Uncovering changes in proteomic signature of rat pelvic floor muscles in pregnancy.

BACKGROUND: Structural and functional changes of the rat pelvic floor muscles during pregnancy, specifically, sarcomerogenesis, increase in extracellular matrix content, and higher passive tension at larger strains protect the integral muscle components against birth injury. The mechanisms underlying these antepartum alterations are unknown. Quantitative proteomics is an unbiased method of identifying protein expression changes in differentially conditioned samples. Therefore, proteomics analysis provides an opportunity to identify molecular mechanisms underlying antepartum muscle plasticity. OBJECTIVE: To elucidate putative mechanisms accountable for pregnancy-induced adaptations of the pelvic floor muscles, and to identify other novel antepartum alterations of the pelvic floor muscles. MATERIALS AND METHODS: Pelvic floor muscles, comprised of coccygeus, iliocaudalis, and pubocaudalis, and nonpelvic limb muscle, tibialis anterior, were harvested from 3-month-old nonpregnant and late-pregnant Sprague-Dawley rats. After tissue homogenization, trypsin-digested peptides were analyzed by ultra-high-performance liquid chromatography coupled with tandem mass spectroscopy using nano-spray ionization. Peptide identification and label free relative quantification analysis were carried out using Peaks Studio 8.5 software (Bioinformatics Solutions Inc., Waterloo, ON, Canada). Proteomics data were visualized using the Qlucore Omics Explorer (New York, NY). Differentially expressed peptides were identified using the multi-group differential expression function, with q-value cutoff set at <0.05. Proteomic signatures of the pelvic floor muscles were compared to nonpelvic limb muscle and between nonpregnant and pregnant states. RESULTS: Unsupervised clustering of the data showed clear separation between samples from nonpregnant and pregnant animals along principal component 1 and between pelvic and nonpelvic muscles along principal component 2. Four major gene clusters were identified segregating proteomic signatures of muscles examined in nonpregnant vs pregnant states: (1) proteins increased in the pelvic floor muscles only; (2) proteins increased in the pelvic floor muscles and tibialis anterior; (3) proteins decreased in the pelvic floor muscles and tibialis anterior; and (4) proteins decreased in the pelvic floor muscles alone. Cluster 1 included proteins involved in cell cycle progression and differentiation. Cluster 2 contained proteins that participate in mitochondrial metabolism. Cluster 3 included proteins involved in transcription, signal transduction, and phosphorylation. Cluster 4 comprised proteins involved in calcium-mediated regulation of muscle contraction via the troponin tropomyosin complex. CONCLUSION: Pelvic floor muscles gain a distinct proteomic signature in pregnancy, which provides a mechanistic foundation for the antepartum physiological alterations acquired by these muscles. Variability in genes encoding these proteins may alter plasticity of the pelvic floor muscles and therefore the extent of the protective pregnancy-induced adaptations. Furthermore, pelvic floor muscles' proteome is divergent from that of the nonpelvic skeletal muscles.

DNA damage signaling mediates the functional antagonism between replicative senescence and terminal muscle differentiation.

The molecular determinants of muscle progenitor impairment to regenerate aged muscles are currently unclear. We show that, in a mouse model of replicative senescence, decline in muscle satellite cell-mediated regeneration coincides with activation of DNA damage response (DDR) and impaired ability to differentiate into myotubes. Inhibition of DDR restored satellite cell differentiation ability. Moreover, in replicative human senescent fibroblasts, DDR precluded MYOD-mediated activation of the myogenic program. A DDR-resistant MYOD mutant could overcome this barrier by resuming cell cycle progression. Likewise, DDR inhibition could also restore MYOD's ability to activate the myogenic program in human senescent fibroblasts. Of note, we found that cell cycle progression is necessary for the DDR-resistant MYOD mutant to reverse senescence-mediated inhibition of the myogenic program. These data provide the first evidence of DDR-mediated functional antagonism between senescence and MYOD-activated gene expression and indicate a previously unrecognized requirement of cell cycle progression for the activation of the myogenic program.

Increased risk of genetic and epigenetic instability in human embryonic stem cells associated with specific culture conditions.

The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging, and feeder-free vs. mouse embryonic fibroblast feeder substrate, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages, we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher rates of cell proliferation, and persistence of OCT4/POU5F1-positive cells in teratomas, with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers, we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53, which was associated with decreased mRNA expression of TP53, as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures, we also observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies.