TGF-β3 attenuates septic cardiomyopathy by reversing cardiomyocyte metabolic reprogramming through Smad7 signaling

TGF-β3 attenuates septic cardiomyopathy by reversing cardiomyocyte metabolic reprogramming through Smad7 signaling | 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 TGF-β3 attenuates septic cardiomyopathy by reversing cardiomyocyte metabolic reprogramming through Smad7 signaling Hongxuan Zhang, Jingqing Xu, Bin Xu, Xiuling Shang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7130728/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jan, 2026 Read the published version in Molecular and Cellular Biochemistry → Version 1 posted 12 You are reading this latest preprint version Abstract BACKGROUND Septic cardiomyopathy (SCM) exhibits over 70% mortality, primarily attributed to cardiomyocyte metabolic reprogramming shifting from oxidative phosphorylation (OXPHOS) to glycolysis. Although TGF-β3 modulates energy metabolism in other tissues, its function in SCM pathogenesis remains unexplored. METHODS In vivo, cecal ligation puncture (CLP) rat models received myocardial injections of TGF-β3-overexpressing or interfering adenovirus. Myocardial injury through histopathology (HE) and apoptosis (TUNEL), and mitochondrial ultrastructure via transmission electron microscopy (TEM). In vitro, primary cardiomyocytes treated with lipopolysaccharide (LPS) were transfected with TGF-β3 overexpression plasmid, with metabolic analyzed using Seahorse XF technology (extracellular acidification rate, ECAR; oxygen consumption rate, OCR). Molecular mechanisms were investigated via Western blotting and co-immunoprecipitation (Co-IP) targeting TGF-β3/Smad7/SKP1 signaling. FINDINGS TGF-β3 was significantly downregulated in SCM. Its overexpression attenuated myocardial injury and apoptosis, improved mitochondrial integrity, and reversed metabolic reprogramming by reducing glycolysis while enhancing OXPHOS. Mechanistically, TGF-β3 promoted Smad7 phosphorylation to inhibit Smad2/3 activation and suppressed SKP1 expression to reduce Smad7 ubiquitination, as confirmed by Co-IP. INTERPRETATION TGF-β3 confers cardioprotection in SCM by reversing metabolic reprogramming through dual regulation of Smad7: enhancing phosphorylation to block Smad2/3 signaling and inhibiting SKP1-mediated ubiquitination to stabilize Smad7. This newly identified TGF-β3/Smad7 axis represents a promising therapeutic target for SCM. Septic cardiomyopathy Metabolic reprogramming TGF-β3 Smad7 Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Sepsis is defined as a dysregulation of the host response to infection leading to life-threatening organ dysfunction[ 1 ]. Globally, the mortality rate of septic patients can be as high as 46.4%[ 2 ], and more severe organ dysfunction is associated with higher mortality[ 1 ]. In septic patients with secondary cardiac dysfunction, known as septic cardiomyopathy (SCM)[ 3 ], the mortality rate can be as high as 70%[ 4 ] causing a severe social and economic burden. The pathogenesis of SCM is complex, and mitochondrial dysfunction plays an critical role[ 5 ]. As the site of the tricarboxylic acid cycle and oxidative phosphorylation, mitochondrial dysfunction is often accompanied by metabolic disturbances. Previous studies have shown that sepsis is associated with decreased oxidative phosphorylation and increased glycolysis in different tissues of the body[ 6 ], i.e., the phenomenon of “metabolic reprogramming” in sepsis. This phenomenon is also present in cardiomyocytes, which are highly dependent on mitochondrial oxidative phosphorylation (OXPHOS) for ATP production. Under physiological conditions, fatty acid oxidation (FAO) dominates to provide substrates for OXPHOS[ 7 ]. In sepsis, the expression of peroxisome proliferator-activated receptors (PPARs) and their γ-coactivator 1 (PGC1α) in cardiomyocytes is suppressed, preventing fatty acid β-oxidation and limiting fatty acid transcription. Consequently, fatty acid β-oxidation is inhibited, and fatty acid transport into mitochondria is restricted, leading to impaired FAO[ 8 ]. In response to the energy crisis, cardiomyocytes are forced to turn to inefficient glycolysis for energy supply, resulting in metabolic reprogramming characterized by a shift from highly efficient OXPHOS to low-efficiency glycolysis[ 5 ]. Therefore, cardiomyocyte metabolic reprogramming is the initiating link in energy depletion, and reversing this metabolic reprogramming represents a highly promising therapeutic strategy and a current research focus in SCM[ 9 , 10 ]. Further elucidation of the mechanisms regulating cardiomyocyte metabolic reprogramming in SCM will help to find effective targets for intervention and thus improve the prognosis of patients. Current research predominantly indicates that the cytokines involved in the pathogenesis of SCM are mainly interleukins, tumor necrosis factor, and complement[ 3 ]. There have been limited studies of the transforming growth factor family (TGF-β) in sepsis, of which TGF-β3 is a member, and its key functions are to regulate immunity and inflammation, and to promote wound fibrosis and healing[ 11 , 12 ]. Several studies have also demonstrated that TGF-β3 has a regulatory effect on energy metabolism, including influencing the transport of various chemicals into and out of mitochondria in keratocytes[ 13 ] and decreasing mitochondrial aerobic respiration in sertoli cells[ 14 ]. However, studies investigating the role of TGF-β3 in energy metabolism within SCM are lacking. The classical mode of action of TGF-β, as a secreted cytokine, is through binding to the TGF-β receptor (TβR) on the cell membrane leading to the recruitment and activation of Smad signaling to transduce signals into the cell[ 15 ]. TGF-β signaling can exert its effects by reprogramming cellular metabolism[ 16 ] and is highly specific in different cells, potentially due to cell-type-specific epigenetic landscapes or interactions among distinct Smad factors[ 17 – 19 ]. This study will therefore aim to clarify the regulatory role of TGF-β3/Smad signaling on metabolic reprogramming in cardiomyocytes by constructing animal and cellular models of SCM, and to elucidate its effect on SCM. This study will provide a theoretical basis for targeting TGF-β3 and metabolic reprogramming as novel diagnostic or therapeutic strategies for SCM. 2. Methods 2.1 Animal model 8-week-old male SD rats were purchased from Beijing Huafukang Biotechnology Co. All experimental procedures were carried out in accordance with the Regulations on the Administration of Laboratory Animals issued by the National Science and Technology Commission and the Implementing Rules for the Administration of Medical Laboratory Animals issued by the Ministry of Health, and approved by the Laboratory Animal Ethics Committee of Jiangxi Zhonghong Boyuan Biotechnology Co. All rats were placed in a temperature-controlled room (22–26°C) with a light/dark cycle of 12 h, relative humidity of 40–70%, and free access to food and water. After 7 days of acclimatization, animals were anesthetized, and a rat sepsis model was established via cecum ligation and puncture (CLP). Following anesthesia, a midline abdominal surgical incision was made to expose the cecum and ligated at 3/5 with a 4 − 0 suture. The cecum was punctured with a 21G needle extruding a small amount of feces, and the abdomen was closed with a 3 − 0 suture. Only the cecum was exposed in the sham operation group. Cardiac ultrasonography was performed at 24, 48, and 72 h postoperatively, and myocardial tissue samples were collected to explore the cardiomyopathic injury in rats at different times after sepsis surgery. To further explore the role of TGF-β3 in vivo, myocardial in situ injection of interfering and overexpressing adenovirus of TGF-β3 was used, and the sepsis cardiomyopathy model was constructed 3 days after adenoviral injection, and myocardial tissues of the whole heart were obtained from rats that were necropsied 48h after modeling. 2.2 HE staining Myocardial tissues of rat whole heart were fixed with 4% paraformaldehyde (PFA), dehydrated by gradient of ethanol solution, paraffin-embedded after xylene transparency, and sectioned. Sections were baked, dewaxed and hydrated, stained with hematoxylin stain for 3–5 min, rinsed with running water, differentiated with 1% hydrochloric acid in alcohol, counterblued with counterblue solution and stained with eosin for 3–5 min, then sections were dehydrated, sealed and observed under a microscope (BX43, Olympus) with panoramic scanning. The scoring criteria were as follows: 0 points, normal tissue; 1. myocardial damaged area < 25%; 2. myocardial damaged area at 25% ~ 50%; 3. myocardial damaged area at 50% ~ 75%; 4. myocardial damaged area at 75% ~ 100%. 2.3 Cell Model Neonatal rats of 1–3 days old, adult normal SD rats and model SD rats were taken, sterilized by 75% alcohol after execution, the hearts were removed in an ultra-clean bench, washed with pre-cooled D-PBS for 3 times, apical tissues were taken, clipped, digested with 0.05% trypsin (T1300, Solarbio) by blowing for 1min, then digested with 0.2% collagenase II ( C8150, Solarbio) at 37℃ for 20min, terminate the digestion and mix, centrifuge at 1500rpm for 5min, discard the supernatant, and culture with DMEM/F12 complete medium (containing 10% FBS + 1% penicillin-streptomycin), and the resulting cell suspension was subjected to differential plating for 60 min and discard the supernatant to leave the adherent cells for further culture. Exploiting the differential adhesion rates of cardiac fibroblasts (which adhere faster) and cardiomyocytes (which remain in suspension). After approximately 60 min, the non-adherent cell suspension (enriched in cardiomyocytes) was carefully transferred to a new culture vessel, and fresh medium was added, and then put in an incubator to continue to cultivate. Immunofluorescence staining was performed to observe α-actin positive expression to identify primary cardiomyocytes. To simulate septic cardiomyopathy in vitro, different concentrations of LPS (5, 10, 20, and 50 µg/mL) were used to stimulate primary cardiomyocytes for 6 h. It was determined that the best modeling effect was achieved at 50 µg/mL. To further investigate the role of TGF-β3, TGF-β3 overexpression vector and empty vector were transfected into cardiomyocytes as follows: when the cell density reached about 70%, the medium was replaced with serum-free medium, and transfection was performed using Lipofectamine 3000, and then the same volume of DMEM/F12 medium (containing 20% FBS) was added after 4h of transfection. LPS stimulation was performed after 48h. 2.4 Transmission electron microscopy Some myocardial tissues or treated cells were taken from the apical portion of the rat heart, fixed with 2.5% glutaraldehyde for 2 h, and again fixed with 1% starvation acid fixative for 2 h. For myocardial tissues, acetone was treated, and the tissues were tissue-embedded with epoxy resin, and then incubated at 37°C overnight, 45°C for 12 h, and 60°C for 48 h, respectively, and then sectioned using an ultramicrotome to a thickness of 70 nm. For cultured cardiomyocytes, fixed cell pellets were embedded in Spurr's 812 epoxy resin. Finally, double staining with 3% uranyl acetate-lead citrate was performed and observed under a transmission electron microscope (HT7800/HT7700, HITACHI, Japan). And the mitochondrial damage was scored, Flameng grading method scoring scale: grade 0 (0 points): normal; grade I (1 point): essentially normal structure, loss of matrix granules (mild swelling, reduced matrix density, cristae separation). Grade II (2 points): mitochondrial swelling (severe reduction in matrix density, cristae separation); matrix hyaline, cristae not broken. Grade III (3 points): mitochondrial cristae broken, matrix coagulated (severe swelling). Grade IV (4 points): loss of inner and outer membrane integrity, vacuolated (severe swelling with cristae fractured, inner and outer membranes ruptured). 2.5 TUNEL assay Paraffin-embedded rat whole heart myocardial tissue, make sections, after baking, dewaxing and hydration, Proteinase K was used for repair, washed thoroughly with PBS three times, PBS around the tissue was sucked off with blotting paper, and a sufficient amount of TUNEL assay solution (C1090, Biunsun) was added dropwise to each slide, and the tissue was incubated at 42°C for 1h under light protection. PBS was used to wash off the excess assay solution, and the specimen was stained with DAPI dropwise for 3min, and the excess DAPI was rinsed with PBS. PBS was used to wash away the excess detection solution, incubate the specimen with DAPI for 3 min, stain the nucleus, and rinse the excess DAPI with PBS; excess liquid was carefully removed from the slides using absorbent paper, sealed with sealing solution containing anti-fluorescence quencher, and the images were collected by observing under the fluorescence microscope. Sections were placed under a scanner (Pannoramic MIDI, 3DHISTECH) to capture images for observation. The images were analyzed for positive score ratio using Image-Pro Plus 6.0 software to quantify the area of positive fluorescence. 2.6 Western Blotting Cardiac tissues or cultured cells were lysed with frozen RIPA lysis buffer (C1053, Applygen), centrifuged at 12,000 r/min for 10 min at 4°C, and the supernatant was harvested.Total protein was quantified by BCA Protein Quantification Kit (E-BC-K318-M, Elabscience), and proteins were separated by SDS-PAGE and transferred to PVDF membranes for immunoblotting. The PVDF membranes were blocked with 5% skimmed milk and then incubated with primary antibodies diluted 1:1000 at 4°C overnight. Primary antibodies used included:Mouse Anti-GAPDH (HC301, TransGen Biotech, 1/2000), Rabbit Anti Smad2/3 (GB111844, Servicebio, 1/1000), Rabbit Anti p-Smad2/3 (AF3367. Affinity, 1/1000), Rabbit Anti Smad7 (AF5147, Affinity, 1/1000), Rabbit Anti p-Smad7 (AF3827, Affinity, 1/1000), Rabbit Anti TGF-β3 (18942-1-AP. Proteintech, 1/1000), Rabbit Anti SKP1 (10990-2-AP, Proteintech, 1/1000). The PVDF membranes were incubated with secondary anti-IgG (H + L) (GB23301/GB23303, Servicebio) at room temperature on the following day for 2 h. The membranes were washed, and the PVDF membranes were wetted with luminescent solution to develop images on an ultra-high sensitivity chemiluminescent imaging system (Tanon-5200, Shanghai Tennent Technology Co., Ltd.) and were quantified using ImageJ V1.8.0 software (NIH, USA). 2.7 Extracellular acidification rate (ECAR) assay and oxygen consumption rate (OCR) assay The Seahorse XF Glycolysis Rate Assay Kit (103344-100, Agilent, USA) was used to detect changes in the cellular glycolysis acid production rate after intervening with TGF-β3, to assess its effect on energy metabolism in cardiomyocytes. The cellular mitochondrial stress test was performed with a Seahorse analyzer (XFe24, Agilent, USA), and measurements were taken at 10-min intervals, and the corresponding drugs were automatically added by the analyzer at the corresponding time points to obtain the acidification rate curve of the cell medium and glycolytic capacity. The Seahorse XF Analyzer assesses mitochondrial respiratory cell function by determining the oxygen consumption rate (OCR) of living cells in multiwell plates. The Agilent Seahorse XF Analyzer measures oxygen consumption rate (OCR) at approximately 5–8 minute intervals. OCR serves as an indicator of mitochondrial respiration. OCR is measured in real time by creating a transient microchamber (~ 7 µL) above a monolayer of cells in a microtiter plate. cellular oxygen consumption (respiration) results in rapid and easily measured changes in the concentration of dissolved oxygen and free protons in the “transient microcompartment”, which can be measured every few seconds by a solid-state sensor probe located 200 µm above the monolayer of cells. The instrument records changes in concentration over 2–5 minute measurement periods and subsequently calculates the OCR. 2.8 Statistical analysis Data are presented as mean ± SEM (n ≥ 3 per group). Differences between two groups were analyzed using Student's t-test. Comparisons among more than two groups were analyzed using one-way or two-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software Inc.). p < 0.05 indicates a statistically significant difference. 3. Results 3.1 Myocardial tissue injury accompanied by metabolic changes in SCM CLP was performed to establish the SCM rat model, and myocardial tissues were harvested at 24h, 48h and 72h after modeling, and HE staining revealed disrupted myocardial tissue structure at each modeling time point, accompanied by inflammatory cell infiltration and fibrosis (Fig. 1 A), confirming successful establishment of the SCM myocardial injury model in rats. The lactate levels in the homogenates of rat myocardial tissues were significantly elevated at all modeling time points, suggesting that there were metabolic changes in myocardial tissues (Fig. 1 B). In addition, the pathological injury score showed that the injury score was highest at 48 hours after modeling, and the lactate level was also highest at 48 hours after modeling. Therefore, the 48-hour time point was selected for subsequent experiments. . 3.2 TGF-β3 showed protective effects against SCM The SCM rat model was constructed by CLP, and the HE staining results showed that myocardial tissue structure was damaged in the SCM model group (Model), inflammatory cell infiltration, and the pathological damage score was elevated; the degree of myocardial damage was reduced in the TGF-β3 overexpression group (TGF-β3 OE), and the pathological damage score was lowered; myocardial damage was aggravated in the TGF-β3 interference group (TGF-β3 sh), and the pathological damage score was further elevated (Fig. 2 A). TUNEL staining revealed that apoptosis was significantly elevated in the SCM model group, the degree of apoptosis was reduced in the TGF-β3 overexpression group, and apoptosis was further aggravated in the TGF-β3 interference group (Fig. 2 B). Transmission electron microscopy results showed that myocardial fibers in the SCM model group were disarranged, the gap was widened, the mitochondrial structure was disrupted, exhibiting swelling, cristae fragmentation, and pleomorphism, and the mitochondrial damage score was significantly increased. The cardiomyocytes in the TGF-β3 overexpression group had markedly improved ultrastructure, mitochondrial morphology normalized, with cristae arranged in parallel arrays, and the mitochondrial damage score was significantly reduced, while cardiomyocytes in the TGF-β3 interference group ultrastructure was further disrupted and mitochondrial damage was aggravated (Fig. 2 C). Collectively, these results demonstrate that TGF-β3 had a protective effect on SCM. 3.3 TGF-β3 reversed cardiomyocyte metabolic reprogramming in SCM In the SCM cell model established by LPS treatment of primary rat cardiomyocytes, the Seahorse assay revealed an increase in the extracellular acidification rate (ECAR) and glycolytic capacity significantly increased after LPS treatment, suggesting that LPS promoted glycolysis in cardiomyocytes, whereas TGF-β3 overexpression resulted in a decrease in the level of extracellular acidification rate (ECAR), and basal glycolysis and glycolytic capacity were markedly reduced, and there was no change in non-glycolytic acidification (Fig. 3 A). In addition, after LPS treatment, oxygen consumption rate (OCR) decreased significantly, while basal and maximal respiration rates of cardiomyocytes decreased, indicating that LPS inhibited mitochondrial aerobic respiration, whereas after TGF-β3 overexpression, OCR increased significantly, while basal and maximal respiration rates recovered, with no change in non-mitochondrial respiration (Fig. 3 B). These findings indicate that there is metabolic reprogramming in cardiomyocytes during SCM, i.e., enhancement of glycolysis and attenuation of oxidative phosphorylation, and TGF-β3 can reverse this phenomenon, thus exerting its protective effects. 3.4 TGF-β3 promotes Smad7 phosphorylation and inhibits SKP1-mediated ubiquitination degradation of Smad7 To elucidate the regulatory mechanism of TGF-β3 on metabolic reprogramming of cardiomyocytes, a SCM rat model was constructed using CLP. First, Firstly, TGF-β3 expression was downregulated in the CLP group (Model) compared with the sham-operated group (Sham), consistent with the trend of TGF-β3 as a protective factor in SCM. Second, Smad2/3 phosphorylation was enhanced in the CLP group (Model) compared to the sham-operated group (Sham), however, TGF-β3 overexpression reversed Smad2/3 phosphorylation, and Smad2/3 phosphorylation was promoted after interference with TGF-β3 (Fig. 4 A). Given that Smad2/3 are R-Smands, we also examined Smad7, an inhibitory Smad (I-Smad) and found that Smad7 phosphorylation was increased after overexpression of TGF-β3, and conversely, Smad7 phosphorylation was inhibited after interference with TGF-β3 (Fig. 4 A). In the SCM cell model established by LPS treatment of primary rat cardiomyocytes, it was also found that TGF-β3 expression level was downregulated, Smad2/3 phosphorylation level was increased, and Smad7 phosphorylation was decreased after LPS stimulation, whereas overexpression of TGF-β3 decreased Smad2/3 phosphorylation level and increased Smad7 phosphorylation level (Fig. 4 B). These results suggest that TGF-β3 plays a role in the inhibitory regulation of Smad2/3 by promoting Smad7 phosphorylation. In addition, we found that TGF-β3 acts on SKP1, a core component of the E3 ubiquitin ligase SCF complex, in cardiomyocytes by IP-MS in a previous study (unpublished), and this experiment confirmed that TGF-β3 overexpression reduced SKP1 expression levels, and conversely, the level of SKP1 expression was increased after interfering with TGF-β3, suggesting that TGF-β3 inhibits SKP1 expression (Fig. 4 A). This further supports the role of SKP1 in mediating Smad7 ubiquitination and degradation. Co-immunoprecipitation (Co-IP) confirmed normal expression of Ubiquitin, Smad7, and GAPDH in the Input lysates across all groups, and showed that TGF-β3 overexpression increased Smad7 expression, an effect counteracted by SKP1 overexpression. The IP results demonstrated successful Ubiquitin pull-down and ubiquitination of Smad7, and it was found that ubiquitinated Smad7 levels decreased following TGF-β3 overexpression, and ubiquitinated Smad7 levels increased following SKP1 overexpression (Fig. 4 C). These results indicate that SKP1 promotes Smad7 ubiquitination and degradation, and that TGF-β3 binding to SKP1 inhibits this process. The above results suggest that TGF-β3 inhibits SKP1-mediated ubiquitination degradation of Smad7. 4. Discussion Metabolic reprogramming was first identified in cancer cells, where cells utilize glycolysis for ATP production even under aerobic conditions, but rather a heavy reliance on inefficient glycolysis[20]. Recently, metabolic reprogramming has also been identified in sepsis: immune cells shift from oxidative phosphorylation to glycolysis, as the main mode of energy supply to facilitate the generation of a large amount of energy for activation in a short period of time; other tissue cells enter a "hibernation-like" state characterized by suppressed physiological functions and metabolic activity[6]. In SCM patients, an initial compensatory hypercontractility phase is often followed by reversible cardiac dysfunction[21,22], mirroring a "hibernation-like" state. This functional impairment aligns with the underlying phenomenon of metabolic reprogramming in cardiomyocytes. Focusing on cardiomyocyte metabolic reprogramming in SCM, this study demonstrates that TGF-β3 reverses cardiomyocyte metabolic reprogramming by reducing the ubiquitination degradation and promoting the phosphorylation of Smad7 through the inhibition of SKP1. The TGF-β family comprises multifunctional peptide cytokines that elicit diverse responses, including promoting inflammation and apoptosis in cardiomyocytes, activating macrophage phagocytosis, and inducing fibrogenesis in fibroblasts[23], thereby influencing cardiac repair, remodeling, and fibrosis[24]. Members of this family, including TGF-β1, TGF-β2, and TGF-β3, play significant roles in regulating energy metabolism, with functional subtype specificity.[16,25-27]. TGF-β1 acts as a key regulator of macrophage metabolism, promoting glycolysis during sepsis[28]; It also induces metabolic reprogramming in fibroblasts, shifting them towards glycolysis and away from mitochondrial OXPHOS to meet the high biosynthetic demands of their profibrotic or proinflammatory states[25]. In contrast, TGF-β3 may counteract pathological fibrosis and reverse metabolic reprogramming, potentially by competing with TGF-β1[29,30]; for example, TGF-β3 heterozygous knockout mice exhibit early renal fibrosis accompanied by insulin resistance and aberrant lipid metabolism[31]; Furthermore, studies confirm that TGF-β2 or TGF-β3, but not TGF-β1, promotes fatty acid oxidation in myotubes and adipocytes[16,32]. Combined with previous findings of upregulated TGF-β1 in myocardial infarction and hypertrophy[33], the specific downregulation of TGF-β3 observed in SCM in this study highlights its distinct role in regulating cardiomyocyte metabolism, and suggests that the downregulation of TGF-β3 may be an important pathologic feature in SCM, providing a rationale for exploring TGF-β3 as a potential diagnostic biomarker or therapeutic target. In this study, we clarified that overexpression of TGF-β3 attenuates myocardial pathological injury, reduces cardiomyocyte apoptosis, and ameliorates mitochondrial structural disruption, demonstrating its cardioprotective effects. Our prior unpublished work focused on mitochondrial dynamics, revealing a key role for AMPK in TGF-β3-mediated mitochondrial biogenesis and autophagy. AMPK is an energy receptor for cellular metabolism[34]. Therefore, the present study focuses on the effect of TGF-β3 on energy metabolism in cardiomyocytes. Direct evidence confirming metabolic reprogramming specifically in SCM cardiomyocytes is relatively limited. The present study confirms the existence of metabolic reprogramming in cardiomyocytes during SCM, which is mainly manifested by enhanced glycolysis and attenuated oxidative phosphorylation. While metabolic reprogramming provides rapid energy compensation short-term, its persistence is detrimental. Consequences include: chronic ATP underproduction, directly impairing contractile function[8]; accumulation of unoxidized lipids, triggering lipotoxic cardiomyocyte damage[9]; metabolite buildup (e.g., lactate), causing microenvironment acidification, immunosuppression, and cellular damage[35], and energy deprivation-induced mitochondrial damage (collapse of membrane potential and respiratory chain disassembly), and the release of mitochondrial DNA fragments activating the NLRP3 inflammasome, creating a vicious cycle of mitochondrial damage, amplified inflammation, and energy depletion[36]. Therefore, reversing metabolic reprogramming represents a promising therapeutic strategy for SCM. This study demonstrates that TGF-β3 overexpression effectively reverses these metabolic abnormalities, constituting a key mechanism underlying its cardioprotective effect. The present study also explored the signaling pathway of TGF-β3 to reverse metabolic reprogramming. While the classical paradigm holds that TGF-β activates downstream profibrotic or inflammatory signaling primarily through Smad2/3 phosphorylation[15]. In the present study, we found that Smad2/3 phosphorylation was reduced, which may be a specific effect of TGF-β3 in a specific pathological setting (SCM). Smad7 belongs to the inhibitory Smad (I-Smads), a classical negative feedback regulator of TGF-β signaling, which competes with receptor-regulated Smads (R-Smads; Smad2/3) for binding to activated TβRI, thereby preventing R-Smad phosphorylation and activation[15]. Smad7 is regulated by non-coding RNAs (ncRNAs) and other post-translational modifications (PTMs)[37]. Phosphorylation, a critical PTM, plays a pivotal role in regulating protein stability and function[38]. However, Smad7 phosphorylation has primarily been studied in the context of TGF-β1 signaling[39,40]. Therefore, the present study verified that TGF-β3 is promotional for Smad7 phosphorylation. Beyond phosphorylation, we investigated mechanisms regulating Smad7 stability, given its impact on TGF-β3 signaling and found that TGF-β3 inhibited SKP1-mediated ubiquitination degradation of Smad7. SKP1 is a core component of the SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase complex[41] , acting as an adaptor bridging CUL1 and F-box proteins, and its activity is essential for regulating the myriad of ubiquitination degradation processes controlled by the SCF complex[42]. Stabilized Smad7 binds activated TβRI more efficiently and competitively, thereby blocking Smad2/3 phosphorylation and activation, consistent with our observed inhibition of Smad2/3 phosphorylation. This study reveals for the first time a novel mechanism by which TGF-β3 exerts cardioprotective effects by regulating the ubiquitinated degradation of Smad7, distinct from previously known mechanisms regulating Smad7. TGF-β3 regulates Smad7 phosphorylation status and, by concurrently enhancing its stability, augments Smad7's capacity to inhibit R-Smad signaling. This study also has some limitations. First, animal and cellular models may not fully recapitulate human disease, and clinical correlation studies are needed. Second, Smad7 can recruit E3 ubiquitin ligases (e.g., Smurf1/2) to degrade activated TβRI receptors and thus inhibit Smad2/3 phosphorylation, a possibility not explored here; whether there is a protein post-translational modification site interaction between Smad7 phosphorylation and ubiquitination needs to be further verified. Finally, the specific molecular targets of TGF-β3 in reversing metabolic reprogramming (e.g., key metabolic enzymes or pathways) remain incompletely defined and require further rigorous validation, such as conditional knockdown studies. This study establishes the reversal of metabolic reprogramming as a novel mechanism for TGF-β3-mediated cardioprotection in SCM. Furthermore, it reveals a previously unrecognized molecular mechanism involving TGF-β3 promotion of Smad7 phosphorylation and inhibition of SKP1-mediated Smad7 ubiquitination and degradation, and deepening our understanding of SCM pathogenesis, particularly the crosstalk between signaling and metabolic regulation. A strength of this study is the combined use of in vivo (rat) and in vitro (cardiomyocyte) models, enabling multi-level investigation from phenotype to molecular mechanism. Future clinical studies should investigate circulating levels of TGF-β3 in SCM patients and their correlation with cardiac function and prognosis, so as to provide new biomarkers for the diagnosis and treatment of SCM. Declarations Author contributions Conceptualization: HZ, XS Data curation: HZ, JX Methodology: HZ, JX Investigation: HZ, JX, BX Visualization: HZ Funding acquisition: XS Software development: BX Writing – original draft: HZ, JX Writing – review & editing: XS Data and materials availability The data that support the findings of this study are available on request from the corresponding author, XS, upon reasonable request. Competing interests Authors declare that they have no competing interests. Acknowledgements This work is supported by Fujian Provincial Health Technology Project(No.2023ZQNZD001). References M. Singer, C.S. Deutschman, C.W. Seymour, M. Shankar-Hari, D. Annane, M. Bauer, R. Bellomo, G.R. Bernard, J.-D. Chiche, C.M. Coopersmith, R.S. Hotchkiss, M.M. Levy, J.C. Marshall, G.S. Martin, S.M. Opal, G.D. Rubenfeld, T. van der Poll, J.-L. Vincent, D.C. 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Cardiology. 18 (2021) 424-434. https://doi.org/10.1038/s41569-020-00492-2. I.A. Hobai, CARDIOMYOCYTE REPROGRAMMING IN ANIMAL MODELS OF SEPTIC SHOCK, Shock. 59 (2023) 200-213. https://doi.org/10.1097/shk.0000000000002024. C. de Ceuninck van Capelle, M. Spit, P. ten Dijke, Current perspectives on inhibitory SMAD7 in health and disease, Critical Reviews in Biochemistry and Molecular Biology. 55 (2020) 691-715. https://doi.org/10.1080/10409238.2020.1828260. Q. Zhong, X. Xiao, Y. Qiu, Z. Xu, C. Chen, B. Chong, X. Zhao, S. Hai, S. Li, Z. An, L. Dai, Protein posttranslational modifications in health and diseases: Functions, regulatory mechanisms, and therapeutic implications, MedComm. 4 (2023) e261. https://doi.org/10.1002/mco2.261. J. Dong, L. Ding, L. Wang, Z. Yang, Y. Wang, Y. Zang, X. Cao, L. Tang, Effects of bradykinin on proliferation, apoptosis, and cycle of glomerular mesangial cells via the TGF-β1/Smad signaling pathway, Turkish journal of biology = Turk biyoloji dergisi. 45 (2021) 17-25. https://doi.org/10.3906/biy-2007-58. J. Mao, Z. Sun, Y. Cui, N. Du, H. Guo, J. Wei, Z. Hao, L. Zheng, PCBP2 promotes the development of glioma by regulating FHL3/TGF-β/Smad signaling pathway, J Cell Physiol. 235 (2020) 3280-3291. https://doi.org/10.1002/jcp.29104. T. Cardozo, M. Pagano, The SCF ubiquitin ligase: insights into a molecular machine, Nature Reviews Molecular Cell Biology. 5 (2004) 739-751. https://doi.org/10.1038/nrm1471. Y. Yoshida, A. Murakami, K. Tanaka, Skp1 stabilizes the conformation of F-box proteins, Biochem Biophys Res Commun. 410 (2011) 24-28. https://doi.org/10.1016/j.bbrc.2011.05.098. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 02 Jan, 2026 Read the published version in Molecular and Cellular Biochemistry → Version 1 posted Editorial decision: Revision requested 02 Oct, 2025 Reviews received at journal 29 Sep, 2025 Reviewers agreed at journal 23 Sep, 2025 Reviews received at journal 21 Sep, 2025 Reviewers agreed at journal 07 Sep, 2025 Reviews received at journal 20 Aug, 2025 Reviewers agreed at journal 04 Aug, 2025 Reviewers agreed at journal 30 Jul, 2025 Reviewers invited by journal 28 Jul, 2025 Editor assigned by journal 27 Jul, 2025 Submission checks completed at journal 15 Jul, 2025 First submitted to journal 15 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7130728","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":494801410,"identity":"e3cf38a1-44e6-4b98-b373-413f59d9895b","order_by":0,"name":"Hongxuan Zhang","email":"","orcid":"","institution":"Department of Critical Care Medicine, Shengli Clinical Medical College of Fujian Medical University, Fuzhou University Affiliated Provincial Hospital, Fujian Provincial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hongxuan","middleName":"","lastName":"Zhang","suffix":""},{"id":494801412,"identity":"121b322b-0071-4e59-9c24-8a3533e58e19","order_by":1,"name":"Jingqing Xu","email":"","orcid":"","institution":"Department of Critical Care Medicine, Shengli Clinical Medical College of Fujian Medical University, Fuzhou University Affiliated Provincial Hospital, Fujian Provincial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jingqing","middleName":"","lastName":"Xu","suffix":""},{"id":494801413,"identity":"887393e1-6c4f-4702-8aab-9c9528747b8b","order_by":2,"name":"Bin Xu","email":"","orcid":"","institution":"Department of Critical Care Medicine, Shengli Clinical Medical College of Fujian Medical University, Fuzhou University Affiliated Provincial Hospital, Fujian Provincial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Xu","suffix":""},{"id":494801414,"identity":"899fcea6-5589-4703-b915-8d943bf365dd","order_by":3,"name":"Xiuling Shang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIie3PsWrDMBCA4TMGZ7lEHeWleYULgSyF+FUUDM2SwWPGQKFZ7AfoW7RbxwsH7WLIGmgHdfHUwWvAQ+VCRsceC9UPp0HoQxKAz/cXQzeWAFSovuz2d8sMIMaReI8hlcOJGzpipAeRaf5eaZN9Lp9l/LblRkCNNgTn124SFPmcDFXpk0zuT4wCcf5NQVF2k1BhYA1JOhFYfNRagE4bCoPHbhKpkeWWgCMZk0DSR3C8m7W3LG8EF8DG3aJ7iMay/YuY+CFKNfMadVllh+IKmebrSteNJErJoebm7lbt0xd7vkIurXaXl7YL9wOAZMghn8/n+6f9AOJZUptZmNndAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Critical Care Medicine, Shengli Clinical Medical College of Fujian Medical University, Fuzhou University Affiliated Provincial Hospital, Fujian Provincial Hospital","correspondingAuthor":true,"prefix":"","firstName":"Xiuling","middleName":"","lastName":"Shang","suffix":""}],"badges":[],"createdAt":"2025-07-15 12:53:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7130728/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7130728/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11010-025-05468-9","type":"published","date":"2026-01-02T15:58:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88197279,"identity":"05d0d806-a3d3-4b3b-a2ad-44faddfd82fb","added_by":"auto","created_at":"2025-08-03 17:38:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2457322,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMyocardial tissue injury accompanied by metabolic changes in SCM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eRepresentative HE staining and pathological damage scores of myocardial tissues from SCM rats at different time points. Each group n=3. Scale bar was 50 um. Differences were assessed using one-way ANOVA and Tukey's multiple comparison test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003eELSIA was performed to detect lactate levels in myocardial tissues of SCM rats at different time points. Each group n=3. Differences were assessed using one-way ANOVA and Tukey's multiple comparison test.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7130728/v1/2112e4011033535bcb876199.png"},{"id":88197034,"identity":"186c1642-2f9e-495d-8928-7b371ce4fdee","added_by":"auto","created_at":"2025-08-03 17:30:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10019581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTGF-β3 showed protective effects against SCM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eRepresentative HE staining and pathological damage scoring of rat myocardial tissue. Each group n=3. Scale bar was 50 um. Differences were assessed using one-way ANOVA and Tukey's multiple comparison test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003eTUNEL staining was used to detect cardiomyocyte apoptosis. Each group n=3. Differences were assessed using one-way ANOVA and Tukey's multiple comparison test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003eTransmission electron microscopy was used to detect mitochondrial damage. Each group n=3. Scale bars are 2um above and 500nm below. red arrows point to mitochondria. Differences were assessed using one-way ANOVA and Tukey's multiple comparison test.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7130728/v1/646ffcfce7847c7bad9f2cf1.png"},{"id":88197031,"identity":"58e2a146-68b3-4067-b549-b05b69e9091a","added_by":"auto","created_at":"2025-08-03 17:30:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":516196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTGF-β3 reversed cardiomyocyte metabolic reprogramming in SCM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eThe extracellular acidification rate (ECAR) of cardiomyocytes was detected by seahorse. Each group n=3. Differences were assessed using one-way ANOVA and Tukey's multiple comparison test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003eOxygen consumption rate (OCR) of cardiomyocytes was assayed by seahorse. Each group n=3. Differences were assessed using one-way ANOVA and Tukey's multiple comparison test.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7130728/v1/510415a91fc0c312d76b7bb1.png"},{"id":88197035,"identity":"a71ca725-e0bb-4bfb-ad9e-3be1122df842","added_by":"auto","created_at":"2025-08-03 17:30:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2845621,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTGF-β3 promotes Smad7 phosphorylation and inhibits SKP1-mediated ubiquitination degradation of Smad7\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eWestern blot was performed to detect the total protein and phosphorylation levels of TGF-β3, SKP1, Smad7 and Smad2/3 in myocardial tissues of SCM rats. Each group n=3. and subsequently analyzed quantitatively by image lab software. Differences were assessed using one-way ANOVA and Tukey's multiple comparison test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003eWestern blot was performed to detect the total protein and phosphorylation levels of TGF-β3, Smad7 and Smad2/3 in SCM primary rat cardiomyocytes. Each group n=3. and subsequently analyzed quantitatively by image lab software. Differences were assessed using one-way ANOVA and Tukey's multiple comparison test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003eImmunoprecipitation assay to detect ubiquitinated degradation of Smad7 in cardiomyocytes.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7130728/v1/9143eb5c76ce1a11e1be028c.png"},{"id":99545389,"identity":"a897706a-218e-49a6-a15f-59b17a5417e1","added_by":"auto","created_at":"2026-01-05 16:06:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18564385,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7130728/v1/d0d8c7e7-6fcd-4565-9f2b-075162bddf5f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"TGF-β3 attenuates septic cardiomyopathy by reversing cardiomyocyte metabolic reprogramming through Smad7 signaling","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSepsis is defined as a dysregulation of the host response to infection leading to life-threatening organ dysfunction[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Globally, the mortality rate of septic patients can be as high as 46.4%[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and more severe organ dysfunction is associated with higher mortality[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In septic patients with secondary cardiac dysfunction, known as septic cardiomyopathy (SCM)[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], the mortality rate can be as high as 70%[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] causing a severe social and economic burden. The pathogenesis of SCM is complex, and mitochondrial dysfunction plays an critical role[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. As the site of the tricarboxylic acid cycle and oxidative phosphorylation, mitochondrial dysfunction is often accompanied by metabolic disturbances. Previous studies have shown that sepsis is associated with decreased oxidative phosphorylation and increased glycolysis in different tissues of the body[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], i.e., the phenomenon of \u0026ldquo;metabolic reprogramming\u0026rdquo; in sepsis. This phenomenon is also present in cardiomyocytes, which are highly dependent on mitochondrial oxidative phosphorylation (OXPHOS) for ATP production. Under physiological conditions, fatty acid oxidation (FAO) dominates to provide substrates for OXPHOS[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In sepsis, the expression of peroxisome proliferator-activated receptors (PPARs) and their γ-coactivator 1 (PGC1α) in cardiomyocytes is suppressed, preventing fatty acid β-oxidation and limiting fatty acid transcription. Consequently, fatty acid β-oxidation is inhibited, and fatty acid transport into mitochondria is restricted, leading to impaired FAO[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In response to the energy crisis, cardiomyocytes are forced to turn to inefficient glycolysis for energy supply, resulting in metabolic reprogramming characterized by a shift from highly efficient OXPHOS to low-efficiency glycolysis[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, cardiomyocyte metabolic reprogramming is the initiating link in energy depletion, and reversing this metabolic reprogramming represents a highly promising therapeutic strategy and a current research focus in SCM[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Further elucidation of the mechanisms regulating cardiomyocyte metabolic reprogramming in SCM will help to find effective targets for intervention and thus improve the prognosis of patients.\u003c/p\u003e\u003cp\u003eCurrent research predominantly indicates that the cytokines involved in the pathogenesis of SCM are mainly interleukins, tumor necrosis factor, and complement[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. There have been limited studies of the transforming growth factor family (TGF-β) in sepsis, of which TGF-β3 is a member, and its key functions are to regulate immunity and inflammation, and to promote wound fibrosis and healing[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Several studies have also demonstrated that TGF-β3 has a regulatory effect on energy metabolism, including influencing the transport of various chemicals into and out of mitochondria in keratocytes[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and decreasing mitochondrial aerobic respiration in sertoli cells[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, studies investigating the role of TGF-β3 in energy metabolism within SCM are lacking. The classical mode of action of TGF-β, as a secreted cytokine, is through binding to the TGF-β receptor (TβR) on the cell membrane leading to the recruitment and activation of Smad signaling to transduce signals into the cell[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. TGF-β signaling can exert its effects by reprogramming cellular metabolism[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and is highly specific in different cells, potentially due to cell-type-specific epigenetic landscapes or interactions among distinct Smad factors[\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis study will therefore aim to clarify the regulatory role of TGF-β3/Smad signaling on metabolic reprogramming in cardiomyocytes by constructing animal and cellular models of SCM, and to elucidate its effect on SCM. This study will provide a theoretical basis for targeting TGF-β3 and metabolic reprogramming as novel diagnostic or therapeutic strategies for SCM.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Animal model\u003c/h2\u003e\u003cp\u003e 8-week-old male SD rats were purchased from Beijing Huafukang Biotechnology Co. All experimental procedures were carried out in accordance with the Regulations on the Administration of Laboratory Animals issued by the National Science and Technology Commission and the Implementing Rules for the Administration of Medical Laboratory Animals issued by the Ministry of Health, and approved by the Laboratory Animal Ethics Committee of Jiangxi Zhonghong Boyuan Biotechnology Co. All rats were placed in a temperature-controlled room (22\u0026ndash;26\u0026deg;C) with a light/dark cycle of 12 h, relative humidity of 40\u0026ndash;70%, and free access to food and water.\u003c/p\u003e\u003cp\u003eAfter 7 days of acclimatization, animals were anesthetized, and a rat sepsis model was established via cecum ligation and puncture (CLP). Following anesthesia, a midline abdominal surgical incision was made to expose the cecum and ligated at 3/5 with a 4\u0026thinsp;\u0026minus;\u0026thinsp;0 suture. The cecum was punctured with a 21G needle extruding a small amount of feces, and the abdomen was closed with a 3\u0026thinsp;\u0026minus;\u0026thinsp;0 suture. Only the cecum was exposed in the sham operation group. Cardiac ultrasonography was performed at 24, 48, and 72 h postoperatively, and myocardial tissue samples were collected to explore the cardiomyopathic injury in rats at different times after sepsis surgery. To further explore the role of TGF-β3 in vivo, myocardial in situ injection of interfering and overexpressing adenovirus of TGF-β3 was used, and the sepsis cardiomyopathy model was constructed 3 days after adenoviral injection, and myocardial tissues of the whole heart were obtained from rats that were necropsied 48h after modeling.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 HE staining\u003c/h2\u003e\u003cp\u003eMyocardial tissues of rat whole heart were fixed with 4% paraformaldehyde (PFA), dehydrated by gradient of ethanol solution, paraffin-embedded after xylene transparency, and sectioned. Sections were baked, dewaxed and hydrated, stained with hematoxylin stain for 3\u0026ndash;5 min, rinsed with running water, differentiated with 1% hydrochloric acid in alcohol, counterblued with counterblue solution and stained with eosin for 3\u0026ndash;5 min, then sections were dehydrated, sealed and observed under a microscope (BX43, Olympus) with panoramic scanning. The scoring criteria were as follows: 0 points, normal tissue; 1. myocardial damaged area\u0026thinsp;\u0026lt;\u0026thinsp;25%; 2. myocardial damaged area at 25% ~ 50%; 3. myocardial damaged area at 50% ~ 75%; 4. myocardial damaged area at 75% ~ 100%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Cell Model\u003c/h2\u003e\u003cp\u003eNeonatal rats of 1\u0026ndash;3 days old, adult normal SD rats and model SD rats were taken, sterilized by 75% alcohol after execution, the hearts were removed in an ultra-clean bench, washed with pre-cooled D-PBS for 3 times, apical tissues were taken, clipped, digested with 0.05% trypsin (T1300, Solarbio) by blowing for 1min, then digested with 0.2% collagenase II ( C8150, Solarbio) at 37℃ for 20min, terminate the digestion and mix, centrifuge at 1500rpm for 5min, discard the supernatant, and culture with DMEM/F12 complete medium (containing 10% FBS\u0026thinsp;+\u0026thinsp;1% penicillin-streptomycin), and the resulting cell suspension was subjected to differential plating for 60 min and discard the supernatant to leave the adherent cells for further culture. Exploiting the differential adhesion rates of cardiac fibroblasts (which adhere faster) and cardiomyocytes (which remain in suspension). After approximately 60 min, the non-adherent cell suspension (enriched in cardiomyocytes) was carefully transferred to a new culture vessel, and fresh medium was added, and then put in an incubator to continue to cultivate. Immunofluorescence staining was performed to observe α-actin positive expression to identify primary cardiomyocytes.\u003c/p\u003e\u003cp\u003eTo simulate septic cardiomyopathy in vitro, different concentrations of LPS (5, 10, 20, and 50 \u0026micro;g/mL) were used to stimulate primary cardiomyocytes for 6 h. It was determined that the best modeling effect was achieved at 50 \u0026micro;g/mL. To further investigate the role of TGF-β3, TGF-β3 overexpression vector and empty vector were transfected into cardiomyocytes as follows: when the cell density reached about 70%, the medium was replaced with serum-free medium, and transfection was performed using Lipofectamine 3000, and then the same volume of DMEM/F12 medium (containing 20% FBS) was added after 4h of transfection. LPS stimulation was performed after 48h.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Transmission electron microscopy\u003c/h2\u003e\u003cp\u003eSome myocardial tissues or treated cells were taken from the apical portion of the rat heart, fixed with 2.5% glutaraldehyde for 2 h, and again fixed with 1% starvation acid fixative for 2 h. For myocardial tissues, acetone was treated, and the tissues were tissue-embedded with epoxy resin, and then incubated at 37\u0026deg;C overnight, 45\u0026deg;C for 12 h, and 60\u0026deg;C for 48 h, respectively, and then sectioned using an ultramicrotome to a thickness of 70 nm. For cultured cardiomyocytes, fixed cell pellets were embedded in Spurr's 812 epoxy resin. Finally, double staining with 3% uranyl acetate-lead citrate was performed and observed under a transmission electron microscope (HT7800/HT7700, HITACHI, Japan). And the mitochondrial damage was scored, Flameng grading method scoring scale: grade 0 (0 points): normal; grade I (1 point): essentially normal structure, loss of matrix granules (mild swelling, reduced matrix density, cristae separation). Grade II (2 points): mitochondrial swelling (severe reduction in matrix density, cristae separation); matrix hyaline, cristae not broken. Grade III (3 points): mitochondrial cristae broken, matrix coagulated (severe swelling). Grade IV (4 points): loss of inner and outer membrane integrity, vacuolated (severe swelling with cristae fractured, inner and outer membranes ruptured).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 TUNEL assay\u003c/h2\u003e\u003cp\u003eParaffin-embedded rat whole heart myocardial tissue, make sections, after baking, dewaxing and hydration, Proteinase K was used for repair, washed thoroughly with PBS three times, PBS around the tissue was sucked off with blotting paper, and a sufficient amount of TUNEL assay solution (C1090, Biunsun) was added dropwise to each slide, and the tissue was incubated at 42\u0026deg;C for 1h under light protection. PBS was used to wash off the excess assay solution, and the specimen was stained with DAPI dropwise for 3min, and the excess DAPI was rinsed with PBS. PBS was used to wash away the excess detection solution, incubate the specimen with DAPI for 3 min, stain the nucleus, and rinse the excess DAPI with PBS; excess liquid was carefully removed from the slides using absorbent paper, sealed with sealing solution containing anti-fluorescence quencher, and the images were collected by observing under the fluorescence microscope. Sections were placed under a scanner (Pannoramic MIDI, 3DHISTECH) to capture images for observation. The images were analyzed for positive score ratio using Image-Pro Plus 6.0 software to quantify the area of positive fluorescence.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Western Blotting\u003c/h2\u003e\u003cp\u003eCardiac tissues or cultured cells were lysed with frozen RIPA lysis buffer (C1053, Applygen), centrifuged at 12,000 r/min for 10 min at 4\u0026deg;C, and the supernatant was harvested.Total protein was quantified by BCA Protein Quantification Kit (E-BC-K318-M, Elabscience), and proteins were separated by SDS-PAGE and transferred to PVDF membranes for immunoblotting. The PVDF membranes were blocked with 5% skimmed milk and then incubated with primary antibodies diluted 1:1000 at 4\u0026deg;C overnight. Primary antibodies used included:Mouse Anti-GAPDH (HC301, TransGen Biotech, 1/2000), Rabbit Anti Smad2/3 (GB111844, Servicebio, 1/1000), Rabbit Anti p-Smad2/3 (AF3367. Affinity, 1/1000), Rabbit Anti Smad7 (AF5147, Affinity, 1/1000), Rabbit Anti p-Smad7 (AF3827, Affinity, 1/1000), Rabbit Anti TGF-β3 (18942-1-AP. Proteintech, 1/1000), Rabbit Anti SKP1 (10990-2-AP, Proteintech, 1/1000). The PVDF membranes were incubated with secondary anti-IgG (H\u0026thinsp;+\u0026thinsp;L) (GB23301/GB23303, Servicebio) at room temperature on the following day for 2 h. The membranes were washed, and the PVDF membranes were wetted with luminescent solution to develop images on an ultra-high sensitivity chemiluminescent imaging system (Tanon-5200, Shanghai Tennent Technology Co., Ltd.) and were quantified using ImageJ V1.8.0 software (NIH, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Extracellular acidification rate (ECAR) assay and oxygen consumption rate (OCR) assay\u003c/h2\u003e\u003cp\u003eThe Seahorse XF Glycolysis Rate Assay Kit (103344-100, Agilent, USA) was used to detect changes in the cellular glycolysis acid production rate after intervening with TGF-β3, to assess its effect on energy metabolism in cardiomyocytes. The cellular mitochondrial stress test was performed with a Seahorse analyzer (XFe24, Agilent, USA), and measurements were taken at 10-min intervals, and the corresponding drugs were automatically added by the analyzer at the corresponding time points to obtain the acidification rate curve of the cell medium and glycolytic capacity.\u003c/p\u003e\u003cp\u003eThe Seahorse XF Analyzer assesses mitochondrial respiratory cell function by determining the oxygen consumption rate (OCR) of living cells in multiwell plates. The Agilent Seahorse XF Analyzer measures oxygen consumption rate (OCR) at approximately 5\u0026ndash;8 minute intervals. OCR serves as an indicator of mitochondrial respiration. OCR is measured in real time by creating a transient microchamber (~\u0026thinsp;7 \u0026micro;L) above a monolayer of cells in a microtiter plate. cellular oxygen consumption (respiration) results in rapid and easily measured changes in the concentration of dissolved oxygen and free protons in the \u0026ldquo;transient microcompartment\u0026rdquo;, which can be measured every few seconds by a solid-state sensor probe located 200 \u0026micro;m above the monolayer of cells. The instrument records changes in concentration over 2\u0026ndash;5 minute measurement periods and subsequently calculates the OCR.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (n\u0026thinsp;\u0026ge;\u0026thinsp;3 per group). Differences between two groups were analyzed using Student's t-test. Comparisons among more than two groups were analyzed using one-way or two-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software Inc.). p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicates a statistically significant difference.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Myocardial tissue injury accompanied by metabolic changes in SCM\u003c/h2\u003e\u003cp\u003eCLP was performed to establish the SCM rat model, and myocardial tissues were harvested at 24h, 48h and 72h after modeling, and HE staining revealed disrupted myocardial tissue structure at each modeling time point, accompanied by inflammatory cell infiltration and fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), confirming successful establishment of the SCM myocardial injury model in rats. The lactate levels in the homogenates of rat myocardial tissues were significantly elevated at all modeling time points, suggesting that there were metabolic changes in myocardial tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In addition, the pathological injury score showed that the injury score was highest at 48 hours after modeling, and the lactate level was also highest at 48 hours after modeling. Therefore, the 48-hour time point was selected for subsequent experiments. .\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.2 TGF-β3 showed protective effects against SCM\u003c/h2\u003e\u003cp\u003eThe SCM rat model was constructed by CLP, and the HE staining results showed that myocardial tissue structure was damaged in the SCM model group (Model), inflammatory cell infiltration, and the pathological damage score was elevated; the degree of myocardial damage was reduced in the TGF-β3 overexpression group (TGF-β3 OE), and the pathological damage score was lowered; myocardial damage was aggravated in the TGF-β3 interference group (TGF-β3 sh), and the pathological damage score was further elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). TUNEL staining revealed that apoptosis was significantly elevated in the SCM model group, the degree of apoptosis was reduced in the TGF-β3 overexpression group, and apoptosis was further aggravated in the TGF-β3 interference group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Transmission electron microscopy results showed that myocardial fibers in the SCM model group were disarranged, the gap was widened, the mitochondrial structure was disrupted, exhibiting swelling, cristae fragmentation, and pleomorphism, and the mitochondrial damage score was significantly increased. The cardiomyocytes in the TGF-β3 overexpression group had markedly improved ultrastructure, mitochondrial morphology normalized, with cristae arranged in parallel arrays, and the mitochondrial damage score was significantly reduced, while cardiomyocytes in the TGF-β3 interference group ultrastructure was further disrupted and mitochondrial damage was aggravated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Collectively, these results demonstrate that TGF-β3 had a protective effect on SCM.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.3 TGF-β3 reversed cardiomyocyte metabolic reprogramming in SCM\u003c/h2\u003e\u003cp\u003eIn the SCM cell model established by LPS treatment of primary rat cardiomyocytes, the Seahorse assay revealed an increase in the extracellular acidification rate (ECAR) and glycolytic capacity significantly increased after LPS treatment, suggesting that LPS promoted glycolysis in cardiomyocytes, whereas TGF-β3 overexpression resulted in a decrease in the level of extracellular acidification rate (ECAR), and basal glycolysis and glycolytic capacity were markedly reduced, and there was no change in non-glycolytic acidification (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In addition, after LPS treatment, oxygen consumption rate (OCR) decreased significantly, while basal and maximal respiration rates of cardiomyocytes decreased, indicating that LPS inhibited mitochondrial aerobic respiration, whereas after TGF-β3 overexpression, OCR increased significantly, while basal and maximal respiration rates recovered, with no change in non-mitochondrial respiration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These findings indicate that there is metabolic reprogramming in cardiomyocytes during SCM, i.e., enhancement of glycolysis and attenuation of oxidative phosphorylation, and TGF-β3 can reverse this phenomenon, thus exerting its protective effects.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.4 TGF-β3 promotes Smad7 phosphorylation and inhibits SKP1-mediated ubiquitination degradation of Smad7\u003c/h2\u003e\u003cp\u003eTo elucidate the regulatory mechanism of TGF-β3 on metabolic reprogramming of cardiomyocytes, a SCM rat model was constructed using CLP. First, Firstly, TGF-β3 expression was downregulated in the CLP group (Model) compared with the sham-operated group (Sham), consistent with the trend of TGF-β3 as a protective factor in SCM. Second, Smad2/3 phosphorylation was enhanced in the CLP group (Model) compared to the sham-operated group (Sham), however, TGF-β3 overexpression reversed Smad2/3 phosphorylation, and Smad2/3 phosphorylation was promoted after interference with TGF-β3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Given that Smad2/3 are R-Smands, we also examined Smad7, an inhibitory Smad (I-Smad) and found that Smad7 phosphorylation was increased after overexpression of TGF-β3, and conversely, Smad7 phosphorylation was inhibited after interference with TGF-β3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In the SCM cell model established by LPS treatment of primary rat cardiomyocytes, it was also found that TGF-β3 expression level was downregulated, Smad2/3 phosphorylation level was increased, and Smad7 phosphorylation was decreased after LPS stimulation, whereas overexpression of TGF-β3 decreased Smad2/3 phosphorylation level and increased Smad7 phosphorylation level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These results suggest that TGF-β3 plays a role in the inhibitory regulation of Smad2/3 by promoting Smad7 phosphorylation.\u003c/p\u003e\u003cp\u003eIn addition, we found that TGF-β3 acts on SKP1, a core component of the E3 ubiquitin ligase SCF complex, in cardiomyocytes by IP-MS in a previous study (unpublished), and this experiment confirmed that TGF-β3 overexpression reduced SKP1 expression levels, and conversely, the level of SKP1 expression was increased after interfering with TGF-β3, suggesting that TGF-β3 inhibits SKP1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). This further supports the role of SKP1 in mediating Smad7 ubiquitination and degradation. Co-immunoprecipitation (Co-IP) confirmed normal expression of Ubiquitin, Smad7, and GAPDH in the Input lysates across all groups, and showed that TGF-β3 overexpression increased Smad7 expression, an effect counteracted by SKP1 overexpression. The IP results demonstrated successful Ubiquitin pull-down and ubiquitination of Smad7, and it was found that ubiquitinated Smad7 levels decreased following TGF-β3 overexpression, and ubiquitinated Smad7 levels increased following SKP1 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These results indicate that SKP1 promotes Smad7 ubiquitination and degradation, and that TGF-β3 binding to SKP1 inhibits this process. The above results suggest that TGF-β3 inhibits SKP1-mediated ubiquitination degradation of Smad7.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMetabolic reprogramming was first identified in cancer cells, where cells utilize glycolysis for ATP production even under aerobic conditions, but rather a heavy reliance on inefficient glycolysis[20]. Recently, metabolic reprogramming has also been identified in sepsis: immune cells shift from oxidative phosphorylation to glycolysis, as the main mode of energy supply to facilitate the generation of a large amount of energy for activation in a short period of time; other tissue cells enter a \u0026quot;hibernation-like\u0026quot; state characterized by suppressed physiological functions and metabolic activity[6]. In SCM patients, an initial compensatory hypercontractility phase is often followed by reversible cardiac dysfunction[21,22], mirroring a \u0026quot;hibernation-like\u0026quot; state. This functional impairment aligns with the underlying phenomenon of metabolic reprogramming in cardiomyocytes. Focusing on cardiomyocyte metabolic reprogramming in SCM, this study demonstrates that TGF-\u0026beta;3 reverses cardiomyocyte metabolic reprogramming by reducing the ubiquitination degradation and promoting the phosphorylation of Smad7 through the inhibition of SKP1.\u003c/p\u003e\n\u003cp\u003eThe TGF-\u0026beta; family comprises multifunctional peptide cytokines that elicit diverse responses, including promoting inflammation and apoptosis in cardiomyocytes, activating macrophage phagocytosis, and inducing fibrogenesis in fibroblasts[23], thereby influencing cardiac repair, remodeling, and fibrosis[24]. Members of this family, including TGF-\u0026beta;1, TGF-\u0026beta;2, and TGF-\u0026beta;3, play significant roles in regulating energy metabolism, with functional subtype specificity.[16,25-27]. TGF-\u0026beta;1 acts as a key regulator of macrophage metabolism, promoting glycolysis during sepsis[28]; It also induces metabolic reprogramming in fibroblasts, shifting them towards glycolysis and away from mitochondrial OXPHOS to meet the high biosynthetic demands of their profibrotic or proinflammatory states[25]. In contrast, TGF-\u0026beta;3 may counteract pathological fibrosis and reverse metabolic reprogramming, potentially by competing with TGF-\u0026beta;1[29,30]; for example, TGF-\u0026beta;3 heterozygous knockout mice exhibit early renal fibrosis accompanied by insulin resistance and aberrant lipid metabolism[31]; Furthermore, studies confirm that TGF-\u0026beta;2 or TGF-\u0026beta;3, but not TGF-\u0026beta;1, promotes fatty acid oxidation in myotubes and adipocytes[16,32]. Combined with previous findings of upregulated TGF-\u0026beta;1 in myocardial infarction and hypertrophy[33], the specific downregulation of TGF-\u0026beta;3 observed in SCM in this study highlights its distinct role in regulating cardiomyocyte metabolism, and suggests that the downregulation of TGF-\u0026beta;3 may be an important pathologic feature in SCM, providing a rationale for exploring TGF-\u0026beta;3 as a potential diagnostic biomarker or therapeutic target.\u003c/p\u003e\n\u003cp\u003eIn this study, we clarified that overexpression of TGF-\u0026beta;3 attenuates myocardial pathological injury, reduces cardiomyocyte apoptosis, and ameliorates mitochondrial structural disruption, demonstrating its cardioprotective effects. Our prior unpublished work focused on mitochondrial dynamics, revealing a key role for AMPK in TGF-\u0026beta;3-mediated mitochondrial biogenesis and autophagy. AMPK is an energy receptor for cellular metabolism[34]. Therefore, the present study focuses on the effect of TGF-\u0026beta;3 on energy metabolism in cardiomyocytes. Direct evidence confirming metabolic reprogramming specifically in SCM cardiomyocytes is relatively limited. The present study confirms the existence of metabolic reprogramming in cardiomyocytes during SCM, which is mainly manifested by enhanced glycolysis and attenuated oxidative phosphorylation. While metabolic reprogramming provides rapid energy compensation short-term, its persistence is detrimental. Consequences include: chronic ATP underproduction, directly impairing contractile function[8]; accumulation of unoxidized lipids, triggering lipotoxic cardiomyocyte damage[9]; metabolite buildup (e.g., lactate), causing microenvironment acidification, immunosuppression, and cellular damage[35], and energy deprivation-induced mitochondrial damage (collapse of membrane potential and respiratory chain disassembly), and the release of mitochondrial DNA fragments activating the NLRP3 inflammasome, creating a vicious cycle of mitochondrial damage, amplified inflammation, and energy depletion[36]. Therefore, reversing metabolic reprogramming represents a promising therapeutic strategy for SCM. This study demonstrates that TGF-\u0026beta;3 overexpression effectively reverses these metabolic abnormalities, constituting a key mechanism underlying its cardioprotective effect.\u003c/p\u003e\n\u003cp\u003eThe present study also explored the signaling pathway of TGF-\u0026beta;3 to reverse metabolic reprogramming. While the classical paradigm holds that TGF-\u0026beta; activates downstream profibrotic or inflammatory signaling primarily through Smad2/3 phosphorylation[15]. In the present study, we found that Smad2/3 phosphorylation was reduced, which may be a specific effect of TGF-\u0026beta;3 in a specific pathological setting (SCM). Smad7 belongs to the inhibitory Smad (I-Smads), a classical negative feedback regulator of TGF-\u0026beta; signaling, which competes with receptor-regulated Smads (R-Smads; Smad2/3) for binding to activated T\u0026beta;RI, thereby preventing R-Smad phosphorylation and activation[15]. Smad7 is regulated by non-coding RNAs (ncRNAs) and other post-translational modifications (PTMs)[37]. Phosphorylation, a critical PTM, plays a pivotal role in regulating protein stability and function[38]. However, Smad7 phosphorylation has primarily been studied in the context of TGF-\u0026beta;1 signaling[39,40]. Therefore, the present study verified that TGF-\u0026beta;3 is promotional for Smad7 phosphorylation. Beyond phosphorylation, we investigated mechanisms regulating Smad7 stability, given its impact on TGF-\u0026beta;3 signaling and found that TGF-\u0026beta;3 inhibited SKP1-mediated ubiquitination degradation of Smad7. SKP1 is a core component of the SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase complex[41] , acting as an adaptor bridging CUL1 and F-box proteins, and its activity is essential for regulating the myriad of ubiquitination degradation processes controlled by the SCF complex[42]. Stabilized Smad7 binds activated T\u0026beta;RI more efficiently and competitively, thereby blocking Smad2/3 phosphorylation and activation, consistent with our observed inhibition of Smad2/3 phosphorylation. This study reveals for the first time a novel mechanism by which TGF-\u0026beta;3 exerts cardioprotective effects by regulating the ubiquitinated degradation of Smad7, distinct from previously known mechanisms regulating Smad7. TGF-\u0026beta;3 regulates Smad7 phosphorylation status and, by concurrently enhancing its stability, augments Smad7\u0026apos;s capacity to inhibit R-Smad signaling.\u003c/p\u003e\n\u003cp\u003eThis study also has some limitations. First, animal and cellular models may not fully recapitulate human disease, and clinical correlation studies are needed. Second, Smad7 can recruit E3 ubiquitin ligases (e.g., Smurf1/2) to degrade activated T\u0026beta;RI receptors and thus inhibit Smad2/3 phosphorylation, a possibility not explored here; whether there is a protein post-translational modification site interaction between Smad7 phosphorylation and ubiquitination needs to be further verified. Finally, the specific molecular targets of TGF-\u0026beta;3 in reversing metabolic reprogramming (e.g., key metabolic enzymes or pathways) remain incompletely defined and require further rigorous validation, such as conditional knockdown studies.\u003c/p\u003e\n\u003cp\u003eThis study establishes the reversal of metabolic reprogramming as a novel mechanism for TGF-\u0026beta;3-mediated cardioprotection in SCM. Furthermore, it reveals a previously unrecognized molecular mechanism involving TGF-\u0026beta;3 promotion of Smad7 phosphorylation and inhibition of SKP1-mediated Smad7 ubiquitination and degradation, and deepening our understanding of SCM pathogenesis, particularly the crosstalk between signaling and metabolic regulation. A strength of this study is the combined use of in vivo (rat) and in vitro (cardiomyocyte) models, enabling multi-level investigation from phenotype to molecular mechanism. Future clinical studies should investigate circulating levels of TGF-\u0026beta;3 in SCM patients and their correlation with cardiac function and prognosis, so as to provide new biomarkers for the diagnosis and treatment of SCM.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: HZ, XS\u003c/p\u003e\n\u003cp\u003eData curation: HZ, JX\u003c/p\u003e\n\u003cp\u003eMethodology: HZ, JX\u003c/p\u003e\n\u003cp\u003eInvestigation: HZ, JX, BX\u003c/p\u003e\n\u003cp\u003eVisualization: HZ\u003c/p\u003e\n\u003cp\u003eFunding acquisition: XS\u003c/p\u003e\n\u003cp\u003eSoftware development: BX\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; original draft: HZ, JX\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; review \u0026amp; editing: XS\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available on request from the corresponding author, XS, upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by Fujian Provincial Health Technology Project(No.2023ZQNZD001).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eM. 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Tanaka, Skp1 stabilizes the conformation of F-box proteins, Biochem Biophys Res Commun. 410 (2011) 24-28. https://doi.org/10.1016/j.bbrc.2011.05.098.\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":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Septic cardiomyopathy, Metabolic reprogramming, TGF-β3, Smad7","lastPublishedDoi":"10.21203/rs.3.rs-7130728/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7130728/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBACKGROUND\u003cbr\u003e\n \u003c/strong\u003eSeptic cardiomyopathy (SCM) exhibits over 70% mortality, primarily attributed to cardiomyocyte metabolic reprogramming shifting from oxidative phosphorylation (OXPHOS) to glycolysis. Although TGF-β3 modulates energy metabolism in other tissues, its function in SCM pathogenesis remains unexplored.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMETHODS\u003cbr\u003e\n \u003c/strong\u003eIn vivo, cecal ligation puncture (CLP) rat models received myocardial injections of TGF-β3-overexpressing or interfering adenovirus. Myocardial injury through histopathology (HE) and apoptosis (TUNEL), and mitochondrial ultrastructure via transmission electron microscopy (TEM). In vitro, primary cardiomyocytes treated with lipopolysaccharide (LPS) were transfected with TGF-β3 overexpression plasmid, with metabolic analyzed using Seahorse XF technology (extracellular acidification rate, ECAR; oxygen consumption rate, OCR). Molecular mechanisms were investigated via Western blotting and co-immunoprecipitation (Co-IP) targeting TGF-β3/Smad7/SKP1 signaling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFINDINGS\u003cbr\u003e\n \u003c/strong\u003eTGF-β3 was significantly downregulated in SCM. Its overexpression attenuated myocardial injury and apoptosis, improved mitochondrial integrity, and reversed metabolic reprogramming by reducing glycolysis while enhancing OXPHOS. Mechanistically, TGF-β3 promoted Smad7 phosphorylation to inhibit Smad2/3 activation and suppressed SKP1 expression to reduce Smad7 ubiquitination, as confirmed by Co-IP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eINTERPRETATION\u003cbr\u003e\n \u003c/strong\u003eTGF-β3 confers cardioprotection in SCM by reversing metabolic reprogramming through dual regulation of Smad7: enhancing phosphorylation to block Smad2/3 signaling and inhibiting SKP1-mediated ubiquitination to stabilize Smad7. This newly identified TGF-β3/Smad7 axis represents a promising therapeutic target for SCM.\u003c/p\u003e","manuscriptTitle":"TGF-β3 attenuates septic cardiomyopathy by reversing cardiomyocyte metabolic reprogramming through Smad7 signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-03 17:30:27","doi":"10.21203/rs.3.rs-7130728/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-02T12:14:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-29T22:37:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"88534582165891877319130969096416240381","date":"2025-09-23T21:29:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-22T01:04:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"70201442922177932583117132616609993841","date":"2025-09-07T18:55:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-20T04:18:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"321714418592018612616736325915734021942","date":"2025-08-04T12:31:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"184267234911028894772893909653952398087","date":"2025-07-30T13:58:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-28T14:01:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-27T23:38:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-15T13:22:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular and Cellular Biochemistry","date":"2025-07-15T12:39:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"111fa6b9-7f5c-42aa-8e52-a0b2974ba53b","owner":[],"postedDate":"August 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-05T16:02:05+00:00","versionOfRecord":{"articleIdentity":"rs-7130728","link":"https://doi.org/10.1007/s11010-025-05468-9","journal":{"identity":"molecular-and-cellular-biochemistry","isVorOnly":false,"title":"Molecular and Cellular Biochemistry"},"publishedOn":"2026-01-02 15:58:27","publishedOnDateReadable":"January 2nd, 2026"},"versionCreatedAt":"2025-08-03 17:30:27","video":"","vorDoi":"10.1007/s11010-025-05468-9","vorDoiUrl":"https://doi.org/10.1007/s11010-025-05468-9","workflowStages":[]},"version":"v1","identity":"rs-7130728","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7130728","identity":"rs-7130728","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}