Mitochondrial biogenesis by NO yields functionally active mitochondria in mammals

Abstract

We recently found that long-term exposure to nitric oxide (NO) triggers mitochondrial biogenesis in mammalian cells and tissues by activation of guanylate cyclase and generation of cGMP. Here, we report that the NO/cGMP-dependent mitochondrial biogenesis is associated with enhanced coupled respiration and content of ATP in U937, L6, and PC12 cells. The observed increase in ATP content depended entirely on oxidative phosphorylation, because ATP formation by glycolysis was unchanged. Brain, kidney, liver, heart, and gastrocnemius muscle from endothelial NO synthase null mutant mice displayed markedly reduced mitochondrial content associated with significantly lower oxygen consumption and ATP content. In these tissues, ultrastructural analyses revealed significantly smaller mitochondria. Furthermore, a significant reduction in the number of mitochondria was observed in the subsarcolemmal region of the gastrocnemius muscle. We conclude that NO/cGMP stimulates mitochondrial biogenesis, both in vitro and in vivo, and that this stimulation is associated with increased mitochondrial function, resulting in enhanced formation of ATP.

Figures

Fig. 1.

NO triggers transcription of genes involved in mitochondrial biogenesis in different mammalian cells through cGMP generation. U937, L6, and PC12 cells were cultured for 6 days with or without 50 μM DETA-NO, 3 mM 8 Br-cGMP, 1 μM BAY 41-2272, or 1 μM ODQ, as indicated. PGC-1α, NRF-1, and Tfam mRNA levels were analyzed by means of quantitative RT-PCR analysis with gene-specific oligonucleotide probes. Specific fluorescence was monitored during the complete amplification process and compared with glyceraldehyde-3-phosphate dehydrogenase (internal control) fluorescence. mRNAs levels were expressed as percentages of increase over the values obtained in untreated cells (n = 5). With all treatments, except those with DETA-NO plus ODQ or ODQ alone, the percentages of increase over basal values were statistically significant (P < 0.01).

Fig. 2.

NO increases mitochondrial content in different mammalian cells through cGMP generation. U937, L6, and PC12 cells were cultured for 6 days with or without 50 μM DETA-NO, 3 mM 8 Br-cGMP, 1 μM BAY 41-2272, or 1 μM ODQ, as indicated. The following parameters were then evaluated: mtDNA (one experiment representative of five; the numbers below the gels show the relative values from the densitometric analysis when control measurements are given a value of 1.0), COX-IV, and Cyt c proteins (prot), detected by immunoblot analysis. Protein levels were expressed as percentages of increase over the values obtained in untreated cells (n = 5). With all treatments, except those with DETA-NO plus ODQ or ODQ alone, the percentages of increase over basal values were statistically significant (P < 0.01).

Fig. 3.

cGMP-dependent mitochondrial biogenesis leads to an increase in oxidative phosphorylation-coupled respiration. U937, L6, and PC12 cells were cultured for 6 days with or without 3 mM 8 Br-cGMP or 1 μM BAY 41-2272, as indicated. O2 consumption was measured in a gas-tight chamber by using an O2 electrode (as described in Materials and Methods in samples; 107 cells per sample) from the various cell preparations in the presence or absence of the mitochondrial uncoupler FCCP (2 μM) or the F1/F0 ATP synthetase inhibitor oligomycin (2.5 μM). O2 consumption values were normalized to the protein content of the cells (n = 6). In each image, the numbers above the bars indicate the fold increase in respiration over basal value induced by cell treatment with 8 Br-cGMP or BAY 41-2272. Asterisks indicate statistical significance, as described in Materials and Methods, vs. controls.

Fig. 4.

cGMP-dependent mitochondrial biogenesis leads to increases in steady-state ATP levels through oxidative phosphorylation. U937, L6, and PC12 cells were cultured for 6 days with or without 3 mM 8 Br-cGMP or 1 μM BAY 41-2272, as indicated. (A) Cellular steady-state ATP levels in the various cell preparations. (B) Steady-state ATP levels, measured in U937 cells in the presence or absence of the complex I inhibitor rotenone (1 μM). Asterisks indicate statistical significance, as described in Materials and Methods, vs. controls (n = 3).