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(Circulation Research. 2005;96:1079.)
© 2005 American Heart Association, Inc.

Cellular Biology

Cardiac Sarcoplasmic Reticulum Calcium Release and Load Are Enhanced by Subcellular cAMP Elevations in PI3K-Deficient Mice

Benoit-Gilles Kerfant^*, Dominica Gidrewicz^*, Hui Sun, Gavin Y. Oudit, Josef M. Penninger, Peter H. Backx

From the Departments of Physiology and Medicine. Heart & Stroke Richard Lewar Centre, and Division of Cardiology, University Health Network, University of Toronto (B.-G.K., D.G., H.S., G.Y.O., P.M.B.) Toronto, Canada.; and the IMBA Institute of Molecular Biotechnology of the Austrian Academy of Sciences (J.M.P.), Vienna, Austria.

Correspondence to Prof Peter H. Backx, DVM, PhD, Fitzgerald Building, Heart and Stroke/Richard Lewar Center, 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 increased cardiac contractility without changes in heart size compared with control mice (ie, PI3K^+/+ or PI3K^+/–). In this study, we show that PI3K^–/– cardiomyocytes have elevated Ca²⁺ transient amplitudes with abbreviated decay kinetics compared with control under field-stimulation and voltage-clamp conditions. When Ca²⁺ transients were eliminated with high Ca²⁺ buffering, L-type Ca²⁺ currents (I_Ca,L), K⁺ currents, and action potential duration (APD) were not different between the groups, whereas, in the presence of Ca²⁺ transients, Ca²⁺-dependent phase of I_Ca,L inactivation was abbreviated and APD at 90% repolarization was prolonged in PI3K^–/– mice. Excitation-contraction coupling (ECC) gain, sarcoplasmic reticulum (SR) Ca²⁺ load, and SR Ca²⁺ release fluxes measured as Ca²⁺ spikes, were also increased in PI3K^–/– cardiomyocytes without detectable changes in Ca²⁺ spikes kinetics. The cAMP inhibitor Rp-cAMP eliminated enhanced ECC and SR Ca²⁺ load in PI3K^–/– without effects in control myocytes. On the other hand, the ß-adrenergic receptor agonist isoproterenol increased I_Ca,L and Ca²⁺ transient equally by 2-fold in both PI3K^–/– and PI3K^+/– cardiomyocytes. Our results establish that PI3K reduces cardiac contractility in a highly compartmentalized manner by inhibiting cAMP-mediated SR Ca²⁺ loading without directly affecting other major modulators of ECC, such as AP and I_Ca,L.

Key Words: heart • PI3K • Ca²⁺ transient • Ca²⁺ spikes • cAMP

	Introduction

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Activation of PhosphoInositide-3 Kinases (PI3Ks)¹ enhances cell survival, proliferation, motility, migration, and secretion, while also regulating cytoskeletal structure and Ca²⁺ signaling.^1–3 Genes encoding PI3Ks are divided into 3 major classes (I, II, and III).⁴ Class I PI3Ks are expressed in the heart and convert phosphatidylinositol-4,5-bis-phosphate (PIP₂) to phosphatidylinositol-3,4,5-tri-phosphate (PIP₃), which acts as a second messenger by recruiting effectors with pleckstrin homology domains.^1,4 Class I PI3Ks are categorized into class I_A PI3Ks, which are typically activated by receptor tyrosine kinases (PI3K and PI3Kß in heart) and class I_B PI3Ks (PI3K), which are primarily activated by Gß subunits of G proteins.^4,5 Selective inhibition of PI3Ks has been shown to potentiate ß2-adrenergic-mediated enhancement of phospholamban (PLN) phosphorylation, contractility, and relaxation in adult rat ventricular cardiomyocytes.⁶ Consistent with these studies, elimination of PI3K in mice increases myocardial contractility as well as basal cAMP levels^7–9 and PLN phosphorylation.^8,9 Moreover, cardiac-specific elimination of PTEN (phosphatase and tensin homologue deleted on chromosome 10), a lipid phosphatase that dephosphorylates PIP₃ to PIP₂, decreases contractility which is prevented by simultaneous ablation of PI3K.⁸ More recently, PI3K was shown to regulate cardiomyocyte contractility by direct activation of a cAMP-degrading phosphodiesterase (PDE3B) in a kinase independent PI3K manner.⁹

PI3K also appears to be important in heart disease. For example, in mice subjected to aortic banding, PI3K activity increases as cardiac function deteriorates,^9,10 whereas PI3K inhibition⁷ or loss of PI3K kinase activity⁹ partially prevents impaired heart function. Moreover, PI3K ablation protects hearts from the detrimental effects of chronic ß-adrenergic¹¹ and platelet-activating factor¹² stimulation. The importance of PI3K in pathogenesis of heart disease is further supported by the observation that suppression of Gq signaling,¹³ in pressure-overload mice, prevents the transition to heart failure while blocking activation of PI3K.

The aim of this study was to elucidate the cellular mechanism(s) responsible for the enhanced cardiac contractility in mice lacking PI3K. Our studies reveal that PI3K elimination increases Ca²⁺ transient amplitude and enhances the efficiency of excitation-contraction coupling (ECC) as a consequence of elevating SR Ca²⁺ content via a cAMP-dependent pathway without affecting L-type Ca²⁺ current (I_Ca,L) or action potential (AP) profile.

	Materials and Methods

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The generation of PI3K^+/+, PI3K^+/–, and PI3K^–/– mice has previously been described.¹⁴ All experiments were performed in accordance with the Canadian Council of Animal Care.

Myocyte Isolation and Measurement of Contractility, Ca²⁺ Transients, and Ca²⁺ Spikes
Ventricular cardiomyocytes were isolated as previously described¹⁵ (see online supplement, available at http://circres.ahajournals.org). For contractility and Ca²⁺ transients the bath solutions contained (in mmol/L) 140 NaCl, 4 KCl, 1 MgCl₂, 1.2 CaCl₂, and 10 HEPES (pH=7.4, NaOH). Contractility was estimated using cell shortening, whereas Ca²⁺ transients were estimated using indo-1,AM^8,9 (see online supplement).

In voltage-clamped studies, cardiomyocytes were loaded with fluo-3 or fluo-5N plus EGTA to record Ca²⁺ transients or Ca²⁺ spikes, respectively. For Ca²⁺ spikes recordings, cardiomyocytes were exciting at 488 nm and the emitted fluorescence at 515 nm was recorded using line-scan mode (IX50, Fluoview, Olympus) as described.¹⁶ The pipette solution contained (in mmol/L) 140 KCl, 5 NaCl, 1 MgCl₂, 7 Mg-ATP, 10 HEPES, 10 D-glucose, 0.75 fluo-5N, 4 EGTA, and 1.55 CaCl₂ ([Ca²⁺]_i=75 nmol/L; pH=7.2 with KOH). Fluorescence signals and I_Ca,L were simultaneously recorded at sampling rates of 10 kHz using voltage protocols and solutions as described (see online supplement).¹⁷

Electrophysiological Recordings and SR Ca²⁺ Content
APs and I_Ca,L were measured with whole-cell patch-clamp technique¹⁸ under current- and voltage-clamp mode, respectively (see online supplement for protocols). For the APs, the pipette solution contained (in mmol/L) 120 K⁺-aspartate, 20 KCl, 1 MgCl₂, 5 NaCl, 5 Mg-ATP, and 10 HEPES (pH set to 7.2 with KOH). I_Ca,L was elicited simultaneously to Ca²⁺ transient. In this case, the recording bath solution contained (in mmol/L) 140 NaCl, 0.5 MgCl₂, 5 CsCl, 5.5 glucose, 5 HEPES, and 1.8 CaCl₂ (pH=7.4, NaOH), whereas the pipette solution contained (in mmol/L) 130 CsCl, 1 MgCl₂, 1 NaH₂PO₄, 3.6 Na₂-phosphocreatine, 2 KCl, 5 MgATP, 0.05 fluo-3 pentapotassium salt, and 10 HEPES (pH=7.2, CsOH).

SR Ca²⁺ content was estimated by integrating the Na⁺–Ca²⁺ exchanger current (I_NCX)¹⁹ induced by brief (10 s) applications of 20 mmol/L caffeine after SR loading protocols involving 8 100-ms depolarizing steps from –80 to +10 mV (1 Hz). The pipette solution contained (in mmol/L) 125 K⁺-aspartate, 20 KCl, 0.5 MgCl₂, 5.0 Na₂-phosphocreatine, 0.4 Na⁺-GTP, 7 MgATP, 0.05 fluo-3, and 10 HEPES, (pH=7.2, KOH) (see online supplement).

Statistics
To test for significance between groups we used either a one-way analysis of variance (ANOVA) followed by the Student Neuman–Keuls test or the Kruskal–Wallis test followed by an unpaired t test when the data were nonparametric or paired t tests when comparing results from the same cardiomyocyte. P<0.05 was considered statistically significant (SPSS). Data are presented as mean±SEM.

	Results

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Contractility, Ca²⁺ Transients, I_Ca,L, SR Ca²⁺ Flux, and SR Ca²⁺ Content
Supplemental Table I establishes that contractility in cardiomyocytes isolated from PI3K^–/– mice was enhanced compared with PI3K^+/– or PI3K^+/+ mice, consistent with our previous results.^8,9 To explore the basis for the enhanced contractility, Ca²⁺ transients were recorded in field-stimulated cardiomyocytes. Ca²⁺ transient amplitudes (ie, ratio of indo-1 fluorescence at 405 nm to 495 nm) at 1 Hz stimulation were larger (P<0.05), whereas times for 50% relaxation were shorter (P<0.05) in PI3K^–/– (0.209±0.01 and 175.9±13.6 ms, n=17) compared with PI3K^+/– cardiomyocytes (0.165±0.01 and 210.98±9.8 ms, n=19) (Figure 1). Similar effects were observed at 3 Hz stimulation (Figure 1). Ca²⁺ transients in PI3K^+/+ myocytes were indistinguishable from PI3K^+/– (see Table).

View larger version (22K): [in this window] [in a new window]   Figure 1. Peak Ca2+ transient amplitudes were increased and Ca2+ transient decay times were shortened in PI3K–/– mice. A, Sample traces of Ca2+ transients, reported as fluorescence ratio of indo-1 (F405/495), recorded under field-stimulation at 1 Hz in PI3K–/– (right) and PI3K+/– (left) cardiomyocytes. B, Bar graph showing averaged peak Ca2+ transient recorded at 1 and 3 Hz in PI3K–/– (black, n=26 at 1 Hz and n=7 at 3 Hz) and PI3K+/– cardiomyocytes (white, n=22 at 1 Hz and n=4 at 3 Hz). C, Bar graph showing corresponding times for 50% decay (50%decay) obtained by fitting F405/495 to a single exponential function at 1 and 3 Hz; *P<0.01.

View this table: [in this window] [in a new window]   Table 1. Mean Ca2+ Transient and Ica,L Amplitudes and Kinetics Measured at 0mV in PI3K –/–, PI3K +/–, and PI3K +/+ Cardiomyocytes in Control Conditions (-Rp-cAMP), After cAMP Inhibition (+Rp-cAMP), and After Isoproterenol Stimulation (ISO)

The previous results show that PI3K^–/– cardiomyocytes have enhanced Ca²⁺ cycling. Although I_Ca,L is a major determinant of SR Ca²⁺ release and loading,²⁰ no increase in I_Ca,L amplitude or kinetics (Figure 2A) was observed in PI3K^–/– cardiomyocytes when pipette solutions contained 4 mmol/L EGTA to eliminate Ca²⁺ transients. Because cell capacitance was similar (P=0.7) between PI3K^–/– (176.3±9.9 pF, n=14) and PI3K^+/– myocytes (169.4±12.1 pF, n=15), I_Ca,L density was unchanged between PI3K^–/– and PI3K^+/– cardiomyocytes at all voltage (Figure 2B). Further, neither the time course of I_Ca,L inactivation (ie, _fast and _slow time constants,¹⁷ data not shown) nor the voltage for 50% steady-state inactivation differed (P>0.9) between PI3K^–/– (–36.9±0.2 mV, n=15) and PI3K^+/– (–36.7±0.1 mV, n=14) myocytes.

View larger version (23K): [in this window] [in a new window]   Figure 2. ICa,L densities and APD recordings in the absence of Ca2+ transients. A, Representative ICa,L traces recorded in PI3K–/– and PI3K+/– cardiomyocytes in response to the protocol shown. B, Current/voltage relationship of mean peak ICa,L density recorded in PI3K–/– (, n=14) and PI3K+/– (, n=15) myocytes. C, Sample APs measured in the presence of 5 mmol/L EGTA and 500 µmol/L CdCl2 in a PI3K–/– (black line) and a PI3K+/– (dashed line) cardiomyocyte. D, Bar graph showing 50% (APD50) and 90% repolarization (APD90) times for APs measured in 10 PI3K–/– (black) and 9 PI3K+/– (white) cardiomyocytes. A train of 15 APs was recorded 5 minutes after breaking into the cardiomyocyte, and the fifteenth steady-state AP was used for analysis.

Modulation of the AP profile as a consequence of K⁺ current changes can also affect cardiomyocyte contractility^21,22 by altering L-type Ca²⁺ channel and Na⁺/Ca²⁺ exchange function. However, no differences in AP duration (APD) were observed at 50% or 90% repolarization between PI3K^–/– and PI3K^+/– cardiomyocytes (Figure 2; supplemental Table II) when APs were recorded in the presence of 500 µmol/L extracellular CdCl₂ plus 5 mmol/L intracellular EGTA to eliminate Ca²⁺ transients, blocking I_Ca,L, and minimize contributions of I_NCX to AP profile. Consistent with these AP measurements, no differences in K⁺ currents (data not shown) and NCX activity (see Figure 4) were detected between PI3K^–/– and PI3K^+/– cardiomyocytes. On the other hand, when I_Ca,L and Ca²⁺ transients were present (achieved by removing Cd²⁺ and lowering pipette EGTA to 0.05 mmol/L), APD was prolonged at 90% but not 50% repolarization in PI3K^–/– compared with PI3K^+/– cardiomyocytes (supplemental Table II), as expected with increased Ca²⁺ transients.^16,22–25

To further investigate the cellular basis for elevated contractility in PI3K^–/– cardiomyocytes, Ca²⁺ transients were recorded simultaneously with I_Ca,L under voltage-clamp conditions (Figure 3A). As in field stimulated myocytes, Ca²⁺ transient amplitudes were elevated at all voltages tested (Figure 3B); at 0 mV the amplitudes in PI3K^–/– (0.35±0.05, n=12) were larger (P<0.01) than either PI3K^+/– (0.17±0.04, n=9) or PI3K^+/+ (0.17±0.02, n=10) mice. Moreover, Ca²⁺ transients in PI3K^–/– cardiomyocytes had reduced (P<0.05) decay times without differences in time to peak in association with reduced (P<0.01) time constants for fast inactivation of I_Ca,L, (which is linked to Ca²⁺-mediated inactivation²⁶) at 0 mV (Table) as well as at other voltages (data not shown). Despite altered inactivation, peak I_Ca,L densities did not differ between PI3K^–/– and PI3K^+/– or PI3K^+/+ myocytes, regardless of voltage (Figure 3C). Because Ca²⁺ transients were elevated without major changes in I_Ca,L, ECC gain, which is estimated by dividing the rate of release of the Ca²⁺ transient (see legend for Figure 3C) by the time integral of I_Ca,L,¹⁷ was markedly enhanced in PI3K^–/– compared with PI3K^+/– or PI3K^+/+ (Figure 3D).

View larger version (24K): [in this window] [in a new window]   Figure 3. Peak Ca2+ transient amplitudes and "CICR gains." A, Representative Ca2+ transients and ICa,L recorded simultaneously at 0 mV in a voltage-clamped PI3K–/– (right), PI3K+/– (middle), and PI3K+/+ (left) cardiomyocyte after stimulation using the protocol shown. Dotted lines represent 0 current levels for ICa,L traces or fluorescence ratios (F/F0) of 1. Averaged Ca2+ transients (B) and ICa,L densities (C) recorded in PI3K–/– (, n=11), PI3K+/– (, n=9), and PI3K+/+ (, n=10) cardiomyocytes. D, CICR gain plotted as a function of voltage where CICR gain=(rate of Ca2+ release)/ICa,L. The rate of Ca2+ release was estimated as the peak (F/F0) divided by the time from depolarization to peak fluorescence. ICa,L is the time integral of ICa,L (pC/pF) during the pulse. *P<0.05, **P<0.01.

Differences in Ca²⁺ transient amplitudes and kinetics seen in PI3K^–/– cardiomyocytes could originate from modifications in amount and time course of SR Ca²⁺ release¹⁶ or from alterations in SR Ca²⁺ uptake rates or both. To directly examine the SR Ca²⁺ release process, Ca²⁺ spikes¹⁶ were measured using the technique of Song et al²⁷ wherein cytosolic Ca²⁺ levels were clamped with high concentrations of Ca²⁺ buffers to known levels. Figure 4A shows typical Ca²⁺ spikes recordings in a PI3K^–/– and a PI3K^+/– myocyte obtained by spatially averaging the fluo-5N fluorescence across the cell following a step depolarization to 0 mV. Figure 4B establishes that SR Ca²⁺ spike peaks were increased (P<0.01) in PI3K^–/– (0.57±0.04, n=7) compared with PI3K^+/– (0.41±0.03, n=9) cardiomyocytes, as were the Ca²⁺ spike integrals for PI3K^–/– (7.8±0.5, n=7) versus PI3K^+/– (6.0±0.4, n=9), consistent with elevated Ca²⁺ transients and ECC gains. On the other hand, no changes were observed in the time to peak of Ca²⁺ spikes for PI3K^–/– (7.9±0.6 ms, n=7) versus PI3K^+/– (8.1±0.5 ms, n=9), demonstrating that SR Ca²⁺ release kinetics were not affected by PI3K ablation.

View larger version (36K): [in this window] [in a new window]   Figure 4. Peak Ca2+ spike and SR Ca2+ load recordings. A, Typical Ca2+ spikes recorded at 0 mV in PI3K–/– and PI3K+/– cardiomyocytes before (left) and after (right) cAMP inhibition (Rp-cAMP 100 µmol/L). Ca2+ spikes were obtained by spatially averaging the line scan fluo-5 signal across the cell after depolarization to 0 mV. B, Bar graph showing both peak Ca2+ spike amplitude (left) and F/F0 integration before and after cAMP inhibition averaged in PI3K–/– (black, n=7 and 9) and PI3K+/– (white, n=9 and 7) cardiomyocytes. *P<0.01. C, sample traces of INCX after rapid application of 20 mmol/L caffeine in PI3K–/– and PI3K+/– cardiomyocytes before and after cAMP inhibition. All cardiomyocytes were held at –80 mV throughout. SR Ca2+ content was estimated by integrating INCX. D, Bar graph showing the average data in PI3K–/– (black bars, n=10 and 15) and PI3K+/– (white bars, n=10 and 13) cardiomyocytes before and after cAMP inhibition; *P<0.05.

The previous results suggest enhanced PI3K^–/– myocyte contractility originates from increased SR Ca²⁺ uptake rates leading to elevated SR Ca²⁺ contents. To test this hypothesis, myocytes were dialyzed with solutions containing 0.05 µmol/L fluo-3 and stimulated at 1 Hz with 8 steps from –80 to +10 mV for 100 ms (see Methods) to allow steady state loading of the SR with Ca²⁺. The myocytes were then superfused with 20 mmol/L caffeine to release Ca²⁺ from the SR which is extruded by forward mode NCX activity (I_NCX).²⁸ Figure 4 shows that time integral of I_NCX (proportional to SR Ca²⁺ content) was increased (P<0.05) in PI3K^–/– (1.64±0.4 pC/pF, n=14) compared with PI3K^+/– cardiomyocytes (1.01±0.1 pC/pF, n=12). By contrast, decay times for caffeine-induced I_NCX did not differ between PI3K^–/– (1368.0±299.4 ms) and PI3K^+/– cardiomyocytes (1332.0±196.1 ms), establishing that I_NCX densities were not detectably altered by PI3K ablation.¹⁹

cAMP Inhibition
The previous results are entirely consistent with recent studies showing that cAMP^7–9,29 levels and PLN phosphorylation are increased in PI3K-deficient cardiomyocytes.^8,9 To test whether elevated cAMP was responsible for increased Ca²⁺ transients and contractility in PI3K^–/– mice, we dialyzed myocytes with 100 µmol/L adenosine-3', 5' cyclic phosphorothioate (Rp-cAMP), a cAMP antagonist. Figure 4 shows that Rp-cAMP had no effect on Ca²⁺ spikes integrals (F/F₀) in PI3K^+/– myocytes while eliminating the differences between PI3K^–/– (6.21±0.4, n=9) and PI3K^+/– (6.27±0.4, n=7) cardiomyocytes. Dialysis with Rp-cAMP also completely abolished increased SR Ca²⁺ content in PI3K^–/– (0.77±0.1 pC/pF, n=15) versus PI3K^+/– (0.75±0.1 pC/pF, n=13) cardiomyocytes while having no effect on the SR Ca²⁺ content in PI3K^+/– (Figure 4). Additionally, Rp-cAMP dialysis reduced the amplitude while slowing the kinetics of Ca²⁺ transients in PI3K^–/– to the baseline levels observed in PI3K^+/– cardiomyocytes with or without Rp-cAMP (Table). These effects on SR Ca²⁺ content and release as well as Ca²⁺ transients were not caused by changes in I_Ca,L because Rp-cAMP had no effect on I_Ca,L in either PI3K^–/– or PI3K^+/– cardiomyocytes (Table). However, in the presence of Ca²⁺ transients, cAMP inhibition eliminated differences in Ca²⁺-dependent inactivation of I_Ca,L to control values in PI3K^–/– mice (Table), suggesting that accelerated inactivation of I_Ca,L was secondary to increased Ca²⁺ transients.

ß-Adrenergic Stimulation
The previous results establish that increases in SR Ca²⁺ content and release in mice lacking PI3K are mediated by cAMP. Because cAMP is typically regulated in myocardium by ß-adrenergic signaling, we examined the effects of the ß-adrenergic agonist isoproterenol (1 µmol/L). Figure 5 shows that application of isoproterenol increased I_Ca,L amplitude and accelerate I_Ca,L inactivation to the same extent in PI3K^–/– and PI3K^+/– cardiomyocytes (as well as PI3K^+/+; Table). Although not shown, the voltage dependence of peak I_Ca,L was shifted in hyperpolarized directions as expected with ß-adrenergic stimulation in all 3 groups of mice. Isoproterenol application also increased Ca²⁺ transients in both PI3K^–/– and PI3K^+/– myocytes by about the same relative amount (Figure 5A and 5C). As a result, Ca²⁺ transients remained elevated and still showed more rapid relaxation kinetics in PI3K^–/– versus PI3K^+/– myocytes with isoproterenol (Table). Taken together, these results suggest that PI3K limits the extent of cAMP elevations in the vicinity of the SR after the stimulation of adenylate cyclase, consistent with its role as an activator of PDE3B in cardiomyocytes.⁹

View larger version (21K): [in this window] [in a new window]   Figure 5. ß-Adrenergic stimulation increased Ca2+ transient and ICa,L amplitudes in PI3K–/– and PI3K+/– cardiomyocytes. A, Representative traces of ICa,L (left) and Ca2+ transients (right) recorded simultaneously at 0 mV before (C) and after isoproterenol (ISO, 1 µmol/L) application in a PI3K–/– and a PI3K+/– cardiomyocyte. Dotted lines represent 0 current levels for ICa,L traces and a fluorescence ratio of 1 for F/F0. B, Mean ICa,L peak amplitude measured at 0 mV in PI3K–/– (black, n=9) and PI3K+/– (white, n=8) cardiomyocytes before and after isoproterenol treatment. C, Mean Ca2+ transient peak amplitude measured simultaneously with ICa,L at 0 mV in PI3K–/– and PI3K+/– cardiomyocytes before and after isoproterenol treatment; P<0.01 (same groups), *P<0.01 (between groups).

	Discussion

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Our results establish that loss of PI3K leads to the increase in cardiomyocyte shortening, Ca²⁺ transients, SR Ca²⁺ release flux, and SR Ca²⁺ load along with enhanced rates of Ca²⁺ transient relaxation and acceleration of Ca²⁺-mediated I_Ca,L inactivation. These functional effects of PI3K ablation were completely abolished by cAMP antagonism using Rp-cAMP. The cAMP-mediated enhancements of cardiac ECC in PI3K^–/– myocytes were not, however, associated with changes in other factors regulating ECC, such as K⁺ currents, APD, and I_Ca,L, when Ca²⁺ transients were suppressed in these myocytes. Collectively, these findings strongly suggest that increases in SR Ca²⁺ uptake rates and loads, as a result of subcellular elevations of cAMP in the vicinity of the SR, are responsible for increased contractility in cardiomyocytes lacking PI3K.^8,9,29 This conclusion is consistent with previous studies showing increased cAMP levels^7–9,29 and PLN phosphorylation^8,9 in PI3K^–/– myocardium. It is conceivable that cAMP-dependent alterations in SR Ca²⁺ release channels also occur and contribute to the increased contractility in PI3K^–/– hearts, although no changes in kinetics of SR Ca²⁺ release fluxes (ie, Ca²⁺ spikes) were observed in our studies. The regulation of cardiac contractility appears to be unique to the PI3K isoform because contractile strength of hearts is unchanged in mice with dominant-negative suppression of PI3K.⁸ Furthermore, PI3K and PI3Kß protein expression, the 2 other main cardiac isoforms in total heart extracts, were unchanged in PI3K^–/– compared with PI3K^+/– mice,⁸ suggesting that compensatory changes in these isoforms are not responsible for the elevated contractility seen in PI3K-deficient mice.

Despite clear reliance of elevated SR Ca²⁺ cycling on cAMP, PI3K^–/– did not have elevated I_Ca,L. These observations were surprising because L-type Ca²⁺ channels are a prototype for cAMP-PKA regulation and because a previous study showed that PI3K inhibitors reduced I_Ca,L in rat neonatal cardiomyocytes.³⁰ Differences between our adult mouse cardiomyocyte studies and previous rat neonatal cardiomyocyte studies are unclear but may be related to the underdevelopment of the SR and T-tubule system in neonatal cardiac myocytes compared with adult myocytes,³¹ although nonspecific actions of PI3K inhibitors might also contribute.^4,32 Regardless, the differential effects of PI3K ablation on SR Ca²⁺ function versus I_Ca,L establishes that PI3K is a critical negative-regulator of cAMP-PKA signaling in microdomains, including the SR within the cardiomyocytes.

The mechanism whereby PI3K regulates cAMP levels in the vicinity of the SR (or in other microdomains of cells) is not entirely clear.³³ In transgenic mice with cardiac-specific overexpression of human AC type 8 cardiac, contractility was increased in association with elevated Ca²⁺ transients and accelerated relaxation but without any alteration of I_Ca,L amplitude caused by a rearrangement of PDE isoforms leading to a strong compartmentation of cAMP.³⁴ In addition, previous studies have established that subcellular compartmentation of cAMP and PKA signaling in cardiomyocytes depends on regional expression of PDEs.³⁵ More recently, studies showed that PI3K stimulates PDE3B isoform by direct protein–protein interactions, independent of PIP₃ generation.⁹ This finding is consistent with previous results showing a lack of effect of PI3K inhibitors on contractility and Ca²⁺ transients in wild-type mice,^32,36 suggesting that the enzymatic generation of PIP₃ is not required for the local regulation of cAMP levels by PI3K. Thus, it appears that PI3K regulates cardiomyocyte contractility and Ca²⁺ transients by directly inhibiting PDE3B and thereby cAMP-PKA signaling in subcellular compartments containing the SR. The absence of I_Ca,L elevations in PI3K^–/– mice suggest that another PDE isoform may regulate cAMP in the vicinity of L-type Ca² channels. Interestingly, connections between PDE3B and PI3K leading to the regulation of cAMP and the control of insulin secretion have been previously reported in pancreatic ß-cells.^37,38 However, a previous study in myocytes has suggested that localization of cAMP-PKA signaling involves the localized regulation of protein phosphatase activity by PI3K.⁶ On the other hand, mice lacking the lipid phosphatase PTEN have elevated cardiac PIP₃ levels along with reduced contractility, supporting the concept that elevated PIP₃ levels can inhibit cardiac contractility.⁸ Clearly, further studies are necessary to fully dissect the molecular mechanisms underlying the subcellular compartmentation of cAMP regulation by PI3K.

Compartmentation of cAMP-PKA signaling in cardiomyocytes has also been hypothesized to help to explain the differential actions of ß2-adrenergic stimulation on contractility versus I_Ca,L^39,40 (reviewed by Steinberg and Brunton⁴¹). Specifically, the acute application of PI3K inhibitors permits ß2-adrenergic stimulation to increase PLN phosphorylation and Ca²⁺ transients amplitude, independent of elevations in cAMP levels, presumably as a consequence of inhibiting the enzymatic activity of the PI3K isoform.⁶ Further studies are clearly warranted to determine the possible role of PI3K and its interaction with PDEs in mediating these ß2-adrenergic actions. It has also been recently shown that inhibition of PI3K with LY294002 significantly enhances ß1-adrenergic–induced increases in I_Ca,L, Ca²⁺ transients, and cardiomyocytes contractility.³⁶ In our studies, ß-adrenergic stimulation increased I_Ca,L amplitude and shifted the current–voltage relationships leftward to the same extent (from the same baseline) in PI3K^–/– myocytes compared with control. ß-Adrenergic stimulation also increased Ca²⁺ transient amplitudes and abbreviated relaxation kinetics in PI3K^–/– and control cardiomyocytes, but Ca²⁺ transients remained elevated with faster kinetics in PI3K^–/– myocytes after isoproterenol treatment. These findings suggest that cAMP levels are submaximally elevated in the SR-containing microdomains of PI3K^–/– cardiomyocytes and that PI3K limits the response of cardiomyocytes to ß-adrenergic at the level of SR, consistent with PI3K’s ability to activate PDE3B.⁹

Although K⁺ currents and APDs (in the absence of Ca²⁺ transients) were unchanged in PI3K-deficient mice, APD₉₀ was prolonged without changes in APD₅₀ when Ca²⁺ transients were present. The precise origin of this AP prolongation is not obvious because during this phase of the AP there is a complex, delicate, and dynamic interaction between depolarizing currents, like I_NCX and I_Ca,L, whose activities are tightly regulated by intracellular Ca²⁺ levels and repolarizing K⁺ currents. Based on previous studies,^42,43 it seems likely that this prolongation originates largely from increased depolarizing I_NCX as a result of elevated Ca²⁺ transients, despite the absence of evidence for increased I_NCX transport densities in the PI3K^–/– (as assessed from the rate of decay of the caffeine-induced I_NCX). On the other hand, although the prolonged APD tends to increase the total Ca²⁺ entry through L-type Ca²⁺ channels,²¹ differences in I_Ca,L are unlikely to be responsible for the increased APD, because I_Ca,L densities (assessed in the absence of Ca²⁺ transients) are unaffected by PI3K ablation. In fact, the rate of I_Ca,L inactivation was accelerated in PI3K^–/– mice, because of elevated Ca²⁺ transients,⁴⁴ when Ca²⁺ transients were present, which would reduce the depolarizing effects of I_Ca,L. Finally, it should be mentioned that the observed APD prolongation will indirectly influence the SR load in PI3K^–/– myocytes by modulating the net Ca²⁺ transported by the NCX and I_Ca,L during each cardiac cycle in a dynamic manner, as summarized in previous studies.⁴⁴ Thus, whereas many factors are likely to complexly modulate SR Ca²⁺ content in PI3K^–/– myocytes, the origin of these changes can ultimately be traced to cAMP-dependent increases in SR Ca²⁺ uptake which in turn affects other Ca²⁺ dependent processes.

A prominent feature of myocardium from diseased hearts is the reduction of Ca²⁺ transients as a result of decreased SR Ca²⁺ load without changes in I_Ca,L density.^45–48 This pattern is opposite of that seen in our PI3K^–/– mice, suggesting that increased PI3K activity may conceivably contribute to the alterations in heart contraction observed in several pathologies^{7,8,10–12,49} by differentially reducing cAMP levels and thereby PKA signaling at the level of the SR. This suggestion is consistent with previous studies showing that PI3K activity and expression are increased in cardiac disease.^9,49 Interestingly, PDE3 and PDE4 gene expression and activities are also enhanced in heart failure.⁵⁰ Increased PI3K in association with increased activity of selected PDE could be expected to work in conjunction with reduced SERCA2a expression to reduce SR Ca²⁺ uptake, leading to impaired Ca²⁺ handling and contractility observed in heart disease.⁴⁸ Clearly, future studies will be required to fully assess the contribution of changes in local cAMP signaling to changes seen in heart disease.

In summary, our results establish that the loss of PI3K leads to increased contractility and Ca²⁺ transients as a result of elevations in SR Ca²⁺ loading. This enhanced contractility likely involves localized elevations of cAMP-PKA signaling and increased PLN phosphorylation at the level of the SR as a result of decreased PDE actions. The PI3K-dependent regulation of contractility by the SR could be an important contributor to the functional changes observed in heart disease.

	Acknowledgments

 
This study was supported by the Canadian Institute for Health Research (CIHR) to P.H.B., who is a Career Investigator with the Heart and Stroke Foundation (HSF) of Ontario. B.G.K. holds a postdoctoral fellowship from the HSF of Canada and the TACTICS-CIHR program at the University of Toronto. G.Y.O. held a postdoctoral fellowship from the CIHR and the TACTICS-CIHR program. J.M.P. is supported by the Austrian National Bank. We thank Alan Prendergast and Rafael Ramirez for technical assistance.

	Footnotes

 
*Both authors contributed equally to this work.

Original received October 28, 2004; resubmission received March 17, 2005; accepted April 14, 2005.

	References

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