Wang, Min

This study uncovers a previously unrecognized role for NAV3 in TGF-β1-driven cardiac fibroblast activation. We show that NAV3 facilitates profibrotic remodeling through noncanonical signaling and cell cycle arrest, independently of SMAD2/3. These findings position NAV3 as a novel regulator of fibroblast phenotype and a potential modulator of cardiac fibrosis.

Background: Cardiac fibrosis is a central pathological process that leads to worse outcomes in various cardiomyopathies. Recent evidence shows that CDCP1 (CUB domain-containing protein 1) modulates myocardial recovery by attenuating human cardiac fibroblast (HCF) proliferation. This study investigates the hypothesis that selective targeting of the CDCP1 cleavage domain can mitigate fibroblast proliferation, thereby offering a novel therapeutic approach to reducing cardiac fibrosis. Methods: A high-throughput in silico screening was performed using the Enamine compound library, focusing on structural regions that include the cleavage and adjacent sites of CDCP1 . A total of 147 candidate compounds were prioritized based on docking scores and binding stability profiles. Selected compounds were subsequently evaluated in vitro using HCF cultures seeded at 3,000 cells/well in 96-well plates. Each compound was administered at 10 μM concentration, and cell proliferation and viability were assessed by time-lapse imaging in the Incucyte® system at 24 and 96 hours. All experiments were done in triplicate with appropriate DMSO vehicle controls. Results: Compound C3 significantly reduced HCF proliferation (Fig B) to 27.3±2.4% relative to DMSO controls (99.9±0.5%, P <0.001) at 96 hours. Detailed structural modeling indicated that C3 binds to the CDCP1 cleavage site (Fig C) with a favorable Glide XP docking score of -7.66 kcal/mol. The interaction is stabilized by hydrogen bonds with Lys365 and Cys374 (Fig D), and supplementary hydrophobic interactions between aromatic rings and aliphatic groups, which contribute to a strong inhibitory effect. Conclusion: Targeting the CDCP1 cleavage domain with compound C3 effectively diminishes HCF proliferation in vitro. Therefore, these results substantiate the therapeutic potential of CDCP1 inhibition in cardiac fibrosis. Ongoing in vivo studies will explore the translational relevance of these findings.

Cardiac fibrosis, a hallmark of heart failure and various cardiomyopathies, represents a complex pathological process that has long challenged therapeutic intervention. High-throughput omics technologies have begun revolutionizing our understanding of the molecular mechanisms driving cardiac fibrosis and are providing unprecedented insights into its heterogeneity and progression. This review provides a comprehensive analysis of how techniques—encompassing genomics, epigenomics, transcriptomics, proteomics, and metabolomics—are providing insight into our understanding of cardiac fibrosis. Genomic studies have identified novel genetic variants and regulatory networks associated with fibrosis susceptibility and progression, and single-cell transcriptomics has unveiled distinct cardiac fibroblast subpopulations with unique molecular signatures. Epigenomic profiling has revealed dynamic chromatin modifications controlling fibroblast activation states, and proteomic analyses have identified novel biomarkers and potential therapeutic targets. Metabolomic studies have uncovered important alterations in cardiac energetics and substrate utilization during fibrotic remodeling. The integration of these multi-omic data sets has led to the identification of previously unrecognized pathogenic mechanisms and potential therapeutic targets, including cell-type-specific interventions and metabolic modulators. We discuss how these advances are driving the development of precision medicine approaches for cardiac fibrosis while highlighting current challenges and future directions in translating multi-omic insights into effective therapeutic strategies. This review provides a systems-level perspective on cardiac fibrosis that may inform the development of more effective, personalized therapeutic approaches for heart failure and related cardiovascular diseases.

Background: Dilated Cardiomyopathy (DCM) is a major cause of heart failure (HF) and carries a high mortality rate. Drug treatment response for DCM-related HF patients is highly variable, with some patients having no response. A pharmacogenomic approach, genome-wide association study (GWAS) followed by biologically functional validation, can potentially identify genetic determinants of prognosis in DCM and might provide insight into novel biology of DCM pharmacotherapy. Methods: A GWAS was performed using DNA from 686 patients with recent onset DCM who were on standard HF therapy using change in left ventricular ejection fraction (LVEF) at a median of 6 months after initial diagnosis. Cultured human cardiac fibroblasts (HCFs) were used to functionally validate the GWAS findings in vitro . RNA-seq followed by pathway analysis after NAV3 knock-down (KD) using siRNA in HCFs were performed to explore potential biological mechanisms. Results: A genetic variant, rs11105445(G>A), identified from the GWAS, mapping to the neuron navigator 3 (NAV3) gene (rs11105445, p=2.37E-07; beta 2.74 ± 0.53) was associated with improvement in LVEF. Functional genomic experiments demonstrated that, both in LV tissue and in cultured fibroblasts, the minor allele A was associated with decreased transcription of NAV3 (pvalue: 0.0335 and 0.0432) suggesting that ↓NAV3 expression might be associated with improvement in LVEF. TGF-β1 can induce HCF transdifferentiation into myofibroblasts and we demonstrate that it also increases NAV3 expression. NAV3 KD which mimics the GWAS variant significantly suppressed TGF-β1 induced HCF transdifferentiation demonstrated by decreased expression of α-smooth muscle actin (ACTA2) and collagen I (COL1A1). RNA-seq after NAV3 KD followed by pathway analysis suggested that NAV3 exerted its effect by regulating ECM and cell cycle processes, especially G1/S transition. We demonstrate that NAV3 KD suppressed TGF-β1 induced G1/S arrest by regulating CDK4, CDK6 and INK4B (cyclin-dependent kinase inhibitor 2B/P15, CDK4 inhibitor). Conclusion: Decreased expression of NAV3 is associated with myocardial recovery in DCM, most likely due to its role in suppressing HCF differentiation by regulating TGF-β1 induced G1/S arrest. The role of NAV3 as a novel therapeutic target in DCM needs to be explored.

Background: Patients with normal resting left ventricular ejection fraction (LVEF) without either coronary artery disease (CAD) or a hypertensive response can paradoxically develop reduced LVEF during exercise stress echocardiography (ESE). The clinical phenotype and outcomes of these patients is unknown. Hypothesis: Patients who paradoxically develop reduced LVEF during exercise may represent a sub-phenotype of heart failure with preserved LVEF (HFpEF). Aims: To describe the baseline characteristics and clinical outcomes of this unique patient population. Methods: Among all ESE performed between January 2003 and December 2022, patients without a hypertensive response to exercise and without CAD by angiogram within 90 days of ESE, who had a resting LVEF ≥50% with a ≥5% LVEF decrease during ESE were identified. All-cause mortality, HF hospitalization, and atrial fibrillation (AF) outcomes were assessed. Kaplan-Meier and Cox regression methods were used to analyze time-to-event outcomes. Results: Among 213,643 stress echocardiograms performed, 134 patients met eligibility criteria (Table 1). The mean age of the population was 66±10 years, 76% were women, and 16% had AF at baseline. Mean LVEF was 58±4% at rest and 43±4% at peak stress. Stress electrocardiogram met criteria for ischemia in 14%. The 10-year all-cause mortality risk was 12.9% (95% CI 5.5-20.3) with 10 (37%) of 27 deaths due to cancer (Figure 1, Panel A). The 10-year estimated risk of HF hospitalization was 17.6% (95% CI 9.0-26.2) (Figure 1, Panel B). Among 112 patients without AF at baseline, the 10-year risk of developing AF was 23.4% (95% CI 13.4-33.4). Conclusions: Patients with exercise-induced reduced LVEF in the absence of obstructive CAD have a high incidence of HF hospitalizations and AF. Given the preponderance of women, risk for AF, HF hospitalizations, and cancer-related deaths, the possibility that this condition is an early sub-phenotype of HFpEF should be investigated.

Background: Electrocardiography-based artificial intelligence (AI-ECG) validated models that detect cardiac disease are increasingly being applied in clinical practice. The utility of such tools to detect insidious cardiac disease in patients with normal baseline left ventricular ejection fraction (LVEF) who paradoxically develop reduced LVEF during exercise stress echocardiography (ESE), and do not have coronary artery disease (CAD) or hypertension, is unknown. Hypothesis: AI-ECG is useful to diagnose insidious cardiac disease and predict clinical outcomes in patients with exercise-induced cardiomyopathy. Aims: To assess the utility of AI ECG in patients with exercise-induced cardiomyopathy. Methods: Among all ESE performed between January 2003 and December 2022, patients without a hypertensive response to exercise and without CAD (confirmed by coronary angiography within 90 days after ESE), with resting LVEF ≥50% and a paradoxical ≥5% LVEF decrease during ESE were identified. A previously validated AI-ECG algorithm that predicts atrial fibrillation (AF), reduced LVEF, and cardiac amyloidosis (CA) was applied to the baseline ECG closest to the time of ESE. The predicted probability of AF, reduced LVEF, and CA if above the published thresholds, was determined. Results: There were 134 patients with exercise-induced cardiomyopathy who were identified. The mean age of this cohort was 66±10 years, 76% were women and 16% had AF at baseline. Mean LVEF was 58±4% at rest and 43±4% at peak stress. The median follow-up period was 6.8 years (IQR 3.0-12.2). Among patients without a baseline history of AF (n=112), AI-ECG identified 29% with a significant probability of AF, which was associated with a subsequent AF diagnosis at univariable analysis (HR 2.505, 95%CI 1.016-6.177, p=0.046). There were 10 patients with an AI-ECG prediction of reduced LVEF among whom 3 were subsequently hospitalized with HF. AI-ECG was positive for CA in 8 and 3 amongst these had subsequent HF hospitalizations (Table). Conclusions: Baseline AI-ECG may help predict subsequent AF and diagnose preclinical amyloid cardiomyopathy in patients who by resting echocardiography do not seem to have cardiac disease but with exercise develop reduced LVEF.

BACKGROUND: Dilated cardiomyopathy (DCM) is a major cause of heart failure and carries a high mortality rate. Myocardial recovery in DCM-related heart failure patients is highly variable, with some patients having little or no response to standard drug therapy. A genome-wide association study may agnostically identify biomarkers and provide novel insight into the biology of myocardial recovery in DCM. METHODS: A genome-wide association study for change in left ventricular ejection fraction was performed in 686 White subjects with recent-onset DCM who received standard pharmacotherapy. Genome-wide association study signals were subsequently functionally validated and studied in relevant cellular models to understand molecular mechanisms that may have contributed to the change in left ventricular ejection fraction. RESULTS: The genome-wide association study identified a highly suggestive locus that mapped to the 5′-flanking region of the CDCP1 (CUB [complement C1r/C1s, Uegf, and Bmp1] domain containing protein 1) gene (rs6773435; P =7.12×10 −7 ). The variant allele was associated with improved cardiac function and decreased CDCP1 transcription. CDCP1 expression was significantly upregulated in human cardiac fibroblasts (HCFs) in response to the PDGF (platelet-derived growth factor) signaling, and knockdown of CDCP1 significantly repressed HCF proliferation and decreased AKT (protein kinase B) phosphorylation. Transcriptomic profiling after CDCP1 knockdown in HCFs supported the conclusion that CDCP1 regulates HCF proliferation and mitosis. In addition, CDCP1 knockdown in HCFs resulted in significantly decreased expression of soluble ST2 (suppression of tumorigenicity-2), a prognostic biomarker for heart failure and inductor of cardiac fibrosis. CONCLUSIONS: CDCP1 may play an important role in myocardial recovery in recent-onset DCM and mediates its effect primarily by attenuating cardiac fibrosis.

BACKGROUND: Common genetic variants are associated with risk for hypertrophic cardiomyopathy and dilated cardiomyopathy and with left ventricular (LV) traits. Whether these variants are associated with myocardial fibrosis, an important pathophysiological mediator of cardiomyopathy, is unknown. METHODS: Multi-Ethnic Study of Atherosclerosis participants with T1-mapping cardiac magnetic resonance imaging in-whom extracellular volume was assessed, and genotyping information was available were included (N=1255). Log extracellular volume (%) was regressed on 50 candidate single nucleotide polymorphisms (previously identified to be associated with hypertrophic cardiomyopathy, dilated cardiomyopathy, and LV traits) adjusting for age, sex, diabetes, blood pressure, and principal components of ancestry. Ancestry-specific results were pooled by fixed-effect meta-analyses. Gene knockdown experiments were performed in human cardiac fibroblasts. RESULTS: The SMARCB1 rs2186370 intronic variant (minor allele frequency: 0.18 in White and 0.50 in Black participants), previously identified as a risk variant for dilated cardiomyopathy and hypertrophic cardiomyopathy, was significantly associated with increased extracellular volume ( P= 0.0002) after adjusting for confounding clinical variables. The SMARCB1 rs2070458 locus previously associated with increased LV wall thickness and mass was similarly significantly associated with increased extracellular volume ( P= 0.0002). The direction of effect was similar in all 4 ancestry groups, but the effect was strongest in Black participants. The variants are strong expression quantitative loci in human LV tissue and associated with genotype-dependent decreased expression of SMARCB1 ( P= 7.3×10 -22 ). SMARCB1 knockdown in human cardiac fibroblasts resulted in increased TGF (transforming growth factor)-β1–mediated α-smooth muscle actin and collagen expression. CONCLUSIONS: Common genetic variation in SMARCB1 previously associated with risk for cardiomyopathies and increased LV wall thickness is associated with increased cardiac magnetic resonance imaging–based myocardial fibrosis and increased TGF-β1 mediated myocardial fibrosis in vitro. Whether these findings suggest a pathophysiologic link between myocardial fibrosis and cardiomyopathy risk remains to be proven.

We demonstrated previously that extracellular vesicles (EVs) released from mesenchymal stem cells (MSCs) play a critical role in angiogenesis. Here, we examine whether this pro-angiogenic efficacy is enhanced in EVs derived from MSCs overexpressing GATA-4 (MSCGATA−4). Methods and Results. EVs were isolated from MSCGATA-4 (EVGATA-4) and control MSCs transduced with an empty vector (EVnull). EVs from both cell types were of the same size and displayed similar molecular markers. Compared with EVnull, EVGATA-4 increased both a tube-like structure formation and spheroid-based sprouting of human umbilical vein endothelial cells (HUVECs). The EVGATA-4 increased the numbers of CD31-positive cells and hemoglobin content inside Matrigel plugs subcutaneously transplanted into mice for 2 weeks. Moreover, EVGATA-4 encapsulated higher levels of let-7 family miRs compared to EVnull. The transfer of exosomal let-7 miRs into HUVECs was recorded with an accompanied down-regulation of thrombospondin-1 (THBS1) expression, a major endogenous angiogenesis inhibitor. The loss-and-gain of function studies of let-7 miRs showed that let-7f knockdown significantly decreased EVGATA-4-mediated vascularization inside Matrigel plugs. In contrast, let-7f overexpression promoted HUVEC migration and tube formation. Conclusion. Our results indicate that EVs derived from genetically modified MSCs with GATA-4 overexpression had increased pro-angiogenic capacity due to the delivery of let-7 miRs that targeted THBS1 in endothelial cells.