Mitochondrial Heme Biosynthesis Research Articles

Endothelin-1 depletion of cartilage oligomeric matrix protein modulates pulmonary artery superoxide and iron metabolism-associated mitochondrial heme biosynthesis.

This study examines if heme biosynthesis-associated iron metabolism is regulated in pulmonary arteries by endothelin-1 (ET1) potentially through modulating cartilage oligomeric matrix protein (COMP) availability. Our studies in organoid-cultured endothelium-rubbed bovine pulmonary arteries (BPAs) observed COMP depletion by siRNA or hypoxia increases NOX2 and superoxide and depletes mitochondrial SOD2. ET1 also increases superoxide in a manner that potentially impairs mitochondrial heme biosynthesis. In this study, organoid culture of BPA with ET1 (10 nM) increases superoxide in the mitochondrial matrix and extramitochondrial regions associated with COMP depletion, and COMP (0.5 μM) inhibited these superoxide increases. As mitochondrial matrix superoxide could impair heme biosynthesis from protoporphyrin IX (PpIX) by decreasing Fe2+ availability and/or ferrochelatase (FECH), we studied ET1, COMP, and COMP siRNA effects on the expression of FECH, transferrin receptor-1 (TfR1, an indicator of iron availability) and soluble guanylate cyclase (sGC, a key heme-dependent protein), and on measurements of PpIX (HPLC) and heme content. ET1 decreased FECH, heme, and sGC, and increased TfR1 and iron. COMP reversed these effects of ET1, and COMP decreased PpIX and increased heme in the absence of ET1. COMP siRNA increased PpIX detection and TfR1 expression and decreased the expression of FECH and sGC. Nitric oxide (spermine NONOate) relaxation of BPA was inhibited by ET1, and this was attenuated by COMP during exposure to ET1. Thus, COMP depletion by ET1 or siRNA modulates pulmonary artery iron metabolism, which results in loss of heme biosynthesis and heme-dependent cGMP mechanisms.

Exercise Preconditioning Prevents Chemotherapy‐Induced Alterations to Transport Proteins Required for Mitochondrial Iron Uptake and Metabolism in the Diaphragm

Doxorubicin (DOX) is an anthracycline antibiotic used in cancer treatment. Unfortunately, the clinical use of this highly efficacious anticancer drug is limited due to the development of respiratory and diaphragm muscle dysfunction in patients. DOX‐induced ventilatory impairment is a debilitating condition that promotes the onset of dyspnea, fatigue and exercise intolerance. While the mechanisms responsible for DOX‐induced respiratory insufficiency are unclear, it has been proposed that DOX myotoxicity develops as a result of increased cellular reactive oxygen species production and mitochondrial iron accumulation. Conversely, endurance exercise training performed prior to DOX administration has been shown to prevent the chemotherapy‐induced diaphragm weakness and mitochondrial dysfunction. While the mechanisms for this protection remain elusive, evidence suggests that exercise may provide protection by attenuating DOX‐induced alterations to proteins required to maintain mitochondrial iron homeostasis. Cause and effect were determined by exposing Sprague‐Dawley rats to a two‐week treadmill preconditioning protocol (5 days/week; 60 min/day; 30 m/min). Twenty‐four hours following the last exercise training period, animals received either saline or DOX (20mg/kg ip) treatment. Our results demonstrate that DOX administration in sedentary animals results in increased expression of the mitochondrial iron importer mitoferrin‐1 and a significant reduction in the cellular iron importer transferrin receptor 1 in the diaphragm. In addition, DOX also stimulated a significant decrease in the expression of sideroflexin 2 and SLC25A28, proteins required for mitochondrial heme biosynthesis. Importantly, exercise training prior to DOX administration prevented the DOX‐induced increase in mitoferrin‐1, and also reduced mitoferrin‐2 below that of sedentary animals treated with DOX. Moreover, diaphragm expression of SLC25A38 was maintained in exercise trained animals receiving DOX. These findings confirm a role for mitochondrial iron accumulation and dysregulation of heme synthesis in the development of DOX‐induced respiratory dysfunction, and demonstrate that exercise preconditioning may ameliorate DOX‐mediated diaphragm muscle pathology through the prevention of mitochondrial iron dysregulation.Support or Funding InformationThis work was supported by NIH R01 HL146443 awarded to AJS

Modulation of Heme Biosynthesis by Ferrochelatase Inhibition Controls Soluble Guanylate Cyclase Expression and Superoxide Production in Bovine Coronary Arteries

Nitric oxide interacts with the Fe2+ heme prosthetic group in soluble guanylate cyclase (sGC) stimulating the conversion of guanosine 5′‐triphosphate to cyclic guanosine 3′,5′‐monophosphate (cGMP). Adequate cGMP production is associated with protective mechanisms against multiple cardiovascular disease processes. Thus, disturbances in this signaling pathway can lead to many pathological consequences. Since there has been minimal investigation on the consequences of altering heme biosynthesis on the cardiovascular system, we investigated if mitochondrial heme biosynthesis is an important factor in controlling the expression and function of sGC and systems influencing superoxide generation and actions. For the purpose of this study, we induced a heme deficiency by inhibiting ferrochelatase (FECH) with N‐methyl protoporphyrin IX (NMPP), potent FECH inhibitor. Bovine coronary arteries (BCA) were treated for 24 hours with 10 μM NMPP. Chemiluminescence (5μM lucigenin) was used to measure superoxide production; western blot analysis was used to measure expression of FECH, sGC and other heme‐containing proteins; and a QuantiChrom heme assay kit was used to measure heme levels. NMPP treatment promoted sGC depletion and attenuation of its activity based on it decreasing protein kinase G‐mediated phosphorylation of VASP and attenuation of relaxation to a nitric oxide donor. NMPP also decreased the expression of heme proteins including cytochrome oxidase subunit 4 and catalase. In addition, superoxide production was significantly elevated in NMPP treated coronary arteries when compared to that in control samples, and scavenging extra mitochondrial superoxide with Tempol and mitochondrial matrix superoxide with MitoTempol attenuated the increase in superoxide. NMPP stimulated increases in superoxide were associated with a decrease expression of mitochondrial FECH expression. Thus, inhibition of FECH increased extra mitochondrial and mitochondrial superoxide and depleted sGC. Our studies suggest that disruption of heme biosynthesis resulting in a loss of cGMP production may serve as a contributing mechanism to the progression of cardiovascular disease.Support or Funding InformationFunded by NIH grants R01HL115124 & R01HL129797

Frataxin-mediated Iron Delivery to Ferrochelatase in the Final Step of Heme Biosynthesis

Human ferrochelatase, a mitochondrial membrane-associated protein, catalyzes the terminal step of heme biosynthesis by insertion of ferrous iron into protoporphyrin IX. The recently solved x-ray structure of human ferrochelatase identifies a potential binding site for an iron donor protein on the matrix side of the homodimer. Herein we demonstrate Hs holofrataxin to be a high affinity iron binding partner for Hs ferrochelatase that is capable of both delivering iron to ferrochelatase and mediating the terminal step in mitochondrial heme biosynthesis. A general regulatory mechanism for mitochondrial iron metabolism is described that defines frataxin involvement in both heme and iron-sulfur cluster biosyntheses. In essence, the distinct binding affinities of holofrataxin to the target proteins, ferrochelatase (heme synthesis) and ISU (iron-sulfur cluster synthesis), allows discrimination between the two major iron-dependent pathways and facilitates targeted heme biosynthesis following down-regulation of frataxin.

Subcellular localization of two types of ferrochelatase in cucumber.

It is widely believed that ferrochelatase (protoheme ferrolyase, EC 4.99.1.1), which catalyzes the insertion of ferrous ion into protoporphyrin IX to form protoheme, exists in both plastids and mitochondria of higher plants. By in vitro import assay with isolated pea (Pisum sativum L.) organelles, it has been proposed that one of two isoforms of ferrochelatase (type 1) is dual-targeted into both plastids and mitochondria, and functions for heme biosynthesis in the both organelles. Recently, however, mitochondrial targeting of ferrochelatase is being disputed since pea mitochondria appeared to accept a variety of chloroplast proteins including the type-1 ferrochelatase of Arabidopsis thaliana (L.) Heynh. To clarify the precise subcellular localization of ferrochelatase in higher plants, here we investigated the subcellular localization of two types of ferrochelatase (CsFeC1 and CsFeC2) in cucumber (Cucumis sativus L.). In cotyledons, a significant level of specific ferrochelatase activity was detected in thylakoid membranes, but only a trace level of activity was detectable in mitochondria. Western blot analysis with specific antibodies showed that anti-CsFeC2 antiserum cross-reacted with plastids in photosynthetic and non-photosynthetic tissues. Anti-CsFeC1 did not cross-react with mitochondria, but CsFeC1 was clearly detectable in plastids from non-photosynthetic tissues. In situ transient-expression assays using green fluorescent protein demonstrated that, as well as CsFeC2, the N-terminal transit peptide of CsFeC1 targeted the fusion protein solely into plastids, but not into mitochondria. These results demonstrated that in cucumber both CsFeC1 and CsFeC2 are solely targeted into plastids, but not into mitochondria. Screening of a cucumber genomic or cDNA library did not allow any other ferrochelatase homologous gene to be isolated. The data presented here imply the reconsideration of mitochondrial heme biosynthesis in higher plants.

Effect of Voluntary Resistance Exercise on Mitochondrial Heme Biosynthesis in Rat Liver.

To evaluate the effects of voluntary resistance exercise training on mitochondrial heme biosynthesis in the liver, we trained adult male Sprague-Dawley rats for 4 or 8 weeks to climb a wire-mesh tower (∅20cm×200cm), and then measured δ-aminolevulinic acid synthetase (ALAS) activity, the rate-limiting enzyme of heme synthesis, and cytochrome c content in their liver. The activity of citrate synthase in the skeletal muscle was also determined to establish whether the training protocol had any aerobic effect. Resistance training resulted in significant increases (p<0.05) in citrate synthase activities in gastrocnemius and forearm muscles, whereas ALAS activity and cytochrome c content in the liver of rats trained for 4 and 8 weeks were the same as those of corresponding sedentary rats. The relative weight of forearm muscle to body mass was significantly greater (p<0.05) in the training group than in the sedentary group. These results suggest that voluntary resistance exercise does not accelerate hepatic heme biosynthesis in spite of increasing aerobic capacity in the skeletal muscles.

Heteroplasmic Point Mutations of Mitochondrial DNA Affecting Subunit I of Cytochrome c Oxidase in Two Patients With Acquired Idiopathic Sideroblastic Anemia

Mitochondrial iron overload in acquired idiopathic sideroblastic anemia (AISA) may be attributable to mutations of mitochondrial DNA (mtDNA), because these can cause respiratory chain dysfunction, thereby impairing reduction of ferric iron (Fe3+) to ferrous iron (Fe2+). The reduced form of iron is essential to the last step of mitochondrial heme biosynthesis. It is not yet understood to which part of the respiratory chain the reduction of ferric iron is linked. In two patients with AISA we identified point mutations of mtDNA affecting the same transmembrane helix within subunit I of cytochrome c oxidase (COX I; ie, complex IV of the respiratory chain). The mutations were detected by restriction fragment length polymorphism analysis and temperature gradient gel electrophoresis. One of the mutations involves a T → C transition in nucleotide position 6742, causing an amino acid change from methionine to threonine. The other mutation is a T → C transition at nt 6721, changing isoleucine to threonine. Both amino acids are highly conserved in a wide range of species. Both mutations are heteroplasmic, ie, they establish a mixture of normal and mutated mitochondrial genomes, which is typical of disorders of mtDNA. The mutations were present in bone marrow and whole blood samples, in isolated platelets, and in granulocytes, but appeared to be absent from T and B lymphocytes purified by immunomagnetic bead separation. They were not detected in buccal mucosa cells obtained by mouthwashes and in cultured skin fibroblasts examined in one of the patients. In both patients, this pattern of involvement suggests that the mtDNA mutation occurred in a self-renewing bone marrow stem cell with myeloid determination. Identification of two point mutations with very similar location suggests that cytochrome c oxidase plays an important role in the pathogenesis of AISA. COX may be the physiologic site of iron reduction and transport through the inner mitochondrial membrane.

Heteroplasmic Point Mutations of Mitochondrial DNA Affecting Subunit I of Cytochrome c Oxidase in Two Patients With Acquired Idiopathic Sideroblastic Anemia

AbstractMitochondrial iron overload in acquired idiopathic sideroblastic anemia (AISA) may be attributable to mutations of mitochondrial DNA (mtDNA), because these can cause respiratory chain dysfunction, thereby impairing reduction of ferric iron (Fe3+) to ferrous iron (Fe2+). The reduced form of iron is essential to the last step of mitochondrial heme biosynthesis. It is not yet understood to which part of the respiratory chain the reduction of ferric iron is linked. In two patients with AISA we identified point mutations of mtDNA affecting the same transmembrane helix within subunit I of cytochrome c oxidase (COX I; ie, complex IV of the respiratory chain). The mutations were detected by restriction fragment length polymorphism analysis and temperature gradient gel electrophoresis. One of the mutations involves a T → C transition in nucleotide position 6742, causing an amino acid change from methionine to threonine. The other mutation is a T → C transition at nt 6721, changing isoleucine to threonine. Both amino acids are highly conserved in a wide range of species. Both mutations are heteroplasmic, ie, they establish a mixture of normal and mutated mitochondrial genomes, which is typical of disorders of mtDNA. The mutations were present in bone marrow and whole blood samples, in isolated platelets, and in granulocytes, but appeared to be absent from T and B lymphocytes purified by immunomagnetic bead separation. They were not detected in buccal mucosa cells obtained by mouthwashes and in cultured skin fibroblasts examined in one of the patients. In both patients, this pattern of involvement suggests that the mtDNA mutation occurred in a self-renewing bone marrow stem cell with myeloid determination. Identification of two point mutations with very similar location suggests that cytochrome c oxidase plays an important role in the pathogenesis of AISA. COX may be the physiologic site of iron reduction and transport through the inner mitochondrial membrane.