Abstract Background Endothelial dysfunction is a central driver of cardiovascular and inflammatory diseases, yet the post-translational mechanisms that preserve endothelial homeostasis remain incompletely understood. Protein neddylation, the covalent conjugation of a ubiquitin-like modifier, regulates diverse cellular processes, yet its physiological role in the vascular endothelium remains unknown. This study investigated whether protein neddylation is required to preserve endothelial identity and vascular homeostasis. Methods We generated tamoxifen-inducible endothelial-specific Nae1 knockout mice to inhibit neddylation and combined bulk RNA sequencing, single-cell and single-nucleus transcriptomics, quantitative proteomics, biochemical analyses, and gain- and loss-of-function approaches to define the role of endothelial neddylation in vascular homeostasis and inflammatory injury. Results Endothelial-specific Nae1 deletion caused rapid mortality associated with vascular leakage, platelet accumulation, inflammation, and multi-organ injury. Multi-omics analyses demonstrated profound loss of endothelial identity, characterized by suppression of core endothelial programs and activation of inflammatory, procoagulant, and pyroptotic pathways. Single-cell analyses revealed progressive endothelial dysfunction culminating in depletion of the endothelial population and remodeling of the vascular niche. Mechanistically, endothelial neddylation deficiency activated gasdermin D (GSDMD)- and gasdermin E (GSDME)-dependent pyroptosis, whereas dual inhibition of GSDMD and GSDME markedly attenuated inflammatory transcriptomic remodeling, vascular injury, hepatocyte death, immune cell infiltration, and platelet accumulation. Translational analyses demonstrated reduced endothelial neddylation in experimental endotoxemia and decreased expression of neddylation pathway components in human atherosclerosis and COVID-19 datasets. Conversely, restoration of endothelial neddylation partially reversed inflammatory endothelial transcriptomic reprogramming in vivo. Conclusions NAE1-dependent protein neddylation is an essential regulator of endothelial identity and vascular integrity. Loss of endothelial neddylation promotes gasdermin-dependent pyroptosis and thrombo-inflammatory vascular injury, whereas restoration of the neddylation pathway mitigates inflammatory endothelial dysfunction. These findings identify endothelial neddylation as a fundamental mechanism maintaining vascular homeostasis and a potential therapeutic target for cardiovascular and inflammatory diseases. Clinical Perspective What Is New? Endothelial-specific deletion of NAE1, the enzyme that initiates protein neddylation, causes rapid mortality in mice from multi-organ tissue damage, cell death, and inflammation. Neddylation loss activates the pyroptosis executioners GSDMD and GSDME, and silencing GSDMD and GSDME together reverses the transcriptomic and tissue-level damage caused by neddylation deficiency. The endothelial neddylation pathway is significantly downregulated in human clinical conditions, including atherosclerosis and COVID-19, as well as in experimental models of systemic inflammation. What Are the Clinical Implications? Reduced neddylation in atherosclerotic human arteries suggests this pathway could serve as a biomarker or intervention point for endothelial dysfunction in cardiovascular disease. GSDMD and GSDME represent potential therapeutic targets for protecting endothelial integrity in vascular and inflammatory diseases, including endotoxemia. Because neddylation inhibitors such as MLN4924 (pevonedistat) are already in clinical use for cancer, these findings suggest a mechanism behind their reported vascular and hepatic toxicity that warrants monitoring.

Constitutive photomorphogenesis mutant 9 (COP9) signalosome (CSN) is composed of eight subunits (CSN1 through CSN8). It acts as an essential regulator of Cullin-RING-ubiquitin ligases (CRLs), which target critical cellular regulators for degradation via the ubiquitin (Ub) proteasome pathway. The role of CSN in adipose tissue development and function has not yet been studied. We sought to determine the role of CSN8, the smallest subunit of the CSN complex, in adipogenesis, adipose tissue maintenance, and metabolic balance. We first found that CSN8 level remained constant during adipogenesis and knocking down CSN8 by CRISPR/Cas9 did not impair adipocyte differentiation. Notably, mice with adipocyte-specific Csn8 gene deletion (Csn8AKO) showed disrupted CSN holo-complex formation and Cullin deneddylation, leading to the loss of white and brown adipose tissue. Csn8AKO mice displayed insulin resistance while maintaining glucose tolerance. They showed increased food intake and a trend toward higher energy expenditure but were cold-intolerant. Bulk RNA sequencing revealed that CSN deficiency caused significant remodeling of white and brown adipose tissues, characterized by adipocyte death and inflammation. Specifically, white and brown adipose tissues lacking CSN8 exhibited marked upregulation of apoptotic and pyroptotic cell death, which was associated with alterations in ubiquitination and proteasome activity. In addition, Csn8AKO mice were protected from high-fat diet-induced adipose tissue expansion but developed notable hepatomegaly, steatosis, and insulin resistance. Taken together, our data highlights that CSN8/CSN is crucial for maintaining protein homeostasis in adipose tissue, promoting adipocyte survival, supporting adipose tissue maintenance, and overall metabolic health.

Ufmylation is a conserved protein post-translational modification that involves the covalent conjugation of the ubiquitin-like modifier UFM1 to target proteins through a dedicated E1–E2–E3 enzymatic cascade. Although ufmylation has been increasingly implicated in a range of human diseases, its role in endothelial cells (ECs) and vascular integrity remains largely unexplored. Here, we identify ufmylation as a critical regulator of vascular development and endothelial function. Bioinformatic analyses reveal that genetic variants within and adjacent to the Ufl1 locus, which encodes the UFM1-specific E3 ligase UFL1, are associated with multiple vascular pathologies. Constitutive, EC-specific deletion of Ufl1 in mice resulted in reduced vascular density and increased vascular permeability, leading to subepidermal hemorrhage, myocardial hypoplasia, growth retardation by embryonic day 16.5, and eventual perinatal lethality. Transcriptomic profiling of isolated ECs demonstrated that loss of UFL1 dysregulated gene programs essential for maintaining endothelial identity and barrier function—findings that were recapitulated in ufmylation-deficient human umbilical vein endothelial cells (HUVECs) following UFL1 or UFM1 silencing. Genetic inhibition of ufmylation in HUVECs suppressed proliferation, increased cell death, impaired tube formation, enhanced migration, and promoted endothelial-to-mesenchymal transition. Furthermore, tamoxifen-induced, EC-specific deletion of Ufl1 in adult mice led to cardiomyopathy as early as six weeks post-induction, characterized by increased left ventricular (LV) mass, LV dilation and reduced ejection fraction. Notably, endothelial ufmylation deficiency markedly exacerbated angiotensin II–induced pathological cardiac remodeling, accompanied by a significant reduction in coronary flow reserve, indicative of coronary microvascular dysfunction. Collectively, these findings establish endothelial ufmylation as an essential determinant of vascular development and homeostasis by promoting angiogenesis, preserving endothelial identity, and protecting against maladaptive cardiac remodeling

Ubiquitin (Ub)-dependent proteolysis is essential for protein homeostasis, and its disruption is increasingly recognized as a pathogenic driver of cardiomyopathy and heart failure. Deubiquitinases (DUBs) counterbalance ubiquitination by removing Ub from substrate proteins, thereby modulating diverse cellular processes. However, the specific roles of individual DUBs in cardiac biology remain largely undefined. This project investigates the cardiac function of OTUD6B, a member of the OTU (Ovarian Tumor) DUB family implicated in cell-cycle control, apoptosis, inflammation, DNA repair, and tumorigenesis. Human OTUD6B variants are associated with intellectual disability and congenital heart defects, yet its mechanistic roles in the heart have remained unexplored. We demonstrate for the first time that OTUD6B is essential for perinatal ventricular development and adult cardiac homeostasis. Mice with germline Otud6b deletion exhibited ventricular septal defects, biventricular wall thinning, and perinatal lethality. Transcriptomic and biochemical analyses revealed defects in cardiomyocyte cell-cycle progression, potentially through dysregulation of cell-cycle regulators such as Cyclin E. To define cardiomyocyte-autonomous functions and assess roles beyond development, we generated cardiomyocyte-specific OTUD6B knockout (CKO) mice (αMHC-Cre). While cardiac function was preserved at 3 months, CKO mice developed reduced ejection fraction by 4 months and progressed to severe dilated cardiomyopathy, myocardial fibrosis, and heart failure by 7 months. Transcriptomic profiling of CKO hearts prior to overt disease uncovered broad suppression of pathways related to oxidative phosphorylation, fatty acid metabolism, and cardiac contraction. GSEA revealed significant downregulation of mitochondrial gene programs. In cultured cardiomyocytes, silencing Otud6b impaired mitochondrial respiration, increased mitochondrial ROS, and markedly reduced CCCP-induced mitochondrial ubiquitination, accompanied by destabilization of PINK1 and Parkin—key regulators of mitophagy. These findings identify OTUD6B as a critical regulator of mitochondrial quality control. To evaluate the pathogenic role of human OTUD6B mutations, we generated a cardiomyocyte-specific knock-in (CKI) model expressing the disease-associated R116* truncation mutant (αMHC-Cre/+:Otud6bKI/flox). CKI mice recapitulated the progressive cardiomyopathy and heart failure phenotype of CKO mice, supporting a causal role for this mutation. In summary, our work establishes OTUD6B as a key regulator of heart development and adult cardiac function, acting in part through maintenance of mitochondrial homeostasis and quality control. These findings further suggest that individuals carrying OTUD6B loss-of-function variants may face elevated cardiovascular risk. The CKI model also provides a powerful platform for mechanistic studies and for developing therapeutic strategies to treat OTUD6B-associated cardiac disease. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Endothelial cell (EC) dysfunction is a primary cause of many cardiovascular disorders. While numerous epigenetic and transcriptional regulators of endothelial homeostasis have been identified, the post-translational mechanisms that preserve EC function and vascular integrity remain poorly understood. Neddylation is a highly conserved post-translational modification that covalently conjugates the ubiquitin-like protein NEDD8 to target substrates through a NEDD8-specific E1–E2–E3 enzymatic cascade. Here, we define the functional importance of neddylation in endothelial integrity and vascular development and identify its key downstream effectors. Constitutive EC-specific deletion of Nae1 , encoding NAE1—the regulatory subunit of the sole NEDD8 E1 enzyme (initiate neddylation process), using Cdh5 -Cre resulted in early embryonic lethality by E11.5 due to defective vasculogenesis. In contrast, tamoxifen-induced deletion of Nae1 in adult mice via Cdh5 - CreERT2 led to loss of EC identity, enhanced EC transdifferentiation, and EC death, resulting in increased vascular permeability across multiple vascular beds, hemorrhage, multi-organ failure (including lung and liver), and premature lethality within ~8 weeks post induction. Cullin family proteins (CUL1-9) are predominantly neddylated in ECs and upon neddylation, serve as scaffolds for the assembly of multi-subunit Cullin-RING ubiquitin ligases (CRLs). Among these, CUL3 is the most highly expressed cullin in ECs. Although mutations in CUL3 and its binding partner LZTR1 are linked to hypertension and vascular leakage, their roles in maintaining vascular function and integrity remain largely unexplored. We found that constitutive EC–specific deletion of Cul3 causes early embryonic lethality by E11.5 with defective vasculogenesis, recapitulating the phenotype observed in NAE1-deficient embryos. In vitro data in HUVECs revealed that depletion of CUL3 impaired wound healing, tube formation and proliferation of ECs but promoted trans well migration. Transcriptomic analysis of HUVECs revealed that loss of CUL3 suppressed endothelial signature gene expression and promoted EC transdifferentiation, findings validated by qPCR, immunoblotting, and complementary biochemical assays. Collectively, our data identify neddylation and its downstream effector CUL3 as critical regulators of vascular development and endothelial function by maintaining EC identity and restraining pathological EC transdifferentiation and cell death.

Compared to men, women are disproportionately affected by hypertension with a significant rise in blood pressure after middle age. Xist is a female-specific gene that plays an essential role in X chromosome inactivation during embryonic development. Beyond embryonic development, the role of Xist remains poorly understood. Xist plays a vital role in autoimmune disease, including lupus, which is usually confounded by hypertension. However, the functional role of Xist in vascular cells during hypertension remains unknown. We hypothesize that vascular smooth muscle-specific deletion of Xist promotes Angiotensin II (Ang II)-induced hypertension and arterial stiffening. We used Sm22-Cre mice to delete Xist (KO) in vascular smooth muscle cells (VSMC). Age-matched female mice (N=8-10/group) were ovariectomized to remove confounding endogenous sex steroid effects. Ang II (490 ng/kg/min) was infused for two weeks to induce hypertension. Day- and night time blood pressure (BP) was measured by radiotelemetry, and arterial stiffness by pulse wave velocity (PWV). Vascular reactivity was assessed by wire myography, while arterial structural remodeling was assessed using pressure myography. Echocardiography was performed to evaluate cardiac function. RT-PCR and immunoblots were used to assess gene expression. GraphPad Prism was used for Multiple t-tests, and P< 0.05 was considered significant. At baseline, systolic BP was higher in floxed mice than in Xist KO mice (120 mmHg vs. 113 mmHg; P< 0.05). We also observed significant decreases in PWV (3.6 m/s vs. 1.8 m/s) and pulse pressure (31 mmHg vs. 22 mmHg) in Xist KO mice compared with floxed mice. Cardiac function indicated decreased ejection fraction in Xist KO mice compared with floxed mice (80% vs 66%). Phenylephrine (PE)-mediated constriction was reduced in Xist KO mice relative to floxed mice (PE 10^-5: 203% vs. 148%; P< 0.01). Aortic stress-strain curves shifted to the right in Xist KO mice compared with floxed mice. Aortic gene expression showed significant downregulation of Xist in both the aorta and the heart. Aortic VSMC contractile genes, including Cnn1, Myh11, and Acta2, were also significantly decreased in Xist KO mice compared with floxed mice. Ren1 mRNA and immunohistology were reduced in the kidney sections of Xist KO mice compared with floxed mice. To determine the impact of Xist deletion on hypertension, we used Ang II infusion. We observed that Ang II hypertension increased systolic BP in Xist KO mice compared to floxed mice (145 mmHg vs. 136 mmHg) and eliminated the differences in PWV, pulse pressure, and PE-mediated vasoconstriction. Ang II hypertension increased fibrosis in the heart and kidney cross-sections in Xist KO mice compared with floxed mice. Our data suggest that VSMC-specific Xist KO is vasoprotective in the absence of hypertension. However, Xist KO and Ang II-induced hypertension promote detrimental effects in cardiac and renal fibrosis compared to floxed mice. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Brown adipose tissue (BAT) metabolizes lipids and glucose to produce heat through nonshivering thermogenesis, a vital process for thermoregulation, maintaining systemic energy balance, and combating obesity. The COP9 (constitutive photomorphogenesis mutant 9) signalosome (CSN), composed of eight subunits (CSN1–CSN8), is crucial for the activity of cullin-RING-ubiquitin (Ub) E3 ligases (CRLs), which target cellular proteins for degradation via ubiquitination. However, its role in BAT function remains largely unknown. Our goal was to explore the role of CSN8, the smallest subunit of the CSN complex, in BAT maintenance, thermogenesis, and obesity. We first examined the BAT phenotype in mice with adipocyte-specific deletion of Csn8 (Csn8f/f; AdipoqCre+, i.e., Csn8AKO). Csn8AKO mice showed BAT atrophy and severe cold intolerance. Bulk RNA-seq of BAT identified 790 upregulated and 1,550 downregulated genes. KEGG analysis revealed increased inflammatory and cell death pathways and decreased metabolic pathways in Csn8AKO BAT. To specifically study CSN8’s role in brown adipocytes, we created mice with brown adipocyte-specific deletion of Csn8 (Csn8f/f; Ucp1-Cre+, i.e., Csn8BKO). Csn8BKO mice showed no differences in body weight, total fat, or lean mass compared to wild-type Csn8f/f mice. However, they displayed BAT atrophy, with approximately 70% reduction in BAT mass by 10 weeks of age in both males and females, and were completely sensitive to cold. Histological analysis showed a lack of multilobular lipid droplets and infiltration by numerous immune cells in Csn8BKO BAT, which were confirmed by lower triglyceride levels, significantly higher expression of inflammatory marker genes, and increased staining for the macrophage marker (MAC2). Furthermore, increased expression of apoptotic and phagocytic genes and proteins in Csn8BKO BAT indicates the occurrence of apoptosis and phagocytosis after CSN8 deletion, which was linked to heightened ubiquitination in Csn8BKO BAT. Surprisingly, Csn8BKO mice did not exhibit worsening of high-fat diet-induced obesity. Instead, their BAT mass was restored and showed a similar degree of whitening as wild-type mice. In conclusion, our findings highlight CSN8 as a crucial player for brown adipocyte survival, BAT maintenance, and thermogenesis. A high-fat diet may affect CSN8's role in brown fat fate and obesity-induced whitening, underscoring its unique function in brown fat. This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases [R01DK135657A1] and the National Heart, Lung and Blood Institute [2R01HL132182]. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Cullin 3 (CUL3) is a central component of CUL3-RING ubiquitin (Ub) ligases (CRL3s), which mediate the ubiquitination and subsequent proteasomal degradation of numerous proteins involved in diverse cellular processes. CUL3 has been shown to regulate the proliferation of liver progenitor cells and the metabolic function of mature hepatocytes. However, its role in perinatal liver development and homeostasis remains largely unexplored. We first generated mice with an embryonic-onset hepatocyte-specific deletion of Cul3 (Cul3f/f; Alb-Cre+, Cul3LKO). Cul3LKO mice were born with lower body weights and showed a significant increase in liver-to-body weight ratio starting at postnatal day 5. They developed rapidly progressive liver failure, and none of them survived beyond four weeks of age. Liver from Cul3LKO mice showed a completely dysregulated architecture, extensive hepatocyte necrosis or dropout, massive lymphocyte infiltration, hepatic rosettes or pseudorosettes, and hemangioma. Most notably, they showed pathological features that partially resembled the human congenital disorder Caroli syndrome. These features included extensive ductal plate malformations, various liver cysts, congenital hepatic fibrosis (scarring of the liver), and hyperbilirubinemia. Molecular analyses revealed a reduction in hepatocyte differentiation markers (HNF4α and CEBPα), along with an increase in cholangiocyte markers (CK7 and CK19) and progenitor markers (AFP, PKM2, and beta-Catenin) in the Cul3LKO liver. Cul3LKO liver demonstrated significantly higher expression of hepatic necroptosis marker proteins, confirming extensive hepatocyte necrosis. All these pathological changes were more prominent in male Cul3LKO mice. Furthermore, CRISPR–Cas9-mediated CUL3 knockout in hepatoblastoma HepG2 cells and pharmacological inhibition of CUL3 neddylation with DI951 in primary hepatocytes recapitulated upregulation of cholangiocyte and progenitor marker expression. In conclusion, loss of CUL3 results in hepatocyte necrosis and cholangiocyte and progenitor cell proliferation, with specific histological and molecular features similar to those observed in human Caroli syndrome. Our study underscores the importance of CUL3 in preserving hepatocyte identity and viability and emphasizes its essential role in regulating hepatocyte and cholangiocyte lineage determination during perinatal liver development. Funding sources: This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases [R01DK135657A1] and the National Heart, Lung and Blood Institute [2R01HL132182]. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Disruption of mitochondrial homeostasis is a root cause of cardiomyopathy and heart failure, but the upstream mechanisms regulating this process remain incompletely understood. Here we report neddylation as a novel post-translational mechanism safeguarding mitochondria integrity in the heart. Neddylation is a reversible post-translational modification that involves the covalent attachment of the ubiquitin-like protein NEDD8 to substrate proteins via NEDD8-specific E1-E2-E3 enzymes. In this study, we targeted NEDD8-activating enzyme 1 (NAE1)- a key subunit of the neddylation E1 enzyme- in adult mouse hearts using low-dose tamoxifen (TAM). Loss of NAE1 led to overt heart failure by 10 weeks post-TAM and ultimately premature death. Transcriptomic analysis of 4-week post-TAM hearts identified perturbations in cardiac metabolism and mitochondrial bioenergetics among other pathways in the mutant hearts, which are validated by biochemical analyses in isolated NAE1-deficient adult cardiomyocytes (CMs) in vitro and hearts in vivo . The deficits in energy are attributed to disrupted mitochondrial dynamics and turnover, as evidenced by massive strikingly elongated mitochondria, the accumulation of mitophagic vesicles and alterations in key regulators such as MFN2, DRP1, PINK1, LC3-II and P62. To understand the contribution of disrupted mitochondrial dynamics, we generated double knockouts (KOs) of NAE1 with either MFN2 (a key mitofusion protein) or DRP1 (a critical mito-fission protein). Intriguingly, MFN2-KO had minimal impact on disease progression of NAE1-deficient mice, while DRP1-KO exhibited accelerated cardiac dysfunction compared to NAE1- or DRP1- single-KO hearts, indicating the pathogenic role of mitochondrial hyperfusion in NAE1-deficient hearts. Furthermore, we found that inhibition of neddylation inactivates cullin family proteins, key scaffold proteins of multi-subunit cullin-RING ubiquitin ligases, thereby inhibiting mitochondrial ubiquitination and consequently their turnover. Taken together, these results uncover a previously unrecognized role of neddylation in balancing mitochondrial dynamics and surveilling mitochondria integrity, highlighting its critical role in sustaining adult heart function.

Deubiquitinases (DUBs) regulate protein stability and various cellular processes by removing ubiquitin from substrate proteins. OTUD6B, a member of the OTU DUB family, has been linked to intellectual disabilities and congenital heart defects through compound heterozygous or homozygous variants in the OTUD6B gene. However, its molecular and pathophysiological roles in the heart remain poorly understood. Here, we demonstrate that OTUD6B is essential for ventricular chamber development and postnatal survival. OTUD6B is ubiquitously expressed in mouse embryos. Hypomorphic Otud6b neo/neo mice, with a 90% reduction in OTUD6B protein, were viable, fertile, and showed no gross abnormalities up to 24 weeks. In contrast, Otud6b -/- mice, with biallelic loss of OTUD6B, exhibited growth retardation and failed to survive beyond postnatal day 1 (P1). Histological analysis revealed significant cardiac defects in Otud6b-/- mice, including ventricular septal defects (VSD) and severe bi-ventricular wall thinning as early as embryonic day 16.5 (E16.5). Echocardiography at E20.5 showed increased cardiac stiffness, a hallmark of heart failure. The severe cardiac phenotype arises from reduced cardiomyocyte proliferation due to defective cell cycle progression, characterized by S-phase arrest and decreased binucleation in OTUD6B-deficient cardiomyocytes. Unbiased transcriptomic analysis of E14.5 hearts reveal perturbations in cell cycle regulation, fatty acid metabolism, myogenesis and cardiomyocyte maturation in Otud6b -/- mice. HIF1α plays a pivotal role in cardiac development by regulating cardiomyocytes proliferation and metabolic transition from glycolysis to oxidative metabolism. Interestingly, overlapping OTUD6b-regulated transcriptome with HIF1a-regulated transcriptome (GSE61209) or HIF1a potential targets (GSE61247) suggest persistent activation of HIF1a signaling in Otud6b -/- hearts. Moreover, homozygous Otud6b knockin mice ( Otud6b KI/KI ) bearing pathogenic variants R145* in OTUD6B gene recapitulated the myocardial hypoplasia, ventricular septal defects, and perinatal lethality observed in Otud6b -/- mice, mirroring cardiac defects seen in patients with R145* mutations. At the molecular level, loss of OTUD6B reduced cyclin D/E protein levels while increasing HIF1a proteins, which may contribute to the abnormal cell cycle progression and disrupted cardiomyocyte metabolism. Loss of OTUD6B did not affect the transcripts of cyclin D/E and HIF1a, suggesting OTUD6B’s role in regulation of their proteolysis, directly or indirectly. Taken together, our findings establish an essential role for OTUD6B in perinatal ventricular chamber development and embryogenesis, likely by fine-tuning cell cycle regulators and HIF1α during heart development. This work is supported by NHLVI 2R01HL124248-06A1 and NIH/NHLBI 1R01HL16505. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Defective cardiac development can lead to congenital heart diseases, the primary cause of congenital mortality. Recent insights into transcriptional regulatory mechanisms have greatly expanded our understanding of the signaling networks that shape cardiac development for postnatal life. However, the role of posttranslational mechanisms in this process remains largely unexplored. Cullin-RING ubiquitin E3 ligases (CRLs) are multi-subunit complexes composed of a catalytic subunit (RBX1/RBX2), a scaffold cullin protein, and a substrate receptor. By facilitating the ubiquitination and degradation of various intracellular proteins, CRLs regulate numerous cellular and pathophysiological processes. Yet, the specific role of CRLs, particularly their catalytic subunit RBX1, in mammalian heart development remains largely unknown. Here, we demonstrate that RBX1 is a dosage-sensitive regulator of cardiac morphogenesis. RBX1 is robustly expressed in fetal mouse hearts but downregulated in adult hearts. Surprisingly, mice with germline deletion of one allele of the RBX1 gene presented severe cardiac morphological abnormalities and died at perinatal stage. To evaluate the cell autonomous role of RBX1 in cardiogenesis, we deleted one allele of RBX1 via a cardiac-specific Cre line Xmlc2-Cre that is highly active beginning at E8.5. The RBX1 haploinsufficient mice presented hypoplastic ventricular myocardium that emerged as early as E13.5 and discernable ventricular septum defects at E18.5. These cardiac morphological abnormalities result in heart failure, evidenced by significantly compromised cardiac contractility revealed by echocardiography and eventually lethality around P5. Interestingly, deletion of one allele of RBX1 via αMHC-Cre, which turns on later than Xmlc2-Cre in cardiomyocytes, were viable, fertile, and showed no gross abnormalities at adulthood, indicating a crucial dosage effect of RBX1 on early cardiac morphogenesis. In contrast, homozygous deletion of RBX1 via αMHC-Cre, resulted in myocardial hypoplasia and biventricular non-compaction by E16.5, culminating in embryonic heart failure and lethality at E18.5. The severe cardiac phenotypes in both models were associated with diminished cardiomyocyte proliferation and cardiomyocyte immaturity, the latter being characterized by disrupted sarcomere assembly, deficits in metabolic transition from glycolysis to oxidative metabolism and impaired ion channel switch (Kir2.1 to HCN4). Mechanistically, loss of RBX1 destabilized cullin proteins and impaired the activities of CRLs, leading to accumulation of Hippo kinases (LATS1/2, MST1, and MST2) and their adaptor proteins (SAV and MOB1). Consequently, YAP failed to enter the nucleus and initiate the expression of genes essential for cardiac morphogenesis. We further reveal that RBX1 targets MOB1 for ubiquitination and degradation. In conclusion, our findings demonstrate the indispensable role of RBX1 in embryonic cardiac development. Sustained and high levels of RBX1 are necessary to regulate the Hippo-YAP pathway thus promoting cardiomyocyte proliferation and maturation, and ventricular chamber development. Our findings also highlight that multiple aspects of embryonic cardiac morphogenesis and function are exquisitely sensitive to small changes in RBX1 expression levels. This work was funded by the American Heart Association Predoctoral Fellowship to J.Z.C (23PRE1012641). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Rapsyn is a scaffold protein that is thought to anchor acetylcholine receptors at the neuromuscular junction (NMJ). We showed that it may be an E3 ligase that regulates NMJ development by neddylation. To obtain genetical evidence, we mutated Nae1 (APP-BP1), an obligatory subunit of the neddylation E1 enzyme specifically in muscle cells. The mutation decreased the stability of acetylcholine receptor (AChR)α, reduced AChR clustering, and impaired NMJ development in Pax7-Cre;Nae1f/f mice (of either sex) and caused neonatal lethality. Moreover, while NMJs were normal in heterozygous mutant mice of Nae1 or rapsyn C366A (a knock-in mutation that eliminates E3 ligase activity), double heterozygous mutant mice (of either sex) displayed NMJ deficits, indicative of a genetic interaction between Nae1 and rapsyn. These results provide genetic evidence for a role of neddylation in NMJ formation and support the notion that rapsyn serves as a neddylation E3 ligase in NMJ formation.

Ufmylation is a novel ubiquitin-like protein modification that plays a critical role in maintaining the homeostasis of different tissues, but its role in the heart remains poorly understood. Here, we showed that mice lacking UFM1 ligase 1 (UFL1), an enzyme essential for ufmylation, in the heart developed peripartum cardiomyopathy. Loss of UFL1 reversed pregnancy-induced adaptive cardiac transcriptome alterations. Moreover, loss of UFL1 triggered excessive endoplasmic reticulum stress, inhibited mitochondrial oxidative metabolism, and caused augmented mTOR signaling, leading to pronounced pathological remodeling and heart failure. These results demonstrate that ufmylation is essential for physiological cardiac remodeling and that disruption of ufmylation predisposes the heart to peripartum cardiomyopathy.

Deubiquitinases (DUBs) regulate protein stability and various cellular processes by removing ubiquitin from substrates. OTUD6B, a member of the OTU DUB family, has been linked to intellectual disabilities and congenital heart defects through compound heterozygous or homozygous variants in the OTUD6B gene. However, its molecular and pathophysiological roles in the heart remain poorly understood. Here, we demonstrate that OTUD6B is essential for ventricular chamber development and postnatal survival. OTUD6B is ubiquitously expressed in mouse embryos. Hypomorphic Otud6b neo/neo mice, with a 90% reduction in OTUD6B protein, were viable, fertile, and showed no gross abnormalities up to 24 weeks. In contrast, Otud6b -/- mice, with biallelic loss of OTUD6B, exhibited growth retardation and failed to survive beyond postnatal day 1 (P1). Histological analysis revealed significant cardiac defects in Otud6b -/ - , including ventricular septal defects (VSD) and severe bi-ventricular wall thinning as early as embryonic day 16.5 (E16.5). Echocardiography at E20.5 indicated signs of heart failure. Transcriptomic analysis of E14.5 hearts revealed perturbations in cell cycle regulation, fatty acid metabolism, myogenesis and cardiomyocyte maturation in Otud6b -/- . The severe cardiac phenotype arises from reduced cardiomyocyte proliferation due to defective cell cycle progression, characterized by S-phase arrest and decreased binucleation in OTUD6B-deficient cardiomyocytes. Moreover, homozygous Otud6b knockin mice ( Otud6b KI/KI ) bearing pathogenic variants R145* in Otud6b gene recapitulated the myocardial hypoplasia, ventricular septal defects, and perinatal lethality observed in Otud6b -/- , mirroring cardiac defects seen in patients with R145* mutations. Taken together, our findings establish an essential role for OTUD6B in perinatal ventricular chamber development, likely by fine-tuning cell cycle regulators during heart development.

Endothelial cells not only form a physical barrier but also play key roles in vascular homeostasis by regulating vascular tone, hemostasis, and inflammatory response. Understanding new mechanisms underlying endothelial homeostasis can unlock novel strategies for combating cardiovascular diseases. Ubiquitin-fold modifier 1 (UFM1) is an ubiquitin-like protein that covalently modifies protein substrates via a highly conserved E1 (UBA5)-E2 (UFC1)-E3 (UFL1) enzymatic cascade. However, the molecular mechanisms by which ufmylation regulates endothelial function remain elusive. Interestingly, we observed that the UFL1 locus is significantly associated with coronary artery diseases, which implies the importance of ufmylation in vascular diseases. Here, we hypothesized that ufmylation in endothelial cells is indispensable for vascular development. We generated pan endothelial-specific E3 (UFL1) knockout (UFL1 ECKO ) mice using Cdh5 Cre mice. UFL1 deficient mice showed perinatal lethality at P0. E16.5 UFL1 ECKO embryos showed severe hemorrhage and disrupted blood vessel formation suggesting defects in angiogenesis. To gain mechanistic insights into the function of ufmylation in endothelial cells, we silenced UFL1 and UFM1 in human umbilical vein endothelial cells (HUVECs). We found that silencing UFL1 and UFM1 impaired cell viability, suppressed cell proliferation and endothelial migration, suggesting an indispensable role of ufmylation in vascular development. Bulk-RNA sequencing of UFL1 and UFM1 deficient HUVECs showed that differentially expressed genes (DEGs) were mostly enriched in cell cycle and inflammatory response gene sets. Tamoxifen-inducible UFL1 ECKO mice show decreased cardiac functions. Overall, our results indicate that ufmylation is essential for endothelial cell homeostasis to maintain angiogenesis. 700000-20300-04250000-12200-61100-NHLBI0027 700000-20300-04250000-12100-64076-AHA00235 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

BACKGROUND Disruption of mitochondrial homeostasis drives cardiomyopathy and heart failure, yet upstream regulatory mechanisms remain poorly defined. Neddylation, a reversible post-translational conjugation of the ubiquitin-like protein NEDD8 by E1/E2/E3 enzymes, is essential for cardiac morphogenesis, but its role in the adult heart is unknown. METHODS We assessed the relevance of neddylation to human cardiac disease by gene set enrichment analysis of ischemic (ICM) and non-ischemic cardiomyopathy (NICM) datasets and by immunoblotting and qPCR of ventricular tissue from patients with ICM or dilated cardiomyopathy (DCM). In adult mice, we induced cardiomyocyte-restricted deletion of the NEDD8-activating enzyme 1 (NAE1) by tamoxifen injection and monitored cardiac function at baseline and after transverse aortic constriction (TAC). Bulk RNA-seq 4 weeks post-tamoxifen was combined with bioenergetic, biochemical, and ultrastructural analyses. To assess mitochondrial dynamics, we generated NAE1/MFN2 and NAE1/DRP1 double-knockout mice. Cullin activity, mitochondrial ubiquitination, and mitophagy were measured in hearts and cultured cardiomyocytes. RESULTS Neddylation pathways were dysregulated in human ICM and NICM datasets and in failing ICM/DCM myocardium. Cardiomyocyte-specific NAE1 deletion caused systolic dysfunction and heart failure by 10 weeks post-tamoxifen, culminating in premature death and exacerbating TAC-induced pressure-overload heart failure. At 4 weeks, NAE1 loss repressed metabolic and mitochondrial bioenergetic programs, reduced ATP production, and impaired respiration. Electron microscopy revealed elongated mitochondria and accumulated mitophagic vesicles, with dysregulation of DRP1, MFN2, PINK1, LC3-II, and p62. DRP1/NAE1 co-deletion accelerated systolic failure relative to either single knockout, whereas MFN2/NAE1 co-deletion did not alter early disease progression, implicating pathogenic mitochondrial hyperfusion. Genetic NAE1 depletion in vivo and pharmacologic NAE1 inhibition in vitro impaired mitophagic vesicle formation and flux, inactivated cullin scaffold proteins, reduced mitochondrial ubiquitination, and blunted mitophagic clearance. CONCLUSIONS Cardiac neddylation preserves adult heart function by coordinating mitochondrial fusion-fission dynamics and sustaining cullin-dependent ubiquitination and turnover of damaged mitochondria. These findings identify neddylation as a key regulator of mitochondrial quality control and link its disruption to human cardiomyopathy. Therapeutically, targeting the neddylation-cullin axis may limit mitochondrial dysfunction, enhance mitophagy, and improve energetic reserve in failing hearts, while neddylation signatures in patient myocardium may help guide stratification and precision therapy for cardiomyopathy. Clinical Perspective What Is New? • Demonstrates for the first time that the NEDD8-activating enzyme (NAE1)driven neddylation pathway is indispensable for maintaining mitochondrial quality control in the adult heart. • Links loss of neddylation to mitochondrial hyperfusion, impaired mitophagy, and rapid progression to heart failure. • Reveals that neddylation promotes cullin-RING ligase-mediated ubiquitination of damaged mitochondria, coupling mitochondrial dynamics with turnover. What Are the Clinical Implications? • Restoring or enhancing cardiac neddylation may represent a novel therapeutic avenue for cardiomyopathies characterized by mitochondrial dysfunction. • Pharmacologic agents that bolster DRP1-dependent fission or activate cullin neddylation could potentially normalize mitochondrial dynamics and improve myocardial energetics. • Conversely, systemic neddylation inhibitors now in oncology trials warrant careful cardiac monitoring, as they may precipitate mitochondrial injury and heart failure. • Circulating or tissue markers of neddylation might help stratify patients at heightened risk for mitochondrial-driven cardiac disease and guide precision therapy.

Disrupting the tightly regulated process of cardiac development can lead to congenital heart diseases, yet the role of posttranslational mechanisms remains largely overlooked. Here, we uncover a novel role for Cullin-RING ubiquitin E3 ligases (CRLs) in perinatal cardiac development. In CRLs, RBX1 functions as the catalytic subunit, partnering with Cullin 1-4 proteins and various substrate-recognition receptors to mediate ubiquitination and degradation of intracellular proteins. RBX1 is highly expressed in fetal hearts but downregulated in adults. Surprisingly, deletion of a single Rbx1 allele in mouse hearts via Xmlc2 Cre , which is active from embryonic day (E) 7.5, was sufficient to cause heart failure and perinatal lethality by postnatal day 5. Heterozygous knockout mice exhibited myocardial hypoplasia in both the trabecular and compact layers, discernible at E13.5 and developed ventricular septal defects by E18.5. Similarly, deletion of one Rbx1 allele via cTnT Cre , which is highly active between E7.5 and E10.5, caused postnatal cardiac dysfunction. In contrast, mice with heterozygous Rbx1 deletion via αMHC Cre , which is highly active in cardiomyocytes during midgestation, were viable and exhibited no cardiac abnormalities. However, homozygous deletion via αMHC Cre led to myocardial hypoplasia and biventricular non-compaction by E14.5, ultimately causing embryonic heart failure and lethality by E18.5. These data suggest that precise temporal regulation of RBX1 levels is essential for cardiac morphogenesis. In all three mutant hearts, the observed cardiac developmental abnormalities are attributed to deficits in cardiomyocyte proliferation and maturation. Transcriptomic analysis revealed disruptions in MEF2 transcriptional networks, which are essential for cardiogenesis, among others in RBX1-deficient hearts. Mechanistically, RBX1 deficiency inactivated CRLs, leading to the accumulation of Hippo kinases (LATS1/2, MST1/2, SAV, and MOB1), which inhibited YAP transactivation and proliferation gene expression. We further demonstrate that RBX1 promotes MOB1 ubiquitination and degradation. These findings highlight that RBX1 acts as a dosage-sensitive regulator of early cardiogenesis, ensuring proper cardiomyocyte proliferation, maturation, and ventricular chamber formation through modulation of the Hippo-YAP pathway.

Ufmylation is a novel protein modification by which protein substrates are covalently modified by the ubiquitin-like protein, UFM1 (ubiquitin-fold modifier 1), in an enzymatic cascade that includes highly conserved E1 (UBA5), E2 (UFC1) and E3 (UFL1) enzymes. Through its ability to regulate the function of cellular proteins, ufmylation controls multiple cellular and pathophysiological events and is implicated in various human diseases. We recently reported that ufmylation is dysregulated by cardiomyopathy in both human and mouse diseased hearts. Inhibition of ufmylation by targeting the E3 ligase, UFL1, in the heart (UFL1 CKO ) evokes endoplasmic reticulum (ER) stress, leading to dilated cardiomyopathy and heart failure under physiological development and hemodynamic stress included by transverse aortic constriction (TAC), demonstrating a critical role for ufmylation in maintaining ER and cardiac homeostasis. TAC surgery created sudden pressure overload, giving the heart little time to adapt; in this study, we tested the role of ufmylation in another widely used cardiac hypertrophy model: sustained adrenergic stimulation through isoproterenol (ISO) administration. Temporal echocardiography demonstrated that UFL1 CKO mice failed to develop compensatory hypertrophy, but significant reduction of cardiac function and chamber dilation. Further analysis identified isoproterenol induced maladaptive hypertrophy, increased fibrosis, and cell death in the UFL1 CKO heart. Mechanistically, adrenergic receptor signaling activation induces ufmylation, while UFL1 CKO disrupted ISO-induced adaptive transcriptomic reprogramming. This further supports the protective role of ufmylation in heart failure since the hallmark characteristic of heart failure is the activation of the sympathetic nervous system which leads to sustained β-adrenergic receptors activation.