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Cardiovascular Research 2004 62(3):509-520; doi:10.1016/j.cardiores.2004.01.029
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

The phosphoinositide 3-kinase inhibitor LY294002 enhances cardiac myocyte contractility via a direct inhibition of Ik,slow currents

Hui Sun, Gavin Y. Oudit, Rafael J. Ramirez, Danny Costantini and Peter H. Backx*

Department of Physiology and Medicine, Heart & Stroke/Richard Lewar Centre and Division of Cardiology, University Health Network, University of Toronto Fitzgerald Building, 150 College St., Toronto, Ontario, Canada M5S 3E2

* Corresponding author. Tel.: +1-416-946-8112, +1-416-528-4003; fax: +1-416-946-8380. Email address: p.backx{at}utoronto.ca

Received 28 August 2003; revised 15 January 2004; accepted 27 January 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Phosphoinositide 3-kinase (PI3K) is a key component in regulating myocardial growth, survival and contractility. LY294002 and wortmannin are two PI3K inhibitors used widely to establish the role of PI3K. The goal of this study was to examine the effects of acute application of LY294002 and wortmannin on cardiac myocyte contractility and underlying machanisms. Methods: Patch-clamp, indo-1 epifluorescence and video-edge detection techniques were used to measure outward K+ currents, action potentials (AP), Ca2+ transients and shortening of myocytes isolated from mouse left ventricular free wall. Results: In field-stimulated myocytes, LY294002 (10 µmol/l) increased Ca2+ transient amplitude by 23%, and cell shortening amplitude by 60% in the absence or presence of wortmannin, while wortmannin alone had no effect. LY294002 (but not wortmannin) prolonged AP duration by specifically inhibiting slowly inactivating K+ currents (i.e., the 4-aminopyrydine-sensitive Ik,slow1 and the tetraethylammonium-sensitive Ik,slow2), leading to an increase in sarcoplasmic reticular Ca2+ levels. It appeared that the AP prolongation was responsible for elevated contractility since AP-clamp of myocytes with prolonged APs (recorded in LY294002-treated myocytes) induced a 29% increase in cell shortening compared with control APs, while LY294002 application did not increase contractility in voltage-clamp studies using either step or AP depolarizations. Conclusions: The putative PI3K inhibitor LY294002 increases Ca2+ release and myocyte contractility via direct inhibition of cardiac Ik,slow and AP prolongation, thus limiting the usefulness of this agent in the analyses of the role of PI3K in heart function.

KEYWORDS Phosphoinositide 3-kinase; LY294002; Cardiac myocyte contractility; Ca2+ transient; Action potential; Ik,slow currents


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Phosphoinositide 3-kinases (PI3Ks) are a family of evolutionarily conserved lipid kinases that mediate the formation of D-3 phosphoinositides which are key components of many signaling pathways regulating a broad range of fundamental cellular processes including cell growth, differentiation, survival and migration in response to extracellular stimuli [1,2]. The availability of PI3K inhibitors such as wortmannin and LY294002 has been crucial in advancing our understanding of the biological functions of PI3K enzymes [3,4]. In the heart, for example, these inhibitors have been used to establish the role of PI3Ks in mediating inotropic [5,6], hypertrophic [7,8] and antiapoptotic responses [9,10] of cardiac myocytes to different stimuli. Wortmannin and LY294002 are structurally distinct antagonists of all three classes of PI3Ks. Wortmannin was originally isolated from Penicillium wortmannii and irreversibly inactivates PI3Ks by covalent modification of the catalytic subunit [4,11,12]. On the other hand, LY294002 is a synthetic compound derived from the broad-spectrum kinase inhibitor quercetin [13] and reversibly inhibits PI3K by competing with ATP for the active site of catalytic subunit p110 [4,13,14].

Recent studies in transgenic mice established that deletion of PI3K{gamma} enhances cardiac contractile function, while dominant-negative inhibition of PI3K{alpha} impairs myocardial growth [15]. Consistent with these mouse studies, acute application of LY294002 increased cell shortening amplitude [15]. In order to elucidate the mechanism(s) of PI3K-mediated inotropic effects, we examined the actions of wortmannin and LY294002 application on K+ current, action potential, Ca2+ transient and cell shortening in isolated mouse myocytes. We found that LY294002, but not wortmannin, increased myocyte shortening which was associated with blockade of Ik,slow currents and action potential prolongation.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Myocyte isolation
Single myocytes were isolated from the left ventricle free wall of C57BL6 male mice (12 weeks old, Charles River) using Type II collagenase (0.4 mg/ml, Boehringer-Mannheim) and Type XIV protease (0.025 mg/ml, Sigma) as previously described [16]. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2. Electrophysiology
Action potentials (AP) and outward K+ currents were recorded with the whole-cell patch clamp technique under current-clamp and voltage-clamp mode, respectively, using an Axopatch 200B amplifier and pClamp 6 software (Axon Instrument). The pipette resistance ranged between 1.2 and 1.8 M{Omega} when filled with a solution containing (mmol/l) 120 potassium aspartate, 20 KCl, 5 NaCl, 1 MgCl2, 5 MgATP, 10 HEPES and 10 EGTA, pH 7.2. Cell capacitance and series resistance were electronically compensated by 80–90%. Myocytes were held at –80 mV, and K+ currents were elicited by 5-s depolarizations to +40 or +60 mV. The fast Na+ current was inactivated by applying a prepulse to –40 mV for 50 ms. Membrane currents were sampled at two different rates (10 kHz for the first 500 ms and 2 kHz for the rest 4.5 s) and filtered at 2 kHz. The bath solution for AP recordings contained (mmol/l) 140 NaCl, 4 KCl, 1 MgCl2, 1.2 CaCl2, 10 HEPES and 10 D-glucose (pH 7.4). For K+ current recordings, the L-type Ca2+ channel was blocked by adding 0.3 mmol/l CdCl2 to the bath solution. In some experiments, NaCl was replaced by 135 mmol/l tetraethylammonium (TEA)-Cl. Experiments were performed at 35.5±0.5 °C.

2.3. Cell shortening measurements
Unloaded myocyte shortening was measured with a video edge detector coupled to a high frequency (240 Hz) charge-coupled camera (Crescent Electronics, UT, USA). Myocytes were voltage-clamped through patch pipettes or field-stimulated at 1 Hz via square pulses (5 ms x 6 V) delivered by a S44 Grass stimulator. The cell motion was sampled at 1 kHz (Felix software, Photon Technologies). The pipette solution for cell shortening (CS) measurement contained (mmol/l) 125 potassium aspartate, 20 KCl, 5 Na2-phosphocreatine, 0.5 MgCl2, 5 MgATP, 0.4 Na2GTP, 10 HEPES and 0.05 EGTA, pH 7.2. The amplitude of CS was measured at steady state. LY294002 or wortmannin was washed in after the steady-state CS was recorded under control conditions. The effects of drugs on CS were measured when the bath solution was completely exchanged for drug-containing solutions and the CS reached the new steady state.

2.4 Intracellular Ca2+ measurements
Ca2+ transients were measured on myocytes loaded with indo 1-AM (Molecular Probe) before and after addition of LY294002. The intracellular indo 1 was excited at 365 nm. Fluorescence emitted at 405 and 495 nm and collected by two matched photomultiplier tubes was filtered at 50 Hz and sampled at 1 kHz (Felix software). The ratio of the intensity of fluorescence emitted at 405 nm over that at 495 nm (R405/495) was calculated off-line after subtraction of background fluorescence and used as an index of intracellular Ca2+ concentrations in this study.

2.5. Chemicals
Wortmannin (Sigma) and LY294002 (Calbiochem) stocks were prepared in DMSO and then diluted to final concentrations in bath solutions before use.

2.6. Data analysis and statistics
The amplitude of cell shortening was expressed as percentage shortening relative to the resting diastolic length. The maximal velocity of shortening (+dL/dt) and the maximal rate of relaxation (–dL/dt) were measured from time derivatives of the cell shortening signal. The AP and current recordings were analyzed using pClamp software (Clampfit 8.2, Axon, CA, USA). The decay phase of outward K+ currents was fitted to a sum of two or three exponentials with Chebyshev method (Clampfit 8.2, Axon, CA, USA). The fits to the data are considered adequate if the correlation coefficient was greater than 0.98.

Statistically significant differences between two means was assessed using pair or unpaired Student's t-tests. A P value<0.05 was considered to indicate significance. Group data are expressed as the mean±S.E.M.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Differential effects of LY294002 and wortmannin on myocyte contractility and action potentials
To examine the role of PI3K in regulating basal myocyte contractility, we applied the PI3K inhibitors LY294002 and wortmannin to freshly isolated, field-stimulated myocytes. Fig. 1A(a) and B(a) shows that application of 10 µmol/l LY294002 increased (P<0.05) the amplitude of steady-state cell shortening from 7.0±0.5% to 11.3±0.7% (n=10) while also enhancing the maximal velocities of shortening and relaxation when myocytes were stimulated at 1 Hz. The effects of higher doses of LY294002 could not be reliably studied due to the frequent appearance of spontaneous contractions, presumably due to excessive SR Ca2+ loading. By contrast, cell shortening was unaltered following the application of PI3K inhibitor wortmannin, at concentrations that are more than 50-fold greater than the IC50 for inhibition of PI3K [13] (Fig. 1A(b) and B(b)). However, the addition of 10 µmol/l LY294002 in the presence of 100 nmol/l wortmannin enhanced myocyte shortening to the same extent (P<0.05) as LY29004 alone (Fig. 1A(c) and B(c)), suggesting that LY294002 increases myocyte contractility by mechanisms independent of PI3K inhibition.


Figure 1
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  		Fig. 1 Effects of LY294002 and wortmannin on cell shortening under field stimulation conditions. (A) Representative recordings of steady-state cell shortening from three different myocytes stimulated at 1 Hz under different conditions: (a) before and 5 min after exposure to 10 µmol/l LY294002 (LY); (b) before and 15 min after application of 200 nmol/l wortmannin (WM); (c) before, 10 min after application of 100 nmol/l wortmannin and 5 min after subsequent addition of 10 µmol/l LY294002 in the presence of wortmannin. (B) Mean percentage cell shortening (CS), maximal velocity of shortening (+dL/dt) and rate of relaxation (–dL/dt) pooled from corresponding experiments shown on (A). (a) n=10; (b) n=6; (c) n=10. *P<0.01 vs. Control; **P<0.01 vs. Control and wortmannin groups.

 
To determine whether the increased myocyte contractility induced by LY294002 was associated with elevated intracellular Ca2+, Ca2+ transients were measured with indo 1. As shown in Fig. 2, addition of 10 µmol/l LY294002 increased (P<0.001) the amplitude of Ca2+ transients by 23% (n=6 cells), suggesting that LY294002 enhances myocyte contractility by increasing Ca2+ release.


Figure 2
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  		Fig. 2 Effects of LY294002 on intracellular Ca2+ transients. (A) Representative recordings of Ca2+ transients in response to field stimulations at 1 Hz obtained from a myocyte before and after bath application of 10 µmol/l of LY294002. R405/495: ratio of indo 1 fluorescence emitted at 405 nm over that at 495 nm. (B) Mean amplitude of Ca2+ transients obtained from six cells in the absence (Control) and presence of 10 µmol/l of LY294002. The amplitude of Ca2+ transients ({Delta}R405/495) was measured as the difference between diastolic and maximal systolic fluorescence R405/495. *P<0.001.

 
Since action potential profile and membrane potential can regulate cardiac contractility via modulation of Ca2+ cycling, we examined the effects of LY294002 on action potentials. Fig. 3A shows that LY294002 markedly prolongs the late phase of action potential repolarization while having no effect on the resting potential, the amplitude of action potential or the early repolarization. Indeed, Fig. 3B establishes that 5 and 10 µmol/l LY294002 prolonged (P<0.05) the time to 90% repolarization (i.e., APD90) but not the time for 50% repolarization (i.e., APD50). These effects of LY294002 on AP were not mediated by PI3K inhibition, since 100 nmol/l wortmannin did not alter AP profile even 15 min after drug application (Fig. 3 and Table 1). On the other hand, application of LY294002 in the presence of 100 nmol/l wortmannin caused similar changes in AP profile as observed with LY294002 alone (Fig. 3A).


Figure 3
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  		Fig. 3 Effects of LY294002 and wortmannin on action potential (AP). (A) Representative APs recorded on current clamp mode (1 Hz). (a) APs recorded before and 5 min after bath application of 10 µmol/l LY294002 (LY). (b) APs recorded in the absence of drugs, 15 min after application of 100 nmol/l wortmannin (WM), and 5 min after subsequent addition of 10 µmol/l LY294002 in the presence of wortmannin. (B) Mean ratio of APD50, APD75 and APD90 obtained in the presence of LY294002 or wortmannin over those obtained on the same cells before application of drugs (Control). n=7 for wortmannin group, n=5 for LY294002 5 µmol/l group and n=8 for LY294002 10 µmol/l group, *P<0.05 vs. Control.

 

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  		Table 1 Effects of wortmannin and LY294002 on mouse ventricle action potentials

 
The results above suggest that changes in AP profile by LY294002 are responsible for the changes in myocyte contractility. To investigate this possibility, we examined the effects of LY294002 on myocyte shortening under voltage-clamp conditions in order to prevent membrane potential changes. Fig. 4A shows representative cell shortening traces recorded in response to 200-ms depolarizations from –80 to 0 mV at 1 Hz before, 5 min after application of 25 µmol/l LY294002 as well as after washout of the drug. In contrast to field-stimulated myocytes, under voltage-clamp conditions, LY294002 application caused a small reduction in cell shortening. This small reduction resulted, at least in part, from a time-dependent rundown of cell contractility since similar reductions in cell shortening were observed in the absence of LY294002 at times corresponding to those when LY294002 was applied (i.e., 10 min after forming whole-cell configuration, Fig. 4B and C). This reduced contractility of voltage-clamped cells might also arise partially from a small measurable (16%) inhibition of L-type Ca2+ currents produced by 25 µmol/l LY294002 application (data not shown).


Figure 4
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  		Fig. 4 Effects of LY294002 on cell shortening under voltage clamp conditions. (A) Representative recordings of cell shortening elicited by 10th step pulse from –80 to 0 mV applied at 1 Hz (top) before, 5 min after application of 25 µmol/l LY294002 (LY) and after washout of the drug. (B) Mean amplitude of cell shortening (CS) normalized with respect to that obtained 5 min after whole-cell formation and before application of LY294002 (Control). n=6. *P<0.05 vs. Control. (C) Mean amplitude of cell shortening recorded at 10 min after forming whole cell configuration (WCC) normalized with respect to that obtained at 5 min after whole-cell access and taken as control. n=5. *P<0.05.

 
To further demonstrate that APD prolongation induced by LY294002 is responsible for the enhanced myocyte contractility observed, we assessed myocyte shortening in response to stimulation with typical steady-state APs recorded from myocytes in the absence (Control AP) or in the presence of 5 µmol/l LY294002 (LY-AP, Fig. 5). LY294002 at 5 µmol/l increased (P<0.05) CS in field-stimulated myocytes (7.0±0.5% vs. 8.9±0.4%, n=5). When myocytes were stimulated with a train of LY APs, cell shortening was increased (P<0.05) by 29% compared to the cell shortening measured in the same myocytes stimulated with Control-APs. The degree of increase in contractility is similar to that observed in field-stimulated myocytes, suggesting that the effects of LY294002 on AP duration are sufficient to explain the increased steady-state myocyte contractility observed with LY294002 application. Changes in action potential profile can alter Ca2+ transients and contractility by modulating triggered release of Ca2+ from the SR, by modifying the SR Ca2+ content or both. To distinguish between these possible mechanisms, we measured myocyte shortening evoked by both a Control-AP and a LY-AP under the conditions when equal SR Ca2+ level was kept fixed by using a train of 10 depolarizing pulses of 100 ms to 0 mV (at 1 Hz). Fig. 5C and D shows that, when the SR Ca2+ was kept constant, the amplitude of cell shortenings elicited by a Control-AP (left) vs. LY-AP (right) was not different, establishing that LY294002 enhances contractility and Ca2+ transients by varying SR Ca2+ load as a result of AP prolongation.


Figure 5
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  		Fig. 5 Effects of LY294002-induced AP prolongation on cell shortening. (A) Representative recordings of steady-state cell shortening evoked by a train of typical APs (1 Hz) recorded in the absence (Control AP) or in the presence of 5 µmol/l LY294002 (LY-AP, top). (B) Mean amplitude of percentage steady-state cell shortening (CS) evoked by Control APs and LY-APs. n=6. *P<0.01. (C) Representative recordings of myocyte shortening evoked by single Control AP or LY-AP (top) following a SR loading protocol consisting of ten 100-ms steps from –80 to 0 mV (1 Hz). (D) Mean amplitude of percentage cell shortening obtained from the experiments shown on (C). n=5.

 
To further establish that APD prolongation is critical for the increased contractility observed following LY294002 addition, we examined the effects of LY294002 on myocyte shortening in response to stimulations with both Control AP and LY-AP (Fig. 6). Similar to the results shown in Fig. 5A and B, stimulation with a train of prolonged LY-APs increased (P<0.01) cell shortening by 23% in a group of five cells compared to the cell shortening measured in the same myocytes stimulated with a train of Control APs. However, bath application of 25 µmol/l LY294002 did not significantly alter cell shortening when myocytes were stimulated with Control or LY-APs. These findings further support our conclusion that LY294002 increases myocyte contractility primarily via prolongation of APD, although effects of LY294002 on other Ca2+ handling proteins or myofilament could still concurrently contribute to the increased myocyte contractility observed.


Figure 6
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  		Fig. 6 Effects of LY294002 on cell shortening under AP-clamped conditions. (A) Representative recordings of steady-state cell shortening evoked by Control AP and LY-AP on the same cell in the absence (–LY294002) and presence (+LY294002) of 25 µmol/l LY294002. (B) Mean amplitude of percentage steady-state cell shortening (CS) evoked by Control APs and LY-APs in the absence (–LY294002) and presence (+ LY294002) of LY294002. n=5. *P<0.01.

 
3.2 Effects of LY294002 on outward K+ currents
Since K+ currents are key determinants of cardiac AP profile, the effects of LY294002 on voltage-dependent K+ currents were examined. Fig. 7A shows representative outward K+ current recordings elicited by 5-s depolarizations to +60 mV in the absence and presence of LY294002 as well as the current that was inhibited by LY294002 (i.e., the LY294002-sensitive current) with the inset displaying currents during the first 120 ms following depolarization. Bath application of 25 µmol/l LY294002 inhibited a large portion of the inactivating outward K+ currents, leaving a very rapidly inactivating component and a sustained component. On average, the peak current, measured as the difference between the maximal outward current and the sustained current remaining at the end of 5-s pulses, was reduced (P<0.05, n=10) from 67.4±7.8 to 45.4±6.9 pA/pF. The sustained component at the end of 5-s pulses was also reduced (P<0.05, n=10) from 6.75±0.93 to 6.07±0.98 pA/pF (n=10). The LY294002-sensitive current density was 48.8±6.7 pA/pF and peaked significantly later than the current recorded prior to LY294002 application (24.9±1.6 ms vs. 4.0±0.2 ms). By contrast, superfusion of myocytes with 100 nmol/l wortmannin did not affect outward K+ currents. Addition of 25 µmol/l LY294002 in the presence of wortmannin inhibited K+ currents to the same extent as LY294002 alone (data not shown), suggesting that reductions of K+ currents by LY294002 were unrelated to inhibition of PI3Ks.


Figure 7
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  		Fig. 7 LY294002 selectively inhibits Ik,slow. (A) Representative current recordings elicited by 5-s depolarizations from –80 to +60 mV in the absence (Control) and presence of 25 µmol/l LY294002 (LY) and the LY294002-sensitive current (LY-sensitive). The LY-sensitive current was obtained by off-line digital subtraction of the current recorded in the presence of LY294002 from the control current. Inset shows the same current traces expanded for the first 120-ms depolarization. (B) Fit of control or LY-sensitive current decay to three- or two-exponential function (dash lines), respectively. The fitting was performed for current decay over 5-s depolarization. Only the first 2-s traces are shown for clarity. (C) Representative current recordings elicited by 5-s depolarizations from –80 to +60 mV in the absence (Control) and presence of both 50 µmol/l 4-AP and 135 mmol/l TEA (4-AP+TEA), together with the 4-AP+TEA-sensitive current obtained by digital subtraction. Inset shows the same traces expanded for the first 120-ms depolarization. (D) Fit of control and 4-AP+TEA-sensitive current decay to a three-exponential function (dash lines).

 
The outward K+ current in mouse ventricular free wall is composed of multiple overlapping components: a rapidly inactivating transient outward K+ current (Ito.f), two slowly inactivating K+ currents (Ik,slow1 and Ik,slow2) and a sustained non-inactivating component (Iss) [17,18]. Two approaches were utilized to determine the K+ current components that are blocked by LY294002. First, we analyzed the decay phase of control currents and LY294002-sensitive currents (Fig. 7B). The decay phase of currents during 5-s pulses in the absence of LY294002 was well fit with a tri-exponential function with time constants of 1802±140 ms ({tau}1), 310±24 ms ({tau}2) and 79±12 ms ({tau}3) which match closely to those reported previously for Ik,slow2 ({tau}1), Ik,slow1 ({tau}2) and Ito,f ({tau}3), respectively [17–19]. The decay of current that is blocked by LY294002 (i.e., LY294002-sensitive current) could be fit with a bi-exponential equation ({tau}1=1500±98 ms and {tau}2=211±8 ms). The slow time constant {tau}1 for the LY294002-sensitive currents was not different from the slowest time constant {tau}1 of control currents, while the faster time constant {tau}2 for the LY294002-sensitive currents had a magnitude closely resembling to the intermediate time constant {tau}2 for the control currents (Table 2), suggesting that LY294002 may block Ik,slow1 and Ik,slow2. This suggestion is consistent with the observation that the amplitude of the two components of LY294002-sensitive currents was also very similar to the corresponding amplitudes of Ik,slow1 and Ik,slow2 identified in the control currents (Table 2).


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  		Table 2 Current decay time constants and current densities obtained in the absence and presence of different agents

 
The above analysis relies on the accurate dissection of multiple overlapping currents, which is generally only valid if LY294002 does not affect the kinetics properties of the currents being blocked. To investigate further whether LY294002 blocks Ik,slow1 and Ik,slow2 currents, we used a combination of 50 µmol/l 4-AP and 135 mmol/l TEA which have been shown (at these concentrations) to block primarily Ik,slow1 and Ik,slow2, respectively [17]. Typical current traces recorded during 5-s depolarizations to +60 mV in the absence and presence of both 4-AP and TEA as well as the corresponding 4-AP + TEA-sensitive current are illustrated on Fig. 7C (inset shows the same recordings for the first 120 ms). The combination of 4-AP and TEA reduced the amplitude of peak current from 56.1±6.2 to 23.2±4.2 pA/pF while also inhibiting Iss from 6.3±0.6 to 3.9±0.5 pA/pF. As is evident, 4-AP plus TEA inhibited a large portion of slowly inactivating outward current, leaving a rapidly inactivating component that is similar to the rapid current remaining in the presence of 25 µmol/l LY294002. These currents blocked by 4-AP plus TEA are virtually identical to the current blocked by LY294002, suggesting that LY294002 blocks both Ik,slow1 and Ik,slow2. Unlike LY294002, the decay of 4-AP + TEA-sensitive currents was fit with a tri-exponential function (Fig. 7D and Table 2), consistent with the previous finding that the mouse Ito,f and Iss were partially inhibited by 4-AP and TEA at these concentrations [17]. These findings suggest that LY294002 selectively inhibits Ik,slow1 and Ik,slow2 currents in mouse myocytes. Consistent with this conclusion, preliminary data in two cells showed that while the combination of 4-AP and TEA reduced the amplitude of peak current by 60% (from 45.3±16.7 to 16.7±0.2 pA/pF), addition of 25 µmol/l LY294002 in the presence of 4-AP and TEA caused only 6% further reduction in current amplitude (from 16.7±0.2 to 14.2±0.2 pA/pF).

3.3 Concentration dependence of Ik,slow inhibition by LY294002
To evaluate the potency of inhibition of Ik,slow by LY294002, we examined the dose–response effects of LY294002 on the current. Fig. 8A presents the outward K+ currents recorded from a myocyte exposed to different concentrations of LY294002. As is evident, increasing concentrations of LY294002 progressively and selectively blocked the slowly inactivating Ik,slow1 and Ik,slow2 currents. The percentage of block is plotted against the concentrations of LY294002 in Fig. 8B. The amplitude of Ik,slow was determined as the difference between the current level at 80 ms of depolarization and Iss at the end of 5-s pulses. These results demonstrate that LY294002 inhibits Ik,slow1 and Ik,slow2 currents with an IC50 of 3.7 µmol/l and a power value of 1.8 (using the logistic function).


Figure 8
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  		Fig. 8 Dose–response relationship of Ik,slow inhibition by LY294002. (A) Typical current recordings elicited by 5-s depolarizations from –80 to +40 mV on one cell in the absence and presence of different concentrations of LY294002 (LY). (B) Percentage block of Ik,slow is plotted against the concentration of LY294002. Smooth line represents the fit of data points to a logistic function, giving rise to an IC50 of 3.7 µmol/l and a power of 1.8. Numbers in parentheses indicate the number of cells studied. The amplitude of Ik,slow was determined as the difference between the current level at 80 ms after the onset of depolarization and the sustained current at the end of 5-s pulse.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
LY294002 and wortmannin are two widely used PI3K inhibitors. In purified enzyme assays, the IC50 of PI3K inhibition was found to be 2–3 nmol/l for wortmannin [11,12] and 1.4 µmol/l for LY294002 [13]. Evidence for an involvement of PI3K in a given biological system including cardiac myocytes has typically been obtained by acutely treating cells with 10–100 nmol/l wortmannin and 5–50 µmol/l LY294002 [1,7,8]. In this study, we found that, at these concentrations, LY294002 exerted very different effects from wortmannin on cardiac myocyte K+ current, AP profile and contractility. Cell shortening of field-stimulated cardiomyocytes was increased by LY294002 but not by wortmannin, suggesting that increased myocyte contractility by LY294002 does not result from PI3K inhibition. Indeed, the positive inotropic effects of LY294002 could be linked to AP prolongation as a result of K+ current inhibition induced by LY294002. This conclusion is supported by the observation that LY294002 did not increase contractility in voltage-clamp studies using either step or AP depolarizations, while stimulation of myocytes with the prolonged APs recorded in LY294002-treated myocytes evoked greater contractions compared with Control APs in the absence or presence of LY294002. Similar differential effects of LY294002 and wortmannin have previously been reported in basophilic leukaemia cells [20] and airway smooth muscle cells [21], although the mechanism for these differences was unclear. Nonspecific effects of LY294002 and wortmannin on L-type Ca2+ channels in chromaffin cells have also been suggested by Warashina [22] in an earlier study.

The inability of wortmannin to affect basal myocyte contraction in our studies agrees with previous results in human myocardial strips [6]. However, the lack of effect of wortmannin was not expected given our previous results showing that a selective deletion of gamma isoform of PI3K increased myocardial contractile function [15]. The lack of effect of wortmannin on myocyte contractility would be expected if the basal level of PI3K activity is low in nonstimulated myocytes as reported previously [6,23], or alternatively, simultaneous inhibition of other PI3Ks (i.e., PI3K{alpha} and PI3Kβ) that are also expressed in myocardium may offset the positive inotropic effects of PI3K{gamma} inhibition. Future studies will be required to fully address the cellular mechanisms accounting for the inability of wortmannin to increase contractility in isolated mouse myocytes.

4.1 Selective inhibition of Ik,slow by LY294002
Outward K+ currents in mouse ventricular free wall are composed of four overlapping and kinetically distinct components, Ito,f, Ik,slow1, Ik,slow2 and Iss, precise details vary between different regions of the heart [17,18]. Studies on genetically engineered mice have demonstrated that Kv1.5 encodes for the faster Ik,slow1 [24,25] while Kv2.1 encodes for the slower Ik,slow2 [19]. Pharmacologically, Ik,slow1 is sensitive to micromolar concentrations of 4-AP, while Ik,slow2 is sensitive to millimolar levels of TEA. In our study, outward K+ currents could be fit with a function containing three exponential components with time constants of 80, 310 and 1800 ms, similar to those reported previously for Ito,f, Ik,slow1 and Ik,slow2, respectively [17,18]. LY294002 preferentially inhibited both Ik,slow1 and Ik,slow2 with a potency (IC50=3.7 µmol/l) much greater than 4-AP blockade of Ik,slow1 (IC50=32 µmol/l [25]) or TEA blockade of Ik,slow2 (IC50=638 µmol/l [18]). Interestingly, the potency of Kv channel inhibition by LY294002 is comparable to that for inhibition of PI3K-dependent superoxide generation in neutrophils (IC50=6.4 µmol/l) [27], although much lower concentrations biochemically inhibit purified PI3K enzymatic activities (IC50=1.4 µmol/l) [13]. The blockade of outward K+ currents by LY294002 was not shared by wortmannin, suggesting that LY294002 inhibits Ik,slow via a PI3K-independent mechanism, possibly by direct channel blockade. Consistent with this suggestion, LY294002 has been shown recently to directly block Kv2.1 currents in heterologouse system [26].

4.2 Functional implications of Ik,slow inhibition in mouse ventricle
The present study revealed that LY294002 prolonged APD90. This effect was attributable to the inhibition of Ik,slow since our AP recordings were obtained in the presence of high EGTA levels (10 mmol/l) in order to minimize inward Na/Ca exchanger currents which can affect late repolarization. Prolongation of APD90 but not APD50 by LY294002 is consistent with the relatively slow kinetic properties of the LY294002-sensitive current which peaked around 25 ms after the onset of depolarization. The effects of LY294002 on AP profile are also consistent with previous pharmacological studies. Specifically, dominant-negative inhibition of Kv2.1 channels (Ik,slow2) caused APD90 prolongation but not APD50 [19]. Similarly, inhibition of Kv1.x-based current using a truncated Kv1.1 proteins increased APD90 but not APD50 in apical myocytes which express Kv1.5 at high levels [28]. On the other hand, replacement of the Kv1.5 gene with the Kv1.1 gene (which is not expressed) in SWAP mice did not affect AP profile, although this was traced to compensatory increases in Ik,slow2 [25].

Prolongation of early repolarization of AP, in rat and mouse, following reduced Ito, strongly enhances the Ca2+ transients and myocyte contractility by increasing the trigger L-type Ca2+ current as well as SR Ca2+ loading [16,30]. Little is known, however, about the role of Ik,slow in the modulation of cardiac contractility. Our findings clearly demonstrate that prolongation of late AP repolarization following Ik,slow inhibition by LY294002 led to an increased myocyte contractility as a result of increases in SR Ca2+ loading. Consistent with this conclusion, inhibition of Ik,slow1 by low levels of 4-AP (50 µmol/l) increase left ventricular systolic pressure in mouse [31]. Changes in the late-phase repolarization via Ik,slow inhibition is expected to reduce Ca2+ extrusion via the Na/Ca exchanger [32] while having little impact on trigger L-type Ca2+ current. The resulting increase in SR Ca2+ content by LY294002 will increase SR Ca2+ release by enhancing the open probability of SR Ca2+ release channels and by increasing the amount of releasable Ca2+ in the absence of changes in trigger ICaL [33,34].

Cardiac myocyte contractility is determined by numbers of factors such as AP profile that controls trans-sarcolemmal Ca2+ influx and efflux, the properties of sarcolemmal and SR Ca2+ handling proteins, the efficiency of excitation–contraction coupling and the properties of myofilaments. Our finding that LY294002 markedly enhanced the Ca2+ transients and contractility of field-stimulated myocytes but did not increase cell shortening under voltage clamp conditions strongly support the conclusion that LY294002 exerts its positive inotropic effects primarily via alteration of AP profile. Consistent with this conclusion, stimulation of myocytes with prolonged APs recorded in the presence of LY294002 elevated contractility to a similar extent as that observed in field-stimulated myocytes treated with LY294002. Nevertheless, it is conceivable that alterations in the activities of other targets affecting Ca2+ cycling (like Na/Ca exchangers) or contractile proteins might also contribute to the increase in contractility observed in the presence of LY294002. It has been shown that the outward Na/Ca exchange current (reverse mode) facilitates late AP repolarization in canine ventricular myocytes and that Na/Ca exchange current inhibition causes APD90 prolongation [29]. The effects of LY294002 on Na/Ca exchange function were not directly examined in our studies. However, if the enhanced contractility observed in the presence of LY294002 was due to Na/Ca exchanger inhibition, a change in contractility would be expected when LY294002 was added in our voltage-clamp studies using either steps or APs, which was not observed. Future studies will be required to directly assess whether LY294002 blocks Na/Ca exchange currents in mouse myocardium.

In contrast to our studies in mouse myocytes, previous studies using rat ventricular myocytes did not observe increases in cell shortening following LY294002 application under basal conditions [5,35]. The inability of LY294002 to enhance contractility in rat probably originate from known molecular differences in K+ currents between mouse and rat myocytes [36]. Additional studies are clearly necessary to explore the basis for these differences between rat and mouse myocytes.

In summary, we found that LY294002 which is widely used as a "selective" PI3K inhibitor inhibits Ik,slow currents in mouse ventricular myocytes via a direct blockade of Ik,slow channels. Selective inhibition of Ik,slow currents by LY294002 slows late phase repolarization in mouse ventricular myocytes, thereby enhancing SR Ca2+ loading, Ca2+ release and contractility. Future studies using LY294002 will need to consider the effects of this agent on K+ currents and membrane repolarization.


   Acknowledgements
 
This work was funded by a CIHR operating grant (MOP-43947) to PHB. PHB is a Career Investigator of the Heart & Stroke Foundation of Ontario. HS is supported by a Diana Meltzer Abramsky Research Fellowship from the Thyroid Foundation of Canada and a Heart & Stroke/Richard Lewar (HSRLCE) fellowship. GYO hold fellowships from the CIHR and the HSRLCE.


   Notes
 
Time for primary review 25 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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