Browsing by Author "Braun, Andrew P."

Now showing 1 - 6 of 6
  • Thumbnail Image
    ItemOpen Access
    Identification and Verification of Differentially Methylated Regions in Cell-Free DNA as a Peripheral Biomarker for Bicuspid Aortic Valve Aortopathy
    (2020-01-08) Maredia, Ashna Karimbhai; Greenway, Steven C.; Fedak, Paul; Bathe, Oliver F.; Braun, Andrew P.
    Bicuspid aortic valve (BAV) is a common congenital cardiac malformation associated with aortopathy for which the progression of aortic dilation is difficult to predict at present. BAV aortopathy has been linked to genetic factors and abnormal hemodynamic flow with regions of elevated wall shear stress (WSS) on the ascending aorta. The dying vascular smooth muscle cells release fragmented DNA into the circulation and this cell-free DNA (cfDNA) could be leveraged as a biomarker for aortopathy. Identification of tissue-specific differentially methylated regions (DMRs) in DNA provides a potential mechanism to identify cfDNA arising from the ascending aorta. The objective is to identify aorta-specific DMRs in the cfDNA of BAV patients as a biomarker for the severity of the aortopathy. We hypothesize that BAV-associated aortopathy leads to increased cell death and increased release of aorta-specific cfDNA correlating with the severity of aortopathy as defined by aortic cell death, elastin degradation and dysregulation of ECM proteins. BAV patient aortic wall samples corresponding to areas of elevated and normal WSS were collected and stained for cell death. Regions of elevated aortic WSS showed greater cell death when compared to regions of normal aortic WSS (p=0.00006). We established a bioinformatic pipeline for the identification of aorta-specific DMRs and they were verified with BAV patient cfDNA. The levels of aorta specific cfDNA of the DMRs on Chr 11, 18 and 22 of BAV patients had a significant correlation with levels of cell death in elevated aortic WSS regions. However, there was no correlation with elastin thickness, ECM concentrations of matrix metalloproteinases (MMP) types 1, 2 and 3, tissue inhibitor of metalloproteinases-1 and transforming growth factor-β1. Further work needs to be done in order to identify more specific aortic DMRs that have stronger correlation to the severity of BAV aortopathy markers with larger cohorts for biomarker validation. The identification of a peripheral biomarker that correlates with tissue disease will be an important advance in the non-invasive diagnosis of BAV-associated aortopathy and potentially help guide clinical decision-making regarding the need for surgical intervention.
  • Thumbnail Image
    ItemOpen Access
    Investigation of Hydrogen Peroxide/Reactive Oxygen Species-Related Signaling on Vasoactive Responses in Myogenic Resistance Arteries
    (2019-03-28) Kendrick, Dylan John; Braun, Andrew P.; Cole, William C.; Slater, Donna M.; Von Der Weid, Pierre Yves
    The study focuses on the investigation of H2O2 and reactive oxygen species as a putative contributor to endothelium-derived hyperpolarization in myogenically-active resistance arteries, and its contribution to the responses evoked by established vasoactive agents. In particular, I hypothesize that H2O2 and/or ROS serve as physiologic, vasoactive agents in myogenically-active resistance arteries in normal tissue and/or in arteries exhibiting endothelial dysfunction (i.e. conditions with reduced NO bioavailability). Using a number of different experimental protocols such as lucigenin molecular assays provided the amount of NADPH-oxidase inhibition in the presence of apocynin, ML171 and VAS2870, where pressure myography experiments showed the response of rat cremaster arteries to external hydrogen peroxide, indicating a role for hydrogen peroxide within the vasculature. Pressure myography experiments also showed the arterial response to the different NADPH-oxidase inhibitors in terms of baseline myogenic tone. Application of apocynin (10µM and 100µM) further constricted the vessels, ML171 resulted in a transient relaxation, with a full relaxation seen with exposure to higher concentrations (0.1µM, 0.3µM, 10µM) of VAS2870. Responses to established vasoactive agents were observed in the presence of the NADPH-oxidase inhibitors, showing reduced responses at ~50% NADPH-oxidase inhibition, with enhanced responses at greater NADPH-oxidase inhibition. The study shows that the three structurally diverse NADPH-oxidase inhibitors differentially affect basal myogenic tone at concentrations (~IC50 values) that produce comparable inhibition of vascular NADPH-oxidase activity.
  • Thumbnail Image
    ItemOpen Access
    The Large Conductance, Calcium-activated K+ (BK) Channel is regulated by Cysteine String Protein
    (Nature Publishing Group, 2013-08-15) Barry D. Kyle; Ahrendt, Eva; Braun, Andrew P.; Braun, Janice E. A.
  • Thumbnail Image
    ItemOpen Access
    Molecular Basis and Regulation of Ca2+ Release Termination and its Role in Cardiomyopathies
    (2017) Liu, Yingjie; Chen, S. R. Wayne; Braun, Andrew P.; French, Robert John
    It is known that sarcoplasmic reticulum (SR) Ca2+ release in cardiac muscle is initiated via cardiac ryanodine receptor (RyR2) through a mechanism called Ca2+-induced Ca2+ release. However, how the SR Ca2+ release is terminated is undetermined. The objective of the current study is to understand the molecular basis and regulation of RyR2-mediated Ca2+ release termination and its role in the pathogenesis of cardiac diseases. Based on recent 3D structural analyses, the NH2-terminal region of RyR2 interacts with the channel domain via the central domain and undergoes dynamic conformational changes during channel gating. It has also been discovered that the NH2-terminal region consists of three distinct domains. HEK293 cell studies on domain deletions and disease mutations demonstrate that the different domains play different roles in RyR2 function. The NH2-terminal region is a major determinant of Ca2+ release activation and termination. Enhanced luminal Ca2+ activation of RyR2 has been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). However, in addition to CPVT, many RyR2 mutations can also cause cardiomyopathies. Knock-in mouse models harboring cardiomyopathy- associated RyR2 mutations have been generated to investigate the causal mechanisms of cardiomyopathies. The exon-3 deletion mouse model exhibited markedly reduced RyR2 expression level and no characteristic phenotype. The RyR2-R420W mouse model showed enhanced susceptibility to CPVT and altered cytosolic Ca2+ transient properties, suggesting that the abnormal cytosolic Ca2+ transient may be a key factor in the pathogenesis of cardiomyopathies. Calmodulin (CaM) is a regulatory protein that binds and inhibits RyR2 in the presence of cytosolic Ca2+. The inhibitory effect depends on the affinity of CaM for Ca2+ and RyR2. CaM mutations affecting either aspect may result in aberrant regulation of RyR2 activity, hence abnormal Ca2+ release termination. Indeed, arrhythmogenic mutations and most newly discovered CaM variants delayed Ca2+ release termination while others enhanced it, indicating that CaM is a major modulator of RyR2-mediated Ca2+ release termination. Overall, Ca2+ release termination is an intrinsic property of RyR2 that can be regulated by modulators such as CaM. Altered Ca2+ release termination is critically involved in the pathogenesis of cardiac diseases.
  • Thumbnail Image
    ItemOpen Access
    Phosphodiesterases mediate neurohumoral regulation of sinoatrial node and atrial arrhythmogenesis
    (2022-06-21) Dorey, Tristan W.; Rose, Robert A.; Phillips, Aaron A.; Braun, Andrew P.
    Each heartbeat originates from a specialized region in the heart called the sinoatrial node (SAN) that is highly regulated by the autonomic nervous system (ANS). Hypertensive heart disease is the most common form of cardiovascular disease and impairs heart rate (HR) regulation. Inability to increase HR in response to various stressors is known as chronotropic incompetence and is linked to increased mortality risk in this population. Nevertheless, the mechanisms for chronotropic incompetence in this setting are unclear. As such, the first aim of this thesis was to characterize the mechanisms for impaired HR regulation in a mouse model of angiotensin II (AngII)-induced hypertensive heart disease. AngII-infused mice had intrinsic SAN dysfunction, increased sympathetic nervous system activity, and reduced SAN responsiveness to ANS stimulation. Mechanistically, reduced SAN responsiveness to sympathetic signaling in AngII-infused mice occurred due to upregulation of the enzyme phosphodiesterase 4D (PDE4D) in SAN myocytes, blunting catecholamine signalling. Inhibiting PDE4D activity and expression increased HR and SAN responses to catecholamine stimulation in AngII-infused mice, suggesting it may be a target for improving sympathetically mediated HR regulation in disease. Atrial fibrillation (AF) is the most common arrhythmia and frequently coexists with SAN dysfunction. While the ANS plays an important role in the generation of SAN and atrial arrhythmogenesis, endocrine factors can also contribute. Natriuretic peptides (NPs) are a family of cardioprotective hormones that act partially through NP receptor B (NPR-B), but NPR-B’s role in SAN function and atrial arrhythmogenesis is poorly understood. Thus, the second aim of this thesis was to characterize the role of NPR-B signaling on HR regulation and AF. NPR-B-deficient mice demonstrated intrinsic SAN dysfunction with reduced in vivo HR secondary to alterations in ion channel function in SAN cells. NPR-B-deficient mice were also highly susceptible to AF due to enhanced atrial catecholamine responses. Correspondingly, atrial NPR-B expression was reduced in humans with AF. Interestingly, activation of NPR-B antagonized catecholamine signaling in both mouse and human atrial myocytes. These studies highlight that NPR-B stimulation may be a novel approach for treatment and prevention of both SAN dysfunction and catecholamine-induced AF through distinct pathways.
  • Thumbnail Image
    ItemOpen Access
    Role of cyclin-dependent kinase 5 (Cdk5) in mitochondrial permeability transition pore (mPTP) opening and intracellular Ca2+ dynamics
    (2020-06-25) Navaneetha Krishnan, Saranya; Lee, Ki-Young; Riabowol, Karl T.; Lees-Miller, Susan P.; Shutt, Timothy; Braun, Andrew P.; Eitzen, Gary A.
    Cyclin-dependent kinase 5 (Cdk5), which plays a role in the development and progression of many human cancers, localizes in the mitochondria, a key determinant of apoptotic cell death. Cdk5 is upregulated in breast cancer cells and Cdk5 loss increases cancer cell sensitivity to chemotherapeutic drugs. However, the molecular mechanism by which Cdk5 loss promotes cell death remains unclear. I hypothesized that Cdk5 loss activates the intrinsic apoptotic pathway in breast cancer cells. I demonstrated that Cdk5-deficient breast cancer cells exhibit increased mitochondrial depolarization, mitochondrial reactive oxygen species (mtROS) levels, and mitochondrial fragmentation that is associated with an increase in both intracellular Ca2+ level and calcineurin activity, and dynamin related protein 1 (DRP1) Ser637 dephosphorylation. To define mitochondria-mediated apoptotic pathway, I utilized various inhibitors of mitochondrial function. Apoptosis is completely prevented by mitochondrial permeability transition pore (mPTP) inhibition, almost fully inhibited by blocking ROS and unaffected by inhibition of mitochondrial fission, suggesting that apoptosis in breast cancer cells due to Cdk5 loss occurs via a novel mPTP-dependent mechanism that acts primarily through ROS increase. Since calcium is the major regulator of mPTP opening, I hypothesized that Cdk5 regulates intracellular calcium homeostasis. Using primary mouse embryonic fibroblasts (MEFs) isolated from Cdk5?/? mouse embryos, I showed that loss of Cdk5 increases inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from internal stores. Cdk5 associates with and phosphorylates the IP3R1 Ca2+ channel at Ser421 and such phosphorylation controls IP3R1-mediated Ca2+ release as loss of Cdk5, and thus loss of IP3R1 Ser421 phosphorylation, triggers an increase in IP3R1-mediated Ca2+ release in Cdk5?/? MEFs. Analysis of subcellular fractions of MEFs demonstrates that Cdk5 localizes in the mitochondria-associated endoplasmic reticulum membrane (MAM) and Cdk5 loss causes increased ER-mitochondria tethering, a process required for Ca2+ transfer from the ER to the mitochondria. Inhibition of ER Ca2+ release or mitochondrial Ca2+ uptake in Cdk5?/? MEFs prevents mPTP opening, indicating that mPTP opening in Cdk5?/? MEFs is due to increased Ca2+ transfer from the ER to the mitochondria. Altogether, our findings suggest that Cdk5 regulates IP3R1-mediated calcium release and mitochondrial Ca2+ homeostasis that are disturbed upon Cdk5 loss, which lead to mPTP opening.