title: Subtractive hybridization for differential gene expression in mechanically unloaded rat heart.
abstract: The objective of this study was to identify differentially expressed genes in the mechanically unloaded rat heart by suppression subtractive hybridization. In male Wistar-Kyoto rats, mechanical unloading was achieved by infrarenal heterotopic heart transplantation. Differentially expressed genes were investigated systematically by suppression subtractive hybridization. Selected targets were validated by Northern blot analysis, real-time RT-PCR, and immunoblot analysis. Maximal ADP-stimulated oxygen consumption (state 3) was measured in isolated mitochondria. Transplantation caused atrophy (heart-to-body weight ratio: 1.6 +/- 0.1 vs. 2.4 +/- 0.1, P < 0.001). We selected 1,880 clones from the subtractive hybridization procedure (940 forward and 940 reverse runs assessing up- or downregulation). The first screen verified 465 forward and 140 reverse clones, and the second screen verified 67 forward and 30 reverse clones. On sequencing of 24 forward and 23 reverse clones, 9 forward and 14 reverse homologies to known genes were found. Specifically, we identified reduced mRNA expression of complex I (-49%, P < 0.05) and complex II (-61%, P < 0.001) of the respiratory chain. Significant reductions were also observed on the respiratory chain protein level: -42% for complex I (P < 0.01), -57% for complex II (P < 0.05), and -65% for complex IV (P < 0.05). Consistent with changes in gene and protein expression, state 3 respiration was significantly decreased in isolated mitochondria of atrophied hearts, with glutamate and succinate as substrates: 85 +/- 27 vs. 224 +/- 32 natoms O.min(-1).mg(-1) with glutamate (P < 0.01) and 59 +/- 18 vs. 154 +/- 30 natoms O.min(-1).mg(-1) with succinate (P < 0.05). Subtractive hybridization indicates major changes in overall gene expression by mechanical unloading and specifically identified downregulation of respiratory chain genes. This observation is functionally relevant and provides a mechanism for the regulation of respiratory capacity in response to chronic mechanical unloading.
authors.affiliation: Dept. of Cardiovascular Surgery, University of Freiburg, Germany.
authors.forename: Heiko
authors.initials: H
authors.lastname: Bugger
authors.forename: Stefanie
authors.initials: S
authors.lastname: Leippert
authors.forename: Daniel
authors.initials: D
authors.lastname: Blum
authors.forename: Peter
authors.initials: P
authors.lastname: Kahle
authors.forename: Bernhard
authors.initials: B
authors.lastname: Barleon
authors.forename: Dieter
authors.initials: D
authors.lastname: Marme
authors.forename: Torsten
authors.initials: T
authors.lastname: Doenst
mesh_terms: D000818:Animals; D001284:Atrophy / Q000235:genetics / Q000503:physiopathology; D001696:Biomechanical Phenomena; D004247:DNA / Q000235:genetics; D015536:Down-Regulation / Q000502:physiology; D004579:Electron Transport / Q000235:genetics* / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology*; D006321:Heart / Q000503:physiopathology*; D016027:Heart Transplantation / Q000502:physiology; D006824:Hybridization, Genetic; D008297:Male; D008929:Mitochondria, Heart / Q000502:physiology; D009206:Myocardium / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011921:Rats, Inbred WKY; D013489:Suppression, Genetic; D016042:Transplantation, Heterotopic
chemical_list: D012333:RNA, Messenger; D004247:DNA