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

Immortalized myogenic cells from congenital muscular dystrophy type1A patients recapitulate aberrant caspase activation in pathogenesis: a new tool for MDC1A research

BACKGROUND: Congenital muscular dystrophy Type 1A (MDC1A) is a severe, recessive disease of childhood onset that is caused by mutations in the LAMA2 gene encoding laminin-alpha2. Studies with both mouse models and primary cultures of human MDC1A myogenic cells suggest that aberrant activation of cell death is a significant contributor to pathogenesis in laminin-alpha2-deficiency. METHODS: To overcome the limited population doublings of primary cultures, we generated immortalized, clonal lines of human MDC1A myogenic cells via overexpression of both CDK4 and the telomerase catalytic component (human telomerase reverse transcriptase (hTERT)). RESULTS: The immortalized MDC1A myogenic cells proliferated indefinitely when cultured at low density in high serum growth medium, but retained the capacity to form multinucleate myotubes and express muscle-specific proteins when switched to low serum medium. When cultured in the absence of laminin, myotubes formed from immortalized MDC1A myoblasts, but not those formed from immortalized healthy or disease control human myoblasts, showed significantly increased activation of caspase-3. This pattern of aberrant caspase-3 activation in the immortalized cultures was similar to that found previously in primary MDC1A cultures and laminin-alpha2-deficient mice. CONCLUSIONS: Immortalized MDC1A myogenic cells provide a new resource for studies of pathogenetic mechanisms and for screening possible therapeutic approaches in laminin-alpha2-deficiency

Individual epigenetic status of the pathogenic D4Z4 macrosatellite correlates with disease in facioscapulohumeral muscular dystrophy

BACKGROUND: Both forms of facioscapulohumeral muscular dystrophy (FSHD) are associated with aberrant epigenetic regulation of the chromosome 4q35 D4Z4 macrosatellite. Chromatin changes due to large deletions of heterochromatin (FSHD1) or mutations in chromatin regulatory proteins (FSHD2) lead to relaxation of epigenetic repression and increased expression of the deleterious double homeobox 4 (DUX4) gene encoded within the distal D4Z4 repeat. However, many individuals with the genetic requirements for FSHD remain asymptomatic throughout their lives. Here we investigated family cohorts of FSHD1 individuals who were either affected (manifesting) or without any discernible weakness (nonmanifesting/asymptomatic) and their unaffected family members to determine if individual epigenetic status and stability of repression at the contracted 4q35 D4Z4 array in myocytes correlates with FSHD disease. RESULTS: Family cohorts were analyzed for DNA methylation on the distal pathogenic 4q35 D4Z4 repeat on permissive A-type subtelomeres. We found DNA hypomethylation in FSHD1-affected subjects, hypermethylation in healthy controls, and distinctly intermediate levels of methylation in nonmanifesting subjects. We next tested if these differences in DNA methylation had functional relevance by assaying DUX4-fl expression and the stability of epigenetic repression of DUX4-fl in myogenic cells. Treatment with drugs that alter epigenetic status revealed that healthy cells were refractory to treatment, maintaining stable repression of DUX4, while FSHD1-affected cells were highly responsive to treatment and thus epigenetically poised to express DUX4. Myocytes from nonmanifesting subjects had significantly higher levels of DNA methylation and were more resistant to DUX4 activation in response to epigenetic drug treatment than cells from FSHD1-affected first-degree relatives containing the same contraction, indicating that the epigenetic status of the contracted D4Z4 array is reflective of disease. CONCLUSIONS: The epigenetic status of the distal 4qA D4Z4 repeat correlates with FSHD disease; FSHD-affected subjects have hypomethylation, healthy unaffected subjects have hypermethylation, and nonmanifesting subjects have characteristically intermediate methylation. Thus, analysis of DNA methylation at the distal D4Z4 repeat could be used as a diagnostic indicator of developing clinical FSHD. In addition, the stability of epigenetic repression upstream of DUX4 expression is a key regulator of disease and a viable therapeutic target

Analysis of Myogenic and Candidate Disease Biomarkers in FSHD Muscle Cells

The UMMS Wellstone Program is a foundation and NIH-funded cooperative research center focusing on identifying biomarkers for facioscapulohumeral muscular dystrophy (FSHD) to gain insight into the molecular pathology of the disease and to develop potential therapies. FSHD is characterized by progressive wasting of skeletal muscles, with weakness often initiating in facial muscles and muscles supporting the scapula and upper arms. While the genetics associated with FSHD are complex, the major form of the disease, FSHD1, is linked to contraction of the D4Z4 repeat region located at chromosome 4q. Recently, a transcript encoded at the distal end of the repeat region, Dux4-fl, normally expressed in embryonic stem cells and germ cells, was also detected in differentiated muscle cells and biopsies from FSHD subjects, giving rise to the hypothesis that DUX4-FL function contributes to muscle weakness. We established a repository of high quality, well-characterized primary and immortalized muscle cell strains from FSHD and control subjects in affected families to provide biomaterials for cell and molecular studies to the FSHD research community. qPCR and immunostaining analyses demonstrate similar growth and differentiation characteristics in cells from FSHD and control subjects within families. We detected Dux4-fl transcript and protein in FSHD cells as recently described; interestingly, we also detected Dux4-fl in muscle cells from a subset of control individuals, suggesting that any Dux4-fl-mediated myopathy would require additional modifying elements. Microarray analysis of FSHD and control muscle cells demonstrated that several genes were upregulated in FSHD cells, including genes that were concurrently identified as downstream targets of Dux4-fl and as candidate FSHD disease genes. Future studies will further characterize the RNA and protein expression of candidate disease genes in cells from FSHD and control subjects, including nonmanifesting subjects with the D4Z4 lesion but no muscle weakness, and utilizing whole transcriptome sequencing (RNAseq) to identify additional candidates

Prdm1 and fast MyHC expression in cultures of E12 hindlimb cells.

<p>Prdm1 immunostaining (Ab from Cell Signaling Technology) was found in the nuclei of both mononucleate cells (<b>A, B</b>) and myotubes (<b>C, D</b>). An additional Prdm1 antibody (Abcam) produced similar nuclear staining of E12 mononucleate cells and myotubes (not shown). As found in previous studies, myotubes formed from E12 myoblasts showed immunostaining with mAb F59 (<b>E</b>), which reacts with all fast MyHC isoforms, but did not stain with mAb S58 (<b>F</b>), which reacts with the slow MyHC2 and slow MyHC3 isoforms. Bar in Panel A = 10 µm for panels A–D and 30 µm for panels E and F.</p

Expression of Prdm1 and fast and slow MyHC isoforms in cultures of E4 hindlimb cultures.

<p>As indicated, individual cultures were analyzed by triple immunostaining for fast MyHC(s) with mAb F59 (A, E, blue fluorescence), Prdm1 (B, F, green fluorescence, Ab from Cell Signaling Technology), and slow MyHC(s) with mAb S58 (C, G, red fluorescence). Merged photographs (D, H) showed that, as found previously, E4 myoblasts formed differentiated, usually mononucleate, cells of two types: those that express only fast MyHC(s) (<i>e.g.</i>, downward pointing arrows in panels E–H) and those that co-express fast and slow MyHCs (upward pointing arrows in all panels). Prdm1 immunostaining was found in the nuclei of both types of differentiated cells. An additional Prdm1 antibody (Abcam) produced similar nuclear staining of E4 cultures (not shown). Bar in Panel A = 20 µm.</p

Prdm1 mRNA and protein were expressed in chicken tissues and muscle cell cultures.

<p><b>A.</b> As indicated, RNAs or proteins were isolated from trunk tissues (Tk), hindlimbs (HL), and forelimbs (FL) of chicken embryos (E4  =  day 4 <i>in ovo</i>) and fetuses (E12), and also from differentiated cultures of mouse C₂C₁₂ myogenic cells (C₂) and primary cultures of E4 and E12 chicken hindlimb cells. RNAs were analyzed by RT-PCR with exon-spanning primers specific for chicken Prdm1 and, as a positive control, GAPDH as indicated. Proteins were analyzed by immunoblotting with antibodies specific for Prdm1 (Cell Signaling Technology) or GAPDH. Bands with the expected ∼240 bp size of the Prdm1 RT-PCR product or the expected ∼100 kDa M_r of the Prdm1 protein were obtained from all chicken embryonic and fetal tissues and from both E4 and E12 chicken myogenic cell cultures. Prdm1 mRNA and protein were not obtained from cells of the mouse C₂C₁₂ myogenic cell line, though GAPDH was found. Specificity of the amplified cDNAs for Prdm1 was confirmed by restriction enzyme analysis (not shown). Bands from some gels were re-arranged for presentation. <b>B.</b> Full-length Prdm1 immunoblot (Ab from Cell Signaling Technology) of the same tissue samples shown in panel A, demonstrating lack of non-specific bands. MW  =  molecular standards with number  =  kDa.</p

Expression of Prdm1 and fast and slow MyHC isoforms in E4 somite explant cultures and the somitic myotome.

<p>Double immunofluorescence analysis for fast MyHC(s) with mAb F59 (<b>A</b>, red fluorescence) and slow MyHC(s) with mAb S58 (<b>B</b>, green fluorescence) showed that fast and slow MyHCs were co-expressed by all differentiated myocytes in somite cultures. In addition, Prdm1 immunostaining (Ab from Cell Signaling Technology) was found in the nuclei of all myocytes, as well as in many MyHC-negative cells (<b>C</b>, merged image, green fluorescence  =  S58, red fluorescence  =  Prdm1, blue fluorescence  =  nuclei). <b>D</b>. Prdm1 (green fluorescence, Ab from Abcam) was expressed throughout the myotome (mt, arrows) of a mature somite at the forelimb bud level at E4. Additional Prdm1 staining was found in some cells of the sclerotome (sct) and neural tube (nt). Arrows indicate Prdm1-positive nuclei within myocytes. Bar in Panel A = 40 µm for panels A and B); 15 µm for panel C; and 50 µm for panel D.</p

Functional domains of the FSHD-associated DUX4 protein

Aberrant expression of the full-length isoform of DUX4 (DUX4-FL) appears to underlie pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). DUX4-FL is a transcription factor and ectopic expression of DUX4-FL is toxic to most cells. Previous studies showed that DUX4-FL-induced pathology requires intact homeodomains and that transcriptional activation required the C-terminal region. In this study, we further examined the functional domains of DUX4 by generating mutant, deletion, and fusion variants of DUX4. We compared each construct to DUX4-FL for (i) activation of a DUX4 promoter reporter, (ii) expression of the DUX4-FL target gene ZSCAN4, (iii) effect on cell viability, (iv) activation of endogenous caspases, and (v) level of protein ubiquitination. Each construct produced a similarly sized effect (or lack of effect) in each assay. Thus, the ability to activate transcription determined the extent of change in multiple molecular and cellular properties that may be relevant to FSHD pathology. Transcriptional activity was mediated by the C-terminal 80 amino acids of DUX4-FL, with most activity located in the C-terminal 20 amino acids. We also found that non-toxic constructs with both homeodomains intact could act as inhibitors of DUX4-FL transcriptional activation, likely due to competition for promoter sites. This article has an associated First Person interview with the first author of the paper