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

Abstract

β1-Adrenergic receptors (β1ARs) 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. 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, intracellular Ca2+ 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 β1ARs 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. 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 lipid rafts alone are not sufficient to explain the difference 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 caveolar lipid rafts.

Caveolin Contributes to the Modulation of Basal and β-Adrenoceptor Stimulated Function of the Adult Rat Ventricular Myocyte by Simvastatin: A Novel Pleiotropic Effect

“…This is consistent with enhanced sensitivity of functional responses to β1-AR stimulation, comparable with data obtained using MBCD to disrupt myocyte caveolae [16]. Increased adenylyl cyclase activity, by removal of the normal inhibitory influence of Cav3 [40], may contribute to this effect.…”

Caveolin-1 regulates P2X7receptor signaling in osteoblasts

“…Since we did not find a direct interaction between P2X 7 R and CAV1, we postulate that the enhanced P2X 7 R signaling in the absence of CAV1 could be due to difference in localization of P2X 7 R on the plasma membrane. Agarwal et al (1) observed similar changes in the functional response of a cell based on the localization of receptors in caveolae in adult cardiac myocytes. They found that the differences in the distribution of the ␤ 1 -adrenegic receptor (␤ 1 -AR) and the E-type prostaglandin receptors on the plasma membrane led to different functional responses, although activation of both of these receptors led to an increase in cAMP production.…”

Sarcolemmal cholesterol and caveolin-3 dependence of cardiac function, ischemic tolerance, and opioidergic cardioprotection

“…Similarly, cholesterol depletion has been reported to reduce myocyte Ca 2ϩ currents and contraction under baseline conditions (6) and inhibit effects of ␤ 2 -AR activity (2,6). Nonetheless, other studies have identified amplified ␤ 1 -AR or ␤ 2 -AR signaling upon myocyte cholesterol depletion, with enhanced PKA/adenylyl cyclase phosphoregulation of target proteins (e.g., phospholamban and troponin I), and activation of Ca 2ϩ currents and myocyte contractile function (1,7,26). While we do not address ␤-AR responsiveness, our data for intact perfused hearts are consistent with inhibitory rather than stimulatory effects of cholesterol depletion on Ca 2ϩ -dependent myocyte function (3,6).…”

“…In isolated cardiomyocytes, cholesterol depletion with methyl-␤-cyclodextrin (M␤CD) induces a negative inotropic effect at rest (6) while enhancing contractile responses to ␤ 2 -adrenergic receptors (ARs) (6,7,26) and ␤ 1 -ARs (1). In coronary smooth muscle, M␤CD impairs endothelin-1 and serotonin responses while enhancing phenylephrine contraction (40).…”