ArfGAP with Dual Pleckstrin Homology Domains 2 Promotes Hypertrophy of Cultured Neonatal Cardiomyocytes

Abstract

Cardiomyocyte hypertrophy is regulated by several factors, including the ADP-ribosylation factor (Arf) family of small G proteins, among others. For instance, ArfGAP with dual pleckstrin homology domains 1 (Adap1) exerts an anti-hypertrophic effect in cultured cardiomyocytes. Its homologous protein, Adap2, is also expressed in the heart but its role remains elusive. To elucidate its function, we investigated the effects of adenoviral-mediated overexpression of Adap2 in cultured neonatal rat ventricular myocytes under both basal and pro-hypertrophic conditions, employing a range of microscopy and biochemical techniques. Despite minimal detection in neonatal rat hearts, Adap2 was found to be well expressed in adult rat hearts, being predominantly localized at the membrane fraction. In contrast to Adap1, overexpression of Adap2 provokes the robust accumulation of β1-integrin at the cellular surface of cultured cardiomyocytes. Interestingly, overexpressed Adap2 relocalizes at the sarcolemma and increases the size of cardiomyocytes upon phenylephrine stimulation, despite attenuating Erk1/2 phosphorylation and Nppa gene expression. Under these same conditions, cardiomyocytes overexpressing Adap2 also express higher level of detyrosinated tubulin, a marker of hypertrophic response. These findings provide new insights into the pro-hypertrophic function of Adap2 in cardiomyocytes.

Keywords: Adap1; Adap2; Centaurin-α; cardiomyocyte; hypertrophy; tubulin detyrosination.

Figure 1

Cardiac expression of homologous Adap proteins in rats. (A), Relative Adap1 and Adap2 mRNA expression levels in neonatal (2- to 3-day-old) and young adult (3- to 4-month-old) Sprague Dawley rat hearts measured by RT-qPCR and normalized to the Rpl30 reporter gene (n = 4 independent isolations). (B), Representative western blots showing Adap1, Adap2, and Gapdh (loading control) protein expression in neonatal rat ventricular myocytes (NRVM) compared to adult rat ventricular myocytes (ARVM) (n = 2 independent isolations). (C), Representative western blots showing the subcellular localization of Adap1, Adap2, Cadherin (membrane marker), and Gapdh (sarcoplasm marker) proteins from fractionated neonatal and young adult rat cardiac ventricles (T: Total, S: Sarcoplasm, M: Membranes). (D), Densitometric analysis of Adap1 and Adap2 protein expression in the corresponding subcellular fractions from young adult rat cardiac ventricles, as shown in (C), and relative to the total extract (n = 3 independent fractionations).

Figure 2

Impact of overexpressing Adap homologous proteins on cell surface expression of β1-integrin in cultured cardiomyocytes. (A), Representative western blots of Adap1 and Adap2 overexpression 72 h after neonatal rat ventricular myocytes (NRVMs) infection with control β-Galactosidase (β-Gal), Adap1, and Adap2 adenoviruses (multiplicity of infection = 50), respectively. (B), Representative western blots showing the cell surface expression of β1-integrin in NRVMs overexpressing either β-Gal, Adap1, or Adap2. (C), Densitometric analysis of β1-integrin cell surface expression relative to total β1-integrin expression in the corresponding conditions, as shown in (B) (n = 3 independent experiments).

Figure 3

Adap2 potentiates phenylephrine-induced cardiomyocyte remodeling. (A), Representative confocal immunofluorescence images showing the cellular localization, 72 h after infection with adenoviruses, of endogenous actin and overexpressed 3xFLAG-ADAP1 and 3xFLAG-Adap2 in neonatal rat ventricular myocytes (NRVMs) incubated with or without phenylephrine (PE, 50 µM) for 48 h (Scale bar = 10 µm). Nuclei and actin filaments were stained with DAPI (blue) and phalloidin-iFluor 647 (red), respectively. (B), Representative immunofluorescence images of α-actinin in NRVMs overexpressing Adap1 or Adap2, as compared to control cells overexpressing β-Galactosidase (β-Gal), and incubated with or without phenylephrine (PE, 50 µM) for 48 h, and using the Operetta high-content analysis system (Perkin Elmer) (Scale bars = 50 µm). Nuclei were stained with DAPI. (C), Cell size measurements of segmented NRVMs from the corresponding conditions are shown in (B) (n = 3 independent experiments).

Figure 4

Adap2 does not amplify the phenylephrine-induced hypertrophic signaling in cardiomyocytes. (A), Representative western blots showing the Erk1/2 pathway activation in neonatal rat ventricular myocytes (NRVMs) overexpressing control β-Galactosidase (β-Gal), Adap1, or Adap2 and incubated with or without phenylephrine (PE, 50 µM) for 48 h. (B), Densitometric analysis of Erk1/2 activation in the corresponding conditions, as shown in (A) (n = 3 independent experiments). (C), Gene expression analysis by RT-qPCR of specific cardiac hypertrophy markers (i.e., Myh7 and Nppa normalized to the Rpl30 reporter gene) in NRVMs overexpressing control β-Gal, Adap1, or Adap2 and incubated with or without PE (50 µM) for 48 h (n = 3 independent experiments).

Figure 5

Adap2 increases microtubule detyrosination in cardiomyocytes. (A), Representative western blots showing the expression of detyrosinated α-tubulin (Detyr-tubulin) in neonatal rat ventricular myocytes (NRVMs) overexpressing control β-Gal, Adap1, or Adap2 and incubated with or without phenylephrine (PE, 50 µM) for 48 h. (B), Densitometric analysis of detyrosinated α-tubulin relative to total α-tubulin in the corresponding conditions, as shown in (A) (n = 3 independent experiments). (C), Representative confocal immunofluorescence images showing the detyrosinated α-tubulin from microtubule cytoskeleton and α-actinin organization in NRVMs overexpressing control β-Gal, Adap1, or Adap2 and incubated with or without PE (50 µM) for 48 h (Scale bar = 10 µm). Nuclei were stained with DAPI (blue).