Association of Serum Galectin-3 Levels and Atrial Remodeling
Association of Serum Galectin-3 Levels and Atrial Remodeling
Thirty-three patients with paroxysmal AF and preserved LV function who were evaluated with cardiac DE-MRI to examine LA anatomy and fibrosis extent before AF ablation were enrolled in this observational study. AF episodes self-terminating within 7 days were defined as paroxysmal AF.
Baseline demographic and clinical characteristics, including age, gender, body mass index (BMI) and smoking history were collected from all patients. A careful history was taken from all patients to exclude any cardiac and noncardiac systemic disease. Data related to the diagnosis of AF including date of first diagnosis, European Heart Rhythm Association (EHRA) score, history of stroke and anticoagulation, rate control drugs and antiarrhythmic medications were also recorded.
Each subject underwent a transthoracic 2D-guided M-mode echocardiogram using commercially available equipment (Vivid S6; GE Healthcare, Horten, Norway). All echocardiographic parameters were measured according to the recommendations of the American Society of Echocardiography by a single cardiologist blinded to the patient characteristics. All patients underwent assessment of LA volume index (LAVI), LV systolic and valvular functions. LA area (LAA) and LA length were measured in the apical 4-chamber and apical 2-chamber views. LA volume (LAV) was derived using the biplane area-length method. Both LAA and LAV were measured at LV end-systole. LAVI was calculated based on the patient's body surface area. LV end-diastolic diameter (LVEDD) and LV ejection fraction (LVEF) were measured using 2D-targeted M-mode echocardiography.
Patients who were pregnant or had a history of hypertension, diabetes mellitus, moderate-severe valvular disease, congenital heart disease, alcohol consumption, abnormal thyroid function, coronary artery disease, renal disease (patients with estimated glomerular filtration <60 mL/min/1.73 m), hepatic disease, autoimmune disease, recent infection, attempted catheter or surgical AF ablation, or contraindication to anticoagulation were excluded from the study. Furthermore, patients who had systolic LV dysfunction (LVEF <50%) were not included in the study. Informed consent was taken from each patient before enrollment. The study was in compliance with the principles outlined in the Declaration of Helsinki and approved by the Institutional Ethics Committee.
Venous blood samples were obtained prior to ablation from the venous sheaths, immediately centrifuged and stored at –80 °C until assayed. Patients were at sinus rhythm during sample collection. The frozen serum samples were rapidly thawed and brought to room temperature and assayed for the presence of human galectin-3 by using enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, Europe/International, Austria), according to the manufacturer's instructions. Serial dilutions of known concentrations of human galectin-3 were used to construct a standard curve of the analytes. The serum levels of galectin-3 from the samples were estimated by extrapolation from a log:log linear regression curve determined from the serially diluted human recombinant galectin-3 ranging from 25 to 0.39 ng/mL.
The time interval between the electrocardiographic P wave and the atrial contraction detected by M-mode or Doppler echocardiography is defined as atrial electromechanical delay. In our study, tissue Doppler echocardiography was performed by transducer frequencies of 3.5–4.0 MHz, adjusting the spectral pulsed Doppler signal filters until a Nyquist limit of 15–20 cm/s and using the minimal optimal gain. The monitor sweep speed was set at 50–100 mm/s to optimize the spectral display of myocardial velocities. The pulsed Doppler sample volume was placed at the lateral and septal sides of the mitral annulus and the right ventricular (RV) tricuspid annulus to obtain tissue Doppler velocities. The time interval from the onset of the P wave on surface ECG to the peak of the late diastolic wave (the A' wave), termed PA', was obtained from the lateral mitral annulus (lateral PA'), septal mitral annulus (the septal PA'), and RV tricuspid annulus (the tricuspid PA'). The difference between the lateral PA' and tricuspid PA' (lateral PA'–tricuspid PA') was defined as the inter-AEMD, while the differences between the septal PA' and the tricuspid PA' and the lateral PA' and the septal PA' were defined as intra-right AEMD and intra-left AEMD, respectively. All echocardiographic measurements were calculated as average of data from 3 beats. All measurements were performed by an experienced cardiologist who was blinded to the LA fibrosis and serum galectin-3 data of all participants.
Studies were performed by a General Electric 1.5-T High Definition scanner (Signa Excite HD™; GE Medical Systems, Waukesha, WI, USA) using a 8-channel phased-array receiver coil. All patients were in sinus rhythm during MRI scan. A contrast-enhanced 3-dimensional (3D) fast low-angle shot MR angiography sequence and a cine true-fast imaging with steady-state precession sequence were used to define the anatomy of the LA and the pulmonary veins. The scan was acquired ~18 (range: 17–20) minutes after contrast agent injection (0.15 mmol/kg i.v., meglumin gadoterad [Dotarem™, Guerbet, Aulnay, France]) using a 3D inversion recovery, respiration-navigated, electrocardiogram (ECG)-gated, gradient echo pulse sequence in the double oblique axial, sagittal, and coronal planes, encompassing the entire LA. Image planes were "double oblique planes" because they did not correspond to true axial, coronal, or sagittal planes, but were oriented based on the long axis of the LV. For each plane, a 3D stack of images were acquired parallel to the 4-chamber view, the 2-chamber view, and the LV short axis for the double oblique axial, sagittal, and coronal planes, respectively. Typical acquisition parameters were: free breathing using navigator gating, a transverse imaging (TI) volume with voxel size 1.25 × 1.25 × 4 mm that was then reconstructed to 0.625 × 0.625 × 2 mm, repetition time (TR)/echo time (TE) = 4.8/2.1 milliseconds, field of view (FOV): 300–340 mm, flip angle: 20°, slice thickness: 4 mm, spacing gap: 0, inversion time (TI) = 280 to 350 milliseconds; bandwith: 224 Hz/pixel, number of excitations: 1 and phase FOV: 0.75. ECG gating was used to acquire a small subset of phase-encoding views during the diastolic phase of the LA cardiac cycle. The time interval between the R-peak of the ECG and the start of data acquisition was defined using the cine images of the LA. The typical value of the interval was 60% of the mean RR interval for patients. Fat saturation was used to suppress the fat signal. The TE of the scan was chosen such that the signal intensity of partial volume fat tissue voxels was reduced allowing improved definition of the LA wall boundary. The TI value for the DE-MRI scan was identified using a scout scan. Typical scan time for the DE-MRI study was between 8 and 12 minutes depending on the subject respiration and heart rate. Raw images were transferred to a workstation for storage and quantitative analysis. LA regions were defined as superior, inferior, anterior (annular), posterior, septal, and lateral walls. In all DE-MRI images, epicardial and endocardial contours were manually drawn around LA myocardium with image display. Threshold for fibrosis identification was determined for each patient individually by using a dynamic threshold algorithm based partly on work in left ventricle. The relative extent of contrast enhancement was quantified within the LA wall using a threshold-based algorithm based on pixel intensity distribution of healthy myocardium and nonviable myocardium (Fig. 1).
(Enlarge Image)
Figure 1.
Contrast-enhancement quantification within the left atrial wall using a threshold-based algorithm based on pixel intensity distribution of healthy myocardium and nonviable. A: Significant (30%) left atrial fibrosis. B: Mild (3%) left atrial fibrosis. Corresponding serum galectin-3 levels are 0.72 and 0.26 ng/mL, respectively.
The images were assessed by a single radiologist experienced in cardiovascular imaging. The observer was completely blinded to the patient information (identity and clinical parameters). To assess the potential effects of intraobserver variability in measurement of delayed enhancement, we randomly selected a subset of 10 patients in whom the responsible user repeated MRI scans in a separate session (median duration between 2 scans was 24 hours). The intraclass correlations for intrareader variability of detected LA wall enhancement were 0.92 for reliability of observations (95% CI: 0.74–0.98) and 0.96 for reliability of the mean (95% CI: 0.85–0.98).
Methods
Study Population
Thirty-three patients with paroxysmal AF and preserved LV function who were evaluated with cardiac DE-MRI to examine LA anatomy and fibrosis extent before AF ablation were enrolled in this observational study. AF episodes self-terminating within 7 days were defined as paroxysmal AF.
Baseline demographic and clinical characteristics, including age, gender, body mass index (BMI) and smoking history were collected from all patients. A careful history was taken from all patients to exclude any cardiac and noncardiac systemic disease. Data related to the diagnosis of AF including date of first diagnosis, European Heart Rhythm Association (EHRA) score, history of stroke and anticoagulation, rate control drugs and antiarrhythmic medications were also recorded.
Each subject underwent a transthoracic 2D-guided M-mode echocardiogram using commercially available equipment (Vivid S6; GE Healthcare, Horten, Norway). All echocardiographic parameters were measured according to the recommendations of the American Society of Echocardiography by a single cardiologist blinded to the patient characteristics. All patients underwent assessment of LA volume index (LAVI), LV systolic and valvular functions. LA area (LAA) and LA length were measured in the apical 4-chamber and apical 2-chamber views. LA volume (LAV) was derived using the biplane area-length method. Both LAA and LAV were measured at LV end-systole. LAVI was calculated based on the patient's body surface area. LV end-diastolic diameter (LVEDD) and LV ejection fraction (LVEF) were measured using 2D-targeted M-mode echocardiography.
Patients who were pregnant or had a history of hypertension, diabetes mellitus, moderate-severe valvular disease, congenital heart disease, alcohol consumption, abnormal thyroid function, coronary artery disease, renal disease (patients with estimated glomerular filtration <60 mL/min/1.73 m), hepatic disease, autoimmune disease, recent infection, attempted catheter or surgical AF ablation, or contraindication to anticoagulation were excluded from the study. Furthermore, patients who had systolic LV dysfunction (LVEF <50%) were not included in the study. Informed consent was taken from each patient before enrollment. The study was in compliance with the principles outlined in the Declaration of Helsinki and approved by the Institutional Ethics Committee.
Measurement of Serum Galectin-3 Levels
Venous blood samples were obtained prior to ablation from the venous sheaths, immediately centrifuged and stored at –80 °C until assayed. Patients were at sinus rhythm during sample collection. The frozen serum samples were rapidly thawed and brought to room temperature and assayed for the presence of human galectin-3 by using enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, Europe/International, Austria), according to the manufacturer's instructions. Serial dilutions of known concentrations of human galectin-3 were used to construct a standard curve of the analytes. The serum levels of galectin-3 from the samples were estimated by extrapolation from a log:log linear regression curve determined from the serially diluted human recombinant galectin-3 ranging from 25 to 0.39 ng/mL.
Electromechanical Time İnterval Measurements
The time interval between the electrocardiographic P wave and the atrial contraction detected by M-mode or Doppler echocardiography is defined as atrial electromechanical delay. In our study, tissue Doppler echocardiography was performed by transducer frequencies of 3.5–4.0 MHz, adjusting the spectral pulsed Doppler signal filters until a Nyquist limit of 15–20 cm/s and using the minimal optimal gain. The monitor sweep speed was set at 50–100 mm/s to optimize the spectral display of myocardial velocities. The pulsed Doppler sample volume was placed at the lateral and septal sides of the mitral annulus and the right ventricular (RV) tricuspid annulus to obtain tissue Doppler velocities. The time interval from the onset of the P wave on surface ECG to the peak of the late diastolic wave (the A' wave), termed PA', was obtained from the lateral mitral annulus (lateral PA'), septal mitral annulus (the septal PA'), and RV tricuspid annulus (the tricuspid PA'). The difference between the lateral PA' and tricuspid PA' (lateral PA'–tricuspid PA') was defined as the inter-AEMD, while the differences between the septal PA' and the tricuspid PA' and the lateral PA' and the septal PA' were defined as intra-right AEMD and intra-left AEMD, respectively. All echocardiographic measurements were calculated as average of data from 3 beats. All measurements were performed by an experienced cardiologist who was blinded to the LA fibrosis and serum galectin-3 data of all participants.
DE-MRI Assessment of LA Fibrosis
Studies were performed by a General Electric 1.5-T High Definition scanner (Signa Excite HD™; GE Medical Systems, Waukesha, WI, USA) using a 8-channel phased-array receiver coil. All patients were in sinus rhythm during MRI scan. A contrast-enhanced 3-dimensional (3D) fast low-angle shot MR angiography sequence and a cine true-fast imaging with steady-state precession sequence were used to define the anatomy of the LA and the pulmonary veins. The scan was acquired ~18 (range: 17–20) minutes after contrast agent injection (0.15 mmol/kg i.v., meglumin gadoterad [Dotarem™, Guerbet, Aulnay, France]) using a 3D inversion recovery, respiration-navigated, electrocardiogram (ECG)-gated, gradient echo pulse sequence in the double oblique axial, sagittal, and coronal planes, encompassing the entire LA. Image planes were "double oblique planes" because they did not correspond to true axial, coronal, or sagittal planes, but were oriented based on the long axis of the LV. For each plane, a 3D stack of images were acquired parallel to the 4-chamber view, the 2-chamber view, and the LV short axis for the double oblique axial, sagittal, and coronal planes, respectively. Typical acquisition parameters were: free breathing using navigator gating, a transverse imaging (TI) volume with voxel size 1.25 × 1.25 × 4 mm that was then reconstructed to 0.625 × 0.625 × 2 mm, repetition time (TR)/echo time (TE) = 4.8/2.1 milliseconds, field of view (FOV): 300–340 mm, flip angle: 20°, slice thickness: 4 mm, spacing gap: 0, inversion time (TI) = 280 to 350 milliseconds; bandwith: 224 Hz/pixel, number of excitations: 1 and phase FOV: 0.75. ECG gating was used to acquire a small subset of phase-encoding views during the diastolic phase of the LA cardiac cycle. The time interval between the R-peak of the ECG and the start of data acquisition was defined using the cine images of the LA. The typical value of the interval was 60% of the mean RR interval for patients. Fat saturation was used to suppress the fat signal. The TE of the scan was chosen such that the signal intensity of partial volume fat tissue voxels was reduced allowing improved definition of the LA wall boundary. The TI value for the DE-MRI scan was identified using a scout scan. Typical scan time for the DE-MRI study was between 8 and 12 minutes depending on the subject respiration and heart rate. Raw images were transferred to a workstation for storage and quantitative analysis. LA regions were defined as superior, inferior, anterior (annular), posterior, septal, and lateral walls. In all DE-MRI images, epicardial and endocardial contours were manually drawn around LA myocardium with image display. Threshold for fibrosis identification was determined for each patient individually by using a dynamic threshold algorithm based partly on work in left ventricle. The relative extent of contrast enhancement was quantified within the LA wall using a threshold-based algorithm based on pixel intensity distribution of healthy myocardium and nonviable myocardium (Fig. 1).
(Enlarge Image)
Figure 1.
Contrast-enhancement quantification within the left atrial wall using a threshold-based algorithm based on pixel intensity distribution of healthy myocardium and nonviable. A: Significant (30%) left atrial fibrosis. B: Mild (3%) left atrial fibrosis. Corresponding serum galectin-3 levels are 0.72 and 0.26 ng/mL, respectively.
The images were assessed by a single radiologist experienced in cardiovascular imaging. The observer was completely blinded to the patient information (identity and clinical parameters). To assess the potential effects of intraobserver variability in measurement of delayed enhancement, we randomly selected a subset of 10 patients in whom the responsible user repeated MRI scans in a separate session (median duration between 2 scans was 24 hours). The intraclass correlations for intrareader variability of detected LA wall enhancement were 0.92 for reliability of observations (95% CI: 0.74–0.98) and 0.96 for reliability of the mean (95% CI: 0.85–0.98).