Phosphorylation and function of cardiac myosin binding protein-C in health and disease

Abstract

During the past 5 years there has been an increasing body of literature describing the roles cardiac myosin binding protein C (cMyBP-C) phosphorylation play in regulating cardiac function and heart failure. cMyBP-C is a sarcomeric thick filament protein that interacts with titin, myosin and actin to regulate sarcomeric assembly, structure and function. Elucidating the function of cMyBP-C is clinically important because mutations in this protein have been linked to cardiomyopathy in more than sixty million people worldwide. One function of cMyBP-C is to regulate cross-bridge formation through dynamic phosphorylation by protein kinase A, protein kinase C and Ca(2+)-calmodulin-activated kinase II, suggesting that cMyBP-C phosphorylation serves as a highly coordinated point of contractile regulation. Moreover, dephosphorylation of cMyBP-C, which accelerates its degradation, has been shown to associate with the development of heart failure in mouse models and in humans. Strikingly, cMyBP-C phosphorylation presents a potential target for therapeutic development as protection against ischemic-reperfusion injury, which has been demonstrated in mouse hearts. Also, emerging evidence suggests that cMyBP-C has the potential to be used as a biomarker for diagnosing myocardial infarction. Although many aspects of cMyBP-C phosphorylation and function remain poorly understood, cMyBP-C and its phosphorylation states have significant promise as a target for therapy and for providing a better understanding of the mechanics of heart function during health and disease. In this review we discuss the most recent findings with respect to cMyBP-C phosphorylation and function and determine potential future directions to better understand the functional role of cMyBP-C and phosphorylation in sarcomeric structure, myocardial contractility and cardioprotection.

Fig. 1.

Schematic diagram of the cardiac sarcomere and arrangement of cMyBP-C. The sarcomere is the basic functional unit of a cardiac muscle’s cross-striated myofibril, which is defined as the boundary between two neighboring Z-lines. The I-band, A-band, H-zone and M-lines are shown. The thick filament protein is composed of myosin, cMyBP-C and titin. cMyBP-C is localized in the inner two-thirds of the A-band, i.e., the C-zone. cMyBP-C is oriented perpendicularly to the long axis of the myosin filaments. The two groups of 7–9 strips of cMyBP-C bands on either side of the H-zone give a characteristic doublet appearance within the A-band. The thin filament is composed of actin monomers, troponins (cTnT and cTnI) and α-TM, which is connected to nebulin in the I-bands.

Fig. 2.

General domain structure of mouse cMyBP-C and identified phosphorylated serines. There are eight Ig (oval) and three fibronectin type-3 domains (hexagonal) numbered C0 to C10. The Pro-Ala–rich region (Pro-Ala), phosphorylated M domain, a 28-amino acid cardiac-specific insertion within the C5 domain and regions that interact with actin, myosin and titin sites are shown. The M domain contains multiple phosphorylation sites that can be phosphorylated by PKA, CaMKII and PKC. The amino acid sequences are conserved between species in the M domain. Domains 7–10 participate in binding of myosin and titin, which are necessary for cMyBP-C stability and sarcomere organization.

Fig. 3.

Dynamic phosphorylation of cMyBP-C’s M domain influences myosin–actin interactions. cMyBP-C facilitates myosin–actin interactions by tethering the fibers together and regulating the ability of the myosin head to bind to the actin filament during cross-bridge cycling. cMyBP-C interacts with the LMM of myosin at the C10 domain and titin at domains C8–C10. The C1-M-C2 domains have been shown to bind to myosin and actin. The capacity of these domains to interact is critical to overall sarcomeric integrity and function. The phosphorylatable M domain facilitates the role of cMyBP-C in cross-bridge formation. In the absence of M domain phosphorylation, the C1-M-C2 domains are tightly bound to myosin S2 region (A), whereas when M domain phosphorylated, C1-M-C2 domains and M domain release their interaction with myosin S2 region and actin, respectively, and changes in the thick filament orientation occur (B). C1 domain binds to actin irrespective of the M domain phosphorylation state. The dynamic phosphorylation of the M domain provides a point of control where the ordering of myosin heads and changes in force production can be regulated. Thin filament proteins include actin, α-TM, cTnI, cTnT and calcium (Ca²⁺)-binding protein cTnC are shown.

Fig. 4.

cMyBP-C connects thick and thin filaments. To determine the localization pattern of cMyBP-C along with thick and thin filament proteins, 3-day-old neonatal rat cardiomyocytes (NRCM) were used in chamber slides In vitro. The healthy NRCM were fixed and stained with the mouse monoclonal anti-α-myosin heavy chain (MF20, University of Iowa, Iowa), rabbit polyclonal anti-C0–C1 region of cMyBP-C [74] and sheep polyclonal anti-α-TM antibodies (Millipore, Billerica, MA). Secondary Alexa Fluor 488 goat anti-mouse IgG antibody for myosin (green color), Alexa Fluor Cy5 goat anti-rabbit IgG antibody for cMyBP-C (blue color) and Alexa Fluor 568 donkey anti-sheep IgG antibody for α-TM (red color) staining were used for immunofluorescence (Molecular Probes, Carlsbad, CA). Cardiomyocytes were analyzed with fluorescent microscopy (PCM-2000 scanner, Nikon, Melville, NY) and SimplePCI software (Compix Inc., Cranberry Township, PA). The rows of figures labeled B are enlarged from the inset in the rows of figures labeled A (60x magnification). Immunofluorescent staining of cMyBP-C (blue color) shows a typical doublet pattern of normal incorporation into the sarcomere. Co-staining with cMyBP-C with either myosin (green) or α-TM (yellow) show the co-localization of cMyBP-C with myosin (blue-green) and α-TM (pink). Co-localization of myosin and α-TM (yellow) confirm the presence of interaction between myosin and thin filament region. Triple staining shows that there is a defined merging of cMyBP-C, myosin and α-TM, but the presence of excess of pink (arrow) confirms the co-localization of cMyBP-C and α-TM. Nonoverlap regions are marked with arrows in respective panels. These data demonstrate that C0–C1 domains may directly interact with thin filament proteins via α-TM and thus connect thick and thin filaments.