Wnt signaling in cardiac development and heart diseases.

The Wnt signaling pathway is a fundamental cellular communication system with extensive implications in various organs including the heart. In cardiac homeostasis, it governs essential processes like cellular proliferation, differentiation, and apoptosis, ensuring the heart's structural and functional integrity from embryonic stages and throughout life. Both canonical and non-canonical Wnt signaling pathways play a critical role during embryonic heart development in a region- and stage-specific manner. Canonical Wnt signaling also plays a significant role in heart diseases such as myocardial infarction and heart failure. However, the role of non-canonical Wnt signaling in heart diseases has not been fully elucidated. Wnt5a is a major ligand that activates non-canonical Wnt pathway, and recent studies start to clarify the role of the Wnt5a signaling axis in cardiac health and disease. In this review, we will briefly summarize the previous findings on the role of Wnt signaling pathways in heart development and diseases, and then focus on the role of Wnt5a signaling in heart failure progression. The multifaceted roles of the Wnt signaling pathway highlight its therapeutic potential for various types of heart diseases.

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.

Wnt5a-YAP signaling axis mediates mechanotransduction in cardiac myocytes and contributes to contractile dysfunction induced by pressure overload.

Non-canonical Wnt signaling activated by Wnt5a/Wnt11 is required for the second heart field development in mice. However, the pathophysiological role of non-canonical Wnt signaling in the adult heart has not been fully elucidated. Here we show that cardiomyocyte-specific Wnt5a knockout mice exhibit improved systolic function and reduced expression of mechanosensitive genes including Nppb when subjected to pressure overload. In cultured cardiomyocytes, Wnt5a knockdown reduced Nppb upregulation induced by cyclic cell stretch. Upstream analysis revealed that TEAD1, a transcription factor that acts with Hippo pathway co-activator YAP, was downregulated both in vitro and in vivo by Wnt5a knockdown/knockout. YAP nuclear translocation was induced by cell stretch and attenuated by Wnt5a knockdown. Wnt5a knockdown-induced Nppb downregulation during cell stretch was rescued by Hippo inhibition, and the rescue effect was canceled by knockdown of YAP. These results collectively suggest that Wnt5a-YAP signaling axis mediates mechanotransduction in cardiomyocytes and contributes to heart failure progression.

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.

Murine models of clonal haematopoiesis to assess mechanisms of cardiovascular disease.

Clonal haematopoiesis (CH) is a phenomenon whereby somatic mutations confer a fitness advantage to haematopoietic stem and progenitor cells (HSPCs) and thus facilitate their aberrant clonal expansion. These mutations are carried into progeny leucocytes leading to a situation whereby a substantial fraction of an individual's blood cells originate from the HSPC mutant clone. Although this condition rarely progresses to a haematological malignancy, circulating blood cells bearing the mutation have the potential to affect other organ systems as they infiltrate into tissues under both homeostatic and disease conditions. Epidemiological and clinical studies have revealed that CH is highly prevalent in the elderly and is associated with an increased risk of cardiovascular disease and mortality. Recent experimental studies in murine models have assessed the most commonly mutated 'driver' genes associated with CH, and have provided evidence for mechanistic connections between CH and cardiovascular disease. A deeper understanding of the mechanisms by which specific CH mutations promote disease pathogenesis is of importance, as it could pave the way for individualized therapeutic strategies targeting the pathogenic CH gene mutations in the future. Here, we review the epidemiology of CH and the mechanistic work from studies using murine disease models, with a particular focus on the strengths and limitations of these experimental systems. We intend for this review to help investigators select the most appropriate models to study CH in the setting of cardiovascular disease.

Somatic Mosaicism in Biology and Disease.

Contrary to earlier beliefs, every cell in the individual is genetically different due to somatic mutations. Consequently, tissues become a mixture of cells with distinct genomes, a phenomenon termed somatic mosaicism. Recent advances in genome sequencing technology have unveiled possible causes of mutations and how they shape the unique mutational landscape of the tissues. Moreover, the analysis of sequencing data in combination with clinical information has revealed the impacts of somatic mosaicism on disease processes. In this review, we discuss somatic mosaicism in various tissues and its clinical implications for human disease.

Repetitive spikes of glucose and lipid induce senescence-like phenotypes of bone marrow stem cells through H3K27me3 demethylase-mediated epigenetic regulation.

Bone marrow-derived endothelial progenitor cells (EPCs) contribute to endothelial repair and angiogenesis. Reduced number of circulating EPCs is associated with future cardiovascular events. We tested whether dysregulated glucose and/or triglyceride (TG) metabolism has an impact on EPC homeostasis. The analysis of metabolic factors associated with circulating EPC number in humans revealed that postprandial hyperglycemia is negatively correlated with circulating EPC number, and this correlation appears to be further enhanced in the presence of postprandial hypertriglyceridemia (hTG). We therefore examined the effect of glucose/TG spikes on bone marrow lineage-sca-1+ c-kit+ (LSK) cells in mice, because primitive EPCs reside in bone marrow LSK fraction. Repetitive glucose + lipid (GL) spikes, but not glucose (G) or lipid (L) spikes alone, induced senescence-like phenotypes of LSK cells, and this phenomenon was reversible after cessation of GL spikes. G spikes and GL spikes differentially affected transcriptional program of LSK cell metabolism and differentiation. GL spikes upregulated a histone H3K27 demethylase JMJD3, and inhibition of JMJD3 eliminated GL spikes-induced LSK cell senescence-like phenotypes. These observations suggest that postprandial glucose/TG dysmetabolism modulate transcriptional regulation in LSK cells through H3K27 demethylase-mediated epigenetic regulation, leading to senescence-like phenotypes of LSK cells, reduced number of circulating EPCs, and development of atherosclerotic cardiovascular disease.NEW & NOTEWORTHY Combination of hyperglycemia and hypertriglyceridemia is associated with increased risk of atherosclerotic cardiovascular disease. We found that 1) hypertriglyceridemia may enhance the negative impact of hyperglycemia on circulating EPC number in humans and 2) metabolic stress induced by glucose + triglyceride spikes in mice results in senescence-like phenotypes of bone marrow stem/progenitor cells via H3K27me3 demethylase-mediated epigenetic regulation. These findings have important implications for understanding the pathogenesis of atherosclerotic cardiovascular disease in patients with T2DM.

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.

The Cancer Therapy-Related Clonal Hematopoiesis Driver Gene Ppm1d Promotes Inflammation and Non-Ischemic Heart Failure in Mice.

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Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis.

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

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.

Influence of different physiological hemodynamics on fractional flow reserve values in the left coronary artery and right coronary artery.

BACKGROUND: Although the left coronary artery (LCA) has a flow profile in that most blood flow occurs during diastole rather than systole, the right coronary artery (RCA) has a flow pattern that is less diastolic dominant. This study assessed whether coronary pressure waveforms distal to stenoses with the same fractional flow reserve (FFR) was the same between the LCA and RCA. METHODS: A total of 347 vessels from 318 patients who underwent FFR measurements were included. Conventional FFR was calculated as the ratio of the mean coronary distal pressure (Pd) to the mean aortic pressure (Pa) at maximal hyperemia. The pressure drop ratios in systole (PDRsystole) and diastole (PDRdiastole) were calculated as the sum of (Pa minus Pd) divided by the sum of Pa at the intracoronary diastolic and systolic pressure phases, respectively. RESULTS: Analysis of covariance of the regression line of correlation between conventional FFR and PDRsystole revealed that the slope was significantly greater in the RCA than in the left anterior descending artery (LAD) and left circumflex artery (LCX) (-0.765, -0.578, and -0.589, p < 0.001). On the other hand, the regression line of correlation between conventional FFR and PDRdiastole found that the slope was significantly greater in the LAD and LCX than in the RCA (-1.349, -1.318, and -1.223, p < 0.001). CONCLUSIONS: The pressure waveform distal to the stenosis differs between the LCA and RCA. In the LCA, the decrease in diastolic pressure mainly contributed to the drop in FFR, whereas in the RCA, it was the decrease in systolic pressure.

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.

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models.

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Heparin Induces the Mobilization of Heart-Derived Multipotent Mesoangioblasts During Cardiac Surgery With Cardiopulmonary Bypass or Cardiac Catheterization.

BACKGROUND: We previously identified circulating mesoangioblasts (cMABs), a subset of mesenchymal stem cells that express cardiac mesodermal markers, in patients undergoing cardiac surgery with cardiopulmonary bypass (CPB). We also found that hepatocyte growth factor (HGF) is upregulated during cardiac surgery with CPB in humans, and induces MAB-like cell mobilization in rodents. These results strongly suggest that heparin induced MAB mobilization via HGF upregulation. Here, we tested this hypothesis in patients undergoing cardiac surgery or cardiac catheterization. We also examined whether human cMABs are derived from the heart.Methods and Results:Plasma HGF levels were determined by ELISA. Mononuclear cells isolated from blood samples were cultured on fibronectin-coated dishes, and outgrowing cMAB colonies were counted. We first confirmed that HGF upregulation and cMAB mobilization were observed before the start of CPB, excluding the possibility that CPB is the primary inducer of cMAB mobilization. We then examined patients undergoing cardiac catheterization and found that heparin significantly increased plasma HGF levels and the number of cMAB colonies in a dose-dependent manner. The results of simultaneous blood sampling from the aortic sinus, coronary sinus, and right atrium were consistent with the notion that human cMABs are derived from the heart. CONCLUSIONS: Human cMABs are mobilized by heparin injection during cardiac surgery or cardiac catheterization, presumably via HGF upregulation.

Percutaneous coronary intervention for left main trunk ostial stenosis in a patient with Takayasu's arteritis.

Takayasu's arteritis with coronary artery involvement is a rare event especially in men. We will report on a male case of Takayasu's arteritis undergoing stent implantation for left main trunk (LMT) ostial stenosis. The case was that of a 25-year-old man who had been diagnosed with Takayasu's arteritis but there was no significant large vessel involvement. He presented with effort angina and a multidetector computed tomography (MDCT) revealed severe ostial stenosis in the LMT. A coronary angiography confirmed this finding and a virtual histology intravascular ultrasound (VH-IVUS) showed fibrous thickening of the intima and media with little necrotic lipid core and calcification. We performed a bare metal stent implantation for this lesion. No restenosis was found in the MDCT at the 6 month follow-up. Our experience suggests that the VH-IVUS is useful for examining the gross structure and component of the coronary vascular wall and for determining the choice of treatment in patients with Takayasu's arteritis.