Caveolae Differentially Control Phosphorylation of Sarcoplasmic Reticular Proteins Following β2 Adrenoceptor Stimulation in the Adult Cardiac Myocyte

Abstract

Caveolae, small flask-like lipid rafts, play a key role in shaping the spatial characteristics of the β2-adrenoceptor cAMP signal and confining this to the sarcolemmal compartment in the adult cardiac myocyte. Here we determine the consequences of disrupting caveolae for the ability of β2 signalling to target sarcoplasmic reticular proteins phospholamban (PLB) and the ryanodine receptor (RyR). Experiments were performed with dissociated adult rat ventricular myocytes. Selective β2 adrenoceptor stimulation was achieved with 10 μM zinterol in the presence of 300 nM CGP20712A (CGP). Disruption of caveolae (using the cholesterol depleting agent methyl-β-cyclodextrin, MBCD) resulted in inotropic and lusitropic responses to β2 stimulation (70.2 ± 9.7% increase in shortening; 13.3 ± 1.3 % decrease in time to half relaxation) which were absent in control cells (n=12-20 myocytes, P<0.001). PLB contributes to inotropic and lusitropic responses via protein kinase A (PKA)-dependent phosphorylation at Ser16. In agreement with functional data, MBCD-treated myocytes showed a marked 561 ± 144% increase in Ser16-phosphorylated PLB in response to β2 stimulation (relative to that in cells exposed to CGP alone) which was absent in control cells (93 ± 31% of that with CGP alone) (n=4, P<0.05). By contrast, we saw no significant increase (P>0.05) in phosphorylation of one of the PKA-targeted sites of RyR, Ser2809, in either control (112 ± 11%) or MBCD-treated (116 ± 18%) myocytes in response to β2 stimulation (n=5). These preliminary data suggest that caveolae selectively control cAMP signals even within the same broad (sarcoplasmic reticular) compartment of the adult cardiac myocyte. Disruption of caveolae allows β2 cAMP-dependent signalling to access a sub-compartment of the sarcoplasmic reticulum which contains PLB, but not one which contains RyR.