Single-Cell RNA-seq of Human Cardiomyocytes Revealed DNA Damage Response as a Novel Predictor for Therapeutic Prognosis in Heart Failure Patients with Dilated Cardiomyopathy

Introduction: The DNA damage response (DDR) pathway is activated upon DNA damage. In mitotic cells, the DDR plays essential role in maintaining genomic stability and preventing cancer formation. DNA damage and activation of the DDR are also observed in the post-mitotic cardiomyocytes of patients with end-stage heart failure, however, their roles in the pathogenesis of heart failure remains elusive. Methods and Results: We performed transverse aortic constriction (TAC) operation to produce mice model of pressure-overload induced heart failure. Alkaline- and neutral- comet assay revealed that unrepaired DNA single-strand break (SSB), not double-strand break, is accumulated in cardiomyocytes of the failing heart. Mice with cardiomyocyte-specific deletion of XRCC1, a scaffold protein essential for SSB repair, exhibited more severe heart failure and higher mortality after TAC operation. Knockdown of Xrcc1 using siRNA produced SSB accumulation in cardiomyocytes and SSB accumulation induced persistent DDR through activation of ataxia telangiectasia mutated (ATM) kinase. Activated ATM also induced nuclear translocation of NF-κB and increased the expression of inflammatory cytokines. Activation of DDR, nuclear translocation of NF-κB, and increased expression of inflammatory cytokines were also observed in the failing heart and were enhanced in the heart of cardiomyocyte-specific XRCC1 knockout mice. Conclusions: Unrepaired DNA SSB accumulates in post-mitotic cardiomyocytes and plays a pathogenic role in pressure overload-induced heart failure. Approaches that promote efficient SSB repair or suppress aberrant activation of DDR pathway may become a novel therapeutic strategy against heart failure.

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LARP7 Protects Against Heart Failure by Enhancing Mitochondrial Biogenesis

Circulation ◽

10.1161/circulationaha.120.050812 ◽

2021 ◽

Author(s):

Huijing Yu ◽

Fang Zhang ◽

Pengyi Yan ◽

Shasha Zhang ◽

Yingmei Lou ◽

...

Keyword(s):

Myocardial Infarction ◽

Heart Failure ◽

Dna Damage ◽

Oxidative Phosphorylation ◽

Cardiac Function ◽

Dna Damage Response ◽

Mitochondrial Biogenesis ◽

Virus Serotype ◽

Damage Response ◽

Human Primate

Background: Heart failure (HF) is among the leading causes of morbidity and mortality, and its prevalence continues to rise. La ribonucleoprotein domain family member 7 (LARP7) is a master regulator that governs the DNA damage response and RNAPII pausing pathway, but the role of it in heart failure pathogenesis is incompletely understood. Methods: We assessed LARP7 expression in human HF, and in non-human primate and mouse HF models. To study the function of LARP7 in heart, we generated global and cardiac-specific LARP7 knockout mice. We acutely abolished LARP7 in mature cardiomyocytes by Cas9-mediated LARP7 somatic knockout. We overexpressed LARP7 in cardiomyocytes using adeno-associated virus serotype 9 (AAV9) and ataxia telangiectasia mutated protein (ATM) inhibitor. The therapeutic potential of LARP7-regulated pathways in heart failure was tested in a mouse myocardial infarction model. Results: LARP7 was profoundly downregulated in failing human hearts and in non-human primate and murine hearts after myocardial infarction (MI). Low LARP7 levels in failing hearts was linked to elevated reactive oxygen species (ROS), which activated the ATM-mediated DNA damage response pathway and promoted LARP7 ubiquitination and degradation. Constitutive LARP7 knockout in mouse resulted in impaired mitochondrial biogenesis, myocardial hypoplasia, and midgestational lethality. Cardiac-specific inactivation resulted in defective mitochondrial biogenesis, impaired oxidative phosphorylation, elevated oxidative stress and HF by 4 months of age. These abnormalities were accompanied by reduced SIRT1 stability and deacetylase activity which impaired SIRT1-mediated transcription of genes for oxidative phosphorylation and energy metabolism and dampened cardiac function. Restoring LARP7 expression after MI by either AAV-mediated LARP7 expression or small molecule ATM inhibitor substantially improved the function of injured heart. Conclusions: LARP7 is essential for mitochondrial biogenesis, energy production and cardiac function by modulating SIRT1 homeostasis and activity. Reduction of LARP7 in diseased hearts due to activation of the ATM pathway contributes to heart failure pathogenesis, and restoring LARP7 in the injured heart confers myocardial protection. These results identify the ATM-LARP7-SIRT1 pathway as a target for therapeutic intervention in heart failure.

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Functional single cell proteomic profiling of cells with abnormal DNA damage response dynamics

10.1101/2021.10.13.464241 ◽

2021 ◽

Author(s):

Pin-Rui Su ◽

Li You ◽

Cecile Beerens ◽

Karel Bezstarosti ◽

Jeroen Demmers ◽

...

Keyword(s):

Dna Damage ◽

Single Cell ◽

Dna Damage Response ◽

Cell Tracking ◽

Single Cells ◽

Proteomic Profiling ◽

Response Dynamics ◽

Isolated Cells ◽

Cellular Dynamics ◽

Damage Response

Tumor heterogeneity is an important source of cancer therapy resistance. Single cell proteomics has the potential to decipher protein content leading to heterogeneous cellular phenotypes. Single-Cell ProtEomics by Mass Spectrometry (SCoPE-MS) is a recently developed, promising, unbiased proteomic profiling techniques, which allows profiling several tens of single cells for >1000 proteins per cell. However, a method to link single cell proteomes with cellular behaviors is needed to advance this type of profiling technique. Here, we developed a microscopy-based functional single cell proteomic profiling technology, called FUNpro, to link the proteome of individual cells with phenotypes of interest, even if the phenotypes are dynamic or the cells of interest are sparse. FUNpro enables one i) to screen thousands of cells with subcellular resolution and monitor (intra)cellular dynamics using a custom-built microscope, ii) to real-time analyze (intra)cellular dynamics of individual cells using an integrated cell tracking algorithm, iii) to promptly isolate the cells displaying phenotypes of interest, and iv) to single cell proteomically profile the isolated cells. We applied FUNpro to proteomically profile a newly identified small subpopulation of U2OS osteosarcoma cells displaying an abnormal, prolonged DNA damage response (DDR) after ionizing radiation (IR). With this, we identified PDS5A and PGAM5 proteins contributing to the abnormal DDR dynamics and helping the cells survive after IR.

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DNA single-strand break-induced DNA damage response causes heart failure

Nature Communications ◽

10.1038/ncomms15104 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 36

Author(s):

Tomoaki Higo ◽

Atsuhiko T. Naito ◽

Tomokazu Sumida ◽

Masato Shibamoto ◽

Katsuki Okada ◽

...

Keyword(s):

Heart Failure ◽

Dna Damage ◽

Dna Damage Response ◽

Strand Break ◽

Single Strand ◽

Single Strand Break ◽

Damage Response

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Moderate and high amounts of tamoxifen in MHC-MerCreMer mice induce a DNA damage response, leading to heart failure and death

Disease Models & Mechanisms ◽

10.1242/dmm.010447 ◽

2013 ◽

Vol 6 (6) ◽

pp. 1459-1469 ◽

Cited By ~ 71

Author(s):

K. Bersell ◽

S. Choudhury ◽

M. Mollova ◽

B. D. Polizzotti ◽

B. Ganapathy ◽

...

Keyword(s):

Heart Failure ◽

Dna Damage ◽

Dna Damage Response ◽

Damage Response

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Abstract 14930: Involvement of Chloride Channel (clic) in Myocardial Fibrosis of Pressure-overload Model Mice and Dilated Cardiomyopathy Patients

Circulation ◽

10.1161/circ.142.suppl_3.14930 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Gaku Oguri ◽

Toshiaki Nakajima ◽

Nomura Seitaro ◽

Nakajima Takahumi ◽

Hironobu Kikuchi ◽

...

Keyword(s):

Heart Failure ◽

Growth Factor ◽

Cardiac Hypertrophy ◽

Dilated Cardiomyopathy ◽

Single Cell ◽

Chloride Channel ◽

Ventricular Myocytes ◽

Pressure Overload ◽

Rna Seq ◽

Single Ventricular Myocytes

Introduction: Pressure overload induces cardiac hypertrophy, electrical remodeling due to changes in various channels, and eventually leads to fibrosis, hardening of the heart, left ventricular diastolic dysfunction, and heart failure. Chloride channels (chloride intracellular channels, Clic) are localized in both plasma membranes and intracellular organelles of various cells, and have recently been reported to be associated with atrial fibrosis. In this study, we examined single-cell RNA-seq analysis of cardiomyocytes for changes in Clic using a pressure overload model due to transverse aortic coarctation (TAC). In addition, a similar analysis was performed on ventricular myocytes in dilated cardiomyopathy. Methods: Single ventricular myocytes were collected from the free wall of the left ventricle after TAC by collagenase treatment in the cardiac hypertrophy phase (3 days after TAC, 1 or 2 weeks (W)) and the heart failure phase (4,8W). The expression of various genes in ventricular myocytes was analyzed for single cells by RNA-seq and compared with sham mice. In addition, a similar study was performed from single ventricular myocytes obtained from dilated cardiomyopathy patients and donors. Results: The expression of myosin heavy chain β (Myh7), a fetal gene, was increased by pressure overload, but the expression of BNP and ANP genes was increased along with the expression of adult Myh6. Regarding Clic, Clic1,4 and 5 increased as compared with Sham mice. Clic1, Clic1 and 4 increased from 3d, and at 8W, Clic1, 4, and 5 also increased . The expression level of these Clic genes is associated with the genes associated with fibrosis (Col4a1, 4a2, 6a2, connective tissue growth factor (CTGF), transforming growth factor-beta 2 (TGFβ2). The KEGG pathway analysis using DAVID for genes with significant associations with Clic1,4, and 5 revealed a strong association with activation of Focal adhesion pathway. Single cell RNA-seq analysis of single ventricular myocytes from patients with dilated cardiomyopathy also showed a increase in CLIC4 and 5 genes compared to healthy donors. Conclusions: Single cell RNA of ventricular myocytes -seq analysis revealed the involvement of chloride channel Clic in myocardial fibrosis and structural remodeling in failing hearts.

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Pathogenic role of DNA single-strand break accumulation and DNA damage response in pressure overload-induced heart failure

Proceedings for Annual Meeting of The Japanese Pharmacological Society ◽

10.1254/jpssuppl.wcp2018.0_or2-4 ◽

2018 ◽

Vol WCP2018 (0) ◽

pp. OR2-4

Author(s):

Atsuhiko T. Naito ◽

Tomoaki Higo ◽

Hiroko Izumi-Nakaseko ◽

Kentaro Ando ◽

Mihoko Hagiwara-Nagasawa ◽

...

Keyword(s):

Heart Failure ◽

Dna Damage ◽

Dna Damage Response ◽

Pressure Overload ◽

Strand Break ◽

Single Strand ◽

Pathogenic Role ◽

Single Strand Break ◽

Damage Response

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DAXX-ATRX-H3.3 Regulation of p53 Chromatin Binding and DNA Damage Response

10.21203/rs.3.rs-966038/v1 ◽

2021 ◽

Author(s):

Nitish Gulve ◽

Zhong Deng ◽

Samantha Soldan ◽

Olga Vladimirova ◽

Jayamanna Wickramasinghe ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Chromatin Binding ◽

Rna Seq ◽

Loss Of Function ◽

Glioblastoma Cells ◽

Histone H3.3 ◽

Alternative Lengthening ◽

Damage Response ◽

Chaperone Complex

Abstract DAXX and ATRX are tumor suppressor proteins that form a histone H3.3 chaperone complex and are frequently mutated in cancers with the alternative lengthening of telomeres (ALT), such as pediatric glioblastoma. Rapid loss of function of either DAXX or ATRX are not by themselves sufficient to induce the ALT phenotype. However, cells lacking DAXX or ATRX can be readily selected for ALT-like features. Here, we show that DAXX and ATRX null glioblastoma cells with ALT-like features have defects in p53 chromatin binding and DNA damage response regulation. RNA-seq analysis of DAXX or ATRX null U87 glioblastoma cells with ALT-like features revealed that p53 pathway is among perturbed. ALT-selected DAXX and ATRX-null cells had aberrant response to DNA damaging agent etoposide. Both DAXX and ATRX-null ALT cells showed a loss of p53 binding at a subset of response elements. Complementation of DAXX null cells with a wild-type DAXX transgene rescued p53 binding and transcription, while the tumor associated mutation L130R that disrupts ATRX binding was incapable of rescuing p53 chromatin binding. We show that histone H3.3 binding is reduced in DAXX-null cells especially at subtelomeric p53 binding sites and telomere repeats. These findings indicate that DAXX and ATRX function to enable p53 chromatin binding through modulation of histone H3.3 binding, especially at sub-telomeric sites.

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