Reversible blockade of complex I or inhibition of PKCβ reduces activation and mitochondria translocation of p66Shc to preserve cardiac function after ischemia

Abstract

Aim: Excess mitochondrial reactive oxygen species (mROS) play a vital role in cardiac ischemia reperfusion (IR) injury. P66Shc, a splice variant of the ShcA adaptor protein family, enhances mROS production by oxidizing reduced cytochrome c to yield H2O2. Ablation of p66Shc protects against IR injury, but it is unknown if and when p66Shc is activated during cardiac ischemia and/or reperfusion and if attenuating complex I electron transfer or deactivating PKCβ alters p66Shc activation during IR is associated with cardioprotection.

Methods: Isolated guinea pig hearts were perfused and subjected to increasing periods of ischemia and reperfusion with or without amobarbital, a complex I blocker, or hispidin, a PKCβ inhibitor. Phosphorylation of p66Shc at serine 36 and levels of p66Shc in mitochondria and cytosol were measured. Cardiac functional variables and redox states were monitored online before, during and after ischemia. Infarct size was assessed in some hearts after 120 min reperfusion.

Results: Phosphorylation of p66Shc and its translocation into mitochondria increased during reperfusion after 20 and 30 min ischemia, but not during ischemia only, or during 5 or 10 min ischemia followed by 20 min reperfusion. Correspondingly, cytosolic p66Shc levels decreased during these ischemia and reperfusion periods. Amobarbital or hispidin reduced phosphorylation of p66Shc and its mitochondrial translocation induced by 30 min ischemia and 20 min reperfusion. Decreased phosphorylation of p66Shc by amobarbital or hispidin led to better functional recovery and less infarction during reperfusion.

Conclusion: Our results show that IR activates p66Shc and that reversible blockade of electron transfer from complex I, or inhibition of PKCβ activation, decreases p66Shc activation and translocation and reduces IR damage. These observations support a novel potential therapeutic intervention against cardiac IR injury.

Figure 1. Phosphorylation of p66^Shc at Ser36 increased during reperfusion (R) after ischemia (I) in isolated guinea pig hearts compared to no ischemia (time controls, TC).

A, upper panel: phosphorylation of p66^Shc at Ser36 from total protein lysate of heart tissue. A, lower panel: total p66^Shc level for loading control. Summary of mean band intensities (B) derived from three independent experiments (n = 3 hearts/group); band intensities were determined by densitometry using imageJ software and the intensities of P-p66 (Ser36) were normalized to the total p66^Shc in each sample. P<0.05: *IR vs. TC and I.

Figure 2. P66^Shc expression levels changed in cytosol and mitochondria during reperfusion after ischemia.

A, upper panel: p66^Shc level in mitochondrial and cytosolic fractions. Anti-VDAC (A, middle panel) and anti-β–actin (A, lower panel) confirm protein loading, mitochondrial (β–actin) and cytosolic purity (VDAC). Summary of mean band intensities (B) derived from three independent experiments (n = 3 hearts/group) (see Fig. 1 for imaging). P<0.05: *IR vs. TC and I20; # IR vs I30; ^a I30R60 vs I30R10 or I30R20.

Figure 3. Phosphorylation of p66^Shc at Ser36 during reperfusion after ischemia was dependent on the duration of ischemia.

A, upper panel: phosphorylation of p66^Shc at Ser36; A, lower panel: total p66^Shc level for loading control. B: Summary of mean band intensities derived from three independent experiments (n = 3 hearts/group) (see Fig. 1 for imaging). P<0.05: *I20 and I30 vs. I5, I10 and TC.

Figure 4. NADH autofluorescence (afu) (A) was higher and DHE fluorescence intensity (O₂ ^−• emission) (B) was lower during ischemia and reperfusion after amobarbital (Amo) treatment compared to IR alone. DHE and NADH were recorded online in isolated hearts at the LV free wall.

n = 6 hearts/group/fluorescence measure. Cardiac contractile and relaxant function: developed LVP (C), diastolic LVP (D), dLVP/dt _min (E) and dLVP/dt _max (F) were improved on reperfusion after amobarbital (Amo) treatment during ischemia. Inset in D shows detailed changes in diastolic LVP during late ischemia and early reperfusion. n = 9 hearts/group/functional measure. All values are means±SEM. TC, time control; IR, ischemia and reperfusion. P<0.05: *IR vs. TC; ^#Amo+IR vs. IR.

Figure 5. IR-induced phosphorylation (P) of p66^Shc at Ser36 was decreased by amobarbital (Amo) after 20 (A) or 30 (B) min ischemia (I) followed by 20 min reperfusion (R).

A, B, upper panel: phosphorylation of p66^Shc at Ser36; equal amounts of p66^Shc loading were verified with anti-ShC antibody (A, B: middle panel). Summary of mean band intensities derived from three independent experiments (n = 3 hearts/group) (A, B: lower panel). IP, immunoprecipitation; TC, time controls (no IR). P<0.05: *IR vs. TC; ^#Amo+IR vs. IR.

Figure 6. IR-induced activation of p66^Shc occurred via the PKCβII signaling pathway.

A: Representative WB of 3 independent experiments of phosphorylation of PKCβII. B: Phosphorylation of p66^Shc at Ser36 during IR with or without hispidin (His) treatment. C: p66^Shc accumulation in mitochondria during IR with or without hispidin (His) treatment. B, C Lower panels show summary of mean band intensities derived from three independent experiments (n = 3 hearts/group) (see Fig. 1 for imaging). P<0.05: *IR vs. TC; ^#IR+His vs. IR.

Figure 7. Inhibiting PKCβ activation reduced cardiac damage after IR.

A: Diastolic LVP without IR (TC; n = 8 hearts/group) and before ischemia, at 30 min of ischemia and at 20 min of reperfusion (IR; n = 12 hearts/group) with or without hispidin (His; n = 10 hearts/group) treatment. B: Infarct size after 30 min of ischemia and 120 min of reperfusion (n = 8 hearts/group) with or without hispidin (His; n = 8 hearts/group) treatment. P<0.05: ^#IR+His vs. IR.