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Biomedical subjects

Louis M Kunkel

Publications and source records attributed to Louis M Kunkel.

At least 19 recordsLinked to original sources
PubMed2006

Modeling human muscle disease in zebrafish.

Zebrafish reproduce in large quantities, grow rapidly, and are transparent early in development. For these reasons, zebrafish have been used extensively to model vertebrate development and disease. Like mammals, zebrafish express dystrophin and many of its associated proteins early in development and these proteins have been shown to be vital for zebrafish muscle stability. In dystrophin-null zebrafish, muscle degeneration becomes apparent as early as 3 days post-fertilization (dpf) making the zebrafish an excellent organism for large-scale screens to identify other genes involved in the disease process or drugs capable of correcting the disease phenotype. Being transparent, developing zebrafish are also an ideal experimental model for monitoring the fate of labeled transplanted cells. Although zebrafish dystrophy models are not meant to replace existing mammalian models of disease, experiments requiring large numbers of animals may be best performed in zebrafish. Results garnered from using this model could lead to a better understanding of the pathogenesis of the muscular dystrophies and the development of future therapies.

Animals↗
PubMed2006

Diagnosis and cell-based therapy for Duchenne muscular dystrophy in humans, mice, and zebrafish.

The muscular dystrophies are a heterogeneous group of genetically caused muscle degenerative disorders. The Kunkel laboratory has had a longstanding research program into the pathogenesis and treatment of these diseases. Starting with our identification of dystrophin as the defective protein in Duchenne muscular dystrophy (DMD), we have continued our work on normal dystrophin function and how it is altered in muscular dystrophy. Our work has led to the identification of the defective genes in three forms of limb girdle muscular dystrophy (LGMD) and a better understanding of how muscle degenerates in many of the different dystrophies. The identification of mutations causing human forms of dystrophy has lead to improved diagnosis for patients with the disease. We are continuing to improve the molecular diagnosis of the dystrophies and have developed a high-throughput sequencing approach for the low-cost rapid diagnosis of all known forms of dystrophy. In addition, we are continuing to work on therapies using available animal models. Currently, there are a number of mouse models of the human dystrophies, the most notable being the mdx mouse with dystrophin deficiency. These mice are being used to test possible therapies, including stem-cell-based approaches. We have been able to systemically deliver human dystrophin to these mice via the arterial circulation and convert 8% of dystrophin-deficient fibers to fibers expressing human dystrophin. We are now expanding our research to identify new forms of LGMD by analyzing zebrafish models of muscular dystrophy. Currently, we have 14 different zebrafish mutants exhibiting various phenotypes of muscular dystrophy, including muscle weakness and inactivity. One of these mutants carries a stop codon mutation in dystrophin, and we have recently identified another carrying a mutation in titin. We are currently positionally cloning the disease-causative mutation in the remaining 12 mutant strains. We hope that one of these new mutant strains of fish will have a mutation in a gene not previously implicated in human muscular dystrophy. This gene would become a candidate gene to be analyzed in patients which do not carry a mutation in any of the known dystrophy-associated genes. By studying both disease pathology and investigating potential therapies, we hope to make a positive difference in the lives of people living with muscular dystrophy.

Animals↗
PubMed2006

Transcriptome-scale similarities between mouse and human skeletal muscles with normal and myopathic phenotypes.

BACKGROUND: Mouse and human skeletal muscle transcriptome profiles vary by muscle type, raising the question of which mouse muscle groups have the greatest molecular similarities to human skeletal muscle. METHODS: Orthologous (whole, sub-) transcriptome profiles were compared among four mouse-human transcriptome datasets: (M) six muscle groups obtained from three mouse strains (wildtype, mdx, mdx5cv); (H1) biopsied human quadriceps from controls and Duchenne muscular dystrophy patients; (H2) four different control human muscle types obtained at autopsy; and (H3) 12 different control human tissues (ten non-muscle). RESULTS: Of the six mouse muscles examined, mouse soleus bore the greatest molecular similarities to human skeletal muscles, independent of the latters' anatomic location/muscle type, disease state, age and sampling method (autopsy versus biopsy). Significant similarity to any one mouse muscle group was not observed for non-muscle human tissues (dataset H3), indicating this finding to be muscle specific. CONCLUSION: This observation may be partly explained by the higher type I fiber content of soleus relative to the other mouse muscles sampled.

Animals↗
PubMed2006

Somitic origin of limb muscle satellite and side population cells.

Repair of mature skeletal muscle is mediated by adult muscle progenitors. Satellite cells have long been recognized as playing a major role in muscle repair, whereas side population (SP) cells have more recently been identified as contributing to this process. The developmental source of these two progenitor populations has been considerably debated. We explicitly tested and quantified the contribution of embryonic somitic cells to these progenitor populations. Chick somitic cells were labeled by using replication-defective retroviruses or quail/chick chimeras, and mouse cells were labeled by crossing somite-specific, Pax3-derived Cre driver lines with a Cre-dependent reporter line. We show that the majority of, if not all, limb muscle satellite cells arise from cells expressing Pax3 specifically in the hypaxial somite and their migratory derivatives. We also find that a significant number of, but not all, limb muscle SP cells are derived from the hypaxial somite. Notably, the heterogeneity in the developmental origin of SP cells is reflected in their functional heterogeneity; somitically derived SP cells are intrinsically more myogenic than nonsomitically derived ones. Thus, we show that the somites, which supply embryonic and fetal myoblasts, are also an important source of highly myogenic adult muscle progenitors.

Animals↗
PubMed2006

Muscle engraftment of myogenic progenitor cells following intraarterial transplantation.

Cell-based therapy continues to be a promising avenue for the treatment of Duchenne muscular dystrophy (DMD), an X-linked skeletal muscle-wasting disease. Recently, we demonstrated that freshly isolated myogenic progenitors contained within the adult skeletal muscle side population (SP) can engraft into dystrophic fibers of nonirradiated mdx(5cv) mice after intravenous transplantation. Engraftment rates, however, have not been therapeutically significant, achieving at most 1% of skeletal muscle myofibers expressing protein from donor-derived nuclei. To enhance the engraftment of transplanted myogenic progenitors, an intraarterial delivery method was adapted from a previously described procedure. Cultured, lentivirus-transduced skeletal muscle SP cells, derived from mdx(5cv) mice, were transplanted into the femoral artery of noninjured mdx(5cv) mice. Based on the expression of microdystrophin or green fluorescent protein (GFP) transgenes in host muscle, sections of the recipient muscles exhibited 5%-8% of skeletal muscle fibers expressing donor-derived transgenes. Further, donor muscle SP cells, which did not express any myogenic markers prior to transplant, expressed the satellite cell transcription factor, Pax7, and the muscle-specific intermediate filament, desmin, after extravasation into host muscle. The expression of these muscle-specific markers indicates that progenitors within the side population can differentiate along the myogenic lineage after intraarterial transplantation and extravasation into host muscle. Given that femoral artery catheterization is a common, safe clinical procedure and that the transplantation of cultured adult muscle progenitor cells has proven to be safe in mice, our data may represent a step toward the improvement of cell-based therapies for DMD and other myogenic disorders.

Animals↗
PubMed2005

The influence of muscle type and dystrophin deficiency on murine expression profiles.

The phenotypic differences among Duchenne muscular dystrophy patients, mdx mice, and mdx(5cv) mice suggest that despite the common etiology of dystrophin deficiency, secondary mechanisms have a substantial influence on phenotypic severity. The differential response of various skeletal muscles to dystrophin deficiency supports this hypothesis. To explore these differences, gene expression profiles were generated from duplicate RNA targets extracted from six different skeletal muscles (diaphragm, soleus, gastrocnemius, quadriceps, tibialis anterior, and extensor digitorum longus) from wild-type, mdx, and mdx(5cv) mice, resulting in 36 data sets for 18 muscle samples. The data sets were compared in three different ways: (1) among wild-type samples only, (2) among all 36 data sets, and (3) between strains for each muscle type. The molecular profiles of soleus and diaphragm separate significantly from the other four muscle types and from each other. Fiber-type proportions can explain some of these differences. These variations in wild-type gene expression profiles may also reflect biomechanical differences known to exist among skeletal muscles. Further exploration of the genes that most distinguish these muscles may help explain the origins of the biomechanical differences and the reasons why some muscles are more resistant than others to dystrophin deficiency.

Animals↗
PubMed2005

Variations in gene expression among different types of human skeletal muscle.

There is a consistent variation in the response of different skeletal muscle groups to mutations in genes known to cause muscular dystrophy, yet these muscles appear histologically similar. To better understand these phenotypic differences, we analyzed gene expression patterns in control muscle specimens obtained from four sites at autopsy: deltoid, quadriceps, gastrocnemius, and tibialis anterior (TA). A total of 35 muscle samples from nine individuals (four pediatric and five geriatric) were studied. Factors potentially influencing gene expression in the different samples included individuality, age, muscle type, gender, cause of death, postmortem interval, and ethnicity. The first three factors, in decreasing order, were found to have a significant impact on the stratification of muscle specimens. A novel analytic method, using a second round of normalization, was used to elicit differences between muscle types. This approach may be extended to a broader survey, potentially elucidating a molecular classification of the skeletal muscles.

Adolescent↗
PubMed2004

Delta-sarcoglycan is required for early zebrafish muscle organization.

Mutations in sarcoglycans (alpha-, beta-, gamma-, and delta-) have been linked with limb girdle muscular dystrophy (LGMD) types 2C-F in humans. We have cloned the zebrafish orthologue encoding delta-sarcoglycan and mapped the gene to linkage group 21. The predicted zebrafish delta-sarcoglycan protein is highly homologous with its human orthologue including conservation of two of the three predicted glycosylation sites. Like other members of the dystrophin-associated protein complex (DAPC), delta-sarcoglycan localizes to the sarcolemmal membrane of the myofiber in adult zebrafish, but is more apparent at the myosepta in developing embryos. Zebrafish embryos injected with morpholinos against delta-sarcoglycan were relatively inactive at 5 dpf, their myofibers were disorganized, and swim bladders uninflated. Immunohistochemical and immunoblotting experiments show that delta-, beta-, and gamma-sarcoglycans were all downregulated in the morphants, whereas dystrophin expression was unaffected. Whereas humans lacking delta-sarcoglycan primarily show adult phenotypes, our results suggest that delta-sarcoglycan plays a role in early zebrafish muscle development.

Amino Acid Sequence↗
PubMed2004

Demystifying SP cell purification: viability, yield, and phenotype are defined by isolation parameters.

Side population (SP) cells isolated from bone marrow, skeletal muscle, and skin have been shown to engraft in dystrophic muscle. However, there have been questions on the phenotypical heterogeneity, tissue of origin, and relationships among SP cell populations extracted from different tissues. Studies on bone marrow SP cells have followed a consistent protocol for their isolation and results obtained are concordant. In contrast, protocols for the isolation of muscle SP cells vary greatly, and consequently reports on their phenotype, differentiation potential and origin have been inconsistent. To address this controversy, we demonstrate that isolation parameters, such as tissue dissociation, cell counting, Hoechst concentration, and stringency in the selection of SP cells, have an effect on the yield, viability, and homogeneity of SP cells derived from bone marrow, skeletal muscle, and skin. In this paper, we demonstrate that SP cells isolated from the bone marrow are distinct from SP cells extracted from skeletal muscle and skin tissues. This study offers an explanation for the controversy surrounding muscle SP cells, provides a detailed standardized protocol for their isolation, and highlights basic guidelines for reproducible and reliable isolation of SP cells from any tissue.

Animals↗
PubMed2004

Systemic delivery of human microdystrophin to regenerating mouse dystrophic muscle by muscle progenitor cells.

Cell-based therapy for Duchenne muscular dystrophy patients and mdx mice has proven to be a safe but ineffective form of treatment. Recently, a group of cells called muscle side population (SP) cells have been isolated based on their ability to efflux the DNA-binding dye Hoechst. To understand the potential of skeletal muscle SP cells to serve as precursors for muscle, SP cells from the two mice strains mdx(5cv) and C57BL/6N were isolated, transduced, and transplanted. Under coculture conditions with myogenic cells, some cells within the SP cell population can give rise to early Pax7-positive satellite cells and other later stage myogenic cells. Transduced SP cells were transplanted via the tail vein and were shown to successfully deliver enhanced GFP and human microdystrophin to the skeletal muscle of nonirradiated mdx(5cv) mice, thus demonstrating their ability to travel through the capillaries and enter into damaged muscle. These results demonstrate that i.v. delivery of genes via SP cells is possible and that these SP cells are capable of recapitulating the myogenic lineage. Because this approach shows definitive engraftment by using autologous transplantation of noninjured recipients, our data may have substantial implications for therapy of muscular dystrophy.

Animals↗
PubMed2004

Transcriptional profile of postmortem skeletal muscle.

Autopsy specimens are often used in molecular biological studies of disease pathophysiology. However, few analyses have focused specifically on postmortem changes in skeletal muscles, and almost all of those investigate protein or metabolic changes. Although some structural and enzymatic changes have been described, the sequence of transcriptional events associated with these remains unclear. We analyzed a series of new and preexisting human skeletal muscle data sets on approximately 12,500 genes and expressed sequence tags (ESTs) generated by the Affymetrix U95Av2 GeneChips from seven autopsy and seven surgical specimens. Remarkably, postmortem specimens (up to 46 h) revealed a significant and prominent upregulation of transcripts involved with protein biosynthesis. Additional upregulated transcripts are associated with cellular responses to oxidative stress, hypoxia, and ischemia; however, only a subset of genes in these pathways was affected. Overexpression was also seen for apoptosis-related, cell cycle regulation/arrest-related, and signal transduction-related genes. No major gene expression differences were seen between autopsy specimens with <20-h and 34- to 46-h postmortem intervals or between pediatric and adult cases. These data demonstrate that, likely in response to hypoxia and oxidative stress, skeletal muscle undergoes a highly active transcriptional, and possibly, translational phase during the initial 46-h postmortem interval. Knowledge of these changes is important for proper interpretation of gene expression studies utilizing autopsy specimens.

Gene Expression Profiling↗
PubMed2004

Specific assembly pathway of sarcoglycans is dependent on beta- and delta-sarcoglycan.

Mutations in sarcoglycans (SG) have been reported to cause autosomal-recessive limb-girdle muscular dystrophy (LGMD) and dilated cardiomyopathy. In skeletal and cardiac muscle, sarcoglycans exist as a complex of four transmembrane proteins (alpha-, beta-, gamma-, and delta-SG). In this study, the assembly of the sarcoglycan complex was examined in a heterologous expression system. Our results demonstrated that the assembly process occurs as a discrete stepwise process. We found that beta-SG appears to play an initiating role and its association with delta-SG is essential for the proper localization of the sarcoglycan complex to the cell membrane. The incorporation of alpha-SG into the sarcoglycan complex occurs at the final stage by interaction with gamma-SG. These findings were supported by chemical cross-linking of endogenous sarcoglycans in cultured myotubes. We have also provided evidence that glycosylation-defective mutations in beta-SG and a common mutation in gamma-SG (C283Y) disrupt sarcoglycan-complex formation. Our proposed model for the assembly and structure of sarcoglycans should generate important insight into their function in muscle as well as their role in muscular dystrophies and cardiomyopathies.

Animals↗
PubMed2004

Beta-synemin localizes to regions of high stress in human skeletal myofibers.

Synemin is an intermediate filament protein shown previously to interact with alpha-dystrobrevin and desmin. Immunoblot analysis detects a beta-synemin protein of 170 kDa in human skeletal muscle and an alpha-synemin protein of 225 kDa in monkey brain. Low-resolution immunohistochemical analysis localizes beta-synemin within muscle along the sarcolemma, whereas confocal microscopic analysis further refines localization to the costamere and muscle Z-lines. In addition to these locations, beta-synemin is also enriched at the neuromuscular and myotendinous junctions, other regions that undergo high stress during myofiber contraction. Based on its localization and its expression pattern, it is proposed that beta-synemin functions as a structural protein involved in maintaining muscle integrity through its interactions with alpha-dystrobrevin, desmin, and other structural proteins.

Adult↗
PubMed2004

Molecular classification of nemaline myopathies: "nontyping" specimens exhibit unique patterns of gene expression.

Nemaline myopathy (NM) is a slowly progressive or nonprogressive neuromuscular disorder caused by mutations in genes encoding skeletal muscle sarcomeric thin filament proteins. It is characterized by great heterogeneity at the clinical, histopathological, and genetic level. Although multiple molecular pathways are commonly affected in all NM patients, little is known about the molecular characteristics of muscles from patients in different NM subgroups. We have analyzed a group of global gene expression data sets for transcriptional patterns characteristic of particular nemaline myopathy classes. Differential expression between disease subgroups was primarily seen in mitochondrial-, structural-, and transcription-related genes. Multiple lines of evidence support the hypothesis that muscles from cases with "nontyping" NM, although clinically classified as typical NM, share a unique pathophysiological state and are characterized by distinct patterns of gene expression. Determination of the specific molecular differences in NM subgroups may eventually lead to improved prognostic determinations and treatment of these patients.

Base Sequence↗
PubMed2003

Expression profiling and identification of novel genes involved in myogenic differentiation.

Skeletal muscle differentiation is a complex, highly coordinated process that relies on precise temporal gene expression patterns. To better understand this cascade of transcriptional events, we used expression profiling to analyze gene expression in a 12-day time course of differentiating C2C12 myoblasts. Cluster analysis specific for time-ordered microarray experiments classified 2895 genes and ESTs with variable expression levels between proliferating and differentiating cells into 22 clusters with distinct expression patterns during myogenesis. Expression patterns for several known and novel genes were independently confirmed by real-time quantitative RT-PCR and/or Western blotting and immunofluorescence. MyoD and MEF family members exhibited unique expression kinetics that were highly coordinated with cell-cycle withdrawal regulators. Among genes with peak expression levels during cell cycle withdrawal were Vcam1, Itgb3, Itga5, Vcl, as well as Ptger4, a gene not previously associated with the process of myogenesis. One interesting uncharacterized transcript that is highly induced during myogenesis encodes several immunoglobulin repeats with sequence similarity to titin, a large sarcomeric protein. These data sets identify many additional uncharacterized transcripts that may play important functions in muscle cell proliferation and differentiation and provide a baseline for comparison with C2C12 cells expressing various mutant genes involved in myopathic disorders.

Animals↗
PubMed2003

Haplotype-based identification of a microsomal transfer protein marker associated with the human lifespan.

We previously reported a genomewide linkage study for human longevity using 308 long-lived individuals (LLI) (centenarians or near-centenarians) in 137 sibships and identified statistically significant linkage within chromosome 4 near microsatellite D4S1564. This interval spans 12 million bp and contains approximately 50 putative genes. To identify the specific gene and gene variants impacting lifespan, we performed a haplotype-based fine-mapping study of the interval. The resulting genetic association study identified a haplotype marker within microsomal transfer protein as a modifier of human lifespan. This same variant was tested in a second cohort of LLI from France, and although the association was not replicated, there was evidence for statistical distortion in the form of Hardy-Weinberg disequilibrium. Microsomal transfer protein has been identified as the rate-limiting step in lipoprotein synthesis and may affect longevity by subtly modulating this pathway. This study provides proof of concept for the feasibility of using the genomes of LLI to identify genes impacting longevity.

Adolescent↗
PubMed2003

Minimal haplotype tagging.

The high frequency of single-nucleotide polymorphisms (SNPs) in the human genome presents an unparalleled opportunity to track down the genetic basis of common diseases. At the same time, the sheer number of SNPs also makes unfeasible genome-wide disease association studies. The haplotypic nature of the human genome, however, lends itself to the selection of a parsimonious set of SNPs, called haplotype tagging SNPs (htSNPs), able to distinguish the haplotypic variations in a population. Current approaches rely on statistical analysis of transmission rates to identify htSNPs. In contrast to these approximate methods, this contribution describes an exact, analytical, and lossless method, called BEST (Best Enumeration of SNP Tags), able to identify the minimum set of SNPs tagging an arbitrary set of haplotypes from either pedigree or independent samples. Our results confirm that a small proportion of SNPs is sufficient to capture the haplotypic variations in a population and that this proportion decreases exponentially as the haplotype length increases. We used BEST to tag the haplotypes of 105 genes in an African-American and a European-American sample. An interesting finding of this analysis is that the vast majority (95%) of the htSNPs in the European-American sample is a subset of the htSNPs of the African-American sample. This result seems to provide further evidence that a severe bottleneck occurred during the founding of Europe and the conjectured "Out of Africa" event.

Algorithms↗