High-intensity Interval Training Enhances Hypoxia-mediated Right Cardiac Mechanics in Sedentary Men

Purpose: Hypoxic exposure increases right ventricular (RV) afterload by triggering pulmonary hypertension, with consequent effects on the structure and function of the right ventricle. Improved myocardial contractility is a critical circulatory adaptation to exercise training. However, the types of exercise that enhance right cardiac mechanics during hypoxic stress have not yet been identied. This study investigated how high-intensity interval training (HIIT) and moderate-intensity continuous training (MICT) inuence right cardiac mechanics during hypoxic exercise (HE). Methods: A total of 54 young and healthy sedentary males were randomly selected to engage in either HIIT (3-min intervals at 40% and 80% of VO 2max , n = 18) or MICT (sustained 60% of VO 2max , n = 18) for 30 min/day and 5 days/week for 6 weeks or were included in a control group (CTL, n = 18) that did not engage in any exercise. Right cardiac mechanics during semiupright bicycle exercise tests under hypoxic conditions (i.e., 50 watts under 12% FIO 2 for 3 min) were measured using two-dimensional speckle-tracking echocardiography. Results: After 6 weeks of training, HIIT was superior to MICT in improving VO 2max . Furthermore, the HIIT group showed reduced pulmonary vascular resistance (PVR) as well as an elevated RV ejection fraction (RVEF) under hypoxia, coupled with a signicant enhancement of the right atrial (RA) reservoir and conduit functions. HIIT is superior to MICT in dilating the right ventricle and ameliorating radial strain rate but reducing radial strain in either systole or diastole. In the correlation analysis, the changes in RVEF were directly associated with improved RA reservoir and conduit functions but inversely associated with the change in radial RV strain caused by HIIT. Conclusion: HIIT is MICT in right cardiac RA conduit and decreasing PVR during HE.


Introduction
According to the Frank-Starling mechanism, the ventricle is able to increase its force of contraction and therefore stroke volume in response to increases in venous return and hence preload. Perturbations in afterload or inotropy move the Frank-Starling curve up or down. 1 Hypoxic exposure induces pulmonary vasoconstriction and hypertension, increases right ventricular (RV) afterload, and induces changes in RV function and dimension 2,3 ; consequently, it limits physical performance during exercise in healthy individuals and patients with respiratory diseases 4,5 . In this study, we used a novel methodology combining both acute hypoxic exposure and exercise stress to generate perturbations to the right ventricle.
Conventionally, it was considered that the right ventricle is exposed to volume overload only due to increased cardiac output during exercise; nevertheless, a recent study further demonstrated the presence of signi cant pressure overload 6,7 . Furthermore, signi cant volume overload may cause enlargement of the right ventricle and right atrium 8,9 with a relatively enhanced longitudinal function in the right ventricle 10 , whereas RV pressure overload primarily affects radial shortening and induces changes in ber orientation in the right ventricle [11][12][13] . Although most studies have focused on the longitudinal motion of the right ventricle, current data suggest that radial motion seems to be a key indicator to assess RV pump function as well 13 . In the concept of mixed hemodynamic RV overload in exercise, these effects can be distinct in different exercise regimens and may also induce functional remodeling. However, few studies have elucidated the distinct effects of different training regimens on radial or longitudinal RV mechanics.
Endurance training is a valuable approach to the management of pulmonary-related disease by augmenting pulmonary vasodilatation 14 . High-intensity interval training (HIIT) has recently been demonstrated to signi cantly attenuate RV dysfunction and reverse RV hypertrophy in a rat model of pulmonary hypertension 15 . However, although several human studies have focused on the left heart, only a few studies have focused on the right heart in association with endurance exercise. Moreover, an effective training strategy that enhances right cardiac mechanics during hypoxic exercise (HE) has not yet been established.
Typically, right cardiac performance is assessed by echocardiography under normoxic environments, which only displays chamber size and wall motion by speckle-tracking echocardiography (STE) with normal estimated pulmonary vascular resistance (PVR) 16 . To comprehensively explore the distinctive mechanical responses to mixed hemodynamic RV overload following various exercise interventions, exercise stress echocardiography (ESE) 17 with STE was performed in a 12% FiO 2 hypoxic environment.
In this study, we hypothesize that acute HE would provoke volume load to enhance RV contractility in longitudinal motion, whereas after 6 weeks of interventions, HIIT, resulting in a signi cantly dilated right ventricle, would be superior to MICT in diminishing PVR and enhancing RA functions, which further affects the right ventricle in both radial and longitudinal motions. This study aimed to compare the effectiveness of HIIT and MICT in improving right cardiac mechanics during HE. It comprehensively clari ed how HIIT (3-min intervals at 40% and 80% of maximal O 2 consumption (VO 2max ) or MICT (sustained 60% of VO 2max ) for 6 weeks affected right cardiac mechanics at rest or during HE in sedentary men using STE-ESE.

Cardiopulmonary tness in normoxia
No signi cant difference in anthropometric parameters or functional capacity among the three groups was found at the onset of the study (Table 1). After the six-week interventions, either HIIT or MICT signi cantly increased stroke volume (SV) and decreased mean arterial pressure (MAP) and total peripheral resistance (TPR) at rest in normoxic conditions (P < 0.05, Table 1). Moreover, these two exercise groups demonstrated improved cardiopulmonary tness by increased work rates, V E , and VO 2 at the ventilation threshold and peak exercise performance (P < 0.05, Table 1). However, HIIT led to a greater improvement in aerobic capacity (VO 2max ) than MICT (P < 0.05, Table 1). No signi cant changes in cardiopulmonary responses to CPET were observed after 6 weeks in the CTL group (Table 1).

Conventional echocardiographic parameters in hypoxia
Under hypoxic conditions, both HIIT and MICT simultaneously lowered TPR and PVR at rest; moreover, only HIIT further decreased RV afterload, PVR ex and RVSP ex during HE (P < 0.05, Table 2). Although both types of training augmented the tricuspid E wave, HIIT was superior to MICT. Furthermore, only HIIT signi cantly elevated RVEF (P < 0.05, Table 2). No signi cant changes in echocardiographic parameters were observed after 6 weeks in the CTL group (Table 2).

RA functions
Before interventions, acute HE increased reservoir and conduit volumes and decreased the booster volume in the right atrium (P < 0.05, Fig Area and dimensions of the right ventricle under hypoxia A bout of acute HE augmented RV FAC% by reducing the systolic RV area (Fig. 1E-1F) in RVD3 (longitudinal) (Fig. 2G-2I). Following the 6-week intervention, HIIT was superior to MICT in enhancing FAC% by a greater diastolic RV area ( Fig. 1D and 1F). Figure 2 further shows details of the dimensions of the right ventricle at rest and during HE. A signi cantly enlarged right ventricle was observed in the HIIT group, as demonstrated by a longer diastole of RVD1 (basal) and RVD2 (middle), when compared to the MICT and CTL groups ( Fig. 2A and 2D). Moreover, compared with no exercise, HIIT led to shorter systoles in both RVD2 and RVD3 during HE (P < 0.05, Fig. 2D and 2G).
Strain and strain rate (SR) in the right atrium and right ventricle under hypoxia Before the interventions, acute HE enhanced both RV longitudinal strain and SR while reducing radial strain (P < 0.05, Table 4). In addition, in the right atrium, both radial and longitudinal strain and SR were increased under HE (P < 0.05, Table 3). Following the 6-week interventions, both HIIT and MICT augmented RA radial strain and systolic/diastolic SR at rest or during HE, whereas only HIIT increased RA longitudinal strain and systolic SR during HE (P < 0.05, Table 3). On the other hand, although HIIT, but not MICT, decreased RV radial strain, the systolic/diastolic SR was ameliorated (P < 0.05, Table 4). Moreover, no signi cant changes were found in radial/longitudinal strain and systolic/diastolic SR in the right atrium or right ventricle at rest or during HE after 6 weeks in the CTL group (P < 0.05, Tables 3 and 4).

Relationship between HE-induced changes in right cardiac mechanical variables following interventions
Here, the change indicates the difference between pretraining and posttraining. No signi cant resting relationship was observed between RA volumes and RVEF following either intervention ( Fig. 3A to 3C). However, HE-induced changes in the RA reservoir ( Fig. 3A; r = 0.60, P < 0.05) and conduit volumes ( Fig.  3B; r = 0.64, P < 0.01) were positively associated with RVEF after HIIT. Furthermore, the HE-induced change in RV radial strain was negatively correlated with the RVEF change in the HIIT group ( Fig. 3D; r = −0.70, P < 0.01). Additionally, in the HIIT group, both resting ( Fig. 3F; r = −0.77, P < 0.01) and HE-induced changes in PVR ( Fig. 3F; r = −0.70, P < 0.01) were inversely associated with RVEF. However, no signi cant correlations were found between HE-induced changes in RVEF and RV longitudinal strain following HIIT ( Fig. 3E). In contrast to the HIIT group, no signi cant correlations were observed among RA reservoir, conduit, and booster pump functions; radial/longitudinal strains and SRs in the right ventricle; and PVR at rest or during HE in the MICT or CTL groups (Fig. 3).

Discussion
This is the rst investigation to clarify the effects of various exercise regimens on right cardiac mechanics during HE using 2D-STE technology. Both HIIT and MICT improve RA reservoir function, while only HIIT enhances RA conduit function to reinforce RV preload. Therefore, HIIT is more e cient than MICT in dilating the chamber of the right ventricle by ameliorating radial SR but reducing radial strain in either systole or diastole. Although both interventions lessen resting RV afterload, PVR and RVSP, only HIIT further diminishes PVR under HE. Notably, the correlation analysis further demonstrated that an augmented RVEF is signi cantly associated with greater RA reservoir and conduit functions and lower PVR following HIIT.
The atrium tends to dilate in response to greater venous return or chronic elevations in ventricular lling pressure when exercising 18 . However, the elevated RA afterload caused by acute hypoxic exposure may cause the ratio of passive reservoir to active contraction to decline 19 . In our longitudinal study, both HIIT and MICT for 6 weeks enhanced reservoir function even during HE to accommodate more venous return. These results correspond with a cross-sectional investigation, i.e., highly dynamic athletes had larger RA reservoir functions for venous return and more blood lling into the right ventricle than less dynamic athletes 20 . As the enhanced tricuspid E wave represented, the ameliorated conduit function in HIIT accelerated RV early lling and enhanced RV preload even under hypoxic stress 21 .
Because of the higher cardiac output demand in the HIIT, volume load-related remodeling may be increased in the HIIT group compared with that in the MICT group 22 . Although most studies have focused on longitudinal motion to generate RV ejection, our study further con rmed that RV dilation primarily occurred in the radial direction instead of the longitudinal direction. Elevated radial motion in uences systolic function via the bellows effect because the free wall of the right ventricle has a larger surface than the tricuspid annular cross-sectional area 23 . Therefore, we speculated that the improvement of ejection fraction during HE is related with not only longitudinal but also radial motion.
Interestingly, suppressed RV radial strain at the onset of HE and following HIIT was noticed. This is in contrast to the traditional viewpoint of enhancing longitudinal strain as the key contributor to overall RV contractility under RV overloading 12 . Regarding RV dilation, the reduced radial strain is considered to further increase wall tension. In this case, we believe that the signi cantly reduced HE-related PVR and dilated right ventricle in HIIT are accommodations to overcome this situation. Brie y, these ndings may represent a consequence of RV remodeling rather than dysfunction in healthy young men. In fact, our ndings are partly similar to athletes' heart characteristics, with a relative decrease in radial shortening with greater RV enlargement and better RVEF 24,25 .
The augmented RV radial strain rate (SR) in the HIIT group may indicate a greater RV contractile e ciency by homeometric autoregulation in response to the decreased radial strain. SR is relatively more independent of HR, structure, and loading conditions than strain and diameter 26 . Hence, SR might better re ect the training responses and appears to be the more accurate parameter in myocardial contractility, especially during exercise 27 . In addition to the context of loading and structure, in some hypoxiasusceptible patients, RV dysfunction has been suggested to be caused by a direct negative inotropic effect of hypoxia on cardiac myocytes and decreased oxygenation 28 . Although the comparison of effects of normoxia and hypoxia on cardiac mechanics is not the main aim of this study, we focused on the fact that both RV diastolic and systolic functions were augmented when facing both exercise and loading stresses after 6 weeks of exercise training.
An elevated PVR is a well-known physiological response to hypoxia 29 . In this investigation, both HIIT and MICT reduced PVR and subsequently decreased RV afterload, thereby improving RVEF in resting conditions. The PVR and RVSP were further reduced during HE in HIIT, thus additionally lowering the afterload when the right ventricle contracted. Previous studies demonstrated that exercise training upregulated endothelial eNOS expression in the pulmonary vasculum 30 . Hassel et al. further revealed that HIIT reduced the muscularization of pulmonary vessels and subsequently attenuated RV dysfunction in COPD mice 15 .
The xed absolute exercise intensity used in HE is due to the concern about the in uence of HE on an altered loading state by the different VO 2max after training. Therefore, our intention is to compare the relative change before and after the intervention rather than the absolute data. In addition, our previous study demonstrated that this protocol is feasible to clarify the LV mechanics during HIIT and MICT 31 .

Limitations Of The Study
As observed in other investigations, the number of men who are young, healthy, and sedentary is limited. Thus, additional clinical evidence is required to extrapolate the present results to patients with abnormal cardiovascular systems, such as those with pulmonary hypertension or right HF, and to analyze potential sex differences 32 .
Because of the thin walls of the right ventricle, the image quality might have highly in uenced the accuracy of our detection. Although our test-retest reliability indicates good imaging quality, it is still important to note the limitations of 2D-STE 33 .
The noninvasive estimation of PVR might not have obtained true absolute data. However, it has been reported that the estimate has an error margin of < 10% relative to the real pressure 34 . Furthermore, using noninvasive echocardiography is more ethical than using invasive catheterization under dynamic conditions, with a much lower risk for the study participants 35 .
The MICT exercise volume is speculated to have been too low to exert a positive effect on cardiac hemodynamic adaptation. The plurality of the positive MICT results suggested that exercise training at least 5 days weekly up to six times daily for a period of at least 12 weeks is necessary 36 .

Conclusion
Typically, right cardiac performance is assessed by echocardiography under normoxic environments, which displays only the chamber size and myocardial motion with a normal PVR. This study further contributes to a greater understanding of RV and RA mechanical responses to hypoxic stress following various exercise interventions by using STE-ESE. The experimental results clearly demonstrate that HIIT with a dilated right ventricle enhances RVEF by increasing the RA reservoir and conduit functions, enhancing RV radial strain rate and decreasing PVR. These ndings provide novel insights into the superior effects of HIIT on contractile e ciency of the right heart during HE by simultaneously increasing preload and decreasing afterload, which might have important implications for exercise training in cardiopulmonary rehabilitation.

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% VO 2max before ve cycles, and each cycle included 3 min at 80% VO 2max with a 3-min active recovery period at 40% VO 2max . Finally, the session was terminated with a 3-min cool-down at 30%  (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 su cient to recover CO following HE 37 . All subjects underwent exercise using a face mask to measure min ventilation (VE), oxygen consumption (VO 2 ), and carbon dioxide production (VCO 2 ) breath by breath using a computer-based system (MasterScreen CPX, CareFusion, USA). After a 5-min baseline resting period, a 2min 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 VO 2 ). The criteria used to de ne VO 2 were as follows: (i) the level of VO 2 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/VO 2 increased without a corresponding increase in the VE-to-VCO 2 ratio, end-tidal PO 2 increased without a decrease in end-tidal PCO 2 , 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 CO 2 scrubber eliminated CO 2 in the air (< 3500 ppm), and the O 2 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 out ow tract (RVOT) was obtained from a modi ed apical four-chamber view, and the ow 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 ow velocities for peak early (E) llings. 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 modi ed apical four-chamber view, as shown in Supplementary Fig. S3 42 . All measurements were independently recorded from three independent image frames, enabling reliable quanti cation.
Speckle-tracking echocardiography of hypoxic exercise (HE) STE was immediately performed after the conventional data were collected completely under hypoxic conditions (12% FiO 2 ) 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 o ine 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 modi ed apical four-chamber view was used to assess STE longitudinal and radial parameters of the right ventricle and right atrium. Brie y, 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 o ine 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 (RA max ) was detected at the end of LV systole just before mitral valve opening, and RA minimum volume (RA min ) 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 RA pre-a , which represents the preload before atrial contraction. Figure 4 shows the schematic RA time-volume curve.
(1) Reservoir volume: the lling or expansion volume, calculated as RA max -RA min.
(2) Conduit volume: the passive emptying volume from venous return during early ventricular diastole, calculated as RA max -RA pre-a .

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. O ine 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 coe cient (ICC), coe cient of variance (CV), and Cronbach alpha value 45 . The threshold for statistical signi cance was set at P < 0.05.