Sympathetic stimulation of adult cardiomyocytes requires association of AKAP5 with a subpopulation of L-type calcium channels.

Methods and Results We used calcium imaging and electrophysiology to examine the sympathetic response of cardiomyocytes isolated from wild type and AKAP5 mutant animals. The β-adrenergic regulation of calcium transients and the phosphorylation of substrates involved in calcium handling were disrupted in AKAP5 knockout cardiomyocytes. The scaffolding protein, AKAP5 (also called AKAP150/79), targets adenylyl cyclase, PKA, and calcineurin to a caveolin 3-associated complex in ventricular myocytes that also binds a unique sub-population of Ca v 1.2 L-type calcium channels. Only the caveolin 3-associated Ca v 1.2 channels are phosphorylated by PKA in response to sympathetic stimulation in wild type heart. However, in the AKAP5 knockout heart the organization of this signaling complex is disrupted, adenylyl cyclase 5/6 no longer associates with caveolin 3 in the T-tubules, and non-caveolin 3 associated calcium channels become phosphorylated after β-adrenergic stimulation although this does not lead to an enhanced calcium transient. The signaling domain created by AKAP5 is also essential for the PKA-dependent phosphorylation of ryanodine receptors and phospholambam.

METHODS The Akap5 −/− allele was generated by gene targeting using 129S1/SvImJ ES cells and standard techniques as previously described 13 . Mice were age-matched littermates of 3 to 6 months of age maintained on a C57Bl/6 background after >10 backcrosses to C57BL/6. Animals were handled in accordance with the guidelines of the University of Washington Institutional Animal Care and Use Committee. Adult cardiomyocytes were isolated and incubated as previously described 18 . Electrophysiology was performed using whole cell patch-clamp as described in online Supplemental Methods . Whole cell [Ca 2+ ] i transients were measured using Fluo-4 loaded cardiomyocytes subjected to field stimulation at 1 Hz. Immunocytochemistry was performed using standard techniques as described in Supplementary Methods . Immunoprecipitation and western blotting were done using heart extracts homogenized in lysis buffer containing (in mmol/L): 10 Na 2 HPO 4 , 150 NaCl, 5 EGTA, 5 EDTA, 5 NaF, with 1% TritonX-100 and 0.5% Na Deoxycholate. The cAMP measurements were performed on isolated cardiomyocytes in laminin coated wells and the cAMP determined with direct cAMP EIA (Assay Design, Ann Arbor, MI, USA). Significant differences were determined by two-way ANOVA and post hoc Student’s unpaired t test, as appropriate. Pearson’s correlation coefficients 19 were calculated using Imaris software (Bitplane AG, Zurich, Switzerland) on nine to 16 images per point.

Supplementary Material 01