Deletion of Dicer in Smooth Muscle Affects Voiding Pattern and Reduces Detrusor Contractility and Neuroeffector Transmission

Abstract

MicroRNAs have emerged as important regulators of smooth muscle phenotype and may play important roles in pathogenesis of various smooth muscle related disease states. The aim of this study was to investigate the role of miRNAs for urinary bladder function. We used an inducible and smooth muscle specific Dicer knockout (KO) mouse which resulted in significantly reduced levels of miRNAs, including miR-145, miR-143, miR-22, miR125b-5p and miR-27a, from detrusor preparations without mucosa. Deletion of Dicer resulted in a disturbed micturition pattern in vivo and reduced depolarization-induced pressure development in the isolated detrusor. Furthermore, electrical field stimulation revealed a decreased cholinergic but maintained purinergic component of neurogenic activation in Dicer KO bladder strips. The ultrastructure of detrusor smooth muscle cells was well maintained, and the density of nerve terminals was similar. Western blotting demonstrated reduced contents of calponin and desmin. Smooth muscle α-actin, SM22α and myocardin were unchanged. Activation of strips with exogenous agonists showed that depolarization-induced contraction was preferentially reduced; ATP- and calyculin A-induced contractions were unchanged. Quantitative real time PCR and western blotting demonstrated reduced expression of Cav1.2 (Cacna1c). It is concluded that smooth muscle miRNAs play an important role for detrusor contractility and voiding pattern of unrestrained mice. This is mediated in part via effects on expression of smooth muscle differentiation markers and L-type Ca2+ channels in the detrusor.

Induction of angiotensin-converting enzyme after miR-143/145 deletion is critical for impaired smooth muscle contractility

“…Using the same mouse model, we also demonstrated impaired depolarizationinduced contraction in the urinary bladder (32). Expression of smooth muscle differentiation markers was, however, hardly affected in the bladder, contrasting with our findings in the aorta.…”

“…Aorta and bladder were excised, cleaned, and quickly frozen in liquid nitrogen. The samples were homogenized in SDS sample buffer (60 mM Tris·HCl pH 6.8, 2% SDS, and 10% glycerol) as previously described (32). The amount of protein was determined using Bio-Rad DC protein assay.…”

“…Several studies have shown that miR-143 and miR-145 are among the most highly expressed miRNAs in differentiated smooth muscle (8,10,11,32,39). They are encoded by a bicistronic miRNA cluster regulated by the transcription factor serum response factor (SRF) and its cofactors myocardin and myocardin-related transcription factors (MRTFs) (11,24,39).…”

Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization

“…Frozen tissue was pulverized using liquid nitrogen as described previously (30). Proteins were detected using commercially available primary antibodies: ␣-actin (1:2000; Sigma-Aldrich, catalog no.…”

MicroRNA MiR-199a-5p Regulates Smooth Muscle Cell Proliferation and Morphology by Targeting WNT2 Signaling Pathway

“…The comparative analysis of the miRNA expression profiles in BPS, bladder cancer, and several inflammatory disorders showed that seven of 31 miRNAs altered in BPS had the same regulatory pattern in inflammatory bowel disease (11), which shares many features with BPS (12). Recently, the role of miRNAs in the bladder smooth muscle was investigated using an induced smooth muscle-specific Dicer knock-out, which caused a significant reduction of miRNA levels, including miR-145, miR-143, miR-22, miR-125b-5p, and miR-27a, leading to a disturbed micturition pattern in vivo (13). In a similar study, the loss of Dicer exacerbated cyclophosphamide-induced bladder overactivity in mice (14).…”