Empagliflozin and intermittent fasting as a strategy to mitigate anthracycline-induced cardiotoxicity

Abstract

Empagliflozin (EMPA) has shown cardioprotective potential by enhancing myocardial energy availability, a mechanism also observed with intermittent fasting strategies, such as time-restricted feeding (TRF). Given the cardiotoxicity of anthracyclines as Doxorubicin (Dox), integrating pharmacological and nonpharmacological interventions is of growing interest in cardio-oncology. This study assessed EMPA, alone or with TRF, in mitigating Dox-induced cardiovascular damage using both an experimental model and a clinical case. Rats were assigned to control, Dox, Dox + EMPA, Dox + TRF, or Dox + EMPA + TRF groups. Treatments included Dox (12 mg/kg), EMPA (10 mg/kg), and TRF (16 h/8 h), for four weeks. In the clinical case, a cancer patient undergoing Dox therapy received EMPA (10 mg/day) followed by TRF for three months. Cardiovascular parameters were measured. In animals, Dox significantly increased systolic, diastolic, and mean arterial pressures, mitigated by EMPA or TRF individually, with no additive effect when combined. EMPA partially normalized Dox-induced P wave and QRS alterations, while EMPA, TRF, or both reduced QTc prolongation. Histology showed Dox-induced myocardial remodeling and inflammation, which were attenuated by all treatments, restoring cardiomyocyte occupancy, reducing extracellular matrix expansion, and decreasing inflammatory infiltrate. All co-treatments prevented Dox-related leukopenia, normalizing leukocyte counts. Mechanistically, EMPA and TRF modulated Dox-induced inflammation in distinct ways: both (alone or combined) lowered TNF, TRF alone produced the strongest IL-1β suppression, the combined treatment increased IL-10 and TGF-β significantly. Clinically, the patient experienced weight loss, stable blood pressure, and cardiovascular stability although a rise in troponin levels. In conclusion, individual EMPA and TRF may help mitigate Dox-induced cardiotoxicity in certain contexts, though further clinical studies are needed to confirm their safety.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-35790-1.

Introduction

Cardiovascular complications from cancer therapies are highly prevalent, making cardiotoxicity the second leading cause of morbidity and mortality among cancer survivors^1,2. Managing patients with both conditions effectively require specialized knowledge and expertise. The first reported cardiovascular complication of anticancer drugs was a decrease in the left ventricular ejection fraction, leading to heart failure (HF) in patients treated with anthracyclines³. Anthracyclines, such as Doxorubicin (Dox), are a class of antineoplastic drugs that are effective in treating lymphomas, leukemias, sarcomas, and breast cancer⁴. However, their clinical use is limited by cardiotoxicity, which can result in ventricular dysfunction and HF^5,6.

The cardiotoxic effects of Dox are highly variable, occurring in up to 50% of patients, depending on preexisting risk factors and drug-specific properties, such as the cumulative dose⁷. Nevertheless, any exposure to Dox carries a risk of inducing cardiac dysfunction⁵. Strategies to mitigate Dox-induced cardiac damage include dose reduction, slow infusion protocols, and specialized formulations such as liposomal preparations^5,6,8. Despite the rapid expansion of cardio-oncology, research in this field remains limited, with significant variability in findings^9,10. Further studies require innovative approaches and adaptive study designs to generate more robust evidence¹¹. The exploration of different pharmacological agents, in combination or not with or without nonpharmacological strategies, could offer a promising new approach for treating this type of cardiotoxicity.

In this matter, the use of inhibitors of sodium-glucose cotransporter 2 (iSGLT2), such as empagliflozin (EMPA), initially used to treat diabetes, has shown benefits to patients at high cardiovascular risk or with established cardiovascular diseases. The EMPA-REG OUTCOME trial demonstrated a 14% reduction in major adverse cardiovascular events and a 35% reduction in HF hospitalizations. These benefits have been consistently confirmed in subsequent clinical trials and meta-analyses, leading to iSGLT2 being recognized as a first-line treatment for diabetic patients with elevated cardiovascular risk and HF^12–15. Moreover, iSGLT2 have emerged as a promising strategy to improve cardiovascular outcomes across various diseases, and their use has been proposed to reduce cardiovascular mortality in cardio-oncology patients¹⁶.

Nonpharmacological adjuvant therapies could also offer promising options for patient treatment. For example, nutritional approaches can play a crucial role in preventing chemotherapy-induced cardiotoxicity, as a healthy diet al.one can reduce the risk of cardiovascular disease^17,18. Specifically, intermittent fasting like time-restricted feeding (TRF) has been shown to improve heart health^19,20. Studies indicate that TRF can improve insulin sensitivity, reduce inflammation, lower triglyceride levels, and decrease blood pressure, all of which contribute to cardiovascular disease prevention²⁰. Notably, this strategy is gaining popularity, with cancer patients increasingly seeking guidance from oncologists regarding its potential benefits for both prevention and treatment^21–23.

One mechanism by which iSGLT2 improve cardiac function is by enhancing myocardial energy availability through increased ketone body production; a pathway similarly stimulated by intermittent fasting²⁴. Although ketone generation does not fully explain the benefits of intermittent fasting, both fasting²⁴ and ketones themselves²⁵ have been proposed to confer cardiovascular protection, including reductions in inflammation and oxidative stress. Based on this, we hypothesized that combining iSGLT2 with TRF could potentiate their cardioprotective actions. This rationale aligns with emerging priorities in cardio-oncology, where patients treated with anthracyclines face substantial risk of cardiotoxicity and where metabolic interventions may be especially valuable, particularly in cancers such as breast, ovarian, and prostate, in which cachexia is uncommon but treatment-related weight gain is frequent^26,27. Thus, even if additive or synergistic effects between iSGLT2 use and fasting remain to be established, their individual benefits and preliminary safety data support further exploration. Establishing biological plausibility and safety constitutes a necessary first step before progressing to randomized clinical trials in this population.

Materials and methods

Study type, ethics approval and consent to participate

This study involved analyses of both animal models (approved by the Animal Use Ethics Committee, protocol no. 002.2023) and patient (approved by the Research Ethics Committee, CAAE: 69864823.6.0000.5134). All animal experiments were carried out following the legislation of the National Council for the Control of Animal Experimentation (CONCEA). This study adheres to internationally accepted standards for animal research, following the 3Rs principle. Additionally, the work was carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki). The study is reported in accordance with ARRIVE guidelines.

Animal model methodology

Female Wistar rats were randomly assigned to five groups: control, Dox, Dox + EMPA, Dox + TRF, and Dox + EMPA + TRF, n = 5 animals per group. Animals were allocated to experimental groups using a random number generator, and investigators conducting the experiments and analyses were blinded to group allocation through the use of independent codes to minimize potential bias. Dox was administered intraperitoneally at a dose of 12 mg/kg weekly as previously described²⁸, EMPA was given orally at 10 mg/kg daily, and TRF followed a 16 h/8 h protocol, with food provided at 8 PM to align with the nocturnal feeding patterns of the rats. Several studies have demonstrated that this fasting window in rodents can elicit metabolic adaptations similar to those observed in humans, including improved insulin sensitivity, reduced inflammatory markers, and enhanced cardiac resilience under stress or baseline conditions^29–32. Moreover, TRF, as we used in our study, is generally well tolerated in humans, with high adherence and limited discomfort caused by this fasting pattern³³. Water was available ad libitum. The treatment lasted for four weeks, after which cardiovascular outcomes were assessed. All animals were housed in institutional animal care facilities on a 12-hour light/dark cycle.

Morphometric parameters

Body weight was recorded weekly. After euthanasia by decapitation, the heart was excised and weighed, after which it was normalized to body weight and tibia length.

Hemodynamic parameters

Systolic, diastolic, and mean arterial pressures were measured by the noninvasive method of tail-cuff plethysmography (CODA, Kent Scientific Corporation, USA). A rubber cuff connected to a calibrated sphygmomanometer was inflated and deflated at 50-second intervals to allow for accurate pressure readings. The experimental rats underwent a 2-day adaptation period before the final measurement. At the end of the experimental protocol, arterial pressure values were averaged from three consecutive measurements taken per animal.

Electrocardiographic (ECG) parameters

For toxicity assessment, ECG monitoring and histological analysis were performed, as previously described for the evaluation of Dox-induced toxicity^34,35. The ECG recordings were acquired via ECG-PC version 2.07 (Brazilian Electronic Technology, TEB, Brazil). The electrodes were positioned on the manubrium, sternum, and both thoracic sides to obtain a comprehensive 12-lead ECG³⁶. Continuous ECG monitoring was performed under isoflurane anesthesia (maintenance dose: 1.5%) to ensure stable physiological conditions throughout the recording process.

Histology

For histological analyses, animals were euthanized by decapitation, and hearts were immediately excised. Tissues were immersion-fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 24 h at 4 °C, dehydrated through graded ethanol, cleared in xylene, and embedded in paraffin. Transverse Sect. (5 μm) were prepared using a microtome (HM335E; Microm, Minneapolis, MN, USA) and stained with hematoxylin-eosin for morphometric evaluation. Three sections per animal were analyzed under a Zeiss Axiovert 100 M microscope, and digital images were processed with ImageJ software (NIH, Bethesda, MD, USA) as previously described³⁷. Morphometric quantification was performed using a standardized grid overlay and cell-counting algorithm, scoring 500 intersection points across five non-overlapping fields per animal (see Supplementary Fig. 1). Assessed parameters included cardiomyocyte occupancy, extracellular matrix, inflammatory cell infiltration, blood vessels, and processing artifacts. Cardiomyocyte cross-sectional nuclear and fiber diameters were also measured from calibrated images. For total morphometry all, measurements were expressed as percentage values.

Hematologic analysis

Complete blood counts were performed using EDTA-anticoagulated whole blood samples within 24 h after collection, including determination of total red blood cell count, hemoglobin concentration, hematocrit, and leukogram. Serum and plasma samples were obtained by centrifugation at 3500 rpm for 15 min, transferred into labeled microtubes, and stored at temperatures below − 20 °C until analysis. Hematological evaluations were performed using an hematology analyzer (Celer, Belo Horizonte, MG, Brazil).

RNA extraction, cDNA synthesis, and qPCR

Total RNA was isolated from cardiac samples using TRIzol Reagent (Invitrogen, USA) at a ratio of 500 µL per 50 mg of tissue, ensuring complete lysis and RNA release. Chloroform (100 µL per sample) was added, followed by centrifugation at 12,000×g for 15 min at 4 °C to allow phase separation. The upper aqueous phase containing RNA was carefully collected, and RNA was precipitated with 250 µL of isopropanol. Samples were incubated for 1 h at -20 °C and centrifuged at 12,000 × g for 10 min at 4 °C. The RNA pellet was washed with 500 µL of 75% ethanol and centrifuged at 7,500 × g for 5 min at 4 °C, air-dried, and resuspended in RNase-free water. RNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Residual genomic DNA was removed by DNase treatment, and complementary DNA (cDNA) was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Relative gene expression was assessed by quantitative PCR (qPCR) on an ABI PRISM 7500 system (Applied Biosystems, Warrington, UK) using SYBR Green PCR Master Mix (Applied Biosystems). All reactions were performed in duplicate. Gene expression levels were calculated using the 2^−ΔΔCt method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene.

Primer sequences:

GAPDH

FW 5′-ACGGCCGCATCTTCTTGTGCA-3′

RV 5′-CGCCCAAATCCGTTCACACCGA-3′

TNF

FW 5′-CCCTCACACTCAGATCATCTTCT-3′

RV 5′-GCTACGACGTGGGCTACA-3′

IL-1β

FW 5′-CTACAGGCTCCGAGATGAACAAC-3′

RV 5′-TCCATTGAGGTGGAGAGCTTTC-3′

IL-10

FW: 5’-GCTCTTACTGACTGGCATGAG-3’

RV: 5’-CGCAGCTCTAGGAGCATGTG-3’

TGF-β

FW 5′-GAGGTCACCCGCGTGCTA-3′

RV 5′-TGTGTGAGATGTCTTTGGTTTTCTC-3′

Clinical study methodology

A quantitative, exploratory case study was conducted. The patient inclusion criteria were as follows: no prior treatment with anthracyclines or iSGLT2, aged between 30 and 70 years, and a glomerular filtration rate (GFR) greater than 30 ml/min/1.73 m². The exclusion criteria included terminal illness, heart failure, any form of diabetes (type 1 or 2), renal failure (GFR < 30 ml/min/1.73 m²), hemodynamic instability, sepsis, or recurrent urinary tract infections. Written informed consent was obtained from the participants following Resolution 466/2012, ensuring ethical conduct in the study. Given the innovative nature of this study, the potential for patient acceptance, and the associated risks, an initial analysis was conducted on a single case.

Patient assessments

Cardiac function was assessed via 12-lead ECG (Micromed Wincardio) while the patient was in the supine position, along with transthoracic echocardiography and longitudinal strain measurements, all of which were performed by certified echocardiographers from the Department of Cardiovascular Imaging. Blood pressure was measured manually via a validated sphygmomanometer. Body weight and abdominal circumference were recorded at each visit.

Blood samples were collected through cubital vein puncture for analysis of blood count, creatinine, urea, venous blood gases, ultrasensitive troponin, B-type natriuretic peptide (BNP), fasting glucose, and glycated hemoglobin. The patient was monitored by a cardiologist and followed an TRF protocol (16 h/8 h). Assessments were performed at baseline and at 1 and 3 months. Symptom prevalence and adherence to the TRF protocol were evaluated via a specific questionnaire.

Statistical analysis

The data are expressed as the means ± standard errors of the means (SEMs). After testing for normality, comparisons between two groups were performed via t test, whereas multiple-group comparisons were analyzed via one-way ANOVA followed by the Newman-Keuls post hoc test or Tukey’s multiple comparisons test. Statistical significance was set at p < 0.05. Analyses were conducted via GraphPad Prism 8.0.

Results

Animal model

After inducing cardiotoxicity with Dox, we assessed the effects of EMPA, TRF, and their combination on morphometric, hemodynamic, and electrocardiographic parameters in the animals (Table 1).

Table 1.

Morphometric, hemodynamic, and electrocardiographic parameters of the animals.

Parameters/groups	Control	Dox	Dox + EMPA	Dox + TRF	Dox + EMPA + TRF
Morphometric
Heart/tibia (mg/mm)	24.0 ± 1.6	23.8 ± 1.1	24.5 ± 1.2	24.8 ± 3.0	23.3 ± 2.1
Heart/body weight (mg/g)	3.6 ± 0.1	3.6 ± 0.2	4.1 ± 0.2	4.1 ± 0.6	3.6 ± 0.2
Nuclei diameter (uM)	3.3 ± 0.1	3.7 ± 0.1 *	3.5 ± 0.1	3.7 ± 0.1	3.6 ± 0.2
Cardiomyocytes diameter (uM)	6.9 ± 0.3	7.4 ± 0.5	6.3 ± 0.1	6.7 ± 0.2	7.1 ± 0.2
Hemodynamic
Systolic pressure (mmHg)	114.8 ± 7.5	165 ± 13.9 *	128.5 ± 7.1 #	120.9 ± 8.4 #	147 ± 11.9
Diastolic pressure (mmHg)	89.6 ± 8.8	127 ± 13.4 *	88.1 ± 14.7 #	80.3 ± 11.5 #	104.8 ± 12.3
Mean Arterial Pressure (mmhg)	97.6 ± 7.8	139.3 ± 13.1 *	101.2 ± 11.5 #	93.4 ± 8.9 #	118.5 ± 12.0
Electrocardiographic
Heart rate	288.6 ± 40.3	361.2 ± 8.6 *	366.2 ± 8.4 *	343.2 ± 12.8 *	370.4 ± 16.6 *
P wave (ms)	30.3 ± 1.4	34 ± 0.8 *	29.5 ± 1.8 #	32.6 ± 1.0	27.9 ± 1.8 #
PR interval (ms)	49.4 ± 4.0	46.5 ± 0.2 *	48.2 ± 1.2	49.1 ± 2.4	48.70 ± 0.7
QRS (ms)	34.2 ± 2.0	42.0 ± 2.6 *	33.4 ± 2.4 #	36.7 ± 3.2	37.7 ± 2.1
T wave (ms)	72.0 ± 4.1	60.8 ± 4.2 *	56.3 ± 4.4	66.3 ± 4.2	62.6 ± 6.1
QTcH	112.9 ± 3.7	122.7 ± 1.6 *	98.2 ± 1.2 * #	111.9 ± 3.2 #	102.3 ± 4.8 * #
RR interval (ms)	182.6 ± 7.8	169.3 ± 4.7	162.8 ± 5.2	172.9 ± 6.9	163.9 ± 7.7
QRS amplitude (mV)	0.1 ± 0.02	0.1 ± 0.02	0.1 ± 0.03	0.2 ± 0.03	0.1 ± 0.018
P amplitude (mV)	0.05 ± 0.008	0.05 ± 0.003	0.05 ± 0.007	0.04 ± 0.005	0.04 ± 0.007
T amplitude (mV)	0.10 ± 0.03	0.06 ± 0.01	0.09 ± 0.007	0.10 ± 0.03	0.08 ± 0.02

Doxorubicin (Dox). Empagliflozin (EMPA). Time Restricted Feeding (TRF); p < 0.05 * in relation to the control group; # in relation to the Dox group. n = 5 animals per group. Means ± standard errors of the means (SEMs). One-way ANOVA followed by the Newman‒Keuls post hoc test.

Morphometric parameters

The heart/tibia ratio was comparable across groups, ranging from 23.3 ± 2.1 to 24.8 ± 3.0 mg/mm. Similarly, the heart/body weight ratio was not significantly different. Cardiomyocyte cross-sectional nuclear and fiber diameters were quantified. While no significant differences were detected among most groups, only the Dox group exhibited a marked increase in nuclear diameter relative to the control group.

Hemodynamic parameters

Systolic pressure was significantly elevated in the Dox group (165 ± 13.9 mmHg) compared with the control group (114.8 ± 7.5 mmHg). Both EMPA and TRF alone significantly attenuated this increase. Diastolic pressure was also greater in the Dox group (127 ± 13.4 mmHg) than in the control group (89.6 ± 8.8 mmHg), with reductions observed in both the EMPA and TRF groups. The mean arterial pressure showed a similar trend. Interestingly, the combined treatment (Dox + EMPA + TRF) did not result in a significant difference among hemodynamic parameters when compared with Dox alone.

Electrocardiographic parameters

The heart rate was significantly higher in the Dox group (361.2 ± 8.6 bpm) than in the control group (288.6 ± 40.3 bpm) and remained elevated with EMPA, TRF, or their combination. The durations of the P wave, QRS complex, and QTc increased after Dox, whereas the duration of the PR interval decreased. Notably, EMPA alone reversed the Dox-induced changes in the P wave and QRS complex. The QTc interval was prolonged in the Dox group but was significantly reduced with EMPA, TRF, or both. Other parameters, including the QRS amplitude, P and T waves, and RR interval, exhibited minor variations but remained within normal ranges across all groups.

Histology

Assessments of myocardial remodeling and inflammation were performed using histological morphometry to gain deeper mechanistic insights. Figure 1A presents representative images. As shown in total morphometry analysis (Fig. 1B), the Dox group exhibited a significant reduction in cardiomyocyte occupancy compared with the control group, accompanied by a marked increase in extracellular matrix content and inflammatory cell infiltration. Treatment with EMPA (Dox + EMPA group) restored cardiomyocyte occupancy to control levels and significantly reduced extracellular matrix expansion and inflammatory infiltrate relative to the Dox group. Both Dox + EMPA and Dox + TRF groups showed significant reductions in extracellular matrix content compared with Dox alone, suggesting beneficial effects on cardiac remodeling. No significant differences were detected among all groups in blood vessel density.

Fig. 1.

Histological morphometry (A) Representative heart images from the experimental groups stained with hematoxylin-eosin. Control cardiac tissue shows no apparent histopathological abnormalities under light microscopy. In the Dox group, signs of inflammatory infiltration and exacerbated deposition of extracellular matrix are observed, suggesting cardiac remodeling and inflammation. In the Dox + EMPA, Dox + TRF, and Dox + EMPA + TRF groups, a less pronounced inflammatory infiltration by mononuclear cells is noted, along with cardiomyocyte degeneration and alterations in the cardiac extracellular matrix, when compared to the Dox group. Scale bar = 10 μm. (B) Morphometric analysis of cardiac tissue. Quantification of cardiomyocytes, extracellular matrix, inflammatory infiltrate, and blood vessels in control and treated groups: Doxorubicin (Dox), Empagliflozin (EMPA), and Time-Restricted Feeding (TRF). *p < 0.05 vs. control group; #p < 0.05 vs. Dox group. n = 5 animals per group. For each animal, 500 grid intersection points were examined across multiple tissue sections. Data are expressed as mean ± standard error of the mean (SEM). Statistical analysis: one-way ANOVA followed by Newman-Keuls post hoc test.

Hematologic analysis

Hematologic analysis provides a sensitive and integrative readout of systemic drug or intervention effects, revealing both therapeutic mechanisms and potential toxicities. Considering this analysis, it was observed that Dox significantly reduced total White Blood Cell count (WBC), Red Blood Cell count (RBC), Hemoglobin (HGB), Hematocrit (HCT), and Mean Corpuscular Volume (MCV) compared with the control group, indicating pronounced myelosuppressive and anemia-inducing effects. Co-treatments Dox + EMPA, Dox + TRF, and Dox + EMPA + TRF effectively prevented the leukopenia observed in the Dox group, restoring WBC values to levels comparable with controls. EMPA alone and EMPA + TRF improved HGB and HCT relative to Dox alone. The combination of EMPA and TRF (Dox + EMPA + TRF) produced the most consistent recovery, with normalization of WBC, RBC, HGB, and HCT values, suggesting additive protective effects on hematopoiesis. Platelet count (PLT), Mean Platelet Volume (MPV), and Plateletcrit (PCT) remained statistically unchanged among all groups. See Table 2 for all data.

Table 2.

Hematologic parameters of the animals.

Parameters/groups		Control	Dox	Dox + EMPA	Dox + TRF	Dox + EMPA + TRF
WBC - White Blood Cell count (total leukocytes)		11.0 ± 1.1	7.4 ± 0.2 *	10.0 ± 1.0 #	10.2 ± 1.2 #	10.0 ± 0.6 #
RBC - Red Blood Cell count (erythrocytes)		8.9 ± 0.3	8.0 ± 0.1 *	8.6 ± 0.3	8.1 ± 0.1	8.9 ± 0.1 #
HGB - Hemoglobin		17.2 ± 0.5	14.4 ± 0.3 *	15.7 ± 0.2 #	15.3 ± 0.1 *	16.6 ± 0.2 #
HCT - Hematocrit		55.9 ± 1.2	48.3 ± 1.1 *	54.6 ± 2.1 #	51.0 ± 0.6 *	52.8 ± 0.7 #
MCV - Mean Corpuscular Volume (average red blood cell size)		64.2 ± 1.2	59.2 ± 0.8 *	62.5 ± 1.0 #	62.5 ± 0.6 #	59.2 ± 1.3 *
MCH - Mean Corpuscular Hemoglobin (average hemoglobin per red blood cell)		18.5 ± 0.3	17.9 ± 0.1	18.9 ± 0.3	18.7 ± 0.2	18.5 ± 0.3
MCHC - Mean Corpuscular Hemoglobin Concentration		29.7 ± 0.3	30.4 ± 0.4	30.2 ± 0.4	30.0 ± 0.6	30.7 ± 0.3
PLT - Platelet count		679.5 ± 22.7	734.3 ± 94.3	757.3 ± 72.2	791.0 ± 12.5	698.5 ± 123.0
MPV - Mean Platelet Volume (average platelet size)		7.5 ± 0.2	7.1 ± 0.2	7.2 ± 0.1	7.4 ± 0.2	7.0 ± 0.04
PCT - Plateletcrit (volume percentage of platelets in blood)		0.53 ± 0.04	0.48 ± 0.1	0.55 ± 0.04	0.58 ± 0.02	0.52 ± 0.04

Doxorubicin (Dox). Empagliflozin (EMPA). Time Restricted Feeding (TRF); p < 0.05 * in relation to the control group; # in relation to the Dox group. n = 5 animals per group. Means ± standard errors of the means (SEMs). One-way ANOVA followed by the Newman‒Keuls post hoc test.

Mechanistic modulation of Dox-triggered inflammatory signaling by EMPA and TRF

To investigate the inflammatory response triggered by Dox treatment and the modulatory effects of EMPA and TRF, the expression of TNF, IL-1β, IL-10, and TGF-β was quantified by RT-qPCR. The red bars in Fig. 2 represent the relative gene expression in the Dox group compared to the control. Dox induced a clear pro-inflammatory profile, with marked increases in TNF (~ 2.7-fold) and IL-1β (~ 1.7-fold), accompanied by a pronounced reduction in IL-10 (in relation to control).

Regarding the effects of the interventions, TNF expression (Fig. 2A) was substantially attenuated in all treated groups. Dox + EMPA produced the strongest reduction (~ 80% vs. Dox), followed by Dox + TRF (~ 65% reduction vs. Dox) and Dox + EMPA + TRF (~ 70% reduction vs. Dox). These findings indicate that both EMPA and TRF effectively mitigate the TNF-mediated inflammatory response induced by Dox.

Fig. 2.

Relative to control mRNA expression of inflammatory, anti-inflammatory, and profibrotic genes in cardiac tissue following Doxorubicin (Dox) treatment and interventions with Empagliflozin (EMPA), and Time-Restricted Feeding (TRF). (A) TNF, (B) IL-1β, (C) IL-10, and (D) TGF-β expression levels normalized to GAPDH. n = 3–5 animals per group. Means ± standard errors of the means (SEM). One-way ANOVA followed by the Tukey’s multiple comparisons test. Statistical significance was set at p < 0.05.

IL-1β expression (Fig. 2B) displayed a distinct pattern. Dox + EMPA had minimal impact on IL-1β levels, whereas Dox + TRF reduced IL-1β expression by nearly 70% vs. Dox. In contrast, the combined Dox + EMPA + TRF treatment did not reduce IL-1β expression relative to Dox, suggesting a non-additive or potentially antagonistic interaction for this cytokine.

IL-10 expression (Fig. 2C) was markedly increased in Dox + EMPA + TRF group compared to Dox. Treatments with TRF or EMPA alone, however, did not significantly modulate IL-10 expression. These data indicate that the combination of treatments with EMPA and TRF potentiate the anti-inflammatory IL-10 response, whereas TRF or EMPA alone exerts only a modest effect in this model.

Dox exposure alone or in combination with EMPA or TRF individually had little effect on TGF-β expression (Fig. 2D). However, the combined Dox + EMPA + TRF treatment led to a significant increase in TGF-β expression, suggesting a unique interaction between these interventions for this marker.

Patient analysis

The selected patient was a 56-year-old female with invasive breast carcinoma who was estrogen receptor-positive and HER2-negative. She had no history of other neoplasms or prior chemotherapy. Bone scintigraphy at the start of therapy indicated a low probability of bone metastases, and chest and abdominal CT scans revealed no evidence of secondary implants. The total dose of Dox administered was 240 mg/m², given in four cycles of 60 mg/m². During the 90-day observation period, the patient experienced the following symptoms and their respective durations: dry mouth (2 days), breath changes (6 days), extreme fatigue (6 days), dizziness (7 days), headache (6 days), and abdominal pain (4 days) (Table 3). None of these symptoms was severe or interfered with adherence to the study protocol. The patient intermittently fasted for 82 out of 90 days and consistently used EMPA without interruption.

Table 3.

Duration of patient symptoms observed during the study period.

Parameter	Days (number)
Dry mouth	2
Breath changes	6
Extreme fatigue	6
Dizziness	7
Headache	6
Abdominal Pain	4

Clinical and biochemical marker findings

The key clinical observations included weight loss and stable blood pressure. The heart rate slightly but not significantly decreased, remaining within normal limits. Echocardiographic analysis revealed no change in the ejection fraction, the primary marker of anthracycline-induced cardiotoxicity. Longitudinal strain, which is correlated with the risk of ejection fraction decline, also remained stable, however, it was persistently outside the reference limits, which is already associated with an increased risk of ventricular dysfunction. Electrocardiographic intervals were within reference values. Laboratory tests revealed stable BNP levels, confirming the absence of heart failure symptoms, whereas troponin was mildly positive, suggesting subclinical myocardial injury. Renal function remained unchanged (Table 4). The main mechanistic findings derived from our data are presented in Fig. 3.

Table 4.

Morphometric. clinical. And biochemistry markers in the patient undergoing Doxorubicin, Empagliflozin, And intermittent Fasting.

Parameter	First day	30 days	90 days	Reference value
Abdominal circumference (cm)	89	95	93	< 88 cm
Weight (kg)	77.7	76	74	< 70 kg
Ejection fraction (%)	59%	60%	58%	54–74%
Longitudinal strain	−17.1	−17.3	−17.1	< −18%
Blood pressure (mmHg)	120 × 80	110 × 70	130 × 80	< 120 × 80
P wave (ms)	80	100	100	< 120 ms
PR interval (ms)	140	160	140	120–200 ms
QRS (ms)	80	80	80	< 120 ms
T wave (ms)	120	120	120	< 200 ms
QT interval (ms)	360	400	400	340-470 ms
QTc (Bazzet)	436	426	426	360–460
QRS amplitude (mV)	0.9	1.0	1.0	< 1.6
P amplitude (mV)	0.15	0.10	0.10	< 0.3
T amplitude (mV)	0.3	0.2	0.3	0.1–0.5
Heart rate (BPM)	88	68	68	60–100
Rhythm	Sinusal	Sinusal	Sinusal	Sinusal
Glycated hemoglobin (%)	5.5	5.5	5.6	< 5.7%
Creatinine (mg/dl)	0.70	0.70	0.75	0.4–1.1
Troponin (ng/mL)	< 3.2	3.2	25.2	< 15.6
Brain Natriuretic Peptide (pg/mL)	24	13	11	< 100

Fig. 3.

Proposed mechanistic interactions between Empagliflozin (EMPA), and Time-Restricted Feeding (TRF), following Doxorubicin (Dox) cardiotoxicity. The diagram summarizes the major pathways modulated by interventions, including synergistic and opposing effects. Abbreviations: SBP, Systolic Blood Pressure; DBP, Diastolic Blood Pressure; MAP, Mean Arterial Pressure; BP, Blood Pressure; CV, Cardiovascular.

Discussion

This study is the first to investigate the combination of a nonpharmacological, dietetic approach, with iSGLT2 to mitigate Dox-induced cardiac stress, using both an animal model and a clinical case report. This innovative strategy introduces new possibilities for cardiotoxicity management, highlighting a potential alternative to conventional treatments. Figure 4 and Supplementary Table 1 depict the potential interactions and mechanistic relationships between iSGLT2 therapy and intermittent fasting, integrating findings from the existing literature.

Fig. 4.

Inhibitors of sodium-glucose cotransporter 2 (iSGLT2), such as Empagliflozin (EMPA), intermittentfasting (IF), and Doxorubicin (Dox)-induced cardiac damage converging pathways. Central cellular processesand mechanisms that are known to be impaired in Dox-induced cardiac damage plus iSGLT2, and IF commoncharacteristics.Oxidative stress and mitochondrial dysfunction: Dox triggers cardiotoxicity via pronounced oxidative stressand mitochondrial dysfunction, driven by redox cycling and excessive generation of reactive oxygen species(ROS) which might disrupt the balance between oxidants and antioxidant defenses. This oxidative burden leadsto loss of mitochondrial membrane potential (ΔΨm), cytochrome c release, disruption of cardiolipin binding,and subsequent ATP depletion due to impaired oxidative phosphorylation (OXPHOS). EMPA enhancesmitochondrial efficiency by reducing ROS production and activating AMP-activated protein kinase (AMPK)and nuclear factor erythroid 2-related factor 2 (Nrf2) pathways, thereby inducing antioxidant enzymes such assuperoxide dismutase 2 (SOD2), catalase, and heme oxygenase-1 (HO-1). These actions might stabilize ΔΨm,preserve mitochondrial integrity, and limit cardiolipin oxidation, maintaining OXPHOS efficiency. IFstrengthens antioxidant defenses also predominantly through Nrf2 activation, upregulating superoxidedismutase 1 and 2 (SOD1/2), catalase, and glutathione peroxidase (GPx). This effect mitigates lipidperoxidation and DNA oxidation, preserving mitochondrial function and cellular integrity.Inflammation and apoptosis: In the nucleus, Dox can intercalate into DNA and stabilizes topoisomerase IIβ(TOP2β)-DNA cleavage complexes, leading to double-stranded DNA breaks (DSBs) and activation of the p53-dependent DNA damage response. Concurrently, Dox-induced ROS accumulation and damaged organellesactivate the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, resulting incleavage of pro-caspase-1, activation of caspase-1, and subsequent maturation and secretion of the proinflammatorycytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18). This sustained inflammatory milieupromotes myocardial fibrosis and extracellular matrix remodeling, which progressively impaired contractility,leading to left ventricular dysfunction. EMPA reduces the expression of pro-apoptotic proteins while increasinglevels of anti-apoptotic proteins, thereby preserving cardiomyocyte viability. In parallel, it inhibits activation ofthe nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway and suppresses NLRP3inflammasome activation, resulting in decreased production of inflammatory cytokines such as interleukin-1β(IL-1β) and tumor necrosis factor-alpha (TNF). IF inhibits activation of NF-κB pathway and NLRP3inflammasome, thereby reducing the production of pro-inflammatory cytokines such as TNF and IL-1β.Energetic metabolism: Dox disrupts cardiac energy metabolism by decreasing the expression and activity ofkey glycolytic enzymes such as hexokinase II (HKII), which detaches from mitochondria, and2phosphofructokinase (PFK). It also downregulates glucose transporters GLUT1 and GLUT4, reducing glucoseuptake. Additionally, Dox impairs the creatine kinase (CK) system. Fatty acid oxidation is suppressed throughdownregulation of PPARα and CPT1, resulting in the accumulation of toxic lipid intermediates like ceramidesand acylcarnitines. Furthermore, Dox inhibits ketone body metabolism by suppressing β-hydroxybutyratedehydrogenase and other ketolytic enzymes, diminishing the heart’s ability to utilize β-hydroxybutyrate as analternative energy source. EMPA supports cardiac energy metabolism by preventing mitochondrial outermembrane permeabilization, thereby preserving mitochondrial integrity and function. It promotes fatty acidoxidation and enhances the utilization of ketone bodies, improving overall mitochondrial ATP production. IFactivates the AMPK and PGC-1α signaling pathways, promoting mitochondrial biogenesis and enhancingelectron transport chain (ETC) efficiency. It inhibits the opening of the mitochondrial permeability transitionpore (mPTP), preserving ΔΨm, and preventing cytochrome c release. Fasting shifts substrate utilization towardfatty acids and ketone bodies, with β-hydroxybutyrate serving not only as an efficient energy source but also asa signaling molecule. β-hydroxybutyrate inhibits histone deacetylases (HDACs), influencing gene expressionand contributing to the cardioprotective and metabolic benefits.Autophagy and immune response: In the early phase of Dox exposure, autophagy is activated as a protectiveresponse, triggered by ROS, an increased AMP/ATP ratio leading to AMPK activation, and DNA damage viap53-dependent pathways. This initiates the ULK1 complex, promoting autophagosome formation and selectiveremoval of damaged mitochondria through mitophagy (PINK1/Parkin pathway). However, in the late orchronic phase, autophagy becomes impaired due to lysosomal dysfunction and defective autophagosomelysosomefusion, leading to the accumulation of autophagic vacuoles and the adaptor protein p62/SQSTM1,with a potential shift toward autosis. Concurrently, damage-associated molecular patterns (DAMPs), such asHMGB1 and mitochondrial DNA, are released from necrotic or apoptotic cells, activating toll-like receptors(TLR4) and NOD-like receptors, thereby promoting inflammation through recruitment of neutrophils andmonocytes into the myocardium. EMPA activates the AMPK/SIRT1 pathway, which supports properautophagy flux and contributes to mitochondrial quality control through partial activation of mitophagy. Thispromotes cellular homeostasis and energy balance in cardiomyocytes. Additionally, EMPA may influenceimmune modulation by shifting macrophage polarization, with some in vivo studies suggesting reduced proinflammatoryM1 and enhanced anti-inflammatory M2 macrophage populations. IF promotes macroautophagy,including mitophagy, primarily through the activation of AMPK, SIRT1, and FoxO3 pathways. This responsetargets dysfunctional organelles for clearance and activates the AMPK-ULK1-Beclin-1 signaling axis, whichinitiates autophagosome formation. The AMPK/ULK1 pathway plays a central role in driving autophagy, whilefasting also helps restore lysosomal function and supports effective autophagosome-lysosome fusion, ensuringproper autophagic flux and cellular homeostasis.

The iSGLT2 and TRF may improve cardiac function by increasing energy availability through enhanced ketone body production and greater lipid oxidation under reduced glucose conditions³⁸. This is relevant because failing hearts frequently exhibit an energy deficit. Supporting this concept, a review by Hubert Kolb et al. (2021)³⁸ showed that increased ketone utilization by cardiomyocytes; whether induced by fasting, ketogenic diets, or βOHB supplementation, triggers an initial mitochondrial stress response that activates adaptive pathways, leading to improved mitochondrial efficiency and antioxidant defenses. Similarly, iSGLT2 appear to engage these mechanisms, with increased ketolysis promoting a protective mitochondrial adaptation that contributes to their cardioprotective effects³⁸.

It is important to highlight that iSGLT2 themselves may cause adverse effects in certain contexts³⁹. In immunocompromised patients for instance, iSGLT2 use has been linked to severe complications, including infections and metabolic acidosis, sometimes resulting in prolonged hospitalizations and high morbidity or mortality⁴⁰. In oncology, careful risk-benefit assessment is essential before introducing new therapies. However, on the other hand, iSGLT2 use was associated with improved outcomes in patients with cancer therapy-related cardiac dysfunction or HF⁴¹.

Currently, studies investigating the combined effects of iSGLT2 with fasting remain poorly explored. One study reported that time-restricted eating (TRE) interventions in individuals with type 2 diabetes using iSGLT2 were safe and well-tolerated, suggesting that iSGLT2 could be suitable candidates for combination with TRE protocols⁴². Nonetheless, dietary interventions such as TRF can be challenging to implement and sustain over the long term, potentially limiting their translation into routine clinical practice. Therefore, pharmacological agents that mimic or enhance the metabolic effects of fasting emerge as attractive, sustainable alternatives. This applies not only in the context of cardio-oncology but also in other metabolic diseases where energy metabolism plays a central role. While iSGLT2 offer cardioprotective benefits^14,15,43, they are also associated with potential side effects, as mentioned. Many of the cardioprotective benefits of iSGLT2 have been well documented in preclinical studies, which have employed various models of cardiotoxicity⁴⁴. However, regulatory agencies such as the Food and Drug Administration and the European Medicines Agency have highlighted several safety concerns since these drugs were introduced to the market³⁹.

Our findings indicate that Dox exposure induced cardiotoxicity in rats, as evidenced by hemodynamic, electrocardiographic, and histologic alterations. While both EMPA and TRF independently attenuated Dox-induced hemodynamic changes, their combined use did not provide additional benefits, in certain contexts. Both TRF and EMPA treatments promoted a partial or complete rescue of electrocardiographic variables in a range that was not different from that of the control group. However, no synergistic benefits could be observed from the combination of both treatments. Importantly, the benefits of each treatment may depend on the degree of cardiac impairment, the metabolic state of the heart, and the duration of Dox exposure, which were not explored in this study. Notably, different studies have shown greater or lesser toxicity to Dox⁴⁵.

Histologically, it was noted that Dox induces adverse myocardial remodeling, marked by reduced cardiomyocyte occupancy, increased extracellular matrix deposition, and heightened inflammatory infiltration, consistent with known anthracycline cardiotoxic mechanisms involving oxidative stress, fibroblast activation, and inflammation⁴⁶. EMPA effectively restored cardiomyocyte occupancy and attenuated fibrosis and inflammation, in line with other reports^47–50. Besides, TRF also reduced extracellular matrix expansion, suggesting metabolic modulation as an additional cardioprotective pathway. The absence of differences in vascular density indicates that microvascular changes may not be prominent at this stage.

Moreover, the hematologic alterations induced by Dox are consistent with its well-known bone marrow cytotoxicity, leading to reduced proliferation of hematopoietic progenitors and subsequent leukopenia and anemia⁵¹. The decrease in neutrophils and red cell indices reflects direct suppression of myeloid and erythroid lineages, which compromises both immune defense and oxygen transport. Treatments with EMPA or TRF appeared to counteract these effects through distinct but potentially complementary mechanisms. As mentioned, EMPA’s benefits may involve reduced systemic inflammation besides improved erythropoietin sensitivity, and enhanced renal oxygen sensing^52,53, while intermittent fasting likely promotes hematological parameters modulation via metabolic reprogramming, attenuation of oxidative stress, and modulation of inflammatory signaling pathways^54,55. Monitoring these markers not only supports safety assessment but also helps distinguish whether effects are cytotoxic, metabolic, or immunomodulatory.

The concurrent normalization of leukocyte counts and attenuation of cardiac inflammation observed with EMPA and TRF likely reflects a coordinated reprogramming of systemic immune homeostasis coupled with localized myocardial effects. Dox induces myelosuppression and peripheral leukopenia through oxidative DNA damage and mitochondrial dysfunction in bone-marrow progenitors, impairing immune surveillance and triggering compensatory pro-inflammatory activation in peripheral tissues, including the heart⁵¹. Mechanistically, iSGLT2 have been shown to modulate immune cell metabolism by shifting monocytes and macrophages toward an anti-inflammatory M2-like phenotype through AMPK, Nrf2, and NF-κB signaling, whereas TRF enhances hematopoietic niche resilience via improved circadian synchronization, autophagy, and redox balance (see Fig. 3 and Supplementary Table). These complementary mechanisms may preserve bone-marrow integrity, enabling restoration of leukocyte counts while simultaneously preventing excessive recruitment of activated immune cells into cardiac tissue. Thus, EMPA and TRF appear to act in concert to recalibrate both systemic and myocardial inflammatory responses, supporting a coordinated cardio-hematologic protection against Dox-induced injury.

At the molecular level, the modulation of TNF, IL-1β, IL-10, and TGF-β expression provides mechanistic support for the observations. As expected, Dox markedly increased TNF expression, contributing to myocardial inflammatory stress^5,6,8. Both EMPA and TRF robustly suppressed TNF, with histological improvements corroborating the reduced inflammatory burden. IL-1β exhibited a distinct pattern: TRF alone produced the most prominent reduction, consistent with fasting-mediated inhibition of inflammasome activation^19,20, particularly suppression of NLRP3 required for IL-1β maturation. In contrast, EMPA alone or in combination did not retain this IL-1β-lowering effect, implying that SGLT2 inhibition may not substantially engage early inflammasome-dependent pathways, in our model. These temporal and pathway-specific differences between TNF and IL-1β regulation underscore the need for deeper mechanistic studies.

The marked induction of IL-10 in the Dox + EMPA + TRF groups further indicates robust activation of anti-inflammatory signaling. IL-10 constrains pro-inflammatory cytokine production, limits macrophage activation, and promotes a shift toward a reparative immune phenotype⁵⁶. A similar profile was observed for TGF-β, in which only the combined intervention significantly modulated its expression. The pronounced increase in the Dox + EMPA + TRF group suggests activation of tissue-remodeling pathways. Although TGF-β is classically associated with fibrosis⁵⁷, the magnitude of its induction in this model likely reflects a controlled, reparative response rather than pathological fibrosis. This is in contrast to the extreme elevations (> 1000-fold) reported in established cardiomyopathies⁵⁸, where TGF-β drives maladaptive extracellular matrix expansion. In our context, the moderate upregulation of TGF-β may support matrix restoration and cytoprotection⁵⁸, secondary to reduced inflammation and improved cardiomyocyte survival. Hence, in our model, the combined treatment seemed to display a synergistic impact in increasing the anti-inflammatory and immunomodulatory responses, rather than in reducing the pro-inflammatory cytokines. Recent studies have demonstrated that EMPA exerts direct immunomodulatory effects, particularly through mechanisms that link metabolic regulation to inflammatory signaling. In models of acute myocardial infarction, EMPA has been shown to attenuate immune activation, prevent endothelial injury, and partially restore cardiac metabolic balance. These findings align with our observations and support the interpretation that EMPA modulates key inflammatory pathways relevant to cardiac injury^59,60. Such immunometabolic interactions are increasingly recognized as central to the progression of heart failure regardless of whether cardiac dysfunction arises from ischemic injury or systemic metabolic disturbance⁶¹.

In addition to the animal model analysis, we conducted a case study using the same drugs and dosages as those used in the preclinical experiment. Clinically, the therapy was well tolerated, with high adherence and minimal side effects, suggesting potential applicability and scalability for larger studies. Notably, weight loss was accompanied by an increase in the abdominal circumference, which may indicate a systemic inflammatory response secondary to chemotherapy, possibly linked to hypercortisolism. This observation underscores the need for close monitoring of metabolic and hormonal parameters during treatment, as these changes could have significant implications for patient prognosis.

Regarding cardiac function, the findings were overall favorable, characterized by preserved ejection fraction, stable longitudinal strain (albeit persistently outside the reference limits and therefore associated with an increased risk of ventricular dysfunction⁶², and normal BNP levels. However, the detection of elevated troponin levels at the end of 90 days indicates some degree of subclinical myocardial damage. This finding is particularly significant, as troponin is a sensitive marker of myocardial injury and can precede severe clinical manifestations⁶³. While this single case study provides a valuable starting point, expanding the analysis to larger cohorts is essential to confirm the reproducibility and safety of these interventions. Our study underscores the importance of integrating clinical data with preclinical models to gain deeper insights into the complexities of cancer therapies. Combining pharmacological and non-pharmacological strategies, such as iSGLT2 and TRF, may not only enhance overall therapeutic efficacy but also enable lower pharmaceutical dosing, potentially reducing toxicity. Conversely, such combinations may lessen the need for strict dietary adherence to achieve benefits, thereby improving clinical feasibility and patient adherence.

Several studies have highlighted the cardioprotective effects of EMPA in the context of Dox-induced cardiotoxicity. For example, preclinical models have demonstrated that EMPA mitigates oxidative stress, reduces inflammation, and preserves mitochondrial function in Dox-treated animals^64–66. On the other hand, findings regarding intermittent fasting are more nuanced in this scenario. While intermittent fasting has been associated with metabolic benefits in various contexts, recent evidence suggests that it may actually exacerbate Dox-induced cardiac injury under specific conditions, potentially due to impaired cellular repair or increased susceptibility to oxidative stress⁶⁷. Hence, caution in this topic is needed. Nonetheless, our study is novel in that it explores the combined use of EMPA and TRF fasting as an early intervention strategy, an approach that, to our knowledge, has not yet been reported in clinical or preclinical settings.

At this point, it is important to note that our study has some limitations. For instance, only female rats were used. Although sex-specific responses to caloric restriction have been previously described^68,69 the use of female animals was intentional and aimed at enhancing translational relevance, as the patient included in the clinical case was also female. Besides this limitation, the patient was not followed beyond 90 days. Since cardiac damage is strongly time-dependent, a longer follow-up could strengthen the clinical relevance and allow for a more robust assessment of sustained treatment effects. However, here an extended monitoring was not feasible due to logistical constraints. Future studies with longer follow-up periods are needed to better clarify the long-term effects of the interventions. While we initially hypothesized that the combination would enhance cardioprotection, our findings did not demonstrate a clear additive benefit in some cardiovascular functions. Several factors may explain this outcome. First, it is possible that both interventions converge on similar metabolic pathways, such as improved mitochondrial function, reduction of oxidative stress, and enhancement of cardiac energy efficiency, thereby reaching a ceiling effect beyond which no further benefit is observed. Second, intermittent fasting may have introduced physiological stress or metabolic shifts that could have counteracted some of the benefits of EMPA, particularly in the context of Dox-induced injury. This notion is supported by recent evidence suggesting that intermittent fasting may exacerbate cardiotoxicity under certain conditions⁶⁷.

It is also important to highlight that, metabolic differences between rodents and humans must be carefully considered when designing and interpreting fasting protocols. For this reason, the 16:8 regimen was selected, as it is one of the most widely studied and validated approaches in both preclinical and clinical research^29–32. TRF, as applied in our study, is generally well tolerated in humans³³, showing high adherence rates and minimal discomfort. Regarding the use of rats as a model to investigate intermittent fasting, several studies have demonstrated that this fasting window induces metabolic adaptations comparable to those observed in humans, including improved insulin sensitivity, reduced inflammatory markers, and enhanced cardiac resilience under both stress and baseline conditions^29–32. Importantly, in our study, the same 16:8 protocol was implemented in both animals and the human participant, aiming to maximize the translational relevance and comparability of our findings.

In conclusion, both EMPA and TRF, when used separately, attenuated several adverse cardiovascular effects of Dox in animal models, highlighting their potential to prevent cardiotoxicity in a context-dependent manner. All treatments restored cardiomyocyte occupancy, limited extracellular matrix expansion, and reduced inflammatory infiltrates. Moreover, they prevented Dox-induced leukopenia, normalizing leukocyte counts. Mechanistically, EMPA and TRF shaped distinct anti-inflammatory profiles: all reduced TNF, TRF most strongly suppressed IL-1β, the combined treatment increased IL-10 and TGF-β. Clinically, the patient preserved echocardiographic, electrocardiographic, and hemodynamic stability despite biochemical evidence of myocardial injury (elevated troponin), without functional impairment and worsening. These findings suggest that EMPA and TRF may serve as viable individual strategies for managing Dox-induced cardiotoxicity. Further experimental and clinical studies are warranted to clarify their mechanisms, optimize therapeutic protocols, and assess their safety and efficacy across diverse patient populations.

Oxidative stress and mitochondrial dysfunction Dox triggers cardiotoxicity via pronounced oxidative stress and mitochondrial dysfunction, driven by redox cycling and excessive generation of reactive oxygen species (ROS) which might disrupt the balance between oxidants and antioxidant defenses. This oxidative burden leads to loss of mitochondrial membrane potential (ΔΨm), cytochrome c release, disruption of cardiolipin binding, and subsequent ATP depletion due to impaired oxidative phosphorylation (OXPHOS). EMPA enhances mitochondrial efficiency by reducing ROS production and activating AMP-activated protein kinase (AMPK) and nuclear factor erythroid 2-related factor 2 (Nrf2) pathways, thereby inducing antioxidant enzymes such as superoxide dismutase 2 (SOD2), catalase, and heme oxygenase-1 (HO-1). These actions might stabilize ΔΨm, preserve mitochondrial integrity, and limit cardiolipin oxidation, maintaining OXPHOS efficiency. IF strengthens antioxidant defenses also predominantly through Nrf2 activation, upregulating superoxide dismutase 1 and 2 (SOD1/2), catalase, and glutathione peroxidase (GPx). This effect mitigates lipid peroxidation and DNA oxidation, preserving mitochondrial function and cellular integrity.

Inflammation and apoptosis In the nucleus, Dox can intercalate into DNA and stabilizes topoisomerase IIβ (TOP2β)-DNA cleavage complexes, leading to double-stranded DNA breaks (DSBs) and activation of the p53-dependent DNA damage response. Concurrently, Dox-induced ROS accumulation and damaged organelles activate the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, resulting in cleavage of pro-caspase-1, activation of caspase-1, and subsequent maturation and secretion of the pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18). This sustained inflammatory milieu promotes myocardial fibrosis and extracellular matrix remodeling, which progressively impair contractility, leading to left ventricular dysfunction. EMPA reduces the expression of pro-apoptotic proteins while increasing levels of anti-apoptotic proteins, thereby preserving cardiomyocyte viability. In parallel, it inhibits activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway and suppresses NLRP3 inflammasome activation, resulting in decreased production of inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF). IF inhibits activation of NF-κB pathway and NLRP3 inflammasome, thereby reducing the production of pro-inflammatory cytokines such as TNF and IL-1β.

Energetic metabolism Dox disrupts cardiac energy metabolism by decreasing the expression and activity of key glycolytic enzymes such as hexokinase II (HKII), which detaches from mitochondria, and phosphofructokinase (PFK). It also downregulates glucose transporters GLUT1 and GLUT4, reducing glucose uptake. Additionally, Dox impairs the creatine kinase (CK) system. Fatty acid oxidation is suppressed through downregulation of PPARα and CPT1, resulting in the accumulation of toxic lipid intermediates like ceramides and acylcarnitines. Furthermore, Dox inhibits ketone body metabolism by suppressing β-hydroxybutyrate dehydrogenase and other ketolytic enzymes, diminishing the heart’s ability to utilize β-hydroxybutyrate as an alternative energy source. EMPA supports cardiac energy metabolism by preventing mitochondrial outer membrane permeabilization, thereby preserving mitochondrial integrity and function. It promotes fatty acid oxidation and enhances the utilization of ketone bodies, improving overall mitochondrial ATP production. IF activates the AMPK and PGC-1α signaling pathways, promoting mitochondrial biogenesis and enhancing electron transport chain (ETC) efficiency. It inhibits the opening of the mitochondrial permeability transition pore (mPTP), preserving ΔΨm, and preventing cytochrome c release. Fasting shifts substrate utilization toward fatty acids and ketone bodies, with β-hydroxybutyrate serving not only as an efficient energy source but also as a signaling molecule. β-hydroxybutyrate inhibits histone deacetylases (HDACs), influencing gene expression and contributing to the cardioprotective and metabolic benefits.

Autophagy and immune response In the early phase of Dox exposure, autophagy is activated as a protective response, triggered by ROS, an increased AMP/ATP ratio leading to AMPK activation, and DNA damage via p53-dependent pathways. This initiates the ULK1 complex, promoting autophagosome formation and selective removal of damaged mitochondria through mitophagy (PINK1/Parkin pathway). However, in the late or chronic phase, autophagy becomes impaired due to lysosomal dysfunction and defective autophagosome-lysosome fusion, leading to the accumulation of autophagic vacuoles and the adaptor protein p62/SQSTM1, with a potential shift toward autosis. Concurrently, damage-associated molecular patterns (DAMPs), such as HMGB1 and mitochondrial DNA, are released from necrotic or apoptotic cells, activating toll-like receptors (TLR4) and NOD-like receptors, thereby promoting inflammation through recruitment of neutrophils and monocytes into the myocardium. EMPA activates the AMPK/SIRT1 pathway, which supports proper autophagy flux and contributes to mitochondrial quality control through partial activation of mitophagy. This promotes cellular homeostasis and energy balance in cardiomyocytes. Additionally, EMPA may influence immune modulation by shifting macrophage polarization, with some in vivo studies suggesting reduced pro-inflammatory M1 and enhanced anti-inflammatory M2 macrophage populations. IF promotes macroautophagy, including mitophagy, primarily through the activation of AMPK, SIRT1, and FoxO3 pathways. This response targets dysfunctional organelles for clearance and activates the AMPK-ULK1-Beclin-1 signaling axis, which initiates autophagosome formation. The AMPK/ULK1 pathway plays a central role in driving autophagy, while fasting also helps restore lysosomal function and supports effective autophagosome-lysosome fusion, ensuring proper autophagic flux and cellular homeostasis.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

JMRF and ILSM contributed with data curation, formal analysis, investigation, methodology, writing – original draft, writing – review and editing; LMK contributed with investigation, methodology. GCM and FAM contributed with qPCR methodology. ADC, ASM and JVJS contributed with investigation, methodology, writing – review and editing, project administration, supervision, validation, visualization. All authors reviewed the manuscript.

Funding

Fundação Educacional Lucas Machado (FELUMA), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Data availability

Data is provided within the manuscript.

Declarations

Competing interests

The authors declare no competing interests.