Differential regulation of cardiac excitation-contraction coupling by cAMP phosphodiesterase subtypes

Aims Multiple phosphodiesterases (PDEs) hydrolyze cAMP in cardiomyocytes, but the functional significance of this diversity is not well understood. Our goal here was to characterize the involvement of three different PDEs (PDE2-4) in cardiac excitation-contraction coupling (ECC). Methods Sarcomere shortening and Ca 2+ transients were recorded simultaneously in and results adult rat ventricular myocytes and ECC protein by PKA was determined by blot Under basal selective inhibition a small but significant increase in Ca transients, sarcomere shortening, and troponin I whereas inhibition had no inhibition, but not or increased phospholamban phosphorylation. Inhibition of either 3 or 4 increased phosphorylation of the myosin binding protein C, had an effect on L-type Ca 2+ channel or ryanodine receptor phosphorylation. Dual inhibition of PDE2 and PDE3 or PDE2 and PDE4 further increased ECC compared to individual PDE inhibition but the most potent combination was obtained when PDE3 and PDE4. This combination also induced a synergistic induction of ECC protein phosphorylation. Submaximal β-adrenergic receptor stimulation increased ECC and this effect was potentiated by individual PDE inhibition with the Identical results were obtained on stimulation or PDE3 inhibition. The were stripped and reprobed with calsequestrin antibodies (CSQ) used as a loading control. A representative blot is shown, and at least 5 separate experiments were performed (n=6 for n=5 for P-MyBP-C, n=6 for P-PLB, n=5 for P-RyR and n=5 for P-Ca 1.2), giving similar results. Phosphorylated proteins/CSQ ratios were quantified, expressed as means ± SEM and normalized to untreated ARVMs (Ctrl). Statistical significance is indicated as: (One-way ANOVA, Newman-Keuls).


Introduction
The cAMP pathway is critical for autonomic regulation of the heart. Sympathetic stimulation increases myocardial contractility mainly through stimulation of β-adrenergic receptors (β-ARs), elevation of intracellular cAMP ([cAMP]i) and activation of the cAMP-dependent protein kinase (PKA). PKA in turn phosphorylates key components of cardiac excitationcontraction coupling (ECC) such as the L-type Ca 2+ channel (Cav1.2), ryanodine receptor type 2 (RyR2), phospholamban (PLB), troponin I (TnI) and myosin-binding protein C (MyBP-C).
The phosphorylation of Cav1.2 and RyR2 leads to enhanced Ca 2+ influx and sarcoplasmic reticulum (SR) Ca 2+ release, contributing to enhanced Ca 2+ transients. PLB phosphorylation increases SR Ca 2+ uptake by the Ca 2+ -ATPase (SERCA2), thus accelerating cardiac relaxation but also increasing SR Ca 2+ load and consequently SR Ca 2+ release. The phosphorylation of TnI and MyBP-C reduces myofilament Ca 2+ affinity and increases crossbridge kinetics. Altogether, these events produce the typical inotropic and lusitropic effects of β-AR stimulation. 1 Intracellular cAMP levels result from the balance between cAMP synthesis by adenylyl cyclases and cAMP degradation by cyclic nucleotide phosphodiesterases (PDEs). PDEs are subdivided into 11 families among which four can hydrolyse cAMP in the heart: PDE1, which is activated by Ca 2+ /calmodulin; PDE2, which is stimulated by cGMP; PDE3, which is inhibited by cGMP; and PDE4. 2 While PDE1 and PDE2 can hydrolyse both cAMP and cGMP, PDE3 preferentially hydrolyses cAMP and PDE4 is specific for cAMP. Another PDE, named PDE8A which specifically hydrolyses cAMP, was shown recently to control ECC in mouse cardiac myocytes. 3 All PDE isoforms except PDE8A 4,5 are inhibited by xanthine derivatives such as 3isobutyl-1-methylxanthine (IBMX), and a number of drugs have been developed as selective PDE inhibitors 6,7 : EHNA 8 and Bay 60-7550 9 for PDE2; milrinone, cilostamide and other cAMP PDEs and cardiac EC coupling -D. Mika et al. 4 bipyridines for PDE3 7 ; rolipram and Ro 20-1724 for PDE4. 7 There is currently no commercially available selective inhibitor of PDE1.
Direct monitoring of cAMP in living cardiac myocytes emphasized the importance of PDE isoforms 2 to 4 for the control of [cAMP]i. In rodent heart, a predominance of PDE4 over other PDEs for the control of cAMP signals generated by stimulation of β-ARs and other GsPCRs was observed. [10][11][12][13][14] PDE4 was shown to associate with β-ARs and ECC proteins, either directly or through scaffold proteins such as β-arrestins or A-kinase anchoring proteins. 15,16 In mouse heart, a specific PDE4 variant (PDE4D3) was shown to control the phosphorylation of RyR2 and regulates diastolic SR Ca 2+ leak. 17 Similarly, a PDE4D variant was found to coimmunoprecipitate with SERCA2 in mouse and to control PLB phosphorylation and Ca 2+ transients decay kinetics. 18 As Cav1.2 is a well characterized target of the cAMP/PKA pathway in heart, the cardiac L-type Ca 2+ channel current (ICa,L) has been frequently used as a functional index of the contribution of PDE isoforms to this pathway. Inhibition of PDE2, PDE3 and PDE4 was shown to increase ICa,L amplitude in a number of species. 12,[19][20][21][22][23][24] Recently, we observed in mouse heart that a PDE4B variant associates with Cav1.2 and controls its activity upon β-AR stimulation. 25 However, important differences exist among species as to whether PDE3 or PDE4 is predominant and whether all three PDE isoforms control the ICa,L amplitude at basal or only upon β-AR stimulation. PDE3 inhibitors were also reported to increase SR Ca 2+ uptake and SR Ca 2+ content, [26][27][28] an effect attributed to an increase in PKA-dependent phosphorylation of PLB.
While it is well established that PDE3 inhibition exerts a direct positive inotropic effect in the heart from large mammals, 29-31 the contribution of other PDE families to the regulation of cardiac contractility remains less clear and often controversial. 32 For instance, while selective inhibition of PDE2 or PDE4 was shown to increase cardiac contractility following β-AR stimulation in some studies, [33][34][35][36][37] other studies failed to demonstrate an effect. 38,39 cAMP PDEs and cardiac EC coupling -D. Mika et al.

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In an attempt to reconcile these contradictory findings, we compared, in the same mammalian cardiac preparation (adult rat ventricular myocytes), the effect of a selective inhibition of PDE2, PDE3 and PDE4 on Ca 2+ transients, sarcomere shortening, and phosphorylation of five key ECC proteins (TnI, Cav1.2, RyR2, PLB and MyBP-C).
Furthermore, we compared for each PDE isoform, the effects observed under basal condition and after sub-maximal β-AR stimulation with isoprenaline. Our study sheds new light on the respective contribution of the three major cardiac PDE isoforms in the regulation of cardiac ECC.  Table 1). IBMX (3-isobutyl-1-methylxanthine) and isoprenaline (Iso) were from Sigma Aldrich (Saint Quentin, France).

Data Analysis
Cell contraction was assessed by the percentage of sarcomere shortening, which is the ratio of twitch amplitude (difference of end-diastolic and peak systolic sarcomere lengths) to enddiastolic sarcomere length (SL). Ca 2+ transient amplitude was assessed by the percentage of variation of the Fura-2 ratio, by dividing the twitch amplitude (difference of end-diastolic and peak systolic ratios) to end-diastolic ratio. Relaxation was assessed by measuring the time-to-50% relaxation from the time to peak shortening, and the Ca 2+ transient decay was evaluated by measuring the time-to-50% decay of the Fura-2 ratio from the time to peak ratio. These parameters were obtained by analyzing the 10 last contractions and calcium transients before addition of the next drug. All parameters were calculated offline on a dedicated software (IonWizard 6x, IonOptix). All results are expressed as mean±SEM. GraphPad Prism software (GraphPad software Inc., La Jolla, CA, USA) was used for statistical analysis. To determine statistical significance with multiple groups, we used a One-way ANOVA followed by a Newman-Keuls test for multiple comparisons. Differences with p values <0.05 were considered as statistically significant. The number of independent experiments performed is indicated in the figure legends.

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To investigate the functional consequences of individual PDE inhibition on basal and β-AR stimulated ECC in ARVMs, Ca 2+ transients and sarcomere shortening were simultaneously recorded in Fura-2-loaded ARVMs. In quiescent myocytes, the average diastolic sarcomere length (SL) was 1.68±0.01 µm (n=95) (Supplemental Table 2). Upon pacing at 0.5 Hz, the average peak amplitude of Ca 2+ transients was 22±0.1% above the diastolic Fura-2 ratio (Supplemental Table 3), whereas sarcomere length decreased by 0.78±0.05% (n=95) (Supplemental Table 1 control). As shown in Supplemental Figure 1, the Iso response on Ca 2+ transients and cell shortening was stable during the 10 min incubation period suggesting that β-AR stimulation by 1 nM Iso does not induce desensitization of the receptors, in our experimental conditions.
In a first series of experiments, the effects of PDE2 inhibition were tested ( Fig. 1 and Supplemental Table 2 (Fig. 1C). These effects were small when compared to that of 1 nM Iso. To examine whether PDE2 modulates ECC under β-AR stimulation, 100 nM Bay was applied on top of Iso.
As shown in Fig. 1A and on the Supplemental Table 2 and 3, the Iso effect was potentiated upon PDE2 inhibition. However, the decay kinetics of the signals were unchanged compared to those obtained under Iso stimulation (Fig. 1C).
cAMP PDEs and cardiac EC coupling -D. Mika et al. 11 We next studied the consequences of a selective PDE3 inhibition on Ca 2+ transients and cell contraction using Cil ( Fig. 2 and Supplemental Table 2 and 3). Application of 1 µM Cil alone induced a significant increase in Ca 2+ transient and sarcomere shortening amplitudes by approximately 2-and 3-fold, respectively ( Fig. 2A and 2B). Cil also significantly accelerated the decay kinetics of both signals (Fig. 2C). This was accompanied by an increase in SR Ca 2+ load with no change in SR fractional Ca 2+ release (Fig. 5). PDE3 inhibition also strongly potentiated the effect of Iso on both parameters, enhancing their amplitude by 30% for Ca 2+ transient and 70% for cell shortening (Fig. 2B), and accelerating their relaxation kinetics (Fig.   2C).
In contrast to the results obtained with PDE2 or PDE3 inhibitors, PDE4 inhibition by 10 µM Ro had no effect on Ca 2+ transients and sarcomere shortening under basal conditions ( Fig. 3A and 3B and Supplemental Table 2 and 3). However, Ro strongly potentiated the effects of Iso (1 nM), further increasing the Ca 2+ transients amplitude and sarcomere shortening by 60% (Fig. 3B). PDE4 inhibition also significantly accelerated the decay kinetics of both signals (Fig. 3C).
We next examined the functional consequences of combinations of selective PDE inhibitors on cell contraction under basal conditions ( Fig.4 and Supplemental Table 2). As shown in Fig. 4A

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to control (Fig. 4C), t1/2 off values of sarcomere shortening were significantly smaller for Bay + Cil as compared to Bay alone (compare Fig. 1C and 4C) but not as compared to Cil alone (compare Fig. 2C and 4C). This suggests that PDE3 has a more prominent role in controlling cell relaxation than PDE2 under basal conditions. Concomitant inhibition of PDE3 and PDE4 by Cil + Ro had a major impact on ECC, increasing the amplitude of Ca 2+ transients 2.5-fold and sarcomere shortening 15-fold (Fig. 4B). This was accompanied by a major acceleration of Ca 2+ transients and cell shortening decay phases, which was significantly larger than with Bay + Cil (Fig. 4C). Furthermore, Cil + Ro doubled SR Ca 2+ load and drastically increased fractional Ca 2+ release from 40% in Ctrl or after PDE3 inhibition, to 80% when both PDE3 and PDE4 were inhibited (Fig. 5). Finally, application of the broad spectrum PDE inhibitor IBMX (100 µM) had similar functional effects as a concomitant inhibition of PDE3 and PDE4.

Regulation of the phosphorylation status of key ECC proteins by PDEs
To get some insights into the molecular mechanisms by which individual PDE families regulate ECC in ARVMs, we examined the consequences of PDE inhibition on the phosphorylation of key ECC proteins. For this, we performed Western blot analysis with phospho-specific antibodies directed against TnI, MyBP-C, PLB, RyR2 and Cav1.2 under basal conditions ( Fig.   6) or after a non maximal β-AR stimulation by Iso (1 nM) (Fig. 7). In line with ECC measurements, Fig. 6 shows that global PDE inhibition with IBMX or concomitant inhibition of PDE3 and PDE4 by Cil + Ro led to a major increase in the phosphorylation of TnI (Fig. 6A), MyBP-C (Fig. 6B), PLB (Fig. 6C), RyR2 (Fig. 6D) and CaV1.2 (Fig. 6E). Individual inhibition of PDE2 and PDE3 as well as simultaneous inhibition of PDE2 and PDE3 or PDE2 and PDE4 slightly but significantly increased the phosphorylation of TnI, whereas inhibition of PDE4 alone did not (Fig. 6A). In contrast, MyBP-C phosphorylation was significantly increased by selective inhibition of PDE2, PDE3 or PDE4 and by their dual inhibition, especially in the case cAMP PDEs and cardiac EC coupling -D. Mika et al.

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of PDE3 and PDE4 (Fig. 6B). Selective PDE3 inhibition by Cil or simultaneous inhibition of PDE2 and PDE3 also increased significantly PLB phosphorylation whereas individual or associated PDE2 and PDE4 inhibition did not (Fig. 6C). The phosphorylation of CaV1.2 and RyR2 was only increased by simultaneous blockade of PDE3 and PDE4 or global PDE inhibition with IBMX ( Fig. 6D and E).

Discussion
It is well established that cardiac ECC is regulated by the cAMP/PKA pathway. Initiation of the pathway takes place at the sarcolemmal membrane by cAMP synthesis through the activity of adenylyl cyclases; termination of the pathway involves the activity of cyclic nucleotide PDEs which are responsible for the hydrolysis of cAMP into 5'-AMP, and phosphatase activity which  shortening (Fig. 4, 6). Other dual combinations of PDE inhibition had much smaller effects on protein phosphorylation and ECC, indicating that PDE3 and PDE4 can compensate for each other, and that their function is partially redundant.
Although PDE2 represents only a few percents of total hydrolytic activity in ARVMs, 12 we show here that it regulates basal and β-AR-stimulated ECC. This is consistent with previous studies showing that PDE2 modulates cAMP levels and β-AR responses in rodent cardiac myocytes. 23,33 In particular, PDE2 modulates the β-AR regulation of the L-type Ca 2+ current (ICa,L) in cardiac myocytes 23 thus providing a possible mechanism for the effects of PDE2 inhibition on Iso-stimulated Ca 2+ transients and contraction. In contrast, the mechanism by which PDE2 regulates basal ECC is less clear since PDE2 inhibition has no effect on basal ICa,L in ARVMs. 23 MyBP-C could participate in the effects of PDE2 on basal ECC by increasing cross bridge cycling and myofilament Ca 2+ sensitivity. 50 In contrast to PDE4, selective PDE3 inhibition increased the amplitude and relaxation kinetics of Ca 2+ transients and sarcomere shortening under basal conditions (Fig. 2), and this was associated with enhanced phosphorylation of PLB, TnI and MyBP-C but not Cav1.2 or RyR2 (Fig. 6). These data are in line with previous findings showing a subcellular localization of PDE3 in SR-enriched membrane fraction 51,52 and the positive effects of PDE3 inhibitors on SR Ca 2+ uptake and SR Ca 2+ content. [26][27][28] This is also consistent with the lack of effect of selective PDE3 inhibition on basal ICa,L in ARVMs. 23 In the present study, we established that PDE2, PDE3 and PDE4 have distinct roles in controlling ECC. In the absence of hormonal stimulation, when intracellular cAMP concentration is low, PDE2 and especially PDE3 are dominant to control cardiac contraction.
PDE3 contribution to the degradation of basal cAMP could be explained by its high affinity for cAMP (Km<1µM). 53 For PDE2, the scheme must be different because of its low affinity for cAMP. Nonetheless, under basal conditions, localized production of cGMP may occur to

Limitations
The present study focused on the role of three different PDE families in controlling the ECC in isolated ARVMs. It is known that the expression level of these PDEs varies among different species and is dependent on the development stage and on the cardiac territory investigated. 32 However, PDE expression is relatively conserved between murine models and human. 58 In mammalian heart, PDE3 and PDE4 remain the two major enzymes degrading cAMP and controlling cardiac ECC. Thus, even if our results might not be fully transposable to any cellular cardiac models or human heart, we unveil how cardiac function is finely tuned by these PDE families. Furthermore, their expression and distribution are altered under pathophysiological conditions. 59 Therefore, the relative contribution of PDE2, PDE3 and PDE4 to compartmentalize cAMP signals and maintain specific PKA phosphorylation of the key ECC           The inhibition constants Ki of each PDE inhibitor are reported in µM for each PDE family.