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(Circulation Research. 1995;76:1011-1019.)
© 1995 American Heart Association, Inc.

Articles

Arachidonic Acid–Dependent Phosphorylation of Troponin I and Myosin Light Chain 2 in Cardiac Myocytes

Derek S. Damron, Ahmad Darvish, LeeAnn Murphy, Wendy Sweet, Christine S. Moravec, Meredith Bond

From the Center for Anesthesiology Research (D.S.D., A.D., W.S., C.S.M.) and the Department of Molecular Cardiology (L.A.M., M.B.), Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio.

Correspondence to Meredith Bond, PhD, Department of Molecular Cardiology/FF10, The Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195.

	Abstract

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Abstract Recent evidence has suggested that arachidonic acid (AA) may be an important signaling molecule in cardiac excitation-contraction coupling. We previously showed that AA and endothelin-1 (ET) inhibit distinct K⁺ channels via protein kinase C–dependent pathways in rat ventricular myocytes. In addition, we demonstrated that Ca²⁺ transients in populations of fura 2–loaded myocytes were potentiated by AA and ET via activation of protein kinase C. In this study, we have used suspensions of [³²P]orthophosphate (³²P_i)–labeled rat ventricular myocytes to study the effects of AA and ET at the level of the myofilaments. After a 10-minute incubation of the labeled cells with phorbol 12-myristate 13-acetate (PMA), AA, or ET in the presence or absence of the protein kinase C inhibitor calphostin C, the myofibrillar proteins were separated by PAGE. Measurement of unloaded cell shortening using video edge detection in single electrically stimulated myocytes was also used to assess the effects of AA and ET on myocyte contractility. Incubation with either PMA, AA, or ET resulted in similar increases in ³²P_i incorporation into troponin I (TnI) and myosin light chain 2 (MLC2), which was inhibited by preincubation with the protein kinase C antagonist calphostin C. In addition, the ability of these agonists to stimulate phosphorylation of TnI or MLC2 did not require extracellular Ca²⁺ or intact intracellular Ca²⁺ stores. The effects of AA and ET together on phosphorylation of TnI or MLC2 were not additive. These results show that AA and ET phosphorylate myofibrillar proteins in intact cardiac myocytes via a calphostin C–inhibitable Ca²⁺-independent pathway and that AA, like ET, stimulates a positive inotropic effect in cardiac myocytes. Thus, phosphorylation of cardiac myofilaments by AA and/or ET, in addition to phosphorylation-dependent inhibition of distinct K⁺ channels, may represent an important signaling pathway by which AA and ET influence the inotropic state of the heart.

Key Words: myocytes • arachidonic acid • endothelin-1 • phosphorylation • myofilaments

	Introduction

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Recent studies in cardiac muscle cells have suggested that arachidonic acid (AA) may be an important signaling molecule in the modulation of cardiac contractile function via alterations in Ca²⁺ cycling.^{1 2} In addition, AA has been shown to stimulate Ca²⁺ release from cardiac sarcoplasmic reticulum (SR) vesicles³ and to both activate⁴ and inhibit² K⁺ channels in cardiac myocytes. AA-dependent activation of Ca²⁺ currents in cardiac myocytes has also been reported.⁵ Therefore, AA may modulate cardiac contractility by altering ion channel activity.

We previously showed that both AA and endothelin-1 (ET) increase action potential duration via protein kinase C (PKC)–dependent inhibition of the transient outward K⁺ current (I_to) and PKC-dependent inhibition of the delayed rectifier K⁺ current (I_K), respectively.² We also demonstrated that the positive inotropic effect of ET was inhibited by the PKC antagonist staurosporine. In isolated perfused hearts, ET has been shown to stimulate both phospholipase C⁶ and phospholipase A₂,⁷ releasing diacylglycerol (DG) and AA, respectively. Both DG and AA are activators of PKC, acting synergistically in some cell types.⁸

Activation of PKC in cardiac muscle cells by phorbol esters has been shown to phosphorylate myofibrillar proteins,^{9 10 11 12} resulting in changes in the sensitivity of the myofilaments to Ca²⁺¹¹ and/or changes in V_max of the actomyosin ATPase.⁹ This is consistent with the finding that phorbol esters and -adrenergic agonists stimulate translocation of some PKC isoforms to the myofilaments in cultured neonatal cardiac myocytes,¹³ whereas other isoforms were shown to translocate to sarcolemmal, perinuclear, or intranuclear regions.¹⁴ In adult rat ventricular myocytes, phorbol esters have been reported to stimulate translocation of the , , and isoforms of PKC to the particulate fraction,¹⁵ whereas ET and -adrenergic agonists¹⁶ stimulate translocation of the isoform to the membrane fraction, although the intracellular site(s) of translocation was not determined.^{15 16} In addition, myocardial ischemia, a condition known to cause accumulation of unesterified AA¹⁷ and increased expression of ET receptors,¹⁸ results in translocation of PKC to the membrane.¹⁹

The role of PKC activation in the regulation of cardiac contractility remains controversial: phorbol esters are known to have pronounced negative inotropic effects^{20 21} in contrast to the well-established positive inotropic effect of ET²² or -adrenergic agonists,²³ which are also known activators of PKC. However, positive inotropic effects of phorbol esters at extremely low concentrations (<1 nmol/L) have also been observed, suggesting perhaps selective activation of specific PKC isoforms.^{24 25} PKC-dependent phosphorylation of Ca²⁺ channels,²⁶ phospholamban,²⁷ the cardiac ryanodine receptor,²⁸ and the SR Ca²⁺-ATPase²⁹ has been demonstrated and may also play a role in mediating inotropic responses in the heart. Therefore, biologically active compounds that activate PKC in cardiac muscle may be involved in the modulation of cardiac contractility via phosphorylation-dependent regulation of the contractile machinery itself and/or phosphorylation of Ca²⁺ regulatory proteins.

Previous studies examining PKC-dependent phosphorylation of myofibrillar proteins have generally focused on isolated myofibrils or permeabilized myocytes.^{9 10 11 12} In the present study, we used intact ventricular myocytes to investigate the effects of AA on cardiac excitation-contraction coupling. Specifically, we investigated whether AA stimulates phosphorylation of myofibrillar proteins in isolated ventricular myocytes. We also investigated whether ET stimulates phosphorylation of cardiac myofibrillar proteins, since ET is known to activate phospholipase C, causing release of DG, an endogenous activator of PKC.^{6 30} The results of this study present the novel findings that both AA and ET stimulate phosphorylation of troponin I (TnI) and myosin light chain 2 (MLC2) in isolated ventricular myocytes.

	Materials and Methods

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Myocyte Preparation
Isolated adult ventricular myocytes from rat heart were obtained as previously described¹ by using a modification of the method described by Altschuld et al.³¹ In brief, the heart was excised, cannulated via the aorta, attached to a modified Langendorff perfusion apparatus, and perfused with oxygenated (95% O₂/5% CO₂) Krebs-Henseleit buffer (KHB), which contained (mmol/L) NaCl 118, KCl 4.8, MgCl₂ 1.2, KH₂PO₄ 1.2, CaCl₂ 1.2, NaHCO₃ 37.5, dextrose 16.5, and pyruvate 7.5, supplemented with 0.68 mmol/L glutamine, 0.1 mmol/L MEM, basal medium Eagle amino acids and vitamin solution, pH 7.35. After 5 minutes of equilibration, the perfusion buffer was changed to a Ca²⁺-free KHB containing collagenase type II (237 U/mL). After collagenase digestion (45 minutes), the ventricles were minced and shaken in KHB, and the resulting cellular digest was washed, filtered, resuspended in phosphate-free HEPES-buffered saline (HBS) containing (mmol/L) NaCl 118, KCl 4.8, MgCl₂ 1.2, CaCl₂ 1.25, dextrose 11, HEPES 25, pyruvate 5, and glutamine 0.68, pH 7.35, and vigorously bubbled immediately before use with 100% O₂. Typically, 6 to 8x10⁶ cells per rat heart were obtained by using this procedure. Viability, as assessed by the percentage of cells retaining a rod-shaped morphology with no blebs or granulations, was routinely between 80% and 90%.

Fura 2 Experiments
Freshly isolated ventricular myocytes suspended in oxygenated HBS (12 mL, 5x10⁵ cells per milliliter) were divided into equal portions (6 mL each) and allowed to settle by gravity on the bench. The supernatant was aspirated, and half was resuspended in 6 mL of KHB, with the other half in 6 mL of KHB lacking Ca²⁺. The cells were washed a second time to ensure removal of all extracellular Ca²⁺ and again pelleted and resuspended in the appropriate buffer. The cell suspensions were placed under 100% O₂ and incubated while gently shaking at room temperature for 1.5 hours, at which time 5 µmol/L fura 2-AM was added to the cells, and the incubation was allowed to continue for an additional 45 minutes. The cells were washed in their respective buffers (ie, KHB±Ca²⁺) to remove extracellular fura 2-AM and resuspended in oxygenated KHB or KHB lacking added Ca²⁺. Fluorescence measurements, designed to examine the size of intracellular Ca²⁺ stores, were performed in populations of myocytes as previously described.² Briefly, 75 µL of the fura 2–loaded cells (1.5x10⁵ cells) were added to the cuvette in 2 mL of the appropriate buffer (KHB or KHB lacking added Ca²⁺, 37°C). EGTA was added to the cuvette to eliminate any potential contribution of extracellular Ca²⁺ to the fluorescence signal. Once the fluorescence signal had stabilized, ionomycin was added to the cuvette to release Ca²⁺ from intracellular stores. The amplitude of the Ca²⁺ transient, evoked by addition of ionomycin to cells that had been incubated in KHB, was compared with the amplitude of the Ca²⁺ transient evoked by addition of ionomycin to cells that had been incubated in Ca²⁺-free KHB.

Labeling of Myofibrils in Intact Ventricular Myocytes With [³²P]Orthophosphate
Phosphorylation of myofibrils in intact rat ventricular myocytes by [³²P]orthophosphate (³²P_i) was performed by using modifications of previously described methods.^{9 32} Isolated ventricular myocytes from one rat heart were suspended in 15 mL of phosphate-free HBS (5x10⁵ cells per milliliter) and incubated with 250 µCi of ³²P_i for 2 hours at room temperature. The cell suspension was gently shaken and maintained under 100% O₂ throughout the entire labeling period. There was no significant loss in myocyte viability (percentage of rod-shaped cells) after the 2-hour incubation period with ³²P_i.

After the labeling period, the cells were pelleted and resuspended in phosphate-free KHB in the presence or absence of extracellular Ca²⁺, divided into 2-mL aliquots, and placed in test tubes with or without calphostin C (1 µmol/L).³³ To ensure that the cells remained viable and maximally oxygenated throughout the incubation, all tubes were purged with 100% O₂, then placed on their sides, and gently agitated for 3 minutes at 37°C in a shaking water bath. After the 3-minute preincubation, either phorbol 12-myristate 13-acetate (PMA), ET, or AA was added to the cell suspension. The phosphatase inhibitor calyculin A (1 nmol/L)³⁴ was also added at this time, and all test tubes were again purged with O₂ and incubated for an additional 10 minutes at 37°C. Controls contained either calphostin C, ethanol (solvent for AA), dimethyl sulfoxide (DMSO, solvent for calphostin C) alone, or both ethanol and DMSO in multiple agonist experiments. The final concentrations of ethanol and/or DMSO together did not exceed 0.1%.

Routinely, myocyte viability was assessed after the incubation periods to ensure that viability remained >75% after the 10-minute incubation at 37°C in the presence of the agonists and inhibitors. Calphostin C has been reported to be cytotoxic to cells at concentrations that inhibit PKC during extended incubation periods (72 hours).³³ Myocyte viability was routinely between 80% and 85% before incubation with calphostin C at 37°C, and we found no significant loss of viability after the 13-minute incubation with this inhibitor (<3%) plus the added agonists. At the end of the incubation, the reactions were terminated, and agonists were removed by rapidly washing the myocytes in physiological saline (4°C, 5 mL) containing protease inhibitors (5 µg/mL pepstatin A, 10 µg/mL leupeptin, 43 µg/mL phenylmethylsulfonyl fluoride, 5 µg/mL antipain, and 5 mmol/L EGTA) and protein phosphatase inhibitors (0.1 µmol/L sodium orthovanadate and 1 nmol/L calyculin A) immediately before suspending the pellet in 2 mL of ice-cold "inhibiting buffer" (mmol/L: KH₂PO₄ 50, NaF 70, and EDTA 5) as described by Holroyde et al.³⁵ Myofibrils were extracted on ice for 30 minutes by adding 1% Triton X-100, plus the protease and phosphatase inhibitors listed above, to the inhibiting buffer. Detergent-extracted myofibrils were pelleted at 5000g by using an Eppendorf microcentrifuge for 5 minutes, and the supernatant was discarded. Examination of the pellet under the light microscope indicated that it was enriched in myofibrils.

SDS-PAGE
PAGE of ³²P-labeled cardiac myofibrils was performed on 12% slab gels according to Laemmli.³⁶ The myofibrillar pellet was solubilized and denatured in sample preparation buffer containing 4% SDS, 0.23 mol/L 2-mercaptoethanol, and 0.2 mol/L Tris-HCl (pH 6.5) at 100°C for 5 minutes. To standardize loading of the gels, protein determinations of the myofibrillar extracts³⁷ were performed on each of the samples before loading the gel. Protein (75 µg) was applied to each lane, and PAGE was performed in the presence of 0.1% SDS at constant current (10 mA) overnight (16 hours). Gels were stained with Coomassie blue, dried the following day, and subjected to phosphor screen autoradiography for quantification of labeled proteins. A PhosphorImager (Molecular Dynamics, Inc) was used to quantify the amount of radioactivity in each band. The extent of ³²P incorporation into protein bands was quantified by using the IMAGEQUANT software package.

Western Blot Analysis of Cardiac Myofibrillar Proteins
After SDS-PAGE of myofibrillar proteins, proteins were transferred (300 mA, 60 minutes) from the gel to a nitrocellulose membrane.³⁸ The remaining protein binding sites on the nitrocellulose membrane were blocked during an overnight incubation with 3% fish gelatin in Tris-saline buffer (pH 7.0). After blocking, nitrocellulose membranes were incubated with rabbit anti-mouse MLC2 antibody (gift from Dr Ken Chien, University of California, San Diego) at a dilution of 1:500 for 2 hours. In a separate experiment, the nitrocellulose membrane containing the myofibrillar proteins was incubated with a monoclonal antibody raised against bovine TnI (gift of Dr Stefano Schiaffino, University of Padua, Italy) at a dilution of 1:500 for 2 hours. After three washes in Tris-saline buffer, the nitrocellulose membranes were incubated with secondary antibody coupled to horseradish peroxidase, at a dilution of 1:2000 for 1 hour. The nitrocellulose membranes were then washed in Tris-saline buffer and placed in horseradish peroxidase substrate until visible bands were observed (1 hour).

Measurement of Myocyte Contractility Using Video Edge Detection
The effects of AA and ET on myocyte contractility were assessed in single electrically stimulated ventricular myocytes. An aliquot of ventricular myocytes was placed in a temperature-regulated (28°C) chamber (Bioptechs, Inc) mounted on the stage of an Olympus CK-2 inverted microscope. The volume of the bath was 2 mL. The cells were superfused continuously with KHB at a flow rate of 2 mL/min and electrically stimulated through bipolar platinum electrodes at a frequency of 0.5 Hz and duration of 5 milliseconds by using an SD9 stimulator (Grass Instruments). Either AA or ET was added to the superfusion solution after a 1- to 2-minute equilibration period and required 30 seconds to reach the myocytes in the chamber. Myocytes were chosen for study according to the following criteria: (1) rod-shaped appearance with clear striations and no membrane blebs, (2) a negative staircase of twitch performance on stimulation from rest, and (3) the absence of frequent (>1/min) spontaneous contractions. Measurements of unloaded cell shortening were monitored with a video edge detector (Crescent Electronics). Light-dark contrast at the cell end provides a marker for measurement of the amplitude and velocity of cell motion. DATA SPONGE software (Bioscience Analysis Software, Ltd) was used for data acquisition and analysis of unloaded cell shortening at a sampling rate of 500 Hz with a 60-Hz filter.

Materials
Collagenase type II was obtained from Worthington Biochemical. Calphostin C and staurosporine were obtained from Research Biochemicals, Inc and were diluted in DMSO to appropriate stock concentrations in single-use vials stored at -70°C. AA (Cayman Chemical Co) was divided into aliquots (25 mmol/L) and diluted in ethanol in N₂-purged airtight glass vials stored at -70°C. A fresh vial was used each day. ET (Peptides International) was diluted into single-use vials in distilled water (100 µmol/L) and stored at -70°C until the day of use. ET and AA were maintained on ice for the duration of the experiment. Triton X-100, Protogel (30% [wt/vol] acrylamide and 0.8% [wt/vol] bis-acrylamide stock solution), and 10x electrophoresis tank buffer (0.25 mol/L Tris, 1.92 mol/L glycine, and 1% SDS) were purchased from National Diagnostics. N,N,N',N'-Tetramethyl-ethylenediamine, protein G, 2-mercaptoethanol, ammonium persulfate, and prestained high and low molecular weight markers were from Biorad Laboratories, Inc.

Experimental Controls and Data Analysis
Untreated controls (control–calphostin C or control–Ca²⁺ rich) were normalized to 100%. Increases in the phosphorylation of TnI and MLC2 in response to PMA, ET, and/or AA were calculated as percent increase in ³²P incorporation (±SEM) of the respective untreated time-matched control. After pretreatment with calphostin C, inhibition of TnI and MLC2 phosphorylation by AA, ET, or PMA was calculated as percent inhibition of ³²P incorporation (±SEM) compared with myocytes treated with PMA, AA, or ET alone. Therefore, percent inhibition refers to percent inhibition of the increase above basal phosphorylation. After results of all trials from 1 day were averaged to give a single value, comparisons between groups were performed by using Student's t test. Differences were considered statistically significant at P<.05. For the contractility study, the amplitude of unloaded cell shortening before the addition of AA or ET was compared with the amplitude of shortening after the addition of agonist. The results are expressed as percent increase over the value before the addition of agonist (±SEM).

	Results

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Identification and ³²P Labeling of Myofibrillar Proteins in Rat Ventricular Myocytes
Based on apparent molecular weight (Fig 1, lane 1) in Coomassie blue–stained 12% SDS-polyacrylamide gels (lane 2), the major myofibrillar proteins were tentatively identified as actin (45 kD), troponin T (TnT, 43 kD), tropomyosin (35 kD), TnI (30 kD), myosin light chain 1 (MLC1, 27 kD), and MLC2 (20 kD). In addition, proteins with molecular weights corresponding to myosin heavy chain (MHC, 205 kD) and C protein (150 kD) were identified (data not shown). After a 2-hour prelabeling period of endogenous pools of ATP with ³²P_i, myocytes were washed and subsequently incubated for 10 minutes at 37°C. Autoradiography of SDS gels of myofibrils from control (untreated) myocytes demonstrated the presence of ³²P incorporation in several myofibrillar proteins, including TnT, tropomyosin, TnI, and MLC2 (Fig 1, lane 3), as well as C protein (data not shown). There was no observable incorporation of ³²P label in actin, MLC1, or MHC.

View larger version (50K): [in this window] [in a new window]   Figure 1. Effect of phorbol 12-myristate 13-acetate (PMA) on the labeling of rat cardiac myofibrillar proteins, in the presence and absence of calphostin C, in intact ventricular myocytes. Myocytes were isolated and labeled with [32P]orthophosphate as described in "Materials and Methods." Myocytes were incubated with or without calphostin C (1 µmol/L) for 3 minutes before the addition of both PMA (1 µmol/L) and calyculin A (1 nmol/L), and the incubation was allowed to continue for 10 minutes at 37°C. Myofibrils were extracted and prepared for PAGE (lanes 1 through 6) as previously described in "Materials and Methods." STDS indicates low molecular weight standards; CB, Coomassie blue staining of myofibrillar proteins; CTRL, untreated time-matched control; PKCI, PKC inhibitor calphostin C; MLC2, myosin light chain 2; MLC1, myosin light chain 1; TnI, troponin I; Tm, tropomyosin; and TnT, troponin T. Similar results were obtained in 11 different experiments. Western blot analysis of phosphorylated myofibrillar proteins is also displayed (lanes 7 and 8). Myofibrillar proteins were transferred from the gel to nitrocellulose strips and incubated in the presence of antibodies directed against TnI or MLC2, as described in "Materials and Methods."

Effect of Phosphatase Inhibition With Calyculin A on the ³²P Labeling of Cardiac Myofibrillar Proteins
Previous studies by others³⁹ have shown that calyculin A at a concentration of 10 nmol/L stimulates incorporation of ³²P into TnI and MLC2 in cardiac muscle cells. Therefore, in order to confirm that the 10-fold lower concentration of calyculin A used in these experiments (1 nmol/L) had no effect on the ³²P labeling of myofibrillar proteins in cardiac muscle cells under control conditions, we investigated the dose-dependent effects of phosphatase inhibition with calyculin A on the labeling of cardiac myofibrillar proteins in intact ventricular myocytes. Addition of calyculin A (1 to 2 nmol/L) for 10 minutes at 37°C had no significant effect on the labeling of myofibrillar proteins in intact cardiac myocytes when compared with time-matched untreated control myocytes (Fig 2). Small increases in the labeling of TnI (12±5.4%) and MLC2 (10±4.8%) were observed with 5 nmol/L calyculin A, but the increase in labeling was not significantly different from untreated control cells (P<.1). However, significant increases in the phosphorylation of TnI (39±6.8%) and MLC2 (48±7.3% increase) were observed with the addition of 10 nmol/L calyculin A when compared with untreated time-matched control cells.

View larger version (37K): [in this window] [in a new window]   Figure 2. Effect of calyculin A on the 32P labeling of cardiac myofibrillar proteins in intact ventricular myocytes. TnT indicates troponin T; Tm, tropomyosin; TnI, troponin I; MLC1, myosin light chain 1; and MLC2, myosin light chain 2. After labeling, myocytes were washed, divided into aliquots, and incubated with calyculin A for 10 minutes at 37°C at the concentrations listed in the figure. Before PAGE, myofibrillar proteins were extracted, homogenized, and denatured as described in "Materials and Methods." Similar results were obtained in seven experiments. In two experiments, calyculin A had no effect at any concentration tested.

Phosphorylation of TnI and MLC2 by PMA
Suspensions of cardiac myocytes were stimulated with PMA in order to activate phorbol ester–sensitive PKC isoforms. In the absence of calyculin A, PMA stimulated a consistent (40% to 60%) increase in TnI phosphorylation, with no apparent effect on other myofibrillar proteins (data not shown). However, when populations of ventricular myocytes were treated with 1 nmol/L calyculin A and PMA together, 1.5-fold and 3-fold increases in the labeling of TnI and MLC2, respectively (Fig 1, lane 5), were observed when compared with calyculin A–treated unstimulated control myocytes (Fig 1, lane 3). Small, insignificant increases in phosphorylation of TnT and tropomyosin were occasionally observed in response to PMA treatment. Therefore, 1 nmol/L calyculin A was used in the following experiments to prevent rapid dephosphorylation of myofibrillar proteins.

Under basal conditions, addition of calphostin C (1 µmol/L) for 3 minutes before the 10-minute incubation resulted in no reduction in the ³²P labeling of TnI compared with control cells. There was also no apparent effect on the labeling of MLC2 (Fig 1, lane 4). The enhanced phosphorylation of TnI in response to the addition of 1 µmol/L PMA was reversed (94.8±5.2% inhibition) in calphostin C–pretreated myocytes (Fig 1, lane 6). Similarly, enhanced phosphorylation of MLC2 in response to PMA treatment was reduced (84±6.5%) by calphostin C. Similar results were obtained in populations of myocytes isolated from hearts of 11 different animals.

Using rabbit anti-mouse MLC2 or a monoclonal antibody raised against TnI as described in "Materials and Methods," we positively identified the proteins phosphorylated by PMA as TnI and MLC2 by using Western blot analysis (Fig 1, lanes 7 and 8).

Phosphorylation of Cardiac Myofibrils by AA and ET
The addition of AA (50 µmol/L) for 10 minutes to ³²P-labeled myocytes resulted in significant increases in phosphorylation of TnI (30 kD) and MLC2 (20 kD) (Fig 3, lane 4), above the phosphorylation level observed in unstimulated control cells (Fig 3, lane 3). Similarly, the addition of ET (250 nmol/L) resulted in an increase in the labeling of TnI and MLC2 (Fig 3, lane 9) above unstimulated control cells. The addition of AA and ET together resulted in a slight increase in the labeling of TnI (9±4.3%) above that observed with either treatment alone (Fig 3, lanes 6 and 8), but this did not achieve statistical significance (n=6, P<.08). Similarly, there was no significant effect of AA and ET together on the labeling of MLC2 when compared with either treatment alone. The results from six such experiments are summarized in Fig 4.

View larger version (49K): [in this window] [in a new window]   Figure 3. Effect of arachidonic acid (AA) and endothelin-1 (ET) on the 32P labeling of cardiac myofibrillar proteins in the presence and absence of calphostin C in intact ventricular myocytes. Myocytes were incubated with or without calphostin C (1 µmol/L) for 3 minutes before the addition of AA (50 µmol/L), ET (250 nmol/L), or both AA and ET together, plus calyculin A (1 nmol/L), for an additional 10 minutes at 37°C. Myofibrils were extracted and prepared for PAGE as previously described. STDS indicates low molecular weight standards; CB, Coomassie blue stain of myofibrillar proteins; CTRL, untreated time-matched control; PKCI, PKC inhibitor calphostin C (1 µmol/L); TnT, troponin T; Tm, tropomyosin; MLC1, myosin light chain 1; and MLC2, myosin light chain 2.

View larger version (20K): [in this window] [in a new window]   Figure 4. Bar graphs summarizing the effect of arachidonic acid (AA), endothelin-1 (ET), or AA and ET on the labeling of rat cardiac myofibrillar proteins. Experiments were performed as described in the legend for Fig 3. Untreated controls (CTRL in the absence of calphostin C [-calphostin C]) were normalized to 100%. Results are expressed as the percent increase/decrease (mean±SEM) in [32P]orthophosphate incorporation compared with untreated time-matched CTRL (-calphostin C). TnI indicates troponin I; MLC2, myosin light chain 2; a, significant difference vs untreated time-matched CTRL (-calphostin C); and b, significant difference vs corresponding value for AA, ET, or AA+ET (solid bars); P<.05. Experiments were repeated on myocyte preparations from six different rat hearts.

Effect of PKC Antagonist Calphostin C on Labeling of TnI and MLC2 by AA and/or ET
To determine if phosphorylation of TnI and MLC2 by AA and ET occurred via activation of PKC, we incubated myocytes in the presence of 1 µmol/L calphostin C for 10 minutes before the addition of AA, ET, or AA and ET together (Fig 3). Pretreatment of the myocytes with 1 µmol/L calphostin C, before stimulation with either AA, ET, or AA plus ET, decreased the labeling of both TnI and MLC2 (Fig 3). Specifically, calphostin C reduced the labeling of TnI in response to stimulation by AA, ET, or AA plus ET by >75% (Fig 3, lanes 5, 7, and 10). Similarly, calphostin C blocked the increase in phosphorylation of MLC2 by AA, ET, or AA plus ET by >90%. A summary of the results obtained from six similar experiments is presented in Fig 4.

Fura 2 Measurements of Intracellular Ca²⁺ Pools
Experiments were performed with isolated myocytes loaded with the Ca²⁺-sensitive fluorescent indicator fura 2 to measure cytosolic free Ca²⁺. These experiments were performed to determine whether prolonged incubation (2 hours) of cardiac myocytes in Ca²⁺-free HBS resulted in depletion of intracellular Ca²⁺ stores, since we planned to perform experiments examining the Ca²⁺ dependence of TnI and MLC2 phosphorylation by AA and ET. Cells incubated in the presence of Ca²⁺ for 2 hours show the presence of a large Ca²⁺ transient in response to the addition of ionomycin, indicative of intact intracellular Ca²⁺ stores (Fig 5). In contrast, cells incubated for 2 hours in the absence of extracellular Ca²⁺ show a very small Ca²⁺ transient in response to ionomycin, which was only 10% to 15% of that observed in cells exposed to HBS containing Ca²⁺.

View larger version (78K): [in this window] [in a new window]   Figure 5. Tracings showing measurement of intracellular Ca2+ stores using fura 2. Cardiac muscle cells were incubated in Ca2+-free or Ca2+-containing HEPES-buffered saline (HBS, 2 hours) during the labeling period with [32P]orthophosphate and then loaded with fura 2-AM, as described in "Materials and Methods." The myocytes were divided into aliquots, and EGTA (1.5 mmol/L) was added to the myocyte suspension (in the cuvette) where indicated. After the fluorescence signal had stabilized, ionomycin (IONO, 5 µmol/L) was added. Ca2+-loaded indicates cells that had been incubated in HBS containing physiological concentrations (1.2 mmol/L) of Ca2+; Ca2+-depleted, cells previously incubated under Ca2+-free conditions. Similar results were obtained in five separate experiments.

Effect of Extracellular Ca²⁺ and Intracellular Ca²⁺ Stores on the Labeling of TnI and MLC2 by PMA, AA, or ET
Incubation of cells in Ca²⁺-free HBS for 2 hours did not significantly affect the labeling of TnI by PMA, AA, or ET compared with ³²P labeling of TnI in myocytes that were suspended in physiological concentrations of extracellular Ca²⁺ (Fig 6) and had intact intracellular Ca²⁺ stores. Under similar conditions, the labeling of MLC2 by PMA, AA, or ET in Ca²⁺-depleted myocytes resuspended in Ca²⁺-free HBS was not significantly different from the extent of ³²P labeling when compared with control incubations in HBS containing Ca²⁺. A summary of results from seven different myocyte preparations is shown in Fig 6.

View larger version (23K): [in this window] [in a new window]   Figure 6. Bar graphs summarizing the effect of Ca2+ removal on the phosphorylation of cardiac myofibrillar proteins. Cardiac myocytes were incubated in the presence or absence of extracellular Ca2+ during the labeling period with [32P]orthophosphate in order to deplete intracellular Ca2+ pools, as described in "Materials and Methods" and in the legend for Fig 3. Myocytes were washed, resuspended in Ca2+-free or Ca2+-added buffer, divided into aliquots, and stimulated with either phorbol 12-myristate 13-acetate (PMA, 1 µmol/L), arachidonic acid (AA, 50 µmol/L), or endothelin-1 (ET, 250 nmol/L) in the presence of calyculin A (1 nmol/L) for 10 minutes at 37°C. Myofibrils were prepared for PAGE as previously described. Results are expressed as the percent increase in 32P incorporation (mean±SEM) into troponin I (TnI) or myosin light chain 2 (MLC2) compared with unstimulated time-matched controls (CTRL). The data presented represent mean values from myocyte preparations from seven different rat hearts.

Effect of AA and ET on Myocyte Contractility
Superfusion of single field-stimulated ventricular myocytes with AA (50 µmol/L) resulted in a 93±26% increase in the amplitude of unloaded shortening above that observed before the addition of AA (Fig 7A). A positive inotropic response was observed in >80% of the cells examined (28 of 34 cells studied). Significant increases in the amplitude of myocyte shortening were also observed with concentrations of AA as low as 12.5 µmol/L and could be reversed by superfusing the myocytes with HBS containing fatty acid–free bovine serum albumin (10 µmol/L, data not shown). The increase in amplitude of myocyte shortening typically reached a plateau within 4 minutes of superfusion with AA. Superfusion of the myocytes with ET (250 nmol/L) resulted in a 71±21% (n=4 cells) increase in myocyte shortening (Fig 7B) and was much slower in onset compared with AA.

View larger version (32K): [in this window] [in a new window]   Figure 7. Tracings showing the effect of arachidonic acid (AA) and endothelin-1 (ET) on unloaded myocyte shortening. The amplitude of myocyte shortening was assessed in single electrically stimulated ventricular myocytes, as described in "Materials and Methods." AA (50 µmol/L) was added to the superfusion buffer where indicated (A). ET (250 nmol/L) was added to the superfusion buffer where indicated (B).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In addition to the regulation of cardiac contractility by neurotransmitters, the inotropic state of cardiac muscle can be influenced by circulating hormones or by paracrine effector molecules from neighboring cells. The purpose of the present study was to further investigate the role of AA as a regulator of contraction in cardiac myocytes and to identify the second messengers involved in its action.

In contrast to earlier studies using, primarily, reconstituted myofibrils^{9 12} or permeabilized preparations of cardiac myocytes^{10 11} to examine the effects of PMA- and/or PKC-dependent activation on phosphorylation of contractile proteins, we have investigated the effects of AA, ET, and PMA on phosphorylation of contractile proteins in freshly isolated ventricular myocytes. In addition, studies by other investigators^{9 10 11 12} have used a mixture of isoforms of PKC isolated from mammalian brain that were incubated with the permeabilized cardiac myocyte preparation^{10 11} or reconstituted myofibrils⁹ in order to study PKC-dependent phosphorylation of cardiac myofilaments. Therefore, isoforms of PKC that are prevalent in brain (ie, and ß isoforms) were present in the in vitro system; however, these may be different from the primary isoform(s) present in adult cardiac muscle cells.^{15 16} Thus, under these conditions, one might observe protein phosphorylation that may not necessarily occur in intact cells.

Phosphatase activity may play an important role in regulating the phosphorylation state of both of these proteins, since higher doses of calyculin A than those used in our experiments (10 nmol/L) stimulated significant increases in the phosphorylation state of MLC2 and TnI in cells that were not treated with agonist (Fig 2). This indicates a rapid turnover of phosphate in both of these proteins in situ, which may play a significant regulatory role in cardiac contractile activity. In fact, phosphatase inhibition with okadaic acid in guinea pig papillary muscle has been demonstrated to induce phosphorylation of myosin light chains, phospholamban, and TnI, resulting in a positive inotropic effect.⁴⁰ In contrast, a rapid acceleration in phosphatase activity following phosphorylation reactions may rapidly remove phosphate incorporated into myofibrillar proteins and may explain the inability of other investigators to observe significant increases in TnI phosphorylation following the addition of DG analogues or PMA to isolated perfused hearts.⁴¹

The role of PKC-dependent phosphorylation of cardiac myofilaments in excitation-contraction coupling in cardiac muscle remains controversial. Phosphorylation of TnI in response to stimulation by phorbol esters in intact cardiac cells and reconstituted myofilaments resulted in a decrease in V_max of actomyosin ATPase, with no shift in Ca²⁺ sensitivity.⁹ Previous studies in intact cardiac muscle cells or ventricular trabeculae have shown that stimulation by PMA or dioctanoylglycerol (DOG) results in a negative inotropic effect (decreased twitch amplitude) associated with either a decrease, or no change, in the sensitivity of the myofilaments to Ca²⁺, as well as a reduction in the amplitude of Ca²⁺ transients.^{20 42} Conversely, treatment of permeabilized cardiac muscle cells with PMA or DOG has been reported to increase Ca²⁺ sensitivity of force development¹⁰ and also to increase rate and amplitude of myocyte cell shortening.^{24 25} Therefore, the role of PMA and DOG as PKC activators and modulators of cardiac muscle cell function remains controversial. Recent reports that PMA can also activate mitogen-activated protein kinase,⁴³ in addition to PKC, further complicate interpretation of these results. The presence of multiple PKC isoforms in cardiac muscle,^{14 44} TnI isoform switching,⁴⁵ and developmental changes in PKC isoforms may also contribute to the observed differences.¹⁵

In addition to phosphorylation of TnI in cardiac muscle cells, AA and ET also stimulate a proportionally greater increase in phosphorylation of MLC2. Similar to phosphorylation of TnI, phosphorylation of MLC2 by AA or ET was inhibited by calphostin C. This suggested that incorporation of ³²P_i into MLC2, in response to AA and ET stimulation, did not occur via activation of myosin light chain kinase but most likely via a PKC-dependent pathway. Therefore, phosphorylation of MLC2 by AA or ET may be involved in modulating cardiac contractility by increasing the sensitivity of the myofibrils to Ca²⁺ and/or by increasing maximal ATPase activity.^{10 12}

Phosphorylation of MLC2 by PKC in rat cardiac myosin and reconstituted myofilaments has been shown to increase actomyosin ATPase activity; however, this effect was reversed by phosphorylation of TnI on the thin filament in a reconstituted model.¹² In addition, studies showed that MLC2 phosphorylation increased the Ca²⁺ sensitivity of isometric force development of skinned ventricular fibers but had no influence on maximal shortening velocity.^{46 47} It has also been shown that the increased Ca²⁺ sensitivity of force development in skinned cardiac muscle cells following MLC2 phosphorylation by myosin light chain kinase could be further enhanced by the addition of PKC.¹⁰ Similarly, maximal Ca²⁺-activated actomyosin ATPase activity was elevated in the presence of myosin light chain kinase and increased further in the presence of both myosin light chain kinase and PKC.¹⁰ Therefore, increases in MLC2 phosphorylation following stimulation by ET or AA may result in increased sensitivity of the myofilaments to Ca²⁺ and/or increased ATPase activity and may explain, in part, the increase in contractility in rat cardiac myocytes in response to AA and ET in the present study.^{48 49} However, it is possible that inhibition of K⁺ channels by AA or ET,² resulting in action potential prolongation and enhanced Ca²⁺ entry, may also play an important role in the modulation of contractility observed in response to AA and ET in the present study.

We also investigated whether there was an additive effect of AA and ET on phosphorylation of TnI and MLC2. Treating the myocytes with AA and ET together resulted in no significant increase in phosphorylation of TnI or MLC2 above that observed after stimulation with either agonist alone, suggesting that both MLC2 and TnI are most likely phosphorylated at the same site in response to stimulation by both AA and ET. Future experiments using two-dimensional gel electrophoresis of tryptic digests of ³²P-labeled cardiac myofilaments will be required to confirm this idea.

Despite effective depletion of the intracellular pools of Ca²⁺ (as judged by a significant decrease in the size of the ionomycin-releasable Ca²⁺ pool) and the absence of extracellular Ca²⁺, PMA, AA, and ET all stimulated phosphorylation of TnI and MLC2 to a similar degree, as observed in cells with intact intracellular Ca²⁺ stores suspended in buffer containing 1.2 mmol/L Ca²⁺. These results thus indicate that Ca²⁺ influx and/or intracellular Ca²⁺ mobilization is not required for phosphorylation of MLC2 or TnI by AA or ET. These data also suggest that activation of myosin light chain kinase, known to be dependent on an increase in Ca²⁺ and the presence of calmodulin, is most likely not involved in mediating phosphorylation of TnI and MLC2 in rat cardiac myocytes in response to AA, ET, or PMA. It is possible that phosphorylation of TnI and MLC2 in response to AA and/or ET is mediated via activation of a Ca²⁺-insensitive isoform of PKC, potentially PKC-, since Bogoyevitch et al¹⁶ have shown that phorbol esters and ET stimulation results in translocation of PKC- to the membrane in adult rat cardiac myocytes.

In the intact cell, physiological agonists are likely to trigger many signaling pathways that modulate cellular events. Therefore, it is important to assess the functional significance of stimulation at the level of the whole cell rather than just at the level of the myofilaments. In the present study, we found that addition of both AA and ET separately to single electrically stimulated myocytes resulted in a positive inotropic effect, as evidenced by an increase in amplitude of myocyte shortening. Other investigators have also obtained similar results with ET.^{48 49}

The positive inotropic effect of AA may be attributable to an increase in the phosphorylation of TnI and/or MLC2, as described in the present study, and/or to increased Ca²⁺ entry as a result of inhibition of I_to, as described in our earlier study.² However, preliminary studies on electrically stimulated Ca²⁺ transients in single myocytes indicate no increase in the amplitude of the cytosolic Ca²⁺ transient during superfusion of isolated cells with AA (D.S. Damron, B. Summers, M. Bond, unpublished data, 1994). Therefore, it is likely that the increase in amplitude of shortening observed in response to AA stimulation may be attributable primarily to an increase in the sensitivity of the myofilaments to Ca²⁺ following phosphorylation of MLC2 and/or TnI.

In conclusion, we have shown that in intact cardiac myocytes, AA and ET stimulate phosphorylation of TnI and MLC2 via Ca²⁺-insensitive pathways that are inhibited by calphostin C. These results also suggest that AA and ET may activate the same Ca²⁺-independent isoform of PKC in intact cardiac muscle cells, since both agents phosphorylate the same myofibrillar proteins and since the increase in phosphorylation of MLC2 or TnI in response to maximal concentrations of AA and ET is similar in magnitude and not additive.

Activation of Ca²⁺-independent PKC isoforms by physiological agonists may be important for transduction of signals that modulate cardiac contractility, since Ca²⁺ is continually cycling in cardiac muscle cells in vivo. These results demonstrate for the first time that AA enhances the contractility of individual muscle cells, which may be mediated, in part, by phosphorylation of the myofibrillar proteins TnI and MLC2.

	Acknowledgments

 
This study was supported by grants HL-41883 (Dr Bond), HL-33713 (Drs Bond and Moravec), and HL-49929 (Dr Moravec) from the National Institutes of Health; an Established Investigator Award from the American Heart Association (Dr Bond); and Cardiovascular Research Support grants from Pfizer (Drs Bond and Moravec). Drs Damron and Darvish are recipients of individual National Research Service awards from the National Institutes of Health (HL-08726 [Dr Damron] and HL-09162 [Dr Darvish]). The authors would like to thank Bradley K. McConnell for his help with the fura 2 experiments. We would also like to thank Drs Ken Chien and Stefano Schiaffino for providing us with antibodies to MLC2 and TnI, respectively.

	Footnotes

 
This manuscript was sent to Leslie A. Leinwand, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received August 22, 1994; accepted February 28, 1995.

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