Joaquin M. Muriel

Use of Autologous Platelet-rich Plasma to Treat Muscle Strain Injuries

Background Standard nonoperative therapy for acute muscle strains usually involves short-term rest, ice, and nonsteroidal anti-inflammatory medications, but there is no clear consensus on how to accelerate recovery. Hypothesis Local delivery of platelet-rich plasma to injured muscles hastens recovery of function. Study Design Controlled laboratory study. Methods In vivo, the tibialis anterior muscles of anesthetized Sprague-Dawley rats were injured by a single (large strain) lengthening contraction or multiple (small strain) lengthening contractions, both of which resulted in a significant injury. The tibialis anterior either was injected with platelet-rich plasma, was injected with platelet-poor plasma as a sham treatment, or received no treatment. Results Both injury protocols yielded a similar loss of force. The platelet-rich plasma only had a beneficial effect at 1 time point after the single contraction injury protocol. However, platelet-rich plasma had a beneficial effect at 2 time points after the multiple contraction injury protocol and resulted in a faster recovery time to full contractile function. The sham injections had no effect compared with no treatment. Conclusion Local delivery of platelet-rich plasma can shorten recovery time after a muscle strain injury in a small-animal model. Recovery of muscle from the high-repetition protocol has already been shown to require myogenesis, whereas recovery from a single strain does not. This difference in mechanism of recovery may explain why platelet-rich plasma was more effective in the high-repetition protocol, because platelet-rich plasma is rich in growth factors that can stimulate myogenesis. Clinical Relevance Because autologous blood products are safe, platelet-rich plasma may be a useful product in clinical treatment of muscle injuries.

Dysferlin stabilizes stress-induced Ca2+ signaling in the transverse tubule membrane

Significance Muscular dystrophies linked to the genetic absence or mutations of dysferlin are currently without a relevant therapy. Dysferlin is thought to mediate membrane repair in skeletal muscle, but its localization and specific functions remain controversial. Here we show that dysferlin is enriched in the transverse tubule membrane of skeletal muscle and demonstrate that, in its absence, mechanical stress leads to calcium-dependent muscle injury. Furthermore, we demonstrate that treatment of dysferlin-deficient muscle with the calcium channel blocker diltiazem reduces in vitro experimental and in vivo contraction-induced muscle damage. As diltiazem is approved for clinical use, our results suggest a potential new therapeutic avenue for patients diagnosed with dysferlinopathies. Dysferlinopathies, most commonly limb girdle muscular dystrophy 2B and Miyoshi myopathy, are degenerative myopathies caused by mutations in the DYSF gene encoding the protein dysferlin. Studies of dysferlin have focused on its role in the repair of the sarcolemma of skeletal muscle, but dysferlin’s association with calcium (Ca2+) signaling proteins in the transverse (t-) tubules suggests additional roles. Here, we reveal that dysferlin is enriched in the t-tubule membrane of mature skeletal muscle fibers. Following experimental membrane stress in vitro, dysferlin-deficient muscle fibers undergo extensive functional and structural disruption of the t-tubules that is ameliorated by reducing external [Ca2+] or blocking L-type Ca2+ channels with diltiazem. Furthermore, we demonstrate that diltiazem treatment of dysferlin-deficient mice significantly reduces eccentric contraction-induced t-tubule damage, inflammation, and necrosis, which resulted in a concomitant increase in postinjury functional recovery. Our discovery of dysferlin as a t-tubule protein that stabilizes stress-induced Ca2+ signaling offers a therapeutic avenue for limb girdle muscular dystrophy 2B and Miyoshi myopathy patients.

Fibulin-1C and Fibulin-1D splice variants have distinct functions and assemble in a hemicentin-dependent manner.

Fibulins are a family of extracellular glycoproteins associated with basement membranes and elastic fibers in vertebrates. Conservation of the fibulin-1 gene throughout metazoan evolution includes fibulin-1C and fibulin-1D alternate splice variants, although little is known about variant specific functions that would justify this striking structural conservation. We have therefore investigated the structure, localization and loss-of-function phenotype specific to both fibulin-1 variants in C. elegans. We find that fibulin-1C has specific roles during pharynx, intestine, gonad and muscle morphogenesis, being required to regulate cell shape and adhesion, whereas fibulin-1D assembles in flexible polymers that connect the pharynx and body-wall-muscle basement membranes. The assembly of fibulin-1C and fibulin-1D in multiple locations is dependent upon the presence of hemicentin, a recently described extracellular member of the immunoglobulin superfamily. We suggest that the distinct developmental roles and hemicentin-dependent assembly for fibulin-1 splice variants demonstrated here may be relevant to fibulin-1 and possibly other fibulin family members in non-nematode species.

Physiology, structure, and susceptibility to injury of skeletal muscle in mice lacking keratin 19-based and desmin-based intermediate filaments

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.

Hemicentins: What have we learned from worms?

Hemicentins are conserved extracellular matrix proteins discovered in Caenorhabditis elegans, with orthologs in all vertebrate species including human and mouse. Hemicentins share a single, highly conserved amino-terminal von Willebrand A domain, followed by a long (>40) stretch of immunoglobulin repeats, multiple tandem epidermal growth factors and a fibulin-like carboxy-terminal module. C. elegans has a single hemicentin gene that has pleiotropic functions in transient cell contacts that are required for cell migration and basement membrane invasion and in stable contacts at hemidesmosome-mediated cell junctions and elastic fiber-like structures. Here, we summarize what is known about the function of hemicentin in C. elegans and discuss implications for hemicentin function in other species.

Myopathic changes in murine skeletal muscle lacking synemin.

Diseases of striated muscle linked to intermediate filament (IF) proteins are associated with defects in the organization of the contractile apparatus and its links to costameres, which connect the sarcomeres to the cell membrane. Here we study the role in skeletal muscle of synemin, a type IV IF protein, by examining mice null for synemin (synm-null). Synm-null mice have a mild skeletal muscle phenotype. Tibialis anterior (TA) muscles show a significant decrease in mean fiber diameter, a decrease in twitch and tetanic force, and an increase in susceptibility to injury caused by lengthening contractions. Organization of proteins associated with the contractile apparatus and costameres is not significantly altered in the synm-null. Elastimetry of the sarcolemma and associated contractile apparatus in extensor digitorum longus myofibers reveals a reduction in tension consistent with an increase in sarcolemmal deformability. Although fatigue after repeated isometric contractions is more marked in TA muscles of synm-null mice, the ability of the mice to run uphill on a treadmill is similar to controls. Our results suggest that synemin contributes to linkage between costameres and the contractile apparatus and that the absence of synemin results in decreased fiber size and increased sarcolemmal deformability and susceptibility to injury. Thus synemin plays a moderate but distinct role in fast twitch skeletal muscle.

Hemicentin Assembly in the Extracellular Matrix Is Mediated by Distinct Structural Modules

Hemicentins are conserved extracellular matrix proteins characterized by a single von Willebrand A (VWA) domain at the amino terminus, a long stretch (>40) of tandem immunoglobulin domains, multiple tandem epidermal growth factors (EGFs), and a single fibulin-like carboxyl-terminal module. In Caenorhabditis elegans, hemicentin is secreted from muscle and gonadal leader cells and assembles at multiple locations into discrete tracks that constrict broad regions of cell contact into adhesive and flexible line-shaped junctions. To determine hemicentin domains critical for function and assembly, we have expressed fragments of hemicentin as GFP tagged fusion proteins in C. elegans. We find that a hemicentin fragment containing the VWA domain can target to multiple assembly sites when expressed under the control of either endogenous hemicentin regulatory sequences or the muscle-specific unc-54 promoter. A hemicentin fragment containing the EGF and fibulin-like carboxyl-terminal modules can co-assemble with existing hemicentin polymers in wild-type animals but has no detectable function in the absence of endogenous hemicentin. The data suggest that the VWA domain is a cell binding domain whose function is to target hemicentin to sites of assembly and the EGF/fibulin-like carboxyl-terminal modules constitute an assembly domain that mediates direct interactions between hemicentin monomers during the hemicentin assembly process.

Production of biosurfactants by Cladosporium resinae

SummaryCladosporium resinae produces extracellular biosurfactants when growing in a hydrocarbon source such as the jet fuel JP8. This production of biosurfactants was observed by the reduction of the surface tension of the aqueous phase of growing medium, and by the increase in emulsion and foaming properties. A partial purification by collapsed foam gave better physical properties by decreasing surface tension and increasing foaming power and stabilization of emulsions. Surface active substances were purified by reversed phase chromatography. Six compounds representing over 75% of fraction containing surface activity were present. This fraction gave an improvement of all surface properties.

Selective assembly of fibulin‐1 splice variants reveals distinct extracellular matrix networks and novel functions for perlecan/UNC‐52 splice variants

Fibulin‐1C and fibulin‐1D splice variants have been conserved throughout metazoan evolution and have distinct functions in Caenorhabditis elegans development. Both splice variants are required for the assembly of hemidesmosome‐mediated mechanosensory neuron and uterine attachments, although the molecular associations that underlie their distinct functions at these locations are not known. Here, we show that the assembly of fibulin‐1C and fibulin‐1D splice variants at these anchorages is dependent upon distinct components of the extracellular matrix (ECM): Fibulin‐1D assembly at uterine and mechanosensory neurons attachments is dependent upon a perlecan/ UNC‐52 splice variant that includes alternately spliced IG8‐IG10, whereas the assembly of fibulin‐1C at mechanosensory neuron attachments is dependent upon laminin/ EPI‐1. These data not only indicate that fibulin‐1C and fibulin‐1D are components of distinct networks of ECM but also demonstrates a novel function for a major class of perlecan splice variants found in C. elegans and mouse. In addition, we demonstrate that overexpression of another ECM protein, collagen XVIII, can suppress gonad morphogenesis defects associated with loss of fibulin‐1C, suggesting that some genetic defects that result in a weakened basement membrane can be compensated by overexpression of genes for ECM components that stabilize basement membranes. Developmental Dynamics 235:2632–2640, 2006.

M142.2 (cut-6), a novel Caenorhabditis elegans matrix gene important for dauer body shape

The cuticle of the nematode Caenorhabditis elegans is a collagenous extracellular matrix which forms the exoskeleton and defines the shape of the worm. We have characterized the C. elegans gene M142.2, and we show that this is a developmentally regulated gene important for cuticle structure. Transgenic worms expressing M142.2 promoter fused to green fluorescent protein showed that M142.2 is expressed in late embryos and L2d predauers, in the hypodermal cells which synthesize the cuticle. The same temporal pattern was seen by RT-PCR using RNA purified from specific developmental stages. A recombinant fragment of M142.2 was expressed in Escherichia coli and used to raise an antiserum. Immunohistochemistry using the antiserum localized M142.2 to the periphery of the alae of L1 and dauers, forming two longitudinal ribbons over the hypodermal cells. Loss-of-function of M142.2 by RNAi resulted in a novel phenotype: dumpy dauers which lacked alae. M142.2 therefore plays a major role in the assembly of the alae and the morphology of the dauer cuticle; because of its similarity to the other cut genes of the cuticle, we have named the gene cut-6.