Forced Expression of Essential Myosin Light Chain Isoforms Demonstrates Their Role in Smooth Muscle Force Production

Huang, Qi; Fisher, Steven A.; Brozovich, Frank V.

doi:10.1074/jbc.274.49.35095

Cited by 28 publications

(20 citation statements)

References 18 publications

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“…2), as predicted from previous studies (12,19,28,29). The resulting higher CCSM tone could make endogenous CCSM relaxation mechanisms less effective and contribute to diabetes-induced ED.…”

Section: Discussionsupporting

confidence: 54%

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Smooth muscle myosin expression, isoform composition, and functional activities in rat corpus cavernosum altered by the streptozotocin-induced type 1 diabetes

Zhang

Kanika

Melman

et al. 2012

American Journal of Physiology-Endocrinology and Metabolism

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Zhang X, Kanika ND, Melman A, DiSanto ME. Smooth muscle myosin expression, isoform composition, and functional activities in rat corpus cavernosum altered by the streptozotocin-induced type 1 diabetes. Am J Physiol Endocrinol Metab 302: E32-E42, 2012. First published September 13, 2011; doi:10.1152/ajpendo.00231.2011.-Diabetes mellitus (DM) is a quite common chronic disease, and the prevalence of erectile dysfunction (ED) is three times higher in this large population. Although diabetes-related ED has been studied extensively, the actin-myosin contractile apparatus was not examined. The mRNAs encoding smooth muscle myosin (SMM) heavy chains (MHC) and essential light chains (LC 17) exist as several different alternatively spliced isoforms with distinct contractile properties. Recently, we provided novel data that blebbistatin (BLEB), a specific myosin II inhibitor, potently relaxed corpus cavernosum smooth muscle (CCSM). In this study, we examine whether diabetes alters SMM expression, alternative splicing, and/or functional activities, including sensitivity to BLEB. By using streptozotocin (STZ)-induced 2-mo diabetic rats, functional activities were tested in vivo by intracavernous pressure (ICP) recording during cavernous nerve stimulation and in vitro via organ bath contractility studies. SMM isoform composition was analyzed by competitive RT-PCR and total SMM, myocardin, and embryonic SMM (SMemb) expression by real-time RT-PCR. Results revealed that the blood glucose level of STZ rats was 407.0 vs. 129.5 mg/dl (control). STZ rats exhibited ED confirmed by significantly increased CCSM contractile response to phenylephrine and decreased ICP response. For STZ rats, SM-B, LC 17a and SM2 isoforms, total SMM, and myocardin expression increased, whereas SM-A, LC17b, and SM1 isoforms were decreased, with SMemb unchanged. BLEB was significantly more effective in relaxing STZ CCSM both in vitro and in vivo. Thus we demonstrated a novel diabetes-specific effect on alternative splicing of the SMM heavy chain and essential light chain genes to a SMM isoform composition favoring a heightened contractility and ED. A switch to a more contractile phenotype was supported further by total SMM expression increase. Moreover, the change in CCSM phenotype was associated with an increased sensitivity to BLEB, which may serve as a novel pharmacotherapy for ED.blebbistatin; cross-bridge cycling; erectile dysfunction; smooth muscle relaxation DIABETES MELLITUS (DM) is a common chronic disease affecting 285 million people, corresponding to 6.4% of the world's adult population in 2010 and expected increase to 7.9% by 2030 (40). The prevalence of erectile dysfunction (ED) is three times higher in diabetic men (28 vs. 9.6%) (17), occurs at an earlier age, and increases with disease duration, being ϳ15% at age 30 yr and rising sharply to 55% at 60 yr in diabetics (30, 31). DM may cause ED by affecting one or a combination of the following: psychological function, central nervous system function, androgen secretion, peripheral nerve activity...

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“…2), as predicted from previous studies (12,19,28,29). The resulting higher CCSM tone could make endogenous CCSM relaxation mechanisms less effective and contribute to diabetes-induced ED.…”

Section: Discussionsupporting

confidence: 54%

“…Several studies have shown that LC 17a seems to increase contraction (19,29). In addition, it is known that the increased relative expression of LC 17a correlates positively with the relative expression of SM-B (3, 13).…”

Section: Discussionmentioning

confidence: 99%

Smooth muscle myosin expression, isoform composition, and functional activities in rat corpus cavernosum altered by the streptozotocin-induced type 1 diabetes

Zhang

Kanika

Melman

et al. 2012

American Journal of Physiology-Endocrinology and Metabolism

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“…Muscle Cells-Primary cultures of chicken gizzard SMCs were isolated from day 15 embryos using a modification of a previously described method (23,24). Briefly, after being minced into fine pieces, the gizzard tissue was incubated in Hanks' balanced salt solution (Cellgro) containing 0.15% (w/v) collagenase type I (Worthington) at 37°C for 20ϳ40 min.…”

Section: Preparation Of Chicken Smoothmentioning

confidence: 99%

Unzipping the Role of Myosin Light Chain Phosphatase in Smooth Muscle Cell Relaxation

Huang

Fisher

Brozovich

2004

Journal of Biological Chemistry

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MBS isoforms could explain differences in tissue sensitivity to NO-mediated vasodilatation.Force regulation in smooth muscle is dependent on the activities of myosin light chain (MLC) 1 kinase and MLC phosphatase (3, 4). The activity of MLC kinase is regulated by Ca 2ϩ -calmodulin (3), whereas MLC phosphatase was originally thought to be constitutively active and unregulated (5). However, there is abundant evidence that the activity of MLC phosphatase can be both inhibited to produce Ca 2ϩ sensitization (reviewed in Refs. 5-7) or an increase in force at a constant [Ca 2ϩ ] and enhanced to produce Ca 2ϩ desensitization (8) or a decrease in force at a constant [Ca 2ϩ ]. NO is the classical agent to produce Ca 2ϩ desensitization (1, 9, 10). Recent evidence (11) suggests that NO produces vasodilatation by activating the soluble pool of guanylate cyclase, which in turn produces cGMP and leads to the activation of type I cGMP-dependent protein kinase (PKGI). PKGI mediates smooth muscle cell relaxation by several mechanisms. It has been demonstrated that PKGI acts on the maxi K ϩ channel to produce hyperpolarization of the smooth muscle (12), decreases Ca 2ϩ flux (13,14), and also activates MLC phosphatase (1, 15) to decrease the level of MLC 20 phosphorylation and to produce smooth muscle relaxation. In addition, PKGI-dependent pathways for vasodilatation may also include phosphorylation of telokin (16, 17) and HSP20 (18).MLC phosphatase is a holoenzyme consisting of a catalytic subunit (PP1c␦); a myosin-binding subunit (MBS), which is also referred to as the myosin-targeting subunit (MYPT1); and a 20-kDa subunit of unknown function (5). The activation of MLC phosphatase by PKGI is hypothesized to be due to a leucine zipper-leucine zipper (LZ-LZ) interaction of the N-terminal LZ of PKGI␣ and the C-terminal LZ of the MBS of MLC phosphatase (1, 2). The MBS has four major isoforms, which are produced by alternative RNA splicing of two different exons (5). Tissue-specific and developmentally regulated alternative splicing of a 123-bp central exon produces a 41-amino acid central insert (19). Alternative splicing of the 31-bp 3Ј-exon is responsible for the expression of LZ ϩ or LZ Ϫ MBS isoforms (5). Specifically, exclusion of the 3Ј-exon shifts the reading frame of the MBS transcript to encode a C-terminal LZ (2).We previously demonstrated that sensitivity to cGMP-mediated relaxation correlates with the relative expression of LZ ϩ / LZ Ϫ MBS isoforms (2), which is consistent with the activation of MLC phosphatase activity resulting from a LZ-LZ interaction of PKGI␣ with the MBS (1). In this study, we tested the hypothesis that cGMP-dependent activation of MLC phosphatase activity and smooth muscle vasodilatation are due to a LZ-LZ interaction of PKGI␣ and MBS by changing the expression of the MBS isoform, in isolation, and determining the effect on cGMP-mediated MLC 20 dephosphorylation in primary cultured smooth muscle cells (SMCs). MATERIALS AND METHODSCloning of the Chicken MBS of the MLC Phosphatase cDNA Fragment-...

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“…Little [41] or no [35] difference has been reported in the actin-activated ATPase activity of myosins enriched in LC17a or LC17b at high actin concentration. A small increase (by a factor of 1.5±2) in response to LC17a, but not LC17b, has also been reported for some parameters of the contractile properties of smooth muscle cells or strips [7,8].…”

Section: Discussionmentioning

confidence: 80%

“…An inverse correlation has been found between relative LC17b content and the maximal shortening velocity of skinned smooth muscle fibers [6] but this result is controversial [1]. Recently, weak but significant LC17 isoform-specific effects on the mechanical properties of smooth muscle cells [7] and strips [8] have been shown. In addition, the two isoforms have different subcellular distributions suggesting that they may have different functions [9].…”

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confidence: 99%

Functional regions in the essential light chain of smooth muscle myosin as revealed by the mutagenesis approach

Quevillon‐Chéruel

Janmot

Nozais

et al. 2000

European Journal of Biochemistry

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The endogenous essential light chain (LC17) of myosin from intestine smooth muscle was replaced with mutated essential light chains prepared using recombinant techniques. Complete exchange was observed with histidinetagged derivatives of LC17a, LC17b and E122A-LC17a (LC17a and LC17b are the usual constituants of smooth muscle myosin), with small changes in the ATPase activity of reconstituted myosins. Much less exchange was observed with the light-chain derivative lacking the last 12 amino acid residues, demonstrating the importance of this segment, which may act as one arm of a pair of pincers to bind the myosin heavy chain.Keywords: isoforms; mutants; myosin light chains; site-directed mutagenesis; smooth muscle.Myosin II is a major contractile protein composed of two heavy chains (< 200 kDa each) and two pairs of light chains (< 20 kDa each). One pair of light chains is described as regulatory (20 kDa) and the other as essential (17 kDa), based on their distinctive chemical and functional features. In smooth muscle myosin, the two 20-kDa regulatory chains (LC20) can be phosphorylated and phosphorylation generates high levels of actin-activated ATPase activity and actin motility. The other pair of myosin light chains consists of the two 17-kDa essential chains (LC17), the functional significance of which is unclear [1]. The regulatory and essential light chains are bound to an 8-nm stretch of myosin heavy chain, mostly a helix. This complex constitutes a regulatory domain [2,3] and is thought to act as a lever arm during force production by muscle motor proteins [4].The essential light chain, LC17, has two isoforms, the ratio of which depends on the tissue. The difference between these two species is five amino acid substitutions in the nine C-terminal residues (Fig. 1). In particular, one isoform, LC17a, has a charged glutamic acid residue at position 142, whereas the other, LC17b, has an alanine residue in the corresponding position [5]. An inverse correlation has been found between relative LC17b content and the maximal shortening velocity of skinned smooth muscle fibers [6] but this result is controversial [1]. Recently, weak but significant LC17 isoform-specific effects on the mechanical properties of smooth muscle cells [7] and strips [8] have been shown. In addition, the two isoforms have different subcellular distributions suggesting that they may have different functions [9].It has been shown that substitution mutations in the essential light chain of nonmuscle myosin from Dictyostelium may lead to decreases in actin-activated ATPase activity and motor function, as assessed by an in vitro motility assay [10].Katoh and co-workers [11,12] recently reported an efficient method for essential light chain exchange in smooth muscle myosin. We used this method to exchange LC17a, LC17b and mutated essential light chains with endogenous light chains from rabbit small-intestine myosin. The recombinant proteins were prepared ( Fig. 1; ELC1±6) with a histidine tag on their N-terminus to facilitate purification. In...

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Forced Expression of Essential Myosin Light Chain Isoforms Demonstrates Their Role in Smooth Muscle Force Production

Cited by 28 publications

References 18 publications

Smooth muscle myosin expression, isoform composition, and functional activities in rat corpus cavernosum altered by the streptozotocin-induced type 1 diabetes

Smooth muscle myosin expression, isoform composition, and functional activities in rat corpus cavernosum altered by the streptozotocin-induced type 1 diabetes

Unzipping the Role of Myosin Light Chain Phosphatase in Smooth Muscle Cell Relaxation

Functional regions in the essential light chain of smooth muscle myosin as revealed by the mutagenesis approach

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