(Poster 163) Impact of Aging on Pelvic Skeletal Muscles in a Lifespan Preclinical Model

Objectives: Aging is one of the key risk factors for pelvic floor muscle (PFM) dysfunction and the associated PFDs. We have previously demonstrated age-related fibrotic degeneration and muscle stem cell (MuSC) pool contracture in aged compared to younger human PFMs but not in the limb muscles of the same cadaveric donors. Given the constraints associated with directly probing human PFMs in vivo, preclinical models suitable for mechanistic studies of the aging impact on PFMs are needed to develop and test scientifically rational interventions. We aimed to determine whether aging effects functionally relevant tissue- and cell-level alterations in rat PFMs.

Methods: The pubocaudalis (PCa) portion of the rat levator ani and the limb muscle-tibialis anterior (TA)-were procured from young (Y; 3-6mo) and old (O; 20-24mo) Fisher rats (N=5/group). Snap-frozen muscles were cryosectioned and fixed in 4% PFA. Collagen content and MuSC reservoir were quantified by incubating slides with anti-Pax7+anti-laminin+DAPI or anti-collagen I+ DAPI, respectively. Collagen I, which comprises 98% of the intramuscular ECM, is the usual target for ascertaining fibrosis in skeletal muscles. Images were captured with KeyenceBZX800 microscope and quantified using ImageJ. Mitochondrial bioenergetics were ascertained using an established respirometry in frozen samples method. Muscles were homogenized, protein concentration was determined by BCA assay, and 5µg of protein/sample (N=2/group) were loaded into the Seahorse XF96 microplate. Wave software was used to quantify the oxygen consumption rates (OCR). Data were compared using GraphPad Prismv10.

Results: Collagen area fraction was significantly higher in PCa (P < 0.01) but not in TA (P=0.53) of older compared to younger animals (Fig.1A). Consistently, MuSC reservoir was significantly reduced in PFMs of older rats (P < 0.001, Fig.1B). OCR sustained by the Complexes I and IV decreased with age in PCa and TA, with more pronounced impairment of mitochondrial function observed in TA (Fig.2).

Conclusions: Age-associated fibrotic degeneration of the rat pelvic but not the limb muscles and contracture of the rat pelvic MuSC reservoir recapitulate the observed human phenotype. Furthermore, our preliminary results demonstrate deterioration of oxygen consumption in aged pelvic and non-pelvic rat muscles. The rat lifespan model is invaluable for investigating molecular changes in PFMs, warranting future larger studies of independent and combinatorial impact of aging, birth injury, and menopause.

Figure 1. (A) Representative images (left) and quantification (right) of collagen I (green) of pelvic and limb muscles in young and old rats. (B) The MuSC reservoir, identified by immunohistochemistry (arrowhead), significantly decreased in pubocaudalis of old compared to young animals. P-values derived from Student t-tests (A) and Mann-Whitney (B).

Figure 2. Representative traces of OCR sustained by Complex I substrate (NADH) and Complex IV substrate (ascorbic acid+TMPD) in young and old animals showing impairment of the mitochondrial function with age. P-values derived from 2-way RM ANOVA/Sidak pairwise comparisons.