The genetic basis of many muscular disorders, including many of the more common muscular dystrophies, is now known. Clinically, the recent genetic advances have improved diagnostic capabilities, but ...they have not yet provided clues about treatment or management. Thanks to better management strategies and therapeutic interventions, however, many patients with a muscular dystrophy are more active and are living longer. Physical therapists, therefore, are more likely to see a patient with a muscular dystrophy, so understanding these muscle disorders and their management is essential. Physical therapy offers the most promise in caring for the majority of patients with these conditions, because it is unlikely that advances in gene therapy will significantly alter their clinical treatment in the near future. This perspective covers some of the basic molecular biological advances together with the clinical manifestations of the muscular dystrophies and the latest approaches to their management.
Intermediate filaments, composed of desmin and of keratins, play important roles in linking contractile elements to each other and to the sarcolemma in striated muscle. Our previous results show that ...the tibialis anterior (TA) muscles of mice lacking keratin 19 (K19) lose costameres, accumulate mitochondria under the sarcolemma, and generate lower specific tension than controls. Here we compare the physiology and morphology of TA muscles of mice lacking K19 with muscles lacking desmin or both proteins double knockout (DKO). K19-/- mice and DKO mice showed a threefold increase in the levels of creatine kinase (CK) in the serum. The absence of desmin caused a larger change in specific tension (-40%) than the absence of K19 (-19%) and played the predominant role in contractile function (-40%) and decreased tolerance to exercise in the DKO muscle. By contrast, the absence of both proteins was required to obtain a significantly greater loss of contractile torque after injury (-48%) compared with wild type (-39%), as well as near-complete disruption of costameres. The DKO muscle also showed a significantly greater misalignment of myofibrils than either mutant alone. In contrast, large subsarcolemmal gaps and extensive accumulation of mitochondria were only seen in K19-null TA muscles, and the absence of both K19 and desmin yielded milder phenotypes. Our results suggest that keratin filaments containing K19- and desmin-based intermediate filaments can play independent, complementary, or antagonistic roles in the physiology and morphology of fast-twitch skeletal muscle.
Obscurin is a large ( approximately 800-kDa), modular protein of striated muscle that concentrates around the M-bands and Z-disks of each sarcomere, where it is well positioned to sense contractile ...activity. Obscurin contains several signaling domains, including a rho-guanine nucleotide exchange factor (rhoGEF) domain and tandem pleckstrin homology domain, consistent with a role in rho signaling in muscle. We investigated the ability of obscurin's rhoGEF domain to interact with and activate small GTPases. Using a combination of in vitro and in vivo approaches, we found that the rhoGEF domain of obscurin binds selectively to rhoA, and that rhoA colocalizes with obscurin at the M-band in skeletal muscle. Other small GTPases, including rac1 and cdc42, neither associate with the rhoGEF domain of obscurin nor concentrate at the level of the M-bands. Furthermore, overexpression of the rhoGEF domain of obscurin in adult skeletal muscle selectively increases rhoA expression and activity in this tissue. Overexpression of obscurin's rhoGEF domain and its effects on rhoA alter the expression of rho kinase and citron kinase, both of which can be activated by rhoA in other tissues. Injuries to rodent hindlimb muscles caused by large-strain lengthening contractions increases rhoA activity and displaces it from the M-bands to Z-disks, similar to the effects of overexpression of obscurin's rhoGEF domain. Our results suggest that obscurin's rhoGEF domain signals at least in part by inducing rhoA expression and activation, and altering the expression of downstream kinases in vitro and in vivo.
Mutations in the gene DYSF, which codes for the protein dysferlin, underlie Miyoshi myopathy and limb-girdle muscular dystrophy 2B in humans and produce a slowly progressing skeletal muscle ...degenerative disease in mice. Dysferlin is a Ca(2+)-sensing, regulatory protein that is involved in membrane repair after injury. To assess the function of dysferlin in healthy and dystrophic skeletal muscle, we generated skeletal muscle-specific transgenic mice with threefold overexpression of this protein. These mice were phenotypically indistinguishable from wild-type, and more importantly, the transgene completely rescued the muscular dystrophy (MD) disease in Dysf-null A/J mice. The dysferlin transgene rescued all histopathology and macrophage infiltration in skeletal muscle of Dysf(-/-) A/J mice, as well as promoted the rapid recovery of muscle function after forced lengthening contractions. These results indicate that MD in A/J mice is autonomous to skeletal muscle and not initiated by any other cell type. However, overexpression of dysferlin did not improve dystrophic symptoms or membrane instability in the dystrophin-glycoprotein complex-lacking Scgd (delta-sarcoglycan) null mouse, indicating that dysferlin functionality is not a limiting factor underlying membrane repair in other models of MD. In summary, the restoration of dysferlin in skeletal muscle fibers is sufficient to rescue the MD in Dysf-deficient mice, although its mild overexpression does not appear to functionally enhance membrane repair in other models of MD.
Little is known about the mechanisms that organize the internal membrane systems in eukaryotic cells. We are addressing this question in striated muscle, which contains two novel systems of internal ...membranes, the transverse tubules and the sarcoplasmic reticulum (SR). Small ankyrin-1 (sAnk1) is an ∼17-kDa transmembrane protein of the SR that concentrates around the Z-disks and M-lines of each sarcomere. We used the yeast two-hybrid assay to determine whether sAnk1 interacts with titin, a giant myofibrillar protein that organizes the sarcomere. We found that the hydrophilic cytoplasmic domain of sAnk1 interacted with the two most N-terminal Ig domains of titin, ZIg1 and ZIg2, which are present at the Z-line in situ. Both ZIg1 and ZIg2 were required for binding activity. sAnk1 did not interact with other sequences of titin that span the Z-disk or with Ig domains of titin near the M-line. Titin ZIg1/2 also bound T-cap/telethonin, a 19-kDa protein of the Z-line. We show that titin ZIg1/2 could form a three-way complex with sAnk1 and T-cap. Our results indicate that titin ZIg1/2 can bind sAnk1 in muscle homogenates and suggest a role for these proteins in organizing the SR around the contractile apparatus at the Z-line.
Myocardial dysfunction contributes to the high mortality of patients with endotoxemia. Although nitric oxide (NO) has been implicated in the pathogenesis of septic cardiovascular dysfunction, the ...role of myocardial NO synthase 3 (NOS3) remains incompletely defined. Here we show that mice with cardiomyocyte-specific NOS3 overexpression (NOS3TG) are protected from myocardial dysfunction and death associated with endotoxemia. Endotoxin induced more marked impairment of Ca(2+) transients and cellular contraction in wild-type than in NOS3TG cardiomyocytes, in part, because of greater total sarcoplasmic reticulum Ca(2+) load and myofilament sensitivity to Ca(2+) in the latter during endotoxemia. Endotoxin increased reactive oxygen species production in wild-type but not NOS3TG hearts, in part, because of increased xanthine oxidase activity. Inhibition of NOS by N(G)-nitro-l-arginine-methyl ester restored the ability of endotoxin to increase reactive oxygen species production and xanthine oxidase activity in NOS3TG hearts to the levels measured in endotoxin-challenged wild-type hearts. Allopurinol, a xanthine oxidase inhibitor, attenuated endotoxin-induced reactive oxygen species accumulation and myocardial dysfunction in wild-type mice. The protective effects of cardiomyocyte NOS3 on myocardial function and survival were further confirmed in a murine model of polymicrobial sepsis. These results suggest that increased myocardial NO levels attenuate endotoxin-induced reactive oxygen species production and increase total sarcoplasmic reticulum Ca(2+) load and myofilament sensitivity to Ca(2+), thereby reducing myocardial dysfunction and mortality in murine models of septic shock.
We describe improved methods for large format, two‐dimensional gel electrophoresis (2DE) that improve protein solubility and recovery, minimize proteolysis, and reduce the loss of resolution due to ...contaminants and manipulations of the gels, and thus enhance quantitative analysis of protein spots. Key modifications are: (i) the use of 7 M urea and 2 M thiourea, instead of 9 M urea, in sample preparation and in the tops of the gel tubes; (ii) standardized deionization of all solutions containing urea with a mixed bed ion exchange resin and removal of urea from the electrode solutions; and (iii) use of a new gel tank and cooling device that eliminate the need to run two separating gels in the SDS dimension. These changes make 2DE analysis more reproducible and sensitive, with minimal artifacts. Application of this method to the soluble fraction of muscle tissues reliably resolves ∼1800 protein spots in adult human skeletal muscle and over 2800 spots in myotubes.
μ-Crystallin, encoded by the CRYM gene, binds the thyroid hormones, T3 and T4. Because T3 and T4 are potent regulators of metabolism and gene expression, and CRYM levels in human skeletal muscle can ...vary widely, we investigated the effects of overexpression of Crym. We generated transgenic mice, Crym tg, that expressed Crym protein specifically in skeletal muscle at levels 2.6–147.5 fold higher than in controls. Muscular functions, Ca2+ transients, contractile force, fatigue, running on treadmills or wheels, were not significantly altered, although T3 levels in tibialis anterior (TA) muscle were elevated ~190-fold and serum T4 was decreased 1.2-fold. Serum T3 and thyroid stimulating hormone (TSH) levels were unaffected. Crym transgenic mice studied in metabolic chambers showed a significant decrease in the respiratory exchange ratio (RER) corresponding to a 13.7% increase in fat utilization as an energy source compared to controls. Female but not male Crym tg mice gained weight more rapidly than controls when fed high fat or high simple carbohydrate diets. Although labeling for myosin heavy chains showed no fiber type differences in TA or soleus muscles, application of machine learning algorithms revealed small but significant morphological differences between Crym tg and control soleus fibers. RNA-seq and gene ontology enrichment analysis showed a significant shift towards genes associated with slower muscle function and its metabolic correlate, β-oxidation. Protein expression showed a similar shift, though with little overlap. Our study shows that μ-crystallin plays an important role in determining substrate utilization in mammalian muscle and that high levels of μ-crystallin are associated with a shift toward greater fat metabolism.
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•μ-Crystallin (Crym) is expressed specifically in transgenic skeletal muscle at highly elevated levels.•T3 is increased ~190 fold in the Tibialis anterior muscle of Crym tg mice.•Small but significant changes in gene and protein expression in tg muscle towards a slow twitch, oxidative phenotype.•Metabolic studies show that Crym tg mice increase their use of fat as an energy source.•Female Crym tg mice gain weight faster on high fat or simple carbohydrate diets than controls.
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