Mitochondrial reactive oxygen species production and respiratory complex activity in rats with pressure overload-induced heart failure

Abstract

We investigated the impact of cardiac reactive oxygen species (ROS) during the development of pressure overload-induced heart failure. We used our previously described rat model where transverse aortic constriction (TAC) induces compensated hypertrophy after 2 weeks, heart failure with preserved ejection fraction at 6 and 10 weeks, and heart failure with systolic dysfunction after 20 weeks. We measured mitochondrial ROS production rates, ROS damage and assessed the therapeutic potential of in vivo antioxidant therapies. In compensated hypertrophy (2 weeks of TAC) ROS production rates were normal at both mitochondrial ROS production sites (complexes I and III). Complex I ROS production rates increased with the appearance of diastolic dysfunction (6 weeks of TAC) and remained high thereafter. Surprisingly, maximal ROS production at complex III peaked at 6 weeks of pressure overload. Mitochondrial respiratory capacity (state 3 respiration) was elevated 2 and 6 weeks after TAC, decreased after this point and was significantly impaired at 20 weeks, when contractile function was also impaired and ROS damage was found with increased hydroxynonenal. Treatment with the ROS scavenger α-phenyl-N-tert-butyl nitrone or the uncoupling agent dinitrophenol significantly reduced ROS production rates at 6 weeks. Despite the decline in ROS production capacity, no differences in contractile function between treated and untreated animals were observed. Increased ROS production occurs early in the development of heart failure with a peak at the onset of diastolic dysfunction. However, ROS production may not be related to the onset of contractile dysfunction.

Figure 1. Scheme for analysis of ROS production

Basal ROS production with pyruvate/malate as complex I substrate (A). Under these conditions, ROS are produced at complex I and at complex III. Succinate as complex II substrate (B), rotenone inhibiting reverse electron flow to complex I, preventing ROS production at this site. Maximal ROS production capacity at complex I with substrate pyruvate/malate and rotenone for complex I inhibition (C).

Figure 2. Mitochondrial respiration

State 3 respiration of cardiac mitochondria after 2, 6, 10 and 20 weeks (w) of pressure overload induced by transverse aortic constriction (filled bars) compared to sham-operated control (open bars) with glutamate (A), pyruvate/malate (B), palmitoylcarnitine/malate (C) or succinate (D) as substrates. Data are mean ± SEM; n = 5–10; *P < 0.05, **P < 0.01, ***P < 0.001 compared to sham-operated animals.

Figure 3. Isolated respiratory chain complex activities

Activities for individual respiratory chain complexes I (A), II (B), III (C) and IV (D) and the combination of complexes I + III (E) and II + III (F) measured in isolated cardiac mitochondria after 2, 6, 10 and 20 weeks of pressure overload (filled bars) and sham-operated control groups (open bars). Data are mean ± SEM; n = 5–9; *P < 0.05 compared to sham-operated animals.

Figure 4. ROS production

ROS production capacity after 2, 6, 10 and 20 weeks of pressure overload (filled bars), compared to sham-operated control (open bars) with substrates pyruvate/malate (A), pyruvate/malate and rotenone (B), or succinate and rotenone (C). ROS production capacity in relation to respiratory capacity: complex I ROS production with pyruvate/malate as substrate compared to pyruvate/malate-based respiration (D), complex I ROS production with pyruvate/malate and rotenone compared to pyruvate/malate-based respiration (E), and complex III ROS production with succinate and rotenone related to succinate-based respiration (F). Data are mean ± SEM; n = 6–13; *P < 0.05; **P < 0.01, ***P < 0.001 compared to sham-operated animals.

Figure 5. ROS damage

Accumulation of 4-hydroxynonenal (A) adducts (4-HNE) and carbonylated proteins (B) as a measure for oxidative damage in pressure overload after 2, 6, 10 and 20 weeks of pressure overload (filled bars), compared to sham-operated control (open bars). Data are mean ± SEM; n = 4–6; *P < 0.05.

Figure 6. Antioxidative therapy

ROS production capacity after 2, 6 and 10 weeks of pressure overload in cardiac mitochondria from PBN-treated (hatched bars) and DNP-treated animals (cross-hatched bars), compared to pressure overload without antioxidant supplement (filled bars) and sham animals (open bars) with substrates pyruvate/malate (A), pyruvate/malate and rotenone (B), or succinate and rotenone (C). D, activities for individual respiratory chain complex I in these animals measured in isolated cardiac mitochondria. Data are mean ± SEM; n = 5–9; different superscripts indicate significant differences.