Transcriptomic and proteomic analysis of global ischemia and cardioprotection in the rabbit heart

Abstract

scriptomic and proteomic analysis of global ischemia and cardioprotection in the rabbit heart. Physiol Genomics 38: 125-137, 2009. First published May 19, 2009 doi:10.1152/physiolgenomics.00033.2009Cardioplegia is used to partially alleviate the effects of surgically induced global ischemia injury; however, the molecular mechanisms involved in this cardioprotection remain to be elucidated. To improve the understanding of the molecular processes modulating the effects of global ischemia and the cardioprotection afforded by cardioplegia, we constructed rabbit heart cDNA libraries and isolated, sequenced, and identified a compendium of nonredundant cDNAs for use in transcriptomic and proteomic analyses. New Zealand White rabbits were used to compare the effects of global ischemia and cardioplegia compared with control (nonischemic) hearts. The effects of RNA and protein synthesis on the cardioprotection afforded by cardioplegia were investigated separately by preperfusion with either ␣-amanitin or cycloheximide. Our results demonstrate that cardioplegia partially ameliorates the effects of global ischemia and that the cardioprotection is modulated by RNA-and protein-dependent mechanisms. Transcriptomic and proteomic enrichment analyses indicated that global ischemia downregulated genes/proteins associated with mitochondrial function and energy production, cofactor catabolism, and the generation of precursor metabolites of energy. In contrast, cardioplegia significantly increased differentially expressed genes/proteins associated with the mitochondrion and mitochondrial function and significantly upregulated the biological processes of muscle contraction, involuntary muscle contraction, carboxylic acid and fatty acid catabolic processes, fatty acid ␤-oxidation, and fatty acid metabolic processes. mitochondrion; ischemia-reperfusion MYOCARDIAL ISCHEMIA-REPERFUSION INJURY occurs as the result of the attenuation or cessation of coronary blood flow such that oxygen delivery to the myocardium is insufficient to meet energy demands. The cessation of myocardial coronary blood flow induces a cascade of cellular events that rapidly alter myocardial cellular homeostasis, leading to cellular dysfunction and/or death and postischemic functional impairment (23).To alleviate the effects of surgically induced ischemia/ reperfusion injury, surgeons use cardioplegia (CP) solutions that allow for the rapid electromechanical arrest of the myocardium through the alteration of cellular electrochemical gradients (40). In a series of studies, we have shown that magnesium-supplemented potassium CP with the addition of diazoxide significantly decreases myocardial cell death and significantly enhances postischemic functional recovery (22,40,43).The mechanisms through which CP affords cardioprotection are complex and cannot be investigated as a single entity; rather, they must be investigated as a system. In previous reports, we and others have used a variety of methods to identify the RNAs and proteins associated with global ischemia (GI) a...

Microarray and proteomic analysis of the cardioprotective effects of cold blood cardioplegia in the mature and aged male and female

“…Total tissue RNA was isolated, and quality and purity were assessed by spectrophotometric analysis and agarose gel electrophoresis as described previously (27). Total tissue ventricular myocardial protein was isolated and quality and purity was assessed by SDS-polyacrylamide gel electrophoresis (PAGE) as described previously (27).…”

“…The rabbit heart closely parallels the human heart, with similar myosin heavy chain phenotype, metabolic rate, and lacking myocardial xanthine oxidase (7,14). The use of 38 mo of age rabbits provides a model that we have previously shown to have significantly increased infarct size, significantly decreased postischemic functional recovery, significantly decreased mitochondrial function, and high energy phosphate preservation and resynthesis following ischemia/reperfusion compared with rabbits 12-15 wk of age (27)(28)(29).In situ blood perfused heart model. Rabbits (n ϭ 72) were sedated with acepromazine (0.75 mg/kg im), and a 21-gage butterfly catheter was placed in the ear and looped and secured in place with tape and Address for reprint requests and other correspondence:…”

Pressure-overload hypertrophy of the developing heart reveals activation of divergent gene and protein pathways in the left and right ventricular myocardium

“…Total tissue RNA was isolated and quality and purity was assessed by spectrophotometric analysis and agarose gel electrophoresis as described previously (32). Total tissue ventricular myocardial protein was isolated, and quality and purity was assessed by SDS-PAGE as described previously (23).…”

“…Microarray analysis was performed as previously described using our nonredundant rabbit heart cDNA compendium consisting of 3,592 nonredundant cDNAs (mean insert size of 1.67 kb) (3,23). Putative identity was assigned only to cDNAs exhibiting matches to Homo sapiens or Oryctolagus cuniculus (rabbit) genes with Eϭ1e-25 (minimum P ϭ 10 -10) and nucleotide sequence identity Ͼ95%.…”

Cardioprotection by gene therapy

“…As of today, genes and signaling pathways by which conditioning exerts its effects on the heart are partially understood, due at least in part to the arbitrarily selected gene targets pursued so far that allowed identifying one or a few genes associated with IRI and with cardioprotection by conditioning. An alternative way to approach this fundamental issue is to use functional genomic approaches to identify genes that are differentially expressed in conditioning [10][11][12]. Genomics refers to the study of multiple genetic variants and/or gene expression profiles simultaneously [13,14].…”