Smooth Muscle Myosin Isoforms Research Articles

Impact of anti-S2 Peptides on myosin S2 Isoforms and HCM Mutants

Myosin subfragment-2 (S2) is an unstable coiled coil that influences the binding of myosin heads to actin. Instability in the myosin S2 could underlie one of the causes for hyper-contractility in Hypertrophic Cardiomyopathy (HCM). Supporting this hypothesis, HCM mutations E924K and E930del in myosin S2 reduced myosin S2 coiled coil stability compared to wild type as measured by Forester's Resonance Energy Transfer (FRET) and Gravitational Force Spectroscopy (GFS). To modulate the stability of the S2, anti-S2 peptides called stabilizer and destabilizer peptides because they are designed to increase and decrease the stability of the MYH7 S2 coiled coil, were tested on a range of human S2 peptide sequences. The anti-S2 peptides were tested on myosin S2 peptides across MYH11 (smooth), MYH7 (cardiac), and MYH2 (skeletal) with GFS and FRET. The results demonstrated that the mechanical stability was increased by stabilizer and decreased by destabilizer across the cardiac and skeletal myosin S2 peptides but not for smooth muscle isoform. Dose-response curves for FRET assays indicated that both peptides bound at nanomolar concentrations to wild type cardiac and skeletal, but not smooth muscle isoforms. The wild type cardiac S2 peptide required more force to unzip than skeletal, smooth, or mutant peptides in the absence of stabilizer peptide. Effectively, the stabilizer peptide improved the stability of both HCM mutants (E924K and E930del). The stabilizer peptide enhanced dimer formation in mutant cardiac, and skeletal myosin S2 peptides, and destabilizer increased flexibility of cardiac and skeletal myosin S2 wild type peptide. This study demonstrates that the structural stability of myosin S2 can be altered by designed small peptides as well as mutations. These anti-S2 peptides share similar specificities and high affinities for the tested striated muscle isoforms but not the smooth muscle myosin isoform.

Blebbistatin modulates prostatic cell growth and contrapctility through myosin II signaling.

To investigate the effect of blebbistatin (BLEB, a selective myosin inhibitor) on regulating contractility and growth of prostate cells and to provide insight into possible mechanisms associated with these actions. BLEB was incubated with cell lines of BPH-1 and WPMY-1, and intraprostatically injected into rats. Cell growth was determined by flow cytometry, and in vitro organ bath studies were performed to explore muscle contractility. Smooth muscle (SM) myosin isoform (SM1/2, SM-A/B, and LC17a/b) expression was determined via competitive reverse transcriptase PCR. SM myosin heavy chain (MHC), non-muscle (NM) MHC isoforms (NMMHC-A and NMMHC-B), and proteins related to cell apoptosis were further analyzed via Western blotting. Masson's trichrome staining was applied to tissue sections. BLEB could dose-dependently trigger apoptosis and retard the growth of BPH-1 and WPMY-1. Consistent with in vitro effect, administration of BLEB to the prostate could decrease rat prostatic epithelial and SM cells via increased apoptosis. Western blotting confirmed the effects of BLEB on inducing apoptosis through a mechanism involving MLC20 dephosphorylation with down-regulation of Bcl-2 and up-regulation of BAX and cleaved caspase 3. Meanwhile, NMMHC-A and NMMHC-B, the downstream proteins of MLC20, were found significantly attenuated in BPH-1 and WPMY-1 cells, as well as rat prostate tissues. Additionally, BLEB decreased SM cell number and SM MHC expression, along with attenuated phenylephrine-induced contraction and altered prostate SMM isoform composition with up-regulation of SM-B and down-regulation of LC17a, favoring a faster contraction. Our novel data demonstrate BLEB regulated myosin expression and functional activity. The mechanism involved MLC20 dephosphorylation and altered SMM isoform composition.

The expression and functional activities of smooth muscle myosin and non-muscle myosin isoforms in rat prostate.

Benign prostatic hyperplasia (BPH) is mainly caused by increased prostatic smooth muscle (SM) tone and volume. SM myosin (SMM) and non‐muscle myosin (NMM) play important roles in mediating SM tone and cell proliferation, but these molecules have been less studied in the prostate. Rat prostate and cultured primary human prostate SM and epithelial cells were utilized. In vitro organ bath studies were performed to explore contractility of rat prostate. SMM isoforms, including SM myosin heavy chain (MHC) isoforms (SM1/2 and SM‐A/B) and myosin light chain 17 isoforms (LC 17a/b), and isoform ratios were determined via competitive RT‐PCR. SM MHC and NM MHC isoforms (NMMHC‐A, NMMHC‐B and NMMHC‐C) were further analysed via Western blotting and immunofluorescence microscopy. Prostatic SM generated significant force induced by phenylephrine with an intermediate tonicity between phasic bladder and tonic aorta type contractility. Correlating with this kind of intermediate tonicity, rat prostate mainly expressed LC 17a and SM1 but with relatively equal expression of SM‐A/SM‐B at the mRNA level. Meanwhile, isoforms of NMMHC‐A, B, C were also abundantly present in rat prostate with SMM present only in the stroma, while NMMHC‐A, B, C were present both in the stroma and endothelial. Additionally, the SMM selective inhibitor blebbistatin could potently relax phenylephrine pre‐contracted prostate SM. In conclusion, our novel data demonstrated the expression and functional activities of SMM and NMM isoforms in the rat prostate. It is suggested that the isoforms of SMM and NMM could play important roles in BPH development and bladder outlet obstruction.

In Vitro Smooth Muscle Hypercontractility Induced by Cd4+ T Cells is Transient

Smooth muscle (SM) hypercontractility is a characteristic of several pathologies, including asthma. Inflammatory cells and/or the cytokines they release may alter contractile protein expression, thereby also altering the SM mechanics. We characterized the maximal unloaded shortening velocity (Vmax), stress (force/cross sectional area), and contractile protein expression of Brown Norway rat airway SM co-cultured with activated CD4+ T cells for 24h and 48h. After 24h incubation, Vmax (mean±SEM) was significantly increased (0.15±0.01 l/s vs. 0.29±0.02 l/s; control vs. T cells, p = 0.0012). However, this alteration in Vmax was transient and disappeared after 48h of co-incubation (0.21±0.01 vs. 0.25±0.01 l/s, p = 0.099). No significant differences in stress were observed at 24h or 48h. Western blot analysis showed a significant increase in the levels of MLCK (0.20±0.03 vs. 0.41± 0.09; control vs. T cells; p<0.05, p=0.018) and the (+)insert SM myosin isoform (0.55±0.04 vs. 0.81±0.05; p = 0.0023) after 24h incubation with the CD4+ T cells. These alterations were also transient; all values returned to baseline after 48h exposure to the CD4+ T cells. Thus, the increase in Vmax was correlated with the increase in MLCK and (+)insert isoform expression. Conversely, there were no differences in levels of total SM myosin heavy chain or α-smooth muscle actin, suggesting there was no increase in muscle mass. These results show that indeed inflammatory cells and/or their mediators can alter SM contractile protein expression and SM function, but that these effects may be transient. This transient behavior may be important to consider when studying SM function in disease.Supported by: CIHR, NIH-RO1HL103405.

Human Tonic and Phasic Smooth Muscle Myosin Isoforms Are Unresponsive to the Loop 1 Insert

Smooth muscle myosin gene products include two isoforms, SMA and SMB, differing by a 7-residue peptide in loop 1 (i7) at the myosin active site where ATP is hydrolyzed. Using chicken isoforms, previous work indicated that the i7 deletion in SMA prolongs strong actin binding by inhibiting active site ingress and egress of nucleotide when compared to i7 inserted SMB. Additionally, i7 deletion inhibits Pi release associated with the switch 2 closed → open transition in actin-activated ATPase. Switch 2 is far from loop 1 indicating i7 deletion has an allosteric effect on Pi release. Chicken SMA and SMB have unknown and robust nucleotide-sensitive tryptophan (NST) fluorescence increments, respectively. Human SMA and SMB both lack NST increments while Pi release in Ca2+ ATPase is not impacted by i7 deletion. The NST reports relay helix movement following conformation change in switch 2 but in the open → closed transition. The NST is common to all known myosin isoforms except human smooth muscle. Other independent works on human SMA and SMB motility indicate no functional effect of i7 deletion. Smooth muscle myosin is a stunning example of species-specific myosin structure/function divergence underscoring the danger in extrapolating disease-linked mutant effects on myosin across species.

Thick Filament Length and Isoform Composition Determine Self-Organized Contractile Units in Actomyosin Bundles

Diverse myosin II isoforms regulate contractility of actomyosin bundles in disparate physiological processes by variations in both motor mechanochemistry and the extent to which motors are clustered into thick filaments. Although the role of mechanochemistry is well appreciated, the extent to which thick filament length regulates actomyosin contractility is unknown. Here, we study the contractility of minimal actomyosin bundles formed in vitro by mixtures of F-actin and thick filaments of nonmuscle, smooth, and skeletal muscle myosin isoforms with varied length. Diverse myosin II isoforms guide the self-organization of distinct contractile units within in vitro bundles with shortening rates similar to those of in vivo myofibrils and stress fibers. The tendency to form contractile units increases with the thick filament length, resulting in a bundle shortening rate proportional to the length of constituent myosin thick filament. We develop a model that describes our data, providing a framework in which to understand how diverse myosin II isoforms regulate the contractile behaviors of disordered actomyosin bundles found in muscle and nonmuscle cells. These experiments provide insight into physiological processes that use dynamic regulation of thick filament length, such as smooth muscle contraction.

Ileal smooth muscle dysfunction and remodeling in cystic fibrosis

Patients with cystic fibrosis (CF) often suffer from gastrointestinal cramps and intestinal obstruction. The CF transmembrane conductance regulator (CFTR) channel has been shown to be expressed in vascular and airway smooth muscle (SM). We hypothesized that the absence of CFTR expression alters the gastrointestinal SM function and that these alterations may show strain-related differences in the mouse. The aim of this study was to measure the contractile properties of the ileal SM in two CF mouse models. CFTR(-/-) and CFTR(+/+) mice were studied on BALB/cJ and C57BL/6J backgrounds. Responsiveness of ileal strips to electrical field stimulation (EFS), methacholine (MCh), and isoproterenol was measured. The mass and the cell density of SM layers were measured morphometrically. Finally, the maximal velocity of shortening (Vmax) and the expression of the fast (+)insert myosin isoform were measured in the C57BL/6J ileum. Ileal hyperreactivity was observed in response to EFS and MCh in CFTR(-/-) compared with CFTR(+/+) mice in C57BL/6J background. This latter observation was not reproduced by acute inhibition of CFTR with CFTR(inh)172. BALB/cJ CFTR(-/-) mice exhibited a significant increase of SM mass with a lower density of cells compared with CFTR(+/+), whereas no difference was observed in the C57BL/6J background. In addition, in this latter strain, ileal strips from CFTR(-/-) exhibited a significant increase in Vmax compared with control and expressed a greater proportion of the fast (+)insert SM myosin isoform with respect to total myosin. BALB/cJ CFTR(-/-) ilium had a greater relaxation to isoproterenol than the CFTR(+/+) mice when precontracted with EFS, but no difference was observed in response to exogeneous MCh. In vivo, the lack of CFTR expression induces a different SM ileal phenotype in different mouse strains, supporting the importance of modifier genes in determining intestinal SM properties.

Generation of a human urinary bladder smooth muscle cell line

We report a cell line (hBSM) established from human urinary bladder wall smooth muscle that maintains most of the phenotypic characteristics of smooth muscle cells. Cells were dissociated from the muscular layer with collagenase (1mg/ml) and collected and grown in M199 supplemented with 10% fetal calf serum and 1% antibiotic-antimycotic. Primary cultures were grown for 2d and small colonies were isolated by placing glass rings around the colonies. These colonies were picked up with a fine-tipped Pasteur pipette and subcultured. This procedure was repeated several times until a culture with a uniform stable morphology was obtained. hBSM cells are elongated with tapered ends, and in high density cultures, they form swirls of cells arranged in parallel. These cells have a doubling time of approximately 72 h. Western blotting and immunofluorescence microscopy revealed stable expression of smooth muscle-specific proteins, including myosin isoforms (N-terminal isoforms SM-A/B and C-terminal isoforms SM1/2), SM22, α-smooth muscle actin, h-caldesmon, Ca(2+)-dependent myosin light chain kinase, and protein kinase G. These cells contract upon exposure to 10μM bethanechol and this contraction is reversible by washing away the drug. Karyotyping showed tetraploidy with a modal chromosome number of 87, with multiple rearrangements. To our knowledge, the hBSM cell line is the first human cell line established from bladder wall smooth muscle that expresses both N- and C-terminal smooth muscle myosin isoforms. This cell line will provide a valuable tool for studying transcriptional regulation of smooth muscle myosin isoforms and effects of drugs on cellular function.

Myosin Isoform-Specificity of Contractile Elements Formed in Reconstituted Actomyosin Bundles

Forces generated by the contraction of actomyosin bundles underlie diverse physiological processes including muscle contraction, cell migration and cytokinesis. While actomyosin force transmission is well understood in the context of organized sarcomeres found in striated muscle, very little is known about the nature of actomyosin force transmission in non-sarcomeric bundles such as those found in smooth and non-muscle cells. We recently showed that contractile elements spontaneously arise during the contraction of disordered actomyosin bundles reconstituted solely from actin filaments and smooth muscle myosin. In turn, the contraction of disordered actomyosin bundles arises from a several contractile elements linked in series resulting in a bundle contraction velocity linearly proportional to bundle length and a length-independent contractile force. The physical properties of the contractile elements, including the rate of contraction, maximum contractile strain and the maximum force generated, depended on the relative quantities of actin and myosin. Here, we demonstrate that reconstituted actomyosin bundles formed with skeletal muscle myosin form qualitatively similar contractile elements with similar dependencies on myosin concentration. However, the physical properties of the contractile units formed with skeletal muscle myosin dramatically differ from those formed with smooth muscle myosin, in a fashion consistent with differences in the myosin mechanochemistry and thick filament geometry of smooth and skeletal muscle myosin. Contractile elements formed by skeletal muscle myosin contract significantly faster, generate more force and can shorten to greater extents than those formed with smooth muscle myosin. Our results demonstrate that both smooth and skeletal muscle myosin isoforms can facilitate the spontaneous assembly of contractile elements within disordered actomyosin bundles and provide insight into the mechanisms underlying contraction in non-sarcomeric actomyosin bundles.

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

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, LC(17b), 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.

Update on Corpus Cavernosum Smooth Muscle Contractile Pathways in Erectile Function: A Role for Testosterone?

Normal erectile function (EF) involves a coordinated relaxation of the arteries that supply the penis and the corpus cavernosum smooth muscle (CCSM), resulting in expansion of the sinusoids and increased intracavernous pressure. But the CCSM spends the majority of its time in the contracted state which is mediated by norepinephrine released from nerve endings and other vasoconstrictors like endothelins released from the endothelium. These agents cause smooth muscle myosin (SMM) phosphorylation by elevating intracellular calcium. When calcium returns to basal levels, the calcium sensitivity increases and prevents myosin dephosphorylation, which involves the RhoA/Rho-kinase (ROK) mechanism, thus maintaining force. Although mounting evidences demonstrate that androgens have a major influence on EF that is not just centrally mediated, this notion remains quite controversial. To summarize the current knowledge on CCSM contractile pathways, the role they play in modulating EF, and the influence of androgens. The article reviews the literature and contains some previously unpublished data on CCSM contraction signaling including the role that androgens are known to play in modulating these pathways. Data from peer-reviewed publications and previously unpublished observations. In addition to downregulation of many pro-erectile molecular mechanisms, decreased testosterone (T) levels upregulate CCSM contractility, including hyperresponsiveness to α-adrenergic agonists, increased SMM phosphorylation, alteration of SMM isoform composition, activation of RhoA/ROK signaling and modulation of sphingosine-1-phosphate regulation of CCSM tone. Decreased T levels upregulate CCSM contractile signaling. Meanwhile, it downregulates CCSM relaxation pathways synergizing to produce erectile dysfunction (ED). Although some urologists and researchers are still skeptical of the influence of androgens on penile erection, understanding these molecular control mechanisms as well as the influence that androgens have on these pathways should provide new evidence supporting the roles of androgens in EF and enhance the discovery of novel targets for drug development to treat ED.

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 ...

Human Bone Marrow-Derived CD133+Cells Delivered to a Collagen Patch on Cryoinjured Rat Heart Promote Angiogenesis and Arteriogenesis

Transplanting hematopoietic and peripheral blood-derived stem/progenitor cells can have beneficial effects in slowing the effects of heart failure. We investigated whether human bone marrow CD133(+)-derived cells (BM-CD133(+) cells) might be used for cell therapy of heart injury in combination with tissue engineering. We examined these cells for: 1) their in vitro capacity to be converted into cardiomyocytes (CMs), and 2) their potential for in vivo differentiation when delivered to a tissue-engineered type I collagen patch placed on injured hearts (group II). To ensure a microvascular network ready for use by the transplanted cells, cardiac injury and patching were scheduled 2 weeks before cell injection. The cardiovascular potential of the BM-CD133(+) cells was compared with that of a direct injection (group I) of the same cells in heart tissue damaged according to the same schedule as for group II. While a small fraction (2 ± 0.5%) of BM-CD133(+)cells cocultured with rat CMs switched in vitro to a CM-like cell phenotype, in vivo-and in both groups of nude rats transplanted with BM-CD133(+)--there was no evidence of any CM differentiation (as detected by cardiac troponin I expression), but there were signs instead of new capillaries and small arterioles. While capillaries prevailed over arterioles in group II, the opposite occurred in group I. The transplanted cells further contributed to the formation of new microvessels induced by the patch (group II) but the number of vessels did not appear superior to the one developed after directly injecting the BM-CD133(+)cells into the injured heart. Although chimeric human-rat microvessels were consistently found in the hearts of both groups I and II, they represented a minority (1.5-2.3%) compared with those of rat origin. Smooth muscle myosin isoform expression suggested that the arterioles achieved complete differentiation irrespective of the presence or absence of the collagen patch. These findings suggest that: 1) BM-CD133(+) cells display a limited propensity for in vitro conversion to CMs; 2) the preliminarily vascularized bioscaffold did not confer a selective homing and differentiation advantage for the phenotypic conversion of BM-CD133(+) cells into CMs; and 3) combined patching and cell transplantation is suitable for angiogenesis and arteriogenesis, but it does not produce better results, in terms of endothelial and smooth muscle cell differentiation, than the "traditional" method of cell injection into the myocardium.

TRANSCRIPTION FACTOR NF-κB MEDIATES THE GENE EXPRESSION OF PROTEINS INVOLVED IN THE REGULATION OF CALCIUM SENSITIZATION PATHWAY IN PARTIAL BLADDER OUTLET OBSTRUCTION IN MICE

Growing clinical evidence suggests that benign prostatic hyperplasia induced partial bladder outlet obstruction is associated with an increased incidence of erectile dysfunction. We determined whether corpus cavernosum smooth muscle from rabbits with partial bladder outlet obstruction show any molecular or functional differences versus controls.Force generation and relaxation of corpus cavernosum smooth muscle 2 weeks after partial bladder outlet obstruction by 125 mM. KCl, phenylephrine and field stimulation were determined. Expression of total smooth muscle myosin and alternatively spliced smooth muscle myosin isoforms were determined by reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative competitive RT-PCR, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Corpus cavernosum smooth muscle from sections of the penis were analyzed morphologically by immunofluorescence microscopy using antibodies to smooth muscle myosin and neurofilament protein.Corpus cavernosum smooth muscle from rabbits with partial bladder outlet obstruction generated 40% to 50% more force than that of sham operated rabbits in response to KCl or phenylephrine and was more difficult to relax. Although quantitative competitive RT-PCR and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that corpus cavernosum smooth muscle from rabbits with partial bladder outlet obstruction expressed only slightly more total smooth muscle myosin at the messenger RNA and protein levels, expression of the high adenosine triphosphatase isoform SM-B increased 2-fold. Morphological examination of corpus cavernosum smooth muscle sections revealed decreased innervation and increased smooth muscle bundle size.We present the novel finding of molecular and functional changes in the corpus cavernosum smooth muscle associated with partial bladder outlet obstruction. Although the inclusion of sham operation ruled out direct injury during surgery, a change in corpus cavernosum smooth muscle innervation induced by nerve compression by the ligature is likely to induce trophic changes in the corpus cavernosum smooth muscle leading to over expression of the SM-B smooth muscle myosin isoform, increased contractility and impaired relaxation.

Ablation of smooth muscle myosin heavy chain SM2 increases smooth muscle contraction and results in postnatal death in mice

The physiological relevance of smooth muscle myosin isoforms SM1 and SM2 has not been understood. In this study we generated a mouse model specifically deficient in SM2 myosin isoform but expressing SM1, using an exon-specific gene targeting strategy. The SM2 homozygous knockout (SM2(-/-)) mice died within 30 days after birth, showing pathologies including segmental distention of alimentary tract, retention of urine in renal pelvis, distension of bladder, and the development of end-stage hydronephrosis. In contrast, the heterozygous (SM2(+/-)) mice appeared normal and reproduced well. In SM2(-/-) bladder smooth muscle the loss of SM2 myosin was accompanied by a concomitant down-regulation of SM1 and a reduced number of thick filaments. However, muscle strips from SM2(-/-) bladder showed increased contraction to K(+) depolarization or in response to M3 receptor agonist Carbachol. An increase of contraction was also observed in SM2(-/-) aorta. However, the SM2(-/-) bladder was associated with unaltered regulatory myosin light chain (MLC20) phosphorylation. Moreover, other contractile proteins, such as alpha-actin and tropomyosin, were not altered in SM2(-/-) bladder. Therefore, the loss of SM2 myosin alone could have induced hypercontractility in smooth muscle, suggesting that distinctly from SM1, SM2 may negatively modulate force development during smooth muscle contraction. Also, because SM2(-/-) mice develop lethal multiorgan dysfunctions, we propose this regulatory property of SM2 is essential for normal contractile activity in postnatal smooth muscle physiology.

Expression of myosin isoforms in the smooth muscle of human corpus cavernosum

The molecular interaction between smooth muscle (SM) myosin and actin in the corpus cavernosum (CC) determines the erectile state of the penis. A key mechanism regulating this interaction and subsequent development and maintenance of force is alternative splicing of SM myosin heavy chain (MHC) and 17 kDa essential SM myosin light chain (MLC) pre-mRNAs. Our aim was to examine the relative SM myosin isoform composition in human CC. Tissue samples were obtained from 18 patients with erectile dysfunction (ED), Peyronie's disease, or both. One specimen was obtained during a transgender operation. Patients then were stratified according to presence of diabetes mellitus, hypertension, ED, or Peyronie's disease, as well as failure of phosphodiesterase-5 (PDE5) inhibitors and history of previous pelvic or penile surgeries, radiation, or both. Our results revealed that all human CC samples expressed only the SM-A isoform. There was a predominance of SM2 isoform mRNA relative to SM1 across all samples, with a mean of 63.8%, which correlated with protein analysis by gel electrophoresis. A statistically significant difference was found between patients who had undergone previous pelvic surgery, radiation, or both and those who did not. The ratio of LC(17b) to LC(17a) was approximately 1:1 for all patients, with a mean of 48.9% LC(17b). Statistical difference was seen in the relative ratio of LC(17b) to LC(17a) among the group who failed conservative therapy with PDE5 inhibitors compared with all others. In conclusion, we determined the SM myosin isoform composition of human CC and present for the first time differences in relative myosin isoform expression among patients with several risk factors contributing to their cause of ED. Our data reflect the fact that alternative splicing events in the MHC and 17 kDa MLC pre-mRNA may be a possible molecular mechanism involved in the altered contractility of the CCSM in patients with ED.

Smooth muscle myosin isoform expression and LC20phosphorylation in innate rat airway hyperresponsiveness

Four smooth muscle myosin heavy chain (SMMHC) isoforms are generated by alternative mRNA splicing of a single gene. Two of these isoforms differ by the presence [(+)insert] or absence [(-)insert] of a 7-amino acid insert in the motor domain. The rate of actin filament propulsion of the (+)insert SMMHC isoform, as measured in the in vitro motility assay, is twofold greater than that of the (-)insert isoform. We hypothesized that a greater expression of the (+)insert SMMHC isoform and greater regulatory light chain (LC(20)) phosphorylation contribute to airway hyperresponsiveness. We measured airway responsiveness to methacholine in Fischer hyperresponsive and Lewis normoresponsive rats and determined SMMHC isoform mRNA and protein expression, as well as essential light chain (LC(17)) isoforms, h-caldesmon, and alpha-actin protein expression in their tracheae. We also measured tracheal muscle strip contractility in response to methacholine and corresponding LC(20) phosphorylation. We found Fischer rats have more (+)insert mRNA (69.4 +/- 2.0%) (mean +/- SE) than Lewis rats (53.0 +/- 2.4%; P < 0.05) and a 44% greater content of (+)insert isoform relative to total myosin protein. No difference was found for LC(17) isoform, h-caldesmon, and alpha-actin expression. The contractility experiments revealed a greater isometric force for Fischer trachealis segments (4.2 +/- 0.8 mN) than Lewis (1.9 +/- 0.4 mN; P < 0.05) and greater LC(20) phosphorylation level in Fischer (55.1 +/- 6.4) than in Lewis (41.4 +/- 6.1; P < 0.05) rats. These results further support the contention that innate airway hyperresponsiveness is a multifactorial disorder in which increased expression of the fast (+)insert SMMHC isoform and greater activation of LC(20) lead to smooth muscle hypercontractility.

Force Generated by Pulmonary Arterial Smooth Muscle Cells: Spatially Distinct Cell Phenotypes from Normal and Hypertensive Arteries

Porcine pulmonary arterial smooth muscle cells (smcs) from inner and outer medial layers have distinct structural phenotypes in both normal and hypertensive pulmonary arteries. We hypothesised that force generated by smcs of the inner and outer media would differ and increase in pulmonary hypertension. Smcs were cultured from the inner (I) 25% and outer (O) 75% of the pulmonary artery wall of 4 normal 14 day old piglets (N) and 4 animals exposed to chronic hypobaric hypoxia from 3 to 14 days (H). The cytoskeletal structure of each cell isolate was studied by immunofluorescence. The basal force/time profile and response to U46619 (thromboxane mimetic) and sodium nitroprusside (SNP) were measured using a Culture Force Monitor. Smcs from all 4 sites expressed smooth muscle actin specific actin and myosin isoforms. Hsmc had shorter actin stress fibres and were less well aligned than Nsmc. HOsmcs had more cell substrate adhesions than other isolates. In a 3D collagen matrix all cell types elongated parallel to the axis of strain. Normal Osmcs exerted a greater force then the NIsmcs (528±49.6 v 349±35 dynes, p<0.05). Rapid relaxation to SNP and increase in tension to U46619 was found in both I and O smcs. In smcs from hypertensive vessels Osmcs showed a striking reduction in contraction compared with NOsmcs (177±43dynes, p<0.05) and a slow rate of force development (p<0.05). Maximum force and rate of force development by HIsmcs were normal. HOsmcs had a stable, abnormal structural phenotype that was unexpectedly associated with decreased not increased force generation. Supported by the British Heart Foundation

505: Effect of Partial Outlet Obstruction on the Expression of Smooth Muscle Myosin Isoforms in Rat Ureter

You have accessJournal of UrologyModerated Poster, Sunday, May 22, 2005, 3:30 - 5:30 pm1 Apr 2005505: Effect of Partial Outlet Obstruction on the Expression of Smooth Muscle Myosin Isoforms in Rat Ureter Paul M. Milhoua, Philip T. Koi, Michael E. DiSanto, and Arnold Melman Paul M. MilhouaPaul M. Milhoua More articles by this author , Philip T. KoiPhilip T. Koi More articles by this author , Michael E. DiSantoMichael E. DiSanto More articles by this author , and Arnold MelmanArnold Melman More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(18)34745-1AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "505: Effect of Partial Outlet Obstruction on the Expression of Smooth Muscle Myosin Isoforms in Rat Ureter." The Journal of Urology, 173(4S), pp. 137–138 © 2016 by American Urological AssociationFiguresReferencesRelatedDetails Volume 173Issue 4SApril 2005Page: 137-138 Advertisement Copyright & Permissions© 2016 by American Urological AssociationMetricsAuthor Information Paul M. Milhoua More articles by this author Philip T. Koi More articles by this author Michael E. DiSanto More articles by this author Arnold Melman More articles by this author Expand All Advertisement PDF downloadLoading ...

REGIONAL ALTERATIONS IN THE EXPRESSION OF SMOOTH MUSCLE MYOSIN ISOFORMS IN RESPONSE TO PARTIAL BLADDER OUTLET OBSTRUCTION

Smooth muscle (SM) myosin (SMM) isoform composition is altered in response to partial bladder outlet obstruction (PBOO). A recent study showed that during PBOO the upper dome region of the bladder is subjected to greater expansion pressure than the base and regional differences in contractility exist in the detrusor of PBOO rabbits. We hypothesized that alteration in SMM isoform composition in response to PBOO may show regional heterogeneity. Detrusor samples were obtained from 9 defined regions of the bladders from dysfunctional PBOO rabbits (greater than 30 voids per 24 hours) and sham operated adult New Zealand White rabbits. Reverse transcriptase-polymerase chain reaction, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting were used to determine the relative levels of SMM isoform expression at the mRNA and protein levels. Contractile responses to bethanechol and KCl were also determined. Myosin isoform expression was uniform throughout the detrusor from sham operated subjects with all regions expressing SM-B almost completely. However, in response to PBOO the dome region showed approximately 70% SM-B and 30% SM-A isoforms, whereas the base region expressed only 35% SM-B and, thus, 65% SM-A. This change also correlated with an approximately 2-fold higher protein level expression of SM-B in the dome region of PBOO rabbit bladders. Expression of the SMemb SMM isoform was significantly increased in PBOO rabbits at the mRNA and protein levels but only in the dome region. Regional differences in SMM isoform expression in the PBOO rabbit bladders correlated with altered contractility. Alteration in SMM isoform composition in response to PBOO shows regional heterogeneity and may be involved in the mechanism responsible for regional localized differences in detrusor contractility in PBOO rabbits.