Hypertrophic cardiomyopathy and the myosin mesa: viewing an old disease in a new light

Abstract

The sarcomere is an exquisitely designed apparatus that is capable of generating force, which in the case of the heart results in the pumping of blood throughout the body. At the molecular level, an ATP-dependent interaction of myosin with actin drives the contraction and force generation of the sarcomere. Over the past six decades, work on muscle has yielded tremendous insights into the workings of the sarcomeric system. We now stand on the cusp where the acquired knowledge of how the sarcomere contracts and how that contraction is regulated can be extended to an understanding of the molecular mechanisms of sarcomeric diseases, such as hypertrophic cardiomyopathy (HCM). In this review we present a picture that combines current knowledge of the myosin mesa, the sequestered state of myosin heads on the thick filament, known as the interacting-heads motif (IHM), their possible interaction with myosin binding protein C (MyBP-C) and how these interactions can be abrogated leading to hyper-contractility, a key clinical manifestation of HCM. We discuss the structural and functional basis of the IHM state of the myosin heads and identify HCM-causing mutations that can directly impact the equilibrium between the 'on state' of the myosin heads (the open state) and the IHM 'off state'. We also hypothesize a role of MyBP-C in helping to maintain myosin heads in the IHM state on the thick filament, allowing release in a graded manner upon adrenergic stimulation. By viewing clinical hyper-contractility as the result of the destabilization of the IHM state, our aim is to view an old disease in a new light.

Keywords: Dilated cardiomyopathy; Hypertrophic cardiomyopathy; Interacting-heads motif; Myosin binding protein C; Myosin mesa; Myosin sequestered state.

Fig. 1

Schematic description of a cardiac sarcomere drawn to scale. a A sarcomere is depicted at its resting length just beginning its contraction. The sarcomere is 2 μm long, and the myosin bipolar thick filaments are 1.6 μm long and constitute the A-band. The actin filaments are 0.8 μm long. The zone containing actin with no myosin overlap is shown as the ½ I-band; the other half of the rightmost I-band is just to the right of the Z-disc in the neighboring sarcomere (not shown). Titin (purple) is attached to the Z-disc and extends to the M-line, where it overlaps with titin from the other half of the sarcomere. Myosin binding protein C (MyBP-C; red) is located in the C-zone. Most MyBP-C molecules shown here are sequestering folded-back myosin heads; four cases are shown where the MyBP-C has dissociated from the myosin heads, freeing them to enter the chemomechanical cycle and allowing the N-terminal domain of MyBP-C to interact with actin. b The total number of myosin molecules in a half-sarcomere directed toward one actin filament in this schematic model is 48. In this schematic model, a maximum of eight of these 48 molecules (17%) are sequestered by MyBP-C (red) holding them in a folded-back state not available for interaction with actin. Of the remaining 40 heads that are not bound to MyBP-C, we show 50% (the percentage regulated by regulatory light chain phosphorylation) of them in a folded-back interacting-heads motif state by head interactions with their own S2 tails and other interactions that prevent them from interacting with actin. This leaves 20 myosin molecules free to interact with actin. If the duty ratio under some load is approximately 0.2, then only approximately four myosin molecules are interacting with a given actin filament at any moment during systolic contraction

Fig. 2

Human β-cardiac myosin structural models and the chemomechanical cycle. a PyMOL-rendered homology model of the full-length human β-cardiac myosin molecule showing the subfragment 1 (S1), proximal subfragment 2 (prox S2), subfragment 2 (S2) and heavy and light meromyosin (HMM and LMM, respectively) domains. The templates used to model the post-stroke structure were obtained from the human β-cardiac myosin motor domain solved by Winkelmann et al. (2015), supplemented with the rigor structure from the squid myosin motor domain (Yang et al. 2007), as described in Nag et al. (2017). The S2 region is a long coiled-coil structure; hence we used the template from the Myosinome database (Syamaladevi et al. 2012). Modeling was done using the MODELLER package. b Simplified chemomechanical cycle of the interaction of myosin heads with actin. Steps of the chemomechanical cycle are: 1 The pre-stroke S1 with bound ADP (D) and inorganic phosphate (P _i) binds to actin (yellow); 2 while bound to actin, the lever arm swings to the right about a fulcrum point (black dot on white star) to the post-stroke position, moving the actin filament to the left (bold blue arrow) with respect to the myosin thick filament; 3 ADP release frees the active site for binding of ATP (T); 4 ATP binding weakens the interaction of the S1 to actin; 5 ATP hydrolysis locks the head into the pre-stroke state. c Homology-modeled human β-cardiac myosin S1 in its pre-stroke state showing various important domains of this subfragment of myosin. ELC essential light chain, RLC regulatory light chain

Fig. 3

Homology-modeled structure of human cardiac MyBP-C. Modeling was as described in Nag et al. (2017). Surface rendition of full-length human cardiac MyBP-C homology-modeled from known structures of the C0, C1, C2, C3, C5 and M2 domains, which were obtained individually using structural homologues from their respective Protein Data Bank (PDB) files. The other domain structures (C4, C6, C7, C8, C9, C10, M1, PA loop) were modeled independently using Ab Initio (Ab Initio Software, Lexington, MA) and template-based prediction methods. The C0–C10 domains were connected C-terminus to N-terminus using PyMOL software (Schrödinger, LLC, New York, NY) to obtain the image shown. There are four serine phosphorylation sites (P) on the M domain (blue) that regulate MyBP-C function (Jia et al. 2010). Interactions with other proteins derived from experimental evidence are shown

Fig. 4

Human β-cardiac myosin structural model in its pre-stroke state showing the myosin mesa residues. a Side view of the myosin mesa with the mesa residues colored pink. The mesa surface is adjacent to two other surfaces of the S1 head, the actin binding domain (yellow residues) and a domain (green residues) that binds to the converter domain of another S1 head in a folded state of myosin discussed later in this review. The converter (light gray) is shown between the mesa and the light chain binding region. Between the converter and the light chain binding region is the pliant region (purple) which consists of six residues and is also a hot spot for HCM mutations. b These three surfaces form a pyramid-like structure with Arg-403 (blue) at its apex. c The positions of seven arginine residues on the mesa, all of which cause hypertrophic cardiomyopathy (HCM) when mutated

Fig. 5

Structural models of the open ‘on state’ and the ‘off state’ of the interacting-heads motif (IHM) of human β-cardiac myosin. The templates used to model the open state were obtained from the human β-cardiac myosin motor domain solved by Winkelmann et al. (2015), supplemented with the rigor structure from the squid myosin motor domain (Yang et al. 2007), as described in Nag et al. (2017). The template used to model the closed state is based on the three-dimensional (3D) reconstruction of tarantula skeletal myosin thick filaments by Alamo et al. (2016) (PDB 3JBH). A short version of myosin HMM, showing only 126 residues of the coiled-coil S2 domain, is illustrated in its ‘on and off states’, which are in equilibrium. The back view (named from the 3D reconstruction of the tarantula thick filament; this side faces the myosin bipolar thick filament) of the IHM state is shown

Fig. 6

Working structural models of the IHM state of human β-cardiac myosin with MyBP-C fragments bound. a The heavy chain residues of the blocked S1 head (on the left) are colored pink (mesa residues), reddish-brown (loop 2), yellow (actin-binding domain), bright green (converter binding domain; barely visible), light gray (converter) and dark gray (all remaining residues). The ELC is colored light brown and the RLC is light green. The color scheme of the free head (on the right) is the same as that of the blocked head, except the main body of the heavy chain is colored medium grey. The green residues correspond to those residues on the blocked head that interact with the converter domain of the free head. The ELC is colored dark brown and the RLC is dark green. b A possible orientation of the C0–C2 domains of MyBP-C is shown, with potential interactions between the C0–C2 domains and the mesa of the free head (on the right) and illustrating potential interactions between proximal S2 and C1–C2 (Gruen and Gautel 1999). The yellow C0 domain is bound to the RLCs (Ratti et al. 2011) and the proline–alanine-rich domain (PA; light pink) connects to the C1 (green)–M (blue)–C2 (magenta) domains, which are on this backside view of the IHM complex. c A possible orientation of the C3–C10 domains of MyBP-C is shown, with potential interactions between the C5–C6 domains and the mesa of the blocked head (on the left) and illustrating potential interactions between proximal S2 and the C3–C6 domains. d Hypothetical model of the interaction of full-length MyBP-C with the IHM state. These structures are working models for experiments going forward

Fig. 7

Structural model of the IHM state of human β-cardiac myosin viewed from the back and the front. a Coloring of domains is the same as in Fig. 6. The back view shows the proximal S2 associating with the mesa of the blocked head in zone 2. Zones 1 and 3 are possible interaction sites for domains of MyBP-C (see Fig. 6). b The front view shows the blocked head converter-binding domain (green) binding to the converter (light grey) of the free head (on the left)

Fig. 8

Schematic drawings of the actin–myosin chemomechanical cycle and hypothesized sequestered states of myosin heads. The color schemes of the myosin heads correspond to those in Fig. 1. Steps of the chemomechanical cycle are as shown in Fig. 2. The heads in the cycle are phosphorylated (~P) on the RLC. S1 heads (orange) that are sequestered into the non-functional IHM state are shown in two states on the left side of the figure: RLC de-phosphorylated and bound to their S2 tail (light cyan), and complexed with de-phosphorylated MyBP-C, which more firmly locks the heads in the IHM state. Note that other than the interactions shown here, many other interactions, such as those involving LMM and titin, are likely involved in the IHM state of myosin, and a common theme for HCM mutations may be that they shift the equilibrium away from the IHM ‘off state’ of the myosin heads to the ‘open state’ in which the heads are functionally accessible for interaction with actin, thus producing the hyper-contractility observed clinically