Final Report Summary - AF IN HUMAN MYOCYTES (Effects of atrial fibrillation on the distribution and frequency of spontaneous calcium release events in human atrial myocytes)
Characterization of the properties, frequency, and spatial distribution of local calcium release events in human atrial myocytes from patients with and without atrial fibrillation:
Atrial fibrillation (AF) has been associated with increased calcium release from the sarcoplasmic reticulum (SR), and we here investigated whether AF affects the distribution of the SR calcium release channel (RyR2), its phosphorylation state, or the distribution of local calcium release events (calcium sparks) in human atrial myocytes from patients with or without AF. For calcium imaging, myocytes were loaded with fluo-4 and patch-clamp technique was used to clamp the membrane potential at -80mV and measure the membrane current and calcium sparks were visualized using resonance scanning confocal microscopy at a frame rate of 90 Hz. RyR2 and calcium sparks were detected using custom-made algorithms. Results obtained shows that, in a total of 424 sparks detected in myocytes from 9 patients with AF and 22 without AF, sparks frequency was significantly higher in myocytes from AF patients (53±23 vs. 8.2±2.2 events/min/cell, p less than 0.01) as expected, and this was due to an increased number of spark sites in AF (7.4±3.1 vs. 1.5±0.4 sites, p less than 0.05). Moreover the distance from the spark to the cell membrane was significantly reduced in AF (from 2.8±0.4 to 1.6±0.3μm; p less than 0.05). The concurrent increase in calcium sparks and shortening of their distance to the sarcolemma may contribute to promote spontaneous membrane depolarizations and increase their amplitude. By contrast, measurement by patch-clamp of the sarcoplasmic reticulum (SR) calcium load showed no significant difference between myocytes from 24 AF patients and 57 patients without AF (7.2±0.7 vs. 8.7±0.4 amol/pF) indicating that the increases spark frequency is not due to an increase of the SR calcium content.
Characterization of the spatial distribution of the ryanodine receptor and proteins that modulate its activity in human atrial myocytes from patients with and without atrial fibrillation:
RyR2 were visualized by using antibodies against the total RyR2 and RyR2 phosphorylated at ser2808 and the analysis of the RyR2 distribution revealed that there was no difference in the density of RyR2 in myoctes from patients with and without AF (346 vs. 413 clusters/cell), nor were there any difference in the density of RyR2 near the cell membrane (closer than 1.5 μm). However, the density of ser2808 clusters increased, resulting in a significantly higher ser2808/RyR ratio in myocytes from AF patients (0.56±0.03 vs. 0.33±0.04 p less than 0.01). These results together with the outcomes shown above indicates that increased RyR2 phosphorylation at ser2808 may contribute to increase the calcium spark frequency in myocytes from patients with AF. Moreover, we studied the possibility that changes in L-type calcium current (ICa,L) and spontaneous calcium release from the SR are linked to alteratered cyclic AMP (cAMP)-dependent signaling modulated through β-adrenergic receptors. Patch-clamp recordings revealed a higher frequency of transient inward currents (ITI) activated by spontaneous calcium release in 46 patients with AF than in 86 without AF (1.9±0.3/min vs. 1.1±0.2/min p less than 0.05) and protein kinase A inhibition with H-89 had a stronger effect in patients with AF (3.2±1.5 to 0.1±0.1/min) than without AF (1.3±0.6 to 0.2±0.1/min). Furthermore, the β-agonist isoproterenol (ISO) increased ITI 13.3-fold in patients without AF but only 4.6-fold in AF. ISO also increased the ratio of ser2808 phosphorylated: total ryanodine receptors. At baseline ICa,L was larger in patients without AF (2.1±0.1 vs. 1.4±0.1 pA/pF in AF, p less than 0.05) and H-89 reduced ICa,L (from 2.4±0.4 to 1.4±0.3pA/pF p=0.001) to levels recorded in AF-patients without and with H-89 (1.4±0.4 and 1.2±0.4pA/pF). Calmodulin kinase II inhibition significanly reduced ICa,L and calcium sparks but had no effect on ITI. Interestingly, analysis of patients treated with beta-blockers showed that carvedilol (but not β-1 blockers) strongly reduced the ITI frequency in patients with AF. We conclude that AF is associated with opposite changes in cAMP-dependent regulation of ICa,L (downregulation) and spontaneous SR calcium release (upregulation). Importantly, patients treated with carvedilol showed no such upregulation, pointing to this treatment as a novel means to prevent abnormal calcium release in AF.
Development of a specific tool to analyze the spatial distribution of the ryanodine receptor and proteins that modulate its activity:
49 human atrial myocytes from 9 patients were labeled with anti-phospho Ser-2808 (Red) and anti-RyR2 (Green) antibodies and detected automatically using a custom made algorithm based on: 1) Enhancing the contrast of both green and red-labeled images using a histogram stretching intensity transformation. 2) Removing background noise by using an adaptive median filter that estimates the noise level. 3) Enhancing the location of all green-labeled RyRs with a 2D Gaussian filter with a standard deviation of 0.5 microns followed by segmentation using a multilevel watershed algorithm. 4) Eliminating non-specific staining by setting the maximal RyR diameter to 1.2 microns. 5) Detection of red-labeled ser2808 phosphorylated RyR2s by checking if a cluster of red pixels was present at the location of each green labeled RyR2. Red clusters that overlapped at least 20% of the area of a green-labeled RyR2 were accepted if the normalized intensity of the overlapping area was above 15%. The superimposition of detected RyR2s on the original confocal image revealed a detection efficiency near 95%, and visual inspection of superimposed original confocal images confirmed that all red ser2808 clusters coincided with a green RyR2. Moreover, stimulation of RyR2 phosphorylation with the beta-adrenergic agonist was used as a positive control, and it significantly increased the ser2808:total RyR2 ratio from 0.32±0.03 to 0.52±0.06 (p less than 0.01). This approach may also be applied to other proteins with a punctate distribution that are modulated by phosphorylation, and should be useful to study how disease affects the distribution of such proteins under basal or phosphorylating conditions.
Effect of adenosine A2A receptors on the beat-to-beat stability of calcium currents and calcium transients in isolated atrial myocytes:
As AF has been associated with adenosine A2A receptor-mediated promotion of spontaneous calcium release from the SR, this project tested how A2AR activation affects the beat-to-beat stability. Patch-clamp and confocal calcium imaging was used to measure calcium currents and transients in human and HL-1 atrial myocytes at increasing stimulation frequencies. Electrical mapping was also performed in perfused porcine atria. Our results showed that protein kinase A inhibition increased the threshold for induction of non-uniform responses (0.84±0.12 to 1.86±0.11 Hz; p less than 0.001) while β-adrenergic stimulation lowered it (1.4±0.1 to 0.6±0.1 Hz; p less than 0.001). A2AR-activation with adenosine lowered the threshold from 1.3±0.2 to 0.6±0.2 Hz (p less than 0.05) which was due to a concurrent stimulation of spontaneous calcium waves. In atrial HL-1 myocyte cultures A2AR activation increased the percentage of non-uniformly responding cells (19±3 to 51±9%; p less than 0.02) and electrical mapping in arterially perfused porcine atria showed that adenosine induced electrical alternans at longer cycle lengths, doubled the fraction of electrodes showing alternation, and increased the amplitude of the alternation. Importantly, prevention of A2AR-activation with exogenous adenosine deaminase selectively increased the threshold for induction of non-uniform responses from 1.3±0.2 to 1.7±0.2 Hz (p=0.01) in atrial myocytes from patients with AF. We can conclude that A2AR-mediated stimulation of spontaneous calcium release destabilizes the beat-to-beat stability of intracellular calcium homeostasis, and that prevention of A2AR activation may be a novel means to stabilize the response of human atrial myocytes during elevation of the beating rate.
IN PARALLEL, THE RESEARCHER HAS PARTICIPATED IN TWO RELATED PROJECTS:
Effect of Post-operative atrial fibrillation on intracellular calcium homeostasis in human atrial myocytes:
Post-operative atrial fibrillation (post-AF) is the most common complication associated with open-heart surgery but the mechanisms leading to post-operative AF remain unclear. We investigated whether calcium handling is different in atrial myocytes from patients with post-AF than in patients without post-AF using perforated whole cell patch-clamp technique to compare L-type Ca2+ current (ICa,L), spontaneous calcium release-induced Na+-Ca2+ exchange current (INCX) frequency, spontaneous INCX amplitude and caffeine releasable SR Ca2+ content in atrial myocytes isolated from samples of right atrial appendage obtained from consenting patients in sinus rhythm undergoing cardiac surgery with post-AF (35 cells from 19 patients) and without post-AF (99 cells from 76 patients). We found no differences in ICa,L density among the two patient groups. By contrast, the spontaneous INCX frequency was significantly increased in myocytes from patients with post-AF at a membrane potential of -50 mV (9.5±0.07 waves/min vs. 4.2±0.02 waves/min in cells from patients without post-AF, p less than 0.05). Moreover, post-AF was associated with increased SR Ca2+ load (6.8±0.33 amol/pF without post-AF vs. 8.7±0.58 amol/pF with post-AF, p less than 0.01). Extended statistical analysis of the data, taking into account potential factors of confusion such as age, left atrial diameter, hypertension, left ventricular ejection fraction and pharmacological treatment of donor patients with beta-adrenergic blockers or ACE-inhibitors or ARB-blockers revealed that post-AF was not an independent factor that affected the spontaneous INCX frequency (p=0.19) or the SR calcium load (p=0.81). None of these potentially confounding factors had a significant independent effect on the INCX frequency or the SR calcium load. We conclude that care should be taken when analyzing electrophysiological data from isolated human atrial myocytes without taking into account the potential influence of factors such as age, concurrent cardiac disease and pharmacological treatments.
Effects of ageing on the regulation and stability of intracellular calcium handling in human atrial myocytes:
Atrial fibrillation increases strongly with old age and this arrhythmia has been associated to alterations in intracellular calcium handling. We therefore tested the hypothesis that ageing per se alters calcium handling in the human atrium. Whole membrane currents were measured in the perforated patch configuration in human atrial myocytes from 74 patients free of atrial fibrillation and with normal left atrial size. Patients were categorized as young (<55 years, n=21), middle aged (between 55 and 75 years, n=42), and old (>75 years, n=17). Protein levels were determined by Western blot. The alpha-subunit of the L-type calcium channel was lower in old patients and aging progressively and significantly (p less than 0.01) reduced the L-type calcium current (ICa,L) from 2.4±0.3 pA/pF in young to 1.4±0.2 pA/pF in old patients (p less than 0.01) even when potentially confounding effects of disease and therapy were taken into account. Time constants for ICa,L inactivation were also slowed from 14.5±0.9 ms in young to 20.9±1.9 ms in old patients (p less than 0.01) for fast ICa,L inactivation, and from 73±3 to 120±12 ms (p less than 0.001) for slow ICa,L inactivation. The SR calcium content decreased from 10.1±0.8 amol/pF in young to 6.4±0.6 amol/pF in old patients (p less than 0.005) and this was accompanied by a significant decrease in both SERCA2 (p less than 0.05) and calsequestrin-2 (p less than 0.05) protein levels. This downregulation of calcium handling contributed to a 3.2-fold reduction of the calcium transient in old versus young patients (p<0.01). By contrast, age had no effect on spontaneous calcium release. These results indicate that ageing is associated with depression of L-type calcium channel current, SR calcium content and calcium transient amplitude, which may favor a progressive decline in right atrial function with age.
Atrial fibrillation (AF) has been associated with increased calcium release from the sarcoplasmic reticulum (SR), and we here investigated whether AF affects the distribution of the SR calcium release channel (RyR2), its phosphorylation state, or the distribution of local calcium release events (calcium sparks) in human atrial myocytes from patients with or without AF. For calcium imaging, myocytes were loaded with fluo-4 and patch-clamp technique was used to clamp the membrane potential at -80mV and measure the membrane current and calcium sparks were visualized using resonance scanning confocal microscopy at a frame rate of 90 Hz. RyR2 and calcium sparks were detected using custom-made algorithms. Results obtained shows that, in a total of 424 sparks detected in myocytes from 9 patients with AF and 22 without AF, sparks frequency was significantly higher in myocytes from AF patients (53±23 vs. 8.2±2.2 events/min/cell, p less than 0.01) as expected, and this was due to an increased number of spark sites in AF (7.4±3.1 vs. 1.5±0.4 sites, p less than 0.05). Moreover the distance from the spark to the cell membrane was significantly reduced in AF (from 2.8±0.4 to 1.6±0.3μm; p less than 0.05). The concurrent increase in calcium sparks and shortening of their distance to the sarcolemma may contribute to promote spontaneous membrane depolarizations and increase their amplitude. By contrast, measurement by patch-clamp of the sarcoplasmic reticulum (SR) calcium load showed no significant difference between myocytes from 24 AF patients and 57 patients without AF (7.2±0.7 vs. 8.7±0.4 amol/pF) indicating that the increases spark frequency is not due to an increase of the SR calcium content.
Characterization of the spatial distribution of the ryanodine receptor and proteins that modulate its activity in human atrial myocytes from patients with and without atrial fibrillation:
RyR2 were visualized by using antibodies against the total RyR2 and RyR2 phosphorylated at ser2808 and the analysis of the RyR2 distribution revealed that there was no difference in the density of RyR2 in myoctes from patients with and without AF (346 vs. 413 clusters/cell), nor were there any difference in the density of RyR2 near the cell membrane (closer than 1.5 μm). However, the density of ser2808 clusters increased, resulting in a significantly higher ser2808/RyR ratio in myocytes from AF patients (0.56±0.03 vs. 0.33±0.04 p less than 0.01). These results together with the outcomes shown above indicates that increased RyR2 phosphorylation at ser2808 may contribute to increase the calcium spark frequency in myocytes from patients with AF. Moreover, we studied the possibility that changes in L-type calcium current (ICa,L) and spontaneous calcium release from the SR are linked to alteratered cyclic AMP (cAMP)-dependent signaling modulated through β-adrenergic receptors. Patch-clamp recordings revealed a higher frequency of transient inward currents (ITI) activated by spontaneous calcium release in 46 patients with AF than in 86 without AF (1.9±0.3/min vs. 1.1±0.2/min p less than 0.05) and protein kinase A inhibition with H-89 had a stronger effect in patients with AF (3.2±1.5 to 0.1±0.1/min) than without AF (1.3±0.6 to 0.2±0.1/min). Furthermore, the β-agonist isoproterenol (ISO) increased ITI 13.3-fold in patients without AF but only 4.6-fold in AF. ISO also increased the ratio of ser2808 phosphorylated: total ryanodine receptors. At baseline ICa,L was larger in patients without AF (2.1±0.1 vs. 1.4±0.1 pA/pF in AF, p less than 0.05) and H-89 reduced ICa,L (from 2.4±0.4 to 1.4±0.3pA/pF p=0.001) to levels recorded in AF-patients without and with H-89 (1.4±0.4 and 1.2±0.4pA/pF). Calmodulin kinase II inhibition significanly reduced ICa,L and calcium sparks but had no effect on ITI. Interestingly, analysis of patients treated with beta-blockers showed that carvedilol (but not β-1 blockers) strongly reduced the ITI frequency in patients with AF. We conclude that AF is associated with opposite changes in cAMP-dependent regulation of ICa,L (downregulation) and spontaneous SR calcium release (upregulation). Importantly, patients treated with carvedilol showed no such upregulation, pointing to this treatment as a novel means to prevent abnormal calcium release in AF.
Development of a specific tool to analyze the spatial distribution of the ryanodine receptor and proteins that modulate its activity:
49 human atrial myocytes from 9 patients were labeled with anti-phospho Ser-2808 (Red) and anti-RyR2 (Green) antibodies and detected automatically using a custom made algorithm based on: 1) Enhancing the contrast of both green and red-labeled images using a histogram stretching intensity transformation. 2) Removing background noise by using an adaptive median filter that estimates the noise level. 3) Enhancing the location of all green-labeled RyRs with a 2D Gaussian filter with a standard deviation of 0.5 microns followed by segmentation using a multilevel watershed algorithm. 4) Eliminating non-specific staining by setting the maximal RyR diameter to 1.2 microns. 5) Detection of red-labeled ser2808 phosphorylated RyR2s by checking if a cluster of red pixels was present at the location of each green labeled RyR2. Red clusters that overlapped at least 20% of the area of a green-labeled RyR2 were accepted if the normalized intensity of the overlapping area was above 15%. The superimposition of detected RyR2s on the original confocal image revealed a detection efficiency near 95%, and visual inspection of superimposed original confocal images confirmed that all red ser2808 clusters coincided with a green RyR2. Moreover, stimulation of RyR2 phosphorylation with the beta-adrenergic agonist was used as a positive control, and it significantly increased the ser2808:total RyR2 ratio from 0.32±0.03 to 0.52±0.06 (p less than 0.01). This approach may also be applied to other proteins with a punctate distribution that are modulated by phosphorylation, and should be useful to study how disease affects the distribution of such proteins under basal or phosphorylating conditions.
Effect of adenosine A2A receptors on the beat-to-beat stability of calcium currents and calcium transients in isolated atrial myocytes:
As AF has been associated with adenosine A2A receptor-mediated promotion of spontaneous calcium release from the SR, this project tested how A2AR activation affects the beat-to-beat stability. Patch-clamp and confocal calcium imaging was used to measure calcium currents and transients in human and HL-1 atrial myocytes at increasing stimulation frequencies. Electrical mapping was also performed in perfused porcine atria. Our results showed that protein kinase A inhibition increased the threshold for induction of non-uniform responses (0.84±0.12 to 1.86±0.11 Hz; p less than 0.001) while β-adrenergic stimulation lowered it (1.4±0.1 to 0.6±0.1 Hz; p less than 0.001). A2AR-activation with adenosine lowered the threshold from 1.3±0.2 to 0.6±0.2 Hz (p less than 0.05) which was due to a concurrent stimulation of spontaneous calcium waves. In atrial HL-1 myocyte cultures A2AR activation increased the percentage of non-uniformly responding cells (19±3 to 51±9%; p less than 0.02) and electrical mapping in arterially perfused porcine atria showed that adenosine induced electrical alternans at longer cycle lengths, doubled the fraction of electrodes showing alternation, and increased the amplitude of the alternation. Importantly, prevention of A2AR-activation with exogenous adenosine deaminase selectively increased the threshold for induction of non-uniform responses from 1.3±0.2 to 1.7±0.2 Hz (p=0.01) in atrial myocytes from patients with AF. We can conclude that A2AR-mediated stimulation of spontaneous calcium release destabilizes the beat-to-beat stability of intracellular calcium homeostasis, and that prevention of A2AR activation may be a novel means to stabilize the response of human atrial myocytes during elevation of the beating rate.
IN PARALLEL, THE RESEARCHER HAS PARTICIPATED IN TWO RELATED PROJECTS:
Effect of Post-operative atrial fibrillation on intracellular calcium homeostasis in human atrial myocytes:
Post-operative atrial fibrillation (post-AF) is the most common complication associated with open-heart surgery but the mechanisms leading to post-operative AF remain unclear. We investigated whether calcium handling is different in atrial myocytes from patients with post-AF than in patients without post-AF using perforated whole cell patch-clamp technique to compare L-type Ca2+ current (ICa,L), spontaneous calcium release-induced Na+-Ca2+ exchange current (INCX) frequency, spontaneous INCX amplitude and caffeine releasable SR Ca2+ content in atrial myocytes isolated from samples of right atrial appendage obtained from consenting patients in sinus rhythm undergoing cardiac surgery with post-AF (35 cells from 19 patients) and without post-AF (99 cells from 76 patients). We found no differences in ICa,L density among the two patient groups. By contrast, the spontaneous INCX frequency was significantly increased in myocytes from patients with post-AF at a membrane potential of -50 mV (9.5±0.07 waves/min vs. 4.2±0.02 waves/min in cells from patients without post-AF, p less than 0.05). Moreover, post-AF was associated with increased SR Ca2+ load (6.8±0.33 amol/pF without post-AF vs. 8.7±0.58 amol/pF with post-AF, p less than 0.01). Extended statistical analysis of the data, taking into account potential factors of confusion such as age, left atrial diameter, hypertension, left ventricular ejection fraction and pharmacological treatment of donor patients with beta-adrenergic blockers or ACE-inhibitors or ARB-blockers revealed that post-AF was not an independent factor that affected the spontaneous INCX frequency (p=0.19) or the SR calcium load (p=0.81). None of these potentially confounding factors had a significant independent effect on the INCX frequency or the SR calcium load. We conclude that care should be taken when analyzing electrophysiological data from isolated human atrial myocytes without taking into account the potential influence of factors such as age, concurrent cardiac disease and pharmacological treatments.
Effects of ageing on the regulation and stability of intracellular calcium handling in human atrial myocytes:
Atrial fibrillation increases strongly with old age and this arrhythmia has been associated to alterations in intracellular calcium handling. We therefore tested the hypothesis that ageing per se alters calcium handling in the human atrium. Whole membrane currents were measured in the perforated patch configuration in human atrial myocytes from 74 patients free of atrial fibrillation and with normal left atrial size. Patients were categorized as young (<55 years, n=21), middle aged (between 55 and 75 years, n=42), and old (>75 years, n=17). Protein levels were determined by Western blot. The alpha-subunit of the L-type calcium channel was lower in old patients and aging progressively and significantly (p less than 0.01) reduced the L-type calcium current (ICa,L) from 2.4±0.3 pA/pF in young to 1.4±0.2 pA/pF in old patients (p less than 0.01) even when potentially confounding effects of disease and therapy were taken into account. Time constants for ICa,L inactivation were also slowed from 14.5±0.9 ms in young to 20.9±1.9 ms in old patients (p less than 0.01) for fast ICa,L inactivation, and from 73±3 to 120±12 ms (p less than 0.001) for slow ICa,L inactivation. The SR calcium content decreased from 10.1±0.8 amol/pF in young to 6.4±0.6 amol/pF in old patients (p less than 0.005) and this was accompanied by a significant decrease in both SERCA2 (p less than 0.05) and calsequestrin-2 (p less than 0.05) protein levels. This downregulation of calcium handling contributed to a 3.2-fold reduction of the calcium transient in old versus young patients (p<0.01). By contrast, age had no effect on spontaneous calcium release. These results indicate that ageing is associated with depression of L-type calcium channel current, SR calcium content and calcium transient amplitude, which may favor a progressive decline in right atrial function with age.