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Sialic acid modulation of cardiac voltage-gated sodium channel gating throughout the developing myocardium

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Title:
Sialic acid modulation of cardiac voltage-gated sodium channel gating throughout the developing myocardium
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Stocker, Patrick J
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University of South Florida
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Ion channels
Cardiomyocytes
N-glycosylation
Development
Neuraminidase
Dissertations, Academic -- Physiology and Biophysics -- Doctoral -- USF
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Abstract:
ABSTRACT: The proper orchestration of voltage-gated ion channel gating is vital to maintaining normal heart rhythms throughout an animal's lifespan. Voltage-gated sodium channels, Nav, are responsible for the initiation of the cardiac action potential, which leads to cardiac systole. Comparison of neonatal ventricular and atrial myocyte Nav gating with adult indicated that the neonatal ventricular Nav gated following a ~10 mV greater depolarization than did atrial or adult ventricular Nav. In this study I questioned whether development- and/or chamber-dependent changes in Nav-associated functional sialic acids could account for these differences. When desialylated with neuraminidase, all gating characteristics for the lower voltage activated atrial and adult ventricular Nav shifted significantly to more depolarized potentials. However, desialylation of the higher voltage activated neonatal ventricular Nav had no effect on channel gating. Furthermore, channels were stripped of^ their N-glycosylation via PNGase-F in an attempt to separate the potential effects of the remaining glycosylation structure on Nav gating. Following treatment, neonatal ventricular Nav gating remained unchanged while atrial and adult ventricular Nav gating again shifted to depolarized potentials nearly identical to those of the neonatal ventricular channel. Immunoblot analyses indicated that atrial and adult ventricular Nav a subunits are more heavily sialylated than the neonatal ventricular a subunit, with approximately 15 more sialic acid residues. The data indicate that differential sialylation of myocyte Nav a subunits is responsible for much of the developmental and chamber-specific remodeling of Nav gating observed here. In addition, the Nav1.5 a subunit can associate with b subunits, also believed to be sialylated. The potential for functional b1 trans sialic acids to further modulate Nav1.5 gating was tested via co-transfection of b1 with the Nav1.5 a subunit into the Pro5 /Lec2 mammalian expression system. Co-transfection revealed that the additional b1 trans sialic acids caused a hyperpolarizing shift in all tested gating parameters. When transfected into neonatal ventricular myocytes, b1 expression revealed no effect, implying that b1 expression alone is not responsible. Together, the myocyte and expression system studies describe a novel mechanism by which Nav gating, and subsequently cardiac excitability, are modulated by the regulated change in channel-associated functional sialic acids.
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Dissertation (Ph.D.)--University of South Florida, 2005.
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by Patrick J. Stocker.
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Sialic Acid Modulation of Cardiac Voltage-Gated Sodium Channel Gating Throughout the Developing Myocardium by Patrick J. Stocker A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Physiology and Biophysics College of Medicine University of South Florida Major Professor: Eric Bennett, Ph.D. Joel Price, Ph.D. Craig A. Doupnik, Ph.D. Daniel Yip, Ph.D. Jahanshah Amin, Ph.D. Date of Approval: 09 / 26 / 2005 Keywords: ion channels, cardiomyocytes, N -glycosylation, development, neuraminidase Copyright 2005, Patrick J. Stocker

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ACKNOWLEDGEMENTS Where does one begin to recognize the contributions of others that helped make my doctoral experience a success? First and foremost I must thank all my family and friends for their countless support, you guys are great. To Dr. Bennett, you were there before I was even accepted as a student and have remained with me through to the end. Without your continued financial and personal investment who knows where I would be today. To Jeanie Harper, we started in Dr. Bennett’s lab together and have become excellent friends, for that I am grateful. To my committee, thank you for volunteering your time and guidance, you were always available when I needed you. To Kathy Zahn and your colleagues, your hard work is much appreciated by myself and all the other graduate students, keep up the good work. To Barbara and Joyce, you could always get a smile out of me. Finally, to Alisha Vogt and family, thanks for your love and friendship through this often stressful experience. To everyone else, thanks and stay strong.

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i TABLE OF CONTENTS LIST OF FIGURES iv LIST OF TABLES vii ABSTRACT viii INTRODUCTION 1 Ion transport 1 Voltage-gated Na+ channels 14 Ion channel glycosylation, sialic acid, and Nav 19 The surface potential theory 23 The cardiac action potential and Nav 24 Cardiac Nav gating in adult and neonatal cardiomyocytes 28 interaction with 29 Nav channel disorders 33 MATERIALS AND METHODS 36 Construction of and cDNAs 36 Pro5, Lec2 cell culture 36 Pro5, Lec2 cell transfection 37 Pro5, Lec2 cell homogenization 37 Neonatal and adult rat cardiomyocyte isolation and culture 38 Neonatal rat ventricular myocyte transfection 39

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ii Cardiac tissue homogenization 39 Electrophysiology 39 Measuring Pro5, Lec2 Na+ currents 40 Measuring whole myocyte Na+ currents 41 Pulse protocols for measuring whole cell Na+ currents 42 Desialylation of rat cardiomyocyte sodium channels 44 Deglycosylation of rat cardiomyocyte sodium channels 44 Antibody production and purification 44 Immunoblots 45 Data analysis 46 RESULTS 47 The ventricular Nav of the newborn gates at more depolarized potentials 47 Gating of three of the four Nav types are sensitive to desialylation – only 50 the neonatal VCM Nav are insensitive Sialic acids are responsible for faster inactivation kinetics only neonatal 61 ventricular Nav inactivation shows no sialic acid sensitivity Sialic acids act to slow the rate of recovery from fast inactivation for all 76 but the neonatal ventricular Nav PNGase-F treatment, removing N -linked glycosylation, results in no 76 significant difference in gating among all four tested Nav The atrial and adult ventricular Nav subunit is apparently more heavily 88 sialylated than the neonatal ventricular Nav Increased functional sialylation of atrial and adult ventricular Nav account 92 for most of the hyperpolarizing shifts in channel gating Nav1.5 expressed with 1 in Pro5 versus Lec2 indicates 1 action on 95 Nav1.5 gating is sialic acid dependent

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iii SDS-PAGE analysis indicates good expression and glycosylation of 1 101 using the Pro5/Lec2 mammalian expression system Transfection with 1 fails to modulate neonatal ventricular Nav gating 101 SDS-PAGE analysis indicates adult ventricular expression of 1 104 exceeds that of atria and neonatal ventricle DISCUSSION 109 Differential sialylation of the subunit is a major contributor to 109 developmental shifts in ventricular Nav gating not observed in atria Chronic versus acute channel regulation 112 Control of Nav gating by differential sialylation appears to be intra and 114 inter tissue specific Acute regulation of Nav gating via 1 expression 116 Potential physiological impact of increased or decreased sialylation and 117 future experiments Summary 122 REFERENCES 127 BIBLIOGRAPHY 139 ABOUT THE AUTHOR End Page

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iv LIST OF FIGURES Figure 1 Ion exchange transport proteins 2 Figure 2 Active transport 4 Figure 3 Secondary active transport of Na+ and glucose in the small 7 Intestine Figure 4 Neuromuscular junction 9 Figure 5 Schematic representation of a voltage-gated Na+ channel 12 Figure 6 A chart outlining the known voltage-gated Na+ channel 15 subunits along with their corresponding anatomical locations Figure 7 Nav1.5 voltage-gated ion channel 16 Figure 8 The three different types of known N -linked glycosylation 21 structures Figure 9 The cardiac action potential and current flow 25 Figure 10 Voltage-gated sodium channel 1 auxiliary subunit 31 Figure 11 Whole cell recordings of isolated adult and neonatal atrial and 48 ventricular myocytes determining voltage-dependent Na v gating characteristics Figure 12 Desialylation of neonatal VCM and neonatal ACM Nav causes 51 a depolarizing shift in ACM steady-state Nav activation neonatal ventricular channels are unaffected Figure 13 Desialylation of adult VCM and adult ACM Nav causes a 53 depolarizing shift in steady-state Nav activation both VCM and ACM channels are similarly affected

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v Figure 14 Desialylation of neonatal VCM and adult VCM Nav causes a 55 depolarizing shift in adult steady-state Nav activation neonatal ventricular channels are unaffected Figure 15 Desialylation of neonatal ACM and adult ACM Nav causes a 57 depolarizing shift in steady-state Nav activation both neonatal and adult ACM channels are similarly affected Figure 16 Desialylation shifts steady-state inactivation curves for neonatal 59 atrial Nav but has no effect on neonatal ventricular Nav inactivation Figure 17 Desialylation similarly shifts steady-state inactivation curves for 62 both adult ventricular and adult atrial Nav Figure 18 Desialylation shifts steady-state inactivation curves for adult 64 ventricular Nav but has no effect on neonatal ventricular Nav inactivation Figure 19 Desialylation similarly shifts steady-state inactivation curves for 66 both neonatal and adult atrial Nav Figure 20 Sialic acid increases the rate of fast inactivation for neonatal 68 atrial Nav but has little effect on neonatal ventricular Nav inactivation rates Figure 21 Sialic acid causes an increase in the rate of fast inactivation for 70 both adult ventricular and adult atrial Nav Figure 22 Sialic acid increases the rate of fast inactivation for adult 72 ventricular Nav but has little effect on neonatal ventricular Nav inactivation rates Figure 23 Sialic acid causes an increase in the rate of fast inactivation for 74 both neonatal atrial and adult atrial Nav Figure 24 Recovery from fast inactivation for neonatal atrial Nav is faster 77 following desialylation neonatal ventricular recovery rate is unaffected by sialic acids Figure 25 Recovery from fast inactivation for adult ventricular and adult 79 atrial Nav are both faster following desialylation

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vi Figure 26 Recovery from fast inactivation for adult ventricular Nav is faster 81 following desialylation neonatal ventricular recovery rate is unaffected by sialic acids Figure 27 Recovery from fast inactivation for neonatal atrial and adult atrial 83 Nav are both faster following desialylation Figure 28 Deglycosylation of atrial and adult ventricular Nav results in 86 gating characteristics nearly identically to those of neonatal ventricular Nav Figure 29 Gel shift analyses indicate that channel sialylation increases 89 throughout the developing ventricle and is greater in the atria versus ventricle of the newborn Figure 30 Functional channel sialic acids can account for much of the 93 developmental and chamber-specific changes in Nav gating parameters Figure 31 1 sialic acids cause a hyperpolarizing shift in Nav1.5 activation 97 and inactivation Figure 32 1 sialic acids increase the rate of fast inactivation while 99 decreasing the rate of recovery from fast inactivation Figure 33 Immunoblot of 1 transfected into Lec2 and Pro5 cells 102 Figure 34 Preliminary results indicate that 1 transfection has no 105 significant impact on neonatal VCM Nav activation or inactivation, potentially due to a lack of subunit sialylation Figure 35 Immunoblot of 1 in neonatal and adult ventricle and atria 107

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vii LIST OF TABLES Table 1 Gating parameters for adult and neonatal cardiomyocyte 124 Nav under conditions of full and reduced sialylation / glycosylation Table 2 Gel shift analyses for neonatal and adult cardiomyocyte sodium 125 channels under conditions of full and reduced glycosylation / sialylation Table 3 Gating parameters for Nav1.5 transfected with or without 1 in the 126 fully sialylating Pro5 or sialylation deficient Lec2 mammalian cell lines

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viii Sialic Acid Modulation Of Cardiac Voltage-Gated Sodium Channel Gating Throughout The Developing Myocardium Patrick J. Stocker ABSTRACT The proper orchestration of voltage-gated ion channel gating is vital to maintaining normal heart rhythms throughout an animal’s lifespan. Voltagegated sodium channels, Nav, are responsible for the initiation of the cardiac action potential, which leads to cardiac systole. Comparison of neonatal ventricular and atrial myocyte Nav gating with adult indicated that the neonatal ventricular Nav gated following a ~10 mV greater depolarization than did atrial or adult ventricular Nav. In this study I questioned whether developmentand/or chamber-dependent changes in Nav-associated functional sialic acids could account for these differences. When desialylated with neuraminidase, all gating characteristics for the lower voltage activated atrial and adult ventricular Nav shifted significantly to more depolarized potentials. However, desialylation of the higher voltage activated neonatal ventricular Nav had no effect on channel gating. Furthermore, channels were stripped of their N -glycosylation via PNGase-F in an attempt to separate the potential effects of the remaining glycosylation structure on Nav gating. Following treatment, neonatal ventricular Nav gating remained unchanged while atrial and adult ventricular Nav gating again shifted to

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ix depolarized potentials nearly identical to those of the neonatal ventricular channel. Immunoblot analyses indicated that atrial and adult ventricular Nav subunits are more heavily sialylated than the neonatal ventricular subunit, with approximately 15 more sialic acid residues. The data indicate that differential sialylation of myocyte Nav subunits is responsible for much of the developmental and chamber-specific remodeling of Nav gating observed here. In addition, the Nav1.5 subunit can associate with subunits, also believed to be sialylated. The potential for functional 1 trans sialic acids to further modulate Nav1.5 gating was tested via co-transfection of 1 with the Nav1.5 subunit into the Pro5/Lec2 mammalian expression system. Co-transfection revealed that the additional 1 trans sialic acids caused a hyperpolarizing shift in all tested gating parameters. When transfected into neonatal ventricular myocytes, 1 expression revealed no effect, implying that 1 expression alone is not responsible. Together, the myocyte and expression system studies describe a novel mechanism by which Nav gating, and subsequently cardiac excitability, are modulated by the regulated change in channel-associated functional sialic acids.

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1 Introduction Ion transport Ion transport from one side of a cell membrane to another is an important element in many processes like nerve transduction, muscle contraction (cardiac, skeletal, smooth), neurotransmitter release, nutrition absorption, and maintenance of the cellular membrane potential. To accomplish this movement of ions many different protein mediated transport mechanisms such as ion exchange, active transport, secondary active transport, leak, stretch-activated, ligand-gated, and voltage-gated ion channels exist. For ion exchange mechanisms, each set of ions move down their electrochemical gradient, produced through the combination of the ion’s concentration gradient and electrical driving force. Some popular examples of these transporters are Na+ / Ca2+, Cl/ HCO3 -, and Na+ / H+ exchangers (figure 1). The transport protein in question associates with its specific ions on each side of the membrane and then the ions are swapped from one side to the other. Active transport is the movement of solutes from one side of the membrane to the other against that solute’s concentration gradient. Fueled by the hydrolysis of ATP, the Na+ / K+ ATPase helps to maintain a cell’s resting membrane potential, regulated by intra and extracellular Na+ and K+ concentrations, by transporting three Na+ outside and two K+ inside the cell (figure 2). Secondary active transport is fueled

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2 Figure 1 : Ion exchange transport proteins

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3 Figure 1 : (continued) Facilitated transport proteins exchange one ion for another from one side of the membrane to the other. A: Na+ / Ca2+ ion exchanger B: Cl/ HCO3 ion exchanger C: Na+ / H+ ion exchanger

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4 Figure 2 : Active transport

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5 Figure 2 : (continued) A schematic example of active transport using the Na+/K+ ATPase to move Na+ and K+ against their concentration gradients. Step 1: Na+ binds to the cytoplasmic open face of the transport protein. Step 2: ATP is hydrolyzed to ADP phosphorylating the protein. Step 3: The Na+ bound phosphorylated protein opens to the outside releasing the Na+. Step 4: K+ binds to the open extracellular face of the phosphorylated transport protein. Step 5: The protein becomes unphosphorylated. Step 6: The unphosphorylated protein opens to cytoplasmic side of the membrane releasing the K+ allowing for the cycle to start again.

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6 by the energy resulting from one solute’s movement from one side of the membrane to the other down its chemical gradient. This supplied energy is used to co-transport other solute molecules against their chemical gradients, as in the case of glucose absorption in the gut and kidneys together with Na+ ions (figure 3). Ion channels are a second type of transmembrane protein that creates permeation pathways for ions to cross membranes. These proteins form aqueous pores in the membrane that, with the use of a selectivity filter, allow specific ions to pass through from one side of the membrane to the other. When an ion is allowed to pass through a channel, it flows down its electrochemical gradient requiring no energy expenditure. Ion channels fall into four general categories based on their mode of activation, leak, stretch-activated, ligandgated, and voltage-gated. Leak channels are exactly that, channels that are believed to be constitutively active. Typically involved in maintaining the resting membrane potential, leak channels are active most of the time creating a permeation pathway for a specific ion (Plant et al. 2005;Tamargo et al. 2004). Stretch-activated channels are believed to open in response to plasma membrane stretch. Once stretched the protein changes conformation to create an aqueous pore through which ions can flow. Their functions are not totally understood but are believed to be involved in the regulation of cell volume (Zagorodnyuk et al. 2005;Kalapesi et al. 2005;Trayanova et al. 2004). Ligandgated channels are thought to be closed at rest; following binding of a ligand to the channel, neurotransmitters for example, the channel opens (figure 4).

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7 Figure 3 : Secondary active transport of Na+ and glucose in the small intestine

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8 Figure 3 : (continued) Co-transport of one molecule accomplished by using the energy released through the transport of a second molecule.

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9 Figure 4 : Neuromuscular junction

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10 Figure 4 : (continued) Close up illustration of the neuromuscular junction showing Acetylcholine being released from the axon and traveling across the synapse to the ligand-gated Ach receptor binding sites to thus result in channel opening and eventual muscle contraction.

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11 Acetylcholine (Ach) receptors at the neuromuscular junction are a good example of ligand-gated ion channels. Ach is released by the motor neuron into the synapse and becomes bound to motor endplate Ach receptors changing them from closed to open. Once Na+ moves down its electrochemical gradient depolarizing the membrane this depolarization then triggers what are known as voltage-gated ion channels. Voltage-gated ion channels are membrane proteins that open in response to a membrane depolarization. Typically, when a cell membrane is at its resting membrane potential, the channel is in its closed conformation. When the membrane becomes more depolarized the channel senses this change in potential, opens, then allows selected ions to pass through. Following the open state most voltage-gated ion channels then rapidly close, voltage-gated K+ channels for example, while others move into a third state called inactivation, voltage-gated Na+ channels for example, rendering it unlikely for the channel to then be reactivated (figure 5). Once the Ach receptor induced Na+ mediated membrane depolarization opens the voltage-gated Na+ channels, Na+ moves down its electrochemical gradient through the open channels into the cell further depolarizing the surrounding membrane. When the membrane potential has become depolarized enough to reach what is known as the threshold potential, an action potential is the result. Once an action potential has been triggered, the membrane depolarization spreads to the surrounding areas and has a similar effect on the voltage-gated ion channels in the area, thus propagating the signal and ultimately resulting in muscle contraction. In addition, the membrane depolarization of the spreading action potential has now also

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12 Figure 5 : Schematic representation of a voltage-gated Na+ channel

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13 Figure 5 : (continued) In segment A is a voltage-gated Na+ channel in the closed state. When moving to segment B the membrane begins to become depolarized being sensed by the channel voltage sensors. In segment C the channel has moved from the closed to the open state allowing Na+ to flow through. Following segment C for some channels, K+ for example, the channel would then move back into the closed state ready to be used again. In this case, the Na+ channel moves into a third, inactivated state (segment D) when a channel inactivating segment blocks the open channel pore. Following membrane repolarization the channel moves back into the closed state, segment A, ready to refire again.

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14 activated voltage-gated K+ channels. This allows K+ to move out of the cell, opposing the inward Na+ current, repolarizing the membrane back to its resting potential and resetting the system to receive and transmit further signals. Voltage-gated Na+ channels Voltage-gated Na+ channels, Nav, are responsible for the initiation and propagation of action potentials in excitable tissues, opening rapidly and then inactivating following a membrane depolarization (Hille, 2001). The orchestrated activation and inactivation of Na+ channels is vital to normal skeletal muscle and neuronal function, and in maintaining normal heart rhythm. To date ten different voltage-gated Na+ channel isoforms have been identified. Nav nomenclature derived to identify the different isoforms is Nav1.1 to Nav1.9 with the tenth being labeled Nax (figure 6) (Goldin et al. 2000). Each Nav isoform shares greater than 50% identity with the other Nav family members. One important fact to note when considering the following description is that, to date, no one has been able to crystallize a voltage-gated Na+ channel in its complete membrane incorporated functional quaternary structure. This current limitation has led to a generally agreed upon structure of functional membrane bound voltage-gated Na+ channels as elucidated from the efforts of channel biologists worldwide. Nav are sodium specific ion channels that are made up of a single polypeptide chain forming the subunit. This chain weaves in and out of the membrane twentyfour times forming 4 distinct domains made up of six membrane-spanning segments each (figure 7). The S4 segment of each domain contains a positively

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15 Figure 6 : A chart outlining the known voltage-gated Na+ channel subunits along with their corresponding anatomical locations (Goldin et al. 2000)

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16 Figure 7 : Nav1.5 voltage-gated ion channel

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17 Figure 7 : (continued) Voltage-gated Na+ channels are made up of four homologous domains each with six membrane spanning alpha helical segments with the fourth alpha helices being the voltage sensors. The protein loops between each S5 and S6 segment are believed to create the channel pore. The intracellular loop between the third and fourth domain is the channel inactivation segment. Represented by pitchfork structures on the large extracellular loop of the first domain are N -linked glycosylation structures.

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18 charged amino acid in every third position. These S4 segments are believed to make up what is referred to as the channel voltage sensor. The S4 amino acids are thought to move from the inner surface of the membrane toward the outer surface, repelled by the membrane depolarization, resulting in the conformation change of the protein moving the channel from the closed to the open state. The way in which the S4 segments are believed to be orientated and their motion in response to a membrane depolarization has been a recent topic of debate. Over the years it has been assumed that the S4 segments are embedded within the quaternary structure in four cylindrically shaped alpha helices surrounding the channel pore. Through work done studying crystallized segments of voltagegated K+ channels, which are homologous to Nav, it has now been suggested that the S4 segments may actually form four paddle-like structures branching to the outside of the channel rather than being embedded within it (Jiang et al. 2003a;Jiang et al. 2003b). Regardless of their orientation, it is still believed that the S4 segments are close to the inner membrane surface at rest moving towards the outer surface following a membrane depolarization. The intracellular linker between the third and the fourth domains is known as the fast inactivation loop, made up of an IMF motif (isoleucine, methionine, phenylalanine). It is this IMF motif of the loop that is believed block the channel pore moving it from the open to inactivated state. During the inactivation period it is unlikely for the channel to become opened again until the system is reset. Finally, for the channel to move from the inactivated back to the closed state allowing the channel to again be activated, the membrane must become hyperpolarized

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19 again. The hyperpolarization of the membrane back to its resting potential drives the channel to reset back to the closed state now ready to be used again. It is important to recognize that this is a simplified description of Nav channel gating and a more thorough explanation of the intricate details surrounding channel gating can be found in many other literary works (e.g., Ion Channels of Excitable Membranes by Bertil Hille). Ion channel glycosylation, sialic acid, and Nav Ion channels are typically glycosylated when in their fully processed form. Protein glycosylation occurs inside the ER and Golgi complex. Some protein glycosylation requires activated carbohydrates as precursors while other forms require oligosaccharide-pyrophosphoryldolichol. Activated carbohydrates are ones that have reacted with UTP, CTP, or GTP, forming an XDP-carbohydrate derivative. Once the reaction is complete, the activated sugar is transported across the ER membrane via a transporter that exchanges XTP for XDPcarbohydrate. Once inside, the carbohydrate is transferred to the growing glycoprotein chain by enzymes called glycosyl transferases. Other sugars are transported into the ER by a carrier lipid (dolichol), which acts as an intermediate in the reaction, forming the oligosaccharide-pyrophosphoryldolichol complex. The overall process of protein glycosylation can typically be broken down into two types, O -linked and N -linked glycosylation. The amino acid side chains to which the carbohydrate group links defines each of the two types. In the case of O -linked glycosylation, the sugars are attached to hydroxyl groups. In the case

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20 of N -linked glycosylation, the sugars are attached to amino groups. With O linked, either a single mannose or acetylgalactosamine residue is transferred from an ER nucleotide carrier to the hydroxyl side chain of a serine, threonine, hydroxyproline, or hydroxylysine amino acid. The completion of the O -linked chain takes place in the Golgi complex with various sugars like acetylgalactosamine, galactose, fucose, and sialic acid. To accomplish N -linked glycosylation, two acetylglucosamine and nine mannose are attached to the ER carrier lipid dolichol. Once assembled the sugar complex is transferred to an asparagine residue that is followed by a serine or threonine. Once attached, the protein is transferred to the Golgi where it becomes a high mannose, hybrid, or complex N -linked glycoprotein through the addition of N-acetylglucosamine, mannose, galactose, and or sialic acid (figure 8). Voltage-gated Na+ channels contain complex N -linked glycosylation structures. The defining characteristic of complex structures is that multiple sialic acid branches cap them. Sialic acids are unique carbohydrates by virtue of the fact that, under normal physiological conditions, they are the only sugars that are charged, carrying a net -1 charge. All of this added negative charge surrounding the voltage-gated channel may potentially exert an effect on channel gating. Up to 35% of the total mass of many Nav may be attributed to carbohydrate (Cohen & Levitt, 1993;Gordon et al. 1988;Roberts & Barchi, 1987;Schmidt & Catterall, 1987;Messner & Catterall, 1985). Up to 45% of these carbohydrates may be composed of sialic acid residues, with around 100 or more sialic acid residues attached to each -subunit (Roberts & Barchi, 1987). Previous reports have

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21 Figure 8 : The three different types of known N -linked glycosylation structures

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22 Figure 8 : (continued) Attached to asparagine residues, there are three different types of N -linked glycosylation structures that each shares the same base structure. Segment A is high mannose N -linked glycosylation that terminates with multiple mannose residues. Segment B is hybrid N -linked glycosylation terminating with a mixture of mannose and galactose residues. Segment C is complex N -linked glycosylation terminating with multiple sialic acid residues.

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23 indicated that sialic acid (SA) may play a role in channel function. Nearly identical depolarizing shifts in Nav1.4 gating were observed using three independent methods to reduce channel sialylation as expressed in CHO cells. Chemical desialylation of the cardiac Nav subunit (Nav1.5) expressed in HEK 293 cells resulted in depolarizing shifts in the voltage of half-activation, Va, while a second study of Nav1.5 expressed in CHO cells reported no SA-dependent shift (Bennett, 2002;Zhang et al. 1999). Because the effects of sialic acid on channel gating vary with the expression system used, studies of Nav function in cardiomyocytes would be the most direct means of addressing whether sialic acid alters cardiac sodium channel gating. The surface potential theory The idea of a surface potential theory has been considered for around 50 years. The theory describes how fixed negative charges, at and around the ion channel associated membrane surface, serve to create a negative surface potential altering the electric field sensed by the channel voltage sensors. This external negative surface potential acts to depolarize the membrane bringing the Nav closer to its threshold potential for activation, thus affecting gating. The establishment of a surface potential can be derived by many factors like sialic acids, charged lipids making up the cell membrane, charged amino acids comprising proteins, and electrolytic screening in solution. Changing [cation]o has been shown to screen a suspected negative surface potential affecting Na+, Ca2+, and K+ channel gating, with increasing concentrations causing channel

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24 activation and inactivation to be shifted in the depolarized direction (Bennett et al. 1997;Hahin & Campbell, 1983;Campbell & Hille, 1976;Dorrscheidt-Kafer, 1976;MOZHAYEV.GN & Naumov, 1970;BLAUSTEI.MP & Goldman, 1968;Hille, 1968;Hagiwara & Naka, 1964;Frankenhaeuser & Hodgkin, 1957;Weidmann, 1955). This phenomenon can be considered the divalent screening effect. This translates into the need for a greater depolarization to enact channel activation in the presence of elevated divalent cations levels. Sialic acid, being negatively charged and so closely associated with the Nav, is an ideal candidate to be a strong contributor to a negative surface potential. The cardiac action potential and Nav The cardiac action potential, the electrical signal that is spread across the atria and ventricles resulting in cardiac systole, is accomplished through the orchestrated opening and closing of voltage-gated sodium, potassium, and calcium channels. The cardiac action potential (figure 9) consists of 5 phases starting with phase 0. At phase 0 a propagating membrane depolarization from neighboring cells elicits the opening of fast current voltage-gated Nav allowing Na+ to rush into the myocyte, iNa, the membrane is depolarized to threshold and an action potential begins. At the same time the Nav are activated, the rapid membrane depolarization causes inward rectifier K+ channels to close, iK1i, and then transient outward K+ channels open resulting in a brief membrane repolarization, iTO, known as phase 1. Also at the start of phase 1, the Nav have now begun to move into the inactivated state ending their role in the action

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25 Figure 9 : The cardiac action potential and current flow

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26 Figure 9 : (continued) Panel A is an example of a cardiac action potential with the five distinct parts labeled from 0 4. Panel B outlines the various ionic currents making up the five different parts of the action potential responsible for its shape and duration.

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27 potential. Phase 2 marks the plateau phase of the cardiac action potential. The plateau phase results from the initial depolarization also causing the opening of Ca2+ channels, iCa, that first counteract the decreasing iTO current. At the same time, delayed rectifier K+ channels are activated, iK, that counteracts the diminishing iCa after iTO stops bringing about the end of the plateau and the beginning of phase 3. During phase 3 the remaining iK hyperpolarizes the cell enough the cause the inward rectifier K+ channels to reopen, iK1, completely repolarizing the cell back to its resting membrane potential maintained during phase 4. The repolarization of the cell triggers the Nav from phase 0 to move from their inactivated to closed states. Now the cell is ready to be refired once more. The primary voltage-gated sodium channels found in cardiac atrial and ventricular myocytes, Nav1.5, are responsible for the initiation and propagation of action potentials resulting in cardiac systole (figure 7). To date, expression of Nav1.1, Nav1.3 Nav1.5 and Nav1.6 have been reported to be expressed in cardiac tissue from animals such as rat, rabbit and mouse (Malhotra et al. 2001;Baruscotti et al. 1997;Rogart et al. 1989). However, research from mice has demonstrated that Nav1.5 appears to be the dominant current producing cardiac Nav involved in the cardiac action potential phase zero (Lei et al. 2004;Maier et al. 2002;Zimmer et al. 2002).

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28 Cardiac Nav gating in adult and neonatal cardiomyocytes While Nav1.5 is the primary current carrying sodium channel expressed in the heart throughout development, previous studies suggested that there are differences between neonatal and adult cardiac ventricular Nav gating (Zhang et al. 1992). This report showed a large hyperpolarizing shift in the voltage dependence of steady-state inactivation and an increase in the rate of inactivation through development. It was speculated that the developmental effects on channel inactivation were attributed to differential sympathetic innervation. Similar results for changing rate of Na+ channel inactivation were reported for chick embryo ventricle through development (Fujii et al. 1988). Previous studies of cardiomyocytes have suggested that channel glycosylation may impact heart function. In rabbit sino-atrial nodal cells and ventricular myocytes, it was shown that treatment with neuraminidase resulted in increased L and T-type Ca2+ current, suggesting that sialic acid is important in the maintenance of proper cardiac cell Ca2+ permeability (Marengo et al. 1998;Fermini & Nathan, 1991). Neuraminidase treatment of adult mouse ventricular myocytes altered the voltage dependence and conductance of transient outward K+ (Ito) and fast inward Na+ (INa) currents and reduced the number of rat cardiac M2 muscarinic agonist-receptor complexes (Ufret-Vincenty et al. 2001a;Ufret-Vincenty et al. 2001b;Haddad et al. 1990). Perhaps some of the differences observed in cardiac Nav gating through development can be attributed to changing levels of channel glycosylation.

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29 interaction with In addition to the varying voltage-gated sodium channel subunits that alone comprise functional pore forming ion channels, there also exists auxiliary subunits often associated with There are currently four different subunits, and two splice variants, that have been identified to interact in one way or another with the varying Nav subunits of excitable tissues (Qin et al. 2003;Yu et al. 2003;Kazen-Gillespie et al. 2000;Morgan et al. 2000;Isom et al. 1995a;McClatchey et al. 1993;Isom et al. 1992). The main function(s) of in the body have yet to be fully understood but are thought to be any number of a range of possible functions. In one study 1, 2, and 3 when co-transfected with Nav1.2 (brain) in HEK293, resulted in Va and Vi, voltage of half-inactivation, being shifted in the depolarized direction. 3 was also shown to increase persistent sodium currents thought to increase the excitability of specific neuronal groups to all of their input by amplifying synaptic summation (Qu et al. 2001). In contrast, when co-transfected with Nav1.3 in CHO cells, 1 and 3 shifted Vi in the hyperpolarized direction. Also, they observed no effect with 2 transfection alone or in combination (Meadows et al. 2002a). 1 -/mice have been used to study the abnormalities that occur in the absence of normal 1 expression. It was noticed that the mice were ataxic with spontaneous seizures and altered levels of expressed Nav1.1 and Nav1.3 in the absence of 1 suggesting a basis for epilepsy. It was also suggested that 1, in these mice, was important in regulating Nav density and localization with an implied role in axo-glial communication at nodes

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30 of Ranvier (Chen et al. 2004). It has also been suggested that 1 and 2 are cell adhesion molecules that recruit ankyrin (Isom, 2001). More specifically in the heart, Nav1.5 and various subunits have also been shown to interact. Using immunocytochemistry, Nav1.5, together with 1 and 2, have been visualized at the intercalated disks (Maier et al. 2004). Another group believes that tyrosine phosphorylation of 1 regulates its localization to the intercalated disks (Malhotra et al. 2004). In the developing mouse heart, 1 mRNA levels were shown to increase at late embryonic and neonatal stages being maximal in the adult with protein again visualized in the intercalated disks (Dominguez et al. 2005). The 1 subunit is made up of a single transmembrane spanning segment with an extracellular N-terminus (figure 10). The extracellular portion of the protein contains four potential N -glycosylation sites and a single immunoglobulin-like fold (Isom et al. 1992). In modulating Nav1.5 channel gating it is generally noted that 1 co-expressed with in various expression systems causes a hyperpolarizing shift in the voltage dependence of channel activation and inactivation (Johnson et al. 2004;Meadows et al. 2002a;Meadows et al. 2002b;Meadows et al. 2001;McCormick et al. 1999;McCormick et al. 1998;Chen & Cannon, 1995;Isom et al. 1995b;McClatchey et al. 1993). However, 1 co-expression in HEK 293 cells, specifically, has resulted in depolarizing shifts in Nav inactivation gating (Cummins et al. 2001;Dhar et al. 2001;Qu et al. 2001). It is thought that at least three of the four N -linked glycosylation sites are glycosylated, and are potentially capped by sialic acids

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31 Figure 10 : Voltage-gated sodium channel 1 auxiliary subunit CO2+ H3N 1outside Inside CO2+ H3N 1outside InsideCO2+ H3N 1outside Inside

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32 Figure 10 : (continued) The voltage-gated sodium channel 1 subunit is made up of a single membrane spanning alpha helix with four predicted potential N-linked glycosylation sites.

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33 (Isom et al., 1992;Messner & Catterall, 1985). Could these sialic acids attached to 1 be responsible for hyperpolarizing shifts observed in Nav1.5 gating? Nav channel disorders Voltage-gated sodium channels are vital for the proper initiation and propagation of action potentials in the body. To reiterate, under normal conditions, Nav experience three conformation changes that define its behavior. Under hyperpolarized conditions the channel is in its closed resting state. Membrane depolarization drives the channel into its open state allowing current flow. Soon after opening and following membrane depolarization the channel moves into the inactivated state blocking current flow. Then, following membrane repolarization, the channel moves back to its resting state. When the inactivation state of the channel comes on too slowly or incompletely then the repolarizing phase of the action potential and the resting membrane potential itself are altered potentially leading to disorders. In skeletal muscle, examples of Nav disorders are paramyotonia congenita and hyperkalemic periodic paralysis (Bouhours et al., 2005;Brancati et al., 2003;Lehmann-Horn et al., 1987a;Lehmann-Horn et al., 1987b). In the central nervous system Nav mutations have been implicated in epilepsies with a variety of phenotypes including generalized epilepsy with febrile seizures plus (GEFS +), severe myoclonic epilepsy in infancy (SMEI), intractable childhood epilepsy with generalized tonic-clonic seizures (ICEGTC), and benign familial neonatal-infantile seizures (BFNIS) (Nagao et al., 2005;Pineda-Trujillo et al., 2005;Yamakawa,

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34 2005;Kanai et al., 2004). In the heart Nav1.5 mutations have been implicated in diseases such as Brugada syndrome, long QT syndrome, dilated cardiomyopathy, arrhythmia, and conduction disorders (McNair et al., 2004;Splawski et al., 2002;Bennett et al., 1995;Wang et al., 1995;Abriel et al., 2001). In addition to these disorders that are linked to various abnormalities associated with the channel protein there are also a host of disorders linked to irregularities in channel glycosylation. These disorders affect both N and Olinked glycosylation and are commonly referred to as congenital disorders of glycosylation, or CDGs. There are twelve N-linked and four O-linked CDGs affecting a wide range of ion channels in the body. Each of these CDGs are caused by protein abnormalities, mostly enzymes, due to specific gene defects. These many CDGs present themselves in a number of different phenotypes. The O-linked CDGs are involved in four different abnormalities known as Ehlers– Danlos syndrome (connective tissue disorder), multiple exostoses syndrome (bone disorder of the epiphyseal plates), Walker–Warburg syndrome (lack of normal folds of the brain, malformations of the cerebellum, abnormalities of the retina, and progressive degeneration and weakness of the voluntary muscles), and muscle–eye–brain disease (muscle weakness and a flaw in neuronal migration resulting in a brain with a bumpy appearance and loss of normal folding). The twelve different N-linked CDGs are separated into two groups. The first group is described by eight assembling defects, labeled CDG-Ia to CDG-Ih, and the second by four processing defects, labeled CDG-IIa to CDG-IId). These

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35 multiple CDGs in turn result in a number of defects such as hypotonia, failure to thrive, developmental delays, hepatopathy, coagulopathy, esotropia, seizures, cerebellar hypoplasia, ataxia, dysarthria, retinis pigmentosa, progressive scoliosis, joint contractures, and absence of puberty in females (Marklova & Albahri, 2004;Jaeken, 2003;Marquardt & Denecke, 2003;Martin & Freeze, 2003;Grunewald et al. 2002). The most common test used to diagnose a CDG is isoelectric focusing of serum transferrin, which contains two N-linked glycosylation sites terminated with sialic acids. When an abnormality is present, the protein is deficient in sialic acid, which changes its IEF signature. Any of the CDGs that ultimately lead to Nav sialic acid glycosylation deficiencies may have a direct effect on cellular excitability.

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36 Materials and Methods Construction of and cDNAs The voltage gated cardiac sodium channel, Nav1.5, was kindly provided in vector pRC-CMV (Invitrogen) by Dr. A. L. George Jr. The voltage gated sodium channel beta subunit, h1, was subcloned into the bicistronic vector, pIRESEGFP (Clontech), to provide use of visual inspection to ensure 1 expression. Pro5, Lec2 cell culture Pro5 and Lec2 cells were grown in T25 flasks (Corning). Pro5 growth medium consisted of MEM (with riboand deoxyribonucleosides, Invitrogen) and 10% FBS. Lec2 growth medium consisted of MEM (without riboand deoxyribonucleosides) and 10% FBS (Mediatech). Each growth medium also contained 100 g / ml-1 streptomycin and 100 U / ml-1 penicillin. Cells were passaged once every three to four days. First, the growth medium was removed, the cells were then rinsed once with PBS (Mediatech), and then 0.5 ml of trypsin / EDTA (0.05% / 0.53mM Mediatech) was added. The cells incubated in the trypsin for about 30 seconds to 1 minute, then the flask was tapped to fully dislodge the cells from the flask. To the 0.5 ml of trypsin was added 4.5 ml of the appropriate growth medium to stop the reaction. Then, an appropriate volume of

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37 this cell suspension was added to a new T25 flask with enough growth medium to equal 5 ml total. Pro5, Lec2 cell transfection Pro5, and Lec2 cell transfections were accomplished as previously described (Johnson et al., 2004). Briefly, cells were seeded onto 35 mm dishes 24 hr before use. The transfection mixture consisted of 1 ml of Opti-mem, 8 l lipofectamine (Invitrogen), and 2.0 – 2.5 g DNA (90% hH1 subunit vector and 10% pGreen Lantern Fluorescent Protein (GFP; Invitrogen) or 1 subunit vector) in which the cells were incubated for 24 hr. The transfection mixture was replaced with the appropriate growth media and the cells were then ready to use 48 hr later. Pro5, Lec2 cell homogenization Cells were first grown to confluence on 100 mm culture dishes. The media was aspirated off and the cells were rinsed twice with ice cold PBS. Then, each dish was incubated for five minutes with 1.5 ml of ice cold homogenization buffer (20 mM tetrasodium pyrophosphate, 20 mM Na2PO4, 1 mM MgCl2, 0.5 mM EDTA, 300 mM sucrose, 0.8 mM benzamidine, 1 mM iodacetamide, 1.1 M leupeptin, 0.7 M pepstatin, 76.8 nM aprotinin). The cells were then scraped off and ground in a dounce tissue grinder (40 strokes). The resulting suspension was centrifuged for 5 minutes at 3,200g at 4 C. The supernatant was then centrifuged for 1 hour at 100,000g at 4 C. The pellet was kept and then

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38 resuspended in 100 l of fresh homogenization buffer and stored at -80 C for future use. Neonatal and adult rat cardiomyocyte isolation and culture Cardiomyocytes were isolated from male neonatal (3-5 days) and adult (10 week +) Sprague-dawley rats. The neonatal cardiomyocyte isolation protocol was adapted from a method previously described (Isenberg & Klockner, 1982). Neonatal rats were anaesthetized with pentobarbital sodium (200mg/kg). Atrial or ventricular tissue was dissected out and washed in 0 Ca2+ Tyrode solution and then separated into individual myocytes by immersion in 0.1% collagenase (Sigma type I) solution for 0.5 hours, centrifuged for 5 minutes @ 160 g, then retreated with fresh collagenase solution for 1 hour. The tissue was then triturated to disperse the myocytes. The myocyte rich collagenase solution was centrifuged @ 160 g for 5 minutes, rinsed with KB solution, centrifuged again, and then plated with culture media. Adult ventricular and atrial myocytes were isolated using a Langendorf perfusion protocol (7-10 ml / minute) adapted from a method previously described (Saint et al., 1992). The adult rats were anaesthetized with pentobarbital sodium (200mg/kg) and their hearts were then dissected out. The whole hearts were first perfused with 0Ca2+ Tyrodes for five minutes, then with 300 units/ml collagenase (Sigma type I) and 7 mg protease (Sigma type XIV) in 0Ca2+ Tyrodes for 12 minutes, followed by 5 minutes of perfusion with 0.1mmol/L Ca2+ Tyrodes. Atrial and ventricular tissue was then cut down and triturated in 0.1mmol/L Ca2+ Tyrodes, run through a 200 m filter,

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39 rinsed 3 times with culture media, and then plated. The growth media consisted of DMEM (Mediatech) supplemented with 25 mM Hepes, 15% FBS, and 100 g / ml-1 streptomycin and 100 U / ml-1 penicillin. Neonatal rat ventricular myocyte transfection Neonatal rat ventricular myocytes were isolated and seeded onto 35 mm dishes as previously described. The transfection mixture consisted of 6 l effectine (Qiagen) with 2 g 1 subunit vector. The transfection mixture was then added to a fresh 1 ml of growth media 3 hours after the cells were isolated. The cells were then used for electrophysiology within 48 hr. Cardiac tissue homogenization First, the appropriate cardiac tissue was dissected out (atrial or ventricular), rinsed of blood in ice cold PBS and placed into a 50 ml conical tube with 4 ml of ice cold homogenization buffer. Using a Polytron homogenizer (Brinkmann Instruments model #PT10/35) set at 10, the tissue was pulsed several times to ensure complete homogenization. The homogenate was then stored at -80 C for future use. Electrophysiology Cells were studied using the patch clamp whole cell recording technique previously described (Bennett, 2002;Bennett et al., 1997). Briefly, an Axon Instruments 200B patch-clamp amplifier with a CV203BU headstage (Axon

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40 Instruments) was used in combination with a Nikon TE200 inverted microscope. The pulse protocols were generated using a Pentium II computer (Dell Computers) running pulse acquisition software (HEKA). The resulting analog signals were filtered at 5 kHz using an eight-pole Bessel filter (9200 LPF; Frequency Devices) and then digitized using the ITC-16 AD/DA converter (Instrutech). A micromanipulator (MP-285 Sutter, Novato, CA, USA) was used to place the electrode on the cell. Electrode glass (Drummond capillary tubes) was pulled using a Sutter (model P-87) electrode puller to resistances of 1-2 M Measuring Pro5, Lec2 Na+ currents The external recording solution used consisted of: (in mmol/L) 224 sucrose, 22.5 NaCl (ph with NaOH), 4 KCl, 2.0 CaCl2, 5 glucose, and 5 Hepes. The internal recording solution consisted of: (in mmol/L) 120 sucrose, 60 CsF, 32.5 NaCl (ph with NaOH), and 5 Hepes. All data was collected between 5-10 minutes after achieving whole cell configuration ensuring complete dialysis. Series resistance was compensated from 95 98% for all the data. Additionally, the smaller current produced by using the low sodium solutions further minimized any remaining series resistance error, resulting in a <1 mV error. Immediately prior to use, each solution was filtered using a 0.2 m Gelman filter. All experiments were performed at room temperature (~22oC).

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41 Measuring whole myocyte Na+ currents The whole cell recording extracellular solution was: (in mmol/L) 5 NaCl, 10 TES, 5 KCl, 1 CaCl2, 5 CsCl, 10 glucose, 115 choline chloride. The electrode solution consisted of (in mmol/L) 120 CsF, 10 TES, 2 MgCl2, 2 CaCl2, 5 NaCl, 20 EGTA. Both the ECF and ICF contained 20 NaCl for the neonatal myocyte ionic strength experiments. To ensure proper space clamping of the cardiomyocyte, several measures were taken: 1) Data were analyzed from cells producing sodium currents of 7nA or less, and reversing at the predicted sodium reversal potential. This required solutions containing only 5 mM NaCl (adult cells cannot be clamped at 20 NaCl). 2) Only myocytes of 76 pF or less were analyzed, to ensure greater ease of space clamp. 3) The slope of the G-V Boltzmann relationships were monitored, and only cells in which the slope > 5 mV were analyzed. Any steeper slope indicated a loss of voltage clamp. 4) All analyzed data were collected between 812 minutes after achieving whole cell configuration. In addition to these precautions, all data were series resistance compensated to 95 98%. If proper compensation was not achieved, the data were not analyzed. Immediately prior to use, each solution was filtered using a 0.2 m Gelman filter. All experiments were performed at room temperature (~22oC).

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42 Pulse protocols for measuring whole cell Na+ currents Pulse protocols were performed as described previously in Bennett, 2002 except that the holding potential used here was -120 mV (rat) and -100 (Pro5, Lec2). Brief descriptions of the protocols follow. Conductance-voltage (G-V) relationship Cells were stepped for 10 ms from their respected holding potentials to various depolarized potentials, ranging from -100 mV to +40 (rat) and +70 mV (Pro5, Lec2) in 10 mV increments. Consecutive pulses were stepped every 1.5 seconds and the data were leak subtracted using the P/4 method, stepping negatively from the -120 mV holding potential. At each test potential, steadystate whole cell conductance was determined by measuring the peak current at that potential and dividing by the driving force (i.e., difference between the membrane potential and the observed reversal potential). Peak conductance as a function of membrane potential was plotted. The maximum sodium conductance for a single cell was determined by fitting the data to a single Boltzmann distribution (equation 1, solving for maximum conductance). The average Va SEM and Ka SEM values were determined from these single Boltzmann distributions. The normalized data were then averaged with those from other cells, and the resultant average conductance-voltage curve was fit via least squares using the following Boltzmann relation: Fraction of maximal conductance = [1+(exp-(V-Va/Ka))]-1, equation (1) where V is the membrane potential, Va is the voltage of half activation, and Ka is the slope factor.

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43 Steady-state inactivation curves (hinf) Cells were first prepulsed for 500 ms from their respected holding potentials to the plotted potentials ranging from -130 mV to -20 mV (rat) and +10 mV (Pro5, Lec2) in 10 mV increments, then to +40 mV for 5 ms, then back to the -120 mV holding potential. Currents from each cell were normalized to the maximum current measured by fitting the data from a single cell to a single Boltzmann distribution (equation 2, solving for maximum current), from which the mean Vi +/SEM and Ki +/SEM values were determined. The normalized data for many cells were then averaged and fit to equation (2), from which the mean voltage of half inactivation (Vi) and slope factor (Ki) parameters for each cell type describing steady-state inactivation for the channel population were calculated. Fraction of maximum current = [1+(exp((V-Vi)/Ki))]-1 equation (2) Measurement of inactivation time constants ( !h) Inactivation time constants for each cell type were determined by fitting to single exponential functions the attenuating portions (90% 10%) of the current traces used to measure the G-V relationships. Recovery from inactivation Cells were held at their respected holding potentials, pulsed to +40 mV (rat) or +60 mV (Pro5, Lec2) for 10 ms, and then stepped to the recovery potential for 1 to 20 ms in 1 ms increments. Following the recovery pulse, the potential was again stepped to +40, or +60 mV for 10 ms. The peak currents measured during the two +40, or +60 mV depolarizations were compared to determine the fractional current measured during the second pulse.

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44 Desialylation of rat cardiomyocyte sodium channels For desialylation experiments, cells plated onto 35 mm dishes were exposed to 1.125 1.500 units neuraminidase (Sigma type X) / 1 ml DMEM for 3 hours. Immediately following treatment, cells were rinsed with and allowed to recover in their appropriate growth media at 3 5 C for 0.5 hours to wash out enzyme and quench further enzymatic reactions. Deglycosylation of rat cardiomyocyte sodium channels For deglycosylation experiments, cells plated onto 35 mm dishes were exposed to 19 20 units PNGase-F (Sigma) / 1 ml DMEM for 12 hours. Immediately following treatment, cells were rinsed with and allowed to recover in their appropriate growth media at 3 5 C for 0.5 hours to wash out enzyme and quench further enzymatic reactions. Antibody production and purification 1Ab was produced as previously described (Bennett, 1999). Site-directed polyclonal antibodies (1Abs) were raised to an 18-mer peptide (pep-1; Diagnostics) corresponding to the highly conserved III-IV linker region (TEEQKKYYNAMKKLGSKK) in vertebrate sodium channels and were affinitypurified on a pep-1-coupled column. 1Abs were raised to a 20-mer peptide, GCLAITSESKENCTGVQVAE. Antibody concentrations were assayed using optical density (O.D.) measurements at a 280-nm wavelength, with the accepted

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45 estimate that a 1.0 O.D. reading approximates 0.8 mg / ml 1Ab (Harlow & Lane, 1988). Immunoblots Cardiomyocyte homogenates were made by first harvesting the appropriate tissues (neonatal and adult cardiac atrial/ventricular muscle). The tissue was placed in ice cold sodium pyrophosphate buffer containing protease inhibitors inhibitors (in mmol/L): 20 tetrasodium pyrophosphate, 20 Na2PO4, 1 MgCl2, 0.5 EDTA, 300 sucrose, 0.8 benzamidine, 1 iodacetamide, 1.1 leupeptin, and 0.7 M pepstatin and 76.8 nM aprotinin, and homogenized using a Polytron homogenizer (Brinkmann Instruments model #PT10/35), set at 10, pulsed several times to ensure complete homogenization, and then stored at -80 C. and 1 samples were mixed with sample buffer (10% glycerol, 5% 2mercaptoethanol, 3% SDS, 12.5% upper Tris buffer), denatured for 2 minutes in boiled water, and then loaded into the lanes of a minigel cast from 4.5% acrylamide. The gel was run for its full length at 75 100 V and then electrophoretically transferred to nitrocellulose paper using a semi-dry transfer cell (Biorad) for 22 minutes at 8 14 V. Cardiac sodium channel and subunits were detected using a site-directed polyclonal antibody previously described (1Ab, 1Ab), (Bennett, 1999) then incubated with donkey anti-rabbit horseradish peroxidase conjugated secondary antibody (Amersham) for 2 hours, and visualized using a Pierce enhanced chemiluminescence kit. Immunoblot gel shift analysis of the -subunit under conditions of full and reduced sialylation

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46 were done by incubating homogenates with 1.35 units neuraminidase / 10 g protein for 3 hours at 37 C. Complete deglycosylation was accomplished via treatment with 20 units PNGase-F (Sigma) / 10 g protein for 12 hours at 37 C. Data analysis All electrophysiological data were analyzed using Pulse / PulseFit (HEKA) and Sigmaplot 2001 (SSPS Inc.) software. Sigmagel (SSPS Inc.) software was used to quantify the immunoblot data.

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47 Results The ventricular Nav of the newborn gates at more depolarized potentials The first step in attempting to determine the effects of channel sialylation / glycosylation on gating is to collect control data. Whole cell currents similar to that shown in Figure 11A were recorded from primary cultures of neonatal and adult ventricular and atrial myocytes. Biophysical analyses of the sodium currents revealed that neonatal and adult atrial and adult ventricular Nav gate following smaller depolarizations than do neonatal ventricular Nav. Figures 11A and B plot the steady-state activation (11A) and inactivation (11B) of the neonatal and adult channels as a function of membrane potential. Both the Va and Vi for the adult ventricle are 9 10 mV more hyperpolarized than those measured for the neonatal ventricular channels. In addition, the Va and Vi for the atrial Nav are similar to the adult ventricular Nav parameters (11A, B). Figure 11C shows the fast inactivation time constants (h) for each. Note that for the neonatal ventricular Na+ current, h generally is slower than for any of the other Nav. The time to Nav recovery from fast inactivation, rec, for all four samples is displayed in figure 11D. rec is slower for all atrial and adult ventricular Nav compared to the neonatal ventricular Nav (11D). Collectively, these data reveal a consistent chamber-specific difference in neonatal cardiac Nav voltage dependent gating, and a developmental hyperpolarization in Nav gating in the ventricle.

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48 Figure 11 : Whole cell recordings of isolated adult and neonatal atrial and ventricular myocytes determining voltage-dependent Na v gating characteristics Inactivation Time Constant (msec)B AMembrane Potential (mV) -100-80-60-40-200Normalized Conductance 0.00 0.25 0.50 0.75 1.00 DRecovery Potential (mV) -140-130-120Recovery Time Constant (msec) 0 5 10 15 20 Time (msec) 05101520Fractional Recovery (I/ Io) 0.00 0.25 0.50 0.75 1.00 CMembrane Potential (mV) -60-50-40-30-20-10 0 2 4 6 8 10 2.0 nA 2.0 msec Prepulse Potential (mV) -140-120-100-80-60-40Normalized Current 0.00 0.25 0.50 0.75 1.00 2.0 msec 1.0 nA 1.0 msec 1.0 nA NV AV, NA, AA AV, AA NV NA

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49 Figure 11 : (continued) Circles: neonatal VCM. Squares: adult VCM. Triangles: neonatal ACM. Inverted Triangles: adult ACM. Panel A: Steady-state activation (G-V relationships). Inset: Whole cell Na + current from an isolated adult VCM. Currents were measured during test pulses of -100 to +40 mV in 10 mV increments from the -120 mV holding potential. Panel B: Steady-state inactivation (h inf ). Data are the average peak current S.E.M. measured during a 5 msec pulse to +40 mV following a 500 msec prepulse to the plotted potentials. Inset: Whole cell Na + currents measured during a series of +40 mV test pulses, each following a prepulse to a larger depolarization ranging from -130 mV to -20 mV in 10 mV increments. panel C: Time constant of fast inactivation (h ). Data are the average h S.E.M. Curves are non-theoretical point-to-point. Inset: Current traces during a 50 mV test pulse. Solid traces: neonatal and adult ACM and adult VCM (NA, AA, and AV, respectively). Dotted trace: neonatal VCM (NV). Panel D: Voltage dependence of the recovery time constants (rec ). Data are the average rec S.E.M. Lines are non-theoretical point-to-point. Inset: Plotted recovery data for all four conditions at a -130 mV recovery potential. Curves are fits of the data to single exponential functions. Solid curves: NA, AA, and AV. Dotted curve: NV. For these and all remaining figures: NA (up arrows), AA (down arrows), NV (circles), AV (squares).

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50 Gating of three of the four Nav types are sensitive to desialylation – only the neonatal VCM Nav are insensitive To determine whether the observed chamber-specific and developmental shifts in Nav gating can be attributed to differences in functional channel sialic acids, cells were desialylated by treatment with neuraminidase. Figure 12B shows that, following neuraminidase treatment, the neonatal atrial channels are sensitive to desialylation, with the Va shifting by 7 mV to more depolarized potentials following neuraminidase treatment. However, identical treatment of the neonatal ventricular Nav had no effect on Nav Va (12A). Desialylation of both the adult atrial and ventricular Nav resulted in similar depolarizing shifts in Va indicating a lack of adult chamber differences in contrast to that seen in neonate (fig. 13). When studied from a developmental point of view, neuraminidase treatment of adult ventricular Nav caused a significant 10 mV depolarizing shift in Va (fig. 14B) when compared to the sialic acid insensitive neonatal ventricular Nav Va (14A). Figure 15A and B show that both the neonatal and adult atrial channels are sensitive to desialylation, with both Va shifting by approximately 7 mV to more depolarized potentials following neuraminidase treatment. Desialylation similarly alters steady-state channel inactivation in a chamberspecific and developmentally dependent manner. Figure 16B shows a significant 5 mV depolarizing shift in neonatal atrial Nav Vi following desialylation, while neonatal ventricular Nav Vi remains unaffected by treatment (16A). Desialylation of both the adult atrial and ventricular Nav resulted in similar depolarizing shifts of Vi demonstrating a lack of adult chamber differences in contrast to that seen in

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51 Figure 12 : Desialylation of neonatal VCM and neonatal ACM Nav causes a depolarizing shift in ACM steady-state Nav activation. Neonatal ventricular channels are unaffected AMembrane Potential (mV) -100-80-60-40-20Normalized Conductance 0.00 0.25 0.50 0.75 1.00 BMembrane Potential (mV) -100-80-60-40-20Normalized Conductance 0.00 0.25 0.50 0.75 1.00

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52 Figure 12 : (continued) G-V relationship for cardiomyocyte Nav neuraminidase. Data are the average normalized peak conductance S.E.M. at a membrane potential. Curves are fits of the data to single Boltzmann distributions. Filled symbols: untreated. Open symbols: treated. Circles: neonatal VCM (A). Triangles: neonatal ACM (B).

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53 Figure 13 : Desialylation of adult VCM and adult ACM Nav causes a depolarizing shift in steady-state Nav activation both VCM and ACM channels are similarly affected AMembrane Potential (mV) -100-80-60-40-20Normalized Conductance 0.00 0.25 0.50 0.75 1.00 BMembrane Potential (mV) -100-80-60-40-20Normalized Conductance 0.00 0.25 0.50 0.75 1.00

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54 Figure 13 : (continued) G-V relationship for cardiomyocyte Nav neuraminidase. Data are the average normalized peak conductance S.E.M. at a membrane potential. Curves are fits of the data to single Boltzmann distributions. Filled symbols: untreated. Open symbols: treated. Squares: Adult VCM (A). Inverted triangles: Adult ACM (B).

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55 Figure 14 : Desialylation of neonatal VCM and adult VCM Nav causes a depolarizing shift in adult steady-state Nav activation neonatal ventricular channels are unaffected BMembrane Potential (mV) -100-80-60-40-20Normalized Conductance 0.00 0.25 0.50 0.75 1.00 AMembrane Potential (mV) -100-80-60-40-20Normalized Conductance 0.00 0.25 0.50 0.75 1.00

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56 Figure 14 : (continued) G-V relationship for cardiomyocyte Nav neuraminidase. Data are the average normalized peak conductance S.E.M. at a membrane potential. Curves are fits of the data to single Boltzmann distributions. Filled symbols: untreated. Open symbols: treated. Circles: neonatal VCM (A). Squares: adult VCM (B).

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57 Figure 15 : Desialylation of neonatal ACM and adult ACM Nav causes a depolarizing shift in steady-state Nav activation both neonatal and adult ACM channels are similarly affected AMembrane Potential (mV) -100-80-60-40-20Normalized Conductance 0.00 0.25 0.50 0.75 1.00 BMembrane Potential (mV) -100-80-60-40-20Normalized Conductance 0.00 0.25 0.50 0.75 1.00

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58 Figure 15 : (continued) G-V relationship for cardiomyocyte Nav neuraminidase. Data are the average normalized peak conductance S.E.M. at a membrane potential. Curves are fits of the data to single Boltzmann distributions. Filled symbols: untreated. Open symbols: treated. Triangles: neonatal ACM (A). Inverted triangles: Adult ACM (B).

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59 Figure 16 : Desialylation shifts steady-state inactivation curves for neonatal atrial Nav but has no effect on neonatal ventricular Nav inactivation APrepulse Potential (mV) -120-100-80-60Normalized Current 0.00 0.25 0.50 0.75 1.00 BPrepulse Potential (mV) -120-100-80-60Normalized Current 0.00 0.25 0.50 0.75 1.00

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60 Figure 16 : (continued) Channel availability (hinf) for cardiomyocyte Nav neuraminidase. Data are the average normalized peak current S.E.M. measured during a 5 ms test pulse to +40 mV following a 500 msec prepulse to the plotted potentials. Curves are fits of the data to single Boltzmann distributions. Filled symbols: untreated. Open symbols: treated. Circles: neonatal VCM (A). Triangles: neonatal ACM (B).

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61 neonate (fig. 17). When developmentally studied, the Vi for adult ventricular Nav is shifted by a depolarizing 13 mV with neuraminidase treatment (fig. 18B), compared to the insensitive neonatal VCM Nav (18A). Figure 19A and B show that desialylation results in similar depolarizing shifts on neonatal and adult atrial Nav Vi, indicating no developmental shift in atrial Nav gating, in contrast to the ventricle. The mean SEM values for all measured voltage dependent gating parameters are listed in Table 1. Sialic acids are responsible for faster inactivation kinetics only neonatal ventricular Nav inactivation shows no sialic acid sensitivity As shown in Figure 11C above, data indicate that atrial and adult ventricular Nav inactivate faster than neonatal ventricular Nav. When comparing neonatal cardiac chambers, figure 20 shows a slowing of the rate of Nav fast inactivation for neuraminidase treated neonatal atria and the neonatal ventricular Nav, again showing no change with treatment. Neonatal ventricular Nav remain insensitive to desialylation. Following neuraminidase treatment of the adult cardiac chambers, desialylation resulted in comparable effects on Nav h kinetics, unlike in the neonate (fig. 21). When comparing the same chambers at two different ages, desialylation of the ventricles indicates a developmental shift in Nav h sialic acid sensitivity (fig. 22). Desialylation of the atria reveals essentially no developmental shift in Nav h (fig. 23).

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62 Figure 17 : Desialylation similarly shifts steady-state inactivation curves for both adult ventricular and adult atrial Nav APrepulse Potential (mV) -120-100-80-60Normalized Current 0.00 0.25 0.50 0.75 1.00 Prepulse Potential (mV) -120-100-80-60Normalized Current 0.00 0.25 0.50 0.75 1.00 B

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63 Figure 17 : (continued) Channel availability (hinf) for cardiomyocyte Nav neuraminidase. Data are the average normalized peak current S.E.M. measured during a 5 ms test pulse to +40 mV following a 500 msec prepulse to the plotted potentials. Curves are fits of the data to single Boltzmann distributions. Filled symbols: untreated. Open symbols: treated. Squares: adult VCM (A). Inverted triangles: adult ACM (B).

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64 Figure 18 : Desialylation shifts steady-state inactivation curves for adult ventricular Nav but has no effect on neonatal ventricular Nav inactivation BPrepulse Potential (mV) -120-100-80-60Normalized Current 0.00 0.25 0.50 0.75 1.00 APrepulse Potential (mV) -120-100-80-60Normalized Current 0.00 0.25 0.50 0.75 1.00

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65 Figure 18 : (continued) Channel availability (hinf) for cardiomyocyte Nav neuraminidase. Data are the average normalized peak current S.E.M. measured during a 5 ms test pulse to +40 mV following a 500 msec prepulse to the plotted potentials. Curves are fits of the data to single Boltzmann distributions. Filled symbols: untreated. Open symbols: treated. Circles: neonatal VCM (A). Squares: adult VCM (B).

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66 Figure 19 : Desialylation similarly shifts steady-state inactivation curves for both neonatal and adult atrial Nav AMembrane Potential -120-100-80-60Normalized Current 0.00 0.25 0.50 0.75 1.00 Membrane Potential -120-100-80-60Normalized Current 0.00 0.25 0.50 0.75 1.00 B

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67 Figure 19 : (continued) Channel availability (hinf) for cardiomyocyte Nav neuraminidase. Data are the average normalized peak current S.E.M. measured during a 5 ms test pulse to +40 mV following a 500 msec prepulse to the plotted potentials. Curves are fits of the data to single Boltzmann distributions. Filled symbols: untreated. Open symbols: treated. Triangles: neonatal ACM (A). Inverted triangles: adult ACM (B).

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68 Figure 20 : Sialic acid increases the rate of fast inactivation for neonatal atrial Nav but has little effect on neonatal ventricular Nav inactivation rates Inactivation Time Constant (ms) AMembrane Potential (mV) -70-60-50-40-30-20-100Inactivation Time Constant (ms) 0 2 4 6 8 10 12 14 BMembrane Potential (mV) -70-60-50-40-30-20-100 0 1 2 3 4 5 6 7

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69 Figure 20 : (continued) Time constant of fast inactivation (h) as a function of membrane potential for cardiomyocyte Nav neuraminidase. Data are the average h S.E.M. Curves are non-theoretical, point-to-point. Filled symbols: untreated. Open symbols: treated. Circles: neonatal VCM (A). Triangles: neonatal ACM (B).

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70 Figure 21 : Sialic acid causes an increase in the rate of fast inactivation for both adult ventricular and adult atrial Nav AMembrane Potential (mV) -70-60-50-40-30-20-100Inactivation Time Constant (ms) 0 2 4 6 8 10 12 14 BMembrane Potential (mV) -70-60-50-40-30-20-100Inactivation Time Constant (ms) 0 1 2 3 4 5 6 7

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71 Figure 21 : (continued) Time constant of fast inactivation (h) as a function of membrane potential for cardiomyocyte Nav neuraminidase. Data are the average h S.E.M. Curves are non-theoretical, point-to-point. Filled symbols: untreated. Open symbols: treated. Squares: adult VCM (A). Inverted Triangles: adult ACM (B).

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72 Figure 22 : Sialic acid increases the rate of fast inactivation for adult ventricular Nav but has little effect on neonatal ventricular Nav inactivation rates AMembrane Potential (mV) -70-60-50-40-30-20-100Inactivation Time Constant (ms) 0 2 4 6 8 10 12 14 BMembrane Potential (mV) -70-60-50-40-30-20-100Inactivation Time Constant (ms) 0 2 4 6 8 10 12 14

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73 Figure 22 : (continued) Time constant of fast inactivation (h) as a function of membrane potential for cardiomyocyte Nav neuraminidase. Data are the average h S.E.M. Curves are non-theoretical, point-to-point. Filled symbols: untreated. Open symbols: treated. Circles: neonatal VCM (A). Squares: adult VCM (B).

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74 Figure 23 : Sialic acid causes an increase in the rate of fast inactivation for both neonatal atrial and adult atrial Nav Inactivation Time Constant (ms) AMembrane Potential (mV) -70-60-50-40-30-20-100 0 1 2 3 4 5 6 7 BMembrane Potential (mV) -70-60-50-40-30-20-100Inactivation Time Constant (ms) 0 1 2 3 4 5 6 7

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75 Figure 23 : (continued) Time constant of fast inactivation (h) as a function of membrane potential for cardiomyocyte Nav neuraminidase. Data are the average h S.E.M. Curves are non-theoretical, point-to-point. Filled symbols: untreated. Open symbols: treated. Triangles: neonatal ACM (A). Inverted Triangles: adult ACM (B).

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76 Sialic acids act to slow the rate of recovery from fast inactivation for all but the neonatal ventricular Nav In line with the previously observed chamber-specific effects of Nav channel sialylation on neonatal gating, figure 24B shows that neonatal atrial Nav rec is once more sensitive to neuraminidase treatment, the ventricular Nav are not (fig. 24A). Upon comparison of treated and untreated adult cardiac chambers, it is noted that again the adult atrial and ventricular Nav rec share similar sensitivities to sialic acid, both becoming faster with treatment (fig. 25). It also seems apparent that a developmental shift in channel sialylation also affects channel rec. When treated with neuraminidase the neonatal VCM Nav rec was consistently unaffected while the slower adult rec became significantly faster and more neonatal like (fig. 26). Furthermore, figure 27 demonstrates for a fourth time a lack of a developmental shift in sialic acid sensitivity with respect to rec when moving from the neonatal to the adult atria, as witnessed with the ventricles. Following desialylation, recovery time decreases for all but the neonatal ventricular Nav. PNGase-F treatment, removing N -linked glycosylation, results in no significant difference in gating among all four tested Nav Desialylation of the atrial and adult ventricular Nav via the use of neuraminidase has consistently accounted for the vast majority of the differences observed in atrial and adult ventricular Nav gating compared to the neonatal

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77 Figure 24 : Recovery from fast inactivation for neonatal atrial Nav is faster following desialylation neonatal ventricular recovery rate is unaffected by sialic acids BRecovery Potential (mV) -140-130-120Recovery Time Constant (ms) 0 5 10 15 20 ARecovery Potential (mV) -140-130-120Recovery Time Constant (ms) 0 5 10 15 20

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78 Figure 24 : (continued) Voltage dependence of the inactivation recovery times for cardiomyocyte Nav neuraminidase treatment. Data are the measured rec as a function of recovery potential. Lines are non-theoretical point-to-point. Filled symbols: untreated. Open symbols: treated. Circles: neonatal VCM (A). Triangles: neonatal ACM (B).

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79 Figure 25 : Recovery from fast inactivation for adult ventricular and adult atrial Nav are both faster following desialylation BRecovery Potential (mV) -140-130-120Recovery Time Constant (ms) 0 5 10 15 20 ARecovery Potential (mV) -140-130-120Recovery Time Constant (ms) 0 5 10 15 20

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80 Figure 25 : (continued) Voltage dependence of the inactivation recovery times for cardiomyocyte Nav neuraminidase treatment. Data are the measured rec as a function of recovery potential. Lines are non-theoretical point-to-point. Filled symbols: untreated. Open symbols: treated. Squares: adult VCM (A). Inverted Triangles: adult ACM (B).

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81 Figure 26 : Recovery from fast inactivation for adult ventricular Nav is faster following desialylation neonatal ventricular recovery rate is unaffected by sialic acids ARecovery Potential (mV) -140-130-120Recovery Time Constant (ms) 0 5 10 15 20 BRecovery Potential (mV) -140-130-120Recovery Time Constant (ms) 0 5 10 15 20

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82 Figure 26 : (continued) Voltage dependence of the inactivation recovery times for ventricular cardiomyocyte Nav neuraminidase treatment. Data are the measured rec as a function of recovery potential. Lines are non-theoretical point-to-point. Filled symbols: untreated. Open symbols: treated. Circles: neonatal VCM (A). Squares: adult VCM (B).

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83 Figure 27 : Recovery from fast inactivation for neonatal atrial and adult atrial Nav are both faster following desialylation ARecovery Potential (mV) -140-130-120Recovery Time Constant (ms) 0 5 10 15 20 BRecovery Potential (mV) -140-130-120Recovery Time Constant (ms) 0 5 10 15 20

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84 Figure 27 : (continued) Voltage dependence of the inactivation recovery times for atrial cardiomyocyte Nav neuraminidase treatment. Data are the measured rec as a function of recovery potential. Lines are non-theoretical point-to-point. Filled symbols: untreated. Open symbols: treated. Triangles: neonatal ACM (A). Inverted Triangles: adult ACM (B).

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85 ventricular Nav. The reason for these differences may be due to the possibility that likely more than just subunit sialylation alone may be impacting channel gating. A second, and simpler explanation may be due in part to inconsistent desialylation of the Nav from sample to sample, experiment to experiment. PNGase-F treatment of cardiac myocyte surface expressed Nav was employed to remove sialic acids, and the remaining N-linked sugar structures, as a second method to try and account for the remaining differences in gating seen after neuraminidase treatment. Figure 28 provides a very clear and to the point comparison of the tested PNGase-F treated atrial and adult ventricular Nav gating parameters with that of the untreated and neuraminidase treated neonatal Nav (no difference observed with PNGase-F treatment for neonatal VCM (curves not shown)). When studying steady-state activation and inactivation it can be noted that deglycosylation resulted in no differences between the atrial and adult ventricular Nav Va and Vi compared to control and desialylated neonatal VCM (figure 28A, B, Table 1). PNGase-F treatment of the atrial and adult ventricular Nav also caused h for these samples to slow to values insignificantly different to those of the control and desialylated neonatal VCM (figure 28C). Additionally, rec for the deglycosylated atrial and adult ventricular Nav became significantly faster, again resulting in virtually identical data for all four samples (figure 28D). In conclusion, these data strongly support the idea that the majority of observed differences between control atrial and adult ventricular Nav, compared to that of control neonatal VCM, could be attributed to differences in channel sialylation, with channel deglycosylation accounting for the remainder.

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86 Figure 28 : Deglycosylation of atrial and adult ventricular Nav results in gating characteristics nearly identically to those of neonatal ventricular Nav Membrane Potential (mV) -60-50-40-30-20-10Inactivation Time Constant (ms) 0 2 4 6 8 10 BPrepulse Potential (mV) -140-120-100-80-60-40Normalized Current 0.00 0.25 0.50 0.75 1.00 DRecovery Potential (mV) -140-130-120Recovery Time Constant (ms) 0 2 4 6 8 10 12 AMembrane Potential (mV) -100-80-60-40-200Normalized Conductance 0.00 0.25 0.50 0.75 1.00 C

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87 Figure 28 : (continued) Panel A: steady-state activation. Panel B: steady-state inactivation. Panel C: fast inactivation kinetics. Panel D: recovery from fast inactivation kinetics. All of the data shown for adult ACM/VCM and neonatal ACM Nav is following treatment with PNGase-F (open symbols), and the neonatal VCM Nav is under control (solid circles) and neuraminidase treated (dotted, open circles) conditions.

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88 The atrial and adult ventricular Nav subunit is apparently more heavily sialylated than the neonatal ventricular Nav Figures 12 28 indicate that gating of the atrial and adult ventricular Nav are more sensitive to desialylation than is neonatal ventricular Nav gating. One possibility is that the neonatal ventricular Nav is the least sialylated of the channels, with differential sialylation occurring throughout the developing ventricle and between the newborn’s atria and ventricle. To test this possibility, a series of immunoblots were run to measure the apparent molecular weight (Mr) of Nav subunits as expressed in the four cardiomyocyte types. Homogenates of atria and ventricles harvested from newborn and adult rats were run on 4.5 % SDS-PAGE gels, blotted, and challenged with the subunit Nav antibody, 1Ab. From these blots, it was apparent that the atrial and adult ventricular Nav subunits were similar in Mr, and were consistently heavier than the neonatal ventricular channel (Figure 29A). The average measured Mr for the atrial and adult ventricular subunit was 251.8 +/0.6 kD (n=41), while the average Mr for the neonatal ventricular subunit measured 246.5 +/0.8 (n=19). Thus, the atrial and adult ventricular Nav subunits were about 5 kD larger on average than the neonatal ventricular Nav subunit. Because the Mr observed for all four homogenates is significantly greater than the predicted Mr for unprocessed Nav1.5, (about 227 kD), the data indicate that all four channel types are glycosylated, with the neonatal ventricular channel less processed than the other three channel types.

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89 Figure 29 : Gel shift analyses indicate that channel sialylation increases throughout the developing ventricle and is greater in the atria versus ventricle of the newborn A Mr(kD) 225 250 Untreated PNGase treated Neuraminidase treated NV SA ~5.3 kD Other SA ~9.1 kD SA = 3.8+/-0.4 kD Other + SA Other -SA NV -SA NV + SANVAVNAAADNVAVNAAA NVAVNAAA 250 NV,AV,NA,AA NV,AV,NA,AAB~230 kD PNGase-F-NVAVNAAA--+ ++ +NVAVNAAA--+ ++ + 250 NV,AV,NA,AA NV,AV,NA,AAC~242 kD Neuraminidase NVAVNAAA--+ ++ +NVAVNAAA--+ ++ +AV,NA,AA ~252 kD NV ~247 kD250**** **1961318n612128 41147

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90 Figure 29 : (continued) Bands detected using Ab at a 1:200 concentration. Lane labels: NV: neonatal ventricle. AV: adult ventricle. NA: neonatal atria. AA: adult atria. Panel A: Immunoblot of ACM and VCM at two developmental stages. Lanes 1, 4, and 7: Molecular weight markers. 5g protein loaded per lane. Panel B: Immunoblot of ACM and VCM PNGase-F treatment. Lanes 1, 6, and 11: Molecular weight markers. 5g protein loaded per untreated lane, 10g protein loaded per treated lane. Panel C: Immunoblot of ACM and VCM neuraminidase treatment. Lanes 1, 6, and 11: Molecular weight markers. 5g protein loaded per untreated lane, 10g protein loaded per treated lane. Panel D: Bar graph of average measured Nav subunit Mr for each homogenate PNGase-F neuraminidase. Each bar represents the mean Mr SEM measured for a series of immunoblots. Dark bars: Untreated. Light bars: PNGase-F treated. Gray bars: Neuraminidase treated. SA: Sialic acid. The number of samples measured is listed at the base of each bar. Significance was determined for the untreated data using a two-tailed student’s t-test comparing the atrial and adult ventricular subunit Mr to the neonatal ventricular subunit Mr. ** = Highly significant (P < 0.01)

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91 To determine more directly whether changes in channel glycosylation are responsible for the observed differences in Mr, myocyte homogenates were treated with the enzyme, PNGase-F. PNGase-F removes N-linked glycosylation at the site of protein attachment. In figure 29B, Mr for all four PNGase-F treated homogenates was reduced to 231.1 0.9 kD (n=20), with no significant difference in Mr measured among the homogenates following PNGase-F treatment. Thus, these data strongly indicate that the neonatal ventricular Nav is ~5 kD less N-glycosylated than the other channel types tested. To examine whether the differences in glycosylation levels observed are due to increases in subunit sialylation, myocyte homogenates were treated with neuraminidase to remove sialic acids specifically. As shown in Figure 29C, the measured Mr for all homogenates was reduced following neuraminidase treatment, suggesting that all cardiac Nav types are sialylated. Consistently, the predicted subunit Mr for all four homogenates were reduced to similar levels following neuraminidase treatment, indicating that the atrial and adult ventricular channels are more sialylated than the neonatal ventricular channel. Figure 29D illustrates the average Mr measured for each Nav under control, PNGase-F, and neuraminidase treated conditions (listed in Table 2). The graph shows that the untreated Mr for the atrial and adult ventricular channels are nearly identical, while the Mr for the untreated neonatal subunit is significantly lower. PNGaseF and neuraminidase treatment results in a similar Mr for all four channel types, indicating that a difference in channel sialylation is primarily responsible for the differences in Nav Mr observed. On average, the sialic acid dependent Mr

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92 (change in Mr with neuraminidase treatment) for the neonatal ventricular Nav subunit is about 5.3 kD, while the average SA-dependent Mr for the other three channel subunits is a much larger, 9.1 kD. Direct comparison of the SAdependent shift in Mr for neonatal VCM versus the other three channel types produced a highly significant, 3.8 +/0.4 kD difference (n=23), all of which can be assigned to channel sialic acids. This 3.8 kD of sialic acids (a 72 % increase from neonatal VCM to ACM and adult VCM) added throughout the developing ventricle and between chambers of the newborn heart is consistent with about 15 more sialic acid residues attached to the atrial and adult ventricular Nav subunits. Increased functional sialylation of atrial and adult ventricular Nav account for most of the hyperpolarizing shifts in channel gating The immunoblot gel shift analyses shown above indicate that the neonatal ventricular Nav subunit is the least sialylated among the four tested cardiomyocyte subunits. As shown in Figures 11-28, the neonatal ventricular channel consistently gates following larger depolarizations compared to the voltage dependent gating of the other channel types. Further, gating of the atrial and adult ventricular Nav all shifted to more depolarized potentials following desialylation, while gating of the neonatal ventricular channel was unaffected. Figure 30 compares the voltage dependent parameters measured for each channel type desialylation / deglycosylation. Note that none of the measured neonatal ventricular Nav gating parameters was significantly altered by

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93 Figure 30 : Functional channel sialic acids can account for much of the developmental and chamber-specific changes in Nav gating parameters NAAANVAVHalf Inactivation Voltage (mV ) -110 -100 -90 -80 -70 NAAANVAV -60 -50 -40 -30 Half Activation Voltage (mV ) NAAANVAV h @ -50 mV (ms) 0 1 2 3 4 5 6 NAAANVAV rec @ -120 mV (ms) 0 5 10 15 20

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94 Figure 30 : (continued) Bar graphs of the measured voltage-dependent Nav gating parameters for each myocyte type neuraminidase. Each bar represents the average measured parameter S.E.M. All values and significance listed in Table 1. Black bars: control. Light gray bars: neuraminidase treated. Dark gray bars: PNGase-F treated. NA: neonatal atria. AA: adult atria. NV: neonatal ventricle. AV: adult ventricle. Panel A: Voltage of half activation (Va) Panel B: Voltage of half inactivation (Vi) Panel C: Time constant of inactivation (h) measured during a -50 mV test pulse. Panel D: Time constant of recovery from inactivation (rec) measured at a –120 mV recovery potential.

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95 desialylation / deglycosylation. However, following desialylation / deglycosylation, all measured gating parameters for the atrial channels, both neonatal and adult, as well as the adult ventricular channel, shifted to depolarized potentials, similar in value to those measured for the neonatal ventricular Nav. Nav1.5 expressed with 1 in Pro5 versus Lec2 indicates 1 action on Nav1.5 gating is sialic acid dependent In order to directly compare the effects of 1 sialylation on Nav1.5 channel gating, the Pro5 / Lec2 CHO cell expression system was used. Pro5 and Lec2 cells are related CHO cells that produce proteins with differing amounts of posttranslationally attached sialic acids (Deutscher et al., 1984;Stanley, 1985;Stanley, 1989). The Pro5 cell is essentially a CHO cell allowing for normal protein sialylation. The Lec2 cell is deficient in the CMP-sialic acid transporter thus sialic acid cannot enter the Golgi and be added to proteins. When Nav1.5 was expressed alone in either of the two cells, whole cell recording indicated that none of the four tested gating parameters (Va, Vi, h, rec) was significantly different from one cell to the other (see Bennett, 2002 for details). This indicates that in the Pro5 / Lec2 cell expression system Nav1.5 gating is not significantly affected by subunit sialylation. That is, the added subunit sialylation accomplished by the Pro5 cell is not sufficient to alter channel gating when compared to the Lec2 cell. To test whether 1 has an effect on

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96 Nav1.5 channel gating and if those effects are sialic acid dependent, the Nav1.5 subunit was co-expressed with 1 in both Pro5 and Lec2 cells. Figure 31 indicates that when the and 1 subunits were co-expressed into the Lec2 cells there was no difference in channel activation, Va (fig 31A), or channel inactivation, Vi (fig 31B), when compared to the subunit alone. When co-expressed in the Pro5 cells, both Va and Vi were shifted in the hyperpolarized direction by 9 and 11 mV respectively. These data indicate that 1’s influence on Nav1.5 steady-state activation and inactivation is sialic acid dependent. The kinetics of channel inactivation and recovery from inactivation were studied next. Figure 32A and B show that the Nav1.5 subunit h and rec were unaffected when co-expressed with 1 in Lec2 cells. Similar to Va and Vi, when and 1 were co-expressed in Pro5 cells, h was faster and rec was slower. 1 sialic acids are indicated to be responsible for the observed changes in h and rec. Together, steady-state and kinetic data gathered from and 1 coexpression experiments in Pro5 versus Lec2 cells indicate that 1 modulation of Nav1.5 gating appears to be sialic acid dependent. All of the values can be found in Table 3. In a final attempt to demonstrate that 1’s influence over Nav1.5 gating is sialic acid dependent, 1 was mutated to remove all four N-glycosylation sites through site-directed mutagenesis. Removal of all four N-glycosylation sites thus removes all sialic acid from the protein. When co-expressed with Nav1.5 in fully sialylating Pro5 cells, the 1 mutant failed to affect Va, similar to wild-type 1 co-

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97 Figure 31 : 1 sialic acids cause a hyperpolarizing shift in Nav1.5 activation and inactivation AB -140-120-100-80-60-40-20 0.00 0.25 0.50 0.75 1.00 -100-80-60-40-20020 0.00 0.25 0.50 0.75 1.00 membrane potential (mV) Prepulse Potential (mV)Normalized Conductance Normalized Current

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98 Figure 31 : (continued) Filled Squares / Solid Lines: transfected into Pro5 cells. Filled Circles / Solid Lines: transfected into Lec2 cells. Filled Squares / Dashed Lines: 1 cotransfected with in Pro5 cells. Open Squares / Dashed Lines: 1 co-transfected with in Lec2 cells. Panel A: Steady-state activation (G-V relationships). Data are the average peak current S.E.M. at a membrane potential and the curves are fits of the data to single Boltzmann relationships. Panel B: Steady-state availability (h inf ) curves. Data are the mean normalized peak current S.E.M. measured during a maximally depolarizing test pulse following a 500 msec prepulse to the plotted potentials. (Johnson et al., 2004)

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99 Figure 32 : 1 sialic acids increase the rate of fast inactivation while decreasing the rate of recovery from fast inactivation AB -60-40-20020Inactivation Time Constant (ms) 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Membrane Potential (mV) -140-120-100 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 Membrane Potential (mV)Recovery Time Constant (ms)

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100 Figure 32 : (continued) Filled Squares / Solid Lines: transfected into Pro5 cells. Filled Circles / Solid Lines: transfected into Lec2 cells. Filled Squares / Dashed Lines: 1 cotransfected with in Pro5 cells. Open Squares / Dashed Lines: 1 co-transfected with in Lec2 cells. Panel A: Time constant of fast inactivation (h ). Data are the average h S.E.M. as a function of membrane potential. Curves are non-theoretical point-topoint.. Panel B: Voltage dependence of the recovery time constants (rec ). Data are the average rec S.E.M. at a -140, -130, and -120 mV recovery potential. Lines are non-theoretical point-to-point. (Johnson et al., 2004)

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101 expressed with in Lec2 cells. Proper surface expression of the 1 mutant was confirmed by tagging with GFP (see Johnson et al., 2004 for details). The addition of the 1 mutant data strongly indicates that 1 modulation of Nav1.5 is sialic acid dependent. SDS-PAGE analysis indicates good expression and glycosylation of 1 using the Pro5 / Lec2 mammalian expression system In order to verify the expression of 1 in transfected Pro5 and Lec2 cells, immunoblot analyses of homogenized transfected Pro5 and Lec2 cells were run. Figure 33 compares 1 transfected Pro5 and Lec2 cells to those of GFP transfected controls. Lanes 2 and 5 show GFP transfected Lec2 and Pro5, respectively, and there is an obvious lack of a band in the predicted 22 37 kd range as previously described (Patton et al., 1994;Sutkowski & Catterall, 1990). Lanes 1 and 4 shows 1 transfected Lec2 and Pro5, respectively, with each lane showing a clear band in the 22 37 kd range indicating good 1 expression throughout. Transfection with 1 fails to modulate neonatal ventricular Nav gating To determine if the difference in Na+ channel gating in the developing ventricle is a 1-induced phenomenon, neonatal VCM were transfected with 1. Whole cell recordings studying Va and Vi for the transfected neonatal VCM were compared to that of untransfected controls. Preliminary data indicates that

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102 Figure 33 : Immunoblot of 1 transfected into Lec2 and Pro5 cells Lec2 Pro5 GFP GFPLec2 Pro5 GFP GFP GFP GFP

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103 Figure 33 : (continued) Lane 1 is 1 transfected into Lec2 cells. Lane 2 is GFP transfected control in Lec2 cells. Lane 3 is a molecular weight marker lane. Lane 4 is 1 transfected into Pro5 cells. Lane 5 is GFP transfected Pro5 cells. Bands detected using 1Ab at a 1:200 concentration.

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104 transfection of 1 has no significant effect on neonatal ventricular Nav Va (fig. 34A). This experiment also demonstrates the same lack of effect on Vi with 1 transfection (fig. 34B). Is it possible that, for some unknown reason at the studied age that 1 is not properly interacting with on a physical level, or, could the apparently reduced capacity of the neonatal ventricle to sialylate proteins be responsible for the lack of effect of transfected 1 on Nav gating, similar to 1 expressed in Lec2? SDS-PAGE analysis indicates adult ventricular expression of 1 exceeds that of atria and neonatal ventricle Our recent work has implicated the 1 sialic acids to be its primary modulatory tool of action on various Nav gating parameters (Johnson et al., 2004). Immunoblot analysis of neonatal and adult rat ventricular and atrial homogenates were done to observe the relative amount of 1 present in each of the four samples for comparative analysis. In figure 35, lanes 2, 3, 4, and 5 are neonatal ventricle, adult ventricle, neonatal atria, and adult atria, respectively. It is clear that in a side-by-side comparison the adult ventricle possesses more 1 protein. The other three samples also contain trace amounts of 1. In the ventricle, 1 appears to be up regulated with development and differentially expressed between cardiac chambers.

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105 Figure 34 : Preliminary results indicate that 1 transfection has no significant impact on neonatal VCM Nav activation or inactivation, potentially due to a lack of 1 subunit sialylation BMembrane Potential (mV) -140-120-100-80-60-40-20Normalized Current 0.00 0.20 0.40 0.60 0.80 1.00 1.20 AMembrane Potential (mV) -120-100-80-60-40-20020Normalized Conductance 0.00 0.20 0.40 0.60 0.80 1.00 1.20 nVCM wo/ 1 = -38.7 1.85 (n=10)nVCM w/ 1 = -36.4 0.97 (n=4)nVCM wo/ 1 = -79.57 1.57 (n=10)nVCM w/ 1 = -79.02 3.79 (n=4)

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106 Figure 34 : (continued) Filled Circles: Untransfected neonatal VCM. Open Circles: Neonatal VCM transfected with 1. Panel A: G-V relationships for neonatal VCM 1. Data are the average normalized peak conductance S.E.M. at a membrane potential. Curves are fits of the data to single Boltzmann distributions. Panel B: Channel availability (hinf) for neonatal VCM 1. Data are the average normalized peak current S.E.M. measured during a 5 ms test pulse to +40 mV following a 500 msec prepulse to the plotted potentials. Curves are fits of the data to single Boltzmann distributions.

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107 Figure 35 : Immunoblot of 1 in neonatal and adult ventricle and atria NVAVNAAA NVAVNAAA NVAVNAAA

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108 Figure 35 : (continued) 1 protein is most prominent in the adult ventricle when compared to the less dense bands seen in neonatal ventricle/atria and adult atria. Lane labels: NV: neonatal ventricle. AV: adult ventricle. NA: neonatal atria. AA: adult atria. Lane 3 is a molecular weight marker. Bands detected using 1Ab at a 1:200 concentration.

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109 Discussion Differential sialylation of the subunit is a major contributor to developmental shifts in ventricular Nav gating not observed in atria Comparison of Nav gating measured from neonatal and adult ventricle indicated that the adult channel gates at potentials about 10 mV more hyperpolarized than does the neonatal channel. In addition, neonatal and adult atrial Nav were tested and, in contrast to ventricle, gated at similar potentials to each other and adult ventricle. More importantly, this demonstrated a difference in Nav channel gating between neonatal atria and ventricle not seen in the adult. In response to these differences, I sought to explore what might be responsible for this Nav remodeling observed throughout development and between cardiac chambers. It was hypothesized that developmentand/or chamberdependent changes in the amount of channel associated functional sialic acids might account for these observed shifts in gating. Neuraminidase treatment was used to remove sialic acids from the cell surface expressed Nav of the ventricular and atrial cardiomyocytes. None of the tested voltage dependent gating parameters for the neonatal ventricular Nav was affected by neuraminidase treatment. When treated, all of the atrial and adult ventricular Nav gating parameters shifted to more depolarized potentials similar in value to those measured for untreated (or treated) neonatal ventricular Nav. Also, neuraminidase treatment resulted in no

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110 significant change in the channel activation slope values, Ka, for any of the tested conditions. This implies no consequence of desialylation on the effective valency of the channel S4 gating charges that move in response to the changing electric field. As a result, desialylation apparently alters the electric field sensed by the channel gating charges, not the charges themselves. These results provided strong evidence that sialic acids, as part of N-glycosylation trees attached to the Nav channel, are responsible for the observed differences in gating. To determine whether sialic acids are fully responsible for the gating differences observed between myocyte chambers and ages, desialylation was achieved via removal of the entire N-linked glycosylation trees attached to the channel. Removal of N-linked sugars through PNGase-F treatment (Figure 28) indicated that all gating parameters for atrial and adult ventricular channels behaved nearly identically to those measured for the neonatal ventricular channel. Similar to what was seen with neuraminidase treatment, PNGase-F treatment again resulted in no significant change in Ka for any of the tested conditions. This indicates no additional outcome of deglycosylation on the effective valency of the channel S4 gating charges that move in response to the changing electric field. The data suggest that the differences in Nav gating between neonatal heart chambers, and throughout ventricular development, can be accounted for by differential glycosylation, with changes in functional sialic acid levels being the most important. That is, functional sialic acids can account for the majority of voltage-dependent gating differences observed, while removal of the full N-glycosylation structures can account for all measured shifts among

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111 Nav. It is possible that two distinct N-glycosylation dependent mechanisms are responsible for the hyperpolarizing shifts in Nav gating. However, it is more likely that while neuraminidase treatment consistently removes functional sialic acids, it may not have removed all functional sialic acids for each cell studied, while PNGase-F treatment was much more effective. If this were the case, the observed SA-dependent effects on gating would be underestimated, resulting in measured parameters that apparently did not fully shift with desialylation. To strengthen the electrophysiological evidence, biochemical analysis was done to determine if atrial and adult ventricular Nav are more heavily sialylated than neonatal ventricular Nav. Immunoblot analysis of the neonatal and adult atria and ventricular Nav subunits revealed that the neonatal ventricular Nav was lighter in weight than the other three. When the homogenates were exposed to PNGase-F, the resultant immunoblot weights for all four samples were now similar to each other, and to the predicted unprocessed weight of Nav1.5 indicating no difference in isoform expression. These results allow us to conclude that the initial difference in Mr observed for the four conditions is due to differences in posttranslational modification. When treated with neuraminidase, gel shift analysis indicated that approximately 15 more sialic acid residues are attached to the atria and adult ventricular Nav than the neonatal ventricle. These data are consistent with the biophysical measurements, suggesting that most of the differences in gating between neonatal ventricular Nav and the Nav expressed in atria and adult ventricle are the result of fewer functional sialic acids attached to the neonatal ventricular subunit. According to these findings the number,

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112 and possibly location, of functional sialic acid residues added to cardiac Nav are regulated as a function of cardiac development and chamber. Potentially this difference is due to changes in the activity level of many of the different sialyltransferase enzymes throughout development. Chronic versus acute channel regulation Differential sialylation may potentially modulate Nav gating through two different mechanisms, acute or chronic. In the case of acute regulation, various Nav subunits, which are differently glycosylated / sialylated, might be expressed at various periods in the life of an excitable cell. This difference in Nav subunit sialylation may directly and differently alter channel gating and cellular excitability. As previously noted, the primary neonatal and adult cardiac Nav subunit isoform is believed to be the same gene product (Nav1.5). However, the primary neonatal skeletal muscle Nav subunit is Nav1.5 while the primary adult skeletal muscle Nav subunit is the Nav1.4 isoform (Cohen & Barchi, 1993;Messner & Catterall, 1985;Miller et al., 1983). In addition, it was shown that the adult skeletal muscle subunit isoform is more heavily glycosylated than the neonatal skeletal muscle subunit isoform (Cohen & Levitt, 1993;Zwerling et al., 1991;Kraner et al., 1989;Gordon et al., 1988;Roberts & Barchi, 1987). To compare these two different Nav isoforms, a study comparing the sialic acid dependent gating of the heavily glycosylated Nav1.4 to the lesser glycosylated Nav1.5 in two types of CHO cell lines was done. When expressed in Pro5 cells (fully sialylating CHO cells) versus Lec2 cells (sialylation deficient CHO cells) it

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113 was shown that Nav1.4 gating is much more sensitive to sialic acids than is Nav1.5 gating. The study went on to localize the functional sialic acid residues to the IS5-S6 loop of Nav1.4 (Bennett, 2002). Thus, channel gating appears to be partially dependent on the “glycosylation signature” of the subunit expressed. This means that skeletal muscle cells, at different periods in their development, may be differently excitable, dependent upon which Nav subunit is expressed at the time. This demonstrates how the number and / or location of sialic acids indeed vary from one subunit to another, producing a potential spectrum of functional sialic acids among Nav subunits that may directly and differently modulate channel gating acutely. The data shown here are consistent with a second “chronic” mechanism by which differential channel sialylation modulates Nav gating. In the case of chronic regulation, the glycosylation signature of the expressed channels of the cell are not modified through the expression of different subunit isoforms, rather via changes in the level of sialyltransferase activity with respect to a single subunit isoform. Altered transferase activity would lead to altered levels of channel functional sialic acids thus affecting cellular excitability. We show here that ventricular Nav gating shifts in the hyperpolarized direction with development, which can be abolished with neuraminidase treatment. Using biochemical means it was shown that in the adult ventricle the Nav subunit is apparently more heavily sialylated than the neonatal ventricular subunit. These data are consistent with a chronic increase in sialyltransferase activity in the

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114 developing ventricular cardiomyocytes. Also, a higher level of sialyltransferase activity in the neonatal atria versus ventricle can explain the increase in atrial subunit functional sialylation. Recently, our laboratory has had Gene Chip analysis performed on mouse atrial and ventricular myocytes done at neonatal and adult ages. These data have yielded results consistent with our idea of chronic regulation. In detail, the up regulation of sialyltransferase expression in the developing ventricle and between chambers of the neonatal heart was shown to be apparent. With increased sialyltransferase activity, more functional sialic acids might be added to the subunit, directly modulating the voltage at which atrial and developing ventricular Nav gate. Control of Nav gating by differential sialylation appears to be intra and inter tissue specific Differential channel sialylation appears to be a mechanism by which cardiac Nav activity may be modulated throughout development or between cardiac chambers. A recent study of Nav1.9 function in rat dorsal root ganglia, also reported developmental regulation of channel sialylation. This study from the Waxman laboratory indicated that an apparent decrease in Nav1.9 sialylation through development as expressed in dorsal root ganglion (DRG) neurons was responsible for a depolarizing shift in the half-inactivation voltage for Nav1.9. More heavily sialylated neonatal Nav1.9 inactivated at more hyperpolarized potentials than did the adult Nav1.9. Channel activation voltage was unaffected by this differential sialylation (Tyrrell et al., 2001). Additionally, NCAM is an example

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115 of another protein, not a voltage-gated ion channel, which is differentially sialylated throughout development, similarly to Nav1.9 becoming less sialylated with age. Reports have indicated that the highly polysialylated neonatal NCAM, with its decreased adhesive function, serves to maintain the integrity of the developing neuroepithelium, while in the adult, the less polysialylated highly adhesive NCAM is used to stabilize the differentiated neural structures in the adult (Breen & Regan, 1988;Sunshine et al., 1987;Mccoy et al., 1985;Finne, 1982;Hoffman et al., 1982;Rothbard et al., 1982). Importantly, this is good evidence to support a possible role of sialic acid in modulating the actions of different elements in multiple unrelated processes. Additionally, these reports and the data shown here lead to a rather provocative and novel finding: chronic differential sialylation evidently modulates Nav gating throughout development in a tissueand intra-tissue (cardiac atria vs. cardiac ventricle) specific manner. The NCAM research showed that the differential sialylation of NCAM throughout the developing neuroepithelium appeared to be vital in the correct construction of the neural pathways. The DRG study showed that only channel inactivation voltage was affected by apparently lower levels of sialic acid attached to the adult Nav1.9. Here, the data indicate that adult ventricular Nav are apparently more heavily sialylated than neonatal ventricular Nav. As a result of this increased sialylation, all tested voltage dependent gating parameters were shown to gate at more hyperpolarized potentials. Speaking strictly of voltagegated ion channels, the opposing changes in Nav1.9 and Nav1.5 sialylation throughout the development of two different cell types suggest that channel

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116 gating may be modulated chronically through enzyme regulated tissue-specific differential sialylation. Furthermore, a lack of changing sialylation with atrial development suggests that the same isoform may be differently chronically regulated, or not, within the same organ system as well. Here we observed essentially uniform hyperpolarizing shifts in all gating parameters with increased sialylation for adult ventricular Nav1.5 while the DRG study reported non-uniform depolarizing shifts for the lesser sialylated adult Nav1.9. Therefore, in addition, these observations suggest that the modulation of Nav gating through chronic differential sialylation may be isoform-specific as well. Acute regulation of Nav gating via 1 expression When one considers how the addition of other elements, such as auxiliary subunits interacting with subunits, and the additional sialic acids they may also bring to the area can affect channel gating, the situation can become even more complicated. Nav1.5 has been shown to interact with 1 and 2 in the heart (Maier et al., 2004;Malhotra et al., 2004). The effects of channel sialylation, more specifically on the Nav individual subunits, and 1, that can be expressed together were examined in two ways. It was hypothesized that the 1 sialic acids may further exert a modulatory effect on channel gating through a novel transregulatory mechanism. To do this the Nav1.5 subunit was expressed with 1 in fullyand non-sialylating CHO cells and 1 alone in neonatal VCM. The expression system studies showed that all of the measured effects of 1 on

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117 channel gating were due to 1 sialic acids (Johnson et al., 2004). Data gathered after transfecting 1 into neonatal rVCM revealed no change in gating from untransfected conditions. This can possibly be explained in two ways, either the transfected 1 subunit did not interact with Nav1.5, or the lesser Nav sialylating neonatal rVCM also fails to fully sialylate 1 thus rendering it nonfunctional. Throughout various excitable tissues it has been shown that 1 processing and expression appears to be developmentally regulated (Sashihara et al., 1996;Sashihara et al., 1995;Yang et al., 1993;Sutkowski & Catterall, 1990). This potential use of auxiliary subunits is another example of chronic and acute channel modulation via differential sialylation. Changing levels of overall channel sialylation through changing levels of sialyltransferase activity will impact subunits chronically, thus regulating channel gating. At the same time, regulation of expression levels may directly impact the level of overall channel associated sialic acids acutely regulating channel gating. As a result of this regulation of subunit sialylation, the excitability of the cell may be affected in a more controlled manner, producing a wide range of differentially sialylated Nav. Unfortunately, the inability to identify in native cells the / complexes in real time, while studying Nav function in real time is a major hurdle in determining the total impact of such varying subunit combinations in vivo. Perhaps the use of a fluorescent tagged antibody specific for 1 can be used to at least confirm, or not, the surface expression of 1 following expression in transfected cardiac myocytes.

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118 Potential physiological impact of increased or decreased sialylation and future experiments Here it is shown that functional sialic acids added to the developing ventricular Nav subunit directly alter channel gating. In addition, neonatal atrial Nav have a greater level of functional sialic acids than do neonatal ventricular Nav. What might be the physiological role for such developmental and spatial remodeling? There are at least two mechanisms by which sialic acid dependent shifts in Nav gating might modulate cardiac Na+ channel activity such that the spreading depolarizing/repolarizing waves across the heart would be affected: 1) A hyperpolarizing shift and slight increase in the window current, and 2) A significant slowing of the rate of recovery from fast inactivation. The distribution of channels between the open and inactivated states at a membrane potential affects the percentage of channels that are persistently active. This distribution can be represented graphically by overlapping G-V and steady-state inactivation curves, and is referred to as the “window current” (Attwell et al., 1979). The intersecting area under the curves represents the voltage range at which a small percentage of channels may be persistently active. There are several reports that show point mutations of SCN1A and SCN5A cause depolarizing shifts in the voltage range of the window current that may be responsible for such maladies as epilepsy and arrhythmias (LQTS) (Spampanato et al., 2003;Abriel et al., 2001;Splawski et al., 2002). It has been proposed that changes that act to depolarize the window current, either through subunit mutation or desialylation, can lead to LQTS by creating persistent inward

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119 sodium current later in phase 2. This inward current will then act against the beginning delayed rectifier outward current, extending phase 2 of the cardiomyocyte action potential and, therefore, increasing the L-Q time period. For the atrial and adult ventricular Nav, the window current will be about 10 mV more hyperpolarized than the neonatal ventricular Nav window current, caused by the sialic acid dependent hyperpolarizing shifts in the G-V and hinf curves for atrial and adult ventricular Nav. This will cause persistent atrial and adult ventricular Nav activity to occur at more hyperpolarized potentials, thus impacting cardiomyocyte excitability in a positive fashion. Potentially this may serve to create a more easily excitable cell, thus protecting the integrity of the spreading action potential, while, at the same time protecting the cells from LQTS abnormalities. The sialic acid dependent slowing of the rate of recovery from fast inactivation likely directly affects cardiac excitability. It is observed here that atrial and adult ventricular Nav recover from fast inactivation more slowly than do the neonatal ventricular Nav. Also, atrial and adult ventricular Nav recovery rates increase with desialylation to values close to or faster than neonatal ventricular Nav recovery rates, indicating that the slower recovery rates are due to an increase in functional sialic acids. This slower recovery rate will alter the effective time between successive depolarizing / repolarizing waves because of the redistribution of Nav among functional states toward a larger population of inactivated channels. That is, at a short time following the initialization of a Nav kinetic cycle, the percentage of atrial and adult ventricular Nav still in the

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120 inactivated state will be greater than the percentage of inactivated neonatal ventricular channels, thus extending the absolute refractory period. This will directly affect the rate at which subsequent depolarizations might lead to activation of significant Nav. Because a larger percentage of atrial and adult ventricular Nav remain inactivated for longer periods of time, these slower recovery rates will act to limit early after depolarizations (EADs), often considered a leading cause of tachycardias. One study, published by Chen et al., showed that certain SCN5A mutations that cause familial idiopathic ventricular fibrillation, IVF, was the result of a 25-30% increase in the Nav1.5 rate of recovery at a -80 mV recovery potential (Chen et al., 1998). In this study, desialylation results in an even greater increase in sodium channel rates of recovery from inactivation than those resulting from the aforementioned SCN5A mutations. Normally, as an action potential is propagated to the left and right ventricles the signal that meets in the middle is cancelled out due to the left and right ventricular myocytes now being in their respective refractory periods. In the case of unidirectional conduction block, myocytes distal to the damaged area causing the block have not been excited by the myocytes proximal to the block. As a result, the normally cancelled out signal is now allowed to propagate from the myocytes distal of the block to the proximal myocytes. Under normal conditions the proximal cells are in their refractory periods, thus preventing reentry through the damaged area of the heart. Under abnormal conditions resulting either from mutation or, in this case, reduced channel sialylation, the proximal myocytes, having recovered faster than normal from inactivation, fail to prevent reentry of the signal leading to

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121 circus movement and potentially fatal ventricular fibrillation. Thus, increased sodium channel sialylation leads to slower rates of recovery from fast inactivation, which will effectively slow the Nav kinetic cycle. This will directly affect whether and what percentage of Nav from an individual myocyte will activate in response to the depolarizing / repolarizing waves propagating across the heart, thereby impacting heart rhythms. Thus, the majority of changes in ventricular Nav gating throughout development and the difference in the newborn’s atrial and ventricular Nav gating can be attributed to differential channel sialylation that directly modulates cardiac voltage-gated sodium channel gating. A physiological role for sialic acids in cardiomyocyte excitability has been demonstrated in a recent study (Ufret-Vincenty et al., 2001a). Cardiac excitability and Nav gating were studied using a heart failure mouse model, a knockout of the muscle LIM protein (MLP). When wild type ventricular myocytes were treated with neuraminidase they produced Na+ currents that gated similarly to those of untreated MLP-/myocytes, which are apparently less sialylated. This suggests that the observed extended action potential repolarization of the MLP-/myocyte was caused in part by lesser sialylated Nav. The knockout and neuraminidase treated wild type myocyte Nav both experienced a reduction and rightward shift in their window current. In addition, the rate of fast inactivation was slowed for desialylated and MLP-/myocytes. Each of these gating effects is consistent with those described here for the SA-insensitive neonatal ventricular Nav, and for all desialylated conditions. The authors concluded that the lesser sialylated Nav of

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122 the MLP-/myocytes might be responsible for the extended action potential duration and increased susceptibility to EADs observed in this study. Thus, differential sialylation of Nav will likely impact cardiac excitability through one or more of the mechanisms described herein. Increased Nav sialylation leads to slower rates of recovery from fast inactivation, effectively extending the refractory period, which should lower susceptibility to EADs. Furthermore, the SA-dependent leftward shift in window current has been implicated in limiting action potential duration. Consequently, increased sialylation may cause the initial wave of excitation to occur at smaller depolarizations, while serving to also limit and control subsequent excitability, thus acting to help modulate and regulate heart rhythm. It is important to realize that this work is merely a small piece of the puzzle that describes cardiac excitability. It has been shown that sialic acid glycosylation may be a strong modulator of cardiac Nav gating. In this study the primary means used to test sialic acid’s influence on gating was with neuraminidase to remove cell surface expressed sialic acid residues, and PNGase-F to remove the N-glycosylation altogether. Other substances, for example swainsonine or castanospermine, that prevent the addition of sialic acids to the protein from inside the cell may also be used as another means to desialylate Nav. Other methods that can be used, similar to this idea of preventing sialylation first rather than removing it later, would be through the use of sialyltransferase deficient knock out mouse models. Not only could such knock out studies investigate specifically the effects of sialic acid on cardiac ion

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123 channels, it could also allow for observations of phenotypic maladies that manifest due to the sialic acid deficiency. It is true that without voltage-gated sodium channels there would be no fast response phase 0 and essentially no cardiac action potential. However, it is impossible to contemplate the cardiac action potential without thinking about the other ion channels involved, potassium and calcium channels. The cardiac iK + is generated by a combination of inward rectifier, delayed rectifier, and transient outward voltage-gated K+ channels. Ltype voltage-gated Ca2+ channels generate the cardiac iCa 2+ forming the myocyte phase 2 plateau, and are responsible for the slow response phase 0 in nodal pacemaker cells. To understand the impact of sialic acid on cardiac excitability we must first understand its impact on these voltage-gated K+ and Ca2+ channels too. Future electrophysiological and biochemical studies, similar to the ones done here, investigating the biophysical effects of, and relative levels of functional sialic acids on these individual channels can be done. Once completed, the individual sialylated vs. unsialylated data for each ion channel can be compiled and perhaps used to run cardiac simulations comparing the two conditions to each other. Action potential measurements done on cardiomyocytes before and after desialylation may further provide more data that can help answer some of the questions regarding the true purposes behind ion channel glycosylation.

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124 Summary Here we see first hand the potential effects of ion channel sialylation with regards to channel gating and cellular excitability. In this example there appears to be a consistent trend in biophysical properties for cardiac Nav when comparing fully sialylated to lesser sialylated channels. In all tested biophysical parameters the existence of greater levels of functional sialic acids acted to create a more readily excitable cell while also appearing to serve a protective function for the heart against various electrical conduction and physical pumping pathologies. It is possible that the atrial Nav are fully sialylated at birth to continually ensure a readily excitable atria providing for maximal ventricular filling to be accomplished in the allotted time before ventricular contraction, a desired optimal result at any atrial age. At the same time the lesser sialylated conditions of the neonatal ventricle, serving to increase recovery from inactivation, may facilitate the more rapid basal heart rate of the smaller neonatal heart. In the adult heart the reason for fully sialylated atrial Nav probably remains similar to that for the neonate. In the adult ventricle, however, this increase in functional channel sialylation, when compared to the neonate, may in fact serve to increase the excitability of the myocytes aiding in the production of ventricular systole. At the same time, due to the substantial increase in necessary conduction length in the larger ventricles, increased functional sialylation may likely aid in the prevention of the action potential from degrading before it fully spreads throughout the ventricles. Finally, and possibly most importantly, increased adult ventricular Nav functional sialylation, through its resultant decrease in Nav rates of recovery from

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125 inactivation, may help to prevent electrical disturbances of the ventricles that may otherwise result in the death of the animal.

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126 Table 1. Gating parameters for adult and neonatal cardiomyocyte Nav under conditions of full and reduced sialylation / glycosylation The mean gating parameter values SEM are listed. Significance was determined using a two-tailed student’s t-test comparing sample groups as follows: 1) Untreated samples were compared to untreated neonatal VCM, 2) Treated samples were compared to: a) their untreated cell type, and b) neonatal VCM. # = Not significant. = Significant (P < 0.02). ** = Highly significant (P < 0.01). Cell n Va (mV) Vi (mV) h @ -50 mV (msec) !rec @ -120 mV (msec) Neonatal VCM Untreated 11 -46.1 1.0 -87.8 1.5 4.0 0.2 7.9 0.3 Neonatal VCM Neuraminidase 11 # -46.8 1.0 # -88.7 1.2 # 4.9 0.4 # 7.9 0.6 Neonatal VCM PNGase-F 6 # -49.5 1.5 # -90.6 1.1 # 3.6 0.3 # 8.4 0.6 Adult VCM Untreated 8 ** -56.9 1.1 ** -97.1 2.7 ** 2.8 0.1 ** 13.7 1.9 Adult VCM Neuraminidase 8 a**; b# -47.3 1.6 a**; b# -83.8 2.1 a**; b# 4.7 0.7 a**; b** 5.4 0.7 Adult VCM PNGase-F 7 a**; b# -48.3 2.0 a**; b# -86.7 2.6 a**; b# 3.7 0.5 a**; b# 7.6 0.7 Neonatal ACM Untreated 17 ** -57.0 0.6 ** -100.0 0.7 ** 2.2 0.1 ** 18.1 1.2 Neonatal ACM Neuraminidase 13 a**; b# -49.9 1.1 a*; b** -95.4 1.8 a**; b** 3.2 0.2 a**; b** 13.2 1.7 Neonatal ACM PNGase-F 6 a**; b# -47.2 2.0 a**; b# -86.3 2.0 a**; b# 4.5 0.6 a**; b# 8.0 0.9 Adult ACM Untreated 13 ** -56.7 1.0 ** -96.7 1.5 ** 2.0 0.1 ** 14.5 1.5 Adult ACM Neuraminidase 11 a**; b# -49.9 1.0 a**; b# -91.3 0.9 a**; b** 3.3 0.2 a**; b# 9.7 0.8 Adult ACM PNGase-F 7 a**; b# -50.0 1.0 a**; b# -88.8 1.9 a**; b# 3.0 0.3 a**; b# 9.9 1.2

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127 Table 2. Gel shift analyses for neonatal and adult cardiomyocyte sodium channels under conditions of full and reduced glycosylation / sialylation Data list the mean Mr +/SEM measured for neonatal and adult cardiomyocyte Nav subunits. For the untreated data and their comparison to the treated homogenates, significance was determined using a two-tailed student’s t-test comparing the atrial and adult ventricular subunit Mr to the neonatal ventricular subunit Mr. ** = Highly significant (P < 0.01). Cell n Untreated n PNGase-F n Neuraminidase Neonatal Ventricle 19 246.5 0.8 6 ** 231.6 1.9 13 ** 241.2 0.7 Adult Ventricle 18 ** 251.8 1.2 6 ** 231.3 2.0 12 ** 242.0 1.1 Neonatal Atria 12 ** 252.2 0.5 4 ** 230.2 1.1 8 ** 243.1 0.6 Adult Atria 11 ** 251.4 0.4 4 ** 230.7 1.2 7 ** 243.3 0.5

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128 Table 3: Gating parameters for Nav1.5 transfected with or without 1 in the fully sialylating Pro5 or sialylation deficient Lec2 mammalian cell lines The data are the mean parameter values S.E.M. Significance was tested using a two-tailed student’s t-test, with each condition being compared to that measured for the fully sialylated subunit alone. ** = highly significant (p < 0.005). = significant (p < 0.1). Cell n Va (mV) Vi (mV) h @ -40 mV (ms) !rec @ -120 mV (ms) Pro5 + 13 -29.0 2.2 -78.7 2.5 2.8 0.4 4.0 0.1 Lec2 + 10 -29.5 1.6 -79.5 1.9 2.4 0.2 4.1 0.1 Pro5 + + 1 11 ** -37.4 1.6 -86.1 3.2 2.0 0.1 ** 5.6 0.3 Lec2 + 1 9 -28.6 0.9 -78.8 1.6 2.9 0.2 4.1 0.1

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141 Bibliography Alberts, Bruce, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. Molecular Biology of the Cell, Fourth Edition New York: Garland Science, 2002. Berne, Robert M., and Matthew N. Levy. Physiology, Fourth Edition St. Louis: Mosby Electronic Production, 1998. Lodish, Harvey, David Baltimore, Arnold Berk, S. Lawrence Zipursky, Paul Matsudaira, and James Darnell. Molecular Cell Biology New York: Scientific American Books, Inc., 1995.

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142 ABOUT THE AUTHOR Patrick John Stocker was born August 29, 1975 in Poughkeepsie, New York. He grew up in Verbank, New York where he attended Millbrook Jr. / Sr. High School where he graduated valedictorian of both his Jr. and Sr. High School classes. He proudly attained the rank of Eagle Scout in the Boy Scouts of America at the age of sixteen. Upon graduating from Sr. High School he traveled abroad as a Rotary Club International Exchange Student to Sweden for a year. In 1994 he was enrolled as a freshman at the State University of New York College at Potsdam, New York where he graduated in 1998 with a B.A. degree majoring in Biology. In 1999 he was accepted to the graduate program at the University of South Florida College of Medicine Department of Physiology and Biophysics majoring in Medical Sciences. In 2001 he received his M.S. in Medical Sciences and was accepted as a candidate for Ph.D. study in the laboratory of Eric Bennett, Ph.D. Finally, in 2005 he successfully completed the requirements necessary to be awarded his Ph.D. in Medical Sciences from the University of South Florida College of Medicine.


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Sialic acid modulation of cardiac voltage-gated sodium channel gating throughout the developing myocardium
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ABSTRACT: The proper orchestration of voltage-gated ion channel gating is vital to maintaining normal heart rhythms throughout an animal's lifespan. Voltage-gated sodium channels, Nav, are responsible for the initiation of the cardiac action potential, which leads to cardiac systole. Comparison of neonatal ventricular and atrial myocyte Nav gating with adult indicated that the neonatal ventricular Nav gated following a ~10 mV greater depolarization than did atrial or adult ventricular Nav. In this study I questioned whether development- and/or chamber-dependent changes in Nav-associated functional sialic acids could account for these differences. When desialylated with neuraminidase, all gating characteristics for the lower voltage activated atrial and adult ventricular Nav shifted significantly to more depolarized potentials. However, desialylation of the higher voltage activated neonatal ventricular Nav had no effect on channel gating. Furthermore, channels were stripped of^ their N-glycosylation via PNGase-F in an attempt to separate the potential effects of the remaining glycosylation structure on Nav gating. Following treatment, neonatal ventricular Nav gating remained unchanged while atrial and adult ventricular Nav gating again shifted to depolarized potentials nearly identical to those of the neonatal ventricular channel. Immunoblot analyses indicated that atrial and adult ventricular Nav a subunits are more heavily sialylated than the neonatal ventricular a subunit, with approximately 15 more sialic acid residues. The data indicate that differential sialylation of myocyte Nav a subunits is responsible for much of the developmental and chamber-specific remodeling of Nav gating observed here. In addition, the Nav1.5 a subunit can associate with b subunits, also believed to be sialylated. The potential for functional b1 trans sialic acids to further modulate Nav1.5 gating was tested via co-transfection of b1 with the Nav1.5 a subunit into the Pro5 /Lec2 mammalian expression system. Co-transfection revealed that the additional b1 trans sialic acids caused a hyperpolarizing shift in all tested gating parameters. When transfected into neonatal ventricular myocytes, b1 expression revealed no effect, implying that b1 expression alone is not responsible. Together, the myocyte and expression system studies describe a novel mechanism by which Nav gating, and subsequently cardiac excitability, are modulated by the regulated change in channel-associated functional sialic acids.
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