|Abstract or Summary
- Ceramides are a group of lipids in the sphingolipid family that are potent cell signaling molecules. Elevations in ceramide levels above the norm generally lead to apoptosis. Evidence from in vitro studies suggests that accumulation of ceramides within mitochondria leads to dysfunction of the mitochondria. This includes inhibition of the electron transport chain and release of reactive oxygen species (ROS). Large elevations of mitochondrial ceramide may also lead to death of the cell as they promote membrane permeability transition, which allows the release of cytochrome c and other apoptogenic factors. With age, cardiac mitochondria show a similar dysfunctional phenotype to that found in conditions of acute ceramide elevation. We therefore hypothesize that ceramides may be accumulating in cardiac mitochondria from aged animals.
To elucidate the characteristics of the sphingolipidome, we developed both mitochondrial isolation techniques and tandem mass spectrometry assays to specifically and sensitively monitor mitochondrial sphingolipids. Using these techniques, it was found that mitochondria contain six distinct ceramide species with highly saturated lipid moieties. Using young (3-6 months old; which corresponds to a post-adolescent human) and old (24 to 28 months old; which corresponds to an elderly human) Fischer 344 rat hearts, we found that aging leads to a significant increase in total mitochondrial ceramides (32%, p < 0.03), with C₁₆-, C₁₈-, and C₂₄:₁-ceramides showing the largest percent increases (72.3%, 73.4%, and 77.7%, respectively, p < 0.05). Furthermore, the age-associated elevation in ceramide levels correlated to a 28% decrease in the activity of complex IV of the electron transport chain (p < 0.05), which could be replicated in vitro by inducing a ceramide accumulation in mitochondria isolated from young animals.
Mitochondria do not contain enzymes for de novo ceramide biosynthesis, rather, these organelles have sphingomyelinases, a family of enzymes that cleave the phosphorylcholine headgroup from nascent pools of sphingomyelin. Specifically, mitochondria contain the magnesium-requiring isoform of sphingomyelinase with a neutral pH optima (nSMase). Recent work has shown that nSMase activity is inversely regulated by glutathione status. Because cardiac mitochondrial glutathione (mGSH) declines by up to 60% with age, we hypothesized that the loss in mGSH leads to an increase in ceramides through the upregulation of nSMase.
To determine whether loss of mGSH plays a role in the regulation of mitochondrial nSMase activity, mGSH levels were depleted by treating freshly isolated hepatocytes with 3-hydroxy-4-pentenoate (3HP). It was found that 3HP rapidly depleted mGSH in a concentration-dependent manner (EC₅₀ = 232 μM, p < 0.05). Moreover, this depletion led to an increase in nSMase activity (24 ± 3% at 250 μM 3HP, p < 0.05), and an increase in total ceramide levels (27%, p < 0.05). These findings suggest that mGSH status plays a critical role in the maintenance of ceramide levels within mitochondria. Furthermore, because nSMase activity is regulated by mGSH levels, we hypothesized that any agent promoting an increase in mGSH would reverse the ceramide accumulation seen in cardiac mitochondria from aged animals.
Lipoic acid (LA) is a naturally occurring dithiol compound used for many years as an anti-inflammatory agent. LA-supplementation has been shown to increase cellular and mGSH by increasing cellular cysteine levels in the aging heart. In order to determine whether LA reverses the age-associated ceramidosis in cardiac mitochondria, young and old F344 rats were pair-fed LA [0.2% (w/w) in the diet] against controls for two weeks and cardiac mitochondria were subsequently isolated and analyzed. It was found that LA-treatment reversed the age-associated decline in mGSH levels [decreased 43% with age (p < 0.05)], and reduced nSMase activity [increased 103% with age (p < 0.05)]. Ceramide levels were reduced [elevated 32% with age (p < 0.03)] so that they were no longer different from young controls and complex IV activity restored t youthful levels [declined 28% with age (p < 0.05)].
In conclusion, this dissertation provides evidence to support a new mechanism that explains, at least in part, the progression of mitochondrial dysfunction in the aging heart, and may also contribute to understanding the age-related loss of cardiomyocytes. It also provides mechanistic insights into the overall health benefits of LA supplementation and supports its use as a safe, natural, and "age-essential" micronutrient.