Acetyl-L-carnitine increases mitochondrial protein acetylation in the aged rat heart

Abstract

Previously we showed that in vivo treatment of elderly Fisher 344 rats with acetylcarnitine abolished the age-associated defect in respiratory chain complex III in interfibrillar mitochondria and improved the functional recovery of the ischemic/reperfused heart. Herein, we explored mitochondrial protein acetylation as a possible mechanism for acetylcarnitine's effect. In vivo treatment of elderly rats with acetylcarnitine restored cardiac acetylcarnitine content and increased mitochondrial protein lysine acetylation and increased the number of lysine-acetylated proteins in cardiac subsarcolemmal and interfibrillar mitochondria. Enzymes of the tricarboxylic acid cycle, mitochondrial β-oxidation, and ATP synthase of the respiratory chain showed the greatest acetylation. Acetylation of isocitrate dehydrogenase, long-chain acyl-CoA dehydrogenase, complex V, and aspartate aminotransferase was accompanied by decreased catalytic activity. Several proteins were found to be acetylated only after treatment with acetylcarnitine, suggesting that exogenous acetylcarnitine served as the acetyl-donor. Two-dimensional fluorescence difference gel electrophoresis analysis revealed that acetylcarnitine treatment also induced changes in mitochondrial protein amount; a two-fold or greater increase/decrease in abundance was observed for thirty one proteins. Collectively, our data provide evidence for the first time that in the aged rat heart in vivo administration of acetylcarnitine provides acetyl groups for protein acetylation and affects the amount of mitochondrial proteins.

Keywords: Acetylcarnitine; Aging; Heart; Mitochondria; Protein acetylation.

Figure 1

Plasma and myocardial acetylcarnitine content of control and acetylcarnitine-treated (intraperitoneal and oral) elderly rats. Mean ± SEM (n=4). The insert shows the myocardial acetylcarnitine content relative to the plasma acetylcarnitine concentration. #: significantly different from control &: significantly different from control and oral acetylcarnitine treatment.

Figure 2

Immunoblot analysis of heart SSM and IFM isolated from saline-treated (CONT.) and orally (ORAL) and intraperitoneally (IP) acetylcarnitine-treated F344 elderly rats. SSM and IFM from three separate experiments were pooled and 50 μg aliquots subjected to SDS-PAGE and immunoblotting using affinity-purified acetyllysine antibodies. The citrate synthase specific activity in the combined mitochondria was as follows: 1890 (cont.), 1550 (oral), and 1718 (IP) nmoles/min/mg mitochondrial protein in SSM and 1891 (control), 1869 (AcCarn-oral) and 2022 (IP-treated) nmoles/min/mg mitochondrial protein in IFM.

Figure 3

SIRT3 expression in SSM and IFM from 6month old adult and 24 month old elderly Fisher 344 rats (n=7). Protein load was normalized to citrate synthase activity (100 mU/lane). &: statistically significant vs. SSM from 6 month old adult; *: statistically significant vs. SSM from 6 month old adult.

Figure 4

Comparison of the effect of acetylation on the activity of enzymes in SSM (open bars) and IFM (shaded bars) isolated from hearts of 24 month-old acetylcarnitine- (n=4) and saline-treated (n=2) Fisher 344 rats. Acyl-CoA dehydrogenase was assayed using palmitoyl- (C16), and 2,6-dimethylheptanoyl-CoA (2,6DMH-CoA) as substrates. Acetylated lysine residues are shown next to the respective columns. Abbreviations used: CS - citrate synthase; ICDH-NAD+ - NAD+-dependent isocitrate dehydrogenase; ICDH-NADP+ - NADP+-dependent isocitrate dehydrogenase; AAT - aspartate aminotransferase; ATPase - complex V; LCAD - long-chain acyl-CoA dehydrogenase. The activities are normalized to values obtained with mitochondria from saline-treated rats and are listed Specific activities (nmoles/min/mg mitochondrial protein) in control SSM: CS=2100; ICDH-NAD⁺=12.6; ICDH-NADP⁺=1616; AAT=3923; ATPase=2064; LCAD-2,6DMH-CoA=187; LCAD-C16-CoA=91. Specific activities (nmoles/min/mg mitochondrial protein) in control IFM: CS=2508; ICDH-NAD⁺=9.7; ICDH-NADP⁺=1941; AAT=4671; ATPase=2673; LCAD-2,6DMH-CoA=139; LCAD-C16-CoA=127.

Figure 5

2-DIGE of rat heart IFM from saline- and acetylcarnitine-treated 24month old Fischer 344 rats. Equal amounts of IFM from three separate experiments were combined and processed for analysis as described under the Methods section. The protein molecular mass scale in kDa is depicted on the y-axis while the x-axis shows the range of isoelectric points. Thirty-one protein spots (numbers in boxes) revealed a two-fold or greater change in protein expression.