Ischaemic preconditioning improves proteasomal activity and increases the degradation of deltaPKC during reperfusion - PubMed (original) (raw)
. 2010 Jan 15;85(2):385-94.
doi: 10.1093/cvr/cvp334. Epub 2009 Oct 10.
Affiliations
- PMID: 19820255
- PMCID: PMC2797452
- DOI: 10.1093/cvr/cvp334
Ischaemic preconditioning improves proteasomal activity and increases the degradation of deltaPKC during reperfusion
Eric N Churchill et al. Cardiovasc Res. 2010.
Abstract
Aims: The response of the myocardium to an ischaemic insult is regulated by two highly homologous protein kinase C (PKC) isozymes, delta and epsilonPKC. Here, we determined the spatial and temporal relationships between these two isozymes in the context of ischaemia/reperfusion (I/R) and ischaemic preconditioning (IPC) to better understand their roles in cardioprotection.
Methods and results: Using an ex vivo rat model of myocardial infarction, we found that short bouts of ischaemia and reperfusion prior to the prolonged ischaemic event (IPC) diminished deltaPKC translocation by 3.8-fold and increased epsilonPKC accumulation at mitochondria by 16-fold during reperfusion. In addition, total cellular levels of deltaPKC decreased by 60 +/- 2.7% in response to IPC, whereas the levels of epsilonPKC did not significantly change. Prolonged ischaemia induced a 48 +/- 11% decline in the ATP-dependent proteasomal activity and increased the accumulation of misfolded proteins during reperfusion by 192 +/- 32%; both of these events were completely prevented by IPC. Pharmacological inhibition of the proteasome or selective inhibition of epsilonPKC during IPC restored deltaPKC levels at the mitochondria while decreasing epsilonPKC levels, resulting in a loss of IPC-induced protection from I/R. Importantly, increased myocardial injury was the result, in part, of restoring a deltaPKC-mediated I/R pro-apoptotic phenotype by decreasing pro-survival signalling and increasing cytochrome c release into the cytosol.
Conclusion: Taken together, our findings indicate that IPC prevents I/R injury at reperfusion by protecting ATP-dependent 26S proteasomal function. This decreases the accumulation of the pro-apoptotic kinase, deltaPKC, at cardiac mitochondria, resulting in the accumulation of the pro-survival kinase, epsilonPKC.
Figures
Figure 1
Ischaemic preconditioning decreases δPKC and increases εPKC levels at cardiac mitochondria during reperfusion. (A) Hearts were hung in Langendorff mode and treated with the listed perfusion protocols. Hearts were then removed, homogenized, fractionated, and the mitochondrial fraction was subjected to western blot analysis with antibodies against the proteins listed in the figure. Values were normalized to adenine nucleotide translocase (ANT), a mitochondrial marker, and expressed as % of normoxia (N) control. (B) Western blot analysis of mitochondrial protein showing that 30 min of ischaemia followed by 60 min of reperfusion (I30R60) resulted in translocation of δPKC to mitochondria (six-folds increase; P < 0.05 vs. N), which was blocked when hearts were preconditioned (IPC) before the global ischaemic event (P < 0.05 vs. I30R60). Likewise, εPKC association with cardiac mitochondria increased ∼7-folds during I30R60 (P < 0.05 vs. N), but in contrast to δPKC, IPC prior to I/R further increased translocation of εPKC to 16-folds over the levels seen in normoxic hearts (P < 0.05 vs. N and I30R60). (C) Western blot analyses showing that the IPC stimulus alone (without I30R60) significantly increased translocation of δPKC (P < 0.05 vs. N) and εPKC translocation (P < 0.05 vs. N) to the mitochondria. *P < 0.05 vs. N, ‡P < 0.05 vs. I30R60) Translocation of δPKC and εPKC to the mitochondria were analysed by one-way analysis of variance with a post-hoc Tukey test. Mitochondrial PKC levels were analysed by Student's _t_-test.
Figure 2
Diminished mitochondrial levels of δPKC following IPC are due to decreased cellular levels of the isozyme. Hearts were hung in Langendorff mode and treated with the listed perfusion protocols in Figure 1. Hearts were then removed, and homogenized, and the total homogenate was subjected to western blot analysis with antibodies against the proteins listed in the figure. Values were normalized to glyceraldehyde phosphate dehydrogenase (GAPDH), a cytosolic protein, and expressed as %N. I30R60 had no significant effect on total levels of either δPKC or εPKC. However, IPC before the global ischaemic event decreased the overall levels of δPKC by ∼80% (P < 0.05 vs. N), but not εPKC levels. *P < 0.05 vs. Normoxia and ‡P < 0.05 vs. I30R60. Cellular δPKC and εPKC levels were analysed by one-way analysis of variance with a post-hoc Tukey test.
Figure 3
Effect of preconditioning on ischaemia-induced loss in ATP-dependent proteasome activity. (A) Cytosolic extracts were prepared from hearts exposed to 70 min of normoxic perfusion (N), 30 min of ischaemia (I30), or three cycles of preconditioning (5 min ischaemia and 5 min reperfusion) followed by 30 min of ischaemia (I30 + IPC) in the absence or presence of the proteasome inhibitor lactacystin or the specific εPKC inhibitor εV1–2. Chymotrypsin-like activity of the proteasome present in the cytoplasmic milieu was evaluated and the specific inhibitor MG-132 (20 μM) was utilized to ensure that measured activities were due to the proteasome (data not shown). The presence of unfolded proteins was evaluated using the slot blot technique with an anti-soluble oligomer antibody. Values representing ATP-dependent proteasome activity and misfolded proteins are presented as a percent of values obtained with samples from hearts exposed to 60 min of normoxic perfusion (N). Values represent the mean ± standard deviation (n = 4). (B) Ischaemia resulted in a 50% decline in ATP-dependent proteasome activity (P < 0.05 vs. N), which was completely reversed by IPC (P < 0.05 vs. I30). The proteasome inhibitor, lactacystin (2 μM) and the specific εPKC inhibitor (1μM εV1–2) both significantly decreased the activity of the proteasome (P < 0.05 vs. I30R60 + IPC; n = 4). (C) I30R60 resulted in an ∼3-fold increase in misfolded proteins which was prevented by IPC (P < 0.05; n = 4). Treatment of hearts with lactacystin or εV1–2 blocked the effect of IPC and increased the accumulation of misfolded proteins. (D) IPC elevated ATP levels by 3.5-fold in hearts that had undergone I30R60, and εV1–2 blocked these effects. (E) Treatment with the εPKC activator (ψεRACK) protected the proteasome from ischaemia-mediated inhibition (P < 0.05 vs. I30). *P < 0.05 vs. Normoxia, +P = 0.05 vs. I30R60, §P < 0.05 vs. I30, ‡P < 0.05 vs. IPC + I30, †P < 0.05 vs. IPC + I30R60; Misfolded protein accumulation and proteasome activity were analysed by one-way analysis of variance with a post-hoc Tukey test. Figure 3D, proteasome activity was analysed by Student's _t_-test.
Figure 4
Inhibition of the proteasome restores δPKC cellular and mitochondrial levels in IPC hearts with a resultant decrease in εPKC levels. (A) Hearts were hung in Langendorff mode and treated with the above-mentioned perfusion protocols. Hearts were then removed, homogenized, and the total homogenate and mitochondrial fractions were subjected to western blot analysis with antibodies against the proteins listed in the figure. Values were normalized to GAPDH (total homogenate) or ANT (mitochondrial fraction) and expressed as % I30R60. IPC before prolonged ischaemia reduced total levels of δPKC by ∼80% (P < 0.05 vs. I30R60). Inhibition of the proteasome with 2 μM lactacystin blocked δPKC degradation (P < 0.05 vs. I30R60 + IPC). Similar to Figure 2, IPC before prolonged ischaemia did not significantly change overall levels of εPKC. However, inhibition of the proteasome increased εPKC levels by ∼2-folds (P < 0.05 vs. I30R60). Inhibition of εPKC activity with εV1–2 did not significantly change the overall levels of either δ or εPKC isozymes (data not shown). (B) As in Figure 2, IPC before I30R60 decreased levels of δPKC at mitochondria by ∼60% (P < 0.05 vs. I30R60). This was completely prevented in hearts treated with 2 μM lactacystin and 1 μM εV1–2 (P < 0.05 vs. I30R60 + IPC). (C) Although δPKC mitochondrial levels were restored, εPKC levels were diminished by 40% relative to I30R60 and by 60% relative to I30R60 + IPC (P < 0.05). Hearts that were treated with 1 μM of a peptide inhibitor of εPKC (εV1–2) during the IPC protocol showed a significant decrease in εPKC mitochondrial levels (P < 0.05 vs. I30R60 + IPC). *P < 0.05 vs. I30R60, ‡P < 0.05 vs. IPC I30R60. Cellular and mitochondrial PKC levels were analysed by the one-way analysis of variance with a post-hoc testing by Tukey.
Figure 5
Inhibition of δPKC degradation restores the apoptotic phenotype seen during reperfusion. Hearts were hung in Langendorff mode and treated with the listed perfusion protocols. Hearts were then removed, homogenized, fractionated, and the cytosolic homogenate was subjected to western blot analysis with antibodies against the proteins listed in the figure. Values were normalized to GAPDH and expressed as % I30R60. (A) IPC before prolonged ischaemia significantly decreased cytochrome c release into the cytosol (P < 0.05 vs. I30R60). Inhibition of the proteasome with 2 μM lactacystin restored cytochrome c release to levels seen during I30R60 (P < 0.05 vs. I30R60 + IPC). (B) Ischaemia alone and perfusion with lactacystin or εV1–2 alone did not result in significant release of cytochrome c into the cytosol. Additionally, as evidenced by a lack of mitochondrial VDAC in the cytosolic fraction, there was little contamination from mitochondrial cytochrome c in this fraction. Enolase was used as a cytosolic loading control. (C) IPC before prolonged ischaemia also increased phosphorylation of the pro-survival kinase, Akt, by ∼3-fold (P < 0.05 vs. I30R60). Inhibition of the proteasome with 2 μM lactacystin decreased phosphorylation back to I30R60 levels (P < 0.05 vs. I30R60 + IPC). *P < 0.05 vs. I30R60, ‡P < 0.05 vs. IPC I30R60. Cytosolic cytochrome c levels and p-Akt were analysed by one-way analysis of variance with a post-hoc Tukey test.
Figure 6
Inhibition of the proteasome reverses the IPC-mediated protective effects on tissue injury. Hearts were hung in Langendorff mode and treated with the listed perfusion protocols. Tissue injury was determined by measuring the release of CPK into the cardiac effluent (total CPK units). Following removal, hearts were sliced and stained with TTC to differentiate between necrotic (stained white) and viable (stained red) tissue (% infarct). (A,B) Hearts subjected to I30R60 showed an increase in both CPK release and myocardial infarction and both were blocked by IPC (reductions of ∼60% for CPK release and ∼40% for infarction, respectively). Perfusion of 2 μM lactacystin during the IPC protocol and for the first 10 min of reperfusion reversed this effect resulting in significantly higher levels of CPK release (P < 0.05 vs. I30R60 + IPC) and myocardial infarction (P < 0.05 vs. I30R60 + IPC). Similar to proteasome inhibition, εPKC inhibition (1 μM εV1–2) also significantly increased both CPK release (P < 0.05 vs. I30R60 + IPC) and myocardial infarction (P < 0.05 vs. I30R60 + IPC). *P < 0.05 vs. I30R60, ‡P < 0.05 vs. IPC I30R60. Total CPK and % infarcted area were analysed by the one-way analysis of variance with a post-hoc Tukey test.
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