Caveolin-3 regulates compartmentation of cardiomyocyte beta2-adrenergic receptor-mediated cAMP signaling.

Heart Failure Model and Cell isolation

All animal surgical procedures and perioperative management were carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health under assurance number A5634-01. All animal surgical procedures and perioperative management conformed to the UK Animals (Scientific Procedures) Act 1986. Adult male Sprague-Dawley rats (250–300 g) underwent proximal coronary ligation to induce myocardial infarction as described [ 33 ]. Anaesthesia was induced by administration of 5% isoflurane for induction and reduced to 2% isoflurane once intubated and ventilated. It was ensured that pain reflexes were absent prior to an incision being made by testing pedal and palpebral reflexes. Preoperatively, buprenorphine was administered subcutaneously at a dose of 0.05 mg/kg to ensure adequate analgesia in addition to anaesthesia. Further doses of buprenorphine were administered as required post-operatively if there was any sign of distress. In addition, enrofloxacin (5 mg/kg) and 0.9 % saline (10 ml/kg) were administered pre-operatively. Cardiac failure was assessed via biometric and echocardiographic means. Heart weight corrected to tibia length provided a measure of hypertrophy. Echocardiography was performed under anaesthesia (2 % isoflurane) in the week of sacrifice. B-Mode echocardiographic images were acquired in the parasternal long axis at 70 Hz using a Visualsonics Vevo 770. This chronic HF model associated with massive hypertrophy and dilation has been previously well characterized by histological and functional analysis [ 33 ]. Isolated failing cardiomyocytes have been shown to have lower β 1 AR and unchanged β 2 AR densities [ 34 ]. Hearts were harvested for cell isolation 16 weeks post myocardial infarction, when a chronic HF phenotype has developed. Rats were sacrificed by cervical dislocation following brief exposure to 5% isoflurane until righting reflex was lost. Myocytes from healthy or failing hearts were isolated by the Langendorff perfusion method [ 34 ], plated on laminin coated coverslips and infected for 48h with Epac2-camps, Cav3 or Cav3DN adenoviral vectors [ 35 ]. For control experiments shown in Figures 3 and 4 , cells were isolated from age-matched sham-operated control animals.

Electron Microscopy

Cardiomyocytes were fixed with 2.5 % glutaraldehyde for 2 to 4h and then centrifuged at 500g for 5 min and the pellet was left overnight. The pellet was washed three times in cacodylate buffer and fixed in 1% osmium-tetroxide, followed by a 5–10 min washing with pure water. A small amount (25 to 50 μl) of liquid 2% agar at 45°C was added to the pellet. Drops were left to solidify on polythene, providing agar blocks with evenly distributed cells. The blocks were dehydrated through a series of graded alcohols, propylene oxide, and embedded in araldite. For low power examination by light microscopy before EM examination, 1μm thick sections were cut and stained with 1% toluidine blue in 1 % borax. For transmission electron microscopy, ultra-thin sections were stained with uranyl acetate and lead citrate. The ultrastructural features of cardiac myocytes, especially the membrane area were examined.

Western blot analysis

ARVMs were transfected with the control (LacZ), Cav3 or Cav3DN adenoviruses (all at MOI 500). 48h later cell were washed once and homogenized in the lysis buffer containing: 300mM sucrose, 150mM NaCl, 1mM EGTA, 2mM CaCl 2 , 1% Triton-100, 10 mM HEPES, pH=7.4. 5 μg protein samples were separated on a 15 % SDS-polyacrylamide gel and blotted onto nitrocellulose membrane (Millipore Corp., Bedford, MA, USA). Membranes were blocked for 1h at room temperature with 5 % non-fat milk in PBS containing 0.05 % Tween-20 and incubated with primary monoclonal Cav3 antibodies (1:5000, Santa Cruz Biotech, USA) overnight at 4°C. After washing, the blots were probed with a 1:5000 dilution of horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Sigma, St Louis, MO, USA) and visualized using the ECL kit (Amersham Biosciences).

Radioligand binding studies

Radioligand binding studies were performed as previously described [ 36 ]. Briefly, isolated cell membranes were incubated for 1h at 30°C with 60–100 pM 125 Icyanopindolol ( 125 I-CYP) (PerkinElmer Life Sciences, Dreieich, Germany) and increasing concentrations of ICI118, 551. Competition binding curves were fitted and analyzed with the Prism software (GraphPad, San Diego, CA).

FRET imaging of cAMP in living cardiac myocytes

FRET was performed in cells infected for 48h with Epac2-camps adenovirus [ 37 ]. Cells were washed once and measured at room temperature in the buffer containing 144mM NaCl, 5.4mM KCl, 1mM MgCl 2 , 2mM CaCl 2 , and 10mM HEPES, pH=7.3. The imaging system was build around the Nikon TE2000 microscope equipped with halogen lamp illuminator pillar, EX436/20 excitation filter combined with DM455 dichroic mirror. Cell fluorescence was split into YFP and CFP channels using the DualView (Optical Insights, equipped with 535/40 and 480/30 emission filters) and monitored by the ORCA-ER CCD camera (Hamamatsu Photonics, Welwyn Garden City, UK). Cell images were analyzed using SimplePCI software (Hamamatsu). FRET ratios were corrected for the bleed through of CFP into the YFP channel and analyzed using the Origin software (OriginLab Corporation, Northhampton, MA). SICM and SICM/FRET experiments SICM/FRET measurements were performed exactly as previously described [ 34 ]. These included scanning of the living cell surface, local ligand application into single T-tubules via the scanning pipette and sub-cellular analysis of the induced cAMP signal by FRET microscopy. For high-resolution SICM, resistance of the pipette tip was around 200MΩ. To calculate the Z-groove index, we measured the maximum length of Z-grooves observed on single SICM images and divided this value by the total estimated Z-groove length as previously described [ 33 ].

Electrophysiological recordings from adult cardiac myocytes

Macroscopic currents were recorded using the whole-cell patch clamp technique with the external recording solution containing 1mM CaCl 2 , 0.5mM MgCl 2 , 5mM HEPES, 140mM choline chloride, 5mM CsCl, 5.5mM glucose, pH 7.4 with CsOH, ~305mOsm, and the internal pipette solution containing 130mM Cs-methanesulphonate, 11mM EGTA, 10mM HEPES, 2mM MgCl 2 , 5mM Mg-ATP, 0.3mM Na-GTP, pH 7.2 with CsOH, ~290mOsm/kg.

Statistical analysis

Data were analyzed using Origin Pro 8.6 software (OriginLab Corporation, Northhampton, MA) and presented as means ± SE from the indicated number of independent experiments, animals or cells isolated from several rats per condition, as indicated in the figure legends. Differences were tested using one-way ANOVA with Bonferroni post-test and considered significant at p