This project focused on extracellular signal-regulated kinase (ERK) and focal adhesion proteins related to ERK activity, and found a novel signaling pathway contributing to aging-related defects in smooth muscle contractility. Previous members of our lab have used ERK inhibitors to demonstrate the role of ERK in smooth muscle contraction. Dr. Nicholson used the ERK inhibitor FR 18024 and noted that, in the presence of this inhibitor phenylephrine (PE) induced a higher stress increase in young mouse aortas compared to old aortas. Inhibition of the kinase ERK abolished this difference. He also quantitated ERK phosphorylation, a marker of ERK activation in PE-stimulated aortas from both young and aged mice and found a significant lower level of phosphorylated-ERK (p-ERK) in aged mouse aortas. I was interested in determining the substrate of ERK that is affected in aging. Caldesmon (CaD) is one of the known substrates of ERK in smooth muscle. More importantly, CaD, as an actin-binding protein, inhibits cross-bridge formation by blocking the interaction between actin and myosin. Thus, I tested the hypothesis that, caldesmon phosphorylation is inhibited in aged mouse aortas. To determine the mechanism by which regulation of ERK activation changes with age, the role of micro-RNAs (miRs) in the regulation ERK phosphorylation was investigated. Transfection of miR-137 and -34a into A7r5 cells resulted in a significantly lower level of p-ERK in response to the phorbol ester DPBA. Further, together with my collaborators I found that transfection of miR-137 and -34a led to significantly decreased focal adhesion protein levels in A7r5 smooth muscle cells, such as paxillin and src. To confirm whether focal adhesion proteins contribute to the impairment of agonist-induced ERK phosphorylation, paxillin siRNA and src inhibitor were used. The results showed that paxillin is required for the phosphorylation of ERK1 and ERK2 and src is required for ERK2 phosphorylation. In conclusion, age-related increases in miR-137 and -34a decrease ERK phosphorylation via downregulation of paxillin and src. The decrease in ERK phosphorylation leads to a decrease in CaD phosphorylation and inhibits contraction. Thus, the thin filament-coupled pathway in differentiated vascular smooth muscle is inhibited in the aged mouse aorta and this leads to aging-associated defects in smooth muscle contractility. / 2021-06-18T00:00:00Z

BACKGROUND:Nemaline myopathy (NM) is a congenital muscle disease associated with weakness and the presence of nemaline bodies (rods) in muscle fibers. Mutations in seven genes have been associated with NM, but the most commonly mutated gene is nebulin (NEB), which is thought to account for roughly 50% of cases.RESULTS:We describe two siblings with severe NM, arthrogryposis and neonatal death caused by two novel NEB mutations: a point mutation in intron 13 and a frameshift mutation in exon 81. Levels of detectable nebulin protein were significantly lower than those in normal control muscle biopsies or those from patients with less severe NM due to deletion of NEB exon 55. Mechanical studies of skinned myofibers revealed marked impairment of force development, with an increase in tension cost.CONCLUSIONS:Our findings demonstrate that the mechanical phenotype of severe NM is the consequence of mutations that severely reduce nebulin protein levels and suggest that the level of nebulin expression may correlate with the severity of disease.

As the genetic causes of Hypertrophic Cardiomyopathy (HCM) have become widely recognized, considerable lag in the development of targeted therapeutics has limited interventions to symptom palliation. This is in part due to an oft-noted finding that similar point mutations within myofilament proteins are known to cause differential disease severity, highlighting the need to understand disease progression at the molecular level. One commonly described pathway in HCM progression is calcium homeostasis dysregulation, albeit little is understood about disruption of the pathway. This dissertation investigated the calcium homeostasis of two clinically relevant murine models of HCM expressing similar point mutations within myofilament proteins. A mutation-specific alteration in the calcium dissociation rate from the cardiac myofilament is proposed to as a primary mechanism of down-stream calcium disruption. Two modes of intervention in down-stream calcium homeostasis were tested to as a means of improving directed therapies in HCM progression. The clinically-utilized diltiazem hydrochloride, an L-type calcium channel blocker, revealed mutation-specific symptom palliation but an inability to target within the disease mechanism itself. Due to this insufficient response to diltiazem, we investigated the role of the calcium-dependent kinase, CaMKII, and its persistent (autonomous) activation resulting from calcium dysregulation. Partial inhibition of the autonomous activation of the kinase was shown to improve functional and morphological indices of failure in calcium-dependent HCM progression. Thus, we conclude a myofilament-linked derangement in calcium homeostasis that potentiates aberrant activation of CaMKII. Moreover, we position the kinase as a nodal point in disease progression and a potential therapeutic target for early, robust management of HCM in the clinical population.

Increases in seawater temperatures have imposed physiological constraints which are partially thought to contribute to recently observed shifts in biogeographic distribution among closely related intertidal ectotherms. For instance, Mytilus galloprovincialis an introduced warm-adapted species from the Mediterranean, has displaced the native cold-adapted congener, M. trossulus, over large latitudinal expanses off the California coast. Several comparative physiological studies have revealed interspecific differences in thermal tolerance, including variation in aerobic metabolism and gape behavior, which suggest the invasive congener is better adapted to acclimate to increasing seawater conditions as predicted due to climate change. However, current analyses seek to discover the cellular process which contribute to thermal plasticity at the level of the whole organism in response to temperature stress. Since proteins represent the primary molecular machinery capable of responding to thermal stress, we quantified the proteomic response of the adductor muscles (AM) of M. galloprovincialis and M. trossulus to acute heat stress. After acclimation to 13°C, we exposed mussels to 24°C, 28°C and 32 °C (at a heating rate of 6C/h), kept mussels at the temperature for 1 h and then added a 24-h recovery period. Posterior adductor muscle samples were then excised and utilized for proteomic analysis. We were able to detect 273 protein spots within M. galloprovincialis and 286 protein spots within M. trossulus. Roughly 33% of these protein spots exhibited significant changes in abundance in response to heat stress within M. trossulus as compared to only 19% in M. galloprovincialis. In both data sets, most proteins changing abundance are part of the cytoskeleton or proteins controlling actin thin filament dynamics and stress fiber formation. Specifically, M. galloprovincialis increased the abundance of proteins involved in thin filament stabilization and cytoskeletal maintenance. In contrast, M. trossulus increased proteins involved in thin filament destabilization and filament turnover. In addition, only M. trossulus increased proteins involved in the cellular stress response at the highest temperature, suggesting its AM proteome is more thermolabile. In return, our results suggest that cytoskeletal architecture is more thermally stable in M. galloprovincialis. The differences in the proteomic responses suggest that M. galloprovincialis is capable of protecting itself from heat stress through valve closure at a higher temperature due to the increase in actin stabilizing proteins.

Thin filament pyrometry is a proven technique used to measure flame temperature by capturing the spectral radiance produced by the immersion of silicon carbide filaments in a hot gas environment. In this study a commercially available CMOS color camera was used, and the spectral response of each color channel was integrated with respect to the assumed graybody radiation spectrum to form a look up table between color ratio and temperature. Interpolated filament temperatures are then corrected for radiation losses via an energy balance to determine the flame temperature. Verification of the technique was performed on the Holthuis and Associates Flat Flame Burner, formerly known as the Mckenna Burner, and the results are directly compared to literature values measured on a similar burner. The results are also supported by radiation corrected measurements taken using a type B thermocouple on the same burner setup. An error propagation analysis was performed to determine which factors contribute the most to the final measurement uncertainty and confidence intervals are calculated for the results. Uncertainty values for a single point measurement were determined to be between ±15 and ±50 K depending on the color ratio and the total uncertainty associated with day-to-day changes in the measurement setup was found to be ±55 K. / Master of Science / Determination of flame temperature is an important aspect of combustion research and is often critical to the evaluation of combustion systems as well as the integration of those systems into more complex devices. In this thesis the technique of thin filament pyrometry was implemented and verified through the use of a well characterized calibration flame. This technique involves placing thin filaments usually made from silicon carbide into the flame and capturing the spectrum of light they emit with a detector. Since the amount of light emitted as well as which wavelengths the light is concentrated in is a strong function of temperature, this methodology may be used to calculate the temperature of the flame. Thin filament pyrometry has the advantage compared to other techniques in that it is extremely cheap to implement and requires no advanced scientific equipment. The SiC filaments have been shown to have a very high resistance to the flame environment and do not face many of the same challenges that can cause problems for other techniques. A statistical analysis of the method implemented in this work was also performed and the expected uncertainty was similar to many of the alternative techniques which necessitate a more complex or expensive setup.

Hypertrophic cardiomyopathy (HCM) is a primary disease of the myocardium. 4-11% of HCM is caused by mutations in cardiac troponin T (cTnT) and 65% of them are within the tropomyosin (TM)-binding TNT1 domain. Two of the known mutational hotspots within TNT1 are in the N and C-terminal domains. Unlike the N-terminal domain; no high-resolution structure exists for the highly conserved C-terminal domain limiting both our ability to understand the functional role of this extended domain in myofilament activation and molecular mechanism(s) of HCM. The Δ160E mutation is an in-frame deletion of a glutamic acid residue at position 160 of cTnT. This TNT1 C-terminal mutation is associated with an especially poor prognosis. The Δ160E mutation is located in a putative "hinge region" immediately adjacent to the unstructured flexible linker connecting the TM-binding TNT1 domain to the Ca²⁺-sensitive TNT2 domain. Unwinding of this α-helical hinge may provide the flexibility necessary for thin filament function. Previous regulated in vitro motility assay (R-IVM) data showed mutation-induced impairment of weak actomyosin binding. Thus, we hypothesized that the Δ160E mutation repositions the flexible linker which impairs weak electrostatic binding and ultimately leads to severe cardiac remodeling. The goal of our studies is two-fold: 1) to gain high-resolution insight into the position of the cTnT linker with respect to the C-terminus of TM, and 2) to identify Δ160E-induced positional changes using Fluorescence Resonance Energy Transfer (FRET) in a fully reconstituted thin filament. To this end, residues in the middle and distal regions of the cTnT linker were sequentially cysteine-substituted (A168C, A177C, A192C and S198C) and labeled with the energy donor IAEDANS. The energy acceptor, DABMI was attached to cysteine 190 (C190) in the C-terminal region of TM and FRET measurements were obtained in the presence and absence of Ca²⁺ and myosin subfragment 1 (S1). An all-atom thin filament model in the Ca²⁺–on state was employed to predict the pathogenic effects of the Δ160E mutation on the structure and the dynamics of the cTnT linker region. Our data suggest that the linker domain runs alongside the C-terminus of TM and is differentially repositioned by calcium, myosin and the Δ160E mutation. The Δ160E mutation moves the linker closer to the C-terminus of TM. The in silico model supported this finding and demonstrated a mutation-induced decrease in linker flexibility. Moreover, the model predicted a pathogenic change in the orientation of the middle region of the linker and in the position of the Ca²⁺-sensitive TNT2 domain and the TM-binding TNT1 domain in response to Δ160E mutation. Collectively, our findings suggest that the Δ160E mutation-induced changes in the structure, position and dynamics of the linker region cause steric blocking of weak myosin binding sites on actin and subsequent impairment of contraction and disruption of sarcomeric integrity. These studies, for the first time, provided information regarding the role of the extended linker in both myofilament activation and disease.

Tropomyosins(Tm) are a group of proteins that regulate the actin filaments in both muscle and non-muscle cells. In mammalian cells four Tm species are found: α-Tm (fast) encoded by α-Tm /TPM1 gene, β-Tm, encoded by β-Tm/ TPM2 gene, α-Tm (slow) encoded by γTm gene/ TPM3 and δ-Tm encoded by δTm / TPM4gene. Mutations in Tm are linked to many cardiac and skeletal diseases like hypertrophic cardiac myopathy (TPM1 and TPM2), familial cardiac myopathy (TPM1) and skeletal diseases like nemaline myopathy (TPM2 and TPM3) along with other sarcomere proteins. The hypothesis on which this study is based is, the isoform composition in both muscle and non-muscle cells adapts in response to disease and physiological changes. A significant part of that adaptation is changes in the thin filament protein isoforms expressed and the post translational modifications of these proteins. In this study Tpm3.12st isoform of γTm and other striated muscle tropomyosin isoforms (Tpm1 and Tpm2) and a non-muscle Tmp4 were characterised using a variety of techniques. The aim was to enhance our understanding of the role of tropomyosin interactions in regards to its efficiency of actin binding capacity as well as its effect on actin polymerisation. Human tropomyosin 3 (Tpm3.12st) was expressed in E. coli to produce recombinant protein with three N-terminal sequence variants (Met, MM and (M)ASM). The proteins were characterised for their binding affinity with actin as this isoform has not been well characterised so far. Its properties are compared with other striated muscle tropomyosin Tpm1.1st and Tpm2.2st and non-muscle Tpm4.1cy. The proteins were purified through ion exchange chromatography and the purity was checked by using SDS-PAGE and UV spectrometry. The molecular weights of the recombinant proteins produced were confirmed by mass spectrometry. Cosedimentation assays were performed for their actin binding affinity using ultracentrifugation. The variant of Tpm3.12st with AS N-terminal extension was found to have similar actin affinity to Tpm1.1st in the range of 0.1-0.8 μM (half saturation). However the variants with Met and MM N-termini bound to actin weakly with high half saturation concentration of ~ 6 μM and ~8 μM tropomyosin respectively. Measurement of actin polymerisation kinetics showed it is affected in presence of tropomyosin. From this study it is shown that tropomyosin accelerates the initiation step in actin polymerisation with varying differences within the isoforms in contrast to several previous studies. There have been very few studies of the effect of tropomyosin on actin polymerisation in the last two decades. This work shows that tropomyosin isoforms have a large and variable role in controlling actin polymerisation and understanding tropomyosin function will need further investigation in this area. This study also developed an ELISA screening method using monoclonal antibodies for identification and quantification of Tpm3.12st which was tested against all the four tropomyosin isoforms. None of the twelve antibodies studied showed reactivity only with Tpm3.12st. From the data analysed it is deduced the amino acid residues in the region of 24-43 shows the prospect of designing a monoclonal antibody specific to Tpm3.12st isoform. Accurate quantification of tropomyosin isoforms is key to understanding their function and the effects of modulation of isoform composition in health and disease. A reverse phase liquid chromatography method was developed which is compatible with the analysis of the thin filament proteome using top-down mass spectrometry. Reverse phase liquid chromatography (RPLC) is one of the most popular methods used in mass spectrometry analysis where proteins are separated based on their hydrophobicity. The RPLC method developed in this study gives an efficient separation of major thin filament proteins along with small soluble proteins that is compatible to use for top down mass spectrometry for identification and quantification of proteins, PTMs and isoform composition. With a minimum amount of 2 mg of tissue using chicken and mouse heart and skeletal muscle samples a buffer system was optimized to extract thin filament proteins. With the optimized RPLC method actin, tropomyosin and troponin complex subunits (TnC, TnI and TnI) were successfully separated and the proteins were identified using SDS-page by comparison with the previous research results. This novel method of extraction and the optimised RPLC method will provide a “bird’s eye view” of thin filament proteome providing information of PTMs of all the proteins together within one single extraction, reducing the time for analysis and the sample size. This has the potential to give insight into tissue, muscle and heart adaptations that could act as a prognostic indicator.

Having an in vivo model of development that develops quickly and efficiently is important for investigators to elucidate the critical steps, components and signaling pathways involved in building a myofibril; hence a compliant in vivo model would provide a pivotal foundation for deciphering muscle disease mechanisms as well as the development of myopathy-related therapeutics. Here, we take advantage of a relatively quick, cost effective, and molecularly pliable developmental model system in the Xenopus laevis (frog) embryo and establish it as an in vivo model to study the roles of sarcomeric proteins during de novo myofibrillogenesis.Using the Xenopus model, we elucidated the functional roles of Leiomodin3 (Lmod3) and Tropomodulin 4 (Tmod4) during de novo skeletal myofibrillogenesis. Tmods have been demonstrated to contribute to thin filament length uniformity by regulating both elongation and depolymerization of actin-thin filaments' pointed-ends. Lmods, which are structurally related to Tmod proteins also localize to actin filament pointed-ends. In situ hybridization studies demonstrated that of their respective families, only tmod4 and lmod3 transcripts were expressed at high levels in skeletal muscle from the earliest stages of development. When reducing their protein levels via morpholino (MO) treatment, thin filament regulation and sarcomere assembly were compromised. Surprisingly, alternate rescues (i.e., lmod3 mRNA co-injected with Tmod4 MO and vice versa) partially restored myofibril structure and actin-thin filament organization. Thus, our results not only indicate that both Tmod4 and Lmod3 are critical for myofibrillogenesis during Xenopus skeletal muscle development, but also revealed that they may share redundant functions during skeletal muscle thin filament assembly.

The cyclical interaction between the force-generating protein myosin and actin is the mechanism responsible for muscle contraction among all muscle types. Cardiac muscle contraction is tightly controlled to ensure that blood pumps effectively and efficiently from the heart to peripheral organs. Mutations in various cardiac proteins can lead to cardiac dysfunction and a number of cardiomyopathies. The first part of this dissertation studies two disease-linked mutations in the regulatory light chain of the cardiac myosin molecule, D166V and K104E, and assesses the kinetic and mechanochemical effects of the mutations via the in vitro motility assay. The data show that D166V mutant myosin force generation is reduced compared to wild type, and exogenous phosphorylation of the mutant light chain rescues force generation. In contrast, the K104E mutation showed no deficit in force production but exhibited increased calcium sensitivity of activation. These results are consistent with contractile defects associated with cardiomyopathies caused by various mutation-induced changes to protein function and mechanism of interaction. The second part uncovers the actin-binding mechanism of one of the chief muscle regulatory proteins tropomyosin. In cardiac and skeletal muscle, tropomyosin and troponin modulate muscle contraction. Tropomyosin binds along the length of actin filaments and blocks myosin-binding sites. Following an excitatory stimulus, calcium binds troponin and causes tropomyosin to shift its position on actin, allowing myosin to bind. The precise mechanism of how tropomyosin monomers with low actin affinity bind to form a stably bound, high affinity chain is unknown. By directly observing fluorescently labeled tropomyosin binding to actin filaments, it was shown that tropomyosin molecules bind randomly along the actin filament. Subsequent monomer binding, and formation of tropomyosin end-to-end bonds, increases the probability of sustained chain growth by decreasing the probability of detachment prior to additional monomer binding. Tropomyosin molecules added to the growing chain at approximately 100 monomers/(μM*s). Different tropomyosin isoforms segregate to distinct functional and structural regions of cells. The last chapter presents data that show spatial segregation of two different tropomyosin isoforms on actin filaments. This suggests that tropomyosin sorting in cells is, at least partly, an intrinsic property of the binding mechanism.

The correct function of cardiac sarcomeric proteins allow for people to maintain quality of life. However, mutations of the cardiac sarcomeric proteins can result in remodeling of the heart which typically results in death. I present a full atomistic cardiac thin filament model that I have developed and three studies that I conducted while at the University of Arizona, while pursuing my doctoral degree in chemistry The goal was to develop the model to be able to study the effects of the mutations on the thin filament proteins. First, I present the long process of developing the model that is still evolving as new information is available. Second, I present the study of two mutants, the troponin T R92L mutant and the tropomyosin D230N mutant. Molecular dynamics was used to simulate the wild–type and mutant versions of the model which resulted in a visualization of the change of interaction between the tropomyosin and troponin, specifically at the overlap region. Third, I present the study of calcium release which is the "gatekeeper" to cardiac contraction. Steered molecular dynamics was utilized to find a previously unseen molecular mechanism that alters the rate of calcium release depending on the mutant. Fourth, I present the study of the mechanism of the tropomyosin transition across the actin filament, in which a longitudinal transition is favored. The studies helped to provide an atomistic level understanding of the cardiac thin filament as well as the methodology to which the mutations disrupt the natural functions of the sarcomeric proteins. The new results of the research can provide new insight into how the effects of the disease causing mutations can be mitigated, potentially extending the life of people with the conditions.