Wnt5a-Mediated Neutrophil Recruitment Has an Obligatory Role in Pressure Overload-Induced Cardiac Dysfunction.

BACKGROUND: Although the complex roles of macrophages in myocardial injury are widely appreciated, the function of neutrophils in nonischemic cardiac pathology has received relatively little attention. METHODS: To examine the regulation and function of neutrophils in pressure overload-induced cardiac hypertrophy, mice underwent treatment with Ly6G antibody to deplete neutrophils and then were subjected to transverse aortic constriction. RESULTS: Neutrophil depletion diminished transverse aortic constriction-induced hypertrophy and inflammation and preserved cardiac function. Myeloid deficiency of Wnt5a, a noncanonical Wnt, suppressed neutrophil infiltration to the hearts of transverse aortic constriction-treated mice and produced a phenotype that was similar to the neutropenic conditions. Conversely, mice overexpressing Wnt5a in myeloid cells displayed greater hypertrophic growth, inflammation, and cardiac dysfunction. Neutrophil depletion reversed the Wnt5a overexpression-induced cardiac pathology and eliminated differences in cardiac parameters between wild-type and myeloid-specific Wnt5a transgenic mice. CONCLUSIONS: These findings reveal that Wnt5a-regulated neutrophil infiltration has a critical role in pressure overload-induced heart failure.

CRISPR-Mediated Gene Editing to Assess the Roles of Tet2 and Dnmt3a in Clonal Hematopoiesis and Cardiovascular Disease.

RATIONALE: Clonal hematopoiesis has been associated with increased mortality and cardiovascular disease. This condition can arise from somatic mutations in preleukemic driver genes within hematopoietic stem/progenitor cells. Approximately 40 candidate driver genes have been identified, but mutations in only 1 of these genes, TET2 (ten-eleven translocation-2), has been shown to casually contribute to cardiovascular disease in murine models. OBJECTIVE: To develop a facile system to evaluate the disease characteristics of different clonal hematopoiesis driver genes using lentivirus vector and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) methodology. Using this methodology, evaluate whether Dnmt3a (DNA [cytosine-5]-methyltransferase 3a)-a commonly occurring clonal hematopoiesis driver gene-causally contributes to cardiovascular disease. METHODS AND RESULTS: Lentivirus vectors were used to deliver Cas9 and guide RNA to introduce inactivating mutations in Tet2 and Dnmt3a in lineage-negative bone marrow cells. After implantation into lethally irradiated mice, these cells were engrafted and gave rise to labeled blood cell progeny. When challenged with an infusion of Ang II (angiotensin II), mice with inactivating mutations in Tet2 or Dnmt3a displayed greater cardiac hypertrophy, diminished cardiac function, and greater cardiac and renal fibrosis. In comparison with Tet2, inactivation of Dnmt3a did not lead to detectable expansion of the mutant hematopoietic cells during the time course of these experiments. Tet2 inactivation promoted the expression of IL (interleukin) 1ß, IL-6, and Ccl5, whereas Dnmt3a inactivation promoted the expression of Cxcl1 (CXC chemokine ligand), Cxcl2, IL-6, and Ccl5 in a lipopolysaccharide-stimulated macrophage cell line. CONCLUSIONS: Experiments using lentivirus vector/CRISPR methodology provided evidence suggesting that inactivating DNMT3A mutations in hematopoietic cells contributes to cardiovascular disease. Comparative analyses showed that inactivation of Tet2 and Dnmt3 was similar in their ability to promote Ang II-induced cardiac dysfunction and renal fibrosis in mice. However, gene-specific actions were indicated by differences in kinetics of hematopoietic stem/progenitor cell expansion and different patterns of inflammatory gene expression.

The Cell Surface Receptors Ror1/2 Control Cardiac Myofibroblast Differentiation.

Background A hallmark of heart failure is cardiac fibrosis, which results from the injury-induced differentiation response of resident fibroblasts to myofibroblasts that deposit extracellular matrix. During myofibroblast differentiation, fibroblasts progress through polarization stages of early proinflammation, intermediate proliferation, and late maturation, but the regulators of this progression are poorly understood. Planar cell polarity receptors, receptor tyrosine kinase-like orphan receptor 1 and 2 (Ror1/2), can function to promote cell differentiation and transformation. In this study, we investigated the role of the Ror1/2 in a model of heart failure with emphasis on myofibroblast differentiation. Methods and Results The role of Ror1/2 during cardiac myofibroblast differentiation was studied in cell culture models of primary murine cardiac fibroblast activation and in knockout mouse models that underwent transverse aortic constriction surgery to induce cardiac injury by pressure overload. Expression of Ror1 and Ror2 were robustly and exclusively induced in fibroblasts in hearts after transverse aortic constriction surgery, and both were rapidly upregulated after early activation of primary murine cardiac fibroblasts in culture. Cultured fibroblasts isolated from Ror1/2 knockout mice displayed a proinflammatory phenotype indicative of impaired myofibroblast differentiation. Although the combined ablation of Ror1/2 in mice did not result in a detectable baseline phenotype, transverse aortic constriction surgery led to the death of all mice by day 6 that was associated with myocardial hyperinflammation and vascular leakage. Conclusions Together, these results show that Ror1/2 are essential for the progression of myofibroblast differentiation and for the adaptive remodeling of the heart in response to pressure overload.

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.

JAK2 V617F -Mediated Clonal Hematopoiesis Accelerates Pathological Remodeling in Murine Heart Failure.

Janus kinase 2 (valine to phenylalanine at residue 617) (JAK2 V617F ) mutations lead to myeloproliferative neoplasms associated with elevated myeloid, erythroid, and megakaryocytic cells. Alternatively these same mutations can lead to the condition of clonal hematopoiesis with no impact on blood cell counts. Here, a model of myeloid-restricted JAK2 V617F expression from lineage-negative bone marrow cells was developed and evaluated. This model displayed greater cardiac inflammation and dysfunction following permanent left anterior descending artery ligation and transverse aortic constriction. These data suggest that JAK2 V617F mutations arising in myeloid progenitor cells may contribute to cardiovascular disease by promoting the proinflammatory properties of circulating myeloid cells.

Tet2-Mediated Clonal Hematopoiesis Accelerates Heart Failure Through a Mechanism Involving the IL-1ß/NLRP3 Inflammasome.

BACKGROUND: Recent studies have shown that hematopoietic stem cells can undergo clonal expansion secondary to somatic mutations in leukemia-related genes, thus leading to an age-dependent accumulation of mutant leukocytes in the blood. This somatic mutation-related clonal hematopoiesis is common in healthy older individuals, but it has been associated with an increased incidence of future cardiovascular disease. The epigenetic regulator TET2 is frequently mutated in blood cells of individuals exhibiting clonal hematopoiesis. OBJECTIVES: This study investigated whether Tet2 mutations within hematopoietic cells can contribute to heart failure in 2 models of cardiac injury. METHODS: Heart failure was induced in mice by pressure overload, achieved by transverse aortic constriction or chronic ischemia induced by the permanent ligation of the left anterior descending artery. Competitive bone marrow transplantation strategies with Tet2-deficient cells were used to mimic TET2 mutation-driven clonal hematopoiesis. Alternatively, Tet2 was specifically ablated in myeloid cells using Cre recombinase expressed from the LysM promoter. RESULTS: In both experimental heart failure models, hematopoietic or myeloid Tet2 deficiency worsened cardiac remodeling and function, in parallel with increased interleukin-1beta (IL-1ß) expression. Treatment with a selective NLRP3 inflammasome inhibitor protected against the development of heart failure and eliminated the differences in cardiac parameters between Tet2-deficient and wild-type mice. CONCLUSIONS: Tet2 deficiency in hematopoietic cells is associated with greater cardiac dysfunction in murine models of heart failure as a result of elevated IL-1ß signaling. These data suggest that individuals with TET2-mediated clonal hematopoiesis may be at greater risk of developing heart failure and respond better to IL-1ß-NLRP3 inflammasome inhibition.