Abstract
The lipid raft concept proposes that membrane environments enriched in cholesterol and sphingolipids cluster certain proteins and form platforms to integrate cell signaling. In cardiac muscle, caveolae concentrate signaling molecules and ion transporters, and play a vital role in adrenergic regulation of excitation–contraction coupling, and consequently cardiac contractility. Proteomic analysis of cardiac caveolae is hampered by the presence of contaminants that have sometimes, erroneously, been proposed to be resident in these domains. Here we present the first unbiased analysis of the proteome of cardiac caveolae, and investigate dynamic changes in their protein constituents following adrenoreceptor (AR) stimulation.Rat ventricular myocytes were treated with methyl-β-cyclodextrin (MβCD) to deplete cholesterol and disrupt caveolae. Buoyant caveolin-enriched microdomains (BCEMs) were prepared from MβCD-treated and control cell lysates using a standard discontinuous sucrose gradient. BCEMs were harvested, pelleted, and resolubilized, then alkylated, digested, and labeled with iTRAQ reagents, and proteins identified by LC-MS/MS on a LTQ Orbitrap Velos Pro. Proteins were defined as BCEM resident if they were consistently depleted from the BCEM fraction following MβCD treatment. Selective activation of α-, β1-, and β2-AR prior to preparation of BCEMs was achieved by application of agonist/antagonist pairs for 10 min in populations of field-stimulated myocytes.We typically identified 600–850 proteins per experiment, of which, 249 were defined as high-confidence BCEM residents. Functional annotation clustering indicates cardiac BCEMs are enriched in integrin signaling, guanine nucleotide binding, ion transport, and insulin signaling clusters. Proteins possessing a caveolin binding motif were poorly enriched in BCEMs, suggesting this is not the only mechanism that targets proteins to caveolae. With the notable exception of the cavin family, very few proteins show altered abundance in BCEMs following AR activation, suggesting signaling complexes are preformed in BCEMs to ensure a rapid and high fidelity response to adrenergic stimulation in cardiac muscle.
Highlights
Rat ventricular myocytes were treated with methyl-cyclodextrin (MCD) to deplete cholesterol and disrupt caveolae
Buoyant caveolin-enriched microdomains (BCEMs) were retrieved from gradient fractions 4 and 5 (Fig. 1B)
Phospholemman and the Na Pump in Cardiomyocyte BCEMs—As an exemplar of a cardiac caveolar resident protein, we investigated the relationship between caveolin 3 and the sodium pump catalytic ␣ and regulatory subunit phospholemman (PLM [30])
Summary
Materials and Antibodies—Unless indicated otherwise, all reagents were obtained from Sigma Chemical Company (Gillingham, UK) and were of the highest grade available. Sucrose Gradient Fractionation of Adult Rat Ventricular Myocytes— Buoyant caveolin-enriched microdomains (BCEMs) were prepared from ARVM homogenized (5 ϫ 6s bursts, Ultra-Turrax, with 20s intervals) and sonicated (6 ϫ 20s bursts, 5 m amplitude, Soniprep) in 500 mM sodium carbonate, pH 11, supplemented with 1 mM DTT, 1 mM EDTA, protease, and phosphatase inhibitors. Ten microliters of sample (a total of 2 g of peptide digest) was loaded with a constant flow of 5 l/min onto an Acclaim PepMap 100 (C18, 100 m ϫ 2 cm) trap column (Dionex Corporation/Thermo Scientific). 24 h later cells were lyzed for immunoprecipitation of the sodium pump catalytic subunit (using monoclonal antibody C464.4, Millipore) in 2 mg/ml C12E10 in PBS supplemented with protease and phosphatase inhibitor cocktails, as well as an additional mixture of chemical phosphatase inhibitors: 5 mM sodium fluoride, 2 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 2 mM sodium glycerophosphate. Co-immunoprecipitation reactions were routinely immunoblotted for membrane proteins not copurifying with the immunoprecipitated protein (not shown)
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