Assessment of in Vivo Muscle Force in the R6/2 Mouse Model of Huntington's Disease Using Newly Designed Force Rig

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Assessment of In Vivo Muscle Force in the R6/2 Mouse Model of Huntington's Disease Using Newly Designed Force Rig

Steven Russell Alan Burke Wright State University

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This Thesis is brought to you for free and open access by the Theses and Dissertations at CORE Scholar. It has been accepted for inclusion in Browse all Theses and Dissertations by an authorized administrator of CORE Scholar. For more information, please contact [email protected]. ASSESSMENT OF IN VIVO MUSCLE FORCE IN THE R6/2 MOUSE MODEL OF HUNTINGTON’S DISEASE USING NEWLY DESIGNED FORCE RIG

A Thesis submitted in partial fulfillment of the requirements for the degree of Master of Science

STEVEN RUSSELL ALAN BURKE B.S., Wright State University, 2017

2020 Wright State University

WRIGHT STATE UNIVERSITY GRADUATE SCHOOL December 2nd, 2020

I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Steven Russell Alan Burke ENTITLED Assessment of In Vivo Muscle Force in the R6/2 Mouse Model of Huntington’s Disease Using Newly Designed Force Rig BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science.

______Andrew Voss, Ph.D. Thesis Director

______Scott Baird, Ph.D. Chair, Biological Sciences Committee on Final Examination:

______Mark Rich, M.D. Ph.D.

______Lynn Hartzler, Ph.D.

______Barry Milligan, Ph.D. Interim Dean of the Graduate School

ABSTRACT

Burke, Steven Russell Alan. MS, Biological Sciences, Wright State University, 2020. Assessment of In Vivo Muscle Force in the R6/2 Mouse Model of Huntington’s Disease Using Newly Designed Force Rig

In this thesis, we develop a system to study in vivo muscle function in a mouse

model of Huntington’s disease that allows for the recording of muscle force by

stimulating the motor nerves or the muscles directly after a nerve block. This allows us to

distinguish between defects in the nerve, such as problems with vesicle release, and

primary muscle defects, such as altered intracellular calcium homeostasis. We

hypothesize that there are primary defects in R6/2 skeletal muscle that are separate from

neurodegeneration or defects in the CNS. In this case, we should see defects in muscle

force generation during nerve stimulation and direct muscle stimulation. If our hypothesis

is shown to be correct, this work would highlight new targets to study for the treatment of

HD. Peripheral treatments of skeletal muscle are a promising area for the treatment of

HD as motor defects are debilitating for patients.

iii

TABLE OF CONTENTS

Page

I. Specific Aims ……………………………………………………………… 1

II. Introduction ……………………………………………………………….. 3 Huntington’s Disease ………………………………………………… 3

Peripheral Defects in Huntington’s Disease …………………………. 6

Skeletal Muscle Physiology …………………………….……..…….. 10

Muscle Force Experiments ……………………………….…..…….... 16

III. Muscle Force System Development …………………………...... 18

Experimental Goals and Considerations …………………………….. 18

In Vivo Muscle Force System Overview ……………………………. 21

The Achilles Platform ……………………………………………….. 27

3D Modeling and Printing …………………………………………… 37

IV. R6/2 Force-Frequency Data ……………………………………………... 43

Force Measurement Methods ………………………………………... 43

Experimental Mice …………………………………………………... 44

Statistics …………………………………………………………….... 44

Force-Frequency Data ………………………………………....…...... 45

V. Discussion ………………………………………………………….…….. 51

Altered Neurotransmission …………………………………….……. 52

TABLE OF CONTENTS

Page

Contractile Machinery Modifications ………………………………... 54

Altered Intracellular Calcium Homeostasis ………………………..… 54

Muscle Fatigue Due to Repetitive Stimulation ………………………. 55

VI. Conclusions ………………………………………………………………. 56

VII. References ………………………………………………………………. 59

LIST OF FIGURES

Page

Figure 1: In Vivo Force System Diagram …………………………………….……… 23

Figure 2: Anesthesia System …………………………………………………….…... 25

Figure 3: Preliminary Force Data Artifacts ……………………………………..…… 29

Figure 4: Hind Limb Immobilization Achilles I & II ……………………………...… 30

Figure 5: Achilles II & III ……………………………………………………………. 31

Figure 6: Hind Limb Support Rods and Rail System ………………………………... 33

Figure 7: Achilles IV ……………………………………………………………….… 34

Figure 8: Achilles Positional Adjustment ………………………………………….… 36

Figure 9: Platform Modeling and 3D Printing ……………………………………….. 38

Figure 10: Diagram of 3D Printer …………………………………………………… 40

Figure 11: Assessment of Platform Improvement …………………………………... 42

Figure 12: Raw Force-Frequency Data ……………………………………………... 47

Figure 13: Nerve Stimulated Specific Force-Frequency …………………………… 48

Figure 14: Muscle Stimulated Force-Frequency ………………………………….... 49

Figure 15: Fusion Index ……………………………………………………………. 50

Figure 16: Reduction in Muscle Force Caused by Altered Neurotransmission ….… 53

I. Specific Aims

Specific Aim 1: To build a muscle force system capable of measuring in vivo muscle force from mice. The most challenging part of developing a muscle force system is designing a platform on which the mouse will be mounted. This platform will have to keep the hind limb stable so that the length of the muscle does not change during the muscle contractions. To achieve this, we will use computer automated design software to design the platform and then 3D print it. Printing the platform will give us the capability to make several prototypes easily and cheaply until the right design is achieved. Once constructed, we will assess the new setup by analyzing the force-frequency relationship and determining the twitch to tetanic ratio, which is the ratio of maximum twitch force to the maximum tetanic contraction. Obtaining this ratio will allow us to determine if there is any unwanted movement in the hind limb due to the design of the platform.

Additionally, we will analyze the shape of the contractile responses to look for sharp changes in force that would indicate movement such as slipping of the suture.

If there is no movement of the hind limb during force measurements, we should obtain a twitch-to-tetanic ratio of ≥30% and the data will tightly fit a Boltzmann sigmoidal curve.

Specific Aim 2: To test for muscle weakness in R6/2 skeletal muscle in vivo using new muscle force system. To test for muscle weakness in R6/2 HD mice, we will expose the sciatic nerve and isolate the plantar flexor muscles (lateral gastrocnemius, medial gastrocnemius, plantaris, and soleus). Isometric muscle contractions will be recorded with a force transducer that is connected to the Achilles tendon with a suture. This approach was chosen so that we could record nerve-stimulated force and subsequently direct muscle-stimulated force after blocking neuromuscular transmission with α- bungarotoxin. This will allow us to differentiate between defects of the nervous system and defects that are present in the muscle tissue itself. Recorded muscle force will be normalized to muscle weight to obtain specific force. This will allow us to compare HD to wild type mice as normalization will account for differences in force that are due to the smaller size of the HD muscles. Force-frequency experiments will be performed first with nerve stimulation, and again after blocking the nerve. First, we will compare WT records to HD to check for muscle weakness. We will also compare nerve vs muscle stimulated recordings to determine if any weakness we observe is a result of a nerve defect or a primary muscle defect.

We hypothesize that HD mice have muscle weakness and therefore, we will observe a significant downward shift in the force-frequency curve when compared to WT.

II. Introduction

HUNTINGTON’S DISEASE

Huntington’s disease (HD) is a genetic degenerative disease that presents with

progressive cognitive and motor defects. The disease affects many areas of the brain, but

most notably the areas known as the striatum (Vonsattel et al., 1985). The striatum is responsible for the control of smooth movement and a person’s mood (Bruce Koeppen and Bruce Stanton, 2017). All the muscles of the body are affected. Throughout the disease, the muscles will atrophy severely (She et al., 2011). Eventually, the affected person will lose control of voluntary movement and will be restricted to a chair. In all cases the disease is fatal. Unfortunately, there is currently no treatment that will prevent or even slow the progression of the disease.

HD has been recognized as a disease as early as 1842. It was named after George

Huntington, who gave his famous description of the disease titled, “On Chorea” in 1872.

Huntington was the first to delineate the disease from other diseases of chorea. There were some other descriptions of HD before George Huntington that noted a hereditary form of chorea, notably by Charles Waters, Johan Christian Lund, and Groving Lyon

(Bates et al., 2014). George Huntington was in a perfect position to make his observations of the disease. He had plenty of experience that was passed to him from his father and his grandfather who were both physicians that had seen HD patients over their years of practice (Bates et al., 2014). After Huntington, interest in the disease spread across America and Europe as well as some South American countries (Marsden, 1975).

Early research of HD was a slow and gradual process until it was better known.

Several international organizations and lay groups, which were developed by family

members of those with the disease, were instrumental in the progress of HD research over

the past 40 years (Bates et al., 2014). The World Federation of Neurology Research

Group on Huntington’s Disease was founded and first met in 1967 and has since evolved

into the World Huntington’s Congress. This organization has held meetings every two

years since its formation and has created a network of researchers around the world that

has resulted in collaboration between researchers from a wide range of disciplines (Bates

et al., 2014). Marjorie Guthrie, the wife of famous American folk singer Woody Guthrie

who famously died from HD in 1967, devoted much of her time promoting HD and

spreading awareness of the disease after Woody’s death (Bates et al., 2014).

HD is inherited in an autosomal dominant fashion so that each child of an affected

parent has a 50% chance of getting the disease (Myers, 2004). HD is an insidious disease.

A person with the gene mutation will start life perfectly normal and then at around

middle age will begin noticing subtle signs and symptoms. The age of onset varies widely

and can be hard to assess due to subtle cognitive changes that can go unnoticed (Duyao et

al., 1993). However, most patients develop noticeable motor symptoms around their

forties and fifties (Myrianthopoulos, 1966; Ridley et al., 1991) such as chorea, rigidity,

dystonia, and bradykinesia (Wells and Ashizawa, 2006).

HD is caused by a mutation in the huntingtin gene, specifically increased CAG

trinucleotide repeats at the 5’ end of the gene (The Huntington’s Disease Collaborative

Research Group, 1993). The gene codes for the huntingtin protein (Htt). It is located on the short arm of chromosome four at position 16.3. The gene consists of a polyQ domain

which codes for a stretch of glutamines in the protein. If the domain is expanded beyond

36 glutamines, the carrier of the gene will develop HD (Myers, 2004). The expanded

CAG repeat in the mutated huntingtin protein forms a polar zipper in the protein that

results in protein aggregation (Perutz, 1995). Protein aggregates in many tissues are a

hallmark of HD pathology (Arrasate and Finkbeiner, 2012; Hoffner and Djian, 2002;

Scherzinger et al., 1997; Wanker, 2000). The age of onset is affected by the glutamine

repeat number. There is an inverse correlation between the two, such that the more CAG

repeats there are the earlier a person develops symptoms (Duyao et al., 1993). Once there

are more than 50 glutamine repeats the carrier will develop the juvenile form of HD

which has an earlier age of onset and death (van Dijk et al., 1986).

Chorea is the main symptom of the disease and is the main sign used for

diagnosis. Chorea consists of involuntary uncontrolled contraction of the muscles. This

results in a clumsy-looking walk and slurred speech that can be mistaken for intoxication

(Bates et al., 2014). Typically, in the early stages of HD, the muscles of the extremities will be affected. As the disease progresses the chorea spreads to more proximal muscles and those of the trunk of the body. When choreic movements begin they are usually so subtle they are unnoticed by others. Eventually, they get worse and it begins to make the affected individual clumsier and will make it more difficult to perform activities of daily living such as walking, speaking, chewing, and swallowing. In later stages of the disease, chorea can be overshadowed by dystonia (random involuntary muscle contractions).

Stress, anxiety, fatigue, and often caffeinated drinks can exacerbate chorea. Chorea eventually becomes so pronounced the affected individual can no longer work and will need family members or in-home caretakers to assist them. Due to disease onset

occurring in an individual’s prime career years, this situation puts significant stress on

their families. Many families will fall down economic ladders as they lose income due to

the affected individual’s inability to work, but also due to the expenses associated with the individual’s care. The leading cause of death in HD is aspiration pneumonia

(Heemskerk and Roos, 2012). This is due to an inability to maintain the coordination of esophageal muscles and the epiglottis. When a person with HD swallows, food can get into the trachea and bacteria can infect the lungs. According to an analysis of 224 HD patients by Heemskerk and Roos, many of the cases where the cause of death could be identified died of aspiration pneumonia (Heemskerk and Roos, 2012).

There is a wide range of cognitive and psychiatric changes associated with HD.

Irritability is increased in HD. Patients can go through bouts where the smallest provocation can result in a sudden outburst of angry or even violent behavior. Most often this aggression is directed toward family or friends most directly involved in the patient’s

care (Bates et al., 2014). Additionally, psychomotor slowing is a feature of HD and has

been correlated to the CAG repeat number (Bamford et al., 1989; Snowden et al., 2002).

Patients will take longer to perform the same tasks that they used to. Also, the perception

of time becomes affected. These changes make it increasingly difficult to manage work

and meet deadlines, especially for those with more demanding and higher stress careers.

PERIPHERAL DEFECTS IN HUNTINGTON’S DISEASE

The most overt and noticeable symptoms of HD are chorea and muscle wasting.

Chorea being the main symptom used for diagnosis of the disease. It is not surprising that

the bulk of the research on HD has been done with the aim of further understanding

neuronal pathologies, especially with the significant psychological and behavioral

changes that accompany HD. It is a prevailing view that most of the peripheral defects found in tissues such as skeletal muscle are secondary to changes in the striatum. That is some changes, such as altered hormonal signaling from the hypothalamus, could alter

physiology in other tissues outside of the CNS. Being a result of something that happened

in the CNS, these pathologic events would be viewed as being secondary pathologies.

However, it is now known that the huntingtin gene is expressed ubiquitously throughout the body (Hoogeveen et al., 1993; Li et al., 1993; Strong et al., 1993; Trottier et al., 1995) and major motor defects are a hallmark of the disease. Even though much of

HD research has focused on the central nervous system (Lo and Hughes, 2011; Strand et al., 2005; The Huntington’s Disease Collaborative Research Group, 1993; Wells and

Ashizawa, 2006), recent work has found many muscle defects that may help explain the motor symptoms of HD (Waters et al., 2013). Supporting that muscle defects may develop separately from neurodegeneration in humans, it has been observed that motor symptoms preceded the cognitive symptoms in a marathon runner (Kosinski et al., 2007) who was a carrier of the HD gene. A study of 960 patients showed that motor symptoms were the earliest disease symptom or sign in 56% of the participants (Marder et al.,

2000).

As previously mentioned, the most noticeable issue with HD skeletal muscle is the fact that it is significantly smaller. HD patients are weaker and skeletal muscle wasting is often observed (Boesgaard et al., 2009; Ribchester et al., 2004), In the R6/2 transgenic mouse model of HD, hind limb muscles are 40% lighter than WT muscles

(Bondulich et al., 2017). It is not clear what the main cause of the loss in muscle mass is.

Some have hypothesized that it is due to metabolic changes or altered myocyte

differentiation and apoptosis (Lodi et al., 2000; She et al., 2011).

It has been previously shown, by our lab, that skeletal muscle is hyperexcitable in

the R6/2 transgenic mouse model of HD, due to reduced resting currents through chloride

channels (ClC-1) and inwardly rectifying potassium channels (Kir) in the muscle

membrane (Waters et al., 2013). A recent publication examined neuromuscular

transmission to assess the relationship between muscle and neuronal defects (Khedraki et

al., 2017). Using voltage-clamp electrophysiology, Khedraki et al. found that

spontaneous miniature endplate currents, nerve-evoked endplate currents, and the quantal content were reduced in R6/2 HD mice.

Peripheral pathology in HD is not exclusive to skeletal muscle. Many if not all other cell types display pathologic changes in physiology, and this has been shown both in mouse models and HD patients. Mutant Htt inclusion bodies are a hallmark of striatal dysfunction. These inclusion bodies have now been found in peripheral tissue as well

(Orth et al., 2003; Trottier et al., 1995; van der Burg et al., 2009). Mutated Htt is known to disrupt gene expression (Chaturvedi et al., 2009; Luthi-Carter et al., 2002; Strand et al.,

2005), which could lead to widespread dysfunction in peripheral tissues.

Cardiac tissue has significant defects in both humans and mice. Reduced cardiac output and decreased myocardial thickness have been shown (Mihm et al., 2007; Pattison et al., 2008), Cardiac failure occurs in 30% of patients with HD (Lanska et al., 1988).

There is also very high expression of Htt in the testes (Strong et al., 1993). This results in decreased concentrations of testosterone and correlates with a worsening of the disease overall (Markianos et al., 2005; Saleh et al., 2009). Additionally, there is a significant

8 disruption of function in the digestive tract. In patients and mice, there is a loss of ghrelin-producing neurons which regulate the sensation of hunger (van der Burg et al.,

2008). Decreased insulin production also occurs which leads to glucose intolerance

(Björkqvist et al., 2005; Duan et al., 2003; Farrer, 1985; Hurlbert et al., 1999; Josefsen et al., 2008; Podolsky et al., 1972; Podolsky and Leopold, 1977). Finally, mitochondrial dysfunction is known in several tissues, including skeletal muscle, and could underly some of the weight loss in HD patients (Arenas et al., 1998; Ciammola et al., 2006;

Turner et al., 2007).

A growing body of evidence is suggesting that some of these peripheral defects are not secondary to defects in the striatum. Testicular degradation develops before reductions in testosterone or gonadotropin-releasing hormone (Van Raams donk et al.,

2007). Gonadotropin-releasing hormone is produced by the hypothalamus. If this were a secondary pathology one would expect to see a reduction in gonadotropin-releasing hormone before dysfunction in the testis. Additionally, concentrations of the luteinizing hormone do not correlate to reductions in testosterone (Markianos et al., 2005). Some studies have even shown that these peripheral defects can contribute to pathology in the brain (Chiang et al., 2007; Martin et al., 2009).

As can be seen HD is a complex disease and many tissues and body systems are impacted. To fully understand this disease, it is important to assess the peripheral defects as they could be novel targets for the treatment of the disease. Our lab is focused on the motor symptoms of HD. As has been discussed, the motor symptoms are debilitating for patients and are the main reason that their lives are unraveled and are ultimately a major cause of death. The main causes of death in HD are aspiration pneumonia and choking

(Lanska et al., 1988; Sørensen and Fenger, 1992) due to the dysfunction of the muscles

involved in swallowing and chewing. In this project, we studied muscle function in the

R6/2 mouse model of HD by performing in vivo measurements of muscle force. In the

following section, we will cover the physiology responsible for muscle contraction at the

cellular level.

SKELETAL MUSCLE PHYSIOLOGY

Skeletal muscle in the body is responsible for generating the force required to

perform work such as moving, breathing, swallowing, talking, facial expression, and so

on. To perform a movement, such as picking up a glass of water, the muscle must receive

an impulse from the nerves that innervate it. The idea that nerves are responsible for

propagating the signal from the brain to the muscles dates back as far as the 2nd century

(Drukarch et al., 2018). However, the “modern” view of nerve conduction known as the

“neuron doctrine” began to be developed in the late 19th century. Luigi Galvani in 1791

made an accidental discovery that electric current can excite nerves and cause muscle

contraction in frog legs (Galvani, 1791). In subsequent experiments, he demonstrated that

there is an electrical force in animal tissue that is responsible for the contraction of

muscles. Using a galvanometer Emil du Bois-Raymond measured the first “action potential” (AP) in frog nerves by noticing a negative voltage deflection when the nerve was excited with respect to a region at rest (Du Bois-Reymond, 1884).

As with many discoveries, the electrochemical basis of nerve conduction and muscle contraction was dependent on incremental advancements of technology and experimental techniques. The development of the light microscope and advancements in histological preparation allowed for early neuroanatomists to visualize neurons in greater

detail. Importantly, Ramón y Cajal, now known as “the father of modern neuroscience”,

utilized staining techniques, developed by Camillo Golgi, that allowed him to make

marvelous depictions of nerves in the 1880s that included the cell body, dendrites, and

axons in stunning detail. His work made it possible for Sherrington to develop the

concept of the synapse in 1897 (Smith, 1996). The prevailing theory at the time was that

nerves formed an interconnected network instead of being discrete separated cells

(Drukarch et al., 2018).

In 1938, John Young performed important experiments showing that the giant axons of the squid were in fact motor neurons (Young, 1938). Their stimulation cause

contraction of the muscles of the mantle of the squid. These giant axons were perfect at

the time to allow for electrical recordings to study the function of these nerves. This was the beginning of the field of electrophysiology. The recording electrodes at the time were too big for intracellular recordings in most other cells. Much of the pioneering work on the electrical properties of nerves were done in these giant axons. Hodgkin and Huxley saw the benefit of the giant axons and for the first-time recorded APs from the inside of the nerve in 1939 (Hodgkin and Huxley, 1939). This work led to the formulation of

Hodgkin and Huxley’s current equation, which allowed for the quantitative assessment of nerve properties under various conditions (Hodgkin and Huxley, 1952a). Hodgkin and

Huxley were ultimately awarded the Nobel Prize for this work in 1963.

The aforementioned Andrew Huxley as well as Hugh Huxley showed coincidently that the actin and myosin proteins that make up a muscle fiber slide across one another to cause muscle shortening (Huxley and Niedergerke, 1954; Huxley and

Hanson, 1954). This work paved the way for the sliding filament theory of muscle

contraction. At the time, the prevailing view was that muscle contracted by the folding or

coiling of various proteins within the muscle fiber.

It was not until the development of the electron microscope that the complete

picture of muscle contraction was resolved. Electron microscopy in conjunction with x-

ray crystallography was used to decipher the 3D structures of the various protein

channels in the muscle membrane. Once these structures were determined their various

functions and gating mechanisms were determined. In the following paragraphs, we will

discuss the physiological events that take place to cause a muscle contraction starting

with the generation of an AP and ending with the process known as excitation- contraction (EC) coupling. We will also discuss the various proteins involved in the process and how they work to ultimately cause muscle contraction and then relaxation.

The electrical impulse that travels along the axon of a nerve is known as an AP

(Hodgkin and Huxley, 1952a). All cells in the body have a membrane composed of a

phospholipid bilayer (Gorter and Grendel, 1925). This membrane is impermeable to

charged particles called ions (Na+, K+, and Ca2+ being the important ones for the process

being discussed here). The ions are held at varying concentrations inside and outside the

cell and create an electrochemical gradient (Bernstein, 1912) that can be harnessed to perform work within the cell. Also present in the cell membrane are various proteins that act as channels for the ions allowing them to move from one side of the membrane to the other down their electrochemical gradient (Danielli and Davson, 1935; Neher and

Sakmann, 1976). The AP that travels down the motor nerve causes a depolarization that is due to the opening of Na+ channels on the membrane (Overton, 1902). The Na+

channels are voltage sensitive meaning that a change in the voltage difference between

the voltage on the outside of the membrane and the inside in the vicinity of that channel

will cause the channel to open and allow Na+ to flow into the cell. This makes the

membrane potential more positive and the other Na+ channels in the vicinity will also

open. This is what allows the AP to propagate down the axon to the nerve terminal. The

AP will originate in nerve bundles located in the ventral horn of the spinal cord (Hong and Etherington, 2011). The impulse travels down the axons until it reaches the nerve terminal which forms a synapse with the muscle fiber membrane known as the neuromuscular junction (NMJ) (Kühne and von Voit, 1886).

Once the impulse reaches the NMJ, voltage-sensitive Ca2+ channels open in response to the AP and cause vesicles in the nerve terminal containing acetylcholine

(ACh) to fuse with the membrane releasing ACh into the interstitial fluid(Couteaux and

Pécot-Dechavassine, 1970; Dale, 1914; Katz and Miledi, 1967). The membrane on the muscle fiber directly below the nerve terminal is known as the synaptic cleft. This area of the membrane has many nicotinic ACh receptors that bind ACh. The binding of ACh to the receptor causes Na+ to enter the muscle fiber and depolarize the muscle membrane

(Fatt and Katz, 1951; Neher and Sakmann, 1976b). This causes APs to propagate down the muscle fibers of the muscle starting a cascade of events that eventually leads to muscle contraction. This whole process is known as excitation-contraction (EC) coupling.

To fully understand this process, we must first look at the organization of muscle starting at the level of the whole muscle and going all the way down to the individual muscle cell.

Muscles are made up of many small fibers that can stretch the length of an entire muscle. The muscle fiber is a single muscle cell. The muscle cell or myocyte is made up of repeating contractile units called a sarcomere (Bowman and Todd, 1840;

Leeuwenhoek, 1710). A sarcomere is a meshwork of different proteins that can work

together to contract in response to the muscle AP. The sarcomere is made up of actin and

myosin proteins which are known as thin and thick filaments, respectively as well as

some other proteins that work as the framework of the sarcomere (Hanson and Huxley,

1953; Huxley and Hanson, 1954). Thick and thin filaments are known as myofibrils.

Actin is anchored to the Z-line of the sarcomere by a protein called titan (Kuroda and

Maruyama, 1976; Wang et al., 1979). The Z-line is the border of the sarcomere and there

is a Z-line on each end of the sarcomere. Many parallel actin filaments stretch from each

Z-line toward the middle of the sarcomere. In between each actin filament are the thick

filaments. They start at the midline of the sarcomere and stretch toward the Z-line. This

organization of myofilaments is what gives skeletal muscle its striated appearance. The

actin filament is a braided fiber of actin molecules (Holmes et al., 1990). The fiber has

many binding sites for myosin. These sites, in the resting state, are covered up by another

protein called tropomyosin. This protein has binding sites for Ca2+ so that when

2+ 2+ 2+ intracellular Ca ([Ca ]i) rises Ca binds to tropomyosin and causes a conformational

change in the protein so that it uncovers the binding sites for myosin (Spudich et al.,

1972; von der Ecken et al., 2015). This will allow myosin to bind to actin so that cross- bridge cycling can begin which will lead to muscle contraction.

Along the muscle fiber membrane, regions of the surface membrane fold inward toward the fiber interior to form narrow tubes. The system is known as the transverse tubular (t-tubular) system and is continuous with the surface membrane (Huxley and

Taylor, 1958; Porter and Palade, 1957). The t-tubules are essential for coordinated

sarcomere shortening across the entire fiber. They allow for the AP to propagate into the

14 center of the fiber so that the entire muscle fiber receives the signal to contract. Also, the t-tubules contain the voltage-sensitive dihydropyridine receptors that are mechanically coupled to L-type Ca2+ channels (ryanodine receptors) on the membrane of the sarcoplasmic reticulum (Curtis and Catterall, 1984; Inui et al., 1987). The sarcoplasmic reticulum is the reservoir for Ca2+ in the myocyte. Once the AP reaches the dihydropyridine receptors they undergo a conformational change that causes the ryanodine receptors to open and allow for the release of Ca2+ into the cytoplasm of the cell (Endo, 2007; Weber and Herz, 1968). This rise in [Ca2+]i ultimately leads to muscle contraction. The t-tubules are lined up over the Z-lines of the sarcomere. This ensures that Ca2+ is released in the vicinity of tropomyosin so that the Ca2+ will bind and uncover the binding sites for myosin.

Myosin has a globular head and a tail domain. (Rayment et al., 1993). The head of the myosin protein contains an ATPase. This enzyme will hydrolyze ATP to ADP and phosphate (Lymn and Taylor, 1971). This chemical reaction is what releases the energy necessary to perform the mechanical work required for the shortening of the sarcomere.

During contraction, this process is happening repeatedly so that all the myosin heads are

“walking” along the actin filament toward the Z-lines. This is known as the sliding filament theory (Finer et al., 1994; Gordon et al., 1966; Huxley and Niedergerke, 1954;

Huxley and Hanson, 1954; Toyoshima et al., 1987). This process will continue until no

2+ more APs come from the motor nerve. At this point [Ca ]i will begin to be pumped back

2+ into the SR via the SERCA pump (Toyoshima et al., 2000). Thus, [Ca ]i will decrease to resting myoplasmic levels. Ca2+ will fall off tropomyosin causing the binding sites for myosin to be covered. ATP binding to the myosin ATPase causes the head of myosin to

unbind from actin. This causes relaxation of the muscle and the return to resting muscle

length and tension.

MUSCLE FORCE EXPERIMENTS

Given the known defects at the neuromuscular junction and in channels on the

muscle fiber membrane of R6/2 mice, we hypothesize that muscle function is affected by

these defects and will, therefore, be weaker than their wild type littermates. To test this

hypothesis, we measured muscle force in vivo in the R6/2 mouse model of HD. It has

been shown using ex vivo techniques that HD muscle is 75% weaker than WT muscle

(Hering et al., 2006). In this study muscle force from extensor digitorum longus (EDL) and soleus muscles (hindlimb dorsiflexor and plantar flexor muscles) was measured using ex vivo techniques. These experiments were performed at room temperature. Our experiments were be performed in vivo at physiological temperatures, which could result in significantly different muscle force as was previously reported. Muscle force is known to be reduced at room temperature (Caremani et al., 2019), and it is unknown if this temperature effect will impact muscle force differently between WT and HD.

Gillian Bates, part of the team that developed the R6/2 mouse line, and a team of researchers tested muscle force in R6/2 mice in 2015 (Mielcarek et al., 2015). They also

showed that muscle force is reduced in HD mice when compared to WT, but only by

25%. They measured muscle force in both the tibialis anterior (TA) and EDL using in

vivo techniques. In this study, raw muscle force was given in grams of force. The muscle

force was not normalized to muscle size in any way. As one would expect a smaller

muscle should produce less force than a larger muscle. So, the question at hand is

16 whether HD is truly weaker than WT once this reduction in muscle mass has been accounted for.

As Hering et al. and Mielcarek et al. have shown differing results, we thought it was worthwhile to revisit muscle force in the R6/2 mice. Both studies used hind limb muscles, but they used only single muscles in their recordings. We plan to use the whole group of plantar flexor muscles that attach to the Achilles tendon. Like the Mielcarek study, we performed in vivo measurements while the mouse is anesthetized. We also recorded muscle force in response to nerve stimulation and direct muscle stimulation after application of α-bungarotoxin (BTX) to assess nerve and muscle function directly.

Finally, after muscle force recordings we muscle weight was collected so that the force records could be normalized to muscle weight to account for differences in muscle size between HD and WT.

To obtain isometric muscle force recordings in vivo, we first built a system to do so. The in vivo system was built using a combination of commercially available equipment as well as a custom platform to which the mouse can be secured during the experiments. It was key to secure the hind limb of the mouse to eliminate any movement during muscle contractions. Any movement during contractions will result in unstable recordings and will prevent the successful detection of peak force during twitch and tetanic contractions. It will also make it difficult to maintain optimal length during the experiment. We utilized both computer automated design software and fused deposition modeling 3D printing to allow for rapid prototyping of the force platform.

III. MUSCLE FORCE SYSTEM DEVELOPMENT

EXPERIMENTAL GOALS AND CONSIDERATIONS

As previously stated, specific aim 1 of this this was to design and build a muscle force system to take isometric force measurements from R6/2 hind limb muscles in vivo.

In this section, we will cover the important factors that were considered before designing the system to achieve this goal. In the following section, the actual components of the muscle force system will be described as well as the design process. Additionally, some preliminary data from the various prototypes will be shown and discussed.

The choice of in vivo recordings was made to study muscle force in conditions that are as close to physiological as possible. Additionally, we wanted to be able to perform nerve as well as direct muscle stimulated muscle contractions after applying

BTX. This technique is important to be able to identify problems related to neurotransmission in the nerve terminal and synaptic cleft. Additionally, comparing direct muscle stimulated force with nerve stimulated force allows for the identification of altered muscle properties directly that are independent of nerve function.

The muscles that we chose to record from are the plantar flexor muscles, which include the lateral and medial gastrocnemius, plantaris, and soleus. These muscles are part of a whole muscle group that attaches to the calcaneal bone by the Achilles tendon.

This prep has the advantage of studying the muscles as a functional group as they are used during locomotion. As stated before, the aspect of performing these experiments in vivo means that the muscle was maintained at 37 ˚C as it would be in the intact animal.

There are known differences in cellular physiology at room temperature vs. physiological

temperature. Performing experiments at room temperature will slow down enzyme

kinetics and could reduce things like myosin light chain phosphorylation which will alter

muscle function. We wanted to minimize these effects in our prep. Finally, in vivo

conditions maintain normal blood perfusion to the muscles as the muscle remains

connected to its blood vessels. In ex vivo experiments the muscles need to be bathed in a

physiological buffer solution. In the prep discussed here, this is unnecessary.

We planned to perform recordings of isometric muscle contractions from the

plantar flexor muscles. To get accurate results of peak muscle force we must keep the

muscle fixed in place and assure there are no movements in either the hind limb or mouse

during the muscle contraction. To achieve this, we went through a lot of trial and error in

our methods of securing the mouse and the hind limb to the platform on which the mouse

is mounted. We also needed a way to achieve the optimal length of the muscles we were

recording from. To do this we mounted the mouse platform to a micromanipulator that

can move the platform so that the muscle is either stretched or relaxed when attached to the lever of the force transducer. Peak muscle force at a given frequency can only be achieved when the muscle is at an optimal length. This is the resting tension of the muscle in which the sarcomeres are arranged in such a way that the most cross-bridge cycling can occur while still having room for the sarcomere to shorten. If during a contraction the mouse or hind limb moves toward the force transducer the length of the muscle will no longer be optimal, and you will no longer be recording the true peak force

of a contraction. Additionally, you will see a drop in the baseline, or resting tension, in

between recordings.

In early experiments we noticed that, not only would the mouse move, but the suture securing the tendon to the lever of the force transducer can also slip. This causes the same issues mentioned in the previous paragraph. However, instead of the limb moving toward the force transducer during the muscle contraction, you are now unable to maintain the proper tension on the muscle and therefore the muscle is no longer at optimal length for the following recordings. The final issue regarding muscle tension and the optimal length is that the suture must be strong enough to oppose the force generated during peak muscle force during tetanic contractions. If the suture is the wrong material or is too small of a gauge it will snap during the strongest contractions. These considerations make the choice of suture and the knots that are used very important for

the accurate assessment of peak muscle force during stimulated contractions.

Our goal was to design a custom platform that is optimized with these

considerations in mind to be used in conjunction with commercially available recording

hardware and software. Before the development of this system, our lab specialized in skeletal muscle electrophysiology. For these experiments, we used a digitizer from

Molecular Devices as well as their suite of recording and analysis software called

pClamp. Due to our familiarity and expertise in setting up and using this equipment we

decided to use Molecular Device’s digitizer and software for the muscle force system.

The only other recording equipment we needed to add was the force measurement hardware. To record muscle force, we needed a force transducer. Most force transducers measure muscle force using an analog signal such as voltage. This signal must be able to be sent to the amplifier and then to the digitizer to be sent to the computer. Additionally, we wanted a force transducer that is capable of recording isometric, isotonic, as well as

eccentric muscle force. Although we are only concerned with isometric force

measurements for this thesis, we wanted the ability to perform other experiments later.

Finally, to complete the system we needed a way to deliver anesthesia to the mice.

It is very important to be able to accurately control the sedation of the mouse during these experiments. These experiments can be well over an hour in length, especially with more complex stimulation protocols. Therefore, we needed a system that can keep a mouse stable under anesthesia for the duration of the experiment. It is also very important to maintain the body temperature of the mouse while it is anesthetized. The core temperature of a mouse is 37 ˚C. We needed our system to be able to measure and maintain the body temperature simultaneously with a temperature probe and a heat source.

IN VIVO MUSCLE FORCE SYSTEM OVERVIEW

The muscle force system can be broken down into five main parts: life support, the surgical platform, the muscle force platform, the data acquisition hardware, and the peripherals. A diagram of the system's main components is shown in figure 1 and each part will be broken down and described in this section.

Arguably, one of the most important parts of this system is life support. As these experiments were performed in vivo, the animals had to be maintained in a sedated state during the entire experiment. Nothing is more important than the welfare of the animal during these experiments. Anesthesia must be delivered in a way so that we can perform our experiments and cause no harm or suffering to the animal. It has been well established how to safely administer anesthesia to animals during experimentation (Kohn,

1997) and all the experiments in this thesis were performed while strictly following procedures and protocols approved by the Wright State IACUC committee. The anesthesia system that was ultimately chosen was the Somnosuite low flow anesthesia machine from Kent Scientific. This system checks all the major boxes that we need with some additional features.

The Somnosuite allows you to deliver isoflurane in a controlled way as well as to monitor and maintain body temperature with a built-in temperature probe and a heating pad. The system has a digital screen that allows you to monitor the isoflurane flow rate and concentration. To deliver isoflurane we are using 99% O2 as opposed to room air, which can also be used with the SomnoSuite. We found that 99% O2 worked better to maintain the HD mice during longer experiments. The SomnoSuite can also send the temperature signal to our digitizer so that we can have a recording of animal temp during our entire experiment. The machine includes a heating pad that can automatically regulate body temp when used with the temperature probe. However, we only use the heating pad to preheat the surgical and force platforms. As you will see later the built-in heating pad is not able to be used during the surgery and force measurements because we place pins through the paws into the platforms. To solve this issue used a lamp with a ceramic heat bulb on a boom arm to provide heat to the mouse while anesthetized. The boom arm allows the heat lamp to be moved from the surgical platform to the muscle force platform as needed. To maintain 37 °C the core temperature of the mouse is monitored with the temperature probe and the height of the lamp is manually adjusted.

Figure 1: In Vivo Force System Diagram The in vivo muscle force system functions through the coordination of five smaller systems; life support, surgical platform, force platform, peripherals, and acquisition hardware. Included is a list of the unique components of each system. The arrows highlight how each of the systems function together to obtain in vivo force data.

One of the great features of the Somnosuite is that it was designed specifically for

small animals. The alternative options, such as traditional veterinary vaporizers are made

to be used with a wide range of animals with a wide range of sizes. This means that for

small animals like mice, conventional vaporizers are very inefficient and put out more

gas than the lungs of the mouse will ever utilize. This is where the low flow aspect of the

Somnosuite is very advantageous. The system drastically reduces costs for isoflurane and

oxygen as much less is used during a given experiment.

A diagram of the anesthesia machine is shown in figure 2. The SomnoSuite uses a

syringe that is placed into a clamp. The syringe is filled with isoflurane which is the

anesthetic that we are using. The plunger of the syringe is depressed automatically by a

block that rides along a lead screw. The lead screw is attached to a motor so that it turns

at a given rate determined by the isoflurane concentration setting. The isoflurane is

pushed through a vaporizer block where it mixes with the O2 which is the carrier for the

isoflurane molecules. The gas is then sent to the lines that are used for inspiration. The

mouse breaths in the gas, whether it is in the induction chamber or through the face mask,

and then the exhaled air returns to the machine through the expiration lines. The waste

gas is sent through a carbon scrubber that filters vapors that are carcinogenic to the

experimenter.

The muscle force platform is where we spent most of our time and energy in the

design stage of this project. This is the only component that had to be made custom for

our experimental purposes. There are some commercially available solutions from various companies, but they are cost-prohibitive and are not tailored to our exact needs.

Therefore, we decided to spend some time designing a custom solution. We started with a

Figure 2: Anesthesia System The anesthesia machine being used in our muscle force system is the SomnoSuite from Kent Scientific. It is a low flow anesthesia system that delivers precise inhalant anesthetics at a rate optimized for small animals like mice. Isoflurane is pumped automatically from a syringe into the vaporizer block and then vaporized anesthetic is sent through the inspiratory tubes (blue arrows). Exhaled gas is sent back to the machine through the expiratory tubes (red arrows). The gas is then sent out the back of the machine and through a carbon scrubber to get rid of the toxic by-products of exhaled isoflurane. The system also features an induction chamber for anesthetizing the animal. It also has a heating pad and temperature probe for monitoring core body temp during experiments.

handmade prototype and ultimately went through three variations of the platform until we

developed the fourth and final version of the platform. The platform was aptly named the

Achilles after the tendon from which we are recording muscle force. The Achilles

platform and its development will be described in detail in the next section.

The data acquisition hardware is the next key component of the muscle force system. The

two main pieces of equipment are the muscle force transducer controller and digitizer.

Aurora Scientific produces a dual-mode lever controller. It can simultaneously record

muscle force and control muscle length. This gives us the ability to perform eccentric and

isotonic force measurements. The 305C is a two-channel controller so we can also use two different force transducers with different force ranges without changing our setup.

We chose this option to be able to have an in vivo system as well as ex vivo. The force transducers are very precise micro stepper motors. They have a variable resistor on the shaft of the motor so that force can be measured in the form of a voltage signal. A small lever is attached to the motor shaft. The tendon of the muscle that is being recorded from pulls on the lever and sends an increasing voltage signal to the digitizer. The digitizer then converts the voltage signal into a digital signal that is sent to the computer. This is ultimately shown in the acquisition software as a force recording.

The digitizer we selected is the Digidata 1550B from Molecular Devices. It has an array of digital inputs and outputs as well as analog inputs and outputs. These allow us to integrate peripheral devices, such as stimulators or temperature controllers, into the system. These can also be triggered with our computer using the digital outputs and can be automated when used with the pClamp software. The recorded signals such as the force measured by the force transducer are sent to the analog inputs of the digitizer. The

1550B has a total of ten inputs and outputs, both analog and digital, which gives us many

options for adding other peripheral hardware in the future.

The only peripheral hardware essential for this project is the pulse generator that we use to stimulate the nerve. The pulse generator that we chose is the S-900 from

Dagan. This pulse generator has many options for creating pulse trains, but we chose it for its ability to be triggered with the computer. To create the various trains for the force- frequency experiments, we have set the simulator to generate a 1 ms pulse and put it on trigger mode. When the generator receives a 5 V triggering pulse from one of the digital outputs of our digitizer it will fire a pulse. In the software, we can send out complex trains of triggering events so that we can stimulate the nerve at any frequency for any amount of time.

Our system also utilizes a temperature controller so that we can perform ex vivo experiments at a physiological temperature. Additionally, we added a differential amplifier that can record muscle compound APs during muscle contractions. This gives us the ability to generate EMG recordings during our in vivo contraction experiments.

The numerous amounts of inputs and outputs on the digitizer gives us plenty of room for expansion in the future.

THE ACHILLES PLATFORM

Now that we have a general overview of the muscle force system and its various components, we will discuss the development of the Achilles platform. We developed four versions of the platform in total. We made incremental improvements with each version. Here, this process will be shown step by step with some preliminary data and the

various features of the final version of the platform will be highlighted in the following

section.

The first version of the Achilles platform was hand made. We started with a simple breadboard and a micromanipulator from Thorlabs (XR25C). The plan for

recording muscle force was to have the mouse in the prone position and the caudal end of the mouse towards the force transducer. The plantar flexor muscles would be exposed,

and the Achilles tendon attached to the lever of the transducer with a suture (6-0 braided silk suture was used with a modified Miller’s knot). To fix the mouse to the platform we chose to use Sylgard (Sylgard 184, Dow Corning, Michigan, US) a silicone elastomer that comes as a two-part kit that you mix and let cure. Once cured it is a pretty solid elastomer with a little bit of give. To keep the mouse in place its limbs can be secured to the Sylgard by placing pins through the paws of the mouse into the Sylgard. The platform would need a cavity in which the Sylgard could be poured and held during the curing process. We decided to use a culture flask which had two advantages. The first was that we could pour the Sylgard into the flask and let it cure. The flask was mounted to the breadboard horizontally and was cut in half so that the mouse could lay on top of a bed of

Sylgard. Secondly, the mouth of the flask gave us a surface onto which we could pin the foot while maintaining its natural position at a 90° angle to the tibia.

Using Achilles I, we were able to collect some preliminary force-frequency data.

However, this data had some key issues that needed to be worked out. First, it was clear that the hindlimb of the mouse needed to be secured with more than just a pin into the

Sylgard through the paw. There was some clear movement that could be detected in our force records. Throughout the recordings breathing artifacts were present due to the hind

Figure 3: Preliminary Force Data Artifacts Achilles I produced force data that was unreliable. Common issues included slippage of the suture securing the tendon to the force transducer. The record shown in panel A is an extreme case of this. The slipping of the suture can be seen by the sudden loss of force in the middle of the trace. Black arrows are pointing to breathing artifacts due to excess movement in the hindlimb. Many of the traces contained significant shifts in the baseline as can be seen in A and B above.

limb not being properly secured (Figure 3A). Additionally, after a contraction, especially

those near the maximum, a baseline shift could be detected by a reduction in resting

tension at the beginning of the following recording (Figure 3A & B). This is unacceptable

as subsequent recordings would no longer be made at the optimal length of the muscles.

To prevent this movement, we came up with a couple of solutions to fix the leg in place

during recordings.

Figure 4: Hind Limb Immobilization Achilles I & II Originally staples over the femur and ankle were pinned into the Sylgard of the platform (A) to secure the hind limb. Later we developed a system utilizing metal straps to secure the ankle and foot to the platform to immobilize the leg during recordings (B).

The first solution that we tried was to use a staple to pin the upper leg to the

Sylgard of the platform (Figure 4A). This worked at first, but as we learned it also created two new problems. The first problem that was noticed was that overtime the Sylgard began to wear out from being punctured repeatedly by the pin. This makes it porous and allows the staple to back out during contractions. This renders the staple ineffective for limiting unwanted movement during muscle contractions. To address this, we came up with a way to pin the leg down in the same manner as the staple while not depending on

30 the Sylgard to keep it in place. We developed a rail system in which we could clamp a rod that had a staple on the end. This rod can then be lowered in place and clamped down so it will not move during contractions. Additionally, we made some metal straps that could be used to secure the foot and ankle (Figure 4B). These improvements were integrated into the second and third versions of the platform, Achilles II and III.

Figure 5: Achilles II & III Achilles II was developed to add the metal straps that are used to secure the foot and ankle of the mouse. Additionally, the rails system and leg clamp rod were added (A). Inserts and holes for fasteners were also added. Finally, epoxy was poured into the bottom before adding Sylgard to reduce the amount of Sylgard being used. Achilles II was eventually streamlined and redesigned in CAD to be 3D printed. The 3D printed Achilles III is shown above (B).

Achilles II was also hand made. We used a micropipette tip container that we trimmed down to size. Additionally, we added holes so that we could place threaded inserts so that we could screw our new straps into place (Figure 5A). This prototype provided the framework for us to make what we thought at the time would be the final version of the platform. Achilles III was 3D modeled in CAD software and then printed using a 3D printer (Figure 5B). This will be discussed in more detail in the paragraphs to follow while discussing the details of Achilles IV. The rail system was made utilizing some basic metal rods as well as some x blocks (UX-6-6) from Thor Labs. The metal rods have tapped holes on the end so they can be secured to the breadboard that the

platform is mounted to. The x blocks allow us to position the rod precisely over the leg so

it can be clamped down. The combination of the new rod system and metal straps

drastically reduced the unwanted movements during muscle contractions. Breathing

artifacts were essentially gone and there were no more shifts in baseline between

recordings.

As mentioned before the stapling technique presented an additional problem.

When we decided to use the staple, we failed to consider the underlying anatomy of the

upper leg. We were placing the staple over the femur. Coincidently, the femoral artery

runs along the femur. It was not until we started clamping down the leg with the rod that

we noticed that blood flow to the lower leg and thus to the plantar flexor muscles was

being constricted. The original staple was backing out and was therefore not constricting

blood flow enough for us to notice. However, the rod being clamped was so effective that

when the leg pushed against it during contractions it was able to cut off the blood flow.

With limited blood getting to the plantar flexor muscles, we noticed force decline

when performing repeated stimulation. At this point, we went back to the drawing board

to address this issue. The first and obvious issue was that the end of the rod was pushing

down on the tissue of the thigh with too much force. To ensure this was no longer an

issue we wanted to abandon clamping the leg directly secure it in place. Instead, we

decided to brace the entire rear end of the mouse. When the muscle pulls on the lever

during a contraction it also pulls on the leg. This causes the whole body of the mouse to

move toward the force transducer. Therefore, we thought that if we could limit the body movement, we would eliminate the movement of the upper leg without having to clamp it

Figure 6: Hind Limb Support Rods and Rail System The hindlimb needed to be secured near the hip as well as near the ankle. The image above shows the rails system we developed to properly secure the caudal end of the mouse and thus the upper leg. Two rods are fixed to a rail system over the platform. The rods can be moved precisely along the x and y-axis of the platform to properly position them at the hip and are secured in place using thumbscrews so that they do not move at all during experiments. Since we were no longer dependent on the Sylgard for securing the hind limb we did not have to worry about baseline shifts in force records after repeated use of the platform. directly. So instead of using one rod clamped at the top, we used two. One was placed on the hip area and the other was placed on the inner thigh and groin area (Figure 6).

The next thing that was done to eliminate the restriction of blood flow to the plantar flexor muscles was to change the design of the platform itself. Blood flow is maintained when the leg is kept in a more natural position. When a mouse is in a prone position the leg is not stretched out flat as it has been in the first three versions of the

Figure 7: Achilles IV In the final version of the force platform an upper platform was added for two reasons. The fist was to raise the body of the mouse higher than the lower hind limb to maintain the natural leg position as well as to increase blood flow. A beveled edge was added to support the upper leg and maintain a bend in the leg while the mouse was in the prone position. The new platform was kept as a separate part to allow for the top to be adjusted to accommodate the various leg sizes of different mice (A). Also, goal with the final design was to reduce the amount of Sylgard used. Instead of one large cavity with Sylgard, there are now two small cavities only where the Sylgard is necessary. The cavity on the upper platform is for pinning the paws. The lower cavity is to for the lower limb to rest on. Mounting posts were modeled on the bottom to allow the platform to be secured to the breadboard and micromanipulator (B).

Achilles platform. The lateral and medial condyles of the femur protrude enough to limit blood flow through the arteries supplying the plantar flexor muscles when the leg is clamped flat. The next modifications to the platform were made to accommodate a natural leg position with the leg being slightly bent. To achieve this, we added a second platform on top of the original platform that is shorter than the base. The front of the upper platform has an angle on its edge so that the upper leg can rest on it. The shin of the leg then rests on a bed of Sylgard. Additionally, Achilles IV only has Sylgard in the areas it is needed as opposed to the entire platform. There is an area for the paws to be pinned down and an area for the lower leg to rest on. Finally, we made the upper platform adjustable to accommodate legs of various sizes.

Importantly, aluminum dovetail extrusions were used so that the platform and

force transducer could be precisely positioned (Figure 8A). The force transducer was

mounted on a carriage that rides on a dovetail that allows it to be positioned at an appropriate height (Figure 8B). The suture needed to be aligned so that it pulls on the lever of the transducer at a 90° angle so that the true muscle force could be measured.

The micromanipulator to which the force platform is mounted to also rides along a dovetail so that it can be adjusted along the x-axis allowing us to align the suture with the force lever (Figure 8C & D).

With these modifications, the Achilles IV has remained the final version of the force platform. Replicable in vivo force data is reliably obtained using this system. The

R6/2 force-frequency data was collected again using this version of the platform. The final data will be presented in the following section of this thesis. First, we will discuss some of the advantages of 3D printing and its other applications with this system. We will also cover some additional considerations that had to be made during the design process as a result of 3D printing the platform.

Figure 8: Achilles Positional Adjustment The Achilles platform and force transducer utilize aluminum dovetail extrusions for precise positional adjustment (A). The force transducer is mounted to carriage that uses a dovetail extrusion that allows the transducer to be positioned at the proper height for force recordings (B). The micromanipulator (C) allows the platform to be positioned along the y-axis by being adjusted with fine movements with the turn of a dial. The dovetail the manipulator is mounted to allows the platform to also be moved along the x-axis so that the hind limb of the mouse can be aligned with the lever of the force transducer (D).

3D MODELING AND PRINTING

To design the Achilles platform before printing, it was first modeled using

parametric modeling. The software used is called Fusion 360 from Autodesk.

Conveniently, their software is free for students. Although it is free, it is a powerful

software. We were able to create a 3D model of our force platform to its exact dimensions. Additionally, the companies that make many of the other components in our

system have made the CAD files of their components available online. After the platform

had been designed, we could model the entire system in the Fusion 360 environment

(Figure 9). Fusion 360 also has the entire McMaster-Carr catalog with CAD models of

their hardware. This was very helpful in determining all the small pieces of hardware we

would need such as the various fasteners, washers, and inserts.

The platform was modeled with the idea in mind that it would be mounted to a

breadboard on the micromanipulator. We modeled mounting posts that can accept heat-

set threaded inserts. Once the part is printed, the inserts can be melted into place using a

soldering iron. The inserts are knurled on the sides so that they create a strong bond with

plastic as it cools. The platform can then be placed on the breadboard and fastened into

place using metric hex screws. We also used this technique for fastening the metal straps

for securing the ankle and foot in place on the platform. The straps we used were cut out

of a sheet of stainless steel and were cut into shape using a rotary tool and cutoff wheel.

Holes were drilled in each end for the screws.

Figure 9: Platform Modeling and 3D Printing The final version of the force platform, Achilles IV, was modeled using Fusion 360 from Autodesk. Many of the companies that make the various components in this system (Thorlabs, Newport, and Misumi) make the CAD files of their products available online. This makes it possible to quickly model the entire system in Fusion 360.

Once the platform had been modeled, it is was time to print the parts. We decided

to print the platform using fused deposition printing. The printer has a hot end that melts

a plastic filament onto a heated print bed. It prints one small layer of the part at a time,

each layer being 200 microns thick. For the printer to do this you have to generate code to

instruct the stepper motors to move the hot end about the print bed and the filament

through the extruder. We used a slicing software called Cura 4 from Ultimaker. The

software slices the part into many 200-micron layers and generates the instructions the printer will use to print the part. An overview diagram of the printer is shown in figure

10.

The Achilles platform was printed using PETG (polyethylene terephthalate with a

glycol modification) filament. There are many filaments available for 3D printing. Our

two main choices were either PLA (polylactic acid) or PETG. PLA is one of the most

widely available filaments. It is popular for hobbyist printers as it is very easy to print

with, especially for beginners. However, it is made from plant starch. This makes it prone

to warping from prolonged heat exposure. We needed a more heat-stable plastic since we are heating the bed during our experiments. PETG filament costs about the same as PLA filament but is more heat stable. It has the same ease of printing as PLA but has the durability of ABS. ABS is widely used in the manufacturing of consumer products that need to be very durable. However, ABS is harder to print with as it needs higher temperatures and an enclosed printing space.

Figure 10: Diagram of 3D Printer The printer used to print the Achilles IV platform utilizes fused deposition modeling technology. The part is printed by a thermoplastic filament being melted through a hot end onto a heated print bed. The printer used in this project is a core XY printer. This means that the hot end moves along the X and Y-axis and the print bed moves along the Z axis. The hot end is on a carriage that moves along aluminum rails. Stepper motors move the hot end around the X and Y-axis using belts. There is also a cooling fan next to the hot end that cools the filament as it is extruded to the filament. Parts are printed in 200-micron layers until the entire part is printed.

Overall, printing our platform has been of great benefit. Not only for this

application but also others around the lab. Now that our lab has the skills to model parts

and print them, we can save significant costs on parts we would otherwise have to buy.

The filament for the printer is very affordable. At the time of writing this, a kg of PETG

is around twenty-five US dollars. Our force platform only takes about 200 g of filament.

Therefore, we can print several platforms with one spool. Making and maintaining parts

in the lab is cheap and easy. Also, once the parts are started on the printer the whole

process is automated. One can leave and work on other things until the part is finished.

As previously discussed, Achilles IV is the final version of the force platform.

Achilles IV is a two-part platform. The base has a small cavity for Sylgard as well as

some spaces for inserts on the top. On the bottom are the mounting posts for securing the

platform to the breadboard. There is also a channel and some fasteners that allow the second part to adjust to different positions and be secured in place. The upper half of the platform is 20 mm thick and has a beveled edge to accommodate the upper leg and

maintain the natural bend in the knee. It has a channel for Sylgard for the front paws to be

pinned to. Once mounted on the breadboard and used in combination with our rail

system, the Achilles IV keeps the mouse and its hind limb very stable. Our force

recordings no longer show any shift in baseline after successive contractions.

Additionally, tetanic muscle force no longer declines after repetitive tetani protocols, as

the platform has been optimized to maintain maximal blood flow (Data not shown as it

was performed after the completion of this thesis)

According to our analysis, we were able to significantly improve the platform.

Figure 11A shows that from Achilles II to Achilles IV the steady decline in resting

Figure 11: Assessment of Platform Improvement Early versions of the force platform produced force records that showed continual baseline decline throughout successive recordings. Achilles II showed significantly more baseline decline throughout force-frequency recordings than Achilles IV (A). Percent decline and twitch to tetanic ratio were both significantly decreases by the improvements made to Achilles IV (B). Significance assessed with Student’s t-test. Values are shown as ± SEM, n = 4 Achilles II & 4 Achilles IV. tension was dramatically reduced. The baseline from our first recording to the last during force-frequency experiments would drop by almost 50% when using Achilles II. We were able to reduce that to just over 15% with the Achilles IV platform (Figure 11B).

Additionally, we saw a significant improvement in our twitch to tetanic ratio and

achieved our goal ratio of 30% (Figure 11C).

IV. R6/2 FORCE-FREQUENCY DATA

FORCE MEASUREMENT METHODS

For force measurements, mice were anesthetized with isoflurane using a low-flow

anesthesia system (SS-01, Kent Scientific, Torrington, CT), with body temperature

maintained at ~35 ℃ using a heat lamp and temperature probe. The plantar flexor

muscles (including the lateral and medial gastrocnemius, plantaris, and soleus muscles)

and sciatic nerve were exposed by removing the surrounding skin. To minimize the

influence of adjacent muscles, the common peroneal and tibial nerves were crushed.

During surgery, the muscles were bathed in physiological saline to prevent them from

drying out. The mice were then transferred to a custom platform filled with Sylgard and

mounted to a micromanipulator (XR25/M, Thorlabs, Newton, NJ). The leg was stabilized

to prevent movement. The proximal end of the plantar flexor muscles (Achilles tendon)

was attached to the lever of the force transducer (300D-305C dual-mode muscle lever,

Aurora Scientific, Ontario, Canada) using 5.0 or 6.0 silk suture and a modified Miller’s

knot. The optimal length was obtained by measuring the maximum twitch force while lengthening the muscle using the micromanipulator.

Nerve-evoked contractions were stimulated with platinum electrodes resting on the sciatic nerve. Direct muscle contractions were stimulated via platinum electrodes positioned above and below the plantar flexor muscles. Stimulus amplitude and the pulse width were determined by ensuring maximal twitch force was achieved. These values were typically ≤5 V at 1 ms or ≤50 V at 1.5 ms for nerve-stimulated and muscle-stimulated contractions, respectively. For muscle stimulated contractions ~150 µl α-bungarotoxin was injected intramuscularly. Nerve stimulated twitch force was assessed until it was reduced

to ≥25% of max. A Dagan S-900 Stimulator and S910 Stimulus Isolation Unit was used

for stimulation. Muscle force was recorded and digitized using pClamp10 software

(Molecular Devices, San Jose, CA). After each experiment, the muscles were quickly

weighed and then frozen in isopentane cooled in liquid nitrogen. The muscles were stored

in a –80 ℃ freezer until needed for further analysis.

EXPERIMENTAL MICE

R6/2 mice were studied at end-stage HD and compared with age-matched

littermates. Selection criteria were based on a combination of behavioral changes and

weight loss based on daily checks after 10 weeks of age. Mice were taken once body weight

loss reached 20% - 25% of max body weight. Animals are housed in the laboratory animal

resources facility at Wright State University. They are kept in cage racks with littermates

of the same sex. Once HD mice reach 10 weeks of age, they are given moist chow on the

cage floor daily.

STATISTICS

A repeated measures ANOVA was run to compare WT vs HD muscle force at

twitch force and 100 Hz. This was performed for both nerve and direct muscle stimulated

force. The repeated measure accounts for potentially correlated results that can arise from

the fact that it is the same mouse being measured at each of the two frequencies. SAS version 9.4 (SAS Institute, Inc., Cary, NC) was used for all analyses. Bonferonni multiple comparison procedure was used to control for a potentially inflated type I error rate

(claiming a significant difference exists when it does not) that can result from making multiple comparisons. This procedure ensures that the type I error rate for the repeated

measures ANOVA is at most α = 0.05. Student’s t-tests were used where otherwise mentioned.

FORCE-FREQUENCY DATA

To complete specific aim 2, a force-frequency protocol was developed so that muscle force could be recorded at increasing frequencies. The protocol for each frequency featured a train of ten 1 ms pulses. Muscle force was recorded at a range of frequencies from 0.3-100 Hz. In all experiments, nerve stimulated contractions were

performed first at all frequencies before moving to direct muscle stimulated contractions.

Throughout all recordings, stimulus amplitude and pulse width were held constant. Once

the platinum stimulating wires were placed on the sciatic nerve, the stimulus threshold

was found by increasing the pulse amplitude until a twitch was generated. The stimulus

was then increased by 20% to ensure that the stimulus remained above the threshold after

multiple rounds of stimulation. The force platform was then adjusted using the

micromanipulator so that optimal muscle length was reached. The optimal length was

determined by stimulating a single twitch and then increasing the tension on the muscle

by moving the platform 10 µm away from the force transducer and then stimulating

another twitch. The peak force is assessed for each twitch while increasing resting muscle

tension. This was repeated until the maximum twitch force was achieved. At this point,

the muscle was rested for ten minutes before proceeding with force-frequency recordings.

A total of 6 WT and 4 HD mice were recorded. Peak force during the train was

taken for each animal and then averaged for each genotype and data are presented as ±

SEM. The average peak force for each frequency was added as a single point on the

force-frequency graph. The points were then fit with a Boltzmann sigmoid curve

according to the following equation ( = ( )/ + , is the initial value and 𝐴𝐴1−𝐴𝐴2 𝑥𝑥−𝑥𝑥 𝑑𝑑𝑑𝑑 1+𝑒𝑒 0 2 1 represents the bottom plateau of the curve,𝑦𝑦 is the final𝐴𝐴 value𝐴𝐴 and represents the upper

2 plateau of the curve, is the midpoint of the𝐴𝐴 curve and represents the frequency of half

0 max or f1/2max). Raw 𝑥𝑥traces are shown for twitch and tetanic contractions (Figure 12A).

As expected, as the stimulation frequency increases the force also increases due to

summation. When looking at raw muscle force, our data is consistent with previous

reports (Hering et al., 2006; Mielcarek et al., 2015). When compared to WT muscle force

HD mice produce significantly less force at low and high frequencies (Figure 11B & C).

However, at 30 Hz there is no significant difference between HD and WT raw force. This

is likely due to the left shift in the force-frequency curve. As such, muscle force at 30 Hz

is closer to maximum tetanic contraction for HD than for WT.

Due to the known atrophy present in HD, it is important to normalize the muscle

force to account for the difference in muscle size. To do this we normalized muscle force

to muscle weight for each animal. Raw traces for specific force are shown in figure 12A.

The normalized muscle force is called specific force. Looking at the specific force of the

R6/2 muscle tells a completely different story than raw force. At all frequencies along the

force-frequency curve, HD produces significantly more force than WT when normalized to muscle weight (Figure 12B & C). In the following section of this thesis, we will

discuss some different possibilities as to why HD muscle is producing more force than

WT muscle. This result was not expected. However, this has not yet been studied using

the in vivo techniques presented here. The only other report on in vivo muscle force in the

R6/2 line showed a reduction in raw force at both twitch and maximum tetanic

Figure 12: Raw Force-Frequency Data: Raw traces for twitch, 30 Hz, and 60 Hz are shown (A). Muscle force increases with stimulation frequency due to summation. WT muscle produces more force than HD at low and high frequencies (B). Twitch force and tetanic force at 100 Hz are significantly reduced in HD. Frequency of ½ max is significantly reduced in HD by 14 Hz compared to WT. All p-values > 0.001, repeated measures ANOVA. Significance shown for frequency of ½ max using a Student’s t- test. Values are shown as ± SEM, n = 6 WT & 4 HD (C).

47 contraction (Mielcarek et al., 2015). However, these results were not normalized and cannot be directly compared to our data.

Muscle stimulated force was slightly lower than nerve stimulated force for both

WT and HD as expected. It is very hard to get 100% of nerve stimulated force when stimulating the muscle directly. This is because the muscle is a very thick tissue compared to the nerve. It takes much less voltage to stimulate the entire nerve maximally.

Figure 13: Nerve Stimulated Specific Force-Frequency Raw traces of specific force are shown for twitch, 30 Hz, and 60 Hz stimulation (A). When normalized to muscle weight, R6/2 muscle produces significantly more force at all frequencies throughout the force-frequency curve, p-value = 0.0032, repeated measures ANOVA (B & C). Values are shown as ± SEM, n = 6 WT & 4 HD.

The direct muscle stimulated contractions also showed higher force than in HD than WT (Figure 13A). However, when looking at the muscle stimulated force-frequency curve for HD, a plateau can be noticed from 60 Hz on. In WT the force continues to climb. We hypothesize that HD muscle is more prone to fatigue. All the different

frequencies were recorded one after another. We started at 0.3 Hz and worked through every frequency until we reached 100 Hz doing all nerve stimulated contraction before moving on to direct muscle stimulation. This means that by the time we started the muscle stimulated contractions we had stimulated ten trains. Once we started direct muscle stimulation and reached 60 Hz, we the muscle had endured 17 trains of 10 pulses each. We believe that the cause of the dip in the muscle stimulated force curve in HD muscle is due to fatigue caused by the preceding trains.

Figure 14: Muscle Stimulated Force-Frequency To test for potential muscle fatigue due to repetitive stimulation the twitch to tetanic ratio was analyzed in nerve and muscle stimulated force (A). Twitch to tetanic ratio was significantly increased (Student’s t-test) in the HD muscle stimulated force-frequency curve. Due to this the muscle stimulated force-frequency curve plateaus at 60 Hz and beyond in HD (B). Values are shown as ± SEM, n = 6 WT & 4 HD.

To test this, we compared the twitch to tetanic ratio of HD to WT for both nerve stimulated contractions and muscle stimulated contractions (Figure 14). We see that there is a significant difference in the twitch to tetanic ratio between HD and WT for direct

muscle stimulated contractions but not for nerve stimulated contractions. Since the

muscle was able to produce enough force to get a lower twitch to tetanic ratio when

stimulated with the nerve, we believe that the muscle has been fatigued due to repeated

stimulation.

Figure 15: Fusion Index Fusion index was analyzed at 50 Hz which is near the middle of the force- frequency curve. This is important because you need a partially fused tetanic contraction to assess the fusion index. Fusion index is a measure of the average Antipeak to the average peak (A). Each peak within the tetanus is averaged as well as the antipeak. Avg fusion index is shown for both HD and WT (B). Values are shown as ± SEM, n = 6 WT & 4 HD. Finally, we noticed a different level of fusion between WT and HD tetanic

contractions. As the frequency of stimulation increases, there is less time for the muscle

to relax in between each stimulus. Therefore, a ten-pulse train at 40 Hz produces more force than a ten pule train at 20 Hz. This is known as summation. The level of fusion in each tetanus can be measured by finding the ratio of the antipeak to the peak. This is called the fusion index. A fusion index of one would be a fully fused tetanic contraction.

We analyzed the fusion index for each frequency in both HD and WT. The fusion index in HD was significantly higher than WT (Figure 15).

V. Discussion

Through the work described in this thesis, we have shown that peripheral defects are important to HD pathology. Our data suggest primary muscle defects may be a key component of the observed muscle defects in HD patients. It is clear that defects in the

CNS are of importance as well to disease progression and symptomology. However, it is

necessary to fully assess defects in all peripheral tissues, not just skeletal muscle.

Furthermore, it is important to look at these defects rigorously and to assess if they are

primary in nature. Peripheral defects would be the key to unlocking accessible targets for

the treatment of HD.

The motor symptoms that occur in patients with HD are debilitating as the disease

progresses. They eventually make it impossible for the affected individual to perform the

simple activities of daily life that most take for granted. If the motor symptoms could be

prolonged or tamed in some way, the quality of life and outlook for the patient would be

greatly improved. Potentially, the life expectancy of those with HD could be increased as

many patients die due to dysfunction in various muscles (muscles of mastication and

swelling, as well as the heart, are most important).

The data presented here makes it clear that there are indeed muscle defects in

R6/2 skeletal muscle function. These defects need to be further explored as we have not

determined the precise mechanism underlying these defects. However, in the following

sections, we will discuss some possible physiological mechanisms that could be altered

based on our data.

ALTERED NEUROTRANSMISSION

Our lab previously assessed neurotransmission in R6/2 mice and found altered

neurotransmission including reduced quantal content and both spontaneous and evoked

endplate potentials (Khedraki et al., 2017). This was shown to be due to a reduced

probability of release of vesicles in the nerve terminal. This data is what led us to

hypothesize that muscle force would be reduced in R6/2 muscle. We observed increased

specific force in response to both nerve and direct muscle stimulated contractions.

Therefore, it is unlikely that altered neurotransmission is impacting muscle force at least

during the brief stimulations that are performed to collect the force-frequency data. If altered neurotransmission were altering the muscle force, we would expect to see the reduced specific force with nerve stimulation but not when stimulating the muscle -/-

directly after application of BTX.

Recently, we published a paper using the force platform that was developed in

this thesis. In this paper, it was shown using BK-/- mice that the absence of BK (big

conductance potassium) channels in the nerve terminal alters neurotransmission (Wang et

al., 2020). This work highlights how our technique can show nerve defects in muscle

force recordings. Nerve stimulated force, in this case, was significantly reduced in BK-/- mice compared to WT littermates (Figure 16) but not direct muscle stimulated force.

Figure 16: Reduction in Muscle Force Caused by Altered Neurotransmission Shown here is an example of how the Achilles IV was put to use to find the cause of muscle weakness in mice lacking big conductance K+ (BK) channels. (A) Representative specific force traces from wild type (black) and BK–/– (red) muscle showing twitches (top panel) and responses to 30 Hz (middle panel) and 60 Hz (bottom panel) stimulation for both nerve stimulation (left column) and direct muscle stimulation (right column). (B) Bar graph showing twitch specific force for nerve and muscle stimulation. Values shown as mean ± SEM, * indicates a p-value < 0.05, one-way ANOVA. (C) Specific force-frequency curves, fitted with a Boltzmann equation, for WT nerve (black solid line) and muscle (black dashed line) stimulation as well as BK–/– nerve (red solid line) and muscle (red dashed line) stimulation. Values are shown as ± SEM, n = 4 WT and 6 BK–/– mice.

CONTRACTILE MACHINERY MODIFICATIONS

Contractile filament alteration could play a role in the increased specific force in

R6/2 muscle. Regulatory proteins are present in both myosin and actin and different

isoforms and levels of expression for both have been shown (Bozzo et al., 2003; Gordon

et al., 2000; Schiaffino and Reggiani, 1996). Myosin light chain (MyLC)

2+ phosphorylation alters contractile filament sensitivity to [Ca ]i (Schoffstall et al., 2006).

When MyLC is phosphorylated, the binding affinity for Ca2+ is increased and force production is increased (Bozzo et al., 2005).

A complete review of all these contractile filament regulatory proteins as well as their transcriptional modifications is beyond the scope of this thesis. However, remodeling of contractile filaments and regulatory proteins could be affecting R6/2 force production. Mutant Htt is known to interact with intracellular proteins and alter gene expression. These protein interactions still need to be explored, especially in skeletal muscle.

ALTERED INTRACELLULAR CALCIUM HOMEOSTASIS

2+ The proper transport and handling of [Ca ]i is very important for muscle

contraction in response to APs. We have talked about the release of Ca2+ through the opening of ryanodine receptors in the SR membrane. Previous reports have shown no change in the expression of ryanodine receptors in R6/2 muscle (Braubach et al., 2014).

Additionally, the SERCA pump responsible for pumping Ca2+ back into the SR should

also be considered. Increased Ca2+ or decreased SERCA pumps or activity could result in

2+ 2+ elevated [Ca ]i. Braubach et al. assessed both and found decreased Ca release with

decreased Ca2+ reuptake into the SR. They did not assess SERCA pump expression.

Interestingly, they also found increased Ca2+ entry from the extracellular space.

Our fusion index data suggest some change in Ca2+ handling during muscle

contraction. Additionally, a leftward shift in the force-frequency curve could be partially

explained by altered Ca2+ handling. Fusion occurs when Ca2+ is not completely pumped

back into the SR when the next stimulus of a train causes the ryanodine receptors to open

again. This gives less time for the force to return to baseline before climbing again in

2+ response to the stimulus. If there is increased [Ca ]i during muscle contraction in R6/2

mice, then more force could be produced at a given frequency than in WT muscle causing

a leftward shift in the force-frequency curve.

MUSCLE FATIGUE DUE TO REPETITIVE STIMULATION

Rarely in biological research does an observed change in physiology happen in

isolation. Increased force output, on one hand, could be viewed as being advantageous,

but on the other hand, it will likely come at a cost. The direct muscle stimulated force-

frequency curve would suggest that this is the case. In R6/2 muscle the force-frequency

curve plateaus at frequencies above 60 Hz, whereas the WT force curve continues to climb. We believe that we caused the R6/2 muscle to fatigue due to the repeated stimulation that was performed to collect the data needed for two force-frequency curves in succession.

The twitch to tetanic data supports this notion. The twitch to tetanic ratio for the nerve stimulated curve in R6/2 muscle was not significantly different than WT. We were

55 able to obtain a ratio of around 30% for both R6/2 and WT during nerve stimulation.

However, the twitch to the tetanic ratio for the muscle stimulated curve in R6/2 was significantly different from WT. Near the end of our experiment R6/2 muscle was producing less tetanic force than it was earlier when we were stimulating the nerve.

Increased muscle weakness after prolonged activity fits with patient symptomology. Patients suffer from motor impersistence (Paulson and Albin, 2011) which is the inability to sustain a voluntary muscular effort. They also suffer from chorea which results in irregular and jerky movements. R6/2 muscle being hyperexcitable

(Waters et al., 2013) and producing more force while being more prone to fatigue could help explain some of these symptoms.

It would be important to perform further studies to assess muscle fatigue in R6/2 muscle. A protocol including repeated rounds of tetanic stimulation could be developed and used to characterize the level of fatigue in response to sustained activity in R6/2 muscle. This type of stimulation protocol better mimics activities such as locomotion.

The brief stimuli of twitches and the short trains used in this study are not as relevant physiologically to sustained activity which is important for tasks such as coordinated movement like walking or chewing food.

VI. Conclusions

Upon successful development of the Achilles in vivo muscle force system, R6/2 muscle function was able to be assessed by generating force-frequency curves. We found that raw muscle force in the R6/2 mouse model of HD is reduced when compared to WT littermates. This data is consistent with previous reports (Hering et al., 2006; Mielcarek et al., 2015). However, when normalized to muscle weight, specific force produced by R6/2 mice is significantly higher than WT. Additionally, increased fusion index suggests that altered myocyte Ca2+ homeostasis could be causing increased specific force in R6/2 muscle. Defects in myocyte Ca2+ homeostasis have been shown in the R6/2 line

(Braubach et al., 2014). Future research must be conducted to determine the specific mechanisms that are responsible for the altered Ca2+ handling during muscle contraction.

Muscle fatigue was detected in the direct muscle stimulated force-frequency data.

The twitch to tetanic ratio was significantly increased in R6/2 muscle suggesting that the plantar flexor muscles were fatigued due to repeated stimulation. Since nerve stimulated contractions were performed prior to direct muscle stimulation, the muscle had contracted over fifteen times at different frequencies. WT muscle had no problem with this level of stimulation and the twitch to tetanic ratio was consistent from nerve stimulation to muscle stimulation in WT muscle. To further assess how prone to fatigue HD muscle is, repeated stimulation protocols should be developed, and muscle force should be assessed.

With motor dysfunction being the primary symptom of HD and a leading cause of mortality, skeletal muscle defects are a promising target for the treatment of symptoms in

HD. This work provides additional support for the continued study of primary peripheral defects in animal models of HD. There is no doubt that cognitive decline and striatal

57 degeneration also play a key role in the progression of HD symptoms, but unfortunately these areas are harder to treat. Even if symptoms could be partially ameliorated and progression of the disease slowed by some percentage, patient outlook could be drastically improved.

An essential tool for the development of the muscle force system used to complete this thesis was the ability to rapidly create various prototypes by utilizing 3D modeling and printing. There is no doubt that the skills obtained throughout this project will be used in future endeavors. It is our hope that 3D printing will continue to be utilized in biological research. Here, we have only scratched the surface of the potential for this tool to help us further understand human disease.

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