Study setting and patients

This double-blind, randomized trial assigned adults with T2D to placebo or 10 mg dapagliflozin daily for 12 months. The study began on 2/26/2019; initial screening of patients and follow up visits occurred at the Clinical Atherosclerosis Research Laboratory at Harborview Medical Center, University of Washington. The study was completed on 11/16/2022.

Inclusion criteria

Age > 18; T2D history > 5 years; Hemoglobin A1c (7–10%), glucose control medication: insulin, metformin, and/or sulfonylurea.

Exclusion criteria

Current use of SGLT2 inhibitor; hypersensitivity to SGLT2 inhibitor; diagnosis of heart failure; contraindications to MRI; eGFR < 45 ml/min/1.73m2; unstable or progressing renal disease; SBP < 100 mmHg; severe hepatic disease (Child-Pugh Class C); active hepatitis B or C, CV disease within 3 months before enrollment (myocardial infarction; CABG, coronary intervention; TIA; stroke, PAD); Bladder cancer; or high risk of diabetic ketoacidosis, high risk of fracture (osteoporosis, osteopenia).

Enrollment and randomization

T2D patients without known heart failure diagnosis (by ICD code) were enrolled in the study. Transthoracic echocardiogram (TTE) was not part of the screening protocol and only a small percentage of the study subjects underwent TTE as per standard of care prior to enrollment. During the enrollment period, 95 patients were assessed for eligibility, 62 patients were randomized, 56 patients completed the study protocol (Fig. 1). Randomization was stratified according to use of glucagon-like peptide (GLP-1) and angiotensin-II receptor blockers (ARBs).

Fig. 1

Trial CONSORT Flow Diagram. MR, magnetic resonance; DM, diabetes mellites

Study intervention

Following screening visit and informed consent, patients were randomized 1:1 to placebo or 10 mg dapagliflozin daily for 12 months. Randomization was performed by the Investigational Drug Services at Harborview Medical Center. During the randomization visit, blood samples were collected for peripheral blood mononuclear cell (PBMC) respiration assessment and plasma samples were collected for cytokine and ketone measurements. Patients also received a baseline CMRI and laboratory evaluation. Patients had clinical visits at 3, 6, 9 months, and at the 12-month visit patients underwent a final CMRI along with blood and plasma sample collection.

Outcomes

Primary outcomes: Changes in global myocardial strain and ECV as assessed by T1 mapping (baseline to 12 months).

Secondary outcomes: Changes in plasma IL-1B, TNFα, IL-6, IL-10, plasma ketones, T2 relaxation time (baseline to 12 months).

Exploratory outcome: PBMC mitochondrial basal and maximal oxygen consumption rate determined by Seahorse XF Analyzer.

Plasma cytokine and Ketone Quantifications

Plasma samples were obtained from whole blood collected in EDTA-containing vacutainers post 2000 g x 10’ at 4 °C and stored in − 80 °C. Plasma concentrations of cytokines were determined by ELISA following manufacturer’s protocol (Biolegend): IL-1B (Cat: 437,004), TNFα (Cat: 430,204), IL-6 (Cat: 430,504), and IL-10 (Cat: 430,601). Plasma concentrations of β-hydroxybutyrate and acetoacetate were determined by EnzyChrom™ Ketone Body Assay Kit following manufacturer’s protocol (BioAssay Systems, Cat: EKBD-100).

PBMC Oxygen Consumption Rate (OCR) Measurement

PBMC was isolated from whole blood collected in acid-citrate-dextrose vacutainers post density gradient (Histopaque-1077, Sigma-Aldrich Cat: 10,771) centrifugation. Freshly isolated PBMCs were resuspended in Seahorse XF medium (Cat: 102353-100) and then plated (106 cells per well) onto Seahorse XFe24 cell culture plate. PBMC mitochondrial respiratory function was assessed by measuring the OCR at basal and maximal stimulated conditions using Seahorse XFe24 Analyzer as described previously [13].

Cardiac magnetic resonance imaging

CMRI examination was done at a 3T clinical whole-body scanner (Ingenia, Phillips®) located at the BioMolecular Imaging Center (BMIC) at the University of Washington, South Lake Union campus. CMRI protocol included: steady state free precession (SSFP) cine imaging to measure heart LV chamber volumes (assessing dilatation and hypertrophy), contractile function and myocardial strain; naïve and post-contrast T1 mapping and ECV fraction to assess changes in diffused myocardial fibrosis; T2 mapping to assess myocardial inflammation; T2* mapping to assess iron deposition; Late gadolinium enhancement for visualize focal fibrosis.

All imaging acquisitions were done with ECG gating and breath hold technique. Imaging parameters are shown in Supplemental Table 1.

Image processing and analysis

Volumetric LV analysis and analysis of quantitative maps (T1, T2, T2*) were performed using Philips IntelliSpace Portal (ISP) software. Volumetric parameters are reported as indexes, after adjustment for body surface area. Variables are compared to normal age specific ranges reported in the literature.

ECV maps were generated offline using MATLAB software. ECV was calculated from native and post-contrast T1 values for blood and myocardial tissue, the partition coefficient lambda (λ), and hematocrit using the following formulas: ECV = λ(1-hematocrit); λ = (1/T1 myocardium post-contrast-1/T1 myocardium-native)/(1/T1 blood post-contrast-1/T1 blood-native).

For precise calculation of parametric mapping values, the ROI was placed in the mid-ventricular short-axis slice of the left ventricle covering the whole circumference of the left ventricle with 1 mm indentation from the LV edge to exclude signal contamination from blood and surrounding tissues [14]. All parametric maps had the same ROI shape and location as shown in Fig. 2B.

Feature tracking was performed using Circle Cardiovascular Software (cvi-42, Circle Cardiovascular Imaging Inc., Calgary, Alberta, Canada) to measure myocardial strain and strain rate from the bSSFP short-axis and long-axis cine images. Long-axis cine images were further used to compute global myocardial longitudinal strain. Short-axis images were used to compute circumferential and radial strain and strain rate. The global values were obtained through averaging the values according to an American Heart Association 17-segment model [15].

Statistical analysis

For systemic inflammatory endpoints (plasma cytokines, plasma ketones, and PBMC OCR), we compared baseline and 1-year post-intervention values in the dapagliflozin and placebo groups. P-values were determined by paired two-tailed t-test. Parametric t-test was used if distribution passes normality test, otherwise non-parametric t-test (Wilcoxon) was used.

For CMRI outcomes, we compared baseline characteristics in the dapagliflozin and placebo groups and expressed age as mean and standard deviation and categorical variables as numbers and percents. The difference between baseline and 1-year results was calculated for the primary outcomes in the dapagliflozin and placebo groups. Within each group, the difference between baseline and 1 year was assessed with the paired t-test. The differences between drug and placebo groups were compared with the Wilcoxon Rank Sum Test.

To adjust for multiple comparisons, the level of statistical significance was set at 0.0125 (0.05/4) for primary outcomes, and 0.0083 (0.05/6) for secondary outcomes.

Ethical oversight

The trial was approved by the Institutional Review Board at the University of Washington.