Triggered arrhythmias can occur as delayed after-depolarizations (DADs), early afterdepolarizations (EADs) or increased automaticity in non-ischemic FHC and are likely related to myocellular Ca2+ signalling and transport (Bers, 2008). DADs are commonly believed to be caused by spontaneous Ca2+ release from the SR that occurs as a consequence of high SR Ca2+ levels. This SR Ca2+ release causes a transient inward current (Iti) that can cause a threshold depolarization leading to an action potential. Several studies suggest that the Na+/ Ca2+ exchanger current (INCX) is responsible for Iti in human ventricular myocytes (Pogwizd et al., 2001). Further to this, Ter Keurs' research group has been investigating myofilament arrhythmogenic Ca2+ release to determine if non-uniform excitationcontraction coupling plays a role in the initiation of extra-systoles that create arrhythmias ((Ter Keurs et al., 2006). Ter Keurs developed a model of non-uniform excitation-contraction using rat trabeculae and exposed a small muscle segment to BDM, a cross-bridge inhibitor (Backx et al., 1995 as cited in Ter Keurs et al., 2006), to recapitulate non-contracting myocardium as found in diseased hearts. Triggered propagating contractions were observed in the border zone of myocardium between non-contractile and contractile tissue when the trabeculae were stimulated to contract. The triggered contractions may be due to a quick release-induced Ca2+ dissociation from cTnC site II, leading to a local Ca2+ surge that is above the threshold for inducing Ca2+-induced Ca2+ release. This mechanism, referred to as "reverse excitation contraction coupling" (RECC), occurs when Ca2+ reuptake mechanisms have sufficiently recovered from the previous contraction during diastole (Boyden & ter Keurs, 2001). In FHC, the myocardium may have focal regions of non-uniformity due to structural anomalies, such as fibrosis or myocardial disarray, or perhaps due to electrical remodelling or gene dosage effects from Tn mutations. Increased Ca2+ binding to cTnC leading to a high Ca2+ buffering capacity may cause a large Ca2+ surge during rapid myofilament shortening during RECC. Hence, RECC may be an underlying pathological mechanism of arrhythmogenesis seen in FHC patients and warrants further investigation. Electrical alternans, in which there is alternating long and short action potential duration (APD), increases the risk of ventricular tachycardia that can degrade to ventricular fibrillation and SCD. The underlying mechanism may to be related to Ca2+ transient amplitude alternans. At high pacing frequencies, slow ryanodine receptor and/or Ca2+ current recovery may result in alternating SR Ca2+ release, due to alternating availability of ryanodine receptors (RyR). Prolonged Ca2+ transient decay rates, as seen in some transgenic FHC animal models may play a role here (see below). Spatially discordant alternans is thought to be a prerequisite to dangerous arrhythmias, as there is a nonsynchronous electrical substrate within the heart (Bers, 2008). Animal models support this hypothesis; increased myofilament Ca2+ sensitivity was associated with an arrhythmogenic substrate in transgenic cTnT mice, despite the absence of structural heart disease (Baudenbacher et al., 2008). Addition of a myofilament Ca2+ sensitizing agent, EMD, resulted in repolarization alternans at high pacing rates, beat to beat variation in APD, shorter effective refractory periods and increased spatial conduction velocity dispersion in wild-type cat and mouse hearts, paralleling the findings as observed in mutant cTnT I79N transgenic mice. Several mechanisms were proposed for the induction of ventricular tachyarrhythmias: increased Ca2+ binding to cTnC resulting in reduced Ca2+ transients with slower decay rates responsible for the shorter APD seen in transgenic I79N cTnT mice, and dysfunctional myocardial relaxation as seen in transgenic mice and in human patients also causing APD shortening. However, transgenic mice have differing

It has proven difficult to elucidate the exact mechanisms linking FHC pathology and arrhythmogenicity in humans with limited access to fresh cardiac tissue from FHC patients. Studies currently utilize tissue from myectomy samples and explanted hearts in which there is profound disease phenotype. Thus, explorations of disease progression in pre-clinical patients are normally limited to non-invasive imaging techniques and electrophysiology. In addition, there is inherent patient referral bias for research studies as most FHC patients seen in surgical referral centres already have profound disease manifestation (Tardiff, 2011).

Stored electrograms from implantable cardiac defibrillators indicate that SCD largely results from sustained ventricular tachycardia and/or ventricular fibrillation (B.J. Maron, 2010). One suggested trigger for SCD is sympathetic excitation, given that the initiating rhythm in many cases is sinus tachycardia, which may underlie the high SCD rate in athletes and subadult FHC populations (Cha et al., 2007). Several studies of HCM patients wearing Holter monitors identified a higher rate of SCD for younger patients (age 30 and under) with nonsustained ventricular tachycardia (NSVT) detected at least once during the 48 hour monitoring period. While NSVT is usually asymptomatic and frequently occurs during periods of increased parasympathetic (vagal) tone, it is associated with an increased SCD risk, especially in children and young adults (Elliott et al., 2000; Monserrat et al., 2003). However, most SCDs occur in patients without ambulatory ECG episodes of NSVT; clearly other contributory factors leading to risk of SCD are at play. A more recent study identified an increased SCD risk with ventricular arrhythmias triggered by exercise (Gimeno, Tome-Esteban et al., 2009), which is more in keeping findings of triggered arrhythmias during sinus tachycardia, implicating sympathetic excitation during physical activity (Cha et al., 2007). This is also in keeping with the *in vitro* experimental evidence of a blunted response to β-adrenergic stimulation through phosphorylation of cTnI (i.e. reduced inhibitory response of phosphorylated cTnI on Ca2+ sensitivity for cTnC) and the resultant diastolic dysfunction as discussed previously.

Cardiac magnetic resonance (CMR) imaging has allowed clinicians to precisely determine myocyte fibrosis and scarring in non-ischemic FHC, including phenotype-negative FHC patients who have experienced life-threatening arrhythmias (Makhoul et al., 2011; Strijack et al., 2008). Detection of fibrosis by late gadolinium enhancement in CMR is associated with increased propensity for VT on ambulatory ECG monitoring (Adabag et al., 2008) and is being considered as a clinical SCD risk marker (Maron, 2010). Fibrosis and scarring may promote localized zones of slowed conduction resulting in re-entrant arrhythmias (Cha et al., 2007). NMR imaging can detect focal or diffuse regions of fibrosis and hypertrophy morphologies that are missed by traditional echocardiography (M. S. Maron, 2009) and will likely continue to improve in resolution leading to improved risk stratification for FHC patients.

Familial Hypertrophic Cardiomyopathy-Related Troponin Mutations and Sudden Cardiac Death 293

sequences generally to inhibit protein expression, either by degradation of the bound mRNA target or by directly inhibiting translation of the mRNA sequence (Bartel, 2004). To date, there are at least 4 miRs shown to be involved in cardiac development, apoptosis and hypertrophy, namely: miR-1, miR-133, miR-208 and miR-499 (van Rooij et al., 2006). Several important target genes for miRs related to cardiac electrophysiology have been identified, including connexin 43 and inwardly-rectifying potassium channel Kir2.1 (Zhao et al., 2007) and miR-1 expression changes have been associated with arrhythmogenesis due to upregulation or down-regulation of these gene products (Girmatsion et al., 2009; Yang et al., 2007). Recent studies are beginning to elucidate the link between miRs and SCD due to arrhythmias by identifying the effects of altered expression levels of miRs in the heart on cardiac conduction and excitability (Callis et al., 2009; Matkovich et al., 2010; Zhao et al., 2007). A database has been developed online (http://www.mir2disease.org/) for miRs

Sudden Infant Death Syndrome (SIDS) refers to the sudden death of an infant under 1 year of age which remains unexplained after a thorough medicolegal investigation (Willinger et al., 1991). Researchers are now considering inherited cardiac arrhythmia syndromes in its etiology. Recent research has revealed that up to 20% of SIDS cases may be associated with inheritable arrhythmia syndromes, such as long QT syndrome (Klaver et al., 2011). Given that FHC is the most common cause of SCD in young individuals, it stands to reason that some infants may die from SCD attributed to FHC. Further to this, as discussed earlier, some Tn mutants, particularly the cTnT mutants, have negligible or mild hypertrophy that may not be observed grossly during the post mortem exam. To date, only one study has screened SIDS cases for FHC mutations (Brion et al., 2009). Their findings of 14 cases with 7 genetic variants from 4 different FHC genes, including cTnT and cTnI, from 140 SIDS tissues suggests that some SIDS cases may be associated with FHC-causing mutations. The relatively recent emergence of FHC-associated Tn (in particular, cTnC) gene mutations make these candidate genes previously unrecognized and perhaps under-represented factors to be considered in future SIDS investigations. Besides SIDS cases, how many FHC mutation positive cases have gone unrecognized in post mortem investigations following the sudden,

Sudden cardiac death affects approximately 1-2% of children and adolescents, and up to 1% of young adults in FHC-affected populations. *In vitro* analysis of single molecule mechanics and reconstituted skinned myocytes have identified intracellular Ca2+ dysregulation, altered myofibrillar Ca2+ sensitivity and altered energy metabolism as potential mechanisms at the sarcomere and cellular level. Animal models incorporating specific FHC mutations have broadened our understanding of the pathogenesis of FHC, including structural and electrophysiological remodelling associated with the arrhythmogenic substrate. There are, however, caveats to using in vitro and animal model analyses, given that some may not necessarily recapitulate the physiological substrate in human FHC patients. Most models, however, share a consistent molecular phenotype, namely increased myofilament Ca2+ sensitivity and increased energetic cost of force development, that underlies the complex and heterogeneous phenotype that exists at the human patient level. Other genetic, environmental and biological factors such as age, lifestyle and other health issues are also likely disease-modifying factors. Research into molecular approaches and post-translational

involved in human disease, including FHC.

unexpected death of children and young adults?

**7. Conclusions**

Myocyte disarray is another common feature in FHC patients and a recent study of myectomy samples from a small pediatric HCM cohort suggests that myocyte disarray has a significantly higher correlation with diastolic dysfunction than either hypertrophy or fibrosis (Menon et al., 2009). Extensive myocyte disarray has been linked to SCD in younger FHC patients (Varnava, Elliott, Mahon et al., 2001), especially those with cTnT mutations (Varnava, Elliott, Baboonian et al., 2001) in the absence of, or with minimal hypertrophy.

#### **6. Future considerations**

Of interest to investigators is the potential role of Ca2+ dysregulation in ER stress pathways*.* The SR in myocardial cells has long been thought to be the cardiac equivalent to ER with its main role as the intracellular regulator of Ca2+ fluxes and hence, excitation-contraction coupling in the heart. Some researchers propose that the SR contains a functional ER "compartment" with physiological roles such as protein synthesis, translocation and integration into membranes, folding and post-translational modifications including glycosylation and Ca2+ homeostasis (Mesaeli et al., 2001), although studies are lacking. ER stress occurs in response to environmental or genetic factors causing ER metabolic disturbances, accumulation of misfolded proteins, oxidative stress and/or depletion of ER Ca2+ stores. The "unfolded protein response" (UPR) is one mechanism by which the ER attempts to reestablish homeostasis by reducing protein expression, by increasing production of chaperones to handle accumulation of misfolded protein, promoting ERassociated degradation to remove misfolded proteins (Schroder & Kaufman, 2005). This initial response of protein synthesis, suppression and upregulation of ER resident chaperones is designed to resolve the ER stress and enhance survival, but if the ER stress is severe or prolonged, the UPR may stimulate apoptosis (cell death). Do FHC mutations play a role in ER stress pathways, such as through Ca2+ dysregulation? Activation of the "fetal gene program" is a response to elevated Ca2+ to increase cardiac efficiency in the stressed heart. This response, unfortunately, also commonly results in detrimental cardiac hypertrophy (Eizirik et al., 2008; Wang et al., 2000). Elevated Ca2+ activates calcineurin A that dephosphorylates the transcription factor NFAT which translocates to the nucleus to stimulate cardiac remodelling and hypertrophy associated with the fetal gene program (Heineke & Molkentin, 2006). Other factors, such as MEF2, GATA and CamKII are also activated which initiate transcription programs associated with hypertrophy, remodelling and heart failure with increased risk of cardiac death (Molkentin et al., 1998). GATA-4 may play a significant role in FHC pathogenesis due to its ability to stimulate transcription of the cardiac-specific Tn genes (Liang et al., 2001; Molkentin & Olson, 1997; Molkentin et al., 1998). Therefore, ER stress may turn on the fetal gene program in response to pathological insult. MicroRNAs (miRs) likely play a role in these regulatory processes with miRNA coding sequences often located within the newly transcribed genes (Eizirik et al., 2008; Wang et al., 2000). The hypothesis of elevated Ca2+ causing transcriptional activation of hypertrophy and perhaps pathological arrhythmia substrates (see below) is provocative and will likely continue to be an area of active investigation.

Research into miRs that play a role in regulation of cardiac function and the recent findings that miR expression is deranged in cardiac disease may help to uncover the pathways to FHC disease progression and arrhythmogenesis. MiRs are short, non-coding RNA sequences that regulate expression of genes involved in orchestrating growth, development, function and stress responses in a spatio-temporal manner. MiRs target specific mRNA sequences generally to inhibit protein expression, either by degradation of the bound mRNA target or by directly inhibiting translation of the mRNA sequence (Bartel, 2004). To date, there are at least 4 miRs shown to be involved in cardiac development, apoptosis and hypertrophy, namely: miR-1, miR-133, miR-208 and miR-499 (van Rooij et al., 2006). Several important target genes for miRs related to cardiac electrophysiology have been identified, including connexin 43 and inwardly-rectifying potassium channel Kir2.1 (Zhao et al., 2007) and miR-1 expression changes have been associated with arrhythmogenesis due to upregulation or down-regulation of these gene products (Girmatsion et al., 2009; Yang et al., 2007). Recent studies are beginning to elucidate the link between miRs and SCD due to arrhythmias by identifying the effects of altered expression levels of miRs in the heart on cardiac conduction and excitability (Callis et al., 2009; Matkovich et al., 2010; Zhao et al., 2007). A database has been developed online (http://www.mir2disease.org/) for miRs involved in human disease, including FHC.

Sudden Infant Death Syndrome (SIDS) refers to the sudden death of an infant under 1 year of age which remains unexplained after a thorough medicolegal investigation (Willinger et al., 1991). Researchers are now considering inherited cardiac arrhythmia syndromes in its etiology. Recent research has revealed that up to 20% of SIDS cases may be associated with inheritable arrhythmia syndromes, such as long QT syndrome (Klaver et al., 2011). Given that FHC is the most common cause of SCD in young individuals, it stands to reason that some infants may die from SCD attributed to FHC. Further to this, as discussed earlier, some Tn mutants, particularly the cTnT mutants, have negligible or mild hypertrophy that may not be observed grossly during the post mortem exam. To date, only one study has screened SIDS cases for FHC mutations (Brion et al., 2009). Their findings of 14 cases with 7 genetic variants from 4 different FHC genes, including cTnT and cTnI, from 140 SIDS tissues suggests that some SIDS cases may be associated with FHC-causing mutations. The relatively recent emergence of FHC-associated Tn (in particular, cTnC) gene mutations make these candidate genes previously unrecognized and perhaps under-represented factors to be considered in future SIDS investigations. Besides SIDS cases, how many FHC mutation positive cases have gone unrecognized in post mortem investigations following the sudden, unexpected death of children and young adults?

#### **7. Conclusions**

292 Cardiomyopathies – From Basic Research to Clinical Management

Myocyte disarray is another common feature in FHC patients and a recent study of myectomy samples from a small pediatric HCM cohort suggests that myocyte disarray has a significantly higher correlation with diastolic dysfunction than either hypertrophy or fibrosis (Menon et al., 2009). Extensive myocyte disarray has been linked to SCD in younger FHC patients (Varnava, Elliott, Mahon et al., 2001), especially those with cTnT mutations (Varnava, Elliott, Baboonian et al., 2001) in the absence of, or with minimal hypertrophy.

Of interest to investigators is the potential role of Ca2+ dysregulation in ER stress pathways*.* The SR in myocardial cells has long been thought to be the cardiac equivalent to ER with its main role as the intracellular regulator of Ca2+ fluxes and hence, excitation-contraction coupling in the heart. Some researchers propose that the SR contains a functional ER "compartment" with physiological roles such as protein synthesis, translocation and integration into membranes, folding and post-translational modifications including glycosylation and Ca2+ homeostasis (Mesaeli et al., 2001), although studies are lacking. ER stress occurs in response to environmental or genetic factors causing ER metabolic disturbances, accumulation of misfolded proteins, oxidative stress and/or depletion of ER Ca2+ stores. The "unfolded protein response" (UPR) is one mechanism by which the ER attempts to reestablish homeostasis by reducing protein expression, by increasing production of chaperones to handle accumulation of misfolded protein, promoting ERassociated degradation to remove misfolded proteins (Schroder & Kaufman, 2005). This initial response of protein synthesis, suppression and upregulation of ER resident chaperones is designed to resolve the ER stress and enhance survival, but if the ER stress is severe or prolonged, the UPR may stimulate apoptosis (cell death). Do FHC mutations play a role in ER stress pathways, such as through Ca2+ dysregulation? Activation of the "fetal gene program" is a response to elevated Ca2+ to increase cardiac efficiency in the stressed heart. This response, unfortunately, also commonly results in detrimental cardiac hypertrophy (Eizirik et al., 2008; Wang et al., 2000). Elevated Ca2+ activates calcineurin A that dephosphorylates the transcription factor NFAT which translocates to the nucleus to stimulate cardiac remodelling and hypertrophy associated with the fetal gene program (Heineke & Molkentin, 2006). Other factors, such as MEF2, GATA and CamKII are also activated which initiate transcription programs associated with hypertrophy, remodelling and heart failure with increased risk of cardiac death (Molkentin et al., 1998). GATA-4 may play a significant role in FHC pathogenesis due to its ability to stimulate transcription of the cardiac-specific Tn genes (Liang et al., 2001; Molkentin & Olson, 1997; Molkentin et al., 1998). Therefore, ER stress may turn on the fetal gene program in response to pathological insult. MicroRNAs (miRs) likely play a role in these regulatory processes with miRNA coding sequences often located within the newly transcribed genes (Eizirik et al., 2008; Wang et al., 2000). The hypothesis of elevated Ca2+ causing transcriptional activation of hypertrophy and perhaps pathological arrhythmia substrates (see below) is provocative and

**6. Future considerations**

will likely continue to be an area of active investigation.

Research into miRs that play a role in regulation of cardiac function and the recent findings that miR expression is deranged in cardiac disease may help to uncover the pathways to FHC disease progression and arrhythmogenesis. MiRs are short, non-coding RNA sequences that regulate expression of genes involved in orchestrating growth, development, function and stress responses in a spatio-temporal manner. MiRs target specific mRNA Sudden cardiac death affects approximately 1-2% of children and adolescents, and up to 1% of young adults in FHC-affected populations. *In vitro* analysis of single molecule mechanics and reconstituted skinned myocytes have identified intracellular Ca2+ dysregulation, altered myofibrillar Ca2+ sensitivity and altered energy metabolism as potential mechanisms at the sarcomere and cellular level. Animal models incorporating specific FHC mutations have broadened our understanding of the pathogenesis of FHC, including structural and electrophysiological remodelling associated with the arrhythmogenic substrate. There are, however, caveats to using in vitro and animal model analyses, given that some may not necessarily recapitulate the physiological substrate in human FHC patients. Most models, however, share a consistent molecular phenotype, namely increased myofilament Ca2+ sensitivity and increased energetic cost of force development, that underlies the complex and heterogeneous phenotype that exists at the human patient level. Other genetic, environmental and biological factors such as age, lifestyle and other health issues are also likely disease-modifying factors. Research into molecular approaches and post-translational

Familial Hypertrophic Cardiomyopathy-Related Troponin Mutations and Sudden Cardiac Death 295

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**15**

*Germany*

Kornelia Jaquet and Andreas Mügge

*University Hospitals of the Ruhr-University of Bochum,*

**Consequences of Mutations in Genes Encoding**

**Cardiac Troponin C, T and I – Molecular Insights**

Cardiac troponin is the main regulatory protein of the thin filament and mediates the Ca2+ sensitivity of the actin-myosin interaction. Troponin forms a heterotrimeric complex composed of the tropomyosin binding subunit (cTnT), the inhibitory subunit (cTnI) and the Ca2+-binding subunit (cTnC). A complex interplay between the cardiac troponin subunits and other thin filament proteins, as tropomyosin (Tm) and actin, is essential to regulate muscle contraction, which can be described by cross bridge cycling on the molecular level. Troponin is located on both sides of the thin filament with a stagger of about 27 Angstroms between two adjacent troponin molecules (Ebashi, 1972; Paul et al., 2009). In the thin filament each troponin binds to one tropomyosin, which covers 7 actin monomers. It is no surprise that mutations in genes encoding proteins, which participate in crossbridge cycling and its regulation, derange interactions and lead to contractile dysfunction and disease. In all three cardiac troponin subunits, changes in amino acid sequence have been identified in families with hypertrophic (HCM), restrictive (RCM) and dilated cardiomyopathy (DCM). Therefore knowledge of structure, function and interactions of the proteins is a prerequisite

One of the main tasks of cardiac troponin T (30-35kDa) is to fix the troponin complex to the thin filament. Furthermore cTnT participates in conferring calcium sensitivity to actin/myosin (Tobacman, 1988). Tobacman also showed that the N-terminal half of cTnT, TnT1 (amino acids 1-158, skeletal muscle numbering), was able to keep the thin filament in the blocked state without TnI. In the blocked state of the thin filament no interaction between actin and myosin is possible, i.e. no force production occurs. Thus cTnT plays an active role in inhibition of actin/myosin interaction in the resting state. It further promotes

Structural information on cTnT is poor. According to EM analysis of thin filaments and low resolution co-crystallization of Tm in complex with cTnT, cTnT is highly asymmetric. It is a 180-202 nm long comma –shaped molecule, with the N-terminal rod like part arranged along the thin filament and a C-terminal more globular domain (Ohtsuki, 1979, Flicker et al., 1982, White et al., 1987). The high resolution crystal structure available for the core troponin complex contains only the less flexible C-terminal part of cTnT, which binds the other two

tropomyosin polymerization and binding of tropomyosin to actin.

**1. Introduction**

to understand dysfunction in disease.

**1.1 Cardiac troponin T (cTnT)**

*Molecular Cardiology & Clinic of Cardiology, St. Josef-Hospital & Bergmannsheil,*

### **Consequences of Mutations in Genes Encoding Cardiac Troponin C, T and I – Molecular Insights**

Kornelia Jaquet and Andreas Mügge

*Molecular Cardiology & Clinic of Cardiology, St. Josef-Hospital & Bergmannsheil, University Hospitals of the Ruhr-University of Bochum, Germany*

#### **1. Introduction**

302 Cardiomyopathies – From Basic Research to Clinical Management

Yang, B., Lin, H., Xiao, J., Lu, Y., Luo, X., Li, B., Wang, Z. (2007). The muscle-specific

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lacking miRNA-1-2. *Cell, 129*(2), 303-317. doi:10.1016/j.cell.2007.03.030

and KCNJ2. *Nature Medicine, 13*(4), 486-491. doi:10.1038/nm1569

microRNA miR-1 regulates cardiac arrhythmogenic potential by targeting GJA1

(2007). Dysregulation of cardiogenesis, cardiac conduction, and cell cycle in mice

Cardiac troponin is the main regulatory protein of the thin filament and mediates the Ca2+ sensitivity of the actin-myosin interaction. Troponin forms a heterotrimeric complex composed of the tropomyosin binding subunit (cTnT), the inhibitory subunit (cTnI) and the Ca2+-binding subunit (cTnC). A complex interplay between the cardiac troponin subunits and other thin filament proteins, as tropomyosin (Tm) and actin, is essential to regulate muscle contraction, which can be described by cross bridge cycling on the molecular level. Troponin is located on both sides of the thin filament with a stagger of about 27 Angstroms between two adjacent troponin molecules (Ebashi, 1972; Paul et al., 2009). In the thin filament each troponin binds to one tropomyosin, which covers 7 actin monomers. It is no surprise that mutations in genes encoding proteins, which participate in crossbridge cycling and its regulation, derange interactions and lead to contractile dysfunction and disease. In all three cardiac troponin subunits, changes in amino acid sequence have been identified in families with hypertrophic (HCM), restrictive (RCM) and dilated cardiomyopathy (DCM). Therefore knowledge of structure, function and interactions of the proteins is a prerequisite to understand dysfunction in disease.

#### **1.1 Cardiac troponin T (cTnT)**

One of the main tasks of cardiac troponin T (30-35kDa) is to fix the troponin complex to the thin filament. Furthermore cTnT participates in conferring calcium sensitivity to actin/myosin (Tobacman, 1988). Tobacman also showed that the N-terminal half of cTnT, TnT1 (amino acids 1-158, skeletal muscle numbering), was able to keep the thin filament in the blocked state without TnI. In the blocked state of the thin filament no interaction between actin and myosin is possible, i.e. no force production occurs. Thus cTnT plays an active role in inhibition of actin/myosin interaction in the resting state. It further promotes tropomyosin polymerization and binding of tropomyosin to actin.

Structural information on cTnT is poor. According to EM analysis of thin filaments and low resolution co-crystallization of Tm in complex with cTnT, cTnT is highly asymmetric. It is a 180-202 nm long comma –shaped molecule, with the N-terminal rod like part arranged along the thin filament and a C-terminal more globular domain (Ohtsuki, 1979, Flicker et al., 1982, White et al., 1987). The high resolution crystal structure available for the core troponin complex contains only the less flexible C-terminal part of cTnT, which binds the other two

Consequences of Mutations in Genes Encoding Cardiac Troponin C, T and I – Molecular Insights 305

alteration in the isoform expression pattern. Differences in the phosphorylation status of sarcomeric proteins, especially of myosin light chain 2 (MLC-2) may be decisive (van der Velden et al., 2003). Also cTnT itself is a phospho protein (Fig. 1) which is constitutively phosphorylated at Ser1 in several species inclusive men due to the action of casein kinase- 2 (Gusev et al., 1980; Risnik & Gusev, 1984, Swiderek et al., 1990). The function of this phosphorylation is not known up to date. It might prevent degradation and/or interaction of the hypervariable region. At least one further reversible phosphorylation site for PKC is located in the C-terminal region of cTnT near the second Ca2+- dependent interaction site for Tm and thus may affect Ca2+- sensitivity of the actin/myosin interaction. Indeed, according to Sumandea et al., reversible phosphorylation of Thr206 (mouse sequence) is critical for function (Sumandea et al., 2003). I*n vitro* experiments showed that phosphorylation by PKCα decreases maximal tension, myofilament Ca2+ -sensitivity, actomyosin ATPase activity and cooperativity (Sumandea et al., 2003). Other protein kinases than PKC, as for example ROCKII (Vahebi et al., 2005), phosphorylate cTnT *in vitro*. The physiological role of cTnT phosphorylation by different protein kinases is not yet clarified. cTnT, however, is not a target of cAMP dependent protein kinase (PKA), which is activated upon ß-adrenergic stimulation. But as recently described by Sumandea et al., (2011) cTnT forms an AKAP for PKA with either regulatory subunit I or II (PKA-RI, PKA-RII) and thus provides a platform for sarcomeric protein phosphorylation upon ß-adrenergic stimulation. Binding of PKA might occur within the amino acid region 202-226 which forms an amphiphilic helix needed for the docking of PKA RI and—II (Feliciello et al., 2001). The interaction site for PKA-R would then be located just near the second interaction site with tropomyosin described by (Jin & Chong, 2010) and the PKC phosphorylation site. This implies that PKA-R binding might be affected by PKC

Fig. 1. Organisation , interaction and phosphorylation sites of cardiac troponin T. Exons

known to contain cardiomyopathy mutations are indicated by numbers

phosphorylation and vice versa.

troponin subunits, cTnI and cTnC (Takeda et al., 2003). There is strong evidence from latest single particle reconstruction studies of the thin filament by Paul et al., (2009), that the Nterminal tail of cTnT points to the M-band of the sarcomere, whereas the core domain of the troponin complex is oriented versus the Z-band.

The N-terminal tail fraction of cTnT contains a hypervariable N-terminal part and a highly conserved region, which is located in the central region of the cTnT molecule (Fig. 1). This conserved region contains the main interaction site for tropomyosin (Biesiadecki et al., 2007, Perry, 1998) comprising 39 amino acids (residues 98-136 (human cardiac sequence)) (Jin & Chong, 2010). The Tm interaction site forms a helix according to Murakami et al., (2008) and binds to the overlapping region of two tropomyosin molecules (Jin & Chong, 2010). Crystal structure of the tropomyosin overlap region and the Tm binding helix reveals the formation of a four helix bundle between tropomyosin ends and cTnT (Murakami et al., 2008). The hypervariable region of the cTnT tail fraction does not bind to tropomyosin and can be truncated without losing binding ability of the rest of the molecule (Zhang et al., 2006). Phylogenetically the hypervariable region might be added to the conserved core region of cTnT (Conserved tail region and C-terminal domain) (Jin & Samanez, 2001; Biesiadecki et al., 2007). Its function is not completely elucidated, but the hypervariable region may play a role as modulator of the core molecule thus subtly affecting binding affinities of Tm and cTnT (Biesiadecki et al, 2007; Feng et al., 2008).

A second interaction site for tropomyosin is located at the beginning of the C-terminal half of cTnT and has lately been analysed by Jin & Chong (2010) using antibodies. They showed that highly conserved amino acid sequences are involved comprising amino acid residues 197-236 (human sequence). This interaction site binds to the middle part of tropomyosin near Cys190. Earlier studies (Pearlstone et al., 1983; Morris & Lehrer, 1984) propose the second interaction with tropomyosin at the very C-terminus of cTnT involving residues 272- 288. Whichever amino acids are included in the cTnT/Tm interactions, in the presence of cTnC binding of the TnT-C-Terminus to tropomyosin near Cys190 is Ca2+ dependent (Chong & Hodges, 1982). Ca2+ weakens its binding to tropomyosin though the molecular mechanism, i.e. conformational changes which lead to alteration in Tm/TnT -binding, is not known yet. The TnT-C-Terminus also contains the binding site for the other two troponin subunits, TnI and TnC. According to the 3 D structure of the core cardiac troponin complex, the helix in the C-terminal region of cTnT (residues 226-271), which is highly conserved (Jin et al., 2008) forms a rigid coiled coil with a cTnI-helix (residues 90-136) and is part of a rigid structure within the troponin complex, called the IT-arm (Takeda et al., 2003). At the Cterminal end of the coiled coil the interaction site for cTnC is located and comprises amino acid residues 256- 270. The organisation of cTnT is summarized in Fig.1.

In cardiac muscle the mammalian cTnT- gene (TNNT2) is composed of 17 exons. Exon 5 is absent in adult cTnT (Cooper & Ordahl, 1985). Exon 5 encodes a 10 amino acid long region within the hypervariable N-terminus of cTnT. This sequence contains several acidic residues and contributes to a more negatively charged cTnT. Such a charge difference may modulate function. Indeed fetal cTnT, which contains exon 5, exhibits a higher Ca2+-sensitivity compared to adult cTnT and shows a higher tolerance towards acidic pH (Gomes et al., 2002). Mainly 4 variants of human cTnT (cTnT1-4) have been described due to alternative splicing of exons 4 and 5, whereby TnT1 and TnT3 are the major isoforms present in fetal and in the adult human cardiac muscle, respectively (Townsend et al., 1995; Anderson et al., 1991). The expression pattern of cTnT isoforms seems to be altered in heart failure and correlates with changes in Ca2+- sensitivity. The modification in Ca2+- sensitivity however, seems not to be caused by an

troponin subunits, cTnI and cTnC (Takeda et al., 2003). There is strong evidence from latest single particle reconstruction studies of the thin filament by Paul et al., (2009), that the Nterminal tail of cTnT points to the M-band of the sarcomere, whereas the core domain of the

The N-terminal tail fraction of cTnT contains a hypervariable N-terminal part and a highly conserved region, which is located in the central region of the cTnT molecule (Fig. 1). This conserved region contains the main interaction site for tropomyosin (Biesiadecki et al., 2007, Perry, 1998) comprising 39 amino acids (residues 98-136 (human cardiac sequence)) (Jin & Chong, 2010). The Tm interaction site forms a helix according to Murakami et al., (2008) and binds to the overlapping region of two tropomyosin molecules (Jin & Chong, 2010). Crystal structure of the tropomyosin overlap region and the Tm binding helix reveals the formation of a four helix bundle between tropomyosin ends and cTnT (Murakami et al., 2008). The hypervariable region of the cTnT tail fraction does not bind to tropomyosin and can be truncated without losing binding ability of the rest of the molecule (Zhang et al., 2006). Phylogenetically the hypervariable region might be added to the conserved core region of cTnT (Conserved tail region and C-terminal domain) (Jin & Samanez, 2001; Biesiadecki et al., 2007). Its function is not completely elucidated, but the hypervariable region may play a role as modulator of the core molecule thus subtly affecting binding affinities of Tm and

A second interaction site for tropomyosin is located at the beginning of the C-terminal half of cTnT and has lately been analysed by Jin & Chong (2010) using antibodies. They showed that highly conserved amino acid sequences are involved comprising amino acid residues 197-236 (human sequence). This interaction site binds to the middle part of tropomyosin near Cys190. Earlier studies (Pearlstone et al., 1983; Morris & Lehrer, 1984) propose the second interaction with tropomyosin at the very C-terminus of cTnT involving residues 272- 288. Whichever amino acids are included in the cTnT/Tm interactions, in the presence of cTnC binding of the TnT-C-Terminus to tropomyosin near Cys190 is Ca2+ dependent (Chong & Hodges, 1982). Ca2+ weakens its binding to tropomyosin though the molecular mechanism, i.e. conformational changes which lead to alteration in Tm/TnT -binding, is not known yet. The TnT-C-Terminus also contains the binding site for the other two troponin subunits, TnI and TnC. According to the 3 D structure of the core cardiac troponin complex, the helix in the C-terminal region of cTnT (residues 226-271), which is highly conserved (Jin et al., 2008) forms a rigid coiled coil with a cTnI-helix (residues 90-136) and is part of a rigid structure within the troponin complex, called the IT-arm (Takeda et al., 2003). At the Cterminal end of the coiled coil the interaction site for cTnC is located and comprises amino

In cardiac muscle the mammalian cTnT- gene (TNNT2) is composed of 17 exons. Exon 5 is absent in adult cTnT (Cooper & Ordahl, 1985). Exon 5 encodes a 10 amino acid long region within the hypervariable N-terminus of cTnT. This sequence contains several acidic residues and contributes to a more negatively charged cTnT. Such a charge difference may modulate function. Indeed fetal cTnT, which contains exon 5, exhibits a higher Ca2+-sensitivity compared to adult cTnT and shows a higher tolerance towards acidic pH (Gomes et al., 2002). Mainly 4 variants of human cTnT (cTnT1-4) have been described due to alternative splicing of exons 4 and 5, whereby TnT1 and TnT3 are the major isoforms present in fetal and in the adult human cardiac muscle, respectively (Townsend et al., 1995; Anderson et al., 1991). The expression pattern of cTnT isoforms seems to be altered in heart failure and correlates with changes in Ca2+- sensitivity. The modification in Ca2+- sensitivity however, seems not to be caused by an

acid residues 256- 270. The organisation of cTnT is summarized in Fig.1.

troponin complex is oriented versus the Z-band.

cTnT (Biesiadecki et al, 2007; Feng et al., 2008).

alteration in the isoform expression pattern. Differences in the phosphorylation status of sarcomeric proteins, especially of myosin light chain 2 (MLC-2) may be decisive (van der Velden et al., 2003). Also cTnT itself is a phospho protein (Fig. 1) which is constitutively phosphorylated at Ser1 in several species inclusive men due to the action of casein kinase- 2 (Gusev et al., 1980; Risnik & Gusev, 1984, Swiderek et al., 1990). The function of this phosphorylation is not known up to date. It might prevent degradation and/or interaction of the hypervariable region. At least one further reversible phosphorylation site for PKC is located in the C-terminal region of cTnT near the second Ca2+- dependent interaction site for Tm and thus may affect Ca2+- sensitivity of the actin/myosin interaction. Indeed, according to Sumandea et al., reversible phosphorylation of Thr206 (mouse sequence) is critical for function (Sumandea et al., 2003). I*n vitro* experiments showed that phosphorylation by PKCα decreases maximal tension, myofilament Ca2+ -sensitivity, actomyosin ATPase activity and cooperativity (Sumandea et al., 2003). Other protein kinases than PKC, as for example ROCKII (Vahebi et al., 2005), phosphorylate cTnT *in vitro*. The physiological role of cTnT phosphorylation by different protein kinases is not yet clarified. cTnT, however, is not a target of cAMP dependent protein kinase (PKA), which is activated upon ß-adrenergic stimulation. But as recently described by Sumandea et al., (2011) cTnT forms an AKAP for PKA with either regulatory subunit I or II (PKA-RI, PKA-RII) and thus provides a platform for sarcomeric protein phosphorylation upon ß-adrenergic stimulation. Binding of PKA might occur within the amino acid region 202-226 which forms an amphiphilic helix needed for the docking of PKA RI and—II (Feliciello et al., 2001). The interaction site for PKA-R would then be located just near the second interaction site with tropomyosin described by (Jin & Chong, 2010) and the PKC phosphorylation site. This implies that PKA-R binding might be affected by PKC phosphorylation and vice versa.

Fig. 1. Organisation , interaction and phosphorylation sites of cardiac troponin T. Exons known to contain cardiomyopathy mutations are indicated by numbers

Consequences of Mutations in Genes Encoding Cardiac Troponin C, T and I – Molecular Insights 307

Grabarek, 2011). Alteration in Ca2+-/Mg2+-binding to sites III and IV might alter the interaction with cTnT and the coiled coil structure. Also the identification of cardiomyopathy causing alterations in this part of cTnC points to the involvement in

The N-terminal EF-hand I is not able to bind Ca2+ due to an insertion of Val and the replacement of two Asp residues involved in Ca2+-coordination by Ala and Leu residues. Therefore there is only one functional active Ca2+-binding site (site II) in the N-terminal domain of cTnC, which is a high affinity Ca2+-specific binding site. Binding and release kinetics of Ca2+ to cTnC within the thin filament are such that Ca2+-binding and release occurs within one contraction cycle (Davis & Tikunova, 2008). Saturation of cTnC with Ca2+ is obtained upon increase in intracellular Ca2+-concentration after influx from sarcoplasmatic Ca2+-store upon membrane depolarization. Therefore site II is named the regulatory Ca2+ binding site. Due to the non functional site I there is no conformational switch from closed to open solely upon Ca2+ -binding as is observed for skeletal muscle TnC and the C-terminal lobe of cTnC (Sia et al., 1997). For the stabilization of the open conformation of the Nterminal lobe cTnI binding is required (Dong et al., 1999; Li et al., 1999). There exist multiple interaction sites for cTnI throughout the complete cTnC molecule. The amphiphilic part of helix 1 in cTnI (residues 43-65) binds via several polar and van der Waals interaction to the C-terminal cTnC lobe and around residue 10 of the N-terminal helix of cTnC. Furthermore residues 93-161 in the C-terminal lobe of cTnC interact with the IT-arm (Takeda et al., 2003), The N-terminal lobe of cTnC forms the Ca2+-dependent binding site for the cTnI switch region (Takeda et al., 2003) and a phosphorylation dependent binding site near the nonfunctional Ca2+-binding loop with the flexible heart specific N-terminal cTnI extension

cTnI, the inhibitory subunit of the troponin complex is a very flexible molecule consisting of helices and random coils. In solution cTnI exhibits no tertiary structure. A specific spatial orientation is only obtained within the ternary troponin complex. cTnI is build up in a

The N-terminal extension of about 31 amino acids (length is dependent on species) is heart specific and resembles the hypervaraible region of cTnT. It contains conserved amino acid stretches; thus at the very N-terminus there is a acidic region (Sadayappan et al., 2008) followed by a proline rich sequence, which forms a polyprolin helix and functions as a rigid spacer to keep the N-terminus extended (Howarth et al., 2007). Then the phosphorylation region follows, which contains two adjacent located serine residues at position 22 and 23 (numbered without starter methionine). Both residues are substrates for PKA (Swiderek et al., 1990; Mittmann et al., 1990). In its dephosphorylated state the N-terminal cTnI arm interacts between residues 10-30 with cTnC around amino acid residue 29 (Fig. 2) (Finley et al., 1999; Gaponenko et al., 1999; Abbott et al., 2000; Ward et al.,2003, 2004; Schmidtmann et al., 2002, 2005). This interaction also seems to stabilize the open conformation of the cTnC-N-terminal lobe (Abbott et al., 2000, 2001; Ward et al., 2004). Bisphosphorylation of the two serine residues 22 and 23 by PKA upon ß- adrenergic stimulation releases the interaction with cTnC. According to Howarth et al, (2007) the bisphosphorylated arm contains a helix

regulation.

(see below) (Schmidtmann et al., 2005).

**1.3 Cardiac troponin I (cTnI)**

**1.3.1 The N-terminal extension**

modular fashion (Fig. 3).

#### **1.2 Cardiac troponin C (cTnC)**

cTnC is the calcium binding subunit of the cardiac troponin complex. Structurally the protein belongs to the EF-hand calcium binding protein super family together with parvalbumin, the first described protein of this family, Calmodulin, skeletal muscle troponin C etc. cTnC is composed of two lobes connected by a flexible linker (Sia et al., 1997) (Fig.2).

Fig. 2. Interaction sites and cardiomyopathy inducing single amino acid exchanges in cTnC

The structure was taken from PDP 1AJ4 based on the work of (Sia et al., 1997). The 3 D structure of cTnC in calcium (green points) saturated from is shown. Helices are given as magenta ribbons. The position of cardiomyopathy mutations is indicated by stars. Interacting proteins are given in blue.

Each lobe contains two parallel EF hands, each of which forms the helix-loop-helix divalent metal binding domain. Helices in a protein have been labeled by capital letters, with A assigned to the most N-terminally located helix. Thus E and F are the loop flanking helices in parvalbumin forming the metal binding motif. The name EF-hand for the helix loop helix metal binding motif is based on this nomenclature. The short helices of about 10-12 residues are arranged perpendicular. The loop is composed of 12 residues essential for calcium coordination in a pentagonal bipyramidal configuration. The residues 1, 3, 5, 7, 9 and 12 (X, Y, Z, -Y, -X, -Z) are involved in Ca2+-coordination. In position 12 there is a conserved glutamate or aspartate residue providing two oxygens for calcium binding (Structure Reference: PDB: 2PMY). The calcium binding residues preferentially have an acidic side chain, but also the protein backbone is involved. In cTnC the two EF hands (III, IV) in the Cterminal lobe are the high affinity Ca2+-, Mg2+-binding sites, which contain metal ions also at low (relaxing) intracellular Ca2+-concentration. This C-terminal domain of cTnC provides the platform for binding of cTnI and of cTnT and therefore is pivotal for the integrity of the troponin complex. Helices of the metal bound C-terminal domain exhibit a hydrophobic pocket, where cTnI is bound (Gasmi-Seabrook et al., 1999). cTnT binds to Calcium sites III and IV at the end of the rigid coiled coil. Besides the structural role of the C-terminal lobe, there is now strong evidence that it plays an active part in thin filament activation (Fuchs & Grabarek, 2011). Alteration in Ca2+-/Mg2+-binding to sites III and IV might alter the interaction with cTnT and the coiled coil structure. Also the identification of cardiomyopathy causing alterations in this part of cTnC points to the involvement in regulation.

The N-terminal EF-hand I is not able to bind Ca2+ due to an insertion of Val and the replacement of two Asp residues involved in Ca2+-coordination by Ala and Leu residues. Therefore there is only one functional active Ca2+-binding site (site II) in the N-terminal domain of cTnC, which is a high affinity Ca2+-specific binding site. Binding and release kinetics of Ca2+ to cTnC within the thin filament are such that Ca2+-binding and release occurs within one contraction cycle (Davis & Tikunova, 2008). Saturation of cTnC with Ca2+ is obtained upon increase in intracellular Ca2+-concentration after influx from sarcoplasmatic Ca2+-store upon membrane depolarization. Therefore site II is named the regulatory Ca2+ binding site. Due to the non functional site I there is no conformational switch from closed to open solely upon Ca2+ -binding as is observed for skeletal muscle TnC and the C-terminal lobe of cTnC (Sia et al., 1997). For the stabilization of the open conformation of the Nterminal lobe cTnI binding is required (Dong et al., 1999; Li et al., 1999). There exist multiple interaction sites for cTnI throughout the complete cTnC molecule. The amphiphilic part of helix 1 in cTnI (residues 43-65) binds via several polar and van der Waals interaction to the C-terminal cTnC lobe and around residue 10 of the N-terminal helix of cTnC. Furthermore residues 93-161 in the C-terminal lobe of cTnC interact with the IT-arm (Takeda et al., 2003), The N-terminal lobe of cTnC forms the Ca2+-dependent binding site for the cTnI switch region (Takeda et al., 2003) and a phosphorylation dependent binding site near the nonfunctional Ca2+-binding loop with the flexible heart specific N-terminal cTnI extension (see below) (Schmidtmann et al., 2005).

#### **1.3 Cardiac troponin I (cTnI)**

306 Cardiomyopathies – From Basic Research to Clinical Management

cTnC is the calcium binding subunit of the cardiac troponin complex. Structurally the protein belongs to the EF-hand calcium binding protein super family together with parvalbumin, the first described protein of this family, Calmodulin, skeletal muscle troponin C etc. cTnC is composed of two lobes connected by a flexible linker (Sia et al., 1997) (Fig.2).

Fig. 2. Interaction sites and cardiomyopathy inducing single amino acid exchanges in cTnC The structure was taken from PDP 1AJ4 based on the work of (Sia et al., 1997). The 3 D structure of cTnC in calcium (green points) saturated from is shown. Helices are given as magenta ribbons. The position of cardiomyopathy mutations is indicated by stars.

Each lobe contains two parallel EF hands, each of which forms the helix-loop-helix divalent metal binding domain. Helices in a protein have been labeled by capital letters, with A assigned to the most N-terminally located helix. Thus E and F are the loop flanking helices in parvalbumin forming the metal binding motif. The name EF-hand for the helix loop helix metal binding motif is based on this nomenclature. The short helices of about 10-12 residues are arranged perpendicular. The loop is composed of 12 residues essential for calcium coordination in a pentagonal bipyramidal configuration. The residues 1, 3, 5, 7, 9 and 12 (X, Y, Z, -Y, -X, -Z) are involved in Ca2+-coordination. In position 12 there is a conserved glutamate or aspartate residue providing two oxygens for calcium binding (Structure Reference: PDB: 2PMY). The calcium binding residues preferentially have an acidic side chain, but also the protein backbone is involved. In cTnC the two EF hands (III, IV) in the Cterminal lobe are the high affinity Ca2+-, Mg2+-binding sites, which contain metal ions also at low (relaxing) intracellular Ca2+-concentration. This C-terminal domain of cTnC provides the platform for binding of cTnI and of cTnT and therefore is pivotal for the integrity of the troponin complex. Helices of the metal bound C-terminal domain exhibit a hydrophobic pocket, where cTnI is bound (Gasmi-Seabrook et al., 1999). cTnT binds to Calcium sites III and IV at the end of the rigid coiled coil. Besides the structural role of the C-terminal lobe, there is now strong evidence that it plays an active part in thin filament activation (Fuchs &

**1.2 Cardiac troponin C (cTnC)**

Interacting proteins are given in blue.

cTnI, the inhibitory subunit of the troponin complex is a very flexible molecule consisting of helices and random coils. In solution cTnI exhibits no tertiary structure. A specific spatial orientation is only obtained within the ternary troponin complex. cTnI is build up in a modular fashion (Fig. 3).

#### **1.3.1 The N-terminal extension**

The N-terminal extension of about 31 amino acids (length is dependent on species) is heart specific and resembles the hypervaraible region of cTnT. It contains conserved amino acid stretches; thus at the very N-terminus there is a acidic region (Sadayappan et al., 2008) followed by a proline rich sequence, which forms a polyprolin helix and functions as a rigid spacer to keep the N-terminus extended (Howarth et al., 2007). Then the phosphorylation region follows, which contains two adjacent located serine residues at position 22 and 23 (numbered without starter methionine). Both residues are substrates for PKA (Swiderek et al., 1990; Mittmann et al., 1990). In its dephosphorylated state the N-terminal cTnI arm interacts between residues 10-30 with cTnC around amino acid residue 29 (Fig. 2) (Finley et al., 1999; Gaponenko et al., 1999; Abbott et al., 2000; Ward et al.,2003, 2004; Schmidtmann et al., 2002, 2005). This interaction also seems to stabilize the open conformation of the cTnC-N-terminal lobe (Abbott et al., 2000, 2001; Ward et al., 2004). Bisphosphorylation of the two serine residues 22 and 23 by PKA upon ß- adrenergic stimulation releases the interaction with cTnC. According to Howarth et al, (2007) the bisphosphorylated arm contains a helix

Consequences of Mutations in Genes Encoding Cardiac Troponin C, T and I – Molecular Insights 309

dependent phosphorylation at Ser22/23 seems not to be impaired by Ser43/45

The C-terminal region of cTnI can functionally be subdivided into the inhibitory region comprising amino acids 137-148, the helical switch region (amino acids 150-159) and the Cterminal mobile region. The inhibitory region binds to actin/tropomyosin in the relaxed state, i.e. when the regulatory Ca2+ binding loop of cTnC contains no Ca2+. Within this region there is another heart specific PKC phosphorylation site at Thr 144. The physiological role of this phosphorylation site is not quite clear (Solaro & Kobayashi, 2011), though investigations by Tachampa et al., (2007) imply that Thr144 is involved in length dependent activation of tension development in thin filament bundles. Phosphorylation might modulate this

Upon Ca2+- saturation of cTnC the inhibitory region is released from actin and the switch region binds to the cTnC-N-terminal lobe inducing the formation of the hydrophic pocket. Strength of interaction is sensible to small conformational changes in the cTnC-N-lobe also affecting Ca2+-binding affinity. The C-terminal mobile region following the switch region is a very important cTnI region for regulation of muscle contraction, though not much is known about this region. It provides a second actin binding site and a tropomyosin binding site. Under relaxing conditions, this mobile region is fixed to tropomyosin (Pirani et al., 2005; Galinska et al., et al., 2008) and actin, and is released from actin/tropomyosin upon Ca2+-saturation of cTnC. Thus the C-Terminus stabilizes the blocked state of the thin filament (Galinska et al., 2010). Probably this effect is intensified by the interaction of cTnT binding to tropomyosin actin. Thus cTnT- N-terminus and cTnI-C-terminus both support the inhibitory region of cTnI in keeping tropomyosin in the blocking position. Hereby troponin complexes on opposite sites cooperate, one complex providing cTnT/tropomyosin interaction the other cTnI/tropomyosin/actin interaction (Paul et al., 2009; Solaro & Kobayashi, 2011). But cardiac TnI is not only involved in regulation of inhibition, but also of activation. Evidence came from investigations of Galinska et al., (2010) using truncated cTnI and normal length cTnI. They showed that the C-terminus is also involved in stabilization of tropomyosin in the active state (Ca2+ - saturated cTnC). Truncation may occur *in vivo* due to proteolysis in myocardial stunning (reversible ischemia/reperfusion injury) (Foster et al.,

A large number of mutations have been detected in genes encoding for the three cTn subunits in patients suffering of cardiomyopathies (tables 1-6). Resulting phenotypes are highly variable, even within a family carrying the same mutation (see below), indicating that modifiers, environment or polymorphisms are involved in disease development. Wang et al., (2005) was the first group who detected a polymorphism in the MYBPC3 (cardiac myosin binding protein C gene) that might be able to modify the expression of hypertrophy. Also combinations of more than one mutation determine the disease development. Therefore it is still impossible to correlate phenotype and genotype, though an immense progress has been made in the understanding of molecular pathogenesis. The goal to understand phenotype development remains. Since the disease is primarily caused by

function probably by loosening the interaction of cTnI inhibitory region with actin.

phosphorylation (Montgomery et al., 2002).

2003) and due to cardiomyopathy mutations.

**2. Cardiomyopathy inducing troponin mutations**

**1.3.3 Regulatory C-terminal region**

comprising amino acids 21-30 being stabilized by salt bridges between phosphate and preceeding arginine residues (Jaquet et al. 1998). The release of the extension from cTnC takes place due to the insertion of negative charges followed by conformational changes. This allows a different interaction, which is directed by the acidic part of the N-terminal cTnI arm and therefore needs a positively charged partner (Sadayappan et al., 2008). Clusters of basic amino acid residues are provided by the regulatory C-terminal domain of cTnI itself, but also an additional interaction with actin cannot be excluded. The release of the N-terminal arm from cTnC leads to a reduction of the Ca2+ -affinity of cTnC, in myofilament Ca2+-sensitivity (Zhang et al 1995; Reiffert et al., 1996) and to enhanced cross bridge cycling (Kentish et al.,2001; Turnbull et al., 2002). There is evidence that the main action occurs via interaction with cTnI, which stabilizes cTnI binding to the thin filament (Sakthivel et al., 2005).

Fig. 3. Organization of cTnI, its interactions (blue) and phosphorylation sites (indicated by )

#### **1.3.2 Constitutive cTnC and cTnT binding sites**

The cTnC binding site is located subsequent to the N-terminal extension. It forms a helix (helix 1 according to the nomenclature of Takeda et al., 2003) which contacts the N-terminal cTnC helix and reaches to the C-terminal lobe of cTnC. It strongly binds to the hydrophobic pocket of the C-terminal cTnC lobe. The C-terminal part of helix 1 also interacts with cTnT via several hydrogen bonds and hydrophobic interactions. This helix is followed by another helical binding site for cTnT (helix 2). Helix 2 forms a coiled coil with a helix located in the C-terminal half of cTnT. The two helices are part of the IT arm (Takeda et al., 2003). Both these binding sites in cTnI are independent on reversible Ca2+ -binding to the regulatory Ca2+ binding loop in cTnC. In helix 1 there are two serine residues (Ser43/45) which are phosphorylated by PKC upon alpha adrenergic stimulation. Ser43/45 are positioned near residue 10 of the N-terminal helix in cTnC-N-terminal lobe as well as near the C-terminal lobe. Thus, phosphorylation might alter these interactions. In mice phosphorylation of these sites by PKC upon α-adrenergic stimulation is responsible for the negative inotropic effect and might be influenced by the cTnT phosphorylation state. The physiological effect of PKA dependent phosphorylation at Ser22/23 seems not to be impaired by Ser43/45 phosphorylation (Montgomery et al., 2002).

#### **1.3.3 Regulatory C-terminal region**

308 Cardiomyopathies – From Basic Research to Clinical Management

comprising amino acids 21-30 being stabilized by salt bridges between phosphate and preceeding arginine residues (Jaquet et al. 1998). The release of the extension from cTnC takes place due to the insertion of negative charges followed by conformational changes. This allows a different interaction, which is directed by the acidic part of the N-terminal cTnI arm and therefore needs a positively charged partner (Sadayappan et al., 2008). Clusters of basic amino acid residues are provided by the regulatory C-terminal domain of cTnI itself, but also an additional interaction with actin cannot be excluded. The release of the N-terminal arm from cTnC leads to a reduction of the Ca2+ -affinity of cTnC, in myofilament Ca2+-sensitivity (Zhang et al 1995; Reiffert et al., 1996) and to enhanced cross bridge cycling (Kentish et al.,2001; Turnbull et al., 2002). There is evidence that the main action occurs via interaction with cTnI, which stabilizes cTnI binding to the thin filament

Fig. 3. Organization of cTnI, its interactions (blue) and phosphorylation sites (indicated by )

The cTnC binding site is located subsequent to the N-terminal extension. It forms a helix (helix 1 according to the nomenclature of Takeda et al., 2003) which contacts the N-terminal cTnC helix and reaches to the C-terminal lobe of cTnC. It strongly binds to the hydrophobic pocket of the C-terminal cTnC lobe. The C-terminal part of helix 1 also interacts with cTnT via several hydrogen bonds and hydrophobic interactions. This helix is followed by another helical binding site for cTnT (helix 2). Helix 2 forms a coiled coil with a helix located in the C-terminal half of cTnT. The two helices are part of the IT arm (Takeda et al., 2003). Both these binding sites in cTnI are independent on reversible Ca2+ -binding to the regulatory Ca2+ binding loop in cTnC. In helix 1 there are two serine residues (Ser43/45) which are phosphorylated by PKC upon alpha adrenergic stimulation. Ser43/45 are positioned near residue 10 of the N-terminal helix in cTnC-N-terminal lobe as well as near the C-terminal lobe. Thus, phosphorylation might alter these interactions. In mice phosphorylation of these sites by PKC upon α-adrenergic stimulation is responsible for the negative inotropic effect and might be influenced by the cTnT phosphorylation state. The physiological effect of PKA

**1.3.2 Constitutive cTnC and cTnT binding sites**

(Sakthivel et al., 2005).

The C-terminal region of cTnI can functionally be subdivided into the inhibitory region comprising amino acids 137-148, the helical switch region (amino acids 150-159) and the Cterminal mobile region. The inhibitory region binds to actin/tropomyosin in the relaxed state, i.e. when the regulatory Ca2+ binding loop of cTnC contains no Ca2+. Within this region there is another heart specific PKC phosphorylation site at Thr 144. The physiological role of this phosphorylation site is not quite clear (Solaro & Kobayashi, 2011), though investigations by Tachampa et al., (2007) imply that Thr144 is involved in length dependent activation of tension development in thin filament bundles. Phosphorylation might modulate this function probably by loosening the interaction of cTnI inhibitory region with actin.

Upon Ca2+- saturation of cTnC the inhibitory region is released from actin and the switch region binds to the cTnC-N-terminal lobe inducing the formation of the hydrophic pocket. Strength of interaction is sensible to small conformational changes in the cTnC-N-lobe also affecting Ca2+-binding affinity. The C-terminal mobile region following the switch region is a very important cTnI region for regulation of muscle contraction, though not much is known about this region. It provides a second actin binding site and a tropomyosin binding site. Under relaxing conditions, this mobile region is fixed to tropomyosin (Pirani et al., 2005; Galinska et al., et al., 2008) and actin, and is released from actin/tropomyosin upon Ca2+-saturation of cTnC. Thus the C-Terminus stabilizes the blocked state of the thin filament (Galinska et al., 2010). Probably this effect is intensified by the interaction of cTnT binding to tropomyosin actin. Thus cTnT- N-terminus and cTnI-C-terminus both support the inhibitory region of cTnI in keeping tropomyosin in the blocking position. Hereby troponin complexes on opposite sites cooperate, one complex providing cTnT/tropomyosin interaction the other cTnI/tropomyosin/actin interaction (Paul et al., 2009; Solaro & Kobayashi, 2011). But cardiac TnI is not only involved in regulation of inhibition, but also of activation. Evidence came from investigations of Galinska et al., (2010) using truncated cTnI and normal length cTnI. They showed that the C-terminus is also involved in stabilization of tropomyosin in the active state (Ca2+ - saturated cTnC). Truncation may occur *in vivo* due to proteolysis in myocardial stunning (reversible ischemia/reperfusion injury) (Foster et al., 2003) and due to cardiomyopathy mutations.

#### **2. Cardiomyopathy inducing troponin mutations**

A large number of mutations have been detected in genes encoding for the three cTn subunits in patients suffering of cardiomyopathies (tables 1-6). Resulting phenotypes are highly variable, even within a family carrying the same mutation (see below), indicating that modifiers, environment or polymorphisms are involved in disease development. Wang et al., (2005) was the first group who detected a polymorphism in the MYBPC3 (cardiac myosin binding protein C gene) that might be able to modify the expression of hypertrophy. Also combinations of more than one mutation determine the disease development. Therefore it is still impossible to correlate phenotype and genotype, though an immense progress has been made in the understanding of molecular pathogenesis. The goal to understand phenotype development remains. Since the disease is primarily caused by

Consequences of Mutations in Genes Encoding Cardiac Troponin C, T and I – Molecular Insights 311

Glu83Lys (E83K) HCM 8 Mogensen et al., (2003) J Med Genet 40: e59 Val85Leu (V85L) HCM 8 Konno et al., (2005) J Intern Med. 258: 216-24.

**Mutation Disease Exon/Intron Reference**  Phe70Leu (F70L) HCM 8 Richard et al., (2003) Circ. 107: 2227-32 Pro77Leu (P77L) HCM 8 Varnava et al., (2001) Circ. 104: 1380-4

Ile79Asn (I79N) HCM , RCM, DCM 8

Arg92Trp (R92W) HCM 9

Arg92Leu (R92L) HCM 9

Arg92Gln (R92Q) HCM 9

Arg94Leu (R94L) HCM 9

Ala104Val (A104V) HCM 9

mutations, it is crucial to improve knowledge on the dysfunctions on molecular level in detail of as many mutants as possible. Thus one might be able to detect common features and mechanisms which might allow detection of a link to phenotype development. Though the molecular effects of only a couple of mutations have been thoroughly investigated a first common rule, namely an enhancement in Ca2+- sensitivity of the myofilament for HCM/RCM-mutants and a decrease in Ca2+- sensitivity for DCM mutants has been stated by Robinson et al., (2007). However it seems to be too simple and does not explain the development of HCM or RCM or the often very low degree of hypertrophy despite large Ca2+ sensitivity changes and susceptibility to malignant arrhythmia. Furthermore, the rule does not apply to all mutations investigated. One problem is that analysis of Ca2+ sensitivity resulted in many opposing statements. Thus for example Nakaura et al., 1999 did not observe enhanced Ca2+ -sensitivity of force in skinned fibres with cTnT-F110I, whereas Hernandez et al., 2005 described enhanced Ca2+ -sensitivity of force development as well as of actomyosin ATPase activity. One main reason lies in the complexity of the systems used, as reconstituted proteins, skinned fibers, myofibrils, isolated cardiomyocytes (adult or neonatal), transgenic animals. In general results obtained with higher organised system sseem to be more reliable. But additionally species differences might account for differing results. For example according to Rust et al., (1999) overexpression of cTnT-I79N in rat cardiomyocytes resulted in suppressed contractile performance, whereas others using mice myofibrils suggested hypercontractility.

#### **2.1 Mutations in TNNT2, the gene encoding cTnT**

Most mutations in TNNT2 detected in patients lead to familial hypertrophic cardiomyopathy (HCM), only a few to restricted cardiomyopathy (RCM) or dilated hypertrophic cardiomyopathy (DCM) (Table 1). Families with DCM mutations in TNNT2 mostly exhibit a severe disease progression with poor prognosis.

#### **2.1.1 HCM inducing mutations**

HCM-mutations in TNNT2 are found in about 10% of the HCM cases, and thus TNNT2 belongs to the more abundant troponin disease genes. There is no known HCM mutation which is located within the hypervariable N-terminal region of cTnT. All mutations identified in patients suffering from HCM are located either near or within the N-terminal main interaction site for tropomyosin (amino acids 79-182) or within the C-terminal half (amino acids 203-288) which contains multiple interaction and putative phosphorylation sites (Table 1; Fig. 1). This distribution of mutations implies that they affect the interaction either with tropomyosin (Tm) and/ or the other troponin subunits and might influence phosphorylation dependent effects. The majority of gene loci, where mutations have been identified in patients encode the N-terminal region of cTnT interacting with the overlap of Tm (Table 1; Fig. 1). Mutations in the Tm binding region of cTnT (amino acids 92-183) destabilize Tm binding to actin filaments (Palm et al., 2001). Mutations within the main Tm binding site further weaken the end-to-end Tm interaction, which is responsible for the cooperativity (Palm et al., 2001). Also a lowered affinity of troponin to actin/Tm could be expected. Indeed cTnT-F110I, located at the C-terminal end of the main Tm binding region, impairs binding of troponin to actin/Tm by altering the dynamic properties of the tail region (Hinkle & Tobacman, 2002). It reduces its flexibility. Flexibility of the cTnT-tail is an important feature for its interaction with Tm overlap region (Hinkle & Tobacman, 2002).

13.

310 Cardiomyopathies – From Basic Research to Clinical Management

mutations, it is crucial to improve knowledge on the dysfunctions on molecular level in detail of as many mutants as possible. Thus one might be able to detect common features and mechanisms which might allow detection of a link to phenotype development. Though the molecular effects of only a couple of mutations have been thoroughly investigated a first common rule, namely an enhancement in Ca2+- sensitivity of the myofilament for HCM/RCM-mutants and a decrease in Ca2+- sensitivity for DCM mutants has been stated by Robinson et al., (2007). However it seems to be too simple and does not explain the development of HCM or RCM or the often very low degree of hypertrophy despite large Ca2+ sensitivity changes and susceptibility to malignant arrhythmia. Furthermore, the rule does not apply to all mutations investigated. One problem is that analysis of Ca2+ sensitivity resulted in many opposing statements. Thus for example Nakaura et al., 1999 did not observe enhanced Ca2+ -sensitivity of force in skinned fibres with cTnT-F110I, whereas Hernandez et al., 2005 described enhanced Ca2+ -sensitivity of force development as well as of actomyosin ATPase activity. One main reason lies in the complexity of the systems used, as reconstituted proteins, skinned fibers, myofibrils, isolated cardiomyocytes (adult or neonatal), transgenic animals. In general results obtained with higher organised system sseem to be more reliable. But additionally species differences might account for differing results. For example according to Rust et al., (1999) overexpression of cTnT-I79N in rat cardiomyocytes resulted in suppressed contractile performance, whereas others using mice

Most mutations in TNNT2 detected in patients lead to familial hypertrophic cardiomyopathy (HCM), only a few to restricted cardiomyopathy (RCM) or dilated hypertrophic cardiomyopathy (DCM) (Table 1). Families with DCM mutations in TNNT2

HCM-mutations in TNNT2 are found in about 10% of the HCM cases, and thus TNNT2 belongs to the more abundant troponin disease genes. There is no known HCM mutation which is located within the hypervariable N-terminal region of cTnT. All mutations identified in patients suffering from HCM are located either near or within the N-terminal main interaction site for tropomyosin (amino acids 79-182) or within the C-terminal half (amino acids 203-288) which contains multiple interaction and putative phosphorylation sites (Table 1; Fig. 1). This distribution of mutations implies that they affect the interaction either with tropomyosin (Tm) and/ or the other troponin subunits and might influence phosphorylation dependent effects. The majority of gene loci, where mutations have been identified in patients encode the N-terminal region of cTnT interacting with the overlap of Tm (Table 1; Fig. 1). Mutations in the Tm binding region of cTnT (amino acids 92-183) destabilize Tm binding to actin filaments (Palm et al., 2001). Mutations within the main Tm binding site further weaken the end-to-end Tm interaction, which is responsible for the cooperativity (Palm et al., 2001). Also a lowered affinity of troponin to actin/Tm could be expected. Indeed cTnT-F110I, located at the C-terminal end of the main Tm binding region, impairs binding of troponin to actin/Tm by altering the dynamic properties of the tail region (Hinkle & Tobacman, 2002). It reduces its flexibility. Flexibility of the cTnT-tail is an important feature for its interaction with Tm overlap region (Hinkle & Tobacman, 2002).

myofibrils suggested hypercontractility.

**2.1.1 HCM inducing mutations**

**2.1 Mutations in TNNT2, the gene encoding cTnT**

mostly exhibit a severe disease progression with poor prognosis.

Consequences of Mutations in Genes Encoding Cardiac Troponin C, T and I – Molecular Insights 313

Arg286Cys (R286C) HCM 16 Richard et al., (2003) Circ. 107: 2227-32 ; Miliou

Arg286His (R286H) HCM 16 Van Driest et al., (2003) Circ. 108: 445-51 ; Van

(R113W) DCM 9 Mogensen et al., (2004) JACC. 44: 2033-40 ;

Ala172Ser (A172S) DCM 11 Stefanelli et al., (2004) Mol Genet Metab. 83:

Arg205Leu (R205L) DCM 13 Mogensen et al., (2004) JACC 44: 2033-40;

Glu96del (ΔE96) RCM 9 Peddy et al., (2006) Pediatrics 117:1830-3; Pinto

Del (ΔN100/ΔE101) RCM 9 Pinto et al., (2011)JBC 286:20901-12 (double

Updated from: Genomics of Cardiovascular Development, Adaptation, and Remodeling. NHLBI Program for Genomic Applications, Harvard Medical School. [*june, 2011 accessed*] and OMIM database. ter designates termination of sequence resulting in a truncated protein, del and Δ are synonyms for a

(D270N) DCM 15 Mirza et al., (2005) JBC. 280 :28498-506

Glu136Lys (E136K) RCM 10 Kaski et al., (2008) Heart 94:1478-84

(W287ter) HCM 16 Richard et al., (2003) Circ. 107: 2227-32.

**Mutation Disease Exon/Intron Reference**

Arg278Cys (R278C) HCM 16

Arg278Pro (R278P) HCM 16

(R141W) DCM 10

Lys210del (ΔK210) DCM 13

deleted amino acid. The one letter code is given in brackets.

Trp287ter

Arg113Trp

Arg141Trp

Asp270Asn

Asn100del/Glu101

Table 1. Mutations in TNNT2

Updated from: Genomics of Cardiovascular Development, Adaptation, and Remodeling. NHLBI Program for Genomic Applications, Harvard Medical School. [*june, 2011 accessed*] and OMIM database. ter designates termination of sequence resulting in a truncated protein, del and Δ are synonyms for a deleted amino acid. The one letter code is given in brackets.

Table 1.  ; Mukherjea et al., (1999) Biochem. 38: 13296-301; Redwood et al., (2000) Circ Res. 86: 1146-52;

Varnava et al., (2001) Circ. 104: 1380-4

et al., (2001) Biophys J. 81: 2827-37

Skvor (2006) Meth Inf Med. 45: 169-72

http://www.cardiogenomics.org

**Mutation Disease Exon/Intron Reference**

Phe110Ile (F110I) HCM 9

Phe110Val (F110V) HCM 9

Arg130Cys (R130C) HCM 10

Glu160del (ΔE160) HCM 11

Glu244Asp (E244D) HCM 14

Lys273Glu (K273E) HCM 15

IVS15+1G>A HCM 15

Consequences of Mutations in Genes Encoding Cardiac Troponin C, T and I – Molecular Insights 315

interestingly I79N, originally listed as HCM mutation, might also cause RCM or DCM even in members of the same family. This indicates that other factors besides the mutation, as for example polymorphisms etc. (see below) are determinant for the development of the specific disease. Also in mice RCM might be evolved by I79N (Table 1). The other two RCM mutations (Table 1), a deletion of an acidic residue (E96del) and replacement of a glutamic acid residue by a basic lysine (E136K) have been detected in children developing RCM with very poor prognosis. E96del poorly inhibited actomyosin activity at low Ca2+ concentrations (Pinto et al., 2008) confirming that the cTnT N-terminus is important for the inhibitory capacity of the cardiac troponin complex. A first double deletion (Table 1) leads to the deletion of two amino acids, located adjacently within the main Tm binding site. In filaments with adult, not fetal cTnT an increase in Ca2+-sensitivity and decrease in

Patients with DCM mutations in cTNNT2 exhibit a malignant prognosis as do also most of the FHC mutations in cTNNT2. Investigations on molecular level are largely missing to date. All patients showed decreased cardiac function (Kamisago et al., 2000) in contrast to enhanced contractility often observed in patients and transgenic animals carrying HCM mutations. In accordance *in vitro* analysis of DCM-mutations revealed a decreased Ca2+ sensitivity (Mirza et al., 2005). Venkatraman et al., 2005 showed that ΔK210, the most prominent example for DCM causing cTnT-variation, decreases Ca2+-sensitivity of force and actomyosin ATPase activity as well as maximal force and ATPase activity not only with adult, but also with fetal cTnT. The deletion of K210 occurs in the second Ca2+ -sensitive tropomyosin binding region near the PKC phosphorylation site and possibly near the binding site for PKA regulatory subunit II. This implies that Ca2+ -regulation as well as PKC dependent phosphorylation of cTnT and/or PKA dependent phosphorylation of other

Mutations in the TNNC1 gene occur seldom. Far less than 1% of the genetic disorders leading to familial cardiomyopathies in patients are due to mutations in TNNC1. Up to date only six HCM and one DCM inducing mutations, all single amino acid exchanges, have been identified (Fig.2; Table 2). A8V, L29Q and C84Y are located in the N-terminal and N122fs, E134D, D145E and G159D in the C-terminal domain of cTnC (Fig. 2). L29Q was the first mutation detected in the cTnC gene (Hoffmann et al., 2001). Since there was only one family showing this mutation, it is not clear if this mutation causes HCM. However, according to Schmidtmann et al., 2005 and Liang et al., 2008 this amino acid replacement has the potential to cause a cardiomyopathy. Replacement of leucine by glutamine destabilizes the interaction of the N-terminal cTnI arm with cTnC (Schmidtmann et al., 2005; Baryshnikova et al., 2008). The release of the N-terminal cTnI arm occurs also upon bisphosphorylation of cTnI after ß-adrenergic stimulation and contributes to a reduction in Ca2+-sensitivity of the actomyosin ATPase activity (see below). Consistent with this effect for L29Q a small reduction of Ca2+- sensitivity has been described (Schmidtmann et al., 2005). However, in the phosphorylated state of cTnI, an enhanced Ca2+-sensitivity of the actomyosin ATPase activity was observed, indicating an altered ß-adrenergic responsiveness. Divergent results might be obtained in systems differing in complexity

cooperativity has been observed so far (Pinto et al., 2011).

sarcomeric proteins might be affected. Detailed information is missing.

**2.2 Mutations in TNNC1, the gene encoding cTnC**

**2.1.3 DCM inducing mutations**

Since the cTnT- N-terminus contributes to the inhibition of the actin/myosin interaction (Tobacman, 1988), one might assume that also inhibition is affected by mutations. Indeed inhibition of is reduced due to cTnT-F110I (Knollmann & Potter, 2001; Gomes et al., 2004). A similar decrease in inhibition has been described for I79N (Yanaga et al., 1999), though this amino acid exchange is positioned N-terminally of the main Tm interaction site. Nevertheless I79N and F110I exhibit several similarities. Thus, Midde et al., 2011 showed that rigor crossbridges in I79N or F110I containing filaments were disordered, indicating that disruption in thin filament structure may lead to severe contractile dysfunction. I79N, as most investigated HCM mutants, enhances the Ca2+- sensitivity of force development and actomyosin ATPase activity, which might contribute to enhanced contractility and higher energy consumption (Lin et al., 1996; Sweeney et al., 1998; Chandra et al., 2005). Furthermore, mouse hearts with I79N or R92Q could not increase cardiac performance upon ß-adrenergic stimulation (Knollmann et al., 2001; Javadpour et al., 2003), performance of I79N transgenic even worsened upon isoproterenol treatment (Sirenko et al., 2006). Such an effect as well as Ca2+ –sensitivity increase has not been observed with a R278C, a mutation located in the cTnT-C-terminus. These findings suggest that the region around amino acids 79 and 92 is important for Ca2+ -signal transmission and affects Ca2+-regulation. Furthermore dysfunction is especially prominent under ß-adrenergic stimulation, which might explain the high risc for cardiac sudden death of these mutations. Causality between enhanced Ca2+-sensitivity and the potential for the development of malignant arrhythmias has been shown by Baudenbacher et al., (2008) for I79N. They described altered action potential duration due to the mutation.

Mutations in TNNT2 gene encoding the C-terminal half of cTnT might affect the interaction with tropomyosin at the second Ca2+ -dependent binding site, as well as binding to cTnI and cTnC. Most mutations in this part lead either to single amino acid exchanges, deletions of single amino acids or to C-terminally truncated cTnT molecules due to splice site mutations. IVS15+1G>A is a splicing donor mutant which might result in two truncated proteins. In one mutant exon 16 is skipped encoding the C-terminal 14 amino acids, in the other mutants seven amino acids replace the C-terminal 28 amino acids encoded by exon 15 and 16 (Thierfelder et al., 1994). Both mutants are able to form a heterotrimeric troponin complex, however their affinity towards cTnI is drastically reduced (Mukherjea et al., 1999). The impaired interaction with cTnI may be the cause for reduced inhibitory capacity of troponin at low Ca2+ concentrations described by Redwood et al., (2000) and accelerated cross bridge kinetics (Stelzer et al., 2004). Furthermore a reduced binding of troponin to the thin filament has been reported dependent on its regulatory states (Knollmann & Potter, 2001; Burhop et al., 2001), indicating that the C-terminal part of cTnT stabilizes binding of cTn to the thin filament and affects Ca2+ -regulation. Increased Ca2+-sensitivity and cooperativity (Nakaura et al., 1999)) and impaired switching off myosin cycling at low Ca2+-concentrations is observed (Burhop et al., 2001). Up to date nearly nothing is known on how cardiomyopathy inducing mutations in TNNT2 influence phosphorylation dependent effects of PKC dependent cTnT or PKA dependent cTnI phosphorylation. According to a study of Nakaura et al., (1999) effects of truncated cTnT were independent on PKA dependent cTnI phosphorylation.

#### **2.1.2 RCM inducing mutations**

There are only few RCM mutations detected in TNNT2 (Table 1). Most RCM mutations in genes encoding for cardiac troponin subunits are found in TNNI3 (see below). However, interestingly I79N, originally listed as HCM mutation, might also cause RCM or DCM even in members of the same family. This indicates that other factors besides the mutation, as for example polymorphisms etc. (see below) are determinant for the development of the specific disease. Also in mice RCM might be evolved by I79N (Table 1). The other two RCM mutations (Table 1), a deletion of an acidic residue (E96del) and replacement of a glutamic acid residue by a basic lysine (E136K) have been detected in children developing RCM with very poor prognosis. E96del poorly inhibited actomyosin activity at low Ca2+ concentrations (Pinto et al., 2008) confirming that the cTnT N-terminus is important for the inhibitory capacity of the cardiac troponin complex. A first double deletion (Table 1) leads to the deletion of two amino acids, located adjacently within the main Tm binding site. In filaments with adult, not fetal cTnT an increase in Ca2+-sensitivity and decrease in cooperativity has been observed so far (Pinto et al., 2011).

#### **2.1.3 DCM inducing mutations**

314 Cardiomyopathies – From Basic Research to Clinical Management

Since the cTnT- N-terminus contributes to the inhibition of the actin/myosin interaction (Tobacman, 1988), one might assume that also inhibition is affected by mutations. Indeed inhibition of is reduced due to cTnT-F110I (Knollmann & Potter, 2001; Gomes et al., 2004). A similar decrease in inhibition has been described for I79N (Yanaga et al., 1999), though this amino acid exchange is positioned N-terminally of the main Tm interaction site. Nevertheless I79N and F110I exhibit several similarities. Thus, Midde et al., 2011 showed that rigor crossbridges in I79N or F110I containing filaments were disordered, indicating that disruption in thin filament structure may lead to severe contractile dysfunction. I79N, as most investigated HCM mutants, enhances the Ca2+- sensitivity of force development and actomyosin ATPase activity, which might contribute to enhanced contractility and higher energy consumption (Lin et al., 1996; Sweeney et al., 1998; Chandra et al., 2005). Furthermore, mouse hearts with I79N or R92Q could not increase cardiac performance upon ß-adrenergic stimulation (Knollmann et al., 2001; Javadpour et al., 2003), performance of I79N transgenic even worsened upon isoproterenol treatment (Sirenko et al., 2006). Such an effect as well as Ca2+ –sensitivity increase has not been observed with a R278C, a mutation located in the cTnT-C-terminus. These findings suggest that the region around amino acids 79 and 92 is important for Ca2+ -signal transmission and affects Ca2+-regulation. Furthermore dysfunction is especially prominent under ß-adrenergic stimulation, which might explain the high risc for cardiac sudden death of these mutations. Causality between enhanced Ca2+-sensitivity and the potential for the development of malignant arrhythmias has been shown by Baudenbacher et al., (2008) for I79N. They described altered action

Mutations in TNNT2 gene encoding the C-terminal half of cTnT might affect the interaction with tropomyosin at the second Ca2+ -dependent binding site, as well as binding to cTnI and cTnC. Most mutations in this part lead either to single amino acid exchanges, deletions of single amino acids or to C-terminally truncated cTnT molecules due to splice site mutations. IVS15+1G>A is a splicing donor mutant which might result in two truncated proteins. In one mutant exon 16 is skipped encoding the C-terminal 14 amino acids, in the other mutants seven amino acids replace the C-terminal 28 amino acids encoded by exon 15 and 16 (Thierfelder et al., 1994). Both mutants are able to form a heterotrimeric troponin complex, however their affinity towards cTnI is drastically reduced (Mukherjea et al., 1999). The impaired interaction with cTnI may be the cause for reduced inhibitory capacity of troponin at low Ca2+ concentrations described by Redwood et al., (2000) and accelerated cross bridge kinetics (Stelzer et al., 2004). Furthermore a reduced binding of troponin to the thin filament has been reported dependent on its regulatory states (Knollmann & Potter, 2001; Burhop et al., 2001), indicating that the C-terminal part of cTnT stabilizes binding of cTn to the thin filament and affects Ca2+ -regulation. Increased Ca2+-sensitivity and cooperativity (Nakaura et al., 1999)) and impaired switching off myosin cycling at low Ca2+-concentrations is observed (Burhop et al., 2001). Up to date nearly nothing is known on how cardiomyopathy inducing mutations in TNNT2 influence phosphorylation dependent effects of PKC dependent cTnT or PKA dependent cTnI phosphorylation. According to a study of Nakaura et al., (1999) effects of

truncated cTnT were independent on PKA dependent cTnI phosphorylation.

There are only few RCM mutations detected in TNNT2 (Table 1). Most RCM mutations in genes encoding for cardiac troponin subunits are found in TNNI3 (see below). However,

potential duration due to the mutation.

**2.1.2 RCM inducing mutations**

Patients with DCM mutations in cTNNT2 exhibit a malignant prognosis as do also most of the FHC mutations in cTNNT2. Investigations on molecular level are largely missing to date. All patients showed decreased cardiac function (Kamisago et al., 2000) in contrast to enhanced contractility often observed in patients and transgenic animals carrying HCM mutations. In accordance *in vitro* analysis of DCM-mutations revealed a decreased Ca2+ sensitivity (Mirza et al., 2005). Venkatraman et al., 2005 showed that ΔK210, the most prominent example for DCM causing cTnT-variation, decreases Ca2+-sensitivity of force and actomyosin ATPase activity as well as maximal force and ATPase activity not only with adult, but also with fetal cTnT. The deletion of K210 occurs in the second Ca2+ -sensitive tropomyosin binding region near the PKC phosphorylation site and possibly near the binding site for PKA regulatory subunit II. This implies that Ca2+ -regulation as well as PKC dependent phosphorylation of cTnT and/or PKA dependent phosphorylation of other sarcomeric proteins might be affected. Detailed information is missing.

#### **2.2 Mutations in TNNC1, the gene encoding cTnC**

Mutations in the TNNC1 gene occur seldom. Far less than 1% of the genetic disorders leading to familial cardiomyopathies in patients are due to mutations in TNNC1. Up to date only six HCM and one DCM inducing mutations, all single amino acid exchanges, have been identified (Fig.2; Table 2). A8V, L29Q and C84Y are located in the N-terminal and N122fs, E134D, D145E and G159D in the C-terminal domain of cTnC (Fig. 2). L29Q was the first mutation detected in the cTnC gene (Hoffmann et al., 2001). Since there was only one family showing this mutation, it is not clear if this mutation causes HCM. However, according to Schmidtmann et al., 2005 and Liang et al., 2008 this amino acid replacement has the potential to cause a cardiomyopathy. Replacement of leucine by glutamine destabilizes the interaction of the N-terminal cTnI arm with cTnC (Schmidtmann et al., 2005; Baryshnikova et al., 2008). The release of the N-terminal cTnI arm occurs also upon bisphosphorylation of cTnI after ß-adrenergic stimulation and contributes to a reduction in Ca2+-sensitivity of the actomyosin ATPase activity (see below). Consistent with this effect for L29Q a small reduction of Ca2+- sensitivity has been described (Schmidtmann et al., 2005). However, in the phosphorylated state of cTnI, an enhanced Ca2+-sensitivity of the actomyosin ATPase activity was observed, indicating an altered ß-adrenergic responsiveness. Divergent results might be obtained in systems differing in complexity

Consequences of Mutations in Genes Encoding Cardiac Troponin C, T and I – Molecular Insights 317

The proximity of A8 and E134 to the same cTnI region underlines findings of Smth et al. (1999) that the cTnC-N-terminal helix is spatially near the C-lobe. Latest kinetical investigations by Pinto et al, (2011a) showed that Ca2+ off rates are delayed for A8V and D145E, both stabilizing cross bridges. Thus structural disturbances at the cTnC-N-terminus not only alter structure of the N- but also of the C-terminal lobe and vice versa and may impair cTnC-cTnI interaction. C84Y is located at the end of the EF hand helix flanking regulatory Ca2+- binding loop at the transition to the central linker region. This residue probably is involved in forming the binding platform for the cTnI switch region and thus might destabilize binding of cTnI in Ca2+ -saturated state of cTnC (Fig. 2). D145E is located in Ca2+ -binding loop IV, in +Z position (see above) indicating that divalent cation binding might be affected (Pinto et al., 2009). Another amino acid exchange in the C-terminal lobe, G159D, is linked to DCM. Consistent with all other investigated DCM causing mutations G159D reduces Ca2+-sensitivity of the actin-myosin interaction and decreases contractility (Mirza et al., 2005). The hydrophobic residues at position 156,157 and 160 make contact to cTnI (Gasmi-Seabrook et al., 1999). Thus the G156D exchange may considerably disturb the hydrophobic interaction with cTnI and also might affect interaction with cTnT. In the troponin complex G159D reduced the opening (Ca2+ binding) and closing rates (Ca2+ dissociation) of the N-terminal domain of cTnC. Alteration in opening rate was also observed for L29Q, indicating that both mutants alter structural transition kinetics (Dong et al., 2008). PKA dependent phosphorylation of cTnI also affects kinetics in that it enhances the closing rate. This effect was abolished by L29Q and G159D implying that phosphorylation signal transduction is impaired as was earlier proposed for L29Q by

Most mutations in the gene encoding cTnI, are located in exon 7 and 8, which encode the regulatory C-terminal region of cTnI a few are located in the N-terminal heart specific extension. In patients they mostly induce either HCM or RCM. There are only three

Only one mutation has been identified in exon 3 encoding part of the heart specific Nterminal cTnI arm (Table 3). This mutation results in an R20C (numbering without starter methionine) amino acid exchange, which is located within the consensus sequence for PKA (Fig. 3). The consensus sequence is present in cTnI in a dublicated form (Mittmann et al., 1992) enabling phosphorylation of two adjacent located serine residues (Ser22, 23) by PKA (Fig. 4). The exchange of the middle arginine in a series of three arginine residues (position 20 or 21 dependent on the inclusion of the starter methionine) impairs phosphorylation of the two serine residues and reduces the phosphorylation effect *in vitro* as shown by Gomes et al. (2005). Since both phosphorylation sites are affected, susceptibility towards proteolysis is enhanced. Phosphorylation protects to a certain extent towards proteolysis (Barta et al., 2003). But also the amino acid exchange itself might enhance susceptibility towards protein degradationas proposed by Gomes et al. (2005). Thus probably impairment of PKA dependent phosphorylation as well as enhanced protein digestion might be the main effect of this mutant protein for disease

Schmidtmann et al. (2005).

development.

**2.3 Mutations in TNNI3, the gene encoding cTnI**

**2.3.1 Mutations in TNNI3 linked to HCM**

mutations identified up to date in TNNI3, which are linked to DCM.

Modified from: OMIM database (http://omim.org); the one letter code is given in brackets; fs designates frameshift.

Table 2. Mutations in TNNC1

(Dweck et al., 2008). Thus, in a higher organized system, namely mouse cardiomyocytes, Liang et al. (2008) described an enhanced Ca2+-sensitivity of force generation dependent on sarcomere length. They showed that also Ca2+- binding affinity to site II was higher in cTnC-L29Q than in cTnC wild type, whereas Ca2+-dissociation rate was not affected. This is in agreement with former findings of Gillis et al., 2005, that residues 2, 28-30 affect Ca2+ binding properties of the regulatory Ca2+-binding site. Replacement of these residues might increase cardiomyocyte contractility. Also the other FHC mutation in TNNC1, leading to A8V, C84Y and D145E replacements in cTnC, enhance Ca2+ -sensitivity of force development (Landstrom et al., 2008). For E134D no influence on Ca2+-sensitivity could be observed. It remains unclear how this mutation leads to contractile dysfunction. Since E134 is located near contact sites of cTnI around Ser43/45 effects of PKC dependent phosphorylation might be influenced. Also A8V in the N-terminal helix of cTnC- N-lobe is located near a contact site with cTnI- Ser43 in H1 of cTnI (Li et al., 2004, Takeda et al., 2003). No data on the impact of these mutations on PKC phosphorylation are available up to date. The proximity of A8 and E134 to the same cTnI region underlines findings of Smth et al. (1999) that the cTnC-N-terminal helix is spatially near the C-lobe. Latest kinetical investigations by Pinto et al, (2011a) showed that Ca2+ off rates are delayed for A8V and D145E, both stabilizing cross bridges. Thus structural disturbances at the cTnC-N-terminus not only alter structure of the N- but also of the C-terminal lobe and vice versa and may impair cTnC-cTnI interaction. C84Y is located at the end of the EF hand helix flanking regulatory Ca2+- binding loop at the transition to the central linker region. This residue probably is involved in forming the binding platform for the cTnI switch region and thus might destabilize binding of cTnI in Ca2+ -saturated state of cTnC (Fig. 2). D145E is located in Ca2+ -binding loop IV, in +Z position (see above) indicating that divalent cation binding might be affected (Pinto et al., 2009). Another amino acid exchange in the C-terminal lobe, G159D, is linked to DCM. Consistent with all other investigated DCM causing mutations G159D reduces Ca2+-sensitivity of the actin-myosin interaction and decreases contractility (Mirza et al., 2005). The hydrophobic residues at position 156,157 and 160 make contact to cTnI (Gasmi-Seabrook et al., 1999). Thus the G156D exchange may considerably disturb the hydrophobic interaction with cTnI and also might affect interaction with cTnT. In the troponin complex G159D reduced the opening (Ca2+ binding) and closing rates (Ca2+ dissociation) of the N-terminal domain of cTnC. Alteration in opening rate was also observed for L29Q, indicating that both mutants alter structural transition kinetics (Dong et al., 2008). PKA dependent phosphorylation of cTnI also affects kinetics in that it enhances the closing rate. This effect was abolished by L29Q and G159D implying that phosphorylation signal transduction is impaired as was earlier proposed for L29Q by Schmidtmann et al. (2005).

#### **2.3 Mutations in TNNI3, the gene encoding cTnI**

Most mutations in the gene encoding cTnI, are located in exon 7 and 8, which encode the regulatory C-terminal region of cTnI a few are located in the N-terminal heart specific extension. In patients they mostly induce either HCM or RCM. There are only three mutations identified up to date in TNNI3, which are linked to DCM.

#### **2.3.1 Mutations in TNNI3 linked to HCM**

316 Cardiomyopathies – From Basic Research to Clinical Management