Cytoskeletal Reorganization Evoked by Rho-associated kinase- and Protein Kinase C-catalyzed Phosphorylation of Cofilin and Heat Shock Protein 27, Respectively, Contributes to Myogenic Constriction of Rat Cerebral Arteries

Cytoskeletal Reorganization Evoked by Rho-associated kinase- and Protein Kinase C-catalyzed Phosphorylation of Cofilin and Heat Shock Protein 27, Respectively, Contributes to Myogenic Constriction of Rat Cerebral Arteries * Received for publication, January 27, 2014, and in revised form, June 3, 2014 Published, JBC Papers in Press, June 9, 2014, DOI 10.1074/jbc.M114.553743 Alejandro Moreno-Domínguez ‡1 , Ahmed F. El-Yazbi ‡1,2 , Hai-Lei Zhu ‡ , Olaia Colinas ‡ ,X.Zoe¨ Zhong ‡ , Emma J. Walsh ‡ , Dylan M. Cole ‡ , Gary J. Kargacin ‡ , Michael P. Walsh §3 , and William C. Cole ‡4 From the Smooth Muscle Research Group, ‡ Departments of Physiology & Pharmacology and § Biochemistry & Molecular Biology, Libin Cardiovascular Institute and Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada Background: The myogenic response of cerebral arteries to intravascular pressure regulates blood flow to the brain. Results: Pressurization reduced smooth muscle G-actin and increased phospho-cofilin and -HSP27 content by a mechanism blocked by ROK or PKC inhibitors. Conclusion: ROK- and PKC-mediated control of cofilin and HSP27 contributes to actin polymerization in myogenic constriction. Significance: Knowledge of cytoskeletal dynamics is crucial for understanding myogenic control of cerebral arterial diameter. Our understanding of the molecular events contributing to myogenic control of diameter in cerebral resistance arteries in response to changes in intravascular pressure, a fundamental mechanism regulating blood flow to the brain, is incomplete. Myosin light chain kinase and phosphatase activities are known to be increased and decreased, respectively, to augment phosphorylation of the 20-kDa regulatory light chain subunits (LC 20 ) of myosin II, which permits cross-bridge cycling and force development. Here, we assessed the contribution of dynamic reorganization of the actin cytoskeleton and thin filament regulation to the myogenic response and serotonin-evoked constriction of pressurized rat middle cerebral arteries. Arterial diameter and the levels of phosphorylated LC 20 , calponin, caldesmon, cofilin, and HSP27, as well as G-actin content, were determined. A decline in G-actin content was observed following pressurization from 10 mm Hg to between 40 and 120 mm Hg and in three conditions in which myogenic or agonist-evoked constriction occurred in the absence of a detectable change in LC 20 phosphorylation. No changes in thin filament protein phosphorylation were evident. Pressurization reduced G-actin content and elevated the levels of cofilin and HSP27 phosphorylation. Inhibitors of Rho-associated kinase and PKC prevented the decline in G-actin; reduced cofilin and HSP27 phosphoprotein content, respectively; and blocked the myogenic response. Furthermore, phosphorylation modulators of HSP27 and cofilin induced significant changes in arterial diameter and G-actin content of myogenically active arteries. Taken together, our findings suggest that dynamic reorganization of the cytoskeleton involving increased actin polymerization in response to Rho-associated kinase and PKC signaling contributes significantly to force generation in myogenic constriction of cerebral resistance arteries. Blood flow to the brain is regulated through the interplay of multiple mechanisms that affect contractile force generation by vascular smooth muscle cells (VSMCs) 5 within the walls of cerebral resistance arteries and arterioles (1–4). Flow can be matched to metabolic demand under dynamic physiological conditions because these vessels develop contractile tone in response to intravascular pressure, referred to as the myogenic response (2). Myogenic tone development allows resistance vessels to achieve a state of partial constriction from which they can dilate or further constrict to alter flow in response to factors in the blood or released from surrounding cells, such as endothelial cells, neurons, and astrocytes (1–3). Our understanding of the molecular basis of myogenic constriction and how it is regulated by these vasoactive factors is incomplete. A core concept of smooth muscle contraction is that crossbridge cycling and force generation are a consequence of thick *This work was supported by Canadian Institutes of Health Research Grants MOP-97988 and MOP-13505; fellowship awards from the Kertland Family Fellowship Fund & Andrew Family Professorship (to A. M.-D.), Alberta Heritage Foundation for Medical Research/Canadian Institutes of Health Research (to A. F. E.), the Spanish Ministry of Education (to O. C.), and Alberta Innovates-Health Solutions (to H.-L. Z.); and a Studentship award from Alberta Innovates-Health Solutions (to X. Z. Z.). 1 These authors contributed equally to this work. 2 Lecturer of Pharmacology at the Faculty of Pharmacy at Alexandria University. 3 Alberta Heritage Foundation for Medical Research Scientist and recipient of a Canada Research Chair (Tier 1) in Vascular Smooth Muscle Research. 4 Andrew Family Professor in Cardiovascular Research. To whom correspondence should be addressed: Smooth Muscle Research Group, Dept. of Physiology and Pharmacology, Libin Cardiovascular Inst. and Hotchkiss Brain Inst., Faculty of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, AB T2N 4N1, Canada. Tel.: 403-220-8885; Fax: 403-270-2211; E-mail: [email protected]. 5 The abbreviations used are: VSMC, vascular smooth muscle cell; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; LC 20 , 20-kDa myosin regulatory light chain subunit; MYPT1, myosin light chain phosphatase targeting subunit 1; RMCA, rat middle cerebral artery; ROK, Rhoassociated kinase; 5-HT, serotonin; HSP, heat shock protein; KRIBB3, 5-(5- ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl)isoxazole; CyA, cyclosporin A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 30, pp. 20939–20952, July 25, 2014 © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. JULY 25, 2014•VOLUME 289•NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 20939 This is an Open Access article under the CC BY license. filament regulation involving phosphorylation of 20-kDa myosin regulatory light chains (LC 20 ), with the level of phospho- LC 20 dependent on the balance of myosin light chain kinase (MLCK) and phosphatase (MLCP) activities (5–8). Activation of actomyosin MgATPase activity and cross-bridge cycling in VSMCs is initiated by phosphorylation of LC 20 at Ser-19 by MLCK. MLCK activity is (Ca 2⫹ ) 4 -calmodulin-dependent, and stimulated by a rise in cytosolic free Ca 2⫹ concentration ([Ca 2⫹ ] i ) caused by membrane potential (E m ) depolarization and Ca 2⫹ influx, or release from internal sarcoplasmic reticulum Ca 2⫹ stores (5, 9). In contrast, LC 20 dephosphorylation by MLCP is inhibited by Rho-associated kinase (ROK)-mediated phosphorylation of the MLCP targeting subunit MYPT1 (10– 12) and PKC-mediated phosphorylation of the 17-kDa PKC- potentiated protein phosphatase 1 inhibitor protein, CPI-17 (13). Appropriate regulation of MLCK and MLCP activities is necessary for the myogenic response. Myogenic constriction is accompanied by: (i) E m depolarization leading to Ca 2⫹ influx through voltage-gated Ca 2⫹ channels (14), increased Ca 2⫹ wave activity (i.e. increased frequency and number of cells exhibiting waves (15), but see (16)), and increased LC 20 phosphorylation (17–19) that is suppressed by the MLCK inhibitor, ML-7 (17); as well as (ii) increased inhibitory MYPT1-T855 phosphorylation that is suppressed by blockers of ROK activity, including Y27632 and H1152, with an associated loss of myogenic constriction (18, 19). Although inhibition of PKC suppresses myogenic constriction (20, 21), no change in CPI-17 phosphorylation was detected, excluding a role for PKC-medi- ated suppression of MLCP activity (18, 19). Although necessary for myogenic constriction, accumulating evidence indicates that mechanisms of MLCK and MLCP regulation are not sufficient (22). Additional mechanisms modulating force development include thin filament regulation (23) and dynamic reorganization of the actin cytoskeleton (24, 25). Calponin and caldesmon are thin filament-associated proteins that regulate smooth muscle contractility by directly inhibiting cross-bridge cycling (23, 26, 27). Phosphorylation of calponin reduces its inhibitory effect on cross-bridge cycling (28) and may account for slow cross-bridge cycling and contraction of smooth muscle cells in the absence of extracellular Ca 2⫹ (29) and when LC 20 phosphorylation is precluded by site-directed mutagenesis (30). Phosphorylation of caldesmon has similarly been linked to force generation in the absence of extracellular Ca 2⫹ (31) and increased LC 20 phosphorylation (32). Whether phosphorylation of calponin and/or caldesmon contributes to the myogenic response is unknown. Several lines of evidence indicate that the cytoskeleton of smooth muscle cells is not a static structure; rather it is dynamically remodeled during contraction and relaxation (22, 24, 25, 33). Evidence of increased actin polymerization in the myogenic response was obtained for rat cerebral and tail arterial preparations (19, 34–37). Also, the myogenic response was affected by compounds that disrupt (cytochalasin D), prevent (latrunculin A or B), or enhance (jasplakinolide) actin polymerization (19, 34–38). Actin filaments of the contractile apparatus are believed to be anchored to the cytoplasmic tails of integrins by a complex of membrane adhesion proteins and to each other at cytosolic dense bodies within the interior of VSMCs (25). Integrins, membrane adhesion complexes, and dense bodies are connected to each other and reinforced by the cortical actin cytoskeleton, and together these elements are thought to distribute and transmit force generated by the contractile apparatus over the cell membrane and to the extracellular matrix. Dynamic reorganization of the actin cytoskeleton during smooth muscle contraction is thought to involve expansion of adhesion protein complexes, severing of existing filaments to provide nucleation sites, and de novo actin polymerization of globular to filamentous actin (G- and F-actin, respectively) within the cortical actin network beneath the cell membrane (25, 39). This is postulated to strengthen the cytoskeleton and enhance force transmission from the contractile apparatus to the cell membrane and extracellular matrix (24, 25). Actin dynamics evoked by exposure to vasoconstrictor agonists involves a spatially restricted pool of nonmuscle ␥ - and ␤ -actin within the cell cortex and at focal adhesions and dense bodies, but not smooth muscle ␣ -actin that interacts with tropomyosin and forms the contractile actin filaments within the core of VSMCs (40, 41). Roles for additional cytoskeletal and membrane adhesion proteins in smooth muscle contraction have also been identified, including proteins such as talin that serve a structural role, and others that facilitate actin dynamics, such as the actin nucleation initiating factor, N-WASp (neuronal Wiskott-Aldrich Syndrome protein), cofilin, HSP20, HSP27, and VASP (vasodilator-stimulated phosphoprotein) (42–47). Here, we employed pressurized rat cerebral arteries to assess the role of thin filament regulation and the contribution and regulation of actin polymerization in the myogenic response. No role for calponin or caldesmon regulation was detected. Force generation caused by actin polymerization was detected independent of a change in LC 20 phosphorylation and crossbridge cycling in the myogenic response and in vasoconstrictor-evoked contraction of myogenic arteries, accounting for ⬃30% of myogenic tone at 120 mm Hg. Evidence supporting a role for two regulators of actin dynamics, cofilin and HSP27, that are known to be regulated by ROK and PKC, respectively, was obtained. EXPERIMENTAL PROCEDURES Ethical Approval—Sprague-Dawley rats (250–275 g; Charles River, Montreal, Canada) were maintained and killed by halothane inhalation and exsanguination according to a protocol approved by the Animal Care Committee of the Faculty of Medicine of the University of Calgary and conforming to the standards of the Canadian Council on Animal Care. A total of 210 rats were employed. Cerebral Arterial Pressure Myography—Rat brains were removed and transferred to ice-cold Krebs’ saline solution containing 120 mMNaCl, 25 mMNaHCO 3 , 4.8 mMKCl, 1.2 mM NaH 2 PO 4 , 1.2 mMMgSO 4 ,11mMglucose, 2.5 mMCaCl 2 (pH 7.4 when aerated with 95% air, 5% CO 2 ). Rat middle cerebral arteries (RMCAs) were dissected free of the surrounding connective tissue and cut into segments of 2–3 mm in length prior to cannulation and mounting in a pressure myograph system (Living Systems, Burlington, VT), as previously described (18). Cytoskeletal Dynamics in Cerebral Arterial Myogenic Response 20940 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 30•JULY 25, 2014 The endothelial layer was disrupted by passing a stream of fine air bubbles through the vessel lumen and confirmed by the loss of vasodilatation to 10 ␮ Mbradykinin. External arterial diameter was measured using edge detection software (IonOptix, Milton, MA). Arteries were equilibrated at 10 mm Hg in warm Krebs’ solution (37 ⫾0.5 °C) for 15 min at the beginning of each experiment prior to an elevation of intraluminal pressure to 60 mm Hg to permit development of myogenic tone within 5–20 min. Arteries were then subjected to two 5-min pressure steps from 20 to 80 mm Hg to evoke a stable level of constriction (vessels exhibiting leaks or a lack of myogenic constriction were discarded). Intraluminal pressure was then set at 10 mm Hg for 10 min prior to the start of each experiment (pressure protocols for individual experiments are given under “Results”). Vessel segments from the same animal were used for control and treatment groups whenever possible to minimize vessel to vessel variability in phosphoprotein and G-actin quantification. Circumferential Wall Stress Calculation—The circumferential wall stress (CWS) was calculated at radius rwithin the arterial wall using the following equation, from Coulson et al. (48), developed for a hollow cylinder subjected to uniform pressure CWS(r)⫽ri 2pi re 2⫺ri 2 冉 1⫹re 2 r2 冊 (Eq. 1) where ris the radius, r i is the internal radius, r e is the external radius, and p i is the internal pressure, respectively. Protein Extraction—Vessels were frozen by rapid transfer to an ice-cold mixture of 10% trichloroacetic acid and 10 mMDTT in acetone prior to washing in acetone containing 10 mMDTT, lyophilization overnight, and storage at ⫺80 °C. Prior to protein extraction, the cannulated ends were removed from each lyophilized vessel segment to avoid inclusion of tissue not subjected to test pressures prior to protein extraction in 60 ␮ lof sample buffer (4% SDS, 100 mMDTT, 10% glycerol, 0.01% bromphenol blue, 60 mMTris-HCl, pH 6.8). Each sample contained material from two to four pooled RMCA segments depending on the protein(s) of interest. Samples were heated at 95 °C for 10 min and rotated overnight at 4 °C prior to gel electrophoresis. Phosphoprotein Western Blotting—A high sensitivity, threestep Western blotting method (18, 49) was used to quantify phosphorylation of LC 20 , calponin (pan-antibody; 1:10,000), caldesmon (anti-S789; 1:250), HSP27 (anti-S82; 1:500), and cofilin (anti-S3; 1:500). Phosphorylated and unphosphorylated LC 20 and calponin were separated by phosphate affinity tag (Phos-tag TM ) SDS-PAGE, transferred to PVDF membranes, and quantified as a percentage of total LC 20 or calponin, respectively (49). Caldesmon, HSP27, and cofilin phosphorylation were detected using phosphoprotein-specific antibodies and normalized to actin content of each sample based on Johnson et al. (18), but the blocking agent was BSA. G-actin Determination—G-actin content was determined for individual RMCA segments as previously described (50). Vessels were rapidly transferred to F-actin stabilization buffer (Cytoskeleton, Denver, CO) containing 50 mMPIPES (pH 6.9), 50 mMKCl, 5 mMMgCl 2 ,5mMEGTA, 5% (v/v) glycerol, 0.1% Nonidet P40, 0.1% Triton X-100, 0.1% Tween 20, 0.1% 2-mer- captoethanol, 0.001% antifoam C and then homogenized in 100 ␮ l of stabilization buffer at room temperature. The homogenate was then centrifuged at 155,000 ⫻gfor1hat22°Cto separate G- and F-actin; 30 ␮ l of the high speed supernatant containing G-actin was carefully removed and added to 30 ␮ lof 2⫻sample buffer. Samples were then heated at 95 °C for 10 min and stored at ⫺20 °C prior to SDS-PAGE and Western blotting using a standard two-step protocol. SDS-PAGE was carried out in 1.5-mm-thick mini-gels (10% acrylamide in the resolving gel with 4.5% acrylamide stacking gel) at 30 mA for 1.5 h in a Mini Protean Cell (Bio-Rad). Following electrophoresis, proteins were transferred to a 0.2- ␮ m nitrocellulose membrane at 100 V for 90 min at 4 °C, in transfer buffer containing 25 mMTris- HCl, 192 mMglycine, and 20% methanol. Blotted membranes were washed in PBS for 5 min, incubated in 0.5% glutaraldehyde in PBS for 15 min to fix proteins on the membrane, and washed (twice for 5 min) with Tris-buffered saline containing Tween 20 (TBST: 20 mMTris-HCl, pH 7.5, 137 mMNaCl, 3 mMKCl, 0.05% Tween 20). Membranes were blocked with 5% nonfat dried milk in TBS and 0.1% Tween 20 (0.1% TBST) for 1 h. After blocking, each membrane was cut at the 35-kDa molecular mass marker; the high molecular mass proteins were incubated with rabbit polyclonal anti-actin (1:1000 dilution), and the low molecular mass proteins were incubated with goat polyclonal anti-SM22 ␣ (1:2000 dilution). Both antibodies were incubated overnight at 4 °C in 1% dry milk in 0.1% TBST. Membranes were washed (four times for 5 min) in 0.1% TBST and incubated for1hin1%drymilk and 0.1% TBST containing anti-rabbit IgG-HRP-conjugated secondary antibody (1:10,000 dilution) or donkey anti-goat IgG-HRP-conjugated secondary antibody (1:5000 dilution), respectively. After incubation with secondary antibody, membranes were washed (five times for 5 min) with TBST and (once for 5 min) with TBS before chemiluminescence signal detection using Amersham Biosciences ECL advanced Western blotting detection kit. The emitted light was detected and quantified with a chemiluminescence imaging analyzer (LAS3000 Mini; Fujifilm Canada, Mississauga, Canada), and images were analyzed with MultiGauge v3.0 software (Fujifilm Canada). Levels of G-actin were normalized to SM22 content, because SM22 is exclusively retained in the supernatant during high speed centrifugation (50) and expressed as a fraction of the level at 10 mm Hg. Chemicals—All chemicals were purchased from Sigma unless indicated otherwise. Latrunculin B and GF109203X were obtained from Enzo Life Sciences (Plymouth Meeting, PA). Tween 20, Coomassie Brilliant Blue R250, TEMED, PVDF, and nitrocellulose membranes were from Bio-Rad. Polyclonal rabbit anti-LC 20 and KRIBB3 (5-(5-ethyl-2-hydroxy-4-me- thoxyphenyl)-4-(4-methoxyphenyl)isoxazole) were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal rabbit antiphospho-caldesmon-S789 was from Upstate (Charlottesville, VA). Polyclonal rabbit anti-phospho-HSP27-S82 was from Cell Signaling (Beverly, MA). Polyclonal goat anti-SM22- ␣ was from Novus Biologicals (Littleton, CO). Polyclonal rabbit anticalponin was prepared as described previously (51). Cyclosporin A, H1152, polyclonal rabbit anti-phospho-cofilin-S3, biotin-conjugated goat anti-rabbit, HRP-conjugated goat antirabbit, and HRP-conjugated donkey anti-goat secondary anti- Cytoskeletal Dynamics in Cerebral Arterial Myogenic Response JULY 25, 2014•VOLUME 289•NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 20941 bodies were from EMD Millipore (Bellerica, MA), and HRP- conjugated streptavidin was from Pierce. Phos-tag TM acrylamide was from NARD Institute Ltd. (Amajasaki City, Japan). Statistical Analysis—All values are presented as means ⫾ S.E., with nvalues indicative of the number of vessels, or in the case of phosphoprotein and G-actin analysis, the number of samples (with two vessels pooled or a single vessel per sample, respectively) studied for each treatment. Statistical difference was determined using paired or unpaired Student’s ttest, analysis of variance, or repeated measures analysis of variance followed by Bonferoni’s post hoc test as required. A pvalue of ⬍ 0.05 was considered to be statistically significant. RESULTS Decreased G-actin Content Associated with Myogenic- or Agonist-evoked Constriction in the Absence of Alterations in LC 20 , Calponin, or Caldesmon Phosphorylation—RMCA constriction under conditions in which a detectable change in the level of LC 20 phosphorylation was not apparent was specifically examined to assess the contribution of thin filament regulatory phosphoproteins and cytoskeletal reorganization to force development. Phospho-LC 20 content of RMCAs pressurized to 100 mm Hg was previously shown to be ⬃50% (18), consistent with the maximal level of 50–55% detected for rat skeletal muscle arterioles pressurized to between 80 and 140 mm Hg (19). Based on these data, the level of phospho-LC 20 was quantified using RMCA segments flash frozen at 10, 80, and 120 mm Hg once vessel diameter had stabilized at the relevant intraluminal pressure (this required 30 s to 3 min in different vessels). Fig. 1A shows a representative recording of arterial diameter during sequential pressure steps from 10 to 80 and 120 mm Hg. Pressure elevation from 80 to 120 mm Hg was associated with a reduction in the diameter of 17 ⫾2.8 ␮ m(n⫽9 vessels; p⬍ 0.05). Fig. 1Bshows a representative Western blot of phosphorylated and unphosphorylated LC 20 at 10, 80, and 120 mm Hg obtained using Phos-tag TM gels, and mean phospho-LC 20 content was expressed as a percentage of total LC 20 at 10, 80, and 120 mm Hg (⫾S.E.; n⫽14, 9, and 7 arterial segments, respectively). No difference in phospho-LC 20 content was observed for segments at 80 and 120 mm Hg, with levels of ⬃50% detected at both pressures (p⬎0.05). Fig. 1 (Cand D) shows representative Western blots and mean (⫾S.E.) levels of calponin and caldesmon phosphorylation at 80 and 120 mm Hg, respectively. No difference in calponin or caldesmon phosphorylation was evident at 80 and 120 mm Hg (n⫽3 per group; p⬎ 0.05 for both). In contrast, a significant reduction in G-actin content was detected. Fig. 1Eshows representative blots of G-actin and SM22 content at 10, 80, and 120 mm Hg, as well as the mean level (⫾S.E.) of G-actin expressed as a fraction of the content at 10 mm Hg for vessels at 10, 80, and 120 mm Hg. Increasing pressure from 10 to 40 mm Hg did not evoke a significant change in G-actin content (data not shown), but a step from 10 to 80 caused G-actin levels to decline by ⬃66%, and a step from 80 to 120 mm Hg caused a further decline in G-actin content of ⬃21% (n⫽8 for 10, 80, and 120 mm Hg, respectively; p⬍0.05). Thus, the increased force generation at 120 compared with 80 mm Hg was associated with a reduction in G-ac- tin content but no change in LC 20 or thin filament regulatory protein phosphorylation. An identical maximal level of LC 20 phosphorylation of ⬃50% was previously detected in RMCAs exposed to 1, 3, or 10 ␮ M 5-HT at 10 mm Hg (52). Based on these data, we quantified the FIGURE1. Myogenicconstriction evoked by pressure elevation from 80 to 120 mm Hg is associated with a decreased G-actin content but does not induce an increase in LC 20 , calponin or caldesmon phosphorylation. A, representative pressure-induced changes in RMCA diameter (left panel) and mean diameter (⫾S.E.; n⫽9 vessels) (right panel) for RMCAs evoked by a step increase in pressure from 80 to 120 mm Hg in normal Krebs’ solution. B, representative Western blot (left panel) and mean level ⫾S.E. of phospho-LC 20 (pLC 20 ;right panel) as a percentage of total LC 20 at 10, 80, and 120 mm Hg (respective nvalues of 14, 9, and 7; one or two arterial segments/sample) with monophosphorylated and unphosphorylated LC 20 separated by Phos-tag TM SDS-PAGE. C, representative Western blot (left panel) and mean level ⫾S.E. of phospho-calponin (pCalp; right panel) as a percentage of total calponin at 80 and 120 mm Hg (n⫽3; one or two arterial segments/sample) with monophosphorylated and unphosphorylated calponin separated by Phos-tag TM SDS-PAGE. D, representative Western blot of phospho-caldesmon (pCald) and corresponding levels of actin in each lane (left) and mean levels ⫾S.E. of normalized (to actin) phosphoprotein content at 120 mm Hg expressed as a fraction of the value at 80 mm Hg (right)(n⫽3; one or two arterial segments/ sample). E, representative Western blot of G-actin and corresponding SM22 content in each lane (left panel) and mean levels ⫾S.E. of G-actin normalized to SM22 at 80 and 120 mm Hg expressed as a fraction of the value at 10 mm Hg (right panel)(n⫽8; one arterial segment per sample). *, significantly different (p⬍0.05) from value at 80 (A)or10mmHg(Band E). **, significantly different (p⬍0.05) from value at 80 mm Hg (E). Cytoskeletal Dynamics in Cerebral Arterial Myogenic Response 20942 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 30•JULY 25, 2014 changes in LC 20 , calponin, and caldesmon phosphoprotein and G-actin content caused by pressurization from 10 to 80 mm Hg in the presence of 5-HT (1 ␮ M), or in the reverse situation, during exposure to 5-HT (1 ␮ M) after stable development of myogenic tone at 80 mm Hg. Fig. 2Ashows representative recordings of arterial diameter for RMCAs treated with 5-HT at 10 mm Hg in the absence or presence of a subsequent pressure step to 80 mm Hg. Notably, the pressure step from 10 to 80 mm Hg after 5-HT treatment evoked myogenic tone development as indicated by the presence of an initial dilation followed by constriction of 25 ⫾5 ␮ m(n⫽6 vessels; p⬍0.05). No difference in LC 20 , calponin, or caldesmon phosphorylation was detected, but a significant 63 ⫾11% decline in the level of G-ac- tin was evident in vessels exposed to 5-HT and then pressurized to 80 from 10 mm Hg (Fig. 2, B(n⫽6 vessels/group) and C–E (n⫽3 vessels/group)). A similar decline in G-actin content in the absence of a change in phosphoprotein content was also associated with the vasoconstriction of RMCAs treated with 5-HT at 80 mm Hg. Fig. 3Ashows representative recordings of RMCA diameter during a step increase in pressure from 10 to 80 mm Hg alone and an identical pressure step followed by 5-HT (1 ␮ M). Although vasoconstriction of ⬃35 ␮ m (215 ⫾8 versus 180 ⫾11 ␮ m; n⫽7 vessels, p⬍0.05) was evident on exposure to 5-HT, no difference in LC 20 , calponin, or caldesmon phosphorylation was detected in the presence compared with the absence of 5-HT at 80 mm Hg (Fig. 3, B(n⫽5 vessels/ group) and CandD(n⫽3 vessels/group); p⬎0.05 in each case). However, the vasoconstriction to 5-HT at 80 mm Hg was associated with a significant decline of 61.5 ⫾5.5% in G-actin content (n⫽4; p⬍0.05). Notably, the values for the final diameter prior to freezing and magnitude of decline in G-actin content at 80 mm Hg in the presence of 5-HT were not different regardless of the order of pressurization and agonist exposure (p⬎0.05). The initial spontaneous development of myogenic tone by RMCAs during equilibration at 60 mm Hg typically requires 5–20 min (Fig. 4A,left panel). Fig. 4Bshows that no detectable difference in the level of LC 20 phosphorylation was apparent when RMCAs were flash frozen prior to tone development (Fig. 4A,right panel) compared with vessels frozen at ⬃20 min when stable myogenic constriction was achieved. In contrast, G-actin content was different before and after tone development at 60 mm Hg (Fig. 4C). Specifically, the level of G-actin detected before tone development was identical to that at 10 mm Hg, but it was ⬃60% lower after stable tone was achieved (p⬍0.05; n⫽ 5 per group). We next determined whether the pressure-evoked decline in G-actin content of RMCAs was sensitive to modulators of the actin cytoskeleton and/or cross-bridge cycling. Treatment with latrunculin B (10 ␮ M) to sequester G-actin did not affect RMCA diameter at 10 mm Hg (data not shown), but it did cause a significant vasodilatation at 80 mm Hg (Fig. 5A). The loss of tone at 80 mm Hg in latrunculin B was not associated with a decline in LC 20 phosphorylation, but a significant ⬃4-fold increase in G-actin content was detected (Fig. 5, Band C). Latrunculin B similarly evoked dilation of vessels treated with 5-HT at 80 mm Hg or pressurized to 80 mm Hg after 5-HT treatment (Fig. 6, Band C), but not in vessels treated with 5-HT at 10 mm Hg (Fig. 6A). Previous work indicated that microcystin (phosphatase inhibitor)-triggered LC 20 phosphorylation in permeabilized rat FIGURE 2. Myogenic constriction at 80 mm Hg in the presence of 5-HT is accompanied by decreased G-actin content, but no change in LC 20 , calponin, or caldesmon phosphorylation. A, representative recordings of RMCA diameter during the application of 5-HT (1 ␮ M) and during a step increase in pressure from 10 to 80 mm Hg following the application of 5-HT. The thick arrow indicates the presence of myogenic constriction in the presence of 5-HT, and thin vertical arrows at the end of each trace indicate the point of flash freezing for biochemical analysis. B, representative Western blot (left panel) and mean level ⫾S.E. of phospho-LC 20 (pLC 20 ;right panel)asa percentage of total LC 20 in 5-HT at 10 mm Hg and at 80 mm Hg in the continued presence of 5-HT (n⫽6; one or two arterial segments/sample) with monophosphorylated and unphosphorylated LC 20 separated by Phos-tag TM SDS-PAGE. C, representative Western blot (left panel) and mean level ⫾S.E. of phospho-calponin (pCalp; right panel) as a percentage of total calponin in 5-HT at 10 mm Hg and at 80 mm Hg in the continued presence of 5-HT (n⫽3; one or two arterial segments/sample) with monophosphorylated and unphosphorylated calponin separated by Phos-tag TM SDS-PAGE. D, representative Western blot of phospho-caldesmon (pCald) and corresponding levels of actin in each lane (left panel) and mean levels ⫾S.E. of normalized (to actin) phosphoprotein content at 80 mm Hg in the presence of 5-HT expressed as a fraction of the value in 5-HT at 10 mm Hg (right panel)(n⫽3; one or two arterial segments/sample). E, representative Western blot of G-ac- tin and corresponding SM22 content in each lane (left panel) and mean levels ⫾S.E. of G-actin normalized to SM22 at 80 mm Hg in the presence of 5-HT expressed as a fraction of the value in 5-HT at 10 mm Hg (right panel)(n⫽3; one arterial segment per sample). *, significantly different (p⬍0.05) from value in 5-HT at 10 mm Hg. Cytoskeletal Dynamics in Cerebral Arterial Myogenic Response JULY 25, 2014•VOLUME 289•NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 20943 mesenteric arteries evoked increased actin polymerization, whereas depolymerization was observed if LC 20 phosphorylation was suppressed with the MLCK inhibitor ML-7, suggesting interplay between the extent of LC 20 phosphorylation and the level of actin polymerization (53). To test this possible mechanism in the myogenic response, we used blebbistatin, which potently inhibits the actin-activated ATPase activity of mammalian smooth muscle myosin II without affecting myosin phosphorylation (54). RMCAs were treated with blebbistatin (20 ␮ M), before or after induction of the myogenic response, and vessel diameter and G-actin content were measured. The addition of blebbistatin after the pressure jump reversed the myogenic response but had no effect on the pressure-induced actin polymerization (Fig. 7A). When added at 10 mm Hg, prior to increasing intraluminal pressure to 120 mm Hg, blebbistatin had no effect on vessel diameter at 10 mm Hg, and the vessel dilated when the pressure was increased to 120 mm Hg in the presence of blebbistatin, i.e. the myogenic response at 120 mm Hg was abolished (Fig. 7B,top panel). Most importantly, as shown in the middle and bottom panels of Fig. 7B, blebbistatin did not affect the pressure-induced actin polymerization. These results suggest that force generation per se was not responsible for the decline in G-actin content; i.e. there was no evidence of interplay between the extent of LC 20 phosphorylation and the level of actin polymerization. Contribution of ROK- and PKC-mediated Cytoskeletal Reorganization Involving Cofilin and HSP27 Phosphorylation in the Myogenic Response of RMCAs—The lack of a direct effect of force generation on G-actin content suggested the possibility that cellular signaling was likely involved in initiating the FIGURE 3. 5-HT-evoked constriction at 80 mm Hg is associated with a decreased G-actin content, but no change in LC 20 , calponin, or caldesmon phosphorylation. A, representative recordings of RMCA diameter during a step increase in pressure from 10 to 80 mm Hg and during a similar increase in pressure followed by 5-HT (1 ␮ M). Vertical arrows at the end of each trace indicate the point of flash freezing for biochemical analysis. B, representative Western blot (left panel) and mean level ⫾S.E. of phospho-LC 20 (pLC 20 ;right panel) as a percentage of total LC 20 at 80 mm Hg ⫾5-HT (n⫽5; one or two arterial segments/sample) with monophosphorylated and unphosphorylated LC 20 separated by Phos-tag TM SDS-PAGE. C, representative Western blot (left panel) and mean level ⫾S.E. of phospho-calponin (pCalp; right panel) as a percentage of total calponin at 80 mm Hg ⫾5-HT (n⫽ 3; one or two arterial segments/sample) with monophosphorylated and unphosphorylated calponin separated by Phos-tag TM SDS-PAGE. D, representative Western blot of phospho-caldesmon (pCald) and corresponding levels of actin in each lane (left panel) and mean levels ⫾S.E. of normalized (to actin) phosphoprotein content in 5-HT at 80 mm Hg expressed as a fraction of the value at 80 mm Hg (right panel)(n⫽3; one or two arterial segments/ sample). E, representative Western blot of G-actin and corresponding SM22 content in each lane (left panel) and mean levels ⫾S.E. of G-actin normalized to SM22 in 5-HT at 80 mm Hg expressed as a fraction of the value at 80 mm Hg (right panel)(n⫽4; one arterial segment per sample). *, significantly different (p⬍0.05) from value at 80 mm Hg. FIGURE 4. Spontaneous development of myogenic tone during equilibration is associated with a decrease in G-actin content, but no change in LC 20 phosphorylation. A, representative recordings of RMCA diameter during equilibration at 60 mm Hg showing spontaneous tone development in Krebs’ solution (after tone, AT) and an identical vessel that was flash frozen prior to tone development (before tone, BT). Vertical arrows indicate flash freezing for biochemical analysis. B, representative Western blot (left panel) and mean levels ⫾S.E. of phospho-LC 20 (pLC 20 ;right panel) as a percentage of total LC 20 at 10, 60 (AT), and 60 (BT)mmHg(n⫽4; one or two arterial segments/sample) with monophosphorylated and unphosphorylated LC 20 separated by Phos-tag TM SDS-PAGE. C, representative Western blot of G-actin and corresponding SM22 content in each lane (left panel) and mean levels ⫾ S.E. of G-actin normalized to SM22 at 60 (AT) and 60 (BT) mm Hg expressed as a fraction of the value at 10 mm Hg (right panel)(n⫽5; one arterial segment per sample). *, significantly different (p⬍0.05) from value at 10 mm Hg. Cytoskeletal Dynamics in Cerebral Arterial Myogenic Response 20944 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 30•JULY 25, 2014 remodeling mechanism. For this reason, we assessed the contribution of ROK and PKC signaling pathways and two potential regulators of cytoskeletal reorganization, cofilin and HSP27, that are downstream of ROK and PKC, respectively. Fig. 8 indicates the effect of ROK and PKC inhibition by H1152 (0.5 ␮ M) and GF109203X (3 ␮ M) treatment, respectively, on the level of G-actin at 120 mm Hg. RMCAs were pressurized to 120 mm Hg, and H1152 or GF109203X was then applied when stable myogenic tone was observed. Both inhibitors caused a loss of myogenic tone and dilatation, as previously reported (18, 19, 52). G-actin content in the presence of H1152 or GF109203X was significantly elevated compared with that of untreated RMCAs at 120 mm Hg and not different from that detected at 10 mm Hg (p⬎0.05; n⫽4 per group), consistent with the view that ROK and PKC mediate cytoskeletal reorganization in the myogenic response. Two cytoskeleton-associated proteins, cofilin and HSP27, were previously identified as potential mediators of ROK- and PKC-de- pendent control of actin polymerization in smooth muscle contraction (44, 55, 56), but their role in the myogenic response of resistance arteries has not been assessed. Fig. 9 shows the pressuredependent changes in, and the effects of ROK and PKC inhibition on, the levels of cofilin-S3 and HSP27-S82 phosphorylation associated with myogenic constriction at 120 mm Hg (n⫽6 per group). Pressurization to 120 from 10 mm Hg was associated with ⬃2.7- and ⬃2.5-fold increased levels of phospho-HSP27-S82 and phospho-cofilin-S3, respectively. GF109203X suppressed the pressure-dependent increase in HSP27-S82 phosphorylation at 120 mm Hg to a level similar to that in untreated vessels at 10 mm Hg (Fig. 9, Aand B). However, GF109203X did not affect phosphocofilin-S3 content (Fig. 9, Aand C). In contrast, suppression of ROK activity with H1152 blocked the pressure-dependent increase in cofilin-S3, but not HSP27-S82 phosphorylation at 120 FIGURE 5. Pressure-evoked reduction in G-actin content is blocked by latrunculin B. A, representative recording of RMCA diameter (left panel) and mean diameter (⫾S.E.; n⫽6; right panel) during a step increase in pressure from 10 to 80 mm Hg followed by sequential treatment with latrunculin B (LatB) (10 ␮ M) and zero extracellular Ca 2⫹ solution (0 Ca 2⫹ )at80mmHg.B, representative Western blot (left panel) and mean level ⫾S.E. of phospho- LC 20 (pLC 20 ;right panel) as a percentage of total LC 20 at 80 mm Hg ⫾latrunculin B (n⫽3; one or two arterial segments/sample) with monophosphorylated and unphosphorylated LC 20 separated by Phos-tag TM SDS-PAGE. C, representative Western blot of G-actin and corresponding SM22 content in each lane (left panel) and mean levels ⫾S.E. of G-actin normalized to SM22 at 80 mm Hg in latrunculin B expressed as a fraction of the value at 80 mm Hg (right panel)(n⫽4; one vessel per sample). *, significantly different (p⬍0.05) from value at 80 mm Hg (Aand C). FIGURE 6. Sequestration of G-actin with latrunculin B is accompanied by fulldilation ofRMCAs treatedwith 5-HTat 80 mm Hg or pressurized to80 mm Hg after 5-HT treatment. A, representative recording of RMCA diameter (left panel) during sequential exposure to 5-HT (1 ␮ M), latrunculin B (LatB) (10 ␮ M), and zero extracellular Ca 2⫹ solution (0 Ca 2⫹ ) at 10 mm Hg and mean diameter (⫾S.E.; n⫽6; right panel) in 5-HT at 10 mm Hg ⫾latrunculin B. B, representative recording of RMCA diameter (left) during a step increase in pressure from 10 to 80 mm Hg followed by sequential treatment with 5-HT (1 ␮ M), latrunculin B (LatB) (10 ␮ M), and zero extracellular Ca 2⫹ solution (0 Ca 2⫹ ) at 80 mm Hg and mean diameter (⫾S.E.; n⫽7; right panel) in 5-HT at 80 mm Hg ⫾latrunculin B. C, representative recording of RMCA diameter (left panel) during treatment with 5-HT followed by a step increase in pressure from 10 to 80 mm Hg, latrunculin B, and zero extracellular Ca 2⫹ solution in the continued presence of 5-HT and mean diameter (⫾S.E.; n⫽7; right panel) in 5-HT at 80 mm Hg ⫾latrunculin B. *, significantly different (p⬍0.05) from the value in the absence of latrunculin B. Cytoskeletal Dynamics in Cerebral Arterial Myogenic Response JULY 25, 2014•VOLUME 289•NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 20945 mm Hg. The level of phospho-cofilin-S3 in H1152 was similar to that in untreated vessels at 10 mm Hg (Fig. 9, Aand C). The present finding of increased cofilin phosphorylation in the myogenic response differs from previous reports showing decreased levels of phospho-cofilin in tracheal and conduit arterial smooth muscle treated with constrictor agonists (44, 56). For this reason, we performed an additional set of experiments in which RMCAs were exposed to 5-HT (1 ␮ M) at 10 mm Hg. The level of phospho-cofilin was increased by 2.4 ⫾0.4-fold in 5-HT compared with untreated vessels (n⫽6 in each group; p⬍0.05; Fig. 10). We also tested the effects of KRIBB3 (10 ␮ M), which inhibits PKC-dependent HSP27 phosphorylation, and cyclosporin A (CyA) (10 ␮ M), a phosphatase 2B (type 2B protein serine/threonine phosphatase (calcineurin)) inhibitor. Fig. 11 shows that KRIBB3 suppressed the pressure-de- pendent increase in HSP27-S82 phosphorylation at 60 mm Hg (Fig. 11, Aand B), with no effect on the phospho-cofilin-S3 content (Fig. 11, Aand C). CyA, on the other hand, enhanced significantly FIGURE 8. Decline in G-actin content in the myogenic response is blocked by inhibitors of PKC- and ROK-dependent signaling. A, representative Western blot of G-actin and corresponding SM22 content in each lane. B, mean levels ⫾S.E. of G-actin normalized to SM22 of RMCAs at 120 mm Hg ⫾ H1152 (0.5 ␮ M) or GF109203X (GF; 3 ␮ M) expressed as a fraction of the value at 10 mm Hg (n⫽4 blots with one arterial segment per sample). *, significantly different (p⬍0.05) from value at 10 mm Hg. FIGURE7. Pressure-evokedreduction inG-actin contentis notaffected by blebbistatin treatment. A, representative recording (top panel) of RMCA diameter during treatment with blebbistatin (Blebb) (20 ␮ M) after a pressure step from 10 to 120 mm Hg. Representative Western blot of G-actin and corresponding SM22 content in each lane (middle panel) and mean levels ⫾S.E. of G-actin normalized to SM22 at 120 mm Hg ⫾blebbistatin expressed as a fraction of the value at 10 mm Hg (bottom panel)(n⫽3; one vessel per sample). B, representative recording (top panel) of RMCA diameter during treatment with blebbistatin before a pressure step from 10 to 120 mm Hg. Representative Western blot of G-actin and corresponding SM22 content in each lane (middle panel) and mean levels ⫾S.E. of G-actin normalized to SM22 at 120 mm Hg in the presence of blebbistatin expressed as a fraction of the value at 10 mm Hg (bottom panel)(n⫽3; one vessel per sample). *, significantly different (p⬍0.05) from value at 10 mm Hg. FIGURE 9. Pressure elevation evokes increased HSP27-S82 and cofilin-S3 phosphorylationviaPKC- andROK-dependentsignaling, respectively. A, representative Western blots of phospho-HSP27 and phospho-cofilin and corresponding levels of actin in each lane. Band C, mean levels ⫾S.E. of normalized (to actin) phosphoprotein content of RMCAs at 120 mm Hg ⫾ H1152 (0.5 ␮ M) or GF109203X (GF;3 ␮ M), respectively, expressed as a fraction of the value at 10 mm Hg (Band C)(n⫽6 blots with three arterial segments/ sample). *, significantly different (p⬍0.05) from value at 10 mm Hg. **, significantly different (p⬍0.05) from value at 120 mm Hg. Cytoskeletal Dynamics in Cerebral Arterial Myogenic Response 20946 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 30•JULY 25, 2014 the pressure-dependent increase in cofilin-S3 but had no effect on HSP27-S82 phosphorylation at this pressure. These modulators induced opposite effects on the myogenic response of RMCAs: addition of KRIBB3 (10 ␮ M) elicited a vasodilation of ⬃35 ␮ m (Fig. 12A),whereasCyAtreatment (10 ␮ M)wasassociated with a reduction in diameter of ⬃50 ␮ m (Fig. 12B). Finally, we assessed the direct contribution of cofilin and HSP27 to the cytoskeletal reorganization of RMCAs during the myogenic response by analyzing the effect of these phosphorylation modulators on the G-actin content at 60 mm Hg (Fig. 13). G-actin content in the presence of KRIBB3 was significantly elevated compared with that of untreated RMCAs at 60 mm Hg, but not different from that at 10 mm Hg, whereas treatment with CyA induced a significant decrease in G-actin content compared with both controls at 10 or 60 mm Hg (p⬍0.05; n⫽ 5 per group). DISCUSSION This study examined the role of thin filament regulatory proteins, calponin and caldesmon, and dynamic reorganization of the actin cytoskeleton in the myogenic response and agonistevoked constriction of pressurized rat cerebral arteries. No change in thin filament protein phosphorylation was apparent, but a decline in G-actin content was observed in three situations of steady-state vasoconstriction not associated with a detectable augmentation of LC 20 phosphorylation. A concomitant increase in the levels of cofilin-S3 and HSP27-S82 phosphorylation and a reduction in G-actin content were detected following pressure elevation, and these changes were blocked by inhibitors of ROK and PKC signaling. Blockade of HSP27 phosphorylation reversed the myogenic response and actin polymerization, whereas enhancement of cofilin phosphorylation induced further vasoconstriction and actin polymerization. Based on calculated levels of circumferential wall stress, we conclude that the fractional contribution of cytoskeletal reorganization to myogenic force development may be substantial, accounting for ⬃30% of maximal force generated at 120 mm Hg that is capable of effectively opposing the dilating influence of intravascular pressure to permit myogenic control of arterial diameter. It is well accepted that LC 20 phosphorylation is required for the activation of actomyosin MgATPase and cross-bridge cycling and that the level of phosphorylated LC 20 determined by the relative activities of MLCK and MLCP is the principal determinant of force generation in smooth muscle (6, 57, 58). However, it is also apparent that the relationship between LC 20 phosphorylation and force production is variable. MLCK activation, LC 20 phosphorylation, and force are tightly coupled to the elevation in [Ca 2⫹ ] i during the initiation of contraction (59, 60), when changes in shortening velocity and force are proportional to the rise in phospho-LC 20 content (57, 58, 61). How- FIGURE 10. 5-HT-evoked increase in cofilin-S3 phosphorylation in RMCAs at 10 mm Hg. Show are representative Western blots of phospho-cofilin and corresponding levels of actin in each lane (upper panel) and mean levels ⫾S.E. of normalized (to actin) phosphoprotein content of RMCAs at 10 mm Hg ⫾ 5-HT (1 ␮ M) expressed as a fraction of the value at 10 mm Hg (lower panel)(n⫽ 6 blots with three arterial segments/sample). *, significantly different (p⬍ 0.05) from value at 10 mm Hg. FIGURE 11. KRIBB3 and cyclosporin A modulate HSP27-S82 and cofilin-S3 phosphorylation, respectively, in pressurized RMCAs. A, representative Western blots of phospho-HSP27 and phospho-cofilin and corresponding levels of actin in each lane. Band C, mean levels ⫾S.E. of normalized (to actin) phosphoprotein content of RMCAs at 60 mm Hg ⫾KRIBB3 (10 ␮ M) or CyA (10 ␮ M), respectively, expressed as a fraction of the value at 10 mm Hg (Band C) (n⫽5 blots with three arterial segments/sample). *, significantly different (p⬍0.05) from value at 10 mm Hg. **, significantly different (p⬍0.05) from value at 60 mm Hg. Cytoskeletal Dynamics in Cerebral Arterial Myogenic Response JULY 25, 2014•VOLUME 289•NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 20947