Abstract Background IPO8 encodes importin-8, a ubiquitously expressed nuclear transport receptor of the importin-² family. Importin-8 translocates cargoes such as proteins and RNAs from the cytosol to the nucleus. We recently identified bi-allelic loss-of-function IPO8 variants as a cause of a syndromic form of thoracic aortic aneurysm (TAA). An Ipo8 knock-out (Ipo8-/-) mouse model on a C57Bl/6N genetic background displayed aortic root and ascending aortic aneurysms from 8 weeks of age onwards. Surprisingly, the identical Ipo8 knock-out mouse on an Sv129 genetic background did not show any aortic aneurysm development. Purpose This project aims to pinpoint the molecular processes promoting aneurysm development in Ipo8-/- C57Bl/6N mice and/or protecting Ipo8-/- Sv129 mice. In the long term, these findings could contribute to the search for novel therapeutic targets. Methods Ipo8-/- and wild-type (WT) C57Bl/6N and Sv129 mice (N=8 per group) were sacrificed at 16 weeks. The aortic root and ascending aorta were isolated and RNA was extracted to perform bulk RNA-sequencing. Results RNA-sequencing data analysis of the four groups pointed out that the largest variation between the groups was due to the genetic background, rather than the mutation itself. Further analysis revealed significant TGF-β superfamily signalling dysregulation in C57Bl/6N as compared to Sv129 Ipo8-/- mice. In particular, we observed a signature of altered BMP signalling in the C57Bl/6N mutants only. While the TGF-β component of the TGF-β superfamily signalling cascade is a well-known contributor to syndromic TAA, relatively little has been reported on dysregulation of BMP signalling. Conclusions RNA-sequencing suggests that dysregulated BMP signalling drives TAA development in a C57Bl/6N Ipo8-/- mouse model. While BMP activity has previously been linked to vascular development and homeostasis, its involvement in TAA represents a novel insight into the disease pathogenesis. Moreover, these findings emphasize the critical role of mouse genetic backgrounds in vascular disease modelling.For image description, please refer to the figure legend and surrounding text.

Loeys-Dietz syndrome (LDS) represents a clinically and genetically heterogeneous group of connective tissue disorders that share features similar to Marfan syndrome, first identified in 2005. Characterized by significant manifestations, such as aortic aneurysms, arterial tortuosity, craniofacial and skeletal anomalies, LDS results from pathogenic variants in key genes of the transforming growth factor-beta signaling pathway. Given its variable expressivity, a multidisciplinary approach to management is critical. The article provides an updated overview of effective management practices since the first LDS primer in 2014. It aims to enhance clinical awareness, inform health care providers, and improve patient outcomes through individualized care strategies for those living with LDS.

Dipeptidyl aminopeptidase–like protein 6 (DPP6) is a subunit of the K v 4 channels that carry the transient-outward current ( I to ) in cardiac Purkinje cells (PCs) and ventricular myocytes (VMs). DPP6 genetic variants have been linked to severe arrhythmia syndromes. Given the influence of other I to subunits on the Na v 1.5-mediated cardiac sodium current ( I Na ), we examined whether DPP6 regulates both I to and I Na . We explored the impact of the DPP6 missense variants c.821G>A and c.637C>T, segregating in families with long-QT syndrome (LQTS), and c.2252C>T and c.1578G>C, associated with J-wave syndromes (JWSs) and unexplained syncope. In human and mouse heart slices, DPP6 localized within 40 nanometers of Na v 1.5. Functionally, DPP6 reduced I Na and increased I to density in transfected Chinese hamster ovary cells. DPP6 variants linked to LQTS and JWSs led to a hypo- and hyperinhibition of I Na , respectively. Conversely, I to was increased by the JWS variants and decreased by the LQTS variants coexpressed with PC (but not VM) I to subunits. These findings were validated in human induced pluripotent stem cell–derived cardiomyocytes. In silico modeling of I Na and I to data into PC and VM single-cell action potentials, subsequently integrated in two-dimensional tissue simulations, produced steep repolarization gradients for LQTS-c.821G>A versus slowed conduction for JWS-c.2252C>T. Noninvasive electrocardiographic imaging, used for advanced clinical phenotyping, showed dispersed and prolonged repolarization in the DPP6 c.821G>A index patient versus right ventricular outflow tract delayed activation of a DPP6 c.2252C>T carrier. In conclusion, DPP6 variants play an important role in the mutually antagonistic regulation of I Na and I to , contributing to cardiac electrophysiology and arrhythmogenesis.

Abstract Background Thoracic aortic aneurysm (TAA), the abnormal widening of the thoracic aorta, can occur in isolation or as part of a connective tissue disorder like Marfan syndrome (MFS). Insulin-growth factor binding protein 2 (Igfbp2) regulates cell proliferation, growth, and extracellular matrix remodeling. Our lab identified significant upregulation of Igfbp2 in the aortic wall of three independent TAA mouse models (including Fbn1C1041G/+ (MFS)), with an 8- to 30-fold increase prior to aneurysm development, suggesting that it might drive TAA establishment. Material and methods We explored the role of upregulated Igfbp2 on TAA development and progression by crossbreeding Igfbp2+/- and Fbn1C1041G/+ mice. This breeding yielded pups belonging to four genotype groups (N = 14 per group): Fbn1+/+/Igfbp2+/-, Fbn1C1041G/+/Igfbp2+/+, Fbn1C1041G/+/Igfbp2+/-, and Fbn1+/+/Igfbp2+/+. Aortic root and ascending aorta diameters of male mice were determined every 4 weeks from 4 to 28 weeks of age via transthoracic echocardiography utilizing a Vevo-2100 system with MS550D transducer. Aortic root and ascending aorta data were analyzed separately using likelihood ratio tests and chi-squared statistics. Post-hoc analysis involved a Tukey’s HSD test. Results Our analysis confirmed exaggerated aortic growth both at the level of the aortic root and the ascending aorta in male Fbn1C1014G/+ versus wildtypes. In contrast, haplo-insufficiency for Igfbp2 alone showed no measurable effect on aortic diameter, nor did it have an impact in combination with an Fbn1C1041G/+ background. Conclusion Haplo-insufficiency for Igfbp2 in Fbn1C1041G/+ mice has no significant impact on TAA development and progression, suggesting that it may not be sufficient to mitigate aneurysm progression.

Dipeptidyl aminopeptidase-like protein 6 (DPP6) is a subunit of the Kv4 channels that carry the transient-outward current (Ito) in cardiac Purkinje cells (PCs) and ventricular myocytes (VMs). DPP6 genetic variants have been linked to severe arrhythmia syndromes. Given the influence of other Ito subunits on the Nav1.5-mediated cardiac sodium current (INa), we examined whether DPP6 regulates both Ito and INa. We explored the impact of the DPP6 missense variants c.821G>A and c.637C>T, segregating in families with long-QT syndrome (LQTS), and c.2252C>T and c.1578G>C, associated with J-wave syndromes (JWSs) and unexplained syncope. In human and mouse heart slices, DPP6 localized within 40 nanometers of Nav1.5. Functionally, DPP6 reduced INa and increased Ito density in transfected Chinese hamster ovary cells. DPP6 variants linked to LQTS and JWSs led to a hypo- and hyperinhibition of INa, respectively. Conversely, Ito was increased by the JWS variants and decreased by the LQTS variants coexpressed with PC (but not VM) Ito subunits. These findings were validated in human induced pluripotent stem cell-derived cardiomyocytes. In silico modeling of INa and Ito data into PC and VM single-cell action potentials, subsequently integrated in two-dimensional tissue simulations, produced steep repolarization gradients for LQTS-c.821G>A versus slowed conduction for JWS-c.2252C>T. Noninvasive electrocardiographic imaging, used for advanced clinical phenotyping, showed dispersed and prolonged repolarization in the DPP6 c.821G>A index patient versus right ventricular outflow tract delayed activation of a DPP6 c.2252C>T carrier. In conclusion, DPP6 variants play an important role in the mutually antagonistic regulation of INa and Ito, contributing to cardiac electrophysiology and arrhythmogenesis.

Marfan syndrome (MFS), a multisystemic connective tissue disorder, shows intra- and interfamilial aortopathy variability. More than 3,000 mutations have been reported in the FBN1 gene, which encodes for fibrillin-1, a glycoprotein component of microfibrils and elastic fibres. Living with MFS affects an individual’s quality of life. Aortic dilatation mainly affects the aortic root and the ascending aorta; however, dissection of the aortic wall can occur either before or after the aortic arch (type A and type B dissections, respectively). There is no doubt that progress in aortic surgery has significantly improved patients’ life expectancy, but conversely, current medical treatment based on beta-blockers (BB) and angiotensin receptor blockers (ARB) only slightly slows aortic growth. Aortopathy is the result of multiple defects in cellular and subcellular processes that affect aortic wall cells and extracellular matrix homeostasis and organisation. Pathomechanisms include dysregulated cell signalling (TGF-β, BMPs, and nitric oxide), altered mechanotransduction, and oxidative stress. Therefore, it is reasonable to consider a multi-target strategy for medical treatment. Here, we suggest incorporating allopurinol as a complementary drug to current medical (BB/ARB) treatment. Its mode of action is based on its strong antioxidant effects, demonstrated in preclinical assays in different MFS mouse models. Importantly, allopurinol is a widely used compound in medical practice for gout, and has been proven safe, economical and efficient in a variety of other cardiovascular diseases. We outline the pros and cons of its administration in MFS aortopathy and extrapolate its potential use to other aortopathies in which oxidative stress is determinant in disease development.

Loeys-Dietz syndrome (LDS) is a rare multisystemic connective tissue disorder characterized by aggressive aortopathy, skeletal, craniofacial, cutaneous and gastrointestinal manifestations. It is caused by pathogenic variants in genes encoding components of the transforming growth factor-beta (TGF-β) signaling pathway, including TGFBR1, TGFBR2, SMAD2, SMAD3, TGFB2 and TGFB3. Preclinical modeling has been instrumental in elucidating LDS pathogenesis and informing therapeutic strategies. In this review, a comprehensive overview of available in vivo and in vitro models used to study LDS is presented. We critically evaluate the extent to which each model recapitulates the diverse LDS phenotypes and discuss their respective advantages and limitations. Attention is given to experimental design and variables that influence phenotype expression, including mouse genetic background, sex differences and variant-specific effects. We further synthetize insights gained from these models into the molecular and cellular mechanisms driving LDS, such as the TGF-β paradox, extracellular matrix dysregulation and immune activation. Lastly, we discuss current and emerging therapeutic strategies. The models described here highlight the critical role of preclinical modeling in advancing our understanding of LDS pathophysiology and therapy, outlining key considerations for future model development and translational application.

BACKGROUND:Shprintzen-Goldberg syndrome (SGS) shares skeletal features with Marfan syndrome (MFS), but differs in its craniofacial and neurodevelopmental features. Cardiovascular features have been specifically investigated in few of the 57 known patients with SGS described in the literature, making it difficult to determine their prevalence and characteristics. METHODS:We reviewed the medical records of an international cohort of 29 patients, with a particular focus on cardiovascular features. Data were compared with those of MFS. RESULTS:The sex ratio was 1.9 and median age was 23 years (range: 4-54). 13 patients (44.8%) had mitral regurgitation (MR), 11 (37.9%) had a thoracic aortic aneurysm (TAA) and 9 (31.1%) had aortic regurgitation (AR). No cases of aortic dissection were reported. None had beta-blockers as a primary prevention of aortic events. The Kaplan-Meier method revealed a 30 years risk of 47%, 33% and 22% for occurrence of MR, TAA and AR, respectively. A statistically significant association was found between variants in the Dachshund Homology Domain and the risk of aortic aneurysm (11/20 vs 0/9, p=0.036). CONCLUSION:Patients with SGS also significantly have cardiovascular manifestations, encouraging the implementation of a follow-up and preventive cardiovascular treatment identical to that of MFS.

Electrocardiograms (ECGs) are widely used to assess cardiac health, but traditional clinical interpretation relies on a limited set of human-defined parameters. While advanced data-driven methods can outperform analyses of conventional ECG features for some tasks, they often lack interpretability. Variational autoencoders (VAEs), a form of unsupervised machine learning, can address this limitation by extracting ECG features that are both comprehensive and interpretable, known as latent factors. These latent factors provide a low-dimensional representation optimised to capture the full informational content of the ECG. The aim of this study was to develop a deep learning model to learn these latent ECG features, and to use this optimised feature set in genetic analyses to identify fundamental determinants of cardiac electrical function. This approach has the potential to expand our understanding of cardiac electrophysiology by uncovering novel phenotypic and genetic relationships. Our novel VAE model was trained on a dataset comprising over one million secondary care median beat ECGs, with external validation in the UK Biobank (UKB). We performed common and rare variant association studies for VAE latent factors and conventional ECG traits on quality-controlled UKB data. Associated genetic variants were compared to loci for conventional ECG parameters available in the UKB and literature. Loci were considered novel if they were not previously associated with ECG traits in the GWAS Catalog and showed no known associations in nearby genes based on literature review. Novel GWAS associations were validated in a withheld subset of the UKB cohort. Additionally, we compared the associations of the VAE latent factors and conventional ECG traits with phenotypic traits, disease codes, and echocardiographic traits. The VAE identified 20 independent latent factors that captured ECG morphology with high accuracy (mean Pearson correlation: 0.95). GWAS of latent factors identified 65 unique loci, including 27 novel regions not associated with conventional ECG parameters in the same dataset. Six novel loci were not associated with the ECG in previous larger GWAS studies, including genes implicated in cardiac function and remodelling. Rare variant analysis identified seven additional genes with links to cardiac electrophysiology and remodelling. Phenotypic analyses revealed stronger and more comprehensive associations for latent factors compared to conventional traits, particularly for echocardiographic measures and cardiac phenotypes. Visualisations of latent factor alterations highlighted the interpretability of this approach. Our study shows that the VAE provides a valuable tool for advancing our understanding of cardiac function and its genetic underpinnings, outperforming traditional approaches in genetic and phenotypic discovery.

Truncating variants in TTN (TTNtv) are present in around 7% of patients with anthracycline-induced cancer therapy-related cardiac dysfunction (CTRCD). In dilated cardiomyopathy, proximal I-band TTNtv (TTNtvI) harbour less pathogenic potential than distant A-band TTNtv (TTNtvA). To assess if a location-dependent effect of TTNtv is at play in an anthracycline-induced CTRCD, in order to refine risk stratification in patients. Distinct isogenic TTNtv hiPSC lines were created using CRISPR/Cas9 starting from an in-house reprogrammed hiPSC line of a healthy female control individual. Exon 48 (E48), located in the I-band, and exon 357 (E357), located in the A-band were targeted, based on variants identified in our CTRCD population. Differentiation of iPSCs to cardiomyocytes was performed using an in-house established protocol, each of the lines was differentiated at least two times. Expression of cardiac markers and TTN was evaluated with immunocytochemistry. For assessment of contractility, bright-field videos of contracting monolayers were obtained at day 27-44 and analysed using Musclemotion software. Next, doxorubicin (DOX) was added for 24 hours (0, 0.1, 1 or 10 µM) and contractility imaging was repeated. After DOX-treatment, apoptosis (as Caspase 3 activity) and calpain activity were assessed using luciferase assays (Caspase-Glo 3/7 Assay and Calpain-GloTM Protease Assay respectively). We created two isogenic hiPSC lines with a heterozygous TTNtvI (E48): E48_C1: c.13836_13853del and E48_C2: c.13836_13838delTTTinsGATACATACAA, and two with a heterozygous TTNtvA (E357): E357_C1: c. 100373delT and E357_C2: c. 100372_100373insT. In all cell lines, 24 hours treatment with DOX resulted in increased nuclear staining for distal titin. Overall, cells became less elongated and shorter segments of striated titin were observed at higher DOX doses, indicating an impact on cell architecture. Apoptosis significantly increased with increasing DOX dose and according to cell line, with lowest values in the isogenic control and highest values in TTNtvA. Similarly, Calpain activity increased with higher DOX dose, and this was most outspoken in TTNtvA, present in TTNtvI, but absent in the control cell line. The impact on contractility however was less clear, as no linear DOX dose related effects were observed. However, DOX-dose and targeted exons still showed a significant interaction (p<0.0001) indicating a different sensibility to DOX according to which exon was targeted. For each DOX dose, TTNtvA and TTNtvI hiPSC-CMs had significantly lower contractility than the control cell line. DOX influenced cell architecture and nuclear presence of distal titin in all cell lines. Heterozygous TTNtvI and TTNtvA hiPSC-CMs show increased apoptosis and calpain activity in response to DOX treatment, suggesting an increased predilection for cardiotoxicity. Effects were more prominent in TTNtvA than in TTNtvI hiPSC-CMs.Figure 1 Figure 2

Up to 40 % of genetic variants identified in inherited arrhythmia syndromes (IAS) are classified as variants of uncertain significance (VUS) due to limited clinical and functional evidence. In Brugada syndrome (BrS), this challenge is further compounded by its polygenic nature, variable expressivity, and incomplete penetrance. Functional characterization in relevant disease models, such as human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), is essential for VUS reclassification. Here, using conventional CRISPR/Cas9, we established two isogenic hiPSC lines harboring the BrS-associated CACNA1C c.989C > T (p.Thr330Met) variant in homozygous and heterozygous configurations to enable future functional assessment.

BACKGROUND:Filamin A (FLNA) is an actin-binding protein involved in cytoskeleton organization and cell migration. Loss-of-function (LOF) variants give rise to a wide variety of symptoms with periventricular nodular heterotopia (PVNH) and epilepsy as the most common features. FLNA deficiency manifests as a multisystemic disorder with abnormalities of connective tissue and involvement of the cardiovascular, pulmonary, gastrointestinal and hematological system. Affected individuals need a multidisciplinary follow-up, but guidelines are lacking. Here, we present findings from a monocentric cross-sectional cohort study as a basis for improving clinical practices and surveillance for individuals with FLNA deficiency. RESULTS:24 index patients with FLNA deficiency were identified. In the cohort, 23 patients exhibited clinical features of PVNH, while one individual presented with congenital pulmonary airway malformation (CPAM). The incidence of clinical features such as epilepsy (84%) and cardiovascular involvement (56%) align with previously published cohorts. Systematic multidisciplinary follow-up, particularly regular cardiological screening, was lacking in a significant number of individuals. Additionally, lesser-known symptoms such as constipation and thrombocytopenia were underreported, highlighting the need for comprehensive phenotypic assessment in FLNA deficiency. CONCLUSION:The incidence of clinical features in this tertiary cohort aligns with existing literature. The absence of uniform and multidisciplinary guidelines hampers effective surveillance and management. Implementation of regular cardiological screening and increased awareness of less overt symptoms could improve medical outcomes for individuals with pathogenic FLNA variants.

Precise gene editing with conventional CRISPR/Cas9 is often constrained by low knock-in (KI) efficiencies (≈ 2-20 %) in human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). This limitation typically necessitates labour-intensive manual isolation and genotyping of hundreds of colonies to identify correctly edited cells. Fluorescence- or antibiotic-based enrichment methods facilitate the identification process but can compromise cell viability and genomic integrity. Here, we present a footprint-free editing strategy that combines low-density seeding with next-generation sequencing (NGS) to rapidly identify cell populations containing precisely modified clones. By optimising the transfection workflow and adhering to CRISPR/Cas9 KI design principles, we achieved high average editing efficiencies of 64 % in hiPSCs (introducing a Brugada syndrome-associated variant) and 51 % in hESCs (introducing a neurodevelopmental disorder (NDD)-associated variant). Furthermore, under suboptimal CRISPR design conditions, this approach successfully identified hESC clones carrying a second NDD-associated variant, despite average KI efficiencies below 1 %. Importantly, genomic integrity was preserved throughout subcloning rounds, as confirmed by Sanger sequencing and single nucleotide polymorphism (SNP) array analysis. Hence, this NGS-based enrichment strategy reliably identifies desired KI clones under both optimal and challenging conditions, reducing the need for extensive colony screening and offering an effective alternative to fluorescence- and antibiotic-based selection methods.

In the modern era, cardiologists managing patients and families with cardiomyopathies need to be familiar with every stage of the diagnostic pathway from clinical phenotyping to the prescription and interpretation of genetic tests. This clinical consensus statement from the ESC Council for Cardiovascular Genomics aims to promote the integration of genetic testing into routine cardiac care of patients with cardiomyopathies, as recommended in the 2023 ESC guidelines for cardiomyopathies. The document describes the types of genetic tests currently available and provides advice on their prescription and for counselling after the return of genetic findings, including the approach in patients and families with variants of unknown significance.

Background Marfan syndrome (MFS) is a systemic disorder of the connective tissue caused by heterozygous mutations in the FBN1 gene, which encodes fibrillin 1, a glycoprotein that constitutes elastic fibers. MFS does not exhibit sexual dimorphism regarding prevalence; however, it remains unknown whether the paternal or maternal inheritance of the FBN1 mutation affects the cardiovascular pathology of the offspring. In this study, we aimed to determine the impact of the parental origin of the FBN1 mutation on the cardiovascular manifestations of the offspring using the Fbn1 C1041G/+ mouse model of MFS. Methods and Results Four experimental groups were generated by crossing wild-type (WT) and MFS mice to obtain WT and MFS offspring from either a paternal (MFS-P) or maternal (MFS-M) MFS parent. Cardiovascular phenotyping of offspring was performed from childhood to adulthood (from one to six months of age), including echocardiography, tail-cuff plethysmography, histopathology, and canonical (pSmad2) and non-canonical (pERK) TGF-β signaling activity in the aortic tissue. At one month of age, both WT and MFS offspring from MFS-M presented lower body weight than those from MFS-P. However, with age, MFS-M offspring became persistently and significantly overweight. Both MFS-P and MFS-M offspring exhibited a significantly increased aortic root diameter compared with WT offspring; however, this enlargement appeared earlier in MFS-M than in MFS-P offspring. These parental and age-related differences in aortic root diameter were accompanied by increased canonical and non-canonical TGF-β signaling. The cardiac ejection fraction was reduced at early ages in both WT and MFS offspring from MFS-M compared with MFS-P, with the difference persisting only in MFS-M offspring at adulthood. Systolic blood pressure was initially lower in MFS-M offspring across both genotypes. However, it progressively increased, resulting in elevated levels in both WT and MFS offspring from MFS-M by six months of age. Conclusion Our results indicate the existence of a gestational maternal MFS environmental factor with an early impact on the aorta and heart of MFS offspring. In adulthood, this becomes normalized in the aorta but not in the heart. ### Competing Interest Statement The authors have declared no competing interest. Ministerio de Ciencia, Innovación y Universidades, https://ror.org/05r0vyz12, PID2023-146296OB-I00