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Am J Physiol Heart Circ Physiol 286: H796-H805, 2004. First published October 16, 2003; doi:10.1152/ajpheart.00546.2003
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Phosphoinositide 3-kinase regulates excitation-contraction coupling in neonatal cardiomyocytes

Susan A. McDowell, Eileen McCall, William F. Matter, Thomas B. Estridge, and Chris J. Vlahos

Cardiovascular Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285

Submitted 10 June 2003 ; accepted in final form 7 October 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 decreased steady-state contraction in neonatal rat ventricular myocytes (NRVM). To determine whether the effect on steady-state contraction could be due to decreased intracellular Ca2+ content, Ca2+ content was assessed with fluorescent plate reader analysis by using the caffeine-releasable Ca2+ stores as an index of sarcoplasmic reticulum (SR) Ca2+ content. Caffeine-releasable Ca2+ content was diminished in a dose-dependent manner with LY-294002, suggesting that the decrease in steady-state contraction was due to diminished intracellular Ca2+ content. Activation of the L-type Ca2+ channel by BAY K 8644 was attenuated by LY-294002, suggesting the effect of LY-294002 is to reduce Ca2+ influx at this channel. To investigate whether additional proteins involved in excitation-contraction (EC) coupling are likewise regulated by PI3K activity, the effects of compounds acting at sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a), the ryanodine receptor, and the Na/Ca exchanger (NCX) were compared with LY-294002. Inhibition of SERCA2a by thapsigargin increased basal Ca2+ levels in contrast to LY-294002, indicating that SERCA2a activity is sustained in the presence of LY-294002. Ryanodine decreased SR Ca2+ content. The additive effect with coadministration of LY-294002 could be attributed to a decrease in Ca2+ influx at the L-type Ca2+ channel. The NCX inhibitor Ni2+ was used to investigate whether the decrease in intracellular Ca2+ content with LY-294002 could be due to inhibition of the NCX reverse-mode activity. The minimal effect of LY-294002 with Ni2+ suggests that the primary effect of LY-294002 on EC coupling occurs through inhibition of PI3K-mediated L-type Ca2+ channel activity.

LY-294002; L-type calcium channel; fluorescent plate reader

EXCITATION-CONTRACTION (EC) coupling in cardiomyocytes involves a tightly regulated system of membranes, channels, receptors, and organelles to rapidly convert the electrical stimulation of the action potential to the chemical stimulation necessary for activating contraction. The propagation of the action potential initiates an inward Ca2+ current through voltage-sensitive L-type Ca2+ channels triggering calcium-induced Ca2+ release from the sarcoplasmic reticulum (SR, the Ca2+ store in the myocyte) via ryanodine receptors, which can be detected by using Ca2+-chelating fluorescent dyes. The resulting increase in intracellular Ca2+ content activates contraction. Relaxation begins with extrusion of Ca2+ from the cell by the Na/Ca exchanger (NCX), and to a lesser degree, by the Ca-ATPase, by reuptake of Ca2+ into the SR by sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) and through mitochondrial uptake (reviewed in Ref. 4).

Although exhibiting functional and structural differences, regulatory proteins involved in EC coupling have been identified in fetal, neonatal, and adult rodent myocytes. L-type Ca2+ channel activity increases in the rodent heart during fetal development, reaching adult levels around the day of birth (8, 11, 27). In contrast to the fetal heart, maturation of the SR during neonatal development positions it as the primary source of Ca2+ for contraction, similar to the adult heart (48). Likewise, ryanodine receptor expression increases in the rat heart during neonatal development (48). NCX expression is highest in the rat fetal heart and declines postnatally (48). The major source of Ca2+ influx into the adult cell is the L-type Ca2+ channel, whereas NCX functioning in the reverse-mode predominates in the fetal heart, decreasing during postnatal development (3, 17). Finally, although the sarcomeric structure of the neonatal rodent myocyte appears less ordered than that of the adult, recent evidence indicates the presence of functional T tubules and Z lines within the cultured neonatal myocyte (49).

Phosphoinositide (PI) 3-kinase (PI3K) isoforms form a family of conserved enzymes classified according to structure, substrate utilization, and source of activation. Isoforms within the mammalian class IA are heterodimers of the catalytic domains (p110{alpha}, p110{beta}, or p110{delta}) and adaptor subunits (p85{alpha}, p85{beta}, or p55{gamma}). The single class IB member is a heterodimer of the catalytic subunit p110{gamma} and adaptor p101. In vitro, class IA and IB isoforms can utilize PI, PI 4-monophosphate [PI(4)P], and PI 4,5-bisphosphate [PI(4,5)P2] to generate PI 3-monophosphate [PI(3)P], PI 3,4-bisphosphate [PI(3,4)P2], and PI 3,4,5-trisphosphate [PI(3,4,5)P3], respectively, by catalyzing the phosphorylation of the inositol ring at the 3'-OH of membrane-bound phosphatidylinositol. Activation of class I isoforms is primarily attributed to receptor tyrosine kinases and G protein-coupled receptors, and more recently, p110{beta} and p110{gamma} activation by G{beta}{gamma}-dimers, putatively present constitutively in cytosolic pools (23), has been characterized (22, 25, 26, 32, 44). Class II isoforms are characterized by a carboxyl-terminal C2 domain and can utilize PI and PI(4)P to mediate growth factor stimulation (1, 2, 50). Class III isoforms are constitutively active, utilizing only PI as a substrate and function to facilitate vesicular trafficking (reviewed in Refs. 7 and 43). The most extensively studied isoforms that have been identified in the adult or neonatal heart are the class I family members p110{alpha}, p110{beta}, and p110{gamma} (reviewed in Ref. 46).

The 3'-phosphorylated lipid products generated by PI3K provide membrane-bound recognition sites for the recruitment of downstream signaling molecules including those that contain pleckstrin homology domains, such as 3'-phospho-inositide-dependent kinase (PDK) and Akt. Recent evidence suggests that the PI3K pathway regulates contractility in the myocyte. In the myocardium, Akt overexpression results in a number of downstream events, including increased contractility, suggesting that PI3K activity can stimulate myocyte contraction (9). Recently, the positive inotropic effect induced by insulin-like growth factor-1 (IGF-1) in the failing human myocardium was found to be mediated by PI3K (47) and, in cardiomyocytes, PI3K activity regulates Ca2+ oscillations in response to purinergic stimulation (6). In vascular myocytes, vasoconstrictor and growth factor stimulation of the L-type Ca2+ channel is mediated by specific PI3K isoforms (25) and, in neuronal cells, Akt potentiates L-type Ca2+ channel stimulation (5). In contrast, the cardiac-specific targeted deletion of the p110{gamma}–/– isoform in mice results in enhanced contractility (10), and in transgenic mice, overexpression of dominant-negative p110{alpha} has no discernable effect on contractility (10, 40). To address the role of PI3K in EC coupling in spontaneously contracting neonatal rat ventricular myocytes (NRVM) in the absence of G protein-coupled receptor or growth factor stimulation, we assessed the effect of agonists and antagonists of regulatory proteins involved in EC coupling in the presence of LY-294002, a reversible inhibitor of PI3K activity (45).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Neonatal rat ventricular myocyte isolation. Myocytes were dissociated from the ventricles of 1- to 3-day-old Sprague-Dawley rats (Harlan Industries; Indianapolis, IN) through repeated digestion in 1.8 mg/ml trypsin (Becton-Dickinson; Franklin Lakes, NJ); 20 mmol/l HEPES (Invitrogen; Carlsbad, CA), 0.02 mg/ml DNAse (Roche) in Hanks buffer solution (Invitrogen), followed by trypsin inactivation using 10% horse serum (HS; Invitrogen). After being preplated (2 h) to remove nonmyocytes, myocytes were plated in Izumo medium for cardiomyocytes (34): 1 µg/ml insulin, 1 µg/ml transferrin, 10 ng/ml sodium selenite, 100 µg/ml ampicillin, 3 mmol/l sodium pyruvate, 15 mmol/l HEPES, 2 g/l bovine serum albumin, 10% HS in DMEM/F12 (Invitrogen), containing 5-bromo-2-deoxuridine (100 µmol/l; Sigma). After 24 h, the medium was replaced with serum-free Izumo medium, containing no 5-bromo-2-deoxuridine. All analyses were performed on day 4 of plating.

The Eli Lilly Institutional Laboratory Animal Care and Use Committee approved all procedures, and animals were housed in a facility approved by the American Association for Accreditation of Laboratory Animal Care.

Steady-state contraction frequency analysis. Contraction frequency analysis was carried out at room temperature with myocytes plated at a density of 1 x 106 cells/well in 6-well plates (Becton-Dickinson). Cell movements were used as the indexes of contraction and were recorded using a video-edge detection system (Crescent Electronics; Sandy, UT). After equilibration (10 min, room temperature) in 1:4 vol/vol DMEM-Tyrode solution (DMEM; Invitrogen) {in mmol/l: 140 NaCl, 6 KCl, 1 MgCl2, 10 glucose, 5 HEPES (pH 7.4) (Sigma); final [Ca2+] = 0.25 mmol/l}, steady-state contraction frequency was determined for an individual cell. The cell under study was then allowed to stabilize for 20 min at each concentration of LY-294002, and two measurements over 30-s periods were made per cell. After the addition of 100 µmol/l LY-294002, the medium was exchanged for that containing 12.5 ng/ml platelet-derived growth factor (PDGF; Calbiochem; San Diego, CA) in place of LY-294002. At 2 min, the frequency of steady-state contractions had returned to control levels.

Fluorescent plate reader analysis. Myocytes (plated at a density of 5 x 104 cells/well in 96-well, poly-D-lysine-coated plates; Becton-Dickinson) were loaded with Fluo-3 AM (dissolved in DMSO and pluronic acid F-127, Molecular Probes; Eugene, OR) in Tyrode buffer containing 0.25, 1.0, or 4.0 mmol/l Ca2+ and 2.5 mmol/l probenecid (Sigma; 1 h, 37°C). Myocytes were then washed three times in calcium-containing Tyrode buffer with probenecid, and basal fluorescence was determined at 480 nM using a fluorometric imaging plate reader (FLIPR, Molecular Devices; Sunnyvale, CA). During incubation (20 min, room temperature) with LY-294002, LY-303511, BAY K 8644, nifedipine, ryanodine, thapsigargin (Calbiochem), or NiCl2 (Sigma) in Ca2+-containing Tyrode buffer with probenecid, fluorescence was measured at 60-s intervals (compounds were dissolved in <0.05% DMSO or EtOH). The EC50 of each compound was determined (data not shown), and this concentration was used for coadministration with LY-294002. After incubation, steady-state SR Ca2+ content was assessed by the rapid application of 10 mmol/l caffeine (Sigma) in calcium-containing Tyrode buffer with probenecid by fluorescence measurements at 0.8-s intervals for 3.8 min. Caffeine-releasable Ca2+ content was calculated for each well as the maximum change in fluorescence over basal fluorescence with each well serving as its own control.

Western blot analysis. NRVM were homogenized and rocked (20 min, 4°C) in 25 mM Tris (pH 7.5) 0.5 mM EGTA (pH 7.4), 2.0 mM EDTA (pH 8.0), 0.5% Triton X-100 containing Complete Mini-Tab (Roche; Indianapolis, IN), and 2 mg/ml microcystin LR (Sigma). Homogenates were clarified by centrifugation (15,000 g for 20 min), and total protein concentration was determined (Pierce), fractionated by SDS-PAGE (4–20%), and transferred to PVDF using the Criterion Blotting System (Bio-Rad; Hercules, CA). Membranes were blocked (1 h) using Odyssey blocking buffer (LI-COR; Lincoln, NE) and incubated in blocking buffer-0.1% Tween 20 (4°C, 16 h) with anti-phospho-Akt AKT473 (Cell Signaling Technology; Beverly, MA), followed with goat anti-rabbit IRDye 600 (1 h, RT; Molecular Probes). Fluorescent signal was detected by using the Odyssey Infrared Imaging System (LI-COR).

Statistical analysis. One-way ANOVA was performed on normally distributed data and is considered statistically significant at P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Spontaneous, steady-state contraction frequency decreases with LY-294002. The steady-state contraction frequency of cultured NRVM was measured in the absence and 20 min after the addition of LY-294002 (Fig. 1). There was an inverse relationship between LY-294002 concentration and steady-state contraction frequency. At 6.25 µmol/l LY-294002, steady-state contraction frequency (8.5 ± 1.5 beats/min) was not different from control (9.5 ± 0.5 beats/min; P > 0.05). At 50 µmol/l LY-294002, steady-state contraction frequency significantly decreased (4.0 ± 1.4 beats/min; P < 0.05), and at 100 µmol/l LY-294002, steady-state contraction ceased. After the addition of 100 µmol/l LY-294002, medium was exchanged for that containing 12.5 ng/ml PDGF in place of LY-294002. By 2 min, the frequency of steady-state contractions had returned to control levels (9.0 ± 1.0 beats/min; P > 0.05), indicating that the LY-294002 effect was reversible and not due to decreased cell viability.



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  		Fig. 1. Spontaneous, steady-state contraction frequency decreases with LY-294002 and is restored with an increase in platelet-derived growth factor (PDGF)-releasable Ca2+. A: steady-state contraction frequency of cultured neonatal rat ventricular myocytes (NRVM) was measured in the absence and presence of LY-294002. After the addition of 100 µmol/l LY-294002, the medium was exchanged for that containing 12.5 ng/ml PDGF in place of LY-294002. By 2 min, the frequency of steady-state contractions had returned to control levels, indicating that the LY-294002 effect was reversible and not due to decreased cell viability. Data are presented as means ± SE for contraction frequency measured twice in 2 cells/treatment. *Significantly different from control, P < 0.05, one-way ANOVA. B: PDGF-releasable Ca2+ content was measured in Fluo 3-AM-loaded NRVM equilibrated (1 h, 37°C) in buffer containing 0.25, 1.0, or 4.0 mmol/l extracellular calcium concentration ([Ca2+]o) by using fluorescent plate reader analysis (FLIPR). Cells were then washed and incubated in vehicle or 50 µmol/l LY-294002 (20 min). Recordings were initiated during the last minute of this incubation, at which time PDGF (50 ng/ml) or buffer was added, and recordings were continued for 5 min. Shown is a representative tracing of one recording (of 16 replicates) of PDGF-stimulated cells in the presence and absence of LY-294002. C: FLIPR data are presented as % basal fluorescence means ± SE measuring the apex from 16 wells/treatment in a 96-well plate. *Statistically less than PDGF alone, P < 0.05, one-way ANOVA. D: LY-294002 inhibits PDGF-stimulated phosphorylation of Akt in NRVM. NRVM were pretreated with 50 µmol/l LY-294002 or vehicle (1 h, 37°C) and treated with vehicle or PDGF (50 ng/ml, 5 min, 37°C), and homogenates were subjected to SDS-PAGE. Blots were probed with anti-phospho Akt, which was detected using Odyssey imaging system.

 

Caffeine-releasable Ca2+ content decreases with PI3K inhibition. To determine whether the observed decrease in steady-state contraction frequency with LY-294002 corresponded to decreased stored Ca2+, the caffeine-releasable Ca2+ content was measured in Fluo 3-AM-loaded NRVM equilibrated in buffer containing 0.25, 1.0, or 4.0 mmol/l extracellular calcium ([Ca2+]o) in the absence or presence of LY-294002 (Fig. 2A). LY-294002 inhibited the caffeine-releasable Ca2+ content in a concentration and [Ca2+]o-dependent manner. LY-294002 attenuated the amplitude of the caffeine-inducible Ca2+ spike while basal fluorescence remained similar to treatment with buffer alone (Fig. 2B). In contrast, LY-303511, an analog of LY-294002 that is completely inactive against PI3K (45), had no effect on Ca2+ content (Fig. 2C), further suggesting a specific role for PI3K in this response.



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  		Fig. 2. Intracellular Ca2+ content decreases with phosphoinositide 3-monophosphate (PI3K) inhibition. Caffeine-releasable Ca2+ content was measured in Fluo 3-AM-loaded NRVM equilibrated (1 h, 37°C) in buffer containing 0.25, 1.0, or 4.0 mmol/l [Ca2+]o using FLIPR. Recordings were made of the last 3 min of this incubation time, at which time LY-294002, vehicle, or LY303511 were added. Incubation then occurred for 20 min at room temperature, at which time caffeine was added. Data are presented as % basal fluorescence means ± SE from 8 wells/treatment in a 96-well plate. {dagger}Statistically less than control, P < 0.05, one-way ANOVA. LY-294002 attenuates amplitude of caffeine-inducible Ca2+ spike in a dose-dependent manner (A), whereas basal fluorescence is similar to buffer alone (B). Data are presented as the mean fluorescence change from 8 wells/treatment in a 96-well plate with 50 µmol/l LY-294002. LY303511, an inactive analog of LY-294002, had no effect on caffeine-releasable Ca2+ content (C). Data are presented as % basal fluorescence means ± SE from 8 wells/treatment in a 96-well plate.

 

L-type Ca2+ channel influx is attenuated with PI3K inhibition. To examine whether the observed decrease in steady-state contraction frequency and in caffeine-releasable Ca2+ content with LY-294002 could be due to inhibition of NRVM L-type Ca2+ channels, the effect of the Ca2+ channel agonist BAY K 8644 (36) in the presence and absence of LY-294002 was examined (Fig. 3). BAY K 8644 increased caffeine-releasable Ca2+ content at 0.25 mmol/l [Ca2+]o (P < 0.05; one-way ANOVA). No additional increase was observed at 1.0 and 4.0 mmol/l [Ca2+]o, suggesting that the SR Ca2+ content is near maximal at these higher extracellular Ca2+ concentrations. LY-294002 coadministered with BAY K 8644 inhibited the caffeine-releasable Ca2+ content, suggesting PI3K regulation of the L-type Ca2+ channel.



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  		Fig. 3. L-type Ca2+ channel influx is attenuated with PI3K inhibition. Caffeine-releasable Ca2+ content was measured in response to the L-type Ca2+ channel agonist BAY K 8644 (0.01 µmol/l; open bars) in the presence or absence of LY-294002. NRVM were equilibrated (1 h) with Fluo 3-AM and 0.25 (A), 1.0 (B), or 4.0 mmol/l [Ca2+]o (C); recordings were made during the last 3 min of this incubation time, at which time BAY K 8644 and LY-294002 were added. Incubation then occurred for 20 min at room temperature, at which time caffeine was added. Caffeine-releasable Ca2+ content was determined using FLIPR analysis. Data are presented as % basal fluorescence means ± SE from 6 wells/treatment in a 96-well plate and are representative of results obtained from at least 3 separate experiments. *Statistically different from caffeine alone; {dagger}statistically different from LY-294002 plus BAY K 8644; {ddagger}statistically different from BAY K 8644 alone; P < 0.05 by one-way ANOVA.

 

Nifedipine and LY-294002 inhibit L-type Ca2+ channel influx. To determine whether the effect of LY-294002 to decrease the steady-state contraction frequency and intracellular Ca2+ content was due to decreased L-type Ca2+ channel activity, the effect of the L-type Ca2+ channel antagonist nifedipine (14) was examined (Fig. 4). Nifedipine decreased caffeine-releasable Ca2+ content at each [Ca2+]o. LY-294002 further inhibited this response throughout the concentration range tested in a dose-dependent manner. Nifedipine inhibition occurs through interaction at the extracellular and transmembrane domains (16, 39, 42). The inhibition observed with LY-294002 in the presence of nifedipine suggests PI3K activity is regulating the L-type Ca2+ channel at a site distinct from nifedipine. Because PI3K is a cytosolic protein that translocates to the membrane during activation, PI3K possibly exerts its effects on the intracellular domain of the channel.



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  		Fig. 4. Nifedipine and LY-294002 inhibit L-type Ca2+ channel influx. Effect of the L-type Ca2+ channel antagonist nifedipine (50 µmol/l; open bars) in the presence or absence of LY-294002 was measured in NRVM. Cells were equilibrated (1 h) in buffer containing Fluo 3-AM with 0.25 (A), 1.0 (B), or 4.0 mmol/l (C) [Ca2+]o; recordings were made during the last 3 min of this incubation time, at which time nifedipine and LY-294002 were added. Incubation then occurred for 20 min at room temperature, at which time caffeine was added. Caffeine-releasable Ca2+ content was determined using FLIPR analysis. Data are presented as % basal fluorescence means ± SE from 6 wells/treatment per 96-well plates and are representative of results obtained from at least 3 separate experiments. *Statistically different from caffeine alone; {dagger}statistically different from LY-294002 plus nifedipine; {ddagger}statistically different from nifedipine alone; P < 0.05 by one-way ANOVA.

 

Inhibition by LY-294002 is distinct from inhibition of SERCA2a by thapsigargin. Another possible mechanism contributing to the observed decrease in caffeine-releasable Ca2+ content with LY-294002 could be through the inhibition of SR Ca2+ uptake at SERCA2a. To examine this possibility, the effect of the SERCA2a inhibitor thapsigargin was assessed (24) (Fig. 5). Thapsigargin alone increased baseline Ca2+ content by 5 min through the inhibition of Ca2+ reuptake into the SR at SERCA2a, possibly with thapsigargin-induced Ca2+ release (Fig. 5A). In contrast, LY-294002 alone had no detectable effect on baseline Ca2+ content (Figs. 5A and 2B). Coadministration of LY-294002 and thapsigargin attenuated the thapsigargin-induced increase in baseline Ca2+ and caffeine-releasable Ca2+ content (Fig 5B). These data would suggest that SR Ca2+ uptake is sustained in the presence of LY-294002, and that PI3K regulation of EC coupling is primarily occurring at another site in the cell rather than at SERCA2a.



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  		Fig. 5. Inhibition by LY-294002 is distinct from inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) by thapsigargin. Inhibition of SERCA2a by thapsigargin (10.0 µmol/l; open bars) in the presence or absence of LY-294002 was assessed in NRVM. Cells were preloaded (1 h) in buffer containing Fluo 3-AM with 0.25, 1.0, or 4.0 mmol/l [Ca2+]o; recordings were made of the last 3 min of this incubation time, at which time thapsigargin and LY-294002 were added. Incubation then occurred for 20 min at room temperature, at which time caffeine was added. Ca2+ content was assessed using FLIPR. Baseline fluorescence increased with thapsigargin inhibition of SERCA2a (solid line), whereas LY-294002 (dashed line) had no effect on baseline fluorescence (A, representative transients at each [Ca2+]o). The inhibition of caffeine-releasable Ca2+ content by LY-294002 with thapsigargin is additive (B). Data are presented as % basal fluorescence means ± SE of 6 wells/treatment in a 96-well plate and are representative of results obtained from at least 3 separate experiments. *Statistically different from caffeine alone; {dagger}statistically different from LY-294002 plus thapsigargin; {ddagger}statistically different from thapsigargin alone; P < 0.05 by one-way ANOVA.

 

To eliminate the possibility that the effect of LY-294002 may be on SERCA2a activity directly, Ca2+ uptake was assessed in adult canine, cardiac-derived microvesicles (Fig. 6). Basal Ca2+ uptake (172 ± 22 counts/min) remained unchanged with 10 µmol/l LY-294002 (210 ± 56 counts/min), whereas quercetin, a nonspecific kinase inhibitor of the same structural class as LY-294002, stimulated Ca2+ uptake (1,256 ± 269 counts/min) as previously reported. Ellagic acid served as a positive control for enhanced SERCA2a activity, releasing phospholamban inhibition of SERCA2a, resulting in a large increase in Ca2+ uptake (1,343 ± 159 counts/min). Taken together with the FLIPR analysis, PI3K activity appears to primarily regulate intracellular Ca2+ content at a site other than SERCA2a.



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  		Fig. 6. Basal Ca2+ uptake by SERCA2a remains unchanged with LY-294002. SERCA2a activity was evaluated by Ca2+ uptake analysis of adult canine, cardiac-derived microvesicles in the absence (buffer) or presence of LY-294002. Quercetin, a nonspecific kinase inhibitor of the same structural class as LY-294002, and the positive control, ellagic acid, increased Ca2+ uptake. Data are presented as means counts per minute ± SE. *Statistically different from buffer alone; {dagger}statistically different from LY-294002 alone; P < 0.05 by one-way ANOVA.

 

Effect of ryanodine with LY-294002 is additive. Ryanodine receptor activity could be modulated directly or indirectly by a decrease in activity at the L-type Ca2+ channel. Therefore, caffeine-releasable Ca2+ content was measured after SR depletion by ryanodine (51) in the absence or presence of LY-294002 (Fig. 7). SR Ca2+ content was diminished with ryanodine at each [Ca2+]o. Coadministration of LY-294002 with ryanodine further decreased caffeine-releasable Ca2+ content in a dose-dependent manner. Because SR Ca2+ uptake at SERCA2a appears to be sustained in the presence of LY-294002 (Figs. 5 and 6), the decrease in caffeine-releasable Ca2+ content appears to result from a combination of diminished influx of Ca2+ into the cell by LY-294002 inhibition (consistent with inhibition of the L-type Ca2+ channel) and by ryanodine-receptor-induced depletion of the SR.



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  		Fig. 7. Effect of ryanodine with LY-294002 is additive. Caffeine-releasable Ca2+ content was measured during sarcoplasmic reticulum depletion by ryanodine (5.0 µmol/l; open bars) in the absence or presence of LY-294002. NRVM were preloaded (1 h, 37°C) with Fluo 3-AM in 0.25 (A), 1.0 (B), or 4.0 mmol/l [Ca2+]o (C); recordings were made during the last 3 min of this incubation time, at which time ryanodine and LY-294002 were added. Incubation then occurred for 20 min at room temperature, at which time caffeine was added, and caffeine-releasable Ca2+ content was measured using FLIPR analysis. Data are presented as % basal fluorescence means ± SE of 6 wells/treatment and are representative of results obtained from at least 3 separate experiments. *Statistically different from caffeine alone; {dagger}statistically different from LY-294002 plus ryanodine; {ddagger}statistically different from ryanodine alone; P < 0.05 by one-way ANOVA.

 

Effect of LY-294002 with NCX inhibition is [Ca2+]o dependent. The observed effect of LY-294002 on steady-state contraction frequency and Ca2+ content could be due to the inhibition of PI3K-mediated, reverse-mode influx. Therefore, the effect of Ni2+, an inhibitor of NCX (13, 20), in the absence and presence of LY-294002 was assessed (Fig. 8). Ni2+ inhibition of NCX decreased the caffeine-releasable Ca2+ content at 0.25 mmol/l [Ca2+]o, suggesting a high level of reverse-mode activity at this extracellular Ca2+ concentration. At 1.0 and 4.0 mmol/l [Ca2+]O, significant reverse-mode NCX activity was not apparent. At 1.0 mmol/l [Ca2+]o, the coaddition of LY-294002 and Ni2+ had a greater effect than LY-294002 alone. Although these data do not exclude the possibility that LY-294002 may be exerting its effect through inhibition of reverse-mode NCX activity, the minimal additive effect of Ni2+ with LY-294002 would suggest that the effect of LY-294002 is primarily restricting influx of Ca2+ into the cell at a site other than NCX.



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  		Fig. 8. Effect of LY-294002 with Na/Ca exchangeer (NCX) inhibition is [Ca2+]o dependent. Inhibition of Ca2+ movement through the NCX by Ni2+ (25.0 µmol/l; open bars) was examined in the absence and presence of LY-294002. NRVM were loaded (1 h, 37°C) with Fluo 3-AM in 0.25 (A), 1.0 (B), or 4.0 mmol/l [Ca2+]o (C); recordings were made of the last 3 min of this incubation time, at which time Ni2+ and LY-294002 were added. Incubation then occurred for 20 min at room temperature, at which time caffeine was added. Caffeine-releasable Ca2+ content was assessed using FLIPR analysis. Data are presented as % basal fluorescence means ± SE of 6 wells/treatment in one 96-well plate and are representative of results obtained from at least 3 separate experiments. *Statistically different from caffeine alone; {dagger}statistically different from LY-294002 plus Ni2+; {ddagger}statistically different from Ni2+ alone; P < 0.05 by one-way ANOVA.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the presence of LY-294002, spontaneous steady-state contraction frequency and caffeine-releasable Ca2+ content decreased in a dose-dependent manner in NRVM. LY-294002 inhibited L-type Ca2+ channel activity stimulated by BAY K 8644, suggesting that LY-294002 exerts its effect on steady-state contraction and Ca2+ content by inhibition of the PI3K activity regulating Ca2+ influx at this channel. Nifedipine-induced inhibition of the L-type Ca2+ channel occurs through interaction with extracellular and transmembrane domains. The further decrease in caffeine-releasable Ca2+ content with nifedipine in the presence of LY-294002 suggests that PI3K regulates the channel at sites other than the dihydropyridine binding site, possibly by phosphorylation events resulting in activation of intracellular domains. Thapsigargin inhibition of SERCA2a increased basal Ca2+; in contrast, LY-294002 had little, if any, effect on basal Ca2+. Ca2+ uptake by SERCA2a assessed in adult canine microvesicles was likewise unchanged in the presence of LY-294002. Taken together, these data suggest that SERCA2a activity is sustained in the presence of LY-294002. Ryanodine, acting at the ryanodine receptor, diminished SR Ca2+, and LY-294002 further decreased the caffeine-releasable Ca2+ content of the cell. This decrease in caffeine-releasable Ca2+ content with LY-294002 plus ryanodine is presumably the result of reduced Ca2+ influx into the cell by LY-294002 at the L-type Ca2+ channel. In addition to the L-type Ca2+ channel, NCX acting in reverse mode is another source of Ca2+ influx into the cell. The effect of LY-294002 plus Ni2+-induced inhibition of NCX was comparable to the effect of LY-294002 alone, again suggesting that the reduction of Ca2+ influx into the cell by LY-294002 is through the inhibition of L-type Ca2+ channel activity.

There is evidence to suggest that the L-type Ca2+ channel can be phosphorylated by protein kinases, resulting in increased channel activity (19). It is therefore conceivable that PI3K may influence the phosphorylation state of specific residues of the L-type Ca2+ channel during steady-state contraction in the myocyte by regulating downstream protein kinases. Although Akt is a candidate downstream kinase of PI3K, phosphorylation of the L-type Ca2+ channel by Akt in cardiomyocytes has yet to be described. However, in neuronal cells, dominant negative Akt inhibited potentiation and constitutively active Akt increased L-type currents (5). Whereas one laboratory (9) has reported that cardiac-restricted overexpression of Akt results in enhanced contractility, at least two other groups have reported that Akt overexpressed in the hearts of transgenic mice have little-to-no changes in contractile function compared with wild-type mice (28, 41) despite showing massive left ventricular hypertrophy. Shioi et al. (41) showed that fractional shortening of the hearts of transgenic mice overexpressing constitutively active Akt was significantly decreased to 37 ± 3%, whereas the cardiac index (the volume of blood that is pumped out of the heart per minute) was not different from that of nontransgenic mice. Matsui et al. (28) also created cardiac-restricted Akt-overexpressing transgenic mice. Two strains of these mice had relatively normal systolic left ventricular function (as measured by ejection fraction) comparable to the findings in nontransgenic mice. Interestingly, three transgenic founders died suddenly with massive cardiac enlargement at ages 9–19 wk (28). Whereas the cause of death was not reported, one can speculate whether the sudden death of these mice may have been due to contractile and/or conductance abnormalities. In any event, whereas the role of Akt in cardiac contraction is somewhat unclear, these findings suggest that other signaling targets downstream of PI3K activity may also be involved, with the activation of PI3K leading to an increase in L-type Ca2+ channel activity.

Candidate signaling targets downstream of PI3K that may function in this role include protein kinase C and {beta}AR kinase 1 ({beta}ARK1). PI3K may exert its effect on steady-state contraction by enhancing phospholipase C{gamma} (PLC{gamma}) activity within the myocyte. The hydrolysis of PI(4,5)P2 by PLC{gamma} results in the formation of inositol 1,4,5-trisphosphate and membrane-bound diacylglycerol. PI3K activity enhances PLC{gamma} hydrolysis of PI(4,5)P2 by recruiting the phospholipase to the cell membrane, increasing substrate availability (33). Protein kinase C, when activated by diacylglycerol, phosphorylates the L-type Ca2+ channel (19). PI3K may be stimulating L-type Ca2+ channel activity through the recruitment of PLC{gamma} to the membrane, increasing diacylglycerol formation and thereby protein kinase C mediated phosphorylation of the L-type Ca2+ channel. PI3K also exerts its effect on contractility through interaction with {beta}ARK1 leading to the endocytosis of the {beta}2AR (10, 18, 29, 30). Experiments to demonstrate changes in the phosphorylation state of the L-type calcium channel in response to LY-294002 are limited by the relatively low abundance of L-type Ca2+ channel protein in these cells.

It has been reported that p110{gamma}–/– mice display hypercontractility (suggesting that p110{gamma} inhibits contractility), whereas p110{alpha}-dominant negative mice display no alteration in contractile function (10, 40). However, cardiac-selective Akt overexpression in mice (CA-Akt) resulted in increased, not decreased, cardiac contractility (9), suggesting that PI3K can exert positive inotropic effects. Interestingly, phosphorylation of Akt remained unchanged in the p110{gamma}–/– mice (10), and relaxation was not enhanced in the CA-Akt transgenic heart (9), suggesting that the hypercontractility observed in the p110{gamma}–/– mice may be facilitated by a pathway distinct from Akt activation and that Akt is acting at a site other than SERCA2a. Indeed, in contrast to the putative Akt pathway, p110{gamma} exerts much of its effect by limiting the activation of SERCA2a (10, 18), presumably through interaction with {beta}ARK1 and endocytosis of the {beta}2AR (29, 30). Recently, IGF-1 was found to exert a positive inotropic effect in the failing human myocardium through PI3K activity (47). Because the growth factor IGF-1 can stimulate PI3K activity through association between the insulin receptor substrate-1 and the p85 subunit of the class IA isoforms (35), this positive inotropic effect is presumably occurring through p110{alpha} or p110{beta}, rather than the class IB family member, p110{gamma}. The differences in contractility in the p110{gamma}–/–, p110{alpha}-dominant negative, and CA-Akt transgenic mice, with IGF-1 in failing myocardium, as well as the inhibition of spontaneous steady-state contraction by LY-294002, suggest that distinct PI3K isoforms exert specific effects on contractility as they do on intracellular Ca2+ content (25, 32, 44). Because G{beta}{gamma}-dimers can stimulate the L-type Ca2+ channel through p110{beta} (25), and constitutive cytosolic pools of {beta}{gamma}-dimers may provide the stimulation of PI3K activity at the L-type Ca2+ channel in NRVM as previously postulated for vascular myocytes (44), the steady-state contraction and regulation of the L-type Ca2+ channel observed in the current report may be mediated by p110{beta}. Contributions by specific isoforms could not be conclusively assessed by using LY-294002 or with wortmannin, which nonselectively inhibit all class I isoforms.

The finding that inhibition of PI3K by LY-294002 reduces spontaneous steady-state contraction frequency appears to be in direct contradiction to a recent report (10), in which single cell contraction amplitude in wild-type cardiomyocytes in the presence of LY-294002 is enhanced. However, the single cell contraction analysis in that report was performed in the presence of theophylline. Theophylline, at the dosage used, inhibits phosphodiesterase activity, limiting cAMP hydrolysis and increasing cAMP accumulation, which results in an increased inotropic effect and accelerated cardiac relaxation, similar to that of {beta}-adrenergic catecholamine stimulation (12, 37, 38). In our study, neither catecholamines nor theophylline were used to investigate the role of PI3K in the regulation of steady-state contraction. Early in the development of L-type Ca2+ channel antagonists, {beta}-adrenergic catecholamines were found to override L-type Ca2+ channel antagonism (14, 15, 21). Thus the L-type Ca2+ channel antagonism of LY-294002 would be overridden in the presence of theophylline. This difference in methods may be responsible for the opposite effects on contraction in wild-type cardiomyocytes reported for LY-294002.

The systematic assessment of intracellular Ca2+ content in the presence of LY-294002 with well-characterized agonists and antagonists of regulatory proteins involved in EC coupling revealed that decreased steady-state spontaneous contraction frequency in NRVM is primarily attributed to inhibition of L-type Ca2+ channel activity. This current work is supported by the recent findings in cardiac-selective Akt overexpressing mice in which cardiac contractility is increased (9) and supports the concept that specific PI3K isoforms exert distinct effects on cardiac contractility (10, 18, 40). Two recent reviews address the role of the PI3K pathway in contractility, as well as data obtained using transgenic models overexpressing (or deleting) various components of this pathway. In the first review (46), it was concluded that PI3K{alpha} modulates heart size, whereas PI3K{gamma} negatively modulates cardiac contractility based on studies of transgenic mice, in which cardiac PI3K{alpha} and/or PI3K{gamma} activities were ablated. A second review by Naga Prasad (31) reached a similar conclusion in that PI3K{gamma} plays a critical role in regulating contractility by negatively regulating adenylyl cyclase activity and phosphorylation of phospholamban, whereas PI3K{alpha} is involved in cardiac growth. Because PI3K{alpha} and PI3K{gamma} have different, and perhaps opposing, roles in regulating cardiomyocyte survival and function, an inhibitor of PI3K{gamma} could be useful in modulating pathological changes associated with calcium handling. Such a compound would improve cardiac relaxation by modulating {beta}2-adrenergic receptor function by increasing PKA activity and phosphorylation of phospholamban. The PI3K inhibitors wortmannin and LY-294002 inhibit both the PI3K{alpha} and PI3K{gamma} isoforms, and any differential effects of these isoforms on contractility may not be distinguished using a general PI3K inhibitor. These current findings build on the previous identification of specific PI3K isoforms shown to mediate agonist-stimulated L-type Ca2+ channel activity in vascular myocytes (25, 44), now linking this role to the maintenance of steady-state spontaneous contraction in NRVM and identifying the L-type Ca2+ channel as a key protein involved in EC coupling regulated by PI3K activity.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. J. Vlahos, Cardiovascular Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285-0520 (E-mail: Vlahos_Chris_J{at}Lilly.com).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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