BACKGROUND AND AIMS:LMNA-related dilated cardiomyopathy (LMNA-DCM) is a progressive genetic disorder characterized by conduction disease, malignant arrhythmias, myocardial fibrosis, and heart failure. Although LMNA mutations have traditionally been associated with cardiomyocyte-intrinsic defects, the mechanisms driving fibrotic remodelling remain incompletely understood. METHODS:Spatial transcriptomics and integrated single-nuclei multiomics were performed on explanted human LMNA-DCM hearts to define endothelial transcriptional and epigenomic states associated with fibrosis. Patient-specific induced pluripotent stem cell-derived endothelial cells, engineered cardiac organoids, and the LMNAH222P/H222P mouse model were used to investigate RUNX1-mediated endothelial-to-mesenchymal transition (EndoMT). Genetic and pharmacological RUNX1 inhibition strategies were evaluated in vitro and in vivo. RESULTS:Endothelial populations exhibiting EndoMT-associated transcriptional and epigenomic signatures were identified in human LMNA-DCM hearts. LMNA induced pluripotent stem cell-derived endothelial cells demonstrated endothelial dysfunction, mesenchymal gene activation, and epigenetic activation of RUNX1 following loss of LMNA-mediated repression. Genetic RUNX1 deletion restored endothelial identity, reversed EndoMT-associated transcriptional programmes, and normalized chromatin accessibility at endothelial regulatory loci. In multicellular cardiac organoids, endothelial RUNX1 activation impaired endothelial-cardiomyocyte signalling and cardiomyocyte contractile function, whereas endothelial-specific RUNX1 deletion restored endothelial and myocardial function. Pharmacological RUNX1 inhibition with Ro24-7429 similarly improved endothelial and cardiomyocyte function in vitro and reduced myocardial fibrosis while preserving cardiac function in LMNAH222P/H222P mice, including after disease onset. CONCLUSIONS:RUNX1-driven EndoMT represents a central mechanism linking LMNA mutations to fibrotic remodelling in LMNA cardiomyopathy. These findings support endothelial transcriptional reprogramming and RUNX1 signalling as potential therapeutic targets in fibrotic cardiomyopathy.

Variants of uncertain significance (VUS) in the LMNA gene represent a major challenge in clinical genetics, as insufficient functional evidence limits their interpretation and clinical decision-making in laminopathies, including dilated cardiomyopathy (DCM). Here, we generated two isogenic induced pluripotent stem cell (iPSC) lines carrying homozygous LMNA variants, c.293A > G (p.Glu98Gly) and c.439G > A (p.Ala147Thr) by prime editing of a healthy donor iPSC line. Both variants are located within Coil 1B domain of lamin A. The edited iPSC lines retain normal morphology, pluripotency, genomic integrity, and trilineage differentiation capacity, providing a valuable platform for functional characterization and potential clinical reclassification of LMNA VUS.

Background: Various measurements around the aortic valve are typically made on computed tomography angiograms (CTAs) before transcathether aortic valve replacement (TAVR) for aortic stenosis (AS), but their collective prognostic inference on periprocedural conduction disturbances (CDs) is not known. Here, we aimed to use unsupervised machine learning (UML) to analyze a multitude of pre-TAVR CTA features and uncover patient subphenotypes with differential risks of CDs. Methods: Twelve nonredundant features involving the aortic valve, aortic root, and ascending aorta were extracted from the CTAs of 660 AS patients. UML of these features using agglomerative hierarchical clustering was performed on separate male and female datasets, with the optimal number of clusters determined by 30 cluster indices. Multivariable logistic regression was conducted to assess the dependence of CDs on cluster type and the latter's incremental prognostic value over conventional risk factors. Results: Three male clusters were optimally identified (M1-M3): M1 was associated with small valve leaflet calcification loads and aortic root dimensions; both M2 and M3 were associated with large valve leaflet calcification loads and a wide aortic root, but the aortic root was shorter in M2 than M3. Two female clusters were optimally determined (F1-F2): F2 was associated with larger valve leaflet calcification loads and aortic root dimensions. By logistic regression analysis, compared to M1 (reference), M2, but not M3, was more associated with CDs (ORM2/M1=2.15, P=0.032; ORM3/M1=2.12, P=0.085), with no difference between M3 and M2 (ORM3/M2=0.986, P=0.974) or between F1 and F2 (ORF2/F1=1.294, P=0.581). Including cluster type as a predictor in a regression model of CDs containing conventional risk factors as covariates improved the goodness-of-fit (P=0.020). Conclusions: UML of pre-TAVR CTAs can reveal subgroups of male patients with differential risks for CDs and improve prognostication over conventional risk factors. UML-augmented pre-TAVR CTAs may help better guide personalized strategies to minimize CDs. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported in part by the Veterans Affairs Palo Alto Health Care System Administration Office Funding Opportunity Announcement and the Stanford Translational Research and Applied Medicine Pilot Grant. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: IRB of Stanford University gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Hypertrophic cardiomyopathy (HCM) is a prevalent inherited cardiac disorder characterized by left ventricular hypertrophy and contractile dysfunction. Mutations in sarcomeric genes, particularly cardiac myosin-binding protein C (MYBPC3), are a leading cause of HCM. Here, we generated two induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells of patients carrying distinct MYBPC3 mutations (c.2490dupT and c.1800delA). Both lines displayed normal morphology, stable karyotypes, robust expression of pluripotency markers, and trilineage differentiation potential. These patient-specific iPSC lines provide a valuable platform for modeling MYBPC3-associated HCM and enable mechanistic and therapeutic studies of inherited cardiac disease

Induced pluripotent stem cells (iPSCs) are a valuable platform for studying human biology and developing patient-specific cellular models. However, individuals of African American ancestry remain underrepresented in existing iPSC repositories, limiting the diversity of available research resources. To address this gap, we generated and characterized two iPSC lines derived from healthy donors of African American ancestry. Both lines exhibited normal morphology, expression of pluripotency markers, trilineage differentiation potential, stable karyotypes, and absence of mycoplasma contamination. Short tandem repeat analysis confirmed donor identity. These well-characterized iPSC lines provide a valuable resource for future studies investigating ancestry-specific genetic and cellular mechanisms relevant to human disease.

Loeys-Dietz syndrome (LDS) is a rare autosomal dominant connective tissue disorder caused by pathogenic variants in genes involved in the TGF-β signaling pathway. Here, we report the generation of a human induced pluripotent stem cell (iPSC) line derived from peripheral blood mononuclear cells (PBMCs) of an LDS patient carrying a heterozygous TGFBR1 mutation (c.679G > A, p.Glu227Lys). The iPSC line exhibits normal morphology, expresses pluripotency markers, maintains chromosomal integrity, and demonstrates trilineage differentiation capacity. This patient-specific iPSC line provides a valuable platform for modeling LDS pathogenesis and investigating vascular disease mechanisms.

Klf9 is a cardiac-enriched transcription factor of the Krüppel-like factor (Klf) family. Klf9 levels decrease during cardiac hypertrophy; however, no studies have examined its transcriptional targets or role in the progression of hypertrophy. Here, we report genome-wide differential Klf9 occupancy during cardiac hypertrophy, with a predominant enrichment at the metabolic gene promoters. Further, using conditional Klf9 knock-in mice subjected to pressure overload for 1 or 2 weeks, we show that restoring Klf9 expression initially inhibits hypertrophy but later leads to early-onset heart failure. We conclude that a decrease in Klf9 is required for metabolic adaptations that support the development of compensatory hypertrophy.

Rare vascular diseases are a diverse group of life-threatening conditions defined by their low prevalence but profound impact on patient morbidity and quality of life. Diagnosing these disorders remains a significant clinical challenge due to their genetic heterogeneity, overlapping phenotypes, and limited patient populations. As such, the development of robust and human-relevant disease models is critical for elucidating pathogenic mechanisms and guiding therapeutic discovery. The advent of human induced pluripotent stem cell (iPSC) technology has opened new avenues for modeling rare vascular diseases by enabling the generation of patient-specific vascular cell types, including endothelial cells, smooth muscle cells, and fibroblasts, and the creation of both two-dimensional cultures and three-dimensional vascular organoids. Together with genome editing and next-generation multiomics, these platforms represent new approach methodologies (NAMs) that allow for detailed investigation of disease biology, facilitate the correction of pathogenic mutations, and enable high-throughput drug screening in a personalized context. In this review, we highlight the advancements in iPSC-derived vascular modeling, discuss the integration of gene editing and multiomics technologies, and explore their transformative potential for uncovering mechanisms and developing precision therapies for rare vascular diseases.

Immune checkpoint inhibitors (ICI) are a pivotal class of immuno-oncology therapeutics that have improved survival rates for patients with cancer. Their widespread usage, however, is limited due to the development of serious and potentially deadly ICI-related side effects. To develop novel diagnostic and therapeutic strategies to identify and treat patients at risk, we need a better understanding of the underlying mechanisms mediating these potentially deadly side effects. To reach this goal, we have generated and validated induced pluripotent stem cells from the peripheral blood mononuclear cells of a melanoma patient who developed ICI-related myasthenia gravis, myositis, and myocarditis overlap syndrome.

The COVID-19 pandemic has revealed that the impact of SARS-CoV-2 infection extends well beyond the acute phase, with long-term sequelae affecting multiple organ systems, most notably, the cardiovascular system. Long COVID, or post-acute sequelae of SARS-CoV-2 infection (PASC), is characterized by persistent symptoms such as fatigue, dyspnea, chest pain, and palpitations, which can last for months or even years after initial recovery. Increasing evidence implicates immune dysregulation, endothelial dysfunction, persistent viral antigens, and coagulopathy as central drivers of cardiovascular complications. Mechanistic studies demonstrate that direct viral infection of cardiac and vascular cells, along with autoantibody formation and cytokine-mediated injury, contribute to myocardial inflammation, fibrosis, and arrhythmias. Sex-based immunological differences and underlying comorbidities further influence individual susceptibility and disease trajectory. Large-scale epidemiological studies have confirmed significantly increased risks of pericarditis, cardiomyopathy, dysrhythmias, and heart failure among COVID-19 survivors. In parallel, the emergence of advanced preclinical platforms, including patient-derived induced pluripotent stem cell (iPSC)-based cardiac organoids, engineered heart tissues, and organ-on-a-chip systems has enabled mechanistic dissection of Long COVID pathophysiology. These human-relevant models, when integrated with clinical datasets and artificial intelligence (AI)-driven analytics, offer powerful tools for biomarker discovery, risk stratification, and precision therapeutic development. This review synthesizes the current understanding of cardiovascular involvement in Long COVID, highlights key mechanistic insights from both clinical and preclinical studies, and outlines future directions for diagnostic and therapeutic innovation.

Dimorphic ion channels, chloride intracellular ion channels (CLIC), are known to regulate several cellular processes, but their implication in cardiac physiology remains unclear. Artificial planar lipid bilayer studies have shown that CLIC2 negatively modulates the activity of cardiac ryanodine receptor 2 (RyR2), implicating the sarcoplasmic/endoplasmic reticulum (SR/ER) Ca 2+ release. In heart failure, RyR2 channel activity is altered causing aberrant SR Ca 2+ release, depletion of SR Ca 2+ stores, and reduced myocardial contractility in heart failure. Hence, the characterization of intrinsic RyR2 modulators will represent a novel target for the prevention and treatment of heart failure. Moreover, CLIC2 protein expression is significantly increased (p<0.05) in end-stage failing human heart samples compared to non-failing heart tissues. Hence using human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM), we assessed the contribution of CLIC2 in the regulation of cellular Ca 2+ homeostasis in cardiac physiology. CLIC2 knockout hiPSC-CM (CLIC2KO-CMs) showed altered electrophysiological properties, where the beat period (0.686±0.018 sec) and field potential duration (134.266±9.07 ms) decreased as compared to age-matched WT hiPSC-CM (WT-CMs) (0.760±0.014sec and 215.37±7.82ms, respectively). Similarly, spike amplitude showed a decrease in CLIC2KO-CMs (0.339 ± 0.046 mV) compared to WT-CMs (0.705±0.069 mV). Moreover, the APD30, APD50, and APD90 in CLIC2KO-CMs (0.109±0.013, 0.132±0.011, 0.186±0.008 sec respectively) were significantly reduced as compared to the WT-CMs (0.173±0.005, 0.204±0.008, 0.264±0.008 sec respectively). Furthermore, the intracellular Ca 2+ measurements revealed an altered Ca 2+ handling in CLIC2KO-CMs, where a decrease in peak amplitude (3.35±0.13) and duration (1179±9.52 ms) was observed compared to the WT-CMs (5.87±0.25 and 3081.38±55.95 ms, respectively). Finally, the caffeine-sensitive Ca 2+ store load in CLIC2KO-CMs showed a marked reduction in cytosolic caffeine transient amplitude, suggesting an increased SR-Ca 2+ leak due to hyperactive RyR2 channels. Overall, our study highlights CLIC2 as an understudied negative modulator of RyR2 involved in the SR/ER Ca 2+ release mechanism that is upregulated in failing human hearts.

Background: Cardiovascular disease (CVD) is the number one cause of death globally, accumulating to over 17 million lives per year. Dilated cardiomyopathy (DCM) is a condition in which the chambers of the heart enlarge and lose their ability to contract, thereby impairing systolic function. The RNA-binding motif protein 20 (RBM20) gene, a nuclear protein that aids in the alternative splicing of genes, targets central cardiac genes like titin (TTN). Irregular splicing of TTN by pathogenic variants in RBM20 is thought to be linked to DCM. Methods: To study this mutation, peripheral blood mononuclear cells were isolated from patients carrying the DCM mutations and reprogrammed using Sendai virus vectors to induced-pluripotent stem cell (iPSC) lines for generation. With these cells, we immunoassayed for the expression of pluripotency markers, SOX2, NANOG, and OCT3/4. Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR), karyotyping, and sequencing were used to confirm the presence of pluripotency markers and RBM20-specific mutations. Results: Mycoplasma free environment confirmed aseptic conditions in cells. The iPSCs transfected with Yamanaka factors displayed typical morphology as cardiomyocytes and expressed key markers after one week. Immunofluorescence (IF) analyses revealed the expression of the markers in iPSCs derived from both patients. IF analyses for OTX2, Brachyun, and SOX17 demonstrated appropriate formation of germ layers: ectoderm, mesoderm, endoderm, respectively. qPCR for SOX2 and NANOG confirmed their expression in both patients carrying RBM20 variants. Sanger sequencing analyses further confirmed the mutations (c.1901G>A and c.3595G>A). Karyotyping analysis in these cell lines with patient specific mutations revealed an absence of abnormalities at the chromosomal level. Thus, successful characterization of iPSC lines from DCM patients carrying different RBM20 mutations was achieved. Conclusion: We established new cell lines with specific RBM20 mutation from DCM patients. These cells mimic disease criteria for DCM and provide a therapeutic rationale for clinical trials and drug development. Future directions will focus on using the iPSC cell lines to assess the pathophysiology of DCM.

Coronary artery vasospasm (CAV) is characterized by transient constriction of epicardial coronary arteries leading to angina. Its disease mechanisms are multifactorial but has centered mostly on endothelial dysfunction and smooth muscle hyperreactivity. To facilitate the investigation of these mechanisms in cell culture, we generated and characterized three induced pluripotent stem cell (iPSC) lines from patients with CAV. These lines demonstrated normal morphology and karyotypes, robust expression of pluripotency markers, and ability for tri-lineage differentiation. Further differentiation of these cell lines into endothelial and smooth muscle cells will allow mechanistic investigation of their relative contributions to CAV in cell culture.

Background: In recent years, induced pluripotent stem cell (iPSC) technologies have represented a promising technology for drug screening, disease modeling, and personalized medicine approaches. From these cell lines, researchers can differentiate iPSCs into cardiomyocytes (iPSC-CMs) and other somatic cell derivatives, which offers a platform to study cell characteristics in a patient-specific manner. The iPSC technology also addresses many challenges of previously used platforms, such as the inherent difference between rodent models and human physiology or the inaccessibility of human cell samples available via surgery or post-mortem. However, by characterizing cell lines from patients of different ancestries, researchers can better understand the race- and gender-specific phenotypes of heart disease. To demonstrate this, our study showed the successful generation of iPSC-CMs from two patients of African American ancestry and their applicability as patient-specific models. Methods: Our group isolated peripheral blood mononuclear cells (PBMCs) from two healthy African American patients, then reprogrammed the cells using Sendai virus vectors into two iPSC lines for generation: GSBi003-A and GSBi004-A. With these lines, reverse transcription-quantitative polymerase chain reactions (RT-qPCR) validated high expression levels of two pluripotency markers: NANOG and SOX2. Immunofluorescence staining confirmed the expression of pluripotency markers: NANOG, OCT3/4, and SOX2. Karyotyping showed normal karyotypes. Trilineage differentiation showed differentiation into all three germ layers: ectoderm, mesoderm, and endoderm. Short tandem repeat analysis verified the two lines’ concordance from their respective donors. Mycoplasma detection confirmed a mycoplasma-free contamination environment. From these methods, we demonstrated the successful characterization of two iPSC lines from two healthy African American patients. Conclusion: Our study validates the potential for patient-specific iPSC lines for drug screening and disease modeling to investigate cardiovascular diseases in a race- and gender-specific context. This platform has promising applications in developing therapeutics for heart disease.

Background: Hypertrophic cardiomyopathy (HCM) is a commonly inherited heart disease characterized by thickened left ventricular walls, leading to contractile dysfunction and potentially fatal arrhythmias. For this reason, an improved understanding of its mechanisms is an important focus for cardiovascular medicine. In particular, the MYBPC3 gene has a definitive disease causation, accounting for 30 to 40% of HCM cases. To investigate this, our study characterized patient lines with MYBPC3 mutations, validating the potential of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) as an in vitro platform for disease modeling and drug testing to advance HCM treatment strategies. Results: To study the MYBPC3 mutations, peripheral blood mononuclear cells (PBMCs) were isolated from two HCM patients carrying different MYBPC3 mutations and reprogrammed using Sendai viruses into two iPSC lines: GSBi001-A and GSBi002-A. From these lines, we completed reverse transcription-quantitative polymerase chain reactions (RT-qPCR) to confirm high expression levels of two pluripotency markers: NANOG and SOX2. Immunofluorescence staining showed the protein expression of three pluripotency markers: NANOG, OCT3/4, and SOX2 ( Fig. 1 ). Karyotyping confirmed normal karyotypes. Trilineage differentiation showed differentiation into all three germ layers. Short tandem repeat analysis confirmed the origin of lines from their donors. Sanger sequencing showed the presence of MYBPC3-specific mutations. Mycoplasma detection indicated negative mycoplasma contamination in both lines. Thus, we successfully characterized two iPSC lines from two HCM patients with MYBPC3 mutations. Conclusion: Our model shows that patient-specific iPSC lines present a promising platform for modeling MYBPC3-associated HCM in vitro, with further applications in therapeutic development for heart disease. Future studies may seek to assess the pathophysiology of HCM.