The mitochondrial bioenergetic phenotype for protection from cardiac ischemia in SUR2 mutant mice

Abstract

The sulfonylurea receptor-2 (SUR2) is a subunit of ATP-sensitive potassium channels (K(ATP)) in heart. Mice with the SUR2 gene disrupted (SUR2m) are constitutively protected from ischemia-reperfusion (I/R) cardiac injury. This was surprising because K(ATP), either sarcolemmal or mitochondrial or both, are thought to be important for cardioprotection. We hypothesized that SUR2m mice have an altered mitochondrial phenotype that protects against I/R. Mitochondrial membrane potential (ΔΨ(m)), tolerance to Ca(2+) load, and reactive oxygen species (ROS) generation were studied by fluorescence-based assays, and volumetric changes in response to K(+) were measured by light scattering in isolated mitochondria. For resting SUR2m mitochondria compared with wild type, the ΔΨ(m) was less polarized (46.1 ± 0.4 vs. 51.9 ± 0.6%), tolerance to Ca(2+) loading was increased (163 ± 2 vs. 116 ± 2 μM), and ROS generation was enhanced with complex I [8.5 ± 1.2 vs. 4.9 ± 0.2 arbitrary fluorescence units (afu)/s] or complex II (351 ± 51.3 vs. 166 ± 36.2 afu/s) substrates. SUR2m mitochondria had greater swelling in K(+) medium (30.2 ± 3.1%) compared with wild type (14.5 ± 0.6%), indicating greater K(+) influx. Additionally, ΔΨ(m) decreased and swelling increased in the absence of ATP in SUR2m, but the sensitivity to ATP was less compared with wild type. When the mitochondria were subjected to hypoxia-reoxygenation, the decrease in respiration rates and respiratory control index was less in SUR2m. ΔΨ(m) maintenance in the SUR2m intact myocytes was also more tolerant to metabolic inhibition. In conclusion, the cardioprotection observed in the SUR2m mice is associated with a protected mitochondrial phenotype resulting from enhanced K(+) conductance that partially dissipated ΔΨ(m). These results have implications for possible SUR2 participation in mitochondrial K(ATP).

Fig. 1.

Mitochondrial membrane potential (ΔΨm). ΔΨm was measured using the potentiometric dye rhodamine. Isolated sulfonylurea receptor-2 (SUR2) mutant (SUR2m) or wild-type (WT) mitochondria (0.5 mg protein) were added to the respiration buffer containing rhodamine (50 nM) and supplemented with oligomycin (5 μg/ml), pyruvate (5 mM), and malate (5 mM) in the presence (A) or absence (B) of ATP (200 μM). In A or B, fluorescence was allowed to stabilize after the addition of mitochondria (mito) and diazoxide (dia, 100 μM) indicated by the arrows. The mitochondria uncoupler carbonylcyanide-4-trifluoromethoxyphenylhydrazone (FCCP, 1 μM) was then added to completely depolarize ΔΨm. ΔΨm was quantified as the percentage of maximum depolarization in the absence or presence of ATP (C). Summary data are presented as means ± SE **P < 0.001 and *P < 0.05. Here and in Figs. 2–7, the no. of experiments is presented as ordered pairs (N,n) where N is the total no. of measurements and n is the no. of different mice used to make the measurements.

Fig. 2.

Mitochondria swelling (change in volume) resulting from K⁺ influx. Mitochondrial (0.5 mg protein) volume was measured as the light (λ; 540 nM) scattered due to mitochondria in K⁺ buffer. The volume increased (i.e., light scattering was decreased) when mitochondria previously in 5 mM K⁺ were suspended in a 140 mM K⁺ respiration buffer supplemented with pyruvate and malate (5 mM each) and ATP (200 μM). The volume (V) just before adding valinomycin (Val; 2 nM) is expressed as the percentage of maximum volume (V_max) induced by Val. A representative experiment (A) and summary data (B) show that swelling (change in volume) was significantly greater for SUR2m in the presence of ATP. The effect of absence of ATP on mitochondria volume is shown in C and D. Representative traces are shown in C. The percentage difference between volumes at time 200 s (V₂₀₀) in the presence of ATP from V₂₀₀ in absence of ATP is plotted in D. Absence of ATP causes significantly greater change in volume (swelling) in WT. The rate of swelling was determined by a negative slope of the one-phase exponential decay curve fitted in C (see text for results). Data from the no. of experiments and mice (N,n) are presented are means ± SE. **P < 0.001 and *P < 0.05.

Fig. 3.

Tolerance to Ca²⁺ overload. Tolerance to Ca²⁺ overload was determined as the amount of Ca²⁺ needed to dissipate the ΔΨm by at least 5% from the baseline in 4 s. Mitochondria (0.5 mg protein) were loaded with rhodamine (50 nM) in the presence of pyruvate and malate (5 mM each), oligomycin (5 μg/ml), and ATP (200 μM). After stabilization of the fluorescence, mitochondria were challenged with 20 μM pulses of Ca²⁺ (indicated by arrows) as shown in the traces in A. Finally, ΔΨm was completely depolarized by adding FCCP (1 μM). The cumulative amount of Ca²⁺ needed to depolarize the mitochondria were quantified (B). Data from the no. of experiments and mice (N,n) are presented as means ± SE. *P < 0.05 compared with WT.

Fig. 4.

Reactive oxygen species (ROS) generation. ROS generation was determined as H₂O₂ generation with Amplex red in the presence of ATP. The mitochondria (0.5 mg protein) were supplemented with pyruvate and malate (5 mM each) (A) or succinate (10 mM) (B). The rate of ROS generation with pyruvate and malate (C) or succinate (D) was quantified as the rate of fluorescence increase by the slope of the curve. H₂O₂ production was blocked by addition of the uncoupler FCCP (1 μM). Data from the no. of experiments and mice (N, n) are presented as means ± SE. *P < 0.05.

Fig. 5.

Effect of metabolic inhibition (MI) on mitochondrial ΔΨm in intact myocytes. Quiescent myocytes were loaded with tetramethylrhodamine ester (TMRE; 50 nM) and perfused with normal Tyrode or Tyrode in which glucose is substituted with 2-deoxyglucose (20 mM) and NaCN (CN, 0.5 mM) as shown in the protocol in A. Dinitrophenol (DNP; 100 μM) was added to completely depolarize the mitochondria at 20 min. Representative images of the cells are shown in A. ΔΨm was estimated from the TMRE fluorescence as the percentage decrease from the maximum (with DNP) at 5 min (normal Tyrode) and at 18 min (after metabolic inhibition) (B). *P < 0.05, **P < 0.001, and ***P < 0.0001.