Prdm1 (Blimp-1) and the Expression of Fast and Slow Myosin Heavy Chain Isoforms during Avian Myogenesis In Vitro

BACKGROUND. 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. PRINCIPAL FINDINGS. In cultures of somitic, embryonic limb, and fetal limb chicken cells, we found that Prdm1 was expressed in all of the differentiated muscle cells that formed, including those that expressed only fast myosin heavy chain isoforms, as well as those that co-expressed both fast and slow myosin heavy chain isoforms. Prdm1 was also expressed in Pax7-positive myoblasts, as well as in non-myogenic cells in the cultures. Furthermore, though all differentiated cells in control somite cultures co-expressed fast and slow myosin heavy chains, antisense knockdown of Prdm1 expression inhibited the formation of these co-expressing cells in somite cultures. CONCLUSIONS. In chicken myogenic cell cultures, Prdm1 was expressed in most Pax7-positive myoblasts and in all differentiated muscle cells, irrespective of the developmental stage of cell donor or the pattern of fast and slow myosin heavy chains expressed in the differentiated cells that were formed. Thus, Prdm1 was expressed in myogenic cells prior to terminal differentiation; and, after differentiation, Prdm1 expression was not limited to cells that expressed slow myosin heavy chain isoforms. In addition, Prdm1 appeared to be required for differentiation of the somitic myocytes, which are the earliest myocytes to form in the avian embryo.National Research Initiative of the United States Department of Agriculture Cooperative State Research, Education, and Extension Service (#2006-35206-16622); National Heart, Lung, and Blood Institute (2R01HL064641

Immortalization of mouse myogenic cells can occur without loss of p16(INK4a), p19(ARF), or p53 and is accelerated by inactivation of Bax

BACKGROUND: 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. RESULTS: We found that immortalization occurred earlier and more frequently when the myogenic cells lacked the pro-apoptotic protein Bax. Furthermore, myogenesis was altered by Bax inactivation as Bax-null cells produced muscle colonies with more nuclei than wild-type cells, though a lower percentage of the Bax-null nuclei were incorporated into multinucleate myotubes. In vivo, both the fast and slow myofibers in Bax-null muscles had smaller cross-sectional areas than those in wild-type muscles. After immortalization, both Bax-null and Bax-positive myogenic cells expressed desmin, retained the capacity to form multinucleate myotubes, expressed p19(ARF )protein, and retained p53 functions. Expression of p16(INK4a), however, was found in only about half of the immortalized myogenic cell lines. CONCLUSIONS: Mouse myogenic cells can undergo spontaneous immortalization via a mechanism that can include, but does not require, loss of p16(INK4a), and also does not require inactivation of p19(ARF )or p53. Furthermore, loss of Bax, which appears to be a downstream effector of p53, accelerates immortalization of myogenic cells and alters myogenesis

The Endosomal Escape Vehicle Platform Enhances Delivery of Oligonucleotides in Preclinical Models of Neuromuscular Disorders

Biological therapeutic agents are highly targeted and potent but limited in their ability to reach intracellular targets. These limitations often necessitate high therapeutic doses and can be associated with less-than-optimal therapeutic activity. One promising solution for therapeutic agent delivery is use of cell-penetrating peptides. Canonical cell-penetrating peptides, however, are limited by low efficiencies of cellular uptake and endosomal escape, minimal proteolytic stability, and toxicity. To overcome these limitations, we designed a family of proprietary cyclic cell-penetrating peptides that form the core of our endosomal escape vehicle technology capable of delivering therapeutic agent-conjugated cargo intracellularly. We demonstrated the therapeutic potential of this endosomal escape vehicle platform in preclinical models of muscular dystrophy with distinct disease etiology. An endosomal escape vehicle-conjugated, splice-modulating oligonucleotide restored dystrophin protein expression in striated muscles in the mdx mouse, a model for Duchenne muscular dystrophy. Furthermore, another endosomal escape vehicle-conjugated, sterically blocking oligonucleotide led to knockdown of aberrant transcript expression levels in facioscapulohumeral muscular dystrophy patient-derived skeletal muscle cells. These findings suggest a significant therapeutic potential of our endosomal escape vehicle conjugated oligonucleotides for targeted upregulation and downregulation of gene expression in neuromuscular diseases, with possible broader application of this platform for delivery of intracellular biological agents

Improving Reproducibility of Phenotypic Assessments in the DyW Mouse Model of Laminin-α2 Related Congenital Muscular Dystrophy

Laminin-α2 related Congenital Muscular Dystrophy (LAMA2-CMD) is a progressive muscle disease caused by partial or complete deficiency of laminin-211, a skeletal muscle extracellular matrix protein. In the last decade, basic science research has queried underlying disease mechanisms in existing LAMA2-CMD murine models and identified possible clinical targets and pharmacological interventions. Experimental rigor in preclinical studies is critical to efficiently and accurately quantify both negative and positive results, degree of efficiency of potential therapeutics and determine whether to move a compound forward for additional preclinical testing. In this review, we compare published available data measured to assess three common parameters in the widely used mouse model DyW, that mimics LAMA2-CMD, we quantify variability and analyse its possible sources. Finally, on the basis of this analysis, we suggest standard set of assessments and the use of available standardized protocols, to reduce variability of outcomes in the future and to improve the value of preclinical research

Of Mice and Measures : A Project to Improve How We Advance Duchenne Muscular Dystrophy Therapies to the Clinic.

A new line of dystrophic mdx mice on the DBA/2J (D2) background has emerged as a candidate to study the efficacy of therapeutic approaches for Duchenne muscular dystrophy (DMD). These mice harbor genetic polymorphisms that appear to increase the severity of the dystropathology, with disease modifiers that also occur in DMD patients, making them attractive for efficacy studies and drug development. This workshop aimed at collecting and consolidating available data on the pathological features and the natural history of these new D2/mdx mice, for comparison with classic mdx mice and controls, and to identify gaps in information and their potential value. The overall aim is to establish guidance on how to best use the D2/mdx mouse model in preclinical studies

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

The most common form of human congenital muscular dystrophy (CMD) is caused by mutations in the laminin-α2 gene. Loss of laminin-α2 function in this autosomal recessive type 1A form of CMD results in neuromuscular dysfunction and, often, early death. Laminin-α2–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-α2–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-α2 deficiency in humans