Early detection of iron overload cardiomyopathy is an important strategy for decreasing the mortality rate of patients with transfusion-dependent thalassemia (TDT). Although cardiac magnetic resonance (CMR) T2* is effective in detecting cardiac iron deposition, it is costly and not generally available. We investigated whether heart rate variability (HRV) can be used as a screening method of iron overload cardiomyopathy in TDT patients. HRV, evaluated by 24-h Holter monitoring, non-transferrin bound iron (NTBI), serum ferritin, left ventricular (LV) ejection fraction (LVEF), and CMR-T2* were determined. Patients with a cardiac iron overload condition had a significantly higher low frequency/high frequency (LF/HF) ratio than patients without a cardiac iron overload condition. Log-serum ferritin (r = −0.41, P=0.008), serum NTBI (r = −0.313, P=0.029), and LF/HF ratio (r = −0.286, P=0.043) showed a significant correlation with CMR-T2*, however only the LF/HF ratio was significantly correlated with LVEF (r = −0.264, P=0.043). These significant correlations between HRV and CMR-T2* and LVEF in TDT confirmed the beneficial role of HRV as a potential early screening tool of cardiac iron overload in thalassemia patients, especially in a medical center in which CMR T2* is not available. A larger number of TDT patients with cardiac iron overload are needed to confirm this finding.

Introduction

Thalassemia is a hereditary hematologic disorder resulting from abnormal production of the globin chains of hemoglobin. These changes can cause a variety of clinical pictures, from normal red blood cell concentration to stillbirth due to severe hemoglobinopathy. Transfusion-dependent thalassemia (TDT) is the most severe form of thalassemia in which regular red blood cell transfusions are a necessity for survival [1]. A definitive treatment of thalassemia is stem cell transplantation, which is limited in its availability due to the difficulty in locating a compatible donor, so regular blood transfusion is still a mainstream treatment of TDT [2]. However, long-term red blood cell transfusions cause peripheral blood iron overload, resulting in deposition and damages in iron-sensitive vital organs, including the heart [3]. Evidence shows that the leading cause of deaths in TDT patients is iron overload cardiomyopathy [4]. Although iron chelators are available, the success rate of chelation is limited because iron overload cardiomyopathy can be reversible only if the initiation of intensive chelation is early enough [5,6]. Therefore, an early detection of iron overload cardiomyopathy is an important strategy in the prevention of mortality in TDT patients.

Several methods including the measurement of both biochemical and radiological parameters have been proposed for use as screening tools for iron overload cardiomyopathy in TDT. Serum ferritin is one of the biomarkers frequently used for iron overload detection [1,7]. However, since serum ferritin is an acute-phase response protein, it has a low specificity for the iron overload condition [1,8]. Non-transferrin bound iron (NTBI) is a low molecular weight and free-form iron which can be detected in plasma only when serum transferrin is fully saturated [9]. NTBI has a high specificity to the iron overload condition, and the correlation between NTBI and vital organ damage has been recently demonstrated [10,11]. Therefore, NTBI can be one of the ideal biochemical parameters used to represent cardiac iron status. However, it has some limitations in generalized usage, specifically the high cost and complicated laboratory procedures [12]. Cardiac MRI T2* (CMR T2*) is a gold-standard diagnostic tool in the recognition of iron overload cardiomyopathy, since it has been shown to have a positive correlation with cardiac function in TDT [13–15]. However, due to its high cost and limited availability, especially in developing countries, CMR T2* might not be the best general screening tool for iron overload cardiomyopathy.

Heart rate variability (HRV), an easily performed, reliable, low cost, and non-invasive cardiac electrophysiologic test, can be used to evaluate cardiac autonomic function in patients with heart disease [16], including post-myocardial infarction [17], heart failure [18], and also iron overload cardiomyopathy in TDT patients [19,20]. Recently, we have demonstrated that depressed HRV has a positive correlation with CMR T2* in non-TDT [12] and pediatric TDT patients [21]. However, these correlations in adult TDT patients have not been elucidated yet. Moreover, those previous studies also had a limitation due to the very low prevalence of iron overload cardiomyopathy, which is defined by CMR T2* ≤20 ms. Therefore, the aim of the present study was to investigate the correlations between HRV parameters and cardiac dysfunctions, including cardiac iron overload status and ventricular contractile function, as well as to compare the diagnostic ability of HRV and biochemical markers such as serum ferritin and plasma NTBI in adult TDT patients. We hypothesized that the existence of a depressed HRV is positively correlated with cardiac iron overload status but negatively correlated with ventricular contractile function, and HRV is a better indicator of cardiac dysfunction in TDT patients than either serum ferritin or NTBI.

Methods

Study protocol

The study protocol was approved by the Institutional Ethics Committee of the Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand and were in accordance with the current version of the Helsinki Declaration. The inclusion criteria included: being diagnosed with TDT, having regular red blood cell transfusions at least once a month after diagnosis, being in the age range from 18 to 50 years and having no infectious diseases or acute illnesses during the investigation. The exclusion criteria included patients with a contraindication to MRI, and clinical evidence of other secondary causes of pulmonary hypertension, including HIV infection, hepatitis virus infection, collagen vascular diseases, cirrhosis, chronic obstructive airway diseases, and acquired heart disease associated with pulmonary hypertension. Patients with neurological diseases causing dysautonomia, severe depression, or chronic inflammatory diseases as well as patients, who were the known cases of the disease that can be the causes of diastolic and systolic cardiomyopathy (e.g. diabetes, hypertension, or coronary heart disease) were also excluded. Medical records of the patients were reviewed to amass epidemiological data, transfusion history, and comorbidities. The body weight, height, and body mass index (BMI) were measured in each patient. Routine laboratory tests, serum ferritin, plasma NTBI, echocardiography, 24-h Holter ECG recording for HRV analysis and CMR T2* were investigated and analyzed at the time of entry. Serum ferritin and plasma NTBI were determined using standard techniques as described previously [21,22].

Echocardiographic studies

Standard 2D and transthoracic Doppler echocardiography was performed at rest in all enroled patients with Philips iE33 (Philips Healthcare, Bothell, WA, U.S.A.). Left ventricular (LV) average S.D. of all 5-min R–R intervals were measured, and LV ejection fraction (LVEF), stroke volume, cardiac output, and fractional shortening (FS) were determined based on the calculation of LV volumes by the method of discs, following the recommendations by the American Society of Echocardiography (ASE), and using apical two- and four-chamber views to measure cardiac function [23,24]. Impaired systolic LV dysfunction (LVEF <55% or LVFS <35%) was also determined in TDT patients [24]. Diastolic function was evaluated from mitral inflow velocities (E-wave, A-wave, E/A ratio) and deceleration time (DT) using pulsed wave (PW) Doppler in the apical four-chamber view. PW tissue-Doppler velocities were acquired at end-expiration, in the apical four-chamber view, with the sample positioned at the lateral mitral annulus, measuring early diastolic (E′) and late diastolic (A′) velocities and calculating the E/E′ ratio. Diastolic function was investigated using E/A ratio, DT, and E/E′ according to the current guidelines [25].

HRV measurement

HRV was evaluated using the SEER Light Holter system (GE Healthcare, Milwaukee, WI, U.S.A.). A lead II ECG was continuously recorded for 24 h. Prior to the HRV measurement, either exercise or caffeine consumption was prohibited in all the participants. The computer software MARS version 7, GE Healthcare, Milwaukee, WI, U.S.A., was used to scan for rhythm disturbance and to detect and label each QRS complex. Excessive noise and artifacts were noted, and ectopy was quantitated. The time-domain analyses including average heart rate, average R–R intervals (NN), standard deviation (S.D.) of the R–R intervals over a 24-h period (SDNN), S.D. of all 5-min mean R–R intervals (SDANN), average S.D. of all 5-min R–R intervals (ASDNN), the percentage of R–R intervals with more than 50-ms variation (pNN50), and the square root of mean squared differences of successive R–R intervals (rMSSD). The frequency domain analyses were determined with the same analytical software using Fast-Fourier transform analysis. The obtained frequency domain indices were the total power (0–0.4 Hz), high-frequency power (HF, 0.15–0.4 Hz) spectral density, low-frequency power (LF, 0.04–0.15 Hz) spectral density, and very LF (0.003–0.04 Hz) spectral density. Total power expresses the magnitude of the entire HRV, whereas HF power reflects the parasympathetic tone, and LF power indicates the sympathovagal interactions [16]. These power spectral densities were described in absolute units (ms2). The designated physician, who operated and fitted the Holter monitor to the patients, was blinded to the patient information. The subjects were fitted with the Holter monitor after blood sample collection at the Holter unit of the Faculty of Medicine, Chiang Mai University. In the present study, none of the patients included in the study took medication such as β-blockers, calcium channel blockers, statins, and ACE inhibitors, which could affect the HRV [26].

MRI

Magnetic resonance acquisition was performed using a 1.5-T MR scanner (GE, HDxt, Milwaukee, WI, U.S.A.), with an eight-channel body array. For the measurements of myocardial iron overload, we used a T2 gradient-echo multi-echo sequence (25-degree flip angle, matrix: 192 × 128 pixels, 38 × 28.5 cm field of view,125-kHz bandwidth, 10.0-mm slice thickness, 1 number of excitations, 4 views per segment, 18.5-ms repetition time). A single mid ventricular short-axis view of the left ventricle was acquired at eight echo times (TEs) (1.7 ms, which increased in 2.0–2.1 ms increments) in a single end-expiratory breath-hold. CMR T2* analysis was performed on a workstation (Advantage Window 4.6, GE Healthcare) using commercial analysis software (StarMap 4.0, GE Healthcare). According to the previous study, the 95% confidence interval of a normal CMR T2* value (>20 ms) was assessed [13,27]. The values 14–20 ms were regarded as mild iron overload, values between 10 and 14 ms were regarded as moderate iron overload, and a T2* less than 10 ms was regarded as severe iron overload [13].

Statistical analysis

Categorical variables were described using percentages of its frequency. Normally distributed numerical variables were presented using arithmetic means and S.D. Non normally distributed variables were modified by taking a natural logarithm, and then presented using geometric mean and S.D. Differences of parameters between patients with and without cardiac iron overload were compared using the independent Student’s ttest. The correlations amongst HRV, biochemical markers, echocardiographic parameters, and CMR T2* were determined using the Pearson’s correlation coefficient. The multivariate linear and binary logistic regression analysis, forward likelihood method, as well as the receiver operative characteristic (ROC) curve were applied to determine independent predictive factors of CMR T2* values. Statistical analyses were performed using SPSS version 13.0 for Windows (SPSS Inc., Chicago, IL, U.S.A.). A P-value of less than 0.05 was considered statistically significant.

Results

Sixty-six TDT patients were enroled in the present study. However, since 7 patients were not evaluated by CMR T2*, only 59 TDT patients were included in a statistical analysis. Nineteen patients (32.2%) were diagnosed with homozygous β-thalassemia. Thirty-eight patients (64.4%) were diagnosed with β-thalassemia/Hemoglobin E. One patient was diagnosed with AE Bart’s disease and the other patient was diagnosed with AE Bart’s Constant Spring (CS) disease. Clinical and biochemical characteristics, echocardiographic and HRV parameters, as well as CMR T2* values of the patients are summarized in Table 1. All continuous variables, except serum ferritin level, were normally distributed. After taking a natural logarithm (ln), the serum ferritin variable was also normally distributed. Therefore, the parametric statistical analysis was applied in all tests. The mean of number of red blood cell transfusions per year in the last 5 years in these subjects was 19.44 times. The number of patients with positive cardiac iron overload, indicated by CMR T2* ≤20 ms, were five, reflecting that the prevalence of cardiac iron overload in the present study was 8.47%. Moreover, when the subjects were divided into two groups, based on the present of cardiac iron overload, the positive cardiac iron overload group had a significantly higher ratio between LF and HF (LF/HF ratio), which indicates a sympathovagal imbalance of the heart, and higher ln-serum ferritin than the negative cardiac iron overload group (1.77 compared with 1.46, P=0.028 and 8.01 compared with 7.26, P=0.010, respectively). On the other hand, there was no difference in clinical characteristics (including age, sex, BMI, and number of red blood cell transfusions per year in the last 5 years), Hb, plasma NTBI, LVEF, or the other HRV parameters between these two groups.

Table 1
Demographic, clinical, biochemical characteristics, cardiac imaging parameters, time-domain and frequency-domain HRV parameters of all transfusion dependent thalassemia TDT patients (n=59) and comparisons between CMR T2* ≤20 and >20 groups
Parameters All patients (n=59) mean (S.D.) CMR T2* ≤20 (n=5) mean (S.D.) CMR T2* >20 (n=54) mean (S.D.) P-value 
Demographic data 
Age (years) 28 (8) 23 (5) 28 (8) 0.178 
Sex (male/female) (%) 39/61 20/80 42/58 0.389* 
Number of transfusions in last 12 months (times) 19 (6) 18 (5) 20 (6) 0.500 
Biochemical parameters 
Hb (g/dl) 7.18 (1.0) 7.3 (1.7) 7.1 (1.0) 0.645 
Hct (%) 23.62 (3.77) 24 (5) 23 (3) 0.892 
Geometric mean of serum ferritin (95% CI) (ng/dL) 1448 (1189, 1764) 3920 (1520, 10011) 1443 (1188, 1754) 0.011 
Plasma NTBI (µM) 6.7 (1.9) 7.41 (2.03) 6.69 (1.92) 0.435 
Cardiac imaging parameters 
LVEF (%) 68 (4.2) 68 (6) 69 (4) 0.594 
LV diastolic volume (ml) 87 (24) 77 (26) 87 (23) 0.318 
E/A ratio 1.6 (0.6) 1.8 (0.4) 1.6 (0.6) 0.594 
TRV max 249 (55) 252 (15) 248 (54) 0.377 
CMR T2* (ms) 38 (12) 7 (3) 41 (9) 0.000 
Positive for cardiac iron overload (CMR T2* ≤20 ms) (%) 8.47    
HRV-frequency domain 
LF (ms2) 12 (5) 11 (4) 12 (8) 0.784 
HF (ms2) 8 (4) 7 (5) 8 (4) 0.498 
LF/HF ratio 1.5 (0.3) 1.8 (0.3) 1.5 (0.3) 0.028 
HRV-time domain 
SDNN (ms) 97 (28) 95 (26) 97 (28) 0.831 
SDANN (ms) 89 (28) 86 (24) 90 (28) 0.763 
ASDNN (ms) 36 (11) 36 (14) 36 (11) 0.954 
rMSSD (ms) 20 (9) 18 (10) 21 (10) 0.256 
Parameters All patients (n=59) mean (S.D.) CMR T2* ≤20 (n=5) mean (S.D.) CMR T2* >20 (n=54) mean (S.D.) P-value 
Demographic data 
Age (years) 28 (8) 23 (5) 28 (8) 0.178 
Sex (male/female) (%) 39/61 20/80 42/58 0.389* 
Number of transfusions in last 12 months (times) 19 (6) 18 (5) 20 (6) 0.500 
Biochemical parameters 
Hb (g/dl) 7.18 (1.0) 7.3 (1.7) 7.1 (1.0) 0.645 
Hct (%) 23.62 (3.77) 24 (5) 23 (3) 0.892 
Geometric mean of serum ferritin (95% CI) (ng/dL) 1448 (1189, 1764) 3920 (1520, 10011) 1443 (1188, 1754) 0.011 
Plasma NTBI (µM) 6.7 (1.9) 7.41 (2.03) 6.69 (1.92) 0.435 
Cardiac imaging parameters 
LVEF (%) 68 (4.2) 68 (6) 69 (4) 0.594 
LV diastolic volume (ml) 87 (24) 77 (26) 87 (23) 0.318 
E/A ratio 1.6 (0.6) 1.8 (0.4) 1.6 (0.6) 0.594 
TRV max 249 (55) 252 (15) 248 (54) 0.377 
CMR T2* (ms) 38 (12) 7 (3) 41 (9) 0.000 
Positive for cardiac iron overload (CMR T2* ≤20 ms) (%) 8.47    
HRV-frequency domain 
LF (ms2) 12 (5) 11 (4) 12 (8) 0.784 
HF (ms2) 8 (4) 7 (5) 8 (4) 0.498 
LF/HF ratio 1.5 (0.3) 1.8 (0.3) 1.5 (0.3) 0.028 
HRV-time domain 
SDNN (ms) 97 (28) 95 (26) 97 (28) 0.831 
SDANN (ms) 89 (28) 86 (24) 90 (28) 0.763 
ASDNN (ms) 36 (11) 36 (14) 36 (11) 0.954 
rMSSD (ms) 20 (9) 18 (10) 21 (10) 0.256 

Significant at P<0.05.

All statistical analyses were performed by independent t test except * by Fisher’s exact test.

Abbreviations: Hb, hemoglobin; Hct, hematocrit; TRV max, maximum velocity of tricuspid regurgitant flow.

The correlation analyses between biochemical markers, echocardiographic data, HRV parameters, and CMR T2* values were determined. None of the clinical characteristics showed a correlation with biochemical markers, HRV parameters, or CMR T2*. None of the HRV parameters showed a significant correlation with log-serum ferritin or plasma NTBI. As regarding the correlation between HRV parameters and CMR T2*, only the LF/HF ratio had a significant negative correlation with CMR T2* values (r = −0.286, P=0.021). Log-serum ferritin and plasma NTBI also had a significant negative correlation with CMR T2* values (r = −0.341, P=0.008 and r = −0.313, P=0.029, respectively). However, only the LF/HF ratio parameter of HRV did have a significant negative correlation with LVEF.

To determine independent predictive factors of the cardiac iron overload status; a multivariate logistic regression analysis, forward likelihood method, was applied. After adjustment by log-serum ferritin and plasma NTBI, LF/HF ratio parameter of HRV was the only independent predictive factor of the cardiac iron overload status (P=0.033, odd ratio =28.43; 95% CI =1.30–621.02). Furthermore, the area under ROC curve was used to demonstrate the predictive accuracy of the parameters. The predicted area under the ROC curve of the LF/HF ratio was in the good level (0.82 ± 0.79; 95% CI =0.67–0.98) while the predicted area under the ROC curve of log-serum ferritin and plasma NTBI were in the fair (0.79 ± 0.9; 95% CI =0.61–0.97) and the poor levels (0.63 ± 0.14; 95% CI =0.37–0.90), respectively. In addition, LF/HF ratio more than 1.49 was the best predictor for CMR T2* ≤20 ms, with 83% sensitivity and 50% specificity and its positive and negative predictive values were 16 and 96%, respectively (Figure 1).

ROC curve of LF/HF ratio parameter of HRV, NTBI and log-serum ferritin for the prediction of cardiac iron deposition determined by CMR T2*

Figure 1
ROC curve of LF/HF ratio parameter of HRV, NTBI and log-serum ferritin for the prediction of cardiac iron deposition determined by CMR T2*
Figure 1
ROC curve of LF/HF ratio parameter of HRV, NTBI and log-serum ferritin for the prediction of cardiac iron deposition determined by CMR T2*

Discussion

The major findings of the present study were that depressed HRV, indicated by increased LF/HF ratio, showed a significant correlation with the presence of cardiac iron overload status, indicated by CMR T2* ≤20 ms. For example, the LF/HF ratio was the only predictive factor determining the present of cardiac iron overload, with the high odds ratio of 28.43. The ROC analysis also demonstrated a good accuracy of LF/HF ratio enabling a prediction of cardiac iron overload status. The LF/HF ratio also had a significant negative correlation with LVEF. On the other hand, biochemical markers, specifically serum ferritin and plasma NTBI levels, had a significant correlation with only cardiac iron overload status. These findings confirmed our hypotheses that depressed HRV is positively correlated with cardiac iron overload status but negatively correlated with ventricular contractile function, and HRV is better than both serum ferritin and NTBI in a giving clear detection of cardiac dysfunction in TDT patients. Previous studies investigated the associations between the HRV and other cardiac parameters in either adult non-TDT (NTDT) cases [12] or pediatric TDT cases [21]. However, this association has never been investigated in adult TDT patients. Our study is the first study demonstrating the correlations between HRV parameters and CMR T2*, as well as LVEF in adult TDT patients. Although most patients with positive cardiac iron overload were female (four out of five, 80%), there was no statistical significance of sexes between positive and negative cardiac iron overload groups. In addition, the multivariate regression analysis also demonstrated an independent significance of the LF/HF ratio and CMR-T2* when adjusted by sex. Therefore, sex might not contribute to the difference between positive and negative cardiac iron overload groups and this correlation could be applicable to both sexes.

The prevalence of cardiac iron overload in the present study was 8.47%, which was lower than that reported in a recent study. Aydinok et al. [28] reported in their multicenter study that the prevalence of cardiac iron overload, indicated by CMR T2* ≤20 ms, was 36.7%. Furthermore, there was absence of cardiac contractile dysfunction in this cross-sectional study. None of these subjects had an LVEF less than 45%. This finding was compatible with the results from the study of Aydinok et al. [28], which also demonstrated the absence of cardiac contractile dysfunction in their study. The decrease in cardiac iron overload prevalence compared with the studies in last 5 years, as well as the absence of cardiac contractile dysfunction in TDT patients, may reflect an effective strategy of early iron chelator administration in order to prevent the cardiac iron overload status in TDT patients [21,29].

In the present study, only the LF/HF ratio, which is a frequency-domain parameter of HRV, was negatively correlated with cardiac iron overload status. This finding emphasized the important findings from our previous studies. In non-TDT patients, we have previously demonstrated that there was a significant negative correlation between the LF/HF parameters of HRV (r = –0.227 and P=0.026), with an odds ratio of 8.71 [12]. On the other hand, our previous study investigated HRV parameters in 101 pediatric TDT patients demonstrated that only SDANN and ASDNN from time-domain parameters and LF and HF, but not the LF/HF ratio, from frequency-domain parameters of HRV showed a correlation with CMR T2* [21]. It is also notable that the correlations between HRV parameters and CMR T2* were weaker than in the present study. This inconsistent finding may be due to the difference in age of the subjects participating in each study. The median age of the subjects in the study of Silvilairat et al. [21] was 15 years while it was 27 years in the present study. Many previous studies demonstrated that HRV parameters including LF/HF ratio were age dependent [30–32]. LF/HF ratio is continuously increased from birth until 6th year, corresponding with a development of sympathetic nervous system [30,31]. After 6th year, LF/HF ratio gradually decreased over time, reflecting an increase in parasympathetic activity [33]. This age-dependent character may be responsible for the inconsistent finding. In addition, this difference may also be responsible for the difference in prevalence of the cardiac iron overload condition because the older the patient gets, the longer he/she lives with anemia. Combining the results from our three correlation studies, we propose that the LF/HF ratio could be the most sensitive HRV parameter for accurate prediction of the cardiac iron overload status in thalassemia patients who are in adult age.

The mechanisms involving in the depression of HRV in TDT patients could mainly be exacerbated by oxidative stress. In thalassemia patients, systemic iron overload occurred via two mechanisms, from excessive intestinal iron absorption and from transfusional siderosis [34]. Excessive iron acquisition causes saturation of transferrin-binding capacity, resulting in the presence of plasma NTBI [3]. NTBI can deposit in various tissues, such as the reticulo-endothelial system of the spleen, liver, bone marrow and in advanced cases, in the cardiac myocytes [9,35,36]. Deposition of NTBI in those tissues causes oxidative stress by enhancing reactive oxygen species (ROS) via the Haber–Weiss and Fenton reaction, leading to cellular injury from lipid peroxidation, damage to cellular proteins as well as nucleic acids, mitochondrial dysfunction, and apoptosis [15,37–39]. Previous studies have shown that the oxidative stress from many pathological conditions, including iron overload, can promote sympathovagal disturbance and cause significantly depressed HRV [40–47]. Therefore, the depressed HRV observed in the present study could be mainly caused by oxidative stress mechanisms.

Interestingly, this is the first study demonstrating the correlations between HRV parameters and CMR T2*, as well as LVEF in adult TDT patients. Only the LF/HF ratio parameter of HRV, but not CMR T2*, was positively correlated with LVEF in the present study. This finding was different from the results of the study of Anderson et al. (2001) [13], which demonstrated a significant correlation between CMR T2* and LVEF. The difference in severity of cardiac iron overload may be responsible for these inconsistent findings. The prevalence of cardiac iron overload in the study by Anderson et al. [13] was ~70%, while it was only 8.47% in the present study. In other cardiac disorders, it was demonstrated that cardiac autonomic dysfunction was more sensitive to oxidative stress than contractile dysfunction [48–51]. Many previous studies demonstrated LF/HF ratio of HRV as the best prognostic factor of mortality in various condition, such as post-myocardial infarction [52], chronic heart failure [53], as well as severe cardiomyopathy [54]. However, only one study did correlated LF/HF with LVEF, and the result did not show a significant correlation (r =0.3, P=0.7) [55]. Since the subjects of these studies had a reduced LVEF at the time HRV was evaluated, the difference in pathophysiology of developing heart failure between intrinsic cardiac diseases (myocardial infarction, cardiomyopathy) and iron overload cardiomyopathy may be responsible for this inconsistence. This evidence addressed that the correlation between LF/HF ratio of HRV and LVEF is conceal if the patients have already a reduced LVEF at the measurement time. Therefore, our hypothesis is that in TDT patients, cardiac autonomic dysfunction occurs earlier, in mild degree of cardiac iron overloads, while cardiac contractile dysfunction occurs later, in more severe cases of cardiac iron overload. To test this hypothesis, a prospective longitudinal cohort clinical study is needed. Moreover, because the prevalence of the condition of cardiac iron overload in current clinical practice is decreasing, HRV might be a potential early screening tool of cardiac iron overload in thalassemia patients, especially in a medical center in which CMR T2* is not available. The proposed mechanism of depressed HRV in TDT, and its correlation with cardiac iron overload status as well as cardiac function is summarized in Figure 2.

The mechanism of depressed HRV, increased serum ferritin, increased plasma NTBI, increased cardiac iron deposition, and decreased cardiac contractile function in TDT patients

Figure 2
The mechanism of depressed HRV, increased serum ferritin, increased plasma NTBI, increased cardiac iron deposition, and decreased cardiac contractile function in TDT patients

In TDT patients, there is an increase in intestinal iron absorption, as well as an increase in red blood cell (RBC) transfusion rate. These changes induce conditions of systemic iron overload, causing an increase in serum ferritin level. Excessive serum iron causes saturation of transferrin, an iron-binding protein, resulting in increased NTBI levels. An increase in NTBI level causes increased ROS, via the Haber–Weiss and Fenton reactions, resulting in increased oxidative stress. Oxidative stress enhances autonomic dysfunction of the heart, resulting in depressed HRV. Meanwhile, an increase in NTBI level enhances cardiac iron deposition, which is represented by decreased CMR T2* values. Eventually, chronic cardiac autonomic dysfunction and abnormal cardiac iron overload impairs cardiac contractile function, resulting in a decrease in the LVEF. The changes in these parameters are time dependent. Serum ferritin and NTBI levels are systemic parameters which occur very early in the condition and their correlation with LVEF, which is a late-stage change, is not significant. On the other hand, HRV, which is a direct change of the heart itself, occurs soon after the increase in oxidative stress, thus is correlated with both CMR T2* and LVEF. Therefore, HRV is possibly a potential screening tool for early detection of conditions of cardiac iron overload in TDT patients.

Figure 2
The mechanism of depressed HRV, increased serum ferritin, increased plasma NTBI, increased cardiac iron deposition, and decreased cardiac contractile function in TDT patients

In TDT patients, there is an increase in intestinal iron absorption, as well as an increase in red blood cell (RBC) transfusion rate. These changes induce conditions of systemic iron overload, causing an increase in serum ferritin level. Excessive serum iron causes saturation of transferrin, an iron-binding protein, resulting in increased NTBI levels. An increase in NTBI level causes increased ROS, via the Haber–Weiss and Fenton reactions, resulting in increased oxidative stress. Oxidative stress enhances autonomic dysfunction of the heart, resulting in depressed HRV. Meanwhile, an increase in NTBI level enhances cardiac iron deposition, which is represented by decreased CMR T2* values. Eventually, chronic cardiac autonomic dysfunction and abnormal cardiac iron overload impairs cardiac contractile function, resulting in a decrease in the LVEF. The changes in these parameters are time dependent. Serum ferritin and NTBI levels are systemic parameters which occur very early in the condition and their correlation with LVEF, which is a late-stage change, is not significant. On the other hand, HRV, which is a direct change of the heart itself, occurs soon after the increase in oxidative stress, thus is correlated with both CMR T2* and LVEF. Therefore, HRV is possibly a potential screening tool for early detection of conditions of cardiac iron overload in TDT patients.

Study limitation

There were several limitations in the present study. First, there was a small sample size in the present study. Second, there was no healthy control group or an additional group with a disease associated with cardiac iron overload from another cause, such as hereditary hemochromatosis. However, because this type of disease is extremely rare in our population, the addition of this group is not possible for the present study. Next, since there were only 5 out of 59 patients who had cardiac iron overload (CMR-T2* ≤20 ms), the correlation found between HRV and cardiac iron overload had the weakest statistical power. Future studies with larger number of TDT patients with cardiac iron overload are needed to confirm this finding. Further prospective longitudinal cohort studies, with larger sample sizes, from more centers, which may include patients with other causes of iron overload and more patients with positive cardiac iron overload, are needed to increase the confidence in the results.

Conclusion

The significant correlations between LF/HF ratio of HRV and CMR-T2* and LVEF in TDT patients indicate that HRV might have the beneficial role as a potential predictor of cardiac iron overload as well as LV contractile function in this group of patients. However, due to weak correlation and small studied population in the present study, larger population of TDT patients with positive cardiac iron overload is needed to confirm this finding.

Author contribution

N.C. and S.C.C. designed the research study and contributed essential materials and reagents. S.P., A.P., A.T., S.K., and S.S. performed the research. S.P., A.P., and S.S. analyzed the data. S.P., S.C.C., and N.C. wrote and revised the manuscript. S.C.C. and N.C. critically reviewed the manuscript. All authors reviewed the final manuscript.

Funding

This work was supported by the Thailand Research Fund [grant number RTA6080003 (to S.C.C.), and RSA5780040 (to A.P.)]; the NSTDA Research Chair Grant from the National Science and Technology Development Agency(to N.C.); and the Chiang Mai University Center of Excellence Award (to N.C.).

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Abbreviations

     
  • ACE

    angiotensin-converting enzyme

  •  
  • ASDNN

    average S.D. of all 5-min R–R intervals

  •  
  • CI

    confidence interval

  •  
  • CMR

    cardiac magnetic resonance

  •  
  • DT

    deceleration time

  •  
  • ECG

    electrocardiography

  •  
  • FS

    fractional shortening

  •  
  • HF

    high-frequency power

  •  
  • HRV

    heart rate variability

  •  
  • LF

    low-frequency power

  •  
  • LF/HF

    low frequency/high frequency

  •  
  • LV

    left ventricular

  •  
  • LVED

    LV end diastolic dimension

  •  
  • LVEF

    LV ejection fraction

  •  
  • LVESD

    LV end systolic dimension

  •  
  • NTBI

    non-transferrin bound iron

  •  
  • pNN50

    the percentage of R–R intervals with more than 50-ms variation

  •  
  • PW

    pulsed wave

  •  
  • rMSSD

    the square root of mean squared differences of successive R–R intervals

  •  
  • ROC

    receiver operative characteristic

  •  
  • ROS

    reactive oxygen species

  •  
  • SDNN

    S.D. of the R–R intervals over a 24-h period

  •  
  • SDANN

    S.D. of all 5-min mean R–R interval

  •  
  • TDT

    transfusion-dependent thalassemia

References

References
1
Koonrungsesomboon
N.
,
Chattipakorn
S.C.
,
Fucharoen
S.
and
Chattipakorn
N.
(
2013
)
Early detection of cardiac involvement in thalassemia: from bench to bedside perspective
.
World J. Cardiol.
5
,
270
279
[PubMed]
2
Rund
D.
and
Rachmilewitz
E.
(
2005
)
Beta-thalassemia
.
N. Engl. J. Med.
353
,
1135
1146
[PubMed]
3
Hershko
C.
(
2010
)
Pathogenesis and management of iron toxicity in thalassemia
.
Ann. N.Y. Acad. Sci.
1202
,
1
9
[PubMed]
4
Borgna-Pignatti
C.
,
Rugolotto
S.
,
De Stefano
P.
,
Zhao
H.
,
Cappellini
M.D.
et al
(
2004
)
Survival and complications in patients with thalassemia major treated with transfusion and deferoxamine
.
Haematologica
89
,
1187
1193
[PubMed]
5
Tanner
M.A.
,
Galanello
R.
,
Dessi
C.
,
Smith
G.C.
,
Westwood
M.A.
et al
(
2008
)
Combined chelation therapy in thalassemia major for the treatment of severe myocardial siderosis with left ventricular dysfunction
.
J. Cardiovasc. Magn. Reson.
10
,
12
[PubMed]
6
Davis
B.A.
and
Porter
J.B.
(
2000
)
Long-term outcome of continuous 24-hour deferoxamine infusion via indwelling intravenous catheters in high-risk beta-thalassemia
.
Blood
95
,
1229
1236
[PubMed]
7
Olivieri
N.F.
,
Brittenham
G.M.
,
Matsui
D.
,
Berkovitch
M.
,
Blendis
L.M.
et al
(
1995
)
Iron-chelation therapy with oral deferipronein patients with thalassemia major
.
N. Engl. J. Med.
332
,
918
922
[PubMed]
8
Orino
K.
,
Lehman
L.
,
Tsuji
Y.
,
Ayaki
H.
,
Torti
S.V.
et al
(
2001
)
Ferritin and the response to oxidative stress
.
Biochem. J.
357
,
241
247
[PubMed]
9
Brissot
P.
,
Ropert
M.
,
Le Lan
C.
and
Loreal
O.
(
2012
)
Non-transferrin bound iron: a key role in iron overload and iron toxicity
.
Biochim. Biophys. Acta
1820
,
403
410
[PubMed]
10
Parkes
J.G.
,
Hussain
R.A.
,
Olivieri
N.F.
and
Templeton
D.M.
(
1993
)
Effects of iron loading on uptake, speciation, and chelation of iron in cultured myocardial cells
.
J. Lab. Clin. Med.
122
,
36
47
[PubMed]
11
Piga
A.
,
Longo
F.
,
Duca
L.
,
Roggero
S.
,
Vinciguerra
T.
et al
(
2009
)
High nontransferrin bound iron levels and heart disease in thalassemia major
.
Am. J. Hematol.
84
,
29
33
[PubMed]
12
Wijarnpreecha
K.
,
Siri-Angkul
N.
,
Shinlapawittayatorn
K.
,
Charoenkwan
P.
,
Silvilairat
S.
et al
(
2015
)
Heart rate variability as an alternative indicator for identifying cardiac iron status in non-transfusion dependent thalassemia patients
.
PLoS ONE
10
,
e0130837
[PubMed]
13
Anderson
L.J.
,
Holden
S.
,
Davis
B.
,
Prescott
E.
,
Charrier
C.C.
et al
(
2001
)
Cardiovascular T2-star (T2*) magnetic resonance for the early diagnosis of myocardial iron overload
.
Eur. Heart J.
22
,
2171
2179
[PubMed]
14
Anderson
L.J.
(
2011
)
Assessment of iron overload with T2* magnetic resonance imaging
.
Prog. Cardiovasc. Dis.
54
,
287
294
[PubMed]
15
Wood
J.C.
(
2014
)
Use of magnetic resonance imaging to monitor iron overload
.
Hematol. Oncol. Clin. North Am.
28
,
747
764
,
[PubMed]
16
Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology
(
1996
)
Heart rate variability. Standards of measurement, physiological interpretation, and clinical use
Eur. Heart J.
17
,
354
381
[PubMed]
17
Harris
P.R.
,
Sommargren
C.E.
,
Stein
P.K.
,
Fung
G.L.
and
Drew
B.J.
(
2014
)
Heart rate variability measurement and clinical depression in acute coronary syndrome patients: narrative review of recent literature
.
Neuropsychiatr. Dis. Treat.
10
,
1335
1347
[PubMed]
18
Chen
W.
,
Zheng
L.
,
Li
K.
,
Wang
Q.
,
Liu
G.
et al
(
2016
)
A novel and effective method for congestive heart failure detection and quantification using dynamic heart rate variability measurement
.
PLoS ONE
11
,
e0165304
[PubMed]
19
Alp
A.
,
Ozdogan
O.
,
Guloglu
C.C.
,
Turker
M.
and
Atabay
B.
(
2014
)
Heart rate variability in beta-thalassaemia major with or without cardiac siderosis
.
Cardiol. Young
24
,
263
267
[PubMed]
20
Rutjanaprom
W.
,
Kanlop
N.
,
Charoenkwan
P.
,
Sittiwangkul
R.
,
Srichairatanakool
S.
et al
(
2009
)
Heart rate variability in beta-thalassemia patients
.
Eur. J. Haematol.
83
,
483
489
[PubMed]
21
Silvilairat
S.
,
Charoenkwan
P.
,
Saekho
S.
,
Tantiworawit
A.
,
Phrommintikul
A.
et al
(
2016
)
Heart rate variability for early detection of cardiac iron deposition in patients with transfusion-dependent thalassemia
.
PLoS ONE
11
,
e0164300
[PubMed]
22
Singh
S.
,
Hider
R.C.
and
Porter
J.B.
(
1990
)
A direct method for quantification of non-transferrin-bound iron
.
Anal. Biochem.
186
,
320
323
[PubMed]
23
Schiller
N.B.
,
Shah
P.M.
,
Crawford
M.
,
DeMaria
A.
,
Devereux
R.
et al
(
1989
)
Recommendations for quantitation of the left ventricle by two-dimensional echocardiography. American Society of Echocardiography Committee on Standards, Subcommittee on Quantitation of Two-Dimensional Echocardiograms
.
J. Am. Soc. Echocardiogr.
2
,
358
367
[PubMed]
24
Aessopos
A.
,
Farmakis
D.
,
Deftereos
S.
,
Tsironi
M.
,
Tassiopoulos
S.
et al
(
2005
)
Thalassemia heart disease: a comparative evaluation of thalassemia major and thalassemia intermedia
.
Chest
127
,
1523
1530
[PubMed]
25
Nagueh
S.F.
,
Appleton
C.P.
,
Gillebert
T.C.
,
Marino
P.N.
,
Oh
J.K.
et al
(
2009
)
Recommendations for the evaluation of left ventricular diastolic function by echocardiography
.
Eur. J. Echocardiogr.
10
,
165
193
[PubMed]
26
Koonrungsesomboon
N.
,
Tantiworawit
A.
,
Phrommintikul
A.
,
Saekho
S.
,
Srichairattanakool
S.
et al
(
2015
)
Heart rate variability for early detection of iron overload cardiomyopathy in beta-thalassemia patients
.
Hemoglobin
39
,
281
286
[PubMed]
27
Shabanian
R.
,
Heidari-Bateni
G.
,
Kocharian
A.
,
Mashayekhi
M.
,
Hosseinzadeh
S.
et al
(
2010
)
Augmentation of left atrial contractile function: a herald of iron overload in patients with beta thalassemia major
.
Pediatr. Cardiol.
31
,
680
688
[PubMed]
28
Aydinok
Y.
,
Porter
J.B.
,
Piga
A.
,
Elalfy
M.
,
El-Beshlawy
A.
et al
(
2015
)
Prevalence and distribution of iron overload in patients with transfusion-dependent anemias differs across geographic regions: results from the CORDELIA study
.
Eur. J. Haematol.
95
,
244
253
[PubMed]
29
Aydinok
Y.
,
Kattamis
A.
,
Cappellini
M.D.
,
El-Beshlawy
A.
,
Origa
R.
et al
(
2015
)
Effects of deferasirox-deferoxamine on myocardial and liver iron in patients with severe transfusional iron overload
.
Blood
125
,
3868
3877
[PubMed]
30
Finley
J.P.
and
Nugent
S.T.
(
1995
)
Heart rate variability in infants, children and young adults
.
J. Auton. Nerv. Syst.
51
,
103
108
[PubMed]
31
Silvetti
M.S.
,
Drago
F.
and
Ragonese
P.
(
2001
)
Heart rate variability in healthy children and adolescents is partially related to age and gender
.
Int. J. Cardiol.
81
,
169
174
[PubMed]
32
Umetani
K.
,
Singer
D.H.
,
McCraty
R.
and
Atkinson
M.
(
1998
)
Twenty-four hour time domain heart rate variability and heart rate: relations to age and gender over nine decades
.
J. Am. Coll. Cardiol.
31
,
593
601
[PubMed]
33
Chen
X.
,
Kintner
D.B.
,
Luo
J.
,
Baba
A.
,
Matsuda
T.
et al
(
2008
)
Endoplasmic reticulum Ca2+ dysregulation and endoplasmic reticulum stress following in vitro neuronal ischemia: role of Na+-K+-Cl- cotransporter
.
J. Neurochem.
106
,
1563
1576
[PubMed]
34
Kremastinos
D.T.
,
Farmakis
D.
,
Aessopos
A.
,
Hahalis
G.
,
Hamodraka
E.
et al
(
2010
)
Beta-thalassemia cardiomyopathy: history, present considerations, and future perspectives
.
Circ. Heart Fail.
3
,
451
458
[PubMed]
35
Gujja
P.
,
Rosing
D.R.
,
Tripodi
D.J.
and
Shizukuda
Y.
(
2010
)
Iron overload cardiomyopathy: better understanding of an increasing disorder
.
J. Am. Coll. Cardiol.
56
,
1001
1012
[PubMed]
36
Murphy
C.J.
and
Oudit
G.Y.
(
2010
)
Iron-overload cardiomyopathy: pathophysiology, diagnosis, and treatment
.
J. Card. Fail.
16
,
888
900
[PubMed]
37
Lekawanvijit
S.
and
Chattipakorn
N.
(
2009
)
Iron overload thalassemic cardiomyopathy: iron status assessment and mechanisms of mechanical and electrical disturbance due to iron toxicity
.
Can. J. Cardiol.
25
,
213
218
[PubMed]
38
Papanikolaou
G.
and
Pantopoulos
K.
(
2005
)
Iron metabolism and toxicity
.
Toxicol. Appl. Pharmacol.
202
,
199
211
[PubMed]
39
Bartfay
W.J.
and
Bartfay
E.
(
2000
)
Iron-overload cardiomyopathy: evidence for a free radical–mediated mechanism of injury and dysfunction in a murine model
.
Biol. Res. Nurs.
2
,
49
59
[PubMed]
40
Kumfu
S.
,
Chattipakorn
S.
,
Chinda
K.
,
Fucharoen
S.
and
Chattipakorn
N.
(
2012
)
T-type calcium channel blockade improves survival and cardiovascular function in thalassemic mice
.
Eur. J. Haematol.
88
,
535
548
[PubMed]
41
Wijarnpreecha
K.
,
Kumfu
S.
,
Chattipakorn
S.C.
and
Chattipakorn
N.
(
2015
)
Cardiomyopathy associated with iron overload: how does iron enter myocytes and what are the implications for pharmacological therapy?
Hemoglobin
39
,
9
17
[PubMed]
42
Kumfu
S.
,
Chattipakorn
S.C.
,
Fucharoen
S.
and
Chattipakorn
N.
(
2016
)
Dual T-type and L-type calcium channel blocker exerts beneficial effects in attenuating cardiovascular dysfunction in iron-overloaded thalassaemic mice
.
Exp. Physiol.
101
,
521
539
[PubMed]
43
Wongjaikam
S.
,
Kumfu
S.
,
Khamseekaew
J.
,
Sripetchwandee
J.
,
Srichairatanakool
S.
et al
(
2016
)
Combined iron chelator and antioxidant exerted greater efficacy on cardioprotection than monotherapy in iron-overloaded rats
.
PLoS ONE
11
,
e0159414
[PubMed]
44
Apaijai
N.
,
Pintana
H.
,
Chattipakorn
S.C.
and
Chattipakorn
N.
(
2012
)
Cardioprotective effects of metformin and vildagliptin in adult rats with insulin resistance induced by a high-fat diet
.
Endocrinology
153
,
3878
3885
[PubMed]
45
Apaijai
N.
,
Pintana
H.
,
Chattipakorn
S.C.
and
Chattipakorn
N.
(
2013
)
Effects of vildagliptin versus sitagliptin, on cardiac function, heart rate variability and mitochondrial function in obese insulin-resistant rats
.
Br. J. Pharmacol.
169
,
1048
1057
[PubMed]
46
Campos
C.
,
Casali
K.R.
,
Baraldi
D.
,
Conzatti
A.
,
Araujo
A.S.
et al
(
2014
)
Efficacy of a low dose of estrogen on antioxidant defenses and heart rate variability
.
Oxid. Med. Cell Longev.
2014
,
218749
[PubMed]
47
Malfitano
C.
,
Barboza
C.A.
,
Mostarda
C.
,
da Palma
R.K.
,
dos Santos
C.P.
et al
(
2014
)
Diabetic hyperglycemia attenuates sympathetic dysfunction and oxidative stress after myocardial infarction in rats
.
Cardiovasc. Diabetol.
13
,
131
[PubMed]
48
Pipatpiboon
N.
,
Sripetchwandee
J.
,
Chattipakorn
S.C.
and
Chattipakorn
N.
(
2015
)
Effects of PPARgamma agonist on heart rate variability and cardiac mitochondrial function in obese-insulin resistant rats
.
Int. J. Cardiol.
201
,
121
122
[PubMed]
49
Pongkan
W.
,
Pintana
H.
,
Sivasinprasasn
S.
,
Jaiwongkam
T.
,
Chattipakorn
S.C.
et al
(
2016
)
Testosterone deprivation accelerates cardiac dysfunction in obese male rats
.
J. Endocrinol.
229
,
209
220
[PubMed]
50
Tanajak
P.
,
Sa-Nguanmoo
P.
,
Wang
X.
,
Liang
G.
,
Li
X.
et al
(
2016
)
FGF21 therapy attenuates left ventricular dysfunction and metabolic disturbance by improving FGF21 sensitivity, cardiac mitochondrial redox homeostasis and structural changes in pre-diabetic rats
.
Acta Physiol. (Oxf.)
[PubMed]
51
Sivasinprasasn
S.
,
Sa-Nguanmoo
P.
,
Pongkan
W.
,
Pratchayasakul
W.
,
Chattipakorn
S.C.
et al
(
2016
)
Estrogen and DPP4 inhibitor, but not metformin, exert cardioprotection via attenuating cardiac mitochondrial dysfunction in obese insulin-resistant and estrogen-deprived female rats
.
Menopause
23
,
894
902
[PubMed]
52
Chattipakorn
N.
,
Incharoen
T.
,
Kanlop
N.
and
Chattipakorn
S.
(
2007
)
Heart rate variability in myocardial infarction and heart failure
.
Int. J. Cardiol.
120
,
289
296
[PubMed]
53
Lucreziotti
S.
,
Gavazzi
A.
,
Scelsi
L.
,
Inserra
C.
,
Klersy
C.
et al
(
2000
)
Five-minute recording of heart rate variability in severe chronic heart failure: correlates with right ventricular function and prognostic implications
.
Am. Heart J.
139
,
1088
1095
[PubMed]
54
Ponikowski
P.
,
Anker
S.D.
,
Chua
T.P.
,
Szelemej
R.
,
Piepoli
M.
et al
(
1997
)
Depressed heart rate variability as an independent predictor of death in chronic congestive heart failure secondary to ischemic or idiopathic dilated cardiomyopathy
.
Am. J. Cardiol.
79
,
1645
1650
[PubMed]
55
Singh
N.
,
Mironov
D.
,
Armstrong
P.W.
,
Ross
A.M.
and
Langer
A.
(
1996
)
Heart rate variability assessment early after acute myocardial infarction. Pathophysiological and prognostic correlates. GUSTO ECG Substudy Investigators. Global Utilization of Streptokinase and TPA for Occluded Arteries
.
Circulation
93
,
1388
1395
[PubMed]
This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY).