REEP5 depletion causes sarco-endoplasmic reticulum vacuolization and cardiac functional defects

The sarco-endoplasmic reticulum (SR/ER) plays an important role in the development and progression of many heart diseases. However, many aspects of its structural organization remain largely unknown, particularly in cells with a highly differentiated SR/ER network. Here, we report a cardiac enriched, SR/ER membrane protein, REEP5 that is centrally involved in regulating SR/ER organization and cellular stress responses in cardiac myocytes. In vitro REEP5 depletion in mouse cardiac myocytes results in SR/ER membrane destabilization and luminal vacuolization along with decreased myocyte contractility and disrupted Ca2+ cycling. Further, in vivo CRISPR/Cas9-mediated REEP5 loss-of-function zebrafish mutants show sensitized cardiac dysfunction upon short-term verapamil treatment. Additionally, in vivo adeno-associated viral (AAV9)-induced REEP5 depletion in the mouse demonstrates cardiac dysfunction. These results demonstrate the critical role of REEP5 in SR/ER organization and function as well as normal heart function and development

Self-renewing resident arterial macrophages arise from embryonic CX3CR1+ precursors and circulating monocytes immediately after birth

Resident macrophages densely populate the normal arterial wall, yet their origins and the mechanisms that sustain them are poorly understood. Here we use gene-expression profiling to show that arterial macrophages constitute a distinct population among macrophages. Using multiple fate-mapping approaches, we show that arterial macrophages arise embryonically from CX3CR1+ precursors and postnatally from bone marrow–derived monocytes that colonize the tissue immediately after birth. In adulthood, proliferation (rather than monocyte recruitment) sustains arterial macrophages in the steady state and after severe depletion following sepsis. After infection, arterial macrophages return rapidly to functional homeostasis. Finally, survival of resident arterial macrophages depends on a CX3CR1-CX3CL1 axis within the vascular niche

PROTEOMIC ANALYSIS OF THE RESIDENT CELL TYPES IN THE HEART: A FOCUS ON THE FIBROBLAST AND ITS SECRETED PROTEOME

The cardiac fibroblast (CF) is a key cellular component of the heart, being reported in some cases as the largest cell population in the heart. CF play a major role in the homeostasis of the tissue, being involved in extracellular matrix (ECM) maintenance and turnover. It is also important in the heart's pathophysiology and stress response as CF are secretory cells that affect other cell types in the heart. This thesis includes studies that investigate how the CF proteome is altered during hypoxic stress. In brief, proteomic analysis of the CF identified 6163 proteins with subsets altered due to hypoxia. Of particular interest was the CF secreted proteome, which when introduced caused an altered response to hypoxia/reoxygenation in cardiomyocytes. Additionally, it was investigated how the CF differed at the proteome level to the other resident cell types in the heart: the cardiomyocyte (CM), endothelial cell (EC), and smooth muscle cell (SMC). This was in an effort to identify alternative cell type-enriched markers to identify the specific cell type contributors of proteome changes in the heart, which is generally performed at the tissue-level, where cellular resolution of these changes is lost. The second set of proteome studies identified 367 FB-enriched, 97 CM-enriched, 340 EC-enriched, and 52 SMC-enriched proteins. We rank ordered cell-specific enrichment of proteins via magnitude and significance of enrichment. With enrichment data being the most pronounced in FB, we then validated expression of multiple highly-ranked candidates (GRB2, AKR1B1, and PRDX1) in myocardial infarction tissue sections. Immunofluorescent staining showed higher expression in the highly fibrotic infarct scar of 4-week post-MI relative to border and non-infarct regions, confirming the cellular enrichment of our candidates. Potential markers of each cell type have been validated by HPA and immunofluorescent imaging of MI.Ph.D.2019-12-19 00:00:0

Hypoxia-Induced Changes in the Fibroblast Secretome, Exosome, and Whole-Cell Proteome Using Cultured, Cardiac-Derived Cells Isolated from Neonatal Mice

Cardiac fibroblasts (CFs) represent a major subpopulation of cells in the developing and adult heart. Cardiomyocyte (CM) and CF intercellular communication occurs through paracrine interactions and modulate myocyte development and stress response. Detailed proteomic analysis of the CF secretome in normal and stressed conditions may offer insights into the role of CF in heart development and disease. Primary neonatal mouse CFs were isolated and cultured for 24 h in 21% (normoxic) or 2% (hypoxic) O₂. Conditioned medium was separated to obtain exosomes (EXO) and EXO-depleted secretome fractions. Multidimensional protein identification technology was performed on secreted fractions. Whole cell lysate data were also generated to provide subcellular context to the secretome. Proteomic analysis identified 6163 unique proteins in total. Statistical (QSpec) analysis identified 494 proteins differentially expressed between fractions and oxygen conditions. Gene Ontology enrichment analysis revealed hypoxic conditions selectively increase expression of proteins with extracellular matrix and signaling annotations. Finally, we subjected CM pretreated with CF secreted factors to hypoxia/reoxygenation. Viability assays suggested altered viability due to CF-derived factors. CF secretome proteomics revealed differential expression based on mode of secretion and oxygen levels in vitro

The Significance of Hepatitis G Virus in Serum of Patients With Sporadic Fulminant and Subfulminant Hepatitis of Unknown Etiology

Excluding acute hepatic failure caused by drugs, the etiology of fulminant hepatitis (FH) remains unknown in many patients. There are conflicting data about a possible pathogenic role for the hepatitis G virus (HGV) in patients with cryptogenic fulminant hepatitis (non–A-E FH). We investigated the presence of circulating HGV in 36 patients with well-documented non–A-E fulminant and 5 patients with subfulminant hepatitis from 3 geographic locations in the United States. Serum HGV RNA was determined by reverse transcriptase-polymerase chain reaction using primers from the NS5 region of the HGV genome. HGV RNA was also measured before and after liver transplantation in 5 patients and at different time points in 7 patients. Serum samples were recoded and reanalyzed for HGV RNA using different primer sets to assess the validity of the HGV RNA assay. HGV was present in serum of 14 of the 36 patients (38.8%) with non–A-E fulminant hepatitis. Twenty percent of patients from the Northeast, 11% of the patients from the Southeast, and 50% from the Mid-Atlantic regions of the United States had circulating HGV RNA. The use of therapeutic blood products was significantly associated with the presence of serum HGV RNA (P < .02). Retesting for HGV RNA with different primers was positive in all but 1 case. HGV RNA is not causally related to non–A-E fulminant hepatitis. The finding of HGV RNA in serum from these patients is likely related to the administration of blood product transfusion after the onset of fulminant hepatitis