Clin Physiol Funct Imaging (2014)

doi: 10.1111/cpf.12172

Identification of cardiac repercussions after intense and prolonged concentric isokinetic exercise in young sedentary people
lon3,4, Caroline Le Goff1, Jean-Francßois Kaux2,3, Vincent Couffignal2, Romain Coubard2, Pierre Me 1 2,3 Etienne Cavalier and Jean-Louis Croisier 1

Department of Clinical Chemistry, University and University Hospital of Liege, 2Department of Motility Sciences, University and University Hospital of Liege, Multidisciplinary Department of Sports Medicine and Traumatology - SPORTS2, University Hospital of Liege, and 4Cardiology Department, University Hospital of Liege, Liege, Belgium 3

Summary Correspondence Caroline Le Goff, Department of Clinical Chemistry, University of Liege, CHU Sart Tilman, Avenue de l’Hoˆpital, B35, 4000 Liege, Belgium E-mail: [email protected]

Accepted for publication Received 23 December 2013; accepted 9 May 2014

Key words cardiac biomarkers; isokinetic; maximal concentric exercise; oxidative stress

Introduction Cardiopathies are the world’s leading cause of mortality and morbidity. Although rare, cardiovascular accidents can occur during intense and infrequent sporting activity, particularly among those who are unaware of their heart condition. The development of cardiospecific biochemical markers has led to a reconsideration of the role of biology in the diagnosis of cardiovascular illnesses. The aim of this study therefore was, through the use of cardiac biomarker assays, to highlight the impact of sustained physical effort in the form of intense and prolonged concentric isokinetic exercise and to research potential cardiovascular risks. Materials and methods Eighteen subjects participated in a maximal concentric isokinetic exercise involving 30 knee flexion–extensions for each leg. Five blood tests were taken to study the kinetics of the cardiac biomarkers. Haemodynamic parameters were measured continuously using a Portapres, and respiratory parameters were measured using a Sensormedics Vmax 29C. Results The results showed significant increases in the creatine kinase, myoglobin, homocysteine and haemoglobin cardiac markers. Evolutionary trends were also observed for the following biomarkers: NT-proBNP, myeloperoxydase and C-reactive protein. All the physiological parameters measured presented statistically significant changes. Conclusion Isokinetic effort leads to the release of cardiac markers in the blood, but these do not exceed the reference values in healthy subjects. Maximal concentric isokinetic exercise does not, therefore, lead to an increased risk of cardiovascular pathologies.

Introduction Cardiovascular illnesses are the leading cause of death in industrialized countries, with a percentage in the order of 30–45%, and they are responsible for the majority of healthcare expenditure (World Health Organization, 2007). Although rare, cardiovascular accidents during physical activity are at the origin of the phenomenon known as ‘sudden death’ (Siegel, 1997; Albert et al., 2000). Biochemical markers, released into the blood, play a particular role in the diagnosis of cardiovascular diseases, in the stratification of risk and in treatment (Le Goff et al., 2012). If during rest under normal conditions, the values of these biomarkers detected in an individual’s blood are higher than the cut-off limits, then the subject presents an

acute risk of developing cardiovascular disease (Le Goff et al., 2012). A range of biomarkers can be useful in terms of highlighting cardiovascular risk. The first, haemoglobin (Hb), is a protein that transports oxygen around the body. Haemoglobin’s affinity for oxygen decreases in the presence of carbon dioxide (CO2), when the blood pH is low, and when temperature increases; in other words, this affinity diminishes while carrying out a physical activity (Costill et al., 2006). Secondly, measuring various muscular and cardiac biomarkers, such as creatine kinase (CK), myoglobin (MYO), N-terminal pro-brain natriuretic peptide (NT-proBNP), troponins (Tn), C-reactive protein (CRP), myeloperoxydase (MPO) and homocysteine (HCY), can be useful in terms of cardiovascular risk assessment.

© 2014 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd

1

2 Cardiac repercussions of concentric exercise, C. Le Goff et al.

CK is an intracellular enzyme involved in energy metabolism. It is present in large quantities in the skeletal muscle (isoenzyme MM), the myocardium (isoenzyme MB) and the brain (isoenzyme BB) and in smaller quantities in visceral tissue. However, an increase in the CKMB/CK ratio is an indicator of heart disease. During prolonged physical effort, CK levels increase and are an indicator of muscular cell destruction (Guezennec et al., 1986; Lefevre, 2005). Myoglobin is a protein that helps transport oxygen and contributes to the build-up of oxygen reserves in the muscles. Myoglobin assays are only informative in the first hours after necrosis. MYO may also be released under any of the following circumstances: surgery, traumatology, rhabdomyolisis, states of shock, muscular ischaemias and myocardial infarctions. Prolonged physical effort, as experienced during a triathlon, releases higher rates of myoglobin, representing levels synonymous with a marker of muscular cell damage (Guezennec et al., 1986; Lefevre, 2005). Another biomarker, NT-proBNP, is becoming increasingly significant in the physiopathology, diagnosis and treatment of certain cardiovascular diseases. In addition to its other functions, NT-proBNP may facilitate early diagnosis of heart failure by detecting an asymptomatic malfunction of the left ventricle or a myocardial ischaemia (Lefevre, 2005). Physical activity may also lead to changes in the plasma levels of the aforementioned biomarkers. The length of the exercise undertaken and the age of the athlete are relevant factors in this regard (Huang et al., 2002). A further cardiac biomarker can be seen in the Tn, which are structural proteins involved in the myocyte contractility system. Troponins may be considered as 100% cardiospecific markers. During prolonged physical activity, an increase observed in cardiac TnT (cTnT) will be due either to cardiomyocyte necrosis or to a temporary and reversible change in membrane permeability (Shave et al., 2002) but would not be influenced by peripheral muscular lesions (Heidenreich et al., 2001; Herrmann et al., 2003; Fortescue et al., 2007). It seems unlikely that a minor increase in the levels of cTnT following prolonged exercise would be due to cardiomyocyte necrosis. Instead, it would be more appropriate to consider the release of cTnT in postexercise periods as an indication of the presence of a reversible lesion in the cardiomyocyte membranes. The presence of such reversible lesions can be linked to a remodelling process (Heidenreich et al., 2001). The arrival of an even more sensitive new generation immunoassay, called high-sensitivity TnT (hsTnT), now enables even minor lesions to be detected at an earlier stage (Lackner, 2014). C-reactive protein is involved during the acute phase of inflammation in the body (Volanakis, 2001). Studies have also highlighted CRP as a risk factor in atherosclerosis (Lloyd-Jones et al., 2006). High-sensitivity doses of CRP (hsCRP) are currently used as a prognostic marker in coronary diseases and as an indicator that preventive measures need to be taken to avoid primary risks. hsCRP can identify subjects presenting a minimal inflammatory reaction, but at a level that is nonetheless

sufficient for it to indicate a pathological risk (Ricker, 2003). To ensure that the risk of coronary heart disease is accurately measured via the hsCRP assay, the interpretation of this assay must be conducted outside of any other clinical or biological inflammatory process. Scientific research has demonstrated that even low intensity leisure activities can significantly reduce rates of CRP (Pitanga & Lessa, 2009; Afshar et al., 2010). Myeloperoxydase is a specific indicator of neutrophil activation and inflammatory activity (Apple et al., 2005). This enzyme is a marker for several pathologies, connected to the activation of neutrophils (for example: cardiovascular diseases, tissue infiltration pathologies requiring intensive care and non-infectious diseases) (McConnico et al., 1999). Although raised levels of MPO indicate an inflammatory response, the correlation with other cardiac markers such as cTnT and NT-proBNP suggests that myocardial damage cannot be ruled out (Marshall et al., 2010). Finally, HCY is a sulphur-containing amino acid. High concentrations of HCY in the plasma can lead to the development of coronary disease, cerebral vascular accidents, thromboembolic accidents or atherosclerosis (Oudi et al., 2010). High concentrations of HCY also represent a graduated and independent risk factor for cardiovascular diseases and overall mortality (Nurk et al., 2002). Several studies have shown that high HCY levels are due to poor lifestyle choices and a lack of physical exercise (Jacques et al., 2001). However, data from the literature remain controversial, due to the lack of studies on the independent relationship between homocysteine, a sedentary lifestyle and physical activity. The objective of our study was to measure the impact of intense physical effort, that is concentric isokinetic fatigue resistant exercise, on healthy, non-trained subjects, by measuring the biological cardiac markers described above.

Methods All tests in the study took place at the University of Liege Hospital Centre (Belgium). Effort tests were conducted in the Cardiology Department, while exercises performed on an isokinetic dynamometer took place in the Physical Medicine and Physical-Therapy-Rehabilitation Department. Biological analyses were conducted in the Clinical Chemistry Department. This study was approved by the Liege Hospital Faculty Ethics Committee (Belgium). The population of this study consisted of 18 healthy male subjects (23
2  4
1 years). The chosen subjects satisfied the following inclusion criteria: having a sedentary lifestyle (or engaging in less than 2 h of sport per week), being aged between 18 and 30, being a non-smoker, having no known prior cardiac or respiratory problems, having no lesions in the lower limbs (ligamentoplasty, meniscectomy) and not taking any medication. The nutritional intake of the volunteers was not an important factor (either before or after the isokinetic exercise) in our study of the cardiac biomarkers. Regularly

© 2014 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd

Cardiac repercussions of concentric exercise, C. Le Goff et al. 3

taking part in an endurance sport such as running or cycling represented a criterion for exclusion from the study. Isokinetic test All subjects were subject to a concentric isokinetic fatigue resistance test using Cybex Norm equipment. An effort test demonstrating their cardiac integrity was conducted in advance. Subjects took part in a general warm-up on an ergonomic bike for 8 min at a resistance of 75 watts and a pedal rate of between 60 and 80 rotations per minute, before moving to the isokinetic dynamometer (Maquet et al., 2002; Hody et al., 2013). The backrest of the equipment was positioned at 90°. The rotational axis of the dynamometer was then aligned with the centre of the external condyle of the femur. Adjustments were made with the knee flexed at 90°. An anteroposterior strap was used to fasten the lower limb, positioned approximately three centimetres above the external malleolus. The angular position was set to 100°, and compensation for the effect of gravity occurred automatically. The seat is equipped with handles, but the subjects were instructed not to use these, so as to avoid interfering with the measurement of physiological parameters. In addition, a researcher was present behind the subject during each series of tests to maintain the torso against the backrest so as to avoid any compensation (Maquet et al., 2002; Hody et al., 2013). After this, the subjects carried out a specific warm-up on the isokinetic dynamometer. This consisted of three series of exercises: (i) five submaximum concentric contractions of the extensor and flexor knee muscles at 120° s 1 allowing the subjects to familiarize themselves with the equipment and to understand the main exercise to be conducted; (ii) five concentric contractions at 120° s 1 of progressive intensity, enabling the subjects to reach their maximum strength; (iii) three concentric contractions at a speed of 180° s 1, in order for subjects to acclimatise themselves to the speed to be used for the test (Hody et al., 2013). The ‘fatigue resistance’ test started 1 min after the end of the specific warm-up. This involved executing a series of 30 flexion–extension knee movements (90° range of motion) at a speed of 180° s 1, for each leg. There was a 5 min break at the end of the first series and before the start of the warm-up for the second series on the other leg. This allowed the researchers to calibrate the test parameters for the leg that had not yet been tested. During the protocol, the subject was strongly encouraged by the researcher to maintain maximum performance; instant representation of the force deployed was displayed on the computer screen, in the form of a curve for each repetition. This enabled the researcher to control individual participation (Hody et al., 2013).

test using a non-invasive complex piece of equipment: the Portapres (Schmidt et al., 1992). This battery-operated portable instrument monitors these parameters through sensors placed on the fingers. These parameter measurements are based partly on the ‘arterial volume-clamp’ method (Penaz, 1992) and partly on a physiological calibration of the arteries in the fingers (Wesseling, 1996). The following physiological parameters were also recorded during the test: blood pressure (recorded using a traditional tensiometer), heart rate (recorded using a heart rate meter watch), respiratory parameters such as ventilation rate (VE), maximum oxygen consumption (VO2max kg 1), the respiratory quotient (RQ), oxygen saturation (SpO2) (recorded using a Sensormedics Vmax 29C; PanGas AG, Dagmersellen, Switzerland) and lactataemia (measured using a YSI 1500 Lactate Sport; YSI, Yellow Springs, OH, USA). Blood parameters Blood tests were taken from the subjects at specific time intervals: just prior to effort (T0), just after (T1), 3 h after (T2), 24 h after (T3) and 48 h after (T4). Analyte assays were conducted in the laboratory, and all automatic analyses were conducted in line with the manufacturer’s specifications. Four analysers were used to perform serial determinations: XE-5000 (Sysmex, Kobe, Japan) for Hb; Modular E170 (Roche Diagnostics, Basel, Switzerland) for CK, MYO, hsTnT, NT-proBNP and hsCRP; Etimax 3000 (Diasorin, Saluggia, Italy) with the Immunodiagnostic kit for MPO; BNII (Siemens, Munich, Germany) for HCY. Statistical analysis Average and standard deviations were calculated for each piece of data. A simple ANOVA test with repeated measurements on the ‘time’ variable for five variability factors enabled variations between the different time intervals to be studied, thus making it possible to determine whether there was any significant changes in biomarker levels. A planned pairwise comparison was conducted to determine variations at different times. Descriptive statistics were used to exploit all the physiological data. Student’s t-tests for matching samples were then conducted to assess variations between physiological data at two different time intervals. The mean levels were compared between the times using an ANOVA-2. Results are presented with Snedecor F-values and P-values. The statistical significance threshold was set at an uncertainty level of 5% (P T2) 23
06  5
13

15
15  0
82** (T0 < T3; T1 > T3) 272
11  123
03** (T0 < T3; T1 < T3; T2 < T3) 35
78  12
01** (T2 < T3)

135
22  89
76

MYO (lg l 1)

29
78  6
07

38
83  19
46** (T0 < T1)

NT-proBNP (pg ml 1)

23
56  25
30

28
06  32
02* (T0 < T1) 0
01  0
00 1
16  1
71

hsTnT (ng ml 1) hs CRP (mg l 1)

0
01  0
00 1
14  1
75

MPO (ng ml 1)

22
39  6
34

HCY (lmol l 1)

9
56  2
02

24
17  6
33* (T0 < T1) 9
99  1
83** (T0 < T1)

Discussion In addition to standard tests assessing maximum muscular strength, the use of isokinetics enables muscle fatigue to be investigated, especially among athletes (Croisier et al., 2013). This form of assessment is very demanding for muscle function. The aim of our study was to ensure that during a maximal concentric isokinetic exercise, subjects did not face any cardiac risk despite the intensity of the effort deployed. Our results are consistent with the literature, which shows, on the one hand, that heart rate increases during physical exercise and, on the other, that it increases sharply at the start then rises more slowly until it reaches an equilibrium value (Costill et al., 2006). We found that blood pressure increased with exercise; it is well known that variations in blood pressure depend in particular on cardiac output and peripheral resistance (Fletcher et al., 1995). In terms of respiratory parameters, even before subjects started their warm-up, it was noted that they all showed RQ values of between 0
77 and 1
02. The average RQ value was 0
87, corresponding to normal metabolic function (Robergs, 2001). By the end of the warm-up and towards the start of the first test, the average RQ value had already risen to 1
06, showing that the subjects’ metabolism was beginning to use glucose as its principal source of energy. Analysis of respiratory gaseous exchange during tests highlighted an increase in ventilation rate and respiratory quotient (VCO2/VO2) closely connected to the production of lactate and the concept of the anaerobic threshold (Karlsson & Saltin, 1970). The anaerobic threshold of around 4 mmol l 1 of lactate (Karlsson & Saltin, 1970) was reached within the first few minutes of recovery from the first isokinetic tests. In line with the literature, we deduced that from the end of the first isokinetic exercise, ATP was no longer being produced in strictly aerobic conditions. Lactate

9,68  1
89** (T1 > T2)

23
67  16
37 0
01  0
00 1
19  1
41 21
83  5
20* (T1 > T3) 9
54  1
60

T4 15
06  0
75** (T0 < T4) 359
94  411
04

57
71  62
56** (T2 > T4) 30
94  28
58* (T0 < T4) 0
01  0
00 1
01  1
09* (T3 > T4) 22
47  7
42 9
14  1
73** (T0 < T4; T1 > T4)

levels continued to increase during the few minutes allowed for recovery. However, to be able to maintain high-intensity effort, the second exercise was conducted while subjects were still experiencing a lactic anaerobic state with a glycolytic energy system. Lactate peak was found to occur well after 5 min posteffort, confirming data found in the literature (Karlsson & Saltin, 1970). The isokinetic exercise undertaken by the subjects in the present study led to a significant difference in their Hb levels over time. The values observed fall within the scope of the reference values (13 g dl 1 < Hb < 17
87 g dl 1). None of the subjects was found to be anaemic (Hb < 13 g dl 1). The reference time was established as the posteffort time (T1), which was statistically significant in comparison with all the other time intervals. The literature highlights the fact that physical exercise reduces plasma volume, leading to early haemoconcentration and, consequently, to an increase in haemoglobin concentration (Costill et al., 2006). Our results also confirm a significant increase in haemoglobin levels during isokinetic effort. Furthermore, we deduced that the subjects were not sufficiently hydrated before the test, thus creating hyperviscosity of the blood during effort, leading to an increase in haemoglobin levels. Such haemoconcentration raises the risk of ischaemia and thrombosis (Costill et al., 2006). Haemoglobin therefore represents a marker of hydration and performance through the volumetric regulation of the blood. With hindsight and with the aim of standardizing the protocol, it would have been beneficial to impose or control the subjects’ drinking levels. CK levels were already above the cut-off values (30 UI l 1 < CK < 175UI l 1) for 20% of the subjects, at the first time interval (T0), before the test. During the isokinetic test, we saw a statistically significant evolution of CK levels throughout the analyses. We can deduce from the results that

© 2014 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd

6 Cardiac repercussions of concentric exercise, C. Le Goff et al.

the increase occurred continually over 24 h until T3, by which time CK levels had doubled, rising from 140 to 280 UI l 1. We then observed a slight, non-significant regression between T3 and T4. CK plays an important role in energy metabolism, and its rate increases during prolonged physical effort (Guezennec et al., 1986). Our results show that intense exercise over a short period can also significantly increase CK levels. CK levels are also known to rise in numerous pathological situations such as in the presence of muscular lesions (Gojanovic et al., 2011). CK levels can also be used to quantify the severity of muscular damage. This may explain the increase in CK levels in some of our subjects, showing an increase of up to five times the initial rate by the end of the test. Our results showed a significant increase in base MYO levels (T0) by T1 and T2, by which points the levels were found to have doubled (from 30 to 73 lg l 1). There then followed a significant reduction between T2–T3, where MYO levels returned to almost normal. After this, the levels tended to rise but not significantly. The reference time for MYO remained 3 h posteffort (T2), which was statistically significant in comparison with all the other time intervals, and where 30% of subjects showed MYO levels that exceeded the cut-off threshold (>72 ng ml 1). It is important to remember that MYO is not cardiospecific but is a component of all skeletal muscle and may thus be released in a range of circumstances, particularly during prolonged physical effort (Guezennec et al., 1986). It can be deduced from our study that simple, short and intense effort also releases MYO, which can be measured by well-defined kinetics. The literature indicates that during myocardial infarction, the kinetics of MYO evolve in such a way as to reach a maximum peak at around 4–12 h postinfarction. By comparison, we observed in our study that the maximum peak was at 3 h by default. We had no references for changes in MYO between 3 and 24 h posteffort. Like CK, MYO is a marker of muscular cell damage (Guezennec et al., 1986), and it may be influenced by the appearance of delayed onset muscle soreness in some subjects (Hody et al., 2011). NT-proBNP assays carried out at the different time intervals showed that all subjects maintained their NT-proBNP levels within the cut-off range (5 qg ml 1 < NT-proBNP < 103 qg ml 1). Following the isokinetic test, we saw that overall, there was no significant difference in the levels of this biomarker during the 48 h of analysis. However, in comparison with initial levels, a significant difference was found in NT-proBNP levels between T1, T2 and T4. The literature highlights that short, intense exercise leads to a rise in this marker, which remains both less prolonged following aerobic exercise and within the cut-off range (Jacques et al., 2001). The results obtained confirm the data in the literature. Intense and prolonged exercise, such as running, leads to a simultaneous large rise in NT-proBNP levels (Huang et al., 2002; Croisier et al., 2003). This increase is felt 24 h after effort, with NT-proBNP levels having been seen to double in another

study (Huang et al., 2002). In our study, no significant difference was found between NT-proBNP levels at T0 and T3. The duration of the exercise therefore represented a relevant factor, as indicated in the literature (Huang et al., 2002). Diabetes is a pathology that can lead to acute secretion of NT-proBNP (Jourdain et al., 2009). In line with this finding, we found in our study that one diabetic subject, present early on in the study, presented NT-proBNP levels at all time intervals that were above the cut-off values (NT-proBNP > 103 qg ml 1). Taking all this into account, clinical measurements of NT-proBNP must therefore be rigorously interpreted, depending on the type and duration of the physical activity and the various other possible factors of influence. Classic TnT assays are not sufficiently sensitive to be able to pick up significant variations in this biomarker during physical exercise, as the detection limit is set at 0
01 gg ml 1 (Le Goff et al., 2012). We therefore used an hsTnT assay, which enables the detection of much lower rates, with norms of around 0
013 to 0
016 ng ml 1. Despite the enhanced level of sensitivity of this test, no statistically significant modification was observed. All values for the marker levels taken for hsTnT were around