INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

INTRACELLULAR SIGNALING SPECIFICITY is determined in part by targeting of signaling cascades to discrete subcellular locations. In heart cells, A-kinase anchoring proteins are important in defining the signaling specificity of protein kinase A (PKA) (11) and may be essential for L-type Ca²⁺ channel regulation (22). There is also evidence for protein kinase C (PKC)- targeting in heart cells (37, 45). Yet much less is known of the phosphatases that oppose the action of such kinases. Biochemical studies (34, 47) suggest that protein phosphatase (PP)1 acts as a phospholamban phosphatase regulating sarcoplasmic reticulum (SR) function, whereas PP2A may regulate myofilament function as a "troponin I phosphatase" (38). Furthermore, immunoprecipitation studies (10, 35, 36) have shown that PP2A is directly associated with the cardiac ryanodine receptor and the ₁-subunit of the L-type Ca²⁺ channel. The fact that these enzymes can be precisely targeted suggests they are important signaling elements. However, the physiological roles of these signaling enzymes in cardiac cells remain to be defined.

Phosphatase inhibitors can reveal the presence of a dynamic balance between protein phosphorylation and dephosphorylation. PP1/PP2A inhibitors, such as okadaic acid and calyculin A, have been shown to stimulate L-type Ca²⁺ channel function in the absence of humoral stimulation (27, 28, 40, 41). Addition of exogenous phosphatases can also reveal such a dynamic balance. A previous study (13) from this laboratory demonstrated that intracellular dialysis of rat ventricular myocytes with either PP1 or PP2A decreased the magnitude of the cytosolic intracellular Ca²⁺ concentration ([Ca²⁺]_i) transient in the steady state without decreasing SR Ca²⁺ load. These studies reveal the importance of phosphatases in local control in the heart. In fact, because PP1/PP2A comprise the majority of serine-threonine phosphatase activity within cells, it might be expected that these enzymes are important regulators of many components of the excitation-contraction (E-C) coupling cascade, including the membrane currents involved in excitation, the proteins involved in uptake and release of Ca²⁺, and the myofilaments that transduce the Ca²⁺ signal into mechanical activity.

In the present study, calyculin A, a potent inhibitor of PP1 and PP2A (30), was used to characterize which components of the E-C coupling cascade are regulated by such a kinase/phosphatase balance in mouse ventricular myocytes. The murine heart model was chosen so that the results could be used to design future studies to exploit transgenic animal models. The goals of this study were as follows: 1) to accurately and precisely measure the activity of PP1 and PP2A and their inhibition by calyculin A in mouse ventricular myocytes, 2) to define the biological role of calyculin A-sensitive PP1 and PP2A in maintaining cardiac contractility in the absence of any receptor-activated signaling, and 3) to identify the specific components of the cardiac E-C coupling cascade whose activities are controlled in this manner. An important conclusion from these studies is that PP1 and/or PP2A, in concert with as-yet-unidentified protein kinase(s), control the level from which receptor-activated signaling pathways regulate contractility. This function is carried out primarily through control of L-type Ca²⁺ current (I_Ca) rather than by acting at all of the steps of the E-C coupling cascade.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Cardiac myocyte preparation. Hearts were removed from adult male CD-1 mice anesthetized with 30 mg/kg ip of pentobarbital sodium (Abbott Laboratories). The aorta was cannulated for Langendorff perfusion, and ventricular myocytes were isolated by a standard enzymatic technique exactly as described previously (14). The cells were maintained at room temperature in HEPES-buffered DMEM with 10% fetal calf serum. All experiments were carried out within 8 h of cell isolation.

Incubation with calyculin A. Cells were preincubated with varying concentrations (100 nM-1 µM) of calyculin A (Alexis Biochemicals) from a 1 mM stock in DMSO. Control cells were preincubated with equal volumes of DMSO alone. Calyculin A was present only during the preincubation period. After 15-30 min of preincubation, the cells were used for either biochemical or functional experiments. A small number of experiments (summarized in Fig. 7) were performed in which cells were exposed to calyculin A and staurosporine during voltage-clamp experiments. Voltage-clamp recordings were made in control, in the presence of calyculin A (100 nM), or with the serine-threonine protein kinase inhibitor staurosporine (300 nM) and then in the presence of both calyculin A and staurosporine. Both agents were added from stock solutions in DMSO, and an equal concentration of DMSO (0.04%) was present in all of the experimental solutions.

Phosphatase assays. Suspensions of myocytes were rapidly washed with 0.9% NaCl and then homogenized in lysis buffer consisting of HEPES-NaOH (20 mM, pH 7.5), NaCl (100 mM), protease inhibitor cocktail (1:400, Sigma P8340), and 0.02% -mercaptoethanol. The cell extracts were centrifuged at 178,000 g for 10 min at 4°C. Protein concentration in the supernatant was determined spectrophotometrically with Bradford's reagent (Bio-Rad) using BSA as the standard.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Specificity of [32P]phosphorylase a and RRATpVA peptide assays. Cytosolic fractions from homogenized myocytes were assayed for phosphatase activity with [32P]phosphorylase a (A) or RRATpVA (B). A: phosphatase activity towards [32P]phosphorylase a in the absence of any inhibitors (total activity), in the presence of 10 nM okadaic acid [OA; inhibits protein phosphatase (PP)2A], 1 µM inhibitor-2 (specific PP1 inhibitor), 500 nM OA (inhibits PP2A and PP1), and inhibitor-2 plus 10 nM OA. Note that both 500 nM OA and the combination of 1 µM inhibitor-2 and OA, which should each completely inhibit PP1 and PP2A, have the same effect on 32Pi release. B: total phosphatase activity toward RRATpVA in the presence and absence of 10 nM OA. Note that 10 nM OA, which is without effect on PP1 (6), inhibits >80% of the 32Pi release from RRATpVA.

1

1

Field stimulation and edge detection. These experiments were conducted at room temperature on a Nikon Diaphot inverted microscope. Cells were superfused with extracellular buffer consisting of 100 mM NaCl, 5 mM KCl, 25 mM NaHCO₃, 25 mM HEPES, 1.0 mM Na₂HPO₄, 1.0 mM MgSO₄, 10 mM D-glucose, and 1.8 mM CaCl₂ at pH 7.4 (NaOH). Cells were field stimulated (Grass S48) at 1 Hz with 5-ms pulses with a magnitude of ×1.5 threshold through platinum wires mounted in the bottom of the experimental chamber. Twitch contractions were recorded using a video-based edge detector (Crescent Electronics), digitized at 500 Hz (PP-50 Lab Patch Panel, Warner Instrument and Axotape, Axon Instruments), and analyzed off-line. Contractions were normalized to the resting cell length.

Field stimulation and [Ca²+]_i transients. Cells were loaded with fluo 3 by incubating a 2-ml cell suspension with 30 µl of 0.5 µg/µl fluo 3-AM stock in 20% (wt/vol) pluronic F-127 in DMSO. The final concentration of fluo 3-AM was 6.6 µM. Fluo 3-loaded cells were field stimulated (1 Hz) as described in Field stimulation and edge detection on a Bio-Rad MRC 600 laser scanning confocal microscope equipped with a Zeiss Neofluor ×63 oil immersion lens (numerical aperture = 1.25). Images were obtained in line-scan mode, processed, and analyzed using COMOS (Bio-Rad) and IDL5.2 (Research Systems) software. To measure [Ca²⁺]_i transients, the fluorescence images were normalized by dividing the fluorescence intensity of each pixel (F) by the average resting fluorescence intensity (F₀) to generate an F/F₀ image. Cell length during each line scan was measured by determining the portion of the scan line that exhibited fluo 3 fluorescence.

Action potential measurements. Action potential measurements were made at 35°C using low-resistance patch type microelectrodes and an Axopatch 200A with a CV202A head stage (Axon Instruments). The filling solution consisted of the following: 110 mM potassium aspartate, 20 mM KCl, 10 mM NaCl, 4 mM MgATP, and 10 mM HEPES at pH 7.3 (with KOH). The extracellular solution was the same as that used for the field stimulation experiments. The cells were stimulated (1 Hz) by applying a brief (2-5 ms) hyperpolarizing current clamp pulse. After release of this pulse, the cells reached threshold and generated an action potential.

Voltage-clamp experiments. These experiments were conducted at 35°C on a Nikon Diaphot 200 inverted microscope that was modified, as previously described (13), for the simultaneous measurement of membrane current and [Ca²⁺]_i. The voltage-clamp amplifier was an Axopatch 200A with a CV202A head stage. Whole cell voltage clamp was carried out using low-resistance (<2 M) patch type microelectrodes. Cells were placed in the experimental chamber and superfused with a solution containing 140 mM NaCl, 5 mM KCl, 10 mM HEPES, 1 mM MgCl₂, 0.33 mM NaH₂PO₄, and 10 mM D-glucose at pH 7.4 (with NaOH). The pipette filling solution used for simultaneous measurements of I_Ca and the cytosolic Ca²⁺ ([Ca²⁺]_i) transient consisted of 130 mM CsCl, 10 mM HEPES, 20 mM TEA chloride, 1.0 mM MgCl₂, 5.0 mM MgATP, and 25 µM K₅indo 1 at pH 7.3 (with CsOH). After whole cell access was attained, a modified superfusate was used, which included CsCl (substituted for KCl), 5 mM 4- aminopyridine, 10 mM TEA chloride to block K⁺ channels, and 10 µM TTX to block Na⁺ channels. Extracellular Ca²⁺ concentration ([Ca²⁺]_o) was 1.8 mM (CaCl₂). I_Ca was recorded from a holding potential of 40 mV. [Ca²⁺]_i was calibrated using a previously described in vitro technique (13). K⁺ currents were recorded using KCl-based intra- and extracellular solutions as described previously (14). [Ca²⁺]_o was 0.1 mM, and nifedipine (0.5 µM) and TTX (10 µM) were included to block I_Ca and Na⁺ current, respectively.

Myofilament Ca²+ sensitivity. These experiments were conducted on the same apparatus used to measure field-stimulated twitch contractions. EGTA-buffered solutions consisting of (in mM) 7 EGTA, 25 imidazole, 1 MgCl₂, 80 KCl, and 4 MgATP at pH 7.0 (with KOH) (29) were prepared at the following Ca²⁺ concentrations (in nM): 1, 100, 200, 300, 400, and 500. The amount of CaCl₂ required to achieve each Ca²⁺ concentration was calculated using a computer program that can account for the binding of Ca²⁺ and other cations to EGTA and the other constituents of the solution (16). CaCl₂ was added from a 250 mM stock prepared using a calcium standard solution (EM Science). An aliquot of cells was placed in the experimental chamber and superfused with solution containing 1 nM Ca²⁺. After several minutes of superfusion, cells were exposed for 1 min to a solution containing 1 nM Ca²⁺ and 10 µM digitonin (Sigma). Preliminary experiments showed that this was sufficient to permeabilize the myocytes. This was followed in succession by 5 min of superfusion with digitonin-free solutions described above (1-500 nM Ca²⁺). During the last minute of exposure in each concentration, when cell length had reached a new steady state, a measurement was taken. Myofilament Ca²⁺ sensitivity was assessed by normalizing cell length at each Ca²⁺ concentration to that at 1 nM Ca²⁺ and plotting the resulting average shortening values versus Ca²⁺ concentration.

Statistics. The experimental values are expressed as means ± SE, with statistical significance (P < 0.05) assessed using Student's paired t-test.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

A recent report (13) from this laboratory demonstrated that increasing the activity of PP1 or PP2A in cardiac cells had selective effects on elements of the E-C coupling cascade. In the present study, we used PP1 and PP2A inhibitors to further explore the selectivity and targeting of phosphatase action. Although there are several PP1/PP2A inhibitors that are frequently used in biochemical studies, it is essential to confirm their efficacy and specificity in intact cells (17). Accordingly, experiments were designed to assess the ability of phosphatase inhibitors to act on these enzymes in intact murine ventricular myocytes, as described in Fig. 1 and METHODS. Initial experiments with okadaic acid revealed that even long incubations (2 h) resulted in only minimal inhibition of PP1 or PP2A (data not shown). This is likely due to its low membrane permeability (17). The effects of calyculin A were quite different, as shown in Fig. 2. A 15-min incubation with 125 nM calyculin A resulted in inhibition of PP1 activity by 52%, whereas 1 µM calyculin A further reduced the activity to 34% of control. Calyculin A proved to be a more potent inhibitor of PP2A under these conditions, reducing the activity to zero at a concentration of 1 µM. Accordingly, calyculin A was used in all of the studies described below.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Inhibition of cytosolic PP1 and PP2A by incubation of isolated mouse ventricular myocytes with calyculin A. Cytosolic fractions from suspensions of homogenized myocytes were assayed for phosphatase activity with a 32P-labeled substrate specific for either PP1 (A) or PP2A (B) (see Fig. 1 and METHODS). A: PP1 activity after preincubation with DMSO alone (control, n = 4), 125 nM calyculin A (n = 3), or 1 µM calyculin A (n = 3). B: the same as for PP2A: control (n = 5), 125 nM calyculin A (n = 4), and 1 µM calyculin A (n = 5).

On the basis of these results, we examined the effects of calyculin A on the twitch contractions of single isolated mouse ventricular myocytes. In fact, calyculin A had a profound effect on the field-stimulated twitch contraction, as shown in Fig. 3. Calyculin A (125 nM) increased the average magnitude of contraction by 106%, from 8.6 ± 0.6% (n = 26) to 17.7 ± 0.7% (n = 17) of resting length. The duration of the contraction, measured as the time from 50% contraction to 50% relaxation, was also greater, increasing by 47% from 109 ± 3 to 159 ± 7 ms. These effects were maximal, because there was no additional effect of 1 µM calyculin A (n = 18) on twitch magnitude (17.5 ± 0.9% of resting length) or duration (156 ± 4 ms). Interestingly, although the twitch was greatly prolonged in calyculin A, there was no effect on the kinetics of twitch relaxation. The average time constant () of relaxation was 39 ± 1 ms in control and 43 ± 3 ms in cells preincubated with calyculin A. The lack of effect of calyculin A on relaxation kinetics is apparent in the normalized contractions shown in Fig. 3B.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Effect of preincubation in calyculin A on cell shortening in field-stimulated mouse ventricular myocytes. A: superimposed twitch contractions from a control cell and a cell preincubated with 1 µM calyculin A. Each tracing is the average of 10 consecutive steady-state twitch contractions. Cell length during the contraction was normalized to resting cell length (RL) and is plotted as %RL vs. time. B: same contractions normalized between resting length and length at peak contraction.

Increased myofilament Ca²⁺ sensitivity could explain the effects of calyculin A on the twitch contraction. Thus chemically skinned isolated mouse ventricular myocytes were used to measure possible increases in myofilament Ca²⁺ sensitivity. After preincubation in DMSO alone or in calyculin A, cells were permeabilized on the microscope stage with 10 µM digitonin. Cell length was measured after equilibration with solutions containing free Ca²⁺ concentrations ranging from 1 to 500 nM. The results are summarized in Fig. 4. Control cells exhibited no change in length as the Ca²⁺ concentration in the superfusate was increased from 1 to 100 nM. However, there was a significant decrease in length, to 99.2 ± 0.3% of resting length (P = 0.009), as the Ca²⁺ concentration was increased from 100 to 200 nM. There were additional incremental decreases in cell length (P < 0.05) as [Ca²⁺]_o was increased to a concentration of 500 nM. The mean cell length at 500 nM Ca²⁺ was 91.9 ± 1.1% of resting length. Above 500 nM Ca²⁺, most cells hypercontracted, presumably because of the absence of a mechanical load. To validate that increases in myofilament Ca²⁺ sensitivity could be detected, the Ca²⁺ dependence of cell length was measured in the presence of caffeine (15, 49). As expected, caffeine significantly increased the extent of shortening at 200 nM Ca²⁺ (P = 0.012), and the effect persisted as [Ca²⁺]_o was increased (P < 0.05). The effects of the PKA activator 8-bromo-cAMP were also examined (25, 42). Cells preincubated with 20 µM 8-bromo-cAMP exhibited less shortening than control cells at all concentrations of Ca²⁺. The effect reached statistical significance at 500 nM Ca²⁺, with shortening to 95.8 ± 0.9% of resting length compared with 91.9 ± 1.1% in control (P = 0.03). Importantly, calyculin A-treated cells did not exhibit an increase in myofilament Ca²⁺ sensitivity. In fact, there was no statistical difference, at any [Ca²⁺] tested, between the extent of shortening in the control cells and those preincubated with calyculin A. Although this technique is limited in its ability to measure myofilament Ca²⁺ sensitivity over the complete range of physiologically relevant Ca²⁺ concentrations, it is very effective in measuring Ca²⁺ sensitivity in the 100-500 nM range, where increases in Ca²⁺ sensitivity are very evident. Thus the results of these experiments reveal that the increase in contraction produced by calyculin A is not due to an increase in steady-state myofilament Ca²⁺ sensitivity.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Effect of calyculin A, caffeine, and 8-bromo-cAMP on myofilament [Ca2+] sensitivity in chemically skinned mouse ventricular myocytes. Cells were skinned (10 µM digitonin) and then exposed to each [Ca2+] in succession from 1 to 500 nM. Cell length at each [Ca2+] was normalized to that at 1 nM [Ca2+]. Shown are plots of normalized cell length vs. [Ca2+] for control cells (n = 9), cells preincubated with 1 µM calyculin A (n = 10), cells in the presence of 5 mM caffeine (n = 3), and cells preincubated with 20 µM 8-bromo-cAMP and superfused with 1 µM 8-bromo-cAMP (n = 3).

It is possible that phosphatase inhibition could lead to targeted changes in one or more of the ionic currents that underlie the mouse ventricular action potential and thus produce the characteristic effects of calyculin A on the twitch. We began our analysis of this hypothesis by examining the effects of calyculin A on the action potential (Fig. 5A). Calyculin A prolonged the duration of the action potential, increasing the time to 50% repolarization from 3.6 ± 0.7 ms (n = 5) to 8.6 ± 1.0 ms (n = 5, P = 0.001) without having any effect on the resting potential (control: 73.5 ± 0.9 mV; calyculin A: 70.6 ± 1.6 mV, P = 0.19). These results suggest that electrophysiological actions of calyculin A are responsible for its characteristic effects on twitch magnitude and duration.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Effect of preincubation in calyculin A on the mouse ventricular action potential and membrane K+ currents. A: representative action potentials from a control cell and a cell preincubated in 100 nM calyculin A. Each waveform is the average of 10 consecutive steady-state action potentials (1 Hz). B: average current-voltage relationships for inward rectifier K+ current (IK1) in control cells (n = 6) and cells preincubated with 1 µM calyculin A (n = 5). The holding potential was 70 mV, and currents were elicited by 300-ms test pulses between 120 and 40 mV. C: representative K+ current recordings from a control cell (left) and a cell preincubated with 1 µM calyculin A (right). Holding potential was 70 mV. Currents were elicited by 1,000-ms test pulses to potentials between 50 and +60 mV at 5-s intervals. Dashed line, zero current level. D: voltage dependence of the transient and sustained components of the mouse ventricular K+ current in control cells (n = 6) and cells preincubated with 1 µM calyculin A (n = 4). The amplitude of the transient component was the difference between the early peak of outward current and the current remaining at the end of the test pulse. The amplitude of the sustained component was the difference between the current remaining at the end of each test pulse and the holding current. E: average recovery from inactivation for the sustained component (top) and the transient component (bottom) of the mouse ventricular K+ current in control (n = 6) and in cells preincubated with 1 µM calyculin A (n = 7). The holding potential was 70 mV. Pairs of 200-ms voltage-clamp pulses to +50 mV were imposed, with interposed rest intervals ranging from 20 to 2,800 ms. There was an interval of 10 s between each pulse pair. The magnitude of each component of the current recorded from a test pulse (Itest) was normalized to that of the preceding conditioning pulse (I0), and the resulting values were plotted vs. the rest interval.

Calyculin A could increase the action potential duration by decreasing the activity of PP1 or PP2A involved in controlling the amplitude of repolarizing K⁺ currents. This was tested with whole cell voltage-clamp experiments on the inward rectifier K⁺ current (I_K1), which is primarily responsible for determining the resting potential, and on the time- and voltage-dependent currents that underlie action potential repolarization. The effect of calyculin A on I_K1 was examined by applying 300-ms test pulses to potentials between 120 and 40 mV from a holding potential of 70 mV. The magnitude of I_K1 was calculated as the difference between the current at the end of the pulse and the holding current. The results are summarized in Fig. 5B, with average current-voltage relationships recorded from control cells (n = 6) and cells preincubated with calyculin A (n = 5). Figure 5B demonstrates that calyculin A is without effect on I_K1.

Figure 5, C-E, summarizes the effects of calyculin A on the voltage- and time-dependent K⁺ currents that are responsible for action potential repolarization. In both control cells and in cells preincubated with calyculin A, the composite K⁺ currents exhibit a large and rapid outward transient, followed by decay to a maintained outward current (Fig. 5C). This is consistent with a recent study (14) from this laboratory and others (19, 51, 53), demonstrating that the composite mouse ventricular K⁺ current consists of contributions from at least three time- and voltage-dependent K⁺ currents, each with distinct activation and inactivation kinetics. Importantly, the average results presented in Fig. 5D indicate that calyculin A had no effect on the voltage dependence of either the transient or maintained components of the composite current. Because the contractile effects of calyculin A were manifest at a stimulation rate of 1 Hz (Fig. 3), it was also necessary to examine the recovery from inactivation of these currents. Recovery from inactivation was examined between 20 and 2,800 ms. Figure 5E shows that the time course of recovery of both the transient and sustained components of the current was unaffected by calyculin A. Thus the K⁺ currents that are important components of the mouse ventricular action potential are insensitive to calyculin A, revealing that localized phosphatase activity is not responsible for maintaining the steady-state amplitude of these currents.

Increased I_Ca could explain the effect of calyculin A on the action potential as well as directly account for the increased contraction magnitude. This possibility was examined under conditions in which I_Ca and cytosolic [Ca²⁺]_i transients were recorded simultaneously. The Ca²⁺-sensitive fluorescent indicator indo 1 was included in the pipette filling solution, and [Ca²⁺]_i was quantified using an in vitro calibration method (13). Figure 6A shows representative original records, and Fig. 6B shows the average voltage dependence of both I_Ca and the [Ca²⁺]_i transient recorded from control cells and cells preincubated with calyculin A. The phosphatase inhibitor increased both I_Ca and the [Ca²⁺]_i transient (P < 0.05) throughout the entire range of test potentials. For example, I_Ca is increased at 0 mV from 7.9 ± 0.6 pA/pF in control to 13.8 ± 0.8 pA/pF with calyculin A. Whereas the magnitude of I_Ca was markedly increased, the kinetics of I_Ca decay, examined between 14 and +28 mV, were unaffected by calyculin A. Currents recorded in both control and calyculin A exhibited biexponential decay, with a fast  of <10 ms and a slower component with a  between 30 and 50 ms. For example, the average fast  at 0 mV was 6.0 ± 0.4 ms (n = 6) in control and 6.5 ± 0.5 ms (n = 5) with calyculin A (P = 0.21). The respective slow  were 39.4 ± 3.4 and 37.0 ± 2.0 ms (P = 0.29).

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Effect of preincubation with calyculin A on L-type Ca2+ current (ICa) and the intracellular [Ca2+] ([Ca2+]i) transient in voltage-clamped mouse ventricular myocytes. A: simultaneously recorded families of [Ca2+]i transients (in µM, top) and ICa (in pA/pF, bottom) from a control cell (left) and a cell preincubated in 1 µM calyculin A (right). Cells were held at 70 mV between test pulses. Test pulses (35 to +60 mV in 7-mV increments) were given from 40 mV after a 1-s voltage ramp from 70 mV. Repolarization was to 70 mV. To maintain sarcoplasmic reticulum Ca2+ loading in a steady state, three 200-ms conditioning pulses to 0 mV were imposed during the 13-s interval between each test pulse. Only the test pulse portions of the data are shown. B: voltage dependence of peak [Ca2+]i (top) and ICa (bottom) in control cells (n = 6) and cells preincubated with 1 µM calyculin A (n = 5). ICa density was calculated by subtracting the steady-state current at the end of each test pulse from the peak current at the beginning of the pulse and then normalizing to cell capacitance.

Like I_Ca, the [Ca²⁺]_i transient was also markedly increased by calyculin A (Fig. 6). For example, the average peak [Ca²⁺]_i at 0 mV was 827 ± 90 nM in control cells and 1,514 ± 185 nM in treated cells. Interestingly, however, the [Ca²⁺]_i transients recorded from voltage-clamped cells preincubated with calyculin A exhibited accelerated decay with respect to control. The average  of decay of [Ca²⁺]_i transients elicited during steady-state drives to 0 mV was 188 ± 17 ms (n = 4) in control cells and 128 ± 17 ms (n = 4) in cells preincubated in calyculin A (P = 0.02). These results demonstrate that calyculin A elicits a marked increase in I_Ca, which in turn produces an increase in the [Ca²⁺]_i transient that activates contraction.

In a dynamic phosphorylation model, the functional effects of phosphatase inhibition are seen through the activity of a counteracting kinase. Thus phosphatase inhibition should be without effect once the kinase activity has been inhibited. Accordingly, the specificity of calyculin A was examined in combination experiments with the broad-spectrum serine-threonine kinase inhibitor staurosporine (46). As shown in Fig. 7B, staurosporine decreased steady-state I_Ca and, importantly, completely blocked the stimulatory action of calyculin A (Fig. 7B). The results of the reverse experiment were also consistent with this model, because staurosporine was completely without effect once the responses to calyculin A were fully developed (Fig. 7C). Thus the effects of calyculin A on I_Ca occur through phosphatase inhibition rather than through a nonspecific effect of this agent on Ca²⁺ channels.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Effect of serial addition of calyculin A (Caly A) and staurosporine (Stau) on ICa. A: superimposed currents recorded during steps from 40 to 7 mV. The control current (Con) was recorded first, followed by the current recorded after 5 min in calyculin A and the current recorded after 5 min in calyculin A and staurosporine. Note the increase during calyculin A and the lack of effect of subsequent addition of staurosporine. B: superimposed currents in which staurosporine was added first, followed by exposure to calyculin A and staurosporine. Note the decreased magnitude in staurosporine and the lack of effect of subsequent addition of calyculin A. C: histogram summarizing the experiments described in A (left, n = 4) and B (right, n = 5).

These data demonstrate that the increase in the twitch contraction seen in Fig. 3 can be explained by an increase in I_Ca brought about specifically by the phosphatase inhibition produced by calyculin A. However, recall the effects of calyculin A described above on the kinetics of [Ca²⁺]_i transient decay (Fig. 6) and twitch contraction (Fig. 3). When these are compared, there is an apparent discrepancy, with calyculin A accelerating the decay of the voltage-clamp-induced [Ca²⁺]_i transient while it prolongs the duration of the field-stimulated twitch contraction.

To address this discrepancy, we measured [Ca²⁺]_i transients and twitch contractions simultaneously in field-stimulated cells. This allowed us to examine the relationships among the magnitudes, durations, and kinetics of the [Ca²⁺]_i transients and resulting twitch contractions. The measurements were made in fluo 3-AM-loaded cells using a laser scanning confocal microscope in the line-scan mode. A representative line-scan image and [Ca²⁺]_i transient (F/F₀) are shown in Fig. 8, and the results are summarized in Table 1. As in cells not loaded with fluorescent dye (Fig. 3), calyculin A markedly increased the magnitude and duration of the field-stimulated twitch contraction. Also, as expected from Fig. 6, calyculin A markedly increased the magnitude of the field-stimulated [Ca²⁺]_i transient. However, despite the fact that the  of decay of the [Ca²⁺]_i transient was accelerated by 25%, the duration of the [Ca²⁺]_i transient, measured as the time to 50% decay (T₅₀), was not significantly different from control. This suggests that calyculin A does not substantially stimulate SR Ca²⁺ uptake.

View larger version (40K): [in this window] [in a new window]   Fig. 8.   Simultaneous measurement of the [Ca2+]i transient [fluorescence intensity of each pixel (F)/average resting intensity (Fo)] and contraction and the effect of preincubation with calyculin A on the [Ca2+]i transient in fluo 3-loaded mouse ventricular myocytes. A: schematic of method for simultaneous measurement of [Ca2+]i and contraction from a confocal line-scan image. A single line on the long axis of the cell is scanned repeatedly at 2-ms intervals. A line-scan image is constructed with time represented horizontally and the position along the scan line represented vertically. During the scan, the cell is field stimulated, resulting in shortening and relengthening. This changes the portion of the image that exhibits fluorescence. The length of the cell during each line scan is determined by measuring the portion of the line that exhibits fluorescence. From that information, a plot of cell length (L) vs. time (t) is constructed. F/F0 is calculated by determining the fluorescence from the same small region of each line scan (F) and dividing by the fluorescence of the same region in the absence of stimulation (F0). From that information, a plot of F/F0 vs. time is constructed. B: representative line-scan image and the twitch contraction record (top) and [Ca2+]i transient (bottom) derived from that image. C: superimposed representative steady-state [Ca2+]i transients from a control cell and a cell preincubated with 100 nM calyculin A. D: same [Ca2+]i transients normalized between resting and peak F/F0 for each.

To test the role of PP1/PP2A in regulating SR Ca²⁺ uptake, we compared the effects of calyculin A (125 nM) with those of the -adrenergic agonist isoproterenol (1 µM). -Adrenergic stimulation is known to greatly stimulate the activity of the SR Ca²⁺ pump. The magnitudes of both the [Ca²⁺]_i transient and contraction were the same in isoproterenol as in calyculin A (Table 1). However, the effects of isoproterenol on the kinetics of the [Ca²⁺]_i transient were quite distinct from those of calyculin A. Whereas calyculin A was without effect on the T₅₀ of the [Ca²⁺]_i transient, isoproterenol decreased this parameter by ~25%. Furthermore, isoproterenol decreased the  of [Ca²⁺]_i decay by ~50%, essentially doubling the effect of calyculin A. Calyculin A and isoproterenol had identical effects on the action potential and I_Ca, and, importantly, isoproterenol was without effect on I_Ca after calyculin A (data not shown). Thus the differences between the effects of calyculin A and isoproterenol on the [Ca²⁺]_i transient kinetics cannot be attributed to different effects on these factors. Taken together, these data suggest that any effects of PP1 or PP2A inhibition on the rate of SR Ca²⁺ uptake are minimal compared with the increase produced by -adrenergic stimulation. However, inhibition of one or both of these phosphatases results in a marked increase in I_Ca, which translates into a substantial increase in the magnitude and duration of contraction.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Phosphatase inhibition has been shown to have a wide variety of effects on cardiac cells. It can modify the responses to adenosine receptor stimulation (39), classic -adrenergic stimulation (26, 27), and ₂-adrenergic stimulation (32). On its own, phosphatase inhibition has also been shown to mimic the cardioprotective effects of ischemic preconditioning (2, 3). With respect to the E-C coupling cascade, Ca²⁺ currents increase during phosphatase inhibition (20, 40, 41, 43, 50), and biochemical measurements have also indicated that myofilament proteins and phospholamban can become phosphorylated (4, 40, 41). Thus because most of the components of the E-C coupling cascade are known to be regulated in some fashion by phosphorylation, it was expected that nearly complete inhibition of the major serine-threonine phosphatase activity in cardiac cells would have a broad impact on all steps of this process. In particular, phosphatase inhibitors might mimic the action of signaling cascades that activate PKC or PKA. -Adrenergic stimulation, acting through PKC, increases myofilament Ca²⁺ sensitivity (21), decreases time- and voltage-dependent K⁺ currents (1, 18), and produces a contractile effect similar to that seen here for calyculin A (18). -Adrenergic stimulation, acting through PKA, produces positive inotropic and lusitropic effects by increasing the magnitude of the Ca²⁺ current (8, 24), increasing SR Ca²⁺ uptake (31, 33), and decreasing myofilament Ca²⁺ sensitivity (21). Unexpectedly, however, the results here reveal that when the major phosphatases in the heart are broadly inhibited by calyculin A, the acute contractile effects cannot be explained by a global effect on the entire E-C coupling cascade. Rather, these new findings raise several questions central to the local steady-state control of E-C coupling by phosphatases in the heart.

Phosphatase inhibition and myofilament Ca²+ sensitivity. To examine potential increases in myofilament Ca²⁺ sensitivity, we utilized an unloaded permeabilized cell model. This method was very effective in measuring steady-state myofilament Ca²⁺ sensitivity over a concentration range in which such increases would be evident. Control experiments with caffeine validated this approach. Because no increases in myofilament Ca²⁺ sensitivity were observed with calyculin A at Ca²⁺ concentrations as high as 500 nM, it cannot account for the inhibitor-evoked increase in the magnitude and duration of contraction. A limitation of this unloaded cell approach is that cell shortening could not be assessed over the complete physiologically relevant Ca²⁺ concentration range. Thus it is not possible to exclude the possibility that phosphatase inhibition with calyculin A actually decreased myofilament Ca²⁺ sensitivity. It is also possible that phosphorylation-dependent changes in dynamic myofilament properties, such as the cross-bridge cycling rate (48), could occur that would not be detected in these steady-state measurements. However, taken together, these studies reveal that PP1/PP2A inhibition does not prolong the twitch duration through an increase in the steady-state Ca²⁺-dependent properties of the myofilaments.

Phosphatase inhibition and repolarizing K+ currents. The similarity between the effects of calyculin A and -adrenergic agonists on contractility and action potential duration (18) suggested that the time- and voltage-dependent repolarizing K⁺ currents are regulated by PP1/PP2A inhibition. In fact, phosphatase inhibitors have been shown to decrease delayed rectifier K⁺ currents in the frog atrium (20). Furthermore, ₁-adrenergic agonists increase the magnitude and duration of contractions (18), prolong action potentials (1, 18), and reduce K⁺ currents in isolated rat ventricular myocytes (1, 18) through activation of PKC (1). The mouse ventricle exhibits a wide variety of time- and voltage-dependent K⁺ currents. There are two transient outward currents, termed I_to,f and I_to,s for their fast and slow inactivation kinetics, respectively (51). In addition, there is a rapidly activating, slowly inactivating current, termed I_K,slow (14, 19, 51, 53), and a noninactivating current, termed I_K,ss (14, 51). In addition, the mouse also possesses I_K1, which is responsible for maintaining the resting potential but has also been shown to affect action potential duration (52). Precise pharmacological separation of these currents is difficult because there are no specific blockers that allow observation of each component in isolation. Kinetic separation of these currents requires the use of very long voltage-clamp pulses. In a recent study (14), we demonstrated that agents that affect any individual K⁺ current will also affect the composite K⁺ current, which can be separated into a transient and a maintained component. In the present study, we report that calyculin A was completely without effect on either the transient or maintained component of the current (Fig. 5D). Furthermore, there was no effect on the recovery from inactivation of either component over the range of 20 ms to nearly 3 s. Thus inhibition of PP1/PP2A has no effect on these K⁺ currents and cannot explain the increase in contractility that we observe (Fig. 3). It is interesting to note that calcineurin, the calcium/calmodulin-dependent phosphatase, does not regulate cardiac K⁺ currents either (12, 14). Thus ventricular K⁺ currents, in contrast to I_Ca, are not locally regulated by any of the major serine-threonine phosphatases.

Phosphatase inhibition and I_Ca. Of the targets studied in this report, the L-type Ca²⁺ channel proved to be the most sensitive to PP1/PP2A inhibition. This might have been expected because phosphatase inhibitors increase I_Ca in both frog atrial and guinea pig ventricular myocytes (20, 23, 27, 28, 40, 41). Furthermore, okadaic acid increases I_Ca in isolated single channels (43, 50), underscoring the role of local PP1 and perhaps PP2A in dynamic control of L-type Ca²⁺ channel activity. In this regard, a recent biochemical study (10) demonstrated that PP2A binds directly to the -subunit of the cardiac L-type Ca²⁺ channel. Taken together, these data demonstrate that in intact cells the L-type Ca²⁺ channel is very sensitive to serine-threonine phosphatase activity in the absence of humoral stimulation. It is also clear that the L-type Ca²⁺ channel is equally sensitive to serine-threonine kinase activity, as demonstrated by the fact that the broad-spectrum kinase inhibitor staurosporine decreased I_Ca and prevented the stimulatory action of calyculin A. It will be important to identify the underlying kinases that oppose the phosphatase action and regulate I_Ca under these conditions.

Phosphatase inhibition and SR Ca²+ uptake. During the course of these studies, analysis of the action of calyculin A revealed conflicting results regarding SR Ca²⁺ uptake. In particular, phosphatase inhibition accelerated the decay of the voltage-clamp-induced [Ca²⁺]_i transient, whereas it markedly prolonged the duration of the field-stimulated twitch contraction. This apparent discrepancy raised uncertainty about the role of serine-threonine phosphatases in regulating SR Ca²⁺ pump activity in the absence of humoral stimulation. It is important to note that the  of [Ca²⁺]_i decay decreases as the magnitude of the [Ca²⁺]_i transient increases (5). Because calyculin A markedly increases peak [Ca²⁺]_i, the concomitant increase in the decay rate of the [Ca⁺]_i transient does not prove that SR Ca²⁺ uptake is activated.