MLS-Stat3E expression in the mitochondria prevents ischemia-induced

The mitochondrial electron transport chain is progressively damaged during ischemia (173, 177, 243, 307). Various reports indicate that the initial site of ischemic damage to the ETC is complex I (52, 225, 254) and with increasing duration of the insult, it progresses to complex III (173) and IV (176). Therefore, as a next step we measured maximal activities of complex I, II and III. Additionally, we tested citrate synthase activity (CS) as a mitochondria-specific marker. CS rates were unchanged between all four experimental groups (Table 3.5), which allowed us to compare the raw data obtained from other enzymatic assays.

As depicted in Table 3.5 and Figure 3.13A, ischemia caused 29% decrease in the maximal activity of NADH:ubiquinone oxidoreductase in mitochondria isolated from wild-type hearts when compared to controls. However, already decreased activity of complex I from transgenic heart mitochondria was not further reduced after ischemia. These results corroborated the decreased rates of glutamate+malate-dependent respiration in WT and the relatively preserved rates in MLS-Stat3E (Table 3.4 and Figure 3.12A).

Complex II activity was not decreased by ischemia in both types of mice (Table 3.5), confirming already reported observations in wild-type mice (175). The basal enzymatic rates in control samples (TC) from MLS-Stat3E group were lower when compared to WT TC but did not decrease further due to the insult. Thus, the reduced rates of respiration with succinate were likely a result of the impaired cytochrome c oxygenase (TMPD/ascorbate rates) or possible complex III damage.

Therefore, we also measured ubiquinone:cytochrome c oxidoreductase activity and did not observe any changes in enzymatic rates caused by ischemia (Table 3.5).

Additionally, we measured the rotenone-insensitive activity of NADH dehydrogenase (NFR), the proximal part of complex I (Figure 2.5 in Materials and Methods). Despite the decreased total (rotenone-sensitive) activity of complex I in WT vs. MLS-Stat3E mitochondria in basal conditions, we did not observe any changes in NFR enzymatic rates between both sample groups (Table 3.5). Moreover, ischemia did not decrease maximal enzymatic rates of NFR in WT cardiac mitochondria, as well as in MLS-Stat3E hearts (Table 3.5). Our data confirmed those

previously published regarding the absence of ischemic damage in NFR (52).

Moreover, these results suggested that the NADH dehydrogenase part of complex I was not a rate-limiting factor for the overall efficiency of the electron transfer through this complex.

To summarize, ischemia reduced rotenone-sensitive complex I activity in wild type heart mitochondria, but not in MLS-Stat3E transgenes. Complex II and III were unaffected. Therefore, observed slower rate of succinate-dependent respiration in ischemic mitochondria was a result of a decrease in complex IV activity or a cytochrome c loss. Therefore, we measured cytochrome c content in mitochondria from hearts subjected to ischemic insult and time controls. Western blot analysis revealed a reduced amount of cytochrome c in WT mitochondria after ischemia (Figure 3.14B). Surprisingly, we did not observe any decrease in MLS-Stat3E samples. According to densitometric measurements, ischemia induced 32% loss of cytochrome c from the wild-type mitochondria (ratio of ischemia/control ranging between 0.48 and 0.78) but only 5% from MLS-Stat3E (ratio: 0.85 – 1.12) (Figure 3.14A). Therefore, the slower rates of TMPD/ascorbate respiration in MLS-Stat3E mitochondria were likely due to the impaired enzymatic activity of complex IV or cardiolipin loss. In contrast, in WT samples the reduced rates could be a result of decreased complex IV activity, cardiolipin loss and the cytochrome c release. Additional measurement of first-order enzymatic rates of complex IV revealed an ischemia-originated defect in cytochrome c oxidase, which resulted in 28% decrease in its activity (Table 3.5). This phenomenon was observed in both WT and MLS-Stat3E mitochondria (Figure 3.13B).

WT types of mitochondria but complex II and III activities are not altered. Hearts of MLS-Stat3E and WT mice were subjected to 45 min of ischemia (ISCH) or kept on ice (time control, TC). Mitochondria were isolated, cholate-solubilized and enzymatic activities of invidual ETC complex and of citrate synthase were determined spectrophotometrically, as described in Materials and Methods. All results are expressed in nmol/min/mg or 1/min/mg in case of complex IV. Bars represent means ± SE, n = 6 mice in each experimental group, except for complex IV measurements n = 4 in each experimental group. Statistically significant differences were annotated as ** for p < 0.001 (WT ISCH vs. WT TC);

 for p < 0.05 (MLS-Stat3E TC vs. WT TC); and MLS-Stat3E ISCH vs. MLS-Stat3E TC as ^ for p < 0.05. All results were compared using two-way ANOVA test followed by pairwise multiple comparison procedure (Holm-Sidak test). CS, citrate synthase; NFR, NADH:ferricyanide reductase.

Figure 3.13. Ischemia causes less damage to complex I activity in MLS-Stat3E mitochondria (A).

Complex IV activity is reduced to the same extent in WT and MLS-Stat3E mitochondria (B).

Hearts of MLS-Stat3E and WT mice were subjected to ischemia (45 min incubation in 1 ml of saline at 37 ^oC with shaking). In parallel, time control (TC) hearts were kept on ice. Mitochondria were isolated and maximal enzymatic rates of complex I (rotenone-sensitive) and complex IV were measured.

Results were expressed in % as a change in complex I and IV activities compared to the time-control activities set as 100%. Each bar represents a mean ± SE. Statistically significant differences were annotated with *. *p < 0.05, Student’s t-test, n = 6 mice for complex I assays, n = 4 for complex IV assays.

Figure 3.14. Ischemia does not induce cytochrome c release from mitochondria with overexpression of MLS-Stat3E. Mitochondria were isolated from ischemic and control hearts of WT and MLS-Stat3E mice according to the protocol described in Materials and Methods. (A-B) Mitochondrial proteins (10 µg) were resolved by SDS-PAGE, transferred to PVDF membrane and immunoblotted against cytochrome c and porin as a control of equal mitochondria loading. (A) Densitometry was measured using ImageJ (NIH, Bethesda, MD) and expressed as ratio of cytochrome c of ISCH sample to cytochrome c of TC sample normalized to the ratio of porin from ISCH to porin from TC. Bars represent means ± SE, n = 4 pairs of ISCH vs. TC hearts in both experimental groups.

Statistically significant differences were annotated as * for p < 0.05, Student’s t-test. (B) An example blot showing probing for cytochrome c. ISCH, ischemia; TC, time control.

3.11. Ischemia does not augment ROS generation from complex I in transgenic