Cardiac myosin regulatory light chain kinase modulates cardiac contractility by phosphorylating both myosin regulatory light chain and troponin I: cMLCK phosphorylates human cardiac troponin I

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Abstract

Heart muscle contractility and performance are controlled by post-translational modifications of sarcomeric proteins. Although myosin regulatory light chain (RLC) phosphorylation has been extensively studied both 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 the current 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 mono-phosphorylates Ser-23 of human cardiac troponin I (cTnI) both 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 both in vitro and in situ, suggesting a species-specific adaptation of cMLCK. Although cMLCK treatment of ventricular trabeculae exchanged with either 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.
Original languageEnglish
JournalJournal of Biological Chemistry
Publication statusAccepted/In press - 21 Feb 2020

Keywords

  • cardiac muscle regulation
  • cardiac myosin light chain kinase
  • troponin I phosphorylation
  • cardiomyocyte
  • heart function
  • posttranslational modification (PTM)
  • calcium signaling
  • myosin regulatory light chain (RLC)

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