Uncovering Changes in Proteomic Signature of Rat Pelvic Floor Muscles in Pregnancy

Abstract 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 molecular 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. Objectives 1. To elucidate putative mechanisms accountable for pregnancy-induced adaptations of the pelvic floor muscles. 2. To identify other novel antepartum alterations of the pelvic floor muscles. Study Design Pelvic floor muscles, comprised of coccygeus, iliocaudalis, and pubocaudalis, and non-pelvic limb muscle, tibialis anterior, were harvested from 3-months old non-pregnant 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 was carried out using Peaks Studio 8.5 software (Bioinformatics solutions Inc., Waterloo, ON, CA). Proteomics data were visualized using the Qlucore Omics Explorer (New York, NY, US). Differentially expressed peptides were identified using the multi-group differential expression function, with q-value cutoff set at Results Unsupervised clustering of the data showed clear separation between samples from non-pregnant and pregnant animals along principal component 1 and between pelvic and non-pelvic muscles along principal component 2. Four major gene clusters were identified segregating proteomic signatures of muscles examined in non-pregnant 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, was comprised of proteins involved in calcium-mediated regulation of muscle contraction via the troponin tropomyosin complex. Conclusions Pelvic floor muscles gain a distinct proteomic signature in pregnancy, which provides a mechanistic foundation for the physiological alterations acquired by these muscles antepartum. Variability in genes encoding these proteins may alter antepartum plasticity of the pelvic floor muscles and, therefore, the extent of the protective pregnancy-induced adaptations. Furthermore, pelvic floor muscles’ proteome is divergent from the non-pelvic skeletal muscles.