As expected, α-tocopherol treatment prevented ROS accumulation and the induction of anti-oxidant genes in the heart (Figure 6A, B and see Figure 8 in [37]).
Consistently, we measured increased lipid peroxidation in both cells and tissues exposed to free heme, which can be rescued by α-tocopherol, an agent able to react with lipid radicals and interrupt the oxidation reaction.
Heme promotes endothelial dysfunction by inducing the expression of adhesion molecules and reducing nitric oxide (NO) availability, which causes vasoconstriction [9-14].
As well as NAC, Hx co-treatment significantly improved systolic Ca2+ transients and accelerated Ca2+ transient decay kinetics, which resulted in a significant improvement of cardiomyocyte contractility (Figure 4A-E).
Moreover, heme-derived ROS induce the proliferation of smooth muscle cells, that participate in vasculopathy associated with atherosclerosis and hypertension [15].
In agreement with data on pro-oxidant effects of heme in neonatal mouse CMs (Figure 1D-F), oxidative stress was significantly increased in adult rat CMs exposed to free heme (see Figure 1 in [37]).
Consistently, the mRNA levels of Glutathione reductase (Gsr), -Glutamylcysteine synthetase (-Gcs) and thioredoxin reductase (Thiored red), anti-oxidant systems important in resistance of cardiac cell to oxidative damage [40, 41], were increased to a higher extent in CMs treated with either albumin heme or heme alone, compared to cells treated with Hx-heme (Figure 1F and see Figure 3 in [37]).
These data collectively indicate that heart free heme accumulating when Hx is lost is responsible for systolic dysfunction.
Excess production of reactive oxygen species (ROS) has been implicated in progression of chronic heart failure as well as in other cardiovascular disorders including ischemia-reperfusion injury and cardiovascular complications of hemolytic diseases [2-7].
Mechanistically, heme-derived ROS may directly modify Ca2+ handling proteins (such as the RyR2 or SERCA2a) [35, 57], and may also activate intracellular stress kinases, such as CaMKII [58], which in turn phosphorylate the same Ca2+ effectors and ultimately exacerbate Ca2+ mishandling.
Hx-heme-treated CMs were protected from heme accumulation, compared to cells treated with albumin-heme or heme alone (Figure 1A), indicating that Hx prevents heme entry in cardiac cells.
Being heme a well-known pro-oxidant agent [17], we then evaluated ROS levels in CMs exposed to heme alone or bound to either Hx or albumin. In the presence of Hx-heme complexes, CMs were protected from ROS formation, if compared to CMs treated with either albumin-heme complexes or heme alone (Figure 1D).
Consistently, HO-1 mRNA and protein levels were higher in hearts from Hx-/- mice than in controls (Figure 2B). Immunohistochemistry for HO-1 on heart sections indicated higher HO-1 expression in cardiomyocytes from Hx-/- mice than in wild-type animals (Figure 2B).
Moreover, in CMs the heme-mediated induction of the iron exporter Ferroportin (Fpn) was abrogated by Hx treatment while the down-regulation of the iron importer Transferrin receptor 1 (TfR1) was significantly reduced by Hx (see Figure 3 in [37]) further indicating that Hx limits heme-iron accumulation within the cell.
Moreover, the mRNA levels of Fpn and Flvcr1a were increased in Hx-/- mice, whereas those of the iron importers Divalent Metal Transporter 1 (Dmt1) and Tfr1 were decreased (see Figure 4 in [37]).
Consistently, Hx treatment blunted heme-mediated induction of the heme exporter Feline Leukemia Virus subgroup C Receptor 1(Flvcr1) [31, 38, 39] in H9c2 cells (see Figure 3 in [37]).
Staining of CMs with a fluorescent probe for specific detection of mitochondrial superoxide (Mito-sox) confirmed lower oxidative stress in cells exposed to Hx-heme than in those exposed to albumin-heme or heme alone (Figure 1E).
Both ROS and lipid peroxidation were increased in the heart of Hx-/- mice compared to that of wild-type animals (Figure 2C, D).
The data were reproduced in H9c2 cells in which we were also able to demonstrate that Hx limits protein nitrosylation, another hallmark of cellular oxidative damage (see Figure 2 in [37]).
Consistently, the oxidative stress responsive gene Gsr was up-regulated in the heart of Hx-/- mice compared to wild-type animals (Figure 2E).
Cardiac contractility was severely impaired in 3 month-old Hx-/- mice compared to wild-type controls, as evidenced by significantly lower fractional shortening (FS) and ejection fraction (EF) (Figure 3A-C).
Consistently, CaMKII-dependent phosphorylation of RyR2 (Ser-2814) and Phospholamban (PLB) (Thr-17) were significantly higher in Hx-/- hearts than in wild-type controls (Figure 5C, D).
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If you find BEL Commons useful in your work, please consider citing: Hoyt, C. T., Domingo-Fernández, D., & Hofmann-Apitius, M. (2018). BEL Commons: an environment for exploration and analysis of networks encoded in Biological Expression Language. Database, 2018(3), 1–11.