Dapagliflozin enhances gut barrier function in rats with chronic myocardial infarction by modulating gut microbiota balance

Introduction

Chronic myocardial infarction (CMI) remains a leading cause of morbidity and mortality worldwide, driven by systemic inflammation, metabolic disturbances, and cardiac dysfunction that ultimately leads to heart failure (1,2). CMI has been reported to not only worsen cardiovascular outcomes but also promote gut microbiota dysbiosis and gut barrier dysfunction through systemic inflammation and oxidative stress-induced apoptosis, highlighting a crucial role for the gut-heart axis (3,4). CMI is associated with chronic gut hypoperfusion, which impairs intestinal barrier integrity and increases permeability, potentially leading to the translocation of bacterial products into systemic circulation (5,6). For instance, patients with CMI exhibit gut dysbiosis accompanied by altered serum, urinary, and fecal metabolite profiles (7). Similarly, mice with CMI demonstrate gut dysbiosis and reduced levels of short-chain fatty acids (SCFAs), which negatively affect immune responses and exacerbate cardiac dysfunction (8). Notably, probiotic treatment in rat models of CMI-induced gut dysbiosis has been shown to reduce infarct size, further supporting the role of gut microbiota in cardiovascular health (9). Emerging preclinical approaches—such as fecal microbiota transplantation (FMT) and probiotic/prebiotic interventions—are being investigated in myocardial infarction (MI)/CMI models, in which the study has shown improvements in gut barrier integrity and cardiac outcomes in a mouse model (10).

Dapagliflozin, a sodium-glucose cotransporter-2 (SGLT2) inhibitor, has shown cardioprotective effects beyond glycemic control in patients with heart failure and other cardiovascular diseases (11,12). Both clinical and preclinical studies indicate that dapagliflozin enhances cardiac function by mitigating oxidative stress, inflammation, fibrosis, and vascular dysfunction, which are key contributors to the pathology of CMI (13,14). Emerging evidence also indicates that dapagliflozin benefits intestinal function; in a rat model of myocardial ischemia-reperfusion injury, it modulates gut dysbiosis and reduces trimethylamine-N-oxide (TMAO) levels, a metabolite implicated in myocardial tissue damage (15). TMAO has been implicated in cardiovascular risk by promoting oxidative stress, endothelial dysfunction, and platelet aggregation, thereby contributing to atherosclerosis (16). In mice with heart failure, dapagliflozin reduces inflammation, myocardial infarct size, and cardiac fibrosis while alleviating gut dysbiosis (17). Additionally, dapagliflozin has been shown to enhance gut barrier integrity by increasing tight junction protein (TJP) expression and reducing intestinal permeability, potentially mitigating systemic inflammation (18). However, the intestinal-tract dimension of dapagliflozin after CMI—and its potential contribution to the gut-heart axis—remains underexplored. We therefore selected dapagliflozin for its well-characterized rodent dosing/safety profile, high SGLT2 selectivity, and translational relevance.

CMI is characterized by persistent low-grade inflammation, prolonged fibrosis, and progressive metabolic dysfunction (19). These alterations may be exacerbated by persistent gut dysbiosis and increased intestinal permeability, perpetuating a cycle of systemic inflammation that further impairs cardiac function. Therefore, investigating how dapagliflozin modulates gut microbiota, metabolic pathways, and inflammatory responses in this context is essential for understanding its therapeutic potential in CMI. In this study, we aimed to investigate the effects of dapagliflozin on the heart, gut microbiota composition, gut barrier function, metabolic pathways, and inflammation in CMI rats. We hypothesized that dapagliflozin alleviates CMI-induced gut dysbiosis, enhances gut barrier integrity, modulates SCFA and TMAO metabolism, and ultimately reduces systemic inflammation and cardiovascular risk. These findings may provide new insights into gut microbiota-mediated mechanisms underlying the cardioprotective effects of dapagliflozin. We present this article in accordance with the ARRIVE reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-25-132/rc).

Methods

Animal preparation

Adult male Wistar rats (350–400 g) were obtained from Nomura Siam International Co., Ltd., Bangkok, Thailand. Experiments were performed under a project license (No. 2566/RT-0006.C2) granted by the Chiang Mai University Animal Care and Use Committee and the Laboratory Animal Center, Office of Research Administration, Chiang Mai University, in compliance with institutional and national guidelines for the care and use of laboratory animals, and the Guide for the Care and Use of Laboratory Animals. All procedures were conducted at the Laboratory Animal Center, Chiang Mai University.

Experimental protocol

A total of 39 male Wistar rats were allocated into two major groups. The sham-operated group (n=13) served as non-MI controls, while the CMI group (n=26) underwent ligation of the left anterior descending (LAD) coronary artery under general anesthesia. Sham-operated controls underwent identical procedures (anesthesia, intubation/ventilation, thoracotomy, and cardiac exposure) without LAD ligation to control for surgery- and anesthesia-related effects. Prior to the surgical procedure, anesthesia was induced via intraperitoneal (i.p.) injection of Xylazine HCl (5 mg/kg; LBS Laboratory Ltd., Part, Bangkok, Thailand) and Zoletil^® (50 mg/kg; Virbac Laboratories, Carros, France) to ensure adequate depth and muscle relaxation. The same anesthetic regimen was used for both sham and CMI procedures, and all outcome measurements were obtained at least 1 week after surgery to avoid transient anesthetic effects. Following induction, all animals were intubated and ventilated with oxygen-enriched air. Anesthesia was maintained with 2% isoflurane (Piramal Critical Care, Bethlehem, PA, USA). Thoracotomy was performed at the fourth intercostal space, and the LAD was permanently ligated approximately 2–3 mm from its origin using 5-0 sterile suture material. Effective occlusion was verified by ST-segment elevation and visible cyanotic discoloration of the anterior left ventricular wall. On postoperative day 8, echocardiography confirmed anterior wall akinesia and a reduction in left ventricular ejection fraction (LVEF). Rats with LVEF >50% or no clear signs of MI were excluded. The confirmed MI rats were then randomly divided into two treatment subgroups (n=13 per group): the vehicle (distilled water) group and the dapagliflozin (1 mg/kg/day, oral) group. Distilled water or dapagliflozin (1 mg/kg) was administered once daily by oral gavage for 70 days to the respective treatment groups. The selected dose (1 mg/kg/day) is widely used in rodent studies in which that dose is equivalent to the human therapeutic dose (~10 mg/day) by standard allometric scaling (13,20). A protocol was prepared before the study without registration. The study protocol is illustrated in the flow diagram shown in Figure 1.

Figure 1 Summary of the experimental protocol. DAPA, dapagliflozin-treated group; MI, myocardial infarction; SHAM, sham-operated control group; VEH, vehicle-treated MI group.

Assessment of cardiac function

LVEF, fractional shortening (FS), and the early to late transmitral inflow (E/A) ratio were measured to assess cardiac function. Briefly, transthoracic echocardiography was performed using a high-frequency ultrasound system (Philips, Amsterdam, Netherlands). LVEF and FS were derived from M-mode images obtained at the level of the left ventricle, while the E/A ratio was calculated from Doppler-derived mitral inflow velocities. These parameters were analyzed to evaluate systolic function (LVEF and FS) and diastolic function (E/A ratio) in each rat.

Colon tissue staining

Colon tissue was fixed in 10% neutral-buffered formalin for 24 hours. After fixation, the samples were dehydrated in a graded ethanol series (75%, 90%, 95%, and three changes of 100%, each for 1 hour), followed by clearing in xylene (three cycles of 30 minutes each). The tissues were then embedded in paraffin by immersing them in two paraffin baths for 1 hour each, followed by a final embedding in a third paraffin bath. Paraffin-embedded samples were sectioned at 5 µm using a microtome (RM2125 RTS, Leica, Wetzler, Germany) and stained with hematoxylin (Sigma, St. Louis, MO, USA) and eosin (Merck, Darmstadt, Germany) for microscopic evaluation. A blinded histological assessment was conducted by researchers and an experienced pathologist.

Western blot analysis

Colon tissues were homogenized using a probe sonicator to extract total protein lysates. Protein concentrations were determined with a colorimetric assay kit (Bio-Rad Laboratories, Berkeley, CA, USA). Equal amounts of protein (20 µg per lane) were resolved by SDS-PAGE and transferred onto a nitrocellulose membrane. Membranes were blocked with 5% non-fat milk for 1 hour at room temperature, followed by overnight incubation at 4 ℃ with a primary antibody diluted 1:1,000. The primary antibodies included actin (sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA), Bax (2772, Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (ab196495, Abcam, Cambridge, UK), caspase-3 (14220, Cell Signaling Technology), claudin-5 (ab15106, Abcam), cleaved-caspase-3 (9661, Cell Signaling Technology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab181602, Abcam), interleukin (IL)-1β (PA5-88078, Thermo Fisher Scientific), IL-6 (ab9324, Abcam), phospho-nuclear factor-κB (p-NF-κB) (3033, Cell Signaling Technology), NF-κB (8242, Cell Signaling Technology), occludin (sc-5562*2, Santa Cruz Biotechnology), and tumor necrosis factor-α (TNF-α) (ab205587, Abcam). After washing, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:1,000; Cell Signaling Technology) for 1 hour at room temperature. Protein-antibody complexes were visualized using a chemiluminescent substrate (Bio-Rad Laboratories), and signal detection was performed on the ChemiDoc Touch system (Bio-Rad Laboratories). Densitometric quantification of the resulting protein bands was carried out using ImageJ software (NIH, Bethesda, MD, USA).

Quantification of plasma SCFAs and TMAO levels

Gas chromatography-mass spectrometry (GC-MS; Agilent Technologies, Santa Clara, CA, USA) was used to quantify plasma SCFAs, including acetic, propionic, butyric, and valeric acids. Plasma TMAO levels were analyzed using liquid chromatography with quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). SCFA and TMAO concentrations were calculated against a standard mixture (Restek, Bellefonte, PA, USA) using MassHunter Quantitative Analysis Software (version 10.1, Agilent Technologies). To correct for batch effects, data normalization was performed using the MetaboDrift software.

Gut microbiome analysis

Bacterial genomic DNA was extracted from 0.25 g of colonic content using the QIAamp PowerFecal Pro DNA Kit (Qiagen, Hilden, Germany), following the manufacturer’s protocol. Amplification of the V3–V4 hypervariable region of the bacterial 16S ribosomal RNA (rRNA) gene was performed using primers 341F and 805R. 16S rRNA libraries were prepared from the resulting amplicons and sequenced using paired-end reads on the Illumina NovaSeq 6000 platform (Novogene, Singapore, Singapore). Microbiome sequencing data were processed using Quantitative Insights into Microbial Ecology 2 (QIIME 2) version 2023.5 as previously described, with a rarefaction depth of 62,915 reads per sample applied prior to diversity analyses. Alpha diversity indices and beta diversity metrics were calculated and visualized following standard procedures. Taxonomic assignment was performed against the SILVA 138 database, and functional predictions were generated with Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2 (PICRUSt2) using MetaCyc annotations.

Statistical analysis

Experimental data were reported as either the median with interquartile range (IQR) or the mean ± standard error of the mean (SEM). For multiple comparisons, the Kruskal-Wallis test followed by Dunn’s test was used for nonparametric data. One-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD) test was applied for parametric data. Statistical significance was defined as P value below 0.05. All statistical analyses and data visualizations were performed using RStudio (Posit Software, Boston, MA, USA) with R version 4.2.3.

Results

Dapagliflozin improved cardiac and gut barrier function in CMI rats

Cardiac function was evaluated by echocardiography at the end of the 10-week treatment period. The vehicle group exhibited cardiac dysfunction, as indicated by a reduction in LVEF, FS, and E/A ratio compared to the sham group (Figure 2). Dapagliflozin treatment significantly improved these cardiac parameters; however, they were not fully restored to sham levels (Figure 2).

Figure 2 Dapagliflozin improves cardiac function in MI rats. (A-C) Dapagliflozin treatment partially restores sympathetic tone in MI rats, as indicated by improved LVEF, FS, and E/A ratio compared to the VEH group. However, values remain lower than the SHAM group. *, P<0.05 vs. SHAM group; ^†, P<0.05 vs. VEH group. DAPA, dapagliflozin-treated group; E/A, early to late transmitral inflow; FS, fractional shortening; LVEF, left ventricular ejection fraction; MI, myocardial infarction; SHAM, sham-operated control group; VEH, vehicle-treated MI group.

To assess the effects of dapagliflozin on CMI-induced intestinal barrier dysfunction, intestinal tissues were analyzed 10 weeks post-MI. Intestinal morphology in the vehicle group showed epithelial damage and crypt loss (Figure 3A). Occludin and claudin-5 are the TJPs, essential for maintaining intestinal barrier integrity (21,22). To evaluate the effect of dapagliflozin on TJP expression, their levels were measured in intestinal tissues. Occludin and claudin-5 levels were significantly decreased in the vehicle group compared to the sham group. However, TJP expression was improved only in the dapagliflozin-treated group (Figure 3B-3D, Appendix 1), suggesting its role in restoring gut barrier integrity.

Figure 3 Dapagliflozin ameliorates intestinal injury and restores TJP in MI rats. (A) Representative H&E images (100× magnification) reveal crypt loss and epithelial damage in MI rats (VEH group), while dapagliflozin treatment preserves intestinal structure. (B-D) Dapagliflozin enhances TJPs (occludin and claudin-5) expression, mitigating MI-induced gut barrier disruption. *, P<0.05 vs. SHAM group; ^†, P<0.05 vs. VEH group. DAPA, dapagliflozin-treated group; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H&E, hematoxylin and eosin; MI, myocardial infarction; SHAM, sham-operated control group; TJP, tight junction protein; VEH, vehicle-treated MI group.

Dapagliflozin ameliorated colonic inflammation and apoptosis in CMI rats

Colonic expression of inflammation- and apoptosis-related proteins was evaluated. Levels of inflammatory markers, including IL-1β, IL-6, TNF-α, and phosphorylated NF-κB/NF-κB ratio, were significantly elevated in the vehicle group compared to the sham group. Dapagliflozin treatment significantly reduced the expression levels of IL-1β, IL-6, and phosphorylated NF-κB/NF-κB compared to the vehicle group (Figure 4A-4D, Appendix 2). The levels of apoptosis-related proteins, including cleaved caspase-3 and the Bax/Bcl-2 ratio, were significantly increased in the vehicle group compared to the sham group (Figure 4E-4G, Appendix 2). However, dapagliflozin treatment significantly reduced the expression of cleaved caspase-3 but not the Bax/Bcl-2 ratio, indicating a partial attenuation of CMI-induced colonic apoptosis.

Figure 4 Dapagliflozin reduces inflammation and apoptosis in MI rats. (A-D) DAPA group significantly reduced inflammatory markers (IL-1β, IL-6, and phospho-NF-kB) compared to the VEH group. (E,F) Dapagliflozin also decreased the apoptotic marker (cleaved caspase-3) compared to the VEH group. (G) Representative Western blot bands of inflammatory and apoptosis markers. *, P<0.05 vs. SHAM group; ^†, P<0.05 vs. VEH group. DAPA, dapagliflozin-treated group; IL, interleukin; MI, myocardial infarction; NF, nuclear factor; SHAM, sham-operated control group; TNF-α, tumor necrosis factor-α; VEH, vehicle-treated MI group.

Dapagliflozin improved gut dysbiosis in CMI rats

To evaluate the effects of dapagliflozin on MI-induced gut dysbiosis, we analyzed microbial composition, diversity, and differentially abundant taxa in CMI rats. Genus-level taxonomy bar plots were generated to illustrate the relative abundance of gut microbiota in each sample (Figure 5A). α-diversity, measured by Faith’s phylogenetic diversity, was significantly higher in the dapagliflozin group than in both the Sham and Vehicle groups (Figure 5B,5C). In contrast, the vehicle group exhibited gut dysbiosis, as evidenced by a significant shift in β-diversity (Figure 5D,5E). The vehicle group showed a significant increase in Holdemania, with reductions in Tyzzerella and Papillibacter relative to the sham group (Figure 6, Table S1). The dapagliflozin group exhibited a distinct shift in gut microbiota composition relative to the vehicle group. This shift was characterized by a significant increase in UCG-007, Peptococcaceae_uncultured, Bilophila, and Bacillus, alongside significant decreases in Gordonibacter, Faecalibaculum, Dorea, Defluvitaleaceae_uncultured, Christensenellaceae_uncultured, Clostridia_vadinBB60_group, and Candidatus_Soleaferrea (Figure 6).

Figure 5 The effects of dapagliflozin on gut microbiome composition and diversity in MI rats. (A) Taxonomy bar plots showing the relative abundance of gut microbiota at the genus level in each sample. (B,C) Alpha diversity indices, including Faith’s PD, were significantly increased in the DAPA group compared to the SHAM and VEH groups. (D,E) Beta diversity analysis demonstrated significant differences in the gut microbiota among the SHAM, VEH, and DAPA groups. *, P<0.05 vs. SHAM group; ^†, P<0.05 vs. VEH group. DAPA, dapagliflozin-treated group; MI, myocardial infarction; PC, principal component; PD, phylogenetic diversity; PERMANOVA, permutational multivariate analysis of variance; SHAM, sham-operated control group; VEH, vehicle-treated MI group.

Figure 6 Differential abundance analysis of gut microbiota composition between groups. DAPA, dapagliflozin-treated group; MI, myocardial infarction; SHAM, sham-operated control group; VEH, vehicle-treated MI group.

Dapagliflozin improved plasma SCFA and TMAO levels in CMI rats

MI is associated with alterations in plasma SCFA and TMAO levels. The plasma concentrations of SCFAs-including acetic acid, butyric acid, propionic acid, and valeric acid-as well as TMAO were analyzed. In the vehicle group, acetic acid levels were significantly increased, while butyric acid levels were significantly decreased compared to the sham group. Dapagliflozin treatment effectively normalized both acetic and butyric acid concentrations to sham levels (Figure 7A-7D), suggesting an enhancement of microbial SCFA production and restoration of gut metabolic homeostasis. Similarly, plasma TMAO levels were significantly increased in the vehicle group compared to the sham group. Dapagliflozin treatment normalized TMAO concentrations to sham group levels (Figure 7E), suggesting its role in modulating gut microbial metabolism to suppress TMAO synthesis.

Figure 7 Effect of dapagliflozin on plasma SCFAs and TMAO in MI rats. (A-D) DAPA group significantly decreased plasma acetic acid levels compared to the VEH group, while significantly increasing plasma butyric acid levels. (E) DAPA group significantly decreased plasma TMAO levels compared to the VEH group. *, P<0.05 vs. SHAM group; ^†, P<0.05 vs. VEH group. DAPA, dapagliflozin-treated group; MI, myocardial infarction; SCFAs, short-chain fatty acids; SHAM, sham-operated control group; TMAO, trimethylamine N-oxide; VEH, vehicle-treated MI group.

Correlation between differentially abundant taxa and host parameters in dapagliflozin-treated CMI rats

To explore the relationship between gut microbiota alterations and dapagliflozin treatment in CMI rats, we conducted a correlation analysis between differentially abundant taxa and key physiological and biochemical parameters. SCFA levels and TJP expression were positively associated with UCG-007 and Candidatus_Soleaferrea but negatively associated with Bilophila, Christensenellaceae_uncultured, Clostridia_vadinBB60_group, Faecalibaculum, Dorea, and Defluvitaleaceae_uncultured. In addition, echocardiographic cardiac function parameters were positively associated with UCG-007 abundance (Figure 8A). These findings suggest that specific gut microbial taxa may contribute to the physiological and biochemical improvements observed in dapagliflozin-treated CMI rats.

Figure 8 Correlation between gut microbiota, biochemical parameters, and metabolic pathway alterations in dapagliflozin-treated rats. (A) Correlation heatmap of differentially abundant taxa and biochemical parameters. Spearman correlation analysis was performed. *, indicates significant correlations at P<0.05. (B) PICRUSt2 analysis shows significantly altered metabolic pathways in DAPA group compared to VEH group. The analysis highlights metabolic pathways are significantly associated with the regulation of SCFAs and TMAO. DAPA, dapagliflozin-treated group; E/A, early to late transmitral inflow; FS, fractional shortening; IL, interleukin; LVEF, left ventricular ejection fraction; MI, myocardial infarction; NF, nuclear factor; PICRUSt2, Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2; SCFAs, short-chain fatty acids; SHAM, sham-operated control group; TMAO, trimethylamine N-oxide; VEH, vehicle-treated MI group.

Alterations of metabolic pathways in dapagliflozin-treated CMI rats

To investigate the impact of dapagliflozin on gut microbial metabolic pathways in CMI rats, PICRUSt2 was employed to predict functional alterations in microbiota-associated metabolism. Significant alterations in key metabolic pathways were observed in the dapagliflozin-treated group compared to the vehicle group. Specifically, pathways involved in SCFA biosynthesis and lipid metabolism were upregulated, suggesting potential benefits for gut barrier function and modulation of inflammation (Figure 8B, Table S2). Conversely, pro-inflammatory signaling was downregulated, suggesting a potential role for dapagliflozin in mitigating metabolic inflammation and cardiovascular risk. These findings suggest that dapagliflozin modulates gut microbial metabolism, potentially contributing to its cardioprotective and anti-inflammatory effects in CMI rats.

Discussion

In this study, we demonstrated that dapagliflozin administration in CMI rats improved cardiac function, preserved gut barrier integrity, reduced colonic inflammation and apoptosis, modulated gut microbiota composition, and altered plasma SCFAs and TMAO levels. These findings are consistent with involvement of the gut-heart axis in CMI rats treated with dapagliflozin. The gut-heart axis describes the bidirectional interaction between gut microbiota, intestinal barrier function, and cardiovascular physiology. In patients with MI, chronic gut hypoperfusion can lead to impaired motility and, in severe cases, paralytic ileus (gastrointestinal paralysis), highlighting how cardiac dysfunction can propagate intestinal injury (3,23). Notably, dapagliflozin enhanced the expression of TJPs. However, these gut-related effects should be considered as one of the contributory factors rather than representing the primary mechanism of SGLT2 inhibitor-mediated cardioprotection, which is predominantly attributed to direct cardiac, renal, and metabolic actions.

TJPs, such as occludin and claudin-5, play an essential role in maintaining gut barrier integrity by regulating paracellular permeability and limiting the translocation of luminal antigens, endotoxins, and pathogenic bacteria into systemic circulation (24). Disruption of these proteins is associated with increased intestinal permeability, commonly known as “leaky gut”, which can subsequently trigger systemic inflammation and exacerbate cardiovascular dysfunction (25,26). In the present study, CMI significantly decreased the expression of occludin and claudin-5 in the intestinal epithelium, which was indicative of gut barrier dysfunction. This observation is consistent with clinical reports showing that gut hypoperfusion and ischemia in patients induce oxidative stress and the release of inflammatory cytokines, ultimately contributing to the downregulation of TJP expression (27,28). Increased gut permeability caused by TJP disruption facilitates the translocation of bacterial products, such as lipopolysaccharide (LPS), into systemic circulation. This, in turn, activates inflammatory pathways and aggravates cardiac dysfunction through the gut-heart axis (29).

Concomitant with TJP disruption, a significant increase in intestinal epithelial apoptosis was observed in CMI rats, as indicated by elevated cleaved caspase-3 expression and an increased Bax/Bcl-2 ratio. Apoptosis plays a pivotal role in gut barrier impairment, as excessive epithelial cell death can result in villous atrophy, crypt loss, and further deterioration of intestinal integrity (30). These apoptotic processes are further exacerbated by pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, which activate death receptor signaling and promote caspase cascade activation. This establishes a vicious cycle in which inflammation induces apoptosis, while apoptosis further exacerbates gut permeability and systemic inflammation. Importantly, dapagliflozin treatment enhanced TJP expression in CMI rats, which likely contributed to reduced epithelial apoptosis and inflammation, indicating a protective mechanism. Consistent with our findings, previous studies demonstrated that dapagliflozin suppressed apoptosis and inhibited the HMGB1/RAGE/NF-κB signaling pathway in rats with colitis (31,32). Moreover, dapagliflozin significantly increased levels of butyric acid, a key microbiome-derived metabolite known for its anti-inflammatory properties and its role in maintaining gut barrier integrity.

Previous reports have shown that butyric acid exerts anti-inflammatory effects by inhibiting the NF-κB signaling pathway, reducing pro-inflammatory cytokine production, and promoting regulatory T cell differentiation (33,34). Additionally, butyrate strengthens the intestinal barrier by enhancing the expression of TJPs (including occludin and claudin family) and reducing epithelial apoptosis via inhibition of histone deacetylases (HDAC) (35,36). These properties may partly explain the observed improvements in gut TJP expression and attenuated intestinal inflammation in dapagliflozin-treated CMI rats.

In contrast, propionic acid levels remained unchanged in our study. However, previous studies have reported elevated propionate in CMI, which may exert deleterious effects through FFAR3-mediated signaling (37-39). Although dapagliflozin has been suggested to restore the dysregulated SCFA profiles, including lowering propionate levels in pathological states, the expression and activation status of cardiac SCFA receptors (FFAR2 and FFAR3) were not evaluated in our study and warrant further investigation.

In contrast, TMAO, another gut microbiota-derived metabolite, has been consistently associated with increased inflammation, endothelial dysfunction, and elevated cardiovascular risk in both preclinical and clinical studies (40-42). Elevated TMAO levels promote the activation of pro-inflammatory pathways, including the NF-κB and NLRP3 inflammasome, which exacerbate systemic inflammation and contribute to cardiovascular pathology (40). In this study, dapagliflozin treatment significantly reduced plasma TMAO levels, suggesting a potential mechanism by which it alleviates inflammation and improves cardiac outcomes. Collectively, these findings highlight the dual role of gut microbial metabolites: SCFAs providing protective effects, while TMAO contributes to disease progression. These results underscore the importance of gut microbiota modulation in mediating the cardioprotective benefits of dapagliflozin.

In this study, CMI-induced gut dysbiosis was characterized by an increase in Holdemania and a decrease in Tyzzerella and Papillibacter, while dapagliflozin treatment restored their abundance to levels comparable to controls, suggesting its role in reversing CMI-induced dysbiosis. A previous study reported that a decrease in Tyzzerella is associated with an increased cardiovascular risk, including acute MI (AMI) (43). Tyzzerella, an SCFA-producing bacterium, contributes to gut barrier function, and its depletion reduces SCFA availability, thereby prompting barrier dysfunction and systemic inflammation (44). Moreover, clinical studies have reported that an increase in Holdemania is associated with atrial fibrillation, potentially linked to impaired lipid metabolism (45,46). Dapagliflozin-treated CMI rats exhibited an increase in beneficial bacteria, including UCG-007 and Bacillus, relative to untreated CMI controls. UCG-007, a member of the Ruminococcaceae family, is a butyrate-producing bacterium that plays a crucial role in improving intestinal barrier integrity, reducing inflammation, and supporting heart function (47,48). Bacillus, a well-known probiotic genus, particularly B. subtilis and B. coagulans, contributes to gut microbiota balance, regulation of inflammatory cytokines, enhancing gut barrier function, and SCFA production (48). Additionally, Bacillus species have been shown to reduce cardiovascular disease risk factors by lowering cholesterol levels through the production of bile salt hydrolase (BSH) in both clinical and animal models (41,49,50). Furthermore, dapagliflozin-treated CMI rats exhibited a gut composition similar to that of healthy controls. Supporting our findings, a previous study in AMI rats treated with dapagliflozin (1.5 mg/kg/day for 28 days) also reported significant shifts in gut microbiota composition, including an increase in beneficial taxa such as Lactobacillus and Bifidobacterium, alongside enhanced TJP expression (51). This restoration may reflect a microbiota adaptation over time, as the acute-phase alterations observed in AMI may have already partially resolved by the chronic stage. Therefore, dapagliflozin-treated CMI rats exhibited a gut microbial profile more closely resembling that of healthy controls. These findings reinforce the notion that dapagliflozin treatment can reverse gut dysbiosis and improve both gut and heart function in MI models.

Moreover, predictive pathway analysis revealed upregulation of lipid metabolism (specifically oleate biosynthesis) and SCFA biosynthesis pathways, notably 1,5-anhydrofructose degradation and lactose and galactose degradation I, in dapagliflozin-treated CMI rats. Upregulation of oleate biosynthesis may be beneficial, as oleic acid (C18:1) exerts cardioprotective effects through attenuation of oxidative stress, inflammation, and dyslipidemia (52,53). Additionally, the enhancement of SCFA biosynthesis pathways aligns with observed increase in butyrate-producing bacteria, reinforcing the functional relevance of our microbial findings. Collectively, these metabolic shifts are consistent with the hypothesis that modulation of the gut microbiota may contribute to the cardioprotective effects observed in CMI rats treated with dapagliflozin.

SGLT2 inhibitors may confer gastrointestinal benefits in the context of cardiovascular disease; however, current evidence suggests these effects are preliminary and mechanistic insights (17,51,54). Accordingly, their clinical significance remains uncertain and is unlikely to alter treatment decisions at present. Nevertheless, emerging data support the plausibility of gut-cardiac interactions, warranting dedicated trials with prespecified gastrointestinal endpoints. Therefore, further studies are still required.

Despite its significant findings, this study has some limitations. First, although PICRUSt2-based predictions offer valuable insights into gut microbiota-associated metabolic pathways, they do not reflect actual metabolite concentrations. Second, only a single dapagliflozin dose (1 mg/kg/day) was tested; dose–response relationships were not evaluated and warrant further investigation. Third, although dapagliflozin modulated the gut microbiota in CMI rats, its mechanistic contribution to the gut-heart axis remains to be elucidated. Future studies should incorporate FMT or antibiotic-induced microbiota depletion models to establish causality. Fourth, the specific contributions of SCFAs, lipid metabolism, and other microbial pathways to the anti-inflammatory effects of dapagliflozin, as well as myocardial FFAR2/FFAR3 expression and downstream SCFA-mediated signaling—warrant further investigation using integrated multi-omics approaches. Lastly, given that this study was performed in a preclinical CMI model, clinical investigations are required to determine whether similar gut microbiota and metabolic alterations occur in humans. Further research, particularly in clinical settings, is necessary to validate these findings and evaluate the therapeutic potential of microbiota-targeted interventions in cardiovascular disease.

Conclusions

Taken together, our study provides new insights into the role of dapagliflozin in modulating the gut-heart axis in CMI. In this model, dapagliflozin treatment was associated with attenuated CMI-induced cardiac dysfunction and gut dysbiosis, improved gut barrier integrity, reduced intestinal apoptosis and inflammation, and changed microbially derived metabolites. Specifically, we observed an increase in SCFA-related biosynthesis pathways and beneficial taxa, alongside with the reduction of pro-inflammatory taxa and plasma TMAO levels, suggesting a shift toward a more balanced, cardioprotective gut microbiota profiles. These findings suggest a potential contribution of gut microbiota-mediated processes to the observed benefits. Further mechanistic studies are warranted.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://atm.amegroups.com/article/view/10.21037/atm-25-132/rc

Data Sharing Statement: Available at https://atm.amegroups.com/article/view/10.21037/atm-25-132/dss

Peer Review File: Available at https://atm.amegroups.com/article/view/10.21037/atm-25-132/prf

Funding: This work was supported by the Distinguished Research Professor Grant from the National Research Council of Thailand (No. N42A660301 to S.C.C.), the Chiang Mai University Centre of Excellence Award (to N.C.), and the CMU Proactive Researcher Program, Chiang Mai University (No. 560/2567 to C.K.).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-25-132/coif). C.K., N.C. and S.C.C. report that this work was supported by the Distinguished Research Professor Grant from the National Research Council of Thailand (No. N42A660301 to S.C.C.), the Chiang Mai University Centre of Excellence Award (to N.C.), and the CMU Proactive Researcher Program, Chiang Mai University (No. 560/2567 to C.K.). The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Experiments were performed under a project license (No. 2566/RT-0006.C2) granted by the Chiang Mai University Animal Care and Use Committee and the Laboratory Animal Center, Office of Research Administration, Chiang Mai University, in compliance with institutional and national guidelines for the care and use of laboratory animals, and the Guide for the Care and Use of Laboratory Animals.

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