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

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 Ca ²⁺ 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.