THE EFFECTS OF CARDIAC BINDING -C AND INORGANIC ON LENGTH-DEPENDENT ACTIVATION

By

MILANA LEYGERMAN

Submitted in partial fulfillment of the requirements

For the degree of Master of Science

Thesis Adviser: Dr. Julian Stelzer

Department of Physiology and Biophysics

CASE WESTERN RESERVE UNIVERSITY

May, 2011

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of ______Milana Leygerman______candidate for the ____Master of Science____degree *.

(signed)______Dr. William Schilling (chair of the committee)

______Dr. Thomas Nosek

______Dr. Margaret Chandler

______Dr. Saptarsi Haldar

______Dr. Andrea Romani

______Dr. Julian Stelzer

(date) ______03/16/2011_____

*We also certify that written approval has been obtained for any proprietary material contained therein. Acknowledgements

I would like to thank my thesis advisor Dr. Julian Stelzer for all his guidance and help during my working on my thesis. I would also like to thank the lab personnel:

Brian Ziese and Dr. Arthur Coulton for their help. Additionally, I would like to thank my thesis committee, including Dr. Nosek, Dr. Andrea Romani, Dr. Schilling, Dr. Chandler, and Dr. Haldar for all their support. Table of Contents

LIST OF FIGURES...... 3

ABSTRACT……………………………………………………………………………..4

INTRODUCTION………………………………………………………………………5

Actin...…………………………………………………………………………………....7

Titin……………………………………………………………………………………....8

Tropomyosin.…………………………………………………………………………….8

Troponins….……………………………………………………………………………..9

Myosin…………………………………………………………………………………...9

Myosin binding protein-C…………………………………………………………….....10

Cross-bridge cycling…………………………………………………………………….12

Frank-Starling Law of the ………………………………………………………...16

Role of inorganic phosphate in cross-bridge cycling…………………………………....17

EXPERIMENTAL DESIGN…………………………………………………………..20

Mouse models…………………………………………………………………………...21

Myocardial preparations………………………………………………………………...21

Experimental apparatus…………………………………………………………………22

Solutions………………………………………………………………………………...23

Phosphate studies……………………………………………………………………….27

RESULTS……………………………………………………………………………....27

DISCUSSION………………………………………………………………………….35

BIBLIOGRAPHY……………………………………………………………………..40

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List of Figures

Figure 1…………………………………………………………………………………..6

Figure 2…………………………………………………………………………………..7

Figure 3……………………………………………………………………………….....12

Figure 4………………………………………………………………………………….14

Figure 5………………………………………………………………………………….15

Figure 6………………………………………………………………………………….17

Figure 7………………………………………………………………………………….19

Figure 8………………………………………………………………………………….23

Figure 9………………………………………………………………………………….23

Figure 10………………………………………………………………………………...25

Figure 11………………………………………………………………………………...26

Figure 12………………………………………………………………………………...30

Figure 13………………………………………………………………………………...31

Figure 14………………………………………………………………………………...32

Figure 15………………………………………………………………………………...32

Figure 16………………………………………………………………………………...33

Figure 17………………………………………………………………………………...34

Figure 18………………………………………………………………………………...34

Figure 19………………………………………………………………………………...35

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The Effects of Cardiac Myosin Binding Protein-C and Inorganic Phosphate on Length Dependent Activation

Abstract

By

MILANA LEYGERMAN

The contractile unit of a striated muscle is called the and is composed of , myosin, , the complex, , the myosin light chains, and cardiac myosin binding protein-C (cMyBP-C). is caused by cross-bridge cycling, which involves the sliding of thick filaments past thin filaments. cMyBP-C is a constituent of the thick filament and is involved in regulation of contraction. in this protein have been known to lead to hypertrophic , an autosomal disease characterized by hypertrophy and fibrosis. Sarcomere length is an important determinant of muscle contractility as increased length results in greater overlap between actin and myosin leading to greater Ca2+-sensitivity and force generation. The Frank-

Starling law of the heart is an important relationship for regulation of contraction and is influenced by sarcomere length. In conditions of , there is a downward and rightward shift of the Frank-Starling relationship where an increase in end-diastolic volume generates a relatively smaller increase in stroke volume insufficient to meet the demands of the heart. Inorganic phosphate plays an important role in muscle

3 contraction as its release from the acto-myosin complex is a crucial step in the cross- bridge cycle that drives muscle contraction. In this study, to elucidate the effects of cMyBP-C and inorganic phosphate on length dependent activation, we utilized a skinned myocardium isolated from a knockout (KO) mouse model that lacks cMyBP-C (cMyBP-

C-/-). A total of 85 mechanical experiments were performed in skinned fibers isolated from WT and KO left ventricles. The results showed that increased sarcomere length increases force generation and Ca2+-sensitivity of force in WT and KO animals. The rate of force development, an index of the speed of cross-bridge function was accelerated with increased sarcomere length in the KO fibers but not WT fibers. Treatment of muscle fibers with a low concentration of inorganic phosphate (1mM) did not have an effect on maximum calcium-activated force at short and long sarcomere length in either WT or KO myocardium, but accelerated rates of force development in both WT and KO muscle fibers. These results imply that the effects of sarcomere length are enhanced in KO myocardium due to the increased proximity of myosin to actin in fibers lacking cMyBP-

C. Low concentrations of inorganic phosphate may accelerate the transitions from weak to strong binding cross-bridge states in both WT and KO myocardium, thereby accelerating rates of force development.

4

Introduction:

The sarcomere (Figure 1) is the contractile unit of striated muscle (skeletal and cardiac) and contains the thin filament, the thick filament, and titin. Muscle contraction and relaxation occurs via the sliding of thick filaments past thin filaments through cross- bridge cycling as can be seen in Figure 2. The thin filament consists of actin, tropomyosin, and the troponin complex, while the thick filament then consists of myosin, an asymmetric dimer that contains a globular head portion ( S1), which is associated with two hetero-dimers light-chain 1 and light-chain 2 (LC-1 and LC-2), a hinged stalk region

(S2) and a rod section (de Tombe et al, 2003). The S1 head portion also consists of the actin binding domain and ATP hydrolysis domain, whereas the myosin rod section is associated with the myosin binding protein-C (MyBP-C) (de Tomble et al, 2003). MyBP-

C is involved in muscle contraction by stabilizing the thick filament and regulating the number of myosin heads available for involvement in a given contractile cycle. Cardiac myosin binding protein-C (cMyBP-C) increases the stiffness of the heart muscle, which slows early contraction but then allows to be sustained so the heart can effectively eject after greater stiffening. Cardiac muscle contraction depends on the properties of cross-bridge cycling, and involves several thin and thick regulatory , which are described below.

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Figure 1. The structure of a sarcomere. This figure was taken from Boron and Boulpaep, Medical

Physiology, 2nd Edition. Reprinted with permission from Walter Boron.

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Figure 2. Muscle contraction. Thick and thin filaments of the sarcomere slide past one another during contraction and relaxation. Tropomyosin overlays the binding sites of actin on myosin, which inhibits contraction. The troponin complex, which has three subunits (-tropomyosin binding, -inhibitory, and -Ca2+ -binding) plays a role in regulating conformational changes of tropomyosin. This figure was taken from de Tombe et al., 2003. Reprinted with permission from Pieter de Tombe and the Journal of Biomechanics.

Key contractile proteins

Actin

Actin is a constituent of the thin filament and is polymerized to a two-stranded helical structure called F-actin. F-actin is composed of G-actin, which represents individual globular actin subunits. Sub-domain 1 of the actin helix is believed to interact with myosin (Miller et al., 1995). Actin is anchored to the Z line of the sarcomere and its interaction with myosin produces force via formation of a cross-bridge. This protein is

7 known to have interactions with other sarcomeric proteins such as titin and , which is a calcium binding homodimer protein.

Titin

Titin is a large sarcomeric protein that extends from Z-line to M-line. A region of titin spans the I band of the sarcomere and can develop passive force in stretched (Granzier and Labeit, 2007) which contributes to the passive tension of the myocardium that determines diastolic filling (Granzier and Labeit, 2007). Titin also plays a role in stabilizing the thick filament during muscle contraction. Studies have suggested that titin may play a role in the sarcomere length-dependent increased Ca2+ sensitivity of active force, which is important for the Frank-Starling law of the heart, either by enhancing acto-myosin interaction through a decrease in interfilament lattice spacing or by increasing strain on the thick filament and influencing cross-bridge movement

(Granzier and Labeit, 2004).

Tropomyosin

Tropomyosin is a constituent of the thin filament and is composed of two alpha helical chains that are coiled around each other and are parallel (Van Buren and Palmer,

2010). This protein spans 40 nm and lies in the groove of actin helical fragment and blocks myosin binding sites on actin during diastole (Van Buren and Palmer, 2010).

When the C-terminal domain of troponin I binds to actin at low diastolic Ca2+ , tropomyosin is in an inhibitory orientation preventing cross-bridge binding (Stehle and

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Iorga, 2010). The troponin complex gets switched on by Ca2+ allowing for tropomyosin to relocate and myosin can then bind to actin (Stehle and Iorga, 2010).

Troponins

The troponin complex consists of three subunits: Troponin I (inhibitory), Troponin

T (tropomyosin binding), and Troponin C (Ca2+ binding). These subunits play a role in the regulation of cross-bridge cycling via interaction with Ca2+ and tropomyosin. TnI is an important myofibrillar constituent during rearrangement of proteins that results in force development (Konhilas et al., 2003). Past studies have shown that TnI plays a role in regulation of both Ca2+ sensitivity and length-dependent activation, which is the basis for the Frank-Starling law of the heart (Konhilas et al., 2003).

Myosin

Myosin is a constituent of the thick filament and is involved in muscle shortening and is regulated by its interaction with actin in ATP-dependent manner. It consists of two heavy chains and four light chains (two essential light chains-ELC, and two regulatory light chains-RLC). The heavy chains of the myosin molecule form the nucleotide-binding constituent as well as a major structural component of the thick filament. Phosphorylation of the regulatory light chain on myosin increases force and the rate of force development, and this effect is more apparent at sub-maximal calcium activations that occur during cardiac muscle systole (Metzger et al., 1989). The S1 region (head and neck region of the heavy chains) consists of binding regions, an ATP binding domain, and an actin binding domain.

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Myosin Binding Protein-C

Cardiac Myosin Binding Protein-C is a sarcomeric thick filament component that interacts with titin, myosin, and actin in order to regulate the assembly, structure and function of a sarcomere (Barefield and Sadayappan, 2009) and is illustrated in Figure 3.

This protein limits cross-bridge cycling rates and can reduce myocyte power output

(Razumova et al., 2008). Due to the fact that this protein slows contraction but allows the force to be sustained longer, detachment of the cross-bridges during muscle contraction is slower. Myosin Binding Protein-C exists in three isoform in striated muscle: cardiac, slow skeletal, and fast skeletal (Ackermann and Kontrogianni-Konstantopoulos, 2010).

Cardiac myosin-binding protein-C is expressed early in development in , along with titin and myosin. The skeletal isoform of MyBP-C are expressed later in development after the expression of titin and myosin, as the slow isoform expression occurs prior to the fast one (Ackermann and Kontrogianni-Konstantopoulos, 2010). The protein is a member of the immunoglobulin superfamily and is located in the C-zone of the A-band of the sarcomere and is bound to thick filaments in distinct stripes (between 7 to 11) (Ackermann and Kontrogianni-Konstantopoulos,2010). The C terminus of myosin binding protein-C (MyBP-C) contains a binding domain with high affinity for myosin subfragment-2 (Stelzer et al., 2006). The myosin molecule then contains sites for MyBP-

C binding within the S2 and light (LMM) fragments (Stelzer et al., 2006).

The cardiac isoform is composed of eight immunoglobulin I-like domains and three fibronectin 3-like domains and is a physiological substrate of cAMP-dependent protein (Flashman et al., 2004). cMyBP-C is contained in two groups separated by a bare H-zone in the C zone within inner two-thirds of the A band (Barefield and

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Sadayappan, 2009). This sarcomeric protein is also able to regulate cross-bridge formation through phosphorylation by , , and Ca2+- -activated kinase II. Phosphorylation of cMyBP-C is associated with regulation of cardiac contraction. Dephosphorylation of this protein is related to development of hypertrophic cardiomyopathy and heart failure. Overall, cMyBP-C encompasses around 2% of the total amount of contractile protein the heart (Barefield and Sadayappan, 2009).

The study of cMyBP-C is important as mutations in this protein can lead to hypertrophic cardiomyopathy and affect more than sixty million people worldwide, and despite the obvious importance of cMyBP-C relatively same little is known about the function of this protein. Hypertrophic cardiomyopathy, an autosomal dominant disease, is characterized by myocyte disarray, left ventricular hypertrophy and interstitial fibrosis.

These disease-causing mutations were originally mapped to the that encodes β- myosin heavy chain on 14q1. The main defect seen with these mutations is a shift in the normal contraction or relaxation of cardiac muscle, which can furthermore promote ventricular remodeling and hypertrophy (Flashman, 2004). cMyBP-C missense mutations can alter an that is outside of the A-band region. Thus, mutants of cMyBP-C can be incorporated into the sarcomere and disturb normal function of a sarcomere. A model of lack of cMyBP-C in the heart elucidates the effects of cMyBP-C on myocardial contraction (Stelzer et al., 2006), and cMyBP-C-/- mice show severe ventricular hypertrophy, systolic dysfunction, manifested as shortened ejection and reduced stroke volume, and diastolic dysfunction seen as a decreased rate of isovolumic relaxation (Stelzer et al., 2006).

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Figure 3. Cardiac myosin binding-protein C localization within the sarcomere. This figure was taken from Schlossarek et al, 2011. Reprinted with permission from the Journal of Molecular and

Cellular Cardiology.

Cross-bridge cycling

Cross-bridge cycling, which is shown in Figure 4, occurs under the strict regulation of Ca2+ release and reuptake via the (Ackermann and

Kontrogianni-Konstantopoulos, 2010), as well as an inward Ca2+ influx through L-type calcium channels. The process of Ca2+ triggering its own release called calcium-induced calcium release (CICR) from the sarcoplasmic reticulum occurs via excitation-

12 contraction coupling, which allows the chambers of the heart to contract and relax

(Kobayashi and Solaro, 2005). The concentration of Ca2+ is around 10-7 M in a relaxed myocardium, which is very low compared to 1-2 x 10-3 M in the extracellular space.

Membrane depolarization by the cardiac action potential leads to the activation of L-type

Ca2+ channels which results in an influx of Ca2+ (Bers, 2002). Ca2+ then binds to and activates the which leads to release of Ca2+ from the sarcoplasmic reticulum (calcium-induced calcium release) resulting in an increase in intracellular Ca2+ up to 10-5 M (Bers, 2002). This new level of Ca2+ activates the and contraction begins with Ca2+ binding to troponin C leading to a conformational change in the tropomyosin complex resulting in the binding of myosin to actin. Myosin will stay in a detached position if it’s still bound by ATP. As the ATP is hydrolyzed to ADP and inorganic phosphate, the rotates to its cocked position before jumping to an actin , forming a cross-bridge. The muscle contraction begins when phosphate dissociates from myosin, triggering a power stroke. The myosin head rotates 45 degrees pulling the actin along with it. After the power stroke is completed, ADP is released and the cross-bridge is stuck in the attached and contracted state. The cycle will start over at the relaxed state once ATP binds to the myosin head (Boron and Boulpaep, Medical

Physiology, 2nd Edition).

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Figure 4: Cardiac muscle cross-bridge cycling. This figure was taken from Boron and Boulpaep,

Medical Physiology, 2nd Edition. Reprinted with permission from Walter Boron.

Sarcomere length influences muscle contractility. The isometric force of contraction depends on the initial length of muscle fiber. Increases in sarcomere length lead to an increase in Ca2+ -sensitivity of force due to increased binding of Ca2+ to low affinity binding site on troponin C and decreased lattice spacing that increases probability of cross-bridge binding (Fuchs and Martyn, 2005). Increased sarcomere length results in greater overlap between actin and myosin leading to greater Ca2+-sensitivity (Fuchs and

Martyn, 2005). This length-tension relationship is shown in Figure 5. Increased myocyte

14 length will reduce interfilament spacing and in turn will increase Ca2+-sensitivity of myofibrillar force generation (Fuchs and Martyn et al., 2005). In contrast, decreased sarcomere length will decrease Ca2+-sensitivity of force generation such that more Ca2+-is required to produce similar force production as in longer sarcomere lengths (Fuchs and

Martyn, 2005, Hanft et al., 2008). The effects of sarcomere length on the cross-bridge cycling kinetics in cardiac muscle are less clear, with some studies showing that increased sarcomere length accelerates cross-bridge kinetics (Stelzer et al., 2006, McDonald et al., 1997,

Fitzsimons et al., 2001), and some showing a slowing of cross-bridge kinetics (Korte et al.,

2007).

Figure 5. Length-tension diagrams. Muscle length influences force development by determining the degree of overlap between actin and myosin filaments. The passive curve shows the tension that is measured at various muscle lengths before muscle contraction. The active tension is the difference between the total and the passive tensions. This figure was taken from Boron and Boulpaep, Medical

Physiology, 2nd Edition. Reprinted with permission from Walter Boron.

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The Frank-Starling law of the heart

The Frank-Starling relationship is essential in regulation of cardiac function and is modulated by the sarcomere length of cardiac muscle (Hanft et al., 2008). This relationship describes how an increase in muscle length increases contractility (Shiels and

White, 2008). This means, that, in normal , increased diastolic volume, which corresponds with increased sarcomere length at the myofilament level (actin-myosin overlap), leads to increased stroke volume (Hanft et al., 2008). This relationship between changes in end-diastolic volume and stroke volume is commonly impaired in end-stage heart failure (Jacob et al., 1992, Scwinger et al., 1994, Holubarsch et al., 1996, Hanft et al., 2008).

Cardiac contractility is known to increase during β-adrenergic stimulation, which leads to increases in the steepness of the Frank-Starling relationship such that greater changes in stroke volume are elicited by changes in end-diastolic volume. However, in conditions of heart failure, there is a downward and rightward shift of the Frank-Starling relationship, compared with a normally functional heart, such that an increase in end- diastolic volume produces a relatively smaller increase in stroke volume, which is inadequate to meet the demands of the circulatory system (Hanft et al., 2008) as demonstrated in Figure 6 below.

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Figure 6. Ventricular function curves showing the Frank–Starling relationship. A change in end- diastolic volume in normal hearts (blue line) leads to a change in stroke volume which is suitable to meet metabolic needs of the heart. The Frank–Starling relationship may be adjusted leftward and upward (green line) in order to yield more stroke volume for a given end-diastolic volume and a greater change in stroke volume with a change in end diastolic volume. This relationship may also be shifted downward and rightward (yellow and red lines), which is seen as heart failure. This figure was taken from Hanft et al., 2008.Reprinted with permission from Cardiovascular Research.

The role of inorganic phosphate in cross-bridge cycling

The byproducts of ATP hydrolysis in cross-bridge cycling are adenosine 5'- diphosphate (ADP) and inorganic phosphate (Pi) ( ). In its ready state, myosin has ADP and inorganic phosphate bound to its nucleotide binding pocket. Myosin binds to actin in the strong-binding state which is coupled to the release of inorganic phosphate. Inorganic phosphate plays an important role in cross-bridge cycling such that the dissociation of Pi from the myosin head triggers a power stroke, which is coupled to a conformational change in which the myosin head bends approximately 45 degrees and pulls the actin filament 11 nm toward the tail of myosin molecule, thereby generating force via transitions from a weak cross-bridge to a strong cross-bridge.

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It has been suggested that phosphate release from acto-myosin- Pi (AM·ADP·Pi) in muscle fibers is reversible and related to force generation. The dissociation for Pi binding to AM·ADP is very fast (White and Taylor,

1976). Cellular concentrations of inorganic phosphate are in the sub-millimolar range

(less than 0.5 mM) even though intracellular Pi may reach such levels as 30 mM during exercise. Thus, Pi dissociation from AM·ADP·Pi is almost irreversible at high phosphate concentrations (Sleep and Hutton, 1980). Since Pi is released in the transition from low- force, weak-binding states to high-force, strong-binding states, the transition to high- force states is impeded by increased Pi (Westerblad et al, 2002). Low phosphate (1 mM) could act like a strong-binding, nonforce-generating analog of myosin subfragment-1

(NEM-S1), accelerating the rate of force development in skinned myocardium without an effect on maximum force (Stelzer et al., 2006). This may be because cardiac muscle is very sensitive to perturbations in cross-bridge function due to its high cooperative nature, i.e., smalls shifts in cross-bridge properties can affect a large number of cross-bridges quickly. Activation of myocardial contraction is a highly cooperative process because initial cross-bridge binding to actin recruits additional cross-binding along with increased

Ca2+-binding to troponin C (Moss et al., 2004). Cooperative recruitment of cross-bridges slow the development of force, which is most evident at low levels of Ca2+-activation when relatively few cross-bridges are bound to actin (Campbell, 1997). Previous experiments have shown that high inorganic phosphate may be used to influence the number of strongly bound cross-bound bridges, decreasing the Ca2+-sensitivity of force via a decrease in the number of strongly bound cross-bridges at different pCas and

18 slowing cooperative recruitment of additional cross-bridges to force-generating states

(Moss et al., 2004).

As Pi reaches high concentrations inside the , fewer cross-bridges would be in high-force states, thereby, diminishing force production (Westerbald et al., 2002). It is also thought to inhibit the transition from the weakly bound to the strongly bound cross bridge, and/or a reduction in the number and force of the strongly bound states (Fitts,

2007). At high concentrations, inorganic phosphate reduces sarcoplasmic reticulum Ca2+ release and reuptake as well. High Pi also increases kinetics by accelerating the reverse rate constant of the transition of low- to high-force state (Fitts, 2007). The kinetic scheme illustrating the two pathways for inorganic phosphate release and rebinding is shown in

Figure 7. Therefore, the rationale for utilizing increased inorganic phosphate was to study kinetics without changes in force by using a small amount of Pi. The cMyBP-C-/- mouse model exhibits faster basal myofilament kinetics that the WT mouse, thus, the transition from weak-to strong-binding state might be faster following treatment with 1 mM inorganic phosphate.

Figure 7. Inorganic phosphate release and rebinding. This figure was taken from Kraft, et al., 2005. Reprinted with permission from PNAS. Copyright (2005) National Academy of Sciences, U.S.A.

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Objectives of the thesis

The aims of this study were to 1) elucidate length-dependent effects of cMyBP-C on muscle contraction seeing that there is an increase in the proximity of myosin to actin at longer sarcomere length, enhancing cross-bridge binding and 2) observe if the accelerated cross-bridge kinetics in the cMyBP-C-/- model are due to changes in a particular cross-bridge step that would be sensitive to inorganic phosphate such as the phosphate release step by treating skinned myocardium from WT and KO hearts with 1 mM inorganic phosphate.

Experimental design and methods:

Specific aims: The specific aims of this project were to 1) measure force and cross-bridge kinetics in skinned myocardium from wild-type and cMyBP-C-/- (null) hearts to determine whether observed contractile changes are related to changes in the proximity of myosin to actin and 2) Perform the experiments described above in skinned myocardium isolated from cMyBP-C-/- (null) hearts to determine whether inorganic phosphate (1 mM) increases cardiac muscle sensitivity and accelerate weak-to strong-state cross-bridge binding.

Experimental approach: A total of 85 skinned fiber experiments were performed in skinned myocardium from 2 distinct groups (WT, cMyBP-C-/-) with and without inorganic phosphate treatment, at short (1.85 μm) and long (2.25 μm) sarcomere lengths. Measurements that were conducted include force-pCa relationships to assess the sensitivity of myofilaments to Ca2+, and the steepness of these relationships was used to assess the of force development. The apparent rates of cross-bridge attachment and detachment were assessed by a stretch and release protocol (ktr). Sarcomere lengths were assessed at the beginning and

20 end of each experiment to make sure that sarcomere length does not deviate within the course of the experiment.

Mouse models: The cMyBP-C null (cMyBP-C-/-) mouse was utilized to study the effects of cMyBP-C ablation on force and kinetics at varied sarcomere length. Gene targeting was used to produce a mouse that lacks cMyBP-C. Knockout mice were created by deletion of 3 to 10 from the endogenous cMyBP-C gene in murine embryonic stem cells followed by breeding of chimeric founder mice to obtain mice heterozygous (+/-) and homozygous (-/-) for the knockout allele (Harris et al., 2002). Wild-type (+/+), cMyBP-C+/-, and cMyBP-C-/- mice were born in agreement with Mendelian inheritance ratios (Harris et al., 2002). cMyBP-C-/- mice, unlike the WT, showed signs of cardiac hypertrophy.

Preparation of muscle fibers: Skinned multicellular preparations were obtained from mouse hearts. To prepare skinned myocardial preparations, the frozen ventricles were thawed and homogenized in relaxing solution for 2 seconds using a Polytron, which yielded multicellular preparations of 100 to 250 µm x 600 to 900 µm. The homogenate was centrifuged at 120g for 1 minute and the resulting pellet was washed with fresh relaxing solution and resuspended in relaxing solution containing 250 g/mL saponin and

1% Triton X-100. These skinned myocardial preparations were allowed to settle for an additional 20 minutes, after which, the fibers were isolated in a glass petri dish with about

50 mL of fresh relaxing solution. The petri dish containing skinned myocardial preparations were set aside on ice if not used.

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Experimental apparatus: The experimental apparatus is shown in Figure 8. Skinned myocardium was transferred from the petri dish to an experimental compartment consisting of fresh relaxing solution. The ends of a preparation were attached to the arms of a motor and force transducer in order to obtain mechanical measurements as shown in

Figure 9. The chamber assembly was then placed on the stage of an inverted microscope fitted with a 40x objective and a closed-circuit television camera in order to assess mean sarcomere length during the course of each experiment. Changes in force and motor position were also sampled at 2.0 kHz using SLControl software and saved to computer files for later analysis. Fiber force during the experiments was recorded on a digital oscilloscope. Sarcomere length as well as fiber length and width were assessed during each experiment with the starting length of 1.85µm. The cross sectional area of the myocardial preparation was obtained from the width of the fiber assuming a circular diameter. The experiments were carried out at 22ºC, and sarcomere length was varied from 1.85 μm to 2.25 μm.

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Cardiac Muscle Mechanics

Apparatus for Mechanical Measurements

Experimental Chamber

100 µm

Permeabilized Force Transducer Motor Myocardial Preparation “Pin” (4-0 Nylon Suture)

“Loops” (10-0 Nylon 100 µm Suture)

Figure 8. Experimental apparatus used for mechanical studies.

Figure 9. A muscle fiber mounted between a force transducer and a motor.

Solutions: Solutions that were used in this study included a relaxing solution which was prepared (in mM) with approximately 100 KCl, 20 imidazole, 4 MgATP, 2 EGTA, and 1

Mg2+ free at pH 7.0 and 22°C. Activating solutions consisted of (in mM) 100 N, N-bis[2- hydroxyethyl]-2-aminoethanesulfonic acid, 15 creatine phosphate, and 5 1, 4- dithiothreitol. pCa 9.0 consisted of 7 EGTA, 5.49 MgCl2, 4.66 ATP, 0.017 CaCl2, and pCa

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4.5 will consist of 7 EGTA, 5.29 MgCl2, 4.67 ATP, and 0.07 EGTA. pCa solutions ranging from 5.5 to 6.1 were prepared by mixing proper volumes of pCa 9.0 and pCa 4.5 solutions.

Force-pCa relationship: In order to measure a force-pCa relationship, each myocardial preparation was allowed to develop steady force in different solution of free calcium. The preparations were initially activated at pCa 4.5 to establish the maximal tension (Po), and the force was then recorded in pCa 9.0 to establish the resting force, which was then subtracted from the total force in activating solutions to yield the Ca2+-activated force (P).

The active force at each pCa was measured as the difference between total steady-state force and passive force determined by applying a 20% slack step to the preparation in a solution of pCa 9.0. Force-pCa relationships were derived by expressing submaximal force (P) at each pCa (range: 6.1–5.5) as a fraction of maximum force (Po) determined at pCa 4.5 (P/Po). The force required for half-maximal activation (pCa50) was determined by fitting the force-pCa data with a Hill equation: P/Po = [Ca2+]nH/(knH + [Ca2+]nH),

2+ where nH is the Hill coefficient and k is the Ca concentration required for pCa50.Force- pCa relationships were arrived at through submaximal force being expressed as a fraction of maximal force (pCa 4.5). The activation of force development was assessed by the steepness of the force-pCa relationship (nH- Hill coefficient for Ca2+ -activated force).

Figure 10 shows a representative force-pCa curve from WT and KO myocardium.

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Figure 10. Force-pCa relationships at short and long sarcomere lengths. Lines were generated by fitting the data to a Hill equation: P/Po = [Ca2+]nH/(knH + [Ca2+]nH),, with nH being the Hill

2+ coefficient and k the [Ca ] required for half-maximal activation (pCa50 ).

Rate of force redevelopment: All multicellular myocardial preparations were transferred from a well containing relaxing solution to a well containing activating solution with different free calcium (pCa 6.1-4.5) and allowed to produce steady-state force. The rate constant of force development (ktr) was acquired by activating the skinned myocardium in solution of pCa 4.5 and further in a series of submaximally activating solutions ranging from pCa 6.1 to pCa 5.5. To measure the rate of force redevelopment, the fiber was activated with Ca2+ and steady isometric tension was allowed to develop, after which the fiber was quickly released to a shorter length to break cross-bridges and then restretched as described in Metzger et al., 1989. The rate of force redevelopment (rate of reattachment of cross-bridges) was measured by determining the time required (ms) to achieve steady state force following the slack-restretch maneuver, such that faster rates of

25 force development were correlated with decreased times (i.e., fewer ms) to achieve steady state force. The slack-restretch maneuver is thought to dissociate cross-bridges from actin, and the following force redevelopment arises from the reattachment of cross- bridges and transition to force-generating states. The rate of force redevelopment after release-restretch maneuver has previously been analyzed using a two-state cross-bridge cycling model with rate constants for transitions between non-force-generating and force- generating states (f), and between force-generating and non-force generating states (g)

(Campbell, 1997). The experiment was run with SLControl software. At the end of an experiment, the myocardial preparation was activated in a solution of pCa 4.5 in order to evaluate decline in the maximal rate of force redevelopment. The value of maximal ktr for each activation was attained by interpolation between the initial and final measurements of maximal ktr (Stelzer et al., 2006). The apparent rate constants of force redevelopment

(ktr) were predicted by linear transformation of the half-time of force redevelopment, i.e., ktr = 0.693/t1/2 (Stelzer et al., 2006). Figure 11 shows representative ktr traces.

a

b

c

Figure 11. Kinetics of muscle contraction. a-high rate of force redevelopment, b-intermediate rate of force redevelopment, and c-low rate of force redevelopment. This figure was taken from Fitzsimons et al., 2001. Reprinted with permission from the Journal of General Physiology.

26

The milliseconds for force redevelopment at different pCas were obtained via analysis of time to peak for each curve. This was done due to the fact that elastic elements due to fibrosis in the KO myocardium increased the residuals of the ktr, making the analysis by exponential fit challenging; therefore we chose to report an analysis of rates with absolute and relative (considered in comparison to the maximum rate) milliseconds.

Phosphate studies: 100 µl of stock pH 7.0 KH2PO4 solution was diluted with 900 µl of relax solution for a total volume of 1 ml. The muscle fiber was incubated for 15 minutes in a 1 mM solution of phosphate.

Statistical Analysis

All data was expressed as mean ±SE. A multifactorial ANOVA was used as a post-hoc test of significance to compare differences in sarcomere length and effects on mechanical parameters of fibers from each mouse line with significance level set at p<0.05.

Results:

Skinned fiber experiments were performed and it was observed that maximal Ca2+

2+ -activated force at pCa 4.5 (Po) and Ca - sensitivity were consistently increased at long sarcomere length for the 2 groups as can be seen in Table 1, confirming that the increase

2+ in Po and Ca - sensitivity after an increase in sarcomere length may be due to an increase in myosin and actin overlap that occurs with increased length. There was an increase in pCa50

27 with increasing SL. Increased inorganic phosphate (1 mM) did not have a major effect on

Po and pCa50 as can be seen in Table 1.

Table 1. Mechanical measurements in skinned muscle fibers from WT and cMyBP-C-/- myocardium at short and long sarcomere lengths

-2 Group Number(n) Prest(mN Po(mN mm ) pCa50 nH mm-2) WT SL 13 0.68±0.1 7.43±1.1 5.75±0.03 3.7±0.6 WT LL 12 1.54±0.4* 12.83±2.7* 5.80±0.01 3.2±0.3 cMyBP-C-/- SL 10 0.88±0.3 7.82±1.3 5.71±0.03 3.6±0.5 cMyBP-C-/- LL 10 1.06±0.3 11.57±2.0* 5.8±0.02 2.9±0.3 WT-Pi SL 10 0.68±0.1 6.18±0.7 5.75±0.03 2.9±0.2 WT-Pi LL 10 2.22±0.2* 12.37±1.6* 5.84±0.02 2.8±0.1 cMyBP-C-/- -Pi SL 10 0.7±0.1 7.71±0.8 5.73±0.02 3.3±0.3 cMyBP-C-/- -Pi LL 10 1.58±0.3* 15.56±2.1* 5.80±0.03 3.0±0.2 2+ Po- maximal Ca -activated force at pCa 4.5 2+ Prest-Ca -independent force at pCa 9.0 nH-Hill coefficient for Ca2+-activated force (steepness) pCa50-pCa required for half-maximal activation *Significantly different compared to SL

Inorganic phosphate accelerated cross-bridge kinetics (both ktr and ms). Pi increased the rate of force redevelopment (ms) more in WT at maximum Ca2+ at both SL and LL. Inorganic phosphate had a smaller effect at maximal Ca2+ concentration in the

KO than the WT myocardium. This can be seen in Table 2. However, at submaximal Ca2+ concentration, Pi had the biggest effects on the KO as there was no big change in the relative milliseconds in the WT with Pi and the increase was much greater in the KO myocardium, which can be seen in Table 3.

Table 2. Ca2+ dependence of the rate of force redevelopment in WT and cMyBP-C-/- myocardium at short and long sarcomere lengths

Group Number(n) ktr max(pCa Relative ktr min(pCa 6.1) Relative ktr 4.5) ktr max min WT SL 13 19.28±1.93 1.0 2.69±0.60 0.14±0.06 WT LL 12 16.41±2.39 1.0 2.71±0.59 0.17±0.07 cMyBP-C-/- SL 10 21.60±1.71 1.0 3.8±1.20¶ 0.18±0.04 cMyBP-C-/- LL 10 22.40±1.79¶ 1.0 3.75±0.25¶ 0.17±0.05 WT-Pi SL 10 21.00±1.05 1.0 7.75±0.56* 0.37±0.01* WT-Pi LL 10 23.10±0.96* 1.0 7.50±0.83* 0.32±0.03* 28 cMyBP-C-/- -Pi SL 10 22.90±1.67 1.0 7.86±0.59* 0.34±0.03* cMyBP-C-/- -Pi LL 10 25.65±0.57* 1.0 10.1±0.41*,¶ 0.39±0.01* ktr -the rate constant of force development *Significantly different compared to without Pi ¶Significantly different compared to WT

Table 3. Ca2+ dependence of the rate of force redevelopment in WT and cMyBP-C-/- myocardium at short and long sarcomere lengths expressed in milliseconds

Group Number ms max(pCa 4.5) Relative ms min(pCa 6.1) Relative ms (n) ms max min WT SL 13 412±29.23 1.0 878±31.77 0.47±0.03 WT LL 12 428±34.90 1.0 911±36.74 0.47±0.02 cMyBP-C-/- SL 10 277±21.50¶ 1.0 857±75.66 0.32±0.03¶ cMyBP-C-/- LL 10 220±12.96¶ 1.0 657±68.24¶ 0.34±0.03¶ WT-Pi SL 10 230±9.24║ 1.0 556±23.06║ 0.41±0.02 WT-Pi LL 10 206±11.59║ 1.0 423±26.30*, ║ 0.49±0.02 cMyBP-C-/- -Pi SL 10 216±9.80* 1.0 428±35.39¶,║ 0.50±0.05¶,║ cMyBP-C-/- -Pi LL 10 177±13.96*,║ 1.0 368±15.69*,¶,║ 0.48±0.03║ *Significantly different compared to SL ¶Significantly different compared to WT ║Significantly different compared to without Pi

The rate constant of force redevelopment after mechanical disruption of force- bearing cross-bridges by release and restretch can be observed as dependent on the level of Ca2+ activation. This can be seen in subsequent figures. At long length, rate of force redevelopment in the WT is greater (fewer milliseconds) with the addition of inorganic phosphate at both short and long sarcomere length as seen in Figure 12.

29

1000

WT SL 800 WT LL WT Pi SL WT Pi LL

600

400

200

Time to redevelopment (ms) force 0 0.0 0.2 0.4 0.6 0.8 1.0 1.2

Relative force (P/Po) Figure 12. Activation dependence of the rate of force development in WT myocardium with and without inorganic phosphate

KO myocardium shows faster rates of force development (fewer milliseconds) and submaximal rates are faster at long sarcomere length. Inorganic phosphate has the biggest effects at submaximal Ca2+ concentration in the KO as can be observed in Figure

13.

30

1000

KO SL 800 KO LL KO Pi SL KO Pi LL 600

400

200

Time toTime redevelopment force (ms) 0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative force (P/Po)

Figure 13. Activation dependence of the rate of force development in KO myocardium with and without inorganic phosphate

Figure 14 shows that time to force redevelopment expressed in relative milliseconds decreases at increased sarcomere length and moreover with addition of inorganic phosphate in the WT myocardium at maximal Ca2+ concentrations. In the KO myocardium, as shown in Figure 15, time to force redevelopment in relative milliseconds is decreased more dramatically at the lowest submaximal Ca2+ concentrations with the addition of inorganic phosphate.

31

1.2

1.0 WT SL WT LL WT Pi SL WT Pi LL 0.8

0.6

0.4

0.2

Time to force redevelopment (Rms) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative force (P/Po) Figure 14. Activation dependence of the rate of force development in WT myocardium with and without inorganic phosphate expressed in relative milliseconds

1.2

1.0 KO SL KO LL KO Pi SL KO Pi LL 0.8

0.6

0.4

0.2

Time to force redevelopment (Rms) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative force (P/Po)

Figure 15. Activation dependence of the rate of force development in KO myocardium with and without inorganic phosphate expressed in relative milliseconds

32

In Figures 16 and 17, it can be observed that time to force redevelopment is Ca2+ - dependent and is consistently decreasing at longer sarcomere length for the KO myocardium. Increased inorganic phosphate (1 mM) further decreases time to force redevelopment, so that the subsequent redevelopment of tension after the slack-restretch maneuver is faster, especially at longer length. This can also be observed in relative milliseconds in Figures 18 and 19 in both WT and KO myocardium.

1000

WT SL WT LL 800 WT Pi SL WT Pi LL

600

400

200

Time to force redevelopment (ms) 0 6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 pCa

Figure 16. Rate-pCa relationships in WT myocardium with and without inorganic phosphate at short and long sarcomere length

33

1000

KO SL KO LL 800 KO Pi SL KO Pi LL

600

400

200

Time to force redevelopment (ms) 0 6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 pCa

Figure 17. Rate-pCa relationships in KO myocardium with and without inorganic phosphate at short and long sarcomere length

1.2

WT SL WT LL 1.0 WT Pi SL WT Pi LL

0.8

0.6

0.4

0.2

Time to force redevelopment(Rms) 6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 pCa

Figure 18. Relative rate-pCa relationships in WT myocardium with and without inorganic phosphate at short and long sarcomere length

34

1.2

KO SL KO LL 1.0 KO Pi SL KO Pi LL

0.8

0.6

0.4

0.2

Time to force redevelopment (Rms) 6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 pCa

Figure 19. Relative rate-pCa relationships in KO myocardium with and without inorganic phosphate at short and long sarcomere length

Discussion:

The goal of this project was to elucidate the length-dependent effects of cMyBP-C on muscle contraction and ascertain if the accelerated cross-bridge kinetics in the cMyBP-C-/- model are due to changes in a particular cross-bridge step that would be sensitive to inorganic phosphate such as the phosphate release step by utilizing increased

(1 mM) inorganic phosphate with WT and cMyBP-C-/- myocardium. It was found that

2+ resting and maximum Ca - activated force both increased at long sarcomere length for the 2 groups. Treatment with low inorganic phosphate (1 mM) did not have a major effect on Po and pCa50, but exhibited accelerated kinetics in both the WT and KO myocardium.

35

The effects of sarcomere length on force in the WT and KO myocardium

There were no differences in change in pCa50 with increasing SL, but there was a trend towards a decrease in the nH (more shallow), indicating that increasing SL favors cross-bridge activation at submaximal Ca2+ concentration likely because myosin heads are closer to actin at long SL. Increasing sarcomere length has been shown to accelerate cooperative cross-bridge recruitment and consequently rates of force development at submaximal Ca2+ activations, but also slow down the rate of cross-bridge detachment

(Stelzer et al., 2006). The increase in Ca2+ -sensitivity after an increase in sarcomere length is likely due to the fact that there is a decrease in interfilament spacing at increased length (Fuchs and Martyn, 2005). Reduced interfilament spacing would in turn increase

Ca2+ -sensitivity of myofibrillar force generation (Fuchs and Martyn et al., 2005). It was observed that kinetics (in milliseconds) were accelerated at long sarcomere length at maximum and submaximal Ca2+ concentrations in the KO, but not WT myocardium. This may be because there is an already higher proximity of myosin to actin in the knockout compared to the wild-type myocardium enhancing the effects of increased sarcomere length.

Low [Pi] does not affect force at SL and LL

Treatment of skinned fibers with low inorganic phosphate (1mM) has shown not to act like high inorganic phosphate as it did not diminish force, but has displayed increased rates of force development. Low phosphate appears to act more like NEM-S1, which is a strong binding derivative of myosin that binds strongly and irreversibly to the thin filament without producing force (Stelzer et al., 2006). In previous studies, low

36 concentrations of NEM-S1 did not have an effect on the Ca2+-sensitivity but have resulted in significant increases in the rate of force development (Stelzer et al., 2006). In this study, resting force refers to the binding of cross-bridges with no Ca2+. The resting force goes up with the addition of low levels of inorganic phosphate, which is more pronounced at longer sarcomere length and can be seen in Table 1. Inorganic phosphate is perhaps associated with more weakly bound cross-bridges.

The effects of inorganic phosphate on kinetics

Low levels of inorganic phosphate may accelerate the rate of cross-bridge recruitment by accelerating the transitions from weak to strong-binding state, an effect which appears to be more pronounced at long sarcomere length. This effect can be explained by the fact that at long sarcomere length, conditions favor cross-bridge binding, meaning that addition of 1 mM inorganic phosphate will decrease the delay in recruitment of cross-bridges and transitions to strongly bound states. In terms of the three state model of muscle activation, blocked, closed, and open (McKillop and Geeves,

1993), cross-bridges can be in unbound, weak, or strong binding states with actin, depending on the position of tropomyosin and the level of Ca2+ (McKillop and Geeves,

1993). Calcium binding to troponin C induces a conformational change and allows tropomyosin to shift from the blocked to a closed state, and strong binding of cross- bridges shifts tropomyosin to an open state allowing, further cooperative cross-bridge binding (McKillop and Geeves, 1993). When tropomyosin is partially moved out of the way (closed state), the cross-bridge is in a weak-binding state. Our data suggests that the transition from the closed to open state is faster after the addition of low levels of

37 inorganic phosphate. Such noticeable effects on kinetics with 1 mM of Pi can be explained by the fact that cardiac muscle is more sensitive to changes in cross-bridge binding in terms of kinetics (Moss et al., 2004) with only a few bound cross-bridges required to affect the rate of cross-bridge cycling.

Pi accelerates kinetics (ktr and ms) probably because it accelerates transitions of cross-bridges from weak to strong but high concentrations of Pi may do the opposite

(which is why force declines as cross-bridges can't complete the power stroke and are

"trapped” in weak binding states, Westerblad, et al, 2002). Pi accelerates ms more in WT at maximum Ca2+ at both SL and LL, because the KO rates at maximal Ca2+ are likely near saturation leaving little room for further acceleration. Because long sarcomere length and inorganic phosphate also increase the residuals of ktr, these values are less reliable than milliseconds for measuring rates. Previous analysis showed that ktr values measured at different Ca2+ concentrations were associated with pronounced relative residual tension right after restretch (Campbell and Holbrook, 2006).

Pi has little effect at maximal Ca2+ concentration in the KO myocardium. 1 mM inorganic phosphate has the greater effects on KO fibers at submaximal Ca2+ because when comparing the relative ms in Table 3, the relative ms did not change much in the

WT with Pi, but it was increased in the KO much more (i.e., rates at pCa 6.1 were almost

50% of maximal rates with Pi compared to ~30-35% of maximal without Pi), suggesting that adding Pi accelerates cross-bridge transitions to strong binding states even more in the KO at submaximal Ca2+. The greater effects of Pi at submaximal Ca2+ suggest that cooperative cross-bridge recruitment in cardiac muscle is an important mechanism for force generation when few cross-bridges are bound to actin. KO fibers are more

38 responsive to Pi because cross-bridge transitions are already favoring weak to strong transitions, and faster rates of force development (Stelzer et al., 2006).

KO at submaximal relative ms at pCa 6.1

It can be clearly observed in Table 3 that KO myocardium is affected more in relative terms by Pi than WT myocardium. The value in relative milliseconds for the KO has increased from 0.32 to 0.50 after the addition of 1mM inorganic phosphate, while the

WT myocardium did not display a similar change. Additionally, there was an increase from 0.34 rms to 0.48 rms at long sarcomere length in the KO myocardium such that rates at pCa 6.1 are approximately 50% of maximal rates. These observations further establish that low levels of inorganic phosphate display similar effects as the addition of

NEM-S1 as this binding derivative has also shown to have a greater effect in the KO

(Stelzer et al., 2006). Low inorganic phosphate accelerates cross-bridge kinetics in the

WT myocardium at pCa 4.5 more because cooperativity is not as crucial at maximum

Ca2+ since most cross-bridges are already bound.

Conclusion

It can be concluded that increased sarcomere length promotes cross-bridge binding to actin and accelerates rates of force generation. Low levels of inorganic phosphate may promote cross-bridge transitions from weak to strong binding states to accelerate rates of force development. The kinetic effects of increased sarcomere length are more pronounced in KO myocardium perhaps because actin and myosin heads are already is close proximity in the absence of cMyBP-C. Inorganic phosphate accelerates rates of force development at low and high Ca2+ activations, with effects at high Ca2+

39 more pronounced in WT myocardium and effects at low Ca2+ more pronounced in KO myocardium. These results suggest that the effects of low inorganic phosphate are mediated by cooperative cross-bridge mechanisms, further enhancing the transitions from weak to strong binding in myocardium lacking cMyBP-C where acto-myosin interactions are favored due to the increased proximity of actin and myosin molecules.

Bibliography:

1. Ackermann, M.A. and A. Kontrogianni-Konstantopoulos. 2010. Myosin-binding

protein-C slow: an intricate subfamily of proteins. Journal of Biomedicine and

Biotechnology. 2010:652065.

2. Barefield D. and S. Sadayappan. 2009. Phosphorylation and Function of Cardiac

Myosin Binding Protein-C in Health and Disease. Journal of Molecular and Cellular

Cardiology. 48:866-875.

3. Bers, D.M. 2002. Cardiac excitation-contraction coupling. Nature 415:198-205.

4. Bodor, G. S., A. E. Oakeley, P.D. Allen, D. L. Crimmins, J. H. Ladenson, and P. A. W.

Anderson. 1997. Troponin I phosphorylation in the normal and failing adult human

heart. Circulation. 96: 1495-1500.

5. Boron, W. and E. Boulpaep. 2009. Medical Physiology. 2nd Edition.

6. Campbell, K. 1997. Rate constant of muscle force redevelopment reflects cooperative

activation as well as cross-bridge kinetics. Biophysical Journal. 72: 254-262.

40

7. Campbell K.S. and A.M. Holbrook. 2006. The rate of tension recovery in cardiac muscle

correlates with the relative residual tension prevailing after restretch. Am J Physiol

Heart Circ Physiol. 292: H2020-H2022.

8. Carorla, O., S. Szilagyi, N. Vignier, G. Salazar, E. Krämer, G. Vassort, L. Carrier, and

A. Lacampagne. 2005. Length and protein kinase A modulations of myocytes in cardiac

myosin binding protein C-deficient mice. Cardiovascular Research. 69: 370-380.

9. Cazorla, O., Vassort G., Garnier D., and J.Y. Le Guennec. 1999. Length modulation of

active force in rat cardiac myocytes: is titin the sensor? J Mol Cell Cardiol. 31(6):1215-

1227.

10. Colson, B.A., T. Bekyarova, M.R. Locher, D. P. Fitzsimons, T. C. Irving, and R. L.

Moss. 2008. Protein kinase A mediated phosphorylation of cMyBP-C increases

proximity of myosin heads to actin in resting myocardium. Circulation Research. 103:

244-251.

11. De Tombe, P.P. and Bers D.M. Cardiac myofilaments: mechanics and regulation. J

Biomech. 36(5):721-730.

12. El-Armouche, A. , L. Pohlmann, S. Schlossarek, J. Starbatty, Y. Yeh, S. Nattel, D.

Dobrev, T. Eschenhagen, and L. Carrier. 2007. Decreased phosphorylation levels of

cardiac myosin-binding protein-C in human and experimental heart failure. Journal of

Molecular and Cellular Cardiology. 43: 223-229.

13. Fentzke, R. C., Buck S.H., Patel J.R., Lin H., Wolska B. M., Stojanovic M.O., Martin

A.F., Solaro R. J., Moss R.L., and J.M. Leiden. 1999. Impaired cardiomyocyte

relaxation and diastolic function in transgenic mice expressing slow skeletal troponin I

in the heart. Journal of Physiology. 517: 143-157.

14. Fitts, R. H. 2007. The cross-bridge cycle and skeletal . Journal of Applied

Physiology. 104: 551-558.

41

15. Fitzsimons, D.P., Patel J.R., Campbell K.S., and R. L. Moss. 2001. Cooperative

mechanisms in the activation dependence of the rate of force development in rabbit

skinned fibers. J Gen Physiol. 117(2):133-148.

16. Fitzsimons, D.P., Patel J.R., and R. L. Moss. 2001. Cross-bridge interaction kinetics in rat

myocardium are accelerated by strong binding of myosin to the thin filament. Journal of

Physiology. 530 (Pt 2): 263-272.

17. Flashman, E., Redwood, C., Moolman-Smook, J., and Watkins, H. 2004. Cardiac

myosin binding protein C: its role in physiology and disease. Circulation Research.

94(10): 1279-1289.

18. Fuchs, F. and D. A. Martyn. 2005. Length-dependent Ca2+ activation in cardiac muscle:

some remaining questions. Journal of Muscle Research and Cell . 26: 199-212.

19. Fukuda, N., Wu Y., Farman G., Irving T. C., Granzier H. 2003. Titin isoform variance

and length dependence of activation in skinned bovine cardiac muscle. Journal of

Physiology. 553: 147-154.

20. Granzier, H. and S. Labeit. 2007. Structure-function relations of the giant elastic protein

titin in striated and cells. Muscle Nerve. 36(6):740-755.

21. Harris, P., Bartley, C.R., Hacker, T. A., McDonald, K.S., Douglas, P.S., Greaser, M.L.,

Powers, P.A., and R.L. Moss. 2002. Hypertrophic cardiomyopathy in cardiac myosin

binding protein-C in knockout mouse. Circulation Research. 90:594.

22. Hanft, L. M., F.S. Korte, and K.S. McDonald. 2008. Cardiac function and modulation of

sarcomeric function by length. Cardiovascular Research. 77: 627-636.

23. Hanft, L.M. and K.S. McDonald. 2009. Sarcomere length dependence of power output is

increased after PKA treatment in rat cardiac myocytes. American Journal of Physiology.

296(5): H1524-H1531.

42

24. Hofmann, P.A. and F. Fuchs. 1987. Effect of length and cross-bridge attachment on

calcium binding to troponin C. American Journal of Physiology. 253: C541-546.

25. Holubarsch, C., T. Ruf, D.J. Goldstein, R.C. Ashton, W. Nickl, B. Pieske et al. 1996.

Existence of the Frank-Starling mechanism in the failing human heart: investigations on

the organ, tissue, and sarcomere levels. Circulation. 94: 683-689.

26. Jacob, R., B. Dierberger, and G. Kissling. 1992. Functional significance of the Frank-

Starling mechanism under physiological and pathophysiological conditions. European

Heart Journal. 13: 7-14.

27. Jacques, A.M., C. O’Neal, A.E. Messer, C. E. Gallon, K. King, W. J. McKenna, V.T.

Tsang, and S.B. Marson. 2008. Myosin binding protein C phosphorylation in normal,

hypertrophic and failing human heart muscle. Journal of Molecular and Cellular

Cardiology. 45: 209-216.

28. Kentish, J.C., McCloskey, D.T., Layland, J., Palmer S., Leiden J.M, Martin A.F., and

R.J. Solaro. 2001. Phosphorylation of troponin I by protein kinase A accelerates

relaxation and crossbridge cycle kinetics in mouse ventricular muscle. Circulation

Research. 88(10): 1059-1065.

29. Kobayashi, T. and R. J. Solaro. 2005. Calcium, thin filaments, and the integrative

biology of cardiac contractility. Annu Rev Physiol. 67: 39-67.

30. Korte, F.S., and K.S. McDonald. 2007. Sarcomere length dependence of rat skinned

cardiac myocyte mechanical properties: dependence on myosin heavy chain. Journal of

Physiology. 581: 725-739.

31. Konhilas, J.P., T.C. Irving, B. M. Wolska, E. E. Jweied, A. F. Martin, R. J. Solaro, and

P. D. de Tombe. 2003. Troponin I in the murine myocardium: influence on length-

dependent activation and interfilament spacing. Journal of Physiology. 547.3: 951-961.

43

32. Kraft, T., Mählmann, E., Mattei,T., and B.Brenner. 2005. Initiation of the power stroke

in muscle: insights from the phosphate analog AIF4. Proc Natl Acad Sci USA.

102(39):13861-6.

33. Layland, J., R. J., Solaro, and A.M. Shah. 2005. Regulation of cardiac contractile

function by troponin I phosphorylation. Cardiovascular research. 66: 12-21.

34. Marston, S. B. and P.P. de Tombe. 2008. Troponin phosphorylation and myofilament

2+ Ca -sensitivity in heart failure: increased or decreased? Journal of Molecular and

Cellular Cardiology. 45: 603-607.

35. McDonald, K.S., M.R. Wolff, and R.L. Moss. 1997. Sarcomere length dependence of

the rate of tension redevelopment and submaximal tension in rat and rabbit skinned

skeletal muscle fibers. Journal of Physiology. 501: 607-621.

36. McKillop, D.F. and M.A. Geeves. 1993. Regulation of the interaction between actin and

myosin sub fragment 1: evidence for three states of the thin filament. Biophysical

Journal. 65(2): 693-701.

37. Messer, A. E., A.M. Jacques, S.B. Marston. 2007. Troponin phosphorylation and

regulatory function in human heart muscle: Dephosphorylation of Ser23/24 on troponin I

could account for contractile defect in end-stage heart failure. Journal of Molecular Cell

Cardiology. 42: 247-259.

38. Metzger, J.M, Greaser M.L, and R.L.Moss. 1989. Variations in cross-bridge attachment

rate and tension with phosphorylation of myosin in mammalian skinned skeletal muscle

fibers. Implications for twitch potentiation in intact muscle. Journal of General

Physiology. 93: 855-883.

39. Miller, C. J., Cheung, P., White. P.,and E. Reisler. 1995. Actin’s view of actomyosin

interface. Biophys J. 68: 50S-54S.

44

40. Moss, R.L., Razumova, M., and D.P. Fitzsimons. 2004. Myosin crossbridge activation

of cardiac thin filaments: implications for myocardial function in health and disease.

Circulation Research. 94: 1290-1300.

41. Muhle-Goll C., Habeck M., Cazorla O., Nilges M., Labeit S., and Granzier H. 2001.

Structural and functional studies of titin’s fn3 modules reveal conserved surface patterns

and binding to myosin S1—a possible role in the Frank-Starling mechanism of the heart.

Journal of Molecular Biology. 313(2): 431-447.

42. Nagayama, T. , E. Takimoto, S. Sadayappan, J.O. Mudd, J. G. Seidman, J.Robbins, and

D.A. Kaas. 2007. Control of in vivo contraction/relaxation kinetics by myosin binding

protein C: protein kinase A phosphorylation dependent and independent regulation.

Circulation Research. 116:2399-2408.

43. Nosek, T.M., Fender, K.Y., and R. E. Godt. 1987. It is diprotonated inorganic phosphate

that depresses force in skinned skeletal muscle fibers.

44. Oakley, C.E., Chamoun, J, Brown, L. J., and Hambly, B.D. 2007. Myosin binding

protein-C: enigmatic regulator of cardiac contraction, International Journal of

Biochemistry and Cell Biology. 39: 2161–2166.

45. Pearson, J.T., M. Shirai, H. Tsuchimochi, D.O. Schwenke, T. Ishida, K. Kangawa, H.

Suga, and N. Yahi. 2007. Effects of sustained length-dependent activation on in situ

cross-bridge dynamics in rat hearts. Biophysical Journal. 93: 4319-4329.

46. Razumova, M.V., Bezold K.L., Tu A.Y., Regnier M., and S.P. Harris. 2008. Contribution

of the myosin binding protein C motif to functional effects in permeabilized rat

trabeculae. J Gen Physiol. 132(5): 575-585.

47. Sadayappan, S. , J.Gulick, H. Osinska, L.A. Martin, H. S. Hahn, G. W. Dorn II, R.

Klevitsky, C.E. Seidman, J. G. Seidman, and J.Robbins. 2005. Cardiac myosin-binding

protein-C phosphorylation and cardiac function. Circulation Research. 97: 1156-1163.

45

48. Sakthivel, S., N.L. Finley, P.R. Rosevear, J.N. Lorenz, J. Gulick, S. Kim, P. VanBuren,

L.A. Martin, J. Robbins. 2005. In vivo and in vitro analysis of cardiac troponin I

phosphorylation. Journal of Biological Chemistry. 280: 703-714.

49. Schlossarek, S., Mearini G., and L.Garrier. 2011. Cardiac myosin binding protein C in

hypertrophic cardiomyopathy: Mechanisms and therapeutic opportunities. J Mol Cell

Cardiol. 50:613-620.

50. Schwinger, R.H., M. Bohm, A. Koch, U. Schmidt, I. Morano, H.J. Eissner et al. 1994.

The failing human heart is unable to use the Frank-Starling mechanism. Circulation

Research. 4: 959-969.

51. Sleep, J.A. and R.L. Hutton. 1980. Exchange between inorganic phosphate and

adenosine 5’-triphosphate in the medium by actomyosin subfragment 1. .

19(7): 1276-1283.

52. Stehle, R. and B. Iorga. 2010. Kinetics of cardiac sarcomeric processes and rate-limiting

steps in contraction and relaxation. J Mol Cell Cardiol. 48(5):843-850.

53. Stelzer, J. E., L. Larsson, D. P. Fitzsimons, and R. L. Moss. 2006. Activation

dependence of stretch activation in mouse skinned myocardium: implications for

ventricular function. The Journal of General Physiology. 127: 95-107.

54. Stelzer, J.E., D. P. Fitzsimons, and R.L. Moss. 2006. Ablation of myosin-binding

protein-C accelerates force development in mouse myocardium. Biophysical Journal.

90:4119-4127.

55. Stelzer J.E, Patel J.R, and R.L. Moss. 2006. Protein kinase A-mediated acceleration of

the stretch activation response in murine skinned myocardium is eliminated by ablation

of cMyBP-C. Circ Res 99:884-90.

46

56. Stelzer J.E., Patel J.R, Walker J.W., and R.L. Moss. 2007. Differential roles of cardiac

myosin-binding protein C and cardiac troponin I in the myofibrillar force responses to

protein kinase A phosphorylation. Circ Res 101:503-11.

57. Shiels, H.A. and E.White. 2008. The Frank-starling mechanism in vertebrate cardiac

mechanism. Journal of Experimental Biology. 211: 2005-2013.

58. Smith, L., C. Tainter, M. Regnier, and D. A. Martyn. 2009. Cooperative cross-bridge

activation of thin filaments contributes to the Frank-Starling mechanism in cardiac

muscle. Biophysical Journal. 96: 3692-3702.

59. Tong, C. W., J.E. Stelzer, M. L. Greaser, P. A. Powers, and R. L. Moss. 2008.

Acceleration of crossbridge kinetics by protein kinase A phosphorylation of cardiac

myosin binding protein C modulates cardiac function. Circulation Research. 103: 974-

982.

60. Westerbald, H., Allen, D. G., Lännergren J. 2002. Muscle fatigue: lactic acid or

inorganic phosphate the major cause? New in Physiological Sciences. 17:17-21.

61. White, H.D. and E.W. Taylor. 1976. Energetics and mechanism of actomyosin adenosine

. Biochemistry. 15(26): 5818-5826.

47