Sympathetic-like-Integrated Engineered Heart Tissue Models AGEs-Induced Adverse Remodeling

Sympathetic-like-Integrated Engineered Heart Tissue Models AGEs-Induced Adverse Remodeling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sympathetic-like-Integrated Engineered Heart Tissue Models AGEs-Induced Adverse Remodeling Yuhong Wang, Yuhong Zhu, Xiang Long, Yuhong Zhu, Tingting Liu, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7937768/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Jan, 2026 Read the published version in Cardiovascular Diabetology → Version 1 posted 12 You are reading this latest preprint version Abstract Background Cardiovascular metabolic diseases (CMDs) are a major contributor to global mortality and disability, yet their pathogenesis remains incompletely understood, partly because existing in vitro models fail to capture disease complexity. Conventional engineered heart tissues (EHT), which typically contain only a limited set of cell types and lack neural components, cannot replicate the intricate neuro-cardiac interactions involved in CMDs. Objective This study aimed to develop a neuron-like-Integrated Engineered Heart Tissue for investigating neuro-cardiac interactions under both physiological and pathological conditions, offering a new tool for CMD research. Methods We constructed a Sympathetic-like-Integrated Engineered Heart Tissue (SIEHT) by incorporating sympathetic-like neuronal cells into EHT. The structural and functional properties of SIEHT were systematically compared with conventional EHT using morphological analysis, immunofluorescence staining, contractility measurements, qPCR, and RNA sequencing. The model was then exposed to advanced glycation end products (AGEs) to assess pathological remodeling through multiple parameters, including cell viability, oxidative stress, structural and functional integrity, and transcriptomic profiles. Results SIEHT exhibited greater structural and functional maturation than EHT, as indicated by improved cardiomyocyte alignment, increased contraction amplitude, and upregulated expression of connexin 43. Transcriptomic analysis revealed enriched pathways associated with multi-system development. Under AGEs-induced pathological conditions, SIEHT demonstrated a more pronounced reduction in cell viability, elevated reactive oxygen species levels, more severe contractile dysfunction, a higher frequency of abnormal spontaneous beating, and greater neural injury relative to controls. Transcriptome profiling further identified significant enrichment of the AGE-RAGE signaling pathway in diabetic complications. Conclusions We successfully established a novel SIEHT model that recapitulates physiological neuro-cardiac interactions and AGEs-induced adverse remodeling across multiple dimensions, providing a powerful and innovative tool for elucidating the pathophysiological mechanisms of neuro-cardiac dysregulation in CMDs. Engineered Heart Tissue iPSC-CM Innervation AGEs Cardiometabolic Diseases Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Cardiometabolic diseases (CMDs), including hypertension, diabetes, and obesity, are among the leading causes of global mortality and disability [ 1 , 2 ]. These conditions pose severe threats to individual health and impose a substantial socioeconomic burden, representing a major challenge to global health [ 3 , 4 ]. The escalating global burden of CMDs underscores the limited understanding of the pathogenesis and progression of these complex disorders, highlighting the urgent need to advance cardiac research and develop more physiologically relevant ex vivo models of complex diseases. Cardiac homeostasis relies on interactions among diverse cell populations. For instance, fibroblasts provide essential structural support and modulate cardiac function; endothelial and vascular smooth muscle cells form microvasculature and larger vessels that facilitate oxygen supply and metabolic waste transport; and the nervous system participates in cardiac development and functional regulation [ 5 – 7 ]. Concurrently, these non-myocyte populations within the heart are implicated in disease processes [ 8 – 10 ]. Studies have shown that type 2 diabetes often leads to microvascular complications and neuro-cardiac dysfunction [ 11 ]. Hyperglycemia activates fibroblasts and promotes cardiac fibrosis [ 12 ]. These findings suggest that non-myocytes may play a profound role in the initiation and progression of cardiac diseases. Neuronal cells constitute a crucial component of the cardiac non-myocyte repertoire, and neuronal activity has been demonstrated to significantly influence the structure and function of cardiac cells [ 13 , 14 ]. In recent years, advances have been made in neuro-cardiac co-culture models [ 15 ]. Yoh-Suke Mukouyama [ 16 ] established a direct co-culture model of sympathetic neurons and cardiomyocytes, demonstrating that the presence of sympathetic neurons upregulates genes associated with contractile function and promotes the formation of gap junctions in cardiomyocytes. Nadja Zeltner [ 17 ] developed a direct co-culture system of cardiomyocytes and parasympathetic neurons. This revealed that beating of the cardiomyocytes was reduced following stimulation of the acetylcholine receptors on the parasympathetic neurons. Albano [ 18 ] engineered an organ-on-a-chip system in which neuronal cells form functional synapses with induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Although these neuro-cardiac co-culture models underscore the functional significance of neuron–cardiomyocyte coupling, they exhibit several limitations. For instance, they lack the spatial signaling provided by the extracellular matrix involved in neuro-cardiac regulation, and fail to recapitulate how alterations in the autonomic nervous system under pathological conditions affect myocardial function. Therefore, it is imperative to develop an advanced neuro-cardiac co-culture model to investigate neuron–cardiomyocyte interactions under both physiological and pathological conditions. This study aims to construct a Sympathetic-like-Integrated Engineered Heart Tissue (SIEHT) as an ex vivo platform for investigating neurocardiac crosstalk. To model the diabetic cardiac microenvironment, we focused on advanced glycation end products (AGEs)—pathogenic mediators that accumulate in diabetes and are implicated in cardiomyopathy [ 19 ]. Using AGEs, we established a pathological model to simulate their impact on neuron-cardiomyocyte interactions. Through multidimensional analysis, this work elucidates the dysregulation of neuro-cardiac modulation induced by pathogenic factors, providing novel scientific insights into the pathophysiology of cardiometabolic diseases. 2. Materials and methods 2.1. Materials The human fibroblast and human induced pluripotent stem cells (iPSC) were obtained from Meisen Chinese Tissue Culture Collections (Zhejiang, China). Undifferentiated PC12 cell line, Horse serum, and Fetal bovine serum (FBS) were purchased from Procell (Wuhan, China). CHIR99021, Wnt-C59, Thiazovivin (Tzv), Accutase™, and mTeSR™1 medium were supplied by MedChemExpress (MCE, USA) and Stem Cell Technologies (USA), respectively. Calcein/PI assays kit, 4% paraformaldehyde solution, 0.3% Triton X-100, 3% bovine serum albumin (BSA), Actin-Tracker Green-488, Actin-Tracker Green-555, LDH Assay Kit, and Reactive Oxygen Species Assay Kit were purchased from Beyotime (Beijing, China). DAPI was from Thermo Fisher Scientific (USA); and both DMEM/F12 and RPMI 1640 media were from Gibco (USA). Anti-Vimentin, Alexa Fluor 488, and Alexa Fluor 555 were from Abcam (USA). Anti-Alpha-actinin, Anti-Alpha-actinin-2, Anti- Troponin I-3, Anti- Beta-1 adrenergic receptor, Anti- Tyrosine Hydrolase and Anti-Connexin 43 were purchased from UpingBio technology Co.,Ltd (Hangzhou, CHINA). Mouse tail collagen type was purchased from Solarbio (Beijing, China). 2.2. Cell culture and CMs differentiation Fibroblasts were cultured in DMEM medium (Gibco, USA) containing 5% FBS (Procell, China) and 1% anti-anti (Gibco, USA), with the medium replaced every 48 hours. PC12 cell differentiation followed an established protocol [ 20 ], in which cells were maintained for 14 days in RPMI 1640 (Gibco, USA) supplemented with 1% horse serum (Procell, China), 1% anti-anti (Gibco, USA), and 50 ng/mL NGF (MCE, USA). Human induced pluripotent stem cells (iPSCs) were plated onto dishes pre-coated with Matrigel (Corning, USA) diluted 1:100 in DMEM/F12 and maintained in mTeSR™1 medium (Stem Cell Technologies, USA) at 37°C under 5% CO₂. When cells reached approximately 85% confluence, they were dissociated using Accutase™ (Stem Cell Technologies, USA) and cultured for 12 hours in mTeSR™1 supplemented with 2 µM Thiazovivin (MCE, USA) to improve survival after passaging. The medium was then replaced with standard mTeSR™1. Cardiomyocyte (CM) differentiation was initiated using established protocols once cells reached ~ 90% confluence by switching to CDM3 [ 21 , 22 ], which contained RPMI 1640 (Gibco, USA), 500 µg/mL recombinant serum albumin (Aladdin, China), and 213 µg/mL L-ascorbic acid 2-phosphate (TargetMol, USA). From days 0 to 2, cells were cultured in CDM3 supplemented with 5 µM CHIR99021 (MCE, USA) to induce mesoderm commitment. On day 2, the medium was changed to CDM3 containing 2 µM Wnt-C59 (MCE, USA), a Wnt pathway inhibitor, to promote cardiac progenitor formation. From day 4 onward, cells were maintained in base CDM3 with medium changes every 48 hours. Spontaneous contractions generally appeared around day 7. To enrich the cardiomyocyte population, a metabolic selection step was performed by culturing the cells in glucose-free medium from days 11 to 13. 2.3. Construction of EHT and SIEHT The SIEHT was constructed as previously described with modifications [ 23 ]. Fibroblasts, day-14 differentiated cardiomyocytes, and PC12 cells treated with NGF for 14 days were digested and resuspended. The cells were mixed at a ratio of cardiomyocytes: fibroblasts: neural cells = 10: 1: 1. A total of approximately 1.0 × 10^6 cells were resuspended in 50 µL of mixing medium. This medium consisted of RPMI 1640 (Gibco, USA), 50 µg/mL NGF (MCE, USA), 2 µM Tzv (MCE, USA), 1% anti-anti (Gibco, USA), 5% horse serum (Procell, China), 500 µg/mL recombinant serum albumin derived from Oryza sativa (Aladdin, China), and 213 µg/mL L-ascorbic acid 2-phosphate trisodium salt (TargetMol, USA). A 5 mg/mL solution of type I rat tail collagen (Solarbio, China) was neutralized with NaOH (Aladdin, China). Subsequently, 50 µL of the cell suspension was mixed with 30 µL of neutralized rat tail collagen and 10 µL of Matrigel (Corning, USA) to form a hydrogel solution. The mixture was transferred into a PDMS mold and incubated at 37°C for 2 hours to form the SIEHT. After gelation, cardiac maintenance medium was carefully added. This medium was composed of RPMI 1640, 50 µg/mL NGF, 1% anti-anti, 5% horse serum, 500 µg/mL recombinant serum albumin, and 213 µg/mL L-ascorbic acid 2-phosphate trisodium salt. The cardiac maintenance medium was replaced every 48 hours. The EHT was constructed similarly using a ratio of cardiomyocytes: fibroblasts = 10: 1, while keeping the total cell number and all other steps identical to the SIEHT protocol. AGEs (Bioss, China) stimulation commenced from day 8, with BSA (Solarbio, China) used as the control treatment. In this study, the neuro-cardiac interactions were modeled by integrating NGF-differentiated PC12 neuron-like cells. 2.4. Immunofluorescence For immunofluorescence staining, samples were first fixed with 4% paraformaldehyde (Beyotime, China) for 15 minutes at room temperature. Following three washes with PBS, permeabilization was carried out using 0.3% Triton X-100 (Beyotime, China) for 30 minutes. Non-specific binding sites were blocked by incubating the samples with 3% bovine serum albumin (BSA, Beyotime, China) for 2 hours at room temperature. The samples were then incubated overnight at 4°C with the following primary antibodies: Anti-Vimentin (Abcam, USA; 1:500), Anti-Alpha-actinin (UpingBio, China; 1:100), Anti-Alpha-actinin-2 (UpingBio, China; 1:100), Anti-Troponin I-3 (UpingBio, China; 1:100), Anti-Beta-1 adrenergic receptor (UpingBio, China; 1:100), Anti-Tyrosine Hydrolase (UpingBio, China; 1:100), and Anti-Connexin 43 (UpingBio, China; 1:100). After extensive washing, appropriate secondary antibodies were applied, followed by a 2-hour incubation at room temperature in the dark. Nuclear counterstaining was performed with DAPI (Thermo Fisher Scientific, USA) for 10 minutes at 37°C, while filamentous actin structures were visualized using Actin-Tracker Green-488 (Beyotime, China). Handle the organization transparently according to previous procedures[ 24 ]. All fluorescence imaging was conducted on a laser scanning confocal microscope (Leica, Germany), and the acquired images were subsequently subjected to analysis using ImageJ software. 2.5. Beating recording The contractile function of EHT/SIEHT constructs was assessed by capturing spontaneous beating events in brightfield videos (Nikon, Japan) and performing automated analysis with ImageJ software to extract key beating parameters [ 25 ]. 2.6. Cell viability and LDH release assay We performed cell viability assays using a Calcein-AM/PI kit (Beyotime, China). According to the manufacturer's instructions, a DPBS staining solution was prepared with 2 µM Calcein-AM and 8 µM PI. At designated time points, constructs were washed twice with DPBS before staining. Subsequently, the Calcein AM/PI working solution was applied, and the samples were incubated at 37°C in the dark for 30 minutes. Following incubation, the constructs were washed three times to remove excess dye. Imaging was performed using a fluorescence microscope (Nikon, Japan). Viable and dead cells were indicated by green and red fluorescence, respectively. Following the acquisition of three random images per sample using ImageJ, the cell viability percentage was derived from the ratio of live cells to the total cell count [ 22 , 26 ]. The lactate dehydrogenase (LDH) release in the culture supernatant was measured to evaluate cytotoxicity, using a commercial assay kit (Beyotime, China) in accordance with the provided protocol. Briefly, the collected supernatant was mixed with the LDH detection reagent and incubated at 37°C for 1 hour. The absorbance of the resulting mixture was then measured at a wavelength of 450 nm using a microplate reader. 2.7. ROS test The intracellular level of reactive oxygen species (ROS) was determined using a commercial ROS Assay Kit (Beyotime, China), following the manufacturer's protocol. In brief, samples were washed three times with DPBS and then incubated with 10 µM 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) in DMEM/F12 for 20 minutes at room temperature, protected from light. This step enables the probe to enter cells and be hydrolyzed to DCFH, which ROS subsequently oxidizes to yield the fluorescent product DCF. After incubation, unincorporated dye was removed by three additional DPBS washes. Fluorescence imaging was performed using a confocal microscope (Leica, Germany) with z-stack acquisition. For each sample, at least three independent fields were captured for analysis. The fluorescence intensity, proportional to the ROS levels, was quantified using ImageJ software. 2.8. RNA sequencing The RNA libraries were sequenced on the Illumina sequencing platform by Genedenovo Biotechnology Co., Ltd (Guangzhou, China). 2.9. Statistic analysis Data are presented as mean ± SD from a minimum of three replicates. Statistical analyses were conducted using SPSS 26 and Origin 2024. Normality was assessed for all datasets. Parametric data were analyzed using an independent t-test (two groups) or one-way ANOVA with LSD post hoc test (multiple groups). Non-parametric data were analyzed with the Kruskal-Wallis H test, followed by Bonferroni-corrected pairwise comparisons. Statistical significance was defined as p < 0.05. Standard methods were applied unless otherwise specified. 3. Results 3.1. Construction of SIEHT To establish a model for neuro-cardiac interactions, we constructed a sympathetic-like neuron-integrated engineered heart tissue using iPSC-CMs, fibroblasts, and NGF-differentiated PC12 cells. Prior to integration, the PC12 cells were confirmed to exhibit sympathetic-like neuronal phenotypes, including neurite outgrowth and tyrosine hydroxylase (TH) expression (Fig. S1 A-C).Light microscopy revealed that the tissue adopted a typical dog-bone-shaped structure (Fig. 1 B). Immunofluorescence staining of sections confirmed the expression of cardiomyocyte-specific proteins (ACTN1, cTNT, CX43), the sympathetic-like neuronal marker TH, and the fibroblast marker Vimentin, verifying the successful coexistence and specific characterization of all three cell types (Fig. 1 C). To determine whether the sympathetic-like neurons formed structural innervation with cardiomyocytes in the SIEHT, we performed whole-mount staining and three-dimensional reconstruction analysis. Morphological observations showed that sympathetic-like neurons were closely associated with cardiomyocytes and exhibited classic features of neural innervation, including variousities and nodal structures (Fig. 1 D, arrows). Furthermore, co-staining analysis of β1-adrenergic receptor (β1-AR) and TH at neuro-cardiac junctions revealed significant signal co-localization. These results confirm the establishment of physical innervation between the sympathetic-like neurons and the cardiomyocytes (Fig. 1 E). We next examined whether this innervation confers functional regulation. Preliminary experiments showed that increasing the proportion of sympathetic-like neurons within a specific range elevated the beating frequency of cardiomyocytes, suggesting a potential regulatory role (Fig. 1 F and S1D). We subsequently performed nicotine stimulation experiments. Because nicotine specifically activates acetylcholine receptors on sympathetic-like neurons to trigger catecholamine release and indirectly modulate cardiac beating, we treated the tissues with low nicotine concentrations. This treatment significantly increased the beating frequency of SIEHT, while no such effect occurred in control EHT lacking sympathetic-like neurons (Fig. 1 G and 1 H). The nicotine response of SIEHT thus depends on sympathetic-like neurons, confirming their functional regulatory role on the engineered cardiac tissue. 3.2. Integration of Sympathetic-like Neurons Promotes Structural and Functional Maturation of SIEHT To elucidate the role of sympathetic-like neurons in engineered myocardial tissue formation, we systematically evaluated the morphogenesis, structural features, and functional maturation of SIEHT, using EHT lacking sympathetic-like neurons as a control (Fig. 2A). Throughout morphogenesis, all tissues compacted from loose cell aggregates into dense, synchronously beating constructs. The SIEHT group, however, displayed distinct dynamic characteristics: during the initial 48 hours of culture, its width was significantly smaller than that of the control, suggesting that sympathetic-like neurons may accelerate cell migration and early aggregation; in later stages, SIEHT width instead surpassed that of the control, indicating the development of a more mature macroscopic structure (Fig. 2B). Cytoskeleton staining was performed to examine tissue microstructure. This analysis revealed significantly higher cardiomyocyte orientation in SIEHT, demonstrating that sympathetic-like neurons promote an anisotropic architecture, a key hallmark of myocardial tissue maturation (Fig. 2C-2E). Functionally, SIEHT exhibited a substantially increased contraction amplitude (Fig. 2F-2H). Moreover, qPCR and immunofluorescence validation confirmed significantly upregulated expression of the gap junction protein CX43 in SIEHT, providing a molecular basis for its enhanced electromechanical coupling (Fig. 2J and 2K). Together, these findings indicate that integrating sympathetic-like neurons not only refines the morphogenetic process and microstructure of engineered myocardial tissue but also markedly advances its structural and functional maturation. Figure 2 Integration of sympathetic-like neurons promotes structural and functional maturation of SIEHT. (A) Schematic diagram of EHT and SIEHT construction. (B) Quantification of tissue width in EHT and SIEHT. (C) Immunofluorescence images of EHT and SIEHT. Shown is a 100 µm scale bar. (D) Cytoskeletal orientation distribution in EHT and SIEHT. (E) Quantification of cytoskeletal orientation in EHT and SIEHT (aligned to the organoid longitudinal axis). (F) Representative contraction traces of EHT and SIEHT. (G) Quantification of contraction amplitude based on contraction traces. (H) Quantification of beats per minute (BPM) based on contraction traces. (J) Immunofluorescence images of EHT and SIEHT. Shown is a 100 µm scale bar. (K) Quantitative analysis of immunofluorescence intensity. (*p < 0.05, **p < 0.01). 3.3. Transcriptomic Analysis of SIEHT Maturation To investigate the potential molecular mechanisms by which sympathetic-like neuron integration promotes the structural and functional maturation of SIEHT, we performed transcriptomic sequencing analysis of SIEHT and control EHT at day 14 of culture. Principal component analysis revealed a clear separation between the two groups at the transcriptional level, indicating systematic differences in their gene expression profiles. Subsequent Gene Ontology (GO) enrichment analysis showed significant activation of pathways related to multi-organ development in SIEHT, providing molecular evidence that SIEHT represents a more complex biomimetic system with higher developmental maturity. Further KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis revealed specific upregulation of the dopaminergic synapse pathway in SIEHT, which is highly consistent with the hypothesis that integrated sympathetic-like neurons regulate cardiac function, providing key mechanistic clues for the observed functional maturation. Figure 3 Transcriptomic changes in EHT and SIEHT. (A) PCA plot. (B) Upregulated pathways identified by Gene Ontology (GO) enrichment analysis. (C) Downregulated pathways identified by GO enrichment analysis. (D) Heatmap of genes involved in the multi-organ development pathway. (E) Upregulated pathways identified by KEGG enrichment analysis. (F) Downregulated pathways identified by KEGG enrichment analysis. (G) Schematic diagram illustrating the mechanism of the dopaminergic synapse pathway. 3.4. Neuronal cells are more sensitive to AGEs damage than cardiomyocytes. Under diabetic and related metabolic abnormal conditions, cardiac autonomic neurons are more vulnerable and susceptible to earlier damage compared to cardiomyocytes, due to their unique metabolic properties and structural characteristics [ 27 – 29 ]. This explains why diabetic cardiac autonomic neuropathy represents one of the most common and earliest complications of diabetes. Currently, the mechanisms underlying diabetic cardiac autonomic nerve injury remain unclear, with the cascade of multiple complex mechanisms and pathways—including hyperglycemia, oxidative stress, and advanced glycation end products—being regarded as primary contributors [ 30 – 32 ]. We selected AGEs as the pathogenic factor and applied them to cardiomyocytes (CMs) and neuronal cells (PC12), respectively (Fig. 4A). During the early stage of AGEs exposure (day 2), we observed that at a concentration of 400 µg/mL, both cardiomyocytes and neuronal cells exhibited severe necrosis and structural abnormalities. However, at an AGEs concentration of 200 µg/mL, the viability and function of cardiomyocytes remained unaffected, whereas neuronal cells showed structural impairment, characterized by irregular cell morphology and shortened neurites (Fig. 4B and S2A, indicated by red boxes). We subsequently examined the long-term effects of AGEs (day 7). At a concentration of 200 µg/mL, neuronal cell viability was significantly reduced. When the AGEs concentration reached 150 µg/mL, neuronal cell viability decreased without obvious structural changes (Fig. 4C, 4D, and S2B, indicated by red boxes). Furthermore, we found that HT22 neuronal cells exhibited a similar sensitivity to AGEs-induced damage. Consequently, an AGEs concentration of 150 µg/mL was established for further investigation. This regimen selectively compromises long-term neuronal function in the absence of acute cytotoxicity, enabling modeling of the progressive neuronal impairment observed in diabetes. Fig. 4 Neurons exhibit higher sensitivity to AGEs damage than cardiomyocytes. (A) Schematic of AGEs-induced damage to cardiomyocytes and neurons. (B) Representative brightfield and live-dead fluorescent images of cardiomyocytes and neurons at the early stage of AGEs exposure. Scale bars = 100 µm. (C) Representative brightfield and live/dead fluorescent images of cardiomyocytes and neurons at the late stage of AGEs exposure. Scale bars = 100 µm. (D) Statistical analysis based on live/dead fluorescent images of cardiomyocytes and neurons. (*p < 0.05, **p < 0.01). 3.5. AGEs cause decreased SIEHT activity and increased ROS production. To evaluate the toxic effects of AGEs on the neuro-cardiac unit at the tissue level, SIEHT exposed to AGEs (SIEHT + AGEs) were designated as the experimental group, while EHT exposed to AGEs (EHT + AGEs) and untreated SIEHT served as controls (Fig. 5A). The results revealed that AGEs exposure significantly reduced the cell viability in both types of engineered cardiac tissues compared to the Control group. Notably, the lactate dehydrogenase (LDH) release was significantly higher in the SIEHT + AGEs group than in the EHT + AGEs group (Fig. 5B–D), suggesting that the integrated sympathetic neuron-like component may exacerbate tissue injury under AGEs-induced stress. The role of oxidative stress in this process was further investigated. As shown in Fig. 5E and 5F, the level of ROS was significantly elevated in the SIEHT + AGEs group compared with the control groups. These findings indicate that enhanced oxidative stress represents a key potential mechanism underlying AGEs-induced functional impairment in SIEHT. Figure 5 AGEs Decrease Cell Viability and Increase ROS Production in SIEHT. (A) Schematic timeline of AGE treatment on SIEHT. (B) Representative live/dead staining images of EHT and SIEHT. Scale bars = 1 mm. (C) Quantitative analysis of cell viability based on live/dead staining. (D) Statistical analysis of LDH release from EHT and SIEHT. (E) Representative ROS staining images of EHT and SIEHT. Scale bars = 100 µm. (F) Quantitative analysis of ROS levels based on ROS staining. (*p < 0.05, **p < 0.01). 3.6. AGEs cause nerve damage in SIEHT We further investigated whether AGEs induced other pathological remodeling, including pathological fibrosis and neural injury. Immunofluorescence staining and quantitative analysis of the fibroblast marker Vimentin revealed no significant activation of fibrosis in any group of engineered myocardial tissues following AGEs treatment (Fig. 6A and 6B). In contrast, analysis of the neural marker TH revealed a distinct toxic effect, characterized by a significant reduction in both the coverage area and fluorescence intensity of TH in the SIEHT + AGEs group (Fig. 6C and 6D). These findings indicate that AGEs primarily impair sympathetic nerve-like components within the tissue, rather than eliciting widespread fibrotic pathological changes through fibroblast activation. Figure 6 AGEs induce neural injury in SIEHT. (A) Fluorescence staining images of EHT and SIEHT. Scale bars = 100 µm. (B) Statistical analysis based on fluorescence staining images. (C) Fluorescence staining images of EHT and SIEHT. Scale bars = 100 µm. (D) Statistical analysis based on fluorescence staining images. (*p < 0.05, **p < 0.01). 3.7. AGEs increase the probability of impaired SIEHT contractile activity and abnormal contractile behavior. With the progression of diabetes, pathological alterations occur in cardiac function and structure in patients, such as systolic and diastolic dysfunction and fibrosis[ 33 , 34 ]. To investigate whether AGEs treatment can simulate this pathological process, we systematically analyzed the contractile and relaxation activities of engineered heart tissues. The results demonstrated that AGEs treatment significantly prolonged the contraction and relaxation times in both EHT and SIEHT, while markedly reducing the contraction amplitude (Fig. 7A and B), indicating that AGEs impair fundamental systolic and diastolic functions of the myocardium, resembling the cardiac dysfunction phenotype observed in diabetic patients. Notably, the frequency of abnormal contraction peaks in the SIEHT + AGEs group was significantly higher than that in the control and EHT + AGEs groups (Fig. 7B), manifesting specifically as either single abnormal peaks or frequent abnormal multi-peak patterns. This specific phenotype suggests that the integrated sympathetic neuron-like component exacerbates AGEs-induced electromechanical instability. Subsequently, key molecular markers of myocardial structure were evaluated. In contrast to the functional impairments described above, no significant differences in myocardial structural proteins were observed among the groups (Fig. 7C and D), indicating that AGEs-induced functional deficits precede structural remodeling. Figure 7 AGEs increase the probability of contractile dysfunction and abnormal contraction behavior in SIEHT. (A) Representative contraction traces of EHT and SIEHT. (B) Statistical analysis based on contraction traces. (C) Fluorescence staining images of EHT and SIEHT. ACTN2: Actinin alpha 2. Scale bars = 100 µm. (D) Fluorescence staining images of EHT and SIEHT. TNNI3: Troponin I3. Scale bars = 100 µm. (*p < 0.05, **p < 0.01). 3.8 Transcriptomic Profiling Reveals Differential Responses to AGEs Stress and the Role of Sympathetic-like Nerves in Pathological Remodeling To further investigate the potential mechanisms underlying the differential responses of EHT and SIEHT under AGEs stress and to elucidate the role of sympathetic-like innervation in pathological remodeling, we performed transcriptome sequencing on EHT + AGEs and SIEHT + AGEs samples. PCA revealed a clear separation between the two groups at the transcriptional level, indicating fundamental differences in their gene expression profiles (Fig. 8 A). Subsequent KEGG pathway enrichment analysis demonstrated that in the SIEHT + AGEs group, the AGE–RAGE signaling pathway closely associated with diabetic complications—was significantly activated, whereas the oxidative phosphorylation pathway was markedly suppressed (Fig. 8 B– 8 D and 8 H). This expression pattern closely mirrors the metabolic characteristics of diabetic cardiomyopathy, suggesting that the SIEHT model more accurately recapitulates the molecular pathological environment of the disease. Concurrently, upregulation of the "glutamatergic synapse" and "axon guidance" pathways was observed, providing key molecular evidence for the active involvement of sympathetic-like nerves in AGEs-induced pathological remodeling. Furthermore, enrichment of pathways related to the "muscle cell cytoskeleton" may be associated with the increased abnormal contraction frequency observed experimentally, although the precise causal relationship requires further validation (Fig. 8 B– 8 G). Additionally, to independently validate the reliability of the SIEHT model in mimicking the diabetic pathological milieu, we compared the transcriptomic profiles of the control and SIEHT + AGEs groups. KEGG enrichment analysis again confirmed significant activation of the AGE–RAGE signaling pathway in diabetic complications in the SIEHT + AGEs group. These results are highly consistent with the aforementioned findings, collectively providing strong evidence that the SIEHT model accurately reproduces key molecular events in diabetic cardiomyopathy. The biomimetic nature and reliability of the model were thus confirmed at the transcriptomic level (Fig. S3A and S3B). 4. Discussion In this study, we developed a novel SIEHT model by integrating sympathetic-like neurons into engineered myocardial tissue for the first time. Neuromodulation of cardiac organoids represents a major focus and challenge in current research. Previous studies have demonstrated the feasibility of forming functional synapses between neurons and cardiomyocytes ex vivo [ 15 , 16 , 35 – 45 ]. Our study serves as an important extension and complement to prior research. At the tissue level, we demonstrate that sympathetic neuron-like cells establish physical contact with cardiomyocytes and exert functional regulatory effects. Consistent with earlier findings, neuronal cells promoted the structural and functional maturation of cardiomyocytes. Interestingly, while Zeltner et al. [ 40 ] proposed that sympathetic nerves facilitate cardiac development via the "norepinephrine" pathway, our data suggest that the role of sympathetic neuron-like cells in myocardial maturation may be mediated through the "dopaminergic synapse formation" signaling pathway. These differences may be attributed to the distinct types of neuronal cells used, implying that different neuronal subtypes may play varying roles during tissue development. Furthermore, our data reveal unique characteristics of sympathetic neuron-like cells during the self-organization of engineered cardiac tissue: the tissue width was significantly smaller in the experimental group than in the control group during early culture stages, but exceeded that of the control group at later stages. This indicates dynamic regulation and a significant role of neuronal cells during the self-organization process. These findings highlight the distinctive contribution of neuronal cells in cardiac development, providing insights for subsequent studies on other neuronal types in development and for constructing more complex organoid models [ 46 , 47 ]. The development of novel organoid models for investigating the pathophysiology of complex diseases represents a current research priority and challenge [ 48 ]. Currently, AGEs-induced diabetic cardiomyopathy modeling remains under development and fails to fully recapitulate the complex pathophysiological alterations of diabetic cardiomyopathy [ 49 , 50 ]. Building upon our previous research, we have further extended the study of engineered heart tissue structure and function. It was observed that under AGEs treatment, the contractile and diastolic functions of SIEHT were significantly impaired, which parallels the clinical manifestations of diabetic cardiomyopathy [ 51 , 52 ]. Concurrently, numerous other methods for constructing diabetic cardiomyopathy models have been reported, such as palmitic acid induction and high-glucose/high-lipid induction [ 48 , 53 – 55 ]. These models simulate complex pathological features of diabetic cardiomyopathy, including oxidative stress, pathological fibrosis, and insulin resistance, thereby providing profound assistance in elucidating the pathophysiology of this disease. However, the advantage of our SIEHT model lies in the additional observation of neural injury and the demonstration that neuronal cells may participate in this pathological process. Furthermore, studies have indicated a complex relationship between diabetes and rhythm disturbances, which cannot be solely attributed to ischemia and autonomic neuropathy [ 56 – 58 ]. Our data demonstrate that AGEs treatment leads to an increased frequency of abnormal contraction peaks in SIEHT, consistent with this theory. The Cai team [ 53 ] reported similar abnormal contraction peaks in EHTs treated with palmitic acid, resembling those in SIEHT; however, such phenomena were not observed in our AGEs-treated EHT group lacking sympathetic innervation. This suggests that diabetes-related cardiac rhythm disturbances may involve multiple targets and pathways, with certain mechanisms potentially implicating neuronal involvement. Nevertheless, the specific mechanisms require further experimental validation. The current study has several limitations. First, although PC12 cells cease proliferation, extend neurites, and express numerous sympathetic neuronal markers (such as TH) upon induction by NGF, they cannot fully replicate all characteristics of sympathetic neurons that have matured in the complex in vivo environment [ 18 , 20 , 59 ]. Additionally, the xenogeneic origin of the PC12 cell line represents a limitation in our model, as it may introduce confounding factors in transcriptomic data. Nevertheless, the use of this well-established cell line is essential for achieving reliable and reproducible sympathetic-like neuronal differentiation within our experimental paradigm. Future studies will prioritize the use of human stem cell-derived neurons to enhance the physiological relevance of the model. Second, although our SIEHT model recapitulates many adverse remodeling phenotypes induced by AGEs, including neural injury, vasomotor dysfunction, and increased abnormal contraction peak frequency, the underlying mechanisms still require extensive experimental exploration and validation. Finally, diabetic cardiomyopathy involves highly complex pathophysiological alterations, including mitochondrial dynamics and insulin resistance [ 60 – 62 ]. As AGEs represent only one of several pathogenic factors in diabetic cardiomyopathy, they cannot fully recapitulate all pathophysiological changes associated with the condition [ 63 ]. The present study focuses on AGE-induced adverse remodeling; therefore, more work is needed to simulate the complete spectrum of diabetic cardiomyopathy characteristics fully. 5. Conclusions In summary, this study developed a novel SIEHT model. This model not only confirmed the critical physiological role of sympathetic-like nerves in promoting the structural and functional maturation of engineered myocardial tissues but also untangled their significant pathological involvement in mediating AGEs-induced remodeling. Collectively, the SIEHT model serves as a robust multidimensional research platform, enabling synchronous analysis of the complex interactions between nerves and cardiomyocytes under both physiological and pathological conditions, thereby providing a novel scientific perspective and research tool for in-depth understanding of the pathophysiological mechanisms of CMDs (Fig. 9 ). Declarations CRediT authorship contribution statement Yu-hong Wang, Xi-ming Zhu: Writing – review & editing, Writing – original draft, Visualization, Validation, Investigation, Data curation, Conceptualization. Xiang Long, Shuo-ji Zhu, Ting-ting Liu, Shu-ting Zhang: Investigation, Validation. Moussa Ide Nasser, Zi-ming Liao, Jia-cheng Shi, Jia-lin Liao, David T.W. Lui: Methodology, Software. Ping Zhu, Bin Yao, Hai-xia Guan: Writing – review & editing, Funding acquisition. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data is available from the corresponding author upon reasonable request. Funding Declaration This research was funded by the Guangdong Major Project of Basic and Applied Basic Research (2023B0303000005) ; Guangdong Provincial Special Support Program for Prominent Talents(2021JC06Y656). References Pastika L, Sau A, Patlatzoglou K, Sieliwonczyk E, Ribeiro AH, McGurk KA, et al. Artificial intelligence-enhanced electrocardiography derived body mass index as a predictor of future cardiometabolic disease. npj Digital Medicine. 2024;7:167. Jia W, Chan JC, Wong TY, Fisher EB. 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22:01:18","extension":"html","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":154452,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7937768/v1/c42277e70742ad04839e8fb8.html"},{"id":95656314,"identity":"dcc37b26-49ab-4c69-8e6e-c1e501d385fa","added_by":"auto","created_at":"2025-11-11 16:18:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1025618,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction and characterization of SIEHT. (A) Schematic diagram illustrating the construction and characterization of SIEHT. (B) Representative brightfield image of SIEHT. Scale bars = 1 cm. (C) Immunofluorescence staining of SIEHT sections. Scale bars = 100 μm. (D) Whole-mount immunofluorescence staining and 3D reconstruction of SIEHT. Scale bars = 20 μm. (E) Whole-mount immunofluorescence staining of SIEHT. Scale bars = 20 μm. (F) Statistical analysis of beats per minute (BPM) in cardiomyocytes co-cultured with different ratios of sympathetic-like neuronal cells. CM: iPSC-derived cardiomyocytes; N: sympathetic-like neuronal cells. (G) Representative contraction traces of EHT and SIEHT before and after nicotine treatment. (H) Comparison of BPM changes in EHT and SIEHT before and after nicotine treatment. (*p \u0026lt; 0.05, **p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7937768/v1/21df4ce5a3000a55c14c0654.png"},{"id":95584157,"identity":"d9058d01-2647-4adc-aab7-6b6a7ad2aa0d","added_by":"auto","created_at":"2025-11-10 22:01:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1257456,"visible":true,"origin":"","legend":"\u003cp\u003eIntegration of sympathetic-like neurons promotes structural and functional maturation of SIEHT. (A) Schematic diagram of EHT and SIEHT construction. (B) Quantification of tissue width in EHT and SIEHT. (C) Immunofluorescence images of EHT and SIEHT. Shown is a 100 μm scale bar. (D) Cytoskeletal orientation distribution in EHT and SIEHT. (E) Quantification of cytoskeletal orientation in EHT and SIEHT (aligned to the organoid longitudinal axis). (F) Representative contraction traces of EHT and SIEHT. (G) Quantification of contraction amplitude based on contraction traces. (H) Quantification of beats per minute (BPM) based on contraction traces. (J) Immunofluorescence images of EHT and SIEHT. Shown is a 100 μm scale bar. (K) Quantitative analysis of immunofluorescence intensity. (*p \u0026lt; 0.05, **p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7937768/v1/fa130c695a7f9e39992c0148.png"},{"id":95584156,"identity":"bece7212-e99e-4a58-9254-e4770fdec101","added_by":"auto","created_at":"2025-11-10 22:01:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1022718,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic changes in EHT and SIEHT. (A) PCA plot. (B) Upregulated pathways identified by Gene Ontology (GO) enrichment analysis. (C) Downregulated pathways identified by GO enrichment analysis. (D) Heatmap of genes involved in the multi-organ development pathway. (E) Upregulated pathways identified by KEGG enrichment analysis. (F) Downregulated pathways identified by KEGG enrichment analysis. (G) Schematic diagram illustrating the mechanism of the dopaminergic synapse pathway.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7937768/v1/194d52af9ccc13bf872f2066.png"},{"id":95584161,"identity":"399fa691-dd88-4f6a-90c4-1bd0ac14e71f","added_by":"auto","created_at":"2025-11-10 22:01:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1039941,"visible":true,"origin":"","legend":"\u003cp\u003eNeurons exhibit higher sensitivity to AGEs damage than cardiomyocytes. (A) Schematic of AGEs-induced damage to cardiomyocytes and neurons. (B) Representative brightfield and live-dead fluorescent images of cardiomyocytes and neurons at the early stage of AGEs exposure. Scale bars = 100 μm. (C) Representative brightfield and live/dead fluorescent images of cardiomyocytes and neurons at the late stage of AGEs exposure. Scale bars = 100 μm. (D) Statistical analysis based on live/dead fluorescent images of cardiomyocytes and neurons. (*p \u0026lt; 0.05, **p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7937768/v1/3f28e6eeb9c172fc7a87ed36.png"},{"id":95656473,"identity":"29001b38-74df-4cbb-b62f-3162eccfb782","added_by":"auto","created_at":"2025-11-11 16:18:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":754307,"visible":true,"origin":"","legend":"\u003cp\u003eAGEs Decrease Cell Viability and Increase ROS Production in SIEHT. (A) Schematic timeline of AGE treatment on SIEHT. (B) Representative live/dead staining images of EHT and SIEHT. Scale bars = 1 mm. (C) Quantitative analysis of cell viability based on live/dead staining. (D) Statistical analysis of LDH release from EHT and SIEHT. (E) Representative ROS staining images of EHT and SIEHT. Scale bars = 100 μm. (F) Quantitative analysis of ROS levels based on ROS staining. (*p \u0026lt; 0.05, **p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7937768/v1/c0409832ac664fe3f78125e5.png"},{"id":95584163,"identity":"5c5ff038-fea1-4c7a-a1b8-63555058ffe0","added_by":"auto","created_at":"2025-11-10 22:01:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1204868,"visible":true,"origin":"","legend":"\u003cp\u003eAGEs induce neural injury in SIEHT. (A) Fluorescence staining images of EHT and SIEHT. Scale bars = 100 μm. (B) Statistical analysis based on fluorescence staining images. (C) Fluorescence staining images of EHT and SIEHT. Scale bars = 100 μm. (D) Statistical analysis based on fluorescence staining images. (*p \u0026lt; 0.05, **p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7937768/v1/ddbd2a7120febda97fa6f97d.png"},{"id":95584175,"identity":"ec99808d-1d2d-452c-80f0-d059e6e1f98e","added_by":"auto","created_at":"2025-11-10 22:01:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1155557,"visible":true,"origin":"","legend":"\u003cp\u003eAGEs increase the probability of contractile dysfunction and abnormal contraction behavior in SIEHT. (A) Representative contraction traces of EHT and SIEHT. (B) Statistical analysis based on contraction traces. (C) Fluorescence staining images of EHT and SIEHT. ACTN2: Actinin alpha 2. Scale bars = 100 μm. (D) Fluorescence staining images of EHT and SIEHT. TNNI3: Troponin I3. Scale bars = 100 μm. (*p \u0026lt; 0.05, **p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7937768/v1/a2d4eb51f97394b33ce37797.png"},{"id":95584165,"identity":"522a7f34-6cc2-400d-8260-aeed4c7b455d","added_by":"auto","created_at":"2025-11-10 22:01:17","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":912294,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic Changes in SIEHT Cells Following AGEs Treatment. (A) PCA plot. (B) KEGG enrichment analysis of upregulated pathways. (C) KEGG enrichment analysis of downregulated pathways. (D) Heatmap of genes in the \"AGE-RAGE signaling pathway in diabetic complications\". (E) Heatmap of genes in the \"cytoskeleton in muscle cell\" pathway. (F) Heatmap of genes in the \"Glutamatergic synapse\" pathway. (G) Heatmap of genes in the \"Axon guidance\" pathway. (H) Heatmap of genes in the \"Oxidative phosphorylation\" pathway.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7937768/v1/240922d4b90d916f88a4e4c7.png"},{"id":95584172,"identity":"8745b77f-f76b-445c-b4b3-81c1aa7742c0","added_by":"auto","created_at":"2025-11-10 22:01:18","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":406537,"visible":true,"origin":"","legend":"\u003cp\u003eSympathetic-like-Integrated Engineered Heart Tissue Models AGEs-Induced Adverse Remodeling\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7937768/v1/0693ba9eb46f5fc9faa2bd68.png"},{"id":100614788,"identity":"1606fe70-fb1f-407f-b462-c56fde2baed2","added_by":"auto","created_at":"2026-01-19 17:25:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9625198,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7937768/v1/08c27e7b-3205-4133-ba36-bcb8b6962c73.pdf"},{"id":95584158,"identity":"ce6b97b7-17f1-46fb-83f7-81e85791670d","added_by":"auto","created_at":"2025-11-10 22:01:17","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":5483951,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7937768/v1/e327ac463cd3ab205f189ff5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sympathetic-like-Integrated Engineered Heart Tissue Models AGEs-Induced Adverse Remodeling","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCardiometabolic diseases (CMDs), including hypertension, diabetes, and obesity, are among the leading causes of global mortality and disability [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These conditions pose severe threats to individual health and impose a substantial socioeconomic burden, representing a major challenge to global health [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The escalating global burden of CMDs underscores the limited understanding of the pathogenesis and progression of these complex disorders, highlighting the urgent need to advance cardiac research and develop more physiologically relevant ex vivo models of complex diseases.\u003c/p\u003e\u003cp\u003eCardiac homeostasis relies on interactions among diverse cell populations. For instance, fibroblasts provide essential structural support and modulate cardiac function; endothelial and vascular smooth muscle cells form microvasculature and larger vessels that facilitate oxygen supply and metabolic waste transport; and the nervous system participates in cardiac development and functional regulation [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Concurrently, these non-myocyte populations within the heart are implicated in disease processes [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Studies have shown that type 2 diabetes often leads to microvascular complications and neuro-cardiac dysfunction [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Hyperglycemia activates fibroblasts and promotes cardiac fibrosis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These findings suggest that non-myocytes may play a profound role in the initiation and progression of cardiac diseases.\u003c/p\u003e\u003cp\u003eNeuronal cells constitute a crucial component of the cardiac non-myocyte repertoire, and neuronal activity has been demonstrated to significantly influence the structure and function of cardiac cells [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In recent years, advances have been made in neuro-cardiac co-culture models [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Yoh-Suke Mukouyama [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] established a direct co-culture model of sympathetic neurons and cardiomyocytes, demonstrating that the presence of sympathetic neurons upregulates genes associated with contractile function and promotes the formation of gap junctions in cardiomyocytes. Nadja Zeltner [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] developed a direct co-culture system of cardiomyocytes and parasympathetic neurons. This revealed that beating of the cardiomyocytes was reduced following stimulation of the acetylcholine receptors on the parasympathetic neurons. Albano [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] engineered an organ-on-a-chip system in which neuronal cells form functional synapses with induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Although these neuro-cardiac co-culture models underscore the functional significance of neuron\u0026ndash;cardiomyocyte coupling, they exhibit several limitations. For instance, they lack the spatial signaling provided by the extracellular matrix involved in neuro-cardiac regulation, and fail to recapitulate how alterations in the autonomic nervous system under pathological conditions affect myocardial function. Therefore, it is imperative to develop an advanced neuro-cardiac co-culture model to investigate neuron\u0026ndash;cardiomyocyte interactions under both physiological and pathological conditions.\u003c/p\u003e\u003cp\u003eThis study aims to construct a Sympathetic-like-Integrated Engineered Heart Tissue (SIEHT) as an ex vivo platform for investigating neurocardiac crosstalk. To model the diabetic cardiac microenvironment, we focused on advanced glycation end products (AGEs)\u0026mdash;pathogenic mediators that accumulate in diabetes and are implicated in cardiomyopathy [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Using AGEs, we established a pathological model to simulate their impact on neuron-cardiomyocyte interactions. Through multidimensional analysis, this work elucidates the dysregulation of neuro-cardiac modulation induced by pathogenic factors, providing novel scientific insights into the pathophysiology of cardiometabolic diseases.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials\u003c/h2\u003e\u003cp\u003eThe human fibroblast and human induced pluripotent stem cells (iPSC) were obtained from Meisen Chinese Tissue Culture Collections (Zhejiang, China). Undifferentiated PC12 cell line, Horse serum, and Fetal bovine serum (FBS) were purchased from Procell (Wuhan, China). CHIR99021, Wnt-C59, Thiazovivin (Tzv), Accutase\u0026trade;, and mTeSR\u0026trade;1 medium were supplied by MedChemExpress (MCE, USA) and Stem Cell Technologies (USA), respectively. Calcein/PI assays kit, 4% paraformaldehyde solution, 0.3% Triton X-100, 3% bovine serum albumin (BSA), Actin-Tracker Green-488, Actin-Tracker Green-555, LDH Assay Kit, and Reactive Oxygen Species Assay Kit were purchased from Beyotime (Beijing, China). DAPI was from Thermo Fisher Scientific (USA); and both DMEM/F12 and RPMI 1640 media were from Gibco (USA). Anti-Vimentin, Alexa Fluor 488, and Alexa Fluor 555 were from Abcam (USA). Anti-Alpha-actinin, Anti-Alpha-actinin-2, Anti- Troponin I-3, Anti- Beta-1 adrenergic receptor, Anti- Tyrosine Hydrolase and Anti-Connexin 43 were purchased from UpingBio technology Co.,Ltd (Hangzhou, CHINA). Mouse tail collagen type was purchased from Solarbio (Beijing, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Cell culture and CMs differentiation\u003c/h2\u003e\u003cp\u003eFibroblasts were cultured in DMEM medium (Gibco, USA) containing 5% FBS (Procell, China) and 1% anti-anti (Gibco, USA), with the medium replaced every 48 hours. PC12 cell differentiation followed an established protocol [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], in which cells were maintained for 14 days in RPMI 1640 (Gibco, USA) supplemented with 1% horse serum (Procell, China), 1% anti-anti (Gibco, USA), and 50 ng/mL NGF (MCE, USA).\u003c/p\u003e\u003cp\u003eHuman induced pluripotent stem cells (iPSCs) were plated onto dishes pre-coated with Matrigel (Corning, USA) diluted 1:100 in DMEM/F12 and maintained in mTeSR\u0026trade;1 medium (Stem Cell Technologies, USA) at 37\u0026deg;C under 5% CO₂. When cells reached approximately 85% confluence, they were dissociated using Accutase\u0026trade; (Stem Cell Technologies, USA) and cultured for 12 hours in mTeSR\u0026trade;1 supplemented with 2 \u0026micro;M Thiazovivin (MCE, USA) to improve survival after passaging. The medium was then replaced with standard mTeSR\u0026trade;1. Cardiomyocyte (CM) differentiation was initiated using established protocols once cells reached\u0026thinsp;~\u0026thinsp;90% confluence by switching to CDM3 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], which contained RPMI 1640 (Gibco, USA), 500 \u0026micro;g/mL recombinant serum albumin (Aladdin, China), and 213 \u0026micro;g/mL L-ascorbic acid 2-phosphate (TargetMol, USA). From days 0 to 2, cells were cultured in CDM3 supplemented with 5 \u0026micro;M CHIR99021 (MCE, USA) to induce mesoderm commitment. On day 2, the medium was changed to CDM3 containing 2 \u0026micro;M Wnt-C59 (MCE, USA), a Wnt pathway inhibitor, to promote cardiac progenitor formation. From day 4 onward, cells were maintained in base CDM3 with medium changes every 48 hours. Spontaneous contractions generally appeared around day 7. To enrich the cardiomyocyte population, a metabolic selection step was performed by culturing the cells in glucose-free medium from days 11 to 13.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Construction of EHT and SIEHT\u003c/h2\u003e\u003cp\u003eThe SIEHT was constructed as previously described with modifications [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Fibroblasts, day-14 differentiated cardiomyocytes, and PC12 cells treated with NGF for 14 days were digested and resuspended. The cells were mixed at a ratio of cardiomyocytes: fibroblasts: neural cells\u0026thinsp;=\u0026thinsp;10: 1: 1. A total of approximately 1.0 \u0026times; 10^6 cells were resuspended in 50 \u0026micro;L of mixing medium. This medium consisted of RPMI 1640 (Gibco, USA), 50 \u0026micro;g/mL NGF (MCE, USA), 2 \u0026micro;M Tzv (MCE, USA), 1% anti-anti (Gibco, USA), 5% horse serum (Procell, China), 500 \u0026micro;g/mL recombinant serum albumin derived from Oryza sativa (Aladdin, China), and 213 \u0026micro;g/mL L-ascorbic acid 2-phosphate trisodium salt (TargetMol, USA). A 5 mg/mL solution of type I rat tail collagen (Solarbio, China) was neutralized with NaOH (Aladdin, China). Subsequently, 50 \u0026micro;L of the cell suspension was mixed with 30 \u0026micro;L of neutralized rat tail collagen and 10 \u0026micro;L of Matrigel (Corning, USA) to form a hydrogel solution. The mixture was transferred into a PDMS mold and incubated at 37\u0026deg;C for 2 hours to form the SIEHT. After gelation, cardiac maintenance medium was carefully added. This medium was composed of RPMI 1640, 50 \u0026micro;g/mL NGF, 1% anti-anti, 5% horse serum, 500 \u0026micro;g/mL recombinant serum albumin, and 213 \u0026micro;g/mL L-ascorbic acid 2-phosphate trisodium salt. The cardiac maintenance medium was replaced every 48 hours. The EHT was constructed similarly using a ratio of cardiomyocytes: fibroblasts\u0026thinsp;=\u0026thinsp;10: 1, while keeping the total cell number and all other steps identical to the SIEHT protocol. AGEs (Bioss, China) stimulation commenced from day 8, with BSA (Solarbio, China) used as the control treatment.\u003c/p\u003e\u003cp\u003eIn this study, the neuro-cardiac interactions were modeled by integrating NGF-differentiated PC12 neuron-like cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Immunofluorescence\u003c/h2\u003e\u003cp\u003eFor immunofluorescence staining, samples were first fixed with 4% paraformaldehyde (Beyotime, China) for 15 minutes at room temperature. Following three washes with PBS, permeabilization was carried out using 0.3% Triton X-100 (Beyotime, China) for 30 minutes. Non-specific binding sites were blocked by incubating the samples with 3% bovine serum albumin (BSA, Beyotime, China) for 2 hours at room temperature.\u003c/p\u003e\u003cp\u003eThe samples were then incubated overnight at 4\u0026deg;C with the following primary antibodies: Anti-Vimentin (Abcam, USA; 1:500), Anti-Alpha-actinin (UpingBio, China; 1:100), Anti-Alpha-actinin-2 (UpingBio, China; 1:100), Anti-Troponin I-3 (UpingBio, China; 1:100), Anti-Beta-1 adrenergic receptor (UpingBio, China; 1:100), Anti-Tyrosine Hydrolase (UpingBio, China; 1:100), and Anti-Connexin 43 (UpingBio, China; 1:100). After extensive washing, appropriate secondary antibodies were applied, followed by a 2-hour incubation at room temperature in the dark.\u003c/p\u003e\u003cp\u003eNuclear counterstaining was performed with DAPI (Thermo Fisher Scientific, USA) for 10 minutes at 37\u0026deg;C, while filamentous actin structures were visualized using Actin-Tracker Green-488 (Beyotime, China). Handle the organization transparently according to previous procedures[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. All fluorescence imaging was conducted on a laser scanning confocal microscope (Leica, Germany), and the acquired images were subsequently subjected to analysis using ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Beating recording\u003c/h2\u003e\u003cp\u003eThe contractile function of EHT/SIEHT constructs was assessed by capturing spontaneous beating events in brightfield videos (Nikon, Japan) and performing automated analysis with ImageJ software to extract key beating parameters [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Cell viability and LDH release assay\u003c/h2\u003e\u003cp\u003eWe performed cell viability assays using a Calcein-AM/PI kit (Beyotime, China). According to the manufacturer's instructions, a DPBS staining solution was prepared with 2 \u0026micro;M Calcein-AM and 8 \u0026micro;M PI. At designated time points, constructs were washed twice with DPBS before staining. Subsequently, the Calcein AM/PI working solution was applied, and the samples were incubated at 37\u0026deg;C in the dark for 30 minutes. Following incubation, the constructs were washed three times to remove excess dye. Imaging was performed using a fluorescence microscope (Nikon, Japan). Viable and dead cells were indicated by green and red fluorescence, respectively. Following the acquisition of three random images per sample using ImageJ, the cell viability percentage was derived from the ratio of live cells to the total cell count [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe lactate dehydrogenase (LDH) release in the culture supernatant was measured to evaluate cytotoxicity, using a commercial assay kit (Beyotime, China) in accordance with the provided protocol. Briefly, the collected supernatant was mixed with the LDH detection reagent and incubated at 37\u0026deg;C for 1 hour. The absorbance of the resulting mixture was then measured at a wavelength of 450 nm using a microplate reader.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. ROS test\u003c/h2\u003e\u003cp\u003eThe intracellular level of reactive oxygen species (ROS) was determined using a commercial ROS Assay Kit (Beyotime, China), following the manufacturer's protocol. In brief, samples were washed three times with DPBS and then incubated with 10 \u0026micro;M 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) in DMEM/F12 for 20 minutes at room temperature, protected from light. This step enables the probe to enter cells and be hydrolyzed to DCFH, which ROS subsequently oxidizes to yield the fluorescent product DCF. After incubation, unincorporated dye was removed by three additional DPBS washes. Fluorescence imaging was performed using a confocal microscope (Leica, Germany) with z-stack acquisition. For each sample, at least three independent fields were captured for analysis. The fluorescence intensity, proportional to the ROS levels, was quantified using ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. RNA sequencing\u003c/h2\u003e\u003cp\u003eThe RNA libraries were sequenced on the Illumina sequencing platform by Genedenovo Biotechnology Co., Ltd (Guangzhou, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Statistic analysis\u003c/h2\u003e\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from a minimum of three replicates. Statistical analyses were conducted using SPSS 26 and Origin 2024. Normality was assessed for all datasets. Parametric data were analyzed using an independent t-test (two groups) or one-way ANOVA with LSD post hoc test (multiple groups). Non-parametric data were analyzed with the Kruskal-Wallis H test, followed by Bonferroni-corrected pairwise comparisons. Statistical significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Standard methods were applied unless otherwise specified.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Construction of SIEHT\u003c/h2\u003e\u003cp\u003eTo establish a model for neuro-cardiac interactions, we constructed a sympathetic-like neuron-integrated engineered heart tissue using iPSC-CMs, fibroblasts, and NGF-differentiated PC12 cells. Prior to integration, the PC12 cells were confirmed to exhibit sympathetic-like neuronal phenotypes, including neurite outgrowth and tyrosine hydroxylase (TH) expression (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-C).Light microscopy revealed that the tissue adopted a typical dog-bone-shaped structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Immunofluorescence staining of sections confirmed the expression of cardiomyocyte-specific proteins (ACTN1, cTNT, CX43), the sympathetic-like neuronal marker TH, and the fibroblast marker Vimentin, verifying the successful coexistence and specific characterization of all three cell types (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eTo determine whether the sympathetic-like neurons formed structural innervation with cardiomyocytes in the SIEHT, we performed whole-mount staining and three-dimensional reconstruction analysis. Morphological observations showed that sympathetic-like neurons were closely associated with cardiomyocytes and exhibited classic features of neural innervation, including variousities and nodal structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, arrows). Furthermore, co-staining analysis of β1-adrenergic receptor (β1-AR) and TH at neuro-cardiac junctions revealed significant signal co-localization. These results confirm the establishment of physical innervation between the sympathetic-like neurons and the cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). We next examined whether this innervation confers functional regulation. Preliminary experiments showed that increasing the proportion of sympathetic-like neurons within a specific range elevated the beating frequency of cardiomyocytes, suggesting a potential regulatory role (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and S1D). We subsequently performed nicotine stimulation experiments. Because nicotine specifically activates acetylcholine receptors on sympathetic-like neurons to trigger catecholamine release and indirectly modulate cardiac beating, we treated the tissues with low nicotine concentrations. This treatment significantly increased the beating frequency of SIEHT, while no such effect occurred in control EHT lacking sympathetic-like neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). The nicotine response of SIEHT thus depends on sympathetic-like neurons, confirming their functional regulatory role on the engineered cardiac tissue.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Integration of Sympathetic-like Neurons Promotes Structural and Functional Maturation of SIEHT\u003c/h2\u003e\u003cp\u003eTo elucidate the role of sympathetic-like neurons in engineered myocardial tissue formation, we systematically evaluated the morphogenesis, structural features, and functional maturation of SIEHT, using EHT lacking sympathetic-like neurons as a control (Fig.\u0026nbsp;2A). Throughout morphogenesis, all tissues compacted from loose cell aggregates into dense, synchronously beating constructs. The SIEHT group, however, displayed distinct dynamic characteristics: during the initial 48 hours of culture, its width was significantly smaller than that of the control, suggesting that sympathetic-like neurons may accelerate cell migration and early aggregation; in later stages, SIEHT width instead surpassed that of the control, indicating the development of a more mature macroscopic structure (Fig.\u0026nbsp;2B). Cytoskeleton staining was performed to examine tissue microstructure. This analysis revealed significantly higher cardiomyocyte orientation in SIEHT, demonstrating that sympathetic-like neurons promote an anisotropic architecture, a key hallmark of myocardial tissue maturation (Fig.\u0026nbsp;2C-2E). Functionally, SIEHT exhibited a substantially increased contraction amplitude (Fig.\u0026nbsp;2F-2H). Moreover, qPCR and immunofluorescence validation confirmed significantly upregulated expression of the gap junction protein CX43 in SIEHT, providing a molecular basis for its enhanced electromechanical coupling (Fig.\u0026nbsp;2J and 2K). Together, these findings indicate that integrating sympathetic-like neurons not only refines the morphogenetic process and microstructure of engineered myocardial tissue but also markedly advances its structural and functional maturation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;2\u003c/b\u003e Integration of sympathetic-like neurons promotes structural and functional maturation of SIEHT. (A) Schematic diagram of EHT and SIEHT construction. (B) Quantification of tissue width in EHT and SIEHT. (C) Immunofluorescence images of EHT and SIEHT. Shown is a 100 \u0026micro;m scale bar. (D) Cytoskeletal orientation distribution in EHT and SIEHT. (E) Quantification of cytoskeletal orientation in EHT and SIEHT (aligned to the organoid longitudinal axis). (F) Representative contraction traces of EHT and SIEHT. (G) Quantification of contraction amplitude based on contraction traces. (H) Quantification of beats per minute (BPM) based on contraction traces. (J) Immunofluorescence images of EHT and SIEHT. Shown is a 100 \u0026micro;m scale bar. (K) Quantitative analysis of immunofluorescence intensity. (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Transcriptomic Analysis of SIEHT Maturation\u003c/h2\u003e\u003cp\u003eTo investigate the potential molecular mechanisms by which sympathetic-like neuron integration promotes the structural and functional maturation of SIEHT, we performed transcriptomic sequencing analysis of SIEHT and control EHT at day 14 of culture. Principal component analysis revealed a clear separation between the two groups at the transcriptional level, indicating systematic differences in their gene expression profiles. Subsequent Gene Ontology (GO) enrichment analysis showed significant activation of pathways related to multi-organ development in SIEHT, providing molecular evidence that SIEHT represents a more complex biomimetic system with higher developmental maturity. Further KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis revealed specific upregulation of the dopaminergic synapse pathway in SIEHT, which is highly consistent with the hypothesis that integrated sympathetic-like neurons regulate cardiac function, providing key mechanistic clues for the observed functional maturation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;3\u003c/b\u003e Transcriptomic changes in EHT and SIEHT. (A) PCA plot. (B) Upregulated pathways identified by Gene Ontology (GO) enrichment analysis. (C) Downregulated pathways identified by GO enrichment analysis. (D) Heatmap of genes involved in the multi-organ development pathway. (E) Upregulated pathways identified by KEGG enrichment analysis. (F) Downregulated pathways identified by KEGG enrichment analysis. (G) Schematic diagram illustrating the mechanism of the dopaminergic synapse pathway.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Neuronal cells are more sensitive to AGEs damage than cardiomyocytes.\u003c/h2\u003e\u003cp\u003eUnder diabetic and related metabolic abnormal conditions, cardiac autonomic neurons are more vulnerable and susceptible to earlier damage compared to cardiomyocytes, due to their unique metabolic properties and structural characteristics [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This explains why diabetic cardiac autonomic neuropathy represents one of the most common and earliest complications of diabetes. Currently, the mechanisms underlying diabetic cardiac autonomic nerve injury remain unclear, with the cascade of multiple complex mechanisms and pathways\u0026mdash;including hyperglycemia, oxidative stress, and advanced glycation end products\u0026mdash;being regarded as primary contributors [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe selected AGEs as the pathogenic factor and applied them to cardiomyocytes (CMs) and neuronal cells (PC12), respectively (Fig.\u0026nbsp;4A). During the early stage of AGEs exposure (day 2), we observed that at a concentration of 400 \u0026micro;g/mL, both cardiomyocytes and neuronal cells exhibited severe necrosis and structural abnormalities. However, at an AGEs concentration of 200 \u0026micro;g/mL, the viability and function of cardiomyocytes remained unaffected, whereas neuronal cells showed structural impairment, characterized by irregular cell morphology and shortened neurites (Fig.\u0026nbsp;4B and S2A, indicated by red boxes). We subsequently examined the long-term effects of AGEs (day 7). At a concentration of 200 \u0026micro;g/mL, neuronal cell viability was significantly reduced. When the AGEs concentration reached 150 \u0026micro;g/mL, neuronal cell viability decreased without obvious structural changes (Fig.\u0026nbsp;4C, 4D, and S2B, indicated by red boxes). Furthermore, we found that HT22 neuronal cells exhibited a similar sensitivity to AGEs-induced damage. Consequently, an AGEs concentration of 150 \u0026micro;g/mL was established for further investigation. This regimen selectively compromises long-term neuronal function in the absence of acute cytotoxicity, enabling modeling of the progressive neuronal impairment observed in diabetes. \u003cb\u003eFig.\u0026nbsp;4\u003c/b\u003e Neurons exhibit higher sensitivity to AGEs damage than cardiomyocytes. (A) Schematic of AGEs-induced damage to cardiomyocytes and neurons. (B) Representative brightfield and live-dead fluorescent images of cardiomyocytes and neurons at the early stage of AGEs exposure. Scale bars\u0026thinsp;=\u0026thinsp;100 \u0026micro;m. (C) Representative brightfield and live/dead fluorescent images of cardiomyocytes and neurons at the late stage of AGEs exposure. Scale bars\u0026thinsp;=\u0026thinsp;100 \u0026micro;m. (D) Statistical analysis based on live/dead fluorescent images of cardiomyocytes and neurons. (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.5. AGEs cause decreased SIEHT activity and increased ROS production.\u003c/h2\u003e\u003cp\u003eTo evaluate the toxic effects of AGEs on the neuro-cardiac unit at the tissue level, SIEHT exposed to AGEs (SIEHT\u0026thinsp;+\u0026thinsp;AGEs) were designated as the experimental group, while EHT exposed to AGEs (EHT\u0026thinsp;+\u0026thinsp;AGEs) and untreated SIEHT served as controls (Fig.\u0026nbsp;5A). The results revealed that AGEs exposure significantly reduced the cell viability in both types of engineered cardiac tissues compared to the Control group. Notably, the lactate dehydrogenase (LDH) release was significantly higher in the SIEHT\u0026thinsp;+\u0026thinsp;AGEs group than in the EHT\u0026thinsp;+\u0026thinsp;AGEs group (Fig.\u0026nbsp;5B\u0026ndash;D), suggesting that the integrated sympathetic neuron-like component may exacerbate tissue injury under AGEs-induced stress. The role of oxidative stress in this process was further investigated. As shown in Fig.\u0026nbsp;5E and 5F, the level of ROS was significantly elevated in the SIEHT\u0026thinsp;+\u0026thinsp;AGEs group compared with the control groups. These findings indicate that enhanced oxidative stress represents a key potential mechanism underlying AGEs-induced functional impairment in SIEHT.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;5\u003c/b\u003e AGEs Decrease Cell Viability and Increase ROS Production in SIEHT. (A) Schematic timeline of AGE treatment on SIEHT. (B) Representative live/dead staining images of EHT and SIEHT. Scale bars\u0026thinsp;=\u0026thinsp;1 mm. (C) Quantitative analysis of cell viability based on live/dead staining. (D) Statistical analysis of LDH release from EHT and SIEHT. (E) Representative ROS staining images of EHT and SIEHT. Scale bars\u0026thinsp;=\u0026thinsp;100 \u0026micro;m. (F) Quantitative analysis of ROS levels based on ROS staining. (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.6. AGEs cause nerve damage in SIEHT\u003c/h2\u003e\u003cp\u003eWe further investigated whether AGEs induced other pathological remodeling, including pathological fibrosis and neural injury. Immunofluorescence staining and quantitative analysis of the fibroblast marker Vimentin revealed no significant activation of fibrosis in any group of engineered myocardial tissues following AGEs treatment (Fig.\u0026nbsp;6A and 6B). In contrast, analysis of the neural marker TH revealed a distinct toxic effect, characterized by a significant reduction in both the coverage area and fluorescence intensity of TH in the SIEHT\u0026thinsp;+\u0026thinsp;AGEs group (Fig.\u0026nbsp;6C and 6D). These findings indicate that AGEs primarily impair sympathetic nerve-like components within the tissue, rather than eliciting widespread fibrotic pathological changes through fibroblast activation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;6\u003c/b\u003e AGEs induce neural injury in SIEHT. (A) Fluorescence staining images of EHT and SIEHT. Scale bars\u0026thinsp;=\u0026thinsp;100 \u0026micro;m. (B) Statistical analysis based on fluorescence staining images. (C) Fluorescence staining images of EHT and SIEHT. Scale bars\u0026thinsp;=\u0026thinsp;100 \u0026micro;m. (D) Statistical analysis based on fluorescence staining images. (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.7. AGEs increase the probability of impaired SIEHT contractile activity and abnormal contractile behavior.\u003c/h2\u003e\u003cp\u003eWith the progression of diabetes, pathological alterations occur in cardiac function and structure in patients, such as systolic and diastolic dysfunction and fibrosis[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. To investigate whether AGEs treatment can simulate this pathological process, we systematically analyzed the contractile and relaxation activities of engineered heart tissues. The results demonstrated that AGEs treatment significantly prolonged the contraction and relaxation times in both EHT and SIEHT, while markedly reducing the contraction amplitude (Fig.\u0026nbsp;7A and B), indicating that AGEs impair fundamental systolic and diastolic functions of the myocardium, resembling the cardiac dysfunction phenotype observed in diabetic patients. Notably, the frequency of abnormal contraction peaks in the SIEHT\u0026thinsp;+\u0026thinsp;AGEs group was significantly higher than that in the control and EHT\u0026thinsp;+\u0026thinsp;AGEs groups (Fig.\u0026nbsp;7B), manifesting specifically as either single abnormal peaks or frequent abnormal multi-peak patterns. This specific phenotype suggests that the integrated sympathetic neuron-like component exacerbates AGEs-induced electromechanical instability. Subsequently, key molecular markers of myocardial structure were evaluated. In contrast to the functional impairments described above, no significant differences in myocardial structural proteins were observed among the groups (Fig.\u0026nbsp;7C and D), indicating that AGEs-induced functional deficits precede structural remodeling.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;7\u003c/b\u003e AGEs increase the probability of contractile dysfunction and abnormal contraction behavior in SIEHT. (A) Representative contraction traces of EHT and SIEHT. (B) Statistical analysis based on contraction traces. (C) Fluorescence staining images of EHT and SIEHT. ACTN2: Actinin alpha 2. Scale bars\u0026thinsp;=\u0026thinsp;100 \u0026micro;m. (D) Fluorescence staining images of EHT and SIEHT. TNNI3: Troponin I3. Scale bars\u0026thinsp;=\u0026thinsp;100 \u0026micro;m. (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003cp\u003e\u003cspan\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e3.8 Transcriptomic Profiling Reveals Differential Responses to AGEs Stress and the Role of Sympathetic-like Nerves in Pathological Remodeling\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the potential mechanisms underlying the differential responses of EHT and SIEHT under AGEs stress and to elucidate the role of sympathetic-like innervation in pathological remodeling, we performed transcriptome sequencing on EHT\u0026thinsp;+\u0026thinsp;AGEs and SIEHT\u0026thinsp;+\u0026thinsp;AGEs samples. PCA revealed a clear separation between the two groups at the transcriptional level, indicating fundamental differences in their gene expression profiles (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA). Subsequent KEGG pathway enrichment analysis demonstrated that in the SIEHT\u0026thinsp;+\u0026thinsp;AGEs group, the AGE\u0026ndash;RAGE signaling pathway closely associated with diabetic complications\u0026mdash;was significantly activated, whereas the oxidative phosphorylation pathway was markedly suppressed (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB\u0026ndash;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eD and \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eH). This expression pattern closely mirrors the metabolic characteristics of diabetic cardiomyopathy, suggesting that the SIEHT model more accurately recapitulates the molecular pathological environment of the disease. Concurrently, upregulation of the \u0026quot;glutamatergic synapse\u0026quot; and \u0026quot;axon guidance\u0026quot; pathways was observed, providing key molecular evidence for the active involvement of sympathetic-like nerves in AGEs-induced pathological remodeling. Furthermore, enrichment of pathways related to the \u0026quot;muscle cell cytoskeleton\u0026quot; may be associated with the increased abnormal contraction frequency observed experimentally, although the precise causal relationship requires further validation (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB\u0026ndash;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eG).\u003c/p\u003e\n\u003cp\u003eAdditionally, to independently validate the reliability of the SIEHT model in mimicking the diabetic pathological milieu, we compared the transcriptomic profiles of the control and SIEHT\u0026thinsp;+\u0026thinsp;AGEs groups. KEGG enrichment analysis again confirmed significant activation of the AGE\u0026ndash;RAGE signaling pathway in diabetic complications in the SIEHT\u0026thinsp;+\u0026thinsp;AGEs group. These results are highly consistent with the aforementioned findings, collectively providing strong evidence that the SIEHT model accurately reproduces key molecular events in diabetic cardiomyopathy. The biomimetic nature and reliability of the model were thus confirmed at the transcriptomic level (Fig. S3A and S3B).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we developed a novel SIEHT model by integrating sympathetic-like neurons into engineered myocardial tissue for the first time. Neuromodulation of cardiac organoids represents a major focus and challenge in current research. Previous studies have demonstrated the feasibility of forming functional synapses between neurons and cardiomyocytes ex vivo [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR36 CR37 CR38 CR39 CR40 CR41 CR42 CR43 CR44\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Our study serves as an important extension and complement to prior research. At the tissue level, we demonstrate that sympathetic neuron-like cells establish physical contact with cardiomyocytes and exert functional regulatory effects. Consistent with earlier findings, neuronal cells promoted the structural and functional maturation of cardiomyocytes. Interestingly, while Zeltner et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] proposed that sympathetic nerves facilitate cardiac development via the \"norepinephrine\" pathway, our data suggest that the role of sympathetic neuron-like cells in myocardial maturation may be mediated through the \"dopaminergic synapse formation\" signaling pathway. These differences may be attributed to the distinct types of neuronal cells used, implying that different neuronal subtypes may play varying roles during tissue development. Furthermore, our data reveal unique characteristics of sympathetic neuron-like cells during the self-organization of engineered cardiac tissue: the tissue width was significantly smaller in the experimental group than in the control group during early culture stages, but exceeded that of the control group at later stages. This indicates dynamic regulation and a significant role of neuronal cells during the self-organization process. These findings highlight the distinctive contribution of neuronal cells in cardiac development, providing insights for subsequent studies on other neuronal types in development and for constructing more complex organoid models [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe development of novel organoid models for investigating the pathophysiology of complex diseases represents a current research priority and challenge [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Currently, AGEs-induced diabetic cardiomyopathy modeling remains under development and fails to fully recapitulate the complex pathophysiological alterations of diabetic cardiomyopathy [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Building upon our previous research, we have further extended the study of engineered heart tissue structure and function. It was observed that under AGEs treatment, the contractile and diastolic functions of SIEHT were significantly impaired, which parallels the clinical manifestations of diabetic cardiomyopathy [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Concurrently, numerous other methods for constructing diabetic cardiomyopathy models have been reported, such as palmitic acid induction and high-glucose/high-lipid induction [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. These models simulate complex pathological features of diabetic cardiomyopathy, including oxidative stress, pathological fibrosis, and insulin resistance, thereby providing profound assistance in elucidating the pathophysiology of this disease. However, the advantage of our SIEHT model lies in the additional observation of neural injury and the demonstration that neuronal cells may participate in this pathological process. Furthermore, studies have indicated a complex relationship between diabetes and rhythm disturbances, which cannot be solely attributed to ischemia and autonomic neuropathy [\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Our data demonstrate that AGEs treatment leads to an increased frequency of abnormal contraction peaks in SIEHT, consistent with this theory. The Cai team [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] reported similar abnormal contraction peaks in EHTs treated with palmitic acid, resembling those in SIEHT; however, such phenomena were not observed in our AGEs-treated EHT group lacking sympathetic innervation. This suggests that diabetes-related cardiac rhythm disturbances may involve multiple targets and pathways, with certain mechanisms potentially implicating neuronal involvement. Nevertheless, the specific mechanisms require further experimental validation.\u003c/p\u003e\u003cp\u003eThe current study has several limitations. First, although PC12 cells cease proliferation, extend neurites, and express numerous sympathetic neuronal markers (such as TH) upon induction by NGF, they cannot fully replicate all characteristics of sympathetic neurons that have matured in the complex in vivo environment [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Additionally, the xenogeneic origin of the PC12 cell line represents a limitation in our model, as it may introduce confounding factors in transcriptomic data. Nevertheless, the use of this well-established cell line is essential for achieving reliable and reproducible sympathetic-like neuronal differentiation within our experimental paradigm. Future studies will prioritize the use of human stem cell-derived neurons to enhance the physiological relevance of the model. Second, although our SIEHT model recapitulates many adverse remodeling phenotypes induced by AGEs, including neural injury, vasomotor dysfunction, and increased abnormal contraction peak frequency, the underlying mechanisms still require extensive experimental exploration and validation. Finally, diabetic cardiomyopathy involves highly complex pathophysiological alterations, including mitochondrial dynamics and insulin resistance [\u003cspan additionalcitationids=\"CR61\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. As AGEs represent only one of several pathogenic factors in diabetic cardiomyopathy, they cannot fully recapitulate all pathophysiological changes associated with the condition [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. The present study focuses on AGE-induced adverse remodeling; therefore, more work is needed to simulate the complete spectrum of diabetic cardiomyopathy characteristics fully.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn summary, this study developed a novel SIEHT model. This model not only confirmed the critical physiological role of sympathetic-like nerves in promoting the structural and functional maturation of engineered myocardial tissues but also untangled their significant pathological involvement in mediating AGEs-induced remodeling. Collectively, the SIEHT model serves as a robust multidimensional research platform, enabling synchronous analysis of the complex interactions between nerves and cardiomyocytes under both physiological and pathological conditions, thereby providing a novel scientific perspective and research tool for in-depth understanding of the pathophysiological mechanisms of CMDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eCRediT authorship contribution statement\u003c/p\u003e\n\u003cp\u003eYu-hong Wang, Xi-ming Zhu: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Visualization, Validation, Investigation, Data curation, Conceptualization. Xiang Long, Shuo-ji Zhu, Ting-ting Liu, Shu-ting Zhang: Investigation, Validation. Moussa Ide Nasser, Zi-ming Liao, Jia-cheng Shi, Jia-lin Liao, David T.W. Lui: Methodology, Software. Ping Zhu, Bin Yao, Hai-xia Guan: Writing \u0026ndash; review \u0026amp; editing, Funding acquisition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDeclaration of competing interest\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eData is available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003eFunding Declaration\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Guangdong Major Project of Basic and Applied Basic Research (2023B0303000005) ; Guangdong Provincial Special Support Program for Prominent Talents(2021JC06Y656). \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePastika L, Sau A, Patlatzoglou K, Sieliwonczyk E, Ribeiro AH, McGurk KA, et al. 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American Journal of Physiology-Endocrinology and Metabolism. 2020;319:E835-E851.\u003c/li\u003e\n\u003cli\u003ePhang RJ, Ritchie RH, Hausenloy DJ, Lees JG, Lim SY. Cellular interplay between cardiomyocytes and non-myocytes in diabetic cardiomyopathy. Cardiovascular Research. 2023;119:668-690.\u003c/li\u003e\n\u003cli\u003eMonea G, Jiritano R, Salerno L, Rubino M, Massimino M, Perticone M, et al. Compromised cardiac autonomic function in non-diabetic subjects with 1 h post-load hyperglycemia: a cross-sectional study. Cardiovascular Diabetology. 2024;23:295.\u003c/li\u003e\n\u003cli\u003eManeechote C, Palee S, Kerdphoo S, Jaiwongkam T, Chattipakorn SC, Chattipakorn N. Modulating mitochondrial dynamics attenuates cardiac ischemia-reperfusion injury in prediabetic rats. Acta Pharmacologica Sinica. 2022;43:26-38.\u003c/li\u003e\n\u003cli\u003eLi Y, Liu Y, Liu S, Gao M, Wang W, Chen K, et al. Diabetic vascular diseases: molecular mechanisms and therapeutic strategies. Signal Transduction and Targeted Therapy. 2023;8:152.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cardiovascular-diabetology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cvdb","sideBox":"Learn more about [Cardiovascular Diabetology](http://cardiab.biomedcentral.com/)","snPcode":"12933","submissionUrl":"https://submission.nature.com/new-submission/12933/3","title":"Cardiovascular Diabetology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Engineered Heart Tissue, iPSC-CM, Innervation, AGEs, Cardiometabolic Diseases","lastPublishedDoi":"10.21203/rs.3.rs-7937768/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7937768/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eCardiovascular metabolic diseases (CMDs) are a major contributor to global mortality and disability, yet their pathogenesis remains incompletely understood, partly because existing in vitro models fail to capture disease complexity. Conventional engineered heart tissues (EHT), which typically contain only a limited set of cell types and lack neural components, cannot replicate the intricate neuro-cardiac interactions involved in CMDs.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eThis study aimed to develop a neuron-like-Integrated Engineered Heart Tissue for investigating neuro-cardiac interactions under both physiological and pathological conditions, offering a new tool for CMD research.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe constructed a Sympathetic-like-Integrated Engineered Heart Tissue (SIEHT) by incorporating sympathetic-like neuronal cells into EHT. The structural and functional properties of SIEHT were systematically compared with conventional EHT using morphological analysis, immunofluorescence staining, contractility measurements, qPCR, and RNA sequencing. The model was then exposed to advanced glycation end products (AGEs) to assess pathological remodeling through multiple parameters, including cell viability, oxidative stress, structural and functional integrity, and transcriptomic profiles.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eSIEHT exhibited greater structural and functional maturation than EHT, as indicated by improved cardiomyocyte alignment, increased contraction amplitude, and upregulated expression of connexin 43. Transcriptomic analysis revealed enriched pathways associated with multi-system development. Under AGEs-induced pathological conditions, SIEHT demonstrated a more pronounced reduction in cell viability, elevated reactive oxygen species levels, more severe contractile dysfunction, a higher frequency of abnormal spontaneous beating, and greater neural injury relative to controls. Transcriptome profiling further identified significant enrichment of the AGE-RAGE signaling pathway in diabetic complications.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eWe successfully established a novel SIEHT model that recapitulates physiological neuro-cardiac interactions and AGEs-induced adverse remodeling across multiple dimensions, providing a powerful and innovative tool for elucidating the pathophysiological mechanisms of neuro-cardiac dysregulation in CMDs.\u003c/p\u003e","manuscriptTitle":"Sympathetic-like-Integrated Engineered Heart Tissue Models AGEs-Induced Adverse Remodeling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-10 22:01:12","doi":"10.21203/rs.3.rs-7937768/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-17T02:53:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-16T18:35:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-11T16:20:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-11T03:13:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"68272708412546520639669853151597809870","date":"2025-11-02T00:37:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"117558239972175507343679167636760948112","date":"2025-11-01T17:39:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4300752869224301179559490967412416963","date":"2025-10-30T16:42:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"299157789598309251322258986385749751715","date":"2025-10-30T16:18:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-30T12:11:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-25T06:09:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-25T05:32:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cardiovascular Diabetology","date":"2025-10-24T07:10:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cardiovascular-diabetology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cvdb","sideBox":"Learn more about [Cardiovascular Diabetology](http://cardiab.biomedcentral.com/)","snPcode":"12933","submissionUrl":"https://submission.nature.com/new-submission/12933/3","title":"Cardiovascular Diabetology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b04edc00-62c9-40d9-a1c6-17d5dfe13fe1","owner":[],"postedDate":"November 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T16:49:37+00:00","versionOfRecord":{"articleIdentity":"rs-7937768","link":"https://doi.org/10.1186/s12933-025-03047-2","journal":{"identity":"cardiovascular-diabetology","isVorOnly":false,"title":"Cardiovascular Diabetology"},"publishedOn":"2026-01-14 16:30:40","publishedOnDateReadable":"January 14th, 2026"},"versionCreatedAt":"2025-11-10 22:01:12","video":"","vorDoi":"10.1186/s12933-025-03047-2","vorDoiUrl":"https://doi.org/10.1186/s12933-025-03047-2","workflowStages":[]},"version":"v1","identity":"rs-7937768","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7937768","identity":"rs-7937768","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}