3.1. Spectrophotometric determination of complex I activity
Representative kinetic traces of complex I activity, monitored in a spectrophotometer as the rate of oxidation of NADH to NAD+ in the presence of UQ10, illustrate differences dependent on the treatment (). In summarized data from 4 hearts from each of the 5 experimental groups () controls (TC) exhibited a NADH oxidation rate of 122±5 nmol/min/mg protein with a large decrease in oxidation rate after I30 to 49±8 nmol/min/mg, but partial restoration to 106±1 nmol/min/mg in the RanI30 group. Similarly, there was a large decrease in complex I activity after IR to 70±4 nmol/min/mg, which was completely restored to 123±7 nmol/min/mg in the RanIR group. No changes in complex I activity were observed in the RanTC group (data not shown). To determine if Ran directly alters mitochondrial complex I activity in fractured mitochondria, in preliminary experiments Ran was added to freeze-thaw fractured mitochondria from the TC, I30 and IR groups, followed by measurement of complex I activity; the result was a slight but insignificant decrease in activity (data not shown).
Fig. 1 A: Representative spectrophotometric assay of mitochondrial complex I activity during cardiac ischemia reperfusion depicting the time points of addition of substrate, enzyme and the inhibitor. B: Summary data shows ischemia alone reduced the activity (more ...)
3.2. Histochemical staining for complex I function in gel
A representative gel (, upper panel) for NBT-oxidoreductase activity, reflecting complex I function with or without IR and Ran, illustrates decreased band density after I30 and IR but a higher band density after RanI30. Summary data (, lower panel) showed that band density decreased after I30 (87±3%) and IR (87±5%) compared to the normalized TC group (100%). Ran treatment before ischemia resulted in a significantly higher band density for RanI30 (95±4%) vs. I30 alone, whereas band density for RanIR (91±4%) was not significantly greater than that for IR alone. The observed band densities were normalized to the total lane intensities.
Fig. 2 Upper panel: Representative histochemical gel staining of complex I activity, measured as NBT-oxidoreductase, during cardiac ischemia reperfusion. Lower panel: Summary data shows ischemia alone resulted in lower staining than in time controls and reperfusion. (more ...)
3.3. Supercomplex assemblies detected by native gels
A representative image of a native gel (, panel A) stained by Coomassie Brilliant Blue illustrates supercomplexes- SC1 and SC2 comprised of complexes I, III and IV as identified by histochemical staining for complex I (panel B) and III and IV (panel C), and respiratory complexes after 2D electrophoresis detected by Western blot using antibodies against the indicated subunits (panel D). The two supercomplexes presumably are composed of multiple copies of respiratory complexes. Summary data of supercomplex assemblies from Coomassie stained gels () showed that band intensity for the supercomplex SC1 (TC =100%) was lower in I30 (86±1 %), IR (88±1%) and RanI30 (87±2%). Ranolazine treatment improved the band intensity of SC1 following IR (96±1%). Band intensity for supercomplex SC2 showed similar results. Compared to TC (100%), there was a significant decrease in band intensity in I30 (77±5%) and IR (85±2%). Ranolazine treatment improved band intensity following I30 (RanI30=93±1%) and IR (RanIR=95±5%). Complex I was not found to associate further with complexes II and V to form other supercomplexes (data not shown). The observed band densities were normalized to the total lane intensities.
Fig. 3 Determination of supercomplex assemblies using native PAGE. Panel A: Coomassie stained gel after native PAGE. SC1 and SC2 indicate supercomplexes composed of complexes I, III and IV, with varying copies of each complex. Components of supercomplex determined (more ...)
3.4. Determination of integrity of complex I subunit NDUFA9 by Western blots
A representative blot for the complex I subunit NDUFA9 (, upper panel) from hearts subjected to IR ± Ran illustrates decreased intensity after I30 and restoration after RanI30 and RanIR. Complex IV subunit I was used as the loading control. Summary data (, lower panel) showed that compared to TC (100%) the anti-NDUFA9 immune reactive band intensity was significantly lower after I30 (84±9%) but was restored after RanI30 (109±7%). Band densities for IR (101±4%) and RanIR (112±7%) groups were not different from the TC group.
Fig. 4 Upper panel: Representative western blot detection of complex I subunit NDUFA9 during cardiac ischemia reperfusion. Lower panel: Summary data shows that ischemia reduced the amount of detectable subunit, indicating a loss of protein or protein damage. (more ...)
3.5. Analysis of acetylation of mitochondrial proteins using ELISA
The optical density for acetylated mitochondrial proteins, as detected by ELISA, showed an overall decrease in all groups compared to TC (76±4.9 arb. units) (data not represented graphically). I30 showed the least acetylation (41.7±3.1 arb. units), whereas IR partially restored the acetylation (57.8±4.6 arb. units). Treatment with Ran partially restored acetylation in both groups (Ran I30 = 56.5±2.7 arb. units; RanIR = 59.8±2.3 arb. units). Values for each treatment group were significantly lower than the TC group, and there was a statistically significant difference (p<0.05) between I30 and RanI30, but not between IR and RanIR groups.
3.6. Electron transfer in Fe-S clusters by electron paramagnetic resonance
Averaged peak intensities (4 hearts per group) of Fe-S resonance signals (in arb. units) showed differences in peak signals dependent on the treatments (). The g=2.023 signal was assigned to S3, the 3Fe-4S cluster of complex II, or to mitochondrial aconitase, g=2.006 to the semi-ubiquinone radical (UQ•), g=1.94 to cluster N1b of complex I, or to cluster SI of complex II, and g=1.89 to cluster N4 of complex I, or to the Rieske center of complex III. Summary data () showed that compared to TC (3.1±1.4 arb. units), neither I30 (2.7±0.8) nor IR (4.2±0.6) significantly altered the g=2.023 signal. However, Ran treatment reduced the g=2.023 signal further after RanI30 (1.6±0.4), but not after RanIR (4.2±1.0). There was a significant increase in the g=2.006 radical after I30 (1.49±0.07), which was lower in TC (0.87±0.09) and after IR (1.06±0.03). Ran treatment also reduced the g=2.006 signal (RanI30 = 1.29±0.09; RanIR = 1.02±0.04). There was also a significant increase in signals pertaining to g=1.94 and 1.89 during ischemia, which returned to TC levels after reperfusion. The contribution at g=1.94 for N4 was verified by the changes noted at g=1.89; i.e. the contribution of N1b and S1 can be estimated after evaluating the contribution of N4. The signal at g=1.94, contributed by N3, was small as estimated by the weak signal at g=1.86, the low field g-value for the signal from N3. Similarly the contribution to the signal at g=1.92 from N2 was weak as determined by the absence of signal from the low field g-value for N2 at 2.05.
Fig. 5 A: Representative traces of EPR signals during cardiac ischemia reperfusion denoting changes in three mitochondrial Fe-S clusters and semiubiquinone. B: Summary data shows the changes in spectral magnitudes of the Fe-S clusters and semiubiquinone. Ischemia (more ...)
3.7. Determination of cardiolipin integrity by HPLC
Representative HPLC traces of cardiolipin integrity were different depending on treatment (). Compared to the cardiolipin standard TC (93±2 arb. units), there were significant decreases in the areas under the curves in I30 (56±21 arb. units) and more so in IR (32±12 arb. units) groups, reflective of damaged cardiolipin. In summary data () the RanI30 group (49±15 arb. units) showed no improvement over the I30 group alone, but the RanIR (69±8 arb. units) group showed a larger area under the peak that is reflective of less fragmented cardiolipin. The number of peaks as detected by HPLC was also higher (reflecting more fragmented species) in I30 and IR groups than in RanI30 and RanIR groups, respectively ().
Fig. 6 A: Representative traces of cardiolipin integrity by HPLC analysis during cardiac ischemia reperfusion. B: Summary data shows that ischemia alone reduced the area under the major peak of cardiolipin, and reperfusion decreased it further. The additional (more ...)
3.8. Improved cardiac function after IR injury with ranolazine
Heart rate, coronary flow, diastolic LVP, and developed (systolic-diastolic) LVP remained unchanged during continuous perfusion of hearts for 60 min (TC, data not displayed) after which time hearts were harvested for examination of complex I function using the methods described above. At the end of 30 min no flow global ischemia, there was no heart beat and diastolic LVP was elevated (); after 10 min reperfusion heart rate and coronary flow were similar to those of the TC in all IR groups. On the other hand, diastolic LVP was more elevated in the IR group than in the RanIR group and developed LVP was more depressed in the IR group than in the RanIR group. These data indicate that Ran had a protective effect on reducing contracture during ischemia and on increasing contractile function on reperfusion. These protective effects were associated with improvements in several assays of complex I function and the integrity of its support structure, cardiolipin.
Cardiac function before, during and after IR injury.