Pig brain homogenates contain smooth muscle myosin and cytoplasmic myosin isoforms

Summary Three myosin isoforms, two of smooth muscle and one of cytoplasmic origin, were found in porcine brain by Western blotting analysis with antibodies specific for smooth and cytoplasmic myosins. The smooth muscle isoforms comprise at least 30% of the total myosin present. Brain tissue is therefore not a suitable source for the isolation of pure cytoplasmic contractile proteins.Abbreviations DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - FITC fluorescein isothiocyanate - PBS phosphate buffered saline - SDS PAGE polyacrylamide electrophoresis in the presence of sodium dodecylsulphate - TRIS tris(hydroxymethyl)aminomethane     

Nonmuscle and smooth muscle myosin isoforms in bovine endothelial cells

A panel of monoclonal antibodies, specific for human platelet (NM-A9, NM-F6, and NM-G2) and for bovine smooth muscle (SM-E7) myosin heavy chains (MHC), were used to study the composition and the distribution of myosin isoforms in bovine endothelial cells (EC), in vivo and in vitro. Using indirect and double immunofluorescence techniques, we have found that in the intact aortic endothelium there is expression of nonmuscle MHC (NM-MHC), exclusively. By contrast, hepatic sinusoidal endothelium as well as cultured bovine aortic EC (BAEC) in the subconfluent phase of growth show coexistence of NM- and smooth muscle MHC (SM-MHC) isoforms. SM myosin immunoreactivity disappears when cultured BAEC become confluent. In this phase of cell growth, NM-MHC isoforms are localized differently within the cells, i.e., in the cytoplasm around the nucleus or in the cortical, submembranous region of EC cytoplasm. A third type of intracellular distribution of NM-MHC immunoreactivity was evident in the cell periphery of binucleated, confluent BAEC. These data indicate that (1) several myosin isoforms are differently distributed in bovine endothelia; and (2) SM myosin expression and the specific subcellular localization of NM myosin isoforms within EC might be regulated by cell-cell interactions.     

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.     

Altered expression of skeletal muscle myosin isoforms in cancer cachexia

Cachexia is commonly seen in cancer and ischaracterized by severe muscle wasting, but little is known about theeffect of cancer cachexia on expression of contractile protein isoforms such as myosin. Other causes of muscle atrophy shift expression ofmyosin isoforms toward increased fast (type II) isoform expression. Weinjected mice with murine C-26 adenocarcinoma cells, a tumor cell linethat has been shown to cause muscle wasting. Mice were killed 21 daysafter tumor injection, and hindlimb muscles were removed. Myosin heavychain (MHC) and myosin light chain (MLC) content was determined inmuscle homogenates by SDS-PAGE. Body weight was significantly lower intumor-bearing (T) mice. There was a significant decrease in muscle massin all three muscles tested compared with control, with the largestdecrease occurring in the soleus. Although no type IIb MHC was detectedin the soleus samples from control mice, type IIb comprised 19% of thetotal MHC in T soleus. Type I MHC was significantly decreased in T vs. control soleus muscle. MHC isoform content was not significantly different from control in plantaris and gastrocnemius muscles. Thesedata are the first to show a change in myosin isoform expression accompanying muscle atrophy during cancer cachexia.

     

Role of myosin phosphatase isoforms in cGMP-mediated smooth muscle relaxation

In vitro experiments showing the activation of the myosin phosphatase via heterophilic leucine zipper interactions between its targeting subunit (MYPT1) and cGMP-dependent protein kinase I suggested a pathway for smooth muscle relaxation (Surks, H. K., Mochizuki, N., Kasai, Y., Georgescu, S. P., Tang, K. M., Ito, M., Lincoln, T. M., and Mendelsohn, M. E. (1999) Science 286, 1583-1587). The relationship between MYPT1 isoform expression and smooth muscle responses to cGMP signaling in vivo has not been explored. MYPT1 isoforms that contain or lack a C-terminal leucine zipper are generated in birds and mammals by cassette-type alternative splicing of a 31-nucleotide exon. The avian and mammalian C-terminal isoforms are highly conserved and expressed in a tissue-specific fashion. In the mature chicken the tonic contracting aorta and phasic contracting gizzard exclusively express the leucine zipper positive and negative MYPT1 isoforms, respectively. Expression of the MYPT1 isoforms is also developmentally regulated in the gizzard, which switches from leucine zipper positive to negative isoforms around the time of hatching. This switch coincides with the development in the gizzard of a cGMP-resistant phenotype, i.e. inability to dephosphorylate myosin and relax in response to 8-bromo-cGMP after calcium activation. Furthermore, association of cGMP-dependent protein kinase I with MYPT1 is detected by immunoprecipitation only in the tissue that expresses the leucine zipper positive isoform of MYPT1. These results suggest that the regulated splicing of MYPT1 is an important determinant of smooth muscle phenotypic diversity and the variability in the response of smooth muscles to the calcium desensitizing effect of cGMP signaling.     

Chloride cotransport in the membrane of earthworm body wall muscles

The resting membrane potential (V(m)) of isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris was studied by glass microelectrodes. The inhibition of chloride permeability by low pH did not affect V(m) of the muscle fibers in isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris which was -48.7 mV (inside negative) at pH 7.3 and -49.1 at pH 5.6. On the other hand, bathing the muscles in Cl(-) and Na(+)-free solutions, or application of the chloride transporter inhibitor furosemide and Na(+)-K(+)-ATPase inhibitor ouabain depolarized the V(m) by 3-5 mV. The effects of a Cl(-) -free solution and ouabain were not additive. This demonstrates relatively small contribution of equilibrium potential for Cl(-) to the resting membrane potential and electrogenic effect of Na(+)K(+)-ATPase which is dependent on the supply of Na(+)(i) ions by furosemide-sensitive and Cl(-)(e)- and Na(+)(e)-dependent electroneutral transport (most probably Na(+)K(+)Cl(-) cotransport).     

Embryonic chicken gizzard: smooth muscle and non-muscle myosin isoforms

Antibodies to smooth muscle and non-muscle myosin allow the development of smooth muscle and its capillary system in the embryonic chicken gizzard to be followed by immunofluorescent techniques. Although smooth muscle development proceeds in a serosal to luminal direction, angiogenetic cell clusters develop independently at the luminal side close to the epithelial layer, and the presumptive capillaries invade the developing muscle in a luminal to serosal direction. The smooth muscle and non-muscle myosin heavy chains in this avian system cannot be separated by SDS polyacrylamide gel electrophoresis and do not show isoform specificity in immunoblotting, unlike the system found in mammals. Only two myosin heavy chains with M_r of 200 and 196 kDa were separable and considerable immunological cross-reactivity was found between the denatured myosin isoform heavy chains.     

Functional characterization of vertebrate nonmuscle myosin IIB isoforms using Dictyostelium chimeric myosin II

The alternatively spliced isoform of nonmuscle myosin II heavy chain B (MHC-IIB) with an insert of 21 amino acids in the actin-binding surface loop (loop 2), MHC-IIB(B2), is expressed specifically in the central nervous system of vertebrates. To examine the role of the B2 insert in the motor activity of the myosin II molecule, we expressed chimeric myosin heavy chain molecules using the Dictyostelium myosin II heavy chain as the backbone. We replaced the Dictyostelium native loop 2 with either the noninserted form of loop 2 from human MHC-IIB or the B2-inserted form of loop 2 from human MHC-IIB(B2). The transformant Dictyostelium cells expressing only the B2-inserted chimeric myosin formed unusual fruiting bodies. We then assessed the function of chimeric proteins, using an in vitro motility assay and by measuring ATPase activities and binding to F-actin. We demonstrate that the insertion of the B2 sequence reduces the motor activity of Dictyostelium myosin II, with reduction of the maximal actin-activated ATPase activity and a decrease in the affinity for actin. In addition, we demonstrate that the native loop 2 sequence of Dictyostelium myosin II is required for the regulation of the actin-activated ATPase activity by phosphorylation of the regulatory light chain.     

Expression of myosin heavy chain isoforms in developing rat muscle spindles

Summary The development of muscle spindles, with respect to the expression of myosin heavy chain isoforms was studied in rat hind limbs from 17 days of gestation up to seven days after birth. Serial cross-sections were labelled with antibodies against slow tonic, slow twitch and neonatal isomyosins, myomesin, laminin and neurofilament protein.At 17–18 days of gestation, a small population of primary myotubes expressing slow tonic myosin were identified as the earliest spindle primordia. These myotubes also expressed slow twitch and, to a lesser extent, neonatal myosin. At 19–20 days of gestation a second myotube became apparent; this staining strongly with anti-neonatal myosin. A day later this secondary myotube acquired reactivity to anti-slow tonic and anti-slow twitch myosins. By birth, a third myotube was present; this staining strongly with anti-neonatal myosin but otherwise unreactive with the other antibodies against myosin heavy chains. Three days after birth a fourth myotube, with identical reactivity to the third one, became apparent. Regional variation in the expression of isomyosins, which was present since birth in the two nuclear bag fibers was further enhanced: the nuclear bag₂ staining strongly with anti-slow tonic and antineonatal in the equatorial region and with decreasing intensity towards the poles, whilst with anti-slow twitch the stainability was low in the equatorial and high in the polar region. The nuclear bag₁ fiber showed a homogeneous staining: high with anti-slow tonic, moderate with anti-neonatal, and displayed stainability to antislow twitch myosin in the polar regions only. No regional variation was found along the chain fiber/myotube. At seven days after birth, the pattern of reactivity was similar to that found in the adult spindles, except for the bag₁ fiber which still expressed neonatal myosin.We show that slow tonic myosin is expressed from early development and it is a reliable marker of developing bag fibers. We suggest that muscle spindles are formed from special cell lineages of which the primary generation myotubes expressing slow tonic myosin represent the primordium of muscle spindles.     

Expression of myosin heavy chain isoforms in developing rat muscle spindles

The development of muscle spindles, with respect to the expression of myosin heavy chain isoforms was studied in rat hind limbs from 17 days of gestation up to seven days after birth. Serial cross-sections were labelled with antibodies against slow tonic, slow twitch and neonatal isomyosins, myomesin, laminin and neurofilament protein. At 17-18 days of gestation, a small population of primary myotubes expressing slow tonic myosin were identified as the earliest spindle primordia. These myotubes also expressed slow twitch and, to a lesser extent, neonatal myosin. At 19-20 days of gestation a second myotube became apparent; this staining strongly with anti-neonatal myosin. A day later this secondary myotube acquired reactivity to anti-slow tonic and anti-slow twitch myosins. By birth, a third myotube was present; this staining strongly with anti-neonatal myosin but otherwise unreactive with the other antibodies against myosin heavy chains. Three days after birth a fourth myotube, with identical reactivity to the third one, became apparent. Regional variation in the expression of isomyosins, which was present since birth in the two nuclear bag fibers was further enhanced: the nuclear bag staining strongly with anti-slow tonic and antineonatal in the equatorial region and with decreasing intensity towards the poles, whilst with anti-slow twitch the stainability was low in the equatorial and high in the polar region. The nuclear bag fiber showed a homogeneous staining: high with anti-slow tonic, moderate with anti-neonatal, and displayed stainability to anti-slow twitch myosin in the polar regions only. No regional variation was found along the chain fiber/myotube.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization and activity of myosin light chain kinase isoforms during the cell cycle

Phosphorylation on Ser 19 of the myosin II regulatory light chain by myosin light chain kinase (MLCK) regulates actomyosin contractility in smooth muscle and vertebrate nonmuscle cells. The smooth/nonmuscle MLCK gene locus produces two kinases, a high molecular weight isoform (long MLCK) and a low molecular weight isoform (short MLCK), that are differentially expressed in smooth and nonmuscle tissues. To study the relative localization of the MLCK isoforms in cultured nonmuscle cells and to determine the spatial and temporal dynamics of MLCK localization during mitosis, we constructed green fluorescent protein fusions of the long and short MLCKs. In interphase cells, localization of the long MLCK to stress fibers is mediated by five DXRXXL motifs, which span the junction of the NH(2)-terminal extension and the short MLCK. In contrast, localization of the long MLCK to the cleavage furrow in dividing cells requires the five DXRXXL motifs as well as additional amino acid sequences present in the NH(2)-terminal extension. Thus, it appears that nonmuscle cells utilize different mechanisms for targeting the long MLCK to actomyosin structures during interphase and mitosis. Further studies have shown that the long MLCK has twofold lower kinase activity in early mitosis than in interphase or in the early stages of postmitotic spreading. These findings suggest a model in which MLCK and the myosin II phosphatase (Totsukawa, G., Y. Yamakita, S. Yamashiro, H. Hosoya, D.J. Hartshorne, and F. Matsumura. 1999. J. Cell Biol. 144:735-744) act cooperatively to regulate the level of Ser 19-phosphorylated myosin II during mitosis and initiate cytokinesis through the activation of myosin II motor activity.     

Further characterization and thiophosphorylation of smooth muscle myosin

(i) Myosin from chicken gizzards was purified by a modification of an earlier procedure (M. N. Malik, 1978,Biochemistry17, 27–32). When this myosin, as well as that prepared by the method of A. Sobieszek and R. D. Bremel (1975,Eur. J. Biochem.55, 49–60), was analyzed by gradient slab gel using the discontinuous buffer system of Neville (1971,J. Biol. Chem.246, 6328–6334), a closely spaced doublet in the heavy chain and four light chains were observed as opposed to one heavy chain and two light chains with the method of Weber and Osborn (1969, J. Biol. Chem.244, 4406–4412). These findings raise the possibility of the existence of myosin isoenzymes in smooth muscle. (ii) The purified gizzard myosin was found to be free of kinase and phosphatase. Phosphorylation or thiophosphorylation of myosin was observed only by exogenously adding kinase. A maximum of 1.2 mol of ³²P/mol of myosin and 2.3 mol of ³⁵S/mol of myosin were obtained. The actin-activated ATPase activity depended upon the extent of thiophosphorylation of myosin; a four- to fivefold increase in the activity was observed when myosin was fully thiophosphorylated. Thiophosphorylated myosin was found to be more stable than phosphorylated myosin.     

Expression and function of COOH-terminal myosin heavy chain isoforms in mouse smooth muscle

Isoforms of the smooth muscle myosin motor, SM1 and SM2, differ in length at the carboxy terminal tail region. Their proportion changes with development, hormonal status and disease, but their function is unknown. We developed mice carrying the myosin heavy chain (MyHC) transgenes SM1, cMyc-tagged SM1, SM2, and V5-tagged SM2, and all transgenes corresponded to the SMa NH₂-terminal isoform. Transgene expression was targeted to smooth muscle by the smooth muscle -actin promoter. Immunoblot analysis showed substantial expression of the cMyc-tagged SM1 and V5-tagged SM2 MyHC protein in aorta and bladder and transgene mRNA was expressed in mice carrying unlabeled SM1 or SM2 transgenes. Despite significant protein expression of tagged MyHCs we found only small changes in the SM1:SM2 protein ratio. Significant changes in functional phenotype were observed in mice carrying unlabeled SM1 or SM2 transgenes. Force in aorta and bladder was increased (72 ± 14%, 92 ± 11%) in SM1 and decreased to 57 ± 1% and 80 ± 3% in SM2 transgenic mice. SM1 transgenic bladders had faster (1.8 ± 0.3 s) and SM2 slower (7.1 ± 0.5 s) rates of force redevelopment following a rapid step shortening. We hypothesize that small changes in the SM1:SM2 ratio could be amplified if they are associated with changes in thick filament assembly and underlie the altered contractility. These data provide evidence indicating an in vivo function for the COOH-terminal isoforms of smooth muscle myosin and suggest that the SM1:SM2 ratio is tightly regulated in smooth muscle tissues. myosin heavy chain; transgenic mice     

Distribution of myosin heavy chain isoforms in non-weight-bearing rat soleus muscle fibers

Talmadge, Robert J., Roland R. Roy, and V. Reggie Edgerton.Distribution of myosin heavy chain isoforms in non-weight-bearing rat soleus muscle fibers. J. Appl.Physiol. 81(6): 2540-2546, 1996.The effects of14 days of spaceflight (SF) or hindlimb suspension (HS) (Cosmos 2044)on myosin heavy chain (MHC) isoform content of the rat soleus muscleand single muscle fibers were determined. On the basis ofelectrophoretic analyses, there was a de novo synthesis of type IIx MHCbut no change in either type I or IIa MHC isoform proportions aftereither SF or HS compared with controls. The percentage of fiberscontaining only type I MHC decreased by 26 and 23%, and the percentageof fibers with multiple MHCs increased from 6% in controls to 32% inHS and 34% in SF rats. Type IIx MHC was always found in combinationwith another MHC or combination of MHCs; i.e., no fibers contained typeIIx MHC exclusively. These data suggest that the expression of thenormal complement of MHC isoforms in the adult rat soleus muscle isdependent, in part, on normal weight bearing and that the absence ofweight bearing induces a shift toward type IIx MHC protein expression in the preexisting type I and IIa fibers of the soleus.