Post‐transcriptional expression of DMT1 in the heart of rat

Abstract

Non-transferrin-bound iron (NTBI) overtaken by heart cells might be a key cause leading to iron-mediated injury in heart disorders. NTBI uptake by heart cells might be mediated by divalent metal transporter 1 (DMT1). The understanding of the role of DMT1 in heart iron metabolism is fundamental for elucidating the cause resulting in excessive iron in the heart. The study was to evaluate effects of age and dietary iron on DMT1 mRNA expression and protein synthesis in rat heart. DMT1 mRNA expression was determined by RT-PCR and sequence analysis, and DMT1 protein by Western blot analysis. DMT1 mRNAs with or without iron-responsive element (IRE) both were found in rat heart. Expression of two forms of DMT1 mRNAs was the lowest at the age of post-natal day (PND) 7, and then increased with the age, reaching the highest at PND196 (non-IRE form) and PND63 (IRE form), respectively. During different ages, the levels of DMT1 (IRE) mRNA were higher than those of DMT1 (non-IRE) mRNA and were significantly correlated with the non-heme iron contents in the heart. After fed a high iron for 6 weeks, the rats had a sixfold elevation in heart iron and 22% (non-IRE from) and 40% (IRE from) reduction in DMT1 protein compared to the controls. A low iron diet for 6-weeks caused cardiac hypertrophy and heart iron deficiency and also an increase in levels of two forms of DMT1 proteins. However, iron status had no significant effect on DMT1 (IRE) and DMT1 (non-IRE) mRNAs expression in the heart, although it can significantly influence heart transferrin receptor (TfR) mRNA expression. The results demonstrated that DMT1 mRNAs expression in the heart is age-dependent and that two forms of DMT1 mRNAs both are regulated by iron on the post-transcriptional level only.

ZIP14 and DMT1 in the liver, pancreas, and heart are differentially regulated by iron deficiency and overload: implications for tissue iron uptake in iron-related disorders

“…The marked up-regulation of DMT1 implies that DMT1 plays an active role in iron acquisition by the heart, perhaps in concert with TfR1, which was similarly up-regulated. Similar to a previous study, 44 the higher DMT1 protein levels in irondeficient heart appear to result from post-transcriptional events because DMT1 mRNA levels were not higher.…”

“…Other studies have found that cardiac DMT1 levels were lower in iron-loaded animals than in controls. 44 Recently, Kumfu et al 43 reported that NTBI uptake by cardiomyocytes was unaffected when the iron uptake activity of DMT1 or TfR1 was blocked, indicating that DMT1 and TfR1 are not required for NTBI uptake into these cells. In iron-deficient heart, DMT1 levels were up-regulated 4-fold whereas ZIP14 levels were not affected.…”

Manganese accumulates primarily in nuclei of cultured brain cells

“…The authenticity of subcellular fractions was confirmed by Western blot analysis as previously described by (Ke et al, 2003), using primary monoclonal antibodies anti-nuclear core complex proteins-clone4 for nuclear fraction, anti-cytochrom-c oxidase for mitochondria, and antilactate dehydrogenase (H-subunit of LDH) clone HH-17 for cytoplasmic fraction (all were purchased from Sigma). A volume of protein extracts (40 µg) of total cell lysate, nuclei, mitochondria or cytoplasm was mixed with an equal volume of 2x sample buffer (0.125M Tris, 4% SDS, 20% glycerol, 0.02% 2-mercaptoethanol, and 0.01% bromophenol Blue), electrophoresed on a 4 -20% Tris-HCl linear gradient ready gels (Bio-Rad, Hercules, CA), and transferred to a PVDF membrane.…”

Upregulation of DMT1 expression in choroidal epithelia of the blood–CSF barrier following manganese exposure in vitro

“…Western blot experiments were carried out as previously described (Ke et al, 2003). In short, cellular proteins were extracted in homogenization buffer containing 20 mM Tris, pH 7.5, 5 mM EGTA, 1% Triton X-100, 0.1% SDS, Protease Inhibitor Cocktail (Calbiochem, San Diego, CA).…”

“…DMT1 is most abundantly expressed in the duodenal epithelia. The expression of DMT1 in other cell types of nearly most of the organs has also been identified including erythrocytes, renal epithelial cells, cardiomyocytes and human placenta epithelia (Ferguson et al, 2001;Georgieff et al, 2000;Ke et al, 2003;Levy et al, 1999). In brain, DMT1 exists in neurons, cerebral capillary endothelial cells that constitute the blood-brain barrier (BBB) and the choroid plexus epithelial cells that comprise the blood-CSF barrier (BCB) (Burdo et al, 2001;Siddappa et al, 2003;Siddappa et al, 2002).…”