Alternative splicing of smooth muscle myosin heavy chains and its functional consequences

The aim of our study was to determine the relation between alternatively spliced myosin heavy chain (MHC) isoforms and the contractility of smooth muscle. The relative amount of MHC with an alternatively spliced insert in the 5′ (amino terminal) domain was determined on the protein level using a peptide-directed antibody (a25K/50K) raised against the inserted sequence (QGPSFAY). Smooth muscle MHC isoforms of both bladder and myometrium but not nonmuscle MHC reacted with a25/50K. Using a quantitative Western-blot approach the amount of 5′-inserted MHC in rat bladder was detected to be about eightfold higher than in normal rat myometrium. The amount of heavy chain with insert was found to be decreased by about 50% in the myometrium of pregnant rats. Although bladder contained significantly more 5′-inserted MHC than myometrium, apparent maximal shortening velocities (Vmax) were comparable, being 0.138 ± 0.012 and 0.114 ± 0.023 muscle length per second of skinned bladder and normal myometrium fibers, respectively. Phosphorylation of myosin light chain 20 induced by maximal Ca²⁺/calmodulin activation was the same in bladder and myometrial fibers. These results suggest that the amount of 5′-inserted MHC is not necessarily associated with contractile properties of smooth muscle. © 1996 Wiley-Liss, Inc.     

Alternative splicing and cycling kinetics of myosin change during hypertrophy of human smooth muscle cells

We investigated in vivo expression of myosin heavy chain (MHC) isoforms, 17 kDa myosin light chain (MLC₁₇), and phosphorylation of the 20 kDa MLC (MLC₂₀) as well as mechanical performance of chemically skinned fibers of normal and hypertrophied smooth muscle (SM) of human myometrium. According to their immunological reactivity, we identified three MHC isoenzymes in the human myometrium: two SM-MHC (SM1 with 204 kDa and SM2 with 200 kDa), and one non-muscle specific MHC (NM with 196 kDa). No cross-reactivity was detected with an antibody raised against a peptide corresponding to a seven amino acid insert at the 25K/50K junction of the myosin head (a-25K/50K) in both normal and hypertrophied myometrium. In contrast, SM-MHC of human myomatous tissue strongly reacted with a-25K/50K. Expression of SM1/SM2/NM (%) in normal myometrium was 31.7/34.7/33.6 and 35.1/40.9/24 in hypertrophied myometrium. The increased SM2 and decreased NM expression in the hypertrophied state was statistically significant (P < 0.05). MHC isoform distribution in myomatous tissue was similar to normal myometrium (35.3/35.3/29.4). In vivo expression of MLC_17a increased from 25.5% in normal to 44.2% in hypertrophied (P < 0.001) myometrium. Phosphorylation levels of MLC₂₀ upon maximal Ca²⁰-calmodulin activation of skinned myometrial fibers were the same in normal and hypertrophied myometrial fibers. Maximal force of isometric contraction of skinned fibers (pCa 4.5, slack-length) was 2.85 mN/mm² and 5.6 mN/mm² in the normal and hypertrophied state, respectively (P < 0.001). Apparent maximal shortening velocity (Vmax_app, extrapolated from the force-velocity relation) of myometrium rose from 0.13 muscle length s ¹ (ML/s) in normal to 0.24 ML/s in hypertrophied fibers (P < 0.001). J. Cell. Biochem, 64:171–181. © 1997 Wiley-Liss, Inc.     

Decreased phosphatase activity, increased Ca2+ sensitivity, and myosin light chain phosphorylation in urinary bladder smooth muscle of newborn mice

Developmental changes in the regulation of smooth muscle contraction were examined in urinary bladder smooth muscle from mice. Maximal active stress was lower in newborn tissue compared with adult, and it was correlated with a lower content of actin and myosin. Sensitivity to extracellular Ca2+ during high-K+ contraction, was higher in newborn compared with 3-wk-old and adult bladder strips. Concentrations at half maximal tension (EC50) were 0.57 +/- 0.01, 1.14 +/- 0.12, and 1.31 +/- 0.08 mM. Force of the newborn tissue was inhibited by approximately 45% by the nonmuscle myosin inhibitor Blebbistatin, whereas adult tissue was not affected. The calcium sensitivity in newborn tissue was not affected by Blebbistatin, suggesting that nonmuscle myosin is not a primary cause for increased calcium sensitivity. The relation between intracellular [Ca2+] and force was shifted toward lower [Ca2+] in the newborn bladders. This increased Ca2+ sensitivity was also found in permeabilized muscles (EC50: 6.10 +/- 0.07, 5.77 +/- 0.08, and 5.55 +/- 0.02 pCa units, in newborn, 3-wk-old, and adult tissues). It was associated with an increased myosin light chain phosphorylation and a decreased rate of dephosphorylation. No difference was observed in the myosin light chain phosphorylation rate, whereas the rate of myosin light chain phosphatase-induced relaxation was about twofold slower in the newborn tissue. The decreased rate was associated with a lower expression of the phosphatase regulatory subunit MYPT-1 in newborn tissue. The results show that myosin light chain phosphatase activity can be developmentally regulated in mammalian urinary bladders. The resultant alterations in Ca2+ sensitivity may be of importance for the nervous and myogenic control of the newborn bladders.     

The B2 alternatively spliced isoform of nonmuscle myosin II-B lacks actin-activated MgATPase activity and in vitro motility

We report the initial biochemical characterization of an alternatively spliced isoform of nonmuscle heavy meromyosin (HMM) II-B2 and compare it with HMM II-B0, the nonspliced isoform. HMM II-B2 is the HMM derivative of an alternatively spliced isoform of endogenous nonmuscle myosin (NM) II-B, which has 21-amino acids inserted into loop 2, near the actin-binding region. NM II-B2 is expressed in the Purkinje cells of the cerebellum as well as in other neuronal cells [X. Ma, S. Kawamoto, J. Uribe, R.S. Adelstein, Function of the neuron-specific alternatively spliced isoforms of nonmuscle myosin II-B during mouse brain development, Mol. Biol. Cell 15 (2006) 2138-2149]. In contrast to any of the previously described isoforms of NM II (II-A, II-B0, II-B1, II-C0 and II-C1) or to smooth muscle myosin, the actin-activated MgATPase activity of HMM II-B2 is not significantly increased from a low, basal level by phosphorylation of the 20 kDa myosin light chain (MLC-20). Moreover, although HMM II-B2 can bind to actin in the absence of ATP and is released in its presence, it cannot propel actin in the sliding actin filament assay following MLC-20 phosphorylation. Unlike HMM II-B2, the actin-activated MgATPase activity of a chimeric HMM with the 21-amino acid II-B2 sequence inserted into the homologous location in the heavy chain of HMM II-C is increased following MLC-20 phosphorylation. This indicates that the effect of the II-B2 insert is myosin heavy chain specific.     

TPA has no influence on the expression of myosin heavy chain isoforms in cultured adult cardiac muscle cells.

The effect of a tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), on the expression of myosin heavy chain isoforms in cultured rat cardiac ventricular muscle cells was studied. The previous preliminary report [Claycomb WC (1988): "Biology of Isolated Adult Cardiac Myocytes." In Clark WA, Decker RS, Borg TK (eds): New York: Elsevier, pp 284-287] indicated that TPA turns off the expression of myosin heavy chain genes in cultured adult cardiac myocytes. Electrophoretic and immunocytochemical analyses were carried out in the present studies. The myosin heavy chain isoform profiles of cardiac myocytes exposed to TPA at concentrations of 50-250 ng/ml culture medium for varying periods were similar to those of controls that were grown in the absence of TPA, showing predominant isoform V1. Immunofluorescence microscopy with monoclonal antibodies to cardiac ventricular isomyosin revealed the structural organization of myosin in TPA-treated cells. The organization of myosin was variable among different myocytes and within a single myocyte. Immunofluorescence microscopy was extended to the examination of the organization of alpha-actinin which did not differ from that of myosin in some myocytes. In contrast to the previous report [Claycomb, 1988], this study has demonstrated that TPA has no influence on the expression of myosin heavy chain isoforms in cultured adult ventricular cardiac muscle cells.     

Smooth muscle myosin isoform expression and LC20 phosphorylation 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.     

1α,25-dihydroxyvitamin D₃ enhances fast-myosin heavy chain expression in differentiated C2C12 myoblasts

We investigated the effect of VD3 (1α,25-dihydroxyvitamin D3) on the proliferating, differentiating and differentiated phases of C2C12 myoblasts, a mouse skeletal muscle cell line. VD3 treatment in 10% FBS (fetal bovine serum) inhibited the proliferation and viability of the cells in a dose-dependent manner. It also dose-dependently increased the percentage of cells in the G0/G1 phase as shown by flow cytometry. In the differentiating phase, VD3 treatment inhibited the formation of myotubes and the expression of total myosin heavy chain at both the mRNA and protein levels. In the differentiated phase, treatment had no significant effect on the amount of total myosin heavy chain, as Western blot analysis with MF20 antibody [DSHB (Developmental Studies Hybridoma Bank)] showed. However, significantly greater expression of fast myosin heavy chain in 1 nM VD3 was found by Western blot analysis with MY-32 (Sigma). Thus VD3 inhibited the proliferation of myoblasts during proliferating and differentiating phases, whereas it increased the expression of the fast myosin heavy chain isoform in the differentiated phase. The data indicate that an adequate concentration of VD3 might have an anabolic effect on differentiated skeletal muscle.     

Regulation by molluscan myosins

Molluscan myosins are regulated molecules that control muscle contraction by the selective binding of calcium. The essential and the regulatory light chains are regulatory subunits. Scallop myosin is the favorite material for studying the interactions of the light chains with the myosin heavy chain since the regulatory light chains can be reversibly removed from it and its essential light chains can be exchanged. Mutational and structural studies show that the essential light chain binds calcium provided that the Ca-binding loop is stabilized by specific interactions with the regulatory light chain and the heavy chain. The regulatory light chains are inhibitory subunits. Regulation requires the presence of both myosin heads and an intact headrod junction. Heavy meromyosin is regulated and shows cooperative features of activation while subfragment-1 is non-cooperative. The myosin heavy chains of the functionally different phasic striated and the smooth catch muscle myosins are products of a single gene, the isoforms arise from alternative splicing. The differences between residues of the isoforms are clustered at surface loop-1 of the heavy chain and account for the different ATPase activity of the two muscle types. Catch muscles contain two regulatory light chain isoforms, one phosphorylatable by gizzard myosin light chain kinase. Phosphorylation of the light chain does not alter ATPase activity. We could not find evidence that light chain phosphorylation is responsible for the catch state.     

Expression of proto-oncogenes and muscle specific genes during cardiac hypertrophy and development in rats and humans

A regulatory interdependence of expression of proto-oncogenes and muscle specific genes observed in smooth muscle was examined in cardiac muscle during normal development and hypertrophy both in rats and humans. During normal development in rats, myosin light chain 2 expression is very low at prenatal stages, while c-fos expression starts from the early stages of embryonic development. In aorta constricted rats c-fos induction occurs within 30 min whereas myosin light chain 2 expression is sufficiently high only after 3 or 4 days of post operative period. In the case of humans, the expression of myosin light chain 2 as well as c-fos occurs at high levels during embryonic development. Similar results were obtained with tissue samples obtained from patients with cardiac abnormalities. Induction of the c-fos gene in cultured myocytes by 12-O-tetradeeanoylphorbol 13-acetate has no influence on the expression of myosin light chain 2. These studies were extended with studies on c-myc and Β-myosin heavy chain gene expression which revealed a similar pattern of expression as that of c-fos and myosin light chain 2. These results have indicated that the expression of proto-oncogenes in cardiac muscle may be independently regulated from the expression of muscle specific genes.     

Molecular genetic characterization of a developmentally regulated human perinatal myosin heavy chain

We have isolated a human cDNA which corresponds to a developmentally regulated sarcomeric myosin heavy chain. RNA hybridization and DNA sequence analysis indicate that this cDNA, called SMHCP, encodes a perinatal myosin heavy chain isoform. The nucleotide and deduced amino acid sequences of the 3.4-kb cDNA insert show strong homology with other sarcomeric myosin heavy chains. The strongest homology is to a previously described 970-bp cDNA encoding a rat perinatal isoform (Periasamy, M., D. F. Wieczorek, and B. Nadal-Ginard. 1984. J. Biol. Chem. 259:13573-13578). The homology between the analogous human and rat perinatal myosin heavy chain cDNAs is maintained through the highly isoform-specific final 20 carboxyl-terminal amino acids, as well as the 3' untranslated region. Ribonuclease protection studies show that the mRNA encoding this isoform is expressed at high levels in 21-wk fetal skeletal tissue and not in fetal cardiac muscle. In contrast to the rat perinatal isoform, which was not found to be expressed in adult hind-leg tissue, the gene encoding SMHCP continues to be expressed in adult human skeletal tissue, but at lower levels relative to fetal skeletal tissue.     

(+)Insert smooth muscle myosin heavy chain (SM-B): from single molecule to human

In smooth muscle, alternative mRNA splicing of a single gene produces four myosin heavy chain (SMMHC) isoforms. Two of these isoforms differ by the presence [(+)insert] or absence [(-)insert] of a seven amino acid insert in the motor domain. This insert enhances the kinetic properties of myosin at the molecular level but its exact role at the cell and tissue levels still has to be elucidated. This review focuses on the expression and biological functions of the (+)insert isoform. Current knowledge is summarized regarding its tissue distribution in animals and humans. Studies at the molecular, cellular and tissue levels that aimed at understanding the contribution of this isoform to smooth muscle mechanical function are presented with a particular focus on velocity of shortening. In addition, the altered expression of the (+)insert isoform in diseases and models of diseases and the compensatory mechanisms that occur when the (+)insert is knocked out are discussed. The need for additional studies on the relationship of this isoform to contractile performance and how expression of this isoform is regulated are also considered.     

Effects of downstream migration on myosin heavy chain expression and dihydropyridine receptor density in farmed smolt of Atlantic salmon

The expression of myosin heavy chain (MHC) subunits and dihydropyridine receptors (DHPRs) from red and white tail muscles of cultured smolts of Atlantic salmon Salmo salar was analysed from samples taken: (1) before the fish were transferred to the river and (2) after the migration distance of c . 50 km. The relative work load of migrating fish, estimated on the basis of swimming speed of Atlantic salmon and discharge rate of the River Simojoki, Finland, was maximally 1700 times higher than that of fish in culture. The data show that after five migration days the density of DHPRs in the muscles of the smolts was increased already. Furthermore, the results indicated a transition from the fast‐to‐slow MHC isoform. Transfer of fish to a natural environment and downstream migration thus had a significant effect on the expression of muscle proteins.     

Myogenesis and histogenesis of skeletal muscle on flexible membranes in vitro

Summary Primary muscle cell cultures consisting of single myocytes and fibroblasts are grown on flexible, optically clear biomembranes. Muscle cell growth, fusion and terminal differentiation are normal. A most effective membrane for these cultures is commercially available Saran Wrap. Muscle cultures on Saran will, once differentiated, contract vigorously and will deform the Saran which is pinned to a Sylgard base. At first, the muscle forms a two-dimensional network which ultimately detaches from the Saran membrane allowing an undergrowth of fibroblasts so that these connective tissue cells completely surround groups of muscle fibers. A three-dimensional network is thus formed, held in place through durable adhesions to stainless steel pins. This three-dimensional, highly contractile network is seen to consist of all three connective tissue compartments seenin vivo, the endomysium, perimysium and epimysium. Finally, this muscle shows advanced levels of maturation in that neonatal and adult isoforms of myosin heavy chain are detected together with high levels of myosin fast light chain 3. Antibody 2E9 to neonatal myosin heavy chain was obtained from Dr. Everett Bandman. MF 20 which reacts with all myosin heavy chain isoforms including the embryonic isoform and MF 14 which reacts specifically with adult myosin heavy chain were obtained from Drs. Bader and Fischman. Antibody to myosin fast light chain 3 was obtained from Dr. Susan Lowey. Antibody to fibronectin was obtained from Dr. Douglas Fambrough. This work was supported by grants to R. C. S. from the Muscular Dystrophy Association and from NIH. Editor's Statement The paper represents a novel and interesting approach to the co-culture of myotubes with fibroblasts which allows three dimensional development of endomysium, perimysium and epimysium and expression of adult-type muscle proteins. Such organogenic development is not normally seen in vitro. The technique should prove useful in elucidating development aspects of muscle cells and their relationship with connective support.     

(+)Insert smooth muscle myosin heavy chain (SM-B) isoform expression in human tissues

Two smooth muscle myosin heavy chain isoforms differ in their amino terminus by the presence [(+)insert] or absence [(–)insert] of a seven-amino acid insert. Animal studies show that the (+)insert isoform is predominantly expressed in rapidly contracting phasic muscle and the (–)insert isoform is mostly found in slowly contracting tonic muscle. The expression of the (+)insert isoform has never been demonstrated in human smooth muscle. We hypothesized that the (+)insert isoform is present in humans and that its expression is commensurate with the organ's functional requirements. We report, for the first time, the sequence of the human (+)insert isoform and quantification of its expression by real-time PCR and Western blot analysis in a panel of human organs. The (+)insert isoform mRNA and protein expression levels are significantly greater in small intestine compared with all organs studied except for trachea and are significantly greater in trachea compared with uterus and aorta. To assess the functional significance of this differential myosin isoform expression between organs, we measured the rate of actin filament movement (_max) when propelled by myosin purified from rat organs, because the rat and human inserts are identical and their remaining sequences show 93% identity. _max exhibits a rank correlation from the most tonic to the most phasic organ. The selective expression of the (+)insert isoform observed among human organs suggests that it is an important determinant of tissue shortening velocity. A differential expression of the (+)insert isoform could also account for altered contractile properties observed in human pathology. phasic and tonic smooth muscle; real-time polymerase chain reaction; in vitro motility assay     

Function of the neuron-specific alternatively spliced isoforms of nonmuscle myosin II-B during mouse brain development

We report that the alternatively spliced isoforms of nonmuscle myosin heavy chain II-B (NHMC II-B) play distinct roles during mouse brain development. The B1-inserted isoform of NMHC II-B, which contains an insert of 10 amino acids near the ATP-binding region (loop 1) of the myosin heavy chain, is involved in normal migration of facial neurons. In contrast, the B2-inserted isoform, which contains an insert of 21 amino acids near the actin-binding region (loop 2), is important for postnatal development of cerebellar Purkinje cells. Deletion of the B1 alternative exon, together with reduced expression of myosin II-B, results in abnormal migration and consequent protrusion of facial neurons into the fourth ventricle. This protrusion is associated with the development of hydrocephalus. Restoring the amount of myosin II-B expression to wild-type levels prevents these defects, showing the importance of total myosin activity in facial neuron migration. In contrast, deletion of the B2 alternative exon results in abnormal development of cerebellar Purkinje cells. Cells lacking the B2-inserted isoform show reduced numbers of dendritic spines and branches. Some of the B2-ablated Purkinje cells are misplaced in the cerebellar molecular layer. All of the B2-ablated mice demonstrated impaired motor coordination.     

鱼类肌球蛋白重链基因研究进展及展望

肌球蛋白是构成鱼类肌肉的主要蛋白之一。肌球蛋白由2条相对分子质量为220×10^3的重链和4条相对分子质量为16×10^3～20×10^3的轻链组成。以往对于肌球蛋白基因的研究大多数集中在高等脊椎动物,而有关鱼类的研究相对薄弱。对鱼类肌球蛋白和肌球蛋白重链基因结构、功能及其表达调节机制等研究进展做了综述分析;同时结合作者的研究实践,探讨了对名贵鱼类肌肉发生和肌球蛋白的进一步研究。     

Temporal expression of TnI fast and slow isoforms in biceps femoris and masseter muscle during pig growth

Biceps femoris (BF) and masseter muscle (MM) are the mixture of slow oxidative and fast-twitch fibres. Compared with MM, BF had the significantly higher expression of myosin heavy chain (MyHC) fast IIx and IIb isoforms (MyHCIIx and MyHCIIb), but lower expression of MyHC slow isoform (MyHCI) and fast IIa isoform (MyHCIIa). The objective of this study was to investigate the expression pattern of troponin I (TnI) slow-twitch isoform (TNNI1) and fast-twitch isoform (TNNI2) in BF and MM of Yorkshire and Meishan pigs which differed significantly in the growth rate. The expression of the TNNI1 and TNNI2 peaked at the postnatal 35 days in Yorkshire pigs and postnatal 60 days in Meishan pigs. The expression of TNNI1 and TNNI2 in Meishan pigs was significantly higher than that in Yorkshire pigs at the foetal 60 days, while the opposite occurred at postnatal 35 days. The expression ratio of TNNI1 relative to TNNI2 favoured TNNI2 expression in BF and MM regardless of Yorkshire and Meishan pigs. TNNI1 expression in MM was significantly higher than that in BF at 60, 120 and 180 days in Meishan pigs and at 120 and 180 days in Yorkshire pigs. On the contrary, no significant difference of TNNI2 expression in BF and MM was found except for Yorkshire pigs of 180 days. This study provided the foundation for future research on TnI isoforms as the model gene to study mechanisms of muscle fibre-specific gene regulation in pigs.     

Structure and function of the essential light chain of myosin

The review summarizes the recent data on the structure and function of the essential light chain of myosin. It is known that the essential light chain of myosin stabilizes the lever arm. Consistent with the model of the shift of the dynamic population of conformations, the conformational flexibility of the essential light chain is emphasized, which opens the way to determining its new functions. It is proposed that the interaction between the C-terminal domain of the essential light chain and the N-terminal subdomain of the heavy chain of myosin may be involved in the coupling of ATP hydrolysis and rotation of the lever arm. The recent data indicate that the isoforms of the essential light chain with the additional N-terminal peptide are capable of interacting with actin and src-homologous domain 3 of myosin. The structural aspects of these interactions and the modulatory role of the isoforms of the essential light chain of myosin are discussed.