Role of mitochondrial permeability transition in taurine deficiency-induced apoptosis

Abstract

It has recently been shown that taurine deficiency leads to impaired respiratory chain function, resulting in reduced ATP generation and enhanced oxidative stress. Because cardiomyopathy develops in taurine-deficient animals, the hypothesis that mitochondrial oxidative stress may contribute to the development of cardiomyocyte dysfunction and cell death was tested. Isolated neonatal cardiomyocytes incubated in medium containing the taurine transport inhibitor, beta-alanine, lost nearly one-half of their cellular taurine content after 48 h. Accompanying the loss of taurine was a time-dependent increase in apoptosis, which was prevented by the mitochondrial permeability transition inhibitor, cyclosporin A. Two taurine-dependent factors, oxidative stress and calcium overload, serve as important regulators of the mitochondrial permeability transition. Although taurine deficiency slowed the removal of calcium from the cytosol, it had no effect on diastolic calcium content and only modestly reduced systolic calcium content, suggesting that calcium overload is not the trigger for mitochondrial permeability transition pore formation. On the other hand, the glutathione redox ratio was significantly altered in the taurine-deficient cardiomyocyte, suggesting that oxidative stress is the primary initiator of mitochondrial permeability transition and apoptosis in the taurine-deficient cardiomyocyte.

Keywords: Apoptosis; Mitochondrial permeability transition; Oxidative stress; Taurine deficiency.

Figure 1)

Relationship between cellular taurine content and apoptosis. Rat neonatal cardiomyocytes were incubated for various time intervals, in a medium containing the taurine transport inhibitor beta-alanine (5 mM). At each time interval, some cells were harvested and examined for taurine content. The number of apoptotic cells was also examined in other cell preparations. Cells incubated for 48 h with the normal medium contained identical taurine levels to cells forgoing the 48 h incubation (the zero-point control). Values shown represent the mean ± SEM of four to five different cell preparations. *Significant difference between the control (zero time point) and the beta-alanine-treated group (P<0.05)

Figure 2)

Cytosolic levels of cytochrome c (cyt c) in taurine-deficient cells. Cardiomyocytes were incubated for 48 h in medium containing or lacking (control) 5 mM beta (β)-alanine. The cells were then harvested and the cytosolic fraction was isolated. Cyt c content of the cytosol was determined by Western blot analysis. Upper panel: A representative gel of cytosolic cyt c and beta-actin content of control and beta-alanine-treated cells. Lower panel: Average levels of cyt c in the cytosol of control and beta-alanine-treated cells. The cyt c data, which were normalized relative to beta-actin content, represent the mean ± SEM of three different cell preparations. *Significant difference between the control and beta-alanine-treated cells (P<0.05)

Figure 3)

Initiation of mitochondrial permeability transition in beta (β)-alanine-treated cells. Cardiomyocytes were incubated for 48 h with either no addition (control), 200 nM cyclosporin A (CsA), 5 mM β-alanine, or 200 nM CsA and 5 mM β-alanine (β-alanine plus CsA). The percentage of apoptotic cells was determined by the annexin V and propidium iodide method. Values shown represent the mean ± SEM of five different cell preparations. *Significant difference between the β-alanine-treated group and the other three groups (P<0.05)

Figure 4)

Effect of taurine deficiency on Ca²⁺ transient. Cells were incubated for 48 h with medium containing or lacking 5 mM beta-alanine. The cells were then loaded with the fluorescent Ca²⁺ probe, Indo-1. Each Ca²⁺ transient parameter is expressed as a percentage of control. Values shown represent mean ± SEM of four to five different cell preparations. *Significant difference from the control value, which was fixed at 100%. [Ca²⁺]_i Intracellular calcium concentration; TR₉₀ Time required to achieve 90% relaxation

Figure 5)

Reduction in reduced glutathione (GSH) and oxidized glutathione (GSSG) ratio in taurine-deficient cells. Cardiomyocytes were incubated for 48 h in medium containing or lacking 5 mM beta (β)-alanine. After harvesting the cells, GSH and GSSG glutathione content was determined. The GSH/GSSG ratio was expressed as a percentage of control. Values shown represent the mean ± SEM of three to four cell preparations. *Significant difference between the control and β-alanine-treated cells (P<0.05)