SM-B Isoform Research Articles

SRSF6 is upregulated in asthmatic horses and involved in the MYH11 SMB expression.

Smooth muscle has a central role in bronchospasm‐induced airway obstruction in asthma. Alternative mRNA splicing of the smooth muscle myosin heavy chain (myh11) gene produces four different isoforms, one of which (SMB) is characterized by the inclusion of the exon5b, which doubles the smooth muscle cells contraction velocity. Deciphering the regulation of the expression levels of the SMB isoform would represent a major step for the understanding of the triggers and pathways leading to airway smooth muscle contraction in asthma. Our objective was therefore, to study the splicing regulation mechanisms of the exon5b in airway smooth muscle cells. Bioinformatics analysis was performed to identify the cis‐regulatory elements present in the exon5b using HSF finder 3 tool. The expression of the corresponding serine/arginine rich protein (SR) genes thus identified was evaluated by quantitative RT‐PCR (qPCR). SRSF1, SRSF6, and hnRNPA1 cis‐acting elements were identified by in silico analysis of the exon5b sequence as splicing regulator candidates. QPCR analyses showed that SRSF1 and SRSF6 are upregulated in ASM cells from asthmatic horses in exacerbation (n = 5) compared to controls (n = 5). The inhibition of the identified splicing factors by small interfering RNA allowed identifying the regulation of the SMB isoform by SRSF6. Our results implicate for the first time the upregulation of SRSF6 and SRSF1 in the asthmatic ASM cells and indicate that SRSF6 induces the exon5b inclusion. This study provides an important first step for the understanding of the triggers and pathways leading to ASM hypercontraction and identifies a possible new target for asthma.

1130 STREPTOZOTOCIN-INDUCED TYPE 1 DIABETES IN RAT ALTERS SMOOTH MUSCLE MYOSIN ISOFORM COMPOSITION TO A MORE CONTRACTILE PHENOTYPE AND INCREASES SENSITIVITY TO THE MYOSIN II SPECIFIC INHIBITOR BLEBBISTATIN

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research1 Apr 20111130 STREPTOZOTOCIN-INDUCED TYPE 1 DIABETES IN RAT ALTERS SMOOTH MUSCLE MYOSIN ISOFORM COMPOSITION TO A MORE CONTRACTILE PHENOTYPE AND INCREASES SENSITIVITY TO THE MYOSIN II SPECIFIC INHIBITOR BLEBBISTATIN Xinhua Zhang, Arnold Melman, and Michael DiSanto Xinhua ZhangXinhua Zhang Camden, NJ More articles by this author , Arnold MelmanArnold Melman Bronx, NY More articles by this author , and Michael DiSantoMichael DiSanto Camden, NJ More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.727AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The mRNAs encoding smooth muscle myosin (SMM) heavy chains (MHC) and the 17 kDa essential light chains (LC17) exist as several different isoforms with distinct contractile properties, produced via alternative splicing mechanisms. Recently, we provided novel data that blebbistatin (BLEB), a specific myosin II inhibitor, potently relaxed both rat and human corpus cavernosum smooth muscle (CCSM)(J Sexual Med, 2009). In this study, we determine if diabetes alters SMM expression, alternative splicing and/or functional activities including sensitivity to BLEB. METHODS Fisher 344 male streptozotocin (STZ)-induced diabetic rats were kept for 2 months. Functional activities were then tested in vivo by intracavernous pressure (ICP) recording and in vitro via organ bath contractility studies. SMM isoform composition was analyzed by competitive RT-PCR and total SMM, myocardin (a transcriptional regulator of SMM) and embryonic SMM (SMemb) expression by Real-time RT-PCR. RESULTS Average blood glucose levels of STZ rats were 407 vs 129.5 mg/dl in rats injected with saline (SHAM). STZ rats showed severe erectile dysfunction (ED) confirmed by decreased ICP response to electrical stimulation of cavernous nerve. For STZ rats, LC17a and SM-B isoforms (both associated with increased contractility and faster shortening velocity) and total SMM and myocardin expression increased while LC17b and SM-A isoforms (both associated with decreased contractility and slower shortening velocity) were decreased with SM1/2 SMM MHC isoforms and SMemb unchanged. Interestingly, BLEB was more effective in relaxing STZ CCSM both in vitro and in vivo. CONCLUSIONS We show for the first time to our knowledge a 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 is further supported by total SMM expression increase. Moreover, we provide novel data that the change in CCSM phenotype is associated with an increased sensitivity to BLEB, which may serve as a novel pharmacotherapy for ED with specificity to the faster type myosin isoforms found in the diabetic rat CCSM. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e454 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Xinhua Zhang Camden, NJ More articles by this author Arnold Melman Bronx, NY More articles by this author Michael DiSanto Camden, NJ More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

A Kinetic Step Involving Loop 1 in Smooth Muscle may Dictate Isoform Specific Differences in ADP Release

The actin sliding velocity and ADP release rates in smooth muscle myosin are modulated by the make-up of a flexible surface loop spanning the active site known as loop 1. There are only two motor domain isoforms of smooth muscle myosin and they differ in the presence (SM-B) or absence (SM-A) of a seven amino acid insert (QGPSFSY) in this loop. The presence of this insert leads to a two-fold increase in both actin sliding velocity and ADP release, although the mechanism for this difference is unknown. To investigate the role of this insert in functional differences between the SM-B and SM-A isoforms of smooth muscle myosin we have inserted a single tryptophan residue into loop 1 of both isoforms. The dynamics of loop 1 have been correlated with the kinetics of ADP release using a combination of steady-state fluorescence measurements (i.e., tryptophan emission, FRET, and acrylamide quenching) and stopped-flow kinetics. Using this approach we have already shown that the long loop SM-B isoform displays an extra step in its ADP release pathway that has not been previously observed. Here, we show that the additional transition seen in the long loop SM-B isoform is not observed in the short loop SM-A isoform upon ADP release. Furthermore, the final ADP release step is twice as slow in the short loop SM-A isoform, suggesting that the unique transition observed in the presence of the insert alters loop 1 dynamics in a way as to facilitate ADP release. This alteration of ADP release constitutes a simple and fundamental way to tune the activity of the motor at the molecular level and mechanical function at the physiological level in smooth muscle.

Loop 1's Role in a Novel Step on the ADP Release Pathway of Smooth Muscle

Smooth muscle myosin has two N-terminal isoforms that result from alternative splicing of loop 1. Loop 1 contains a seven amino acid insert (QGPSFSY) in one isoform (SM-B) that is absent in the other (SM-A). It has been shown that the presence of the insert causes a two-fold increase in the rate of in-vitro actin sliding velocity and actin-activated ATPase activity (Rovner et al., 1997, Muscle Res Cell Motil 18:103). Based on these results and its proximity to the active site, it was hypothesized that loop 1 plays a role in modulating the release of ADP (Spudich, 1994, nature 372:515). However, little is known about the conformation of loop 1 in different nucleotide states, as it is absent in crystal structures. To further investigate the role of loop 1 in modulating ADP release we have inserted a single tryptophan residue into the interior of loop 1 in the SM-B isoform to monitor its dynamics. In combination with stopped-flow kinetics to monitor the release rate of mant-ADP from the motor domain, we have observed three steps in the ADP release mechanism, one of which is a unique transition that occurs before ADP release and following opening of the active site. Significantly, this previously undetected kinetic step appears to arise from a specific change in the state of loop 1. This is the first time a role of loop 1 in the ADP release mechanism has been directly identified and may account for the functional differences observed between two isoforms of smooth muscle myosin.

Preferential Myosin Heavy Chain Isoform B Expression May Contribute to the Faster Velocity of Contraction in Veins versus Arteries

Smooth muscle myosin heavy chains occur in 2 isoforms, SMA (slow) and SMB (fast). We hypothesized that the SMB isoform is predominant in the faster-contracting rat vena cava compared to thoracic aorta. We compared the time to half maximal contraction in response to a maximal concentration of endothelin-1 (ET-1; 100 nM), potassium chloride (KCl; 100 mM) and norepinephrine (NE; 10 µM). The time to half maximal contraction was shorter in the vena cava compared to aorta (aorta: ET-1 = 235.8 ± 13.8 s, KCl = 140.0 ± 33.3 s, NE = 19.8 ± 2.7 s; vena cava: ET-1 = 121.8 ± 15.6 s, KCl = 49.5 ± 6.7 s, NE = 9.0 ± 3.3 s). Reverse-transcription polymerase chain reaction supported the greater expression of SMB in the vena cava compared to aorta. SMB was expressed to a greater extent than SMA in the vessel wall of the vena cava. Western analysis determined that expression of SMB, relative to total smooth muscle myosin heavy chains, was 12.5 ± 4.9-fold higher in the vena cava compared to aorta, while SMA was 4.9 ± 1.2-fold higher in the aorta than vena cava. Thus, the SMB isoform is the predominant form expressed in rat veins, providing one possible mechanism for the faster response of veins to vasoconstrictors.

Changes in the Expression of Smooth Muscle Myosin Heavy Chain mRNA following Partial Bladder Obstruction or Spinal Cord Injury in Rat: A Preliminary Study

Purpose: The smooth muscle myosin heavy chain (SMMHC) isoform composition has been actively researched in a partial bladder obstruction (PBO) or spinal cord injury (SCI) model. Even though rat is an ideal animal for studying bladder physiology, there were very few reports about the changes of the SMMHC isoforms in the PBO or SCI injured bladder of rat. We developed two polymerrase chain reaction (PCR) primer sets to amplify the isoforms and we applied the primers to the PBO and SCI rat models. Materials and Methods: Female rats had their bladder necks surgically obstructed or they were subjected to spinal cord injury. Six weeks after the event, the bladders were excised. The expression of the C-terminal (SM1 and SM2) and N-terminal (SM-A and SM-B) isoforms of SMMHC was analyzed by performing reverse transcriptase-PCR (RT-PCR). Results: The control bladder showed only the SM-B isoform in the C-terminal. However, the bladder after SCI showed an increased SM-A to SM-B ratio. In case of PBO, the ratios were variable. A decreased SM1 expression was noted in the PBO and SCI groups when compared to the control group (p<0.05). Conclusions: Our female rat models for PBO or SCI demonstrates changes in the expression of smooth muscle myosin heavy chain isoforms. We will apply this primer set for studying of rat muscular physiology in PBO or SCI model. (Korean J Urol 2007;48:522-526)

Smooth, slow and smart muscle motors.

Smooth muscle is a slow and economical muscle with a large variability in contractile properties. This review describes results regarding the relation between expression of myosin isoforms and the contraction of smooth muscle. The focus of the review is on studies of the organised contractile system in the smooth muscle tissue. The role of the myosin heavy chain variants formed by alternative splicing in the myosin heavy chain tail (SM1, SM2 isoforms) and head (SM-A SM-B isoforms) regions, as well as the role of essential light chains (LC17a, LC17b isoforms) for the variability of contractile properties are discussed. Smooth muscle also has the ability to alter its contractile properties in response to altered functional demands in vivo, e.g. during hypertrophic growth of urinary bladder, intestine, uterus and vessels and in response to altered hormone levels. These alterations involve changes in myosin expression and altered contractile kinetics. Non-muscle myosin has been shown to have a contractile function in some smooth muscle tissues and recent data on the kinetic properties of non-muscle myosin filaments in smooth muscle tissue are described.

Generation of a cell line with smooth muscle phenotype from hypertrophied urinary bladder.

We have established a cell line from hypertrophied rabbit urinary bladder smooth muscle (SM) that stably expresses SM myosin (SMM). These cells, termed BSM, are spindle shaped and form swirls, similar to the "hills and valleys" described for cultured aortic SM cells. Western blotting revealed that BSM expresses the amino-terminal SMM heavy chain isoform SM-B, the carboxy-terminal SM1 and SM2 isoforms, and SM alpha-actin. In addition, they express cGMP-dependent protein kinase G, made by contractile SM cells in vitro but not by noncontractile cells synthesizing extracellular matrix. Immunofluorescence studies indicate a homogeneous population of cells expressing alpha-actin and SMM, including the SM-B isoform, and karyotyping demonstrates a stable 4N chromosomal pattern. These cells also express calcium-dependent myosin light chain kinase and phosphatase activity and contract in response to the muscarinic agonist bethanechol. To our knowledge, BSM is the first visceral SM cell line that expresses the SM-B isoform and might serve as a useful model to study the transcriptional regulation of tissue-specific SMM isoforms in differentiation and pathological SM.

Estrogen modulates the expression of myosin heavy chain in detrusor smooth muscle.

The effect of low serum estrogen levels on urinary bladder function remains poorly understood. Using a rabbit model, we analyzed the effects of estrogen on the expression of the isoforms of myosin, the molecular motor for muscle contraction, in detrusor smooth muscle. Expression of myosin heavy chain (MHC) isoforms, which differ in the COOH-terminal (SM1 and SM2) and the NH(2)-terminal (SM-A and SM-B) regions as a result of alternative splicing of the mRNA at either the 3'- or 5'-ends, was analyzed in age-matched female rabbits that were sham operated, ovariectomized (Ovx), and given estrogen after ovariectomy (4 rabbits/group). Ovx rabbits showed a significant decrease in the overall MHC content per gram of wet detrusor smooth muscle compared with controls (P < 0.04), which was reversed by estrogen replacement (P < 0.02). MHC content, as a proportion of total milligram of protein in the bladder tissue extracted, was also increased in estrogen-treated Ovx rabbits. Quantitative competitive RT-PCR revealed 1.72-, 2.63-, and 5.82 x 10(6) copies of MHC mRNA/100 ng total mRNA in Ovx, control, and estrogen-treated rabbits, respectively (P < 0.01). RT-PCR analysis using oligonucleotides specific for the region containing the SM1/SM2 MHC alternative splice sites indicated a lower SM2-to-SM1 ratio in estrogen-treated compared with control and Ovx rabbits (P < 0.05). Similarly, SDS-PAGE analysis of extracted myosin from estrogen-treated rabbits revealed a significantly lower SM2-to-SM1 isoform ratio compared with control and Ovx rabbits (P < 0.05). Expression of the SM-A and SM-B isoforms was not affected. These results indicate that myosin content is increased upon estrogen replacement in Ovx rabbits and that the abundance of SM1 relative to SM2 is greater in estrogen-treated rabbits compared with normal and Ovx rabbits. These data suggest that estrogen affects alternative splicing at the 3'-end of the MHC pre-mRNA to increase the proportion of SM1 vs. SM2.

Single rabbit stomach smooth muscle cell myosin heavy chain SMB expression and shortening velocity.

Isolated single smooth muscle cells (SMCs) from different regions of the rabbit stomach were used to determine a possible correlation between unloaded shortening velocity and smooth muscle (SM) myosin heavy chain (MHC) S1 head isoform composition (SMA, no head insert; SMB, with head insert). alpha-Toxin-permeabilized isolated single cells were maximally activated to measure unloaded shortening velocity and subsequently used in an RT-PCR reaction to determine the SMA/SMB content of the same cell. SM MHC SMA and SMB isoforms are uniquely distributed in the stomach with cells from the fundic region expressing little SMB (38.1 +/- 7.3% SMB; n = 16); cells from the antrum express primarily SMB (94.9 +/- 1.0% SMB; n = 16). Mean fundic cell unloaded shortening velocity was 0.014 +/- 0.002 cell lengths/s compared with 0.036 +/- 0.002 for the antrum cells. Unloaded shortening velocity in these cells was significantly correlated with their percent SMB expression (r2 = 0.58). Resting cell length does not correlate with the percent SMB expression (n = 32 cells). Previously published assays of purified or expressed SMA and SMB heavy meromyosin show a twofold difference in actin filament sliding speed in in vitro motility assays. Extrapolation of our data to 0-100% SMB would give a 10-fold range of shortening velocity, which is closer to the approximately 20-fold range reported from various SM tissues. This suggests that mechanisms in addition to the MHC S1 head isoforms regulate shortening velocity.

Smooth muscle myosin heavy chain isoforms and their role in muscle physiology.

Unlike vertebrate skeletal muscle, smooth muscle myosin heavy chain isoforms are encoded by a single gene. Alternative splicing of the primary transcript from a single gene generates four smooth muscle myosin heavy chain isoforms. These isoforms differ both at the carboxyl terminus (SM1 and SM2 isoforms) and at the amino terminus (SM-A and SM-B isoforms). The smooth muscle myosin heavy chain isoforms are differentially expressed during smooth muscle development and in different smooth muscle cell types. The mechanical properties of smooth muscle may be correlated with the myosin heavy chain content/isoform expression. However, the precise function of each smooth muscle myosin heavy chain isoform to muscle contraction remains to be determined. This review mainly focuses on the molecular basis of smooth muscle myosin heavy chain isoform diversity, its expression during development and disease, and its role in muscle physiology.

Smooth-muscle myosin heavy-chain SM-B isoform expression in developing and adult rat lung.

The smooth-muscle cells composing the vasculature and airways of the lung display a variety of contractile protein phenotypes. To date, however, it has remained unclear how these phenotypes might contribute differentially to contractile activity. To address this issue, we made monospecific rabbit polyclonal antibodies against the difference peptide for the SM-B smooth-muscle myosin heavy chain (SMMHC) and used these to investigate the distribution of the SM-B isoform in lung. SM-B has a seven-amino acid insert in the head region that is known to result in a higher actin-activated adenosine triphosphatase activity and in vitro motility. During development, reactivity is first seen in the trachea and bronchi of saccular lung at the time of birth, when other SMMHC isoforms also are present. Immunoreactivity spreads distally through the airways as development proceeds, reaching the level of alveolar septae in the adult. Although the smaller vessels of the pulmonary vasculature react strongly with the SM-B antibody, reactivity is infrequently observed in large pulmonary vessels. Adult tracheal smooth muscle is highly and more uniformly reactive, commensurate with its relatively high maximal velocity of shortening. The differential expression of the SM-B isoform in vascular and airway smooth muscles demonstrated in this study may provide the molecular basis for functional differences between these smooth-muscle cell types and may provide one mechanism for adapting contractility in response to physiologic stresses in the lung.

Microvessel precursor smooth muscle cells express head-inserted smooth muscle myosin heavy chain (SM-B) isoform in hyperoxic pulmonary hypertension.

The present study analyzes smooth muscle myosin heavy chain (SMMHC) expression as lung microvascular precursor smooth muscle cells (PSMCs), cells derived from fibroblasts and intermediate cells (immature SMCs), acquire a smooth muscle phenotype in an in vivo model of pulmonary hypertension (PH). Because of the unique contractile properties of the SMMHC isoform SM-B, we analyzed its expression in the microvessels (<100 micrometers diameter) and in larger vessels (100-700 micrometers) quantitatualy by the labeled [strept]avidin-biotin technique (day 1-28), and related this to cell phenotype by transmission microscopy and protein A-gold labeling (at day 28). Airway SMCs of the normal and hypertensive lung uniformly expressed SM-B whereas vascular SMC expression was heterogeneous. Thus, in some large arteries (and veins) SMCs contained cells expressing SM-B while in others all the cells were immunonegative. Microvascular cells expressing SM-B (arteries and veins) were rare in normal lung and numerous in PH, increasing as wall muscle developed in smaller segments with time. As in large vessels, some microvessels had immunopositive cells and others only negative ones. Ultrastructural analysis confirmed that the SMCs of bronchial vessels, and the septal SMCs adjoining alveolar ducts, contained dense filament arrays decorated with SM-B. While the PSMC processes of the normal lung contained sparse filaments decorated with SM-B, these cells expressed dense filament arrays in PH. Fibroblasts migrating to align around the microvessels also expressed SM-B but in the absence of a filament network. For the first time, we demonstrate in vivo that newly developed microvascular PSMCs express the SMMHC SM-B isoform in PH.

Myosin heavy chain isoform expression in rat smooth muscle development.

Smooth muscle myosin heavy chains (MHCs), the motor proteins that power smooth muscle contraction, are produced by alternative splicing from a single gene. The smooth muscle MHC gene is capable of producing four isoforms by utilizing alternative splice sites located at the regions encoding the carboxy terminus and the junction of the 25- and 50-kDa tryptic peptides. These four isoforms, SM1A, SM1B, SM2A, and SM2B, are a combination of one of two heavy chains containing different carboxy-terminal tails (1 or 2) without (A) or with (B) an additional motif in the myosin head. In the present study, using RNA analysis and isoform-specific antibodies, we demonstrate the expression patterns of MHC isoforms during development in rat smooth muscle tissues. RNase protection analysis indicates that the mRNAs for SMA and SMB isoforms, which differ by a 21-nucleotide insertion in the region encoding the S1 head region of the myosin molecule, are differentially expressed during development in a highly tissue-specific manner. Smooth muscle MHC transcripts are first detectable in developing rat smooth muscle tissues at 17 days of fetal development. The SMB mRNA is shown to be expressed in smooth muscle from fetal bladder, intestine, and stomach and from neonatal aorta; however, it is not expressed in cultured smooth muscle cells from rat aorta. The SMA mRNA is also present at all stages of development in the smooth muscles examined; however, it is much less abundant than SMB mRNA in most fetal smooth muscles. We show here that the SMB isoform, which contains a unique seven-amino acid insertion at the junction of the 25- and 50-kDa tryptic peptides, is present in conjunction with SM1 and SM2 tails on immunoblots of smooth muscle from stomach, intestine, bladder, and uterus and is expressed during development in a pattern distinct from that of the SM1 and SM2 tail isoforms.

Myosin Heavy Chain (SMMHC) SM-B Isoform Is Differentially Expressed in Developing and Adult Smooth Muscle Tissues of the Rat

inILD(n=15), and rarefocal staining (<1%) was seen in normal subjects (n=8). However inPPH,APN was posi¬ tive for6of6patients inthemedia andadventitia ofsmall, medium, andlarge vessels, butforonly 3of6patients in theEN layer ofsmall, medium, andlarge vessels; APN staining was present intheadventitia andmedia ofthe plexiform lesions foronly 1of6patients and was absent in theEN oftheplexiform lesions forall 6patients. NEPand ACEwere present inall cell types innormal subjects and inpatients withILD, butthestaining distribution differed fromPPH.NEP was seen inall cell types andvessels in PPH,with theexception that NEPstaining inEN cells of plexiform lesions was absent in 8 of9 patients (score=0.08), which was less thantheNEP scoresfor media(1.4) andadventitia (3.94) oftheplexiform lesions (p<0.0001). ACE staining inplexiform EN was signifi¬ cantly less than inplexiform media(1.0) oradventitia (1.1) (p<0.0012). NEP andACEstaining intheEN ofplexi¬ formlesions was less thanthat intheEN ofsmall, medium, andlarge vessels (p<0.0001 andp<0.0078, respectively) (Fig 1). Nested reversetranscriptase polymerase chain reaction forpeptidases wasperformed on PPHlungs andthose of normal subjects. Representative products weresequenced toconfirm identity. NEPandAPNproducts were present inPPHandnormal lungs butACEmessage waspresent in normallungs only. We have an experimental modelfor plexiformlike lesions invitrobyapplying shear stress to HUVECs ina CellMax apparatus (Quad Instrument; Cellco, NJ). Ourpreliminary findings show an absence of peptidase expression inplump EN oftheplexiformlike lesions stained, as compared tooccasional staining in spindle EN.Whereas expression ofall three peptidases is