Isoflurane protects cardiomyocytes and mitochondria by immediate and cytosol-independent action at reperfusion

Abstract

Background and purpose: The volatile anaesthetic isoflurane protects the heart from ischaemia and reperfusion (I/R) injury when applied at the onset of reperfusion [anaesthetic postconditioning (APoC)]. However, the mechanism of APoC-mediated protection is unknown. In this study, we examined the effect of APoC on mitochondrial bioenergetics, mitochondrial matrix pH (pH(m)) and cytosolic pH (pH(i)), and intracellular Ca(2+).

Experimental approach: Cardiac mitochondria from Wistar rats were isolated after in vivo I/R with or without APoC (1.4%-vol isoflurane, 1 minimum alveolar concentration), and mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential (DeltaPsi(m)), and oxygen consumption were assessed. In isolated cardiomyocytes and isolated mitochondria I/R injury was produced in vitro, with or without APoC (0.5 mM isoflurane). Intracellular Ca(2+), pH(m), pH(i) and DeltaPsi(m) were monitored with SNARF-1, TMRE and fluo-4, respectively. Myocyte survival was assessed when APoC was induced at pH 7.4 and 7.8. In isolated mitochondria oxygen consumption and ATP synthesis were measured.

Key results: In vivo APoC protected against mPTP opening, slowed mitochondrial respiration and depolarized mitochondria. APoC decreased the number of hypercontracted cardiomyocytes at pH 7.4, but not at pH 7.8. APoC attenuated intracellular Ca(2+) accumulation, maintained lower pH(m), and preserved DeltaPsi(m) during reoxygenation. Isoflurane did not affect the regulation of cytosolic pH. In mitochondria, APoC preserved ATP production rate and respiration.

Conclusions and implications: At reperfusion, APoC inhibited mitochondrial respiration, depolarized mitochondria and acidified pH(m). These events may lead to inhibition of mPTP opening and, consequently, to preserved DeltaPsi(m) and ATP synthesis. This reduces intracellular Ca(2+) overload and cell death.

Figure 5

Isoflurane does not modulate NHE exchanger and carbonic anhydrase activities. (A) Representative images showing whole-cell loading of SNARF-1. (B) Representative trace of pH_i recovery from the cardiomyocyte following exposure to NH₄Cl (10 mM) in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered Tyrode solution, pH_o 7.4. The trace demonstrates that after two consecutive NH₄Cl pulses, pH_i recovery from an intracellular acid load was not different in the presence of isoflurane. (C) Summary for recovery of pH_i from NH₄Cl -induced acidosis in the presence and subsequent absence of 0.5 mM isoflurane. (D) Effect of isoflurane on carbonic anhydrase activity following intracellular acid loading induced by application of 5% CO₂/HCO^-₃. The magnitude of acidosis was not different in the presence of isoflurane. Data are presented as means ± standard deviation. Traces are representative from at least five measurements. SNARF-1, 5-(and-6)-carboxy SNARF-1, acetoxymethyl ester; NHE, Na⁺-H⁺ exchanger, CA, carbonic anhydrase.

Figure 1

Isoflurane postconditioning in vivo protects mitochondria from ischaemia and reperfusion (I/R) injury. (A) Original recordings of ΔΨ_m in mitochondria from control and APoC hearts in the presence of pyruvate/malate. (B) Summarized data showing ΔΨ_m relative to maximum depolarization. (C) The amount of Ca²⁺ necessary to open the mPTP in sham, control and APoC mitochondria. Values are means ± standard deviation. *P < 0.05 versus sham, #P < 0.05 versus control, n= 9. ΔΨ_m, mitochondrial membrane potential; mPTP, mitochondrial permeability transition pore; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; S2, state 2 respiration; APoC, isoflurane postconditioning.

Figure 2

Isoflurane postconditioning protects isolated cardiomyocytes from H/R injury. (A) Representative images of isolated cardiomyocyte changes during H/R. During reoxygenation after hypoxia cardiomyocytes became spherical, hypercontracted and exhibited membrane blebs. APoC protected cardiomyocytes against H/R injury at pH 7.4, but the protection was lost when APoC was performed at pH 7.8. (B) Summary graph showing the percentage of cell survival after 35 min of reoxygenation. Data are means ± standard deviation; *P < 0.05 versus time control; #P < 0.05 versus ApoC; n= 5. TC, time control; H/R, hypoxia and reoxygenation; APoC, isoflurane postconditioning.

Figure 3

APoC decreases cytosolic Ca²⁺ overload in cardiomyocytes during reoxygenation. (A) Representative image of isolated cardiomyocytes loaded with fluo-4, fluorescent indicator for intracellular Ca²⁺. Under hypoxic conditions intracellular Ca²⁺ increased relative to pre-hypoxic values and then recovered at reoxygenation. During reoxygenation, Ca²⁺ remained low at early reoxygenation, but then increased. APoC decreased cytosolic Ca²⁺ overload during reoxygenation. (B) Summarized data for intracellular Ca²⁺ during hypoxia and reoxygenation. Standard deviations (SDs) are omitted for clarity in APoC group. Values are means ± SD; *P < 0.05 versus time control; #P < 0.05 versus control; n= 17. APoC, isoflurane postconditioning.

Figure 4

APoC prevents rapid pH_m recovery in cardiomyocytes at reoxygenation. (A) Representative images showing selective mitochondrial loading of SNARF-1. SNARF-1 colocalizes with mitochondrial flavoproteins (FP). (B) pH_m decreased during hypoxia as shown by a decrease in SNARF-1 ratio. Upon reoxygenation, pH_m quickly recoverd to baseline levels. Treatment of cardiomyocytes with isoflurane at the beginning of reoxygenation preserved acidic intracellular pH_m during reoxygenation as compared with the control group. (C) The panel shows representative time lapse of baseline pH_m recording. Baseline pH_m was reduced upon addition of isoflurane into superfusing solution, and this effect was reversible after anaesthetic washout. (D) Administration of the mitochondrial uncoupler 2-4-dinitrophenol (DNP) prevented any further effect of isoflurane on pH_m. Data are presented as means ± standard deviation, *P < 0.05 versus APoC; n= 20. APoC, isoflurane postconditioning; SNARF-1, 5-(and-6)-carboxy SNARF-1, acetoxymethyl ester.

Figure 6

APoC preserves ΔΨ_m in isolated myocytes throughout reoxygenation. (A) Representative recording of ΔΨ_m changes during H/R. Hypoxia caused a significant decrease in the standard deviation of TMRE fluorescence indicating mitochondrial depolarization whereas reoxygenation caused an increase in the standard deviation indicating recovery of ΔΨ_m. Treatment of cardiomyocytes with isoflurane at the beginning of reoxygenation preserved ΔΨ_m as compared with control cells. (B) Summary data for control and APoC groups at 10 min of reoxygenation. (C) Effect of isoflurane on mitochondrial flavoprotein fluorescence, and summary data (inset). (D) Representative trace of ΔΨ_m in isolated mitochondria. Isoflurane induced slight depolarization of ΔΨ_m. Data are means ± standard deviation; n= 15; n= 6 in flavoprotein and isolated mitochondria experiments. *P < 0.05 versus control. APoC, isoflurane postconditioning, TMRE, tetramethylrhodamine ethyl ester. FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone.

Figure 7

In vitro APoC protects isolated mitochondria from H/R injury. (A) Typical recordings showing respiration under baseline condition and after reoxygenation in control and isoflurane-postconditioned mitochondria. Oxygen consumption was initiated by addition of pyruvate/malate (PM), accelerated by addition of ADP (state 3 respiration), and decelerated after all ADP was consumed (state 4 respiration). Following H/R, state 3 respiration was preserved in APoC-treated mitochondria. (B) After H/R, state 3 respiration decreased in all groups; however, state 3 respiration in APoC-treated mitochondria was better preserved as compared with control and preconditioned mitochondria. (C) Respiratory control ratio (RCR) was better maintained in APoC treated mitochondria, whereas preconditioning did not improve RCR. (D) The rate of ATP production was decelerated after reoxygenation. However, ATP synthesis in APoC-treated mitochondria was better preserved as compared with control and preconditioned mitochondria. Values are mean ± SD. *P < 0.05 versus baseline or APoC; #P < 0.05 versus control or APC. C, control; APoC, isoflurane postconditioning; APC, isoflurane preconditioning; open circle, control; solid circle, isoflurane postconditioning, n= 7 per group.