Age-matched subjects with T2D (n=7) and healthy controls (n=7) were included. In total, fifteen men volunteered for the study. One person was excluded in the T2D group due to lack of parameters confirming T2D. Written informed consent was obtained prior to data collection. The study was approved by the Regional Comittee for Medical Research Ethics Central Norway, REK Central (REC ID 2016/1596) and is in conformance with the statement of ethical principles for medical research outlined in the Declaration of Helsinki. All methods were performed in accordance with the relevant guidelines and in agreement with the approval from the ethical committee. The study is registered in ClinicalTrials.gov Identifier: NCT02998008 (First posted: 20/12/2016).
Test procedure and protocols
Participant registration and timeline
Day 1. All participants completed a questionnaire from a medical doctor evaluating the health status and medical consent for training and cardiopulmonary exercise testing. Blood pressure was measured following 10 minutes of rest in a chair. Three tests were performed and the average of the latter two was used. Thereafter, height was measured in cm, followed by an Inbody scan (Inbody, AU). Subsequently, a Holter electrocardiogram (ECG) monitored the cardiac electrical activity 24 hours prior to, during and 24 hours after exercise. Lastly, a blood sample was collected from the participants at the end of day 1.
Day 2. Echocardiography was first performed immediately before the single bout of exhaustive exercise consisting of 4x4 minutes interval exercise and one last bout performed as a ramp protocol to assess peak oxygen uptake (VO2peak). After the exercise all participants rested for 30 minutes before a second echocardiography was done. At the end of day 2, 30 minutes after the second echocardiography, a second blood sample was collected.
Day 3. Participants ended Holter ECG monitoring and one last blood sample was collected 24 hours after the exercise test.
Interval training and maximal oxygen uptake
The exercise was performed as a 4x4 minutes high intensity interval training. The intervals were conducted at about 90% of VO2peak and were followed by 2-minute breaks with active recovery at 60%. We then added a fifth interval performed as a ramp protocol with increments in speed and/or inclination to measure VO2peak. The cardiopulmonary exercise testing (CPET) to measure VO2peak was performed at the NeXt Move core facility at NTNU - Norwegian University of Science and Technology. All participants were regularly offered water during and after exercise to avoid hypovolemia. As diabetic patients sometimes exhibit physical limitations, experienced personnel determined the best in dividual CPET regimen during a 6-minute warm-up on the treadmill (Woodway PPS55, USA Inc., Waukesha, WI, USA), by detecting functional walking or running speed and inclination, as well as subjective moderate aerobic intensity based on rated perceived exertion (RPE Borg scale 6-20). Subjects were then fitted with a heart rate monitor (H7, Polar Electro, Kempele, Finland) and facemask (7450 Series V2 CPET mask, Hans Rudolph Inc., Shawnee, KS, USA). During an initial period of 4 minutes at fixed submaximal workload serving as an extended warm-up, work economy measurements were made.
Maximal oxygen uptake (VO2max) was defined using the following criteria: 1) VO2 levelling off (<2 ml min–1 kg–1) despite increase in workload and 2) Respiratory exchange ratio ≥1.05. If these criteria were not met, the term VO2peak was used. A subject’s VO2peakwas defined as the mean of the three successive highest VO2 registrations achieved during the CPET. For simplicity, the term VO2peak is used for all patients.
An individualized ramp protocol was used, until either exhaustion or fulfilment of the criteria for VO2max or VO2peak Workload was gradually increased, and gas measurements were recorded every tenth second using a mixing chamber ergospirometry system (Metalyzer II, Cortex Biophysik Gmbh, Leipzig, Germany).
Echocardiographic recordings and analyses of the different chambers followed the recommendation by the American and European societies of Echocardiography 42. Transthoracic echocardiography (TTE) was performed by one experienced cardiologist. All participants were examined in the left-lateral decubitus position. A Vivid E95 scanner with a phased-array transducer (M5S) (GE Ultrasound, Horten, Norway) was used. Echocardiographic data were stored digitally and analyzed after end of study inclusion by the same cardiologist. All echocardiograms were acquired with the operator blinded to group assignment to avoid any bias in the analyses. All analyses were performed by the same operator blinded to group assignment and whether the echocardiogram was recorded before or after the exhaustive exercise session. All measurements reflect the average of three cardiac cycles, as recommended for patients in sinus rhythm. The measurements are reported as absolute values and not indexed to body surface area.
Grey-scale two-dimensional (2D) views were recorded from the parasternal border in short- and long-axis, and the apical position in 4-chamber, 2-chamber, and long-axis views. Separate recordings were made to optimize the volumetric measurements of the specific chambers, and similarly care was taken to avoid foreshortening and misalignment. Linear measurements of the LV myocardium and dimensions were done in parasternal long-axis recordings at end-diastole and end-systole immediately below the level of the mitral valve leaflet tips. The fractional shortening was calculated by the change in LV dimension divided by the end-diastolic dimension. Left atrial (LA) and LV volumes were measured by the summation of discs method in 4- and 2-chamber views by tracing of the endocardial border. LV EF was calculated as the percentage ejected blood volume during systole using biplane method of disc summation (Simpson’s method). Right atrial (RA) and RV volumes were estimated from RV focused 4-chamber views by the area-length method. The dimension of the RV was measured in RV focused 4-chamber views at the basal and mid-ventricular level. Tricuspid annular plane systolic excursion was measured by reconstructed motion mode aligned to the movement of the basal right ventricular free wall.
Color coded Doppler mode was recorded through all valvular orifices and vessels to identify pathology as regurgitations and stenoses.
Blood flow was recorded by spectral Doppler with sample volume; a) at tip of the mitral leaflet and in the presence of mitral regurgitation aligned to the regurgitant jet, b) in the distal LV outflow tract, c) through the aortic valve, d) at tip of the tricuspid valve and in presence of tricuspid regurgitation aligned to the regurgitant jet, e) in the RV outflow tract. For all measurements care was taken to align the ultrasound beam to the blood flow direction. The mitral inflow peak early (E) and late (A) diastolic velocities and the early diastolic deceleration time was measured, and the E/A ratio was calculated.
Color tissue Doppler cine-loops were recorded in the apical 4-chamber, 2-chamber and long-axis views, and RV focused view. Target frame-rate for the color tissue Doppler recordings was 100 fps. Care was taken to align the ultrasound beam to the myocardial wall. Peak systolic (S’) and early diastolic (e’) mitral annular velocities were measured at the base of the six myocardial walls by color tissue Doppler, and the average values are used as measurements of the LV myocardial velocities. Pulsed-wave tissue Doppler velocity curves were recorded from the basal part of the left and right ventricle, at the septal and lateral points (near the insertion of the mitral valve) and from the RV free wall (near the insertion of the tricuspid valve). e’ was measured at the base of the septal and anterolateral wall by pulsed-wave tissue Doppler and the average was used for calculation of the E/e’ ratio. The tricuspid annular peak systolic and early diastolic velocities were measured by color tissue Doppler, as well as pulsed-wave Doppler, in the basal part of the right ventricular free wall.
Blood samples were analyzed following standard operating procedures at St. Olavs Hospital. Glucose, hemoglobin A1c (HbA1c), total cholesterol, low density lipoprotein (LDL) cholesterol, TnT and insulin C peptide were all obtained before the training. Glucose and TnT were also obtained 1 hour and 24 hours post workout.
Body composition and weight:
Body composition was measured using the validated bioelectrical impedance unit, Inbody 720 (Biospace, Seoul, Korea) 43. In this machine, four pairs of electrodes are implanted into the handles and floor scale of the analyzer. Before testing, subjects had fasted for minimum two hours. They were encouraged to go to the toilet right before entering the scale. The subjects stood five minutes in upright position before entering the scale. They were barefoot. Due to the electrical impulse, people with pacemaker were not tested. Height, age, and gender were plotted om the scale-display. After two minutes, weight (kg), body mass index (BMI), muscle mass (kg), bodyfat % and visceral fat (cm2) was measured by the scale. The device was auto-calibrated once a week when the machine was turned off.
A 48-hour ambulatory, continuous ECG recording (DigiTrak XT, Phillips Healthcare, Andover, MA) was used the 24 hours before and the 24 hours after the exercise session. Supraventricular and ventricular premature beats and arrhythmias were counted by the vendor specific software, but manually controlled by a trained physician.
Student’s t-test (independent samples) was applied to compare different parameters between groups. Paired t-tests were used to compare changes within each group before and after exercise. Heart rhythm was not normally distributed, and thus, group differences were analyzed by Mann-Whitney two-tailed test (exact p-values) at pre- and post-exercise. Differences in TnT and glucose in blodsamples between T2D and control at baseline, 1 hour- and 24 hours post exercise was performed using a mixed-effect model for repeated measures corrected with Šídák's multiple comparisons test. With TnT as a markers cardiac stress we performed separate post hoc analyses to determine if individuals with rise in TnT following exercise also had more severe alterations in cardiac function than individuals with no elevation in TnT. Data from individuals with increased TnT (i.e., TnT >10 ng/L) was therefore additionally analyzed as a separate population and compared to all individuals with no rise inn TnT. Analyses was performed using GraphPad Prism (version 9.2.0)