Eyestalk ablation has little effect on actin and myosin heavy chain gene expression in adult lobster skeletal muscles

The organization of skeletal muscles in decapod crustaceans is significantly altered during molting and development. Prior to molting, the claw muscles atrophy dramatically, facilitating their removal from the base of the claw. During development, lobster claw muscles exhibit fiber switching over several molt cycles. Such processes may be influenced by the secretion of steroid molting hormones, known collectively as ecdysteroids. To assay the effects of these hormones, we used eyestalk ablation to trigger an elevation of circulating ecdysteroids and then quantified myofibrillar mRNA levels with real-time PCR and myofibrillar protein levels by SDS-PAGE. Levels of myosin heavy chain (MHC) and actin proteins and the mRNA encoding them were largely unaffected by eyestalk ablation, but in muscles from intact animals, myofibrillar gene expression was modestly elevated in premolt and postmolt animals. In contrast, polyubiquitin mRNA was significantly elevated (about 2-fold) in claw muscles from eyestalk-ablated animals with elevated circulating ecdysteroids. Moreover, patterns of MHC and actin gene expression are significantly different among slow and fast claw muscles. Consistent with these patterns, the three muscle types differed in the relative amounts of myosin heavy chain and actin proteins. All three muscles also co-expressed fast and slow myosin isoforms, even in fibers that are generally regarded as exclusively fast or slow. These results are consistent with other recent data demonstrating co-expression of myosin isoforms in lobster muscles.     

Role of the ubiquitin-proteasome pathway in sepsis-induced muscle catabolism

Several lines of evidence suggest that the ubiquitin-proteasome pathway is involved in sepsis-induced muscle catabolism. The gene expression of ubiquitin and several of the proteasome subunits was increased in muscle from both septic rats and patients. In other studies, the activity of isolated 20S proteasomes was stimulated in septic muscles. Sepsis-induced increase in muscle total and myofibrillar protein breakdown was inhibited with specific proteasome blockers. Although the ubiquitin-proteasome pathway is upregulated in septic muscle, it is still unclear how the myofibrillar proteins actin and myosin are ubiquitinated and become substrates for the 26S proteasome. Recent studies suggest that a calcium-dependent, calpain-mediated process releases myofilaments from the Z-disks during sepsis. It is possible that this process exposes destabilizing N-terminal residues on actin and myosin, making them suitable substrates for the N-end rule pathway involving the 14 kD ubiquitin-conjugating enzyme E214k and the ubiquitin-protein ligase E3.     

Regenerative defect in vastus lateralis muscle of patients with chronic obstructive pulmonary disease

Background

Impaired skeletal muscle regeneration could contribute to the progression of muscle atrophy in patients with chronic obstructive pulmonary disease (COPD).

Methods

Satellite cells and myogenesis-related proteins were compared between healthy subjects and patients with COPD, with or without muscle atrophy. Satellite cells were isolated and cultured to assess their proliferative and differentiation aptitudes.

Results

Although satellite cell numbers in muscle samples were similar between groups, the proportion of muscle fibers with central nuclei was increased in COPD. In muscle homogenates, increased expression of MyoD and decreased expression of myogenin and MRF4 were observed in COPD. In cultured satellite cells of patients with COPD, increased protein content was observed for Pax7, Myf5 (proliferation phase) and myogenin (differentiation phase) while myosin heavy chain protein content was significantly lower during differentiation.

Conclusion

In COPD, the number of central nuclei was increased in muscle fibers suggesting a greater number of attempts to regenerate muscle tissue than in healthy subjects. Myogenesis signaling was also altered in muscle homogenates in patients with COPD and there was a profound reduction in the differentiation potential in this population as indicated by a reduced ability to incorporate myosin heavy chain into newly formed myotubes. Collectively, these results indicate that skeletal muscle regenerative capacity termination is impaired in COPD and could contribute to the progression of muscle atrophy progression in this population.     

Direct Regulation of Striated Muscle Myosins by Nitric Oxide and Endogenous Nitrosothiols

Background

Nitric oxide (NO) has long been recognized to affect muscle contraction [1], both through activation of guanylyl cyclase and through modification of cysteines in proteins to yield S-nitrosothiols. While NO affects the contractile apparatus directly, the identities of the target myofibrillar proteins remain unknown. Here we report that nitrogen oxides directly regulate striated muscle myosins.

Principal Findings

Exposure of skeletal and cardiac myosins to physiological concentrations of nitrogen oxides, including the endogenous nitrosothiol S-nitroso-L-cysteine, reduced the velocity of actin filaments over myosin in a dose-dependent and oxygen-dependent manner, caused a doubling of force as measured in a laser trap transducer, and caused S-nitrosylation of cysteines in the myosin heavy chain. These biomechanical effects were not observed in response to S-nitroso-D-cysteine, demonstrating specificity for the naturally occurring isomer. Both myosin heavy chain isoforms in rats and cardiac myosin heavy chain from human were S-nitrosylated in vivo.

Significance

These data show that nitrosylation signaling acts as a molecular “gear shift” for myosin—an altogether novel mechanism by which striated muscle and cellular biomechanics may be regulated.     

Ubiquitin and actin expression in claw muscles of land crab,Gecarcinus lateralis,and American lobster,Homarus americanus: differential expression of ubiquitin in two slow muscle fiber types during molt-induced atrophy

The closer muscle of large-clawed decapod crustaceans undergoes a proecdysial (premolt) atrophy to facilitate withdrawal of the appendage at ecdysis. This atrophy involves the activation of both calcium-dependent (calpains) and ubiquitin (Ub)/proteasome-dependent proteolytic systems that break down proteins to reduce muscle mass. Moreover, the large slow-twitch (S(1)) fibers undergo a greater atrophy than the small slow-tonic (S(2)) fibers. Both polyUb mRNA and Ub-protein conjugates increase during claw muscle atrophy. In this study in situ hybridization and RT-PCR were used to determine the temporal and spatial expression of polyUb and alpha-actin. A cDNA encoding the complete sequence of lobster muscle alpha-actin was characterized; a probe synthesized from the cDNA provided a positive control for optimizing RT-PCR and in situ hybridization. PolyUb was expressed at low levels in claw closer muscle from anecdysial (intermolt) land crab. By early proecdysis (premolt; stage D(0)), polyUb mRNA levels increased in medial fibers that insert along the midline of the apodeme, with greater expression in S(1) than S(2), while levels remained low in peripheral fibers. By late proecdysis, polyUb mRNA decreased in central fibers, while mRNA increased in peripheral S(1) fibers. In contrast, alpha-actin was expressed in lobster claw muscles at relatively constant levels during the intermolt cycle. These results suggest that Ub/proteasome-dependent proteolysis contributes to enhanced turnover of myofibrillar proteins during claw closer muscle atrophy. Furthermore, atrophy is not synchronous within the muscle; it begins in medial fibers and then progresses peripherally.     

In vitro non-enzymatic glycosylation of myofibrillar proteins

• 1.1. Glycation is non-enzymatic modification of proteins by sugars in which not only structural but also biological properties of proteins are altered.
• 2.2. Our in vitro experiments show that incubation of myofibrillar proteins with ribose results in sugar attachment to proteins and at the same time myofibrillar ATPase activity is lowered.
• 3.3. DETAPAC, aminoguanidine and 2-mercaptoethanol all partially block myofibrillar protein glycation and ATPase activity is less inactivated.
• 4.4. The dependence of ATPase activity of myofibrils incubated with ribose on the amount of 2-mercaptoethanol present suggests that also modification of SH groups is involved in enzyme inactivation.
• 5.5. Electrophoretic studies revealed that heavy chains of myosin, actin, and tropomyosins are proteins which are mainly glycated in vitro.

     

Getting out of a Tight Squeeze--Enzymatic Regulation of Claw Muscle Atrophy in Molting

The claw closer muscle of the land crab, Gecarcinus lateralis,undergoes a cyclical atrophy and restoration during the intervalbetween ecdyses. During proecdysis (stage D₀), 30–60%of the muscle protein is degraded, which reduces tissue massand facilitates withdrawal of the propodus at ecdysis. Proteinis resynthesized as the muscle grows back to its previous sizeduring metecdysis. This atrophy is specific to the claws andcan be accentuated by multiple limb autotomy. Crustacean musclescontain five cytosolic proteinases that degrade myofibrillarproteins. Four of these constitute a family of enzymes requiringCa²⁺ for activity. These calcium-dependent proteinases (CDPs)hydrolyze myofibrillar proteins in vitro and in situ and showincreased activity in atrophic claw muscles, which suggeststhat CDPs play an important role in myofibrillar protein metabolism.The fifth enzyme is a multicatalytic proteinase (MCP), a multisubunitproteolytic complex that degrades a wide range of peptide andprotein substrates. The catalytic properties of the complexare altered with low concentrations of sodium dodecyl sulfateor by brief heating at 60°C. Only the heat-activated formdegrades myofibrillar proteins. Since the CDPs hydrolyze contractileproteins about 30-fold more rapidly than the heat-activatedMCP, the MCP probably has a more limited or specialized functionin molt-induced claw muscle atrophy.     

Regulation of muscle gene expression: The accumulation of messenger RNAs coding for muscle-specific proteins during myogenesis in a mouse cell line

The expression of RNA sequences coding for myofibrillar proteins has been followed during terminal differentiation in a mouse skeletal muscle cell line. Cloned complementary DNA probes hybridizing with the actins, skeletal muscle α-actin, myosin heavy chain and the myosin alkali light chains were employed in Northern blotting experiments with total cellular poly (A)-containing RNA extracted from the cultures at different times after plating. At the same times, parallel cultures were pulse-labelled with [³⁵S]methionine and the pattern of newly synthesized proteins was analysed by two-dimensional gel electrophoresis. Synthesis of skeletal muscle α-actin and of the myosin alkali light chains (LC_lemb, LC₁, LC₃) was not detectable in dividing myoblast cultures. From the onset of cell fusion, the synthesis of myosin heavy chain, LC_lemb and α-actin increases with similar kinetics. Synthesis of LC₃ (and trace amounts of LC_1F) is detectable and subsequently increases at later stages of myotube formation. The corresponding messenger RNAs coding for myosin heavy chain and skeletal muscle α-actin are first detectable immediately before the initiation of myofibrillar protein synthesis. mRNAs coding for the non-muscle actins are accumulated in myoblasts and diminish after cell fusion. Comparisons between muscle mRNAs depend on the relative sensitivities of the different probes, reflecting mainly their homology with the isoform of the actin or myosin multigene family expressed. Quantitative analysis of Northern blots gives an estimated increase in skeletal muscle α-actin mRNA, with an homologous probe, of at least 130-fold with a minimum level of detection of 40 to 80 molecules per cell. Accumulation of this species and of the myosin heavy chain mRNA follows similar kinetics. mRNA coding for LC₃, the principal myosin light chain detected with the probe, appears to accumulate to a lesser extent initially, paralleling synthesis of the corresponding protein. These results using cloned probes demonstrate a close temporal correlation between muscle mRNA accumulation and protein synthesis during terminal myogenesis in this muscle line.     

Actin mediated calcium dependency of actomyosin in a myxomycete

A fraction obtained from $\text{Physarum polycephalum}$ by differential centrifugation displays magnesium adenosine triphosphatase activity; at low ionic strength (0.07 M KCl) the rate at which ATP¹ is split in 0.1 mM CaCl₂ is from 1.5 to 6.6 times the rate in 1 mM EGTA¹. Both actin and myosin are present in this fraction. On SDS gels several polypeptide bands are present in the range of 39,000 daltons to 14,000 daltons as well as those of actin and myosin. The addition of desensitized rabbit muscle actin to the fraction increased the rate of ATP splitting in EGTA, thereby decreasing the EGTA inhibition 30–50%. We conclude that actomyosin regulation by calcium in this acellular slime mould is, at least in large part, mediated through actin.     

The contractile apparatus of striated muscle in the course of atrophy and regeneration

Summary The ultrastructure of the contractile apparatus of the rat soleus muscle during the course of denervation atrophy was investigated. It was found that the ratio of thin to thick filaments increased in myofibrils of atrophying muscle fibers. Elevation of the ratio was observed as early as the second day after denervation, and became more pronounced with the progress of atrophy. Parallel measurements of the amounts of actin and myosin in the myofibrils and in the muscle protein extracts revealed a lower proportion of myosin heavy chains to actin in the fractions from denervated muscles, compared with the control values. Both the electron-microscopic observations and the biochemical evaluation of the actin content of the muscle, suggests that the elevated ratio of thin to thick filaments seen in the course of the muscle atrophy appears as the result of an earlier and more intensive disappearance of thick filaments. Thin filaments disappeared more slowly, in parallel to the decrease in muscle weight.On the basis of the results presented a mechanism of progress of simple atrophy of muscle in suggested.     

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.     

Accumulation of myosin, actin, tropomyosin, and alpha-actinin in cultured muscles cells.

In an effort to understand the conditions that promote the assembly of myofibrillar proteins in muscle cells, the temporal sequence of accumulation of four myofibrillar proteins, actin, myosin, tropomyosin, and α-actinin, was monitored during the period of de novo assembly of myofibrils in differentiating muscle cells. Isotope dilution experiments indicated that all four proteins were accumulated simultaneously. Therefore, assembly of myofibrils may be occurring in the presence of a full complement of myofibrillar proteins.     

Degradation of myofibrillar proteins by a calpain-like proteinase in the arm muscle of Octopus vulgaris

The effects of a calpain-like proteinase (CaDP) isolated from the arm muscle of Octopus vulgaris on the myofibrils and myofibrillar proteins isolated from the same tissue were examined. Our studies clearly showed that treatment of intact myofibrils with CaDP in the presence of 5 mM Ca²⁺ results in the degradation of the major myofibrillar proteins myosin, paramyosin, and actin. From the isolated α- and β-paramyosins only β-paramyosin is degraded by CaDP in the presence of 5 mM Ca²⁺ producing three groups of polypeptides of 80, 75, and 60 kDa, respectively. The degradation rate depends on the proteinase to substrate ratio, temperature, and time of proteolysis and is inhibited by the endogenous CaDP inhibitory factor (CIF), as well as by various known cysteine proteinase inhibitors (E-64, leupeptin, and antipain). From the other myofibrillar proteins examined myosin, but not actin, is degraded by CaDP; myosin heavy chain (MHC, 200 kDa) is degraded by CaDP producing four groups of polypeptides of lower molecular masses (155, 125, 115, and 102 kDa, respectively); the degradation rate depends on the incubation time and the proteinase to substrate ratio. Furthermore, CaDP undergoes limited autolysis in the presence of both the exogenous casein and the endogenous β-paramyosin producing two large active fragments of 52 and 50.6 kDa, respectively; CIF reversibly inhibits this CaDP autolysis. Accepted: 26 May 2000     

Redistribution of myosin heavy chain mRNA in the midregion of stretched muscle fibers

Summary Myosin mRNA distribution was compared to the distribution of striations, nuclei, and cytoskeletal components in normal fibers and in fibers undergoing growth and repair processes in response to stretch. Plantarflexion of rabbit lower hindlimb for 4 or 6 days resulted in a 35% increase in weight of the tibialis anterior muscle. Slow myosin expression in stretched fibers increased such that the proportion of fibers shifted from the fast type towards an intermediate type. Semi-quantitative in situ hybridization revealed a large increase in concentration of slow myosin mRNA in stretched fibers. Polysomes translating myosin heavy chain were excluded from the intact myofibrillar lattice. Significant increases of myosin mRNA concentration occurred only in the outer 8 m subsarcolemmal annulus of these stretched fibers (P<0.001) where myofibril formation also was evident. In some fibers, stretch caused myofibrillar disorder where nuclei became centrally located, and focal concentrations of myosin mRNA also occurred. We discuss mechanisms for mRNA accumulation and favor free diffusion to loosely packed cytoplasmic regions where myosin is needed for myofibrillar growth and repair.     

Caldesmon and thin-filament regulation of muscle contraction

Smooth muscle contraction is regulated by phosphorylation of myosin and also possibly by the actin associated protein, caldesmon. The properties of caldesmon are discussed and compared with those of tropomyosin-troponin, the well characterized actin-based regulatory system of striated muscle. Caldesmon functions quite differently from tropomyosin-troponin. Under relaxing conditions tropomyosin-troponin does not affect the binding of myosin subfragment-1 to actin. In contrast, caldesmon strongly inhibits the binding of subfragment-1 to actin in the presence of ATP. This inhibition of binding parallels the decrease in ATPase activity that occurs as the caldesmon concentration is increased. Caldesmon has the opposite effect on the two headed myosin subfragment, heavy meromyosin. The apparent binding of skeletal heavy meromyosin increases slightly as the caldesmon concentration is increased, although the rate of ATP hydrolysis is inhibited. It is suggested that in the presence of caldesmon, myosin·ATP does not bind to the productive actin binding site but interacts with a distinct site on actin-caldesmon. This could lead to both an inhibition of ATP hydrolysis and an inrease in resting stiffness of relaxed smooth muscle.     

Regulation of myosin sliding alongChara actin bundles by native skeletal muscle tropomyosin

Summary Native tropomyosin from rabbit skeletal muscle introduced by intracellular perfusion intoChara cells inhibited the cytoplasmic streaming irrespective of the Ca²⁺ concentration. To find the action site of native tropomyosin inChara, the cytoplasmic streaming was reconstituted by introducing isolated endoplasm into actin donorChara cells from which native endoplasm had been removed. The reconstituted streaming was inhibited by pretreatment of the actin donor cells with native tropomyosin but not by that of the endoplasm, suggesting that the native tropomyosin inhibited the cytoplasmic streaming by binding toChara actin bundles. Staining of the actin bundles with FITC-labeled native tropomyosin also showed that the native tropomyosin could bind to the actin bundles. Streaming reconstituted fromChara actin bundles and skeletal muscle myosin was insensitive to Ca²⁺, but became sensitive on application of the native tropomyosin.Abbrevations APW artificial pond water - ATP adenosine 5-triphosphoric acid - BSA bovine serum albumin - EDTA ethylene diamine tetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethylether) N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - FITC-NTM fluorescein isothiocyanate-labeled native tropomyosin - NTM native tropomyosin     

Diversity of native myosin and myosin heavy chain in fish skeletal muscles

• 1.1. Polymorphism of native myosin and myosin heavy chain (MHC) of fish skeletal muscles was analysed by pyrophosphate and SDS-gel electrophoreses.
• 2.2. Depending on the species, three or four myosin isoforms were detected in the white muscle, one or two isoforms in the pure red muscle, and four isomyosins were found in the red muscle composed of red and pink (intermediate) fibres.
• 3.3. It is suggested that all main types of fish muscle fibre (red, intermediate and white) differ in myosin isoform content.
• 4.4. Myosin heavy chain of the red muscle is a distinct protein from that of the white muscle. However, structural differences between these proteins vary among species.

     

Mode of action of rabbit skeletal muscle cathepsin B towards myofibrillar proteins and the myofibrillar structure.

1. The mode of degradation of myofibrillar proteins and the structural changes in myofibrils due to the action of cathepsin B highly purified from rabbit skeletal muscle were studied. 2. Cathepsin B degraded myosin heavy chain, actin and troponin T, but not alpha-actinin, tropomyosin, troponin I or troponin C among myofibrillar proteins. 3. Cathepsin B optimally degraded myosin heavy chain, actin and troponin T at around pH 5. Degradation of myosin heavy chain produced 6 fragments, 180,000, 150,000, 87,000, 81,000, 75,000 and 69,000 Da, respectively. Actin was hydrolyzed into fragments of 41,000, 38,000 and 30,000 Da. Troponin T was degraded into fragments of 21,000, 12,000 and 10,000 Da. 4. Cathepsin B caused the fragmentation of myofibrils and disturbance of the lateral arrangement of myofibrils. 5. Cathepsin B partly disintegrated the Z-line and the M-line, and induced disordering of the arrangement of filaments in the I-band.     

Inhibition of the relative movement of actin and myosin by caldesmon and calponin.

Contractile activity of myosin II in smooth muscle and non-muscle cells requires phosphorylation of myosin by myosin light chain kinase. In addition, these cells have the potential for regulation at the thin filament level by caldesmon and calponin, both of which bind calmodulin. We have investigated this regulation using in vitro motility assays. Caldesmon completely inhibited the movement of actin filaments by either phosphorylated smooth muscle myosin or rabbit skeletal muscle heavy meromyosin. The amount of caldesmon required for inhibition was decreased when tropomyosin is present. Similarly, calponin binding to actin resulted in inhibition of actin filament movement by both smooth muscle myosin and skeletal muscle heavy meromyosin. Tropomyosin had no effect on the amount of calponin needed for inhibition. High concentrations of calmodulin (10 microM) in the presence of calcium completely reversed the inhibition. The nature of the inhibition by the two proteins was markedly different. Increasing caldesmon concentrations resulted in graded inhibition of the movement of actin filaments until complete inhibition of movement was obtained. Calponin inhibited actin sliding in a more "all or none" fashion. As the calponin concentration was increased the number of actin filaments moving was markedly decreased, but the velocity of movement remained near control values.