Piezo1 Knockdown Activates PI3K/AKT and Enhances SPP1 to Drive M2 Macrophage Polarization and Reduce Cardiac Inflammation

Piezo1 Knockdown Activates PI3K/AKT and Enhances SPP1 to Drive M2 Macrophage Polarization and Reduce Cardiac Inflammation | 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 Article Piezo1 Knockdown Activates PI3K/AKT and Enhances SPP1 to Drive M2 Macrophage Polarization and Reduce Cardiac Inflammation Yunhan Zhang, Ying Zhang, Jiaoyan Song, Ziwen Zhao, Wenhao Ju, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6737272/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Piezo1 plays a key role in the immune response during sepsis. To date, our understanding of the role of Piezo1 in inflammatory diseases has mostly been limited to influencing vasomotor function and regulating inflammatory infiltration. Whether and how Piezo1 in macrophages is involved in developing septic cardiac dysfunction has never been explored. Here, we have successfully established a mouse model with macrophage-specific knockdown of the Piezo1. The intraperitoneal injection of lipopolysaccharide (LPS) resulted in a significant increase in cardiac macrophage infiltration, as well as an increase in the expression of inflammatory factors and the inflammatory response. However, macrophage-specific knockdown of Piezo1 impaired this response, leading to an increment in macrophage polarization towards the M2 type and the decreased inflammatory response. As a result, myocardial injury caused by sepsis was attenuated. We have also demonstrated that the PI3K/AKT pathway is significantly activated after Piezo1 knockdown, resulting in reduced myocardial dysfunction. Our data indicate that macrophage-specific knockdown of Piezo1 can influence macrophage polarization and thus exert cardioprotective effects in a murine model of sepsis, providing potential ideas and targets for the treatment of infectious cardiac dysfunction. Biological sciences/Genetics Health sciences/Cardiology Piezo1 Macrophage Sepsis-induced Cardiomyopathy Cardiac Dysfunction PI3K/AKT pathway Figures Figure 1 Figure 2 Figure 3 Introduction Sepsis is a systemic inflammatory response characterized by excessive production of inflammatory cytokines, oxidative stress, and multiple organ dysfunction 1 . One severe sequel of this multiorgan impairment is sepsis-induced cardiomyopathy (SCM), characterized by myocardial depression and ensuing high mortality due to cardiac dysfunction. This condition arises from an overwhelming secretion of inflammatory mediators and chemokines during the septic response, inflicting damage upon myocardial cells 2 . Macrophages are well-known for their pivotal roles in preserving homeostasis, defending against pathogens, and repairing tissue damage 3 . They are capable of differentiating into various functional phenotypes in different environments and tissues. These phenotypes include the classical activation phenotype (M1 type), which has a pro-inflammatory effect, and the alternative activation phenotype (M2 type), which has a repair effect 4 , 5 . Furthermore, endotoxin, a key component of the outer membrane of gram-negative bacteria and a principal instigator of sepsis, provokes macrophage activation and drives polarization toward the M1 phenotype. This polarization leads to an overproduction of inflammatory cytokines like interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin 1β (IL-1β), thereby fueling an inflammatory cascade that exacerbates damage to cardiomyocytes 6 . Thus, inhibiting macrophage activation is crucial in reducing myocardial injury during sepsis. Piezo1 is a mechanically activated ion channel with a high affinity for calcium 3 . It is evolutionarily conserved and involved in the development, differentiation, and growth of multiple tissues. It responds to diverse mechanistic stimuli across numerous cell types 7 , with a distinctive significance in the regulation of inflammatory infiltration subjected to hemodynamic stress. Activation of the PI3K/AKT phosphorylation pathway is manifested by upregulation of the expression of pathway-associated proteins and upregulation of the phosphorylation level of proteins. Activation of the pathway promotes macrophage polarization to the M2 type, with an increase in the proportion of macrophages of the M2 type and enhanced anti-inflammatory effects 8 . Recent studies have revealed that macrophages demonstrate significant expression of Piezo1, which is pivotal to their involvement in mediating inflammatory responses 9 – 11 . In this study, we employed a classical sepsis model induced by lipopolysaccharide (LPS) to investigate the role of Piezo1 in regulating macrophage responses during sepsis 12 . Our research findings suggest that macrophage polarization relies on the function of Piezo1. Following the knockdown of Piezo1, the application of LPS resulted in an enhanced phosphorylation of the PI3K/AKT pathway, concomitant with an upregulation in the expression of SPP1. This shift correlated with an increased polarization of macrophages towards the M2 phenotype, leading to a reduction in myocardial injury. We showed that macrophages lacking Piezo1 holistically reduce inflammation responses. This suggests the regulatory role of Piezo1 and the possibility of Piezo1 as a potential therapeutic target in Sepsis-induced cardiomyopathy. Materials and methods Animals The mice needed for the experiment were purchased from Shanghai Model Organisms Center, Inc. Wild-type, Piezo1 flox/flox and Piezo1 flox/flox Lyz2-Cre C57BL/6J mice, all male and aged eight weeks, were maintained in specific pathogen-free environments and sustained on a standard mouse chow diet. The impact and conclusions regarding gender differences in SCM have not yet gained widespread recognition. Consequently, the use of male mice remains the predominant choice. All animal experiments were done by the Guide for the Care and Use of Laboratory Animals published by the Ministry of the People’s Republic of China (1998) and approved by the Institutional Committee on Animal Care of Capital Medical University. The animal research was sanctioned by the Institutional Animal Care and Use Committee (IACUC), under the approval number A5095F17-EA0D-4377-88F7-FED2CE3A2ED2. All methods were performed in accordance with the relevant guidelines and regulations, and follow the ARRIVE guidelines. Genotype is characterized by PCR, and the primer sequences are in Table S1 . Echocardiography Using a 30 MHz probe small animal ultrasonic biological microscope (Vevo 3100, FUJIFILM Visual Sonics Inc, Toronto, Canada). Under isoflurane anesthesia, the long-axis section shows the maximum section of the left ventricle. Switch to M-mode ultrasound to measure left ventricular structure and systolic function, covering at least 25 cardiac cycles to ensure sufficient data for accurate analysis. Western blot analysis BMDMs were exposed to a lysis buffer, a combination of RIPA lysis buffer and a 1% protease inhibitor. The lysate was spun at 14000 rpm for 15 min, and the supernatant was obtained. The proteins were denatured through the use of LDS sample buffer and sample reducing agent at 70°C for 10 min before each sample was loaded into a well of a 4–12% Bis-Tris gel (all from Invitrogen). The membranes were blocked by using 5% skim milk for 1 h. After washing, the membranes were probed with a primary antibody at 4 ℃ overnight. Then the membranes were washed and probed with secondary antibodies at room temperature for 1 h. The membrane was then washed in TBST, immersed in a western HRP substrate solution, and then imaged. The antibodies are in Table S2, Uncropped blots are provided in the Source Data file. Peritoneal Macrophage Isolation Inject mice intraperitoneally with 3% thioglycollate broth (1 mL) once daily for three days. Collect macrophages: a) Euthanize mice via cervical dislocation post-anesthesia and sanitize with 75% ethanol for 3–5 min. b) In a biosafety cabinet, make an abdominal incision, expose the peritoneum, and inject 5 mL chilled PBS. Massage gently and rest for 3–5 min. c) Aspirate peritoneal fluid for centrifugation. Purify and culture: Spin at 1000 rpm for 5 min, resuspend pellet in RPMI-1640, and incubate at 37°C for 2–3 hours to allow adhesion. Wash to remove non-adherent cells, obtaining purified macrophages. BMDM Isolation Harvesting Bones: a) Euthanize the mouse post-anesthesia via cervical dislocation, and sterilize in 75% ethanol for 5 minutes. Remove and clean femurs and tibiae, preserving bone integrity. b) Sterilize bones in 75% ethanol for 5 minutes, then rinse in cold PBS. Marrow Extraction and Macrophage Induction: a) Cut ends of bones, flush out marrow using a syringe, and break up clumps with a pipette. b) Filter cells through a 70µm strainer, centrifuge at 1500 rpm for 5 minutes, and remove supernatant. c) Lyse red blood cells, let stand for 5 minutes, wash with cold PBS, centrifuge, and discard supernatant. d) Resuspend cells in chilled macrophage medium, and seed onto culture plates. *During culture, replace BMDM medium every 2–3 days post-washing with PBS. Harvest after 7-day culture. Flow Cytometry The cells were then resuspended in an EP tube and centrifuged at 500g for 10 minutes to discard the supernatant. Next, the cells were resuspended with CD16/32, incubated on ice for 10 minutes. Then the cells were washed. Add F4/80 and CD86 antibodies with a fixative solution and incubate for 30 minutes. After washing, add the CD206 antibody in a membrane-breaking solution and incubate for another 30 minutes. Finally, resuspend the cells in PBS. Subsequently, analyze the sample using the BD FACSVerse™ flow cytometer. For data analysis, employ FlowJo v10.10 software with gating strategies as depicted in Fig. S3. Quantification and Statistical Analysis All data are representative of at least three independent experiments. All statistical analyses were performed using Prism9.5.1 (GraphPad). The data are presented as the mean ± SD as indicated in the legends. Survival data were analyzed by the Kaplan–Meier statistical method. A p-value < 0.05 was considered statistically significant. Results Cardiac function decreased in mice with sepsis after LPS stimulation, and this impairment was ameliorated after the knockdown of Piezo1 in macrophages To investigate the impact of LPS on cardiac function, we divided male C57BL/6J mice, wild-type and eight-weeks-age, into 4 groups and administered LPS via intraperitoneal injection at progressive doses of 0 mg/kg (n = 16), 10 mg/kg (n = 16), 12 mg/kg (n = 16), and 15 mg/kg (n = 17). These doses corresponded to survival rates of 100%, 75%, 56.25%, and 17.65% (Fig. 1 B). Opting for a dose of 12 mg/kg LPS for our sepsis model, we were able to maintain mouse survival rates at 50%-70%. Before LPS injection, baseline cardiac assessments were performed using echocardiography. A follow-up echocardiographic evaluation was conducted 6–8 hours post-LPS administration. Cardiac function was quantitatively assessed by recording and analyzing M-mode echocardiograms (Fig. 1 A). Our findings indicate that intraperitoneal administration of LPS precipitates myocardial damage. Echocardiographic analysis revealed substantial degradation in cardiac function, with marked left ventricular systolic dysfunction observed notably in the LPS-treated mice (Fig. S1 ), and Piezo1 levels were notably increased through Western blot analysis in the LPS-treated mice (Fig. S2). This was characterized by decreased myocardial contraction amplitudes and a pronounced augmentation in myocyte edema in the LPS-treated mice (Fig. 1 C). One study has discovered that specific knockout of Piezo1 in macrophages can protect the mouse liver and decelerate the progression of fibrosis 13 . To investigate the role of Piezo1 in sepsis-induced cardiomyopathy, we generated macrophage-specific Piezo1 knockdown C57BL/6J mice (Fig. 1 D). Piezo1 f/f and Piezo1 f/f Lyz2-Cre (Piezo1-cKO) mice were used, and we first harvested peritoneal macrophages. Through Western blot analysis, we confirmed the effective deletion of Piezo1, ultimately demonstrating a knockdown efficiency of 74% (Fig. 1 E and F). Then, we constructed a model of sepsis in eight-week-old male Piezo1 f/f (n = 41) and Piezo1-cKO (n = 32) mice. The sepsis yielded a survival rate of approximately 65.63% in Piezo1-cKO mice, but only approximately 56.10% of Piezo1 f/f mice ( p = 0.0911) (Fig. 1 G). Before administering LPS, mice with Piezo1 knockdown did not exhibit mortality, and there were no significant differences in body weight or cardiac function between Piezo1 f/f and Piezo1-cKO groups. However, following LPS treatment, both Piezo1 f/f and Piezo1-cKO groups exhibited a significant reduction in body weight, LVEF, and FS (p < 0.0001; p = 0.0001; p < 0.0001, respectively). Notably, the Piezo1 f/f group displayed worse cardiac function, with LVEF and FS being significantly lower compared to the Piezo1-cKO group (Fig. 1 H and I). Additionally, the Piezo1 f/f group presented with an increased cardiac mass, as evidenced by an elevated heart weight-to-tibia length ratio (p = 0.0286) (Fig. 1 J). Serological assays also showed a greater increase in cTnI levels in the Piezo1 f/f group (p = 0.0022) (Fig. 1 K). Our results indicate that targeted knockdown of Piezo1 in macrophages may impart a cardioprotective advantage in septic mice, potentially mitigating mortality rates associated with sepsis-induced cardiomyopathy. Piezo1 knockdown alleviates myocardial cell damage via induction of M2 macrophage polarization First, the hematoxylin-eosin (HE) staining revealed disorganized myocardial fibers, partial fiber rupture, and degeneration, with striations becoming blurred or vanishing (vacuolization), interstitial edema, and inflammatory cell infiltration following LPS treatment (Fig. 2 A). Furthermore, the transmission electron microscopy revealed disorganized myocardial fibers, myofibrillar separation, twisting of the Z lines, shortened distances between Z lines, disorder of mitochondria within myocardial cell, swelling, diminished mitochondrial cristae, and necrotic changes including partial vacuolization (Fig. 2 B). But these damages were significantly mitigated in the Piezo1-cKO group compared to the Piezo1 f/f group. Then we isolated bone marrow-derived macrophages for culture, and under the microscope, there was no evident differentiation before LPS treatment. After 36 hours of exposure to LPS, the differentiation ratio was around 60%, with no significant difference between the Piezo1 f/f and Piezo1-cKO groups (p = 0.7807). Based on the morphology of the macrophages, M1 and M2 phenotypes were distinguished: the Piezo1-cKO group exhibited a higher proportion of M2 macrophages among the differentiated cells (32.91% vs. 17.87%, p = 0.0002) (Fig. 2 C). This was further substantiated by flow cytometry, which identified macrophages with the F4/80 marker and M2 macrophages with the CD206 marker, with the Piezo1-cKO group showing a higher ratio of M2 macrophages among differentiated cells (43.7% vs. 28.1%) (Fig. 2 D). To ascertain if the abundance of M2 macrophages within myocardial tissue paralleled our in vitro observations, we performed fluorescence staining with CD68 and CD206. The Piezo1-cKO group exhibited a stronger CD206 expression (p = 0.0486) with cellular morphology more representative of the archetypal M2 macrophage phenotype (Fig. 2 E). We contend that silencing Piezo1 in macrophages induces a skewing toward an M2 anti-inflammatory phenotype, concomitantly diminishing myocardial inflammation and alleviating cardiomyocyte injury, thus safeguarding cardiac functionality. Macrophage Piezo1 Knockdown Activates the PI3K/AKT Pathway and Promotes M2 Polarization To investigate the mechanism by which Piezo1 regulates macrophage polarization, we conducted transcriptomic sequencing. It was found that compared to the control group, macrophages with Piezo1 knockdown showed significant upregulation in 511 genes and downregulation in 74 genes (p < 0.05). The differential gene expression clustering heat map was consistent with these changes (Fig. 3 A and B). Notably, as a factor that can drive macrophage polarization toward the M2 phenotype 14 – 16 , the SPP1 gene was also significantly upregulated (p = 5.3E-07). Further, KEGG enrichment analysis indicated a significant accumulation of genes related to the PI3K/AKT signaling pathway (Fig. 3 C) 17 – 19 . To validate the sequencing analysis results, we isolated and cultured bone marrow-derived macrophages from mice and performed Western blot experiments. Initially, it was observed that SPP1 levels were notably increased in the Piezo1 knockdown group (Fig. 3 D and E). Within the PI3K/AKT pathway, despite AKT levels being roughly constant, phosphorylated AKT (P-AKT) and downstream signaling molecule p-S6 were significantly elevated (Fig. 3 E). This implies that in mice with Piezo1 knockdown, the PI3K/AKT pathway was significantly upregulated following LPS treatment. Our research has indicated that both the upregulation of SPP1 and the PI3K/AKT pathway can promote macrophage polarization towards the M2 phenotype, playing an anti-inflammatory role. We also verified changes in cardiac macrophage-derived inflammatory cytokines; in the hearts of Piezo1 knockdown mice, pro-inflammatory cytokines IL-1β and IL-6, primarily derived from M1 macrophages, were markedly decreased, whereas the anti-inflammatory cytokines IL-10, predominantly from M2 macrophages, was increased (Fig. 3 F). These changes reflect that the knockdown of Piezo1 activates the PI3K/AKT pathway, promotes the expression of SPP1, and causes macrophage polarization towards the M2 phenotype. Consequently, the anti-inflammatory protective capacity of macrophages is enhanced, inflammation levels are reduced, and cardiac function is protected (Fig. 3 G). Discussion To examine the effects of macrophage-specific knockdown of Piezo1 on sepsis-induced cardiac dysfunction following LPS infection, our study employed a classical sepsis model induced by LPS, which ensured a mortality rate of 50–70% in mice. We successfully generated macrophage-specific Piezo1 knockdown mice to utilize within this framework. In these genetically modified mice, loss of Piezo1 conferred a markedly improved physiological state characterized by lower mortality rates, reduced weight loss, less cardiac edema, better cardiac function, and enhanced myocardial contractility after LPS infection. Histopathological findings further suggested that the diminishment of macrophage infiltration following Piezo1 deletion is likely attributable to Piezo1's regulatory role in macrophage proliferation and infiltration 20 , 21 . Additionally, upregulation of Piezo1 may lead to increased ROS production and cellular apoptosis 22 . Consequently, it can be inferred that the functional alterations in macrophages due to Piezo1 knockdown exerted a protective effect in mice. The postulate has been substantiated by HE staining and electron microscopy, which confirmed an improved condition of the myocardium after Piezo1 deletion. Previous research has categorized macrophages into two subsets, M1 and M2 23 , with M2 being associated with more pronounced anti-inflammatory effects 24 , 25 . We proceeded to assess macrophage polarization within our study. Initially, we conducted conventional morphological observations and flow cytometric analyses of bone marrow-derived macrophages, which revealed an increase in the M2 macrophage population. Subsequently, immunofluorescence staining for macrophage subsets within the cardiac tissue demonstrated a significantly higher expression of CD206, an M2 marker. Based on these findings, we can tentatively conclude that Piezo1 may play a role in promoting the differentiation of macrophages towards the M2 phenotype. To dissect the underlying mechanisms, we performed transcriptomic analysis on six mice and observed significant differences in gene expression following Piezo1 knockdown. SPP1, originally characterized as a pro-inflammatory cytokine secreted by T cells, is a multifunctional glycoprotein that is also expressed in an array of tissue-resident macrophages. This molecule plays a pivotal role in the phagocytic clearance of apoptotic cells, the chemotactic response, and the directed migration of macrophages 26 . A study highlights that SPP1 fosters M2 macrophage polarization through its interaction with αvβ3 integrin and CD44 receptors 16 . Notably, SPP1 and the PI3K/AKT pathway have been implicated in macrophage polarization in prior studies 27 . Some researchers have reported that a reduction in SPP1 correlates with a decrease in factors associated with M2 macrophage polarization, and others have considered SPP1 as a key target for macrophage phenotype identification and a prognostic factor for cancer outcomes 14 , 28 . Upon assessing protein expression changes, we discerned that Piezo1 knockdown resulted in upregulated expression of SPP1, accompanied by enhanced activation of the PI3K/AKT signaling pathway, both of which promote differentiation towards the M2 macrophage phenotype. The interplay between SPP1 and the PI3K/AKT pathway has been recognized in previous studies. One study in prostate cancer revealed that high SPP1 expression maintained PI3K/AKT pathway activation 29 , while another study on bone fracture healing found that activation of the PI3K/AKT pathway stimulated M2 macrophage polarization 8 . Although we have verified that Piezo1 knockdown could cause the polarization of macrophages towards M2, this may also relate to the role of Piezo1 in stiffness sensing and subsequent regulation of macrophage polarization 30 . Moreover, Piezo1 knockdown appears to reduce the phagocytic activity of macrophages, which could be another consequence of the shift toward the M2 phenotype. We cannot exclude the possibility that this phagocytic reduction might inhibit polarization towards the M1 phenotype, contributing to an increased proportion of the M2 phenotype. To further elucidate the intricate mechanistic details, it may be necessary to conduct single-cell sequencing to analyze different cell types and to perform a more comprehensive dissection of the underlying processes. Previous studies have authenticated the regulatory role of Piezo1 in modulating macrophage function and influencing the levels of inflammation 9 , 31 , 32 . Our study corroborated these findings by observing changes in myocardial inflammation, which align with the anti-inflammatory M2 macrophage phenotype. Specifically, the pro-inflammatory cytokines IL-1β and IL-6 were significantly diminished, whereas the anti-inflammatory cytokine IL-10 was elevated 12 . This pattern of cytokine expression indicates that myocardial cells were shielded from inflammatory damage, thereby exhibiting improved cardiac function. Our investigation indicates that Piezo1 knockdown in macrophages activates the PI3K/AKT pathway and drives the expression of SPP1, which in turn polarizes macrophages towards the M2 phenotype. Within myocardial tissues, these macrophage alterations not only lower inflammation levels but also boost their anti-inflammatory capacity, thereby conferring cardioprotection and preserving cardiac function in mice, suggesting Piezo1 might as a potential therapeutic target in the clinic intervention. Abbreviations LPS lipopolysaccharide SCM sepsis-induced cardiomyopathy IL-6 inflammatory cytokines like interleukin 6 TNF-α tumor necrosis factor-alpha IL-1β interleukin 1β Piezo1-cKO Piezo1 f/f Lyz2-Cre HE hematoxylin-eosin P-AKT phosphorylated AKT Declarations Funding sources This research was supported by National Natural Science Foundation of China (82300301) and Beijing Natural Science Foundation (7232077) and the Basic Research Project of Yunnan Provincial Science and Technology Department (202401AY070001-020). Authors' contributions YHZ, YZ, YJT and JYS: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Conceptualization. ZWZ, WHJ and HZ: Investigation, Formal analysis. HLL, YBM and ZWN: Writing – review & editing. QZG, SL, JQP and TYS: Validation. YZW, BHP, MH, CYL, ZLL and DLZ: Writing – review & editing, Validation, Supervision, Resources, Project administration, Funding acquisition, Formal analysis, Conceptualization. Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Source data are provided in this paper. Ethical Approval and Consent to participate This study has been approved by the Institutional Committee on Animal Care of Capital Medical University (Protocol number A5095F17-EA0D-4377-88F7-FED2CE3A2ED2). Consent for publication Not applicable. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors are thankful for the experimental platform provided by the State Key Laboratory of Cardiovascular Disease, China & Fuwai Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College. References Jia, L. et al. 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Supplementary Files 20250703SUPPLEMENTALMATERIAL.docx Cite Share Download PDF Status: Published Journal Publication published 08 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 13 Aug, 2025 Reviews received at journal 23 Jul, 2025 Reviews received at journal 20 Jul, 2025 Reviewers agreed at journal 10 Jul, 2025 Reviewers agreed at journal 10 Jul, 2025 Reviewers agreed at journal 10 Jul, 2025 Reviewers invited by journal 10 Jul, 2025 Submission checks completed at journal 09 Jul, 2025 First submitted to journal 28 Jun, 2025 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6737272","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":483465896,"identity":"70c91e2a-88bb-4c41-a24a-fc0a42aee6b4","order_by":0,"name":"Yunhan Zhang","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences, Affiliated Cardiovascular Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yunhan","middleName":"","lastName":"Zhang","suffix":""},{"id":483465897,"identity":"5a416c0c-d599-4105-a80a-b33585c002c2","order_by":1,"name":"Ying Zhang","email":"","orcid":"","institution":"Chinese PLA General Hospital, National Clinical Research Center for Geriatric diseases","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Zhang","suffix":""},{"id":483465898,"identity":"7c9c89a3-c07a-4da9-99f3-012824273c88","order_by":2,"name":"Jiaoyan Song","email":"","orcid":"","institution":"Peking Union Medical College Hospital (PUMCH), Chinese Academy of Medical Sciences (CAMS)","correspondingAuthor":false,"prefix":"","firstName":"Jiaoyan","middleName":"","lastName":"Song","suffix":""},{"id":483465900,"identity":"ea06fc19-b41e-4856-94c2-a92f5b67cce4","order_by":3,"name":"Ziwen Zhao","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences, Affiliated Cardiovascular Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ziwen","middleName":"","lastName":"Zhao","suffix":""},{"id":483465901,"identity":"5f79c0bd-7cf8-418b-8777-00f69b5f4c65","order_by":4,"name":"Wenhao Ju","email":"","orcid":"","institution":"Peking Union Medical College Hospital (PUMCH), Chinese Academy of Medical Sciences (CAMS)","correspondingAuthor":false,"prefix":"","firstName":"Wenhao","middleName":"","lastName":"Ju","suffix":""},{"id":483465902,"identity":"278efebe-34bc-4a56-9764-1635a55d53af","order_by":5,"name":"Hao Zhang","email":"","orcid":"","institution":"Peking Union Medical College Hospital (PUMCH), Chinese Academy of Medical Sciences (CAMS)","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Zhang","suffix":""},{"id":483465903,"identity":"63ed684c-b7d2-4f48-ae82-d1c6cea94ee1","order_by":6,"name":"Shuang Li","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences, Affiliated Cardiovascular Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shuang","middleName":"","lastName":"Li","suffix":""},{"id":483465906,"identity":"9da0089c-a7a6-4d96-bf4f-7413415503bf","order_by":7,"name":"Hanlu Li","email":"","orcid":"","institution":"Peking Union Medical College Hospital (PUMCH), Chinese Academy of Medical Sciences (CAMS)","correspondingAuthor":false,"prefix":"","firstName":"Hanlu","middleName":"","lastName":"Li","suffix":""},{"id":483465908,"identity":"54c3b12b-8f5f-415a-a370-9d4975f070e0","order_by":8,"name":"Qiuzhe Guo","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences, Affiliated Cardiovascular Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qiuzhe","middleName":"","lastName":"Guo","suffix":""},{"id":483465910,"identity":"037e46da-4eb9-4ab7-b111-ecef27ad0c5a","order_by":9,"name":"Yinbo Ma","email":"","orcid":"","institution":"Peking Union Medical College Hospital (PUMCH), Chinese Academy of Medical Sciences (CAMS)","correspondingAuthor":false,"prefix":"","firstName":"Yinbo","middleName":"","lastName":"Ma","suffix":""},{"id":483465912,"identity":"27eda1a7-5e2f-4feb-aa57-b9c16d935a3b","order_by":10,"name":"Zhangwei Nong","email":"","orcid":"","institution":"Peking Union Medical College Hospital (PUMCH), Chinese Academy of Medical Sciences (CAMS)","correspondingAuthor":false,"prefix":"","firstName":"Zhangwei","middleName":"","lastName":"Nong","suffix":""},{"id":483465913,"identity":"42c258c4-8478-4ac3-b7ba-4ed19459202e","order_by":11,"name":"Tianyuan Shen","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences, Affiliated Cardiovascular Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tianyuan","middleName":"","lastName":"Shen","suffix":""},{"id":483465914,"identity":"ca35e29e-65a6-4bdb-9b5d-e09a80841554","order_by":12,"name":"Yuanzheng Wang","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences, Affiliated Cardiovascular Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuanzheng","middleName":"","lastName":"Wang","suffix":""},{"id":483465915,"identity":"4784f10b-1867-4032-96a5-4cb945a965f9","order_by":13,"name":"Boheng Pang","email":"","orcid":"","institution":"Peking Union Medical College Hospital (PUMCH), Chinese Academy of Medical Sciences (CAMS)","correspondingAuthor":false,"prefix":"","firstName":"Boheng","middleName":"","lastName":"Pang","suffix":""},{"id":483465916,"identity":"581cce93-b6de-44ac-9bc6-5d6372325f21","order_by":14,"name":"Min Hao","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences, Affiliated Cardiovascular Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Hao","suffix":""},{"id":483465917,"identity":"cf6ab125-f7eb-4311-839c-0dcc39f73fb9","order_by":15,"name":"Chunyuan Luo","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences, Affiliated Cardiovascular Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chunyuan","middleName":"","lastName":"Luo","suffix":""},{"id":483465918,"identity":"1d9f682d-63c9-4b5d-bb7f-6b6f3bb9885c","order_by":16,"name":"Zhiling Luo","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences, Affiliated Cardiovascular Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhiling","middleName":"","lastName":"Luo","suffix":""},{"id":483465919,"identity":"13c03cb2-3ed5-4d72-9e71-b82453df4fb2","order_by":17,"name":"Yinjiang Tang","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences, Affiliated Cardiovascular Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yinjiang","middleName":"","lastName":"Tang","suffix":""},{"id":483465920,"identity":"9857bee2-cd6a-4bc0-aa2b-858a5ca34697","order_by":18,"name":"Donglin Zhuang","email":"","orcid":"","institution":"Peking Union Medical College Hospital (PUMCH), Chinese Academy of Medical Sciences (CAMS)","correspondingAuthor":false,"prefix":"","firstName":"Donglin","middleName":"","lastName":"Zhuang","suffix":""},{"id":483465921,"identity":"1d96acde-3e9e-4af4-ad09-328354700e15","order_by":19,"name":"Jianqiu Pei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYFACxgaDBCDFxsDY+OCDgQ0PP3sD0VqYDxvOqEiTkew5QLR1bGnCPGcO2xjccMCvzpy9uaHgQc0d2T6JHDMG3rbzPAw3GBg/fMzBrcWy5yDQYceeGbcBtTyQbLvNwzi7gVly5jbcWgxuJAK1sB1OBGoxNzAEamGWOcDGzItPy/2HQC3/wFrMJBLbzvGwSSQQ0HIDGGKJbSAtaWkSB84c4OEhpMWyB+iwxL7Dxm08jw8bNlQk80jwHGzG6xdz9uPPDH98Oyw7vz2x8fEfAzt7++PNBz98xOcwYHwAMTBCEWLIbOxamB8QVjYKRsEoGAUjGgAAnS9YJZwJwsoAAAAASUVORK5CYII=","orcid":"","institution":"Capital Medical University","correspondingAuthor":true,"prefix":"","firstName":"Jianqiu","middleName":"","lastName":"Pei","suffix":""}],"badges":[],"createdAt":"2025-05-24 06:53:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6737272/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6737272/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-34794-7","type":"published","date":"2026-01-08T15:58:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86680376,"identity":"ca71a3c0-ba73-4173-b4ef-89821d504ad0","added_by":"auto","created_at":"2025-07-14 13:02:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6546568,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction of sepsis model and pathological indicator alterations in macrophage Piezo1 knockdown mice. (A) Schematic representation of the construction of a sepsis model in mice. (B) Dose-dependent acute mortality curves in mice following LPS induction. (C) WGA staining reveals myocardial cell swelling induced by LPS treatment (p=0.0005). (D) Genotype identification results for Piezo1\u003csup\u003ef/f\u003c/sup\u003e and lyz2-Cre transgenic mice. (E) Western blot analysis of Piezo1 protein in macrophages from Piezo1 gene knockdown mice. (F) Quantitative analysis of Piezo1 protein expression. (G) Comparison of 24-hour post-LPS injection survival rates. (H) Echocardiographic images before and after LPS injection, with comparison of LVEF and FS. (I) Comparison of the degree of weight loss following LPS treatment. (J) Changes in the ratio of heart size to tibial length after LPS treatment. (K) Alterations in cTnI levels before and after LPS treatment.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6737272/v1/dd7315d519cff008eda0eb16.png"},{"id":86680394,"identity":"e26da19e-e20c-4efd-819c-72d3db671523","added_by":"auto","created_at":"2025-07-14 13:02:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":37950585,"visible":true,"origin":"","legend":"\u003cp\u003eCardiac histopathological changes and macrophage characterization. (A) H\u0026amp;E staining of cardiac tissues before and after LPS administration, vacuolization of myocytes (\"↑\") and myofibrillar rupture (\"*\"). (B) Electron micrographs of myocardial ultrastructure post-LPS treatment, depicting mitochondria (\"mit\"), myofibers (\"m\"), cristae of mitochondria (\"↑\"), and mitochondrial vacuolation (\"*\"). (C) Microscopic imaging of BMDMs along with statistical analysis of typical cell phenotypes. (D) Flow cytometric enumeration of BMDMs. (E) Fluorescent staining and quantification of CD68 and CD206 in cardiac tissue sections.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6737272/v1/cf88d2f895c775bf4c853b8f.png"},{"id":86680375,"identity":"5673115e-9f9b-475d-85a4-84d3401fbb04","added_by":"auto","created_at":"2025-07-14 13:02:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3076611,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism of Piezo1 knockdown macrophages in alleviating Sepsis-induced cardiomyopathy. (A) Differentially expressed genes across various experimental conditions. (B) Heatmap illustrating clustering of differential gene expression. (C) KEGG pathway enrichment analysis. (D) Volcano plot visualizing differential gene expression. (E) Western blot results for bone marrow-derived macrophages. (F) Western blot results for cardiac tissue. (G) Conceptual diagram depicting cardioprotection mediated by Piezo1 knockdown-induced M2 macrophage polarization.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6737272/v1/ed701a42a2d1c11c684f4b43.png"},{"id":100069365,"identity":"154ba196-a242-4eb8-b487-18fde43fef7c","added_by":"auto","created_at":"2026-01-12 16:13:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":65526320,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6737272/v1/b030d2dd-bd4c-48cf-b78a-21ae2a41535c.pdf"},{"id":86680378,"identity":"9544f943-6506-4527-a384-5e3a3cf7ce14","added_by":"auto","created_at":"2025-07-14 13:02:33","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2818668,"visible":true,"origin":"","legend":"","description":"","filename":"20250703SUPPLEMENTALMATERIAL.docx","url":"https://assets-eu.researchsquare.com/files/rs-6737272/v1/59aba62d0234cb3623ba8f6d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Piezo1 Knockdown Activates PI3K/AKT and Enhances SPP1 to Drive M2 Macrophage Polarization and Reduce Cardiac Inflammation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSepsis is a systemic inflammatory response characterized by excessive production of inflammatory cytokines, oxidative stress, and multiple organ dysfunction\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. One severe sequel of this multiorgan impairment is sepsis-induced cardiomyopathy (SCM), characterized by myocardial depression and ensuing high mortality due to cardiac dysfunction. This condition arises from an overwhelming secretion of inflammatory mediators and chemokines during the septic response, inflicting damage upon myocardial cells\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Macrophages are well-known for their pivotal roles in preserving homeostasis, defending against pathogens, and repairing tissue damage\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. They are capable of differentiating into various functional phenotypes in different environments and tissues. These phenotypes include the classical activation phenotype (M1 type), which has a pro-inflammatory effect, and the alternative activation phenotype (M2 type), which has a repair effect\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Furthermore, endotoxin, a key component of the outer membrane of gram-negative bacteria and a principal instigator of sepsis, provokes macrophage activation and drives polarization toward the M1 phenotype. This polarization leads to an overproduction of inflammatory cytokines like interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin 1β (IL-1β), thereby fueling an inflammatory cascade that exacerbates damage to cardiomyocytes\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Thus, inhibiting macrophage activation is crucial in reducing myocardial injury during sepsis.\u003c/p\u003e\u003cp\u003ePiezo1 is a mechanically activated ion channel with a high affinity for calcium\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. It is evolutionarily conserved and involved in the development, differentiation, and growth of multiple tissues. It responds to diverse mechanistic stimuli across numerous cell types\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, with a distinctive significance in the regulation of inflammatory infiltration subjected to hemodynamic stress. Activation of the PI3K/AKT phosphorylation pathway is manifested by upregulation of the expression of pathway-associated proteins and upregulation of the phosphorylation level of proteins. Activation of the pathway promotes macrophage polarization to the M2 type, with an increase in the proportion of macrophages of the M2 type and enhanced anti-inflammatory effects\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Recent studies have revealed that macrophages demonstrate significant expression of Piezo1, which is pivotal to their involvement in mediating inflammatory responses\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we employed a classical sepsis model induced by lipopolysaccharide (LPS) to investigate the role of Piezo1 in regulating macrophage responses during sepsis\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Our research findings suggest that macrophage polarization relies on the function of Piezo1. Following the knockdown of Piezo1, the application of LPS resulted in an enhanced phosphorylation of the PI3K/AKT pathway, concomitant with an upregulation in the expression of SPP1. This shift correlated with an increased polarization of macrophages towards the M2 phenotype, leading to a reduction in myocardial injury. We showed that macrophages lacking Piezo1 holistically reduce inflammation responses. This suggests the regulatory role of Piezo1 and the possibility of Piezo1 as a potential therapeutic target in Sepsis-induced cardiomyopathy.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eAnimals\u003c/p\u003e\u003cp\u003eThe mice needed for the experiment were purchased from Shanghai Model Organisms Center, Inc. Wild-type, Piezo1\u003csup\u003eflox/flox\u003c/sup\u003e and Piezo1\u003csup\u003eflox/flox\u003c/sup\u003e Lyz2-Cre C57BL/6J mice, all male and aged eight weeks, were maintained in specific pathogen-free environments and sustained on a standard mouse chow diet. The impact and conclusions regarding gender differences in SCM have not yet gained widespread recognition. Consequently, the use of male mice remains the predominant choice.\u003c/p\u003e\u003cp\u003e All animal experiments were done by the Guide for the Care and Use of Laboratory Animals published by the Ministry of the People\u0026rsquo;s Republic of China (1998) and approved by the Institutional Committee on Animal Care of Capital Medical University. The animal research was sanctioned by the Institutional Animal Care and Use Committee (IACUC), under the approval number A5095F17-EA0D-4377-88F7-FED2CE3A2ED2. All methods were performed in accordance with the relevant guidelines and regulations, and follow the ARRIVE guidelines. Genotype is characterized by PCR, and the primer sequences are in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eEchocardiography\u003c/p\u003e\u003cp\u003eUsing a 30 MHz probe small animal ultrasonic biological microscope (Vevo 3100, FUJIFILM Visual Sonics Inc, Toronto, Canada). Under isoflurane anesthesia, the long-axis section shows the maximum section of the left ventricle. Switch to M-mode ultrasound to measure left ventricular structure and systolic function, covering at least 25 cardiac cycles to ensure sufficient data for accurate analysis.\u003c/p\u003e\u003cp\u003eWestern blot analysis\u003c/p\u003e\u003cp\u003eBMDMs were exposed to a lysis buffer, a combination of RIPA lysis buffer and a 1% protease inhibitor. The lysate was spun at 14000 rpm for 15 min, and the supernatant was obtained. The proteins were denatured through the use of LDS sample buffer and sample reducing agent at 70\u0026deg;C for 10 min before each sample was loaded into a well of a 4\u0026ndash;12% Bis-Tris gel (all from Invitrogen). The membranes were blocked by using 5% skim milk for 1 h. After washing, the membranes were probed with a primary antibody at 4 ℃ overnight. Then the membranes were washed and probed with secondary antibodies at room temperature for 1 h. The membrane was then washed in TBST, immersed in a western HRP substrate solution, and then imaged. The antibodies are in Table S2, Uncropped blots are provided in the Source Data file.\u003c/p\u003e\u003cp\u003ePeritoneal Macrophage Isolation\u003c/p\u003e\u003cp\u003eInject mice intraperitoneally with 3% thioglycollate broth (1 mL) once daily for three days.\u003c/p\u003e\u003cp\u003eCollect macrophages: a) Euthanize mice via cervical dislocation post-anesthesia and sanitize with 75% ethanol for 3\u0026ndash;5 min. b) In a biosafety cabinet, make an abdominal incision, expose the peritoneum, and inject 5 mL chilled PBS. Massage gently and rest for 3\u0026ndash;5 min. c) Aspirate peritoneal fluid for centrifugation.\u003c/p\u003e\u003cp\u003ePurify and culture: Spin at 1000 rpm for 5 min, resuspend pellet in RPMI-1640, and incubate at 37\u0026deg;C for 2\u0026ndash;3 hours to allow adhesion. Wash to remove non-adherent cells, obtaining purified macrophages.\u003c/p\u003e\u003cp\u003eBMDM Isolation\u003c/p\u003e\u003cp\u003eHarvesting Bones: a) Euthanize the mouse post-anesthesia via cervical dislocation, and sterilize in 75% ethanol for 5 minutes. Remove and clean femurs and tibiae, preserving bone integrity. b) Sterilize bones in 75% ethanol for 5 minutes, then rinse in cold PBS.\u003c/p\u003e\u003cp\u003eMarrow Extraction and Macrophage Induction: a) Cut ends of bones, flush out marrow using a syringe, and break up clumps with a pipette. b) Filter cells through a 70\u0026micro;m strainer, centrifuge at 1500 rpm for 5 minutes, and remove supernatant. c) Lyse red blood cells, let stand for 5 minutes, wash with cold PBS, centrifuge, and discard supernatant. d) Resuspend cells in chilled macrophage medium, and seed onto culture plates.\u003c/p\u003e\u003cp\u003e*During culture, replace BMDM medium every 2\u0026ndash;3 days post-washing with PBS. Harvest after 7-day culture.\u003c/p\u003e\u003cp\u003eFlow Cytometry\u003c/p\u003e\u003cp\u003eThe cells were then resuspended in an EP tube and centrifuged at 500g for 10 minutes to discard the supernatant. Next, the cells were resuspended with CD16/32, incubated on ice for 10 minutes. Then the cells were washed. Add F4/80 and CD86 antibodies with a fixative solution and incubate for 30 minutes. After washing, add the CD206 antibody in a membrane-breaking solution and incubate for another 30 minutes. Finally, resuspend the cells in PBS. Subsequently, analyze the sample using the BD FACSVerse\u0026trade; flow cytometer. For data analysis, employ FlowJo v10.10 software with gating strategies as depicted in Fig. S3.\u003c/p\u003e\u003cp\u003eQuantification and Statistical Analysis\u003c/p\u003e\u003cp\u003eAll data are representative of at least three independent experiments. All statistical analyses were performed using Prism9.5.1 (GraphPad). The data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD as indicated in the legends. Survival data were analyzed by the Kaplan\u0026ndash;Meier statistical method. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eCardiac function decreased in mice with sepsis after LPS stimulation, and this impairment was ameliorated after the knockdown of Piezo1 in macrophages\u003c/p\u003e\u003cp\u003eTo investigate the impact of LPS on cardiac function, we divided male C57BL/6J mice, wild-type and eight-weeks-age, into 4 groups and administered LPS via intraperitoneal injection at progressive doses of 0 mg/kg (n\u0026thinsp;=\u0026thinsp;16), 10 mg/kg (n\u0026thinsp;=\u0026thinsp;16), 12 mg/kg (n\u0026thinsp;=\u0026thinsp;16), and 15 mg/kg (n\u0026thinsp;=\u0026thinsp;17). These doses corresponded to survival rates of 100%, 75%, 56.25%, and 17.65% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Opting for a dose of 12 mg/kg LPS for our sepsis model, we were able to maintain mouse survival rates at 50%-70%. Before LPS injection, baseline cardiac assessments were performed using echocardiography. A follow-up echocardiographic evaluation was conducted 6\u0026ndash;8 hours post-LPS administration. Cardiac function was quantitatively assessed by recording and analyzing M-mode echocardiograms (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Our findings indicate that intraperitoneal administration of LPS precipitates myocardial damage. Echocardiographic analysis revealed substantial degradation in cardiac function, with marked left ventricular systolic dysfunction observed notably in the LPS-treated mice (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and Piezo1 levels were notably increased through Western blot analysis in the LPS-treated mice (Fig. S2). This was characterized by decreased myocardial contraction amplitudes and a pronounced augmentation in myocyte edema in the LPS-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eOne study has discovered that specific knockout of Piezo1 in macrophages can protect the mouse liver and decelerate the progression of fibrosis\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. To investigate the role of Piezo1 in sepsis-induced cardiomyopathy, we generated macrophage-specific Piezo1 knockdown C57BL/6J mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Piezo1\u003csup\u003ef/f\u003c/sup\u003e and Piezo1\u003csup\u003ef/f\u003c/sup\u003eLyz2-Cre (Piezo1-cKO) mice were used, and we first harvested peritoneal macrophages. Through Western blot analysis, we confirmed the effective deletion of Piezo1, ultimately demonstrating a knockdown efficiency of 74% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and F).\u003c/p\u003e\u003cp\u003eThen, we constructed a model of sepsis in eight-week-old male Piezo1\u003csup\u003ef/f\u003c/sup\u003e (n\u0026thinsp;=\u0026thinsp;41) and Piezo1-cKO (n\u0026thinsp;=\u0026thinsp;32) mice. The sepsis yielded a survival rate of approximately 65.63% in Piezo1-cKO mice, but only approximately 56.10% of Piezo1\u003csup\u003ef/f\u003c/sup\u003e mice (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0911) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Before administering LPS, mice with Piezo1 knockdown did not exhibit mortality, and there were no significant differences in body weight or cardiac function between Piezo1\u003csup\u003ef/f\u003c/sup\u003e and Piezo1-cKO groups. However, following LPS treatment, both Piezo1\u003csup\u003ef/f\u003c/sup\u003e and Piezo1-cKO groups exhibited a significant reduction in body weight, LVEF, and FS (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; p\u0026thinsp;=\u0026thinsp;0.0001; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively). Notably, the Piezo1\u003csup\u003ef/f\u003c/sup\u003e group displayed worse cardiac function, with LVEF and FS being significantly lower compared to the Piezo1-cKO group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH and I). Additionally, the Piezo1\u003csup\u003ef/f\u003c/sup\u003e group presented with an increased cardiac mass, as evidenced by an elevated heart weight-to-tibia length ratio (p\u0026thinsp;=\u0026thinsp;0.0286) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). Serological assays also showed a greater increase in cTnI levels in the Piezo1\u003csup\u003ef/f\u003c/sup\u003e group (p\u0026thinsp;=\u0026thinsp;0.0022) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK).\u003c/p\u003e\u003cp\u003eOur results indicate that targeted knockdown of Piezo1 in macrophages may impart a cardioprotective advantage in septic mice, potentially mitigating mortality rates associated with sepsis-induced cardiomyopathy.\u003c/p\u003e\u003cp\u003ePiezo1 knockdown alleviates myocardial cell damage via induction of M2 macrophage polarization\u003c/p\u003e\u003cp\u003eFirst, the hematoxylin-eosin (HE) staining revealed disorganized myocardial fibers, partial fiber rupture, and degeneration, with striations becoming blurred or vanishing (vacuolization), interstitial edema, and inflammatory cell infiltration following LPS treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Furthermore, the transmission electron microscopy revealed disorganized myocardial fibers, myofibrillar separation, twisting of the Z lines, shortened distances between Z lines, disorder of mitochondria within myocardial cell, swelling, diminished mitochondrial cristae, and necrotic changes including partial vacuolization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). But these damages were significantly mitigated in the Piezo1-cKO group compared to the Piezo1\u003csup\u003ef/f\u003c/sup\u003e group. Then we isolated bone marrow-derived macrophages for culture, and under the microscope, there was no evident differentiation before LPS treatment. After 36 hours of exposure to LPS, the differentiation ratio was around 60%, with no significant difference between the Piezo1\u003csup\u003ef/f\u003c/sup\u003e and Piezo1-cKO groups (p\u0026thinsp;=\u0026thinsp;0.7807). Based on the morphology of the macrophages, M1 and M2 phenotypes were distinguished: the Piezo1-cKO group exhibited a higher proportion of M2 macrophages among the differentiated cells (32.91% vs. 17.87%, p\u0026thinsp;=\u0026thinsp;0.0002) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This was further substantiated by flow cytometry, which identified macrophages with the F4/80 marker and M2 macrophages with the CD206 marker, with the Piezo1-cKO group showing a higher ratio of M2 macrophages among differentiated cells (43.7% vs. 28.1%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eTo ascertain if the abundance of M2 macrophages within myocardial tissue paralleled our in vitro observations, we performed fluorescence staining with CD68 and CD206. The Piezo1-cKO group exhibited a stronger CD206 expression (p\u0026thinsp;=\u0026thinsp;0.0486) with cellular morphology more representative of the archetypal M2 macrophage phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eWe contend that silencing Piezo1 in macrophages induces a skewing toward an M2 anti-inflammatory phenotype, concomitantly diminishing myocardial inflammation and alleviating cardiomyocyte injury, thus safeguarding cardiac functionality.\u003c/p\u003e\u003cp\u003eMacrophage Piezo1 Knockdown Activates the PI3K/AKT Pathway and Promotes M2 Polarization\u003c/p\u003e\u003cp\u003eTo investigate the mechanism by which Piezo1 regulates macrophage polarization, we conducted transcriptomic sequencing. It was found that compared to the control group, macrophages with Piezo1 knockdown showed significant upregulation in 511 genes and downregulation in 74 genes (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The differential gene expression clustering heat map was consistent with these changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). Notably, as a factor that can drive macrophage polarization toward the M2 phenotype\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, the SPP1 gene was also significantly upregulated (p\u0026thinsp;=\u0026thinsp;5.3E-07). Further, KEGG enrichment analysis indicated a significant accumulation of genes related to the PI3K/AKT signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC)\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. To validate the sequencing analysis results, we isolated and cultured bone marrow-derived macrophages from mice and performed Western blot experiments. Initially, it was observed that SPP1 levels were notably increased in the Piezo1 knockdown group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and E). Within the PI3K/AKT pathway, despite AKT levels being roughly constant, phosphorylated AKT (P-AKT) and downstream signaling molecule p-S6 were significantly elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). This implies that in mice with Piezo1 knockdown, the PI3K/AKT pathway was significantly upregulated following LPS treatment. Our research has indicated that both the upregulation of SPP1 and the PI3K/AKT pathway can promote macrophage polarization towards the M2 phenotype, playing an anti-inflammatory role. We also verified changes in cardiac macrophage-derived inflammatory cytokines; in the hearts of Piezo1 knockdown mice, pro-inflammatory cytokines IL-1β and IL-6, primarily derived from M1 macrophages, were markedly decreased, whereas the anti-inflammatory cytokines IL-10, predominantly from M2 macrophages, was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eThese changes reflect that the knockdown of Piezo1 activates the PI3K/AKT pathway, promotes the expression of SPP1, and causes macrophage polarization towards the M2 phenotype. Consequently, the anti-inflammatory protective capacity of macrophages is enhanced, inflammation levels are reduced, and cardiac function is protected (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo examine the effects of macrophage-specific knockdown of Piezo1 on sepsis-induced cardiac dysfunction following LPS infection, our study employed a classical sepsis model induced by LPS, which ensured a mortality rate of 50\u0026ndash;70% in mice. We successfully generated macrophage-specific Piezo1 knockdown mice to utilize within this framework. In these genetically modified mice, loss of Piezo1 conferred a markedly improved physiological state characterized by lower mortality rates, reduced weight loss, less cardiac edema, better cardiac function, and enhanced myocardial contractility after LPS infection. Histopathological findings further suggested that the diminishment of macrophage infiltration following Piezo1 deletion is likely attributable to Piezo1's regulatory role in macrophage proliferation and infiltration\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Additionally, upregulation of Piezo1 may lead to increased ROS production and cellular apoptosis\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Consequently, it can be inferred that the functional alterations in macrophages due to Piezo1 knockdown exerted a protective effect in mice. The postulate has been substantiated by HE staining and electron microscopy, which confirmed an improved condition of the myocardium after Piezo1 deletion.\u003c/p\u003e\u003cp\u003ePrevious research has categorized macrophages into two subsets, M1 and M2\u003csup\u003e23\u003c/sup\u003e, with M2 being associated with more pronounced anti-inflammatory effects\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. We proceeded to assess macrophage polarization within our study. Initially, we conducted conventional morphological observations and flow cytometric analyses of bone marrow-derived macrophages, which revealed an increase in the M2 macrophage population. Subsequently, immunofluorescence staining for macrophage subsets within the cardiac tissue demonstrated a significantly higher expression of CD206, an M2 marker. Based on these findings, we can tentatively conclude that Piezo1 may play a role in promoting the differentiation of macrophages towards the M2 phenotype.\u003c/p\u003e\u003cp\u003eTo dissect the underlying mechanisms, we performed transcriptomic analysis on six mice and observed significant differences in gene expression following Piezo1 knockdown. SPP1, originally characterized as a pro-inflammatory cytokine secreted by T cells, is a multifunctional glycoprotein that is also expressed in an array of tissue-resident macrophages. This molecule plays a pivotal role in the phagocytic clearance of apoptotic cells, the chemotactic response, and the directed migration of macrophages\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. A study highlights that SPP1 fosters M2 macrophage polarization through its interaction with αvβ3 integrin and CD44 receptors\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Notably, SPP1 and the PI3K/AKT pathway have been implicated in macrophage polarization in prior studies\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Some researchers have reported that a reduction in SPP1 correlates with a decrease in factors associated with M2 macrophage polarization, and others have considered SPP1 as a key target for macrophage phenotype identification and a prognostic factor for cancer outcomes\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Upon assessing protein expression changes, we discerned that Piezo1 knockdown resulted in upregulated expression of SPP1, accompanied by enhanced activation of the PI3K/AKT signaling pathway, both of which promote differentiation towards the M2 macrophage phenotype. The interplay between SPP1 and the PI3K/AKT pathway has been recognized in previous studies. One study in prostate cancer revealed that high SPP1 expression maintained PI3K/AKT pathway activation\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, while another study on bone fracture healing found that activation of the PI3K/AKT pathway stimulated M2 macrophage polarization\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Although we have verified that Piezo1 knockdown could cause the polarization of macrophages towards M2, this may also relate to the role of Piezo1 in stiffness sensing and subsequent regulation of macrophage polarization\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Moreover, Piezo1 knockdown appears to reduce the phagocytic activity of macrophages, which could be another consequence of the shift toward the M2 phenotype. We cannot exclude the possibility that this phagocytic reduction might inhibit polarization towards the M1 phenotype, contributing to an increased proportion of the M2 phenotype. To further elucidate the intricate mechanistic details, it may be necessary to conduct single-cell sequencing to analyze different cell types and to perform a more comprehensive dissection of the underlying processes.\u003c/p\u003e\u003cp\u003ePrevious studies have authenticated the regulatory role of Piezo1 in modulating macrophage function and influencing the levels of inflammation\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Our study corroborated these findings by observing changes in myocardial inflammation, which align with the anti-inflammatory M2 macrophage phenotype. Specifically, the pro-inflammatory cytokines IL-1β and IL-6 were significantly diminished, whereas the anti-inflammatory cytokine IL-10 was elevated\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. This pattern of cytokine expression indicates that myocardial cells were shielded from inflammatory damage, thereby exhibiting improved cardiac function.\u003c/p\u003e\u003cp\u003eOur investigation indicates that Piezo1 knockdown in macrophages activates the PI3K/AKT pathway and drives the expression of SPP1, which in turn polarizes macrophages towards the M2 phenotype. Within myocardial tissues, these macrophage alterations not only lower inflammation levels but also boost their anti-inflammatory capacity, thereby conferring cardioprotection and preserving cardiac function in mice, suggesting Piezo1 might as a potential therapeutic target in the clinic intervention.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eLPS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; lipopolysaccharide\u003c/p\u003e\n\u003cp\u003eSCM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;sepsis-induced cardiomyopathy\u003c/p\u003e\n\u003cp\u003eIL-6 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;inflammatory cytokines like interleukin 6\u003c/p\u003e\n\u003cp\u003eTNF-α \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;tumor necrosis factor-alpha\u003c/p\u003e\n\u003cp\u003eIL-1β \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;interleukin 1β\u003c/p\u003e\n\u003cp\u003ePiezo1-cKO \u0026nbsp; \u0026nbsp;Piezo1\u003csup\u003ef/f\u003c/sup\u003eLyz2-Cre\u003c/p\u003e\n\u003cp\u003eHE \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;hematoxylin-eosin\u003c/p\u003e\n\u003cp\u003eP-AKT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;phosphorylated AKT\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u0026nbsp;sources\u003c/h2\u003e\n\u003cp\u003eThis research was supported by National Natural Science Foundation of China (82300301) and Beijing Natural Science Foundation (7232077) and the Basic Research Project of Yunnan Provincial Science and Technology Department (202401AY070001-020).\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026apos; contributions\u003c/h2\u003e\n\u003cp\u003eYHZ, YZ, YJT and JYS: Writing \u0026ndash; original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Conceptualization.\u003c/p\u003e\n\u003cp\u003eZWZ, WHJ and HZ: Investigation, Formal analysis.\u003c/p\u003e\n\u003cp\u003eHLL, YBM and ZWN: Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eQZG, SL, JQP and TYS: Validation.\u003c/p\u003e\n\u003cp\u003eYZW, BHP, MH, CYL, ZLL and DLZ: Writing \u0026ndash; review \u0026amp; editing, Validation, Supervision, Resources, Project administration, Funding acquisition, Formal analysis, Conceptualization.\u003c/p\u003e\n\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request. Source data are provided in this paper.\u003c/p\u003e\n\u003ch2\u003eEthical Approval and Consent to participate\u003c/h2\u003e\n\u003cp\u003eThis study has been approved by the Institutional Committee on Animal Care of Capital Medical University (Protocol number A5095F17-EA0D-4377-88F7-FED2CE3A2ED2).\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eThe authors are thankful for the experimental platform provided by the State Key Laboratory of Cardiovascular Disease, China \u0026amp; Fuwai Hospital, Chinese Academy of Medical Sciences \u0026amp; Peking Union Medical College.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJia, L. et al. 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Immunol.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 1149336. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2023.1149336\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2023.1149336\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Piezo1, Macrophage, Sepsis-induced Cardiomyopathy, Cardiac Dysfunction, PI3K/AKT pathway","lastPublishedDoi":"10.21203/rs.3.rs-6737272/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6737272/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePiezo1 plays a key role in the immune response during sepsis. To date, our understanding of the role of Piezo1 in inflammatory diseases has mostly been limited to influencing vasomotor function and regulating inflammatory infiltration. Whether and how Piezo1 in macrophages is involved in developing septic cardiac dysfunction has never been explored. Here, we have successfully established a mouse model with macrophage-specific knockdown of the Piezo1. The intraperitoneal injection of lipopolysaccharide (LPS) resulted in a significant increase in cardiac macrophage infiltration, as well as an increase in the expression of inflammatory factors and the inflammatory response. However, macrophage-specific knockdown of Piezo1 impaired this response, leading to an increment in macrophage polarization towards the M2 type and the decreased inflammatory response. As a result, myocardial injury caused by sepsis was attenuated. We have also demonstrated that the PI3K/AKT pathway is significantly activated after Piezo1 knockdown, resulting in reduced myocardial dysfunction. Our data indicate that macrophage-specific knockdown of Piezo1 can influence macrophage polarization and thus exert cardioprotective effects in a murine model of sepsis, providing potential ideas and targets for the treatment of infectious cardiac dysfunction.\u003c/p\u003e","manuscriptTitle":"Piezo1 Knockdown Activates PI3K/AKT and Enhances SPP1 to Drive M2 Macrophage Polarization and Reduce Cardiac Inflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 13:02:28","doi":"10.21203/rs.3.rs-6737272/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-13T10:37:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-23T14:37:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-20T22:26:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"219789400298495285713879685577584938234","date":"2025-07-10T11:00:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60323981380629665622467574560902340710","date":"2025-07-10T09:33:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"312831206068555184263331131538020774159","date":"2025-07-10T09:05:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-10T08:57:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-09T10:36:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-28T13:43:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c0057aab-c3b2-4ae8-a307-84a7367a6294","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51335529,"name":"Biological sciences/Genetics"},{"id":51335530,"name":"Health sciences/Cardiology"}],"tags":[],"updatedAt":"2026-01-12T16:03:26+00:00","versionOfRecord":{"articleIdentity":"rs-6737272","link":"https://doi.org/10.1038/s41598-025-34794-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-08 15:58:09","publishedOnDateReadable":"January 8th, 2026"},"versionCreatedAt":"2025-07-14 13:02:28","video":"","vorDoi":"10.1038/s41598-025-34794-7","vorDoiUrl":"https://doi.org/10.1038/s41598-025-34794-7","workflowStages":[]},"version":"v1","identity":"rs-6737272","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6737272","identity":"rs-6737272","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}