Her-2 expression is not altered in ß2-/- mice. CONCLUSIONS • 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. * *p < 0.001 SYBR-RT-PCR