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

PLoS One. 2015 Oct 29;10(10):e0141130. doi: 10.1371/journal.pone.0141130. eCollection 2015.

Abstract

Background & aims: Cardiac 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 results: By 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%.

Conclusions: We 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing
  • Amino Acid Sequence
  • Animals
  • Cardiac Myosins / metabolism*
  • Gene Expression
  • Gene Order
  • Isoenzymes
  • Male
  • Mice
  • Molecular Sequence Data
  • Myocardial Contraction*
  • Myocardium / metabolism
  • Myocytes, Cardiac / metabolism
  • Myosin-Light-Chain Kinase / chemistry
  • Myosin-Light-Chain Kinase / genetics
  • Myosin-Light-Chain Kinase / metabolism*
  • Phosphorylation
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Rats
  • Receptors, Adrenergic, alpha-1 / metabolism*
  • Sequence Alignment
  • Species Specificity
  • Ventricular Function

Substances

  • Isoenzymes
  • RNA, Messenger
  • Receptors, Adrenergic, alpha-1
  • Myosin-Light-Chain Kinase
  • Cardiac Myosins

Grant support

This work was supported in part grants-in-aids for Scientific Research from the Ministry of Education, Science, Technology, Sports and Culture, Japan (19390211 to M.I., 25460655 to T.I., 24591113 to R.O.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.