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Mitochondrial import of PKCepsilon is mediated by HSP90: a role in cardioprotection from ischaemia and reperfusion injury.

ABSTRACT: AIMS:Protein kinase C epsilon (PKCepsilon) is critical for cardiac protection from ischaemia and reperfusion (IR) injury. PKCepsilon substrates that mediate cytoprotection reside in the mitochondria. However, the mechanism enabling mitochondrial translocation and import of PKCepsilon to enable phosphorylation of these substrates is not known. Heat shock protein 90 (HSP90) is a cytoprotective protein chaperone that participates in mitochondrial import of a number of proteins. Here, we investigated the role of HSP90 in mitochondrial import of PKCepsilon. METHODS AND RESULTS:Using an ex vivo perfused rat heart model of IR, we found that PKCepsilon translocates from the cytosol to the mitochondrial fraction following IR. Immunogold electron microscopy and mitochondrial fractionation demonstrated that following IR, mitochondrial PKCepsilon is localized within the mitochondria, on the inner mitochondrial membrane. Pharmacological inhibition of HSP90 prevented IR-induced interaction between PKCepsilon and the translocase of the outer membrane (Tom20), reduced mitochondrial import of PKCepsilon, and increased necrotic cell death by approximately 70%. Using a rational approach, we designed a 7-amino acid peptide activator of PKCepsilon, derived from an HSP90 homologous sequence located in the C2 domain of PKCepsilon (termed psiepsilonHSP90). Treatment with this peptide (conjugated to the cell permeating TAT protein-derived peptide, TAT(47-57)) increased PKCepsilon-HSP90 protein-protein interaction, enhanced mitochondrial translocation of PKCepsilon, increased phosphorylation and activity of an intra-mitochondrial PKCepsilon substrate, aldehyde dehydrogenase 2, and reduced cardiac injury in ex vivo and in vivo models of myocardial infarction. CONCLUSION:Our results suggest that HSP90-mediated mitochondrial import of PKCepsilon plays an important role in the protection of the myocardium from IR injury.

SUBMITTER: Budas GR

PROVIDER: S-EPMC2936125 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Mitochondrial import of PKCepsilon is mediated by HSP90: a role in cardioprotection from ischaemia and reperfusion injury.

Budas Grant R GR Churchill Eric N EN Disatnik Marie-Hélène MH Sun Lihan L Mochly-Rosen Daria D

Cardiovascular research 20100616 1

<h4>Aims</h4>Protein kinase C epsilon (PKCepsilon) is critical for cardiac protection from ischaemia and reperfusion (IR) injury. PKCepsilon substrates that mediate cytoprotection reside in the mitochondria. However, the mechanism enabling mitochondrial translocation and import of PKCepsilon to enable phosphorylation of these substrates is not known. Heat shock protein 90 (HSP90) is a cytoprotective protein chaperone that participates in mitochondrial import of a number of proteins. Here, we inv ...[more]

PMID: 20558438

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Project description:Brief episodes of ischaemia and reperfusion render the heart resistant to subsequent prolonged ischaemic insult, termed ischaemic preconditioning. Here, we hypothesized that transient non-ischaemic stress by hypertrophic stimulation would induce endogenous cardioprotective signalling and enhance cardiac resistance to subsequent ischaemic damage. Transient transverse aortic constriction (TAC) or Ang-Ⅱ treatment was performed for 3-7 days in male mice and then withdrawn for several days by either aortic debanding or discontinuing Ang-Ⅱ treatment, followed by subsequent exposure to regional myocardial ischaemia by in situ coronary artery ligation. Following ischaemia/reperfusion (I/R) injury, myocardial infarct size and apoptosis were markedly reduced and contractile function was significantly improved in the TAC preconditioning group compared with that in the control group. Similar results were observed in mice receiving Ang-Ⅱ infusion. Mechanistically, TAC preconditioning enhanced ALDH2 activity, promoted AMPK activation and improved mitochondrial energy metabolism by increasing myocardial OXPHOS complex expression, elevating the mitochondrial ATP content and improving viable myocardium glucose uptake. Moreover, TAC preconditioning significantly mitigated I/R-induced myocardial iNOS/gp91phox activation, inhibited endoplasmic reticulum stress and ameliorated mitochondrial impairment. Using a pharmacological approach to inhibit AMPK signalling in the presence or absence of preconditioning, we demonstrated AMPK-dependent protective mechanisms of TAC preconditioning against I/R injury. Furthermore, treatment with adenovirus-encoded ALDH2 partially emulated the actions of hypertrophic preconditioning, as evidenced by improved mitochondrial metabolism, inhibited oxidative stress-induced mitochondrial damage and attenuated cell death through an AMPK-dependent mechanism, whereas genetic ablation of ALDH2 abrogated the aforementioned actions of TAC preconditioning. The present study demonstrates that preconditioning with hypertrophic stress protects the heart from I/R injury via mechanisms that improve mitochondrial metabolism, reduce oxidative/nitrative stress and inhibit apoptosis. ALDH2 is obligatorily required for the development of cardiac hypertrophic preconditioning and acts as the mediator of this process.

Project description:AimsNo effective therapy is available in clinics to protect the heart from ischaemia/reperfusion (I/R) injury. Endothelial cells are activated after I/R, which may drive the inflammatory response by releasing ATP through pannexin1 (Panx1) channels. Here, we investigated the role of Panx1 in cardiac I/R.Methods and resultsPanx1 was found in cardiac endothelial cells, neutrophils, and cardiomyocytes. After in vivo I/R, serum Troponin-I, and infarct size were less pronounced in Panx1-/- mice, but leukocyte infiltration in the infarct area was similar between Panx1-/- and wild-type mice. Serum Troponin-I and infarct size were not different between mice with neutrophil-specific deletion of Panx1 and Panx1fl/fl mice, suggesting that cardioprotection by Panx1 deletion rather involved cardiomyocytes than the inflammatory response. Physiological cardiac function in wild-type and Panx1-/- hearts was similar. The time to onset of contracture and time to maximal contracture were delayed in Panx1-/- hearts, suggesting reduced sensitivity of these hearts to ischaemic injury. Moreover, Panx1-/- hearts showed better recovery of left ventricle developed pressure, cardiac contractility, and relaxation after I/R. Ischaemic preconditioning failed to confer further protection in Panx1-/- hearts. Panx1 was found in subsarcolemmal mitochondria (SSM). SSM in WT or Panx1-/- hearts showed no differences in morphology. The function of the mitochondrial permeability transition pore and production of reactive oxygen species in SSM was not affected, but mitochondrial respiration was reduced in Panx1-/- SSM. Finally, Panx1-/- cardiomyocytes had a decreased mitochondrial membrane potential and an increased mitochondrial ATP content.ConclusionPanx1-/- mice display decreased sensitivity to cardiac I/R injury, resulting in smaller infarcts and improved recovery of left ventricular function. This cardioprotective effect of Panx1 deletion seems to involve cardiac mitochondria rather than a reduced inflammatory response. Thus, Panx1 may represent a new target for controlling cardiac reperfusion damage.

Project description:AimsGenetic and pharmacological inhibition of mitochondrial fission induced by acute myocardial ischaemia/reperfusion injury (IRI) has been shown to reduce myocardial infarct size. The clinically used anti-hypertensive and heart failure medication, hydralazine, is known to have anti-oxidant and anti-apoptotic effects. Here, we investigated whether hydralazine confers acute cardioprotection by inhibiting Drp1-mediated mitochondrial fission.Methods and resultsPre-treatment with hydralazine was shown to inhibit both mitochondrial fission and mitochondrial membrane depolarisation induced by oxidative stress in HeLa cells. In mouse embryonic fibroblasts (MEFs), pre-treatment with hydralazine attenuated mitochondrial fission and cell death induced by oxidative stress, but this effect was absent in MEFs deficient in the mitochondrial fission protein, Drp1. Molecular docking and surface plasmon resonance studies demonstrated binding of hydralazine to the GTPase domain of the mitochondrial fission protein, Drp1 (KD 8.6±1.0 µM), and inhibition of Drp1 GTPase activity in a dose-dependent manner. In isolated adult murine cardiomyocytes subjected to simulated IRI, hydralazine inhibited mitochondrial fission, preserved mitochondrial fusion events, and reduced cardiomyocyte death (hydralazine 24.7±2.5% vs. control 34.1±1.5%, P=0.0012). In ex vivo perfused murine hearts subjected to acute IRI, pre-treatment with hydralazine reduced myocardial infarct size (as % left ventricle: hydralazine 29.6±6.5% vs. vehicle control 54.1±4.9%, P=0.0083), and in the murine heart subjected to in vivo IRI, the administration of hydralazine at reperfusion, decreased myocardial infarct size (as % area-at-risk: hydralazine 28.9±3.0% vs. vehicle control 58.2±3.8%, P<0.001).ConclusionWe show that, in addition to its antioxidant and anti-apoptotic effects, hydralazine, confers acute cardioprotection by inhibiting IRI-induced mitochondrial fission, raising the possibility of repurposing hydralazine as a novel cardioprotective therapy for improving post-infarction outcomes.

Project description:The alterations in mitochondrial protein glycosylation in myocardial ischaemia reperfusion (I/R) injury are still unclear. Therefore, based on a lectin microarray and liquid chromatograph-mass spectrometer/mass spectrometer (LC‒MS/MS) technology combined with a bioinformatics analysis, we studied the changes in mitochondrial protein glycosylation during I/R injury. This study revealed significant differences in mitochondrial glycoprotein during I/R injury. Compared with the sham operation group, the model group, which underwent ischaemia for 30 min, showed a high expression of glycan structures recognized by lectins, such as WFA, PTL-I, LTL, GSL-I, SBA and SNA, and a low expression of glycan structures recognized by ConA, VVA and RCA120. The model group, which underwent ischaemia for 45 min, showed a high expression of glycan structures recognized by LTL and SNA and a low expression of glycan structures recognized by ECA. Further analysis showed that the Siaα2-6Gal/N-acetylgalactosamine (GalNAc) structures recognized by SNA were significantly increased. In total, 91 differential proteins were identified by LC‒MS/MS, and 8 hub genes were screened by Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and protein interaction analyses. Compared with the Gene Expression Omnibus (GEO) database genes, two differential genes, Pros1 and Vtn, were obtained. Pros1 is a key regulator of the inflammatory response and vascular injury response. The Vtn gene variant is associated with the risk of myocardial infarction. This study is expected to provide a new method for the treatment of I/R injury and could provide new ideas for the postoperative prognosis of patients. Highlights • Changes in the mitochondrial glycoprotein glycan profile in Myocardial Ischaemia Reperfusion Injury.• Expression of Siaα2-6Gal/GalNAc structures recognized by SNA increased in the process of I/R injury.• Proteins identified by LC‒MS/MS in the sham-operated group and the 30-min ischaemia group.• By comparison with the GEO database, two candidate genes, Pros1 and Vtn, were obtained.

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Mitochondrial import of PKCepsilon is mediated by HSP90: a role in cardioprotection from ischaemia and reperfusion injury.

Publications

Mitochondrial import of PKCepsilon is mediated by HSP90: a role in cardioprotection from ischaemia and reperfusion injury.

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