T18/S19 diphosphorylation of myosin regulatory light chain impairs pulmonary artery relaxation in monocrotaline-induced pulmonary hypertensive rats

Abstract

Abstract Phosphorylation of Ser19 (S19-p) on the myosin regulatory light chain (MLC2) is critical for arterial contraction. It has shown that elevated rho A-dependent kinase (ROCK) activity or decreased MLC phosphatase (MLCP) activity leads to further phosphorylation of Thr18 (T18/S19-pp), which has been linked to vasospastic diseases. However, this phenomenon has not yet been studied in the context of pulmonary arterial hypertension (PAH). In monocrotaline-induced PAH (PAH-MCT) rat model, we observed a significant delay in pulmonary artery (PA) relaxation following high potassium-induced contraction, which persisted even with the use of a L-type calcium channel blocker or in a calcium-free solution. Immunoblot analysis showed increased levels of both S19-p and T18/S19-pp in unstimulated PAs from PAH-MCT rats. Proteomics analysis revealed a reduction in soluble guanylate cyclase (sGC) and protein kinase G (PKG) levels, and immunoblotting confirmed decreased levels of MYPT1 (a component of MLCP) and increased ROCK in PAH-MCT. In the control PAs, the pharmacological inhibition of sGC with ODQ resulted in a prominent delay of relaxation and increased T18/S19-pp as like PAH-MCT. The delayed relaxation and the T18/S19-pp in PAH-MCT was reversed by ROCK inhibitor, Y27632, while not by membrane permeable 8-Br-cGMP. The delayed relaxation and T18/S19-diP in the ODQ-treated control PA were also reversed by Y27632. Taken together, the increased T18/S19-pp leads to the decreased ability of PA to relax in PAH-MCT rats. This is caused by the decreased sGC and MLCP, and increased ROCK. Targeting and inhibiting ROCK may therefore be an effective strategy for pharmacological treatment of PAH.