Bladder smooth muscle organ culture preparation maintains the contractile phenotype

Abstract

Smooth muscle cells, when subjected to culture, modulate from a contractile to a secretory phenotype. This has hampered the use of cell culture for molecular techniques to study the regulation of smooth muscle biology. The goal of this study was to develop a new organ culture model of bladder smooth muscle (BSM) that would maintain the contractile phenotype and aid in the study of BSM biology. Our results showed that strips of BSM subjected to up to 9 days of organ culture maintained their contractile phenotype, including the ability to achieve near-control levels of force with a temporal profile similar to that of noncultured tissues. The technical aspects of our organ culture preparation that were responsible, in part, for the maintenance of the contractile phenotype were a slight longitudinal stretch during culture and subjection of the strips to daily contraction-relaxation. The tissues contained viable cells throughout the cross section of the strips. There was an increase in extracellular collagenous matrix, resulting in a leftward shift in the passive length-tension relationship. There were no significant changes in the content of smooth muscle-specific α-actin, calponin, h-caldesmon, total myosin heavy chain, protein kinase G, Rho kinase-I, or the ratio of SM1 to SM2 myosin isoforms. Moreover the organ cultured tissues maintained functional voltage-gated calcium channels and large-conductance calcium-activated potassium channels. Therefore, we propose that this novel BSM organ culture model maintains the contractile phenotype and will be a valuable tool for the use in cellular/molecular biology studies of bladder myocytes.

In vivo roles for myosin phosphatase targeting subunit‐1 phosphorylation sites T694 and T852 in bladder smooth muscle contraction

“…For embryonic mouse bladder smooth muscle, we confirmed that MYPT1 T852 and CPI-17 are phosphorylated by K + depolarization or muscarinic receptor stimulation, which could be reduced by ROCK and PKC inhibitors, respectively. The constitutive MYPT1 T694 phosphorylation was not inhibited by ROCK or PKC inhibitors, similar to other reports for bladder smooth muscle (Wang et al 2009(Wang et al , 2012. Additionally, treatment of bladder tissues with calyculin A did not further increase MYPT1 T694 phosphorylation.…”

“…The constitutive MYPT1 T694 phosphorylation was not inhibited by ROCK or PKC inhibitors, similar to other reports for bladder smooth muscle (Wang et al . , ). Additionally, treatment of bladder tissues with calyculin A did not further increase MYPT1 T694 phosphorylation.…”

Carbachol-induced signaling through Thr696-phosphorylation of myosin phosphatase-targeting subunit 1 (MYPT1) in rat bladder smooth muscle cells

“…Their results suggested that phosphorylation of Thr696 was more stable compared with that of Thr853, and it may facilitate Thr853 phosphorylation. However, investigations from Chen et al [25] indicated that MYPT1 T852 and CPI-17 are phosphorylated by muscarinic receptor stimulation, which could be reduced by ROCK and PKC inhibitors respectively, and that most MYPT1 Thr694 was phosphorylated constitutively, similar to other reports for bladder smooth muscle [15,26]. So even in studies using the same smooth muscle preparation, the results can vary depending on the laboratory reporting the findings.…”

Increased expression of desmin and vimentin reduces bladder smooth muscle contractility via JNK2

“…The bladder neck, trigone, base region, the urothelium, and submucosa were removed and the bladders were divided in the midsagittal plane, and cut into longitudinal strips. The bladders were cut into longitudinal strips approximately 2 mm wide and 4‐5 mm long and the muscle strips were prepared for adenovirus transduction as described previously . Briefly, the strips were mounted onto polymerized silicone elastomer (Dow Corning, Midland, MI, USA) using 0.1 mm stainless pins.…”

“…The bladders were cut into longitudinal strips approximately 2 mm wide and 4-5 mm long and the muscle strips were prepared for adenovirus transduction as described previously. 26 Briefly, the strips were mounted onto polymerized silicone elastomer (Dow Corning, Midland, MI, USA) using 0.1 mm stainless pins. The pins were positioned such that the strips were slightly stretched longitudinally, while raised 2-3 mm above the surface of the elastomer.…”