Combined use of stress echocardiography and cardiopulmonary exercise testing to assess exercise intolerance in patients treated for acute myocardial infarction

Exercise intolerance after acute myocardial infarction (AMI) is a predictor of worse prognosis, but its causes are complex and poorly studied. This study assessed the determinants of exercise intolerance using combined stress echocardiography and cardiopulmonary exercise testing (CPET-SE) in patients treated for AMI. We prospectively enrolled patients with left ventricular ejection fraction (LV EF) ≥40% for more than 4 weeks after the first AMI. Stroke volume, heart rate, and arteriovenous oxygen difference (A-VO2Diff) were assessed during symptom-limited CPET-SE. Patients were divided into four groups according to the percentage of predicted oxygen uptake (VO2) (Group 1, <50%; Group 2, 50–74%; Group 3, 75–99%; and Group 4, ≥100%). Among 81 patients (70% male, mean age 58 ± 11 years, 47% ST-segment elevation AMI) mean peak VO2 was 19.5 ± 5.4 mL/kg/min. A better exercise capacity was related to a higher percent predicted heart rate (Group 2 vs. Group 4, p <0.01), higher peak A-VO2Diff (Group 1 vs. Group 3, p <0.01) but without differences in stroke volume. Peak VO2 and percent predicted VO2 had a significant positive correlation with percent predicted heart rate at peak exercise (r = 0.28, p = 0.01 and r = 0.46, p < 0.001) and peak A-VO2Diff (r = 0.68, p <0.001 and r = 0.36, p = 0.001) but not with peak stroke volume. Exercise capacity in patients treated for AMI with LV EF ≥40% is related to heart rate response during exercise and peak peripheral oxygen extraction. CPET-SE enables non-invasive assessment of the mechanisms of exercise intolerance.


Introduction
Exercise intolerance after acute myocardial infarction (AMI) is common and it indicates a poor prognosis [1][2][3][4][5].In a previous study of 2,896 patients with newly diagnosed ischemic heart disease, which included 1,064 patients post-AMI, exercise capacity assessed before cardiac rehabilitation was significantly decreased at roughly 60% of age-matched values of healthy

Study sample
We prospectively enrolled consecutive patients aged >18 years who underwent percutaneous coronary intervention for their first AMI, between October 2015 and January 2019, at our cardiology department.
The flow chart of the study is presented in Fig 1 .The study exclusion criteria were: previous AMI, history or presence of symptomatic congestive heart failure, permanent atrial fibrillation or atrial flutter, chronic obstructive pulmonary disease, heart surgery, peripheral nerve or musculoskeletal disorder, peripheral vascular disease with intermittent claudication, stroke with residual deficits, LV EF <40% at least 4 weeks after AMI, residual coronary artery stenosis (>50%) after percutaneous coronary intervention, anemia (hemoglobin <12 g/dL), decompensated thyroid disease, chronic kidney disease (creatinine clearance <30 mL/min), severe valvular diseases, pulmonary hypertension, hypertrophic cardiomyopathy with left ventricular outflow tract obstruction, exercise-induced ischemia, pulmonary limitations of exercise (breathing reserve at peak exercise <15%), respiratory exchange ratio (RER) at peak exercise <1.05, poor echocardiographic acoustic window, and lack of informed consent.
We collected data on demographic characteristics, medical history, and treatments as baseline characteristics.Self-assessed physical activity prior to AMI was categorized as low, moderate, or high according to the International Physical Activity Questionnaire [23].

Cardiopulmonary exercise testing
We performed symptom-limited cardiopulmonary exercise tests using a Schiller Cardiovit CS-200 (Schiller, Baar, Switzerland) and an Ergo Spiro adapter (Ganshorn, Niederlauer, Germany) with patients on a semi-supine cycle ergometer eBike EL (Ergoline GmbH, Bitz, Germany).Volumetric and gas calibration was performed daily before the tests.Volumetric calibration for current temperature, relative air humidity, and atmospheric pressure was performed with a standard 2-L syringe.Gas calibration was performed using a standard gas mixture containing 15% oxygen, 6% carbon dioxide, and 79% nitrogen.In all cases, we used a ramp protocol with an incremental load of 12.5 watts/min.All patients were familiar with the exercise protocol and were encouraged to exercise at maximal effort (�8 points using the 10-point Borg scale) [24].All exercise tests were supervised and analyzed according to current guidelines [25][26][27][28].During the stress test, we assessed the clinical and hemodynamic status of the patient, recorded 12-lead electrocardiograms, and recorded ventilation and gas exchange parameters.The peak VO 2 was averaged from the highest 20 s of exercise, in mL/kg/min.Maximum predicted VO 2 was calculated according to the Wasserman/Hansen equations [29].The anaerobic threshold was calculated using a dual method approach (V-slope and ventilatory equivalent methods).Other analyzed cardiopulmonary exercise testing parameters included oxygen uptake to work rate increment ratio (ΔO 2 /ΔWR), ventilatory efficiency (VE/VCO 2 slope), and breathing reserve at peak exercise, calculated as the percentage of maximum voluntary ventilation: [(maximum voluntary ventilation − minute ventilation at peak exercise) / maximum voluntary ventilation] × 100.The systolic and diastolic blood pressure and heart rate were recorded at rest and at peak exercise during the test, and the chronotropic index and percentage of maximum predicted heart rate at peak exercise was calculated [27].The maximum predicted heart rate was calculated as 220-age in years [30].Heart rate recovery was calculated as the difference between peak heart rate and heart rate at the first minute of recovery.Recorded electrocardiographic parameters included the presence or absence of ischemic changes, arrhythmias and conduction abnormalities according to the American Heart Association [27].

Stress echocardiography
Echocardiography was performed at rest and at peak exercise using a VIVID 9 ultrasound machine (General Electric Medical System, Horten, Norway).Exercise echocardiography was carried out simultaneously with cardiopulmonary exercise testing.Resting echocardiograms were recorded in semi-recumbent position before starting the exercise.Exercise echocardiographic images were recorded at peak exercise, before effort termination.Two-dimensional images were recorded in standard views.Left ventricular volumes were measured in 4-and 2chamber apical views and LV EF was calculated using the modified Simpson's rule [31].Left ventricular systolic (s') and early diastolic (e') myocardial velocities were evaluated using pulsed-tissue Doppler at the basal segments of the interventricular septum and lateral wall and were presented as averaged values.Regional wall motion was assessed and graded using a 4-point scale, where 1 represented normal and 4 represented dyskinetic motion in a 16-segment model and was expressed as a wall motion score index (WMSI).Mitral flow was assessed as early mitral inflow velocity (E), late (atrial) inflow velocity (A), and deceleration time using a pulse-wave Doppler sample volume between the mitral leaflet tips [32].In cases with E to A fusion at peak exercise, diastolic function was assessed in the early recovery phase.Stroke volume was calculated by multiplying the area of the left ventricular outflow tract at rest by the left ventricular outflow tract velocity-time integral (in pulsed-wave Doppler averaged from three cardiac cycles at rest and at peak exercise).Right ventricular systolic function was assessed by evaluating tricuspid annular plane systolic excursion (TAPSE) and right ventricular systolic myocardial velocity (RV s') in the 4-chamber apical view.The A-VO 2 Diff was calculated using the Fick equation as follows: VO 2 /cardiac output calculated from echocardiography [12,14,15].Measurements and recordings of echocardiographic parameters were performed according to the American Society of Echocardiography and the European Association of Echocardiography recommendations [31][32][33][34].Images were analyzed off-line using EchoPAC PC software v.110.0.x (GE Healthcare).
All CPET-SE examinations were performed and interpreted by one cardiologist experienced in stress echocardiography and cardiopulmonary exercise testing.

Statistical analyses
Data are presented as mean (± standard deviation) or median (IQR) for continuous variables.Categorical variables are presented as numbers (percentages).Normality for all continuous variables was tested using the Shapiro-Wilk test.Group comparisons between continuous variables were performed using Welch's t-test or Mann-Whitney U test, and the Fisher exact test or χ2 (chi-squared) test for categorical variables.Analysis of variance was used for multiple group comparisons of normally distributed numeric data.

Ethical statement
This study was conducted in conformance with the requirements set out in the Declaration of Helsinki.All patients provided written informed consent to participate.The study and all its protocols were approved by the Institutional Ethics Committee of the Centre of Postgraduate Medical Education Bioethical Committee (protocol code 16/PB/2015, approved on February 25, 2015).

Baseline characteristics
Of 102 eligible patients, 81 patients (57 male, mean age 58 ± 11 years) who had undergone after CPET-SE were enrolled into analysis.Among these, 47% had AMI with and 53% had AMI without ST segment elevation.The included patients were mostly in Killip class 1.Only 3 patients in the group 3 were in Killip class 2. Patients in Group 4 were mostly women (p = 0.045 for Group 2 vs. Group 4) and had lower BMI (p = 0.011 for Group 2 vs. Group 4).Clinical characteristics are presented in Table 1.Almost 31% of the study sample had diabetes mellitus or impaired glucose tolerance, 67% had hypertension, and 47% were smokers.All patients were on optimal medical therapy for AMI, with 70 patients on beta-blockers.Betablockers were not withheld before the exercise tests.

Combined stress echocardiography and cardiopulmonary exercise testing
Cardiopulmonary parameters in groups according to the percentage of predicted VO 2 are presented in Table 2.The median (IQR) time from AMI to CPET-SE was 42  days without significant differences between groups.The mean peak VO 2 was 19.5 ± 5.4 mL/kg/min (20.6 ± 4.8 mL/kg/min for men and 16.8 ± 5.8 for women).The median (IQR) RER at peak exercise was 1.14 (1.07-1.21).Mean breathing reserve at peak exercise was 53 ± 12%, and no participants had a breathing reserve of <15% at peak exercise.The heart rate response to exercise (percent predicted heart rate) was the highest in Group 4 (p <0.01 for Group 2 vs. Group 4).The ΔO 2 /ΔWR was the lowest in Group 1 (p <0.001 for Group 1 vs. Group 3 and Group 4).
The peak A-VO 2 Diff was lowest in Group 1 (p <0.01 for Group 1 vs. Group 3).Mild mitral regurgitation at peak exercise was most frequent in Group 3. No other significant differences in resting and stress echocardiography parameters were noticed (Table 3).

Determinants of exercise capacity
Peak VO 2 and percent predicted VO 2 had a significant positive correlation with percent predicted heart rate at peak exercise (r = 0.28, p = 0.01 and r = 0.46, p < 0.001) and peak A-VO 2 Diff (r = 0.68, p <0.001 and r = 0.36, p = 0.001) but not with peak stroke volume (Fig 2  and heart rate at peak exercise, chronotropic index, ΔO 2 /ΔWR, VE/VCO 2 slope, A-VO 2 Diff, and LV s' at peak exercise and also for percent predicted VO 2 and heart rate at peak exercise, percent predicted heart rate, chronotropic index, ΔO 2 /ΔWR, and A-VO 2 Diff at peak exercise.

Discussion
Our study revealed that in post-AMI patients with LV EF �40%, exercise capacity is related to heart rate response during exercise and peak peripheral oxygen extraction.Impaired extraction of oxygen by working muscles plays a significant contribution in the most compromised patients.
Several causes of exercise intolerance, leading to low peak VO 2 , have been described; they include Fick variables, such as stroke volume, heart rate, A-VO 2 Diff, and non-Fick variables, such as motivation; pulmonary, central, and peripheral nervous system diseases; peripheral arterial or vein pathologies; or bone and joint abnormalities [35].We excluded patients with non-Fick variables that led to exercise termination from our study.Heart rate and A-VO 2 Diff have the largest dynamic response during exercise.Both parameters can increase up to 2.5-fold, contrary to stroke volume, which increases up to 1.4-fold during maximal exercise [36].Among these parameters, stroke volume and heart rate during exercise reflect cardiac function, while A-VO 2 Diff reflects peripheral oxygen extraction.

Exercise capacity after acute myocardial infarction
The exercise capacity in our study group was similar to that of previous studies on post-AMI patients entering cardiac rehabilitation.In an observational study of 12,169 male rehabilitation candidates tested on a cycle ergometer, the mean peak VO 2 was 20.5 ± 5.2 mL/kg/min [1].In a similar study of 2,380 women, the mean peak VO 2 was 15.4 ± 4.0 mL/kg/min [2].Moreover, in another group of 2,896 patients who exercised on a treadmill after AMI and before cardiac rehabilitation, the mean peak VO 2 was 20.4 ± 6.6 mL/kg/min and 14.7 ± 4.2 mL/kg/min in men and women, respectively [3].These studies focused on the prognostic significance of exercise capacity, but not on the mechanisms leading to low peak VO 2 .

Contributors to exercise intolerance
To the best of our knowledge, no previous study described mechanisms of exercise intolerance in patients treated for AMI with LV EF � 40% using CPET-SE.Heart rate response and peripheral factors were previously found to be the main contributors to reduced exercise capacity, in healthy subjects and in patients with heart failure but with preserved LV EF (HFpEF) or midrange LV EF (HFmrEF).A small study of 14 subjects with normal cardiac function and 16 patients with HFpEF examined effort intolerance using CPET-SE, with noninvasively calculated A-VO 2 Diff.They found that heart rate and A-VO 2 Diff, but not stroke volume, at peak exercise were the most significant independent predictors of peak VO 2 .However, among patients with HFpEF, diastolic dysfunction was also found to be a determinant of peak VO 2 [15].
In another study of 48 patients with HFpEF assessed with CPET-SE, both reduced cardiac output and calculated A-VO 2 Diff contributed significantly to exercise intolerance.In this study, the strongest independent predictor of peak VO 2 was the change in A-VO 2 Diff from rest to peak exercise [14].
In a study of 169 subjects (healthy controls and heart failure patients with a wide range of LV EF) assessed with CPET-SE, where A-VO 2 Diff was calculated noninvasively based on Fick's equation, multivariate analysis revealed that peak VO 2 was predominantly influenced by peripheral factors (such as A-VO 2 Diff) in patients with HFmrEF and HFpEF, whereas it was influenced by decreased stroke volume in patients with reduced LV EF [19].
In another, recently published study involving patients with hypertension with and without HFpEF, reduced peak VO 2 was found to be related to decreased calculated peak A-VO 2 Diff [20].Like our study, a study of 278 patients with various degrees of heart failure showed that A-VO 2 Diff was a significant contributor to exercise capacity in the most compromised group (percent predicted VO 2 <50%) [37].
Similar mechanisms of exercise intolerance were also found in a study with cardiopulmonary exercise testing with invasive hemodynamic monitoring.Directly measured peak A-VO 2 Diff has been found to be the major exercise-limiting factor in patients with HFpEF [38].
In our study, chronotropic response parameters, such as the percentage of predicted heart rate and the chronotropic index were highest in the group with the highest exercise capacity.Although the majority of our patients were on beta-blockers, we did not analyze beta-blocker doses.Our recent research suggested that exercise capacity is related to chronotropic response during exercise, rather than to the beta-blocker doses [39].Aerobic training could improve chronotropic responses to exercise by adaptation of autonomic function [40], and could improve A-VO 2 Diff by enhancing endothelial function and skeletal muscle deoxygenation [41,42].
Although previous studies have demonstrated the influence of diastolic dysfunction on exercise capacity [43,44], our study did not support these findings.In our study, elevated E/e' ratio >14 at peak exercise was recorded in patients with better exercise capacity (2 patients in Group 3, and 1 patient in Group 4).The lack of significant impact of diastolic function on exercise capacity could be related to the underrepresentation of such patients in our sample.
In patients treated for AMI, functional impairment may be caused by exercise-induced functional mitral regurgitation [45,46].In our studied group, no significant deterioration of mitral regurgitation was noticed during exercise.Mild mitral regurgitation during exercise was most frequent in Group 3, but only 7 patients had moderate, and none had severe mitral regurgitation.

Combined stress echocardiography and cardiopulmonary exercise testing
The increased accessibility of CPET-SE provides the opportunity for assessment of cardiac and peripheral factors of exercise intolerance.However, CPET-SE is not methodologically standardized.The use of a cycle ergometer in a semi-recumbent position has been suggested to offer improved echocardiographic evaluation [16,18].
Combined use of stress echocardiography and cardiopulmonary exercise testing allows the identification of central (low peak stroke volume, chronotropic incompetence) or peripheral (low peak A-VO 2 Diff) mechanisms of exercise intolerance.Two representative patients from our study, patient A with reduced and patient B with good exercise capacity, are presented in S2 Table .The combination of cardiopulmonary exercise testing with exercise stress echocardiography is a valuable diagnostic tool and its clinical utility has been proven in the diagnostic evaluation of many cardiac diseases, including heart failure; cardiomyopathies; pulmonary arterial hypertension; valvular heart disease, and coronary artery disease [14,15,19,20,[47][48][49]. Furthermore, CPET-SE provides additional information in the case of patients who do not have heart failure, but have unexplained exercise dyspnea [50].Exercise pulmonary hypertension due to mitral regurgitation or left ventricular dysfunction can also lead to effort intolerance [46,48].Furthermore, elevated left ventricular filling pressure during exercise in patients with exercise intolerance and without diastolic dysfunction at rest can be identified by CPET-SE [47].Because resting LV EF is weakly correlated with exercise capacity, there is a need to clarify other parameters contributing to exercise performance, including left and right ventricular contractile reserve, interventricular dependence, diastolic function, left atrial function, as well as peripheral factors [46].

Limitations
This study has several limitations.We only included patients who were able to exercise.The mode of exercise used, a cycle ergometer in a semi-recumbent position, could cause lowerextremity muscle fatigue, particularly in untrained patients, and can lead to lower peak VO 2 values compared to treadmill or to supine cycle ergometer.Respiratory movements at peak exercise can cause difficulties in image acquisition, and due to the angle-dependency of Doppler-measured velocities, could lead to underestimation of the calculated stroke volume.
A particular feature of our study was the noninvasive assessment of cardiac and peripheral mechanisms of exercise limitation.Noninvasively assessed A-VO 2 Diff, as calculated using Fick's equation, is strongly related to oxygen uptake and cardiac output and rather reflects "non-cardiac" contributors to oxygen uptake and should not be equated to invasively measured A-VO 2 Diff.However, this method has been used in previous non-invasive studies [15,17,19,20,37,45], and calculated A-VO 2 Diff values were similar to those measured in invasive studies [38,51].
Our study applies only to patients with LV EF � 40%.In patients with reduced LV EF, parameters of heart injury and stroke volume could play a more significant role in exercise intolerance.Furthermore, we recruited a relatively small group of patients from a single center.Some patients did not consent to participate in the study, introducing selection bias.

Conclusions
Patients who have been treated for AMI, even those without reduced LV EF, remain at a risk of developing symptomatic heart failure [52].Identification of factors responsible for exercise intolerance is crucial for their evaluation and management.CPET-SE enables non-invasive assessment of the mechanisms of exercise intolerance.In such patients, the heart rate response during exercise and peak peripheral oxygen extraction have the most marked effect on exercise intolerance.Our findings can help in clinical decision-making and can guide therapy to improve exercise capacity.Further studies with a larger group of post-AMI patients with various degrees of left ventricular dysfunction and with directly measured A-VO 2 Diff are needed to confirm our findings.
Note: Values represent mean ± SD, median (IQR) or number (%).P2Y 12 inhibitors: clopidogrel or ticagrelor.Creatinine clearance was calculated according to Cockrofta Group 2 vs. Group 4 b Group 2 vs. Group 3 � for non-significant presented the lowest p value.https://doi.org/10.1371/journal.pone.0255682.t001 ). Spearman correlations for peak VO 2 , percent predicted VO 2 and clinical and CPET-SE data are presented in S1 Table.Moderately to strong correlations were found for peak VO 2
Note: Values represent mean ± SD, median (IQR) or number (%).Abbreviations: AT, anaerobic threshold; DBP, diastolic blood pressure, FEV 1, forced expiratory volume in the first second; IVC, inspiratory vital capacity; RER, respiratory exchange ratio; SBP, systolic blood pressure; VCO 2 , carbon dioxide production; VD/VT, physiological dead space to tidal volume ratio; VE/VCO 2 slope, minute ventilation to carbon dioxide production slope; VO 2 , oxygen uptake; ΔO 2 /ΔWR, oxygen uptake to work rate increment.Bonferroni post hoc test (by Group) for significant p value: a Group 3 vs.Group 4 b all Groups c Group 1 vs. Group 2 and the Group 3 vs.Group 4 d Group 1 vs. Group 3; and Group 1 vs. Group 4, p = 0.026 for the Group 2 vs Group 4 e Group 1 vs. Group3 f Group 1 vs. Group 2; Group 1 vs. Group 3 g Group 2 vs. Group 4 � for non-significant presented the lowest p value.
Bonferroni post hoc test (by Group) for significant p value: a Group 1 vs. Group 3 b Group 2 vs. Group 3 � for non-significant presented the lowest p value.https://doi.org/10.1371/journal.pone.0255682.t003