AKAP-Lbc mobilizes a cardiac hypertrophy signaling pathway.

In situ analysis of PKD activity Our working hypothesis is that AKAP-Lbc mediated activation of PKD potentiates the hypertrophic response. Accordingly, overexpression of AKAP-Lbc enhanced activation of the kinase as assessed by immunoblot using anti-phospho-PKD Ser 744/748 antibodies when phenylephrine, endothelin or PDBu was used as the agonist ( Fig 5A & Supplementary data S7 ). The time course of these events was further evaluated using a genetically encoded fluorescence based D-Kinase Activity Reporter (DKAR) ( Kunkel et al., 2007 ). PKD phosphorylation of a consensus substrate sequence (LSRQL T AAVSE) results in a decrease in Fluorescence Resonance Energy Transfer (FRET) as measured by dynamic changes in the YFP/CFP ratio ( Fig. 5B ). A nuclear localization signal (PKKKRKVEDA) was engineered into the parent DKAR construct. This allowed us to selectively monitor PKD activity in the nucleus where HDAC5 is a likely substrate. ET-1 was applied to NRVM at time zero and fluorescent images were collected at 30 sec intervals for 90 minutes ( Fig 5C–E ). Increased PKD activity was scored as a decrease in the YFP/CFP ratio in a defined area of the nucleus. Half-maximal responses were reached within 44 ± 3 min (n=11) following application of ET-1 in NRVM expressing nDKAR alone ( Fig 5C upper panels, Fig 5D blue line & Fig 5E column 1). However, the rate and magnitude of the DKAR response was increased in NRVM expressing AKAP-Lbc-mCherry showing a half maximal activation within 25 ± 3 min (n=15, Fig 5C lower panels, Fig 5D orange line & Fig 5E column 2). Control experiments confirmed that the nDKAR FRET ratio was reversed upon application of Gö6976 (500 nM), the most selective, commercially available PKD inhibitor ( Fig 5D ). Hence AKAP-Lbc acts as a catalyst to favor PKD activation and apparently increases the pool of active enzyme in the nucleus. Both factors drive NRVM toward the nuclear exclusion of HDAC5. Additional imaging experiments confirmed that pre-treatment of NRVM with Gö6976 blocks agonist-induced nuclear export of HDAC5-RFP when compared to controls ( Fig 5F ).

Experimental Procedures All Western blotting, RII overlays, immunoprecipitations and phosphorylation experiments were performed as described previously ( Westphal et al., 1999 ). Cell culture, immunocytochemistry and live cell imaging Cos7 cells were cultured on glass coverslips. All transfections were carried out using Effectene reagent (Qiagen, Valencia, CA). Neonatal rat cardiomyocytes were cultured as described in (Pare et al, 2005). After 1 day in culture, cells were treated with vehicle or agonist for a further 48hrs at 37°C, prior to lysis and evaluation of AKAP-Lbc expression by Western blot or RT-PCR.

Preparation of primary

NRVM and confocal microscopy experiments, NRVM were as described in ( Dodge-Kafka et al., 2005 ). Cells were fixed in 3.7% paraformaldehyde in PBS followed by staining for α-actinin or ANF. Secondary antibodies used were from Jackson Immunoresearch. To determine the effect of shRNA expression on cellular hypertrophy, myocytes were co-transfected with rat AKAP-Lbc shRNA-GFP or control (human AKAP-Lbc) shRNA plasmid and pEYFP (Clontech). For rescue experiments FLAG-human AKAP-Lbc constructs were used. Anti-actinin was used to stain sarcomeric Z-disks to distinguish myocytes from contaminating fibroblasts. Images were acquired using a BioRad MRC1024 confocal microscope. For all live cell imaging experiments NRVM were cultured on glass coverslips coated with 1% gelatin (Sigma). For ectopic expression of protein, NRVM were electroporated using a modified Amaxa Nucleofector protocol. After incubation at 37 °C for 18–48 h post electroporation, cells were washed twice with Hank’s balanced salt solution, mounted in a Ludin chamber (Life Imaging Services) and imaged using a Leica AS MDW workstation. Images were acquired as described in ( Dodge-Kafka et al., 2005 ). Cells were treated with ET-1 (100 nM working concentration) for 60 mins prior to treatment with the PKC and PKD inhibitor Go 6976 (500 nM) to validate that we were measuring changes in PKD activity rather than other basophilic kinases. Nuclear phosphatase activity was inhibited by pretreatment with calyculin A (10 nM). Additional experiments were performed using cells that were pretreated with the CaMK inhibitor KN93 (5 μM working concentration for 30 mins) to ensure that nDKAR was not detecting CaMK activity in NRVM.

Statistical analyses

All data are expressed as mean ± standard error of the mean. Differences in quantitative variables were examined by one-way analysis of variance (ANOVA), or an unpaired two- tailed t test. A P value < 0.05 was considered significant (*), a P value < 0.01 was considered very significant (**) and a P value < 0.001 was considered extremely significant (***). All analyses were performed using InStat. Note: Experimental details for RT-PCR, expression constructs used, in vitro HDAC5 phosphorylation, 14-3-3 binding assays and MEF2-luciferase assays are included in the Supplementary data .

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