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Circulation, Volume 150, Issue Suppl_1, Page A4144583-A4144583, November 12, 2024. Donor-derived cell-free DNA (dd-cfDNA) has been increasingly used to detect acute rejection (AR). We aimed to compare our institutional dd-cfDNA results to previously published adult and pediatric dd-cfDNA AR cutoffs. We also hypothesized that in the absence of AR, elevated dd-cfDNA would be associated with CAV and positive DSA.Patients (pt) < 18 years at transplant with >1 dd-cfDNA between 2021-2023 were included. Using dd-cfDNA levels from this cohort, sensitivity, specificity, NPV, and PPV were calculated. False positives and false negatives (FN) were determined using published dd-cfDNA thresholds. AR was defined as decision-to-treat with increased immunosuppression, which was independent of dd-cfDNA in our cohort. In pt without AR,t-test was used to compare the means of dd-cfDNA levels in pt with and without DSA. χ2testing was then performed to evaluate the association between dd-cfDNA levels above and below 0.2% and the presence/absence of DSA and CAV. DSA was defined as allele-specific DSA identified by single antigen bead with mean fluorescence intensity >1000, and CAV as any disease by angiography.There were 379 samples among 163 pt, a median of 2 samples per pt, and 32 samples obtained at time of AR. Performance of dd-cfDNA in our cohort vs published dd-cfDNA thresholds is shown in Table 1. The FN rate ranged from 16 to 37% as the dd-cfDNA threshold increased. Mean dd-cfDNA was higher in patients with positive DSA versus those without (0.83% vs 0.19%, p0.2% were associated with a higher prevalence of positive DSA (n=66) (48% vs 13%, p

Circulation, Volume 150, Issue Suppl_1, Page A4146311-A4146311, November 12, 2024. Introduction/Background:A 58-year-old man with inotrope dependent heart failure was declined for allotransplant and MCS at multiple centers and was transplanted with a 10 gene-edited porcine cardiac xenograft under FDA expanded access Investigational New Drug (eIND) approval. After requiring perioperative transfusions, by post operative day (POD) 13, endomyocardial biopsy revealed antibody and complement deposition without damage or cellular infiltrate. Hemodynamic decompensation on POD 29 prompted biopsy revealing antibody mediated rejection (AMR). The patient required ECMO support POD31 and chose comfort care POD 40.Research Questions/Hypothesis:The cardiac xenograft doubled in mass from implant to autopsy. Donor derived cell free DNA (cfDNA) correlated temporally with the rejection findings. These processes are hypothesized to correlate.Approach:Routine TTE measurements of diastolic LV posterior wall dimension (LVPWd), intraventricular septal dimension (IVSd), LV mass, and LV mass index were taken. Donor derived cfDNA was measured weekly and PRN (Care Dx Inc., Brisbane, CA). Pearson correlation coefficient analysis was conducted in GraphPad Prism 10 (Dotmatics, Boston, MA).Results/Data:POD 26 TTE measurements indicated thickening graft. POD 30 cfDNA began elevating. cfDNA correlated moderately with LVPWd (r = 0.43, p=0.4) and IVSd (r= 0.5, p=0.31) less so with LV mass (r= 0.28, p=0.59) and LV mass index (r=0.34, p=0.51)Conclusions:Xenograft cardiac thickening measured on TTE preceded elevation in cfDNA. Both were moderately correlated and temporally corresponded to biopsy diagnosed AMR and ultimate graft failure. Future xenograft surveillance will likely benefit from multimodal approach.

Circulation, Volume 150, Issue Suppl_1, Page A4132149-A4132149, November 12, 2024. Background:Heart failure (HF) with reduced ejection fraction (HFrEF) is associated with increased inflammatory response that contributes to progressive worsening of the HF state. Potential endogenous triggers of this process include damage-associated molecular patterns (DAMPs). DAMPs originate from within cells following tissue stress or injury and activate pattern recognition receptors of the immune system, triggering an inflammatory response. Mitochondria are the major cell organelles that release DAMPs into the circulation. Chronic HF is associated with multi-organ mitochondrial dysfunction and cell injury and death.Purpose:The present study sought to quantify differences in mitochondrial DNA (mtDNA) DAMPs in the setting of HFrEF in humans and dogs compared to normal humans and dogs.Methods:Plasma samples were obtained from 9 HFrEF patients with left ventricular ejection fraction (LVEF) ≤35% and 9 age-matched healthy subjects, and from 6 dogs with coronary microembolization-induced HF (LVEF ≤35%) and 6 normal dogs. DNA fragments were isolated from 1 ml of plasma using a commercially available kit. Quantitative PCR and specifically designed primers were used to measure the amount of DNA in plasma fromCOX1(cytochrome c oxidase subunit 1),ND1(NADH dehydrogenase subunit 1) andND6(NADH dehydrogenase subunit 6) using an Applied Biosystems 7500 Fast-PCR unit. The relative abundances of plasma mtDNA DAMPs were expressed as threshold cycles (CT).Results:Compared to normal human subjects, mtDNA DAMPs levels ofCOX1,ND1andND6in plasma of HFrEF patients was 4-fold, 9-fold and 8-fold greater, respectively. Similarly, compared to normal dogs, mtDNA DAMPs levels ofCOX1,ND1andND6in plasma of HFrEF dogs was increased 48-fold, 12-fold and 4-fold, respectively.Conclusions:These findings indicate a marked increase of mtDNA DAMPs in plasma of humans and dogs with HFrEF. The increase of mtDNA DAMPs is consistent with the elevated systemic inflammatory state of HFrEF. The results underscore mitochondrial abnormalities as integral players in the progressive worsening of HFrEF.

Circulation, Volume 150, Issue Suppl_1, Page A4147025-A4147025, November 12, 2024. Heart failure (HF) treatment advances have improved outcomes but HF heterogeneity leads to ~50% response to standard therapies, emphasizing a need to further study mechanisms of disparate outcomes. One serious disparity is increased cardiovascular disease risk and mortality in African Americans (AA) compared to Caucasian Americans (CA). Race is a social construct and thus poor proxy for physiology; hence, our lab studies epigenetics, stable yet reversible changes to DNA serving as a possible interface of how environment impacts gene regulation. We’ve previously implicated cardiac DNA methylation as a new indicator of socio-economic disparities in HF outcomes. Related to HF, US adult obesity rates are 40% and similar disparities exist with AA having higher rates, and studies on obesity show how inequity leads to disparate rates. Thus, our secondary analysis investigates the role of obesity, through its impact on cardiac epigenetics and transcription, as an underlying environment/molecular driver within HF disparities.Multi-omics data of left ventricular assist device placement biopsies of age-matched males were from our previous publication’s public dataset. Cardiac DNA methylation (850k-array) analyzed via minfi, and differential gene expression (RNA-seq) via DESeq2.Two-year survival is decreased in non-obese (NO) vs. obese (OB) patients (p

Circulation, Volume 150, Issue Suppl_1, Page A4145247-A4145247, November 12, 2024. Background:Plasma donor-derived cell-free DNA (dd-cfDNA) has emerged as a biomarker for acute heart transplant rejection as a potential adjunct to endomyocardial biopsy (EMBx). However, an established threshold for dd-cfDNA in acute cardiac transplant rejection is lacking, with prior studies employing different study specific thresholds. This meta-analysis aims to assess the overall diagnostic accuracy of dd-cfDNA across various cutoffs and provide an optimal threshold.Method:PubMed and Web of Science were searched for relevant publications reporting on the use of dd-cfDNA for the detection of acute cardiac transplant rejection defined as acute cellular rejection (ACR) and/or antibody-mediated rejection (AMR), with EMBx as the reference standard. Pooled sensitivity and specificity were estimated across different thresholds of dd-cfDNA across studies. The optimal dd-cfDNA threshold was estimated by specificity and 1-sensitivity in each data point pooled with fractional polynomial models, maximizing the Youden index between these models.Results:Out of 247 screened papers, 13 studies were included, comprising 12,017 samples from 3,723 patients. Twelve studies used both ACR and AMR and one study used only ACR for the definition of transplant rejection. The thresholds of dd-cfDNA varied across studies, ranging from 0.1 to 0.35 (Figure A). The overall pooled sensitivity and specificity for detecting acute transplant rejection were 0.68 (95% CI: 0.58-0.77) and 0.82 (95% CI: 0.74-0.88) respectively. The areas under the hierarchical modeling-based summary receiver-operating characteristics (sROC) curves were 0.82 (95% CI: 0.78-0.85). Specificity increased with higher thresholds while sensitivity decreased (Figure A and B). The optimal dd-cfDNA threshold derived from combining all included studies was 0.218.Conclusion:Dd-cfDNA assay demonstrates high diagnostic accuracy for acute cardiac transplant rejection, particularly notable for its high specificity. However, there exists significant inconsistency in dd-cfDNA thresholds across studies. Increasing the threshold leads to a trade-off between increased specificity and decreased sensitivity. A cutoff of 0.22 may be considered for dd-cfDNA screening for heart transplant rejection.

Circulation, Volume 150, Issue Suppl_1, Page A4147327-A4147327, November 12, 2024. Introduction:The Donor-Derived Cell-Free DNA-Outcomes AlloMap Registry (D-OAR) study has highlighted the significant capability of dd-cfDNA, showcasing a 97% negative predictive value (NPV) for identifying both acute cellular rejection (ACR) and antibody-mediated rejection (AMR) at a dd-cfDNA threshold of 0.20%. This development has greatly enhanced the range of biomarkers available to clinicians, confirming dd-cfDNA as a reliable clinical biomarker for monitoring heart transplant (HT) recipients. Short and long-term outcomes for African American HT recipients are worse. Given these findings, our study aims to investigate baseline differences in the levels and positivity rates of dd-cfDNA stratified by race in a negative biopsy cohort.Methods:We conducted a single-center retrospective review of all HT recipients from 2019 to 2023 with at least one valid combined dd-cfDNA sample drawn between April 2019 and July 2023. Multiorgan transplant recipients and those without a paired biopsy result were excluded. A positive biopsy result was defined as >= ISHLT grade 1R/1B or pAMR >0. Dd-cfDNA (AlloSure, AS) result was considered positive if ≥0.12%, and as a sub-analysis using a threshold ≥0.20%. A positive peripheral GEP (pGEP, AlloMap, AM) test was defined as >30 in the first five months post-transplant and >34 afterward. Samples associated with a positive biopsy result were excluded. We further stratified our cohort based on race.Results:Of 643 paired dd-cfDNA/GEP samples from 227 patients, the median age was 55 years, and 27% were female. The cohort was stratified into four categories: 53% were white, 18.8% were AA, 20.4% were Hispanic/Latino, and 7.8% were of other races. We observed no baseline differences in the positive dd-cfDNA levels (p=0.39) and positivity rates of dd-cfDNA at a threshold of 0.12% and 0.20% (p=0.08, p=0.06) or GEP positivity rates (p=0.52) between the 4 race categories in our surveillance cohort (Figure 1). Further comparison of patients treated for rejection based on non-invasive monitoring did not reveal a difference.Conclusions:In our surveillance cohort, dd-cfDNA levels and positivity rates were not different between AA and white HT recipients. Further prospective randomized trials are needed to elucidate potential race-related differences in dd-cfDNA levels.

Circulation, Volume 150, Issue Suppl_1, Page A4144019-A4144019, November 12, 2024. Introduction:Donor fraction (DF) cell-free DNA (cfDNA) is an emerging tool for non-invasive rejection surveillance in heart transplantation (HTx). Little is known about the significance of DF and/or total cfDNA (TcfDNA) in the first month post HTx. We explored the relationship between early cfDNA results and later clinical events in adult and pediatric HTx recipients.Aims:1. Explore the relationship between early cfDNA levels and late events after HTx 2. Describe the decline in DF during the first month post HTxMethods:Retrospective data from the multicenter prospective blinded DTRT study (DNA-based transplant rejection test) was used. DF and TcfDNA results from samples drawn post HTx day 1 (35d to 1 year post HTx. Captured events include cardiac arrest, mechanical circulatory support or death. Exclusions included absent TcfDNA, multiorgan Tx, any PTLD, or other cancer in previous 2 yrs. Cell-free DNA values were compared across event groups and time windows using GEE (generalized estimating equation) with Max Likelihood Estimation.Results:190 subjects had 566 samples drawn < 35 days post HTx. Median age was 17.8 yrs with a range of 26 days - 73.4 yrs; 51.8% were pediatric. 16 subjects (8.4%) had events bridging or following day 35 post HTx; 23 with events prior to day 35 were included as non-event subjects. TcfDNA was significantly higher in event subjects on days 4, 7 and 28 post HTx. [Fig 1]. There was no difference in DF between event and non-event subjects. Median (IQR) DF at 14 days was 0.26% (0.20, 0.39) and 0.19% (0.13, 0.29) at 28 days.Conclusions:Elevated TcfDNA, even in the setting of normal DF, may be a useful early marker to identify patients at risk for later events in the first year post HTx. The majority of patients reach low DF levels by 28 days post HTx suggesting an opportunity to incorporate cfDNA earlier into rejection surveillance protocols.

Circulation, Volume 150, Issue Suppl_1, Page A4146137-A4146137, November 12, 2024. Introduction:Durable left ventricular assist devices (LVADs) improve survival in heart failure but carry high risks of infection, hemocompatibility-related adverse events (HRAE), and death. Current clinical tools use subjective assessments or do not incorporate changes in end-organ injury, limiting their prognostic value with these complications. Plasma cell-free DNA (cfDNA), a potential marker for systemic and tissue-specific injury, holds promise to enhance patient risk stratification and prognosis.Hypothesis:LVAD implantation decreases systemic and end-organ injury, measured by cfDNA.Goal:We aimed to assess changes in global and organ-specific injury patterns with LVAD implantation via cfDNA, and to determine if pre-LVAD cfDNA levels predict post-LVAD complications.Methods:We prospectively recruited 40 LVAD candidates and 40 healthy controls. Pre- and post-LVAD plasma samples were analyzed for nuclear cfDNA (ncfDNA) and mitochondrial cfDNA (mtcfDNA) using digital droplet PCR. Tissue-specific cfDNA levels were measured using bisulfite sequencing and a deconvolution algorithm with a DNA methylation library. Wilcoxon sign-rank tests were used to compare cfDNA levels, and log-rank tests were used to compare outcomes. The clinical outcomes were HRAE-free survival (major bleeding, stroke, device thrombosis, death), and post-LVAD infection. Random forest machine learning analysis ranked tissue-specific cfDNA to identify the tissue patterns of those who develop HRAE and those who develop infection.Results:Prior to LVAD implantation, patients had 4-fold higher ncfDNA and 2-fold higher mtcfDNA compared to healthy controls. After LVAD placement, ncfDNA (18980 vs. 10228 copies/mL, p < .001) and mtcfDNA levels (1051882 vs 579609 copies/mL, p < .001) nearly halved, with significant reductions in innate immune and liver cfDNA (Figure). High pre-LVAD ncfDNA levels were associated with worse HRAE-free survival (p = .035) and infection (p < .001). Tissue sources important in determining HRAEs were distinct from those related to infections.Conclusion:LVAD placement significantly improves systemic and tissue-specific injury, measured by reductions in cfDNA. While pre-LVAD cfDNA levels predict both post-LVAD HRAEs and infections, the cfDNA tissue contributions influencing these outcomes are distinct. Future studies will need to validate these findings and compare cfDNA to other predictors of poor LVAD outcomes.

Circulation, Volume 150, Issue Suppl_1, Page A4143940-A4143940, November 12, 2024. Introduction:Ischemia/reperfusion (I/R) injury occurs after coronary revascularization, contributing to infarct size. Circulating mitochondrial DNA (mtDNA) levels are elevated in acute myocardial infarction (MI) patients, and act as Damage Associated Molecular Patterns (mtDNA DAMP), which are recognized by the Toll-like receptor 9 (TLR9), initiating pro-inflammatory responses. Prior studies have shown that loss of TLR9 prevents I/R injury in isolated mouse hearts. However, mtDNA DAMP levels have not been measured in ST-elevation MI (STEMI) patients, and whether blocking TLR9 in mice can reduce I/R injury remains unknown.Hypothesis:MtDNA DAMP levels serve as markers of STEMI related cardiac injury. Blocking the activation of TLR9 will decrease cardiac I/R injury.Methods:MtDNA DAMP levels in serum were measured pre- and 24 hours post- PCI in 55 STEMI patients and 37 healthy controls by qPCR. To evaluate the role of TLR9 on I/R injury, ODN2088 was used to block TLR9 receptor, wild type and TLR9 germline KO mice were subjected to close-chest I/R surgery with minimal systemic inflammation. The cardiac systolic function and infarct size were assessed. Immune cells were isolated from the injured left ventricle and spleens and detected by flow cytometry.Results:Pre- PCI mtDNA DAMP levels were increased ~200 folds in STEMI patients compared to healthy controls. After PCI, the elevated mtDNA DAMP levels reduced significantly, while the troponin T levels increased, suggesting mtDNA is an early marker of MI. Compared with negative ODN, ODN2088 treatment at reperfusion reduced infarct size and total leukocytes, myeloid cells, neutrophils and TNF-α+cells, and a trend of reduced IL-1β+cells, and there was no difference in IL-6+cells, total macrophages and residential macrophages. Loss of TLR9 in male and female mice significantly reduced infarct size by ~40% and preserved the systolic function. Meanwhile, there is no difference between genders.Conclusions:Circulating mtDNA DAMP level is an early marker of STEMI and may predict the success of PCI. Blocking the mtDNA DAMP-TLR9 signaling pathway during reperfusion significantly reduces I/R injury, indicating it is a viable therapy to mitigate cardiac I/R injury after prompt coronary revascularization.

Circulation, Volume 150, Issue Suppl_1, Page A4143732-A4143732, November 12, 2024. Background:Variants of mitochondrial DNA (mtDNA) may exist in heteroplasmy and have been associated with increased risk of mortality and cancer. While variation in mtDNA has been associated with cardiomyopathy in mitochondrial disease, the association between mtDNA heteroplasmy and cardiovascular disease (CVD) in the general population has not been well studied.Methods:We quantified mtDNA heteroplasmy from whole genome sequencing data in 476,434 participants in the UK Biobank. We defined a variant allele frequency of 5–95% as heteroplasmic. We functionally characterized mtDNA single nucleotide variants using a constraint-based score, mitochondrial local constraint score sum (MSS). We evaluated the associations of MSS and incident CVD, defined as fatal or non-fatal myocardial infarction (MI) or ischemic stroke (IS), using Cox proportional hazards models adjusted for demographic, lifestyle, and clinical factors in 463,705 participants free of CVD. We further tested the interactions with clonal hematopoiesis of indeterminant potential (CHIP) and leukocyte telomere length (LTL) in the association between heteroplasmy and CVD. We performed additional analysis stratified by MSS for each complex/region in the mtDNA, and subgroup analysis by sex and C-reactive protein (CRP; ≥3mg/L). Lastly, we performed analysis separately for MI and IS.Results:During a median (1stand 3rdquartiles) follow-up of 13.6 (13.1–14.5) years, there were 24,723 incident CVD events (16,959 MI and 9671 IS cases). 28.3% of the study population had 1 or more heteroplasmies. We found that higher MSS was associated with CVD (adjusted hazard ratio [aHR] for a 1-unit increase in MSS 1.08; 95% confidence interval 1.00–1.17). In particular, a higher MSS was associated with MI (aHR 1.13; 1.02–1.24) but not with IS (aHR 1.03; 0.90–1.17). The association between MSS and MI was stronger for fatal MI (aHR 1.52; 1.10–2.10) than for non-fatal MI (aHR 1.10; 0.99–1.21). When stratified by complex/region, higher MSS in Complex I increased the risk of MI by 27% (aHR 1.27; 1.06–1.53) but not in other complexes/regions. Although LTL was inversely associated with CVD outcomes, there were no associations with CHIP. Furthermore, there was no interactions by LTL, CHIP, sex, and CRP in the association between MSS and CVD outcomes.Conclusion:These results indicate that mitochondria may have a functional role in the development of CVD, particularly MI, and MSS may serve as a biomarker for CVD risk.

Circulation, Volume 150, Issue Suppl_1, Page A4143847-A4143847, November 12, 2024. Introduction:Our previous studies have shown that sustained activation of the DNA damage response (DDR) in cardiomyocytes leads to p53/p21 activation and cardiac dysfunction. Although the DDR generally involves molecules in DNA replication and repair pathways, the non-proliferative nature of cardiomyocytes suggests a cardio-specific DDR mechanism. However, our understanding of DDR in cardiomyocytes remains limited. Here, we aim to use CRISPR interference (CRISPRi) knockdown screens to identify genes critically involved in DDR regulation in human cardiomyocytes. We hypothesize that identifying these gene clusters may allow us to develop methods to prevent cardiac dysfunction by suppressing DDR in cardiomyocytes.Methods and Results:We established a human iPS cell line stably expressing dCas9-KRAB, which allows CRISPRi-mediated gene knockdown, and differentiated the cells into cardiomyocytes. The resulting human iPS cell-derived cardiomyocytes (hiPSCMs) showed the achievement of approximately 80% knockdown efficiency after gRNA transfection. We stimulated the hiPSCMs with H2O2and quantitatively evaluated the expression levels of the DDR markers γH2AX and p21 by immunostaining using the Operetta®high content imaging system. The DDR markers showed a significant concentration-dependent increase in response to H2O2administration. For arrayed CRISPRi screening, we constructed a gRNA library targeting 437 DDR-related genes. Using this library, we knocked down each DDR-related gene in hiPSCMs followed by H2O2stimulation. We quantified the expression levels of DDR markers by calculating the fluorescence intensity ratios relative to control after gene knockdown, and standardized them to calculate Z scores for all 437 genes. The screening successfully revealed the differential impact of each gene knockdown on γH2AX and p21 expression. We identified 71 genes that significantly affected their expression (Z-score < -1 or > 1). Mapping these genes to DDR pathways highlighted the differential impact of gene knockdown within the same pathway, and stratified their importance in cardiomyocytes.Conclusions:Arrayed CRISPR screening using hiPSCMs revealed differential functional significance of DDR-related genes in cardiomyocytes, identifying 71 genes of particularly significant importance. These findings provide a critical understanding of the cardio-specific DDR pathway and important clues for establishing an appropriate method to suppress DDR in the failing heart.

Circulation, Volume 150, Issue Suppl_1, Page A4141912-A4141912, November 12, 2024. Introduction:Pulmonary arterial hypertension (PAH) is characterized by vasoconstriction and vascular remodeling of the distal pulmonary arteries (PAs) leading to progressive elevation of PA pressure, right heart failure and death. Extensive proliferation and resistance to apoptosis of PA smooth muscle cells (PASMCs) is perhaps the most prominent feature of PAH accounting for the histopathological changes seen in this disease. With the recognition of this, direct targeting of vascular remodeling may represent a promising strategy.Goal:To illuminate new actionable targets involved in pulmonary vascular remodeling in PAH.Methods:Molecular, biochemical and pharmacological approaches were used to identify Never In Mitosis A-Related Kinase 2 (NEK2) and study its role in pulmonary vascular remodeling in PAHResults:We performed a comparative RNA-sequencing analysis between control and PAH-PASMCs and then integrated our dataset with publicly available datasets conducted on comparable samples. Among the genes that were concordantly up-regulated in PAH-PASMCs, we identified NEK2, a kinase and druggable target implicated in the progression of mitosis. Using Western blot (WB) and immunofluorescence (IF), we confirmed that NEK2 is upregulated in PASMCs and distal PAs of PAH patients and animal models (Sugen/Hypoxia- and monocrotaline-exposed rats). Pharmacological (NCL0017509) and molecular (siNEK2) inhibition of NEK2 reduced PAH-PASMCs proliferation, as assessed by IF (Ki67 labeling, EdU incorporation, p