UNIVERSITY OF CALGARY. Titin Regulation of Active and Passive Force in Skeletal Muscle. Michael Mark DuVall A THESIS

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1 UNIVERSITY OF CALGARY Titin Regulation of Active and Passive Force in Skeletal Muscle by Michael Mark DuVall A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN BIOMEDICAL ENGINEERING CALGARY, ALBERTA AUGUST, 2015 Michael Mark DuVall 2015

2 Abstract History dependent properties of muscle contraction such as force enhancement and force depression are not readily explainable by contemporary theories describing how muscles function. The lack of clear mechanistic understanding makes these phenomena difficult to reconcile within the current muscle framework of the sliding filament and crossbridge theories. Experimental evidence has been mounting however, to overturn this antiquated two filament (actin and myosin) based paradigm for one that incorporates a third filament called titin which may serve to explain history dependence. Evidence now suggests that titin can increase its stiffness in response to contractile conditions, thus being critically involved in both passive (low calcium) and active (high calcium) muscle function. The mechanisms behind this are still debated, but two potential ideas for titin based force regulation during active stretch are: (A) an increase in the inherent stiffness of the molecular chain through calcium binding to titin immunoglobulin (Ig) domains or (B) a decrease in titin s elastic length via transient attachment to actin. Our Ig domain work indicated that calcium had a biochemical effect that further manifested in a mechanical stiffening when Ig domains were stretched with an atomic force microscope in the presence of calcium. The magnitude of this calcium stiffening was about 20 % which lends support to this titin-based stiffening being important for active muscle contraction. Titin-actin interaction was investigated by fluorescently labeling sites on titin within muscle myofibrils where this interaction was speculated to occur. Upon muscle activation, no titin-actin interaction was seen, but rather unexpectedly a titin myosin interaction consistently developed. This led to the formulation of our Titin Entanglement Hypothesis based on observations of entangled I-band titin segments with myosin crossbridges during periods of spatial overlap between the two. This rendered the spring abbreviated and thus able to generate more force upon ii

3 active stretch, until a release threshold was reached. This entanglement mechanism along with calcium stiffening of Ig domains has the potential to explain the history dependent properties mentioned above, and may serve to help understand the role of titin in active muscle contraction. iii

4 I would like to thank the following people: Acknowledgements First and foremost Dr. Walter Herzog for the opportunity, the support, the mentorship and the challenges associated with pursuing this degree. The lessons learned will long outlast my time here, and for that I am deeply appreciative. Amanda Lottermoser for being a role model for real life, and the source of many laughs and impossible optimism. I do not know how you do it. Rosalie Kolstad, for being the best Program Coordinator and the unsung reason behind my graduate school adventure. Enough kind words do not exist. The lab members that make the HPL what it is. Specifically, Dr. Tim Leonard and Azim Jinha for their endless support and patience both technically and philosophically. Kaleena Johnston, Gudrun Schappacher-Tilp, Krysta Powers for the critical reading of this document and for making the lab a fun place to work. The many office mates I have had the pleasure of working alongside over the years in the Super Office. Especially Bernd Friesenbichler, Lisa Wong, Neal Austin, Brandon Hisey, Ryan Madden, Marc Bomhoff, and Nicole Schrier. Thank you all. My supervisory committee: Dr. Matthias Amrein, Dr. Douglas Syme (previously), and Dr. Venus Joumaa for providing support and ideas that helped me learn to be a better researcher. My examining committee, Dr. Henk Granzier for traveling and lecturing in addition to critically reviewing this work. Dr. Tannin Schmidt, who remains one of the most approachable people I have met, and Dr. Mike Walsh for sharing an interest and enthusiasm for this work. Funding provided by Alberta Innovates Health Solutions, The Canadian Institutes of Health Research, Natural Sciences and Engineering Research Council of Canada and the Canada Research Chairs Program for Molecular and Cellular Biomechanics, which helped make this work possible. Dr. Jane Clarke, for providing us with the I27 plasmid and Dr. Marion Greaser for sharing the titin 891 antibody. iv

5 Table of Contents Abstract... ii Acknowledgements... iv Table of Contents...v List of Tables... vii List of Figures and Illustrations... viii List of Symbols, Abbreviations and Nomenclature... xiv CHAPTER ONE: INTRODUCTION...1 CHAPTER TWO: BACKGROUND Striated Skeletal Muscle History Dependence Residual Force Enhancement after Stretch Force Depression after Shortening Passive Force Enhancement after Deactivation Titin...12 CHAPTER THREE: TITIN IMMUNOGLOBULIN DOMAINS IN THE PRESENCE OF CALCIUM Introduction Materials and Methods Protein Expression and Purification Fluorescence Spectroscopy Atomic Force Microscopy Results Fluorescence Spectroscopy Atomic Force Microscopy Discussion Conclusion Footnote...32 CHAPTER FOUR: TITIN PASSIVE AND ACTIVE BEHAVIOUR Introduction Purpose Hypothesis Methods Microscope and Setup Myofibril Preparation Solutions Antibody Labels Protocol Stretch Speed Experiments Immunofluorescent Sarcomere Tracking Algorithm Statistics Results...48 v

6 4.5.1 Stress Label Tracking Passive Labels Active Labels Individual Myofibrils Individual Sarcomeres Long Length Activation Stretch Then Activate Stretch Speed Experiments Individual Sarcomere Turning Points Discussion Stress Data Titin-Actin The Titin Entanglement Hypothesis (TEH) Antibody Movement Predictions Entanglement Correlation Stress-Entanglement Correlation Stress at Release Active Long, Active Optimal Purely Isometric Force Enhancement Stretch Magnitude and Length Dependence Speed Independence Force Depression Passive Force Enhancement Winding Filament Hypothesis Beyond Filament Overlap TEH Literature Support Conclusion CHAPTER FIVE: THESIS CONCLUSION Limitations Future Work REFERENCES CHAPTER SIX: APPENDIX Force Measurement Optical Measurement Protocol Immunofluorescence Tracking Algorithm Tracking of 9D Solutions: Chemical Abbreviations pca and Dilution Primary Antibodies Secondary Labels vi

7 List of Tables Table 4.1 Myofibril Diameter Comparisons Table 4.2 Solutions used in the preparation (rigor), relaxation (relaxing) and activation (activating) of skeletal myofibrils Table 4.3 Algorithm data revealing the total number of myofibrils (and sarcomeres) analyzed and how many were included in further analysis vii

8 List of Figures and Illustrations Figure 2.1 Hierarchical arrangement of skeletal muscle from whole muscle to the subcellular myofibril. Adapted from (Herzog, 2007). Bottom: striation pattern of skeletal muscle under the light microscope highlighting the dark refractive A-bands and light I-bands. Scale bar 2 µm Figure 2.2 Relationship between muscle length and force as dictated by the proportion of overlap between thick myosin containing filaments (black) and thin actin containing filaments (red) within the muscle sarcomere. The filament overlap schematic is presented to the right, with the letters corresponding to the force at that approximate length. Inset: physical connections called crossbridges link myosin to actin and are responsible for generating the force associated with muscle contraction through head rotation. Adapted from (Gordon et al., 1966) and (A. Huxley & Simmons, 1971) Figure 2.3 A) History dependent residual force enhancement (RFE) associated with active muscle lengthening, and force depression (FD) associated with active muscle shortening, compared to the isometric (same final muscle length) condition (thick dashed line). Shaded areas in A) from left to right represent activation, stretch (RFE) or shortening (FD) and steady state achievement. B) Passive force enhancement (PFE) after muscle deactivation. Shaded areas in B) only apply to the active stretch (blue line) and isometric condition (thick dashed line), and represent the same as in A). The last box in B) indicates muscle deactivation. Comparison of the passive stretch (orange) to the deactivated force enhanced (blue) in the last phase of B) illustrates the long lasting calcium independent increase in force. Adapted from (Herzog, 2007) Figure 2.4 Sarcomere schematic illustrating the arrangement of the actin containing thin filaments, the myosin containing thick filaments and I-band titin linking the two. Skeletal titin is composed of proximal and distal Ig domains, as well as the N2A and PEVK sequences that work together to develop passive force during muscle stretch. Adapted from (Powers et al., 2014) Figure 2.5 The winding filament hypothesis in which titin s N2A region binds to actin in the presence of calcium limiting any straightening of titin in the region proximal to N2A. With active force generation, actin is translated and rotated which causes titin to wind around actin (C). With further active stretch, titin s abbreviated elastic region contributes more force which is stored in the PEVK region (D). From (Nishikawa et al., 2011), used with permission Figure 3.1 Fluorescence intensity measurements of the tryptophan microenvironment within the I27 protein. Upon calcium addition (1 mm), there was a change in the fluorescence emission spectrum indicating an alteration in the internal tryptophan environment, which was further depressed in the presence of excess calcium (10 mm). With the subsequent removal of calcium using the chelator EDTA, the fluorescence improved toward baseline levels indicating a reversibility to the environmental perturbation. Inset: One I27 domain with the core tryptophan exaggerated within the beta barrel structure (Protein Data Bank code: 1TIT). From (DuVall et al., 2013), used with permission viii

9 Figure 3.2 Data represent mean ± SEM. Peaks 1 5 correspond to a sample of 47 and 55 for the control and calcium conditions respectively, contributing the most heavily to the overall trends in force and persistence length. A) Force produced for each respective unfolding peak in the control and calcium condition. The addition of calcium resulted in a consistent 40 pn increase in force for the first 5 peaks compared to the control group (** P value < 0.001), maintaining a significant difference for peaks 6 and 7 (* P value < 0.05). B) The PL increased with unfolding until peak four and then approximated a plateau. This was reflected equally well in both conditions being significant for peaks 2 6 (* P value < 0.05). Peak 1 results deviate most from the WLC model fit, which could account for the relative insignificance (P value = 0.061). From (DuVall et al., 2013), used with permission Figure 3.3 Raw force traces of seven unfolding events (of a possible eight) in the control (left) and calcium (right) condition. Broken line corresponds to 200 pn in force, while the curved line displays the fit of the WLC model to the ascending half of each forced unfolding event. The unfolding distance (xu) measured according to the WLC model indicates the mean unfolding distance for control (24.76 nm ± 0.13 nm) and calcium (25.25 nm ± 0.13 nm) conditions. Distance was not found to have a peak dependence, but was significant between conditions (*P value = 0.005). From (DuVall et al., 2013), used with permission Figure 4.1 A myofibril captured beween a rigid glass needle and force transducing cantilever (inset). This is contained within a fluid bath to which a laser and quadrant detector are coupled to record force Figure 4.2 A) Approximate antibody localization within rabbit psoas titin. Main segments are the proximal Ig, N2A, PEVK, and distal Ig. Red serves to indicate inextensible parts, yellow for fibronectin and green and blue for unique sequences. Adapted from (W. A. Linke et al., 1998). B) Phase contrast (Pc), Fluorescence (Fluo.) and merged images (Merged) of myofibrils labeled with titin F146 and myomesin antibodies. Markers were placed on each fluorescent band in order to monitor the movement of the M-line (M) and the antibody to antibody distance across the Z-line (Ab Ab). Additionally, Ab M was recorded for each half sarcomere. C) Fluorescent images of the three antibodies used (F146, 9D10 and 891) in conjunction with myomesin at the M-line, at resting length Figure 4.3 Myofibril video frames during passive stretch (left) and the digitized rendering (right) from an average sarcomere length between 2.0 and 2.5 µm. The digitized rendering separates the segments by colour, with red representing titin antibodies across the Z-line, and grey being epitope to M-line for each half sarcomere Figure 4.4 Average passive and active stress production ± SEM for labeled and unlabeled myofibrils (* Active results, p < 0.05 between active labeled and unlabeled; Passive results, p < between 2.1 and 3.5 µm labeled and unlabeled. p = 0.03 at 2.0 µm for the passive labeled and unlabeled comparison) ix

10 Figure 4.5 Average passive antibody tracking for titin F146, 9D10 and 891 relative to the Z- line (bottom three traces, left vertical axis) and M-line (top three traces, right vertical axis) ± SEM Figure 4.6 Average active antibody tracking for titin F146, 9D10 and 891 relative to the Z- line (bottom three traces, left vertical axis) and M-line (top three traces, right vertical axis) ± SEM Figure 4.7 Binned myofibril behaviour of F146 labeled titin undergoing active stretch relative to the Z-line ± SEM. Inset: the point immediately before and after activation for 14 myofibrils. The activation does not markedly alter the F146 distance from the Z-line. Inset axes are the same as the outset Figure 4.8 Binned myofibril behaviour of F146 labeled titin undergoing active stretch relative to the M-line ± SEM. Dashed line represents the A-band edge where the myosin thick filament terminates. Inset: the point immediately before and after activation for 14 myofibrils. The activation resulted in the F146 antibody moving towards the M-line. Inset axes are the same as the outset Figure 4.9 Binned myofibril behaviour of 9D10 labeled titin undergoing active stretch relative to the Z-line ± SEM. Inset: the point immediately before and after activation for nine myofibrils. Inset axes are the same as the outset Figure 4.10 Binned myofibril behaviour of 9D10 labeled titin undergoing active stretch relative to the M-line ± SEM. Dashed line represents the A-band edge where the myosin thick filament terminates. Inset: the point immediately before and after activation for nine myofibrils. Inset axes are the same as the outset Figure 4.11 Binned myofibril behaviour of 891 labeled titin undergoing active stretch relative to the Z-line ± SEM. Inset: the point immediately before and after activation for four myofibrils. Inset axes are the same as the outset Figure 4.12 Binned myofibril behaviour of 891 labeled titin undergoing active stretch relative to the M-line ± SEM. Dashed line represents the A-band edge where the myosin thick filament terminates. Inset: the point immediately before and after activation for four myofibrils. Inset axes are the same as the outset Figure 4.13 Location of turning point plateaus ± SEM for binned F146 labeled sarcomeres relative to the Z-line undergoing active stretch. Inset: sarcomere turning points undergoing passive stretch. Inset axes are the same as the outset Figure 4.14 Location of turning point plateaus ± SEM for binned F146 labeled sarcomeres relative to the M-line undergoing active stretch. Inset: sarcomere turning points undergoing passive stretch. Inset axes are the same as the outset. Dashed line represents the A-band edge where the myosin thick filament terminates x

11 Figure 4.15 Location of turning point plateaus ± SEM for binned 9D10 labeled sarcomeres relative to the Z-line undergoing active stretch. Inset: sarcomere turning points undergoing passive stretch. Inset axes are the same as the outset Figure 4.16 Location of turning point plateaus ± SEM for binned 9D10 labeled sarcomeres relative to the M-line undergoing active stretch. Inset: sarcomere turning points undergoing passive stretch. Inset axes are the same as the outset. Dashed line represents the A-band edge where the myosin thick filament terminates Figure 4.17 Location of turning point plateaus ± SEM for binned 891 labeled sarcomeres relative to the Z-line undergoing active stretch. Inset: sarcomere turning points undergoing passive stretch. Inset axes are the same as the outset Figure 4.18 Location of turning point plateaus ± SEM for binned 891 labeled sarcomeres relative to the M-line undergoing active stretch. Inset: sarcomere turning points undergoing passive stretch. Inset axes are the same as the outset. Dashed line represents the A-band edge where the myosin thick filament terminates Figure 4.19 Long length activation (blue circles) comparison to purely passive stretch (green triangles). Note that when myofibrils were stretched from resting length and activated shortly thereafter (red diamonds), no real change was seen from the case where a myofibril was activated at a similarly long length. Dashed line represents the A-band edge where the myosin thick filament terminates. Long length activation resulted in much longer contour length attainment than in the passive case relative to the Z-line (extra 600 nm) which may be associated with Ig domain unfolding at long length (inset).. 67 Figure 4.20 Stretch then activate experiments of four F146 labeled myofibrils activated dynamically at near optimal lengths. Inset: for each of the four myofibrils, the point immediately before and after activation was plotted. The activation brings the antibody towards the M-line, but not typically into the A-band area. The inset axes remain the same as the outset Figure 4.21 Titin F146 labeled myofibril tracking at three different speeds relative to the Z- line (bottom three traces, left vertical axis) and M-line (top three traces, right vertical axis) ± SEM Figure 4.22 Average stress produced by F146 labeled myofibrils at three different speeds ± SEM (* p < 0.05; ; p < 0.001) Figure 4.23 Titin F146 labeled myofibril tracking at three different speeds relative to the Z- line (bottom three traces, left vertical axis) and M-line (top three traces, right vertical axis) ± SEM. These began as a passive stretch and were activated shortly thereafter Figure 4.24 Average stress produced by F146 labeled myofibrils at three different speeds ± SEM. Only the active results are plotted for the stretch then activate experiments (* p < 0.05) xi

12 Figure 4.25 A) Location of turning point plateaus ± SEM for binned F146 labeled sarcomeres relative to the Z-line for the activate then stretch dataset after pooling the velocity data. B) Turning points relative to the M-line. Dashed line represents the A-band edge where the myosin thick filament terminates Figure 4.26 A) Location of turning point plateaus ± SEM for binned F146 labeled sarcomeres relative to the Z-line from the stretch then activate dataset after pooling the velocity data. B) Turning points relative to the M-line. Dashed line represents the A-band edge where the myosin thick filament terminates Figure 4.27 Proposed Titin Entanglement Hypothesis (TEH). The PEVK to Z-line distance would increase greatly if some site on titin were tangled with myosin crossbridges (Bottom left half sarcomere), while the PEVK would be less than 0.8 µm from the M- line. This would not be the case if titin were bound to actin around the N2A element (green box) (Bottom right half sarcomere). Figure adapted from (Powers et al., 2014) Figure 4.28 F146 label tracking for six sarcomeres from one myofibril undergoing active stretch. A) Little movement is seen between the antibody and the M-line with time until about 18 seconds. This anchoring occurs right at the A-band edge for each of these sarcomeres. B) Dramatic elongation occurred asynchronously between the Z-line and F146 with time, before a plateauing event. C) Sarcomere length trace for each of the serial sarcomeres Figure 4.29 Relationship between the sarcomere length at which active stretch began and the sarcomere length at plateau for all active labeled myofibrils (n = 36). A significant positive correlation was seen, irrespective of which label was considered Figure 4.30 Stress produced ± SEM of labeled myofibrils binned by the activation onset sarcomere length Figure 4.31 Stress observed for the three titin labeled myofibril groups at titin-myosin separation (plateau). The median is presented within each box, with the boundaries corresponding to the first and third quartile while the whiskers represent the range Figure 4.32 Active stress dependence on sarcomere length at active stretch ± SEM. With the same magnitude stretch, the initial sarcomere length at stretch onset served to influence the stress potential Figure 4.33 Sarcomere treatment groups as originally presented by Yasuda et al., (1995), used with permission. a) Intact sarcomere at resting length. b) Rigor-gelsolin group where I-band actin is removed and then stretched c) in rigor. d) Relaxation of group c) translated into freeing of A-band actin into the I-band in most cases (see right half of panel d). e) Group c, receiving further gelsolin treatment under contraction conditions Figure 6.1 Force-time traces for a passive (blue) and active (red) myofibril stretch plotted in line with motor movement (black, right vertical axis). Dotted vertical line represents activation timing for the active stretch trace while the dashed line represents stretch onset (motor movement) for both stretch traces xii

13 Figure 6.2 9D10 tracking for six actively stretched sarcomeres to which an algorithm was applied to mark the plateauing behaviour in each sarcomere. The length relative to the M-line or Z-line was plotted on the same vertical axis for simplicity. When the location of the plateau differed by more than 300 nm, it was rejected (red box) Figure 6.3 Stress comparison of passive unlabeled myofibrils and Qdot 525 labeled myofibrils ± SEM Figure 6.4 Label tracking comparison of passive labeled Alexa 488 and Qdot 525 myofibrils ± SEM. Although the Qdot label tracking was obtained from four myofibrils, each point was typically representative of only one value from one myofibril Figure 6.5 Repeated A) passive stretch and B) active stretch from resting length showing labels move consistently with stretch relative to the Z-line (left vertical axis) and M-line (right vertical axis) xiii

14 List of Symbols, Abbreviations and Nomenclature Symbol Definition WLC Worm Like Chain Model RFE Residual Force Enhancement PFE Passive Force Enhancement FD Force Depression SL Sarcomere Length TEH Titin Entanglement Hypothesis PL Persistence Length AFM Atomic Force Microscope Ig Immunoglobulin I27 Titin Immunoglobulin Domain 27 xiv

15 Chapter One: Introduction Muscles continue to be a source of curiosity for our inquisition, as even today discrepancies persist between theoretical expectations and experimental results. This has largely been due to the dynamic and adaptive nature inherent within muscles, making it difficult to uniformly characterize the mechanisms of muscular change. Attempts often start with investigations into the smallest subunits of muscle to resolve at what level this experimental divergence occurs and to further characterize the underlying mechanisms. As technological advancements occur, the potential to explore these intricacies is expanded, often yielding new insights in the process. To this end, light, and later electron microscopes presented observations of striated muscle in the nineteenth century that challenged previous notions about a single folding contractile substance (A. Huxley, 1980). As no satisfactory theory incorporating these observations was presented, they were deemed inconsequential and appeared lost from memory (A. Huxley, 1980). This serves to illustrate one of the cautions in burgeoning fields, when experimental observations contradict some well-entrenched paradigms of the time. Ultimately, a rediscovery was made in the mid-twentieth century that carried the field towards an understanding of that same striation pattern of muscle observed a century earlier. Two independent Huxleys (Andrew and Hugh) suggested the refractive A-band corresponded to the myosin containing thick filament, while the light I-band pattern reflected the actin containing thin filament. Importantly, the changes in the striation pattern depended on the length of the fiber but the A-band width was constant under nearly all conditions (A. Huxley & Niedergerke, 1954; H. Huxley & Hanson, 1954). This filament sliding mechanism of actin past myosin was then linked to the presence of independent force generators distributed evenly in the area of overlap between the two (A. Huxley, 1957). These force generators, called crossbridges, convert a high energy phosphate bond of ATP into 1

16 asynchronous mechanical translation of actin towards the center of the muscle sarcomere, resulting in muscle shortening through filament sliding. The mathematics behind the observations soon followed (A. Huxley & Simmons, 1971), entrenching the sliding filament and crossbridge theories with our understanding of muscle contraction. Testable predictions derived from these theories subsequently led to a framework associating observations of muscle length with isometric steady-state force (Gordon, Huxley, & Julian, 1966). Despite the predictive power of this force-length relationship for isometric conditions with maximal activation, the dynamic nature of muscle does not readily fit with current theories when muscles change length or experience rapid calcium flux. History dependence in the context of muscle mechanics is an umbrella term that describes the importance of contractile conditions before, during and/or after contraction. Broadly, these can be separated into at least three conditions: 1. Force enhancement after stretch 2. Force depression after shortening 3. Passive force enhancement after deactivation These conditions are complex and differ from the isometric in their own unique ways. Proposed mechanisms have tried to reconcile them collectively, but it was quickly discovered that they are not simply the opposite of one another (force depression is not the opposite of force enhancement), nor are they entirely associated with crossbridge force generators (Herzog & Leonard, 2000). One tempting speculation that has emerged is the existence of an elastic element that engages upon muscle activation, stiffening the muscle sarcomere independently of crossbridges (Leonard & Herzog, 2010). At the level of the sarcomere this spring is called titin, the last of the filaments to be characterized in muscle. Conceptually, a spring can change its stiffness in different ways. During this thesis work, we investigated two such mechanisms: a 2

17 calcium stiffening effect in which ion binding may stabilize the titin chain and contribute to a decrease in bending rigidity (persistence length). Secondly, an abbreviation to the spring contour length through transient interaction with neighboring filaments during active sarcomeric stretch. Chapter two begins with a review of relevant observations and theories required to understand the framework these experiments were conducted under. Chapter three is a published manuscript detailing the calcium interaction with titin immunoglobulin domains. Chapter four evaluates any titin truncation that may occur during muscle activation. Finally, chapter five summarizes the work done in this thesis, discusses some underlying limitations and presents directions of future inquiry. 3

18 Chapter Two: Background As the knowledge of muscle biomechanics improves, so does the applicability of this understanding to muscle related injuries and diseases. This background highlights the development of ideas that have proven able to withstand experimental and theoretical scrutiny, leading to our contemporary understanding of muscle contraction. 2.1 Striated Skeletal Muscle Skeletal muscles are composed of fascicle bundles which themselves are made of even smaller muscle fibers or cells. These muscle fibers terminate into structural elements called myofibrils, which contain the contractile units of skeletal muscle (Figure 2.1). In the mid-twentieth century, striated muscle myofibril observations of dark A-bands alternating with light I-bands led to the revelation that these striations could be explained by the overlap of thin actin containing filaments and thick myosin containing filaments. In back to back papers, two independent Huxleys proposed that the change in the striation pattern was associated with a sliding of the filaments past one another (A. Huxley & Niedergerke, 1954; H. Huxley & Hanson, 1954). Quite controversially, this meant a constancy of the A-band width (myosin filament), which contradicted the folding of proteins associated with A-band shrinkage as an explanation for muscle contraction. 4

19 Figure 2.1 Hierarchical arrangement of skeletal muscle from whole muscle to the subcellular myofibril. Adapted from (Herzog, 2007). Bottom: striation pattern of skeletal muscle under the light microscope highlighting the dark refractive A-bands and light I-bands. Scale bar 2 µm. This sliding of filaments was later detailed through the attachment of thermally driven physical connections called crossbridges extending from myosin to actin. This crossbridge theory was dependent on rate constants of attachment and detachment based on distance from an equilibrium position (A. Huxley, 1957). Subsequent addendums to the crossbridge theory added degrees of freedom to the model through series elasticity and transition states for the crossbridge head based on potential energy (A. Huxley & Simmons, 1971). With the utilization of a high energy phosphate to rotate the myosin ATPase, the molecular mechanisms of active muscle contraction 5

20 had become synonymous with the sliding filament (A. Huxley & Niedergerke, 1954; H. Huxley & Hanson, 1954) and crossbridge theories (A. Huxley, 1957; A. Huxley & Simmons, 1971; H. Huxley, 1969). A subtlety proposed in the original formulation of the sliding filament theory was that overlap dictates force production in a predictable fashion such that tension should decline linearly as the fiber is stretched from optimal length to loss of filament overlap (A. Huxley & Niedergerke, 1954). This relationship was successfully demonstrated when Gordon, Huxley and Julian (1966) showed that filament overlap permits crossbridge formation which is the sole determinant of isometric steady-state active force reflected in what is now known as the force-length relationship (Figure 2.2). The crossbridge head has the capacity to interact with actin at different potential energy states (inset, Figure 2.2), translating into the power stroke and ultimately contractile force (A. Huxley & Simmons, 1971). Maximal force is achieved when the overlap between actin and myosin is such that the greatest opportunities for crossbridge attachment exist (point C, Figure 2.2). Moving down the ascending limb (point B), the proportion of overlap decreases as interference is presented from adjacent half thin-actin filaments removing possibilities for crossbridge attachment. With even more shortening (point A), the myosin thick filament may be compressed as its ends make contact with both Z-lines (vertical black bars marking the end of the sarcomere) inhibiting crossbridge functionality, in addition to the actin overlap interference described for point B. 6

21 Figure 2.2 Relationship between muscle length and force as dictated by the proportion of overlap between thick myosin containing filaments (black) and thin actin containing filaments (red) within the muscle sarcomere. The filament overlap schematic is presented to the right, with the letters corresponding to the force at that approximate length. Inset: physical connections called crossbridges link myosin to actin and are responsible for generating the force associated with muscle contraction through head rotation. Adapted from (Gordon et al., 1966) and (A. Huxley & Simmons, 1971). On the descending limb (point D), the proportion of overlap and thus force, decreases in an expected fashion as fewer force generating sites are available. This force-length relationship has great predictive power for maximally activated isometric contractions in which the muscle length does not change, and has the capacity to be explained by the sliding filament theory. However, muscle function is often associated with a dynamic motion in which muscles shorten or lengthen to orchestrate desired movements. These concentric (shortening) or eccentric (lengthening) contractions lack the same predictability as the isometric steady-state from which classic theories were derived, in part because they are affected by the history of contraction. 7

22 2.2 History Dependence The history of a contraction can greatly influence the force outcome in a manner that has yet to be fully realized or understood, but has been seen for as long as the other theories have been in existence (Abbott & Aubert, 1952). These history dependent properties for the purposes of the studies done here, include: 1. Residual force enhancement after stretch 2. Force depression after shortening 3. Passive force enhancement after deactivation These will be explored individually now Residual Force Enhancement after Stretch When a muscle is activated at a shorter length, and then stretched actively to a final length, it can produce more force than a muscle activated isometrically at that final length (Figure 2.3) at steady state. This mechanism called residual force enhancement (RFE) has been documented in whole muscles (Herzog & Leonard, 2000), all the way down to individual sarcomeres (Leonard, DuVall, & Herzog, 2010), for more than half a century (Abbott & Aubert, 1952) which contradicts the predictive capacity of current theories which are based on the notion that muscle force magnitude is theoretically confined at a particular length. In an attempt to reconcile these observations under the crossbridge theory, the sarcomere length non-uniformity theory has been proposed based on the premise that there are populations of sarcomeres that are stronger or weaker than others (Morgan, 1994). This originated as an extension of the creep that has been seen upon activation thought to be due to the instability of the descending limb (point D, Figure 2.2) (Gordon et al., 1966). According to Morgan (1994), active muscle stretch causes the 8

23 stronger sarcomeres to shorten (or creep up the descending limb) while the weaker ones are forced to lengthen and are eventually caught by the elastic properties of muscle when they pop. To evaluate this notion, Leonard, DuVall & Herzog (2010) employed a technically challenging experimental design precluding the possibility of two distinct populations of sarcomeres by showing force enhancement is possible in a single mechanically isolated sarcomere. Although this and other observations to the contrary have been published (RFE on the ascending limb for example (Herzog & Leonard, 2002)), the sarcomere length non-uniformity theory persists as a candidate for explaining RFE, in part because it offers little opposition to current theories (Herzog, 2004). Qualities of RFE include a stretch magnitude (Abbott & Aubert, 1952; Herzog & Leonard, 2002) and muscle length dependence (Edman, Elzinga, & Noble, 1982; Joumaa, Leonard, & Herzog, 2008), but an overall independence of stretch speed (Edman, Elzinga, & Noble, 1978; Sugi & Tsuchiya, 1988) Force Depression after Shortening History dependence has also been seen during experiments where a muscle was activated at a longer length than the isometric condition, and allowed to quickly shorten to the same corresponding muscle length (Herzog & Leonard, 1997) (Figure 2.3, A). A lower force resulted when juxtaposed with the steady state isometric at the same final muscle length, suggesting that optimal contractile conditions were not met. Mechanisms proposed for force depression (FD) have once again been associated with the development of sarcomere length non-uniformities or a change associated with actin that renders some crossbridges unable to contribute force when compared to those in the isometric steady-state (Maréchal & Plaghki, 1979). This latter mechanism has received support when stiffness measurements (reflecting the proportion of 9

24 attached crossbridges) revealed a decrease correlating with the amount of force depression (Lee & Herzog, 2003; Sugi & Tsuchiya, 1988). This is thought to arise due to strain development in the actin thin filament specifically in the newly formed region of overlap created upon active shortening (Sugi & Tsuchiya, 1988) however, the mechanism behind FD is still debated. Qualities of FD include a dependence on stretch amplitude, muscle length before and after shortening, stretch speed and muscle activation (Herzog & Leonard, 1997; Maréchal & Plaghki, 1979). A) B) Figure 2.3 A) History dependent residual force enhancement (RFE) associated with active muscle lengthening, and force depression (FD) associated with active muscle shortening, compared to the isometric (same final muscle length) condition (thick dashed line). Shaded areas in A) from left to right represent activation, stretch (RFE) or shortening (FD) and steady state achievement. B) Passive force enhancement (PFE) after muscle deactivation. Shaded areas in B) only apply to the active stretch (blue line) and isometric condition (thick dashed line), and represent the same as in A). The last box in B) indicates muscle deactivation. Comparison of the passive stretch (orange) to the deactivated force enhanced (blue) in the last phase of B) illustrates the long lasting calcium independent increase in force. Adapted from (Herzog, 2007). 10

25 2.2.3 Passive Force Enhancement after Deactivation While investigating active sarcomere stretching associated with force enhancement, it was discovered that residual force enhancement was caused by at least two different components (Herzog & Leonard, 2002). The active component resulted in a consistent increase in force associated with active stretch, best exemplified on the ascending limb of the force length relationship where no passive force can develop (Herzog & Leonard, 2002). The passive component was observed at longer lengths when a passive element was engaged. Since passive force in the isolated myofibrillar preparation is virtually exclusively caused by the structural protein titin (Horowits, Maruyama, & Podolsky, 1989), Herzog & Leonard (2002) suggested that titin might help to explain the experimental force enhancement with stretch that current muscle theories could not. In evaluating this idea, the same active stretch experiments were performed with a deactivation at the end (Joumaa, Rassier, Leonard, & Herzog, 2007), which revealed passive force enhancement was % larger than the purely passive stretch where no activation was done (B, Figure 2.3). Narrowing down the source of this increased force, stretching titin containing myofibrils in the presence of calcium but absence of crossbridges was seen to increase the stiffness of intact myofibrils (Joumaa, Rassier, Leonard, & Herzog, 2008); however, this was too small to explain all of the passive force enhancement seen. Although titin was critical, active force generation through crossbridge interaction prior to deactivation was also required for this two component history dependence to manifest. 11

26 2.3 Titin The existence of an elastic mechanism capable of producing its own passive force upon stretch, independent of crossbridges, predated its actual discovery. When myosin removal still allowed stretched psoas myofibrils to return back to resting length (H. Huxley & Hanson, 1954) ideas arose for ways to explain this important reversibility intrinsic to muscle. Different terms had arisen to reconcile observations that required such an element to exist, including S-filaments (H. Huxley & Hanson, 1954), connectin (Maruyama, Murakami, & Ohashi, 1977), gap filaments (Horowits, Kempner, Bisher, & Podolsky, 1986), elastic filaments (Funatsu, Higuchi, & Ishiwata, 1990) and more recently titin. Titin was a relative late comer to the understanding of muscle mechanics, being discovered in many species about 25 years after the sliding filament theory was published (Maruyama et al., 1977). Electron microscopy work initially failed to detect titin within the sarcomere lattice owing in part to its similarity in size with the parallel thin actin filament. Upon actin removal however, the visualization of a connecting filament occurred, which propelled explorations into titin (Funatsu et al., 1990). Titin continues to be the largest protein discovered, with a molecular size of MDa depending on the muscle isoforms expressed (Freiburg et al., 2000). Although this protein is not limited to muscle, its importance is particularly seen within the muscle context. 12

27 Figure 2.4 Sarcomere schematic illustrating the arrangement of the actin containing thin filaments, the myosin containing thick filaments and I-band titin linking the two. Skeletal titin is composed of proximal and distal Ig domains, as well as the N2A and PEVK sequences that work together to develop passive force during muscle stretch. Adapted from (Powers et al., 2014). Titin spans between the Z-line and the M-line of sarcomeres, connecting the myosin thick filaments with the Z-line and supplying a structural framework for active muscle contraction (H. L. Granzier & Labeit, 2006). A-band titin is virtually inextensible at physiological sarcomere lengths, due to myosin anchorage supplied by myosin binding protein-c (Kampourakis, Yan, Gautel, Sun, & Irving, 2014). The extensible behaviour of titin is derived from the I-band region (Figure 2.4), which serves to restore muscles to their resting length, prevent damage that may arise from over-stretch and maintain sarcomere homogeneity and stability by centering myosin (D. Labeit et al., 2003). The extensible region of titin is subdivided into segments which behave cooperatively to define titin s elastic properties. For skeletal muscle, these include the proximal and distal immunoglobulin (Ig) regions, the PEVK region, and the N2A sequence (Figure 2.4). The proximal and distal Ig segments are different enough mechanically and spatially to warrant separate classification (H. Li et al., 2002). Differential splicing of the titin gene results in muscle specific isoforms of varying lengths (W. Linke & Fernandez, 2002), which translates into unique elastic behaviours in different muscles (Freiburg et al., 2000; W. A. Linke et al., 2002). The number of tandem Ig domains found in titin for example ranges from 37 in cardiac to 90 in 13

28 skeletal muscle (Lu, Isralewitz, Krammer, Vogel, & Schulten, 1998), which allows muscles to behave very differently upon stretch. As skeletal sarcomeres are stretched from resting length towards the descending limb (between µm for psoas muscle) the tandemly arranged folded immunoglobulin domains are forced to straighten contributing little to passive force (W. A. Linke, Ivemeyer, Mundel, Stockmeier, & Kolmerer, 1998). Further stretch recruits the PEVK element of titin, rich in proline (P), glutamate (E), valine (V) and lysine (K), generating passive force in the process (Tatsumi, Maeda, Hattori, & Takahashi, 2001). The PEVK region has a very charged structure due to the amino acid side chains that predominate, which is thought to prevent any stable tertiary structure from arising (Gao, Lu, & Schulten, 2001; Lu et al., 1998). In addition to isoform variation, titin s force production is further complexed through dynamic adjustments to short term events such as calcium flux (D. Labeit et al., 2003) or phosphorylation (Anderson, Bogomolovas, Labeit, & Granzier, 2010; H. L. Granzier & Labeit, 2006; Yamasaki et al., 2002) and long term diseased states or atrophy (Fujita, Labeit, Gerull, Labeit, & Granzier, 2004; Legerlotz, Matthews, McMahon, & Smith, 2009; Udaka et al., 2008). Proposed mechanisms behind these adjustments have at times resulted in contradictory findings (see Chapter 4, discussion) making it challenging to understand titin s dynamic behaviour and to further relate it to history dependent properties. Explorations into history dependence suggest rapid stiffening of muscle occurs, which from a mechanistic perspective yields at least two interesting hypotheses in which a change in titin stiffness could be used to explain RFE: (A) an increase in the inherent stiffness of the molecular chain through calcium binding to titin Ig domains, or (B) a decrease in titin s elastic length via attachment to actin. 14

29 Calcium flux into and out of the sarcomere, as required for active muscle contraction, could serve as a rapid way to alter spring stiffness. It has been seen that titin is capable of stiffening its elastic response in a calcium dependent manner, but this has largely been relegated to the PEVK region (D. Labeit et al., 2003; Watanabe, Muhle-Goll, Kellermayer, Labeit, & Granzier, 2002). As I-band titin is composed primarily of Ig domains, we examined these in the presence and absence of calcium in order to evaluate any calcium-ig dependent force regulation ability (Hypothesis A). This was first done biochemically to see if an interaction could be detected, and then mechanically to determine the relevance and magnitude of any calcium stiffening of titin Ig domains. Additionally, experimental evidence has suggested that titin may undergo an electrostatic interaction with actin during muscle activation that could abbreviate the length of the extensible region (W. A. Linke et al., 1997; W. A. Linke et al., 2002; Yamasaki et al., 2001). The location, exact mechanism and magnitude are unknown, making it difficult to ascribe history dependent properties to titin-actin behaviour. Recently, the winding filament hypothesis has served to partly address this void by providing a detailed description of titin s capacity to interact with actin. Notably, calcium influx may result in titin s N2A region binding to actin and subsequent myosin crossbridge rotation not only translates actin towards the center of the sarcomere, but also rotates it resulting in titin winding on the actin thin filament (Figure 2.5) (Nishikawa et al., 2011). Currently, no direct evidence exists to support the winding of titin on actin (Nishikawa et al., 2011), which would require a visualization of titin s behaviour in the sarcomere relative to the intact arrangement of parallel filaments. 15

30 Figure 2.5 The winding filament hypothesis in which titin s N2A region binds to actin in the presence of calcium limiting any straightening of titin in the region proximal to N2A. With active force generation, actin is translated and rotated which causes titin to wind around actin (C). With further active stretch, titin s abbreviated elastic region contributes more force which is stored in the PEVK region (D). From (Nishikawa et al., 2011), used with permission. Our group has now developed an immunofluorescence setup amenable to tracking the location of titin specific antibodies in order to evaluate titin stretch behaviour under different contractile conditions, and determine whether actin interaction is a prominent feature (Hypothesis B). The evaluation of hypothesis A (Ig domain calcium interaction) and hypothesis B (titin-actin interaction) will be explored in Chapters 3 and 4, respectively. The work outlined in this thesis was almost exclusively conducted by the author. This included the recombinant production, purification, dialysis and lyophilization of titin I27 proteins. 16

31 Subsequently, the fluorescence spectroscopy of I27 was done with the assistance of Dr. Jessica Gifford, and the atomic force microscopy of I27 was possible with training received from Dr. Matthias Amrein. The myofibril setup was optimized and fine-tuned by the author and all experiments involving the myofibrils were done by the author. Analysis of all data in this thesis was done by the author and statistical advice was provided by Dr. Tak Fung. Sarcomere selection was done using an algorithm developed by Dr. Gudrun Schappacher-Tilp. Conceptual design, interpretation and technical information was provided by Dr. Walter Herzog, Dr. Tim Leonard and Azim Jinha. 17

32 Chapter Three: Titin Immunoglobulin Domains in the Presence of Calcium The following is based on the published manuscript: Altered Mechanical Properties of Titin Immunoglobulin Domain 27 in the Presence of Calcium (DuVall, Gifford, Amrein, & Herzog, 2013). 3.1 Introduction Titin, initially called connectin (Maruyama et al., 1977), is a sarcomeric protein responsible for passive force (elasticity) in muscle sarcomeres (W. Linke & Fernandez, 2002; W. A. Linke et al., 1999). Passive force develops due to titin s resistance to stretch when muscles elongate, in contrast to active force which is produced from actin and myosin based crossbridges (A. Huxley, 1957; A. Huxley & Simmons, 1971; H. Huxley, 1969) causing muscles to shorten. Titin has been implicated in numerous mechanical roles such as sensing stretch (H. L. Granzier & Labeit, 2006) and opposing sarcomeric extension (Wang, McCarter, Wright, Beverly, & Ramirez-Mitchell, 1991), both ultimately contributing to the prevention of stretch induced muscle damage. Titin s dynamic ability to produce passive force is illustrated with elements adjusting to short term changes in calcium flux (D. Labeit et al., 2003) and phosphorylation (Anderson et al., 2010; H. L. Granzier & Labeit, 2006; Yamasaki et al., 2002), as well as long term diseased states (H. Granzier, Labeit, Wu, & Labeit, 2002; H. L. Granzier & Labeit, 2006) and differential isoform splicing conditions (H. L. Granzier & Labeit, 2006). By altering its mechanical properties, titin can adapt its passive force capability permitting tuning in accordance with muscle demands. Although the complex mechanisms of titin s passive response have been widely studied, the entirety of titin s capacity to regulate force remains to be characterized. From a physiological 18

33 perspective, an important mechanism has been calcium binding to select domains of titin, resulting in a rapid yet transient change in mechanical properties. This feature has been observed at the myofibril (Joumaa et al., 2008), muscle fiber (D. Labeit et al., 2003) and myocardial levels (Fujita et al., 2004), reflecting the macroscopic manifestations that occur with calcium interaction on the subcellular level. Experiments performed in vitro to evaluate calcium based force regulation have shown that the PEVK region, abundant in the amino acids proline (P), glutamate (E), valine (V) and lysine (K), has a calcium responsiveness localized to the glutamate rich portion that renders titin stiffer (Fujita et al., 2004; D. Labeit et al., 2003). Other regions such as the numerous immunoglobulin (Ig) domains have also been speculated to possess a calcium binding potential (Trombitás et al., 1998), but this has largely been inconclusive. Watanabe et al., (2002) investigated interaction of distal region titin Ig domains over a physiological range of calcium concentrations (pca ) concluding that calcium had no observable effect on the unfolding force. This contrasts work done by others (Coulis et al., 2008; Lu et al., 1998), revealing an unclear understanding of titin calcium interaction. With the prevalence of Ig domains in titin and their biochemical potential for ligand binding, we used a homopolymeric approach to reevaluate the Ig domains as a calcium responsive element that could further elucidate the regulation of titin based passive force. In this study we observed calcium regulated changes in the biochemical and mechanical properties of the individual recombinant Ig domain 27 (I27) from the distal I band region of human cardiac muscle titin. By breaking titin down into its fundamental elements, we can explore how they behave independently under different conditions, as the molecular basis of titin s extensibility can be understood as the sum of its components (Scott, Steward, Fowler, & 19

34 Clarke, 2002). We speculate that calcium binding to titin Ig domains may function as a rapid protective mechanism to further stiffen titin and oppose stretch related muscle damage. 3.2 Materials and Methods Protein Expression and Purification The plasmid (T1 I27) encoding eight human cardiac titin Ig domain 27 repeats was generously donated by Dr. Clarke (Cambridge University, Cambridge, UK) (Steward, Toca-Herrera, & Clarke, 2002). The T1 I27 plasmid, was inserted into the genome of chemically competent Escherichia coli of the C41 (DE3) strain (Lucigen Corp. Middleton, WI, USA). One isolated colony was selected and grown up to an OD600 of 0.6 at 37 C and 250 RPM in 2X YT medium (Sigma-Aldrich, Oakville, ON, Canada). Induction was performed using 0.9 mm IPTG (Sigma- Aldrich, Oakville, ON, Canada) for eight hours at 28 C and 250 RPM. The amino acid sequence (Improta, Politou, & Pastore, 1996) included two C terminal cysteines used for attachment to a gold coated surface and 6 N terminal histidines for purification purposes (Best, Fowler et al., 2003; Lu et al., 1998). The bacteria were lysed using buffer (50 mm Tris HCl, 1 mm EGTA, 1 µg/ml Leupeptin) in conjunction with a mechanical French Press pre cooled to 4 C. Proteins were purified using a standard histidine nickel charged column chromatography kit (Novagen, Madison, WI, USA), leaving the His6 tag intact. Fractions were collected and evaluated using a 12% SDS PAGE separating gel and 4.5% stacking gel (Laemmli, 1970). Gels were visualized with silver stain. Pure fractions were dialyzed against 50 mm Tris HCl, 150 mm NaCl, lyophilized (Thermo ModulyoD 115, Milford, MA, USA) and were stored at - 80 C. Isoelectric points were calculated with the MW/PI calculator at (Gasteiger et al., 2005). 20

35 3.2.2 Fluorescence Spectroscopy Fluorescence spectroscopy was conducted utilizing internal tryptophan excitation with a Varian Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies Canada Inc. Mississauga, ON, Canada). Using a wavelength of 295 nm, we selectively excited the π electrons of the tryptophan ring by surpassing the excitation wavelength of other aromatic ring containing side chains (Albani, 2007). Eleven combinations of calcium (1mM and 10mM CaCl2), protein (I27), water, buffer (50 mm Tris, 100 mm KCl, 5 mm DTT ph 7.5) and EDTA were tested for calibration and experimentation purposes. At least ten traces were recorded for each sample and buffer condition tested (11 total). Following manual mixing in a 1 cm pathlength cuvette, dilution effects were accounted for and an average was plotted. The buffer baseline (without I27) was subtracted from all spectra to obtain the final emission graph Atomic Force Microscopy Atomic force microscopy (AFM) was utilized to stretch mechanically single molecules. Proteins (~ 30 µg/ml) adsorbed to freshly gold coated glass slides for 15 minutes, however this worked equally well with new glass slides lacking gold plating. Unbound proteins were washed away with two changes of relaxing buffer solution (24 mm Imidazole, 200 mm KCl, 4 mm MgCl2, 1 mm EGTA, ph 7.4). The Ig domains were pulled in a relaxing solution or activation solution (relaxing solution adjusted to a final calcium concentration of pca = 4). Molecules were stretched at 1 µm/sec using an AFM tip (Si3N4 Biolever, BL RC 150 VB C1, Asylum Research, Santa Barbara, CA, USA) in conjunction with a custom JPK Nanowizard II force robot, JPK software (Berlin, Germany) and inverted Zeiss microscope (Göttingen, Germany). All cantilevers were calibrated at room temperature in solution according to the thermal noise 21

36 method and separate cantilevers were used for calcium and control experiments. Typical cantilever stiffness was 25 pn/nm. The AFM unfolding peaks in a force trace were selected and analyzed according to Best et al., (2003). Specifically, these curves contained a minimum of five and a maximum of eight unfolding events, excluding the final protein detachment from the cantilever or glass. The last detachment peak was typically different in amplitude from all preceding peaks, and was always excluded from analysis. Traces were only selected in which the peak to peak unfolding distance was regular from one peak to the next, with marginal non-specific interactions between the sample, tip and surface. The force, persistence length (PL) and unfolding distance were recorded for each peak, as well as positional information. PL, a measure of polymer stiffness, was derived from the ascending slope of the force trace when fit to the worm like chain (WLC) model (Bustamante, Marko, Siggia, & Smith, 1994; Marko & Siggia, 1995). Equation 3.1 WLC Model FA z 1 1 kbt L z 4(1 ) 2 4 L A one way and two way analysis of variance (ANOVA) was conducted to compare the calcium and control conditions, as well as the peak order dependence of unfolding events. 22

37 3.3 Results Fluorescence Spectroscopy With the addition of 1 mm calcium to the I27 protein in buffer solution, the emission spectra resulted in a drop in fluorescence intensity (Figure 3.1). Subsequent addition of excess calcium (10 mm) resulted in a larger depression reflecting an augmented calcium effect on the tryptophan core within the I27 protein. With addition of a calcium chelating agent (10 mm EDTA), there was a restoration in the fluorescence intensity although not entirely to baseline levels. EDTA and I27 together were indistinguishable from the baseline I27 fluorescence (data not shown). Figure 3.1 Fluorescence intensity measurements of the tryptophan microenvironment within the I27 protein. Upon calcium addition (1 mm), there was a change in the fluorescence emission spectrum indicating an alteration in the internal tryptophan environment, which was further depressed in the presence of excess calcium (10 mm). With the subsequent removal of calcium using the chelator EDTA, the fluorescence improved toward baseline levels indicating a reversibility to the environmental perturbation. Inset: One I27 domain with the core tryptophan exaggerated within the beta barrel structure (Protein Data Bank code: 1TIT). From (DuVall et al., 2013), used with permission. 23

38 3.3.2 Atomic Force Microscopy In Figure 3.2, there was an immediate difference in the unfolding force with calcium addition (P value < 0.001). This difference paralleled the control condition with a consistent separation of approximately 40 pn for the most robust dataset collected (peaks 1 5). Importantly, peaks one to five contain the largest number of force traces (N = 55 and 47 for calcium and control, respectively) translating into the most characteristic force peak relationship. The observed difference between conditions remained for peaks 6 and 7 as well (P value < 0.05). The cantilever tip rarely made contact with the free end of the protein resulting in a low frequency of observing eight unfolding force traces (N = 2 and 3 for calcium and control, respectively). Figure 3.2B indicates the persistence length change with calcium addition. Overall, this translated into an increased polymeric stiffness with calcium, reflected as a shorter persistence length. This difference ran parallel to the control case and changed with further domain unfolding in a significant manner (P value < 0.05 for peaks 2 6). The peak to peak distance was compared under the two conditions, with a significant difference arising due to the presence of calcium (P value = 0.005) (Figure 3.3). Although there was no peak effect, calcium resulted in an increased distance between peaks. Collectively, 321 calcium and 276 control peaks were analyzed. 24

39 Figure 3.2 Data represent mean ± SEM. Peaks 1 5 correspond to a sample of 47 and 55 for the control and calcium conditions respectively, contributing the most heavily to the overall trends in force and persistence length. A) Force produced for each respective unfolding peak in the control and calcium condition. The addition of calcium resulted in a consistent 40 pn increase in force for the first 5 peaks compared to the control group (** P value < 0.001), maintaining a significant difference for peaks 6 and 7 (* P value < 0.05). B) The PL increased with unfolding until peak four and then approximated a plateau. This was reflected equally well in both conditions being significant for peaks 2 6 (* P value < 0.05). Peak 1 results deviate most from the WLC model fit, which could account for the relative insignificance (P value = 0.061). From (DuVall et al., 2013), used with permission. 25

40 Figure 3.3 Raw force traces of seven unfolding events (of a possible eight) in the control (left) and calcium (right) condition. Broken line corresponds to 200 pn in force, while the curved line displays the fit of the WLC model to the ascending half of each forced unfolding event. The unfolding distance (xu) measured according to the WLC model indicates the mean unfolding distance for control (24.76 nm ± 0.13 nm) and calcium (25.25 nm ± 0.13 nm) conditions. Distance was not found to have a peak dependence, but was significant between conditions (*P value = 0.005). From (DuVall et al., 2013), used with permission. 3.4 Discussion Following recombinant protein production of I27, we conducted fluorescence spectroscopy utilizing the emission spectra of a single tryptophan within each of the eight serially linked I27 domains (8 total). Given that calcium is generally responsible for changes in protein structure upon binding, intrinsic fluorophores such as tryptophan serve as excellent detectors of environmental change (Borgia, Williams, & Clarke, 2008; Coulis et al., 2004). Introduction of 1 mm calcium to the I27 protein in solution depressed fluorescence intensity, reflecting a change in the environment surrounding the core tryptophan amino acid (Figure 3.1, inset). With additional calcium (10 mm), the fluorescence intensity diminished further, suggesting the calcium interaction with the I27 domains is concentration dependent. Subsequently, a reversibility of this interaction was visualized in the partial restoration of 26

41 fluorescence intensity upon calcium chelation with 10 mm EDTA. This incomplete recovery may be resolved by considering that not all calcium bound to I27 was successfully removed with the 10 mm concentration of chelator used (Coulis et al., 2004). The proximity of the internal tryptophan to calcium is known to affect the sensitivity to environmental perturbations (Lakowicz, 2006), thus the single buried tryptophan within I27 (Improta et al., 1996) may explain the marginal fluorescence change with addition and removal of calcium. It should be noted that no denaturant was used to unfold chemically the I27 domains. A conformational change in I27 attributed to calcium may ultimately manifest in a modulation of mechanical stability (Borgia et al., 2008). This can steady a protein (like I27) against mechanical stress and increase the force required to unfold the protein (Ainavarapu, Li, Badilla, & Fernandez, 2005). These biochemical changes with calcium have particular importance for a protein such as titin that must resist force in vivo, where calcium interactions are a regular occurrence. An atomic force microscope (AFM) specialized for stretching single molecules was used to evaluate any unfolding force, stiffness or distance change with calcium. Observing the general force trends in Figure 3.2A, specifically for the most robust dataset collected for peaks 1 5, it can be seen that the calcium addition translates into a sustained 40 pn increase in the forces required to unfold the domains. We propose that the difficulty in obtaining force traces with eight unfolding events presents a sampling difference that could account for the more irregular pattern observed for peak eight (Figure 3.2A). Homopolymer unfolding behavior for individual domains such as the eight I27 studied here, are typically assumed to be identical within thermal noise for a defined retraction speed. However, measuring the passive elastic response of one folded domain within a chain is complicated by the 27

42 polymeric nature of the arrangement used. Monte Carlo simulation work has suggested that grouping all the unfolding forces in a polymer chain together may obscure subtleties in the mechanical unfolding data (Zinober et al., 2002) such as the intricacies revealed in Figure 3.2A when the unfolding forces are plotted per peak rather than an average across peaks. Notably, an order dependence of force on peak number arose suggesting that force has a complex non linear function dependence on the unfolding event number (Zinober et al., 2002). We believe that this force dependence on peak number transpired for three reasons: a) a probability component for polymer unfolding which decreases as more domains unfold, b) a compliance component affecting polymeric stiffness, which increases with unfolding (Best et al., 2003) and c) a calcium dependent stabilization contributing to the steeper slope for the calcium condition (36 pn change), compared to the control (27 pn change). Assuming that all domains in a chain experience calcium interaction equivalently, then calcium explains a net difference of 9 pn of force with peak number between conditions. Figure 3.2A and B are obtained from the well characterized worm like chain (WLC) model (Bustamante et al., 1994; Marko & Siggia, 1995) used to describe the relationship between polymer extension and the entropic force that develops. Briefly, the model predicts that the stiffness of a polymer, measured as a persistence length (PL), is inversely related to the force produced (Ottenheijm & Granzier, 2010). An entropic spring is known to become softer with unfolding as an element of compliance is continually introduced (Zinober et al., 2002), which translates into an increase in the PL (decrease in stiffness). Taken together, a two way ANOVA of group (calcium and control) and peak number revealed that the force and PL were significantly different (P value < ). Notably, both control and calcium experiments were conducted in the presence of 4mM MgCl2 to address divalent ion presence. 28

43 Watanabe et al., (2002) studied fragments of titin including the domain used here (I27) 1 reporting that there were no differences in the unfolding forces or the persistence lengths in the presence of high calcium ( pca ~3.0). However, this fails to describe our data (Figure 3.2A and B). This discrepancy may reside in the different protein constructs used and the analysis that followed. Immunoglobulin domains within the I band extensible region of titin are characterized according to variations in size, sequence and composition (Improta et al., 1996; Marino et al., 2005) resulting in naturally diverse unfolding forces. Employing a heteropolymeric approach will introduce a variability that can mask persistence length or unfolding force differences. This complexity has been proposed by others (Gao et al., 2001; Marszalek et al., 1999), making the elastic properties of heterogeneous domains difficult or even impossible to identify (Fisher, Oberhauser, Carrion-Vazquez, Marszalek, & Fernandez, 1999). Furthermore, grouping the mean force and PL obtained for each force trace (not peak) implicitly assumes all domains are equivalent, which is not the case for mixed modular polymers. The peak unfolding distance was obtained by using the WLC model in conjunction with a molecular ruler function in the JPK software package. Simply, the molecular ruler connected one WLC fit to the next, near the peak force as shown in Figure 3.3. This proved to be more consistent than connecting one saw-toothed peak to the next, which has to contend with stochastic fluctuations in force which naturally over or underestimate the distance. Upon calcium addition, the length of the unfolding between peaks increased, likely associated with the increased fractional extension due to the elevated forces seen in the presence of calcium. The additional 40 pn of force upon stretch with calcium elongates the protein domain farther such that the experimental unfolding distance (xu = nm) approaches the theoretical (xu = nm), assuming each amino acid contributes 0.34 nm (Brockwell et al., 2002) and there are 89 29

44 residues. With the physiological pulling speed of 1 µm/sec (W. Linke & Grützner, 2008), the gradual increase in unfolding distance did not manifest in a peak order dependence, but was averaged and found to be significant (P value = 0.005). Our control measurements for unfolding distance, force and persistence length were in good approximation with experimental literature values (Carrion-Vazquez et al., 1999) suggesting AFM consistency, but literature values did not encapsulate our calcium measurements within error. In the presence of calcium, the experimental conditions used here suggest that the same domains become inherently more stable, providing support for the notion that calcium indeed binds to the I27 protein domain as has been suggested theoretically (Lu et al., 1998). This may be in part due to the high proportion of glutamates in the amino acid sequence of I27 (12.4%), which has been shown to be a prerequisite for the PEVK region of titin to bind calcium (D. Labeit et al., 2003). Additionally, the theoretical isoelectric point was calculated to be 5.07 using ProtParam (Gasteiger et al., 2005), which at physiological ph suggests a strong negative net charge identical to that of the PEVK region (pi=5.06) (Tatsumi et al., 2001). Calcium regulated stabilization may add structural integrity and thus delay or even preclude domain unfolding in order to maintain the strong passive elastic response of titin seen in cardiac muscle, should unfolding prove possible. The subsequent unbinding of calcium may occur with the assistance of the ubiquitous calcium binding protein S100A (Yamasaki et al., 2001), permitting a transient response in titin s passive force development. In this manner, the calcium ions present in the sarcomeric environment following cycles of active muscle contraction can then additionally function in passive force regulation ascribed to titin Ig domains. It should be mentioned that without detailed mutagenic studies evaluating a proposed binding site on I27 (see (Lu et al., 1998)) we cannot verify a calcium binding location on I27. However, 30

45 the stabilization interaction between calcium and I27 persists and should be considered of importance. Given that no other immunoglobulin domain has been characterized to the same extent as I27, it remains to be determined if the calcium interaction seen here can be extrapolated to other titin Ig domains, although there is recent support for this in the proximal Ig region (Coulis et al., 2008). Should more domains be stabilized by calcium, unfolding would be further restricted to pathophysiological stretches, indicating an exceptional mechanism for rapid adjustment of passive force by titin. 3.5 Conclusion In the presence of calcium the I27 protein chain is stabilized resulting in novel mechanical properties. More force is required to unfold the domains, jointly due to decreased probability of unfolding, increased compliance and calcium necessitated stabilization. Furthermore, the distance between unfolding peaks increases, approaching the theoretical contour length of the extended protein chain. Finally, by plotting these novel characteristics along their respective peak numbers, it becomes apparent that the unfolding forces and persistence lengths follow a non linear, peak dependent pattern often overlooked in AFM experimental work. This work has led us to suggest that individual Ig domains within titin are capable of increasing the inherent stiffness of this molecular spring upon calcium interaction. The mechanical properties of these domains are an important determinant of how titin is able to resist stretch, leading to a rapid physiological mechanism of passive force regulation. 31

46 3.5.1 Footnote 1 The numbering scheme used is according to (S. Labeit & Kolmerer, 1995). I27 in skeletal muscle was later renamed I91 according to (Bang et al., 2001) when titin was completely sequenced. 32

47 Chapter Four: Titin Passive and Active Behaviour 4.1 Introduction The contractile explanation for skeletal muscle function can largely be attributed to the sliding filament (A. Huxley & Niedergerke, 1954; H. Huxley & Hanson, 1954) and cross-bridge theories (A. Huxley, 1957; A. Huxley & Simmons, 1971; H. Huxley, 1969). These theories subsequently led to the formulation of the force-length relationship (Gordon et al., 1966) which relates observations of muscle length with maximal isometric force. However, for as long as these theories have been in existence, there has been experimental evidence to the contrary (Abbott & Aubert, 1952). The term residual force enhancement (RFE) is used to describe the increase in isometric steady-state force when a muscle is actively stretched, relative to the corresponding (same length and activation) isometric steady-state case (Abbott & Aubert, 1952; Edman et al., 1982). When an activated muscle is stretched (termed eccentric contraction), the force produced can exceed theoretical force predictions at the corresponding muscle length. For the right stretch conditions, the isometric force following stretch can exceed the purely isometric force produced at any muscle length (Leonard et al., 2010). This means that a mechanism exists within muscle enabling it to surpass the maximal force potential by hundreds of percent simply by coupling muscle activation and stretch. To date, a convincing explanation for this departure from theory has failed to materialize into an understanding of how muscles can accomplish this. What has emerged are a series of indirect observations that highlight possible candidates to explain RFE. The fact that RFE has been observed in preparations ranging from whole muscle (Herzog & Leonard, 2000) down to the individual contractile units called sarcomeres (Leonard et al., 2010), suggests that the mechanism behind RFE is a property inherent to sarcomeres. As RFE is associated with a stretch, the molecular spring within sarcomeres called titin is a tempting 33

48 suspect for explaining this phenomenon. Supporting this notion further, stretched muscles are capable of producing higher forces than the isometric case without an increase in the energetic cost (Joumaa & Herzog, 2013; Linari, Woledge, & Curtin, 2003), which could relate to energy storage in elastic elements such as titin. Titin links the M-line to the Z-line within the sarcomere and is thought to maintain sarcomere symmetry. Only the I-band region of titin is functionally extensible, with the exception of the approximately 100 nm region adjacent to the Z-line where titin remains firmly bound to actin (W. A. Linke et al., 1997; W. Linke & Fernandez, 2002; Trombitas & Granzier, 1997). The extensible region of skeletal titin can be divided into proximal and distal immunoglobulin (Ig) domains which flank two adjacent sequences called the N2A and the PEVK. Titin has traditionally been considered a passive elastic element, maintaining independence as the sole contributor to passive force at the sarcomeric level (Horowits et al., 1989). However, recent RFE evidence suggests that titin is also a potent regulator of active force (Leonard & Herzog, 2010) capable of tuning its mechanical properties (stiffness) in response to different contractile conditions. From a mechanistic perspective, a fast and impactful way to increase titin stiffness would be to adjust the free extensible spring length. Stretching a shorter titin spring within the sarcomere would generate more force than an unabbreviated one, which could account for the experimental departures from theoretical predictions associated with RFE. Inconclusive observations of titin interaction with filaments in the sarcomere warrant further investigation into the existence of titin truncation during active stretch. Given that titin interaction with actin at the N-terminal end has been observed (Trombitas & Granzier, 1997), it became natural to inquire about this coupling occurring in other locations. 34

49 4.2 Purpose The purpose of this work was to observe titin s extensible segments by using fluorescent markers on different parts of titin within the sarcomere during passive and active stretch. 4.3 Hypothesis We hypothesize that titin has the ability to transiently interact with neighboring actin filaments upon muscle activation rendering this spring abbreviated. This actin interaction is not expected to occur in the passive sarcomere, which would be reflected by a different degree of antibody translocation during active and passive stretch. 4.4 Methods Microscope and Setup An inverted Olympus microscope (IX-81, Olympus, Japan) with epifluorescence was used with a 100 X oil immersion objective (NA 1.3, Olympus, Japan) and the 2 X optivar. Other objectives included: 4 X (NA 0.13), 10 X (NA 0.3), 20 X (NA 0.5) and 40 X (NA 0.75) (Olympus, Japan). Antibody excitation was accomplished with an X-Cite system with foot pedal shutter (Excelitas Technologies Corp., MA, USA) at 20% intensity. The image was recorded using a chargecoupled device camera (Retiga 4000DC, B.C., Canada) with an optical resolution of nm per pixel. Manipulation of the myofibril was accomplished with several micromanipulators: model: MMN-1, NMN-21, MMO 203 (Narishige Co. Ltd., Japan) and Newport 461 as well as Newport M-642 series (CA, USA) to which glass pipettes were inserted. These glass pipettes were formed from a 5 µl capacity capillary tube (model: , VWR Inc, ON., Canada) pulled to a sharp point using a pipette puller (model # 720, Kopf Instruments Ltd., CA, USA). 35

50 The motor movement to stretch the myofibrils was performed using a Physik Instrumente GmbH & Co (Karlsruhe, Germany) piezo disk translator and E controller. Force measurements were accomplished by using a 660 nm laser (Schäfter-Kirchoff, Hamburg, Germany) and quadrant detector (H-series receiver module and PS1 amplifier, Electro-optical Systems, PA, USA) that measured cantilever deflection in real time at 200 Hz (Figure 4.1). The force resolution was estimated to be less than one nn, calculated from the standard deviation of the baseline signal. Cantilevers were gold coated on the reflex side to increase signal (HYDRA6R NG, AppNano, CA, USA) and had a stiffness of N/m. Motor control, laser spot detection and data acquisition was done through custom written software in LabView (National Instruments Corp., TX, USA). Myofibril activation was done with a modified Warner Instruments six channel (CT, USA) gravity fed control system with a 25 ml activating solution reservoir. All equipment was isolated on a high performance workstation (Kinetic Systems Inc., MA., USA). Figure 4.1 A myofibril captured beween a rigid glass needle and force transducing cantilever (inset). This is contained within a fluid bath to which a laser and quadrant detector are coupled to record force. 36

51 4.4.2 Myofibril Preparation Ethical approval for all experiments was granted by the Life and Environmental Sciences Animal Care Committee at the University of Calgary. Six month old New Zealand White rabbits were euthanized, the psoas muscle was freshly harvested into strips, fixed in place with sutures to maintain in vivo sarcomere lengths and chemically skinned overnight according to Joumaa et al., (2007). Skinned psoas muscles were then stored in a rigor/glycerol mix (1:1) for a minimum of ten days to allow for chemical membrane degradation. Solutions were supplemented with a Complete protease inhibitor cocktail (Roche Diagnostics, QB, Canada) to minimize protein degradation. Myofibrils were prepared fresh on the day of experiments by homogenizing a sliver (1 mm diameter by 3 mm length) of psoas muscle in rigor using a mechanical blender (Model PRO250, Pro Scientific, Oxford, CT, USA). Unlabeled experiments were done after 30 minutes to allow the myofibrils to settle. For labeled myofibrils, a drop of homogenized tissue was added to a glass slide and the density of the solution was visually inspected. The top µl was siphoned off according to the myofibrillar density and added to 3 ml of fresh rigor. Primary antibodies were added to this tube, and rotated for 20 min at 4 C. Following this, secondary antibodies were introduced for another 20 min at 4 C, and 200 µl of the final solution was deposited on a fresh glass slide within a fluid cell to which 3.5 ml of relaxing solution was added. All experiments were performed at room temperature. There was little difference in diameter between the myofibril groups (Table 4.1). 37

52 Table 4.1 Myofibril Diameter Comparisons Myofibril Group Diameter ± S.D. (µm) Unlabeled 1.64 ± 0.23 F146 Labeled 1.71 ± D10 Labeled 1.71 ± Labeled 1.71 ± Solutions The three solutions utilized were based on those published by Joumaa et al., (2007). Notable changes included reduced concentrations of EGTA and calcium in both the relaxing and activating solutions. Rigor was prepared as published originally. All solutions were adjusted to a ph of 7.02 (Corning Pinnacle 530, Corning Inc., NY, USA) with 40% hydrochloric acid or 10 M potassium hydroxide. A full list of chemical abbreviations is located in the Appendix. 38

53 Table 4.2 Solutions used in the preparation (rigor), relaxation (relaxing) and activation (activating) of skeletal myofibrils. Relaxing: Concentration (mm) MOPS 10 Kprop 64.4 Mgprop Na 2 SO K 2 EGTA 2 ATP 7 Creatine Pi 10 Rigor: Concentration (mm) Tris 50 NaCl 100 KCl 2 MgCl 2 2 EGTA 10 Activating: Concentration (mm) MOPS 10 Kprop 45.1 Mgprop Na 2 SO Na 2 EGTA 1 ATP 7 Creatine Pi 10 CaCl Antibody Labels All experiments were conducted using a double labeling approach in which each of the three titin primary antibodies was paired individually with a primary myomesin antibody at the M-line in the middle of the sarcomere for reference. Myofibrils labeled only with secondary antibody showed no fluorescence. Typical dilutions of antibodies were: 9D10 (1:20), F146 (1:35), 891 (1:80), myomesin (1:65) and Alexa 488 goat anti-mouse (1:70). Primary antibody 891 was provided by Dr. Marion Greaser (Madison, WI, USA). This mouse monoclonal epitope has not previously been published but has been thought to bind in a similar location to the well described titin T11 antibody (Fürst, Osborn, Nave, & Weber, 1988). Using electron micrographs, the location of 891 has been suggested to be 0.88 ± 0.03 micro[meter] from the M-line (Greaser, 2015). 39

54 The IgM monoclonal antibody 9D10 developed by Dr. Marion Greaser was obtained from the Developmental Studies Hybridoma Bank under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biology. 9D10 serves as a PEVK identifying antibody, with one or more bands being possible in each half sarcomere between exon 112 and 225 (Trombitás, Greaser, French, & Granzier, 1998; Trombitás et al., 1998). The IgG1 mouse monoclonal myomesin primary antibody mmac myomesin B4, specific for myomesin in the center of the sarcomere, was developed by Dr. Jean-Claude Perriard and obtained from the Developmental Studies Hybridoma Bank. F146.9B9, henceforth known as F146, was an IgG1 mouse monoclonal from Enzo Life Sciences Inc. (NY, USA). As the immunogen sequences are considered proprietary, the exact epitope location could not be ascertained from the manufacturer. Alexa Fluor 488 goat anti-mouse highly cross-adsorbed IgG was used as the secondary antibody (Invitrogen, CA, USA), which worked equally well for both IgG and IgM primary antibodies (W. A. Linke et al., 1996). As the exact location of each antibody epitope is not known, assumptions have been made to estimate and quantify their location within psoas tissue. Literature values for psoas muscle at approximately 3.5 µm reflect the contour length potential of folded proximal plus distal, and straightened PEVK segments. For this study, we approximated the size of individual folded domains fully straightened to be 5 nm (Freiburg et al., 2000; Trombitás et al., 1998; Trombitás et al., 2000) which matches with literature calculations of 4.6 ± 1.7 nm (Oberhauser, Marszalek, Carrion-Vazquez, & Fernandez, 1999) and NMR observations of 4.4 nm (Improta et al., 1998). The PEVK region was assumed to have a length of 0.38 nm per residue (Freiburg et al., 2000). 40

55 We assumed no differences existed between rat and rabbit psoas muscle (W. A. Linke et al., 1996) and further assumed negligible contribution of the N2A sequence to titin elongation. Figure 4.2 A) Approximate antibody localization within rabbit psoas titin. Main segments are the proximal Ig, N2A, PEVK, and distal Ig. Red serves to indicate inextensible parts, yellow for fibronectin and green and blue for unique sequences. Adapted from (W. A. Linke et al., 1998). B) Phase contrast (Pc), Fluorescence (Fluo.) and merged images (Merged) of myofibrils labeled with titin F146 and myomesin antibodies. Markers were placed on each fluorescent band in order to monitor the movement of the M-line (M) and the antibody to antibody distance across the Z- line (Ab Ab). Additionally, Ab M was recorded for each half sarcomere. C) Fluorescent images of the three antibodies used (F146, 9D10 and 891) in conjunction with myomesin at the M-line, at resting length. Given the antibody locations along titin fit to literature values, F146 served as the best marker for a boundary separating titin s unique segments (Figure 4.2A). The contour length from Z-line center to the distal end of the PEVK was then estimated as: 41

56 100 nm (Ig domains linking the inextensible proximal region to the center of the Z-line) 250 nm (50 proximal Ig domains at 5 nm per Ig domain) 304 nm (800 PEVK residues at 0.38 nm per residue) 654 nm is the predicted maximum folded and straightened contour length at a sarcomere length of 3.5 µm in psoas muscle between the distal Ig- PEVK boundary and the center of the Z-line. For antibody 9D10, we assume a binding of the antibody at two-thirds of the straightened PEVK contour length (203 nm) marking the location at approximately 553 nm from the center of the Z- line at a sarcomere length of 3.5 µm while maintaining a folded Ig domain arrangement. The 22 Ig domains in the distal Ig region also affect the onset of titin elasticity. These account for 110 nm in contour length when maximally straightened, with the possibility of a few extra domains present at the A/I junction (W. Linke, Stockmeier, Ivemeyer, Hosser, & Mundel, 1998; W. A. Linke et al., 1998). This would fit the 891 antibody to between 650 nm (distal-pevk boundary) to 760 (A/I junction boundary) at 3.5 µm based on our element spacing assumptions Protocol A glass needle was dipped in glue (Dow Corning 3145 and :1 mixture, MI, USA) and was then used to distribute glue onto the glass needle attached to the motor, as well as the force transducing cantilever, both in solution. A single myofibril was then attached at either end to the pre-glued motorized needle and cantilever. Once a myofibril was mounted, the laser was aligned on the back of the cantilever using a custom quadrant centering program (LabView, National Instruments Corp., TX, USA). The LabView experimental protocol program was then run which adjusted the speed and amplitude of stretch for the number of sarcomeres present with a nominal 42

57 two micrometer stretch amplitude per sarcomere. The passive stretch protocol included a ten second pause, after which the motor moved at 0.1 µm/sar/sec, or about 5 % of the sarcomere resting length per second. At the end of stretch, a two second pause was done to permit syncing of force and video data, after which the motor returned to its original position. Prior to active stretch onset, there was a twenty second pause to ensure full activation of the myofibril, after which the motor operated under the same speed and amplitude conditions as the passive stretch. The myofibril was activated by a directed stream of activation solution (Colomo, Piroddi, Poggesi, te Kronnie, & Tesi, 1997) which was moved into position only prior to active stretch experiments. The myofibril remained activated for the duration of stretch and was not deactivated at any point during active experiments. Camera exposure time was set to about 200 msec (five frames per second) with the exception of the stretch speed experiments in which the 0.2 µm/sar/sec had an exposure time of 100 msec and 0.05 µm/sar/sec had an exposure time of 400 msec to maintain image quality Stretch Speed Experiments The speed experiments were only done using the F146 labels which were tracked at three different speeds (0.05, 0.1 and 0.2 µm/sar/sec) for two activation conditions. These two conditions were: activate then stretch and stretch then activate. The stretch then activate experiments began passively at resting length and were activated dynamically during stretch at various sarcomere lengths and speeds. Activate then stretch experiments occurred as described above, at three different speeds. 43

58 4.4.6 Immunofluorescent Sarcomere Tracking Sarcomere banding patterns were manually digitized using ImageJ software (1.47V, NIH, MA, USA). A marker was placed on the centroid of each fluorescent band and the translocation behaviour was plotted relative to sarcomere length or time for each sarcomere/myofibril (Figure 4.3). As the videos were dynamic and the banding patterns not always uniform in intensity and direction, it was deemed more appropriate to track bands manually rather than with pixel intensity profile tracking. Additionally, some frames were briefly out of focus and band pattern skewing could occur with extreme stretch which further supported a requirement of user input. Sarcomeres were defined as the centroid of one A-band tagged with a myomesin antibody, to the centroid of the next consecutive A-band myomesin. This has a few key advantages over measurement from Z-line to Z-line. Tracking of markers was clearer when the bands were further apart from each other. In phase contrast unlabeled experiments, Z-lines become difficult to resolve at longer sarcomere lengths, thus any impact of switching to A-band tracking later was precluded. Finally, and somewhat fortuitously, measurements of antibodies relative to the M-line revealed interesting titin behaviour that may have been overlooked if the measurement was taken across the entire A-band. The distance from the label on titin to the center of the Z-line was taken as half the distance from one titin antibody to the other titin antibody on the adjacent half sarcomere. Importantly, the same sarcomeres were tracked throughout the stretch as a reflection of the overall myofibril behaviour, and every effort was made to capture two measurements for each 100 nm bin to increase precision. Myofibril stretch could then be plotted as a series of consecutive segments detailing titin s location relative to the M-line and Z-line (Figure 4.3). 44

59 Figure 4.3 Myofibril video frames during passive stretch (left) and the digitized rendering (right) from an average sarcomere length between 2.0 and 2.5 µm. The digitized rendering separates the segments by colour, with red representing titin antibodies across the Z-line, and grey being epitope to M-line for each half sarcomere Algorithm Myofibril data was further broken down to investigate individual sarcomere behaviour during passive and active stretch. To minimize any bias in determining the location of observable changes in antibody tracking with stretch, an algorithm was designed. We used a three line model to describe the distance from one epitope on titin to the other (across the Z-line) as a function of time. For any two given time points, t1 t2, we calculated three lines by linear regression: a. From the start of the experiment to ݐ ଵ b. From t1 to t2 c. From t2 to the end of the experiment 45

60 Two constraints were applied: the last line has a slope of zero, and the first and second lines meet at point t1, while the second and third lines meet at t2. Of all possible combinations of t1 and t2 we selected those two points with the overall minimum mean square error to represent the plateau or turning point. This turning point reflected a change in the label tracking behaviour in which the distance relative to the Z-line or M-line began to become very small (epitope to Z-line distance was no longer increasing appreciably) or very large (epitope to M-line distance began to increase dramatically). For the distance from the M-line to the titin epitope, we used a two line model. Sarcomere length was the independent variable which proved to be most stable, and the mean value of the epitope to M-line distance was the dependent variable. For any given sarcomere length, we calculated two lines by linear regression: a. From the sarcomere length at the start of the experiment to point s1 b. From s1 to the sarcomere length at the end of the experiment The only constraint applied was that the two lines meet at sarcomere length s1. Of all possible values, we selected the sarcomere length with the overall minimum mean square error to represent the turning point. This algorithm looks at each point in the sarcomere trace, and highlights where the pattern changed abruptly. If the sarcomere length of this plateau was different by more than 300 nm between the titin antibody to M-line and the titin antibody to titin antibody distance, then that sarcomere was withdrawn as the plateau break was not prominent enough to discriminate the event in both cases successfully (see Appendix). The success rate in classifying the turning point for each sarcomere in each myofibril based on the above criteria is summarized below. 46

61 Table 4.3 Algorithm data revealing the total number of myofibrils (and sarcomeres) analyzed and how many were included in further analysis. Antibody Myofibrils (Sarcomeres) Post Filtering Success Rate (%) Active F (200) 22 (149) 75 9D10 9 (91) 9 (75) (30) 5 (21) 70 Passive F (173) 13 (63) 36 9D10 12 (91) 11 (72) (76) 6 (34) 45 As passive traces often did not reach sarcomere lengths that were long enough to exhibit a turning point, the algorithm could not be applied successfully to many passive traces. For a similar reason, the algorithm was less effective in highlighting the turning points for the stretch then activate experiments described below Statistics A t-test was used for determining possible differences in passive stress labeled and unlabeled myofibrils, and for possible differences in active stress for labeled and unlabeled results (α = 0.05). A one-way ANOVA was used to look at any stress or label movement differences at three different speeds of stretch. Any significant difference between speed and conditions was further investigated using Tukey s post-hoc test to identify where the differences existed. The same was done with the stretch then activate speed experiments. 47

62 A linear regression was performed between the sarcomere length at active stretch onset and the sarcomere length where plateau occurred, independent of which label was considered. 4.5 Results Stress A comparison of passive and active myofibrillar stress with and without labels is presented in Figure 4.4. These are pooled labeled and unlabeled myofibrils that were passively and actively stretched, with the exception of those collected in the speed subset of experiments (below). Stress data collection was absent from three labeled myofibrils due to technical problems with force collection, while the label tracking remained unaffected Active Unlabeled (n = 11) Passive Unlabeled (n = 26) Active Labeled (n = 36) Passive Labeled (n = 35) Stress (nn/µm 2 ) 200 * Sarcomere Length (µm) Figure 4.4 Average passive and active stress production ± SEM for labeled and unlabeled myofibrils (* Active results, p < 0.05 between active labeled and unlabeled; Passive results, p < between 2.1 and 3.5 µm labeled and unlabeled. p = 0.03 at 2.0 µm for the passive labeled and unlabeled comparison). 48

63 The passive labeled myofibril stress was statistically different from the unlabeled data from sarcomere lengths of 2.0 to 3.5 µm. The stress began earlier and increased steeper with stretch than in the unlabeled case, and further continued to increase approaching that of the active (un)labeled stress. Active labeled myofibrils showed an impaired contractile ability at short sarcomere lengths below 2.0 µm, compared to the unlabeled myofibrils Label Tracking A comparison of the pooled data of all myofibrils in their respective label groups undergoing passive and active stretch is presented in Figure 4.5 and Figure 4.6, respectively. This is further broken down into binned myofibril groups, and then binned individual sarcomeres. 49

64 Passive Labels (n = 7) 1.6 Z-Line to Epitope Distance (µm) D10 (n = 12) F146 (n = 18) M-Line to Epitope Distance (µm) Sarcomere Length (µm) 0 Figure 4.5 Average passive antibody tracking for titin F146, 9D10 and 891 relative to the Z-line (bottom three traces, left vertical axis) and M-line (top three traces, right vertical axis) ± SEM. The 9D10 antibody moved away from the Z-line least (left vertical axis) and accordingly moved away from the M-line most (right vertical axis), indicating it is the most proximal (closest to the Z-line) primary titin antibody tested. The F146 antibody is intimately positioned on the distal side of 9D10, likely at the PEVK-distal Ig boundary. As PEVK is not thought to increase in contour length significantly until passive force begins to develop, one would not expect noteworthy separation of the two PEVK labels until at least 2.6 µm (W. A. Linke et al., 1998). Primary antibody 891 is localized to the distal Ig domain region, thus separates from the PEVK labels with stretch almost immediately. 50

65 Active Labels (n = 5) 1.6 Z-Line to Epitope Distance (µm) D10 (n = 9) F146 (n = 23) M-Line to Epitope Distance (µm) Sarcomere Length (µm) 0 Figure 4.6 Average active antibody tracking for titin F146, 9D10 and 891 relative to the Z-line (bottom three traces, left vertical axis) and M-line (top three traces, right vertical axis) ± SEM. The active experiments showed differences from the passive case although these must be examined carefully given the optical resolution employed. The labels are generally more packed together and show a different pattern relative to each other than with passive stretch. If we assume the half A-band thick myosin filament is 0.8 µm in size, then at short sarcomere lengths all three labels appeared to have entered into the A-band area (are found below 0.8 µm - top three traces, right vertical axis). As 9D10 is the most proximal label used in this study, it is found to re-enter the I-band area (increase beyond 0.8 µm) of the half sarcomere first, followed later by the F146 label and finally the 891 distal Ig label. In the passive case, 9D10 and F146 appeared close spatially, but in the active case, F146 followed 891 and their separation does not seem to 51

66 change by the same proportion as in the passive case. The difference between 891 and F146 in the passive case is about 180 nm at 3.5 µm whereas it is about 90 nm in the active case, which is within the detectable optical resolution limit of our system Individual Myofibrils As all of these myofibrils were activated from resting length, it would seem that a prerequisite for some of the more dramatic departures from the passive case occurred when shortening preceded active stretch. When individual myofibrils were sorted into 200 nm bins according to active stretch onset, a pattern emerged: the shorter the sarcomere length at the onset of active stretch, the earlier the flattening out, or pseudo plateau occurred relative to the Z-line (Figure 4.7). 52

67 Figure 4.7 Binned myofibril behaviour of F146 labeled titin undergoing active stretch relative to the Z-line ± SEM. Inset: the point immediately before and after activation for 14 myofibrils. The activation does not markedly alter the F146 distance from the Z-line. Inset axes are the same as the outset. The activation resulted in little change in the F146 distance relative to the Z-line (inset, Figure 4.7), but a systematic change was seen in the distance relative to the M-line (inset, Figure 4.8). The distance from F146 to the M-line in the binned myofibril data was smaller than 0.8 µm at the start of stretch for all bins (Figure 4.8), corroborating the pooled myofibril data that the I-band part of titin had entered the A-band part of the sarcomere (Figure 4.6). 53

68 Figure 4.8 Binned myofibril behaviour of F146 labeled titin undergoing active stretch relative to the M-line ± SEM. Dashed line represents the A-band edge where the myosin thick filament terminates. Inset: the point immediately before and after activation for 14 myofibrils. The activation resulted in the F146 antibody moving towards the M-line. Inset axes are the same as the outset. Interestingly, some of the individual myofibril data had M-line to F146 distances that were shorter than 0.8 µm before activation (Inset, Figure 4.8), which could reflect natural titin migration into this area. There were instances where the passive tracking data also displayed entry into the A-band region at resting length ( µm), suggesting this may be a regular occurrence. 54

69 (n = 2) Z-Line to Epitope Distance (µm) (n = 5) (n = 2) 0.2 9D (n = 9) Sarcomere Length (µm) Figure 4.9 Binned myofibril behaviour of 9D10 labeled titin undergoing active stretch relative to the Z-line ± SEM. Inset: the point immediately before and after activation for nine myofibrils. Inset axes are the same as the outset. In the case of 9D10, the activation does not show a systematic change in the distance from the Z- line (inset, Figure 4.9). 55

70 Figure 4.10 Binned myofibril behaviour of 9D10 labeled titin undergoing active stretch relative to the M-line ± SEM. Dashed line represents the A-band edge where the myosin thick filament terminates. Inset: the point immediately before and after activation for nine myofibrils. Inset axes are the same as the outset. The activation shortened the 9D10 distance from the M-line (inset, Figure 4.10) but to a smaller extent than that for the F146 antibody (inset, Figure 4.8). 56

71 Z-Line to Epitope Distance (µm) (n = 1) (n = 2) (n = 1) (n = 1) 0.2 (n = 4) Sarcomere Length (µm) Figure 4.11 Binned myofibril behaviour of 891 labeled titin undergoing active stretch relative to the Z-line ± SEM. Inset: the point immediately before and after activation for four myofibrils. Inset axes are the same as the outset. The activation of 891 resulted in minor shortening in the antibody distance from the Z-line in three of four myofibrils (inset Figure 4.11) and a marked decrease in the distance from the M- line (inset, Figure 4.12). 57

72 Figure 4.12 Binned myofibril behaviour of 891 labeled titin undergoing active stretch relative to the M-line ± SEM. Dashed line represents the A-band edge where the myosin thick filament terminates. Inset: the point immediately before and after activation for four myofibrils. Inset axes are the same as the outset. 9D10 did not appear to be anchored in the same manner as F146 or 891, which permitted it to reenter the I-band much earlier than the other two antibodies. The translocation of labels for F146 and 891 relative to the M-line (Figure 4.8 and Figure 4.12, respectively) was flatter than the case for 9D10 (Figure 4.10) for the binned myofibrils data, reflecting little movement with stretch between 891 and F146 from the M-line. The opposite was true for 891 and F146 relative to the Z-line. This could mean that 9D10 represents part of the PEVK that is only on the periphery to some more distal interaction of titin and part of the thick filament. On average, the 9D10 antibody moved least into the A-band area upon activation, which supports this notion (Inset Figure 4.10, compared to Inset Figure 4.8 and Inset Figure 4.12). 58

73 Individual Sarcomeres Given the average data for binned myofibrils, it then became interesting to look at the individual sarcomere behaviour within each myofibril. To analyze the active labeled stretch experiments for individual sarcomeres in an unbiased fashion, an algorithm was applied to each tracked individual sarcomere within each myofibril. This algorithm indicated at which point the antibody translocation behaviour changed, based on the criteria described above (section 4.4.7). Briefly, sarcomeres were removed that had not undergone a far enough stretch where a characteristic plateau was seen, or that were classified inconsistently between traces relative to the M-line and Z-line. This proved not to change the overall myofibril behavior when the binned myofibril data were compared to the binned sarcomere data (Figure 4.7/Figure 4.8 compared to Figure 4.13/Figure 4.14, respectively) and further suggested this behaviour is well represented at both the myofibril and sarcomere level. The occurrence of a plateau was marked on each individual sarcomere extension trace, and the value of this intersection relative to the Z-line and M-line was quantified. The plateaus were then characterized using the same sarcomere bins, based on sarcomere length at stretch onset. This was used to evaluate any significance relative to the attainment of the fully straightened contour length, or the edge of the A-band which was assumed to not change. For reference, the contour length of F146 was estimated as 654 nm, 9D10 was an estimated 553 nm and the 891 antibody was an average of 705 nm from the Z-Line at a sarcomere length of 3.5 µm (Figure 4.5) which assumes folded but straightened Ig domains. 59

74 Z-Line to Epitope Distance at Plateau (µm) (n = 30) (n = 51) (n = 41) (n = 13) (n = 3) 2.5 (n = 11) Active F Passive F Sarcomere Length at Plateau (µm) (n = 3) (n = 23) (n = 32) (n = 4) (n = 1) Figure 4.13 Location of turning point plateaus ± SEM for binned F146 labeled sarcomeres relative to the Z-line undergoing active stretch. Inset: sarcomere turning points undergoing passive stretch. Inset axes are the same as the outset. The passive plot (inset, Figure 4.13) indicated that most sarcomeres clustered around the 3.5 µm sarcomere length at 650 nm away from the Z-line revealing they are not affected by starting length in the same manner as the active data in the same figure. Note that 55 of 63 sarcomeres are presented in the green and purple circles for the passive inset above. The active sarcomere stretch data showed a wide spread of distances attained relative to the Z- line, further illustrating a heavy dependence on active stretch onset much like the myofibril binned data. 60

75 Figure 4.14 Location of turning point plateaus ± SEM for binned F146 labeled sarcomeres relative to the M-line undergoing active stretch. Inset: sarcomere turning points undergoing passive stretch. Inset axes are the same as the outset. Dashed line represents the A-band edge where the myosin thick filament terminates. Most sarcomere plateau turning points relative to the M-line clustered around the A-band edge during active stretch, which could suggest that the plateau is related to some interaction occurring at this location. Note that 122 of 149 sarcomeres are accounted for by the blue, red and green circles approximately 800 nm from the M-line. The same was not seen in the passive case (Inset, Figure 4.14). 61

76 Z-Line to Epitope Distance at Plateau (µm) (n = 4) (n = 21) (n = 37) (n = 12) (n = 1) Active 9D (n = 38) (n = 32) (n = 2) 0.2 Passive 9D Sarcomere Length at Plateau (µm) Figure 4.15 Location of turning point plateaus ± SEM for binned 9D10 labeled sarcomeres relative to the Z-line undergoing active stretch. Inset: sarcomere turning points undergoing passive stretch. Inset axes are the same as the outset. The passive 9D10 data relative to the Z-line showed a broad contour length attainment when binned by resting length stretch onset (inset, Figure 4.15). This is typically seen beyond the predicted contour length for this tissue and label and occurred at sarcomere lengths longer than expected, which suggests there may be some inherent variability to the localization of this antibody within rabbit psoas tissue (see Appendix). The active stretch plateaus reflect an equally large spread of distances from the Z-line as the passive stretch, however this typically occurred at shorter sarcomere lengths. 62

77 Figure 4.16 Location of turning point plateaus ± SEM for binned 9D10 labeled sarcomeres relative to the M-line undergoing active stretch. Inset: sarcomere turning points undergoing passive stretch. Inset axes are the same as the outset. Dashed line represents the A-band edge where the myosin thick filament terminates. The active case indicates less separation between 9D10 and the M-line than in the passive case (inset, Figure 4.16). Only the shortest 9D10 active stretch sarcomere bin coincided with the A- band edge, suggesting a great deal of shortening is required prior to active stretch before plateau behaviour can be related to any A-band edge interaction. The other active bins suggest an abbreviation to the distance between 9D10 and the M-line when compared to the passive inset, but not a consistent dependence on the A-band edge as seen with F146 (Figure 4.14). 63

78 Z-Line to Epitope Distance at Plateau (µm) (n = 4) (n = 6) (n = 7) (n = 3) (n = 1) Active (n = 5) (n = 27) (n = 2) Passive Sarcomere Length at Plateau (µm) Figure 4.17 Location of turning point plateaus ± SEM for binned 891 labeled sarcomeres relative to the Z-line undergoing active stretch. Inset: sarcomere turning points undergoing passive stretch. Inset axes are the same as the outset. The passive 891 data (Figure 4.17, inset) showed a clustering around 3.5 µm at an epitope to Z- line distance around 800 nm which is larger than predicted from sequence data (section 4.4.4), but is in line with the predicted distance from the M-line (Figure 4.18) estimated by others (Greaser, 2015). A slightly shorter distance from the Z-line was reached during active stretch, but occurred at shorter sarcomere lengths than in the passive case. Four myofibrils greatly exceeded the contour length prediction of 705 nm, which suggests the region between the 891 distal Ig antibody and the center of the Z-line was stretched a great deal to accommodate sarcomere lengthening. 64

79 Figure 4.18 Location of turning point plateaus ± SEM for binned 891 labeled sarcomeres relative to the M-line undergoing active stretch. Inset: sarcomere turning points undergoing passive stretch. Inset axes are the same as the outset. Dashed line represents the A-band edge where the myosin thick filament terminates. Like F146, the turning points for 891 appeared to cluster around the A-band edge in 17 of 21 actively stretched sarcomeres. This also occurred at shorter sarcomere lengths than during passive stretch (inset, Figure 4.18). Comparing the turning points at different SL bins for the three titin antibodies, it was seen that the A-band edge was a prominent feature for antibodies F146 and 891. It would seem that these two antibodies plateau in the epitope distance-elongation curves at close to 0.8 µm in most instances as they may be dependent on an interaction with the thick filament edge. Any sarcomere stretch beyond these turning points translated into a large displacement of the antibody relative to the M-line, with little contribution in the region between the antibody and the 65

80 Z-line. The 9D10 antibody had a different pattern in that it appeared to move away from both the M-line and the Z-line with active stretch. Given observations of titin s distal region interacting with the A-band area of the sarcomere, several exploratory experiments were performed to better understand this behaviour Long Length Activation As I-band titin appeared to localize to the A-band with active shortening before the onset of stretch, it became prudent to activate at a sarcomere length that maintained titin in a straightened configuration thus limiting titin s ability to migrate into the A-band. To ensure this transpired, activation was performed at 2.7 and 2.9 µm (Figure 4.19). 66

81 Figure 4.19 Long length activation (blue circles) comparison to purely passive stretch (green triangles). Note that when myofibrils were stretched from resting length and activated shortly thereafter (red diamonds), no real change was seen from the case where a myofibril was activated at a similarly long length. Dashed line represents the A-band edge where the myosin thick filament terminates. Long length activation resulted in much longer contour length attainment than in the passive case relative to the Z-line (extra 600 nm) which may be associated with Ig domain unfolding at long length (inset). Holding titin at an extended configuration can lead to time dependent Ig domain unfolding (Trombitás et al., 1998), so these experiments were done two different ways: initially activating at this final long length and then stretching actively, or stretching passively from resting length and then activating dynamically when the stretch had reached the approximate desired long length. The stretch-then-activate experiment at 2.7 µm showed an identical pattern to the long length activation at 2.9 µm, which suggested that the mass unfolding observed was not explainable by experimentally holding the myofibril taut at 2.9 µm for a period of time before the experiment. This long length activation was compared with an exemplary passive myofibril 67

82 stretch (green triangles, Figure 4.19). The slopes of the epitope migration between passive and active were not the same, but the overall indifference to the A-band edge was maintained. As the passive trace approximated the contour length expected for this antibody, we have no reason to suspect that any significant proximal Ig domain unfolding occurred with passive stretch below 3.5 µm Stretch Then Activate Given the similarity between passive and active data at long length which did not reflect PEVK or distal Ig entry into the A-band, an experiment was conducted where the stretch started as a passive condition and activation solution was directed at the myofibril after five seconds of initial passive stretch. This subset of experiments was designed such that activation could occur at near optimal sarcomere lengths where titin was freely extensible to varying degrees and the PEVK was largely contracted, but that hindered significant shortening of sarcomeres from occurring upon activation as the stretch had already commenced (Figure 4.20). 68

83 Figure 4.20 Stretch then activate experiments of four F146 labeled myofibrils activated dynamically at near optimal lengths. Inset: for each of the four myofibrils, the point immediately before and after activation was plotted. The activation brings the antibody towards the M-line, but not typically into the A-band area. The inset axes remain the same as the outset. The degree of movement of F146 relative to the M-line is much less than that of the active experiments for F146 at the same sarcomere lengths (inset, Figure 4.8), which suggests that spatial overlap between titin s I-band and A-band is important for interaction and may be even more favourable with a larger degree of active shortening preceding active stretch. 69

84 4.5.5 Stretch Speed Experiments Speed of stretch was investigated at three speeds, for two conditions: activate then stretch experiments at 0.05, 0.1 and 0.2 µm/sar/sec and stretch then activate experiments (same speeds) using F146 as the only titin primary label. The F146 labeled myofibril behaviour is presented in Figure 4.21 with the corresponding stress in Figure 4.22, at different speeds of active stretch. When myofibrils were activated, allowed to shorten, and then stretched at different speeds, the labels reflected no significant differences due to stretch speed. The plateauing occurred consistently at about 2.6 µm between all stretch speeds. Z-Line to Epitope Distance (µm) F µm/sar/sec (n = 8) µm/sar/sec (n = 23) 0.2 M-Line to Epitope Distance (µm) 0.2 µm/sar/sec (n = 7) Sarcomere Length (µm) 0 Figure 4.21 Titin F146 labeled myofibril tracking at three different speeds relative to the Z-line (bottom three traces, left vertical axis) and M-line (top three traces, right vertical axis) ± SEM. 70

85 µm/sar/sec (n = 8) Stress (nn/µm 2 ) µm/sar/sec (n = 23) 0.2 µm/sar/sec (n = 6) * * 100 F Sarcomere Length (µm) Figure 4.22 Average stress produced by F146 labeled myofibrils at three different speeds ± SEM (* p < 0.05; ; p < 0.001). Speed of stretch was significant for stress between groups at a length of 1.9 µm but the post-hoc test was not able to establish which groups were different (p = between 0.1 µm/sar/sec and 0.2 µm/sar/sec). At 3.1 µm, the significant differences were between 0.1 µm/sar/sec and 0.05 µm/sar/sec. Beyond 3.1 µm, the differences were between 0.1 µm/sar/sec and the other two speeds. Stretch then activate experiments were also done at different speeds of stretch to see if the pattern was different when stretch began passively, and was activated in line with dynamic stretch. These data are presented in Figure 4.23 and Figure 4.24, respectively. 71

86 Z-Line to Epitope Distance (µm) F µm/sar/sec (n = 4) 0.1 µm/sar/sec (n = 5) 0.2 µm/sar/sec (n = 4) Sarcomere Length (µm) M-Line to Epitope Distance (µm) Figure 4.23 Titin F146 labeled myofibril tracking at three different speeds relative to the Z-line (bottom three traces, left vertical axis) and M-line (top three traces, right vertical axis) ± SEM. These began as a passive stretch and were activated shortly thereafter. Activation time was adjusted for speed to ensure it occurred at approximately the same overall sarcomere length but varied to a degree from one experiment to the next. Only the active part of the experiment was plotted, and once again revealed no significant differences for label tracking at different speeds. Note that no plateauing behaviour was exhibited in this subset of experiments, which differed from the case where activation was done before stretch onset (Figure 4.21). Additionally, the label tracking did not reflect consistent F146 entry into the space associated with the A-band, as transpired in the activate then stretch label tracking (Figure 4.21). 72

87 µm/sar/sec (n = 4) µm/sar/sec (n = 5) Stress (nn/µm 2 ) µm/sar/sec (n = 4) ** * F Sarcomere Length (µm) Figure 4.24 Average stress produced by F146 labeled myofibrils at three different speeds ± SEM. Only the active results are plotted for the stretch then activate experiments (* p < 0.05). Significant differences were observed at five sarcomere length stress bins, always between the 0.2 µm/sar/sec and 0.05 µm/sar/sec speeds Individual Sarcomere Turning Points The "activate then stretch and stretch then activate experiments proved not to be speed dependent for most stress bins sampled, and not significant for any of the label tracking at different speeds. As such, sarcomeres were arranged in bins based on when activation occurred, irrespective of the speed of stretch. The activate then stretch experiments followed the F146 active experiments closely, which were done at 0.1 µm/sar/sec (Figure 4.13 and Figure 4.14). 73

88 A) B) Z-Line to F146 Distance at Plateau (µm) F (n = 38) (n = 32) (n = 14) (n = 3) 2.5 (n = 1) Sarcomere Length at Plateau (µm) Figure 4.25 A) Location of turning point plateaus ± SEM for binned F146 labeled sarcomeres relative to the Z-line for the activate then stretch dataset after pooling the velocity data. B) Turning points relative to the M-line. Dashed line represents the A-band edge where the myosin thick filament terminates. A) B) Z-Line to F146 Distance at Plateau (µm) (n = 26) (n = 25) (n = 8) (n = 14) (n = 3) 3.0 (n = 1) F Sarcomere Length at Plateau (µm) Figure 4.26 A) Location of turning point plateaus ± SEM for binned F146 labeled sarcomeres relative to the Z-line from the stretch then activate dataset after pooling the velocity data. B) Turning points relative to the M-line. Dashed line represents the A-band edge where the myosin thick filament terminates. 74

89 At the individual sarcomere level, the pattern for the activate then stretch experiments (Figure 4.25) is maintained when compared to the larger dataset presented above (Figure 4.13 and Figure 4.14). Once again, plateau behaviour coincided with the termination of the A-band in 87 of 88 sarcomeres from this subset of experiments. Correspondingly, the distance between the F146 antibody and the Z-line varied, dependent on the amount of shortening that occurred prior to active stretch. The stretch then activate experiments appeared qualitatively different than the aforementioned subset, indicating some sarcomeres cluster around the 650 nm distance from the Z-line at close to 3.5 µm, but others display more elongation at longer sarcomere lengths. The distance from the M-line appeared similar to that seen with purely passive stretch (inset, Figure 4.14), but different than when activation preceded stretch (Figure 4.25). 4.6 Discussion Stress Data Antibody introduction into the lattice spacing of sarcomeres at resting length likely resulted in crosslinking among the filaments as supported by the qualitatively different shape of the stress data of all labeled myofibrils contrasted with the unlabeled. This could occur between the greater than one thousand parallel titins interacting in each half sarcomere (H. Li et al., 2002; W. A. Linke et al., 1998), or between titin and other parallel filaments. Experiments directed at exploring this further revealed that primary antibody labeled myofibrils were indistinguishable from the unlabeled myofibrils both in the stress produced and the lack of fluorescence (data not shown), which is in agreement with previous observations (W. A. Linke et al., 1996). It was not until the introduction of the secondary labels that stress was affected, but any decrease in the 75

90 concentration of the secondary antibody compromised the fluorescence signal quality. This labeling artifact contrasted with work done by Telley et al., (2006) who found no change in mechanical properties associated with labeling α-actinin and myomesin in rabbit psoas using a modified Alexa 488 kit, but was in line with the approximately two fold reduction in active force due to Z-line antibody treatment seen by Shimamoto et al., (2009). Although the lattice structure was affected to some degree, the primary outcome measure was the label tracking behaviour of titin segments. It has been reported that labeling the sarcomere with antibodies before passive stretch was virtually the same as labeling after passive stretch, which suggests that antibodies do not significantly impede titin s movement during elongation despite having an effect on stiffness (W. A. Linke et al., 1996). The similarity of passive elongation curves for different titin antibodies in the immunofluorescence and more detailed electron microscopy technique in which labels are applied after stretch, also lends support to the unconstrained dynamic tracking behaviour (W. Linke et al., 1998; W. A. Linke et al., 1998). To further evaluate this crosslinking effect on the elongation pattern of antibodies in our study, experiments were conducted using inorganic quantum dots (Qdots) as a secondary fluorophore that proved not to affect the elastic properties of the myofibril (see Appendix). The fluorescent Qdots conjugated to the same F146 antibody moved similarly to the crosslinked Alexa 488 discussed throughout this chapter, suggesting the label tracking was largely unaffected by the introduction of either secondary antibodies into the lattice spacing. Additionally, a labeled myofibril stretched twice passively or actively with a ten minute break in between, showed that sarcomeres could shorten back to resting length and that labels on titin moved consistently with repeated stretch, further supporting free label movement in these experiments (see Appendix). 76

91 4.6.2 Titin-Actin As RFE is seen at the level of the sarcomere, it can be deduced that the interaction partners responsible for RFE must also be present in the sarcomere, if RFE is not purely explainable by sarcomere length non-uniformities. The most popular idea that could render titin abbreviated, and thus potentially responsible for increased force associated with RFE, is a titin-actin interaction. Various constructs have been created in vitro with varying degrees of success in establishing a physical, likely electrostatic (Forbes et al., 2005; Kimura, Maruyama, & Huang, 1984; Nagy et al., 2004) connection between titin and actin. A motility inhibition of actin on myosin ascribed to titin has been seen with recombinant cardiac titin (Kulke et al., 2001; Q. Li, Jin, & Granzier, 1995), recombinant skeletal PEVK (Nagy et al., 2004) and native skeletal titin (Kellermayer & Granzier, 1996). However, recombinant cardiac PEVK resulted in an increased motility in the presence of calcium (Kulke et al., 2001) while native titin showed a calcium dependent inhibition (Kellermayer & Granzier, 1996). Meanwhile, recombinant skeletal PEVK interaction with actin was not affected by calcium using a solid surface binding assay (Nagy et al., 2004). Contrastingly, native skeletal PEVK fragments actually increased the sliding velocity of reconstituted thin filaments on heavy meromyosin which differentiated itself entirely from the motility studies described above (Niederländer, Raynaud, Astier, & Chaussepied, 2004). A PEVK-actin interaction was observed with one methodology and not another within the same study (Gutierrez-Cruz, Van Heerden, & Wang, 2001), thus further questioning the strength and qualities of a titin-actin interaction. Probing a broader titin-actin interaction using recombinant protein fragments, Linke et al., (1997) only found binding in the Z-line region of cardiac titin. This contrasted other groups having found titin-actin interaction elsewhere with the same methodology (Jin, 1995; Q. Li et al., 1995). More contemporary thinking supports the notion that 77

92 the titin PEVK domain binding to actin may be tissue dependent with cardiac isoforms completing this more readily than skeletal ones (W. A. Linke et al., 2002; Niederländer et al., 2004; Yamasaki et al., 2001). Comparisons between skeletal and cardiac muscle PEVK regions with actin argue that only the cardiac isoform is able to bind actin at physiological ionic strengths (Yamasaki et al., 2001), although there are exceptions (W. A. Linke et al., 1997). The relevance of this possible abbreviation of titin in cardiac tissue where active stretching does not occur (Nishikawa et al., 2011), is still unclear. In one of the earliest studies on the topic, a weak interaction between titin and actin was seen by Kimura et al., (1984) that was easily disrupted by a weak mechanical force, suggesting that this interaction has little impact on contraction in myofibrils and, by extension, would not be able to withstand the forces observed during RFE experiments. Recently, the winding filament hypothesis has been proposed that incorporates a winding of titin on actin through myosin crossbridge twisting of actin (Nishikawa et al., 2011), which then cleverly explains how titin could generate such large forces associated with history dependence, but this has not been carefully tested to date. These many contradictory findings and speculative ideas draw into question what is actually happening to titin in an intact sarcomere arrangement during passive and active muscle stretch and has served as the impetus for the current work. As the PEVK region has largely been suggested as the site for titin-actin interaction (Kulke et al., 2001; Nagy et al., 2004; Niederländer et al., 2004), antibodies were used that characterized this region in greater detail. When myofibrils were activated in this study, an abbreviation to this titin spring was seen, but remarkably in the reverse pattern to what we hypothesized above (section 4.3). As no sufficient explanation could be found for these observations, a new theory was 78

93 developed in order to explain our experimental observations and offer predictions for future experiments The Titin Entanglement Hypothesis (TEH) The titin entanglement hypothesis (TEH) is predicated on the idea that spatial overlap between I- band titin and A-band myosin creates opportunities for entanglement when thick filament activation results in crossbridge extension. The myosin heads laying along the thick filament backbone under relaxing (passive) conditions could offer ample lattice space at resting sarcomere lengths for extensible titin migration into and out of the A-band area (Gutierrez-Cruz et al., 2001; Reconditi et al., 2011), which was seen in our data. With passive stretch, we observed unimpeded label movement away from the Z-line, as did Gutierrez-Cruz (2001). Importantly, this was not the case for active stretch performed in this study. We consistently observed titin s distal Ig-PEVK region becoming anchored at the A-band edge with active stretch only, likely through association with myosin crossbridges. Upon muscle activation, the opportunity for myosin activation from nearly parallel along the backbone up to about 60 to the filament axis (Reconditi et al., 2011), offers an intriguing way in which titin may become ensnared during a period of spatial overlap. This appears even more favourable when some degree of sarcomere shortening is permitted prior to the commencement of active stretch, which dually serves to increase the likelihood of such a spatial overlap between extensible I-band titin and myosin and further may increase the likelihood of entanglement due to natural crossbridge rotation through a titin filament network. It is tempting to speculate that titin was drawn into the A-band area through active crossbridge rotation as spatial overlap between I-band titin and A-band myosin was sometimes created upon shortening at sarcomere lengths where it previously was not 79

94 overlapping. However, marginal change occurred in the epitope to Z-line distance upon activation (insets: Figure 4.7; Figure 4.9; Figure 4.11) suggesting this mechanism may be more of a pushing of titin into the A-band area where subsequent anchoring ensued. The titin entanglement observed in this study would likely occur as a mechanical consequence of moving crossbridge heads through a dense titin meshwork, and not a biochemical attraction of the myosin heads for I-band titin (Q. Li et al., 1995; Murayama, Nakauchi, Kimura, & Maruyama, 1989; Nagy et al., 2004; Niederländer et al., 2004). The proportion of overlap between the A- band and distal PEVK end varied but has been seen to infiltrate by as much as 200 nm per half sarcomere upon activation, which could serve as another mechanism of force loss with shortening on the ascending limb of the force length relationship. Once a threshold specific for the contractile conditions of each sarcomere was attained, this ensnared portion of titin was released from the A-band and extension occurred predominantly between the titin epitope and the M-line. The titin-entanglement-hypothesis has several testable features that can separate it from other theories (namely titin actin interactions) proposed to explain titin s behaviour during eccentric contraction. These predictions will be discussed individually below Antibody Movement Predictions 1. If a region of titin had become entangled with myosin upon activation, then there would be little to no movement of that region relative to the M-line at distances of 0.8 µm and below, with considerable movement between that region and the Z-line with active stretch. 80

95 In the passive case, the movement of labels would approximately follow straightening and elongation dictated by the cumulative behaviour of unique segment contour and persistence lengths, since the myosin crossbridges are not activated and therefore titin presumably does not get entangled. For a titin-actin interaction to occur with active stretch, one could expect to observe little movement of segments corresponding to proximal Ig domains and perhaps part of the PEVK, while the distal Ig region would elongate dramatically (Top right and bottom right half sarcomere, Figure 4.27). As this was not seen at the myofibril (Figure Figure 4.12) nor sarcomere level (Figure Figure 4.18), it could be suggested that the binding of titin does not occur immediately with calcium introduction as thought (Nishikawa et al., 2011), but may require some straightening of titin first to open up binding sites for such an interaction (Gutierrez-Cruz et al., 2001). This too, however, does not fit well with the data collected here, when titin was straightened at long lengths (sections and below). Figure 4.27 Proposed Titin Entanglement Hypothesis (TEH). The PEVK to Z-line distance would increase greatly if some site on titin were tangled with myosin crossbridges (Bottom left half sarcomere), while the PEVK would be less than 0.8 µm from the M-line. This would not be the case if titin were bound to actin around the N2A element (green box) (Bottom right half sarcomere). Figure adapted from (Powers et al., 2014). 81