Endocytosis machinery is required for beta1-adrenergic receptor-induced hypertrophy in neonatal rat cardiac myocytes

Abstract

Aims: Cardiac hypertrophy by activation of the beta-adrenergic receptor (beta AR) is mediated more efficiently by the beta1-AR than by the beta2-AR. We investigated the signalling mechanism by which the beta1-AR mediates cardiac hypertrophy.

Methods and results: Experiments were performed in cultured neonatal rat cardiomyocytes. Hypertrophy was determined by the protein/DNA content and atrial natriuretic factor transcription. Phosphorylation of Akt and Src was assessed by immunoblotting. Isoproterenol (ISO, 10 microM), a non-selective beta-AR agonist, caused selective downregulation of the beta1-AR (control beta1 vs. beta2: 35 vs. 65%, Bmax 78 +/- 4 fmol/mg; 4 h, 10 vs. 90%, 61 +/- 5 fmol/mg). Concanavalin A (Con A, 0.5 microg/mL), an inhibitor of endocytosis, prevented downregulation of beta1-ARs by ISO treatment (4 h, 35 vs. 65%, 73 +/- 8 fmol/mg), suggesting that beta1-ARs selectively undergo endocytosis. Interference with beta1-AR endocytosis by Con A, carboxyl terminal peptide of beta-AR kinase-1, dominant negative (DN) beta-arrestin-1, or DN dynamin inhibited beta-adrenergic hypertrophy, suggesting that the endocytosis machinery plays a key role in mediating beta-adrenergic hypertrophy. Activation of Akt by the beta1-AR was blocked by inhibition of the endocytosis machinery, suggesting that endocytosis mediates activation of Akt. Akt plays a critical role in beta-adrenergic hypertrophy, since DN Akt blocked ISO-induced hypertrophy. beta-Adrenergic activation of Akt is mediated by Src, which associates with the endocytosis machinery and is necessary and sufficient to mediate beta-adrenergic hypertrophy.

Conclusion: Activation of the endocytosis machinery is required for activation of Akt, which, in turn, critically mediates beta1-AR-induced cardiac hypertrophy.

Figure 1

Concanavalin A negatively affects isoproterenol-induced cardiac hypertrophy. (A) Cultured neonatal rat cardiac myocytes were treated with isoproterenol (10 µM) or foetal bovine serum (FBS, 20%) for 48 h in the presence or absence of concanavalin A (Con A, 0.5 µg/mL) and protein/DNA was determined. Experiments were performed in triplicate six times. **P < 0.01, ***P < 0.001 vs. control. (B) Cardiac myocytes were transfected with ANF-Luc and SV40-β-galactosidase. Twenty-four hours after transfection, myocytes were stimulated with isoproterenol (10 µM) in the presence or absence of concanavalin A (0.5 µg/mL) for an additional 24 h. Experiments were performed in triplicate six times. **P < 0.001 vs. respective control. (C) Myocytes were pre-treated with IBMX for 30 min and then stimulated with isoproterenol (10 μM) for 15 min in the presence or absence of concanavalin A (0.5 µg/mL). The level of cAMP was determined by RIA. The values of cAMP were adjusted by the mean protein content. Experiments were performed in triplicate three to five times. **P < 0.05 vs. control. (D) Myocytes were transfected with pFR-Luc (1 µg/mL), GAL4-CREB, and the SV40-β-galactosidase. After 24 h, cells were stimulated with isoproterenol for 24 h with or without concanavalin A. Luciferase activities were normalized by those of β-galactosidase. Experiments were performed in triplicate three times. **P < 0.05 vs. control, #P < 0.01 vs. isoproterenol.

Figure 2

Gβγ plays an essential role in isoproterenol-induced atrial natriuretic factor transcription. (A) Neonatal rat cardiac myocytes were transfected with atrial natriuretic factor-luciferase (ANF-Luc), expression plasmids encoding Gβ2 plus Gγ2 and the SV40-β-galactosidase. Experiments were performed in duplicate five times. *P < 0.05, **P < 0.001 vs. control. (B) Cardiac myocytes were transfected with ANF-Luc, an expression plasmid encoding β-ARK-CT, and the SV40-β-galactosidase. Some myocytes were stimulated with isoproterenol (10 µM) for 24 h. Experiments were performed in duplicate five times. **P < 0.001 vs. control. (C) Myocytes were transduced with control adenovirus or adenovirus harbouring β-ARK-CT. Myocytes were stimulated with isoproterenol (10 µM) for 48 h. Protein/DNA in control virus-transduced myocytes without isoproterenol stimulation was expressed as 1. Experiments were performed in triplicate three times. *P < 0.01 vs. control virus-transduced myocytes without isoproterenol stimulation.

Figure 3

Dominant negative β-arrestin-1 and dominant negative dynamin inhibit isoproterenol-induced cardiac hypertrophy. (A) Cardiac myocytes were transfected with ANF-Luc and SV40-β-galactosidase, together with expression vector encoding dominant negative β-arrestin-1. Twenty-four hours after transfection, myocytes were stimulated with isoproterenol (10 µM) for an additional 24 h. Luciferase activities normalized by β-galactosidase activities were expressed as relative to unstimulated samples transfected with 0.5 µg of pcDNA3.1 (Cont). Experiments were performed in duplicate six times. **P < 0.001 vs. control. (B) Myocytes were transduced with either control virus or adenovirus harbouring dominant negative dynamin. Myocytes were then stimulated with isoproterenol (10 µM) for 48 h. Protein/DNA in control virus-transduced myocytes without isoproterenol stimulation was expressed as 1. **P < 0.01 vs. control virus-transduced myocytes without isoproterenol stimulation. Experiments were performed in duplicate six times. (C) Cardiac myocytes were transfected with ANF-Luc and SV40-β-galactosidase. Twenty-four hours after transfection, myocytes were stimulated with isoproterenol (10 µM). Cardiac myocytes were also transduced with either control virus or adenovirus harbouring dominant negative dynamin. Myocytes were then stimulated with isoproterenol (10 µM) for 24 h. Luciferase activities were normalized by those of β-galactosidase. Experiments were performed in duplicate six times. *P < 0.05, **P < 0.01 vs. control.

Figure 4

Isoproterenol-induced activation of Akt is mediated by β₁-AR and concanavalin A-dependent mechanisms and plays an important role in mediating cardiac hypertrophy. (A) Cardiac myocytes were treated with isoproterenol (10 µM) for 30 min with or without betaxolol (10 µM) or ICI115,881 (10 µM), and activation of Akt was quantitated using an antibody against Serine 473-phosphorylated Akt. Lanes 3 and 4, betaxolol treated. Lanes 5 and 6, ICI115,881 treated. Lanes 2, 4, and 6, isoproterenol treated. (B) Cardiac myocytes were treated with isoproterenol (10 µM) for 10 or 30 min in the presence or absence of concanavalin A (Con A, 0.5 µg/mL), and the activated form of Akt was quantitated using an antibody against Serine 473-phosphorylated Akt. The results shown are representative of three experiments. (C) Myocytes were transduced with either control adenovirus (Lac Z) or adenovirus harbouring β-ARK-CT. Myocytes were stimulated with isoproterenol (10 µM) for 30 min, and activation of Akt was quantitated using phopho-specific antibody. In (A)–(C), the results shown are representative of three experiments. (D) Cardiac myocytes were transduced with control adenovirus or adenovirus harbouring dominant negative Akt (DN Akt). Myocytes were stimulated with isoproterenol (10 µM) for 48 h. Protein/DNA in control virus-transduced myocytes without isoproterenol stimulation was expressed as 1. Experiments were performed in duplicate six times. **P < 0.01 vs. control.

Figure 5

Src mediates isoproterenol-induced activation of Akt and cardiac hypertrophy. (A) Cardiac myocytes were stimulated with isoproterenol (10 µM), and activation of Src was determined by anti-Tyrosine 418 phospho-specific Src antibody. Levels of Src phosphorylation were expressed relative to unstimulated samples. Experiments were performed in triplicate three times. (B) Cardiac myocytes were stimulated with isoproterenol (10 µM) with or without PP1 (1 µM) for 10 or 30 min. Activated forms of Akt were quantitated using an antibody against phospho-Serine 473 Akt. The result shown is representative of three experiments. (C) and (D) Cardiac myocytes were treated with or without PP1 (10 nM) for 30 min and then stimulated with isoproterenol (10 µM) in the presence or absence of PP1 for an additional 48 h (C) or 1 h (D). (C) Protein/DNA in control myocytes without PP1 treatment or isoproterenol stimulation was expressed as 1. Experiments were performed in duplicate six times. (D) Re-organization of polymerized actin was detected by phalloidin staining. The result shown is representative of three experiments. (E) and (F) Cardiac myocytes were transfected with ANF-Luc and SV40-β-galactosidase, together with expression vector encoding dominant negative Src (E) or constitutively active Src (F). The total amount of plasmid was adjusted to 0.5 µg by adding pcDNA3.1. Twenty-four hours after transfection, some myocytes were stimulated with isoproterenol (10 µM) for 24 h. Luciferase activities normalized by β-galactosidase activities were expressed relative to unstimulated samples with 0.5 µg of pcDNA3.1 (Control). Experiments were performed in duplicate six to nine times.

Figure 6

A hypothesis regarding the role of the endocytosis machinery in β-adrenergic cardiac hypertrophy. Ligand binding to the β₁-AR leads to activation of Src and the endocytosis machinery. Activation of the endocytosis machinery causes activation of Akt, which in turn mediates cardiac hypertrophy. It is also possible that endocytosis and hypertrophy are regulated in parallel by common upstream signalling mechanisms. A, betaxolol; B, β-ARK-CT; C, dominant negative β-arrestin-1; D, dominant negative Src and PP1; E, concanavalin A and dominant negative dynamin.