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Mitochondrial Changes in β0-Thalassemia/Hb E Disease.

Khungwanmaythawee K, Sornjai W, Paemanee A, Jaratsittisin J, Fucharoen S, Svasti S, Lithanatudom P, Roytrakul S, Smith DR - PLoS ONE (2016)

Bottom Line: While a number of factors are known that moderate presentation, these factors do not account for the full spectrum of presentation.Differences in relative mitochondria number and gene expression were seen primarily in day 10 cells.The evidence presented here suggest that there are inherent differences in these cells as early as the erythroid progenitor cell stage, and that maximum deficit is seen coincident with high levels of globin gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biosciences, Mahidol University, Salaya campus, 25/25 Phuttamontol Sai 4, Salaya, Nakorn Pathom, 73170, Thailand.

ABSTRACT
The compound β°-thalassemia/Hb E hemoglobinopathy is characterized by an unusually large range of presentation from essentially asymptomatic to a severe transfusion dependent state. While a number of factors are known that moderate presentation, these factors do not account for the full spectrum of presentation. Mitochondria are subcellular organelles that are pivotal in a number of cellular processes including oxidative phosphorylation and apoptosis. A mitochondrial protein enriched proteome was determined and validated from erythroblasts from normal controls and β°-thalassemia/Hb E patients of different severities. Mitochondria were evaluated through the use of mitotracker staining, analysis of relative mitochondrial genome number and evaluation of mitochondrial gene expression in addition to assay of overall cellular redox status through the use of alamarBlue assays. Fifty differentially regulated mitochondrial proteins were identified. Mitotracker staining revealed significant differences in staining between normal control erythroblasts and those from β°-thalassemia/Hb E patients. Differences in relative mitochondria number and gene expression were seen primarily in day 10 cells. Significant differences were seen in redox status as evaluated by alamarBlue staining in newly isolated CD34+ cells. Mitochondria mediate oxidative phosphorylation and apoptosis, both of which are known to be dysregulated in differentiating erythrocytes from β°-thalassemia/Hb E patients. The evidence presented here suggest that there are inherent differences in these cells as early as the erythroid progenitor cell stage, and that maximum deficit is seen coincident with high levels of globin gene expression.

No MeSH data available.


Related in: MedlinePlus

Mitochondria in erythroid precursor cells: quantity.Erythroid precursor cells on days 7, 10 and 14 of culture were (a and b) stained with mitotracker and examined by (a) confocal microscopy or (b) flow cytometry. (a) Representative merged confocal images are shown, and individual fields and merges are shown in Fig D in S1 File. (b) Tabulated flow cytometry data is shown, and individual scatterplots are shown in Fig E in S1 File. (c) Real-time quantitative PCR was used to determine relative mitochondria numbers. Data is normalized to nuclear genome through the ferroportin 1A gene. (b and c) white bars represent normal controls, grey bars are from mild and black bars are from severe β°-thalassemia/Hb E patients. Error bars show ± S.E.M. * p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001.
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pone.0153831.g003: Mitochondria in erythroid precursor cells: quantity.Erythroid precursor cells on days 7, 10 and 14 of culture were (a and b) stained with mitotracker and examined by (a) confocal microscopy or (b) flow cytometry. (a) Representative merged confocal images are shown, and individual fields and merges are shown in Fig D in S1 File. (b) Tabulated flow cytometry data is shown, and individual scatterplots are shown in Fig E in S1 File. (c) Real-time quantitative PCR was used to determine relative mitochondria numbers. Data is normalized to nuclear genome through the ferroportin 1A gene. (b and c) white bars represent normal controls, grey bars are from mild and black bars are from severe β°-thalassemia/Hb E patients. Error bars show ± S.E.M. * p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001.

Mentions: Mature red blood cells do not contain mitochondria [17] and it is well established that mitochondria are lost by the process termed mitophagy (reviewed by Mortensen and colleagues [18]), and it is generally believed that mitochondria are lost during reticulocyte maturation [17]. To directly observe mitochondria during erythroblast differentiation cells on days 7, 10 and 14 were stained with mitotracker and observed under a confocal microscope. Results showed markedly different patterns of staining between cells from normal controls and those from β°-thalassemia/Hb E patients (Fig 3). This was most clearly observed on day 7 of culture whereby the signal in normal control cells was predominantly perinuclear, while in cells from β°-thalassemia/Hb E patients the signal was largely colocalized with the nuclei (Fig 3).


Mitochondrial Changes in β0-Thalassemia/Hb E Disease.

Khungwanmaythawee K, Sornjai W, Paemanee A, Jaratsittisin J, Fucharoen S, Svasti S, Lithanatudom P, Roytrakul S, Smith DR - PLoS ONE (2016)

Mitochondria in erythroid precursor cells: quantity.Erythroid precursor cells on days 7, 10 and 14 of culture were (a and b) stained with mitotracker and examined by (a) confocal microscopy or (b) flow cytometry. (a) Representative merged confocal images are shown, and individual fields and merges are shown in Fig D in S1 File. (b) Tabulated flow cytometry data is shown, and individual scatterplots are shown in Fig E in S1 File. (c) Real-time quantitative PCR was used to determine relative mitochondria numbers. Data is normalized to nuclear genome through the ferroportin 1A gene. (b and c) white bars represent normal controls, grey bars are from mild and black bars are from severe β°-thalassemia/Hb E patients. Error bars show ± S.E.M. * p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4836671&req=5

pone.0153831.g003: Mitochondria in erythroid precursor cells: quantity.Erythroid precursor cells on days 7, 10 and 14 of culture were (a and b) stained with mitotracker and examined by (a) confocal microscopy or (b) flow cytometry. (a) Representative merged confocal images are shown, and individual fields and merges are shown in Fig D in S1 File. (b) Tabulated flow cytometry data is shown, and individual scatterplots are shown in Fig E in S1 File. (c) Real-time quantitative PCR was used to determine relative mitochondria numbers. Data is normalized to nuclear genome through the ferroportin 1A gene. (b and c) white bars represent normal controls, grey bars are from mild and black bars are from severe β°-thalassemia/Hb E patients. Error bars show ± S.E.M. * p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001.
Mentions: Mature red blood cells do not contain mitochondria [17] and it is well established that mitochondria are lost by the process termed mitophagy (reviewed by Mortensen and colleagues [18]), and it is generally believed that mitochondria are lost during reticulocyte maturation [17]. To directly observe mitochondria during erythroblast differentiation cells on days 7, 10 and 14 were stained with mitotracker and observed under a confocal microscope. Results showed markedly different patterns of staining between cells from normal controls and those from β°-thalassemia/Hb E patients (Fig 3). This was most clearly observed on day 7 of culture whereby the signal in normal control cells was predominantly perinuclear, while in cells from β°-thalassemia/Hb E patients the signal was largely colocalized with the nuclei (Fig 3).

Bottom Line: While a number of factors are known that moderate presentation, these factors do not account for the full spectrum of presentation.Differences in relative mitochondria number and gene expression were seen primarily in day 10 cells.The evidence presented here suggest that there are inherent differences in these cells as early as the erythroid progenitor cell stage, and that maximum deficit is seen coincident with high levels of globin gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biosciences, Mahidol University, Salaya campus, 25/25 Phuttamontol Sai 4, Salaya, Nakorn Pathom, 73170, Thailand.

ABSTRACT
The compound β°-thalassemia/Hb E hemoglobinopathy is characterized by an unusually large range of presentation from essentially asymptomatic to a severe transfusion dependent state. While a number of factors are known that moderate presentation, these factors do not account for the full spectrum of presentation. Mitochondria are subcellular organelles that are pivotal in a number of cellular processes including oxidative phosphorylation and apoptosis. A mitochondrial protein enriched proteome was determined and validated from erythroblasts from normal controls and β°-thalassemia/Hb E patients of different severities. Mitochondria were evaluated through the use of mitotracker staining, analysis of relative mitochondrial genome number and evaluation of mitochondrial gene expression in addition to assay of overall cellular redox status through the use of alamarBlue assays. Fifty differentially regulated mitochondrial proteins were identified. Mitotracker staining revealed significant differences in staining between normal control erythroblasts and those from β°-thalassemia/Hb E patients. Differences in relative mitochondria number and gene expression were seen primarily in day 10 cells. Significant differences were seen in redox status as evaluated by alamarBlue staining in newly isolated CD34+ cells. Mitochondria mediate oxidative phosphorylation and apoptosis, both of which are known to be dysregulated in differentiating erythrocytes from β°-thalassemia/Hb E patients. The evidence presented here suggest that there are inherent differences in these cells as early as the erythroid progenitor cell stage, and that maximum deficit is seen coincident with high levels of globin gene expression.

No MeSH data available.


Related in: MedlinePlus