This study evaluates rostafuroxin, a selective ouabain inhibitor, for its potential to antagonize electrophysiological derangements in ouabain overdosing. Methodologically, the study uses the iCE model, previously introduced as a suitable 3 R-compliant cardiovascular research platform. We developed and validated a comprehensive electrophysiological workflow in iCEs to perform our investigations. Ouabain increased heart rate variability, induced arrhythmia and electrolyte imbalances in iCEs, whereas rostafuroxin largely protected them from these effects.

Background/Objectives: Neuroblastoma cells are phenotypically plastic, transitioning between mesenchymal and adrenergic states. Core functional genes (e.g., YAP1) mark the mesenchymal state, which is linked to unfavorable prognosis. We and others previously demonstrated relapse-specific Hippo-YAP pathway activation in matched primary/relapsed neuroblastomas. Here we explored the role of YAP1 in neuroblastoma aggressiveness and response to therapy. Methods: RT-qPCR and immunoblotting assessed YAP1 expression in neuroblastoma cell lines. RNA-sequencing detected YAP1-dependent gene signatures in Tet-ON SK-N-AS and SH-EP neuroblastoma cell models expressing wildtype YAP1 or constitutively activated YAP1^S127A. Data from cell models were compared with our published YAP1 expression data from neuroblastomas. Efficacy of commonly used chemotherapeutics was comparatively analyzed in the cell models. Results: YAP1 expression showed marked variability across a panel of neuroblastoma cell lines, assessed by mRNA analysis in 10 cell lines and protein analysis in a subset of 9 cell lines. RNA sequencing in constitutively activated YAP1^S127A mutant and wildtype YAP1 models detected 2162 and 1837 significantly differentially expressed genes in the SK-N-AS and SH-EP backgrounds, respectively. Continuously activating YAP1 in SK-N-AS cells upregulated mesenchymal signature genes and mesenchymal-associated transcription factors. Gene expression influenced by YAP1 activity in the cell models significantly overlapped with YAP1-associated genes (e.g., CYR61 and SPRY4) in published tumor data. Functionally, YAP1^S127A expression rendered neuroblastoma cells resistant to chemotherapy. Conclusions: Findings corroborate the idea of a mechanistic role for YAP1 in neuroblastoma adrenergic to mesenchymal reprogramming and therapy resistance. The YAP1-mediated plastic switch towards a mesenchymal expression state in neuroblastoma cells may provide the molecular basis for novel therapeutic avenues.

BACKGROUND: Pulmonary hypertension due to left heart disease (PH-LHD) is a common complication of myocardial infarction (MI) and heart failure (HF). Myeloid inflammation is a major hallmark of both the local cardiac and systemic post-MI response, with resident and recruited monocyte-derived macrophages orchestrating each step of the ensuing inflammatory cascade, from clearance of dead cardiomyocytes to scar tissue formation. Similarly, myeloid inflammation is a key feature of the PH lung, driving remodeling of small pulmonary vessels. We thus hypothesized that MI-induced systemic myeloid inflammation contributes directly to the development of PH-LHD. METHODS: We assessed expression of inflammatory adhesion molecules, as markers of vascular inflammatory activation and myeloid cell recruitment pathways, in patients with pulmonary arterial hypertension, PH-LHD, as well as in a PH-LHD rat model. We then induced MI and subsequent HF in 8- to 10-week-old female C57BL/6J mice via permanent ligation of the left anterior descending coronary artery. HF mice were then treated with a CCR2 (C-C chemokine receptor 2) antagonist to block monocyte recruitment and the development of PH-LHD was assessed at 6 weeks post-MI by a combination of invasive hemodynamics, flow cytometry, echocardiography, and lung histology. RESULTS: Expression of key inflammatory adhesion molecules, including VCAM1 and SELE , was increased in PH-LHD lungs from humans and rats. Following early post-MI development of HF with reduced ejection fraction, mice exhibited increased numbers of circulating monocytes, lung monocytes, and lung interstitial macrophages, as well as PH-LHD by 6 weeks, characterized by right ventricular hypertrophy, elevated right ventricular pressures, and lung vascular remodeling of small-to-medium-sized pulmonary vessels. Administration of the selective CCR2 antagonist RS102895 from day 10 post-MI to reduce monocyte tissue recruitment attenuated lung vascular remodeling, right ventricular systolic and diastolic pressures, right ventricular hypertrophy, and lung adhesion molecule expression at 6 weeks, compared with vehicle-treated controls, without improving the underlying left-sided HF. CONCLUSIONS: This proof-of-concept study demonstrates a causal role for CCR2-dependent myeloid cell recruitment in the progression from MI-induced HF to secondary pulmonary vascular disease.

Sensitive detection of minimal residual disease (MRD) remains a major unmet need in high-risk neuroblastoma. MYCN amplification, a hallmark of high-risk disease, typically occurs on extrachromosomal DNA (ecDNA), but the potential of ecDNA-associated genomic rearrangements for individualized MRD monitoring has not been fully exploited. Here, we applied neuroblastoma-specific hybrid capture-based panel sequencing to identify patient-unique breakpoints within MYCN amplicons, and used Circle-seq and Nanopore sequencing to resolve the extrachromosomal amplicon structure in representative samples. Analysis of 8 neuroblastoma cell lines and 22 primary tumors identified 69 tumor-specific breakpoints. Those selected for assay development were validated by breakpoint-specific PCR and Sanger sequencing. Breakpoints detected in primary tumors remained detectable at relapse, supporting their stability as MRD markers. Breakpoint-specific real-time quantitative PCR and droplet digital PCR detected these junctions in bone marrow aspirates with high specificity and reached sensitivities down to a tumor DNA fraction of 10^-6. We applied this approach to 53 serial bone marrow aspirates from 14 patients with high-risk neuroblastoma to monitor MRD dynamics, resolving treatment response and molecular persistence. In six samples, breakpoint-positive DNA was detected in bone marrow that was negative by conventional cytology and immunocytology, highlighting the added value of molecular monitoring. Together, these findings establish ecDNA breakpoint-based detection as a strategy for MRD assessment in neuroblastoma, that is, in principle, applicable to any ecDNA-amplified oncogene.

BACKGROUND: The role of leukocytes in the pathogenesis of right ventricular (RV) dysfunction (RVD) associated with heart failure with preserved ejection fraction (HFpEF) remains poorly defined, partially due to the lack of suitable small animal models. Here, we followed a translational research approach by establishing a murine HFpEF model developing manifest RVD and analyzed human HFpEF cohorts to study the mechanistic link between leukocytes and RVD in HFpEF. METHODS: Young (80-week-old) male and female C57BL/6J mice were divided into 4 experimental groups: chow, HFpEF (L-NAME [Nω-nitro-L-arginine methyl ester] plus a high-fat diet), chronic hypoxia (10% O ₂ ), and HFpEF plus hypoxia (RV-HFpEF). Biventricular function and myeloid cell dynamics were assessed across groups. To test whether myeloid cells are causally involved in the development of RV remodeling in HFpEF, we treated RV-HFpEF mice with a colony-stimulating factor 1 receptor inhibitor to deplete myeloid cells. RESULTS: RV-HFpEF resulted in left ventricular diastolic dysfunction, indicated by increased E/E′ ratio, reduced global longitudinal peak strain, smaller end-diastolic diameters, and increased isovolumetric relaxation time compared with chow. RV-HFpEF animals developed RV hypertrophy and RVD, evident as increased Fulton index as well as elevated RV systolic pressure and reduced tricuspid annular plane systolic excursion, respectively. Total leukocyte, monocyte, and macrophage counts were elevated in RV tissue from RV-HFpEF mice compared with RV tissue from chow mice and left ventricular tissue from young and aged RV-HFpEF animals. These findings were confirmed by unbiased proteomic analyses of RV tissue from RV-HFpEF mice, demonstrating an increased abundance of proteins involved in activation of the innate immune system, macrophage chemotaxis, and leukocyte migration when compared with left ventricular tissue and the other experimental groups. Fate-mapping experiments revealed that recruited monocyte-derived macrophages became the main source of total cardiac macrophages in RV tissue from RV-HFpEF mice. Depletion of myeloid cells was associated with lower RVSP profiles in RV-HFpEF mice compared with controls. In RV biopsies from patients with HFpEF, we found increased expression of adhesion molecules, fibrotic markers, and inflammatory transcripts, as well as an association between RVD and CD68 ⁺ cells. CONCLUSIONS: We demonstrate that dysregulated myeloid cell dynamics are associated with, and directly contribute to, the pathogenesis of RVD associated with HFpEF in humans and mice.