Hypertrophic cardiomyopathy disease results from disparate impairments of cardiac myosin function and auto-inhibition

Abstract

Hypertrophic cardiomyopathies (HCM) result from distinct single-point mutations in sarcomeric proteins that lead to muscle hypercontractility. While different models account for a pathological increase in the power output, clear understanding of the molecular basis of dysfunction in HCM is the mandatory next step to improve current treatments. Here, we present an optimized quasi-atomic model of the sequestered state of cardiac myosin coupled to X-ray crystallography and in silico analysis of the mechanical compliance of the lever arm, allowing the systematic study of a large set of HCM mutations and the definition of different mutation classes based on their effects on lever arm compliance, sequestered state stability, and motor functions. The present work reconciles previous models and explains how distinct HCM mutations can have disparate effects on the motor mechano-chemical parameters and yet lead to the same disease. The framework presented here can guide future investigations aiming at finding HCM treatments.

Fig. 1

Myosin mesa and sequestered state. a Schematic representation of the motor cycle and the regulation of the β-cardiac myosin activity. On the left, when the motor detaches from the track upon ATP binding, the motor adopts the post-rigor (PR) state in which the lever arm is down and the motor has poor affinity for F-actin. During the recovery stroke, repriming of the lever arm leads to the pre-powerstroke (PPS) state in which hydrolysis can occur. The swing of the lever arm (powerstroke) upon reattachment of the motor to F-actin is coupled with the release of hydrolysis products. The nucletotide-free or rigor state has the highest affinity for F-actin. On the right, scheme of the sequestered state that is formed during relaxation. According to the mesa hypothesis, HCM mutations disrupt the sequestered state, increasing the number of myosin heads available to produce force. According to the Hypercontractile hypothesis, HCM mutations alter the myosin motor activity. b The mesa (purple dashed lines) is a long and flat surface of the myosin head composed of several myosin subdomains conserved in myosin IIs. c Electron density map of the human cardiac filament obtained from negative staining (EMDB code EMD-2240). In the relaxed state, interactions between the blocked head (BH) and the free head (FH) of the myosin 2 dimer stabilize an asymmetric configuration. Extra densities of the filament correspond to other components of the thick filament, the cardiac myosin MyBP-C, and titin. Location of the mesa for each head indicates that the FH mesa (*) interacts with the BH while the BH mesa (#) is buried and interacts with components of the thick filament

Fig. 2

Crystal structures of β-cardiac myosin and description of the converter/ELC interface. a X-ray structure of β-cardiac myosin S1 complexed with MgADP in the post-rigor state (PR-S1). b Interface between the converter and the ELC as found in the PR state. This interface involves mainly electrostatic interactions between a cluster of negatively charged residues of the ELC (D136; E135 and E139) and positively charged residues of the heavy chain (R723 and R780). Side chains of interacting residues (sticks) and polar interactions (yellow lines) are represented. Four converter mutations studied in this work (R719W; R723G; I736T; G741R) are colored in light blue. c Structure alignment of converters of the Myosin 2 superfamily. MYH7: bovine (Bos taurus) β-cardiac myosin; ScMyo2: bay scallop (Argopecten irradians) Myosin 2; SqMyo2: longfin inshore squid (Dorytheutis pealeii) Myosin 2; SmMyo2: chicken (Gallus gallus) gizzard smooth muscle myosin 2. d Superimposition of several Myo2 converter structures: from PR-S1 colored in green; from SqMyo2 (PDB code 3I5F, pink); from SmMyo2 (PDB code 1BR1, orange); from ScMyo2 in the PPS (PDB code 1QVI, purple) and Rigor (PDB code 1SR6, cyan) states. e Location of the top loop (deep teal blue) and the side loop (sand yellow) in the cartoon representation of the crystal structure of the β-cardiac myosin PR-S1 (gray) with the converter colored in light green and the ELC colored in light pink. The top loop is part of the converter/ELC interface

Fig. 3

Dynamics of the converter/ELC interface. Schematic representation of the results from the molecular dynamics simulations for the wild-type (WT) and the three HCM mutants that have been analyzed in silico: R719W, R723G, G741R. On the left, a “putty representation” of the β-cardiac myosin converter and first IQ (aa 701–806) bound to the ELC. RMS fluctuations during 30 ns simulations are represented with RMS. Scale ranging from 0.6 (in yellow) to 5.8 Å (in red). On the center, the structure of the interface between the ELC, the converter, and the pliant region is represented, as well as the position of key residues maintaining the interface and its plasticity. In each structure, the position of the residue mutated is labeled in red. On the right, a schematic representation of the region containing the converter, the ELC, and the lever arm is displayed. The different populations of the top loop allowed by the dynamics of this region are drawn and the nature of the interactions between the converter and the ELC is also represented. In each case a state is represented opaque and the others are in transparency in order to best compare the different populations (positions of the top loop are colored differently). On the center and on the left, the myosin subdomains are colored differently: the converter in green; the IQ region in cyan; and the ELC in light pink. a WT, b R719W, c R723G, d G741R

Fig. 4

An optimized model of the sequestered state of β-cardiac myosin. a β-cardiac myosin sequestered state modeled from the cardiac myosin S1 PPS structure. This model results from optimization of the intra-head interactions that occur upon formation of the IHM with two asymmetric heads: the free head (FH) and the blocked head (BH). b Detailed analysis of a region of the interface involving the FH-converter and the BH-U50 subdomain. Note that the top loop plays a major role. c Surface of the FH-mesa and the FH-converter involved in interactions with the BH. d Surface of the BH-mesa and the BH-converter involved in interactions with the FH. A putative surface of interaction with the partner MyBP-C is also represented. The regions of the interface that are part of the mesa are labeled with a * and a # on the FH and on the BH, respectively. e Schematic representation analyzing the prerequisites to form the IHM. On top, different myosin conformations illustrate the differences in position of the lever arm in the PR and the PPS states. Two heads in the PPS conformation are shown in the sequestered state. On the center, a schematic representation of the two states adopted by the heavy meromyosin (HMM) is shown. On the bottom, the different conformations of the top loop present in the PR crystal structure and in the BH and the FH of the sequestered state are displayed in cartoon representation. To form the sequestered state, the lever arms of the FH and BH heads must adopt conformations, in particular for the top loop. Note, however, that the pliant region remains close to the conformation of the WT PPS structure. Dynamics at the ELC/converter interface of the two heads of the myosin dimer is thus important to promote sequestration of the heads

Fig. 5

Structural and functional consequences of 178 HCM mutations. a The chart pie represents the proportion of mutations belonging to six classes depending on their effect on the structure, the function and the stability of the IHM. These six classes are: mutations destabilizing the IHM (green); mutations destabilizing the IHM and the motor function (yellow); mutations destabilizing the PPS conformation and the IHM (orange); mutations that alter the protein function (blue); mutations that alter the protein function and the protein stability (purple); and mutations that mildly affect protein function or stability (black). b Positions of the HCM mutations on the PPS structure of the β-cardiac myosin (PDB code: 5N69). Mutations are represented as balls colored depending on their predicted consequences, following the color code defined in a. c Cartoon representation of the PPS structure of the β-cardiac myosin with all the subdomains colored. TR transducer, ELC essential light chain

Fig. 6

Antagonistic effects of omecamtiv mecarbil (OM) and blebbistatin (BS) on the sequestered state. Schematic representation of the effects of the inhibitor BS and the activator OM. On top: BS occupies a pocket within the motor domain core, close to the active site. Occupation of this pocket by BS allows to stabilize the PPS conformation without any effect on the local compliance of the lever arm required to form the sequestered state. Thus, BS binding is compatible with the IHM and favors the formation of the sequestered state. On the bottom: OM occupies a pocket at the interface between the converter and the N-ter subdomain. This pocket is expected to decrease the compliance of the converter/ELC interface and constrain the lever arm in a particularly primed position. There is thus a loss in the compliance required to form the sequestered state if OM is bound to the heads of the Myo2 dimer. This explains why OM is incompatible with the formation of the sequestered state