Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
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SHR Neuro Krebs Kardio Lipid Stoffw Microb
Bugger, H; Leippert, S; Blum, D; Kahle, P; Barleon, B; Marme, D; Doenst, T.
Subtractive hybridization for differential gene expression in mechanically unloaded rat heart.
Am J Physiol Heart Circ Physiol. 2006; 291(6): H2714-H2722.
Doi: 10.1152/ajpheart.00445.2005
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PubMed
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- Führende Autor*innen der Med Uni Graz
- Bugger Heiko Matthias
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- 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.
- Find related publications in this database (using NLM MeSH Indexing)
- Animals -
- Atrophy - genetics
- Atrophy - physiopathology
- Biomechanical Phenomena -
- DNA - genetics
- Down-Regulation - physiology
- Electron Transport - genetics
- Electron Transport - physiology
- Gene Expression Regulation - physiology
- Heart - physiopathology
- Heart Transplantation - physiology
- Hybridization, Genetic -
- Male -
- Mitochondria, Heart - physiology
- Myocardium - metabolism
- Myocardium - pathology
- RNA, Messenger - genetics
- Rats -
- Rats, Inbred WKY -
- Suppression, Genetic -
- Transplantation, Heterotopic -
- Find related publications in this database (Keywords)
- suppression subtractive hybridization
- respiratory chain gene
- mitochondria