Table of Contents
Study setting and patients
In this double-blind, randomized trial, adult patients with type 2 diabetes received placebo or dapagliflozin 10 mg once daily for 12 months. The study began on February 26, 2019, and patients' initial screening and follow-up visits were conducted at the Clinical Atherosclerosis Laboratory at the University of Washington Harborview Medical Center. The study was completed on November 16, 2022.
Inclusion Criteria
Age > 18 years, history of type 2 diabetes > 5 years, hemoglobin A1c (7–10%), glycemic control medications: insulin, metformin, and/or sulfonylureas.
Exclusion criteria
Current use of SGLT2 inhibitors, hypersensitivity to SGLT2 inhibitors, diagnosis of heart failure, contraindication to MRI, eGFR < 45 ml/min/1.73m2; unstable or progressive renal disease, SBP < 100 mmHg, severe liver disease (Child-Pugh class C), active hepatitis B or C within 3 months prior to enrollment, cardiovascular disease (myocardial infarction, CABG, coronary intervention, transient ischemic attack, stroke, PAD), bladder cancer, or high risk of diabetic ketoacidosis, high risk of fracture (osteoporosis, osteopenia).
Enrollment and Randomization
Patients with type 2 diabetes without a known diagnosis of heart failure (by ICD code) were enrolled in the study. Transthoracic echocardiography (TTE) was not part of the screening protocol, and only a few subjects underwent TTE according to standard of care before enrollment. During the enrollment period, 95 patients were assessed for eligibility, 62 patients were randomized, and 56 patients completed the study protocol (Figure 1). Randomization was stratified according to the use of glucagon-like peptide (GLP-1) and angiotensin II receptor blockers (ARBs).
CONSORT study flow diagram. MR, magnetic resonance, DM, diabetes
Study interventions
After a screening visit and informed consent, patients were randomized 1:1 to receive placebo or dapagliflozin 10 mg 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) respiratory assessment and plasma samples were collected for cytokine and ketone measurements. Patients also underwent baseline CMRI and clinical assessments. Patients underwent clinical visits at 3, 6, and 9 months, and at the 12-month visit, patients underwent a final CMRI and collection of blood and plasma samples.
Achievement
Primary endpoint: Change in global myocardial strain and ECV assessed by T1 mapping (from baseline to 12 months).
Secondary endpoints: Changes in plasma IL-1B, TNFα, IL-6, IL-10, plasma ketones, and T2 relaxation times (from baseline to 12 months).
Exploratory Results: Basal and maximal oxygen consumption rates of PBMC mitochondria measured by the Seahorse XF Analyzer.
Plasma cytokine and ketone quantification
Plasma samples were obtained from whole blood drawn after 2000 gx 10 min at 4 °C in vacutainers containing EDTA and stored at -80 °C. Plasma concentrations of cytokines were measured by ELISA according to the 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 measured with EnzyChrom™ Ketone Body Assay Kits according to the manufacturer's protocol (BioAssay Systems, Cat: EKBD-100).
PBMC oxygen consumption rate (OCR) measurement
PBMCs were isolated from whole blood collected in acid citrate dextrose vacutainers after density gradient centrifugation (Histopaque-1077, Sigma-Aldrich Catalog No.: 10,771). Freshly isolated PBMCs were resuspended in Seahorse XF medium (Cat. No.: 102353-100) and then plated (106 PBMC mitochondrial respiratory function was assessed by measuring OCR under basal and maximally stimulated conditions using a Seahorse XFe24 Analyzer as previously described. [13].
Cardiac magnetic resonance imaging
CMRI examinations were performed on a 3T clinical whole-body scanner (Ingenia, Phillips®) at the Biomolecular Imaging Center (BMIC) at the University of Washington South Lake Union campus. The CMRI protocol included steady-state free precession (SSFP) cine imaging to measure cardiac left ventricular volumes (assessing dilation and hypertrophy), systolic function, and myocardial strain, naïve and post-contrast T1 mapping and ECV fraction to assess diffuse myocardial fibrosis changes, T2 mapping to assess myocardial inflammation, T2* mapping to assess iron deposition, and late gadolinium enhancement to visualize focal fibrosis.
All image acquisitions were performed with ECG gating and breath-hold technique. Imaging parameters are shown in Supplementary 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 indices after adjustment for body surface area. Variables are compared with age-specific normal ranges reported in the literature.
ECV maps were generated offline using MATLAB software. ECV was calculated from the native and post-contrast T1 values of blood and myocardial tissue, the partition coefficient lambda (λ), and hematocrit using the following formulas: ECV = λ(1-hematocrit); λ = (1/T1 post-myocardial contrast – 1/T1 myocardial native)/(1/T1 blood post-contrast – 1/T1 blood native).
To accurately calculate parametric mapping values, the ROI was placed on a mid-ventricular short-axis slice of the left ventricle, covering the entire circumference of the left ventricle with a recess of 1 mm from the edge of the left ventricle to exclude signal contamination from blood and surrounding tissues. [14]As shown in Figure 2B, all parametric maps had the same ROI shape and location.
Circle Cardiovascular Software (cvi-42, Circle Cardiovascular Imaging Inc., Calgary, Alberta, Canada) was used to perform feature tracking and measure myocardial strain and strain rate from short-axis and long-axis cine images of bSSFP. Long-axis cine images were further used to calculate global longitudinal myocardial strain. Short-axis images were used to calculate circumferential and radial strain and strain rate. Global values were obtained by averaging values according to the American Heart Association 17-segment model. [15].
Statistical analysis
Systemic inflammatory endpoints (plasma cytokines, plasma ketones, PBMC OCR) were compared between baseline and 1-year post-intervention values in the dapagliflozin and placebo groups. P values were determined by paired two-tailed t-tests. Parametric t-tests were used if the distributions passed normality tests, and nonparametric t-tests (Wilcoxon) were used if they did not.
For CMRI results, baseline characteristics were compared between the dapagliflozin and placebo groups, with age expressed as mean and standard deviation, and categorical variables as numbers and percentages. Differences between baseline and 1-year outcomes were calculated for primary outcomes between the dapagliflozin and placebo groups. Within each group, differences between baseline and 1-year outcomes were assessed with paired t-tests. Differences between drug and placebo groups were compared with Wilcoxon rank-sum tests.
To adjust for multiple comparisons, the statistical significance level was set at 0.0125 (0.05/4) for the primary outcome and 0.0083 (0.05/6) for the secondary outcomes.
Ethical oversight
The study was approved by the University of Washington Institutional Review Board.
