Heterogeneity of bladder myocytes in vitro: Modulation of myosin isoform expression

We studied the expression of myosin heavy chain isoforms differing at the N-terminal (SM-A, SM-B) and the C-terminal (SM1, SM2) regions and non-muscle myosin heavy chain II-A and II-B (NMMHC II-A and B) in newborn and adult rabbit bladder smooth muscle cells (SMCs) and in cultures of enzymatically dissociated neonatal detrusor. RT-PCR analyses revealed that 94.5 ± 3.27% of MHC transcripts of the adult bladder SMCs contained the 21-nucleotide insert (SM-B) compared with 83.8 ± 3.2% in the newborn bladder, with the remainder of the mRNA being non-inserted (SM-A). In 3, 7, and 10 days of primary culture (proliferating, confluent, and post-confluent, respectively) and up to 4 subculture passages, bladder myocytes expressed predominantly SM-A. Immunofluorescence microscopy revealed heterogeneity in cultured myocytes, i.e. SM-B positive cells coexisting with negatively stained cells. In adult bladder, the C-terminal isoforms SM1 and SM2 represented, 43.1 ± 4.3% and 56.89% ± 4.3% of the mRNA, respectively, while newborn bladders expressed 72.5 ± 7% SM1 and 27.5 ± 7% SM2. Upon culturing, cells predominantly expressed SM1 at both the mRNA and protein levels. NMMHC II-A was expressed by both adult and newborn bladders and in culture, whereas NMMHC II-B was expressed at low levels only in newborn bladders, but upregulated in culture. These data indicate that bladder myocytes in vitro undergo modulation with relative overexpression of SM-A and SM1 and upregulation of NMMHC II-B. Information on the mechanisms responsible for this modulation in vitro might provide an understanding of the nature of altered myosin isoform expression associated with smooth muscle dysfunction in certain bladder diseases.