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
]. 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
]. 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 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
], which increases ROS formation, oxidative stress, and altered energy metabolism upon its accumulation in membranes [21
Generally, acute inflammatory stimuli generate ceramide at the plasma membrane or endoplasmic reticulum by sphingomyelin hydrolysis or de novo
synthesis, respectively [23
]. Recent evidence shows that mitochondria may also be an important site of sphingolipid action [22
]. 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
]. 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 is able to adversely affect electron transport chain (ETC) activity, heighten ROS appearance, and also initiate mitochondrial-mediated apoptosis [21
]. 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
]. 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
], thus providing a rationale that LA may reverse at least certain aspects of mitochondrial decay by opposing ceramidosis.