AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 289: H1736-H1743, 2005. First published May 20, 2005; doi:10.1152/ajpheart.00318.2004
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Modulation of myosin phosphatase targeting subunit and protein phosphatase 1 in the heart

Ravi Rajashree, Bradford C. Blunt, and Polly A. Hofmann

Department of Physiology, University of Tennessee School of Medicine, Memphis, Tennessee

Submitted 30 March 2004 ; accepted in final form 12 May 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Myosin light chain 2 (LC2) phosphorylation is of both physiological and pathological importance to myocardial function. The phosphatase that directly dephosphorylates LC2 is a type 1 protein phosphatase (PP1) that contains a catalytic subunit that complexes with a myosin-binding phosphatase targeting subunit (MYPT). The goal of the present study was to examine the role of MYPT in the regulation of PP1 in ventricular myocytes. In the first part of the study, regional distribution of MYPT expression and phosphorylation were determined in unstimulated hearts. The pattern of MYPT phosphorylation was inversely related to the LC2 phosphorylation spatial gradient as described by Epstein and colleagues (Davis JS, Hassanzadeh S, Winitsky S, Lin H, Satorius C, Vemuri R, Aletras AH, Wen H, and Epstein ND. Cell 107: 631–641, 2001). In the second part of the study, adult rat isolated ventricular myocytes were exposed to an {alpha}-adrenergic receptor agonist, and properties of MYPT, PP1, and LC2 were studied. We found MYPT associates with cardiac myofilaments, and this association increases upon {alpha}-adrenergic receptor stimulation. Activation of {alpha}-adrenergic receptors also led to a decrease in the PP1-myofilament association. Furthermore, {alpha}-adrenergic receptor stimulation results in phosphorylation of MYPT and LC2 and an increase in myocyte Ca2+ sensitivity of tension that all depend on Rho kinase activation. These data support the hypothesis that {alpha}-adrenergic receptor activation works through Rho kinase to phosphorylate MYPT, and phosphorylated MYPT dissociates from PP1 so that PP1 is no longer physically associated with LC2. Hence, we propose a pathway for the dynamic modulation of LC2 phosphorylation through receptor-dependent phosphorylation of MYPT, and a spatial gradient of LC2 phosphorylation under basal conditions that occurs due to varied levels of phosphorylation of MYPT in ventricles.

light chain 2; {alpha}-adrenergic receptor; Rho kinase; ventricular myocytes; isometric tension

MYOSIN REGULATORY LIGHT CHAIN [also known as myosin light chain 2 (LC2)] phosphorylation leads to activation of smooth muscle and modulation of striated muscle contraction. This modulation was initially documented in studies that correlated the extent of LC2 phosphorylation to an increase in tension at submaximum Ca2+ concentration ([Ca2+]) in permeabilized myocardial preparations (14, 26). Recent studies (21) demonstrate that this effect may be much greater than previously recognized, with phosphorylation of LC2 potentially causing a 20–30% increase in cardiac muscle isometric tension generation. LC2 phosphorylation has also been shown to contribute to the positive inotropic response seen upon exposure to {alpha}-adrenergic receptor agonists in heart (1, 9). Furthermore, a spatial gradient of LC2 phosphorylation may contribute to the beating heart's torsion that results in the wringing out of blood (4). Taken together these studies indicate that LC2 phosphorylation is of functional consequence in heart. However, control of the phosphorylation state of myocardial LC2 in heart has not been well studied.

Factors and signaling pathways that influence the phosphorylation state of LC2 have been well studied in smooth muscle (reviewed in Refs. 17, 18, 27). It is well established that phosphorylation of LC2 occurs through Ca2+-activated myosin light chain kinase (MLCK) in both smooth and striated muscle. The phosphatase that directly dephosphorylates LC2 is a type 1 protein phosphatase (PP1; Ref. 3), and PP1 appears to have numerous pathways that regulate its activity. The PP1 catalytic subunit complexes with a myosin-binding phosphatase-target subunit 1 (MYPT1) and a 20-kDa subunit of unknown function in smooth muscle. MYPT localizes PP1 to its substrate to cause an increase in localized phosphatase activity. At a given [Ca2+], the force produced by smooth muscle increases through Rho kinase or ZIP-like kinase phosphorylation of MYPT1 (reviewed in Ref. 18). Part of the basis of this increase in force is that the phosphorylation of MYPT1 causes the dissociation of MYPT1 from the PP1 catalytic subunit and decreases PP1 activity associated with LC2 in smooth muscle. Studies also support the idea that a decrease in PP1 activity can occur through a Rho kinase-dependent phosphorylation of a protein that associates with MYPT1 in smooth muscle. Cyclic GMP is also known to inhibit Rho kinase and MYPT phosphorylation, and increase PP1 localized activity (22). Hence, MYPT1 is a key player in regulating smooth muscle PP1 activity. However, the role of MYPT in the regulation of PP1 in ventricular myocytes is not as well studied.

Both MYPT1 and MYPT2 isoforms are expressed in human heart (8), whereas the {delta}-isoform of PP1 copurifies with MYPT in heart myofibrils (16). Myocardial Rho kinase activation has a positive inotropic effect in atrial tissue (28) and increases the phosphorylation of LC2 in left ventricle (11). These studies and the many studies done on smooth muscle MYPT led us to hypothesize that cardiac MYPT is phosphorylated by Rho kinase upon {alpha}-adrenergic receptor stimulation. Furthermore, phosphorylated MYPT decreases PP1 localized to the cardiac myofilaments and leads to an increase in phosphorylated LC2 and altered functional properties. Finally, in addition to the dynamic modulation of MYPT, we wished to look at MYPT regional expression under basal conditions. Epstein and colleagues (4) demonstrated that a gradient of LC2 phosphorylation exists from apex to base and from epicardium to endocardium in heart. Thus we hypothesized that a gradient of cardiac MYPT expression or phosphorylation might exist.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation of ventricular myocytes. Animals used in this study were treated in accordance with The American Physiological Society's "Guiding Principles in the Care and Use of Animals," and protocols were approved by the University of Tennessee Institutional Animal Care and Use Committee. Collagenase isolated ventricular myocytes from adult rats were obtained according to the protocol of Liu and Hofmann (13). Typically, we obtain a 2 ml pellet of cells per rat heart with a 70% yield of 3:1 (length-to-width ratio) rectangular myocytes. Only those isolations that provided >60% rod-shaped myocytes were used.

Regional MYPT expression and phosphorylation. Tissue samples were obtained from the apex, base, endocardium, and epicardium of left ventricle to determine whether any regional variations exist in the levels of MYPT expression or in the extent of MYPT phosphorylation. One-third of the inner and outer mid-ventricular free walls were analyzed for comparison of endocardium vs. epicardium. To minimize variance, the center portion of the free wall was not used. Transverse slices of the left ventricular apex and the upper third of the free wall were analyzed for comparison of apex vs. base. Samples were homogenized and diluted such that protein loads were equal on SDS-PAGE analysis. However, to fully normalize for any minor differences in protein load, Western blot immuno-signals were analyzed relative to two separate, randomly selected bands on Coomassie stain gels. For Western blots, we used an antibody to a peptide that corresponds to amino acids 723–840 of MYPT1 and reacts with both phosphorylated and nonphosphorylated MYPT (catalog no. 612164; BD Biosciences) and an antibody to a peptide that corresponds to amino acids 691–701 with phosphorylation of Thr696 of MYPT1 (catalog no. 07-251; Upstate Biotechnology).

Determination of phosphorylated MYPT in cells. Ventricular myocytes were pretreated with the Rho kinase inhibitor Y-27632 (Y-27; 10 µM) or vehicle for 20 min, which was followed by addition of 100 nM isoproterenol and 10 µM phenylephrine plus 3 µM propranolol or 100 nM 17-phenyl trinor prostaglandin E2 (PGE2; Cayman Chemical) for 3 min. A 3-min receptor agonist treatment was used based on past observations that a similar duration of phenylephrine stimulation alters cardiac myocyte function (19). The cellular reactions were stopped with urea-containing sample buffer. Western blotting was done using an antibody to phosphorylated MYPT (see Regional MYPT expression and phosphorylation).

Time-dependent MYPT and PP1 localization to myofilaments. Ventricular myocytes were treated with 10 µM phenylephrine plus 3 µM propranolol or vehicle for 3 min. Washed cells were then placed for 30 min in ice-cold lysis buffer. This buffer contained 0.1% Triton X-100, 250 mM NaCl, 5 mM EDTA, and 1 mM mercaptoethanol. Subsequent to lysis, cells were centrifuged at 12,000 g for 10 min at 4°C, and the particulate was retained. This protocol, which uses Triton and 250 mM NaCl, allows for isolation of MYPT strongly associated with the particulate/myofilaments. To some extent, 250 mM NaCl solubilized myosin thick filaments.

To determine whether MYPT associates with the nuclei and/or myofilaments in the particulate fraction, MYPT immunofluorescence microscopy was carried out. Ventricular myocytes were isolated and suspended on a coverslip. Cells were fixed with paraformaldehyde- and ethanol-containing solutions. After extensive washings, cells were permeabilized with 0.2% Triton X-100 in saline. After washes, the permeabilized cells were incubated with primary antibody for 2 h at room temperature using antibodies to MYPT (1:10 dilution) or phosphate buffer without primary antibody as a negative control. Cells were then exposed to the secondary antibody conjugated to rhodamine for 20 min at room temperature. After cells were washed with a 0.2% Triton X-containing phosphate buffer, an antifade solution was applied (catalog no. 2828; Molecular Probes). Images were collected with a cooled charge-couple device camera on a Nikon Eclipse E300 inverted microscope using MetaMorph software. It should be noted that the antibody to MYPT does recognize both nonphosphorylated and phosphorylated MYPT and may recognize MYPT1 and MYPT2 due to the high degree of sequence homology in MYPT1 and MYPT2 (8).

Determination of cytosolic PP1. Ventricular myocytes were pretreated with 10 µM Y-27 or vehicle for 20 min; this was followed by addition of 10 µM phenylephrine plus 3 µM propranolol for 3 min. Cells were exposed to cytosolic lysis buffer for 5 min. Cytosolic lysis buffer contained 0.05% digitonin, 10 mM KCl, 1 mM DTT, 1 mM EDTA, 1.5 mM MgCl2, 10 mM HEPES, pH 7.5, and 25% glycerol. After a 12,000 g centrifugation for 15 min, the supernatant containing the cytosolic components was retained. Western blotting was done using an antibody to a peptide that corresponds to amino acids 315–326 on human PP1-{delta} (catalog 07-270; Upstate Biotechnology).

Determination of phosphorylated LC2. Immunoblotting and autoradiography were used to determine the phosphorylation state of LC2. For cells undergoing autoradiography, 30 µCi of H3-32PO4 (37 mBQ; catalog no. NEX053; PerkinElmer) was combined with 120 µl of cells in solution for 60–80 min before drug treatments to allow time for 32P incorporation into cellular ATP. Ventricular myocytes were then pretreated with 10 µM Y-27 or vehicle for 15 min, which was followed by addition of either 1 nM or 100 nM calyculin A to all groups for a 5- to 10-min incubation in both autoradiographic and immunoblotting protocols. Calyculin A was added since phosphorylation of LC2 is difficult to demonstrate without attenuation of background phosphatase activity in ventricular myocytes (1; data not shown). After these pretreatments, cells were stimulated with 10 µM phenylephrine plus 3 µM propranolol for 3 min. The cellular reactions were stopped with non-urea Laemmli sample buffer that contained 50 mM NaF and 5 mM EDTA. Western blotting was done using an antibody to a peptide that corresponds to a short amino acid sequence of phosphorylated LC2 of human origin (catalog no. 12896; Santa Cruz Biotechnology). This antibody reacts with Thr18 and Ser19 phosphorylated LC2 but not with nonphosphorylated LC2. Radioactive phosphate incorporation into LC2 was determined by exposing dried SDS gels to X-Omat film (Eastman Kodak; Rochester, NY) for 12–48 h. Variability in reactivity of the phosphorylated LC2 antibody, related to the antibody lot number, made it necessary to also measure phosphorylated LC2 using 32P. The consistently robust signal we obtained from 32P of LC2 suggests that cardiac LC2 was being phosphorylated rather than trace amounts of LC2 from contaminating cell types.

Isometric tension as a function of [Ca2+]. Cells were treated with 1 nM calyculin A, 10 µM Y-27, or vehicle and 10 µM phenylephrine plus 3 µM propranolol or vehicle as described above. The preincubation of all cells in 1 nM calyculin A was done to attenuate the PP2a basal activity. A recent study using isolated ventricular myocytes indicates that the IC50 of PP1 inhibition is ~125 nM, whereas the IC50 of PP2a inhibition is at a significantly lower calyculin A concentration (5). Hence, any LC2 phosphorylation brought about by our treatments was prolonged by primarily inhibiting the basal PP2a activity.

Treated cells were pelleted and incubated for 7 min on ice in a skinning solution that contained 50 mM KH2PO4, 70 mM NaF, 5 mM EDTA, 1% Triton X-100, 5 µg/ml pepstatin A, 10 µg/ml leupeptin, 40 µg/ml PMSF, 5 mM EGTA, 0.1 µM vanadate, and 1 nM calyculin A. After tritonization, the demembranated cells were washed and placed on ice for use up to 8 h later. Myocyte mechanical measurements were carried out using procedures and equipment identical to those described by Lester et al. (12). In brief, myocytes were attached via glue (Great Stuff foam; Insta-Foam; Marietta, GA)-coated glass micropipettes to a piezoelectric translator and force transducer. Cells were set at an initial sarcomere length of ~2.2 µm in low-[Ca2+] solution. Myocytes were observed and photographed for later analysis of sarcomere length and sarcomere length uniformity. Cells that were able to maintain a constant and uniform sarcomere length in a solution with pCa 4.5 and pH 7.0 were used. A tension-pCa relationship was obtained by initially measuring force during maximal activation (pCa 4.5) followed by contractions at randomly chosen submaximal pCa values and then measuring force again at pCa 4.5 to assess any decline in cell performance. Maximum active tension was calculated as the difference between force generated in a pCa 4.5 solution and the passive tension measurement obtained in a pCa 9.0 solution. Only those cells that initially generated >2.0 g/mm2 maximum active tension were included in the data analysis. Maximum tension, the Ca2+ sensitivity of tension (pCa50), and degree of cooperative activation (as judged by the steepness of the tension-pCa relationship) were assessed from the tension-pCa relationship. Maximum tension per cross-sectional area was calculated assuming a cell cross-sectional area with a cell depth equal to 60% of cell width (7). Composition of the pCa solutions can be found in Lester et al. (12).

Statistical analysis. For studies in which data was normalized to paired vehicle-treated cells, a two-way ANOVA using complete block design without replication and appropriate post hoc tests were completed. For studies examining the effects of treatment over time, a two-way ANOVA was followed by Tukey's test. P < 0.05 was selected as indicating significance. Power calculations (P = 0.80) indicate a significant difference of ≥10% between groups can be identified in maximum tension normalized to cross-sectional area results given the typical variability and number of cells utilized.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Regional MYPT expression and phosphorylation in adult rat heart. Total MYPT and phosphorylated MYPT were determined from the base, apex, endocardium, and epicardium of adult rat hearts. No difference in expression of total MYPT was observed in the base and endocardial regions compared with the apex and epicardium, respectively (Fig. 1). The level of phosphorylated MYPT was higher in the apex compared with the base of heart. To normalize for minor protein-load variations, the Coomassie-stained density of a random protein band was determined, and immunoreactive densities of total MYPT and phosphorylated MYPT are expressed relative to this protein load. Similar results were obtained when MYPT or phosphorylated MYPT densities were normalized to a second Coomassie-stained band.



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  		Fig. 1. Typical Western blots (A) and cumulative analysis (B) of total myosin-binding phosphatase targeting subunit (MYPT) and phosphorylated MYPT (P-MYPT) in left ventricular apex, base, endocardium (Endo), and epicardium (Epi) samples of adult rat hearts. Densities of total MYPT expression and P-MYPT were normalized to the Coomassie-stained density of a protein band on SDS-PAGE gels loaded with aliquots from the same samples as the Western blots, i.e., a normalization for any variations in relative protein load. Data are averages ± SE; n = 7 hearts; *P < 0.05 compared with Apex.

 
Phosphorylation of MYPT by membrane receptor agonists. Isolated ventricular myocytes were treated with 10 µM phenylephrine, phenylephrine plus 3 µM propranolol, 100 nM isoproterenol, 100 nM PGE2, or vehicle. Stimulation of {alpha}-adrenergic receptors with phenylephrine in combination with propranolol (a {beta}-adrenergic receptor antagonist) increased the phosphorylation of MYPT compared with vehicle-treated cells (Fig. 2). {beta}-Adrenergic receptor stimulation with isoproterenol and PGE2 receptor stimulation with PGE2 also increased the phosphorylation of MYPT (Fig. 2).



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  		Fig. 2. Activation of {alpha}-adrenergic receptors with phenylephrine plus propranolol (Phe/Prop), the PGE2 receptors with 17-phenyl trinor PGE2, and the {beta}-adrenergic receptors with isoproterenol (Iso) increased the levels of phosphorylated MYPT in isolated ventricular myocytes as observed in representative Western blots (A) and a normalized cumulative analysis (B). Aliquots of samples used in the representative Western blots (top) were also run on an SDS gel that was Coomassie stained to demonstrate equality of protein load (bottom; actin region of the gel shown). Cumulative data (B) are an average of data from cells treated for 3 min with phenylephrine plus propranolol (n = 15 isolations), 3 min with isoproterenol (n = 3 isolations), and 3 or 5 min with PGE2 (n = 3 isolations/group). Data are normalized to paired vehicle-treated cells and are presented as averages ± SE; *P < 0.05 compared with Control.

 
Role of Rho kinase in phosphorylation of MYPT. To test for the involvement of Rho kinase in MYPT phosphorylation, ventricular myocytes were pretreated with Y-27 (a Rho kinase inhibitor) or vehicle for 20 min before stimulation with phenylephrine plus propranolol or PGE2. Rho kinase inhibition with Y-27 significantly blocked the increase in phosphorylation of MYPT upon {alpha}-adrenergic or PGE2 receptor stimulation and decreased basal phosphorylated MYPT levels (Fig. 3).



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  		Fig. 3. Rho kinase inhibition with Y-27632 (Y-27) blocked the phenylephrine/propranolol-induced and 17-phenyl trinor PGE2-induced increases in phosphorylated MYPT in isolated ventricular myocytes as observed in representative Western blots (A) and a normalized cumulative analysis (B). Data are normalized to paired vehicle-treated cells (controls) and are averages of 3–8 isolations ± SE; *P < 0.05 compared with Control.

 
Cellular translocation of MYPT and PP1-{delta}. Ventricular myocytes were stimulated with phenylephrine plus propranolol for various periods of time, and the time course of {alpha}-adrenergic-induced translocation of MYPT (Fig. 4A) to the particulate/myofilament nuclei-rich fraction was established. Stimulation of {alpha}-adrenergic receptors significantly increased the MYPT level in the insoluble particulate fraction. A corresponding decrease in the MYPT level in the supernatant was seen at the 10-min time point but not at 6- and 15-min time points (data not shown). Immunofluorescence microscopic analysis revealed that MYPT in the particulate fraction is primarily associated with myofilaments rather than nuclei (Fig. 4B).



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  		Fig. 4. Time-dependent translocation of MYPT to the particulate fraction in ventricular myocytes treated for 3, 6, 10, and 15 min with phenylephrine plus propranolol or vehicle (A). Cells were lysed and fractionated using a protocol optimized for myofilament isolation (see MATERIALS AND METHODS). Representative examples of fluorescence micrographs (B) of myofilaments in the absence (left) and presence (right) of a primary antibody to MYPT with a secondary antibody conjugated to rhodamine are shown. Cumulative data are averages of 3 isolations ± SE; *P < 0.05 compared with 3-min control.

 
Phenylephrine plus propranolol blocked the time-dependent translocation of PP1-{delta} to the particulate fraction in control-treated myocytes (Fig. 5). Specifically, after 15 min of treatment with phenylephrine and propranolol, PP1 in the particulate fraction was significantly lower than in controls (Fig. 5). In addition, {alpha}-adrenergic receptor stimulation with phenylephrine plus propranolol for 3 min increased the level of PP1-{delta} in the soluble 0.05% digitonin fraction of ventricular myocytes (cytosol; Fig. 6). Addition of Y-27 (the Rho kinase inhibitor) 20 min before {alpha}-adrenergic receptor stimulation attenuated the {alpha}-adrenergic-induced increase in the level of cytosolic PP1-{delta}. A trend (not significant) was also observed in some cases for Y-27 to decrease basal levels of cytosolic PP1 (for example, Fig. 6A).



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  		Fig. 5. Time-dependent translocation of type 1 protein phosphatase (PP1)-{delta} to the particulate fraction in ventricular myocytes treated with phenylephrine plus propranolol or vehicle for the times indicated. Cells were lysed and fractionated using a protocol optimized for myofilament isolation (see MATERIALS AND METHODS). Cumulative data are averages of 3 isolations ± SE; *P < 0.05 compared with 3-min Control.

 


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  		Fig. 6. Phenylephrine/propranolol increased the level of PP1 in the cytosolic fraction of cardiac myocytes as observed in representative Western blots (A) and a normalized cumulative analysis (B). Rho kinase inhibition with Y-27 attenuated the phenylephrine-induced increase in cytosolic PP1. Cytosolic fractions were collected after 0.05% digitonin lysis of cells. Cumulative data are averages of 5 isolations ± SE; *P < 0.05 compared with Control.

 
Phosphorylation of LC2. Stimulation of {alpha}-adrenergic receptors with phenylephrine plus propranolol increased the phosphorylation of LC2 in membrane-intact ventricular myocytes pretreated with 100 nM calyculin A (Fig. 7). This increase in the phosphorylation of LC2 was attenuated by pretreatment of cells with the Rho kinase inhibitor Y-27.



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  		Fig. 7. Phenylephrine/propranolol increased the level of phosphorylated myosin light chain 2 (P-LC2) in isolated ventricular myocytes as determined by cumulative analysis of immunoreactivity to a phosphorylated LC2 antibody (A) and 32P incorporation into myosin light chain 2 (LC2; B). Typical examples of phosphorylated LC2 and 32P-LC2 along with Coomassie stain of corresponding gels (inset) are included. Rho kinase inhibition with Y-27 blocked the phenylephrine-induced increase in phosphorylated LC2. Cumulative data were normalized to appropriate controls; i.e., either the untreated control or Y-27-pretreated groups. Aliquots of samples used were also run on SDS gels and were Coomassie stained to demonstrate equality of protein load. Cumulative data for immunoblots are averages of 6 isolations ± SE, whereas 32P autoradiography data are averages from 4 isolations ± SE; *P < 0.05 compared with Controls.

 
Isometric tension as a function of [Ca2+]. Stimulation of {alpha}-adrenergic receptors with phenylephrine plus propranolol increased the phosphorylation of LC2 by 66 ± 30% as determined by immunoblotting in skinned ventricular myocytes pretreated with 1 nM calyculin A (n = 4 isolations). Both the skinning procedures and the lower calyculin A concentration contributed to the decrease in the level of LC2 phosphorylation compared with the data in Fig. 7 (an approximate threefold increase in phosphorylated LC2 is shown in Fig. 7A). This modest increase in the phosphorylation of LC2 at low calyculin A concentration and after tritonization was fully blocked by pretreatment of cells with the Rho kinase inhibitor Y-27.

The relationship between isometric tension as a function of [Ca2+] was established in cells pretreated with 1 nM calyculin A followed by addition of Y-27 or vehicle, phenylephrine plus propranolol or vehicle, and skinning. Phenylephrine plus propranolol caused an increase in active tension from pCa 6.0 to 5.4 (Fig. 8A). The cumulative pCa at which 50% tension is observed (pCa50) for control cells was 5.70 ± 0.02 (n = 7 cells), whereas the pCa50 for phenylephrine plus propranolol-treated cells was 5.80 ± 0.02 (n = 9 cells). Maximum isometric tension, pCa 4.5, was not significantly different between groups. The phenylephrine-induced increase in submaximum isometric tension was blocked by inhibition of Rho kinase using Y-27 (Fig. 8B). The cumulative pCa50 for Y-27-treated cells was 5.72 ± 0.03 (n = 5 cells), whereas the pCa50 for Y-27 and phenylephrine plus propranolol-treated cells was 5.76 ± 0.04 (n = 7 cells). The slope (Hill coefficient) of the tension-pCa relationship was not significantly influenced by any treatments.



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  		Fig. 8. Cumulative plot of relative isometric tension as a function of pCa for ventricular myocytes that were {alpha}-adrenergic receptor stimulated with phenylephrine/propranolol (A) or were pretreated for Rho kinase inhibition with Y-27 plus phenylephrine/propranolol (B) and skinned. Relative tensions were normalized to maximum active force produced at pCa 4.5. Each group is an average ± SE. Curves were fit to the cumulative data using the Hill equation with coefficients determined as follows: control, pCa50 5.71 and slope 1.65; phenylephrine/propranolol, pCa50 5.81 and slope 1.74; Y-27, pCa50 5.72 and slope 1.77; and Y-27 + phenylephrine/propranolol, pCa50 5.76 and slope 1.67.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Figure 9 depicts one way that {alpha}-adrenergic receptor activation may decrease PP1 activity and, as such, increase the phosphorylation of LC2 in ventricular myocytes. This hypothesized pathway is based on four observations of the present study. First, {alpha}-adrenergic receptor stimulation leads to a phosphorylation of MYPT that depends on Rho kinase activation in ventricular myocytes. This can be mimicked by a known Rho kinase activator. Second, MYPT binding to cardiac myofilaments is altered upon {alpha}-adrenergic receptor stimulation and results in a decrease in the association of PP1 with the myofilaments. Third, inhibition of Rho kinase blocks the {alpha}-adrenergic-dependent increase in phosphorylation of LC2. Fourth, basal levels of LC2 phosphorylation correlate with the extent of MYPT phosphorylation. Our studies also demonstrate that activation of this pathway increases myofilament Ca2+ sensitivity of isometric tension.



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  		Fig. 9. Signaling pathway consistent with the findings of the present study in adult rat ventricular myocytes. MLCK, myosin light chain kinase; adren receptor, adrenergic receptor.

 
Previous studies established that there is a spatial gradient of LC2 phosphorylation in heart (4). Phosphorylated LC2 is greater in the epicardium compared with the endocardium and greater at the apex compared with the base of the heart (4). Furthermore, levels of MLCK expression correlate with the LC2 phosphorylation pattern (4). The regional nature of LC2 phosphorylation, with corresponding Ca2+ sensitization of contraction, may facilitate torsion in the heart (4). In the present study, we establish that the distribution of phosphorylated MYPT (non-PP1 binding and nontargeting) is higher at the apex compared with the base of the heart under basal conditions. Hence, increased MLCK expression (4) and an increased basal level of MYPT phosphorylation (present study) both contribute to the increase in LC2 phosphorylation at the apex of the heart.

Activation of {alpha}-adrenergic receptors increases MLCK activity and phosphorylation of LC2 to contribute to the {alpha}-adrenergic-induced positive inotropic response of heart (1, 9). Activation of {alpha}-adrenergic receptors is also known to phosphorylate MYPT and lead to a reduction in PP1 activity and an increase in phosphorylation of LC2 in smooth muscle (18, 25). The present study used ventricular myocytes to demonstrate that {alpha}-adrenergic receptor stimulation also leads to phosphorylation of MYPT and an increase in phosphorylation of LC2 in heart. These observations are consistent with a phosphorylated MYPT-dependent decrease in localized PP1 enzymatic activity in cardiac myocytes. However, this does not exclude the possible involvement of other known PP1 modulators such as protein phosphatase inhibitor protein 1 (6, 10).

The increase in phosphorylation of MYPT by the {beta}-adrenergic receptor agonist isoproterenol was unexpected. PKA does not directly phosphorylate the Thr696 of MYPT. However, the PKA effects can occur through cross-activation of PKG phosphorylation sites as has been shown to be the case in some smooth muscle studies (reviewed in Ref. 23). In addition, PKA is known to phosphorylate inhibitor protein 1, which causes the inhibition of PP1 (10) and increases PP1 target-protein phosphorylation such as phosphorylated MYPT. Thus indirect mechanisms could account for the PKA-dependent increase in phosphorylated MYPT.

The {alpha}-adrenergic-dependent phosphorylation of MYPT leads to the dissociation of MYPT from PP1 so that PP1 is found in the cytosol of smooth muscle (2, 27). Our data are consistent with this, as we found an increase in PP1 in the cytosolic fraction after {alpha}-adrenergic-dependent phosphorylation of MYPT in ventricular myocytes. Furthermore, MYPT association with the myofilament increased upon {alpha}-adrenergic stimulation. Finally, in vehicle-treated but not {alpha}-adrenergic-stimulated cells, we observed an increase in PP1 and MYPT associated with the myofilaments over time. Although we do not understand why this unstimulated, time-dependent translocation of PP1 and MYPT occurred, it may in part explain why assays of PP1 activity have historically had such a high degree of variability.

An {alpha}-adrenergic receptor-dependent increase in LC2 phosphorylation and isometric tension at submaximum [Ca2+] were obtained after pretreatment of cells with calyculin A. Calyculin A is a serine/threonine phosphatase inhibitor that was used in our studies to dampen basal phosphatase activity. This is necessary to observe changes in LC2 phosphorylation (Ref. 1; data not shown) and suggests that phosphatase levels are typically high in resting cells. However, calyculin A would also alter the dynamic phosphorylation state of a number of other phosphatase target proteins. This is a limitation in the present study. To minimize this concern, all groups (including the control group) were treated with calyculin A before any experimental treatments. Thus the relative change due specifically to phenylephrine or Y-27 could be documented with respect to LC2 phosphorylation and isometric tension at submaximum [Ca2+].

The increase in Ca2+ sensitivity of myocardial tension after {alpha}-adrenergic receptor activation was fully blocked by Rho kinase inhibition. Previous work has indicated that {alpha}-adrenergic receptor activation increases MLCK activity through an increase in intracellular Ca2+ and activation of calmodulin. These changes in MLCK activity are probably not present in our study, since quiescent ventricular myocytes were exposed to the {alpha}-adrenergic receptor agonist. Thus dynamic changes in intracellular [Ca2+] do not occur. In addition, the Ca2+-activated phosphatase PP2b (calcineurin) would be expected to have a lower activity in quiescent cells. The decreases in Ca2+-dependent enzymes such as MLCK and PP2b may have fortuitously allowed us to record changes in phosphorylated MYPT and phosphorylated LC2. However, caution is needed when applying our conclusions to the in vivo state in which [Ca2+] is cycling on a beat-to-beat basis, since our results were obtained in conditions of low, steady-state [Ca2+].

Myocardial {alpha}-adrenergic receptor-G{alpha}q activation also leads to Rho kinase activation (20), and inhibition of Rho kinase attenuates the positive inotropic effect of {alpha}-adrenergic stimulation in heart (1, 9). We found that Rho kinase inhibition blocks the {alpha}-adrenergic-dependent phosphorylation of MYPT, translocation of PP1, phosphorylation of LC2, and increase in Ca2+ sensitivity of isometric tension. A PGE2 receptor agonist that is also a positive inotropic agent and activator of Rho kinase in atria (28) caused a Rho kinase-dependent increase in the phosphorylation of MYPT in ventricular myocytes. Taken together, these data indicate that Rho kinase decreases PP1 activity through MYPT phosphorylation and leads to an increase in the phosphorylation of LC2 in cardiac myocytes and a subsequent increase in Ca2+ sensitivity of isometric tension. It should be noted that our studies do not exclude other pathways, such as MLCK activation, from contributing to the effects of {alpha}-adrenergic receptor stimulation. In fact, we propose that both increased MLCK and decreased PP1 activity contribute to the positive inotropic effects of {alpha}-adrenergic G protein-coupled receptor activation in heart (1, 9) and may contribute to the localized, differential force requirements needed to wring blood from the heart as it beats (4). In addition, a number of studies demonstrate that ischemic and failing hearts have altered PP1 activity (15). For example, rapid pacing-induced failure leads to an increase in RhoA expression and phosphorylation of LC2 that allows for a Ca2+-sensitizing effect upon {alpha}-adrenergic stimulation (24). Thus we hypothesize that the pathway identified in the present study may play a role in some or all of these functional effects due to altered LC2 phosphorylation in heart.


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This study was supported by National Institutes of Health Grant HL-48839 (to P. A. Hofmann).


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Hofmann, Dept. of Physiology, Univ. of Tennessee School of Medicine, 894 Union Ave., Memphis, Tennessee 38163 (E-mail: phofmann{at}physio1.utmem.edu)

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


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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