Download Heart rate recovery after maximal exercise is associated with

Am J Physiol Heart Circ Physiol 291: H459 –H466, 2006.
First published February 24, 2006; doi:10.1152/ajpheart.01193.2005.
Heart rate recovery after maximal exercise is associated with acetylcholine
receptor M2 (CHRM2) gene polymorphism
Arto J. Hautala,1,2 Tuomo Rankinen,1 Antti M. Kiviniemi,2,3 Timo H. Mäkikallio,3,4
Heikki V. Huikuri,3 Claude Bouchard,1 and Mikko P. Tulppo2,3
1
Pennington Biomedical Research Center, Human Genomics Laboratory, Louisiana State University System,
Baton Rouge, Louisiana; 2Merikoski Rehabilitation and Research Center, Oulu; 3Division of Cardiology,
Department of Medicine, University of Oulu, Oulu; and 4Lapland Central Hospital, Rovaniemi, Finland
Submitted 11 November 2005; accepted in final form 19 February 2006
genotype; exercise training; cardiovascular autonomic function
DELAYED HEART RATE (HR) recovery after physical exercise has
been shown to be a powerful independent predictor of mortality in healthy subjects and in different patient populations
(9 –11, 25, 33, 49). The balance between the vagal and sympathetic activities mediates the changes in HR during recovery
from physical exercise. Acetylcholine released from cardiac
vagal nerve endings triggers a chronotropic effect by decreasing HR (6, 8). It is well established that attenuated vagal
reactivation after the termination of exercise is involved in
slow HR recovery (4, 11, 12, 24, 35). However, marked
interindividual differences have been observed in HR recovery
rates even in healthy subjects, suggesting a possibility of a
Address for reprint requests and other correspondence: A. J. Hautala, Dept.
of Exercise and Medical Physiology, Merikoski Rehabilitation and Research
Centre, P.O. Box 404, Kasarmintie 13, FIN-90101, Oulu, Finland (e-mail:
[email protected]).
http://www.ajpheart.org
genetically determined background for postexercise HR behavior (39, 40).
Regular endurance training shifts the cardiac autonomic
balance toward vagal dominance (3, 7, 13, 17, 19, 47). Cardiac
vagal activity increases markedly even after 2 wk of regular
training (30, 50). Previous studies have also shown that
postexercise HR recovery improves after endurance training in
sedentary healthy males (46) and in patients with coronary
artery disease (18). However, a substantial heritable component may also be involved in the regulation of HR behavior in
response to training (40, 41), and this may partly explain the
large interindividual variation in HR recovery.
Muscarinic acetylcholine receptors play a fundamental role
in cardiac function via vagally mediated regulation of the
autonomic nervous system. The human heart expresses predominantly muscarinic acetylcholine receptor subtype M2
(CHRM2) (6, 8). Fisher et al. (14) showed that vagally induced
bradycardiac responses were totally abolished in CHRM2deficient mice in vivo, suggesting the exclusive role of
CHRM2 in HR regulation (14). Therefore, we tested the
hypothesis that, among healthy individuals, the CHRM2 gene
polymorphisms are associated with 1-min HR recovery after
peak exercise in the sedentary state and after endurance training.
METHODS
Subjects
The subjects were recruited by newspaper ads, which attracted 355
replies. All smokers, subjects with body mass index (BMI) ⬎ 32
kg/m2, subjects who did regular physical training more than two times
per week, and subjects with diabetes mellitus, asthma, or cardiovascular disorders were excluded. We invited 108 subjects to our laboratory (Dept. of Exercise and Medical Physiology, Merikoski Rehabilitation and Research Centre, Oulu, Finland) for more specific
assessment. The number of subjects was selected on the basis of a
priori power analysis to give 80% power to the responsiveness of
maximal aerobic power [peak oxygen consumption (V̇O2 peak)] after 2
wk of endurance training. According the previous studies (16, 30, 31,
50), 8% improvement in V̇O2 peak can be expected after short-term
endurance training with SD of approximately ⫾7%, giving the estimate of needing at least 10 subjects for both the intervention and the
control group. The subjects were randomized into a training group
(n ⫽ 90) and a control group (n ⫽ 18). The unbalanced study design
was used to particularly assess the contribution of genotypes to actual
training effect. The average heterozygosity and allele frequencies of
DNA sequence variation of interest are available from the National
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0363-6135/06 $8.00 Copyright © 2006 the American Physiological Society
H459
Downloaded from ajpheart.physiology.org on November 2, 2009
Hautala, Arto J., Tuomo Rankinen, Antti M. Kiviniemi, Timo
H. Mäkikallio, Heikki V. Huikuri, Claude Bouchard, and Mikko
P. Tulppo. Heart rate recovery after maximal exercise is associated
with acetylcholine receptor M2 (CHRM2) gene polymorphism. Am J
Physiol Heart Circ Physiol 291: H459 –H466, 2006. First published
February 24, 2006; doi:10.1152/ajpheart.01193.2005.—The determinants of heart rate (HR) recovery after exercise are not well known,
although attenuated HR recovery is associated with an increased risk
of cardiovascular mortality. Because acetylcholine receptor subtype
M2 (CHRM2) plays a key role in the cardiac chronotropic response,
we tested the hypothesis that, in healthy individuals, the CHRM2 gene
polymorphisms might be associated with HR recovery 1 min after the
termination of a maximal exercise test, both before and after endurance training. The study population consisted of sedentary men and
women (n ⫽ 95, 42 ⫾ 5 yr) assigned to a training (n ⫽ 80) or control
group (n ⫽ 15). The study subjects underwent a 2-wk laboratorycontrolled endurance training program, which included five 40-min
sessions/wk at 70 – 80% of maximal HR. HR recovery differed between the intron 5 rs324640 genotypes at baseline (C/C, ⫺33 ⫾ 10;
C/T, ⫺33 ⫾ 7; and T/T, ⫺40 ⫾ 11 beats/min, P ⫽ 0.008). Endurance
training further strengthened the association: the less common C/C
homozygotes showed 6 and 12 beats/min lower HR recovery than the
C/T heterozygotes or the T/T homozygotes (P ⫽ 0.001), respectively.
A similar association was found between A/T transversion at the
3⬘-untranslated region of the CHRM2 gene and HR recovery at
baseline (P ⫽ 0.025) and after endurance training (P ⫽ 0.005). These
data suggest that DNA sequence variation at the CHRM2 locus is a
potential modifier of HR recovery in the sedentary state and after
short-term endurance training in healthy individuals.
H460
HEART RATE RECOVERY AND CHRM2 GENE POLYMORPHISM
Center for Biotechnology Information (NCBI) database. These frequencies were used to estimate the hypothetical number of subjects of
each genotype group at the present population. During the study, 13
subjects dropped out (10 from the training group and 3 from the
control group) because of personal or health-related problems. Finally, 80 subjects (36 men and 44 women) performed the endurance
training program. The control group consisted of 15 subjects (9 men
and 6 women). The Ethical Committee of the Northern Ostrobothnia
Hospital District, Oulu, Finland, approved the protocol.
Experimental Design
AJP-Heart Circ Physiol • VOL
Table 1. Baseline characteristics of subjects
Phenotype
Endurance (n ⫽ 80)
Control (n ⫽ 15)
Age, yr
BMI, kg/m2
DBP, mmHg
SBP, mmHg
V̇O2peak, l/min
Peak respiratory exchange ratio
Maximal HR, beats/min
HR recovery, beats/min
HR overnight, beats/min
SDNN, ms
LF power, ln ms2
HF power, ln ms2
LF-to-HF ratio
41⫾5
25⫾3
78⫾8
123⫾12
2.43⫾0.70
1.25⫾0.07
181⫾10
36⫾9
59⫾7
101⫾23
7.26⫾0.60
6.61⫾0.82
2.26⫾1.29
41⫾5
25⫾2
76⫾5
124⫾14
2.66⫾0.70
1.25⫾0.06
180⫾11
36⫾9
57⫾11
110⫾27
7.28⫾0.55
6.60⫾0.76
2.25⫾1.25
Values are means ⫾ SD; n, number of subjects. Heart rate (HR) variability
is measured overnight (from midnight to 6 AM). BMI, body mass index; DBP
and SBP, diastolic and systolic blood pressure, respectively; V̇O2peak, peak O2
uptake; LF, low frequency; HF, high frequency; SDNN, standard deviation of
all R-R intervals.
291 • JULY 2006 •
www.ajpheart.org
Downloaded from ajpheart.physiology.org on November 2, 2009
Sequence of tests. On their first laboratory day, the subjects completed a health status questionnaire, gave written informed consent,
and were assessed for BMI. Resting electrocardiogram (12-lead ECG)
was recorded to confirm their cardiac health status, and overnight R-R
intervals were recorded to evaluate autonomic regulation by the HR
variability method. On the second day, blood pressure was recorded
and maximal exercise testing performed. Use of alcohol or strenuous
physical activity was not allowed during the test days and on the two
preceding days.
The endurance training period was 2 wk, including five consecutive
sessions per week (Monday–Friday). The short intervention period
was chosen on the basis of the previous studies, which shows that a
well-controlled, short-term training intervention increased significantly V̇O2 peak (16, 20, 30, 31, 45, 50) and cardiac vagal activity (30,
50). At the end of the training intervention, all measures were repeated
48 h after the last training session. The control group was tested
similarly to the training group, and they were asked to maintain their
habitual physical activity level during the 2-wk period. Finally, blood
samples were collected 2 days after the last exercise testing in the
laboratory of Internal Medicine at the University Hospital of Oulu.
Assessment of V̇O2 peak, maximal HR, and HR recovery. The subjects performed a graded maximal exercise test on an 839E Monark
cycle ergometer (Stockholm, Sweden). The test was started at 25 W,
and work rate was increased by 25 W every 2 min until exhaustion.
Pedaling was started at 50 rpm and increased by 5 rpm up to 90 rpm,
to achieve maximal effort. The subjects were encouraged to continue
cycling until they could no longer maintain the required pace, at which
time the test was terminated. After the termination of the test, the
subjects were asked to remain seated on the bicycle, and HR recording
was continued for 6 min. They were not allowed to talk or move
during the first minute after the test, which was followed by a 5-min
cool-down period at a work rate of 25 W. HR recovery was determined as maximal HR (mean of 10 s) ⫺ HR at 1 min after exercise
(mean of 5 s). Ventilation, gas exchange (M909 Ergospirometer,
Medikro, Kuopio, Finland), and HR (Cardiolife TEC-7721K, Nihon
Kohden, Tokyo) were monitored continuously during the protocol.
The highest oxygen uptake value measured during the test (1-min
collection) was taken as the V̇O2 peak. All subjects fulfilled the criteria
for V̇O2 peak given in the literature (i.e., respiratory exchange ratio
⬎1.1 or maximal HR within ⫾10 beats of the age-appropriate reference value) (21). HR was recorded during the exercise test and
recovery with a Polar R-R Recorder (Polar Electro, Kempele, Finland)
and saved in a computer for further analysis with the HEARTS
software (Heart Signal, Kempele, Finland).
Assessment of resting blood pressure. Resting systolic (SBP) and
diastolic blood pressures (DBP) were assessed by using an electronic
sphygmomanometer (Omron M4, Omron Healthcare). The subjects
lay in the supine position in a quiet room for at least 10 min before the
blood pressure measurements. The blood pressure measurements were
performed in a supine position at the same time of the day before and
after the training program.
Assessment of HR variability. The R-R intervals were recorded
overnight (from midnight to 6 AM) with a Polar R-R Recorder at an
accuracy of 1 ms and saved in a computer for further analysis of HR
variability with the Hearts software. All R-R intervals were edited by
visual inspection on the basis of ECG portions to exclude all undesirable beats, which accounted for ⬍2% in every subject’s recording.
The details of this analysis and the filtering technique have been
described previously (22). The subjects were asked to go to bed before
midnight and to stay in bed until 6 AM on the R-R interval recording
days.
The mean HR and the SD of all R-R intervals were used as
time-domain measures of HR variability. An autoregressive model
was used to estimate the power spectrum densities of R-R interval
variability. Low-frequency power (LF, 0.04 – 0.15 Hz) and highfrequency power (HF, 0.15– 0.4 Hz) were calculated from the segments of the 512 R-R interval overnight recording (36). A logarithmic
transformation to the natural base was performed on both spectral
components of HR variability.
Exercise training. The endurance training program consisted of
cycling on an 818E Monark cycle ergometer for 40 min (Stockholm,
Sweden). Each exercise session consisted of a 5-min warm-up period
(cycling at 50-W and 75-W resistance for women and men, respectively), followed by 30 min of cycling at a resistance that elicited a HR
of 70 – 80% of maximal HR, and ended with a 5-min cool-down
period (cycling at 50-W and 75-W resistance for women and men,
respectively). Exercise intensity was closely monitored using a Polar
Electro HR monitor A1, and the mean HR of each training session was
recorded. Endurance exercise training was planned on the basis of the
recommendations of American College of Sports Medicine (2). A
professional instructor supervised each training session.
Determination of genotypes. EDTA blood samples were obtained
from all subjects, and DNA was isolated by using simple salting-out
procedure. Six single-nucleotide polymorphisms (SNPs) were selected from the NCBI dbSNP database: rs2278098 in intron 3 (1780
bp downstream from exon 3), rs2061174 in intron 4 (25,573 from
exon 4), rs324640 and rs324650 in intron 5 (10,571 and 5,906 bp,
respectively, upstream of exon 6), rs8191992 in the 3⬘-untranslated
region of exon 6, and rs1378650 located 152 bp downstream of exon
6. These SNPs were chosen on the basis of their clinical relevance
(48) and their capturing role of common genetic variation at the
CHRM2 locus. The SNPs were genotyped by using template-directed
dye-terminator incorporation with the fluorescence polarization detection (FP-TDI) method. A DNA sequence containing the SNP was
amplified with PCR, and after cleaning with shrimp alkaline phosphatase and exonuclease I, the PCR product was used as a template in
the AcycloPrime-FP reaction. The SNP detection primers were designed so as to locate their 3⬘-end immediately upstream of the
polymorphic (SNP) site. The SNP detection PCR reaction utilized a
mutant thermostable polymerase and a pair of AcycloTerminators
H461
HEART RATE RECOVERY AND CHRM2 GENE POLYMORPHISM
Table 2. Allele frequencies and pairwise linkage disequilibriums of CHRM2 SNPs
SNP
rs2278098
rs2278098
rs2061174
rs324640
rs324650
rs8191992
rs1378650
0.01
0.01
0.01
0.01
0.01
rs2061174
rs324640
rs324650
rs8191992
rs1378650
MAF
MAP
1.00
0.11
0.86
0.11
0.86
1.00
0.44
0.94
0.96
0.96
0.26
0.78
0.89
0.89
0.95
A:0.07
G:0.15
C:0.39
T:0.39
A:0.37
A:0.32
136013199
136118655
136146251
136150916
136158563
136162406
0.21
0.21
0.28
0.24
1.00
0.82
0.58
0.82
0.58
0.74
D⬘ values are shown in the upper triangle (in boldface), and r2 values in the lower triangle (in italics). MAF, minor allele frequencies; MAP, location of the
single nucleotide polymorphism (SNP) in base pairs (bp) on chromosome 7. CHRM2, acetylcholine receptor subtype MZ gene.
Statistical Analyses
Chi-square test was used to verify whether the observed genotype
frequencies were in Hardy-Weinberg equilibrium, and pairwise linkage disequilibrium (LD) between the SNPs was assessed using the
ldmax program available in the GOLD software package (1). The
normal Gaussian distribution of the data was verified by the Kolmo-
gorov-Smirnov goodness-of-fit test. The difference in change in
V̇O2 peak after training within the training and control group was
analyzed by a two-factor ANOVA for repeated measures with time
and interventions followed by post hoc analysis (Student’s paired
t-test). The associations between the CHRM2 gene polymorphisms,
the end-point phenotypes, and the associations with the haplotypes
were tested with an analysis of covariance by using the general linear
model procedure of the SAS software package. The phenotypes were
adjusted for age, sex, and BMI. The training response phenotypes
were adjusted in addition to the baseline value of the phenotype.
Pearson’s correlation coefficients were calculated to study the associations between the HR phenotypes, HR variability phenotypes,
physical performance phenotypes, and resting blood pressure before
and after the training program. Pearson’s correlation analysis was also
used to test the relationships between HR recovery and the other
measured phenotypes separately within the three genotype groups
Table 3. Associations between CHRM2 rs324640 genotype and hemodynamic phenotypes, V̇O2peak, and HR variability
overnight (from midnight to 6 AM) before and after a 2-wk endurance training program
CHRM2 rs324640 Genotype: Endurance (n ⫽ 80)
Baseline
Men/Women
Age, yr
DBP, mmHg
SBP, mmHg
V̇O2peak, l/min
HRmax, beats/min
HRrec, beats/min
HR, beats/min
SDNN, ms
LF power, ln ms2
HF power, ln ms2
LF-to-HF ratio
Posttraining
C/C (n ⫽ 13)
C/T (n ⫽ 39)
T/T (n ⫽ 28)
GLM P value
7/6
42⫾7 (31–50)
82⫾8
129⫾12
2.40⫾0.80
184⫾10
33⫾10
62⫾7
99⫾27
7.19⫾0.74
6.23⫾0.77
3.06⫾1.68
16/23
42⫾5 (31–51)
78⫾8
124⫾12
2.43⫾0.69
180⫾11
33⫾7
58⫾7
104⫾26
7.35⫾0.62
6.73⫾0.85
2.08⫾0.97
13/15
41⫾6 (33–51)
75⫾7
120⫾12
2.45⫾0.69
181⫾10
40⫾11
59⫾6
99⫾19
7.19⫾0.50
6.61⫾0.78
2.13⫾1.38
0.649
0.049
0.138
0.452
0.393
0.008
0.058
0.461
0.278
0.183
0.064
C/C (n ⫽ 13)
C/T (n ⫽ 39)
T/T (n ⫽ 28)
7/6
16/23
13/15
78⫾8
125⫾13
2.58⫾0.84
184⫾6
30⫾9
59⫾7
105⫾29
7.35⫾0.72
6.41⫾0.89
3.33⫾1.68
76⫾8
122⫾12
2.63⫾0.74
180⫾10
36⫾8
57⫾7
106⫾28
7.43⫾0.71
6.79⫾0.90
2.14⫾1.11
72⫾6
118⫾9
2.64⫾0.72
182⫾9
42⫾13
57⫾6
107⫾30
7.36⫾0.71
6.74⫾0.92
2.22⫾1.36
0.045
0.196
0.311
0.408
0.001
0.284
0.955
0.587
0.442
0.063
C/T (n ⫽ 10)
T/T (n ⫽ 5)
GLM P value
6/4
3/2
75⫾8
122⫾11
2.73⫾0.80
178⫾8
33⫾7
55⫾12
109⫾34
7.33⫾0.43
6.56⫾0.96
2.69⫾1.82
77⫾3
120⫾7
2.55⫾0.85
181⫾17
37⫾5
62⫾6
101⫾17
7.64⫾0.29
6.64⫾0.83
3.02⫾1.78
GLM P value
CHRM2 rs324640 Genotype: Control (n ⫽ 15)
C/C (n ⫽ 0)
Men/Women
Age, yr
DBP, mmHg
SBP, mmHg
V̇O2peak, l/min
HRmax, beats/min
HRrec, beats/min
HR, beats/min
SDNN, ms
LF power, ln ms2
HF power, ln ms2
LF-to-HF ratio
C/T (n ⫽ 10)
T/T (n ⫽ 5)
GLM P value
6/4
41⫾4 (35–46)
76⫾5
124⫾16
2.66⫾0.70
177⫾9
35⫾8
55⫾11
113⫾30
7.16⫾0.58
6.58⫾0.85
2.12⫾1.40
3/2
42⫾6 (32–50)
75⫾3
123⫾9
2.66⫾0.77
185⫾13
39⫾11
62⫾8
105⫾23
7.57⫾0.40
6.66⫾0.56
2.57⫾0.81
0.765
0.571
0.911
0.975
0.290
0.411
0.768
0.696
0.105
0.224
0.685
C/C (n ⫽ 0)
0.539
0.626
0.573
0.923
0.210
0.998
0.838
0.118
0.222
0.379
Values are means ⫾ SD; n, number of subjects; age ranges are in parenthesis. The phenotypes were adjusted for age, sex, and BMI. GLM, general linear model;
HRmax, maximal HR; HRrec, HR recovery. Baseline, before 2-wk endurance training program; posttraining, after 2-wk endurance training program.
AJP-Heart Circ Physiol • VOL
291 • JULY 2006 •
www.ajpheart.org
Downloaded from ajpheart.physiology.org on November 2, 2009
labeled with R110 and TAMRA (AcycloPrime-FP SNP kit,
PerkinElmer Life Sciences, Boston, MA), representing the possible
alleles for the SNP of interest. Allele detection was done by measuring
the changes in fluorescence polarization after excitation of the samples by plane-polarized light by using a Victor2 FP Plate Reader
(PerkinElmer Life Sciences) on a 384-well plate format. Allele calling
was done by using the SNPscorer genotyping software (PerkinElmer
Life Sciences). Haplotypes were constructed using the SHEsis software package (38).
H462
HEART RATE RECOVERY AND CHRM2 GENE POLYMORPHISM
(rs324640 in Intron 5; C/C, C/T, T/T). Values are given as means ⫾
SD. The SAS statistical software package (SAS Institute) was used for
the analyses.
RESULTS
the CHRM2 SNPs (e.g., for rs324640 SNP the improvement of
V̇O2 peak was 7.5 ⫾ 7.2% for C/C, 8.2 ⫾ 7.2% for C/T, and
8.0 ⫾ 5.7% for T/T, P ⫽ 0.924).
Association Between CHRM2 SNPs and HR Recovery
Training
All the subjects in the training group maintained the training
frequency of 10 times during 2 wk. The intensity of training
was 74 ⫾ 3% of maximal HR, and the mean duration of
exercise was 40 ⫾ 3 min/session. The change in V̇O2 peak was
⫹8.1 ⫾ 5.7% (P ⬍ 0.001) for the training group and ⫺0.5 ⫾
7.0% (P ⫽ 0.941) for the control group. There were no
differences in the change of V̇O2 peak between the genotypes in
Maximal HR was not significantly associated with the
CHRM2 SNPs. However, HR recovery differed significantly
between the rs324640 genotypes at baseline (C/C, ⫺33 ⫾ 10;
C/T, ⫺33 ⫾ 7; and T/T, ⫺40 ⫾ 11 beats/min, P ⫽ 0.008, Fig.
1A, Table 3). The association was even stronger (P ⫽ 0.001)
after the 2-wk endurance training period: the C/C homozygotes
showed 6 and 12 beats/min lower HR recovery than the C/T
heterozygotes and the T/T homozygotes, respectively (Fig.
1B). The change in HR recovery after the training program also
differed between the rs324640 genotypes (C/C, ⫺3 ⫾ 7; C/T,
2 ⫾ 7; and T/T, 2 ⫾ 8 beats/min, P ⫽ 0.038). Moreover,
rs8191992 SNP was associated with HR recovery: the A/A
homozygotes and the A/T heterozygotes showed lower HR
recovery values than the T/T homozygotes both at baseline
(P ⫽ 0.025, Table 4) and after the endurance training program
(P ⫽ 0.005, Table 4). The SNPs rs2278098, rs2061174, and
rs1378650 were not associated with HR recovery.
Association Between CHRM2 SNPs and HR Variability
Overnight HR was not significantly associated with CHRM2
SNPs. However, the LF-to-HF ratio showed significantly
Table 4. Associations between the CHRM2 rs8191992 genotype and the hemodynamic phenotypes, V̇O2peak, and HR
variability overnight (from midnight to 6 AM) before and after a 2-wk endurance training program
CHRM2 rs8191992 Genotype: Endurance (n ⫽ 80)
Baseline
Men/Women
Age, yr
DBP, mmHg
SBP, mmHg
V̇O2peak, l/min
HRmax, beats/min
HRrec, beats/min
HR, beats/min
SDNN, ms
LF power, ln ms2
HF power, ln ms2
LF-to-HF ratio
Posttraining
A/A (n ⫽ 13)
A/T (n ⫽ 36)
T/T (n ⫽ 31)
GLM P value
7/6
43⫾6 (33–51)
81⫾8
129⫾12
2.52⫾0.71
186⫾11
33⫾9
60⫾7
102⫾26
7.30⫾0.69
6.32⫾0.90
3.12⫾1.66
14/22
41⫾5 (31–51)
79⫾8
124⫾12
2.32⫾0.66
180⫾11
34⫾7
59⫾7
105⫾26
7.33⫾0.66
6.72⫾0.84
2.09⫾0.99
15/16
40⫾4 (33–51)
75⫾7
120⫾12
2.48⫾0.70
181⫾10
39⫾11
59⫾6
97⫾19
7.18⫾0.49
6.60⫾0.76
2.10⫾1.32
0.252
0.102
0.211
0.853
0.053
0.025
0.435
0.242
0.329
0.420
0.036
A/A (n ⫽ 13)
A/T (n ⫽ 36)
T/T (n ⫽ 31)
7/6
14/22
15/16
79⫾8
125⫾13
2.72⫾0.74
184⫾8
32⫾9
58⫾6
106⫾28
7.41⫾0.68
6.41⫾0.89
3.44⫾2.27
77⫾9
122⫾12
2.51⫾0.74
180⫾10
35⫾9
57⫾7
107⫾30
7.43⫾0.74
6.81⫾0.92
2.13⫾1.16
72⫾6
118⫾9
2.68⫾0.73
182⫾9
41⫾12
57⫾6
106⫾29
7.34⫾0.68
6.72⫾0.88
2.19⫾1.30
0.022
0.174
0.786
0.176
0.005
0.595
0.828
0.576
0.536
0.046
A/T (n ⫽ 8)
T/T (n ⫽ 7)
GLM P value
4/4
2/5
74⫾7
122⫾11
2.93⫾0.75
175⫾8
35⫾5
52⫾8
118⫾30
7.44⫾0.40
6.81⫾0.87
2.36⫾1.65
78⫾8
120⫾8
2.36⫾0.76
183⫾14
34⫾8
65⫾10
91⫾22
7.38⫾0.46
6.30⫾0.91
3.35⫾1.85
GLM P value
CHRM2 rs8191992 Genotype: Control (n ⫽ 15)
A/A (n ⫽ 0)
Men/Women
Age, yr
DBP, mmHg
SBP, mmHg
V̇O2peak, l/min
HRmax, beats/min
HRrec, beats/min
HR, beats/min
SDNN, ms
LF power, ln ms2
HF power, ln ms2
LF-to-HF ratio
A/T (n ⫽ 8)
T/T (n ⫽ 7)
GLM P value
4/4
41⫾4 (35–46)
77⫾6
126⫾17
2.85⫾0.65
175⫾9
37⫾6
51⫾9
119⫾28
7.34⫾0.48
6.75⫾0.85
2.20⫾1.58
2/5
42⫾5 (32–50)
74⫾3
120⫾9
2.45⫾0.73
185⫾11
35⫾11
64⫾8
99⫾23
7.20⫾0.68
6.40⫾0.63
2.31⫾0.75
0.740
0.913
0.697
0.507
0.087
0.972
0.069
0.490
0.837
0.607
0.936
A/A (n ⫽ 0)
Values are means ⫾ SD; n, number of subjects; age ranges are in parenthesis. The phenotypes were adjusted for age, sex, and BMI.
AJP-Heart Circ Physiol • VOL
291 • JULY 2006 •
www.ajpheart.org
0.700
0.758
0.257
0.197
0.934
0.088
0.256
0.923
0.568
0.599
Downloaded from ajpheart.physiology.org on November 2, 2009
The baseline characteristics of the subjects are shown in
Table 1, and a summary of the minor allele frequencies, as well
as the pairwise LD of the CHRM2 SNPs, is shown in Table 2.
The minor allele frequencies were similar between men and
women in all CHRM2 SNPs (e.g., for rs324640 C:0.40 for men
and C:0.39 for women). SNPs rs2278098 and rs2061174
showed low LDs with other CHRM2 SNPs, whereas moderate
to high pairwise LDs were observed between the SNPs located
in the vicinity of the coding exon 6 of the CHRM2 gene.
Because the intron 5 SNPs rs324640 and rs324650 were in
complete LD (r2 ⫽ 1.0), they yielded identical results. Therefore, only the results for rs324640 are presented. All genotype
frequencies were in Hardy-Weinberg equilibrium.
HEART RATE RECOVERY AND CHRM2 GENE POLYMORPHISM
H463
gotes (Fig. 2F) showed a strong positive correlation [r ⫽
0.517, 95% confidence interval (CI) from 0.20 to 0.74],
whereas the C/C homozygotes (Fig. 2B) showed a negative
correlation (r ⫽ ⫺0.353, CI from ⫺0.72 to 0.17) and the C/T
heterozygotes (Fig. 2D) an intermediate correlation (r ⫽ 0.087,
CI from ⫺0.22 to 0.38).
Haplotype Analysis
DISCUSSION
Fig. 1. Individual and mean values of heart rate recovery before (baseline; A)
and after (posttraining; B) 2 wk of controlled endurance training program
according to the acetylcholine receptor subtype M2 (CHRM2) gene rs324640
polymorphism in healthy subjects. The association tests between the rs324640
genotype and heart rate recovery are adjusted for age, sex, and body mass
index. bpm, Beats/min.
higher values in the rs8191992 A/A homozygotes than in the
other genotypes both before (P ⫽ 0.036) and after (P ⫽ 0.046)
endurance training (Table 4). The rs324640 C/C homozygotes
showed a similar trend (P ⫽ 0.064 and P ⫽ 0.063, respectively), but the difference was not statistically significant (Table 3).
Association Between CHRM2 SNPs and Blood Pressure
Resting DBP was significantly higher in the rs324640 C/C
homozygotes than in the other genotypes both before (P ⫽
0.049) and after (P ⫽ 0.045) the training program (Table 3).
Similar trends were also observed for SBP, but the differences
did not reach statistical significance.
Correlation Between SBP and HR Recovery Between
rs324640 Genotypes
There were no statistically significant correlations between
HR recovery, HR variability, and the other hemodynamic
phenotypes before (e.g., HR recovery vs. HF power, r ⫽ 0.016;
and HR recovery vs. LF-to-HF ratio, r ⫽ ⫺0.064) or after the
training program (e.g., HR recovery vs. HF power, r ⫽
⫺0.033; and HR recovery vs. LF-to-HF ratio, r ⫽ ⫺0.135).
However, the relationship between resting SBP and HR recovery varied considerably between the rs324640 genotypes, especially in the trained state (Fig. 2): the common T/T homozyAJP-Heart Circ Physiol • VOL
The novel finding of the present study was the association of
the CHRM2 gene polymorphism with HR recovery after maximal exercise. These data suggest that, among healthy individuals, DNA sequence variation at the CHRM2 locus is a potential modifier of HR recovery both in the sedentary state and
after a short-term endurance training program. Because
postexercise HR recovery has been shown to be an independent
predictor of mortality both in healthy subjects and in various
patient groups, the present results may provide important clues
to understanding the molecular mechanisms underlying the
regulation of HR recovery and its association with adverse
cardiovascular events.
Physiological Background of HR Recovery
CHRM2 plays a fundamental role in cardiac autonomic
regulation (14). Activation of cardiac vagal efferents leads to
release of acetylcholine, which acts on cardiac CHRM2 to
decrease HR (6, 8). Several studies have shown that the main
physiological mechanism underlying postexercise cardiodeceleration is vagal reactivation (4, 10, 24, 25, 35). A recent study
by Smith and colleagues (43) showed that mongrel dogs
vulnerable to ventricular fibrillation had reduced HR recovery
compared with dogs resistant to malignant arrhythmias. The
differences in HR recovery and cardiac vagal activity were
eliminated by administration of atropine, which confirmed the
dominant role of vagal activity in the control of HR after
exercise (43). Furthermore, acetylcholine receptor resistance in
rats resulted in reduced cardiac muscarinic receptor function,
leading to cardiovagal insufficiency (34). As a novel finding of
the present study, we found that genetic variation at the
CHRM2 locus is associated with interindividual variability in
the modulation of HR after maximal exercise.
The role of HR recovery as a clean index of cardiac vagal
function has been questioned because the sympathetic withdrawal is also involved (37). In the present study, HR recovery
291 • JULY 2006 •
www.ajpheart.org
Downloaded from ajpheart.physiology.org on November 2, 2009
The CHRM2 haplotypes were constructed by using the
“best” option of the SHEsis software haplotyping function. The
haplotype analyses confirmed the associations detected with
individual SNPs. The haplotype consisting of the minor alleles
of the rs324640 and rs8191992 variants (CA) was associated
with lower HR recovery values in the trained state (P ⫽ 0.007).
The minor allele haplotype also showed a stronger association
with the LF-to-HF ratio both at baseline (P ⫽ 0.004) and after
the training period (P ⫽ 0.007) compared with the association
detected in rs8191992 alone. The common allele (TT) haplotype of the rs324640 and rs8191992 variants was associated
with higher HR recovery both before (P ⫽ 0.003) and after the
training program (P ⫽ 0.003). Furthermore, the TT haplotype
showed lower values of resting DBP at the baseline (P ⫽
0.037) and after the training (P ⫽ 0.011).
H464
HEART RATE RECOVERY AND CHRM2 GENE POLYMORPHISM
did not associate with HF power of R-R intervals, which is the
most commonly used index of cardiac vagal outflow. Analysis
of HF power from ambulatory R-R interval recordings includes
also some methodological problems, e.g., the saturation of HF
power during the very high vagal outflow (26). This may be
one reason for the low association between HR recovery and
ambulatory measured HF power. Second, the CHRM2 gene
polymorphisms were slightly but significantly associated with
LF-to-HF ratio, known as an index of sympathovagal balance.
Taken together, it is possible that both branches of autonomic
regulation contribute to the magnitude of HR recovery after
maximal exercise.
Association Between CHRM2 SNPs and Central
Nervous System
The central nervous system plays an important role in
cardiovascular regulation (15). Locally released acetylcholine
acts through CHRM2 within the ventrolateral medulla in the
brain, increasing the activity of sympathetic preganglionic
neurons and consequently elevating blood pressure and HR
(15, 27, 28). It was previously observed that the same SNPs of
AJP-Heart Circ Physiol • VOL
the CHRM2 gene as reported in the present study showed a
strong association with alcohol dependence, depressive disorder, and various biopotential waves, some of which are also
associated with cardiovascular dysfunctions (48). Our subjects
were healthy but were not assessed for alcohol dependence or
depressive disorders. However, we were able to confirm the
association between the CHRM2 polymorphisms and early
cardiovascular dysfunction in various autonomic markers by,
for instance, verifying slow HR recovery in untrained and
trained states and an elevated LF-to-HF ratio in long-term R-R
interval recording.
Effects of Training on HR Recovery
Endurance training has been suggested to protect the heart
against harmful cardiac events by increasing vagal tone (5).
Cardiac vagal activity has been shown to increase after 2 wk of
regular endurance training (30, 50). Previous studies on sedentary healthy men and patients with severe coronary artery
disease have reported endurance training induced improvement
in cardiac autonomic regulation, quantified as improved
postexercise HR recovery (18, 46). In the present study, the
291 • JULY 2006 •
www.ajpheart.org
Downloaded from ajpheart.physiology.org on November 2, 2009
Fig. 2. Associations between heart rate recovery
and resting systolic blood pressure before (baseline;
A, C, and E) and after (posttraining; B, D, and F) 2
wk of controlled endurance training program in the
CHRM2 rs324640 genotypes: C/C homozygotes (A
and B), C/T heterozygotes (C and D), and T/T
homozygotes (E and F). CI, confidence interval.
HEART RATE RECOVERY AND CHRM2 GENE POLYMORPHISM
minor allele homozygotes of the rs324640 and rs8191992
variants showed less optimal HR recovery than the common
allele homozygotes, both in the sedentary state and especially
after the 2-wk training program. Furthermore, the determinant
of sympathovagal balance, i.e., LF-to-HF ratio, showed an
association with the minor allele (CA) haplotype of the
rs324640 and rs8191992 variants, confirming the contribution
of the CHRM2 gene locus to the autonomic regulation of the
heart.
Role of Acetylcholine Receptors in Blood
Pressure Regulation
Limitations
The present study was limited by the fact that a relatively
small number of subjects were examined, which may emphasize the preliminary nature of the results. Therefore, the results
must obviously be confirmed in other populations. Second, to
date, there is no functional evidence of the associated CHRM2
SNPs, although the role of CHRM2 in the regulation of human
HR is evident at the receptor level.
Implications
The importance of HR recovery after both maximal (10, 25)
and submaximal exercise (11) as a predictor of mortality has
been well established in large population-based cohorts. The
independent predictive value is evident both in healthy subjects
(25) and in different patient groups (10, 32, 33, 44). Indeed, the
American Heart Association has proposed that HR recovery
could be used to assess the risk for developing clinical coronary disease among asymptomatic subjects (29). In this respect, the findings of the present study may have clinical
relevance and contribute to our understanding of the genetic
background of HR recovery after exercise. In summary, DNA
sequence variation in the CHRM2 gene locus is a potential
modifier of postexercise HR recovery in healthy individuals.
AJP-Heart Circ Physiol • VOL
Whether the same findings apply to other ethnic groups and to
cardiac patients remains to be confirmed in future studies.
GRANTS
This research was funded by grants from the Finnish Ministry of Education
(Helsinki, Finland), the Medical Council of the Academy of Finland (Helsinki,
Finland), the Seppo Säynäjäkangas Foundation, the Juho Vainio Foundation,
and the Olga and Vilho Linnamo Foundation. We appreciate the technical and
financial support received from Polar Electro (Kempele, Finland) and the
generous help from Heart Signal (Kempele, Finland).
REFERENCES
1. Abecasis GR and Cookson WO. GOLD— graphical overview of linkage
disequilibrium. Bioinformatics 16: 182–183, 2000.
2. ACSM. American College of Sports Medicine Position Stand. The recommended quantity and quality of exercise for developing and maintaining cardiorespiratory and muscular fitness, and flexibility in healthy adults.
Med Sci Sports Exerc 30: 975–991, 1998.
3. Al-Ani M, Munir SM, White M, Townend J, and Coote JH. Changes
in R-R variability before and after endurance training measured by power
spectral analysis and by the effect of isometric muscle contraction. Eur
J Appl Physiol Occup Physiol 74: 397– 403, 1996.
4. Arai Y, Saul JP, Albrecht P, Hartley LH, Lilly LS, Cohen RJ, and
Colucci WS. Modulation of cardiac autonomic activity during and immediately after exercise. Am J Physiol Heart Circ Physiol 256: H132–H141,
1989.
5. Billman GE. Aerobic exercise conditioning: a nonpharmacological antiarrhythmic intervention. J Appl Physiol 92: 446 – 454, 2002.
6. Brodde OE and Michel MC. Adrenergic and muscarinic receptors in the
human heart. Pharmacol Rev 51: 651– 690, 1999.
7. Carter JB, Banister EW, and Blaber AP. The effect of age and gender
on heart rate variability after endurance training. Med Sci Sports Exerc 35:
1333–1340, 2003.
8. Caulfield MP. Muscarinic receptors— characterization, coupling and
function. Pharmacol Ther 58: 319 –379, 1993.
9. Cheng YJ, Lauer MS, Earnest CP, Church TS, Kampert JB, Gibbons
LW, and Blair SN. Heart rate recovery following maximal exercise
testing as a predictor of cardiovascular disease and all-cause mortality in
men with diabetes. Diabetes Care 26: 2052–2057, 2003.
10. Cole CR, Blackstone EH, Pashkow FJ, Snader CE, and Lauer MS.
Heart-rate recovery immediately after exercise as a predictor of mortality.
N Engl J Med 341: 1351–1357, 1999.
11. Cole CR, Foody JM, Blackstone EH, and Lauer MS. Heart rate
recovery after submaximal exercise testing as a predictor of mortality in a
cardiovascularly healthy cohort. Ann Intern Med 132: 552–555, 2000.
12. Curfman GD and Hillis LD. A new look at cardiac exercise testing.
N Engl J Med 348: 775–776, 2003.
13. De Meersman RE. Respiratory sinus arrhythmia alteration following
training in endurance athletes. Eur J Appl Physiol Occup Physiol 64:
434 – 436, 1992.
14. Fisher JT, Vincent SG, Gomeza J, Yamada M, and Wess J. Loss of
vagally mediated bradycardia and bronchoconstriction in mice lacking M2
or M3 muscarinic acetylcholine receptors. FASEB J 18: 711–713, 2004.
15. Giuliano R, Ruggiero DA, Morrison S, Ernsberger P, and Reis DJ.
Cholinergic regulation of arterial pressure by the C1 area of the rostral
ventrolateral medulla. J Neurosci 9: 923–942, 1989.
16. Goodman JM, Liu PP, and Green HJ. Left ventricular adaptations
following short-term endurance training. J Appl Physiol 98: 454 – 460,
2005.
17. Gregoire J, Tuck S, Yamamoto Y, and Hughson RL. Heart rate
variability at rest and exercise: influence of age, gender, and physical
training. Can J Appl Physiol 21: 455– 470, 1996.
18. Hao SC, Chai A, and Kligfield P. Heart rate recovery response to
symptom-limited treadmill exercise after cardiac rehabilitation in patients
with coronary artery disease with and without recent events. Am J Cardiol
90: 763–765, 2002.
19. Hautala AJ, Makikallio TH, Kiviniemi A, Laukkanen RT, Nissila S,
Huikuri HV, and Tulppo MP. Heart rate dynamics after controlled
training followed by a home-based exercise program. Eur J Appl Physiol
92: 289 –297, 2004.
20. Hickson RC, Hagberg JM, Ehsani AA, and Holloszy JO. Time course
of the adaptive responses of aerobic power and heart rate to training. Med
Sci Sports Exerc 13: 17–20, 1981.
291 • JULY 2006 •
www.ajpheart.org
Downloaded from ajpheart.physiology.org on November 2, 2009
Altered autonomic modulation of HR has been shown to be
associated with elevated blood pressure (23) and early stage of
hypertension among normotensive men (42). Systemic administration of acetylcholine and other muscarinic agonists lowers
blood pressure, presumably because of vasodilatation caused
by the activation of muscarinic acetylcholine receptors located
on vascular endothelial cells (14). Fisher et al. (14) reported
recently that the blood pressure response in CHRM3-deficient
mice was greatly attenuated after the administration of muscarinic agonist compared with wild-type control mice. Furthermore, they observed that the CHRM2-mediated decrease in HR
was the major factor responsible for the pronounced reduction
in blood pressure after the administration of muscarinic agonist, suggesting a dominant role of CHRM receptors in the
control of blood pressure level (14). In our healthy normotensive series, we observed that rs324640 C/C homozygotes also
tended to have higher resting blood pressure values before and
after endurance training. Furthermore, the relationship between
resting blood pressure and postexercise HR recovery varied
markedly across the rs324640 genotypes, especially after endurance training. Taken together, these results suggest that the
CHRM2 gene locus may contribute to interindividual variation
in blood pressure levels. However, this hypothesis should be
confirmed in larger populations.
H465
H466
HEART RATE RECOVERY AND CHRM2 GENE POLYMORPHISM
AJP-Heart Circ Physiol • VOL
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
interval dynamics from childhood to senescence: comparison of conventional and new measures based on fractals and chaos theory. Circulation
100: 393–399, 1999.
Savin WM, Davidson DM, and Haskell WL. Autonomic contribution to
heart rate recovery from exercise in humans. J Appl Physiol 53: 1572–
1575, 1982.
Shi YY and He L. SHEsis, a powerful software platform for analyses of
linkage disequilibrium, haplotype construction, and genetic association at
polymorphism loci. Cell Res 15: 97–98, 2005.
Singh JP, Larson MG, Manolio TA, O’Donnell CJ, Lauer M, Evans
JC, and Levy D. Blood pressure response during treadmill testing as a risk
factor for new-onset hypertension. The Framingham Heart Study. Circulation 99: 1831–1836, 1999.
Singh JP, Larson MG, O’Donnell CJ, Tsuji H, Corey D, and Levy D.
Genome scan linkage results for heart rate variability (the Framingham
Heart Study). Am J Cardiol 90: 1290 –1293, 2002.
Singh JP, Larson MG, O’Donnell CJ, Tsuji H, Evans JC, and Levy D.
Heritability of heart rate variability: the Framingham Heart Study. Circulation 99: 2251–2254, 1999.
Singh JP, Larson MG, Tsuji H, Evans JC, O’Donnell CJ, and Levy D.
Reduced heart rate variability and new-onset hypertension: insights into
pathogenesis of hypertension: the Framingham Heart Study. Hypertension
32: 293–297, 1998.
Smith LL, Kukielka M, and Billman GE. Heart rate recovery after
exercise: a predictor of ventricular fibrillation susceptibility after myocardial infarction. Am J Physiol Heart Circ Physiol 288: H1763–H1769,
2005.
Spies C, Otte C, Kanaya A, Pipkin SS, Schiller NB, and Whooley MA.
Association of metabolic syndrome with exercise capacity and heart rate
recovery in patients with coronary heart disease in the heart and soul
study. Am J Cardiol 95: 1175–1179, 2005.
Spina RJ, Chi MM, Hopkins MG, Nemeth PM, Lowry OH, and
Holloszy JO. Mitochondrial enzymes increase in muscle in response to
7–10 days of cycle exercise. J Appl Physiol 80: 2250 –2254, 1996.
Sugawara J, Murakami H, Maeda S, Kuno S, and Matsuda M. Change
in post-exercise vagal reactivation with exercise training and detraining in
young men. Eur J Appl Physiol 85: 259 –263, 2001.
Tulppo MP, Hautala AJ, Makikallio TH, Laukkanen RT, Nissila S,
Hughson RL, and Huikuri HV. Effects of aerobic training on heart rate
dynamics in sedentary subjects. J Appl Physiol 95: 364 –372, 2003.
Wang JC, Hinrichs AL, Stock H, Budde J, Allen R, Bertelsen S, Kwon
JM, Wu W, Dick DM, Rice J, Jones K, Nurnberger JI Jr, Tischfield
J, Porjesz B, Edenberg HJ, Hesselbrock V, Crowe R, Schuckit M,
Begleiter H, Reich T, Goate AM, and Bierut LJ. Evidence of common
and specific genetic effects: association of the muscarinic acetylcholine
receptor M2 (CHRM2) gene with alcohol dependence and major depressive syndrome. Hum Mol Genet 13: 1903–1911, 2004.
Watanabe J, Thamilarasan M, Blackstone EH, Thomas JD, and
Lauer MS. Heart rate recovery immediately after treadmill exercise and
left ventricular systolic dysfunction as predictors of mortality: the case of
stress echocardiography. Circulation 104: 1911–1916, 2001.
Yamamoto K, Miyachi M, Saitoh T, Yoshioka A, and Onodera S.
Effects of endurance training on resting and post-exercise cardiac autonomic control. Med Sci Sports Exerc 33: 1496 –1502, 2001.
291 • JULY 2006 •
www.ajpheart.org
Downloaded from ajpheart.physiology.org on November 2, 2009
21. Howley ET, Bassett DR Jr, and Welch HG. Criteria for maximal oxygen
uptake: review and commentary. Med Sci Sports Exerc 27: 1292–1301,
1995.
22. Huikuri HV, Linnaluoto MK, Seppanen T, Airaksinen KE, Kessler
KM, Takkunen JT, and Myerburg RJ. Circadian rhythm of heart rate
variability in survivors of cardiac arrest. Am J Cardiol 70: 610 – 615, 1992.
23. Huikuri HV, Ylitalo A, Pikkujamsa SM, Ikaheimo MJ, Airaksinen
KE, Rantala AO, Lilja M, and Kesaniemi YA. Heart rate variability in
systemic hypertension. Am J Cardiol 77: 1073–1077, 1996.
24. Imai K, Sato H, Hori M, Kusuoka H, Ozaki H, Yokoyama H, Takeda
H, Inoue M, and Kamada T. Vagally mediated heart rate recovery after
exercise is accelerated in athletes but blunted in patients with chronic heart
failure. J Am Coll Cardiol 24: 1529 –1535, 1994.
25. Jouven X, Empana JP, Schwartz PJ, Desnos M, Courbon D, and
Ducimetiere P. Heart-rate profile during exercise as a predictor of sudden
death. N Engl J Med 352: 1951–1958, 2005.
26. Kiviniemi AM, Hautala AJ, Seppanen T, Makikallio TH, Huikuri HV,
and Tulppo MP. Saturation of high-frequency oscillations of R-R intervals in healthy subjects and patients after acute myocardial infarction
during ambulatory conditions. Am J Physiol Heart Circ Physiol 287:
H1921–H1927, 2004.
27. Kubo T. Cholinergic mechanism and blood pressure regulation in the
central nervous system. Brain Res Bull 46: 475– 481, 1998.
28. Kubo T, Taguchi K, Sawai N, Ozaki S, and Hagiwara Y. Cholinergic
mechanisms responsible for blood pressure regulation on sympathoexcitatory neurons in the rostral ventrolateral medulla of the rat. Brain Res Bull
42: 199 –204, 1997.
29. Lauer M, Froelicher ES, Williams M, and Kligfield P. Exercise testing
in asymptomatic adults: a statement for professionals from the American
Heart Association Council on Clinical Cardiology, Subcommittee on
Exercise, Cardiac Rehabilitation, and Prevention. Circulation 112: 771–
776, 2005.
30. Lee CM, Wood RH, and Welsch MA. Influence of short-term endurance
exercise training on heart rate variability. Med Sci Sports Exerc 35:
961–969, 2003.
31. Mier CM, Turner MJ, Ehsani AA, and Spina RJ. Cardiovascular
adaptations to 10 days of cycle exercise. J Appl Physiol 83: 1900 –1906,
1997.
32. Nishime EO, Cole CR, Blackstone EH, Pashkow FJ, and Lauer MS.
Heart rate recovery and treadmill exercise score as predictors of mortality
in patients referred for exercise ECG. JAMA 284: 1392–1398, 2000.
33. Nissinen SI, Makikallio TH, Seppanen T, Tapanainen JM, Salo M,
Tulppo MP, and Huikuri HV. Heart rate recovery after exercise as a
predictor of mortality among survivors of acute myocardial infarction.
Am J Cardiol 91: 711–714, 2003.
34. Padley JR, Overstreet DH, Pilowsky PM, and Goodchild AK. Impaired
cardiac and sympathetic autonomic control in rats differing in acetylcholine receptor sensitivity. Am J Physiol Heart Circ Physiol 289: H1985–
H1992, 2005.
35. Pierpont GL, Stolpman DR, and Gornick CC. Heart rate recovery
post-exercise as an index of parasympathetic activity. J Auton Nerv Syst
80: 169 –174, 2000.
36. Pikkujamsa SM, Makikallio TH, Sourander LB, Raiha IJ, Puukka P,
Skytta J, Peng CK, Goldberger AL, and Huikuri HV. Cardiac interbeat