Regulation of Pelvic Muscle Stem Cells in Pregnancy Using Rat Pre-clinical Model

Objectives: Pelvic floor muscle (PFM) birth injury is a key risk factor for PFDs. Thus, it is imperative to understand what segregates women who do and do not sustain PFM birth injury. Using the rat model, we have shown that pregnancy-induced sarcomerogenesis protects PFMs from parturition-associated mechanical injury. Muscle stem cells (MuSCs) mediate skeletal muscle plasticity in response to various physiological cues, such as hormonal changes during pregnancy. Here we aimed to determine how pregnancy-associated hormonal milieu impacts pelvic MuSC fate along the gestational continuum.

Methods: Serum and PFMs were collected from 3-month-old Sprague-Dawley non-(NP), mid-(MP), and late-(LP) pregnant rats. In vivo MuSC number (Pax7+) and proliferation (Pax7+/EdU+, EdU injected 24h before tissue harvest) were assessed using immunohistochemistry in PFM cross-sections. MuSCs were isolated through FACS and timelapse microscopy was employed to assess cell division dynamics. MuSC gene expression was assessed through qRT-PCR. The impact of pregnancy systemic milieu and the major pregnancy-associated hormones were ascertained by treating MuSCs plated for 72h with either serum collected from NP, MP, LP animals or estriol, or progesterone.

Results: In vivo MuSC proliferation was significantly increased in MP compared to both NP and LP states, while LP showed decreased proliferation compared to NP cells (Fig.A). Consistently, time to first division of isolated MuSCs was decreased in MP but increased in LP relative to NP state (Fig.B). Gene expression analysis revealed a significant decrease in genes associated with maintenance of MuSC quiescence in MP but also in LP (Fig.C). To identify putative drivers of changes in MuSC fate across NP-LP continuum, we treated MuSCs isolated from PFMs of NP, MP, and LP animals with NP, MP, and LP serum. No differences were observed after treatment with serum from NP or MP rats; however, serum from LP animals caused profound cell death on MuSCs from NP, MP, or LP rats (Fig.D). While estriol did not alter MuSC proliferation in vitro, progesterone induced a reduction in cell proliferation compared to untreated cells (Fig.E).

Conclusions: MP MuSCs undergo concordant transcriptional and phenotypical changes that lead to increased proliferation. Interestingly, a novel pseudo-quiescent MuSC state, characterized by phenotypical quiescence and transcriptional activation, was discovered in LP. Our initial perturbations to identify specific factors responsible for MuSC states did not recapitulate either the in vivo findings or in vitro response to LP serum, indicating that alone progesterone, and estriol cannot explain the observed changes in MuSCs. Ongoing studies include combinatorial hormonal treatments and serum fractionation analysis.

Figure Legend: (A) Quantification of Pax7+/EdU+ cells in tissues (One-wayANOVA; n=3-8); (B) Brightfield images and quantification of time of cell division (One-wayANOVA; n=3); (C) Gene expression analysis (One-wayANOVA; n=2-4); (D) Immunofluorescence image of EdU+ cells and quantification (Two-wayANOVA; n=6); (E) Hormonal treatment quantification (One-wayANOVA; n=4).