Emergence of Mechanical Oscillations from Stochastic Simulations of a Discrete Chemical Thermodynamic Model of Force Generation

This dissertation provides a thermodynamic framework, to investigate collective behaviors and emergent mechanisms of muscle contraction. It emphasizes the need for a chemical thermodynamic approach to properly characterize muscle mechanics by presenting facts and historical context to show the inadequacies of the molecular mechanics’ approach. A simple and comprehensive model is proposed to explain experimental findings that conventional models of muscle contraction are unable to explain by integrating quantifiable features backed by studies. This study extends our understanding of ensemble muscle function and addresses important issues in the field of muscle research by integrating theory, experimentation, and computer simulations

Preparation and Properties of Isolated Z-disks

Z-disks form the boundaries of the sarcomeres, the basic contractile units of muscle cells. Within the Z-line thin filaments containing mainly actin interdigitate and are crosslinked by α-actinin. Ends of the giant proteins titin and nebulin are also anchored in the Z-disk. The Z-line was originally thought to have the purely mechanical function of transmitting contractile force along the myofibrils. However, more recently, the Z-disk has emerged as a highly dynamic structure involved in stress sensing and important signaling pathways that govern muscle homeostasis. In order to fully understand how the Z-disk functions a detailed description of its molecular organization is essential. Even though the structure the structure of the Z-disk has been studied by electron microscopy techniques its molecular organization is known only in outline to a resolution of about 5 nm, whereas at least 3 nm is required to begin distinguishing protein shapes and to accurately dock crystal structure. Reports describing the isolation of intact Z-disks from insect indirect flight muscle date from 30-40 years ago, but these preparations have not been subjected to modern electron microscopy techniques. We improved the existing methods for the isolation of the Z-disk from honeybee flight muscle and investigated its structure using cryo-electron tomography and subtomogram averaging. The preliminary data indicate that the resolution was improved when compared with past studies of plastic sectioned muscle. We have also investigated the protein composition of the preparations to monitor the components that are washed away during preparation. Methods for the isolation of intact Z-disks from vertebrate muscle are not available. We explored strategies for isolating Z-disks from skeletal and cardiac muscle. Even though such a preparation has not been achieved we present promising approaches that, with optimization, should enable isolation of Z-disks from vertebrate muscle

The Effect of Muscle Length on Post-Tetanic Potentiation of skMLCK-/- and C57BL/6 Mouse EDL Muscles

Post-tetanic potentiation of force in fast skeletal muscle is inversely related to muscle or sarcomere length, diminishing at longer lengths. This relationship has been mainly attributed to the structural effects of the primary mechanism of potentiation, phosphorylation of the regulatory light chain (RLC) of myosin, which is catalyzed by skeletal myosin light chain kinase (skMLCK). The purpose of this thesis was to compare the relationship between isometric twitch force potentiation and muscle or sarcomere length in fast twitch extensor digitorum longus (EDL) muscles from wildtype and skMLCK-/- mice. It was hypothesized that in addition to reduced potentiation, skMLCK-/- muscles without the ability to phosphorylate the RLC would also display an altered length-dependence of potentiation compared to wildtype muscles with RLC phosphorylation. The main finding was that although twitch potentiation was greater in WT muscles at all lengths, the relationship between potentiation and muscle length was similar in both WT and skMLCK-/- muscles. This indicates that the length-dependence of potentiation cannot necessarily be attributed to RLC phosphorylation. Thus, additional mechanisms, possibly related to Ca2+ handling, thick filament mechanosensing and length-dependent activation may participate in the length-dependence of potentiation displayed by murine fast muscle models

Phenotypic and evolutionary variation in fish myofibrillar proteins

Chapter 1 General Introduction: The general introduction initially presents the major landmarks in muscle research of the last 3 millennia. The proteins of the contractile apparatus and their role in muscle contraction are described. There is then a description of how contractile proteins are known to alter through the expression of isoforms. Finally, a description of fish muscle and its fibre types is given, followed by the aims of the thesis. Chapter 2 Materials and Methods: An account is given of the materials and methods used throughout this thesis. This includes myofibril preparation and electrophoretic techniques. The techniques described are one and two dimensional polyacrylamide gel electrophoresis (PAGE), iso-electric focusing (lEF), peptide mapping, and one and two dimensional alkali-urea PAGE. The methods used to fix, stain and store the gels are then given, followed by the protocol used for Western blotting. Finally, the analysis of proteins bands is described. Chapter 3 Temperature and the plasticity of myofibrillar proteins during ontogeny in the Atlantic herring (Clupea harengus L.): Many aspects of development are influenced by temperature. The aim of this study was to investigate the effect of rearing temperature on the development and myofibrillar protein expression during ontogeny in the Atlantic herring (Clupea harengus L.). Chapter 4 The effect of body size on the myofibrillar protein composition of fast muscle fibres in the short horn sculpin (Myoxocephalus scorpius L.): The contractile properties of muscle vary with body length in fish. The aim of this study was determine if the proteins altered with body size in the short-horn sculpin (Myoxocephalus scorpius L.). Furthermore, could changes in protein expression be related to differences in contractile properties. Chapter 5 The myofibrillar proteins of Antarctic and sub-Antarctic fish: The myosins of Antarctic fish are specialised for function at low temperature. The aim of this study was to determine if the myofibrillar proteins present in Antarctic fish were highly conserved for function in this stable, low temperature environment. The variation in protein structure from myotomal fast muscles and the m. adductor profundis between five Antarctic fish species from two genera was compared with five sub-Antarctic species from four genera. The myofibrillar proteins of both the Antarctic and sub-Antarctic species showed a high degree of similarity between fish within the same genera. However, the isoforms present were considerably different between genera in both the Antarctic and sub-Antarctic species. Furthermore, the extent of the variation in protein isoforms between genera of the Antarctic fish was similar to that of the sub-Antarctic fish. This suggests that divergence in the tertiary structure of myosins from these species has occurred and that the Antarctic fish myofibrillar proteins are not highly conserved. Chapter 6 General Discussion: The major findings of the thesis are discussed in relation to the expression of myofibrillar proteins, with reference to further studies. (Abstract shortened

Role of the Drosophila Melanogaster Indirect Flight Muscles in Flight and Male Courtship Song: Studies on Flightin and Mydson Light Chain - 2

Complex behaviors using wings have facilitated the insect evolutionary success and diversification. The Drosophila indirect flight muscles (IFM) have evolved a highly ordered myofilament lattice structure and uses oscillatory contractions by pronounced stretch activation mechanism to drive the wings for high powered flight subject to natural selection. Moreover, the IFM is also utilized during small amplitude wing vibrations for species-specific male courtship song (sine and pulse), an important Drosophila mating behavior subject to sexual selection. Unlike flight, the contractile mechanism and contribution of any muscle gene in courtship song is not known. To gain insight into how separate selection regimes are manifested at the molecular level, we investigated the effect on flight and mating behaviors of mutations in two contractile proteins essential for IFM functions: an IFM-specific protein, flightin (FLN), known to be essential for structural and mechanical integrity of the IFM, and a ubiquitous muscle protein, myosin regulatory light chain (MLC2), known to enhance IFM stretch activation. Comparison of FLN sequences across Drosophila spp., reveal a dual nature with the N-terminal region (63 aa) evolving faster (dN/dS=0.4) than the rest of the protein (dN/dS=0.08). A deletion of the N-terminal region (fln�N62) resulted in reduced IFM fiber stiffness, oscillatory work and power output leading to a decreased flight ability (flight score: 2.8±0.1 vs 4.2±0.4 for fln+ rescued control) despite a normal wing beat frequency. Interestingly, the FLN N-terminal deletion reduced myofilament lattice spacing and order suggesting that this region is required to improve IFM lattice for enhancing power output and flight performance. Moreover, fln�N62 males sing the pulse song abnormally with a longer interpulse interval (IPI, 56±2.5 vs 37±0.7 ms for fln+) and a reduced pulse duty cycle (PDC, 2.6±0.2 vs 7.3±0.2 % for fln+) resulting in a 92% reduction in their courtship success. This suggested that FLN N-terminal region fine-tunes sexually selected song parameters in D. melanogaster, possibly explaining its hypervariability under positive selection. That FLN N-terminal region is not essential but required to optimize IFM functions of both flight and song, indicate that FLN could be an evolutionary innovation for IFM-driven behaviors, possibly through its role in lattice improvement. Mutations of the highly conserved MLC2 [N-terminal 46 aa deletion (Ext), disruption of myosin light chain kinase phosphorylations (Phos), and the two mutations put together (Dual)] are known to impair or abolish flight through severe reductions in acto-myosin contractile kinetics and magnitude of the stretch activation response. Unlike FLN, these MLC2 mutations do not show a pleitropic effect on flight and song. Flight abolished Phos and Dual mutants are capable of singing suggesting that these mutations affect song minimally compared to flight. Moreover, unlike FLN, none of these mutations affect interpulse interval, the most critical sexually selected song parameter in Drosophila. Also, in contrary to the known additive effects of Ext and Phos in the Dual mutant on flight wing beat frequency, a subtractive effect on sine song frequency is found in this study. That mutations in MLC2 are manifested differently for song and flight suggest that stretch activation plays a minimal or no role in song production. The results in this study suggest that the conserved regions of FLN and MLC2 are essential to support underlying IFM contractile structure and function necessary for flight, whereas the fast evolving FLN N-terminal region optimizes IFM\u27s biological performance in flight and species-specific song possibly under positive selection regime