Elsevier

Biochemical Pharmacology

Volume 91, Issue 3, 1 October 2014, Pages 323-336
Biochemical Pharmacology

Trimetazidine prevents palmitate-induced mitochondrial fission and dysfunction in cultured cardiomyocytes

https://doi.org/10.1016/j.bcp.2014.07.022Get rights and content

Abstract

Metabolic and cardiovascular disease patients have increased plasma levels of lipids and, specifically, of palmitate, which can be toxic for several tissues. Trimetazidine (TMZ), a partial inhibitor of lipid oxidation, has been proposed as a metabolic modulator for several cardiovascular pathologies. However, its mechanism of action is controversial. Given the fact that TMZ is able to alter mitochondrial metabolism, we evaluated the protective role of TMZ on mitochondrial morphology and function in an in vitro model of lipotoxicity induced by palmitate. We treated cultured rat cardiomyocytes with BSA-conjugated palmitate (25 nM free), TMZ (0.1–100 μM), or a combination of both. We evaluated mitochondrial morphology and lipid accumulation by confocal fluorescence microscopy, parameters of mitochondrial metabolism (mitochondrial membrane potential, oxygen consumption rate [OCR], and ATP levels), and ceramide production by mass spectrometry and indirect immunofluorescence. Palmitate promoted mitochondrial fission evidenced by a decrease in mitochondrial volume (50%) and an increase in the number of mitochondria per cell (80%), whereas TMZ increased mitochondrial volume (39%), and decreased mitochondrial number (56%), suggesting mitochondrial fusion. Palmitate also decreased mitochondrial metabolism (ATP levels and OCR), while TMZ potentiated all the metabolic parameters assessed. Moreover, pretreatment with TMZ protected the cardiomyocytes from palmitate-induced mitochondrial fission and dysfunction. TMZ also increased lipid accumulation in cardiomyocytes, and prevented palmitate-induced ceramide production. Our data show that TMZ protects cardiomyocytes by changing intracellular lipid management. Thus, the beneficial effects of TMZ on patients with different cardiovascular pathologies can be related to modulation of the mitochondrial morphology and function.

Introduction

Metabolic syndrome and obesity are characterized by imbalanced caloric intake and expenditure, increasing the risk of diabetes and cardiovascular diseases, including heart failure [1]. Obese, diabetic and heart failure patients have increased plasma free fatty acids [2], which can elicit toxic effects (lipotoxicity) leading to cell death in several models, including cardiomyocytes [3]. Although the specific mechanism for lipotoxicity is not fully understood, sphingolipids may play a role because palmitate, the most abundant saturated free fatty acid, is the obligate substrate for initiating de novo ceramide synthesis [4]. We have shown that ceramides induce mitochondrial fission and cell death [5] by altering mitochondrial function and Ca2+ buffering capacity [6] in cultured cardiomyocytes. Interestingly, chemical inhibition of ceramide synthesis protects adult cardiomyocytes from palmitate-induced apoptosis [7].

Mitochondrial dynamics control mitochondrial shape and function by coordinating the opposing processes of mitochondrial fusion and fission [8], [9]. Recent evidence suggests a role for mitochondrial dynamics in lipotoxicity [10], [11]. In pancreatic β-cells, a complex mixture of free fatty acids (including palmitate) caused apoptosis [10], and in skeletal muscle palmitate induced mitochondrial dysfunction and insulin resistance [11]. Interestingly, in both studies the prevention of mitochondrial fission protected the cells from damage. In cardiomyocytes, lipotoxicity has been partially addressed but the role of mitochondrial dynamics in this process is unknown.

The heart uses different metabolic substrates to fuel the contractile machinery, and the importance of this energetic balance in the myocardium becomes particularly evident during heart failure [2]. Trimetazidine (TMZ), an antianginal drug used in Europe and Asia, has been proposed as a metabolic modulator for heart failure treatment. TMZ decreases free fatty acid-oxidation probably by partial inhibition of the β-oxidation enzyme long chain 3-ketoacyl-CoA thiolase (3-CAT) [12], and carnitine-palmitoyl-transferase 1 (CPT1) [13], thereby increasing glucose utilization for ATP production [14]. At the mitochondrial level, TMZ decreases the activity of complex I of the electron transport chain during ischemia [15], decreasing oxygen consumption, production of reactive oxygen species (ROS) [16] and preserving mitochondrial integrity [17], allowing conservation of mitochondrial membrane potential (Ψmt) [18], thus preventing the opening of the mitochondrial permeability transition pore [19] and apoptosis [20].

Lipotoxicity is linked to mitochondrial dysfunction and ROS production [21], sharing some of the features of ischemic damage. Because diabetes, heart failure, and other cardiac pathologies are associated with increased plasma free fatty acids and cardiac lipotoxicity [1], we sought to evaluate the protective effect of TMZ on palmitate lipotoxicity using a cultured cardiomyocyte model system. We evaluated changes in mitochondrial morphology, metabolism, and ceramide production. To our knowledge, this corresponds to the first report of protective effects of TMZ on palmitate-induced lipotoxicity in cardiomyocytes, thus expanding our understanding of its mechanism of action [22].

Section snippets

Materials

Antibodies against DRP-1 and HSP70 were from BD Biosciences and Affinity BioReagents, respectively. Antibodies for MFN-2 and OPA-1 were from Abcam. Antibodies against C16-ceramide and FIS-1 were from Enzo Life Sciences. Tetramethylrhodamine-methyl-ester (TMRM), mitotracker Green-FM (MTG), HCS-LipidTOX Green, Hoechst 33342, the antibodies Alexa-543 conjugated anti-mouse IgG and Alexa-488 conjugated anti-rabbit IgG, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester

Palmitate induces mitochondrial fission in cardiomyocytes

Several reports have shown toxic effects of palmitate, ranging from mitochondrial fragmentation and dysfunction [11] to cell death [3], [7]. However, the underlying mechanism is not fully understood, and as such, we sought to evaluate whether palmitate alters mitochondrial morphology and function in our conditions. Fig. 1A shows mitochondrial fission caused by palmitate, evidenced by a significant increase in the percentage of cells with fragmented appearance and the number of mitochondria per

Discussion

The present study shows that palmitate induced mitochondrial fission and dysfunction in neonatal cardiomyocytes, which can be prevented by the β-oxidation inhibitor TMZ. Our data suggest that TMZ induces the accumulation of lipids into lipid droplets, thus sequestering the excess of palmitate in a metabolically neutral reservoir, hindering the production of ceramides. These results may expand our knowledge and comprehension of the action mechanism of TMZ, a matter that is still controversial.

Funding

This work was supported by “Comisión Nacional de Investigación Científica y Tecnológica (CONICYT)” grants ANILLO 1111 [to S.L., M.C. and P.C.], FONDAP 15130011 [to S.L., M.C., H.E.V., L.G. and P.F.C.] and FONDECYT [1120212 to S.L and 1090727 to P.C.]. Conicyt scholarship 21130200 (CLC) holds a doctoral scholarship from CONICYT-CHILE. V.P. thanks Conicyt scholarship 74120010 (VP) and the AHA fellowship 13POST16520009 for her postdoctoral funding. DAB is supported by NIH DK084669 and the

Conflicts of interests

None declared.

Acknowledgments

We thank Fidel Albornoz and Gindra Latorre for their excellent technical assistance.

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