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−1with glutamate ( P < 0.01) and 59 ± 18 vs. 154 ± 30 natoms O·min−1·mg−1with 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.