Author ORCID Identifier

https://orcid.org/0000-0003-4824-4779

Document Type

Thesis

Date of Award

12-2021

Degree Name

Master of Science (MS)

Department

Basic Biomedical Science

First Advisor

Xuejun Wang

Abstract

Background: Within a large subset of heart failure, cardiac ubiquitin-proteasome system (UPS) inadequacy is causative. A vital UPS regulator is the COP9 signalosome (CSN). The CSN holocomplex is formed by 8 unique protein subunits (COPS1~COPS8) and regulates Cullin-RING ligases via Cullin deneddylation. Cardiomyocyte-restricted knockout (cko) of Cops8 causes massive cardiomyocyte necroptosis via the RIPK1-RIPK3-MLKL pathway, resulting in dilated cardiomyopathy (DCM) and shortened lifespan in mice. CSN’s deneddylase resides in COPS5 and Cops5-cko has not been explored. It is important to investigate how the necroptotic pathway within cardiomyocytes is activated in Cops8-cko mice as cardiomyocyte necroptosis has been shown to play an important role in cardiac pathophysiology. Protein kinase Cδ (PKCδ) regulates cardiomyocyte survival but its potential involvement in cardiomyocyte necroptosis remains unknown. To fill these gaps, we conducted the present study to examine the hypothesis that Cullin deneddylation suppresses cardiomyocyte necroptosis but PKCδ promotes the activation of cardiomyocyte canonical necroptotic pathway in Cops8-cko mice. Methods and Results: Cops8-cko, Cops5-cko, or Cops8+Cops5 double cko (Double-cko) initiated in adult mice were achieved using a tamoxifen-inducible Cre-LoxP system. Echocardiography performed 21 days after tamoxifen withdrawal showed no significant difference between 2 control groups: Myh6-MerCreMer transgenic mice (MCM) and Cops5-floxed/Cops8-floxed mice. Compared with MCM, all cko groups displayed DCM, but the severity in the Cops5-cko and Double-cko groups were similarly greater than that in the Cops8-cko group. Kaplan-Meier survival analyses revealed that the post-tamoxifen lifespans of Cops5-cko and Double-cko mice (median, 42 days) were comparably shortened but significantly shorter than that of Cops8-cko mice (81 days). We observed a greater proportion of cardiomyocytes positive for Evans blue dye uptake and greater increases of myocardial CD45 proteins in Cops5-cko and double-cko mice than in Cops8-cko mice. Both Cops8-cko and Cops5-cko mice exhibited a 2.5-fold upregulation of myocardial PKCδ. Coupling heterozygous germ-line knockout of PKCδ with perinatal Cops8-cko decreased the activation of the RIP1-RIP3-MLKL pathway, attenuated cardiac malfunction, and delayed mouse premature death induced by Cops8-cko. Conclusions: Cullin-deneddylation activity is required for the CSN to suppress cardiomyocyte necroptosis; upregulation of PKCδ contributes to activation of the RIPK1-RIPK3-MLKL pathway by Cops8 deficiency in cardiomyocytes in mice.

Subject Categories

Molecular Biology

Keywords

MOLECULAR MECHANISMS, CARDIOMYOCYTE NECROPTOSIS, MICE COP9 SIGNALOSOME

Number of Pages

60

Publisher

University of South Dakota

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