DNMT3A clonal hematopoiesis-driver mutations induce cardiac fibrosis by paracrine activation of fibroblasts.

Hematopoietic mutations in epigenetic regulators like DNA methyltransferase 3 alpha (DNMT3A), play a pivotal role in driving clonal hematopoiesis of indeterminate potential (CHIP), and are associated with unfavorable outcomes in patients suffering from heart failure (HF). However, the precise interactions between CHIP-mutated cells and other cardiac cell types remain unknown. Here, we identify fibroblasts as potential partners in interactions with CHIP-mutated monocytes. We used combined transcriptomic data derived from peripheral blood mononuclear cells of HF patients, both with and without CHIP, and cardiac tissue. We demonstrate that inactivation of DNMT3A in macrophages intensifies interactions with cardiac fibroblasts and increases cardiac fibrosis. DNMT3A inactivation amplifies the release of heparin-binding epidermal growth factor-like growth factor, thereby facilitating activation of cardiac fibroblasts. These findings identify a potential pathway of DNMT3A CHIP-driver mutations to the initiation and progression of HF and may also provide a compelling basis for the development of innovative anti-fibrotic strategies.

Single-nuclear transcriptome profiling identifies persistent fibroblast activation in hypertrophic and failing human hearts of patients with longstanding disease.

AIMS: Cardiac fibrosis drives the progression of heart failure in ischaemic and hypertrophic cardiomyopathy. Therefore, the development of specific anti-fibrotic treatment regimens to counteract cardiac fibrosis is of high clinical relevance. Hence, this study examined the presence of persistent fibroblast activation during longstanding human heart disease at a single-cell resolution to identify putative therapeutic targets to counteract pathological cardiac fibrosis in patients. METHODS AND RESULTS: We used single-nuclei RNA sequencing with human tissues from two samples of one healthy donor, and five hypertrophic and two failing hearts. Unsupervised sub-clustering of 7110 nuclei led to the identification of 7 distinct fibroblast clusters. De-convolution of cardiac fibroblast heterogeneity revealed a distinct population of human cardiac fibroblasts with a molecular signature of persistent fibroblast activation and a transcriptional switch towards a pro-fibrotic extra-cellular matrix composition in patients with established cardiac hypertrophy and heart failure. This sub-cluster was characterized by high expression of POSTN, RUNX1, CILP, and a target gene adipocyte enhancer-binding protein 1 (AEBP1) (all P < 0.001). Strikingly, elevated circulating AEBP1 blood level were also detected in a validation cohort of patients with confirmed cardiac fibrosis and hypertrophic cardiomyopathy by cardiac magnetic resonance imaging (P < 0.01). Since endogenous AEBP1 expression was increased in patients with established cardiac hypertrophy and heart failure, we assessed the functional consequence of siRNA-mediated AEBP1 silencing in human cardiac fibroblasts. Indeed, AEBP1 silencing reduced proliferation, migration, and fibroblast contractile capacity and α-SMA gene expression, which is a hallmark of fibroblast activation (all P < 0.05). Mechanistically, the anti-fibrotic effects of AEBP1 silencing were linked to transforming growth factor-beta pathway modulation. CONCLUSION: Together, this study identifies persistent fibroblast activation in patients with longstanding heart disease, which might be detected by circulating AEBP1 and therapeutically modulated by its targeted silencing in human cardiac fibroblasts.

The RNA editor ADAR2 promotes immune cell trafficking by enhancing endothelial responses to interleukin-6 during sterile inflammation.

Immune cell trafficking constitutes a fundamental component of immunological response to tissue injury, but the contribution of intrinsic RNA nucleotide modifications to this response remains elusive. We report that RNA editor ADAR2 exerts a tissue- and stress-specific regulation of endothelial responses to interleukin-6 (IL-6), which tightly controls leukocyte trafficking in IL-6-inflamed and ischemic tissues. Genetic ablation of ADAR2 from vascular endothelial cells diminished myeloid cell rolling and adhesion on vascular walls and reduced immune cell infiltration within ischemic tissues. ADAR2 was required in the endothelium for the expression of the IL-6 receptor subunit, IL-6 signal transducer (IL6ST; gp130), and subsequently, for IL-6 trans-signaling responses. ADAR2-induced adenosine-to-inosine RNA editing suppressed the Drosha-dependent primary microRNA processing, thereby overwriting the default endothelial transcriptional program to safeguard gp130 expression. This work demonstrates a role for ADAR2 epitranscriptional activity as a checkpoint in IL-6 trans-signaling and immune cell trafficking to sites of tissue injury.

Long-term cardiac pathology in individuals with mild initial COVID-19 illness.

Cardiac symptoms are increasingly recognized as late complications of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in previously well individuals with mild initial illness, but the underlying pathophysiology leading to long-term cardiac symptoms remains unclear. In this study, we conducted serial cardiac assessments in a selected population of individuals with Coronavirus Disease 2019 (COVID-19) with no previous cardiac disease or notable comorbidities by measuring blood biomarkers of heart injury or dysfunction and by performing magnetic resonance imaging. Baseline measurements from 346 individuals with COVID-19 (52% females) were obtained at a median of 109 days (interquartile range (IQR), 77-177 days) after infection, when 73% of participants reported cardiac symptoms, such as exertional dyspnea (62%), palpitations (28%), atypical chest pain (27%) and syncope (3%). Symptomatic individuals had higher heart rates and higher imaging values or contrast agent accumulation, denoting inflammatory cardiac involvement, compared to asymptomatic individuals. Structural heart disease or high levels of biomarkers of cardiac injury or dysfunction were rare in symptomatic individuals. At follow-up (329 days (IQR, 274-383 days) after infection), 57% of participants had persistent cardiac symptoms. Diffuse myocardial edema was more pronounced in participants who remained symptomatic at follow-up as compared to those who improved. Female gender and diffuse myocardial involvement on baseline imaging independently predicted the presence of cardiac symptoms at follow-up. Ongoing inflammatory cardiac involvement may, at least in part, explain the lingering cardiac symptoms in previously well individuals with mild initial COVID-19 illness.

Longer leukocyte telomere length is associated with myeloid inflammation and increased mortality after transcatheter aortic valve replacement.

Aims: Inflammatory activation of leukocytes may limit prognosis of patients (pts) with severe aortic valve stenosis (AS) undergoing transcatheter aortic valve replacement (TAVR). Leukocyte telomere length (LTL) is a marker of proliferative capacity and inflammatory responsiveness but the impact of LTL on the prognosis in AS remains elusive. The aim of this study was to analyse the association of LTL with inflammatory markers and prognosis of pts undergoing TAVR. Methods and results: LTL was analysed using quantitative real-time PCR in 285 consecutive pts (median age 82 years) undergoing TAVR and correlated with 18-month all-cause mortality. C-reactive protein was significantly elevated in pts with the longest LTL (P = 0.017), paralleled by increased procalcitonin (PCT) serum levels (P = 0.0006). This inflammatory reaction was accompanied by increased myeloid cells in the highest LTL tertile, mainly a rise in circulating neutrophils (P = 0.0025) and monocytes (P = 0.01). Multivariate analysis revealed LTL (HR 2.6, 95%CI 1.4-5.1, P= 0.004) and PCT levels (HR 4.3, 95%CI 1.7-11.0, P = 0.003) as independent predictors of mortality. Conclusions: Longer LTL is associated with increased mortality after TAVR. This might be explained by enhanced proliferative capacity of cells resulting in myeloid and systemic inflammation. Our findings suggest that targeting the specific inflammation pathways could present a novel strategy to augment survival in selected patients with degenerative aortic stenosis.

Post-myocardial infarction heart failure dysregulates the bone vascular niche.

The regulation of bone vasculature by chronic diseases, such as heart failure is unknown. Here, we describe the effects of myocardial infarction and post-infarction heart failure on the bone vascular cell composition. We demonstrate an age-independent loss of type H endothelium in heart failure after myocardial infarction in both mice and humans. Using single-cell RNA sequencing, we delineate the transcriptional heterogeneity of human bone marrow endothelium, showing increased expression of inflammatory genes, including IL1B and MYC, in ischemic heart failure. Endothelial-specific overexpression of MYC was sufficient to induce type H bone endothelial cells, whereas inhibition of NLRP3-dependent IL-1ß production partially prevented the post-myocardial infarction loss of type H vasculature in mice. These results provide a rationale for using anti-inflammatory therapies to prevent or reverse the deterioration of bone vascular function in ischemic heart disease.

Single Nuclei Sequencing Reveals Novel Insights Into the Regulation of Cellular Signatures in Children With Dilated Cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is a leading cause of death in children with heart failure. The outcome of pediatric heart failure treatment is inconsistent, and large cohort studies are lacking. Progress may be achieved through personalized therapy that takes age- and disease-related pathophysiology, pathology, and molecular fingerprints into account. We present single nuclei RNA sequencing from pediatric patients with DCM as the next step in identifying cellular signatures. METHODS: We performed single nuclei RNA sequencing with heart tissues from 6 children with DCM with an age of 0.5, 0.75, 5, 6, 12, and 13 years. Unsupervised clustering of 18 211 nuclei led to the identification of 14 distinct clusters with 6 major cell types. RESULTS: The number of nuclei in fibroblast clusters increased with age in patients with DCM, a finding that was confirmed by histological analysis and was consistent with an age-related increase in cardiac fibrosis quantified by cardiac magnetic resonance imaging. Fibroblasts of patients with DCM >6 years of age showed a profoundly altered gene expression pattern with enrichment of genes encoding fibrillary collagens, modulation of proteoglycans, switch in thrombospondin isoforms, and signatures of fibroblast activation. In addition, a population of cardiomyocytes with a high proregenerative profile was identified in infant patients with DCM but was absent in children >6 years of age. This cluster showed high expression of cell cycle activators such as cyclin D family members, increased glycolytic metabolism and antioxidative genes, and alterations in ß-adrenergic signaling genes. CONCLUSIONS: Novel insights into the cellular transcriptomes of hearts from pediatric patients with DCM provide remarkable age-dependent changes in the expression patterns of fibroblast and cardiomyocyte genes with less fibrotic but enriched proregenerative signatures in infants.

Telomerase as a Therapeutic Target in Cardiovascular Disease.

Shortened telomeres have been linked to numerous chronic diseases, most importantly coronary artery disease, but the underlying mechanisms remain ill defined. Loss-of-function mutations and deletions in telomerase both accelerate telomere shortening but do not necessarily lead to a clinical phenotype associated with atherosclerosis, questioning the causal role of telomere length in cardiac pathology. The differential extranuclear functions of the 2 main components of telomerase, telomerase reverse transcriptase and telomerase RNA component, offer important clues about the complex relationship between telomere length and cardiovascular pathology. In this review, we critically discuss relevant preclinical models, genetic disorders, and clinical studies to elucidate the impact of telomerase in cardiovascular disease and its potential role as a therapeutic target. We suggest that the antioxidative function of mitochondrial telomerase reverse transcriptase might be atheroprotective, making it a potential target for clinical trials. Graphic Abstract: A graphic abstract is available for this article.

Single-cell RNA-sequencing reveals profound changes in circulating immune cells in patients with heart failure.

AIMS: Identification of signatures of immune cells at single-cell level may provide novel insights into changes of immune-related disorders. Therefore, we used single-cell RNA-sequencing to determine the impact of heart failure on circulating immune cells. METHODS AND RESULTS: We demonstrate a significant change in monocyte to T-cell ratio in patients with heart failure, compared to healthy subjects, which were validated by flow cytometry analysis. Subclustering of monocytes and stratification of the clusters according to relative CD14 and FCGR3A (CD16) expression allowed annotation of classical, intermediate, and non-classical monocytes. Heart failure had a specific impact on the gene expression patterns in these subpopulations. Metabolically active genes such as FABP5 were highly enriched in classical monocytes of heart failure patients, whereas ß-catenin expression was significantly higher in intermediate monocytes. The selective regulation of signatures in the monocyte subpopulations was validated by classical and multifactor dimensionality reduction flow cytometry analyses. CONCLUSION: Together this study shows that circulating cells derived from patients with heart failure have altered phenotypes. These data provide a rich source for identification of signatures of immune cells in heart failure compared to healthy subjects. The observed increase in FABP5 and signatures of Wnt signalling may contribute to enhanced monocyte activation.

Value of prognostic nutritional index for survival prediction in trans-catheter aortic valve replacement compared to other common nutritional indexes.

BACKGROUND: Nutritional status predicts outcomes after TAVR. Predictive value of Prognostic Nutritional Index (PNI) was investigated in patients undergoing TAVR, and compared to other nutritional indexes. METHODS: A cohort of 114 patients undergoing TAVR in a high-volume centre was studied. A prospective 1-year follow-up was completed. PNI was estimated as follows: (10 × serum albumin[g/dl])+(0.005 × total lymphocytes [1000/µl]). One-year survival was compared in patients with PNI above vs below median; Kaplan-Meier curves were created. A multivariate analysis was used to assess predictive value of PNI for 1-year mortality. ROC curves were used to assess discrimination by PNI, and to compare it with Geriatric Nutritional Risk Index (GNRI) and Body Mass Index (BMI). RESULTS: Mean age was 82.2 years, 59.6% were male. Mean PNI was 46 ± 5. Pre-procedurally, no differences were found between patients with high vs. low PNI. One-year mortality was significantly higher in patients with low PNI values (19/57 vs. 4/57; p < .001). Complications did not differ. A higher PNI predicted 1-year survival, even after adjusting for clinical factors (model 1: HR 0.8, 95% CI 0.7-0.9, p < .0001) and laboratory parameters (NT-proBNP, IL-6, CRP, eGFR, cystatin C, haemoglobin) (model 2: HR 0.8, 95% CI 0.7-0.9, p < .05). ROC curves revealed a stronger predictive value for PNI (AUC 0.80) compared to GNRI (0.77) and BMI (0.6). The optimal cut-off for PNI was 45. CONCLUSION: PNI is a useful and practical nutritional marker reflecting malnutrition and inflammation prior to the intervention, and strongly predicts 1-year survival. PNI seems to be a better prognostic marker than BMI or GNRI after TAVR.

Clonal haematopoiesis in chronic ischaemic heart failure: prognostic role of clone size for DNMT3A- and TET2-driver gene mutations.

AIMS: Somatic mutations of the epigenetic regulators DNMT3A and TET2 causing clonal expansion of haematopoietic cells (clonal haematopoiesis; CH) were shown to be associated with poor prognosis in chronic ischaemic heart failure (CHF). The aim of our analysis was to define a threshold of variant allele frequency (VAF) for the prognostic significance of CH in CHF. METHODS AND RESULTS: We analysed bone marrow and peripheral blood-derived cells from 419 patients with CHF by error-corrected amplicon sequencing. Cut-off VAFs were optimized by maximizing sensitivity plus specificity from a time-dependent receiver operating characteristic (ROC) curve analysis from censored data. 56.2% of patients were carriers of a DNMT3A- (N = 173) or a TET2- (N = 113) mutation with a VAF >0.5%, with 59 patients harbouring mutations in both genes. Survival ROC analyses revealed an optimized cut-off value of 0.73% for TET2- and 1.15% for DNMT3A-CH-driver mutations. Five-year-mortality was 18% in patients without any detected DNMT3A- or TET2 mutation (VAF < 0.5%), 29% with only one DNMT3A- or TET2-CH-driver mutations above the respective cut-off level and 42% in patients harbouring both DNMT3A- and TET2-CH-driver mutations above the respective cut-off levels. In carriers of a DNMT3A mutation with VAF ≥ 1.15%, 5-year mortality was 31%, compared with 18% mortality in those with VAF < 1.15% (P = 0.048). Likewise, in patients with TET2 mutations, 5-year mortality was 32% with VAF ≥ 0.73%, compared with 19% mortality with VAF < 0.73% (P = 0.029). CONCLUSION: The present study defines novel threshold levels for clone size caused by acquired somatic mutations in the CH-driver genes DNMT3A and TET2 that are associated with worse outcome in patients with CHF.

Clonal Hematopoiesis-Driver DNMT3A Mutations Alter Immune Cells in Heart Failure.

RATIONALE: Clonal hematopoiesis driven by mutations of DNMT3A (DNA methyltransferase 3a) is associated with increased incidence of cardiovascular disease and poor prognosis of patients with chronic heart failure (HF) and aortic stenosis. Although experimental studies suggest that DNMT3A clonal hematopoiesis-driver mutations may enhance inflammation, specific signatures of inflammatory cells in humans are missing. OBJECTIVE: To define subsets of immune cells mediating inflammation in humans using single-cell RNA sequencing. METHODS AND RESULTS: Transcriptomic profiles of peripheral blood mononuclear cells were analyzed in n=6 patients with HF harboring DNMT3A clonal hematopoiesis-driver mutations and n=4 patients with HF and no DNMT3A mutations by single-cell RNA sequencing. Monocytes of patients with HF carrying DNMT3A mutations demonstrated a significantly increased expression of inflammatory genes compared with monocytes derived from patients with HF without DNMT3A mutations. Among the specific upregulated genes were the prototypic inflammatory IL (interleukin) IL1B (interleukin 1B), IL6, IL8, the inflammasome NLRP3, and the macrophage inflammatory proteins CCL3 and CCL4 as well as resistin, which augments monocyte-endothelial adhesion. Silencing of DNMT3A in monocytes induced a paracrine proinflammatory activation and increased adhesion to endothelial cells. Furthermore, the classical monocyte subset of DNMT3A mutation carriers showed increased expression of T-cell stimulating immunoglobulin superfamily members CD300LB, CD83, SIGLEC12, as well as the CD2 ligand and cell adhesion molecule CD58, all of which may be involved in monocyte-T-cell interactions. DNMT3A mutation carriers were further characterized by increased expression of the T-cell alpha receptor constant chain and changes in T helper cell 1, T helper cell 2, T helper cell 17, CD8+ effector, CD4+ memory, and regulatory T-cell-specific signatures. CONCLUSIONS: This study demonstrates that circulating monocytes and T cells of patients with HF harboring clonal hematopoiesis-driver mutations in DNMT3A exhibit a highly inflamed transcriptome, which may contribute to the aggravation of chronic HF.

Inflammatory signatures are associated with increased mortality after transfemoral transcatheter aortic valve implantation.

AIMS: Systemic inflammatory response, identified by increased total leucocyte counts, was shown to be a strong predictor of mortality after transcatheter aortic valve implantation (TAVI). Yet the mechanisms of inflammation-associated poor outcome after TAVI are unclear. Therefore, the present study aimed at investigating individual inflammatory signatures and functional heterogeneity of circulating myeloid and T-lymphocyte subsets and their impact on 1 year survival in a single-centre cohort of patients with severe aortic stenosis undergoing TAVI. METHODS AND RESULTS: One hundred twenty-nine consecutive patients with severe symptomatic aortic stenosis admitted for transfemoral TAVI were included. Blood samples were obtained at baseline, immediately after, and 24 h and 3 days after TAVI, and these were analysed for inflammatory and cardiac biomarkers. Myeloid and T-lymphocyte subsets were measured using flow cytometry. The inflammatory parameters were first analysed as continuous variables; and in case of association with outcome and area under receiver operating characteristic (ROC) curve (AUC) ≥ 0.6, the values were dichotomized using optimal cut-off points. Several baseline inflammatory parameters, including high-sensitivity C-reactive protein (hsCRP; HR = 1.37, 95% CI: 1.15-1.63; P < 0.0001) and IL-6 (HR = 1.02, 95% CI: 1.01-1.03; P = 0.003), lower counts of Th2 (HR = 0.95, 95% CI: 0.91-0.99; P = 0.009), and increased percentages of Th17 cells (HR = 1.19, 95% CI: 1.02-1.38; P = 0.024) were associated with 12 month all-cause mortality. Among postprocedural parameters, only increased post-TAVI counts of non-classical monocytes immediately after TAVI were predictive of outcome (HR = 1.03, 95% CI: 1.01-1.05; P = 0.003). The occurrence of SIRS criteria within 48 h post-TAVI showed no significant association with 12 month mortality (HR = 0.57, 95% CI: 0.13-2.43, P = 0.45). In multivariate analysis of discrete or dichotomized clinical and inflammatory variables, the presence of diabetes mellitus (HR = 3.50; 95% CI: 1.42-8.62; P = 0.006), low left ventricular (LV) ejection fraction (HR = 3.16; 95% CI: 1.35-7.39; P = 0.008), increased baseline hsCRP (HR = 5.22; 95% CI: 2.09-13.01; P < 0.0001), and low baseline Th2 cell counts (HR = 8.83; 95% CI: 3.02-25.80) were significant predictors of death. The prognostic value of the linear prediction score calculated of these parameters was superior to the Society of Thoracic Surgeons score (AUC: 0.88; 95% CI: 0.78-0.99 vs. 0.75; 95% CI: 0.64-0.86, respectively; P = 0.036). Finally, when analysing LV remodelling outcomes, ROC curve analysis revealed that low numbers of Tregs (P = 0.017; AUC: 0.69) and increased Th17/Treg ratio (P = 0.012; AUC: 0.70) were predictive of adverse remodelling after TAVI. CONCLUSIONS: Our findings demonstrate an association of specific pre-existing inflammatory phenotypes with increased mortality and adverse LV remodelling after TAVI. Distinct monocyte and T-cell signatures might provide additive biomarkers to improve pre-procedural risk stratification in patients referred to TAVI for severe aortic stenosis.

Outcomes of Cardiovascular Magnetic Resonance Imaging in Patients Recently Recovered From Coronavirus Disease 2019 (COVID-19).

IMPORTANCE: Coronavirus disease 2019 (COVID-19) continues to cause considerable morbidity and mortality worldwide. Case reports of hospitalized patients suggest that COVID-19 prominently affects the cardiovascular system, but the overall impact remains unknown. OBJECTIVE: To evaluate the presence of myocardial injury in unselected patients recently recovered from COVID-19 illness. DESIGN, SETTING, AND PARTICIPANTS: In this prospective observational cohort study, 100 patients recently recovered from COVID-19 illness were identified from the University Hospital Frankfurt COVID-19 Registry between April and June 2020. EXPOSURE: Recent recovery from severe acute respiratory syndrome coronavirus 2 infection, as determined by reverse transcription-polymerase chain reaction on swab test of the upper respiratory tract. MAIN OUTCOMES AND MEASURES: Demographic characteristics, cardiac blood markers, and cardiovascular magnetic resonance (CMR) imaging were obtained. Comparisons were made with age-matched and sex-matched control groups of healthy volunteers (n = 50) and risk factor-matched patients (n = 57). RESULTS: Of the 100 included patients, 53 (53%) were male, and the mean (SD) age was 49 (14) years. The median (IQR) time interval between COVID-19 diagnosis and CMR was 71 (64-92) days. Of the 100 patients recently recovered from COVID-19, 67 (67%) recovered at home, while 33 (33%) required hospitalization. At the time of CMR, high-sensitivity troponin T (hsTnT) was detectable (greater than 3 pg/mL) in 71 patients recently recovered from COVID-19 (71%) and significantly elevated (greater than 13.9 pg/mL) in 5 patients (5%). Compared with healthy controls and risk factor-matched controls, patients recently recovered from COVID-19 had lower left ventricular ejection fraction, higher left ventricle volumes, and raised native T1 and T2. A total of 78 patients recently recovered from COVID-19 (78%) had abnormal CMR findings, including raised myocardial native T1 (n = 73), raised myocardial native T2 (n = 60), myocardial late gadolinium enhancement (n = 32), or pericardial enhancement (n = 22). There was a small but significant difference between patients who recovered at home vs in the hospital for native T1 mapping (median [IQR], 1119 [1092-1150] ms vs 1141 [1121-1175] ms; P = .008) and hsTnT (4.2 [3.0-5.9] pg/dL vs 6.3 [3.4-7.9] pg/dL; P = .002) but not for native T2 mapping. None of these measures were correlated with time from COVID-19 diagnosis (native T1: r = 0.07; P = .47; native T2: r = 0.14; P = .15; hsTnT: r = -0.07; P = .50). High-sensitivity troponin T was significantly correlated with native T1 mapping (r = 0.33; P < .001) and native T2 mapping (r = 0.18; P = .01). Endomyocardial biopsy in patients with severe findings revealed active lymphocytic inflammation. Native T1 and T2 were the measures with the best discriminatory ability to detect COVID-19-related myocardial pathology. CONCLUSIONS AND RELEVANCE: In this study of a cohort of German patients recently recovered from COVID-19 infection, CMR revealed cardiac involvement in 78 patients (78%) and ongoing myocardial inflammation in 60 patients (60%), independent of preexisting conditions, severity and overall course of the acute illness, and time from the original diagnosis. These findings indicate the need for ongoing investigation of the long-term cardiovascular consequences of COVID-19.

CMV-independent increase in CD27-CD28+ CD8+ EMRA T cells is inversely related to mortality in octogenarians.

Cytomegalovirus (CMV) seropositivity in adults has been linked to increased cardiovascular disease burden. Phenotypically, CMV infection leads to an inflated CD8 T-lymphocyte compartment. We employed a 8-colour flow cytometric protocol to analyse circulating T cells in 597 octogenarians from the same birth cohort together with NT-proBNP measurements and followed all participants over 7 years. We found that, independent of CMV serostatus, a high number of CD27-CD28+ CD8 EMRA T-lymphocytes (TEMRA) protected from all-cause death after adjusting for known risk factors, such as heart failure, frailty or cancer (Hazard ratio 0.66 for highest vs lowest tertile; confidence interval 0.51-0.86). In addition, CD27-CD28+ CD8 EMRA T-lymphocytes protected from both, non-cardiovascular (hazard ratio 0.59) and cardiovascular death (hazard ratio 0.65). In aged mice treated with the senolytic navitoclax, in which we have previously shown a rejuvenated cardiac phenotype, CD8 effector memory cells are decreased, further indicating that alterations in T cell subpopulations are associated with cardiovascular ageing. Future studies are required to show whether targeting immunosenescence will lead to enhanced life- or healthspan.

High-Content Immunophenotyping and Hierarchical Clustering Reveal Sources of Heterogeneity and New Surface Markers of Human Blood Monocyte Subsets.

OBJECTIVE: Blood monocyte subsets are emerging as biomarkers of cardiovascular inflammation. However, our understanding of human monocyte heterogeneity and their immunophenotypic features under healthy and inflammatory conditions is still evolving. RATIONALE: In this study, we sought to investigate the immunophenome of circulating human monocyte subsets. METHODS: Multiplexed, high-throughput flow cytometry screening arrays and computational data analysis were used to analyze the expression and hierarchical relationships of 242 specific surface markers on circulating classical (CD14++CD16-), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++) monocytes in healthy adults. RESULTS: Using generalized linear models and hierarchical cluster analysis, we selected and clustered epitopes that most reliably differentiate between monocyte subsets. We validated existing transcriptional profiling data and revealed potential new surface markers that uniquely define the classical (e.g., BLTR1, CD35, CD38, CD49e, CD89, CD96), intermediate (e.g., CD39, CD275, CD305, CDw328), and nonclassical (e.g., CD29, CD132) subsets. In addition, our analysis revealed phenotypic cell clusters, identified by dendritic markers CMRF-44 and CMRF-56, independent of the traditional monocyte classification. CONCLUSION: These results reveal an advancement of the clinically applicable multiplexed screening arrays that may facilitate monocyte subset characterization and cytometry-based biomarker selection in various inflammatory disorders.

Clonal haematopoiesis in patients with degenerative aortic valve stenosis undergoing transcatheter aortic valve implantation.

AIMS: Clonal haematopoiesis of indeterminate potential (CHIP), defined as the presence of an expanded somatic blood cell clone without other haematological abnormalities, was recently shown to increase with age and is associated with coronary artery disease and calcification. The most commonly mutated CHIP genes, DNMT3A and TET2, were shown to regulate inflammatory potential of circulating leucocytes. The incidence of degenerative calcified aortic valve (AV) stenosis increases with age and correlates with chronic inflammation. We assessed the incidence of CHIP and its association with inflammatory blood cell phenotypes in patients with AV stenosis undergoing transfemoral aortic valve implantation (TAVI). METHODS AND RESULTS: Targeted amplicon sequencing for DNMT3A and TET2 was performed in 279 patients with severe AV stenosis undergoing TAVI. Somatic DNMT3A- or TET2-CHIP-driver mutations with a VAF ≥ 2% were detected in 93 out of 279 patients (33.3%), with an age-dependent increase in the incidence from 25% (55-69 years) to 52.9% (90-100 years). Patients with DNMT3A- or TET2-CHIP-driver mutations did not differ from patients without such mutations in clinical parameters, concomitant atherosclerotic disease, blood cell counts, inflammatory markers, or procedural characteristics. However, patients with DNMT3A- or TET2-CHIP-driver mutations had a profoundly increased medium-term all-cause mortality following successful TAVI. Differential myeloid and T-cell distributions revealed pro-inflammatory T-cell polarization in DNMT3A-mutation carriers and increased pro-inflammatory non-classical monocytes in TET2-mutation carriers. CONCLUSION: This is the first study to show that acquired somatic mutations in the most commonly mutated CHIP-driver genes occur frequently in patients with severe degenerative AV stenosis, are associated with increased pro-inflammatory leucocyte subsets, and confer a profound increase in mortality following successful TAVI.

Association of Mutations Contributing to Clonal Hematopoiesis With Prognosis in Chronic Ischemic Heart Failure.

Importance: Somatic mutations causing clonal expansion of hematopoietic cells (clonal hematopoiesis of indeterminate potential [CHIP]) are increased with age and associated with atherosclerosis and inflammation. Age and inflammation are the major risk factors for heart failure, yet the association of CHIP with heart failure in humans is unknown. Objective: To assess the potential prognostic significance of CHIP in patients with chronic heart failure (CHF) owing to ischemic origin. Design, Setting, and Participants: We analyzed bone marrow-derived mononuclear cells from 200 patients with CHF by deep targeted amplicon sequencing to detect the presence of CHIP and associated such with long-term prognosis in patients with CHF at University Hospital Frankfurt, Frankfurt, Germany. Data were analyzed between October 2017 and April 2018. Results: Median age of the patients was 65 years. Forty-seven mutations with a variant allele fraction (VAF) of at least 0.02 were found in 38 of 200 patients with CHF (18.5%). The somatic mutations most commonly occurred in the genes DNMT3A (14 patients), TET2 (9 patients), KDM6A (4 patients), and BCOR (3 patients). Patients with CHIP were older and more frequently had a history of hypertension. During a median follow-up of 4.4 years, a total of 53 patients died, and 23 patients required hospitalization for heart failure. There was a significantly worse long-term clinical outcome for patients with either DNMT3A or TET2 mutations compared with non-CHIP carriers. By multivariable Cox proportional regression analysis, the presence of somatic mutations within TET2 or DNMT3A (HR, 2.1; 95% CI, 1.1-4.0; P = .02, for death combined with heart failure hospitalization) and age (HR, 1.04; 95% CI, 1.01-1.07 per year; P = .005) but not a history of hypertension remained independently associated with adverse outcome. Importantly, there was a significant dose-response association between VAF and clinical outcome. Conclusions and Relevance: Our data suggest that somatic mutations in hematopoietic cells, specifically in the most commonly mutated CHIP driver genes TET2 and DNMT3A, may be significantly associated with the progression and poor prognosis of CHF. Future studies will have to validate our findings in larger cohorts and address whether targeting specific inflammatory pathways may be valuable for precision medicine in patients with CHF carrying specific mutations encoding for CHIP.