Introduction
Hypertrophic cardiomyopathy (HCM) is a common structural anomaly of the myocardium that is unexplained by an underlying condition such as hypertension. The main findings in HCM are varying degrees of ventricular and/or septal hypertrophy, myocyte disarray and increased myocardial fibrosis (Maron et al., 1995). There is significant variation in the clinical manifestation among patients, from asymptomatic, to mild dyspnea upon exertion, to substantive heart failure. While many individuals will present with clinical symptoms, including a cardiac murmur related to outflow tract obstruction, in some families, diagnosis is not established until the sudden death of, or incidental finding of hypertrophy within, a family member. Transthoracic echocardiography has traditionally been the clinician’s primary tool for determination of asymmetric hypertrophy of the left interventricular septum, with or without left ventricular outflow tract obstruction. Given the heterogeneity in severity of disease and penetrance within HCM-affected families, it is important to rule out other secondary causes of hypertrophy, such as hypertension or aortic stenosis. Diagnosis can be difficult, especially in elite athletes who may present with physiological left ventricular hypertrophy (Maron, 2009). Clinically identifiable HCM has a prevalence of 1:500 in young adults in the general population, making it the most common genetic cardiovascular disease in many countries (Maron et al., 1995).
Although familial hypertrophic cardiomyopathy (FHC) was first described clinically more than half a century ago (Teare, 1958), it was only about 20 years ago that the underlying molecular causes of FHC began to be established, with the finding of a mutation in the beta-myosin heavy chain (MYH7) gene (Geisterfer-Lowrance et al., 1990). Since this seminal discovery, there have been more than 900 different mutations identified in over 20 FHC candidate genes (Tester & Ackerman, 2009). Historically, attempts to establish the link between genotype and phenotype were based on studying FHC cohorts with severe, well established disease with cardiac remodelling and in some patients, progression to end-stage cardiac dilatation and failure. It is increasingly apparent that focussing on the end phenotype as a link to genotype is problematic; families are highly heterogeneous in their disease presentation with, in many cases, low penetrance (at least on echocardiography diagnoses) and with novel mutations not seen in other families. There are few large FHC-affected families, leading to linkage analysis difficulties. When a pathogenic FHC mutation is uncovered in the proband, genetic testing of all first degree relatives is highly recommended. When other family members are genotyped, mutation-positive relatives can be closely monitored for disease progression (Colombo et al., 2008).
Up to 60% of patients with a high index of suspicion for FHC are found to have a genetic mutation in one of the FHC-susceptibility genes. A subset of FHC patients do not have identifiable mutations, perhaps because of reduced screening sensitivity that does not incorporate deep intronic sequencing, identify large insertions or deletions in the known candidate genes or include non-hot spot encoding regions. In addition, some patients may have mutations in as-yet unrecognized candidate genes (Rodriguez et al., 2009). The majority of documented FHC mutations occur as single nucleotide substitutions or "missense" mutations, although nucleotide deletions and insertions have also been identified. Insertions and deletions can potentially truncate the gene product by causing a shift in the reading frame leading to a premature stop codon. Mutations that occur at exon/intron boundaries can cause splice anomalies, leading to abnormal and potentially dysfunctional protein products (Wheeler et al., 2009).
Two prominent hypotheses have been developed to explain how sarcomere protein mutations cause the FHC phenotype: first is the "poison polypeptide" hypothesis, in which a single mutant protein disrupts the function of the entire sarcomere unit in a dominant negative manner (Thierfelder et al., 1994). The mutant protein is translated and incorporated into the sarcomere, where it can impair contraction. The second hypothesis is that sarcomeric protein mutations can lead to haploinsufficiency, in which mutations disrupt one copy of the gene, leaving the wild-type gene copy to produce the protein product in inadequate quantities for a balanced sarcomere unit (Thierfelder et al., 1994). In this situation, there is a 50% reduction in peptide concentration due to disruption in translation or trafficking of the mutant. Inadequate levels of incorporated wild-type protein create an imbalance in thin filament stoichiometry.
Sudden cardiac death in FHC
FHC is the most common cause of sudden cardiac death (SCD) in young people, affecting approximately 1-2% of children and adolescents, and up to 1% of young adults in HCM community cohorts (Elliott et al., 2000; Maron, 2002). Although SCD is considered rare in competitive athletes (1 in 200,000), HCM is associated with nearly one third of such occurrences (Maron, 2003). Children have the highest SCD rates of FHC patients suggesting that early onset can result from a more severe phenotype that includes lethal arrhythmias (Maron et al., 1999; Ostman-Smith et al., 2008). The highest mortality rates are seen in children aged 9 to 14 years, averaging 7.2%. The SCD risk peaks in girls at 10 to 11 years of age and occurs in boys at 15 to 16 years of age, leading to some researchers to propose that the surge in androgens that occurs prior to puberty may be associated with rapid disease progression and increased SCD risk (Ostman-Smith et al., 2008). There is a male preponderance for FHC-related SCD, especially among athletes (Maron et al., 1996). FHC patients with 2 or more mutations (Hershberger, 2010; Van Driest et al., 2004), and homozygous mutation patients, have more severe disease phenotypes with higher penetrance and greater incidence of SCD over single mutation patients (Ho et al., 2000; Ingles et al., 2005). Modifier gene polymorphisms such as angiotensin I converting enzyme (ACE) D allele, (Marian et al., 1993) and lifestyle/environmental factors such as diet, exercise, body mass and hypertension may also affect the FHC phenotype. With such complexities in disease manifestation, SCD risk assessment has been problematic. Younger age at onset, history of syncope with exertion, history of SCD within close relatives, severity of symptoms and degree of ventricular and septal wall thickness have been used in risk stratification algorithms; however, many risk factor studies involved non-genotyped patients with sometimes conflicting or confusing results and frequently with no single risk factor being identified. Prognosis for genotyped patients varies with the gene and in many cases, specific mutations within a gene; however, the mechanisms by which such mutations have an increased propensity for sudden death in some individuals, while in others appear to be relatively benign, are not well understood. The primary prevention risk factors for SCD in FHC include family history of SCD, unexplained recent syncope, runs of non-sustained ventricular tachycardia on ambulatory 24 hour Holter monitor, hypotensive response to exercise and severe left ventricular wall thickness (over 30 mm) (Maron, 2010). With respect to the latter, mild ventricular hypertrophy, however, does not correlate with low SCD risk, especially with thin filament mutations, as discussed later.
The role of the troponin complex in cardiac dynamics
The focus of this topic is on three genes that encode the troponin complex found within the sarcomere; TNNT2, encoding cardiac troponin T, TNNI3, encoding cardiac troponin I and TNNC1, encoding troponin C. These genes encode the cardiac troponin genes that are unique from their skeletal counterparts and have evolved to help regulate excitation-contraction coupling in the heart. The troponin (Tn) proteins are part of a thin filament regulatory unit of the sarcomere. Cardiac troponin C (cTnC) is the Ca2+-binding subunit that acts as a cytosolic Ca2+ sensor, cardiac troponin I (cTnI) is the inhibitory subunit that inhibits contraction when intracellular Ca2+ levels are below activation levels and cardiac troponin T (cTnT) is the subunit responsible for attaching the troponin complex to the thin filament via binding with tropomyosin (Tm) and believed responsible for movement of Tm on the thin filament modulating binding of the myosin head to actin. The subunits are arranged in a 1:1:1 stoichiometric ratio along the thin filament with one Tn:Tm complex bound to every seven actin monomers. Actin monomers are arranged in a double helix oriented in parallel to myosin-containing thick filaments. These protein-protein configurations allow for thin filament activation (Figure 1), which in turn facilitates cross-bridge cycling through the action of myosin binding to actin and the production of force (Gordon et al., 2000).
Takeda and collaborators (Takeda et al., 2003) successfully crystallized the globular core of the Tn complex, which revealed that the complex is highly flexible, an inherent feature crucial to its role in heart muscle contraction. The structure consists of two domains: the regulatory head composed of the N-terminus of TnC (residues 3 – 84) and two a-helices of TnI (denoted as H3 and H4, residues 150 – 188), and the highly conserved IT arm composed of the C-terminus of TnC (residues 93-161), two a-helices of Tnl (H1 and H2, residues 42-136) and two a-helices of TnT (H3 and H4, residues 203-271). Although crystallography allowed most of the Tn complex structure to be observed, some regions remain unresolved, including the inhibitory region of TnI, and both the N- and C-terminal regions of TnT. These regions are likely highly flexible, allowing them to bind to other thin filament proteins (i.e. actin) to modulate thin filament activation. The primary role of the regulatory domain is as the "Ca2+ sensor", while the rigid IT domain appears to be sensitive to myosin binding during contraction (Sun, Bradmeier & Irving, 2006, as cited in Willott et al., 2010).
Fig. 1. A schematic representation of cardiac troponin in relation to the thin filament in the absence and presence of Ca2+. The inhibitory region of TnI (IR), and the N-terminal and C-terminal domains of TnT are not clearly observed in the crystal structure, likely due to their inherent flexibility (see text). The red dots represent Ca2+ ions. The figure is adapted from Takeda, 2003 (Li, 2009).
Most researchers believe that a "3 state model" exists to explain myofilament contraction. Interestingly, it was the study of how various mutations disrupt these interactions that lead to further development and confirmation of the 3 state model (Gordon et al., 2000). During diastole, the ventricles fill with blood to their end-diastolic volumes. The sarcomeres are stretched to longer lengths but without developing significant diastolic pressures. Cross-bridge cycling is physically blocked by the Tm:Tn complex at this stage and is referred to as the "blocked" or "B" state. Recently it has been postulated that perhaps only 50% of the cross-bridges are sterically blocked. The rest may be in a weakly-bound non-force generating state that facilitates the transition of cross-bridge cycling into the systolic state. There are two actin-binding regions on cTnI that play an essential role in diastole. There is an inhibitory region (residues 137-148) and a downstream helix (H3, residues 150-159) that tightly binds to actin, which along with cTnT, anchor Tm into the blocking position (see review by Parmacek & Solaro, 2004).
Calcium initially enters the cell mainly through L-type Ca2+ channels and initiates Ca2+-induced Ca2+ release from the sarcoplasmic reticulum. As cytosolic Ca2+ levels rise, the sarcomeres develop tension that increases ventricular isovolumic pressure until the aortic and pulmonary valves open. Blood is expelled from the ventricles by sarcomeres shortening to their end-systolic lengths. At the subcellular level, myofilament activation begins with Ca2+ binding to cTnC site II, exposing a hydrophobic region at the N lobe of cTnC and creating a new binding site for cTnI. Cardiac TnI then dissociates from actin and binds tightly to the hydrophobic region of cTnC, causing a cascade of protein-protein interactions that allows Tm to move closer into the thin filament groove. This stage is referred to as the "closed" or "C" state. This movement exposes myosin binding sites on actin and also appears to alter thin filament structure, allowing more cross-bridges to occur and moving Tm further into the thin filament groove (thus shifting into the "open" or "M" state). Positive feedback may arise from bound cross-bridges causing an increased affinity for Ca2+ by cTnC (Pan & Solaro, 1987 as cited in Solaro & Kobayashi, 2011). At basal states of contractility, only 25% of available cTnC regulatory (site II) Ca2+ binding sites are occupied due to low cytosolic Ca2+ levels, resulting in a substantial cardiac reserve for recruitment of blocked cross-bridges when required.
After the valves close, the sarcomeres are quiescent as the ventricles prepare for refilling. This relaxation phase is highly dependent upon the rate of cytosolic Ca2+ removal, the offloading of Ca2+ from cTnC, and the cross-bridges returning to the weakly bound or blocked state. Phosphorylation of the thin filament proteins, in particular the N-terminus of cTnI, plays a crucial role in drawing upon cardiac reserve, cross-bridge cycling rate and hence, relaxation, in a signaling cascade initiated by P-adrenergic stimulation (see review by Tardiff, 2011). These mechanisms are critically important when increased heart rate is required during exercise. Considering that SCD in young FHC patients frequently occurs during exercise (Cha et al., 2007), thin filament mutations may have an inhibitory effect on phosphorylation signalling and cardiac reserve as well as other cross-bridge cycling effects.
Mechanisms of Sudden Cardiac Death in FHC troponin mutations
Various mechanisms for SCD due to FHC have been suggested including arrhythmias arising from sinus nodal and atrioventricular nodal conduction abnormalities, and tachycardia due to re-entrant depolarization pathways from myocardial disarray and fibrosis, abnormal Ca2+ homeostasis, ventricular diastolic dysfunction or left ventricular outflow tract obstruction (Fatkin & Graham, 2002). With several underlying mechanisms leading to SCD, research is only beginning to define the link between the underlying molecular pathology and arrhythmogenesis in FHC-associated troponin mutations. One emerging issue is that studying patients with well established disease for the purpose of linking phenotype to genotype has proven extremely difficult. Like all monogenic disorders, there are myriad disease modifiers including genetic, environmental and lifestyle factors that influence disease progression and severity in a manner that is poorly understood. What is also becoming apparent is that ventricular hypertrophy, fibrosis and obstructive disease are likely compensatory FHC features and based on complex signalling cascades arising from pathologies within the sarcomere, as discussed later in this topic. Perhaps longitudinal studies of patients prior to the onset of structural disease may uncover mutation-specific disease progression that parallels the molecular and biophysical effects observed in in vitro experiments, animal models and in silico predictions (Tardiff, 2011). There is likely a less complex phenotype in FHC patients in the early disease stages allowing a more discernable link between genotype and phenotype. A study of preclinical FHC patients provides evidence for this hypothesis (Ho et al., 2002). In their study, most FHC cohorts presented with one common phenotype, namely prolonged diastolic relaxation on echocardiography, despite patients having different mutations within different genes. This approach may also benefit treatment outcomes for preclinical FHC cohorts with targeted mutation-specific treatment to attenuate disease progression. It makes sense to treat pre- symptomatic FHC patients long before gross phenotype becomes established. Diltiazem, a calcium channel blocker, normalized Ca2+ regulation and attenuated ventricular hypertrophy in a mouse model (Semsarian et al., 2002) and formed the basis of an ongoing clinical trial, in which preclinical FHC patients receive diltiazem therapy while being monitored for disease progression (http://clinicaltrials.gov/ct2/show/NCT00319982). Recent approaches to identifying the pathophysiology of FHC mutations includes investigation of the dynamic properties of cross-bridge cycling at the molecular level and how Tn mutations disrupt precise molecular movements. Such high resolution investigations commonly incorporate computational approaches to examine protein flexibility and to predict changes in protein mobility caused by mutations, using molecular dynamics simulation programs such as GROMACS (Van Der Spoel et al., 2005) and CHARMM (Brooks et al., 2009). Another approach is Nuclear Magnetic Resonance (NMR) imaging, allowing investigators to compare recombinant wild-type and mutated Tn complexes in different metal-binding states to measure conformational changes (Lassalle, 2010).
Troponin T mutations
Since the identification of cTNNT2 in 1993 as the first Tn-based gene associated with FHC (Thierfelder et al., 1993), cTnT mutations have been extensively studied and account for up to 15% of all FHC mutations (Watkins et al., 1995). To date, there are at least 68 cTnT mutations identified associated with FHC (Willott et al., 2010), with a subset that present with a high frequency of SCD and/or ventricular arrhythmia in humans (Table 1). Alternative splicing of exons 4 and 5 of the cTNNT2 gene in the human heart results in four temporally regulated isoforms: one adult isoform (TnT3) and 3 fetal isoforms (TnT1,TnT2 and TnT4). The variable cTnT N-domain contributes to the Ca2+ sensitivity of force development and the presence of fetal isoforms in adult myofilaments has been associated with increased myofilament Ca2+ sensitivity and diastolic dysfunction (Gomes et al., 2002 as cited in Gomes et al., 2004). Tn complexes with fetal isoforms TnT1 and TnT2 (containing exon 5) have a reduced inhibition of actomyosin ATPase activity compared with the adult TnT3 isoform which suggests that the TnT isoforms have varying ability to modulate cross-bridge cycling and hence, cardiac contraction (Gomes et al., 2002). These findings are noteworthy in that increased myofilament Ca2+ sensitivity and diastolic dysfunction occur with many FHC causing Tn mutations; however, studies investigating the expression of cTnT isoforms in diseased hearts and a possible contributory role in altered contractile performance remain unresolved with no as-yet obvious correlation between fetal isoform TnT4 expression and Ca2+ sensitivity in diseased hearts (see review by Parmacek & Solaro, 2004).
The majority of cTnT mutations occur within the two structurally poorly resolved regions with a clustering of mutations within residues 69 to 110. There are three "hot spots" occurring at residues 92, 94 and 110, of which R92L and F110I have been associated with high rates of SCD and/or ventricular arrhythmia (Table 1). Another mutational "hotspot" occurs at residues 160 to 163, which is found within a highly charged and a highly conserved sequence from 157 to 166. This region is believed to be a flexible linker between H1 and H2 and whose structure has so far eluded resolution. Closer to the C terminus is a scattering of mutations associated with dilated cardiomyopathy (DCM) as well as several FHC mutations. It is believed that residues in this particular region affect Ca2+ sensitivity via allosteric interactions with the cTnC C domain, although actual evidence is lacking (Tardiff, 2011).
|
Troponin subunit |
Mutation(s) |
SCD or Ventricular Arrhythmia |
References |
|
cTnT |
 |
SCD |
(Gimeno et al., 2009; Knollmann & Potter, 2001; |
|
Moolman et al., 1997; |
|||
|
Thierfelder et al., 1993; |
|||
|
Thierfelder et al., 1994) |
|||
|
F110I |
Ventricular arrhythmia |
(Watkins et al., 1995) |
|
|
cTnl |
 |
SCD |
(Ashrafian et al., 2003; Niimura et al., 2002; Van Driest et al., 2003) |
|
R141Q, G203R |
Ventricular arrhythmia |
(Alcalai et al., 2008; Ashrafian et al., 2003) |
|
|
cTnC |
Q122AfsX30¥ |
SCD |
(Chung et al., 2011) |
Table 1. Troponin mutations associated with SCD and ventricular arrhythmia. *A denotes deletion of the noted residue causing an in-frame mutation; T denotes a splice donor site mutation that removes 28 residues at the C terminus and replaces them with 7 nonsense codons resulting in a truncated cTnT mutant; ¥ denotes a nucleotide duplication (G) at position 363, causing a frame-shift substitution on residue 122 (Q122A) and a premature stop codon (X) at residue 30 resulting in a truncated cTnC mutant.
