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

Mardjaneh Karbalaei Sadegh,Mari Ekman,Catarina Rippe,Sebastian Albinsson,Karl Swärd,Bengt Uvelius,Partha Mukhopadhyay

doi:10.1371/journal.pone.0035882

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.

Highlights

Emptying of the urinary bladder depends on coordinated contraction of the detrusor and relaxation of the urethra [1,2]
Acetylcholine is central among the transmitters released from neural varicosities in the detrusor, and the muscarinic Gq-coupled M3 receptor is primarily responsible for cholinergic detrusor activation [5]
In the present study we used smooth muscle-specific and Tamoxifen-inducible knockout of Dicer to evaluate the importance of miRNAs for urinary bladder function in mice

Summary

Introduction

Emptying of the urinary bladder depends on coordinated contraction of the detrusor and relaxation of the urethra [1,2]. Contraction follows the release of neurotransmitters from motor nerves that are dispersed in the muscle bundles [3]. This results in a rapid elevation of the sarcoplasmic Ca2+ concentration, activation of myosin light chain kinase, phosphorylation of myosin, and, after a brief delay, force development [4]. Acetylcholine is central among the transmitters released from neural varicosities in the detrusor, and the muscarinic Gq-coupled M3 receptor is primarily responsible for cholinergic detrusor activation [5]. In addition to muscarinic mechanisms, purinergic signalling plays a role [7], and the relative contribution of purinergic versus cholinergic excitation varies between species and in pathological situations. For example, the relative size of the muscarinic component of neurogenic activation decreases with age and in bladder disturbances [7,8,9,10]; this so called ‘‘atropine resistance’’ is accompanied by an increase in the relative dependence on purinergic activation

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