Vascular Reactivity in Heart Failure

Karim, Syed M.; Rhee, Albert Y.; Given, Allison M.; Faulx, Michael D.; Hoit, Brian D.; Brozovich, Frank V.

doi:10.1161/01.res.0000142736.39359.58

Cited by 45 publications

(40 citation statements)

References 45 publications

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“…2D). These results are similar to our previous results, which demonstrated that 1C2 recognizes all four naturally occurring MYPT1 isoforms (MYPT1 CIϩ/LZϩ, MYPT1 CIϩ/LZϪ, MYPT1 CIϪ/LZϩ, (14).…”

Section: Resultssupporting

confidence: 93%

MYPT1 mutants demonstrate the importance of aa 888–928 for the interaction with PKGIα

Given¹,

Ogut²,

Brozovich³

2007

American Journal of Physiology-Cell Physiology

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286: 1583-1587, 1999), but we previously showed that PKGI␣ interacts with LZ-positive (LZϩ) and LZ-negative (LZϪ) MYPT1 isoforms (13). Interestingly, PKGI␣ is known to preferentially bind to RR and RK motifs (WR Dostmann et al. Proc Natl Acad Sci USA 97: 14772-14777, 2000), and there is an RK motif within the aa 888 -928 sequence of MYPT1 in LZϩ and LZϪ isoforms. Thus, to localize the domain of MYPT1 important for the MYPT1-PKGI␣ interaction, we designed four MYPT1 fragments that contained both the aa 888 -928 sequence and the downstream LZ domain (MYPT1FL), lacked both the aa 888 -928 sequence and the LZ domain (MYPT1TR), lacked only the aa 888 -928 sequence (MYPT1SO), or lacked only the LZ domain (MYPT1TR2). Using coimmunoprecipitation, we found that only the fragments containing the aa 888 -928 sequence (MYPT1FL and MYPT1TR2) were able to form a complex with PKGI␣ in avian smooth muscle tissue lysates. Furthermore, mutations of the RK motif at aa 916 -917 (R 916 K 917 ) to AA decreased binding of MYPT1 to PKGI␣ in chicken gizzard lysates; these mutations had no effect on binding in chicken aorta lysates. However, mutation of R -calmodulin activation of MLC kinase has been proposed to be the primary event for regulation of MLC 20 phosphorylation, and MLC phosphatase was thought to be constitutively active (12,22). However, recent studies have shown that MLC phosphatase is an important target for the physiological regulation of vascular tone; MLC phosphatase activity can be stimulated to produce Ca 2ϩ desensitization and inhibited to produce Ca 2ϩ sensitization (reviewed in Refs. 12 and 22).MLC phosphatase is a trimeric enzyme consisting of a 130-kDa myosin targeting subunit (MYPT1), a 37-kDa catalytic subunit (PP1c␦), and a 20-kDa subunit of unknown function (M20) (12). MYPT1, the primary site for regulation of phosphatase activity, has multiple binding and phosphorylation sites, which allow its function to be tightly controlled. Multiple pathways feed into this regulation, including G protein-coupled signaling and nitric oxide (NO) (reviewed in Refs. 12 and 22).NO-mediated vasodilatation is a fundamental response of the vasculature and is the classical model for Ca 2ϩ desensitization (9, 10). NO diffuses into the smooth muscle cells and activates guanylate cyclase, thereby increasing the production of cGMP. This second messenger can then bind to and activate type I␣ cGMP-dependent protein kinase (PKGI␣), which interacts with multiple targets within the smooth muscle cell, including the L-type Ca 2ϩ channel (8), maxi-K ϩ channels (1), sarcoplasmic reticulum channels (20), telokin (4, 25), and MYPT1 (13,23,24), all of which have been demonstrated to produce smooth muscle relaxation.MYPT1 (Fig. 1A) contains two putative COOH-terminal coiled-coil (CC) domains at aa 715-746 and aa 929 -969, which have 60% and 81% probabilities of CC formation, respectively (16), and a third domain at aa 1013-1039, which is known as the leucine zipper (LZ) (12). These sequences are numbered according to the M133 MYPT1 is...

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Section: Resultssupporting

confidence: 93%

MYPT1 mutants demonstrate the importance of aa 888–928 for the interaction with PKGIα

Given¹,

Ogut²,

Brozovich³

2007

American Journal of Physiology-Cell Physiology

Self Cite

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“…47 In coronary artery ligation-induced congestive heart failure, resistance to NO-mediated relaxation is attributed to the decreased expression of the LZ-positive MYPT1 isoform. 12 In the present study, we did not detect any change either in the overall MYPT1 mRNA expression or in the relative abundance of LZ-positive MYPT1 isoform in apoE-KO mice during atherosclerosis progression. Instead, a reduction in MYPT1 may result from increased protein degradation, which adds a new dimension to the regulation of MYPT1 expression at the level of protein stability.…”

Section: Discussioncontrasting

confidence: 72%

“…In the present study, our results showed that peroxynitrite treatment induced MYPT1 phosphorylation at both Thr696 and Thr850 in Modulation of MYPT1 abundance and differential expression of spliced variants occurs during development, as well as under physiological and pathological conditions. 9,12,14,15,47,48 In rat uterus, MYPT1 mRNA levels are decreased during the later stages of pregnancy. 15 Hypoxia exposure increases MYPT1 protein levels in the rat aorta.…”

Section: Discussionmentioning

confidence: 99%

“…9 In vitro and in vivo evidence showed that the LZ motif present in the C-terminal of MYPT1 is required for the activation of MP by type I PKG. [10][11][12] The sensitivity of smooth muscle to cGMP/PKG-mediated relaxation is proportional to the expression ratio of LZ þ / LZ À MYPT1. [10][11][12] In smooth muscle cells, the relationship between MP activity and the phosphorylation of MYPT1 has been extensively studied.…”

mentioning

confidence: 99%

“…[10][11][12] The sensitivity of smooth muscle to cGMP/PKG-mediated relaxation is proportional to the expression ratio of LZ þ / LZ À MYPT1. [10][11][12] In smooth muscle cells, the relationship between MP activity and the phosphorylation of MYPT1 has been extensively studied. 13 However, changes in MYPT1 abundance have only been reported by a few studies.…”

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confidence: 99%

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ROS-mediated downregulation of MYPT1 in smooth muscle cells: a potential mechanism for the aberrant contractility in atherosclerosis

Cheng

Tsai

et al. 2013

Laboratory Investigation

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Reactive oxygen species (ROS) mediates the aberrant contractility in hypertension. Abnormal contractility occurs in atherosclerotic vessels but changes in proteins that regulate contractility remain poorly understood. Myosin phosphatase (MP) activity, which regulates smooth muscle relaxation, is regulated by the phosphorylation of its regulatory subunit, MP targeting subunit 1 (MYPT1). In the present study, we examined the roles of ROS in MP subunit expression both in cultured human aortic smooth muscle cells (HASMCs) and during atherosclerosis progression in apolipoprotein E-knockout (apoE-KO) mice. Furthermore, the effect of decreased MYPT1 on actin cytoskeleton and cell migration activity was assessed in HASMCs. Short hairpin RNA-mediated knockdown of MYPT1 increased stress fibers and attenuated platelet-derived growth factor-induced cell migration in HASMCs. Superoxide anion-inducing agent LY83583 downregulated MYPT1 mRNA and protein levels, but did not affect the phosphorylation of MYPT1 and catalytic subunit of MP, PP1d. The LY83583-induced decrease in MYPT1 was abolished by co-treating with superoxide dismutase or by inhibiting NADPH oxidase with diphenyleneiodonium. Treatment of peroxynitrite, but not hydrogen peroxide (H 2 O 2 ), downregulated MYPT1 protein expression and induced MYPT1 phosphorylation without affecting mRNA levels. Co-treatment with a proteasome inhibitor, MG-132, eliminated peroxynitrite-induced MYPT1 downregulation. In apoE-KO mice, MYPT1 protein, but not mRNA, levels were markedly decreased in 16-week-and 24-week-old mice. Oral estrogen treatment, which was previously shown to decrease aortic ROS levels, upregulated aortic MYPT1 expression. Moreover, reduction in MYPT1 expression correlated with increased aortic sensitivity toward vasoconstrictors. These results suggested that during atherosclerosis progression oxidative stress mediates the downregulation of MYPT1, which may inhibit smooth muscle cell migration and contribute to the aberrant contractility.Laboratory Investigation (2013) 93, 422-433; doi:10.1038/labinvest.2013 published online 18 February 2013 KEYWORDS: atherosclerosis; contractility; myosin phosphatase; reactive oxygen species; smooth muscle At the molecular level, phosphorylation of the 20-kDa myosin light chains (MLC 20 ) is a key determinant for smooth muscle contraction. MLC 20 phosphorylation levels are determined by the activity ratio between myosin light chain kinase (MLCK) and myosin phosphatase (MP). 1 Although MLCK activation depends on cytoplasmic calcium concentration, MP activity is subject to modulation by various signaling molecules. 2 MP is a heterotrimer comprised of a 37-to 38-kDa catalytic subunit (PP1d), a 110-to 130-kDa regulatory subunit (MP targeting subunit 1; MYPT1), and a 20-kDa subunit of unknown function. The phosphorylation of MYPT1 by protein kinases is a major regulatory mechanism for MP that results in either the inhibition or enhancement of MP activity. 3 Small GTPase RhoA and its effecter, Rho-kinase, inhibit MP activity through ...

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