Mutations in the LMNA gene, encoding lamin A/C (LMNA) protein, cause a diverse array of diseases referred to as laminopathies. Dilated cardiomyopathy (DCM) is common and the main cause of death in laminopathies. LMNA mutations are also the second most common causes of familial DCM. DCM due to LMNA mutations (hereafter, LMNA-DCM) has a poor prognosis and a high incidence of sudden cardiac death. The underpinning mechanism(s) of LMNA-DCM is unknown. Hence, there is no effective therapy for LMNA-DCM. We have shown that LMNA binds to about 300 genomic regions in human cardiac myocytes, which are referred to as Lamin-Associated Domains (LADs). LADs comprise about 20% of the genome and several hundred genes. We show that LADs are redistributed in LMNA-DCM, resulting in Gain of LADs (GoL) and Loss of LADs (LoL). LoL is associated with active transcription, whereas GoL suppresses gene expression. At the mechanistic level, Preliminary data show that LADs are lost at the binding motifs for CTCF protein. CTCF insulates transcriptionally active chromatin loops and recruits topoisomerase 2B (TOP2B) to cut the compact chromatin and open the loops for active transcription. In parallel with these findings, we show that double stranded DNA breaks (DSBs), induced by TOP2B, are increased. We also show that expression of DSB repair genes is suppressed in LMNA-DCM, partly because of GoL. Consequently, DSBs are released into the cytoplasm and sensed by CGAS protein, which activates DNA damage response (DDR) and expression of genes involved in cell death, senescence, fibrosis, and cardiac dysfunction, phenotypic features of human LMNA-DCM. We propose to study the mechanisms responsible for increased DSBs and impaired DSB repair, and determine therapeutic effects of targeting the DDR pathway in LMNA-DCM.
In aim 1, we will test the hypothesis that LoL leads to active transcription, induction of DSBs, and stalled TOP2B, whereas GoL suppresses transcription (hence, no DSBs). To test this hypothesis, we will map genome-wide DSB sites at the nucleotide level by END-Seq technique, compare distribution density of DSBs at LoL, GoL, and non-LAD regions in control and LMNA-DCM hearts, and determine stalling of TOP2B at the DSBs by ChIP-Seq.
In aim 2, we will test the hypothesis that DSB repair is impaired in LMNA-DCM partly because GoL suppresses expression of key repair genes and in. part because recruitment of the repair enzymes to the DSB sites is impaired. Recruitment and assembly of the selected repair proteins at the DSBs will be analyzed by ChIP-Seq to map the binding motifs to DSB sites. Interactions between LMNA and the repair proteins will be determined by immunoprecipitation.
In aim 3, the DDR pathway will be blocked genetically and pharmacologically by inhibiting CGAS, the key sensor of the cytoplasmic DNA, in two mouse models of LMNA-DCM. The ensuing effects on survival, cardiac function, gene expression, DDR activation, fibrosis, senescence, and apoptosis will be determined. The findings could delineate the mechanisms of increased DSBs and determine therapeutic effects of targeting of the DDR in LMNA-DCM.

Public Health Relevance

Genetic defects, including defects in Lamin A/C gene (LMNA) are important causes of heart failure and premature death. We have identified DNA damage as the underpinning mechanism in heart failure caused by LMNA mutations. We propose to delineate the responsible mechanisms and determine therapeutic benefits of targeting the DNA damage response pathway in prevention, attenuation, and reversal of heart failure.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL132401-05
Application #
10049013
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Schwartz, Lisa
Project Start
2016-03-15
Project End
2024-07-31
Budget Start
2020-08-01
Budget End
2021-07-31
Support Year
5
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Type
Schools of Medicine
DUNS #
800771594
City
Houston
State
TX
Country
United States
Zip Code
77030
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