Proteomic Analysis of Pharmacological Preconditioning

Abstract

Abstract-Ischemic preconditioning is characterized by resistance to ischemia reperfusion injury in response to previous short ischemic episodes, a protective effect that can be mimicked pharmacologically. The underlying mechanism of protection remains controversial and requires greater understanding before it can be fully exploited therapeutically. To investigate the overall effect of preconditioning on the myocardial proteome, isolated rabbit ventricular myocytes were treated with drugs known to induce preconditioning, adenosine or diazoxide (each at 100 mol/L for 60 minutes). Their protein profiles were then compared with vehicle-treated controls (nϭ4 animals per treatment) using a multitiered 2D gel electrophoresis approach. Of 28 significantly altered protein spots, 19 nonredundant proteins were identified (5 spots remained unidentified). The majority of these proteins are involved in mitochondrial energetics, including subunits of tricarboxylic acid cycle enzymes and oxidative phosphorylation complexes. These changes were not indiscriminate, with only a small number of enzymes or complex subunits altered, indicating a very specific and targeted affect of these 2 preconditioning mimetics. Among the changes were shifts in the extent of posttranslational modification of 4 proteins. One of these, the adenosine-induced phosphorylation of the ATP synthase ␤ subunit, was fully characterized with the identification of 5 novel phosphorylation sites. This proteomics approach provides an overall assessment of the cellular response to pharmacological treatment with adenosine and diazoxide and identifies a distinct subset of enzymes and protein complex subunit that may underlie the preconditioned phenotype. Key Words: ATP synthase Ⅲ phosphorylation Ⅲ preconditioning Ⅲ proteomics I schemic preconditioning (PC) is an endogenous cytoprotective process whereby cells become profoundly resistant to ischemia/reperfusion injury following brief episodes of conditioning ischemia. The initial report of PC described a 76% reduction in cell death in PC compared with control hearts. 1 Protection was found to be transient, lasting 1 to 3 hours following the conditioning trigger, 1,2 and is now known to be biphasic, resuming after 24 hours to protect for a second phase of 3 to 4 additional days. 3,4 First described in the heart, PC is a ubiquitous phenomenon that has now been demonstrated in every mammalian tissue type and species examined to date, 5 including humans. 6,7 PC can also be initiated pharmacologically. Like ischemic PC, pharmacological PC triggers biphasic protection and inhibitors of pharmacological PC block ischemic PC, 5 indicating commonality in their underlying mechanistic properties. A broad range of PC mimetic agents have been identified, encompassing such diverse functions as G proteincoupled receptor (GPCR) agonists (eg, adenosine), potassium channel openers (eg, diazoxide), sodium/hydrogen exchange inhibitors, and volatile anesthetics. 8 Preservation of the preconditioned phenotype across this array of trigger...

Human ATP synthase beta is phosphorylated at multiple sites and shows abnormal phosphorylation at specific sites in insulin-resistant muscle

“…Here, we demonstrate that human ATPsyn-β is phosphorylated at multiple specific sites in vivo. These results extend our previous work [21] and that of several other studies that have used various phospholabelling techniques or phosphopeptide identification by MS, which have provided indirect evidence for serine/ threonine/tyrosine phosphorylation of ATPsyn-β in yeast, plants and mammalian tissues and cell lines [27][28][29][30][31][32][33][34][35].…”

“…Recently, five specific phosphorylation sites were assigned to ATPsyn-β in rabbit heart muscle using phosphopeptide enrichment and MS/MS technology [27]. However, although all sites were targeted in our analyses, of these we could only confirm the phosphorylation at Thr312 in human skeletal muscle.…”

“…However, although all sites were targeted in our analyses, of these we could only confirm the phosphorylation at Thr312 in human skeletal muscle. In addition to Thr213 [21] and Thr312 [27], we identified five novel phosphorylation sites at Thr475, Tyr230, Tyr269, Tyr361 and Tyr395, respectively. These data suggest the possibility that reversible phosphorylation of human ATPsyn-β at specific serine/threonine/tyrosine residues regulates the catalytic activity of ATP synthase, and hence ATP synthesis.…”

The 12S rRNA A1555G mutation in the mitochondrial haplogroup D5a is responsible for maternally inherited hypertension and hearing loss in two Chinese pedigrees

“…40 An inefficient metabolism caused by mitochondrial dysfunctions in skeletal and vascular smooth muscles may lead to the elevation of systolic blood pressure and therefore may be involved in the development of hypertension. [41][42][43] In summary, our investigation provides the first direct evidence that the known 12S rRNA A1555G mutation leads to both of hearing loss and hypertension. The A1555G mutation should be added to the list of inherited factors for future molecular diagnosis for hypertension.…”

Predict, prevent and personalize: Genomic and proteomic approaches to cardiovascular medicine

“…Several studies (47-49) of ischemia-reperfusion models have identified alterations in several functional groups: sarcomeric and cytoskeletal proteins, redox regulation, energy metabolism and the stress response. To further understand the preconditioned phenotype, proteomic analysis of pharmacological protection to ischemiareperfusion injury revealed that the majority of the proteins involved are involved in mitochondrial energetics (50). Myocardial subproteome analysis of a Rac-1-expressing mouse with lethal hypertrophy identified differential expression of the creatine kinase M-chain, tubulin beta-chain, manganese superoxide dismutase and malate dehydrogenase (51).…”