Sex-specific cardiac cardiolipin remodelling after doxorubicin treatment
© Moulin et al. 2015
Received: 14 July 2015
Accepted: 6 October 2015
Published: 15 October 2015
Imbalance in lipid metabolism and membrane lipid homeostasis has been observed in numerous diseases including heart failure and cardiotoxicity. Growing evidence links phospholipid alterations especially cardiolipins (CLs) to defects in mitochondrial function and energy metabolism in heart failure. We have shown recently that doxorubicin cardiotoxicity is more severe in male than female Wistar rats. We aimed to study whether this sex specificity is linked to differences in cardiac phospholipid profiles.
Adult male and female rats were injected 2 mg/kg doxorubicin weekly for 7 weeks. Cardiac phospholipid molecular species were determined by liquid chromatography coupled with mass spectrometry fragmentation (LC)/MSn. Sex difference in phosphatidylethanolamine and phosphatidylcholine species containing docosahexaenoic and docosapentaenoic acyl chains was observed, females having more than males. In both sexes, doxorubicin induced an important loss of the main CL(18:2)4, while the level of monolysocardiolipin MLCL(18:2)3 remained stable. However, a severe remodelling appeared in treated rats with the longest CL acyl chains in doxorubicin-treated females, which might compensate for the loss of tetra-linoleoyl CL. The level of oxidized cardiolipin was not particularly increased after doxorubicin treatment. Finally, expression of genes involved in the biosynthesis of fatty acid appeared to be decreased in doxorubicin-treated males.
These results emphasize for the first time the cardiac remodelling in the phospholipid classes after doxorubicin treatment. These observations suggest that doxorubicin has a sex-specific impact on the heart phospholipidome especially on cardiolipin, an essential mitochondrial lipid. Further studies are needed to better understand the roles of lipids in the anthracycline cardiotoxicity and sex differences, but phospholipid cardioprotection seems a valuable new additive therapeutic strategy for anthracycline cardiotoxicity.
KeywordsCardiolipin Sex-specific Heart failure Doxorubicin LC/MSn
Doxorubicin (DOXO), a widespread anti-cancer agent, has clinical limited use due to important deleterious cardiac side effects that ultimately lead to heart failure (HF). How anthracyclines induce both anti-cancer activity and cardiotoxicity are not completely understood . Numerous studies have shown the interaction of doxorubicin with membranes, particularly cardiolipin (CL), causing membrane fluidity alteration and enzyme activity deficiency [2–4]. We have recently observed that after 7 weeks of doxorubicin treatment, male rats but not females had high mortality rate, signs of heart failure, and altered mitochondrial function, and we identified a significant decrease in total cardiolipin content specifically in the heart of treated males .
Phospholipids (PLs), a fundamental class of biological compounds, are the main component of membranes which represents a biological and physical barrier but also a site for interaction and signal transduction. Fatty acid (FA) composition of PL influences membrane characteristics such as membrane fluidity, microdomain formation, transport system, and membrane-bound enzyme activities (for review, see ). Obviously, deregulation of PL is involved in numerous diseases such as cancer, neurological disorders, hepatic disorders, and cardiovascular diseases (reviewed in [7, 8]).
Cardiolipin, a signature PL of the inner membrane of the mitochondria (around 20 % of the lipid composition), is involved in numerous functional and structural features of membrane-bound mitochondrial proteins and is linked to important mitochondrial processes, including apoptosis, mitochondrial dynamics, contact sites formation and assembly, and function of mitochondrial membrane proteins for an effective oxidative phosphorylation (reviewed in [9–12]). CL is a unique dimeric phospholipid containing two phosphatidyl moieties bridged by a glycerol and four fatty acyl chains. The FA composition is also highly specific and is predominantly composed of 18:2 acyl chains with tetra-linoleoyl cardiolipin (L4CL) being the most abundant . CL composition depends on de novo synthesis of nascent CL followed by complex processes of acyl chain remodelling. The fatty acyl chain composition of CL is critical for mitochondrial function and cardiac pathology. Alteration in CL content and composition has been associated with numerous disorders like diabetes, thyroid status, aging, and heart failure .
HF is characterized by the inability of the heart to fulfill the metabolic requirements of the organism. Among the characteristics of HF, the state of energy depletion [15, 16], alterations of the mitochondrial function, and biogenesis  have been well described, but changes in cardiac PL composition are less addressed. However, cardiolipin may play a role in mitochondrial alterations in heart failure . Indeed, decreased cardiolipin content, altered cardiolipin synthesis and remodelling , reduction in 18:2 CL species [10, 18, 19], and modified expression or activity of cardiac biosynthesis enzymes  have been reported in numerous diseases. The importance of CL in heart failure is exemplified by the Barth syndrome, a severe X-linked genetic disorder which is characterized by CL alterations, mitochondrial defects, and heart failure and which results from mutations in the Tafazzin gene, an acyltransferase involved in CL remodelling . Finally, CLs are susceptible to oxidative damages because they are present in the mitochondria which are the main source of reactive oxygen species (ROS) and because they contain double bonds that are susceptible to oxidation. CL oxidation has been shown to induce decreased activity and organisation of respiratory chain complexes and to favor apoptosis (for review, see ).
As we have observed, a decrease in total cardiolipin content in treated males but no females; the objective of the present study was thus to explore in more detail the cardiac phospholipidome after doxorubicin treatment in adult rats and to understand whether CL remodelling participates in the sex-specific cardiac alterations.
An expanded method section is available in the Additional file 1.
Doxorubicin animal model
A chronic model of doxorubicin  by weekly intravenous injection of 2 mg/kg doxorubicin or saline solution during 7 weeks in 11-week-old male and female Wistar rats (Janvier Labs) was chosen as previously described . Animal experimental procedures were approved by the Animal Ethics Committee of Paris Sud University. Investigations were done in accordance with European Community legislation relating to the care and use of animals (Directive 2010/63/EU) and the corresponding French legislation (French decree 2013-118 du 1er février 2013).
A Folch method  was used to extract the lipids from 10 to 20 mg of heart homogenized in phosphate-buffered saline (PBS). Each group was composed of four animals; one technical repeat was done for a non-treated male and a doxorubicin-treated male. Total lipids were extracted by adding 1.5 mL of methanol and 3 mL of chloroform to the tissue suspension. After centrifugation at 1000g for 10 min, the lower phase containing total lipids was collected and evaporated to dryness at room temperature under nitrogen gas. The samples were resuspended in 100 μL of chloroform per 10 mg of heart and subsequently analyzed.
Cardiolipins were oxidized following the method of Paradies . Briefly, cardiolipins in PBS were mixed in 1 mL of oxygenated buffer containing 20 μM ferrous sulfate, 120 μM ADP, and 200 μM ascorbic acid for 15 min at 37 °C before being extracted by a Folch method.
For the phospholipid analysis, each sample was duplicated, except the technical repeat for a non-treated male. Chromatographic system from Thermo Fisher Scientific included a Dionex U 3000 RSLC system with two quaternary pumps, an autosampler, and a column oven. The RSLC system was coupled online to a charged aerosol detector Corona CAD Ultra for the quantitative part of the study and a LTQ Orbitrap Velos Pro for the lipidomic study (all from Thermo Fisher).
Chromatographic program with the following mobile phase compositions: mixture of heptane and isopropanol 98:2 (v/v) (phase A); mixture of chloroforme and isopropanol 65:35 (v/v) (phase B); mixture of methanol and water 95:5 (v/v) (phase C); isopropanol (phase D)
The LC effluent was split after the analytical column with aid of a tee; thus, 250 μL/min entered to the Corona CAD and 150 μL/min entered the mass spectrometer. To increase the flow entering the Corona CAD and maintain good aerosol stability, absolute ethanol was added with a second mixing tee with a flow of 200 μL/min.
The LTQ Orbitrap Velos Pro is equipped with an H-ESI II probe. Spray voltage was set at 3.3 kV. Heater temperature of the probe was set at 200 °C. Sheath gas, auxiliary gas, and sweep gas flow rates were set at 20, 8, and 0 (arbitrary unit), respectively. Capillary temperature was set at 325 °C and S-lens RF level at 60 %. Analysis was performed in negative mode to obtain structural nformation on phospholipids fatty chains. This mass spectrometer is equipped with two analyzers: a double linear ion trap (LTQ) for fragmentation at low resolution and an orbital trap for high-resolution detection. The detection was performed either in full mass spectrometry (MS) scan with 100,000 resolution and data dependant MS2 and MS3 with collision-induced dissociation in the CID fragmentation (collision energy set at 35).
Phospholipid species identification
The chromatographic method used separates the phospholipids by polar head. Retention time is a crucial information for polar head identification and compared to commercial standards. Then, species can be observed under each chromatographic peak. PL identification was performed as described  using high-resolution mass detection in full scan mode and MS2/MS3 fragmentation in data dependant mode. MSn fragmentation has not always been performed by the instrument due to very small amount of some ions. When no MSn data is available, PLs are mentioned as, e.g., PL(34:1). Otherwise, PL are mentioned as, e.g., PL(16:0-18:1). Sn1 and sn2 position of the acyl chains were not determined. Oxidized cardiolipin identification was performed as described by Kim et al. [28, 29].
Phospholipid species representation
Total phospholipid amount was determined with Corona CAD detection by calculating the concentration of each PL class (in micrograms per milliliter) compared to calibration ranges of commercial standards. PL species intensities were determined by taking the mean relative intensity of m/z ions over the chromatographic peak of each PL class.
2D separation of CL
The sample’s cardiolipin was collected from 11 to 12 min, dried over nitrogen, and dissolved in 2D phase mobile (2-propanol:water:triethylamine:acetic acid 45:5:0.25:0.25). A Dionex C18 column (Acclaim RSLC 120, 2.2 μm, 120 Å, 2.1 × 150 mm) was used under isocratic solvent condition. The column temperature was thermostatically controlled at 40 °C. Flow was maintained at 200 μL/min on a Dionex U 3000 RSLC system, and MS and MS/MS acquisitions via LTQ Orbitrap Velos Pro were performed on the most abundant mass ions.
MS raw data were submitted to chromatogram builder, alignment and gap filling using the MzMine 2.10 LC/MS toolbox . SIMCA-P 12.0 was used to analyze the data generated by mass spectrometry (principal component analysis (PCA) and OPLS-DA). Data are expressed as mean ± SEM. Two-way ANOVA (homogenous variance verified by Levene test for all parameters except Cds1 and Acsl1) was used to study the global effect of sex and treatment, followed by Student-Newman-Keuls post hoc test to determine individual differences. Statistical significance for doxorubicin effect was defined as * for p < 0.05, ** for p < 0.01, and *** for p < 0.001 and for sex effect as § for p < 0.05, §§ for p < 0.01, and §§§ for p < 0.001.
Doxorubicin treatment affects cardiac phospholipid content differently between males and females
Sex-specific cardiolipin remodelling after doxorubicin treatment
Low level of oxidized cardiolipin after 7 weeks of doxorubicin treatment in males
Cardiolipin biosynthesis is altered in males and females treated with doxorubicin
In support of our previous work showing male-specific doxorubicin cardiotoxicity , this study describes for the first time the cardiac phospholipidome after doxorubicin treatment in both sexes. The role of phospholipids in the heart and the sex impact has been poorly explored, despite essential structure and signal transduction functions. After chronic doxorubicin treatment in male and female adult rats, we characterized doxorubicin and sex-specific phospholipid remodelling as best discriminating component by PCA.
From the 1980s, alterations of heart phospholipid content have been described in many cardiovascular diseases. Decrease in linoleic acid and increases in arachidonic and docosahexaenoic acid have been observed in myocardial phospholipids of rats and cats after TAC  and chronic coronary artery ligation . A link has been made between alteration in PL and the severity of heart failure. PE, PC, PI, and PS, the main classes of PL, are present in different membranes of cardiomyocytes (sarcolemma, RS, mitochondria). These phospholipids are involved in various signal transduction pathways; they can be split by phospholipases into inositol triphosphate and diacylglycerol that function as second messengers. As we used heart tissue, we cannot discriminate the sarcolemma PL from the intracellular membrane organelles PL. Additional investigations are needed to determine the exact composition of the different membrane organelles. Thus, we will focus the discussion on CL because there are specific to the inner mitochondrial membrane and appears critical for mitochondrial function and cardiac pathology .
Several studies have highlighted the essential role of the mitochondria in heart failure and in anthracycline cardiotoxicity [5, 35–37]. The energetic depletion linked to mitochondrial defect is often related to a reduction in mitochondrial mass. However, when maximal oxygen consumption decreased and activities of key enzymes of energy metabolism (e.g., CS, CK, and COX) remained stable with no change in protein level of the peroxisome proliferator-activated receptor gamma co-activator 1 (PGC-1), the orchestrator for mitochondrial biogenesis, mitochondrial energetic deficiency has been suggested to be linked to a disruption of local environment [5, 38]. One key parameter for optimal and functional mitochondria is cardiolipin. Not surprisingly, CL alterations such as content, distribution, oxidation, and acyl chain composition have been reported in many diseases including Barth syndrome, Parkinson disease, and heart failure . As previously described , the main molecular species of cardiolipin is the L4CL (18:2)4, representing around 55 % of total CL in male and female rat hearts. This level was reduced to 35 % in both sexes after doxorubicin treatment. Decrease of linoleic acid in cardiolipin has also been observed in ischemic, dilated, diabetic cardiomyopathies, and in senescent heart [20, 39, 40]. Moreover, most of the time, increase in 22:6 docosahexaenoic acid was noticed [20, 39, 40]. Hearts from doxorubicin-treated males presented an increase in CL containing 22:6 acyl chain, and this was highly exacerbated for the females. Moreover, some CL species appeared in DOXO-treated animals that were not present in controls also with a significantly higher content in females, indicating the occurrence of an alternate form of CL remodelling after DOXO treatment. Such a cardiac long acyl chain PL remodelling has been reported in heart failure and aging [32, 41]. One important point is that all these studies showing acyl chain CL remodelling were obtained in male animals/patients, and no comparison was made with females. This remodelling in males might be a compensatory mechanism but still insufficient to correct CL alterations. Because the total CL level was not reduced in DOXO-treated females and because mitochondrial function was not altered , it can be inferred that the significant increase in longer acyl chain (20:2, 20:3, 20:4, 22:5, and 22:6) could have compensated for the loss of (18:2)4 CL thus preserving the local environment for normal mitochondrial function. Indeed, calcium retention, cytochrome c sequestration, membrane fusion and fission, respiratory chain, and apoptosis are all dependent on CL content [9–12].
Because one of the controversial molecular mechanisms of anthracycline-induced cardiotoxicity is oxidative stress [42–44], we used a recently developed two-dimensional LC-MS/MS [28, 29] to analyze oxidized CL. Various conditions can cause cardiolipin oxidation, among them cytochrome c/H2O2 and oxygenated Fe2+ buffer (hydroxyl radical generated by the Fenton and the Haber-Weiss) reactions [28, 31, 45]. As doxorubicin leads to accumulation of iron in the mitochondria , we used ferrous sulfate buffer for CLox-positive control. We did not observe a high level of CLox in doxorubicin-treated males. Indeed, around 0.05 % of (18:2)4 containing 4 to 7 oxygens was detected. This result is in accordance with our previous data showing no obvious global protein oxidation . Obviously, oxidative stress is species-, time-, and drug concentration-dependent, in other words model-dependent [42–44, 46–48]. It seems that while high level of CLox has been observed in acute stress, level of CLox could be moderate in chronic stress. After traumatic brain injury in postnatal day 17 rats, the content of oxidized CL species increased 20-fold at the expense of non-oxidized species . Chronic western diet of LDLR−/− (low density lipoprotein receptor knockout) mice during 16 weeks induced a 0.1 % significant increase of oxidized cardiolipin in the liver mitochondria . Therefore, we can hypothesize that either chronic doxorubicin treatment with moderate doses induces only low levels of CLox or that CLox may be produced early after injection but rapidly discarded to avoid mitochondrial dysfunction.
In DOXO-treated males but not females, we observed an early decrease in PGC-1β . Recently, PGC-1α/β, key transcriptional regulators of mitochondrial metabolism, has been implicated in the regulation of cardiac phospholipid biosynthesis . Despite grossly normal cardiac function of PGC-1α/β heart−/−, mitochondrial ultrastructure abnormalities were observed . PGC-1α/β hearts−/− were characterized by normal level of MLCL but reduced levels of most PE, PC, and CL and a decreased level of CDP-diacylglycerol synthase Cds-1 showing that Cds-1 was a direct target of PGC-1. We observed that Cds-1 was highly downregulated after doxorubicin in both sexes and even in basal level for females. In PGC-1α/β heart −/−, a reduction in PL-containing 22:5 and 22:6 acyl chains was observed  whereas in our model an increase was noticed. Thus, PL remodelling in DOXO cardiotoxicity can hardly be explained by PGC-1 downregulation.
It has been proposed that remodelling of CL can be linked to poly-unsaturated fatty acid metabolism . An interesting feature of female cardiac PLs is the presence of species with long acyl chains not only in CL but in other PL classes. Elevated long-chain omega 3 fatty acids in females under the same diet than males have been reported in numerous studies . This sex difference has been linked in part to estrogens, FA remodelling enzymes, and peroxisome proliferator-activated receptor alpha (PPARα) activity. In the liver, but not the heart, gene expression of Δ6- and Δ5-desaturases and Elongases, enzymes involved in the production of long-chain poly-unsaturated FAs from 18:2, is greater in females . In our study, supply of essential fatty acid (α-linolenic acid 18:3 and linoleic acid 18:2) from diet was the same for males and females treated or not with doxorubicin. However, in males, the specific decrease of Elongase5 and the tendency to decrease for Δ6-desaturase were more suggestive of impaired rather than improved production of long-chain fatty acids from α-linolenic acid 18:3 and linoleic acid 18:2. Expression of these enzymes has been shown to be under the control of an estrogen-mediated PPARα activation. Interestingly, we have previously observed that PPARα was less downregulated in doxorubicin-treated females . Regarding FA pathway, the gene expression of Cd36, a regulator of the first step, i.e., uptake of FA across the plasma membrane, remained stable after DOXO in both sexes. However, Acsl1, the major acyl-CoA synthethase isoform in the heart that converts long-chain fatty acid into acyl-CoA thioesters plus AMP was also less downregulated in females. Interestingly, ACSL1 in the liver was described to play an important role in directing FA into pathways of phospholipid synthesis and away from cholesterolesterification and β-oxidation . After doxorubicin treatment, we have shown that multiple steps of β-oxidation (MCAD and CPT1) were downregulated similarly in both sexes . It appeared that FA pathway of PL synthesis rather than β-oxidation seems more preserved in females that have a better cardiac function (Additional file 1: Figure S3). In line with all these results, we have previously observed that females treated with doxorubicin have intensified liver hepatomegaly and elevated circulating level of triglycerides compared to males . Similar to our results, Julicher et al. also observed a more important hepatomegaly after doxorubicin treatment in females . We can hypothesize that a protective mechanism linked to lipid metabolism is activated only in females.
Finally, sex difference in cardiotoxicity of doxorubicin has not been extensively studied. However, some clinical and experimental studies have observed that females develop less unwanted cardiac side effects after doxorubicin treatment than males [5, 55–59]. The mechanism of female cardioprotection is far from being completely understood. Among the proposed pathways involved were higher oxidative defenses capacity in females , increased level of cardiac mast cells activity in males , and better mitochondrial metabolism and bioenergetic protection in females . Interestingly, interactions between doxorubicin and negatively charged phospholipid and more specifically cardiolipin have been demonstrated by several groups [2, 4]. Cardiolipins are essential for respiratory chain functioning, ATP generation, and energy transfer. The heart is a highly energy-consuming organ, and cardiac contraction is linearly related to mitochondrial respiration thus anthracycline-mediated cardiolipin alterations would be a determinant factor in mitochondrial alterations and cardiotoxicity [5, 37, 44, 60]. We have now shown that cardiolipin remodelling is probably important in the sexual dimorphism of doxorubicin-induced cardiotoxicity. Additional studies are necessary to better understand the roles of lipids in the anthracycline cardiotoxicity and sex differences.
In this study, we showed that (1) a decrease of global phospholipids was only observed in doxorubicin-treated males, (2) the level of PE and PC species containing long acyl chains was more important in females than in males with or without doxorubicin, (3) doxorubicin mediated a decline of PE and PC containing one linoleoyl acyl chain in both sexes, (4) significant loss of L4CL was comparable in treated males and females while (18:2)3 MLCL remained stable, (5) the level of most long-chain CL and MLCL species was more important specifically for doxorubicin-treated females, (6) CL oxidation did not appear to be a key parameter in this chronic treatment, and (7) sex differences were found in gene expression of FA biosynthesis after doxorubicin treatment. Altogether, the cardiac phospholipidomic analysis has highlighted an interesting sex-specific PL molecular species remodelling (Table 2).
This study has unraveled a significant sexual dimorphism in the cardiac phospholipid content after a chronic cardiotoxic stress. A cause-and-effect relationship between altered phospholipid content and sex differences in doxorubicin-induced cardiotoxicity remains to be established. Further studies are needed to decode the male-specific cardiotoxicity to be able to develop new therapeutic strategies.
tetra-linoleoyl cardiolipin (18:2)4
reactive oxygen species
We thank Valérie Domergue and Pauline Robert (Animal facility of UMS IPSIT) for their help with the animals and Claudine Deloménie (Transcriptomic platform of UMS IPSIT) for her expertise in molecular biology. We thank Philippe Mateo and Jean-Louis Paul for helpful discussion, Frédéric Joubert for critical reading, and Rodolphe Fischmeister for continuous support.
Our laboratory is a member of the Laboratory of Excellence LERMIT. This work is supported by a grant from the “Fondation pour la Recherche Médicale” (DPM2012-125,554), Université Paris-Sud (ERM), and CORDDIM and MALINF-DIM from Région Ile de France. MM is recipient of a fellowship from the “Fondation ARC.” RVC is an emeritus scientist at CNRS.
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