Genetic Ablation of Tbk1, a key regulator of DNA Damage Response, in Lamin A/C associated dilated cardiomyopathy
Description: Dilated cardiomyopathy (DCM) caused by mutations in LMNA gene is an autosomal inherited cardiomyopathy with high mortality rates. We show that in cardiac myocytes from human DCM (caused by pathogenic mutations in LMNA) samples, redistribution of Lamin associated domains (LADs) result in suppression of genes involved in DNA damage repair pathway. Consistent with these findings human hearts with DCM showed increased DNA damage and induction of cytosolic DNA damage sensor CGAS. Cardiac myocyte specific Lmna deleted mouse model show increased DNA damage response pathway proteins indicated by TBK1 and CGAS. TANK binding kinase 1 (TBK1), a serine threonine kinase, component of CGAS-CGAMP-STING signaling pathway, and is a downstream effector of DNA damage induced inflammation. However, the effect of TBK1 suppression on DNA damage response pathway and rescue of DCM phenotype is unknown. Hence in this study we propose to identify the upstream regulatory mechanisms involved in TBK1 activation and to determine phenotypic and molecular effects of Tbk1 inactivation in Lmna deficient mouse model. In Aim 1 we will identify upstream regulatory mechanisms involved in activation of TBK1 by assessing a) cytoplasmic accumulation of dsDNA b) activated CGAS c) STING activation. To ascertain the role of STING in TBK1 activation, we will knockdown STING, by AAV9 mediated shRNA approach invivo. In Aim2 we will assess the phenotypic and molecular effects of Tbk1 deletion on DNA damage induced inflammation in Lmna deficient mouse model, by inducible cardiac myocyte specific Cre deleter mice (Myh6-CreMCM:LmnaF/F:Tbk1F/F). We will determine the effect of Tbk1 deletion on survival by Kaplan-Meier survival curves, cardiac function by serial echocardiography, apoptosis by TUNEL assay, fibrosis by Picosirius red staining and myocyte cross sectional area by WGA staining. At molecular level the effect of Tbk1 deletion on gene expression mediated by Interferon regulatory factor 3 (IRF3)/NFkB, will be determined by, ChIP-seq and RNA-seq to identify the direct targets rescued by Tbk1 deletion in the Myh6-CreMCM:LmnaF/F mice. Taken together this study will unequivocally determine the upstream regulatory mechanism involved in TBK1 activation and the effect of Tbk1 deletion on rescue of the phenotypic and molecular defects observed in DCM. The findings from this study will provide the basis for therapeutic intervention of DNA damage induced inflammation in DCM with mutations in LMNA.
Funder: American Heart Association
Amount: $231,000
Funding type: fellowship
Funder scheme: Career Development Award
Start date: 2021-01-01
End date: 2022-08-01
Investigators: Sirisha Marreddy, Sirisha Marreddy
Institution: The University of Texas Health Science Center at Houston
Funded outputs count: 3
Funder award ID: 20CDA35310229
OpenAlex ID: G2958251564