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     

Effects of estradiol on phenylephrine contractility associated with intracellular calcium release in rat aorta

The ability of estradiol to affect phenylephrine-induced contraction and the subsequent increase in resting tone, associated with capacitative Ca²⁺ entry across the plasma membrane, was evaluated in rat aortic rings incubated in Ca²⁺-free solution. The incubation with estradiol (1–100 nM, 5 min) inhibited both the phenylephrine-induced contraction and the IRT. Neither cycloheximide (1 µM; inhibitor of protein synthesis) nor tamoxifen (1 µM; blocker of estrogenic receptors) modified the effects of estradiol. Estradiol (100 µM) also blocked the contractile response to serotonin (10 µM) but not to caffeine (10 mM). In addition, estradiol (100 µM) inhibited the contractile responses to cyclopiazonic acid (1 µM; selective Ca²⁺-ATPase inhibitor) associated with capacitative Ca²⁺ influx through non-L-type Ca²⁺ channels. Finally, estradiol inhibited the Ca²⁺-induced increases in intracellular free Ca²⁺ (after pretreatment with phenylephrine) in cultured rat aorta smooth muscle cells incubated in Ca²⁺-free solution. In conclusion, estradiol interfered in a concentration-dependent manner with Ca²⁺-dependent contractile effects mediated by the stimuli of ₁-adrenergic and serotonergic receptors and inhibited the capacitative Ca²⁺ influx through both L-type and non-L-type Ca²⁺ channels. Such effects are in essence nongenomic and not mediated by the intracellular estrogenic receptor. estrogen; ₁-adrenergic agonists     

Immunohistochemical studies on the distribution of cellular myosin II isoforms in brain and aorta.

The distribution of nonmuscle myosin isoforms in brain and aorta was studied by using polyclonal antibodies against two synthetic peptides selected from a region near the carboxyl terminus of bovine brain (peptide IIB) and human macrophage (peptide IIA) myosin. Immunoblots of brain homogenates and purified myosin showed two major bands stained by anti-peptide IIB (MIIB1 and MIIB2) and a minor band stained by anti-peptide IIA (MIIA2). Polyclonal anti-human platelet myosin antibodies did not react with MIIB isoforms. In cryosections from bovine, rat, and mouse brains, anti-peptide IIB stained most neuronal cells. In bovine cryosections, glial staining was also observed. In contrast, anti-peptide IIA and anti-platelet myosin antibodies primarily stained blood vessels. In bovine aorta, the anti-peptide antibodies recognized four bands, MIIB3, MIIB4, MIIA1, and MIIA2. Only MIIA2 was recognized by anti-human platelet myosin antibodies. In bovine aorta cryosections, anti-peptide IIB stained smooth muscle cells in tunica intima and tunica media but did not stain endothelial cells. Anti-peptide IIA stained smooth muscle cells in the tunica media, and endothelial cells of vaso vasorum but not of aorta. Only polyclonal anti-platelet myosin antibodies stained the endothelial cells of aorta tunica intima. These results indicate that multiple isoforms of cellular myosins exist in mammals, that these isoforms are expressed in a cell specific manner, and that the major myosin isoforms isolated from whole brain originate from neurons and, at least in bovine brain, from glia, but not from blood vessels.     

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)     

Effects of regression of cardiac hypertrophy on myocardial contractility and ventricular myosin isoenzymes

The effects of regression of cardiac hypertrophy on myocardial contractility and ventricular myosin isoenzymes were investigated in rats with renovascular hypertension. Six-week-old male Wistar rats were made hypertensive by constriction of one renal artery with a silver clip. Regression of cardiac hypertrophy was induced following the lowering of blood pressure by nephrectomy on the affected side 5–6 weeks after constriction of the renal artery and was maintained for 5–6 weeks. In contrast, myocardial hypertrophy was induced by 10–11 weeks of the hypertensive state. Isometric developed tension of isolated left ventricular papillary muscles was measured, while they were being perfused with Tyrode solution. Left ventricular myosin isoenzymes were separated by pyrophosphate gel electrophoresis. The ventricular to body weight ratio of the nephrectomized group was significantly lower than that of the hypertensive group, although it was greater than that of age-matched normal control rats. There were no significant differences in the isometric developed tension among three groups, the nephrectomized, hypertensive, and normal control rats. However, dT/dtmax tended to decrease in the hypertensive rats and recovered to normal in the nephrectomized rats. The left ventricular myosin isoenzyme pattern was shifted toward VM-3 in hypertensive rats and was shifted back toward VM-1 again in nephrectomized rats.     

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.     

Effect of extraction of myosin binding protein C on contractility of rat heart

Human hearts with reduced or mutant myosin binding protein C (MyBP-C) undergo hypertrophy and dilation, suggesting that reduction or alteration of MyBP-C interferes with normal contraction. Extraction of 60-70% of MyBP-C over 1 h from a mechanically disrupted cardiac myocyte has been shown to increase Ca sensitivity but does not appear to impair development of maximum Ca-activated force (Fmax). To determine whether loss of MyBP-C over a longer period of time will decrease force development in a reversible manner, MyBP-C has been extracted from chemically skinned rat cardiac trabeculae for 1-4 h, and force production, Ca sensitivity, and thick filament structure were measured. Although extraction of MyBP-C for 1 h did not alter Fmax, after 4 h, myosin heads became disordered and Fmax decreased. At this point, incubation of the trabeculae with rat cardiac MyBP-C in a relaxing solution reversed the decline in Fmax and most of the change in order of myosin heads. Extraction of MyBP-C appears to produce a change in the orientation of myosin heads that is associated with a decreased ability of the contractile system to develop force.     

Two isoforms of 17-kDa essential light chain of aorta media smooth muscle myosin

Aorta myosin contains two kinds of light chain, 20-kDa phosphorylatable light chain and 17-kDa essential light chain (LC17). Purified myosin from porcine aorta media showed 3 distinct light chain bands on polyacrylamide gel electrophoresis (PAGE) in the presence of urea (urea-PAGE). The mobilities of the faster two components did not change after incubation of the myosin with a myosin light chain kinase. Gel slices containing the faster two bands were separately subjected to PAGE in the presence of sodium dodecylsulfate. Both components showed identical mobility with that of LC17. The two components were designated as LC17a and LC17b in increasing order of mobility on urea-PAGE. They were isolated by DEAE-Toyopearl ion exchange column chromatography. The amino acid compositions of LC17a and LC17b were similar to each other, but the contents of Ser, Met, Ile, and His were distinctly different. These results suggest that the two components are isoforms. The ratio of the content of each isoform (LC17a: LC17b) in the purified porcine aorta myosin was 39:61, and essentially the same ratio was found with washed muscle homogenate of porcine aorta. Then washed aorta muscle homogenates of rabbit and rat were examined. Two bands having similar mobilities to those of porcine homogenate were also found in urea-PAGE. The ratios of the two components were 31:69 and 66:34, respectively, for rabbit and rat. Aorta smooth muscle thus may contain many types of isomyosin.     

Role of 17-kDa essential light chain isoforms of aorta smooth muscle myosin.

Aorta smooth muscle myosin contains two kinds of 17-kDa essential light chain, LC17nm (nonmuscle-type) and LC17gi (gizzard-type) [Hasegawa, Y., Ueda, Y., Watanabe, M., & Morita, F. (1992) J. Biochem. 111, 798-803]. The LC17 isoforms were released from porcine aorta myosin by incubation at 46 degrees C. The rate of release was 1.5 to 2 times higher with LC17gi than with LC17nm. Aorta myosins containing the two LC17 isoforms in various ratios could be reconstituted. The actin-activated ATPase activity was measured as a function of LC17nm content. The Vm value was lower with myosin which contained more LC17nm. The apparent dissociation constant for F-actin, Km, was 20-fold less with myosin which contained 81% LC17nm than myosin which contained 23% LC17nm. A similar difference in the dissociation constants of myosin for F-actin was observed in the presence of adenylyl imidodiphosphate. The role of LC17nm appears to be to make aorta myosin suitable for maintaining the muscle tension with a low expenditure of energy. The isoform-dependent difference in the F-actin-binding affinities of myosin seems partly due to the difference in the affinities of LC17 isoforms themselves for F-actin. We found that the isolated LC17nm itself could bind with F-actin with a dissociation constant of 64 microM, but LC17gi could not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for isoforms of the phosphorylatable myosin light chain in rat uterus

The phosphorylatable myosin light chain of rat uterus was resolved into four spots by two-dimensional gel electrophoresis. Antibody against the 20 kDa light chain from smooth muscle myosin recognized these four spots. Purified light chain exhibited four spots on a diagonal upon isoelectrofocusing in both first and second dimensions, proving that these spots are not due to artifactual charge modification. Accordingly, the four spots contain light chain isoforms derived from myosin.     

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.

     

Expression of myosin isoforms during notexin-induced regeneration of rat soleus muscles

Myosin isozymes and their fiber distribution were studied during regeneration of the soleus muscle of young adult (4-6 week old) rats. Muscle degeneration and regeneration were induced by a single subcutaneous injection of a snake toxin, notexin. If reinnervation of the regenerating muscle was allowed to occur (functional innervation nearly complete by 7 days), then fiber diameters continued to increase and by 28 days after toxin treatment they attained the same values as fibers in the contralateral soleus. If the muscles were denervated at the time of toxin injection, the early phases of regeneration still took place but the fibers failed to continue to increase in size. Electrophoresis of native myosin showed multiple bands between 3 and 21 days of regeneration which could be interpreted as indicating the presence of embryonic, neonatal, fast and slow myosins in the innervated muscles. Adult slow myosin became the exclusive from in innervated regenerates. In contrast, adult fast myosin became the predominant form in denervated regenerating muscles. Immunocytochemical localization of myosin isozymes demonstrated that in innervated muscles the slow form began to appear in a heterogeneous fashion at about 7 days, and became the major form in all fibers by 21-28 days. Thus, the regenerated muscle was almost entirely composed of slow fibers, in clear contrast to the contralateral muscle which was still substantially mixed. In denervated regenerating muscles, slow myosin was not detected biochemically or immunocytochemically whereas fast myosin was detected in all denervated fibers by 21-28 days. The regenerating soleus muscle therefore is clearly different from the developing soleus muscle in that the former is composed of a uniform fiber population with respect to myosin transitions. Moreover the satellite cells which account for the regeneration process in the soleus muscle do not appear to be predetermined with respect to myosin heavy chain expression, since the fibers they form can express either slow or fast isoforms. The induction of the slow myosin phenotype is entirely dependent on a positive, extrinsic influence of the nerve.     

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.     

Effects of anethole and structural analogues on the contractility of rat isolated aorta: Involvement of voltage-dependent Ca2+-channels

Anethole is a naturally occurring aromatic oxidant, present in a variety of medicinal plant extracts, which is commonly used by the food and beverage industry. Despite its widespread occurrence and commercial use, there is currently little information regarding effects of this compound on the vasculature. Therefore the actions of anethole on the contractility of rat isolated aorta were compared with those of eugenol, and their respective isomeric forms, estragole and isoeugenol. In aortic rings precontracted with phenylephrine (PE; 1 microM), anethole (10(-6) M-10(-4) M) induced contraction in preparations possessing an intact endothelium, but not in endothelium-denuded tissues. At higher concentrations (10(-3) M-10(-2) M), anethole-induced concentration-dependent and complete relaxation of all precontracted preparations, irrespective of whether the endothelium was intact or not, an action shared by eugenol, estragole and isoeugenol. The contractile and relaxant effects of anethole in PE-precontracted preparations were not altered by L-NAME (10 microM) or indomethacin (10 microM), indicating that neither nitric oxide nor prostaglandins were involved in these actions. The mixed profile of effects was not confined to PE-mediated contraction, since similar responses were obtained to anethole when tissues were precontracted with 25 mM KCl. Anethole and estragole (10(-6)-10(-4) M), but not eugenol or isoeugenol, increased the basal tonus of endothelium-denuded aortic rings, an action that was abolished by VDCC blockers nifedipine (1 microM) and diltiazem (1 microM), or by withdrawal of extracellular Ca(2+). Our data suggest complex effects of anethole on isolated blood vessels, inducing contraction at lower doses, mediated via opening of voltage-dependent Ca(2+)-channels, and relaxant effects at higher concentrations that are shared by structural analogues.