SGLT2i Therapy Prevents Anthracycline-Induced Cardiotoxicity in a Large Animal Model by Preserving Myocardial Energetics

Abstract

Background: Anthracycline-induced cardiotoxicity (AIC) is characterized by a disruption in myocardial metabolism.

Objectives: The authors used a large animal model to test sodium-glucose cotransporter inhibitor therapy to prevent AIC.

Methods: Female large white pigs (n = 36) were used to identify the most translational AIC regimen: 6 triweekly intravenous doxorubicin injections (1.8 mg/kg each). Another group of 32 pigs were randomized (1:1:2) to doxorubicin plus empagliflozin 20 mg, doxorubicin plus empagliflozin 10 mg, or doxorubicin control. Pigs were serially examined using multiparametric cardiac magnetic resonance and magnetic resonance spectroscopy. At the end of the 21-week follow-up period, blood samples were obtained to measure myocardial metabolic substrate extraction, and the left ventricle was harvested and processed for analysis using metabolomics, transmission electron microscopy, mitochondrial respirometry, and histopathology.

Results: Final left ventricular ejection fraction (LVEF), the prespecified primary outcome, was significantly higher in pigs receiving 20 mg empagliflozin than in the doxorubicin control group (median 57.5% [Q1-Q3: 55.5%-60.3%] vs 47.0% [Q1-Q3: 40.8%-47.8%]; P = 0.027). Final LVEF in pigs receiving 10 mg empagliflozin was 51% (Q1-Q3: 46.5%-55.5%; P = 0.020 vs 20 mg empagliflozin). The incidence of AIC events was 0%, 50%, and 72% in the empagliflozin 20 mg, empagliflozin 10 mg, and doxorubicin control groups, respectively. Empagliflozin 20 mg treatment resulted in enhanced ketone body consumption by the myocardium, preserved magnetic resonance spectroscopy-measured cardiac energetics, and improved mitochondrial structure and function on transmission electron microscopy and respirometry. These changes were more modest with the 10-mg empagliflozin dose.

Conclusions: Sodium-glucose cotransporter-2 inhibitor therapy with empagliflozin exerts a dose-dependent cardioprotective effect against AIC. The improved LVEF was accompanied by enhanced ketone body consumption, improved cardiac energetics, and preserved mitochondrial structure and function.

Keywords: anthracycline; cardio-oncology; cardiomyopathy; cardioprotection; doxorubicin cardiotoxicity; heart failure; imaging; ketosis; metabolism; myocardial energetics; pigs; sodium glucose contransporter-2 inhibitors; treatment.

Graphical abstract

Figure 1

Study Design See text for a detailed description of study designs. AIC = anthracycline-induced cardiotoxicity; Dox = doxorubicin; Empa = empagliflozin; SGLT2i = sodium-glucose cotransporter-2 inhibitor.

Figure 2

Results of Study 1 (A) In vivo trajectories for cardiac magnetic resonance–derived left ventricular ejection fraction (LVEF) in each doxorubicin (Dox) dose group, together with (B) Kaplan-Meier curves for humanitarian sacrifice. Discontinuous vertical lines indicate the end of Dox therapy at 15 weeks. (C) End of follow-up water content in the anterior myocardial wall. Colored dots represent individual animals. Black dots and lines indicate median (Q1-Q3). Representative hematoxylin and eosin histologic images are shown. Insets show low-magnification views. Scale bar, 100 μm. ∗P < 0.05. Data were compared using analysis of variance for parametric data, followed by Bonferroni correction (for paired comparisons) and Tukey correction (for unpaired comparisons) for multiple comparisons. For nonparametric data, the Wilcoxon rank sum test was used.

Figure 3

Cardiac Function and AIC Prevalence in the Randomized Preclinical Trial (A) LVEF (%) from baseline to study end. Discontinuous vertical lines indicate the end of doxorubicin therapy. (B) LVEF (%) values at 21-week follow-up examination for each group. Colored dots represent individual animals, and colored shading represents the density. Box plots show median (Q1-Q3), and whiskers show 1.5 times the IQR. **P < 0.01. (C) Representative end of follow-up cine cardiac magnetic resonance (CMR) images of systole and diastole for each group. (D) End of follow-up AIC incidence in each experimental group according to the 2022 European Society of Cardiology cardio-oncology guideline definition. Data were compared using analysis of variance for parametric data, followed by Bonferroni correction (for paired comparisons) and Tukey correction (for unpaired comparisons) for multiple comparisons. For nonparametric data, the Wilcoxon rank sum test was used. Abbreviations as in Figures 1 and 2.

Figure 4

Myocardial Tissue Characterization (A) Myocardium T2 values at end of follow-up. Colored dots represent individual animals, colored shading represents density, and black dots represent outliers. Box plots show median (Q1-Q3), and whiskers show 1.5 times the IQR. ∗P < 0.05. (B) Billingham score (calculated as percentage of cardiomyocyte vacuolization: 0, <0.5%; 1, 0.5%-1%; 2, 1%-3%; 3, 3%-5; and 4, >5%) at end of follow-up. (C) T1 mapping–based extracellular volume fraction (ECV). Box plots as in A. ∗∗P < 0.01. (D) Percentage collagen fractional area in the LV quantified from Sirius red–stained sections. (E) Representative histologic images of hematoxylin and eosin (H-E) staining to reveal cardiomyocyte vacuolization (top row) and Sirius red staining to detect fibrosis (bottom row). Paired images per condition show low-magnification (5×) and high-magnification (20×) views (scale bars, 100 and 50 μm). (F) Cardiomyocyte cross-sectional area (square micrometers). (G) Deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)–positive cells (%). Colored dots indicate individual animals. Black dots and lines indicate median (Q1-Q3). ∗∗P < 0.01. (H) Quantification of protein levels (fold increased) for p-FOXO3A, p-TFEB, and p-Akt. Data were compared using analysis of variance for parametric data, followed by Bonferroni correction (for paired comparisons) and Tukey correction (for unpaired comparisons) for multiple comparisons. For nonparametric data, the Wilcoxon rank sum test was used. CTRL = control; GraSE = gradient spin echo; other abbreviations as in Figure 1.

Figure 5

Mitochondrial and Metabolic Evaluation (A) dATP/phosphocreatine (PCr) area over the study for the 3 groups and at the end of the study. Mitochondrial (Mito) area (B), number (C), sphericity index (D), and cristae density (E) in transmission electron microscopic (TEM) images. (F) Representative ultrastructural TEM images for each group. (G) Complex I oxidative phosphorylation (OXPHOS CI) respiration capacity. (H) Oroboros representative curves. Tukey box plot and individual colored plots indicated as previous figures. Black square and error bars represent median (Q1-Q3), colored bars represent mean and SE from (G). ∗P < 0.05 and ∗∗P < 0.01. Data were compared using analysis of variance for parametric data, followed by Bonferroni correction (for paired comparisons) and Tukey correction (for unpaired comparisons) for multiple comparisons. For nonparametric data, the Wilcoxon rank sum test was used. ADP = adenosine diphosphate; dATP = deoxyadenosine triphosphate; FCCP = carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; PM = pyruvate, malate; ROX = respiratory rate oxygenation index; other abbreviations as in Figure 1.

Figure 6

Metabolic Substrate Extraction of Myocardium and Tissue Metabolomics Metabolic substrate gradients between plasma from aorta and coronary sinus corrected by CMR quantitative perfusion for (A) triglycerides, (B) glucose, and (C) ketone bodies. Tukey box plots represent median (Q1-Q3) (box and lines), the remainder of the data distribution (whiskers), and outliers (individual dots). Myocardial tissue metabolomic quantification for (D) fatty acids, (E) glucose and (F) ketone bodies. Bars represent means and SE, and colored dots represent individual data per group. Data were compared using Student’s t-test for parametric data and the Wilcoxon rank sum test for nonparametric data. GCMS = gas chromatography–mass spectrometry; other abbreviations as in Figure 1.

Central Illustration

SGLT2i Therapy Prevents Anthracycline-Induced Cardiotoxicity in a Dose-Dependent Manner in a Large Animal Model In a pig model of anthracycline cardiotoxicity, empagliflozin exerted a dose-dependent cardioprotective effect. Empagliflozin 20 mg/d was associated with preserved systolic cardiac function, improved cardiac energetics, heavy ketone body consumption, and preserved mitochondrial structure and function. Empagliflozin 10 mg exerted a much milder cardioprotective effect. SGLT2i = sodium-glucose cotransporter-2 inhibitor.