do-s-don-ts-preparation-muscle-cryosections-for-histological

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

Boston University

In anurans, circulating levels of androgens influence certain secondary sexual characteristics that are expressed only during the breeding season. We studied the contractile properties of external oblique muscles (used to power sound production) in a species of North American gray tree frog, Hyla chrysoscelis, during the breeding season and also in testosterone-treated captive males and females after the breeding season. Compared with the muscles of breeding-season males, the trunk muscles of postbreeding-season males have 50% less mass, 60% longer twitches, and 40% slower shortening velocities. Testosterone levels similar to those found in breeding-season male hylid frogs restore the contractile speed and mass of male trunk muscles and also convert the small slow trunk muscles of females into larger fast-contracting muscles. We conclude that androgens likely play a key role in altering the contractile properties of these muscles in males during the annual cycle, allowing them to operate in the breeding season at the frequencies required to produce the characteristic rapidly pulsed calls of this species. Females as well as nonbreeding-season males do not produce advertising calls, and therefore the slower muscles found in these animals may allow more economic operation of these muscles. The effects of testosterone on female trunk muscles indicate the potential of this hormone in contributing to the sexual dimorphism in size and contractile properties of these muscles, but this dimorphism is likely due to the interaction of more than one hormone.

Season and testosterone affect contractile properties of fast calling muscles in the gray tree frog Hyla chrysoscelis.

Upon serial passaging of mouse skeletal muscle cells, a small number of cells will spontaneously develop the ability to proliferate indefinitely while retaining the ability to differentiate into multinucleate myotubes. Possible gene changes that could underlie myogenic cell immortalization and their possible effects on myogenesis had not been examined.

Immortalization of mouse myogenic cells can occur without loss of p16INK4a, p19ARF, or p53 and is accelerated by inactivation of Bax.

The most common form of human congenital muscular dystrophy (CMD) is caused by mutations in the laminin-alpha2 gene. Loss of laminin-alpha2 function in this autosomal recessive type 1A form of CMD results in neuromuscular dysfunction and, often, early death. Laminin-alpha2-deficient skeletal muscles in both humans and mice show signs of muscle cell death by apoptosis. To examine the significance of apoptosis in CMD1A pathogenesis, we determined whether pathogenesis in laminin-alpha2-deficient (Lama2(-/-)) mice could be ameliorated by inhibiting apoptosis through either (a) inactivation of the proapoptosis protein Bax or (b) overexpression of the antiapoptosis protein Bcl-2 from a muscle-specific transgene. We found that both of these genetic interventions produced a several-fold increase in the lifespan of Lama2(-/-) mice. Bax inactivation also improved postnatal growth rate and myofiber histology and decreased fixed contractures of Lama2(-/-) mice. Thus, Bcl-2 family-mediated apoptosis contributes significantly to pathogenesis in the mouse model of CMD1A, and antiapoptosis therapy may be a possible route to amelioration of neuromuscular dysfunction due to laminin-alpha2 deficiency in humans.

Inhibition of apoptosis improves outcome in a model of congenital muscular dystrophy.

Multiple types of mononucleate cells reside among the multinucleate myofibers in skeletal muscles and these mononucleate cells function in muscle maintenance and repair. How neuromuscular disease might affect different types of muscle mononucleate cells had not been determined. In this study, therefore, we examined how two neuromuscular diseases, dystrophin-deficiency and laminin-alpha2-deficiency, altered the proliferation and composition of different subsets of muscle-derived mononucleate cells.

Diseased muscles that lack dystrophin or laminin-alpha2 have altered compositions and proliferation of mononuclear cell populations.

Although genetic mutations that are responsible for most of the inherited neuromuscular diseases have been identified, the molecular and cellular mechanisms that cause muscle and nerve depletion are not well understood and therapies are lacking. Histological studies of many neuromuscular diseases indicated that loss of motor-nerve and/or skeletal-muscle function might be due to excessive cell death by apoptosis. Recent studies have confirmed this possibility by showing that pathology in mouse models of amyotrophic lateral sclerosis, congenital muscular dystrophy, oculopharyngeal muscular dystrophy and collagen-VI deficiency, but not Duchenne muscular dystrophy, is significantly ameliorated by genetic or pharmacological interventions that have been designed to inhibit apoptosis. Thus, apoptosis greatly contributes to pathology in mouse models of several neuromuscular diseases, and appropriate anti-apoptosis therapy might therefore be beneficial for the corresponding human diseases.

The role of apoptosis in neuromuscular diseases and prospects for anti-apoptosis therapy.

Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-kappaB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration.

Congenital muscular dystrophy type 1A is an autosomal recessive disease that is caused by loss-of-function mutations in the laminin-alpha2 gene, and results in motor nerve and skeletal muscle dysfunction. In a previous study, we used genetic modifications to show that inappropriate induction of apoptosis was a significant contributor to pathogenesis in a laminin-alpha2-deficient mouse model of congenital muscular dystrophy type 1A. To identify a possible pharmacological therapy for laminin-alpha2 deficiency, we designed this study to determine whether treatment with minocycline or doxycycline, which are tetracycline derivatives reported to have antiapoptotic effects in mammals, would significantly increase lifespan and improve neuromuscular function in laminin-alpha2-deficient mice.

Pathology is alleviated by doxycycline in a laminin-alpha2-null model of congenital muscular dystrophy.

Multiple types of fast and slow skeletal muscle fibers form during early embryogenesis in vertebrates. In zebrafish, formation of the earliest slow myofibers in fin muscles requires expression of the zinc-finger transcriptional repressor Prdm1 (also known as Blimp1). To further understand how the role of Prdm1 in early myogenesis may vary through evolution and during development, we have now analyzed Prdm1 expression in the diverse types of myotubes that form in culture from somitic, embryonic, and fetal chicken myoblasts.

Prdm1 (Blimp-1) and the expression of fast and slow myosin heavy chain isoforms during avian myogenesis in vitro.

MDC1A, the second most prevalent form of congenital muscular dystrophy, results from laminin-α2 chain deficiency. This disease is characterized by extensive muscle wasting that results in extremely weak skeletal muscles. A large percentage of children with MDC1A are faced with respiratory as well as ambulatory difficulties. We investigated the effects of overexpressing insulin-like growth factor-1 (IGF-1) as a potential therapeutic target for the disease in the Lama2(Dy-w) mouse, a model that closely resembles human MDC1A. IGF-1 transgenic Lama2(Dy-w) mice showed increased survivability, body weight and muscle weight. In addition, these mice showed better ability to stand up on their hind limbs: a typical exploratory behavior seen in healthy mice. Histology and immunohistochemistry analyses revealed increased regenerative capacity and proliferation in IGF-1 transgenic Lama2(Dy-w) muscles. Western blot analysis showed increased phosphorylation of Akt and ERK1/2, both known to enhance myogenesis. Additionally, we saw increases in the expression of the regeneration markers MyoD, myogenin and embryonic myosin (myosin heavy chain 3, MYH3). We conclude that overexpression of IGF-1 in Lama2(Dy-w) mice increases lifespan and improves their overall wellbeing mainly through the restoration of impaired muscle regeneration, as fibrosis or inflammation was not impacted by IGF-1 in this disease model. Our results demonstrate that IGF-1 has a promising therapeutic potential in the treatment of MDC1A.

Muscle-specific expression of insulin-like growth factor 1 improves outcome in Lama2Dy-w mice, a model for congenital muscular dystrophy type 1A.

The Myomatrix 2012 conference held April 22-24th, 2012 at the University of Nevada, Reno convened 73 international participants to discuss the dynamic relationship between muscle and its matrix in muscular dystrophy with a specific focus on congenital muscular dystrophy. Seven sessions over 2½ days defined three central themes: (1) the role of extracellular matrix proteins and compartments in development and specifically in congenital muscular dystrophy (CMD) (2) the role of extracellular matrix signaling and adhesion to membrane receptors and (3) the balance and interplay between inflammation and fibrosis as drivers of altered matrix stiffness, impaired regeneration and progressive dystrophy. This report highlights major conference findings and the translational roadmap as defined by conference attendees.

Report on the Myomatrix Conference April 22-24, 2012, University of Nevada, Reno, Nevada, USA.

Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is an autosomal recessive disorder caused by mutations in the laminin-α2 gene (OMIM: 607855). Currently, no treatment other than palliative care exists for this disease. In our previous work, genetic interventions in the Lama2(Dy-w) mouse model for MDC1A demonstrated that limited regeneration and uncontrolled apoptosis are important drivers of this disease. However, targeting one of these disease drivers without addressing the other results in only partial rescue of the phenotype. The present study was designed to determine whether utilizing a combinatorial treatment approach can lead to a more profound amelioration of the disease pathology. To accomplish this task, we generated Bax-null Lama2(Dy-w)mice that overexpressed muscle-specific IGF-1 (Lama2(Dy-w)Bax(-/-)+IGF-1tg). Further to test the translational potential of IGF-1 administration in combination with Bax inhibition, we treated Lama2(Dy-w)Bax(-/-) mice postnatally with systemic recombinant human IGF-1 (IPLEX™). These two combinatorial treatments lead to similar, promising outcomes. In addition to increased body and muscle weights, both transgenic overexpression and systemic administration of IGF-1 combined with Bax-inhibition resulted in improved muscle phenotype and locomotory function that were nearly indistinguishable from wild-type mice. These results provide a fundamental proof of concept that justifies the use of a combination therapy as an effective treatment for MDC1A and highlights a compelling argument toward shifting the paradigm in treating multifaceted neuromuscular diseases.

Triggering regeneration and tackling apoptosis: a combinatorial approach to treating congenital muscular dystrophy type 1 A.

MDC1A is a congenital neuromuscular disorder with developmentally complex and progressive pathologies that results from a deficiency in the protein laminin α2. MDC1A is associated with a multitude of pathologies, including increased apoptosis, inflammation and fibrosis. In order to assess and treat a complicated disease such as MDC1A, we must understand the natural history of the disease so that we can identify early disease drivers and pinpoint critical time periods for implementing potential therapies.

Article	Year
Season and testosterone affect contractile properties of fast calling muscles in the gray tree frog Hyla chrysoscelis. American journal of physiology. Regulatory, integrative and comparative physiology\| PubMed ID: 12595277	2003
Immortalization of mouse myogenic cells can occur without loss of p16INK4a, p19ARF, or p53 and is accelerated by inactivation of Bax. BMC cell biology\| PubMed ID: 14711384	2004
Inhibition of apoptosis improves outcome in a model of congenital muscular dystrophy. The Journal of clinical investigation\| PubMed ID: 15578095	2004
Diseased muscles that lack dystrophin or laminin-alpha2 have altered compositions and proliferation of mononuclear cell populations. BMC neurology\| PubMed ID: 15817132	2005
The role of apoptosis in neuromuscular diseases and prospects for anti-apoptosis therapy. Trends in molecular medicine\| PubMed ID: 16650805	2006
TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration. The EMBO journal\| PubMed ID: 17124496	2006
Pathology is alleviated by doxycycline in a laminin-alpha2-null model of congenital muscular dystrophy. Annals of neurology\| PubMed ID: 19086074	2009
Prdm1 (Blimp-1) and the expression of fast and slow myosin heavy chain isoforms during avian myogenesis in vitro. PloS one\| PubMed ID: 20376350	2010
Muscle-specific expression of insulin-like growth factor 1 improves outcome in Lama2Dy-w mice, a model for congenital muscular dystrophy type 1A. Human molecular genetics\| PubMed ID: 21441569	2011
Report on the Myomatrix Conference April 22-24, 2012, University of Nevada, Reno, Nevada, USA. Neuromuscular disorders : NMD\| PubMed ID: 22800409	2013
Triggering regeneration and tackling apoptosis: a combinatorial approach to treating congenital muscular dystrophy type 1 A. Human molecular genetics\| PubMed ID: 23773998	2013
Dysregulation of matricellular proteins is an early signature of pathology in laminin-deficient muscular dystrophy. Skeletal muscle\| PubMed ID: 25075272	2014

Mahasweta Girgenrath

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