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The Departments of Physiology and Medicine. Heart & Stroke Richard Lewar Centre
Division of Cardiology, University Health Network, University of Toronto (B.-G.K., D.G., H.S., G.Y.O., P.M.B.) Toronto, Canada.
the IMBA Institute of Molecular Biotechnology of the Austrian Academy of Sciences (J.M.P.), Vienna, Austria.
Abstract
We recently showed that phosphoinositide-3-kinase-eCdeficient (PI3KeC/eC) mice have increased cardiac contractility without changes in heart size compared with control mice (ie, PI3K+/+ or PI3K+/eC). In this study, we show that PI3KeC/eC 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 (ICa,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 ICa,L inactivation was abbreviated and APD at 90% repolarization was prolonged in PI3KeC/eC mice. Excitation-contraction coupling (ECC) gain, sarcoplasmic reticulum (SR) Ca2+ load, and SR Ca2+ release fluxes measured as Ca2+ spikes, were also increased in PI3KeC/eC cardiomyocytes without detectable changes in Ca2+ spikes kinetics. The cAMP inhibitor Rp-cAMP eliminated enhanced ECC and SR Ca2+ load in PI3KeC/eC without effects in control myocytes. On the other hand, the -adrenergic receptor agonist isoproterenol increased ICa,L and Ca2+ transient equally by 2-fold in both PI3KeC/eC and PI3K+/eC 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 ICa,L.
Key Words: heart PI3K Ca2+ transient Ca2+ spikes cAMP
Introduction
Activation of PhosphoInositide-3 Kinases (PI3Ks)1 enhances cell survival, proliferation, motility, migration, and secretion, while also regulating cytoskeletal structure and Ca2+ signaling.1eC3 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 IA PI3Ks, which are typically activated by receptor tyrosine kinases (PI3K and PI3K in heart) and class IB PI3Ks (PI3K), which are primarily activated by G subunits of G proteins.4,5 Selective inhibition of PI3Ks has been shown to potentiate 2-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 levels7eC9 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 -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 (ICa,L) or action potential (AP) profile.
Materials and Methods
The generation of PI3K+/+, PI3K+/eC, and PI3KeC/eC 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,AM8,9 (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 ICa,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 ICa,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). ICa,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+eCCa2+ exchanger current (INCX)19 induced by brief (10 s) applications of 20 mmol/L caffeine after SR loading protocols involving 8 100-ms depolarizing steps from eC80 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 NeumaneCKeuls test or the KruskaleCWallis 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
Contractility, Ca2+ Transients, ICa,L, SR Ca2+ Flux, and SR Ca2+ Content
Supplemental Table I establishes that contractility in cardiomyocytes isolated from PI3KeC/eC mice was enhanced compared with PI3K+/eC 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 PI3KeC/eC (0.209±0.01 and 175.9±13.6 ms, n=17) compared with PI3K+/eC 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+/eC (see Table).
The previous results show that PI3KeC/eC cardiomyocytes have enhanced Ca2+ cycling. Although ICa,L is a major determinant of SR Ca2+ release and loading,20 no increase in ICa,L amplitude or kinetics (Figure 2A) was observed in PI3KeC/eC cardiomyocytes when pipette solutions contained 4 mmol/L EGTA to eliminate Ca2+ transients. Because cell capacitance was similar (P=0.7) between PI3KeC/eC (176.3±9.9 pF, n=14) and PI3K+/eC myocytes (169.4±12.1 pF, n=15), ICa,L density was unchanged between PI3KeC/eC and PI3K+/eC cardiomyocytes at all voltage (Figure 2B). Further, neither the time course of ICa,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 PI3KeC/eC (eC36.9±0.2 mV, n=15) and PI3K+/eC (eC36.7±0.1 mV, n=14) myocytes.
Modulation of the AP profile as a consequence of K+ current changes can also affect cardiomyocyte contractility21,22 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 PI3KeC/eC and PI3K+/eC cardiomyocytes (Figure 2; supplemental Table II) when APs were recorded in the presence of 500 eol/L extracellular CdCl2 plus 5 mmol/L intracellular EGTA to eliminate Ca2+ transients, blocking ICa,L, and minimize contributions of INCX 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 PI3KeC/eC and PI3K+/eC cardiomyocytes. On the other hand, when ICa,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 PI3KeC/eC compared with PI3K+/eC cardiomyocytes (supplemental Table II), as expected with increased Ca2+ transients.16,22eC25
To further investigate the cellular basis for elevated contractility in PI3KeC/eC cardiomyocytes, Ca2+ transients were recorded simultaneously with ICa,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 PI3KeC/eC (0.35±0.05, n=12) were larger (P<0.01) than either PI3K+/eC (0.17±0.04, n=9) or PI3K+/+ (0.17±0.02, n=10) mice. Moreover, Ca2+ transients in PI3KeC/eC 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 ICa,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 ICa,L densities did not differ between PI3KeC/eC and PI3K+/eC or PI3K+/+ myocytes, regardless of voltage (Figure 3C). Because Ca2+ transients were elevated without major changes in ICa,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 ICa,L,17 was markedly enhanced in PI3KeC/eC compared with PI3K+/eC or PI3K+/+ (Figure 3D).
Differences in Ca2+ transient amplitudes and kinetics seen in PI3KeC/eC 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 PI3KeC/eC and a PI3K+/eC 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 PI3KeC/eC (0.57±0.04, n=7) compared with PI3K+/eC (0.41±0.03, n=9) cardiomyocytes, as were the Ca2+ spike integrals for PI3KeC/eC (7.8±0.5, n=7) versus PI3K+/eC (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 PI3KeC/eC (7.9±0.6 ms, n=7) versus PI3K+/eC (8.1±0.5 ms, n=9), demonstrating that SR Ca2+ release kinetics were not affected by PI3K ablation.
The previous results suggest enhanced PI3KeC/eC 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 eol/L fluo-3 and stimulated at 1 Hz with 8 steps from eC80 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 (INCX).28 Figure 4 shows that time integral of INCX (proportional to SR Ca2+ content) was increased (P<0.05) in PI3KeC/eC (1.64±0.4 pC/pF, n=14) compared with PI3K+/eC cardiomyocytes (1.01±0.1 pC/pF, n=12). By contrast, decay times for caffeine-induced INCX did not differ between PI3KeC/eC (1368.0±299.4 ms) and PI3K+/eC cardiomyocytes (1332.0±196.1 ms), establishing that INCX densities were not detectably altered by PI3K ablation.19
cAMP Inhibition
The previous results are entirely consistent with recent studies showing that cAMP7eC9,29 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 PI3KeC/eC mice, we dialyzed myocytes with 100 eol/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+/eC myocytes while eliminating the differences between PI3KeC/eC (6.21±0.4, n=9) and PI3K+/eC (6.27±0.4, n=7) cardiomyocytes. Dialysis with Rp-cAMP also completely abolished increased SR Ca2+ content in PI3KeC/eC (0.77±0.1 pC/pF, n=15) versus PI3K+/eC (0.75±0.1 pC/pF, n=13) cardiomyocytes while having no effect on the SR Ca2+ content in PI3K+/eC (Figure 4). Additionally, Rp-cAMP dialysis reduced the amplitude while slowing the kinetics of Ca2+ transients in PI3KeC/eC to the baseline levels observed in PI3K+/eC 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 ICa,L because Rp-cAMP had no effect on ICa,L in either PI3KeC/eC or PI3K+/eC cardiomyocytes (Table). However, in the presence of Ca2+ transients, cAMP inhibition eliminated differences in Ca2+-dependent inactivation of ICa,L to control values in PI3KeC/eC mice (Table), suggesting that accelerated inactivation of ICa,L was secondary to increased Ca2+ transients.
-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 -adrenergic signaling, we examined the effects of the -adrenergic agonist isoproterenol (1 eol/L). Figure 5 shows that application of isoproterenol increased ICa,L amplitude and accelerate ICa,L inactivation to the same extent in PI3KeC/eC and PI3K+/eC cardiomyocytes (as well as PI3K+/+; Table). Although not shown, the voltage dependence of peak ICa,L was shifted in hyperpolarized directions as expected with -adrenergic stimulation in all 3 groups of mice. Isoproterenol application also increased Ca2+ transients in both PI3KeC/eC and PI3K+/eC 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 PI3KeC/eC versus PI3K+/eC 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
Discussion
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 ICa,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 PI3KeC/eC myocytes were not, however, associated with changes in other factors regulating ECC, such as K+ currents, APD, and ICa,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 levels7eC9,29 and PLN phosphorylation8,9 in PI3KeC/eC myocardium. It is conceivable that cAMP-dependent alterations in SR Ca2+ release channels also occur and contribute to the increased contractility in PI3KeC/eC 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 PI3K protein expression, the 2 other main cardiac isoforms in total heart extracts, were unchanged in PI3KeC/eC compared with PI3K+/eC 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, PI3KeC/eC did not have elevated ICa,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 ICa,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 ICa,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 ICa,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 proteineCprotein 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 ICa,L elevations in PI3KeC/eC 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 -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 2-adrenergic stimulation on contractility versus ICa,L39,40 (reviewed by Steinberg and Brunton41). Specifically, the acute application of PI3K inhibitors permits 2-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 2-adrenergic actions. It has also been recently shown that inhibition of PI3K with LY294002 significantly enhances 1-adrenergiceCinduced increases in ICa,L, Ca2+ transients, and cardiomyocytes contractility.36 In our studies, -adrenergic stimulation increased ICa,L amplitude and shifted the currenteCvoltage relationships leftward to the same extent (from the same baseline) in PI3KeC/eC myocytes compared with control. -Adrenergic stimulation also increased Ca2+ transient amplitudes and abbreviated relaxation kinetics in PI3KeC/eC and control cardiomyocytes, but Ca2+ transients remained elevated with faster kinetics in PI3KeC/eC myocytes after isoproterenol treatment. These findings suggest that cAMP levels are submaximally elevated in the SR-containing microdomains of PI3KeC/eC cardiomyocytes and that PI3K limits the response of cardiomyocytes to -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 INCX and ICa,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 INCX as a result of elevated Ca2+ transients, despite the absence of evidence for increased INCX transport densities in the PI3KeC/eC (as assessed from the rate of decay of the caffeine-induced INCX). On the other hand, although the prolonged APD tends to increase the total Ca2+ entry through L-type Ca2+ channels,21 differences in ICa,L are unlikely to be responsible for the increased APD, because ICa,L densities (assessed in the absence of Ca2+ transients) are unaffected by PI3K ablation. In fact, the rate of ICa,L inactivation was accelerated in PI3KeC/eC mice, because of elevated Ca2+ transients,44 when Ca2+ transients were present, which would reduce the depolarizing effects of ICa,L. Finally, it should be mentioned that the observed APD prolongation will indirectly influence the SR load in PI3KeC/eC myocytes by modulating the net Ca2+ transported by the NCX and ICa,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 PI3KeC/eC 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 ICa,L density.45eC48 This pattern is opposite of that seen in our PI3KeC/eC mice, suggesting that increased PI3K activity may conceivably contribute to the alterations in heart contraction observed in several pathologies7,8,10eC12,49 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.
Both authors contributed equally to this work.
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