Heart muscle contractility and performance are controlled by posttranslational modifications of sarcomeric proteins. Although myosin regulatory light chain (RLC) phosphorylation has been studied extensively in vitro and in vivo, the precise role of cardiac myosin light chain kinase (cMLCK), the primary kinase acting upon RLC, in the regulation of cardiomyocyte contractility remains poorly understood. In this study, using recombinantly expressed and purified proteins, various analytical methods, in vitro and in situ kinase assays, and mechanical measurements in isolated ventricular trabeculae, we demonstrate that human cMLCK is not a dedicated kinase for RLC but can phosphorylate other sarcomeric proteins with well-characterized regulatory functions. We show that cMLCK specifically monophosphorylates Ser23 of human cardiac troponin I (cTnI) in isolation and in the trimeric troponin complex in vitro and in situ in the native environment of the muscle myofilament lattice. Moreover, we observed that human cMLCK phosphorylates rodent cTnI to a much smaller extent in vitro and in situ, suggesting species-specific adaptation of cMLCK. Although cMLCK treatment of ventricular trabeculae exchanged with rat or human troponin increased their cross-bridge kinetics, the increase in sensitivity of myofilaments to calcium was significantly blunted by human TnI, suggesting that human cTnI phosphorylation by cMLCK modifies the functional consequences of RLC phosphorylation. We propose that cMLCK-mediated phosphorylation of TnI is functionally significant and represents a critical signaling pathway that coordinates the regulatory states of thick and thin filaments in both physiological and potentially pathophysiological conditions of the heart.
|Number of pages||13|
|Journal||Journal of Biological Chemistry|
|State||Published - Apr 3 2020|
Bibliographical noteFunding Information:
This study was supported by British Heart Foundation Basic Science Interme-diate Fellowship FS/16/3/31887 (to T. K.), Senior Basic Science Fellowship FS/15/1/31071 (to Y.-B. S.), and British Heart Foundation Chair of Molecular Cardiology CH/08/001 (to M. G. and B. B.) The authors declare that they have no conflicts of interest with the contents of this article.
© 2020 Sevrieva et al. Published by The American Society for Biochemistry and Molecular Biology, Inc.
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology