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. 2007 Jun 12;104(24):10140-5.
doi: 10.1073/pnas.0701099104. Epub 2007 May 30.

The A-kinase anchoring protein (AKAP)-Lbc-signaling complex mediates alpha1 adrenergic receptor-induced cardiomyocyte hypertrophy

Aline Appert-Collin  1 , Susanna Cotecchia, Monique Nenniger-Tosato, Thierry Pedrazzini, Dario Diviani
Affiliations

The A-kinase anchoring protein (AKAP)-Lbc-signaling complex mediates alpha1 adrenergic receptor-induced cardiomyocyte hypertrophy

Aline Appert-Collin et al. Proc Natl Acad Sci U S A. .

Abstract

In response to various pathological stresses, the heart undergoes a pathological remodeling process that is associated with cardiomyocyte hypertrophy. Because cardiac hypertrophy can progress to heart failure, a major cause of lethality worldwide, the intracellular signaling pathways that control cardiomyocyte growth have been the subject of intensive investigation. It has been known for more than a decade that the small molecular weight GTPase RhoA is involved in the signaling pathways leading to cardiomyocyte hypertrophy. Although some of the hypertrophic pathways activated by RhoA have now been identified, the identity of the exchange factors that modulate its activity in cardiomyocytes is currently unknown. In this study, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor activity, is critical for activating RhoA and transducing hypertrophic signals downstream of alpha1-adrenergic receptors (ARs). In particular, our results indicate that suppression of AKAP-Lbc expression by infecting rat neonatal ventricular cardiomyocytes with lentiviruses encoding AKAP-Lbc-specific short hairpin RNAs strongly reduces both alpha1-AR-mediated RhoA activation and hypertrophic responses. Interestingly, alpha1-ARs promote AKAP-Lbc activation via a pathway that requires the alpha subunit of the heterotrimeric G protein G12. These findings identify AKAP-Lbc as the first Rho-guanine nucleotide exchange factor (GEF) involved in the signaling pathways leading to cardiomyocytes hypertrophy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
PE selectively up-regulates AKAP-Lbc expression in cardiomyocytes. (A) Real-time PCR analysis of the mRNA expression of various Rho-GEFs was performed on total RNA samples extracted from rat NVMs that were left untreated or that were stimulated for 24 h with 10−4 M PE. (B) Northern blots were prepared with 20 μg of total RNAs extracted from rat NVMs that were left untreated or were stimulated for 6 h and 24 h with 10−4 M PE. Blots were hybridized with cDNA probes corresponding to the first 500 nucleotides of the rat AKAP-Lbc. (C) Real-time PCR analysis of AKAP-Lbc mRNA expression was performed on total RNA samples extracted from rat NVMs that were left untreated or were stimulated for 6 h and 24 h with 10−4 M PE or isoproterenol. (D) Real-time PCR analysis of AKAP-Lbc and ANF mRNA expression was performed on total RNA samples extracted from C57B6 mice that were left untreated or infused for 14 days with 100 μg/kg/day of PE. (E) shRNA-mediated knockdown of AKAP-Lbc expression. NVMs were infected with lentiviruses encoding GFP (control), or lentiviruses encoding both GFP and wild-type or mutated AKAP-Lbc shRNAs at a moi of 50. Seventy-two hours after infection cells were incubated for 24 h with or without 10−4 M PE. RNAs were extracted and AKAP-Lbc expression analyzed by real-time PCR. Results are the mean ± SE of three independent experiments.
Fig. 2.
Fig. 2.
Silencing of AKAP-Lbc expression inhibits α1-AR-mediated Rho activation in cardiomyocytes. (A) Rat NVMs were infected with lentiviruses encoding GFP (control), both GFP and wild-type or mutated AKAP-Lbc shRNAs, or the Gα12 G228A mutant at a moi of 50. Seventy-two hours after infection, cells were incubated for 15 min with or without 10−4 M PE. Cell lysates were incubated with GST-RBD beads. The bound RhoA was detected with a monoclonal anti-RhoA antibody (Top). The relative amount of total RhoA and Gα12 G228A mutant in the cell lysates were assessed by using a monoclonal antibody against RhoA (Middle) and a polyclonal antibody against Gα12 (Bottom), respectively. (B) Quantitative analysis of the GTP-RhoA associated with RBD beads was obtained by densitometry. The amount of RhoA bound to RBD was normalized to the RhoA content of cell extracts. Results are expressed as mean ± SE of four independent experiments. Statistical significance was analyzed by paired Student's test. ∗, P < 0.05 as compared with Rho-GTP levels measured in PE stimulated cardiomyocytes infected with control lentiviruses.
Fig. 3.
Fig. 3.
Silencing of AKAP-Lbc expression inhibits α1-AR-mediated cardiomyocytes hypertrophy. (A) Rat NVMs were infected with lentiviruses encoding GFP (control), both GFP and wild-type or mutated AKAP-Lbc shRNAs, or the Gα12 G228A mutant at a moi of 50. Seventy-two hours after infection, cells were incubated for 24 h in the absence or presence of 10−4 M PE. Cells infected with control lentiviruses were treated with or without 2 μg/ml of C3 botulinum toxin before the incubation with PE. Cells were then fixed, permeabilized, and incubated with anti-α-actinin monoclonal antibodies as well as rhodamine-conjugated anti-mouse secondary antibodies. GFP expression was visualized directly by fluorescent excitation at 490 nm. (B) Mean cell surface area (± SE) of cardiomyocytes infected and treated as indicated in A. The cell surface area was determined on a total of 150 GFP-positive cardiomyocytes derived from five independent experiments by using the Image J software. Statistical significance was analyzed by paired Student's test. ∗, P < 0.05 as compared with the cell surface area measured in PE stimulated cardiomyocytes infected with control lentiviruses.
Fig. 4.
Fig. 4.
Silencing of AKAP-Lbc expression inhibits α1-AR-mediated induction of hypertrophic markers. (A) Cardiomyocytes were infected and treated with PE as indicated in Fig. 3. The relative expression of α-actinin, β-tubulin, and Gα12 G228A mutant in the cell lysates was assessed by using a monoclonal antibody against α-actinin (Top), β-tubulin (Middle), and a polyclonal antibody against Gα12 (Bottom), respectively. (B) Real-time PCR analysis of ANF and skeletal α-actin (Ska) mRNA expression was performed on total RNA samples extracted from rat ventricular cardiomyocytes that were infected and treated with PE as indicated in Fig. 3. All results are expressed as mean ± SE of three independent experiments. Statistical significance was analyzed by paired Student's test. ∗, P < 0.05 as compared with ANF or Ska levels measured in PE-stimulated cells infected with control lentiviruses. (C and D) Cardiomyocytes were infected with lentiviruses expressing AKAP-Lbc shRNAs and subsequently transfected with the empty Flag vector or with the cDNA encoding the Flag-tagged silencing resistant mutant of AKAP-Lbc. Forty-eight hours after transfection, cells were incubated for 24 h in the absence or presence of PE. Cells were then fixed, permeabilized, and incubated with anti-Flag monoclonal antibodies as well as rhodamine-conjugated anti-mouse secondary antibodies to detect the expression of the Flag-AKAP-Lbc mutant. GFP expression was visualized directly by fluorescent excitation at 490 nm. The mean cell surface area (±SE) of cardiomyocytes was determined as indicated in Fig. 3B. Statistical significance was analyzed by paired Student's test. ∗, P < 0.05 as compared with the cell surface area measured in PE-stimulated cardiomyocytes infected with lentiviruses encoding AKAP-Lbc shRNAs.
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