Hypoxia/reoxygenation of isolated rat heart mitochondria causes cytochrome c release and oxidative stress; evidence for involvement of mitochondrial nitric oxide synthase

Abstract

The objective of the present study was to delineate the molecular mechanisms for mitochondrial contribution to oxidative stress induced by hypoxia and reoxygenation in the heart. The present study introduces a novel model allowing real-time study of mitochondria under hypoxia and reoxygenation, and describes the significance of intramitochondrial calcium homeostasis and mitochondrial nitric oxide synthase (mtNOS) for oxidative stress. The present study shows that incubating isolated rat heart mitochondria under hypoxia followed by reoxygenation, but not hypoxia per se, causes cytochrome c release from the mitochondria, oxidative modification of mitochondrial lipids and proteins, and inactivation of mitochondrial enzymes susceptible to inactivation by peroxynitrite. These alterations were prevented when mtNOS was inhibited or mitochondria were supplemented with antioxidant peroxynitrite scavengers. The present study shows mitochondria independent of other cellular components respond to hypoxia/reoxygenation by elevating intramitochondrial ionized calcium and stimulating mtNOS. The present study proposes a crucial role for heart mitochondrial calcium homeostasis and mtNOS in oxidative stress induced by hypoxia/reoxygenation.

Figure 1. H/R induces cytochrome c release, lipid peroxidation and protein carbonylation

A) Cytochrome c release from the mitochondria incubated under normoxia (Norm) or lowered oxygen concentration (O₂ (μM); 50, 100, 200; Hypoxia) or hypoxia/reoxygenation (H/R). B) Cytochrome c release from mitochondria incubated at 50 μM O₂ hypoxia/reoxygenation (H/R) while mtNOS was stimulated by Ca²⁺ (10 μM; Ca²⁺), inhibited by L-NMMA (L-NMMA), or mitochondria were supplemented with peroxynitrite scavengers Trolox (Trolox) or GME (GME). C) Lipid peroxidation (LPO) and protein carbonylation (PC) were measured in mitochondria treated under hypoxia (Hypoxia) or hypoxia/reperfusion (H/R) in the absence of L-NMMA, Trolox or GME (LPO; PC) or presence of L-NMMA (LPO NMMA; PC NMMA), Trolox (LPO TROLOX; PC TROLOX) or GME (LPO GME; PC GME). Numbers on the horizontal axes represent oxygen concentration in μM). Norm represents mitochondria sample incubated 40 min under normoxia (air) and Ctrl represents freshly isolated mitochondria. *Significantly different from control.

Figure 2. H/R stimulates mtNOS activity, and inactivates mitochondrial enzymes

In all panels, Ctrl represents freshly isolated mitochondria without treatment, H/R represents mitochondria treated under H/R, H/R + NMMA represents mitochondria treated under H/R while mtNOS was inhibited by L-NMMA, and hypoxia represents mitochondria treated under hypoxia. A) mtNOS activity determined by radioassay. B) mtNOS activity of samples as under panel A measured using chemiluminescence assay. C) Aconitase, MnSOD, and creatine kinase (mtCK) activities. D) SCOT activity. *Significantly different from control.

Figure 3. mtNOS activity and [Ca²⁺]_m

A) Heart mtNOS activity measured in the following samples: Ctrl: freshly isolated mitochondria; Ctrl+NMMA: mtNOS was inhibited by L-NMMA; 20 μM Ca, 40 μM Ca, 80 μM Ca: when [Ca²⁺]_m was elevated by providing mitochondria with 20, 40, or 80 μM Ca²⁺; Ca + NMMA: mitochondria were provided with 40 μM Ca²⁺ and mtNOS was inhibited by L-NMMA, Ca + RR: mitochondria were provided with 40 μM Ca²⁺ and mitochondrial Ca²⁺ uptake was inhibited by ruthenium red; Ca + Rot: mitochondria were provided with 40 μM Ca²⁺ and mitochondrial Ca²⁺ uptake was prevented by collapsing Δψ with rotenone. mtNOS activity was also measured when mitochondria were supplemented with L-arginine in the absence (L-arg) or presence of ruthenium red (L-arg + RR). The effect of MgCl₂ on mtNOS activity in the absence (Mg) or presence of Ca²⁺ or L-arg (Mg + Ca, Mg + L-arg) is shown. B) mtNOS activity determined using NO-sensitive fluorescent dye, DAF-2. NO was determined in freshly isolated mitochondria (Ctrl) or when mitochondria were supplemented with 40 μM Ca²⁺(Ca), ruthenium red. (RR), RR plus 40 μM Ca²⁺ (RR + Ca) or 40 μM Ca²⁺ and L-NMMA (Ca + L-NMMA). *Significantly different from control. ^#Significantly different from 40 μM Ca²⁺.

Figure 4. H/R, [Ca²⁺]_m and Δψ

A) [Ca²⁺]_m was measured for untreated mitochondria (Ctrl), mitochondria treated under hypoxia (Hypox) or hypoxia-reoxygenation (H/R). Mitochondria samples (mito) were added to the cuvette, and after 1 minute rotenone and carbonyl cyanide m-chlorophenylhydrazone (rot/cccp) were added to collapse the Δψ to allow [Ca²⁺]_m equilibrate with buffer. Where indicated, EGTA was added. B) [Ca²⁺]_m was measured as in panel A for broken mitochondria (BM) untreated (Ctrl), treated under hypoxia (Hypox) or H/R (H/R). Where indicated, 5 μM Ca²⁺ (Ca) or EGTA (EGTA) was added. C) Δψ was measured at 511–533 nm using Safranin. The Δψ was supported by K⁺-succinate (succ) in the presence of rotenone (rot). At the end of the test, carbonyl cyanide m-chlorophenylhydrazone (cccp) was added to ensure the test. D) Real-time and simultaneous detection of oxygen concentration ([O₂]) and intramitochondrial Ca²⁺ ([Ca²⁺]_m) during H/R. Where indicated, reoxygenation (Reox) was performed.

Scheme I. Hypoxia and H/R chamber

A) Schematic representation of the in vitro hypoxia/reoxygenation system consisting of a tightly sealed thermostated chamber with fine tubes allowing to purge N₂ or air, or adding the mitochondria samples. The chamber is also equipped with an oxygen sensor. B) Schematic [O₂] trace detected by the oxygen sensor during hypoxia and reoxygenation. In a typical experiment, 1 ml of air saturated buffer was added to a chamber, 100 μl of buffer was removed and the remaining buffer was purged with N₂ (N₂) until the [O₂] measured with the oxygen electrode (oxygen electrode) reached the desired concentration. Then mitochondria (mitochondria) were added to the chamber and collected after 40 minutes. C) In a fluorescence cuvette buffer was added and purged with N₂ (N₂) until oxygen concentration detected by oxygen sensor ([O₂] sensor) was reached the desired concentration. Then Fura-2-loaded mitochondria were added to a cuvette (mito) and [Ca²⁺]_m was detected throughout the hypoxia and reoxygenation.

Scheme II. Mitochondria: structure, intra-organelle calcium homeostasis, and mtNOS

Mitochondria consist of the inner (IM) and the outer membrane (OM), the matrix, and the intermembrane space (IMS). The IM carries the respiratory chain. The chain consists of four respiratory complexes (I, II, III, IV) embedded in the IM, coenzyme Q (ubiquinone; Q) and ATP synthase that is often referred to complex V (V). Electrons (e⁻) enter the chain through oxidation of NADH at complex I or FADH₂ at complex II and flow down the chain to complex IV to reduce O₂ to H₂O. Coupled to the electron flow protons are extruded from the matrix into the IMS. This proton extrusion establishes a transmembrane potential (Δψ). The Δψ is the driving force for mitochondria to take up Ca²⁺. Mitochondria take up relatively large quantities of Ca²⁺, however, the intramitochondrial ionized calcium concentration ([Ca²⁺]_m) is tightly maintained low by precipitating the [Ca²⁺]_m to non-ionized calcium pools, the matrix electron-dense granules. Mitochondria produce NO via mitochondrial NO-synthase (mtNOS) that is Ca²⁺-sensitive, i.e., increased [Ca²⁺]_m stimulates mtNOS activity. Hypoxia/reoxygenation (H/R) alters intramitochondrial calcium homeostasis by shifting the mitochondrial nonionized/ionized calcium equilibrium in the favor of the ionized form. Elevated [Ca²⁺]_m stimulates mtNOS-derived NO synthesis. Mitochondrial NO readily reacts with superoxide anion (O₂⁻) generated by mitochondrial respiratory chain to produce the powerful oxidizing species, peroxynitrite (ONOO⁻). ONOO⁻ releases cytochrome c (cyto c), induces oxidative moidification of mitochondrial lipids and proteins including MnSOD, and releases cytochrome c.