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Autophagy is a bulk degradation process in which cytosolic proteins and organelles are degraded through lysosomes. To evaluate autophagic flux in cardiac myocytes, we generated adenovirus and cardiac-specific transgenic mice harboring tandem fluorescent mRFP-GFP-LC3. Starvation significantly increased the number of mRFP-GFP-LC3 dots representing both autophagosomes and autolysosomes per cell, suggesting that autophagic flux is increased in cardiac myocytes. H2O2 significantly increased autophagic flux, which was attenuated in the presence of N-2-mercaptopropionyl glycine (MPG), an antioxidant, suggesting that oxidative stress stimulates autophagy in cardiac myocytes. Myocardial ischemia/reperfusion (I/R) increased both autophagosomes and autolysosomes, thereby increasing autophagic flux. Treatment with MPG attenuated I/R-induced increases in oxidative stress, autophagic flux, and Beclin-1 expression, accompanied by a decrease in the size of myocardial infarction (MI)/area at risk (AAR), suggesting that oxidative stress plays an important role in mediating autophagy and myocardial injury during I/R. MI/AAR after I/R was significantly reduced in beclin1+/− mice, whereas beclin1+/− mice treated with MPG exhibited no additional reduction in the size of MI/AAR after I/R. These results suggest that oxidative stress plays an important role in mediating autophagy during I/R, and that activation of autophagy through oxidative stress mediates myocardial injury in response to I/R in the mouse heart. Antioxid. Redox Signal. 14, 2179–2190.
Autophagy is a bulk degradation process in which cytosolic proteins and organelles are sequestrated by double-membrane structures of unknown origin called autophagosomes, transferred to lysosomes, and degraded by proteases therein (14). Autophagy is generally an adaptive mechanism by which amino acids and fatty acids are recycled for ATP production, and damaged organelles, protein aggregates, and exogenous pathogens, all of which could lead to cellular malfunction and cell death, are removed (15). However, excessive activation of autophagy can be toxic and may even induce cell death, termed type II programmed cell death (15).
Increasing lines of evidence suggest that autophagy is activated during various pathologic conditions in the heart (25). For example, both chronic and acute myocardial ischemia activate autophagy in the myocardium (18, 33), and autophagy also is activated during cardiac hypertrophy, atrophy, and heart failure (25). However, the function of autophagy in the heart appears to be stimulus dependent. For example, we showed previously that although autophagy can be protective during acute myocardial ischemia and chronic hibernation, it can be detrimental during myocardial reperfusion after a short period of ischemia [ischemia/reperfusion (I/R)] (18, 32). Because autophagy plays an important role in mediating survival and death of cardiac myocytes in a stimulus-dependent manner, it is important to determine whether autophagy is stimulated during various pathophysiologic conditions in vivo and to evaluate its functional significance.
Importantly, evaluating whether autophagy is stimulated is not an easy task, especially at the organ level in vivo. Autophagy is a dynamic process involving nucleation, autophagosome formation, fusion of autophagosomes to lysosomes, and degradation in lysosomes, each of which is governed by multiple signaling mechanisms (22). Although the assessment of autophagosome formation at a single time point may be used for evaluating the extent of autophagy, the results could be misleading because the extent of autophagosome formation often dissociates from the level of autophagic flux (14). Conversely, tandem fluorescent mRFP-GFP-LC3 (tf-LC3), recently developed by Kimura et al. (13), allows one to evaluate the extent of autophagosome and autolysosome formation simultaneously, because LC3 puncta labeled with both GFP and mRFP represent autophagosomes, whereas those labeled with mRFP alone represent autolysosomes. As GFP-LC3 mice developed by Mizushima and colleagues (23) have been a very useful tool for assessing the level of autophagosome formation in mice in vivo, we reasoned that transgenic mice harboring tf-LC3 could also be a useful tool for conveniently evaluating the extent of autophagic flux in organs in vivo.
Here we generated transgenic mice with cardiac-specific expression of tf-LC3 (Tg-tf-LC3) and evaluated the effect of I/R on autophagic flux in the heart. Accumulating lines of evidence suggest that oxidative stress plays an important role in mediating autophagy in various cell types and organs (12). Because oxidative stress is an important cause of myocardial injury at the time of reperfusion (17), and because our previous results suggest that activation of autophagy during the reperfusion phase is detrimental (18), we hypothesized that oxidative stress activates autophagy during I/R and that activation of autophagy by oxidative stress plays an important role in mediating I/R injury in the heart. We tested (a) whether tf-LC3 can be used to evaluate autophagic flux in cardiac myocytes, (b) whether Tg-tf-LC3 can be used to evaluate the extent of autophagy during I/R in the mouse heart in vivo, and (c) whether oxidative stress plays an essential role in mediating autophagy and myocardial injury during I/R in the mouse heart in vivo.
Antibodies used in the study include those against LC3 (MBL Intl. Corp.; PD014), p62 (ARP Inc.; 03-GP62-C), Beclin1 (BD Biosciences; 612112), and α-Tubulin (Sigma-Aldrich; T6199).
Primary cultures of left ventricular cardiac myocytes were prepared from 1-day-old Crl: (WI) BR-Wistar rats (Harlan Laboratories), as described previously (18). A cardiac myocytes–enriched fraction (>95%) was obtained by centrifugation through a discontinuous Percoll gradient (18). After 24h of culture in gelatin-coated culture dishes, the media were changed to serum-free Dulbecco's modified Eagle's medium (DMEM)/F-12 (Mediatech Inc.). For amino acid deprivation (AAD), myocytes were washed 3 times with phosphate-buffered saline (PBS) and incubated with amino acid–free, serum-free Hanks Balanced Salt Solution (HBSS; Mediatech Inc.) for 2h, as described previously (18).
The tf-LC3 plasmid construct (13) (courtesy of Dr Tamotsu Yoshimori, Osaka University, Osaka, Japan) was used to generate an adenovirus (Ad-tf-LC3) by using shuttle vector pDC316 and the Admax system (Microbix). Ad-LacZ was used as the control. Cardiac myocytes were transduced with 15 multiplicities of infection (MOIs) of adenovirus for 24h.
Protein lysates were prepared from myocytes cultured in 6-cm culture dishes by using boiled (95°C for 2min) 2×SDS sample buffer containing 4% SDS, 20% glycerol, 120mM Tris-HCl (pH 6.8), 0.01% bromophenol blue, and 5% β-mercaptoethanol. The protein samples were immediately boiled again at 95°C for 3min.
Heart-tissue homogenates were prepared by using RIPA buffer containing 50mM Tris-HCl (pH 8.0), 150mM NaCl, 0.1% SDS, 1% Igepal CA-630, and 0.5% sodium deoxycholate with protease inhibitors (Sigma; P8340) at a 1:400 dilution. After determining the protein concentration with BCA Assay (Thermo Scientific), equal amounts of proteins were loaded on SDS-PAGE gels with 6×sample buffer containing 0.35 M Tris-HCl (pH 6.8), 10.28% SDS, 36% glycerol, 0.01% bromophenol blue, and 5% β-mercaptoethanol.
In vivo samples containing 35μg of proteins were subjected to SDS-PAGE, as described previously (18). Densitometric analyses of the blots were carried out by using the public domain ImageJ program (NIH, Baltimore, MD).
Cardiac myocytes cultured on coverslips were transduced with Ad-tf-LC3 at 15 MOI. Twenty-four hours after adenovirus transduction, the cells were washed with PBS, fixed with 4% paraformaldehyde, mounted with a reagent containing 4′,6-diamidino-2-phenylindole (DAPI) (Vectashield; Vector Laboratories, Inc.), and viewed under a fluorescence microscope (Nikon Eclipse E800). The number of GFP and mRFP dots was determined by manual counting of fluorescent puncta in five fields from three different myocyte preparations with a 60×objective. The nuclear number was evaluated by counting the number of DAPI-stained nuclei in the same field. The number of dots per cell was obtained by dividing the total number of dots by the number of nuclei in each microscopic field.
For in vivo determination of the number of fluorescent LC3 dots, fresh heart slices were embedded with Tissue-Tek OCT compound (Sakura Finetechnical Co., Ltd.) and frozen at −80°C. Sections 10μm thick were obtained from the frozen tissue samples by using a cryostat (CM3050 S; Leica), air-dried for 30min, fixed by washing in 95% ethanol for 10min, mounted by using a reagent containing DAPI, and viewed under a fluorescence microscope.
The method used to identify the presence of oxidative stress with dihydroethidium (DHE) fluorescence was described previously (1).
Transgenic mice with cardiac-specific expression of tf-LC3 (Tg-tf-LC3) were generated on an FVB background with the murine α-Myosin Heavy Chain promoter, kindly provided by Dr. J. Robbins (Children's Hospital, Cincinnati, OH). Non-transgenic (NTg) mice were used as controls. Beclin-1–heterozygous knockout (beclin1+/−) mice (courtesy of Dr. Beth Levine, University of Texas Southwestern, Dallas, TX) were described previously (18). For starvation studies, Tg-tf-LC3 mice were starved for 24h, following which the mice were sacrificed. All protocols concerning the use of animals were approved by the Institutional Animal Care and Use Committee at the University of Medicine and Dentistry of New Jersey.
Cardiac myocytes were treated with 200μM hydrogen peroxide (H2O2) for 1h and 1mM N-2-mercaptopropionyl glycine (MPG) for 4h to induce oxidative stress and antioxidant treatment, respectively.
The method of inducing I/R in mice was described previously (18). Mice generated on an FVB background were subjected to ischemia for 45min and to reperfusion for either 2 or 24h. Mice generated on a C57BL/6 background were subjected to 30-min ischemia followed by reperfusion for 2 or 24h. MPG, 100mg/kg, was injected intraperitoneally into mice 24h and 1h before occlusion of the artery.
The method used to determine the area at risk (AAR) and infarct size after I/R by using triphenyltetrazolium chloride (TTC) staining was described previously (18).
Data are expressed as mean±SEM. Statistical analyses between groups of two were conducted with the unpaired Student t test. Groups of three or more were analyzed with one-way ANOVA, followed by the Newman–Keuls multiple comparison test. A value of p<0.05 was considered statistically significant.
Autophagic flux being a dynamic process, it is insufficient to evaluate only autophagosome formation to determine the extent of autophagy. It is imperative to distinguish between increased autophagosome formation and decreased autophagosome clearance (14). tf-LC3 staining is a very useful tool for evaluating the extent of autophagic flux, as it helps to differentiate between autophagosomes and autolysosomes (13). GFP fluorescence is quenched in the acidic pH of the lysosomal compartment, thereby limiting the use of GFP-LC3 to the identification of autophagosomes. However, mRFP continues to fluoresce, and mRFP-LC3 can be used to identify both autophagosomes and autolysosomes. By using tf-LC3 and determining the number of red dots that overlay green dots and appear yellow in merged images, the number of autophagosomes can be evaluated. The red dots that do not overlay green dots and appear red in merged images indicate autolysosome formation (13).
To evaluate autophagic flux in cultured cardiac myocytes, we generated an adenovirus harboring tf-LC3 (Ad-tf-LC3). Cardiac myocytes were transduced with Ad-tf-LC3 for 24h. Expression of Ad-tf-LC3 does not affect the level of endogenous LC3 (Fig. 1A). We used amino acid deprivation (AAD) as a model to study the regulation of autophagic flux in cultured cardiac myocytes. Expression of LC3-II was significantly increased after 2h of AAD (Fig. 1B). Expression of p62, a polyubiquitin-binding protein known to be sequestered and degraded during autophagy (2, 26), was significantly decreased after AAD, indicating enhanced autophagy (Fig. 1B). We then evaluated the extent of autophagosome and autolysosome formation by using fluorescent LC3 puncta. The numbers of GFP and mRFP dots per cell were both significantly increased after AAD (Fig. 1C and D). In the merged images, more free red dots than yellow dots were seen, indicating significantly increased autolysosome formation compared with autophagosomes (Fig. 1E), and suggesting that AAD increases autophagic flux. Thus, the results of the analysis using Ad-tf-LC3 well corroborate those obtained with conventional methods.
To evaluate the level of autophagic flux in the heart in vivo with tf-LC3, we generated transgenic mice with cardiac-specific expression of tf-LC3 (Tg-tf-LC3). Expression of tf-LC3 does not affect the level of endogenous LC3 at baseline or in response to starvation for 24h in vivo (Fig. 2A). Starvation increased the level of endogenous LC3-II in both Tg-tf-LC3 and non-transgenic (NTg) control mice (Fig. 2A). Starvation also increased both green and red dots in Tg-tf-LC3 mice, and the merged images showed that starvation significantly increased both yellow dots representing autophagosome formation and red dots representing autolysosome formation in the mouse heart (Fig. 2B–D). These results suggest that Tg-tf-LC3 mice are useful for evaluating autophagic flux in the heart in vivo.
Oxidative stress plays an important role in mediating myocardial cell death in response to stress, such as I/R (6). Oxidative stress also stimulates autophagy in some cell types (6, 12). To evaluate the effect of oxidative stress on autophagy, we treated cardiac myocytes with 200μM H2O2 for 1h. H2O2 treatment significantly increased LC3-II and decreased p62, suggesting that oxidative stress stimulates autophagy in cardiac myocytes (Fig. 3A, B). In cardiac myocytes transduced with Ad-tf-LC3, H2O2 increased the number of green and red dots, and the merged images showed that H2O2 increased both yellow dots representing autophagosomes and red dots representing autolysosomes (Fig. 3C, D). These results suggest that oxidative stress enhances autophagic flux in cultured myocytes.
To understand further the role of oxidative stress in mediating autophagy in cardiac myocytes, we used the antioxidant MPG. MPG is an amino acid derivative that contains a sulfhydryl or thiol group, and is known to act as a free radical scavenger (21). Treatment of cardiac myocytes with 1mM MPG for 4h significantly attenuated H2O2-induced increases in LC3-II and decreases in p62 accumulation (Fig. 3A, B), and significantly reduced both autophagosome and autolyososome formation, as evaluated by Ad-tf-LC3 (Fig. 3C, D). Taken together, the antioxidant, MPG, inhibits oxidative stress–induced autophagy in cardiac myocytes.
Reperfusion of the ischemic myocardium increases production of reactive oxygen species (ROS), which play an important role in mediating reperfusion injuries (19). In mouse hearts subjected to 45min of ischemia followed by 2h of reperfusion, DHE fluorescence, an indicator of superoxide, was significantly increased (Fig. 4A). However, when mice were injected intraperitoneally with 100mg/kg of the antioxidant, MPG, 24h and 1h before ischemia followed by reperfusion, the amount of oxidative stress was significantly decreased (Fig. 4A).
We showed previously that autophagy is robustly increased during I/R and has a detrimental effect (18). To evaluate the role of oxidative stress in mediating autophagy during I/R, mice were subjected to I/R in the presence or absence of MPG. As previously reported, I/R significantly increased LC3-II and decreased p62 accumulation, indicative of increased autophagy (Fig. 4B, C). Although treatment with MPG increased the level of LC3-II, it also increased accumulation of p62, indicating that autophagic flux is inhibited (Fig. 4B, C). To evaluate further the formation of autophagosomes and autolysosomes in cardiac myocytes during I/R, Tg-tf-LC3 mice were subjected to I/R in the presence and absence of MPG. I/R significantly increased red and green puncta, with more autolysosome formation, as evidenced by red puncta, than autophagosome formation, indicated by yellow puncta in merged images (Fig. 4D–F). Treatment with MPG partially decreased both green and red dots after I/R (Fig. 4D, E). Overlaid images showed that MPG significantly decreased I/R-induced increases in yellow dots and red dots, with a more prominent effect on red dots, indicating that MPG significantly decreased autophagic flux during I/R (Fig. 4F). These results suggest that an MPG-sensitive mechanism, most likely oxidative stress, plays an important role in mediating autophagy in response to I/R in the heart.
We showed previously that Beclin-1, a key molecule mediating autophagy, is significantly upregulated during I/R, and that autophagy is induced in a Beclin-1–dependent manner (18). Although MPG did not affect expression of Beclin-1 at baseline (data not shown), MPG significantly attenuated I/R-induced upregulation of Beclin-1 (Fig. 4G, H). Beclin-1 forms a complex with class III phosphatidylinositol 3-kinase and other autophagy-related proteins, and is localized to the pre-autophagosomal structure mediating vesicle nucleation (18, 28). However, Beclin-1 does not affect the conjugation of LC3-I with phosphatidylethanolamine, leading to LC3-II formation (18, 29). This may explain why treatment with MPG did not affect LC3-II accumulation, while significantly inhibiting autophagosome formation.
MPG plays a cytoprotective role during I/R in dog and mouse hearts (11, 20, 21). In mouse hearts subjected to 45-min ischemia and 24-h reperfusion, although no significant difference was found in the area at risk (AAR) (Fig. 5A, C), the size of myocardial infarction (MI)/AAR, as evaluated by triphenyltetrazolium chloride staining, was significantly decreased in the presence of MPG (Fig. 5A, B), suggesting that oxidative stress plays an important role in mediating I/R injury in our model, consistent with previous results (18).
Because MPG treatment inhibited both autophagic flux and myocardial injury, and because stimulation of autophagy during I/R could be detrimental (18), the MPG-induced reduction in infarct size may be mediated through its inhibition of autophagy. To test this hypothesis, we used beclin1 heterozygous knockout mice (beclin1+/−), in which autophagy during I/R is significantly attenuated (18). As shown previously, beclin1+/− mice exhibited a significantly smaller size of MI/AAR after I/R (Fig. 5A, B), suggesting that Beclin-1–induced autophagy after I/R is detrimental (18). Although MPG significantly reduced the size of MI/AAR in control mice, it did not further reduce the size of MI/AAR in beclin1+/− mice (Fig. 5A, B). Neither MPG nor downregulation of Beclin-1 significantly affected the size of AAR (Fig. 5C). These results are consistent with the notion that MPG inhibits I/R injury through suppression of autophagy. We confirmed that I/R-induced upregulation of Beclin-1 is suppressed both by MPG and in beclin1+/− mice, either alone or in combination (Fig. 5D).
In this study, we showed that tf-LC3 can be used to evaluate the level of autophagic flux in cardiac myocytes both in vitro and in vivo. By using Tg-tf-LC3, we demonstrated that I/R increases autophagic flux in cardiac myocytes through an oxidative stress–dependent mechanism in vivo.
tf-LC3 is a useful tool for evaluating the formation of both autophagosomes and autolysosomes simultaneously in cardiac myocytes. The results of our experiments with tf-LC3 showed that AAD increases both autophagosomes and autolysosomes, an observation also supported by conventional indexes of autophagy, including increases in LC3-II and decreases in p62 accumulation in cardiac myocytes. Although autophagosomes accumulate when fusion between autophagosomes and lysosomes is inhibited, a concurrent increase in both autophagosomes and autolysosomes indicates that autophagic flux is stimulated (14). Although autophagic flux can be evaluated by comparing the extent of autophagosome accumulation in the presence or absence of inhibitors of lysosomal fusion or degradation or both, such interventions are usually toxic, which would confound analyses of the role of autophagy in mediating biologic functions in vivo (14, 24). Tg-tf-LC3 mice are useful not only in that the extent of autophagic flux can be monitored in vivo, but also in that one can investigate the effect of various interventions on formation of autophagosomes and autolysosomes independently.
Terada and colleagues (23, 31) previously reported double-transgenic mice generated by cross-breeding cardiac-specific mCherry-LC3 mice with systemic GFP-LC3 mice. Compared with those mice, our transgenic mice with cardiac-specific expression of a single transgene, tf-LC3, have several advantages. First, because either green or red color can be emitted from the same LC3 molecule in Tg-tf-LC3, more-accurate quantification of autophagosomes and autolysosomes can be made than in the cross between cardiac-specific mCherry-LC3 and systemic GFP-LC3 mice, in which the number of mCherry-LC3 and GFP-LC3 molecules are not identical and GFP-LC3 is not cardiac myocyte specific. Second, the mouse model reported by Terada requires cross-breeding between mCherry-LC3 mice and GFP-LC3 mice, whereas our model does not, because both GFP and mRFP are expressed in a single transgene. Finally, because tf-LC3 is expressed in a cardiac myocyte–specific manner in Tg-tf-LC3, our model allows one to quantitate the extent of autophagic flux specifically in cardiac myocytes.
With our unique mouse model, we here provide evidence that oxidative stress plays an important role in mediating autophagy during reperfusion (Fig. 6). Reperfusion causes various cellular responses relevant to autophagy, including oxidative stress, mitochondrial permeability transition pore (mPTP) opening, endoplasmic reticulum stress, Ca2+ overloading, and mitochondrial damage (7, 17, 30). However, to our knowledge, the role of oxidative stress in mediating increases in autophagic flux during I/R has not been clearly demonstrated. Our results suggest that increases in oxidative stress are both necessary and sufficient for inducing autophagy during I/R in cardiac myocytes.
Oxidative stress affects autophagy through multiple mechanisms. During starvation, H2O2 directly oxidizes HsAtg4 and inhibits its cysteine protease activity, thereby causing increased LC3 lipidation and autophagy in cancer cell lines (27). Oxidative stress affects lysosomal membrane permeability through disulfide bond formation in the lysosomal membrane proteins (6, 12). Autophagy is also activated as a compensatory response to damages in intracellular organelles (12). For example, increases in oxidative stress in mitochondria induce opening of the mPTP, leading to depolarization of the mitochondrial membrane potential, swelling, and damage to mitochondrial proteins (3, 5, 9). Cyclophilin D, a component of the mPTP, is involved in starvation-induced mitophagy in cardiac myocytes (4). BH3-only proteins, such as Bnip3, are upregulated during mPTP opening and mediate autophagy during I/R (10, 30). Oxidative stress also triggers ER stress, which, in turn, induces autophagy (17, 30).
Although the molecular mechanism by which oxidative stress induces autophagy during myocardial reperfusion remains to be elucidated, we showed previously that Beclin-1 is dramatically upregulated during the reperfusion phase in the mouse heart, which, in turn, plays an essential role in mediating increases in autophagy (18). Because MPG treatment significantly inhibited upregulation of Beclin-1 during the reperfusion phase, it is possible that oxidative stress induces autophagy through upregulation of Beclin-1. Whether the dramatic upregulation of Beclin-1 during the reperfusion phase is mediated through a transcriptional mechanism, and, if so, which transcription factor is involved in this process, remains to be elucidated.
Our results showed that MPG treatment inhibited formation of both autophagosomes and autolysosomes during reperfusion. Interestingly, we noted that the formation of the latter is more strongly affected. Although the precise mechanism by which this regulation takes place is yet to be identified, it is possible that mechanisms mediating autolysosome formation, such as the fusion of autophagosomes and lysosomes, may be sensitive to oxidative stress. Interestingly, a study by Li et al. (16) showed that MPG neutralizes 3-aminopropanol, a neurotoxin formed during cerebral ischemia that damages the lysosome.
Although oxidative stress activates various mechanisms leading to an increase in death of cardiac myocytes (19), we propose that oxidative stress induces reperfusion injury in part through stimulation of autophagy. Several lines of evidence support our hypothesis. First, suppression of oxidative stress inhibits autophagy during the reperfusion phase, and a similar extent of autophagy suppression by Beclin-1 downregulation was sufficient to reduce the level of reperfusion injury. Second, although MPG significantly reduced the size of MI/AAR in control mice, it did not further reduce the size of MI/AAR when autophagy is suppressed in beclin1+/− mice, consistent with the notion that oxidative stress regulates cell survival/death through regulation of autophagy. Whether stimulation of autophagy can be detrimental during reperfusion is controversial (8, 18). Although our results indicating that MPG suppresses both autophagy and myocardial injury support the notion that autophagy during reperfusion is detrimental, further studies are needed to elucidate the functional significance of autophagy during I/R.
At present, the contribution of autophagic cell death to overall myocardial injury is unknown. Because I/R-induced increases in apoptosis are attenuated in beclin1+/− mice, it is possible that autophagy and apoptosis may be linked. Although mPTP opening may initially induce both autophagy and apoptosis, severe depletion of ATP caused by mPTP opening may lead to necrosis as well. Whether strong activation of lysosomal degradation by autophagy contributes to necrotic cell death during I/R remains to be elucidated.
In summary, our results suggest that tf-LC3 is a useful tool for investigating the level of autophagic flux in the heart and the cardiac myocytes therein. With Tg-tf-LC3 mice, we showed that oxidative stress plays an important role in stimulating autophagic flux, which contributes to myocardial injury during I/R in vivo. We propose that suppression of oxidative stress during the reperfusion phase prevents myocardial injury in part through suppression of excessive autophagy.
We thank Drs. Tamotsu Yoshimori and Beth Levine for tf-LC3 and beclin1+/− mice, respectively. We thank Daniela Zablocki for critical reading of the manuscript.
This work was supported in part by U.S. Public Health Service grants HL 59139, HL67724, HL69020, HL91469, AG27211, and HL102738, and by Foundation of Leducq Transatlantic Network of Excellence.
No competing financial interests exist.