ACKNOWLEDGEMENTS
Many people have contributed in various ways to this master thesis and the results within it. I would like to thank all the members of our ion channel group and Professor Bentzen’s lab, particularly Professor Bo Hjorth Bentzen and Dr. Pia Rengtved Lundegaard, Dr. Mark, xx and xx, who helped me so much along my thesis. I am also grateful to all my colleagues in clinical cardiac research at the National Hospital of the University of Copenhagen. My work in this dissertation would not be possible without their help and assistance and constant collaboration and collegiality across the University of Copenhagen and National hospital.

First and foremost, I would like to express my supervisors, Professor Bo Hjorth Bentzen and Dr. Pia Rengtved Lundegaard, who have supported and guide me throughout my Master thesis project with their encouragement, patience and insightful discussions.
I am deeply grateful to Professor Bo Hjorth Bentzen, my esteemed teacher and leader, who got me involved in this project and made this work real, for his patient and invaluable assistance and advice during the ECG studies, and for his kind input. I would like to thank Dr. Pia Rengtved Lundegaard for the opening of the world of molecular biology and work with zebrafish; willingness to share her experience and expertise with patience and support and always being positive; and for her great friendship.

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I would also like to thank the Professor xx and his lab for their GWAS studies. Without their research, this project will not exist. The relationship between titin and atrial fibrillation will not be revealed.

I thank Mark for his valuable advice and helped so much on ECG studies with his moral support.
I thank xx for technical assistance during all my progra
ABSTRACT
Atrial fibrillation (AF) is the most common form (one in four) of cardiac arrhythmia and cause serious complications such as stroke, heart failure and death. AF is mainly initiated and sustained by ectopic foci and re-entry circuits with associated substrates, respectively. These arrhythmogenic substrates are produced from the combinational effects of risk factors (i.e. age, myocardial infarction), and co-morbidities (i.e. heart failure) with structural and electrical remodelling. The genetic component of AF has been elucidated in recent years through next-generation exome sequencing and genome-wide association studies (GWAS) which related a number of genetic variants and common single-nucleotide polymorphisms (SNPs) to AF. A nationwide study has found 24 families where at least three family members have lone atrial fibrillation. Exome sequencing of these families revealed that Titin-truncating variant (TTNtv) is highly associated with the development of AF. Titin protein has been termed the third myofilament of cardiac muscle, spans half of the length at sarcomere from the Z-disc to M-line, together with actin-based thin filaments (I-band) and myosin-based thick filaments (A-band).

This project used zebrafish to investigate consequence of loss-function mutation on ttn.2 isoform in zebrafish, using the whole mount in situ hybridisation and electrocardiogram (ECG). In situ hybridization results indicated that only ttn.2 are expressed in cardiac myocyte, while ttn.1 are mainly located at the tail of zebrafish. ECG experiment data suggest that truncating mutations in ttn.2 cause an electrical defect (prolonged PR interval and QRS complex) in zebrafish larvae. The electron microscopy of zebrafish larvae found an abnormal sarcomere structure with missing M-line and blurred Z-disc. Combining those results and GWAS studies, TTNtv during the development of zebrafish heart causes disrupted zebrafish sarcomere structure and results in electrical changes relevant for AF. Accordingly, the loss of proper titin function lead to abnormal sarcomere assembly and result in irregular electrical properties of the heart that may play a crucial role in the development of AF.
CHAPTER 1?INTRODUCTION
A normally functioning heart is essential for survival. Electrical disturbance of the heart causes cardiac arrhythmia. The most common arrhythmia is atrial fibrillation, but the molecular mechanisms underlying the disorder are still not fully understood. Recent research highlights the importance of genetics in the pathogenesis of the disease. In a nationwide study, we have found 24 families where at least three family members have lone atrial fibrillation. Exome sequencing of these families revealed that Titin-truncating variant is highly associated with the development of AF. To study the effects of titin mutations on heart function, I have used zebrafish as a model organism. The following section will introduce basic cardiac physiology, the mechanism of AF, titin and zebrafish as a model organism

1.1?Human Heart Action Potential
The human heart is a mechanical pump with the function of assuring pulmonary and systemic blood circulation. The contraction of the right atrium and ventricle pumps deoxygenated blood into the pulmonary artery sending it on through the blood vessels of the lungs. After passing through the circulation of the lungs, the blood having been recharged with oxygen while removing carbon dioxide and returned to the left atrium through the pulmonary veins. The left atrium and ventricle then pump the blood throughout the body, providing the tissues with oxygen, nutrients and chemical mediators. Those regular contractions of the heart are mediated by a specialised muscle cell group called cardiomyocyte which is controlled by the electrical impulses, known as action potential (AP), and propagate though excitation-contraction coupling (EC coupling). Excitation-contraction coupling is the process of contraction of myofibrils by muscle action potential in the muscle fibre.

At the molecular level, the action potential is mediated by the opening and closing of ion channels in the cardiac cell membrane. Generally, the action potential can be divided into five phases (Figure 1.1). In phase zero, opening of fast sodium channels allows a massive influx of sodium ions into the cytoplasm, which depolarizes the membrane. In phase one, there is a rapid repolarization because of the opening of potassium channels which allows the efflux of positively charged potassium ions outside the cells. At the same time, fast sodium channels are suddenly closed (inactivated). In phase two, a more or fewer plateaus phase is produced. The voltage-sensitive calcium channels are opened to facilitate the influx of positively charged calcium ions to balance the repolarizing effects of potassium efflux. In phase three, there is a rapid repolarization caused by suddenly closing of calcium channel which leaves the potassium efflux current unopposed towards the membrane voltage to resting membrane potential. Finally, in phase four, the resting membrane potential is set primarily by the inward rectifying potassium channels (1).

Figure 1.1 Action potential of a ventricular cardiomyocyte. Numbers indicate different phases of the action potential (2).

1.2?Electrocardiogram
The heart generates an electrical stimulus that propagates through the heart’s conduction system. The conduction system of the heart governs the regular pumping activity through several critical parts — i.e. the sinoatrial node (SA) pacemaker cells which are located in the right atrium, near the entrance of superior vena cava, functions to initiate the heartbeat and provide an electric impulse, that spreads across the atria causing depolarization of the muscle cells and stimulate them to contract (i.e. atrial systole); the atrioventricular (AV) node which pass the impulse from the atrium onto atrioventricular bundle; i.e. the AV bundle or bundle of His. the bundle branches and Purkinje fibres which provides fast electrical activation of the ventricles. It regularly beats about 100,000 times/day.

This electrical activity of the heart can be recorded known as an electrocardiogram or ECG (EKG) using skin electrodes from which a composite recording is produced from all the action potential through nodes and cells of the myocardium that constitute the electrical activity of the heart. Thus, each event of the cardiac electrical cycle corresponds segments of the ECG. Also, the cycle of ECG (Figure 1.2) trace repeats itself with every heartbeat.

In a healthy heart, ECG starts from the P wave which reflects the production of action potential signal from SA node, spread throughout the atria and cause depolarization. A flat line following P wave as PR segment represents the time of signals travels from the SA node to the AV node. Then the signal pass the AV node, enters the bundle of His and spreads through bundle branches which conduct the impulses toward the apex of the heart and passed onto Purkinje fibers along the ventricle walls. These cause the depolarization of ventricular contractile fibers and ventricular systole which representing as QRS complex on ECG. Q wave represents as the depolarization of the interventricular septum. R wave corresponds to the depolarization of the main mass of the ventricles. S wave is produced by the last phase of ventricular depolarization. Finally, a state described as ventricular diastole happen as the signal passes out of the ventricles, relaxation and recovering starts on the ventricular walls. The dome-shaped T wave on the ECG represents this ventricular repolarization. On the ECG, the QT interval depicts the time it takes for both depolarization and repolarization of the ventricles to occur.

Figure 1.2 ECG waves and intervals (2).

1.3?Atrial Fibrillation
Disturbances of the normal cardiac rhythm are categorized as cardiac arrhythmias. These encompass a variety of disorders. Arrhythmias could present anywhere in this system and can manifest as tachycardia (fast heart rate) and bradycardia (slow heart rate). Arrhythmias affect both the ventricle (e.g. ventricular fibrillation) and the atrial (e.g. atrial fibrillation) to cause sudden cardiac death and stroke respectively. This project is focused on the most common type of arrhythmia, i.e. atrial fibrillation.

Atrial fibrillation is the common form (one in four) of cardiac arrhythmia characterized by rapid and uncoordinated electrical activation of the atria. These abnormal electrical activities could be identified through ECG, i.e. waves appear more chaotic and random including irregular R-R interval and abnormal P waves (Figure 1.3) (3). Furthermore, AF could lead to ineffective atrial contraction and decreased cardiac output (3) (Figure1.1). As a result, the ventricular filling is reduced, and blood stasis occurs in the atria and causes a clot to form, which predispose to heart failure and thromboembolic stroke, respectively. AF increases the risk of stroke by 5-fold, and it is estimated that 15% of all strokes are attributable to atrial fibrillation. The lifetime risks for development of AF are 1 in 4 for men and women 40 years of age and older. 33 million people worldwide were affected by atrial fibrillation (3, 4, 5, 6, 7). The data from 1990 and 2010 shows that the prevalence of AF is doubled and estimated to at least double in the next 50 years (8).

Figure 1.3 irregular heart beat in AF (142)

While the causes of AF are often unknown, there are numbers of things that increase the risk of getting AF. Some of these include age, family history, smoking, hypertension, obesity, diabetes, mitral stenosis, restrictive cardiomyopathy and hypertrophic cardiomyopathy. The more of these risk factors the patient have, the more likely the patient might experience an AF. These diseases could reduce the diastolic ventricular filling and lead to the further decrease in cardiac output and exacerbation of AF (3). Therefore, it is essential to have it diagnosed early and cared for appropriately as AF can get worse overtime and harder to return to normal. However, the etiology of AF remains poorly understood, which contribute to the current lack of highly effective therapies (8, 9).

1.3.1?Classification of AF
Regardless of the underlying mechanism, paroxysmal, persistent and permanent AF are three type of AF clinically classified by the different duration of episodes. AF terminates spontaneously within seven days of onset should be considered as paroxysmal AF. While persistent AF could be stained more than seven days and need the help of cardioversion (either with drug or direct current cardioversion) to restore to normal heart rhythm. The term permanent AF is used to describe the patient who has continuous AF lasting for last one year. Moreover, the term “lone or idiopathic AF” normally could be applied to younger patients in whom “subsequent investigation (clinical and echocardiographic evidence) shows that heart disease is absent (such as hypertension or other systemic diseases)” (2,3,10 11,12).

1.3.2?Mechanism of AF
The mechanism of AF is complex and incompletely understood. Ectopic and re-entry activity are two major mechanisms leading to AF (Figure 1.4) (13). The origins of these ectopic activities are normally from the left-atrial posterior wall and the pulmonary veins (13). The ectopic activity could result from a delayed afterdepolarizations (DADs). DADs are referring to as the extra depolarization in phase 4 (after full repolarization) that can trigger a tachycardia. Re-entry impulse could be triggered by this premature ectopic activity and maintained due to shorting of action potential duration (APD), slow conduction, conduction barriers and reduced refractory period (14). However, to be self-sustain of this re-entry activity still needs a susceptible substrate (13,15). Recent research revealed that these arrhythmogenic substrates are produced from the combinational effects of risk factors (i.e. age, myocardial infarction, volume overload and gene mutations), and co-morbidities (i.e. heart failure, hypertension and valvular disease) caused structural and electrical remodelling (13,15). These abnormalities could alter the atrial tissue and lead to the abnormal electric impulses. (3,10,14)

Figure 1.4 Fundamental mechanisms of AF

1.3.2.1?ectopic activity of AF
The ectopic activity of AF is normally caused by electrical remodelling in ionic current. Intracellular Ca2+ concentration, ryanodine receptor channel type 2 and Na+-Ca2+ exchanger are vital in this mechanism (14,16).

1.3.2.1.1?role of Ca2+ in ectopic activity of AF
During normal rhythm, the cardiac action potential is initiated from SA node. The membrane depolarization causes the voltage gated Ca2+ channel to open and allow Ca2+ to influx into the cell through the activation of L-type Ca2+ current (Ica,L) during the action potential plateau phase (14,16,17). This calcium entry triggers a large calcium release which stored in the sarcoplasmic reticulum (SR) by ryanodine receptor channel type 2 (RyR2) (16,18,19). Therefore, the hypersensitivity of the RyR2 could increase the Ca2+ leak from SR into the cytoplasm and increase the intracellular Ca2+ concentration. The high intracellular Ca2+ concentration allow Ca2+ to bind to the myofilament protein troponin C. This activates the actin-myosin contractile machinery; the thin and thick myofilaments slide against each other in the presence of ATP (released from mitochondria) to produce contraction of the ventricular cardiac muscle fiber (Figure 1.5) (20). During relaxation, the Ca2+I need to reduce quickly, and this happens via three different efflux channels. The Ca2+ ions are either released from the cell entirely or are sent into the SR for storage. The latter occurs by the SR Ca2+ ATPase pump (SERCA) that is present on the SR uniformly found throughout the myocytes, and to a lesser extent by the sarcolemmal Ca2+ ATPase and the mitochondrial Ca2+ uniporter, and the former happens primarily through the Na+/Ca2+ exchanger (three Na+ ions into the cell accompany with outflux of one Ca2+ ion) (AF BASIC 75) (Figure 2.3) (20). Normally, these inward Na+ current depoalrize the membrane, however, RyR2 dysfunction could cause more inward Na+ current and generates DADs. When these DADs reach the threshold potential, an action potential will be fired and producing ectopic firing of AF(13,14,15,16,21). Data from several animal models suggest that excessive RyR2 activation are highly associated with the initiation of AF (16,18).

1.3.2.2?re-entry of AF
Leading circle and spiral wave are two main hypothesis of re-entry circuits to explain the arrhythmic mechanism (14,16,21,23,24,25). These mechanisms are associated with both electrical (functional substrate) and structural (anatomical substrates) remodelling. Electrical remodelling causes the action potential to change shape and shorten. It is caused by the decrease of L-type Ca2+ current, Na+ and increase of inward K+ current. (13,15). Most of the structural remodelling changes in atrium cause oxidative stress which modulates several channel proteins and eventually leads to tissue fibrosis.

Whether the electrical signal propagates from cardiomyocyte to cardiomyocyte depends on if the cardiomyocyte the electrical signal meets is refractory. Under normal circumstances, the signal is spread in every direction and the tissue from where it came is left refractory and so cannot be reactivated. If people have an area of heart tissue that is not conducting the signal cannot propagate through this, but has to propagate around; and if the path length of this circle is long enough the electrical impulse will encounter tissue that is no longer refractory and hence can be re-excited. This can initiate a self-sustainable electrical signal that will continue to propagate and cause re-entry arrhythmia. Allessie et al. (1977) developed this leading circle theory in functional re-entry, and it is still the predominant theory on how arrhythmia occurs in AF (26). Hence, the electrical remodelling that takes place in AF with shortening of the action potential therefore makes the heart more prone to AF as it shortens the refractory period. Likewise, structural remodelling with scar tissue formation sets a substrate for the re-entry circle to circle around.

1.3.2.2.1?electric remodelling in re-entry
The role of Ca2+ in re-entry
As atrial depolarization and heart rate increases in AF, Ca2+ start to accumulate inside cardiomyocyte, resulting in activation of Ca2+/calmodulin system. This system is a molecular feedback system that could detect the intracellular concentration of Ca2+ and activates calcineurin. Calcineurin is a complex of serine/threonine phosphatase with a catalytic A subunit and a regulatory B subunit function to dephosphorylating numbers of cytoplasmic proteins. Nuclear factor of activated T cell (NFAT)c3/4 is regulatory domains which translocate into the nucleus that could also be dephosphorylated by calcineurin and then affect the gene transcription (14,15). This reduces the transcription of Cav1.2 L-type calcium channel (CACNA1C) and the L-type Ca2+ current. This has been demonstrated in both animal models and isolated human atrial myocytes (13,27,28). The influx of Ca2+ (ICA,L) is reduced by 73% in AF patient atrium cardiomyocyte compared to normal (29). Similar reductions of this Ica,L also found in dog models (30) and other human reports (31). Reduction of L-type Ca2+ current leads to a shortening of action potential duration and the atrial effective refractory period leading to contractile dysfunction and re-entry in AF (14).

The role of Na+ in re-entry
Na+ current and channels play an essential role in initiating of an action potential and cardiac excitation-contraction coupling (32). Alternation in Na+ current may also contribute to the mechanism of re-entry in AF. Reductions of the density of Nav 1.5 subunit (i.e. SCA5A) (at mRNA and protein expression level) and peak Ina were identified in dog and isolated myocardium, respectively (15,16,29,32,33). Bosch et al. also reported the reduction of Ina in human AF (29). Peak Na+ current (INa) is a large inward current mainly through the SCN5A. Peak INa is a fast current that only needs several milliseconds to rise and decay during an action potential (34). Reduction in peak INa could slow the conduction of action potential and promote the re-entry in AF (16).

In addition to the decrease of peak INa, late or persistent INa is increased due to elevated Nav1.1 (16,32). In contrast to peak Ina, late INa is a much smaller inward current that flows much longer time and throughout the AP plateau (16). Due to different properties between peak and late INa, late INa plays the more crucial role in the contribution of intracellular Na+ concentration (23,32). Thus, increase late Ina could also elevate the intracellular Na+ concentration. This could active the NCX but in the negative direction (reverse mode) which less Ca2+ could be exchanged out and more Ca2+ influx during diastole (23,32). Overload of diastolic Ca2+ could further cause contractile dysfunction electrical instability in AF (32).

The role of K+ in re-entry
Electrical remodelling that altering the repolarization phase in action potential of cardiomyocyte could also lead to reduced atrial refractoriness and APD. The inward rectifier K+ current (Ik1 and Ik,AcH) are intimately associated with re-entry in AF (14,35). This elevated Ik1 current in left atrium cardiomyocyte is mainly due to upregulation of the Kir2.1 (KCNJ2) channel which is the major channel for Ik1 current (14,36,37). Moreover, the gene expression of Kir 2.1 is associated with the microRNA-26 (miR-26) and miR-1 levels in both AF animal model and human AF (37). These repolarization abnormalities are caused by Ik1 rectification and associated with Ca2+ overload and Ca2+-calcineurin-NFAT system (23,39). Thus in AF, activation of this Ca2+-calcineurin-NFAT system results from downregulation of miR-1 and miR-26, leading to upregulation of Kir 2.1 and increases of K+ current, and ultimately cause reduced atrial refractoriness and re-entry waves. Some researchers also demonstrated that other Ik1 current channels such as Kir 2.2 and Kir2.3 were also upregulated which contributing to the increased Ik1 (23,38).

1.3.2.2.2?Structural remodelling in re-entry
The atrial myocardial structure can also be affected by AF causing structural remodelling (3,10,14,40). Changes in tissue properties (atrial fibrosis), atrial size and cellular ultrastructure are the salient feature that associated with AF-induced structural remodelling, as well as a consequence of other diseases such as heart failure, hypertension and valvular heart diseases. The association of other features with AF structural remodellings such as ageing (14,41), myocardial infarction (MI) (AF basic 42) and volume overload (43) are also demonstrated in both animal models and human clinical experiments.

Atrial fibrosis
The progression and development of atrial fibrosis function as a substrate for AF and is a hallmark of AF structural remodelling (44,45). The development of atrial fibrosis in AF patient is closely related to the severity of AF. Advanced atrial fibrosis in AF patient could further reduce the function of atrial and the effectiveness of the anti-arrhythmic drug, promoting to permanent AF (14,40,44,46,). The pathogenesis of the fibrosis is the accumulation of extracellular matrix (ECM) proteins including collagen (47). The correlation between AF and increased collagen deposition and extracellular matrix (ECM) volume has been documented in several studies (44,45,47). ECM not only provides the role of scaffolding for myocytes and the maintenance of the heart structure but also as a participant in the heart activation conduction. Therefore, changes in the balance between collagen synthesis and degradation could change the ECM volume and affect the conduction of the action potential in the heart (47,48). Various factors such as oxidative stress and cell stretch act synergistically with profibrotic signalling molecules such as Angiotensin II, aldosterone, and TGF-?1 to change the properties of the cardiac fibroblast which are responsible for the synthesis of ECM, collagen production, promote the proliferation and differentiation of the fibroblast into cardiomyocytes and the development of atrial fibrosis (14, 15, 44, 48, 49, 50, 51, 52, 53). A physical conduction barrier is formed by fibrosis, resulting in conduction block that could lead to re-entry arrhythmia (53).

Atrial size
Increasing the size of the atrium has long been thought as risk factors of AF (12, 14, 54). AF was common in patients with a left atrial dimension larger than 40 mm (80 of 148 or 54%, P36 hpf/30 min) and washed two times in PBS-T for 5 minutes each before postfix for 20 minutes in 4% PFA/PBS at room temperature followed by two times wash in PBS-T (5 minutes each). Embryos were subsequently pre-hybridized in pre-warmed Hyb+ at least 1 hour at 67 in the heating block. Then, the Hyb+ was replaced by probe solution (1?l of RNA probe in 400 ?l Hyb+) and incubates overnight at 67. The day after incubation, probe solution was removed, and the embryos were washed with wash buffer 1-4 for 1 hour each at 67, followed by two times washes (10 minutes each) in PBS-T at room temperature. The embryos were then blocked in blocking solution (10% sheep serum/PBS-T) at room temperature for at least 1 hour. After blocking, the anti-DIG-AP antibody was diluted in blocking solution and replaced with the normal blocking solution and incubate overnight at 4. The embryo was washed four times in PBS-T (15 min each) in the following day and transferred into a 24-well plate. Embryos were then washed two times with staining buffer for 10 minutes each and replaced by BCIP/NBT solution. When staining of an embryo is bright enough, the reaction was stopped by the post-fixing with 4% PFA/PBS for 20 minutes. These stained embryos could be dehydrated into MeOH and leave in 100% MeOH at least overnight. Embryos have to be washed through a series of glycerol/H2O (20%,40%,60%,80%) and incubate for several hours before observing it under the microscope.
2.1.4? Electrophysiology
2.1.4.1? Micropipette electrode and recording equipment preparation
The micropipette electrode used for the ECG recordings was made from a borosilicate glass capillary (3.5 3-000-203-G/X Drummond) that are pulled by a horizontal DMZ Universal Puller (Zeitz, Germany) to produce a high resistance micropipette electrode. The micropipette was filled with potassium acetate as pipette solution and assembled by suspending a 0.25 mm chloride-coated silver wire which connected to the amplifier through a gold pin. The ECG signal is recorded between the micropipette electrode and a Ag/AgCl ground electrode positioned in the recording chamber as indicated in Figure xx. x. The working platform and all of the recording equipment was encased on an air table in a grounded Faraday cage to decrease the background noise (Figure 2.2). The signal was acquired using a Multiclamp 700B amplifier (Axon Instruments, Inc.) in current-clamp configuration through a shielded cable and connected to a PC which is located outside the cage. The amplifier was operated with 10 kHz Bessel; 0.1 Hz AC. The analogue output signals were digitized and recorded at 1 kHz by using a data acquisition device (Pclamp10.2).

Figure 2.2: Equipment encased on an air table in a grounded Faraday cage
2.1.4.2? In vivo ECG recording
The ECG data were collected from zebrafish larvae at 3-5 dpf. After 15 min of anaesthesia (no gill movement), the individual zebrafish larva was transferred to the ECG recording plate which contains 3 ml of E3 buffer. All larvae were positioned in the same position and explanted under dissection microscope during measuring (as Figure 2.3). The ECG signals were recorded in E3 solution using prepared borosilicate glass micropipette. The experiment was performed at 24-25°C. The micropipette was positioned above the heart on the skin surface of larvae and adjusted by micromanipulator to change the position and record the maximum signal readings. Each larva was recorded with the ECG signals for one and a half min.

Figure 2.3 ECG recording on a 3 days postfertilizations (dpf) larva. The micropipette was positioned on the ventricle part of the contracting larvae heart.

2.1.5? Data analysis
2.1.5.1? ECG analysis
ECG data were exported to and analysed using LabChart 8 (ADInstruments). Between 10-50 consecutive beats were averaged and the ECG intervals were determined on this averaged signal in order to reduce the influence of noise. The interpretations of the ECG were based on the thorough description provided in (Crowcombe et al., 2016). The ECG configurations A and B corresponding to electrode position 1 and 3 (Figure 2.4) were used for the analysis. Examples of ECG recordings can be seen in in (Figure 2.4). The analysis of the ECG signal was done before zebrafish were genotyped.

Figure 2.4 A: Representative recording from position 1 (atrial recording) showing the key ECG features: P wave, QRS complex, T wave, PR interval and QT interval B: A representative recording from position 3 (ventricular recording) C: Close-up ventral view of electrode positions over the heart. The signals from positions 1 and 3 were measured at the same time on the same zebrafish, that from position 2 was measured on a different animal and hence no voltage scale is shown. A positive deflection in the ECG is caused by the depolarization wave moving towards the recording electrode (188). D: Examples of ECG recordings (electrode position 1). E: Examples of ECG recordings (electrode position 3).

2.1.5.2? Statistics
A total of 107 larvae were included in the ECG analysis (17 for each day, i.e. n=17). Results are presented as mean ± SEM. All data were checked for the normal distribution by QQ plot, Kolmogorov-Smirnov test and Shapiro-Wilk test. If results followed the normal distribution then compared with Student’s t-test; the Wilcoxon signed rank test will be used if the data did not follow the normal distribution. A value of p 0.8) especially TTN (176). Linkage disequilibrium refers to the phenomenon that the frequency of simultaneous enrollment of two genes in different loci is significantly higher than the expected random frequency in a specific population. As Figure 4.2 illustrated, TTN contain a high recombination rate associated with PR interval. Their study also showed that TTN not only associated with prolonged PR interval but also associated with increased AF risk (176). The similar hypothesis was identified by Christophersen, I. E et al., 2017 in Japanese. Therefore, the effect of the TTN gene variants on the PR interval and AF is enormous, and our experiment supports this hypothesis in zebrafish larvae models.

Table 4.1 Description of ten high LD PR loci among those of European descent (176)
SNP Chr Position (bp, hg18) Closest Gene
rs6599250 3 38759033 SCN10A
rs10154914 3 38607634 SCN5A
rs12127701 1 109639787 MYBPHL
rs11264339 1 153407272 KRTCAP2
rs397637 1 226519951 OBSCN
rs922984 2 179324132 TTN
rs7638853 3 186833400 SENP2
rs3733409 4 187864587 FAT1
rs7729395 5 102128475 PAM
rs365990 14 22931651 MYH6

Figure 4.2 Regional association plots of specific loci associated with PR interval. Each SNP is plotted with respect to its chromosomal location (x axis) and its P value (y axis on the left). The blue line indicates the recombination rate (y axis on the right) at that region of the chromosome. Blue outlined squares mark non-synonymous SNPs. Green triangles depict association results of the African Americans meta-analysis, only SNPs with P ; 0.1 are shown. (176)

In addition to the prolonged PR interval, the zebrafish larvae model also confirmed the association between TTNvt and QRS complex prolongation. Brody, J. A., et al. confirmed that some of the genes associated with PR prolongation were also associated with prolonged QRS duration; however, the TTN gene is not included (176).
Surprisingly, the results of zebrafish ECG data showed that there was a significant slowing of the heart rate in the 3 day TTNtv larvae as compared to wt (P

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