The stress-responsive protein REDD1 drives diabetic myocardial injury via activation of autophagy-dependent ferroptosis

Diabetic cardiomyopathy (DCM), a lethal cardiovascular complication of diabetes, lacks effective therapies. Regulated in development and DNA damage response 1 (REDD1), a stress-responsive gene implicated in diabetic pathologies, was investigated for its role in autophagy and ferroptosis during DCM progression. Diabetic mice (high-fat diet/streptozotocin-induced) and high glucose (HG)-exposed human AC16 cardiomyocytes were utilized. REDD1 expression was analyzed via RT-qPCR/western blot. Cardiac function, fibrosis (H&E/Masson staining), metabolic parameters (blood glucose, insulin resistance), autophagy (LC3-II/p62, immunofluorescence), and ferroptosis (iron overload, lipid peroxidation, Mito-FerroGreen) were assessed. REDD1 was upregulated in diabetic hearts and HG-treated cardiomyocytes. REDD1 ablation in mice attenuated hyperglycemia, restored cardiac function, reduced hypertrophy/fibrosis, and suppressed autophagy/ferroptosis. In vitro, REDD1 knockdown enhanced cardiomyocyte viability (CCK-8 assay) and mitigated injury (lactate dehydrogenase release). Mechanistically, REDD1 silencing reduced ferroptosis, which was dependent on autophagy inhibition, as both rapamycin (autophagy activator) and Erastin (ferroptosis inducer) partially reversed the protective effects of REDD1 siRNA. These findings identify REDD1 as a critical mediator of DCM via autophagy-driven ferroptosis, offering a novel therapeutic target for diabetic cardiovascular complications. The stress-responsive protein REDD1 drives diabetic myocardial injury via activation of autophagy-dependent ferroptosis Yongjun Hu 1#, Siao Wen 1#, Wen Xiao 1, Xu Xie 2, Yutao Zhang 2, Ziqin Liu 2, Huiping You 2, Xin Zhong 2 * 1.Department of Cardiology, Hunan Provincial People’s Hospital (The First Affiliated Hospital of Hunan Normal University), Changsha 410005, China. 2.Department of Ultrasonic Medicine, Hunan Provincial People’s Hospital (The First Affiliated Hospital of Hunan Normal University), Changsha 410005, China. #Co-first authors have equal contributions to the work *Corresponding author: Xin Zhong, Department of Ultrasonic Medicine, Hunan Provincial People’s Hospital (The First Affiliated Hospital of Hunan Normal University), No. 61 Jiefang Road, Furong District, Changsha 410005, China. Tel: +86-13787076369 mail: [email protected] Diabetic cardiomyopathy (DCM), a lethal cardiovascular complication of diabetes, lacks effective therapies. Regulated in development and DNA damage response 1 (REDD1), a stress-responsive gene implicated in diabetic pathologies, was investigated for its role in autophagy and ferroptosis during DCM progression. Diabetic mice (high-fat diet/streptozotocin-induced) and high glucose (HG)-exposed human AC16 cardiomyocytes were utilized. REDD1 expression was analyzed via RT-qPCR/western blot. Cardiac function, fibrosis (H&E/Masson staining), metabolic parameters (blood glucose, insulin resistance), autophagy (LC3-II/p62, immunofluorescence), and ferroptosis (iron overload, lipid peroxidation, Mito-FerroGreen) were assessed. REDD1 was upregulated in diabetic hearts and HG-treated cardiomyocytes. REDD1 ablation in mice attenuated hyperglycemia, restored cardiac function, reduced hypertrophy/fibrosis, and suppressed autophagy/ferroptosis. In vitro, REDD1 knockdown enhanced cardiomyocyte viability (CCK-8 assay) and mitigated injury (lactate dehydrogenase release). Mechanistically, REDD1 silencing reduced ferroptosis, which was dependent on autophagy inhibition, as both rapamycin (autophagy activator) and Erastin (ferroptosis inducer) partially reversed the protective effects of REDD1 siRNA. These findings identify REDD1 as a critical mediator of DCM via autophagy-driven ferroptosis, offering a novel therapeutic target for diabetic cardiovascular complications. Key words: Autophagy; cytotoxicity; diabetes mellitus; ferroptosis; myocardial injury; REDD1

Diabetes mellitus (DM) is an endocrine disorder hallmarked by long-term hyperglycemia. As projected by the International Diabetes Federation, the global prevalence of diabetes will reach a staggering 800 million by 2045 (1). Diabetic cardiomyopathy (DCM) is an irreversible cardiovascular complication linked to diabetes, labeling specific cardiac manifestation with impaired cardiac contraction and relaxation in the absence of the well-established cardiovascular risk factors such as coronary and/or valvular complications, hypertension and dyslipidemia (2). DCM is a substantial contributor to end-stage heart failure (HF), representing a relevant cause of mortality fatalities among the diabetic population (3). As a result, it is pressing to thoroughly elucidate the underlying mechanisms of DCM and to develop effective interventions that could delay the course of DCM. Mechanically, both autophagy and ferroptosis are implicated in the pathogenesis of DCM (4, 5). Ferroptosis is a necrotic cell death modality distinguished by iron overload-driven lipid peroxidation accumulation (6). The maintenance of iron homeostasis is essential for proper cardiac function (7) and excessive iron deposition has been reported to augment the risk of DCM progression (8). Moreover, the crosslink between autophagy and ferroptosis has been increasingly discovered in the occurrence and development of human diseases (9). Therefore, autophagy and ferroptosis have emerged as critical targets for the prevention and treatment of DCM. Regulated in development and DNA damage response 1 (REDD1, also known as DDIT4/RTP801 ) is a hypoxia and stress response gene that is transcriptionally up-regulated in response to a variety of adverse physiological events (10). REDD1 is overexpressed during the diabetic process and can be viewed as a promising therapeutic target for diabetic complications (11). Notably, REDD1 deletion has been unveiled to prevent cardiac function deficits in diabetic mice (12). Specifically, REDD1 can also act as a regulator of autophagy (10). However, the impacts of REDD1 on ferroptosis in the process of DCM remain indistinct. Thereafter, both in vitro and in vivo DCM models were developed here to comprehensively expose the potential contribution of REDD1 to autophagy and ferroptosis in DCM.

Animals and DCM development The 8-week-old male mice of the C57BL/6J strain were caged for one week in a temperature- (22±2°C) and humidity-controlled (55±10%) animal care facility under a photoperiod of 12 h light/12 h darkness and allowed food and water ad libitum. After being given high-fat diet for 3 weeks, the mice were treated by streptozotocin (STZ; 85 mg/kg) via intraperitoneal injection twice within 72 h (13). Mice were considered diabetic when 6 h fasting blood glucose levels exceed 11.6 mmol/L. Mice receiving a normal diet and intraperitoneal injection of the same volume of buffer solution were used as the Control group. Then diabetic mice were then assigned into 3 other groups at random: DCM group, DCM+Lv-shRNA-REDD1 group and DCM+Lv-shRNA-NC group. In the latter two groups, 3 × 10 7 TU GFP-expressing shRNA lentivirus for REDD1 or the scramble control lentivirus designed by Genewiz (Beijing, China) was respectively intramuscularly injected into the apex and anterolateral wall of mice for 8 weeks (14, 15). Body weight was recorded and blood glucose levels were measured with an Ascensia Contour Glucometer once a week. Animals were sacrificed by anesthetization with pentobarbital sodium followed by cervical dislocation. Mice blood samples were harvested and heart tissues were dissected for the ensuing assays. This study was granted an animal license by the Animal Ethics Committee of Hunan Provincial People’s Hospital (Approval no. 2023-14). Cell culture, treatment and siRNA transfection Human cardiomyocyte cell line AC16 was grown in DMEM/F12 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and propagated at 37°C and 5% CO 2 atmosphere. Cells were then respectively exposed to 5.5 mmol/l glucose and 33 mmol/l glucose as the Control group and high glucose (HG) group (16). After being synthesized by Biotend Biotechnology Company (Shanghai, China), REDD1 siRNA (siRNA-REDD1-1/2) or the scramble negative control (siRNA-NC) complexed with ViaFect™ Transfection Reagent (Promega, Madison, USA) was transfected into AC16 cells. Besides, cells were pretreated by 100 nM autophagy activator rapamycin (17) or 1 mmol/L ferroptosis activator Erastin (18). Biochemical analyses After 20 min of centrifugation of blood samples at 1000 × g, the serum lactate dehydrogenase (LDH), creatine kinase MB (CK-MB), cardiac troponin I (cTnI), triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C) levels were respectively assayed by Lactate Dehydrogenase (LDH) Activity Assay Kit, Mouse TNNI3/cTn-I(Troponin I Type 3, Cardiac) ELISA Kit and Mouse CKMB(Creatine Kinase MB Isoenzyme) ELISA Kit, Triglyceride (TG) Colorimetric Assay Kit and High-density Lipoprotein Cholesterol (HDL-C) Colorimetric Assay Kit. The corresponding absorption was surveyed under a microplate reader. Hematoxylin and Eosin ( H&E) staining and Masson’s trichrome staining After being immobilized in 4% paraformaldehyde for immobilization and blocked by paraffin, the 5-µm thick samples passed through xylene and a gradient of ethanol for clearance and rehydration, followed by H&E staining and Masson’s trichrome staining. The cardiac morphology and fibrosis were observed applying an inverted microscope. Immunofluorescence staining After being treated by 4% paraformaldehyde and 0.5% Triton X-100, the heart tissues or AC16 cells were saturated with PBS harboring 5% BSA and hatched with LC3 antibody overnight at 4°C prior to the addition of secondary antibody conjugated with Alexa Fluor® 488 for 1 h at room temperature. Cell nuclei were identified following the supplementation of DAPI. The fluorescent-positive areas were photographed under a fluorescence microscope. Iron assay kit The mice heart tissues were centrifuged at 12000 × g for 10 min to assay total iron level using Total Iron Colorimetric Assay Kit. The spectrophotometric analysis at 593 nm was finally performed with a microplate reader. Evaluation of oxidative stress parameters The mice heart tissues were centrifuged at 10000 × g for 5 min and AC16 cells were centrifuged at 300 × g for 5 min to assay malondialdehyde (MDA) content with MDA Assay Kit. After centrifugation at 8000 × g for 10 min, glutathione (GSH) content in tissues or cell supernatants was examined with GSH Quantification Kit II. The spectrophotometric analysis at 532 and 450 nm was finally performed with a microplate reader. Immunohistochemistry After being immobilized in 4% paraformaldehyde for immobilization and blocked by paraffin, the 5-µm thick samples passed through xylene and a gradient of ethanol for clearance and rehydration. Then the sections were treated by GPX4 primary antibody at 4°C overnight and secondary antibody conjugated with horseradish peroxidase (HRP) for half an hour after being quenched in 3% H 2 O 2 and placed in 5% BSA for 20 min. Diaminobenzidine was used to shadow and hematoxylin was used to redye. The positively stained areas were captured under a light microscope. Cell Counting Kit-8 (CCK-8) After being implanted into the 96-well plates (2 × 10 4 cells per well), AC16 cells were reacted with 10 μl CCK-8 reagent for 2 h as described by the manufacturer. Finally, cells were placed in the microplate reader for the wavelength reading at 450 nm . Lactose dehydrogenase (LDH) assay kit AC16 cells were centrifuged at 400 × g for 5 min to assay LDH leakage using Lactate Dehydrogenase (LDH) Cytotoxicity Colorimetric Assay Kit. The spectrophotometric analysis at 450 nm was finally performed with a microplate reader. Mito-FerroGreen detection After corresponding treatment and transfection, AC16 cells were labeled by 5 μmol/L Mito-FerroGreen at 37℃ for half an hour. The fluorescence images were captured under a fluorescent microscope. Reverse transcription-quantitative PCR (RT-qPCR) After being purified from mice heart tissues and AC16 cells utilizing RNAzol reagent, total RNA was converted to cDNA with the aid of TransScript First-Strand cDNA Synthesis kit. PCR reaction was carried out using SYBR Green PCR Kit on a Roche Light Cycler 96 System. Relative REDD1 expression were calibrated in terms of 2 -ΔΔCt approach, with GAPDH as a normalizer. Western blotting After protein isolation from mice heart tissues and AC16 cells utilizing RIPA buffer and protein amount determination adopting BCA method, the sample proteins were divided by 12% SDS-PAGE, loaded and passed to the PVDF membranes. The membranes placed in 5% BSA were treated by primary antibodies at 4°C overnight and secondary antibody conjugated with horseradish peroxidase (HRP) for 2 h. The membrane was finally sink in the ECL reagent and the signal intensity was quantified with Image J software. Statistics The data were applied to graphic charting employing GraphPad Prism version 8.0. Means and standard deviation (SD) were calculated for all data points. Differences in the tested variables among different groups were determined by Student’s t-test, and one-way ANOVA followed by Tukey’s test. Statistical significance was accepted as p < 0.05.

Myocardial REDD1 expression was elevated in diabetic mice To identify the role of REDD1 in DM, diabetic mice were firstly developed. As demonstrated in H&E staining, morphologically, mice of the DCM group exhibited substantial myocardial disarray with a large number of vacuoles and distortions in comparison with the Control group (Fig. 1A). Also, the serum levels of cardiac biomarkers including CK-MB, cTnI and LDH were discovered to be distinctly elevated in DCM group relative to the Control group (Fig. 1B), indicating significant myocardial injury during diabetic modeling. RT-qPCR and western blotting results illuminated that REDD1 mRNA expression and protein expression were both upregulated in the heart tissues of diabetic mice (Fig. 1C-D). REDD1 ablation normalized body weight, reduced blood glucose and improved insulin resistance in diabetic mice To explore the influences of REDD1 on the progression of DCM, REDD1 was cardiac-specifically knocked down. As analyzed by RT-qPCR and western blotting, REDD1 expression was notably depleted after lentiviral transfection in the myocardial tissues of diabetic mice (Fig. 2A-B), presenting the efficiency of Lv-shRNA-REDD1. During the experiments, the mice of the control group gained body weight continuously. Compared with the Control group, mice body weight in the DCM group was remarkably increased from week 1 to week 4 of lentiviral transfection and was declined from week 4 to week 8. Moreover, the body weight of mice in the DCM+Lv-shRNA-REDD1 was notably increased by contrast with the DCM+Lv-shRNA-NC group (Fig. 2C). Besides, the blood glucose level of mice in the Control group was stable, which was markedly fluctuated during diabetic modeling. After REDD1 was knocked down, blood glucose level was stably decreased (Fig. 2D). Additionally, DCM mice exhibited dramatically elevated serum TG and reduced serum HDL-C levels and downregulation of REDD1 resulted in the decline on TG level and the increase on HDL-C level (Fig. 2E), suggesting that REDD1 improved insulin resistance in diabetic mice. REDD1 ablation ameliorated myocardial injury and cardiac hypertrophy in diabetic mice Meanwhile, the myocardial structural damage in DCM mice was significantly alleviated by deficiency of REDD1, displaying a more orderly arrangement (Fig. 3A). Masson trichrome staining demonstrated that collagen was noticeably accumulated in the cardiac tissues of DCM mice and the degree of fibrosis was reduced by treatment with Lv-shRNA-REDD1 (Fig. 3B). Expectedly, the serum CK-MB, cTnI and LDH contents in DCM mice were evidently lowered when REDD1 was depleted (Fig. 3C). REDD1 ablation suppressed autophagy and ferroptosis in the cardiac tissues of diabetic mice As observed in immunofluorescence staining, LC3 fluorescence intensity was intensified in the myocardial tissues of diabetic mice and then was weakened again by silencing of REDD1 (Fig. 4A). Western blotting analysis also delineated that LC3II/I, Beclin1 expressions were upregulated while p62 expression was downregulated in the hearts of diabetic mice. When REDD1 was silenced in diabetic mice, LC3II/I, Beclin1 expressions were strongly declined while p62 expression was boosted in the myocardial tissues (Fig. 4B). In addition, total iron level, MDA activity were noted to be distinctly increased and GSH activity was reduced in the cardiac tissues of diabetic mice, which were all reverted by interference with REDD1 (Fig. 4C-D). Through immunohistochemistry staining and western blotting, ferroptosis-related proteins were also detected. It was found that SLC7A11 and GPX4 expressions were both descending and ACSL4 expression was ascending in the heart tissues of diabetic mice. However, REDD1 disruption inversely raised SLC7A11 and GPX4 expressions and lowered ACSL4 expression in the heart tissues of diabetic mice (Fig. 4E-F). REDD1 deletion potentiated AC16 cell viability and alleviated cell injury upon exposure to HG For further verification, AC16 cells were subjected to HG conditions and the viability of AC16 cells was significantly eliminated under the circumstance (Fig. 5A). On the contrary, LDH leakage was exacerbated in AC16 cells treated by HG, suggesting the existence of cell toxicity (Fig. 5B). Consistently, REDD1 was also highly expressed at both mRNA and protein level in HG-exposed AC16 cells (Fig. 5C-D). After the construction and transfection of REDD1 interference plasmids, REDD1 was overtly depleted and the notable interference efficacy of siRNA-REDD1-1 was determined which was then applied to the follow-up assays (Fig. 5E-F). Upon knockdown of REDD1, the viability of HG-exposed AC16 cells was potentiated (Fig. 5G). The LDH leakage caused by HG treatment was also prevented by REDD1 down regulation (Fig. 5H). REDD1 deletion inactivated autophagy-dependent ferroptosis in HG-exposed AC16 cells Observably, HG challenge amplified LC3 fluorescence intensity that was diminished in REDD1-silencing AC16 cells (Fig. 6A). HG-stimulated AC16 cells also demonstrated elevated LC3II/I, Beclin1 expressions and lowered p62 expression. After REDD1 was depleted, LC3II/I, Beclin1 expressions were repressed and p62 expression was raised (Fig. 6B). Moreover, iron level, MDA activity were elevated and GSH activity was inhibited in AC16 cells challenged with HG. Relative to the HG+siRNA-NC group, iron level, MDA activity were strikingly eliminated again and GSH activity was promoted in HG+siRNA-REEDD1 group, which were all partially restored by pretreatment with autophagy activator rapamycin (Fig. 6C-D). The data from immunofluorescence and western blotting manifested that SLC7A11 and GPX4 expressions were lessened while ACSL4 expression was boosted in HG-injured AC16 cells. In AC16 cells stimulated with HG, insufficiency of REDD1 elevated SLC7A11, GPX4 expressions and eliminated ACSL4 expression. Compared with the HG+siRNA-REEDD1 group , SLC7A11, GPX4 expressions were declined again and ACSL4 expression was increased in the HG+siRNA-REEDD1+Rap group (Fig. 6E-F). Autophagy-dependent ferroptosis was required for the impacts of REDD1 on the viability and toxicity of HG-induced AC16 cells Combined with the above findings, it was speculated that REDD1 might function in DCM via mediating autophagy-dependent ferroptosis. To identify the potential mechanism, ferroptosis activator Erastin was also utilized. REDD1 absence facilitated AC16 cell viability that was impaired upon HG challenge and this effect was partially abolished by pretreatment with rapamycin or Erastin (Fig. 7A). Also, HG-elicited LDH leakage release in AC16 cells was hampered by REDD1 downregulation. Similarly, the cytotoxicity of REDD1-depleting AC16 cells exposed to HG was further aggravated by the addition of rapamycin or Erastin (Fig. 7B).

Myocardial autophagy and ferroptosis are causative factors in diabetes‐induced cardiac dysfunction (19, 20). STZ is a highly selective islet β-cytotoxic agent that can exert similar clinical and pathological changes in mice and rats to those in DCM patients, such as insulin deficiency, hyperglycemia, left ventricular function, along with myocardial performance (21, 22). Hyperglycaemia represents one of the major metabolic alterations in diabetes and the primary pathogenic factor of DCM (23). Therefore, in our study, diabetes was induced in mice via a high-fat diet combined with STZ injection and in human cardiomyocyte cell line AC16 exposed to HG. Extensive studies have underlined the cardioprotective effects of REDD1 inhibition (24-26). Our experimental results unveiled the novel insight into the efficacy of REDD1 knockdown in mitigating cardiac damage and fibrosis, autophagy and ferroptosis in diabetic mice and hampering the viability loss and cytotoxicity of HG-exposed cardiomyocytes. Mechanistically, REDD1 interference decreased autophagy level, and thus inactivated ferroptosis to obstruct cell viability loss and cytotoxicity in cardiomyocytes in response to HG. Herein, after modeling, myocardial disarray occurred and the serum levels of cardiac biomarkers including CK-MB, cTnI and LDH were raised, suggesting the substantial myocardial injury during the process of diabetes. In response to the characteristically high levels of REDD1, REDD1 inhibition has been revealed to retard the progression of diabetic complications via a plethora of mechanisms (27, 28). Herein, we found that REDD1 expression abundance was increased in both DCM mouse hearts and HG-stimulated AC16 cells, which was coincident with previous investigation reporting the upregulation of REDD1 in HG-treated AC16 cells (29). As proposed by Lee et al, REDD1-deficient mice exhibit reduced weight gain and lower blood glucose (30). We consistently observed that REDD1 knockout restored mice body weight, reduced blood glucose level, downregulated TG level and upregulated HDL-C level, further introducing the inhibitory role of REDD1 interference in IR. Importantly, REDD1 deletion can protect against inflammatory response in both HG-challenged cardiomyocytes and in the hearts of obese mice (29). Recent research has claimed that REDD1 contributes to cardiac defects in diabetic mice (12). In this research, REDD1 disruption was proved to decrease cardiac injury and hypertrophy in vivo and prevent cell viability loss and LDH leakage in vitro. All these findings underlined the potential protective role of REDD1 silencing in DCM. As a highly conserved catabolic process, autophagy delivers damaged organelles and cytoplasmic components to lysosomes for degradation and recirculation, and maintains energy balance and cell survival (30). It is generally believed that autophagy is crucial to maintaining normal heart morphology and function (31). Preclinical studies have observed that autophagic activity is disturbed and can play either cardiac protective or detrimental role in type 1 and type 2 diabetes (32). In detail, autophagy in the heart is enhanced and the reduction of autophagy may be an adaptive response that can prevent myocardial injury in type 1 diabetes. However, autophagy in the heart is suppressed and in type 2 diabetes. In our hands, LC3II/I, Beclin1 expressions were elevated and p62 expression was declined in both heart tissues of diabetic mice and HG-stimulated AC16 cells, suggesting the activation of autophagy in DCM here . Since mammalian target of rapamycin (mTOR) is commonly known as a pharmacologic target for autophagy regulation (33), there is convincing evidence suggesting that REDD1 can activate autophagy, as a negative regulator of mTOR signaling in response to cellular stress. It is worth noting that REDD1 can exaggerate autophagy to protect against cardiac hypertrophy (24), cardiac dysfunction (34) and drive myocardial ischemia/reperfusion injury (35). Our research concentrated on the impacts of REDD1 on autophagy in DCM and it turned out that after REDD1 was silenced, LC3II/I, Beclin1 expressions were suppressed and p62 expression was raised in both heart tissues of diabetic mice and HG-stimulated AC16 cells, newly expanding the promoting role of REDD1 in autophagy in DCM . Further, autophagy enhancer rapamycin compensated the effects of REDD1 deficiency on the viability and LDH release in HG-induced AC16 cells. Intriguingly, ferroptosis is recognized as an autophagic cell death process and there is a complex crosstalk between ferroptosis and autophagy through complex feedback loops (9, 36). Ferroptosis is extensively implicated in DCM (5, 37) and abnormal metabolism of Fe 2+ not only increases the risk of insulin resistance and diabetes (38) but also causes cardiovascular diseases in diabetic subjects (39). MDA is a main end product of lipid peroxidation resulting from the compromise of GSH-dependent antioxidant system (40, 41). SLC7A11 and GPX4 both function as importers of cystine and synthesizers of GSH to reduce lipid peroxidation and suppress ferroptosis (41, 42). The lipid metabolizing enzyme ACSL4 serves as a promoter of ferroptosis by converting free AA into arachidonoyl‐CoA11 to generate lipid hydroperoxides (42). Through investigation, REDD1 ablation was observed to lower iron level, reduce MDA activity, ACSL4 expression and elevate GSH activity, SLC7A11, GPX4 expressions in both heart tissues of diabetic mice and HG-stimulated AC16 cells, which identified the novel role of REDD1 in the ferroptotic event in DCM. Under the circumstance, the effects of REDD1 deficiency on ferroptosis could be mimicked by rapamycin. Furthermore, ferroptosis activator Erastin could partially revert the effects of REDD1 deficiency on the viability and LDH release in HG-induced AC16 cells, corroborating the integrated role of autophagy-dependent ferroptosis in the action mechanism of REDD1 in DCM.

REDD1 deletion could attenuate DCM, which might be partly attributed by the resistance to autophagy-dependent ferroptosis. Targeting autophagy-mediated ferroptosis may represent a therapeutic strategy for the clinical management of DCM. This study may also broaden the understanding of REDD1’s therapeutic potential in heart diseases. Ethics statement This study was granted an animal license by the Animal Ethics Committee of Hunan Provincial People’s Hospital (Approval no. 2023-14). Funding This study was supported by the Natural Science Foundation of Hunan Province (2024JJ9307).

1. Sun, H., Saeedi, P., Karuranga, S., Pinkepank, M., Ogurtsova, K., Duncan, B. B. et al. (2022) IDF Diabetes Atlas: Global, regional and country-level diabetes prevalence estimates for 2021 and projections for 2045 Diabetes Res Clin Pract 183, 1091192. Dillmann, W. H. (2019) Diabetic Cardiomyopathy Circ Res 124, 1160-11623. Park, J. J. (2021) Epidemiology, Pathophysiology, Diagnosis and Treatment of Heart Failure in Diabetes Diabetes Metab J 45, 146-1574. Li, J., Xie, Y., Zheng, S., He, H., Wang, Z., Li, X. et al. (2024) Targeting autophagy in diabetic cardiomyopathy: From molecular mechanisms to pharmacotherapy Biomed Pharmacother 175, 1167905. Zhao, Y., Pan, B., Lv, X., Chen, C., Li, K., Wang, Y. et al. (2023) Ferroptosis: roles and molecular mechanisms in diabetic cardiomyopathy Front Endocrinol (Lausanne) 14, 11406446. Jiang, X., Stockwell, B. R., and Conrad, M. (2021) Ferroptosis: mechanisms, biology and role in disease Nat Rev Mol Cell Biol 22, 266-2827. Lakhal-Littleton, S. (2019) Mechanisms of cardiac iron homeostasis and their importance to heart function Free Radic Biol Med 133, 234-2378. Yan, X., Xie, Y., Liu, H., Huang, M., Yang, Z., An, D. et al. (2023) Iron accumulation and lipid peroxidation: implication of ferroptosis in diabetic cardiomyopathy Diabetol Metab Syndr 15, 1619. Zhou, B., Liu, J., Kang, R., Klionsky, D. J., Kroemer, G., and Tang, D. (2020) Ferroptosis is a type of autophagy-dependent cell death Semin Cancer Biol 66, 89-10010. Kim, J. Y., Kwon, Y. G., and Kim, Y. M. (2023) The stress-responsive protein REDD1 and its pathophysiological functions Exp Mol Med 55, 1933-194411. Sunilkumar, S., and Dennis, M. D. (2024) REDD1 Is a Promising Therapeutic Target to Combat the Development of Diabetes Complications: A Report on Research Supported by Pathway to Stop Diabetes Diabetes 73, 1553-156212. Stevens, S. A., Sunilkumar, S., Subrahmanian, S. M., Toro, A. L., Cavus, O., Omorogbe, E. V. et al. (2024) REDD1 Deletion Suppresses NF-κB Signaling in Cardiomyocytes and Prevents Deficits in Cardiac Function in Diabetic Mice Int J Mol Sci 25, 13. Tian, J. H., Wu, Q., He, Y. X., Shen, Q. Y., Rekep, M., Zhang, G. P. et al. (2021) Zonisamide, an antiepileptic drug, alleviates diabetic cardiomyopathy by inhibiting endoplasmic reticulum stress Acta Pharmacol Sin 42, 393-40314. Wang, P., Wang, D., Yang, Y., Hou, J., Wan, J., Ran, F. et al. (2020) Tom70 protects against diabetic cardiomyopathy through its antioxidant and antiapoptotic properties Hypertens Res 43, 1047-105615. Ouyang, C., Huang, L., Ye, X., Ren, M., and Han, Z. (2022) HDAC1 Promotes Myocardial Fibrosis in Diabetic Cardiomyopathy by Inhibiting BMP-7 Transcription Through Histone Deacetylation Exp Clin Endocrinol Diabetes 130, 660-67016. Zhang, P., Wu, H., Lou, H., Zhou, J., Hao, J., Lin, H. et al. (2024) Baicalin Attenuates Diabetic Cardiomyopathy In Vivo and In Vitro by Inhibiting Autophagy and Cell Death Through SENP1/SIRT3 Signaling Pathway Activation Antioxid Redox Signal 10.1089/ars.2023.045717. Yao, M., Wang, J., Zhang, J., Guo, Y., Ni, Z., Jia, X. et al. (2023) Asiaticoside attenuates oxygen-glucose deprivation/reoxygenation-caused injury of cardiomyocytes by inhibiting autophagy J Appl Toxicol 43, 789-79818. Bai, Y. T., Xiao, F. J., Wang, H., Ge, R. L., and Wang, L. S. (2021) Hypoxia protects H9c2 cells against Ferroptosis through SENP1-mediated protein DeSUMOylation Int J Med Sci 18, 1618-162719. Dewanjee, S., Vallamkondu, J., Kalra, R. S., John, A., Reddy, P. H., and Kandimalla, R. (2021) Autophagy in the diabetic heart: A potential pharmacotherapeutic target in diabetic cardiomyopathy Ageing Res Rev 68, 10133820. Liu, P., Zhang, Z., Cai, Y., Li, Z., Zhou, Q., and Chen, Q. (2024) Ferroptosis: Mechanisms and role in diabetes mellitus and its complications Ageing Res Rev 94, 10220121. Furman, B. L. (2015) Streptozotocin-Induced Diabetic Models in Mice and Rats Curr Protoc Pharmacol 70, 5.47.41-45.47.2022. Marino, F., Salerno, N., Scalise, M., Salerno, L., Torella, A., Molinaro, C. et al. (2023) Streptozotocin-Induced Type 1 and 2 Diabetes Mellitus Mouse Models Show Different Functional, Cellular and Molecular Patterns of Diabetic Cardiomyopathy Int J Mol Sci 24, 23. Jia, G., Whaley-Connell, A., and Sowers, J. R. (2018) Diabetic cardiomyopathy: a hyperglycaemia- and insulin-resistance-induced heart disease Diabetologia 61, 21-2824. Liu, C., Xue, R., Wu, D., Wu, L., Chen, C., Tan, W. et al. (2014) REDD1 attenuates cardiac hypertrophy via enhancing autophagy Biochem Biophys Res Commun 454, 215-22025. Huang, P., Bai, L., Liu, L., Fu, J., Wu, K., Liu, H. et al. (2021) Redd1 knockdown prevents doxorubicin-induced cardiac senescence Aging (Albany NY) 13, 13788-1380626. Li, P., Lin, N., Guo, M., Huang, H., Yu, T., and Zhang, L. (2019) REDD1 knockdown protects H9c2 cells against myocardial ischemia/reperfusion injury through Akt/mTORC1/Nrf2 pathway-ameliorated oxidative stress: An in vitro study Biochem Biophys Res Commun 519, 179-18527. Sunilkumar, S., Yerlikaya, E. I., Toro, A. L., Miller, W. P., Chen, H., Hu, K. et al. (2022) REDD1 Ablation Attenuates the Development of Renal Complications in Diabetic Mice Diabetes 71, 2412-242528. Mu, L., Chen, N., Chen, Y., Yang, Z., Zhou, H., Song, S. et al. (2022) Blocking REDD1/TXNIP Complex Ameliorates HG-Induced Renal Tubular Epithelial Cell Apoptosis and EMT through Repressing Oxidative Stress Int J Endocrinol 2022, 607391129. Stevens, S. A., Gonzalez Aguiar, M. K., Toro, A. L., Yerlikaya, E. I., Sunilkumar, S., VanCleave, A. M. et al. (2023) PERK/ATF4-dependent expression of the stress response protein REDD1 promotes proinflammatory cytokine expression in the heart of obese mice Am J Physiol Endocrinol Metab 324, E62-e7230. Lee, D. K., Kim, T., Byeon, J., Park, M., Kim, S., Kim, J. et al. (2022) REDD1 promotes obesity-induced metabolic dysfunction via atypical NF-κB activation Nat Commun 13, 630331. Sciarretta, S., Maejima, Y., Zablocki, D., and Sadoshima, J. (2018) The Role of Autophagy in the Heart Annu Rev Physiol 80, 1-2632. Kanamori, H., Takemura, G., Goto, K., Tsujimoto, A., Mikami, A., Ogino, A. et al. (2015) Autophagic adaptations in diabetic cardiomyopathy differ between type 1 and type 2 diabetes Autophagy 11, 1146-116033. Kim, Y. C., and Guan, K. L. (2015) mTOR: a pharmacologic target for autophagy regulation J Clin Invest 125, 25-3234. Huang, P., Fu, J., Chen, L., Ju, C., Wu, K., Liu, H. et al. (2019) Redd1 protects against post‑infarction cardiac dysfunction by targeting apoptosis and autophagy Int J Mol Med 44, 2065-207635. Gao, C., Wang, R., Li, B., Guo, Y., Yin, T., Xia, Y. et al. (2020) TXNIP/Redd1 signalling and excessive autophagy: a novel mechanism of myocardial ischaemia/reperfusion injury in mice Cardiovasc Res 116, 645-65736. Liu, J., Kuang, F., Kroemer, G., Klionsky, D. J., Kang, R., and Tang, D. (2020) Autophagy-Dependent Ferroptosis: Machinery and Regulation Cell Chem Biol 27, 420-43537. Song, Z., Wang, J., and Zhang, L. (2024) Ferroptosis: A New Mechanism in Diabetic Cardiomyopathy Int J Med Sci 21, 612-62238. Miao, R., Fang, X., Zhang, Y., Wei, J., Zhang, Y., and Tian, J. (2023) Iron metabolism and ferroptosis in type 2 diabetes mellitus and complications: mechanisms and therapeutic opportunities Cell Death Dis 14, 18639. Zhang, X., Sun, J., Wang, J., Meng, T., Yang, J., and Zhou, Y. (2023) The role of ferroptosis in diabetic cardiovascular diseases and the intervention of active ingredients of traditional Chinese medicine Front Pharmacol 14, 128671840. Tsikas, D. (2017) Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and relatives in biological samples: Analytical and biological challenges Anal Biochem 524, 13-3041. Ursini, F., and Maiorino, M. (2020) Lipid peroxidation and ferroptosis: The role of GSH and GPx4 Free Radic Biol Med 152, 175-18542. Chen, X., Li, J., Kang, R., Klionsky, D. J., and Tang, D. (2021) Ferroptosis: machinery and regulation Autophagy 17, 2054-2081 Figure legends Figure 1 Myocardial REDD1 expression was elevated in diabetic mice. (A) H&E staining determined cardiac morphology. (B) Biochemical kits evaluated cardiac biomarkers. (C) RT-qPCR and (D) western blotting examined REDD1 expression. ***P<0.001 vs. Control. Figure 2 REDD1 ablation normalized body weight, reduced blood glucose and improved insulin resistance in diabetic mice. (A) RT-qPCR and (B) western blotting examined REDD1 expression after lentiviral transfection. (C) Mice body weight. (D) Mice blood glucose levels. (E) Biochemical kits evaluated IR markers. *P<0.05, **P<0.01, ***P<0.001 vs. Control; #P<0.05, ##P<0.01, ###P<0.001 vs. DCM+Lv-shRNA-NC. Figure 3 REDD1 ablation ameliorated myocardial injury and cardiac hypertrophy in diabetic mice. (A) H&E staining determined cardiac morphology. (B) Masson trichrome staining determined cardiac fibrosis. (C) Biochemical kits evaluated cardiac biomarkers. ***P<0.001 vs. Control; ###P<0.001 vs. DCM+Lv-shRNA-NC. Figure 4 REDD1 ablation suppressed autophagy and ferroptosis in the cardiac tissues of diabetic mice. (A) Immunofluorescence staining measured LC3 expression. (B) Western blotting examined proteins related to autophagy. (C) Iron assay kit determined total iron level. (D) Assay kits determined MDA and GSH activities. (E) Immunohistochemistry staining measured GPX4 expression. (F) Western blotting examined proteins related to ferroptosis. **P<0.01, ***P<0.001 vs. Control; ##P<0.01, ###P<0.001 vs. DCM+Lv-shRNA-NC. Figure 5 REDD1 deletion potentiated AC16 cell viability and alleviated cell injury upon exposure to HG. (A) CCK-8 ascertained cell viability. (B) LDH assay kit ascertained cell injury. (C) RT-qPCR and (D) western blotting examined REDD1 expression. (E) RT-qPCR and (F) western blotting examined REDD1 expression after plasmid transfection. ***P<0.001 vs. siRNA-NC. (G) CCK-8 ascertained cell viability. (H) LDH assay kit ascertained cell injury. **P<0.01, ***P<0.001 vs. Control; ##P<0.01 vs. HG+siRNA-NC. Figure 6 REDD1 deletion inactivated autophagy-dependent ferroptosis in HG-exposed AC16 cells. (A) Immunofluorescence staining measured LC3 expression. (B) Western blotting examined proteins related to autophagy. (C) Mito-FerroGreen assay detected intracellular iron. (D) Assay kits determined MDA and GSH activities. (E) Immunohistochemistry staining measured GPX4 expression. (F) Western blotting examined proteins related to ferroptosis. ***P<0.001 vs. Control; ##P<0.01, ###P<0.001 vs. HG+siRNA-NC; @@P<0.01, @@@P<0.001 vs. HG+siRNA-REDD1. Figure 7 Autophagy-dependent ferroptosis was required for the impacts of REDD1 on the viability and toxicity of HG-induced AC16 cells. (A) CCK-8 ascertained cell viability. (B) LDH assay kit ascertained cell injury. ***P<0.001 vs. Control; ###P<0.001 vs. HG+siRNA-NC; @P<0.05, @@P<0.01 vs. HG+siRNA-REDD1. Information & Authors Information Version history Copyright This work is licensed under a Non Exclusive No Reuse License.

Authors Metrics & Citations Metrics Article Usage 255views 108downloads Citations Download citation Yongjun Hu, Siao Wen, Wen Xiao, et al. The stress-responsive protein REDD1 drives diabetic myocardial injury via activation of autophagy-dependent ferroptosis. Authorea. 17 April 2025. DOI: https://doi.org/10.22541/au.174489134.49875039/v1 DOI: https://doi.org/10.22541/au.174489134.49875039/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.