Original Article

Pivotal Role of Rho-Associated Kinase 2 in Generating the Intrinsic Circadian Rhythm of Vascular ContractilityClinical Perspective

Toshiro Saito, Mayumi Hirano, Tomomi Ide, Toshihiro Ichiki, Noriyuki Koibuchi, Kenji Sunagawa, Katsuya Hirano
https://doi.org/10.1161/CIRCULATIONAHA.112.135608
Circulation. 2013;127:104-114
Originally published January 2, 2013

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Abstract

Background—The circadian variation in the incidence of cardiovascular events may be attributable to the circadian changes in vascular contractility. The circadian rhythm of vascular contractility is determined by the interplay between the central and peripheral clocks. However, the molecular mechanism of the vascular intrinsic clock that generates the circadian rhythm of vascular contractility still remains largely unknown.

Methods and Results—The agonist-induced phosphorylation of myosin light chain in cultured smooth muscle cells synchronized by dexamethasone pulse treatment exhibited an apparent circadian oscillation, with a 25.4-hour cycle length. The pharmacological inhibition and knockdown of Rho-associated kinase 2 (ROCK2) abolished the circadian rhythm of myosin light chain phosphorylation. The expression and activity of ROCK2 exhibited a circadian rhythm in phase with that of myosin light chain phosphorylation. A clock gene, RORα, activated the promoter of the ROCK2 gene, whereas its knockdown abolished the rhythmic expression of ROCK2. In the mouse aorta, ROCK2 expression exhibited the circadian oscillation, with a peak at Zeitgeber time 0/24 and a nadir at Zeitgeber time 12. The myofilament Ca2+ sensitization induced by GTPγS and U46619, a thromboxane A2 analog, at Zeitgeber time 0/24 was greater than that seen at Zeitgeber time 12. The circadian rhythm of ROCK2 expression and myofilament Ca2+ sensitivity was abolished in staggerer mutant mice, which lack a functional RORα.

Conclusions—ROCK2 plays a pivotal role in generating the intrinsic circadian rhythm of vascular contractility by receiving a cue from RORα. The ROCK2-mediated intrinsic rhythm of vascular contractility may underlie the diurnal variation of the incidence of cardiovascular diseases.

Introduction

The cardiovascular system displays circadian rhythms in some physiological parameters, including blood pressure. In addition, the occurrence of coronary artery events, such as myocardial infarction and angina pectoris, has a circadian variation with a peak during the morning.1,2 These changes may be attributable to the diurnal variation of sympathetic nerve activity, plasma fibrinolytic activity, platelet aggregability, or vascular contractility.3,4 The circadian changes in the vascular tone and reactivity to adrenergic receptor agonists have been well documented.5 However, the precise mechanism underlying the circadian rhythm of vascular contractility has not been fully elucidated.

Editorial see p 19

Clinical Perspective on p 114

In mammals, the diurnal rhythm of biological processes is driven by the biological clock system. The molecular mechanism of the biological clock is based on the transcriptional-translational autoregulatory feedback loops composed of a set of clock genes.6 The transcription factors CLOCK and BMAL1 work as a heterodimer to activate the transcription of the Cry and Per genes.6 Once CRY and PER have reached a critical concentration, they repress the transactivation of CLOCK-BMAL1 and inhibit their own transcription.6 Additional loops involving other clock genes, such as Rev-erb and Ror, interact with and modulate this central loop.6 The circadian oscillation of the biological clock is then dictated by the rhythmic expression of genes, which generate the rhythm of physiological processes. The circadian rhythm of peripheral tissues is determined by the interplay between the central and peripheral clock mechanisms. The central clock influences the rhythm of the peripheral tissues by generating the circadian rhythm of neurohumoral cues.6 Recently, the peripheral clock system has been shown to dominate in regulating the expression of several important genes in different organs under certain conditions.7,8 However, the role of the vascular intrinsic clock in the regulation of vascular contractility and its molecular mechanisms still remain elusive.

The present study thus aimed to clarify whether there is any intrinsic diurnal oscillation in the vascular contractility and, if so, to elucidate the underlying molecular mechanism. The importance of the endothelium in regulating the physiological vascular tone is well recognized; however, the smooth muscle also plays a fundamental role in determining the vascular contractility.9,10 Our investigations were initiated with cultured vascular smooth muscle cells to exclude any influence of the central clock or external cues. Because the phosphorylation of myosin light chain (MLC) plays a central role in the regulation of smooth muscle contraction,11 the existence of circadian oscillation of MLC phosphorylation was first investigated. Accordingly, a clock gene RORα and its regulation of the expression of Rho-associated kinase 2 (ROCK2) were found to generate the circadian oscillation of MLC phosphorylation. The physiological significance of this clock mechanism in the regulation of vascular contractility was then evaluated using staggerer mutant mice, which lack a functional RORα.12 As a result, the present study elucidated, for the first time, a pivotal role of ROCK2 in generating the intrinsic circadian rhythm of vascular contractility.

Methods

An expanded Methods sections is available in the online-only Data Supplement.

Cell Culture and Dexamethasone Pulse Treatment

The porcine coronary artery smooth muscle cells (PCSMCs) and porcine aortic smooth muscle cells were cultured in DMEM containing 10% FBS until semiconfluence for 3 to 4 days before experimental use. The cells at semiconfluence were incubated with 100 nmol/L of dexamethasone in growth medium for 2 hours to induce a synchronized circadian rhythm.

Animals

The original staggerer mutant mice, B6C3Fe-a/a-Rorasg mice (Jackson Laboratory, Bar Harbor, ME), were adjusted by crossing these mice with C57BL/6J inbred mice (CLEA Japan, Tokyo, Japan) for >10 generations.13 The study protocol was approved by the animal care and use committee of Kyushu University. The animals were treated in accordance with the guidelines stipulated by the committee.

Real-Time Polymerase Chain Reaction Analysis

The total RNA was extracted from cultured cells and subjected to a real-time polymerase chain reaction analysis with FastStart SYBR Green Master kit using a LightCycler (Roche, Basel, Switzerland). The expression level of each mRNA was normalized to the level of β-actin obtained from the corresponding reverse transcription product.

Western Blot Analysis

The proteins from cultured smooth muscle cells or mice aortas were extracted in the lysis buffer and subjected to Western blot analysis. The immune complex was detected with an ECL plus detection kit (GE Healthcare, Buckinghamshire, United Kingdom). The light emission was detected and analyzed with a ChemiDoc XRS-J instrument and the computer program Quantity One (BioRad). The density of the immunoreactive band was normalized to that of the corresponding actin band to adjust for any possible variations in sample loading.

Phos-tag SDS-PAGE Analysis of the MLC Phosphorylation

MLC phosphorylation was evaluated with Phos-tag SDS-PAGE analysis, as described previously.14 The level of MLC phosphorylation (PO4 mol/MLC mol) was calculated as follows:

MLC phosphorylation = (P-MLC + PP-MLC × 2)/(MLC + P-MLC + PP-MLC)

where MLC, P-MLC, and PP-MLC indicate the optical density of the non-, mono-, and di-phosphorylated forms of MLC.

Transfection of Small Interfering RNA

Small interfering RNAs (siRNAs) were synthesized with a 3′-UU overhang by Dharmacon (Lafayette, CO). The siRNAs were transfected with the HVJ Envelope Vector Kit GenomONE (Ishihara Sangyo, Osaka, Japan).

Pull-Down Assay for the GTP-Bound Forms of RhoA

The GTP-bound form of RhoA in the cell extract was recovered using a RhoA-binding domain of ROCK2 as a (His)6-tagged pull-down probe and Ni2+-nitrilotriacetic acid resin. Equal amount of the resin eluates and cell lysates were subjected to an immunoblot analysis with an anti-RhoA antibody, as described above.

Luciferase Assay

The activity of the rock2 promoter (−1337 to +74) was evaluated in PCSMCs 32 hours after transfection, using the dual-luciferase reporter assay system (Promega). The activity of firefly luciferase was normalized to that of Renilla luciferase, and the value obtained with the empty vector was considered to be 1.

Tension Measurement in the α-Toxin–Permeabilized Preparations of Mouse Aortas

The aortic rings were permeabilized with 5000 U/mL of staphylococcal α-toxin (Sigma, St. Louis, MO),14 and subjected to the studies. The entire procedure, from euthanization to tension measurement, was completed within 1.5 to 2.0 hours.

Data Analysis

The data are expressed as the mean±SEM of the indicated number of experiments or mice. Either the Steel test or Student t test was used to determine the statistical significance of the differences among groups or between 2 groups, respectively, as indicated in the Figure legends. A value of P<0.05 was considered to be statistically significant.

Results

Circadian Changes in the Response of MLC Phosphorylation in Vascular Smooth Muscle Cells

The circadian rhythms of PCSMCs were synchronized by 2-hour pulse treatment with dexamethasone. The mRNA expression levels of the bmal1, rev-erbα, per1, and cry1 genes exhibited oscillatory changes that were consistent with those described in previous reports, thus indicating the successful synchronization of the circadian rhythm (Figure S1, available in the online-only Data Supplement).6,15

The lysate of PCSMCs exhibited 3 immunoreactive bands on Phos-tag SDS-PAGE (Figure 1A). The lowest band corresponded with the purified unphosphorylated MLC. The middle band corresponded with the purified monophosphorylated MLC, both of which were detected by an antibody specific for P-MLCSer19 (Figure 1A). The uppermost band was detected by an antibody specific for PP-MLCThr18+Ser19 (Figure 1A). Thrombin and endothelin 1 were used as contractile stimuli in the present study, because they have been suggested to play a pathological role in cardiovascular diseases.16,17 Thrombin (1 U/mL) and endothelin 1 (100 nmol/L) transiently increased MLC phosphorylation with a peak at 2 minutes after the simulation (Figure 1B).

Figure 1.
Figure 1.

The evaluation of myosin light chain (MLC) phosphorylation with Phos-tag SDS-PAGE. A, Immunoblot detection of the purified MLC with and without phosphorylation by MLC kinase (MLCK) and the lysates of porcine coronary artery smooth muscle cells (VSMC) with the anti-MLC antibody (total MLC) and the antibodies specific to the monophosphorylated MLC (P-MLCSer19) and diphosphorylated MLC (PP-MLCThr18+Ser19), after Phos-tag SDS-PAGE. The cell lysates were obtained 2 minutes after the stimulation of cells with 1 U/mL of thrombin. B, The time courses of MLC phosphorylation induced by 1 U/mL of thrombin (n=3) and 100 nmol/L of endothelin 1 (n=3) were analyzed with Phos-tag SDS-PAGE, followed by immunoblot detection with the anti-MLC antibody. Representative immunoblot images of β-actin are shown below those for MLC detection. All of the data are expressed as the mean±SEM. #P<0.05 (Student t test).

After the dexamethasone pulse treatment, the levels of MLC phosphorylation both at rest and 2 minutes after thrombin stimulation transiently increased with a peak at 4 to 8 hours and then returned within 20 to 24 hours to levels seen at 0 hours (Figure 2A). The level of MLC phosphorylation recorded 2 minutes after thrombin stimulation, but not the resting level, exhibited an oscillatory change, with peaks at 36 hours and 60 hours and a nadir at 48 hours (Figure 2A). The frequency analysis by an autoregressive model revealed that it had a 25.4-hour cycle length (Figure 2B). An analysis using the antibodies specific for PP-MLCThr18+Ser19 and P-MLCSer19 revealed a similar oscillatory change in the levels of PP-MLCThr18+Ser19 but not P-MLCSer19 (Figure 2C).

Figure 2.
Figure 2.

The circadian changes in the thrombin-induced phosphorylation of myosin light chain (MLC) in porcine coronary artery smooth muscle cells. A and C, The representative immunoblots and summaries of the circadian changes in the MLC phosphorylation as analyzed by Phos-tag SDS-PAGE (A; n=3 for thrombin [+], n=5 for thrombin [−]) and conventional SDS-PAGE (C; n=3). A maximum of 13 samples could be analyzed in 1 gel. The vertical bars between immunoblot images indicate the border between different gels. B, The mean values of MLC phosphorylation in A were subjected to an analysis of frequency with an autoregressive model. The samples were obtained before (A) and 2 minutes after the stimulation with 1 U/mL of thrombin (A and C) at the indicated times after synchronization of the circadian rhythm. The immunoblot detection was performed with the anti-MLC antibody in A or antibodies specific for the MLC monophosphorylated (P-MLCSer19) and the MLC diphosphorylated (PP-MLCThr18+Ser19) in C. In A, immunoblot images of β-actin are shown below those for MLC detection. In C, the level of phosphorylation, as normalized to the level of total MLC, was expressed as a relative value to that obtained at 48 hours. All of the data are expressed as the mean±SEM.

Role of ROCK2 in the Circadian Oscillation of the MLC Phosphorylation in Vascular Smooth Muscle Cells

MLC kinase (MLCK), protein kinase C (PKC), ROCK, and Zipper-interacting kinase (ZIPK) play a major role in the regulation of MLC phosphorylation.10,18 The inhibitors of MLCK (10 µmol/L of ML-9), PKC (1 µmol/L of GF109203), and ROCK (3 µmol/L of Y27632) were applied 10 minutes before and during thrombin stimulation. The treatment with these inhibitors had no effect on the cell viability (Table S1, available in the online-only Data Supplement). All 3 of the inhibitors suppressed the thrombin-induced MLC phosphorylation to a similar level (≈0.5 PO4 mol/MLC mol) at 24 hours after synchronization (Figure 3A). Only ROCK inhibitor abolished the circadian oscillation of MLC phosphorylation (Figure 3A). In line with these observations, the knockdown of ROCK2, but not MLCK or ZIPK, abolished the circadian oscillation of the MLC phosphorylation (Figure 3B). The level of expression of ROCK2, MLCK, and ZIPK after siRNA-mediated knockdown was 55.4 ± 6.3% (n=7), 50.9 ± 5.1% (n=6), and 64.9 ± 4.3% (n=9), respectively, of that seen with the control siRNA (Figure 3B).

Figure 3.
Figure 3.

The involvement of Rho-associated kinase 2 (ROCK2) in the circadian changes in the thrombin-induced myosin light chain (MLC) phosphorylation in porcine coronary artery smooth muscle cells. A, The circadian changes in the MLC phosphorylation induced by 1 U/mL of thrombin in the presence of 10 µmol/L of ML-9, 1 µmol/L of GF109203, and 3 µmol/L of Y27632 (n=3). The cells were treated with inhibitors for 10 minutes before and during thrombin stimulation at the indicated times after synchronization of the circadian rhythm. B, Representative immunoblots of MLC kinase (MLCK), Zipper-interacting kinase (ZIPK), and ROCK2 in the cells transfected with control small interfering RNA (siRNA) or targeted siRNA and the Phos-tag SDS-PAGE analysis of the effects of the knockdown of MLCK, ZIPK, and ROCK2 on the circadian changes in the MLC phosphorylation induced by 1 U/mL of thrombin (n=3–4). The open, single, and double arrowheads indicate non-, mono-, and di-phosphorylated MLC, respectively. All of the data are expressed as the mean±SEM; #P<0.05 (Student t test).

ROCK phosphorylates MYPT1, a noncatalytic subunit of MLC phosphatase, at Thr696 and Thr853 in human MYPT1, whereas other kinases, including ZIPK, phosphorylate MYPT1 at Thr696.18,19 The phosphorylation of Thr853 thus reflects the circadian oscillation of ROCK activity. The phosphorylation of Thr853 induced by thrombin exhibited circadian oscillation with peaks at 36 and 60 hours and a nadir at 48 hours (Figure 4A). This circadian pattern was similar to that seen with the thrombin-induced MLC phosphorylation (Figure 4A). The phosphorylation of Thr696 exhibited no apparent circadian oscillation (Figure 4A).

Figure 4.
Figure 4.

The circadian changes in the activity and the expression of Rho-associated kinase 2 (ROCK2) in porcine coronary artery smooth muscle cells. A, The circadian changes in the level of MYPT1 phosphorylation at residues corresponding with Thr853 (n=5) and Thr696 (n=4) in human MYPT1 obtained 2 minutes after the stimulation with 1 U/mL of thrombin. B, The representative immunoblot detection of ROCK2 and summaries of circadian changes in the level of ROCK2 protein (left; n=5) and mRNA (right; n=4). C, The circadian patterns of the protein levels of MLC kinase (MLCK), protein kinase C (PKC)-∝, and Zipper-interacting kinase (ZIPK; n=3). D, The circadian pattern of the activity of RhoA as evaluated by the level of the GTP-bound form of RhoA in the total RhoA (GTP-RhoA/total RhoA) obtained 2 minutes after the stimulation with 1 U/mL of thrombin (n=4). The values of GTP-RhoA/total RhoA were obtained after normalizing the levels of GTP-RhoA and total RhoA to those of the pull down probe and actin, respectively. The data are expressed as the relative values to those obtained at 48 hours (A and B) and 24 hours (C and D). All of the data are expressed as the mean±SEM; #P<0.05 vs 48 hours (Steel test; A and B, left). ##P<0.01 (Student t test; B, right).

The level of expression of ROCK2 mRNA and protein exhibited circadian changes (Figure 4B). The pattern of the change in the level of ROCK2 protein was similar to that seen with the phosphorylation of MLC and MYPT1 (Thr853). The level of rock2 mRNA oscillated with a phase that had a peak ≈4 hours earlier than the protein expression (Figure 4B). The expression of the ROCK2 protein also exhibited similar circadian changes in porcine aortic smooth muscle cell, with peaks at 32 to 36 hours and 60 hours (Figure S2, available in the online-only Data Supplement). In contrast, the level of the protein expression of MLCK, PKCα, and ZIPK exhibited no apparent circadian oscillation (Figure 4C). The level of the GTP-bound form of RhoA seen after thrombin stimulation also remained constant (Figure 4D).

Circadian Changes in the Phosphorylation of MLC and MYPT1 After Endothelin 1 Stimulation in Vascular Smooth Muscle Cells

The levels of PP-MLCThr18+Ser19 and P-MYPT1Thr853 seen 2 minutes after stimulation with 100 nmol/L of endothelin 1 exhibited a similar circadian change to those seen with thrombin stimulation, with peaks at 36 and 60 hours and a nadir at 48 hours (Figure S3A and S3B, available in the online-only Data Supplement). The levels of P-MLCSer19 and P-MYPT1Thr696 exhibited no apparent circadian changes (Figure S3A and S3B, available in the online-only Data Supplement). These observations seen with endothelin 1 stimulation were similar to those seen with thrombin stimulation.

Role of RORα in the Circadian Changes in the Transcription of ROCK2 in Vascular Smooth Muscle Cells

Among the regulatory elements for the known clock gene products, 2 ROR response elements (ROREs) separated by 102 to 108 nucleotides are preserved in the promoter region of the rock2 gene among various mammalian species (Table S2, available in the online-only Data Supplement). REV-ERBα, REV-ERBβ, RORα, RORβ, and RORγ are all capable of binding to RORE.20,21 The luciferase promoter assay with the −1337 to +74 nucleotide region of the human rock2 gene showed that RORα and RORγ, but not RORβ, REV-ERBα or REV-ERBβ, increased the promoter activity >5-fold the level seen with control vectors in PCSMCs (Figure 5A). The endogenous expression of RORα exhibited circadian changes, with a phase that peaked ≈4 hours earlier than the ROCK2 protein expression (Figure 5B versus Figure 4B), whereas the expression of RORγ showed no apparent circadian oscillation (Figure 5B). The suppression of the RORα expression by siRNA abolished the circadian rhythm of the ROCK2 expression, whereas a control siRNA had no effect (Figure 5C).

Figure 5.
Figure 5.

RORα regulation of the circadian expression of Rho-associated kinase 2 (ROCK2) in porcine coronary artery smooth muscle cells. A, The effects of REV-ERBα, REV-ERBβ, RORα, RORβ, and RORγ on the activity of the human rock2 promoter, as evaluated by the luciferase activity. pCMV6xl4 was used as a vector for REV-ERBα and RORα, whereas pCMV6xl5 was used for REV-ERBβ, RORβ, and RORγ (n=3). The data are expressed as the relative values to those obtained with the corresponding empty vectors. B, The circadian changes of the endogenous expression of RORα and RORγ (n=3). The data were expressed as relative values to those obtained at 48 hours. C, The effects of the knockdown of RORα on the circadian changes in the expression of the ROCK2 protein (n=4). The data are expressed as the relative values to those obtained with a control small interfering RNAs (siRNAs) at 44 hours. All of the data are expressed as the mean±SEM; #P<0.05, ##P<0.01 (Student t test).

RORα-Mediated Regulation of Circadian Changes in ROCK2 Expression and Myofilament Sensitivity to Ca2+ in the Mouse Aorta

The expression of ROCK2 protein (Figure 6A and 6B) but not ROCK1 protein (Figure S4, available in the online-only Data Supplement) in the aortas of wild-type mice kept under a 12-hour dark-light cycle exhibited a circadian oscillation, with a peak at Zeitgeber time (ZT) 0/24, which corresponds to ≈36 hours after dexamethasone pulse treatment in the cultured cells.6,15 The expression of RORα also exhibited a circadian change, with a phase peak 4 hours earlier than that of ROCK2 (Figure 6A and 6B). In the aortas of staggerer mice, which lack a functional RORα, the ROCK2 expression did not show any apparent circadian changes (Figure 6A and 6B). Instead, the level of ROCK2 protein in staggerer mice was similar to that seen at the nadir (ZT12) in the aortas of wild-type mice (Figure 6C). The level of MLCK was also similar between wild-type and staggerer mice at ZT12 (Figure 6C).

Figure 6.
Figure 6.

The absence of the circadian changes in the expression of Rho-associated kinase 2 (ROCK2) in the aortas of staggerer mice. A and B, The representative immunoblots (A) and summaries (B) showing the circadian patterns of the expression of the RORα and ROCK2 proteins in the aortas of wild-type and staggerer (Sg/Sg) mice that were housed under 12-hour light (open bar) and 12-hour dark (closed bar) cycle conditions. The expression levels are expressed as the relative values to those obtained at Zeitgeber time 12 (ZT12). All of the data are expressed as the mean±SEM (n=5–6 for RORα in wild-type; n=6–7 for ROCK2 in wild-type; n=6–7 for ROCK2 in Sg/Sg). #P<0.05, ##P<0.01 (Student t test). C, The expression of ROCK2 and myosin light chain (MLC) kinase (MLCK) proteins in the aortas of wild-type and Sg/Sg mice at ZT12 was directly compared on the same gel.

ROCK plays an important role in modulating the myofilament Ca2+ sensitivity.11,18 The functional relevance of the RORα-mediated circadian expression of ROCK2 was evaluated using the α-toxin–permeabilized preparations, which allowed us to directly evaluate the myofilament Ca2+ sensitivity by examining the contraction at a fixed concentration of Ca2+. GTPγS, a nonhydrolyzable GTP analog, was used to induce the myofilament Ca2+ sensitization.14

First, the Ca2+-dependent contraction was examined by increasing the Ca2+ concentrations in a stepwise manner in the absence of any simulation of Ca2+ sensitivity. The pCa2+-tension relationship of this contraction did not differ between ZT0 and ZT12 in both wild-type and staggerer mice (Figure 7A, GTPγS [−]). However, the pCa2+-tension relationship seen in staggerer mice shifted to the left of that seen in the wild-type mice (Figure 7A, GTPγS [−]). The diurnal change in the Ca2+-induced contraction became prominent in the presence of GTPγS in the wild-type mice (Figure 7A, GTPγS [+]). The aortic ring preparations were first contracted with 0.3, 0.5, or 1.0 µmol/L of Ca2+ and then stimulated with 10 µmol/L of GTPγS (Figure 7B). The level of tension obtained with 10 µmol/L of GTPγS and either 0.3 or 0.5 µmol/L of Ca2+ at ZT0 was significantly greater than that of the corresponding contraction seen at ZT12 in the wild-type mice (Figure 7A, GTPγS [+]). However, the contraction seen with 10 µmol/L of GTPγS and 1 µmol/L of Ca2+, which reached closer to the maximal level of contraction, was similar at ZT0 and ZT12. In contrast, there was no significant difference in the GTPγS-induced contraction between ZT0 and ZT12 in the staggerer mice (Figure 7A, GTPγS [+]).

Figure 7.
Figure 7.

The circadian changes in the Ca2+ sensitivity of the contractile apparatus in the α-toxin–permeabilized preparations of aortas of wild-type and staggerer mice. A, The pCa2+-tension curves of the contractions induced by stepwise increases in the Ca2+ concentrations in the absence of GTPγS (GTPγS [−]) and the contractions induced by 10 µmol/L of GTPγS and 0.3, 0.5, or 1.0 µmol/L of Ca2+ (GTPγS [+]), in the wild-type (n=4–5) and staggerer (Sg/Sg; n=4–5) mice at Zeitgeber time (ZT) 0 and 12. B, Representative recordings showing the experimental protocol used to examine the contractions induced by 10 µmol/L of GTPγS and 0.3, 0.5, or 1.0 µmol/L of Ca2+. The level of tension obtained with different Ca2+ concentrations in either the presence or absence of GTPγS was expressed as a percentage, whereas the levels of tension obtained in Ca2+-free solution and with 10 µmol/L of Ca2+ were assigned a value of 0% and 100%, respectively. All of the data are expressed as the mean±SEM; #P<0.05 vs ZT12.

Circadian Changes in Myofilament Sensitivity to Ca2+and MLC Phosphorylation Induced by U46619 in the Mouse Aorta

The present study examined whether the circadian change in myofilament Ca2+ sensitivity was observed for the receptor-mediated contraction. The Ca2+-sensitizing effect of a thromboxane A2 analog, U46619, was examined at ZT0 and ZT12 in the α-toxin permeabilized aortic ring preparations. The contraction was induced by 1 µmol/L of U46619 in the presence of 10 µmol/L of GTP during the 0.5-µmol/L Ca2+-induced contraction, as reported previously (Figure 8A).22 The extent of the contraction seen at ZT0 was significantly greater than that seen at ZT12 in wild-type mice (Figure 8A). In contrast, there was no significant difference in the U46619-induced contraction between ZT0 and ZT12 in the staggerer mice (Figure 8A).

Figure 8.
Figure 8.

The circadian changes in the Ca2+ sensitivity and myosin light chain (MLC) phosphorylation induced by U46619 in the α-toxin–permeabilized preparations of aortas of wild-type and staggerer mice. Representative recordings and summaries of the contractions (A) and MLC phosphorylation (B) induced by 1 µmol/L of U46619 in the presence of 10 µmol/L of GTP during the 0.5-µmol/L Ca2+-induced contractions in wild-type (n=4) and staggerer (n=4) mice at Zeitgeber time (ZT) 0 and 12. The increase in tension induced by U46619 (Δ) was expressed as a percentage of the tension obtained with 10 µmol/L of Ca2+. MLC phosphorylation was analyzed using Phos-tag SDS-PAGE. All of the data are expressed as the mean±SEM. #P<0.05; NS, not significantly different vs ZT12.

In accordance with the diurnal change in the Ca2+-sensitizing effect of U46619, the U46619-induced MLC phosphorylation also exhibited a circadian change (Figure 8B). MLC phosphorylation was analyzed 5 and 25 minutes after the simulation with 1 µmol/L of U46619 in the presence of 10 µmol/L of GTP during the 0.5-µmol/L Ca2+-induced contractions. The MLC phosphorylation at ZT0 at both 5 and 25 minutes was significantly greater than that seen at ZT12 in the wild-type mice (Figure 8B). In contrast, there was no significant difference in the U46619-induced MLC phosphorylation between ZT0 and ZT12 in the staggerer mice (Figure 8B).

Discussion

The present study elucidated, for the first time, the existence of the vascular clock mechanism intrinsic to the smooth muscle that generates the circadian oscillation of the myofilament Ca2+ sensitivity with a peak at the beginning of the light phase, in a manner independent of external cues. The present study further delineated the signaling pathway from the clock gene to the circadian rhythm of the myofilament Ca2+ sensitivity. The circadian oscillation of the expression of a clock gene, RORα, appears to be translated to the oscillation of ROCK2 transcription, which in turn generates the oscillation of MLC phosphorylation in response to contractile stimuli. ROCK2-mediated potentiation of MLC phosphorylation is an important mechanism underlying myofilament Ca2+ sensitivity.23,24 Myofilament Ca2+ sensitivity plays a critical role in determining the extent of the vascular response to contractile stimuli and vascular contractility.18,24 As a result, the present study suggests ROCK2 to be a key oscillator generating the circadian rhythm of myofilament Ca2+ sensitivity and vascular contractility. The circadian oscillation of the MLC phosphorylation induced by thrombin and endothelin 1 was consistently observed by the 2 different analyses. The Phos-tag SDS-PAGE analysis allowed us to perform a stoichiometric evaluation of MLC phosphorylation for each time point in a self-contained manner, thus minimizing the influence of the variations in the amount of proteins loaded. The analysis with phosphor-specific antibodies further revealed that the circadian oscillation of the agonist-induced MLC phosphorylation was mainly attributed to PP-MLCThr18+Ser19. In the Phos-tag SDS-PAGE analysis, both the resting and stimulated levels of MLC phosphorylation transiently increased during the 4 to 8 hours after the dexamethasone pulse treatment. Pulse treatment with dexamethasone has been shown to induce an early surge of the expression of various genes, including not only clock genes, such as per1, but also c-fos or β-actin, which do not show any oscillation.25,26 Therefore, the transient increase in MLC phosphorylation may be attributable to the expression of not only clock-controlled genes but also unrelated genes that can affect the MLC phosphorylation. As a result, most of the analyses of circadian rhythm were performed starting from 24 hours after the dexamethasone pulse treatment.

Because the circadian oscillation of MLC phosphorylation was observed with both thrombin and endothelin 1, the mechanisms regulating MLC phosphorylation common to both agonists are likely responsible for the oscillation. MLCK is a major kinase that phosphorylates MLC in a Ca2+-dependent manner,27 whereas ROCK, ZIPK, and integrin-linked kinase phosphorylate MLC in a Ca2+-independent manner.18 Any of these kinases could induce PP-MLCThr18+Ser19.18,28 On the other hand, the dephosphorylation of MLC is mainly catalyzed by a type 1 phosphatase consisting of 3 subunits.29 The activity of this MLC phosphatase is suppressed either when MYPT1 is phosphorylated by ROCK, ZIPK, or integrin-linked kinase or when an inhibitor protein, CPI-17, is phosphorylated by ROCK or PKC.18,29 The pharmacological inhibitors of MLCK, PKC, and ROCK suppressed the thrombin-induced MLC phosphorylation to a similar level. However, only the ROCK inhibitor abolished the circadian oscillation of the MLC phosphorylation. The knockdown of MLCK, ZIPK, and ROCK2 suppressed the thrombin-induced MLC phosphorylation to a similar level at a nadir, whereas only ROCK2 knockdown abolished the circadian oscillation. It should be noted that an ≈50% reduction of ROCK2 expression was sufficient to abolish the circadian rhythm of MLC phosphorylation. The specificity of this phenomenon was supported in that only ROCK2 knockdown was effective in suppressing the circadian rhythm of MLC phosphorylation, whereas the degree of knockdown was similar among the 3 kinases.

There are 2 isoforms of ROCK, ROCK1 and ROCK2, which share 65% overall homology at the amino acid level.30 ROCK2 is the major isoform in gizzard smooth muscle, and ROCK2 plays a predominant role in the regulation of vascular smooth muscle contraction.28,31 An in silico analysis revealed that the promoter regions of the mammalian rock1 genes lack any regulatory element for the known clock genes. Indeed, no obvious circadian rhythm was observed for the ROCK1 protein expression in the aorta of wild-type mice (Figure S4, available in the online-only Data Supplement). Therefore, ROCK2 is suggested to play a key role in the circadian rhythm of MLC phosphorylation.

The present study further demonstrates that the circadian oscillation of the expression and activity of ROCK2 correlate with the circadian rhythm of the MLC phosphorylation. ROCK is known to phosphorylate MYPT1 at both Thr696 and Thr853 in humans, with a 3-fold preference for Thr853 over Thr696.19 Thr696 is also phosphorylated by other kinases, including ZIPK and integrin-linked kinase.18,19 Therefore, the phosphorylation of Thr853 more accurately indicates the activity of ROCK. The observations of the present study thus indicated that the rhythm of ROCK2 expression was correlated with the rhythm of activity (the phosphorylation of Thr853). The circadian oscillation of Thr853 phosphorylation and the in-phase oscillation of MLC phosphorylation were similarly observed after stimulation with thrombin and endothelin 1. A key role of ROCK2 in generating the circadian rhythm of the MLC phosphorylation is thus consistent as a mechanism common to both agonists. ROCK can modulate MLC phosphorylation either by inhibiting MLC phosphatase activity via the phosphorylation of MYPT1 or CPI-17 or by directly phosphorylating MLC.18,29 However, the basal level of MLC phosphorylation did not shown any apparent circadian oscillation. The circadian oscillation of MLC phosphorylation was apparently attributed to the agonist stimulation. Furthermore, the substrate specificity of ROCK for MYPT1 (Michaelis constant value, 0.1–0.2 µmol/L) was higher than that for MLC (2.5–5.0 µmol/L).31 It is therefore conceivable that ROCK2 generates the circadian oscillation of MLC phosphorylation mainly through the inhibition of the MLC phosphatase activity.

We concluded that the oscillation of ROCK2 expression was regulated by a clock gene, RORα, based on the following observations: the luciferase promoter assay demonstrated that the human rock2 promoter was responsive to RORα and RORγ. The expression of RORα, but not RORγ, exhibited circadian oscillation in phase with that of ROCK2 mRNA. The rhythmic expression of ROCK2 was abolished by knocking down the expression of RORα. The 2 ROREs, which are separated by a 102- to 108-nucleotide interval, are well preserved in the promoter regions of the mammalian rock2 genes (Table S2, available in the online-only Data Supplement). RORE is known to be responsible for gene expression during the dark phase.20 This role of RORE is consistent with the observation that the expression of ROCK2 protein peaked at 36 and 60 hours after the dexamethasone pulse treatment, both of which correspond with the transition from the dark phase to the light phase.6,15 There are 3 isoforms of ROR, and the rhythmic expression of each isoform shows tissue specificity.12,21 RORα expression is rhythmic in white adipose tissue but not in brown adipose tissue, liver, or muscle, whereas RORγ expression is rhythmic specifically in brown adipose tissue and liver.21 RORα thus appears to play a major role in the transcriptional regulation of the circadian rhythm of ROCK2 expression in vascular smooth muscle.

The physiological significance of the observations in the cultured smooth muscle cells was demonstrated by using the aortas of staggerer mice, which lack a functional RORα.12 The use of the aorta is supported by the observation that the circadian oscillation of ROCK2 expression was similarly observed in both PCSMCs and porcine aortic smooth muscle cells. As a result, the RORα-mediated circadian expression of ROCK2 was demonstrated to occur both in vivo and in culture. The functional significance of the oscillation of ROCK2 expression in the regulation of smooth muscle contraction was demonstrated by using α-toxin–permeabilized preparations. The contraction of smooth muscle is regulated by Ca2+ signaling and the change in the myofilament Ca2+ sensitivity.11 ROCK plays an important role in modulating the myofilament Ca2+ sensitivity.11,18 The use of permeabilized preparations allowed the focused investigation on the myofilament Ca2+ sensitivity and thereby enabled the successful detection of the diurnal changes in the myofilament Ca2+ sensitivity as a consequence of the circadian oscillation of ROCK2 expression. The results indicated that the myofilament Ca2+ sensitivity increased in association with an increase in MLC phosphorylation when the ROCK2 expression reached a peak (ZT0/24) and decreased when the ROCK2 expression reached a nadir (ZT12). These diurnal changes were abolished in the staggerer mice. In contrast, there were no significant diurnal changes in the Ca2+-dependent contractile mechanism. These findings thus suggest that the RORα-mediated circadian oscillation of ROCK2 expression is translated specifically to the oscillation of the myofilament Ca2+ sensitivity by modulating the MLC phosphorylation.

It was noticed that the Ca2+-tension relationship of the Ca2+-induced contraction in the staggerer mice shifted to the left of that obtained in the wild-type mice. The precontractions induced by Ca2+ before the application of GTPγS or U46619 in the staggerer mice were higher than those seen in the wild-type mice. These observations suggest that the activity of some Ca2+-dependent contractile mechanisms was enhanced in staggerer mice. However, the level of MLCK expression in the staggerer mice was similar to that seen in the wild-type mice. The precise mechanism underlying this enhancement of the Ca2+-induced contraction in staggerer mice remains to be investigated.

In conclusion, the present study revealed that the vascular intrinsic clock mechanism involving RORα generates the circadian rhythm of myofilament Ca2+ sensitivity. ROCK2 was identified as a key oscillator generating the circadian rhythm of the response of MLC phosphorylation and myofilament Ca2+ sensitivity. It is conceivable that the interplay between the central and the peripheral clocks plays an important role in determining the circadian rhythm of vascular contractility. How this vascular intrinsic rhythm of myofilament Ca2+ sensitivity interplays with the external cues from the central clock mechanism remains to be elucidated. Furthermore, the intrinsic rhythm of the myofilament Ca2+ sensitivity peaks at the beginning of the light phase, when the occurrence of cardiovascular diseases also peaks. The increased ROCK activity is suggested to play an important role in the pathogenesis of coronary vasospasm in both animal models and patients with vasospastic angina.10,32,33 The ROCK-mediated oscillation of myofilament Ca2+ sensitivity is therefore suggested to underlie the onset of cardiovascular events. However, such a pathological role of the vascular clock mechanism remains to be investigated.

Acknowledgments

We appreciate technical support from the Research Support Center, Graduate School of Medical Sciences, Kyushu University, and Dr Brian Quinn for linguistic comments and help with the article.

Sources of Funding

This study was supported in part by Grants-in-Aid for Scientific Research (Nos. 18100006, 23220013, and 24591118) from the Japan Society for the Promotion of Science, Health, and Labour; Sciences Research Grants for Research on Medical Devices for Improving Impaired QOL and Health (H20-007) and for Clinical Research (H21-013) from the Ministry of Health, Labour, and Welfare of Japan; and a grant from the Yokoyama Rinsho Yakuri Foundation.

Disclosures

None.

Footnotes

  • Received December 20, 2011.
  • Accepted October 22, 2012.

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Clinical Perspective

The cardiovascular system displays circadian rhythms in some physiological parameters, including blood pressure. The occurrence of pathological events, such as myocardial infarction and angina pectoris, also exhibits circadian variation. The circadian changes in vascular contractility underlie these physiological and pathological circadian events. The present study elucidated the details of the vascular intrinsic clock mechanism that regulate vascular contractility. The most prominent achievement of the present study is the identification of ROCK2 as a clock-regulated gene. The circadian oscillation of the expression of a clock gene, RORα, is translated to the oscillatory expression of ROCK2, which in turn generates the oscillation of myofilament Ca2+ sensitivity and vascular contractility. ROCK2 plays an important role in the regulation of smooth muscle contraction, especially under pathological setting. Furthermore, ROCK2 regulates smooth muscle growth and contributes to the development of vascular lesions. Therefore, it remains to be investigated how this intrinsic vascular clock is, if at all, modified under pathological conditions and how it contributes to the pathogenesis and pathophysiology of cardiovascular diseases, such as hypertension, coronary vasospasm, or atherosclerosis. In addition, vascular function is regulated by the interplay between the central and peripheral clocks. How these 2 clock systems cross-talk to each other and how they regulate vascular function as an integrated system remain to be elucidated. The present study thus provides a novel conceptual insight into vascular biology regarding the circadian regulation of vascular contractility and thereby contributes to understanding the pathogenesis of cardiovascular disease and developing new therapeutic strategies.

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  • Pivotal Role of Rho-Associated Kinase 2 in Generating the Intrinsic Circadian Rhythm of Vascular ContractilityClinical Perspective
    Toshiro Saito, Mayumi Hirano, Tomomi Ide, Toshihiro Ichiki, Noriyuki Koibuchi, Kenji Sunagawa and Katsuya Hirano
    Circulation. 2013;127:104-114, originally published January 2, 2013
    https://doi.org/10.1161/CIRCULATIONAHA.112.135608
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