Patrick W. Reed

Absence of keratin 19 in mice causes skeletal myopathy with mitochondrial and sarcolemmal reorganization

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. We examined the contractile properties and morphology of fast-twitch skeletal muscle from mice lacking keratin 19. Tibialis anterior muscles of keratin-19-null mice showed a small but significant decrease in mean fiber diameter and in the specific force of tetanic contraction, as well as increased plasma creatine kinase levels. Costameres at the sarcolemma of keratin-19-null muscle, visualized with antibodies against spectrin or dystrophin, were disrupted and the sarcolemma was separated from adjacent myofibrils by a large gap in which mitochondria accumulated. The costameric dystrophin-dystroglycan complex, which co-purified with γ-actin, keratin 8 and keratin 19 from striated muscles of wild-type mice, co-purified with γ-actin but not keratin 8 in the mutant. Our results suggest that keratin 19 in fast-twitch skeletal muscle helps organize costameres and links them to the contractile apparatus, and that the absence of keratin 19 disrupts these structures, resulting in loss of contractile force, altered distribution of mitochondria and mild myopathy. This is the first demonstration of a mammalian phenotype associated with a genetic perturbation of keratin 19.

Costameres: repeating structures at the sarcolemma of skeletal muscle.

Costameres, structures at the plasma membrane of skeletal muscle, are present in a rectilinear array that parallels the organization of the underlying contractile apparatus. Costameres have three major functions: to keep the plasma membrane, or sarcolemma, aligned and in register with nearby contractile structures; to protect the sarcolemma against contraction-induced damage; and to transmit some of the forces of contraction laterally, to the extracellular matrix. These functions require that costameres link the contractile apparatus through the membrane to the extracellular matrix. Mutations to key components of costameres cause these structures to lose their rectilinear organization and can result in muscle weakness or death. This article summarizes the evidence that costameres are composed of large complexes of integral and peripheral membrane proteins that are linked to the contractile apparatus by intermediate filaments and to the extracellular matrix by laminin. They also present evidence that costameres are altered when key costameric components are missing, as in a murine form of muscular dystrophy.

Extensive mononuclear infiltration and myogenesis characterize recovery of dysferlin-null skeletal muscle from contraction-induced injuries

We studied the response of dysferlin-null and control skeletal muscle to large- and small-strain injuries to the ankle dorsiflexors in mice. We measured contractile torque and counted fibers retaining 10-kDa fluorescein dextran, necrotic fibers, macrophages, and fibers with central nuclei and expressing developmental myosin heavy chain to assess contractile function, membrane resealing, necrosis, inflammation, and myogenesis. We also studied recovery after blunting myogenesis with X-irradiation. We report that dysferlin-null myofibers retain 10-kDa dextran for 3 days after large-strain injury but are lost thereafter, following necrosis and inflammation. Recovery of dysferlin-null muscle requires myogenesis, which delays the return of contractile function compared with controls, which recover from large-strain injury by repairing damaged myofibers without significant inflammation, necrosis, or myogenesis. Recovery of control and dysferlin-null muscles from small-strain injury involved inflammation and necrosis followed by myogenesis, all of which were more pronounced in the dysferlin-null muscles, which recovered more slowly. Both control and dysferlin-null muscles also retained 10-kDa dextran for 3 days after small-strain injury. We conclude that dysferlin-null myofibers can survive contraction-induced injury for at least 3 days but are subsequently eliminated by necrosis and inflammation. Myogenesis to replace lost fibers does not appear to be significantly compromised in dysferlin-null mice.

Abnormal expression of mu-crystallin in facioscapulohumeral muscular dystrophy

To identify proteins expressed abnormally in facioscapulohumeral muscular dystrophy (FSHD), we extracted soluble proteins from deltoid muscle biopsies from unaffected control and FSHD patients and analyzed them using two-dimensional electrophoresis, mass spectrometry and immunoblotting. Muscles from patients with FSHD showed large increases over controls in a single soluble, 34 kDa protein (pI=5.08) identified by mass spectrometry and immunoblotting as mu-crystallin (CRYM). Soluble fractions of biopsies of several other myopathies and muscular dystrophies showed no appreciable increases in mu-crystallin. Mu-crystallin has thyroid hormone and NADPH binding activity and so may influence differentiation and oxidative stress responses, reported to be altered in FSHD. It is also linked to retinal and inner ear defects, common in FSHD, suggesting that its up-regulation may play a specific and important role in pathogenesis of FSHD.

Sarcolemmal Reorganization in Facioscapulohumeral Muscular Dystrophy

We examined the sarcolemma of skeletal muscle from patients with facioscapulohumeral muscular dystrophy (FSHD1A) to learn if, as in other murine and human muscular dystrophies, its organization and relationship to nearby contractile structures are altered.

Physiological and histological changes in skeletal muscle following in vivo gene transfer by electroporation

Electroporation (EP) is used to transfect skeletal muscle fibers in vivo, but its effects on the structure and function of skeletal muscle tissue have not yet been documented in detail. We studied the changes in contractile function and histology after EP and the influence of the individual steps involved to determine the mechanism of recovery, the extent of myofiber damage, and the efficiency of expression of a green fluorescent protein (GFP) transgene in the tibialis anterior (TA) muscle of adult male C57Bl/6J mice. Immediately after EP, contractile torque decreased by ∼80% from pre-EP levels. Within 3 h, torque recovered to ∼50% but stayed low until day 3. Functional recovery progressed slowly and was complete at day 28. In muscles that were depleted of satellite cells by X-irradiation, torque remained low after day 3, suggesting that myogenesis is necessary for complete recovery. In unirradiated muscle, myogenic activity after EP was confirmed by an increase in fibers with central nuclei or developmental myosin. Damage after EP was confirmed by the presence of necrotic myofibers infiltrated by CD68+ macrophages, which persisted in electroporated muscle for 42 days. Expression of GFP was detected at day 3 after EP and peaked on day 7, with ∼25% of fibers transfected. The number of fibers expressing green fluorescent protein (GFP), the distribution of GFP+ fibers, and the intensity of fluorescence in GFP+ fibers were highly variable. After intramuscular injection alone, or application of the electroporating current without injection, torque decreased by ∼20% and ∼70%, respectively, but secondary damage at D3 and later was minimal. We conclude that EP of murine TA muscles produces variable and modest levels of transgene expression, causes myofiber damage due to the interaction of intramuscular injection with the permeabilizing current, and that full recovery requires myogenesis.

The sarcolemma in the largemyd mouse

In the Largemyd mouse, dystroglycan is incompletely glycosylated and thus cannot bind its extracellular ligands, causing a muscular dystrophy that is usually lethal in early adulthood. We show that the Largemyd mutation alters the composition and organization of the sarcolemma of fast‐twitch skeletal muscle fibers in young adult mice. Costameres at the sarcolemma of the tibialis anterior muscle of Largemyd mice contain reduced levels of several membrane cytoskeletal proteins, including dystrophin and β‐spectrin. In the quadriceps, longitudinally oriented costameric structures tend to become thickened and branched. More strikingly, proteins of the dystrophin complex present between costameres in controls are absent from Largemyd muscles. We propose that the absence of the dystrophin complex from these regions destabilizes the sarcolemma of the Largemyd mouse and thereby contributes to the severity of its muscular dystrophy. Thus, the positioning of sarcolemmal proteins may have a profound effect on the health of skeletal muscle. Muscle Nerve, 2004

Optimization of large gel 2D electrophoresis for proteomic studies of skeletal muscle

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.

Crystallin-gazing: unveiling enzymatic activity

binding protein (Vie et al. 1997), originally discovered in the ocular lens of marsupials and subsequently found in other tissues including brain, cochlea, kidney, heart, skeletal muscle and other tissues. Its crystal structure has been solved (Cheng et al. 2007). Mutations in l-crystallin, including one that inhibits binding of thyroid hormone, cause non-syndromic deafness in man (Abe et al. 2003; Oshima et al. 2006). Altered expression of l-crystallin also occurs in other human diseases, such as Alzheimer’s disease, hyperglycemia, heart failure, facioscapulohumeral muscular dystrophy and schizophrenia (Reed et al. 2007; Sommer et al. 2010; Martins-de-Souza et al. 2011), as well as in murine mutants of superoxide dismutase 1 (Fukada et al. 2007), studied as models of amyotrophic lateral sclerosis. l-Crystallin is thought to bind to thyroid hormone in the cytoplasm of cells (Suzuki et al. 2007) and may deliver the hormone to the nucleus (Mori et al. 2002), where it plays an important role in regulating gene expression. Until the current report by Hallen et al. (2011), no enzymatic function for l-crystallin has been reported. In this study, Hallen et al. (2011) show for the first time that l-crystallin has ketimine reductase activity. They use mass spectroscopy to demonstrate that l-crystallin is essentially identical to ketimine reductase purified from lamb forebrain, and show further that a purified, recombinant form of human l-crystallin has a specific activity and pH optimum in ketimine reductase assays similar to the purified mammalian enzyme. Like the latter, it can catalyze the conversion of several different cyclic ketimines to their reduced products. Remarkably, the authors report that the most common form of thyroid hormone, 3,5,3¢-triiodothyronine (T3), strongly inhibits catalytic activity at sub-micromolar concentrations. The authors point out that the purified l-crystallin accounts for only 0.19% of the total enzymatic activity measured in the crude brain extract. No other fractions obtained during the purification were identified as being enriched in ketimine reductase activity. This raises the possibility, acknowledged in the paper, that there may be other proteins with ketimine reductase activity in mammalian brain tissue. Alternatively, the enzyme may be unstable or subject to post-translational modifications that can alter its activity and yield. If the enzymatic function of l-crystallin is indeed associated with any or all of the diseases mentioned above, it will be important to understand the relationships linking stability and catalytic activity to binding of substrates and key ligands, including NADH, NADPH and T3. The report also raises many questions about the roles of l-crystallin as an enzyme and as a thyroid hormone binding protein. Do the known variants of l-crystallin have similar enzymatic activities? Does l-crystallin have a similar affinity for NADH and NADPH in vitro, but as a primarily cytoplasmic enzyme does it utilize NADPH preferentially in situ? What are its affinities for the different forms of thyroid hormone that are biologically active, and do all of them inhibit catalytic activity as effectively as T3? What are its preferences for substrates in different tissues, and does its catalytic activity in some way regulate signaling and gene expression by thyroid hormones? If there are indeed other forms of ketimine reductase in mammalian tissues, is l-crystallin’s major function enzymatic, or, as suggested by the point mutation that inhibits T3 binding and causes deafness (Oshima et al. 2006), is its more likely role, at least in some tissues, to bind thyroid hormone and thereby regulate its effects on transcription? Future studies that couple assays of enzymatic activity to appropriate cellular and molecular manipulations could address these questions. The dependence of the binding and catalytic activities of l-crystallin on metabolism are also likely to be significant. To take only one possibility, many of the diseases mentioned above are associated with hypoxia, which typically causes acidosis. Hallen et al. (2011) show that l-crystallin’s