Effects of cholesterol depletion on compartmentalized cAMP responses in adult cardiac myocytes

Abstract

β(1)-Adrenergic receptors (β(1)ARs) and E-type prostaglandin receptors (EPRs) both produce compartmentalized cAMP responses in cardiac myocytes. The role of cholesterol-dependent lipid rafts in producing these compartmentalized responses was investigated in adult rat ventricular myocytes. β(1)ARs were found in lipid raft and non-lipid raft containing membrane fractions, while EPRs were only found in non-lipid raft fractions. Furthermore, β(1)AR activation enhanced the L-type Ca(2+) current, intracellular Ca(2+) transient, and myocyte shortening, while EPR activation had no effect, consistent with the idea that these functional responses are regulated by cAMP produced by receptors found in lipid raft domains. Using methyl-β-cyclodextrin to disrupt lipid rafts by depleting membrane cholesterol did not eliminate compartmentalized behavior, but it did selectively alter specific receptor-mediated responses. Cholesterol depletion enhanced the sensitivity of functional responses produced by β(1)ARs without having 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 compartments that include microdomains associated with caveolar lipid rafts and a freely diffusible Epac2-based probe to monitor total cytosolic cAMP. β(1)AR and EPR activation elicited responses detected by both FRET probes. However, cholesterol depletion only affected β(1)AR responses detected by the PKA probe. These results indicate that lipid rafts alone are not sufficient to explain the difference between β(1)AR and EPR responses. They also suggest that β(1)AR regulation of myocyte contraction involves the local production of cAMP by a subpopulation of receptors associated with caveolar lipid rafts.

Fig. 1

Effect of the β-adrenergic receptor (βAR) agonist isoproterenol (Iso) and the E-type prostaglandin receptor (EPR) agonist PGE1 on the L-type Ca²⁺ current (I_Ca-L) in rat ventricular myocytes. A, Time course of changes in I_Ca-L amplitude and corresponding sample current traces (inset) under control conditions (a) and following exposure to 30 nM Iso (b). B, Time course of changes in I_Ca-L amplitude and corresponding sample current traces (inset) under control conditions (a), and following exposure to 10 μM PGE1 (b) and 30 nM Iso (c). C, Average increase in I_Ca-L amplitude recorded in the presence of 10 μM PGE1 or 30 nM Iso (**p < 0.05). D, Western blot of ventricular myocyte membrane fractions obtained by sucrose density centrifugation. Caveolin-3 (Cav-3) was used as a marker of caveolae-containing buoyant membranes (fraction 5); β-adaptin was used as a marker of heavy non-caveolar membranes (fractions 9–12).

Fig. 2

Effect of cholesterol depletion on L-type Ca²⁺ current (I_Ca-L) responses to isoproterenol (Iso) and PGE1. Time courses of change in I_Ca-L amplitude and corresponding sample current traces (inset). A, Untreated cell under control conditions (a), and following exposure to 1 nM (b) and 30 nM (c) Iso. B, MβCD-treated cell under control conditions (a), and following exposure to 1 nM (b) and 30 nM (c) Iso. C, MβCD-treated cell in the presence of the selective β₁-adrenergic receptor antagonist CGP20712A (CGP, 100 nM) (a), and following exposure to CGP plus 1 nM Iso (b) and 1 μM Iso plus the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 100 μM) (c). D, MβCD-treated cell under control conditions (a), and following exposure to 10 μM PGE1 (b) and 30 nM Iso (c). E, Average change in I_Ca-L amplitude in untreated and MβCD-treated myocytes (**p < 0.05, ns = not significant, see text for n numbers). F, Effect of MβCD treatment on membrane cholesterol content detected by filipin staining.

Fig. 3

Effect of cholesterol depletion on intracellular Ca²⁺ ([Ca²⁺]_i) transient and cell shortening responses to isoproterenol (Iso) and PGE1. A, Cell shortening elicited by electric field stimulation before (black traces) and after (gray traces) exposure to 1 nM Iso (upper panels) and 10 μM PGE1 (lower panels) in untreated cells (left hand panels) and MβCD-treated cells (right hand panels). Scale bars: 200 ms, 4% resting of cell length. B, [Ca²⁺]_i transients elicited by electric field stimulation before (black traces) and after (gray traces) exposure to 1 nM Iso (upper panels) and 10 μM PGE1 (lower panels) in untreated cells (left hand panels) and MβCD-treated cells (right hand panels). Scale bars: 200 ms, 0.4 relative units. C, Average cell shortening responses expressed as the change relative to resting cell length in untreated and MβCD-treated cells (upper panel); average change in time to half (t_0.5) relaxation of cell shortening in control and MβCD-treated cells (lower panel). D, Average [Ca²⁺]_i transient responses expressed as the change in fura-2 fluorescence ratio relative to baseline in untreated and MβCD-treated cells (upper panel); average change in t_0.5 decay of the [Ca²⁺]_i transient in control and MβCD-treated cells (lower panel). Responses to 1 nM Iso were recorded in the presence of 100 nM ICI 118,551, a selective β₂ receptor antagonist (**p < 0.01, ***p < 0.001, ns = not significant; see text for n numbers).

Fig. 4

Effect of cholesterol depletion on intracellular cAMP response to β-adrenergic receptor stimulation detected by the type II PKA biosensor. Time course of changes in FRET response (ΔR/R₀) and corresponding pseudocolor images recorded under control conditions (a), and following exposure to 0.3 nM (b) and 30 nM Iso (c) in an untreated cell (A) and a MβCD-treated cell (B). Scale bar, 10 μm. C, Average changes in FRET responses in untreated and MβCD-treated cells. D, Average changes in FRET response to 0.3 nM Iso in MβCD-treated cells recorded in the presence of the β₁ receptor antagonist CGP20712A (CGP, 100 nM) or the β₂ receptor antagonist ICI 118,551 (ICI, 100 nM) (**p < 0.05, ns = not significant).

Fig. 5

Effect of cholesterol depletion on intracellular cAMP response to β-adrenergic receptor stimulation detected by the Epac2 biosensor. Time course of changes in FRET response (ΔR/R₀) and corresponding pseudocolor images under control conditions (a), and following exposure to 1 nM (b) and 30 nM Iso (c) in an untreated cell (A) and a MβCD-treated cell (B). Scale bar, 10 μm. C, Average change in FRET responses in untreated cells and MβCD-treated cells (ns = not significant).

Fig. 6

Effect of cholesterol depletion on intracellular cAMP response to PGE1. Time course of FRET response detected by Epac2-based biosensor (A, B) or type II PKA-based biosensor (D, E) following exposure to 10 μM PGE1 in untreated cells (A, D) and MβCD-treated cells (B, E). Average changes in FRET response to 10 μM PGE1 detected by Epac2-based biosensor (C) and PKA-based biosensor (F) in untreated and MβCD-treated cells (ns = not significant).