Rho-kinase-mediated pathway induces enhanced myosin light chain phosphorylations in a swine model of coronary artery spasm

Objective: We recently demonstrated in our swine model of coronary artery s pasm that enhanced myosin light chain (MLC) phosphorylations (both MLC mono - and diphosphorylations) play a central role in the pathogenesis of the sp asm. However, the molecular mechanism for and the phosphorylation sites for the enhanced MLC phosphorylations were unknown. In the present study, we a ddressed these points using hydroxyfasudil, a novel inhibitor of protein ki nases, which we found preferentially inhibits Rho-kinase. Methods: The spec ificity of the inhibitory effects of hydroxyfasudil on Rho-kinase, MLCK, MR CK beta and PKC were examined by kinase assay in vitro. The left porcine co ronary artery was chronically treated with interleukin-1 beta (IL-1 beta, 2 .5 mu g). Two weeks after the operation, coronary artery vasomotion was exa mined both in vivo and in vitro. MLC phosphorylations were examined by West ern blot analysis and the sites for the phosphorylations by anti-phosphoryl ated MLC antibodies that identified the monophosphorylation site as Ser19 a nd diphophorylation sites as Ser19/Thr18 of MLC. Results: Inhibitory effect s of hydroxyfasudil was at least 100 times more potent for Rho-kinase as co mpared with other protein kinases tested. Intracoronary serotonin (10 mu g/ kg) caused coronary hyperconstriction at the IL-1 beta-treated site in vivo , which was dose-dependently inhibited by hydroxyfasudil (p<0.01). The coro nary segment taken from the spastic site also showed hypercontractions to s erotonin in vitro, which were again dose-dependently inhibited by hydroxyfa sudil (p<0.01). Western blot analysis showed that MLC monophosphorylation w as significantly greater in the spastic segment than in the control segment , while MLC diphosphorylation was noted only at the spastic segment (p<0.01 ). The sites for the mono- and diphosphorylated MLC were identified as the monophosphorylated site Ser19 and diphosphorylated sites Ser19/Thr18 of MLC , respectively. Both types of MLC phosphorylations at the spastic segment w ere markedly inhibited by hydroxyfasudil (p<0.01). Conclusion: These result s indicate that hydroxyfasudil sensitive Rho-kinase-mediated pathway appear s to mediate the enhanced MLC phosphorylations (on Ser19 and Ser19/Thr18 re sidues) and plays a central role in the pathogenesis of coronary artery spa sm. (C) 1999 Elsevier Science B.V. All rights reserved.