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First published online 19 December 2006
doi: 10.1242/jcs.03318

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A new activating role for CO in cardiac mitochondrial biogenesis

Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA

View larger version (83K): [in this window] [in a new window]   Fig. 1. Metabolic and mitochondrial responses to CO in heart. (A) Basal VO2 and VCO2 in Wt mice before and after CO exposure (1 hour, 1250 ppm). Gray bars indicate steady-state O2 uptake and white bars CO2 production. Values are the mean + s.e., n= 4 mice (P<0.05 less than pre-exposure control and *P<0.05 higher than pre-exposure control by repeated measures ANOVA). (B) 2D-PAGE of mouse cardiac mitochondria. Heart mitochondrial preparations of equal yield (2.5% of original protein) from a control and a CO-exposed mouse were carried simultaneously through 2D separation. The five respiratory complexes (I to V) showed approximately 1.5- to 2.2-fold increases in protein content 24 hours after CO (densitometry not shown). (C) Respiration rates in cardiac mitochondria of controls or mice 24 hours after CO exposure. State-4–State-3 per mg protein were similar except for higher succinate use after CO (mean + s.e., n=4; *P<0.05). (D) Low-temperature difference spectra (77°K) of cardiac mitochondria showing CO–cytochrome-a3 complex formation (CO-a3) and selective cytochrome b-c1 reduction (Cyt bc1). (E) TEM of longitudinal sections of hearts of control (left) and 24 hours after CO exposure (center) in Wt mice. Biogenesis is seen after CO in interfibrillar mitochondria (8000x magnification). Enlargement (right panel) shows budding (arrowhead) and dividing (arrows) mitochondria characteristic of mitochondrial biogenesis (22,000x magnification). The increase in mitochondrial density was approximately 30% 24 hours after treatment with CO by point counting.

View larger version (29K): [in this window] [in a new window]   Fig. 2. Cardiac CO, PGC-1, TFAM and Pol expression. (A) Cellular CO content and PGC-1 mRNA levels. (Left) Dose-dependent increases in left ventricular CO content in blood-free tissue of Wt mice exposed to CO for 1 hour (mean + s.e. in pmol/mg tissue, n=4; *P<0.05). Hypoxia (HH, 1 hour) was the control. (Middle) Cardiac mRNA levels for PGC-1 in Wt mice by real-time RT-PCR 6 hours after CO exposure. Hypoxia (HH, 1 hour) with 5 hours recovery was the control (pmol/mg tissue, n=4 per group; *P<0.05). (Right) Dose plot for CO content and cardiac PGC-1 mRNA. (B) Pol (left) and TFAM protein (middle) in Wt and eNOS–/– mouse hearts by western blot analysis. -actin was the loading control. Histograms are the mean + s.e. for n=4; #P<0.05 compared with control for both strains. The right panel shows mtDNA copy number in Wt and eNOS–/– mice 24 hours after treatment with CO. The top band corresponds to the 571-bp mtDNA and the bottom band to the 332-bp competitive DNA fragment of the sample. Values are the mean + s.e., n=4 for each strain, *P< 0.05).

View larger version (35K): [in this window] [in a new window]   Fig. 3. CO activates gene expression of cardiac mitochondrial biogenesis in mice. Real time RT-PCR for Wt and eNOS–/– mouse heart after 1-hour treatment with CO. (A) mRNA levels for PGC-1; (B) NRF1; (C) NRF2 subunit; (D) Tfam. (E,F) No increase of PGC-1 or NRF1 mRNA in Wt heart after hypoxia (HH) or hyperbaric oxygen together with (HBO+CO). Values are the mean + s.e. normalized to GAPDH and multiplied by total RNA (mg/wet weight); *P<0.05 between strains at baseline; **P<0.05 compared with time zero; #P<0.05 compared with time zero and Wt.

View larger version (39K): [in this window] [in a new window]   Fig. 4. CO activation of cardiac p38 MAP kinase and PI3-K/Akt. (A, top) Western blot of phosphorylated p38 (P-p38) in Wt and eNOS–/– mice after treatment with CO (1250 ppm, 1 hour) with or without p38 inhibition (SB20). (Middle) Graphs indicate time courses for PGC-1, NRF1 and Tfam mRNA expression by real-time RT-PCR in Wt and eNOS–/– mice after treatment with CO with or without the p38 inhibitor (SB20) Values are the mean ± s.e. normalized to GAPDH multiplied by total RNA mg/wet weight (*P< 0.05 Wt vs eNOS–/–). Inhibition of p38 did not attenuate the CO effect in either strain. (Bottom) Gel showing cardiac mtDNA copy number by competitive PCR in Wt and eNOS–/– mice 24 hours after treatment with CO. The top band corresponds to the 571 bp of target mtDNA and the bottom band to the 332 bp DNA fragment. p38 inhibition did not alter the copy number. (B, top) Western blot of unphosphorylated Akt and phosphorylated Akt (pAkt) demonstrating an increase in the ratio of pAkt:Akt in Wt and eNOS–/– mice after treatment with CO. (Middle) Western blot of cardiac unphosphorylated Bad and phosphorylated Bad (pBAD), showing the pBad:Bad ratio in Wt and eNOS–/– mice after treatment with CO demonstrating phosphorylation of the mitochondrial anti-apoptotic protein. (Bottom) Western blot of Akt and pAkt showing the pAkt:Akt ratio in Wt heart after treatment with CO with or without hyperbaric oxygen (HBO), hypoxia (HH) or the p38 inhibitor (SB203580). Akt phosphorylation was not caused by hypoxia, and was prevented by hyperbaric oxygen but not by p38 inhibition (n=3).

View larger version (64K): [in this window] [in a new window]   Fig. 5. Cellular CO and mitochondrial biogenesis in H9c2 cardiomyocytes. (A, left) Effect of DCM on CO content in H9c2 cardiomyocytes. Peak CO levels occur 6 hours after 100 µM DCM. (Inset) Dose-response at 6 hours, showing that CO release is blocked by cytochrome P450 inhibition with SKF-525A. (Middle) CO stimulates cGMP content of H9c2 cells (P<0.05). (Right) Increase in functional mitochondria by MTT assay in CO-treated (100 µM DCM) vs control cells adjusted for protein (mean+ s.e., nine wells, three experiments). (B) Western blot for SOD2 in H9c2 cells at 6 hours and 24 hours with 100 µM DCM. SOD2 protein increased tenfold (densitometry not shown) and induction was inhibited by SKF-525A. (C) Oxidant localization in H9c2 cells after CO exposure. CSLM and colocalization of the oxidant probe, CC-1 with mitochondrial-selective dye, Mito-Tracker, in control H9c2 cells (A-C), cells exposed to CO (100 µM DCM) for 6h (D-F), or 24 hours (G-I) or after pre-incubation with cytochrome P450 inhibitor (SKF-525A) followed by DCM (J-M). Mitochondria localized with MitoTracker (A,D,G,J); CC-1 localization (B,E,H,L). Merged images for MitoTracker and CC-1 (C,F,I,M), yellow/orange signal indicates colocalization to mitochondria. (D) PI3-K/Akt activation in H9c2 cells by CO. Western blot is shown for pAkt/Akt. Lanes 1-4 show time course after treatment with CO, lanes 5-7 show effects of inhibitors of guanylate cyclase (ODQ 50 µM), p38 (SB20, 50 µM) or PI3-K/Akt (LY29, 50 µM) ODQ attenuated the response, SB20 had no effect and LY29 (and Wortmannin, not shown) was inhibitory. Lanes 8-10 demonstrate loss of pAkt response to CO in mtCAT transfected cells. (E) Western blot analysis of TFAM, NRF1, PGC-1, and NRF2 protein after treatment with CO in H9c2 cells relative to -actin. Lanes 1-4 are time course after treatment with CO (100 µM DCM); lanes 5-7 show the effects of inhibitors of guanylate cyclase (ODQ 50 µM), p38 (SB20, 50 µM) or PI3-K/Akt (LY29, 50 µM); ODQ and SB20 had no effect, but LY29 (and Wortmannin, not shown) inhibited expression of all four proteins. (F) Increases in the mtDNA content in H9c2 cells after treatment with CO (100 µM DCM). Gel shows competitive PCR data. Top band, 571-bp mtDNA amplified from each sample; bottom, 332-bp competitive DNA fragment. The increase in mtDNA was blocked by P450 inhibitor (SKF-525A), cGMP inhibitor (ODQ) or PI3-K/Akt inhibitor (LY29) but not by p38 inhibitor (SB20) at the concentrations indicated above.

View larger version (43K): [in this window] [in a new window]   Fig. 6. PGC-1, NRF1 and TFAM induction in cardiomyocytes. (A) PGC-1 associates with NRF1 in H9c2 cells after treatment with CO. Equal protein was immunoprecipitated (IP) with anti-PGC-1 or IgG (control), separated by SDS-PAGE, and blotted with anti-NRF1. After a 6-hour treatment with CO (100 mM DCM), PGC-1 partners with NRF1 and is abrogated by PI3-K/Akt inhibition (LY29). Loading was evaluated with 10% of the final immunoprecipitate on gels stained with Coomassie Blue. Data are representative of n=3. (B) ChIP assay for NRF1 and NRF2 binding to the Tfam promoter in H9c2 cells. Input lanes show PCR product prior to immunoprecipitation (loading control). DNA was analyzed by PCR with specific primers for Tfam promoter. CO (DCM 100 µM) increased binding of both transcription factors to Tfam promoter. After mtCAT transfection, CO had no significant effect on NRF1. (C) Mitochondrial BrdU incorporation after CO in cardiomyocytes. Confocal images of MitoTracker Green and Cy3 immunostaining for BrdU were obtained by BrdU labeling (red) as indicated followed by double-label confocal microscopy. Control H9c2 cells (A-C); cells exposed to CO (DCM 100 µM) for 6h followed by washing and 6h incubation (D-F). Panels A and D show mitochondria with MitoTracker (A,D); Red Cy3 immunostaining for BrdU (B,E). merged images of MitoTracker and Red Cy3 (C,F). Cytochrome P450 inhibitor (SKF-525A) abrogated CO-induced BrdU incorporation. Yellow/orange signal (G-I) indicates mitochondrial BrdU localization.

View larger version (20K): [in this window] [in a new window]   Fig. 7. Schematic of the mitochondrial respiratory chain showing the effect of CO binding (x) at the terminal oxidase on hydrogen peroxide (H2O2) production. CO slows the rate of electron transport (solid arrows), enabling electrons to accumulate, including at complex III. Complex III and the proton motive Q cycle have been expanded to show sites of ubi-semiquinone (Q·-) formation from which electrons can be donated to produce superoxide (·O2-) (dotted arrows). Complex III O2 availability is also increased by CO, which promotes ·O2-production and its conversion to H2O2 by SOD2 (MnSOD). SOD2 induction may increase the extra-mitochondrial H2O2 leak rate. Cyt, cytochrome; ISP, iron sulfur protein; Q, oxidized coenzyme; QH2, reduced coenzyme Q. The details of the Q cycle, including the transmembrane proton flux, have been omitted for clarity.