Background: Andersen-Tawil syndrome type 1 (ATS1) is caused by loss-of-function mutations in KCNJ2, which encodes the inward rectifier K+ channel Kir2.1, a key determinant of IK1. Impaired Kir2.1 destabilizes membrane excitability and predisposes to ventricular arrhythmias. Most ATS1 variants disrupt channel regulation by phosphatidylinositol 4,5-bisphosphate (PIP2), but whether specific mutations confer differential arrhythmic risk remains unclear. Objective: To determine whether ATS1 variants disrupting Kir2.1-PIP2 interactions define distinct arrhythmic risk profiles and establish a mechanistically informed framework for risk stratification. Methods: We performed a pooled patient-level analysis of 225 ATS1 patients carrying KCNJ2 variants impairing Kir2.1-PIP2 interaction. Inclusion of 22 clinical and electrocardiographic variables were used to identify mutation-specific risk profiles and predictors for arrhythmia risk. The approach was validated in a multicenter cohort of 20 ATS1 patients. Functional validation was performed using patient-derived iPSC-CMs, cardiac-targeted mouse models, and structural in silico analyses. Results: ATS1 variants segregated into three discrete clusters corresponding to high-, intermediate-, and low-risk arrhythmic phenotypes, establishing a mutation-dependent hierarchy of arrhythmic risk. Regression analyses identified six variables independently associated with severe arrhythmic outcomes. Patient-derived iPSC-CM demonstrated graded impairment of electrical propagation and arrhythmia susceptibility, with a hierarchy in conduction velocity, CV:Control > R82W > R218W > G215D). Cardiac-targeted ATS1 mouse models reproduced the clinical risk stratification. Structural modeling showed that high-risk variants localize near the channel pore and disrupt Kir2.1-PIP2 interactions through mutation-specific mechanisms. Conclusions: ATS1 caused by Kir2.1-PIP2-disrupting variants is not a uniform disorder but comprises biologically distinct subgroups with predictable differences in arrhythmic severity. Integrating genetics, functional phenotyping, and structural modeling provides a mechanistically grounded framework for ATS1 risk stratification and precision therapy development. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported in part by La Caixa Banking Foundation [project code LCF/PR/HR19/52160013]; grants PI20/01220 and PI23/01039 of the public call Proyectos de Investigación en Salud 2020 and 2023 [PI-FIS-2020] funded by Instituto de Salud Carlos III (ISCIII) and the Biobanco La Fe (B.0000723); MCIU grant BFU2016-75144-R and PID2020-116935RB-I00, and co-funded by Fondo Europeo de Desarrollo Regional (FEDER); and Fundación La Marató de TV3 [736/C/2020]. We also receive support from the European Union's Horizon 2020 [grant agreement GA-965286]. CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence [grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033]. We also receive support from the European Union's Horizon 2020 Research and Innovation programme [grant agreement GA-965286]. L.K.G. held an FPI contract [PRE2018-083530], Ministerio de Economía y Competitividad de España co-funded by Fondo Social Europeo, attached to Project SEV-2015-0505-18-2. AIM-M held a FPU contract (FPU20/01569) from Ministerio de Universidades. MLVP held contract PEJD-2019-PRE/BMD- 15982 funded by Consejería de Educación e Investigación de la Comunidad de Madrid y Fondo Social Europeo. MMM holds a PID2021-126423OB-C22 Grant by Agencia Estatal de Investigación. MGR holds a PID2022-137214OB-C22 Grant funded by Agencia Estatal de Investigación and MICIU/AEI/10.13039/501100011033 by ERDF/EU. ADA held a Garantía Juvenil contract from Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid co-funded by Fondo Social Europeo. JMR holds a FPU contract (FPU22/03253) from Ministerio de Universidades. IMC holds a PFIS contract (FI21/00243) funded by Instituto de Salud Carlos III and Fondo Social Europeo Plus (FSE+), co-funded by the European Union. ### 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: iPS-CMs protocols were approved by local ethics committees and the Comunidad de Madrid (PROEX 111.4/20, 226.5/23).The study was approved by the CNIC and Carlos III Institute ethics committees. Mice were reared and housed in accordance with institutional guidelines and regulations. (EU Directive 103 2010/63EU and Recommendation 2007/526/EC, enforced in Spanish law under Real 104 Decreto 53/2013; and PROEX 111.4/20 and 226.5/23 from the Comunidad Autónoma de Madrid). 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 The data supporting the findings of this study are available within the article and its Supplementary Materials. Additional data underlying the results presented in this manuscript are available from the corresponding author upon reasonable request

Abstract Angiogenesis is essential for embryonic brain development and tissue repair, yet the mechanisms that spatiotemporally coordinate endothelial behavior to ensure balanced vascular remodeling remain elusive. Here, we identify the glycosylphosphatidylinositol (GPI)-anchored protease MT4-MMP as a critical, context-dependent determinant of angiogenic growth. Global loss of MT4-MMP transiently impairs vascular network formation in the embryonic hindbrain, whereas endothelial-specific deletion triggers an aberrant angiogenesis characterized by increased vessel density, branching, and a profound loss of vascular organization. This dual phenotype reveals MT4-MMP as a fundamental coordinator of neurovascular development. Consistently, MT4-MMP expression was dynamically regulated during wound repair, and its absence amplifies angiogenesis and accelerates wound closure in adult skin, highlighting its role in maintaining vascular homeostasis postnatally. Mechanistically, MT4-MMP-deficient endothelial cells exhibit impaired polarization and sustained, rather than transient, VEGFA-induced ERK activation. We identify NRP1 as a novel substrate of MT4-MMP and demonstrate that MT4-MMP-mediated NRP1 cleavage restricts NRP1 surface availability to tune the intensity of VEGFA signaling. Furthermore, pharmacological blockade of VEGFA–NRP1 binding partially rescues the vascular defects caused by endothelial MT4-MMP loss in vivo . Together, these findings uncover the MT4-MMP/NRP1 axis as a pivotal control point that prevents aberrant vessel expansion, establishing membrane-anchored proteolysis as a primary regulator across developmental and reparative contexts. Teaser The proteolytic constraint exerted by MT4-MMP dictates neurovascular development and wound repair through the spatial control of NRP1-VEGFA signaling.

Acute myocardial infarction remains a leading cause of morbidity and mortality worldwide. Pharmacogenetic and chronotherapeutic approaches are increasingly applied to optimize therapy in chronic cardiovascular diseases. While gene variants are known to influence long-term drug efficacy, their role in modulating drug-induced cardioprotection in acute conditions such as myocardial infarction is unclear. Similarly, the impact of circadian timing on cardioprotective responses remains insufficiently defined. To address these questions, we evaluated metoprolol as a model cardioprotective agent. Here we examine, in a non-pre-specified exploratory analysis of the METOCARD-CNIC trial (NCT01311700), the influence of ADRB1 Arg389Gly polymorphism and the time of AMI onset on metoprolol efficacy. We found that metoprolol reduced infarct size only in patients homozygous for the ADRB1 Arg389 allele, consistent with its genotype-dependent inhibition of neutrophil migration. In-silico docking and binding studies revealed unstable interactions of metoprolol with the Gly389 variant of ADRB1. Moreover, metoprolol was associated with reduced infarct size when AMI onset occurred between 6:00 and 12:00 h. Restricted cardioprotection to the light phase was confirmed in male mice and in neutrophil-specific Adrb1-knockout models. Collectively, these findings highlight the critical roles of genetic background and circadian timing in shaping the efficacy of acute cardioprotective therapies, supporting the rationale for personalized interventions in acute myocardial infarction.

Aims Short QT syndrome type 3 (SQTS3) is a rare arrhythmogenic disease caused by gain-of-function mutations in KCNJ2, the gene coding the inward rectifier potassium channel Kir2.1. We used a multidisciplinary approach and investigated arrhythmogenic mechanisms in an in-vivo model of de-novo mutation Kir2.1(E299V) identified in a patient presenting an extremely abbreviated QT interval and paroxysmal atrial fibrillation. Methods and results We used intravenous adeno-associated virus-mediated gene transfer to generate mouse models, and confirmed cardiac-specific expression of Kir2.1(WT) or Kir2.1(E299V). On ECG, the Kir2.1(E299V) mouse recapitulated the QT interval shortening and the atrial-specific arrhythmia of the patient. The PR interval was also significantly shorter in Kir2.1(E299V) mice. Patch-clamping showed extremely abbreviated action potentials in both atrial and ventricular Kir2.1(E299V) cardiomyocytes due to a lack of inward-going rectification and increased I-K1 at voltages positive to -80 mV. Relative to Kir2.1(WT), atrial Kir2.1(E299V) cardiomyocytes had a significantly reduced slope conductance at voltages negative to -80 mV. After confirming a higher proportion of heterotetrameric Kir2.x channels containing Kir2.2 subunits in the atria, in-silico 3D simulations predicted an atrial-specific impairment of polyamine block and reduced pore diameter in the Kir2.1(E299V)-Kir2.2(WT) channel. In ventricular cardiomyocytes, the mutation increased excitability by shifting I-Na activation and inactivation in the hyperpolarizing direction, which protected the ventricle against arrhythmia. Moreover, Purkinje myocytes from Kir2.1(E299V) mice manifested substantially higher I-Na density than Kir2.1(WT), explaining the abbreviation in the PR interval. Conclusion The first in-vivo mouse model of cardiac-specific SQTS3 recapitulates the electrophysiological phenotype of a patient with the Kir2.1(E299V) mutation. Kir2.1(E299V) eliminates rectification in both cardiac chambers but protects against ventricular arrhythmias by increasing excitability in both Purkinje-fiber network and ventricles. Consequently, the predominant arrhythmias are supraventricular likely due to the lack of inward rectification and atrial-specific reduced pore diameter of the Kir2.1(E299V)-Kir2.2(WT) heterotetramer.

Aortic stenosis (AS) exposes the left ventricle (LV) to pressure overload leading to detrimental LV remodeling and heart failure. In animal models of cardiac injury or hemodynamic stress, bone morphogenetic protein-7 (BMP7) protects LV against remodeling by counteracting TGF-β effects. BMP receptor 1A (BMPR1A) might mediate BMP7 antifibrotic effects. Herein we evaluated BMP7-based peptides, THR123 and THR184, agonists of BMPR1A, as cardioprotective drugs in a pressure overload model. We studied patients with AS, mice subjected to four-week transverse aortic constriction (TAC) and TAC release (de-TAC). The LV of AS patients and TAC mice featured Bmpr1a downregulation. Also, pSMAD1/5/(8)9 was reduced in TAC mice. Pre-emptive treatment of mice with THR123 and THR184, during the four-week TAC period, normalized pSMAD1/5/(8)9 levels in the LV, attenuated overexpression of remodeling-related genes (Col 1α1, β-MHC, BNP), palliated structural damage (hypertrophy and fibrosis) and alleviated LV dysfunction (systolic and diastolic). THR184 administration, starting fifteen days after TAC, halted the ongoing remodeling and partially reversed LV dysfunction. The reverse remodeling after pressure overload release was facilitated by THR184. Both peptides diminished the TGF-β1-induced hypertrophic gene program in cardiomyocytes, collagen transcriptional activation in fibroblasts, and differentiation of cardiac fibroblasts to myofibroblasts. Molecular docking suggests that both peptides bind with similar binding energies to the BMP7 binding domain at the BMPR1A. The present study results provide a preclinical proof-of-concept of potential therapeutic benefits of BMP7-based small peptides, which function as agonists of BMPR1A, against the pathological LV remodeling in the context of aortic stenosis.

Smooth muscle is an essential component of the intestine, both to maintain its structure and produce peristaltic and segmentation movements. However, very little is known about other putative roles that smooth muscle cells may have. Here, we show that smooth muscle cells may be the dominant suppliers of BMP antagonists, which are niche factors essential for intestinal stem cell maintenance. Furthermore, muscle-derived factors render epithelium reparative and fetal-like, which includes heightened YAP activity. Mechanistically, we find that the membrane-bound matrix metalloproteinase MMP17, which is exclusively expressed by smooth muscle cells, is required for intestinal epithelial repair after inflammation- or irradiation-induced injury. Furthermore, we propose that MMP17 affects intestinal epithelial reprogramming after damage indirectly by cleaving diffusible factor(s) such as the matricellular protein PERIOSTIN. Together, we identify an important signaling axis that establishes a role for smooth muscle cells as modulators of intestinal epithelial regeneration and the intestinal stem cell niche.

Aims Clinical guidelines recommend early intravenous beta-blockers during ongoing myocardial infarction; however, it is unknown whether all beta-blockers exert a similar cardioprotective effect. We experimentally compared three clinically approved intravenous beta-blockers. Methods and results Mice undergoing 45 min/24 h ischaemia-reperfusion (I/R) received vehicle, metoprolol, atenolol, or propranolol at min 35. The effect on neutrophil infiltration was tested in three models of exacerbated inflammation. Neutrophil migration was evaluated in vitro and in vivo by intravitat microscopy. The effect of beta-blockers on the conformation of the beta 1 adrenergic receptor was studied in silico. Of the tested beta-blockers, only metoprolol ameliorated I/R injury [infarct size (IS) = 18.0% +/- 0.03% for metoprolol vs. 35.9% +/- 0.03% for vehicle; P < 0.01]. Atenolot and propranolol had no effect on IS. In the three exacerbated inflammation models, neutrophil infiltration was significantly attenuated only in the presence of metoprolol (60%, 50%, and 70% reductions vs. vehicle in myocardial I/R injury, thioglycolate-induced peritonitis, and lipopolysaccharide-induced acute lung injury, respectively). Migration studies confirmed the particular ability of metoprolol to disrupt neutrophil dynamics. In silico analysis indicated different intracellular beta 1 adrenergic receptor conformational changes when bound to metoprolol than to the other two beta-blockers. Conclusions Metoprolol exerts a disruptive action on neutrophil dynamics during exacerbated inflammation, resulting in an infarct-limiting effect not observed with atenolol or propranolol. The differential effect of beta-blockers may be related to distinct conformational changes in the beta 1 adrenergic receptor upon metoprolol binding. If these data are confirmed in a clinical trial, metoprolol should become the intravenous beta-blocker of choice for patients with on-going infarction. [GRAPHICS] .

Adult cardiac progenitor/stem cells (CPC/CSC) are multipotent resident populations involved in cardiac homeostasis and heart repair. Assisted by complementary RNAseq analysis, we defined the fraction of the CPC proteome associable with specific functions by comparison with human bone marrow mesenchymal stem cells (MSC), the reference population for cell therapy, and human dermal fibroblasts (HDF), as a distant reference. Label-free proteomic analysis identified 526 proteins expressed differentially in CPC. iTRAQ analysis confirmed differential expression of a substantial proportion of those proteins in CPC relative to MSC, and systems biology analysis defined a clear overrepresentation of several categories related to enhanced angiogenic potential. The CPC plasma membrane compartment comprised 1,595 proteins, including a minimal signature of 167 proteins preferentially or exclusively expressed by CPC. CDH5 (VE-cadherin), OX2G (OX-2 membrane glycoprotein; CD200), GPR4 (G protein-coupled receptor 4), CACNG7 (calcium voltage-gated channel auxiliary subunit gamma 7) and F11R (F11 receptor; junctional adhesion molecule A; JAM-A; CD321) were selected for validation. Their differential expression was confirmed both in expanded CPC batches and in early stages of isolation, particularly when compared against cardiac fibroblasts. Among them, GPR4 demonstrated the highest discrimination capacity between all cell lineages analyzed.

Pathological angiogenesis contributes to cancer progression and chronic inflammatory diseases. In inflammatory bowel disease, the microvasculature expands by intussusceptive angiogenesis (IA), a poorly characterized mechanism involving increased blood flow and splitting of pre-existing capillaries. In this report, mice lacking the protease MT1-MMP in endothelial cells (MT1(i Delta)(EC)) presented limited IA in the capillary plexus of the colon mucosa assessed by 3D imaging during 1% DSS-induced colitis. This resulted in better tissue perfusion, preserved intestinal morphology, and milder disease activity index. Combined in vivo intravital microscopy and lentiviral rescue experiments with in vitro cell culture demonstrated that MT1-MMP activity in endothelial cells is required for vasodilation and IA, as well as for nitric oxide production via binding of the C-terminal fragment of MT1-MMP substrate thrombospondin-1 (TSP1) to CD47/alpha v beta 3 integrin. Moreover, TSP1 levels were significantly higher in serum from IBD patients and in vivo administration of an anti-MT1-MMP inhibitory antibody or a nonamer peptide spanning the alpha v beta 3 integrin binding site in TSP1 reduced IA during mouse colitis. Our results identify MT1-MMP as a new actor in inflammatory IA and a promising therapeutic target for inflammatory bowel disease.

Matrix metalloproteinases are involved in vascular remodeling. Little is known about their immune regulatory role in atherosclerosis. Here we show that mice deficient for MT4-MMP have increased adherence of macrophages to inflamed peritonea, and larger lipid deposits and macrophage burden in atherosclerotic plaques. We also demonstrate that MT4-MMP deficiency results in higher numbers of patrolling monocytes crawling and adhered to inflamed endothelia, and the accumulation of Mafb+ apoptosis inhibitor of macrophage (AIM)+ macrophages at incipient atherosclerotic lesions in mice. Functionally, MT4-MMP-null Mafb+ AIM+ peritoneal macrophages express higher AIM and scavenger receptor CD36, are more resistant to apoptosis, and bind acLDL avidly, all of which contribute to atherosclerosis. CCR5 inhibition alleviates these effects by hindering the enhanced recruitment of MT4-MMP-null patrolling monocytes to early atherosclerotic lesions, thus blocking Mafb+ AIM+ macrophage accumulation and atherosclerosis acceleration. Our results suggest that MT4-MMP targeting may constitute a novel strategy to boost patrolling monocyte activity in early inflammation.

Ventricular chambers are essential for the rhythmic contraction and relaxation occurring in every heartbeat throughout life. Congenital abnormalities in ventricular chamber formation cause severe human heart defects. How the early trabecular meshwork of myocardial fibres forms and subsequently develops into mature chambers is poorly understood. We show that Notch signalling first connects chamber endocardium and myocardium to sustain trabeculation, and later coordinates ventricular patterning and compaction with coronary vessel development to generate the mature chamber, through a temporal sequence of ligand signalling determined by the glycosyltransferase manic fringe (MFng). Early endocardial expression of MFng promotes Dll4-Notch1 signalling, which induces trabeculation in the developing ventricle. Ventricular maturation and compaction require MFng and Dll4 downregulation in the endocardium, which allows myocardial Jag1 and Jag2 signalling to Notch1 in this tissue. Perturbation of this signalling equilibrium severely disrupts heart chamber formation. Our results open a new research avenue into the pathogenesis of cardiomyopathies.

Rationale: Aortic dissection or rupture resulting from aneurysm causes 1% to 2% of deaths in developed countries. These disorders are associated with mutations in genes that affect vascular smooth muscle cell differentiation and contractility or extracellular matrix composition and assembly. However, as many as 75% of patients with a family history of aortic aneurysms do not have an identified genetic syndrome.Objective: To determine the role of the protease MMP17/MT4-MMP in the arterial wall and its possible relevance in human aortic pathology.Methods and Results: Screening of patients with inherited thoracic aortic aneurysms and dissections identified a missense mutation (R373H) in the MMP17 gene that prevented the expression of the protease in human transfected cells. Using a loss-of-function genetic mouse model, we demonstrated that the lack of Mmp17 resulted in the presence of dysfunctional vascular smooth muscle cells and altered extracellular matrix in the vessel wall; and it led to increased susceptibility to angiotensin-II-induced thoracic aortic aneurysm. We also showed that Mmp17-mediated osteopontin cleavage regulated vascular smooth muscle cell maturation via c-Jun N-terminal kinase signaling during aorta wall development. Some features of the arterial phenotype were prevented by re-expression of catalytically active Mmp17 or the N-terminal osteopontin fragment in Mmp17-null neonates.Conclusions: Mmp17 proteolytic activity regulates vascular smooth muscle cell phenotype in the arterial vessel wall, and its absence predisposes to thoracic aortic aneurysm in mice. The rescue of part of the vessel-wall phenotype by a lentiviral strategy opens avenues for therapeutic intervention in these life-threatening disorders.

The Notch signaling pathway has a key role in shaping the developing heart. Guillermo Luxán et al . identify two human mutations in the gene encoding the Notch pathway protein MIB1 that cause a type of cardiomyopathy, left ventricular noncompaction. The authors show that mice lacking Mib1 in the myocardium have a similar type of cardiomyopathy and analyze how MIB1 deficiency leads to defective ventricular development.

Mature leaves of dwarf elder (Sambucus ebulus L.) contain the non-toxic type 2 ribosome-inactivating protein ebulin 1 (Girbés et al., 1993b, J. Biol. Chem. 268: 18195–18199). We have now found that the green fruits of dwarf elder contain both free and polymerized forms of ebulin (ebulin f) and a new homo-dimeric D-galactose-binding lectin (SELfd). Polymerized material containing ebulin and lectin is composed of aggregates of variable relative molecular mass, some of them being close to 250 000. These aggregate forms are maintained in part by reducible disulphide bridges and reconstitute from reductant-free dialyzed material previously reduced with 2-mercaptoethanol. Direct incubation of free ebulin f with the free SELfd did not lead to polymerization, thus indicating that polymerization triggers some kind of substantial and perhaps catalyzed change in the structure of these proteins. Ebulin-containing polymerized material reacts with anti-ebulin f antibodies. Our results indicate that ebulin f is a fruit-form of ebulin 1. In contrast to green fruits, mature fruits lack both polymerized material and ebulin f, thus indicating some kind of reserve role for them in green fruits. Polymerization of ebulin and the dimeric lectin may represent a novel means of storing the non-toxic type 2 ribosome-inactivating proteins and lectins found in highly metabolic tissues, such as green fruits.