Deletion of SM-B, the high ATPase isoform of myosin, upregulates the PKC-mediated signal transduction pathway in murine urinary bladder smooth muscle

Abstract

Detrusor smooth muscle (DSM) hypertrophy induced by partial bladder outlet obstruction (PBOO) is associated with changes in the NH2-terminal myosin heavy chain isoform from predominantly SM-B to SM-A, alteration in the Ca2+ sensitization pathway, and the contractile characteristics from phasic to tonic in rabbits. We utilized the SM-B knockout (KO) mouse to determine whether a shift from SM-B to SM-A without PBOO is associated with changes in the signal transduction pathway mediated via PKC and CPI-17, which keeps the myosin phosphorylation (MLC20) level high by inhibiting the myosin phosphatase. DSM strips from SM-B KO mice generated more force in response to electrical field stimulation, KCl, carbachol, and phorbol 12,13-dibutyrate than that of age-matched wild-type mice. There was no difference in the ED50 for carbachol but the maximum response was greater for the SM-B KO mice. DSM from SM-B KO mice revealed increased mass and hypertrophy. The KO mice also showed an overexpression of PKC-alpha, increased levels of phospho-CPI-17, and an elevated level of IP3 and DAG upon stimulation with carbachol. Two-dimensional gel electrophoresis revealed an increased level of MLC20 phosphorylation in response to carbachol. Together, these changes may be responsible for the higher level of force generation and maintenance by the DSM from the SM-B KO bladders. In conclusion, our data show that ablation of SM-B is associated with alteration of PKC-mediated signal transduction and CPI-17-mediated Ca2+ sensitization pathway that regulate smooth muscle contraction. Interestingly, similar changes are also present in PBOO-induced DSM compensatory response in the rabbit model in which SM-B is downregulated.

Fig. 1.

A: mean bladder weights for wild-type (WT; mean = 25 ± 0.074) and SM-B knockout (KO; mean = 33.0 ± 0.09), n = 10, P < 0.05. The average body weight of WT and KO mice was similar. B: representative photomicrographs (×40) showing 5-μm cross sections stained with Masson's trichrome for both WT and SM-B KO bladders. A much higher density of muscle fibers can be observed in the SM-B KO compared with the WT bladders with significantly less intermuscular connective tissue matrix in the KO.

Fig. 2.

A: summary data for the carbachol concentration-response curve for WT (n = 5) and SM-B KO (n = 5). The force data for each point represent the mean and SE of strips of bladder smooth muscle. There was no difference in the ED₅₀ but the E_max of 0.47 ± 0.059 g/mm² for the WT was significantly less than that of the SM-B KO (0.71 ± 0.12) g/mm², P < 0.05. B: representative tracings of the raw data for WT, SM-B KO, and when superimposed, respectively.

Fig. 3.

A: maximal response to electrical field stimulation (EFS; 70 V, 32 Hz, 1-ms duration), KCl (125 mM), PDBU (3 μM), and carbachol (20 μM). In all cases, the SM-B KO bladder strips responded with higher force than the WT bladders, P < 0.05. B: representative tracings of the summary data in A for KCl, EFS, and PDBu. Note the higher levels of force maintenance in the SM-B KO bladders compared with WT.

Fig. 4.

Western blot for PKC-α (A), CPI-17 (B), and Phospho-CPI-17 (C). PKC-α was significantly elevated in SM-B KO bladders (8.87 ± 0.34) ODUs compared with WT bladders (5.0 ± 0.43) ODUs, P < 0.05. There was no difference in the total CPI-17 level (B); however, the level of phospho-CPI-17 (C) was higher in the SM-B KO mice (5.04 ± 0.95) compared with the WT mice (2.74 ± 0.78) ODUs, n = 4, P < 0.05. D: results for ROKα, ROKβ, and RhoA. The data reveal no significant difference between WT and SM-B KO bladders.

Fig. 5.

A: time course of MLC₂₀ phosphorylation at 5, 15, and 30 s after activation with 20 μM carbachol. The mean level of phosphorylation at each time point was 26.4 ± 2.9, 30.0 ± 3.2, and 30.0 ± 3.5%, respectively, for WT bladders and 39 ± 5.1, 38 ± 4.4, and 31 ± 3.3% for SM-B KO bladders, P < 0.05, n = 4. B: representative 2-dimensional gels showing unphosphorylated and phosphorylated MLC₂₀ at each time point for WT and SM-B KO bladders, respectively.

Fig. 6.

A: total IP₃ production at rest (L₀) and when stimulated with 25 μM carbachol. The IP₃ levels were higher in the SM-B KO bladders both at rest and when activated by carbachol, P < 0.05. B: results for total IP production. There was no difference in the IP production in the absence of stimulation while carbachol (25 μM) caused an increase in the SM-B KO compared with the WT, P < 0.05.