Role of TNNI3K in Cardiovascular Disease and Prospects for Therapy

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Understanding cardiac electrical phenotypes in the genomic era

Milano, A.

Publication date 2015 Document Version Final published version

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Download date:27 Sep 2021 Chapter 4

TNNI3K in cardiovascular disease and prospects for therapy

under revision at the Journal of Molecular and Cellular Cardiology

Annalisa Milano, Elisabeth. M. Lodder, Connie. R. Bezzina

57 Chapter 4

Abstract

Cardiovascular diseases are an important cause of morbidity and mortality worldwide and the global burden of these diseases continues to grow. Therefore new therapies are urgently needed. The role of protein kinases in disease, including cardiac disease, is long recognized, making kinases important therapeutic targets. We here review the knowledge gathered in the last decade about troponin I-interacting kinase (TNNI3K), a kinase with cardiac-restricted expression that has been implicated in various cardiac phenotypes and diseases including heart failure, cardiomyopathy, ischemia/reperfusion injury and conduction of the cardiac electrical impulse.

Key words: Tnni3k, heart failure, ischemia/reperfusion, arrhythmia, kinase

58 Role of TNNI3K in cardiovascular disease and prospects for therapy

Introduction

Cardiovascular diseases are an important cause of morbidity and mortality world-wide and the global burden of these diseases continues to grow1. Among the cardiovascular disorders, heart failure is a common disease affecting over 23 million worldwide. Mortality risk increases steadily after a diagnosis of heart failure and 20-30% of patients die within 1 year after receiving the diagnosis2. Similarly, atrial fibrillation, which is the most commonly observed sustained cardiac arrhythmia and which is associated with heart failure and stroke, is similarly increasing in prevalence with the aging of the population3. Thus, new therapeutic approaches for prevention and treatment of 4 cardiovascular diseases are urgently needed. Protein kinases catalyze the phosphorylation of proteins as a means of signal transduction. Catalysis by protein kinases brings about the transfer of a phosphate group from ATP onto the hydroxyl group of serine or threonine (in the case of serine/threonine protein kinases), or tyrosine residues (in the case of tyrosine kinases) in target proteins to modulate their function. The human kinome is vast and protein kinases constitute approximately 2% of human genes (>500 genes)4; they can be grouped into families based on their molecular target5. The role of protein kinases in disease, including cardiac disease, is long recognized, making kinases important targets for therapy6. The function of protein kinases can be inhibited by numerous strategies including competition for ATP binding, prevention of binding to anchoring proteins, and binding to substrate interaction sites5,6. Although the generation of highly selective kinase inhibitors is challenging (for example because of the similarity in the ATP-binding pocket across different kinases), targeting protein kinases with cardiac-restricted expression is an attractive goal for therapy of cardiac diseases as targeting such kinases limits unwanted off-target effects in other organs. We here review knowledge about troponin I-interacting kinase (TNNI3K), a kinase with cardiac-restricted expression that has in the last ten years been implicated in various cardiac phenotypes. These include progression of cardiomyopathy7, hypertrophy8,9, conduction of the cardiac electrical impulse10 and ischemia/reperfusion injury, making TNNI3K an interesting target for the development of specific antagonists for the treatment of cardiac disease.

TNNI3K: Gene and Transcript

The cDNA for TNNI3K was first isolated from a human adult heart cDNA library11. In humans the TNNI3K gene is located on chromosome 1 (1p31.1) and at the moment of writing eight TNNI3K transcripts were annotated in Ensembl, of which four are protein coding12,13. In humans, the longest TNNI3K transcript encodes a 92kD protein of 835 amino acids. Some studies have indicated the presence of conjoined transcripts in human heart14, resulting from the transcription of TNNI3K and the neighboring gene FPGT; 6

59 Chapter 4 such transcripts (of which 5 are protein coding) are annotated in Ensembl. The longest FPGT-TNNI3K transcript encodes a 105kD protein of 949 amino acids. Experimental evidence of the function of such fusion transcripts is thus far lacking14. No such fusion transcripts have been reported or detected in the murine heart (Ensembl GRCm38 and unpublished data from our group). Analysis of TNNI3K mRNA expression in multiple human tissues revealed that expression is restricted to the heart, being highly expressed in both adult and fetal heart, and expressed in left and right atria and ventricles11. A more recent study reported similar findings except for a slight expression in brain and testis15. The latter study additionally demonstrated that within the heart TNNI3K expression is limited to cardiomyocytes15. The TNNI3K gene is highly conserved and there are only a few differences between humans and other species including mice. The mouse Tnni3k cDNA was cloned and the basal promoter was characterized16. Here a series of reporter studies uncovered Mef2c binding sites as critical cis-acting elements in the regulation of Tnni3k expression. Mef2c is a member of the MADS (MCM1-agamous-deficiens-serum response factor) superfamily of transcription factors17 and is involved amongst others in myogenesis18 and in the development of structures derived from the second heart field19. The role of Mef2c binding in control of Tnni3k expression was confirmed in the same study through experiments in primary rat cardiomyocytes. In these cells transfection with Mef2c antisense morpholinos resulted in reduced Tnni3k expression16. Tnni3k may be a direct target of Mef2c transcriptional regulation, as shown by electrophoretic mobility shift assay (EMSA) in primary rat neonatal cardiomyocytes nuclear extracts16.

TNNI3K Protein: Domain structure, kinase activity, binding partners and localization

A homology search and phylogenetic analysis showed that the overall protein domain structure of TNNI3K is similar to that of integrin-linked kinase (ILK) that regulates signaling pathways controlling cardiac growth, contractility and repair11. TNNI3K contains three main recognizable domains (Figure 1): a domain containing ankyrin (ANK) repeats at the N-terminal part of the protein, an active protein kinase domain and a C-terminal serine-rich domain11. Most likely TNNI3K interacts with its partners via the ANK domain as this domain is an established protein-binding target of proteins in other pathways20 TNNI3K has also been shown to be able to form homo-dimers or oligomers21 and an in silico analysis has uncovered putative interaction interfaces at which TNNI3K monomers interact during dimerization or oligomerization22. In spite of intense research on TNNI3K, the identification of downstream phosphorylation targets of TNNI3K remains scarce. Similarly knowledge on interacting partners that may be involved in TNNI3K-mediated signaling as well as data on the subcellular localization of TNNI3K which might provide hints concerning its function

60 Role of TNNI3K in cardiovascular disease and prospects for therapy FIGURE 1A FIGURE 1A

Anyrin repeat domain Protein kinase domain Serine-rich domain Anyrin repeat domain Protein kinase domain Serine-rich domain

FIGURE 1B Dilata+on and heart failure FIGURE 1B Dilata+on and heart failure

Hypertrophy Cardiac conduc+on Hypertrophy TNNI3K Cardiac conduc+on TNNI3K

? ? Ischemia/reperfusion injury Viral myocardi+s Ischemia/reperfusion injury Viral myocardi+s Figure 1. Tnni3k and cardiac disease: (A) Schematic representation of the domain structure of TNNI3k. (B) The cardiac phenotypes in which TNNI3K has been implicated. While genetic studies in mice have identified the chromosomal locus harbouring Tnni3k as a susceptibility locus for viral myocarditis, whether this effect is mediated by Tnni3k is yet unknown.

is largely lacking, limiting insight into the exact molecular function of this protein in myocardial biology. The fact that TNNI3K is a functional kinase has been principally established through studies investigating the auto-phosphorylation of the protein9,21. The domain structure of TNNI3K suggested that it is a dual-function kinase with both tyrosine and serine/threonine kinase activity. This was confirmed inin vitro studies that identified multiple auto-phosphorylation sites involving tyrosine as well as serine and threonine residues9,21. These sites are mainly located in the ANK-repeat and serine-rich domains of the protein. Studies conducted in mice over-expressing human TNNI3K (DBA/2J-hTNNI3K mice) and mice over-expressing a kinase-dead version of human TNNI3K (DBA/2J-TNKD mice; due to residue 490 Lys>Arg substitution at the canonical ATP binding site of the kinase domain) demonstrated that auto- phosphorylation of TNNI3K also occurs in vivo9. In an early study, candidate proteins that interact with TNNI3K have been identified by means of a yeast two-hybrid screen using the C-terminal serine-rich domain of the protein as bait11. Among the candidate proteins identified in this screen

61 Chapter 4 the most frequently observed was cardiac troponin I (cTnI), hence the name troponin-I- interacting kinase. The interaction with cTnI was confirmed by co-immunoprecipitation of over-expressed cTnI and TNNI3K in a heterologous expression system11. In a series of experiments conducted in this system as well as in rat cardiomyocytes, the same research group later demonstrated that TNNI3K can phosphorylate cTnI at residues Ser43 and Thr143, sites also involved in protein kinase C-mediated phosphorylation of cTnI23. In the same study the authors additionally implicated this phosphorylation in the regulation of cardiomyocyte contractility, where phosphorylation promotes contractility23. Another study implicated TNNI3K in the phosphorylation of cTnI residues Ser22/Ser238. The latter study also demonstrated the necessity of the C-terminal part of TNNI3K in mediating binding to cTnI8. Taken together this data suggests a direct role of TNNI3K in regulation of cardiac contractility. Another yeast two-hybrid screen, this time using the N-terminal ankyrin- containing domain of TNNI3K as bait, followed by confirmatory immunoprecipitation in an over-expression cellular system, led to the identification of the mitochondrial protein anti-oxidant protein 1 (AOP-1, also called peroxiredoxin 3) as another possible interaction partner of TNNI3K24. AOP-1 functions as a thioredoxin-dependent 25 peroxidase that scavenges ROS, such as H2O2 . This study also suggested that AOP- 1 may down-regulate the auto-phosphorylation of TNNI3K24. Based on the diverse functions of TNNI3K, discussed below, it is most likely that many targets and interaction partners remain to be discovered. Studies that have investigated the subcellular localization of TNNI3K have provided conflicting findings. Immunohistochemical studies in mice over-expressing hTNNI3K (DBA/2J-hTNNI3K mice) and C57BL/6J mice using an antibody raised against the C-terminus of the protein demonstrated localization of TNNI3K to the sarcomeric Z-disk9. Besides its structural role in the sarcomere, the Z-disc is recognized to serve as a nodal point for signaling in general and mechanosensation and mechanotransduction in particular26, and also links to the t-tubular system and the sarcoplasmic reticulum27. This localization was also observed in DBA/2J-TNKD mice, suggesting that the kinase activity of TNNI3K is not required for its Z-disk localization9. Vagnozzi et al., reported localization at the nucleus in neonatal rat ventricular myocytes. Studies conducted in human heart have shown both nuclear and cytoplasmic localization, although the relative intensity of cytoplasmic staining differed between the two studies that demonstrated this11,22.

TNNI3K in cardiac function and disease

Quantitative Trait Locus mapping studies in mice Robust relations between Tnni3k and distinct cardiac phenotypes have been found through quantitative trait locus (QTL) genetic mapping studies conducted in

62 Role of TNNI3K in cardiovascular disease and prospects for therapy different inbred strains of mice. These studies have linked the Tnni3K locus on mouse chromosome 3 to cardiac function and survival28,29 and the electrocardiographic PR- interval28 (Figure 1B). QTL mapping studies in mice have also identified theTnni3k locus as a susceptibility locus for viral myocarditis caused by Coxsackievirus B3 infection; however while Tnni3k is considered a candidate for this effect, evidence in support of its role as the causal gene within the locus is as yet lacking29,30. The principle of the QTL mapping approaches used in these studies is depicted in Figure 2. QTL mapping is based on the idea that one can identify a genetic locus that controls a phenotype of interest through the identification of genetic markers linked to the locus in a segregating population with known pedigree31 . Although not known at the 4 outset, the studies identifying the Tnni3k locus as a modulator of cardiac function32,33 and PR-interval28 all exploited the fact that the endogenous levels of Tnni3k mRNA and protein vary widely among different strains of inbred mice, ranging from robust levels in some strains (e.g. the C57BL/6J, WSB/Eij, AKR/J and 129P2 strains) to a total lack/undetectable expression in others (e.g. the FVB/N and DBA/2J strains)7,10,34. Although the latter mice effectively represent Tnni3k-null mice, of note, wild-type mice that naturally lack or that express Tnni3k do not show baseline cardiac abnormalities as determined by echocardiographic and electrocardiographic (ECG) measurements7,10.

TNNI3K in hypertrophy and heart failure While initial in vitro work suggested a cardioprotective role for increased levels of TNNi3K35 more recent work has provided strong evidence that increased levels of TNNI3K have detrimental effects on cardiac function. The Tnni3k locus was first linked to cardiac function (as determined by echocardiographic parameters) and survival by Marchuk, Rockman and co-workers in two independent genetic mapping studies, 32,33 each utilizing a different mouse N2 cross . Both of these studies were conducted in cardiomyopathy-sensitized mice due to the cardiac-specific overexpression of the Ca2+ binding protein calsequestrin (Csq, Csqtg mice)36. Additionally for genetic mapping, they both used inbred mouse lines that displayed marked differences in cardiac function and survival as a consequence of Csq over-expression, namely DBA/2J, with a less severe phenotype, and C57Bl632 or AKR/J33, with a more severe phenotype. This variability in disease severity in the setting of disease-sensitization mediated by Csq overexpression, alongside the genetic differences between the mouse strains used, enhanced the possibility of genetic locus discovery. Following the initial reports that identified the chromosomal interval harboring Tnni3k as a modulator of cardiac function in the setting of Csq overexpression, the same investigators conducted a series of elegant studies that led to the unequivocal identification of Tnni3k as the causal gene within the interval7. One crucial observation they made was the large variability in Tnni3k mRNA and protein expression in cardiac tissue from the inbred mice used in the genetic mapping studies, with high Tnni3k levels being observed

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FIGURE 2A Parental X strains Strain 1 Strain 2

F1 Strain 2

N2 Strain 2 repeated backcross (with Strain 2 [recipient strain]

N2 mice segrega+ng trait-‐modifying alleles used in QTL mapping for ≥10 genera+ons)

congenic

N10+ FIGURE 2B Parental X strains Strain 1 Strain 2

X F1 Intercross F1 F1

F2 mice segrega+ng trait-‐modifying alleles used in QTL mapping

Figure 2. Mouse breeding strategies: (A) Production of second filial (F2) generation mice for quantitative trait locus (QTL) mapping as used by Scicluna et al28 and Aly et al.29. (B) Backcross strategy for generation of N2 mice for QTL mapping (as used by Suzuki etal.32, Wheeler et al. 200533 and Wiltshire et al.30) and for generation of a congenic mouse strain such as the DBA/2J.AKR/J congenic strain produced by Wheeler et al 20097.

64 Role of TNNI3K in cardiovascular disease and prospects for therapy in the susceptible strains (AKR/J, C57Bl6) as opposed to low/undetectable levels in the protected strain (DBA/2J). This they attributed to defective splicing of Tnni3k leading to a frameshift that was associated with decreased Tnni3k mRNA by a mechanism of nonsense-mediated decay in mice with undetectable Tnni3k expression. The in vivo role of TNNI3K was subsequently investigated by cardiac-specific over-expression of human TNNI3K in DBA/2J mice (DBA/2J-hTNNI3K, which show no detectable endogeneous mouse Tnni3k) (Table 1). Crossing these DBA/2J-hTNNI3K mice with DBA/2J-Csqtg ‘sensitizer mice’ led to a marked acceleration in the development of heart failure, with severe systolic dysfunction, chamber dilatation and a profound reduction in survival in double transgenic mice (overexpressing hTNNI3K and Csq) compared to 4 single transgenic DBA/2J-Csqtg mice7. This provided evidence that differences in Tnni3k expression underlie the heart failure modifier locus7. These investigators also generated a DBA/2J.AKR/J congenic mouse strain by repeated backcrossing into DBA/2J (Figure 2A). This strain essentially harbored a 20Mb chromosomal interval containing Tnni3k of the AKR/J strain in the setting of the DBA/2J genome. By crossing this DBA/2J. AKR/J congenic line with DBA/2J-Csqtg mice, these investigators could demonstrate that even natural levels of Tnni3k (as offered by the AKR/J-derived Tnni3k alleles), are associated with decreased survival and acceleration of cardiomyopathy in the setting of Csq-overexpression7. Transverse aortic constriction (TAC, which induces left ventricular hypertrophy in response to pressure overload) in the DBA/2J-hTNNI3K transgenic mice confirmed the detrimental effect of TNNI3Kh expression outside the context of Csq-mediated sensitization7. Post-TAC DBA/2J-hTNNI3K mice showed greater diastolic and systolic dysfunction and reduced fractional shortening compared to wild- type DBA/2J littermates.

Table 1: Glossary of mice generated and investigated in the various studies

Mouse Details Reference DBA/2J-hTNNI3K Overexpression of hTNNI3K in DBA/2J mice; wt DBA/2J 33 mice do not have detectable murine Tnni3k C57BL/6J-hTNNI3K Overexpression of hTNNI3K in C57Bl6 mice; wt C57Bl6 8,15 mice express robust levels of murine Tnni3k FVB/N-hTNNI3K Overexpression of hTNNI3K in FVB/N mice; wt FVB/N 15 mouse strain mice do not have detectable murine Tnni3k DBA/2J-TKND Overexpression of kinase-dead hTNNI3K in DBA/2J mice; 9 wt DBA/2J mice do not have detectable murine Tnni3k DBA/2J-Csqtg Overexpression of murine calsequestrin in DBA/2J mice 33 DBA/2J-hTNNI3K-Csq Overexpression of murine calsequestrin and hTNNI3K in 33 (double transgenic) DBA/2J mice DBA/2J.AKR/J Congenic mice generated by back-crossing DBA/2J mice 33 congenic mouse (recipient strain) with AKR/J mice (donor strain) for >10 generations; resultant congenic mice contain a 20Mb region from AKR/J chromosome 3 in the setting of a DBA/2J genetic background

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A subsequent in-depth characterization of DBA/2J-hTNNI3K mice by the same investigators and comparison of these mice with another line that they generated overexpressing the kinase-dead version of TNNI3K (TNKD, with kinase activity ablated by the 490Lys->Arg substitution; here referred to as DBA/2J-TKND mice) provided additional important insight9. Heart weight to body weight ratio was increased and cardiomyocytes were enlarged in DBA/2J-hTNNI3K mice compared to DBA/2J mice. Furthermore hearts from DBA/2J-hTNNI3K mice were slightly dilated and showed increased deposition of fibrosis. Consistent with the hypertrophic phenotype, mRNA levels of Nppa (which encodes the atrial natriuretic peptide, ANF) and Nppb (which encodes the brain natriuretic peptide, BNP) were elevated in DBA/2J-hTNNI3K mice. However, the phosphorylation status of p38, ERK1/2 and AKT, established effectors transducing cardiac remodeling signals, was unchanged, suggesting that TNNI3K may not regulate directly these effectors. Examination of the ultrastructure of cardiomyocytes uncovered a decreased resting sarcomere length in DBA/2J-hTNNI3K mice. Furthermore, DBA/2J-hTNNI3K mice showed a trend towards an increase of the N2BA to N2B titin isoform ratio9 similar to that observed in human DCM37. Interestingly, although not as severe as in DBA/2J-hTNNI3K mice, cardiac mass and cardiomyocyte size was also increased in DBA/2J-TKND mice compared to wild- type DBA/2J littermates. This implies that while the kinase activity of TNNI3K is involved in mediating cardiac remodeling, effects of TNNI3K on cardiac remodeling that are independent of its kinase activity may also exist. Post-TAC, progression of left ventricular dysfunction was similar in DBA/2J-TNKD and the wild-type DBA/2J mice (the latter are protected by virtue of absent Tnni3k expression)9. While this implies that kinase activity is essential to the disease accelerating properties of TNNI3K in pressure overload-induced cardiomyopathy7,9, histological investigation of the heart was not conducted and thus a greater deterioration of tissue structure in DBA/2J-TNKD mice as compared to wild-type DBA/2J mice cannot be ruled out. Of note, levels of phosphorylated TNNI3K were increased post-TAC in DBA/2J-hTNNI3K mice compared to non-TAC DBA/2J-hTNNI3K controls and DBA/2J-TNKD mice post- TAC, indicating that the kinase activity of TNNI3K is elevated by signals induced by mechanical stress, and that TNNI3K further accelerates the cardiac dysfunction during pressure overload9. A mouse model with cardiac over-expression of hTNNI3K was also established by Wang and co-workers, in this case in a C57BL/6J mouse genetic background8. Interestingly these investigators uncovered signs of hypertrophy in mice with a high level of over-expression of the transgene but not in mice with intermediate and low expression. This comparison was however only restricted to a gross analysis involving estimation of heart weight and left ventricular weight to body weight ratio and subsequent in-depth analysis was only conducted in high-expression mice. Mice expressing high levels of TNNI3K displayed concentric hypertrophy and while cardiomyocytes where enlarged,

66 Role of TNNI3K in cardiovascular disease and prospects for therapy no necrosis, cardiomyocyte disarray or interstitial fibrosis was seen. Importantly, enhanced contractility and diastolic function was observed. Despite the fact that both achieved over-expression of hTNNI3K in heart by means of the α-MHC promoter in the transgene construct, the compensated hypertrophy phenotype in this model is at variance with that reported by Tang and co-workers9 in DBA/2J-hTNNI3K mice. These differences may be due to a number of factors among which are the absolute level of expression of the transgene achieved in the different models and aspects of mouse genetic background. With respect to the latter, the C57BL6/6J strain used by Wang and co-workers8 has murine Tnni3k expression which lacks in the DBA/2J strain used by Tang et al.9. 4 A study conducted in rats showed differences in the expression of Tnni3k in myocardium post-TAC, where Tnni3k mRNA expression first decreased at the first day post-TAC and then increased steadily up to the 15th day of measurement post-TAC8. Tnni3k also showed a dynamic patter of expression in a rat cardiomyocyte model with endothelin-1 induced hypertrophy38. TNNI3K was reported to induce hypertrophy in neonatal rat ventricular cardiomyocytes in the latter study38.

TNNI3K in ischemia/reperfusion injury A study by Vagnozzi et al.15 provided evidence for a role of Tnni3k in mediation of ischemic-reperfusion injury (I/R), a pathology that is associated with increased oxidative stress, mitochondrial dysfunction, and cardiomyocyte death39. In this setting p38 mitogen activated protein kinase (MAPK) was identified as a key downstream effector of Tnni3k. In a series of ischemia/reperfusion studies conducted in mice over-expressing hTNNI3K (FVB/N strain, which do not naturally express murine Tnni3k), in mice with inducible cardiomyocyte-specific knock-out ofTnni3k (C57BL6 strain, which naturally express Tnni3k), and in a neonatal rat ventricular cardiomyocyte model of ischemic injury, these investigators provided convincing evidence that TNNI3K worsens I/R injury. They demonstrated that this effect is mediated, at least in part, through the overproduction of mitochondrial reactive oxygen species (mROS) with ensuing mitochondrial and bioenergetic impairment, and is dependent on activation of the mitogen-activated protein kinase p38. By administering active-site binding inhibitors of TNNI3K that they developed (GSK854 and GSK329) to wild-type C57BL6 mice at reperfusion following ischemia (mimicking clinical intervention in patients), they went on to show reduction of infarct size following I/R. They also demonstrated long-term beneficial effects of Tnni3k inhibition during reperfusion coupled to sustained inhibition thereafter, in C57Bl6 mice after a severe ischemic insult. In this model of human acute coronary syndrome, GSK854 inhibition of Tnni3k reduced left ventricular dysfunction and limited progressive remodeling, with improvement in cardiac function, dimensions and a reduction in cardiac fibrosis and cardiomyocyte hypertrophy. Although the mechanism by which TNNI3K activates p38 MAPK remains unclear, taken together

67 Chapter 4 these data provide an approach for treating ischemia–perfusion injury in a clinical setting40. In this respect, the exclusive localization of TNNI3K in the heart makes it a particularly interesting target as opposed to targeting other effectors, such as p38, which are more broadly expressed and which regulate numerous biological processes.

TNNI3K in cardiac conduction In 2012 our group linked Tnni3k to conduction of the cardiac electrical impulse, particularly in control of the electrocardiographic PR-interval which represents the time between the beginning of electrical activation of the atria and the beginning of activation of the ventricles10. Our identification ofTnni3k as a gene influencing cardiac conduction started with a QTL mapping approach28 similar to that used by Marchuk, Rockman and 32,33 co-workers , but in our case conducted in an F2 mouse population (Figure 2B). Our study employed conduction disease-sensitized F2 mice; this sensitization was achieved by virtue of the Scn5a-1798insD (insertion of an aspartic acid (D) at codon 1798 within the murine Scn5a gene)41. This mutation is homologous to the SCN5A-1795insD mutation we had identified in a large Dutch family presenting with conduction disease and Long QT Syndrome42. SCN5A encodes the major sodium channel isoform in heart. It mediates the depolarization phase of the cardiac action potential and thereby plays a major role in cardiac conduction.

F2 mice were generated by crossing Scn5a-1798insD mutant mice of the 129P2 strain with Scn5a-1798insD mutant mice of the FVB/NJ strain, where these two inbred mouse strains vary in their susceptibility to cardiac conduction disease43. By QTL mapping we mapped a locus controlling the PR-interval on mouse chromosome 3, overlapping with the locus previously linked to cardiac function and survival by Marchuk, Rockman and co-workers32,33. As genetic variation within a locus may affect a given phenotype through effects on expression levels of neighbouring genes, we next used cardiac tissue from the same F2 mice to map genetic loci controlling cardiac gene expression (commonly called expression QTLs, eQTLs), including Tnni3k. This uncovered an eQTL controlling Tnni3k transcript levels overlapping the PR-interval QTL on chromosome 3. Furthermore, Tnni3k transcript abundance showed a strong positive correlation with the PR-interval in the F2 mice. Taken together this made Tnni3k a prime candidate for the effect observed on the PR-interval at the locus10. To confirm the hypothesis that the PR interval is modulated by TNNI3K we compared the PR-interval between DBA/2J-hTNNI3K transgenic mice and wild-type DBA/2J mice (naturally lacking Tnni3k expression), showing that hTNNI3K over-expression resulted in a marked increase in the PR interval. A comparison of the PR-interval in DBA/2J.AKR/J congenic mice (with natural levels of Tnni3k as offered by the AKR/J- derived Tnni3k allele) with wild-type DBA/2J mice, showed PR-intervals comparable to those of AKR/J mice in DBA/2J.AKR/J congenic mice, demonstrating that natural levels of Tnni3k are associated with a longer PR-interval in comparison with the absence

68 Role of TNNI3K in cardiovascular disease and prospects for therapy of Tnni3k10. While altogether this data provided a robust link between Tnni3k and cardiac conduction, as for Tnni3k-mediated mechanisms of cardiac dysfunction and hypertrophy, the molecular mechanism whereby increased levels of Tnni3k delay cardiac conduction await elucidation.

TNNI3K in human cardiac disease Recently, using an approach combining linkage analysis and exome sequencing, Theis and co-workers22 reported on a TNNI3K mutation (TNNI3K-G526D) co-segregating with a cardiac phenotype in a family. This family presented with a phenotype consisting 4 of variably expressed atrial tachyarrhythmia, conduction system disease and dilated cardiomyopathy. The G526D mutation identified in this family concerns a conserved residue in TNNI3K, which although located in the kinase domain of the protein is not predicted to directly impact functional sites within the ATP-binding catalytic domain. However a peptide consisting of the mutated kinase domain (with 526D) aggregated in vitro, as opposed to a peptide consisting of the wild-type kinase domain (with 526G) that was soluble. In silico protein modeling showed that while the G>D substitution is expected to have a minimal effect on the monomeric protein structure, it alters the relative affinity of docking configurations during oligomerization. The energetically favorable interactions reported for the mutant protein, also with wild-type TNNI3K, would allow for a dominant-negative effect of the mutant, which would be in line with the autosomal dominant inheritance in the family. Immunohistochemical analysis on a right ventricular endomyocardial biopsy from the proband using two separate antibodies targeting respectively the C-terminal and N-terminal domains of the protein showed reduced TNNI3K staining in the cytoplasm. Ultrastructural analysis of this biopsy by electron microscopy uncovered features of degenerative myopathy with abnormal mitochondria, amorphous inclusions in the cytoplasm and nuclei, and myofilament loss. The aggregation observed in vitro for the mutant and the reduced staining for TNNI3K in myocardium from the proband could point to decreased bioavailability or a loss-of-function defect for the TNNI3K-G526D mutation. Such a mechanism is however difficult to reconcile with findings from rodent studies that have largely identified protective effects for loss of TNNI3K function in heart, including faster cardiac conduction and less severe cardiomyopathy. Clearly, more research on this mutation is warranted to understand the mechanism whereby it leads to the phenotype observed in patients. Furthermore, analysis of TNNI3K in additional patient sets may uncover more mutations that could increase our insight into the role of this gene in human cardiac disease. TNNI3K mRNA expression was found to be higher in cardiac tissue samples from patients with end-stage idiopathic dilated cardiomyopathy compared to healthy controls44. In another study, TNNI3K protein levels were shown to be increased in a set of left ventricular samples from failing hearts of patients with ischemic cardiomyopathy

69 Chapter 4 undergoing transplantation compared to non-failing control hearts15. Although both studies were conducted in a very small sample set, they suggest that elevated TNNI3K levels may play a role in human cardiac disease. Yet whether these are causal or adaptive changes remains unknown. Recent human genetic association studies, including genome-wide association studies, have identified robust association between genetic variation at the chromosomal locus harboring TNNI3K with variation in body mass index45,46. Considering the cardiac-specific expression ofTNNI3K and the fact that another gene exists at this locus which appears to be a more likely candidate, namely FPGT (encoding fucose- 1-phosphate guanylyltransferase, involved in the turnover of L-fucose in the liver)47, TNNI3K may not be a likely causal gene mediating this effect. On the other hand, the role of the TNNI3K-FPGT fusion transcript in relation to phenotypes mapping to this locus may need to be taken into account. Clearly more studies are needed to understand the genetic mechanism at this locus and any possible involvement of TNNI3K.

Concluding remarks

In the last decade, studies largely conducted in mice have uncovered TNNI3K as a cardiac- specific kinase that plays an important role in a broad spectrum of cardiac pathologies. However, in spite of the robust links that have been laid between TNNI3K and distinct cardiac phenotypes, insight into the molecular mechanisms whereby TNNI3K mediates its effects is very scarce. In particular information concerning upstream regulators and downstream effectors of TNNI3K is lacking and their identification is likely to be an important goal of future research on TNNI3K. Such in-depth knowledge on TNNI3K is necessary as its cardiac-specific expression makes it an attractive target for therapy in human cardiac disease. Furthermore, future research in humans, including genetic studies, may provide an increased insight into the role of TNNI3K in human cardiac disease, which has only just started to be uncovered and awaits confirmatory studies.

Acknowledgements Work by the authors is funded by the Netherlands CardioVascular Research Initiative (Dutch Heart Foundation, Dutch Federation of University Medical Centres, the Netherlands Organisation for Health Research and Development and the Royal Netherlands Academy of Sciences) PREDICT project. Disclosure None

70 Role of TNNI3K in cardiovascular disease and prospects for therapy

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