Enhancement of the recycling and activation of beta-adrenergic receptor by Rab4 GTPase in cardiac myocytes

Abstract

We investigate the role of Rab4, a Ras-like small GTPase coordinating protein transport from the endosome to the plasma membrane, on the recycling and activation of endogenous beta-adrenergic receptor (beta-AR) in HL-1 cardiac myocytes in vitro and transgenic mouse hearts in vivo. Beta1-AR, the predominant subtype of beta-AR in HL-1 cardiac myocytes, was internalized after stimulation with isoproterenol (ISO) and fully recycled at 4 h upon ISO removal. Transient expression of Rab4 markedly facilitated recycling of internalized beta-AR to the cell surface and enhanced beta-AR signaling as measured by ISO-stimulated cAMP production. Transgenic overexpression of Rab4 in the mouse myocardium significantly increased the number of beta-AR in the plasma membrane and augmented cAMP production at the basal level and in response to ISO stimulation. Rab4 overexpression induced concentric cardiac hypertrophy with a moderate increase in ventricle/body weight ratio and posterior wall thickness and a selective up-regulation of the beta-myosin heavy chain gene. These data provide the first evidence indicating that Rab4 is a rate-limiting factor for the recycling of endogenous beta-AR and augmentation of Rab4-mediated traffic enhances beta-AR function in cardiac myocytes.

FIGURE 1. Cell surface expression and internalization of β-AR in HL-1 cardiac myocytes

A, cell surface expression of total β-AR, β₁-AR, and β₂-AR measured by intact cell ligand binding. HL-1 myocytes were cultured in 12-well dishes and incubated with [³H]CGP12177 as described under “Experimental Procedures.” Cell surface expression of β₁-AR and β₂-AR was determined in the presence of atenolol and ICI118,551, respectively. Nonspecific binding obtained in the presence of alprenolol was substracted from the value presented. The data shown are the percentage of the total β-AR binding (2242 ± 151 cpm) and are presented as the means ± S.E. of six separate experiments. B, time-dependent internalization of β-AR. HL-1 cells were exposed to 10 μM ISO at 37 °C for different time periods and the β-AR binding sites left at the cell surface were determined as described in A. C, dose-dependent internalization of β-AR. HL-1 cells were stimulated with different concentrations of ISO for 30 min and the cell surface expression of β-AR was measured. In B and C, the data are presented as percentage of β-AR obtained in absence of ISO (2382 ± 212 cpm) and presented as means ± S.E. of six independent determinations. *, p < 0.05 versus the data obtained in the absence of ISO.

FIGURE 2. Effect of transient expression of Rab4 on the recycling of internalized β-AR in HL-1 myocytes

A, immunoblot analysis of Rab4 expression. HL-1 myocytes were transiently transfected with FLAG-tagged Rab4 or the pcDNA3 vector (Control). Rab4 expression was determined by Western blotting using anti-FLAG (upper panel) and Rab4 antibodies (lower panel). Rab4 antibodies detected both exogenous and endogenous Rab4. B, effect of Rab4 on the recycling of ISO-mediated internalized β-AR. HL-1 cells were transiently transfected for 48 h with Rab4 (squares) or the pcDNA3 vector (triangles), stimulated with ISO (10 μM) for 30 min, and allowed to recover for 15, 30, 60, 120, and 240 min at 37 °C. Cell surface expression of β-AR was measured by ligand binding as described in the legend of Fig. 1. β-AR expression at the cell surface are 2382 ± 212 cpm in the absence of ISO and 952 ± 317 cpm after 30 min exposure to ISO. The data shown are the percentage of β-AR recycling in the cell transfected with pcDNA3 and recovered for 240 min after ISO stimulation and presented as the means ± S.E. of six independent experiments. *, p < 0.05 versus control at the same time points.

FIGURE 3. Effect of transient expression of Rab4 on ISO-stimulated cAMP production in HL-1 myocytes

HL-1 myocytes cultured on 12-well plates were transiently transfected with FLAG-tagged Rab4 (squares) or the pcDNA3 vector (triangles) and stimulated with increasing concentrations of ISO (from 10⁻⁹ to 10⁻⁵ M) for 10 min. cAMP concentrations were determined as described under “Experimental Procedures.” Rab4 expression did not significantly influence cAMP production at basal level (Rab4-transfected cells: 381 ± 136 and pcDNA3-transfected cells: 452 ± 67 pmol/well, n = 4, p > 0.05). The data shown are fold increased over the basal and presented as the means ± S.E. of four independent determinations. *, p < 0.05 versus control at the same ISO concentration.

FIGURE 4. Effect of transgenic expression of Rab4 in the myocardium on the density of β-AR in the plasma membrane

A, immunoblot analysis of Rab expression levels in hearts from transgenic mice overexpressing Rab4 and NTG mice. Fifty μg of total homogenate prepared from ventricles of four Rab4 and four NTG mice was separated by 12% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. Rab4 expression levels were detected by Western blotting using anti-Rab4 and anti-FLAG antibodies and Rab1 and Rab5 expression by Rab-isoform specific antibodies. B, immunoblot analysis of the ER marker calregulin, the Golgi marker GM130, and the plasma membrane marker Na⁺-K⁺-ATPase in cytosolic and membrane fractions. Myocardial cytosolic and plasma membrane fractions were prepared by homogenization and centrifugation at 10,000 × g as described under “Experimental Procedures.” Twenty-five μg of protein from the cytosolic and membrane fractions was analyzed. The blots shown are representatives of three separate experiments. C, specific [¹²⁵I]iodocyanopindolol binding to membrane fractions prepared from Rab4 and NTG mouse ventricles. Twenty-five μg of membrane protein was incubated with the nonselective β-AR ligand [¹²⁵I]iodocyanopindolol (400 pM) in a total volume of 500 μl of binding buffer for 1 h. Specific binding was performed in duplicate and nonspecific binding determined in the presence of 20 μM alprenolol. Receptor density was expressed as fmol/mg membrane protein and presented as the means ± S.E. (n = 6). *, p < 0.05 versus NTG. D, Expression of G_s, G_i, Gβ, and GRK2 in four Rab4 and four NTG mouse hearts. Fifty μg of total homogenate prepared from mouse ventricles was analyzed.

FIGURE 5. cAMP production in Rab4 transgenic and NTG mice

A, ventricular membrane fractions from Rab4 transgenic and NTG mice were prepared as described in the legend of Fig. 4. cAMP production in response to stimulation with ISO (10 μM) and forskolin (100 μM) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM) was measured as described under “Experimental Procedures.” The data shown are the means ± S.E. of four Rab4 transgenic and four NTG mice. *, p < 0.05 versus respective NTG. B, ISO-induced cAMP production in NTG and Rab4 transgenic mice. The data are presented as fold increase over the basal values in ventricle membrane fractions (n = 4; *, p < 0.05 versus NTG).

FIGURE 6. Effect of Rab4 overexpression on cardiac hypertrophy and function

A, ventricle/body weight in Rab4 transgenic and NTG mice. B, representative cross-sections of hearts stained with hematoxylin and eosin from Rab4 transgenic and NTG mice at 22 weeks of ages. The heart from Rab4 transgenic mouse shows concentric remodeling with thicker left ventricular wall and smaller left ventricular chamber. C, histological analysis of left ventricles stained with Masson’s trichrome from transgenic and NTG mice, showing decreased myocyte number per microscopic field and enlarged myocyte size in Rab4 transgenic mice. D, effect of Rab4 overexpression on the expression of cardiac hypertrophy-associated genes. Representative RNA dot blots from two NTG and two Rab4 transgenic mice are shown (left panel). Total RNA was extracted from ventricles of NTG and transgenic mice. RNA dot blotting was carried out as described under “Experimental Procedures” using 3 μg of RNA per dot. Quantitative data of mRNA expression normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (right panel) are shown. E, echocardiographic analysis of posterior wall thickness of NTG and Rab4 transgenic mouse hearts. F, left ventricular fractional shortening in NTG and Rab4 transgenic mice. Values are the means ± S.E. (n = 7–12; *, p < 0.05 versus NTG).