A CaMKII/PDE4D negative feedback regulates cAMP signaling

cAMP production and protein kinase A (PKA) are the most widely studied steps in β-adrenergic receptor (βAR) signaling in the heart; however, the multifunctional Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is also activated in response to βAR stimulation and is involved in the regulation of cardiac excitation-contraction coupling. Its activity and expression are increased during cardiac hypertrophy, in heart failure, and under conditions that promote arrhythmias both in animal models and in the human heart, underscoring the clinical relevance of CaMKII in cardiac pathophysiology. Both CaMKII and PKA phosphorylate a number of protein targets critical for Ca²⁺ handling and contraction with similar, but not always identical, functional consequences. How these two pathways communicate with each other remains incompletely understood, however. To maintain homeostasis, cyclic nucleotide levels are regulated by phosphodiesterases (PDEs), with PDE4s predominantly responsible for cAMP degradation in the rodent heart. Here we have reassessed the interaction between cAMP/PKA and Ca²⁺/CaMKII signaling. We demonstrate that CaMKII activity constrains basal and βAR-activated cAMP levels. Moreover, we show that these effects are mediated, at least in part, by CaMKII regulation of PDE4D. This regulation establishes a negative feedback loop necessary to maintain cAMP/CaMKII homeostasis, revealing a previously unidentified function for PDE4D as a critical integrator of cAMP/PKA and Ca²⁺/CaMKII signaling.During cardiac excitation-contraction coupling (ECC), Ca²⁺ elevation throughout the cell promotes myofilament sliding, which generates contractile force. This process is highly regulated by positive and negative regulatory circuits, the most critical being the sympathetic nervous system that acts via activation of the β-adrenergic (βAR)/cAMP/PKA signaling pathway. During βAR stimulation, PKA phosphorylates and activates key proteins involved in ECC and Ca²⁺ handling. These proteins include L-type Ca²⁺ channels and ryanodine receptors (RyR), leading to enhanced Ca²⁺ influx and consequent sarcoplasmic reticulum (SR) Ca²⁺ release; phospholamban (PLB), increasing SR Ca²⁺ uptake by the Ca²⁺ ATPase (SERCA), thereby accelerating cardiac relaxation; and contractile proteins, increasing cell contraction. Collectively, these events produce the typical inotropic and lusitropic effects of βAR stimulation (1).This βAR/cAMP/PKA pathway is only one of the components involved in regulating cardiac function, however. Data accumulated in the last decade have revealed that Ca²⁺/calmodulin-dependent kinase II (CaMKII) is equally important to the regulation of cardiac function under physiological and pathological conditions (2–7). Most of the functions distal to cAMP and PKA are regulated by CaMKII as well (8). Proteins critical for ECC are substrates for both PKA and CaMKII, and phosphorylation at different sites often produces similar changes in protein function. Whereas CaMKII is directly regulated by Ca²⁺, its activity is indirectly regulated by cAMP (9). In response to βAR stimulation, Epac (Exchange Protein directly Activated by cAMP) activates CaMKII (10–14). This regulation is critical in pathophysiological conditions in which CaMKII expression and activation may be elevated, such as hypertrophy, heart failure, and arrhythmias (2, 3). Despite the wealth of data available, the exact mechanisms integrating CaMKII activity with cAMP/PKA signaling remain unclear.An established concept in cell signaling is that freely diffusible cAMP is not distributed uniformly throughout the cell, but is compartmentalized to generate specificity and to allow PKA regulation in distinct subdomains (15). Phosphodiesterases (PDEs), the enzymes that degrade cAMP, have emerged as ubiquitous and important modulators of cAMP/PKA signaling in specific cellular compartments, including cardiac myocytes (16, 17). They are part of macromolecular complexes that include PKAs and A-kinase anchoring proteins (AKAPs). In contrast to the large body of data linking PDEs to PKA regulation, whether PDEs regulate CaMKII is unclear. We and others have shown that genetic ablation of PDE4s, the isoform responsible for cAMP degradation in the heart, disrupts ECC via PKA-mediated alteration in Ca²⁺ handling (18–20); however, the possibility that some of the effects are mediated by CaMKII has not been investigated.For proper homeostasis, cAMP signals are constrained by feedback mechanisms required to tightly regulate cyclic nucleotide levels under basal or stimulated conditions, with the feedback regulation of PDE4s a preeminent example. PDE4s are activated by PKA phosphorylation, providing a negative feedback mechanism by which cAMP regulates its own level (21, 22). Given the cAMP-dependent, Epac-mediated CaMKII activation, we surmise that feedback mechanisms linking cAMP and CaMKII must be operating in cardiac myocytes as well. In the present study, we reassessed the interaction between cAMP signaling and CaMKII. We demonstrate that CaMKII activity constrains basal and βAR-activated cAMP signaling. These effects are mediated, at least in part, by CaMKII regulation of PDE4D, revealing an additional function of this enzyme as a critical integrator of cAMP/PKA and Ca²⁺/CaMKII signaling.