Racial and socioeconomic disparity associates with differences in cardiac DNA methylation among men with end-stage heart failure.

Heart failure (HF) is a multifactorial syndrome that remains a leading cause of worldwide morbidity. Despite its high prevalence, only half of patients with HF respond to guideline-directed medical management, prompting therapeutic efforts to confront the molecular underpinnings of its heterogeneity. In the current study, we examined epigenetics as a yet unexplored source of heterogeneity among patients with end-stage HF. Specifically, a multicohort-based study was designed to quantify cardiac genome-wide cytosine-p-guanine (CpG) methylation of cardiac biopsies from male patients undergoing left ventricular assist device (LVAD) implantation. In both pilot (n = 11) and testing (n = 31) cohorts, unsupervised multidimensional scaling of genome-wide myocardial DNA methylation exhibited a bimodal distribution of CpG methylation found largely to occur in the promoter regions of metabolic genes. Among the available patient attributes, only categorical self-identified patient race could delineate this methylation signature, with African American (AA) and Caucasian American (CA) samples clustering separately. Because race is a social construct, and thus a poor proxy of human physiology, extensive review of medical records was conducted, but ultimately failed to identify covariates of race at the time of LVAD surgery. By contrast, retrospective analysis exposed a higher all-cause mortality among AA (56.3%) relative to CA (16.7%) patients at 2 yr following LVAD placement (P = 0.03). Geocoding-based approximation of patient demographics uncovered disparities in income levels among AA relative to CA patients. Although additional studies are needed, the current analysis implicates cardiac DNA methylation as a previously unrecognized indicator of socioeconomic disparity in human heart failure outcomes.NEW & NOTEWORTHY A bimodal signature of cardiac DNA methylation in heart failure corresponds with racial differences in all-cause mortality following mechanical circulatory support. Racial differences in promoter methylation disproportionately affect metabolic signaling pathways. Socioeconomic factors are associated with racial differences in the cardiac methylome among men with end-stage heart failure.

Genome-wide DNA methylation encodes cardiac transcriptional reprogramming in human ischemic heart failure.

Ischemic cardiomyopathy (ICM) is the clinical endpoint of coronary heart disease and a leading cause of heart failure. Despite growing demands to develop personalized approaches to treat ICM, progress is limited by inadequate knowledge of its pathogenesis. Since epigenetics has been implicated in the development of other chronic diseases, the current study was designed to determine whether transcriptional and/or epigenetic changes are sufficient to distinguish ICM from other etiologies of heart failure. Specifically, we hypothesize that genome-wide DNA methylation encodes transcriptional reprogramming in ICM. RNA-sequencing analysis was performed on human ischemic left ventricular tissue obtained from patients with end-stage heart failure, which enriched known targets of the polycomb methyltransferase EZH2 compared to non-ischemic hearts. Combined RNA sequencing and genome-wide DNA methylation analysis revealed a robust gene expression pattern consistent with suppression of oxidative metabolism, induced anaerobic glycolysis, and altered cellular remodeling. Lastly, KLF15 was identified as a putative upstream regulator of metabolic gene expression that was itself regulated by EZH2 in a SET domain-dependent manner. Our observations therefore define a novel role of DNA methylation in the metabolic reprogramming of ICM. Furthermore, we identify EZH2 as an epigenetic regulator of KLF15 along with DNA hypermethylation, and we propose a novel mechanism through which coronary heart disease reprograms the expression of both intermediate enzymes and upstream regulators of cardiac metabolism such as KLF15.

The role of fibrinolytic genes and proteins in the development of allograft vascular disease.

BACKGROUND: We have previously shown that lack of plasminogen activator inhibitor-1 (PAI-1) expression in donor tissue greatly increases intimal proliferation (IP) after allogeneic transplantation. We sought to determine the relative role of PAI-1 and other fibrinolytic proteins in the development of IP. METHODS: We used an abdominal aortic transplant model in mice to investigate IP in 3 groups of 6 recipients. In the isograft group, CBA/J strain mice were donors and recipients, donors for allograft group were C57BL/6J mice, and for the allograft/knockout group, C57BL/6J PAI-1 knockout mice. All groups received weekly injections of anti-CD8/CD4 monoclonal antibodies. IP was calculated at 50 days, and sections were analyzed for fibrinolytic proteins, messenger RNA (mRNA) and PAI-1 activity using immunohistochemistry (IHC), in situ hybridization (ISH), reverse transcription-polymerase chain reaction (RT-PCR), and Western blot analysis. RESULTS: Significantly more IP developed in the allograft/knockout group vs the isograft (p < 0.001) and the allograft groups (p = 0.003). There was marked intimal expression of tissue plasminogen activator (tPA), urokinase PA (uPA), and uPA receptor (uPAR) proteins and mRNA in the allograft and allograft/knockout groups vs the isograft group. Allografts also showed significant intimal staining for PAI-1 protein and mRNA. RT-PCR demonstrated a stepwise increase in profibrinolytic protein mRNA from isograft to allograft to allograft/knockout groups, particularly uPA (p = 0.02) and uPAR (p = 0.016). Western blot data showed complementary findings. PAI-1 activity was persistently present in isograft and allograft animals, only. Intimas in allograft and allograft/knockout groups were primarily smooth muscle cells. CONCLUSIONS: PAI-1 reduces IP by limiting smooth muscle cell activity, with little change in matrix composition likely by modulating profibrinolytic protein expression.

Transforming growth factor-beta polymorphisms and cardiac allograft rejection.

BACKGROUND: Cytokine gene polymorphisms regulate cytokine expression. We analyzed transforming growth factor-beta (TGF-beta) allelic variation in codon 25 in susceptibility to acute rejection episodes in cardiac transplant recipients. METHODS: Between June 1997 and December 2001, 123 de novo heart transplants were performed at UAB with analysis based on 109 patients. Clinical and laboratory data were recorded at intervals up to 1 year post-transplant. Recipient genotypes for TGF-beta (codon 25) were determined using polymerase chain reaction (PCR) sequence-specific primers. Correlations between TGF-beta genotypes and acute rejection were made using Kaplan-Meier plots and parametric hazard models. RESULTS: Of the patients enrolled, 72% had at least one rejection and 46% had multiple rejections in the first year post-transplant. Among those > or =55 years of age at transplant, patients with the GG genotype had significantly fewer rejections as compared to those with the CC or GC genotype (1.25 vs 2.5, p < 0.01). There was no difference in risk of rejection between the genotype groups among patients <50 years of age at transplant (p = 0.43). Similar results were observed when we used time to cumulative Grade 2R or greater rejection as the outcome. CONCLUSION: The GG TGF-beta genotype may protect against acute rejection in older recipients during the first year after transplant.

Alterations in the fibrinolytic cascade post-transplant: evidence of a bimodal expression pattern.

BACKGROUND: Changes in the fibrinolytic system that occur after cardiac transplantation (CTx) and the factors that influence such changes have been poorly described, yet may be important in determining the varying morphologic features of transplant-related coronary artery disease (TxCAD). METHODS: Baseline demographic as well as serial clinical information and plasma fibrinolytic levels were prospectively recorded pre-CTx and at multiple time-points post-CTx in 110 de novo cardiac transplant recipients. RESULTS: We noted a biphasic change in fibrinolytic activity over the first year of CTx with an early immediate decline in plasminogen activator inhibitor-1 (PAI-1) activity (p > 0.001) matched with stable PAP (plasmin) activity corresponding to an "enhanced" fibrinolytic state early post-CTx. This was followed by a significant increase at 6 months (p = 0.004) and 1 year (p < 0.001) in PAI-1 activity concomitant with a significant decline in PAP after 3 months (p = 0.005 at 3 months, p < 0.001 at 6 months, p < 0.001 at 1 year) corresponding to an "impaired" fibrinolytic state late post-CTx. CONCLUSION: The biphasic nature of the fibrinolytic system observed herein may account for the varying morphologic features of TxCAD.

Identification of elements essential for transcription in Brugia malayi promoters.

Little is known concerning promoter structure in the filarial parasites. Recently, transient transfection methods have been developed for the human filarial parasite Brugia malayi. These methods have been employed to localize the promoter for the 70kDa heat shock protein (BmHSP70) to a region extending 394nt upstream from the initiating codon of the BmHSP70 open reading frame. Replacement mutagenesis was used to define the elements necessary for BmHSP70 promoter activity in detail. Four domains, ranging in size from six to 22 nucleotides, were found to be necessary for full promoter activity. The two most distal domains encoded a binding site for the heat shock transcription factor and a putative binding site for the GAGA transcription factor, motifs that are found in many other HSP70 promoters. However, none of the essential domains contained sequences typical of cis elements that are usually found in the core domain of a eukaryotic promoter. The largest essential domain was located at positions -53 to -32, and included the splice leader addition site. These data suggest that the regulatory domains of the BmHSP70 promoter were similar to those found in other eukaryotes, but that the core promoter domain exhibited features that appeared to be distinct from those found in most other well-characterized eukaryotic promoters. An analysis of two additional promoters of B.malayi highly transcribed genes suggests that they also lack features commonly found in most eukaryotic core promoters, suggesting that the unique features of the BmHSP70 core promoter are not confined to this gene.

Donor PAI-1 expression inhibits the intimal response of early allograft vascular disease.

BACKGROUND: The development of allograft vascular disease (AVD) may be related to altered expression of the fibrinolytic system. We determined the extent to which plasminogen activator inhibitor type 1 (PAI-1) expression in donor tissue influences intimal proliferation (IP) in a mouse model of AVD. METHODS: We utilized an end-to-end abdominal aortic transplant model in mice to investigate the development of IP in 3 groups of 6 recipients. Group A (negative control) utilized C57BL/6J strain mice as both donors and recipients. In Groups B (positive control) and C, C57BL/6J mice were vessel donors and CBA/J mice were recipients. Both groups received intraperitoneal anti-CD4 and anti-CD8 monoclonal antibodies (250 microg/week for 5 weeks). Group C recipients, however, were transplanted with vessels from C57BL/6J PAI-1 knockout mice. Animals were killed at 50 days. Transplanted aortas were removed and intimal areas calculated using morphometric analysis. RESULTS: Group A (mean intimal area 6421 +/- 8507 microm(2)) demonstrated very little IP in comparison to the other groups. IP was significantly higher in Group B (mean intimal area 56357 +/- 35629 microm(2)) than Group A (p = 0.008). Group C (mean intimal area 288195 +/- 123279 microm(2)) demonstrated significantly more intimal proliferation than either Groups A or B (vs B, p = 0.003; vs A, p < 0.001). The significance of these results is maintained if intimal thickness is measured as a stand-alone reference for the intimal response. CONCLUSIONS: Lack of PAI-1 expression in donor tissue greatly exaggerates the extent of IP after allogeneic transplantation and suggests that PAI-1 is important in limiting the early phase of AVD.