New Isoform of Cardiac Myosin Light Chain Kinase and the Role of Cardiac Myosin Phosphorylation in α1-Adrenoceptor Mediated Inotropic Response

Abstract

Background & AimsCardiac myosin light chain kinase (cMLCK) plays an obligatory role in maintaining the phosphorylation levels of regulatory myosin light chain (MLC2), which is thought to be crucial for regulation of cardiac function. To test this hypothesis, the role played by ventricular MLC2 (MLC2v) phosphorylation was investigated in the phenylephrine-induced increase in twitch tension using the naturally-occurring mouse strain, C57BL/6N, in which cMLCK is down regulated.Methods and ResultsBy Western blot and nanoLC-MS/MS analysis, cMLCKs with molecular mass of 61-kDa (cMLCK-2) and/or 86-kDa were identified in mice heart. Among various mouse strains, C57BL/6N expressed cMLCK-2 alone and the closest relative strain C57BL/6J expressed both cMLCKs. The levels of MLC2v phosphorylation was significantly lower in C57BL/6N than in C57BL/6J. The papillary muscle twitch tension induced by electrical field stimulation was smaller in C57BL/6N than C57BL/6J. Phenylephrine had no effect on MLC2v phosphorylation in either strains but increased the twitch tension more potently in C57BL/6J than in C57BL/6N. Calyculin A increased papillary muscle MLC2v phosphorylation to a similar extent in both strains but increased the phenylephrine-induced inotropic response only in C57BL/6N. There was a significant positive correlation between the phenylephrine-induced inotropic response and the levels of MLC2v phosphorylation within ranges of 15–30%.ConclusionsWe identified a new isoform of cMLCK with a molecular mass of 61kDa(cMLCK-2) in mouse heart. In the C57BL/6N strain, only cMLCK-2 was expressed and the basal MLC2v phosphorylation levels and the phenylephrine-induced inotropic response were both smaller. We suggest that a lower phenylephrine-induced inotropic response may be caused by the lower basal MLC2v phosphorylation levels in this strain.

Mylk3null C57BL/6N mice develop cardiomyopathy, whereasNntnull C57BL/6J mice do not

“…Instead, we identify a mutation in Mylk3 in B6N mice that abolishes protein expression and likely causes DCM, whereas MYLK3-replete B6J mice are unaffected. Interestingly, the same Mylk3 variant (g.chr8:85365179A>T; c.-5T>A) was previously described and, although that study found no evidence of DCM at an early time point (16 wk), it did show the B6N heart papillary muscles contracted with less force than those from B6J hearts (44).…”

“…A previous study generated Mylk3 −/− mice and showed they develop a similar phenotype of dilated ventricles, reduced fractional shortening, and reduced ejection fraction, whereas their phenotype is similarly nonlethal (28). As the cardiac isoform of the family of myosin light chain kinases, MYLK3 phosphorylates MYL2 and potentially autophosphorylates itself (44,48). MYLK3 expression is also important for sarcomere assembly.…”

Thick Filament Protein Network, Functions, and Disease Association

“…Thus, the differential phosphorylation profile of MYL2 in response to β1-adrenergic stimulation may be due to the different regions examined in the two studies. Moreover, the phosphorylation status of MYL2 is not altered in response to α1-adrenergic signaling (200, 547). …”

Role of myosin light chain phosphatase in cardiac physiology and pathophysiology

“…Thus, care must be used to prevent RLC dephosphorylation when extracting RLC from quick-frozen heart muscle. A standard method of minimizing protein dephosphorylation [21, 29, 30, 34, 41, 46, 75-77], involves thawing frozen powdered heart in 10% trichloroacetic acid to denature and precipitate all proteins in the tissue sample, including kinases, phosphatases, and proteases [78]. …”