TNF-induced mitochondrial damage: a link between mitochondrial complex I activity and left ventricular dysfunction

Abstract

Mitochondrial damage is implicated in the progression of cardiac disease. Considerable evidence suggests that proinflammatory cytokines induce oxidative stress and contribute to cardiac dysfunction. This study was conducted to determine whether a TNF-induced increase in superoxide (O(2)(*)(-)) contributes to mitochondrial damage in the left ventricle (LV) by impairing respiratory complex I activity. We employed an electron paramagnetic resonance (EPR) method to measure O(2)(*)(-) and oxygen consumption in mitochondrial respiratory complexes, using an oxygen label. Adult male Sprague-Dawley rats were divided into four groups: control, TNF treatment (ip), TNF+ apocynin (APO; 200 micromol/kg bw, orally), and TNF+ Tempol (Temp; 300 micromol/kg bw, orally). TNF was injected daily for 5 days. Rats were sacrificed, LV tissue was collected, and mitochondria were isolated for EPR studies. Total LV ROS production was significantly higher in TNF animals than in controls; APO or Temp treatment ameliorated TNF-induced LV ROS production. Total mitochondrial ROS production was significantly higher in the TNF and TNF+ APO groups than in the control and TNF+ Temp groups. These findings suggest that TNF alters the cellular redox state, reduces the expression of four complex I subunits by increasing mitochondrial O(2)(*)(-) production and depleting ATP synthesis, and decreases oxygen consumption, thereby resulting in mitochondrial damage and leading to LV dysfunction.

Fig. 1

(A) Total ROS production in LV tissue. EPR spectra and their graphic interpretations are given. TNF administration significantly increased total ROS production in LV tissue. APO or Temp significantly decreased total LV ROS production. All values are presented as means ± SEM (*p< 0.05, control vs TNF; #p< 0.05, TNF vs TNF+ APO; *$p< 0.05 TNF vs TNF+ Temp). (B) Superoxide production in LV tissue. EPR spectra and their graphic interpretations are given. TNF administration significantly increased total superoxide production in LV tissue. APO or Temp significantly decreased total tissue superoxide production. All values are presented as means ± SEM (*p< 0.05, control vs TNF; #p< 0.05, TNF vs TNF+ APO; $p< 0.05, TNF vs TNF+ Temp).

Fig. 2

Mitochondrial purity as determined with transmission electron microscopy.

Fig. 3

(A) Total ROS production in LV mitochondria. EPR spectra and their graphic interpretations are shown. TNF administration significantly increased total ROS production in LV mitochondria. Note the similarities between the TNFand the TNF+ APO groups. Temp significantly decreased mitochondrial ROS. All values are presented as means ± SEM (*p< 0.05, control vs TNF; $p< 0.05, TNF vs TNF+ Temp; @p< 0.05, TNF+ APO vs TNF+ Temp). (B) Superoxide production in LV mitochondria. Significantly higher superoxide production was noted in the TNF group compared to the control and TNF+ Temp groups. In the graphical representation, all values are presented as means ± SEM (*p< 0.05, control vs TNF; $p< 0.05, TNF vs TNF+ Temp; @p< 0.05, TNF+ APO vs TNF+ Temp). (C) Hydrogen peroxide production in LV mitochondria. Significantly lower rates of hydrogen peroxide production were found in the TNF+ Temp and control groups compared to the TNF and TNF+ APO groups. All values are presented as means ± SEM (*p< 0.05, control vs TNF; $p< 0.05 TNF vs TNF+ Temp; @p< 0.05, TNF+ APO vs TNF+ Temp).

Fig. 4

Complex I enzyme activities (total ROS production) in LV mitochondria. Note the significant decreases in the activity levels of complex I in the TNF and TNF+ APO groups and the restoration of activity in the TNF+ Temp group. All values are presented as means ± SEM (*p< 0.05, control vs TNF; #p< 0.05, TNF vs TNF+ APO).

Fig. 5

(A) MnSOD and (B) oxygen consumption in LV mitochondria. MnSOD/oxygen consumption was significantly lower in the TNF and TNF+ APO groups compared to the control and TNF+ Temp groups. All values are presented as means ± SEM (*p< 0.05, control vs TNF; #p< 0.05, TNF vs TNF+ APO; $p < 0.05, TNF vs TNF+ Temp; @p< 0.05, TNF+ APO vs TNF+ Temp).

Fig. 6

(A) Effect of TNF on expression of mitochondrial respiratory complex I subunits. Significant decreases in the expression of mitochondrial complex I 17-, 20-, 30-, and 39-kDa subunits were observed in the TNF and TNF+ APO groups compared to the control and TNF+ Temp groups. (B) Densitometry values are represented as relative intensities in mean arbitrary units calculated from the Western blots shown in (A). All values are presented as means ± SEM (*p< 0.05, control vs TNF; #p< 0.05, TNF vs TNF+ APO; $p< 0.05, TNF vs TNF+ Temp; @p < 0.05, TNF+ APO vs TNF+ Temp).

Fig. 7

(A) ATP levels and (B) ATP/ADP ratio in LV mitochondria. TNF administration significantly decreased ATP levels in the TNF and TNF+ APO groups compared to the control and TNF+ Temp groups. All values are presented as means ± SEM (*p< 0.05, control vs TNF; #p< 0.05, TNF vs TNF+ APO; $p< 0.05, TNF vs TNF+ Temp; @p< 0.05, TNF+ APO vs TNF+ Temp).