Comprehensive Genomic Analysis Identifies a Diverse Landscape of Sideroblastic and Nonsideroblastic Iron-Related Anemias with Novel and Pathogenic Variants in an Iron-Deficient Endemic Setting.

Inherited iron metabolism defects are possibly missed or underdiagnosed in iron-deficient endemic settings because of a lack of awareness or a methodical screening approach. Hence, we systematically evaluated anemia cases (2019 to 2021) based on clinical phenotype, normal screening tests (high-performance liquid chromatography, α gene sequencing, erythrocyte sedimentation rate, C-reactive protein, and tissue transglutaminase), and abnormal iron profile by targeted next-generation sequencing (26-gene panel) supplemented with whole-exome sequencing, multiplex ligation probe amplification/mitochondrial DNA sequencing, and chromosomal microarray. Novel variants in ALAS2, STEAP3, and HSPA9 genes were functionally validated. A total of 290 anemia cases were screened, and 41 (14%) enrolled for genomic testing as per inclusion criteria. Comprehensive genomic testing revealed pathogenic variants in 23 of 41 cases (56%). Congenital sideroblastic anemia was the most common diagnosis (14/23; 61%), with pathogenic variations in ALAS2 (n = 6), SLC25A38 (n = 3), HSPA9 (n = 2) and HSCB, SLC19A2, and mitochondrial DNA deletion (n = 1 each). Nonsideroblastic iron defects included STEAP3-related microcytic anemia (2/23; 8.7%) and hypotransferrenemia (1/23; 4.3%). A total of 6 of 22 cases (27%) revealed a non-iron metabolism gene defect on whole-exome sequencing. Eleven novel variants (including variants of uncertain significance) were noted in 13 cases. Genotype-phenotype correlation revealed a significant association of frameshift/nonsense/splice variants with lower presentation age (0.8 months versus 9 years; P < 0.01) compared with missense variants. The systematic evaluation helped uncover an inherited iron defect in 41% (17/41) of cases, suggesting the need for active screening and awareness for these rare diseases in an iron-deficient endemic population.

Gene expression analysis revealed downregulation of complement receptor 1 in clonal B cells in cold agglutinin disease.

Cold agglutinin disease (CAD) is a rare B-cell lymphoproliferative disorder of the bone marrow, manifested by autoimmune hemolytic anemia caused by binding of monoclonal IgM autoantibodies to the I antigen. Underlying genetic changes have previously been reported, but their impact on gene expression profile has been unknown. Here, we define differentially expressed genes in CAD B cells. To unravel downstream alteration in cellular pathways, gene expression by RNA sequencing was undertaken. Clonal B-cell samples from 12 CAD patients and IgM-expressing memory B cells from 4 healthy individuals were analyzed. Differential expression analysis and filtering resulted in 93 genes with significant differential expression. Top upregulated genes included SLC4A1, SPTA1, YBX3, TESC, HBD, AHSP, TRAF1, HBA2, RHAG, CA1, SPTB, IL10, UBASH3B, ALAS2, HBA1, CRYM, RGCC, KANK2, and IGHV4-34. They were upregulated at least 8-fold, while complement receptor 1 (CR1/CD35) was downregulated 11-fold in clonal CAD B cells compared to control B cells. Flow cytometry analyses further confirmed reduced CR1 (CD35) protein expression by clonal CAD IgM+ B cells compared to IgM+ memory B cells in controls. CR1 (CD35) is an important negative regulator of B-cell activation and differentiation. Therefore, reduced CR1 (CD35) expression may increase activation, proliferation, and antibody production in CAD-associated clonal B cells.

Antagonism of ALAS1 by the Measles Virus V protein contributes to degradation of the mitochondrial network and promotes interferon response.

Viruses have evolved countless mechanisms to subvert and impair the host innate immune response. Measles virus (MeV), an enveloped, non-segmented, negative-strand RNA virus, alters the interferon response through different mechanisms, yet no viral protein has been described as directly targeting mitochondria. Among the crucial mitochondrial enzymes, 5'-aminolevulinate synthase (ALAS) is an enzyme that catalyzes the first step in heme biosynthesis, generating 5'-aminolevulinate from glycine and succinyl-CoA. In this work, we demonstrate that MeV impairs the mitochondrial network through the V protein, which antagonizes the mitochondrial enzyme ALAS1 and sequesters it to the cytosol. This re-localization of ALAS1 leads to a decrease in mitochondrial volume and impairment of its metabolic potential, a phenomenon not observed in MeV deficient for the V gene. This perturbation of the mitochondrial dynamics demonstrated both in culture and in infected IFNAR-/- hCD46 transgenic mice, causes the release of mitochondrial double-stranded DNA (mtDNA) in the cytosol. By performing subcellular fractionation post infection, we demonstrate that the most significant source of DNA in the cytosol is of mitochondrial origin. Released mtDNA is then recognized and transcribed by the DNA-dependent RNA polymerase III. The resulting double-stranded RNA intermediates will be captured by RIG-I, ultimately initiating type I interferon production. Deep sequencing analysis of cytosolic mtDNA editing divulged an APOBEC3A signature, primarily analyzed in the 5'TpCpG context. Finally, in a negative feedback loop, APOBEC3A an interferon inducible enzyme will orchestrate the catabolism of mitochondrial DNA, decrease cellular inflammation, and dampen the innate immune response.

ALAS1 associated with goat kidding number trait was regulated by the transcription factor ASCL2 to affect granulosa cell proliferation.

ALAS1 is a member of the α-oxoamine synthase family, which is the first rate-limiting enzyme for heme synthesis and is important for maintaining intracellular heme levels. In the ovary, ALAS1 is associated with the regulation of ovulation-related mitochondrial P450 cytochromes, steroid metabolism, and steroid hormone production. However, there are few studies on the relationship between ALAS1 and reproductive traits in goats. In this study, a mutation located in the promoter region of ALAS1 (g.48791372C>A) was found to be significantly (p < 0.05) associated with the kidding number of Yunshang black goats. Specifically, the mean kidding number in the first three litters and the kidding numbers of all three litters were significantly (p < 0.05) higher in individuals with the CA genotype or AA genotype than in those with the CC genotype. To further investigate the regulatory mechanism of ALAS1, the expression of ALAS1 in goat ovarian tissues with different genotypes was verified by real-time quantitative PCR. The results showed that the expression of ALAS1 was significantly higher in the ovaries of individuals with AA genotype than those with AC and CC genotypes (p < 0.01), and the expression trend of transcription factor ASCL2 was consistent with ALAS1. Additionally, the ALAS1 g.48791372C>A mutation created a new binding site for the transcription factor ASCL2. The luciferase activity assay indicated that the mutation increased the promoter activity of ALAS1. Overexpression of the transcription factor ASCL2 induced increased expression of ALAS1 in goat granulosa cells (p < 0.05). The opposite trend was shown for the inhibition of ASCL2 expression. The results of real-time quantitative PCR, EdU and Cell Counting Kit-8 assays indicated that the transcription factor ASCL2 increased the proliferation of goat granulosa cells by mediating the expression of ALAS1. In conclusion, the transcription factor ASCL2 positively regulated the transcriptional activity and expression levels of ALAS1, altering granulosa cell proliferation and the kidding number in goats.

Identification of crucial genes and metabolites regulating the eggshell brownness in chicken.

BACKGROUND: Protoporphyrin IX (Pp IX) is the primary pigment for brown eggshells. However, the regulatory mechanisms directing Pp IX synthesis, transport, and genetic regulation during eggshell calcification in chickens remain obscure. In this study, we investigated the mechanism of brown eggshell formation at different times following oviposition, using White Leghorn hens (WS group), Rhode Island Red light brown eggshell line hens (LBS group) and Rhode Island Red dark brown eggshell line hens (DBS group). RESULTS: At 4, 16 and 22 h following oviposition, Pp IX concentrations in LBS and DBS groups were significantly higher in shell glands than in liver (P < 0.05). Pp IX concentrations in shell glands of LBS and DBS groups at 16 and 22 h following oviposition were significantly higher than WS group (P < 0.05). In comparative transcriptome analysis, δ-aminolevulinate synthase 1 (ALAS1), solute carrier family 25 member 38 (SLC25A38), ATP binding cassette subfamily G member 2 (ABCG2) and feline leukemia virus subgroup C cellular receptor 1 (FLVCR1), which were associated with Pp IX synthesis, were identified as differentially expressed genes (DEGs). RT-qPCR results showed that the expression level of ALAS1 in shell glands was significantly higher in DBS group than in WS group at 16 and 22 h following oviposition (P < 0.05). In addition, four single nucleotide polymorphisms (SNPs) in ALAS1 gene that were significantly associated with eggshell brownness were identified. By identifying the differential metabolites in LBS and DBS groups, we found 11-hydroxy-E4-neuroprostane in shell glands and 15-dehydro-prostaglandin E1(1-) and prostaglandin G2 2-glyceryl ester in uterine fluid were related to eggshell pigment secretion. CONCLUSIONS: In this study, the regulatory mechanisms of eggshell brownness were studied comprehensively by different eggshell color and time following oviposition. Results show that Pp IX is synthesized de novo and stored in shell gland, and ALAS1 is a key gene regulating Pp IX synthesis in the shell gland. We found three transporters in Pp IX pathway and three metabolites in shell glands and uterine fluid that may influence eggshell browning.

ANTXR1 Regulates Erythroid Cell Proliferation and Differentiation through wnt/ß-Catenin Signaling Pathway In Vitro and in Hematopoietic Stem Cell.

Erythropoiesis is a highly complex and sophisticated multistage process regulated by many transcription factors, as well as noncoding RNAs. Anthrax toxin receptor 1 (ANTXR1) is a type I transmembrane protein that binds the anthrax toxin ligands and mediates the entry of its toxic part into cells. It also functions as a receptor for the Protective antigen (PA) of anthrax toxin, and mediates the entry of Edema factor (EF) and Lethal factor (LF) into the cytoplasm of target cells and exerts their toxicity. Previous research has shown that ANTXR1 inhibits the expression of γ-globin during the differentiation of erythroid cells. However, the effect on erythropoiesis from a cellular perspective has not been fully determined. This study examined the role of ANTXR1 on erythropoiesis using K562 and HUDEP-2 cell lines as well as cord blood CD34+ cells. Our study has shown that overexpression of ANTXR1 can positively regulate erythrocyte proliferation, as well as inhibit GATA1 and ALAS2 expression, differentiation, and apoptosis in K562 cells and hematopoietic stem cells. ANTXR1 knockdown inhibited proliferation, promoted GATA1 and ALAS2 expression, accelerated erythrocyte differentiation and apoptosis, and promoted erythrocyte maturation. Our study also showed that ANTXR1 may regulate the proliferation and differentiation of hematopoietic cells, though the Wnt/ß-catenin pathway, which may help to establish a possible therapeutic target for the treatment of blood disorders.

Construction of 5-aminolevulinic acid synthase variants by cysteine-targeted mutation to release heme inhibition.

5-Aminolevulinic acid (5-ALA), a vital precursor for the biosynthesis of tetrapyrrole compounds, has been widely applied in agriculture and medicine, while extremely potential for the treatment of cancers, corona virus disease 2019 (COVID-19) and metabolic diseases in recent years. With the development of metabolic engineering and synthetic biology, the biosynthesis of 5-ALA has attracted increasing attention. 5-Aminolevulinic acid synthase (ALAS), the key enzyme for 5-ALA synthesis in the C4 pathway, is subject to stringent feedback inhibition by heme. In this work, cysteine-targeted mutation of ALAS was proposed to overcome this drawback. ALAS from Rhodopseudomonas palustris (RP-ALAS) and Rhodobacter capsulatus (RC-ALAS) were selected for mutation and eight variants were generated. Variants RP-C132A and RC-C201A increased enzyme activities and released hemin inhibition, respectively, maintaining 82.5% and 81.9% residual activities in the presence of 15 µM hemin. Moreover, the two variants exhibited higher stability than that of their corresponding wild-type enzymes. Corynebacterium glutamicum overexpressing RP-C132A and RC-C201A produced 14.0% and 21.6% higher titers of 5-ALA than the control, respectively. These results strongly suggested that variants RP-C132A and RC-C201A obtained by utilizing cysteine-targeted mutation strategy released hemin inhibition, broadening their applications in 5-ALA biosynthesis.

A Secondary Metabolic Enzyme Functioned as an Evolutionary Seed of a Primary Metabolic Enzyme.

The antibiotic alaremycin has a structure that resembles that of 5-aminolevulinic acid (ALA), a universal precursor of porphyrins, and inhibits porphyrin biosynthesis. Genome sequencing of the alaremycin-producing bacterial strain and enzymatic analysis revealed that the first step of alaremcyin biosynthesis is catalysed by the enzyme, AlmA, which exhibits a high degree of similarity to 5-aminolevulinate synthase (ALAS) expressed by animals, protozoa, fungi, and α-proteobacteria. Site-directed mutagenesis of AlmA revealed that the substitution of two amino acids residues around the substrate binding pocket transformed its substrate specificity from that of alaremycin precursor synthesis to ALA synthesis. To estimate the evolutionary trajectory of AlmA and ALAS, we performed an ancestral sequence reconstitution analysis based on a phylogenetic tree of AlmA and ALAS. The reconstructed common ancestral enzyme of AlmA and ALAS exhibited alaremycin precursor synthetic activity, rather than ALA synthetic activity. These results suggest that ALAS evolved from an AlmA-like enzyme. We propose a new evolutionary hypothesis in which a non-essential secondary metabolic enzyme acts as an 'evolutionary seed' to generate an essential primary metabolic enzyme.

ISCA2 deficiency leads to heme synthesis defects and impaired erythroid differentiation in K562 cells by indirect ROS-mediated IRP1 activation.

Iron­sulfur (Fe-S) clusters have been shown to play important roles in various cellular physiological process. Iron­sulfur cluster assembly 2 (ISCA2) is a vital component of the [4Fe-4S] cluster assembly machine. Several studies have shown that ISCA2 is highly expressed during erythroid differentiation. However, the role and specific regulatory mechanisms of ISCA2 in erythroid differentiation and erythroid cell growth remain unclear. RNA interference was used to deplete ISCA2 expression in human erythroid leukemia K562 cells. The proliferation, apoptosis, and erythroid differentiation ability of the cells were assessed. We show that knockdown of ISCA2 has profound effects on [4Fe-4S] cluster formation, diminishing mitochondrial respiratory chain complexes, leading to reactive oxygen species (ROS) accumulation and mitochondrial damage, inhibiting cell proliferation. Excessive ROS can inhibit the activity of cytoplasmic aconitase (ACO1) and promote ACO1, a bifunctional protein, to perform its iron-regulating protein 1(IRP1) function, thus inhibiting the expression of 5'-aminolevulinate synthase 2 (ALAS2), which is a key enzyme in heme synthesis. Deficiency of ISCA2 results in the accumulation of iron divalent. In addition, the combination of excessive ferrous iron and ROS may lead to damage of the ACO1 cluster and higher IRP1 function. In brief, ISCA2 deficiency inhibits heme synthesis and erythroid differentiation by double indirect downregulation of ALAS2 expression. We conclude that ISCA2 is essential for normal functioning of mitochondria, and is necessary for erythroid differentiation and cell proliferation.

Structural basis for dysregulation of aminolevulinic acid synthase in human disease.

Heme is a critical biomolecule that is synthesized in vivo by several organisms such as plants, animals, and bacteria. Reflecting the importance of this molecule, defects in heme biosynthesis underlie several blood disorders in humans. Aminolevulinic acid synthase (ALAS) initiates heme biosynthesis in α-proteobacteria and nonplant eukaryotes. Debilitating and painful diseases such as X-linked sideroblastic anemia and X-linked protoporphyria can result from one of more than 91 genetic mutations in the human erythroid-specific enzyme ALAS2. This review will focus on recent structure-based insights into human ALAS2 function in health and how it dysfunctions in disease. We will also discuss how certain genetic mutations potentially result in disease-causing structural perturbations. Furthermore, we use thermodynamic and structural information to hypothesize how the mutations affect the human ALAS2 structure and categorize some of the unique human ALAS2 mutations that do not respond to typical treatments, that have paradoxical in vitro activity, or that are highly intolerable to changes. Finally, we will examine where future structure-based insights into the family of ALA synthases are needed to develop additional enzyme therapeutics.

Azacitidine is a potential therapeutic drug for pyridoxine-refractory female X-linked sideroblastic anemia.

X-linked sideroblastic anemia (XLSA) is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Treatment of XLSA is mainly supportive, except in patients who are pyridoxine responsive. Female XLSA often represents a late onset of severe anemia, mostly related to the acquired skewing of X chromosome inactivation. In this study, we successfully generated active wild-type and mutant ALAS2-induced pluripotent stem cell (iPSC) lines from the peripheral blood cells of an affected mother and 2 daughters in a family with pyridoxine-resistant XLSA related to a heterozygous ALAS2 missense mutation (R227C). The erythroid differentiation potential was severely impaired in active mutant iPSC lines compared with that in active wild-type iPSC lines. Most of the active mutant iPSC-derived erythroblasts revealed an immature morphological phenotype, and some showed dysplasia and perinuclear iron deposits. In addition, globin and HO-1 expression and heme biosynthesis in active mutant erythroblasts were severely impaired compared with that in active wild-type erythroblasts. Furthermore, genes associated with erythroblast maturation and karyopyknosis showed significantly reduced expression in active mutant erythroblasts, recapitulating the maturation defects. Notably, the erythroid differentiation ability and hemoglobin expression of active mutant iPSC-derived hematopoietic progenitor cells (HPCs) were improved by the administration of δ-aminolevulinic acid, verifying the suitability of the cells for drug testing. Administration of a DNA demethylating agent, azacitidine, reactivated the silent, wild-type ALAS2 allele in active mutant HPCs and ameliorated the erythroid differentiation defects, suggesting that azacitidine is a potential novel therapeutic drug for female XLSA. Our patient-specific iPSC platform provides novel biological and therapeutic insights for XLSA.

Heme-dependent recognition of 5-aminolevulinate synthase by the human mitochondrial molecular chaperone ClpX.

The caseinolytic mitochondrial matrix peptidase chaperone subunit (ClpX) plays an important role in the heme-dependent regulation of 5-aminolevulinate synthase (ALAS1), a key enzyme in heme biosynthesis. However, the mechanisms underlying the role of ClpX in this process remain unclear. In this in vitro study, we confirmed the direct binding between ALAS1 and ClpX in a heme-dependent manner. The substitution of C108 P109 [CP motif 3 (CP3)] with A108 A109 in ALAS1 resulted in a loss of ability to bind ClpX. Computational disorder analyses revealed that CP3 was located in a potential intrinsically disordered protein region (IDPR). Thus, we propose that conditional disorder-to-order transitions in the IDPRs of ALAS1 may represent key mechanisms underlying the heme-dependent recognition of ALAS1 by ClpX.

A Drug-Drug Interaction Study Evaluating the Effect of Givosiran, a Small Interfering Ribonucleic Acid, on Cytochrome P450 Activity in the Liver.

Givosiran (trade name GIVLAARI) is a small interfering ribonucleic acid that targets hepatic delta-aminolevulinic acid synthase 1 (ALAS1) messenger RNA for degradation through RNA interference (RNAi) that has been approved for the treatment of acute hepatic porphyria (AHP). RNAi therapeutics, such as givosiran, have a low liability for drug-drug interactions (DDIs) because they are not metabolized by cytochrome 450 (CYP) enzymes, and do not directly inhibit or induce CYP enzymes in the liver. The pharmacodynamic effect of givosiran (lowering of hepatic ALAS1, the first and rate limiting enzyme in the heme biosynthesis pathway) presents a unique scenario where givosiran could potentially impact heme-dependent activities in the liver, such as CYP enzyme activity. This study assessed the impact of givosiran on the pharmacokinetics of substrates of 5 major CYP450 enzymes in subjects with acute intermittent porphyria (AIP), the most common type of AHP, by using the validated "Inje cocktail," comprised of caffeine (CYP1A2), losartan (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A4). We show that givosiran treatment had a differential inhibitory effect on CYP450 enzymes in the liver, resulting in a moderate reduction in activity of CYP1A2 and CYP2D6, a minor effect on CYP3A4 and CYP2C19, and a similar weak effect on CYP2C9. To date, this is the first study evaluating the DDI for an oligonucleotide therapeutic and highlights an atypical drug interaction due to the pharmacological effect of givosiran. The results of this study suggest that givosiran does not have a large effect on heme-dependent CYP enzyme activity in the liver.

The ubiquitous mitochondrial protein unfoldase CLPX regulates erythroid heme synthesis by control of iron utilization and heme synthesis enzyme activation and turnover.

Heme plays a critical role in catalyzing life-essential redox reactions in all cells, and its synthesis must be tightly balanced with cellular requirements. Heme synthesis in eukaryotes is tightly regulated by the mitochondrial AAA+ unfoldase CLPX (caseinolytic mitochondrial matrix peptidase chaperone subunit X), which promotes heme synthesis by activation of δ-aminolevulinate synthase (ALAS/Hem1) in yeast and regulates turnover of ALAS1 in human cells. However, the specific mechanisms by which CLPX regulates heme synthesis are unclear. In this study, we interrogated the mechanisms by which CLPX regulates heme synthesis in erythroid cells. Quantitation of enzyme activity and protein degradation showed that ALAS2 stability and activity were both increased in the absence of CLPX, suggesting that CLPX primarily regulates ALAS2 by control of its turnover, rather than its activation. However, we also showed that CLPX is required for PPOX (protoporphyrinogen IX oxidase) activity and maintenance of FECH (ferrochelatase) levels, which are the terminal enzymes in heme synthesis, likely accounting for the heme deficiency and porphyrin accumulation observed in Clpx-/- cells. Lastly, CLPX is required for iron utilization for hemoglobin synthesis during erythroid differentiation. Collectively, our data show that the role of CLPX in yeast ALAS/Hem1 activation is not conserved in vertebrates as vertebrates rely on CLPX to regulate ALAS turnover as well as PPOX and FECH activity. Our studies reveal that CLPX mutations may cause anemia and porphyria via dysregulation of ALAS, FECH, and PPOX activities, as well as of iron metabolism.

Plasmid-Free System and Modular Design for Efficient 5-Aminolevulinic Acid Production by Engineered Escherichia coli.

5-Aminolevulinic acid (ALA) is an essential intermediate for many organisms and has been considered for the applications of medical especially in photodynamic therapy of cancer recently. However, ALA production via chemical approach is complicated; hence, microbial manufacturing has received more attentions. In this study, a modular design to simultaneously express ALA synthase from Rhodobacter sphaeroides (RshemA), a non-specific ALA exporter (RhtA), and chaperones was first developed and discussed. The ALA production was significantly increased by coexpressing RhtA and RshemA. Besides, ALA was enhanced by the cofactor pyridoxal phosphate (PLP) which was supplied by expressing genes of pdxK and pdxY or direct addition. However, inclusion bodies of RshemA served as an obstacle; thus, chaperones DnaK and GroELS were introduced to reform the conformation of proteins and successfully improved ALA production. Finally, a plasmid-free strain RrGI, as the robust chassis, was established and a 6.23-fold enhancement on ALA biosynthesis and led to 7.47 g/L titer and 0.588 g/L/h productivity under the optimal cultural condition.

Skeletal muscle heme oxygenase-1 activity regulates aerobic capacity.

Physical exercise has profound effects on quality of life and susceptibility to chronic disease; however, the regulation of skeletal muscle function at the molecular level after exercise remains unclear. We tested the hypothesis that the benefits of exercise on muscle function are linked partly to microtraumatic events that result in accumulation of circulating heme. Effective metabolism of heme is controlled by Heme Oxygenase-1 (HO-1, Hmox1), and we find that mouse skeletal muscle-specific HO-1 deletion (Tam-Cre-HSA-Hmox1fl/fl) shifts the proportion of muscle fibers from type IIA to type IIB concomitant with a disruption in mitochondrial content and function. In addition to a significant impairment in running performance and response to exercise training, Tam-Cre-HSA-Hmox1fl/fl mice show remarkable muscle atrophy compared to Hmox1fl/fl controls. Collectively, these data define a role for heme and HO-1 as central regulators in the physiologic response of skeletal muscle to exercise.

Effects of Doxorubicin on Heme Biosynthesis and Metabolism in Cardiomyocyte. / Os Efeitos da Doxorrubicina na Biossíntese e no Metabolismo do Heme em Cardiomiócitos.

BACKGROUND: Doxorubicin is associated with cardiotoxicity and late cardiac morbidity. Heme is related to cellular oxidative stress. However, its specific regulation in cardiomyocytes under doxorubicin effects has not yet been documented. OBJECTIVE: This study seeks to evaluate the changing profiles of rate-limiting enzymes in the heme metabolism pathway under the effect of doxorubicin. METHODS: H9c2 cardiomyocytes were incubated with doxorubicin at different concentrations (1,2,5,10µM respectively). The real-time PCR and Western Blot were used to determine the mRNA and protein expression for four pivotal enzymes (ALAS1, ALAS2, HOX-1, and HOX-2) regulating cellular heme metabolism, as well as the levels of heme were detected by ELISA. p<0.01 was considered significant. RESULTS: This study observed a dose-dependent changing pattern in heme levels in H9c2 cells with the highest level at the 5µM concentration for doxorubicin, which occurred synchronously with the highest upregulation level of ALAS1, as well as the degradative enzymes, HOX-1, and HOX-2 in mRNA and protein expression. By contrast, ALAS2, contrary to the increasing concentrations of doxorubicin, was found to be progressively down-regulated. CONCLUSION: The increase in ALAS1 expression may play a potential role in the heme level elevation when H9c2 cardiomyocyte was exposed to doxorubicin and may be a potential therapeutic target for doxorubicin-induced myocardial toxicity. (Arq Bras Cardiol. 2021; 116(2):315-322).

FUNDAMENTO: A doxorrubicina está associada à cardiotoxicidade e à morbidade cardíaca tardia. O heme está relacionado ao stress oxidativo celular. Entretanto, sua regulação específica em cardiomiócitos sob os efeitos de doxorrubicina ainda não foi documentada. OBJETIVO: Nosso objetivo é avaliar as alterações de enzimas limitantes de velocidade no caminho metabólico do heme sob o efeito de doxorrubicina. MÉTODOS: Cardiomiócitos H9c2 com doxorrubicina em concentrações diferentes (1, 2, 5, 10µM respectivamente). Os testes de PCR em tempo real e Western Blot foram usados para determinar a expressão de proteína e mRNA para quatro enzimas cruciais (ALAS1, ALAS2, HOX-1, e HOX-2) que regulam o metabolismo do heme celular, e os níveis de heme foram detectados por ELISA. Um p<0,01 foi considerado significativo. RESULTADOS: Observamos um padrão com alteração dependendo da dose nos níveis de heme nas células H9c2 com o nível mais alto na concentração de 5µM de doxorrubicina, o que ocorreu sincronicamente com o nível mais alto de regulação para cima de ALAS1, bem como as enzimas degenerativas HOX-1 e HOX-2 na expressão de proteína e mRNA. Em contraste, observamos que a ALAS2 foi regulada para baixo gradualmente, inversamente proporcional às concentrações de doxorrubicina. CONCLUSÃO: O aumento da expressão de ALAS1 pode ter um papel na elevação do nível do heme quando o cardiomiócito H9c2 for exposto à doxorrubicina, e pode ser um alvo terapêutico para a toxicidade miocárdica induzida por doxorrubicina. (Arq Bras Cardiol. 2021; 116(2):315-322).

[Knockdown of ALAS2 Affects Erythroid Differentiation by Down-regulating Mitophagy Receptor BNIP3L].

AbstractObjective: To investigate the effect of ALAS2 downregulation on the expression of BNIP3L and erythroid differentiation in K562 cells. METHODS: The expression of ALAS2 was down-regulated by transfection of lentivirus, then quantitative real-time PCR was performed to detect the transfection efficiency. Flow cytometry analysis was applied to evaluate apoptosis of cells, erythroid differentiation, mitochondrial membrane potential and reactive oxygen species (ROS) level. Western blot was used to detect the BNIP3L expression, Co-immunoprecipitation was performed to analyze the relationship between ALAS2 and BNIP3L. RESULTS: Compared with sh-NC group, knockdown of ALAS2 induced downregulation of BNIP3L mRNA and protein expression(P<0.01) and erythroid related transcription factors GATA1, Nrf2 expression, as well as reduction of ROS level(P<0.05). Mitochondrial membrane potential of control (sh-NC) group was lower than that of shALAS2 group(P<0.05), but there was no significant change of cell apoptotic rate in two groups. CD71highCD235ahigh + CD71lowCD235ahigh cells of sh-NC and shALAS2 groups were 53.5%, 92.9% at 96 h after hemin induction, respectively. No direct action between ALAS2 and BNIP3L was observed. CONCLUSION: The intracellular heme level can affect the expression of BNIP3L which may be related with the regulation of ROS and transcription factors GATA1 and Nrf2. Higher BNIP3L facilitates cell differentiation but lower BNIP3L is favorable for cells survival.