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

SUR2m mice.

Studies were conducted in accordance with guidelines set forth by the University of Wisconsin, Madison, the Animal Welfare Act regulations, and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The studies were approved by the Institutional Animal Care and Use Committee at the University of Wisconsin. Male FVB WT or SUR2m mice (n = 40 each) aged 8–12 wk were used. SUR2m mice were previously generated by targeted disruption of exons 14–18 encoding the first nucleotide binding fold (5). Detailed methods are available in Supplemental Fig. S1 (Supplemental data for this article may be found on the American Journal of Physiology: Heart and Circulatory Physiology website).

Isolation of cardiomyocytes and mitochondria.

Cardiac ventricular cell mitochondria were isolated by homogenization and differential centrifugation as described previously (18). Protein concentration was assessed, and mitochondria were used within 4 h. For all measurements, 0.5 mg mitochondrial protein/ml was used. Ventricular myocytes were isolated by enzymatic dissociation with 0.2 mg/ml Liberase blendzyme (Roche Diagnostic) as reported previously (27) with some modifications.

ΔΨ_m and tolerance to Ca²⁺ upload.

ΔΨ_m was monitored spectrophotometrically (λ_ex 503 nm, λ_em 527 nm) using rhodamine 123 (5 nM) as described (22) in the presence or absence of ATP (200 μM) and oligomycin (5 μg/ml). Diazoxide (0.1 mM), Gly (20 μM), and carbonylcyanide-4-trifluoromethoxyphenylhydrazone (FCCP; 1 μM) were added when indicated. ΔΨ_m was expressed as the percentage of rhodamine 123 fluorescence relative to the fluorescence after FCCP. Tolerance to Ca²⁺ loading was assessed in the presence of ATP (200 μM) by adding Ca²⁺ in 20-μM increments and determining the cumulative amount of Ca²⁺ required to cause at least 5% depolarization within 4 s. Mitochondria were suspended in EGTA-free buffer.

Measurement of K⁺ influx by mitochondrial swelling.

Mitochondrial swelling was determined as change in mitochondria volume. As mitochondria volume increases the light scattering decreases. A change in mitochondria volumes was measured as change in light (λ; 540 nM) scattering, in response to K⁺, in the presence or absence of ATP (200 μM) as described previously (10). Initially, the isolated mitochondria were stored in low K⁺ (5 mM) without ATP. The mitochondria volume increased when mitochondria were moved from low-K⁺ (5 mM) to a high-K⁺ (140 mM) buffer with or without ATP. ATP or vehicle was added at time 0. In some experiments, valinomycin (Val; 2 nM) was added to induce the maximal volume (V_max) at the times indicated. In the presence of ATP, the swelling was expressed as percentage change in volume (V) just before addition of Val normalized to the V_max. The effect of ATP on swelling was determined as the percentage difference in volume at 200 s (V₂₀₀) in the presence of ATP from the V₂₀₀ in the absence of ATP.

Mitochondrial H₂O₂ release rate.

Rates of H₂O₂ release were measured spectrophotometrically (λ_ex 530 nm, λ_em 583 nm) using Amplex red (12.5 μM) in the presence of 0.1 units/ml horseradish peroxidase (3) with complex I substrates (pyruvate and malate, 5 mM each) or complex II substrate succinate (10 mM). H₂O₂ production was blocked by FCCP (1 μM). Rates of H₂O₂ were expressed as the slope of the curves (afu/s) after addition of substrates.

Mitochondrial O₂ consumption.

O₂ consumption in mitochondria (0.5 mg/ml) was measured polarographically as described (12). State 2 respiration was initiated by adding succinate (10 mM) and rotenone (2 μM) or with pyruvate and malate (5 mM each), state 3 with 250 μM ADP, and state 4 respiration was measured after complete phosphorylation of ADP to ATP. Hypoxia (0% O₂) and reoxygenation was achieved by closing and opening of the chamber, respectively.

Effect of metabolic inhibition on myocytes.

ΔΨ_m was determined with tetramethylrhodamine ester (TMRE, 25 nM, λ_ex 543 nm and λ_em 594 nm) as described (18). Myocytes were perfused with normal Tyrode for 5 min and then with a Tyrode in which glucose was substituted with deoxyglucose (20 mM) and NaCN (0.5 mM) (2-DG/CN Tyrode) to cause metabolic inhibition for 15 min. TMRE fluorescence was measured to determine ΔΨ_m, and 2,4-dinitrophenol (DNP, 0.1 mM) was added at the end to completely depolarize the mitochondria.

Statistical analysis.

Data were expressed as means ± SE. Two groups of data were compared by nonparametric unpaired Student's t-test, and more than two groups were analyzed by one-way ANOVA following a Bonferroni posttest. The values were considered statistically significant when P < 0.05.

ΔΨ_m in isolated mitochondria.

ΔΨ_m was measured in the presence or absence of 200 μM ATP; mitochondria were treated with oligomycin and supplemented with a metabolic substrate (Fig. 1). Representative traces show that ΔΨ_m of SUR2m mice were less polarized than WT with ATP present (Fig. 1A), but the ΔΨ_m of SUR2m tended to be more polarized than WT with ATP absent (Fig. 1B). Summary data (Fig. 1C) show the significant differences. In the absence of ATP, the change in ΔΨ_m of WT was greater (∼13%) than the change in SURm mice (∼5%), suggesting that, although ΔΨ_m in SUR2m was sensitive to ATP, ΔΨ_m was more sensitive to ATP in WT. Furthermore, in the presence of ATP, diazoxide decreased the ΔΨ_m of WT but did not affect the ΔΨ_m of SUR2m (Fig. 1A). In absence of ATP, Gly (20 μM) increased the ΔΨ_m in WT but not in SUR2m mitochondria (Supplemental Fig. S1). These results on ΔΨ_m are consistent with a diazoxide-, Gly-, and ATP-sensitive K⁺ conductance in WT mitochondria that has been disrupted in SUR2m.

K⁺ influx in the mitochondria and matrix swelling.

K⁺ influx in the matrix causes a partial depolarization, increased ion exchange, and increased water intake in mitochondria, causing swelling. This volumetric change in response to increased extramitochondrial K⁺ has been a standard method to assess K⁺ influx in mitochondria (7). Volume changes, in the presence of ATP, in mitochondria from SUR2m and WT mice are shown in Fig. 2A. Val, a K⁺ ionophore, was added at the end to induce maximum volume to allow normalization. Swelling was expressed as the percentage of volume (V) just before addition of Val normalized to the V_max and summarized in Fig. 2B. In the presence of ATP, swelling was significantly greater in SUR2m mitochondria than WT mitochondria. The effect of ATP on swelling was determined as the difference in V₂₀₀ in the absence of ATP from V₂₀₀ in the presence of ATP (Fig. 2C). At the end of 200 s, the difference in volumes (swelling) was significantly greater in WT compared with SUR2m (Fig. 2D). Thus the swelling was sensitive to ATP in mitochondria from both WT and SUR2m mice but was more sensitive in WT mitochondria. The rate of swelling was determined, as rate constant (k), from the one-phase exponential decay curves shown in Fig. 2C. In the presence of ATP, the “k” of WT mitochondria [0.002 ± 0.002 arbitrary units (AU)/s] trended to be less than that of SUR2m mitochondria (0.010 ± 0.003 AU/s); however, the data did not reach a significant difference. In the absence of ATP, the k increased in WT mitochondria by 17.5 times, whereas in SUR2m mitochondria it increased by only 0.2 times. Maximum swelling in WT mitochondria, in the absence of ATP, reached saturation levels within 100 s in contrast to the SUR2m mitochondria, which swelled more slowly. The data suggest that mitochondrial swelling depended on an ATP-induced increased K⁺ flux more strongly in WT than in SUR2m mitochondria.

Sensitivity of ΔΨ_m to Ca²⁺ load.

Mitochondrial depolarization decreases the electrochemical gradient for the uptake of Ca²⁺ in the matrix and dissipates the capacity of mitochondria to load Ca²⁺ in the matrix (13). To test resistance to Ca²⁺ loading, mitochondria from SUR2m and WT mice were challenged with 20-μM pulses of Ca²⁺ concentrations until the occurrence of dissipation of ΔΨ_m (as described in materials and methods), as a measure of tolerance to mitochondrial Ca²⁺ uptake (Fig. 3). Representative traces (Fig. 3A) and the summary data (Fig. 3B) show that, in mitochondria from WT mice, the Ca²⁺ concentration required to cause depolarization was 47 μM lower than the concentration required for the SUR2m mice. These data show that tolerance to Ca²⁺ overload was greater in the SUR2m mitochondria, consistent with a protected phenotype.

ROS generation in the mitochondria.

Increase in signaling ROS is associated with the protection against I/R injury by activating protective pathways (17). To estimate ROS generation, we measured production of H₂O₂ in the presence of ATP when the mitochondria were supplemented with complex I substrate (Fig. 4A) or complex II substrate (Fig. 4B). The rate of ROS generation reported as change in afu per second (Fig. 4, C and D) was significantly greater for SUR2m mitochondria compared with WT for both complex I substrates (Fig. 4C) and for complex II substrate (Fig. 4D) (P < 0.05). Thus, at resting state, SUR2m mitochondria demonstrated increased basal production of ROS, which could serve in protective signaling.

Mitochondrial respiration and hypoxia-reoxygenation.

The less-polarized ΔΨ_m of SUR2m mitochondria, increased resistance to Ca²⁺ overload, and increased ROS generation observed in SUR2m were consistent with the protective mitochondria phenotype demonstrated in other models of protection (2, 6, 18, 24). To test resistance to hypoxia, changes in the mitochondria respiration rates from normoxia to posthypoxia-reoxygenation were determined. O₂ consumption was measured at state 2, 3, and 4 as an index of respiration rate and is summarized in Table 1. The respiration control index (RCI) was determined as a ratio of state 3 and 4 respiration rates to estimate coupling in these mitochondria. The prehypoxic RCI for WT and SUR2m mitochondria respiring with succinate, or pyruvate and malate, were >2.5, indicating coupled mitochondria. The respiration rates and RCI of the freshly isolated SUR2m mitochondria, respiring on succinate or pyruvate and malate, were greater at normoxia after isolation than the WT mitochondria (P = not significant). This may represent a protection conferred by SUR2m mitochondria after the stress of the isolation procedure. After hypoxia-reoxygenation, RCI was reduced in both WT and SUR2m (Table 1), but the decrease was greater in the WT mitochondria compared with SUR2m. Individually, the decrease in state 3 and 4 respiration rates due to hypoxia-reoxygenation in WT mitochondria was greater than the decrease in SUR2m mitochondria. Together these data suggest that the respiration was better preserved in SUR2m mitochondria after hypoxia-reoxygenation compared with WT mitochondria.

ΔΨ_m in intact myocytes and protection against metabolic inhibition.

Cytosolic components of myocytes may modulate the functions of mitochondria; therefore, we measured mitochondrial ΔΨ_m in intact myocytes. To evaluate the effect of metabolic inhibition, the TMRE-loaded myocytes were perfused with 2-DG/CN Tyrode. Representative images of TRME fluorescence at 5 min (with normal Tyrode), at 10 and 18 min with 2-DG/CN-Tyrode, and at 20 min when DNP was added to completely depolarize the mitochondria are shown in Fig. 5A. ΔΨ_m was measured as the percentage change in fluorescence from the baseline, and summary data are presented in Fig. 5B. In WT or SUR2m myocytes, TMRE fluorescence decreased with time when perfused with the 2-DG/CN-Tyrode. However, after 18 min, the decrease in TMRE florescence was greater in the WT compared with the SUR2m myocytes (Fig. 5B). DNP addition completely dissipated the mitochondria ΔΨ_m. These data show a strong preservation of cardiac mitochondrial function after a strong metabolic challenge as shown by preserved ΔΨ_m in the SUR2m mice myocyte.

Mitochondria from SUR2m mice have a favorable bioenergetic phenotype for protection.

Results from this study give insight into the mechanisms by which SUR2m mice unexpectedly showed a constitutively protected myocardial phenotype. Compared with WT mice, mitochondria from SUR2m hearts were slightly depolarized at rest (Fig. 1), had greater volume in the presence of ATP (Fig. 2), were more resistant to Ca²⁺ overload (Fig. 3), and had a basal level of mildly increased ROS production (Fig. 4), all consistent with a favorable bioenergetic phenotype for protection (18). In addition, mild ROS increase activates protective signaling in the mitochondria, although this is debatable in the literature (18). In addition, the function of SUR2m mitochondria, as assessed by RCI, recovered better after hypoxia reperfusion compared with the WT mitochondria (Table 1). The prehypoxic RCI of the SUR2m mitochondria was better compared with the RCI of the WT mitochondria, and, individually, the prehypoxic respiration rates were also faster in SUR2m mitochondria, consistent with the partially depolarized state of these mitochondria to start with. This suggests that the mitochondria from the SUR2m mice were protected from the stress of the isolation procedure. Moreover, the posthypoxic state 3 and state 4 respiration recovered better in the SUR2m mitochondria compared with the WT mitochondria (Table 1), indicating that more WT mitochondria were uncoupled and underwent hypoxia- and reoxygenation-induced injury. The change observed following anoxia-reoxygenation was mainly due to a decrease in state 3 respiration, whereas state 4 was unaltered. This suggests that the major point of the damage might be the respiratory chain, whereas inner membrane integrity was not different between the two groups. The protective phenotype of SUR2m mitochondria was also seen in the intact myocytes where the ΔΨ_m of SUR2m myocytes were maintained longer than in the WT myocyte after metabolic inhibition (Fig. 5). All of these observations are consistent with a constitutive protective bioenergetic mitochondrial phenotype in SUR2m mice.

Is there a diazoxide- and WT-insensitive mitochondrial K_ATP in cells lacking full-length SUR2?

The slightly dissipated ΔΨ_m of the SUR2m at rest suggests a net change in the balance of the K⁺/H⁺ conductance that sets ΔΨ_m (21). Although we cannot exclude a decrease in H⁺ conductance or gradient as the mechanism for this effect, the ΔΨ_m data (Fig. 1) and the volume data (Fig. 2) are more consistent with an increased K⁺ influx (10, 14) in the SUR2m mitochondria. Moreover, ΔΨ_m of SUR2m mitochondria was more depolarized in the absence of ATP compared with the ΔΨ_m in the presence of ATP (Fig. 1), indicating that SUR2m ΔΨ_m had some sensitivity to ATP. ATP synthase was inhibited with oligomycin; therefore, this ΔΨ_m sensitivity to ATP was not due to ATP synthase-induced ion flux but was more likely caused by a membrane conductance. Moreover, swelling experiments indicated an ATP-sensitive K⁺ flux in the SUR2m mitochondria. However, diazoxide did not change the ΔΨ_m in SUR2m mitochondria, suggesting that the diazoxide-sensitive SUR2 elements in these mitochondria were absent. Taken together, the results showed that, in SUR2m mitochondria, both ΔΨ_m (Fig. 1) and volume (Fig. 2) are influenced by K⁺ influx, sensitive to ATP (but less so than WT), and are insensitive to diazoxide.

A diazoxide- and Gly-sensitive mitochondrial K_ATP (mitoK_ATP) has been thought to be important for cardioprotection (20); if it is absent from SUR2m then how can this importance be reconciled? Our recent studies showed that, in the SUR2m mouse, the full-length SUR2 is absent, but short-form variants of SUR2 in various sizes (28, 55, and 66 kDa) persist and may account for a low-density Gly-insensitive sarcK_ATP found in SUR2m cardiomyocytes (23). A 55-kDa SUR2 variant unique to the mitochondrial fraction was cloned, and, when mitochondrial targeting sequences were removed, it was coexpressed with Kir6.x pores and produced a Gly-insensitive K_ATP in a heterlogous expression system (23) with an ATP sensitivity much less than conventional K_ATP. The shortened structure of this short SUR2 form lacks transmembrane segments 1–6 and nucleotide-binding domain 1, which are reported to confer diazoxide sensitivity (1). We speculate that the short-form variants, and the 55-kDa form in particular, retained in the SUR2m mitochondria form a type of mitoK_ATP that is both insensitive to diazoxide and is sensitive to ATP but with a decreased sensitivity compared with WT. These mitoK_ATP are responsible for the ATP-sensitive ΔΨ_m and swelling observed in the SUR2m mitochondria. Moreover, at physiological concentrations of ATP, these mitoK_ATP channels in SUR2m would remain open and cause a persistent potassium leak that underlies the protective phenotype of a partially dissipated ΔΨ_m and subsequent effects on volume, mitochondrial Ca²⁺ homeostasis, and ROS signaling.

Implications for mitoK_ATP and mechanisms of protection.

Studies on mitochondrial function have indicated several players that may be involved in cardioprotection (21) and that must be considered in interpreting the results of this study. These include the flux of anions through an inner membrane anion channel, transport of nucleotides through adenine nucleotide transporters, anion and metabolite flux by voltage-dependent anion channels, and energy transfer via phosphates through the creatinine/kinase system. It is unlikely that such mechanisms are perturbed in the SUR2m mice because SUR2 has not been shown to directly interact with these mechanisms, but this possibility cannot be excluded. mitoK_ATP conductances were not measured directly in this study, and the relative role of the different SUR2 variants, if any, remains to be studied. However, these results support the idea that one of the SUR2 variants, perhaps the long-form SUR2 present in WT but lacking in SUR2m, contributes and is necessary for many diazoxide-sensitive effects previously implicated as mitoK_ATP. The SUR2m mouse has the advantage of providing a nonpharmacological model that provides evidence for a role for SUR2 in protection, and this is particularly important with the implication that the short-form SUR2 variants might form mitoK_ATP and sarcK_ATP with altered pharmacology. If such channels exist or can be induced in WT, it will require reinterpretation of IPC and protection from I/R that relied on K_ATP pharmacology. We speculate that preconditioning stimuli may trigger a change in SUR2 regulation of mitoK_ATP that produces a temporary protected phenotype in WT. This speculation and possible relevance to clinically relevant protection mechanisms remains to be determined.