Mitochondrial Buffering of cAMP in Adult Cardiac Myocytes

Abstract

Compartmentation of the diffusible second messenger cAMP plays a key role in producing unique responses to different G‐protein coupled receptors. It has been proposed that this can only happen if the movement of cAMP within the cell occurs at rates well below those of free diffusion. To directly test this hypothesis we used Raster Image Correlation Spectroscopy (RICS) to measure the diffusion coefficient of fluorescently labeled cAMP, 8‐[Pharos‐450]‐cAMP (P450‐cAMP) in live cells. Confocal imaging showed that incubation with the membrane permeable P450‐cAMP led to a distinct longitudinal pattern of fluorescence accumulation in adult rat ventricular myocytes. This was in contrast to the uniform distribution of cAMP‐free dye (P450), suggesting that P450‐cAMP binds to immobilized target. Interestingly, expression of PKA type II regulatory subunit labeled with CFP (RII‐CFP) exhibited a pattern similar to P450‐cAMP, which corresponded with the extra‐myofibrillar space populated by mitochondria, as verified by labeling with MitoTracker Red. RICS analysis found the diffusion coefficient (μm2/s) of P450‐cAMP (9.7±1.6, n=37) to be significantly slower than that of P450 (17.6 ± 1.73, n=14), but similar to that of RII‐CFP (12.1±1.85, n=11). Furthermore, Ht31, a peptide that disrupts interactions between RII and A kinase anchoring proteins (AKAPs), caused the distribution pattern of both P450‐cAMP and RII‐CFP to become more diffuse. Correspondingly, the diffusion coefficients for P450‐cAMP (34.4±4.37, n=15) and RII‐CFP (32.0±4.53, n=8) increased significantly, while that of P450 did not. These data suggest that the movement of cAMP in adult cardiac myocytes is limited by interactions with PKA RII subunits associated specifically with mitochondria.