Biosynthesis of Heme and Cholorophylls, 183-232, Green Pub.
Methods for H9c2 cell culturing, lentiviral and adenovirial transduction, heme and total iron content determination, enzyme activity analyses, microscopy and ROS quantification, mRNA and protein level measurements, 14C-glycine incorporation, Ca2+ transient analysis, cell death quantification, NRCM isolation, as well as MI surgery and tissue harvesting in mice are described in the detailed methods provided in the . Data are expressed as mean ± SEM. Statistical significance was assessed with the unpaired Student t test; a P value of less than 0.05 was considered statistically significant.
Heme Synthesis Pathway Made Simple! - YouTube
One potential explanation for the defect in heme synthesis with ABCB10 shRNA and its reversal by the addition of exogenous ALA is a reduction in mitochondrial bioenergetic function due to increased ROS levels, and subsequent deficiency in succinyl CoA, a necessary substrate for the synthesis of ALA and heme. To address this possibility, we supplemented cells with membrane-permeable methyl-succinate and measured the effects of ABCB10 knockdown on heme production. Methyl-succinate addition led to a small, but significant increase in heme levels in the control cells, but failed to restore heme content in ABCB10 shRNA cells (), suggesting that the defect observed with ABCB10 knockdown is not due to succinyl CoA deficiency.
Since ABCB10 knockdown is associated with a defect in hemoglobinization and differentiation of erythropoietic lineage both in vitro and in vivo, we assessed the effects of ALA supplementation on these parameters in mouse erythroid leukemia (MEL) cells with ABCB10 knockdown. We used siRNA approach to knock down ABCB10 in MEL cells and achieved about 50% reduction in its mRNA and protein levels (). Consistent with our hypothesis, ABCB10 knockdown reduced both heme content and differentiation of MEL cells (), while addition of exogenous ALA reversed these defects (). These findings suggest that ABCB10 also facilitates mitochondrial ALA synthesis or export in erythroid progenitor cells, and this function of ABCB10 is necessary for erythrocytic differentiation.
Heme Synthesis : Synthesis of Porphyrin Molecule
Overexpression of ABCB10 had no effect on mitochondrial or total cellular heme levels in H9c2 cells (). However, cellular and mitochondrial heme content was significantly reduced by ABCB10 knockdown in H9c2 cells () and in neonatal rat cardiomyocytes (NRCM, ). The reduction in heme was not due to a decrease in non-heme mitochondrial iron levels, which were unaffected by ABCB10 downregulation (). It is important to note that mitochondrial heme levels were reduced, not increased, by ABCB10 knockdown, in contrast to the commonly stated hypothesis that ABCB10 functions in heme export.
Heme Synthesis : This is the Synthesis of Porphyrin molecule
Consistent with a reduction in heme content, the activities of several heme-containing enzymes were decreased with ABCB10 knockdown (), while their mRNA and protein levels remained unchanged (). On the other hand, we observed no change in the activities or mRNA levels of heme-containing enzymes with ABCB10 overexpression ( and ), which is in agreement with unaltered heme content in these cells. Thus, our results suggest that ABCB10 regulates the heme biosynthetic pathway, but does not appear to be involved in heme export out of the mitochondria.
BIOSYNTHESIS OF HEME - Microbial Iron Metabolism - …
In this study, we show that ABCB10 plays a role in the early steps of heme synthesis and ALA export into the cytoplasm in cardiac myoblasts, providing evidence contrary to the commonly held hypothesis that ABCB10 may function as a heme exporter. We report that downregulation of ABCB10 impairs heme synthesis and the activities of heme-containing proteins in cardiac cells, and that addition of ALA, but not overexpression of ALAS2, reverses these defects.
Porphyrin and Heme Synthesis and Bilirubin Metabolism
To determine if hypoxia-driven upregulation of ABCB10 bears physiological consequences, we assessed ROS levels and cell death in cardiac cells subjected to 48 hours of hypoxia followed by 30 minutes of reoxygenation with and without knockdown of ABCB10. Failure to upregulate ABCB10 in hypoxia due to siRNA treatment was associated with increased oxidative stress, as determined by the superoxide-sensitive dye MitoSox (). The accumulation of MitoSox in the mitochondria was not due to changes in mitochondrial membrane potential, as TMRE staining did not differ between the groups (). Moreover, cell death was significantly increased in hypoxic cells in which ABCB10 upregulation was suppressed (), and further augmented by addition of hydrogen peroxide as a source of oxidative stress (). Thus, in contrast to forced overexpression of ABCB10 in normoxia which does not alter heme synthesis, physiologic induction of ABCB10 in hypoxia is necessary to support increased rates of heme synthesis, and failure to maintain adequate ABCB10 levels compromises cell survival.