Myosin motor domains carrying mutations implicated in early or late onset hypertrophic cardiomyopathy have similar properties.

Hypertrophic cardiomyopathy (HCM) is a common genetic disorder characterized by left ventricular hypertrophy and cardiac hyper-contractility. Mutations in the ß-cardiac myosin heavy chain gene (ß-MyHC) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to this disease remain incompletely understood. Predicting the severity of any ß-MyHC mutation is hindered by a lack of detailed examinations at the molecular level. Moreover, because HCM can take ≥20 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations that result in early onset disease would have more severe changes in function than do later onset mutations. Here, we performed steady-state and transient kinetic analyses of myosins carrying one of seven missense mutations in the motor domain. Of these seven, four were previously identified in early onset cardiomyopathy screens. We used the parameters derived from these analyses to model the ATP-driven cross-bridge cycle. Contrary to our hypothesis, the results indicated no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted occupancy of the force-holding actin·myosin·ADP complex at [Actin] = 3 Kapp along with the closely related duty ratio (the fraction of myosin in strongly attached force-holding states), and the measured ATPases all changed in parallel (in both sign and degree of change) compared with wildtype (WT) values. Six of the seven HCM mutations were clearly distinct from a set of previously characterized DCM mutations.

Muscle structure influences utrophin expression in mdx mice.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by mutations in the dystrophin gene. To examine the influence of muscle structure on the pathogenesis of DMD we generated mdx4cv:desmin double knockout (dko) mice. The dko male mice died of apparent cardiorespiratory failure at a median age of 76 days compared to 609 days for the desmin-/- mice. An ∼ 2.5 fold increase in utrophin expression in the dko skeletal muscles prevented necrosis in ∼ 91% of 1a, 2a and 2d/x fiber-types. In contrast, utrophin expression was reduced in the extrasynaptic sarcolemma of the dko fast 2b fibers leading to increased membrane fragility and dystrophic pathology. Despite lacking extrasynaptic utrophin, the dko fast 2b fibers were less dystrophic than the mdx4cv fast 2b fibers suggesting utrophin-independent mechanisms were also contributing to the reduced dystrophic pathology. We found no overt change in the regenerative capacity of muscle stem cells when comparing the wild-type, desmin-/-, mdx4cv and dko gastrocnemius muscles injured with notexin. Utrophin could form costameric striations with α-sarcomeric actin in the dko to maintain the integrity of the membrane, but the lack of restoration of the NODS (nNOS, α-dystrobrevin 1 and 2, α1-syntrophin) complex and desmin coincided with profound changes to the sarcomere alignment in the diaphragm, deposition of collagen between the myofibers, and impaired diaphragm function. We conclude that the dko mice may provide new insights into the structural mechanisms that influence endogenous utrophin expression that are pertinent for developing a therapy for DMD.

Two Classes of Myosin Inhibitors, Para-nitroblebbistatin and Mavacamten, Stabilize ß-Cardiac Myosin in Different Structural and Functional States.

In addition to a conventional relaxed state, a fraction of myosins in the cardiac muscle exists in a low-energy consuming super-relaxed (SRX) state, which is kept as a reserve pool that may be engaged under sustained increased cardiac demand. The conventional relaxed and the super-relaxed states are widely assumed to correspond to a structure where myosin heads are in an open configuration, free to interact with actin, and a closed configuration, inhibiting binding to actin, respectively. Disruption of the myosin SRX population is an emerging model in different heart diseases, such as hypertrophic cardiomyopathy, which results in excessive muscle contraction, and stabilizing them using myosin inhibitors is budding as an attractive therapeutic strategy. Here we examined the structure-function relationships of two myosin ATPase inhibitors, mavacamten and para-nitroblebbistatin, and found that binding of mavacamten at a site different than para-nitroblebbistatin populates myosin into the SRX state. Para-nitroblebbistatin, binding to a distal pocket to the myosin lever arm near the nucleotide-binding site, does not affect the usual myosin SRX state but instead appears to render myosin into a new, perhaps much more inhibited, 'ultra-relaxed' state. X-ray scattering-based rigid body modeling shows that both mavacamten and para-nitroblebbistatin induce novel conformations in human ß-cardiac heavy meromyosin that diverge significantly from the hypothetical open and closed states, and furthermore, mavacamten treatment causes greater compaction than para-nitroblebbistatin. Taken together, we conclude that mavacamten and para-nitroblebbistatin stabilize myosin in different structural states, and such states may give rise to different functional energy-sparing states.

Molecular and cellular adaptations to chronic myotendinous strain injury in mdx mice expressing a truncated dystrophin.

Myotendinous strain injury is the most common injury of human skeletal muscles because the majority of muscle forces are transmitted through this region. Although the immediate response to strain injury is well characterized, the chronic response to myotendinous strain injury is less clear. Here we examined the molecular and cellular adaptations to chronic myotendinous strain injury in mdx mice expressing a microdystrophin transgene (microdystrophin(DeltaR4-R23)). We found that muscles with myotendinous strain injury had an increased expression of utrophin and alpha7-integrin together with the dramatic restructuring of peripheral myofibrils into concentric rings. The sarcolemma of the microdystrophin(DeltaR4-R23)/mdx gastrocnemius muscles was highly protected from experimental lengthening contractions, better than wild-type muscles. We also found a positive correlation between myotendinous strain injury and ringed fibers in the HSA(LR) (human skeletal actin, long repeat) mouse model of myotonic dystrophy. We suggest that changes in protein expression and the formation of rings are adaptations to myotendinous strain injury that help to prevent muscle necrosis and retain the function of necessary muscles during injury, ageing and disease.

Sequencing protocols to genotype mdx, mdx(4cv), and mdx(5cv) mice.

Currently available polymerase chain reaction (PCR) genotyping methods for point mutations in the mouse dystrophin gene can lead to false positives and result in wasted time and money due to breeding or treating the wrong mice. Here we describe a simple and accurate method for sequencing the point mutations in mdx, mdx(4cv), and mdx(5cv) mice. This method clearly distinguishes between wildtype, heterozygous, and mutant transcripts, and thereby time and money can be saved by avoiding false positives.

Brain alpha-dystroglycan displays unique glycoepitopes and preferential binding to laminin-10/11.

alpha-Dystroglycan was quantitatively enriched from mammalian brain based on its uniform reactivity with Vicia villosa agglutinin and resolved into sub-populations possessing or lacking the sulfated glucuronic acid epitope recognized by monoclonal antibody HNK-1. We generated a new monoclonal antibody specific for a glycoepitope on brain alpha-dystroglycan but absent from alpha-dystroglycan expressed in all other tissues examined. Finally, we found that laminin-10/11 preferentially bound to brain alpha-dystroglycan compared to skeletal muscle alpha-dystroglycan. Our results suggest that tissue-specific glycosylation modifies the laminin binding specificity of alpha-dystroglycan.

Enhanced laminin binding by alpha-dystroglycan after enzymatic deglycosylation.

Carbohydrate modifications are clearly important to the function of alpha-dystroglycan but their composition and structure remain poorly understood. In the present study, we describe experiments aimed at identifying the alpha-dystroglycan oligosaccharides important for its binding to laminin-1 and carbohydrate-dependent mAbs (monoclonal antibodies) IIH6 and VIA4(1). We digested highly purified skeletal muscle alpha-dystroglycan with an array of linkage-specific endo- and exoglycosidases, which were verified for action on alpha-dystroglycan by loss/gain of reactivity for lectins with defined glyco-epitopes. Notably, digestion with a combination of Arthrobacter ureafaciens sialidase, beta(1-4)galactosidase and beta-N-acetylglucosaminidase substantially degraded SiaAalpha2-3Galbeta1-4GlcNAcbeta1-2Man glycans on highly purified alpha-dystroglycan that nonetheless exhibited enhanced IIH6, VIA4(1) and laminin-1 binding activity. Additional results indicate that alpha-dystroglycan is probably modified with other anionic sugars besides sialic acid and suggest that rare alpha-linked GlcNAc moieties may block its complete deglycosylation with currently available enzymes.

Crystal structure and cell surface anchorage sites of laminin alpha1LG4-5.

The laminin G-like (LG) domains of laminin-111, a glycoprotein widely expressed during embryogenesis, provide cell anchoring and receptor binding sites that are involved in basement membrane assembly and cell signaling. We now report the crystal structure of the laminin alpha1LG4-5 domains and provide a mutational analysis of heparin, alpha-dystroglycan, and galactosylsulfatide binding. The two domains of alpha1LG4-5 are arranged in a V-shaped fashion similar to that observed with laminin alpha2 LG4-5 but with a substantially different interdomain angle. Recombinant alpha1LG4-5 binding to heparin, alpha-dystroglycan, and sulfatides was dependent upon both shared and unique contributions from basic residues distributed in several clusters on the surface of LG4. For heparin, the greatest contribution was detected from two clusters, 2719RKR and 2791KRK. Binding to alpha-dystroglycan was particularly dependent on basic residues within 2719RKR, 2831RAR, and 2858KDR. Binding to galactosylsulfatide was most affected by mutations in 2831RAR and 2766KGRTK but not in 2719RKR. The combined analysis of structure and activities reveal differences in LG domain interactions that should enable dissection of biological roles of different laminin ligands.

Core 1 glycans on alpha-dystroglycan mediate laminin-induced acetylcholine receptor clustering but not laminin binding.

Although unique O-linked oligosaccharides on alpha-dystroglycan are important for binding to a variety of extracellular ligands, the function(s) of more generic carbohydrate structures on alpha-dystroglycan remain unclear. Recent studies suggest a role for glycoconjugates bearing the core 1 disaccharide Galbeta(1-3)GalNAc in acetylcholine receptor (AChR) clustering on the surface of muscle cells. Here, we report experiments demonstrating that the core 1-specific lectin jacalin almost completely abrogated laminin-induced AChR clustering in C2C12 myotubes and that alpha-dystroglycan was the predominant jacalin-binding protein detected in C2C12 myotube lysates. Although jacalin likely inhibited laminin-induced AChR clustering by directly binding to alpha-dystroglycan, jacalin had no effect on laminin binding to the myotube surface or to alpha-dystroglycan. Like jacalin, peanut agglutinin lectin also binds the core 1 disaccharide but not when it is terminally sialylated as expressed on alpha-dystroglycan. We show that C2C12 alpha-dystroglycan bound to peanut agglutinin only after digestion with neuraminidase. Simultaneous treatment of myotubes with neuraminidase and endo-O-glycosidase diminished alpha-dystroglycan binding to peanut agglutinin and inhibited neuraminidase-induced AChR clustering. We conclude that sialylated core 1 oligosaccharides of alpha-dystroglycan are important for laminin-induced AChR clustering and that their function in this process is distinct from the established role of alpha-dystroglycan oligosaccharides in laminin binding.

Molecular dissection of the alpha-dystroglycan- and integrin-binding sites within the globular domain of human laminin-10.

The adhesive interactions of cells with laminins are mediated by integrins and non-integrin-type receptors such as alpha-dystroglycan and syndecans. Laminins bind to these receptors at the C-terminal globular domain of their alpha chains, but the regions recognized by these receptors have not been mapped precisely. In this study, we sought to locate the binding sites of laminin-10 (alpha5beta1gamma1) for alpha(3)beta(1) and alpha(6)beta(1) integrins and alpha-dystroglycan through the production of a series of recombinant laminin-10 proteins with deletions of the LG (laminin G-like) modules within the globular domain. We found that deletion of the LG4-5 modules did not compromise the binding of laminin-10 to alpha(3)beta(1) and alpha(6)beta(1) integrins but completely abrogated its binding to alpha-dystroglycan. Further deletion up to the LG3 module resulted in loss of its binding to the integrins, underlining the importance of LG3 for integrin binding by laminin-10. When expressed individually as fusion proteins with glutathione S-transferase or the N-terminal 70-kDa region of fibronectin, only LG4 was capable of binding to alpha-dystroglycan, whereas neither LG3 nor any of the other LG modules retained the ability to bind to the integrins. Site-directed mutagenesis of the LG3 and LG4 modules indicated that Asp-3198 in the LG3 module is involved in the integrin binding by laminin-10, whereas multiple basic amino acid residues in the putative loop regions are involved synergistically in the alpha-dystroglycan binding by the LG4 module.