SGLT2 Inhibitors Act Independently of SGLT2 to Confer Benefit for HFrEF in Mice

Sodium-glucose co-transporter 2 inhibitors (SGLT2i) reduce heart failure (HF) hospitalization rates and death in patients with HF with reduced ejection fraction (HFrEF).¹ Despite rapid adoption of SGLT2i in HF standard-of-care guidelines, the mechanism of their benefit is unknown. The efficacy of SGLT2i in HF is much greater than can be explained by the known modest, pleiotropic effects (e.g. lowering hemoglobin A1c, weight loss, and reduction in systolic blood pressure). This plausibly suggests an off-target mechanism or indirect actions such as SGLT2i-induced ketogenesis and re-programming cardiac substrate utilization.^2,3 This study sought to determine whether SGLT2i exerts therapeutic benefit via on- or off-target mechanisms in a mouse model of HFrEF, and address whether ketosis contributes to the beneficial action in HF.

We conducted empagliflozin (empa) dose-ranging studies in C57BL/6NJ (NNT “wild-type”) male mice to approximate efficacious doses used in human clinical trials.⁴ When admixed in laboratory rodent diet (LabDiet, #5001), a dose of 50 mg/kg/day resulted in a peak (dark cycle) plasma concentration equivalent to published human data (Fig. 1A). Within three days, treatment results in a trend toward reduced plasma glucose, increased fasting ketosis (8h fast, light cycle), and profound glucosuria, without significant weight loss.

Figure 1. Pharmacologic and genetic SGLT2 inhibition have similar metabolic but divergent cardiac protective effects in HFrEF in mice.

(A, left) Peak and trough plasma concentrations of empagliflozin (empa) dose-ranging in WT mice for 2 weeks (n=5-8 per group). (A, right) Fasted blood glucose and beta-hydroxybutyrate (BHB) levels, urine glucose, and body weight (n=5-8 per group) in WT mice treated with 50 mg/kg/day empa for 2 weeks. (B, left) QT-PCR and representative western blot showing SGLT2 reduction in tissue. (B, right) Fasted blood glucose and BHB levels, urine glucose (n=6 per group), and body weight in WT and gKO littermates at age 8-10 weeks (n=12-18 per group). (C) Plasma metabolites and (D) cardiac BHB concentration, targeted cardiac TCA cycle intermediates and cardiac acylcarnitine profile from 6-7 hour fasted WT +/− empa and WT vs gKO littermates (n= 8-16 per group). (E) Experimental schema, Kaplan-Meier curve, fasting plasma BHB and (F) echo and gross morphologic endpoints for sham and TAC/MI +/− empa WT and gKO male littermates (n=10-12 sham, 20-26 HF per group). All data are presented as mean ± SEM and analyzed by Student’s two-tailed t-test (A-D) or 2-way ANOVA/Tukey multiple comparisons test (D – ACs and F) using Prism 9. P values shown. Abbreviations: BiV/TL, biventricular weight to tibia length ratio; EDV/ESV, end-diastolic/systolic volume; EF, ejection fraction; GLS, global longitudinal strain; GRS, global radial strain; LVMI, left ventricular mass index (to pre-surgical weight); TCA, tricarboxylic acid; WMSI, wall motion score index. Schema in A, B and E created with BioRender.com.

We compared the metabolic and cardiac phenotype of SGLT2i-treated mice with mice lacking SGLT2. Generalized SGLT2 knockout (gKO) mice on a C57BL/6NJ background were born in expected Mendelian ratios, developed normally, and had no baseline cardiac phenotype (data not shown), though they were smaller compared to WT littermates (Fig. 1B). Empa treatment and gKO had similar effects on circulating metabolic parameters (Fig. 1B). We hypothesized that reduced insulin/glucagon ratios lead to increased lipolysis, subsequent ketogenesis, and changes in cardiac fuel delivery. Insulin/glucagon ratios trended lower in gKO but not empa-treated mice (Fig. 1C). This difference may be due to the fasting condition at sample collection or other mechanisms driving ketosis in the context of SGLT2 inhibition. Circulating non-esterified fatty acids (NEFA) were increased in both conditions, consistent with increased lipolysis. Targeted metabolomics of left ventricle (LV) myocardium showed increased ketone body beta-hydroxybutyrate (BHB) in both empa-treated and gKO conditions, consistent with the observed ketonemia (Fig. 1D). No differences in gene expression for ketone oxidation enzymes (Bdh1, 3-hydroxybutyrate dehydrogenase 1; Oxct1, succinyl-CoA-3-oxaloacid CoA transferase) were observed (data not shown). A subset of myocardial TCA cycle intermediates was increased in a similar pattern in both conditions. Lastly, abundance of medium- and long-chain acylcarnitines in the LV were significantly increased (2-way ANOVA, p<0.004) with empa treatment and gKO which may indicate increased fatty acid utilization or a pathway “bottlenecking” within the β-oxidation spiral. Taken together, these results indicate that gKO phenocopies the metabolic effects of SGLT2i treatment.

To test the impact of genetic and pharmacologic SGLT2 inhibition in HFrEF, 8—10 weeks old SGLT2 WT and gKO male littermates underwent sham surgery or trans-aortic constriction with apical myocardial infarction (TAC/MI)⁵ and were randomized to empa treatment on post-operative day 3 (Fig. 1E). Empa treatment and gKO genotype did not affect body weight (data not shown) or survival, but increased plasma BHB to a comparable magnitude (Fig. 1E, right). Six weeks following TAC/MI, empa-treated WT mice demonstrated improved function and reduced pathological remodeling (Fig. 1F). In contrast, gKO HFrEF mice had no difference in EF, strain, or pathologic remodeling compared to WT controls. Moreover, empa treatment of the gKO mice produced the same degree of benefit for cardiac functional and remodeling endpoints observed in treated WT controls.

In conclusion, generalized SGLT2 deficiency phenocopies key metabolic effects of pharmacological SGLT2i treatment with empa in mice including increased ketosis, lipolysis, and evidence of myocardial substrate utilization shifts. Despite similar metabolic profiles, gKO did not recapitulate cardiac protection of empa therapy in an established HFrEF model. Beneficial effects of SGLT2i treatment in gKO mice conclusively demonstrate that in a physiologically relevant pre-clinical model of HFrEF, SGLT2i can exert therapeutic benefits via off-target pharmacology.³ The degree of ketosis we observed differs from prior reports, likely related to unmasking in a fasting state, but argues against myocardial ketone body oxidation as a therapeutic mechanism.^2,3 Further work remains to identify specific cardiac or extra-cardiac targets of SGLT2i that confer beneficial effects in HF.

Availability of Data

Data that support the findings of this study are available from the corresponding author upon reasonable request.