Effects of Cholesterol Depletion on Compartmentalized cAMP Responses in Adult Cardiac Myocytes

Abstract

The role of cholesterol-dependent lipid rafts in producing compartmentalized responses of β1-adrenergic receptors (β1ARs) and E-type prostaglandin receptors (EPRs) was investigated in adult rat ventricular myocytes. β1ARs were found in lipid raft and non-lipid raft containing membrane fractions, while EPRs were only found in non-lipid raft fractions. Furthermore, β1AR activation enhanced the L-type Ca2+ current (ICa-L), intracellular Ca2+ [Ca2+]i transient, and myocyte shortening, while EPR activation had no effect. Cholesterol depletion by treatment with methyl-β-cyclodextrin (MβCD) did not eliminate compartmentalized behavior but significantly enhanced the sensitivity of functional responses produced by β1ARs. In MβCD-treated cells, 1 nM isoproterenol increased ICa-L by 49 +/- 8.3% (n = 5) as compared to 15 +/- 5% (n = 13) in control cells. These responses were blocked by the specific β1AR antagonist, 100 nM CGP20712A. Similarly, β1AR activation led to an increase in cell shortening and [Ca2+]i transient from 63 +/- 13% and 18 +/- 4.1% in control cells (n = 17) to 188 +/- 19.5% and 35 +/- 4.3% in MβCD-treated cells (n = 22) respectively. Cholesterol depletion failed to elicit any effect on EPR activation. Changes in cAMP activity were also measured in intact cells using two different FRET-based biosensors: a type II PKA-based probe to monitor cAMP in subcellular compartment that include caveolar lipid raft microdomains and a freely diffusible Epac2-based probe. β1AR and EPR activation elicited responses detected by both FRET probes. However, cholesterol depletion only affected β1AR responses detected by the PKA probe. These results indicate that caveolar lipid rafts alone are not sufficient to explain the differences between β1AR and EPR responses. They also suggest that β1AR regulation of myocyte contraction involves the local production of cAMP by a subpopulation of receptors associated with lipid rafts.