The important role of phosphorylation of CPI-17 at threonine 38 on vascular smooth muscle contraction and maintain normal blood pressure using CPI-17 genetically modified mice

Abstract

Background: CPI-17, an endogenous myosin phosphatase inhibitory protein(Protein kinase C activated phosphatase inhibitor), is considered a key molecule for Ca2+-sensitization of contractile apparatus. However, it is still unclear how CPI-17 plays an important role for blood pressure regulation in vivo. Here, we successfully generated CPI-17 KO mice (KO) and phospho-inactive mutant CPI-17 at threonine 38 to alanine knocked-in mice (TA) using CRISPR/Cas 9 system. Aim: We tried to clarify physiological functions of CPI-17 on vascular contractility and blood pressure using these genetically modified mice. Methods: Smooth muscle contraction of isolated thoracic aorta was isometrically measured. Phosphorylations of Myosin light chain (MLC), CPI-17 and MYPT1 were measured by Western blot. Mean blood pressure (MBP) was measured by telemetry system. Results: High concentration of KCl (High K+)-induced contractions and MLC-phosphorylation were not different in the aorta isolated from wild-type (WT), KO and TA mice. Phorbol 12, 13-dibutyrate (PDBu) induced a sustained contraction in WT aorta with significant increment of phosphorylation of CPI-17 and MLC, whereas no contraction showed in TA and KO aorta. Phenylephrine (PE)-induced contraction in WT aorta was significantly stronger than KO and TA aorta in accordance with changes in MLC phosphorylation. The phosphorylation level of MYPT1 at T853 was tended to increase in WT, KO and TA aorta, when the muscle was stimulated with PE. The phosphorylation level of MYPT1 at T696 was phosphorylated even in the resting state and did not change by stimulation with PE in WT, KO and TA aorta. MBP was significantly decreased in KO and TA mice even with a compensatory increase in heart rate. Conclusion: Phosphorylation of CPI-17 at threonine 38 was essential for force generation of vascular tissue and maintenance of physiological blood pressure in vivo.