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Circulation Research. 1996;78:553-563

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(Circulation Research. 1996;78:553-563.)
© 1996 American Heart Association, Inc.

Articles

Thrombin Receptor Actions in Neonatal Rat Ventricular Myocytes

Tianrong Jiang, Valery Kuznetsov, Elena Pak, HongLu Zhang, Richard B. Robinson, Susan F. Steinberg

From the Departments of Medicine (S.F.S.) and Pharmacology (T.J., V.K., E.P., H.Z., R.B.R., S.F.S.), Columbia University, New York, NY.

Correspondence to Susan F. Steinberg, MD, Associate Professor of Medicine and Pharmacology, Department of Medicine, Columbia University, College of Physicians and Surgeons, 630 West 168 St, New York, NY 10032.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Abstract Previous studies established that thrombin stimulates phosphoinositide hydrolysis and modulates contractile function in neonatal rat ventricular myocytes. The present study further defines the signaling pathways activated by the thrombin receptor and their role in thrombin's actions in cardiac myocytes. The thrombin receptor–derived agonist peptide (TRAP, a portion of the tethered ligand created by thrombin's proteolytic activity) stimulates the rapid and transient accumulation of inositol bis- and tris-phosphates (IP2 and IP3, respectively), which is followed by the more gradual and sustained accumulation of inositol monophosphate (IP1). TRAP elicits a larger and more sustained accumulation of IP1 than does thrombin. Thrombin and TRAP also activate mitogen-activated protein kinase (MAPK) in cultured neonatal rat ventricular myocytes. Differences in the kinetics and magnitude of thrombin- and TRAP-dependent inositol phosphate (IP) accumulation are paralleled by differences in the kinetics and magnitude of thrombin- and TRAP-dependent activation of MAPK. Pretreatment with phorbol 12-myristate 13-acetate (PMA) to downregulate protein kinase C (PKC) attenuates thrombin- and TRAP-dependent activation of MAPK, although small and equivalent effects of thrombin and TRAP to stimulate MAPK persist in PMA-pretreated cells. These results support the notion that the thrombin receptor activates MAPK through PKC-dependent and PKC-independent pathways and that the incremental activation of MAPK by TRAP over that induced by thrombin is the consequence of enhanced activation through the PKC limb of the phosphoinositide lipid pathway. TRAP also increases the beating rate of spontaneously contracting ventricular myocytes and elevates cytosolic calcium in myocytes electrically driven at a constant basic cycle length. The effects of TRAP to modulate contractile function and elevate intracellular calcium are not inhibited by tricyclodecan-9-yl-xanthogenate (D609, to block TRAP-dependent IP accumulation) or pretreatment with PMA (to downregulate PKC). The TRAP-dependent rise in intracellular calcium also is not inhibited by verapamil or removal of extracellular calcium but is markedly attenuated by depletion of sarcoplasmic reticular calcium stores by caffeine. Patch-clamp experiments demonstrate that TRAP elevates intracellular calcium in cells held at a membrane potential of -70 mV. Taken together, these results support the conclusion that the thrombin receptor modulates contractile function by mobilizing intracellular calcium through an IP3-independent mechanism and that this response does not require activation of voltage-gated ion channels.


Key Words: thrombin receptors • inositol phosphates • mitogen-activated protein kinase • Ca2+ • automaticity


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Thrombin is a multifunctional serine protease that is formed at the site of vascular injury and activates a variety of cellular events important in hemostasis as well as the inflammatory and proliferative responses. Thrombin evokes multiple biological responses in many cell types, including platelets, endothelial cells, smooth muscle cells, and fibroblasts.1 2 3 4 5 6 7 These responses result from activation of the cell surface thrombin receptor, which couples via heterotrimeric G proteins to traditional intracellular effector response mechanisms, including PLC, PLA2, and adenylyl cyclase.5 8 9 10 Thrombin also increases tyrosine phosphorylation,2 5 11 activates MAPK,12 13 and stimulates AP-1 transcriptional activity.14 However, the precise mechanism by which thrombin receptors couple to these responses remains unknown.

Recent studies by Coughlin and colleagues15 have provided critical insights into the mechanism for thrombin-dependent cellular activation. Using molecular cloning techniques, these investigators have identified a receptor for thrombin that is a single polypeptide chain having seven predicted membrane-spanning domains and a long extracellular amino-terminal extension that contains a thrombin cleavage site.15 16 These structural features suggested that the thrombin receptor resembles other G protein–coupled receptors but has a unique activation mechanism. Studies in several cell systems support a model in which thrombin binds to and cleaves the N-terminal ectodomain of its receptor, thereby revealing a new N-terminal sequence that serves as a "tethered ligand." In particular, thrombin's cellular actions require a proteolytically active form of thrombin,17 and synthetic peptides corresponding to the N-terminal tetradecapeptide newly exposed by thrombin mimic thrombin's actions in many cell types.5 8 17

We previously demonstrated that thrombin stimulates phosphoinositide hydrolysis, elevates cytosolic calcium ion concentration, increases automaticity, prolongs repolarization, and enhances afterdepolarizations in cardiac myocytes.18 Thrombin also induces several of the characteristics of the cardiac hypertrophic phenotype, including increased cell size, enhanced sarcomeric organization, and increased atrial natriuretic factor expression.19 In view of recent insights into the mechanism by which thrombin activates cells, we initiated studies with TRAP. The goals of the present study were (1) to determine whether the thrombin receptor can be implicated in the rapid contractile response to thrombin in cardiac myocytes, (2) to determine whether the hypertrophic stimuli thrombin and TRAP activate MAPK in cardiac myocytes, and (3) to examine the role of second messenger molecules generated via the phosphoinositide pathway in thrombin receptor–dependent responses in cardiomyocytes.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Preparation of Cultured Neonatal Rat Ventricular Myocytes
Cardiac myocytes were isolated from the ventricles of 2-day-old Wistar rats by a trypsin dispersion procedure and were cultured for 5 days in the presence of 10% fetal calf serum according to a protocol described previously.20 For experiments measuring phosphoinositide hydrolysis, MAPK activation, and intracellular cAMP accumulation, cells were grown in multiwell tissue culture dishes. Measurements of cell shortening and cytosolic calcium were performed on myocytes grown on round fibronectin-coated glass coverslips (0.1-mm thickness, 31-mm diameter; Biophysica Technologies, Inc) in 35-mm culture dishes. Although the culture technique includes a preplating step that effectively decreases fibroblast contamination, it is well known that a small number of cells with proliferative capability such as cardiac fibroblasts persist in the myocardial cell cultures. Proliferation of these cells was further curtailed with an irradiation protocol.18

Measurement of IP Accumulation
IP accumulation was measured according to standard techniques as described previously.18 Briefly, neonatal myocytes were cultured in multiwell (6x35-mm) dishes in the presence of 3 µCi [3H]myoinositol for 72 hours to label membrane phosphoinositides. The monolayer culture was washed extensively with HEPES-buffered saline to remove unincorporated radioisotopes. After a 10-minute preincubation in the presence of 10 mmol/L LiCl, experimental protocols were initiated by the addition of 1 mL HEPES-buffered saline containing 10 mmol/L LiCl and the indicated test agents. After incubation for the indicated time intervals at room temperature, the buffer was rapidly aspirated, and 1.5 mL acidified chloroform/methanol/6 mol/L HCl (500:1000:3) was added to the cell monolayer. Cells were harvested with a rubber policeman, and lipids were extracted for 30 minutes at room temperature. Chloroform (0.5 mL) and H2O (1 mL) were added, and the mixture was vortexed and then centrifuged at 1000g for 2 minutes to separate the phases. The aqueous phase was transferred to Dowex anion-exchange columns, and IPs were eluted sequentially according to standard methods.21 Results are reported as counts per minute (with background radioactivity subtracted).

Measurements of MAPK Activity in SDS-Polyacrylamide Gels Containing MBP
Cells were maintained in serum-free medium (1:1 DMEM/F-12 medium, GIBCO BRL) supplemented as described by Mohamed et al22 for 4 days for studies of MAPK activation. After exposure of cells to agonists for the times indicated, cells were washed three times in ice-cold calcium/magnesium-free Dulbecco's PBS and then scraped into ice-cold extraction buffer (20 mmol/L ß-glycerophosphate, 20 mmol/L NaF, 2 mmol/L EDTA, 0.2 mmol/L Na3VO4, 10 µg/mL aprotinin, 25 µg/mL leupeptin, 50 µg/mL phenylmethanesulfonyl fluoride, and 0.3% [vol/vol] ß-mercaptoethanol, pH 7.5). Homogenates were centrifuged for 10 minutes at 10 000g, and the supernatants were diluted in SDS-PAGE sample buffer, boiled, and stored at -80°C for subsequent assay of MAPK activity. Cell extracts were resolved on 10% SDS-polyacrylamide gels containing 0.5 mg/mL MBP. After electrophoresis, SDS was removed from the gel by washing the gels three times for 20 minutes each with 20% (vol/vol) 2-propanol in 50 mmol/L Tris-HCl, pH 8.0, followed by three additional washes (20 minutes each) with 5 mmol/L ß-mercaptoethanol in 50 mmol/L Tris-HCl, pH 8.0. Proteins were denatured by treating the gels twice (30 minutes each) with 50 mL of 6 mol/L guanidine HCl, 5 mmol/L ß-mercaptoethanol, and 50 mmol/L Tris-HCl, pH 8.0, and then renatured by washing overnight at 4°C in five changes of 50 mmol/L Tris-HCl, pH 8.0, containing 0.04% (vol/vol) Tween 40 and 5 mmol/L ß-mercaptoethanol. After preincubation of the gels at 20°C for 1 hour in 40 mmol/L HEPES, 2 mmol/L dithiothreitol, and 10 mmol/L MgCl2, pH 8.0, in situ phosphorylation of MBP was carried out by incubating the gels at 20°C for 1 hour in 15 mL of 40 mmol/L HEPES, 0.5 mmol/L EGTA, 10 mmol/L MgCl2, and 50 µmol/L [{gamma}-32P]ATP (5 µCi/mL, 25 µCi per gel), pH 8.0. The reaction was stopped by washing the gels in a 5% trichloroacetic acid solution containing 10 mmol/L sodium pyrophosphate. The buffer was changed repeatedly until the radioactivity of the solution was equivalent to background, and the gel was dried and then subjected to autoradiography. The combined intensities of the signals derived from the 42- and 44-kD MAPK species were quantified using a PhosphorImager 445SI (Molecular Dynamics).

Measurement of cAMP Accumulation
Intracellular cAMP was measured essentially as described previously.18 Briefly, neonatal myocytes grown in 22.1-mm multiwell dishes were preincubated for 60 minutes at room temperature with 10 mmol/L theophylline. Where indicated, 100 µmol/L D609 was added to the cells 12 hours before the agonist. Assays were performed for 5 minutes at room temperature and were terminated by removal of the incubation buffer and addition of 1 mL ethanol. Each condition was performed on two wells and was assayed for cAMP in quadruplicate. The alcohol-fixed cell extract was boiled for 3 minutes, cooled, brought to original volume with ethanol, and stored at -80°C. Aliquots of the supernatant were dried under a stream of nitrogen, and cAMP in the residue was determined using a radioimmunoassay (New England Nuclear/Du Pont Co).

Measurement of Cytosolic Free Calcium and Cell Shortening
Methods for the photometric measurement of cytosolic calcium in fura 2–loaded cultured neonatal ventricular myocytes have been previously published.18 In brief, neonatal myocyte monolayer cultures on a coverslip were loaded with fura 2 by incubation in Tyrode's solution containing 3 µmol/L of the acetoxymethyl ester form of fura 2 (fura 2-AM) and 1.5 µL/mL of 25% (wt/vol in dimethyl sulfoxide) Pluronic F-127 (BASF Wyandotte Corp) for 20 minutes at room temperature. Myocytes were rinsed with fresh Tyrode's solution and maintained for at least 15 minutes at room temperature to allow for deesterification of the dye.

The device used for monitoring the fluorescence of intracellular fura 2 (Photon Technologies, Inc) alternately illuminates the cells with 340- and 380-nm light while measuring emission at 520 nm. The sampling rate for collection of ratio values was 100 Hz. Cytosolic free calcium ion concentration theoretically can be calculated from the fura 2 fluorescence ratio at the two excitation wavelengths. Although numerous approaches to calibrate intracellular fura 2 have been presented, it is extremely difficult to entirely circumvent uncertainties in the calibration because of the potential compartmentalization of the dye in fura 2-AM–loaded cells, differences in the spectral properties of fura 2 in cells and in buffer solutions, and fluorescence changes due to cell contracture during calibration protocols.23 24 Because of these unavoidable uncertainties and because fura 2 fluorescence provides an extremely sensitive indicator of changes in intracellular calcium during experimental protocols (the primary focus of these studies), we have reported intracellular calcium as the fura 2 fluorescence ratio.

Glass beads (2.1±0.5 µm, Duke Scientific Corp) were added to the neonatal monolayer cultures to provide high-contrast spots for tracking cell motion. To monitor cell motion, the cells were simultaneously illuminated with red light, and a dichroic mirror (630-nm cutoff) in the emission path deflected the cell image to a video optical system (Crescent Electronics), which tracked motion of the high-contrast microsphere attached to the myocyte surface along a raster line segment of the image during electrically stimulated contractions. The analogue voltage output from the motion detector was calibrated to convert to microns of motion. The motion signal was obtained at a rate of 60 Hz and reflected the motion of the same myocyte simultaneously monitored with fura 2 for calcium. The signal was digitized and stored along with the fluorescence data. Measurements of changes in cell length are not possible in neonatal monolayer cultures, because these cells lack a single axis of myofibrillar alignment. Therefore, these studies on individual cultured neonatal myocytes report cell shortening.

Studies were performed using a three-compartment superfusion chamber modified so that a coverslip formed the bottom of the chamber. The chamber was placed on the stage of a Zeiss inverted microscope, and the cells were visualized with a x40 oil immersion Neofluor objective. The cells were superfused with Tyrode's solution gassed with 95% O2/5% CO2 at a rate of 1 mL/min. Experiments were performed at room temperature. Where indicated, myocytes were paced by electrical field stimulation at 1 Hz using platinum wires embedded in the walls of the superfusion chamber throughout the experimental protocol to avoid changes in cell calcium or shortening due to the chronotropic actions of TRAP. Myocytes were exposed to a bolus of agonist by introducing 50 µL of superfusion buffer containing five times the desired final concentration of drug into the initial prechamber. The fluid rapidly mixes with the inflow stream and thereby is diluted as it enters the main 1x1-cm central compartment of the chamber, which is designed to maintain a constant volume of {approx}250 µL. In preliminary experiments, we established that vehicle, delivered in an identical fashion, does not affect the rate, calcium transient, or twitch. This protocol does not permit measurements under steady state conditions but provides the means to rapidly deliver agonist to the myocytes in the experimental chamber.

Electrophysiological Recording Methods
For combined patch-clamp and fura 2 measurements, 5-day-old monolayer cultures were resuspended by a 1- to 3-minute exposure to 0.1% trypsin, preplated for 45 minutes to reduce fibroblast contamination, and then replated onto protamine sulfate–coated round coverslips. They were studied 2 to 8 hours later, by which time they had reattached and formed single isolated spheres. A coverslip of cells was loaded with fura 2 by incubation in Tyrode's solution containing 3 µmol/L of fura 2-AM and 1.5 µL/mL of 25% (wt/vol in dimethyl sulfoxide) Pluronic F-127 for 20 minutes at room temperature and placed in the superfusion chamber described above. Voltage clamp was carried out using the perforated-patch method25 to minimize disruption of cytosolic components. The pore-forming antibiotic nystatin (Sigma Chemical Co) was dissolved in dimethyl sulfoxide at 50 mg/mL, added to the pipette solution (mmol/L: potassium glutamate 120, KCl 20, MgCl2 1, Na2ATP 4, EGTA 0.5, mannitol 25, and HEPES 10, pH 7.2) at a final concentration of 200 to 250 µg/mL, and dissolved by ultrasonication. The entire pipette, including the tip, was filled with the nystatin-containing solution.26 Pipette resistance in external solution was 2 to 4 M{Omega}.

After forming a seal against the cell, pores typically started to form within 5 minutes. Series resistance during the experiment was typically 20 to 40 M{Omega}. Voltage clamp was carried out using an Axopatch 200 amplifier, Digidata 1200 A/D interface, and Axotape 2.0 software (Axon Instruments) running on a 486 computer. The current record was simultaneously digitized on the second computer running the PTI software and stored along with the fluorescence data. Current records were normalized by cell capacitance, as measured by the capacity compensation circuit of the amplifier.

The cell was initially held at a potential of -70 mV. At the start of data acquisition, a brief depolarization to 0 mV was imposed to confirm that the particular cell being studied could generate a voltage-dependent calcium transient. The cell was then maintained at -70 mV for the duration of the experiment. Holding current and fura 2 ratio were measured during superfusion with control Tyrode's solution for {approx}2 minutes, followed by solution containing 300 µmol/L TRAP for 30 to 45 seconds, and then returned to control solution.

Statistics
For measurements of intracellular calcium and cell motion, six successive transients were superimposed and averaged. Amplitude and width at half-maximal amplitude were measured for both the calcium and motion transients. For studies of calcium and contractile function, all data represent results of experiments on cells from at least two separate cultures. For studies of IP and cAMP accumulation, experiments were performed in triplicate. All biochemical studies were replicated at least twice on different myocyte preparations. Data are presented as mean±SEM, and statistical comparisons were made using Student's t test for paired observations or ANOVA for multiple comparisons as indicated. Significance was defined at the P<.05 level.

Materials
TRAP (SFLLRN) was purchased from Bachem. Thrombin was purchased from Sigma. D609 was purchased from Kamiya Biomedical Co. Fura 2-AM was purchased from Molecular Probes. PMA was purchased from LC Services. All other chemicals were reagent grade and were obtained from standard chemical suppliers.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
*Results
down arrowDiscussion
down arrowReferences
 
Biochemical Signaling Mechanisms Activated by the Thrombin Receptor
Our previous studies established that thrombin stimulates a rapid and transient accumulation of IP3 and IP2, which is followed by a more gradual and sustained accumulation of IP1 in cultured neonatal rat ventricular myocytes.18 Fig 1Down illustrates that this action is mimicked by TRAP. IP1, IP2, and IP3 accumulation in response to TRAP is time dependent. The peaks in IP2 and IP3 accumulation occur within the first minute. Both in magnitude and kinetics, these responses are similar to those induced by thrombin. Although the effects of thrombin and TRAP to elevate IP2 and IP3 appear modest, the agonist-induced increases in radioactivity in these fractions represent twofold to fivefold increases over the very low radioactivity recovered in these fractions in basal unstimulated cells. At early time points (during the first 5 minutes), TRAP also elicits an increase in IP1 accumulation that is similar in magnitude to the response induced by thrombin. However, in contrast to the response to thrombin, which subsequently wanes, prolonged exposure to TRAP results in a large increase in IP1 accumulation, which is progressive for at least 60 minutes. The effect of TRAP to stimulate the formation of IP1, IP2, and IP3 is concentration dependent, with an EC50 of {approx}20 µmol/L (Fig 2Down).



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  		Figure 1. Kinetics of TRAP and thrombin-dependent [3H]IP accumulation in cultured ventricular myocytes. [3H]Inositol-labeled myocytes were stimulated with 100 µmol/L TRAP or 1 U/mL thrombin for the indicated time intervals and were extracted, and IP metabolites were separated by Dowex anion-exchange chromatography. Results are expressed as counts per minute over the corresponding controls for triplicate determinations from three separate experiments (mean±SEM).



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  		Figure 2. TRAP causes a concentration-dependent increase in IP accumulation in cardiac myocytes. [3H]Inositol-labeled myocytes were incubated for 10 minutes (for IP1) or 1 minute (for IP2 and IP3) with the indicated concentrations of TRAP or 1 U/mL thrombin and were extracted, and IP metabolites were isolated by Dowex anion-exchange chromatography. Results are expressed as counts per minute over the corresponding controls for triplicate determinations from three separate experiments (mean±SEM). The response to a higher concentration of TRAP (3 mmol/L) was identical to that elicited by 300 µmol/L TRAP, indicating that a maximal response was attained with 300 µmol/L TRAP.

It recently has become evident that a variety of extracellular stimuli that activate the phosphoinositide signaling pathway and induce hypertrophy in cardiac myocytes activate MAPK,27 28 29 a family of serine/threonine kinases that play a pivotal role in signal transduction and transcriptional regulation by phosphorylating a large array of downstream substrates including kinases (S6 kinase) and transcription factors (c-Jun, c-Myc).29 30 31 To determine whether thrombin and/or TRAP-dependent activation of phosphoinositide hydrolysis also is associated with the activation of MAPK in cardiac myocytes, we assessed "in-the-gel" MBP phosphorylation in lysates from agonist-stimulated cells. Fig 3Down illustrates the effects of thrombin and TRAP to activate MAPK at agonist concentrations that lead to equivalent initial stimulation of phosphoinositide hydrolysis (1 U/mL and 300 µmol/L, respectively). In each case, MAPK species with molecular masses of 42 and 44 kD are detectably activated by 1 minute and maximally activated by 5 minutes. Other higher molecular weight renaturable proteins also phosphorylate MBP in this assay, but the intensity of MBP phosphorylation by these proteins is not detectably changed by exposure to thrombin or TRAP. The phosphorylation is dependent upon the presence of MBP; no signal was detected in a control experiment in which MBP was omitted from the gel (data not shown). Of note, the effect of TRAP to activate MAPK is consistently larger and more persistent than the response elicited by thrombin.



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  		Figure 3. The kinetics of MAPK activation by TRAP and thrombin in cultured neonatal ventricular myocytes. Cardiac myocytes were treated with 300 µmol/L TRAP or 1 U/mL thrombin for the indicated times. Extracts were prepared and assayed for MAPK activity with MBP as substrate, as described in "Materials and Methods." Top, Results of a representative experiment are shown. Each lane was from the same gel and was exposed for the same duration. Bottom, Data (mean±SEM, n=5 independent experiments on separate cultures) are expressed relative to MAPK activity in control cells.

Given the importance of the PLC/PKC pathway as a mechanism for heterotrimeric G protein–coupled receptor activation of MAPK, we postulated that the incremental activation of MAPK by TRAP over that induced by thrombin might be the consequence of the more robust and persistent activation of PLC by TRAP than by thrombin. To investigate the role of PKC as a mediator of thrombin- and TRAP-dependent activation of MAPK, we took advantage of the actions of the tumor-promoting phorbol ester PMA. This substance acutely mimics the effects of endogenously produced diacylglycerol to activate PKC but, with chronic exposure, leads to the downregulation of phorbol ester–sensitive isoforms of PKC and a concomitant loss of responsiveness to agonist activation of this pathway. Previous studies from our laboratory established that cultured neonatal rat ventricular myocytes express conventional ({alpha}) and novel ({delta} and {varepsilon}) isoforms of PKC, which are completely (PKC{alpha} and PLC{delta}) or substantially (PKC{varepsilon}, {approx}80%) depleted from the cell after prolonged treatment with PMA (24 hours).32 Fig 4Down illustrates that PMA activates MAPK in control cells, and this response is virtually abolished in cells exposed to PMA for 24 hours, providing confirmation that activation through the phorbol ester–sensitive isoforms of PKC is prevented by the PMA pretreatment protocol. Pretreatment with PMA to downregulate PKC also markedly attenuates both TRAP- and thrombin-dependent activation of MAPK. The effect of PMA to block thrombin receptor–dependent activation of MAPK in cardiac myocytes cannot be explained by a decrease in cell surface thrombin receptor expression (as has been shown to occur in other cell systems33 ), since IP accumulations in response to TRAP and thrombin are not significantly affected by the PMA pretreatment protocol (data not shown). The decrease in thrombin receptor–dependent activation of MAPK in cells chronically treated with PMA also is not due to loss of functional MAPK molecules, since the effect of fibroblast growth factor to activate MAPK through a receptor tyrosine kinase is equivalent in control and PMA-pretreated cells (5.16±0.45- and 4.90±0.57-fold over basal, respectively). Finally, although pretreatment with PMA for 24 hours virtually abolishes subsequent PMA-dependent activation of MAPK, small but statistically significant effects of TRAP and thrombin to activate MAPK persist in PMA-pretreated cells; the magnitude of the residual activation of MAPK by TRAP and thrombin in PMA-pretreated cells does not differ. Taken together, these results provide evidence that TRAP and thrombin activate MAPK via PKC-dependent and PKC-independent signaling mechanisms and that the PKC-dependent pathway for activation of MAPK is more effectively activated by TRAP than by thrombin.



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  		Figure 4. The role of PKC in TRAP- and thrombin-dependent activation of MAPK. Myocytes were preincubated for 24 hours in the absence or presence of 1 µmol/L PMA. Cells were then challenged with PMA (300 nmol/L), TRAP (300 µmol/L), or thrombin (1 U/mL) for 5 minutes. After the preparation of extracts in SDS-PAGE sample buffer, samples were subjected to SDS-PAGE in 10% polyacrylamide gels containing 0.5 mg/mL MBP, and in situ phosphorylation of MBP was assayed as described in "Materials and Methods." Data (mean±SEM, n=7 independent experiments on separate cultures) are expressed relative to MAPK activity in unstimulated cells, which was similar in the control and PMA-pretreated cultures. TRAP and thrombin significantly activate MAPK in both control and PMA-pretreated myocytes. TRAP-dependent activation of MAPK is significantly greater than thrombin-dependent activation of MAPK only in control myocytes (P<.05); in PMA-pretreated cells, these responses do not differ.

Cellular Actions of TRAP
The functional consequences of myocyte stimulation with TRAP were explored. TRAP profoundly increases the spontaneous beating rate of cultured neonatal rat ventricular myocytes. Fig 5Down illustrates a typical experiment depicting the increased automaticity in response to 300 µmol/L TRAP, and the complete data are summarized in Fig 6Down. The effect of TRAP to increase the spontaneous beating rate is concentration dependent (EC50, {approx}30 µmol/L; maximal response, {approx}300 µmol/L) and more rapid as the concentration of agonist is increased. Recovery from these effects of TRAP occurs within 3 to 4 minutes of washout. These effects of TRAP to modulate spontaneous automaticity are equivalent to the actions previously reported for thrombin.18



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  		Figure 5. TRAP increases the automatic beating rate and intracellular calcium in ventricular myocytes. A representative tracing illustrates the fura 2 fluorescence ratio from a single spontaneously contracting myocyte in a monolayer culture before and after the addition of TRAP (300 µmol/L) to the superfusate in the prechamber (at the arrow). The figure depicts results obtained in a single cell that are representative of the group of 10 cells studied with this concentration of TRAP according to this protocol. In this particular cell, an increase in automaticity clearly precedes the rise in intracellular calcium. This was observed in 7 of the 10 cells studied according to this protocol.



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  		Figure 6. TRAP induces a concentration-dependent increase in the spontaneous beating rate of cardiac myocytes. The average spontaneous beating rate of myocytes is shown before and after exposure to increasing concentrations of TRAP (added at the arrow) to the superfusate in the prechamber. Concentrations are indicated as follows: 3 µmol/L ({circ}), 30 µmol/L ({blacktriangledown}), and 300 µmol/L ({bullet}) (mean±SEM, n=4 for each).

In most cells (70%), the effect of TRAP to modulate the contractile rate precedes the rise in intracellular calcium ion concentration (see Fig 5Up). This difference in the kinetics of onset of these responses could indicate that the effect of TRAP to increase cytosolic calcium is secondary to an increase in contractile rate. Alternatively, it is possible that TRAP increases intracellular calcium and enhances automaticity through independent mechanisms. To unambiguously determine whether TRAP exerts a direct effect on cellular calcium metabolism, independent of its effect to increase contractile rate, these experiments were repeated in cells electrically driven at a basic cycle length of 1000 milliseconds. This rate is sufficiently rapid to maintain a constant beating rate during exposure to TRAP. Fig 7Down illustrates a typical experiment demonstrating that TRAP induces a rapid and striking increase in both diastolic and peak systolic calcium ion concentration in electrically driven myocytes. TRAP-dependent increases in intracellular calcium also are observed in cells driven at faster and slower rates (basic cycle lengths, 500 and 2000 milliseconds; data not shown). The increase in intracellular calcium is dose dependent, is completely reversed during a 3- to 4-minute washout (as was previously reported for thrombin18 ), and is not accompanied by any significant change in the kinetics of the calcium transient (Table 1Down). Moreover, although TRAP markedly increases the amplitude of the calcium transient, there are no associated changes in the amplitude or the kinetics of the contraction (Table 1Down). In fact, the amplitude of cell shortening tends to decrease with more prolonged exposure to TRAP (see Fig 7Down, bottom panel, tracing c), although this decrease is not statistically significant. This result is surprising, since a rise in intracellular calcium generally results in increased contractile function. Indeed, Table 2Down demonstrates that even modest increases in the amplitude of the intracellular calcium transient induced by stepwise increments in extracellular calcium are associated with substantial increases in the amplitude of the motion transient. Taken together, the data presented in Tables 1Down and 2Down indicate that TRAP alters the relationship between the amplitude of the calcium transient and the amplitude of the motion transient in neonatal myocytes.



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  		Figure 7. Representative tracings of the effect of a bolus of 300 µmol/L TRAP to increase the amplitude of the calcium transient in a neonatal rat ventricular myocyte. The top panel presents the calcium transients (as measured by the fura 2 fluorescence ratio) and the simultaneously recorded contractions during continuous electrical stimulation at 1 Hz and exposure to a bolus of 300 µmol/L TRAP. Note that cell shortening is recorded as microns of motion of a microsphere on the cell surface. The drift in baseline position during exposure to TRAP is not meaningful, since relative cell motion, rather than cell length, is measured in these studies. The gap in the records represents the time for data storage between files. The bottom panel illustrates signal-averaged transients obtained at the times indicated by the letters in the records in the top panel and shows the control condition (tracing a) and the response to the bolus of TRAP (tracings b and c). The motion of only one portion of the cell is measured. Therefore, in the bottom panel, for purposes of comparison, the position of the microsphere before electrical stimulation (diastole) is set to zero, and motion relative to the diastolic position before (tracing a) and after the bolus administration of TRAP (tracings b and c) is reported.


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  		Table 1. TRAP Increases the Amplitude of the Calcium, but Not the Motion, Transient


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  		Table 2. Rise in Extracellular Calcium Is Associated With Increase in Amplitude of Intracellular Calcium Transient and Motion Transient

Role of the Phosphoinositide Signaling Pathway in the Contractile Response to TRAP
Receptor-dependent activation of phosphoinositide hydrolysis is associated with changes in cardiac contractile function. However, the precise role of second messenger molecules generated by this pathway in the modulation of contractile function has been very difficult to determine. Accordingly, we used two approaches to investigate the role of the phosphoinositide signaling cascade in the cellular actions of TRAP. First, cells were pretreated for 24 hours with PMA to downregulate PKC. Table 3Down shows that pretreatment with PMA does not attenuate the effect of TRAP to increase the spontaneous contractile rate and to elevate cytosolic calcium. These results suggest that the effects of TRAP to influence contractile function are not mediated by phorbol ester–sensitive isoforms of PKC.


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  		Table 3. Effects of TRAP to Increase Automaticity and Cytosolic Calcium Are Not Attenuated by Pretreatment With PMA

As a second strategy to investigate the role of the phosphoinositide signaling pathway in the cellular response to TRAP, we used the antiviral and antitumoral xanthate compound D609, which has been shown to effectively inhibit PLC activity.34 35 This alternative approach to block the PKC limb of the phosphoinositide signaling pathway was required, since the long-term effects of PMA may not be limited to downregulation of PKC. Moreover, by inhibiting PLC activity, D609 broadens the assessment to include the functional role of the IP3 limb of the phosphoinositide signaling pathway in the cellular response to thrombin receptor activation. Fig 8ADown verifies that D609 effectively inhibits TRAP-dependent stimulation of IP accumulation. In separate experiments, we demonstrated that ß-adrenergic receptor–dependent stimulation of cAMP accumulation is similar in control and D609-treated cells (207.6±25.5 and 240.4±30.7 pmol cAMP per well over the corresponding controls, respectively; n=3). These results demonstrating that the effect of the ß-adrenergic receptor agonist isoproterenol to stimulate cAMP accumulation is completely spared by the xanthate compound establish that the inhibitory effect of D609 is specific for the phosphoinositide signaling pathway. Fig 8BDown and 8CDown demonstrates that under conditions in which TRAP-dependent IP accumulation is profoundly inhibited by D609, the effect of TRAP to increase automaticity in spontaneous myocytes and increase cytosolic calcium in electrically driven myocytes persists. These results provide evidence that TRAP enhances automaticity and increases cytosolic calcium through a mechanism that is not dependent upon the action of any phosphoinositide-derived second messenger molecules.



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  		Figure 8. D609 inhibits the TRAP-dependent increase in IP accumulation but not TRAP-dependent changes in automaticity and intracellular calcium. Cells were preincubated for 12 hours without or with 100 µmol/L D609. Biochemical and functional responses were evaluated in the absence (open bars) or continued presence (hatched bars) of D609 throughout the duration of the experimental protocol. A, [3H]Inositol-labeled myocytes were incubated with or without 300 µmol/L TRAP, and IP3 accumulation (at 1 minute) and IP1 accumulation (at 30 minutes) were assessed as described in "Materials and Methods." Results are expressed as counts per minute over the corresponding controls for triplicate determinations from a single experiment, which is representative of three experiments on separate cultures. B, Myocytes were exposed to 300 µmol/L TRAP, and the effect on the spontaneous beating rate is shown. Results represent the response 30 seconds after the bolus administration of TRAP and are expressed as percent change from control (mean±SEM, n=4). C, Myocytes driven at a basic cycle length of 1000 milliseconds were exposed to a bolus of 300 µmol/L TRAP, and the effect on the calcium transient amplitude is shown. Results represent the response 30 seconds after the bolus administration of TRAP and are expressed as percent change from control (mean±SEM, n=5). Effects of thrombin to enhance automaticity and elevate intracellular calcium also persisted in D609-pretreated cells (data not shown).

Role of Voltage-Sensitive Calcium Channels in the Calcium Response to TRAP
Thrombin has been shown to stimulate calcium entry through voltage-dependent calcium channels in frog myocytes,36 guinea pig myocytes,37 and rat portal vein smooth muscle cells.38 To examine the possibility that the TRAP-induced rise in cytosolic calcium is due to the activation of voltage-sensitive calcium channels, myocytes were treated with the calcium channel antagonist verapamil, and the effect of TRAP was evaluated. Fig 9Down, top, illustrates that verapamil markedly attenuates electrically stimulated calcium transients but does not prevent the TRAP-induced rise in cytosolic calcium. Electrically stimulated calcium transients were completely abolished when cells were exposed to verapamil in a superfusion buffer devoid of any added calcium, but the TRAP-dependent rise in cytosolic calcium persisted (Fig 9Down, middle). In contrast, when caffeine was used to deplete intracellular calcium stores, the TRAP-dependent increase in cytosolic calcium was markedly attenuated (Fig 9Down, bottom). These results are most consistent with the conclusion that the thrombin receptor–dependent rise in calcium predominantly reflects the release of calcium from intracellular stores.



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  		Figure 9. Effects of verapamil, low extracellular calcium, and caffeine on the calcium response to TRAP. The effect of TRAP (300 µmol/L), delivered at the arrow as a bolus to the superfusate in the prechamber, on the fura 2 fluorescence tracing is shown in cells pretreated with 1 µmol/L verapamil (top), 1 µmol/L verapamil in superfusion buffer without any added calcium (middle), or 20 mmol/L caffeine (bottom). Cells were field-stimulated at 1 Hz throughout the recording interval. The gaps in the tracings represent the time for data storage between files. During the periods marked by the thick horizontal lines, the time scale is expanded 10-fold. The figures depict results obtained in single experiments that are representative of the group of 13 (top), 8 (middle), or 9 (bottom) cells studied.

Perforated-patch voltage-clamp experiments were carried out to further probe the mechanism for thrombin receptor–dependent modulation of calcium in cardiac myocytes. Fig 10Down illustrates that even when the membrane potential is held at -70 mV to prevent activation of voltage-gated ion channels, TRAP still induces a transient elevation of intracellular calcium. In three of four cells, the elevation in calcium was associated with a measurable increase in inward current (-5.22±1.11 pA/pF; mean capacitance, 27±4 pF). Of note, the one cell without a detectable change in current in association with the rise in intracellular calcium had the smallest increase in intracellular calcium. To determine whether the inward current observed in these experiments is an initiating event or is the response to the rise in calcium (eg, Na+-Ca2+ exchange current), two cells were treated with caffeine to deplete intracellular calcium stores. Under these conditions, TRAP did not induce any detectable changes in calcium or holding current, consistent with the observed current being a secondary event.



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  		Figure 10. Effect of TRAP on intracellular calcium and holding current in a cell voltage-clamped at a membrane potential of -70 mV. The perforated-patch technique was used to voltage-clamp a single rat ventricular myocyte loaded with fura 2 using the AM form of the dye (see "Materials and Methods"). The cell was held at a constant potential of -70 mV throughout the experiment. During the time indicated by the horizontal bar, the superfusion solution contained 300 µmol/L TRAP. The upper tracing displays the increase in the 340/380 fura 2 fluorescence ratio caused by TRAP superfusion; the lower tracing displays the simultaneous increase in net inward current, normalized to cell capacitance (23 pF for this cell).

Taken together, these results provide evidence that TRAP stimulates the release of calcium from intracellular stores. Although a critical role for transsarcolemmal influx of calcium to trigger intracellular calcium release cannot be excluded, the data demonstrating that the effect of TRAP to elevate intracellular calcium can occur in the presence of verapamil or when cells are held at -70 mV argue that the increase in calcium does not require activation of a voltage-gated calcium current.


*    Discussion
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 up arrowIntroduction
 up arrowMaterials and Methods
up arrowResults
*Discussion
down arrowReferences
 
Results of the present study establish that the thrombin receptor agonist peptide TRAP mimics thrombin's actions to stimulate phosphoinositide hydrolysis, activate MAPK, enhance automaticity, and elevate intracellular calcium in neonatal rat ventricular myocytes. These findings implicate the thrombin receptor, recently identified in cultured neonatal rat ventricular myocytes,19 in thrombin's cellular actions in these cells. These studies also provide insights into the role of second messenger molecules generated via the phosphoinositide pathway in thrombin receptor–dependent signaling in the heart. Results reported herein indicate that the thrombin receptor–dependent activation of MAPK is mediated, in part, by diacylglycerol activation of PKC. In contrast, phosphoinositide-derived second messenger molecules appear to play no role in thrombin receptor–dependent modulation of cardiac contractile function.

In our attempt to define the biochemical events regulated by the thrombin receptor, we observed that the nature of the thrombin receptor activation mechanism profoundly influences thrombin receptor coupling to downstream biochemical signaling cascades. Proteolytic activation of the thrombin receptor by thrombin results in the rapid and transient stimulation of phosphoinositide hydrolysis and activation of MAPK. In contrast, activation of the thrombin receptor by peptide agonist (TRAP) leads to a more robust and sustained stimulation of phosphoinositide hydrolysis and activation of MAPK. The similar kinetics for thrombin receptor–dependent stimulation of phosphoinositide hydrolysis and activation of MAPK suggests a relationship between these responses. This is supported by the observation that the incremental activation of MAPK by TRAP is abolished by pretreatment with PMA to downregulate phorbol ester–sensitive isoforms of PKC. Although the PKC-dependent pathway for MAPK activation appears to be more effectively activated by TRAP than by thrombin, both agonists have a similar (albeit smaller) effect to activate MAPK in PMA-pretreated cells. This suggests that an additional (more minor) PKC-independent pathway for MAPK stimulation is activated similarly by thrombin and TRAP. This is not surprising, given the evidence from several laboratories that the phosphoinositide signaling mechanism is one of several converging signal transduction pathways activated by thrombin. For example, in nonmyocytes thrombin stimulates tyrosine phosphorylation of various proteins,2 5 11 including the GAP protein, which activates Ras.2 Functional Ras, in turn, has been shown to be required for thrombin-induced mitogenesis.39 40 In cardiac myocytes, thrombin receptor activation induces hypertrophy and augments the expression of atrial natriuretic factor (one of a group of embryonic genes induced during myocyte hypertrophy) through a mechanism that appears to involve both PKC and protein tyrosine kinases.19 Thus, studies in diverse cell types are consistent with the notion that the integrated response to thrombin is dependent upon multiple converging signaling pathways.12 In this context, one might anticipate that the more robust and sustained activation of phosphoinositide hydrolysis and MAPK by TRAP than by thrombin would be associated with important functional consequences. However, both thrombin and TRAP have been reported to be equivalently effective inducers of atrial natriuretic factor expression in cultured cardiac myocytes.19 Nevertheless, the MAPK cascade has been postulated to play an important role in the hypertrophic response of cardiac myocytes, and the possibility that the dissimilar patterns of MAPK activation by TRAP and thrombin lead to distinct hypertrophic responses requires further study.

We also have attempted to elucidate the precise signaling mechanism(s) by which the thrombin receptor modulates cardiac contractile function. Our initial studies demonstrated that the kinetics of thrombin- and TRAP-dependent stimulation of phosphoinositide hydrolysis differ, whereas the kinetics of the automaticity and/or calcium responses to these agonists cannot be distinguished. Although this could suggest that the biochemical and functional responses are unrelated, this type of a comparison likely is not entirely valid; the biochemical experiments were performed on cells incubated in the presence of agonist for prolonged intervals, whereas measurements of calcium and contractile function were performed under conditions in which the cells were subjected to a rapid and transient bolus of agonist. Therefore, additional studies were required to determine whether the phosphoinositide signaling pathway mediates thrombin receptor–dependent changes in calcium and contractile function. There are several lines of evidence arguing that activation of the PLC/PKC pathway is neither necessary nor sufficient for thrombin-induced modulation of contractile function. First, prolonged treatment with PMA, which downregulates most isoforms of PKC, does not attenuate the contractile response to TRAP. Second, the effect of TRAP to stimulate the biochemical response and to modulate contractile function can be dissociated using D609. Third, norepinephrine is an even more robust and persistent activator of phosphoinositide hydrolysis than is thrombin but induces only a relatively minor increase in automaticity and does not increase intracellular calcium in these cells.41 Taken together, these results are the first to convincingly argue that second messenger molecules generated via phosphoinositide hydrolysis do not play an essential role in the rapid thrombin receptor–induced changes in cardiac contractile function. In view of previous evidence that thrombin increases calcium channel current in cardiac myocytes36 and smooth muscle cells38 and prolongs the duration of the action potential of Purkinje myocytes through a pathway that is sensitive to disruption by the calcium channel antagonist nisoldipine,18 we also considered the possibility that thrombin modulates contractile function by directly triggering the activation of a voltage-sensitive calcium channel. However, results reported herein establish that an influx of extracellular calcium through voltage-dependent calcium channels is not essential for the thrombin-dependent rise in cytosolic calcium (although we have not completely ruled out the possibility that such an action could contribute to the overall response to TRAP in actively beating myocytes). It is also noteworthy that when caffeine is used to prevent the TRAP-induced release of calcium from the sarcoplasmic reticulum, the effect of TRAP to increase the net inward current also is lost. This suggests that a net ion flux via a voltage-independent pathway (eg, a ligand-gated ion channel) does not initiate the TRAP-dependent rise in calcium. Thus, the thrombin receptor modulates contractile function through a mechanism that is distinct from the phosphoinositide signaling cascade and activation of a voltage-sensitive ion channel. The nature of the stimulus that links thrombin receptor activation to changes in contractile function remains elusive. It is possible that a signaling molecule such as phosphatidic acid, which has been reported to mobilize intracellular calcium42 and could be formed in D609-treated cells as a result of receptor-dependent hydrolysis of phosphatidylcholine by PLD,43 plays a role in this process. In this regard, cultured neonatal myocytes will provide a particularly useful model for future investigations designed to define the potential role of this and other signaling processes in the events that bridge the gap between thrombin receptor activation and changes in contractile function.

TRAP increases the amplitude of the calcium transient without increasing the amplitude of cell shortening in cultured neonatal rat ventricular myocytes. These results could suggest that TRAP activates a mechanism(s) that depresses myofibrillar calcium sensitivity. However, an alternative interpretation of the results is possible. Since cultured myocytes form a sheet of interconnecting cells that lack a single major axis of myofibrillar alignment, measurements of resting cell length are not possible. Therefore, the amplitude of cell shortening can be influenced by changes in contractile function during systole as well as diastolic contracture due to severe calcium overload. In contrast, the rod-shaped geometry of adult rat ventricular myocytes permits measurements of actual cell length at rest and during contraction and are well suited to distinguish these processes. Although we recognize that age-dependent differences in the contractile response to TRAP may exist and confound the interpretation of these studies, we have initiated studies on adult ventricular myocytes for this purpose. Recent experiments indicate that TRAP increases twitch amplitude in adult rat ventricular myocytes (authors' unpublished data, 1995). Assuming that similar mechanisms are operative in cultured neonatal myocytes, these data suggest that activation of the thrombin receptor leads to a positive inotropic response but that the magnitude of this response may be blunted in myocyte cultures due to concomitant changes in diastolic cell length.

It is difficult to envision a mechanism whereby thrombin would come into direct contact with myocardial cells and influence contractile function in the normal heart. We previously speculated that thrombin might assume importance as a humoral mediator of electrophysiological events in the setting of hemorrhagic infarction where the endothelial barrier is broken.18 However, Goldstein et al44 recently presented evidence that acute ischemia induced by thrombotic coronary occlusions results in a higher incidence of malignant ventricular arrhythmias than does nonthrombotic balloon occlusion, despite equivalent amounts of jeopardized myocardium. The implication of these studies is that factors derived from the thrombus or induced during thrombotic occlusion could be arrhythmogenic, even in the absence of direct hemorrhage into the myocardium. The notion that thrombin, produced during coronary thrombosis, directly influences myocytes in the ischemic regions of the heart is particularly intriguing in view of our recent finding that the actions of thrombin to stimulate phosphoinositide hydrolysis and enhance IP3 accumulation are exaggerated in hypoxic myocytes.45 Whether this translates into exaggerated contractile responses to thrombin in hypoxic myocytes remains to be determined. Experimental evidence that the direct and exaggerated effects of thrombin on the ischemic myocardium contribute importantly to the genesis of malignant arrhythmias would provide an important rationale for the development of therapeutic interventions designed to block thrombin's actions at its receptor on cardiac myocytes.


*    Selected Abbreviations and Acronyms
 
D609 = tricyclodecan-9-yl-xanthogenate
IP, IP1, IP2, IP3 = inositol phosphate, monophosphate, bis-phosphate, and tris-phosphate
MAPK = mitogen-activated protein kinase
MBP = myelin basic protein
PKC = protein kinase C
PLA2, PLC, PLD = phospholipase A2, C, and D
PMA = phorbol 12-myristate 13-acetate
TRAP = thrombin receptor–derived agonist peptide


*    Acknowledgments
 
This study was supported by US Public Health Service–National Heart, Lung, and Blood Institute grants HL-49537 and HL-43731.

Received October 25, 1995; accepted December 15, 1995.


*    References
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*References
 

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