Subjects
The investigation was performed in accordance with the principles of the Declaration of Helsinki and was approved by the Institutional Review Board of the Chang Gung Memorial Hospital in Taiwan. A total of 54 sedentary males were recruited. We recruited males who were nonsmokers; did not take medications or vitamins; did not have any cardiopulmonary/hematological risks; and, most importantly, had a sedentary lifestyle (without regular exercise; exercise frequency ≤ once weekly, duration < 20 min). Informed consent was obtained from all subjects after the experimental procedures were explained. These subjects were randomly divided into the HIIT (n = 18), MICT (n = 18), and control (CTL, n = 18) groups. All subjects arrived at the testing center at 9:00 AM to eliminate any possible circadian effect.
Training protocols
Both the HIIT and MICT groups performed exercise regimens on a stationary bicycle ergometer (Corival 400, Lode) 5 times a week for 6 weeks. For comparison, the CTL group did not undergo any exercise, but their physical activity and daily diet were carefully monitored and recorded. HIIT subjects warmed up for 3 min at 30% VO2max before five cycles, and each cycle included 3 min at 80% VO2max with a 3-min active recovery period at 40% VO2max. Finally, the session was terminated with a 3-min cool-down at 30% VO2max. The MICT group underwent the same warm-up and cool-down phases as the HIIT group, but the training periods were 30 min at 60% VO2max. Both training protocols were isovolumetric with the same duration (i.e., HIIT exercise volume: {6 min [40% VO2max + 80% VO2max] × 5 cycles} = MICT exercise volume: 30 min [60% VO2max]). To achieve the desired exercise intensity, each subject used a heart rate (HR) monitor (Tango, SunTech Medical). During the exercise, the work rate of the bicycle ergometer was continuously adjusted to match the exercise intensity with the target HR. The percentage of HR reserve (%HRR) is widely considered to be equivalent to the percentage of VO2 reverse for exercise prescription purposes. Accordingly, the target HR of HIIT and MICT were calculated using the following equations:
Peak HR = 220 − age (1)
%HHR = % (peak HR − resting HR) + resting HR (2)
Target HR of HIIT = 3 − minute intervals at 40% HHR and 80% HRR (3)
Target HR of MICT = sustained 60% HRR (4)
The groups were asked to record their daily activities and nutritional intake using the short form of the International Physical Activity Questionnaire and a written diet record, respectively. Subjects were asked to refrain from regular extra exercise until the end of the study. Moreover, all subjects completed the experiments with a participant compliance rate of 100%.
Cardiopulmonary exercise test
To assess aerobic capacity, a cardiopulmonary exercise test (CPET) on a cycle ergometer (Corival 400, Lode B.V., Netherlands) was performed 2 days before and after the intervention, which is sufficient to recover CO following HE 37. All subjects underwent exercise using a face mask to measure min ventilation (VE), oxygen consumption (VO2), and carbon dioxide production (VCO2) breath by breath using a computer-based system (MasterScreen CPX, CareFusion, USA). After a 5-min baseline resting period, a 2-min warm-up period (60 rpm, unloaded pedaling) was initiated, followed by incremental work (30 W elevation for each 3 min) until exhaustion (i.e., progressive exercise to VO2). The criteria used to define VO2 were as follows: (i) the level of VO2 increased by <2 mL/kg/min over at least 2 min; (ii) HR exceeded its predicted maximum; (iii) the respiratory exchange ratio exceeded 1.2; and (iv) the venous lactate concentration was >8 mM. These criteria were consistent with the American College of Sports Medicine guidelines for exercise testing 38. During CPET, continuous monitoring of 12-lead electrocardiography, blood pressure, and pulse oxygen saturation was performed. In addition, the ventilation threshold was determined when VE/VO2 increased without a corresponding increase in the VE-to-VCO2 ratio, end-tidal PO2 increased without a decrease in end-tidal PCO2, or a deviation from linearity for VE.
Conventional echocardiography
A standard echocardiographic examination according to the American Society of Echocardiography guidelines was performed at each stage 39. Each subject underwent echocardiography 4 days before and after the intervention in an air-conditioned normobaric hypoxia chamber (Colorado Mountain Room, USA) 40. The hypoxia chamber was maintained at a temperature of 22°C ± 0.5°C with a relative humidity of 60% ± 5%; a CO2 scrubber eliminated CO2 in the air (< 3500 ppm), and the O2 concentration was set at 12%, which corresponded to an altitude of 4460 m. All subjects were positioned at a 30° semiupright position oriented in a left lateral 60° semisupine position and secured to the echocardiography table (Angio with Echo Cardiac Stress Table, Lode B.V., Netherlands). The parameters were measured using the Siemens ACUSON SC2000™ ultrasound system (Siemens Healthineers, Germany) with the 4V1C probe (4.5 MHz). Images of subjects with regular breathing patterns and no breath holding were captured at end expiration. The RV outflow tract (RVOT) was obtained from a modified apical four-chamber view, and the flow immediately proximal to the pulmonary artery valve during systole was detected to calculate both maximal velocity and pulsed-wave blood VTI. Doppler imaging was used to measure peak tricuspid annular velocities through the cardiac cycle in early diastole (E’) and diastolic transmitral blood flow velocities for peak early (E) fillings. Tricuspid annular plane systolic excursion was measured by placing an M-mode cursor through the tricuspid annulus and measuring peak systolic motion. The RA pressure (RAP) was estimated from the inferior vena cava (IVC) size during inspiration and during forced inhalation at rest. The IVC diameter was measured just proximal to the entrance of the hepatic veins. Pulmonary vascular resistance (PVR) was calculated using the formula PVR = ([TR velocity/RVOT VTI] × 10 + 0.16), which has shown a good correlation with invasively derived PVR 41. All data were recorded over three cycles, and the averages were calculated. RV basal cavity diameter (RVD1), mid-cavity diameter (RVD2), RV longitudinal diameter (RVD3), and RV area at end-diastole and end-systole were evaluated in the modified apical four-chamber view, as shown in Supplementary Fig. S3 42. All measurements were independently recorded from three independent image frames, enabling reliable quantification.
Speckle-tracking echocardiography of hypoxic exercise (HE)
STE was immediately performed after the conventional data were collected completely under hypoxic conditions (12% FiO2) as previously described 31. Resting images were acquired after the subject was placed in the aforementioned position for 10 min. The exercise images were conducted using semirecumbent cycling with a 50-W resistance for 3 min and acquired at the third minute of cycling to ensure that subjects had reached a steady-state HR (i.e., HR changes <10 bpm within 10 s and <110–120 bpm) 43. Three consecutive cardiac cycles were evaluated for each acquisition. The 2D-STE analysis was performed offline by the same echocardiographer, who was blinded to the group allocation and image sequence, using semiautomatic strain software (ACUSON SC2000™ system, Siemens Healthineers, Germany).
A modified apical four-chamber view was used to assess STE longitudinal and radial parameters of the right ventricle and right atrium. Briefly, after manual tracing, the end-systolic RV endocardial border, a region of interest, was automatically generated; its width and position were manually readjusted to include the entire myocardial wall when it showed poor-quality tracking by visual assessment. The software automatically divides the right ventricle into a 6-segment model as a more robust analysis recommend by Muraru et al. 32, whereas the right atrium was automatically divided into a 3-segment model. The RV strain and SR were calculated using the average peak segmental values displayed by the software using a 6-segment model. The compliance rate of this study was 100%, and no subject was excluded due to inadequate images.
Volumetric analysis in RA function
RA volumes were assessed offline using semiautomatic strain software (Siemens ACUSON SC2000™ ultrasound system, Siemens Medical Solutions USA Inc., Mountain View, CA) on dedicated 2D-STE sets in the apical four-chamber view. The border-tracing process was similar to the abovementioned STE protocol. RA maximum volume (RAmax) was detected at the end of LV systole just before mitral valve opening, and RA minimum volume (RAmin) was acquired at the end of LV diastole just after mitral valve closure. Atrial function is most often assessed using 2D volumetric analysis, such as reservoir, conduit, and booster pump functions. The volume immediately before atrial contraction (onset of P wave) is denoted as RApre-a, which represents the preload before atrial contraction. Figure 4 shows the schematic RA time-volume curve.
(1) Reservoir volume: the filling or expansion volume, calculated as RAmax – RAmin.
(2) Conduit volume: the passive emptying volume from venous return during early ventricular diastole, calculated as RAmax – RApre-a.
(3) Booster volume: the RA stroke volume (SV), calculated as RApre-a –RAmin.
Test-retest reliability
A subgroup (n = 20) was assessed for test-retest variability in RV radial and longitudinal strains. Each participant had two separate echocardiograms using the same set of 2D-STE images under normoxic conditions that were approximately 24 hours apart to reduce the impact of physiological variation. The echocardiographer was blinded to the original images and used a standard echocardiographic protocol for each acquisition. Offline analyses were randomized by the same echocardiographer and performed using available software (Siemens ACUSON SC2000™ ultrasound system, Siemens Medical Solutions USA Inc., Mountain View, CA) 44.
Statistical analysis
Quantitative data were expressed as the mean ± SEM. Data analysis was performed using IBM SPSS Statistics V22.0. Experimental results were analyzed by repeated-measure ANOVA and Bonferroni post hoc tests to compare aerobic capacity and cardiac mechanics at the beginning of the study and after 6 weeks of intervention. Linear regression analyses were performed using Pearson’s method to assess univariate associations between echocardiographic data. Intra-reproducibility was assessed using the intraclass correlation coefficient (ICC), coefficient of variance (CV), and Cronbach alpha value 45. The threshold for statistical significance was set at P < 0.05.