Edited by: P. Bryant Chase, Florida State University, United States
Reviewed by: Margaret Westfall, University of Michigan, United States; Lewis J. Watson, University of Pikeville, United States
*Correspondence: Thiago M. Fidale
This article was submitted to Striated Muscle Physiology, a section of the journal Frontiers in Physiology
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Cardiotoxicity is one of the most significant adverse effects of the oncologic treatment with doxorubicin, which is responsible for a substantial morbid and mortality. The occurrence of heart failure with ventricular dysfunction may lead to severe cardiomyopathy and ultimately to death. Studies have focused on the effects of leucine supplementation as a strategy to minimize or revert the clinical condition of induced proteolysis by several clinical onsets. However, the impact of leucine supplementation in heart failure induced by doxorubicin is unknown. Therefore, the objective of this work is to evaluate the effects of leucine supplementation on the cardiotoxicity in the heart of rats treated with doxorubicin. Rats treated with a 7.5 mg/kg cumulative dose of doxorubicin for 14 days presented a dilatation of the left ventricle (LV), and a reduction of the ejection fraction (FE). The 5% supplementation of leucine in the rats' food prevented the malfunctioning of the LV when administered with doxorubicin. Some alterations in the extracellular matrix remodeling were confirmed by the increase of collagen fibers in the doxorubicin group, which did not increase when the treatment was associated with leucine supplementation. Leucine attenuates heart failure in this experimental model with doxorubicin. Such protection is followed by the maintenance of interstitial collagen fibers.
Doxorubicin is an antineoplastic agent from the group of anthracyclines, commonly used in a wide range of neoplasms treatment (Adão et al.,
Efforts to reduce cardiotoxicity include alterations in the dexamethasone molecule and/or reduction of the therapeutic dose exposure. Alternatively, myocardial uptake of dexamethasone was reduced after the introduction of liposome encapsulation and the simultaneous administration of the iron chelator, dexrazoxane was employed to minimize reactive oxygen formation (Batist,
The regulation of the metabolism, hypertrophy and cell apoptosis in heart failure is complex, but evidence suggests that these processes can be modulated by vital macronutrients in the diet, including amino acids (Layman,
Many of the underlying mechanisms of LV dysfunction and induced by doxorubicin, are shared by other cardiac insufficiency forms. Studies using rats show that leucine reduced arrhythmia and heart failure in experimental myocardial ischemia, and can serve as an alternative energetic substrate, by providing metabolic intermediaries directly to the tricarboxylic acid (Marazzi et al.,
Thirty-six Wistar male rats, 2–3 months old, and weighing 260 ± 14 g were used (Supplementary Table
Diets were offered for an adaptation period of 14 days before the doxorubicin treatment. During the 14 days of treatment, and for a 14-days period after the last injection of doxorubicin, adding up to 42 days of diet. Composition of the standard diet (SD) included a minimum concentration of 1.54 g/100 g (1.5%) leucine, according to the American Institute of Nutrition (AIN-93G) (Reeves et al.,
For the treatment, 10 rats of the DOXO group received, during 2 weeks and three times a week, intraperitoneal injections of doxorubicin hydrochloride reaching the 7.5 mg/Kg cumulative dose, as proposed in previous study (Campos et al.,
Each heart was divided into four pieces (base, middle proximal, distal middle and apex) by making three transverse cuts. These pieces were fixed in a 10% formalin solution for 48 h. Material processing followed the stages of dehydration, diaphanization, bath and embedding in paraffin. Next, the middle proximal segment was sectioned (5 um) and then stained for collagen using Picrosirius Red. The histological cuts stained in Sirius red were analyzed in the optical microscope
After obtaining the photomicrographs, the quantification of the total collagen area, given in percentage, was obtained with the aid of the threshold tool of the software ImageJ1.6.0_24. This tool allows limiting the percentage of collagen area deposited in the extracellular matrix in relation to the total area of the evaluated field.
The experimental results were expressed as mean ± standard deviation and the normality of the data was tested using the Shapiro-Wilk test. For the normal data, the comparisons among groups was done through analysis of variance (ANOVA), and after that, a Tukey's test or Newman-Keuls Multiple Comparison Test was performed, just in case. Statistical analysis was done with a GraphPad statistical package Prism (5.0 version). Statistical significance was established when the value of
No deaths occurred in any of the groups during the experiment. The animals treated with doxorubicin showed less movement and developed periocular and nasal exudation, as well as reddened lesions in the legs similar to drug dermatitis.
Firstly, the protector role of the leucine induced cardiotoxicity by doxorubicin in rats was tested. In order to achieve this purpose, 2 groups of 10 rats were injected with the 10-mg/kg cumulative doses during 2 weeks, one of the groups also received in its diet a 5% leucine supplementation. Fourteen days past the final treatment, DOXO group presented a higher diameter of the left ventricle at the end of systole (LVEDS) 4.61 ± 0.1 mm, and at the end of diastole (LVEDD) 6.72 ± 0.3 mm, when compared to Control group (LVEDS) 3.39 ± 0.7 mm (
Leucine prevents ventricular dilation and reduces the ejection fraction produced by doxorubicin. Echocardiographic images with dilation and reduction of LV ejection fraction induced by doxorubicin and protected leucine products in the leucine+DOXO group. *
Changes in the extracellular matrix remodeling were confirmed by a greater quantity of collagen fibers in the DOXO group 4.55 ± 2.6 mg/mg, when compared to the control group 2.85 ± 1.3 mg/mg (
Leucine preserves cardiac structure and increase of collagen fibers produced by doxorubicin. Samples of histopathological images with increased amount of collagen fibers in the extracellular matrix induced by doxorubicin and the protective effects of leucine in the leucine+DOXO group. *
Doxorubicin is used in antineoplasic therapies, yet it can cause left ventricular dysfunction and heart failure (Raschi et al.,
Cardiotoxicity related to doxorubicin is a multifactorial process, sustained by energy collapse, disturbance of homeostasis and suppression of specific sarcomere cardiac genes, apoptosis of cardiomyocytes followed by remodeling and dilated cardiomyopathy (Minotti et al.,
The results of the current study are in agreement with results of previous studies (Campos et al.,
Rats treated with different cumulative doses of doxorubicin, administered over 8 weeks, totaling 8, 12, and 16 mg/kg had a higher area of fibrosis in the animals' heart when compared to the animals in the control group (Oliveira et al.,
An earlier study (Ito et al.,
Previous studies have pointed out to cardiac protection with leucine as a therapy strategy in different models of systolic dysfunction, suggesting that leucine acts as a nutritional sign to stimulate protein synthesis in cardiac muscle, increasing the availability of the eukaryotic initiation factor (eIF4B) and activating via p70S6K and mTOR cardiac, a fact that can have attenuated damages observed in the doxorubicin group (Escobar et al.,
Leucine may protect against cardiotoxicity by preventing the loss of post-translational efficiency and/or developmental autophagy. Both of the efficiency and onset of autophagy are postulated to develop in response to doxorubicin, and are putative contributors to the cardiotoxicity.
Our study has some limitations. Doxorubicin is administered in patients with cancer, while our study investigates experimental cardiotoxicity in rats that did not have this disease. This study design may have influenced our results, despite the focus of our work being the specific identification of the effect of leucine in heart failure and quantity of collagen fiber induced by doxorubicin. Leucine may not adequately protect against cardiotoxicity when doxorubicin is administered at levels that produce cancer-related lethality, moreover, the present study did not monitor the consumption of food and water. Another aspect is that this study did not aim at identifying the possible pathways of doxorubicin in which leucine may have acted and improved the animals' cardiac conditions. In this sense, new studies should be conducted, in order to map protein synthesis pathways and cellular death, aiming at identifying possible interventions of leucine in cardiac cells.
The design of the study may have influenced our results, although the focus of our work was the specific identification of the leucine effect on heart failure and the amount of collagen fiber induced by doxorubicin. Another aspect is that this study did not aim to identify the possible pathways of action of doxorubicin in which leucine may have acted and improved the cardiac conditions of the animals. In this sense, new studies must be carried out in order to map pathways of protein synthesis and cell death, in order to identify possible leucine interventions in cardiac cells.
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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