Abstract

   Rationale: Dilated cardiomyopathy (DCM) is a severe cardiac condition
   characterized by ventricular dilation and systolic dysfunction, often
   leading to heart failure. While the DNA damage response (DDR) pathway
   is increasingly implicated in DCM pathogenesis, the precise mechanisms
   linking DDR activation to specific pathological features like adverse
   extracellular matrix (ECM) remodeling and fibrosis remain poorly
   understood. Interferon-inducible protein 16 (IFI16), a known DNA sensor
   involved in DDR and inflammatory signaling, emerges as a potential
   mediator in this process. This study aimed to investigate the role of
   the DDR-IFI16 axis in DCM, specifically exploring its connection to ECM
   dysregulation and cardiac dysfunction, and to evaluate its potential as
   a therapeutic target.

   Methods: W This study integrated bioinformatics analyses of human
   cardiac transcriptomic datasets with experimental validation in a
   doxorubicin-induced murine DCM model. Cardiac function was assessed by
   echocardiography. Key molecular pathways were investigated using qPCR,
   ELISA, and enrichment analyses. Mechanistic roles were tested via
   pharmacological DDR inhibition in vivo and targeted IFI16 siRNA
   knockdown in vitro, followed by analysis of fibrosis, cell viability,
   and cytotoxicity markers.

   Results: Bioinformatic analyses consistently revealed activation of DDR
   and cytosolic DNA sensing pathways across human iPSC-CM models and ex
   vivo DCM heart tissue. WGCNA identified a key gene module strongly
   associated with DCM, co-enriched for DDR, DNA replication, and
   ECM/TGF-β signaling pathways. Single-cell RNA-seq analysis confirmed
   significant IFI16 upregulation in human DCM samples. High IFI16
   expression strongly correlated with pathways governing 'Extracellular
   matrix organization' and key fibrotic genes. Experimental validation in
   the doxorubicin mouse model confirmed DDR activation. Crucially, in
   vivo treatment with the DDR inhibitor NU7441 significantly attenuated
   IFI16 upregulation, ameliorated cardiac dysfunction, and decreased
   cardiac fibrosis markers. Complementarily, in vitro knockdown of IFI16
   significantly reduced pro-fibrotic markers, increased cell viability,
   and decreased cell injury.

   Conclusions: Our findings delineate a novel pathogenic axis in DCM
   where nuclear stress-induced DDR activation drives the upregulation of
   the DNA sensor IFI16. IFI16 acts as a critical mediator linking DDR
   signaling to pathological ECM remodeling and fibrosis. Pharmacological
   inhibition of the upstream DDR pathway effectively mitigates IFI16
   induction, attenuates cardiac fibrosis, and improves cardiac function.
   This study identifies the DDR-IFI16-ECM remodeling axis as a crucial
   contributor to DCM pathogenesis and highlights its potential as a
   therapeutic target for mitigating adverse cardiac remodeling and
   dysfunction.

   Keywords: DCM, DNA damage response, IFI16, fibrosis, ECM

Introduction

   The maintenance of genomic integrity is a fundamental requirement for
   cellular survival and proper tissue function. The DNA damage response
   (DDR) is a sophisticated and highly coordinated network that has
   evolved to detect, signal, and repair a wide array of DNA lesions,
   ensuring the faithful transmission of genetic information [47]^1. When
   this intricate network is compromised, it can lead to genomic
   instability and contribute to the development of various human diseases
   [48]^2. Interferon-inducible protein 16 (IFI16) is an intracellular
   sensor that recognizes both self and non-self DNA, playing a crucial
   role in the innate immune system, inflammation, and the regulation of
   cell death pathways. Dilated cardiomyopathy (DCM) is a condition
   characterized by the enlargement and weakening of the heart muscle,
   leading to impaired pumping ability and potentially heart failure,
   arrhythmias, and sudden cardiac death. The causes of DCM are diverse,
   encompassing genetic factors, viral infections, toxic exposures, and
   autoimmune responses.

   The DNA damage response (DDR) is a fundamental cellular process that
   serves as a guardian of the genome, ensuring the integrity and accurate
   inheritance of genetic information [49]^2. It is a complex and highly
   regulated network composed of numerous interconnected components that
   work in concert to detect, signal, and repair a vast array of DNA
   lesions [50]^1. This intricate system is essential for maintaining
   cellular homeostasis and preventing the accumulation of genetic errors
   that can lead to disease. Emerging research suggests a complex
   interplay between DNA damage, inflammation, and the development of DCM
   [51]^3. DNA damage can trigger inflammatory pathways, and chronic
   inflammation is known to contribute to the myocardial dysfunction
   observed in DCM.

   Studies have shown that myocardial DNA damage is prevalent in DCM
   patients, with significant DNA strand breaks observed in cardiomyocytes
   [52]^4. This damage is associated with interstitial fibrosis and a
   progressive loss of cardiac function. Besides, patients with pathogenic
   variants in sarcomere genes exhibit less DNA damage and a better
   treatment response compared to those with non-sarcomere gene variants
   [53]^5. This suggests that DNA damage is a mediator between genotype
   and treatment outcomes in DCM [54]^5. Mechanismically, endogenous DNA
   damage leads to oxidative stress and apoptosis in cardiac myocytes,
   contributing to DCM. The chronic activation of p53 and increased
   oxidative stress are key mechanisms driving this process [55]^6.
   However, the key regulator links DDR and DCM have not been fully
   understood.

   Interferon-inducible protein 16 (IFI16) is a key player in cellular
   responses to both pathogenic threats and cellular stress. IFI16 plays a
   crucial role in the DNA damage response and immune signaling pathways.
   It is a member of the HIN-200 family and acts as a DNA sensor,
   participating in various cellular processes, including the regulation
   of cell growth, apoptosis, and immune responses. IFI16's involvement in
   DNA damage response is particularly significant due to its interactions
   with key proteins such as BRCA1 and p53 [56]^7^, [57]^8, which are
   essential for maintaining genomic stability and initiating apoptosis in
   response to DNA damage. Additionally, FI16 is implicated in the
   activation of cell cycle checkpoints in response to DNA damage, as
   evidenced by its involvement in the phosphorylation of p53 and
   accumulation of p21WAF1, which are critical for cell cycle arrest
   [58]^8. IFI16, with its capacity to sense DNA and activate inflammatory
   signaling, presents a potential molecular link between DNA damage and
   the pathogenesis of DCM [59]^9. Furthermore, defects in the DDR system
   can lead to genomic instability and cellular stress, which may
   contribute to the deterioration of cardiac function in DCM [60]^10. The
   convergence of these biological processes, all potentially influenced
   by IFI16, underscores the need for a comprehensive investigation into
   their interconnections. This report aims to explore the relationship
   between the DDR, IFI16, and DCM, focusing on IFI16's potential role as
   a mediator and marker, and to identify therapeutic implications arising
   from their interplay.

Methods

Mice

   Procedures involving animals were carried out adhering to guidelines
   sanctioned by the Institutional Animal Care and Use Committee (IACUC)
   at China Academy of Chinese Medical Sciences. To establish a dilated
   cardiomyopathy (DCM) phenotype, C57BL/6 male rodents (20-25 g) were
   administered intraperitoneal (i.p.) doses of doxorubicin hydrochloride.
   The dosage was 5 mg/kg, given weekly across a six-week duration,
   utilizing a protocol adapted from prior research. Subjects in the
   control group received i.p. delivery of a matching volume of PBS
   following an identical timing regimen. For inhibiting the DNA Damage
   Response (DDR), certain mice received treatment with NU7441
   (SelleckChem, Cat# S2638) [61]^11^, [62]^12, recognized as a selective
   blocker for the DNA-dependent protein kinase catalytic subunit
   (DNA-PKcs). The inhibitor was prepared in a solution containing 10%
   DMSO to reach the target strength. This compound was given through the
   intraperitoneal route, utilizing a dosage of 3 mg/kg. Delivery of
   either NU7441 or its carrier solution started simultaneously alongside
   the initial doxorubicin dose and occurred each day throughout the
   entire doxorubicin regimen. Upon concluding the intervention phase,
   heart performance was evaluated using echocardiography [63]^13^,
   [64]^14. Afterward, animals were humanely sacrificed for harvesting
   organ samples designated for subsequent examinations, including
   quantitative PCR and ELISA, to quantify markers related to DDR and
   fibrosis [65]^15^-[66]^18.

Echocardiography

   Evaluation of left ventricular (LV) metrics was carried out via
   echocardiography on non-anesthetized animals employing a GE Vivid 7
   Dimension device coupled with a GE i13L 10-14 MHz probe. For examining
   LV diastolic performance, Doppler ultrasound techniques (pulse wave or
   tissue modes) were acquired using the Vevo3100 Imaging System (Fujifilm
   Visual Sonics) while subjects were under isoflurane anesthesia. Left
   ventricular measurements were subsequently calculated as the mean value
   derived from three sequential cardiac cycles [67]^19^, [68]^20.

Cell culture

   The HL-1 cardiomyocyte line was propagated using a specialized Claycomb
   formulation (Sigma-Aldrich), fortified with 10% fetal bovine serum,
   penicillin (100 U/ml), streptomycin (100 μg/ml), norepinephrine (0.1
   mM), plus L-glutamine (2 mM). Growth surfaces were pre-treated with a
   combination containing fibronectin and gelatin (both Sigma-Aldrich) to
   enhance cellular attachment. Propagation occurred within an incubator
   providing humidity, set to 37 °C and supplying 5 % carbon dioxide,
   offering ideal conditions for proliferation and viability [69]^21^,
   [70]^22. For experimental procedures, cardiomyocytes were plated into
   standard 6-well formats using an initial cell number calculated to
   achieve 70-80 % surface coverage when interventions began. Upon
   reaching the target density, cultures were exposed to doxorubicin
   (Sigma-Aldrich) at a 5 μm/L. This agent was derived from a concentrated
   stock dissolved in either sterile H₂O or phosphate-buffered saline
   (PBS) and introduced straight to the growth media, ensuring the carrier
   solvent level stayed under 0.1 %. Parallel control cultures were
   administered a matching amount of the carrier solution alone [71]^23^,
   [72]^24. This exposure period lasted for two days within the normal
   incubator environment. Subsequently, cellular material was collected
   for subsequent investigations aimed at evaluating indicators and
   characteristics linked to the dilated cardiomyopathy condition
   [73]^25^, [74]^26.

Total RNA isolation and real-time PCR analysis

   Cellular ribonucleic acid extraction was accomplished employing the
   TransZol Up Plus RNA Kit (TransGen Biotech, Beijing, China; Cat#
   ER501-01). The extracted RNA subsequently served as a template for
   conversion into complementary DNA (cDNA) utilizing random hexamers
   alongside SuperScript III reverse transcriptase (Thermo Fisher
   Scientific, Waltham, MA, USA; Cat# 12574035). Quantitative PCR (qPCR)
   analysis was conducted on an ABI 7900HT platform (Life Technologies)
   using reaction mixtures containing TransStart® Top Green qPCR SuperMix
   (TransGen Biotech, Beijing, China; Cat# AQ131-01) [75]^27^, [76]^28.
   Cycling parameters applied during qPCR runs involved an initial 10 min
   hold at 95 °C for enzyme activation, succeeded by 40 amplification
   rounds comprised of: denaturing at 95 °C for 30 s, annealing at 60 °C
   for 30 s, and extension at 72 °C for 30 s. The presence of solitary
   peaks upon dissociation curve assessment verified amplification product
   specificity [77]^29^, [78]^30. 18S ribosomal RNA served as the internal
   reference for transcript quantification, and relative expression
   changes were determined employing the 2^-ΔΔCt approach for comparisons
   [79]^31^, [80]^32. Oligonucleotides targeting specific genes utilized
   in the qPCR assays included: Bax, Fwd: 5'-TGAAGACAGGGGCCTTTTTG-3', Rev:
   5'-AATTCGCCGGAGACACTCG-3'; Ogg1, Fwd: 5'-CTGCCTAGCAGCATGAGACAT-3', Rev:
   5'-CAGTGTCCATACTTGATCTGCC-3'; Xpc, Fwd: 5'-TCCAGGGGACCCCACAAAT-3', Rev:
   5'-GCTTTTTGGGTGTTTCTTTGCC-3'; Ku70, Fwd: 5'-ATGTCAGAGTGGGAGTCCTAC-3',
   Rev: 5'-TCGCTGCTTATGATCTTACTGGT-3'; Chk2, Fwd:
   5'-TGACAGTGCTTCCTGTTCACA-3', Rev: 5'-GAGCTGGACGAACCCTGATA-3'; Col1a1,
   Fwd: 5'-GCTCCTCTTAGGGGCCACT-3', Rev: 5'-CCACGTCTCACCATTGGGG-3';
   β-actin: Fwd: 5'-TCTGGCACCACACCTTCTA-3', Rev:
   5'-AGGCATACAGGGACAGCAC-3'.

siRNA transfection

   For in vitro gene silencing studies, HL-1 cells were cultured in
   Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10 % fetal
   bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin and
   maintained in a humidified incubator at 37 °C with 5 % CO[2]. Cells
   were seeded into 6-well plates at a density of 1 x 10^5 cells/well for
   6-well plates and allowed to adhere for approximately 24 h.
   Subsequently, cells were transfected with either a validated small
   interfering RNA targeting IFI16 (si-IFI16) or a non-targeting control
   siRNA (si-Ctrl) at a final concentration of 25 nM. Transfection was
   performed using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen)
   following the manufacturer's instructions [81]^33^-[82]^35. Briefly,
   siRNA and the transfection reagent were individually diluted in
   Opti-MEM I Reduced Serum Medium (Gibco), then combined and incubated
   for 10-20 minutes at room temperature to allow complex formation before
   being added dropwise to the cells in fresh culture medium. After 6 h of
   incubation, the transfection medium was replaced with fresh complete
   growth medium. At 72 h post-transfection, cells were collected for mRNA
   analysis (Col1a1, Col1a3), or cell culture supernatants were collected
   for TGF-β activity measurement, or cells were subjected to MTT assays
   for viability assessment and LDH release assays for cytotoxicity
   measurement according to standard protocols [83]^36^, [84]^37.

ELISA assays

   Following enumeration, cells were dispensed into 96-well format dishes
   and subsequently incubated for one day. After this incubation period,
   conditioned media was harvested [85]^38^, [86]^39. Passage through 0.45
   μm filters then occurred in order to remove any non-adherent cells or
   debris. Levels of both Transforming Growth Factor-beta (TGF-β) and
   gamma-H2AX protein present within the collected media were quantified
   employing dedicated enzyme-linked immunosorbent assay kits sourced from
   Abcam (Cambridge, UK). Every specimen was assessed using multiple
   independent biological preparations, with triplicate measurements
   performed for each preparation [87]^40^, [88]^41.

MTT and LDH assays

   HL-1 cardiomyocytes underwent cultivation followed by exposure to
   doxorubicin at 5 μm/L concentration over a two-day period, creating an
   in vitro system mimicking aspects of dilated cardiomyopathy (DCM)
   [89]^42^, [90]^43. To evaluate metabolic activity, after the drug
   exposure duration, the MTT compound was introduced straight into the
   well contents, reaching a 0.5 mg/ml final concentration. Incubation
   proceeded at 37 °C for three to 4 h, allowing violet formazan
   precipitate formation. Afterwards, the overlaying liquid was aspirated,
   and the precipitate was dissolved using dimethyl sulfoxide (DMSO);
   optical density readings taken at 570 nm via a plate spectrophotometer
   served for determining relative cell survival [91]^44^, [92]^45.

   Concurrently, cytotoxicity assessment via LDH release was conducted by
   gathering the conditioned media following the same drug exposure
   period. Extracellular lactate dehydrogenase activity was measured
   utilizing a purchased LDH assay kit per the supplier's guidelines. This
   involved combining the gathered media with the kit's reagent solution
   (containing NADH and a tetrazolium salt); generation of the colored
   formazan derivative was then assessed via absorbance reading at 490
   nanometers. Untreated control groups were administered a matching
   amount of the carrier solution. Each assay condition was performed
   three times independently for robust data analysis [93]^46^, [94]^47.

Raw data processing

   This investigation utilized public repositories on Gene Expression
   Omnibus (GEO) under accession numbers [95]GSE5406 and [96]GSE197850.
   The [97]GSE197850 dataset comprised samples from 4 cardiomyocytes
   representing dilated models alongside 4 corresponding control heart
   cells. Meanwhile, dataset [98]GSE5406 contained eighty-six heart tissue
   specimens derived from DCM patients plus sixteen specimens from
   non-diseased hearts [99]^48^, [100]^49. Information regarding genes
   associated with DNA damage responses (DARGs) was sourced through the
   GeneCards resource [101]^50^, [102]^51.

Data filtering and processing

   Precise transcript abundance information was obtained through custom
   Perl scripts implementing alignment and ordering procedures on the gene
   expression datasets. Subsequently, a normalization step was performed
   on both the [103]GSE5406 and [104]GSE197850 cohorts [105]^52^,
   [106]^53. Specifically, the "limma" package within R software handled
   the pre-processing plus normalization tasks for the initial gene count
   tables derived from the aforementioned GEO repositories
   [107]^54^-[108]^57.

Co-expression gene identification and enrichment analyses

   Application of the Weighted Gene Co-expression Network Analysis (WGCNA)
   approach enabled gene grouping into modules, and subsequently,
   relationships connecting these gene groups with specific
   characteristics were evaluated [109]^58^, [110]^59. Network structures
   based on co-expression were built employing the upper quartile of genes
   exhibiting the highest variance within the [111]GSE5406 cohort data.
   The dynamic tree cutting technique, utilizing a merging threshold set
   at 0.25, facilitated the consolidation of modules. Subsequently,
   modules displaying the highest correlation with relevant traits were
   identified and characterized. Functional enrichment investigations were
   performed utilizing the R software package 'clusterProfiler'.
   Furthermore, Gene Set Enrichment Analysis (GSEA) was employed to
   discover relevant biological processes or routes.

ScRNA seq analysis

   The single-cell RNA sequencing (scRNA-seq) dataset [112]GSE121893 was
   acquired via the Gene Expression Omnibus (GEO) repository. Subsequent
   analysis of this scRNA-seq data was carried out in the R environment,
   utilizing primarily version 4.3.0.1 of the Seurat toolkit for sample
   processing. Initial quality control involved removing 12 individual
   cells exhibiting feature counts below 200 or exceeding 5,000 detected
   genes [113]^60^-[114]^62. Following library size normalization
   (accomplished via NormalizeData), the two thousand features showing
   greatest variability were selected employing the FindVariableFeatures
   routine. Expression values for these variable features were
   subsequently scaled utilizing the ScaleData command. Principal
   component analysis (PCA) dimensionality reduction was conducted based
   on the scaled variable genes (executed via RunPCA), aiming to condense
   the feature space within the single-cell dataset. The Harmony algorithm
   implementation in R served for integrating batch effects across the
   eighteen distinct samples [115]^63^, [116]^64. Uniform Manifold
   Approximation and Projection (UMAP) visualizations were produced based
   on the Harmony-corrected dimensions (computed with RunUMAP).
   Correspondingly, cell populations were grouped via a graph clustering
   approach utilizing the Harmony-integrated space, applying a resolution
   parameter of 0.7. Employment of the FindAllMarkers command allowed
   identification of distinguishing transcripts for every cell group.
   Subsequently, population types were assigned by consulting published
   cell type signatures alongside the CellMarker version 2.0 resource.
   Comparisons of gene expression profiles between specified populations
   were performed via the FindMarkers tool, utilizing the Wilcoxon
   rank-sum statistical test. The Benjamini-Hochberg procedure was applied
   for false discovery rate adjustment on the results [117]^65^, [118]^66.

Statistics

   Assessment of data normality employed the Shapiro-Wilk procedure.
   Evaluating differences between two independent groups involved an
   unpaired, two-sided Student's t-test, incorporating Welch's adjustment
   if variances were found to be unequal. Alternatively, the Mann-Whitney
   U test was applied when comparing two sets of data lacking a normal
   distribution pattern. When assessing differences across three or more
   groups, appropriate analysis of variance (one-way, two-way, or two-way
   repeated measures) was utilized, succeeded by post-hoc analysis using
   Sidak's correction for multiple comparisons. For non-parametric
   multi-group comparisons (if any group deviated from normality), the
   Kruskal-Wallis H test was employed, followed by Dunn's post-hoc test
   for pairwise assessments [119]^67^, [120]^68. Results are presented
   showing the mean value plus or minus the standard error of the mean
   (SEM). A threshold for statistical significance was established at a
   P-value below 0.05. All statistical computations were executed using
   GraphPad Prism software, version 10.

Results

Activation of DNA damage sensing and inflammatory pathways in an iPSC-CM
model of dilated cardiomyopathy

   To investigate the transcriptional landscape associated with
   cardiomyopathy and the potential activation of DNA Damage Response
   (DDR), we analyzed high-throughput RNA-sequencing data from the
   publicly available [121]GSE197850 dataset. This dataset profiled human
   induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs),
   providing an in vitro model system. Our analysis specifically compared
   the transcriptomes of the dilated cardiomyopathy (DCM) model group (n =
   4) against the control iPSC-CM group (n = 4) within this dataset.
   Following data preprocessing and normalization procedures to ensure
   robust inter-sample comparability (Figure [122]1A-B), differential
   expression analysis was performed. This identified a substantial number
   of differentially expressed genes (DEGs) distinguishing the DCM model
   iPSC-CMs from the controls, revealing a distinct disease-associated
   transcriptional signature visualized by heatmap clustering and volcano
   plot analysis (Figure [123]1C), including significant alterations in
   genes like ACTA1 and NPPB. To elucidate the functional implications of
   these transcriptional changes, Gene Set Enrichment Analysis (GSEA) was
   conducted. Notably, this analysis demonstrated a significant enrichment
   of the "Cytosolic DNA-sensing" pathway within the DCM model group
   (Figure [124]1D), strongly suggesting the activation of DDR mechanisms
   in these cardiomyocytes. Furthermore, consistent with a potential
   interplay between cellular stress and inflammation, pathways related to
   immune activation, such as "NF-kappa B signaling," "TNF signaling,"
   "Toll-like receptor signaling," and "Cytokine-cytokine receptor
   interaction," were also found to be significantly enriched (Figure
   [125]1D-F). Taken together, these bioinformatic analyses of iPSC-CM
   provide compelling evidence for significant transcriptomic alterations
   in this in vitro model of DCM, characterized by the pronounced
   activation of pathways involved in DNA damage sensing and
   pro-inflammatory signaling.

Figure 1.

   [126]Figure 1
   [127]Open in a new tab

   Bioinformatic analysis of RNA-seq data from [128]GSE197850 reveals
   activation of DNA damage sensing and inflammatory pathways in a human
   iPSC-CM model of dilated cardiomyopathy (DCM). (A, B) Box plots
   illustrating the distribution of expression values for each sample from
   the [129]GSE197850 dataset before (A) and after (B) normalization. Each
   box represents the interquartile range (IQR), with the central line
   indicating the median. Whiskers extend to 1.5 times the IQR.
   Normalization aligned median expression levels across samples. (C)
   Differential gene expression analysis between the DCM model group and
   control induced pluripotent stem cell-derived cardiomyocytes
   (iPSC-CMs). (D, E, F) Gene Set Enrichment Analysis (GSEA) plots showing
   pathways significantly enriched in the DCM model group compared to
   controls. Plots illustrate the enrichment score (ES) reflecting the
   degree to which a gene set is overrepresented at the extremes of the
   ranked gene list. Representative enriched pathways are shown, including
   (D) "Cytosolic DNA-sensing" and inflammatory pathways, (E) additional
   immune/inflammatory pathways like "TNF signaling", and (F) pathways
   such as "NF-kappa B signaling" and "Toll-like receptor signaling".
   Peaks indicate enrichment of the respective gene set.

Enrichment of DNA replication, repair, and cell cycle pathways in the iPSC-CM
DCM model

   Further investigation into the molecular consequences of DNA Damage
   Response (DDR) activation in the DCM model iPSC-CMs from the
   [130]GSE197850 dataset revealed significant alterations in related
   cellular processes. GSEA demonstrated a highly significant positive
   enrichment for the "DNA replication" pathway in the DCM group compared
   to controls (Figure [131]2A), suggesting an aberrant activation of DNA
   synthesis machinery. This finding was supported by the differential
   expression patterns of specific protein-coding and non-coding genes
   potentially involved in these pathways, as visualized by heatmap
   clustering which distinguished DCM samples from controls (Figure
   [132]2B), and further illustrated in the volcano plot highlighting
   numerous significantly altered genes (Figure [133]2C). Comprehensive
   pathway enrichment analyses provided additional detail, revealing
   significant overrepresentation of gene sets associated not only with
   "DNA replication" and "DNA repair" (including specific mechanisms like
   "Double-Strand Break Repair via Homologous Recombination"), but also
   intimately linked processes such as "DNA Damage Response," cell cycle
   control (e.g., "G2/M checkpoints," "G1 to S cell cycle control," "Cell
   cycle"), and key regulatory pathways like the "ATM pathway" and "p53
   activity regulation" (Figure [134]2D-E). Interestingly, enrichment
   related to "Beta regulation of extracellular matrix" was also detected
   (Figure [135]2D), potentially linking the observed DDR to extracellular
   remodeling processes relevant to DCM. Taken together, these results
   indicate a complex molecular phenotype in the DCM model iPSC-CMs
   characterized by the concurrent upregulation of DNA replication, DNA
   repair, and cell cycle control pathways alongside DDR activation.

Figure 2.

   [136]Figure 2
   [137]Open in a new tab

   Enrichment of DNA replication, DNA damage response, and cell cycle
   pathways in the iPSC-CM model of dilated cardiomyopathy from
   [138]GSE197850. (A) Gene Set Enrichment Analysis (GSEA) plot showing
   significant enrichment of the "DNA replication" pathway in the DCM
   model group compared to control induced pluripotent stem cell-derived
   cardiomyocytes (iPSC-CMs). The plot displays the enrichment score (ES)
   profile, the positions of gene set members on the ranked list of genes,
   and the corresponding Normalized Enrichment Score (NES), P-value, and
   Adjusted P-value. (B) Heatmap illustrating the expression patterns of
   selected genes, including lncRNAs, protein-coding genes, and scRNAs
   potentially involved in DNA damage response (DDR) and replication
   pathways. Rows represent genes, columns represent individual samples
   from Control (Cont) and DCM groups. Expression levels are indicated by
   color intensity, and data are clustered hierarchically. (C) Volcano
   plot visualizing differentially expressed genes (DEGs) between the DCM
   model and control iPSC-CMs. Points are colored based on significance
   thresholds. (D, E) Bubble plots summarizing pathway enrichment analysis
   results for DEGs identified between DCM and control groups. The x-axis
   indicates the Rich Factor, bubble size corresponds to the number of
   DEGs in the pathway, and bubble color represents the statistical
   significance. Key enriched pathways related to (D) DNA strand
   elongation, cell cycle checkpoints (G2/M, G1 to S), DNA replication
   pre-initiation, ATM pathway, p53 regulation, and beta regulation of
   extracellular matrix, and (E) DNA-templated DNA replication, DNA
   recombination, double-strand break repair, DNA damage response, and
   chromatin remodeling are shown.

DDR activation and suppressed DNA replication characterize human DCM hearts

   To further validate the activation of DDR in human DCM using ex vivo
   tissue, we analyzed the publicly available [139]GSE5406 dataset. This
   dataset comprises expression profiles generated using Affymetrix Human
   Genome U133A microarrays on human left ventricular myocardium. Samples
   were obtained during cardiac transplantation from patients with
   advanced DCM (including idiopathic and ischemic etiologies) and from
   non-failing (NF) donor hearts used as controls. Following quality
   control and array data normalization procedures to ensure comparability
   across samples (Figure [140]3A-B), differential expression analysis was
   performed comparing DCM tissues to NF controls. This analysis
   identified a substantial number of DEGs that effectively distinguished
   the DCM samples from the NF group, as demonstrated by hierarchical
   clustering (Figure [141]3C) and volcano plot visualization (Figure
   [142]3D), indicating significant transcriptomic remodeling in the
   diseased human heart. Subsequent GSEA revealed key perturbed biological
   pathways. Notably, significant positive enrichment of the "Cytosolic
   DNA-sensing pathway" (Figure [143]3F) and the "Chemical
   carcinogenesis-DNA adducts" pathway (Figure [144]3G) was observed in
   DCM tissues relative to controls, providing strong evidence for DDR
   activation and response to DNA damage in the failing human myocardium.
   Conversely, GSEA revealed a significant negative enrichment of the "DNA
   Replication" pathway (Figure [145]3E) in DCM hearts, suggesting a
   suppression of DNA synthesis pathways in this ex vivo pathological
   context. Therefore, analysis of the human heart tissue microarray
   dataset confirms the activation of DNA damage sensing pathways in
   advanced human DCM, which occurs concomitantly with a downregulation of
   DNA replication processes.

Figure 3.

   [146]Figure 3

   [147]Figure 3
   [148]Open in a new tab

   Activation of DNA damage sensing pathways and suppression of DNA
   replication revealed by analysis of human heart tissue transcriptomes
   from [149]GSE5406. (A, B) Box plots depicting the distribution of
   expression values from Affymetrix microarray data for each human left
   ventricular myocardium sample in the [150]GSE5406 dataset, shown before
   (A) and after (B) data normalization. Normalization ensured
   comparability across arrays. (C) Heatmap displaying hierarchical
   clustering of samples (columns) and significant differentially
   expressed genes (DEGs) (rows) identified between dilated cardiomyopathy
   (DCM) and non-failing (NF) control tissues. Sample group annotation and
   gene biotypes are indicated. (D) Volcano plot illustrating DEGs between
   DCM and NF groups. (E, F, G) Gene Set Enrichment Analysis (GSEA) plots
   comparing DCM versus NF control tissues. GSEA revealed (E) significant
   negative enrichment (downregulation) of the "DNA Replication" pathway,
   (F) significant positive enrichment (activation) of the "Cytosolic
   DNA-sensing pathway", and (G) significant positive enrichment of the
   "Chemical carcinogenesis - DNA adducts" pathway in the DCM samples.

WGCNA identifies a key co-expression module linking DDR and ECM/fibrosis
pathways in DCM

   To identify networks of co-expressed genes associated with DCM, specify
   which term is appropriate for your samples] and gain insights into
   their biological roles, we performed Weighted Gene Co-expression
   Network Analysis (WGCNA) on the transcriptomic data comparing diseased
   samples with normal controls. Initial sample clustering confirmed data
   quality and identified potential outliers (Figure [151]4A). The WGCNA
   algorithm identified 15 distinct modules of co-expressed genes,
   delineated by unique colors following hierarchical clustering and
   dynamic tree cutting (Figure [152]4B). Correlating module eigengenes
   (MEs), which represent the overall expression profile of each module,
   with clinical traits revealed several modules significantly associated
   with the disease state (Figure [153]4C). Notably, the black module
   demonstrated the strongest positive correlation with DCM/IHF status,
   while the turquoise and cyan modules were significantly negatively
   correlated. Further investigation of the highly significant black
   module showed a strong positive correlation between Module Membership
   (MM) and Gene Significance (GS) for DCM (Figure [154]4D-E), indicating
   that genes most central to the black module network are also highly
   associated with the disease phenotype (Figure [155]4D-E). Functional
   enrichment analysis of genes within the black module identified
   significant overrepresentation of pathways related to DNA Damage
   Response (DDR), including "DNA damage response," "ATM-dependent DNA
   damage response," and "DNA repair," as well as "DNA replication"
   (Figure [156]4F). Importantly, pathways crucial for extracellular
   matrix (ECM) remodeling and fibrosis, namely the "TGF-beta signaling
   pathway" and "TGF-beta regulation of extracellular matrix," were also
   significantly enriched within this module. Taken together, these WGCNA
   findings highlight specific gene co-expression networks correlated with
   DCM, with the most significantly associated module (black) integrating
   genes involved in both DDR/DNA replication processes and pathways
   governing ECM remodeling and fibrosis.

Figure 4.

   [157]Figure 4
   [158]Open in a new tab

   Weighted gene co-expression network analysis (WGCNA) identifies modules
   associated with dilated cardiomyopathy enriched in DNA damage response
   and extracellular matrix pathways. (A) Sample clustering dendrogram
   based on gene expression profiles used for WGCNA, facilitating the
   identification of potential outliers. Samples are indicated by their
   GSM IDs. (B) Hierarchical clustering dendrogram of genes based on
   topological overlap matrix (TOM) dissimilarity. Branches of the
   dendrogram represent clusters of highly co-expressed genes. Below the
   dendrogram, colors indicate distinct gene co-expression modules
   identified using the dynamic tree cut algorithm. (C) Heatmap
   illustrating the correlation between module eigengenes (MEs) and
   clinical traits (DCM/IHF vs. Control). Each cell represents the
   correlation coefficient and the corresponding P-value for a specific
   module-trait relationship. (D, E) Scatter plots showing the
   relationship between Gene Significance (GS) and Module Membership (MM)
   within the black module. (D) GS for control status versus MM in the
   black module. (E) GS for DCM status versus MM in the black module.
   Correlation coefficients (cor) and P-values are indicated for each
   plot. (F) Bubble plot displaying results of pathway enrichment analysis
   performed on genes within the black module. The x-axis represents the
   Rich Factor, bubble size indicates the number of genes enriched in the
   pathway, and color intensity corresponds to the significance. Key
   enriched pathways related to DNA damage response, DNA replication,
   TGF-beta signaling, and extracellular matrix regulation are shown.

Upregulation of DNA damage response markers in experimental
doxorubicin-induced cardiomyopathy

   To experimentally validate the bioinformatic findings suggesting DNA
   Damage Response (DDR) activation in Dilated Cardiomyopathy (DCM), we
   employed a murine model induced by chronic doxorubicin administration
   (5 mg/kg weekly, 6 weeks) and compared results to saline-treated
   controls. Analysis of cardiac tissue from this model revealed
   significant alterations in key DDR markers. A primary indicator of DNA
   double-strand breaks, phosphorylated histone H2AX (γ-H2AX), showed
   significantly increased activity levels in the DCM group compared to
   controls (Figure [159]5H). Consistent with checkpoint activation, the
   mRNA expression of checkpoint kinase 2 (Chk2) was significantly
   upregulated (Figure [160]5G). Furthermore, components of multiple DNA
   repair pathways exhibited increased mRNA expression in DCM hearts,
   including Ogg1 (base excision repair, Figure [161]5C), Xpc (nucleotide
   excision repair, Figure [162]5D), Brca1 (homologous recombination,
   Figure [163]5E), and Ku70 (non-homologous end joining, Figure [164]5F).
   We also observed significant upregulation of downstream DDR effectors,
   including the p53-target genes p21, involved in cell cycle arrest
   (Figure [165]5B), and the pro-apoptotic gene Bax (Figure [166]5A).
   Collectively, these results from the doxorubicin-induced DCM model
   demonstrate significant activation across multiple facets of the DDR
   pathway - including damage sensing, checkpoint signaling, DNA repair
   mechanisms, and downstream effectors - providing strong experimental
   validation for DDR activation in this cardiomyopathy context.

Figure 5.

   [167]Figure 5
   [168]Open in a new tab

   Experimental validation of DNA damage response (DDR) pathway activation
   in a doxorubicin-induced mouse model of dilated cardiomyopathy (DCM).
   (A-G) Relative mRNA expression levels of DDR-related genes in cardiac
   tissue from control (Cont) and doxorubicin-induced DCM mice, determined
   by quantitative PCR (qPCR). Genes measured include (A) Bax, (B) p21,
   (C) Ogg1, (D) Xpc, (E) Brca1, (F) Ku70, and (G) Chk2. Data are
   presented as fold change relative to the control group. (H) Relative
   activity levels of phosphorylated histone H2AX (γ-H2AX) in cardiac
   tissue from Control (Cont) and DCM mice Data are presented as fold
   change relative to the control group. For all panels, individual data
   points represent biological replicates (individual animals). Data are
   shown as mean ±SEM. *p < 0.05 vs Control group.

Single-cell RNA-seq analysis pipeline reveals cellular heterogeneity in
cardiac samples

   To complement insights gained from bulk transcriptomic analyses
   ([169]GSE197850 iPSC-CM RNA-seq and [170]GSE5406 human heart tissue
   microarrays) and to investigate cellular heterogeneity in DCM, we
   performed single-cell RNA sequencing (scRNA-seq) analysis. The data
   processing workflow is outlined in Figure [171]6. Initial quality
   control involved filtering cells based on standard metrics, including
   the number of detected genes (nFeature_RNA), transcript counts
   (nCount_RNA), and percentage of mitochondrial/ribosomal reads
   (percent.mt/percent.rb), ensuring the removal of low-quality cells or
   potential doublets (Figure [172]6A-F). To address potential batch
   effects, data integration was performed using Harmony] (Figure
   [173]6G). Principal Component Analysis (PCA) was applied for
   dimensionality reduction, and significant principal components (PCs)
   capturing meaningful biological variation were selected based on
   statistical evaluation and variance contribution (Figure [174]6H-I).
   Utilizing these significant PCs, unsupervised graph-based clustering
   identified distinct cell populations within the cardiac cellular milieu
   (Figure [175]6J). This comprehensive scRNA-seq processing pipeline
   yielded well-defined cell clusters, providing a necessary foundation
   for detailed downstream analyses at single-cell resolution, such as
   examining cell-type-specific expression patterns of genes like IFI16 in
   the context of DCM.

Figure 6.

   [176]Figure 6
   [177]Open in a new tab

   Processing, quality control, and clustering of single-cell RNA
   sequencing data. (A-F) Quality control (QC) metrics for the scRNA-seq
   dataset. (A, B) Scatter plots showing the relationship between the
   number of detected genes (nFeature_RNA), total transcript counts
   (nCount_RNA), and percentage of ribosomal reads (percent.rb) per cell.
   (C, D, F) Violin plots illustrating the distribution of nFeature_RNA,
   nCount_RNA, and percent.rb across cells. (E) Visualization of the
   percentage of mitochondrial reads (percent.mt). These metrics were used
   to filter out low-quality cells and potential doublets. (G) Scatter
   plot displaying cells projected onto Harmony coordinates, illustrating
   the results of data integration to correct for potential batch effects.
   (H) Quantile-Quantile (QQ) plot comparing the distribution of P-values
   for principal components (PCs) against the uniform distribution,
   assessing PC significance. Significant PCs deviate from the diagonal
   line. Listed P-values indicate the significance of the top 20 PCs. (I)
   Elbow plot showing the standard deviation associated with each
   principal component (PC), used to determine the optimal number of PCs
   for downstream dimensionality reduction and clustering. (J)
   Visualization of unsupervised clustering results based on significant
   PCs. Node size corresponds to the number of cells in the cluster, and
   node color represents cluster identity.

Single-cell analysis identifies cardiac cell populations with enriched
DDR/ECM pathways relevant to IFI16 activity

   Following the initial processing and clustering of the single-cell RNA
   sequencing (scRNA-seq) data (Figure [178]6), we proceeded to identify
   and characterize the major cell populations present. Visualization
   using Uniform Manifold Approximation and Projection (UMAP) revealed
   distinct cell clusters, with contributions from different experimental
   conditions or sample origins clearly delineated (Figure [179]7A-D).
   Based on the expression of established marker genes, we annotated the
   primary cell clusters (Figure [180]7B), identifying populations
   including endothelial cells, cardiomyocytes, tissue stem cells,
   macrophages, and smooth muscle cells, whose spatial distribution is
   shown on the UMAP plot (Figure [181]7E). Heatmaps confirmed the
   expression of key marker genes defining these annotated cell types
   (Figure [182]7F) and highlighted the top differentially expressed genes
   characterizing each distinct cluster (Figure [183]7C). Importantly,
   functional enrichment analysis performed revealed significant
   enrichment of pathways central to our investigation (Figure [184]7G).
   Pathways related to DNA Damage Response (DDR), such as "DNA damage
   response" and "ATM-dependent DNA damage response," along with "DNA
   replication," were significantly enriched. Concurrently, pathways
   associated with fibrosis and extracellular matrix (ECM) remodeling,
   including the "TGF-beta signaling pathway" and "TGF-beta regulation of
   extracellular matrix," were also prominently enriched. These
   single-cell level findings, demonstrating the co-enrichment of DDR and
   ECM/fibrosis-related pathways within the cardiac cellular landscape,
   provide a context for understanding the potential role of genes like
   IFI16 (previously observed to be upregulated in bulk DCM samples and
   associated with ECM organization) within specific cell populations
   contributing to DCM pathology.

Figure 7.

   [185]Figure 7
   [186]Open in a new tab

   Single-cell RNA sequencing analysis identifies distinct cardiac cell
   populations and reveals enrichment of DNA damage response and
   extracellular matrix pathways. (A) Uniform Manifold Approximation and
   Projection (UMAP) plots displaying the integrated single-cell landscape
   ('Combined') and the distribution of cells originating from different
   sample conditions/locations ('HF_LA_CM', 'HF_LA_NCM', 'HF_LV_CM',
   'HF_LV_NCM'). (B) Heatmap illustrating the expression scores of marker
   genes used to define initial cell clusters (Clusters 0-7) and assign
   preliminary cell type identities (Labels) such as Endothelial cells,
   Cardiomyocytes, and Tissue stem cells. (C) Heatmap showing the Z-score
   normalized expression of the top differentially expressed genes across
   all identified final cell clusters (Identity 0-13), highlighting gene
   signatures that distinguish each cluster. (D) UMAP plot displaying the
   identified cell clusters (Clusters 0-7) mapped onto the two-dimensional
   embedding space. (E) UMAP plot showing the distribution of major
   annotated cell types, including Tissue stem cells, Endothelial cells,
   Smooth muscle cells, and Macrophages. (F) Heatmap depicting the Z-score
   normalized expression of canonical marker genes across the major
   annotated cell types, confirming cell identity assignments. (G) Bubble
   plot summarizing results from pathway enrichment analysis. The plot
   displays enriched pathways related to DNA Damage Response (DDR), DNA
   replication, TGF-beta signaling, and extracellular matrix regulation.
   The x-axis represents the Rich Factor, bubble size corresponds to the
   number of genes (GeneNumber) in the pathway, and color intensity
   indicates statistical significance.

Elevated IFI16 expression associates with extracellular matrix remodeling
pathways in DCM

   To further explore the role of IFI16 within a high-resolution cellular
   context, we utilized the publicly available single-cell RNA sequencing
   (scRNA-seq) dataset [187]GSE121893, derived from human left atrial and
   ventricular heart tissues, including samples from patients with dilated
   cardiomyopathy (DCM). Consistent with findings in bulk datasets,
   analysis within [188]GSE121893 confirmed the significant upregulation
   of the DNA sensor IFI16 in diseased samples/conditions compared to
   controls (Figure [189]8A, B). To investigate the functional
   consequences associated with this upregulation within the
   [190]GSE121893 dataset, we stratified cells based on IFI16 expression
   levels and performed differential gene expression analysis. This
   identified numerous significantly differentially expressed genes (DEGs)
   between IFI16-High and IFI16-Low groups, as visualized by volcano plot
   (Figure [191]8C) and heatmap (Figure [192]8D). Notably, correlation
   analysis within this scRNA-seq data revealed strong positive
   co-expression between IFI16 and key genes involved in extracellular
   matrix (ECM) remodeling and fibrosis, such as COL1A1, COL3A1, FN1, and
   TGFB1 (Figure [193]8E). Functional enrichment analysis of these DEGs
   using Gene Ontology (GO) showed highly significant enrichment for
   'Extracellular matrix organization' (GO:0030198), among other terms
   (Figure [194]8F), while KEGG analysis highlighted pathways including
   phagosome activity (Figure [195]8G). Furthermore, Gene Set Enrichment
   Analysis (GSEA) comparing the IFI16-High versus IFI16-Low profiles
   derived from the [196]GSE121893 data demonstrated significant positive
   enrichment for gene sets related to ECM components in the IFI16-High
   group (Figure [197]8H). Gene Set Variation Analysis (GSVA) further
   explored these connections, revealing significant associations between
   IFI16 expression levels and the activity scores of numerous biological
   processes and molecular functions within the [198]GSE121893 dataset
   (Figure [199]8I, J). Taken together, analysis of the [200]GSE121893
   scRNA-seq data reinforces that elevated IFI16 expression is associated
   with a transcriptomic signature strongly linked to ECM organization and
   fibrosis pathways at the cellular level in human heart tissue,
   suggesting a role for IFI16 in connecting DNA damage sensing to adverse
   cardiac remodeling in DCM.

Figure 8.

   [201]Figure 8
   [202]Open in a new tab

   Elevated IFI16 expression is associated with extracellular matrix
   remodeling pathways in human cardiac scRNA-seq data ([203]GSE121893).
   (A, B) Violin plots demonstrating significantly higher expression
   levels of IFI16 in diseased ('Treat'/DCM) compared to Control samples
   derived from the [204]GSE121893 dataset. (C) Volcano plot visualizing
   differentially expressed genes (DEGs) between sample groups stratified
   by high versus low IFI16 expression. Red points indicate significantly
   upregulated genes; green points indicate significantly downregulated
   genes in the IFI16-High group. (D) Heatmap displaying the Z-score
   normalized expression levels of significant DEGs across samples grouped
   by low versus high IFI16 expression, illustrating distinct
   transcriptomic profiles. (E) Correlation heatmap showing pairwise
   Pearson correlation coefficients between the expression levels of IFI16
   and selected key genes, including those involved in extracellular
   matrix (ECM) remodeling (e.g., COL1A1, COL3A1, FN1, TGFB1). Red
   indicates positive correlation; blue indicates negative correlation.
   (F) Circular plot summarizing Gene Ontology (GO) enrichment analysis
   results for DEGs identified between IFI16-High and IFI16-Low groups.
   Enriched Biological Process terms are shown, with "Extracellular matrix
   organization" highlighted. Segments may indicate logFC direction of
   constituent genes. (G) Circular plot summarizing Kyoto Encyclopedia of
   Genes and Genomes (KEGG) pathway enrichment analysis results for the
   DEGs. Enriched pathways are displayed. (H) Gene Set Enrichment Analysis
   (GSEA) plot showing positive enrichment of an ECM-related gene set in
   the IFI16-High expression group compared to the IFI16-Low group. (I, J)
   Bar plots displaying Gene Set Variation Analysis (GSVA) results,
   illustrating the association between IFI16 expression levels and the
   activity scores of various gene sets categorized by Biological Process
   (I) and Molecular Function (J). Red bars indicate positive association;
   green bars indicate negative association.

Inhibition of DDR with NU7441 reduces IFI16 levels and ameliorates cardiac
dysfunction in doxorubicin-induced DCM

   To investigate whether inhibiting the DNA Damage Response (DDR) could
   ameliorate cardiac dysfunction and affect IFI16 levels in our
   doxorubicin-induced Dilated Cardiomyopathy (DCM) model, mice were
   treated with the DNA-PKcs inhibitor NU7441 or vehicle (PBS). As
   anticipated, DCM mice treated with PBS (DCM+PBS) exhibited
   significantly elevated IFI16 activity and mRNA expression compared to
   control mice treated with PBS (Cont+PBS) (Figure [205]9A, B). Notably,
   concurrent treatment with NU7441 (DCM+NU7441) significantly attenuated
   the doxorubicin-induced increase in both IFI16 activity and mRNA levels
   compared to the DCM+PBS group (Figure [206]9A, B). NU7441 treatment did
   not alter basal IFI16 levels in control animals (Cont+NU7441 vs
   Cont+PBS). Functionally, echocardiography confirmed severe cardiac
   impairment in the DCM+PBS group, characterized by significantly reduced
   left ventricular ejection fraction (LVEF, Figure [207]9C) and
   fractional shortening (LVFS, Figure [208]9D), impaired diastolic
   function (indicated by significantly decreased E/A and e'/a' ratios,
   and increased E/e' ratio; Figure [209]9E, G, F respectively), and
   cardiac dilation (increased LVDd and LVSd; Figure [210]9H, I) compared
   to Cont+PBS animals. Remarkably, administration of NU7441 significantly
   mitigated the cardiac dysfunction induced by doxorubicin. Compared to
   the DCM+PBS group, DCM+NU7441 mice displayed significantly improved
   LVEF and LVFS, ameliorated diastolic function parameters, and reduced
   left ventricular diastolic and systolic dimensions (LVDd, LVSd) (Figure
   [211]9C-I). NU7441 administration did not significantly affect cardiac
   function parameters in control animals. These results indicate that
   pharmacological inhibition of DDR via NU7441 significantly reduces
   IFI16 upregulation and ameliorates cardiac dysfunction and adverse
   remodeling in the doxorubicin-induced DCM model.

Figure 9.

   [212]Figure 9
   [213]Open in a new tab

   Pharmacological inhibition of DNA-PKcs with NU7441 attenuates IFI16
   upregulation and ameliorates cardiac dysfunction in doxorubicin-induced
   dilated cardiomyopathy (DCM). (A, B) Relative IFI16 activity levels (A)
   and mRNA expression levels (B) in cardiac tissue from the four
   experimental groups: Control mice treated with PBS (Cont+PBS), Control
   mice treated with NU7441 (Cont+NU7441), DCM mice treated with PBS
   (DCM+PBS), and DCM mice treated with NU7441 (DCM+NU7441). Data are
   presented as fold change relative to the Cont+PBS group. (C-I)
   Echocardiographic assessment of cardiac function and dimensions in the
   four experimental groups. Parameters measured include: (C) Left
   Ventricular Ejection Fraction (LVEF, %), (D) Left Ventricular
   Fractional Shortening (LVFS, %), (E) Ratio of early (E) to late (A)
   diastolic filling velocity (E/A ratio), (F) Ratio of early diastolic
   filling velocity (E) to early diastolic mitral annular velocity (e')
   (E/e' ratio), (G) Ratio of early (e') to late (a') diastolic mitral
   annular velocity (e'/a' ratio), (H) Left Ventricular internal Diameter
   in diastole (LVDd, mm), and (I) Left Ventricular internal Diameter in
   systole (LVSd, mm). For all panels, individual data points represent
   biological or technical replicates as appropriate. Data are shown as
   mean ±SEM. *Indicates significant difference (p < 0.05) between the
   groups connected by the bracket/line.

Inhibition of DDR in vivo and IFI16 knockdown in vitro attenuate pro-fibrotic
and cell damage markers

   Following the observation that DDR inhibition with NU7441 improved
   cardiac function and reduced IFI16 levels in vivo, we further
   investigated the effects of targeting the DDR pathway and IFI16 on
   cardiac fibrosis and injury markers. In the doxorubicin-induced DCM
   mouse model, treatment with NU7441 significantly reduced the cardiac
   expression of key fibrosis-related markers compared to vehicle-treated
   DCM animals (DCM+PBS). Specifically, NU7441 administration resulted in
   significantly lower mRNA levels of Galectin-3, Col1a3, and Col1a1, as
   well as decreased TGF-β activity (Figure [214]10A-D). To directly
   assess the contribution of IFI16, we utilized siRNA-mediated knockdown
   in vitro. Compared to control siRNA (si-Ctrl), cells with IFI16
   knockdown (si-IFI16) exhibited significantly reduced mRNA expression of
   Col1a1 and Col1a3, along with lower TGF-β activity (Figure [215]10E-G).
   Moreover, silencing IFI16 led to a significant increase in cell
   viability as assessed by MTT assay (Figure [216]10H) and a significant
   decrease in cell injury indicated by reduced lactate dehydrogenase
   (LDH) release (Figure [217]10I). Collectively, these findings
   demonstrate that both pharmacological inhibition of DDR in vivo and
   direct knockdown of IFI16 in vitro can suppress pro-fibrotic markers.
   Furthermore, reducing IFI16 levels directly mitigated markers of cell
   injury and enhanced viability in vitro, supporting a detrimental role
   for the DDR-IFI16 axis in contributing to both fibrosis and cellular
   damage in the context of cardiomyopathy.

Figure 10.

   [218]Figure 10
   [219]Open in a new tab

   Inhibition of DDR pathway or IFI16 knockdown reduces markers of cardiac
   fibrosis and cell injury. (A-D) Effects of DNA-PKcs inhibitor NU7441 on
   markers of cardiac fibrosis in the doxorubicin-induced mouse model of
   Dilated Cardiomyopathy (DCM). Relative mRNA expression levels of (A)
   Galectin-3, (B) Col1a3, (D) Col1a1, and relative activity levels of (C)
   TGF-β were measured in cardiac tissue from four experimental groups:
   Control mice treated with PBS (Cont+PBS), Control mice treated with
   NU7441 (Cont+NU7441), DCM mice treated with PBS (DCM+PBS), and DCM mice
   treated with NU7441 (DCM+NU7441). Data are presented as fold change
   relative to the Cont+PBS group. (E-I) Effects of siRNA-mediated
   knockdown of IFI16 (si-IFI16 used in label, likely intended si-IFI16)
   compared to control siRNA (si-Ctrl) on markers of fibrosis, cell
   viability, and cell injury in vitro. Measurements include: Relative
   mRNA expression levels of (E) Col1a1 and (G) Col1a3; (F) Relative TGF-β
   activity; (H) Relative cell viability assessed by MTT assay; and (I)
   Relative cell injury assessed by LDH release assay. Data are presented
   as fold change relative to the si-Ctrl group. For all panels,
   individual data points represent biological or technical replicates as
   appropriate. Data are shown as mean ±SEM. *Indicates significant
   difference (p < 0.05) between the groups connected by the bracket/line.

Discussion

   Dilated cardiomyopathy (DCM) represents a major clinical challenge,
   characterized by progressive cardiac dysfunction and adverse
   remodeling, often culminating in heart failure. While diverse
   etiologies contribute to DCM, converging evidence suggests that
   compromised genomic integrity and the ensuing DNA damage response (DDR)
   play a significant, potentially unifying role in its pathogenesis. Our
   study provides compelling multi-level evidence, spanning human
   iPSC-derived cardiomyocytes (iPSC-CMs), ex vivo human heart tissue, in
   vivo murine models, and single-cell transcriptomics, that establishes a
   critical pathogenic axis linking DDR activation to interferon-inducible
   protein 16 (IFI16)-mediated extracellular matrix (ECM) remodeling in
   DCM. We demonstrate that IFI16, a known DNA sensor, acts as a pivotal
   mediator coupling nuclear stress signals to pro-fibrotic pathways, and
   crucially, that targeting the upstream DDR pathway can attenuate IFI16
   induction and ameliorate cardiac dysfunction.

   Emerging evidence points towards a significant connection between
   defects in the DDR and the development of DCM. The DDR is a critical
   cellular mechanism for maintaining genomic stability, and its
   impairment has been implicated in the pathogenesis of various diseases,
   including potentially those affecting the heart [220]^10. Given the
   genetic heterogeneity of DCM, it is plausible that mutations in genes
   associated with DCM could directly or indirectly impact the efficiency
   and fidelity of DDR pathways [221]^69. Our investigation consistently
   revealed robust activation of DDR pathways across disparate models of
   DCM. In human iPSC-CMs modeling DCM, we observed significant enrichment
   of cytosolic DNA sensing pathways alongside broad inflammatory
   signaling cascades, consistent with cellular stress responses.
   Intriguingly, these in vitro models also exhibited upregulation of DNA
   replication and cell cycle control pathways, potentially reflecting an
   aberrant proliferative or stress-induced attempt at repair in
   pluripotent stem cell derivatives [222]^70^-[223]^73. In contrast,
   analysis of ex vivo human heart tissue from patients with advanced DCM
   corroborated the activation of DNA sensing and damage pathways but
   revealed a significant suppression of DNA replication
   [224]^74^-[225]^77. This divergence likely reflects differences between
   the in vitro stem cell model and the complex, terminally differentiated
   environment of the failing adult human heart, where suppression of
   replication might indicate cell cycle arrest or senescence driven by
   persistent damage. Nonetheless, the common thread across these
   platforms was the clear activation of DDR sensing mechanisms. We
   further validated this experimentally in a doxorubicin-induced murine
   DCM model, demonstrating significant increases in the DNA double-strand
   break marker γ-H2AX, checkpoint kinase Chk2, multiple DNA repair
   pathway components (Ogg1, Xpc, Brca1, Ku70), and downstream effectors
   like p21 and Bax [226]^78^-[227]^81. These findings align with previous
   reports showing increased DNA damage in DCM patient biopsies and its
   correlation with prognosis and genotype, solidifying DDR activation as
   a core feature of the disease.

   A particularly strong link has been observed in the context of
   mutations in the LMNA gene, a well-known cause of severe and
   progressive DCM [228]^69. Studies [229]^82^-[230]^85 have demonstrated
   that LMNA mutations, which disrupt the structural integrity of the
   nuclear envelope, can lead to increased DNA damage and the activation
   of DDR pathways within cardiac myocytes [231]^69. Research has shown
   that the deletion of the Lmna gene in fibroblasts can cause
   senescence-associated DCM by activating the double-stranded DNA damage
   response [232]^86. This underscores the critical role of nuclear
   envelope integrity in maintaining genomic stability and preventing
   cardiac disease. Beyond genetic mutations, cellular stress and the
   accumulation of DNA damage appear to be significant factors in the
   development and progression of DCM across various etiologies. Increased
   DNA damage and activation of DDR pathways have been observed in
   cardiovascular disease and heart failure. Conditions like oxidative
   stress can elevate internal DNA stressors in cardiomyopathies [233]^3.
   Notably, the extent of myocardial DNA damage has been found to
   correlate with treatment response and prognosis in DCM patients
   [234]^5. This suggests that the accumulation of DNA damage in
   cardiomyocytes, whether due to impaired repair or increased stress, is
   a critical aspect of DCM pathogenesis.

   IFI16 is not only a sensor of foreign DNA but also plays an active role
   within the DDR pathways [235]^87. The expression of the IFI16 gene is
   itself induced by the activation of the DDR in response to DNA damage,
   indicating its involvement in this cellular defense mechanism [236]^87.
   IFI16 interacts with key DDR proteins, most notably the tumor
   suppressor p53 [237]^87. This interaction is crucial for amplifying the
   DDR, as IFI16 promotes the phosphorylation and activation of p53 in
   response to DNA damage [238]^87. By enhancing p53 activity, IFI16
   contributes to cell cycle arrest and apoptosis, critical processes for
   preventing the propagation of damaged cells [239]^87. Interestingly,
   IFI16 has also been shown to inhibit DNA repair in certain contexts,
   potentially potentiating the effects of type-I interferons. This
   suggests a complex and context-dependent role for IFI16 in modulating
   the cellular response to DNA damage. Furthermore, IFI16 can activate
   ATM-p53 DDR signaling and inhibit telomerase activity [240]^87, and it
   is involved in the activation of the inflammasome in response to DNA
   damage, linking the DDR to inflammatory pathways [241]^87. IFI16 shows
   a preference for binding to quadruplex DNA structures over other DNA
   forms. This binding affinity suggests a role for IFI16 in stabilizing
   these structures, which may be relevant in DNA damage responses and
   cellular processes such as proliferation [242]^88.

   A key advance of our study is the identification of IFI16 as a central
   player connecting DDR activation to detrimental cardiac remodeling
   [243]^89^, [244]^90. While IFI16 is recognized for its role in innate
   immunity and sensing viral/bacterial DNA, and its interaction with DDR
   components like p53 is established, its specific function in linking
   DDR to cardiac fibrosis has remained largely unexplored. Our Weighted
   Gene Co-expression Network Analysis (WGCNA) on human DCM tissue was
   instrumental, identifying a gene module highly correlated with DCM
   status that was co-enriched for both DDR/DNA replication and critical
   ECM/fibrosis pathways, including TGF-β signaling. This suggested an
   integrated network response. Subsequent analysis of human cardiac
   single-cell RNA-seq data provided cellular resolution, confirming
   significant IFI16 upregulation in DCM conditions. Strikingly,
   stratifying cells by IFI16 expression revealed that high IFI16 levels
   were strongly associated with a transcriptomic signature enriched for
   'Extracellular matrix organization' and correlated positively with key
   fibrotic genes like COL1A1, COL3A1, FN1, and TGFB1. This robust
   correlation at the single-cell level strongly implicates IFI16 in
   orchestrating pro-fibrotic gene programs within the diseased heart
   [245]^91^-[246]^95, moving beyond its canonical role as a purely immune
   sensor in this context [247]^96^-[248]^99. This contrasts with studies
   primarily focusing on other DNA sensors like cGAS in cardiac pathology
   and highlights a potentially distinct or complementary role for IFI16
   in driving cardiac fibrosis secondary to DNA damage.

   Research specifically examining the direct relationship between IFI16
   and DCM is still an emerging area. However, several studies provide
   insights into potential connections [249]^100^-[250]^103.
   Transcriptomic analyses of heart tissue from patients with LMNA-related
   DCM have revealed increased DNA damage and activation of cytosolic
   pattern recognition pathways, which are thought to involve DNA sensors
   such as IFI16 [251]^104^, [252]^105. These findings suggest that in the
   context of LMNA mutations, the resulting nuclear instability might lead
   to the release of DNA into the cytoplasm, where it could be sensed by
   IFI16, potentially triggering downstream inflammatory signaling
   [253]^106^, [254]^107. IFI16's involvement in PANoptosis, a
   pro-inflammatory form of programmed cell death, also suggests a
   potential link to DCM [255]^9. Given the role of inflammation and
   cardiomyocyte death in DCM pathogenesis, IFI16's participation in these
   processes could be significant. One study indicated that the activation
   of cytosolic pattern recognition in cardiomyocytes in LMNA-related DCM
   was independent of cGAS, another major cytosolic DNA sensor, implying
   that other sensors, possibly including IFI16, might be more relevant in
   this specific context [256]^108^, [257]^109. While direct and
   conclusive evidence specifically linking IFI16 to DCM is still
   developing, these studies, along with IFI16's known roles in
   inflammation and DNA sensing in other cardiovascular conditions like
   abdominal aortic aneurysm and in response to viral infections, suggest
   a potential involvement in the pathogenesis of DCM across different
   etiologies [258]^110^, [259]^111.

   The causal link within the proposed DDR-IFI16-ECM axis was
   substantiated through targeted interventions. Pharmacological
   inhibition of DNA-PKcs, a key DDR kinase, using NU7441 in our
   doxorubicin-induced DCM model yielded remarkable results
   [260]^112^-[261]^115. NU7441 treatment not only confirmed the
   dependence of IFI16 upregulation on DDR signaling by significantly
   reducing its expression and activity but also led to a significant
   amelioration of cardiac dysfunction [262]^116^-[263]^119. This included
   improvements in systolic function, diastolic parameters, and a
   reduction in adverse ventricular remodeling. Furthermore, this
   functional recovery was associated with a significant reduction in
   cardiac fibrosis markers, including Galectin-3, Col1a3, Col1a1, and
   TGF-β activity, upon NU7441 treatment [264]^120^, [265]^121.
   Complementing these in vivo findings, direct siRNA-mediated knockdown
   of IFI16 in vitro mirrored the anti-fibrotic effects, significantly
   reducing Col1a1, Col1a3, and TGF-β activity. Importantly, IFI16
   silencing also directly enhanced cardiomyocyte viability and reduced
   cell injury markers, supporting a direct detrimental role of IFI16
   beyond just promoting fibrosis [266]^122^, [267]^123. Together, these
   interventional data provide strong functional evidence that disrupting
   the DDR-IFI16 cascade, either upstream via DDR inhibition or directly
   at the level of IFI16, can mitigate both cardiac fibrosis and
   cardiomyocyte injury, leading to improved cardiac function.

   This study significantly advances our understanding by proposing a
   novel mechanism where nuclear stress, manifested as DNA damage, is
   transduced via the IFI16 sensor to drive pathological ECM remodeling—a
   hallmark of DCM progression [268]^124^-[269]^127. The identification of
   this specific DDR-IFI16-ECM axis offers a potential therapeutic
   vulnerability. Our demonstration that inhibiting DNA-PKcs with NU7441
   effectively counters both IFI16 induction and DCM progression in vivo
   highlights the potential utility of targeting DDR components as a
   therapeutic strategy [270]^128^, [271]^129. While DDR inhibitors are
   primarily explored in oncology, our findings suggest their potential
   repurposing for cardiac diseases characterized by significant DNA
   damage, although careful consideration of systemic and off-target
   effects would be paramount. Alternatively, developing strategies to
   specifically target IFI16 activity or its downstream signaling
   pertinent to ECM regulation in the heart could offer a more focused
   therapeutic approach.

   Certain limitations warrant consideration [272]^130^-[273]^133. Our in
   vivo work primarily utilized a doxorubicin-induced model, which, while
   relevant for chemotherapy-induced cardiomyopathy and robustly
   activating DDR, may not fully recapitulate the heterogeneity of human
   DCM etiologies (e.g., genetic, viral). Future studies validating these
   findings in genetic models (like LMNA-mutant mice) or other
   stress-induced DCM models would strengthen the conclusions
   [274]^134^-[275]^137. While our scRNA-seq analysis implicated multiple
   cell types in expressing DDR/ECM pathway components, further
   investigation using cell-type-specific manipulations is needed to
   dissect the precise contribution of cardiomyocytes versus fibroblasts
   or immune cells to the IFI16-mediated fibrotic response [276]^138^,
   [277]^139. Elucidating the exact molecular steps connecting IFI16
   activation to TGF-β pathway induction and collagen synthesis represents
   a critical next step.

   In conclusion, our comprehensive study integrates bioinformatics,
   experimental models, and functional interventions to uncover a
   pathogenic cascade in DCM initiated by DNA damage response activation,
   mediated by the DNA sensor IFI16, and culminating in detrimental ECM
   remodeling and cardiac dysfunction. We identify IFI16 as a critical
   node linking nuclear stress to fibrosis and demonstrate that
   interrupting this axis via DDR inhibition holds therapeutic promise.
   These findings not only provide novel mechanistic insights into DCM
   pathogenesis but also highlight the DDR-IFI16 interface as a potential
   target for future therapeutic development aimed at halting or reversing
   adverse cardiac remodeling in patients with dilated cardiomyopathy.

Acknowledgments