Bmal1 mediates nucleolin phase separation and prevents sepsis-induced myocardial dysfunction

Bmal1 mediates nucleolin phase separation and prevents sepsis-induced myocardial dysfunction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bmal1 mediates nucleolin phase separation and prevents sepsis-induced myocardial dysfunction Qingfang Han, Huiling Zhang, Yan Xu, Tonggan Lu, Yu Zhang, Xinlan Lv, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8078806/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Sepsis-induced myocardial dysfunction (SIMD) is a life-threatening complication of sepsis with high mortality, however, its underlying molecular mechanisms remain poorly understood. Brain and muscle ARNT-like protein 1 (Bmal1), a core circadian regulator, plays a well-established role in cardiovascular physiology, yet its function in SIMD has not been fully elucidated. Nucleolin (Ncl), a key nucleolar protein critical for ribosome biogenesis, exhibits liquid–liquid phase separation (LLPS) and may mediate cardiomyocyte stress responses. Methods SIMD models were established in mice by cecal ligation and puncture (CLP) and in H9C2 cardiomyocytes using lipopolysaccharide (LPS). Cardiac function was assessed via echocardiography. Molecular interactions were investigated using co-immunoprecipitation (Co-IP), molecular docking, and fluorescence recovery after photobleaching (FRAP). Ribosome biogenesis and nucleolar function were evaluated through AgNOR staining, sucrose gradient centrifugation, and Ribo-Halo assays. Results Bmal1 expression was significantly downregulated in both in vivo and in vitro SIMD models. Bmal1 deficiency exacerbated cardiac dysfunction, amplified inflammatory responses, and disrupted ribosome biogenesis. We identified a direct interaction between Bmal1 and Ncl and demonstrated that Bmal1 regulates Ncl expression and dynamics. Bmal1 silencing impaired Ncl’s LLPS, rRNA synthesis, and ribosome assembly. Furthermore, LPS-induced SIMD disrupted Ncl’s LLPS, while Bmal1 overexpression restored ribosome biogenesis. Conclusions Bmal1 deficiency aggravates SIMD by impairing Ncl’s LLPS and ribosomal biogenesis. Our findings reveal a novel Bmal1–Ncl’s LLPS axis that regulates ribosome biogenesis under septic stress, highlighting its potential as a therapeutic target for SIMD. SIMD LLPS Bmal1 Nucleolin Ribosome biogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Sepsis is a systemic inflammatory response syndrome caused by infection, characterized by acute organ failure. It is one of the most serious diseases, and related deaths account for 19.7% of all deaths in the world [ 1 , 2 ]. Despite advances in antimicrobial treatment, the death of sepsis continues to rise due to the aging population and the increased prevalence of antibiotic resistant pathogens [ 3 ]. Sepsis-induced myocardial dysfunction (SIMD), a major cause of death in septic patients, occurs in up to 70% of cases and is characterized by impaired myocardial contractility and reduced cardiac output [ 4 , 5 ]. Elevated levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), along with decreased left ventricular ejection fraction, have been observed in sepsis [ 4 ]. The pathogenesis of SIMD involves multiple mechanisms. Myocardial ischemia has been implicated in disease progression [ 6 , 7 ], while gastrin/CCKBR signaling in macrophages has been shown to attenuate SIMD by suppressing TLR4/NF-κB–mediated inflammation [ 8 ]. Additionally, malonylation of VDAC2 triggers mitochondrial ferroptosis in septic hearts [ 9 ]. These diverse pathways highlight the complexity of SIMD and the associated challenges in treatment development [ 10 ]. Therefore, there is an urgent need to uncover novel mechanisms of SIMD to guide the development of effective therapies. Brain and muscle ARNT-like protein 1 (Bmal1) is a core circadian clock protein whose loss abolishes circadian rhythms. It heterodimerizes with Clock to form the Bmal1/Clock transcription factor complex, which activates the transcription of clock-controlled genes by binding to E-box elements (CACGTG) in their promoter regions [ 11 ]. This activation is counteracted by repressors such as Period (PER1/2/3) and Cryptochrome (CRY1/2), forming a self-sustaining transcriptional-translational feedback loop that governs the 24-hour circadian cycle [ 11 , 12 ]. Notably, Bmal1 has been shown to exert protective effects in the cardiovascular system. Myocardial infarction (MI) involves cardiomyocyte death as a central cause of cardiac dysfunction, and MI mortality exhibits circadian variation [ 13 ]. Bmal1 deficiency in cardiomyocytes exacerbates MI outcome, while its dysregulation promotes inflammation and increases the burden of cardiovascular diseases (CVD) [ 14 ]. In clinical SIMD studies, Bmal1 expression is significantly downregulated [ 15 ]. Similarly, it is observed that Bmal1 function is markedly inhibited in H9C2 cardiomyocytes, an in vitro model of SIMD induced by lipopolysaccharide (LPS) [ 16 ]. However, the mechanism by which Bmal1 regulates SIMD progression remains unclear. A recent study revealed that Bmal1 deletion impairs pre-rRNA processing and ribosome biogenesis [ 17 ]. The nucleolus, a membraneless nuclear compartment, serves as the primary site for ribosome biogenesis, including rRNA transcription, processing, and ribosome assembly [ 18 ]. Mutations in RNA polymerase I have been shown to disrupt ribosome biogenesis in cardiomyocytes, ultimately contributing to CVD. Nevertheless, the role of ribosome biogenesis in SIMD has not yet been explored. Nucleolin (Ncl) is a major nucleolar protein involved in multiple stages of ribosome biogenesis, including RNA Pol I-mediated transcription and ribosome assembly [ 19 ]. It also supports cell proliferation and exhibits cardioprotective properties [ 20 ]. Previous work indicates that Bmal1 influences Ncl transcription in H9C2 cardiomyocytes. Moreover, co-immunoprecipitation coupled with LC-MS/MS identified a direct interaction between Bmal1 and Ncl [ 21 ], suggesting that Bmal1 may regulate ribosome biogenesis via Ncl. Still, it remains unknown whether the Bmal1–Ncl axis influences SIMD through ribosome biogenesis. Liquid–liquid phase separation (LLPS) is a widespread biophysical process [ 22 ] that drives the formation of membraneless organelles (MLOs) [ 23 ]. Through LLPS, macromolecules condense into liquid-like droplets that concentrate specific biomolecules and facilitate compartmentalized cellular functions [ 11 , 12 ]. Growing evidence indicates that LLPS not only regulates normal physiological processes but may also contribute to organ dysfunction [ 24 ]. Interestingly, Bmal1 can form nuclear condensates that modulate rhythmic gene expression and metabolic activity [ 25 , 26 ], and Ncl also exhibits LLPS potential. However, the functional relevance of Bmal1–Ncl co-condensation via LLPS in SIMD has yet to be elucidated. In this study, we investigate the role and mechanistic basis of Bmal1 in SIMD. Given the predicted LLPS propensity of Ncl, we assess whether Ncl-driven LLPS and ribosome biogenesis are disrupted during SIMD progression, and whether Bmal1 is required for maintaining Ncl’s LLPS. Our findings may offer novel mechanistic insights and identify potential therapeutic strategies for SIMD. Results The role of Bmal1 in SIMD The advantage of the CLP (cecal ligation and puncture) model lies in its ability to inoculate the peritoneum with a mixed microbial community derived from the animal’s own intestinal flora, in conjunction with necrotic tissue. This triggers immune and biochemical responses that closely resemble those seen in human sepsis. At 24 hours after CLP, surviving mice in the CLP group and controls underwent echocardiography, which revealed a significant reduction in both ejection fraction (EF) and fractional shortening (FS) (Fig. 1 A, B). In mice with SIMD, serum levels of creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH) were elevated (Fig. S1A, B), while ANP and BNP levels were also markedly increased (Fig. S1C, D), indicating aggravated myocardial injury. Histological examination at 24 hours post-modeling showed disrupted myocardial fibers, along with inflammatory cell infiltration (Fig. S1E). Taken together, these results confirm the successful establishment of a mouse model of SIMD. To investigate the role of Bmal1 in SIMD beyond circadian regulation, we established mouse models and collected heart tissue samples from both control and CLP mice 24 hours post-modeling. Results showed a significant reduction in both Bmal1 protein expression and mRNA levels in the heart tissues of CLP mice at the 24-hour time point (Fig. 1 C, D). To further explore the mechanism of SIMD, we treated H9C2 cells with 1 µg/mL LPS to establish an in vitro sepsis model. After LPS treatment, Western blot and qRT-PCR analyses revealed a consistent downregulation of Bmal1, mirroring the trend observed in vivo (Fig. 1 E, F). These findings suggest that Bmal1 expression is suppressed following the onset of SIMD, indicating its potential involvement in the pathogenesis of this condition. The impact of Bmal1 deficiency on sepsis In order to investigate the role of Bmal1 in SIMD, we generated cardiomyocyte-specific, tamoxifen-inducible Bmal1 knockout mice (Bmal1-cKO). For this purpose, 6- to 8-week-old male αMHC Cre/WT /Bmal1 Flox/Flox mice were injected intraperitoneally with tamoxifen on days 1 and 3. Subsequent experiments were conducted 10 days later. We established CLP models using both Bmal1-cKO mice and wild-type controls, and performed echocardiography on surviving mice 24 hours after surgery. The results showed that, compared with control CLP mice, Bmal1-cKO CLP mice exhibited reduced ejection fraction (EF) and fractional shortening (FS) (Fig. 2A, B), along with significantly impaired cardiac function. Moreover, other markers of myocardial injury—ANP, BNP, CK-MB, and LDH—were markedly elevated (Fig. 2C–F), indicating that Bmal1 deficiency exacerbates SIMD. In vitro , we transfected H9C2 cells with si-Bmal1 and then induced sepsis using LPS. Upon Bmal1 silencing, both the protein and mRNA levels of the inflammatory factors TNF-α and IL-18 were elevated (Fig. 2G–J), along with increased IL-6 expression in the culture medium (Fig. 2K). LDH release was also significantly enhanced (Fig. 2L), indicating that reduced Bmal1 expression exacerbates SIMD. The role of nucleoli in SIMD The nucleoli are mainly responsible for rRNA synthesis and ribosome assembly, and are also involved in quality control of phase separation proteins and stem cell differentiation [ 27 ]. The functions of nucleoli play an important role in CVD, and there have been no reports of nucleoli involvement in SIMD. To investigate whether the nucleoli are affected in SIMD, we first measured the expression levels of ribosomal proteins and rRNA synthesis levels in SIMD model. The results showed a significant decrease in the expression of ribosomal proteins RPS3 and RPL29 (Fig. 3 A, B), as well as a decrease in rRNA synthesis (Fig. 3 C, D). EU (not Edu) staining is a technique used to monitor the overall transcription level within cells, and can also be used for studying nucleolar function. The results showed that overall transcription levels decreased after LPS induction (Fig. 3 E). Argyrophilic nucleolar organizing regions (Ag NORs) can reflect nucleolar function by regulating rRNA transcription and processing, directly participating in nucleolar function and structural integrity. The number and distribution of Ag NORs also reflect cellular metabolism and proliferation levels. We observed Ag NORs i n vivo through silver staining to evaluate nucleolar function. In the hearts of SIMD mice, we observed a decrease in the number of Ag NORs (Fig. 4 A). Similarly, we found a decrease in the number of Ag NORs induced by LPS in H9C2 cells (Fig. 4 B). Sucrose density gradient centrifugation is used to study the functional status of ribosomes. By analyzing the distribution of ribosomes in different density components, the translation activity of cells and the functional integrity of ribosomes can be evaluated. After LPS induction, ribosome-dimer aggregation occurred and mature 80S ribosomes were significantly reduced (Fig. 4 C). In addition, FBL (Fibrillarin) is a nucleolar protein located in the DFC region of the nucleoli, which can effectively indicate the size and morphology of the nucleoli (Fig. 4 D). We also found significant changes in the size and morphology of the nucleoli after LPS induction. After LPS treatment, the abundance of newly formed RPS3 and RPL29 proteins was significantly inhibited (Fig. 4 E), which is consistent with the results of Western blotting. The above results indicate that the morphology and function of the nucleoli are affected after the occurrence of SIMD. Bmal1 interacts with Ncl Ncl, a key structural component of the nucleolus, is essential for its primary function and structural integrity. To determine whether Bmal1 directly interacts with Ncl, we first performed molecular docking, which suggested potential structural binding between the two proteins (Fig. 5 A). We further validated this protein-protein interaction in H9C2 cells using co-immunoprecipitation (Co-IP) assays (Fig. 5 B). To investigate whether Bmal1 regulates Ncl expression, we examined Ncl levels under Bmal1 modulation. Silencing Bmal1 led to a decrease in both Ncl protein and mRNA levels (Fig. 5 C, D). Consistent with this, Ncl expression was also reduced in Bmal1 cKO mice (Fig. 5 E, F). Conversely, Bmal1 overexpression significantly increased Ncl protein levels (Fig. 5 ). These results demonstrate that Bmal1 not only interacts with Ncl but also positively regulates its expression. Bmal1 is required for the dynamic behavior and organization of Ncl To computationally evaluate the LLPS potential of Ncl, we employed the online predictor PhaseP ( http://predict.phasep.pro/ ), which returned a high score, indicating a strong propensity for phase separation (Fig. 6 A). Intrinsically disordered regions (IDRs)—typically enriched in aromatic and polar residues as well as multivalent interaction motifs—promote LLPS by facilitating weak noncovalent interactions, including hydrophobic, hydrogen bonding, and electrostatic forces [ 28 ]. In silico sequence analysis revealed the presence of IDRs in Ncl (Fig. 6 B), domains often associated with biomolecular condensation. Together, these bioinformatic results suggest that Ncl has a high intrinsic propensity to undergo LLPS, prompting us to further examine its biophysical properties in cellular contexts. To assess Ncl’s LLPS within the nucleolus, we transfected H9C2 cells with an Ncl-EGFP plasmid and performed fluorescence recovery after photobleaching (FRAP). After photobleaching, the fluorescence of Ncl-EGFP recovered to over 70% of the pre-bleach level (Fig. 6 C), reflecting high mobility and dynamic exchange consistent with LLPS in the nucleolus. In contrast, upon Bmal1 silencing, FRAP analysis showed that Ncl-EGFP fluorescence did not recover and was even subject to quenching (Fig. 6 D), indicating that Bmal1 depletion disrupts the nucleolar dynamics of Ncl’s LLPS (Fig. 6 E). Bmal1 affects nucleolar function Ncl, an essential nucleolar protein, plays a critical role in ribosome biogenesis. Previous studies have reported that NCL coexists with proteins such as LIN28 and FBL in a common complex to help maintain nucleolar structural integrity [ 29 , 30 ]. Given our findings that Bmal1 interacts with Ncl and influences its dynamic properties, we further investigated whether Bmal1 regulates ribosome biogenesis via Ncl. At the cellular level, Bmal1 knockdown resulted in reduced rRNA synthesis (Fig. 7 A). Consistent with this, EU staining indicated a declining trend in overall transcription levels (Fig. 7 B). The number of AgNORs also decreased (Fig. 7 C), and Western blot analysis revealed a reduction in ribosomal protein synthesis (Fig. 7 D). Importantly, Bmal1 silencing led to decreased production of newly synthesized RPS3 and RPL29, as well as enhanced degradation of both proteins, as demonstrated by Ribo-Halo assay (Fig. 7 E). The distribution and dynamics of Ncl in SIMD To determine the dynamics of Ncl in SIMD, we transfected LPS-induced SIMD cell model with an Ncl-EGFP plasmid, and performed fluorescence recovery after photobleaching (FRAP). The results showed that Ncl fluorescence failed to recover beyond 50%, even after an extended recovery period, and direct fluorescence quenching was observed in a few cells (Fig. 8 A). Immunofluorescence analysis revealed that LPS treatment induced morphological changes in Ncl similar to those seen after si-Bmal1 treatment, indicating disrupted Ncl distribution under septic stress (Fig. 8 B). These findings suggest that Ncl’s LLPS is markedly altered in SIMD. The parallel effects of Bmal1 deficiency and LPS stress on Ncl dynamics underscore the pathophysiological relevance of this Bmal1-Ncl’s LLPS regulatory axis, with Ncl dysfunction emerging as a common pathological endpoint that promotes nucleolar impairment and contributes to SIMD progression. Discussion In this study, we demonstrate that SIMD leads to decreased Bmal1 expression and impaired ribosome biogenesis in mouse hearts. We further establish that reduced Bmal1 expression exacerbates SIMD pathology by disrupting Ncl’s LLPS and ribosome biogenesis (Fig. 9 ). These results identify the Bmal1–Ncl’s LLPS axis as a novel and therapeutically relevant target for SIMD. Growing clinical evidence underscores the importance of circadian rhythms in sepsis. Data from the UK Biobank indicate that healthy sleep patterns can reduce sepsis risk [ 31 ], while sepsis patients with pre-existing circadian rhythm disorders exhibit significantly higher mortality and heart failure rates [ 2 ]. Notably, the circadian rhythm of Bmal1 expression is blunted in sepsis patients [ 15 ], suggesting that Bmal1 downregulation may contribute to SIMD progression. Supporting this, a recent study showed that LPS-induced ferroptosis in cardiomyocytes follows a diurnal pattern mediated by the Bmal1/AKT/Nrf2 axis, further emphasizing the role of circadian disruption in SIMD [ 32 ]. Consistent with these observations, our study revealed the decreased expression of Bmal1 in vitro SIMD model, and demonstrated that Bmal1 deficiency exacerbates SIMD in our cardiac-specific Bmal1 knockout mice. Our findings suggest that Bmal1 represents a potential therapeutic target for SIMD. The nucleolus, one of the first described membrane-less organelles (MLOs), utilizes LLPS to dynamically regulate cell cycle progression and stress responses [ 33 , 34 ]. Nucleolar dysfunction is increasingly implicated in diverse pathologies, including cancer, neurodegenerative diseases, and aging [ 35 ]. For instance, in neurological disorders, impaired rRNA synthesis triggers nucleolar structural alterations and activates stress response pathways, ultimately leading to apoptosis and neuronal damage [ 36 ]. In cardiovascular contexts, cardiomyocytes from patients with ischemic or dilated cardiomyopathy exhibit damaged nucleolar organizer regions (AgNOR) [ 37 ]. However, the role of nucleoli in SIMD had remained unexplored. Here, we report a significant reduction in AgNORs in septic hearts and provide evidence of impaired ribosome biogenesis in both in vivo and in vitro SIMD models, indicated by increased ribosome collisions. Using the Ribo-Halo system, we further demonstrated that SIMD leads to decreased synthesis and increased degradation of ribosomal proteins. Nucleolin (Ncl) has recognized cardioprotective functions, mitigating cell death and reducing infarct size [ 38 ]. Ncl-deficient zebrafish exhibit severe cardiomyocyte defects, impaired cardiac development, and abnormal ventricular remodeling [ 39 ]. Additionally, Ncl is involved in regulating key inflammatory cytokines such as IL-6 and IL-1β [ 40 ], underscoring its importance in myocardial injury response. In our study, we found that Bmal1 expression is significantly reduced in SIMD and identified a novel interaction between Bmal1 and Ncl through molecular docking and co-immunoprecipitation (Co-IP) experiments. LLPS is fundamental to nucleolar organization and function. Proteins like LIN28A require RNA-binding domains (RBDs) and intrinsically disordered regions (IDRs) for nucleolar LLPS, and aberrant phase separation can disrupt cellular translation and metabolism [ 29 ]. Similarly, the METTL3/METTL14 complex maintains nucleolar LLPS and function by targeting SUV39H1/H2 for degradation, thereby preventing aberrant H3K9me3 accumulation [ 41 ]. Fibrillarin (FBL), a canonical nucleolar marker, relies on LLPS to sustain leukemia cell expansion and stem/progenitor cell self-renewal [ 42 ]. In this study, we report for the first time that Ncl’LLPS is significantly disrupted in an LPS-induced SIMD cell model, demonstrating that SIMD impairs this critical process. Ncl regulates multiple stages of ribosome biogenesis, including rDNA transcription, RNA Polymerase I activity, and ribosome assembly [ 43 , 44 ]. It collaborates with proteins like FBL to ensure proper ribosome assembly [ 45 ] and interacts with signaling molecules to fine-tune the speed and efficiency of ribosome production [ 46 ]. Our study provides the first evidence that the Bmal1/Ncl interaction is essential for maintaining normal ribosome biogenesis. Conclusion In summary, using a cecal ligation and puncture (CLP) mouse model of SIMD, we identified a significant decrease in Bmal1 expression, increased ribosome collisions, and impaired ribosome biogenesis following SIMD onset. The disrupted interaction between Bmal1 and Ncl in septic cardiomyocyte nucleoli exacerbates cardiac dysfunction. We observed marked impairment in the recovery of Ncl’s LLPS in SIMD, which is further aggravated by Bmal1 silencing. Collectively, these findings highlight the Bmal1–Ncl’s LLPS axis as a promising novel target for both mechanistic investigation and therapeutic intervention in SIMD. These results not only reveal a novel Bmal1–Ncl’s LLPS regulatory pathway in cardiac function but also position this axis as a compelling therapeutic target for SIMD. Methods In the present study, all animal and cell samples in this study were collected within a strict 4-hour window between Zeitgeber Time (ZT) 4 and ZT 8, a period widely recognized as corresponding to the peak expression of Bmal1. To ensure data accuracy, sample collection was strictly standardized in timing, thereby minimizing potential variations due to circadian rhythm fluctuations. Generation of SIMD model in mice The C57BL/6J mice used in this study were purchased from Suzhou Inovio New Drug Research Center Co., Ltd. and housed in the Laboratory Animal Center of Soochow University. Cecal ligation and puncture (CLP) was performed on male SPF-grade C57BL/6J mice, aged 8 weeks and weighing 25–30 g. The mice were randomly divided into two groups: a control group and a model group. The model group underwent cecal ligation and puncture, while the control group received only laparotomy and suture treatment[ 47 ]. Construction of the SIMD model in vitro H9C2 cardiomyocytes and 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS at 37°C in a 5% CO 2 atmosphere. H9C2 cells were treated with 1 µg/mL LPS to construct the SIMD model in vitro . A concentration of 1 µg/mL LPS was selected based on previous literature and our own dose-response experiments, which showed that this concentration robustly induced inflammatory responses and cardiomyocyte injury without causing excessive cell death within 24 hours. Evaluation of cardiac function Cardiac function was assessed via echocardiography using a 13 MHz transducer (VisualSonics). The left ventricular ejection fraction (EF) and fraction shorting (FS) were measured to evaluate heart performance. All procedures and analyses were conducted by a researcher blinded to the treatment groups to ensure unbiased results[ 46 ]. EU proliferation assay Cell proliferation was evaluated using EU Cell Proliferation Assay kit (Epizyme, Shanghai, China). Following various treatments, H9C2 cardiomyocytes were incubated in fresh medium containing 10 µM EU for 2 h. Subsequently, H9C2 cells were washed with PBS, fixed with 4% paraformaldehyde for 30 mn, and permeabilized with 0.5% Triton X-100 for 10 min. Cell nuclei were stained with DAPI for 15 minutes. The percentage of EU-positive cells was then determined using fluorescence microscopy. RNA extraction and real-time PCR Total RNA was extracted using the Trizol reagent (TaKaRa Biotech, Japan). cDNA synthesis was performed using the Prime Script™ RT reagent kit (TaKaRa Biotech, Japan). The relative expression levels of target genes were quantified using SYBR® Premix Ex Taq™ (TliRNaseH Plus) (TaKaRa Biotech, Japan) in a real-time PCR assay conducted on the ABI StepOnePlus Real-Time PCR System (Foster City, CA, USA). Western blot Total proteins were extracted using RIPA buffer (Beyotime Biotechnology, China) and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to polyvinylidene fluoride (PVDF) membranes (Millipore ISEQ00010). The membranes were initially incubated with primary antibodies, then with corresponding secondary antibodies. The primary antibodies used were: Nucleolin (14547) and Bmal1 (14020) from Cell Signaling Technology (Danvers, MA, USA); RPL29 (Cat#15799-1-AP) from Proteintech Group (Chicago, IL, USA); GAPDH (Cat#AC002) from ABclonal (Wuhan, China); and RPS3 (Cat#DF3684) from Affinity Biopharma (Shanghai, China). Protein signals were detected using an ECL chemiluminescence kit (Biological Industries) and visualized on a BioRad luminescent imaging system. Molecular docking analysis The docking model between Bmal1 and Ncl was generated using the HDOCK server ( http://hdock.phys.hust.edu.cn/ ) [ 48 ]. Full-length structures of human Bmal1 (UniProt ID: Q9EPW1) and Ncl (UniProt ID: P13383) were retrieved from the UniProt database and submitted for docking. HDOCK employs a hybrid algorithm combining template-based and ab initio docking to rapidly predict interactions. The top-ranked docking model, selected based on the HDOCK score (a composite measure of binding energy), yielded a score of − 242.52. The resulting complex was visualized using PyMOL Molecular Graphics System (Schrödinger, LLC). Co-Immunoprecipitation (Co-IP) Co-immunoprecipitation was performed using the PiereceTM Classic Magnetic IP/Co-IP Kit (Thermofisher, USA) following the manufacture’s protocol. Briefly, cell lysates were incubated with the selected antibody overnight at 4°C. The antigen-antibody complex was then bound to Protein A/G magnetic beads for 1 h at room temperature. The beads were washed twice with IP Lysis/Wash Buffer and once with purified water. Finally, the antigen-antibody complex was eluted and analyzed by western blot. Ribosome-biogenesis AgNOR staining Silver staining of NORs in control cells and Ncl siRNA treated cells was performed using standard AgNOR staining procedures. Briefly, following fixation, H9C2 cardiomyocytes or cardiac tissue sections were stained with freshly prepared AgNOR staining solution for 30 minutes. After staining, the samples were rinsed twice in ddH 2 O and mounted for analysis. Images were captured using bright-field microscopy. Ribo-Halo assay The RPS3-HaloTag7 or RPL29-HaloTag7 donor plasmids, along with their respective gRNAs were designed and constructed by UBIGENE Biotechnology (Guangzhou, China). Ribosomes were labeled according to a previously established protocol[ 49 ]. Briefly, 293T cells were seeded in 24-well plates and transfected with either the RPS3-HaloTag7 donor plasmid and its corresponding gRNA, or the RPS29-HaloTag7 donor plasmid and gRNA, to generate Ribo-Halo cells. To label pre-existing ribosomes, 100 nM TMR-Halo ligand was added and incubated for 1 h. The cells were then washed with 1 ml of fresh DMEM and incubated for 5 min in the dark to remove the remaining excess TMR-ligand. Following this, the cells were transfected with Ncl siRNA, and treated with 50 nM R110 Halo ligand for 24 h to label newly synthesized ribosomes. The proportion of labeled Halo-cells was analyzed using fluorescence microscopy. Ribo-disome assay Ribosome was isolated using a sucrose gradient based on a previously established protocol. Cells were grown to 70–80% confluency and treated with actidione for 10 min. After treatment, cells were washed with PBS and lysed in a lysis buffer. The lysates were then layered onto a 10%-35% sucrose gradients centrifuged at 40,000 rpm, 4°C for 2 h using a Beckmann Coulter ultracentrifuge. Ribosome profiles were assessed by measuring absorbance at 260 nm. Fluorescence recovery after photobleach (FRAP) analysis The Ncl-EGFP plasmid was constructed by Fenghui Biotechnology (Changsha, China). H9C2 cells were transfected with the Ncl-EGFP plasmid. Fluorescence recovery after photobleach experiments in H9C2 cells were performed on confocal microscope with an oil immersion objective. The imaging was performed using a Zeiss LSM 880 confocal microscope equipped with a 63× oil objective lens. A region of interest (ROI) was selected and photobleached using a high-intensity 488 nm pulse. The recovery of fluorescence within the ROI was monitored over time by acquiring sequential images at 10 s intervals. Fluorescence recovery curves were generated by measuring the mean fluorescence intensity within the ROI and were analyzed using ZEISS ZEN 3.8 to determine the rate and extent of recovery. Statistical analysis Data were presented as mean ± SEM. Multiple group comparisons were performed using ANOVA followed by Tukey’s post-hoc test. For comparisons between two groups, two-tailed t-tests were conducted. A p-value < 0.05 was considered statistically significant. The statistical significance levels are defined as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant. Abbreviations AgNOR Argyrophilic nucleolar organizing regions ANP Atrial natriuretic peptide BNP Brain natriuretic peptide Bmal1 Brain and muscle ARNT-like protein 1 cKO Conditional knockout CLP Cecal ligation and puncture Co-IP Co-immunoprecipitation EF Ejection fraction EU 5-Ethynyl uridine FRAP Fluorescence recovery after photobleaching FS Fractional shortening FBL Fibrillarin IDR Intrinsically disordered region LLPS Liquid-liquid phase separation LPS Lipopolysaccharide LDH Lactate dehydrogenase Ncl Nucleolin RPS3 Ribosomal protein S3 RPL29 Ribosomal protein L29 SIMD Sepsis-induced myocardial dysfunction Declarations Funding This work was supported by the National Natural Science Foundation of China (82370264, 82570312, 81870194 and 91849122 to Y Li). Hubei Key Laboratory of Embryonic Stem Cell Research, Hubei University of Medicine (No. 2025ESOF001 to Y Zhang). Project of State Key Laboratory of Radiation Medicine and Protection, Soochow University (No. GZK12023023 to Y Li). A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. This work was supported by the National Key Research and Development Program of China (2022YFE0209700 to X Yu). Author contributions QH, HZ designed the study, analyzed data, and wrote the manuscript. TL, YZ, XL, AL, and ZZ performed experiments and collected data. YX, BL and XY interpreted the data and revised the manuscript. YL conceived and designed the study, analyzed data, and wrote the manuscript. All authors read and approved the final manuscript. Data availability The authors declared that all data supporting the conclusions of this research is available. Ethics approval and consent to participate Our research was approved by Soochow University. Our research has received approval from the ethics committee of the Institute Cardiovascular Science of Soochow University (approval number: 201903A177). 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Wang Y, Tu J, Wu W, Xu Y, Li Y, Pan X, Liu B, Lu T, Han Q, Zhang H et al : The orchestration of cell-cycle reentry and ribosome biogenesis network is critical for cardiac repair . Theranostics 2024, 14 (10):3927-3944. Wang H, Li Y, Jiang S, Liu N, Zhou Q, Li Q, Chen Z, Lin Y, Chen C, Deng Y: LncRNA xist regulates sepsis associated neuroinflammation in the periventricular white matter of CLP rats by miR-122-5p/PKCη Axis . Front Immunol 2023, 14 :1225482. Yan Y, Tao H, He J, Huang SY: The HDOCK server for integrated protein-protein docking . Nat Protoc 2020, 15 (5):1829-1852. An H, Ordureau A, Körner M, Paulo JA, Harper JW: Systematic quantitative analysis of ribosome inventory during nutrient stress . Nature 2020, 583 (7815):303-309. Additional Declarations No competing interests reported. Supplementary Files FigureS1.tif Fig. S1. Construction of SIMD model by CLP in mice A Serum CK-MB content detection in Sham group and CLP group, n=4. B Detection of serum LDH content in Sham group and CLP group, n=4. C The expression of ANP mRNA in the hearts of Sham group and CLP group, n=3. D The expression of BNP mRNA in the hearts of Sham group and CLP group, n=3. E Sham group and CLP group underwent cardiac HE staining, n=4. **p < 0.01, ***p < 0.001, ****p < 0.0001. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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07:39:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2791599,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Bmal1 on cardiac function in SIMD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Cardiac ultrasound was performed to assess cardiac function in Control+CLP and Bmal1-cKO+CLP mice (n=4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e Echocardiographic measurements of ejection fraction (EF) and fractional shortening (FS) in Control+CLP and Bmal1 cKO+CLP groups (n=4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e Serum CK-MB levels in Control+CLP and Bmal1-cKO+CLP groups (n=4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e Serum LDH levels in Control+CLP and Bmal1-cKO+CLP groups (n=4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE \u003c/strong\u003eBNP mRNA expression in cardiac tissues of 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(n=4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eL\u003c/strong\u003e LDH levels in the supernatant of si-NC and si-Bmal1 groups after LPS treatment (n=4). ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8078806/v1/e1154f3adbb0ba8cb21f8e72.png"},{"id":97222216,"identity":"d87b23e9-196a-410f-8e16-ae7d7c8b24d4","added_by":"auto","created_at":"2025-12-02 07:39:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4779264,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges of nucleolar function in the SIMD model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Protein expression levels of RPS3 and RPL29 in the Sham and CLP groups (n=3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB \u003c/strong\u003eProtein expression levels of RPS3 and RPL29 in the Control and LPS groups (n=3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC \u003c/strong\u003erRNA expression levels in the Sham and CLP groups (n=3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e rRNA expression levels in the Control and LPS groups (n=3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE\u003c/strong\u003e EU staining in the Control and LPS groups (n=4).\u003c/p\u003e\n\u003cp\u003e*p \u0026lt; 0.05, **p \u0026lt; 0.01, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8078806/v1/dbeac9022d5b642423926a7f.png"},{"id":97222201,"identity":"b9888421-0805-4ef9-9d38-03a563c8ece6","added_by":"auto","created_at":"2025-12-02 07:39:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":10695179,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlterations in nucleolar function in the SIMD 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07:39:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2468522,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteraction between Bmal1 and Ncl in H9C2 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Predicted protein-protein docking model and interaction sites between Bmal1 and Ncl.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e Co‑immunoprecipitation (Co‑IP) analysis of Bmal1 and Ncl interaction (n = 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e Ncl protein expression in si‑NC and si‑Bmal1 groups (n=4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e Ncl mRNA expression in Control and Bmal1 cKO groups (n=3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE\u003c/strong\u003e Ncl mRNA expression in si‑NC and si‑Bmal1 groups (n=3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF\u003c/strong\u003e Ncl 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Bmal1 interacts with Ncl to modulate nucleolar activity, and its deficiency results in impaired rRNA synthesis, increased ribosomal collisions, and disrupted ribosome biogenesis. These impairments further exacerbate the pathological progression of SIMD.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-8078806/v1/384ad03ccd37457004834cec.png"},{"id":100610201,"identity":"40b7c1cb-6afd-4699-bcef-aeb2c2202a79","added_by":"auto","created_at":"2026-01-19 16:31:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":44091771,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8078806/v1/094db243-8795-487e-822e-a383e24828e4.pdf"},{"id":97222186,"identity":"352ff5df-8cae-48ad-871a-8c2a1b777c52","added_by":"auto","created_at":"2025-12-02 07:39:17","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16512772,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1. Construction of SIMD model by CLP in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Serum CK-MB content detection in Sham group and CLP group, n=4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e Detection of serum LDH content in Sham group and CLP group, n=4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e The expression of ANP mRNA in the hearts of Sham group and CLP group, n=3. \u003cstrong\u003eD\u003c/strong\u003e The expression of BNP mRNA in the hearts of Sham group and CLP group, n=3. \u003cstrong\u003eE\u003c/strong\u003e Sham group and CLP group underwent cardiac HE staining, n=4. **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8078806/v1/a48304680104fc64ec579822.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bmal1 mediates nucleolin phase separation and prevents sepsis-induced myocardial dysfunction","fulltext":[{"header":"Background","content":"\u003cp\u003eSepsis is a systemic inflammatory response syndrome caused by infection, characterized by acute organ failure. It is one of the most serious diseases, and related deaths account for 19.7% of all deaths in the world [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite advances in antimicrobial treatment, the death of sepsis continues to rise due to the aging population and the increased prevalence of antibiotic resistant pathogens [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSepsis-induced myocardial dysfunction (SIMD), a major cause of death in septic patients, occurs in up to 70% of cases and is characterized by impaired myocardial contractility and reduced cardiac output [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Elevated levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), along with decreased left ventricular ejection fraction, have been observed in sepsis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The pathogenesis of SIMD involves multiple mechanisms. Myocardial ischemia has been implicated in disease progression [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], while gastrin/CCKBR signaling in macrophages has been shown to attenuate SIMD by suppressing TLR4/NF-κB\u0026ndash;mediated inflammation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Additionally, malonylation of VDAC2 triggers mitochondrial ferroptosis in septic hearts [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These diverse pathways highlight the complexity of SIMD and the associated challenges in treatment development [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, there is an urgent need to uncover novel mechanisms of SIMD to guide the development of effective therapies.\u003c/p\u003e\u003cp\u003eBrain and muscle ARNT-like protein 1 (Bmal1) is a core circadian clock protein whose loss abolishes circadian rhythms. It heterodimerizes with Clock to form the Bmal1/Clock transcription factor complex, which activates the transcription of clock-controlled genes by binding to E-box elements (CACGTG) in their promoter regions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This activation is counteracted by repressors such as Period (PER1/2/3) and Cryptochrome (CRY1/2), forming a self-sustaining transcriptional-translational feedback loop that governs the 24-hour circadian cycle [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNotably, Bmal1 has been shown to exert protective effects in the cardiovascular system. Myocardial infarction (MI) involves cardiomyocyte death as a central cause of cardiac dysfunction, and MI mortality exhibits circadian variation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Bmal1 deficiency in cardiomyocytes exacerbates MI outcome, while its dysregulation promotes inflammation and increases the burden of cardiovascular diseases (CVD) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In clinical SIMD studies, Bmal1 expression is significantly downregulated [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Similarly, it is observed that Bmal1 function is markedly inhibited in H9C2 cardiomyocytes, an \u003cem\u003ein vitro\u003c/em\u003e model of SIMD induced by lipopolysaccharide (LPS) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, the mechanism by which Bmal1 regulates SIMD progression remains unclear.\u003c/p\u003e\u003cp\u003eA recent study revealed that Bmal1 deletion impairs pre-rRNA processing and ribosome biogenesis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The nucleolus, a membraneless nuclear compartment, serves as the primary site for ribosome biogenesis, including rRNA transcription, processing, and ribosome assembly [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Mutations in RNA polymerase I have been shown to disrupt ribosome biogenesis in cardiomyocytes, ultimately contributing to CVD. Nevertheless, the role of ribosome biogenesis in SIMD has not yet been explored.\u003c/p\u003e\u003cp\u003eNucleolin (Ncl) is a major nucleolar protein involved in multiple stages of ribosome biogenesis, including RNA Pol I-mediated transcription and ribosome assembly [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. It also supports cell proliferation and exhibits cardioprotective properties [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Previous work indicates that Bmal1 influences Ncl transcription in H9C2 cardiomyocytes. Moreover, co-immunoprecipitation coupled with LC-MS/MS identified a direct interaction between Bmal1 and Ncl [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], suggesting that Bmal1 may regulate ribosome biogenesis \u003cem\u003evia\u003c/em\u003e Ncl. Still, it remains unknown whether the Bmal1\u0026ndash;Ncl axis influences SIMD through ribosome biogenesis.\u003c/p\u003e\u003cp\u003eLiquid\u0026ndash;liquid phase separation (LLPS) is a widespread biophysical process [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] that drives the formation of membraneless organelles (MLOs) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Through LLPS, macromolecules condense into liquid-like droplets that concentrate specific biomolecules and facilitate compartmentalized cellular functions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Growing evidence indicates that LLPS not only regulates normal physiological processes but may also contribute to organ dysfunction [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Interestingly, Bmal1 can form nuclear condensates that modulate rhythmic gene expression and metabolic activity [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and Ncl also exhibits LLPS potential. However, the functional relevance of Bmal1\u0026ndash;Ncl co-condensation \u003cem\u003evia\u003c/em\u003e LLPS in SIMD has yet to be elucidated.\u003c/p\u003e\u003cp\u003eIn this study, we investigate the role and mechanistic basis of Bmal1 in SIMD. Given the predicted LLPS propensity of Ncl, we assess whether Ncl-driven LLPS and ribosome biogenesis are disrupted during SIMD progression, and whether Bmal1 is required for maintaining Ncl\u0026rsquo;s LLPS. Our findings may offer novel mechanistic insights and identify potential therapeutic strategies for SIMD.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eThe role of Bmal1 in SIMD\u003c/h2\u003e\u003cp\u003eThe advantage of the CLP (cecal ligation and puncture) model lies in its ability to inoculate the peritoneum with a mixed microbial community derived from the animal\u0026rsquo;s own intestinal flora, in conjunction with necrotic tissue. This triggers immune and biochemical responses that closely resemble those seen in human sepsis. At 24 hours after CLP, surviving mice in the CLP group and controls underwent echocardiography, which revealed a significant reduction in both ejection fraction (EF) and fractional shortening (FS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). In mice with SIMD, serum levels of creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH) were elevated (Fig. S1A, B), while ANP and BNP levels were also markedly increased (Fig. S1C, D), indicating aggravated myocardial injury. Histological examination at 24 hours post-modeling showed disrupted myocardial fibers, along with inflammatory cell infiltration (Fig. S1E). Taken together, these results confirm the successful establishment of a mouse model of SIMD.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo investigate the role of Bmal1 in SIMD beyond circadian regulation, we established mouse models and collected heart tissue samples from both control and CLP mice 24 hours post-modeling. Results showed a significant reduction in both Bmal1 protein expression and mRNA levels in the heart tissues of CLP mice at the 24-hour time point (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). To further explore the mechanism of SIMD, we treated H9C2 cells with 1 \u0026micro;g/mL LPS to establish an in vitro sepsis model. After LPS treatment, Western blot and qRT-PCR analyses revealed a consistent downregulation of Bmal1, mirroring the trend observed in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, F). These findings suggest that Bmal1 expression is suppressed following the onset of SIMD, indicating its potential involvement in the pathogenesis of this condition.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eThe impact of Bmal1 deficiency on sepsis\u003c/h3\u003e\n\u003cp\u003eIn order to investigate the role of Bmal1 in SIMD, we generated cardiomyocyte-specific, tamoxifen-inducible Bmal1 knockout mice (Bmal1-cKO).\u003c/p\u003e\n\u003cp\u003eFor this purpose, 6- to 8-week-old male αMHC\u003csup\u003eCre/WT\u003c/sup\u003e/Bmal1\u003csup\u003eFlox/Flox\u003c/sup\u003e mice were injected intraperitoneally with tamoxifen on days 1 and 3. Subsequent experiments were conducted 10 days later. We established CLP models using both Bmal1-cKO mice and wild-type controls, and performed echocardiography on surviving mice 24 hours after surgery. The results showed that, compared with control CLP mice, Bmal1-cKO CLP mice exhibited reduced ejection fraction (EF) and fractional shortening (FS) (Fig. 2A, B), along with significantly impaired cardiac function. Moreover, other markers of myocardial injury—ANP, BNP, CK-MB, and LDH—were markedly elevated (Fig. 2C–F), indicating that Bmal1 deficiency exacerbates SIMD.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e, we transfected H9C2 cells with si-Bmal1 and then induced sepsis using LPS. Upon Bmal1 silencing, both the protein and mRNA levels of the inflammatory factors TNF-α and IL-18 were elevated (Fig. 2G–J), along with increased IL-6 expression in the culture medium (Fig. 2K). LDH release was also significantly enhanced (Fig. 2L), indicating that reduced Bmal1 expression exacerbates SIMD.\u003c/p\u003e\n\u003ch3\u003eThe role of nucleoli in SIMD\u003c/h3\u003e\n\u003cp\u003eThe nucleoli are mainly responsible for rRNA synthesis and ribosome assembly, and are also involved in quality control of phase separation proteins and stem cell differentiation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The functions of nucleoli play an important role in CVD, and there have been no reports of nucleoli involvement in SIMD. To investigate whether the nucleoli are affected in SIMD, we first measured the expression levels of ribosomal proteins and rRNA synthesis levels in SIMD model. The results showed a significant decrease in the expression of ribosomal proteins RPS3 and RPL29 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B), as well as a decrease in rRNA synthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). EU (not Edu) staining is a technique used to monitor the overall transcription level within cells, and can also be used for studying nucleolar function. The results showed that overall transcription levels decreased after LPS induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eArgyrophilic nucleolar organizing regions (Ag NORs) can reflect nucleolar function by regulating rRNA transcription and processing, directly participating in nucleolar function and structural integrity. The number and distribution of Ag NORs also reflect cellular metabolism and proliferation levels. We observed Ag NORs i\u003cem\u003en vivo\u003c/em\u003e through silver staining to evaluate nucleolar function. In the hearts of SIMD mice, we observed a decrease in the number of Ag NORs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Similarly, we found a decrease in the number of Ag NORs induced by LPS in H9C2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Sucrose density gradient centrifugation is used to study the functional status of ribosomes. By analyzing the distribution of ribosomes in different density components, the translation activity of cells and the functional integrity of ribosomes can be evaluated. After LPS induction, ribosome-dimer aggregation occurred and mature 80S ribosomes were significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In addition, FBL (Fibrillarin) is a nucleolar protein located in the DFC region of the nucleoli, which can effectively indicate the size and morphology of the nucleoli (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). We also found significant changes in the size and morphology of the nucleoli after LPS induction. After LPS treatment, the abundance of newly formed RPS3 and RPL29 proteins was significantly inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), which is consistent with the results of Western blotting. The above results indicate that the morphology and function of the nucleoli are affected after the occurrence of SIMD.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eBmal1 interacts with Ncl\u003c/h3\u003e\n\u003cp\u003eNcl, a key structural component of the nucleolus, is essential for its primary function and structural integrity. To determine whether Bmal1 directly interacts with Ncl, we first performed molecular docking, which suggested potential structural binding between the two proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We further validated this protein-protein interaction in H9C2 cells using co-immunoprecipitation (Co-IP) assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo investigate whether Bmal1 regulates Ncl expression, we examined Ncl levels under Bmal1 modulation. Silencing Bmal1 led to a decrease in both Ncl protein and mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). Consistent with this, Ncl expression was also reduced in Bmal1 cKO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F). Conversely, Bmal1 overexpression significantly increased Ncl protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These results demonstrate that Bmal1 not only interacts with Ncl but also positively regulates its expression.\u003c/p\u003e\n\u003ch3\u003eBmal1 is required for the dynamic behavior and organization of Ncl\u003c/h3\u003e\n\u003cp\u003eTo computationally evaluate the LLPS potential of Ncl, we employed the online predictor PhaseP (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://predict.phasep.pro/\u003c/span\u003e\u003cspan address=\"http://predict.phasep.pro/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which returned a high score, indicating a strong propensity for phase separation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Intrinsically disordered regions (IDRs)\u0026mdash;typically enriched in aromatic and polar residues as well as multivalent interaction motifs\u0026mdash;promote LLPS by facilitating weak noncovalent interactions, including hydrophobic, hydrogen bonding, and electrostatic forces [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In silico sequence analysis revealed the presence of IDRs in Ncl (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), domains often associated with biomolecular condensation. Together, these bioinformatic results suggest that Ncl has a high intrinsic propensity to undergo LLPS, prompting us to further examine its biophysical properties in cellular contexts.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo assess Ncl\u0026rsquo;s LLPS within the nucleolus, we transfected H9C2 cells with an Ncl-EGFP plasmid and performed fluorescence recovery after photobleaching (FRAP). After photobleaching, the fluorescence of Ncl-EGFP recovered to over 70% of the pre-bleach level (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), reflecting high mobility and dynamic exchange consistent with LLPS in the nucleolus. In contrast, upon Bmal1 silencing, FRAP analysis showed that Ncl-EGFP fluorescence did not recover and was even subject to quenching (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), indicating that Bmal1 depletion disrupts the nucleolar dynamics of Ncl\u0026rsquo;s LLPS (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eBmal1 affects nucleolar function\u003c/h2\u003e\u003cp\u003eNcl, an essential nucleolar protein, plays a critical role in ribosome biogenesis. Previous studies have reported that NCL coexists with proteins such as LIN28 and FBL in a common complex to help maintain nucleolar structural integrity [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Given our findings that Bmal1 interacts with Ncl and influences its dynamic properties, we further investigated whether Bmal1 regulates ribosome biogenesis \u003cem\u003evia\u003c/em\u003e Ncl. At the cellular level, Bmal1 knockdown resulted in reduced rRNA synthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Consistent with this, EU staining indicated a declining trend in overall transcription levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The number of AgNORs also decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), and Western blot analysis revealed a reduction in ribosomal protein synthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Importantly, Bmal1 silencing led to decreased production of newly synthesized RPS3 and RPL29, as well as enhanced degradation of both proteins, as demonstrated by Ribo-Halo assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eThe distribution and dynamics of Ncl in SIMD\u003c/h3\u003e\n\u003cp\u003eTo determine the dynamics of Ncl in SIMD, we transfected LPS-induced SIMD cell model with an Ncl-EGFP plasmid, and performed fluorescence recovery after photobleaching (FRAP). The results showed that Ncl fluorescence failed to recover beyond 50%, even after an extended recovery period, and direct fluorescence quenching was observed in a few cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eImmunofluorescence analysis revealed that LPS treatment induced morphological changes in Ncl similar to those seen after si-Bmal1 treatment, indicating disrupted Ncl distribution under septic stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). These findings suggest that Ncl\u0026rsquo;s LLPS is markedly altered in SIMD. The parallel effects of Bmal1 deficiency and LPS stress on Ncl dynamics underscore the pathophysiological relevance of this Bmal1-Ncl\u0026rsquo;s LLPS regulatory axis, with Ncl dysfunction emerging as a common pathological endpoint that promotes nucleolar impairment and contributes to SIMD progression.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrate that SIMD leads to decreased Bmal1 expression and impaired ribosome biogenesis in mouse hearts. We further establish that reduced Bmal1 expression exacerbates SIMD pathology by disrupting Ncl\u0026rsquo;s LLPS and ribosome biogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e). These results identify the Bmal1\u0026ndash;Ncl\u0026rsquo;s LLPS axis as a novel and therapeutically relevant target for SIMD.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGrowing clinical evidence underscores the importance of circadian rhythms in sepsis. Data from the UK Biobank indicate that healthy sleep patterns can reduce sepsis risk [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], while sepsis patients with pre-existing circadian rhythm disorders exhibit significantly higher mortality and heart failure rates [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Notably, the circadian rhythm of Bmal1 expression is blunted in sepsis patients [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], suggesting that Bmal1 downregulation may contribute to SIMD progression. Supporting this, a recent study showed that LPS-induced ferroptosis in cardiomyocytes follows a diurnal pattern mediated by the Bmal1/AKT/Nrf2 axis, further emphasizing the role of circadian disruption in SIMD [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Consistent with these observations, our study revealed the decreased expression of Bmal1 \u003cem\u003ein vitro\u003c/em\u003e SIMD model, and demonstrated that Bmal1 deficiency exacerbates SIMD in our cardiac-specific Bmal1 knockout mice. Our findings suggest that Bmal1 represents a potential therapeutic target for SIMD.\u003c/p\u003e\u003cp\u003eThe nucleolus, one of the first described membrane-less organelles (MLOs), utilizes LLPS to dynamically regulate cell cycle progression and stress responses [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Nucleolar dysfunction is increasingly implicated in diverse pathologies, including cancer, neurodegenerative diseases, and aging [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. For instance, in neurological disorders, impaired rRNA synthesis triggers nucleolar structural alterations and activates stress response pathways, ultimately leading to apoptosis and neuronal damage [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In cardiovascular contexts, cardiomyocytes from patients with ischemic or dilated cardiomyopathy exhibit damaged nucleolar organizer regions (AgNOR) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, the role of nucleoli in SIMD had remained unexplored. Here, we report a significant reduction in AgNORs in septic hearts and provide evidence of impaired ribosome biogenesis in both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e SIMD models, indicated by increased ribosome collisions. Using the Ribo-Halo system, we further demonstrated that SIMD leads to decreased synthesis and increased degradation of ribosomal proteins.\u003c/p\u003e\u003cp\u003eNucleolin (Ncl) has recognized cardioprotective functions, mitigating cell death and reducing infarct size [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Ncl-deficient zebrafish exhibit severe cardiomyocyte defects, impaired cardiac development, and abnormal ventricular remodeling [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Additionally, Ncl is involved in regulating key inflammatory cytokines such as IL-6 and IL-1β [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], underscoring its importance in myocardial injury response. In our study, we found that Bmal1 expression is significantly reduced in SIMD and identified a novel interaction between Bmal1 and Ncl through molecular docking and co-immunoprecipitation (Co-IP) experiments.\u003c/p\u003e\u003cp\u003eLLPS is fundamental to nucleolar organization and function. Proteins like LIN28A require RNA-binding domains (RBDs) and intrinsically disordered regions (IDRs) for nucleolar LLPS, and aberrant phase separation can disrupt cellular translation and metabolism [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Similarly, the METTL3/METTL14 complex maintains nucleolar LLPS and function by targeting SUV39H1/H2 for degradation, thereby preventing aberrant H3K9me3 accumulation [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Fibrillarin (FBL), a canonical nucleolar marker, relies on LLPS to sustain leukemia cell expansion and stem/progenitor cell self-renewal [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In this study, we report for the first time that Ncl\u0026rsquo;LLPS is significantly disrupted in an LPS-induced SIMD cell model, demonstrating that SIMD impairs this critical process.\u003c/p\u003e\u003cp\u003eNcl regulates multiple stages of ribosome biogenesis, including rDNA transcription, RNA Polymerase I activity, and ribosome assembly [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. It collaborates with proteins like FBL to ensure proper ribosome assembly [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] and interacts with signaling molecules to fine-tune the speed and efficiency of ribosome production [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Our study provides the first evidence that the Bmal1/Ncl interaction is essential for maintaining normal ribosome biogenesis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, using a cecal ligation and puncture (CLP) mouse model of SIMD, we identified a significant decrease in Bmal1 expression, increased ribosome collisions, and impaired ribosome biogenesis following SIMD onset. The disrupted interaction between Bmal1 and Ncl in septic cardiomyocyte nucleoli exacerbates cardiac dysfunction. We observed marked impairment in the recovery of Ncl’s LLPS in SIMD, which is further aggravated by Bmal1 silencing. Collectively, these findings highlight the Bmal1–Ncl’s LLPS axis as a promising novel target for both mechanistic investigation and therapeutic intervention in SIMD. These results not only reveal a novel Bmal1–Ncl’s LLPS regulatory pathway in cardiac function but also position this axis as a compelling therapeutic target for SIMD.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eIn the present study, all animal and cell samples in this study were collected within a strict 4-hour window between Zeitgeber Time (ZT) 4 and ZT 8, a period widely recognized as corresponding to the peak expression of Bmal1. To ensure data accuracy, sample collection was strictly standardized in timing, thereby minimizing potential variations due to circadian rhythm fluctuations.\u003c/p\u003e\u003ch2\u003eGeneration of SIMD model in mice\u003c/h2\u003e\u003cp\u003eThe C57BL/6J mice used in this study were purchased from Suzhou Inovio New Drug Research Center Co., Ltd. and housed in the Laboratory Animal Center of Soochow University. Cecal ligation and puncture (CLP) was performed on male SPF-grade C57BL/6J mice, aged 8 weeks and weighing 25–30 g. The mice were randomly divided into two groups: a control group and a model group. The model group underwent cecal ligation and puncture, while the control group received only laparotomy and suture treatment[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eConstruction of the SIMD model\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eH9C2 cardiomyocytes and 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS at 37°C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. H9C2 cells were treated with 1 µg/mL LPS to construct the SIMD model \u003cem\u003ein vitro\u003c/em\u003e. A concentration of 1 µg/mL LPS was selected based on previous literature and our own dose-response experiments, which showed that this concentration robustly induced inflammatory responses and cardiomyocyte injury without causing excessive cell death within 24 hours.\u003c/p\u003e\u003ch2\u003eEvaluation of cardiac function\u003c/h2\u003e\u003cp\u003eCardiac function was assessed \u003cem\u003evia\u003c/em\u003e echocardiography using a 13 MHz transducer (VisualSonics). The left ventricular ejection fraction (EF) and fraction shorting (FS) were measured to evaluate heart performance. All procedures and analyses were conducted by a researcher blinded to the treatment groups to ensure unbiased results[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003eEU proliferation assay\u003c/h2\u003e\u003cp\u003eCell proliferation was evaluated using EU Cell Proliferation Assay kit (Epizyme, Shanghai, China). Following various treatments, H9C2 cardiomyocytes were incubated in fresh medium containing 10 µM EU for 2 h. Subsequently, H9C2 cells were washed with PBS, fixed with 4% paraformaldehyde for 30 mn, and permeabilized with 0.5% Triton X-100 for 10 min. Cell nuclei were stained with DAPI for 15 minutes. The percentage of EU-positive cells was then determined using fluorescence microscopy.\u003c/p\u003e\u003ch2\u003eRNA extraction and real-time PCR\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted using the Trizol reagent (TaKaRa Biotech, Japan). cDNA synthesis was performed using the Prime Script™ RT reagent kit (TaKaRa Biotech, Japan). The relative expression levels of target genes were quantified using SYBR® Premix Ex Taq™ (TliRNaseH Plus) (TaKaRa Biotech, Japan) in a real-time PCR assay conducted on the ABI StepOnePlus Real-Time PCR System (Foster City, CA, USA).\u003c/p\u003e\u003ch2\u003eWestern blot\u003c/h2\u003e\u003cp\u003eTotal proteins were extracted using RIPA buffer (Beyotime Biotechnology, China) and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to polyvinylidene fluoride (PVDF) membranes (Millipore ISEQ00010). The membranes were initially incubated with primary antibodies, then with corresponding secondary antibodies. The primary antibodies used were: Nucleolin (14547) and Bmal1 (14020) from Cell Signaling Technology (Danvers, MA, USA); RPL29 (Cat#15799-1-AP) from Proteintech Group (Chicago, IL, USA); GAPDH (Cat#AC002) from ABclonal (Wuhan, China); and RPS3 (Cat#DF3684) from Affinity Biopharma (Shanghai, China). Protein signals were detected using an ECL chemiluminescence kit (Biological Industries) and visualized on a BioRad luminescent imaging system.\u003c/p\u003e\u003ch2\u003eMolecular docking analysis\u003c/h2\u003e\u003cp\u003eThe docking model between Bmal1 and Ncl was generated using the HDOCK server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://hdock.phys.hust.edu.cn/\u003c/span\u003e\u003cspan address=\"http://hdock.phys.hust.edu.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Full-length structures of human Bmal1 (UniProt ID: Q9EPW1) and Ncl (UniProt ID: P13383) were retrieved from the UniProt database and submitted for docking. HDOCK employs a hybrid algorithm combining template-based and ab initio docking to rapidly predict interactions. The top-ranked docking model, selected based on the HDOCK score (a composite measure of binding energy), yielded a score of − 242.52. The resulting complex was visualized using PyMOL Molecular Graphics System (Schrödinger, LLC).\u003c/p\u003e\u003ch2\u003eCo-Immunoprecipitation (Co-IP)\u003c/h2\u003e\u003cp\u003eCo-immunoprecipitation was performed using the PiereceTM Classic Magnetic IP/Co-IP Kit (Thermofisher, USA) following the manufacture’s protocol. Briefly, cell lysates were incubated with the selected antibody overnight at 4°C. The antigen-antibody complex was then bound to Protein A/G magnetic beads for 1 h at room temperature. The beads were washed twice with IP Lysis/Wash Buffer and once with purified water. Finally, the antigen-antibody complex was eluted and analyzed by western blot.\u003c/p\u003e\u003ch2\u003eRibosome-biogenesis AgNOR staining\u003c/h2\u003e\u003cp\u003eSilver staining of NORs in control cells and Ncl siRNA treated cells was performed using standard AgNOR staining procedures. Briefly, following fixation, H9C2 cardiomyocytes or cardiac tissue sections were stained with freshly prepared AgNOR staining solution for 30 minutes. After staining, the samples were rinsed twice in ddH\u003csub\u003e2\u003c/sub\u003eO and mounted for analysis. Images were captured using bright-field microscopy.\u003c/p\u003e\u003ch2\u003eRibo-Halo assay\u003c/h2\u003e\u003cp\u003eThe RPS3-HaloTag7 or RPL29-HaloTag7 donor plasmids, along with their respective gRNAs were designed and constructed by UBIGENE Biotechnology (Guangzhou, China). Ribosomes were labeled according to a previously established protocol[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Briefly, 293T cells were seeded in 24-well plates and transfected with either the RPS3-HaloTag7 donor plasmid and its corresponding gRNA, or the RPS29-HaloTag7 donor plasmid and gRNA, to generate Ribo-Halo cells. To label pre-existing ribosomes, 100 nM TMR-Halo ligand was added and incubated for 1 h. The cells were then washed with 1 ml of fresh DMEM and incubated for 5 min in the dark to remove the remaining excess TMR-ligand. Following this, the cells were transfected with Ncl siRNA, and treated with 50 nM R110 Halo ligand for 24 h to label newly synthesized ribosomes. The proportion of labeled Halo-cells was analyzed using fluorescence microscopy.\u003c/p\u003e\u003ch2\u003eRibo-disome assay\u003c/h2\u003e\u003cp\u003eRibosome was isolated using a sucrose gradient based on a previously established protocol. Cells were grown to 70–80% confluency and treated with actidione for 10 min. After treatment, cells were washed with PBS and lysed in a lysis buffer. The lysates were then layered onto a 10%-35% sucrose gradients centrifuged at 40,000 rpm, 4°C for 2 h using a Beckmann Coulter ultracentrifuge. Ribosome profiles were assessed by measuring absorbance at 260 nm.\u003c/p\u003e\u003ch2\u003eFluorescence recovery after photobleach (FRAP) analysis\u003c/h2\u003e\u003cp\u003eThe Ncl-EGFP plasmid was constructed by Fenghui Biotechnology (Changsha, China). H9C2 cells were transfected with the Ncl-EGFP plasmid. Fluorescence recovery after photobleach experiments in H9C2 cells were performed on confocal microscope with an oil immersion objective. The imaging was performed using a Zeiss LSM 880 confocal microscope equipped with a 63× oil objective lens. A region of interest (ROI) was selected and photobleached using a high-intensity 488 nm pulse. The recovery of fluorescence within the ROI was monitored over time by acquiring sequential images at 10 s intervals. Fluorescence recovery curves were generated by measuring the mean fluorescence intensity within the ROI and were analyzed using ZEISS ZEN 3.8 to determine the rate and extent of recovery.\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData were presented as mean ± SEM. Multiple group comparisons were performed using ANOVA followed by Tukey’s post-hoc test. For comparisons between two groups, two-tailed t-tests were conducted. A p-value \u0026lt; 0.05 was considered statistically significant. The statistical significance levels are defined as follows: *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, ns: not significant.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAgNOR Argyrophilic nucleolar organizing regions\u003c/p\u003e\n\u003cp\u003eANP Atrial natriuretic peptide\u003c/p\u003e\n\u003cp\u003eBNP Brain natriuretic peptide\u003c/p\u003e\n\u003cp\u003eBmal1 Brain and muscle ARNT-like protein 1\u003c/p\u003e\n\u003cp\u003ecKO Conditional knockout\u003c/p\u003e\n\u003cp\u003eCLP Cecal ligation and puncture\u003c/p\u003e\n\u003cp\u003eCo-IP Co-immunoprecipitation\u003c/p\u003e\n\u003cp\u003eEF Ejection fraction\u003c/p\u003e\n\u003cp\u003eEU 5-Ethynyl uridine\u003c/p\u003e\n\u003cp\u003eFRAP Fluorescence recovery after photobleaching\u003c/p\u003e\n\u003cp\u003eFS Fractional shortening\u003c/p\u003e\n\u003cp\u003eFBL Fibrillarin\u003c/p\u003e\n\u003cp\u003eIDR Intrinsically disordered region\u003c/p\u003e\n\u003cp\u003eLLPS Liquid-liquid phase separation\u003c/p\u003e\n\u003cp\u003eLPS Lipopolysaccharide\u003c/p\u003e\n\u003cp\u003eLDH Lactate dehydrogenase\u003c/p\u003e\n\u003cp\u003eNcl Nucleolin\u003c/p\u003e\n\u003cp\u003eRPS3 Ribosomal protein S3\u003c/p\u003e\n\u003cp\u003eRPL29 Ribosomal protein L29\u003c/p\u003e\n\u003cp\u003eSIMD Sepsis-induced myocardial dysfunction\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (82370264, 82570312,\u0026nbsp;81870194 and 91849122 to Y Li). Hubei Key Laboratory of Embryonic Stem Cell Research, Hubei University of Medicine (No. 2025ESOF001 to Y Zhang). Project of State Key Laboratory of Radiation Medicine and Protection, Soochow University (No. GZK12023023 to Y Li). A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. This work was supported by the National Key Research and Development Program of China (2022YFE0209700 to X Yu).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQH, HZ designed the study, analyzed data, and wrote the manuscript. TL, YZ, XL, AL, and ZZ performed experiments and collected data. YX, BL and XY interpreted the data and revised the manuscript. YL conceived and designed the study, analyzed data, and wrote the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared that all data supporting the conclusions of this research is available.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur research was approved by Soochow University. Our research has received approval from the ethics committee of the Institute Cardiovascular Science of Soochow University (approval number: 201903A177).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have reviewed and approved the final version of the manuscript\u003c/p\u003e\n\u003cp\u003efor publication.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo conflict of interest was declared by the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRudd KE, Johnson SC, Agesa KM, Shackelford KA, Tsoi D, Kievlan DR, Colombara DV, Ikuta KS, Kissoon N, Finfer S\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eGlobal, regional, and national sepsis incidence and mortality, 1990-2017: analysis for the Global Burden of Disease 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\u003cstrong\u003e583\u003c/strong\u003e(7815):303-309.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"SIMD, LLPS, Bmal1, Nucleolin, Ribosome biogenesis","lastPublishedDoi":"10.21203/rs.3.rs-8078806/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8078806/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eSepsis-induced myocardial dysfunction (SIMD) is a life-threatening complication of sepsis with high mortality, however, its underlying molecular mechanisms remain poorly understood. Brain and muscle ARNT-like protein 1 (Bmal1), a core circadian regulator, plays a well-established role in cardiovascular physiology, yet its function in SIMD has not been fully elucidated. Nucleolin (Ncl), a key nucleolar protein critical for ribosome biogenesis, exhibits liquid\u0026ndash;liquid phase separation (LLPS) and may mediate cardiomyocyte stress responses.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eSIMD models were established in mice by cecal ligation and puncture (CLP) and in H9C2 cardiomyocytes using lipopolysaccharide (LPS). Cardiac function was assessed \u003cem\u003evia\u003c/em\u003e echocardiography. Molecular interactions were investigated using co-immunoprecipitation (Co-IP), molecular docking, and fluorescence recovery after photobleaching (FRAP). Ribosome biogenesis and nucleolar function were evaluated through AgNOR staining, sucrose gradient centrifugation, and Ribo-Halo assays.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eBmal1 expression was significantly downregulated in both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e SIMD models. Bmal1 deficiency exacerbated cardiac dysfunction, amplified inflammatory responses, and disrupted ribosome biogenesis. We identified a direct interaction between Bmal1 and Ncl and demonstrated that Bmal1 regulates Ncl expression and dynamics. Bmal1 silencing impaired Ncl\u0026rsquo;s LLPS, rRNA synthesis, and ribosome assembly. Furthermore, LPS-induced SIMD disrupted Ncl\u0026rsquo;s LLPS, while Bmal1 overexpression restored ribosome biogenesis.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eBmal1 deficiency aggravates SIMD by impairing Ncl\u0026rsquo;s LLPS and ribosomal biogenesis. Our findings reveal a novel Bmal1\u0026ndash;Ncl\u0026rsquo;s LLPS axis that regulates ribosome biogenesis under septic stress, highlighting its potential as a therapeutic target for SIMD.\u003c/p\u003e","manuscriptTitle":"Bmal1 mediates nucleolin phase separation and prevents sepsis-induced myocardial dysfunction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-02 07:38:57","doi":"10.21203/rs.3.rs-8078806/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b61788b2-e633-411f-96af-2642d37a485a","owner":[],"postedDate":"December 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T15:29:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-02 07:38:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8078806","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8078806","identity":"rs-8078806","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}