Alanine mutation of the targeting subunit of the myosin phosphatase, MYPT1 at threonine 696 reduces cGMP-reactivity of murine femoral arteries

Abstract

The femoral artery (FA) is the largest vessel of the hind limb circulation whose proper tone-regulation ensures adequate blood supply of muscle tissue. We investigated whether alanine mutation of the targeting subunit of myosin-light-chain-phosphatase (MLCP), MYPT1 at threonine696 (MYPT1-T696A/+) affects reactivity of young and old FAs (y-FAs and o-FAs) to activation of nitric-oxide/soluble-guanylate-cyclase/protein-kinase-G cascade (NO/sGC/PKG). Contractile responses of the vessels were measured by wire myography. Phosphorylation of the regulatory-light-chain of myosin at serine19 (MLC₂₀-S19), MLCP-inhibitory subunit, MYPT1-T696, the PKG-sensitive site of MYPT1, S668 (MYPT1-S668) as well as the regulatory phosphorylation eNOS at T1177 (eNOS-T1177) were determined in arterial homogenates by western blot. In FAs from all ages and genetic groups, MYPT1-T696-mutation did not alter vascular diameter and the reactivity to the thromboxaneA₂-analogue U46619 and the RhoA-associated kinase inhibitor Y27632. By contrast, the mutation attenuated the relaxing effect of exogenous NO (DEA-NONOate) in y-FAs and the effect of a direct sGC-activation by cinaciguat in both age groups. MYPT1-T696-mutation also attenuated acetylcholine induced relaxation, but only in o-FAs. Accordingly, only in old MYPT-T696A/+-FAs alanine mutation diminished acetylcholine effect on MLC₂₀-S19- and MYPT1-T696. Interestingly, neither regulatory eNOS-T1177 phosphorylation nor MYPT1-S668 were altered by MYPT1-T696 mutation or aging. These findings suggest that alanine mutation of MYPT1-T696 diminishes the ability of NO/cGMP/PKG-system to relax FAs in old age. Our data support the view that well balanced phosphorylation of MYPT1 and in particular at the T696-residue of the protein is essential for the proper vascular reactivity, especially in elderly.