Journal
FASEB JOURNAL
Volume 32, Issue 4, Pages 2095-2109Publisher
FEDERATION AMER SOC EXP BIOL
DOI: 10.1096/fj.201700794R
Keywords
CRISPR/Cas9; aorta; radio telemetry; PKC; MYPT1
Categories
Funding
- Ministry of Education, Japan [24248050, 25221205, 26712025]
- Salt Science Research Foundation [1645]
- Grant for Joint Research Project of the Institute of Medical Science, The University of Tokyo [235]
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PKC-potentiated phosphorylation-dependent inhibitory protein of protein phosphatase 1 (CPI-17), an endogenous myosin phosphatase inhibitory protein, is considered a key molecule for Ca2+ sensitization of the contractile apparatus. Here, we have used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 to generate CPI-17-deficient [knockout (KO)] and threonine 38 (T38)-phospho-resistant mice [threonine mutant into alanine (TA)], and then effects of CPI-17 on vascular contractility in vitro and mean blood pressure (MBP) in vivo were investigated. In isolated thoracic aorta, phorbol 12, 13-dibutyrate induced a sustained contraction of wild-type (WT) mice, whereas no contraction showed from TA or KO mice. A high concentration of KCl solution-induced contraction was not different between transgenic and WT mice. In contrast, phenylephrine (PE)-induced contractions in both mutant strains were significantly smaller than those of WT mice in association with a low level of myosin phosphorylation, suggesting that at least part of PE-induced contraction is regulated by phosphorylation of CPI-17 at T38. Finally, the physiologic role of CPI-17 in the regulation of blood pressure was investigated using radio telemetry. MBP was decreased significantly in both transgenic mice, even with a compensatory increase in heart rate. In summary, we generated KO and constitutively phospho-resistant mouse models of CPI-17 for the first time. p-CPI-17 at T38, possibly by PKC, could be important to maintain vascular contractility and blood pressure in vivo.
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