Protective mitophagy in human hypoxic cardiomyocytes: mechanistic insights into SGLT2 inhibitor Dapagliflozin cardioprotection in ischemic injury

Protective mitophagy in human hypoxic cardiomyocytes: mechanistic insights into SGLT2 inhibitor Dapagliflozin cardioprotection in ischemic injury | 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 Protective mitophagy in human hypoxic cardiomyocytes: mechanistic insights into SGLT2 inhibitor Dapagliflozin cardioprotection in ischemic injury Yi-Hsiung Lin, Shih-Jie Jhuo, Wei-Chung Tsai, Yi-Hsueh Liu, Nai-Yu Chi, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9239009/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Background Acute myocardial infarction (AMI) remains a leading cause of mortality worldwide, with cardiomyocyte death under hypoxic stress being a critical pathogenic mechanism. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) demonstrate cardiovascular benefits beyond glycemic control, yet their protective mechanisms in ischemic injury remain incompletely understood. Methods This study investigated dapagliflozin’s effects on human cardiomyocytes derived from induced pluripotent stem cell (iPSC-CMs) subjected to hypoxic stress. iPSC-CMs were applied to hypoxia with or without dapagliflozin treatment, followed by RNA-sequencing, qPCR validation, Western blotting, immunofluorescence, flow cytometry, and autophagy flux assays. Seahorse energy analyzer was used for cardiomyocyte metabolism analysis. Results RNA-sequencing revealed that dapagliflozin reversed hypoxia-induced transcriptomic alterations, particularly in 66 genes associated with mitochondrial dysfunction. Dapagliflozin activated the AMPK/SIRT1/SIRT3 signaling axis, upregulated autophagy/mitophagy markers (LC3B, BECN1, Parkin). Seahorse cardiomyocyte metabolism analysis results demonstrated that dapagliflozin improved iCM mitochondrial function by restoring mitochondrial membrane potential, mitophagy capability and reduced hypoxia-related apoptosis. Autophagy inhibition with bafilomycin A1 abolished cardioprotection, confirming autophagy-dependent mechanisms. Conclusions These findings suggest that dapagliflozin protects cardiomyocytes through enhanced SIRT1/3-associated mitochondrial quality control and adaptive autophagy, providing mechanistic evidence supporting SGLT2 inhibitors' therapeutic potential in mitigating myocardial ischemic injury. SGLT2 inhibitor dapagliflozin AMI SIRT AMPK Autophagy Mitophagy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction SGLT2 inhibitors are now known to be beneficial in the treatment of heart failure. However, its efficacy in other cardiovascular diseases remains unclear, especially in the treatment of acute myocardial infarction (AMI). Our previous studies have shown that AMI leads to a reduction in the SIRT family in vivo, which in turn induces intracellular mechanisms detrimental to cardiomyocytes[ 1 ]. In vitro experiments demonstrated that SGLT2i dapagliflozin was effective in reversing hypoxic stress-induced myocardial injury, and increased SIRT1/3 family, which protected ventricular myocardial cells from myocardial injury caused by hypoxic stress. The present study aimed to further elucidate the mechanism of dapagliflozin-activated intracellular protection against hypoxic stress-induced myocardial cell death after exposing to hypoxic stress. Autophagy is an intracellular biodegradation process that breaks down and recycles cellular components. It is also a cellular self-protection mechanism that removes pathogens, damaged proteins, cells, organs and other items from cells to maintain normal cellular function[ 2 , 3 ]. Macroautophagy begins as the ULK complex gathers near the endoplasmic reticulum to start the process. Next, ATG proteins (ATG5, ATG12, ATG16L, ATG9) form a phagophore assembly site to encapsulate damaged cell components. The membrane extends, closing into a double-membrane autophagosome. This autophagosome fuses with a lysosome to form an autolysosome. Finally, contents are degraded into molecules like amino acids and fatty acids for recycling. [ 4 – 6 ]. The main features of cellular autophagy are selective or non-selective degradation of cytoplasmic components (e.g., damaged mitochondria, protein aggregates, etc.) and maintenance of cellular homeostasis and adaptation to stress, which is the main mechanism of cellular protection. Mitophagy selectively removes damaged mitochondria, helping regulate cellular energy and lower oxidative stress. Mitochondrial dysfunction contributes to diseases like heart disease, neurodegeneration, and metabolic syndrome[ 7 ]. In heart failure, damaged mitochondria reduce energy in heart cells and raise ROS, causing cell death and poor cardiac function. Mitophagy protects the heart by clearing abnormal mitochondria and aiding mitochondrial renewal[ 8 ]. Recent studies indicate that mitochondrial quality control and mitophagy activity are imbalanced in heart failure patients[ 9 , 10 ]. Existing research demonstrates that excessive inhibition of mitophagy exacerbates cardiac structural abnormalities, while excessive activation may lead to excessive mitochondrial clearance and myocardial energy deficiency[ 11 ]. Regulating mitophagy affects cardiovascular disease progression and offers promising therapeutic potential. Understanding mitophagy is vital for developing precision medicine targeting mitochondrial quality, and adjusting mitophagy may help restore cardiac function and slow disease. Many diseases are associated with autophagy and mitophagy disorder, including neurodegenerative diseases[ 12 , 13 ], cardiovascular diseases[ 3 , 14 , 15 ], musculoskeletal diseases[ 16 , 17 ], metabolic syndromes[ 18 ], liver diseases and various cancer[ 19 , 20 ]. Increasing evidence suggests that mitophagy is associated with the development of cardiovascular diseases and may serve as a viable therapeutic target[ 15 , 21 ]. Mitophagy plays a crucial role in maintaining cardiac morphology and function, as well as in the progression of hypertension[ 22 ]. Moderate mitophagy can slow the progression of Heart failure (HF), while insufficient autophagy/mitophagy (e.g., through the knockout of ATG5 and ATG7 in animal models) leads to increased hypertrophic cardiomyocytes and worsened cardiac function[ 9 , 23 , 24 ]. Conversely, excessive autophagy can accelerate cardiac dysfunction, particularly in advanced stages of hypertension, where damaged organelles and harmful factors accumulate, leading to further deterioration of heart function[ 25 ]. Currently, there is no clear evidence that SGLT2 inhibitors improve outcomes or induce remission in acute myocardial infarction. Most large clinical trials have demonstrated their benefits in reducing cardiovascular events—such as cardiovascular death, heart failure hospitalization, nonfatal myocardial infarction, and renal deterioration—mainly in patients with diabetes or high cardiovascular risk. The EMPA-REG OUTCOME and CANVAS trials showed significant reductions in major adverse cardiovascular events (MACE), including cardiac death, nonfatal stroke, and nonfatal myocardial infarction. However, results for stroke and myocardial infarction alone have often failed to reach statistical significance. Thus, current evidence does not support SGLT2 inhibitors as essential agents for reperfusion therapy or as effective treatment after acute myocardial infarction. Mitophagy is showed to play crucial role for maintaining mitochondrial quality and energy balance in cardiomyocytes. The removal of damaged mitochondria helps reduce oxidative stress and cell apoptosis, thereby protecting cardiomyocytes from ischemic or injury exacerbation. Verifying the ability of SGLT2 inhibitors to induce mitophagy in cardiomyocytes is essential to fully understand their cardioprotective mechanisms and offers new avenues for mitophagy-targeted cardiovascular therapies. Materials and Methods Human iPSC-derived cardiomyocyte (iCM) maintains The human isogenic-induced pluripotent stem cell (iPSC) lines were obtained from the NCFB Human iPS Cells Service Consortium (clone number IBMS-iPSC-001-02, characterization and validation: https://catalog.bcrc.firdi.org.tw/resources/images/temp/17526382101992i) and maintained in Stemflex medium at 37°C in a 5% CO 2 atmosphere. For cardiomyocyte differentiation, iPSC-01-02 cells were initially derived using CHIR 99021, B-27 minus insulin, and serum-free RPMI. CHIR 99021 was removed on day 3 and replaced with IWR-1 for days 4-6. On day 7, cells were cultured using B-27 plus insulin and glucose serum-free RPMI. On day 21, cells were dispersed and re-pleated with 0.05% trypsin-EDTA. Ischemia-reperfusion (IR) injury procedure Hypoxic cell culture was performed in 6 well plates or culture dishes in a tank contained 1% O 2 gas mixture to create low oxygen and hypoxic stress. Re-gas every 1-3 hours to remove O 2 from the culture medium. SGLT2 inhibitor Dapagliflozin (DAPA) administration iCM cells were treated with DAPA at concentrations as previously published. After incubation in low oxygen, cells are treated with low (2.5µM) and high dose (5 μM) for 48 hours to observe cell morphology. The cell supernatant is removed and washed twice with PBS before the cells are scraped for subsequent experiments. mRNA collection and qRT-PCR analysis Total RNA was isolated using the Nucleospin RNA kit (Macherey-Nagel, USA), the cell lysate was applied to the RNA column and centrifuged at 15,000 rpm. Membrane desalting buffer was added to the RNA column and centrifuged. When the DNAase reaction is complete, the RNA-containing columns are transferred to new tubes for final purification of the RNA samples. Finally, the RNA sample is solubilized with RNase-free HO and transferred to a new 1.5 ml tube. All RNA samples were stored at -80°C before complementary DNA (cDNA) was synthesized using reverse transcription reagent (Applied Biosystem, USA). The prepared cDNA was then introduced into a 96-well quantitative polymerase chain reaction (qPCR) to analyze gene expression. Protein preparation and western blotting Use SDS polyacrylamide gel electrophoresis (SDS-PAGE) to separate protein lysates based on gel chemistry, perform wet transfer, and prepare transfer buffer. Use PVDF transfer membrane and follow manufacturer's instructions for dry membrane preparation. Pre-soak in ethanol (100%) for 30 seconds, rinse briefly in deionized water, and equilibrate in transfer buffer for 5 minutes. Incubate the membrane with sufficient volume of Blocking Buffer with agitation for 30-60 minutes at room temperature. After diluting the primary antibody in blocking buffer, react overnight and incubate with the appropriate secondary antibody. Primary antibody used in the current study including LC3B Antibody (#2775, Cell Signaling Technology, MA, US), LAMP2 Rabbit mAb (#34141), Beclin-1 (D40C5) Rabbit mAb (#3495), Parkin Antibody (#2132), phosphor-AMPK(07-681, Sigma-Aldrich, MO, US), SIRT1 (TA809834, Origene, MD, USA)and SIRT3(TA330278). ECL-stimulated chemiluminescence is then used to monitor the expression of the target protein. Autophagy and mitophagy detection Cells were stained with AO for acidic vesicular organelles (AVOs; red fluorescence) as well as cytoplasm and nucleus (green fluorescence) as indicated in the instructions. iCM cells were treated with hypoxic stress w/o dapagliflozin, respectively, and red/green fluorescence of AO was observed using a fluorescence microscope. The mitophagy detection kit (Dojindo, #MD01, Kumamoto City, Japan) includes Mtphagy Dye and Lyso Dye for detecting mitophagy in live iCM. Mtphagy Dye selectively accumulates in intact mitochondria and exhibits increased fluorescence upon mitophagy induction, while Lyso Dye stains lysosomes to confirm fusion with mitochondria. iCM were incubated with Mtphagy Dye, PBS washed for 15 mins, and exposed to mitophagy-inducing agents, hypoxia incubation and dapagliflozin treatment. Fluorescence microscopy was then used to detect the mitophagy signal and colocalization with lysosomes. Biological pathway analysis High-quality read alignments were mapped to the human reference genome (grch38.p7) and subsequently imported into Ingenuity's IPA software. Following this, feature Counts were utilized to assess participation across various mechanisms, and performance metrics were quantified using RLE, TMM, and FPKM methods. The screening for differentially expressed genes employed a q value threshold of less than 0.05. The analysis was based solely on the Ingenuity knowledge base, which included genes and took into account both direct and indirect relationships. The identified results pertinent to the dataset encompassed categories such as Diseases and Disorders, Molecular and Cellular Functions, Physiological Phylogenetics and Functions, alongside Top Canonical Pathways, with all findings significant at P < 0.05. Immunofluorescence staining and microscopy analysis The analysis of cardiomyocyte cells is conducted using immunofluorescence assays. Primary antibodies targeting specific antigens in the cells are diluted in the range of 1:100 to 1:200, as indicated in the user menu. Following this preparation, the samples are washed and incubated with DAPI-conjugated secondary antibodies that interact with the various primary antibodies. The final visualization of the cells occurs through the fluorescence microscope (Olympus BX51, Olympus, Japan). Statistics All data will be presented as mean ± standard error of the mean (SEM). The software used for statistical analysis will be SPSS version 13.0 (SPSS13.0 Software Inc., USA), which will facilitate the establishment of one-way analysis of variance (ANOVA) to determine differences among groups. To conduct comparisons between the various groups, Duncan's multi-range test will be employed. A p-value of less than 0.05 will be deemed statistically significant. Results RNA sequencing analyses of hypoxia toxicity and the therapeutic effect of dapagliflozin in iPSC-derived cardiomyocyte The present study was conducted with the objective of verifying the effect of hypoxia on cardiomyocytes and discussing the protective effect of SGLT2i dapagliflozin on cardiomyocytes against hypoxia. To this end, an in vitro model of hypoxic stress was established using iCM. The experiments were carried out with the aim of verifying the cardiomyocyte-specific marker MYL4 using human iPSC-derived cardiomyocytes (iCM, Fig. 1 A). We followed analyzed the gene clusters in cardiomyocytes affected by hypoxia and 5 µM dapagliflozin treatment. As shown in Fig. 1 B, the results showed the RNA-sequencing gene expression clustering from human iCM treated with different conditions (hypoxia, different doses of dapagliflozin, and a normal control). Hypoxia treatment revealed significant downregulation of most genes (green), while dapagliflozin treatment (Hy+Dapa 2.5 µM and Hy+Dapa 5 µM) showed a recovery of most hypoxia-affected genes (from green to red), with this trend varying with dose. Overall, dapagliflozin significantly reversed hypoxia-induced gene expression dysregulation in a dose-dependent manner, demonstrating its potential in alleviating hypoxia-induced cardiomyocyte damage. Among these differential expressed genes (DEGs), hypoxic stress upregulated 2054 genes and downregulated 2384 genes in iCM. RNA sequencing analysis of volcano pot showed that 2384 genes significantly downregulated and 2054 genes upregulated compared with normoxic controls. Subsequent treatment with 5 µM dapagliflozin under hypoxic conditions further affected gene expression, with 1852 genes downregulated and 2098 genes upregulated compared with hypoxia alone (Fig. 1 C). These results suggest that dapagliflozin administration substantially modulates hypoxia-induced gene expression profiles, indicating its potential role in regulating adaptive and protective cellular pathways under ischemic stress. Furthermore, we found that, dapagliflozin reversed those that were up- /down-regulated by hypoxic stress; 1717 of the 2054 genes re-stored by dapagliflozin treatment which decreased by hypoxic stress, and similarly, dapagliflozin decreased 1387 of the 2384 genes that up-regulated by hypoxic stress (Fig. 1 D). We further analyzed the pathways involved using IPA and found that dapagliflozin mainly enhanced a catalog of genes, including those regulating the skeletal and muscle development systems, and reversed genes related to cell death pathways (Fig. 1 E). In addition, KEGG network analysis revealed the signaling pathways involved in the genes regulated by dapagliflozin, among which the most affected signaling pathways were those related to cardiomyocyte metabolic pathways, cell apoptosis, and lysosome-related signaling pathways (Fig. 1 F). These results demonstrate that dapagliflozin treatment effectively reverses the cellular damage caused by hypoxic stress and pathway modulation in human cardiomyocyte. Dapagliflozin reverses the potential mechanism of hypoxia-induced mitochondrial damage in cardiomyocytes In the IPA analysis, we identified genes regulated by dapagliflozin that are associated with mitochondrial dysfunction. Among the 66 gene cluster heatmap showed the regulation of hypoxic stress and dapagliflozin in mitochondrial dysfunction-associated gene, and it was found that most of them were inverted by dapagliflozin treatment and tended to be similar to the normal gene expression of the control group (Fig. 2 A). Further analysis using the KEGG pathway showed that dapagliflozin suppressed most of the NDUFA/NDUFB family signaling regulator and most of the COX family gene expression up-regulated by hypoxic stress. In addition, the mitochondrial-encoding gene MT-CO family, which was also up-regulated by hypoxic stress, was also suppressed and returned to normal levels (Fig. 2 B). We then verified these results using qPCR, which found that hypoxic stress increased gene involved in mitochondrial fission in iCM, indicating that the disturbance of mitochondrial dynamic while exposure to hypoxia. However, it showed that dapagliflozin reduced the genes up-regulated by hypoxic stress, including ND1, NDUFA12, A4, A7, A8, PSEN2, UQCRFS1 and VAC1 (Fig. 2 C). Figure 2 D showed that, in the Seahorse assay, hypoxia modestly reduced basal and stress-induced oxygen consumption rate (OCR) in iPSC-derived cardiomyocytes compared with normoxia, indicating impaired mitochondrial respiratory reserve and oxidative phosphorylation capacity under low oxygen tension. In contrast, dapagliflozin-treated hypoxic cells largely preserved OCR dynamics across the sequential mitochondrial stressors, suggesting partial restoration of mitochondrial respiratory flexibility and ATP-generating capacity. Extracellular acidification rate (ECAR) was slightly increased by hypoxia, consistent with a compensatory shift toward glycolytic metabolism, whereas dapagliflozin did not further augment ECAR, indicating that its protective effect was not driven by additional glycolytic activation (Fig. 2 E). Together, these data suggest that dapagliflozin primarily maintains mitochondrial oxidative metabolism rather than enhancing glycolytic flux, thereby supporting more balanced bioenergetic adaptation of cardiomyocytes under hypoxic stress. Dapagliflozin activates cellular autophagy mechanism and flux in cardiomyocytes IPA pathway analysis also showed that dapagliflozin activated the mechanism of cytosolic autophagy in cells, as shown in the heat map, dapagliflozin reversed the up-/down-regulation of many genes due to hypoxic stress (Fig. 3 A). KEGG pathway analysis revealed that the expression of key regulators, including SIRT1, AMPK and cytosolic autophagy-associated regulators such as LC3B, the ATG family and BECN1, were all up-regulated by dapagliflozin treatment ( Figure. 3B ). Using qPCR to verify gene expression, we found that most autophagy-related genes were not affect or only a slight increased (LC3B and BECN1) by hypoxic stress, but were significantly up-regulated by dapagliflozin treatment, which were further enhanced by dapagliflozin in gene expression (Fig. 3 C). We also examined the upstream regulators of cellular mechanisms and found that, in addition to AMPK, the expression of TGFß1/2 were increased by dapagliflozin treatment (Fig. 3 D). Figure 3 E showed a protein–protein interaction network constructed from differentially expressed genes related to mitochondrial dysfunction and autophagy/mitophagy in hypoxic iCMs with or without dapagliflozin treatment. Autophagy- and mitophagy-associated regulators clustered in the lower module, whereas genes involved in mitochondrial bioenergetics and structural integrity were enriched in the upper module. Within this integrated network, SIRT1 and SIRT3 emerged as central hubs that bridge the autophagy/mitophagy subnetwork with the mitochondrial dysfunction cluster, linking deacetylase signaling to mitochondrial quality control. The prominent positioning and connectivity of SIRT1 and SIRT3 highlight their pivotal roles as coordinators of stress-adaptive autophagy and mitochondrial homeostasis in dapagliflozin-treated hypoxic cardiomyocytes. Dapagliflozin enhances autophagy and autophagy-related regulators in cells Since gene expression analysis indicated that dapagliflozin can initiate and enhance autophagic flux in cardiomyocytes, we then verified the production of autophagosomes in cardiomyocyte. As shown in Fig. 4 A, we used acridine orange (AO) staining to detect acidic vesicular organelles (AVOs; red fluorescence) as well as cytoplasm and nucleus (green fluorescence) of cardiomyocytes in normal cardiomyocytes, hypoxic cultured, and low concentration (2.5µM) dapagliflozin-treated under hypoxic conditions, respectively. We found that under hypoxic conditions, the expression of AVOs in cardiomyocytes was increased but not significant. After treatment with dapagliflozin, the number of AVOs in cardiomyocytes was significantly increased and expressed a strong AVOs staining. Bafilomycin A1 (Baf-A1), an inhibitor of the late-stage of autophagy, was applied to block the fusion of autophagosomes with lysosomes to verify the regulation of dapagliflozin in cellular autophagy. It showed that with the additional Baf-A1 treatment increased the AVO staining, suggest the blockade of autophagy progression stop in the late-phase. To further verify the emergence of autophagic cells, we used the Autophagy Staining Kit to explore the occurrence of autophagy. A fluorescent probe monodansylcadaverine (MDC) is designed to incorporate into multilamellar bodies by both an ion trapping mechanism and the interaction with membrane lipids while autophagy occurrence. Using the MDC probe, it allows us to detect the iCM undergoing autophagy. As shown in Fig. 4 B, we found that the MDC fluorescence response tended to increase after hypoxic treatment, but not significant. However, the addition of 2.5µM dapagliflozin treatment significantly increased the MDC-responsive cells, which indicated that dapagliflozin induced amount of autophagy in iCM. We then investigated the effect of dapagliflozin on cell survival when inducing autophagy in iCM. We follow used the flow cytometry and iCM stained with 7AAD/Annexin V double staining to analyze the apoptosis after hypoxia incubation and dapagliflozin treatment. We found that the secondary apoptosis of iCM under the hypoxic stress was approximately 11%. After 2.5µM dapagliflozin treatment, the apoptotic iCMs were reduced to 4%, which was comparable to that of the control group. The combined treatment of dapagliflozin and Baf-A1 increased the iCM apoptosis to 20%. The results demonstrated the cardio-protective effect of dapagliflozin in iCM (Fig. 4 C). We also investigated the mitochondrial autophagy (mitophagy) responses under hypoxic stress and dapagliflozin treatment via chemiluminescent staining. As shown in Fig. 4 D, fluorescence imaging results indicate that lysosomal fluorescence signals were nearly absent in the normal control group. However, after 72 hours of hypoxia-induced culture, increased expression of lysosomal and mitophagy signals was detected. Interestingly, upon adding 2.5 µM dapagliflozin, a significant increase in signal intensity was observed, particularly in lysosomes. This suggests dapagliflozin may enhance mitophagy by upregulating mitophagy activity. Baf-A1 was also used to test the effect that dapagliflozin induces mitophagy in hypoxia-iCM cells, and it showed the further increased lysosomal signaling upon Baf-A1 co-treatment. However, this was accompanied by reduced iCM cell survival. These findings support dapagliflozin's role in activating an anti-hypoxic mechanism through mitophagy in iCM cells. Related protein expression changes were then conducted. As shown in Fig. 4 E, the immunoblot analysis demonstrates the expression changes of LC3B-I/II, LAMP2, and Parkin in human iCM under hypoxia, dapagliflozin, and bafilomycin A1 (Baf-A1) treatments. Hypoxia markedly increased LC3B-II and Parkin expression, indicative of enhanced autophagic and mitophagic activity. Dapagliflozin further augmented LC3B-II and Parkin accumulation, especially in the presence of Baf-A1, suggesting increased autophagic flux rather than impaired autophagosome degradation. Interestingly, LAMP2 expression, a lysosomal marker, decreased under hypoxia but was upregulated by dapagliflozin co-treatment, particularly with Baf-A1, reflecting restored lysosomal biogenesis or function. These results indicate that dapagliflozin promotes autophagic and lysosomal activity, and enhances mitophagy in human cardiomyocytes subjected to hypoxic stress. Dapagliflozin-initiated SIRT1 and AMPK and LC3B colocalization of cytosolic autophagy proteins The previous results showed that dapagliflozin treatment reversed and enhanced iCM autophagy, we follow investigated the associated upstream regulators Sirt1, Sirt3 and AMPK activation after hypoxia and dapagliflozin treatment. As shown in Fig. 5 A, hypoxia alone decreased Sirt1, Sirt3, and pAMPK expression compared to controls. Dapagliflozin treatment under hypoxic conditions restored Sirt1 and pAMPK levels, with the highest increase observed when combined with Baf-A1, indicating activation of the SIRT1/AMPK pathway. Sirt3 was also partially restored following dapagliflozin treatment. These findings suggest that dapagliflozin enhances SIRT1/SIRT3/AMPK and associated signaling in human cardiomyocytes during hypoxic stress, potentially contributing to improved cell survival. Then changes in the expression and localization of SIRT1 and AMPK, LC3B was examined by immunofluorescence staining. As shown in Fig. 5 B, we found the co-localization of SIRT1 and phosphorylated AMPK in control cardiomyocyte. However, SIRT1 was significantly decreased and AMPK slightly increased in the condition of hypoxic stress. Both of which were significantly increased and re-colocalized in the cells after 2.5µM dapagliflozin treatment. Similarly, it was found that LC3B was not significantly altered under hypoxic stress, including its intracellular location, whereas dapagliflozin significantly increased the expression of SIRT1 and LC3B and some of them were co-localized in the nucleus (Fig. 5 C). These results indicate that hypoxic stress causes cell death but does not affect cellular autophagy, but dapagliflozin provides intracellular stress resistance by initiating autophagy and increasing its flux. Discussion In this study, we developed iPSC-derived cardiomyocytes to examine the effects of hypoxic stress on mitochondrial damage and the regulation of autophagy in human cardiomyocytes. AMI induces severe hypoxia and ischemia, ultimately causing cardiomyocyte death. Whether SGLT2i provide substantial benefit in AMI remains unclear. Our previous findings in AMI mouse models showed that hypoxic stress leads to myocardial injury and reduced expression of the SIRT family, activating pathways linked to myocardial incapacitation, fibrosis, hypertrophy, and cell damage—risk factors for subsequent MI and heart failure. Importantly, we demonstrated that the SGLT2i dapagliflozin attenuates hypoxia-induced myocardial injury by re-activating the SIRT1-dependent signaling pathway, thereby preventing hypoxia-driven apoptosis [ 1 ]. In the current study, we have uncovered the potential benefits of dapagliflozin in correcting human cardiomyocyte mitochondrial dysfunction and enhancing autophagic/ mitophagic flux to reverse myocardial death/apoptosis, which together promote myocardial survival. New therapeutic approaches are needed to protect the myocardium from the devastating effects of acute ischemia and reperfusion injury (IRI) occurred in AMI and subsequent heart failure (HF)[ 26 ]. In this regard, cardiac mitochondria play a dual role as arbiters of cell survival and death after AMI. During periods of acute myocardial ischemia, the deprivation of oxygen and nutrients leads to a metabolic shift towards anaerobic glycolysis within the cells[ 27 ]. This process results in the production of lactate, an accumulation of protons, and a decrease in pH[ 28 ]. In our case, hypoxia disrupts mitochondrial gene expression and impairs oxidative phosphorylation in iPSC-derived cardiomyocytes, as evidenced by altered OXPHOS transcripts and reduced respiratory reserve in Seahorse assays. Dapagliflozin partially restores mitochondrial gene networks, preserves OCR without excessive ECAR increase, and upregulates AMPK–SIRT1/SIRT3–autophagy/mitophagy regulators, thereby enhancing mitochondrial quality control and reversing hypoxia-induced mitochondrial dysfunction. This could further lead to an overload of calcium within the mitochondria, the induction of oxidative stress, a rapid correction of pH, and the opening of the mitochondrial permeability transition pore (MPTP)[ 29 ]. This series of events culminates in mitochondrial dysfunction and the subsequent death of the cardiomyocytes[ 30 ]. The present findings from in vitro experiments using iPSC-derived cardiomyocytes corroborate RNA-seq results from AMI mouse models. These studies revealed reduced expression of genes linked to mitochondrial fusion and increased expression of fission-related genes, indicating disrupted mitochondrial balance in hypoxic cardiomyocytes. Notably, dapagliflozin largely reversed the hypoxia-induced changes, supporting the restoration of mitochondrial function. Our previous work also showed that another SGLT2i, empagliflozin, regulates mitochondrial biosynthesis and fusion/fission balance by activating genes such as Mfn2, Ryr1/2, Vdac, Ifit1 , and Ppid etc [ 31 ]. The current findings extend beyond this, as we have uncovered a series of gene clusters that are regulated by dapagliflozin and ultimately mitigate AMI-induced impairment of cardiomyocyte mitochondrial function. This finding further corroborates our earlier observation that dapagliflozin mitigates the effect of ROS generation in cardiomyocytes. In this study, we investigated the capability of autophagy in AMI-iCM and found that it induces mitochondrial calcium overload, oxidative stress, and cardiomyocyte death. A key factor may be disrupted contact between the sarcoplasmic reticulum and mitochondria, forming mitochondria-associated membranes (MAMs). Although the exact mechanisms remain unclear, these changes promote myocardial damage, with Ca²⁺ playing a central role in AMI pathophysiology [ 32 , 33 ]. Autophagy, as a response to acute hypoxic stress, plays an important role in immunity and the pathogenesis of AMI, which is similar to the reports regarding the autophagy activated while myocardial ischemia[ 34 , 35 ]. The newest finding reveals the involved key genes associated with autophagy, including SQSTM1, UBC, MFN2, ATG5 , and TOMM20 , and the employment of machine learning algorithms, such as Random Forest and Support Vector Machine Regression Feature Elimination, for AMI diagnosis[ 36 ]. Besides, dapagliflozin has been shown to enhance mitophagy and mitochondrial quality control mechanisms, which play a critical role in maintaining cellular energy homeostasis and survival under stress conditions such as hypoxia. Our findings that dapagliflozin reduces mitochondrial dysfunction-related gene expression while upregulating key regulatory proteins including TGFB1/2, SIRT1/3, AMPK, LC3B, BECN1, SQSTM1, and ATGs are consistent with recent studies demonstrating dapagliflozin’s ability to activate mitophagy pathways and improve mitochondrial integrity. Especial SIRT1 and SIRT3 that occupy a central position at the intersection of mitochondrial quality control and stress-responsive autophagy/mitophagy signaling. As NAD + -dependent deacetylases, they modulate key mitochondrial and autophagy regulators, thereby linking cellular redox and energy status to the selective removal of damaged mitochondria and the maintenance of respiratory competence. Dapagliflozin treatment coordinated upregulation and network centrality of SIRT1/3 strongly suggest that this drug exerts its cardioprotective effects, at least in part, by engaging a SIRT1/3-dependent axis. For example, Shen et al. reported dapagliflozin protects cardiac function through FUNDC1-dependent mitophagy activation[ 37 ]. Another study by Gao et al. found dapagliflozin ameliorates mitochondrial injury in cardiomyocytes by restoring mitochondrial complexes and reducing reactive oxygen species[ 38 ]. These molecular effects contribute to enhanced cell survival in hypoxic environments, supporting the view that dapagliflozin promotes cellular resilience by modulating mitophagy and mitochondrial quality control. Moreover, these studies confirm dapagliflozin’s capacity to enhance mitophagy rather than non-specific general autophagy, emphasizing selective mitochondrial turnover as a key cardioprotective mechanism. By analyzing DEGs in the AMI dataset and intersecting them with autophagy-related gene sets, it was identified a subset of 12 key autophagy-related DEGs (MRDEGs) enriched including AGPS, CA2, CAT, LTA4H, MYO9B, PRDX6, PYGB, SIRT3, TFEB, TOM1, UBA52 , and UBB , which may serve as potential diagnostic risk factors for AMI[ 36 ]. Consistent with our findings, cellular autophagy was modestly activated in human cardiomyocyte under ischemic stress. Beyond the 12 key MRDEGs identified, our model further revealed over 30 additional genes regulated by dapagliflozin, highlighting its broader impact on autophagy-related pathways. Our results also showed activation of AMPK/SIRT-related pathways and initiation of autophagy in LC3B- and BECN1-dependent cells, whereas activation of the AMPK pathway and inhibition of the Rheb/mTORC1 pathway are critical for autophagy initiation, and disruption of these mechanisms may exacerbate myocardial injury[ 39 ].. Besides, both autophagy mitophagy can be initiated independently of the AMPK pathway to activate BECN1-associated autophagy in a ROS-dependent manner during this process[ 40 , 41 ]. Therefore, dapagliflozin appears to initiate autophagy and mitochondrial autophagy via both ROS- and SIRTs/AMPK pathways. The use of autophagy inhibitors, such as 3-methyladenosine and Bafilomycin A1, has been shown to exacerbate myocardial damage[ 42 , 43 ]. Our results with the additional Bafilomycin A1 showed that with higher autophagic and mitophagic response. However, Bafilomycin A1 also led to increased iCM death. In hypoxic cardiomyocytes, LC3B-II, Sirt1, and phosphorylated AMPK expression increased with dapagliflozin, Bafilomycin A1, or their combination compared with hypoxia alone. These findings suggest that dapagliflozin protects cardiomyocytes under hypoxic stress by enhancing autophagic flux, and that activation of autophagy may promote cell survival during myocardial ischemia. In hypoxic conditions, mitophagy selectively removes damaged mitochondria to maintain mitochondrial quality and quantity, a finding consistent with previous reports on cellular stress responses. Our study confirmed hypoxia-induced mitochondrial dysfunction and highlighted dapagliflozin’s role in upregulating Parkin, suggesting its potential in stimulating mitophagy to mitigate hypoxic injury. Despite the absence of observable LC3B-mitochondria colocalization, this likely reflects limitations in the hypoxia model and timing. Additionally, we showed dapagliflozin enhances autophagic flux via the AMPK/SIRT1/LC3B pathway, improving cardiomyocyte viability under hypoxic stress. These insights align with growing evidence of dapagliflozin’s cardioprotective mechanism involving mitochondrial quality control, contributing to its beneficial off-target effects in acute myocardial infarction. Future work should explore in vivo effects and time-course dynamics to optimize therapeutic use. This integrated perspective furthers understanding of mitophagy and autophagy's roles in cardiac health and highlights dapagliflozin as a promising modulator for mitochondrial homeostasis under stress. Study Highlights This study demonstrated that dapagliflozin significantly alleviated hypoxia-induced mitochondrial dysfunction and apoptosis in human iPSC-derived cardiomyocytes and improved cell survival by restoring the SIRTs/AMPK signaling pathway. RNA-sequencing and functional analyses revealed that dapagliflozin reversed hypoxia-induced gene expression changes, promoted autophagy and mitochondrial autophagy, and regulated mitochondrial quality control by increasing the activity of key proteins such as LC3B and Parkin. Pharmacological data showed that dapagliflozin-activated autophagy and mitochondrial autophagy exhibited stronger protective effects against myocardial injury, demonstrating the beneficial effects of dapagliflozin in cardiac tissue. Declarations Acknowledgements Special thanks to the Center for Lipid Biosciences at Kaohsiung Medical University Hospital for providing research resources and space. Author Contributions P.-C.H., C.-Y.C., and Y.-H.L. (Yi-Hsiung Lin) wrote the manuscript; P.-C.H., C.-Y.C., and Y.-H.L. (Yi-Hsiung Lin) conceptualized the study; Y.-H.L. (Yi-Hsiung Lin), J.-S.J., and N.-Y.C. performed the research; Y.-H.L. (Yi-Hsiung Lin) developed the methodology and software; W.-T.W., Y.-H.L. (Yi-Hsiung Lin), and T.-H.L. conducted formal analysis; Y.-H.L. (Yi-Hsiung Lin), C.-Y.C., W.-T.W., T.-H.L., Y.-H.L. (Yi-Hsueh Liu), and T.-C.H. validated the results; W.-T.L. and S.-H.S. provided resources and supervised the project; P.-C.H., J.-S.J., and Y.-H.L. (Yi-Hsiung Lin) administered the project; P.-C.H. and Y.-H.L. (Yi-Hsiung Lin) acquired funding; P.-C.H., W.-C.T., C.-Y.C., and Y.-H.L. (Yi-Hsiung Lin) reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript. Declaration Of Competing Interest The authors declare that they have no financial interests related to the material in the manuscript. This research was conducted independently of any business or financial relationships that could be construed as a potential conflict of interest. Funding This research was funded by Kaohsiung Medical University Hospital (KMUH111-1R76, KMUH112-2M03, and KMUH113-3M63), Kaohsiung Medical University (KMU-TC114A02) and National Science and Technology Council, Taiwan (NSTC 112-2320-B-037-016). Availability of data and materials The datasets generated and analyzed during this study have not been made publicly available because the corresponding paper has not yet been published; however, they may be obtained from the corresponding author upon reasonable request. Ethics approval and consent to participate Not applicable. Patient consent for publication Not applicable. References Lin, Y.H., et al., The Beneficial Effect of the SGLT2 Inhibitor Dapagliflozin in Alleviating Acute Myocardial Infarction-Induced Cardiomyocyte Injury by Increasing the Sirtuin Family SIRT1/SIRT3 and Cascade Signaling. Int J Mol Sci, 2024. 25 (15). Wu, Y., et al., Autophagy-modulating biomaterials: Multifunctional weapons to promote tissue regeneration. Cell Communication and Signaling, 2024. 22 (1): p. 124. Pan, W., et al., Comprehensive view of macrophage autophagy and its application in cardiovascular diseases. Cell proliferation, 2024. 57 (1): p. e13525. Yamamoto, H. and T. Matsui, Molecular mechanisms of macroautophagy, microautophagy, and chaperone-mediated autophagy. Journal of Nippon Medical School, 2024. 91 (1): p. 2-9. Yu, Z., et al., The role of macroautophagy in substance use disorders. Annals of the New York Academy of Sciences, 2025. 1543 (1): p. 68-78. Cheng, Y., et al., Baicalin alleviates angiotensin II‐induced cardiomyocyte apoptosis and autophagy and modulates the AMPK/mTOR pathway. Journal of Cellular and Molecular Medicine, 2024. 28 (9): p. e18321. Prasun, P., Mitochondrial dysfunction in metabolic syndrome. Biochimica et biophysica acta (BBA)-Molecular basis of disease, 2020. 1866 (10): p. 165838. Zhang, X., et al., Roles of Autophagy, Mitophagy, and Mitochondria in Left Ventricular Remodeling after Myocardial Infarction. Reviews in Cardiovascular Medicine, 2025. 26 (3): p. 28195. Tang, Y., et al., Autophagy protects mitochondrial health in heart failure. Heart Failure Reviews, 2024. 29 (1): p. 113-123. Ravindran, R. and Å.B. Gustafsson, Mitochondrial quality control in cardiomyocytes: safeguarding the heart against disease and ageing. Nature Reviews Cardiology, 2025: p. 1-16. Paraskevaidis, I., et al., Heart Failure: A Deficiency of Energy—A Path Yet to Discover and Walk. Biomedicines, 2024. 12 (11): p. 2589. Nixon, R.A. and D.C. Rubinsztein, Mechanisms of autophagy–lysosome dysfunction in neurodegenerative diseases. Nature Reviews Molecular Cell Biology, 2024. 25 (11): p. 926-946. Zhang, L., L. Dai, and D. Li, Mitophagy in neurological disorders. Journal of neuroinflammation, 2021. 18 (1): p. 297. Mohiuddin, O., et al., The Role of Autophagy in Heart Disease. International Journal of Drug Discovery and Pharmacology, 2024: p. 100021-100021. Ajoolabady, A., et al., Mitophagy in cardiovascular diseases: molecular mechanisms, pathogenesis, and treatment. Trends in molecular medicine, 2022. 28 (10): p. 836-849. Zhang, H., et al., Aging and Autophagy: Roles in Musculoskeletal System Injury. Aging and Disease, 2025. 16 (3): p. 2. Zhang, K., et al., Targeting Mitophagy as a Potential Therapeutic Approach for Age‐Related Bone Diseases. Advanced Therapeutics, 2024. 7 (7): p. 2400078. Zhao, J., et al., New insights into the interplay between autophagy, gut microbiota and insulin resistance in metabolic syndrome. Biomedicine & Pharmacotherapy, 2024. 176 : p. 116807. Wu, H., et al., The upregulation of Annexin A2 by TLR4 pathway facilitates lipid accumulation and liver injury via blocking AMPK/mTOR-mediated autophagy flux during the development of non-alcoholic fatty liver disease. Hepatology international, 2024. 18 (4): p. 1144-1157. Zhang, W., et al., TMX2 potentiates cell viability of hepatocellular carcinoma by promoting autophagy and mitophagy. Autophagy, 2024. 20 (10): p. 2146-2163. Forte, M., et al., Mitophagy modulation for the treatment of cardiovascular diseases. European Journal of Clinical Investigation, 2024. 54 (8): p. e14199. Ma, Y., et al., Mitophagy in hypertension-mediated organ damage. Frontiers in cardiovascular medicine, 2024. 10 : p. 1309863. Ljubojević-Holzer, S., et al., Loss of autophagy protein ATG5 impairs cardiac capacity in mice and humans through diminishing mitochondrial abundance and disrupting Ca2+ cycling. Cardiovascular research, 2022. 118 (6): p. 1492-1505. Yan, F. and L. Bao, The Role of Mitophagy in Cardiac Metabolic Remodeling of Heart Failure: Insights of Molecular Mechanisms and Therapeutic Prospects. Journal of Cardiovascular Translational Research, 2025: p. 1-13. Li, J., et al., Meteorin-like (METRNL) attenuates hypertensive induced cardiac hypertrophy by inhibiting autophagy via activating BRCA2. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 2024. 1870 (4): p. 167113. Sagris, M., et al., Myocardial ischemia–reperfusion injury: unraveling pathophysiology, clinical manifestations, and emerging prevention strategies. Biomedicines, 2024. 12 (4): p. 802. Ao-Di, F., et al., Advances in macrophage metabolic reprogramming in myocardial ischemia-reperfusion. Cellular Signalling, 2024: p. 111370. Karunarathna, I., et al., Advances in the Understanding and Management of Lactic Acidosis . 2024, ResearchGate. Available at https://www. researchgate. net/publication/383476212. Endlicher, R., et al., The mitochondrial permeability transition pore—current knowledge of its structure, function, and regulation, and optimized methods for evaluating its functional state. Cells, 2023. 12 (9): p. 1273. Baev, A.Y., et al., Mitochondrial permeability transition, cell death and neurodegeneration. Cells, 2024. 13 (7): p. 648. Jhuo, S.J., et al., Sodium Glucose Cotransporter 2 (SGLT2) Inhibitor Ameliorate Metabolic Disorder and Obesity Induced Cardiomyocyte Injury and Mitochondrial Remodeling. Int J Mol Sci, 2023. 24 (7). Agyapong, E.D., et al., Calcium signaling from sarcoplasmic reticulum and mitochondria contact sites in acute myocardial infarction. Journal of Translational Medicine, 2024. 22 (1): p. 552. Kowalczyk, P., et al., Effect of Oxidative Stress on Mitochondrial Damage and Repair in Heart Disease and Ischemic Events. International Journal of Molecular Sciences, 2024. 25 (22): p. 12467. Peng, Y., et al., Crosstalk among reactive oxygen species, autophagy and metabolism in myocardial ischemia and reperfusion stages. Aging and disease, 2024. 15 (3): p. 1075. Wang, K., et al., Protective effects of tetramethylpyrazine on myocardial ischemia/reperfusion injury involve NLRP3 inflammasome suppression by autophagy activation. Biochemical Pharmacology, 2024. 229 : p. 116541. Sheng, Z., et al., Identification of mitophagy-related key genes and their correlation with immune cell infiltration in acute myocardial infarction via bioinformatics analysis. Frontiers in Cardiovascular Medicine, 2025. 11 : p. 1501608. Zhao, N., et al., Pterostilbene attenuates oxidative stress induced by hydrogen peroxide in MAC-T cells through activating PINK1/Parkin-mediated mitophagy. Italian Journal of Animal Science, 2024. 23 (1): p. 758-768. Gan, L., et al., The biology of exosomes and exosomal non-coding RNAs in cardiovascular diseases. Frontiers in Pharmacology, 2025. 16 : p. 1529375. Zhu, Z., et al., THSWD upregulates the LTF/AMPK/mTOR/Becn1 axis and promotes lysosomal autophagy in hepatocellular carcinoma cells by regulating gut flora and metabolic reprogramming. International Immunopharmacology, 2025. 148 : p. 114091. Zamanian, M.Y., et al., Effects of metformin on cancers in experimental and clinical studies: Focusing on autophagy and AMPK/mTOR signaling pathways. Cell Biochemistry and Function, 2024. 42 (4): p. e4071. Bi, D., et al., The regulatory mechanisms of mitophagy and oxidative stress in androgenetic alopecia. Cellular Signalling, 2025: p. 111862. Horng, C.-T., et al., Koelreuteria Formosana extract induces growth inhibition and cell death in human Colon carcinoma cells via G2/M arrest and LC3-II activation-dependent autophagy. Nutrition and Cancer, 2017. 69 (1): p. 44-55. Box, A.H., S.-M. Kim, and D.J. Demetrick, AKT loss in human epithelial cells treated with severe hypoxia. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 2010. 1803 (8): p. 951-959. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 30 Apr, 2026 Reviews received at journal 24 Apr, 2026 Reviews received at journal 11 Apr, 2026 Reviews received at journal 06 Apr, 2026 Reviewers agreed at journal 02 Apr, 2026 Reviewers agreed at journal 01 Apr, 2026 Reviewers agreed at journal 01 Apr, 2026 Reviewers invited by journal 30 Mar, 2026 Editor assigned by journal 27 Mar, 2026 Submission checks completed at journal 27 Mar, 2026 First submitted to journal 26 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9239009","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":615125374,"identity":"8ac2f0fb-04ae-4f4a-8746-4998b18da3b1","order_by":0,"name":"Yi-Hsiung Lin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYJCCAwwMFjwM7A1ApoEF0VokeBh4DoC0SBBtEVClRAKUQQjwt59OPFxQISFjLvn86oYfBRJAke4E/Mafyd1weMYZCR7L2TllN3uADpM4c3YDXi0GDEAtvG0SPAa3c9Ju8AC1GEjkEtDC/xaq5eaZtJt/iNIiAbPlBvux20TZInEDaAsP0C8GZ3LYbssYSPAQ9At/f+7mzzwVNvYGx48/u/nmj40cf3svfi1IgMcATBKrHATYH5CiehSMglEwCkYQAABBV0VpeAEksgAAAABJRU5ErkJggg==","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yi-Hsiung","middleName":"","lastName":"Lin","suffix":""},{"id":615125375,"identity":"8a258988-4e23-48d4-b1d8-521e98aa1c57","order_by":1,"name":"Shih-Jie Jhuo","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shih-Jie","middleName":"","lastName":"Jhuo","suffix":""},{"id":615125376,"identity":"eb569e71-3004-445d-a518-d65e344556d7","order_by":2,"name":"Wei-Chung Tsai","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wei-Chung","middleName":"","lastName":"Tsai","suffix":""},{"id":615125377,"identity":"7717d08f-e800-4e5c-8590-44afafc25b2c","order_by":3,"name":"Yi-Hsueh Liu","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yi-Hsueh","middleName":"","lastName":"Liu","suffix":""},{"id":615125378,"identity":"af0de167-e1ce-4b00-8aac-bbe7f7ea6aae","order_by":4,"name":"Nai-Yu Chi","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Nai-Yu","middleName":"","lastName":"Chi","suffix":""},{"id":615125379,"identity":"7d3334de-af04-43b2-bcd7-b2c301d69007","order_by":5,"name":"Tien-Chi Huang","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tien-Chi","middleName":"","lastName":"Huang","suffix":""},{"id":615125380,"identity":"841f0de5-450c-4a1b-a2cf-21744e54fc16","order_by":6,"name":"Wei-Tsung Wu","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wei-Tsung","middleName":"","lastName":"Wu","suffix":""},{"id":615125381,"identity":"baaf573c-e332-4c3e-8b08-3c19f6ea8570","order_by":7,"name":"Tsung-Hsien Lin","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tsung-Hsien","middleName":"","lastName":"Lin","suffix":""},{"id":615125382,"identity":"3090ae22-3eec-4677-b13f-036822479e89","order_by":8,"name":"Wen-Ter Lai","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wen-Ter","middleName":"","lastName":"Lai","suffix":""},{"id":615125383,"identity":"bf5bfdb3-51ee-478f-b4b8-c505a0a2b383","order_by":9,"name":"Sheng-Hsiung Sheu","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Sheng-Hsiung","middleName":"","lastName":"Sheu","suffix":""},{"id":615125384,"identity":"4ece681c-73d7-4253-a211-8b3b6ade68f7","order_by":10,"name":"Chun-Yuan Chu","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chun-Yuan","middleName":"","lastName":"Chu","suffix":""},{"id":615125385,"identity":"90986b1d-fef9-4ceb-a2c8-58141fba8714","order_by":11,"name":"Po-Chao Hsu","email":"","orcid":"","institution":"Kaohsiung Medical University","correspondingAuthor":false,"prefix":"","firstName":"Po-Chao","middleName":"","lastName":"Hsu","suffix":""}],"badges":[],"createdAt":"2026-03-27 02:08:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9239009/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9239009/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105954830,"identity":"678775d6-302b-4b7b-8eda-bdbe6d052f94","added_by":"auto","created_at":"2026-04-01 19:44:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":702383,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNA sequencing results and bioinformatic analysis of hypoxia-cultured human iPSC-derived cardiomyocyte(iCM).\u003c/strong\u003e (A) Atrial myocyte biomarker MYL4 of iCM confirmation. N=3 (B) RNA-sequencing analysis and gene clustered-heatmap that regulated by hypoxic stress exposure and dapagliflozin treatment. N=3 (C)Volcano pots showing the number of differentially expressed genes (DEGs) that increased and decreased after hypoxic stress and dapagliflozin treatment. (D) Venn diagram showing the DEG counts that overlapped between the genes that increased and decreased after hypoxic stress and 5µM dapagliflozin 48h-treatment. Up_/Down_Hypoxia: DEGs regulated by hypoxia, Up_/Down_ Hy+Dapa: DEGs regulated by hypoxia and 5µM dapagliflozin incubation. (E) Heatmap showing that DEGs were involved in the bio-functional categories of intracellular disorders related to myocardial function. (F) KEGG networks analysis revealed the co-relation between pathways that affected in hypoxia-iCM treated with dapagliflozin.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9239009/v1/6c980576f9e7ead3d274d7a4.png"},{"id":106093522,"identity":"d1ff2161-d04d-430a-8558-dc7ea4306272","added_by":"auto","created_at":"2026-04-03 11:37:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":456153,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of mitochondrial dysfunction genes in hypoxia and dapagliflozin-treated iCM. (A)Heatmap showing the distribution of mitochondria-associated gene clusters of iPSC-derived cardiomyocyte (iCM) affected by hypoxic stress and dapagliflozin (Dapa) treatment. N=3 (B) KEGG pathway analysis revealed the involvement of DEG in mitochondrial function and predicted regulatory mechanisms. (C) qPCR validation of gene expression alterations under hypoxic stress and dapagliflozin treatment. (D)Oxygen consumption rate (OCR) and (E)extracellular acidification rate (ECAR) measurements in iPSC-derived cardiomyocytes showed mitochondrial respiratory capacity and glycolytic activity in hypoxia- and dapagliflozin treated- iCM. N=3, # \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 compared with hypoxia group.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9239009/v1/56a6a9a81806a3282e002197.png"},{"id":105954834,"identity":"1b0eab30-780a-4c19-aa25-47f06562b585","added_by":"auto","created_at":"2026-04-01 19:44:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":720331,"visible":true,"origin":"","legend":"\u003cp\u003eHypoxic stress induces autophagy in iCM cells. (A) Regulation and changes of autophagy-related genes in iCM cells. (B) KEGG pathway analysis shows that hypoxic stress induces the initiation of autophagy in cardiomyocytes. (C) qPCR was performed to verify that autophagy-related genes were regulated by hypoxic stress and dapagliflozin. N=3 (D)Verification of genetic changes in the upstream mechanism of cellular autophagy. (E)Protein–protein interaction analysis of regulators related to mitochondrial dysfunction and autophagy/mitophagy reveals two interconnected modules, linking mitochondrial quality control pathways with bioenergetic regulators. Scoring their key roles in coordinating dapagliflozin-induced adaptive responses in hypoxic iCM. N=3, * \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, # \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 compared with hypoxia group.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9239009/v1/c2c8be6d0dbe60cffc358245.png"},{"id":106093722,"identity":"fc59e569-d52c-4503-9035-65c1a953b0ae","added_by":"auto","created_at":"2026-04-03 11:38:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":567879,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of autophagy and mitophagy in iCM cells under hypoxic stress and dapagliflozin treatment. (A)Acridine orange (AO) staining was used to detect autophagy in hypoxic stress and 2.5µM dapagliflozin-treated iCM. N=3 (B) Autophagic iCM evaluation by using Monodansylcadaverine (MDC)-staining. MDC is detectable using a UV (350 nm) laser and filter at 525 nm. N=7 (C)Flow cytometry was used for detection of apoptotic iCM incubated w/o hypoxia and 2.5µM dapagliflozin treatment. (N=4) The right panel was the quantitated result of apoptotic iCM under indicated treatment. (D) Investigation of the mitophagy response in iCM cells under hypoxia and following dapagliflozin treatment, utilizing chemical fluorescence staining to detect lysosomal and mitophagy dye fluorescence images. N=3 (E) Upstream regulator of mitophagy in iCM in response to hypoxia and 2.5µM dapagliflozin treatment. N=3 * \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, # \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 compared with the hypoxia group.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9239009/v1/1cf98a6c66ed7eabd72c1aa2.png"},{"id":105954832,"identity":"89924d5d-3373-4f43-ad3d-63148fdfdf70","added_by":"auto","created_at":"2026-04-01 19:44:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":576027,"visible":true,"origin":"","legend":"\u003cp\u003eDapagliflozin treatment altered autophagy and mitophagy flux and associated Sirt-AMPK -LC3B expression of iCM under hypoxic stress. (A) The protein expression of autophagy and mitophagy flux upstream regulator examination of 5µM dapagliflozin in iCM. Baf-A1 (Bafilomycin A1) was used as the late-stage inhibitor of autophagy by inhibiting the combination of autophagosome and lysosome. Immunofluorescence co-staining was conducted to examine the results of dapagliflozin-treated iCM on the initiation of (B) Sirt1-AMPK phosphorylation and (C) Sirt1-LC3B double staining. N=3, * \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ## \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 compared with the hypoxia group.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9239009/v1/ad51be12511363aaab5192fc.png"},{"id":106402036,"identity":"0b38ca1b-eb1c-4aa3-9008-80aadb20c974","added_by":"auto","created_at":"2026-04-08 09:10:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3943406,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9239009/v1/607f227e-5bb9-4a03-814c-520e39d093be.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Protective mitophagy in human hypoxic cardiomyocytes: mechanistic insights into SGLT2 inhibitor Dapagliflozin cardioprotection in ischemic injury","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSGLT2 inhibitors are now known to be beneficial in the treatment of heart failure. However, its efficacy in other cardiovascular diseases remains unclear, especially in the treatment of acute myocardial infarction (AMI). Our previous studies have shown that AMI leads to a reduction in the SIRT family in vivo, which in turn induces intracellular mechanisms detrimental to cardiomyocytes[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In vitro experiments demonstrated that SGLT2i dapagliflozin was effective in reversing hypoxic stress-induced myocardial injury, and increased SIRT1/3 family, which protected ventricular myocardial cells from myocardial injury caused by hypoxic stress. The present study aimed to further elucidate the mechanism of dapagliflozin-activated intracellular protection against hypoxic stress-induced myocardial cell death after exposing to hypoxic stress.\u003c/p\u003e \u003cp\u003eAutophagy is an intracellular biodegradation process that breaks down and recycles cellular components. It is also a cellular self-protection mechanism that removes pathogens, damaged proteins, cells, organs and other items from cells to maintain normal cellular function[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Macroautophagy begins as the ULK complex gathers near the endoplasmic reticulum to start the process. Next, ATG proteins (ATG5, ATG12, ATG16L, ATG9) form a phagophore assembly site to encapsulate damaged cell components. The membrane extends, closing into a double-membrane autophagosome. This autophagosome fuses with a lysosome to form an autolysosome. Finally, contents are degraded into molecules like amino acids and fatty acids for recycling. [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The main features of cellular autophagy are selective or non-selective degradation of cytoplasmic components (e.g., damaged mitochondria, protein aggregates, etc.) and maintenance of cellular homeostasis and adaptation to stress, which is the main mechanism of cellular protection.\u003c/p\u003e \u003cp\u003eMitophagy selectively removes damaged mitochondria, helping regulate cellular energy and lower oxidative stress. Mitochondrial dysfunction contributes to diseases like heart disease, neurodegeneration, and metabolic syndrome[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In heart failure, damaged mitochondria reduce energy in heart cells and raise ROS, causing cell death and poor cardiac function. Mitophagy protects the heart by clearing abnormal mitochondria and aiding mitochondrial renewal[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Recent studies indicate that mitochondrial quality control and mitophagy activity are imbalanced in heart failure patients[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Existing research demonstrates that excessive inhibition of mitophagy exacerbates cardiac structural abnormalities, while excessive activation may lead to excessive mitochondrial clearance and myocardial energy deficiency[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Regulating mitophagy affects cardiovascular disease progression and offers promising therapeutic potential. Understanding mitophagy is vital for developing precision medicine targeting mitochondrial quality, and adjusting mitophagy may help restore cardiac function and slow disease.\u003c/p\u003e \u003cp\u003eMany diseases are associated with autophagy and mitophagy disorder, including neurodegenerative diseases[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], cardiovascular diseases[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], musculoskeletal diseases[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], metabolic syndromes[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], liver diseases and various cancer[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Increasing evidence suggests that mitophagy is associated with the development of cardiovascular diseases and may serve as a viable therapeutic target[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Mitophagy plays a crucial role in maintaining cardiac morphology and function, as well as in the progression of hypertension[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Moderate mitophagy can slow the progression of Heart failure (HF), while insufficient autophagy/mitophagy (e.g., through the knockout of ATG5 and ATG7 in animal models) leads to increased hypertrophic cardiomyocytes and worsened cardiac function[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Conversely, excessive autophagy can accelerate cardiac dysfunction, particularly in advanced stages of hypertension, where damaged organelles and harmful factors accumulate, leading to further deterioration of heart function[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, there is no clear evidence that SGLT2 inhibitors improve outcomes or induce remission in acute myocardial infarction. Most large clinical trials have demonstrated their benefits in reducing cardiovascular events\u0026mdash;such as cardiovascular death, heart failure hospitalization, nonfatal myocardial infarction, and renal deterioration\u0026mdash;mainly in patients with diabetes or high cardiovascular risk. The EMPA-REG OUTCOME and CANVAS trials showed significant reductions in major adverse cardiovascular events (MACE), including cardiac death, nonfatal stroke, and nonfatal myocardial infarction. However, results for stroke and myocardial infarction alone have often failed to reach statistical significance. Thus, current evidence does not support SGLT2 inhibitors as essential agents for reperfusion therapy or as effective treatment after acute myocardial infarction. Mitophagy is showed to play crucial role for maintaining mitochondrial quality and energy balance in cardiomyocytes. The removal of damaged mitochondria helps reduce oxidative stress and cell apoptosis, thereby protecting cardiomyocytes from ischemic or injury exacerbation. Verifying the ability of SGLT2 inhibitors to induce mitophagy in cardiomyocytes is essential to fully understand their cardioprotective mechanisms and offers new avenues for mitophagy-targeted cardiovascular therapies.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eHuman iPSC-derived cardiomyocyte (iCM) maintains\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe human isogenic-induced pluripotent stem cell (iPSC) lines were obtained from the NCFB Human iPS Cells Service Consortium (clone number IBMS-iPSC-001-02, characterization and validation: https://catalog.bcrc.firdi.org.tw/resources/images/temp/17526382101992i) and maintained in Stemflex medium at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. For cardiomyocyte differentiation, iPSC-01-02 cells were initially derived using CHIR 99021, B-27 minus insulin, and serum-free RPMI. CHIR 99021 was removed on day 3 and replaced with IWR-1 for days 4-6. On day 7, cells were cultured using B-27 plus insulin and glucose serum-free RPMI. On day 21, cells were dispersed and re-pleated with 0.05% trypsin-EDTA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIschemia-reperfusion (IR) injury procedure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHypoxic cell culture was performed in 6 well plates or culture dishes in a tank contained 1% O\u003csub\u003e2\u003c/sub\u003e gas mixture to create low oxygen and hypoxic stress. Re-gas every 1-3 hours to remove O\u003csub\u003e2\u003c/sub\u003e from the culture medium.\u003c/p\u003e\n\u003cp\u003eSGLT2 inhibitor Dapagliflozin (DAPA) administration\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eiCM cells were treated with DAPA at concentrations as previously published. After incubation in low oxygen, cells are treated with low (2.5\u0026micro;M) and high dose (5 \u0026mu;M) for 48 hours to observe cell morphology. The cell supernatant is removed and washed twice with PBS before the cells are scraped for subsequent experiments.\u003c/p\u003e\n\u003cp\u003emRNA collection and qRT-PCR analysis\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated using the Nucleospin RNA kit (Macherey-Nagel, USA), the cell lysate was applied to the RNA column and centrifuged at 15,000 rpm. Membrane desalting buffer was added to the RNA column and centrifuged. When the DNAase reaction is complete, the RNA-containing columns are transferred to new tubes for final purification of the RNA samples. Finally, the RNA sample is solubilized with RNase-free HO and transferred to a new 1.5 ml tube. All RNA samples were stored at -80\u0026deg;C before complementary DNA (cDNA) was synthesized using reverse transcription reagent (Applied Biosystem, USA). The prepared cDNA was then introduced into a 96-well quantitative polymerase chain reaction (qPCR) to analyze gene expression.\u003c/p\u003e\n\u003cp\u003eProtein preparation and western blotting\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUse SDS polyacrylamide gel electrophoresis (SDS-PAGE) to separate protein lysates based on gel chemistry, perform wet transfer, and prepare transfer buffer. Use PVDF transfer membrane and follow manufacturer\u0026apos;s instructions for dry membrane preparation. Pre-soak in ethanol (100%) for 30 seconds, rinse briefly in deionized water, and equilibrate in transfer buffer for 5 minutes. Incubate the membrane with sufficient volume of Blocking Buffer with agitation for 30-60 minutes at room temperature. After diluting the primary antibody in blocking buffer, react overnight and incubate with the appropriate secondary antibody. Primary antibody used in the current study including LC3B Antibody (#2775, Cell Signaling Technology, MA, US), LAMP2 Rabbit mAb (#34141), Beclin-1 (D40C5) Rabbit mAb (#3495), Parkin Antibody (#2132), phosphor-AMPK(07-681, Sigma-Aldrich, MO, US), SIRT1 (TA809834, Origene, MD, USA)and SIRT3(TA330278). ECL-stimulated chemiluminescence is then used to monitor the expression of the target protein.\u003c/p\u003e\n\u003cp\u003eAutophagy and mitophagy detection\u003c/p\u003e\n\u003cp\u003eCells were stained with AO for acidic vesicular organelles (AVOs; red fluorescence) as well as cytoplasm and nucleus (green fluorescence) as indicated in the instructions. iCM cells were treated with hypoxic stress w/o dapagliflozin, respectively, and red/green fluorescence of AO was observed using a fluorescence microscope.\u0026nbsp;\u003cbr\u003e\u0026nbsp;The mitophagy detection kit (Dojindo, #MD01, Kumamoto City, Japan) includes Mtphagy Dye and Lyso Dye for detecting mitophagy in live iCM. Mtphagy Dye selectively accumulates in intact mitochondria and exhibits increased fluorescence upon mitophagy induction, while Lyso Dye stains lysosomes to confirm fusion with mitochondria. iCM were incubated with Mtphagy Dye, PBS washed for 15 mins, and exposed to mitophagy-inducing agents, hypoxia incubation and dapagliflozin treatment. Fluorescence microscopy was then used to detect the mitophagy signal and colocalization with lysosomes.\u003c/p\u003e\n\u003cp\u003eBiological pathway analysis\u003c/p\u003e\n\u003cp\u003eHigh-quality read alignments were mapped to the human reference genome (grch38.p7) and subsequently imported into Ingenuity\u0026apos;s IPA software. Following this, feature Counts were utilized to assess participation across various mechanisms, and performance metrics were quantified using RLE, TMM, and FPKM methods. The screening for differentially expressed genes employed a q value threshold of less than 0.05. The analysis was based solely on the Ingenuity knowledge base, which included genes and took into account both direct and indirect relationships. The identified results pertinent to the dataset encompassed categories such as Diseases and Disorders, Molecular and Cellular Functions, Physiological Phylogenetics and Functions, alongside Top Canonical Pathways, with all findings significant at P \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003eImmunofluorescence staining and microscopy analysis\u003c/p\u003e\n\u003cp\u003eThe analysis of cardiomyocyte cells is conducted using immunofluorescence assays. Primary antibodies targeting specific antigens in the cells are diluted in the range of 1:100 to 1:200, as indicated in the user menu. Following this preparation, the samples are washed and incubated with DAPI-conjugated secondary antibodies that interact with the various primary antibodies. The final visualization of the cells occurs through the fluorescence microscope (Olympus BX51, Olympus, Japan).\u003c/p\u003e\n\u003cp\u003eStatistics\u003c/p\u003e\n\u003cp\u003eAll data will be presented as mean \u0026plusmn; standard error of the mean (SEM). The software used for statistical analysis will be SPSS version 13.0 (SPSS13.0 Software Inc., USA), which will facilitate the establishment of one-way analysis of variance (ANOVA) to determine differences among groups. To conduct comparisons between the various groups, Duncan\u0026apos;s multi-range test will be employed. A p-value of less than 0.05 will be deemed statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing analyses of hypoxia toxicity and the therapeutic effect of dapagliflozin in iPSC-derived cardiomyocyte\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe present study was conducted with the objective of verifying the effect of hypoxia on cardiomyocytes and discussing the protective effect of SGLT2i dapagliflozin on cardiomyocytes against hypoxia. To this end, an in vitro model of hypoxic stress was established using iCM. The experiments were carried out with the aim of verifying the cardiomyocyte-specific marker MYL4 using human iPSC-derived cardiomyocytes (iCM, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). We followed analyzed the gene clusters in cardiomyocytes affected by hypoxia and 5 \u0026micro;M dapagliflozin treatment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, the results showed the RNA-sequencing gene expression clustering from human iCM treated with different conditions (hypoxia, different doses of dapagliflozin, and a normal control). Hypoxia treatment revealed significant downregulation of most genes (green), while dapagliflozin treatment (Hy+Dapa 2.5 \u0026micro;M and Hy+Dapa 5 \u0026micro;M) showed a recovery of most hypoxia-affected genes (from green to red), with this trend varying with dose. Overall, dapagliflozin significantly reversed hypoxia-induced gene expression dysregulation in a dose-dependent manner, demonstrating its potential in alleviating hypoxia-induced cardiomyocyte damage. Among these differential expressed genes (DEGs), hypoxic stress upregulated 2054 genes and downregulated 2384 genes in iCM. RNA sequencing analysis of volcano pot showed that 2384 genes significantly downregulated and 2054 genes upregulated compared with normoxic controls. Subsequent treatment with 5 \u0026micro;M dapagliflozin under hypoxic conditions further affected gene expression, with 1852 genes downregulated and 2098 genes upregulated compared with hypoxia alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These results suggest that dapagliflozin administration substantially modulates hypoxia-induced gene expression profiles, indicating its potential role in regulating adaptive and protective cellular pathways under ischemic stress. Furthermore, we found that, dapagliflozin reversed those that were up- /down-regulated by hypoxic stress; 1717 of the 2054 genes re-stored by dapagliflozin treatment which decreased by hypoxic stress, and similarly, dapagliflozin decreased 1387 of the 2384 genes that up-regulated by hypoxic stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). We further analyzed the pathways involved using IPA and found that dapagliflozin mainly enhanced a catalog of genes, including those regulating the skeletal and muscle development systems, and reversed genes related to cell death pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). In addition, KEGG network analysis revealed the signaling pathways involved in the genes regulated by dapagliflozin, among which the most affected signaling pathways were those related to cardiomyocyte metabolic pathways, cell apoptosis, and lysosome-related signaling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). These results demonstrate that dapagliflozin treatment effectively reverses the cellular damage caused by hypoxic stress and pathway modulation in human cardiomyocyte.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDapagliflozin reverses the potential mechanism of hypoxia-induced mitochondrial damage in cardiomyocytes\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn the IPA analysis, we identified genes regulated by dapagliflozin that are associated with mitochondrial dysfunction. Among the 66 gene cluster heatmap showed the regulation of hypoxic stress and dapagliflozin in mitochondrial dysfunction-associated gene, and it was found that most of them were inverted by dapagliflozin treatment and tended to be similar to the normal gene expression of the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Further analysis using the KEGG pathway showed that dapagliflozin suppressed most of the NDUFA/NDUFB family signaling regulator and most of the COX family gene expression up-regulated by hypoxic stress. In addition, the mitochondrial-encoding gene MT-CO family, which was also up-regulated by hypoxic stress, was also suppressed and returned to normal levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). We then verified these results using qPCR, which found that hypoxic stress increased gene involved in mitochondrial fission in iCM, indicating that the disturbance of mitochondrial dynamic while exposure to hypoxia. However, it showed that dapagliflozin reduced the genes up-regulated by hypoxic stress, including ND1, NDUFA12, A4, A7, A8, PSEN2, UQCRFS1 and VAC1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD showed that, in the Seahorse assay, hypoxia modestly reduced basal and stress-induced oxygen consumption rate (OCR) in iPSC-derived cardiomyocytes compared with normoxia, indicating impaired mitochondrial respiratory reserve and oxidative phosphorylation capacity under low oxygen tension. In contrast, dapagliflozin-treated hypoxic cells largely preserved OCR dynamics across the sequential mitochondrial stressors, suggesting partial restoration of mitochondrial respiratory flexibility and ATP-generating capacity. Extracellular acidification rate (ECAR) was slightly increased by hypoxia, consistent with a compensatory shift toward glycolytic metabolism, whereas dapagliflozin did not further augment ECAR, indicating that its protective effect was not driven by additional glycolytic activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Together, these data suggest that dapagliflozin primarily maintains mitochondrial oxidative metabolism rather than enhancing glycolytic flux, thereby supporting more balanced bioenergetic adaptation of cardiomyocytes under hypoxic stress.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDapagliflozin activates cellular autophagy mechanism and flux in cardiomyocytes\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIPA pathway analysis also showed that dapagliflozin activated the mechanism of cytosolic autophagy in cells, as shown in the heat map, dapagliflozin reversed the up-/down-regulation of many genes due to hypoxic stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). KEGG pathway analysis revealed that the expression of key regulators, including SIRT1, AMPK and cytosolic autophagy-associated regulators such as LC3B, the ATG family and BECN1, were all up-regulated by dapagliflozin treatment (\u003cb\u003eFigure. 3B\u003c/b\u003e). Using qPCR to verify gene expression, we found that most autophagy-related genes were not affect or only a slight increased (LC3B and BECN1) by hypoxic stress, but were significantly up-regulated by dapagliflozin treatment, which were further enhanced by dapagliflozin in gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). We also examined the upstream regulators of cellular mechanisms and found that, in addition to AMPK, the expression of TGF\u0026szlig;1/2 were increased by dapagliflozin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE showed a protein\u0026ndash;protein interaction network constructed from differentially expressed genes related to mitochondrial dysfunction and autophagy/mitophagy in hypoxic iCMs with or without dapagliflozin treatment. Autophagy- and mitophagy-associated regulators clustered in the lower module, whereas genes involved in mitochondrial bioenergetics and structural integrity were enriched in the upper module. Within this integrated network, SIRT1 and SIRT3 emerged as central hubs that bridge the autophagy/mitophagy subnetwork with the mitochondrial dysfunction cluster, linking deacetylase signaling to mitochondrial quality control. The prominent positioning and connectivity of SIRT1 and SIRT3 highlight their pivotal roles as coordinators of stress-adaptive autophagy and mitochondrial homeostasis in dapagliflozin-treated hypoxic cardiomyocytes.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDapagliflozin enhances autophagy and autophagy-related regulators in cells\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSince gene expression analysis indicated that dapagliflozin can initiate and enhance autophagic flux in cardiomyocytes, we then verified the production of autophagosomes in cardiomyocyte. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, we used acridine orange (AO) staining to detect acidic vesicular organelles (AVOs; red fluorescence) as well as cytoplasm and nucleus (green fluorescence) of cardiomyocytes in normal cardiomyocytes, hypoxic cultured, and low concentration (2.5\u0026micro;M) dapagliflozin-treated under hypoxic conditions, respectively. We found that under hypoxic conditions, the expression of AVOs in cardiomyocytes was increased but not significant. After treatment with dapagliflozin, the number of AVOs in cardiomyocytes was significantly increased and expressed a strong AVOs staining. Bafilomycin A1 (Baf-A1), an inhibitor of the late-stage of autophagy, was applied to block the fusion of autophagosomes with lysosomes to verify the regulation of dapagliflozin in cellular autophagy. It showed that with the additional Baf-A1 treatment increased the AVO staining, suggest the blockade of autophagy progression stop in the late-phase. To further verify the emergence of autophagic cells, we used the Autophagy Staining Kit to explore the occurrence of autophagy. A fluorescent probe monodansylcadaverine (MDC) is designed to incorporate into multilamellar bodies by both an ion trapping mechanism and the interaction with membrane lipids while autophagy occurrence. Using the MDC probe, it allows us to detect the iCM undergoing autophagy. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, we found that the MDC fluorescence response tended to increase after hypoxic treatment, but not significant. However, the addition of 2.5\u0026micro;M dapagliflozin treatment significantly increased the MDC-responsive cells, which indicated that dapagliflozin induced amount of autophagy in iCM. We then investigated the effect of dapagliflozin on cell survival when inducing autophagy in iCM. We follow used the flow cytometry and iCM stained with 7AAD/Annexin V double staining to analyze the apoptosis after hypoxia incubation and dapagliflozin treatment. We found that the secondary apoptosis of iCM under the hypoxic stress was approximately 11%. After 2.5\u0026micro;M dapagliflozin treatment, the apoptotic iCMs were reduced to 4%, which was comparable to that of the control group. The combined treatment of dapagliflozin and Baf-A1 increased the iCM apoptosis to 20%. The results demonstrated the cardio-protective effect of dapagliflozin in iCM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). We also investigated the mitochondrial autophagy (mitophagy) responses under hypoxic stress and dapagliflozin treatment via chemiluminescent staining. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, fluorescence imaging results indicate that lysosomal fluorescence signals were nearly absent in the normal control group. However, after 72 hours of hypoxia-induced culture, increased expression of lysosomal and mitophagy signals was detected. Interestingly, upon adding 2.5 \u0026micro;M dapagliflozin, a significant increase in signal intensity was observed, particularly in lysosomes. This suggests dapagliflozin may enhance mitophagy by upregulating mitophagy activity. Baf-A1 was also used to test the effect that dapagliflozin induces mitophagy in hypoxia-iCM cells, and it showed the further increased lysosomal signaling upon Baf-A1 co-treatment. However, this was accompanied by reduced iCM cell survival. These findings support dapagliflozin's role in activating an anti-hypoxic mechanism through mitophagy in iCM cells. Related protein expression changes were then conducted. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, the immunoblot analysis demonstrates the expression changes of LC3B-I/II, LAMP2, and Parkin in human iCM under hypoxia, dapagliflozin, and bafilomycin A1 (Baf-A1) treatments. Hypoxia markedly increased LC3B-II and Parkin expression, indicative of enhanced autophagic and mitophagic activity. Dapagliflozin further augmented LC3B-II and Parkin accumulation, especially in the presence of Baf-A1, suggesting increased autophagic flux rather than impaired autophagosome degradation. Interestingly, LAMP2 expression, a lysosomal marker, decreased under hypoxia but was upregulated by dapagliflozin co-treatment, particularly with Baf-A1, reflecting restored lysosomal biogenesis or function. These results indicate that dapagliflozin promotes autophagic and lysosomal activity, and enhances mitophagy in human cardiomyocytes subjected to hypoxic stress.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDapagliflozin-initiated SIRT1 and AMPK and LC3B colocalization of cytosolic autophagy proteins\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe previous results showed that dapagliflozin treatment reversed and enhanced iCM autophagy, we follow investigated the associated upstream regulators Sirt1, Sirt3 and AMPK activation after hypoxia and dapagliflozin treatment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, hypoxia alone decreased Sirt1, Sirt3, and pAMPK expression compared to controls. Dapagliflozin treatment under hypoxic conditions restored Sirt1 and pAMPK levels, with the highest increase observed when combined with Baf-A1, indicating activation of the SIRT1/AMPK pathway. Sirt3 was also partially restored following dapagliflozin treatment. These findings suggest that dapagliflozin enhances SIRT1/SIRT3/AMPK and associated signaling in human cardiomyocytes during hypoxic stress, potentially contributing to improved cell survival. Then changes in the expression and localization of SIRT1 and AMPK, LC3B was examined by immunofluorescence staining. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, we found the co-localization of SIRT1 and phosphorylated AMPK in control cardiomyocyte. However, SIRT1 was significantly decreased and AMPK slightly increased in the condition of hypoxic stress. Both of which were significantly increased and re-colocalized in the cells after 2.5\u0026micro;M dapagliflozin treatment. Similarly, it was found that LC3B was not significantly altered under hypoxic stress, including its intracellular location, whereas dapagliflozin significantly increased the expression of SIRT1 and LC3B and some of them were co-localized in the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results indicate that hypoxic stress causes cell death but does not affect cellular autophagy, but dapagliflozin provides intracellular stress resistance by initiating autophagy and increasing its flux.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003c/p\u003e\u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn this study, we developed iPSC-derived cardiomyocytes to examine the effects of hypoxic stress on mitochondrial damage and the regulation of autophagy in human cardiomyocytes. AMI induces severe hypoxia and ischemia, ultimately causing cardiomyocyte death. Whether SGLT2i provide substantial benefit in AMI remains unclear. Our previous findings in AMI mouse models showed that hypoxic stress leads to myocardial injury and reduced expression of the SIRT family, activating pathways linked to myocardial incapacitation, fibrosis, hypertrophy, and cell damage—risk factors for subsequent MI and heart failure. Importantly, we demonstrated that the SGLT2i dapagliflozin attenuates hypoxia-induced myocardial injury by re-activating the SIRT1-dependent signaling pathway, thereby preventing hypoxia-driven apoptosis [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. In the current study, we have uncovered the potential benefits of dapagliflozin in correcting human cardiomyocyte mitochondrial dysfunction and enhancing autophagic/ mitophagic flux to reverse myocardial death/apoptosis, which together promote myocardial survival.\u003c/p\u003e \u003cp\u003eNew therapeutic approaches are needed to protect the myocardium from the devastating effects of acute ischemia and reperfusion injury (IRI) occurred in AMI and subsequent heart failure (HF)[\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. In this regard, cardiac mitochondria play a dual role as arbiters of cell survival and death after AMI. During periods of acute myocardial ischemia, the deprivation of oxygen and nutrients leads to a metabolic shift towards anaerobic glycolysis within the cells[\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. This process results in the production of lactate, an accumulation of protons, and a decrease in pH[\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. In our case, hypoxia disrupts mitochondrial gene expression and impairs oxidative phosphorylation in iPSC-derived cardiomyocytes, as evidenced by altered OXPHOS transcripts and reduced respiratory reserve in Seahorse assays. Dapagliflozin partially restores mitochondrial gene networks, preserves OCR without excessive ECAR increase, and upregulates AMPK–SIRT1/SIRT3–autophagy/mitophagy regulators, thereby enhancing mitochondrial quality control and reversing hypoxia-induced mitochondrial dysfunction. This could further lead to an overload of calcium within the mitochondria, the induction of oxidative stress, a rapid correction of pH, and the opening of the mitochondrial permeability transition pore (MPTP)[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. This series of events culminates in mitochondrial dysfunction and the subsequent death of the cardiomyocytes[\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. The present findings from in vitro experiments using iPSC-derived cardiomyocytes corroborate RNA-seq results from AMI mouse models. These studies revealed reduced expression of genes linked to mitochondrial fusion and increased expression of fission-related genes, indicating disrupted mitochondrial balance in hypoxic cardiomyocytes. Notably, dapagliflozin largely reversed the hypoxia-induced changes, supporting the restoration of mitochondrial function. Our previous work also showed that another SGLT2i, empagliflozin, regulates mitochondrial biosynthesis and fusion/fission balance by activating genes such as \u003cem\u003eMfn2, Ryr1/2, Vdac, Ifit1\u003c/em\u003e, and \u003cem\u003ePpid etc\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. The current findings extend beyond this, as we have uncovered a series of gene clusters that are regulated by dapagliflozin and ultimately mitigate AMI-induced impairment of cardiomyocyte mitochondrial function. This finding further corroborates our earlier observation that dapagliflozin mitigates the effect of ROS generation in cardiomyocytes.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the capability of autophagy in AMI-iCM and found that it induces mitochondrial calcium overload, oxidative stress, and cardiomyocyte death. A key factor may be disrupted contact between the sarcoplasmic reticulum and mitochondria, forming mitochondria-associated membranes (MAMs). Although the exact mechanisms remain unclear, these changes promote myocardial damage, with Ca²⁺ playing a central role in AMI pathophysiology [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. Autophagy, as a response to acute hypoxic stress, plays an important role in immunity and the pathogenesis of AMI, which is similar to the reports regarding the autophagy activated while myocardial ischemia[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. The newest finding reveals the involved key genes associated with autophagy, including \u003cem\u003eSQSTM1, UBC, MFN2, ATG5\u003c/em\u003e, and \u003cem\u003eTOMM20\u003c/em\u003e, and the employment of machine learning algorithms, such as Random Forest and Support Vector Machine Regression Feature Elimination, for AMI diagnosis[\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]. Besides, dapagliflozin has been shown to enhance mitophagy and mitochondrial quality control mechanisms, which play a critical role in maintaining cellular energy homeostasis and survival under stress conditions such as hypoxia. Our findings that dapagliflozin reduces mitochondrial dysfunction-related gene expression while upregulating key regulatory proteins including TGFB1/2, SIRT1/3, AMPK, LC3B, BECN1, SQSTM1, and ATGs are consistent with recent studies demonstrating dapagliflozin’s ability to activate mitophagy pathways and improve mitochondrial integrity. Especial SIRT1 and SIRT3 that occupy a central position at the intersection of mitochondrial quality control and stress-responsive autophagy/mitophagy signaling. As NAD\u003csup\u003e+\u003c/sup\u003e-dependent deacetylases, they modulate key mitochondrial and autophagy regulators, thereby linking cellular redox and energy status to the selective removal of damaged mitochondria and the maintenance of respiratory competence. Dapagliflozin treatment coordinated upregulation and network centrality of SIRT1/3 strongly suggest that this drug exerts its cardioprotective effects, at least in part, by engaging a SIRT1/3-dependent axis. For example, Shen et al. reported dapagliflozin protects cardiac function through FUNDC1-dependent mitophagy activation[\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. Another study by Gao et al. found dapagliflozin ameliorates mitochondrial injury in cardiomyocytes by restoring mitochondrial complexes and reducing reactive oxygen species[\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. These molecular effects contribute to enhanced cell survival in hypoxic environments, supporting the view that dapagliflozin promotes cellular resilience by modulating mitophagy and mitochondrial quality control. Moreover, these studies confirm dapagliflozin’s capacity to enhance mitophagy rather than non-specific general autophagy, emphasizing selective mitochondrial turnover as a key cardioprotective mechanism. By analyzing DEGs in the AMI dataset and intersecting them with autophagy-related gene sets, it was identified a subset of 12 key autophagy-related DEGs (MRDEGs) enriched including \u003cem\u003eAGPS, CA2, CAT, LTA4H, MYO9B, PRDX6, PYGB, SIRT3, TFEB, TOM1, UBA52\u003c/em\u003e, and \u003cem\u003eUBB\u003c/em\u003e, which may serve as potential diagnostic risk factors for AMI[\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]. Consistent with our findings, cellular autophagy was modestly activated in human cardiomyocyte under ischemic stress. Beyond the 12 key MRDEGs identified, our model further revealed over 30 additional genes regulated by dapagliflozin, highlighting its broader impact on autophagy-related pathways.\u003c/p\u003e \u003cp\u003eOur results also showed activation of AMPK/SIRT-related pathways and initiation of autophagy in LC3B- and BECN1-dependent cells, whereas activation of the AMPK pathway and inhibition of the Rheb/mTORC1 pathway are critical for autophagy initiation, and disruption of these mechanisms may exacerbate myocardial injury[\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e].. Besides, both autophagy mitophagy can be initiated independently of the AMPK pathway to activate BECN1-associated autophagy in a ROS-dependent manner during this process[\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]. Therefore, dapagliflozin appears to initiate autophagy and mitochondrial autophagy via both ROS- and SIRTs/AMPK pathways. The use of autophagy inhibitors, such as 3-methyladenosine and Bafilomycin A1, has been shown to exacerbate myocardial damage[\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]. Our results with the additional Bafilomycin A1 showed that with higher autophagic and mitophagic response. However, Bafilomycin A1 also led to increased iCM death. In hypoxic cardiomyocytes, LC3B-II, Sirt1, and phosphorylated AMPK expression increased with dapagliflozin, Bafilomycin A1, or their combination compared with hypoxia alone. These findings suggest that dapagliflozin protects cardiomyocytes under hypoxic stress by enhancing autophagic flux, and that activation of autophagy may promote cell survival during myocardial ischemia.\u003c/p\u003e \u003cp\u003eIn hypoxic conditions, mitophagy selectively removes damaged mitochondria to maintain mitochondrial quality and quantity, a finding consistent with previous reports on cellular stress responses. Our study confirmed hypoxia-induced mitochondrial dysfunction and highlighted dapagliflozin’s role in upregulating Parkin, suggesting its potential in stimulating mitophagy to mitigate hypoxic injury. Despite the absence of observable LC3B-mitochondria colocalization, this likely reflects limitations in the hypoxia model and timing. Additionally, we showed dapagliflozin enhances autophagic flux via the AMPK/SIRT1/LC3B pathway, improving cardiomyocyte viability under hypoxic stress. These insights align with growing evidence of dapagliflozin’s cardioprotective mechanism involving mitochondrial quality control, contributing to its beneficial off-target effects in acute myocardial infarction. Future work should explore in vivo effects and time-course dynamics to optimize therapeutic use.\u003c/p\u003e \u003cp\u003eThis integrated perspective furthers understanding of mitophagy and autophagy's roles in cardiac health and highlights dapagliflozin as a promising modulator for mitochondrial homeostasis under stress.\u003c/p\u003e \u003c/div\u003e \u003cp\u003e\u003c/p\u003e "},{"header":"Study Highlights","content":"\u003cp\u003eThis study demonstrated that dapagliflozin significantly alleviated hypoxia-induced mitochondrial dysfunction and apoptosis in human iPSC-derived cardiomyocytes and improved cell survival by restoring the SIRTs/AMPK signaling pathway. RNA-sequencing and functional analyses revealed that dapagliflozin reversed hypoxia-induced gene expression changes, promoted autophagy and mitochondrial autophagy, and regulated mitochondrial quality control by increasing the activity of key proteins such as LC3B and Parkin. Pharmacological data showed that dapagliflozin-activated autophagy and mitochondrial autophagy exhibited stronger protective effects against myocardial injury, demonstrating the beneficial effects of dapagliflozin in cardiac tissue.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecial thanks to the Center for Lipid Biosciences at Kaohsiung Medical University Hospital for providing research resources and space.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP.-C.H., C.-Y.C., and Y.-H.L. (Yi-Hsiung Lin) wrote the manuscript; P.-C.H., C.-Y.C., and Y.-H.L. (Yi-Hsiung Lin) conceptualized the study; Y.-H.L. (Yi-Hsiung Lin), J.-S.J., and N.-Y.C. performed the research; Y.-H.L. (Yi-Hsiung Lin) developed the methodology and software; W.-T.W., Y.-H.L. (Yi-Hsiung Lin), and T.-H.L. conducted formal analysis; Y.-H.L. (Yi-Hsiung Lin), C.-Y.C., W.-T.W., T.-H.L., Y.-H.L. (Yi-Hsueh Liu), and T.-C.H. validated the results; W.-T.L. and S.-H.S. provided resources and supervised the project; P.-C.H., J.-S.J., and Y.-H.L. (Yi-Hsiung Lin) administered the project; P.-C.H. and Y.-H.L. (Yi-Hsiung Lin) acquired funding; P.-C.H., W.-C.T., C.-Y.C., and Y.-H.L. (Yi-Hsiung Lin) reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration Of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no financial interests related to the material in the manuscript. This research was conducted independently of any business or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Kaohsiung Medical University Hospital (KMUH111-1R76, KMUH112-2M03, and KMUH113-3M63), Kaohsiung Medical University (KMU-TC114A02) and National Science and Technology Council, Taiwan (NSTC 112-2320-B-037-016).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during this study have not been made publicly available because the corresponding paper has not yet been published; however, they may be obtained from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLin, Y.H., et al., \u003cem\u003eThe Beneficial Effect of the SGLT2 Inhibitor Dapagliflozin in Alleviating Acute Myocardial Infarction-Induced Cardiomyocyte Injury by Increasing the Sirtuin Family SIRT1/SIRT3 and Cascade Signaling.\u003c/em\u003e Int J Mol Sci, 2024. \u003cstrong\u003e25\u003c/strong\u003e(15).\u003c/li\u003e\n\u003cli\u003eWu, Y., et al., \u003cem\u003eAutophagy-modulating biomaterials: Multifunctional weapons to promote tissue regeneration.\u003c/em\u003e Cell Communication and Signaling, 2024. \u003cstrong\u003e22\u003c/strong\u003e(1): p. 124.\u003c/li\u003e\n\u003cli\u003ePan, W., et al., \u003cem\u003eComprehensive view of macrophage autophagy and its application in cardiovascular diseases.\u003c/em\u003e Cell proliferation, 2024. \u003cstrong\u003e57\u003c/strong\u003e(1): p. e13525.\u003c/li\u003e\n\u003cli\u003eYamamoto, H. and T. Matsui, \u003cem\u003eMolecular mechanisms of macroautophagy, microautophagy, and chaperone-mediated autophagy.\u003c/em\u003e Journal of Nippon Medical School, 2024. \u003cstrong\u003e91\u003c/strong\u003e(1): p. 2-9.\u003c/li\u003e\n\u003cli\u003eYu, Z., et al., \u003cem\u003eThe role of macroautophagy in substance use disorders.\u003c/em\u003e Annals of the New York Academy of Sciences, 2025. \u003cstrong\u003e1543\u003c/strong\u003e(1): p. 68-78.\u003c/li\u003e\n\u003cli\u003eCheng, Y., et al., \u003cem\u003eBaicalin alleviates angiotensin II‐induced cardiomyocyte apoptosis and autophagy and modulates the AMPK/mTOR pathway.\u003c/em\u003e Journal of Cellular and Molecular Medicine, 2024. \u003cstrong\u003e28\u003c/strong\u003e(9): p. e18321.\u003c/li\u003e\n\u003cli\u003ePrasun, P., \u003cem\u003eMitochondrial dysfunction in metabolic syndrome.\u003c/em\u003e Biochimica et biophysica acta (BBA)-Molecular basis of disease, 2020. \u003cstrong\u003e1866\u003c/strong\u003e(10): p. 165838.\u003c/li\u003e\n\u003cli\u003eZhang, X., et al., \u003cem\u003eRoles of Autophagy, Mitophagy, and Mitochondria in Left Ventricular Remodeling after Myocardial Infarction.\u003c/em\u003e Reviews in Cardiovascular Medicine, 2025. \u003cstrong\u003e26\u003c/strong\u003e(3): p. 28195.\u003c/li\u003e\n\u003cli\u003eTang, Y., et al., \u003cem\u003eAutophagy protects mitochondrial health in heart failure.\u003c/em\u003e Heart Failure Reviews, 2024. \u003cstrong\u003e29\u003c/strong\u003e(1): p. 113-123.\u003c/li\u003e\n\u003cli\u003eRavindran, R. and \u0026Aring;.B. Gustafsson, \u003cem\u003eMitochondrial quality control in cardiomyocytes: safeguarding the heart against disease and ageing.\u003c/em\u003e Nature Reviews Cardiology, 2025: p. 1-16.\u003c/li\u003e\n\u003cli\u003eParaskevaidis, I., et al., \u003cem\u003eHeart Failure: A Deficiency of Energy\u0026mdash;A Path Yet to Discover and Walk.\u003c/em\u003e Biomedicines, 2024. \u003cstrong\u003e12\u003c/strong\u003e(11): p. 2589.\u003c/li\u003e\n\u003cli\u003eNixon, R.A. and D.C. Rubinsztein, \u003cem\u003eMechanisms of autophagy\u0026ndash;lysosome dysfunction in neurodegenerative diseases.\u003c/em\u003e Nature Reviews Molecular Cell Biology, 2024. \u003cstrong\u003e25\u003c/strong\u003e(11): p. 926-946.\u003c/li\u003e\n\u003cli\u003eZhang, L., L. Dai, and D. Li, \u003cem\u003eMitophagy in neurological disorders.\u003c/em\u003e Journal of neuroinflammation, 2021. \u003cstrong\u003e18\u003c/strong\u003e(1): p. 297.\u003c/li\u003e\n\u003cli\u003eMohiuddin, O., et al., \u003cem\u003eThe Role of Autophagy in Heart Disease.\u003c/em\u003e International Journal of Drug Discovery and Pharmacology, 2024: p. 100021-100021.\u003c/li\u003e\n\u003cli\u003eAjoolabady, A., et al., \u003cem\u003eMitophagy in cardiovascular diseases: molecular mechanisms, pathogenesis, and treatment.\u003c/em\u003e Trends in molecular medicine, 2022. \u003cstrong\u003e28\u003c/strong\u003e(10): p. 836-849.\u003c/li\u003e\n\u003cli\u003eZhang, H., et al., \u003cem\u003eAging and Autophagy: Roles in Musculoskeletal System Injury.\u003c/em\u003e Aging and Disease, 2025. \u003cstrong\u003e16\u003c/strong\u003e(3): p. 2.\u003c/li\u003e\n\u003cli\u003eZhang, K., et al., \u003cem\u003eTargeting Mitophagy as a Potential Therapeutic Approach for Age‐Related Bone Diseases.\u003c/em\u003e Advanced Therapeutics, 2024. \u003cstrong\u003e7\u003c/strong\u003e(7): p. 2400078.\u003c/li\u003e\n\u003cli\u003eZhao, J., et al., \u003cem\u003eNew insights into the interplay between autophagy, gut microbiota and insulin resistance in metabolic syndrome.\u003c/em\u003e Biomedicine \u0026amp; Pharmacotherapy, 2024. \u003cstrong\u003e176\u003c/strong\u003e: p. 116807.\u003c/li\u003e\n\u003cli\u003eWu, H., et al., \u003cem\u003eThe upregulation of Annexin A2 by TLR4 pathway facilitates lipid accumulation and liver injury via blocking AMPK/mTOR-mediated autophagy flux during the development of non-alcoholic fatty liver disease.\u003c/em\u003e Hepatology international, 2024. \u003cstrong\u003e18\u003c/strong\u003e(4): p. 1144-1157.\u003c/li\u003e\n\u003cli\u003eZhang, W., et al., \u003cem\u003eTMX2 potentiates cell viability of hepatocellular carcinoma by promoting autophagy and mitophagy.\u003c/em\u003e Autophagy, 2024. \u003cstrong\u003e20\u003c/strong\u003e(10): p. 2146-2163.\u003c/li\u003e\n\u003cli\u003eForte, M., et al., \u003cem\u003eMitophagy modulation for the treatment of cardiovascular diseases.\u003c/em\u003e European Journal of Clinical Investigation, 2024. \u003cstrong\u003e54\u003c/strong\u003e(8): p. e14199.\u003c/li\u003e\n\u003cli\u003eMa, Y., et al., \u003cem\u003eMitophagy in hypertension-mediated organ damage.\u003c/em\u003e Frontiers in cardiovascular medicine, 2024. \u003cstrong\u003e10\u003c/strong\u003e: p. 1309863.\u003c/li\u003e\n\u003cli\u003eLjubojević-Holzer, S., et al., \u003cem\u003eLoss of autophagy protein ATG5 impairs cardiac capacity in mice and humans through diminishing mitochondrial abundance and disrupting Ca2+ cycling.\u003c/em\u003e Cardiovascular research, 2022. \u003cstrong\u003e118\u003c/strong\u003e(6): p. 1492-1505.\u003c/li\u003e\n\u003cli\u003eYan, F. and L. Bao, \u003cem\u003eThe Role of Mitophagy in Cardiac Metabolic Remodeling of Heart Failure: Insights of Molecular Mechanisms and Therapeutic Prospects.\u003c/em\u003e Journal of Cardiovascular Translational Research, 2025: p. 1-13.\u003c/li\u003e\n\u003cli\u003eLi, J., et al., \u003cem\u003eMeteorin-like (METRNL) attenuates hypertensive induced cardiac hypertrophy by inhibiting autophagy via activating BRCA2.\u003c/em\u003e Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 2024. \u003cstrong\u003e1870\u003c/strong\u003e(4): p. 167113.\u003c/li\u003e\n\u003cli\u003eSagris, M., et al., \u003cem\u003eMyocardial ischemia\u0026ndash;reperfusion injury: unraveling pathophysiology, clinical manifestations, and emerging prevention strategies.\u003c/em\u003e Biomedicines, 2024. \u003cstrong\u003e12\u003c/strong\u003e(4): p. 802.\u003c/li\u003e\n\u003cli\u003eAo-Di, F., et al., \u003cem\u003eAdvances in macrophage metabolic reprogramming in myocardial ischemia-reperfusion.\u003c/em\u003e Cellular Signalling, 2024: p. 111370.\u003c/li\u003e\n\u003cli\u003eKarunarathna, I., et al., \u003cem\u003eAdvances in the Understanding and Management of Lactic Acidosis\u003c/em\u003e. 2024, ResearchGate. Available at https://www. researchgate. net/publication/383476212.\u003c/li\u003e\n\u003cli\u003eEndlicher, R., et al., \u003cem\u003eThe mitochondrial permeability transition pore\u0026mdash;current knowledge of its structure, function, and regulation, and optimized methods for evaluating its functional state.\u003c/em\u003e Cells, 2023. \u003cstrong\u003e12\u003c/strong\u003e(9): p. 1273.\u003c/li\u003e\n\u003cli\u003eBaev, A.Y., et al., \u003cem\u003eMitochondrial permeability transition, cell death and neurodegeneration.\u003c/em\u003e Cells, 2024. \u003cstrong\u003e13\u003c/strong\u003e(7): p. 648.\u003c/li\u003e\n\u003cli\u003eJhuo, S.J., et al., \u003cem\u003eSodium Glucose Cotransporter 2 (SGLT2) Inhibitor Ameliorate Metabolic Disorder and Obesity Induced Cardiomyocyte Injury and Mitochondrial Remodeling.\u003c/em\u003e Int J Mol Sci, 2023. \u003cstrong\u003e24\u003c/strong\u003e(7).\u003c/li\u003e\n\u003cli\u003eAgyapong, E.D., et al., \u003cem\u003eCalcium signaling from sarcoplasmic reticulum and mitochondria contact sites in acute myocardial infarction.\u003c/em\u003e Journal of Translational Medicine, 2024. \u003cstrong\u003e22\u003c/strong\u003e(1): p. 552.\u003c/li\u003e\n\u003cli\u003eKowalczyk, P., et al., \u003cem\u003eEffect of Oxidative Stress on Mitochondrial Damage and Repair in Heart Disease and Ischemic Events.\u003c/em\u003e International Journal of Molecular Sciences, 2024. \u003cstrong\u003e25\u003c/strong\u003e(22): p. 12467.\u003c/li\u003e\n\u003cli\u003ePeng, Y., et al., \u003cem\u003eCrosstalk among reactive oxygen species, autophagy and metabolism in myocardial ischemia and reperfusion stages.\u003c/em\u003e Aging and disease, 2024. \u003cstrong\u003e15\u003c/strong\u003e(3): p. 1075.\u003c/li\u003e\n\u003cli\u003eWang, K., et al., \u003cem\u003eProtective effects of tetramethylpyrazine on myocardial ischemia/reperfusion injury involve NLRP3 inflammasome suppression by autophagy activation.\u003c/em\u003e Biochemical Pharmacology, 2024. \u003cstrong\u003e229\u003c/strong\u003e: p. 116541.\u003c/li\u003e\n\u003cli\u003eSheng, Z., et al., \u003cem\u003eIdentification of mitophagy-related key genes and their correlation with immune cell infiltration in acute myocardial infarction via bioinformatics analysis.\u003c/em\u003e Frontiers in Cardiovascular Medicine, 2025. \u003cstrong\u003e11\u003c/strong\u003e: p. 1501608.\u003c/li\u003e\n\u003cli\u003eZhao, N., et al., \u003cem\u003ePterostilbene attenuates oxidative stress induced by hydrogen peroxide in MAC-T cells through activating PINK1/Parkin-mediated mitophagy.\u003c/em\u003e Italian Journal of Animal Science, 2024. \u003cstrong\u003e23\u003c/strong\u003e(1): p. 758-768.\u003c/li\u003e\n\u003cli\u003eGan, L., et al., \u003cem\u003eThe biology of exosomes and exosomal non-coding RNAs in cardiovascular diseases.\u003c/em\u003e Frontiers in Pharmacology, 2025. \u003cstrong\u003e16\u003c/strong\u003e: p. 1529375.\u003c/li\u003e\n\u003cli\u003eZhu, Z., et al., \u003cem\u003eTHSWD upregulates the LTF/AMPK/mTOR/Becn1 axis and promotes lysosomal autophagy in hepatocellular carcinoma cells by regulating gut flora and metabolic reprogramming.\u003c/em\u003e International Immunopharmacology, 2025. \u003cstrong\u003e148\u003c/strong\u003e: p. 114091.\u003c/li\u003e\n\u003cli\u003eZamanian, M.Y., et al., \u003cem\u003eEffects of metformin on cancers in experimental and clinical studies: Focusing on autophagy and AMPK/mTOR signaling pathways.\u003c/em\u003e Cell Biochemistry and Function, 2024. \u003cstrong\u003e42\u003c/strong\u003e(4): p. e4071.\u003c/li\u003e\n\u003cli\u003eBi, D., et al., \u003cem\u003eThe regulatory mechanisms of mitophagy and oxidative stress in androgenetic alopecia.\u003c/em\u003e Cellular Signalling, 2025: p. 111862.\u003c/li\u003e\n\u003cli\u003eHorng, C.-T., et al., \u003cem\u003eKoelreuteria Formosana extract induces growth inhibition and cell death in human Colon carcinoma cells via G2/M arrest and LC3-II activation-dependent autophagy.\u003c/em\u003e Nutrition and Cancer, 2017. \u003cstrong\u003e69\u003c/strong\u003e(1): p. 44-55.\u003c/li\u003e\n\u003cli\u003eBox, A.H., S.-M. Kim, and D.J. Demetrick, \u003cem\u003eAKT loss in human epithelial cells treated with severe hypoxia.\u003c/em\u003e Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 2010. \u003cstrong\u003e1803\u003c/strong\u003e(8): p. 951-959.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"clinical-and-experimental-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"clem","sideBox":"Learn more about [Clinical and Experimental Medicine](https://www.springer.com/journal/10238)","snPcode":"10238","submissionUrl":"https://submission.nature.com/new-submission/10238/3","title":"Clinical and Experimental Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"SGLT2 inhibitor; dapagliflozin, AMI, SIRT; AMPK, Autophagy, Mitophagy","lastPublishedDoi":"10.21203/rs.3.rs-9239009/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9239009/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAcute myocardial infarction (AMI) remains a leading cause of mortality worldwide, with cardiomyocyte death under hypoxic stress being a critical pathogenic mechanism. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) demonstrate cardiovascular benefits beyond glycemic control, yet their protective mechanisms in ischemic injury remain incompletely understood.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis study investigated dapagliflozin\u0026rsquo;s effects on human cardiomyocytes derived from induced pluripotent stem cell (iPSC-CMs) subjected to hypoxic stress. iPSC-CMs were applied to hypoxia with or without dapagliflozin treatment, followed by RNA-sequencing, qPCR validation, Western blotting, immunofluorescence, flow cytometry, and autophagy flux assays. Seahorse energy analyzer was used for cardiomyocyte metabolism analysis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eRNA-sequencing revealed that dapagliflozin reversed hypoxia-induced transcriptomic alterations, particularly in 66 genes associated with mitochondrial dysfunction. Dapagliflozin activated the AMPK/SIRT1/SIRT3 signaling axis, upregulated autophagy/mitophagy markers (LC3B, BECN1, Parkin). Seahorse cardiomyocyte metabolism analysis results demonstrated that dapagliflozin improved iCM mitochondrial function by restoring mitochondrial membrane potential, mitophagy capability and reduced hypoxia-related apoptosis. Autophagy inhibition with bafilomycin A1 abolished cardioprotection, confirming autophagy-dependent mechanisms.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings suggest that dapagliflozin protects cardiomyocytes through enhanced SIRT1/3-associated mitochondrial quality control and adaptive autophagy, providing mechanistic evidence supporting SGLT2 inhibitors' therapeutic potential in mitigating myocardial ischemic injury.\u003c/p\u003e","manuscriptTitle":"Protective mitophagy in human hypoxic cardiomyocytes: mechanistic insights into SGLT2 inhibitor Dapagliflozin cardioprotection in ischemic injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-01 19:44:35","doi":"10.21203/rs.3.rs-9239009/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-30T06:30:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-25T00:33:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-12T01:50:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-07T01:43:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"337726994899853864894391185941122983710","date":"2026-04-03T01:58:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"111560602813403956940489750380482150840","date":"2026-04-02T02:03:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"333809153309066133659544763562461037980","date":"2026-04-01T12:33:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-30T06:36:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-27T10:00:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-27T10:00:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical and Experimental Medicine","date":"2026-03-27T01:50:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"clinical-and-experimental-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"clem","sideBox":"Learn more about [Clinical and Experimental Medicine](https://www.springer.com/journal/10238)","snPcode":"10238","submissionUrl":"https://submission.nature.com/new-submission/10238/3","title":"Clinical and Experimental Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6fc4fee9-b5f4-46c4-9a48-7e349cf2d03f","owner":[],"postedDate":"April 1st, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-04-30T06:30:01+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-30T06:39:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-01 19:44:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9239009","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9239009","identity":"rs-9239009","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}