Cardiac myosin binding protein C (cMyBP-C) regulates cross-bridge cycling kinetics and thereby fine-tunes the rate of cardiac muscle contraction and relaxation. Its effects on cardiac kinetics are modified by phosphorylation. Three phosphorylation sites (Ser275, Ser284, Ser304) have been identified in vivo, all located in the cardiac-specific M-domain of cMyBP-C. However recent work has shown that up to four phosphate groups are present in human cMyBP-C.
To identify and characterize additional phosphorylation sites in human cMyBP-C.
Methods and results
Cardiac MyBP-C was semi-purified from human heart tissue. Tandem mass-spectrometry analysis identified a novel phosphorylation site on serine 133 in the proline-alanine (Pro-Ala) rich linker sequence between the C0 and C1 domains of cMyBP-C. Unlike the known sites, Ser133 was not a target of protein kinase A. In silico kinase prediction revealed glycogen synthase kinase 3β (GSK3β) as the most likely kinase to phosphorylate Ser133. In vitro incubation of the C0C2 fragment of cMyBP-C with GSK3β showed phosphorylation on Ser133. In addition, GSK3β phosphorylated Ser304, although the degree of phosphorylation was less compared to PKA-induced phosphorylation at Ser304. GSK3β treatment of single membrane-permeabilized human cardiomyocytes significantly enhanced the maximal rate of tension redevelopment.
GSK3β phosphorylates cMyBP-C on a novel site, which is positioned in the Pro-Ala rich region and increases kinetics of force development, suggesting a non-canonical role for GSK3β at the sarcomere level. Phosphorylation of Ser133 in the linker domain of cMyBP-C may be a novel mechanism to regulate sarcomere kinetics.