Cardiac myosin binding protein-C (cMyBP-C) is important in heart contractions. Mutations in cMyBP-C are known to cause familial hypertrophic cardiomyopathy, and samples taken from failing hearts have revealed cMyBP-C becomes dephosphorylated, although the exact causes have not been studied.1 To better understand which kinases are involved with cMyBP-C phosphorylation, Kooij et al. (2013) sought to identify cMyBP-C phosphorylation sites in human heart tissue.2 They obtained samples from participants with explanted donor hearts and from patients exhibiting end-stage heart failure, including those with idiopathic dilated cardiomyopathy.
In the Journal of Molecular and Cellular Cardiology, Kooij et al. describe the two methods they employed to characterize cMyBP-C. In method one, researchers subjected pooled samples to strong-cation exchange chromatography fractionation. In the second method, the team used sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). To enrich the tryptic phosphopeptides, all samples were subjected to titanium dioxide affinity chromatography. Samples were analyzed using high resolution mass spectrometry on an LTQ-Orbitrap (Thermo Scientific) mass spectrometer.
Results revealed the N-terminus side of cMyBP-C was extensively phosphorylated. Seventeen phosphorylation sites were identified in vivo, including nine sites that had not been identified previously. Sixteen phosphorylation sites were also identified in an in vitro kinase incubation. The strong-cation exchange method identified more phosphorylation sites in the cMyBP-C phosphoproteome when compared with the results of SDS-PAGE. The Kooij group suggests that the discrepancy in the identified phosphorylation sites may be caused by a difference in the amount of tissue required by each method, with 1 mg of pooled protein used in the strong-cation exchange, versus just 30 µg in gel electrophoresis. However, both methods were able to identify the three most common phosphorylation sites: Ser284, Ser286 and Thr290.
In samples taken from individuals with healthy hearts, Ser284 was the most highly phosphorylated, yet in failing hearts, phosphorylation at the Ser284 site was decreased. A statistical analysis implicated Ser284 (*p < 0.05) as important for regulating heart contractions. Ser286 and Thr290 were also among the most highly phosphorylated and were located near each other in the regulatory M-domain of cMyBP-C; they may be important for regulating actin binding. Although more research is needed, this work represents a step toward discovering how cMyBP-C dephosphorylation may be related to heart failure.
1. James, J., and Robbins, J. (2011) “Signaling and myosin-binding protein C,” The Journal of Biological Chemistry, 286(12) (pp. 9913–19).
2. Kooij, V., et al. (2013, July) “Characterization of the cardiac myosin binding protein-C phosphoproteome in healthy and failing human hearts,” Journal of Molecular and Cellular Cardiology, 60 (pp. 116–20).
Post Author: Emily Humphreys. As an undergraduate studying biology at the University of Utah, Emily balanced a heavy class schedule while working long hours in a lab studying eye development. Following graduation, she became involved in infectious disease and aging research involving SNPS.
While she enjoyed the thrill of research, Emily has since traded bench work for science journalism.
She has spent the last year writing about new developments involved in proteomics research, and now food testing.
When she isn’t writing,she can be found playing outside with her kids.