Cardiac β-myosin variants cause hypertrophic (HCM) or dilated (DCM) cardiomyopathy by disrupting sarcomere contraction and relaxation. The locations of variants on isolated myosin head structures predict contractility effects but not the prominent relaxation and energetic deficits that characterize HCM. During relaxation, pairs of myosins form interacting-heads motif (IHM) structures that with other sarcomere proteins establish an energy-saving, super-relaxed (SRX) state. Using a human β-cardiac myosin IHM quasi-atomic model, we defined interactions sites between adjacent myosin heads and associated protein partners, and then analyzed rare variants from 6112 HCM and 1315 DCM patients and 33,370 ExAC controls. HCM variants, 72% that changed electrostatic charges, disproportionately altered IHM interaction residues (expected 23%; HCM 54%, p=2.6×10−19; DCM 26%, p=0.66; controls 20%, p=0.23). HCM variant locations predict impaired IHM formation and stability, and attenuation of the SRX state - accounting for altered contractility, reduced diastolic relaxation, and increased energy consumption, that fully characterizes HCM pathogenesis.https://doi.org/10.7554/eLife.24634.001
Vertebrate skeletal and cardiac muscles produce force through a chemo-mechanical cycle that contracts and relaxes the sarcomere, the contractile unit in all muscle cells. Sarcomeres are composed of inter-digitating thick filaments that contain myosin, comprising a globular head (subfragment 1; S1) and α-helical tail, and thin filaments that contain actin. Interactions between myosin and actin generate the contractile force underpinning sarcomere contraction.
Myosin heads comprise a motor domain (MD) that hydrolyses ATP, a regulatory domain (RD) that includes the essential (ELC) and regulatory (RLC) light chains, and a proximal S2 subfragment. Myosin tails are packed in antiparallel formation and shape the thick filament’s backbone, from which myosin heads protrudes every 14.3 nm as crowns formed by three pairs of heads. In some crowns (Zoghbi et al., 2008) the S2 fragment interacts with myosin binding protein-C (MyBP-C), another thick filament protein that modulates sarcomere activity. Contraction occurs through conformational changes in the myosin MD that accompany ATP hydrolysis, increasing affinity for actin in the thin filament, and resulting in a powerstroke that transfers force by rotation of the myosin lever domain around the converter domain.
After contraction, myosin heads rebind ATP, detach from actin, and assume a relaxed pre-powerstroke state (Llinas et al., 2015). During relaxation, two myosin heads pack together to form an interacting-heads motif (IHM; [Woodhead et al., 2005]). Consecutive IHMs interact along helices that protrude from the thick filament backbone and limit potential interactions between myosin heads and actin (Alamo et al., 2008; Jung et al., 2008). The IHM forms only in relaxed muscle and is a highly conserved structure throughout the evolution of myosins among different species, in different muscle types (smooth, skeletal, and cardiac), and non-muscle myosins (Alamo et al., 2016).
The IHM appears to form through key interactions (Figure 1A) involving the actin-binding region of one myosin head (denoted as ‘blocked’) and the converter region of the partner ‘free’ head (Woodhead et al., 2005; Alamo et al., 2008; Jung et al., 2008). IHM priming occurs when the blocked head assumes a pre-powerstroke state (Llinas et al., 2015) and docks onto its own S2 by intra-molecular interactions. IHM anchoring occurs by inter-molecular interactions between the docked, blocked head and the neighboring myosin’s tail, and intra-molecular interactions with its ELC. IHM stabilization occurs by intra-molecular interactions between the free myosin head and the anchored blocked head (Alamo et al., 2008; Brito et al., 2011; Alamo et al., 2016). IHM scaffolding interactions between myosin heads and both ELCs and RLCs further support its three-dimensional shape.
The dynamic structure of the IHM (Brito et al., 2011) enables properties that are not observed in isolated S1 myosin fragments (Figure 1A): (i) reduced ATPase activity in blocked heads and intermittently in the free head; (ii) mobility of the free head, that sways forth and back by breaking and restoring stabilizing IHM interactions (Alamo et al., 2008; Brito et al., 2011; Sulbarán et al., 2013; Espinoza-Fonseca et al., 2015; Alamo et al., 2015; Yamaguchi et al., 2016; Alamo et al., 2016); and (iii) release of extra free heads upon activation and blocked heads for force potentiation (Brito et al., 2011). Consistent with these properties, negatively stained smooth muscle myosins show that free heads are flexible, detaching from their partner blocked heads and adopting different motor domain orientations (Burgess et al., 2007); that swaying free heads support the sliding of F-actin filaments along relaxed thick filaments (Brito et al., 2011); and that thick filament respond to calcium activation producing force immediately (Linari et al., 2015).
The asymmetric IHM structure imposes functional consequences on the sarcomere. The blocked myosin head has a pre-powerstroke state (Xu et al., 2003; Zoghbi et al., 2004; Llinas et al., 2015) while docked onto its S2, so ATPase activity is sterically inhibited, and the actin-binding interface is positioned on the converter domain of the free head, so that it cannot bind actin. By contrast, the partnered free myosin head can sway, hydrolyze ATP, and bind actin, producing disordered relaxation (DRX; [McNamara et al., 2016]). In addition, the asymmetric conformations of myosins could also impact RLC phosphorylation, which modulates the rate and extent of force development in cardiac muscle (Toepfer et al., 2016). The RLC associated with the free head is more accessible to protein kinase C phosphorylation (Alamo et al., 2008), which promotes swaying (Brito et al., 2011) and potential actin interactions.
The IHM structure could account for super-relaxation (SRX; [Stewart et al., 2010]). When the free myosin head docks onto the blocked head, its ATPase is also inhibited through stabilization of converter domain movements that are needed to achieve strong actin binding (Wendt et al., 2001). The dual enzymatic inhibition of SRX is reflected by a low ATP turnover rate, detected by quantitative epi-fluorescence in skeletal (Stewart et al., 2010; Naber et al., 2011; McNamara et al., 2016) and cardiac (Hooijman et al., 2011) relaxed muscle fibers, and is proposed as an energy saving mechanism (Cooke, 2011). Muscle relaxation can therefore be characterized by two myosin head IHM conformations and ATPase activities (Figure 1A): the DRX state with swaying free myosin heads and ATPase rate comparable to that observed in single myosin molecules, and the SRX state with a highly inhibited (five-fold lower) ATPase rate (Stewart et al., 2010; Naber et al., 2011; McNamara et al., 2016; Hooijman et al., 2011).
Hypertrophic (HCM) and dilated (DCM) cardiomyopathies are heart muscle diseases caused by variants in genes encoding sarcomere proteins (Geisterfer-Lowrance et al., 1990; Seidman and Seidman, 1991; Konno et al., 2010), most commonly MYH7 (encoding cardiac β-myosin heavy chain; MHC) and MYPBC3 (encoding MyBP-C) and rarely in MYL2 and MYL3 that encode the ELC and RLC, respectively. Distinct variants in sarcomere genes also cause DCM, albeit less frequently. Variant location is not highly predictive of whether it will trigger HCM or DCM (Colegrave and Peckham, 2014).
HCM hearts exhibit hyperdynamic contraction, impaired relaxation, and increased energy demands (Ashrafian et al., 2011). Impaired relaxation (clinically denoted as diastolic dysfunction) is a major cause of HCM symptoms and arrhythmias. Increased energy demands in HCM hearts are thought to contribute to cardiomyocyte death and progression to heart failure. Previous studies demonstrate that HCM MYH7 variants increase myofibrillar Ca2+-sensitivity and sarcomere power, therein explaining hyperdynamic contraction (Seidman and Seidman, 2001; Tyska et al., 2000; Moore et al., 2012; Spudich, 2014; Marston, 2011; Harris et al., 2011; Ashrafian et al., 2011; Spudich et al., 2016), but the mechanisms accounting for diminished relaxation and excessive energy requirements remain poorly understood. By contrast, DCM MYH7 variants decrease Ca2+-sensitivity and reduce motor domain functions (Debold et al., 2007; Moore et al., 2012; Spudich, 2014; Marston, 2011; Spudich et al., 2016), therein accounting for diminished contractile performance, the prototypic feature of DCM. Unlike HCM, diastolic dysfunction is uncommon early in the clinical course of DCM (Lakdawala et al., 2012) but emerges with myocardial remodeling.
Disease-associated MYH7 variants cause single amino acid (aa) substitutions, and as such the locations of these substitutions inform physiologically important domains in myosin. Because myosin sequences are highly conserved across species, the impact of MYH7 variants on MD properties have been studied in the context of a variety of vertebrate molecular myosin structures. Early analyses, employing the chicken skeletal S1 myosin (Rayment et al., 1995, 1993), demonstrated that HCM variants perturbed MD residues involved in nucleotide-binding, actin-binding, or the relay, converter, and lever. More recently additional HCM variants were located onto the myosin mesa (Homburger et al., 2016), a newly characterized flat broad (>20 nm2) structural feature that is proposed to bind MyBP-C (Spudich, 2015; Spudich et al., 2016). The mesa is prominent in the pre-stroke S1 contractile state, but substantially reduced following the powerstroke (Homburger et al., 2016).
The tarantula striated IHM quasi-atomic model PDB 3DTP (Alamo et al., 2008) complements these structures by defining interactions between adjacent myosin heads and possible interactions with IHM-associated protein partners and the backbone. Harnessing this model with other structures (human S2 crystal PDB 2FXM model [Blankenfeldt et al., 2006] and chicken smooth muscle myosin PDB 1I84 model [Liu et al., 2003]), the analyses of a few selected HCM variants (Blankenfeldt et al., 2006; Alamo et al., 2008; Moore et al., 2012) hint that these may impact residues involved in IHM intra- and intermolecular interactions.
Here we assessed the structural pathogenesis of HCM and DCM by comprehensive analyses of the largest series of genotyped cardiomyopathy cases, comprising 7427 individuals with clinical-grade sequencing (Walsh et al., 2017). Modeling a human homologous β-cardiac myosin IHM model (PDB 5TBY, newly reported here) to the newest tarantula IHM quasi-atomic structure PDB 3JBH (Alamo et al., 2016), we first identified putative interaction sites, then examined the locations of all variants categorized as pathogenic (PVs) and likely pathogenic (LPVs) in MYH7, MYL2, and MYL3. We demonstrate that HCM and DCM variants differentially impact myosin MD functions and specific IHM interactions, data that informs the mechanisms for failed contractile power in DCM and the markedly compromised relaxation and energetics in HCM (Ashrafian et al., 2003, 2011).
We modeled a homologous human β-cardiac myosin IHM structure (Figure 2A and Video 1; deposited as entry PDB 5TBY) based on the tarantula striated muscle IHM quasi-atomic cryo-EM structure (PDB 3JBH, Alamo et al., 2016; Materials and methods). Although resolution of the IHM structure as derived by cryo-EM does not define specific atomic contacts or individual side chain densities, this model nonetheless identifies molecular interactions.
The new model fitted well to the 28 Å resolution 3D-reconstruction for the negatively stained human cardiac thick filament (EMD-2240)(Al-Khayat et al., 2013) (Figure 2A). In addition, the predicted SAXS profile of PDB 5TBY is consistent with X-ray solution scattering of squid HMM in Ca2+-free (EGTA) conditions (Figure 2B and Figure 2—figure supplement 1) and the computed scattering profile for the model closely agrees with the measured scattering profile of squid heavy meromyosin (HMM) (Gillilan et al., 2013). Figure 2C shows the residuals (relative deviation) between the squid HMM data and the computed 5TBY scattering profile over the range of scattering angles (red line). The χ goodness of fit parameters reported by the FoXS program are 1.5 for 3JBH and 1.85 for 5TBY respectively (Schneidman-Duhovny et al., 2013). Relative deviation between the 5TBY and 3JBH calculated scattering profiles (dashed line) falls below the noise level of the experimental data (red line) consequently the two models cannot be distinguished. Extending the computations to wider angles (q = 1.0 Å-1; see Figure 2B and Figure 2—figure supplement 1), revealed that the tarantula PDB 3JBH and the human models are essentially identical above q = 0.4 Å-1 and unlikely to be distinguished by future SAXS and wide angle X-ray solution scattering experiments in that regime. The SAXS data in this angle range provide support for the overall shape, placement, and orientation of IHM domains.
The PDB 5TBY model also closely matches crystal structures of fragments for human β-cardiac S1 MD fragment (PDB 4DB1) (Figure 2—figure supplement 2A and Video 2) and S2 (PDB 2FXM)(Blankenfeldt et al., 2006) (Figure 2—figure supplement 2B and Video 3). Moreover, this model includes important myosin loops that improve the analyses of IHM interactions, and that are absent from the only other IHM model used for cardiac muscle (PDB 3DTP; Alamo et al., 2008). In the PDB 5TBY model, the blocked and free myosin heads are in the pre-powerstroke state (Xu et al., 2003; Zoghbi et al., 2004; Llinas et al., 2015) and bent at the ‘pliant region’ (Houdusse et al., 2000) (see Figure 2—figure supplement 2C and Video 4). The blocked head is in a pre-powerstroke state (Llinas et al., 2015) (similar to the Mg·ADP-AlF4, PDB 1BR1; [Liu et al., 2003; Alamo et al., 2008]) while the free head, which is also in a pre-powerstroke state, shows a less angled lever arm than the blocked head (see Figure 2—figure supplement 2C). In the absence of a human β-cardiac IHM crystal atomic structure, the homologous human β-cardiac myosin IHM model PDB 5TBY (Figure 2A and Video 1) provides a robust option for analyses of cardiomyopathy variants on IHM interactions.
All sarcomere PVs and LPVs previously identified in 6112 HCM and 1315 DCM cases (Walsh et al., 2017) were analyzed. Among HCM cases, 40 MYH7 PVs substituted 39 distinct aa (two distinct variants encoded an identical substitution) at 33 positions and 95 LPVs led to 95 substitutions at 87 sites (Tables 1 and 2). Thirty-eight PVs altered S1 and S2 residues, and two altered LMM residues. Four further HCM PVs occurred in ELC (n = 2) or RLC (n = 2). Among DCM cases, one MYH7 PV and 26 LPVs altered 26 distinct aa residues (Table 3). We considered how these variants impacted residues involved in motor domain functions (Supplementary file 1) and in pre-specified IHM interactions (Supplementary file 2).
We compared the distributions of MYH7 HCM and DCM variants at functional MD residues involved in the nucleotide-binding pocket (39 aa), actin binding (60 aa), converter (68 aa), or relay (27 aa), that together make up 10% of the myosin heavy chain (194 aa; Supplementary file 1). MYH7 variants were not distributed uniformly, but were over-represented at functional MD residues both in HCM (16/40 PVs (40%), 4-fold enrichment above 10% expected, p=6.14e-07; 23/95 LPVs (24%), p=5.11e-05; combined 39/135 (29%), p=7.9e-9; Tables 1, 2 and 4) and DCM (7/27 variants (26%), p=0.022; Tables 3 and 5). However these impacted distinct regions. HCM variants were enriched in residues of the converter (n = 21/135 (16%), expected 3.5%, p=1.25e-08; Supplementary file 3) while 5/27 DCM variants altered nucleotide-binding pocket residues (19%, expected 2.0%, p=0.00019; Supplementary file 4). This supports previous observations that HCM and DCM variants alter distinct functional motor domains (Debold et al., 2007; Spudich et al., 2016), leading to altered myocardial contraction (Figure 1).
We next defined the distribution of HCM and DCM variants on 447 MYH7 residues involved in major IHM interactions (Supplementary file 2; Alamo et al., 2008, Alamo et al., 2016). Priming interactions (‘f’ and ‘g’, involving 113 aa) dock the blocked head onto its own S2. Anchoring (156 aa) associates the blocked head and ELC to the neighboring myosin tail via interactions ‘i’, ‘h’, and ‘j’. Stabilizing (189 aa) docks the free head onto the blocked head via interactions ‘a’, ‘d’, and ‘e’. Scaffolding (120 aa) supports the IHM three-dimensional structure (RLC-MHC and ELC-MHC interactions). Additional RLC-RLC interactions regulate the IHM (Alamo et al., 2016). As residues can participate in several interactions, HCM and DCM variants that alter multi-interactive residues can impact formation of the IHM in several ways.
In comparison to expectation, HCM PVs were enriched among the 23% of myosin residues (447 of 1935) involved in the IHM structure, with thirty-one of 40 (78%) MYH7 PVs altering IHM residues (3.35-fold enrichment; p=5.25e-13; Table 1 Supplementary file 3a, Figure 3—figure supplement 1A). We validated this estimate by analyzing LPVs (Table 2): Forty-two of 95 LPVs (44%) similarly impacted IHM interactions (1.9-fold enrichment; p=7.04e-06; Supplementary file 3b). Based on the significant enrichment of HCM variants in residues involved in IHM interactions (Table 4) we infer that disruption of the IHM contributes to HCM pathophysiology (Figure 1B).
As cardiomyopathy variants can alter distinct interactions when located on the blocked head versus the free head, we assessed HCM PVs and LPVs exposed to these different environments (Figure 3A and Video 5, Supplementary file 3f). Thirty-nine of 135 PVs or LPVs alter residues involved in free head interactions (29%, expected 8.8%, p=1.93e-11), 52 altered residues involved in blocked head interactions (39%, expected 19%, p=9.32e-08), and 14 altered residues involved in tail interactions (10%, expected 3.6%, p=3.57e-04). HCM variants that disrupt priming and anchoring residues would prevent the docking of the blocked head to the S2, delaying IHM formation (Figure 1B, left), while variants that alter stabilizing residues will tend to release the free head from the partner blocked head (Figure 1B, right). HCM PVs and LPVs (Supplementary file 3a, b) were particularly enriched in IHM stabilizing interactions (p=5.37e-16 vs. expectation; Table 4, Figure 3—figure supplement 4). These 48 variants (Figure 3, pink) include 11 variants affecting stabilizing residues when located on the blocked head (p=2.01e-2) and 31 variants affecting stabilizing residues when located on the free head (p=2.58e-12). Twenty-four HCM variants (Figure 3A, orange and Figure 3—figure supplement 3) altered blocked head residues involved in IHM anchoring interactions (p=2.1e-4). Five of eight anchoring variants (involved in ‘i’ interactions; Figure 3—figure supplement 3) affect residues that also participate in another IHM interaction (Tables 1 and 2), we cannot discern whether one or both interactions are more relevant to HCM pathogenesis. HCM variants were only modestly enriched in IHM priming interactions (p=2.6e-3; Table 4, Figure 3A, green and Figure 3—figure supplement 2), and affected nine residues on the blocked head and 14 tail residues, including three residues that also participate in stabilizing interactions (Supplementary file 3d). In addition, 24 MYH7 variants (Figure 3A, white; Figure 3—figure supplement 5) altered residues that scaffold the MHC and ELC or RLC (p=3.0e-6; Supplementary file 3d), that were particularly enriched in MHC blocked head residues affecting ELC interactions (p=1.19e-07).
All four HCM PVs in MYL2 (n = 2) and MYL3 (n = 2) also affected IHM residues. The MYL3 variants altered ELC residues involved in anchoring (orange) and scaffolding (white) the blocked head (Table 1 and Figure 3A), and residues involved in stabilizing (pink) and scaffolding (white) the free head. MYL2 variants altered residues on the RLC involved in scaffolding (Figure 3A, white). These variants also altered residues involved in RLC-RLC regulation (Figure 3A, yellow) of the blocked and free heads.
HCM variants therefore overlie distinct interaction sites when found in the context of the free or blocked head. As the same residue may be involved in more than one interaction, HCM variants have the potential to disrupt multiple IHM properties. Yet while MD and IHM residues overlap, particularly in the converter region, that 73 of 135 MYH7 PVs and LPVs (54%) alter IHM interaction residues, as do all HCM variants in MYL2 and MYL3, challenges the paradigm that these act primarily through the impairment of myosin motor functions (Rayment et al., 1995). As the IHM structure allows myosin to adopt the SRX state, we suggest that HCM PVs and LPVs that perturb dynamic IHM interactions would likely alter physiologic relaxation by the heart, and account for diastolic dysfunction, which is the major component of HCM pathophysiology.
Parallel analyses of all DCM PVs and LPVs (n = 27) in MYH7 (Tables 3 and 5, Figure 3B and Figure 3—figure supplement 1B) showed that these were over-represented at MD residues, but were not enriched in residues across all IHM interactions (p=0.65) nor among residues involved in interactions by the blocked head, free head, or tail (p≥0.25). Within specific components of IHM interactions (Supplementary file 4a, b), five DCM variants altered IHM priming (Figure 3B, green and Video 6), albeit with only nominal significance (p=0.019). Additional DCM variants altered blocked head residues involved in IHM stabilizing interaction (pink) and ELC scaffolding of blocked and free head residues (white), but these were not enriched over expectation. This data show that residues within IHM interaction sites are differentially impacted by variants that cause the distinct pathophysiologies of HCM and DCM.
Given the overall limited numbers of myosin DCM PV and LPVs, we also assessed two previously reported DCM MYH7 PVs, S532P and F764L (Schmitt et al., 2006) that were not identified in the cardiomyopathy cohort studied here. S532P alters a blocked head residue (Figure 3B, side chain green), but is not involved in IHM interactions by the free head. F764L alters a blocked head residue that is involved in anchoring and a free head residue involved in IHM stabilization (Figure 3B, side chain orange and pink, respectively).
A far smaller proportion of myosin DCM PV and LPVs altered residues involved in IHM interactions (Figure 1C) than HCM variants. Together with the observed enrichment at MD functional domains shown above, this implies that DCM variants predominantly affect myosin MD properties, which is consistent with the predominant clinical manifestations of DCM MYH7 variants - reduced contractile force but normal diastolic relaxation (Lakdawala et al., 2012).
An electrostatic charge difference between the wild type and variant residue, Δq, is expected to further destabilize IHM inter- and intra-molecular interactions (Tables 1–3). Among HCM PVs that alter IHM interacting residues, 22/31 altered the charge (observed 71%, expected 49%, p=0.019): nine (29%) produced charge-gain (+2 > Δq > 0) and thirteen (42%) produced charge-loss (−2 < Δq < 0).
Charge changing HCM PVs are involved in multiple IHM interactions (Table 1). Four of five HCM PVs that alter IHM priming residues change the charge, including R870H, which is located on the S2 ‘kink’ resulting in S2 bending which shapes the overall IHM (Figure 3—figure supplement 2, ‘g’ interaction). Nine of 12 PVs that alter IHM anchoring residues (Figure 3—figure supplement 3, ‘i’ and ‘j’) cause a charge change. Nineteen of 24 PVs (79%) change the charge of IHM stabilization residues, particularly those involved in ‘d’ interactions (Figure 3—figure supplement 4).
Both MYL2 HCM PVs are charge changing and could destabilize MHC-RLC and RLC-RLC interactions (Figure 3A). MYL2 R58Q resides in the loop between RLC helices B and C (Figure 3—figure supplement 5) that form the RLC-RLC interface. Spectroscopic studies of the RLC-RLC interface demonstrate that piperine destabilizes the SRX state, possibly in part by divalent cation binding (Nogara et al., 2016b2016b, 2016a). As the E22K variant abolishes RLC phosphorylation and markedly reduces calcium-binding affinity (Szczesna et al., 2001), while the charge change associated with the R58Q variant would alter helical structures involved in the RLC-RLC interface, both variants should destabilize the SRX state. Consistent with this conclusion, ex vivo analyses of hearts from MYL2 R58Q transgenic mice demonstrate higher ATPase rates and incomplete relaxation (Greenberg et al., 2009).
Five of seven (71%) DCM PVs and LPVs affecting IHM interacting residues also have electrostatic charge changes. Variant R904C should make the S2 Ring 1 more negative (−2) and strengthen IHM priming (‘f.2’) when the blocked head docks onto its S2, therein decreasing ATPase activity. By contrast, a nearby HCM variant (D906G Figure 3—figure supplement 2) makes the S2 Ring 1 more positive (+1) which weakens IHM priming (‘f.2’) with resultant increase in expected ATP turnover and numbers of released swaying free heads. Therefore the predicted reciprocal biophysical consequences of adjacent MYH7 variants on the IHM provide potential mechanisms to explain how nearby, indistinguishable groups variants with opposite charge changes cause the distinct pathophysiology of HCM and DCM (Figure 1B vs. C).
The blocked myosin head structure in the IHM is similar to its pre-powerstroke structure (Figure 2—figure supplement 2C and Video 4) that contains a prominent flat myosin surface, denoted as the mesa (Spudich, 2015; Spudich et al., 2016; Homburger et al., 2016). The mesa is comprised of 277 aa, of which approximately half are IHM interacting residues. Based on the dynamic IHM structure, we expect that myosin can assume two mesa structures, corresponding to the blocked, docked head and the free head.
Fifty-two of 135 HCM variants (18 PVs and 34 LPVs; p=3.9e-12 versus expected), but only 3 DCM variants (p=0.79), reside on the mesa (Tables 1–3, Supplementary file 5, Figure 4 and Video 7). Thirty-six HCM variants alter mesa residues that also participate in IHM interactions (4.1-fold above expectation, p=2.38e-13). When located on the blocked head, HCM mesa variants altered residues involved in priming (n = 9), anchoring and scaffolding (n = 15), and stabilizing (n = 6). When located on the free head HCM residues altered IHM stabilization (n = 21) and scaffolding (n = 1). By disrupting blocked S1 head interactions with S2 (‘f.1’ with S2 Ring 1 and ‘g’ with the S2 kink; Figure 3—figure supplement 2) that are crucial for establishing SRX, and stabilizing interactions between the blocked and free heads, HCM mesa variants could compromise relaxation and increase free heads available for force potentiation. The model that disruption of the IHM contributes to HCM pathophysiology is therefore entirely compatible with recent data suggesting an important role for the mesa. We expect that variants residing on the mesa (Spudich, 2015) and that are involved in IHM interactions (Alamo et al., 2008; Jung et al., 2008; Luther et al., 2011) may also impact MyBP-C interactions.
Having identified a relationship between cardiomyopathy variants and residues identified as IHM interaction sites, we considered whether this information improved clinical variant interpretation. We compared the prevalence of all rare (ExAC global allele frequency <0.0001) missense variants in the 7427 cardiomyopathy subjects (Walsh et al., 2017) with 33,364 ancestry-matched ExAC subjects. We calculated disease odds ratios (under the assumption that each individual carries at most one rare variant, since individual-level genotypes are not available in ExAC, see Material and methods) and the etiologic fraction (EF, the proportion of variants in cases that are disease-causing), which represents the probability that a rare variant found on genetic testing in a cardiomyopathy subject is pathogenic (HCM, Supplementary file 6, DCM, Supplementary file 7). We calculated these measures for the whole protein, and for pre-specified sub-regions: the myosin head, MD functional sites, the mesa, a previously-described cluster of 29 amino acids in the converter region (Homburger et al., 2016), and the IHM interactions described here.
We identified 326 rare (global ExAC allele frequency <0.0001) variants in 854 of 6112 HCM patients (14%), resulting in 324 distinct aa substitutions at 300 residues. In the ExAC reference sample, 253 rare variants in 449 of 33,364 non-Finnish European subjects (1.4%), resulted in 252 distinct substitutions at 220 residues. Only 50 ExAC variants altered IHM residues (20%, expected 23%, p=0.23) and these were not enriched in specific interactions.
Regional analyses identified portions of the protein that are highly enriched in HCM, yielding a high probability that variants found in HCM patients are causative, with sufficient confidence for clinical application. As expected, these regions include the mesa (EF = 0.98) and MD functional sites (EF = 0.99), particularly the converter domain (EF = 0.99). Interpretive confidence is even higher for certain specific IHM interactions. Notably, rare variants involved in blocked head IHM priming (‘g’) and stabilizing (‘d.1’) residues were absent from ExAC but present in 62 HCM cases (p<e-200), while variants in five other IHM interactions were exceedingly rare in ExAC (EF ≥0.99). These regions accounted for ~44% of variants found in HCM cases (combined prevalence, 6%), but less than 4% of variants in ExAC (combined prevalence, 0.048%), indicating that variants within these sites have extraordinarily high prior probabilities of pathogenicity. Irrespective of our conclusion that these variants act by disrupting IHM interactions, these analyses robustly demonstrate that variants that alter a subset of MYH7 residues are strongly predictive of HCM and clinically actionable.
Parallel comparisons of 58 rare variants in 68 of 1315 DCM patients (5.2%) indicated that a large proportion of those within the myosin head are pathogenic (EF = 0.75), but that this is insufficient to report novel variants as LPV without additional evidence of pathogenicity. Variants at functional MD residues have a high probability of pathogenicity (EF = 0.96). Among DCM rare variants that altered residues involved in IHM interactions (EF = 0.84), only those that altered IHM priming residues (EF = 0.96) achieved high probabilities of pathogenicity.
Experimental preparations of relaxed cardiac muscles (Hooijman et al., 2011) display two rates of ATP hydrolysis (slow turnover time, 138–144s; fast turnover time 20 s) that are respectively attributed to docked blocked heads and transiently docked free heads (the SRX state) and swaying free heads in the DRX state (Alamo et al., 2016). As such, the proportion of myosin heads in the SRX or DRX state impacts cardiac muscle expenditure of ATP and energy efficiency, parameters that are critical for continuous pumping.
We calculated a theoretical estimate of the populations of relaxed cardiac myosin heads in DRX or SRX (Material and methods) based on the assumptions that (a) the IHM structure accounts for the SRX and that (b) activation of cardiac muscle only impacts free heads in the DRX state while docked blocked heads remain parallel to the thick filament. The latter assumption is supported by fluorescence analyses of RLC in relaxed rat heart fibers, that show both parallel and perpendicular alignment to the thick filament (Kampourakis and Irving, 2015) and by evidence that cardiac myosin heads remain in the SRX state with tetanic fiber activation (Hooijman et al., 2011). Based on these assumptions, the asymmetric configuration of myosin heads in the cardiac IHM could yield savings of one ATP molecule when the free head is docked to blocked head (SRX) that would be lost when swaying free heads increase ATP turnover (DRX, Figure 1A). As such HCM variants in MYH7, MYL2, and MYL3 that impair IHM interactions or that destabilize the RLC-RLC interface (Figure 3—figure supplement 5) such as occurs with RLC phosphorylation, would reduce SRX:DRX proportions, consume more ATP, and promote free head swaying with potential myosin-actin interactions and force production.
Biophysical and biochemical analyses of the sarcomere’s chemomechanical cycle have defined the molecular basis of force production by striated muscles and illuminated how this is perturbed by cardiomyopathy mutations. Identification of dynamic IHM intra- and inter-molecular interactions that produce populations of relaxed myosins in DRX and SRX states provides additional fundamental insights into sarcomere biology that informs the molecular pathogenesis of HCM and DCM.
We demonstrate that similar proportions of HCM and DCM variants (26–29%) impact myosin residues with MD functions, albeit with distinctly different distributions. DCM variants were enriched only in the nucleotide-binding site (p=0.00019) but were notably absent from the converter domain, a region where residues also function in IHM interactions. By contrast, HCM variants are highly enriched in the converter domain (p=1.25e-8) and on the mesa (p=3.89e-12), domains that both participate in MD functions (HCM variant enrichment, p=7.9e-9) and IHM interactions (HCM variant enrichment, p=7.95e-15). Indeed, HCM variants were 1.19-fold enriched in residues involved only in MD functions, 1.74-fold enriched in residues involved only in IHM interactions, and 4.17-fold enriched in residues that participate in both, providing an account for why HCM PVs and LPVs, unlike DCM variants, have broad effects throughout the contraction and relaxation phases of the cardiac cycle (Figure 1B).
While the location of HCM variants within functional MD residues of myosin, in vitro motility assays, and single molecule biophysical studies (Tyska et al., 2000; Spudich, 2014; Spudich et al., 2016) provide mechanisms for abnormal contractility, these do not account for the prevalent and profound relaxation deficits that characterize HCM. By contrast, evidence that HCM variants in MYH7, MYL2, and MYL3 alter residues that support the dynamic properties of IHM provide mechanisms by which these disrupt cardiac relaxation and energetics (Figure 1A, Figure 3—figure supplement 5; see Material and methods). HCM variants that impair IHM interactions would reduce SRX:DRX proportion, promote additional myosin-actin interactions, and consume more ATP, resulting in altered relaxation, energetics and force production.
Previous studies of selected cardiomyopathy variants, that employed early thick filament and IHM structures, suggested that these might impact IHM formation (Blankenfeldt et al., 2006; Alamo et al., 2008; Moore et al., 2012). Our study advances these conclusions in several ways. Capitalizing on a novel human β-cardiac myosin IHM PDB 5TBY structure we considered if cardiomyopathy residues altered specific IHM interactions. Second, we studied a comprehensive and unselected set of PVs and LPVs that were independently identified in a large cardiomyopathy cohort (7427 HCM and DCM patients) and compared these to variants in 33,370 ExAC controls. We found that only HCM variants were significantly enriched in many IHM interacting residues, most notably in those that stabilize the IHM (p=5.37e-16 vs. expectation). As most HCM variants resulted in a charge change (Tables 1 and 2), these are expected to adversely affect IHM interactions. Taken together, these data provide strong support that HCM variants perturb cardiac relaxation by disrupting IHM interactions. A corollary to this conclusion is that the IHM interacting residues identified by the PDB 5TBY structure are physiologically valid.
We recognize several limitations in our analyses. First, there are neither atomic nor near-atomic structures of the human cardiac IHM. Although a near-atomic (0.6 nm) structure of the Lethocerus thick filament backbone has been reported (Hu et al., 2016), the resolution for the IHM is comparable (2.0 nm) to the tarantula structure employed here, perhaps because the intrinsic flexibility of swaying free heads limits higher resolution (Brito et al., 2011; Sulbarán et al., 2013). Second, using homologous MYH7 sequences, we converted the tarantula (PDB 3JBH) into a human (PDB 5TBY) IHM model, which fits well into the human beta-cardiac 3D-reconstruction (Figure 2a). While capitalizing on the high evolutionary conservation between human MYH7 and tarantula striated myosin (60% amino acid identity), especially residues involved in IHM interactions (Alamo et al., 2016), we expect that future models will refine the resolution of interactions identified in the PDB 5TBY model. Third, the tarantula striated muscle lacks MyBP-C and titin, molecules that may interact with IHMs to form helices that protrude from vertebrate thick filaments and limit interactions between myosin heads and actin (Alamo et al., 2008; Jung et al., 2008; Luther et al., 2011). The locations of several HCM variants hint that these may have additional functional consequences that impair interactions between consecutive IHMs. Fourth, while our data support the conclusion that the structural states defined by the IHM are responsible for the DRX and SRX functional states, these cannot be proven to be synonymous.
We also expect that high-resolution human cardiac thick filament structures will extend these insights to other HCM genes and variants. MyBP-C harbors many HCM variants that reduce protein levels (Harris et al., 2011) and evoke functional consequences similar to MyBP-C phosphorylation: increase calcium sensitivity of active force and acceleration of rigor force and rigor stiffness development (Kunst et al., 2000). The C1C2 fragment of MyBP-C (Gruen and Gautel, 1999) is predicted to interact with the second of three negatively charged rings (Blankenfeldt et al., 2006) on the myosin S2 domain (Figure 2A and Figure 4, labeled as R1-3), and Ring 2 residues participate in stabilizing the IHM. Charge inverting (+2) variants on Ring 2 (E924K and E930K in Table 1 and Figure 3—figure supplement 4 ‘a’; E927K and E935K, described in (Blankenfeldt et al., 2006) could disrupt MyBP-C binding (Lee et al., 2015), and destabilize the IHM, as would HCM variants that deplete MyBP-C protein levels. Moreover, destabilization of the IHM could account for the significant reduction of SRX that occurs in mice lacking MyBP-C (McNamara et al., 2016). With the enrichment of MYH7 variants in IHM interacting residues reported here, we suggest that altered physiologic ratios of SRX and DRX populations is the mechanism by which HCM disrupts cardiac relaxation.
HCM and DCM are dominant disorders and sarcomeres contain an admixture of normal and mutated myosin proteins. As MYH7 DCM variants primarily impact functional MD residues (e.g. nucleotide binding and hydrolysis), these will depress contractility in proportion to the summed force produced by myosins with and without variants (Schmitt et al., 2006; Spudich et al., 2016), with concurrent reduction in ATP consumption. The resultant under-performance of sarcomeres containing DCM variants promotes compensatory ventricular dilation (Frank-Starling mechanism) to maintain circulatory demands (Figure 1C).
By contrast, (Schmitt et al., 2006; Spudich et al., 2016) HCM variants impact both MD functions and IHM properties. Any pair of myosins involved in asymmetric IHM interactions may contain zero, one, or two mutated residues with singular or dual consequences when located on the blocked or free head (Tables 1 and 2, Figure 3). The 135 HCM PVs and LPVs described here will alter 39 functional MD residues on each head and 73 IHM interacting residues, located on the blocked or free heads or S2 domain (Supplementary file 3f). Notably, 41 of these 135 variants altered only one IHM interaction, while 32 altered residues involved in at least two interactions simultaneously, typically a different one for each head (Tables 1 and 2). We suggest that the effects of HCM variants on contractility, their consequences on MD functions, and the multiple effects on IHM interactions, confers a complexity that can explain the dissimilar results obtained when myosin mutations are studied in the context of single myosin molecules that only assess MD functions and myofibril experiments that also interrogate the IHM (Moore et al., 2012). For example, R453C can simultaneously affect IHM priming (‘g’ interaction) when located on the blocked head (Figure 3—figure supplement 2) and stabilization (‘d.1’) when on the free head (Figure 3—figure supplement 4). Biomechanical assays of myosin R453C shows decreased actin-sliding velocity and ATPase, but increased force production (Sommese et al., 2013). These dichotomous results may be explained by the IHM paradigm (Figures 1B and 3A). Located near the switch 2 with the nucleotide-binding pocket, R453C would impair S1 functions that are assayed by single molecule in vitro analyses. When located on the blocked head (‘g’, Figure 3—figure supplement 2) the charge change of R453C (Δq = −2) would destabilize blocked myosin heads and increase the number of heads (the released blocked head and the associated partner free heads) that are available for force potentiation. As such, the increase in sarcomere force observed with many HCM variants likely reflects the combined effect on MD functions and IHM interactions that increase myosin populations in DRX and reduce those in SRX states.
HCM variants will further impact cardiac physiology because of the relationship between the proportion of myosins in the DRX and SRX states and energy consumption (Figure 1B). Swaying free myosin heads in the DRX state consume 5-fold greater ATP than in the SRX state (Hooijman et al., 2011), and physiologic SRX:DRX levels during relaxation may reduce energy consumption and support life-long continuous activity. By disrupting IHM interactions, HCM variants would reciprocally increase the proportion of myosins in the DRX state and decreased the proportion in the SRX state – resulting in increased ATP consumption and metabolic demands, yielding energy deficiencies that characterize human HCM hearts (Ashrafian et al., 2003). These observations also provide a plausible mechanistic explanation for why MYK-461, an ATPase inhibitor, improved the pathophysiology in HCM mouse models (Green et al., 2016).
In summary, the integrated interpretation of HCM and DCM variants on myosin MD functions and dynamic IHM interactions provides a fuller understanding of sarcomere biology in health and disease. While DCM variants evoke primary deficits in MD functions (Figure 1C), HCM variants perturb both MD functions and IHM interactions (Figure 1B). By disrupting IHM priming, anchoring, stability, and scaffolding interactions, HCM variants decrease populations of docked myosin heads (SRX) and increase swaying heads (DRX) that are available for force production, thereby promoting hypercontractility. The reduction of myosin populations in SRX would perturb diastolic relaxation and increase ATP consumption, causing energy deficiencies in HCM hearts (Ashrafian et al., 2003). Notably, these are primary biophysical deficits produced directly by HCM variants that are unrelated to secondary compensatory changes observed in other heart diseases. Clinical observations support this conclusion: hyperdynamic contraction, diastolic dysfunction (Ho et al., 2002), and energy deficiency (Jung et al., 1998; Crilley et al., 2003) precedes hypertrophic remodeling in young HCM variant mutation carriers. We conclude that the identification of sarcomere protein residues involved in relaxation provide new opportunities to target the IHM to improve HCM pathophysiology and other heart diseases with diastolic dysfunction. By extension of these analyses to skeletal myopathies we anticipate further insights into critical roles of the highly conserved IHM in the relaxation of striated muscles.
We first generated a new homologous human β-cardiac myosin IHM quasi-atomic model, and used this to identify residues at sites of IHM interactions. These interaction sites were predefined from structural models, without prior knowledge of the location or distributions of HCM or DCM variants. We then examined the locations of a large series of clinically defined cardiomyopathy variants (Walsh et al., 2017). All these independently classified HCM and DCM PVs or LPVs were included in our statistical analyses. Finally, for predefined MYH7 functional regions we compared the total prevalence rare variants (not stratified by clinical consequence) in cardiomyopathy cases and a reference population to derive unbiased and quantitative estimates of clinical interpretability. Code to reproduce statistical analyses (in R) and figures (in Chimera) is available on GitHub (Ware, 2017).
Sequences include human β-cardiac MHC (P12883), ELC (P08590) and RLC (P10916); chicken skeletal muscle MHC (P13538) and tarantula (Aphonopelma sp.) skeletal muscle ELC (GenBank KT390185), RLC (PDB KT390186) (Zhu et al., 2009) and MHC (GenBank KT619079) (Alamo et al., 2016). Alignments of the human β-cardiac, chicken skeletal and tarantula striated MHC, ELC and RLC that are shown in Figure 3—figure supplements 6–9 were generated with Clustal (Larkin et al., 2007) over the JalView platform (Waterhouse et al., 2009).
Human β-cardiac myosin ELC, RLC and MHC (MD and S2) were modeled as described (Alamo et al., 2016). The human β-cardiac myosin interacting-heads motif (Figure 2A and Video 1) has been deposited into the Research Collaboratory for Structural Bioinformatics Protein Data Bank (PDB 5TBY). A rigid fitting of this model was done against the human cardiac negatively stained thick filament 2.8 nm resolution 3D-reconstruction (EMD-2240) (Al-Khayat et al., 2013) using the Chimera ‘Fit in Map’ option (Figure 2A and Video 1) (Pettersen et al., 2004).
Sample preparation and data collection details for squid HMM are previously reported (Gillilan et al., 2013). Scattering intensity, I(q), is in arbitrary units, with q = 4π sin(θ)/λ in units of Å−1. The wavelength λ = 1.2563 Å and the scattering angle is 2θ as measured from the direct beam. Scattering profiles for PDB 5TBY and tarantula PDB 3JBH were computed using both FoXS (Schneidman-Duhovny et al., 2013) and CRYSOL (Svergun et al., 1995) to compare agreement, especially at scattering angles q > 0.3 Å-1. As described previously, CRYSOL required more than the default number of harmonics (Svergun et al., 1995) for agreement with FoXS in the WAXS regime and the algorithms agreed on the location and magnitude of the difference between the PDB models despite a minor baseline shift (not shown). Currently neither of these methods model flexibility, an effect that could also introduce baseline shifts at wider angles. The FoXS calculations reported here fit both the computed profiles to the squid HMM experimental data using the default parameters of hydration layer, excluded volume, and background adjustment (Gillilan et al., 2013). In the case of Figure 2B,C the ‘profile offset’ optimization was performed to obtain best fit with the data at widest angles (Schneidman-Duhovny et al., 2013). The goodness of fit parameter χ, reported by FoXS, is defined as
where is the experimental scattering profile measured at M points with error given by Model profiles, , that agree with the data to within expected noise levels give χ values close to unity. (Gillilan et al., 2013; Schneidman-Duhovny et al., 2013; Svergun et al., 1995)
The analysis of the interactions was done as described (Alamo et al., 2016), using the ‘Protein interfaces, surfaces and assemblies’ (PISA) service at the European Bioinformatics Institute (http://www.ebi.ac.uk/pdbe/prot_int/pistart.html) (Krissinel and Henrick, 2007). Interactions were described as intra- or inter-molecular according to the customary convention used in IHM studies (Woodhead et al., 2005; Alamo et al., 2016).
The atomic coordinates of the human β-cardiac myosin IHM quasi-atomic model have been deposited into the Research Collaborators for Structural Bioinformatics as PDB entry 5TBY.
Variants found in samples from 7427 individuals referred for genetic testing with a referral diagnosis of HCM or DCM, sequenced in accredited diagnostic laboratories with clinical-grade variant interpretation (3826 individuals sequenced at Oxford Medical Genetics Laboratories (Walsh et al., 2017), and 3668 individuals from the Partners HealthCare Laboratory of Molecular Medicine (Pugh et al., 2014; Alfares et al., 2015) were used in our analysis. In total, 6112 HCM patients had MYH7 sequencing, including 4185 also with MYL2 and MYL3 sequencing, while 1315 DCM cases had MYH7 sequencing and 543 DCM also with MYL2 and MYL3 sequencing. First, all curated variants reported as pathogenic by either laboratory (HCM PV, Table 1) were assessed in the mapping analyses (Figure 3A, Supplementary file 3a, Figure 3—figure supplements 1–5). Variants reported as likely pathogenic (HCM LPV, Table 2, Supplementary file 3b, Figure 3A) were then analyzed for replication. Subsequent analyses of specific interactions include HCM PVs and LPVs combined (Table 4, Supplementary file 3a-d). Given the smaller number of variants in DCM, and that PV and LPV behaved similarly in HCM, all DCM PV and LPV were considered together (Tables 3 and 5, Supplementary file 4, Figure 3B).
To determine whether variants were over-represented in a region of interest (Supplementary files 1, 2), the proportion of variants (Supplementary files 3–5) that fell within the region was compared with the proportion that would be expected under a uniform distribution (given by the length of the region / total protein length). Proportions were compared using a binomial test, implemented in R (Ware, 2017).
In order to confirm the importance of the pre-specified IHM interaction sites (Supplementary file 2), and to determine the strength of the disease association for variants at different sites, we compared the total prevalence of rare variants in the case series described above (irrespective of clinical laboratory classification) with the prevalence amongst reference samples from the Exome Aggregation Consortium (ExAC; Lek et al., 2016); Supplementary files 6, 7). Rare (ExAC global AF <0.0001) missense variants were extracted from the ExAC vcf (version 0.3.1). Analysis was restricted to the ExAC sub-population of European ancestry, as the case series is drawn from a predominantly Caucasian background (Walsh et al., 2017). The median number of ExAC samples genotyped at each variant site was used as the ExAC population size (33,364), and prevalence was compared using the binomial test (Ware, 2017). A disease odds ratio (OR) was estimated (under the assumption that each rare variant was found in a distinct individual), and the etiological fraction (EF) was calculated as (OR – 1)/OR (Ware, 2017). The EF, a commonly used method in epidemiology, estimates the proportion of affected variant carriers in which the variant caused the disease, which can also be interpreted as the probability that an individual rare variant, found in a proband, is responsible for the disease (Greenland and Robins, 1988; Robins and Greenland, 1989; Cole and MacMahon, 1971; Walsh et al., 2017).
The sites of 43 curated HCM aa substitutions (Table 1), 95 LPV substitutions (Table 2), and 27 DCM aa substitutions (PVs plus LPVs, Table 3) are shown in Figure 3 and Figure 3—figure supplements 1–5. UCSF Chimera software version 10.2 (Pettersen et al., 2004) was used to build Figures 2A, 3 and 4 and Figure 3—figure supplements 1–5. The Chimera sessions (.py) used for making Figure 2A and Figure 2—figure supplement 2, Figure 3 and Figure 3—figure supplements 1–5 and Figure 4 are available in GitHub (Ware, 2017). A copy is archived at https://github.com/elifesciences-publications/eLife_Alamo2017.
In order to determine what proportion of missense changes might be expected to be charge-changing, we determined all possible single nucleotide variant events for MYH7 (1935 codons x nine possible SNV codon changes = 17,415 events). For 12,850 possible missense substitution events, we then determined the charge change: 2681 would lead to a negative charge change, 3604 to a positive charge change, and 6565 would be charge neutral, yielding an expectation that 49% of missense substitutions might be charge changing.
The proportion of skeletal muscle myosins in the SRX or DRX states has been deduced from the population of heads parallel or perpendicular to the filament axis, using fluorescence polarization from bifunctional rhodamine probes bound to the RLC. In relaxed rabbit psoas muscle, approximately 70% of all myosin heads are parallel to the fiber axis, while ~30% of all myosin heads are perpendicular (Fusi et al., 2015), implying that the head population comprises 50% blocked heads that are all parallel, 20% docked free heads that are parallel and 30% swaying free heads that are perpendicular. According to the relaxed IHM state model for skeletal muscle (Alamo et al., 2016), the blocked head remains docked while the free head sways away for a time ts and is docked only for a time td. Therefore for a total time t = ts + td, the swaying duty cycle Ds is defined as the ratio ts/t. Ds can be estimated in the relaxed skeletal muscles from measurements of the RLC orientation using fluorescence polarization from bifunctional rhodamine probes bound to the skeletal RLC and cardiac RLC (Fusi et al., 2015) (Kampourakis and Irving, 2015). In relaxed demembranated rabbit psoas muscle ~70% of the heads have their RLC orientation roughly parallel to the fiber axis, similar to those in the IHM structure, while the RLC orientation of the remainder ~30% of heads is more perpendicular, which is inconsistent with the IHM structure (Fusi et al., 2015). For the estimation of Ds and the SRX/DRX ratio we assume a population of N myosin molecules, so as each myosin molecule has one free (FH) and one blocked (BH) head, the sub-populations of free (NFH) and blocked (NBH) heads sum NBH + NFH = 2N. The populations of heads measured in the parallel conformation is Npar = NBH-par + NFH-par, while the populations of heads measured in the perpendicular conformation is Nper = NBH-per + NFH-per. In the relaxed state, blocked heads are docked so NBH-per = 0, therefore Nper = NFH-per. As a result the number of heads in the perpendicular conformation Nper = NFH-per = (ts/t)NFH = DsNFH = DsN while the number of free heads in the parallel conformation is NFH-par = NFH - NFH-per = NFH - (ts/t)NFH = NFH -DsNFH = (1-Ds)N. Therefore the number of heads in the parallel conformation is Npar = NBH-par + NFH-par = NBH-par+ (1 - Ds)N, and as NBH-par = N because blocked heads are docked in the relaxed state, then Npar = (2-Ds)N; in contrast the number of heads in the perpendicular conformation is Nper = DsN. Therefore the relative number of heads in the parallel and perpendicular conformations are respectively Npar/2N = (2 - Ds)N/2N = (2- Ds)/2 and Nper/2N = DsN/2N = Ds/2. Finally the ratio R of heads in the parallel and perpendicular conformation is R= Npar/Nper= (2-Ds)/Ds so we conclude that Ds = 2/(1+R). Therefore in skeletal muscle R = 0.70/0.3 = 2.3 so Ds = 0.60. Assuming that FHs spend 60% of the cycle released and swaying, and 40% docked on the blocked head the estimated ratio of skeletal muscle heads in the SRX and DRX states is SRX/DRX = (2 – Ds)/Ds = 2.3, predicting an estimated 2.3-fold reduction of ATP hydrolysis. As skeletal muscle myosin heads rapidly transition out of SRX into DRX, such as occurs with tetanic activation (Hooijman et al., 2011) (Brito et al., 2011), the changing population of myosin SRX and DRX heads in skeletal muscle achieves energy-saving imposed by the IHM structure but retains the ability to rapidly shift myosin into DRX for force potentiation.
Assessment of the population of cardiac myosin heads based on parallel or perpendicular alignment to the filament axis is more complicated because fluorescence analyses of RLC in relaxed ventricular fibers from rat hearts show both orientations (Kampourakis and Irving, 2015). However, because previous studies show that cardiac myosin heads remain in the SRX state with tetanic fiber activation (Hooijman et al., 2011), we deduced that activation of cardiac muscle only impacts free heads in the DRX state; docked blocked heads in the SRX state are not recruited. Supporting this idea, there is rapid re-ordering and presumably re-forming of the IHM following cardiac contraction, a finding that suggests that the IHM must be regenerated during the relaxation phase that follows every heartbeat (Huxley et al., 1982) (Yuan et al., 2015). Based on the assumption that IHM and SRX states are one and the same in cardiac muscle, we calculated the corresponding Ds and SRX/DRX ratios from the fluorescent analysis results of (Kampourakis and Irving, 2015) that showed both parallel and perpendicular orientations. By further assuming that the myosin parallel and perpendicular populations are roughly similar, a conservative estimate of cardiac Ds is 80% and the SRX/DRX ratio is 1.5. This conservative estimate of Ds = 0.8 was considerable higher than skeletal muscle, that is, most free heads are not interacting with the blocked head, with a smaller SRX/DRX of 1.5. Based on these observations, we suggest that cardiac IHM swaying duty cycle Ds should be considerable higher than skeletal muscle, that is, most free heads are not interacting with the blocked head. With these assumptions, the asymmetric configuration of myosin heads in the cardiac IHM would yield savings of one ATP molecule when the free head is docked to blocked head (SRX) that would be lost when swaying free heads increase ATP turnover (DRX, Figure 1A). Moreover, HCM variants in MYH7, MYL2, and MYL3 that impair IHM regulating interactions, like phosphorylation-dependent destabilization of the RLC-RLC interface (Figure 3—figure supplement 7) would reduce SRX:DRX proportions and consume more ATP and promote additional myosin-actin interactions and force.
Hypertrophic cardiomyopathy:a paradigm for myocardial energy depletionTrends in Genetics 19:263–268.https://doi.org/10.1016/S0168-9525(03)00081-7
A molecular model of phosphorylation-based activation and potentiation of tarantula muscle thick filamentsJournal of Molecular Biology 414:44–61.https://doi.org/10.1016/j.jmb.2011.09.017
Structures of smooth muscle myosin and heavy meromyosin in the folded, shutdown stateJournal of Molecular Biology 372:1165–1178.https://doi.org/10.1016/j.jmb.2007.07.014
Structural implications of β-cardiac myosin heavy chain mutations in human diseaseThe Anatomical Record 297:1670–1680.https://doi.org/10.1002/ar.22973
Hypertrophic cardiomyopathy due to sarcomeric gene mutations is characterized by impaired energy metabolism irrespective of the degree of hypertrophyJournal of the American College of Cardiology 41:1776–1782.https://doi.org/10.1016/S0735-1097(02)03009-7
Hypertrophic and dilated cardiomyopathy mutations differentially affect the molecular force generation of mouse alpha-cardiac myosin in the laser trap assayAJP: Heart and Circulatory Physiology 293:H284–H291.https://doi.org/10.1152/ajpheart.00128.2007
Regulatory light chain mutations associated with cardiomyopathy affect myosin mechanics and kineticsJournal of Molecular and Cellular Cardiology 46:108–115.https://doi.org/10.1016/j.yjmcc.2008.09.126
Conceptual problems in the definition and interpretation of attributable fractionsAmerican Journal of Epidemiology 128:1185–1197.https://doi.org/10.1093/oxfordjournals.aje.a115073
Time-resolved X-ray diffraction studies of the myosin layer-line reflections during muscle contractionJournal of Molecular Biology 158:637–684.https://doi.org/10.1016/0022-2836(82)90253-4
Head-head and head-tail interaction: a general mechanism for switching off myosin II activity in cellsMolecular Biology of the Cell 19:3234–3242.https://doi.org/10.1091/mbc.E08-02-0206
Phosphorylation of myosin regulatory light chain controls myosin head conformation in cardiac muscleJournal of Molecular and Cellular Cardiology 85:199–206.https://doi.org/10.1016/j.yjmcc.2015.06.002
Inference of macromolecular assemblies from crystalline stateJournal of Molecular Biology 372:774–797.https://doi.org/10.1016/j.jmb.2007.05.022
Subtle abnormalities in contractile function are an early manifestation of sarcomere mutations in dilated cardiomyopathyCirculation: Cardiovascular Genetics 5:503–510.https://doi.org/10.1161/CIRCGENETICS.112.962761
Orientation of myosin binding protein C in the cardiac muscle sarcomere determined by domain-specific immuno-EMJournal of Molecular Biology 427:274–286.https://doi.org/10.1016/j.jmb.2014.10.023
Refined model of the 10S conformation of smooth muscle myosin by cryo-electron microscopy 3D image reconstructionJournal of Molecular Biology 329:963–972.https://doi.org/10.1016/S0022-2836(03)00516-3
How do mutations in contractile proteins cause the primary familial cardiomyopathies?Journal of Cardiovascular Translational Research 4:245–255.https://doi.org/10.1007/s12265-011-9266-2
Ablation of cardiac myosin binding protein-C disrupts the super-relaxed state of myosin in murine cardiomyocytesJournal of Molecular and Cellular Cardiology 94:65–71.https://doi.org/10.1016/j.yjmcc.2016.03.009
Slow myosin ATP turnover in the super-relaxed state in tarantula muscleJournal of Molecular Biology 411:943–950.https://doi.org/10.1016/j.jmb.2011.06.051
UCSF Chimera--a visualization system for exploratory research and analysisJournal of Computational Chemistry 25:1605–1612.https://doi.org/10.1002/jcc.20084
Estimability and estimation of excess and etiologic fractionsStatistics in Medicine 8:845–859.https://doi.org/10.1002/sim.4780080709
Effects of hypertrophic and dilated cardiomyopathy mutations on power output by human β-cardiac myosinJournal of Experimental Biology 219:161–167.https://doi.org/10.1242/jeb.125930
The myosin mesa and a possible unifying hypothesis for the molecular basis of human hypertrophic cardiomyopathyBiochemical Society Transactions 43:64–72.https://doi.org/10.1042/BST20140324
CRYSOL – a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinatesJournal of Applied Crystallography 28:768–773.https://doi.org/10.1107/S0021889895007047
X-ray diffraction analysis of the effects of myosin regulatory light chain phosphorylation and butanedione monoxime on skinned skeletal muscle fibersAmerican Journal of Physiology - Cell Physiology 310:ajpcell.00318.2015–.00318.2700.https://doi.org/10.1152/ajpcell.00318.2015
Constitutive phosphorylation of cardiac myosin regulatory light chain prevents development of hypertrophic cardiomyopathy in miceProceedings of the National Academy of Sciences 112:E4138–E4146.https://doi.org/10.1073/pnas.1505819112
Helical order in tarantula thick filaments requires the "closed" conformation of the myosin headJournal of Molecular Biology 342:1223–1236.https://doi.org/10.1016/j.jmb.2004.07.037
Jeffrey RobbinsReviewing Editor; Cincinnati Children's Hospital Medical Center, University of Cincinnati, United States
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
Thank you for submitting your article "Effect of Myosin Variants on Interacting-Heads Motif Explain Distinct Hypertrophic and Dilated Cardiomyopathy Phenotypes" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Harry Dietz as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Sharlene Day (Reviewer #1); Anne Houdusse (Reviewer #3).
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. In general, all reviewers and the Reviewing Editor found the study very interesting but some concerns were raised that need to be resolved. With important modifications, in particular by recognizing the limits of the current knowledge of the interactions in the IHM and the difficulties and inherent limitations of evaluating how a single mutation can impact interactions and function, the article will be of interest for people in the field since there has not been a global description of how the current model of the IHM interactions can result in HCM or DCM disease.
In an attempt to understand the functional consequences of different hypertrophic and dilated cardiomyopathy mutations in human cardiac β myosin heavy chain, the manuscript integrates data from protein modeling using a quasi-atomic model, mutations found in the patient population and protein-protein interactions of the head region in an attempt to establish mechanistic bases for structure-function. Major conclusions are reached concerning the structural pathogenesis of the hypertrophic and dilated cardiomyopathy mutations utilizing a large cardiomyopathy database comprised of 7,427 individuals. The authors use a low resolution model of the interacting heads motif that was previously published to globally identify which residues mutated in these diseases are involved in stabilization interactions. The data show that the hypertrophic and dilated cardiomyopathic variants differentially affect the motor domain and interacting heads motif of the protein, potentially informing the mechanistic interpretations of how the different mutations influence sarcomeric function and power development at the molecular level.
1) A major concern on the part of multiple reviewers was that the relatively low resolution of the published data, and the paucity of rigorously defined interactive residues are not commensurate with the "definite" tone taken by the authors at multiple places in the manuscript when stating their conclusions. That is, there is too much emphasis throughout the manuscript on trying to establish dogma and generalize conclusions. For example, in paragraph three of the Discussion section, the authors speculate that mutations in titin and MYBPC3 could also impact IHM properties and alter relaxation. The majority of mutations in these 2 genes are truncating mutations that are hypothesized to act primarily by loss of function. And since the residues in MYBPC3 and titin that interact with S2 are not known, it's difficult to predict which missense variants would affect these interactions. Proposing that disruption of IHM formation or stability is a common mechanism for mutations in all thick filament genes for HCM seems to be a bit of a leap. It is not clear from the current paragraph, subsection “HCM variants implicated in MyBP-C binding disrupt IHM interactions”, whether the authors here mostly report on what has been previously proposed or whether they have themselves performed a study that agrees with previous reports. In particular, they should report whether the EM model can provide a proposition on the residues likely involved in interaction with MyBP-C and if it is restricted to the second charged ring on the S2. In fact, the impact of MyBP-C on the IHM should also be considered. While cMyBP-C may stabilize the IHM (Zhogbi, Woodhead, Moss and Craig, 2008) and SRX state (McNamara, et al., 2016) the sites of interaction are still highly debated. The structure proposed by Alamo et al. is based on a small subset of contacts in the literature and highly speculative at best. The authors might consider removing this section to avoid further confusion.
The reviewers raise the very legitimate question as to why, in previous manuscripts, the authors state that their previous models didn't have the resolution needed to draw such conclusions. What has led to their changing their minds? In the absence of a convincing argument that addresses this concern, they have simply used a large database to formulate hypotheses that now can be rigorously tested. The limitations of the work presented in this manuscript need to be clearly and explicitly stated in the Discussion.
2) Alamo et al. state "Although there is limited resolution of the cardiac interconnecting inter-molecular interactions between sequential IHMs along the helices of myosin heads protruding from the thick filament backbone, we note that 12 HCM PVs and LPVs are also positioned to alter inter-IHM interactions data not shown) and limit swaying free heads from actin interactions." Can the authors clarify what they believe the resolution to be for these inter-IHM and intra-IHM interactions? Would the inter- and intra-IHM contacts be affected by the fact that the tarantula striated muscle IHM quasi-atomic cryo-EM structure comes from a thick filament with a 4 start helix while the human cardiac thick filament is a 3 start helix?
3) The manuscript makes the assumption that IHM first observed in smooth muscle and then in tarantula muscle is the structural state responsible for the SRX kinetic state first described by Cooke, 2011, in skeletal and then cardiac muscles. While I realize this assumption may be correct, can the authors point to clear experimental evidence showing that IHM and SRX states are one and the same in cardiac muscle as it pertains to the literature?
4) SAXS and model (Figure 2B): The authors compare theoretical scattering curves from their homology model and experimental scattering curve from squid HMM (subsection “Comparisons of the human β-cardiac myosin IHM quasi-atomic PDB 5TBY with existing models”). In order to better compare the two curves, the authors should provide a χ2 and residual curves (resulting from the comparison of the two scattering curves). The authors do not demonstrate with this comparison whether different possible models that could be built within the EM map would be distinguished. The limitation of the model for defining precisely the contact regions within the IHM should be discussed further. The sentence “Although resolution of the IHM structure as derived by cryo-EM does not define specific atomic contacts or individual side chain densities, this model nonetheless identifies molecular interactions.”, is too ambiguous to indicate how confident one can be on the position of various regions that the authors describe as being involved in IHM contacts. For example, what is really known about what forms the RLC/RLC interface (subsection “Charge Change by HCM and DCM variants Impacts the IHM” third paragraph, on MYL2 R58Q)? Is there sufficient resolution to indicate which residues form the interface between these two side chains?
5) The mutations studied here are not clearly identified from the figures and tables. The statistics mentioned in the text (for example subsection “HCM variants altering IHM interactions”) cannot always allow the identification of the specific mutations they correspond to – as an example, in paragraph three: which are the five of eight variants involved in'i' interactions that affect residues that also participate in another IHM interaction? The authors should better introduce how the mutations were classified, which mutations belong with which category – rather than providing% on whether or not they belong to a particular subclass. This could be improved possibly by the figure legend of the table and a clearer reference to the table in which the residues beyond these numbers are presented. It seems important also to describe how many mutations do not fit in these conclusions to evaluate the limits of the prediction this study can provide.
6) Energetic consequences of SRX and DRX states: This part of the result doesn't really exploit much of the authors' model or mutations. It is not clear what the authors are trying to add here that has not been discussed in previous publications or would be relevant with what is presented above. In any case, for the demonstration/calculations/ definition of Ds, the authors should explicitly say that all the parameters and calculations are based on an assumption: the blocked head remains in a parallel configuration and that only SRX or DRX configurations of the myosin is possible (one head blocked / the other one freely able to dock or stay free).
The authors should provide substantial support for this important assumption that the blocked head always stays in the parallel configuration. In any case, this model doesn't seem appropriate here when mutations that would destabilize the blocked head are considered.
All the parameters introduced here do not really make a point relevant about DCM or HCM mutations that justify their Introduction in the paper, as currently written.https://doi.org/10.7554/eLife.24634.038
- Jonathan G Seidman
- Christine E Seidman
- Raúl Padrón
- James S Ware
- James S Ware
- Richard E Gillilan
- Jonathan G Seidman
- Christine E Seidman
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank Dr. Neal Epstein for initial suggestions regarding HCM variants on interaction ‘a’, Dr. Tom Kirchhausen for early help in this project, Dr. Hind AL-Khayat for permission to use the human cardiac thick filament 3D-map (EMD- 2240), and Dr. Gustavo Márquez for his manuscript assistance. This work was supported by the Leducq Foundation (11-CVD-01; CES and JGS), the Wellcome Trust (107469/Z/15/Z; JSW), the MRC (JSW), NHLBI (HL084553; JGS) and the Howard Hughes Medical Institute (CES and RP). Centro de Biología Estructural del Mercosur (www.cebem-lat.org) (to RP) Cornell High Energy Synchrotron Source (CHESS) is supported by the NSF and NIH/NIGMS via NSF award DMR-1332208, and the MacCHESS resources are supported by NIGMS award GM-103485 (to REG). We dedicate this paper to the memory of Dr. Guillermo Whittembury.
- Jeffrey Robbins, Reviewing Editor, Cincinnati Children's Hospital Medical Center, University of Cincinnati, United States
- Received: December 24, 2016
- Accepted: May 5, 2017
- Version of Record published: June 13, 2017 (version 1)
© 2017, Alamo et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.