BACKGROUND: We identified 4 humans with PBS having X-linked, pathogenic FLNA missense mutations, including the severe FLNA p.C2160R gain-of-function mutation disrupting critical regulatory protein binding sites. The FLNA protein is highly expressed in detrusor smooth muscle cells (SMC); this cytoplasmic protein is crucial for SMC regulation, facilitating bidirectional force transmission between actin and integrins. To elucidate mechanistic causality, analogous FlnaC2152R/Y mice were generated using Crispr-Cas9, allowing assessment of this mutation’s impact on bladder form and function. METHODS: Homozygous FlnaC2152R/C2152R dams were crossed with hemizygous FlnaC2152R/Y male mice. 2-month-old adult male offspring (WT and FlnaC2152R/Y) were evaluated for bladder anatomy, gene expression, and function. 2-hour awake suprapubic cystometry measured bladder pressure and voiding events (Cystometry Lab Station, Med-Associates), 24-hour metabolic cage natural fill measured voided urine volume and frequency (UroVoid system, Med-Associates), and 4-hour void spot assay (VSA) quantified urine area during day versus nighttime. Ex-vivo organ bath assays assessed bladder strip contractility using electrical field stimulation (EFS) and cholinergic and purinergic agonists. Bladder bulk RNA-seq compared WT and FlnaC2152R/Y male mice transcriptomes. β-III-tubulin and VAChT expression were measured via western blot (WB). Mann-Whitney was used to test group differences with p<0.05 considered significant (GraphPad software). RESULTS:Comparing adult WT to FlnaC2152R/Y male mice, FlnaC2152R/Y bladders had significant changes, including 28% increased weight and 40% increased detrusor thickness (Figure-1A). On bladder natural filling (urovoid), despite similar urine production rates, FlnaC2152R/Y male mice had 172% larger mean voided volumes per void, and 57% fewer voids per 24hrs (Figure-1B). In contrast, supraphysiological filling rate cystometry revealed 20% lower mean maximum pressure per void, and 106% more voids in FlnaC2152R/Y bladders, suggesting altered bladder responsivity (Figure-1C). Bladder contractility assays demonstrated normally functioning cholinergic and purinergic pathways suggesting normal muscle function, but FlnaC2152R/Y bladders showed a 24% decrease in EFS response at 50Hz, indicating diminished bladder neural input (Figure-1D). Consistently, FlnaC2152R/Y bladders showed significantly reduced neuronal markers by WB (22% β-III-tubulin and 27% VAChT) (Figure-1E). Gene ontology analysis from bladder bulk RNA-seq revealed in FlnaC2152R/Y mouse bladders downregulation of extracellular matrix and muscle cell differentiation, and upregulation of circadian rhythm genes (Figure-1F), which are known to affect daytime vs nighttime voiding frequency. When comparing daytime vs nighttime circadian rhythm voiding, since mice are nocturnal animals, WT mice had 65% of void events (urovoid) with 2X larger urine spots (VSA) during the night. In contrast, FlnaC2152R/Y mice lost these voiding differences between day and night, suggesting this FLNA mutation impacts bladder circadian rhythm (Figure-1G). CONCLUSIONS: Adult male mutant mice carrying the analogous human PBS missense mutation FlnaC2152R/Y exhibited PBS-like thick-walled bladder with functionally large capacity and infrequent voiding. These bladders show alteration of circadian rhythm gene expression with altered day/night voiding patterns. Collectively, these results highlight the complex interplay between structural, functional, and molecular factors contributing to PBS bladder dysfunction in FlnaC2152R/Y mice. NIH-R01DK100483, DK127589 PI:Baker AUA/UCF Research Scholar Award PI:Amado
