Role of mitochondrial pka in compartmentation of cAMP signaling in cardiac ventricular myocytes.

Abstract

In cardiac ventricular myocytes, both β1 and β2-adrenergic receptors (βARs) produce an increase in the rate and force of contraction by stimulating cAMP production. However, only β1ARs enhance the rate of relaxation by regulating the activity of the SERCA2 pump found in the distal scaroplasmic reticulum. Previously we found that β1, but not β2AR production of cAMP could be detected by Epac2-αKAP, a FRET-based biosensor targeted to the the SERCA2 signaling complex, demonstrating that compartmentation of cAMP signaling contributes to the difference in functional responses. While inhibition of phosphodiesterase (PDE) activity was found to influence this behavior, in silico studies have suggested that intracellular cAMP must also be slowed by other mechanisms in order for this to occur. Consistent with this prediction, using raster image correlation spectroscopy (RICS), we found that the intracellular diffusion coefficient of fluorescently labeled cAMP is actually 30 fold slower than the rate of free diffusion. Furthermore, this slowing of diffusion was found to be due to the buffering effect of protein kinase A (PKA), specifically PKA anchored to the outer membrane of mitochondria. To directly test the hypothesis that this buffering effect contributes to cAMP compartmentation, we used adenoviral-mediated knockdown to target two key mitochondrial A kinase anchoring proteins (AKAPs), D-AKAP1 and D-AKAP2. Following knockdown of either AKAP alone, we found that the cAMP diffusion coefficient was significantly increased compared to control. Furthermore, knocking down the expression of D-AKAP1 enhanced β2AR stimulation of cAMP production detected by Epac2-αKAP. These results support the conclusion that buffering of cAMP mobility by PKA anchored to mitochondria influences cAMP dynamics and contributes to compartmentation of β2AR signaling in cardiac ventricular myocytes.