Clonal Hematopoiesis in Clinical and Experimental Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Clonal hematopoiesis (CH), which results from an array of nonmalignant driver gene mutations, can lead to altered immune cell function and chronic disease, and has been associated with worse outcomes in patients with heart failure (HF) with reduced ejection fraction. However, the role of CH in the prognosis of HF with preserved ejection fraction (HFpEF) has been understudied. This study aimed to characterize CH in patients with HFpEF and elucidate its causal role in a murine model. METHODS: Using a panel of 20 candidate CH driver genes and a variant allele fraction cutoff of 0.5%, ultradeep error-corrected sequencing identified CH in a cohort of 81 patients with HFpEF (mean age, 71±6 years; ejection fraction, 63±5%) and 36 controls without a diagnosis of HFpEF (mean age, 74±7 years; ejection fraction, 61.5±8%). CH was also evaluated in a replication cohort of 59 individuals with HFpEF. RESULTS: Compared with controls, there was an enrichment of TET2-mediated CH in the HFpEF patient cohort (12% versus 0%, respectively; P=0.02). In the HFpEF cohort, patients with CH exhibited exacerbated diastolic dysfunction in terms of E/e' (14.9 versus 11.7, respectively; P=0.0096) and E/A (1.69 versus 0.89, respectively; P=0.0206) compared with those without CH. The association of CH with exacerbated diastolic dysfunction was corroborated in a validation cohort of individuals with HFpEF. In accordance, patients with HFpEF, an age ≥70 years, and CH exhibited worse prognosis in terms of 5-year cardiovascular-related hospitalization rate (hazard ratio, 5.06; P=0.042) compared with patients with HFpEF and an age ≥70 years without CH. To investigate the causal role of CH in HFpEF, nonconditioned mice underwent adoptive transfer with Tet2-wild-type or Tet2-deficient bone marrow and were subsequently subjected to a high-fat diet/L-NAME (Nω-nitro-l-arginine methyl ester) combination treatment to induce features of HFpEF. This model of Tet2-CH exacerbated cardiac hypertrophy by heart weight/tibia length and cardiomyocyte size, diastolic dysfunction by E/e' and left ventricular end-diastolic pressure, and cardiac fibrosis compared with the Tet2-wild-type condition. CONCLUSIONS: CH is associated with worse heart function and prognosis in patients with HFpEF, and a murine experimental model of Tet2-mediated CH displays greater features of HFpEF.

Endothelial cell cycle state determines propensity for arterial-venous fate.

During blood vessel development, endothelial cells become specified toward arterial or venous fates to generate a circulatory network that provides nutrients and oxygen to, and removes metabolic waste from, all tissues. Arterial-venous specification occurs in conjunction with suppression of endothelial cell cycle progression; however, the mechanistic role of cell cycle state is unknown. Herein, using Cdh5-CreERT2;R26FUCCI2aR reporter mice, we find that venous endothelial cells are enriched for the FUCCI-Negative state (early G1) and BMP signaling, while arterial endothelial cells are enriched for the FUCCI-Red state (late G1) and TGF-ß signaling. Furthermore, early G1 state is essential for BMP4-induced venous gene expression, whereas late G1 state is essential for TGF-ß1-induced arterial gene expression. Pharmacologically induced cell cycle arrest prevents arterial-venous specification defects in mice with endothelial hyperproliferation. Collectively, our results show that distinct endothelial cell cycle states provide distinct windows of opportunity for the molecular induction of arterial vs. venous fate.

Retrospective analysis of somatic mutations and clonal hematopoiesis in astronauts.

With planned deep space and commercial spaceflights, gaps remain to address health risks in astronauts. Multiple studies have shown associations between clonal expansion of hematopoietic cells with hematopoietic malignancies and cardiometabolic disease. This expansion of clones in the absence of overt hematopoietic disorders is termed clonal hematopoiesis (CH) of indeterminate potential (CHIP). Using deep, error-corrected, targeted DNA sequencing we assayed for somatic mutations in CH-driver genes in peripheral blood mononuclear cells isolated from de-identified blood samples collected from 14 astronauts who flew Shuttle missions between 1998-2001. We identified 34 nonsynonymous mutations of relatively low variant allele fraction in 17 CH-driver genes, with the most prevalent mutations in TP53 and DNMT3A. The presence of these small clones in the blood of relatively young astronaut cohort warrants further retrospective and prospective investigation of their clinical relevance and potential application in monitoring astronaut's health.

Hematopoietic loss of Y chromosome leads to cardiac fibrosis and heart failure mortality.

Hematopoietic mosaic loss of Y chromosome (mLOY) is associated with increased risk of mortality and age-related diseases in men, but the causal and mechanistic relationships have yet to be established. Here, we show that male mice reconstituted with bone marrow cells lacking the Y chromosome display increased mortality and age-related profibrotic pathologies including reduced cardiac function. Cardiac macrophages lacking the Y chromosome exhibited polarization toward a more fibrotic phenotype, and treatment with a transforming growth factor ß1-neutralizing antibody ameliorated cardiac dysfunction in mLOY mice. A prospective study revealed that mLOY in blood is associated with an increased risk for cardiovascular disease and heart failure-associated mortality. Together, these results indicate that hematopoietic mLOY causally contributes to fibrosis, cardiac dysfunction, and mortality in men.

TP53-mediated therapy-related clonal hematopoiesis contributes to doxorubicin-induced cardiomyopathy by augmenting a neutrophil-mediated cytotoxic response.

Therapy-related clonal hematopoiesis (t-CH) is often observed in cancer survivors. This form of clonal hematopoiesis typically involves somatic mutations in driver genes that encode components of the DNA damage response and confer hematopoietic stem and progenitor cells (HSPCs) with resistance to the genotoxic stress of the cancer therapy. Here, we established a model of TP53-mediated t-CH through the transfer of Trp53 mutant HSPCs to mice, followed by treatment with a course of the chemotherapeutic agent doxorubicin. These studies revealed that neutrophil infiltration in the heart significantly contributes to doxorubicin-induced cardiac toxicity and that this condition is amplified in the model of Trp53-mediated t-CH. These data suggest that t-CH could contribute to the elevated heart failure risk that occurs in cancer survivors who have been treated with genotoxic agents.

Overexpression of an ethylene-forming ACC oxidase (ACO) gene precedes the Minute Hilum seed coat phenotype in Glycine max.

BACKGROUND: To elucidate features of seed development, we investigated the transcriptome of a soybean isoline from the germplasm collection that contained an introgressed allele known as minute hilum (mi) which confers a smaller hilum region where the seed attaches to the pod and also results in seed coat cracking surrounding the hilum region. RESULTS: RNAs were extracted from immature seed from an extended hilum region (i.e., the hilum and a small ring of tissue surrounding the hilum in which the cracks form) at three different developmental stages:10-25, 25-50 and 50-100 mg seed fresh weight in two independent replicates for each stage. The transcriptomes of these samples from both the Clark isoline containing the mi allele (PI 547628, UC413, ii R t mi G), and its recurrent Clark 63 parent isoline (PI 548532, UC7, ii R T Mi g), which was used for six generations of backcrossing, were compared for differential expression of 88,648 Glyma models of the soybean genome Wm82.a2. The RNA sequence data obtained from the 12 cDNA libraries were subjected to padj value < 0.05 and at least two-fold expression differences to select with confidence genes differentially expressed in the hilum-containing tissue of the seed coat between the two lines. Glyma.09G008400 annotated as encoding an ethylene forming enzyme, ACC oxidase (ACO), was found to be highly overexpressed in the mi hilum region at 165 RPKMs (reads per kilobase per million mapped reads) compared to the standard line at just 0.03 RPKMs. Evidence of changes in expression of genes downstream of the ethylene pathway included those involved in auxin and gibberellin hormone action and extensive differences in expression of cell wall protein genes. These changes are postulated to determine the restricted hilum size and cracking phenotypes. CONCLUSIONS: We present transcriptome and phenotypic evidence that substantially higher expression of an ethylene-forming ACO gene likely shifts hormone balance and sets in motion downstream changes resulting in a smaller hilum phenotype and the cracks observed in the minute hilum (mi) isoline as compared to its recurrent parent.

Tet2-mediated clonal hematopoiesis in nonconditioned mice accelerates age-associated cardiac dysfunction.

Clonal hematopoiesis of indeterminate potential is prevalent in elderly individuals and associated with increased risks of all-cause mortality and cardiovascular disease. However, mouse models to study the dynamics of clonal hematopoiesis and its consequences on the cardiovascular system under homeostatic conditions are lacking. We developed a model of clonal hematopoiesis using adoptive transfer of unfractionated ten-eleven translocation 2-mutant (Tet2-mutant) bone marrow cells into nonirradiated mice. Consistent with age-related clonal hematopoiesis observed in humans, these mice displayed a progressive expansion of Tet2-deficient cells in multiple hematopoietic stem and progenitor cell fractions and blood cell lineages. The expansion of the Tet2-mutant fraction was also observed in bone marrow-derived CCR2+ myeloid cell populations within the heart, but there was a negligible impact on the yolk sac-derived CCR2- cardiac-resident macrophage population. Transcriptome profiling revealed an enhanced inflammatory signature in the donor-derived macrophages isolated from the heart. Mice receiving Tet2-deficient bone marrow cells spontaneously developed age-related cardiac dysfunction characterized by greater hypertrophy and fibrosis. Altogether, we show that Tet2-mediated hematopoiesis contributes to cardiac dysfunction in a nonconditioned setting that faithfully models human clonal hematopoiesis in unperturbed bone marrow. Our data support clinical findings that clonal hematopoiesis per se may contribute to diminished health span.

RNA-Seq profiling of a defective seed coat mutation in Glycine max reveals differential expression of proline-rich and other cell wall protein transcripts.

The plant cell wall performs a number of essential functions including providing shape to many different cell types and serving as a defense against potential pathogens. The net pattern mutation creates breaks in the seed coat of soybean (Glycine max) because of ruptured cell walls. Using RNA-Seq, we examined the seed coat transcriptome from three stages of immature seed development in two pairs of isolines with normal or defective seed coat phenotypes due to the net pattern. The genome-wide comparative study of the transcript profiles of these isolines revealed 364 differentially expressed genes in common between the two varieties that were further divided into different broad functional categories. Genes related to cell wall processes accounted for 19% of the differentially expressed genes in the middle developmental stage of 100-200 mg seed weight. Within this class, the cell wall proline-rich and glycine-rich protein genes were highly differentially expressed in both genetic backgrounds. Other genes that showed significant expression changes in each of the isoline pairs at the 100-200 mg seed weight stage were xylem serine proteinase, fasciclin-related genes, auxin and stress response related genes, TRANSPARENT TESTA 1 (TT1) and other transcription factors. The mutant appears to shift the timing of either the increase or decrease in the levels of some of the transcripts. The analysis of these data sets reveals the physiological changes that the seed coat undergoes during the formation of the breaks in the cell wall.

MGOS: development of a community annotation database for Magnaporthe oryzae.

Magnaporthe oryzae causes rice blast disease, which is the most serious disease of cultivated rice worldwide. We previously developed the Magnaporthe grisea-Orzya sativa (MGOS) database as a repository for the M. oryzae and rice genome sequences together with a comprehensive set of functional interaction data generated by a major consortium of U.S. researchers. The MGOS database has now undergone a major redesign to include data from the international blast research community, accessible with a new intuitive, easy-to-use interface. Registered database users can manually annotate gene sequences and features as well as add mutant data and literature on individual gene pages. Over 900 genes have been manually curated based on various biological databases and the scientific literature. Gene names and descriptions, gene ontology annotations, published and unpublished information on mutants and their phenotypes, responses in diverse microarray analyses, and related literature have been incorporated. Thus far, 362 M. oryzae genes have associated information on mutants. MGOS is now poised to become a one-stop repository for all structural and functional data available on all genes of this critically important rice pathogen.