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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24:464.)
© 2004 American Heart Association, Inc.

Vascular Biology

Transduction of the N-Terminal Fragments of MYPT1 Enhances Myofilament Ca²⁺ Sensitivity in an Intact Coronary Artery

Katsuya Hirano; Dmitry N. Derkach; Mayumi Hirano; Junji Nishimura; Shosuke Takahashi; Hideo Kanaide

From the Division of Molecular Cardiology, Research Institute of Angiocardiology (K.H., D.N.D., M.H., J.N., H.K.), the Department of Anesthesia and Critical Care Medicine (S.T.), and the Kyushu University COE Program on Lifestyle-Related Diseases (H.K.), Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

Correspondence to Dr Hideo Kanaide, Professor, Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail kanaide{at}molcar.med.kyushu-u.ac.jp

Objective— The region of the 110 kDa regulatory subunit (MYPT1) of smooth muscle myosin phosphatase involved in the regulation of contraction was determined under physiological conditions.

Methods and Results— Using HIV Tat protein-mediated protein transduction, the N-terminal fragments of MYPT1 were introduced to the intact porcine coronary arterial strips. Pre-incubation with 3 µmol/L TAT-MYPT1^1–374, a construct containing the Tat peptide and the residues 1 to 374 of MYPT1, for 15 minutes augmented (2.4-fold) the subsequent contraction induced by adding 1.25 mmol/L of extracellular Ca²⁺ under 118 mmol/L K⁺ depolarization, with no augmentation of the [Ca²⁺]_i elevation. The deletion of the Tat peptide, MYPT1^1–374, abolished the augmenting effect. TAT-MYPT1^1–296 demonstrated a weaker but significant augmentation (1.7-fold). However, TAT-MYPT1^1–171, TAT-MYPT1^39–374, TAT-MYPT1^39–296, and TAT-MYPT1^297–374 had no augmenting activity. The myosin light chain phosphorylation level as a function of extracellular Ca²⁺ concentrations was shifted to the left in the strips pretreated with TAT-MYPT1^1–374 compared with the control.

Conclusions— Region 1 to 296 was the minimal region involved in the enhancement of contraction, and region 297 to 374 played a supplemental role. These results suggested that the interaction mainly between catalytic subunit and MYPT1 play a critical role in the regulation of the endogenous myosin phosphatase in intact smooth muscle.

Key Words: myosin • phosphatase • smooth muscle • contraction • protein transduction

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