Keywords :
Animals; Calcium/metabolism; Cyclic AMP/metabolism; Cyclic GMP/genetics/metabolism; Cyclic Nucleotide Phosphodiesterases, Type 2/genetics/metabolism; Ion Transport/physiology; Membrane Microdomains/enzymology/genetics; Mice; Mice, Knockout; Multienzyme Complexes/genetics/metabolism; Muscle Proteins/genetics/metabolism; Myocardial Contraction/physiology; Myocytes, Cardiac/enzymology; Nitric Oxide Synthase Type I/genetics/metabolism; Plasma Membrane Calcium-Transporting ATPases/genetics/metabolism; Signal Transduction/physiology
Abstract :
[en] Identification of the signaling pathways that regulate cyclic nucleotide microdomains is essential to our understanding of cardiac physiology and pathophysiology. Although there is growing evidence that the plasma membrane Ca(2+)/calmodulin-dependent ATPase 4 (PMCA4) is a regulator of neuronal nitric-oxide synthase, the physiological consequence of this regulation is unclear. We therefore tested the hypothesis that PMCA4 has a key structural role in tethering neuronal nitric-oxide synthase to a highly compartmentalized domain in the cardiac cell membrane. This structural role has functional consequences on cAMP and cGMP signaling in a PMCA4-governed microdomain, which ultimately regulates cardiac contractility. In vivo contractility and calcium amplitude were increased in PMCA4 knock-out animals (PMCA4(-/-)) with no change in diastolic relaxation or the rate of calcium decay, showing that PMCA4 has a function distinct from beat-to-beat calcium transport. Surprisingly, in PMCA4(-/-), over 36% of membrane-associated neuronal nitric-oxide synthase (nNOS) protein and activity was delocalized to the cytosol with no change in total nNOS protein, resulting in a significant decrease in microdomain cGMP, which in turn led to a significant elevation in local cAMP levels through a decrease in PDE2 activity (measured by FRET-based sensors). This resulted in increased L-type calcium channel activity and ryanodine receptor phosphorylation and hence increased contractility. In the heart, in addition to subsarcolemmal calcium transport, PMCA4 acts as a structural molecule that maintains the spatial and functional integrity of the nNOS signaling complex in a defined microdomain. This has profound consequences for the regulation of local cyclic nucleotide and hence cardiac beta-adrenergic signaling.
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