Investigation of Biomarker Response to SGLT2 Inhibition in Heart Failure (SiN-HF)

Investigation of Biomarker Response to SGLT2 Inhibition in Heart Failure (SiN-HF) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Investigation of Biomarker Response to SGLT2 Inhibition in Heart Failure (SiN-HF) Patrick Savage, Katie Linden, Lana Dixon, David Grieve, Chris Watson This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7301417/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Dec, 2025 Read the published version in Cardiovascular Drugs and Therapy → Version 1 posted 6 You are reading this latest preprint version Abstract Introduction Following several landmark trials, sodium glucose co-transport (SGLT) 2 inhibitors, have been established as a guideline directed therapy for heart failure (HF). Moreover, their benefit has been established across the spectrum of left ventricular (LV) dysfunction. Much remains unclear regarding their mechanism of action with current evidence implicating pathways involvement of inflammatory, autophagic and anti-fibrotic pathways. Aim We therefore sought to evaluate the effects of SGLT2 inhibition on cardiac biomarkers, myocardial remodelling and patient reported outcomes in heart failure. Methods This was a 26-week, single-arm prospective evaluation of the effects of SGLT2 inhibition on novel biomarkers, myocardial remodeling and patient reported outcomes in patients with heart failure. Baseline echocardiography, serum analysis (standard care and novel biomarkers) and quality of life (QoL) metrics were assessed prior to SGLT2i therapy and at 26-week follow-up. Novel biomarkers were analysed using enzyme-linked immunosorbent assays. Data were analysed using SPSS (IBM SPSS Statistics, Version 28.0). Clinical Trials.gov identifier: NCT06140251 . Results Forty-six patients were recruited with forty patients undergoing biomarker analysis (mean age 67.2+/-8.2years: 68.3% female). Mean LV ejection fraction (LVEF) at baseline was 45.3+/9.8% (ischaemic aetiology: 40.0%, diabetic: 5%). At a median follow-up of 196 days, soluble suppression of tumorigenicity 2 (sSt2) fell significantly (mean difference − 13.5pg/ml [95% CI: -17.9 to − 8.9: p < 0.001]), with no significant change in interleukin (IL)1-β, IL-4, IL-6 or insulin growth factor binding protein 1 (IGFBP-1) (all p = ns). Interestingly, delta change in IL-6 modestly correlated with change in global longitudinal strain (GLS) (%) (r=-0.43, p = 0.012). Change in GLS (%) was not correlated with other novel cardiac biomarkers. Conclusion In our cohort we found that sSt2, a protein implicated in cardiac fibrosis, was suppressed by SGLT2i. Additionally, we observed that suppression of IL-6, a marker of inflammation, correlated with reverse cardiac remodeling. These data support the implication of SGLT2i in suppression of fibrotic and inflammatory pathways. More exploration of these associations is warranted. HEART FAILURE SGLT2 Inhibitors BIOMARKERS ECHOCARDIOGRAPHY Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Since the publication of the now landmark EMPA-REG OUTCOME (Empagliflozin Cardiovascular Outcome Event Trial in Type 2 Diabetes Mellitus Patients) study, nearly a decade ago, SGLT2 inhibitors have emerged as a new pillar of heart failure (HF) therapy. Unique to this drug class, their cardio-protective effects appear to extend across the spectrum of left ventricular (LV) dysfunction (1,2). Their place in current guideline HF therapy has been consolidated by multiple positive placebo-controlled Randomised Controlled Trials (RCTs) and re-iterated in data from several robust meta-analyses (3–5). More-over, their plethora of effects appear to extend beyond the realm of HF with incorporation into diabetic, renal and atherosclerotic cardiovascular disease (ASCVD) guidelines also (6–8). Despite the ubiquity of positive RCT data, much remains unclear regarding their mechanisms of action (9). The early hypothesis that their cardio-protective effects were mediated simply by glycosuria causing diuresis and CV risk reduction were soon refuted, once it was seen their effects occurred independently of baseline HbA1c levels (10). In addition to mediating general CV risk reduction from weight loss and improved blood pressure control, several end-organ effects have been observed with SGLT2 inhibition, including haemoconcentration, erythropoiesis, natriuresis and improved myocardial energetics (11–13). Indeed, cardiac imaging data has also demonstrated that SGLT2 inhibition has direct effects on reverse cardiac remodeling (14–16). Although we can extrapolate from previous RCT data as to the mechanisms driving this remodeling, much remains unclear. Of emerging interest, is the role of SGLT2 inhibition in modification of both inflammatory and fibrotic pathways in HF (17). Both these processes are known to be involved in driving negative remodeling in HF and early pre-clinical data has implicated SGLT2 inhibition in modification of several of these pathways. Of particular interest, are several key pro-inflammatory cytokines such as interleukin (IL)-1β, IL-4 and IL-6 in addition to soluble suppression of tumorigenicity 2 ( sSt2) and insulin growth factor binding protein 1 (IGFBP-1), which are involved in cardiac fibrosis (3,18,19). High levels of IL-4 are associated with cardiac fibrosis and endothelial dysfunction and have been implicated in the development of atherosclerosis (20). IL-4 induces oxidative stress mediators including cytokines, chemokines and several adhesion molecules, in addition to promoting cardiac fibrosis by production of cardiac monocyte chemoattractant protein 1 (MCP-1) and fibroblasts, in addition to activating Reactive Oxygen Species (ROS) mediated expression of the transcription factor activator protein (AP) 1 and collagen-1a in cardiac fibroblasts. IL-6 has been implicated in atherosclerosis, heart failure and stroke (21,22). Interestingly a meta-analysis of SGLT2 inhibitor use in diabetic patients demonstrated that lower levels of IL-6 are associated with SGLT2 inhibitor use, a finding also noted in early studies of patients with CKD (18,23). IL-1β is a key pro-inflammatory cytokine produced by activated macrophages which is a driver of multiple pro-inflammatory processes and has been implicated in the progression of atherosclerosis, HF and myocardial infarction. In several in vitro studies IL-1β expression has been shown to be suppressed by SGLT2 inhibition (24,25). With respect to cardiac fibrosis, sSt2 is a protein secreted in response to activation of myocardial stretch and is known to stimulate cardiac fibrosis and hypertrophy with high circulating levels seen in patients with heart failure (26). With respect to SGLT2 inhibition in heart failure, no clinical studies have been conducted to date exploring its relationship to sSt2, highlighting its novelty as a potential mechanistic pathway. Additionally, IGFBP-1, a protein which binds to and inhibits IGF-1 and is implicated in positive cardiac remodeling, has been highlighted as another possible protein of interest within a proteomic sub-study of the EMPORER trials (27). Therefore, the purpose of this study was to evaluate the impact of SGLT2 inhibition on novel cardiac biomarkers in patients with HF, in addition to evaluating its effects on reverse cardiac remodeling as determined by echocardiography. Secondly, we sought to evaluate the effects of SGLT2 inhibition on standard of care biomarkers in addition to quality of life outcomes (QoL)’s. METHODS Study design This was a 26-week, open label, single-arm prospective evaluation of the effects of SGLT2 inhibition on cardiac biomarkers, myocardial remodelling and patient reported outcomes in patients with heart failure. The trial protocol, consent, recruitment procedure and enrolment were approved by the UK Research Ethics Committee with local governance approval, the full details of which are available in the ( Supplement ). In brief, adult patients with stable symptoms and otherwise on optimal medical therapy who were eligible for commencement on a SGLT2 inhibitor for the treatment of heart failure as per standard care guidelines were considered. The diagnosis of HF was confirmed with recent echocardiography < 1 year with a left ventricular ejection fraction (LVEF) 50% LVEF with objective echocardiographic evidence of cardiac dysfunction (left atrial [LA) volume index > 34ml/m2, E/e’ ratio > 9, tricuspid regurgitation [TR] velocity > 2.8m/s, pulmonary arterial systolic pressure [PASP] > 25mmHg or left ventricular hypertrophy [LVH]) as outlined in current ESC Heart failure guidance. Key exclusion criteria included: hospitalisation for heart failure within 4 weeks prior to enrolment, EGFR < 25 mL/min/1.73m2 at screening, type 1 diabetes, suspected cardiac amyloid, myo- or pericarditis or infiltrative cardiomyopathy. Patients identified with heart failure (both reduced ejection fraction and preserved), on otherwise optimally tolerated standard therapy and were candidates for treatment with SGLT2 inhibition were identified from a local heart failure database, and local heart failure clinics. Following signed, informed consent and screening, patients underwent baseline assessment including clinical evaluation, completion of KCCQ-12 score, biomarker sampling and echocardiography, followed by commencement of a SGLT2 inhibitor as per standard care. The SGLT2 inhibitor used was at the discretion of the prescribing clinician. At 26 weeks these data points were repeated. The study was conducted in accordance with Good Clinical Practice and the Declaration of Helsinki and is registered as Clinical Trials.gov identifier: NCT06140251. Endpoints The full study objectives and endpoints are detailed in Table 1 . In brief, the primary study outcome evaluated whether SGLT2 inhibition in heart failure affects changes in novel cardiac biomarkers. This was an exploratory evaluation of novel cardiac pathways which may serve to establish, as of yet unknown, therapeutic mechanisms of action of SGLT2 inhibition in heart failure. Secondary outcomes evaluate changes in standard of care biomarkers in response to SGLT2 inhibition in heart failure. Additionally, we sought to evaluate changes in echocardiographic parameters and quality of life (QoL) heart failure outcomes following SGLT2 inhibitor therapy and evaluate the relationship between these clinical parameters and novel and standard care biomarkers. Novel biomarker analysis As part of biomarker evaluation, peripheral venous blood sampling was performed prior to SGLT2i commencement and at follow-up, with the samples subsequently centrifuged at 2500 g for 10 min with subsequent aliquoting and storage − 80 ∘C. Enzyme-linked immunosorbent assay (ELISA) analysis was performed to quantify serum levels of proteins of interest, specifically sST2, IGFBP-1, IL-β, IL-4 and IL-6. All assays were performed using assay kits supplied by R&D systems and were performed according to the manufacturer’s instructions. Echocardiography Echocardiography was performed at baseline and at follow-up by a British Society Echocardiography (BSE) accredited operator to include the full standard BSE dataset with averages of measurements taken on sequential cardiac cycles. All images were obtained using a Phillips EPIC™ CVX model echocardiography machine. Left ventricular internal diameter at end diastole (LVIDd) and end systole (LVIDs), interventricular septum diameter (IVSd) and left ventricular posterior wall thickness in diastole (LVPWd) measurements will be obtained in the parasternal long axis (PSAX) views. Left atrial (LA) volume, left ventricular volumes and LVEF will be calculated using biplane volumes and indexed to body surface area (BSA). Where image quality is sub-optimal for Simpson’s biplane assessment of LVEF, a visual estimate will be given. The maximum velocity of early filling (E-wave) during diastole (EVmax), ratio of early ventricular diastolic (E-wave) and atrial filling (A-wave), and deceleration time of the mitral E-wave (DT), will be measured in the apical four chamber view (A4C) using pulsed wave doppler at the level of the mitral valve (MV) leaflet tips at end-expiration. Additionally, the velocity of early myocardial relaxation (e′), velocity of myocardial tissue during ventricular systole (s’) and E to early diastolic mitral annular tissue velocity (E/e′) will be measured in the A4C view using tissue doppler imaging. Global two-dimensional speckle tracking will be performed in the two, three and four chamber views to obtain values for global longitudinal strain (GLS). The endocardial border will be traced automatically along the region of interest at end systole and adjusted as appropriate. Peak GLS will be calculated as an average of the peak strain from the three projections. In the case of sub-optimal image quality, the data will be excluded from the final analysis. Statistical analysis Continuous variables are expressed as mean+/-SD with categorical variable expressed as n (%), unless otherwise stated. Normality was assessed using the Shapiro–Wilk test and visually assessed using quantile plots. Differences in categorical data were assessed using a X 2 test and differences between groups for continuous data assessed using a two-tailed paired student t-test if normally distributed and Wilcoxon signed-rank test if non-normally distributed. Bivariate correlation was assessed using Pearson linear correlation or Spearman rank correlation, if non-normally distributed. Analysis performed using SPSS V28.0 (IBM). Full details of statistical methods and power calculations for this study are provided in the Supplement. RESULTS Demographics A total of 46 patients meeting the study inclusion criteria were enrolled in the study, with 40 patients included in the final analysis. Full recruitment details are depicted in Fig. 1 . At enrolment, the mean age was 67.2+/-8.2 years (29.3% female) and mean LVEF was 45.3+/-9.8%. The majority of patients were non-diabetic (95%) with a mean NYHA score of 2.4+/0.5 and a median duration from initial HF diagnosis of 74.1 months (36.4 to 115.7). All patients received SGLT2i with dapagliflozin with a median duration of follow-up following initiation of 6.8 months (6.4 to 7.1) A full description of baseline demographics and comorbidities are detailed in Table 2 . Additionally, a full description of baseline clinical parameters and QoL metrics are available in the Appendix (A1) . Novel Biomarker analysis The delta change in five novel biomarkers in response to SGLT2 inhibition were assessed in our population, these included IGFBP1, sSt2, IL-1β, IL-4 and IL-6 ( Table 3 ). Of these, IL-4 was not detectable in our cohort. There was a significant decrease in levels of sSt2 following SGLT2 inhibition (mean difference − 13.5pg/mL (95% CI: -8.9 to -17.9; p < 0.001) however, there was no significant difference in IGFBP-1 (+ 3.5µg/mL [95% CI: -8.0 to 1.2; p = 0.141]), IL-6 (+ 1.5pg/mL [95% CI: -3.4 to 0.3; p = 0.10]) or L-1β (+ 0.21 pg/ml [95% CI -0.4 to + 0.04: p = 0.09]) (Fig. 2 a). Biomarker correlation with GLS (%) All patients underwent baseline and follow-up echocardiographic assessment. Of these, 36 patients had windows sufficient for interval global longitudinal strain analysis. At follow-up, there was no significant difference in GLS (%) (13.9+/-3.8% to 14.1+/-3.7%, p = 0.803) following SGLT2 inhibition. Additionally, in our cohort, duration of therapy did not appear to be associated with degree of GLS % improvement (r -0.1, p = ns). A full description of echocardiographic findings are detailed in the Appendix (A2) . Of the novel biomarkers assessed, IL-6 reduction over time was noted to be modestly correlated with delta change in absolute GLS (%) (r = 0.43, p = 0.012) (Fig. 3 a). No difference in demographics or comorbidities at baseline were noted between patients who did or did not experience reverse cardiac remodeling (+ RCR, defined as ≥ 10% relative improvement in GLS%). Notably, patients who had + RCR had a lower LVEF at baseline (41.9 vs 47.2%, p = 0.02) ( Appendix , A3 ). Interestingly, in patients who had a ≥ 10% relative improvement in GLS following SGLT2 inhibition, IL-6 was noted to fall (5.7+/-3.9 pg/ml to 5.0+/-2.9 pg/ml, p = 0.20) whereas in patients who did not see improvements in GLS, IL-6 levels rose (4.6+/-2.7 to 5.9+/-5.9, p = 0.2); however, neither of these trends reached statistical significance. Furthermore, change in GLS (%) was not correlated with other novel cardiac biomarkers tested following six months SGLT2 inhibitor therapy. Novel Biomarker prediction of improvement in GLS (%) At a median follow-up of 196 days, +RCR was noted in 13 patients (mean relative improvement + 24.0+/-18.3%). Baseline serum sSt2 levels significantly predicted + RCR (AUC 0.773; 95% CI: 0.62–0.96: p = 0.008) with a Youden Index cut-off value of 37.3ng/L yielding an 83% sensitivity. Both baseline serum IL-6 and IGFBP1 did not predict + RCR (AUC 0.563; 95% CI 0.36–0.77: p = 0.10) and (AUC 0.515; 95% CI 0.32–0.71: p = 0.88), respectively (Fig. 4 ). Sub-analysis of novel biomarker correlation with LVEF (%) Of the patients who underwent echocardiographic assessment, 36 had windows sufficient for Simpsons biplane analysis. Although this study was not powered to detect changes in LVEF, when assessing patients with impaired LVEF at baseline (n = 29), a significant improvement in LVEF was noted following SGLT2 inhibition (41.8+/-6.7% to 44.1+/-9.1%, p < 0.001). Interestingly, sSt2 was modestly negatively correlated with improvement in LVEF (r= -0.412, p = 0.02) (Fig. 3 b). Neither IL-6 nor IGFBP-1 were correlated with LVEF improvement (p = 0.2) and (p = 0.146) respectively. Standard biomarkers The delta change in standard of care biomarkers in response to SGLT2 inhibition was also assessed ( Table 4 ). A significant increase in haematocrit (41.4+/-3.9% to 43.7+/-3.9%, p < 0.001) and haemoglobin (14.1+/-1.4 g/dL to 14.8+/-1.4 g/dL, p < 0.001) was noted however no significant change in NT-proBNP (+ 174.1 ng/L [95% CI: -38.1 to 33.1; p = 0.441]) or HbA1c (+ 4.7 mmol/mol [95% CI: -13.6 to 4.1; p = 0.746)] was seen (Fig. 2 b). Additionally, no significant correlations were noted between any of the markers tested and change in GLS % following SGLT2i. A full description of these data are available in the Appendix (A4). QoL outcomes At six months follow-up, KCCQ-12 score improved significantly (43.3+/-13.4 to 52.6+/-10.4, p < 0.001). No correlation was noted between delta change in KCCQ-12 score and GLS% (r = 0.03, p = 0.459) or LVEF % (r-0.001, p = 0.934). Additionally, duration of therapy did not appear to influence KCCQ-12 (r = 0.113, p = 0.487). Furthermore, no correlation was noted between sSt2 (r = 0.06, p = 0.702), IL-6 (r = − 0.06, p = 0.692) or IGFBP-1 (r=-0.243, p = 0.169) and QOL score, nor was any association noted between any standard of care biomarker and symptom improvement as defined by KCCQ-12 ( Appendix 5 ). DISCUSSION SGLT2 inhibitors are now a pillar of guideline directed HF care (2). Unlike traditional HF therapies they do not appear to interact with the renin-angiotensin system but rather may exert their pluripotent effects via alternative mechanisms (28,29). Indeed, their proven cardio-protective effects, which are not fully explained by their effects on natriuresis, blood pressure and blood glucose alone, have led to several hypotheses as to their mechanism of action. Much interest surrounds their potential modulatory effects on inflammatory and anti-fibrotic pathways with implication of specific cytokines such as IL-1β, IL-4 and IL-6 along with the proteins IGFBP-1 and sSt2 (9,23,27). In this study, we have demonstrated that the novel cardiac protein sSt2 is suppressed by SGLT2 inhibition and correlated with improvements in LVEF. Furthermore, baseline sSt2 exhibits a predictive utility to identify which patients treated with SLGT2i will experience improvement in cardiac function, as determined by a ≥ 10% improvement in GLS. sSt2 is a member of the IL-1 receptor family and has an important role in mediating inflammatory responses. Its production is stimulated by myocardial stretch with higher levels associated with higher mortality in CVD and HF (26,30). It acts by binding and inhibiting the action of IL-33, which normally acts to protect against Ang-2 driven adverse cardiac remodeling. In the context of our study, these data suggest that SGLT2 inhibition suppresses this protein, thereby attenuating activation of pro-fibrotic and pro-inflammatory pathways. Interestingly, although overall IL-6 levels were not significantly affected by SGLT2 inhibition, a modest positive correlation was noted with improvements in GLS. Additionally, there seemed to be a dichotomous signal with IL-6 trending up in patients with no improvements in GLS and down in those with improvements in GLS. Albeit, this trend was non-significant, in conjunction with the significant correlation data it may serve to highlight a possible signal which may become clearer with a larger sample size or possibly longer duration of therapy. IL-6 is a pro-inflammatory cytokine associated with coronary artery disease, insulin resistance and endothelial dysfunction. Animal studies have demonstrated that SGLT2 inhibition promotes HKII and ERK1/2 mediated suppression of IL-6 levels with early observational clinical data re-iterating these findings (31). Indeed, in a mouse model of HF, chronic activation of IL-6 has been demonstrated to promote LVH with subsequent deletion reversing these effects (32). This has led to much interest in targeting IL-6 as a therapy in patients with myocardial dysfunction, notably in the recent study, ASSAIL-MI, where administration of tocilizumab (a biologic IL-6 inhibitor) reduced infarct size and improved viable myocardial tissue post STEMI as demonstrated via cardiac MRI (33). In our study, the trend observed in IL-6 taken in context with sSt2 provides a suggestion that SGLT2i may promote reverse cardiac remodeling by suppression of inflammatory and pro-fibrotic pathways. To our knowledge, this has not been previously demonstrated in this context. Despite promising pre-clinical data, in our cohort, IGFBP-1 did not change significantly following SGLT2 inhibition nor was it correlated with or have any predictive utility for identifying any marker of cardiac remodeling. We did not detect IL-4 in either pre or post treatment samples. IL-1β was only detectable in half our cohort with so significant change in levels following SGLT2 inhibitor therapy. In our population, we saw significant improvement in QoL metrics, however these were not correlated with novel biomarkers or echocardiographic markers of reverse cardiac remodeling. Moreover, despite the significant increases in haemoglobin and haematocrit (which plausibly may improve preload, cardiac output and oxygenation), neither of these markers were correlated with symptom improvement. This finding is reflected in data from the landmark SGLT2i trials such as DAPA-HF and EMPORER-REDUCED, where the symptomatic benefits of SGLT2 inhibition were noted very early (within weeks), before biomarker changes (10,34). Therefore, it is plausible that these occur independently to observed echocardiographic or biomarker changes and again, a longer duration of follow-up may facilitate a meaningful change in these parameters. LIMITATIONS There are several limitations to this study. Firstly, given it is a prospective observational study it is exposed to selection bias and risk of confounding factors. Furthermore, general interpretation of this data is limited to association of effect and not causation. Secondly, this study was powered to detect changes in IL-6, sSt2 and GLS, and not to detect changes in standard of care biomarkers nor other echocardiographic findings such as LVEF. Thirdly, our follow-up period was limited to six months, which may not be sufficient to fully realise echocardiographic changes secondary to SGLT2 inhibition. Fourthly, our median time from HF diagnosis was over six years therefore the majority of patients in this group had well established HF, with limited scope for reverse cardiac remodeling. It is plausible that this may have limited the potential impact of SGLT2 inhibition on both novel biomarkers and echocardiographic parameters. < SUMMARY In this study of stable HF patients treated with SGLT2 inhibition, we have demonstrated an inhibitory effect on the novel cardiac protein Sst2 which was modestly correlated with improvements in LVEF. Additionally, we noted a modest positive correlation between IL-6 and improvements in global longitudinal strain. These effects appeared to be independent of symptom improvement and baseline demographics. Collectively, these data support the implication of SGLT2 inhibition in suppression of pro-fibrotic and pro-inflammatory pathways in patients with HF. Declarations Sources of funding Patrick Savage is a clinical research fellow investigating the effects of SGLT2 inhibition in heart failure. His work is supported in the form of charitable funding received from the Belfast Heart Trust Fund. Chris Watson is supported by British Heart Foundation grant investigating novel therapeutics for diabetes and heart failure (PG/20/10424). Conflicts of interest No conflicts of interest to declare. Availability of data and material Anonymised data will be made available upon reasonable request. Code availability Not applicable. Authors' contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Patrick Savage. The first draft of the manuscript was written by Patrick Savage and all authors commented on previous versions of the manuscript. All authors read, contributed and approved the final manuscript. Ethical approval & consent The study was conducted in accordance with Good Clinical Practice and the Declaration of Helsinki. Ethical approval was approved by the UK Research Ethics and Research Ethics Committee (22/NW/0348). This trial is registered as Clinical Trials.gov identifier: NCT06140251. Consent to participate Informed, written consent was obtained from all individual participants included in the study . Consent to publish Consent to publish the results of the study were obtained. References Zinman B, Wanner C, Lachin JM, Fitchett D, Bluhmki E, Hantel S, et al. Empagliflozin, Cardiovascular Outcomes, and Mortality in Type 2 Diabetes. N Engl J Med. 2015 Nov 26;373(22):2117–28. McDonagh TA, Metra M, Adamo M, Gardner RS, Baumbach A, Böhm M, et al. 2023 Focused Update of the 2021 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure. Eur Heart J. 2023 Aug 25;ehad195. Vaduganathan M, Docherty KF, Claggett BL, Jhund PS, Boer RA de, Hernandez AF, et al. 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Empagliflozin Inhibits IL-1β-Mediated Inflammatory Response in Human Proximal Tubular Cells. Int J Mol Sci. 2021 May 11;22(10):5089. Dudek M, Kałużna-Oleksy M, Migaj J, Sawczak F, Krysztofiak H, Lesiak M, et al. sST2 and Heart Failure—Clinical Utility and Prognosis. J Clin Med. 2023 Apr 26;12(9):3136. Zannad F, Ferreira JP, Butler J, Filippatos G, Januzzi JL, Sumin M, et al. Effect of empagliflozin on circulating proteomics in heart failure: mechanistic insights into the EMPEROR programme. Eur Heart J. 2022 Dec 21;43(48):4991–5002. Nassif ME, Windsor SL, Tang F, Khariton Y, Husain M, Inzucchi SE, et al. Dapagliflozin Effects on Biomarkers, Symptoms, and Functional Status in Patients With Heart Failure With Reduced Ejection Fraction. Circulation. 2019 Oct 29;140(18):1463–76. Tanaka A, Hisauchi I, Taguchi I, Sezai A, Toyoda S, Tomiyama H, et al. Effects of canagliflozin in patients with type 2 diabetes and chronic heart failure: a randomized trial (CANDLE). ESC Heart Fail. 2020 Aug;7(4):1585–94. Watson CJ, Gallagher J, Wilkinson M, Russell-Hallinan A, Tea I, James S, et al. Biomarker profiling for risk of future heart failure (HFpEF) development. J Transl Med. 2021 Feb 9;19(1):61. Uthman L, Kuschma M, Römer G, Boomsma M, Kessler J, Hermanides J, et al. Novel Anti-inflammatory Effects of Canagliflozin Involving Hexokinase II in Lipopolysaccharide-Stimulated Human Coronary Artery Endothelial Cells. Cardiovasc Drugs Ther. 2021 Dec;35(6):1083–94. Zhao L, Cheng G, Jin R, Afzal MR, Samanta A, Xuan YT, et al. Deletion of Interleukin-6 Attenuates Pressure Overload-Induced Left Ventricular Hypertrophy and Dysfunction. Circ Res. 2016 Jun 10;118(12):1918–29. Broch K, Anstensrud AK, Woxholt S, Sharma K, Tøllefsen IM, Bendz B, et al. Randomized Trial of Interleukin-6 Receptor Inhibition in Patients With Acute ST-Segment Elevation Myocardial Infarction. J Am Coll Cardiol. 2021 Apr 20;77(15):1845–55. Packer M, Anker SD, Butler J, Filippatos G, Pocock SJ, Carson P, et al. Cardiovascular and Renal Outcomes with Empagliflozin in Heart Failure. N Engl J Med. 2020 Oct 8;383(15):1413–24. Tables Tables 1 to 4 are available in the Supplementary Files section. Supplementary Files Table.1.CVDT.tiff Table 1: Study objectives and associated clinical endpoints. sST2:soluble suppression of tumorigenicity 2 protein, IGFBP1: Insulin-like growth factor-binding protein, IL-1B: Interleukin-1 beta, IL-4: Interleukin-4, IL-6: Interleukin-6, CRP: C-reactive protein, HbA1c: glycosylated haemoglobin, SGLT2i: Sodium-glucose co-transport 2 inhibitor, GLS: Global longitudinal strain, LVEF: Left ventricular ejection fraction, LVESVi: Left ventricular end systolic volume index, LVEDVi: Left ventricular end diastolic volume index, LAVi: Left atrial volume index. Table2.Tiff.CVDT.png Table 2: Baseline demographics of study population. Data presented as n (%) with data denoted by * representing median (IQR). Data denoted by ** expressed as mean+/-SD BMl: Body mass index, COPD: Chronic obstructive pulmonary disease, DM: Diabetes mellitus, HF: Heart failure, NYHA: New York Heart Association. Table.3.CVDT.Novel.tiff Table 3: Change in novel biomarkers following six months SGLT2 inhibitor therapy. Data presented as mean (IQR) with differences in means assessed using a paired two tailed t-test with significance defined as p<0.05. *Data displayed as mean+/-SD. **Insufficient levels of IL-4 were detected precluding a paired analysis. SGLT2i: Sodium-glucose co-transport 2 inhibitor, sST2: soluble suppression of tumorigenicity 2 protein, IGFBP1: Insulin-like growth factor-binding protein, IL-1B: Interleukin-1 beta, IL-4: Interleukin-4, IL-6: Interleukin-6. Table.4.standard.CVDT.tiff Table 4: Change in standard care biomarkers following six months SGLT2 inhibitor therapy. Data presented as mean (SD), *Data presented as mean (IQR). Differences in means assessed using a paired two tailed t-test with significance defined as p<0.05. CI: Confidence interval, CRP: C-reactive protein, HbA1c: glycosylated haemoglobin, NT-proBNP: NT-pro brain natriuretic peptide, SGLT2i: Sodium-glucose co-transport 2 inhibitor. Protocol.SiN.HF.Paper.CV.disease.Therapy.docx SiN.HF.Appendix.CVDT.docx Cite Share Download PDF Status: Published Journal Publication published 05 Dec, 2025 Read the published version in Cardiovascular Drugs and Therapy → Version 1 posted Editorial decision: Major Changes Required 25 Aug, 2025 Reviewers agreed at journal 13 Aug, 2025 Reviewers invited by journal 13 Aug, 2025 Editor invited by journal 13 Aug, 2025 Editor assigned by journal 13 Aug, 2025 First submitted to journal 12 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7301417","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":499929982,"identity":"804cdfae-e2a3-49a4-9652-9f7c5ed04a07","order_by":0,"name":"Patrick Savage","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYBADHvv2BgZmCJuNOC1yBjwHSNRibCCRQKQWc/7DzyR+/LJJ3C75eJt0AYOdPINEWgJeLZYz0swke/vSEnfOTiuTnsGQbNggkXYArxaDG0A38fYcTmy4nWMmzcPAnMAgkd6AX8v5458N//b8T2y4eQakpZ4ILQdyDB/z/DhgbHCDB6TlMFALAYdZzsgpfCzbkCwn2ZNWbD3D4LhhG8+zBLxazPmPbzj45o8dDz/74Y23Cyqq5fnZ0wzwOwxEMLbB2AZERCTEwD9I7FEwCkbBKBgF6AAA89lCm1QPYu4AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-3518-5984","institution":"Queen's University Belfast","correspondingAuthor":true,"prefix":"","firstName":"Patrick","middleName":"","lastName":"Savage","suffix":""},{"id":499929983,"identity":"9c729da9-1e82-454f-a433-2b3d03831b3e","order_by":1,"name":"Katie Linden","email":"","orcid":"","institution":"Queen's University 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14:00:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7301417/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7301417/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10557-025-07800-3","type":"published","date":"2025-12-05T15:57:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89633736,"identity":"b3d4a2dd-a4f3-4b7e-ae16-9bd56b4cdf5b","added_by":"auto","created_at":"2025-08-22 06:59:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":567839,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure1.Tiff.CVDT.png","url":"https://assets-eu.researchsquare.com/files/rs-7301417/v1/6c796cc018b826a5029306de.png"},{"id":89633749,"identity":"9d6e5761-1b51-4056-b9b7-55226fbc5456","added_by":"auto","created_at":"2025-08-22 06:59:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":164322,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure2.CVDT.Tiff.png","url":"https://assets-eu.researchsquare.com/files/rs-7301417/v1/e30c1f8a93a90552284fd4b6.png"},{"id":89633741,"identity":"8eb00800-4ffd-4a01-b238-a028ce5f59a8","added_by":"auto","created_at":"2025-08-22 06:59:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":551067,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure3.CVDT.png","url":"https://assets-eu.researchsquare.com/files/rs-7301417/v1/61865d97825c42633b10482c.png"},{"id":89634503,"identity":"d9945d76-e5ac-422a-83bc-ebb45f9520d5","added_by":"auto","created_at":"2025-08-22 07:07:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":474776,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure.4.cvdt.png","url":"https://assets-eu.researchsquare.com/files/rs-7301417/v1/592a84f819d2ecd0f2194fc2.png"},{"id":97724075,"identity":"fad03fd4-3f2b-4b2f-b4ab-3d48aec05136","added_by":"auto","created_at":"2025-12-08 16:11:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2567958,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7301417/v1/a2cb6e74-af95-4e2f-8c27-77ccd89127ba.pdf"},{"id":89634500,"identity":"9ad8fa50-7f22-4b1d-af32-cf9054f85fb8","added_by":"auto","created_at":"2025-08-22 07:07:23","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":247282,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1: \u003c/strong\u003eStudy objectives and associated clinical endpoints.\u003c/p\u003e\n\u003cp\u003esST2:soluble suppression of tumorigenicity 2 protein, IGFBP1: Insulin-like growth factor-binding protein, IL-1B: Interleukin-1 beta, IL-4: Interleukin-4, IL-6: Interleukin-6, CRP: C-reactive protein, HbA1c: glycosylated haemoglobin, SGLT2i: Sodium-glucose co-transport 2 inhibitor, GLS: Global longitudinal strain, LVEF: Left ventricular ejection fraction, LVESVi: Left ventricular end systolic volume index, LVEDVi: Left ventricular end diastolic volume index, LAVi: Left atrial volume index.\u003c/p\u003e","description":"","filename":"Table.1.CVDT.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7301417/v1/24d9c2409d04af25ab54dca8.tiff"},{"id":89633738,"identity":"19cd649c-ffea-4672-884f-723b022db29b","added_by":"auto","created_at":"2025-08-22 06:59:23","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":119486,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 2: \u003c/strong\u003eBaseline demographics of study population. Data presented as \u003cem\u003en\u003c/em\u003e (%) with data denoted by * representing median (IQR). Data denoted by ** expressed as mean+/-SD\u003c/p\u003e\n\u003cp\u003eBMl: Body mass index, COPD: Chronic obstructive pulmonary disease, DM: Diabetes mellitus, HF: Heart failure, NYHA: New York Heart Association.\u003c/p\u003e","description":"","filename":"Table2.Tiff.CVDT.png","url":"https://assets-eu.researchsquare.com/files/rs-7301417/v1/4ba1e0dc54d95fadf862130d.png"},{"id":89634502,"identity":"19582032-d05b-471f-b9a5-faf154a52b91","added_by":"auto","created_at":"2025-08-22 07:07:23","extension":"tiff","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":147118,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 3: \u003c/strong\u003eChange in novel biomarkers following six months SGLT2 inhibitor therapy. Data presented as mean (IQR) with differences in means assessed using a paired two tailed t-test with significance defined as p\u0026lt;0.05. *Data displayed as mean+/-SD. **Insufficient levels of IL-4 were detected precluding a paired analysis.\u003c/p\u003e\n\u003cp\u003eSGLT2i: Sodium-glucose co-transport 2 inhibitor, sST2: soluble suppression of tumorigenicity 2 protein, IGFBP1: Insulin-like growth factor-binding protein, IL-1B: Interleukin-1 beta, IL-4: Interleukin-4, IL-6: Interleukin-6.\u003c/p\u003e","description":"","filename":"Table.3.CVDT.Novel.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7301417/v1/5670b1f671fb740c2fa7b5fe.tiff"},{"id":89633746,"identity":"d96f846f-a5a4-4e4a-b650-9eab0fe5f33c","added_by":"auto","created_at":"2025-08-22 06:59:23","extension":"tiff","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":198362,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 4: \u003c/strong\u003eChange in standard care biomarkers following six months SGLT2 inhibitor therapy. Data presented as mean (SD), *Data presented as mean (IQR). Differences in means assessed using a paired two tailed t-test with significance defined as p\u0026lt;0.05.\u003c/p\u003e\n\u003cp\u003eCI: Confidence interval, CRP: C-reactive protein, HbA1c: glycosylated haemoglobin, NT-proBNP: NT-pro brain natriuretic peptide, SGLT2i: Sodium-glucose co-transport 2 inhibitor.\u003c/p\u003e","description":"","filename":"Table.4.standard.CVDT.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7301417/v1/c85fef7721284a32121151ac.tiff"},{"id":89634504,"identity":"f657df16-ccc1-4b25-88c3-f1a87c623c7a","added_by":"auto","created_at":"2025-08-22 07:07:23","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":227012,"visible":true,"origin":"","legend":"","description":"","filename":"Protocol.SiN.HF.Paper.CV.disease.Therapy.docx","url":"https://assets-eu.researchsquare.com/files/rs-7301417/v1/a7469d9f2a8c085acc4fbbfd.docx"},{"id":89633759,"identity":"41c326fe-46cd-4652-99db-bb7536a14ac9","added_by":"auto","created_at":"2025-08-22 06:59:23","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":373637,"visible":true,"origin":"","legend":"","description":"","filename":"SiN.HF.Appendix.CVDT.docx","url":"https://assets-eu.researchsquare.com/files/rs-7301417/v1/33cb97311f06a9e18301b6d2.docx"}],"financialInterests":"","formattedTitle":"Investigation of Biomarker Response to SGLT2 Inhibition in Heart Failure (SiN-HF)","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eSince the publication of the now landmark \u003cem\u003eEMPA-REG OUTCOME\u003c/em\u003e (Empagliflozin Cardiovascular Outcome Event Trial in Type 2 Diabetes Mellitus Patients) study, nearly a decade ago, SGLT2 inhibitors have emerged as a new pillar of heart failure (HF) therapy. Unique to this drug class, their cardio-protective effects appear to extend across the spectrum of left ventricular (LV) dysfunction (1,2). Their place in current guideline HF therapy has been consolidated by multiple positive placebo-controlled Randomised Controlled Trials (RCTs) and re-iterated in data from several robust meta-analyses (3\u0026ndash;5). More-over, their plethora of effects appear to extend beyond the realm of HF with incorporation into diabetic, renal and atherosclerotic cardiovascular disease (ASCVD) guidelines also (6\u0026ndash;8).\u003c/p\u003e\u003cp\u003eDespite the ubiquity of positive RCT data, much remains unclear regarding their mechanisms of action (9). The early hypothesis that their cardio-protective effects were mediated simply by glycosuria causing diuresis and CV risk reduction were soon refuted, once it was seen their effects occurred independently of baseline HbA1c levels (10). In addition to mediating general CV risk reduction from weight loss and improved blood pressure control, several end-organ effects have been observed with SGLT2 inhibition, including haemoconcentration, erythropoiesis, natriuresis and improved myocardial energetics (11\u0026ndash;13). Indeed, cardiac imaging data has also demonstrated that SGLT2 inhibition has direct effects on reverse cardiac remodeling (14\u0026ndash;16). Although we can extrapolate from previous RCT data as to the mechanisms driving this remodeling, much remains unclear.\u003c/p\u003e\u003cp\u003eOf emerging interest, is the role of SGLT2 inhibition in modification of both inflammatory and fibrotic pathways in HF (17). Both these processes are known to be involved in driving negative remodeling in HF and early pre-clinical data has implicated SGLT2 inhibition in modification of several of these pathways. Of particular interest, are several key pro-inflammatory cytokines such as interleukin (IL)-1β, IL-4 and IL-6 in addition to soluble suppression of tumorigenicity 2 ( sSt2) and insulin growth factor binding protein 1 (IGFBP-1), which are involved in cardiac fibrosis (3,18,19).\u003c/p\u003e\u003cp\u003eHigh levels of IL-4 are associated with cardiac fibrosis and endothelial dysfunction and have been implicated in the development of atherosclerosis (20). IL-4 induces oxidative stress mediators including cytokines, chemokines and several adhesion molecules, in addition to promoting cardiac fibrosis by production of cardiac monocyte chemoattractant protein 1 (MCP-1) and fibroblasts, in addition to activating Reactive Oxygen Species (ROS) mediated expression of the transcription factor activator protein (AP) 1 and collagen-1a in cardiac fibroblasts. IL-6 has been implicated in atherosclerosis, heart failure and stroke (21,22). Interestingly a meta-analysis of SGLT2 inhibitor use in diabetic patients demonstrated that lower levels of IL-6 are associated with SGLT2 inhibitor use, a finding also noted in early studies of patients with CKD (18,23).\u003c/p\u003e\u003cp\u003eIL-1β is a key pro-inflammatory cytokine produced by activated macrophages which is a driver of multiple pro-inflammatory processes and has been implicated in the progression of atherosclerosis, HF and myocardial infarction. In several in vitro studies IL-1β expression has been shown to be suppressed by SGLT2 inhibition (24,25).\u003c/p\u003e\u003cp\u003eWith respect to cardiac fibrosis, sSt2 is a protein secreted in response to activation of myocardial stretch and is known to stimulate cardiac fibrosis and hypertrophy with high circulating levels seen in patients with heart failure (26). With respect to SGLT2 inhibition in heart failure, no clinical studies have been conducted to date exploring its relationship to sSt2, highlighting its novelty as a potential mechanistic pathway. Additionally, IGFBP-1, a protein which binds to and inhibits IGF-1 and is implicated in positive cardiac remodeling, has been highlighted as another possible protein of interest within a proteomic sub-study of the EMPORER trials (27).\u003c/p\u003e\u003cp\u003eTherefore, the purpose of this study was to evaluate the impact of SGLT2 inhibition on novel cardiac biomarkers in patients with HF, in addition to evaluating its effects on reverse cardiac remodeling as determined by echocardiography. Secondly, we sought to evaluate the effects of SGLT2 inhibition on standard of care biomarkers in addition to quality of life outcomes (QoL)\u0026rsquo;s.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy design\u003c/h2\u003e\u003cp\u003eThis was a 26-week, open label, single-arm prospective evaluation of the effects of SGLT2 inhibition on cardiac biomarkers, myocardial remodelling and patient reported outcomes in patients with heart failure. The trial protocol, consent, recruitment procedure and enrolment were approved by the UK Research Ethics Committee with local governance approval, the full details of which are available in the (\u003cb\u003eSupplement\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e In brief, adult patients with stable symptoms and otherwise on optimal medical therapy who were eligible for commencement on a SGLT2 inhibitor for the treatment of heart failure as per standard care guidelines were considered. The diagnosis of HF was confirmed with recent echocardiography\u0026thinsp;\u0026lt;\u0026thinsp;1 year with a left ventricular ejection fraction (LVEF)\u0026thinsp;\u0026lt;\u0026thinsp;50% on echocardiography, or if\u0026thinsp;\u0026gt;\u0026thinsp;50% LVEF with objective echocardiographic evidence of cardiac dysfunction (left atrial [LA) volume index\u0026thinsp;\u0026gt;\u0026thinsp;34ml/m2, E/e\u0026rsquo; ratio\u0026thinsp;\u0026gt;\u0026thinsp;9, tricuspid regurgitation [TR] velocity\u0026thinsp;\u0026gt;\u0026thinsp;2.8m/s, pulmonary arterial systolic pressure [PASP]\u0026thinsp;\u0026gt;\u0026thinsp;25mmHg or left ventricular hypertrophy [LVH]) as outlined in current ESC Heart failure guidance.\u003c/p\u003e\u003cp\u003eKey exclusion criteria included: hospitalisation for heart failure within 4 weeks prior to enrolment, EGFR\u0026thinsp;\u0026lt;\u0026thinsp;25 mL/min/1.73m2 at screening, type 1 diabetes, suspected cardiac amyloid, myo- or pericarditis or infiltrative cardiomyopathy.\u003c/p\u003e\u003cp\u003ePatients identified with heart failure (both reduced ejection fraction and preserved), on otherwise optimally tolerated standard therapy and were candidates for treatment with SGLT2 inhibition were identified from a local heart failure database, and local heart failure clinics. Following signed, informed consent and screening, patients underwent baseline assessment including clinical evaluation, completion of KCCQ-12 score, biomarker sampling and echocardiography, followed by commencement of a SGLT2 inhibitor as per standard care. The SGLT2 inhibitor used was at the discretion of the prescribing clinician. At 26 weeks these data points were repeated. The study was conducted in accordance with Good Clinical Practice and the Declaration of Helsinki and is registered as Clinical Trials.gov identifier: \u003cb\u003eNCT06140251.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEndpoints\u003c/h3\u003e\n\u003cp\u003eThe full study objectives and endpoints are detailed in \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e. In brief, the \u003cem\u003eprimary study outcome\u003c/em\u003e evaluated whether SGLT2 inhibition in heart failure affects changes in novel cardiac biomarkers. This was an exploratory evaluation of novel cardiac pathways which may serve to establish, as of yet unknown, therapeutic mechanisms of action of SGLT2 inhibition in heart failure.\u003c/p\u003e\u003cp\u003e\u003cem\u003eSecondary outcomes\u003c/em\u003e evaluate changes in standard of care biomarkers in response to SGLT2 inhibition in heart failure. Additionally, we sought to evaluate changes in echocardiographic parameters and quality of life (QoL) heart failure outcomes following SGLT2 inhibitor therapy and evaluate the relationship between these clinical parameters and novel and standard care biomarkers.\u003c/p\u003e\n\u003ch3\u003eNovel biomarker analysis\u003c/h3\u003e\n\u003cp\u003eAs part of biomarker evaluation, peripheral venous blood sampling was performed prior to SGLT2i commencement and at follow-up, with the samples subsequently centrifuged at 2500 g for 10 min with subsequent aliquoting and storage \u0026minus;\u0026thinsp;80 ∘C. Enzyme-linked immunosorbent assay (ELISA) analysis was performed to quantify serum levels of proteins of interest, specifically sST2, IGFBP-1, IL-β, IL-4 and IL-6. All assays were performed using assay kits supplied by R\u0026amp;D systems and were performed according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003ch3\u003eEchocardiography\u003c/h3\u003e\n\u003cp\u003eEchocardiography was performed at baseline and at follow-up by a British Society Echocardiography (BSE) accredited operator to include the full standard BSE dataset with averages of measurements taken on sequential cardiac cycles. All images were obtained using a Phillips EPIC\u0026trade; CVX model echocardiography machine.\u003c/p\u003e\u003cp\u003eLeft ventricular internal diameter at end diastole (LVIDd) and end systole (LVIDs), interventricular septum diameter (IVSd) and left ventricular posterior wall thickness in diastole (LVPWd) measurements will be obtained in the parasternal long axis (PSAX) views. Left atrial (LA) volume, left ventricular volumes and LVEF will be calculated using biplane volumes and indexed to body surface area (BSA). Where image quality is sub-optimal for Simpson\u0026rsquo;s biplane assessment of LVEF, a visual estimate will be given.\u003c/p\u003e\u003cp\u003eThe maximum velocity of early filling (E-wave) during diastole (EVmax), ratio of early ventricular diastolic (E-wave) and atrial filling (A-wave), and deceleration time of the mitral E-wave (DT), will be measured in the apical four chamber view (A4C) using pulsed wave doppler at the level of the mitral valve (MV) leaflet tips at end-expiration. Additionally, the velocity of early myocardial relaxation (e\u0026prime;), velocity of myocardial tissue during ventricular systole (s\u0026rsquo;) and E to early diastolic mitral annular tissue velocity (E/e\u0026prime;) will be measured in the A4C view using tissue doppler imaging. Global two-dimensional speckle tracking will be performed in the two, three and four chamber views to obtain values for global longitudinal strain (GLS). The endocardial border will be traced automatically along the region of interest at end systole and adjusted as appropriate. Peak GLS will be calculated as an average of the peak strain from the three projections. In the case of sub-optimal image quality, the data will be excluded from the final analysis.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eContinuous variables are expressed as mean+/-SD with categorical variable expressed as \u003cem\u003en\u003c/em\u003e (%), unless otherwise stated. Normality was assessed using the Shapiro\u0026ndash;Wilk test and visually assessed using quantile plots. Differences in categorical data were assessed using a X\u003csup\u003e2\u003c/sup\u003e test and differences between groups for continuous data assessed using a two-tailed paired student t-test if normally distributed and Wilcoxon signed-rank test if non-normally distributed. Bivariate correlation was assessed using Pearson linear correlation or Spearman rank correlation, if non-normally distributed. Analysis performed using SPSS V28.0 (IBM). Full details of statistical methods and power calculations for this study are provided in the Supplement.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eDemographics\u003c/h2\u003e\u003cp\u003eA total of 46 patients meeting the study inclusion criteria were enrolled in the study, with 40 patients included in the final analysis. Full recruitment details are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. At enrolment, the mean age was 67.2+/-8.2 years (29.3% female) and mean LVEF was 45.3+/-9.8%. The majority of patients were non-diabetic (95%) with a mean NYHA score of 2.4+/0.5 and a median duration from initial HF diagnosis of 74.1 months (36.4 to 115.7). All patients received SGLT2i with dapagliflozin with a median duration of follow-up following initiation of 6.8 months (6.4 to 7.1) A full description of baseline demographics and comorbidities are detailed in \u003cb\u003eTable\u0026nbsp;2\u003c/b\u003e. Additionally, a full description of baseline clinical parameters and QoL metrics are available in the \u003cb\u003eAppendix (A1)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eNovel Biomarker analysis\u003c/h3\u003e\n\u003cp\u003eThe delta change in five novel biomarkers in response to SGLT2 inhibition were assessed in our population, these included IGFBP1, sSt2, IL-1β, IL-4 and IL-6 (\u003cb\u003eTable\u0026nbsp;3\u003c/b\u003e). Of these, IL-4 was not detectable in our cohort.\u003c/p\u003e\u003cp\u003eThere was a significant decrease in levels of sSt2 following SGLT2 inhibition (mean difference \u0026minus;\u0026thinsp;13.5pg/mL (95% CI: -8.9 to -17.9; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) however, there was no significant difference in IGFBP-1 (+\u0026thinsp;3.5\u0026micro;g/mL [95% CI: -8.0 to 1.2; p\u0026thinsp;=\u0026thinsp;0.141]), IL-6 (+\u0026thinsp;1.5pg/mL [95% CI: -3.4 to 0.3; p\u0026thinsp;=\u0026thinsp;0.10]) or L-1β (+\u0026thinsp;0.21 pg/ml [95% CI -0.4 to +\u0026thinsp;0.04: p\u0026thinsp;=\u0026thinsp;0.09]) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eBiomarker correlation with GLS (%)\u003c/h2\u003e\u003cp\u003eAll patients underwent baseline and follow-up echocardiographic assessment. Of these, 36 patients had windows sufficient for interval global longitudinal strain analysis. At follow-up, there was no significant difference in GLS (%) (13.9+/-3.8% to 14.1+/-3.7%, p\u0026thinsp;=\u0026thinsp;0.803) following SGLT2 inhibition. Additionally, in our cohort, duration of therapy did not appear to be associated with degree of GLS % improvement (r -0.1, p\u0026thinsp;=\u0026thinsp;ns). A full description of echocardiographic findings are detailed in the \u003cb\u003eAppendix (A2)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eOf the novel biomarkers assessed, IL-6 reduction over time was noted to be modestly correlated with delta change in absolute GLS (%) (r\u0026thinsp;=\u0026thinsp;0.43, p\u0026thinsp;=\u0026thinsp;0.012) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). No difference in demographics or comorbidities at baseline were noted between patients who did or did not experience reverse cardiac remodeling (+\u0026thinsp;RCR, defined as \u0026ge;\u0026thinsp;10% relative improvement in GLS%). Notably, patients who had\u0026thinsp;+\u0026thinsp;RCR had a lower LVEF at baseline (41.9 vs 47.2%, p\u0026thinsp;=\u0026thinsp;0.02) (\u003cb\u003eAppendix\u003c/b\u003e, \u003cb\u003eA3\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eInterestingly, in patients who had a\u0026thinsp;\u0026ge;\u0026thinsp;10% relative improvement in GLS following SGLT2 inhibition, IL-6 was noted to fall (5.7+/-3.9 pg/ml to 5.0+/-2.9 pg/ml, p\u0026thinsp;=\u0026thinsp;0.20) whereas in patients who did not see improvements in GLS, IL-6 levels rose (4.6+/-2.7 to 5.9+/-5.9, p\u0026thinsp;=\u0026thinsp;0.2); however, neither of these trends reached statistical significance. Furthermore, change in GLS (%) was not correlated with other novel cardiac biomarkers tested following six months SGLT2 inhibitor therapy.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eNovel Biomarker prediction of improvement in GLS (%)\u003c/h2\u003e\u003cp\u003eAt a median follow-up of 196 days, +RCR was noted in 13 patients (mean relative improvement\u0026thinsp;+\u0026thinsp;24.0+/-18.3%). Baseline serum sSt2 levels significantly predicted\u0026thinsp;+\u0026thinsp;RCR (AUC 0.773; 95% CI: 0.62\u0026ndash;0.96: p\u0026thinsp;=\u0026thinsp;0.008) with a Youden Index cut-off value of 37.3ng/L yielding an 83% sensitivity. Both baseline serum IL-6 and IGFBP1 did not predict\u0026thinsp;+\u0026thinsp;RCR (AUC 0.563; 95% CI 0.36\u0026ndash;0.77: p\u0026thinsp;=\u0026thinsp;0.10) and (AUC 0.515; 95% CI 0.32\u0026ndash;0.71: p\u0026thinsp;=\u0026thinsp;0.88), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eSub-analysis of novel biomarker correlation with LVEF (%)\u003c/h2\u003e\u003cp\u003eOf the patients who underwent echocardiographic assessment, 36 had windows sufficient for Simpsons biplane analysis. Although this study was not powered to detect changes in LVEF, when assessing patients with impaired LVEF at baseline (n\u0026thinsp;=\u0026thinsp;29), a significant improvement in LVEF was noted following SGLT2 inhibition (41.8+/-6.7% to 44.1+/-9.1%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Interestingly, sSt2 was modestly negatively correlated with improvement in LVEF (r= -0.412, p\u0026thinsp;=\u0026thinsp;0.02) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Neither IL-6 nor IGFBP-1 were correlated with LVEF improvement (p\u0026thinsp;=\u0026thinsp;0.2) and (p\u0026thinsp;=\u0026thinsp;0.146) respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStandard biomarkers\u003c/h2\u003e\u003cp\u003eThe delta change in standard of care biomarkers in response to SGLT2 inhibition was also assessed (\u003cb\u003eTable\u0026nbsp;4\u003c/b\u003e). A significant increase in haematocrit (41.4+/-3.9% to 43.7+/-3.9%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and haemoglobin (14.1+/-1.4 g/dL to 14.8+/-1.4 g/dL, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was noted however no significant change in NT-proBNP (+\u0026thinsp;174.1 ng/L [95% CI: -38.1 to 33.1; p\u0026thinsp;=\u0026thinsp;0.441]) or HbA1c (+\u0026thinsp;4.7 mmol/mol [95% CI: -13.6 to 4.1; p\u0026thinsp;=\u0026thinsp;0.746)] was seen (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Additionally, no significant correlations were noted between any of the markers tested and change in GLS % following SGLT2i. A full description of these data are available in the \u003cb\u003eAppendix (A4).\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eQoL outcomes\u003c/h2\u003e\u003cp\u003eAt six months follow-up, KCCQ-12 score improved significantly (43.3+/-13.4 to 52.6+/-10.4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). No correlation was noted between delta change in KCCQ-12 score and GLS% (r\u0026thinsp;=\u0026thinsp;0.03, p\u0026thinsp;=\u0026thinsp;0.459) or LVEF % (r-0.001, p\u0026thinsp;=\u0026thinsp;0.934). Additionally, duration of therapy did not appear to influence KCCQ-12 (r\u0026thinsp;=\u0026thinsp;0.113, p\u0026thinsp;=\u0026thinsp;0.487). Furthermore, no correlation was noted between sSt2 (r\u0026thinsp;=\u0026thinsp;0.06, p\u0026thinsp;=\u0026thinsp;0.702), IL-6 (r\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.06, p\u0026thinsp;=\u0026thinsp;0.692) or IGFBP-1 (r=-0.243, p\u0026thinsp;=\u0026thinsp;0.169) and QOL score, nor was any association noted between any standard of care biomarker and symptom improvement as defined by KCCQ-12 (\u003cb\u003eAppendix 5\u003c/b\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e SGLT2 inhibitors are now a pillar of guideline directed HF care (2). Unlike traditional HF therapies they do not appear to interact with the renin-angiotensin system but rather may exert their pluripotent effects via alternative mechanisms (28,29). Indeed, their proven cardio-protective effects, which are not fully explained by their effects on natriuresis, blood pressure and blood glucose alone, have led to several hypotheses as to their mechanism of action. Much interest surrounds their potential modulatory effects on inflammatory and anti-fibrotic pathways with implication of specific cytokines such as IL-1β, IL-4 and IL-6 along with the proteins IGFBP-1 and sSt2 (9,23,27).\u003c/p\u003e\u003cp\u003eIn this study, we have demonstrated that the novel cardiac protein sSt2 is suppressed by SGLT2 inhibition and correlated with improvements in LVEF. Furthermore, baseline sSt2 exhibits a predictive utility to identify which patients treated with SLGT2i will experience improvement in cardiac function, as determined by a\u0026thinsp;\u0026ge;\u0026thinsp;10% improvement in GLS.\u003c/p\u003e\u003cp\u003esSt2 is a member of the IL-1 receptor family and has an important role in mediating inflammatory responses. Its production is stimulated by myocardial stretch with higher levels associated with higher mortality in CVD and HF (26,30). It acts by binding and inhibiting the action of IL-33, which normally acts to protect against Ang-2 driven adverse cardiac remodeling. In the context of our study, these data suggest that SGLT2 inhibition suppresses this protein, thereby attenuating activation of pro-fibrotic and pro-inflammatory pathways.\u003c/p\u003e\u003cp\u003eInterestingly, although overall IL-6 levels were not significantly affected by SGLT2 inhibition, a modest positive correlation was noted with improvements in GLS. Additionally, there seemed to be a dichotomous signal with IL-6 trending up in patients with no improvements in GLS and down in those with improvements in GLS. Albeit, this trend was non-significant, in conjunction with the significant correlation data it may serve to highlight a possible signal which may become clearer with a larger sample size or possibly longer duration of therapy. IL-6 is a pro-inflammatory cytokine associated with coronary artery disease, insulin resistance and endothelial dysfunction. Animal studies have demonstrated that SGLT2 inhibition promotes HKII and ERK1/2 mediated suppression of IL-6 levels with early observational clinical data re-iterating these findings (31). Indeed, in a mouse model of HF, chronic activation of IL-6 has been demonstrated to promote LVH with subsequent deletion reversing these effects (32). This has led to much interest in targeting IL-6 as a therapy in patients with myocardial dysfunction, notably in the recent study, ASSAIL-MI, where administration of tocilizumab (a biologic IL-6 inhibitor) reduced infarct size and improved viable myocardial tissue post STEMI as demonstrated via cardiac MRI (33). In our study, the trend observed in IL-6 taken in context with sSt2 provides a suggestion that SGLT2i may promote reverse cardiac remodeling by suppression of inflammatory and pro-fibrotic pathways. To our knowledge, this has not been previously demonstrated in this context.\u003c/p\u003e\u003cp\u003eDespite promising pre-clinical data, in our cohort, IGFBP-1 did not change significantly following SGLT2 inhibition nor was it correlated with or have any predictive utility for identifying any marker of cardiac remodeling. We did not detect IL-4 in either pre or post treatment samples. IL-1β was only detectable in half our cohort with so significant change in levels following SGLT2 inhibitor therapy. In our population, we saw significant improvement in QoL metrics, however these were not correlated with novel biomarkers or echocardiographic markers of reverse cardiac remodeling. Moreover, despite the significant increases in haemoglobin and haematocrit (which plausibly may improve preload, cardiac output and oxygenation), neither of these markers were correlated with symptom improvement. This finding is reflected in data from the landmark SGLT2i trials such as DAPA-HF and EMPORER-REDUCED, where the symptomatic benefits of SGLT2 inhibition were noted very early (within weeks), before biomarker changes (10,34). Therefore, it is plausible that these occur independently to observed echocardiographic or biomarker changes and again, a longer duration of follow-up may facilitate a meaningful change in these parameters.\u003c/p\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eLIMITATIONS\u003c/h2\u003e\u003cp\u003eThere are several limitations to this study. Firstly, given it is a prospective observational study it is exposed to selection bias and risk of confounding factors. Furthermore, general interpretation of this data is limited to association of effect and not causation. Secondly, this study was powered to detect changes in IL-6, sSt2 and GLS, and not to detect changes in standard of care biomarkers nor other echocardiographic findings such as LVEF. Thirdly, our follow-up period was limited to six months, which may not be sufficient to fully realise echocardiographic changes secondary to SGLT2 inhibition. Fourthly, our median time from HF diagnosis was over six years therefore the majority of patients in this group had well established HF, with limited scope for reverse cardiac remodeling. It is plausible that this may have limited the potential impact of SGLT2 inhibition on both novel biomarkers and echocardiographic parameters.\u003c/p\u003e\u003c/div\u003e\u003c"},{"header":"SUMMARY","content":"\u003cp\u003eIn this study of stable HF patients treated with SGLT2 inhibition, we have demonstrated an inhibitory effect on the novel cardiac protein Sst2 which was modestly correlated with improvements in LVEF. Additionally, we noted a modest positive correlation between IL-6 and improvements in global longitudinal strain. These effects appeared to be independent of symptom improvement and baseline demographics. Collectively, these data support the implication of SGLT2 inhibition in suppression of pro-fibrotic and pro-inflammatory pathways in patients with HF.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSources of funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatrick Savage is a clinical research fellow investigating the effects of SGLT2 inhibition in heart failure. His work is supported in the form of charitable funding received from the Belfast Heart Trust Fund. Chris Watson is supported by British Heart Foundation grant investigating novel therapeutics for diabetes and heart failure (PG/20/10424).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnonymised data will be made available upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Patrick Savage. The first draft of the manuscript was written by Patrick Savage and all authors commented on previous versions of the manuscript. All authors read, contributed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval \u0026amp; consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with Good Clinical Practice and the Declaration of Helsinki. Ethical approval was approved by the UK Research Ethics and Research Ethics Committee (22/NW/0348). This trial is registered as Clinical Trials.gov identifier: NCT06140251. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed, written consent was obtained from all individual participants included in the study\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsent to publish the results of the study were obtained.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZinman B, Wanner C, Lachin JM, Fitchett D, Bluhmki E, Hantel S, et al. Empagliflozin, Cardiovascular Outcomes, and Mortality in Type 2 Diabetes. N Engl J Med. 2015 Nov 26;373(22):2117\u0026ndash;28.\u003c/li\u003e\n\u003cli\u003eMcDonagh TA, Metra M, Adamo M, Gardner RS, Baumbach A, B\u0026ouml;hm M, et al. 2023 Focused Update of the 2021 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure. Eur Heart J. 2023 Aug 25;ehad195.\u003c/li\u003e\n\u003cli\u003eVaduganathan M, Docherty KF, Claggett BL, Jhund PS, Boer RA de, Hernandez AF, et al. SGLT2 inhibitors in patients with heart failure: a comprehensive meta-analysis of five randomised controlled trials. The Lancet. 2022 Sep 3;400(10354):757\u0026ndash;67.\u003c/li\u003e\n\u003cli\u003eBenham JL, Booth JE, Sigal RJ, Daskalopoulou SS, Leung AA, Rabi DM. Systematic review and meta-analysis: SGLT2 inhibitors, blood pressure and cardiovascular outcomes. Int J Cardiol Heart Vasc. 2021 Feb 10;33:100725.\u003c/li\u003e\n\u003cli\u003eZannad F, Ferreira JP, Pocock SJ, Anker SD, Butler J, Filippatos G, et al. SGLT2 inhibitors in patients with heart failure with reduced ejection fraction: a meta-analysis of the EMPEROR-Reduced and DAPA-HF trials. Lancet Lond Engl. 2020 Sep 19;396(10254):819\u0026ndash;29.\u003c/li\u003e\n\u003cli\u003eRoddick AJ, Wonnacott A, Webb D, Watt A, Watson MA, Staplin N, et al. UK Kidney Association Clinical Practice Guideline: Sodium-Glucose Co-transporter-2 (SGLT-2) Inhibition in Adults with Kidney Disease 2023 UPDATE. BMC Nephrol. 2023 Oct 25;24(1):310.\u003c/li\u003e\n\u003cli\u003eVrints C, Andreotti F, Koskinas KC, Rossello X, Adamo M, Ainslie J, et al. 2024 ESC Guidelines for the management of chronic coronary syndromes. Eur Heart J. 2024 Sep 29;45(36):3415\u0026ndash;537.\u003c/li\u003e\n\u003cli\u003eSGLT-2 inhibitors | Prescribing information | Diabetes - type 2 | CKS | NICE [Internet]. [cited 2025 Apr 30]. Available from: https://cks.nice.org.uk/topics/diabetes-type-2/prescribing-information/sglt-2-inhibitors/\u003c/li\u003e\n\u003cli\u003ePacker M. Critical Reanalysis of the Mechanisms Underlying the Cardiorenal Benefits of SGLT2 Inhibitors and Reaffirmation of the Nutrient Deprivation Signaling/Autophagy Hypothesis. Circulation. 2022 Nov;146(18):1383\u0026ndash;405.\u003c/li\u003e\n\u003cli\u003eMcMurray JJV, Solomon SD, Inzucchi SE, K\u0026oslash;ber L, Kosiborod MN, Martinez FA, et al. Dapagliflozin in Patients with Heart Failure and Reduced Ejection Fraction. N Engl J Med. 2019 Nov 21;381(21):1995\u0026ndash;2008.\u003c/li\u003e\n\u003cli\u003eSavage P, Dixon L, Grieve D, Watson C. SGLT2 Inhibition in Heart Failure: Clues to Cardiac Effects? Cardiol Rev. 2024 Jan 8;\u003c/li\u003e\n\u003cli\u003eXiao L, Nie X, Cheng Y, Wang N. Sodium-Glucose Cotransporter-2 Inhibitors in Vascular Biology: Cellular and Molecular Mechanisms. Cardiovasc Drugs Ther. 2021 Dec;35(6):1253\u0026ndash;67.\u003c/li\u003e\n\u003cli\u003eHammer A, Niessner A, Sulzgruber P. Early Initiation of SGLT2 Inhibitors in Acute Heart Failure: a Focus on Diuresis and Renal Protection. Cardiovasc Drugs Ther. 2025 Jun;39(3):687\u0026ndash;90.\u003c/li\u003e\n\u003cli\u003eSavage P, Watson C, Coburn J, Cox B, Shahmohammadi M, Grieve D, et al. Impact of SGLT2 inhibition on markers of reverse cardiac remodelling in heart failure: Systematic review and meta-analysis. ESC Heart Fail. 2024 Dec;11(6):3636\u0026ndash;48.\u003c/li\u003e\n\u003cli\u003eVerma S, Mazer CD, Yan AT, Mason T, Garg V, Teoh H, et al. Effect of Empagliflozin on Left Ventricular Mass in Patients With Type 2 Diabetes Mellitus and Coronary Artery Disease: The EMPA-HEART CardioLink-6 Randomized Clinical Trial. Circulation. 2019 Nov 19;140(21):1693\u0026ndash;702.\u003c/li\u003e\n\u003cli\u003eBrown AJM, Gandy S, McCrimmon R, Houston JG, Struthers AD, Lang CC. A randomized controlled trial of dapagliflozin on left ventricular hypertrophy in people with type two diabetes: the DAPA-LVH trial. Eur Heart J. 2020 Sep 21;41(36):3421\u0026ndash;32.\u003c/li\u003e\n\u003cli\u003eAlsereidi FR, Khashim Z, Marzook H, Gupta A, Al-Rawi AM, Ramadan MM, et al. Targeting inflammatory signaling pathways with SGLT2 inhibitors: Insights into cardiovascular health and cardiac cell improvement. Curr Probl Cardiol. 2024 May;49(5):102524.\u003c/li\u003e\n\u003cli\u003eKoshino A, Schechter M, Sen T, Vart P, Neuen BL, Neal B, et al. Interleukin-6 and Cardiovascular and Kidney Outcomes in Patients With Type 2 Diabetes: New Insights From CANVAS. Diabetes Care. 2022 Nov 1;45(11):2644\u0026ndash;52.\u003c/li\u003e\n\u003cli\u003eKim SR, Lee SG, Kim SH, Kim JH, Choi E, Cho W, et al. SGLT2 inhibition modulates NLRP3 inflammasome activity via ketones and insulin in diabetes with cardiovascular disease. Nat Commun. 2020 May 1;11(1):2127.\u003c/li\u003e\n\u003cli\u003eHunter CA, Jones SA. IL-6 as a keystone cytokine in health and disease. Nat Immunol. 2015 May;16(5):448\u0026ndash;57.\u003c/li\u003e\n\u003cli\u003eMehta NN, deGoma E, Shapiro MD. IL-6 and Cardiovascular Risk: A Narrative Review. Curr Atheroscler Rep. 2024;27(1):12.\u003c/li\u003e\n\u003cli\u003eSu JH, Luo MY, Liang N, Gong SX, Chen W, Huang WQ, et al. Interleukin-6: A Novel Target for Cardio-Cerebrovascular Diseases. Front Pharmacol. 2021 Aug 24;12:745061.\u003c/li\u003e\n\u003cli\u003eGohari S, Ismail-Beigi F, Mahjani M, Ghobadi S, Jafari A, Ahangar H, et al. The effect of sodium-glucose co-transporter-2 (SGLT2) inhibitors on blood interleukin-6 concentration: a systematic review and meta-analysis of randomized controlled trials. BMC Endocr Disord. 2023 Nov 24;23:257.\u003c/li\u003e\n\u003cli\u003eMancini SJ, Boyd D, Katwan OJ, Strembitska A, Almabrouk TA, Kennedy S, et al. Canagliflozin inhibits interleukin-1\u0026beta;-stimulated cytokine and chemokine secretion in vascular endothelial cells by AMP-activated protein kinase-dependent and -independent mechanisms. Sci Rep. 2018 Mar 27;8:5276.\u003c/li\u003e\n\u003cli\u003ePirklbauer M, Sallaberger S, Staudinger P, Corazza U, Leierer J, Mayer G, et al. Empagliflozin Inhibits IL-1\u0026beta;-Mediated Inflammatory Response in Human Proximal Tubular Cells. Int J Mol Sci. 2021 May 11;22(10):5089.\u003c/li\u003e\n\u003cli\u003eDudek M, Kałużna-Oleksy M, Migaj J, Sawczak F, Krysztofiak H, Lesiak M, et al. sST2 and Heart Failure\u0026mdash;Clinical Utility and Prognosis. J Clin Med. 2023 Apr 26;12(9):3136.\u003c/li\u003e\n\u003cli\u003eZannad F, Ferreira JP, Butler J, Filippatos G, Januzzi JL, Sumin M, et al. Effect of empagliflozin on circulating proteomics in heart failure: mechanistic insights into the EMPEROR programme. Eur Heart J. 2022 Dec 21;43(48):4991\u0026ndash;5002.\u003c/li\u003e\n\u003cli\u003eNassif ME, Windsor SL, Tang F, Khariton Y, Husain M, Inzucchi SE, et al. Dapagliflozin Effects on Biomarkers, Symptoms, and Functional Status in Patients With Heart Failure With Reduced Ejection Fraction. Circulation. 2019 Oct 29;140(18):1463\u0026ndash;76.\u003c/li\u003e\n\u003cli\u003eTanaka A, Hisauchi I, Taguchi I, Sezai A, Toyoda S, Tomiyama H, et al. Effects of canagliflozin in patients with type 2 diabetes and chronic heart failure: a randomized trial (CANDLE). ESC Heart Fail. 2020 Aug;7(4):1585\u0026ndash;94.\u003c/li\u003e\n\u003cli\u003eWatson CJ, Gallagher J, Wilkinson M, Russell-Hallinan A, Tea I, James S, et al. Biomarker profiling for risk of future heart failure (HFpEF) development. J Transl Med. 2021 Feb 9;19(1):61.\u003c/li\u003e\n\u003cli\u003eUthman L, Kuschma M, R\u0026ouml;mer G, Boomsma M, Kessler J, Hermanides J, et al. Novel Anti-inflammatory Effects of Canagliflozin Involving Hexokinase II in Lipopolysaccharide-Stimulated Human Coronary Artery Endothelial Cells. Cardiovasc Drugs Ther. 2021 Dec;35(6):1083\u0026ndash;94.\u003c/li\u003e\n\u003cli\u003eZhao L, Cheng G, Jin R, Afzal MR, Samanta A, Xuan YT, et al. Deletion of Interleukin-6 Attenuates Pressure Overload-Induced Left Ventricular Hypertrophy and Dysfunction. Circ Res. 2016 Jun 10;118(12):1918\u0026ndash;29.\u003c/li\u003e\n\u003cli\u003eBroch K, Anstensrud AK, Woxholt S, Sharma K, T\u0026oslash;llefsen IM, Bendz B, et al. Randomized Trial of Interleukin-6 Receptor Inhibition in Patients With\u0026nbsp;Acute ST-Segment Elevation Myocardial Infarction. J Am Coll Cardiol. 2021 Apr 20;77(15):1845\u0026ndash;55.\u003c/li\u003e\n\u003cli\u003ePacker M, Anker SD, Butler J, Filippatos G, Pocock SJ, Carson P, et al. Cardiovascular and Renal Outcomes with Empagliflozin in Heart Failure. N Engl J Med. 2020 Oct 8;383(15):1413\u0026ndash;24.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cardiovascular-drugs-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cdty","sideBox":"Learn more about [Cardiovascular Drugs and Therapy](https://www.springer.com/journal/10557)","snPcode":"10557","submissionUrl":"https://submission.nature.com/new-submission/10557/3","title":"Cardiovascular Drugs and Therapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"HEART FAILURE, SGLT2 Inhibitors, BIOMARKERS, ECHOCARDIOGRAPHY","lastPublishedDoi":"10.21203/rs.3.rs-7301417/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7301417/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eIntroduction\u003c/h2\u003e\u003cp\u003e Following several landmark trials, sodium glucose co-transport (SGLT) 2 inhibitors, have been established as a guideline directed therapy for heart failure (HF). Moreover, their benefit has been established across the spectrum of left ventricular (LV) dysfunction. Much remains unclear regarding their mechanism of action with current evidence implicating pathways involvement of inflammatory, autophagic and anti-fibrotic pathways.\u003c/p\u003e\u003ch2\u003eAim\u003c/h2\u003e\u003cp\u003eWe therefore sought to evaluate the effects of SGLT2 inhibition on cardiac biomarkers, myocardial remodelling and patient reported outcomes in heart failure.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThis was a 26-week, single-arm prospective evaluation of the effects of SGLT2 inhibition on novel biomarkers, myocardial remodeling and patient reported outcomes in patients with heart failure. Baseline echocardiography, serum analysis (standard care and novel biomarkers) and quality of life (QoL) metrics were assessed prior to SGLT2i therapy and at 26-week follow-up. Novel biomarkers were analysed using enzyme-linked immunosorbent assays. Data were analysed using SPSS (IBM SPSS Statistics, Version 28.0). Clinical Trials.gov identifier: \u003cem\u003eNCT06140251\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eForty-six patients were recruited with forty patients undergoing biomarker analysis (mean age 67.2+/-8.2years: 68.3% female). Mean LV ejection fraction (LVEF) at baseline was 45.3+/9.8% (ischaemic aetiology: 40.0%, diabetic: 5%). At a median follow-up of 196 days, soluble suppression of tumorigenicity 2 (sSt2) fell significantly (mean difference \u0026minus;\u0026thinsp;13.5pg/ml [95% CI: -17.9 to \u0026minus;\u0026thinsp;8.9: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001]), with no significant change in interleukin (IL)1-β, IL-4, IL-6 or insulin growth factor binding protein 1 (IGFBP-1) (all p\u0026thinsp;=\u0026thinsp;ns). Interestingly, delta change in IL-6 modestly correlated with change in global longitudinal strain (GLS) (%) (r=-0.43, p\u0026thinsp;=\u0026thinsp;0.012). Change in GLS (%) was not correlated with other novel cardiac biomarkers.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eIn our cohort we found that sSt2, a protein implicated in cardiac fibrosis, was suppressed by SGLT2i. Additionally, we observed that suppression of IL-6, a marker of inflammation, correlated with reverse cardiac remodeling. These data support the implication of SGLT2i in suppression of fibrotic and inflammatory pathways. More exploration of these associations is warranted.\u003c/p\u003e","manuscriptTitle":"Investigation of Biomarker Response to SGLT2 Inhibition in Heart Failure (SiN-HF)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 06:59:18","doi":"10.21203/rs.3.rs-7301417/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Changes Required","date":"2025-08-26T03:36:09+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-08-13T23:09:19+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-13T11:30:42+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Cardiovascular Drugs and Therapy","date":"2025-08-13T08:07:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-13T05:28:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cardiovascular Drugs and Therapy","date":"2025-08-12T05:00:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cardiovascular-drugs-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cdty","sideBox":"Learn more about [Cardiovascular Drugs and Therapy](https://www.springer.com/journal/10557)","snPcode":"10557","submissionUrl":"https://submission.nature.com/new-submission/10557/3","title":"Cardiovascular Drugs and Therapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f38df69c-95a0-4150-a414-a6818119060a","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T16:06:20+00:00","versionOfRecord":{"articleIdentity":"rs-7301417","link":"https://doi.org/10.1007/s10557-025-07800-3","journal":{"identity":"cardiovascular-drugs-and-therapy","isVorOnly":false,"title":"Cardiovascular Drugs and Therapy"},"publishedOn":"2025-12-05 15:57:14","publishedOnDateReadable":"December 5th, 2025"},"versionCreatedAt":"2025-08-22 06:59:18","video":"","vorDoi":"10.1007/s10557-025-07800-3","vorDoiUrl":"https://doi.org/10.1007/s10557-025-07800-3","workflowStages":[]},"version":"v1","identity":"rs-7301417","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7301417","identity":"rs-7301417","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}