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Knockdown of Death-Associated Protein Expression Induces Global Transcriptome Changes in Proliferating and Differentiating Muscle Satellite Cells.

ABSTRACT: Death-associated protein (DAP) undergoes substantial changes in expression during turkey skeletal muscle development, decreasing from the 18 day embryonic stage to 1 day posthatch, and again from 1 day posthatch to 16 weeks of age. These changes suggest that DAP plays an important role at critical stages of the developmental process. The objective of this study was to elucidate the role of DAP in muscle development by examining the effect of reduced DAP expression on global gene expression in proliferating and differentiating turkey pectoralis major muscle satellite cells. Small interfering RNA was used to knock down expression of DAP and the transcriptome was subsequently profiled using a turkey skeletal muscle long oligonucleotide microarray. Microarray data were corroborated using quantitative real-time PCR. In proliferating cells, 458 loci, resulting in 378 uniquely annotated genes, showed differential expression (false discovery rate, FDR < 0.05). Pathway analysis highlighted altered eukaryotic translational initiation factors (eIFs) signaling, protein ubiquitination, sirtuin signaling, and mechanistic target of rapamycin (mTOR) signaling as the primary pathways affected in the knockdown proliferating cells. The findings underpinned the potential DAP involvement in cell proliferation of turkey satellite cells through the coordination between protein synthesis and cell cycle. In differentiating cells, 270 loci, accounting for 189 unique genes, showed differential expression (FDR < 0.05). Decreased expression of genes encoding various myofibrillar proteins and proteins involved in sarcoplasmic reticulum calcium flux suggests that DAP may affect regulation of calcium homeostasis and cytoskeleton signaling. This study provides the first evidence that reduced expression of DAP significantly alters the transcriptome profile of pectoralis major muscle satellite cells, thereby reducing proliferation and differentiation.

SUBMITTER: Horton KA

PROVIDER: S-EPMC7457014 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Knockdown of Death-Associated Protein Expression Induces Global Transcriptome Changes in Proliferating and Differentiating Muscle Satellite Cells.

Horton Katherine A KA Sporer Kelly R B KRB Tempelman Robert J RJ Malila Yuwares Y Reed Kent M KM Velleman Sandra G SG Strasburg Gale M GM

Frontiers in physiology 20200814

Death-associated protein (DAP) undergoes substantial changes in expression during turkey skeletal muscle development, decreasing from the 18 day embryonic stage to 1 day posthatch, and again from 1 day posthatch to 16 weeks of age. These changes suggest that DAP plays an important role at critical stages of the developmental process. The objective of this study was to elucidate the role of DAP in muscle development by examining the effect of reduced <i>DAP</i> expression on global gene expressio ...[more]

PMID: 32922311

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Project description:Background: Exposure of poultry to extreme temperatures during the critical period of post-hatch growth can seriously affect muscle development and thus compromise subsequent meat quality. This study was designed to characterize transcriptional changes induced in turkey muscle satellite cells by thermal challenge during differentiation. Our goal is to better define how thermal stress alters breast muscle ultrastructure and subsequent development. Results: Skeletal muscle satellite cells previously isolated from the Pectoralis major muscle of 7-wk-old male turkeys (Meleagris gallopavo) from two breeding lines: the F-line (16 wk body weight-selected) and RBC2 (randombred control line) were used in this study. Cultured cells were induced to differentiate at 38°C (control) or thermal challenge temperatures of 33 or 43°C. After 48 h of differentiation, cells were harvested and total RNA was isolated for RNAseq analysis. Analysis of 39.9 Gb of sequence found 89% mapped to the turkey genome (UMD5.0, annotation 101) with average expression of 18,917 genes per library. In the cultured satellite cells, slow/cardiac muscle isoforms are generally present in greater abundance than fast skeletal isoforms. Statistically significant differences in gene expression were observed among treatments and between turkey lines, with a greater number of genes affected in the F-line cells following cold treatment whereas more differentially expressed (DE) genes were observed in the RBC2 cells following heat treatment. Many of the most significant pathways involved signaling, consistent with ongoing cellular differentiation. Regulation of Ca2+ homeostasis appears to be significantly affected by temperature treatment, particularly cold treatment. Conclusions: Satellite cell differentiation is directly influenced by temperature at the level of gene transcription with greater effects attributed to selection for fast growth. At lower temperature, muscle-associated genes in the satellite cells were among the genes with the greatest down regulation consistent with slower differentiation and smaller myotubes. Fewer expression differences were observed in the differentiating cells than previously observed for proliferating cells. This suggests the impact of temperature on satellite cells occurs primarily at early points in satellite cell activation.

Project description:We have examined the expression of 13 proto-oncogenes in proliferating and terminally differentiated cardiac and skeletal muscle. Total RNA was prepared from intact ventricular cardiac-muscle tissue and from purified ventricular cardiac-muscle cells of neonatal and adult rats and from cultured proliferating and terminally differentiated L6A1 rat skeletal-muscle cells. cDNA probes for histone H4, thymidine kinase, myosin heavy chain and M-creatine kinase were used to assess cellular proliferation and differentiation. Oncogenes c-myc, c-raf, c-erb-A, c-ras-H, c-ski, and c-sis were expressed in both proliferating and differentiated cardiac muscle tissue and cells, whereas c-myb expression was not observed in either. c-src was expressed only in neonatal cardiac muscle tissue and cells. c-fms, c-abl, and c-ras-K were expressed in tissue from both neonatal and adult animals but only in purified cells from neonatal animals. c-fes/fps was expressed only in neonatal cardiac muscles cells. c-fos expression was not observed in cardiac-muscle tissue from either neonatal or adult rats, but surprisingly was abundantly expressed in freshly isolated cardiac-muscle cells from animals of both ages. These results emphasize that biochemical analysis using intact cardiac-muscle tissue may not necessarily reflect muscle-specific cell processes. They also show that the expression of c-fos can be activated by the cell isolation procedure. c-myc, c-ski, c-ras-H, c-ras-K, c-abl, c-raf and c-erb-A were expressed in both proliferating and terminally differentiated skeletal-muscle cells, whereas c-myb, c-fos, c-src and c-fms transcripts were observed only in proliferating cells. c-fes/fps and c-sis were not expressed in dividing or fused skeletal-muscle cells. These results demonstrate unique tissue and cell-specific patterns of proto-oncogene expression and suggest that these genes may be involved with the regulation of cellular proliferation and terminal differentiation in striated muscle.

Project description:Background:Sarcopenia, the age-related loss of skeletal muscle, is a side effect of androgen deprivation therapy (ADT) for prostate cancer patients. Resident stem cells of skeletal muscle, satellite cells (SCs), are an essential source of progenitors for the growth and regeneration of skeletal muscle. Decreased androgen signaling and deficits in the number and function of SCs are features of aging. Although androgen signaling is known to regulate skeletal muscle, the cellular basis for ADT-induced exacerbation of sarcopenia is unknown. Furthermore, the consequences of androgen deprivation on SC fate in adult skeletal muscle remain largely unexplored. Methods:We examined SC fate in an androgen-deprived environment using immunofluorescence and fluorescence-activated cell sorting (FACS) with SC-specific markers in young castrated mice. To study the effects of androgen deprivation on SC function and skeletal muscle regenerative capacity, young castrated mice were subjected to experimental regenerative paradigms. SC-derived-cell contributions to skeletal muscle maintenance were examined in castrated Pax7CreER/+; ROSA26mTmG/+ mice. SCs were depleted in Pax7CreER/+; ROSA26DTA/+ mice to ascertain the consequences of SC ablation in sham and castrated skeletal muscles. Confocal immunofluorescence analysis of neuromuscular junctions (NMJs), and assessment of skeletal muscle physiology, contractile properties, and integrity were conducted. Results:Castration led to SC activation, however this did not result in a decline in SC function or skeletal muscle regenerative capacity. Surprisingly, castration induced SC-dependent maintenance of young skeletal muscle. The functional dependence of skeletal muscles on SCs in young castrated mice was demonstrated by an increase in SC-derived-cell fusion within skeletal muscle fibers. SC depletion was associated with further atrophy and functional decline, as well as the induction of partial innervation and the loss of NMJ-associated myonuclei in skeletal muscles from castrated mice. Conclusion:The maintenance of skeletal muscles in young castrated mice relies on the cellular contributions of SCs. Considering the well-described age-related decline in SCs, the results in this study highlight the need to devise strategies that promote SC maintenance and activity to attenuate or reverse the progression of sarcopenia in elderly androgen-deprived individuals.

Dataset Information

Knockdown of Death-Associated Protein Expression Induces Global Transcriptome Changes in Proliferating and Differentiating Muscle Satellite Cells.

Publications

Knockdown of Death-Associated Protein Expression Induces Global Transcriptome Changes in Proliferating and Differentiating Muscle Satellite Cells.

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