Circulation Research

Glutathione peroxidase 4 (GPX4) is an antioxidant enzyme important for the reduction of toxic lipid peroxide products. Previous studies revealed the importance of mouse Gpx4 in protecting cardiomyocytes from ferroptosis and, subsequently, the development of cardiovascular disease. In this paper, we investigate the transcriptional consequences of cardiac-specific deletion of Gpx4 in mice and compare this response with that observed in human cardiomyopathy. The findings in this study highlight the importance of GPX4 in maintaining both structural and functional stability of the heart and identify key pathway changes resulting from excessive ferroptosis in cardiac tissue. By overlapping common transcriptional programs perturbed in this animal model and human cardiomyopathy, our findings identify putative mechanisms through which ferroptosis contributes to the development and progression of heart disease. These studies may help guide future cardiovascular therapeutics targeting ferroptosis-dependent pathways.

Smooth muscle (SM) contraction is well known to be regulated by the reversible phosphorylation of the myosin regulatory light chain. However, SM force generation and relaxation are often uncoupled from myosin phosphorylation levels (e.g. the latch-state), indicating that additional regulatory mechanisms must be at play. The precise effects of the actin binding protein caldesmon (CaD) on SM force production and relaxation remain ambiguous, largely due to contradictory findings in experiments performed at the tissue level. To date, there are no studies that have measured the effects of CaD on force and relaxation at the molecular level. Here, we use a laser-trap assay to measure the force produced by SM myosin molecules in the presence and absence of CaD. Measurements were performed before and during myosin dephosphorylation, thus simulating SM contraction and relaxation in-vitro. We demonstrate that CaD inhibits force generation, most likely through competitive inhibition of actomyosin binding while simultaneously introducing a resistive load via tethering of actin and myosin. We also establish CaD as a potentiator of relaxation, increasing force decay rate during myosin dephosphorylation. Finally, we show that CaD directly modulates the dependence of myosin-actin mechanics on myosin phosphorylation levels. These findings refine our understanding of SM regulation, highlighting CaD not merely as a passive structural stabilizer, but as a critical regulatory component of force development and relaxation. Ultimately, understanding these mechanical functions offers new perspectives on pathophysiologies involving SM, such as asthma, hypertension, and gastrointestinal disorders, potentially guiding targeted therapeutic strategies. SIGNIFICANCE STATEMENTSmooth muscle (SM) is responsible for controlling the internal diameter of blood vessels and viscera. Understanding the precise regulation of SM relaxation by actin-binding proteins remains a fundamental lacuna in physiology. Using a molecular mechanics chamber to manipulate the biochemical milieu during active measurements, we demonstrate, for the first time at the molecular level, that caldesmon (CaD) acts as a mechanical modulator that inhibits force generation and accelerates relaxation of SM myosin ensembles. Our results provide a molecular basis for resolving previous contradictory findings reported in tissue-level experiments. Ultimately, understanding the role of contractile and regulatory proteins of SM will provide the basis for understanding SM disorders, such as hypertension and asthma, and guide the development of targeted therapeutic strategies.

Genetic cardiomyopathies consist of a heterogeneous group of myocardial disorders caused by variants that disrupt key regulators of cardiac structure and function. Variants in PLN, encoding phospholamban (PLN), the main inhibitor of the sarco/endoplasmic reticulum Ca{superscript 2}-ATPase 2a (SERCA2a), have been linked to both dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM). Among these, the PLN Arg14del (R14del) variant is the most prevalent. PLN R14del cardiomyopathy is characterized by the accumulation of large perinuclear PLN aggregates in cardiomyocytes of end-stage heart failure tissue. However, the mechanisms driving PLN aggregate formation and their role in disease progression remain unresolved. Using a humanized plna R14del zebrafish model, left ventricular tissue from end-stage PLN R14del cardiomyopathy patients and pharmacological modeling in wild type (WT) cardiac slices, we demonstrate that previously described PLN aggregates represent accumulated sarcoplasmic reticulum (SR)-derived PLN-containing vesicles that form due to impaired SERCA2a activity and increased cytosolic Ca{superscript 2} levels. Furthermore, these SR-derived vesicles often localize adjacent to lysosomes. Interestingly, Ca2+ dysregulation in plna R14del hearts leads to reduced lysosomal function, resulting in SR-derived vesicle accumulation at the microtubule organizing center (MTOC). This perinuclear accumulation induces microtubule aster formation and subsequent cellular disorganization, including sarcomere misalignment and nuclear deformation. Strikingly, reactivation of lysosomal function through fasting reduces SR-derived vesicle accumulation, restores microtubule integrity, and rescues cellular organization in plna R14del zebrafish hearts. Together, these findings identify impaired lysosomal clearance of SR-derived vesicles and the resulting microtubule disorganization as key pathological mechanisms driving PLN R14del cardiomyopathy. Additionally, our results highlight lysosomal reactivation as a promising potential therapeutic strategy to halt or reverse PLN R14del cardiomyopathy progression. Main findingsO_LIPLN aggregates in PLN R14del cardiomyopathy represent SR-derived vesicles formed due to Ca{superscript 2} dysregulation. C_LIO_LIThese SR-derived vesicles often localize perinuclearly at the microtubule organizing center (MTOC), where they are positioned adjacent to lysosomes. C_LIO_LICa2+ dysregulation leads to lysosomal dysfunction which drives vesicle accumulation responsible for microtubule remodeling and pathological cellular rearrangements. C_LIO_LILysosomal reactivation restores vesicle clearance and rescues cardiomyocyte structure. C_LI

BACKGROUNDExcessive trabeculations and myocardial crypts are recurrent features across cardiomyopathies, yet their developmental origins and clinical significance remain poorly defined. To reveal the link between cardiac morphogenesis and disease, we generated humanized mouse models carrying patient-derived MYBPC3 frameshift mutations associated with overlapping hypertrophic cardiomyopathy (HCM) and left ventricular non-compaction (LVNC). METHODSWe applied CRISPR-Cas9 to introduce distinct MYBPC3 frameshift alleles into the mouse genome and performed comprehensive phenotypic and transcriptomic profiling from fetal life through adulthood. RESULTSAdult homozygous Mybpc3 frameshift mutant mice like humans displayed hallmark HCM; however, without LVNC. Fetal and neonatal mutant hearts exhibited markedly enlarged ventricular trabeculae and crypts that progressed postnatally into the observed adult hypertrophy. Transcriptomic analysis revealed stage-specific dysregulation of oxidative metabolism, nonsense-mediated decay (NMD), and cell cycle pathways, peaking at postnatal days 1 and 7, indicating that these stages represent critical time points in disease onset. The persistent NMD signature, also observed in phenotype-negative heterozygotes, suggests a compensatory stress response. Enlarged trabeculae exhibited 2-fold increased trabecular cardiomyocyte proliferation, reversing the normal compact-trabecular proliferative gradient and leading to impaired ventricular compaction in neonates. Hey2CreERT2 lineage tracing demonstrated invasion of Hey2+ compact cardiomyocytes into the trabeculae and ectopic trabecular expression of the Prdm16 transcription factor, indicating defective ventricular wall patterning and maturation. Postnatally, Hey2+-derived cardiomyocytes became restricted to the outer/compact myocardium in mutants, while the inner/trabecular myocardium underwent accelerated hypertrophy concurrent with Prdm16 downregulation. Mice with a Mybpc3 missense variant also exhibited Hey2+ myocardial lineage expansion into trabeculae but no increased proliferation, implicating additional mechanisms beyond Hey2 regulation. Postnatal Prdm16 restoration, via transgenic expression in Mybpc3-null mice effectively attenuated hypertrophy, establishing a causal link between Mybpc3 loss, Prdm16 decline, and pathological remodeling. CONCLUSIONSMybpc3 governs ventricular wall maturation by regulating cardiomyocyte proliferation, patterning, and maturation, partly via Prdm16. Disruption of these developmental programs precedes and drives adult HCM, highlighting a developmental role for sarcomeric proteins, and revealing postnatal Prdm16 modulation as an antihypertrophic therapeutic strategy.

IntroductionVariants in PRKAG2 cause hypertrophic cardiomyopathy (HCM) and conduction disturbances. While prior studies associated PRKAG2-related hypertrophy with increased glycogen storage, many HCM phenotypes remain unexplained. We aimed to uncover how PRKAG2 variants induce myocyte hypertrophy and electrical changes during early cardiac development. MethodsWe generated transgenic zebrafish expressing wild-type (TgWT) or pathogenic variant (TgR299Q) Prkag2 cDNA under a myocardium-specific promoter, and examined cardiac electrophysiology, contractile function, and cytoarchitecture during cardiogenesis and in adult hearts. ResultsTgR299Q fish showed hypertrophic cardiomyocytes and progressive contractile abnormalities, recapitulating human HCM phenotypes. Cardiomyocyte glycogen was elevated in adult but not embryonic hearts. Despite the absence of glycogen accumulation at 6-day post-fertilization, TgR299Q hearts showed electrical abnormalities, including reduced conduction velocity and prolonged action potential and Ca2+ transient durations. We observed decreased AMPK phosphorylation in the TgR299Q hearts. However, AMPK activation did not rescue the electrophysiological abnormalities in TgR299Q. Proximity ligation assays and co-immunoprecipitation identified a physical interaction between AMPK{gamma}2 and myosin, enhanced by the R299Q variant and accompanied by increased AMPK{gamma}2 localization to the myofilament. Na/Ca{superscript 2} exchanger (NCX) inhibition increased Ca2+ duration and diastolic Ca2+ in TgWT but not TgR299Q hearts, indicating reduced free cytosolic Ca2+ for NCX-mediated extrusion in TgR299Q. These findings suggest that enhanced AMPK{gamma}2-myosin interaction may promote myofilament Ca{superscript 2} retention, thereby prolonging Ca{superscript 2} transient duration and APD in the mutant. Notably, the myosin inhibitor mavacamten reduced AMPK{gamma}2-myosin interaction in TgR299Q hearts, and both mavacamten and vmhcl knockdown rescued the early electrophysiological abnormalities. ConclusionsThe PRKAG2 variant altered cardiac excitability, contractility, and Ca2+ handling during cardiogenesis, independent of glycogen accumulation. Enhanced interactions between AMPK{gamma}2 and myosin contributed to these early changes. Our study revealed a novel link between cellular energy sensing and contractile machinery, with therapeutic potential for modulating contractile function in cardiomyopathies.

Ventricular myosin light chain-1 (MLC1v) is a key structural and function-modulating component of the {beta}-cardiac myosin ({beta}M-II) motor complex. Single-point mutations in MLC1v are linked to severe forms of hypertrophic cardiomyopathy (HCM) and sudden cardiac death (SCD) at a young age. However, the molecular mechanisms underlying the motor dysfunction responsible for HCM phenotype development are not fully understood. Here, we investigated native {beta}M-II motors isolated from septal myectomy sample of an HCM patient, harboring a rare homozygous mutation in MLC1v (A57D). Using a pure population of mutant motors (MUT), and sensitive single-molecule functional analysis approach, we directly assessed the primary functional alterations in {beta}M-II bearing A57D MLC1v mutation. In optical trap single-molecules measurements, the mutant motors displayed increased actomyosin (AM) interaction duration in strongly bound state (ton), corresponding to 3-fold reduced AM detachment rate than wild type myosin (WT). The MUT myosin also generated a shorter powerstroke size ({delta}). Ensemble average analysis of AM interaction events demonstrated that both the first powerstroke ({delta}1) associated with Pi release and the second powerstroke ({delta}2) linked to ADP release were reduced in MUT myosin. Moreover, the increased actomyosin cross-bridge stiffness in the AM.ADP state was observed for MUT compared to WT motors. Consistent with slower AM detachment rate and shorter stroke size, reconstituted human mutant {beta}M-II displayed slower actin filament gliding speed. Alterations in sarcomere-level mechanics included increased Ca2+ sensitivity of force generation and prolonged relaxation time, as predicted by FiberSim modelling. Molecular dynamics simulations indicated that the substitution of alanine by aspartate altered MLC1v interactions with myosin heavy chain (MyHC) and light chain 2 (MLC2v), affecting the curvature and flexibility of the lever arm. Overall, these studies establish the molecular mechanism underlying the primary myosin dysfunction due to A57D MLC1v mutation and further highlight the crucial role of MLC1v-mediated regulation of myosin function. Understanding the precise changes in the mutant myosins biomechanical properties is directly relevant to comprehending the initial triggers for pathological cardiac remodeling in HCM patients and designing tailored therapeutic interventions.

BackgroundDiabetic vascular complications are driven by endothelial dysfunction, yet the role of 3D genome organization in this process is unknown. We sought to define the alterations in chromatin architecture in diabetic endothelium and identify the key regulators involved. MethodsWe generated a high-resolution 3D epigenomic atlas of diabetic endothelial cells from mouse models and human subjects using H3K27ac HiChIP, complemented by ChIP-seq, ATAC-seq, and RNA-seq. A human cohort was used to assess protein expression in diabetic versus non-diabetic endothelial cells. To identify JUNB-interacting proteins, we performed rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME), with protein-protein interaction validated by co-immunoprecipitation. Functional validation was performed using in vitro, ex vivo, and in vivo approaches, including endothelial-specific knockdown in a diabetic hindlimb ischemia model. ResultsMulti-omics profiling revealed extensive enhancer reprogramming in diabetic endothelium, with AP-1 binding motifs being consistently and selectively enriched in downregulated enhancers across three distinct diabetic models. Analysis of a human cohort confirmed significantly reduced JUNB protein levels in diabetic endothelial cells. We identified widespread disruption of JUNB-anchored enhancer-promoter interactions, which underlies transcriptional repression of key endothelial genes. RIME and co-immunoprecipitation established the E3 ubiquitin ligase RBBP6 as a direct JUNB interactor that promotes its polyubiquitination and proteasomal degradation in response to hyperglycemia. Human cohort analysis further showed reciprocal elevation of RBBP6 in diabetic endothelial cells. Either JUNB overexpression or RBBP6 knockdown restored enhancer-promoter connectivity, reactivated vasoprotective transcriptional programs, and rescued endothelial function. Critically, endothelial-specific knockdown of Rbbp6 in diabetic mice restored endothelium-dependent vasorelaxation and improved perfusion recovery after hindlimb ischemia, independent of systemic glucose levels. ConclusionsOur study unveils a novel mechanism whereby hyperglycemia induces enhancer reprogramming and disrupts endothelial 3D genome architecture through RBBP6-mediated degradation of JUNB. The RBBP6-JUNB axis is established as a crucial link between metabolic stress and epigenomic reprogramming in vascular disease, presenting a promising therapeutic target for diabetic vasculopathy.

Myocardial infarction (MI) triggers a systemic neutrophil response, yet the roles of distinct neutrophil subsets in cardiac remodeling remain unclear. Studying this requires murine models that accurately mirror human neutrophil dynamics. Here, we show that a minimally invasive intact-chest MI model is more pathophysiologically relevant than the standard open-chest approach for investigating post-MI immune responses. In the open-chest model, surgical trauma disrupts bone marrow homeostasis, releases large numbers of immature neutrophils, and masks MI-specific immune mechanisms. In contrast, the intact-chest model preserves bone marrow integrity and induces only a modest rise in circulating immature neutrophils, closely reflecting MI patient profiles. We further demonstrate that accumulation of immature neutrophils in the infarcted heart exacerbates cardiac dysfunction. Beyond neutrophils, the overall cardiac immune landscape differs markedly between both models. Collectively, our findings establish the intact-chest model as superior for studying post-MI inflammation and reveal immature neutrophils as mediators of adverse cardiac remodeling.

BackgroundHeart failure with preserved ejection fraction (HFpEF) is a major clinical challenge characterized by diastolic dysfunction. Left ventricular stiffening and inflammation are hallmarks of HFpEF, yet the contribution of extracellular matrix (ECM) stiffness and the immune-stromal mechanisms driving ECM stiffening in cardiometabolic HFpEF remain poorly understood. MethodsWe used the murine "2-hit model" of cardiometabolic HFpEF, in which the combination of high fat diet and hypertension induced by L-NAME causes diastolic dysfunction. We evaluated diastolic function by echocardiography and ECM mechanics by uniaxial tensile testing of decellularized cardiac tissue. Functional in vivo studies included genetic depletion of T cells, interferon-{gamma} (IFN{gamma}) knockout mice, and pharmacological lysyl oxidase inhibition. We combined co-cultures of CD4+ T cells and cardiac fibroblasts (CFB) with mechanical testing of cardiac ECM and molecular biology to elucidate cellular and molecular mechanisms. ResultsLeft ventricular ECM stiffness strongly correlated with impaired diastolic function in experimental cardiometabolic HFpEF. Cardiac CD4 T cell infiltration was required for ECM stiffening and upregulation of lysyl oxidase enzymes in CFB. CD4+ T cell-derived IFN{gamma} was both necessary and sufficient to induce LOXL3 in CFB, which increased ECM stiffness in vitro. Mechanistically, IFN{gamma} signaling activated hypoxia-inducible factor-1 (HIF1) in CFB, driving LOXL3 expression and subsequent collagen crosslinking. Genetic or pharmacologic disruption of this IFN{gamma}-HIF1-LOXL3 axis in vivo attenuated adverse ECM remodeling and improved diastolic function. ConclusionsCD4 T cells promote pathological ECM stiffening in cardiometabolic HFpEF through IFN{gamma}-mediated, LOXL3-dependent ECM crosslinking by CFB. Targeting this immune-stromal pathway may offer a novel therapeutic strategy for HFpEF.

BackgroundVascular smooth muscle cells (VSMCs) play a central role in atherosclerotic coronary artery disease (CAD). Oxidised low-density lipoprotein cholesterol (ox-LDL) induces VSMCs dysfunction but the underlying molecular mechanisms are unclear. CAD genome-wide association studies (GWAS) have identified hundreds of disease-associated loci but their biological roles remain poorly defined. We hypothesised that ox-LDL drives pro-atherogenic changes in VSMCs by altering gene regulatory programs involving causal CAD variants. MethodsEx-vivo human coronary VSMCs were exposed to ox-LDL and profiled using RNA-seq, ATAC-seq, and H3K27ac ChIPmentation. Enhancer-gene links were inferred by integrating these data with Hi-C using the Activity-by-Contact (ABC) model. Variant effect predictions were done using AlphaGenome and key target genes functionally tested by CRISPR/Cas9 knockout. ResultsOx-LDL induced widespread transcriptional reprogramming in coronary VSMCs, with 1,487 upregulated and 1,864 downregulated genes (FDR < 0.05). Single-cell RNA-seq meta-analysis demonstrated that ox-LDL-associated programmes enriched in pro-inflammatory and synthetic-inflammatory VSMC clusters in vivo. ATAC-seq identified [~]22k differentially accessible regions following ox-LDL exposure (FDR < 0.05). Integration of ATAC-seq, H3K27ac, and Hi-C using the ABC framework showed that ox-LDL-driven chromatin remodelling was concentrated at distal enhancers, which linked to 2,008 differentially expressed genes via 4,243 peak-gene connections. ABC enhancers were significantly enriched for CAD variants compared with non-vascular disease controls, with stronger enrichment in dynamically accessible enhancers. AlphaGenome predicted larger regulatory effects of prioritised CAD variants in smooth muscle cells than in a non-vascular comparator, and motif analyses indicated allele-dependent transcription factor binding at prioritised enhancer variants. Locus-level prioritisation nominated candidate enhancer-mediated mechanisms at the SPECC1L and MAP1S loci, and CRISPR knockout of the target genes GUCD1 and BACH1 rescued ox-LDL-induced growth arrest/senescence phenotypes in human coronary artery VSMCs. ConclusionsOur unbiased multi-omics framework shows that ox-LDL rewires VSMC regulatory programmes that influence CAD genetic risk. Enhancer-gene mapping refines effector-gene assignment at CAD loci and prioritises regulatory targets in coronary VSMCs.

AimsHypoplastic left heart syndrome (HLHS) is a severe congenital heart disease characterized by ventricular hypoplasia and impaired cardiac function. Clinically, inhaled nitric oxide (NO) therapy is used to reduce pulmonary vascular resistance and improve cardiopulmonary stability in HLHS patients. However, whether NO signaling contributes to HLHS pathogenesis remains unknown. Cytoglobin (CYGB) is a heme protein traditionally thought to limit NO bioavailability. Unexpectedly, our recent work shows that CYGB/Cygb enhances NO signaling through activation of the nitric oxide synthase-soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) signaling pathway. In zebrafish embryos, Cygb-dependent NO signaling is required for normal cilia motility and for the establishment of correct cardiac laterality. Here, our aim was to determine whether Cygb-dependent NO-sGC signaling linked to cilia function regulates cardiac morphogenesis and contributes to ventricular hypoplasia in HLHS. Methods and ResultsWe found that loss of Cygb (cygb2) in zebrafish disrupts NO-sGC signaling during cardiogenesis, altering cardiac progenitor organization and migration within the anterior lateral plate mesoderm (ALPM). Disruption of these processes impairs heart tube morphogenesis, thereby producing a compact ventricle with increased wall thickness despite preserved cardiomyocyte number, reduced ventricle size and decreased stroke volume, recapitulating key features of HLHS. Genetic disruption of the sGC -subunit (gucy1a1) and pharmacological NO scavenging phenocopy the cygb2 mutant phenotype, resulting in reduced cGMP levels, compact ventricular architecture and decreased stroke volume (SV). Consistently, restoration of NO-sGC signaling in cygb2 mutants rescues early cardiac progenitor patterning, ventricular morphology and SV. ConclusionsThese findings identify Cygb-dependent NO-sGC signaling as a critical developmental pathway for ventricular development and performance, temporally linking cardiac progenitor dynamics to cilia-dependent signaling associated with left-right patterning. This study further suggests that pharmacological activation of sGC may provide a therapeutic strategy for hypoplastic ventricular disease. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=187 HEIGHT=200 SRC="FIGDIR/small/711730v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@e266corg.highwire.dtl.DTLVardef@fca897org.highwire.dtl.DTLVardef@1a06fc2org.highwire.dtl.DTLVardef@93acd_HPS_FORMAT_FIGEXP M_FIG C_FIG

Mutations in the spliceosomal gene PRPF8 are associated with a range of human diseases. Studies in mouse and zebrafish suggest that Prpf8 also has a developmental function. Here, using a Prpf8 mutant mouse line isolated from a chemical induced mutagenesis screen, we uncover a previously unrecognised and essential role for Prpf8 in heart development, consistent with the embryonic lethality observed in Prpf8N1531S homozygous mutants. Prpf8N1531S mutant embryos display severe defects in ventricular trabeculation and compact zone formation, accompanied by increased cardiomyocyte proliferation specifically in the compact zone. Mutant embryonic hearts also exhibit disrupted cellular organisation, altered cytoskeletal architecture and changes in extracellular matrix protein expression. Notably, these cardiac abnormalities were exacerbated in embryos exhibiting cardiac looping defects. Transcriptomic analysis identified multiple aberrantly spliced transcripts in Prpf8N1531S mutant embryos, among which the cardiac transcription factor Tead1 was selected as a key functional candidate due to it known role in cardiac ventricle wall developemnt. Tead1 mis-splicing generated an in-frame, lower molecular weight protein isoform, associated with reduced overall TEAD1 expression. The Tead1 mis-spliced isoform showed altered nuclear localisation and dysregulation of TEAD1-dependent gene network important for heart development, including known cardiac sarcomeric genes. In addition, we observed reduced levels of the intracellular domain of the NOTCH1 receptor (NICD1), indicating impaired Notch signalling.. These findings suggest that impaired TEAD1-dependent transcription and Notch signalling contribute to abnormal cardiac trabeculation and compact zone development, highlighting a critical role for Prpf8 in maintaining proper heart development through the regulation of cardiac transcription factor expression and associated signalling networks. This study offers new mechanistic insights into congenital heart diseases linked to spliceosomal gene mutations.

Disturbances of neurovascular coupling (NVC) contribute to metabolic derailment and neurological symptoms associated with epilepsy. While postictal arterial constriction can be alleviated by inhibitors of voltage gated calcium channels (VGCCs), less is known regarding seizure-associated electrical signals in higher-order capillaries and their role in determining pericyte tone during seizures. Here we investigated electrical signaling within the ex vivo neurovascular unit (NVU) derived from rat and human brain tissue. We focused on electrical signal transduction between pericytes and endothelial cells and the potential role of VGCCs in vasomotion. Using dye coupling and paired patch-clamp recordings, we showed that morphologically heterogeneous groups of mid-capillary pericytes build a functional syncytium with endothelial cells. Coupling was asymmetric, allowing for directed propagation of electrical signals. Regardless of their morphology, mid-capillary pericytes responded with depolarization and constriction to metabotropic receptor (GPCR) activation (by thromboxane, norepinephrine and UDP-glucose). However, depolarization via the patch pipette induced neither Ca2+-influx nor constriction, suggesting lack of contribution of VGCCs to vasomotion. On inducing epileptiform activity, A2a adenosine receptors and inwardly rectifying potassium channels hyperpolarized the capillary syncytium, followed by repeated depolarizations due to seizure-associated potassium increase in the parenchyma. Thus, while mid-capillary pericytes are contractile, their tone does not rely on their membrane potential and VGCCs. However, syncytial coupling allows for transmission of seizure-associated hyper- and depolarizing signals to upstream feeding arterioles. Significance statementElectro-metabolic signaling is a mechanism, which couples neuronal metabolic activity to local blood flow, by generation and conduction of hyperpolarizing electrical signals in the vasculature. Repeated seizures are followed by postictal hypoperfusion, suggesting disturbances in this signaling mechanism. Due to the inaccessibility of mid capillary pericytes, little is known about how seizure-associated electrical signals modulate local capillary tone. O_LIRat and human mid-capillary pericytes are contractile and actively regulate capillary diameter upon GPCR activation. C_LIO_LIWhile GPCR-induced vasoconstriction is associated with depolarization of the pericytes, depolarization via the patch pipette induces neither constriction nor intracellular Ca2+ increases. C_LIO_LIDespite differences in their morphology, mesh and thin strand pericytes participate in a common electrical syncytium along with the capillary endothelial cells both in rat and in human tissue. C_LIO_LISignal transmission at electrical synapses between pericyte-pericyte and pericyte-endothelial cell pairs is asymmetric, suggesting a preferred direction of propagation of electrical signals. C_LIO_LIActivation of A2a adenosine receptors and Kir channels mediate capillary hyperpolarization prior to the onset of seizures, which is followed by seizure-associated depolarization due to extracellular potassium accumulation. C_LI

Increased literature support the pathogenetic role of dysfunctional energetic metabolism in the setup and progression of organ damage and failure. Genetic diseases often offer the possibility to investigate pathogenetic mechanisms. In particular, excessive cardiac damage is the most frequent cause of mortality in Fabry disease (FD), a genetic condition caused by deficient -galactosidase A (GLA) activity, leading to globotriaosylceramide (Gb3) accumulation. Beyond Gb3 storage, metabolic alterations and mitochondrial dysfunction, supported by in vitro evidence or studies in other tissues, may contribute to FD cardiomyopathy. This study investigated, for the first time, the mechanisms of mitochondrial involvement in FD, its role in determining cardiac manifestations, and its potential as a therapeutic target. We used a humanized FD mouse model (R301Q-Tg/GLA knockout), along with derived embryonic fibroblasts and neonatal and adult cardiomyocytes, to assess mitochondrial function across the lifespan. FD cells showed impaired mitophagy, reduced mitochondrial respiration, and increased reactive oxygen species production. Importantly, this mitochondrial dysfunction exacerbated the lysosomal deficit in FD cells, forming a vicious cycle. In cardiomyocytes, these alterations progressed with age, leading to the accumulation of dysfunctional mitochondria, energetic failure, and, in adult hearts, terminal mitochondrial damage and apoptosis. These events ultimately result in cardiac remodeling and dysfunction, including hypertrophy and diastolic impairment. Indeed, L-arginine supplementation, which promotes NO/PGC-1-dependent mitochondrial rescue, prevented the development of cardiac abnormalities in FD mice. Our findings identify early mitochondrial dysfunction as a key driver of FD cardiomyopathy and support mitochondrial targeting, including L-arginine supplementation, as a promising adjuvant therapeutic strategy. The mechanistic link between lysosomal dysfunction, altered mitochondrial turnover, and energetic collapse emerges as a key targetable pathway in organ damage, extending beyond FD. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/718770v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@927153org.highwire.dtl.DTLVardef@4e3f03org.highwire.dtl.DTLVardef@10af094org.highwire.dtl.DTLVardef@1388b3c_HPS_FORMAT_FIGEXP M_FIG C_FIG Cardiac manifestations vs mitochondrial alterations in Fabry disease: the visible tip and the hidden base of the icebergCardiac manifestations in hR301Q Tg/KO mice become evident from 9 months of age. However, mitochondrial homeostasis is perturbed much earlier (neonatal to young stages), with impaired mitophagy, reduced mitochondrial respiration and membrane potential, increased ROS production and PGC-1 downregulation. At later stages, from 6 months of age, mitochondrial dysfunction progresses and begins to impact cellular energetics, as indicated by reduced ETC expression and the onset of energetic deficit (ATP reduction). The resulting energetic collapse, together with progressive mitochondrial leakage, leads to cardiomyocyte hypertrophy, apoptosis, and dysfunction, which become detectable from 9 months of age, when clinical signs emerge. These findings support a mechanistic model in which 1) lysosomal incompetence due to GLA deficit is the initiating event inducing impairment of mitophagy; 2) Unsuccessful mitophagy, induces downregulation of PGC-1a-dependent mitogenesis; 3) exhausted mitochondria accumulate, inducing energetic collapse (able to exacerbate lysosomal dysfunction and further perturb mitophagy in a vitious cycle); 4) ultimate mitochondrial leakage induces Cytochrome C release and apoptosis activation. This cascade of molecular events is responsible for clinical manifestations, and mitochondrial targeting prevents cardiac organ damage. Significance statementFabry disease is a rare genetic disorder in which cardiac complications are a major cause of death, yet underlying mechanisms remain unclear. Here, we identify mitochondrial dysfunction as an early pathogenic event associated with impaired mitophagy, whereby defective mitochondrial quality control both results from and exacerbates lysosomal dysfunction, creating a self-reinforcing cycle that drives disease progression. Using a humanized model, we demonstrate that mitochondrial dysfunction is a key determinant of cardiac phenotype in vivo, driving energetic failure, oxidative stress, and cardiac damage. Importantly, L-arginine treatment restores mitochondrial function and prevents cardiac abnormalities. Our findings define a broadly relevant pathogenic axis linking lysosomal dysfunction, mitophagy failure, and mitochondrial impairment, that lead to impaired energetic metabolism and consequent cardiac hypertrophy, independently from GB3 accumulation. The implications of our study go beyond Fabry disease and support the therapeutic targeting of cellular energy homeostasis to prevent and treat organ damage and failure in chronic diseases. IMPORTANTO_LIManuscripts submitted to Review Commons are peer reviewed in a journal-agnostic way. C_LIO_LIUpon transfer of the peer reviewed preprint to a journal, the referee reports will be available in full to the handling editor. C_LIO_LIThe identity of the referees will NOT be communicated to the authors unless the reviewers choose to sign their report. C_LIO_LIThe identity of the referee will be confidentially disclosed to any affiliate journals to which the manuscript is transferred. C_LI GUIDELINESO_LIFor reviewers: https://www.reviewcommons.org/reviewers C_LIO_LIFor authors: https://www.reviewcommons.org/authors C_LI CONTACTThe Review Commons office can be contacted directly at: office@reviewcommons.org

BackgroundArrhythmogenic cardiomyopathy (ACM) is a heritable nonischemic cardiomyopathy and a leading cause of sudden cardiac death. Although inflammation is a pathological hallmark of ACM, the contribution of peptidylarginine deiminase 4 (PAD4)-dependent neutrophil extracellular trap (NET) formation and myeloperoxidase (MPO) to disease progression remains poorly defined. MethodsTo define the role of PAD4-dependent NETosis and MPO signaling in ACM disease progression homozygous desmoglein-2 mutant (Dsg2mut/mut) mice were utilized. We employed genetic and pharmacological approaches to determine the efficacy of targeting PAD4 and MPO on cardiac function, arrhythmogenic burden, myocardial fibrosis, inflammatory signaling, and gap junction integrity. Cardiac phenotyping included echocardiography, electrocardiography, histology, inflammatory profiling, and biochemical assays. ResultsMarkers of PAD4-dependent NETosis were elevated in Dsg2mut/mut hearts as early as 4 weeks of age, prior to cardiac dysfunction. Genetic deletion of Pad4 significantly preserved left ventricular function, reduced ectopics, attenuated myocardial fibrosis, and suppressed proinflammatory and profibrotic cytokines. MPO levels were increased in Dsg2mut/mut hearts, and genetic ablation of Mpo preserved cardiac function, reduced arrhythmic burden, prevented myocardial fibrosis, and restored connexin-43 phosphorylation and localization. Furthermore, pharmacological MPO-inhibition improved cardiac function, reduced arrhythmias, and attenuated inflammatory signaling, though myocardial fibrosis was not fully prevented. Notably, hearts from patients with ACM demonstrated increased MPO signal in both cardiomyocytes and non-cardiomyocyte populations compared with donor controls. ConclusionsPAD4-dependent NETosis and MPO signaling are key drivers of inflammation, fibrosis, and arrhythmogenesis in early disease onset in ACM. Targeting neutrophil-mediated pathways represents a promising therapeutic strategy to mitigate disease progression in ACM. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIPAD4-dependent NET formation is activated early in ACM and directly contributes to myocardial inflammation, fibrosis, arrhythmias, and cardiac dysfunction. C_LIO_LIGenetic ablation of Pad4 or Mpo preserves cardiac function, reduces arrhythmogenic burden, and attenuates proinflammatory and profibrotic signaling in a Dsg2 mutant model of ACM. C_LIO_LIPharmacological inhibition of MPO improves cardiac function and electrical stability, identifying neutrophil-derived pathways as modifiable drivers of disease. C_LI What Are the Clinical Implications?O_LINeutrophil-mediated inflammation represents a clinically relevant mechanism in ACM that may be targeted without global immunosuppression. C_LIO_LIMPO inhibition may offer a novel disease-modifying strategy to reduce arrhythmias and preserve cardiac function in patients with ACM. C_LIO_LINeutrophil- and NET-associated biomarkers may improve early risk stratification and therapeutic decision-making in genetically susceptible individuals. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/718596v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@8e9712org.highwire.dtl.DTLVardef@16043f5org.highwire.dtl.DTLVardef@10d8ea2org.highwire.dtl.DTLVardef@10f3566_HPS_FORMAT_FIGEXP M_FIG C_FIG (A) Signaling pathway for PAD4-dependent NETosis. (B) Illustration of neutrophil undergoing NETosis resulting in the release of MPO and DNA histone complexes. (C) Effects of MPO release on cardiac tissue of ACM mice

Atherosclerotic plaques accumulate low-density lipoprotein (LDL) together with antibodies targeting LDL and its apolipoprotein B (apoB) component. Given the association between IgG and plaque vulnerability, we hypothesized that apoB-specific immune complexes actively promote plaque destabilization. Using immunohistochemistry in carotid endarterectomy specimens, we quantified antibody deposition across morphologically defined plaque regions, and measured apoB reactivity and immune complex levels in matched plaque and plasma samples. IgG deposition was strongly associated with thin fibrous caps, reduced collagen content, and higher overall plaque vulnerability. Symptomatic patients exhibited increased apoB-specific IgG and reduced apoB-IgG immune complexes within plaques, indicating enhanced IgG recycling and heightened inflammatory activity. The neonatal Fc receptor (FcRn) was predominantly expressed by CD163+ macrophages, and mediated antibody recycling, LDL uptake, and production of tumor necrosis factor (TNF) and matrix metalloproteinase-9 (MMP-9) in vitro. Plaque FcRn expression increased with age and correlated with mediators of vulnerability, including collagen-degrading enzymes and pro-inflammatory cytokines. Ex vivo treatment of human plaques with a clinically used FcRn-blocking monoclonal antibody reduced IgG recycling and suppressed TNF and MMP-9 production. These findings identify FcRn-dependent antibody recycling as a contributor to inflammatory plaque vulnerability and highlight FcRn as a potential therapeutic target in atherosclerosis.

Pathologic cardiac hypertrophy requires increased protein synthesis, but the mechanosensors that link membrane stretch to translational control remain poorly understood. Polycystin-1 (PC1), encoded by PKD1, has been proposed as a cardiac mechanosensor, with its C-terminal tail (PC1-CT) promoting hypertrophy in rodent cardiomyocytes. However, its subcellular localization and downstream signaling remain incompletely defined, especially in human cardiomyocytes. Here, we examined endogenous PC1 C-terminus localization and the effects of adenoviral PC1-CT overexpression in human iPSC-derived ventricular cardiomyocytes (hiPSC-CMs) and adult mouse ventricular myocytes. Immunofluorescence revealed a striking striated pattern for both endogenous PC1 C-terminus (detected with a PC1-CT antibody) and the overexpressed PC1-CT fragment. In hiPSC-CMs, the PC1 C-terminus localized between the -actinin bands. In contrast, in adult cardiomyocytes, the overexpressed protein colocalized with -actinin and desmin, suggesting that PC1-CT sarcomeric distribution depends on cardiomyocyte maturation. We performed RNA-seq to assess transcriptional responses downstream of PC1-CT overexpression in hiPSC-CMs relative to LacZ controls. Gene Set Enrichment Analysis (GSEA) revealed enrichment of gene sets related to ribosome biogenesis, RNA processing, and protein synthesis, while classical hypertrophic markers remained unchanged. Pathway analysis suggested increased PI3K activity. PC1-CT overexpression increased phosphorylation of Akt, ERK, S6K1, and ribosomal protein S6 without altering 4EBP1 phosphorylation, suggesting preferential activation of the mTOR-S6K1-S6 branch. Pharmacological studies showed that pan-PI3K inhibition abolished S6 phosphorylation, whereas MEK blockade did not affect it; pertussis toxin and PI3K{gamma}-selective inhibitors also did not affect S6, suggesting a Gi/o-independent PI3K/Akt signaling driving mTOR-S6K1-S6 activation. Collectively, these data identify a sarcomere-associated pool of PC1-CT that engages PI3K-Akt-mTOR-S6K1-S6 signaling to enhance transcriptional programs related to ribosome biogenesis and protein synthesis, without activating a canonical hypertrophic gene program. These findings reveal a mechanistic link between PC1-CT and cardiomyocyte growth.

While dysbiosis and inflammation were previously implicated in cardiovascular diseases, the circuits of how microbiota drives distant perivascular innervation, neuroinflammation and atherosclerosis remains unknown. Here, we report that IL-17RC signaling in intestine protects from atherosclerosis controlling intestinal barrier and microbiota, and loss of IL-17RC in intestinal epithelial cells alters microbiota, enhances perivascular innervation and aortic inflammation, augmenting the disease. Neuronal outgrowth is functionally dependent on microbiota and is essential for neuroinflammation and augmentation of atherosclerosis as chemical denervation reduces inflammation, macrophage activation and disease progression. Microbiota-dependent IL-17A producing {gamma}{delta} T cells accumulate in aorta to promote neuronal outgrowth and activation that can be reversed by {gamma}{delta} T cell blockade. Perivascular neuron activation is further dependent on cell autonomous IL-17 signaling as IL-17RC ablation in sympathetic neurons protected mice from microbiota-driven atherosclerosis. Together, our data illuminate how intestinal cytokine signaling distantly restrains neuroimmune interactions in aorta and uncovers a novel link between IL-17 signaling, microbiota, perivascular innervation and neuroimmune pro-inflammatory crosstalk instrumental for atherosclerosis progression. SummaryIL-17RC signaling regulates intestinal dysbiosis and perivascular neuronal outgrowth that modulates inflammation in atherosclerosis.

Cardiac neural crest cells (CNCCs) contribute to key cardiac structures during embryonic development. Disruption of CNCC patterning or function can lead to congenital heart defects. Here, we investigate whether hemodynamic perturbation alters CNCC behavior in chick embryos. We use the left atrial ligation model to modify intracardiac blood flow in the early common-atrium, common-ventricle heart and track retrovirally labelled CNCCs for lineage tracing and single-cell transcriptomic analysis. Results revealed a significant reduction of CNCC derivatives in major cardiac regions, including the pharyngeal arch arteries and myocardium, in flow-perturbed embryos compared with controls. Notably, despite reduced CNCC numbers in the PAAs, their relative proportion increased, suggesting retention within the PAAs and delayed differentiation. Transcriptional analysis shows the expression of CNCC post-migratory markers (HAND1, FOXC2, GATA6, and TBX2) were consistently downregulated at 4, 24, and 48 hours after LAL. Together, these findings indicate that hemodynamic perturbation impairs CNCC migration and differentiation while preserving their capacity to contribute to mature cardiac structures.

Microtubule detyrosination and re-tyrosination on the C-terminus of -tubulin are mediated by the vasohibin (VASH)-small vasohibin-binding protein (SVBP) complex and tubulin tyrosine ligase (TTL), respectively. Elevated levels of detyrosinated -tubulin (dTyr-tub) are observed in heart failure, and reducing this modification improves cardiac function, suggesting that clinically used heart failure therapies may modulate microtubule detyrosination. We investigated whether sacubitrilat and valsartan, the active components of the angiotensin receptor-neprilysin inhibitor LCZ696, influence dTyr-tub levels in endothelin-1 (ET1)-induced hypertrophy in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). While both sacubitrilat and valsartan prevented hypertrophy, only sacubitrilat prevented ET1-induced dTyr-tub accumulation. RNA sequencing revealed that sacubitrilat normalized several ET1-induced dysregulated pathways. Sacubitrilat slightly increased cyclic guanosine 3,5-monophosphate (cGMP) levels and lowered dTyr-tub, whereas inhibition or knockdown of the cGMP-dependent protein kinase 1 (PRKG1) increased dTyr-tub level. Mechanistically, PRKG1 alpha phosphorylated native VASH1. Incubation of microtubules with the VASH1-SVBP complex containing wild-type VASH1 increased detyrosination, while incubation of the complex containing a VASH1 phosphomimic, in which seven C-terminal serine residues were mutated to glutamate (VASH1-7E) did not. Consistently, overexpression of VASH1-7E gave rise to lower dTyr-tub level than overexpression of a non-phosphorylatable form of VASH1 (VASH1-7A) in hiPSC-CMs deficient in VASH1. In conclusion, these findings identify a cGMP-PRKG1-VASH1 signaling axis that reduces microtubule detyrosination in cardiomyocytes. Our work provides mechanistic insight into how neprilysin inhibition may contribute to therapeutic benefit in heart failure. One Sentence SummaryWe establish a neprilysin-cGMP-PRKG1-VASH1 signaling axis that reduces microtubule detyrosination in cardiomyocytes.