Muscle-specific expression of insulin-like growth factor I counters muscle decline in mdx mice

The Rho GTPase and Fyn tyrosine kinase have been implicated previously in positive control of keratinocyte cell-cell adhesion. Here, we show that Rho and Fyn operate along the same signaling pathway. Endogenous Rho activity increases in differentiating keratinocytes and is required for both Fyn kinase activation and increased tyrosine phosphorylation of beta- and gamma-catenin, which is associated with the establishment of keratinocyte cell-cell adhesion. Conversely, expression of constitutive active Rho is sufficient to promote cell-cell adhesion through a tyrosine kinase- and Fyn-dependent mechanism, trigger Fyn kinase activation, and induce tyrosine phosphorylation of beta- and gamma-catenin and p120ctn. The positive effects of activated Rho on cell-cell adhesion are not induced by an activated Rho mutant with defective binding to the serine/threonine PRK2/PKN kinases. Endogenous PRK2 kinase activity increases with keratinocyte differentiation, and, like activated Rho, increased PRK2 activity promotes keratinocyte cell-cell adhesion and induces tyrosine phosphorylation of beta- and gamma-catenin and Fyn kinase activation. Thus, these findings reveal a novel role of Fyn as a downstream mediator of Rho in control of keratinocyte cell-cell adhesion and implicate the PRK2 kinase, a direct Rho effector, as a link between Rho and Fyn activation.     

Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The absence of dystrophin induces an abnormal increase of sarcolemmal calcium influx through cationic channels in adult skeletal muscle fibers from dystrophic (mdx) mice. We observed that the activity of these channels was increased after depletion of the stores of calcium with thapsigargin or caffeine. By analogy with the situation observed in nonexcitable cells, we therefore hypothesized that these store-operated channels could belong to the transient receptor potential channel (TRPC) family. We measured the expression of TRPC isoforms in normal and mdx adult skeletal muscles fibers, and among the seven known isoforms, five were detected (TRPC1, 2, 3, 4, and 6) by RT-PCR. Western blot analysis and immunocytochemistry of normal and mdx muscle fibers demonstrated the localization of TRPC1, 4, and 6 proteins at the plasma membrane. Therefore, an antisense strategy was used to repress these TRPC isoforms. In parallel with the repression of the TRPCs, we observed that the occurrence of calcium leak channels was decreased to one tenth of its control value (patch-clamp technique), showing the involvement of TRPC in the abnormal calcium influx observed in dystrophic fibers.     

Muscle contraction: energy rate equations in relation to efficiency and step-size distance

We derive the energy rate equation for muscle contraction. Our equation has only two parameters m, the maintenance heat rate and 1/S, the shortening heat coefficient. The impulsive model (previously described in earlier papers) provides a physical basis for parameter 1/S as well as for constants a and b in Hill’s force–velocity equation. We develop new theory and relate the efficiency and the step-size distance to our energy rate equation. Correlation between the efficiency and the step-size distance is established. The various numbers are listed in Table 1: we use data from five different muscles in the literature. In summary, our analysis strongly supports the impulsive model as the correct model of contraction.     

Models in which many cross-bridges attach simultaneously can explain the filament movement per ATP split during muscle contraction

Contractile filaments in skeletal muscle are moved by less than 2 nm for each ATP used. If just one cross-bridge is attached to each thin filament at any instant then this distance represents the fundamental myosin cross-bridge step size (i.e. the distance one cross-bridge moves a thin filament in one ATP-splitting cycle). However, most contraction models assume many cross-bridges are attached at any instant along each thin filament. The purpose of this study was to establish whether the net filament sliding per ATP used could be explained quantitatively in terms of a cross-bridge model in which multiple cross-bridges are attached along each thin filament. It was found that the relationship between net filament sliding per ATP split and the load against which the muscle shortens is compatible with such a model and furthermore predicts that the cross-bridge step size is between 7.5 and 12.5 nm over most of the range of loads. These values were similar for different muscle fibre types.     

Comparative analysis of Dp427-deficient mdx tissues shows that the milder dystrophic phenotype of extraocular and toe muscle fibres is associated with a persistent expression of beta-dystroglycan

The cell biological hypothesis of Duchenne muscular dystrophy assumes that deficiency in the membrane cytoskeletal element dystrophin triggers a loss in surface glycoproteins, such as beta-dystroglycan, thereby rendering the sarcolemmal membrane more susceptible to micro-rupturing. Secondary changes in ion homeostasis, such as increased cytosolic Ca2+ levels and impaired luminal Ca2+ buffering, eventually lead to Ca2+-induced myonecrosis. However, individual muscle groups exhibit a graded pathological response during the natural time course of x-linked muscular dystrophy. The absence of the dystrophin isofom Dp427 does not necessarily result in a severe dystrophic phenotype in all muscle groups. In the dystrophic mdx animal model, extraocular and toe muscles are not as severely affected as limb muscles. Here, we show that the relative expression and sarcolemmal localization of the central trans-sarcolemmal linker of the dystrophin-glycoprotein complex, beta-dystroglycan, is preserved in mdx extraocular and toe fibres by means of two-dimensional immunoblotting and immunofluorescence microscopy. Thus, with respect to improving myology diagnostics, the relative expression levels of beta-dystroglycan appear to represent reliable markers for the severity of secondary changes in dystrophin-deficient fibres. Immunoblotting and enzyme assays revealed that mdx toe muscle fibres exhibit an increased expression and activity of the sarcoplasmic reticulum Ca2+-ATPase. Chemical crosslinking studies demonstrated impaired calsequestrin oligomerization in mdx gastrocnemius muscle indicating that abnormal calsequestrin clustering is involved in reduced Ca2+ buffering of the dystrophic sarcoplasmic reticulum. Previous studies have mostly attributed the sparing of certain mdx fibres to the special protective properties of small-diameter fibres. Our study suggests that the rescue of dystrophin-associated glycoproteins, and possibly the increased removal of cytosolic Ca2+ ions, might also play an important role in protecting muscle cells from necrotic changes.     

The impact of the paper of Edsall and Mehl (1940) on actin and actomyosin research

The paper of Edsall and Mehl, ‘The effect of denaturing agents on myosin, II. Viscosity and double refraction of flow’, J. Biol. Chem. 133 (1940) 409–429, inspired our research on actin and actomyosin. It led to the specific purification of actin with magnesium ions and to the demonstration of the central role of the Mg²⁺-activated actomyosin ATPase in contraction of live muscle.     

An unexpectedly large working stroke from chymotryptic fragments of myosin II

Recent structural evidence indicates that the light chain domain of the myosin head (LCD) bends on the motor domain (MD) to move actin. Structural models usually assume that the actin-MD interface remains static and the possibility that part of the myosin working stroke might be produced by rotation about the acto-myosin interface has been neglected. We have used an optical trap to measure the movement produced by proteolytically shortened single rabbit skeletal muscle myosin heads (S-1(A1) and S-1(A2)). The working stroke produced by these shortened heads was more than that which the MD-LCD bend mechanism predicts from the full-length (papain) S-1’s working stroke obtained under similar conditions. This result indicates that part of the working stroke may be caused by motor action at the actin-MD interface.     

Filamin 2 (FLN2): A muscle-specific sarcoglycan interacting protein

Mutations in genes encoding for the sarcoglycans, a subset of proteins within the dystrophin-glycoprotein complex, produce a limb-girdle muscular dystrophy phenotype; however, the precise role of this group of proteins in the skeletal muscle is not known. To understand the role of the sarcoglycan complex, we looked for sarcoglycan interacting proteins with the hope of finding novel members of the dystrophin-glycoprotein complex. Using the yeast two-hybrid method, we have identified a skeletal muscle-specific form of filamin, which we term filamin 2 (FLN2), as a gamma- and delta-sarcoglycan interacting protein. In addition, we demonstrate that FLN2 protein localization in limb-girdle muscular dystrophy and Duchenne muscular dystrophy patients and mice is altered when compared with unaffected individuals. Previous studies of filamin family members have determined that these proteins are involved in actin reorganization and signal transduction cascades associated with cell migration, adhesion, differentiation, force transduction, and survival. Specifically, filamin proteins have been found essential in maintaining membrane integrity during force application. The finding that FLN2 interacts with the sarcoglycans introduces new implications for the pathogenesis of muscular dystrophy.     

Heterogeneity in growth and differentiation characteristics in male and female satellite cells isolated from turkey lines with different growth rates

The effects of growth- and gender-related differences on satellite cell proliferation and differentiation were investigated using satellite cells isolated from the pectoralis major muscle of a turkey line selected for increased 16-week body weight (F-line) and its unselected randombred control (RBC2-line). Proliferation rates within the F- and RBC2-lines did not differ between sexes. The F-line male and female satellite cells when compared to the RBC2-line male and female satellite cells proliferated at a faster rate. Differentiation rates were increased for the F-line male cells compared to both the F-line female and RBC2-line male satellite cells. No difference in differentiation rate was noted within the RBC2-line satellite cells. For satellite cells from females, the RBC2-line differentiated faster than the F-line. Morphological data on myotube length and the number of nuclei per myotube supported the differentiation data in that F-line male satellite cells had the longest myotubes with the most nuclei, there was no significant difference between myotubes within the RBC2-line, and female-derived myotubes from the RBC2-line were longer than those of the F-line by 96 h of fusion. These data are suggestive of both growth- and gender- related differences in satellite cell proliferation and differentiation.