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

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. C(16)-, C(18)-, and C(24:1)-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.

Figure 1. Asymmetric distribution of cardiac mitochondrial ceramides

Ceramide content of purified intact mitochondria and inner mitochondrial membrane (IMM) as quantified by LC-MS/MS. Both fractions contain six ceramide isoforms ranging from 16- to 24- carbon units in length, with C₂₄-ceramide being the predominant species in intact mitochondria. C₁₆-, C₁₈-, and C_24:1- ceramide are found in near-equivalent quantities in the IMM as compared to intact mitochondria, whereas C₂₀-, C₂₂, and C₂₄-ceramide are present in much lower quantities. This suggests that these latter ceramides are primarily found in the outer mitochondrial membrane. Data represent the means ± SEM, n = 4.

Figure 2. LA treatment decreases mitochondrial ceramides

Young and old rats were fed (R)-α-lipoic acid (LA; 0.2% [w/w]) or a control diet for two weeks. Mitochondria were isolated, lipids extracted and analyzed by LC-MS/MS. Total ceramide was increased in cardiac mitochondria from aged rats; LA restored ceramides to levels seen in young animals but resulted in no alteration of ceramide levels in young rats. Data represent the means ± SEM, n = 4; an asterisk (*) denotes a significant difference by Student's t-test between old and young control animals, p < 0.03.

Figure 3. LA treatment restores Complex IV activity in cardiac mitochondria from aged animals

Isolated cardiac mitochondria from LA-supplemented or non-supplemented rats were assayed for Complex IV activity. Enzymatic activity declined with age and was restored by LA to the levels seen in young animals. Data represent the means ± SEM, n = 4; an asterisk (*) denotes a significant difference from old controls, p ≤ 0.05.

Figure 4. LA restores neutral sphingomyelinase (nSMase) activity in mitochondria from old animals to youthful levels

Cardiac mitochondria from young and old rats fed LA or the control diet for two weeks were assayed for nSMase activity. nSMase activity significantly increased with age and was restored to youthful levels by LA treatment. Data represent the means ± SEM, n = 4; an asterisk (*) denotes a significant difference, p < 0.01.

Figure 5. LA markedly increases mitochondrial glutathione levels that otherwise decrease with age

GSH levels were monitored in mitochondria from young and old rats fed LA or the control diet. GSH content significantly decreased with age and was restored to youthful levels by LA treatment. Data represent the means ± SEM, young, n = 3; old, n = 4; an asterisk (*) denotes a significant difference, p ≤ 0.05.