Aerobic exercise improves ventricular remodeling by promoting macrophages to phagocytose dying cardiomyocytes in heart failure model

Aerobic exercise improves ventricular remodeling by promoting macrophages to phagocytose dying cardiomyocytes in heart failure model | 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 Aerobic exercise improves ventricular remodeling by promoting macrophages to phagocytose dying cardiomyocytes in heart failure model Yuqin Shen, Xiaoling Liu, Chun Li, Yuxuan Fan, Zhongyan Zhou, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4420177/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 Heart failure (HF), the terminal stage of various forms of cardiovascular disease, is a severe disorder characterized by pathological cardiac fibrosis, ventricular remodeling, and reduced heart function. Chamber remodeling is the basic pathological mechanisms of HF. Aerobic exercise training (AET) as one of the non-pharmacological treatments of cardiac rehabilitation, has become one of the important therapeutic means for the long-term management of chronic HF, but how AET can improve the process in HF has not been well clarified. This study aims to determine the role of AET in pathological cardiac remodeling in HF and its potential mechanisms. We identified AET promoting the clearance of apoptosis cardiomyocytes by boosting interactions of cardiomyocytes-macrophages in HF. Lgmn was associated with the efferocytosis elevation of macrophages by AET. In addition, AET, improving the ventricular remodeling and strengthening heart function ultimately, upregulation of the anti-inflammatory mediators and downregulationof the proinflammatory mediators by boosting the expression of Lgmn in chronic repair stage of HF.Our results link AET to efferocytosis elevation of macrophages in the chronic repair stage of heart injury and identify AET as a significant prevention and therapeutic of ventricular remodeling in HF to mediate proper inflammation resolution and cardic function increase. Health sciences/Medical research/Drug development Health sciences/Diseases/Cardiovascular diseases/Heart failure Health sciences/Pathogenesis/Inflammation/Chronic inflammation heart failure aerobic exercise training apoptotic cardiomyocytes macrophages Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Heart failure (HF) is a heterogeneous clinical syndrome stemming from cardiac overload and injury with considerable morbidity and mortality 1 , 2 , 3 . The incorporation of various medications such as angiotensin-converting enzyme inhibitors (ACEI), angiotensin II receptor blockers (ARBs), angiotensin receptor/neprilysin inhibitors (ARNIs), beta-blockers, and aldosterone antagonists has marked a substantial decrease in mortality rates within the field of cardiology. Despite of these, the quality of life (QoL) and social engagement of patients with HF still experience a downward trend. Hence, additional and effective strategies to enhance heart function are imperative. Ventricular remodeling is a basic pathological process of HF and is characterized by pathological cardiomyocyte hypertrophy, apoptosis, fibroblast proliferation, myofibroblast formation, and interstitial fibrosis 4 , 5 . Aerobic exercise training (AET) as one of the non-pharmacological treatments of cardiac rehabilitation, has become one of the important therapeutic means for the long-term management of chronic HF 6 , 7 , 8 . AET has been proven to be effective in mitigating the detrimental cardiac remodeling 9 , 10 , enhancing one's cardiopulmonary function, decreasing readmission and mortality, and boosting social engagement and QoL 11 , 12 , 13 . Despite the observed benefits, the specific mechanisms responsible for these benefits have not yet been fully elucidated. Several research works have shed light on certain mechanisms through which AET mitigates pathological ventricular remodeling in HF, notably including the inhibition of the TGF-β/smad2 signaling cascade and the TGF-β/TIMP-1/MMP-1 signaling pathway to reduce myocardial fibrosis 14 , the activation of the β3-AR-NOS-NO signaling cascade to counteract oxidative stress 15 , thereby attenuating myocardial hypertrophy. Anwen Yin et al. 10 recently identified that AET stimulates the secretion of C-terminal of coiled-coil domain-containing protein 80 (CCDC80tide), which selectively binds to active kinase-active form of Janus kinase 2 (JAK2), inhibiting its ability to phosphorylate and activate signal transducer and activator of transcription 3 (STAT3). This interaction helps protect mice from against angiotensin II (Ang II)-induced cardiac hypertrophy and remodeling. Carolin Lerchenmüller's team 16 discovered that CBP/p300-interacting transactivators with E/D-rich-carboxylterminal domain4 (CITED4), a cardiac protein, guards against cardiac remodeling by modulating mTOR activity and miRNAs. Nonetheless, the existing research has been predominantly centered on macroscopic tissue-level assessments, thereby lacking of comprehending the underlying molecular dynamics at the cellular stratum. To address this inquiry, we developed models of HF by ligating the left anterior descending (LAD) coronary artery, and subsequently investigated the therapeutic effects and underlying cellular mechanisms of AET on pathological ventricular remodeling in vivo, leveraging single-nucleus RNA sequencing (snRNA-Seq) technology. Recent advancements in snRNA-Seq tech have facilitated the acquisition of high-resolution, cell-type-specific cardiac transcriptomic data, thereby allowing for a more nuanced understanding of cellular heterogeneity within the heart, avoiding the problems of bulk RNA sequencing that analyze mixed cells from different stages and potentially obscure critical molecular events and signals in specific cell populations. As anticipated, HF mice exhibited increased ventricular remodeling and cardiac dysfunction, but those treated with AET showed lessened ventricular remodeling and improved heart function. Notably, our study has demonstrated that a 4-week regimen of AET not only effectively increases the typically diminished macrophage population observed in HF but also substantially amplifies the intricate interplay between these immune cells and cardiomyocytes within the affected cardiac milieu. This enhancement in cellular communication is instrumental in augmenting the efficiency of efferocytosis, thereby facilitating the clearance of in vivo accumulated apoptotic cardiomyocytes during HF. Furthermore, we revealed that cardamom protein (legumain, Lgmn) played a crucial role in AET's promotion of the effective phagocytosis of apoptotic cardiomyocytes and contributed to the appropriate resolution of inflammation. Investigating AET's regulatory mechanisms can demystify its relationship with heart disease, advancing more effective treatments. Methods Mice Wild-type (WT) C57BL6/J mice (male, 6 weeks old; GemPharmatech Laboratory Animal, Shanghai, China) were used in this study. This study and all animal procedures conformed to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH publication No. 85 − 23, revised 1996) and were approved by the Experimental Animal Welfare and Ethics Committee, Shanghai University of Chinese Medicine (PZSHUTCM210312008). Models of HF were established by ligation of the left anterior descending (LAD) coronary artery Models of HF were established by ligation of the LAD coronary artery. LAD operation was performed on 6-week-old WT mice as described previously 17 .Briefly, the mice were anesthetized with 4% chloral hydrate using intraperitoneal injection. Subsequently, we placed the endotracheal tube below the glottis and connected the tube with the ventilator (HX-101E, Techman Software, Chengdu, China) to assist the breathing mice. Then, a cut was made between the third and fourth intercostal space, and a 7 − 0 nylon suture was used for permanent ligation of the LAD. Animals, dead within the first 24 h after the surgery, were excluded from the analysis. Sham mice received the same operation without coronary artery ligation. At the specified time points, mice were euthanized by cervical dislocation, and tissues were then collected for analyses. Transthoracic echocardiography Transthoracic echocardiography assesses heart geometry, systolic and diastolic function, as described earlier 18 .The Vevo2100 ultrasound system (VisualSonics, Toronto, Canada) were used. Mice were lightly anesthetized with 0.5% isoflurane until the heart rate stabilized at 400–500 beats per minute, and placed on a ECG platform. Parasternal long-axis images were obtained in two-dimensional B mode at the appropriate scanning head position to determine the maximum LV length, and the sampling line was placed at the maximum cross-section of the left ventricle to guide the recording of continuous M-mode echocardiography. Automatic calculation of left ventricular ejection fraction (LVEF), fractional shortening (FS), left ventricular end-systolic dimension (LVESD), and left ventricular end-diastolic diameter (LVEDD) from at least three distinct frames for each mouse. Aerobic exercise training program HF mice were trained with a treadmill ( Slope 0°), with the speed of 12 m/ min, the training time of 30 min/time, five times per week, for 4 weeks. Meanwhile, WT mice received fed conventionally without specific exercise training. 10X sample processing and single nucleus RNA sequencing (SnRNA-Seq) Single nucleus suspensions were mixed-extracted from three biological replicate mice at indicated time points after LAD with Nuclear Separation Kit (Shanghai Biotechnology Corporation) according to the manufacturer’s instructions. The concentration of nuclear suspension was adjusted with the corresponding NB solution according to subsequent experiments. About 50,000 nuclei were loaded into one channel of the Single Cell Chip M for each sample using the Single Cell 3’ HT kit (10X Genomics) for Gel bead Emulsion generation in the Chromium X system. Following capture and lysis, cDNA was synthesized and amplified for 14 cycles. 50 ng of the amplified cDNA were used for each sample to construct Illumina sequencing libraries. Sequencing was performed on the NextSeq500 Illumina sequencing platform following 10x Genomics instructions for reads generation. Bioinformatic analyses Raw data processing Raw sequencing data (bcl-files) were converted to fastq files with the Illumina bcl2fastq tool, integrated into the CellRanger (10X Genomics) suite (version 2.1.1). The CellRanger analysis pipeline was used to generate a digital gene expression matrix starting from raw data. Pre-build mouse genome (version mm10-1.2.0) was used as genome reference. The CellRanger count module was used to map reads with default settings and sequence length set to r1-length = 26 and r2-length = 50. Global mapping statistics, such as Estimated Number of Cells, Mean Reads per Cell and Median Genes per Cell for each experimental condition are reported in Supplementary Table S1 . Quality control (QC) and normalization of SnRNA-seq data For further data pre-processing, filtering and QC were done using the Seurat package (v3.1.2). For each specimen, genes with counts in fewer than 10 nuclei were discarded to exclude random noise. Nuclei were filtered for genes (500 < nFeature_RNA < 5000), the proportion of mitochondrial genes (percent.mt < 0.15) and the proportion of ribosomal genes (percent.ribo < 0.05) to remove poor-quality nuclei potentially ascribed to doublets or other technical noise. For normalization, unique molecular identifier (UMI) counts for all nuclei were scaled by library size (total UMI counts), multiplied by 10,000 and transformed to a log scale. Unsupervised dimensional reduction and single cell clustering Highly variable genes (HVGs) were identified using the function FindVariableFeatures in Seurat, and 2000 HVGs were selected for each sample. Nuclei of all samples were integrated via canonical correlation analysis implemented in Seurat to correct potential batch effects and identify shared cell states across samples. Subsequently, the scaled data were subjected to linear dimensional reduction through principal component analysis (PCA). Using the first 40 PCA components, a shared nearest neighbor graph of the nuclei was calculated, followed by clustering and visualization with t-distributed stochastic neighbor embedding (t-SNE), which were performed using the FindClusters and RunTSNE functions, respectively. The clustering was done using a resolution of 0.4. Marker identification and Cluster annotation In this study, a targeted clustering analysis was performed within the most abundant cell types. The annotations of cell identity on each cluster were defined by the expression of known marker genes. The DotPlot function (Seurat package) was used to visualize the average expression of the known markers related to specific cell types, based on the overall expression profile of the nuclei, regardless of dropout events. On this basis, we employed Pecam1, Cdh5, Emcn, and Flt1 to classify “Endothelial cell”; Ttn, Myh6, Tnnt2, and Atp2a2 for “Cardiomyocyte”; Col1a2, Col8a1, Pdgfra, and Fstl1 for “Fibroblast”; Rgs5, Abcc9, and Pdgfrb for “Pericyte”; Myh11, Acta2, and Synpo2 for “Myofibroblast”; Cd163, Mrc1, and C1qa for “Macrophage”; Cd3e, Cd8a, Il2ra, and Tcf7 for “T cell”; Pax5, Cd22, Cd19, and Blnk for “B cell”. We performed the function check markers (Garnett package) to evaluate the ambiguity score and the relative number of cells for each cell type. Differentially expressed gene (DEG) analysis For the identification of DEGs in AET-HF and HF groups, we calculated the log2 fold change (log2FC) between these groups using the Seurat FindMarkers function. The significance of the difference was determined using Wilcoxon test with the statistical threshold of adjusted P-value 0.25. Functional enrichment analyses The upregulated and downregulated genes identified for each group (AET-HF or HF group) were used in subsequent functional enrichment analyses using R-based application enrichR, respectively. EnrichGO was applied to identify genes enriched in biological process (BP), molecular function (MF), and cellular component (CC). Meanwhile, enrichKEGG was applied to detect genes enriched in pathway maps, such as metabolic pathways, signal transduction, protein interaction, and other network-related pathways. All terms with a P-value (Benjamini or Benjamini– Hochberg adjusted) less than 0.05 were considered significant and ranked by adjusted P-value or the number of genes identified in the group. Cell-cell communication analysis CellCall was performed to infer inter- and intracellular communication pathways by integrating paired ligand-receptor and transcription factor (TF) activity among all cell types. The L (ligand) -R (receptor) -TF axis dataset was extracted from the KEGG pathway analysis. This method infers the potential interaction strength between two cell subsets based on gene expression level, and provides the significance through permutation test. Only those with a P- value < 0.01 were used for the prediction of cell-cell communication between any two cell types. In addition, CellCall embeds a pathway activity analysis method to help explore the main pathways involved in communication between certain cell types. In brief, the activity of pathway i was quantified according to the pathway activity score nPASi, based on Jaccard similarity coefficient, and the significance of pathway activity was estimated by hypergeometric testing. Immunofluorescence For immunofluorescence, hearts were collected and encased in optimal cutting temperature (OCT) compound (Sakura, Torrance, CA, USA). Frozen slices (7-µm thickness) were obtained and sealed with diluted donkey serum for 1 h at 25°C, followed by overnight incubation with primary antibody at 4°C. Visualized signal of secondary antibody (Invitrogen, Carlsbad, CA, USA) conjugated with Alexa. The following primary antibodies were used in these experiments: CD68 (AB53444, 1:300; Abcam, Cambridge, England), TNNI3 (#SAB2502170, 1:300; Sigma-Aldrich, St. Louis, MO, USA), Lgmn (sc-133234, 1:300; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Myoglobin (AB77232, 1:300; Abcam, Cambridge, England). Sections were viewed and photographed using a confocal laser scanning microscope (LEICA TCS SP8, Oberkochen, Germany). Terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labeling (TUNEL) assay TUNEL staining was performed with an In Situ Apoptosis Detection kit (Yeasen, Shanghai, China), according to the manufacturer’s instructions. Images were obtained using a laser scanning confocal microscope (LEICA TCS SP8, Oberkochen, Germany). Histological Analysis Hearts collected after mice were perfused with cold PBS, fixed with 4% paraformaldehyde for 24 hours, paraffin or OCT embedding, paraffin embedding interval of 6µm, OCT embedding interval of 8µm, and continuous sections were performed. Continuous sections were stained with Masson's trichrome 19 for detection of myocardial fibrosis. The images were captured by a Leica microscope (DM6000B, Leica, Germany). In order to quantify myocardial fibrosis, 10 visual fields were randomly selected from 3 heart sections to calculate the percentage of Masson’s trichromatic positive staining area in the total myocardial area. Western Blot Analysis Cell particles and heart tissues samples were homogenized in RIPA Lysis Buffer (Beyotime, Shanghai, China) containing proteinase and phosphatase inhibitor cocktail. Protein concentrations were determined by a BCA kit (23225, Pierce, USA). Protein (30 to 50 mg total protein) was isolated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE; Biorad, Hercules, California, USA) gels, and then transferred to polyvinylidene difluoride (PVDF) membranes. Subsequently, in TBS-T, the membranes were blocked in 5% non-fat dried milk at room temperature for 2 hours and incubated with indicated primary antibodies at 4°C overnight according to the requirements of each experiment. The primary antibodies used in the study were: TNNI3 (#SAB2502170, 1:2000; Sigma-Aldrich, St. Louis, MO, USA), Lgmn (sc-133234, 1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Myoglobin (AB77232, 1:2000; Abcam, Cambridge, England) and GAPDH (HRP60004,1:10000; Proteintech,San Diego, MO, USA). The second day, it was washed three times with TBS-T for 10 min, and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:3000) for 2 hours at room temperature. After washing in TBS-T for another three times, all membranes were detected using a chemiluminescent system, and signal intensities were analyzed with an Amersham Imager 600 (GE Healthcare, USA). Experiments were repeated three times and the target protein level was quantified by Image J and normalized to internal control. Statistical Analysis Results are presented as mean ± SEM for at least 3 independent assays unless otherwise noted. Data normality was determined by the Shapiro-Wilk test. The Student t test was used for 2-sample comparisons; 1-way ANOVA with Turkey post hoc tests was used for comparisons between multiple groups; and 2-way ANOVA was used for comparisons between multiple groups when there were 2 experimental factors. Nonnormal data were analyzed by Mann-Whitney U test or Kruskal-Wallis test with Dunn multiple comparisons. A value of P < 0.05 was considered statistically different. GraphPad Prism 8 and SPSS 22 for Windows was used to perform all statistical analyses. Results AET improved ventricular remodeling and cardiac function in HF To unravel the functional alterations in HF, we first constructed chronic HF mouse model through LAD surgery, and wild-type mice served as control. To investigated the pathological features in HF after myocardial ischemia mice, we compared the severity of cardiac function whether subjected to LAD surgery. Echocardiography confirmed the cardiac abnormalities in the ischemia-induced HF mice compared with WT mice, reflecting in the significant differences of echocardiographic parameters (Fig. 1 A- 1 C). Cardiac function was analyzed before and after 4-week AET (Fig. 1 A). The echocardiographic analyses revealed expansion of LV dilatation (LV end-systolic diameter, LVESD), reduction in left ventricular ejection fraction (LVEF), and fractional shortening (FS) in the ischemia-induced HF mice compared with WT mice (Fig. 1 B and 1 C), indicating deteriorated cardiac function in HF mice. Taken together, these findings indicate that compared to mice with normal cardiac function, ischemia-induced HF mice exhibit notable increases in ventricular remodeling, leading to heart function exacerbation. To test whether AET can enhance the cardiac function of HF mice, we performed echocardiography, which displayed effective improvement on cardiac function through the echocardiographic parameters in HF mice after AET(Fig. 1 A- 1 C). The left ventricular ejection fraction (LVEF) and fractional shortening (FS) aggrandized obviously in the AET mice compared with HF mice (Fig. 1 A- 1 C), manifesting AET significantly improved cardiac function in HF mice. In addition, AET improved the severity of myocardial fibrosis in HF mice as determined by masson’s trichrome staining (Fig. 1 D). To sum up, AET, as a possible treatment, can meet the needs of HF patients for improving ventricular remodeling and myocardial fibrosis, thereby improving cardiac function in the chronic repair stage of heart injury. AET promoted the clearance of increased apoptotic cardiomyocytes during HF To investigated the key molecular events and regulators controlling cardiac ventricular remodeling of HF by AET, we performed single-nucleus RNA sequencing (snRNA-Seq) and compared the difference of RNAs between HF without and with AET in mice. We firstly generated whole transcriptomes of the heart tissues at single-nucleus resolution using 10x Genomics technology, reaching a median depth of 25,426 reads/nucleus and 988 genes/nucleus. After removing low-quality nuclei, a total of 46955 single nuclei (29,714 single nuclei for HF group, and 17241 for control group), which aggregated into 26 clusters on the basis of transcriptional similarity, depicted the cardiac cell atlas and transcriptional heterogeneity (Fig. 2 A, S1A, S1B). To identify principal cell types involved in heart remodeling, we cataloged these single nuclei into 8 distinct cell lineages annotated with canonical marker gene expression, thus defining cardiomyocytes, endothelial cells, fibroblasts, macrophages, T cells/NK cells, B cells, myofibroblasts, and pericytes (Fig. 2 B and 2 C). To gain insights into the changes of cardiomyocytes between ischemia-induced HF and Ctrl, we compared the transcriptomic signatures from the hearts of mice with or without LAD surgery, and found that several cardiomyocyte subtypes increased dramatically in ischemia-induced HF (Fig. 2 D). To clarify these interesting subsets, we dissected cell-type heterogeneity by performing cell subtype analysis. Sub-clustering of cardiomyocyte revealed 9 clusters, among which, subcluster 0 highlighted the tremendous growth of cell number(Figure S1 C and S1D). Each subtype possessed cardiomyocytes from three mice at the same time point, suggesting the reliability and reproducibility of our data. To elucidate the nature of these specific cardiomyocytes, we first investigated the pro-apoptotic, anti-apoptotic and necrosis marker genes expression in different groups. After screening the expression profiles of these specific cardiomyocytes, ischemia-induced HF showed statistically credible increases in apoptotic cells (Fas, Casp4 and Casp8), and necrosis cells (Mb, Ldha and Ckm) compared to WT mice, whereas the anti-apoptotic genes (Bcl2, Mcl1 and Sirt1) expression levels decreased markedly (Fig. 2 E to 2 G). Furthermore, TUNEL staining and western blot confirmed increased cardiomyocyte apoptosis in the hearts of ischemia-induced HF mice (Fig. 2 H and 2 I). Taken together, these findings show that apoptotic cardiomyocytes increased notably in HF. To investigate the function of AET during ventricular remodeling in vivo, ischemia-induced HF mice were subjected to train with a treadmill for 4 weeks. Meanwhile, WT-HF mice were fed conventionally without specific exercise training. As anticipated, in HF mice after AET compared to the HF mice without AET, sub-clustering analysis of cardiomyocyte demonstrated a considerable decrease in quantity of apoptotic cardiomyocytes, which principally concentrated upon subcluster 0 and 8 (Fig. 2 D, S1C and S1D). In addition, The expression levels of canonical marker genes of apoptotic (Fas, Casp4 and Casp8) and necrosis (Mb, Ldha and Ckm) that involved in apoptotic cardiomyocytes experienced a noteworthy reduction in AET mice compared to WT-HF mice, in contrast, the anti-apoptotic genes (Bcl2, Mcl1 and Sirt1) expression levels displayed a remarkable upregulation (Fig. 2 E to 2 G).Furthermore, western blot and TUNEL confirmed decreased cardiomyocyte apoptosis in the hearts of HF mice after AET (Fig. 2 H to 2 I). To sum up, AET, as a possible treatment, can meet the needs of HF patients for improving the efficiency of clearing apoptotic cardiomyocytes, thereby improving ventricular remodeling and cardiac function. Cell-cell communication between cardiomyocytes and macrophages increases in HF mice treated with AET To explore the underlying mechanisms of AET in clearing the apoptotic cardiomyocytes in HF mice, we then applied the latest cell-cell communication analytic algorithm to our dataset, and observed an overall alteration in intercellular crosstalk. We found that the overall communication quantity was increased in HF sample compared with control sample, with the most pronounced changes between the cardiomyocytes and macrophages (Fig. 3 A). Interactions of cardiomyocytes-macrophages increased both in HF mice with or without AET, supporting the role of efferocytosis by macrophages as a key event in the clearance of the apoptotic cardiomyocytes during ischemia-induced HF. Moreover, to identify whether the amount of macrophages had changed in different groups, we predicted the macrophage count through the single-nucleus transcriptomic signatures, and found that macrophages decreased in HF mice while increased dramatically in HF mice with AET (Fig. 3 B). To test this, using cardiomyocyte marker cardiac troponin I (TNNT3) and macrophage marker CD68, we examined macrophage and cardiomyocyte colocalization in myocardial tissue sections isolated from WT, HF with or without AET mice, respectively. We then calculated the percentage of cardiac resident macrophages to assess if AET enhances macrophage function in myocardial tissue (Fig. 3 C and 3 D).As anticipated, in the heart tissue from HF mice, but not normal mice, the number of macrophages (CD68 + ) significantly decreased and that they were in contact with cardiomyocytes (TNNT3 + ) (Fig. 3 C), whereas the number of macrophages (CD68 + ) increased remarkably in the hearts of HF mice with AET, displaying the more obvious induction of the percentage of these cardiomyocyte-containing macrophages compared to the HF mice (Fig. 3 D).Besides, AET can notably increase the function of macrophages (CD68 + ) that clear the necrosis cardiomyocytes (Myoglobin + ) in HF (Fig. 3 E).Together, these findings highlight the presence of multiple functional macrophages and their potential roles in efficient-clearance of apoptotic cardiomyocytes in HF mice after AET. Lgmn is critical for AET to enhance macrophage efferocytosis and resolve inflammation in HF To elucidate the role of macrophage-mediated efferocytosis in ischemia-induced HF and its effect on AET-HF, we first investigated its putative genes expression involved in endocytosis and intracellular trafficking in mouse myocardial tissues. We compared the transcriptomic signatures of macrophages in the heart tissues of WT, HF, and AET mice, observing that the expression of these genes involved in efferocytosis (Lgmn, Fcgr4, Fcgr1, Tlr7, and Msr1) was reduced in HF mice and increased significantly in AET mice (Fig. 4 A). Because of its high and specific expression in macrophages 1 , Lgmn was considered as a major candidate gene to explore the overall function changes in macrophages’ clearance of apoptotic cardiomyocytes in HF mice after AET. Thus, we this time focus mainly on the differences in Lgmn. Lgmn expression levels decreased obviously in myocardial tissues of HF mice compared with the WT mice, whereas the expressions confirmed to increase in HF mice after AET (Fig. 4 B to 4 E, S2A to S2C). Double-immunofluorescence staining for Lgmn along with CD68 confirmed that Lgmn was expressed predominately by cardiac CD68 + macrophages and AET can induce its expression in ischemia-induced HF (Fig. 4 F). Ventricular remodeling is a complex process with diverse manifestations, including not only myocardial fibrosis,but also inflammatory cell infiltration. Since the Lgmn-dependent phagocytosis was responsible for inflammation resolution 1 , we first examined the genes expression of anti-inflammatory mediators interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1), as well as proinflammatory mediators interleukin1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) by the global single-nucleus transcriptome analysis in HF mice with or without AET, and then confirmed by expression detection assay. Gene expression analyses and verification of the anti-inflammatory mediators and proinflammatory mediators confirmed delayed inflammation resolution in the ischemia-induced HF mice after LAD surgery compared with the WT mice, whereas the AET promoted the post-HF inflammation resolution (Fig. 4 G and 4 H). Taken together, these findings indicate that AET accelerates the efferocytosis of macrophages mediating proper inflammation resolution, and Lgmn is required for AET to clear the apoptotic cardiomyocytes by macrophages in the chronic repair stage of heart injury. Discussion The process of ventricular remodeling underlies the fundamental pathology of HF, with a multifaceted etiology contributing to its complexity 20 , 21 , 22 . The application of AET in HF elicits a multitude of beneficial outcomes, such as mitigating myocardial hypertrophy and myocardial fibrosis, thereby ameliorating pathological ventricular remodeling 23 , 24 . The precise mechanism by which AET enhances ventricular remodeling in HF remains elusive and is a subject of ongoing research. We penetrated some of mysteries between AET and cardiac ventricular remodeling, confirming that AET plays an important role in effectively mitigating the remarkable accumulation of apoptotic cardiomyocytes associated with HF. Single-nucleus sequencing detected a substantial upsurge in certain apoptotic cardiomyocyte subsets within the HF, which were markedly diminished following AET. Studies have shown that 1 , 25 , inadequate clearance of apoptotic cardiomyocytes during cardiac injury triggers secondary necrosis, precipitating additional damage, causing the loss of adjacent, non-regenerative cardiomyocytes. Therefore, maintaining cardiac function necessitates a mechanism that ensures the efficient removal of apoptotic cardiomyocytes. The number of apoptotic cardiomyocytes is a crucial determinant of poor remodeling of the injured heart, which also related to the clearance efficiency of necrotic and apoptotic cardiomyocytes during cardiac injury 1 , 26 . The effective clearance of dead cells and the formation of scar tissue promote the regression of inflammation and prevent widespread cell death, thereby helping preserve heart integrity and slowing the progression of myocardial injury. In a state of homeostasis, apoptosis stands as the prevalent form of cell demise, and the effective removal of the resulting cellular remnants is crucial for averting the buildup of debris that may precipitate detrimental inflammatory reactions 27 , 28 , 29 . This clearance is facilitated by a specialized process termed efferocytosis 27 , 30 , 31 , which is the engulfment and degradation of apoptotic cells by phagocytes. Efferocytosis, elimination of dead or apoptotic cells from viable tissues, is a highly programmed and vital process to maintain the healthy function of the organism. As one of the specialized phagocytes, macrophages show effective efferocytosis during myocardial injury, thus preventing the secondary necrosis of apoptotic cells to release protease, oxide, antibodies 32 , which cause inflammation. Currently, there is a dearth of research examining the influence of AET on the process of Efferocytosis. It is therefore crucial to explore the potential impact of AET on the clearance of apoptotic cardiomyocytes by macrophages through efferocytosis and to determine its subsequent effects on the improvement of ventricular remodeling. We have observed that AET has the potential to not only augment the diminished macrophage population associated with HF but also to markedly improve the interplay between these immune cells and cardiomyocytes within the failing heart. AET plays a crucial role in macrophage-mediated efferocytosis processing of apoptotic cardiomyocytes, particularly in the chronic phase of cardiac injury, contributing to the proper resolution of inflammation, improving ventricular remodeling, and ultimately leading to improvements in cardiac function. Consequently, AET is vital for preventing the accumulation of apoptotic cells during cardiac injury, avoiding the excessive release of apoptotic cardiomyocyte contents, and facilitating the timely resolution of inflammation, all of which are crucial for preserving the integrity of cardiac function. Analyzing single-nucleus sequencing data, we identified that Lgmn, a gene highly specific to macrophages and an essential lysosomal enzyme 1 , was among those differentially expressed in HF mice post-exercise, playing a notable role in efferocytosis. Therefore, we further explored the role of Lgmn in the promotion of efferocytosis by AET. As anticipated, AET markedly elevated Lgmn expression in macrophages of HF, preserved the anti-inflammatory/pro-inflammatory immune equilibrium, and prevented additional immune-mediated cardiac damage. Previous studies 1 during the acute phase of MI have similarly demonstrated that a deficiency in Lgmn hampers the capacity of cardiac macrophages to effectively remove and degrade apoptotic cardiomyocytes post-MI. Impaired efferocytosis results in the accumulation of inflammogenic material and disruption of efferocytosis-dependent anti-inflammatory signaling within resident cardiac macrophages 1 . Lgmn is evidently essential for the removal of apoptotic cardiomyocytes and the preservation of cardiac function during cardiac injury. Ventricular remodeling in HF is governed by intricate pathological mechanisms, with the cardiac microenvironment being significantly influenced by a diverse array of cellular components. While the present investigation is predominantly centered on cardiomyocytes, it is prudent to acknowledge the possible contributions of other cellular constituents. Subsequent research endeavors should aim to conduct a more comprehensive analysis of the diverse cell types involved to elucidate the precise mechanisms by which AET exerts its effects in the multifaceted cellular milieu of HF. Concurrently, ventricular remodeling in the setting of HF is intricately linked to the extent of myocardial fibrosis. The specific mechanisms influencing myocardial fibrosis, including but not limited to endothelial-to-mesenchymal transition (EndoMT) 33 , 34 , 35 , must be considered and further explored to fully comprehend the multifactorial nature of this process. Future research endeavors are imperative for delving into the potential intercellular interactions mediated by AET and their implications on the ventricular remodeling process in HF. Conclusions AET, as an efficient prevention and treatment of HF, repairs the proper expression and function of Lgmn and macrophage efferocytosis pathways, making the optimal clearance of dying cardiomyocytes, mediating proper inflammation resolution, improving ventricular remodeling, and strengthening cardic function in the chronic repair stage of heart injury. Therefore, AET, as a measure to boost Lgmn and heighten efferocytosis, is a feasible approach for enhancing cardiac protection during HF. Abbreviations HF heart failure AET aerobic exercise training Lgmn legumain LAD left anterior descending SnRNA-Seq single-nucleus RNA sequencing QoL quality of life TUNEL terminal deoxynucleotidyl transferasemediated dUTP-biotin nick-end labeling WT wild-type Declarations Acknowledgments Xiaoling Liu, Chun Li, Yuxuan Fan and Zhongyan Zhou contributed equally to this article. All authors gave final approval and agreed to be accountable for the integrity and accuracy of all aspects of the work. Sources of Funding This study was supported by the Key Research and Development Special Project of the Autonomous Region（2022B03023-3）and Key Supported Discipline of Health System in Shanghai (2023ZDFC0302) . Conflict of Interest None. References Jia D., et al. Cardiac Resident Macrophage-Derived Legumain Improves Cardiac Repair by Promoting Clearance and Degradation of Apoptotic Cardiomyocytes After Myocardial Infarction. Circulation 145, 1542–1556 (2022). Sun H., et al. Risk prediction model construction for post myocardial infarction heart failure by blood immune B cells. Front Immunol 14, 1163350 (2023). Alvarez, C.K., Cronin, E., Baker, W.L. & Kluger, J. Heart failure as a substrate and trigger for ventricular tachycardia. J Interv Card Electrophysiol 56, 229–247 (2019). Pilz PM., et al. Large and Small Animal Models of Heart Failure With Reduced Ejection Fraction. Circulation Research 130, 1888–1905 (2022). Sygitowicz, G., Maciejak-Jastrzębska, A. & Sitkiewicz, D. MicroRNAs in the development of left ventricular remodeling and postmyocardial infarction heart failure. Pol Arch Intern Med 130, 59–65 (2020). McDonagh T.A., et al. 2021 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure. European Heart Journal 42, 3599–3726 (2021). Heidenreich P.A., et al. 2022 AHA/ACC/HFSA Guideline for the Management of Heart Failure: A Report of the American College of Cardiology/American Heart Association Joint Committee on Clinical Practice Guidelines. Circulation 145, (2022). van der Meer, P., Gaggin, H.K. & Dec G.W. ACC/AHA Versus ESC Guidelines on Heart Failure: JACC Guideline Comparison. J Am Coll Cardiol 73, 2756–2768 (2019). Chen H., et al. Exercise training maintains cardiovascular health: signaling pathways involved and potential therapeutics. Signal Transduct Target Ther 7, 306 (2022). Yin A., et al. Exercise-derived peptide protects against pathological cardiac remodeling. EBioMedicine 82, 104164 (2022). Adams, V. & Linke, A. Impact of exercise training on cardiovascular disease and risk. Biochim Biophys Acta Mol Basis Dis 1865, 728–734 (2019). Tucker, W.J., Fegers-Wustrow, I., Halle, M., Haykowsky, M.J., Chung, E.H. & Kovacic, J.C. Exercise for Primary and Secondary Prevention of Cardiovascular Disease: JACC Focus Seminar 1/4. J Am Coll Cardiol 80, 1091–1106 (2022). Bozkurt B., et al. Cardiac Rehabilitation for Patients With Heart Failure: JACC Expert Panel. J Am Coll Cardiol 77, 1454–1469 (2021). Zhou J. Cycling and heart failure: A 2-sample Mendelian randomization. Medicine 103, e37619 (2024). Kwak, H.B., Kim, J.h., Joshi, K., Yeh, A., Martinez, D.A. & Lawler, J.M. Exercise training reduces fibrosis and matrix metalloproteinase dysregulation in the aging rat heart. FASEB J 25, 1106–1117 (2011). Lerchenmüller C., et al. CITED4 Protects Against Adverse Remodeling in Response to Physiological and Pathological Stress. Circulation Research 127, 631–646 (2020). Gao E., et al. A novel and efficient model of coronary artery ligation and myocardial infarction in the mouse. Circulation Research 107, 1445–1453 (2010). Gao, S., Ho, D., Vatner, D.E. & Vatner, S.F. Echocardiography in Mice. Curr Protoc Mouse Biol 1, 71–83 (2011). Bi H.L., et al. The deubiquitinase UCHL1 regulates cardiac hypertrophy by stabilizing epidermal growth factor receptor. Sci Adv 6, eaax4826 (2020). Yerra V.G., et al. Pressure overload induces ISG15 to facilitate adverse ventricular remodeling and promote heart failure. J Clin Invest 133, (2023). Wan J., et al. Astragaloside IV derivative HHQ16 ameliorates infarction-induced hypertrophy and heart failure through degradation of lncRNA4012/9456. Signal Transduct Target Ther 8, 414 (2023). Zheng X., et al. Fibulin7 Mediated Pathological Cardiac Remodeling through EGFR Binding and EGFR-Dependent FAK/AKT Signaling Activation. Advanced Science (Weinheim, Baden-Wurttemberg, Germany) 10, e2207631 (2023). Liu K., et al. Exercise training ameliorates myocardial phenotypes in heart failure with preserved ejection fraction by changing N6-methyladenosine modification in mice model. Front Cell Dev Biol 10, 954769 (2022). Sun J., et al. Protective effect of urotensin II receptor antagonist urantide and exercise training on doxorubicin-induced cardiotoxicity. Scientific Reports 13, 1279 (2023). Rahnavard M., et al. Curcumin ameliorated myocardial infarction by inhibition of cardiotoxicity in the rat model. J Cell Biochem 120, 11965–11972 (2019). Patil M., et al. Novel Mechanisms of Exosome-Mediated Phagocytosis of Dead Cells in Injured Heart. Circulation Research 129, 1006–1020 (2021). Mehrotra, P. & Ravichandran, K.S. Drugging the efferocytosis process: concepts and opportunities. Nat Rev Drug Discov 21, 601–620 (2022). Boada-Romero, E., Martinez, J., Heckmann, B.L. & Green, D.R. The clearance of dead cells by efferocytosis. Nat Rev Mol Cell Biol 21, 398–414 (2020). Gao J., et al. SIRT3 Regulates Clearance of Apoptotic Cardiomyocytes by Deacetylating Frataxin. Circulation Research 133, 631–647 (2023). Gerlach B.D., et al. Efferocytosis induces macrophage proliferation to help resolve tissue injury. Cell Metab 33, (2021). Raymond M.H., et al. Live cell tracking of macrophage efferocytosis during Drosophila embryo development in vivo. Science (New York, NY) 375, 1182–1187 (2022). Jung S.H., et al. Spatiotemporal dynamics of macrophage heterogeneity and a potential function of Trem2hi macrophages in infarcted hearts. Nat Commun 13, 4580 (2022). Bischoff J. Endothelial-to-Mesenchymal Transition. Circulation Research 124, 1163–1165 (2019). Kovacic J.C., et al. Endothelial to Mesenchymal Transition in Cardiovascular Disease: JACC State-of-the-Art Review. J Am Coll Cardiol 73, 190–209 (2019). Li, Y., Lui, K.O. & Zhou, B. Reassessing endothelial-to-mesenchymal transition in cardiovascular diseases. Nat Rev Cardiol 15, 445–456 (2018). Additional Declarations (Not answered) Supplementary Files SupplementaryInformation.docx 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. 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-4420177","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":315221830,"identity":"d8e83b12-30e8-4cdd-83e9-a2f7317e3be5","order_by":0,"name":"Yuqin Shen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYJCCAx8qauTY2JsPEK2D8eCMM8eM+XiOJRCthfkwbwtz4jyJHAXi1MvPSGA4zNvAlt7GkMPA8KNiG2EtBjcSGA7O3SGT28Zw9gBjz5nbRGiRSGA48PYMW24bY18CM2MbEVpADjvA28aczsbMY0CcFgaQw4BaEtjYiNVicOYBAyiQDdt42BIOEuUX+fYE5g/AqJSXn//44IMfFcQ4TCD/A5x9gAj1QMBPpLpRMApGwSgYwQAATiQ+nOLceOEAAAAASUVORK5CYII=","orcid":"","institution":"Department of Rehabilitation, Tongji Hospital Affiliated to Tongji University, Tongji University School of medicine","correspondingAuthor":true,"prefix":"","firstName":"Yuqin","middleName":"","lastName":"Shen","suffix":""},{"id":315221831,"identity":"a19dfa84-7caf-49f9-947c-4abe06c57599","order_by":1,"name":"Xiaoling Liu","email":"","orcid":"","institution":"Department of Rehabilitation, Tongji Hospital Affiliated to Tongji University, Tongji University School of medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiaoling","middleName":"","lastName":"Liu","suffix":""},{"id":315221832,"identity":"f38388c0-f626-4575-bb92-7eaead528280","order_by":2,"name":"Chun Li","email":"","orcid":"","institution":"Tongji Hospital, Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Chun","middleName":"","lastName":"Li","suffix":""},{"id":315221833,"identity":"1362d5f2-3934-46e6-a039-eeee5886e9a3","order_by":3,"name":"Yuxuan Fan","email":"","orcid":"","institution":"Department of Rehabilitation, Tongji Hospital Affiliated to Tongji University, Tongji University School of medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuxuan","middleName":"","lastName":"Fan","suffix":""},{"id":315221834,"identity":"4b615ff6-08f1-4abc-8997-712693438116","order_by":4,"name":"Zhongyan Zhou","email":"","orcid":"","institution":"Longhua hospital, Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhongyan","middleName":"","lastName":"Zhou","suffix":""},{"id":315221835,"identity":"1ba329ee-abb6-4a14-870c-157ad3371d9a","order_by":5,"name":"Wenjuan Xiu","email":"","orcid":"","institution":"Department of Rehabilitation, Tongji Hospital Affiliated to Tongji University, Tongji University School of medicine","correspondingAuthor":false,"prefix":"","firstName":"Wenjuan","middleName":"","lastName":"Xiu","suffix":""},{"id":315221836,"identity":"53651145-9106-4f5a-aaad-0f1f1b40049c","order_by":6,"name":"Baopeng Tang","email":"","orcid":"","institution":"The First Affiliated Hospital of Xinjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Baopeng","middleName":"","lastName":"Tang","suffix":""},{"id":315221837,"identity":"673428ac-efc6-4002-9098-8ec45b48b2e5","order_by":7,"name":"Lemin Wang","email":"","orcid":"","institution":"Department of Rehabilitation, Tongji Hospital Affiliated to Tongji University, Tongji University School of medicine","correspondingAuthor":false,"prefix":"","firstName":"Lemin","middleName":"","lastName":"Wang","suffix":""},{"id":315221838,"identity":"36501fe1-ffd3-4659-b24f-a6ab8bb2383d","order_by":8,"name":"Haoming Song","email":"","orcid":"","institution":"Department of General Practice, Tongji Hospital, School of Medicine, Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Haoming","middleName":"","lastName":"Song","suffix":""},{"id":315221839,"identity":"7114d4af-a530-44e0-8259-ff443e8b05ab","order_by":9,"name":"Jingyi Tang","email":"","orcid":"","institution":"Longhua hospital, Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jingyi","middleName":"","lastName":"Tang","suffix":""},{"id":315221840,"identity":"d301783b-a313-4966-8b35-8e76899ffbea","order_by":10,"name":"Siguang Li","email":"","orcid":"","institution":"Tongji Hospital, Tongji University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Siguang","middleName":"","lastName":"Li","suffix":""},{"id":315221841,"identity":"e181fdc1-c784-42c0-bc08-b77f5a9ff7bf","order_by":11,"name":"Lixia Lu","email":"","orcid":"","institution":"Tongji Hospital, Department of Ophthamology, Tongji Eye Institute, Tongji University School of Medicine Department of Regenerative Medicine, and Department of Pharmacology","correspondingAuthor":false,"prefix":"","firstName":"Lixia","middleName":"","lastName":"Lu","suffix":""}],"badges":[],"createdAt":"2024-05-14 15:06:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4420177/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4420177/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59566554,"identity":"2a438051-3129-43e1-b0c1-77bb6404c304","added_by":"auto","created_at":"2024-07-03 09:16:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2106690,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAET improved ventricular remodeling and cardiac function in HF.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e, Representative parasternal long-axis views, short-axis views, and M-mode images before and after 4-week AET. \u003cstrong\u003eB\u003c/strong\u003e, Echocardiographic analysis the difference value of left ventricular ejection fraction (LVEF), fractional shortening (FS), left ventricular end-systolic dimension (LVESD) after 4-week AET (n=10), *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05. \u003cstrong\u003eC\u003c/strong\u003e, Echocardiographic analysis of LVEF, FS before and after 4-week AET (n=10). Data are expressed as mean±SEM, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. \u003cstrong\u003eD\u003c/strong\u003e, Representative Masson's trichrome staining of cardiac tissue obtained from WT mice after LAD or sham operation, HF mice with AET.Scale bar, 25 μm. Quantitative analysis of the severity of myocardial fibrosis (n = 10, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 by 1-way ANOVA followed by Bonferroni post hoc analysis).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4420177/v1/a5e346603346ca5101d12fcc.png"},{"id":59566551,"identity":"83920762-45bd-4685-9db7-08d03b2c85dc","added_by":"auto","created_at":"2024-07-03 09:16:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2052585,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAET promoted the clearance of increased apoptotic cardiomyocytes during HF.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e, T-Distributed Stochastic Neighbor Embedding (t-SNE) projection showing 25,426 single nuclei isolated from hearts at different group. Nuclei were marked by cluster number. \u003cstrong\u003eB\u003c/strong\u003e, t-SNE showing 25,426 single nuclei isolated from ischemia-induced HF mice. Cell types were determined according to the expression of known markers. \u003cstrong\u003eC\u003c/strong\u003e, Bubble diagram showing differentially expressed genes in each cell type. \u003cstrong\u003eD\u003c/strong\u003e, The distribution characteristics of all cells type (display from WT mice after LAD operation, HF mice with AET). \u003cstrong\u003eE\u003c/strong\u003e, The pro-apoptotic genes expression in different mice. \u003cstrong\u003eF\u003c/strong\u003e,The anti-apoptotic genes expression in different mice. \u003cstrong\u003eG\u003c/strong\u003e, The necrosis marker genes expression in different mice. \u003cstrong\u003eH\u003c/strong\u003e, Western blot shows the differences of the expression of Myoglobin and TNNI3 among groups. \u003cstrong\u003eI\u003c/strong\u003e, Representative photomicrographs of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and nuclear DAPI staining of cardiomyocyte marker cardiac troponin I (TNNI3)–positive cardiomyocytes obtained from WT, HF, and HF with AET mice. White arrows point out TUNEL-positive (green) cardiomyocyte (red) nuclei (blue; scale bar, 50 μm). Percentage of TUNEL-positive cardiomyocytes after LAD surgery, compared to WT mice (n=10; *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 vs WT by 1-way ANOVA followed by Bonferroni post hoc analysis).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4420177/v1/3d2b85e3810e635181ff192d.png"},{"id":59567241,"identity":"edaf567c-c28d-4fc9-8259-91677cd1c80a","added_by":"auto","created_at":"2024-07-03 09:24:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3515553,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell-cell communication between cardiomyocytes and macrophages increases in HF mice treated with AET.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e, Alterations in cell-cell interactions in WT, HF with or without AET mice. \u003cstrong\u003eB\u003c/strong\u003e, Information of nuclei proportion in each cell type. \u003cstrong\u003eC\u003c/strong\u003e, Immunofluorescence shows colocalization of CD68+ macrophages with TNNI3+ cardiomyocytes (white). Scale bar, 50 μm. \u003cstrong\u003eD\u003c/strong\u003e, Analysis of internalization of cardiomyocyte-derived proteins in macrophages. Macrophages that stain positive for cardiomyocyte TNNI3 are scored as having internalized cardiomyocyte-derived proteins (n=10, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs WT MI by Mann-Whitney U test). \u003cstrong\u003eE\u003c/strong\u003e, Immunofluorescence shows colocalization of CD68+ macrophages with Myoglobin+cardiomyocytes (white),*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4420177/v1/6e37017012c50253ac6e583b.png"},{"id":59566549,"identity":"1a040349-c388-457e-a194-128bab3b28f4","added_by":"auto","created_at":"2024-07-03 09:16:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1691484,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLgmn is critical for AET to enhance macrophage efferocytosis and resolve inflammation in HF.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e, Some genes differentially expressed between groups related to macrophage phagocytosis. \u003cstrong\u003eB-C\u003c/strong\u003e,Expression of Lgmn in WT, HF with or without AET mice showed by bubble diagram and t-SNE. \u003cstrong\u003eD-E\u003c/strong\u003e, Legumain (Lgmn) expression levels in myocardial tissues were analyzed at different mice (n=10, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01,***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 by Mann-Whitney U test). \u003cstrong\u003eF\u003c/strong\u003e, Immunofluorescence showed colocalization of CD68+ macrophages with Lgmn (yellow). Scale bar, 20 μm. \u003cstrong\u003eG\u003c/strong\u003e, Expression of anti-inflammatory mediators interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) and proinflammatory mediators interleukin1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in WT, HF with or without AET mice showed by violin diagram. \u003cstrong\u003eH\u003c/strong\u003e, Expression of TGF-β, IL-1β, TNF-α, and IL-6 in blood were performed at different mice (n =10, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01,***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 by 1-way ANOVA followed by Bonferroni post hoc analysis).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4420177/v1/325b2292ec77ddb2cbb3f7da.png"},{"id":60778337,"identity":"01a77967-74ef-4253-9d2d-cd033d39d02a","added_by":"auto","created_at":"2024-07-21 23:08:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9771116,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4420177/v1/e6e7086b-de5d-44d0-91b7-9047639a2404.pdf"},{"id":59566552,"identity":"2ca721a9-5ddd-4d6e-8f8d-d1e4e7a6d9a7","added_by":"auto","created_at":"2024-07-03 09:16:57","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":392120,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4420177/v1/f477b19e541e2ab09d7ceab1.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Aerobic exercise improves ventricular remodeling by promoting macrophages to phagocytose dying cardiomyocytes in heart failure model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHeart failure (HF) is a heterogeneous clinical syndrome stemming from cardiac overload and injury with considerable morbidity and mortality\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The incorporation of various medications such as angiotensin-converting enzyme inhibitors (ACEI), angiotensin II receptor blockers (ARBs), angiotensin receptor/neprilysin inhibitors (ARNIs), beta-blockers, and aldosterone antagonists has marked a substantial decrease in mortality rates within the field of cardiology. Despite of these, the quality of life (QoL) and social engagement of patients with HF still experience a downward trend. Hence, additional and effective strategies to enhance heart function are imperative.\u003c/p\u003e \u003cp\u003eVentricular remodeling is a basic pathological process of HF and is characterized by pathological cardiomyocyte hypertrophy, apoptosis, fibroblast proliferation, myofibroblast formation, and interstitial fibrosis \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Aerobic exercise training (AET) as one of the non-pharmacological treatments of cardiac rehabilitation, has become one of the important therapeutic means for the long-term management of chronic HF \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. AET has been proven to be effective in mitigating the detrimental cardiac remodeling\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, enhancing one's cardiopulmonary function, decreasing readmission and mortality, and boosting social engagement and QoL\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Despite the observed benefits, the specific mechanisms responsible for these benefits have not yet been fully elucidated.\u003c/p\u003e \u003cp\u003eSeveral research works have shed light on certain mechanisms through which AET mitigates pathological ventricular remodeling in HF, notably including the inhibition of the TGF-β/smad2 signaling cascade and the TGF-β/TIMP-1/MMP-1 signaling pathway to reduce myocardial fibrosis\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, the activation of the β3-AR-NOS-NO signaling cascade to counteract oxidative stress\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, thereby attenuating myocardial hypertrophy. Anwen Yin et al.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e recently identified that AET stimulates the secretion of C-terminal of coiled-coil domain-containing protein 80 (CCDC80tide), which selectively binds to active kinase-active form of Janus kinase 2 (JAK2), inhibiting its ability to phosphorylate and activate signal transducer and activator of transcription 3 (STAT3). This interaction helps protect mice from against angiotensin II (Ang II)-induced cardiac hypertrophy and remodeling. Carolin Lerchenm\u0026uuml;ller's team \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e discovered that CBP/p300-interacting transactivators with E/D-rich-carboxylterminal domain4 (CITED4), a cardiac protein, guards against cardiac remodeling by modulating mTOR activity and miRNAs. Nonetheless, the existing research has been predominantly centered on macroscopic tissue-level assessments, thereby lacking of comprehending the underlying molecular dynamics at the cellular stratum.\u003c/p\u003e \u003cp\u003eTo address this inquiry, we developed models of HF by ligating the left anterior descending (LAD) coronary artery, and subsequently investigated the therapeutic effects and underlying cellular mechanisms of AET on pathological ventricular remodeling in vivo, leveraging single-nucleus RNA sequencing (snRNA-Seq) technology. Recent advancements in snRNA-Seq tech have facilitated the acquisition of high-resolution, cell-type-specific cardiac transcriptomic data, thereby allowing for a more nuanced understanding of cellular heterogeneity within the heart, avoiding the problems of bulk RNA sequencing that analyze mixed cells from different stages and potentially obscure critical molecular events and signals in specific cell populations. As anticipated, HF mice exhibited increased ventricular remodeling and cardiac dysfunction, but those treated with AET showed lessened ventricular remodeling and improved heart function.\u003c/p\u003e \u003cp\u003eNotably, our study has demonstrated that a 4-week regimen of AET not only effectively increases the typically diminished macrophage population observed in HF but also substantially amplifies the intricate interplay between these immune cells and cardiomyocytes within the affected cardiac milieu. This enhancement in cellular communication is instrumental in augmenting the efficiency of efferocytosis, thereby facilitating the clearance of in vivo accumulated apoptotic cardiomyocytes during HF. Furthermore, we revealed that cardamom protein (legumain, Lgmn) played a crucial role in AET's promotion of the effective phagocytosis of apoptotic cardiomyocytes and contributed to the appropriate resolution of inflammation. Investigating AET's regulatory mechanisms can demystify its relationship with heart disease, advancing more effective treatments.\u003c/p\u003e \u003c/div\u003e"},{"header":"Methods","content":" \u003cp\u003eMice\u003c/p\u003e \u003cp\u003eWild-type (WT) C57BL6/J mice (male, 6 weeks old; GemPharmatech Laboratory Animal, Shanghai, China) were used in this study. This study and all animal procedures conformed to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH publication No. 85\u0026thinsp;\u0026minus;\u0026thinsp;23, revised 1996) and were approved by the Experimental Animal Welfare and Ethics Committee, Shanghai University of Chinese Medicine (PZSHUTCM210312008).\u003c/p\u003e \u003cp\u003eModels of HF were established by ligation of the left anterior descending (LAD) coronary artery\u003c/p\u003e \u003cp\u003eModels of HF were established by ligation of the LAD coronary artery. LAD operation was performed on 6-week-old WT mice as described previously\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.Briefly, the mice were anesthetized with 4% chloral hydrate using intraperitoneal injection. Subsequently, we placed the endotracheal tube below the glottis and connected the tube with the ventilator (HX-101E, Techman Software, Chengdu, China) to assist the breathing mice. Then, a cut was made between the third and fourth intercostal space, and a 7\u0026thinsp;\u0026minus;\u0026thinsp;0 nylon suture was used for permanent ligation of the LAD. Animals, dead within the first 24 h after the surgery, were excluded from the analysis. Sham mice received the same operation without coronary artery ligation. At the specified time points, mice were euthanized by cervical dislocation, and tissues were then collected for analyses.\u003c/p\u003e \u003cp\u003eTransthoracic echocardiography\u003c/p\u003e \u003cp\u003eTransthoracic echocardiography assesses heart geometry, systolic and diastolic function, as described earlier\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.The Vevo2100 ultrasound system (VisualSonics, Toronto, Canada) were used. Mice were lightly anesthetized with 0.5% isoflurane until the heart rate stabilized at 400\u0026ndash;500 beats per minute, and placed on a ECG platform. Parasternal long-axis images were obtained in two-dimensional B mode at the appropriate scanning head position to determine the maximum LV length, and the sampling line was placed at the maximum cross-section of the left ventricle to guide the recording of continuous M-mode echocardiography. Automatic calculation of left ventricular ejection fraction (LVEF), fractional shortening (FS), left ventricular end-systolic dimension (LVESD), and left ventricular end-diastolic diameter (LVEDD) from at least three distinct frames for each mouse.\u003c/p\u003e \u003cp\u003eAerobic exercise training program\u003c/p\u003e \u003cp\u003eHF mice were trained with a treadmill ( Slope 0\u0026deg;), with the speed of 12 m/ min, the training time of 30 min/time, five times per week, for 4 weeks. Meanwhile, WT mice received fed conventionally without specific exercise training.\u003c/p\u003e \u003cp\u003e10X sample processing and single nucleus RNA sequencing (SnRNA-Seq)\u003c/p\u003e \u003cp\u003e Single nucleus suspensions were mixed-extracted from three biological replicate mice at indicated time points after LAD with Nuclear Separation Kit (Shanghai Biotechnology Corporation) according to the manufacturer\u0026rsquo;s instructions. The concentration of nuclear suspension was adjusted with the corresponding NB solution according to subsequent experiments. About 50,000 nuclei were loaded into one channel of the Single Cell Chip M for each sample using the Single Cell 3\u0026rsquo; HT kit (10X Genomics) for Gel bead Emulsion generation in the Chromium X system. Following capture and lysis, cDNA was synthesized and amplified for 14 cycles. 50 ng of the amplified cDNA were used for each sample to construct Illumina sequencing libraries. Sequencing was performed on the NextSeq500 Illumina sequencing platform following 10x Genomics instructions for reads generation.\u003c/p\u003e \u003cp\u003eBioinformatic analyses\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eRaw data processing\u003c/strong\u003e \u003cp\u003eRaw sequencing data (bcl-files) were converted to fastq files with the Illumina bcl2fastq tool, integrated into the CellRanger (10X Genomics) suite (version 2.1.1). The CellRanger analysis pipeline was used to generate a digital gene expression matrix starting from raw data. Pre-build mouse genome (version mm10-1.2.0) was used as genome reference. The CellRanger count module was used to map reads with default settings and sequence length set to r1-length\u0026thinsp;=\u0026thinsp;26 and r2-length\u0026thinsp;=\u0026thinsp;50. Global mapping statistics, such as Estimated Number of Cells, Mean Reads per Cell and Median Genes per Cell for each experimental condition are reported in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eQuality control (QC) and normalization of SnRNA-seq data\u003c/strong\u003e \u003cp\u003eFor further data pre-processing, filtering and QC were done using the Seurat package (v3.1.2). For each specimen, genes with counts in fewer than 10 nuclei were discarded to exclude random noise. Nuclei were filtered for genes (500\u0026thinsp;\u0026lt;\u0026thinsp;nFeature_RNA\u0026thinsp;\u0026lt;\u0026thinsp;5000), the proportion of mitochondrial genes (percent.mt\u0026thinsp;\u0026lt;\u0026thinsp;0.15) and the proportion of ribosomal genes (percent.ribo\u0026thinsp;\u0026lt;\u0026thinsp;0.05) to remove poor-quality nuclei potentially ascribed to doublets or other technical noise. For normalization, unique molecular identifier (UMI) counts for all nuclei were scaled by library size (total UMI counts), multiplied by 10,000 and transformed to a log scale.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eUnsupervised dimensional reduction and single cell clustering\u003c/strong\u003e \u003cp\u003eHighly variable genes (HVGs) were identified using the function FindVariableFeatures in Seurat, and 2000 HVGs were selected for each sample. Nuclei of all samples were integrated via canonical correlation analysis implemented in Seurat to correct potential batch effects and identify shared cell states across samples. Subsequently, the scaled data were subjected to linear dimensional reduction through principal component analysis (PCA). Using the first 40 PCA components, a shared nearest neighbor graph of the nuclei was calculated, followed by clustering and visualization with t-distributed stochastic neighbor embedding (t-SNE), which were performed using the FindClusters and RunTSNE functions, respectively. The clustering was done using a resolution of 0.4.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMarker identification and Cluster annotation\u003c/strong\u003e \u003cp\u003eIn this study, a targeted clustering analysis was performed within the most abundant cell types. The annotations of cell identity on each cluster were defined by the expression of known marker genes. The DotPlot function (Seurat package) was used to visualize the average expression of the known markers related to specific cell types, based on the overall expression profile of the nuclei, regardless of dropout events. On this basis, we employed Pecam1, Cdh5, Emcn, and Flt1 to classify \u0026ldquo;Endothelial cell\u0026rdquo;; Ttn, Myh6, Tnnt2, and Atp2a2 for \u0026ldquo;Cardiomyocyte\u0026rdquo;; Col1a2, Col8a1, Pdgfra, and Fstl1 for \u0026ldquo;Fibroblast\u0026rdquo;; Rgs5, Abcc9, and Pdgfrb for \u0026ldquo;Pericyte\u0026rdquo;; Myh11, Acta2, and Synpo2 for \u0026ldquo;Myofibroblast\u0026rdquo;; Cd163, Mrc1, and C1qa for \u0026ldquo;Macrophage\u0026rdquo;; Cd3e, Cd8a, Il2ra, and Tcf7 for \u0026ldquo;T cell\u0026rdquo;; Pax5, Cd22, Cd19, and Blnk for \u0026ldquo;B cell\u0026rdquo;. We performed the function check markers (Garnett package) to evaluate the ambiguity score and the relative number of cells for each cell type.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDifferentially expressed gene (DEG) analysis\u003c/strong\u003e \u003cp\u003eFor the identification of DEGs in AET-HF and HF groups, we calculated the log2 fold change (log2FC) between these groups using the Seurat FindMarkers function. The significance of the difference was determined using Wilcoxon test with the statistical threshold of adjusted P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and log2|FC| \u0026gt; 0.25.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFunctional enrichment analyses\u003c/strong\u003e \u003cp\u003eThe upregulated and downregulated genes identified for each group (AET-HF or HF group) were used in subsequent functional enrichment analyses using R-based application enrichR, respectively. EnrichGO was applied to identify genes enriched in biological process (BP), molecular function (MF), and cellular component (CC). Meanwhile, enrichKEGG was applied to detect genes enriched in pathway maps, such as metabolic pathways, signal transduction, protein interaction, and other network-related pathways. All terms with a P-value (Benjamini or Benjamini\u0026ndash; Hochberg adjusted) less than 0.05 were considered significant and ranked by adjusted P-value or the number of genes identified in the group.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCell-cell communication analysis\u003c/strong\u003e \u003cp\u003eCellCall was performed to infer inter- and intracellular communication pathways by integrating paired ligand-receptor and transcription factor (TF) activity among all cell types. The L (ligand) -R (receptor) -TF axis dataset was extracted from the KEGG pathway analysis. This method infers the potential interaction strength between two cell subsets based on gene expression level, and provides the significance through permutation test. Only those with a P- value\u0026thinsp;\u0026lt;\u0026thinsp;0.01 were used for the prediction of cell-cell communication between any two cell types. In addition, CellCall embeds a pathway activity analysis method to help explore the main pathways involved in communication between certain cell types. In brief, the activity of pathway i was quantified according to the pathway activity score nPASi, based on Jaccard similarity coefficient, and the significance of pathway activity was estimated by hypergeometric testing.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eImmunofluorescence\u003c/p\u003e \u003cp\u003eFor immunofluorescence, hearts were collected and encased in optimal cutting temperature (OCT) compound (Sakura, Torrance, CA, USA). Frozen slices (7-\u0026micro;m thickness) were obtained and sealed with diluted donkey serum for 1 h at 25\u0026deg;C, followed by overnight incubation with primary antibody at 4\u0026deg;C. Visualized signal of secondary antibody (Invitrogen, Carlsbad, CA, USA) conjugated with Alexa. The following primary antibodies were used in these experiments: CD68 (AB53444, 1:300; Abcam, Cambridge, England), TNNI3 (#SAB2502170, 1:300; Sigma-Aldrich, St. Louis, MO, USA), Lgmn (sc-133234, 1:300; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Myoglobin (AB77232, 1:300; Abcam, Cambridge, England). Sections were viewed and photographed using a confocal laser scanning microscope (LEICA TCS SP8, Oberkochen, Germany).\u003c/p\u003e \u003cp\u003eTerminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labeling (TUNEL) assay\u003c/p\u003e \u003cp\u003eTUNEL staining was performed with an In Situ Apoptosis Detection kit (Yeasen, Shanghai, China), according to the manufacturer\u0026rsquo;s instructions. Images were obtained using a laser scanning confocal microscope (LEICA TCS SP8, Oberkochen, Germany).\u003c/p\u003e \u003cp\u003eHistological Analysis\u003c/p\u003e \u003cp\u003eHearts collected after mice were perfused with cold PBS, fixed with 4% paraformaldehyde for 24 hours, paraffin or OCT embedding, paraffin embedding interval of 6\u0026micro;m, OCT embedding interval of 8\u0026micro;m, and continuous sections were performed. Continuous sections were stained with Masson's trichrome\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e for detection of myocardial fibrosis. The images were captured by a Leica microscope (DM6000B, Leica, Germany). In order to quantify myocardial fibrosis, 10 visual fields were randomly selected from 3 heart sections to calculate the percentage of Masson\u0026rsquo;s trichromatic positive staining area in the total myocardial area.\u003c/p\u003e \u003cp\u003eWestern Blot Analysis\u003c/p\u003e \u003cp\u003eCell particles and heart tissues samples were homogenized in RIPA Lysis Buffer (Beyotime, Shanghai, China) containing proteinase and phosphatase inhibitor cocktail. Protein concentrations were determined by a BCA kit (23225, Pierce, USA). Protein (30 to 50 mg total protein) was isolated by SDS\u0026ndash;polyacrylamide gel electrophoresis (SDS-PAGE; Biorad, Hercules, California, USA) gels, and then transferred to polyvinylidene difluoride (PVDF) membranes. Subsequently, in TBS-T, the membranes were blocked in 5% non-fat dried milk at room temperature for 2 hours and incubated with indicated primary antibodies at 4\u0026deg;C overnight according to the requirements of each experiment. The primary antibodies used in the study were: TNNI3 (#SAB2502170, 1:2000; Sigma-Aldrich, St. Louis, MO, USA), Lgmn (sc-133234, 1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Myoglobin (AB77232, 1:2000; Abcam, Cambridge, England) and GAPDH (HRP60004,1:10000; Proteintech,San Diego, MO, USA). The second day, it was washed three times with TBS-T for 10 min, and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:3000) for 2 hours at room temperature. After washing in TBS-T for another three times, all membranes were detected using a chemiluminescent system, and signal intensities were analyzed with an Amersham Imager 600 (GE Healthcare, USA). Experiments were repeated three times and the target protein level was quantified by Image J and normalized to internal control.\u003c/p\u003e \u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eResults are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM for at least 3 independent assays unless otherwise noted. Data normality was determined by the Shapiro-Wilk test. The Student t test was used for 2-sample comparisons; 1-way ANOVA with Turkey post hoc tests was used for comparisons between multiple groups; and 2-way ANOVA was used for comparisons between multiple groups when there were 2 experimental factors. Nonnormal data were analyzed by Mann-Whitney U test or Kruskal-Wallis test with Dunn multiple comparisons. A value of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically different. GraphPad Prism 8 and SPSS 22 for Windows was used to perform all statistical analyses.\u003c/p\u003e "},{"header":"Results","content":"\u003cp\u003eAET improved ventricular remodeling and cardiac function in HF\u003c/p\u003e \u003cp\u003eTo unravel the functional alterations in HF, we first constructed chronic HF mouse model through LAD surgery, and wild-type mice served as control. To investigated the pathological features in HF after myocardial ischemia mice, we compared the severity of cardiac function whether subjected to LAD surgery. Echocardiography confirmed the cardiac abnormalities in the ischemia-induced HF mice compared with WT mice, reflecting in the significant differences of echocardiographic parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Cardiac function was analyzed before and after 4-week AET (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The echocardiographic analyses revealed expansion of LV dilatation (LV end-systolic diameter, LVESD), reduction in left ventricular ejection fraction (LVEF), and fractional shortening (FS) in the ischemia-induced HF mice compared with WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), indicating deteriorated cardiac function in HF mice. Taken together, these findings indicate that compared to mice with normal cardiac function, ischemia-induced HF mice exhibit notable increases in ventricular remodeling, leading to heart function exacerbation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo test whether AET can enhance the cardiac function of HF mice, we performed echocardiography, which displayed effective improvement on cardiac function through the echocardiographic parameters in HF mice after AET(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The left ventricular ejection fraction (LVEF) and fractional shortening (FS) aggrandized obviously in the AET mice compared with HF mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), manifesting AET significantly improved cardiac function in HF mice. In addition, AET improved the severity of myocardial fibrosis in HF mice as determined by masson\u0026rsquo;s trichrome staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). To sum up, AET, as a possible treatment, can meet the needs of HF patients for improving ventricular remodeling and myocardial fibrosis, thereby improving cardiac function in the chronic repair stage of heart injury.\u003c/p\u003e \u003cp\u003eAET promoted the clearance of increased apoptotic cardiomyocytes during HF\u003c/p\u003e \u003cp\u003eTo investigated the key molecular events and regulators controlling cardiac ventricular remodeling of HF by AET, we performed single-nucleus RNA sequencing (snRNA-Seq) and compared the difference of RNAs between HF without and with AET in mice. We firstly generated whole transcriptomes of the heart tissues at single-nucleus resolution using 10x Genomics technology, reaching a median depth of 25,426 reads/nucleus and 988 genes/nucleus. After removing low-quality nuclei, a total of 46955 single nuclei (29,714 single nuclei for HF group, and 17241 for control group), which aggregated into 26 clusters on the basis of transcriptional similarity, depicted the cardiac cell atlas and transcriptional heterogeneity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, S1A, S1B). To identify principal cell types involved in heart remodeling, we cataloged these single nuclei into 8 distinct cell lineages annotated with canonical marker gene expression, thus defining cardiomyocytes, endothelial cells, fibroblasts, macrophages, T cells/NK cells, B cells, myofibroblasts, and pericytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo gain insights into the changes of cardiomyocytes between ischemia-induced HF and Ctrl, we compared the transcriptomic signatures from the hearts of mice with or without LAD surgery, and found that several cardiomyocyte subtypes increased dramatically in ischemia-induced HF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). To clarify these interesting subsets, we dissected cell-type heterogeneity by performing cell subtype analysis. Sub-clustering of cardiomyocyte revealed 9 clusters, among which, subcluster 0 highlighted the tremendous growth of cell number(Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC and S1D). Each subtype possessed cardiomyocytes from three mice at the same time point, suggesting the reliability and reproducibility of our data. To elucidate the nature of these specific cardiomyocytes, we first investigated the pro-apoptotic, anti-apoptotic and necrosis marker genes expression in different groups. After screening the expression profiles of these specific cardiomyocytes, ischemia-induced HF showed statistically credible increases in apoptotic cells (Fas, Casp4 and Casp8), and necrosis cells (Mb, Ldha and Ckm) compared to WT mice, whereas the anti-apoptotic genes (Bcl2, Mcl1 and Sirt1) expression levels decreased markedly (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE to \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Furthermore, TUNEL staining and western blot confirmed increased cardiomyocyte apoptosis in the hearts of ischemia-induced HF mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Taken together, these findings show that apoptotic cardiomyocytes increased notably in HF.\u003c/p\u003e \u003cp\u003eTo investigate the function of AET during ventricular remodeling in vivo, ischemia-induced HF mice were subjected to train with a treadmill for 4 weeks. Meanwhile, WT-HF mice were fed conventionally without specific exercise training. As anticipated, in HF mice after AET compared to the HF mice without AET, sub-clustering analysis of cardiomyocyte demonstrated a considerable decrease in quantity of apoptotic cardiomyocytes, which principally concentrated upon subcluster 0 and 8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, S1C and S1D). In addition, The expression levels of canonical marker genes of apoptotic (Fas, Casp4 and Casp8) and necrosis (Mb, Ldha and Ckm) that involved in apoptotic cardiomyocytes experienced a noteworthy reduction in AET mice compared to WT-HF mice, in contrast, the anti-apoptotic genes (Bcl2, Mcl1 and Sirt1) expression levels displayed a remarkable upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE to \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG).Furthermore, western blot and TUNEL confirmed decreased cardiomyocyte apoptosis in the hearts of HF mice after AET (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH to \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). To sum up, AET, as a possible treatment, can meet the needs of HF patients for improving the efficiency of clearing apoptotic cardiomyocytes, thereby improving ventricular remodeling and cardiac function.\u003c/p\u003e \u003cp\u003eCell-cell communication between cardiomyocytes and macrophages increases in HF mice treated with AET\u003c/p\u003e \u003cp\u003eTo explore the underlying mechanisms of AET in clearing the apoptotic cardiomyocytes in HF mice, we then applied the latest cell-cell communication analytic algorithm to our dataset, and observed an overall alteration in intercellular crosstalk. We found that the overall communication quantity was increased in HF sample compared with control sample, with the most pronounced changes between the cardiomyocytes and macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Interactions of cardiomyocytes-macrophages increased both in HF mice with or without AET, supporting the role of efferocytosis by macrophages as a key event in the clearance of the apoptotic cardiomyocytes during ischemia-induced HF. Moreover, to identify whether the amount of macrophages had changed in different groups, we predicted the macrophage count through the single-nucleus transcriptomic signatures, and found that macrophages decreased in HF mice while increased dramatically in HF mice with AET (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo test this, using cardiomyocyte marker cardiac troponin I (TNNT3) and macrophage marker CD68, we examined macrophage and cardiomyocyte colocalization in myocardial tissue sections isolated from WT, HF with or without AET mice, respectively. We then calculated the percentage of cardiac resident macrophages to assess if AET enhances macrophage function in myocardial tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).As anticipated, in the heart tissue from HF mice, but not normal mice, the number of macrophages (CD68\u003csup\u003e+\u003c/sup\u003e) significantly decreased and that they were in contact with cardiomyocytes (TNNT3\u003csup\u003e+\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), whereas the number of macrophages (CD68\u003csup\u003e+\u003c/sup\u003e) increased remarkably in the hearts of HF mice with AET, displaying the more obvious induction of the percentage of these cardiomyocyte-containing macrophages compared to the HF mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).Besides, AET can notably increase the function of macrophages (CD68\u003csup\u003e+\u003c/sup\u003e) that clear the necrosis cardiomyocytes (Myoglobin\u003csup\u003e+\u003c/sup\u003e) in HF (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).Together, these findings highlight the presence of multiple functional macrophages and their potential roles in efficient-clearance of apoptotic cardiomyocytes in HF mice after AET.\u003c/p\u003e \u003cp\u003eLgmn is critical for AET to enhance macrophage efferocytosis and resolve inflammation in HF\u003c/p\u003e \u003cp\u003eTo elucidate the role of macrophage-mediated efferocytosis in ischemia-induced HF and its effect on AET-HF, we first investigated its putative genes expression involved in endocytosis and intracellular trafficking in mouse myocardial tissues. We compared the transcriptomic signatures of macrophages in the heart tissues of WT, HF, and AET mice, observing that the expression of these genes involved in efferocytosis (Lgmn, Fcgr4, Fcgr1, Tlr7, and Msr1) was reduced in HF mice and increased significantly in AET mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Because of its high and specific expression in macrophages\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, Lgmn was considered as a major candidate gene to explore the overall function changes in macrophages\u0026rsquo; clearance of apoptotic cardiomyocytes in HF mice after AET. Thus, we this time focus mainly on the differences in Lgmn. Lgmn expression levels decreased obviously in myocardial tissues of HF mice compared with the WT mice, whereas the expressions confirmed to increase in HF mice after AET (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB to \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, S2A to S2C). Double-immunofluorescence staining for Lgmn along with CD68 confirmed that Lgmn was expressed predominately by cardiac CD68\u003csup\u003e+\u003c/sup\u003e macrophages and AET can induce its expression in ischemia-induced HF (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eVentricular remodeling is a complex process with diverse manifestations, including not only myocardial fibrosis,but also inflammatory cell infiltration. Since the Lgmn-dependent phagocytosis was responsible for inflammation resolution\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, we first examined the genes expression of anti-inflammatory mediators interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1), as well as proinflammatory mediators interleukin1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) by the global single-nucleus transcriptome analysis in HF mice with or without AET, and then confirmed by expression detection assay. Gene expression analyses and verification of the anti-inflammatory mediators and proinflammatory mediators confirmed delayed inflammation resolution in the ischemia-induced HF mice after LAD surgery compared with the WT mice, whereas the AET promoted the post-HF inflammation resolution (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Taken together, these findings indicate that AET accelerates the efferocytosis of macrophages mediating proper inflammation resolution, and Lgmn is required for AET to clear the apoptotic cardiomyocytes by macrophages in the chronic repair stage of heart injury.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe process of ventricular remodeling underlies the fundamental pathology of HF, with a multifaceted etiology contributing to its complexity\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The application of AET in HF elicits a multitude of beneficial outcomes, such as mitigating myocardial hypertrophy and myocardial fibrosis, thereby ameliorating pathological ventricular remodeling\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The precise mechanism by which AET enhances ventricular remodeling in HF remains elusive and is a subject of ongoing research. We penetrated some of mysteries between AET and cardiac ventricular remodeling, confirming that AET plays an important role in effectively mitigating the remarkable accumulation of apoptotic cardiomyocytes associated with HF. Single-nucleus sequencing detected a substantial upsurge in certain apoptotic cardiomyocyte subsets within the HF, which were markedly diminished following AET. Studies have shown that\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, inadequate clearance of apoptotic cardiomyocytes during cardiac injury triggers secondary necrosis, precipitating additional damage, causing the loss of adjacent, non-regenerative cardiomyocytes. Therefore, maintaining cardiac function necessitates a mechanism that ensures the efficient removal of apoptotic cardiomyocytes.\u003c/p\u003e \u003cp\u003eThe number of apoptotic cardiomyocytes is a crucial determinant of poor remodeling of the injured heart, which also related to the clearance efficiency of necrotic and apoptotic cardiomyocytes during cardiac injury\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The effective clearance of dead cells and the formation of scar tissue promote the regression of inflammation and prevent widespread cell death, thereby helping preserve heart integrity and slowing the progression of myocardial injury. In a state of homeostasis, apoptosis stands as the prevalent form of cell demise, and the effective removal of the resulting cellular remnants is crucial for averting the buildup of debris that may precipitate detrimental inflammatory reactions\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. This clearance is facilitated by a specialized process termed efferocytosis\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, which is the engulfment and degradation of apoptotic cells by phagocytes. Efferocytosis, elimination of dead or apoptotic cells from viable tissues, is a highly programmed and vital process to maintain the healthy function of the organism. As one of the specialized phagocytes, macrophages show effective efferocytosis during myocardial injury, thus preventing the secondary necrosis of apoptotic cells to release protease, oxide, antibodies\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, which cause inflammation. Currently, there is a dearth of research examining the influence of AET on the process of Efferocytosis. It is therefore crucial to explore the potential impact of AET on the clearance of apoptotic cardiomyocytes by macrophages through efferocytosis and to determine its subsequent effects on the improvement of ventricular remodeling.\u003c/p\u003e \u003cp\u003eWe have observed that AET has the potential to not only augment the diminished macrophage population associated with HF but also to markedly improve the interplay between these immune cells and cardiomyocytes within the failing heart. AET plays a crucial role in macrophage-mediated efferocytosis processing of apoptotic cardiomyocytes, particularly in the chronic phase of cardiac injury, contributing to the proper resolution of inflammation, improving ventricular remodeling, and ultimately leading to improvements in cardiac function. Consequently, AET is vital for preventing the accumulation of apoptotic cells during cardiac injury, avoiding the excessive release of apoptotic cardiomyocyte contents, and facilitating the timely resolution of inflammation, all of which are crucial for preserving the integrity of cardiac function.\u003c/p\u003e \u003cp\u003eAnalyzing single-nucleus sequencing data, we identified that Lgmn, a gene highly specific to macrophages and an essential lysosomal enzyme\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, was among those differentially expressed in HF mice post-exercise, playing a notable role in efferocytosis. Therefore, we further explored the role of Lgmn in the promotion of efferocytosis by AET. As anticipated, AET markedly elevated Lgmn expression in macrophages of HF, preserved the anti-inflammatory/pro-inflammatory immune equilibrium, and prevented additional immune-mediated cardiac damage. Previous studies\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e during the acute phase of MI have similarly demonstrated that a deficiency in Lgmn hampers the capacity of cardiac macrophages to effectively remove and degrade apoptotic cardiomyocytes post-MI. Impaired efferocytosis results in the accumulation of inflammogenic material and disruption of efferocytosis-dependent anti-inflammatory signaling within resident cardiac macrophages\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Lgmn is evidently essential for the removal of apoptotic cardiomyocytes and the preservation of cardiac function during cardiac injury.\u003c/p\u003e \u003cp\u003eVentricular remodeling in HF is governed by intricate pathological mechanisms, with the cardiac microenvironment being significantly influenced by a diverse array of cellular components. While the present investigation is predominantly centered on cardiomyocytes, it is prudent to acknowledge the possible contributions of other cellular constituents. Subsequent research endeavors should aim to conduct a more comprehensive analysis of the diverse cell types involved to elucidate the precise mechanisms by which AET exerts its effects in the multifaceted cellular milieu of HF. Concurrently, ventricular remodeling in the setting of HF is intricately linked to the extent of myocardial fibrosis. The specific mechanisms influencing myocardial fibrosis, including but not limited to endothelial-to-mesenchymal transition (EndoMT) \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, must be considered and further explored to fully comprehend the multifactorial nature of this process. Future research endeavors are imperative for delving into the potential intercellular interactions mediated by AET and their implications on the ventricular remodeling process in HF.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eAET, as an efficient prevention and treatment of HF, repairs the proper expression and function of Lgmn and macrophage efferocytosis pathways, making the optimal clearance of dying cardiomyocytes, mediating proper inflammation resolution, improving ventricular remodeling, and strengthening cardic function in the chronic repair stage of heart injury. Therefore, AET, as a measure to boost Lgmn and heighten efferocytosis, is a feasible approach for enhancing cardiac protection during HF.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eHF\u003c/strong\u003e heart failure\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAET\u003c/strong\u003e aerobic exercise training\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLgmn\u003c/strong\u003e legumain\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLAD\u003c/strong\u003e left anterior descending\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSnRNA-Seq\u003c/strong\u003e single-nucleus RNA sequencing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQoL\u003c/strong\u003e quality of life\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTUNEL\u003c/strong\u003e terminal deoxynucleotidyl transferasemediated dUTP-biotin nick-end labeling\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWT\u003c/strong\u003e wild-type\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiaoling Liu, Chun Li, Yuxuan Fan and Zhongyan Zhou contributed equally to this article. All authors gave final approval and agreed to be accountable for the integrity and accuracy of all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSources of Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Key Research and Development Special Project of the Autonomous Region（2022B03023-3）and Key Supported Discipline of Health System in Shanghai (2023ZDFC0302) .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJia D., \u003cem\u003eet al.\u003c/em\u003e Cardiac Resident Macrophage-Derived Legumain Improves Cardiac Repair by Promoting Clearance and Degradation of Apoptotic Cardiomyocytes After Myocardial Infarction. Circulation 145, 1542\u0026ndash;1556 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun H., \u003cem\u003eet al.\u003c/em\u003e Risk prediction model construction for post myocardial infarction heart failure by blood immune B cells. Front Immunol 14, 1163350 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlvarez, C.K., Cronin, E., Baker, W.L. \u0026amp; Kluger, J. Heart failure as a substrate and trigger for ventricular tachycardia. J Interv Card Electrophysiol 56, 229\u0026ndash;247 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePilz PM., \u003cem\u003eet al.\u003c/em\u003e Large and Small Animal Models of Heart Failure With Reduced Ejection Fraction. Circulation Research 130, 1888\u0026ndash;1905 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSygitowicz, G., Maciejak-Jastrzębska, A. \u0026amp; Sitkiewicz, D. MicroRNAs in the development of left ventricular remodeling and postmyocardial infarction heart failure. Pol Arch Intern Med 130, 59\u0026ndash;65 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcDonagh T.A., \u003cem\u003eet al.\u003c/em\u003e 2021 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure. European Heart Journal 42, 3599\u0026ndash;3726 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeidenreich P.A., \u003cem\u003eet al.\u003c/em\u003e 2022 AHA/ACC/HFSA Guideline for the Management of Heart Failure: A Report of the American College of Cardiology/American Heart Association Joint Committee on Clinical Practice Guidelines. Circulation 145, (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan der Meer, P., Gaggin, H.K. \u0026amp; Dec G.W. ACC/AHA Versus ESC Guidelines on Heart Failure: JACC Guideline Comparison. J Am Coll Cardiol 73, 2756\u0026ndash;2768 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen H., \u003cem\u003eet al.\u003c/em\u003e Exercise training maintains cardiovascular health: signaling pathways involved and potential therapeutics. Signal Transduct Target Ther 7, 306 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYin A., \u003cem\u003eet al.\u003c/em\u003e Exercise-derived peptide protects against pathological cardiac remodeling. EBioMedicine 82, 104164 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdams, V. \u0026amp; Linke, A. Impact of exercise training on cardiovascular disease and risk. Biochim Biophys Acta Mol Basis Dis 1865, 728\u0026ndash;734 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTucker, W.J., Fegers-Wustrow, I., Halle, M., Haykowsky, M.J., Chung, E.H. \u0026amp; Kovacic, J.C. Exercise for Primary and Secondary Prevention of Cardiovascular Disease: JACC Focus Seminar 1/4. J Am Coll Cardiol 80, 1091\u0026ndash;1106 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBozkurt B., \u003cem\u003eet al.\u003c/em\u003e Cardiac Rehabilitation for Patients With Heart Failure: JACC Expert Panel. J Am Coll Cardiol 77, 1454\u0026ndash;1469 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou J. Cycling and heart failure: A 2-sample Mendelian randomization. Medicine 103, e37619 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKwak, H.B., Kim, J.h., Joshi, K., Yeh, A., Martinez, D.A. \u0026amp; Lawler, J.M. Exercise training reduces fibrosis and matrix metalloproteinase dysregulation in the aging rat heart. FASEB J 25, 1106\u0026ndash;1117 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLerchenm\u0026uuml;ller C., \u003cem\u003eet al.\u003c/em\u003e CITED4 Protects Against Adverse Remodeling in Response to Physiological and Pathological Stress. Circulation Research 127, 631\u0026ndash;646 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao E., \u003cem\u003eet al.\u003c/em\u003e A novel and efficient model of coronary artery ligation and myocardial infarction in the mouse. Circulation Research 107, 1445\u0026ndash;1453 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao, S., Ho, D., Vatner, D.E. \u0026amp; Vatner, S.F. Echocardiography in Mice. Curr Protoc Mouse Biol 1, 71\u0026ndash;83 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBi H.L., \u003cem\u003eet al.\u003c/em\u003e The deubiquitinase UCHL1 regulates cardiac hypertrophy by stabilizing epidermal growth factor receptor. Sci Adv 6, eaax4826 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYerra V.G., \u003cem\u003eet al.\u003c/em\u003e Pressure overload induces ISG15 to facilitate adverse ventricular remodeling and promote heart failure. J Clin Invest 133, (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan J., \u003cem\u003eet al.\u003c/em\u003e Astragaloside IV derivative HHQ16 ameliorates infarction-induced hypertrophy and heart failure through degradation of lncRNA4012/9456. Signal Transduct Target Ther 8, 414 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng X., \u003cem\u003eet al.\u003c/em\u003e Fibulin7 Mediated Pathological Cardiac Remodeling through EGFR Binding and EGFR-Dependent FAK/AKT Signaling Activation. Advanced Science (Weinheim, Baden-Wurttemberg, Germany) 10, e2207631 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu K., \u003cem\u003eet al.\u003c/em\u003e Exercise training ameliorates myocardial phenotypes in heart failure with preserved ejection fraction by changing N6-methyladenosine modification in mice model. Front Cell Dev Biol 10, 954769 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun J., \u003cem\u003eet al.\u003c/em\u003e Protective effect of urotensin II receptor antagonist urantide and exercise training on doxorubicin-induced cardiotoxicity. Scientific Reports 13, 1279 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahnavard M., \u003cem\u003eet al.\u003c/em\u003e Curcumin ameliorated myocardial infarction by inhibition of cardiotoxicity in the rat model. J Cell Biochem 120, 11965\u0026ndash;11972 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatil M., \u003cem\u003eet al.\u003c/em\u003e Novel Mechanisms of Exosome-Mediated Phagocytosis of Dead Cells in Injured Heart. Circulation Research 129, 1006\u0026ndash;1020 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMehrotra, P. \u0026amp; Ravichandran, K.S. Drugging the efferocytosis process: concepts and opportunities. Nat Rev Drug Discov 21, 601\u0026ndash;620 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoada-Romero, E., Martinez, J., Heckmann, B.L. \u0026amp; Green, D.R. The clearance of dead cells by efferocytosis. Nat Rev Mol Cell Biol 21, 398\u0026ndash;414 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao J., \u003cem\u003eet al.\u003c/em\u003e SIRT3 Regulates Clearance of Apoptotic Cardiomyocytes by Deacetylating Frataxin. Circulation Research 133, 631\u0026ndash;647 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGerlach B.D., \u003cem\u003eet al.\u003c/em\u003e Efferocytosis induces macrophage proliferation to help resolve tissue injury. Cell Metab 33, (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaymond M.H., \u003cem\u003eet al.\u003c/em\u003e Live cell tracking of macrophage efferocytosis during Drosophila embryo development in vivo. Science (New York, NY) 375, 1182\u0026ndash;1187 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJung S.H., \u003cem\u003eet al.\u003c/em\u003e Spatiotemporal dynamics of macrophage heterogeneity and a potential function of Trem2hi macrophages in infarcted hearts. Nat Commun 13, 4580 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBischoff J. Endothelial-to-Mesenchymal Transition. Circulation Research 124, 1163\u0026ndash;1165 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKovacic J.C., \u003cem\u003eet al.\u003c/em\u003e Endothelial to Mesenchymal Transition in Cardiovascular Disease: JACC State-of-the-Art Review. J Am Coll Cardiol 73, 190\u0026ndash;209 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, Y., Lui, K.O. \u0026amp; Zhou, B. Reassessing endothelial-to-mesenchymal transition in cardiovascular diseases. Nat Rev Cardiol 15, 445\u0026ndash;456 (2018).\u003c/span\u003e\u003c/li\u003e\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":"heart failure, aerobic exercise training, apoptotic cardiomyocytes, macrophages","lastPublishedDoi":"10.21203/rs.3.rs-4420177/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4420177/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHeart failure (HF), the terminal stage of various forms of cardiovascular disease, is a severe disorder characterized by pathological cardiac fibrosis, ventricular remodeling, and reduced heart function. Chamber remodeling is the basic pathological mechanisms of HF. Aerobic exercise training (AET) as one of the non-pharmacological treatments of cardiac rehabilitation, has become one of the important therapeutic means for the long-term management of chronic HF, but how AET can improve the process in HF has not been well clarified. This study aims to determine the role of AET in pathological cardiac remodeling in HF and its potential mechanisms. We identified AET promoting the clearance of apoptosis cardiomyocytes by boosting interactions of cardiomyocytes-macrophages in HF. Lgmn was associated with the efferocytosis elevation of macrophages by AET. In addition, AET, improving the ventricular remodeling and strengthening heart function ultimately, upregulation of the anti-inflammatory mediators and downregulationof the proinflammatory mediators by boosting the expression of Lgmn in chronic repair stage of HF.Our results link AET to efferocytosis elevation of macrophages in the chronic repair stage of heart injury and identify AET as a significant prevention and therapeutic of ventricular remodeling in HF to mediate proper inflammation resolution and cardic function increase.\u003c/p\u003e","manuscriptTitle":"Aerobic exercise improves ventricular remodeling by promoting macrophages to phagocytose dying cardiomyocytes in heart failure model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-03 09:16:52","doi":"10.21203/rs.3.rs-4420177/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":"4200fe03-eea1-4589-a66b-fcd4433ae512","owner":[],"postedDate":"July 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":33331184,"name":"Health sciences/Medical research/Drug development"},{"id":33331185,"name":"Health sciences/Diseases/Cardiovascular diseases/Heart failure"},{"id":33331186,"name":"Health sciences/Pathogenesis/Inflammation/Chronic inflammation"}],"tags":[],"updatedAt":"2024-07-21T23:00:20+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-03 09:16:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4420177","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4420177","identity":"rs-4420177","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}