Doctor of Philosophy, The Ohio State University, 2022, Molecular, Cellular and Developmental Biology

Arrhythmias account for approximately 250,000 deaths in the U.S annually, with nearly half being associated with heart disease. Arrhythmogenic disorders are broken down into a variety of subcategories, with the vast majority being primarily caused by either activity changes or variants in ion channels/exchangers. Arrhythmogenic cardiomyopathy (ACM) is a unique form of heart disease that is primarily hereditary, where variants in genes encoding structural proteins are the most frequent cause of disease formation. Variants within desmosomal genes are one of the leading predisposing factors to ACM, primarily characterized by fibro-fatty infiltration in the ventricular myocardium with an increased propensity for ventricular arrhythmias. This frequently results in sudden cardiac death, even prior to the detection of any cardiac structural abnormalities. Previous work on a familial ACM variant in desmoplakin (DSP) (p.R451G) identified a post-translational degradation of DSP that stemmed from increased sensitivity to the protease calpain, a pattern identified in additional pathogenic variants. Despite these findings, incomplete penetrance within most familial ACM cases complicates understanding of the associated molecular pathways, as well as determining the external factors that contribute to disease development. While the generation of murine models have significantly contributed to the understanding of disease progression, most utilized knock-out or transgenic techniques, limiting the potential translational impact. Our group has developed one of the first mouse models of ACM derived from a human variant by introducing the murine equivalent of the R451G variant into endogenous desmoplakin (DspR451G/+). Mice homozygous for this variant displayed embryonic lethality. While DspR451G/+ mice were viable with reduced expression of DSP, no presentable arrhythmogenic phenotype was identified at baseline. Following acute stress through catecholaminergic challenge, DspR451G/+ mic (open full item for complete abstract)

Committee: Peter Mohler (Advisor); Maegen Borzok (Committee Member); Federica Accornero (Committee Member); Thomas Hund (Committee Member); Brandon Biesiadecki (Committee Member) Subjects: Cellular Biology; Physiology

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

Cardiovascular disease is the leading cause of death in the United States, claiming nearly 800,000 lives each year. Regardless of the underlying cardiovascular dysfunction, nearly 50% of these patients die of sudden cardiac arrest caused by arrhythmia. Development and sustainment of cardiac arrhythmia begins with dysfunction of excitability and structure at the cellular level. Therefore, in order to improve therapeutic options for these patients, a basic understanding of the molecular mechanisms regulating cardiac cellular excitability and structure is required. Decades of research have demonstrated that intracellular scaffolding polypeptides known as ankyrins are critical for the regulation of cellular excitability and structure in multiple cell types. Ankyrin-G (ANK3) is critical for regulation of action potentials in neurons and lateral membrane development in epithelial cells. Given its central importance for cellular physiology in excitable and non-excitable cell types, we hypothesized that functional ankyrin-G expression is critical for proper cardiac function. To test this hypothesis in vivo, we generated cardiac-specific ankyrin-G knockout (cKO) mice. In the absence of ankyrin-G, mice display significant reductions in membrane targeting of the voltage-gated sodium channel Nav1.5. This disruption in turn causes severely reduced whole cell sodium current, leading to significant conduction abnormalities, bradycardia, and ventricular arrhythmia and atrioventricular nodal block following infusion of NaV channel antagonists. In addition to regulating cardiac excitability, we also demonstrate a critical role for ankyrin-G in the regulation of the cardiomyocyte cytoarchitecture. Specifically, ankyrin-G cKO mice show disrupted cellular distribution of the desmosomal protein plakophilin-2 (PKP2) at baseline. In a setting of pressure overload-induced heart failure we observed severe disruptions to the cellular localization of PKP2. Further, as desmosomes mediate the in (open full item for complete abstract)

Committee: Peter Mohler (Advisor); Noah Weisleder (Committee Chair); Thomas Hund (Committee Member); Philip Binkley (Committee Member) Subjects: Cellular Biology; Physiology

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