Actin Capping Protein
An Essential Element in Protein Kinase Signaling to the Myofilaments
Actin capping protein (CapZ) binds the barbed ends of actin at sarcomeric Z-lines. In addition to anchoring actin, Z-discs bind protein kinase C (PKC). Although CapZ is crucial for myofibrillogenesis, its role in muscle function and intracellular signaling is unknown. We hypothesized that CapZ downregulation would impair myocardial function and disrupt PKC-myofilament signaling by impairing PKC–Z-disc interaction. To test these hypotheses, we examined transgenic (TG) mice in which cardiac CapZ protein is reduced. Fiber bundles were dissected from papillary muscles and detergent extracted. Some fiber bundles were treated with PKC activators phenylephrine (PHE) or endothelin (ET) before detergent extraction. We simultaneously measured Ca2+-dependent tension and actomyosin MgATPase activity. CapZ downregulation increased myofilament Ca2+ sensitivity without affecting maximum tension or actomyosin MgATPase activity. Maximum tension and actomyosin MgATPase activity were decreased after PHE or ET treatment of wild-type (WT) muscle. Fiber bundles from TG hearts did not respond to PHE or ET. Immunoblot analysis revealed an increase in myofilament-associated PKC-ε after PHE or ET exposure of WT preparations. In contrast, myofilament-associated PKC-ε was decreased after PHE or ET treatment in TG myocardium. Protein levels of myofilament-associated PKC-β were decreased in TG ventricle. C-protein and troponin I phosphorylation was increased after PHE or ET treatment in WT and TG hearts. Basal phosphorylation levels of C-protein and troponin I were higher in TG myocardium. These results indicate that downregulation of CapZ, or other changes associated with CapZ downregulation, increases cardiac myofilament Ca2+ sensitivity, inhibits PKC-mediated control of myofilament activation, and decreases myofilament-associated PKC-β.
An exciting and developing concept in cellular biology is the idea that elements of the cytoskeleton not only form an internal super structure of the cell, but also participate in cell signaling. Cytoskeletal proteins are likely to transmit force generated by the molecular motors of the sarcomere to sensors that modify cellular activity as diverse as nuclear transcription and ion channel conductance. Moreover, cytoskeletal elements may serve as scaffolding for molecular signaling cascades, providing sites for localization and anchoring of signaling molecules. In the experiments reported in this article, we have tested these ideas by using a transgenic mouse model in which the function of a strategically located cytoskeletal element, actin capping protein, has been modified.
Actin capping proteins (CPs) are heterodimers composed of an α- and β-subunit (CP-β), and exist as an isoform population that displays tissue- and subcellular-specific patterns of localization. Vertebrate cells express three α-subunit isoforms, each encoded by a separate gene, and three β-isoforms, produced by alternate splicing of messenger RNA from one gene. Although CP-β1 and CP-β2 bind actin with nearly identical affinities and kinetics in vitro,1 each has a distinct function that cannot be substituted for by the other in cardiac myocytes.2 The subcellular distribution of the α-subunits is unknown. However, in cardiac myocytes actin capping protein composed of the β1-subunit is confined to the Z-line, whereas β2-containing actin capping protein localizes to the intercalated disc and cell periphery.3
The function of CP-β1 is to cap the barbed ends of the thin filaments and anchor them to the Z-line, a role that is critical for normal muscle development. CP-β1 assembles at Z-lines before the formation of actin filaments in developing myotubes.4 Disruption of actin–CP-β1 interaction impairs myofibrillogenesis5 and produces gross myofibrillar disarray.2 Although the importance of CP-β1 in the development of normal muscle architecture is well established, its role in muscle function has not been investigated. Transgenic mice in which the levels of CP-β1 are specifically and significantly diminished in the heart develop cardiac hypertrophy and die between 7 and 26 days postnatal.2 The high lethality of this model indicates that CP-β1 is critical for normal cardiac performance. Thus, our first aim was to understand how the sarcomeric disruption associated with the decrease in CP-β1 alters myofilament function.
Disrupting the interaction of CP-β1 with the Z-line may affect signaling through the protein kinase C (PKC) cascade by interrupting binding of PKC with its Z-line–associated anchoring proteins. PKC is a family of serine-threonine kinases that translocate to isoform-specific, subcellular locations on activation. Anchoring proteins, termed receptors for activated C-kinase (RACK), fix active PKC near its target to facilitate phosphorylation. Modulation of the interaction between PKC-isozymes and their respective RACKs alters the effectiveness of PKC.6–8⇓⇓ On activation, PKC-ε translocates and binds to cardiac myofilaments.9,10⇓ Confocal microscopy has revealed that most active PKC-ε is localized near the Z-lines,11,10⇓ an area that has been found to contain a putative PKC adaptor protein.12 Prevention of normal Z-line development may hinder the interaction of PKC-RACK that is necessary for modulation of myofilament activity. Our second aim was to determine if the Z-line disruption associated with a reduction in CP-β1 attenuates PKC signaling in murine myocardium.
Our results indicate that the downregulation of CP-β1, or associated changes in CP-β2 or tropomodulin, increases myofilament Ca2+ sensitivity, without affecting maximum tension or actomyosin MgATPase activity. Moreover, we found that the reduction in CP-β1 abolished PKC-dependent regulation of myofilament function. Despite the absence of functional changes after PKC activation, PKC-dependent phosphorylation of troponin I was observed in myocardium deficient in CP-β1, indicating an uncoupling of myofilament phosphorylation and functional control.
Materials and Methods
Generation of a Mouse Model
Single actin capping protein subunits are unstable and nonfunctioning.13–15⇓⇓ Overexpression of one isoform replaces the endogenous isoform.2 To decrease cardiac CP-β1, we generated a mouse model in which CP-β2 was overexpressed, lowering the level of the Z-line–associated CP-β1 in the functional α/β heterodimer. Previous work has shown that this approach reduces the amount of CP-β1 associated with the Z-line without causing CP-β2 to localize to the Z-discs.2 Transgenic mice that are deficient in the Z-line–associated CP-β1 are henceforth referred to as “CapZ mice.” CapZ mice were homozygous for the transgene allele.
Isolation of Papillary Fiber Bundles
Hearts were excised from 3- to 6-month-old mice that had been anesthetized with ether. Hearts were rinsed free of blood in ice-cold saline (0.9% NaCl), and left ventricular papillary muscles quickly dissected. Some papillary muscles were treated with receptor agonists/antagonists, whereas others were transferred to a dish containing ice-cold high relaxing solution (HR) and cut into fiber bundles approximately 100 μm in diameter. Triton X-100 (1% v/v final concentration; Sigma) was added to permeabilize the fiber bundles.
Isometric Tension and Actomyosin MgATPase Activity
Measurement of isometric tension and actomyosin MgATPase activity was done according to the methods of de Tombe and Stienen.16 Sarcomere length was set to 2.3 μm. Only those fibers able to generate greater than 80% of initial tension in their final contraction were kept for analysis.
Ventricular strips were dissected from hearts and treated with receptor agonists/antagonists. After agonists/antagonist treatment, ventricular tissue was homogenized in ice-cold HR containing 1% Triton X-100 v/v and myofibrils isolated according to a modified procedure from Huang et al.10 Sample loading buffer was added and the samples boiled for 5 minutes.
Samples were resolved on sodium-dodecyl sulfate-polyacrylamide gels (SDS-PAGE) using 4% stacking and 8% resolving gels. Proteins were transferred to nitrocellulose membranes and probed with monoclonal antibodies for PKC-ε (1:1000), PKC-δ (1:1000), PKC-α (1:1000) (Santa Cruz Biotechnology), or PKC-β (1:500) (BD Transduction Laboratories). Density of PKC bands was determined using NIH Image software.
Back-Phosphorylation of Myofilament Proteins
Back-phosphorylation was done using a modified protocol from Karczewski et al.17 Ventricular strips were dissected from hearts and treated with receptor agonists/antagonists. Back-phosphorylation was done using recombinant PKC-ε made according to the procedure of Medkova and Cho.18 Reactions were carried out for 75 minutes at 30°C, which was sufficient for maximum back-phosphorylation (data not shown). Proteins were visualized by Coomassie staining to ensure equal loading of gels and exposed overnight in a phosphor screen. Band density of all agonist/antagonist treatment lanes were divided by control density to give a drug-to-control ratio for each isolation.
Tropomodulin and Actin Protein Levels
Control samples from back-phosphorylation studies were used to determine tropomodulin and actin protein levels. Actin was visualized by Coomassie staining and tropomodulin by immunoblotting with an anti-tropomodulin antibody (Dr M.A. Sussman, The Children’s Hospital and Research Foundation, Cincinnati, Ohio). Band density was measured with NIH Image software.
Left ventricular papillary fibers or ventricular strips were placed in oxygenated (95% O2/5% CO2) Krebs-Henseleit solution at room temperature. Fibers and ventricular strips were treated with 10 μmol/L phenylephrine plus 1 μmol/L propranolol (α-adrenergic receptor/PKC activation), 100 nmol/L endothelin-1 (endothelial type A (ETA) receptor/PKC activation), or were untreated (control) for 5 minutes. To inhibit PKC activation, some preparations were pretreated with chelerythrine chloride (2 μmol/L). After treatment, papillary fiber bundles were permeabilized (1% Triton X-100 v/v final concentration).
High relaxing (HR) solution contained 10 mmol/L EGTA, 25 μmol/L CaCl2, 20 mmol/L MOPS, 50 mmol/L KCl, 6.8 mmol/L MgCl2, 12 mmol/L phosphocreatine, 5 mmol/L Na2ATP, 5 mg/mL leupeptin, 12.5 mg/mL pepstatin, and 250 mmol/L PMSF, pH 7.0. Relaxing solution contained 8.37 mmol/L MgCl2, 5.80 mmol/L Na2ATP, 20 mmol/L EGTA, and 42.5 mmol/L potassium propionate, pH 7.1. Preactivating solution contained 7.78 mmol/L MgCl2, 5.80 mmol/L Na2ATP, 0.50 mmol/L EGTA, 19.5 mmol/L HDTA, and 43.6 mmol/L potassium propionate, pH 7.1. Activating solution contained 7.63 mmol/L MgCl2, 5.87 mmol/L Na2ATP, 20 mmol/L Ca2+-EGTA, and 43.6 mmol/L potassium propionate, pH 7.1. Relaxing, preactivating, and activating solutions also contained 900 μmol/L NADH, 100 mmol/L N, N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid, 5 mmol/L sodium azide, 10 mmol/L phospho(enol)pyruvate, 4 mg/mL pyruvate kinase (500 U/mg), 0.24 mg/mL lactate dehydrogenase (870 U/mg), 10 μmol/L oligomycin B, 200 μmol/L P1P5-di(adenosine-5′)pentaphosphate, and 100 μmol/L leupeptin. Sample loading buffer contained 20% glycerol, 2% SDS, 0.025 mg/mL bromophenol blue, 7.1 mmol/L β-mercaptoethanol, and 125 mmol/L Tris-HCl, pH 6.8.
All values are presented as mean±SEM, and values of P<0.05 were the criteria for statistical significance. Data from agonist/antagonist-treated fibers were analyzed using a 1-way ANOVA and post hoc Dunnett’s t test. Basal characteristics were analyzed with a Student’s t test.
All animals were handled in accordance with the guidelines of the Animal Care Committee at the University of Illinois, Chicago.
Mechanoenergetic Characteristics of Left Ventricular Papillary Fiber Bundles From Transgenic Mice With Decreased Expression of CP-β1
To determine if a decreased level of CP-β1 leads to altered cardiac function, we examined the effects of downregulated CP-β1 expression on Ca2+ activation of the myofilaments and the ratio of force generation to actomyosin MgATPase rate. Data in Figure 1 compare the relations between (A) isometric tension and (B) actomyosin MgATPase activity and free [Ca2+] for wild-type controls and transgenic myofilaments. Compared with wild-type, papillary fiber bundles from CapZ mice demonstrated a significant increase in Ca2+ sensitivity for both parameters, as is evidenced by the leftward shift in the curves and decreased EC50 values (Table 1). There were no significant changes in the slope (ie, Hill coefficient) of either curve. Isometric tension and actomyosin MgATPase activity at maximally activating Ca2+ concentration were not different between wild-type or CapZ papillary fiber bundles. Tension cost, the relation between the rate of ATP consumption and level of steady-state tension development, was unaffected with the reduction of CP-β1 (Table 1).
Effects of CP-β1 Reduction on PKC-Mediated Changes in Myofilament Activity
In a second set of experiments, we tested whether the alterations associated with a reduction in CP-β1 modified the cellular response to activation of the PKC pathway. Our approach was to activate PKC in wild-type and CapZ mice, and to examine the effects on myofilament activation and ATP consumption. Stimulation of PKC-coupled α-adrenergic or ETA receptors decreased maximum isometric tension and actomyosin MgATPase activity in wild-type fiber bundles, without altering myofilament Ca2+ sensitivity (Figure 2; Table 2). Tension cost was not affected by any agonist/antagonist treatment in wild-type fiber bundles (Figure 3).
In contrast to the case with wild-type controls, PKC activation did not alter activity in fiber bundles from CapZ mouse hearts (Figure 4). Neither maximum isometric tension nor maximum actomyosin MgATPase activity was reduced with α-adrenergic or ETA-receptor activation (Table 2). Tension cost was not affected by any of the agonist/antagonist treatment in CapZ mouse fiber bundles (Table 2).
Immunoblot Analysis of PKC Activation
To determine if the translocation of PKC isoforms to the myofilaments was affected by CP-β1 downregulation, we examined the myofilament association of PKC-α, -β, -δ, and -ε after agonist/antagonist treatment by Western blot analysis (Figure 5). There was an increase in PKC-ε protein levels in the myofilament fraction after α-adrenergic and ETA-receptor stimulation of wild-type myocardium. By contrast, the same agonists/antagonists decreased PKC-ε protein levels in the myofilament fraction of CapZ murine myocardium. Both α-adrenergic and ETA-receptor activation decreased myofilament-associated PKC-β in wild-type ventricular tissue. Myofilament-associated PKC-β was undetectable in 4 out of 6 control CapZ mouse hearts. Endothelin increased the amount of PKC-δ protein in the myofilament fraction of wild-type hearts, whereas phenylephrine plus propranolol had no effect. Myofilament-associated PKC-δ was decreased in transgenic hearts after α-adrenergic or ETA-receptor activation. α-Adrenergic and ETA-receptor stimulation decreased the amount of PKC-α in the myofilament fraction of both transgenic and wild-type ventricles.
Phosphorylation of Myofilament Proteins
Phosphorylation levels of myofilament proteins were determined using a back-phosphorylation assay. As such, lighter bands reflected relatively low incorporation of 32P and indicate higher levels of phosphorylation. α-Adrenergic and ETA-receptor stimulation increased the phosphorylation of troponin I in both wild-type and transgenic hearts. Endothelin also increased myosin-binding C-protein and troponin T phosphorylation in CapZ mouse hearts. The PKC inhibitor chelerythrine chloride abolished endothelin and α-adrenergic receptor–dependent changes in myofilament protein phosphorylation of both wild-type and transgenic mice without altering basal phosphorylation levels (data not shown). Basal phosphorylation levels of myosin binding C-protein, troponin T, and troponin I were higher in CapZ control myocardium as compared with wild-type controls.
Actin and Tropomodulin Protein Levels
Actin protein levels were not different between wild-type and CapZ hearts (data not shown). The expression of tropomodulin was 15±6% higher in CapZ transgenic myocardium, compared with wild-type (data not shown). Because tropomodulin binds the pointed ends of both cytoskeletal and sarcomeric actin filaments, we are uncertain if these results reflect changes in the capping of cytoskeletal or sarcomeric actin or both.
Our results provide the first evidence that a reduction in the expression of CP-β1 increases myofilament Ca2+ sensitivity and disrupts PKC-dependent myofilament regulation. These data provide new insights into the functional significance of CP-β1 and support the hypothesis that a localized region at the thin filament–Z-line interface is critical for the transmission of signals in the PKC pathway.
Previous studies2,5⇓ reported that reduced CP-β1 disrupts sarcomere assembly, leading to cardiac hypertrophy and juvenile lethality. Given the severe nature of this defect some functional impairment would be expected to be manifest along with the architectural disarray. The present study, however, found no overtly detrimental mechanical or energetic characteristics in CapZ mice. One explanation for the apparent discrepancy between the structural and functional data may be the different founder lines used in each study. Hart and Cooper2 characterized two transgenic lines (TG-wt β2 No. 2 and TG-wt β2 No. 3) in which CP-β1 was decreased by 37% and 64%, respectively, as compared to wild-type levels. By contrast, our study used mice derived from a line (TG-wt β2 No. 1)2 in which CP-β1 levels were decreased by 7%. The line with moderate overexpression produced a less severe phenotype and modest functional changes. Evidence in support of this hypothesis includes the relative viability of each founder line. Whereas Hart and Cooper2 reported a 100% death rate by day 26 postnatal, the transgenic line of the current study shows no increase in mortality (M.C. Hart and J.A. Cooper, unpublished observations, 2002). The different levels of transgene expression in each line correlates with the severity of phenotype: lines with higher levels of β2-overexpression had a more severe phenotype; the line with the lowest level of expression displayed a modest phenotype. This correlation supports the assumption that the phenotype is due to CP-β2 overexpression and not a secondary mutation.
Single subunits of CP are unstable and show little or no function in vitro and in vivo.13 Therefore, even though the overexpression of CP-β2 produces a 2-fold increase in the level of β2-subunits, free β2-subunits are not expected to have any biological function in the cardiomyocyte. Immunofluorescence studies done by Hart and Cooper2 showed no significant changes in the amount of CP-β2 localized at the cell periphery and intercalated discs. Thus, overexpression of β2-subunits reduces CP-β1 incorporation at the Z-line, without affecting CP elsewhere in the cell. We cannot exclude with absolute certainty the hypothesis that free β2-subunits have an effect on cardiomyocyte function on the basis of some alternative mechanism; however, we are aware of no data that support the existence of such a mechanism.
Data from the present study demonstrate that downregulation of CP-β1 was associated with an increase in Ca2+ sensitivity of both myofilament isometric tension and actomyosin MgATPase activity with no change in maximum isometric tension or actomyosin MgATPase activity. The mechanism underlying the increase in myofilament Ca2+ sensitivity has not been definitively identified. One possible explanation involves the disruption of PKC-dependent myofilament regulation in these transgenic mice. The inability of PKC to effectively control myofilament activation may remove a regulator of Ca2+ sensitivity, thereby allowing the myofilaments to function with increased sensitivity to Ca2+.
In our present study, the stimulation of α-adrenergic or endothelial receptors did not alter myofilament Ca2+ sensitivity. Others have found changes in myofilament Ca2+ sensitivity after PKC activation.19,20⇓ One explanation for these discrepancies may be the presence of multiple PKC isoforms in cardiac muscle. The stimulation of membrane receptors can activate a variety of PKC isozymes, each one of which may induce distinct effects. Even when identical PKC agonists are used, variations in treatment time can lead to different PKC isoform activation profiles with assorted temporal patterns.21
An important and novel finding of our study was that stimulation of PKC-coupled α-adrenergic or ETA receptors in CapZ mice had no effect on myofilament activity. In contrast, α-adrenergic or ETA receptor activation in wild-type preparations decreased both maximum tension and maximum actomyosin MgATPase activity.22 Mochly-Rosen23 has proposed that anchoring proteins for PKC (called RACKs) are essential for the mediation of PKC-dependent effects. On activation, PKC-ε translocates to the myofilaments, producing a pattern consistent with Z-line binding.9–11⇓⇓ Downregulation of CP-β1 disrupts normal Z-line formation, resulting in shortened or even absent Z-lines.2 Without normal Z-line structure, RACK binding to Z-lines or Z-line–associated proteins may be disrupted, diminishing the ability of PKC to translocate near myofilament substrates. We demonstrate here for the first time an important role for cardiac actin capping protein in the association of PKC with cardiac myofilaments.
Whereas the downregulation of cardiac actin capping protein did not prevent the association of PKC-ε, -α, or -δ with the myofilament subcellular fraction, it did correlate with alterations in the amount of activated PKC-ε that translocated to the myofilament fraction, as well as a reduction in basal myofilament-associated PKC-β levels. The reason for the disappearance of PKC-ε from the myofilament fraction on activation of α-adrenergic or ETA receptors is unknown. One possibility is that the downregulation of CP-β1 reduces the binding affinity between PKC-ε and its Z-line anchor, such that it may be displaced by another signaling molecule activated by phenylephrine or endothelin. Among the intracellular signaling molecules downstream of α-adrenergic or ETA receptor activation that bind to cardiac myofilaments are calcineurin24–26⇓⇓ and the focal adhesion kinase-p130Cas complex.27 Whether any or all of these signaling molecules competes with PKC-ε for binding sites on cardiac Z-discs is unknown.
The results of the present study demonstrate an increase in the phosphorylation of troponin I and myosin binding C-protein in both wild-type and transgenic myocardium after α-adrenergic or ETA receptor stimulation. In wild-type hearts, these changes correspond with alterations in myofilament activation, whereas transgenic myocardium demonstrated no correlative functional differences. To our knowledge this uncoupling between changes in myofilament phosphorylation levels and function has not been reported before. We suggest two possible mechanisms that may account for this phenomenon. First, long-range changes in actin structure are induced by a number of actin binding proteins including actin capping protein.28–30⇓⇓ Changes in actin structure have been shown to modify the interaction between actin-troponin and/or actin-tropomyosin31 as well as myofilament function.31–33⇓⇓ Saeki and Wakabayashi32 have suggested that the changes in myofilament function associated with alterations in actin conformation may not be due to the structural modifications in actin per se, but rather changes in the interaction between actin and troponin-tropomyosin. Such changes in actin-troponin-tropomyosin interaction might reduce the effectiveness through which the phosphorylation of troponin regulates myofilament activity, to the extent where troponin phosphorylation causes no discernible function differences.
A second mechanism involves the ordered phosphorylation of myofilament proteins. It has previously been reported that the PKA-dependent reduction in myofilament Ca2+ sensitivity requires the phosphorylation of both N-terminal phosphorylation sites on troponin I.34,35⇓ The addition of the second phosphate group to serine 23 in the N-terminus is considerably slower than the phosphorylation of serine 24.34,35⇓ As such, a PKA-dependent increase in troponin I phosphorylation might precede any functional changes if the phosphorylation of serine 23 was significantly slower. Winegrad and colleagues have published a series of studies in which they propose a similar importance of ordered phosphorylation for myosin binding C-protein.36 We suggest that in cardiac CapZ deficient mice, the phosphorylation of troponin I and myosin binding C-protein might be ineffective at controlling myofilament activation because of the inability to phosphorylate the full compliment of residues required for functional regulation.
Intracellular signaling through PKC is a vital mechanism of myocardial regulation. Activation of PKC results in a wide range of effects, including compensated and decompensated myocardial hypertrophy,37,38⇓ as well as the cardioprotection of preconditioning.39,40⇓ Given the key role PKC occupies in the heart, manipulation of PKC activation might serve as a useful target for the management of myocardial dysfunction. However, the diverse nature of PKC requires that such treatment be specifically directed in order to minimize unintended effects. The precise management of PKC activity necessitates a full understanding of how activated PKC interacts with specific substrates. The results of this study demonstrate a crucial role for the Z-line–associated actin capping protein in the transduction of PKC signals to the myofilaments. These novel findings suggest that the Z-line–associated actin capping protein might serve as a useful target in the management of myofilament function by PKC.
This work was supported by NIH research grants RO1-HL52322 (P.P.d.T.), R37 HL 22231 (R.J.S.), PO1-HL62426 (P.P.d.T. and R.J.S.), and R01-GM38542 (J.A.C.). M.P.S. was supported by NIH T32 07692 and F32 HL 10409. W.G.P. is a postdoctoral fellow of the American Heart Association (Midwest Affiliate) and received the 2001 Richard J. Bing Young Investigator Award from the International Society for Heart Research for parts of this work. M.C.H. was supported by an American Heart Association postdoctoral fellowship and was a member of the Lucille P. Markey Pathway of Human Pathophysiology, Washington University School of Medicine.
Original received January 18, 2002; revision received May 22, 2002; accepted May 22, 2002.
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