Role of Oxidative Stress in Mediating Elevated Atrial ... - TSpace

Role of Oxidative Stress in Mediating Elevated Atrial Fibrillation by Tumor Necrosis Factor-Alpha

S. Moniba Mirkhani

A thesis submitted in conformity with the requirements for the degree of Masters of Science Graduate Department of Physiology University of Toronto

© Copyright by S. Moniba Mirkhani 2012

Role of Oxidative Stress in Mediating Elevated Atrial Fibrillation by Tumor Necrosis Factor-Alpha S. Moniba Mirkhani Master of Science Department of Physiology University of Toronto 2012

Abstract Atrial fibrillation (AF) , the most common arrhythmia encountered in clinical practice, is a major source of morbidity and mortality, and is highly associated with inflammation and oxidative stress. In the present study, we show that acute exposure of mice atrial tissue to tumor necrosis factor-α (TNF-α) increases susceptibility to AF. We further show that acute exposure to TNF-α led to increased spontaneous sarcoplasmic reticulum (SR) calcium release and generated triggered activities in isolated mice atrial myocytes. This increase in spontaneous SR calcium activity was found to be due to elevated reactive oxygen species production from mitochondria and NADPH oxidase sources triggered by TNF-α. Hence we concluded that acute exposure to TNF-α leads to elevated oxidative stress that increases spontaneous SR Ca2+ release and triggered activity through which it can lead to AF induction and maintenance.

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Acknowledgments I would like to first extend my gratitude to my supervisor Dr. Peter H. Backx for providing continuous support and guidance, both academic and personal, throughout my graduate studies and for challenging me to be independent and believe in myself. His exemplary passion for science and expertise has aided in my development as a young scientist and has made me appreciate what true research is and what it takes to become a good scientist. I would also like to thank my committee members Dr. Scott Heximer and Dr. Steffen-Sebastian Bolz, who were always available for consultation. Their valuable comments and suggestions have guided me throughout this process. I would like to specially thank Dr. Roozbeh Aschar-Sobbi for his limitless patience and guidance in teaching me experimental techniques and his continuous support and invaluable discussions regarding my project. I would also like to genuinely thank my friend and colleague Dr. Sanja Beca for her continuous support and guidance. Her understanding and encouragements kept me motivated throughout the past two years of my graduate studies. I would also like to thank Wallace Yang for his valuable technical support and contribution to this project. I am also grateful to all the former and current members of Dr. Backx’s laboratory, which have made my work environment friendly, helpful and productive: Dr. Gerrie Farman, Dr. KyoungHan Kim, Anna Rosen, Dr. Brian Panama, Jack Liu, Farzad Izaddoustdar, Dr. Roman Pekhletski, Elena Pekhletski, Dongling Zhao, Bill Liang, Adam Korogyi and Nazar Polidovitch. I would also like to thank my friends Amin and Sanaz for their continuous support.

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Lastly, and most importantly, I would like to thank my family. I would like to remember my father who is always in my heart and who his memories, give me courage and motivation to peruse my goals in life. He has always been and will be, both my personal and academic role model throughout my life. I would also like to deeply extend my gratitude to my mother for her unconditional love, sacrifice and limitless support throughout my life and for believing me in any endeavors I have taken in life, which is what has helped me succeed. I am blessed for having such great parents and to them I dedicate this thesis. Finally, my lovely sisters Melika and Mahta thank you for believing me and supporting me throughout every step I have taken in life.

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Table of Contents Acknowledgments.......................................................................................................................... iii Table of Contents ............................................................................................................................ v List of Figures .............................................................................................................................. viii List of Abbreviations ..................................................................................................................... ix Chapter 1 Introduction .................................................................................................................... 1 1 Introduction ................................................................................................................................ 2 1.1 Atrial Fibrillation ................................................................................................................ 2 1.1.1

Mechanisms of AF .................................................................................................. 3

1.1.2

The electrophysiological basis of AF ..................................................................... 5

1.2 Cardiac calcium Regulation ................................................................................................ 8 1.2.1

The role of calcium in cellular function .................................................................. 8

1.2.2

Cardiac excitation-contraction coupling overview ................................................. 8 1.2.2.1 Ca2+-induced-Ca2+ release (CICR) ........................................................... 9 1.2.2.2 Termination of the CICR, removal of Ca2+ from the cytosol of the cardiac myocyte ...................................................................................... 12

1.2.3

Calcium propagation in ventricles and atrial myocytes ........................................ 13

1.2.4

Calcium abnormalities in AF ................................................................................ 14

1.3 Inflammation and Atrial Fibrillation ................................................................................. 16 1.3.1

TNF-alpha ............................................................................................................. 16 1.3.1.1 TNF-α and heart function ....................................................................... 17

1.4 Oxidative stress and Atrial Fibrillation ............................................................................. 19 1.4.1

Oxidative stress ..................................................................................................... 19

1.4.2

The association between TNF-α, oxidative stress and heart function .................. 24 v

1.5 Synopsis ............................................................................................................................ 27 Technical Contribution and Acknowledgement ........................................................................... 28 Chapter 2 Materials and Methods ................................................................................................. 29 2 Materials and Methods ............................................................................................................. 30 2.1 Experimental Animals ...................................................................................................... 30 2.2 Atrial Isolation and Optical Imaging ................................................................................ 30 2.3 Isolation of Atrial Myocytes ............................................................................................. 31 2.4 Calcium Sparks Measurements Using Confocal Microscopy .......................................... 32 2.4.1

Signal processing and data analysis ...................................................................... 34

2.5 Reactive Oxygen Species Measurements ......................................................................... 36 2.6 L-Type Calcium Current Measurements Using Whole Cell Patch-Clamp Technique ..... 37 2.7 Statistical Analysis ............................................................................................................ 37 Chapter 3 Results .......................................................................................................................... 38 3 Results ...................................................................................................................................... 39 3.1 Acute exposure to TNF- α promotes rotor formation in isolated atria ............................. 39 3.2 TNF-α Increases Spontaneous SR Ca2+ Activity (Ca2+ Sparks) ....................................... 41 3.2.1

TNF-α Leads to Increased Oxidative Stress ......................................................... 51

3.3 TNF- α induced mitochondrial ROS leads to increased frequency of Ca2+ sparks ........... 52 3.4 TNF- α induced NADPH oxidase activity leads to increased frequency of Ca2+ sparks .. 55 3.5 TNF- α does not effect L-type calcium current ................................................................ 57 Chapter 4 Discussion .................................................................................................................... 58 4 Discussion ................................................................................................................................ 59 Chapter 5 Future Directions .......................................................................................................... 74 5 Future Directions ...................................................................................................................... 75 vi

5.1 Determine the level of ROS in TNF-α treated cells in the presence of apocynin and mitoTEMPO...................................................................................................................... 75 5.2 Determine the role of SR Ca2+ load in the generation of the spontaneous Ca2+ activities that were observed in TNF-α treated cell. ......................................................... 75 5.3 Determine the role of IP3Rs in the generation of the spontaneous Ca2+ activities that were observed in TNF-α treated cell................................................................................. 75 5.4 Determine the underlying cause of the spontaneous Ca2+ transients observed in TNF-α treated cells. ...................................................................................................................... 76 5.5 Determine the effect of TNF-α induced oxidative stress in triggering spontaneous SR Ca2+ release in isolated atrial tissue................................................................................... 76 References ..................................................................................................................................... 77

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List of Figures Figure 2.1 Mouse Atrial Isolation ................................................................................................. 31 Figure 2.2: Confocal images signal processing. ........................................................................... 35 Figure 3.1: Acute exposure to TNF- α promotes rotor formation in isolated atria. ...................... 40 Figure 3.2: Spontaneous Ca2+ activity........................................................................................... 43 Figure 3.3: Confocal images of Ca2+ spark in atrial myocytes. .................................................... 44 Figure 3.4: TNF- α increases SR Spontaneous Ca2+ activity (Ca2+ sparks). ................................. 47 Figure 3.5 : TNF-α leads to generation of Ca2+ waves in isolated atrial myocytes. .................... 48 Figure 3.6: TNF-α induced spontaneous Ca2+ transient in isolated atrial myocytes. ................... 49 Figure 3.7: TNF-α induces spontaneous Ca2+ transients and Ca2+ waves in isolated atrial myocytes. ...................................................................................................................................... 50 Figure 3.8: TNF-α increased ROS production in isolated atrial myocytes. .................................. 51 Figure 3.9: TNF-α induced mitochondrial ROS stimulated spontaneous SR Ca2+ release in isolated atrial myocytes................................................................................................................. 54 Figure 3.10: TNF-α induced NADPH oxidase ROS led to increased frequency of Ca2+sparks in isolated atrial myocytes................................................................................................................. 56 Figure 3.11: TNF-α does not affect L-type calcium current. ....................................................... 57

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List of Abbreviations AF

- Atrial Fibrillation

Ang-II

- Angiotensin-II

AP

- Action Potential

APD

- Action Potential Duration

AV

- Atrioventricular

CICR

- Calcium-Induced Calcium-Release

CPVT

- Catecholaminergic Polymorphic Ventricular Tachycardia

CSQ

- Calsequestrin

DAD

- Delayed Afterdepolarization

DHPR

- Dihydropyridine-sensitive Receptor

EAD

- Early Afterdepolarizations

E-C

- Excitation-Contraction

ETC

- Electron Transport Chain

IL

- Interleukin

IP3

- Inositol-triphosphate

IP3R

- Inositol-triphosphate Receptor

IκB

- Inhibitory kappa B

j-SR

- Junctional SR

LPS

- Lipopolysaccharide

LTCC

- L-Type Calcium Channel

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NADPH

- Nicotineamide Adenine Dinucleotide Phosphate

NCX

- Na+/ Ca2+ exchanger

NF-κB

- Nuclear Factor kappa B

NF-κB

- Nuclear factor kappa B

nj-SR

- Non-junctional SR

PLN

- Phospholamban

PV

- Pulmonary Vein

RET

- Reverse Electron Flow

ROS

- Reactive Oxygen Species

RyR

- Ryanodine Receptor

SAN

- Sinoatrial Node

sarcKATP

- Sarcolemmal ATP- Sensitive K+ Channel

SERCA

- Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPase

SOD

- Superoxide Dismutase

SR

- Sarcoplasmic reticulum

TACE

- TNF-α converting enzyme

TNF- α

- Tumor Necrosis Factor – α

TNFR

- TNF-α receptors

t-tubules

- Transverse Tubules

x

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Chapter 1 Introduction

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Introduction

1.1 Atrial Fibrillation Atrial fibrillation (AF) is the most common arrhythmia encountered in clinical practice. It leads to reduced quality of life in patients with and without heart disease as well as increased mortality 1

. AF, which is characterized by rapid and uncoordinated activation of the atrium, increases death

rates by two to three folds, compared to individuals with no AF 2, 3. The occurrence and prevalence of AF increases with age, affecting >5% of the population over the age of 65 3, 4. AF leads to increased risk of stroke, impaired cardiac performance and heart failure 2, 5. Several cardiac and non-cardiac disorders predispose individuals to AF including hypertension, coronary artery disease, mitral valve disease, sleep apnea, hyperthyroidism, inflammation and diabetes 4, 610

. AF may also occur in individuals with no indication of heart disease or arrhythmia, a

condition called as “Lone AF” 11. Normally, the heart rate is controlled by the sinoatrial node (SAN) (sinus rhythm). The atria and ventricles undergo coordinated and regular depolarization/repolarization in order to maximize the heart function to meet the body’s metabolic demands. During AF however, electrical impulses that originate from areas other than the SAN in particular pulmonary veins (PV) 12 overcome the SAN electrical impulses leading to rapid and disorganized beating of the atria. The response of the ventricles to AF depends on fibrillating rate of the atria and the filtering function of the atrioventricular (AV) node, which atrial impulses must pass before depolarizing the ventricles. Irregular impulses to the ventricles can lead to increased heart rate of 150 beats per minute as opposed to the normal heart rate of 60 beats per minute during rest 13 .AF can be classified into paroxysmal AF (episodes lasting more than 2 minutes and fewer than 7 days) or chronic AF (episodes lasting more than 7 days) 14. AF is believed to begin with isolated

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paroxysmal (non sustained) events which can lead to sustained chronic AF by causing electrophysiological changes in the atria (i.e. AF-begets AF) 15, 16. Paroxysmal AF may be recognized by sensation of palpitation, chest discomfort, fatigue and light-headedness 17, however in sustained chronic AF palpitation decreases and AF may become asymptomatic and the uncontrolled rapid ventricular response itself can lead to severe chronic heart failure, especially in patients unaware of the arrhythmia 13, 17, 18. AF is treated by both pharmacological and non-pharmacological therapies. Drug therapy is the main treatment option for AF. Antiarrhythmic drugs, which mainly control rhythm by changing the cardiac electrophysiological properties have been widely used for management of AF however, the majority of these drugs are not atrial-specific and have been associated with ventricular arrhythmias or long-term organ toxicity 19-21. Non-pharmacological treatment modalities such as ablation therapy and implantable devices that can detect and terminate electrical discharges have also been effective to some extent in managing AF 22. However, both drug therapy and non-pharmacological modalities are still limited when it comes to treatment of AF and this has prompted investigators to search for improved treatment options for AF 21, 22 . Hence understanding the underlying mechanism of AF can aid in development of new or improved treatment modalities.

1.1.1

Mechanisms of AF

At present there are three different theories regarding the mechanism of AF. AF could be due to: 1) rapidly firing, spontaneously active atrial ectopic foci (hyperectopia theory) 2) a single reentry circuit (“mother wave”) with fibrillating conduction 3) multiple functional re-entry circuits in the atria (multiple wavelet theory). Hence re-entry is an important concept in AF mechanism. Reentry, a disorder of impulse propagation, occurs when an impulse repeatedly travels around an

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abnormal circuit, often referred to as wavelets. When ectopic beat reaches a refractory tissue in one direction it might be able to propagate in the faster recovering tissue in the other direction, which can lead to generation of re-entrant rotors (abnormal re-entry electrical circuits). This selfsustained sequence of rapid depolarization- repolarization cycles can lead to rapid and frequent activation of the atria, hence AF 23-25. For a long time the multiple re-entry circuit theory was accepted as the dominant mechanism underlying atrial fibrillation with the “wavelength of re-entry” being an important component of this theory 26, 27. The wavelength is the product of refractory period (~action potential duration) and the conduction velocity. At a given conduction velocity the wavelength is the distance an impulse travels in one refractory period. Therefore in order to sustain re-entry the distance that an electrical impulse travels must be larger or equal to the wavelength. If the circuit size that the signal travels is smaller than the wavelength, the signal will arrive at the initial point, which is still in the refractory period and it cannot depolarize the tissue hence the signal will die. So the wavelength is the shortest distance that can maintain re-entry. Thus, to have one or more rotors in the atria the wavelength of the atria should be less than the physical dimension of the atria. Prolonging the refractory period (thereby the wavelength) is one approach to managing AF 23, 24. Further investigation during the past few years have observed rapidly firing atrial foci and single small re-entry circuit as possible mechanisms responsible for developing AF 12, 28, 29 suggesting that each of the above mentioned mechanism of AF are likely to occur in particular circumstances. In the process of AF certain functional, structural, electrical and biochemical changes would have to occur in the atria, referred to as remodeling. Remodeling provides the required substrate (capable of sustaining arrhythmia) for AF 15, 24, 30. In addition, an appropriately timed stimulus

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(trigger) is also required to induce re-entry and as a result arrhythmia. Triggers are diverse and include stimulations from the sympathetic and parasympathetic system, premature atrial beats, tachycardia, as well as ectopic beats, in particular those originating from the pulmonary veins are shown to have a prominent role in initiating AF 12, 31-33. These ectopic beats upon reaching the appropriate substrate can lead to either multiple-circuit re-entry or single circuit re-entry. As mentioned the atria that underwent remodeling provides the appropriate substrate for AF 24. In order to appreciate the physiological mechanism by which remodeling promotes AF it is important to understand the electrophysiological and molecular basis of atrial remodeling that leads to AF.

1.1.2

The electrophysiological basis of AF

In order for reentry to be sustained resulting in AF a number of changes have to be present. Action potential (AP) is a measure of the membrane potential across the myocyte sarcolemma. AP oscillates from around -80mv at rest, to +50 as it depolarizes and repolarizes back to resting membrane potential of around -80 mV. As AP propagates from cell to cell, it activates Ca2+ channels and initiates myocyte contraction. The closing and opening of a series of voltage and time dependent ion channels that conduct depolarizing (inward) and repolarizing (outward) currents determine the shape of atrial AP. The inward rectifier K+ current (IK1) is the background current responsible for maintaining the resting membrane potential between -70 and -80 mV. When an impulse reaches the cell a large inward Na current (INa) gives a rise to the rapid upstroke (phase 0) of AP and depolarizes the cell to around +40 mV. Partial repolarization (phase 1) is caused by the transient outward current (Ito), followed by a plateau (phase 2), maintained by a balance of depolarizing inward Ca2+ current (ICa) current and the outward K+ current. The latter is composed of series of K+ currents, delayed rectifiers (IK) that activate in a

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time dependent-manner and consist of three ultra-rapid (IKur), rapid (IKr) and slow (IKs) components. The final rapid repolarization (phase 3) is mainly due to the background IK1 and IKr current, which return the membrane potential to the resting values at phase 4. At this phase some additional currents such as the acetylcholine-activated inward rectifier (IK,Ach) or the ATPdependent inward rectifier (IK,ATP) also contribute to the final repolarization of the cell. The (Na+,K+)-ATPase maintains the concentration gradient of Na+ and K+ across the cell membrane by extruding 3 Na+ in exchange for moving in 2 K+. Another transporter, Na+/ Ca2+ exchanger (NCX) is responsible for removing the Ca2+ that entered the cell during the plateau phase of the AP. The plateau phase of the action potential, which determines the AP duration (APD) results from a balance between the inward Ca2+ and the outward K+ current and NCX current. Ca2+ entering the atrial cells through L-type Ca2+ channels (LTCC) (Cav 1.3 and Cav 1.2) 34, 35 along with the increased atrial beating frequency seen in AF, leads to progressive Ca2+ loading of the cell 36. It has been documented that rapidly paced canine atria show a reduction in L-type Ca2+current (ICa, L) 37, 38and so does atrial cells from AF patients 39. This seems to be an adaptive response to the arrhythmia-induced calcium overload aimed at restoring the Ca2+ concentration to the normal levels 38, 39. However, this reduction in ICa, L decreases APD, thus promoting the induction of reentry and hence AF 40. An increase in K+ current during chronic AF, in particular the background IK1 current, due to increased expression of the Kir2.1-subunit has also been reported 41, 42. Evidence from both clinical 43 and basic science studies44, 45 suggests that the autonomic nervous system plays an important role in the incidence of AF. Paroxysmal AF occuring under vagotonic conditions and are clinically referred to as “vagal AF” 46since episodes appear at rest, sleep, after meals, during sleep or with exercise. Furthermore, the inward rectifier IK,ACh is activated by the

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acetylcholine released from the vagal system leading to APD abbreviation 47, 48. It has been postulated that an increase in IK,ACh , similar to those seen in athletes with enhanced vagal activity promotes AF by maintaining re-entrant rotors 49-53. It has been shown that atrial conduction velocity also plays a role in maintaining the re-entrant rotors. Atrial conduction is determined by a balance between the current passing through the plasmalemmal Na+ channels and current passing through gap junctions to other cells 54, 55. In this case, re-entry is favored by slowed conduction, which can happen due to a decrease in the source current, i.e. INa and/or impaired connexin (proteins forming the gap junctions) function. Both have been seen in AF and the reduction in INa has been mainly linked to decreased expression of the alpha subunits of the cardiac Na+ channel55-58. In addition to electrical remodeling, structural remodeling has been strongly implicated in AF induction. Not only do frequent episodes of AF cause structural changes in atrial tissue (structural remodeling) to sustain AF, but preexisting structural abnormalities increase susceptibility to AF. Structural changes associated with AF include interstitial fibrosis, dilatation and hypertrophy 59, 60. Mice with overexpression of transforming growth factor β1 have increased interstitial fibrosis in the atrial myocardium. This increased fibrosis alone is sufficient to increase the risk of AF induction by alteration in atrial conductance. Hence, fibrosis provides a substrate that reinforces rotor formation resulting in AF 60. Other signaling pathways such as the reninangiotensin system and tumor necrosis factor – α (TNF- α)/ nuclear factor kappa B (NF-κB) pathway are also associated with increased fibrosis 59, 61, 62. Atrial structural changes have been mainly linked to stretch, oxidative stress and inflammation. Although the mechanism of stretch induced remodeling is not very clear, it can lead to elevated oxidative stress and inflammatory cell infiltrations, both of which are known to be associated with AF63-65.

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In addition to the electrical and structural remodeling that happens during AF, abnormalities in intracellular Ca2+ handling is also involved in the underlying mechanism of AF in particular ectopic beats that are known to trigger AF. Cardiac excitation-contraction coupling and Ca2+ abnormalities in AF will be discussed in detail in the following Section.

1.2 Cardiac calcium Regulation 1.2.1

The role of calcium in cellular function

Ca2+ is the most widely utilized second messenger in the body 66. Ca2+ signaling plays a role in variety of cellular processes such as fertilization, cell differentiation and proliferation, gene expression, muscle contraction, secretion and apoptosis. Ca2+ signaling is under very tight spatial and temporal regulation within the cell, which enables regulation of diverse biological processes. Ca2+ concentration within the cell is tightly regulated through activity of different Ca2+ channels, pumps, transporters and Ca2+ binding proteins. In cardiomyocytes, concentration of the Ca2+ is tightly regulated on a beat-to-beat basis. The rise in intracellular Ca2+ concentration during electrical excitation of the myocyte leads to contraction of the myocardium in a process known as excitation-contraction (E-C) coupling67. In this section the mechanism of Ca2+ handling within atrial myocytes and the Ca2+ handling abnormalities in AF will be addressed.

1.2.2

Cardiac excitation-contraction coupling overview

The heart rate is under the control of the autonomic nervous system. Heart contraction is controlled by the spontaneous depolarization of pacemaker cells. Auto-depolarization of pacemaker cells from resting values of -60 to -70mV is mainly attributed to gradual decrease in repolarizing K+ currents, along with increase in depolarizing INa and ICa , until a threshold is reached. Any trigger above threshold leads to opening of voltage gated Na+ channels. This leads to INa that is responsible for the rapid upstroke of the cardiac AP. The increase in INa as a result

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activates voltage-dependent LTCC (dihydropyridine-sensitive receptor, DHPR) located on the sarcolemma resulting in Ca2+ entry into the cell down the electrochemical gradient during the plateau phase of AP. However this inward ICa is small and not sufficient on its own to begin contraction. ICa instead triggers the release of much larger Ca2+ from the intracellular Ca2+ store, the sarcoplasmic reticulum (SR), by activating the SR Ca2+-sensitive Ca2+ release channels (ryanodine receptors; RyRs). This processes is commonly referred to as calcium induced-calcium release (CICR) 68. This is achieved by close proximity of RyRs in the SR membranes to LTCC in invaginations of the plasma membrane called transverse tubules (t-tubules). Ca2+ entry through DHPRs activates an individual cluster of RyRs on the SR membrane, which as a result dramatically amplifies the release of more Ca2+ from the neighboring RyRs 69, 70. This leads to a coordinated release of calcium which following depolarization generates a cell-wide intracellular Ca2+ transient of adequate magnitude to generate contraction 71.

1.2.2.1

Ca2+-induced-Ca2+ release (CICR)

RyRs were named after plant alkaloid ryanodine, because of their high affinity to this plant 72. Single channel studies of RyRs have identified these receptors as poor selective cation channel 73

. These channels have a very high conductance for both divalent and monovalent cations 74, 75,

which makes these receptors suitable for rapid and considerable Ca2+ release from the SR during EC coupling 76. So far three tissue-specific isoforms of RyRs have been identified: the RyR1 isoform is mostly found in skeletal muscle, RyR2 is the predominant isoform in cardiac cells and RyR3 RyR3 is more ubiquitously expressed at low levels in different cell types including muscle cells 77. RyR is made of four ~565 kilodalton (KDa) subunits, forming a large tetramer complex with a total molecular weight of 2300 KDa; 78. The activity of these channels is controlled by both cytosolic and luminal SR Ca2+. The RyR shows a bell-shaped activation/inactivation curve

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which depends on the intracellular Ca2+ concentration ([Ca2+]i ).The open probability of RyRs gradually increases with Ca2+ concentrations from 1-10µM and gets inhibited with Ca2+ concentrations from 1-10mM 79. The activity of RyR channels is mainly regulated by a number of endogenous molecules like Mg2+ or ATP and associated proteins such as FKBP12 (FK 506-binding protein) and calmodulin. Cytoplasmic ATP stimulates, while magnesium has inhibitory effects on RyRs in millimolar range 79-81. FKBP12 is believed to act as a regulatory protein and stabilizes RyR channel (in particular maintenance of RyR closed state) and couples the gating of adjacent RyR tetramers 82. Calsequestrin (CSQ) is the major Ca2+-binding protein in skeletal and cardiac muscle 83. The total SR Ca2+ concentration can reach as high as 10mM. However, majority of this Ca2+ gets bound to CSQ and results in an intraluminal [Ca2+]free at 0.6-1.0 mM. This [Ca2+]free helps maintain the Ca2+ gradient and the electrochemical driving force for Ca2+ fluxes through the SR membrane 84. CSQ is attached to the junctional side of the SR membrane, which contacts with RyR, triadin and junctin 85. The precise mechanism underlying regulation of CICR through the interaction between the RyR channel and CSQ, triadin and junctin is still elusive. Most likely scenario includes gradual regulation of RyR2 open probability with respect to total SR Ca2+ content. Higher luminal Ca2+ concentrations lead to leaky RyRs, while lower luminal SR Ca2+ concentrations slowly reduce RyR activity. Inevitably, complete emptying of the SR Ca2+ content leads to almost complete inhibition of the RyR activity 86-89 The SR and the sarcolemmal membrane come into very close proximity of each other and it has been suggested that ~100 RyRs group with 10-25 DHPRs and form a local SR Ca2+ unit called junction (or couplon) 90. The combination of confocal microscopy with Ca2+ fluorescence imaging has allowed recording of Ca2+ release from spatially discrete individual clusters of

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RyRs, called Ca2+ sparks or spontaneous local Ca2+ transients 91. Synchronous activation of a cluster of about 6-20 RyRs leads to generation of Ca2+ sparks 91-93. Analysis of spark properties have shown sparks to have a brief lifetime of ~30ms and typically span 1.5 – 2.2 μm in length (measured as the full width at half maximum amplitude) 91, 94, 95. Ca2+ sparks are believed to be the elementary events and fundamental units of Ca2+ release during both rest and E-C coupling 95

. During E-C coupling, spatio-temporal recruitment of Ca2+ sparks, triggered by AP by

simultaneous activation of ICa leads to generation of whole-cell Ca2+ transient. Clusters of RyRs can also become activated (without ICa) during rest and produce Ca2+ sparks because the resting [Ca2+]i (~100nM) seems to be sufficient to activate RyRs, but at lower rates 95, 96. The SR [Ca2+ ] at rest is at the range of 0.3 to 1.0 mM, during normal contractile function 97 however, under conditions of SR Ca2+ overload the SR [Ca2+] can reach to as high as 5mM 98. This increases in SR Ca2+ load, leads to increased frequency of spontaneous Ca2+ release (Ca2+ sparks), which may generate Ca2+-activated transient depolarizing inward current. Delayed afterdepolarization (DADs) can form as a result of this current and the generated DADs can lead to arrhythmias 99 100

. Other than the SR Ca2+ overload that can lead to elevated spark frequency increased RyRs

leakiness can also elevate spark frequency. One such factor that increases SR Ca2+ leak from RyRs is reactive oxygen species (ROS) 101.The redox state of the cell is also known to affect RyR activity, hence SR Ca2+ release 101. RyR2 tetramer has a total of 346 cysteines and their highly reactive sulfhydryl group is susceptible to redox modifications. These thiol groups are in a reduced state and oxidation of these groups increases RyR activity and as a result induces Ca2+ release from the SR 102, 103. In conclusion, CICR is a positive feedback mechanism that leads to a synchronized transient increase in [Ca2+]i. Muscle relaxation is achieved by closure of the SR Ca2+ release channels and

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rapid removal of Ca2+ from the cytoplasm. Cytosolic Ca2+ is mainly sequestered into the SR via the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), which is regulated by phospholamban (PLN) and consumes the energy of ATP to pump Ca2+ back into the lumen of the SR. Some Ca2+ gets extruded across the cell membrane through the NCX. These two pathways are the prominent means by which Ca2+ is removed from the cytosol. Alternate Ca2+ transporters such as the plasmalemmal Ca2+-ATPase and mitochondrial Ca2+ uniporter are also involved in removing Ca2+ though in much lower levels104.

1.2.2.2

Termination of the CICR, removal of Ca2+ from the cytosol of the cardiac myocyte

To maintain steady state, i.e. to prevent loss or gain of Ca2+ (Ca2+ overload) it is very important that the amount of Ca2+ that enters the cell during contraction is equal the amount of Ca2+ that is extruded during relaxation and the Ca2+ that is released from SR is transferred back to the SR 105. SERCA whose under strong sympathetic control is the most important factor in regulating the cardiac muscle relaxation rate and the amount of Ca2+ accumulated in the SR 106. Increased sympathetic stimulation leads to increased Ca2+ transport across the SR membrane 107. This is a particularly important adaptive mechanism that is necessary for dealing with the consequences of increased heart rate 108. NCX activity is highly dependent on voltage (reversal potential of ~35mV) and generally functions to efflux Ca2+. However if the [Na+]i increases it may transiently function in the reverse mode. For example inhibition of the Na+/K+ pump by cardiotonic glycosides or prolonged APD leads to NCX activation in reverse mode, which can lead to Ca2+ influx during phase 2 of the cardiac AP and trigger dangerous early afterdepolarizations (EADs) and generate arrhythmia 109-111.

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1.2.3

Calcium propagation in ventricles and atrial myocytes

Structural differences between ventricular and atrial myocytes result in different calcium propagation during E-C coupling between these two types of cells. The ventricular myocyte is characterized by the presence of a well-developed three-dimensional network of sarcolemmal membrane invaginations, t-tubules. These invaginations occur at the z-line. DHPRs are located on the sarcolemmal membrane along the t-tubules 112. Ventricular SR is composed of three structurally distinct regions: the network SR, the peripheral junctional SR (j-SR) and the specialized non-junctional SR (nj-SR). The network SR is composed of anastomosing network that surrounds the entire length of the myofibril. The j-SR is located in close opposition or attached to the sarcolemma or t-tubules, in contrast to nj-SR that is not physically associated with either the sarcolemma or the t-tubules 113, 114. Both j-SR and nj-SR have RyRs anchored to their membrane. In cardiac muscles, there is a peripheral coupling between the t-tubules and the j-SR via “feet structures” (containing RyRs) called dyadic coupling. The dyadic structures, the fundamental functional unit for E-C coupling, ensure close proximity of DHPRs and RyRs. This allows simultaneous activation of SR Ca2+ release units during an AP and as a result rapid and homogenous propagation of excitation throughout the entire ventricular cells 70, 114, 115. This is contrary to the spatiotemporal organization of E-C coupling in atria. E-C coupling and Ca2+ transient propagation is more complex in atria compared to ventricles. Unlike ventricular myocytes that have a well-developed t-tubule system, atrial myocytes lack a fully developed t-tubule system and DHPRs and j-SR coupling occurs mostly around the periphery of the cell. Ventricular myocytes exhibit a homogenous Ca2+ transient but the Ca2+ transient in atrial cells has two components. In atrial cells [Ca2+]i initially rises in the periphery of the cell (junctional SR) , without any rise in the center of the myocytes. Subsequently, Ca2+

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propagates from the peripheral region to the center (where the nj-SR is located) reaching its peak 116

. It has been suggested that the two components of the atrial myocyte [Ca2+]i transient could be

due to initial activation of the LTCCs that induce Ca2+ release from the j-SR in the periphery. The subsarcolemmal elevation in [Ca2+]i leads to a secondary Ca2+ release from nj-SR in the center (not coupled to LTCCs) activating more Ca2+ release, eventually leading to whole Ca2+ transient117. Another factor that might play a role in different Ca2+ propagation in atrial and ventricular myocytes is the role of inositol-triphosphate (IP3) dependent Ca2+ release. Evidence suggests that in atria both receptor mediated IP3 production and expression level of IP3 receptors (IP3Rs) (that are located in junctional SR near the RyRs) are higher compared to ventricles, which leads to enhanced frequency of Ca2+ sparks 118, 119. Therefore IP3-dependent Ca2+ release may play an important but yet undefined function in the process of E-C coupling in atrial myocytes 120.

1.2.4

Calcium abnormalities in AF

Atrial remodeling provides the suitable vulnerable substrate for re-entry and hence AF to occur. However, a trigger is also typically required to act on the substrate to initiate and maintain reentry. This trigger is mainly due to abnormal and spontaneous electrical activity denoted as ectopic beats (because it arises from area other than the SA node), ectopic foci12, 121. These ectopic foci interrupt the normal sinus rhythm and, if appropriately timed, ectopic beats can initiate reentrant rotors, a process referred to as “kindling”12, 122. Ectopic beats are known to originate from several areas of the atria, but the cardiomyocytes in the pulmonary vein seem to be an important source of ectopic beats that can trigger paroxysms of AF 12. The underlying cause of ectopic beats is not very clear but it has been suggested to arise from EADs and DADs, which occur due to abnormal calcium handling122, 123. The Ca2+ entering the cell during systole is

15

generally removed via the action of SERCA and NCX in order to balance the cytosolic Ca2+ load. If this balance is interrupted it can lead to elevated diastolic intracellular Ca2+ . This Ca2+ can in turn activate NCX current in forward mode and produce an inward Na+ current during Ca2+ extrusion. This inward current can depolarize the cell and generate DADs 99, 124. Therefore DADs are mainly due to spontaneous Ca2+ release from the SR through either RyRs or IP3Rs during diastole 124, 125, whereas EADs are generally due to L-type Ca2+ or Na+ current reactivation 122, 123. If these afterdepolarizations become larger than the threshold potential, they can cause myocytes to generate (“fire”) AP 123. These events can occur either in the form of single ectopic event (triggered arrhythmia) or can occur repeatedly (seen as tachycardia).The protein levels of NCX1 has shown to increase in patients with chronic AF 126. Elevated diastolic Ca2+ level typically occurs as a result of spontaneous SR Ca2+ release, which usually occurs as a result of elevated SR Ca2+ load or as a result of enhanced tendency of RyR2 channels to open for any given SR Ca2+ load. 127, 128. The high atrial frequency in AF leads to elevated Ca2+ load in the atrial myocytes despite the compensatory decreases in ICa that occurs in AF 36. SERCA activity is regulated by phospholamban (PLN), an endogenous inhibitor of SERCA, that once phosphorylated it can no longer inhibit SERCA. PLN hyperphosphorylation occurs in AF, which leads to enhanced SERCA activity to reduce the diastolic Ca2+ load. However, this is at the expense of promoting SR Ca2+ overload, that itself can lead to DADs 126. In addition to SR Ca2+ overload, RyR open probability is also increased in AF. This is mainly due to hyperphosphorylation of RyRs that enhances FKBP12.6 dissociation and as a result increases SR Ca2+ leak during diastole 121. Redox modification of the RyR subunit is also known to increase RyR leakiness under conditions of tachycardia 129. This increased RyR leakiness can lead to increased spontaneous Ca2+ release events (Ca2+ sparks), hence DADs125 . Another factor that might be involved in generation of DADs is IP3- mediated calcium releases from the SR. IP3R type 2 (IP3R2) and

16

RyR2 are co-localized in the subsarcolemal area of atrial cell 125; thus, IP3 dependent SR Ca2+ release may lead to Ca2+ leak via RyRs. This can result in an increase in intracellular diastolic Ca2+ level and generate depolarizing NCX currents, which can lead to DADs124, 125, 130. In atria both receptor mediated IP3 production and expression level of IP3Rs are higher compared to ventricles118, 119. Further, there is a strong expression of angiotensin-II (Ang-II), a major inducer of IP3 production131, 132. There is also emerging evidence that in chronic AF Ca2+ release through IP3R increases significantly and that atrial tachycardia remodeling induces IP3R2 expression 133, 134

. Together, these suggest that IP3-related Ca2+ signaling can also play a prominent role in AF.

1.3 Inflammation and Atrial Fibrillation The exact mechanism underlying AF are not known. Studies have shown ischemia 135, atrial dilation59 and inflammation can increase susceptibility to AF 136, 137. The first evidence for association of atrial remodeling with inflammation came from a histological study by Frustaci et al. 138 in which atrial biopsies from 12 patient with lone AF were found to have a high prevalence of inflammatory infiltrates, myocytes necrosis, and fibrosis compared to biopsies from the control group. Since then several studies have tried to evaluate the role of inflammation in genesis and maintenance of AF. Furthermore, acute inflammation seems to play a prominent role in post-operative AF (the most common complication associated with cardiac surgery) as patients exhibit pronounced increase in their white blood cell counts139. Studies have shown that AF correlates with the level of inflammatory cytokines such as C-reactive protein, interlukin-6 (IL-6)136, 140, and of great interest to our study, TNF- α 61.

1.3.1

TNF-alpha

TNF-α is a potent proinflammatory cytokine. Proinflammatory cytokines are intercellular signaling polypeptides that besides increasing their own production increase synthesis of other

17

inflammatory mediators such as eicosanoids, platelet activating factor, and oxidative radical. These cytokines are produced during inflammatory processes from multiple sources and have a variety of targets and functions. TNF-α is mainly produced by lymphocytes and macrophages; it is also produced by resident cardiac macrophages, cardiomyocytes 141, and vascular smooth muscle 142. TNF-α is a type II protein that is initially produced in the cytosol as propeptide called pro-TNF-α (26KDa) and gets arranged as a homotrimer protein in the cell membrane (transmembrane TNF-α). The membrane-integrated form of TNF-α is cleaved by the matrix metalloproteinase, TNF-α converting enzyme (TACE) through the ‘shedding’ process to the 17 KDa soluble form of TNF-α143, 144. Both forms are capable of binding to TNF-α receptors type 1 (TNFR1) and type 2 (TNFR2), triggering alterations in cytosolic protein synthesis and activating different kinases and signaling pathways. Both receptor subtypes are present in cardiac cells 145. TNF-α synthesis is regulated at both transcriptional and translational levels. At transcriptional level, NF-κB plays an important role in TNF-α synthesis. Typically in unstimulated cells NF-κB exist in an inactive state in the cytosol sequestered by inhibitory kappa B (IκB) proteins. Phosphorylation of IκB by a multimeric complex IκB kinase leads to degradation of IκB. As a result the liberated NF-κB translocates to the nucleus and alters transcription of target genes, including TNF-α. Multiple stimuli such as oxidative stress, lipopolysaccharide (LPS), IL-6, IL-1, and TNF-α (through a positive feedback loop) can induce IκB kinase activity, as a result NF-κB activation146, 147. Therefore, NF-κB plays a prominent role in TNF-α production and in fact inhibition of NF-κB has been shown to completely block LPS-induced TNF-α production148. At the translational level, TACE is the prominent regulator of TNF-α production 143.

1.3.1.1

TNF-α and heart function

TNF-α has been implicated in the pathogenesis of many cardiovascular diseases such as

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myocardial infarction, chronic heart failure, atherosclerosis and sepsis- associated cardiac dysfunction 149-151. Studies have shown TNF-α to be a prominent determinant of myocardial apoptosis, hypertrophy, arrhythmias, contractile dysfunction (in particular decreased contractility), cardiac dilation and fibrosis 152-155. However, TNF-α actions in the heart are complex and like any other compensatory response in heart disease it causes both beneficial and detrimental effects. The kind of effect could depend on the type and duration of the disease initiating stimuli or the type of receptor, hence the pathway that gets activated by TNF-α. For instance, the dichotomous TNF-α effect in heart failure is due to the two subtypes of TNFRs, with TNFR1 aggravating ventricular remodeling, hypertrophy, NF-κB signaling, inflammation and apoptosis and TNFR2 ameliorating these consequences in HF 156. This suggests differential roles for the two TNFRs, therefore TNF-α also has beneficial effects (e.g. in innate and adaptive immune response), which may explain the disappointing results that were obtained in randomized clinical trials in HF patients treated with TNF-α antagonist. Unexpectedly rather than having beneficial effects, a time and dose related increase in death and hospitalization was obtained157. Of particular interest to our study is the effect of TNF-α on arrhythmia, in specific AF. Studies have shown that there is a link between inflammation, AF and the TNF-α that is released during immune response. Biopsies from right atrial tissue of patients with valvular disease with AF were found to have higher NF-κB activity and as a result higher concentrations of TNF-α and IL6 with more fibrosis compared to patients with valvular disease with sinus rhythm. Indicating the potential role of TNF-α in structural remodeling (fibrosis) required for the incidence and maintenance of AF 61. Mice with chronic overexpression of TNF-α (TNF-α 1.6) exhibit both ventricular and atrial arrhythmias which seems to be calcium dependent62, 158. Telemetry

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recordings from these mice showed both atrial flutter and atrial fibrillation that were not seen in the control mice. In ex vivo optical mapping studies of isolated atria from TNF-α 1.6 mice reentrant atrial arrhythmias were observed that were not seen in the atria from control mice. Further, atrial myocytes isolated from TNF-α 1.6 mice had ~3 times higher incidence of spontaneous SR Ca2+ release (both Ca2+ sparks and waves) compared to control myocytes that resulted in DADs, which if reached threshold can produce spontaneous AP that can trigger arrhythmia. Thus TNF-α has the potential to contribute arrhythmia through both fibrosis and altered Ca2+ handling61, 62. TNF-α along with IL-1 has also been implicated in increased susceptibility of ventricular myocytes to arrhythmias through increased Ca2+ leak from the SR 159

. As mentioned earlier, ectopic beats arising from the PV cardiomyocytes play a prominent

role in the development of paroxysmal AF. Abnormal automaticity, triggered activity and abnormal calcium handling may contribute to PV arrhythmogenesis 12, 123, 160. TNF-α has shown to induce abnormal calcium homeostasis in PV cardiomyocytes and as a result increase PV arrhythmogenicity 161.

1.4 Oxidative stress and Atrial Fibrillation 1.4.1

Oxidative stress

In addition to inflammation imbalanced cellular redox state, oxidative stress, seems to also play a role in development of AF 162. ROS are intermediates of the reduction of O2 to water and are composed of superoxide anion (O2-), hydrogen peroxide (H2O2) and hydroxyl radicals (OH-). ROS are derived from multiple sources such as the mitochondrial electron transport chain, xanthine oxidase, cytochrome P450-based enzymes and the nicotineamide adenine dinucleotide/ phosphate (NADH/ NADPH) oxidase 163. Small amounts of ROS usually serve as signaling molecules under physiological conditions, however an excess amount of ROS are potentially

20

harmful and stimulate pathologic cellular processes, such as altered cardiac ion homeostasis and structural remodeling 164, 165. To protect cellular function from ROS, cells have developed various enzymatic (e.g., superoxide dismutase, catalase, and glutathione peroxidase) and nonenzymatic redox-defense system (e.g., glutathione, antioxidant vitamins such as vitamins E and C) that detoxify ROS 163. Oxidative stress has been implicated in a number of cardiovascular diseases such as ischemia-reperfusion injury 166, hypertrophy 167, heart failure 168, ventricular fibrillation 169, and coronary artery disease 170. Elevated oxidative stress has also shown to be related to pathogenesis of arrhythmias 169, including AF 162. Studies have shown elevated oxidative stress in tissue samples obtained from AF patients and that antioxidant treatments are beneficial in atrial tachycardia remodeling and post-operative AF 171, 172. ROS sources can be divided in to two categories. One includes sources that produce ROS purposely as part of a defense mechanism or signal transduction. The other, are sources that unintentionally as part of the biological process produce ROS as byproducts or waste 173. An example of the first category is the NADPH oxidase complex that is a significant source of cardiac ROS. The NADPH oxidase of the cardiovascular system is a membrane bound enzyme that catalyzes the production of superoxide from oxygen and NADPH (as the electron donor) according to the following reaction 174: NADPH + 2O 2 → NADP + + H + + 2O 2− This enzyme is found in professional phagocytes such as neutrophils 175, eosinophils, monocytes and macrophages 176 and non-phagocytic cells including endothelium, kidney, spleen 177, vascular smooth muscle cells 167, 178, and cardiomyocytes 167. The structure of the NADPH oxidase is complex and consist of a membrane-associated heterodimeric flavocytochrome b558

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comprised of the highly glycosylated gp91phox subunit and a smaller p22phox subunit. The enzyme also has three regulatory cytosolic subunit p47phox, p67phox, p40phox and small molecular weight G protein rac1 or rac2. The p22phox subunit is present in almost all cell types but this is not the case for the gp91phox subunit. Homologous isoforms of gp91phox subunit encoded by different genes called Nox1-5 have been identified in different cell types, with gp91phox being recognized as Nox2 179. Cardiomyocytes are known to express both Nox2 and Nox4 180, 181. In order for the NADPH oxidase to become active, several of the cytosolic regulatory subunits are required to translocate to the membrane subunits of the enzyme to assemble the complete oxidase 179. The NADPH oxidase is considered as a significant source of cardiac ROS and it is activated by a variety of stimuli such as G-protein coupled receptor agonist (e.g., angiotensin II and endothelin1), growth factors, cytokines (e.g., TNF- α, IL-1), mechanical forces (e.g., shear stress), ischemia and hypoxia 179. Another important and major source of ROS is the mitochondria, which are the second category of ROS sources. The major biological function of the mitochondria is to generate ATP via the process of oxidative phosphorylation. Briefly the metabolic substrates of the heart e.g., glucose and fatty acids are oxidized to acetyl-CoA, which leads to the production of the reducing equivalents NADH and FADH2 during Krebs cycle that subsequently enter the mitochondrial electron transport chain (ETC) to generate ATP. There are four complex catalyzes in the ETC which contain a number of oxidation-reduction cofactors. Electrons for the ETC are provided either by NADH at complex I or by succinate at complex II. Electrons flow from NADHubiquinone oxidoreductase (complex I) or succinate dehydrogenase (complex II) to ubiquinone (coenzyme Q). Ubiquinone (Q) is a mobile lipid soluble carrier that mediates electron transfer from complexes I and II to cytochrome bc1 complex (complex III). Before Q becomes fully

22

reduced to ubiquinol (QH2) it passes through a ubisemiquinone anion (.Q- ) intermediate that plays an important role in the ETC. Reduced ubiquinone, QH2, transfer electrons to cytochrome c, through the Q cycle. The Q cycle of complex III is a two-step oxidation process. During the first step, QH2 donates one electron to cytochrome c1 of complex III, forming .Q- and releasing two protons to the intermembrane space. However during the subsequent step, a second QH2 again transfers one electron to produce another reduced cytochrome c1, while the second electron is used to reduce the .Q- that was generated during the first step, through cytochrome b. This step also releases two protons to the inner membrane space but also uses two protons from the matrix to produce QH2. Therefore during Q cycle two QH2 molecule are oxidized and one QH2 molecule is formed. Electrons migrate from the mobile cytochrome c electron carrier of complex III to cytochrome c oxidase (complex IV) and ultimately to molecular oxygen leading to production of water. At complexes I, III and IV protons are transferred from the matrix to the intermembrane space, establishing a proton motive force composed of both pH gradient and electrical potential across the inner membrane. The mitochondrial ATP synthase uses this proton motive force to transfer protons into the matrix and as a result generate ATP182-184. Even though the process of oxidative phosphorylation is a very efficient process but there are always some electrons that leak during the transfer process and generate superoxide, making mitochondria a major site of ROS production. Electron leak is known to occur at three main sites in the electron transport chain: complex I, and the matrix and inter membrane side of complex III 184

. The extent of electron leakage through each of these sites is regulated by different means.

Superoxide production at complex I is mainly favored when the NADH/NAD+ couple is almost entirely reduced, in other words when there is a high NADH/NAD+ ratio in the mitochondria 185. Hence inhibition of the ETC by damage, ischemia, loss of cytochrome c, or by rotenone, which

23

is a blocker of complex I, will increase the NADH/NAD+ ratio and lead to superoxide formation185, 186. Complex I can also produce superoxide by reverse electron flow (RET), which occurs when succinate is available as a substrate. RET also occurs when the mitochondria is not making ATP and hence there is a high proton motive force and a reduced coenzyme Q pool available that forces electrons to flow back from the QH2 molecule to complex I and reduce NAD+ to NADH. This increases the NADH/NAD+ ratio that leads to enhanced superoxide production by complex I 187, 188 . Hence, complex I superoxide production is favored by low electron flow and reduced electron transport chain. However not all mitochondrial inhibitors increase ROS production under reducing conditions. For the Q cycle to take place at complex III, supply of substrate to drive reduction of Q to QH2 and continuous availability of downstream electron carriers is required. Therefore, blocking complex III with the inhibitor antimycin increases superoxide production by this complex. This is because antimycin binds to cytochrome b of complex III increasing the steady state concentration of ubisemiquinone increasing chances of electron leakage to oxygen and hence formation of superoxide 189. However, unlike complex I inhibition of complex III at points that reduces electron flow into the Q-cycle by rotenone decreases superoxide production. Furthermore, inhibition of electron acceptors such as the Rieske Fe-S protein (which is responsible for transferring electrons from QH2 to cytochrome c1 during Q cycle) with myxothiazol or inhibition of points downstream of complex III by cyanide or cytochrome c depletion also decreases superoxide production182, 189. Therefore ROS production by each of these complexes require different and in fact opposite conditions. The harmful effects of ROS produced in mitochondria are to a large extent avoided by a variety of antioxidant systems. The superoxide produced in the matrix gets converted to H2O2 by a specific superoxide dismutase (SOD) found in the matrix of mitochondria called manganese

24

SOD (MnSOD). The resultant H2O2 can lead to production of OH- radicals that are highly reactive in the presence of transition metals 190. Hence, glutathione peroxidase a major antioxidant in the mitochondria detoxifies H2O2 to water and oxidized glutathione in the matrix, which gets reduced back to glutathione by the reducing power of NADPH 190. Multiple pathways detoxify the superoxide produced in the inner membrane space. An SOD containing copper and zinc (Cu, Zn-SOD) instead of magnesium converts superoxide to H2O2 191. The inner membrane space also contains cytochrome c, which can get reduced by superoxide, and generate oxygen in the inner membrane space and transfer the electron back to the ETC and contributes to energy production192. However, some of the superoxide in the inner membrane space may also escape into the cytoplasm through voltage dependent anion channel located in the outer membrane, making mitochondria a source of cytoplasmic superoxide193. Even though under normal conditions there is a balance between antioxidants and the ROS being produced, but under some pathological conditions the antioxidant defense system becomes inadequate and ineffective leading to oxidative stress. This can have detrimental effects and eventually lead to apoptosis and cell death 184.

1.4.2

The association between TNF-α, oxidative stress and heart function

Both ROS and TNF-α related pathways are known to interact, hence effecting cardiac function. Oxidative stress stimulates inflammation, activating downstream targets such as NF-κB and MAP kinase pathways194-196. Activation of these factors induces a variety of cytokines such as TNF-α 146. On the other hand pro-inflammatory cytokine TNF-α activates oxidative stress pathways and increases ROS production197. This leads to a “vicious circle” in which oxidative stress intensifies proinflammatory responses that in turn stimulates ROS production creating a perpetuating cycle163.

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TNF-α upregulates transcription of the NADPH oxidase enzyme components such as Nox2, p47phox, p67phox and p22phox, which leads to elevated superoxide production198-200. TNF-α induced ROS regulates many important cellular events such as apoptosis and necrosis 194, 197 201. The NADPH oxidase-derived ROS is capable of modulating various signaling pathways and redox – sensitive proteins. Reports have shown NADPH oxidase to be involved in pathophysiological processes such as endothelial dysfunction, inflammation, cardiomyocyte hypertrophy, apoptosis, cardiac remodeling and in particular AF 179, 202. In a swine model of AF, superoxide production was increased by elevated NADPH oxidase and xanthine oxidase activity after one week of rapid atrial pacing. Treatment with the NADPH oxidase inhibitor, apocynin, decreased superoxide levels in the left atrial appendage by 91%. This increased in NADPH oxidase activity was accompanied by 6.9 fold increase in rac1 protein subunit 203. A significant elevation in superoxide production due to increased NAPDH oxidase activity was also observed in a group of 170 patients with postoperative AF 204. Furthermore, Ang II stimulates superoxide production by NADPH oxidase. Patients with AF are known to have increased Ang II receptors. Since Ang II receptor blockers are effective in AF treatments, it is speculated that this beneficial effect might be partly due to NADPH oxidase inhibition167, 205, 206. All these suggest that NADPH oxidases may play an important role in AF and could be a potential target for treatment of AF. TNF-α also increases mitochondrial ROS production 207. There is growing number of studies that have shown mitochondria play an important role in genesis of arrhythmias 208, 209 210. Increased mitochondrial superoxide production seems to be associated with AF 204. Mitochondria can cause arrhythmia by influencing the sarcolemmal ATP- sensitive K+ channel (sarcKATP) 211. These channels are heteromultimers that are regulated by the cellular ATP/ADP ratios where intracellular ATP inhibits and ADP, Pi, Mg activate these channels 212. Under conditions of

26

oxidative stress these channels become activated and produce inwardly rectifying background ATP-sensitive K+ current (IK,ATP) 213. The functions of sarcKATP channels are important during oxidative stress. The activity of these channels protects against the metabolic insult that ischemia-reperfusion injury imposes, by reducing Ca2+ transient preventing Ca2+ overload and the associated energy depletion (since Ca2+ handling is energy consuming) 214. However, this leads to increased potassium current through these channels. If enough channels open they can significantly reduce the action potential duration and refractoriness leading to cell inexcitability, 211 210

. Hence they can play a role in development or stabilization of arrhythmias such as AF.

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1.5

Synopsis

AF is believed to begin with isolated paroxysmal events which through causing electrophysiological changes in the atria can lead to sustained chronic AF (i.e. AF-begets AF) 15

.While the mechanism underlying paroxysmal AF are poorly understood, AF induction

generally requires a vulnerable substrate subject to electrical events that promote re-entry and/or rotor formation (i.e. kindling). Appropriately timed spontaneous electrical events, such as those that occur with triggered electrical activity associated with EADs and DADs are of particular relevance for AF induction 55. Previous studies have shown that chronic overexpression of TNFα in mice increases spontaneous Ca2+ release (i.e. DADs) and increase susceptibility to AF62. It is however unclear whether these changes in TNF-α over-expression mice result from acute effects of TNF-α or chronic remodeling. Studies have shown that TNF- α alters calcium homeostasis and increases SR Ca2+ leak129, 159, 161.TNF- α has also shown to induce oxidative stress through both mitochondria and increased NADPH oxidase activity207. On the other hand, ROS are known to affect RyRs function and stimulate RyRs channel activity129. All these suggest that a strong association exists between inflammation, oxidative stress, calcium handling and AF. Therefore, we hypothesize that: TNF- α alters Ca2+ homeostasis in atrial myocytes through elevated oxidative stress via an increase in sarcoplasmic reticulum spontaneous Ca2+ release (Ca2+ sparks), ultimately leading to delayed afterdepolarizations (DADs), premature atrial beats and triggered arrhythmias.

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Technical Contribution and Acknowledgement I would like to acknowledge the following people for contributing to my MSc thesis project: 1. Dr. Roozbeh Aschar-Sobbi

-

Performed the recordings from isolated atrial tissue with TNF-α and the ICa, L recording from isolated atrial myocytes. Provided instruction for atrial cell isolation. Worked on implementing the highly light sensitive 2000 fps confocal system and the Yokagawa spinning disk and the Cascade camera. Also, was a great source of intellectual and technical support.

2. Wallace Yang

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Increased signal-to-noise ratios to levels that signal can be processed and visualized. Implemented the highly-light-sensitive 2000 fps confocal system. Integration the system of the Yokagawa spinning disk, the Cascade camera, the Hamamatsu image intensifier and the associated electronic implementation, the lens relay and housing design, and emission filter optimization. Developed tools to process and visualize data wrote macros to extend Spark Master to 2 dimensional analysis from 1 dimension, developed 2 dimensional program in java, analogous to Spark Master, to analyze and visualize in 2 dimensions

I was responsible for the principle planning of all studies and the primary data collection of: Spontaneous Ca2+ sparks measurements in isolated atrial myocytes, ROS measurements as well as analysis and interpretation of all experimental data. I also helped in integration of the confocal system and the Yokagawa spinning disk, the Cascade camera, the Hamamatsu image intensifier and the associated electronic implementation, the lens relay and housing design, and emission filter optimization.

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Chapter 2 Materials and Methods

30

2

Materials and Methods

2.1 Experimental Animals Seven-ten week old male CD-1 mice (Charles River Laboratories, Wilmington, MA, USA) were used for experiments. All mice were housed in standard cages with proper ventilation in temperature- and humidity controlled rooms with 12- hour light-dark cycles in the Department of Comparative Medicine animal facility at the University of Toronto. All experimental protocols confirmed the standards of the Canadian Council on Animal Care.

2.2 Atrial Isolation and Optical Imaging For atrial preparations, seven-ten week old male CD-1 mice were given an IP injection of 0.2 ml heparin (1000 IU/ml) to prevent blood clotting. Mice were anesthetized using isoflurane and sacrificed by cervical dislocation. The thorax was opened by midsternal incision and the heart was placed into warm Tyrode solution consisting of (in mmol/L): 140 NaCl, 5.4 KCl, 1.2 KH2PO4, 5 HEPES, 5.5 glucose, 1 MgCl2, 1.8 CaCl2 and 1000u/ml heparin. The lungs, thymus, fat and ventricular tissue were dissected away, leaving a preparation with interconnected left and right atria (Figure 2.1). The tissue was then pinned to the bottom of a Sylgaurd-coated petri dish (Figure 2.1), after which the tissue was stained with 10 mM di-4AminoNaphthylEthenylPyridinium (Di-4-ANEPPS) in warm Tyrode’s solution for 10 minutes. After the incubation period, the tissue was superfused with Krebs solution with the following composition (in mmol/L): 118 NaCl, 4.2 KCl, 1.2 KH2PO4, 1.5 CaCl2, 1.2 MgSO4, 23 NaHCO3, 20 glucose, 2 Na-pyruvate, continuously bubbled with carbogen (95% O2-5% CO2). The tissue was illuminated using a mercury light source filtered through a 543 ± 22 nm band-pass filter. The fluorescence light emitted from the preparation was filtered through a 645 ± 75 bandpass filter and the change in fluorescence in the tissue was recorded using a high speed CCD-camera

31

(Cascade: 128+; Photometrics, AZ, USA). AF propensity was assessed by rapid stimulation protocol of the atria (consisting of 15 pulses with15 ms duration followed by 3 extra stimuli with coupling down to 15ms) applied through a platinum wire attached to the surface of the right or left atrial appendage. Simultaneous electrocardiogram (ECG) recordings were also taken to aid in the identification of AF. AF was measured in atria pretreated with TNF-α (100ng/ml) for 30 minutes and in the presence of carbachol (1μM) or in the presence of carbachol alone. AF was defined as appearance of reentrant rotors on optical images that lasted longer than 1 sec after the end of the stimulation protocol.

Figure 2.1 Mouse Atrial Isolation

2.3 Isolation of Atrial Myocytes Male CD-1 (7-10 weeks) mice were administrated 0.2 ml IP injection of heparin (1000 IU/ml) to prevent blood clotting and were given 5 minutes for it to be absorbed. They were then sacrificed after 5 minutes under isoflurane via cervical dislocation. The thorax was opened by midsternal incision and the heart was rapidly removed and transferred to a bath of modified horizontal Langendorff apparatus in cold Ca2+-free Tyrode solution containing (in mmol/L) 130 NaCl, 5.4 KCl, 0.4 NaH2PO4, 0.5 MgCl2, 22 D-glucose, 25 HEPES with pH adjusted to 7.4 with NaOH, where aorta was cannulated on a blunted 20-gage needle attached to a bubble trap and waterjacket heated perfusion system. Once the heart was mounted it was retrogradely perfused on the

32

horizontal Langendorff apparatus with Ca2+-free Tyrode solution. The solution was bubbled with 100% O2. The temperature was maintained at 37°C. The hearts were perfused at a constant flow rate of 3.5 ml/min, for 10 minutes with the Ca2+-free solution followed with the same Ca2+ solution containing 1 mg/ml type II collagenase (225 U/ml, Worthington Biochemical Corporation, Lakewood, NJ, USA) and 0.3U/ml of elastase for approximately 7-8 minutes. Once the digestion was complete the left atrial appendage was removed and gently dissociated in a high K+ solution (modified KB) containing (in mmol/L) 100 potassium glutamate, 10 potassium aspartate, 25 KCl, 10 KH2PO4, 2MgSO4, 20 taurine, 5 creatine, 0.5 EGTA, 20 D-glucose, 5 HEPES, and 0.1% BSA, with pH adjusted to 7.4 with KOH. Atrial myoctes were stored in KB solution at room temperature (22-25°C) until used in calcium imaging studies, 70-80% of the cells were Ca2+ tolerant.

2.4 Calcium Sparks Measurements Using Confocal Microscopy Calcium sparks were measured with Yokogawa fluorescence laser scanning confocal microscopy. Ca2+ sparks in the cells were recorded using a high speed EMCCD-camera (Cascade: 128+; Photometrics, AZ, USA) attached to an image intensifier (V6886U-05-P46, Hamamatsu; Ikawa city, Japan) with custom relay housing interfaced with ImagePro Plus 5.1 software resulting in an image resolution of 16-bit. Since Ca2+ sparks are quick events, a fast acquisition camera was required, hence cascade 128+, which captures 510 frames/sec at full resolution was used. However, due to the fast speed the signal that was captured was reduced. In order to amplify the signal intensity an image intensifier was attached to the camera and a lens relay was positioned between the image intensifier and the camera to pass the image from the image intensifier to the camera. The image intensifier was powered by a high voltage power

33

supply (C6706-20, Hamamatsu, Ikawa city, Japan). 100 x oil immersion objective was used to visualize the sparks. Isolated atrial myocytes were incubated with 20ng/ml recombinant TNF- α (mouse) (SigmaAldrich, Oakville, Ontario, Canada) in the calcium free Tyrodes for one hour at room temperature and for the controls the same was done to take in to account the effect of time on the cells. The cells were then loaded with 1.1µM of the calcium indicator Fluo-3 AM (AnaSpec; CA, USA) (for 15 minutes at room temperature). Cells were continuously superfused with Tyrode solution containing (in mmol/L) 140 NaCl, 5.4 KCl, 1 MgCl2, and 0.5 CaCl2, 10 D-glucose and10 HEPES, with pH adjusted to 7.4 with NaOH. Fifteen minutes was allowed for deesterification of the dye. Fluo-3 AM was excited at 488 nm with the laser and the fluorescence was measured at 526 nm wavelengths. Atrial myocytes were electrically paced using two field platinum electrodes (1Hz, 10-12 V, 2ms duration of each pulse) attached to a Grass S44 stimulator (Grass instrument Co, MA, USA). Occurrence of Ca2+ sparks was measured during the resting period of each cell after the stimulation was stopped. Images were acquired at 2ms intervals. In another set of experiments the role of NADPH oxidase in production of Ca2+ sparks was assessed using the NADPH oxidase inhibitor, apocynin (Sigma-Aldrich Canada Inc.). Both TNFα and control cells were pretreated with apocynin (100μM) for 15 minutes. Since apocynin effects are reversible, apocynin was added to the superfusion Tyrode solution. In order to determine the role of ROS generated by mitochondria in the production of calcium sparks both control and TNF-α treated cells were pretreated with the mitochondrial targeted superoxide scavenger, MitoTEMPO (25nM) (Enzo Life Sciences, NY, USA), for 15 minutes.

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MitoTEMPO was also added to the superfusion Tyrode solution. Ca2+ sparks were recorded and measured as described above.

2.4.1

Signal processing and data analysis

The recordings were processed and analyzed using imageJ (NIH, USA,http://rsbweb.nih.gov/ij/). Each recording was 4000 frames and the occurrence of Ca2+ sparks was measured in the last 1000 frames, i.e. the resting period (Figure 2.2A) The automated measurement of Ca2+ sparks was done using a plugin for ImageJ called “SparkMaster” 215. Manual analysis of sparks is time consuming, prone to errors and also investigator bias, therefore SparkMaster, which is a program for automated Ca2+ spark analysis has been widely used by investigators studying elementary Ca2+ release events. SparkMaster is a threshold-based algorithm that identifies Ca2+ sparks based on their deviation from the background noise 215. SparkMaster is capable of analyzing images captured with the confocal line-scan microscopy, but since our recordings were captured in two dimensional (2D) mode, we converted the 2D image of the cell into linescan images (2ms/scan; pixel size 0.39 μm) (Figure 2.2B) using a macro derived from the pseudo-linescan function of ImageJ (courtesy of Wallace Yang). Ca2+ spark frequencies are expressed as number of observed sparks per 100μm of the linescan images (Sparks * S-1 * (100 μm)-1 ). In order to visualize Ca2+ sparks and Ca2+ wave propagation in the whole cell (2D), a modified algorithm of SparkMaster program was used (courtesy of Wallace Yang).

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A)

B) x t

C)

Figure 2.2: Confocal images signal processing. A) A representative trace of Ca2+ transient obtained from a control cell. The occurrence of Ca2+ spark was measured in the last 1000 frames of the recording, i.e. the last 2 seconds indicated by red line. B) 2D image obtained from a cell. The 2D image was converted into line scan images. The yellow line was moved throughout the entire cell and C) each line was analyzed for occurrence of Ca2+ spark over the period of last 1000 frames.

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2.5 Reactive Oxygen Species Measurements ROS were measured in isolated atrial myocytes using the cell permeable 5-(and-6)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA; Invitrogen Canada Inc.). H2DCFDA once inside the cells gets hydrolyzed by intracellular esterases to the nonfluorescent compound dichlorodihydrofluorescein (H2DCF). When H2DCF comes in contact with ROS it oxidizes to the fluorescent compound dichlorofluorescein (DCF) with excitation of 488nm and emission of >525nm. Images were acquired using Yokogawa fluorescence laser scanning confocal microscope. Images were captured using QuantEM: 512SC camera (Photometrics, AZ, USA) with 16 bit resolution interfaced with MetaMorph. Isolated TNF- α treated (20ng/ml for one hour) and control atrial myocytes were incubated for 15 minutes with CM-H2DCFDA (3μM) at room temperature and 30 minutes was allowed for deesterification of the dye, while superfused with Tyrode solution. Cells were then filled stimulated (1Hz 10-12 V, 2ms duration of each pulse) to ensure viability. Since the CM- H2DCFDA dye itself is susceptible to photo-oxidation the laser was set at the lowest intensity 216 .For each cell four recording were captured in the following order: recording #1, laser was set at minimum intensity (dye is a mixture of H2DCF+DCF), recording # 2 As mentioned above the CM-H2DCFDA itself is susceptible to photo-oxidation, hence we used this characteristic to assess the total amount of dye present in the cell. In order to achieve this, we increased the laser intensity to maximum level until the dye reached saturation and there was no further increase in intensity. Increasing the laser to maximum causes complete conversion of H2DCF to DCF, which gives an estimation of the total amount of dye present in the cell , recording # 3, laser intensity was reduced down to minimum level and the recording was captured immediately after recording #2 and finally recording #4, which was taken 5 minutes after recording number#2, during which the total dye was fully activated. This ensures that when recordings #1(H2DCF+ DCF) and number #4(DCF)

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are compared they are both at the same energy level (same laser intensity) In order to calculate the amount of ROS in each cell the intensity ratio of recording # 1 to recording # 4 was calculated. The images were background corrected and analyzed using ImageJ. H2O2 was used as positive control.

2.6 L-Type Calcium Current Measurements Using Whole Cell Patch-Clamp Technique For recording left atrial appendage ICa,L, cells were allowed to settle on bottom of recording chamber for 5 minutes and were continuously superfused with external buffer containing (at rate of 2ml/min): 140 mM NaCl, 4 mM CsCl, 1.2 mM CaCl2, 1mM MgCl2, 10 mM HEPES, 10 mM Glucose, pH adjusted to 7.4. Membrane currents were recorded at room temperature (22-23oC) using whole cell patch-clamp technique using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, USA) and digitized using Digidata 1322A and pClamp 8 software (Molecular Devices, Sunnyvale, USA). Patch pipettes were pulled from borosilicate glass (with filament, 1.5 mm OD, 0.75 mm ID, Sutter Instruments Company). These pipette had a resistance of 4-6 MΩ. The pipette solution contained 125 mM CsCl, 20 mM TEA-Cl, 10 mM EGTA, 10 mM HEPES, 4 mM Mg-ATP, pH adjusted to 7.3 with CsOH. The membrane potential was held at -75 mV after forming whole cell configuration. Sodium currents were inactivated by applying a prepulse to 40 mV for 50 ms before membrane potentials were stepped between -50 mV to +50 mV for a duration of 500 ms to elicit Ca2+ currents.

2.7 Statistical Analysis Summary data are expressed as mean ± S.E.M (the standard error of the mean). Significance and difference of multiple groups was determined using unpaired student t-test or two-way ANOVA. Differences at