Caveolae compartmentalise β2-adrenoceptor signals by curtailing cAMP production and maintaining phosphatase activity in the sarcoplasmic reticulum of the adult ventricular myocyte

Abstract 

Inotropy and lusitropy in the ventricular myocyte can be efficiently induced by activation of β1-, but not β2-, adrenoceptors (ARs). Compartmentation of β2-AR-derived cAMP-dependent signalling underlies this functional discrepancy. Here we investigate the mechanism by which caveolae (specialised sarcolemmal invaginations rich in cholesterol and caveolin-3) contribute to compartmentation in the adult rat ventricular myocyte. Selective activation of β2-ARs (with zinterol/CGP20712A) produced little contractile response in control cells but pronounced inotropic and lusitropic responses in cells treated with the cholesterol-depleting agent methyl-β-cyclodextrin (MBCD). This was not linked to modulation of L-type Ca²⁺ current, but instead to a discrete PKA-mediated phosphorylation of phospholamban at Ser¹⁶. Application of a cell-permeable inhibitor of caveolin-3 scaffolding interactions mimicked the effect of MBCD on phosphorylated phospholamban (pPLB) during β2-AR stimulation, consistent with MBCD acting via caveolae. Biosensor experiments revealed β2-AR mobilisation of cAMP in PKA II signalling domains of intact cells only after MBCD treatment, providing a real-time demonstration of cAMP freed from caveolar constraint. Other proteins have roles in compartmentation, so the effects of phosphodiesterase (PDE), protein phosphatase (PP) and phosphoinositide-3-kinase (PI3K) inhibitors on pPLB and contraction were compared in control and MBCD treated cells. PP inhibition alone was conspicuous in showing robust de-compartmentation of β2-AR-derived signalling in control cells and a comparatively diminutive effect after cholesterol depletion. Collating all evidence, we promote the novel concept that caveolae limit β2-AR-cAMP signalling by providing a platform that not only attenuates production of cAMP but also prevents inhibitory modulation of PPs at the sarcoplasmic reticulum. This article is part of a Special Issue entitled “Local Signaling in Myocytes”.

Abbreviations: AKAP, A kinase anchoring protein, ARVM, adult rat ventricular myocyte, β-AR, beta adrenoceptor, C3SD, caveolin-3 scaffolding domain, cAMP, cyclic adenosine 3′-5′-monophosphate, Cav-3, caveolin-3, CGP, CGP20712A, EHNA, erythro-9-(2-hydroxy-3-nonyl)adenine, Epac, exchange protein activated by cAMP, FRET, fluorescence resonance energy transfer, FSK, forskolin, HIV, human immunodeficiency virus, IBMX, 3-isobutyl-1-methylxanthine, I_Ca,L, L-type Ca²⁺ current, Iso, isoproterenol bitartrate, MBCD, methyl-β-cyclodextrin, PDE, phosphodiesterase, PKA, protein kinase A, PLB, phospholamban, pPLB, Ser¹⁶ phosphorylated PLB, PP, protein phosphatases, RyR, ryanodine receptor, TAT, trans-activating transcriptional activator, TnI, troponin I, ZNT, zinterol hydrochloride

Keywords: Caveolae, Cyclic AMP, Compartmentation, β2-adrenoceptor, Phosphatase