Hypothesis / aims of study
Men with benign prostatic hyperplasia (BPH) and benign prostatic obstruction (BPO) often present with lower urinary tract symptoms (LUTS) including overactive bladder (OAB). OAB is often associated with detrusor overactivity (DO). DO is the urodynamic diagnosis of involuntary spontaneous bladder contractions which can cause the symptoms of OAB. Although the symptomatic diagnosis of OAB does not always correlate with DO, DO is an objective, measureable characteristic of bladder dysfunction. Previously, using next generation sequencing (NGS) we determined mRNA and miRNA expression profiles in biopsies of BPO patients. Here we sought to investigate the specific regulation of beta3-adrenergic receptor and other components of detrusor contractile apparatus, including phospholamban in a larger patients’ cohort and in bladder cell-based models.
Study design, materials and methods
Bladder dome biopsies of controls (n=10) and BPO patients with DO (n=22) were collected and total RNA isolated. Primary smooth muscle (SMC) and urothelial (UE) cells were cultured for several passages. UE cells were differentiated with serum and Ca2+. Regulation of selected genes was studied by qRT-PCR.
Our previous NGS data (n=6 subjects per group) revealed significant up-regulation of phospholamban (PLN), and down-regulation of ADRA2A and ADRB3, encoding alpha 2A and beta 3 adrenoceptors. Here we confirm these findings by RT-qPCR in a larger patients’ group. We investigated the expression of ADRB3 mRNA in bladder layers. We reliably detected ADRB3 in patients’ biopsies and in differentiated smooth muscle, but not in urothelium, or cultures of SMC or UE. Importantly, in DO patients SM markers MYH11 and ACTA2 were elevated in line with increased detrusor contractility, indicating that the observed ADRB3 down-regulation was not the result of SM de-differentiation. PLN is targeted my microRNA miR-374a-5p, significantly reduced in DO patients, whereas both ADRA2A and ADRB3 are targeted by the calcium-regulated miR-212-5p, increased in DO.
Interpretation of results
Phospholamban (PLN) is an inhibitor of the sarcoplasmic reticulum (SR) calcium-ATPase (SERCA), and its SM-specific upregulation in animal models significantly increases bladder contractility responses to carbachol. Our results indicate that PLN up-regulation and ADRB3 down-regulation in DO smooth muscle might contribute to myogenic overactivity. PLN increase in SM might lead to calcium overload, which induces miR-212-5p and might cause a down-regulation of its target ADRB3, inducing a feed-forward loop which results in bladder overactivity.