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Doxorubicin is a widely used chemotherapy drug, but its application is associated with cardiotoxicity. Free radical generation and mitochondrial dysfunction are thought to contribute to doxorubicin-induced cardiac failure. Angiotensin-converting enzyme (ACE) inhibitors are commonly used as cardioprotective agents and have recently been shown in clinical studies to be efficacious in the prevention of anthracycline induced heart failure. Here we evaluated a mechanism for these protective effects by testing the ability of the ACE inhibitor enalapril to preserve mitochondrial function in a model of chronic doxorubicin treatment in rats.
Sprague Dawley rats were divided into three groups and followed for a total of 10 weeks: a) control-untreated, b) Doxorubicin treated (Dox), and c) Doxorubicin + Enalapril treated (DE). Doxorubicin was administered via intraperitoneal injection at weekly intervals from week 2 through week 7. Enalapril was administered in the drinking water of the DE group for the study duration.
Doxorubicin treatment produced a significant loss in left ventricular contractility (P< 0.05), decrease in mitochondrial function via impairment of state-3 respiration, decrease in the cytosolic fraction of ATP, and up-regulation of free radical production. Enalapril significantly attenuated the decrease in percent fractional shortening (P< 0.05) and prevented the doxorubicin-associated reduction in respiratory efficiency and cytosolic ATP content (P< 0.05). Importantly, enalapril also abolished the robust doxorubicin-induced increase in free radical formation.
Administration of enalapril attenuates doxorubicin-induced cardiac dysfunction via preservation of mitochondrial respiratory efficiency and reduction in doxorubicin-associated free radical generation.
Doxorubicin, an anthracycline, is a widely used cytotoxic agent for the treatment of human neoplasms such as leukemias and Hodgkin’s lymphoma. However, administration of doxorubicin is known to cause cardiomyopathy, eventually leading to congestive heart failure that almost invariably develops in patients receiving cumulative doses of doxorubicin over 550 mg/m2 1. As a result of doxorubicin therapy, a growing number of patients in the United States survive childhood and adult cancers only to develop severe cardiac dysfunction following treatment 2. These patients have few surgical options for treatment as they suffer from a combination of both dilated and restrictive cardiomyopathies, and make poor heart transplant candidates due to their history of cancer. Development of medical therapies that aid in the management of cardiac dysfunction is thus paramount for the long term survival of these individuals.
A number of previous studies have implicated free radical formation and oxidative stress as central mechanisms underlying the cardiotoxic effects of doxorubicin 3. As a result, many experimental drug treatments to ameliorate cardiotoxicity of doxorubicin have focused on the protective effects of antioxidants and metal chelators, which minimize free radical damage 4. While these substances have shown some benefit, they are difficult to administer due to the challenge of achieving constant plasma concentrations of antioxidant drugs and their poor uptake by the heart 5.
ACE inhibitors (ACEI) are a class of drugs that are routinely administered in the clinic and have clearly shown positive therapeutic profiles for the treatment of heart failure caused by a number of cardiovascular diseases 6. ACEI possess free radical scavenger and antioxidant properties 7, and have recently been shown in two prospective clinical studies to be efficacious in the prevention of anthracycline induced heart failure when administered early after chemotherapy regimens 8, 9. Importantly, administration of ACEI in these trials and preclinical studies has not been linked with an increased rate of recurrent malignancy 1, 10. However, only a limited number of studies have investigated the mechanisms by which ACE inhibition can prevent anthracycline-induced cardiotoxicity 7, 11. Importantly, the potential effects ACE inhibitors may have upon prevention of anthracycline induced mitochondrial dysfunction remain unknown. To answer these questions, in this study, we set out to: a) investigate in vivo the possible protective effects of the ACE inhibitors (enalapril) against doxorubicin-induced cardiac toxicity in a longitudinal model of treatment, and b) determine if the improved cardiac function from ACE inhibitor therapy is due to improved mitochondrial function and reduced free radical generation. Our results show that the concurrent administration of ACE inhibitors with doxorubicin treatment not only ameliorates cytotoxic effects of doxorubicin, but also prevents doxorubicin-induced free radical formation and preserves mitochondrial respiratory efficiency and cellular ATP content.
Twenty four female Sprague Dawley rats at ~10 weeks of age were obtained from Charles River laboratories. The animals were divided into 3 groups: a) control-untreated (n=8), b) Doxorubicin treated (Dox) (n=8), and c) Doxorubicin + Enalapril treated (DE) (n=8). Both Dox and DE groups received doxorubicin at a cumulative dose of 25 mg/kg, administered weekly via intraperitonial (IP) injection for six weeks. Enalapril pre-treatment for the DE group was started one week before administration of the first doxorubicin injection and continued throughout the study and for an additional three weeks after the last doxorubicin injection (Figure 1A). The dosage of enalapril was calculated based upon a previous study by Sanbe et al. and set at a dose of 10 mg/kg/day for each animal as previously described 12. A detailed description is included in the Supplemental Methods.
Echocardiography studies were performed prior to treatment and immediately before animal sacrifice to determine left ventricular (LV) fractional shortening, using a 14.7-MHz transducer on a Sequoia C512 Echocardiography system (Siemens, Malvern, PA, USA). A detailed description is included in the Supplemental Methods.
Heart and liver specimens were fixed in 4% paraformaldehyde and stained with hematoxylin and eosin for analysis by light microscopy. A detailed description is included in the Supplemental Methods.
Caspase-3 and caspase-9 activities were measured using fluorometric protease assay kits: (Caspase-3/CPP32, Caspase-9/Mch6, Biovision, Mountain View, CA, USA). A complete description is included in the Supplemental Methods.
Mitochondrial and cytosolic protein fractions were isolated using differential centrifugation as previously described. A detailed description is included in the Supplemental Methods.
Mitochondrial oxygen consumption was measured at 37°C by polarography, with a Clark-type oxygen electrode (Oxytherm, Hansatech, Norfolk, UK) under identical conditions (same mitochondria, buffer composition, and substrate concentrations) to H2O2 production measurements. The rate of mitochondrial H2O2 production was assayed in freshly isolated mitochondria by a fluorometric method described by Barja et. al 13. A complete description is included in the Supplemental Methods.
Cytosolic homogenate isolated from heart were used immediately after isolation to determine ATP content using a bioluminescence ATP kit from Sigma (Sigma-Aldrich Inc, St. Louis, MO). A complete description is included in the Supplemental Methods.
Data was analyzed using a one-way analysis of variance (ANOVA) with Newman–Keuls post hoc comparison. Significance was set at P< 0.05. All data are represented as average ± standard error mean (SEM).
Rats were studied longitudinally over 10 weeks (Figure 1A). Control animals were compared to rats receiving doxorubicin (Dox) and rats receiving doxorubicin with enalapril (DE). There was a 100% survival rate in both control and DE groups up to 10 weeks. In contrast, the Dox group experienced only a 75% survival rate (2 deaths) (Figure 1B). These data show that a 10-week model of doxorubicin administration causes significant toxicity resulting in mortality, and validates the time course for intervention.
Doxorubicin treatment was observed to cause a significant decrease in cardiac function, as quantified by depressed fractional shortening of the LV via echocardiography (baseline prior to Dox treatment: 37.62 ± 0.77, post-treatment control: 39.28 ± 0.83 vs Dox: 24.92 ± 3.08 %, P< 0.05). Enalapril treatment significantly attenuated loss of systolic function in the DE group (DE: 31.42 ± 1.06 %, P< 0.05 vs Dox) (Figure 2A–B). These findings confirm previous findings including two recent human clinical studies showing that simultaneous administration of enalapril can significantly blunt the deleterious cardiotoxic effects of doxorubicin on heart function 7–9, 14.
Doxorubicin not only reduced contractility in treated animals but also caused pathologic changes as observed by histology. Histological sections of the heart in Dox animals showed numerous scattered foci of myofibrillar degeneration with associated contraction bands (Figure 2C). By comparison, DE hearts showed only elongated cardiomyocytes. Interestingly, the livers in both Dox and DE treatments showed gross enlargement, thickened and inflamed capsules, indications of venous congestion, and hepatocyte cytopathology with regenerative changes.
The decline in cardiac function and histologic changes of the myocardium caused by doxorubicin are not fully understood. Apoptosis has been previously implicated as a culprit in doxorubicin-induced cardiac dysfunction 15. Here we found that apoptosis levels as measured by Caspase-3 activity in the cytosol of Dox and DE rat hearts were significantly higher than those in control animals (control: 122.2 ± 8.7 vs Dox: 165.5 ± 19.8 vs DE: 169.0 ± 9.2 FU/mg protein, P< 0.05). Caspase-9 activity was also elevated in Dox and DE rats compared to controls (control: 196.4 ± 15.1 vs Dox: 251.6 ± 11.8 vs DE: 252.9 ± 16.0 FU/mg protein, P< 0.05) (Figure 3A). Comparison of caspase-3 activity with that of caspase-9 revealed a significant positive correlation (r = 0.65, P< 0.01) (Figure 3B), providing evidence for induction of the mitochondrial mediated pathway of apoptosis. These data corroborate previous findings that apoptosis levels are increased in the myocardium by doxorubicin, and show that the increase in cardiac apoptosis is not significantly attenuated by enalapril.
As enalapril was observed to improve systolic function following doxorubicin treatment but did not prevent apoptosis, we sought to determine if protection against anthracyline induced toxicity by ACE inhibitors was due to an alternative mechanism. Several recent clinical studies have suggested that ACE inhibition may be efficacious in the prevention of anthracycline induced heart failure by protection of mitochondrial function 8, 9. However, these reports did not show causality between anti-oxidant properties of enalapril and improvement in systolic function in patients post-chemotherapy. To test whether doxorubicin significantly affects mitochondrial processes and whether enalapril can ameliorate these changes, we evaluated oxygen consumption in intact mitochondria isolated from the hearts of Dox, DE, and control animals. Addition of pyruvate/malate (P/M) was used to simulate state 4, the inactive state of mitochondria, and addition of P/M with ADP as substrates was used to simulate state 3, the active state of mitochondria 16.
Treatment with doxorubicin alone (Dox group) decreased oxygen consumption levels in both state 3 and state 4 (Figure 4A). This effect was particularly pronounced in state 3, the ATP producing state of mitochondria where oxygen consumption typically increases robustly. While doxorubicin alone decreased mitochondrial oxygen consumption by 2.5 fold in state 3 compared to control animals, the addition of enalapril (DE group) completely reversed this effect (control: 47.66 ± 4.15, Dox: 18.94 ± 4.67, DE: 50.44 ± 6.90 nmoles O2/min mg protein, P< 0.05 for control vs Dox and DE vs Dox, P=NS for control vs DE) (Figure 4A). Doxorubicin also significantly lowered oxygen consumption in state 4 mitochondria, the inactive state of mitochondria in which ADP phosphorylation does not occur. As with state 3, enalapril completely abolished this effect. These findings suggest that enalapril can prevent mitochondrial dysfunction caused by doxorubicin toxicity by allowing mitochondrial state 3 and state 4 respiration to be maintained at control levels.
Correlation of active to inactive mitochondrial coupling was further determined by calculating the respiratory control ratio (RCR), defined as state 3 oxygen consumption/state 4 oxygen consumption. By calculating the RCR, we found that doxorubicin not only decreased inactive (state 4) and active (state 3) mitochondrial respiration but also lowered the functional coupling of oxygen consumption at baseline (state 4) with the efficiency of energy production by ADP phosphorylation (state 3). Addition of enalapril significantly ameliorated the disproportional decrease in RCR and impairment of mitochondrial metabolic activity induced by doxorubicin (control RCR: 6.747 ± 0.428, Dox RCR: 3.666 ± 0.791, DE RCR: 5.458 ± 0.516, P< 0.05 for control vs Dox, P=NS for DE vs control) (Figure 4A).
To test whether impairment of oxidative respiration affected mitochondrial output, we next evaluated ATP production in the hearts of animals treated with doxorubicin. ATP produced by mitochondria is exported into the cytosol for cellular function. ATP content was therefore measured in the cytosolic homogenate of heart tissue from animals in control, Dox, and DE groups. Total ATP content in the cytosolic homogenate was found to be significantly depleted in doxorubicin-treated rats as compared to control animals (control: 27.87 ± 2.37, Dox: 14.44 ± 3.92 fmoles ATP/mg protein, P< 0.05) (Figure 4B). The addition of enalapril completely reversed this effect, raising the cytosolic ATP content to near control levels in the DE group of animals (control: 27.87 ± 2.37, DE: 24.24 ± 1.66 fmoles ATP/mg protein, P=NS). This outcome suggests that the impairment of mitochondrial respiratory efficiency by doxorubicin results in depletion of ATP for cellular use, and that enalapril is able to reverse this effect by preserving normal levels of oxidative respiration.
Finally, as several previous studies have shown that dysfunction of mitochondrial respiration and production of ATP are linked to increased generation of reactive oxygen species (ROS) 17, we assessed levels of oxidative stress in the hearts of control, Dox, and DE animals. ROS production was measured as the amount of H2O2 released from the mitochondria in an incubation medium supplemented with pyruvate/malate (P/M) to evaluate total basal mitochondrial ROS generation 13. Absolute values for basal levels of H2O2 production were not significantly different between control, Dox, and DE animals (control: 0.313 ± 0.028 vs Dox: 0.345 ± 0.039 vs DE: 0.353 ± 0.037 nmoles H2O2/min mg protein, P=NS) (Figure 5A). While these results may seem to suggest that doxorubicin administration is not associated with increased oxidative stress, these findings actually indicate the opposite–that free radical generation is elevated in the hearts of animals treated with doxorubicin. This is due to the consideration that H2O2 generation is typically proportional to mitochondrial O2 consumption 20. As levels of O2 consumption were observed to be much lower in mitochondria of Dox animals as compared to that of control and DE rats (Figure 4A), we would normally anticipate H2O2 production in the Dox group to be proportionately low as well. Hence, elevation of H2O2 production in animals receiving doxorubicin to levels observed in control and DE groups indicate significant impairment in the efficiency of mitochondria of Dox animals to avoid free radical leakage from the respiratory chain (Figure 5A).
This is best illustrated by calculation of the free radical leak (FRL%) which is defined as the proportion of electrons that flow out of sequence in the electron transport chain (ETC) to reduce O2 to hydrogen peroxide instead of reaching cytochrome oxidase to reduce O2 to water. Because 2 electrons are needed to reduce 1 mole of O2 to hydrogen peroxide, whereas 4 electrons are required to reduce 1 mole of O2 to water, the fraction of electrons that form ROS may be calculated by dividing the rate of H2O2 production by two times the rate of O2 consumption and multiplying the result by 100 18. Calculation of the FRL% revealed a significant difference in ETC dysfunction between control and Dox animals (control: 2.29 ± 0.27 vs Dox: 3.54 ± 0.60 %, P< 0.05) (Figure 5B). Importantly, enalapril treatment abolished ETC dysfunction caused by doxorubicin (DE: 1.98 ± 0.26 %, P< 0.05 vs Dox). These findings demonstrate that anthracycline administration causes leakage of free radicals, and that enalapril can ameliorate this negative effect.
The efficacy of doxorubicin as an anti-cancer drug has led to the search for diverse strategies to prevent its cytotoxic effects 19. Although many candidate compounds have been tested, surprisingly little research has examined the possible effect of ACE inhibitors on doxorubicin toxicity. Two recent clinical studies have demonstrated early treatment of anthracycline induced cardiotoxicity with ACE inhibition significantly ameliorates development of heart failure 8, 9. While the authors of these studies do not present a definite mechanism for these results, they strongly postulate the anti-oxidant properties of enalapril are a primary mechanism by which ACE inhibition prevents against anthracycline cardiotoxicity. Here we demonstrate that enalapril does have a protective effect upon mitochondrial function in doxorubicin-treated rats by: a) maintaining mitochondrial O2 consumption at control levels, b) preventing the depletion of cellular ATP content, and c) lowering the mitochondrial free radical leak.
It is important to recognize that treatment by ACE inhibition has been shown to have a number of beneficial effects upon the cardiovascular system. These effects include reduction in mean arterial pressure, preload, and afterload which contribute toward preservation of cardiac contractility 20. The systolic improvements observed in animals treated with enalapril in this study are almost certainly due in part to the beneficial effects of enalapril upon the hemodynamic aspects of cardiac filling and output. In this study, we now provide evidence for an additional mechanism of ACE inhibitors to ameliorate doxorubicin cardiotoxicity via the preservation of mitochondrial function and downregulation of free radical generation. Rats treated with both enalapril and doxorubicin were observed to have higher levels of respiratory efficiency, minimal depletion of cytosolic ATP, and less oxidative stress as compared to animals receiving doxorubicin alone. Enalapril was able to attenuate the harmful effects of doxorubicin to near control levels in each of these respects, and prevented deterioration in cardiac function and histopathologic grading.
Previous research has suggested that at the subcellular level, mitochondria may be a primary target of doxorubicin-induced cardiotoxicity. Specifically, it has been shown that doxorubicin-treated hearts exhibit mitochondrial abnormalities, including a decrease in respiratory efficiency, inhibition of electron transport complexes, and disruption of calcium homeostasis 21–23. In accordance with previous findings, we found that doxorubicin caused a robust decrease in state 3 respiration, which resulted in an overall robust decrease in the mitochondrial respiratory efficiency. The important observation here is that enalapril prevented the decrease in state 4 and, more importantly, in state 3 oxygen consumption and RCR, thereby maintaining mitochondrial bioenergetics at normal levels. Similarly, we found that cellular ATP levels were significantly lowered following doxorubicin treatment, but that enalapril was able to maintain cellular ATP levels at near control values.
We did not detect any differences among groups in the amount of basal H2O2 produced and released by isolated mitochondria respiring with either P/M or succinate. However, these observations in combination with lower RCR and respiration rates of mitochondria isolated from Dox animals reinforced the hypothesis that doxorubicin induces ETC dysfunction (defined as the loss of ETC complex enzymatic activity and/or complex content). As support for these ideas, although basal H2O2 production was not different, the calculated FRL% differed significantly among groups. Specifically, doxorubicin induced a significant rise in FRL%, whereas enalapril completely abolished this effect, maintaining the FRL% at control values. These results demonstrate that enalapril is capable of attenuating the fraction of electrons flowing out of sequence in the ETC, which ultimately reduce oxygen to form free radicals. In addition to causing cardiomyocyte dysfunction, generation of O2− has also been linked to dysfunction of the coronary vasculature through inactivation of NO produced by endothelial cells and formation of ONOO-. Reduction in the bioavailability of NO is significant to the development of heart failure as the anti-inflammatory and vasodilation properties of NO are critical to maintaining cardiac function 24, 25.
In summary, this is the first study to assess the effects of ACE inhibitors on mitochondrial bioenergetics and recovery following chronic doxorubicin treatment. By maintaining the efficiency of mitochondrial respiration and cellular ATP content to near control levels, our results show that concurrent treatment with enalapril improves cardiac function in rats exposed to doxorubicin. Furthermore, the lowered potential of these mitochondria to produce ROS may contribute to the attenuation of dysfunction. Moreover, as has been previously suggested 15, we also found that cardiomyocyte apoptosis is only one contributing factor to the decreased cardiac function during doxorubicin cardiotoxicity. Lastly, these outcomes illustrate the importance of maintaining intact mitochondrial bioenergetics as a defense mechanism against doxorubicin cardiotoxicity, even in the case when part of the organ is lost or atrophied. Our findings in conjunction with recent clinical reports that early treatment of enalapril following doxorubicin administration prevents development of heart failure 8, 9, support a priori treatment of patients undergoing anthracyclin chemotherapy with ACEI rather than after heart failure has been diagnosed.
This work was supported in part by grants from the BWF CAMS, CA BCRP 14IB-0039 (JCW), NIH HL 095571 (JCW), and HHMI Fellowship (ASL)
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