The landscape of somatic mutation in normal colorectal epithelial cells.

The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions that lead to cancer1. However, our understanding of the earliest phases of colorectal neoplastic changes-which may occur in morphologically normal tissue-is comparatively limited, as for most cancer types. Here we use whole-genome sequencing to analyse hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed; some of these were ubiquitous and continuous, whereas others were only found in some individuals, in some crypts or during certain periods of life. Probable driver mutations were present in around 1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium. Colorectal cancers exhibit substantially increased mutational burdens relative to normal cells. Sequencing normal colorectal cells provides quantitative insights into the genomic and clonal evolution of cancer.

The 2022 Banff Meeting Lung Report.

The Lung Session of the 2022 16th Banff Foundation for Allograft Pathology Conference-held in Banff, Alberta-focused on non-rejection lung allograft pathology and novel technologies for the detection of allograft injury. A multidisciplinary panel reviewed the state-of-the-art of current histopathologic entities, serologic studies, and molecular practices, as well as novel applications of digital pathology with artificial intelligence, gene expression analysis, and quantitative image analysis of chest computerized tomography. Current states of need as well as prospective integration of the aforementioned tools and technologies for complete assessment of allograft injury and its impact on lung transplant outcomes were discussed. Key conclusions from the discussion were: (1) recognition of limitations in current standard of care assessment of lung allograft dysfunction; (2) agreement on the need for a consensus regarding the standardized approach to the collection and assessment of pathologic data, inclusive of all lesions associated with graft outcome (eg, non-rejection pathology); and (3) optimism regarding promising novel diagnostic modalities, especially minimally invasive, which should be integrated into large, prospective multicenter studies to further evaluate their utility in clinical practice for directing personalized therapies to improve graft outcomes.

Report of the 2022 Banff Heart Concurrent: Focus on non-human leukocyte antigen antibodies in rejection and the pathology of "mixed" rejection.

The Banff Heart Concurrent Session, held as part of the 16th Banff Foundation for Allograft Pathology Conference at Banff, Alberta, Canada, on September 21, 2022, focused on 2 major topics: non-human leukocyte antigen (HLA) antibodies and mixed rejection. Each topic was addressed in a multidisciplinary fashion with clinical, immunological, and pathology perspectives and future developments and prospectives. Following the Banff organization model and principles, the collective aim of the speakers on each topic was to • Determine current knowledge gaps in heart transplant pathology • Identify limitations of current pathology classification systems • Discuss next steps in addressing gaps and refining classification system.

Graft function and incidence of cardiac allograft vasculopathy in donation after circulatory-determined death heart transplant recipients.

Experience in donation after circulatory-determined death (DCD) heart transplantation (HTx) is expanding. There is limited information on the functional outcomes of DCD HTx recipients. We sought to evaluate functional outcomes in our cohort of DCD recipients. We performed a single-center, retrospective, observational cohort study comparing outcomes in consecutive DCD and donation after brain death (DBD) HTx recipients between 2015 and 2019. Primary outcome was allograft function by echocardiography at 12 and 24 months. Secondary outcomes included incidence of cardiac allograft vasculopathy, treated rejection, renal function, and survival. Seventy-seven DCD and 153 DBD recipients were included. There was no difference in left ventricular ejection fraction at 12 months (59% vs 59%, P = .57) and 24 months (58% vs 58%, P = .87). There was no significant difference in right ventricular function at 12 and 24 months. Unadjusted survival between DCD and DBD recipients at 5 years (85.7% DCD and 81% DBD recipients; P = .45) was similar. There were no significant differences in incidence of cardiac allograft vasculopathy (odds ratio 1.59, P = .21, 95% confidence interval 0.77-3.3) or treated rejection (odds ratio 0.60, P = .12, 95% confidence interval 0.32-1.15) between DBD and DCD recipients. Post-transplant renal function was similar at 1 and 2 years. In conclusion, cardiac allografts from DCD donors perform similarly to a contemporary population of DBD allografts in the medium term.

A novel injury site-natural antibody targeted complement inhibitor protects against lung transplant injury.

Complement is known to play a role in ischemia and reperfusion injury (IRI). A general paradigm is that complement is activated by self-reactive natural IgM antibodies (nAbs), after they engage postischemic neoepitopes. However, a role for nAbs in lung transplantation (LTx) has not been explored. Using mouse models of LTx, we investigated the role of two postischemic neoepitopes, modified annexin IV (B4) and a subset of phospholipids (C2), in LTx. Antibody deficient Rag1-/- recipient mice were protected from LTx IRI. Reconstitution with either B4 or C2nAb restored IRI, with C2 significantly more effective than B4 nAb. Based on these information, we developed/characterized a novel complement inhibitor composed of single-chain antibody (scFv) derived from the C2 nAb linked to Crry (C2scFv-Crry), a murine inhibitor of C3 activation. Using an allogeneic LTx, in which recipients contain a full nAb repertoire, C2scFv-Crry targeted to the LTx, inhibited IRI, and delayed acute rejection. Finally, we demonstrate the expression of the C2 neoepitope in human donor lungs, highlighting the translational potential of this approach.

Emphysema-associated Autoreactive Antibodies Exacerbate Post-Lung Transplant Ischemia-Reperfusion Injury.

Chronic obstructive pulmonary disease-associated chronic inflammation has been shown to lead to an autoimmune phenotype characterized in part by the presence of lung autoreactive antibodies. We hypothesized that ischemia-reperfusion injury (IRI) liberates epitopes that would facilitate preexisting autoantibody binding, thereby exacerbating lung injury after transplant. We induced emphysema in C57BL/6 mice through 6 months of cigarette smoke (CS) exposure. Mice with CS exposure had significantly elevated serum autoantibodies compared with non-smoke-exposed age-matched (NS) mice. To determine the impact of a full preexisting autoantibody repertoire on IRI, we transplanted BALB/c donor lungs into NS or CS recipients and analyzed grafts 48 hours after transplant. CS recipients had significantly increased lung injury and immune cell infiltration after transplant. Immunofluorescence staining revealed increased IgM, IgG, and C3d deposition in CS recipients. To exclude confounding alloreactivity and confirm the role of preexisting autoantibodies in IRI, syngeneic Rag1-/- (recombination-activating protein 1-knockout) transplants were performed in which recipients were reconstituted with pooled serum from CS or NS mice. Serum from CS-exposed mice significantly increased IRI compared with control mice, with trends in antibody and C3d deposition similar to those seen in allografts. These data demonstrate that pretransplant CS exposure is associated with increased IgM/IgG autoantibodies, which, upon transplant, bind to the donor lung, activate complement, and exacerbate post-transplant IRI.

Catastrophic sickling crisis in patient undergoing cardiac transplantation with sickle cell trait.

There is debate in the literature regarding management of patients with sickle cell trait (SCT) undergoing cardiac surgery, since it is recognized that cardiopulmonary bypass presents many precipitating risk factors for a sickling crisis. Despite this, many report successful outcomes without any modification to perioperative management. A 49-year-old woman with SCT (HbS 38%) with postpartum cardiomyopathy underwent cardiac transplantation. The patient was cooled to 34.0°C and retrograde cold blood cardioplegia was infused continuously. The cold ischemic time was 219 minutes and warm ischemic time 46 minutes. After weaning from bypass, she developed global cardiac dysfunction requiring veno-arterial extracorporeal membrane oxygenation. The circuit suddenly stopped, requiring emergency reinstitution of bypass; the circuit had clotted. Transesophageal-echocardiogram revealed thrombus within the left atrium and ventricle. There was no recovery of cardiac function and the patient developed multiorgan failure. At postmortem there was extensive myocardial infarction with evidence of widespread catastrophic intravascular red-cell sickling. This case highlights the danger of complacency in patients with SCT, offering a learning opportunity for the cardiothoracic community to highlight the most serious complication that can occur in this group of patients. We have learned that SCT and cardiac surgery is not a benign combination.

Germinal center humoral autoimmunity independently mediates progression of allograft vasculopathy.

The development of humoral autoimmunity following organ transplantation is increasingly recognised, but of uncertain significance. We examine whether autoimmunity contributes independently to allograft rejection. In a MHC class II-mismatched murine model of chronic humoral rejection, we report that effector antinuclear autoantibody responses were initiated upon graft-versus-host allorecognition of recipient B cells by donor CD4 T-cells transferred within heart allografts. Consequently, grafts were rejected more rapidly, and with markedly augmented autoantibody responses, upon transplantation of hearts from donors previously primed against recipient. Nevertheless, rejection was dependent upon recipient T follicular helper (TFH) cell differentiation and provision of cognate (peptide-specific) help for maintenance as long-lived GC reactions, which diversified to encompass responses against vimentin autoantigen. Heart grafts transplanted into stable donor/recipient mixed haematopoietic chimeras, or from parental strain donors into F1 recipients (neither of which can trigger host adaptive alloimmune responses), nevertheless provoked GC autoimmunity and were rejected chronically, with rejection similarly dependent upon host TFH cell differentiation. Thus, autoantibody responses contribute independently of host adaptive alloimmunity to graft rejection, but require host TFH cell differentiation to maintain long-lived GC responses. The demonstration that one population of helper CD4 T-cells initiates humoral autoimmunity, but that a second population of TFH cells is required for its maintenance as a GC reaction, has important implications for how autoimmune-related phenomena manifest.

Donor pretreatment with nebulized complement C3a receptor antagonist mitigates brain-death induced immunological injury post-lung transplant.

Donor brain death (BD) is an inherent part of lung transplantation (LTx) and a key contributor to ischemia-reperfusion injury (IRI). Complement activation occurs as a consequence of BD in other solid organ Tx and exacerbates IRI, but the role of complement in LTx has not been investigated. Here, we investigate the utility of delivering nebulized C3a receptor antagonist (C3aRA) pretransplant to BD donor lungs in order to reduce post-LTx IRI. BD was induced in Balb/c donors, and lungs nebulized with C3aRA or vehicle 30 minutes prior to lung procurement. Lungs were then cold stored for 18 hours before transplantation into C57Bl/6 recipients. Donor lungs from living donors (LD) were removed and similarly stored. At 6 hours and 5 days post-LTx, recipients of BD donor lungs had exacerbated IRI and acute rejection (AR), respectively, compared to recipients receiving LD lungs, as determined by increased histopathological injury, immune cells, and cytokine levels. A single pretransplant nebulized dose of C3aRA to the donor significantly reduced IRI as compared to vehicle-treated BD donors, and returned IRI and AR grades to that seen following LD LTx. These data demonstrate a role for complement inhibition in the amelioration of IRI post-LTx in the context of donor BD.

Targeting pathogenic postischemic self-recognition by natural IgM to protect against posttransplantation cardiac reperfusion injury.

BACKGROUND: Natural IgM antibodies represent a class of innate pattern recognition receptors that recognize danger-associated molecular patterns expressed on stressed or dying cells. They play important roles in tissue homeostasis by disposing of prenecrotic cells and suppressing inflammation. However, ischemic insult leads to a pathogenic level of IgM binding and complement activation, resulting in inflammation and injury. We investigate the role of self-reactive IgM in the unique setting of transplantation where the donor organ undergoes both cold and warm ischemia and global ischemic insult. METHODS AND RESULTS: By transplanting hearts from wild-type donor mice into antibody-deficient mice reconstituted with specific self-reactive IgM monoclonal antibodies, we identified neoepitopes expressed after transplantation and demonstrated a key role for IgM recognition of these epitopes in graft injury. With this information, we developed and characterized a therapeutic strategy that exploited the postischemia recognition system of natural antibodies. On the basis of neoepitope identification, we constructed an anti-annexin IV single-chain antibody (scFv) and an scFv linked to Crry, an inhibitor of C3 activation (scFv-Crry). In an allograft transplantation model in which recipients contain a full natural antibody repertoire, both constructs blocked graft IgM binding and complement activation and significantly reduced graft inflammation and injury. Furthermore, scFv-Crry specifically targeted to the transplanted heart and, unlike complement deficiency, did not affect immunity to infection, an important consideration for immunosuppressed transplant recipients. CONCLUSIONS: We identified pathophysiologically important epitopes expressed within the heart after transplantation and described a novel translatable strategy for targeted complement inhibition that has several advantages over currently available approaches.

Diagnostic yield of molecular autopsy in patients with sudden arrhythmic death syndrome using targeted exome sequencing.

AIMS: The targeted genetic screening of Sudden Arrhythmic Death Syndrome (SADS) probands in a molecular autopsy has a diagnostic yield of up to 35%. Exome sequencing has the potential to improve this yield. The primary aim of this study is to examine the feasibility and diagnostic utility of targeted exome screening in SADS victims, utilizing familial clinical screening whenever possible. METHODS AND RESULTS: To determine the feasibility and diagnostic yield of targeted exome sequencing deoxyribonucleic acid (DNA) was isolated from 59 SADS victims (mean age 25 years, range 1-51 years). Targeted exome sequencing of 135 genes associated with cardiomyopathies and ion channelopathies was performed on the Illumina HiSeq2000 platform. Non-synonymous, loss-of-function, and splice-site variants with a minor allele frequency <0.02% in the NHLBI exome sequencing project and an internal set of control exomes were prioritized for analysis followed by <0.5% frequency threshold secondary analysis. First-degree relatives were offered clinical screening for inherited cardiac conditions. Seven probands (12%) carried very rare (<0.02%) or novel non-sense candidate mutations and 10 probands (17%) had previously published rare (0.02-0.5%) candidate mutations-a total yield of 29%. Co-segregation fully confirmed two private SCN5A Na channel mutations. Variants of unknown significance were detected in a further 34% of probands. CONCLUSION: Molecular autopsy using targeted exome sequencing has a relatively low diagnostic yield of very rare potentially disease causing mutations. Candidate pathogenic variants with a higher frequency in control populations are relatively common and should be interpreted with caution.

Donor brain death exacerbates complement-dependent ischemia/reperfusion injury in transplanted hearts.

BACKGROUND: Brain death (BD) can immunologically prime the donor organ and is thought to lead to exacerbated ischemia/reperfusion injury after transplantation. Using a newly developed mouse model of BD, we investigated the effect of donor BD on posttransplantation cardiac ischemia/reperfusion injury. We further investigated the therapeutic effect of a targeted complement inhibitor in recipients of BD donor hearts and addressed the clinical relevance of these studies by analyzing human heart biopsies from BD and domino (living) donors. METHODS AND RESULTS: Hearts from living or BD donor C57BL/6 mice were transplanted into C57BL/6 or BALB/c recipients. Recipient mice were treated with the complement inhibitor CR2-Crry or vehicle control (n=6). Isografts were analyzed 48 hours after transplantation for injury, inflammation, and complement deposition, and allografts were monitored for graft survival. Human cardiac biopsies were analyzed for complement deposition and inflammatory cell infiltration. In the murine model, donor BD exacerbated ischemia/reperfusion injury and graft rejection, as demonstrated by increased myocardial injury, serum cardiac troponin, cellular infiltration, complement deposition, inflammatory chemokine and cytokine levels, and by decreased graft survival. CR2-Crry treatment of recipients significantly reduced all measured outcomes in grafts from both BD and living donors compared with controls. Analysis of human samples documented the relevance of our experimental findings and revealed exacerbated complement deposition and inflammation in grafts from BD donors compared with grafts from living donors. CONCLUSIONS: BD exacerbates posttransplantation cardiac ischemia/reperfusion injury in mice and humans and decreases survival of mouse allografts. Furthermore, targeted complement inhibition in recipient mice ameliorates BD-exacerbated ischemia/reperfusion injury.

TNF, acting through inducibly expressed TNFR2, drives activation and cell cycle entry of c-Kit+ cardiac stem cells in ischemic heart disease.

TNF, signaling through TNFR2, has been implicated in tissue repair, a process that in the heart may be mediated by activated resident cardiac stem cells (CSCs). The objective of our study is to determine whether ligation of TNFR2 can induce activation of resident CSCs in the setting of ischemic cardiac injury. We show that in human cardiac tissue affected by ischemia heart disease (IHD), TNFR2 is expressed on intrinsic CSCs, identified as c-kit(+)/CD45(-)/VEGFR2(-) interstitial round cells, which are activated as determined by entry to cell cycle and expression of Lin-28. Wild-type mouse heart organ cultures subjected to hypoxic conditions both increase cardiac TNF expression and show induced TNFR2 and Lin-28 expression in c-kit(+) CSCs that have entered cell cycle. These CSC responses are enhanced by exogenous TNF. TNFR2(-/-) mouse heart organ cultures subjected to hypoxia increase cardiac TNF but fail to induce CSC activation. Similarly, c-kit(+) CSCs isolated from mouse hearts exposed to hypoxia or TNF show induction of Lin-28, TNFR2, cell cycle entry, and cardiogenic marker, α-sarcomeric actin (α-SA), responses more pronounced by hypoxia in combination with TNF. Knockdown of Lin-28 by siRNA results in reduced levels of TNFR2 expression, cell cycle entry, and diminished expression of α-SA. We conclude that hypoxia-induced c-kit(+) CSC activation is mediated by TNF/TNFR2/Lin-28 signaling. These observations suggest that TNFR2 signaling in resident c-kit(+) CSCs induces cardiac repair, findings which provide further understanding of the unanticipated harmful effects of TNF blockade in human IHD.

Correcting common OCT artifacts enhances plaque classification and identification of higher-risk plaque features.

BACKGROUND: Optical coherence tomography (OCT) is used widely to guide stent placement, identify higher-risk plaques, and assess mechanisms of drug efficacy. However, a range of common artifacts can prevent accurate plaque classification and measurements, and limit usable frames in research studies. We determined whether pre-processing OCT images corrects artifacts and improves plaque classification. METHODS: We examined both ex-vivo and clinical trial OCT pullbacks for artifacts that prevented accurate tissue identification and/or plaque measurements. We developed Fourier transform-based software that reconstructed images free of common OCT artifacts, and compared corrected and uncorrected images. RESULTS: 48 % of OCT frames contained image artifacts, with 62 % of artifacts over or within lesions, preventing accurate measurement in 12 % frames. Pre-processing corrected >70 % of all artifacts, including thrombus, macrophage shadows, inadequate flushing, and gas bubbles. True tissue reconstruction was achieved in 63 % frames that would otherwise prevent accurate clinical measurements. Artifact correction was non-destructive and retained anatomical lumen and plaque parameters. Correction improved accuracy of plaque classification compared against histology and retained accurate assessment of higher-risk features. Correction also changed plaque classification and prevented artifact-related measurement errors in a clinical study, and reduced unmeasurable frames to <5 % ex-vivo and ~1 % in-vivo. CONCLUSIONS: Fourier transform-based pre-processing corrects a wide range of common OCT artifacts, improving identification of higher-risk features and plaque classification, and allowing more of the whole dataset to be used for clinical decision-making and in research. Pre-processing can augment OCT image analysis systems both for stent optimization and in natural history or drug studies.

Assessing the role of phosphorylated S6 ribosomal protein in the pathological diagnosis of pulmonary antibody-mediated rejection.

BACKGROUND: Pulmonary antibody-mediated rejection is still a challenging diagnosis as C4d immunostaining has poor sensitivity. Previous studies have indicated that the phosphorylated S6 ribosomal protein, a component of the mammalian target of rapamycin (mTOR) pathway, is correlated with de novo donor-specific antibodies in lung transplantation. The objective of this study was to evaluate the phosphorylation of S6 ribosomal protein as a surrogate for antibody-mediated rejection diagnosis in lung transplant patients. METHODS: This multicentre retrospective study analyzed transbronchial biopsies from 216 lung transplanted patients, 114 with antibody-mediated rejection and 102 without (19 with acute cellular rejection, 17 with ischemia/reperfusion injury, 18 with infection, and 48 without post-transplant complications). Immunohistochemistry was used to quantify phosphorylated S6 ribosomal protein expression in macrophages, endothelium, epithelium, and inter-pathologist agreement was assessed. RESULTS: Median phosphorylated S6 ribosomal protein expression values were higher in antibody-mediated rejection cases than in controls for all cell components, with the highest sensitivity in macrophages (0.9) and the highest specificity in endothelial expression (0.8). The difference was mainly significant in macrophages compared to other post-lung transplantation complications. Inter-pathologist agreement was moderate for macrophages and endothelium, with higher agreement when phosphorylated S6 ribosomal protein expression was dichotomized into positive/negative. The inclusion of phosphorylated S6 ribosomal protein in the diagnostic algorithm could have increased antibody-mediated rejection certainty levels by 25%. CONCLUSIONS: The study supports the role of the mTOR pathway in antibody-mediated rejection-related graft injury and suggests that tissue phosphorylation of S6 ribosomal protein could be a useful surrogate for a more accurate pathological diagnosis of lung antibody-mediated rejection.

Blocking lymphotoxin signaling abrogates the development of ectopic lymphoid tissue within cardiac allografts and inhibits effector antibody responses.

Tertiary lymphoid organs (TLOs) may develop within allografts, but their contribution to graft rejection remains unclear. Here, we study a mouse model of autoantibody-mediated cardiac allograft vasculopathy to clarify the alloimmune responses mediated by intragraft TLOs and whether blocking lymphotoxin-ß-receptor (LTßR) signaling, a pathway essential for lymphoid organogenesis, abrogates TLO development. TLOs (defined as discrete lymphoid aggregates associated with high endothelial venules) were detectable in 9 of 13 heart allografts studied and were predominantly B cell in composition, harboring germinal-center activity. These are most likely manifestations of the humoral autoimmunity triggered in this model after transplantation; TLOs did not develop if autoantibody production was prevented. Treatment with inhibitory LTßR-Ig fusion protein virtually abolished allograft TLO formation (mean TLOs/heart: 0.2 vs. 2.2 in control recipients; P=0.02), with marked attenuation of the autoantibody response. Recipients primed for autoantibody before transplantation rejected grafts rapidly, but this accelerated rejection was prevented by postoperative administration of LTßR-Ig (median survival time: 18 vs. >50 d, respectively, P=0.003). Our results provide the first demonstration that TLOs develop within chronically rejecting heart allografts, are predominantly B cell in origin, and can be targeted pharmacologically to inhibit effector humoral responses.

Apoptosis of vascular smooth muscle cells induces features of plaque vulnerability in atherosclerosis.

Vascular smooth muscle cell (VSMC) apoptosis occurs in many arterial diseases, including aneurysm formation, angioplasty restenosis and atherosclerosis. Although VSMC apoptosis promotes vessel remodeling, coagulation and inflammation, its precise contribution to these diseases is unknown, given that apoptosis frequently accompanies vessel injury or alterations to flow. To study the direct consequences of VSMC apoptosis, we generated transgenic mice expressing the human diphtheria toxin receptor (hDTR, encoded by HBEGF) from a minimal Tagln (also known as SM22alpha) promoter. Despite apoptosis inducing loss of 50-70% of VSMCs, normal arteries showed no inflammation, reactive proliferation, thrombosis, remodeling or aneurysm formation. In contrast, VSMC apoptosis in atherosclerotic plaques of SM22alpha-hDTR Apoe-/- mice induced marked thinning of fibrous cap, loss of collagen and matrix, accumulation of cell debris and intense intimal inflammation. We conclude that VSMC apoptosis is 'silent' in normal arteries, which have a large capacity to withstand cell loss. In contrast, VSMC apoptosis alone is sufficient to induce features of plaque vulnerability in atherosclerosis. SM22alpha-hDTR Apoe-/- mice may represent an important new model to test agents proposed to stabilize atherosclerotic plaques.

Selenoprotein deficiency disorder predisposes to aortic aneurysm formation.

Aortic aneurysms, which may dissect or rupture acutely and be lethal, can be a part of multisystem disorders that have a heritable basis. We report four patients with deficiency of selenocysteine-containing proteins due to selenocysteine Insertion Sequence Binding Protein 2 (SECISBP2) mutations who show early-onset, progressive, aneurysmal dilatation of the ascending aorta due to cystic medial necrosis. Zebrafish and male mice with global or vascular smooth muscle cell (VSMC)-targeted disruption of Secisbp2 respectively show similar aortopathy. Aortas from patients and animal models exhibit raised cellular reactive oxygen species, oxidative DNA damage and VSMC apoptosis. Antioxidant exposure or chelation of iron prevents oxidative damage in patient's cells and aortopathy in the zebrafish model. Our observations suggest a key role for oxidative stress and cell death, including via ferroptosis, in mediating aortic degeneration.

Distinct epigenomic features in end-stage failing human hearts.

BACKGROUND: The epigenome refers to marks on the genome, including DNA methylation and histone modifications, that regulate the expression of underlying genes. A consistent profile of gene expression changes in end-stage cardiomyopathy led us to hypothesize that distinct global patterns of the epigenome may also exist. METHODS AND RESULTS: We constructed genome-wide maps of DNA methylation and histone-3 lysine-36 trimethylation (H3K36me3) enrichment for cardiomyopathic and normal human hearts. More than 506 Mb sequences per library were generated by high-throughput sequencing, allowing us to assign methylation scores to ≈28 million CG dinucleotides in the human genome. DNA methylation was significantly different in promoter CpG islands, intragenic CpG islands, gene bodies, and H3K36me3-enriched regions of the genome. DNA methylation differences were present in promoters of upregulated genes but not downregulated genes. H3K36me3 enrichment itself was also significantly different in coding regions of the genome. Specifically, abundance of RNA transcripts encoded by the DUX4 locus correlated to differential DNA methylation and H3K36me3 enrichment. In vitro, Dux gene expression was responsive to a specific inhibitor of DNA methyltransferase, and Dux siRNA knockdown led to reduced cell viability. CONCLUSIONS: Distinct epigenomic patterns exist in important DNA elements of the cardiac genome in human end-stage cardiomyopathy. The epigenome may control the expression of local or distal genes with critical functions in myocardial stress response. If epigenomic patterns track with disease progression, assays for the epigenome may be useful for assessing prognosis in heart failure. Further studies are needed to determine whether and how the epigenome contributes to the development of cardiomyopathy.

Targeted complement inhibitors protect against posttransplant cardiac ischemia and reperfusion injury and reveal an important role for the alternative pathway of complement activation.

Ischemia reperfusion injury (IRI) is an unavoidable event during solid organ transplantation and is a major contributor to early graft dysfunction and subsequent graft immunogenicity. In a therapeutic paradigm using targeted complement inhibitors, we investigated the role of complement, and specifically the alternative pathway of complement, in IRI to heart isografts. Mouse heterotopic isograft heart transplants were performed in C57BL/6 mice treated with a single injection of either CR2-Crry (inhibits all complement pathways) or CR2-fH (inhibits alternative complement pathway) immediately posttransplantation. Transplanted hearts were harvested at 12 and 48 h for analysis. Both inhibitors resulted in a significant reduction in myocardial IRI, as measured by histology and serum cardiac troponin I levels. Furthermore, compared with untreated controls, both inhibitors reduced graft complement deposition, neutrophil and macrophage infiltration, adhesion molecule expression (P-selectin, E-selectin, and I-CAM-1), and proinflammatory cytokine expression (TNF-α, IL-1ß, KC, and MCP-1). The reduction in myocardial damage and cellular infiltration was not significantly different between CR2-Crry- and CR2-fH-treated mice, although adhesion molecule and cytokine levels were significantly lower in CR2-Crry-treated mice compared with CR2-fH-treated mice. In conclusion, the alternative complement pathway plays a major contributing role in myocardial IRI after heart transplantation, and local (targeted) complement inhibition has the potential to provide an effective and safe therapeutic strategy to reduce graft injury. Although total complement blockade may be somewhat more efficacious in terms of reducing inflammation, specific blockade of the alternative pathway is likely to be less immunosuppressive in an already immunocompromised recipient.