The HCM-causing Y235S cMyBPC mutation accelerates contractile function by altering C1 domain structure

Abstract

Mutations in cardiac myosin binding protein C (cMyBPC) are a major cause of hypertrophic cardiomyopathy (HCM). In particular, a single amino acid substitution of tyrosine to serine at residue 237 in humans (residue 235 in mice) has been linked to HCM with strong disease association. Although cMyBPC truncations, deletions and insertions, and frame shift mutations have been studied, relatively little is known about the functional consequences of missense mutations in cMyBPC. In this study, we characterized the functional and structural effects of the HCM-causing Y235S mutation by performing mechanical experiments and molecular dynamics simulations (MDS). cMyBPC null mouse myocardium was virally transfected with wild-type (WT) or Y235S cMyBPC (KO^Y235S). We found that Y235S cMyBPC was properly expressed and incorporated into the cardiac sarcomere, suggesting that the mechanism of disease of the Y235S mutation is not haploinsufficiency or poison peptides. Mechanical experiments in detergent-skinned myocardium isolated from KO^Y235S hearts revealed hypercontractile behavior compared to KO^WT hearts, evidenced by accelerated cross-bridge kinetics and increased Ca²⁺ sensitivity of force generation. In addition, MDS revealed that the Y235S mutation causes alterations in important intramolecular interactions, surface conformations, and electrostatic potential of the C1 domain of cMyBPC. Our combined in vitro and in silico data suggest that the Y235S mutation directly disrupts internal and surface properties of the C1 domain of cMyBPC, which potentially alters its ligand-binding interactions. These molecular changes may underlie the mechanism for hypercontractile cross-bridge behavior, which ultimately results in the development of cardiac hypertrophy and in vivo cardiac dysfunction.

Keywords: Cardiac muscle; Contractile function; Cross-bridge kinetics; In silico modeling; Molecular dynamics simulation.

Figure 1.. Multiple sequence alignment of cardiac isoform of myosin binding protein C from 25 species with corresponding NCBI and UniProtKB Accession IDs.

Homo sapiens (human, AAI51212.1), Mus musculus (house mouse, O70468), Aotus nancymaae (Nancy Ma's night monkey, XP_021521618.1), Bos taurus (cattle, AAI19826.1), Canis lupus familiaris (dog, ABB72024.1), Carlito syrichta (Philippine tarsier, XP_021564270.1), Castor canadensis (North American beaver, XP_020034741.1), Cavia porcellus (guinea pig, XP_003465195.2), Danio rerio (zebrafish, NP_001037814.2), Delphinapterus leucas (beluga whale, XP_022447963.1), Enhydra lutris kenyoni (sea otter, XP_022362351.1), Felis catus (cat, XP_019667955.1), Gallus gallus (red junglefowl, NP_990447.2), Ictidomys tridecemlineatus (thirteen-lined ground squirrel, XP_021585283.1), Labrus bergylta (Ballan wrasse, XP_020488153.1), Meriones unguiculatus (Mongolian gerbil, XP_021496537.1), Myotis davidii (vesper bat, ELK23828.1), Neomonachus schauinslandi (Hawaiian monk seal, XP_021540477.1), Pan paniscus (bonobo, XP_003815230.1), Physeter catodon (sperm whale, XP_007113025.1), Rattus norvegicus (brown rat, NP_001099960.1), Rhincodon typus (whale shark, XP_020385793.1), Trichechus manatuslatirostris (West Indian manatee, XP_004387787.1), Xenopus laevis (African clawed frog, AAL15871.1) and Xenopus tropicalis (Western clawed frog, NP_001106379.1). Region of homology modeling using 3CX2 is shown with a double sided black arrow. Secondary structures are shown on top of the alignment with colored arrows (β sheets) and rectangles (3¹⁰-helix). The tyrosine residue 235 is conserved 100% across 25 species.

Figure 2.. Determination of protein expression and PKA-mediated phosphorylation of cMyBPC and other sarcomeric proteins.

(a) Representative Western blot and quantification of cMyBPC showing total protein expression. (b) Representative gels shown are stained by Pro-Q (left) for total protein phosphorylation, and the same gel is shown for total protein level (right) stained with Coomassie Blue. Relative protein phosphorylation (phosphorylated signal/total protein signal) was calculated for each protein and is expressed as % of NTG -PKA values for that protein. Values are expressed as mean ± S.E.M., from four hearts in each group. * P <0.05, different from non–PKA-treated samples from the same group.

Figure 3.. Immunohistochemical analysis.

Confocal microscopy (×96 magnification) was used to demonstrate the localization of α-actinin (red) and cMyBPC (green) in non-transgenic (NTG), cMyBPC ^−/− (KO), and cMyBPC^−/− myocardium treated with lentiviruses encoding the wild type cMyBPC (KO^WT) and Y235S mutant cMyBPC (KO^Y235S). Scale bar: 5 μm.

Figure 4.. Force-pCa relationship between KO^WT and KO^Y235S myocardium.

The KO^Y235S myocardium shows a left-ward shift in the pCa curve, indicating increased calcium sensitivity. A total of 15 skinned myocardial preparations (3 fibers each from 5 hearts) were used for both groups.

Figure 5.. Structural parameters of the C1 domain.

(a) Representative cartoon structure of mouse C1 domain template-based homology model created using the 3CX2 X-ray crystallographic structure. The first and last residue of the domain, N- and C- terminal ends, secondary structure nomenclature and residue Y235 of the C1 domain are shown. PyMOL was used to render the image. (b) 500 ns RMSD trajectory plot and Gaussian distribution of wild type (WT) and mutant (Y235S) C1 domain homology model of mouse cMyBPC. Only hydrogen atoms were excluded from the RMSD calculation. MDS of both models show gradual equilibration and stabilization to about 3.0 A. The structures were stable until the end of the simulation. Various RMSD peaks indicate natural fluctuations due to sidechain movements. (c) Averaged RMSF per residue of C1 domain. Highlighted residues are statistically significant. Values are expressed as mean ± S.D., * P<0.05. VMD was used to obtain data and render the image.

Figure 6.. Representative intramolecular hydrogen bond and hydrophobic interactions.

WT and Y235S models are shown in gray and red cartoon representations, respectively. Hydrogen bonding interactions of Y235 in (a) WT and (b) Y235S models are shown. The connection between S235 and S231 is broken in the Y235S mutant model. The VDW radius of those two residues are also shown as a wire mesh, colored by atom charge. Green dashes indicate conventional hydrogen bonds (distance threshold=2.5Å). The intricate networks of hydrophobic interactions are represented in (c) WT and (d) Y235S models. It is zoomed into the hydrophobic core of the C1 domain. Key residues are shown as stick figures. Pink dashes indicate alkyl-alkyl or pi-alkyl hydrophobic interactions (distance threshold=0.5Å of van der Waals radii) and blue dashes indicate pi-pi or amide-pi stacking interactions (distance threshold=6.5A). Distance measurements defined by the RING2.0 software were used. Many of the core-stabilizing interactions are ablated in the Y235S model.

Figure 7.. Three representative intramolecular salt bridge trajectory plots for K188-D196, R236-E238, and R158-D246.

The left panels show combined WT and Y235S trajectory plots of salt bridge bond distance vs. time. The dotted yellow lines at 4Å indicate the upper boundary of salt bridge formation. The right panels show the 3D positions of the above salt bridges K188-D196, R236-E238, and R158-D246. The residues are labeled and colored in CPK.

Figure 8.. Secondary structure profile.

A sample secondary structure per residue (spanning residues D149 to E256) for the whole 500 ns trajectory graphed in VMD plug-in Timeline, with DSSP classification of secondary structure of WT (left) and Y235S (right). The black ovals show transient changes as well as changes in residues T229 to G233 and F250 to L252 in the Y235S model. VMD was used to obtain data and render the image.

Figure 9.. C1 domain surface features and domain conformation

(a) Averaged SASA per residues spanning D149 to E256 for the WT and Y235S C1 domains. MDS results showed that there were 11 residues with significant changes in SASA as a result of the Y235S mutation. Values are expressed as mean ± S.D., * P<0.05. (b) A volume slice image of WT (left) and Y235S (right). It shows the solvent accessible volume bulging into the hydrophobic core of the C1 domain. Residue 235 is shown in VDW. VMD was used to obtain data and render the image.

Figure 10.. Representative electrostatic potentials maps and potentials

(a) Electrostatic potential mapped onto solvent accessible surface for WT (upper left) and Y235S mutant (upper right) C-terminal end of C1 domains. The corresponding cartoon representation of WT (gray) and Y235S (red) are shown in the lower images. (b) Positive (top) and negative (bottom) electrostatic isocontour images of WT and Y235S overlaid on SASA. N-terminal end of C1 domain is on the left side of each image. (c) A representative image of electric field lines of WT and Y235S C-terminal ends. Blue, white, and red represent positive, neutral, and negative charges, respectively. Backbone structure of WT (gray) and Y235S (red) are shown for the electric potential, isocontour and field lines. All three figures show that there are alterations of charge density and electric potential in the C-terminal end of the mutant Y235S model.

Figure 11.. Theoretical model of the altered XB kinetics due to the Y235S mutation.

Left panels show the XB formation of the WT cMyBPC: 1). In the relaxation phase, cMyBPC inhibits XB formation by tethering to myosin S1 or S2. 2). Potentiation of the thin filament causes some myosin molecules to bind actin weakly. cMyBPC aids in the thin filament activation but the XB recruitment rate is kept at a low rate due to cMyBPC’s initial inhibition of myosin. 3). At high levels of activation, cMyBPC interacts with actin to open myosin binding site on actin and myosin molecules undergo cycles of power strokes. However, the drag on the thin filament by cMyBPC decreases shortening velocity, slows cycling rate and increases XB stiffness. 4). Myosin detaches from the thin filament, post power stroke. The detachment rate is slowed because cMyBPC is still attached to actin, keeping the myosin binding sites open. 5). The XB’s return to the relaxation phase where myosin is inhibited by cMyBPC and actin filament is deactivated. Right panels show the corresponding phases of XB formation in the Y235S cMyBPC: 1). With the introduction of the Y235S mutation in the C1 domain of cMyBPC, proper interactions with myosin S1 and S2 may be ablated. Therefore, cMyBPC does not properly bind and inhibit myosin S1 or S2. Consequently, myosin molecules may be physically closer to actin, priming them for faster and greater magnitude of XB formation. 2). At low levels of Ca²⁺ activation, we see an increase in the rate of XB recruitment (k_df) of myosin in the mutant Y235S cMyBPC, because its inhibition on myosin is lifted. Although cMyBPC is also not able to properly interact with actin, we see an increase in pCa₅₀ and decrease in nH (increase in Ca²⁺ sensitivity) because Ca²⁺ and myosin are more potent activators of the thin filament. 3). At high Ca²⁺ levels, the lack of cMyBPC-actin interactions lead to increased force generation and accelerated cycling rate, increased shortening velocity, and decreased XB stiffness. 4). After the power stroke, cMyBPC is unable to keep myosin binding sites open for myosin to sustain the interaction with actin, contributing to an accelerated XB detachment (k_rel), as well as increased magnitude of XB detachment. 5). The XBs are unbound and return to their relaxed states. However, the myosin molecules are not properly inhibited by cMyBPC so that they are again primed for subsequent XB formation.