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First published online November 23, 2005
doi: 10.1242/10.1242/jcs.02675

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The mAKAP complex participates in the induction of cardiac myocyte hypertrophy by adrenergic receptor signaling

Genevieve C. Pare^1,*, Andrea L. Bauman^1,*, Molly McHenry¹, Jennifer J. Carlisle Michel¹, Kimberly L. Dodge-Kafka² and Michael S. Kapiloff^1,

¹ Department of Pediatrics, Department of Cell and Developmental Biology, Heart Research Center, Oregon Health and Science University, NRC5 3181 S.W. Sam Jackson Park Road, Portland, OR 97239, USA
² Calhoun Center for Cardiology University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA

View larger version (29K): [in a new window]   Fig. 1. The mAKAP complex includes the PKA substrate RyR2 and CaNAß. (A) Myocyte cultures were treated for 30 minutes with 1 mmol/l diBu-cAMP or 10 µmol/l Iso. mAKAP-associated RyR2 co-immunoprecipitated with VO54 mAKAP serum (lanes 2-4), but not preimmune control serum (lane 1). The immunoprecipitates were back-phosphorylated with recombinant PKA catalytic subunit and [-32P]ATP (top panel). RyR2 was detected by immunoblotting with a RyR2 monoclonal antibody (bottom panel). The bar chart shows relative back-phosphorylation (mean ± s.e.m.). *P<0.003 compared to control; n=5; ANOVA, P<0.001. (B) Immune complexes were precipitated from adult rat heart extracts with non-specific mouse IgG (lane 1), mAKAP monoclonal antibody 720 (lane 2), and goat anti-CaNAß (lane 3). CaNAß (top panel), nesprin-1 (middle panel) and mAKAP (bottom panel) were detected by immunoblotting. Relative molecular mass markers are indicated. (C) Immune complexes were precipitated from adult rat heart extracts with non-specific goat IgG (lane 1) and goat anti-CaNAß antibody (lane 2). mAKAP (top panel), RyR2 (middle panel) and CaNAß (bottom panel) were detected by immunoblotting. n=3.

View larger version (74K): [in a new window]   Fig. 2. Ryanodine receptor and CaN activities are required for induction of cardiac myocyte hypertrophy by isoproterenol. (A) Primary myocytes were cultured for 2 days in a minimal medium containing either no drug (a), 10 µmol/l Iso (d-f), 50 µmol/l Ry (b,e) and/or 400 nmol/l CsA (c,f). Cells were fixed and stained with antibody for the sarcomeric Z-disk protein -actinin (grayscale). Scale bar in a indicates 20 µm. (B) Mean cell surface area (± s.e.m.). *P<0.04 relative to the value for cells treated with Iso alone; n3 independent myocyte preparations; ANOVA, P=0.01.

View larger version (45K): [in a new window]   Fig. 3. mAKAP is involved in the induction of adrenergic-stimulated myocyte hypertrophy. (A) Following co-transfection with a GFP expression vector and an expression vector for either control (left panels) or mAKAP siRNA (right panels), primary myocytes were cultured for 2 days in minimal medium containing either no drug (a-f), 10 µmol/l Iso (g-l), or 100 µmol/l PE (m-r). Cells were stained with mAKAP VO56 (red, b,e,h,k,n,q) and -actinin (blue, c,f,i,l,o,r) antibodies. The nuclei of transfected, GFP-expressing myocytes (green) are indicated with arrowheads on the mAKAP panels. Bar, 20 µm. (B) The level of mAKAP expression in myocytes infected with control or mAKAP siRNA-expressing adenoviruses were assayed by immunoblotting with VO56 mAKAP antibody. Total protein was detected by Ponceau S stain; the major band is myosin. (n=3) (C) Mean cell surface area (± s.e.m.) for GFP-labeled cells co-expressing either the control siRNA, the mAKAP siRNA, or the mAKAP siRNA and exogenous, myc-tagged mAKAP protein (mAKAP rescue). *P=0.04, **P=0.01, ***P=0.003 for the samples compared, n>150 with myocytes derived from 7 independent cultures; ANOVA, P=0.004 and 0.02 for Iso- and PE-treated data, respectively.

View larger version (16K): [in a new window]   Fig. 4. mAKAP RNAi inhibits protein synthesis. To detect new protein synthesis, myocytes were infected with control or mAKAP siRNA-expressing adenovirus. Myocytes were cultured for 18 hours in medium containing [3H]leucine and either no agonist (No Drug), 10 µmol/l Iso, or 100 µmol/l PE and 1 µmol/l propanolol (PE). Total [3H]leucine incorporation was determined. Data are normalized to the control siRNA, untreated sample (bar 1). *P0.05; n4 independent myocyte preparations.

View larger version (33K): [in a new window]   Fig. 5. mAKAP RNAi inhibits ANF expression. (A) Myocytes were infected with control (a-c) or mAKAP (d-f) siRNA-expressing adenovirus. The cells were treated with 100 µmol/l PE and 1 µmol/l propanolol for 48 hours (a-f) or left untreated (not shown), and then stained with monoclonal mAKAP antibody (red, a,d), rabbit ANF antibody (green, b,e) and Hoechst DNA stain (shown in the merged images in c,f). Bar, 20 µm. (B) The fraction of myocytes showing peri-nuclear, Golgi-type ANF staining is indicated for treated and non-treated myocytes. Data are average expression ± s.e.m. *P=0.03; n=4.

View larger version (53K): [in a new window]   Fig. 6. mAKAP-bound PKA is important for the transduction of signals that induce cardiac myocyte hypertrophy. (A-C) Following co-transfection with expression vectors for GFP and mAKAP siRNA (a-d), primary myocytes were cultured for 2 days in minimal medium containing either no drug (A), 10 µmol/l Iso (B), or 100 µmol/l PE (C). Additional myocytes were co-transfected with a third expression plasmid for either myc-tagged mAKAP WT (e-h) or mAKAP Del PKA BD (i-l). Cells were stained with myc-tag (blue, b,f,j) and mAKAP VO56 (red, c,g,k) antibodies. The nuclei of transfected, GFP-expressing myocytes (green) are marked with arrowheads on the mAKAP and myc panels. Bar, 20 µm. (D) mAKAP WT and mAKAP Del PKA BD protein were expressed by adenoviral infection of COS-7 cells and purified by immunoprecipitation. PKA binding activity of the WT and mutant protein was assayed by RII overlay assay using recombinant His-tagged RII protein and HRP-conjugated anti-His antibody. Equal loading was demonstrated by immunoblotting the same filter with VO54 mAKAP antibody (n=3). (E) Mean cell surface area (±s.e.m.) for myocytes cultured as in A. *P<0.015, **P<0.0001 for the samples compared; n>115 from 6 independent cultures; ANOVA, P<10-4 for the entire data set, and Iso- and PE-treated data alone.

View larger version (37K): [in a new window]   Fig. 7. The mAKAP complex contributes to NFATc1 activation. (A) Primary myocytes were co-transfected with an expression vector for Flag-tagged NFATc1 and either the control (a,b) or mAKAP (c,d) siRNA expression plasmid. The myocytes were treated for 2 days with no drug, 10 µmol/l Iso, 100 µmol/l PE, 400 nmol/l CsA and/or 100 nmol/l KT5720. Representative cells cultured in Iso-containing medium are shown. Cells were fixed and stained with antibodies for the Flag-tag (green, a,c) and -actinin (red, b,d) and with Hoechst 33258 DNA stain (blue, b,d). Bar, 20 µm. (B) The fraction of transfected myocytes with predominately nuclear NFATc1 (mean±s.e.m.). *P<0.05, **P<0.002 in comparison to the similarly treated control siRNA sample, n6; ANOVA, P<10-4 for entire data set and Iso- and PE-treated data alone.

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