Empagliflozin effects on iron metabolism as a possible mechanism for improved clinical outcomes in non-diabetic patients with systolic heart failure

doi:10.21203/rs.3.rs-2615895/v1

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Empagliflozin effects on iron metabolism as a possible mechanism for improved clinical outcomes in non-diabetic patients with systolic heart failure

https://doi.org/10.21203/rs.3.rs-2615895/v1

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published 26 Oct, 2023

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Sodium-glucose cotransporter-2 (SGLT2) inhibitors improve clinical outcomes in patients with heart failure (HF), but mechanisms of action are incompletely understood. In the EMPATROPISM trial, empagliflozin reversed cardiac remodeling and increased physical capacity in stable non-diabetic patients with systolic HF. EMPATROPISM-FE explored post hoc in this cohort, who had a high prevalence of iron deficiency, whether treatment effects were related to changes in iron metabolism. Here we show that myocardial iron content estimated by cardiac magnetic resonance T2* quantification increased after initiation of empagliflozin, but not placebo (between-group difference p = 0.01). T2* changes were significantly correlated with changes in left ventricular volumes, mass and ejection fraction, peak oxygen consumption and 6-minute walking distance; concomitant changes in red blood cell indices were consistent with augmented hematopoiesis. Our findings suggest that effects of empagliflozin on iron metabolism may be a potential mechanism mediating its beneficial clinical effects in patients with HF.

Clinical Trial Registration: www.clinicaltrials.gov; NCT03485222

Health sciences/Cardiology/Cardiovascular biology/Cardiovascular diseases/Heart failure

Biological sciences/Cell biology/Membrane trafficking

heart failure

empagliflozin

iron metabolism

reverse remodeling

exercise capacity

hematopoiesis

Sodium-glucose cotransporter-2 (SGLT2) inhibitors rapidly reduce the risk of cardiovascular death or hospitalization for patients with heart failure (HF),^1–3 but mechanisms of action of these agents have remained incompletely understood.^4,5

Among multiple other effects, data from both animal and human studies suggest that SGLT2 inhibitors enhance myocardial energetics,^5–8 and may reverse cardiac remodeling and improve exercise capacity,^7,9−13 irrespective of diabetes status. Several mechanistic trials reported changes in laboratory iron markers, red blood cell (RBC) indices or erythropoietin levels^14–20 and, more recently, proteomics research reported complex proteome shifts induced by SGLT2-inhibitors, implicating modifications of iron homeostasis proteins and erythropoietin, among other changes.^21,22 Similarly, secondary analyses from large outcome trials suggested that SGLT2 inhibitors increase iron mobilization and use, and enhance hematopoiesis.^17,18,20 Given the high prevalence and adverse prognostic significance of iron deficiency and anemia in people with HF,^23–26 it is important to better understand medications affecting these pathways, including possible biological links between changes in iron status and the beneficial clinical effects of SGLT2 inhibitors.

Using cardiac magnetic resonance imaging (CMR), the Are the ‘cardiac benefits’ of empagliflozin independent of its hypoglycemic activity? (ATRU-4; EMPATROPISM) trial showed that 6 months’ treatment with empagliflozin induced reverse cardiac remodeling and improved functional capacity during exercise testing.¹¹ The EMPATROPISM-FE substudy was designed post hoc: (1) to determine whether treatment with empagliflozin is associated with changes in myocardial iron content as estimated by parametric CMR T2* quantification, and in laboratory markers of iron status, RBC indices, hepcidin and erythropoietin; and (2) to explore whether changes in myocardial iron content correlate with the changes in LV structure and function and in exercise capacity observed in the EMPATROPISM trial.¹¹

Study Patients

Recruitment took place between May 2018, and August 2019. The last patient completed follow-up on February 14, 2020. One patient in the placebo group died, three patients were lost to follow-up and none received iron supplementation, meaning that 80 of the original 84 EMPATROPISM study participants were eligible for the EMPATROPISM-FE substudy.

The study population had a mean age of 62 ± 12 years and 60% were men. A high proportion of minorities was enrolled, and the majority had ischemic HF etiology. In both study arms, 18% of patients had chronic kidney disease (estimated glomerular filtration rate [eGFR] < 60 ml/min/1,73m²), but average eGFR was within the normal range. Baseline patient characteristics were also similar with respect to demographic and clinical features, comorbidities, prescription rates of GRMT, and device therapies (Table 1).

Table 1

Patient Demographic and Clinical Characteristics at Baseline
Variable	Empagliflozin (n = 40)	Placebo (n = 40)
Age, years	64 ± 11	60 ± 13
Male sex, n (%)	25 (63)	23 (57)
Ethnicity, n (%)
Caucasian	15 (38)	6 (15)
Hispanic/Latino	18 (45)	22 (55)
African /American	7 (18)	9 (23)
Asian	0	3 (8)
Systolic blood pressure, mmHg	126.5 ± 19.9	120.8 ± 18.3
Diastolic blood pressure, mmHg	77.3 ± 9.5	76.9 ± 12.3
Heart rate (ECG), beats/min	73.7 ± 16.6	77.5 ± 14.8
Body mass index, kg/m²	29.3 ± 6.0	30.1 ± 6.0
Heart Failure Features and Comorbidities, n (%)
Ischemic etiology	23 (57)	19 (49)
Hospitalization for heart failure (last 12 months)	13 (33)	12 (30)
NYHA class I/II	31 (78)	36 (90)
NYHA class III	9 (23)	4 (10)
Hypertension	32 (80)	26 (67)
Atrial fibrillation	8 (20)	7 (18)
CKD	7 (18)	7 (18)
Heart Failure Therapies, n (%)
ACEi/ARB/ARNi	35 (88)	31 (79)
ARNi	19 (48)	14 (36)
Βeta-blocker	34 (85)	36 (92)
Loop diuretic	20 (50)	23 (59)
Thiazide diuretic	3 (8)	1 (3)
MRA	10 (25)	12 (31)
Pacemaker or ICD	6 (15)	4 (10)
CRT	1 (3)	1 (3)
Values are mean ± standard deviation, or number of patients (%).
ARB indicates angiotensin receptor blocker; ACEi, angiotensin converting enzyme inhibitor; ARNi, angiotensin receptor neprilysin inhibitor; CKD, chronic kidney disease (estimated glomerular filtration rate < 60 mL/min/1.73 m²); CRT, cardiac resynchronization therapy – pacemaker; ECG, electrocardiogram; HF, heart failure; ICD, implantable cardioverter defibrillator; MRA, mineralocorticoid receptor antagonist; NYHA, New York Heart Association.

Changes in Myocardial T2*

Baseline T2* values were similar in both study arms (Table 2). After 6 months, T2* had significantly decreased in the empagliflozin group (adjusted change [95% CI] − 1.3 [–2.1, − 0.5] ms), while there was no significant change from baseline in the placebo group (adjusted change [95% CI]: +0.2 [–0.6, 1.0] ms). The overall treatment effect on parametric T2* for empagliflozin versus placebo was − 1.5 ms (95% CI − 2.7, − 0.4) (Table 2, Fig. 1, right).

Table 2

Observed Efficacy Outcomes
Outcome	Empagliflozin (n = 40)		Placebo (n = 40)
Outcome	Baseline	6 months	Baseline	6 months
Cardiac Magnetic Resonance Imaging
T2*, ms	41.9 ± 4.8	40.5 ± 5.3	41.0 ± 4.9	41.2 ± 5.2
LVEDV, mL	219.8 ± 75.8	194.6 ± 69.7	210.4 ± 68.9	208.9 ± 72.8
LVESV, mL	143.6 ± 66.3	117.0 ± 60.0	135.1 ± 54.8	134.5 ± 58.9
LVEF, %	36.2 ± 8.2	42.2 ± 9.2	36.5 ± 8.0	36.3 ± 8.5
LV mass, g	135.2 ± 45.2	122.0 ± 40.6	131.8 ± 54.4	135.8 ± 61.0
Cardiopulmonary Exercise Testing
PeakVO₂, mL/kg/min	15.3 ± 4.3	16.4 ± 4.4	14.5 ± 3.9	14.0 ± 4.2
6-min walking distance, m	419.8 ± 93.4	501.3 ± 100.0	451.8 ± 100.6	417.3 ± 110.9
Laboratory Markers of Iron Status and Inflammation
Iron, µmol/L	6.2 (4.8–9.9)	6.6 (4.8–8.4)	7.4 (4.8–8.5)	7.8 (5.3–9.4)
Iron deficiency* (by iron level), n (%)	35 (92)	37 (97)	34 (89)	34 (92)
Transferrin, mg/dL	248.8 (233.0-273.5)	259.0 (230.5-293.5)	264.8 (228.5-292.5)	260.5 (214.5-285.5)
Transferrin saturation, %	11.2 (7.5–15.8)	10.5 (7.5–14.7)	10.9 (7.1–13.9)	12.0 (9.2–15.3)
Iron deficiency* (by transferrin saturation), n (%)	36 (95)	35 (92)	34 (89)	34 (92)
sTfR, ng/mL	1393 (1167–1678)	1436 (1266–1787)	1522 (1182–1789)	1338 (1149–1711)
Ferritin, µg/L	108.1 (144.7-177.3)	74.2 (40.7-134.1)	83.2 (47.5-128.9)	89.9 (54.5-122.6)
Ferritin < 100 µg/L**, n (%)	18 (47)	24 (63)	23 (61)	23 (62)
Hepcidin, µg/L	9.3 (4.3–23.5)	6.8 (2.7–13.8)	7.3 (3.7–15.3)	6.7 (4.9–11.2)
hsCRP, mg/dL	1.9 (0.9–4.3)	2.0 (0.8–4.6)	2.7 (1.3–6.3)	2.4 (1.1–7.3)
Red Blood Cell Indices and Hematocrit
Hemoglobin, g/dL	13.2 ± 1.6	13.7 ± 1.8	13.4 ± 1.6	13.5 ± 1.7
Anemia^§ (by hemoglobin), n (%)	13 (33)	9 (23)	11 (28)	8 (20)
Hematocrit, (%)	40.1 ± 4.6	41.7 ± 5.4	41.0 ± 5.0	41.1 ± 5.0
Anemia^§ (by hematocrit), n (%)	11 (28)	8 (20)	9 (23)	8 (20)
RBC count, x10¹²/L	4.5 ± 0.5	4.7 ± 0.6	4.6 ± 0.7	4.6 ± 0.6
MCV, fL	89.1 ± 6.2	89.4 ± 6.4	89.0 ± 6.0	90.2 ± 6.0
MCHC, g/dL	33.0 ± 0.6	32.8 ± 0.7	32.8 ± 0.8	33.0 ± 0.7
MCH, pg	29.4 ± 2.3	29.5 ± 2.2	29.2 ± 2.3	29.8 ± 2.2
RBC distribution width, %	14.2 (13.5–15.4)	14.4 (13.8–15.8)	14.4 (13.8–16.4)	14.0 (13.4–15.1)
Erythropoietin
Erythropoietin concentration, mU/mL	10.1 (7.6–15.7)	10.3 (6.4–15.4)	10.9 (9.0-15.6)	9.5 (6.9–14.5)
Values are mean ± standard deviation, median (interquartile range), or number of patients (%).
*Iron deficiency was defined as plasma iron level ≤ 13 µmol/L or transferrin saturation < 20%.
**Ferritin < 100 µg/L also indicates iron deficiency in patients with heart failure.
^§Anemia was defined as hemoglobin < 13 g/dL (< 12 g/dL) in men (women) or hematocrit < 39% (< 36%) in men (women).
ECG indicates electrocardiogram; hsCRP, high sensitive C-reactive protein; IQR, interquartile range; LV mass, left ventricular mass; LVEDV, left ventricular end-diastolic volume; LVEF, left ventricular ejection fraction, LVESV, left ventricular end-systolic volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; Peak VO₂, peak oxygen consumption; RBC, red blood cell; sTfR, soluble transferrin receptor; SD, standard deviation.

Changes in Myocardial T2* versus Changes in Left Ventricular Structure and Function and in Exercise Capacity

Baseline measures of LV structure and function did not differ between study arms. At the 6-month follow-up, LV volumes and mass had decreased, and LVEF had improved in patients treated with empagliflozin, but not in the placebo group (Table 2). Treatment effects (95% CI) for empagliflozin versus placebo were as follows: LVEDV − 23 (–34, − 11) mL, p < 0.001; LVESV − 25 (–35, − 16) mL, p < 0.001; LV mass − 17 (–24, − 11) g, p < 0.001; and LVEF + 6.1 (4.2, 8.0) %, p < 0.001. Across study groups, correlations were found between the magnitude of changes in myocardial T2* values and changes in LVEDV (r = 0.50, p < 0.001), LVESV (r = 0.47, p < 0.001) and LV mass (r = 0.27, p = 0.020), and decreases in myocardial T2* correlated with improvements in LVEF (r=–0.35, p = 0.003, Fig. 2A).

Baseline peak VO₂ and 6-minute walking distance did not differ significantly between treatment groups (Table 2). At the 6-month follow-up, exercise capacity had improved in patients on empagliflozin and declined in those from the placebo group (Table 2). The treatment effect (95% CI) for change in peak VO₂ with empagliflozin (n = 31) versus placebo (n = 22) was + 1.7 (0.4, 3.1) mL/kg/min, p = 0.011, and there was an inverse correlation between the magnitude of T2* changes and changes in peak VO₂ across study groups (r=–0.64, p < 0.001) (Fig. 2B, left). The treatment effect (95% CI) for empagliflozin compared with placebo on 6-minute walking distance (assessed in all patients) was 112 (82, 141) m, p < 0.001. The magnitude of T2* changes was, albeit weakly, also inversely correlated with changes in 6-minute walking distance (r=–0.25, p = 0.027) (Fig. 2B, right).

Changes in Markers of Iron Status and Inflammation, RBC Indices, Hepcidin and Erythropoietin

There were no baseline between-group differences in markers of iron status and inflammation, RBC indices, hepcidin or erythropoietin (Table 2). Overall, 69 patients (91% of the study population) had a plasma iron level ≤ 13 µmol/L and 70 (92%) had a TSAT < 20%, and were therefore classified as having iron deficiency.²⁵ Plasma ferritin was < 100 µg/L in 41 patients (54%). Depending on the definition (hemoglobin < 13/<12 g/dL in men/women or hematocrit < 39%/<36% in men/women), anemia was present in 24 patients (30%) or 20 patients (25%) at baseline. Distribution of patients with iron deficiency and/or anemia was similar in the two treatment groups (Table 2).

In patients treated with empagliflozin, there was a 12% increase in sTfR concentration at the 6-month follow-up, and mean ferritin and hepcidin had declined to 76% and 68% of baseline values, respectively, while iron and transferrin levels, TSAT and hsCRP were unchanged (Table 2, Fig. 3). In the placebo group, no changes occurred in any of these markers. Figure 3 shows the treatment effects.

Six months of treatment with empagliflozin was associated with increases in hemoglobin, RBC count and hematocrit, while these parameters were unchanged from baseline in the placebo group (Table 2, Fig. 4). Conversely, no major changes from baseline in MCV, MCH and MCHC occurred after 6 months treatment with empagliflozin, while all these indices increased from baseline to 6 months in the placebo group (Table 2, Fig. 4). RBC distribution width tended to increase in the empagliflozin group and to decrease in the placebo group. Figure 4 shows the treatment effects.

Baseline erythropoietin levels correlated directly with sTfR concentrations and inversely with hemoglobin, RBC count and hematocrit (Figure S3A). Similar, but weaker associations were found between changes of these variables after 6 months’ treatment (Figure S3B). No significant treatment effects on erythropoietin were observed (Table 2, Fig. 4). There were no correlations between baseline T2* values, laboratory iron markers, hepcidin or hsCRP levels or of changes of these variables after 6 months’ treatment, nor were the changes in T2* related to baseline iron status, hepcidin or hsCRP levels (Table S1).

Key findings from EMPATROPISM-FE are as follows. First, there was a significant decrease in CMR-derived myocardial parametric T2* values after 6 months’ treatment with empagliflozin, indicating repletion of myocardial iron content. Second, the magnitude of myocardial iron repletion correlated with previously reported improvements in variables reflecting cardiac remodeling and physical capability during treatment with empagliflozin.¹¹ These relationships were consistent across study groups, and increases in parametric T2* values (more frequently seen in placebo-treated patients) were associated with progressive adverse remodeling. Although some correlations were not strong and causality remains unproven, these data indicate a possible link between cardiac dysfunction and myocardial iron depletion. Third, treatment with empagliflozin was associated with changes in various laboratory iron markers, which together suggested increased mobilization and utilization of stored iron.²⁰ Finally, changes and trends observed in RBC indices collectively implied augmented hematopoiesis.

Although most EMPATROPISM-FE participants had iron deficiency at baseline, 6 months’ treatment with empagliflozin was associated with further depletion of systemic iron stores. Our study shows replenishment of myocardial iron content in most patients receiving empagliflozin, but the specific molecular pathways of this effect remain elusive. Beyond the effects of empagliflozin, myocardial iron content is likely to be modulated by multiple other regulatory mechanisms implicated in both systemic and intracellular iron homeostasis.^27,28

Iron is an essential cofactor for the biogenesis of enzymes, lipids and proteins, and for multiple oxidative metabolism processes, erythropoiesis, and oxygen transport and storage.^27,29 Experimental data suggest that myocardial iron deficiency leads to progressive cardiac remodeling and hypertrophy, and impaired mitochondrial respiration.^30,31 Multiple factors may contribute to the high prevalence of iron deficiency among people with HF including reduced dietary intake, attenuated absorption due to systemic inflammation and intestinal congestion, drug effects, blood loss or genetic disposition.^23,27,29 In the clinical setting, dysregulated iron homeostasis with low TSAT and elevated sTfR levels has been linked to increased norepinephrine levels.³² Neurohormonal activation is thought to also induce changes in key regulatory molecules of cellular iron homeostasis, that result in downregulation of mRNA expression of transferrin receptor 1 and inactivation of iron regulatory proteins, thereby inducing intracellular iron deficiency and mitochondrial dysfunction.^28,33,34 There is robust evidence to suggest that myocardial iron content is not closely related to laboratory markers of systemic iron status, which may help explain why in EMPATROPISM-FE myocardial iron content as assessed by T2* and laboratory iron markers and their changes did neither correlate at baseline nor during treatment with empagliflozin versus placebo.^33,35,36

A recent randomized trial in iron deficient patients with HF and reduced LVEF demonstrated that intravenous administration of ferric carboximaltose (FCM) reduced parametric T2* in CMR mapping, consistent with an increase in myocardial iron content.³⁷ Baseline parametric myocardial T2* values were similar to those in the current study. However, at days 7 and 30, the decrease in T2* in the FCM group was more than twice that observed during treatment with empagliflozin in EMPATROPISM-FE. While LVEF did not differ between treatment groups at day 7, the decrease in T2* correlated with improvements in LVEF at day 30, whereas 6-MWT remained unchanged.³⁷ Although there was a greater decrease in T2* with FCM compared with empagliflozin, functional improvements in EMPATROPISM-FE were greater, and accompanied by structural reverse remodeling and improved exercise capacity. Therefore, replenishing myocardial iron stores using FCM may not alter the pathophysiological mechanisms underlying cellular iron deficiency in HF.^28,33,34

FCM increases ferritin and TSAT. In contrast, 6 months’ treatment with empagliflozin was associated with a substantial decline in ferritin, a trend towards lower TSAT, and higher transferrin and iron levels in EMPATROPISM-FE. Our results expand on similar observations in non-HF patients with type 2 diabetes treated with SGLT2 inhibitors,^14–16 and are consistent with changes in laboratory markers of iron metabolism after 12 months’ exposure to dapagliflozin in a recent analysis from the DAPA-HF (Dapagliflozin and Prevention of Adverse Outcomes in Heart Failure) study.²⁰ Although in agreement with guidelines, the definition of iron deficiency used in DAPA-HF has recently been challenged. This is because, compared with the prevalence of iron deficiency based on bone marrow staining, both TSAT < 20% or an iron level ≤ 13 µmol/L (thresholds used to define iron deficiency in EMPATROPISM-FE) do better at identifying the condition and predicting prognosis.²⁵ The use of different definitions limits the ability to directly compare the two studies. Nevertheless, although iron status variables indicated greater severity of iron deficiency in patients from EMPATROPISM-FE compared with those from DAPA-HF, the changes in laboratory iron markers during treatment were of similar magnitude in both studies. Together, these results suggest that improvements in cellular iron availability and metabolism occurred irrespective of iron status, which may help explain why beneficial clinical effects were observed in both iron-replete and iron deficient DAPA-HF patients.²⁰

Complementary to findings by Ghanim et al, who reported an increase in transferrin receptor-1 mRNA expression in patients with diabetes on dapagliflozin,¹⁵ and to observations from DAPA-HF,²⁰ we found that sTfR levels had increased after 6 months’ treatment with empagliflozin. The rise in sTfR in EMPATROPISM-FE co-occurred with increases in hemoglobin, RBC count and hematocrit, changes in other RBC indices and a wider RBC distribution width, all consistent with augmented erythropoiesis.

Mazer et al reported similar, albeit more pronounced changes in RBC indices in patients with type 2 diabetes after 6 months of treatment with empagliflozin.¹⁴ More severe iron deficiency, as reflected by substantially lower baseline iron levels and TSAT in EMPATROPISM-FE participants, could have limited their erythropoietic response. Together, results indicate that increases in hemoglobin and/or hematocrit, as consistently reported from large SGLT2 inhibitor outcome trials,^17,18 likely reflect augmented erythropoiesis rather than only hemoconcentration (an alternative mechanism of rise in hematocrit).^17,18 Mediation analysis identified increases in hemoglobin and/or hematocrit as the most important correlates of the clinical benefits derived from empagliflozin in patients with diabetes,³⁸ and increases in RBC mass might also have contributed to the favorable effects of SGLT2 inhibitors on prognosis by improving tissue oxygen delivery. our results raise the alternative possibility that effects of empagliflozin on iron metabolism (not measured in these trials) could have acted as an additional mediator.

Several previous studies reported a transient early increase in erythropoietin levels after initiation of an SGLT2 inhibitor, but treatment effects lost statistical significance after prolonged treatment.^{14–16, 19} Our observation of a non-significant increase in erythropoietin at 6-month follow-up is consistent with this. Erythropoietin originates mainly from fibroblasts in the renal cortex and governs erythropoiesis and maintenance of RBCs by a feedback mechanism between bone marrow and kidneys. Levels increase proportionally to the degree of anemia and activate hematopoiesis, which in turn increases sTfR levels.^29,39 Inverse correlations between baseline erythropoietin levels and hemoglobin, RBC count and hematocrit and of their changes after 6 months’ treatment, and a correlation between baseline erythropoietin and sTfR levels in EMPATROPISM-FE participants reflect this regulatory principle. Thus, gradual improvements in RBC indices might explain the decline in erythropoietin levels with more prolonged SGLT2 inhibitor exposure.^14,15

In line with Ghanim et al,¹⁵ who reported that dapagliflozin reduced hepcidin levels in patients with diabetes, and with findings from DAPA-HF,²⁰ hepcidin levels decreased in EMPATROPISM-FE participants on empagliflozin, although the between-group difference was only of borderline statistical significance. One possible explanation is depletion of systemic iron stores, because hepatic hepcidin production is transcriptionally downregulated with increased iron utilization, allowing stored iron to be released into the circulation and increasing absorption of iron by enterocytes.²⁹ However, hepcidin levels are modulated by multiple factors (e.g. inflammation), so the current findings relate to the specific population studied but possibly not to other groups with different characteristics.^23,27,29,31

In EMPATROPISM-FE, we demonstrate for the first time that, even in the presence of iron deficiency, treatment with the SGLT2 inhibitor empagliflozin was associated with myocardial iron repletion in most study participants and that increased myocardial iron content was associated with reversal of cardiac remodeling and improved physical capabilities, and co-occurred with changes in RBC indices suggestive of augmented hematopoiesis (although none of the patients received iron supplementation). Recent proteomics research and evidence from other clinical trials corroborate the relevance of our findings.^{14,15,20−22} Although increased iron uptake and utilization in metabolizing tissues following treatment with an SGLT2 inhibitor requires further confirmation, available evidence suggests a potential synergy with therapeutic iron supplementation to replenish deficient iron stores, further enhance myocardial energetics, and ameliorate anemia. Whether iron uptake by enterocytes is enhanced by SGLT2 inhibitors and whether other organ-specific effects of SGLT2 inhibitors may also be attributable to improved cellular iron availability and use deserves further study.

Strengths and Limitations

The first report of an association between an increase in myocardial iron content and reversal of cardiac remodeling plus improved physical capabilities, and the multi-ethnic descent of the participants are strengths of this study. Conversely, the variety of ethnicities represented in this population could limit generalizability due to the impact of inter-ethnic differences in dietary habits and genetic makeup on iron deficiency prevalence and severity.^23,40

Several other potential limitations should also be noted. First, EMPATROPISM-FE was designed post hoc relying on previously stored images and biomaterials from a relatively small, single-site randomized trial. The modest sample size did not provide sufficient power for analysis of all efficacy outcomes and precluded subgroup analyses. Small sample size and lack of adjustment for multiple comparisons also increase the risk of type I error. However, consistent changes and trends in a variety of laboratory iron markers and in RBC indices provide support for the validity and clinical relevance of our findings. Second, although CMR parametric T2* quantification is currently considered the method of choice for myocardial tissue iron assessment, it is better established for determining cardiac iron overload rather than assessment of myocardial iron depletion.⁴¹ Third, some patients had missing CPET assessments, and therefore observed changes may not be representative of the entire study population. However, CPET results were consistent with those of the 6MWT, which was performed by all patients. Fourth, correlations between changes in iron/iron indices and left ventricular size/function were modest, and the post hoc nature of this analysis does not allow definitive conclusions to be drawn regarding this association. Finally, as in all studies, inclusion and exclusion criteria, especially restriction of the sample to patients with reduced LVEF, limit generalizability.

Treatment with empagliflozin for 6 months in non-diabetic patients with stable HF and reduced LVEF, who had a high prevalence of iron deficiency, increased myocardial iron content, further depleted iron stores and may activate hematopoiesis. Changes in CMR-derived parametric T2* values correlated with those in cardiac structure and function, and in exercise capacity. These data suggest that the effects of empagliflozin on mobilization of stored iron, iron availability and iron utilization in cardiomyocytes and, possibly, other cells, and in erythropoiesis could be one potential mechanism mediating the rapid beneficial effects of SGLT2 inhibitors on clinical outcomes. Given the modest sample size and post hoc design of EMPATROPISM-FE, confirmation of the study findings in a prospective multicenter trial is warranted.

Study Design

EMPATROPISM (NCT03485222) was a single-site, investigator-initiated, double-blind, placebo-controlled trial designed and conducted in accordance with Good Clinical Practice standards. The protocol was approved by the Institutional Review Board of Icahn School of Medicine at Mount Sinai. Before enrolment, all patients provided written informed consent. Full details of EMPATROPISM study design and primary results have been published.^11,42

Study Participants

EMPATROPISM study inclusion and exclusion criteria have been reported previously.^11,42 Briefly, male and female outpatients aged ≥ 18 years with an established diagnosis of HF and LVEF < 50% were eligible if they had been on stable pharmacological and device therapy for ≥ 3 months. Key exclusion criteria were: any history of diabetes; acute coronary syndrome or cardiac surgery within the last three months; estimated glomerular filtration rate < 30 mL/min/1.73m²; systolic blood pressure < 90 mmHg; contraindications to CMR (e.g., CMR-incompatible cardiac devices). EMPATROPISM participants were eligible for EMPATROPISM-FE if they completed 6-month follow-up including CMR and did not receive any iron supplementation within 6 months before or during the trial.

Study Procedures

After provision of informed consent, eligible patients were randomized 1:1 to empagliflozin 10 mg/day or matching placebo added to guideline-directed medical HF therapy (GDMT) using a secure web-based system stratified with block sizes of four.^11,42 Assessments at baseline and after 6 months on study medication included physical examination, standard laboratory testing, full RBC count, CMR, cardiopulmonary exercise testing (CPET), and 6-minute walk test (6MWT). At both time points, biomaterials were collected and immediately stored at − 80ºC. Interim visits for safety assessments and drug dispensing were scheduled at one and 3 months.

Cardiac Magnetic Resonance Imaging

CMR was performed on a 1.5-T magnet (Magnetom Avanto FIT, Siemens HealthCare, Erlangen, Germany) using 32-element phased-array coils as receivers. Images were acquired during end-expiratory breath-holds and with electrocardiographic gating. Short-axis cine images of both ventricles were obtained from base to apex with a steady-state free precession sequence. In addition, a mid-ventricular short-axis image was acquired using a multi-echo gradient T2* sequence with 8 echo times from 2.59 to 21 ms. Image analysis was performed using commercial software (cvi42, Circle Cardiovascular Imaging, Calgary, Alberta, Canada). From cine images, LV volumes, LVEF, and LV mass were quantified as previously reported.¹¹ Parametric T2* quantification was performed following recent recommendations for quantitative myocardial tissue iron assessment (Fig. 1, left).⁴³ A region of interest was placed in the septal myocardium of the mid-ventricular short axis image, carefully avoiding scar tissue (as determined by late gadolinium enhancement imaging). To quantify T2*, a mono-exponential decay model and nonlinear algorithm were applied for curve fitting of mean signal intensities of increasing echo time images. All CMR analyses were performed after completion of the EMPATROPISM study. Results are reported as mean values of measurements by two investigators (CGSG and JARI). Both were blinded to study time point and treatment allocation. Reproducibility of T2* measurements was strong (inter-rater reliability [95% confidence interval (CI)] at baseline: 0.87 [0.80–0.91] and at 6-month follow-up: 0.88 [0.82–0.92], Figure S1).

Exercise Testing

To minimize variability, the same sequence of exercise testing was chosen at baseline and at 6-month follow-up, and examinations were supervised by the same personnel. Procedural details have been previously reported,¹¹ and are detailed in Supplemental Methods.

Patients underwent CPET in a fasting state. Upright incremental bicycle ergometry (Lode, Groningen, The Netherlands) with respiratory gas analysis (Med Graphics Ultima O2, MGC Diagnostics Corporation, Saint Paul, Minnesota) was used. Peak oxygen consumption (VO₂) was defined as the highest 30-second average of oxygen consumption. The 6MWT was performed according to American Thoracic Society guidelines.⁴⁴ Patients were instructed to walk as fast and perform as many steps as possible over a 6-minute period, without encouragement. The total walking distance was recorded.

Laboratory Assessments

Red blood cell (RBC) indices (hemoglobin, mean cell volume [MCV], mean corpuscular hemoglobin [MCH], mean corpuscular hemoglobin concentration [MCHC], RBC distribution width) and hematocrit were determined at baseline and at 6-month follow-up. Duplicate measurements of laboratory markers of iron status (iron, transferrin, transferrin saturation [TSAT], soluble transferrin receptor [sTfR], ferritin), inflammation (high-sensitive C-reactive protein [hsCRP]), hepcidin and erythropoietin were performed after completion of the EMPATROPISM study in the Biomarker Laboratory of University Heart & Vascular Center Hamburg, using stored EDTA plasma samples. Coefficients of variation (CV) were calculated for all laboratory markers. Mean values of both measurements are reported. Intra-class correlations were excellent (between 0.97 and 1.00, Figure S2). See Supplemental Methods for detailed descriptions of analysis methods, assay ranges and quality assurance.

Efficacy Measures

The primary endpoint of the EMPATROPISM study was change in LV end-diastolic volume (EDV) from baseline to the 6-month follow-up. Secondary outcomes included changes in LV end-systolic volume (ESV), and in LVEF, LV mass, peak VO₂, and 6-minute walking distance. The main outcome of the EMPATROPISM-FE substudy was the change in CMR-derived parametric T2* values as a surrogate of myocardial iron content.⁴³ This was then correlated with the primary and secondary outcome measures. Additional substudy endpoints included changes in laboratory markers of iron status (iron, transferrin, TSAT, sTfR, ferritin) and inflammation (hsCRP), and changes in RBC indices, hepcidin and erythropoietin.

Sample Size and Statistical Analysis

The number of participants in EMPATROPISM-FE was a consequence of the sample size calculation for EMPATROPISM, which was powered for the primary endpoint and comprised 84 patients.¹¹

Continuous variables are reported as mean ± standard deviation or median (quartiles), and categorical variables as absolute and relative frequencies. Inter-rater reliability was estimated for repeated measurements of T2* (two investigators) and intra-class correlations determined for repeated laboratory measurements. Continuous variables with skewed distributions were log-transformed. Analyses include all patients with available data, with no imputation for missing values. All treatment effects are reported as baseline-adjusted difference from, or multiple of, baseline values as appropriate.

Treatment effects on T2* (primary outcome), the measures of LV structure and function, exercise capacity, laboratory iron markers, hsCRP, RBC indices, hepcidin and erythropoietin levels were estimated using baseline-adjusted linear regression models.

Correlations between changes in parametric T2* values and changes in measures of LV structure and function, and exercise capacity were calculated and visualized within study groups. When within-group correlations did not differ statistically, correlation coefficients across both study arms were also reported. In addition, correlations between parametric T2* values and baseline laboratory iron markers, hepcidin and hsCRP and between changes in these variables after 6 months’ treatment were examined, as well as correlation coefficients between erythropoietin levels, laboratory iron markers and measures of hematopoiesis and their changes.

All statistical analyses were carried out at a two-sided 0.05 level of significance. Nominal p-values are reported without correction for multiplicity. Statistical analyses were conducted using Stata Statistical Software, version 17 (Stata Corp, USA).

Author to whom correspondence and requests for materials should be addressed

Christiane E. Angermann, M.D, Professor of Medicine/Cardiology, Comprehensive Heart Failure Centre, Würzburg, Am Schwarzenberg 15, 97078 Würzburg, Germany. Tel: +49 931 201 46361; Fax: +49 931 201 646360. E-mail: [email protected]

Sources of Funding

Funding of the investigator-initiated EMPATROPISM trial was provided by Boehringer Ingelheim & Lilly Company. The EMPATROPISM FE substudy was supported by Comprehensive Heart Failure Centre, University of Würzburg, Germany (BMBF grant 01EO1004), Athero-Thrombosis Research Unit, Icahn School of Medicine at Mount Sinai, New York, USA, and University Center for Cardiovascular Science, partner site of DZHK, and Institute of Medical Biometry and Epidemiology, University of Hamburg-Eppendorf, Germany

Acknowledgements

CEA acknowledges grant support from German Ministry for Education and Research (BMBF, grants 01GI0205 and 01GI1202A). TZ is supported by the German Center for Cardiovascular Research (DZHK, partner site projects 81Z1710101 and 81Z1710102). LMSG was supported by a postdoctoral fellowship of the German Research Foundation (DFG, GE 3179/1-1). CM is supported by the German Research Foundation (DFG; SFB 894, SFB 1525) and by the German Center for Cardiovascular Research (DZHK; project EX-22).

Author contributions

CEA: Research conception and study design, acquisition of funding, study coordination, data analysis and interpretation, manuscript drafting, revision of manuscript.

CGSG and JARI: Data acquisition, analysis and interpretation, critical revision of manuscript.

SS: Statistical analysis plan, data analysis and interpretation, manuscript drafting, critical revision of manuscript.

TZ: Laboratory biomarker analysis and quality control, critical revision of manuscript.

LMSG: Data analysis and interpretation, manuscript drafting, critical revision of manuscript.

CM and GE: Research conception and study design, critical revision of manuscript.

JS: Data acquisition, critical revision of manuscript.

SF and VF: Critical revision of manuscript.

JB: Research conception and study design, acquisition of funding, critical revision of manuscript.

Disclosures

CEA reports grant support, personal fees and/or non-financial support from Abbott, Astra-Zeneca, Boehringer Ingelheim & Eli Lilly Company, Medtronic, Novo Nordisk, Novartis, ResMed, Thermo Fisher and Vifor, all outside of the submitted work. CM reports personal fees from Amgen, Abbott, AstraZeneca, Berlin Chemie, Boehringer Ingelheim & Eli Lilly Company, Bristol Myers Squibb, Edwards, Novartis, Novo Nordisk, Pharmacosmos, and Servier, all outside the submitted work. TZ reports funding from Vifor for research-related biobanking, outside the submitted work. SF reports significant honoraria from AMGEN Europe, AstraZeneca, Bayer Vital, Boehringer Ingelheim, Bristol-Meyers Squibb GmbH, Daiichi Sankyo, MSD, Novartis, Pfizer, Sanofi, Servier, and Vifor, all outside the submitted work. GE reports personal fees and/or grant support from Abbott, Bayer, Boehringer Ingelheim & Eli Lilly Company, Novartis, ResMed and Vifor. JB reports grant support by Boehringer Ingelheim & Eli Lilly Company. All other authors report no disclosures.

Data Sharing Statement

Deidentified data that support the findings of this study are available from the corresponding author on reasonable request. Because of the modest sample size and single-center nature of the study, creating a higher potential for reidentification, data will be provided only to qualified researchers with training in human subject confidentiality protocols.

Butler, J., et al. Effect of empagliflozin in patients with heart failure across the spectrum of left ventricular ejection fraction. Eur Heart J 43, 416-426 (2022).
Jhund, P.S., et al. Dapagliflozin across the range of ejection fraction in patients with heart failure: a patient-level, pooled meta-analysis of DAPA-HF and DELIVER. Nat Med 28, 1956-1964 (2022).
Zelniker, T.A., et al. SGLT2 inhibitors for primary and secondary prevention of cardiovascular and renal outcomes in type 2 diabetes: a systematic review and meta-analysis of cardiovascular outcome trials. Lancet 393, 31-39 (2019).
Lopaschuk, G.D. & Verma, S. Mechanisms of Cardiovascular Benefits of Sodium Glucose Co-Transporter 2 (SGLT2) Inhibitors: A State-of-the-Art Review. JACC Basic Transl Sci 5, 632-644 (2020).
Packer, M. Critical Reanalysis of the Mechanisms Underlying the Cardiorenal Benefits of SGLT2 Inhibitors and Reaffirmation of the Nutrient Deprivation Signaling/Autophagy Hypothesis. Circulation 146, 1383-1405 (2022).
Thirunavukarasu, S., et al. Empagliflozin Treatment Is Associated With Improvements in Cardiac Energetics and Function and Reductions in Myocardial Cellular Volume in Patients With Type 2 Diabetes. Diabetes 70, 2810-2822 (2021).
Santos-Gallego, C.G., et al. Empagliflozin Ameliorates Adverse Left Ventricular Remodeling in Nondiabetic Heart Failure by Enhancing Myocardial Energetics. J Am Coll Cardiol 73, 1931-1944 (2019).
Croteau, D., et al. Effects of Sodium-Glucose Linked Transporter 2 Inhibition With Ertugliflozin on Mitochondrial Function, Energetics, and Metabolic Gene Expression in the Presence and Absence of Diabetes Mellitus in Mice. J Am Heart Assoc 10, e019995 (2021).
Lee, M.M.Y., et al. Effect of Empagliflozin on Left Ventricular Volumes in Patients With Type 2 Diabetes, or Prediabetes, and Heart Failure With Reduced Ejection Fraction (SUGAR-DM-HF). Circulation 143, 516-525 (2021).
Omar, M., et al. Associations of Empagliflozin With Left Ventricular Volumes, Mass, and Function in Patients With Heart Failure and Reduced Ejection Fraction: A Substudy of the Empire HF Randomized Clinical Trial. JAMA Cardiol 6, 836-840 (2021).
Santos-Gallego, C.G., et al. Randomized Trial of Empagliflozin in Nondiabetic Patients With Heart Failure and Reduced Ejection Fraction. J Am Coll Cardiol 77, 243-255 (2021).
Palau, P., et al. Short-term effects of dapagliflozin on maximal functional capacity in heart failure with reduced ejection fraction (DAPA-VO(2) ): a randomized clinical trial. Eur J Heart Fail 24, 1816-1826 (2022).
Zhang, N., et al. Effect of sodium-glucose cotransporter-2 inhibitors on cardiac remodelling: a systematic review and meta-analysis. Eur J Prev Cardiol 28, 1961-1973 (2022).
Mazer, C.D., et al. Effect of Empagliflozin on Erythropoietin Levels, Iron Stores, and Red Blood Cell Morphology in Patients With Type 2 Diabetes Mellitus and Coronary Artery Disease. Circulation 141, 704-707 (2020).
Ghanim, H., et al. Dapagliflozin Suppresses Hepcidin And Increases Erythropoiesis. J Clin Endocrinol Metab 105, dgaa057 (2020).
Thiele, K., et al. Effects of empagliflozin on erythropoiesis in patients with type 2 diabetes: Data from a randomized, placebo-controlled study. Diabetes Obes Metab 23, 2814-2818 (2021).
Docherty, K.F., et al. Effect of dapagliflozin on anaemia in DAPA-HF. Eur J Heart Fail 23, 617-628 (2021).
Ferreira, J.P., et al. Impact of anaemia and the effect of empagliflozin in heart failure with reduced ejection fraction: findings from EMPEROR-Reduced. Eur J Heart Fail 24, 708-715 (2022).
Fuchs Andersen, C., et al. Effects of empagliflozin on erythropoiesis in heart failure: data from the Empire HF trial. Eur J Heart Fail, Nov 14. doi: 10.1002/ejhf.2735. Online ahead of print (2022).
Docherty, K.F., et al. Iron Deficiency in Heart Failure and Effect of Dapagliflozin: Findings From DAPA-HF. Circulation 146, 980-994 (2022).
Ferrannini, E., et al. Mechanisms of Sodium-Glucose Cotransporter 2 Inhibition: Insights From Large-Scale Proteomics. Diabetes Care 43, 2183-2189 (2020).
Zannad, F., et al. Effect of empagliflozin on circulating proteomics in heart failure: mechanistic insights into the EMPEROR programme. Eur Heart J 43, 4991-5002 (2022).
Alnuwaysir, R.I.S., Hoes, M.F., van Veldhuisen, D.J., van der Meer, P. & Beverborg, N.G. Iron Deficiency in Heart Failure: Mechanisms and Pathophysiology. J Clin Med 11, 125 (2021).
Masini, G., et al. Criteria for Iron Deficiency in Patients With Heart Failure. J Am Coll Cardiol 79, 341-351 (2022).
Grote Beverborg, N., et al. Definition of Iron Deficiency Based on the Gold Standard of Bone Marrow Iron Staining in Heart Failure Patients. Circ Heart Fail 11, e004519 (2018).
Anand, I.S. & Gupta, P. Anemia and Iron Deficiency in Heart Failure: Current Concepts and Emerging Therapies. Circulation 138, 80-98 (2018).
Jayakumar, D., KK, S.N. & Periandavan, K. Triad role of hepcidin, ferroportin, and Nrf2 in cardiac iron metabolism: From health to disease. J Trace Elem Med Biol 69, 126882 (2022).
Haddad, S., et al. Iron-regulatory proteins secure iron availability in cardiomyocytes to prevent heart failure. Eur Heart J 38, 362-372 (2017).
Ghafourian, K., Shapiro, J.S., Goodman, L. & Ardehali, H. Iron and Heart Failure: Diagnosis, Therapies, and Future Directions. JACC Basic Transl Sci 5, 300-313 (2020).
Hoes, M.F., et al. Iron deficiency impairs contractility of human cardiomyocytes through decreased mitochondrial function. Eur J Heart Fail 20, 910-919 (2018).
Kobak, K.A., et al. Structural and functional abnormalities in iron-depleted heart. Heart Fail Rev 24, 269-277 (2019).
Moliner, P., et al. Association Between Norepinephrine Levels and Abnormal Iron Status in Patients With Chronic Heart Failure: Is Iron Deficiency More Than a Comorbidity? J Am Heart Assoc 8, e010887 (2019).
Maeder, M.T., Khammy, O., dos Remedios, C. & Kaye, D.M. Myocardial and systemic iron depletion in heart failure implications for anemia accompanying heart failure. J Am Coll Cardiol 58, 474-480 (2011).
Tajes, M., et al. Neurohormonal activation induces intracellular iron deficiency and mitochondrial dysfunction in cardiac cells. Cell Biosci 11, 89 (2021).
Hirsch, V.G., et al. Cardiac iron concentration in relation to systemic iron status and disease severity in non-ischaemic heart failure with reduced ejection fraction. Eur J Heart Fail 22, 2038-2046 (2020).
Leszek, P., et al. Myocardial iron homeostasis in advanced chronic heart failure patients. Int J Cardiol 159, 47-52 (2012).
Núñez, J., et al. Noninvasive Imaging Estimation of Myocardial Iron Repletion Following Administration of Intravenous Iron: The Myocardial-IRON Trial. J Am Heart Assoc 9, e014254 (2020).
Fitchett, D., et al. Mediators of the improvement in heart failure outcomes with empagliflozin in the EMPA-REG OUTCOME trial. ESC Heart Fail 8, 4517-4527 (2021).
Beguin, Y. Soluble transferrin receptor for the evaluation of erythropoiesis and iron status. Clin Chim Acta 329, 9-22 (2003).
Kang, W., et al. Ethnic Differences in Iron Status. Adv Nutr 12, 1838-1853 (2021).
Lakhal-Littleton, S., et al. An essential cell-autonomous role for hepcidin in cardiac iron homeostasis. Elife 5, e19804 (2016).
Santos-Gallego, C.G., et al. Rationale and Design of the EMPA-TROPISM Trial (ATRU-4): Are the "Cardiac Benefits" of Empagliflozin Independent of its Hypoglycemic Activity? Cardiovasc Drugs Ther 33, 87-95 (2019).
Messroghli, D.R., et al. Clinical recommendations for cardiovascular magnetic resonance mapping of T1, T2, T2* and extracellular volume: A consensus statement by the Society for Cardiovascular Magnetic Resonance (SCMR) endorsed by the European Association for Cardiovascular Imaging (EACVI). J Cardiovasc Magn Reson 19, 75 (2017).
ATS statement: guidelines for the six-minute walk test. Am J Respir Crit Care Med 166, 111-117 (2002).

Yes there is potential Competing Interest. CEA reports grant support, personal fees and/or non-financial support from Abbott, Astra-Zeneca, Boehringer Ingelheim & Eli Lilly Company, Medtronic, Novo Nordisk, Novartis, ResMed, Thermo Fisher and Vifor, all outside of the submitted work. CM reports personal fees from Amgen, Abbott, AstraZeneca, Berlin Chemie, Boehringer Ingelheim & Eli Lilly Company, Bristol Myers Squibb, Edwards, Novartis, Novo Nordisk, Pharmacosmos, and Servier, all outside the submitted work. TZ reports funding from Vifor for research-related biobanking, outside the submitted work. SF reports significant honoraria from AMGEN Europe, AstraZeneca, Bayer Vital, Boehringer Ingelheim, Bristol-Meyers Squibb GmbH, Daiichi Sankyo, MSD, Novartis, Pfizer, Sanofi, Servier, and Vifor, all outside the submitted work. GE reports personal fees and/or grant support from Abbott, Bayer, Boehringer Ingelheim & Eli Lilly Company, Novartis, ResMed and Vifor. JB reports grant support by Boehringer Ingelheim & Eli Lilly Company. All other authors report no disclosures.

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Empagliflozin effects on iron metabolism as a possible mechanism for improved clinical outcomes in non-diabetic patients with systolic heart failure

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Abstract

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Introduction

Results

Study Patients

Changes in Myocardial T2*

Changes in Markers of Iron Status and Inflammation, RBC Indices, Hepcidin and Erythropoietin

Discussion

Strengths and Limitations

Conclusions

Methods

Study Design

Study Participants

Study Procedures

Cardiac Magnetic Resonance Imaging

Exercise Testing

Laboratory Assessments

Efficacy Measures

Sample Size and Statistical Analysis

Declarations

References

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Supplementary Files

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