THE ANATOMICAL RECORD 297:1670–1680 (2014)

Structural Implications of b-Cardiac Myosin Heavy Chain Mutations in Human Disease MELANIE COLEGRAVE AND MICHELLE PECKHAM* Faculty of Biological Sciences, School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom

ABSTRACT Over 500 disease-causing point mutations have been found in the human b-cardiac myosin heavy chain, many quite recently with modern sequencing techniques. This review shows that clusters of these mutations occur at critical points in the sequence and investigates whether the many studies on these mutants reveal information about the function of this proC 2014 Wiley Periodicals, Inc. tein. Anat Rec, 297:1670–1680, 2014. V

Key words: b-cardiac myosin; cardiac disease; skeletal muscle disease; point mutation

INTRODUCTION Mutations in sarcomeric proteins are well known to cause hypertrophic cardiomyopathy (HCM; Ho, 2012). The two most common proteins to be affected are the heavy chain of b-cardiac myosin, and myosin-binding protein C (MyBPC), but many other sarcomeric proteins, including cardiac actin, myosin light chains, tropomyosin, and troponin, have also been implicated in hypertropic cardiomyopathy, leading to the idea that the development of this disease is due to a problem with sarcomere organization and contractile function. b-Cardiac myosin comprises two heavy chains, and four light chains, of which two are essential, and two are regulatory. It is expressed in cardiac and slow skeletal muscle and the interaction of the myosin head (or crossbridge) with actin is responsible for muscle contraction. Structurally, this myosin is similar to the other skeletal muscle myosin isoforms in the subfamily of Class 2 myosins. It contains an amino-terminal motor domain, which binds to actin and to nucleotide (ATP, ADP, and Pi). Hydrolysis of ATP and subsequent release of Pi, followed by ADP drives muscle contraction. Following the motor domain is the neck, an a-helix to which the two light chains bind. This domain forms the lever, which amplifies small movements in the motor domain into a large movement. The motor domain and lever together make up the myosin head [Subfragment 1 (S-1)]. The lever runs into the tail, which is composed of Subfragment 2 (S-2), and light meromyosin (LMM). In the tail, the two a-helical C 2014 WILEY PERIODICALS, INC. V

heavy chains dimerize to form a coiled–coil. Repeating patterns of charge in LMM are responsible for selfassembly of myosin molecules into filaments in a highly precise manner, in which there are nine myosin heads every 42.9 nm, and a thick filament contains precisely 296 molecules. The thick filaments are incorporated into the muscle sarcomere where they interdigitate between actin filaments allowing myosin crossbridges to interact with actin in a stereospecific manner. The majority (62%) of the HCM mutations in b-cardiac myosin are found in the motor domain and lever, with the remainder evenly distributed in the S-2

Abbreviations used: DCM 5 dilated cardiomyopathy; ELC 5 essential light chain; HCM 5 hypertrophic cardiomyopathy; LDM, Laing’s distal myopathy; LMM 5 light meromyosin (myosin tail); MSM 5 myosin storage myopathy; MyBPC 5 myosin-binding protein C; RLC 5 regulatory light chain; S-1 5 Subfragment 1 (myosin motor domain); S-2 5 Subfragment 2; SM 5 skeletal myopathy. Additional Supporting Information may be found in the online version of this article. *Correspondence to: Michelle Peckham, University of Leeds, School of Molecular and Cellular Biology, Faculty of Biological Sciences, Miall Building, Leeds, LS2 9JT, United Kingdom, Fax: 0044(0)1133434228. E-mail: [email protected] Received 10 February 2014; Accepted 9 April 2014. DOI 10.1002/ar.22973 Published online in Wiley Online Library (wileyonlinelibrary. com).

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and LMM regions of myosin (Figs. 2 and 3, Supporting Information Table 1). Approximately 20% of all the known mutations reside in the filament-forming region of the molecule, LMM, the first of which were described in 2002 (Blair et al., 2002). In this review, we have analyzed the positions of the known disease causing mutations, and show that they cluster to important areas of the myosin structure, we summarize some of the available information on the effects of these mutants and discuss how this information reveals important details about the way this motor protein works.

Structure of S-1: A Brief Overview Although to date there are no published crystal structures for the b-cardiac myosin motor domain, the crystal structures for a number of other myosins (smooth, skeletal, and nonmuscle dictyostelium myosin; Smith and Rayment, 1995; Dominguez et al., 1998; Houdusse et al., 1999, 2000) have been solved. An unpublished structure of the motor domain for b-cardiac myosin has also been uploaded to the protein database (4DB1). These and other structures in the database were used to create a model structure of the b-cardiac myosin motor domain and light chain binding region using iTASSER (Fig. 1B). The structure of the motor domain, originally described in 1993 (Rayment et al., 1993), consists of a number of a-helices, surrounding a core of seven-stranded b-sheet, which is close to the nucleotide-binding pocket. The convention of naming the three main subdomains as the N-terminal 25 kDa, the central 50 kDa, and the C-terminal 20 kDa domain arises from earlier work, in which trypsin was found to cleave S-1 into these three domains. The N-terminal region contains the 25 kDa domain, which consists of an “SH3”-like fold, and the remainder of this domain contributes to six of the seven strands in the central b-sheet, and the “GESGAGKT” motif for the well-conserved phosphate binding loop at the base of the nucleotide-binding pocket. The junction between the 25 kDa and central 50 kDa domain lies within Loop 1, just after the phosphate-binding loop. This domain is separated into two functional domains known as the upper and lower 50 kDa domains. It contains two motifs termed Switch 1 and Switch 2, originally identified in RAS GTP proteins (Smith and Rayment, 1996). These two loops are close together when ATP is bound, and move apart after phosphate is released and ADP is bound, thus conveying information about the nucleotide binding state to the rest of the molecule. Loop 1 lies above the nucleotide-binding pocket, and is has been suggested to modulate the ATPase kinetics of the motor, and in particular the rate of ADP release (Uyeda et al., 1994). The actin binding regions are predominantly found in regions of the 50 kDa domain (Fig. 1B), with most in the ˚ ) EM lower 50 kDa domain. A recent high resolution (8 A reconstruction of myosin bound to actin filaments decorated with tropomyosin (Behrmann et al., 2012) identified that the interaction between myosin and actin involves several regions on the motor domain. Loop 2 and helix W or HW, and the CM loop (in the upper 50 kDa subdomain), interact with Subdomains 1 and 3 of actin. Loop 3 (in the 50 kDa subdomain) interacts with Subdomain 1 of the neighboring actin. A further helix– loop–helix (HR–loop–HS, in the 50 kDa subdomain)

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interacts with Subdomains 1 and 3 of actin, and with Subdomain 2 of the neighboring actin. Binding to F-actin induces a closure of the cleft between the upper and lower 50 kDa domains. The N-terminal region of the 20 kDa domain forms a long helix (the relay helix) that interacts with residues in both the lower 50 and 25 kDa domains, running from the actin-binding region to the fifth strand of the b-sheet. It then forms a turn and broken helix, in which there are two reactive thiols (SH1 and SH2), followed by the converter domain and short so-called pliant region and the remainder of this domain forms a further long a helix to which the essential and regulatory light chains (RLCs) bind, forming the lever (Fig. 1B). Differences in surface loops and domains within the motor modify the function of the motor allowing each isoform to adapt its performance according to the specific muscle environment in which it is expressed (Onishi et al., 2006).

Mutations in MYH7 and Disease Mutations in b-cardiac myosin heavy chain (MYH7) cause both cardiac and skeletal muscle diseases. The first mutation to be linked to disease was the R403Q mutation in the myosin heavy chain, which causes HCM (Geisterfer-Lowrance et al., 1990). Since then many other mutations have been found in the heavy chains that also cause HCM (Supporting Information Table 1). A search of the publications database (up to the end of December 2013), together with mutations listed on the Human Genome Mutation Database show that there are almost 400 mutations described for the MYH7 gene that cause HCM. This compares to just over 200 mutations around 5 years ago (Buvoli et al., 2008), described on the website MyoMAPR (http://bmf.colorado.edu/myomapr/). It is likely that even the 4001 mutations identified in this work are simply a snapshot and numbers will continue to increase over the next few years, making it difficult to keep free on-line repositories up-todate. It is important to note that it is not always clear whether the identified mutation is causal, or whether it increases susceptibility to the disease and other factors are additionally required. Once mutations are included that cause the other heart diseases such as dilated cardiomyopathy (DCM; McNally et al., 2013), endocardial fibroelastosis (Kamisago et al., 2006), left ventricular noncompaction (Hoedemaekers et al., 2013), and Ebstein’s anomaly (van Engelen et al., 2013), and others that cause skeletal muscle disease such as Laing’s distal myopathy (LDM; Lamont et al., 2006), myosin storage myopathy (MSM; Laing et al., 2005), hyaline body myopathy (Bohlega et al., 2004), and multiminicore disease (Cullup et al., 2012), over 500 mutations have been identified. Very few of these mutations have been investigated at the molecular level. The majority of those that have been investigated cause HCM. HCM affects at least 1 in 500 of the population (Ho, 2012) and is the most common cause of sudden death in the under-30 age group (Maron et al., 2004). DCM is the second most common disease arising from mutations in the MYH7 gene and his disease affects 1 in 2,500 people (Taylor et al., 2006). Further mutations are linked to the skeletal muscle myopathies, LDM; a disease that causes early onset weakness (Lamont et al.,

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Fig. 1. (A) Schematic of the three main regions of myosin: subfragment 1 (S-1), subfragment 2 (S-2), and light meromyosin (LMM). (B) A model structure of the b-cardiac myosin motor domain (constructed using I-Tasser; Roy et al., 2010). Helices are shown in red, and key functional regions of the motor are labelled (for more info; see text).

(C) A representation of the folded form of myosin, showing the two heads (blocked and free) and how they fold back and interact with the S-2 region of myosin (coiled–coil). The three charged rings are indicated (open circles; based on 3DTP, Alamo et al., 2008).

2006), and MSM, that manifests as subsarcolemmal hyaline bodies in Type 1 muscle fibers and proximal muscle weakness (Shingde et al., 2006).

While the HCM phenotype is initially characterized by hyperdynamic behavior and impaired diastolic relaxation of hearts from patients, secondary contractile

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Fig. 2. Graph showing the pattern of mutations that occur along the length of the b-cardiac myosin heavy chain amino acid sequence. The total numbers of mutations for each stretch of 50 amino acids (for S-2 and S-2) and for 100 amino acid stretches (for LMM) are shown. Key functional regions of the motor are identified. Mutations for HCM, DCM and SM (skeletal myopathies) are shown.

dysfunction is observed in many human samples that can mask the initial effect of the mutation (Elliott and McKenna, 2004; Force et al., 2010). This can make it challenging to discover the effects of the mutation and if they are causal for disease. Moreover, research using patient tissue does not always consider how much mutant myosin is expressed compared to normal myosin in hearts from patients. One study has suggested that the levels of mutant myosin can be low (20% or less) of the total myosin (Nier et al., 1999) for two different mutations (V606M, G584R). A second study has shown that the relative amounts of mutant mRNA can vary from 67% (R723G) to 29% (V606M) of the total mRNA (Tripathi et al., 2011). Assuming this translates to levels of protein, clearly different mutations may be expressed at different levels in the hearts of patients, and this can contribute to the expression of the disease phenotype. Finally, an intriguing recent finding is that the relative amounts of wild type and mutant myosin can vary from

cell to cell, which could result in a variation in force output from cell to cell, helping to contribute to the myocyte disarray that is typical of this disease (Montag et al., 2014). The many studies of the first HCM mutation to be discovered (R403Q) also help to demonstrate how our ideas about the effects of mutations have developed. This mutation is in the myosin head in the cardiomyopathy loop, a region of myosin that binds to actin, and would be expected to affect the interaction between myosin and actin. The first investigation of the R403Q mutation, using b-cardiac myosin purified from cardiac and soleus muscle biopsies (and thus a mixture of mutant and wildtype myosin), demonstrated that this mutation decreased the velocity of actin filament movement of actin filaments in “in vitro” motility assays by 50% (Cuda et al., 1997). A study of the force generation of slow fibers isolated from patients with the R403Q mutation showed that isometric tension was reduced (by

Fig. 3. Mutations in S-1 and S-2 found in HCM, DCM, skeletal myopathies and other cardiomyopathies (see Supporting Information Table 1 for a list of the mutations). In Subfragment 1, functional regions of the motor and lever are indicated. In Subfragment 2, the

acidic and basic residues are indicated in red and blue, respectively, a skip residue is in green, and the heptad repeat of the coiled–coil sequence is indicated below the amino acid residues. “P” indicates a mutation to proline, D indicates a “stop” codon.

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18%), and that about 50% of the myosin expressed was the mutated R403Q isoform (Malinchik et al., 1997). After 3 years, again using myosin purified from heart and muscle biopsies, it was demonstrated that this mutation increased the velocity by 30% (Palmiter et al., 2000). This later study also showed, through single molecule measurements, that the mutant myosin motor spent a lower average time (ton) attached to actin during its ATPase cycle, detaching faster than normal myosin, explaining the faster velocity. A subsequent study, in which the R403Q mutation was introduced into smooth muscle myosin that was recombinantly expressed using the baculovirus system again showed an increased motility, and a reduced length of attachment time (Yamashita et al., 2000). Further studies using isolated myofibrils from the hearts of patients with the R403Q mutations showed that the myofibrils generated a lower maximal isometric tension compared to those from a normal heart, as well as faster rates of tension generation and relaxation (Belus et al., 2008). Their data showed that the turnover rate for myosin crossbridges was faster for the R403Q mutation. The three-fold increase in the rate of crossbridge detachment for the R403Q mutant myofibrils mostly accounts for the observed lower maximal isometric tension. As each crossbridge cycle uses one molecule of ATP, the energetic costs for contraction are also likely to be higher for the R403Q mutant, in that less force is generated per ATP. Thus, while it appears that there is an apparent gain of function for the R403Q mutation, in that velocity increases, and rates of force generation and relaxation also increase, there is also an increased energetic cost for tension generation and force output is reduced, and it is these features that are likely to elicit the hypertrophic phenotype, at least for the R403Q mutation. More recent experiments using myofibrils, for a range of different mutations in b-cardiac myosin heavy chain, myosin binding C-protein, tropomyosin and troponin (Witjas-Paalberends et al., 2013) again showed reduced force output for mutants. While it remains to be seen whether there really is a common trend for the effects of mutations in sarcomeric proteins in general on force output, the conclusion from this study was that all eleven mutations in b-cardiac myosin heavy chain studied resulted in a hypocontractile phenotype. This comprehensive study also found that cardiomyocyte hypertrophy was accompanied by a decrease in myofibrillar density, suggesting that these factors also contribute to the decrease in maximal force of cardiomyocytes, but they conclude that these may be secondary effects with hypocontractility at the myofibrillar level being the initial trigger, at least for mutations in b-cardiac myosin heavy chain. This may be different to mutations in other sarcomeric proteins, such as troponin, that have been suggested to result in hypercontractility due to changes in Ca21 sensitivity (Redwood et al., 1999; Marston, 2011).

Mutations in S-1 Analysing all the mutations (Supporting Information Table 1), we observed that 62% of all mutations in b-cardiac myosin heavy chain associated with HCM are found in S-1, which is composed of the motor domain

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and lever (Figs. 2 and 3). This is similar to that described in an earlier analysis of the distribution of mutations in the b-cardiac myosin heavy chain (Buvoli et al., 2008). Even though almost 20% of the 838 residues (myosin S-1) harbor one or more mutations, and we have investigated 500 mutations, it is still possible that these may only be a small fraction of those that exist (Hamady et al., 2010) and it is clear that the b-cardiac myosin heavy chain is highly sensitive to mutations (Buvoli et al., 2008). From a molecular point of view the location and nature of these mutations can provide an important insight into the structure and function of the myosin motor protein. These mutations must confer subtle alterations in muscle function otherwise they would be lethal. An analysis of the positions of these mutations against their amino acid location shows several “hot spots” or peaks where mutations are more common, and these tend to be in key functional areas (Fig. 2). Analysing the positions of mutations in S-1 shows that there are significant numbers of mutations in Loop 1, in which half of the residues are associated with missense mutations. In regions of the myosin that interact with actin, mutations are found most commonly in the HCM loop, in which almost half of the residues have been shown to be mutated in HCM. Mutations in the relay helix, and the proximal region of the converter are also common, as well as in the plaint region, and the lever. Interestingly, although we have analyzed over twice as many mutations, the mutation hotspots are somewhat similar to those of an earlier, similar study (Buvoli et al., 2008). It seems likely that this pattern is genuine, and that the majority of the identified mutations are likely to affect force output either by affecting aspects of the ATPase cycle, the interaction of myosin with actin, or movement of the lever. Areas where mutations are less common could highlight areas where a mutation could cause a lethal phenotype, but it is more likely that residue changes in these regions do not alter the nature of the protein enough to cause disease.

Mutations in S-2 20% of the mutations in b-cardiac myosin heavy chain associated with HCM are found in S-2 (Supporting Information Table 1 and Fig. 2). S-2 is composed of coiled– coil. There are 82 HCM mutations and 17 DCM mutations. The proximal region of S-2 (Residues 838–877) contains 27 (or 27%) of all of the mutations in S-2, of which most cause HCM. Structural studies of the S-2 have revealed that the first 12 residues of one of the chains in this region of coiled–coil are disordered while the other straightens without the coiled–coil interactions (Blankenfeldt et al., 2006). This allows the S-2 to be flexible and for the heads of myosin to be nonsymmetrical, important in allowing the heads to point away from the myosin filament. In this proximal region there appear to be some intra-helical interactions between charged residues (E, K, and R), for example, R845-E848 (i,i 1 3) and E854-R858 (i,i 1 4). This is somewhat reminiscent of the proximal region of the coiled–coil domain of kinesin (reviewed in Peckham, 2011), in which the two helices in this region of the coiled–coil are able to separate, but maintain their helical structure due to charged intrahelical interactions. A charge change in R845 (to E)

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causes HCM and could potentially abolish one of these intrahelical interactions. Mutations in R858 are common (4), although none is a change to E. As this proximal region is not involved in interacting with the motor domain or other protein, the cluster of mutations in this region is likely to affect helix stability. Two further clusters of mutations occur in the first two of three negatively charged rings (10% and 13%, respectively) found in the proximal region of S-2 (Blankenfeldt et al., 2006) (Figs. 2 and 3). Mutations in the first charged ring may affect the ability of the motor domain to “park” the myosin heads onto the thick filament between contractions. Cardiac myosin adopts a compact form in the filament, where the two myosin heads contact each other, and fold back to interact with S-2 (Zoghbi et al., 2008). In this “parked” myosin, one head is “free” and one head is blocked (Fig. 1C). It has been suggested that there is an interaction between positively charged residues in Loop 2 in the motor domain in the blocked head and the first negatively charged ring of S-2 (Blankenfeldt et al., 2006). This interaction is likely to be important in maintaining the relaxed conformation of myosin in the thick filament, and mutations in the first charged ring may lead to more disordered heads, and perhaps some dysregulation of contraction. There is also an interaction between the HCM loop in the “free” head and S-2. The second charged ring is likely to be a site of interaction for a myosin binding protein, cMyBPC (cardiac MyBPC; Gruen and Gautel, 1999). Both R870H (“a” position in the coiled–coil) and E924K (“f” position) mutations reduce cMyBPC binding to the S-2 domain (Gruen and Gautel, 1999), and E924K, E927K, E930K, and E935K mutations are all found in the second charged ring, and are thought to reduce binding of cMyBPC to this region. cMyBPC is found in stripes every 43 nm (equivalent to nine myosin molecules) along the A-band in skeletal muscle, and cMyBPC is thought to modulate contraction. Thus, mutations in the second charged ring have the potential to affect the interaction of cMyBPC with S-2, and thus modulation of contraction by cMyBPC.

Mutations in LMM The LMM of myosin is a parallel coiled–coil formed by two a-helices and this part of the molecule incorporates into the thick filaments of the muscle sarcomere. The remaining 17% of mutations that cause HCM lie within LMM (Figs. 2 and 4, Supporting Information Table 1). In this region there are 65 mutations that cause HCM, 22 that cause DCM, and 22 skeletal myopathy (SM) mutations have been described for LMM. The variation in the types of disease that these mutations cause is interesting because their location in the tail and filament forming region of myosin suggests that the way they cause dysfunction might be different from those that occur in the motor. There is also the possibility that in the heart, there could be a change in isoform redistribution, where mutant b-cardiac myosin heavy chain is exchanged for a-cardiac myosin heavy chain, as has been demonstrated for tropomyosin mutations (Ochala et al., 2008). Mutations are again clustered into two regions in LMM (Fig. 2). There is a broad peak from 1,200–1,450

(which contains mostly HCM mutations) and a second peak in the last 200 residues of the tail (which contains mutations for HCM, DCM, and SM). The region of the tail thought to bind myomesin (1506–1674, Obermann et al., 1997) and MyBPC (1554 and 1581, Flashman et al., 2007) seems to have a reduced incidence of mutations. Does this reflect the fact that if these areas are altered and these proteins cannot bind then the effect is a lethal phenotype? In contrast, the broader peak, close to the C-terminus contains the region of LMM to which titin is thought to bind. Moreover, this region contains the so-called assembly competent domain which is required for filament formation, although mutations in this region are not as common. Mutations in LMM have generally been investigated by determining the ability of the coiled–coil to form normally, and the ability of the mutant rods to form thick filaments and incorporate successfully into the sarcomere. An essential characteristic of the coiled–coil is a repeating heptad of the sequence abcdefg where a and d are usually hydrophobic and are buried by the interaction of the two alpha-helices (Parry et al., 2008). Skip residues interrupt the repeating heptad sequence, at residues T1188, E1385, E1582, and G1807 and these are thought to correspond with bends in the myosin tail in isolated molecules (Offer, 1990). Mutations occur in each of the residues in the heptad repeats, although mutations in the “d” position are not so common. Single point mutations in full length LMM (E1356K, L1793P, R1845W, E1886K, H1901L, R1500P, and R1500W) have been shown not to markedly destabilize the structure of a full-length coiled–coil, although they can modify thermal stability (Armel and Leinwand, 2009, 2010a, 2010b). However, other mutations (N1327K, E1356K, R1382W, E1555K, and R1768K and R1500W) investigated in smaller fragments of just a few heptad repeats do affect helicity (Wolny et al., 2013). That study also suggested that the region close to N1327K might be an important region for coiled–coil formation (Wolny et al., 2013), and this region is close to a mutation “hot spot” in the LMM tail for HCM. Three of those mutants (N1327K, E1356K, and E1555K) showed reduced incorporation into sarcomeres in rat adult cardiomyocytes (when expressed as full length b-cardiac myosin heavy chain tagged with eGFP, Wolny et al., 2013). This suggests that small effects on helicity can affect the ability of myosin to incorporate into thick filaments in vivo. Two mutations implicated in LDM (R1500P and L1706P) mutants formed aggregates when a GFP-tagged myosin rod domains were expressed in cells (Buvoli et al., 2012), These data suggest that subtle changes in the stability of the coiled–coil might influence the incorporation of myosin into muscle filaments. However, proline residues are not found in coiled coils, as they induce a sharp bend into the a-helical structure that is incompatible with coiled–coil formation. Thus mutations to proline in S-2 or LMM would be expected to disrupt the coiled–coil structure at least locally. Differences in stability of the coiled–coil arising from these mutations could then cause compensatory changes in the heart muscle in HCM and DCM, and result in aggregation of myosin outside the muscle sarcomeres in the skeletal muscle diseases LDM and MSM leading to muscle weakness.

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Fig. 4. Mutations in LMM associated with heart and skeletal muscle disease (see Supporting Information Table 1 for a list of mutations).

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RLC and Essential Light Chain Mutations Mutations that lead to HCM have also been discovered in both the regulatory and essential light chains (ELCs), with 16 reported mutations in the RLC (MYL2) and 15 in the ELC (MYL3) in the HGMD database (December 2013). The light chains both bind to the neck of myosin S-1 and are important in stabilizing the lever arm of the myosin motor. They are both EF-hand proteins similar in structure to calmodulin (Grabarek, 2011). The ELC has lost the ability to bind calcium while RLC usually only binds magnesium at its N-terminal EF-hand. It appears that the RLC mutations might affect the function of the myosin protein directly as they have all been shown to decrease the force of the motor (Kerrick et al., 2009; Greenberg et al., 2010) and so may be involved in compensatory cardiomyopathy. One RLC mutation D166V also increased calcium sensitivity of muscle fibers (Kerrick et al., 2009). The ELC mutations studied have all shown a lower affinity for the heavy chain IQ domain, where the light chain is expected to bind (Lossie et al., 2012). Lower incorporation into sarcomeres was also seen with one mutant. This suggests that force generation of the motor might be affected by altering the elastic properties of the lever arm. In addition, the RLC has been shown to interact with LMM in compact folded molecules, where Cys109 can be cross-linked to residues 1,555 and 1,584 in smooth muscle (reviewed in Sellers and Knight, 2007). It has been suggested that basic residues close to the N-terminal domain of the RLC could interact with acidic residues in LMM in this region. Interestingly, the E1555K mutation in HCM, reduces the local net charge of this region of the molecule by 4, and cardiac myosin has also shown to be able to form a compact molecule (Jung et al., 2008), perhaps to enable transport newly formed myosin to its correct place in the muscle sarcomere. Thus one further possibility to consider is that some mutations in the RLC may affect the formation of a compact structure.

DISCUSSION The aim of this review was to investigate the location of mutations along the b-cardiac myosin heavy chain and has identified that mutations tend to appear in functional hotspots. This analysis is timely because many mutations have been discovered in the last 2–3 years due to the development of high throughput sequencing. The location of the mutations tells us very little without reference to the structure of the protein and the cellular and biochemical studies that have looked at the functional effects of mutations. Slowly these studies can reveal information about the function of b-cardiac myosin in the muscle sarcomere. These mutants while revealing interesting information about the structure and function of b-cardiac myosin may also in time help develop treatments for these devastating diseases. Although the number of mutants that have been investigated to date are only a small proportion of the total number so far discovered, they demonstrate that there are a wide range of ways in which the mutants cause disease and these can be different even in the same disease. The most studied mutants are from HCM and these show alterations in the function of the isolated motor, regulation of the sarcomere and the

ability to incorporate into filaments. So it is clear that HCM at least occurs from a very diverse range of causes. This review has looked at the consequences of some mutations on the function of the motor protein and the muscle sarcomere, but there are other methods by which mutations can cause disease. One of these, haploinsufficiency, has been described for cMyBPC, another gene implicated in HCM (Pezzoli et al., 2012), in which one copy of the gene is inactivated by the mutation. Most of the mutations are heterozygous but two homozygous mutants have been investigated (Keller et al., 2004; Alpert et al., 2005). The ratio of the mutant protein that is actually expressed with the wild type in heterozygous patients has been shown to vary and is not 50% but often higher (Becker et al., 2007). The ratio of mutant to wild-type protein affects the function of muscle fibers and is directly related to the clinical features of the affected patient (Di Domenico et al., 2012); an increased ratio of mutant protein presents as a more severe disease. The amount of mutant protein expressed has been shown to be the same in the left and right ventricles in one patient, so in this case the thickening of the left ventricle wall cannot be due to the mutant protein alone (Borchert et al., 2010). A study investigating prehypertrophic mutation carriers suggests the at least for the R403W and A797T mutants a deficit of ATP might be the cause (Revera et al., 2008). In terms of functional studies of all these mutants much remains to be discovered. Mutations in some regions of b-cardiac myosin heavy chain have not really been investigated. From our graph of the locations of the mutations it is surprising that the pliant region of myosin has not been investigated especially as this is a critical point in the myosin motor where the lever arm hinges and has a high number of mutations. The limitations on investigating b-cardiac myosin heavy chain mutations because of the difficulty in expressing recombinant protein are slowly reducing, as assays require less protein and novel methods are being devised to study this fascinating protein.

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