Role of Mitochondria and Endoplasmic Reticulum in Taurine-Deficiency-Mediated Apoptosis

Abstract

Taurine is a ubiquitous sulfur-containing amino acid found in high concentration in most tissues. Because of its involvement in fundamental physiological functions, such as regulating respiratory chain activity, modulating cation transport, controlling inflammation, altering protein phosphorylation and prolonging lifespan, taurine is an important nutrient whose deficiency leads to severe pathology and cell death. However, the mechanism by which taurine deficiency causes cell death is inadequately understood. Therefore, the present study examined the hypothesis that overproduction of reactive oxygen species (ROS) by complex I of the respiratory chain triggers mitochondria-dependent apoptosis in hearts of taurine transporter knockout (TauTKO) mice. In support of the hypothesis, a 60% decrease in mitochondrial taurine content of 3-month-old TauTKO hearts was observed, which was associated with diminished complex I activity and the onset of mitochondrial oxidative stress. Oxidative damage to stressed mitochondria led to activation of a caspase cascade, with stimulation of caspases 9 and 3 prevented by treatment of 3-month-old TauTKO mice with the mitochondria specific antioxidant, MitoTempo. In 12 month-old, but not 3-month-old, TauTKO hearts, caspase 12 activation contributes to cell death, revealing a pathological role for endoplasmic reticulum (ER) stress in taurine deficient, aging mice. Thus, taurine is a cytoprotective nutrient that ensures normal mitochondrial and ER function, which is important for the reduction of risk for apoptosis and premature death.

Keywords: apoptosis; caspase cascade; endoplasmic reticulum stress; mitochondria; mitochondria encoded proteins; oxidative stress; respiratory chain; tRNALeu(UUR).

Figure 1

Reduced mitochondrial taurine content of taurine transporter (TauTKO) hearts. Following isolation of the mitochondrial fraction from homogenized hearts, extracts were prepared by precipitating protein. Mitochondrial taurine content was then measured. Values shown represent means ± SEM of 5–7 different hearts. * p < 0.05. WT, the wild-type.

Figure 2

ND6 is reduced in TauTKO hearts. The mitochondrial fraction was isolated from homogenized hearts and then subjected to western blot analyses. The left panel shows representative gels for ND1, ND2, ND3, ND4, ND6, Cyt b (cytochrome b) and COX I (cytochrome c oxidase I), with succinate dehydrogenase (SDH) serving as the loading control. The right panel shows the means ± SEM of the mitochondrial protein/SDH ratio of 6–9 different hearts. Values are expressed relative to wild-type (WT), where WT is fixed at 100%. * p < 0.05.

Figure 3

Taurine depletion decreases complex I activity. Isolated mitochondrial fractions of WT and TauTKO hearts were assayed for the activities of complexes I–V. Values of individual complexes shown represent means ± SEM of 4–6 different hearts. * p < 0.05.

Figure 4

Taurine depletion causes oxidative stress. (A) Reduced (GSH) and oxidized (GSSG) glutathione content were determined and the data expressed as the glutathione redox state (GSH/GSSG), with values shown representing means ± SEM of 6–8 hearts. * p < 0.05; (B) Aconitase activity of WT and TauTKO mitochondria were assayed and normalized relative to SD activity. Values represent means ± SEM of 4–6 hearts. * p < 0.05; (C) Following preparation of total heart lysates and the mitochondrial fraction, proteins were derivatized with 2,4-dinitrophenylhydrazine and then subjected to western blot analysis of carbonylated proteins. The top panels show representative gels of carbonylated proteins of total lysate and the mitochondrial fraction. Values shown in the bottom panel represent means ± SEM for relative cellular and mitochondrial carbonylated protein content from 4–5 hearts. Values are expressed relative to WT, where WT is fixed at 1.0. * p < 0.05.

Figure 5

Taurine depletion induces apoptosis. Total heart lysates of 3-month-old TauTKO and WT hearts were subjected to western blot analyses of the active forms of caspase 9, caspase 3 and caspase 12, as well as the inactive pro-caspase forms of the three proteases (A–E). In (A), the data are expressed as the ratio of active caspase 9/pro-caspase 9 while in (B), the data are depicted as the ratio of cleaved caspase 3/pro-caspase 3. In (C), the data are expressed as cleaved PARP levels. In (D), the ratio of cleaved/pro-caspase 12 is shown. In (E), TauTKO and WT mice were treated with the mitochondria-specific antioxidant, MitoTempo, for 7 days and changes in the levels of the inactive pro-caspase 3 zymogen were determined. Each panel contains a representative gel. The representative bands for WT and TauTKO were spliced from one original gel and the splice junction is indicated by the black splicing line. Values shown represent means ± SEM of 6–9 hearts. All values are expressed relative to WT, where WT is fixed at 1.0. * p < 0.05.

Figure 5

Taurine depletion induces apoptosis. Total heart lysates of 3-month-old TauTKO and WT hearts were subjected to western blot analyses of the active forms of caspase 9, caspase 3 and caspase 12, as well as the inactive pro-caspase forms of the three proteases (A–E). In (A), the data are expressed as the ratio of active caspase 9/pro-caspase 9 while in (B), the data are depicted as the ratio of cleaved caspase 3/pro-caspase 3. In (C), the data are expressed as cleaved PARP levels. In (D), the ratio of cleaved/pro-caspase 12 is shown. In (E), TauTKO and WT mice were treated with the mitochondria-specific antioxidant, MitoTempo, for 7 days and changes in the levels of the inactive pro-caspase 3 zymogen were determined. Each panel contains a representative gel. The representative bands for WT and TauTKO were spliced from one original gel and the splice junction is indicated by the black splicing line. Values shown represent means ± SEM of 6–9 hearts. All values are expressed relative to WT, where WT is fixed at 1.0. * p < 0.05.

Figure 6

Unfolded protein response (UPR) is not activated in TauTKO hearts at an early age. Total lysates from 3-month-old WT and TauTKO mice treated with or without MitoTempo were subjected to western blot analysis of (A) phosphorylated PERK and ATF4 (B) spliced XBP-1 and phosphorylated-IRE1 and spliced XBP-1 and (C) GRP78. Each panel contains a representative gel and summation data expressed as means ± SEM of 6–9 hearts. Values are expressed relative to WT, where WT is fixed at 1.0. * p < 0.05.

Figure 7

CHOP and UPR caspase cascade are activated in older TauTKO hearts. Total heart lysates from 12-month-old TauTKO and WT mouse hearts were subjected to western blot analysis of (A) CHOP, (B) caspase 12, (C) caspase 3 and (D) PARP. Each panel contains a representative gel and summation data expressed as means ± SEM of 6–9 hearts. The representative bands for WT and TauTKO shown in B were spliced from one original gel and the splice junction is indicated by the black splicing line. All values are expressed relative to WT, where WT is fixed at 1.0. * p < 0.05.

Figure 8

Mechanisms underlying taurine deficiency-mediated myocardial cell death.