Phosphodiesterase 4 and Phosphatase 2A Differentially Regulate cAMP/Protein Kinase A Signaling for Cardiac Myocyte Contraction under Stimulation

Activation of the adrenergic receptor ( AR) induces a tightly controlled cAMP/protein kinase A (PKA) activity to ensure an agonist dose-dependent and saturable contraction response in animal heart. We have found that stimulation of 1AR by isoproterenol induces maximal contraction responses at the dose of 1 M in cardiac myocytes; however, cAMP accumulation continues to increase with higher agonist concentrations. Dose-dependent cAMP accumulation is tightly controlled by negative regulator phosphodiesterase 4 (PDE4) that hydrolyzes cAMP. At 1 nM isoproterenol, cAMP accumulation is minimal because of the hydrolysis of cAMP by PDE4, which leads to a small increase in PKA phosphorylation of phospholamban and troponin I (TnI), and contraction responses. Inhibition of PDE4 activity with rolipram enhances cAMP accumulation, yields maximal PKA phosphorylation of phospholamban and TnI, and myocyte contraction responses. In contrast, at 10 M isoproterenol, despite the negative effect of PDE4, cAMP accumulation is sufficient for maximal PKA phosphorylation of phospholamban and TnI. Inhibition of PDE4 with rolipram enhances cAMP accumulation, but not PKA phosphorylation and contraction responses. It is interesting that activities of both PKA and protein phosphatase 2A (PP2A) are enhanced under 1AR activation with 10 M isoproterenol, and PP2A is recruited to PKA/A kinase-anchoring protein complex. Inhibition of PP2A with okadaic acid further enhances the phosphorylation of phospholamban and TnI as well as contraction responses induced by 10 M isoproterenol. Therefore, PP2A plays a key role in limiting PKA phosphorylation of phospholamban and TnI for myocyte contraction responses under 1AR stimulation. adrenergic receptors ( ARs) regulate cardiac contraction to enhance cardiac output in response to sympathetic nerve activity. It is well known that cardiac contraction is a saturable process, which is essential to prevent the heart from undergoing fibrillation or cardiac arrest. Among the adrenergic receptors expressed in myocardium, 1AR serves as the primary receptor subtype in both human and murine hearts and is responsible for regulating cardiac contraction. Activated 1ARs couple to Gs proteins to stimulate adenylyl cyclases, which synthesize second-messenger cAMP to activate PKA (Lefkowitz, 2007). PKA phosphorylates a wide range of substrates to enhance contraction, including L-type calcium channels and phospholamban for regulating cytosolic calcium concentration and troponin I (TnI) for myofibril shortening (Xiang and Kobilka, 2003; Xiao et al., 2006). The Gs/cAMP/PKA system serves as a tightly controlled axis to conduct 1AR signaling using negative regulators such as phosphodiesterases (PDEs) and protein phosphatases to finetune the output of signaling transduction. PDEs hydrolyze cAMP to attenuate cAMP/PKA activity. Accumulating evidences have shown that intracellular cAMP induced by AR signaling is regulated by different PDE enzymes in magnitude, time, and space, which are responsible for transiently increasing local PKA activity for a set of specific substrates (Kapiloff, 2002; Cooper, 2005; Mongillo and Zaccolo, 2006; Conti and Beavo, 2007; Houslay et al., 2007). PDEs 3 and 4 account for more than 90% of specific activity for cAMP hydrolysis in animal hearts (Richter et al., 2005). In particular, PDE4D isoforms have been shown to associate with ARs and regulate the receptor-induced cAMP accumulation in cardiac myocytes (Perry et al., 2002; MonThis work was supported by grants from the National Institutes of Health and the American Heart Association (to Y.X.). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.108.049718. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: AR, -adrenergic receptor; TnI, troponin I, PDE, phosphodiesterase; AKAP, A kinase anchoring proteins; IBMX, 3-isoproterenolbutyl-1-methylxanthine; PKA, protein kinase A; PKI, protein kinase A inhibitor; KO, knockout; PP2A, protein phosphatase 2A. 0026-895X/08/7405-1453–1462$20.00 MOLECULAR PHARMACOLOGY Vol. 74, No. 5 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 49718/3398098 Mol Pharmacol 74:1453–1462, 2008 Printed in U.S.A. 1453 http://molpharm.aspetjournals.org/content/suppl/2008/08/15/mol.108.049718.DC1 Supplemental material to this article can be found at: at A PE T Jornals on Jne 5, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 5, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 5, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 5, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on Jne 5, 2017 m oharm .aspeurnals.org D ow nladed from gillo et al., 2004; Xiang et al., 2005; Richter et al., 2008). However, recent studies suggest that cAMP accumulation is not in linear relation to the myocyte contraction responses induced by AR signaling. When the function of PDE4 is disrupted by genetic deletion or pharmacological inhibition, AR-induced cAMP can be greatly enhanced by more than 3-fold (Xiang et al., 2005). However, the higher cAMP accumulation fails to promote equivalent increases in contraction responses. We propose that further regulation downstream of cAMP accumulation plays a rate-limiting role in the contraction responses. PKA is one of the major targets of cAMP involved in cardiac myocyte contraction. Upon binding cAMP, PKA is activated to phosphorylate a wide range of downstream proteins for myocyte contraction. In contrast, protein phosphatases dephosphorylate phosphorylated proteins. In cardiac tissues, protein phosphatase 2A (PP2A) has been identified as one of the major phosphatases associated with protein contraction machinery under AR stimulation (Marks, 2001; Zhou et al., 2007). Phosphatases are often associated with scaffold protein A kinase anchoring proteins (AKAPs), which also anchor PKA holoenzymes. The locally bound PKA and phosphatase thus act together for tight regulation on phosphorylation of substrates. Thus, the tightly controlled PKA phosphorylation of substrates may be necessary to prevent myocytes from undergoing supermaximal contraction under AR stimulation with high concentrations of agonist or when cAMP hydrolysis is perturbed. We hypothesize that increased PP2A activity plays a key role to prevent hyperphosphorylation of proteins for cardiac myocyte contraction responses under AR stimulation. Here, we explored agonist dose-dependent myocyte contraction induced by 1AR signaling in neonatal and adult cardiac myocytes. We find that myocyte contraction stimulated by 1AR signaling is a saturable process and is differentially controlled by PDE (on cAMP levels) and PP2A (on protein phosphorylation by PKA) downstream of receptor/G protein-uncoupling. At submaximal doses, PDE4 ensures a tight control of cAMP accumulation at minimal levels, which leads to a small increase of PKA activity and phosphorylation of contractile proteins for contraction. In contrast, at saturated doses, despite hydrolysis of cAMP by PDE4, activation and recruitment of PP2A to PKA complexes prevent hyperphosphorylation of contractile proteins under incremental cAMP/PKA activities, which ensures saturated contraction responses in cardiac myocytes. Materials and Methods Measurements of Cell Contraction. Adult mouse ventricular myocytes were isolated from hearts of 2to 3-month-old male 2ARknockout (KO) FVB mice via a modified enzymatic technique (Zhou et al., 2000). Spontaneously beating neonatal cardiac myocytes were isolated from newborn pups from 2AR-KO mice as described previously (Devic et al., 2001). We have characterized previously 1ARs as the major AR subtype responsible for adrenergic stimulation in cardiac myocytes. Thus, 2AR-KO myocytes serve as an ideal model system to analyze activation of 1AR signaling by isoproterenol, a AR-specific agonist without the complication of signaling induced by endogenous 2AR. Adult myocytes were placed in a dish with HEPES buffer (Zhou et al., 2000) and electrically stimulated at 30 V/cm at 1 Hz at room temperature. Cell length was recorded with a charge-coupled device camera. Cell contraction shortening was analyzed by Metamorph software (Molecular Devices, Sunnyvale, CA) and normalized as the increase over the basal levels after being fitted to a sigmoidal curve. The maximal shortening was normalized to the baseline value or plotted as a percentage of the maximal response stimulated by 10 M forskolin. Measurement of spontaneous neonatal cardiac myocyte contraction rate was carried out as described previously (Devic et al., 2001). The responses in myocyte contraction velocity after drug treatments was analyzed by Metamorph software (Spinale et al.,