(R)-α-Lipoic acid treatment restores ceramide balance in aging rat cardiac mitochondria

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Abstract

Inflammation results in heightened mitochondrial ceramide levels, which cause electron transport chain dysfunction, elevates reactive oxygen species, and increases apoptosis. As mitochondria in aged hearts also display many of these characteristics, we hypothesized that mitochondrial decay stems partly from an age-related ceramidosis that heretofore has not been recognized for the heart. Intact mitochondria or their purified inner membranes (IMM) were isolated from young (4–6 mo) and old (26–28 mo) rats and analyzed for ceramides by LC–MS/MS. Results showed that ceramide levels increased by 32% with age and three ceramide isoforms, found primarily in the IMM (e.g. C16-, C18-, and C24:1-ceramide), caused this increase. The ceramidosis may stem from enhanced hydrolysis of sphingomyelin, as neutral sphingomyelinase (nSMase) activity doubled with age but with no attendant change in ceramidase activity. Because (R)-α-lipoic acid (LA) improves many parameters of cardiac mitochondrial decay in aging and lowers ceramide levels in vascular endothelial cells, we hypothesized that LA may limit cardiac ceramidosis and thereby improve mitochondrial function. Feeding LA [0.2%, w/w] to old rats for two weeks prior to mitochondrial isolation reversed the age-associated decline in glutathione levels and concomitantly improved Complex IV activity. This improvement was associated with lower nSMase activity and a remediation in mitochondrial ceramide levels. In summary, LA treatment lowers ceramide levels to that seen in young rat heart mitochondria and restores Complex IV activity which otherwise declines with age.

Introduction

Mitochondria from aged tissue undergo a systematic decline in overall function, which manifests in the heart as an increased rate of reactive oxygen species (ROS) formation, concomitant oxidative damage, and impaired electron transport [1], [2], [3], [4], [5], [6], [7], [8]. All of these factors limit the ability of mitochondria to meet cellular energy needs. Interestingly, these mitochondrial traits of aging are also evident, albeit more severely, in inflammatory pathologies [9], [10], [11]. As it is recognized that the aging heart is subjected to a low-grade chronic inflammation, it is reasonable to argue that inflammatory bio-factors may be involved in both the initiation and the progression of mitochondrial decay. One such bio-factor that appears to be a hallmark of pro-inflammatory conditions is ceramide, a pro-apoptotic and growth arrest sphingolipid [12], [13], [14], [15], [16], [17], [18], [19], [20], which increases ROS formation, oxidative stress, and altered energy metabolism upon its accumulation in membranes [21], [22].

Generally, acute inflammatory stimuli generate ceramide at the plasma membrane or endoplasmic reticulum by sphingomyelin hydrolysis or de novo synthesis, respectively [23], [24], [25], [26], [27], [28], [29]. Recent evidence shows that mitochondria may also be an important site of sphingolipid action [22], [30], [31], [32], [33]. Our laboratory recently showed that cardiac mitochondria normally contain a variety of sphingolipids, including sphingomyelin and ceramide [34]. Mitochondria from other organs also contain ceramide as well as neutral sphingomyelinase (nSMase), which hydrolyzes sphingomyelin to ceramide [35], [36], [37]. This suggests that mitochondria have the means to alter ceramide levels in response to pro-inflammatory stimuli. Moreover, in vitro experiments suggest that even small elevations of mitochondrial ceramide are able to adversely affect electron transport chain (ETC) activity, heighten ROS appearance, and also initiate mitochondrial-mediated apoptosis [21], [22], [31], [32], [33]. Thus, age-associated inflammation of the heart and mitochondrial decay may be connected via ceramidosis (i.e., the accumulation of ceramide). If so, this would provide a novel target for therapies to improve cardiac mitochondrial function and bioenergetics, which otherwise decline with age.

Despite this potential association, the role that ceramide plays in age-related mitochondrial decay has not been studied. Because many of the phenotypes of mitochondrial dysfunction can be plausibly linked to ceramidosis, the goal of the current study was to determine ceramide levels in interfibrillary mitochondria isolated from young and old rat hearts. Moreover, as mitochondria are double-membraned organelles, we further pursued the hypothesis that ceramide accumulation would be evident in the inner mitochondrial membrane (IMM) and adversely affect ETC activity. Lastly, if mitochondrial ceramides were indeed found to become elevated with age, a contingent goal was to determine whether anti-inflammatory agents could remediate any ceramide accumulation, thereby ameliorating the mitochondrial aging phenotype.

With regard to this latter contingent goal, our laboratory and others showed that the dithiol compound, (R)-α-lipoic acid (LA) may act as a potent anti-inflammatory and anti-oxidant agent at pharmacological doses [38], [39], [40], [41], [42]. Moreover, we recently reported that when old rats were treated with LA, age-associated increases in nSMase activity were limited and ceramide imbalance in aortic endothelia was remediated [43]. We have also previously shown that LA lowers indices of mitochondrial dysfunction [43], [44], [45], thus providing a rationale that LA may reverse at least certain aspects of mitochondrial decay by opposing ceramidosis.

Section snippets

Chemicals and antibodies

Digitonin, genistein, Subtilisin A (type VIII), Triton X-100, and Tween 20 were from Sigma–Aldrich (St. Louis, MO). Bovine serum albumin (fraction V, fatty acid free) was obtained from EMD Biosciences (La Jolla, CA). Purified ceramide standards were purchased from Avanti Polar Lipids (Alabaster, AL). NBD-sphingomyelin and NBD-ceramide were purchased from Life Technologies (Carlsbad, CA). Rabbit polyclonal antibody to the voltage-dependent anion channel protein (VDAC) and mouse monoclonal

Profile of cardiac mitochondrial sphingolipids

In keeping with our previous work [34], both intact cardiac mitochondria and purified IMM contained six ceramide isotypes with N-acyl-chain lengths varying from 16- to 24-carbon units (Fig. 1). The ceramides were predominantly saturated, with only one species, C24:1-ceramide, containing an unsaturated N-acyl side-chain. C24-ceramide was the predominant isoform found in cardiac mitochondria and comprised 38% of the total ceramide pool. Quantifying ceramides in the IMM showed that C16-, C18-, and

Discussion

To our knowledge, this is the first study showing that cardiac mitochondrial ceramides increase with age. Even though the accumulation was seemingly modest, nevertheless, it may be sufficient to adversely affect mitochondrial function. Tissue analysis following ischemia/reperfusion injury [55], myocardial infarct [56], [57], Type II diabetes [58], as well as in vitro experiments where isolated mitochondria were treated with ceramide-laden liposomes [21], [22], reinforce the view that perturbing

Conclusions

In conclusion, this paper shows that the age-related decay in cardiac mitochondria may stem from an accumulation of ceramide, a pro-apoptotic signaling lipid shown to induce mitochondrial dysfunction. Furthermore, we show that a two-week feeding of LA is able to restore ceramide levels to that seen in cardiac mitochondria from young animals. These results thus highlight the role of LA as an age-essential micronutrient, which may be an effective adjunct in preventing or reversing

Acknowledgements

The authors would like to thank Alan W. Taylor of the Oxidative and Nitrosative Stress Core Lab, Linus Pauling Institute, for expert analysis of mitochondrial ceramides. We would also like to thank Alix Gitelman, Ph.D., for help with the statistical analysis. The research was funded by the National Institute on Aging, grant number 2R01AG017141. We also acknowledge the facilities service cores of the NIEHS (NIEHS ES00240).

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