Over expression of smooth muscle specific caldesmon by transfection and intermittent agonist induced contraction alters cellular morphology and restores differentiated smooth muscle phenotype.
OBJECTIVE
The thin filament associated protein h-caldesmon (h-CaD) modulates actin myosin interaction and contraction. Bladder outlet obstruction and detrusor hypertrophy are associated with the over expression of the nonmuscle CaD isoform l-CaD. It implies a poorly differentiated state of bladder myocytes and cytoskeletal remodeling in detrusor hypertrophy. We determined if h-CaD expression can be increased in a unique bladder smooth muscle (BSM) cell line derived from obstructed rabbit bladder smooth muscle that over expresses l-CaD. We examined whether the genetic restoration of h-caldesmon is possible in bladder smooth muscle cells by transfection or by agonist mediated contraction and whether this manipulation would alter cellular morphology.


METHODS
BSM cells were transfected with chicken h-CaD cDNA inserted into a mammalian vector. In another experiment BSM cells underwent intermittent bethanechol induced stimulation. h-CaD mRNA and protein were quantified with reverse transcriptase-polymerase chain reaction and Western blot analyses. Cell morphology was assessed using phase, video and confocal microscopy after double immunostaining with antibodies against alpha-actin and caldesmon.


RESULTS
Reverse transcriptase-polymerase chain reaction using primers specific for the transfected vector and h-CaD cDNA confirmed stable transfection of cells and increased content of h-CaD mRNA. Following bethanechol induced intermittent contraction Western blotting revealed 80% relative over expression of h-CaD in treated transfected cell lines (p <0.05) and 74% (not significant) in treated nontransfected controls. Confocal immunofluorescence microscopy revealed CaD in the cytoplasmic filaments co-localized to alpha-actin in the main cell body and perinuclear region in transfected cells, in contrast to the diffuse, irregular distribution of these filaments in control cells.


CONCLUSIONS
A unique bladder myocyte cell line was successfully and stably transfected with h-CaD cDNA. We show that agonist induced intermittent contraction preferentially increases h-CaD expression, the predominant CaD in nonobstructed bladder smooth muscle, and the restoration of h-CaD alters cell morphology and the organization of cytoplasmic filaments in cells derived from obstructed rabbit detrusor musculature.
Shukla
Aseem R
AR
Division of Pediatric Urology, Children's Hospital of Philadelphia, Pennsylvania 19104, USA. shukla@email.chop.edu
Nguyen
Trang
T
Zheng
Yongmu
Y
Zderic
Stephen A
SA
DiSanto
Michael
M
Wein
Alan J
AJ
Chacko
Samuel
S
D018723:Bethanechol / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics*; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D010641:Phenotype; D014162:Transfection; D001743:Urinary Bladder / Q000166:cytology / Q000378:metabolism*
D002148:Calmodulin-Binding Proteins; D018723:Bethanechol