All ETDs from UAB

Advisory Committee Chair

John C Chatham

Advisory Committee Members

Richard B Marchase

Chenbei Chang

Martin E Young

Suzanne Oparil

Document Type


Date of Award


Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine


In non-excitable cells, STIM1 and Orai1 are the primary mediators of store-operated Ca2+ entry (SOCE); however little is known about their roles in cardiomyocyte Ca2+ handling. We have previously shown that acute increases in the levels of O-GlcNAcylation (the attachment of O-linked N- acetylglucosamine (O-GlcNAc) to target proteins) are cardioprotective through attenuating Ca2+-mediated injury and increased O-GlcNAcylation blunts SOCE in cardiomyocytes. Therefore, the goals here were to determine the regulation of STIM1 and STIM1-mediated SOCE by O-GlcNAcylation in cardiomyocytes. Immunohistochemical analysis demonstrated the co-localization of STIM1 with SERCA and RyR in adult cardiomyocytes. NRVMs were transfected with the eYFP-tagged STIM1 and/or mCherry-tagged Orai1 adenovirus overnight and then visualized for STIM1 puncta and STIM1-Orai1 coupling by confocal microscopy. Under basal conditions STIM1 occurred as a reticular network while Orai1 was predominantly localized on the plasma membrane, consistent with their distribution in non-excitable cells; depletion of ER Ca2+ stores with 2mM EGTA and 5µM thapsigargin (TG) resulted in the dynamic STIM1 puncta formation (<3 min) in ~90% of cells, followed by a co-localization of STIM1 and Orai1 (<5 min). Treatment with glucosamine (GlcN) or thiamet-G (TMG) to increase overall O-GlcNAcylation levels significantly attenuated STIM1 puncta formation in a dose-dependent manner. Ca2+ imaging with Fluo-4 Am demonstrates that SOCE in NRVMs is highly Ca2+-selective and significantly attenuated by both GlcN and TMG. Immunoprecipitation of STIM1 followed by anti-O-GlcNAc immunoblot suggests that STIM1 is a target for O-GlcNAcylation and its O-GlcNAcylation level increased with GlcN or TMG treatment. These data indicate that the molecular machinery responsible for SOCE in non-excitable cells is present in cardiomyocytes and acute increases of O-GlcNAcylation levels significantly attenuate the response of STIM1 to ER Ca2+ depletion. This leads to the provocative hypothesis that STIM1-mediated SOCE is an important contributor to reperfusion injury in the heart, and that O-GlcNAc-mediated cardioprotection is due to inhibition of this pathway. Further, since SOCE is a fundamental mechanism underlying Ca2+ signaling in most cells and tissues, it is possible that STIM1 represents a nexus between protein O-GlcNAcylation and Ca2+-dependent signaling pathways.



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