Isoflurane modulates cardiac mitochondrial bioenergetics by selectively attenuating respiratory complexes

Abstract

Mitochondrial dysfunction contributes to cardiac ischemia-reperfusion (IR) injury but volatile anesthetics (VA) may alter mitochondrial function to trigger cardioprotection. We hypothesized that the VA isoflurane (ISO) mediates cardioprotection in part by altering the function of several respiratory and transport proteins involved in oxidative phosphorylation (OxPhos). To test this we used fluorescence spectrophotometry to measure the effects of ISO (0, 0.5, 1, 2mM) on the time-course of interlinked mitochondrial bioenergetic variables during states 2, 3 and 4 respiration in the presence of either complex I substrate K(+)-pyruvate/malate (PM) or complex II substrate K(+)-succinate (SUC) at physiological levels of extra-matrix free Ca(2+) (~200nM) and Na(+) (10mM). To mimic ISO effects on mitochondrial functions and to clearly delineate the possible ISO targets, the observed actions of ISO were interpreted by comparing effects of ISO to those elicited by low concentrations of inhibitors that act at each respiratory complex, e.g. rotenone (ROT) at complex I or antimycin A (AA) at complex III. Our conclusions are based primarily on the similar responses of ISO and titrated concentrations of ETC. inhibitors during state 3. We found that with the substrate PM, ISO and ROT similarly decreased the magnitude of state 3 NADH oxidation and increased the duration of state 3 NADH oxidation, ΔΨm depolarization, and respiration in a concentration-dependent manner, whereas with substrate SUC, ISO and ROT decreased the duration of state 3 NADH oxidation, ΔΨm depolarization and respiration. Unlike AA, ISO reduced the magnitude of state 3 NADH oxidation with PM or SUC as substrate. With substrate SUC, after complete block of complex I with ROT, ISO and AA similarly increased the duration of state 3 ΔΨm depolarization and respiration. This study provides a mechanistic understanding in how ISO alters mitochondrial function in a way that may lead to cardioprotection.

Keywords: Cardiac IR injury; Cardioprotection; Electron transport chain; Isoflurane; Mitochondrial bioenergetics; Volatile anesthetic.

Figure 1

Schema depicting the mitochondrial electron transport chain (ETC) protein complexes of the mitochondrial inner membrane (IMM), along with their specific inhibitors: rotenone (ROT), malonate (MAL), antimycin A (AA), potassium cyanide (KCN), oligomycin (OMN), and atractyloside (ATR). Reducing equivalents, NADH and FADH₂, are generated via the tricarboxylic acid (TCA) cycle in the matrix, while electrons are transferred through the complexes to the final O₂ acceptor. At complex IV, coupled with this transfer is the pumping of protons (H⁺) into the intermembrane space (IMS) by complex I, complex III, and complex IV, which generate the H⁺ gradient used by ATP synthase (complex V) to phosphorylate ADP to ATP. Depending on the substrate used, K⁺-pyruvate/malate (PM) or K⁺-succinate (SUC), isolated mitochondria respire differentially using NADH and FADH₂ to produce ATP; this is coupled to forward or reverse electron transfer (FET or RET) depending on the reduction potential across each complex and across the IMM. (✧) represents potential targets of isoflurane (ISO) on the ETC components explored in this study.

Figure 2

Normalized mitochondrial redox state (NADH/NADH+NAD⁺) during states 2, 3 and 4 respiration with different substrates and either ISO or a blocker of ETC complexes. State 2 was induced with 10 mM PM (A–C) or SUC (D–F) and state 3 with 250 µM ADP; state 4 is after phosphorylation of all ADP to ATP. ISO, ROT, or AA was added after PM or SUC and before ADP. ADP induced a transient decrease in the reduced state of NADH. The insets summarize effects of ISO compared to ROT and AA on duration of ADP-induced state 3 NADH oxidation (S3 time, s). Note similarities and differences of responses to each drug. NADH transients were normalized on a scale of 0–1 i.e. between fully oxidized to fully reduced states by adding excess FCCP or ROT, respectively. Data represent mean ± SD of 3 replicates.

Figure 2

Normalized mitochondrial redox state (NADH/NADH+NAD⁺) during states 2, 3 and 4 respiration with different substrates and either ISO or a blocker of ETC complexes. State 2 was induced with 10 mM PM (A–C) or SUC (D–F) and state 3 with 250 µM ADP; state 4 is after phosphorylation of all ADP to ATP. ISO, ROT, or AA was added after PM or SUC and before ADP. ADP induced a transient decrease in the reduced state of NADH. The insets summarize effects of ISO compared to ROT and AA on duration of ADP-induced state 3 NADH oxidation (S3 time, s). Note similarities and differences of responses to each drug. NADH transients were normalized on a scale of 0–1 i.e. between fully oxidized to fully reduced states by adding excess FCCP or ROT, respectively. Data represent mean ± SD of 3 replicates.

Figure 2

Normalized mitochondrial redox state (NADH/NADH+NAD⁺) during states 2, 3 and 4 respiration with different substrates and either ISO or a blocker of ETC complexes. State 2 was induced with 10 mM PM (A–C) or SUC (D–F) and state 3 with 250 µM ADP; state 4 is after phosphorylation of all ADP to ATP. ISO, ROT, or AA was added after PM or SUC and before ADP. ADP induced a transient decrease in the reduced state of NADH. The insets summarize effects of ISO compared to ROT and AA on duration of ADP-induced state 3 NADH oxidation (S3 time, s). Note similarities and differences of responses to each drug. NADH transients were normalized on a scale of 0–1 i.e. between fully oxidized to fully reduced states by adding excess FCCP or ROT, respectively. Data represent mean ± SD of 3 replicates.

Figure 2

Normalized mitochondrial redox state (NADH/NADH+NAD⁺) during states 2, 3 and 4 respiration with different substrates and either ISO or a blocker of ETC complexes. State 2 was induced with 10 mM PM (A–C) or SUC (D–F) and state 3 with 250 µM ADP; state 4 is after phosphorylation of all ADP to ATP. ISO, ROT, or AA was added after PM or SUC and before ADP. ADP induced a transient decrease in the reduced state of NADH. The insets summarize effects of ISO compared to ROT and AA on duration of ADP-induced state 3 NADH oxidation (S3 time, s). Note similarities and differences of responses to each drug. NADH transients were normalized on a scale of 0–1 i.e. between fully oxidized to fully reduced states by adding excess FCCP or ROT, respectively. Data represent mean ± SD of 3 replicates.

Figure 2

Normalized mitochondrial redox state (NADH/NADH+NAD⁺) during states 2, 3 and 4 respiration with different substrates and either ISO or a blocker of ETC complexes. State 2 was induced with 10 mM PM (A–C) or SUC (D–F) and state 3 with 250 µM ADP; state 4 is after phosphorylation of all ADP to ATP. ISO, ROT, or AA was added after PM or SUC and before ADP. ADP induced a transient decrease in the reduced state of NADH. The insets summarize effects of ISO compared to ROT and AA on duration of ADP-induced state 3 NADH oxidation (S3 time, s). Note similarities and differences of responses to each drug. NADH transients were normalized on a scale of 0–1 i.e. between fully oxidized to fully reduced states by adding excess FCCP or ROT, respectively. Data represent mean ± SD of 3 replicates.

Figure 2

Normalized mitochondrial redox state (NADH/NADH+NAD⁺) during states 2, 3 and 4 respiration with different substrates and either ISO or a blocker of ETC complexes. State 2 was induced with 10 mM PM (A–C) or SUC (D–F) and state 3 with 250 µM ADP; state 4 is after phosphorylation of all ADP to ATP. ISO, ROT, or AA was added after PM or SUC and before ADP. ADP induced a transient decrease in the reduced state of NADH. The insets summarize effects of ISO compared to ROT and AA on duration of ADP-induced state 3 NADH oxidation (S3 time, s). Note similarities and differences of responses to each drug. NADH transients were normalized on a scale of 0–1 i.e. between fully oxidized to fully reduced states by adding excess FCCP or ROT, respectively. Data represent mean ± SD of 3 replicates.

Figure 3

Normalized mitochondrial membrane potential (ΔΨ_m) during states 2, 3 and 4 respiration with different substrates and after adding either ISO or a blocker of ETC complexes. ISO, ROT, or AA after PM (A–C) or SUC (D–F) and before ADP, which induced a transient depolarization. The insets summarize effects of ISO compared to ROT and AA on duration of ADP-induced state 3 decreases in ΔΨ_m (S3 time, s). Note similarities and differences of responses to each drug. ΔΨ_m transients were normalized with respect to maximal (100 %) depolarization obtained with added CCCP. Data represent mean ± SD of 3 replicates.

Figure 3

Normalized mitochondrial membrane potential (ΔΨ_m) during states 2, 3 and 4 respiration with different substrates and after adding either ISO or a blocker of ETC complexes. ISO, ROT, or AA after PM (A–C) or SUC (D–F) and before ADP, which induced a transient depolarization. The insets summarize effects of ISO compared to ROT and AA on duration of ADP-induced state 3 decreases in ΔΨ_m (S3 time, s). Note similarities and differences of responses to each drug. ΔΨ_m transients were normalized with respect to maximal (100 %) depolarization obtained with added CCCP. Data represent mean ± SD of 3 replicates.

Figure 3

Normalized mitochondrial membrane potential (ΔΨ_m) during states 2, 3 and 4 respiration with different substrates and after adding either ISO or a blocker of ETC complexes. ISO, ROT, or AA after PM (A–C) or SUC (D–F) and before ADP, which induced a transient depolarization. The insets summarize effects of ISO compared to ROT and AA on duration of ADP-induced state 3 decreases in ΔΨ_m (S3 time, s). Note similarities and differences of responses to each drug. ΔΨ_m transients were normalized with respect to maximal (100 %) depolarization obtained with added CCCP. Data represent mean ± SD of 3 replicates.

Figure 3

Normalized mitochondrial membrane potential (ΔΨ_m) during states 2, 3 and 4 respiration with different substrates and after adding either ISO or a blocker of ETC complexes. ISO, ROT, or AA after PM (A–C) or SUC (D–F) and before ADP, which induced a transient depolarization. The insets summarize effects of ISO compared to ROT and AA on duration of ADP-induced state 3 decreases in ΔΨ_m (S3 time, s). Note similarities and differences of responses to each drug. ΔΨ_m transients were normalized with respect to maximal (100 %) depolarization obtained with added CCCP. Data represent mean ± SD of 3 replicates.

Figure 3

Normalized mitochondrial membrane potential (ΔΨ_m) during states 2, 3 and 4 respiration with different substrates and after adding either ISO or a blocker of ETC complexes. ISO, ROT, or AA after PM (A–C) or SUC (D–F) and before ADP, which induced a transient depolarization. The insets summarize effects of ISO compared to ROT and AA on duration of ADP-induced state 3 decreases in ΔΨ_m (S3 time, s). Note similarities and differences of responses to each drug. ΔΨ_m transients were normalized with respect to maximal (100 %) depolarization obtained with added CCCP. Data represent mean ± SD of 3 replicates.

Figure 3

Normalized mitochondrial membrane potential (ΔΨ_m) during states 2, 3 and 4 respiration with different substrates and after adding either ISO or a blocker of ETC complexes. ISO, ROT, or AA after PM (A–C) or SUC (D–F) and before ADP, which induced a transient depolarization. The insets summarize effects of ISO compared to ROT and AA on duration of ADP-induced state 3 decreases in ΔΨ_m (S3 time, s). Note similarities and differences of responses to each drug. ΔΨ_m transients were normalized with respect to maximal (100 %) depolarization obtained with added CCCP. Data represent mean ± SD of 3 replicates.

Figure 4

Normalized mitochondrial membrane potential (ΔΨ_m) during states 2, 3 and 4 respiration with SUC+ROT and either ISO or a blocker of ETC. ROT (1 µM) was given before other drugs to maximally block complex I. ISO (A), AA (B), MAL (C), or ATR (D) was added after SUC (A–D) and before adding ADP, which induced a transient depolarization. The insets summarize effects of ISO compared to AA, MAL, and ATR on duration of state 3 ADP-induced decreases in ΔΨ_m (S3 time, s). Note similarity and differences of responses to each drug. ΔΨ_m transients were normalized with respect to maximal (100 %) depolarization obtained with added CCCP. Data represents mean ± SD of 3 replicates.

Figure 4

Normalized mitochondrial membrane potential (ΔΨ_m) during states 2, 3 and 4 respiration with SUC+ROT and either ISO or a blocker of ETC. ROT (1 µM) was given before other drugs to maximally block complex I. ISO (A), AA (B), MAL (C), or ATR (D) was added after SUC (A–D) and before adding ADP, which induced a transient depolarization. The insets summarize effects of ISO compared to AA, MAL, and ATR on duration of state 3 ADP-induced decreases in ΔΨ_m (S3 time, s). Note similarity and differences of responses to each drug. ΔΨ_m transients were normalized with respect to maximal (100 %) depolarization obtained with added CCCP. Data represents mean ± SD of 3 replicates.

Figure 4

Normalized mitochondrial membrane potential (ΔΨ_m) during states 2, 3 and 4 respiration with SUC+ROT and either ISO or a blocker of ETC. ROT (1 µM) was given before other drugs to maximally block complex I. ISO (A), AA (B), MAL (C), or ATR (D) was added after SUC (A–D) and before adding ADP, which induced a transient depolarization. The insets summarize effects of ISO compared to AA, MAL, and ATR on duration of state 3 ADP-induced decreases in ΔΨ_m (S3 time, s). Note similarity and differences of responses to each drug. ΔΨ_m transients were normalized with respect to maximal (100 %) depolarization obtained with added CCCP. Data represents mean ± SD of 3 replicates.

Figure 4

Normalized mitochondrial membrane potential (ΔΨ_m) during states 2, 3 and 4 respiration with SUC+ROT and either ISO or a blocker of ETC. ROT (1 µM) was given before other drugs to maximally block complex I. ISO (A), AA (B), MAL (C), or ATR (D) was added after SUC (A–D) and before adding ADP, which induced a transient depolarization. The insets summarize effects of ISO compared to AA, MAL, and ATR on duration of state 3 ADP-induced decreases in ΔΨ_m (S3 time, s). Note similarity and differences of responses to each drug. ΔΨ_m transients were normalized with respect to maximal (100 %) depolarization obtained with added CCCP. Data represents mean ± SD of 3 replicates.

Figure 5

Mitochondrial O₂ concentration (nmol•min⁻¹) during states 2, 3 and 4 respiration with different substrates and either ISO or a blocker of ETC. ISO, ROT, or AA was added after PM (A–C) or SUC (D–F) and before ADP. Adding ADP induced a transient depolarization. The insets summarize effects of added ISO, ROT and AA on state 3 and state 4 respiration rates (nmol•min⁻¹•mg⁻¹protein). Note similarity and differences of responses to each drug. Data represent mean ± SD of 3 replicates.

Figure 5

Mitochondrial O₂ concentration (nmol•min⁻¹) during states 2, 3 and 4 respiration with different substrates and either ISO or a blocker of ETC. ISO, ROT, or AA was added after PM (A–C) or SUC (D–F) and before ADP. Adding ADP induced a transient depolarization. The insets summarize effects of added ISO, ROT and AA on state 3 and state 4 respiration rates (nmol•min⁻¹•mg⁻¹protein). Note similarity and differences of responses to each drug. Data represent mean ± SD of 3 replicates.

Figure 5

Mitochondrial O₂ concentration (nmol•min⁻¹) during states 2, 3 and 4 respiration with different substrates and either ISO or a blocker of ETC. ISO, ROT, or AA was added after PM (A–C) or SUC (D–F) and before ADP. Adding ADP induced a transient depolarization. The insets summarize effects of added ISO, ROT and AA on state 3 and state 4 respiration rates (nmol•min⁻¹•mg⁻¹protein). Note similarity and differences of responses to each drug. Data represent mean ± SD of 3 replicates.

Figure 5

Mitochondrial O₂ concentration (nmol•min⁻¹) during states 2, 3 and 4 respiration with different substrates and either ISO or a blocker of ETC. ISO, ROT, or AA was added after PM (A–C) or SUC (D–F) and before ADP. Adding ADP induced a transient depolarization. The insets summarize effects of added ISO, ROT and AA on state 3 and state 4 respiration rates (nmol•min⁻¹•mg⁻¹protein). Note similarity and differences of responses to each drug. Data represent mean ± SD of 3 replicates.

Figure 5

Mitochondrial O₂ concentration (nmol•min⁻¹) during states 2, 3 and 4 respiration with different substrates and either ISO or a blocker of ETC. ISO, ROT, or AA was added after PM (A–C) or SUC (D–F) and before ADP. Adding ADP induced a transient depolarization. The insets summarize effects of added ISO, ROT and AA on state 3 and state 4 respiration rates (nmol•min⁻¹•mg⁻¹protein). Note similarity and differences of responses to each drug. Data represent mean ± SD of 3 replicates.

Figure 5

Mitochondrial O₂ concentration (nmol•min⁻¹) during states 2, 3 and 4 respiration with different substrates and either ISO or a blocker of ETC. ISO, ROT, or AA was added after PM (A–C) or SUC (D–F) and before ADP. Adding ADP induced a transient depolarization. The insets summarize effects of added ISO, ROT and AA on state 3 and state 4 respiration rates (nmol•min⁻¹•mg⁻¹protein). Note similarity and differences of responses to each drug. Data represent mean ± SD of 3 replicates.

Figure 6

Mitochondrial O₂ concentration (nmol•min⁻¹) during states 2, 3 and 4 respiration with substrate SUC and either ISO or a blocker (AA) of ETC. ROT (1 µM) was given before succinate to maximally block complex I and RET. The insets summarize effects of added ISO, ROT, and AA on state 3 and state 4 respiration rates (nmol•min⁻¹•mg⁻¹protein). Note similarities and differences of responses to each drug. Data represent mean ± SD of 3 replicates.

Figure 6

Mitochondrial O₂ concentration (nmol•min⁻¹) during states 2, 3 and 4 respiration with substrate SUC and either ISO or a blocker (AA) of ETC. ROT (1 µM) was given before succinate to maximally block complex I and RET. The insets summarize effects of added ISO, ROT, and AA on state 3 and state 4 respiration rates (nmol•min⁻¹•mg⁻¹protein). Note similarities and differences of responses to each drug. Data represent mean ± SD of 3 replicates.

Figure 7

Schema depicting ADP-induced changes in magnitude and duration of mitochondrial bioenergetic variables (NADH, ΔΨ_m, respiration), and presumed changes in electron transfer activity (ETA) along the ETC before and after ISO, are compared with ROT or AA to induce attenuation of complex I and complex III with NAD-linked substrate PM (A), FAD-linked substrate SUC (B), and substrate SUC+ROT after complete inhibition of complex I with 1 µM ROT (C). Small arrows () indicate the relative number of electrons transported during FET before and after attenuation of complex I or complex III. Broken arrows () near complex I (CI) and complex III (CIII) indicate mild inhibition of complexes and the solid arrow () near complex I shows complete inhibition of complex I with ROT (1 µM). Complexes I-IV (CI-IV); PM, K⁺-pyruvate/malate; SUC, K⁺-succinate; ISO, isoflurane; ROT, rotenone; AA, antimycin A; FET, forward electron transfer; RET, reverse electron transfer.

Figure 7

Schema depicting ADP-induced changes in magnitude and duration of mitochondrial bioenergetic variables (NADH, ΔΨ_m, respiration), and presumed changes in electron transfer activity (ETA) along the ETC before and after ISO, are compared with ROT or AA to induce attenuation of complex I and complex III with NAD-linked substrate PM (A), FAD-linked substrate SUC (B), and substrate SUC+ROT after complete inhibition of complex I with 1 µM ROT (C). Small arrows () indicate the relative number of electrons transported during FET before and after attenuation of complex I or complex III. Broken arrows () near complex I (CI) and complex III (CIII) indicate mild inhibition of complexes and the solid arrow () near complex I shows complete inhibition of complex I with ROT (1 µM). Complexes I-IV (CI-IV); PM, K⁺-pyruvate/malate; SUC, K⁺-succinate; ISO, isoflurane; ROT, rotenone; AA, antimycin A; FET, forward electron transfer; RET, reverse electron transfer.

Figure 7

Schema depicting ADP-induced changes in magnitude and duration of mitochondrial bioenergetic variables (NADH, ΔΨ_m, respiration), and presumed changes in electron transfer activity (ETA) along the ETC before and after ISO, are compared with ROT or AA to induce attenuation of complex I and complex III with NAD-linked substrate PM (A), FAD-linked substrate SUC (B), and substrate SUC+ROT after complete inhibition of complex I with 1 µM ROT (C). Small arrows () indicate the relative number of electrons transported during FET before and after attenuation of complex I or complex III. Broken arrows () near complex I (CI) and complex III (CIII) indicate mild inhibition of complexes and the solid arrow () near complex I shows complete inhibition of complex I with ROT (1 µM). Complexes I-IV (CI-IV); PM, K⁺-pyruvate/malate; SUC, K⁺-succinate; ISO, isoflurane; ROT, rotenone; AA, antimycin A; FET, forward electron transfer; RET, reverse electron transfer.

Figure 8

Schema depicting electron transfer from substrate SUC to downstream and upstream complex I and complex III, respectively. Electrons from SUC pass through FADH₂ to the Q pool, which transfers electrons to the Q site of complex I. Within complex I, electrons transferred to the Q site can either go in the reverse direction via RET through FeS clusters and FMN to generate NADH from NAD⁺ or go in the forward direction via FET to produce QH₂; RET vs. FET is dependent on the standard reduction potential across complex I. Binding of ISO, like ROT, may inhibit the transfer of electrons from the QH₂ of complex I to the Q pool to complex III. Solid arrows () show electrons transported in the forward direction (FET) and broken arrows () show electrons transported in the reverse direction (RET). Question marks (?) show the possible indirect mechanisms of attenuation in FET either via attenuation of electron transfer from Q site of complex I to Q pool or succinate generated accumulation of oxaloacetate due to ROT/ISO mediated decrease in NADH oxidation.