Author + information
- Received August 6, 2010
- Revision received January 7, 2011
- Accepted January 11, 2011
- Published online May 24, 2011.
- Shu-Guang Zhu, MD, PhD⁎,
- Rakesh C. Kukreja, PhD⁎,⁎ (, )
- Anindita Das, PhD⁎,
- Qun Chen, MD, PhD⁎,
- Edward J. Lesnefsky, MD⁎,† and
- Lei Xi, MD⁎,⁎ ()
- ↵⁎Reprint requests and correspondence:
Dr. Lei Xi or Rakesh C. Kukreja, Virginia Commonwealth University, Division of Cardiology, Box 980204, 1101 East Marshall Street, Room 7-020C/D, Richmond, Virginia 23298-0204
Objectives The aim of this study was to test the hypothesis that long-term dietary nitrate supplementation protects against doxorubicin-induced cardiomyopathy by improving ventricular function and reducing mitochondrial respiratory chain damage.
Background Doxorubicin is a powerful anthracycline antibiotic used to treat divergent human neoplasms. Its clinical use is limited because of severe cardiotoxic side effects. Dietary nitrate and nitrite are essential nutrients for maintenance of steady-state tissue levels of nitric oxide and may play a therapeutic role in diseases associated with nitric oxide insufficiency or dysregulation. Dietary nitrate and nitrite supplementation alleviates myocardial injury caused by ischemia-reperfusion and cardiac arrest–resuscitation.
Methods Adult male CF-1 mice were given a single dose of doxorubicin (15 mg/kg intraperitoneally), and left ventricular contractile function was assessed 5 days later using both echocardiography and pressure-volume Millar catheterization. A nitrate supplementation regimen (1 g/l sodium nitrate in drinking water) was started 7 days before doxorubicin injection and continued thereafter. Cardiomyocyte necrosis and apoptosis, tissue lipid peroxidation, and plasma nitrate and nitrite levels were assessed. In addition, mitochondrial complex I activity, oxidative phosphorylation capacity, and hydrogen peroxide generation were determined in parallel experiments.
Results Doxorubicin caused impairment of ventricular contractility and cell death, which were significantly reduced by nitrate supplementation (p < 0.05). These cardioprotective effects were associated with a significant decrease in tissue lipid peroxidation. Nitrate supplementation significantly preserved mitochondrial complex I activity and oxidative phosphorylation and attenuated hydrogen peroxide generation after doxorubicin treatment.
Conclusions Long-term oral intake of inorganic nitrate attenuates doxorubicin-induced ventricular dysfunction, cell death, oxidative stress, and mitochondrial respiratory chain damage. Nitrate could be a promising therapeutic agent against doxorubicin-induced cardiotoxicity.
Doxorubicin (DOX) (with trade names such as Adriamycin and Rubex) is a broad-spectrum and potent anthracycline antibiotic that has been used to treat a variety of human neoplasms (1). Despite its anticancer efficacy, the clinical use of DOX is often limited by dose-dependent cardiotoxicity, which may lead to irreversible dilated cardiomyopathy and heart failure (2). The cardiotoxicity of DOX involves increased oxidative stress in cardiomyocytes, alteration of mitochondrial energetics, and a direct effect on deoxyribonucleic acid. However, the optimal therapeutic approaches for protection against DOX cardiotoxicity have not been identified. This is because most of the tested agents (such as antioxidants and beta-adrenergic receptor blockers) have pronounced clinical disadvantages, including a decline in high-density lipoprotein level, an inability to prevent mortality, weight loss, and potentiation of myelosuppression (3).
Nitric oxide (NO) is essential for the integrity of the cardiovascular system, and decreased production and/or bioavailability of NO lead to the development of cardiovascular disorders (4) and heart failure (5). NO protects against ischemia-reperfusion (I/R) injury induced by ischemic or pharmacological preconditioning (6), which could involve inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) (7,8). The role of NO in protection against DOX-induced cardiotoxicity with the phosphodiesterase-5 inhibitor sildenafil was suggested by our group (9). Moreover, a possible role of NO in protection against DOX-induced cardiotoxicity was indicated by beta-adrenergic blockers (10) or statins (11), because these drugs increase iNOS and eNOS messenger ribonucleic acid and protein levels in myocardium (12) and cardiomyocytes (13).
In addition to L-arginine-NOS-dependent NO synthesis, the NOS-independent mechanisms of NO generation also convert nitrate to nitrite to NO in vivo via both enzymatic reduction (e.g., xanthine oxidoreductase ) and nonenzymatic acidic disproportionation (15). There are numerous colonies of bacteria in the mammalian oral cavity and intestine that can reduce nitrate to nitrite in vivo (16). The nitrate-nitrite-NO pathway is a complementary system to the NOS-dependent pathway for ensuring sufficient NO production, especially under pathophysiological situations, including hypoxia and acidosis, when the oxygen-dependent NOS activity is compromised (17,18). Because a substantial portion of nitrate and nitrite in the blood and tissue is derived from dietary sources, the dietary supplementation of nitrate or nitrite protects against myocardial I/R injury (19,20) and cardiac arrest–resuscitation, reduces hypertension (21), and improves endothelial and platelet function as well as exercise performance (22).
In this context, the present study was designed to test the hypothesis that long-term nitrate supplementation may protect against ventricular dysfunction and cardiomyocyte loss from DOX-induced cardiomyopathy. We also investigated whether mitochondria are protected by nitrate, because DOX accumulates mainly in mitochondria and cell nuclei (23). Because DOX enhances reactive oxygen species (ROS) generation in cardiac mitochondria (24,25), we studied the effect of nitrate and nitrite in reducing oxidative stress in the mitochondria. Because of the potential role of transient blockade of the mitochondrial complex I in nitrite-induced anti-ischemic protection (26), we also investigated the effect of nitrate on DOX-induced damage of the mitochondrial electron transport chain (ETC).
Adult male CF-1 outbred mice were purchased from Harlan Sprague Dawley, Inc. (Frederick, Maryland). The animal experimental protocol was approved by the Institutional Animal Care and Use Committee of the Virginia Commonwealth University.
As illustrated in Online Figure 1A, mice were administered a single dose of DOX intraperitoneally (15 mg/kg dissolved in saline; DOX group, n = 8) or volume-matched saline (0.2 ml; control group, n = 8). Another group of mice (nitrate + DOX group, n = 8) received sodium nitrate (NaNO3) added to their drinking water at a concentration of 1 g/l (12 mmol/l) for 7 days before DOX injection on day 8. The nitrate treatment regimen was continued throughout the post-DOX period. A control group of mice were given nitrate alone (nitrate group, n = 8). This oral dose of NaNO3 has been shown to be cardioprotective against I/R injury (19). Five days later (on day 13), left ventricular (LV) function was assessed on echocardiography under light anesthesia and subsequently with a Millar catheter (Millar Instruments, Inc., Houston, Texas) inserted into the LV cavity under surgical anesthesia.
To determine the efficacy of nonoral nitrate, a subset of mice were subcutaneously implanted with a micro-osmotic pump (ALZET model 1002, ALZET Osmotic Pumps, Inc., Cupertino, California), which continuously delivered NaNO3 solution (1 g/l concentration) or saline (as sham controls) at a rate of 0.25 μl/h for 13 days. The nitrate-infused and saline-infused mice received DOX injection (15 mg/kg intraperitoneally) on day 8 (n = 7 per group).
Measurement of DOX cytotoxicity in vitro
As illustrated in Figure 1B, mice with or without 7 days of nitrate supplementation were sacrificed on day 8, and cardiomyocytes were isolated and exposed to 1 μmol/l DOX by adding to cell culture medium. Myocyte necrosis and apoptosis were quantified 18 h later using trypan blue exclusion and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assays.
Echocardiographic assessment of ventricular contractile function
Echocardiography was performed using the Vevo770 imaging system (VisualSonics Inc., Toronto, Ontario, Canada).
Measurement of LV hemodynamic parameters
After echocardiographic assessment, a microtip pressure-volume catheter transducer (Millar Instruments, Inc.) was inserted into the right carotid artery and advanced into the LV cavity. LV systolic and end-diastolic pressures, the maximal slope of the systolic pressure increment (+dP/dtmax) and diastolic pressure decrement (−dP/dtmax), heart rate, and aortic blood pressure were recorded on a beat-by-beat basis.
Measurement of plasma nitrate and nitrite
Blood samples were collected from mice that underwent 4 different treatment conditions (n = 6 per group) and centrifuged to collect the plasma. Nitrate and nitrite were measured with a Sievers NO analyzer (model 280NOA, GE Analytical Instruments, Boulder, Colorado).
Measurement of mitochondrial oxidative phosphorylation and enzyme activities
Oxidative phosphorylation was studied in the isolated mitochondria using glutamate and malate (complex I) and succinate (complex II) as substrates to localize defects within the ETC. Enzyme activities of complex I (nicotinamide adenine dinucleotide [NADH]:duroquinone oxidoreductase) and NADH dehydrogenase (proximal segment of complex I) were determined.
Assessment of lipid peroxidation
Lipid peroxidation in the cardiac tissues was determined by measuring malondialdehyde and 4-hydroxyalkenals using a colorimetric assay kit (Bioxytech LPO-586, OXIS International, Beverly Hills, California).
Measurement of hydrogen peroxide
The rate of hydrogen peroxide (H2O2) generation in mitochondria (n = 5 per group) was determined using oxidation of the fluorogenic indicator Amplex Red (Invitrogen Corporation, Carlsbad, California) in the presence of horseradish peroxidase.
Data are presented as group mean ± SEM. Statistical analysis was performed using 1-way analysis of variance with subsequent Student-Newman-Keuls post hoc tests for pairwise comparisons among the 4 treatment groups. Unpaired t tests were used to compare 2 treatment groups in the study with subcutaneous infusion of nitrate. p values <0.05 were considered statistically significant.
Please see the Online Appendix for a complete description of the methods.
Nitrate supplementation ameliorates DOX-induced LV contractile dysfunction
LV ejection fraction and fractional shortening decreased significantly in the DOX group compared with the control group after 5 days of treatment (Fig. 1), which improved with nitrate supplementation before and after DOX injection (p < 0.05, nitrate + DOX vs. DOX; n = 8 per group) (Fig. 1). Similar results were obtained with invasive measurements on Millar catheterization. DOX-induced LV systolic (Figs. 2A and 2C) and diastolic (Figs. 2B and 2D) dysfunction was significantly reduced by nitrate supplementation. In addition, heart rate (Fig. 2E) and mean aortic blood pressure (Fig. 2F) were decreased by DOX administration and were partially attenuated by nitrate (p < 0.05, nitrate + DOX vs. DOX; n = 8 per group). Nitrate alone had no effect on these functional parameters (Figs. 2A to 2F).
Nitrate supplementation enhances plasma levels of nitrate and nitrite
Oral nitrate supplementation enhanced plasma nitrate (Fig. 3A) and nitrite (Fig. 3B) compared with the control group (p < 0.05). DOX treatment also elevated plasma nitrate levels (likely through the enhanced NOS-dependent NO synthesis triggered by DOX). In contrast, nitrite levels did not increase in DOX-treated groups with or without nitrate supplementation (Figs. 3A and 3B), suggesting impeded nitrate-to-nitrite conversion. Overall NO oxidation products (NOx) were increased in the nitrate and nitrate + DOX groups compared with the control and DOX groups (p < 0.05) (Fig. 3C).
Nitrate supplementation reduces DOX-induced cardiomyocyte death in vitro
The number of both trypan blue–positive (necrotic) and TUNEL-positive (apoptotic) cardiomyocytes increased after 18 h of exposure to 1 μmol/L DOX in vitro (p < 0.01, control vs. DOX; n = 4 per group) (Fig. 4). Basal cell viability was not altered with nitrate supplementation. However, cardiomyocytes from nitrate-treated mice were more resistant to DOX-induced cytotoxicity, as indicated by reductions in necrosis (Fig. 4A) and apoptosis after 18 h of DOX exposure (Fig. 4B) compared with the DOX group (p < 0.01 for necrosis, p < 0.05 for apoptosis).
Nitrate supplementation preserves oxidative phosphorylation in mitochondria
DOX interferes with mitochondrial respiration at several levels of the ETC. It diverts electrons from complex I of the respiratory chain to generate the semiquinone free radical, initiating a futile redox cycle leading to a stimulation of oxygen free radical generation (27,28). To demonstrate the effect of nitrate supplementation on oxidative phosphorylation after DOX treatment, cardiac mitochondria were isolated from treated groups (Online Fig. 1A). DOX treatment markedly decreased oxidative phosphorylation when glutamate and malate were used as complex I substrates. The rate of succinate oxidation was not altered in the DOX-treated group (n = 6 per group) (Fig. 5A). Nitrate supplementation restored oxidative phosphorylation with glutamate and malate as substrates, showing protection of complex I against DOX-induced damage (Fig. 5A).
Nitrate supplementation prevents DOX-induced damage of mitochondrial NADH dehydrogenase
To further localize the site of the defect in complex I, enzyme activities of complex I (NADH/duroquinone oxidoreductase) and NADH dehydrogenase (the proximal part of complex I) were measured in the homogenates. DOX treatment decreased the activities of complex I and its NADH dehydrogenase (p < 0.05 vs. control; n = 5 per group) (Fig. 5B). Nitrate supplementation protected both complex I and its NADH dehydrogenase against DOX-induced damage.
Nitrate supplementation reduces lipid peroxidation
Lipid peroxidation in the DOX group was increased by 38% compared with the control group (n = 6, p < 0.05) (Fig. 6A), which was completely suppressed by nitrate supplementation (p > 0.05, nitrate + DOX group vs. control group) (Fig. 6A). Nitrate supplementation alone had no effect on the oxidative markers.
Nitrate supplementation limits DOX-induced H2O2 generation
H2O2 was increased in mitochondria isolated from DOX-treated mice compared with the control group when glutamate and malate were used as a complex I substrate (p < 0.05) (Fig. 6B). Nitrate significantly decreased H2O2 generation when combined with DOX. Nitrate alone had no effect on mitochondrial H2O2 generation. Similarly, nitrate supplementation attenuated DOX-induced H2O2 generation when succinate and rotenone were used as a complex II substrate (Fig. 6B).
Subcutaneous infusion of nitrate fails to prevent DOX-induced LV contractile dysfunction
To determine if a nonoral route of nitrate supplementation could protect against DOX cardiotoxicity, we implanted micro-osmotic pumps subcutaneously in 2 groups of DOX-treated mice (n = 7 in each group) receiving continuous infusion of either nitrate (NaNO3, 1 g/l concentration) or saline for the same 13-day duration of oral nitrate supplementation. LV systolic (Online Figs. 2A and 2C) and diastolic (Online Figs. 2B and 2D) functional parameters were not improved by subcutaneous infusion of nitrate (p > 0.05, DOX vs. nitrate + DOX). The subcutaneous infusion route increased plasma levels of nitrate (p < 0.05, DOX vs. nitrate + DOX) (Online Fig. 2E), while nitrite levels remained unchanged (Online Fig. 2F).
DOX-induced cardiomyopathy remains a clinical dilemma in oncology and cardiology practice (3) and has severely limited the therapeutic potential of this potent anticancer drug (2). We examined the effects of long-term oral supplementation of nitrate against DOX-induced cardiomyopathy using a comprehensive approach to demonstrate protection at the level of intact organ, isolated cells, and mitochondria. Nitrate supplementation caused significant preservation of LV contractile function and decreased cardiomyocyte death through mechanisms involving inhibition of lipid peroxidation, reduction of mitochondrial complex I damage, and attenuation of H2O2 generation in mice treated with DOX. These results suggest that nitrate supplementation could be a potentially novel treatment modality in alleviating DOX-induced cardiotoxicity in patients with cancer.
Cardioprotective effects of nitrate supplementation: role of NO and NOS
Nitrite and nitrate have traditionally been considered the inert end products of NO metabolism, with limited intrinsic biological activity (29). Nitrate and nitrite have recently emerged at the forefront of NO biology because they represent a major storage form of NO in blood and tissues (30). It has been increasingly appreciated that nitrate and nitrite can be reduced to NO under tissue ischemia or hypoxia and acidosis (18,30–32). At extremely low tissue pH and partial pressure of oxygen during myocardial ischemia, nitrite may be reduced to NO by acidic disproportionation (15,33) or by the enzymatic reduction of xanthine oxidoreductase (34). The NOS-independent pathway of NO production is an important alternative system under pathological conditions such as myocardial ischemia, because NO production through this mechanism is critically influenced by various cofactors and presence of oxygen. Therefore, mobilization of the nitrate-nitrite-NO pathway is an important alternative means to produce NO, which in turn limits tissue injury under pathophysiological conditions. Particularly, dietary nitrate or nitrite supplementation was recently found to be beneficial against myocardial I/R injury (19,20), cardiac arrest–resuscitation, and hypertension (21). The present study has extended the potential use of nitrate supplementation in reducing myocardial damage caused by DOX. Our results provide conclusive evidence that long-term nitrate supplementation significantly reduced DOX-induced LV contractile dysfunction (Figs. 1 and 2), depressed central aortic pressure (Fig. 2F), and reduced cardiomyocyte necrotic and apoptotic death (Fig. 4). Most important, these cardioprotective effects are associated with concomitant increases in plasma levels of nitrate and nitrite (i.e., NOx) (Fig. 3C), an indicator of enhanced NO production.
The role of NOS-dependent versus NOS-independent NO generation in the nitrate-induced protection against DOX-induced cardiomyopathy remains largely elusive. Previous studies have suggested a detrimental role of iNOS-derived NO in DOX-induced cardiomyopathy (35,36). Conversely, NO is a ubiquitous signaling molecule in protection against myocardial I/R injury by ischemic or pharmacological pre-conditioning (6). Additionally, anthracyclines produce distinct and largely irreversible changes in levels of phosphate metabolites and substantial acidosis in the heart (37), which could potentially trigger NOS-independent protection induced by nitrite. Although the dietary nitrite supplementation–induced cardioprotection against I/R injury has been reported to be eNOS independent (38), it is unclear whether this is the case with DOX-induced myocardial dysfunction. Further studies are necessary to delineate the potential role of NOS in nitrate-mediated protection against DOX cardiomyopathy.
Another interesting observation was that DOX elevated plasma nitrate levels without significant changes in nitrite levels (with or without nitrate supplementation). The reason is not clear, but it could presumably have resulted from the enhanced NOS-dependent NO synthesis induced by DOX treatment (Figs. 3A and 3B). This would suggest impeded nitrate-to-nitrite conversion, possibly due to the antibiotic property of DOX, which may have hindered bacterial nitrate-to-nitrite conversion after DOX treatment. Nevertheless, overall NOx were significantly increased in the nitrate + DOX group compared with the DOX group (p < 0.05) (Fig. 3C), suggesting an association between enhanced NO production and nitrate-induced cardioprotection. It is noteworthy that although nitrate supplementation resulted in a relatively small change in nitrite levels (Fig. 3B), it caused large increases in NOx levels (Fig. 3C). The reason is not clear, although it is possible that a substantial amount of NO could be generated via the NOS-dependent pathway, because both iNOS and eNOS were up-regulated in the heart by nitrate supplementation (unpublished data). Future studies are needed to examine the role of each of the NOS isoforms (i.e., iNOS, eNOS, and neuronal NOS) in nitrate-induced cardioprotection against DOX cardiotoxicity.
Role of nitrate in protection of mitochondrial complex I
DOX-induced cardiomyopathy occurs primarily via the generation of ROS in mitochondria (24,25), a mechanism that is separate from its antineoplastic activity (39). Complex I and complex III of the mitochondria are the key sites for ROS generation (40), and DOX enhances ROS generation through its bioreductive activation, which converts DOX to a semiquinone radical via univalent reduction (24). Complex I, especially NADH dehydrogenase (the initial segment of complex I), is the key site for DOX bioreductive activation in cardiac cells (27,28). The increased generation of ROS from the ETC in turn damages mitochondria and induces cell injury (40,41). The cardiotoxicity of DOX is apparently dose dependent: A low concentration of DOX selectively damages NADH dehydrogenase by increasing oxidative stress, whereas a high concentration of DOX induces nonoxidative inactivation of the complexes at the ETC through the formation of a DOX-cardiolipin complex (24,42). The present study showed that DOX impairs complex I by decreasing NADH dehydrogenase activity (Fig. 5), which is protected by nitrate supplementation. These data suggest that the attenuated damage at the mitochondrial complex I level may at least partially explain the nitrate-induced protection against cardiotoxicity. Moreover, these results are conceptually consistent with the nitrosylation of complex I through NO generation by nitrate or nitrite (26,43). Complex I nitrosylation results in the decrease of its activity (44), thereby providing an acute, protective partial blockade of electron transport (41,44). The transient blockade of complex I during stress, including I/R, protects complex I activity and reduces cardiomyocyte injury (26). We propose that the analogous blockade of electron transport during DOX exposure protects against mitochondrial and cardiac damage.
Nitrate supplementation suppresses DOX-induced oxidative stress
DOX-enhanced tissue lipid peroxidation (Fig. 6A) as well as mitochondrial H2O2 generation (Fig. 6B) were effectively diminished by long-term nitrate intake. These novel observations may represent a key mechanistic explanation for the reduction of DOX-induced cardiotoxicity by nitrate supplementation.
Oral administration of nitrate is critical for cardioprotection
An interesting observation in the present study is the necessity of the oral route for nitrate-induced protection against DOX cardiotoxicity. The long-term subcutaneous infusion of nitrate failed to significantly improve DOX-induced LV dysfunction (Online Figs. 2A to 2D), despite the comparable plasma nitrate levels between the oral and subcutaneous routes of administration (Fig. 3A, Online Fig. 2E). These results suggest that the bacterial bioconversion and reduction of nitrate in the mouth and lower gastrointestinal tract are critical for initiation and/or maintenance of the cardioprotective signals induced by nitrate supplementation. Therefore, an oral route of inorganic nitrate administration is more effective in protection against DOX-induced cardiomyopathy.
We have provided the first evidence for the efficacy of long-term dietary nitrate supplementation to attenuate DOX-induced ventricular dysfunction, cell death, oxidative stress, and mitochondrial respiratory chain damage. These results are potentially important in designing clinical trials on nitrate supplementation as a prophylactic pharmacological intervention to attenuate DOX-induced cardiomyopathy in patients with cancer.
For a detailed Methods section and Online Figures 1 and 2, please see the online version of this article.
This study was supported in part by grants from the National Institutes of Health (HL51045, HL79424, and HL93685 to Dr. Kukreja and AG15885 to Dr. Lesnefsky), the American Heart Association (National Scientist Development Grant 0530157N to Dr. Xi and Mid-Atlantic Affiliate Beginning Grant-in-Aid 0765273U to Dr. Das), and Medical Research Service, U.S. Department of Veterans Affairs (Merit Review Award to Dr. Lesnefsky). The authors have reported that they have no relationships to disclose.
- Abbreviations and Acronyms
- endothelial nitric oxide synthase
- electron transport chain
- hydrogen peroxide
- inducible nitric oxide synthase
- left ventricular
- nicotinamide adenine dinucleotide
- sodium nitrate
- nitric oxide
- nitric oxide oxidation products
- nitric oxide synthase
- reactive oxygen species
- terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling
- Received August 6, 2010.
- Revision received January 7, 2011.
- Accepted January 11, 2011.
- American College of Cardiology Foundation
- Liu X.,
- Chen Z.,
- Chua C.C.,
- et al.
- Xi L.,
- Jarrett N.C.,
- Hess M.L.,
- Kukreja R.C.
- Guo Y.,
- Jones W.K.,
- Xuan Y.T.,
- et al.
- Fisher P.W.,
- Salloum F.,
- Das A.,
- Hyder H.,
- Kukreja R.C.
- Riad A.,
- Bien S.,
- Westermann D.,
- et al.
- Atar S.,
- Ye Y.,
- Lin Y.,
- et al.
- Webb A.,
- Bond R.,
- McLean P.,
- Uppal R.,
- Benjamin N.,
- Ahluwalia A.
- Bryan N.S.,
- Calvert J.W.,
- Elrod J.W.,
- Gundewar S.,
- Ji S.Y.,
- Lefer D.J.
- Tsuchiya K.,
- Kanematsu Y.,
- Yoshizumi M.,
- et al.
- Chaiswing L.,
- Cole M.P.,
- Ittarat W.,
- Szweda L.I.St. Clair D.K.,
- Oberley T.D.
- Shiva S.,
- Sack M.N.,
- Greer J.J.,
- et al.
- Davies K.J.,
- Doroshow J.H.
- Doroshow J.H.,
- Davies K.J.
- Lauer T.,
- Preik M.,
- Rassaf T.,
- et al.
- Gladwin M.T.,
- Raat N.J.,
- Shiva S.,
- et al.
- Bryan N.S.,
- Rassaf T.,
- Maloney R.E.,
- et al.
- Tiravanti E.,
- Samouilov A.,
- Zweier J.L.
- Godber B.L.,
- Doel J.J.,
- Sapkota G.P.,
- et al.
- Weinstein D.M.,
- Mihm M.J.,
- Bauer J.A.
- Pacher P.,
- Liaudet L.,
- Bai P.,
- et al.
- Meyers P.A.,
- Gorlick R.,
- Heller G.,
- et al.
- Chen Q.,
- Camara A.K.,
- Stowe D.F.,
- Hoppel C.L.,
- Lesnefsky E.J.
- Selemidis S.,
- Dusting G.J.,
- Peshavariya H.,
- Kemp-Harper B.K.,
- Drummond G.R.
- Burwell L.S.,
- Nadtochiy S.M.,
- Tompkins A.J.,
- Young S.,
- Brookes P.S.