PhD, University of Cincinnati, 2022, Medicine: Molecular, Cellular and Biochemical Pharmacology

Heart failure is one of the leading causes of death globally, and the function of a failing heart is often worsened by the presence of pathological cardiac hypertrophy and fibrosis. RNA-binding proteins (RBPs) are gaining interest as emerging players in the mechanisms of adverse cardiac remodeling and can exert affect broad effects on the transcriptome through RNA splicing and stabilization/destabilization of target RNA transcripts. HuR (HuR; gene name ELAVL1) is a ubiquitously expressed RBP that is highly expressed in multiple cell types in the heart, although little is known about its physiological function. Immunohistochemistry of HuR expression in the myocardium indicates that HuR expression is increased in failing human hearts. Using a pressure-overload model of heart failure (Transverse Aortic Constriction, TAC), these studies demonstrate that a cardiomyocyte-specific deletion of HuR, as well as pharmacological inhibition of HuR, are cardioprotective in preserving cardiac function and decreasing cardiac fibrosis post-TAC. Interestingly, global pharmacological inhibition of HuR in every cell type within the heart, produced a much stronger dampening of adverse cardiac remodeling than the cardiomyocyte-specific knockout. This indicates that HuR could be modulating remodeling in cell types other than cardiac myocytes. Therefore, the follow-up studies went on to assess the role of HuR in cardiac fibroblasts in vitro. Using isolated primary mouse cardiac fibroblasts and a series of myofibroblast activation assays including scratch assays, collagen gel contraction assays, and picrosirius staining, these studies determined that HuR is necessary for fibroblast activation and myofibroblast activity. In order to determine which directly-bound RNA targets of HuR are important in fibroblasts activation, a photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) was performed to identify mRNA transcripts that were enriched for HuR binding fol (open full item for complete abstract)

Committee: Michael Tranter Ph.D. (Committee Member); Onur Kanisicak PhD (Committee Member); Thomas Thompson Ph.D. (Committee Member); Sakthivel Sadayappan Ph.D. (Committee Member); Jo El Schultz Ph.D. (Committee Member); Evangelia Kranias Ph.D. (Committee Member) Subjects: Organismal Biology

PhD, University of Cincinnati, 2020, Medicine: Molecular Genetics, Biochemistry, and Microbiology

Cardiac fibroblasts (CFs) are essential to normal heart function through maintenance of extracellular matrix (ECM). Following injury, CFs can undergo acute activation to increase ECM production and support the injured heart, but chronic activation can cause excess ECM deposition resulting in fibrosis. Although the extent of CF activation determines whether a supportive scar or detrimental fibrosis will ensue, the nuances of CF activation and differentiation are understudied. CFs differentiate into myofibroblasts via two mechanisms: biochemical cues, or mechanical cues such as increased environmental rigidity. Gene expression is a widely-used means of documenting CF activation. Tissue resident CFs express genes such as transcription factor-21 (Tcf21). Upon activation, they express genes including Acta2 which encodes smooth muscle a-actin (aSMA) protein. While these expression profiles have been verified in vivo, other presumed CF characteristics are derived from in vitro studies, often conducted on plastic tissue culture plates (TCPs). As TCPs are 6 orders of magnitude stiffer than cardiac tissue, mechanosensitive CFs cultured on TCPs may display non-physiological biology. Therefore, in vitro studies utilizing TCPs may present non-physiologically relevant results. Though studies utilizing TCPs may be flawed, in vitro studies are important for understanding CF biology as they provide a controlled yet easily manipulated environment. To circumvent the issues of TCP stiffness and provide an environment of physiological stiffness, we generated two novel gelatin-based hydrogel cell culture systems containing either HPMA or NIPAAm crosslinking polymers. Both can be made to mimic the stiffness of a healthy or fibrotic heart and later softened through cysteine treatment, which breaks disulfide bonds between polymers. Both hydrogel systems were biocompatible and demonstrated stiffness-dependent CF activation, measured by cell area and aSMA expression. Notably, specific ce (open full item for complete abstract)

Committee: Jeff Molkentin Ph.D. (Committee Chair); William Miller Ph.D. (Committee Member); David Wieczorek Ph.D. (Committee Member); Katherine Yutzey Ph.D. (Committee Member) Subjects: Cellular Biology

PhD, University of Cincinnati, 2018, Medicine: Molecular, Cellular and Biochemical Pharmacology

Heart failure (HF) is the leading cause of morbidity and mortality in the United States and is characterized by progressive pathologic remodeling, fibrosis and deteriorating cardiac function. Cardiac fibrosis occurs due to an imbalance in the production and degradation of the extracellular matrix (ECM). Cardiac fibroblasts (CF) are largely responsible for the secretion of ECM proteins in the heart. Upon injury or pathologic stimulation, CF transition to a pathologic myofibroblast phenotype, leading to excess production of ECM proteins and pro-inflammatory cytokines. Elevated expression of matrix metalloproteinases (MMPs), proteolytic enzymes responsible for maintenance and degradation of the ECM, is common in HF. Specifically, MMP-13, one of the major interstitial collagenases in both mice and humans, is known to be upregulated in human HF patients as well as animal models of HF. However, few studies have been performed to elucidate the role of MMP-13 in the heart. Further, while CF are considered the main cellular source of MMPs in the heart, MMP-13 has been shown to be expressed in multiple cell types. Therefore, the goal of this dissertation was to determine the role of MMP-13 in a long-term model of HF as well as examine the cell specific source and role of MMP-13 in the heart. Utilizing a selective MMP-13 inhibitor, we show, for the first time, a role for MMP-13 in the myofibroblast phenotype and in the development and progression of ventricular remodeling and failure after injury. Specifically, inhibition of MMP-13 attenuates myofibroblast invasion, migration and proliferation, suggesting that MMP-13 inhibition reduces the pathologic phenotype of these cells. Additionally, mice that were treated with the MMP-13 inhibitor 4 weeks after injury display significant improvement in cardiac function compared to control animals after cardiac injury with concomitant attenuation in cardiac hypertrophy. Further, mice treated with the MMP-13 inhibitor have significa (open full item for complete abstract)

Committee: Burns Blaxall Ph.D. (Committee Chair); Evangelia Kranias Ph.D. (Committee Member); Jo El Schultz Ph.D. (Committee Member); Katherine Yutzey Ph.D. (Committee Member) Subjects: Pharmacology

PhD, University of Cincinnati, 2017, Medicine: Molecular, Cellular and Biochemical Pharmacology

Heart failure, the final clinical manifestation of numerous cardiovascular maladies, is a devastating disease characterized by interstitial fibrosis, chamber remodeling and reduced ventricular compliance. Elevated myocardial sympathetic stimulation, a hallmark of heart failure, induces pathological signaling through G protein βγ subunits that results in the activation and membrane recruitment of G protein-coupled receptor kinase 2 (GRK2). In the failing heart, diminished cardiac output triggers a vicious cycle of persistent sympathetic stimulation accompanied by perpetual maladaptive signaling due in large part to GRK2-mediated β-adrenergic receptor desensitization and loss of responsiveness. We recently identified and validated the novel small molecule inhibitor gallein that disrupts the Gβγ-GRK2 interaction. Here, we investigated the therapeutic potential of gallein in murine ischemia-reperfusion (I/R) injury, a clinically relevant model of ischemic heart failure. We found that pharmacological disruption of Gβγ-GRK2 signaling post-I/R offered significant protection against cardiac dysfunction and remodeling. Moreover, we observed that gallein treatment significantly ameliorated fibrotic infarct expansion and the expression of several pro-fibrotic markers, which likely contributed to the overall improvement in ventricular contractility. One principal objective was to decipher the cellular specificity of gallein by ablating GRK2 in various resident cell populations of the heart. While the salutary properties of cardiomyocyte-specific ablation of GRK2 have been extensively documented, our goal was to explore the potential cardiomyocyte-independent cardioprotective properties of gallein. While GRK2 ablation in cardiomyocytes offered modest protection against cardiac dysfunction and remodeling, it was only with concurrent gallein treatment that significant cardioprotection was achieved. These findings suggested functional significance for Gβγ-GRK2 inhibition in c (open full item for complete abstract)

Committee: Burns Blaxall Ph.D. (Committee Chair); Evangelia Kranias Ph.D. (Committee Member); William Miller Ph.D. (Committee Member); Jeff Molkentin Ph.D. (Committee Member); Jo El Schultz Ph.D. (Committee Member) Subjects: Pharmacology

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

Volume overload (VO) induced heart failure results from an increase in blood volume (preload) to the heart. The heart responds to increases in hemodynamic load through compensative remodeling. VO has a distinct pattern of remodeling compared to pressure overload induced heart failure, which results in fibrosis. VO results in a net decrease in extracellular matrix (ECM). This loss of ECM contributes to the progression of the disease due to the loss of structural integrity. Since cardiac fibroblasts (CFs) are the main cells responsible for maintaining ECM in the heart, we characterized the in vitro phenotype of CFs isolated from a rat VO model, aortocaval fistula (ACF). Compared to sham operated animals, ACF fibroblasts displayed a phenotype that we described as “hypofibrotic”. ACF CFs secreted relatively less collagen and profibrotic molecules, such as a-smooth muscle actin (aSMA) and connective tissue growth factor (CTGF). Interestingly, ACFs produce approximately twice as much transforming growth factor-ß1 (TGF-ß), a key profibrotic stimulus, as their sham counterparts. However, there were no changes in the canonical TGF-ß pathway that could account for the hypofibrotic phenotype observed in ACF fibroblasts. Since others have shown that the cytoskeleton and the Rho/ROCK pathway play a role in fibroblast phenotype, we characterized the actin cytoskeleton in sham and ACF fibroblasts. We found that ACF CFs have significantly less F-actin than sham CFs. We were able to show that it is possible the actin cytoskeleton might account for phenotypic differences in CFs by chemically altering the amounts of F-actin and G-actin. When the cells were treated with a ROCK inhibitor, which allows F-actin to depolymerize into G-actin, CFs displayed a more hypofibrotic phenotype. Conversely, enhancement of F-actin with jasplakinolide treatment forced the CFs to have a profibrotic phenotype. Numerous studies have linked substrate modulus with effects on the cytoskeleton. S (open full item for complete abstract)

Committee: Keith Gooch PhD (Advisor); Jun Liu PhD (Committee Member); Pamela Lucchesi PhD (Committee Member); Aaron Trask PhD (Committee Member) Subjects: Biomedical Engineering

PHD, Kent State University, 2010, College of Arts and Sciences / School of Biomedical Sciences

Cardiac fibroblasts (CFs) are the major non-contractile cells present in the myocardium and are the primary producers of the extracellular matrix (ECM) in the heart. Cardiac fibrosis, an accumulation of ECM, is often the result of overactive fibroblasts. Cardiac fibroblasts are activated by numerous stimuli including ECM components, circulating hormones such as angiotensin II (ANG II), and hyperglycemia. Excessive proliferation, migration, and differentiation of fibroblasts contribute to the fibrosis, and therefore it is crucial to study the mechanisms that regulate these processes to unveil new targets aimed at preventing prolonged fibroblast activation and fibrosis. The overall goal of this project is to understand how the ECM, ANG II, and diabetes affect cardiac fibroblast activation. To achieve this goal, Aim 1 will determine the influence of collagen types I, III, VI on fibroblast and myofibroblast migration. Aim 2 will establish the specific PKC isoform mediating angiotensin II-induced ERK 1/2 phosphorylation. Aim 3 is designed to explore how hyperglycemia alters cardiac fibroblast proliferation, migration, myofibroblast differentiation and whether changes in ECM composition can alter the function of cardiac fibroblasts in high glucose media. Finally, Aim 4 will determine the influence of type 1 diabetes on fibroblast activation in the in vivo setting. This dissertation reveals that type III collagen and to a lesser extent, type I collagen promote migration whereas fibroblasts and myofibroblasts plated on collagen VI exhibit delayed migration. This study demonstrates that ANG II stimulates ERK1/2 phosphorylation via activation of distinct parallel signaling pathways mechanistically controlled by intracellular Ca2+ and PKC delta. This study also reveals that hyperglycemia induces CF proliferation and accelerates myofibroblast differentiation in vitro and that early stage diabetic hearts contain highly proliferative fibroblasts, but have significantly fewer my (open full item for complete abstract)

Committee: J. Gary Meszaros PhD (Committee Chair); Ian Bratz PhD (Committee Member); Derek Damron PhD (Committee Member); Daniel Ely PhD (Committee Member); Bansidhar Datta PhD (Committee Member) Subjects: Biomedical Research; Cellular Biology

PHD, Kent State University, 2006, College of Biomedical Sciences

Cardiac fibroblasts (CFs) are the major non-contractile cells present in the myocardium. The primary responsibility of CFs is to regulate the synthesis and secretion of extracellular matrix (ECM) proteins. Excessive proliferation and differentiation of CFs during cardiovascular pathologies directly leads to the development of cardiac fibrosis, a condition characterized by a stiffening of the myocardium, which has significant effects on cardiac function. Resveratrol (RES), a component of red wine, has been identified as an agent having considerable cardioprotective properties. The first Specific Aim of my dissertation research was designed to investigate the effects of RES on CF function. I concluded that 25 microM RES significantly inhibited CF proliferation and myofibroblast differentiation, thus identifying RES as a potential anti-fibrotic agent. A common characteristic of cardiovascular diseases is the activation of the renin-angiotensin system, resulting in increased circulating angiotensin II (ANG II). ANG II directly stimulates CF proliferation, myofibroblast differentiation, and de novo synthesis of collagens and other ECM proteins and therefore, an elevation in systemic ANG II is a major contributing factor in the progression of cardiac fibrosis. The intracellular signaling pathways mediating the effects ANG II signaling are most well characterized in vascular smooth muscle cells (VSMCs). Proliferation of CFs by ANG II is dependent on the ERK 1/2 signaling pathway, but the exact pathway from the ANG II type 1 receptor to ERK 1/2 has not yet been elucidated in adult CFs. Through studies outlined in the second Aim, I determined that ANG II-induced ERK 1/2 activation is dependent on both intracellular calcium and protein kinase C (PKC). I also concluded that ANG II stimulation did not induce transactivation of the epidermal growth factor receptor, a finding which distinguishes ANG II signaling in adult CFs from that in neonatal CFs and VSMCs. In addition, I det (open full item for complete abstract)

Committee: J Meszaros (Advisor) Subjects: Biology, Cell