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(Circulation Research. 2007;101:400.)
© 2007 American Heart Association, Inc.

Cellular Biology

PI3K Is Required for PDE4, not PDE3, Activity in Subcellular Microdomains Containing the Sarcoplasmic Reticular Calcium ATPase in Cardiomyocytes

Benoit-Gilles Kerfant, Dongling Zhao, Ilka Lorenzen-Schmidt, Lindsay S. Wilson, Shitian Cai, S. R. Wayne Chen, Donald H. Maurice, Peter H. Backx

From the Departments of Physiology and Medicine, the Heart & Stroke Richard Lewar Centre, and the Division of Cardiology at the University Health Network (B.-G.K., D.Z., I.L.-S., P.H.B.), University of Toronto; the Departments of Pharmacology & Toxicology (L.S.W., D.H.M.), Queen’s University, Kingston; and the Departments of Physiology and Biophysics (S.C., S.R.W.C.), University of Calgary, Canada.

Correspondence to Peter H. Backx, DVM, PhD, Professor, 150 College St, Toronto, ON, M5S 3E2, Canada. E-mail p.backx{at}utoronto.ca

	Abstract

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We recently showed that phosphoinositide-3-kinase-–deficient (PI3K^–/–) mice have enhanced cardiac contractility attributable to cAMP-dependent increases in sarcoplasmic reticulum (SR) Ca²⁺ content and release but not L-type Ca²⁺ current (I_Ca,L), demonstrating PI3K locally regulates cAMP levels in cardiomyocytes. Because phosphodiesterases (PDEs) can contribute to cAMP compartmentation, we examined whether the PDE activity was altered by PI3K ablation. Selective inhibition of PDE3 or PDE4 in wild-type (WT) cardiomyocytes elevated Ca²⁺ transients, SR Ca²⁺ content, and phospholamban phosphorylation (PLN-PO₄) by similar amounts to levels observed in untreated PI3K^–/– myocytes. Combined PDE3 and PDE4 inhibition caused no further increases in SR function. By contrast, only PDE3 inhibition affected Ca²⁺ transients, SR Ca²⁺ loads, and PLN-PO₄ levels in PI3K^–/– myocytes. On the other hand, inhibition of PDE3 or PDE4 alone did not affect I_Ca,L in either PI3K^–/– or WT cardiomyocytes, whereas simultaneous PDE3 and PDE4 inhibition elevated I_Ca,L in both groups. Ryanodine receptor (RyR₂) phosphorylation levels were not different in basal conditions between PI3K^–/– and WT myocytes and increased in both groups with PDE inhibition. Our results establish that L-type Ca²⁺ channels, RyR₂, and SR Ca²⁺ pumps are regulated differently in distinct subcellular compartments by PDE3 and PDE4. In addition, the loss of PI3K selectively abolishes PDE4 activity, not PDE3, in subcellular compartments containing the SR Ca²⁺-ATPase but not RyR₂ or L-type Ca²⁺ channels.

Key Words: cardiomyocytes • PI3K • PDE3 • PDE4 • excitation-contraction-coupling

	Introduction

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Phosphoinositide 3-kinases (PI3Ks) are a family of enzymes that have the unique capacity to function as both lipid and protein kinases. PI3Ks have been divided into 3 classes (I, II, and III) based on their substrate specificity, mode of activation, and molecular structure.¹ PI3K and PI3K, which are members of PI3K subclass IA and IB, respectively, are the two main isoforms expressed in cardiomyocytes.¹ Whereas PI3K regulates heart growth,² PI3K has emerged as an important regulator of cardiac contractility and cardiac excitation-contraction coupling (ECC)^3–7 by regulating basal cAMP levels^3–5,8 and phospholamban (PLN) phosphorylation.^3,5 We recently established that cardiomyocytes lacking PI3K have cAMP-dependent increases in sarcoplasmic reticulum (SR) Ca²⁺ load and release without changes in L-type Ca²⁺ current (I_Ca,L),^7,9 which is a prototype for regulation by cAMP-dependent protein kinase A (PKA) (see review⁸). Although the basis for the cAMP compartmentation in PI3K-deficient mice is unclear, phosphodiesterases (PDEs) are a family of enzymes that are critical components of macromolecular complexes involved in the subcellular compartmentation of cAMP-PKA signaling^8,10–12 by locally hydrolyzing cAMP or cGMP.^13,14 For instance, cardiac-specific overexpression of human adenylate cyclase type 8 (AC8) in mice enhances cAMP levels, contractility, and Ca²⁺ transient, without affecting I_Ca,L¹⁵ because of PDE-based compartmentation of cAMP hydrolysis.¹⁰ Although the dominant PDEs regulating cAMP-PKA in heart are PDE3A and several members of the PDE4 family,^16,17 a recent study reported that PDE3B activity is eliminated in PI3K^–/–-deficient hearts^5,6 and that PI3K stimulates PDE3B,^5,18 even though PDE3B is primarily expressed in vascular smooth muscle cells and not in cardiomyocytes.^13,19

The aim of this study was to elucidate the involvement of PDEs in the regulation of L-type Ca²⁺ channels and SR Ca²⁺ handling by PI3K. Our results demonstrate that both PDE3 and PDE4 activities are required in mouse myocardium to maintain cAMP-PKA signaling at baseline levels within microdomains containing SR Ca²⁺ pumps and RyR₂, but not in microdomains surrounding L-type Ca²⁺ channels. In addition, the loss of PI3K abolishes PDE4, but not PDE3B, activity in microdomains containing SR Ca²⁺ pumps.

	Materials and Methods

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Mouse Usage and Cardiomyocytes Isolation
The generation of WT and PI3K^–/– mice has previously been described²⁰ (see supplemental materials, available online at http://circres.ahajournals.org).

Electrophysiology and SR Ca²⁺ Content
I_Ca,L was measured at room temperature (22°C, 1 mL min^–1) with whole-cell patch-clamp technique²¹ under voltage-clamp mode, whereas the SR Ca²⁺ content was estimated by integrating the Na⁺-Ca²⁺ exchanger current (I_NCX) induced by brief (10 sec) applications of 20 mmol/L caffeine, as previously described⁷ (see supplemental materials).

Fluorescence
Voltage-clamped myocytes were loaded via the patch-pipette with fluo-3, 5K⁺ salt to record Ca²⁺ transients simultaneously to I_Ca,L, as previously described⁷ (see supplemental materials).

Western Blots
The phosphorylation levels of PLN and cardiac RyR₂ were measured as previously described.^3,22 We measured RyR₂ phosphorylation at position S2030 because, unlike S2808, S2030 shows very low baseline phosphorylation.²² (see supplemental materials).

Statistics
Statistical significance was assessed using a 1-way analysis of variance (ANOVA) followed by either a Student Neuman-Keuls test or a Kruskal-Wallis test when the data were nonparametric. Paired t tests were used when comparing results from the same cardiomyocyte. P<0.05 was considered statistically significant. Data are presented as mean±SEM.

	Results

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Because PDEs are critical determinants of subcellular cAMP-PKA compartmentation,^10–12 we examined whether the major PDEs isoforms regulating cAMP in myocardium (ie, PDE3 and PDE4)^24,25 mediate the effects of PI3K on cAMP compartmentation in vicinity of the SR. Figure 1 shows that perfusion of myocytes with milrinone for 8 minutes, at doses capable of selectively inhibiting PDE3,^13,26 increased amplitudes and accelerated decay rates (Table) of Ca²⁺ transients in both WT and PI3K^–/– myocytes, establishing that PDE3 is active in PI3K^–/–. To confirm that the effects of milrinone were selective,²⁶ the PDE3 inhibitor, cilostazol, at doses that selectively inhibit PDE3,²⁶ yielded identical results to milrinone (supplemental Table I). Furthermore, 1 µmol/L milrinone had no effect, whereas the effects of 100 µmol/L milrinone were indistinguishable from 10 µmol/L milrinone (supplemental Table I).

View larger version (27K): [in this window] [in a new window]   Figure 1. Effects of PDE3 inhibition on Ca2+ transients and ICa,L. A, Representatives traces of ICa,L (bottom) and Ca2+ transients (top) recorded simultaneously at 0 mV before (Ctr) and after 10 µmol/L milrinone (Mil) using the voltage protocol shown. Dotted lines represent 0 current for ICa,L traces and a F/F0 ratio of 1. B, Peak ICa,L (bottom) and F/F0 (top) as a function of voltage recorded in PI3K–/– (circles, n=6) and WT (squares, n=6) cardiomyocytes, before (unfilled) and after (filled) milrinone application. *P<0.01 (same group); P<0.01 (between WT and KO).

View this table: [in this window] [in a new window]   Table 1. Mean Ca2+ Transient and ICa,L Amplitudes and Kinetics Measured at 0 mV in PI3K–/– and WT Cardiomyocytes Before and After Specific PDE3 (Mil 10 µmol/L), Specific PDE4 (Rol, 10 µmol/L), Combined PDE3 and PDE4 (Mil+Rol), or all PDEs (IBMX, 50 µmol/L) Inhibition

In contrast, rolipram application, at doses that selectively inhibit PDE4,¹³ had no effect on Ca²⁺ transients in PI3K^–/– myocytes, while enhancing the amplitude and decay rates of fluo-3 fluorescence in WT myocytes to levels indistinguishable from untreated PI3K^–/– myocytes (Figure 2 and Table). These findings suggest that PDE4 activity is lost in PI3K^–/– myocytes. As expected from pharmacological studies,¹⁹ 1 µmol/L rolipram had no effect (supplemental Table I), whereas 100 µmol/L rolipram had similar effects to those observed with 10 µmol/L (Table S1), demonstrating specificity for PDE4 inhibition.

View larger version (25K): [in this window] [in a new window]   Figure 2. Effects of PDE4 inhibition on Ca2+ transients and ICa,L. A, Representatives traces of ICa,L (bottom) and Ca2+ transients (top) recorded simultaneously at 0 mV before (Ctr) and after 10 µmol/L rolipram (Rol) perfusion. B, Peak ICa,L (bottom) and F/F0 (top) as a function of voltage recorded in PI3K–/– (circles, n=7) and WT (squares, n=7) cardiomyocytes, before (unfilled) and after (filled) rolipram application *P<0.01 (same group); P<0.01 (between WT and KO).

The changes in F/F₀ ratios induced by PDE inhibition were not related to cell shortening (which was as high as 12%, data not shown), because cell shortening leads to small underestimations of the Ca²⁺ transient amplitudes (ie, about 6%, data not shown). Thus, the elevated cell shortening observed with PDE3 and PDE4 inhibition cannot underlie the increased amplitudes or accelerated kinetics of the F/F₀ ratios observed (see supplemental materials).

To assess the effects of PDEs inhibition on I_Ca,L, we measured I_Ca,L both at 8 minutes after membrane rupture in the absence of drug and again after 8 minutes of drug application (ie, 16 minutes after membrane rupture). PDE3 inhibition did not affect (P>0.5) I_Ca,L amplitudes (Figure 1B) and did not change either the maximum conductance of I_Ca,L (ie, G_max) in WT myocytes (112±14 pS/pF control versus 98±10 pS/pF milrinone, n=7) and PI3K^–/– myocytes (117±5 pS/pF control versus 111±9 pS/pF milrinone, n=8) or voltage for half-maximal activation (V_1/2) of I_Ca,L (supplemental Table I). Milrinone did, however, accelerate the Ca²⁺-dependent phase of I_Ca,L inactivation (ie, _fast) in both groups (Table), consistent with increased Ca²⁺ transients^7,8 (see below). The absence of I_Ca,L changes were not caused by rundown because no differences in I_Ca,L were observed when comparisons were made between treated and untreated myocytes 16 minutes after membrane rupture (supplemental Figure I).

PDE4 inhibition also did not affect I_Ca,L (Figure 2B). Indeed, rolipram did not change (P>0.5) either G_max of I_Ca,L in WT myocytes (107±4 pS/pF control versus 94±6 pS/pF rolipram, n=6) and PI3K^–/– (115±9 pS/pF control versus 101±11 pS/pF rolipram, n=8) myocytes or V_1/2 of I_Ca,L (Table S1). Rolipram did, however, abbreviate _fast in WT myocytes, but not in PI3K^–/– myocytes (Table), because of secondary effects on Ca²⁺ transients (See below). Again, no differences in I_Ca,L amplitudes were observed 16 minutes after membrane rupture between myocytes treated with PDE4 inhibitors and untreated myocytes (supplemental Figure I).

Because SR Ca²⁺ release strongly affects I_Ca,L via Ca²⁺-mediated inactivation, the effects of PDEs inhibition could be partially masked by simultaneous changes in Ca²⁺ transients. Indeed, the fast component of the inactivation rate of I_Ca,L (ie, _fast), which reflects Ca²⁺-mediated inactivation, correlated strongly with Ca²⁺ transient amplitudes (Table). Thus, I_Ca,L measurements were repeated in myocytes dialyzed with 4 mmol/L EGTA to suppress SR Ca²⁺ release.²⁷ Although EGTA does not eliminate Ca²⁺-mediated inactivation,²⁸ it did abolish differences in _fast as expected.²⁷ With EGTA in the pipette, PDE3 or PDE4 inhibition alone did not affect I_Ca,L amplitudes or G_max in either WT or PI3K^–/– myocytes (data not shown).

The results above suggest that PDEs regulate baseline cAMP levels in subcellular compartments containing the SR, but not L-type Ca²⁺ channels, and that PI3K is required for PDE4 activity in the SR compartment. To more directly investigate SR function, SR Ca²⁺ content was measured in myocytes dialyzed with clamped free Ca²⁺ concentrations (75 nmol/L). After 8 minutes of dialysis, caffeine (20 mmol/L) was applied to induce SR Ca²⁺ release, allowing SR Ca²⁺ content to be measured by integrating the forward-mode NCX current (I_NCX).²⁹ Figure 3 shows that, without PDEs inhibitors, the I_NCX was higher (P<0.05) in PI3K^–/– compared with WT myocytes. Milrinone increased I_NCX in both groups, whereas PDE4 inhibition only increased I_NCX in WT myocytes. Decay times for caffeine-induced I_NCX were not different between the groups (data not shown), suggesting that I_NCX densities were not detectably altered by PI3K ablation.

View larger version (48K): [in this window] [in a new window]   Figure 3. Effects of PDE4 inhibition on SR Ca2+ content and PLN phosphorylation. A, INCX recordings at –80 mV after rapid application of 20 mmol/L caffeine before (left) and after PDE3 (middle) or PDE4 (right) inhibition. B, Bar graph of the integrated INCX in PI3K–/– (black bars) and WT (white bars) cardiomyocytes before (Ctr, filled-bars) and after (hatched-bars) PDE3 or PDE4 inhibition. C, Western Blots for PLN-PO4 (top) and PLN (bottom) expression in PI3K–/– (right) and WT (left) cardiomyocytes before and after treatment with 10 µmol/L milrinone or 10 µmol/L rolipram. D, Bar graph summarizing PLN-PO4/PLN ratios in PI3K–/– and WT cardiomyocytes. *P<0.05, **P<0.01 (same group); P<0.05 (between WT and KO).

Consistent with the observed differences in SR Ca²⁺ content, the PLN-PO₄/PLN ratio was elevated in PI3K^–/– compared with WT myocytes (Figure 3C and 3D). PDE3 inhibition increased PLN-PO₄/PLN in both groups, whereas PDE4 inhibition enhanced this ratio in WT myocytes only. Although alterations in PLN-PO₄ can explain the functional differences between the various groups, RyR₂ phosphorylation could also contribute to enhanced SR Ca²⁺ release.³⁰ Under basal conditions, RyR₂-PO₄ levels at position S2030, expressed as a fraction of the maximal phosphorylation achieved with isoproterenol treatment, did not differ (P=0.6) between WT (0.06±0.02, n=4) and PI3K^–/– (0.04±0.02, n=4) myocytes (Figure 4). On the other hand, PDE3 or PDE4 inhibition alone increased (P<0.05) RyR₂-PO₄ levels about 2 to 3 times in both groups, revealing that the loss of PI3K does not abolish PDE4 activity in junctional SR microdomains containing RyR₂, unlike SERCA2a-containing compartments.

View larger version (28K): [in this window] [in a new window]   Figure 4. Effects of PDE4 inhibition on RyR2 phosphorylation. A, Western blots for RyR2 and RyR2-PO4 measured at serine-2030 for PI3K–/– (n=4) and WT (n=4) mice, in basal conditions (Ctr) and after 8 minutes of milrinone (Mil) or rolipram (Rol) treatment. B, RyR2-PO4-2030 for PI3K–/– and WT mice normalized to maximum levels of phosphorylation achieved with 1 µmol/L of isoproterenol; *P<0.01 (same groups).

Because cAMP inhibitors reduce Ca²⁺ transients and SR Ca²⁺ content in PI3K^–/– myocytes,^7,8 but not WT, our findings support a model (Figure 6A) where both PDE3 and PDE4 activities are required to maintain cAMP at basal levels in a common microdomains containing SERCA2a-PLN, with PI3K being necessary for PDE4 activity. This model predicts that Ca²⁺ transients in WT and PI3K^–/– myocytes should be indistinguishable when PDE3 and PDE4 are inhibited simultaneously. However, Figure 5A shows that Ca²⁺ transients in WT myocytes treated with combined PDE3 and PDE4 inhibitors were not different from those with PDE3 or PDE4 inhibition. Moreover, with combined PDE3 and PDE4 inhibition, Ca²⁺ transients in WT myocytes were below those observed in PI3K^–/– myocytes. On the other hand, Ca²⁺ transients in PI3K^–/– myocytes with combined PDE3 and PDE4 inhibition were the same as those with PDE3 inhibition alone. These observations could be explained if other PI3K-regulated PDEs also influence cAMP levels in the SR subdomain. However, Ca²⁺ transients with global inhibition of PDEs using IBMX (50 µmol/L) were identical to those observed when PDE3 and PDE4 were inhibited simultaneously (Table). Taken together, these results suggest that multiple microdomains with distinct cAMP regulation exist in the SR subcellular compartment (see Discussion).

View larger version (26K): [in this window] [in a new window]   Figure 5. Effects of combined inhibition of PDE3 and PDE4 on Ca2+ transients and ICa,L. A, Peak ICa,L (bottom) and F/F0 (top) as a function of voltage recorded in PI3K–/– (circles, n=8) and WT (squares, n=6) cardiomyocytes before (empty symbols) and after Mil+Rol perfusion (filled symbols). B, ICa,L-voltage relationship recorded with 4 mmol/L EGTA in PI3K–/– (circles, n=8) and WT (squares, n=7) cardiomyocytes, before (empty symbols) and after Mil+Rol perfusion (filled symbols). *P<0.01, **P<0.001 (same group); P<0.01 (between WT and KO).

When Ca²⁺ transients were present, combined inhibition of PDE3 and PDE4 did not increase (P=0.6) G_max for I_Ca,L in WT myocytes but increased G_max (P<0.05) in PI3K^–/– (Figure 5A). With EGTA in the pipette, combined PDE3 and PDE4 inhibition increased (P<0.001) G_max by 63±9% and shifted (P<0.001) V_1/2 from –12.5±0.8 mV to–20.7±0.9 mV in PI3K^–/–, while increasing (P<0.05) G_max by only 15±10% and modestly shifting (P<0.05) V_1/2 from–11.4±0.8 mV to –17.6±2.0 mV in WT myocytes. The larger increases in I_Ca,L observed when EGTA was included in the pipette (Figure 5B) may be related to reductions in either Ca²⁺-dependent inhibition of AC isoforms³¹ or Ca²⁺-mediated I_Ca,L inactivation, as discussed below.

	Discussion

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Previously we established that elevated cardiac contractility in PI3K^–/– mice results from increased SR Ca²⁺ cycling without changes in I_Ca,L.^3,7,8 In this study we found that both PDE3 and PDE4 activities are required in mouse myocardium to maintain cAMP-PKA signaling at baseline levels within microdomains containing SR Ca²⁺ pumps and RyR₂, but not in microdomains surrounding L-type Ca²⁺ channels. In addition, the loss of PI3K abolishes PDE4 activity in microdomains containing SR Ca²⁺ pumps.

The increased SR Ca²⁺ content as well as elevated Ca²⁺ transient amplitudes and relaxation rates observed with either PDE3 and PDE4 inhibition in WT myocytes are consistent with previous studies.^{13,19,26,32–35} The Ca²⁺ changes with PDE inhibition appeared to result from local cAMP elevations because they were tightly linked to increased PLN-PO₄/PLN ratios without alterations in I_Ca,L. It is conceivable that increased RyR₂-PO₄ levels also contribute to the changes in Ca²⁺ homeostasis observed with PDE inhibition because RyR₂ phosphorylation enhances SR Ca²⁺ leak¹² and release.³⁰ However, RyR₂ phosphorylation is predicted to decrease SR Ca²⁺ loads and to have little effect on Ca²⁺ transient relaxation, contrary to our observations. Importantly, because PDE4 and PDE3 inhibition alone had similar effects on Ca²⁺ cycling and because cAMP antagonists don’t affect Ca²⁺ cycling in mouse myocytes at baseline,^7,8 our results establish that PDE3 and PDE4 enzymes are equally important in maintaining the cAMP at basal levels in microdomains containing SERCA2a-PLN. This conclusion is consistent with the high expression levels of PDE3A and PDE4D we observed in mouse cardiomyocytes, as reported previously^13,19 as well as with the ability of PDE4D to coimmunoprecipitate with SERCA2a (supplemental Figure II). Moreover, PDE3A can be isolated from cardiac SR microsomal fractions,^36,37 and PDE3 inhibition increases SERCA2a activity^36,37 in cardiomyocytes.

A rather unexpected finding in our studies was the inability of combined PDE3 and PDE4 to alter Ca²⁺ transients beyond that observed with PDE3 or PDE4 inhibition alone. These observations could be explained by several mechanisms that are not mutually exclusive. For example, other PDEs isoforms could assist in the locally controlling cAMP hydrolysis. But this seems unlikely, because global PDE inhibition with IBMX produced effects identical to those observed with combined PDE3-PDE4 inhibition. Alternatively, it is also conceivable that the interactions between the PDEs are complicated via feedback regulation involving increased PDE3 and PDE4 activities by PKA-dependent phosphorylation.^13,19,23,38 However, this type of interaction should enhance, not reduce, the effects of combined or global inhibition of PDEs. A more plausible explanation would be that, under basal conditions, more than 1 local pool of cAMP exists in microdomains containing target SERCA2a-PLN effectors. The precise characteristics of cAMP regulation in these pools are undoubtedly complex depending on the distribution and enzyme kinetics of PDE3 and PDE4 as well as the local cAMP production,³⁹ which is also highly compartmentalized.⁴⁰ For example, the EC50 for cAMP hydrolysis by PDE3 (ie, 0.1 to 0.5 µmol/L) is 10-fold higher than for PDE4 (0.5 to 4 µmol/L),^13,19 thereby allowing PDE3 to dominate cAMP hydrolysis at low cAMP concentrations compared with PDE4. Clearly, further studies will be needed to fully identify the molecular and structural basis for the subcellular regulation of SR function by PDE3 and PDE4.

Our results demonstrate that Ca²⁺ transients, SR Ca²⁺ content, and PLN-PO₄ levels were identical between PI3K^–/– myocytes under basal conditions and WT myocytes after PDE4 inhibition, and were unaffected in PI3K^–/– myocytes by PDE4 inhibition either under baseline conditions or when PDE3 was inhibited. These observations reveal that PDE4 activity is absent in the SR subdomains containing SERCA2a-PLN of PI3K^–/– myocytes. These results are surprising because previous studies concluded that the loss of PI3K protein, but not of its enzymatic activity, abolishes PDE3B activity in the myocardium.⁵ However, we were unable to detect PDE3B expression in mouse cardiomyocytes (supplemental Figure II), consistent with previous reports,^13,19 and a recent study found that PI3K binding does not activate PDE3B.¹⁸ Furthermore, ablation of the lipid phosphatase PTEN increases cardiac PIP₃ and impairs cardiac contractility³ and these effects of PTEN disruption were abolished by PI3K deletion,³ suggesting that PIP₃ generation by PI3K is an important factor in the regulation of cardiac contractility.

Interestingly, Ca²⁺ transients, SR Ca²⁺ loads, and PLN-PO₄ were greater in PI3K^–/– myocytes after PDE3 inhibition than in WT myocytes with combined PDE3-PDE4 inhibition. Given the inability of combined PDE3-PDE4 inhibition in WT cardiomyocytes to alter Ca²⁺ cycling and PLN-PO₄ levels beyond that observed with PDE3 or PDE4 inhibition alone, one would anticipate that PDE3 inhibition in PI3K^–/– myocytes would have minimal impact. These unanticipated findings could arise if PDE4 activity was not completely inhibited in WT myocytes by 10 µmol/L rolipram.⁴¹ However, 10-fold higher doses of rolipram, which fully inhibit PDE4,⁴¹ had no further effect on Ca²⁺ transient in WT myocytes. Alternatively as discussed above, these results could reflect the presence of multiple cAMP pools with complex distribution patterns of PDEs and cAMP production. In fact, our results in WT and PI3K^–/– mice support a model wherein there are two pools of cAMP in SR microdomains containing SERCA2a/PLN (Figure 6). In one pool, both PDE3 and PDE4 activities are required to maintain cAMP levels at basal levels. In the second pool, PDE3 activity is necessary to stave off elevations in cAMP arising from increased local cAMP production, as shown previously in PI3K-deficient myocardium as a consequence of reduced local G-i activity⁶ and increased AC activity.

View larger version (52K): [in this window] [in a new window]   Figure 6. PI3K regulates cAMP in cardiomyocytes by controlling PDE4 activity in subcellular SR domain. A, Our working model for cAMP regulation by PI3K, PDE3, and PDE4. PDE4D binds to SERCA2a. B, The loss of PI3K induces local increases in cAMP by eliminating PDE4 activity in microdomains containing SERCA2a-PLN, but not L-type Ca2+ channels-RyR2. PI3K loss also appears to increase cAMP level possibly by regulating AC via G-i.

Although no differences in I_Ca,L amplitudes were observed between PI3K^–/– and WT myocytes, the inactivation rates of I_Ca,L.^7,8 were accelerated in PI3K^–/– myocytes, which can be attributed to increased Ca²⁺-mediated inactivation secondary to elevated Ca²⁺ transients.⁴² To eliminate the complicating effects of Ca²⁺ transients on I_Ca,L, we dialyzed myocytes with high levels of EGTA. Although EGTA does not eliminate Ca²⁺-mediated inactivation,²⁸ it successfully eliminated the effects of SR Ca²⁺ release on I_Ca,L in our studies. When WT myocytes were dialyzed with EGTA, PDE3 or PDE4 inhibition alone did not affect I_Ca,L whereas combined PDE3-PDE4 inhibition induced modest I_Ca,L increases (15% at –10mV), as reported previously.^16,17 Combined PDE3-PDE4 inhibition in WT myocytes shifted V_1/2 to negative voltages without affecting the G_max, verifying that V_1/2 and G_max for I_Ca,L can be separately regulated.⁴³ Thus, in microdomains containing L-type Ca²⁺ channels, either PDE3 or PDE4 activity alone is sufficient to maintain cAMP at basal levels. Several explanations could account for these observations. For example, because basal I_Ca,L is unchanged by cAMP antagonists,^7,8 the inability of PDE3 or PDE4 inhibition alone to alter I_Ca,L could arise because of low basal cAMP production in I_Ca,L microdomains or because high cAMP levels are required to enhance I_Ca,L, or both. In this respect, it is conceivable that cAMP levels were in fact elevated in I_Ca,L microdomains but did not rise to levels required to modulate I_Ca,L. Alternatively, the I_Ca,L microdomains could possess high activity of other PDEs isoforms, although this seems unlikely because IBMX enhanced I_Ca,L by similar amounts as combined PDE4 and PDE3 inhibition in WT myocytes (data not shown).

The pattern of I_Ca,L regulation in PI3K^–/– myocytes was similar to WT myocytes, confirming that cAMP is regulated in I_Ca,L microdomains by both PDE3 and PDE4. Interestingly, because PDE3 inhibition alone did not increase I_Ca,L, we conclude that PDE4 activity is not abolished by PI3K ablation in I_Ca,L microdomains, unlike subcompartments containing SERCA2a. The molecular basis for these differences between the SERCA2a and I_Ca,L subdomains is unclear, but we have found in preliminary studies that neither PDE3B nor PDE4D coimmunoprecipitate with the 1C subunit of the L-type Ca²⁺ channel. Moreover, combined PDE3-PDE4 inhibition increased I_Ca,L in PI3K^–/– far more (2-fold) than in WT myocytes, suggesting that, as in microdomains containing SERCA2a, microdomains containing I_Ca,L in PI3K^–/– myocytes have higher cAMP levels, possibly resulting from decreased G-i mediated inhibition of AC activity.⁶ Alternatively, PDE3 and PDE4 families have multiple subisoforms which can be localized differentially in cardiomyocytes.¹⁷ Unfortunately, no pharmacological tools exist currently to dissect contributions of separate members of the PDE3 or PDE4 families. The molecular and anatomical basis for PDEs regulation of I_Ca,L will clearly require further studies.

Another important target of cAMP-dependent PKA phosphorylation is RyR₂,²² which can be phosphorylated at positions S2808 and S2030.²² Previous results have established that, unlike S2808, S2030 shows very low baseline phosphorylation levels²² and is phosphorylated by PKA, not CaMKinase. Therefore, we assessed the RyR₂ phosphorylation status at position S2030. As with PLN-PO₄ and Ca²⁺ homeostasis, PDE3 and PDE4 are both required to maintain basal RyR₂-PO₄ levels in WT myocytes. These findings are not unexpected given the colocalization of these proteins in the SR, particularly the junctional SR.³⁰ However, unlike SERCA2a-PLN and Ca²⁺ homeostasis, PDE4 activity in subcellular compartments containing the RyR₂ does not require PI3K, establishing that PI3K’s involvement in the PDE4-dependent regulation of the SR function is unique to SERCA2a-PLN. This seems somewhat surprising because PDE4D coassembles with both SERCA2a and RyR₂,¹² and it will be interesting to determine whether PI3K is in the macromolecular complex of signaling molecules that associates with RyR₂.³⁰

Our results have several important clinical implications. For example, a prominent feature of diseased myocardium is reduced Ca²⁺ transients resulting from decreased SR Ca²⁺ uptake, without changes in I_Ca,L density.^44–46 This pattern is the opposite to that seen in PI3K^–/– mice suggesting that increased PI3K activity may contribute to impaired contractility in heart disease^3,4,47–50 by stimulating PDE4 activity and reducing Ca²⁺ uptake in the SR. This suggestion is consistent with the elevated PI3K activity and expression,^5,47 as well as increased PDE4 activity, observed in heart disease.⁵¹ These effects are expected to accentuate the impaired Ca²⁺ handling and contractility in heart disease⁴⁶ originating from β-adrenergic receptor downregulation, enhanced G-i activity, and reduced SERCA2a expression. Clearly, future studies will be required to fully assess the contribution of PI3K-mediated changes in local cAMP to the pathogenesis of heart disease.

	Acknowledgments

 
We thank Dr Anthony Gramolini for PLN antibodies and Dr Robert Rose for helpful comments.

Sources of Funding

This study was supported by Canadian Institutes for Health Research (CIHR) operating grant to P.H.B. P.H.B. and D.H.M. are Career Investigators with the Heart and Stroke Foundation (HSF) of Ontario. B.-G.K. held fellowships from HSF of Canada and TACTICS-CIHR program (University of Toronto).

Disclosures

None.

	Footnotes

 
Original received August 9, 2006; resubmission received May 23, 2007; revised resubmission received June 19, 2007; accepted June 26, 2007.

	References

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