Functions of Fibroblast Growth Factor Homologous Factor 2 in Excitable Tissues

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Functions of Fibroblast Growth Factor Homologous Factor 2 in
Excitable Tissues, Is A Well-Researched Topic, It Is To Be Used As A Guide Or Framework For Your Research.

Abstract

Purpose: Fibroblast Growth Factor Homologous Factors (FHFs) are a group of proteins known to associate with and modulate voltage-gated sodium channels (Nav) in excitable cells. The four FHF genes are differentially expressed in specific cell-types, with FHF2 expressed prominently in the hippocampus, cerebral cortex, heart and dorsal
root ganglia. Due to previous unavailability of an Fhf2 knockout mouse, this gene’s functions have been understudied in comparison to other those encoding other FHFs. The purpose of this research has been to better understand the normal physiological functions of FHF2 at the cellular and system levels in the heart, sensory nervous system and central nervous system. These studies also include functional analysis of a point mutation in FHF2 that causes early-onset epileptic encephalopathy (EOEE) in humans. Methods: A stable fertile Fhf2KO mouse line was derived and used for phenotypic observation, in vivo
experimentation, tissue harvest and primary cell culture for imaging, protein analysis and electrophysiological recordings. Whole-cell patch clamp electrophysiology on primary cultured cardiomyocytes and dorsal root ganglion (DRG) cells supplemented with ectopic expression experiments in transfected HEK and Neuro2A cells was used to determine the modulatory effects of various FHF2 isoforms on various sodium channels. Results: FHF2 is required for proper cardiac function specifically under hyperthermic conditions. In the absence of FHF2, there is a breakdown in cardiac conduction due to the impairment of cardiomyocyte action potential generation. Without FHF2 there is a hyperpolarizing shift in cardiac Nav1.5 steady-state inactivation decreasing Nav1.5 availability.

This reduces overall available sodium conductance at resting potential
which, when paired with accelerated inactivation brought about by elevated temperatures, suppresses excitability. FHF2 is also necessary for topical heat nociception, as Fhf2KO mice do not respond to noxious heat. The nociceptive deficit is reproduced in mice where Fhf2 disruption is restricted to peripheral sensory neurons. Our experiments support an
underlying mechanism that parallels the cardiac phenotype, as the absence of FHF2 induces hyperpolarizing shifts in the predominant sodium channel isoforms Nav1.7 and Nav1.8 expressed in DRG nociceptors. Reduced Nav1.7 availability is again exacerbated
by more rapid channel inactivation at elevated temperature, which correlates with reversible temperature-sensitive conduction failure in Fhf2KO peripheral sensory fibers. Lastly, in light of recently discovered missense mutations in the A-type first exon of the FHF2 gene in several people suffering from EOEE, I have shown that these mutations
prevent anti-excitatory FHF2A-mediated Nav1.6 fast-onset long-term inactivation without impairing the pro-excitatory modulation of Nav1.6 steady-state inactivation, likely leaving CNS neurons hyperexcitable and susceptible to the epileptogenic phenotype.

Table of Contents

i – Title Page
ii – Copyright
iii – Approval Page
iv – Abstract
vi – Acknowledgements
ix – Table of Contents
xv – List of Figures & Tables
xvi – Abbreviations
Chapter 1: Introduction ———————————————————————- – 1 –
1.1 Fibroblast Growth Factor Homologous Factors ————————— – 1 –
1.1.1 FGFs vs. FHFs ——————————————————– – 1 –
1.1.2 Genes & Expression Patterns ————————————— – 1 –
1.1.3 Isoform Specific Functions —————————————– – 2 –
1.2 Fibroblast Growth Factor Homologous Factor 2 ————————- – 4 –
1.2.1 Gene & Expression Pattern —————————————– – 4 –
1.2.2 Known Functions to Date ——————————————- – 5 –
1.2.3 Direct FHF2 Links to Disease ————————————– – 6 –
1.3 Other FHF Links to Human Disease —————————————– – 6 –
1.3.1 Mutated FHF1 in Epileptic Encephalopathy ———————- – 6 –
1.4 Voltage-gated Sodium channels ———————————————– – 7 –
1.4.1 General Structure & Isoforms ————————————– – 8 –
1.4.2 Voltage-gated Sodium Channel Gating & Types of Inactivation —
——————————————————————– – 10 –
1.4.2.1 Fast Inactivation ——————————————— – 12 –
1.4.2.2 Slow Inactivation ——————————————- – 13 –
1.4.2.3 Long-term Inactivation ————————————- – 14 –
1.4.2.4 Transient Open-channel Block —————————- – 14 –

1.5 Voltage-gated Sodium Channels in Cardiac Function & Heart Disease —
—————————————————————————————- – 15-
1.5.1 Nav in Cardiac Function ——————————————- – 15 –
1.5.1.1 The Heart —————————————————– – 15 –
1.5.1.2 The Cardiac Conduction System ————————– – 16 –
1.5.1.3 Nav 1.5 ——————————————————– – 18 –
1.5.1.4 Post-translational Modifications and Interacting Proteins —-
—————————————————————– – 18 –
1.5.2 Nav Associated Heart Disease ———————————— – 20 –
1.5.2.1 Long QT Syndrome —————————————- – 20 –
1.5.2.2 Brugada Syndrome —————————————– – 21 –
1.5.2.3 Sick Sinus Syndrome ————————————– – 22 –
1.5.2.4 Progressive Cardiac Conduction Disease —————- – 22 –
1.6 Pain Sensation & Sensory Conduction ———————————— – 23 –
1.6.1 Noxious Heat Pain Sensory Acquisition & Pathway ———– – 24 –
1.6.2 Transient Receptor Protein Cation Channel V1 ————— – 26 –
1.6.3 Role of Peripheral Sensory Nervous System Navs in Nociception —
————————————————————————- – 27 –
1.6.3.1 Nav1.7 ——————————————————– – 27 –
1.6.3.2 Nav1.8 ——————————————————– – 28 –
1.6.3.3 Nav1.9 ——————————————————– – 29 –
1.7 Voltage-Gated Sodium Channelopathies and Epilepsy —————- – 30 –
1.8 Preview to Dissertation Research ——————————————- – 32 –
Chapter 2: Materials & Methods ——————————————————— – 33 –
2.1 Plasmids & Mutagenesis ——————————————————- – 33 –
2.1.1 Nav Plasmids ——————————————————– – 33 –
2.1.2 FHF2 Plasmids —————————————————— – 33 –
2.2 Transfection ———————————————————————- – 33 –
2.2.1 Nav and FHF2 Expression in HEK 293T Cells —————– – 33 –
2.2.2 Nav and FHF2A Expression in Neuro2A Cells —————- – 34 –

2.3 Embryonic Stem Cells, Blastocyst Transplantation & Pronuclear
Injection ———————————————————————- – 34 –
2.4 Mouse Breeding & Genotyping ———————————————- – 35 –
2.5 Antibodies Used —————————————————————– – 37 –
2.6 Western-blot & Co-Immunoprecipitation ——————————— – 37 –
2.7 Immunofluorescence ———————————————————– – 38 –
2.8 Electrophysiological Recordings of Sodium Currents in HEK293T Cells —
——————————————————————————————- – 39 –
2.8.1 V-Clamp Protocols ————————————————- – 40 –
2.9 Isolation of Mouse Ventricular Cardiomyocytes ————————- – 41 –
2.10 Electrophysiological Recordings of Isolated Ventricular Cardiomyocytes
——————————————————————————————- – 42 –
2.10.1 I-Clamp Protocols ————————————————– – 43 –
2.10.2 V-Clamp Protocols for Sodium Current ————————- – 43 –
2.11 Isolation of Dorsal Root Ganglion Sensory Neurons ——————- – 44 –
2.12 Electrophysiological Recordings of Isolated Dorsal Root Ganglion
Sensory Neurons ——————————————————————— – 45 –
2.12.1 V-Clamp ————————————————————- – 45 –
2.12.2 I-Clamp Protocols ————————————————– – 46 –
2.13 Epilepsy Recordings ———————————————————- – 47 –
2.13.1 V-Clamp Protocols ————————————————- – 47 –
2.14 Statistical Analysis ———————————————————— – 48 –
Chapter 3: Derivation of Transgenic FHF2 Mouse Lines —————————- – 49 –
3.1 Derivation of FHF2Targeted Mice ———————————————- – 49 –
3.2 Derivation of FHF2KO Mice ————————————————— – 51 –
3.3 Derivation of FHF2Con Mice ————————————————- -53 –
Chapter 4: Absence of FHF2 Causes Temperature-Sensitive Cardiac Conduction
Failure ————————————————————————————— – 54 –

4.1 The Absence of FHF2 Results in a Temperature Sensitive Cardiac
Conduction Failure —————————————————————– – 56 –
4.1.1 Fhf2KO Mice Have Temperature-Induced Cardiac Conduction
Block ————————————————————————– – 56 –
4.1.2 Hearts & Cardiomyocytes of Fhf2KO Mice are Structurally Normal
———————————————————————— – 57 –
4.1.3 Temperature Sensitive Fhf2KO Phenotype is Heart Autonomous —-
——————————————————————————– – 58 –
4.1.4 Flecainide Sensitivity and Slowed Cardiac Conduction Implicates
Nav Dysfunction in the Fhf2KO Heart ———————————– – 58 –
4.1.5 Fhf2KO Cardiomyocytes Fail to Fire Action Potentials at Elevated
Temperatures —————————————————————– – 59 –
4.2 Hyperthermic Cardiac Conduction Failure in Fhf2KO Mice Altered
Inactivation Gating of Cardiac Sodium Channels —————————- – 61 –
4.2.1 Absence of FHF2 Causes Hyperpolarized Shift in Voltage
Dependence of Nav1.5 Steady-State Inactivation ———————– – 61 –
4.2.2 Absence of FHF2 and Elevated Temperatures Accelerate Nav1.5
Open and Closed-State Inactivation ————————————— – 63 –
4.3 Computational Modeling Verifies Accelerated Nav Inactivation Causes
the Observed Phenotype at Elevated Temperature————————— – 66 –
4.4 Summary ————————————————————————- – 69 –
Chapter 5: FHF2 Necessary for Heat Nociception and Hyperthermic Conduction of
Action-Potentials Through Dorsal Root Ganglion Sensory Axons —————– – 75 –
5.1 Fhf2KO Mice Have a Temperature Specific Nociceptive Deficit ——- – 75 –
5.1.1 Fhf2KO Mice Do Not Respond to Noxious Heat Stimulation – – 75 –

5.1.2 Fhf2KO Sensory Deficits are Temperature Specific ———— – 75 –
5.1.3 Inability to Respond to Noxious Heat is Due to a Failure
Specifically at the Level of the Peripheral Nervous System ———– – 76 –
5.1.4 Sensory Innervation is Unaltered in Fhf2KO Mice ———— – 77 –
5.2 Altered Inactivation Gating of Peripheral Neuronal Sodium Channels in
the Absence of FHF2 —————————————————————- – 79 –
5.2.1 Absence of FHF2 Alters Voltage Dependence of Steady State
Nav1.7 Inactivation and Rate of Nav1.7 Inactivation ——————- – 79 –
5.2.2 Fhf2KO DRG Sensory Neurons Display Hyperpolarizing Shift in
Inactivation of TTX-Resistant Sodium Channels ———————– – 80 –
5.3 Accelerated Nav Inactivation in Fhf2KO Nociceptors Suppresses Action
Potential Conduction at Elevated Temperature —————————— – 83 –
5.3.1 Acutely Dissociated Fhf2KO DRG Heat-Sensitive Nociceptors
Have Unimpaired Heat-Induced Excitability —————————- – 83 –
5.3.2 Fhf2KO Sensory Nerve Fibers Fail to Conduct Action Potentials at
Elevated Temperatures —————————————————— – 85 –
5.4 Summary ————————————————————————- – 88 –
Chapter 6: Human FHF2 Missense Mutations Alter Sodium Channel Long-Term
Inactivation and Induce Epileptic Encephalopathy ———————————– – 90 –
6.1 Discovery FHF2 Point Mutations in Epileptic Patients —————– – 92 –
6.1.1 FHF2 Point Mutations in Epileptic Patients Align with Sites
Crucial to FHF2A Function ———————————————— – 95 –

6.2 FHF2A Epileptogenic Mutants Retain Some Wild-type Functions — -97 –
6.2.1 FHF2AR11C and FHF2AR14T Retain Ability to Bind Directly to Nav
———————————————————————————– – 97 –
6.2.2 FHF2AR11C and FHF2AR14T Retain Ability to Modulate Voltage
Dependence of Nav1.6 Inactivation ————————————— – 98 –
6.3 FHF2A Epileptogenic Mutations Impair Nav Long-term Inactivation —–
——————————————————————————————- – 99 –
6.4 Summary ———————————————————————— – 103 –
Chapter 7: Discussion ———————————————————————- – 106 –
7.1 Main Findings —————————————————————— – 106 –
7.2 Role of FHF2 in Cardiac Function —————————————– – 107 –
7.3 Role of FHF2 in Nociception ———————————————— – 108 –
7.4 Role of FHF2 in Epilepsy —————————————————- – 110 –
7.5 Limitations & Future Directions ——————————————- – 112 –
References ———————————————————————————— – 115 –
Appendix 1: Park et al Nat Comm.
Appendix 2: Dover et al Nat. Comm.

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YourPastQuestions Brand

Additional information

Author

Christopher Marra

No of Chapters

7

No of Pages

170

Reference

YES

Format

PDF

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