Doctor of Philosophy, The Ohio State University, 2022, Biomedical Sciences

Muscle function consists of two critical components. The first is the level of force that is developed, and the underlying mechanisms, are well studied and well, albeit not completely, understood. The second component is kinetics, i.e., the speed at which force is developed and dissipates, which is less extensively studied, and less well understood. This work takes a multi-faceted approach in studying contractile kinetics in striated and smooth muscle in health and disease. In striated muscle, muscles function at sub-maximal levels in vivo, whereas maximal tetanic contractions are most commonly used to assess and report skeletal muscle function in muscular dystrophy studies. At submaximal activation, both the force and kinetics of contraction and relaxation are heavily impacted by the kinetics of the single twitch. To investigate the effect of muscle disease on twitch contraction kinetics, isolated diaphragm, and extensor digitorum longus (EDL) muscles of 10-, 20-week, "het" (dystrophin deficient and utrophin haplo-insufficient), and 52-week mdx (dystrophin deficient) mice were analyzed and compared to wild-type controls. We observed that twitch contractile kinetics are dependent on muscle type, age, and disease state. Differences in kinetics yielded greater statistical significance compared to previously published maximal tetanic force measurements, thus, using kinetics as an outcome parameter could potentially allow for use of smaller experimental groups in future study designs. We applied this knowledge to investigate if kinetics can be regulated via troponin-C (TnC) Ca2+ sensitivity. To investigate this key question, we measured the EDL and soleus force-frequency response (FFR) in a murine model with a TnC mutation (expressed after TnCL48QAAV or TnCD73NAAV injection) that causes an increased calcium sensitivity (L48Q) or a decreased calcium sensitivity (D73N) and compared the FFR to the wildtype counterparts. The kinetics of the twitch contraction are im (open full item for complete abstract)

Committee: Paul Janssen (Advisor); Philip Binkley (Committee Member); Mark Ziolo (Committee Member); Jill Rafael-Fortney (Committee Member); Douglas Kniss (Committee Member) Subjects: Physiology

Doctor of Philosophy, The Ohio State University, 2017, Molecular, Cellular and Developmental Biology

The basic unit of contractile machinery in cardiac and skeletal muscle is the sarcomere. This array of proteins contains thick filaments made up of myosin motors that bind actin in thin filaments to generate tension. In all types of striated muscle calcium binds the C subunit of troponin on the thin filaments. When bound by calcium, troponin C initiates a series of events to remove inhibition of myosin binding sites on actin. In myopathies mutations in sarcomeric, calcium handling, or structural lead to malfunction of the muscle and disease. The gap in our understanding lies in how mutations in such proteins affect the fundamental function of muscle, and how changes in basic muscle function lead to disease. Therefore, it is important to develop new methods to distinguish between benign and pathologic mutations and their respective effects on muscle function. Many experimental paradigms are used to study how myopathy-causing mutations affect muscle. Simpler systems (like isolated myofilaments) have fewer variables but are missing the regulatory processes present in intact muscle. Mouse models maintain regulatory processes but do not always recapitulate human disease phenotypes. Zebrafish are excellent models for studying muscle physiology and function for reasons including: a manipulable genome, a large ratio of muscle to body size, and amenabilities to chemical treatment and live imaging. In addition, their small size permits stimulation of the entire trunk musculature simultaneously. I improved techniques by developing an assay to directly measure muscle strength. Many previous methods analyzed images of swimming larvae. Using anesthetized fish, I bypassed motor input and electrically stimulated trunk musculature. I developed a companion assay to measure cross-sectional area (CSA) of muscle. I characterized contractile strength in developing wild-type muscle and distinguished between muscle phenotypes in morphant zebrafish. We characterized m (open full item for complete abstract)

Committee: Paul Janssen (Advisor); Sharon Amacher (Committee Member); Christine Beattie (Committee Member); Jon Davis (Committee Member) Subjects: Biomedical Research; Biophysics; Cellular Biology; Molecular Biology; Physiology

Doctor of Philosophy, The Ohio State University, 2016, Biophysics

Troponin C (TnC) is a calcium-sensing switch that regulates contraction and relaxation in skeletal and cardiac muscle. Mutations in troponin (TnC, troponin I (TnI), troponin T (TnT)) that alter the apparent Ca2+ binding properties of TnC have been implicated in several cases of cardiomyopathy. Further studies have focused on TnC as a target for pharmacological intervention, and a recently engineered Ca2+-sensitized TnC variant has been shown to enhance contractility in mice with myocardial infarction. Previous studies have shown that the Ca2+ binding properties of TnC depend not only on troponin interactions but also upon interactions of many other myofilament proteins. Furthermore, many of these proteins are targets for phosphorylation and other post-translational modifications that alter the apparent Ca2+ binding properties of TnC. In this regard, TnC is not merely a simple switch, but a central hub receiving input from several other proteins. Studies have shown that while isolated TnC has a low Ca2+ binding affinity, the Tn complex has a high Ca2+ binding affinity. Thin filaments containing the Tn complex and actin/tropomyosin have an intermediate affinity which is restored to high affinity similar to that of the Tn complex by the addition of S1 myosin. One of the major questions we sought to answer was what could account for the differences in Ca2+ affinity between these different states of TnC. Furthermore, studies demonstrated that several TnI and TnT modifications altered the Ca2+ properties of thin filaments but did not change the Tn complex properties. Based on previous studies, we know that TnC-TnI interactions play a prominent role in modulating TnC's Ca2+ sensitivity via stabilization of TnC's Ca2+ binding. We hypothesize that since TnC and TnI are proximally confined on the Tn complex, they are able sense one another more than if they were separated in solution (i.e. they experience a high effective concentration of each other). With reg (open full item for complete abstract)

Committee: Jonathan Davis PhD (Advisor); Brandon Biesiadecki PhD (Committee Member); Mark Ziolo PhD (Committee Member); Thomas Hund PhD (Committee Member) Subjects: Biophysics; Physiology

Doctor of Philosophy, The Ohio State University, 2016, Biomedical Sciences

Heart failure is a leading cause of morbidity and mortality in the United States and worldwide. Failing hearts are characterized by the inability to pump sufficient blood to meet the nutrient demands of the body, in which cardiac contraction and relaxation is often compromised. Proper contraction and relaxation of the heart are dependent on numerous dynamic properties including the myofilament response to calcium. Thus, one strategy to modify the contractile dynamics of the heart is to manipulate the calcium binding properties of the myofilament. The heart contains a natural mechanism to modulate the myofilament response to calcium through sarcomeric protein phosphorylation. However, even with a substantial body of literature having investigated the functional effects of different phosphorylations in isolation, the intricate interplay between a number of phosphorylations together to affect the myofilament response to calcium remains poorly understood. At the level of the myofilament, the troponin complex is a critical molecular switch that transduces the calcium activating signal into contraction. Troponin I (TnI), the inhibitory subunit of the complex, is phosphorylated as a key mechanism to alter the calcium regulation of contraction. The canonical phosphorylation of TnI at serine-23/24 (Ser-23/24) is a significant modulator of this regulation, and may be fine-tuned by other TnI phosphorylation events. A novel TnI phosphorylation at tyrosine 26 (Tyr-26) was identified in the human heart and shown to decrease in heart failure. We first sought to investigate the functional properties elicited by TnI Tyr-26 phosphorylation on thin filament regulation as well as its functional integration with TnI Ser-23/24 phosphorylation. Our results demonstrate TnI Tyr-26 with actual phosphate and phosphomimetic substitution decreases thin filament calcium sensitivity and calcium sensitive force development. TnI Tyr-26 phosphomimetics also accelerate calcium dissociation fro (open full item for complete abstract)

Committee: Brandon Biesiadecki (Advisor) Subjects: Biomedical Research

Doctor of Philosophy, The Ohio State University, 2014, Integrated Biomedical Science Graduate Program

Heart disease, with an anticipated $316 billion in economic expenses, affects one in three adults and is the leading cause of death in the United States. In the diseased heart a multitude of cellular changes occur, either as a compensatory mechanism to deter the modifications brought on as a result of cardiac disease or a direct result of the pathophysiology. One such avenue for uncovering the molecular mechanisms underlying cardiac disease and their functional changes is to study post-translational modification (PTM) of proteins. While extensive work has been done characterizing phosphorylation of cardiac contractile regulatory proteins, this work has been conducted investigating the modifications in isolation. Despite the fact that a single phosphorylation site may be sufficient to alter function, the additive functional effect of multiple phosphorylation sites to crosstalk inter-molecularly and change function differently than that evoked by a single phosphorylation must be taken into consideration. Initially, we sought to determine if the metabolic regulatory kinase AMPK phosphorylates cardiac troponin I (cTnI) at Ser-150 in vivo to alter cardiac contractile function directly at the level of the myofilament. Rabbit cardiac myofibrils separated by two-dimensional isoelectric focusing subjected to a Western blot with a cTnI phosphorylation-specific antibody demonstrate that cTnI is endogenously phosphorylated at Ser-150 in the heart. Treatment of myofibrils with the AMPK holoenzyme increased cTnI Ser-150 phosphorylation within the constraints of the muscle lattice. Compared with controls, cardiac fiber bundles exchanged with troponin containing cTnI pseudo-phosphorylated at Ser-150 demonstrate increased sensitivity of calcium-dependent force development, blunting of both PKA-dependent calcium desensitization, and PKA-dependent increases in length dependent activation. We next wanted to investigate the effect of ischemic pH on Ser-150 and Ser-23/24 phosphor (open full item for complete abstract)

Committee: Brandon Biesiadecki PhD (Advisor); Jonathan Davis PhD (Committee Member); Michael Freitas PhD (Committee Member); Mark Ziolo PhD (Committee Member) Subjects: Biochemistry; Biomedical Research; Biophysics

Doctor of Philosophy, The Ohio State University, 2013, Integrated Biomedical Science Graduate Program

Vascular smooth muscle alpha-actin (SMA) is an indicator of myofibroblast differentiation, as well as one of several fetal contractile protein isoforms re-expressed in adult cardiomyocytes in response to mechanical stress-injury. The stress-response protein, Y-box binding protein-1 (YB-1) binds SMA mRNA and regulates its translational activity. Our central hypothesis is that YB-1 drives maladaptive SMA expression in injury-activated myofibroblasts by modulating the packaging, delivery, and translational efficiency of its cognate mRNA. In a mouse model for cardiac fibrosis, we observed that accumulation of fetal SMA protein in cardiac sarcomeres was associated with accumulation of punctate YB-1 deposits which localized to perinuclear regions as well as polyribosome-enriched cytosol proximal to cardiac intercalated discs. Samples from both fibrotic mouse hearts as well as SMA positive endomyocardial biopsies from human heart transplant patients were enriched with high molecular weight, heat-denaturing resistant YB-1 oligomers migrating in the range of 100-250 kDa during reducing SDS-PAGE. Based on these intriguing observations, which suggested that YB-1 oligomer formation may be associated with the packaging and translation control of SMA mRNA, we examined the regulatory aspects of YB-1 oligomerization using a model system based on isolated human pulmonary fibroblasts (hPFBs). Activation of SMA gene expression in hPFBs by TGFbeta1 was associated with formation of denaturation-resistant YB-1 oligomers with selective affinity for a SMA exon-3 translation-silencer sequence. We discovered that YB-1 is a substrate for the protein-crosslinking enzyme transglutaminase 2 (TG2) that catalyzes calcium-dependent formation of covalent gamma-glutamyl-isopeptide linkages in response to reactive oxygen signaling. TG2 transamidation reactions using intact cells, cell lysates, and recombinant YB-1 revealed covalent crosslinking of the 50 kDa YB-1 polypeptide into protein olig (open full item for complete abstract)

Committee: Arthur Strauch PhD (Advisor); Denis Guttridge PhD (Committee Member); Lai-Chu Wu PhD (Committee Member); Mark Ziolo PhD (Committee Member) Subjects: Biochemistry; Biomedical Research; Cellular Biology; Molecular Biology

PhD, University of Cincinnati, 2001, Medicine : Molecular, Cellular and Biochemical Pharmacology

The type-1 phosphatase (PP1) functionally antagonizes kinase activity by dephosphorylating phosphoproteins. PP1 and its endogenous regulator, inhibitor-1 (I-1), have been suggested to play important roles in cardiovascular function. Previous studies have shown that the SR-associated PP1 activity is increased in failing human hearts and this study uniquely demonstrated that this increase in PP1 activity may be due to inactivation (dephosphorylation) of I-1. To investigate the significance of I-1 inactivation in the failing human heart, cardiac function was assessed in mice lacking I-1, (I-1(-/-)). Both basal and β-adrenergic stimulated cardiac contractility was decreased in I-1(-/-) hearts. The attenuated β-adrenergic response was associated with increased (23%) PP1 activity and dephosphorylation of phospholamban (PLB) and the ryanodine receptor (RyR) in I-1(-/-) hearts. The decreased cardiac function in I-1(-/-) mice was most likely not due to alterations in vascular smooth muscle function, since ablation of I-1 was largely without effect on contractile properties assessed in aorta or portal vein. The importance of PP1 in human heart failure was demonstrated by adenoviral infection of failing human cardiomyocytes with a constitutively active I-1 protein, which resulted in enhanced mechanical and calcium handling properties under β-adrenergic stimulation. These findings suggest that interference with the increased PP1 activity in the failing human heart may be a potential therapeutic strategy in treating this disease. Finally, the functional significance of increases in SR-associated PP1 activity was examined, using transgenic mice overexpressing the α-isoform of PP1's catalytic subunit (PP1c) in the heart. The cardiac phenotype of mice exhibiting increased (~3-fold) SR-associated PP1c activity, resembling the increases in human heart failure, were characterized. The PP1c mice exhibited diminished basal and isoproterenol stimulated cardiac contractility, which was as (open full item for complete abstract)

Committee: Evangelia Kranias (Advisor) Subjects: Health Sciences, Pharmacology

PhD, University of Cincinnati, 2000, Medicine : Molecular, Cellular and Biochemical Pharmacology

Phospholamban is a phosphoprotein in the cardiac sarcoplasmic reticulum (SR), which regulates the apparent affinity of the SR Ca2+-ATPase (SERCA2) for Ca2+. Phospholamban, in its dephosphorylated form, inhibits SERCA2 activity, and phosphorylation removes this inhibition. Transgenic approaches were utilized in this dissertation to further elucidate the role of phospholamban's regulation of SERCA2, in vivo, under normal and pathophysiological conditions, both in the presence and absence of b-agonist stimulation. 1) Phospholamban ablation has been shown to be associated with an attenuated response to b-agonist stimulation. However, it is not known if the b-adrenergic response of phospholamban-deficient hearts would be affected under altered thyroid conditions. Hypo- and hyperthyroidism were induced in phospholamban-deficient and wild-type mice, and their responses to isoproterenol stimulation were examined in work-performing hearts. A close linear correlation was observed between the magnitude of the contractile parameter response and the PLB/SERCA2 ratio in hypo-, eu- and hyperthyroid wild-type hearts. However, no response to b-agonist stimulation was observed in phospholamban-deficient hearts with altered thyroid conditions, indicating that the changes in the thyroid states of these hearts do not influence the effects of isoproterenol on cardiac function. 2) The PLB/SERCA2 stoichiometric ratio under saturating conditions is not known. 1.8-fold, 2.6-fold, 3.7-fold and 4.7-fold overexpression of a non-phosphorylatable form of phospholamban, relative to SERCA2, resulted in maximal inhibition of SERCA2's apparent affinity for Ca2+ at phospholamban expression levels of 2.6-fold or higher, indicating that saturation of the PLB/SERCA2 ratio occurs at 2.6:1, in vivo. 3) The contribution of phospholamban versus the other key cardiac phosphoproteins during b-adrenergic stimulation is not known. To determine phospholamban's contribution indirectly, a non-phosphorylatable form (open full item for complete abstract)

Committee: Evangelia Kranias (Advisor) Subjects:

PhD, University of Cincinnati, 2004, Medicine : Molecular, Cellular and Biochemical Pharmacology

The major function of the sarcoplasmic reticulum (SR) in Ca 2+ homeostasis and contractility in cardiac and slow-twitch skeletal muscles is tightly regulated by the SR Ca 2+ ATPase (SERCA2a) and its crucial inhibitor phospholamban (PLN). The present dissertation investigated: 1) the role of PLN in cardiac muscle with specific emphasis on evaluation of the efficacy of PLN inhibition on contractile dysfunction and remodeling; and 2) the physiological significance of PLN in slow-twitch skeletal muscle. 1) In cardiac muscle, mounting evidence has shown depressed SR Ca 2+ cycling is a hallmark feature of failing heart. Normalization of Ca 2+ cycling by ablation or inhibition of the SERCA2a inhibitor PLN has prevented cardiac failure in experimental dilated cardiomyopathy. However, the potential benefits of restoring SR function on primary cardiac hypertrophy, a common antecedent of heart failure, remain unknown. We therefore tested the efficacy of PLN ablation in preventing the ventricular failure of Gαq overexpression induced cardiac hypertrophy. PLN ablation normalized the characteristically prolonged cardiomyocyte Ca 2+ transients and enhanced unloaded fractional shortening with no change in SR Ca 2+ pump abundance in the Gαq overexpressor. Despite “rescue” of cardiomyocyte mechanical function and Ca 2+ signaling, there was no parallel improvement in in vivo cardiac function. Furthermore, PLN ablation was unable to alleviate Gαq-induced hypertrophic remodeling. These findings indicated restoration of SR Ca 2+ cycling by PLN ablation was not sufficient to prevent the ventricular failure of the Gαq-induced cardiac hypertrophy. 2) In transgenic mice with PLN specific overexpression in slow-twitch skeletal muscle, the PLN protein levels and the PLN/SERCA2a ratio in transgenic soleus were comparable to those in cardiac muscle. The isometric-twitch contractile performance was significantly depressed in PLN overexpressing soleus, but isopreterenol stimulation relieved the in (open full item for complete abstract)

Committee: Dr. Evangelia Kranias (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2012, Integrated Biomedical Science Graduate Program

There are three main physiological governing mechanisms for cardiac contractility; the force-frequency response, the length-tension response, and the beta-adrenergic response. One of the major hallmarks of heart failure is a decrease in the force of contraction with increasing frequency. Here we utilize the ex vivo isolated cardiac muscle in an attempt to discuss the physiological role of the force-frequency response in health. We aim to determine the functional attributes of the normal heart by studying alterations in contractility, calcium transient amplitude, and phosphorylation status following changes in frequency, alterations in beta-adrenergic stimulation, and chronic stretch. All experiments were conducted on isolated muscle preparations extracted from the right ventricular free wall of the male New Zealand white rabbit. Our hope is to increase the understanding of the manipulations and alterations that occur in the context of the force-frequency response in health. We first determined the direct changes in the calcium and force response on a beat-to-beat basis during an instantaneous change in frequency. Because heart failure is rarely a spontaneous event, determining the minute changes in calcium transient amplitude as it relates to force production provides insight into the intricate balance that occurs at every beat. In this study we were able to highlight the dynamic relationship between force and calcium during the process of force stabilization at different frequencies. In additional studies, we determined the effects of beta-adrenergic stimulation in conjunction to the force-frequency response. This work aimed to combine the force-frequency response with the beta-adrenergic response in a controlled manner. These experiments highlighted a modulation of the force-frequency response during beta-adrenergic stimulation, suggesting an important role for beta-stimulation in immediate contractile alterations which have an inhibitory effect on the response (open full item for complete abstract)

Committee: Paul Janssen PhD (Advisor); Candice Askwith PhD (Committee Member); Jonathan Davis PhD (Committee Member); Mark Ziolo PhD (Committee Member) Subjects: Physiology

Doctor of Philosophy, The Ohio State University, 2010, Biophysics

The Ca2+ sensitivity of cardiac muscle force development can be modulated by physiological or patho-physiological stimuli such as phosphorylation or disease related protein modification. Since troponin C (TnC) is the Ca2+ sensor for cardiac muscle contraction, TnC's Ca2+ binding properties could be affected by these physiological or patho-physiological related protein modifications. Thus, in this study, we examined the effect of mimicking phosphorylation and disease related protein modifications in TnI and TnT on the Ca2+ binding properties of TnC in a physiologically relevant biochemical model system, i.e. reconstituted thin filaments. Our results demonstrated that these protein modifications alter thin filament Ca2+ binding in a way generally consistent with their effect on myofilament Ca2+ sensitivity. Besides steady state Ca2+ binding affinity, the kinetic rate of Ca2+ dissociation from thin filament was also modulated by these physiologically relevant protein modifications. Generally, the protein modifications that sensitize thin filament Ca2+ binding usually slow the rate of Ca2+ dissociation from thin filament, while those that desensitize thin filament Ca2+ binding usually accelerate the rate of Ca2+ dissociation from thin filament. Experimental evidence suggests that the symptoms of some cardiac dysfunctions may be caused by the aberrant Ca2+ binding. Thus, correcting the aberrant Ca2+ binding might improve cardiac function. To achieve this goal, we have engineered TnC constructs with a wide, yet adjustable, range of Ca2+ binding sensitivities by modulating the negatively charged residues in the Ca2+ chelating loop and/or by replacing key hydrophobic amino acids in the regulatory domain of TnC with polar Gln. We were able to correct both the increased and decreased thin filament Ca2+ sensitivities caused by the disease associated proteins via replacing the wild type TnC with specifically engineered TnC constructs. Additionally, engineered TnC constructs ca (open full item for complete abstract)

Committee: Jonathan Davis (Advisor); Jack Rall (Committee Member); Peter Reiser (Committee Member); Paul Janssen (Committee Member) Subjects:

Master of Science, The Ohio State University, 2010, Biophysics

Many factors play a role in regulating striated muscle contraction; however, the relative importance of the thin filament in this process is still under poorly understood. In this work, the role of troponin C (TnC) on the rate of skeletal muscle contraction and as a regulator of the Ca2+ sensitivity of cardiac force production were studied. To investigate how TnC might affect the rate of contraction, skeletal TnC constructs with altered Ca2+ binding properties were reconstituted into single skinned psoas fibers from rabbits to assess the Ca2+ dependence of force development and the rate of force redevelopment (ktr) at 15°C. This procedure resulted in a sensitization of both force and ktr to Ca2+ for V43QTnC, whereas T70DTnC and I60QTnC desensitized force and ktr to Ca2+, with I60QTnC causing a greater desensitization. In addition, T70DTnC and I60QTnC depressed both maximal force (Fmax) and maximal ktr. Even though V43QTnC and I60QTnC had drastically different effects on the Ca2+ binding properties of TnC, they both exhibited decreases in the cooperativity of force production and elevated ktr at force levels less than 30% Fmax compared to wild-type TnC. However, at matched force levels greater than 30% Fmax, ktr was similar for all the TnC constructs. These results suggest that the TnC mutants primarily affected ktr through modulating the level of thin filament activation and not by altering intrinsic cross-bridge cycling properties. To corroborate these results, NEMS-1, a non force generating cross-bridge analogue that activates the thin filament, fully recovered maximal ktr for the I60QTnC at low [Ca2+]. Additionally, a protocol was developed to passively exchange whole human Tn into rat skinned cardiac trabeculae to study how TnC works in conjunction with other thin filament proteins to regulate the Ca2+ sensitivity of force production. To this end, the disease related abnormal Ca2+ sensitivities resulting from a truncation of TnI (residues 1-192) and a single re (open full item for complete abstract)

Committee: Jonathan Davis (Advisor); Jack Rall (Committee Member); Peter Reiser (Committee Member) Subjects: Biophysics

Doctor of Philosophy, The Ohio State University, 2007, Biophysics

Cardiac muscle contraction is initiated by Ca2+ binding to troponin C (TnC), which triggers conformational changes on the thin filament, allowing the myosin heads (or crossbridges) to attach to actin and the thick filaments to slide along the thin filaments. This study investigated the influence of the thin filament Ca2+ binding affinity on modulating the rate of contraction at various levels of Ca2+ activation in rat skinned cardiac trabeculae at 15°C. The rate of contraction was assessed as the rate of force (tension) redevelopment, or ktr. Our novel approach was to directly change the level of thin filament Ca2+ activation by incorporating into trabeculae cardiac TnC mutants with various Ca2+ binding affinities. The TnC mutants V44QTnCF27W and F20QTnCF27W, when reconstituted into trabeculae, increased and decreased, respectively, the Ca2+ sensitivity of force production, both in conditions of normal or accelerated crossbridge cycling induced by the presence of added inorganic phosphate (Pi). The rates of contraction at submaximal levels of Ca2+ activation were increased or decreased, respectively, when the muscle was sensitized or desensitized to Ca2+, but the rates of contraction at saturating Ca2+ activation were similar. When crossbridges cycle faster in the presence of Pi, enhancing or reducing the thin filament Ca2+ activation can still increase or decrease the rate of contraction at submaximal levels of force production, but the rates of contraction at maximal levels of Ca2+ activation were similar for trabeculae reconstituted with TnC mutants. This study indicates that the rate of cardiac muscle contraction is modulated, at submaximal levels of Ca2+ activation, by the Ca2+ binding properties of TnC, and at maximal levels of Ca2+ activation, by the kinetics of crossbridge cycling. These results have physiological relevance and possible clinical applications considering that, on a beat-to-beat basis, the heart contracts at submaximum Ca2+ activation. In hea (open full item for complete abstract)

Committee: Jack Rall (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2005, Molecular, Cellular, and Developmental Biology

In this dissertation, we examine the in vivo functions of two cell junction proteins, CASK and Claudin-5. CASK is a member of the membrane associated guanylate kinase family of proteins, while Claudin-5 is a protein that has been most thoroughly characterized as a component of tight junctions. This dissertation describes investigations of the CASK protein in skeletal muscle at the neuromuscular junction (NMJ) and Claudin-5 in cardiomyocytes We show that the CASK protein is present in skeletal muscle and is predominantly localized to the primary gutter of the NMJ, with a small amount of presynaptic localization. Immunoprecipitations reveal that CASK interacts in vivo with Dlg. We also examined the CASK protein in the C2C12 myogenic cell line and found that CASK localization is developmentally regulated and determined that CASK is recruited to the NMJ by a mechanism with is independent of that which recruits acetylcholine receptors. Finally, we generated two lines of transgenic mice, which overexpress a full-length or truncated version of the CASK protein. Overexpression of either the full-length or truncated CASK protein does not result in any morphological or functional abnormalities of the skeletal muscle or NMJ. However, overexpression of either CASK protein results in a loss of CASK protein presynaptically at the NMJ. Finally, overexpression of either CASK protein did not alter the expression or localization of Dlg. This dissertation also details an examination of the Claudin-5 protein in cardiac muscle from wild-type mice and two mouse models of cardiomyopathy, the mdx and dko mouse. We show that the Claudin-5 protein is present in normal cardiac muscle and is localized to the lateral membranes of cardiomyocytes. We also demonstrate that Claudin-5 protein expression and localization is unaltered in hearts from mdx mice, but is downregulated in dko hearts, as compared to normal hearts. Expression levels of cell junction proteins present at the intercalated disc w (open full item for complete abstract)

Committee: Jill Rafael-Fortney (Advisor) Subjects:

Master of Science, University of Akron, 2010, Biology

The transcription factor Sry is expressed in male embryos where it initiates testis development. It is also transcribed in several adult tissues of various species. Tissue specific differences in the expression profile of the 6 or 7 rat Sry loci have been reported in adult male testis, adrenal, and kidney of the SHR/y strain, indicating likely differential locus functions. The promoter of Acsl3, an enzyme in fatty acid metabolism, contains putative Sry response elements, implicating Sry as a potential regulator of Acsl3 gene activity. It was hypothesized that Sry is expressed in adult cerebral cortex, ventricle, atrium, aorta, and skeletal muscle in adult normotensive WKY, borderline hypertensive SHR/y, and hypertensive SHR male rats with differential locus expression in cerebral cortex and ventricle. Further, total Sry transcript expression was expected to correlate with Acsl3 transcript expression. RNA isolated from skeletal muscle, atrium, aorta, ventricle, and cerebral cortex of 15-20 week old WKY, SHR/y, and SHR male rats was reverse transcribed to cDNA. Total Sry and Acsl3 transcript expression was determined by real-time PCR using Sry and Acsl3 specific primers. Individual Sry locus expression in ventricle and cerebral cortex was determined by fragment analysis using standard PCR and fluorescent-tagged primers. Sry transcripts were detected in all five tissues. Total Sry expression in cerebral cortex was between 3.9 and 42.3 fold (P<0.05) greater than in aorta, skeletal muscle, and ventricle in all three strains. Within each tissue, proportional expression of individual Sry loci differed (P<0.05): Sry1, Sry2, and Sry3A/3C differed in WKY cerebral cortex; Sry1 differed from Sry2 and Sry3/3A/3C in SHR cerebral cortex and SHR/y cerebral cortex; and Sry1 differed from Sry2 in WKY ventricle and SHR/y ventricle. Within each strain, tissue specific differences were present for Sry1 and Sry2 in all three strains; Sry3A/3C in WKY; and Sry3/3A/3C in SHR/y. Strain prop (open full item for complete abstract)

Committee: Amy Milsted Dr. (Advisor) Subjects: Molecular Biology

PhD, University of Cincinnati, 0, Medicine: Molecular and Developmental Biology

Congenital cardiac malformations are the most common birth defects in the United States, and cardiovascular disease (CVD) is the leading cause of death in the U.S. and worldwide. Therefore, identifying conserved molecular mechanisms that regulate cell fate in cardiac development and disease is of great clinical significance. During embryonic heart development, epicardium-derived cells (EPDCs) invade the myocardium and differentiate into fibroblasts and vascular smooth muscle (SM) cells, which support the coronary vessels. Work described in this dissertation demonstrates that the transcription factors Tcf21/Pod1, WT1, NFATC1, and Tbx18 are expressed in overlapping and distinct epicardial and EPDC populations. Retinoic acid (RA) signaling promotes Tcf21 and WT1 expression, while inhibiting EPDC differentiation into SM. Loss of Tcf21 in mice leads to epicardial blistering, increased SM differentiation on the surface of the heart, and a paucity of interstitial fibroblasts, with neonatal lethality. On the surface of the myocardium, SM marker expression is increased in Tcf21-deficient EPDCs, demonstrating premature SM differentiation. Increased SM differentiation also is observed in Tcf21-deficient lung and kidney mesenchyme. Together, these data demonstrate a critical role for Tcf21 in controlling mesenchymal progenitor cell differentiation into SM and fibroblast lineages during cardiac development. The role of Tcf21 in adult cardiac homeostasis or disease, however, is unknown. In order to define the role of Tcf21 in cardiac fibrosis, its expression was examined in mouse models of heart disease as well as in human congestive heart failure (CHF) tissue. Mouse models of ischemia and pressure overload were examined. Studies detailed in this dissertation demonstrate that ischemic injury leads to increased subepicardial cells positive for WT1, Tbx18, and Tcf21 in mice and human hearts. Mice subjected to pressure overload have extensive coronary perivascular fi (open full item for complete abstract)

Committee: Katherine Yutzey Ph.D. (Committee Chair); Jo El Schultz Ph.D. (Committee Member); John Shannon Ph.D. (Committee Member); Stephanie Ware M.D. Ph.D. (Committee Member); Aaron Zorn Ph.D. (Committee Member) Subjects: Developmental Biology