5physiologic measurements suggest that the absence of erbB2 abrogates the activation of Erk1/2 by Ucn 2 and mitigates the potent enhancement of left ventricular function by this GPCR agonist. heart-specific erbB2 mutant mice. Here we demonstrate that erbB2, but not EGF receptor, is essential for MAPK activation induced by multiple GPCR agonists in cardiac myocytes. erbB2 is immunocomplexed with a GPCR and is transactivated after ligand treatment (8)]. Second, Erk1/2 plays a cardioprotective role against impaired erbB2 signaling and the cardiotoxicity observed with Sodium Tauroursodeoxycholate the chemotherapeutic agent doxorubicin, which is frequently used in conjunction with herceptin Sodium Tauroursodeoxycholate in breast cancer patients (11, 12). We have previously shown that erbB2-deficient cardiac myocytes are highly sensitive to doxorubicin (13). Heterozygous NRG1 (NRG1+/?) mutant mice are more susceptible to doxorubicin-induced heart failure. Doxorubicin treatment results in decreased phosphorylated Erk1/2 levels in Sodium Tauroursodeoxycholate NRG1+/? mice compared with controls (14). Finally, GPCR agonists are important in cardiac homeostasis (15). For example, impaired -AR stimulation with decreased expression and coupling of -AR subtypes is a hallmark of heart failure (15). Doxorubicin administration to 2-AR mutant mice results in altered Erk1/2 activation and decreased contractile function (16). Mice overexpressing MEK1, an upstream activator of Erk1/2, display enhanced cardiac contractility (17). Urocortin (Ucn) 2, acting through its GPCR, corticotropin-releasing factor receptor (CRFR) 2, is cardioprotective against ischemia by activation of the Erk1/2 (18) and enhances cardiac contractility in a heart failure model (muscle-specific LIM protein-deficient mice) (19). Results To determine the role of erbB2 in GPCR-mediated Erk1/2 activation, adult cardiac myocytes were prepared from control and heart-specific erbB2 mutant mice. Consistent with the idea that erbB2 is required for NRG1 signaling, we found that NRG1-induced Erk1/2 activation is abrogated in erbB2-deficient cardiac myocytes as compared with controls (Fig. 1= 10 controls; = 6 mutants) (= 8 controls; = 9 mutants) (= 10 controls; = 9 mutants) (= 5 controls; = 5 mutants). ?, 0.05; ???, 0.005. We then focused our subsequent studies on the interaction of erbB2 with 2-AR or CRFR2. To verify that the loss of Erk1/2 activation in response to the 2-AR agonist ISO and the CRFR2 agonist Ucn 2 was not due to a loss in receptor expression, Sodium Tauroursodeoxycholate we tested the ability of ISO and Ucn 2 to elevate cAMP levels in myocytes. Both ISO and Ucn 2 increased cAMP levels in control and erbB2-deficient myocytes (Fig. 1and and and and (25). Ucn 2 does not activate Erk1/2 in nontransfected SYF cells because they do not express CRFR2 (Fig. 8). Cotransfection of erbB2 and CRFR2 in SYF cells resulted in Erk1/2 activation in response to Ucn 2 (Fig. 4activation of Erk1/2 in the heart, wild-type and heart-specific erbB2 mutants were studied before and after infusion with Ucn 2. There were no significant differences between basal levels of Erk1/2 in wild-type and heart-specific erbB2 Sodium Tauroursodeoxycholate mutants. Administration of Ucn 2 to control mice resulted in a significant increase in Erk1/2 activation. In contrast, infusion of Ucn 2 to heart-specific erbB2 mutant mice did not result in an elevation of Erk1/2 phosphorylation (Fig. 5physiologic measurements suggest that the absence of erbB2 abrogates the activation of Erk1/2 by Ucn 2 and mitigates the potent enhancement of left ventricular function by this GPCR agonist. These results suggest that erbB2 is required for mediating the contractile response of a GPCR agonist, such as Ucn 2, correlate to the observations described above. Open in a separate window Fig. 5. erbB2 is required for Ucn 2-induced Erk1/2 activation and Rabbit polyclonal to HYAL2 left ventricular responsiveness. ( 0.05 wild type compared with erbB2 mutant basal or Ucn 2 responses; ?, 0.05 Ucn 2 response vs. basal wild type; #, 0.05 erbB2 mutant response to Ucn 2 compared with basal; @, 0.05 wild type response to Ucn 2 vs. erbB2 mutant response. Discussion In the present study we provide evidence that erbB2 is required for Erk1/2 activation induced by multiple GPCR ligands in the heart (Fig. 1(25). We show that EGF activates Erk1/2 in erbB2-deficient cardiac myocytes (Fig. 1model in which multiple parameters of left ventricular function were shown to be impaired in heart-specific erbB2 mutant mice under basal and GPCR agonist (Ucn 2)-stimulated conditions (Fig. 5). These data provide an correlate to our observations demonstrating that erbB2 is required for mediating certain aspects of GPCR-dependent cardiac homeostasis. It will be of interest to test our model with additional GPCR ligands in different cell types where erbB2 is known to be expressed. For example, we show that erbB2 forms a complex with 2-AR in the brain (Fig. 2test. Intracellular cAMP levels were measured by an RIA as described previously (18). Transient Transfection and Immunoprecipitation. COS7, B82L, and SYF cells were transfected with pcDNA3 or various expression plasmids. For immunoprecipitation, cells and tissues were lysed with RIPA buffer, precleared by incubation with protein A/G-agarose.