Abstract-Two mechanisms are proposed to account for the inhibition of myosin phosphatase (MP) involved in Ca 2ϩ sensitization of vascular muscle, ie, phosphorylation of either MYPT1, a target subunit of MP or CPI-17, an inhibitory phosphoprotein. In cultured vascular aorta smooth muscle cells (VSMCs), stimulation with angiotensin II activated RhoA, and this was blocked by pretreatment with 8-bromo-cGMP. VSMCs stimulated by angiotensin II, endothelin-1, or U-46619 significantly increased the phosphorylation levels of both MYPT1 (at Thr696) and CPI-17 (at Thr38). The angiotensin II-induced phosphorylation of MYPT1 was completely blocked by 8-bromo-cGMP or Y-27632 (a Rho-kinase inhibitor), but not by GF109203X (a PKC inhibitor). In contrast, phosphorylation of CPI-17 was inhibited only by GF109203X. Y-27632 dramatically corrected the hypertension in N -nitro-L-arginine methyl ester (L-NAME)-treated rats, and this hypertension also was sensitive to isosorbide mononitrate. The level of the active form of RhoA was significantly higher in aortas from L-NAME-treated rats. Expression of RhoA, Rho-kinase, MYPT1, CPI-17, and myosin light chain kinase were not significantly different in aortas from L-NAME-treated and control rats. Activation of RhoA without changes in levels of other signaling molecules were observed in three other rat models of hypertension, ie, stroke-prone spontaneously hypertensive rats, renal hypertensive rats, and DOCA-salt rats. These results suggest that independent of the cause of hypertension, a common point in downstream signaling and a critical component of hypertension is activation of RhoA and subsequent activation of Rho-kinase. A lthough the mechanisms underlying development of hypertension are not established, one critical feature observed in most cases of hypertension is increased peripheral resistance, implying enhanced constriction of the relevant vessels. 1 A major determinant of vascular tone is the level of myosin light chain (MLC) phosphorylation that is controlled by the Ca 2ϩ /calmodulin-dependent myosin light chain kinase (MLCK) and by myosin phosphatase (MP). 2 However, there is no fixed relationship between cytosolic Ca 2ϩ and MLC phosphorylation and this can vary depending on conditions. An increased phosphorylation and tension can occur at suboptimal Ca 2ϩ levels and is referred to as Ca 2ϩ sensitization and occurs frequently after stimulation by many agonists. 3 A major contributor to this effect is inhibition of MP and over the last decade this was shown to be operating via a small GTPase RhoA-linked pathway. [3][4][5][6] There is a strong evidence to implicate Rho-kinase as a downstream target in the RhoA-linked pathway, 6,7 and it has been shown that Rho-kinase inhibitors, such as Y-27632, block the agonist-induced Ca 2ϩ sensitization in smooth muscle. 8 At a molecular level, one mechanism responsible for inhibition of MP is that phosphorylation by Rho-kinase of the target subunit of MP, MYPT1, at Thr696 (for the human isoform) inhibits activity of the catalytic subunit. 7...
RhoA is commonly activated in the aorta in various hypertensive models, indicating that RhoA seems to be a molecular switch in hypertension. The molecular mechanisms for RhoA activation in stroke-prone spontaneously hypertensive rats (SHRSP) were here investigated using cultured aortic smooth muscle cells (VSMC).
Objective-Rho/Rho-kinase pathway plays pivotal roles in cardiovascular diseases including arteriosclerosis and hypertension. Recently it has become evident that C-reactive protein (CRP), a powerful marker for cardiovascular events, has direct proatherothrombotic effects on vascular cells. However, its molecular mechanism has not been fully investigated. We examined the involvement of Rho/Rho-kinase signaling in CRP-induced plasminogen activator inhibitor-1 (PAI-1) expression in bovine aortic endothelial cells (BAECs). Methods and Results-PAI-1 expression was determined by Western blotting. RhoA activation was determined by an affinity pull-down assay using Rho-binding fragment of rhotekin. NF-B activity was determined using the luciferase reporter gene. A ccumulating evidence suggests that inflammation plays a significant role in the development and progression of atherosclerosis. 1,2 Several plasma markers of inflammation have been evaluated as potential tools for the prediction of the risk of future cardiovascular events and, of these, the most reliable and accessible for clinical use is currently highsensitive C-reactive protein (CRP). 3,4 Recently, CRP was shown to elicit a multitude of effects on endothelial biology favoring proinflammatory and proatherosclerotic phenotypes. These include the expression of adhesion molecules, the stimulation of the release of inflammatory chemokine/cytokine, and the reduction of nitric oxide bioactivity and prostacyclin release in endothelial cells. 4,5 CRP was also able to increase the expression and activity of plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitors and a marker of impaired fibrinolysis and atherothrombosis, in human aortic endothelial cells. 6 The atherothrombotic effects on PAI-1 expression by CRP were confirmed in vivo by the result that human CRP-transgenic mice (CRP-tg) showed a prothrombotic phenotype, as evidenced by higher rates of thrombotic occlusion after arterial injury. 7 CRP was also shown to possess similar proatherogenic properties toward vascular smooth muscle cells (VSMCs) and monocyte-macrophages, 5 strongly indicating that CRP is not only a biomarker of atherosclerotic events but is also a mediator of atherosclerosis.The molecular mechanisms involved in CRP-induced gene expression remain unknown, although one possible mechanism for this is via the activation of nuclear factor (NF)-B, 8 -10 a key mediator of atherosclerosis. 11,12 Rho/Rho-kinase signaling factors, initially identified as key molecules for Ca 2ϩ sensitization of smooth muscle contraction, 13,14 are known to be involved in the signal cascades related to inflammation including the NF-B pathway, 15 and several experimental models revealed the involvement of this signaling in the development of vascular remodeling and atherosclerosis. 16 The aim of the present investigation is to clarify whether the Rho/Rho-kinase signaling pathway is involved in PAI-1 expression by CRP in bovine aorta endothelial cells (BAECs). We demonstrated in this study tha...
Background: Phosphorylation of the regulatory light chain of myosin (MLC) has roles in cardiac function. In vitro, myosin phosphatase target subunit 2 (MYPT2) is a strongly suspected regulatory subunit of cardiac myosin phosphatase (MP), but there is no in-vivo evidence regarding the functions of MYPT2 in the heart. Methods and Results:Transgenic mice (Tg) overexpressing MYPT2 were generated using the α-MHC promoter. Tg hearts showed an increased expression of MYPT2 and concomitant increase of the endogenous catalytic subunit of type 1 phosphatase (PP1cδ), resulting in an increase of the MP holoenzyme. The level of phosphorylation of ventricular MLC was reduced. The pCa-tension relationship, using β-escin permeabilized fibers, revealed decreased Ca 2+ sensitization of contraction in the Tg heart. LV enlargement with associated impairment of function was observed in the Tg heart and ultrastructural examination showed cardiomyocyte degeneration. Conclusions:Overexpression of MYPT2 and the increase in PP1cδ resulted in an increase of the MP holoenzyme and a decrease in the level of MLC phosphorylation. The latter induced Ca 2+ desensitization of contraction and decreased LV contractility, resulting in LV enlargement. Thus, MYPT2 is truly the regulatory subunit of cardiac MP in-vivo and plays a significant role in modulating cardiac function. (Circ J 2010; 74: 120 - 128)
Myosin phosphatase (MP) is a major phosphatase responsible for the dephosphorylation of the regulatory light chain of myosin II. MYPT1, a target subunit of smooth and nonmuscle MP, is responsible for activation and regulation of MP. To identity the physiological roles of MP, we have generated MYPT1-deficient mice by gene targeting. The heterozygous mice showed no changes in expression levels of MYPT1 and no distinct phenotype compared to wild-type mice was observed. None of the F2 mice were homozygous for the MYPT1 deletion, indicating that the targeted disruption of the MYPT1 gene resulted in embryonic lethality. The point of embryonic lethality is before 7.5 dpc. These findings indicate that MYPT1 is essential for mouse embryogenesis.
A 71-year-old male was referred to another hospital for dizziness. A bradycardia -tachycardia syndrome and Cor triatriatum were detected, and an operation to resect the membrane in the left atrium and implant a pacemaker epicardially was performed. However, no suitable site could be found on either atria and therefore, a single chamber ventricular pacemaker was implanted. In the electrophysiological study performed in our hospital, we could not detect any atrial potentials in either atria, excluding the region close to the His bundle (HB) and within coronary sinus (CS), in spite of extensive catheter mapping. A regular atrial rhythm with a cycle length of 820 ms, which was synchronous with the rate of the QRS complex on the surface ECG, was recorded only at the HB. Meanwhile, the CS catheter recording exhibited regular focal activity with a cycle length of 150 ms, and this focal activity did not conduct to the atrium close to the HB. Furthermore, this activity was dissociated from the ventricular activity recorded from the CS catheter. During an isoproterenol infusion, an atrial tachycardia with a cycle length of 380 ms was recorded only at the HB, and the twelve-lead ECG exhibited a regular tachycardia with the same cycle length as this tachycardia. Meanwhile, the focal activity within the CS persisted without any change in the cycle length. These findings suggested that there was dissociation between the right atrium (RA) and CS. Furthermore, partial atrial standstill was observed in both atria, excluding the RA close to the atrio-ventricular (AV) node and area within the CS. These rare electrophysiological features were considered to play an important role in the genesis of a simultaneous combination of the two tachycardias at their respective sites.
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