Abstract 15670: Caveolin3 Depletion Modifies β2AR-cAMP Responses at Both Cellular and Sub-Cellular Levels in Cardiomyocytes
The two β-adrenergic receptor (βAR) subtypes on the surface of adult rat ventricular cardiomyocytes (ARVMs) play different roles in regulating cardiac function. Previously, we found that β1AR-cAMP signaling is distributed throughout the entire cell membrane whereas β2AR-cAMP signaling is localized exclusively in the transverse (T)-tubules. β2ARs, but not β1ARs, have also been shown to be associated with caveolae. The β2AR gives rise to a spatially confined cAMP signal in contrast with the β1AR. We intended to investigate whether caveolin-3 (Cav3), the main scaffolding protein component of cardiomyocyte caveolae, regulates the character of cellular β2AR-cAMP signals. Wildtype Cav3 or a Cav3 dominant-negative mutant (Cav3DN) constructs were co-expressed in ARVMs together with the Förster resonance energy transfer (FRET)-based cAMP sensor Epac2-camps using adenoviral vectors. FRET and scanning ion conductance microscopy were used to locally stimulate β2ARs and measure cytosolic cAMP levels. Cav3 overexpression increased the number of caveolae and decreased the magnitude of the β2AR-cAMP signal (from 3.9±0.3 % to 2.2±0.3%, p≤0.05 n=7-12 cells %raw FRET) but not the β1AR-cAMP signal (from 9.7±0.4% to 9.8±0.4%, N.S n=14-15 cells %raw FRET). Conversely, Cav3DN expression resulted in an increased β2AR-cAMP signal (from 3.2±0.4% to 5.6±0.4%, p≤0.05 n=12-13 cells %raw FRET). This did not alter the whole-cell L-type calcium current. In Cav3DN-expressing ARVMs, β2AR response could only be generated in T-tubules. However, the normally spatially confined β2AR-cAMP signal became diffuse, similar to the diffuse β2AR-cAMP response observed in failing cardiomyocytes. Overexpression of Cav3 in failing ARVMs normalized the β2AR-cAMP response in ∼50% of cells. This included both return of spatial confinement of the β2AR-cAMP response and re-confinement of the sites of response induction to T-tubular areas. Our results show that, in ARVMs, Cav3 regulates β2AR-cAMP signaling, playing a crucial role in the compartmentation of cAMP signals in healthy cells. We suggest this is necessary for the β2AR to maintain its subtype specific character. In failing ARVMs, Cav3 overexpression can partially restore the disrupted localization of these receptors.
- © 2012 by American Heart Association, Inc.