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In the 20 yrs since the discovery of the first mutation linked to familial hypertrophic cardiomyopathy (HCM) an astonishing number of mutations affecting numerous sarcomeric proteins have been described. Among the most prevalent of these are mutations that affect thick filament binding proteins including the myosin essential and regulatory light chains and cardiac myosin binding protein-C (cMyBP-C). However, despite the frequency with which myosin binding proteins, especially cMyBP-C, have been linked to inherited cardiomyopathies, the functional consequences of mutations in these proteins and the mechanisms by which they cause disease are still only partly understood. The purpose of this review is to summarize the known disease-causing mutations that affect the major thick filament binding proteins and to relate these mutations to protein function. Conclusions emphasize the impact that discovery of HCM causing mutations has had on fueling insights into the basic biology of thick filament proteins and reinforce the idea that myosin binding proteins are dynamic regulators of the activation state of the thick filament that contribute to the speed and force of myosin driven muscle contraction. Additional work is still needed to determine the mechanisms by which individual mutations induce hypertrophic phenotypes.
In the 20 years since the landmark study of Geisterfer-Lowrance et al.1 that first identified the R403Q mutation in the gene encoding the β-cardiac myosin heavy chain gene (MYH7) as causative for hypertrophic cardiomyopathy (HCM), hundreds of additional mutations in 10 different sarcomeric genes have been linked to the disease (for reviews see2, 3). Like the original R403Q mutation, the majority of these mutations affect the β-myosin heavy chain (MHC), the molecular motor that drives cardiac muscle contraction and that is the primary component of sarcomere thick filaments. However, a glimpse into the vast genetic heterogeneity that underlies HCM was evident even as Geisterfer-Lowrance and colleagues made their initial discovery because another report by the same group demonstrated that HCM in two other pedigrees was not related to MHC genes4. In prescient commentary Geisterfer-Lowrance et al. proposed that loci encoding myosin-associated peptides might also cause disease1. Although it would be another 5 yrs before mutations in myosin binding proteins were eventually identified5, 6, their initial prediction has been overwhelmingly validated and mutations in myosin binding proteins of the thick filament, especially cardiac myosin binding protein-C (cMyBP-C), are now known to occur with a frequency comparable to β-MHC mutations. Collectively, these mutations cause cardiomyopathy, sudden cardiac death or heart failure in millions of people worldwide7. Yet, in contrast to the well-established role of β-MHC in driving muscle contraction, the regulatory significance of the myosin binding proteins, including the myosin essential and regulatory light chains and cMyBP-C, in cardiac contraction is still far from complete. The discovery of HCM mutations in these proteins has therefore provided both an urgent impetus as well as the means to advance our understanding of the basic biology of myosin binding proteins as the functional consequences of HCM mutations become known. The purpose of this review is to highlight disease-causing mutations in the myosin binding proteins of the thick filament as first presaged by Geisterfer-Lowrance and then to highlight how insights from HCM mutations have contributed to understanding of thick filament protein function. The picture that is emerging reinforces the idea that myosin binding proteins are dynamic regulators of the activation state of the thick filament that act in concert with β-MHC to fine-tune the strength and speed of cardiac contraction.
Myosin is a hexameric protein that consists of two heavy chains (MHC) and two pairs of light chains. The heavy chains dimerize forming a coiled-coil helix that makes up the light meromyosin or “rod” segment of myosin. Each MHC unwinds from its partner near its N-terminus and folds separately into one of the two catalytic S1 cross-bridge “heads” of a myosin molecule. At the tail end of each S1 head the MHC forms a long 8.5 nm α-helical segment, the lever arm domain, whose movement transduces energy from the hydrolysis of ATP into motion and force. Two light chains, i.e., one each essential light chain (ELC) and regulatory light chain (RLC), bind in tandem to the lever arm domain and thus occupy key positions that not only stabilize the lever arm8 but also act to position the myosin heads relative to the myosin rod and the actin filament9. By orienting the myosin heads at this juncture, the light chains thus can modulate the speed and force of contraction as in cardiac muscle10 or, in the case of regulated myosins such as those of smooth and invertebrate muscle, to turn contraction “on” or “off” altogether in response to a rise in intracellular Ca2+ as Ca2+ either binds directly to a trimeric complex formed by the ELC, RLC, and MHC (invertebrate muscles) or activates a Ca2+/calmodulin-dependent myosin light chain kinase that phosphorylates RLC (smooth muscle).
A single thick filament consists of ~300 myosin molecules (each consisting of 2 MHC, 2 ELC, and 2 RLC) that assemble with their rods facing inward to form a bipolar filament. Multiple thick filaments are aligned to create A (anisotropic)-bands such that thick filament rod segments delineate a bare zone devoid of cross-bridges (the H-zone) centered at the M-line in sarcomeres (Figure 1). Additional myosin binding proteins bind along the length of the thick filament starting at the M-line and repeat outward toward the Z-lines at intervals every ~42 nm, or roughly coincident with every third crown of myosin heads emerging from the thick filament11. Myosin binding protein-C (MyBP-C)12, the best known of these myosin binding proteins, is localized to a series of 9 of these positions on each side of the A-band11, 13. Thus, cMyBP-C is present at a limited stoichiometry with respect to myosin with ~1 MyBP-C for every 9–10 myosin molecules (~37 MyBP-C molecules per thick filament) or 2–4 MyBP-C molecules at each position12. At each position MyBP-C is anchored to the thick filament through three domains at its C-terminus that bind to both myosin and titin14–16. Titin, the giant protein that spans each half sarcomere from Z-disk to M-line, also binds to myosin along the length of the thick filament16.
While the positions of ELC and RLC relative to myosin are known from the S1 crystal structure8, the precise arrangement of cMyBP-C with respect to myosin is still uncertain. The C-terminus of cMyBP-C has alternately been proposed to encircle the thick filament in a trimeric collar arrangement17 or to extend linearly along the thick filament backbone18. In addition, a second binding site near the N-terminus of MyBP-C can also bind to myosin S2, the hinge segment that joins the catalytic heads to the myosin rod19. If so, then MyBP-C, like ELC and RLC, binds to myosin near the critical junction where the lever arm domain meets the myosin rod. Binding of cMyBP-C at this position has been proposed to limit the extension of myosin heads away from the thick filament backbone, thereby limiting interactions of MyBP-C with actin20. On the other hand, the same region of cMyBP-C can also bind reversibly to actin21, consistent with previous observations that MyBP-C binds to actin and thin filaments22, 23 and with conclusions from modeling of X-ray diffraction data suggesting that MyBP-C interacts with thin filaments in muscle18. If so, then cMyBP-C interactions with actin could be similar to those proposed for myosin ELC since interactions of the ELC N-terminus with the thin filament may also limit cross-bridge cycling and shortening velocity24. In either case, it is clear that thick filament associated proteins are well positioned to influence myosin cross-bridge kinetics.
Mutations that cause HCM have been found in genes encoding all major thick filament binding proteins including ELC and RLC, cMyBP-C and titin. Of these, mutations in MYBPC3, the gene encoding cMyBP-C, are by far the most common with a total number and frequency comparable to that of mutations affecting β-MHC2. Together mutations in genes encoding cMyBP-C and β-MHC thus account for >60% of HCM cases with an identified genetic cause. To date, 197 MYBPC3 mutations have been reported (Figure 2 and Online Table I) which are responsible for disease phenotypes ranging from asymptomatic to progressive hypertrophy and heart failure to sudden cardiac death (SCD). Compared to mutations in β-MHC, however, a greater number of the cMyBP-C mutations are associated with an overall more benign clinical course, a later average age at onset of symptoms, and lower incidence of SCD2. Nonetheless cMyBP-C mutations are a significant cause of morbidity and mortality for millions of people worldwide. For instance, a single cMyBP-C founder mutation in people of South Asian descent renders ~4.5% of the Indian population (>40 million people) up to 7 times more at risk for development of cardiac dysfunction and heart failure than non-carriers7. While individuals affected with this mutation are typically symptom free until the third decade of life, 90% of older individuals eventually develop cardiac complications. Another founder mutation (Trp729fs) is prevalent in Dutch families25. By contrast, mutations in ELC, RLC, and titin are much less frequent and occur in less than 5% of HCM patients26. Indeed, only a single mutation in TTN, the gene encoding titin, has been linked to HCM27. On the other hand, mutations in TTN are more frequently associated with inherited dilated cardiomyopathy (DCM) i.e., hypertrophy characterized by ventricular chamber dilation, wall thinning and impaired systolic function28. By contrast, the prevalence and number of MYBPC3 mutations linked to DCM is low2 and ELC and RLC mutations have thus far not yet been linked to DCM.
Two ELC isoforms are expressed in mammalian hearts, ventricular ELC (also referred to as VLC1, ELCv, or MLC-1v) encoded by the MYL3 gene and atrial ELC (ALC1, ELCa, or MLC1a ) encoded by the MYL4 gene (for reviews see24, 29, 30). To date, HCM mutations have only been linked to the ventricular isoform (MYL3). Mutations in MYL3 were first linked to HCM in 1996 by Poetter et al.31 who identified 2 missense mutations (M149V, R154H) in conserved residues of exon 4. The mutations were associated with marked papillary muscle hypertrophy and an unusual pronounced mid-ventricular wall thickening that was evident as early as childhood in some patients. Arad et al.32 also reported that the M149V mutation was associated with an unusual hypertrophy localized to the cardiac apex. Three other VLC1 missense variants were identified in exons 3 (E56G, A57G) and 4 (E143K)33–35. However, while homozygous inheritance of the latter was associated with mid-ventricular and apical hypertrophy and sudden cardiac death at young ages, inheritance of a single mutant allele was apparently benign and not associated with a disease phenotype35.
All 5 VLC1 mutations thus far linked to HCM are clustered in exons 3 and 4 of MYL3 which encode 2 of the 4 EF-hand domains of ELC36. Both ELC and RLC are members of the EF-hand superfamily of Ca2+ binding proteins but, unlike RLC, ELC isoforms have lost the ability to independently bind divalent cations. Yet, the prevalence of HCM mutations within the EF-hand domains suggests that even though the ability of divalent ions to regulate ELC has been lost through evolution24, that these domains are still critical for the proper structure and function of VLC1. Consistent with this idea, cardiac biopsies from patients affected by the M149V variant showed altered contractile function with enhanced myosin activity evident as increased actin sliding velocity in motility assays31. It will be necessary to determine whether the remaining VLC1 variants similarly increase myosin activity, since ectopic expression of atrial ELC (ALC1) also increases contractility but has been reported to be an adaptive response in stress sed or diseased human myocardium37, 38. In this respect, transgenic replacement of VLC1 with ALC1 in mouse hearts increased unloaded shortening velocity in permeabilized fibers and increased whole heart contraction and relaxation rates, but did not induce hypertrophy39.
Initial modeling of the M149V mutation in transgenic mice was promising since expression of a human genomic DNA fragment encoding the VLC1 gene and the M149V mutation recapitulated the human phenotype including pronounced papillary muscle and mid-ventricular hypertrophy40. However, in another study where the homologous mutation was expressed in a mouse ELC cDNA (mouse M158V), the mutation failed to induce hypertrophic growth despite increasing Ca2+ sensitivity of force and myofibrillar ATPase rates and reducing maximum power output41. Similar results were obtained in transgenic rabbits where the mutation was expressed on a predominantly β-MHC background similar to that of human myocardium42 (Table 1). Reasons for the differing results between the initial results of Vemuri et al40 and subsequent transgenic models have not been resolved, but could serve to highlight some of the difficulties in extrapolating functional results across species in proteins that are tuned to different hemodynamic loads and heart rates.
HCM mutations in the gene encoding ventricular RLC (MYL2) were first identified in 1996 in parallel with mutations in MYL331 and a total of 10 variants have been identified since then. Similar to MYL3 variants, several of the MYL2 mutations were initially associated with an unusual mid ventricular hypertrophy. Of the three missense mutations first identified within highly conserved residues of RLC (A13T, E22K, and P95A (originally reported as P95R)), two (A13T and E22K) were associated with pronounced mid cavity obstruction31. Another N47K variant was also associated with mid ventricular hypertrophy in a Danish family43. However, the mid ventricular phenotype appears variable since other family members showed a more typical HCM phenotype as did other patients with the A13T mutation43. Additional MYL2 variants include a splice site acceptor mutation (IVS6-1G>C) predicted to produce a truncation within the MHC binding site and an L103E missense substitution, but it was unclear whether the latter was causative in disease or simply a rare polymorphism and it has not been reported since43. Other MYL2 mutations include a second splice acceptor site mutation (IVS5-2A>G) and missense variants D166V (originally reported as D166L)33, F18L, and R58Q44. Whereas most RLC mutations appear to lead to mild or benign phenotypes, the R58Q, D166V, and IVS5-2 mutations are associated with malignant phenotypes evident at earlier ages and/or with sudden cardiac death33, 44–46.
The RLCv HCM mutations map primarily to two functional domains within the protein. The first is a EF hand domain (residues 37–48) that retains the ability to bind Ca2+ and Mg2+ 8, 47 and the second is the highly conserved serine 15 which is phosphorylated in vivo by myosin light chain kinase. N47K and R58Q are located within or near the divalent ion binding site, whereas A13T, F18L, and E22K may be in closer proximity to S15. Because the structure of the RLC N-terminus containing S15 was not resolved in the S1 crystal structure8, the positions of A13T, F18L, and E22K are not known with certainty.
Although the functional significance of divalent ion binding is not well understood, phosphorylation of RLC in smooth muscle is the trigger that activates contraction. In cardiac muscle RLC phosphorylation augments force and the rate of force development and recently was shown to affect the overall timing of ventricular systole9, 10, 48. The precise mechanisms by which phosphorylation exerts these effects have remained elusive despite intensive study, but it now seems clear that RLC phosphorylation contributes to activation by disrupting myosin S1 head-head interactions that stabilize the compact 10S “off” state of smooth muscle myosin49–51. Importantly, the head-head interactions, in which one myosin head is positioned so that its actin-binding site is blocked by interactions with the converter and ELC domains of a second head, appear not to be unique to smooth muscle myosins but are instead common characteristics of invertebrate and vertebrate myosins that are assembled into thick filaments16, 51–53. These findings thus provide a structural basis for recent as well as long-standing observations that RLC phosphorylation in thick filaments increases cross-bridge disorder and shifts the distribution of cross-bridge mass away from thick filaments and toward thin filaments9, 54, 55. To date, structural studies have not yet investigated whether RLC HCM mutations affect thick filament structure or cross-bridge distribution, but it is an intriguing possibility that warrants additional study.
While the impact of RLC HCM mutations on thick filament structure is largely unexplored, HCM mutations do appear to affect both the ability of RLC to bind Ca2+ and its ability to be phosphorylated. Moreover, because variants near the phosphorylation site can affect Ca2+ binding and vice versa, analysis of HCM mutations has suggested that communication between these two sites could be important for RLC function. For instance, in one study it was reported that 5 RLC HCM mutations (A13T, F18L, E22K, R58Q, and P95A) all decreased the Ca2+ binding properties of recombinant RLC in solution47. The most dramatic effects were observed for the E22K and R58Q mutations where binding was reduced 17 fold (E22K) or abolished completely (E58Q). However, Ca2+ binding to the R58Q RLC could be restored by phosphorylation, whereas the E22K mutant could no longer be phosphorylated at all. Phosphorylation of wild-type RLC decreased its Ca2+ binding affinity, but phosphorylation of A13T had the opposite effect and increased Ca2+ binding. Thus, effects of Ca2+ binding and phosphorylation appear functionally linked. Furthermore, because exchange of a non-divalent ion binding mutant (D47A) into skeletal muscle fibers decreased Ca2+ sensitivity of force and cross-bridge cycling kinetics (ktr), the ability of RLC to bind divalent ions appears important for the contractile effects of RLC56, 57. Consistent with this idea, when HCM mutants in (rat) RLC were exchanged into permeabilized skeletal muscle fibers, Ca2+ sensitivity of tension was also affected. The E22K mutant significantly increased Ca2+ sensitivity of force, whereas other mutations either decreased Ca2+ sensitivity of force (G13T -rat and F18L) or had no effect (P95A).
The ability of RLC HCM mutations to alter Ca2+ sensitivity of tension and actomyosin interactions has been explored in vivo using transgenic mouse models. Transgenic expression of all variants investigated thus far has led to altered contractile function at the cellular or organ levels (Table 1). However, the direction and magnitude of the effects have been variable and difficult to correlate with Ca2+ binding results observed in vitro. For instance, similar to results in exchanged fibers, transgenic expression of human E22K RLC in mouse hearts led to increased Ca2+ sensitivity of force and ATPase rates in permeabilized fibers58, although contradictory results were later reported by the same group in a separate study showing that there was no effect of the mutation on Ca2+ sensitivity of force59. The E22K mutation had no or negligible effect on cross-bridge kinetics59, 60, but significantly decreased maximal ATPase rates and maximal force in skinned fibers along with decreases in both the magnitude and duration of force and Ca2+ transients in intact fibers59. Based on these results the authors proposed that by reducing affinity of the Ca2+ binding of RLC47 the E22K mutation effectively converted RLC into a delayed Ca2+ binding buffer similar to the low affinity site of TnC59. However, in contrast to the latter results, the duration of Ca2+ transients was increased in fibers from transgenic mice expressing N47K or R58Q variants61. Other functional effects including prolonged force transients, increased Ca2+ sensitivity of force and ATPase rates were evident in R58Q but not N47K fibers61. Transgenic expression of the D166V mutation which is associated with a malignant phenotype in humans had the most pronounced effects on force, including prolonged force transients in intact fibers and increased Ca2+ sensitivity of force in permeabilized myocytes62.
Despite the extensive functional changes in cellular and myofilament contractile properties following transgenic expression of RLC HCM variants, transgenic RLC HCM models have thus far failed to recapitulate human hypertrophic phenotypes (Table 1). For instance, ejection fraction and fractional shortening were unchanged in E22K transgenic hearts and histological analysis of E22K cardiac sections suggested only mild thickening of inter-ventricular septa58 which was only somewhat reminiscent of the mid-cavity hypertrophy reported for humans with the E22K mutation31, 45. However, echocardiography failed to show significant increases in chamber dimensions58. Similarly, none of the other transgenic models of RLC HCM variants led to overt cardiac hypertrophic phenotypes (Table 1). It is unclear why this should be the case, but could suggest that the degree of contractile dysfunction caused by these mutations is not sufficient to otherwise impair cell energetics63 or to trigger a hypertrophic growth response in mice. Nonetheless, more extensive alterations of RLC, such as truncation of the RLC N-terminus, are sufficient to reduce force and cause compensatory hypertrophy in transgenic mice despite overall preserved cardiac function64.
Mutations in MYBPC3, the gene encoding cMyBP-C, were first reported in 1995 in separate studies by Watkins and colleagues5 and Bonne and colleagues6. Together their discoveries marked the 4th gene locus linked to HCM, behind β-MHC, cardiac troponin T (TnT) and α-tropomyosin. In both studies the mutations identified were unusual in that they were not single amino acid substitutions typical of the majority of variants in other genes linked to HCM. For instance, in the study by the Seidman group5 two cMyBP-C mutations were identified. The first was a splice donor site mutation (G→C transversion) predicted to result in exon skipping, loss of the final 213 amino acids of cMyBP-C, a reading frameshift and insertion of a premature termination codon. The second mutation was an 18 nucleotide duplication near the C-terminus of the molecule causing insertion of 6 repeated amino acids. Both mutations were predicted to disrupt the binding of cMyBP-C to MHC since the primary MHC binding site is localized within the C-terminal domain of cMyBP-C14. A third variant, occurring in two unrelated French families, was reported by the Swartz group6. The mutation was a 3′ splice acceptor site mutation (A→G transition), which like the first two mutations, was predicted to lead either to a truncated protein lacking the protein’s C-terminal domains or potentially to a null allele. These initial observations defined a continuing trend and thus far of 197 mutations linked to MYBPC3 (Figure 2 and Supplemental Data Table 1), the majority are splice site donor/acceptor or other insertion/deletion mutations that are predicted to lead to reading frameshifts, premature termination codons, and truncated proteins.
The prevalence of MYBPC3 mutations expected to encode truncated proteins led to initial supposition that truncated cMyBP-C proteins act as dominant negative “poison polypeptides” within the sarcomere. The conclusion was further supported by observations that transgenic expression of truncated cMyBP-C proteins which lacked C-terminal domains including the myosin and titin binding sites showed aberrant localization in sarcomeres, increased Ca2+ sensitivity of tension, and reduced power output in permeabilized myocytes65, 66 (Table 1). Additional, albeit indirect, support for the idea that HCM truncation mutations caused dominant negative effects came from numerous studies in which recombinant truncated proteins containing cMyBP-C N-terminal regulatory domains affected actomyosin interactions in permeabilized fibers, in motility assays, and in solution ATPase assays67–69. However, despite this early supportive evidence from transgenic mouse models and biochemical studies, analysis of human biopsies has failed to reveal truncated proteins in myocardium from HCM patients70–73. The puzzling lack of expected shortened proteins is now attributed to cell surveillance mechanisms, including nonsense mediated decay (NMD) of mRNA transcripts that contain premature termination codons and/or ubiquitin-mediated proteasomal (UPS) degradation of misfolded proteins, since such mechanisms protect the cell from potentially deleterious effects of truncated proteins74. Therefore, alternative disease mechanisms, such as haploinsufficiency (i.e., reduced amounts of protein due a mutant or null allele) or other impairments of cell homeostasis, e.g., due to processing, degradation, and disposal of mutant proteins, are now favored to account for the development of HCM linked to truncation mutations75.
Direct evidence in support of haploinsufficiency was obtained in two recent reports showing that the total amount of cMyBP-C was reduced in myocardium from HCM patients relative to non-failing donor myocardium or HCM patients without mutations in cMyBP-C72, 73. Reduced cMyBP-C was associated with contractile deficits including decreased maximal force production and increased Ca2+ sensitivity of tension in myocardium of affected patients72. However, the observed increase in Ca2+ sensitivity of tension was likely secondary to reduced troponin I phosphorylation rather than a direct result of reduced cMyBP-C72. Somewhat surprisingly, however, the amount of cMyBP-C protein was also reduced in myocardial samples from patients with single amino acid substitutions in cMyBP-C73. The latter result was unexpected since it implies that at least some missense variants also increase the susceptibility of cMyBP-C to degradation. If so, then common pathways could contribute to HCM development for at least some truncation and missense cMyBP-C variants. As discussed further below, it is possible that both truncated proteins or missense mutations that cause improper protein folding enhance degradation of cMyBP-C thereby causing haploinsufficiency or otherwise impairing UPS function such that changes in cell homeostasis trigger hypertrophy75, 76. In support of this idea, cMyBP-C variants were found to be preferred substrates for UPS-mediated degradation75. However, it remains to be demonstrated whether haploinsufficiency per se (i.e., reduced cMyBP-C) or other cell derangements (e.g., as a result of UPS impairment) or some combination of these two factors are sufficient to cause HCM.
The recent recognition that haploinsufficiency may be causative in human disease has focused attention on transgenic models with reduced cMyBP-C expression. Three such models have been produced that result in functional null alleles (Table 1). The first two include gene targeted C-terminal77 or N-terminal deletions78 that lead to reading frameshifts and premature termination codons, but in neither case were truncated proteins detected. Thus, these models result in functional null alleles potentially similar to those proposed for frameshifts and truncation mutations in human disease. A third null allele model was created by ablating the MYBPC3 transcription start site79. In all three models the homozygous cMyBP-C knockout mice show pronounced hypertrophic phenotypes with wall thickening and chamber dilation evident at early ages. Notably, heterozygous mice did not develop hypertrophy, although mild septal thickening was reported at older ages in some mice79.
Functional analyses of the different knockout models have yielded overall similar results revealing altered contractile properties in homozygous knockout mice from myofilament to whole animal levels. Most notably, loss of cMyB-C is associated with marked acceleration of cross-bridge kinetics evident in permeabilized fibers as increased rates of tension redevelopment (ktr), loaded and unloaded shortening velocities, myofilament power output, and rates of stretch activation80–82. By contrast, maximal force was unchanged and Ca2+ sensitivity of tension was either not affected or modestly affected77–79, 83. Collectively these and other results have led to the conclusion that cMyBP-C normally functions to limit cross-bridge kinetics, potentially by stabilizing a post power stroke state or by functioning as an internal cross-bridge load84 similar to that initially proposed by Hofmann et al.85 Importantly, reversal of these inhibitory effects is thought to contribute to increased cardiac contractility in response to β-adrenergic stimulation following PKA phosphorylation since knockout of cMyBP-C and PKA phosphorylation have similar effects to accelerate cross-bridge cycling86, 87. Consistent with this idea, cross-bridge cycling is not increased in response to PKA in myocytes from transgenic mice that lack cMyBP-C PKA phosphorylation sites (S→A mutations)88, whereas cross-bridge kinetics are accelerated in transgenic mice that express cMyBP-C phosphomimetic proteins (S→D mutations)89.
The precise mechanisms by which cMyBP-C slows cross-bridge kinetics have yet to be fully resolved, but reversible interactions with myosin S290 or actin21 seem likely. In support of the former, binding of N-terminal domains of cMyBP-C to myosin S2 is abolished when recombinant cMyBP-C proteins are phosphorylated by PKA in vitro91. Release of myosin heads from the thick filament could thus contribute to acceleration of myosin cross-bridge kinetics in response to either cMyBP-C knockout orβ-adrenergic stimuli 92, 93. On the other hand, reversible interactions of cMyBP-C with the thin filament could also account for accelerated cross-bridge kinetics since N-terminal domains of cMyBP-C bind actin21 and slow apparent cross-bridge detachment rates in motility and ATPase assays independent of the presence of myosin S268, 94. Additional experiments are necessary to resolve this point since binding of the N-terminus of cMyBP-C to neither myosin S2 nor actin has yet been demonstrated in sarcomeres.
While both cMyBP-C knockout and cMyBP-C phosphorylation produce nearly identical enhancements of cross bridge kinetics, the two are not strictly equivalent. This is most evident when considering that homozygous knockout of cMyBP-C causes profound cardiac hypertrophy, whereas constitutive phosphorylation of cMyBP-C using phosphomimetic transgenes (S→D mutations) rescues the hypertrophic phenotype and further protects against ischemia-reperfusion injuries95. The latter suggests cMyBP-C phosphorylation may be a significant factor in modulating disease progression in heart failure or HCM because phosphorylation of cMyBP-C is reduced in these states (reviewed in96). However, other PKA independent effects of cMyBP-C are also apparent at the whole heart level in cMyBP-C knockout mice. In this regard, contractile deficits, including reduced fractional shortening, reduced ejection fraction, and prolonged relaxation rates, are prevalent at the cellular and whole heart levels despite accelerated cross-bridge kinetics and increased power output at the myofilament level78, 82, 97, 79. Global contractile deficits have been reported for other models of cardiac hypertrophy, but cMyBP-C knockout mice also show an usual acceleration of the time course of ventricular stiffening and an abbreviated period of systolic ejection that appears unique to loss of cMyBP-C that could account for reduced ejection fractions82. Notably, the only other protein associated with a change in the time course of cardiac contraction is cardiac RLC where expression of a non-phosphorylatable RLC transgene caused slowing of time course of stiffening48. However, the ability of cMyBP-C to slow systolic ejection appears unrelated to its phosphorylation state because the slower time course of stiffening can be restored by transgenic expression of a non-phosphorylatable cMyBP-C construct (S→A mutations) or by a wild-type transgene with or without PKA phosphorylation98. These results thus suggest cMyBP-C exerts PKA independent effects on actomyosin interactions that influence the timing of systole independent of phosphorylation. In this respect, a proline-alanine rich region at the N-terminus of cMyBP-C99 and the C1 domain100 have been shown to have activating effects on contraction and so it is possible that these domains contribute to the maintenance of systolic ejection in vivo.
Although homozygous knockout of cMyBP-C causes contractile dysfunction and hypertrophy, haploinsufficiency or other impairments of protein processing have still proven challenging to model in heterozygous cMyBP-C mice. For instance, cMyBP-C protein is only modestly reduced in heterozygous mice (~80–100% of wild-type levels) and the mice show no functional impairments80, 101. Thus post-transcriptional mechanisms can virtually completely compensate for a null allele and achieve near stoichiometric replacement of cMyBP-C in sarcomeres since transgenic expression of as little as 40% of a wild-type transgene on a cMyBP-C knockout background was sufficient to rescue hypertrophy102. The situation appears to differ in humans since ~70% of the normal cMyBP-C content is present in heterozygous individuals that develop HCM phenotypes72, 73. The lack of disease phenotypes in heterozygous mice has also confounded attempts to link impaired processing of mutant proteins (distinct from haploinsufficiency) to HCM. For instance, a G>A transition engineered into the donor splice site of exon 6 produced 3 different mRNA transcripts in knock-in mice, but protein expression from each was either modest or not detected103. The heterozygous mice lacked an overt phenotype suggesting that processing and degradation of the mutant transcripts or proteins from a single allele was not detrimental. It is possible that species differences and the state of the cell’s protein synthesis/degradation pathways can change with age or stress and affect the extent of impairment and functional consequences of protein processing104, 105.
While truncation/frameshift/splice variants account for the greatest proportion of cMyBP-C HCM mutations, a substantial minority (>40%) are variants that cause single amino acid substitutions (Figure 3). These variants in general are associated with more benign clinical outcomes than truncation mutations, but a range of phenotypes from benign to severe has been reported2. They occur throughout the length of cMyBP-C with the notable exception of a linker sequence rich in proline and alanine residues between domains C0 and C1 (Figure 3). The region may be relatively tolerant to non-conserved substitutions since there is considerable sequence variation across species106. Nonetheless, in mammals the overall percentage of prolines and alanines in the sequence scales inversely with heart rate, suggesting a conserved regulatory function for the region106. By contrast, all other domains of cMyBP-C have multiple missense variants linked to HCM and “hot spots” are often apparent (Figure 3). For instance, domain C3 has the greatest number of missense mutations and the R502W mutation in domain C3 is among the most prevalent HCM mutations occurring in 2.4% of HCM patients107. A R502Q variant has also been reported108. Other hotspots within C3 are R495 having three variants, R495W, R495Q, and R495G and G523 with two variants G523R and G523W.
The functional significance of the individual missense mutations or hot spots is not yet known, but potentially they could directly disrupt cMyBP-C function and/or interactions with other proteins or they could alter cMyBP-C structure. In this respect, the C0 through C10 domains of cMyBP-C have tertiary structures that bear homology to either immunoglobulin (Ig) or fibronectin (Fn)-like folds109 and it is possible that missense HCM variants alter proper domain folding. For instance, the N755K mutation in C5 results in almost complete unfolding of the domain110, 111. Variants in nearby residues (V757M and G758D) could have similar effects since C5 itself has a low intrinsic thermodynamic stability due in part to a cardiac-specific elongation of the central CD-loop110. Accordingly, disruption of domain architecture could either directly impair cMyBP-C function or potentially lead to haploinsufficiency or other cell impairments similar to those proposed for truncation mutations if misfolded cMyBP-C molecules were targeted for UPS disposal. On the other hand, it is likely that at least some of the missense variants disrupt cMyBP-C function or impair protein-protein interactions without grossly affecting domain structure. For instance, HCM mutations of charged residues on the surface of C5 including R654H and R668H do not appear to affect domain stability but instead influence intermolecular interactions with domain C8110, 111, 17. Similarly, domains C7-C10 are involved in protein-protein interactions that anchor cMyBP-C to the thick filament and to titin14, whereas domains C1 and C2 bind to myosin S219, 90 or actin112. Disruptions of these interactions could impair cMyBP-C function and lead to hypertrophy.
In the case of C1, C2, and C5 it has been possible to map HCM-causing mutations onto their three dimensional structures since these have been solved using NMR or X-ray crystallography90, 110, 113, 114. For C1, missense mutations cluster either towards its C-terminus near the point where C1 joins the adjacent regulatory M-domain or along a β-sheet on an opposing face of C190, 114. The prevalence of mutations affecting surface-exposed residues suggests that mutations disrupt protein-protein interactions rather than disturbing Ig-domain folding90. Consistent with this idea, HCM mutations in the C-terminus of C1 weaken binding interactions with myosin S290. Somewhat surprisingly, NMR analysis also revealed that C1 contains a Zinc binding domain, but it is unclear whether C1 binds Zinc in vivo due to its low concentration in cardiac myocytes90. Nonetheless, the Q208H HCM mutation is predicted to increase Zn binding affinity. 3 missense mutations (G416S, E441K, E451Q) also occur on surface exposed loops of C2, suggesting that they also affect protein interactions. E451, the last residue in the domain, is thought to contribute to binding of C2 to myosin S2113.
Little information is yet available regarding the functional impact of cMyBP-C substitution mutations. To date there have been no studies on the mechanical properties of myocytes obtained either from patients carrying cMyBP-C missense mutations (although as mentioned previously mechanical experiments have been performed using myocardium from patients with truncation mutations72) or from rodent models with engineered missense mutations. Thus far, a single knock-in mouse model has been created to carry a point mutation in cMyBP-C. However, little or no missense protein (E264K) was expressed in that model because the G>A transition occurs in an exon splice site and leads to alternative processing of the transcript103. Additional engineered models of cMyBP-C missense mutations are therefore needed to probe the functional effects of these variants. On the other hand, two naturally occurring cMyBP-C missense mutations have been discovered that cause HCM in domestic cats. The first is an A31P substitution in the cardiac-specific C0 domain in the Maine Coon cat breed115 and the second is an R820W substitution in domain C6 of the Ragdoll breed116. These cat models thus offer a unique opportunity to investigate the functional effects of cMyBP-C missense mutations in a context more closely resembling the human disease since the disease is prevalent in heterozygous animals and the clinical phenotype is similar to HCM in humans, including systolic anterior motion, outflow tract obstruction, and sudden cardiac death. Furthermore, an R820W variant was recently reported in humans and was associated with rare left ventricular non-compaction in addition to HCM117 while a second R820Q variant was associated with HCM that progresses to a “burn-out” phase of DCM in the elderly118. Although there is not yet a human counterpart of the A31P mutation, it may still provide critical insights into how missense mutations can lead to reduced expression of cMyBP-C because myocytes from A31P affected cats show an anomalous reduction of cMyBP-C115 similar to that reported for human missense mutations73. Furthermore, as shown in Figure 4, the A31P variant is also expressed and incorporated into sarcomeres of affected cats, raising the possibility that A31P cMyBP-C functions as a dominant negative within the sarcomere. Investigations into the cellular processing and functional effects of the A31P mutation should therefore yield useful insights toward distinguishing between haploinsufficiency, impaired protein processing, or poison polypeptide mechanisms in the development of HCM linked to mutations in cMyBP-C.
Functional analyses of HCM mutations have revealed unique insights into the molecular mechanisms by which the myosin binding proteins of the thick filament affect actomyosin interactions and have thereby advanced our understanding of underlying causes of disease as well as our basic understanding of contractile protein function. The discovery of cMyBP-C mutations linked to HCM5, 6 has been especially important because prior to this few clues regarding the function of cMyBP-C were available. Recognition that mutations of cMyBP-C are a prevalent cause of HCM has thus provided a primary driving force toward understanding the role of cMyBP-C in cardiac contraction. Studies of knockout and transgenic models have solidly established the role of cMyBP-C in modulating cross-bridge cycling kinetics, shortening velocity, and myocyte power output80, 82, 84, 119. Thus, cMyBP-C like other thick filament myosin binding proteins can be viewed as a regulator of contraction that contributes to tuning the overall speed and efficiency of contraction and relaxation. Because loss of cMyBP-C is likely to be an important mechanism causing cardiac hypertrophy in people72, 73, it will be of interest to determine whether similar changes in contractile kinetics underlie the development of HCM in patients and animal models with reduced cMyBP-C.
Despite the significant progress made in the past 20 years toward the identification and functional analysis of the underlying genetic causes of HCM, fundamental questions still remain. Chief among these are how the vast allelic and locus diversity underlying HCM mutations can predict functional outcomes and individual prognoses and how different mutations can lead to final common pathway(s) that culminate either in HCM or that cause arrhythmias and sudden death96. In these respects, progress has been relatively slow since the initial discovery of Geisterfer-Lowrance1. One difficulty has been has been that rodent models engineered to carry thick filament HCM mutations, for example ELC and RLC mutations, have consistently failed to recapitulate human hypertrophic phenotypes or have done so only when a mutation is inherited in a homozygous state (e.g., cMyBP-C knockout). Reasons for the difficulties in translating human mutations into rodent models are not immediately apparent, but suggest that sufficient species-specific or hemodynamic differences exist between mouse and human hearts that mouse models may not be adequate to completely bridge the gap between identification of contractile deficits and the identification of signals that initiate pathological growth. It will thus be especially important to investigate functional effects of myosin binding protein mutations in human myocardium as well as in novel large animal models of disease where the functional effects of these proteins have been tuned to optimize contractile efficiency and power output of larger hearts.
In summary, the initial discovery of an HCM-causing mutation in β-MHC and the prediction that myosin associated peptides would also be affected1 provided the opening chapter linking sarcomeric proteins to disease and has thereby focused renewed attention on the basic physiological functions of these proteins. The final chapters in this story will no doubt include resolution of a plot arc that can fully account for the diversity of genetic causes that lead to final common disease pathways and yet cause individual phenotypes. In the meantime, we are “in the thick of it” in pursuit of how mutations in myosin binding proteins affect thick filament function and contribute to cardiac contraction in health and disease.
Sources of Funding
This work was supported by NIH HL080367 and a UC Davis New Research Initiative Award (to S.P.H.) and a National Defense Science and Engineering Graduate Fellowship to K.L.H.
The authors thank Dr. Mark Kittleson for feline myocardium and Elaine Hoye for expert technical assistance in performing immunofluorescence imaging experiments.
Subject Codes:  Hypertrophy;  Animal models of human disease;  Genetics of cardiovascular disease;  Contractile function.