Skeletal muscle myogenesis is regulated by G protein-coupled receptor kinase 2

G protein-coupled receptor kinase 2 （GRK2） is an important serine/threonine-kinase regulating different membrane receptors and intraceUular proteins. Attenuation of Drosophila Gprk2 in embryos or adult flies induced a defective differentiation of somatic muscles, loss of fibers, and a flightless phenotype. In vertebrates, GRK2 hemizygous mice contained less but more hypertrophied skeletal muscle fibers than wild-type littermates. In C2C12 myoblasts, overexpression of a GRK2 kinase-deficient mutant （K220R） caused precocious differentiation of ceUs into immature myotubes, which were wider in size and contained more fused nuclei, while GRK2 overexpression blunted differentiation. Moreover, p38MAPK and Akt pathways were activated at an earlier stage and to a greater extent in K220R-expressing cells or upon kinase downregulation, while the activation of both kinases was impaired in GRK2-overexpressing cells. The impaired differentiation and fewer fusion events promoted by enhanced GRK2 levels were recapitulated by a p38MAPK mutant, which was able to mimic the inhibitory phosphorylation of p38MAPK by GRK2, whereas the blunted differentiation observed in GRK2-expressing clones was rescued in the presence of a constitutively active upstream stimulator of the p38MAPK pathway. These results suggest that balanced GRK2 function is necessary for a timely and complete myogenic process.     

Skeletal myogenesis by human embryonic stem cells

We have examined the myogenic potential of human embryonic stem （hES） cells in a xeno-transplantation animal model. Here we show that precursors differentiated from hES cells can undergo myogenesis in an adult environment and give rise to a range of cell types in the myogenic lineage. This study provides direct evidences that hES cells can regenerate both muscle and satellite cells in vivo and are another promising cell type for treating muscle degenerative disorders in addition to other myogenic cell types.     

Skeletal myogenesis by human primordial germ cell-derived progenitors

We have isolated and cultured human primordial germ cells (PGCs) from early embryos. The PGCs expressed embryonic germ (EG) cell-specific surface markers, including Oct4 and Nanos. We derived a cell population from these PGCs that we termed embryoid body-derived (EBD) cells. EBD cells can be extensively expanded in vitro for more than 50 passages and express lineage markers from all three primary germ layers. The myogenic potential of the EBD cells was examined both in vitro and in vivo.In vitro, the EBD cells can be induced to form multinucleated myotubes, which express late skeletal muscle-specific markers, including MHC and dystrophin, when exposed to human galectin-1. In vivo, the EBD cells gave rise to all the myogenic lineages, including the skeletal muscle stem cells known as satellite cells. Strikingly, these cells were able to partially restore degenerated muscles in the SCID/mdx mouse, an animal model of the Duchenne’s muscular dystrophy. These results indicate the EBD cells may be a promising source of myogenic stem cells for cell-based therapies for muscle degenerative disorders.     

Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca²⁺/calmodulin-dependent serine–threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca²⁺ binding to calmodulin forming a (Ca²⁺)₄•calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca²⁺ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca²⁺/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca²⁺-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.     

Enhanced skeletal muscle contraction with myosin light chain phosphorylation by a calmodulin-sensing kinase

Repetitive low frequency stimulation results in potentiation of twitch force development in fast-twitch skeletal muscle due to myosin regulatory light chain (RLC) phosphorylation by Ca(2+)/calmodulin (CaM)-dependent skeletal muscle myosin light chain kinase (skMLCK). We generated transgenic mice that express an skMLCK CaM biosensor in skeletal muscle to determine whether skMLCK or CaM is limiting to twitch force potentiation. Three transgenic mouse lines exhibited up to 22-fold increases in skMLCK protein expression in fast-twitch extensor digitorum longus muscle containing type IIa and IIb fibers, with comparable expressions in slow-twitch soleus muscle containing type I and IIa fibers. The high expressing lines showed a more rapid RLC phosphorylation and force potentiation in extensor digitorum longus muscle with low frequency electrical stimulation. Surprisingly, overexpression of skMLCK in soleus muscle did not recapitulate the fast-twitch potentiation response despite marked enhancement of both fast-twitch and slow-twitch RLC phosphorylation. Analysis of calmodulin binding to the biosensor showed a frequency-dependent activation to a maximal extent of 60%. Because skMLCK transgene expression is 22-fold greater than the wild-type kinase, skMLCK rather than calmodulin is normally limiting for RLC phosphorylation and twitch force potentiation. The kinase activation rate (10.6 s(-1)) was only 3.6-fold slower than the contraction rate, whereas the inactivation rate (2.8 s(-1)) was 12-fold slower than relaxation. The slower rate of kinase inactivation in vivo with repetitive contractions provides a biochemical memory via RLC phosphorylation. Importantly, RLC phosphorylation plays a prominent role in skeletal muscle force potentiation of fast-twitch type IIb but not type I or IIa fibers.     

Mitosis-specific phosphorylation of myosin light chain kinase

Cell cytosol preparations from mitotic HeLa cells exhibit a kinase activity that phosphorylates myosin light chain kinase (MLCK). This MLCK kinase activity is apparently distinct from the known MLCK kinases, including cAMP-dependent protein kinase, cGMP-dependent protein kinase, Ca(2+)-activated phospholipid-dependent protein kinase, or Ca(2+)-calmodulin-dependent protein kinase II, based on the following criteria. First, the MLCK kinase activity of mitotic cells does not respond to a variety of characteristic activators or inhibitors of these known kinases. Second, one- and two-dimensional peptide maps have revealed that the site of phosphorylation by the MLCK kinase of mitotic cells differs from those by these known kinases. The mitotic MLCK kinase phosphorylates MLCK at a threonine residue at a ratio of up to 1 mol of phosphate/mol of chicken gizzard MLCK. The MLCK kinase is mitosis-specific because mitotic cell extracts show much higher phosphorylation activity than nonmitotic cell extracts.     

Insulin-mediated tyrosine phosphorylation of myosin heavy chain and concomitant enhanced association of C-terminal SRC kinase during skeletal muscle differentiation

In this study, we present for the first time: (1) evidence regarding tyrosine phosphorylation of myosin heavy chain, (2) evidence that insulin can phosphorylate myosin, (3) association of myosin with Csk, a signalling molecule, (4) modulation of this association by insulin, and (5) evidence that these interactions are associated with skeletal muscle differentiation.     

MicroRNA-323-3p promotes myogenesis by targeting Smad2

Skeletal muscle is an important and complex organ with multiple biological functions in humans and animals. Proliferation and differentiation of myoblasts are the key steps during the development of skeletal muscle. MicroRNA (miRNA) is a class of 21-nucleotide noncoding RNAs regulating gene expression by combining with the 3′-untranslated region of target messenger RNA. Many studies in recent years have suggested that miRNAs play a critical role in myogenesis. Through high-throughput sequencing, we found that miR-323-3p showed significant changes in the longissimus dorsi muscle of Rongchang pigs in different age groups. In this study, we discovered that overexpression of miR-323-3p repressed myoblast proliferation and promoted differentiation, whereas the inhibitor of miR-323-3p displayed the opposite results. Furthermore, we predicted Smad2 as the target gene of miR-323-3p and found that miR-323-3p directly modulated the expression level of Smad2. Then luciferase reporter assays verified that Smad2 was a target gene of miR-323-3p during the differentiation of myoblasts. These findings reveal that miR-323-3p is a positive regulator of myogenesis by targeting Smad2. This provides a novel mechanism of miRNAs in myogenesis.     

Effects of relaxin on rat uterine myosin light chain kinase activity and myosin light chain phosphorylation

Isometrically suspended uteri from estrogen-primed rats were stimulated with prostaglandin F2 alpha and then exposed to relaxin. Relaxin-dependent decreases in the ratio of phosphorylated to total myosin light chains (MLC) and in MLC kinase activity, measured in the presence of 0.5 mg/ml of uterine myosin and the absence and presence of Ca2+-calmodulin (CaM), were observed. The time-course and concentration-response of these biochemical effects of relaxin paralleled the hormone-induced inhibition of uterine contractile activity. Relaxin treatment resulted in a change in the requirements of MLC kinase for Ca2+, CaM, and myosin. Titrations of MLC kinase activity showed a shift in K50 values for Ca2+ from 82 to 260 nM and for CaM from 2.2 to 25 nM in extracts from control and relaxin-treated tissues, respectively. The myosin Km values of MLC kinase from control and relaxin-treated tissues were 0.33 and 0.71 mg/ml, respectively. Under optimal assay conditions (100 microM Ca2+, 1 microM CaM, and 1.2 mg/ml of myosin) the activities of MLC kinase in both extracts were identical, regardless of hormone concentration or exposure time. These data suggest that relaxin-treatment results in a change in the affinity of MLC kinase for its substrate and modulator and that relaxin inhibits uterine contractile activity by a mechanism which involves a decrease in MLC kinase activity and, in turn, a decrease in phosphorylation of the 20,000-dalton light chains of myosin.     

Selective phosphorylation of myosin light chain in intact skeletal muscle

The phosphorylation of myosin light chain was quantitated in fast and slow chicken skeletal muscles and in frog sartorius and semitendinosus muscles. The phosphate content of light chain was determined either as moles [³²P]phosphate per mole of light chain in ³²P-labeled muscles or as percentage phosphorylated light chain of the total P-light chain, measured by densitometry after separating the phospho and dephospho forms of P-light chain with two-dimensional gel electrophoresis. Both methods revealed that the percentage of total P-light chain which was phosphorylated did not exceed 50% either in maximally tetanized or caffeine-contracted skeletal muscle. This suggests that one of the two P-light chains is selectively phosphorylated in skeletal muscle.     

Autophosphorylation of skeletal muscle myosin light chain kinase.

Ca2+/calmodulin-dependent myosin light chain kinase phosphorylates the regulatory light chain of myosin. Rabbit skeletal muscle myosin light chain kinase also catalyzes a Ca2+/calmodulin-dependent autophosphorylation with a rapid rate of incorporation of 1 mol of 32P/mol of kinase and a slower rate of incorporation up to 1.52 mol of 32P/mol. Autophosphorylation was inhibited by a peptide substrate that has a low Km value for myosin light chain kinase. Autophosphorylation at both rates was concentration-independent, indicating an intramolecular mechanism. There were no significant changes in catalytic properties toward light chain and MgATP substrates or in calmodulin activation properties upon autophosphorylation. After digestion with V8 protease, phosphopeptides were purified and sequenced. Two phosphorylation sites were identified, Ser 160 and Ser 234, with the former associated with the rapid rate of phosphorylation. Both sites are located amino terminal of the catalytic domain. These results indicate that the extended "tail" region of the enzyme can fold into the active site of the kinase.     

Protein kinase C modulates in vitro phosphorylation of the smooth muscle heavy meromyosin by myosin light chain kinase

Protein kinase C phosphorylates different sites on the 20,000-Da light chain of smooth muscle heavy meromyosin (HMM) than did myosin light chain kinase (Nishikawa, M., Hidaka, H., and Adelstein, R. S. (1983) J. Biol. Chem. 258, 14069-14072). Although protein kinase C incorporates 1 mol of phosphate into 1 mol of 20,000-Da light chain when either HMM or the whole myosin molecule is used as a substrate, it catalyzes the incorporation of up to 3 mol of phosphate/mol of 20,000-Da light chain when the isolated light chains are used as a substrate. Threonine is the major phosphoamino acid resulting from phosphorylation of HMM by protein kinase C. Prephosphorylation of HMM by protein kinase C decreases the rate of phosphorylation of HMM by myosin light chain kinase due to a 9-fold increase of the Km for prephosphorylated HMM compared to that of unphosphorylated HMM. Prephosphorylation of HMM by myosin light chain kinase also results in a decrease of the rate of phosphorylation by protein kinase C due to a 2-fold increase of the Km for HMM. Both prephosphorylations have little or no effect on the maximum rate of phosphorylation. The sequential phosphorylation of HMM by myosin light chain kinase and protein kinase C results in a decrease in actin-activated MgATPase activity due to a 7-fold increase of the Km for actin over that observed with phosphorylated HMM by myosin light chain kinase but has little effect on the maximum rate of the actin-activated MgATPase activity. The decrease of the actin-activated MgATPase activity correlates well with the extent of the additional phosphorylation of HMM by protein kinase C following initial phosphorylation by myosin light chain kinase.     

A novel lncRNA promotes myogenesis of bovine skeletal muscle satellite cells via PFN1-RhoA/Rac1

Myogenesis, the process of skeletal muscle formation, is a highly coordinated multistep biological process. Accumulating evidence suggests that long non-coding RNAs (lncRNAs) are emerging as a gatekeeper in myogenesis. Up to now, most studies on muscle development-related lncRNAs are mainly focussed on humans and mice. In this study, a novel muscle highly expressed lncRNA, named lnc23, localized in nucleus, was found differentially expressed in different stages of embryonic development and myogenic differentiation. The knockdown and over-expression experiments showed that lnc23 positively regulated the myogenic differentiation of bovine skeletal muscle satellite cells. Then, TMT 10-plex labelling quantitative proteomics was performed to screen the potentially regulatory proteins of lnc23. Results indicated that lnc23 was involved in the key processes of myogenic differentiation such as cell fusion, further demonstrated that down-regulation of lnc23 may inhibit myogenic differentiation by reducing signal transduction and cell fusion among cells. Furthermore, RNA pulldown/LC-MS and RIP experiment illustrated that PFN1 was a binding protein of lnc23. Further, we also found that lnc23 positively regulated the protein expression of RhoA and Rac1, and PFN1 may negatively regulate myogenic differentiation and the expression of its interacting proteins RhoA and Rac1. Hence, we support that lnc23 may reduce the inhibiting effect of PFN1 on RhoA and Rac1 by binding to PFN1, thereby promoting myogenic differentiation. In short, the novel identified lnc23 promotes myogenesis of bovine skeletal muscle satellite cells via PFN1-RhoA/Rac1.     

Identification of a phosphorylation site on skeletal muscle myosin light chain kinase that becomes phosphorylated during muscle contraction.

A protein phosphorylated efficiently in vitro by MAP kinase-activated protein kinase-2 (MAPKAP-K2) was purified from skeletal muscle extracts and identified as the calcium/calmodulin-dependent myosin light chain kinase (MLCK). The phosphorylation site was mapped to Ser(161), a residue shown previously to be autophosphorylated by MLCK. The residue equivalent to Ser(161) became phosphorylated in vivo when rat hindlimbs were stimulated electrically. However, phosphorylation was triggered within seconds, whereas activation of MAPKAP-K2 required several minutes. Moreover, contraction-induced Ser(161) phosphorylation was similar in wild-type or MAPKAP-K2-/- mice. These results indicate that contraction-induced phosphorylation is probably catalyzed by MLCK and not MAPKAP-K2. Ser(161) phosphorylation induced the binding of MLCK to 14-3-3 proteins, but did not detectably affect the kinetic properties of MLCK. The sequence surrounding Ser(161) is unusual in that residue 158 is histidine. Previously, an arginine located three residues N-terminal to the site of phosphorylation was thought to be critical for the specificity of MAPKAP-K2.