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dc.contributor.advisorElrod, John W.
dc.creatorLambert, Jonathan Paul
dc.date.accessioned2020-10-27T15:14:03Z
dc.date.available2020-10-27T15:14:03Z
dc.date.issued2019
dc.identifier.urihttp://hdl.handle.net/20.500.12613/1682
dc.description.abstractMitochondrial Calcium (mCa2+) overload is a central event in myocardial-ischemia reperfusion (IR) injury that leads to metabolic derangement as well as activation of the mitochondrial permeability transition pore (mPTP). mPTP activation results in necrosis and loss of cardiomyocytes which results in acute death in some individuals while survivors are prone to developing heart failure and are predisposed to recurrent infarction events. mCa2+ has also long been known to activate cellular bioenergetics implicating mCa2+ in the highly metabolically demanding state of cardiac contractility. The mitochondrial calcium uniporter channel (mtCU) is a multi-subunit complex that resides in the inner mitochondrial membrane and is required for mitochondrial Ca2+ (mCa2+) uptake. Mitochondrial Calcium Uniporter B (MCUB, CCDC109B gene), a recently identified paralog of MCU, is reported to negatively regulate mCa2+ uptake; however, its precise regulation of uniporter function and contribution to cardiac physiology remain unresolved. Size exclusion chromatography of mitochondria isolated from ventricular tissue revealed MCUB was undetectable in the high-molecular weight (MW) fraction of sham animals (~700kD, size of functional mtCU), but 24 hours following myocardial ischemia-reperfusion injury (IR) MCUB was clearly observed in the high-MW fraction. To investigate how MCUB contributes to mtCU regulation we created a stable MCUB-/- HeLa cell line using CRISPR-Cas9n. MCUB deletion increased histamine-mediated mCa2+ transient amplitude by ~50% versus Wild-Type (WT) controls (mito-R-GECO1). Further, MCUB deletion increased mtCU capacitance (patch-clamp) and rate of [mCa2+] uptake. Fast protein liquid chromatography (FPLC) fractionation of the mtCU revealed that loss of MCUB increased MCU incorporation into the high-MW complex suggesting stoichiometric replacement and overall increase in functional mtCU complexes. Next, we generated a cardiac-specific, tamoxifen-inducible MCUB mouse model (CAG-CAT-MCUB x MCM; MCUB-Tg) to examine how the MCUB/MCU ratio regulates mtCU function and contributes to cardiac physiology. MCUB-Tg mice were infected with AAV9-mitycam (genetic mCa2+ reporter) and adult cardiomyocytes were isolated to record [mCa2+] transients during pacing using live cell imaging. Increasing the MCUB/MCU ratio decreased [mCa2+] peak amplitude by ~30% and significantly reduced the [mCa2+] uptake rate. FPLC assessment revealed MCUB was undetectable in the high-MW fraction of MerCreMer controls, but enriched in MCUB-Tg hearts. MCUB incorporation into the mtCU decreased the overall size of the uniporter and reduced the presence of channel gatekeepers, MICU1/2. Immunoprecipitations suggest that MCUB directly interacts with MCU but does not bind MICU1/2. These results suggest that MCUB replaces MCU in the mtCU and thereby modulates the association of MICU1/2 to regulate channel gating. Cardiomyocytes isolated from MCUB-Tg hearts displayed decreased maximal respiration and reserve capacity, which correlated with a severe impairment in isoproterenol-induced contractile reserve (LV invasive hemodynamics). MCUB-Tg cardiac mitochondria were resistant to Ca2+-induced mitochondrial swelling suggesting MCUB limits mitochondrial permeability transition. Further, MCUB-Tg mice subjected to in vivo myocardial IR revealed a ~50% decrease in infarct size per area-at-risk suggesting increased MCUB expression prevents mCa2+ overload and limits cell death. These data suggest that MCUB regulation of the mtCU is an endogenous compensatory mechanism to decrease mCa2+ overload during ischemic injury, but this expression is maladaptive to cardiac energetic responsiveness and contractility.
dc.format.extent170 pages
dc.language.isoeng
dc.publisherTemple University. Libraries
dc.relation.ispartofTheses and Dissertations
dc.rightsIN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available.
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/
dc.subjectBiology
dc.subjectBiology, Molecular
dc.subjectCellular Biology
dc.subjectCalcium
dc.subjectMitochondria
dc.titleMCUB REGULATES THE MOLECULAR COMPOSITION OF THE MITOCHONDRIAL CALCIUM UNIPORTER CHANNEL TO LIMIT MITOCHONDRIAL CALCIUM OVERLOAD DURING STRESS
dc.typeText
dc.type.genreThesis/Dissertation
dc.contributor.committeememberTilley, Douglas G.
dc.contributor.committeememberRamirez, Servio H.
dc.contributor.committeememberKishore, Raj
dc.contributor.committeememberFinkel, Toren
dc.description.departmentBiomedical Sciences
dc.relation.doihttp://dx.doi.org/10.34944/dspace/1664
dc.ada.noteFor Americans with Disabilities Act (ADA) accommodation, including help with reading this content, please contact scholarshare@temple.edu
dc.description.degreePh.D.
refterms.dateFOA2020-10-27T15:14:03Z


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