Heme oxygenase-1 regulates cardiac mitochondrial biogenesis via Nrf2-mediated transcriptional control of nuclear respiratory factor-1 - PubMed (original) (raw)

Heme oxygenase-1 regulates cardiac mitochondrial biogenesis via Nrf2-mediated transcriptional control of nuclear respiratory factor-1

Claude A Piantadosi et al. Circ Res. 2008.

Abstract

Heme oxygenase (HO)-1 is a protective antioxidant enzyme that prevents cardiomyocyte apoptosis, for instance, during progressive cardiomyopathy. Here we identify a fundamental aspect of the HO-1 protection mechanism by demonstrating that HO-1 activity in mouse heart stimulates the bigenomic mitochondrial biogenesis program via induction of NF-E2-related factor (Nrf)2 gene expression and nuclear translocation. Nrf2 upregulates the mRNA, protein, and activity for HO-1 as well as mRNA and protein for nuclear respiratory factor (NRF)-1. Mechanistically, in cardiomyocytes, endogenous carbon monoxide (CO) generated by HO-1 overexpression stimulates superoxide dismutase-2 upregulation and mitochondrial H(2)O(2) production, which activates Akt/PKB. Akt deactivates glycogen synthase kinase-3beta, which permits Nrf2 nuclear translocation and occupancy of 4 antioxidant response elements (AREs) in the NRF-1 promoter. The ensuing accumulation of nuclear NRF-1 protein leads to gene activation for mitochondrial biogenesis, which opposes apoptosis and necrosis caused by the cardio-toxic anthracycline chemotherapeutic agent, doxorubicin. In cardiac cells, Akt silencing exacerbates doxorubicin-induced apoptosis, and in vivo CO rescues wild-type but not Akt1(-/-) mice from doxorubicin cardiomyopathy. These findings consign HO-1/CO signaling through Nrf2 and Akt to the myocardial transcriptional program for mitochondrial biogenesis, provide a rationale for targeted mitochondrial CO therapy, and connect cardiac mitochondrial volume expansion with the inducible network of xenobiotic and antioxidant cellular defenses.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Nuclear Nrf2 accumulation and cardiac protection by CO. CO breathing in mice induces HO-1 mRNA expression and enzyme activity in Wt heart after 24 hours (A). Cardiac Nrf2 nuclear localization by confocal microscopy after CO is enhanced at 12 hours (B), confirmed by nuclear protein Western blot (C). Cardiac Nrf2 mRNA increases 3-fold within 6 hours and remains elevated for 12 hours after CO (D). *P<0.05 compared with preexposure control (n=4). CO given at days 0 and 7 after Dox (E) prevents the decline in right and left ventricular ejection fraction at 14 days by Doppler ultrasound.

Figure 2

Figure 2

Nrf2 expression in mouse cardiomyocytes. HL-1 cells exposed to DCM/CO (50 _μ_mol/L) increase nuclear Nrf2 content by >5-fold along with cytoplasmic Keap1 dissociation that is blocked by mitochondrial-targeted catalase (mCAT) (A). B, DCM/CO increases Nrf2, HO-1, and SOD2 mRNA levels by 3- to 4-fold, whereas Nrf2 silencing abrogates the mRNA responses and scrambled siRNA has no effect. *P<0.05 compared with control (n=4). C, DCM/CO increases HO-1 enzyme activity by >10-fold, also abrogated by Nrf2 silencing but not by scrambled siRNA. *P<0.05 (n=4). D, Top, HL-1 cells treated with DCM/CO (50 _μ_mol/L) or overexpressing HO-1 increase Akt phosphorylation and nuclear Nrf2 accumulation. mCAT inhibits HO-1-induced Akt activation and Nrf2 nuclear translocation. D, Bottom, PI3-kinase inhibition shows Akt primarily regulates Nrf2 translocation, not exclusion of the Bach1 nuclear repressor. DCM/CO produces greater Akt activity but less nuclear Bach1, whereas Akt inhibition does not affect nuclear Bach1 but reduces Nrf2 translocation. Bach1 siRNA increases nuclear Nrf2 with stable Akt activity.

Figure 3

Figure 3

HO-1 increases nuclear NRF-1 and Nrf2 occupancy of NRF-1 ARE promoter motifs. Active HO-1 increases NRF-1 expression and nuclear protein by confocal microscopy in HL-1 cells (A). Cotransfection with HO-1 and active Akt containing an amino-terminal myristoylation signal (myr-Akt) produces a further 3-fold increase in nuclear NRF-1 protein (A and B) accompanied by 8- to 10-fold increases in Tfam expression (B, right) as well as mtDNA copy number (C). A search of the NRF-1 promoter for Nrf2-binding sites 10 kb upstream of the TSS identified multiple ARE motifs investigated by ChIP (D). Arrows indicate orientation of each ARE motif relative to the ARE consensus. Gray boxes are motifs meeting the consensus sequence and black boxes show active motifs. The graph shows DNA enrichment by Nrf2 ChIP at each NRF-1 ARE motif. Values along the abscissa indicate positions of DNA regions amplified by qPCR. Black bars depict significant DNA enrichment after DCM/CO at positions −1400, −3289, 3386, and −9829. Nrf2 binding increased 11 to 13-fold at each site. Values represent the means±SEM of independent experiments performed in triplicate (*P<0.05).

Figure 4

Figure 4

Nrf2/HO-1 induction of NRF-1 activates mitochondrial biogenesis and prevents doxorubicin-induced cardiomyocyte apoptosis. Nuclear Nrf2 and NRF-1 expression were evaluated in H9c2 cardiomyoblasts without and with HO-1 overexpression or silencing compared with readouts of 2 nuclear-encoded downstream genes, Tfam and PolRMT, that regulate mitochondrial mRNA levels and with which Dox interferes (A, top). HO-1 overexpression doubles mitochondrial Tfam and PolRMT protein and attenuates Dox interference (A, middle). A, Bottom, demonstrates effective HO-1 silencing at 24 and 48 hours. Mitochondrial functional protection by HO-1 is reflected by enhanced MTT reduction and citrate synthase (CS) expression (B) and mRNA for mitochondrial-encoded subunits COX I and ND1 (C). Dox-induced changes in Mitotracker green show organelle modification from a fine reticulum to vesicles and aggregates within 24 hours (D, top). In contrast, mitochondrial density in cells overexpressing HO-1 is greater than control, and changes in mitochondrial structure occur sporadically after Dox (D, bottom). E, Dox-induced caspase 3 cleavage; HO-1 overexpression limits and HO-1 silencing exacerbates caspase 3 cleavage. Dox also decreases mitochondrial Bcl-XL and increases mitochondrial phosphorylated Bad (Ser-112) protein, which is counteracted by HO-1 overexpression. HO-1 silencing reverses the effects of HO-1 expression on mitochondrial pBad, Bid, and Bcl-XL.

Figure 5

Figure 5

HO-1/CO, mitochondrial biogenesis, and cardiomyocyte survival. Mitochondrial damage and cell loss after Dox is more severe in Akt1−/− mice expressing mitochondrial GFP (Akt1−/−/GFP) than in Wt mice (A), and CO protects mitochondria and rescues cells in Wt but not Akt1−/− mice. Left ventricular myocardial sections stained with hematoxylin/eosin show interstitial edema, focal necrosis, and myofibrillar degeneration 7 days after Dox. Damage is more extensive in Akt−/− than Wt mice and ameliorated by CO in Wt but not Akt1−/− mice. B, TUNEL staining of heart sections from Wt and Akt1−/− mice before and 7 days after Dox. Large numbers of TUNEL-stained nuclei are noted after Dox, and CO protection is limited to the Wt. Supplemental Figure I shows the quantification of cardiac mitochondrial fluorescence in the GFP mice.

Figure 6

Figure 6

CO and the Akt-GSK3_β_ relationship. In immunoprecipitation studies (A), Nrf2 serine/threonine phosphorylation, as well as Bach1 phosphorylation, decreases after CO (Nrf2 and Bach1 motifs for GSK3_β_ are inhibited by Akt). In contrast to Wt mice, neither Akt nor GSK3_β_ is phosphorylated in Akt1−/− mice after CO (B). Also, nuclear translocation of NRF-1 induced by CO in Wt mice is absent in Akt1−/− mice (C).

Figure 7

Figure 7

Working diagram of the HO-1/CO/Nrf2 pathway involved in the regulation of mitochondrial biogenesis. Nrf2 constitutively docks with Keap1, which sequesters it in the cytoplasm, allowing it to undergo ubiquitination and degradation (upper left). HO-1/CO increases mitochondrial H2O2 (lower left), which stabilizes Keap1 and results in Nrf2 gene expression and nuclear translocation. Nuclear Nrf2 undergoes binding to AREs in the Hmox1, SOD2, and NRF-1 promoters (far right). HO-1 and SOD2 amplify the mitochondrial H2O2 signal, whereas NRF-1 entry into the nucleus drives transcription for Tfam, PolRMT, and other genes of mitochondrial biogenesis.

Comment in

References

    1. Ashrafian H, Watkins H. Reviews of translational medicine and genomics in cardiovascular disease: new disease taxonomy and therapeutic implications cardiomyopathies: therapeutics based on molecular phenotype. J Am Coll Cardiol. 2007;49:1251–1264. - PubMed
    1. Suliman HB, Carraway MS, Ali AS, Reynolds CM, Welty-Wolf KE, Piantadosi CA. The CO/HO system reverses inhibition of mitochondrial biogenesis and prevents murine doxorubicin cardiomyopathy. J Clin Invest. 2007;117:3730–3741. - PMC - PubMed
    1. Maines MD. Heme oxygenase: function, multiplicity, regulatory mechanisms, and clinical applications. FASEB J. 1988;2:2557–2568. - PubMed
    1. Ryter SW, Alam J, Choi AM. Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications. Physiol Rev. 2006;86:583–650. - PubMed
    1. Dulak J, Deshane J, Jozkowicz A, Agarwal A. Heme oxygenase-1 and carbon monoxide in vascular pathobiology: focus on angiogenesis. Circulation. 2008;117:231–241. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources