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- Alvarado, Gerardo, et al.
(author)
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Heme-induced contractile dysfunction in Human cardiomyocytes caused by oxidant damage to thick filament proteins.
- 2015
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In: Free Radical Biology & Medicine. - : Elsevier BV. - 0891-5849. ; 89:sep 23, s. 248-262
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Journal article (peer-reviewed)abstract
- Intracellular free heme predisposes to oxidant-mediated tissue damage. We hypothesized that free heme causes alterations in myocardial contractility via disturbed structure and/or regulation of the contractile proteins. Isometric force production and its Ca(2+)-sensitivity (pCa50) were monitored in permeabilized human ventricular cardiomyocytes. Heme exposure altered cardiomyocyte morphology and evoked robust decreases in Ca(2+)-activated maximal active force (Fo) while increasing Ca(2+)-independent passive force (Fpassive). Heme treatments, either alone or in combination with H2O2, did not affect pCa50. The increase in Fpassive started at 3µM heme exposure and could be partially reversed by the antioxidant dithiothreitol. Protein sulfhydryl (SH) groups of thick myofilament content decreased and sulfenic acid formation increased after treatment with heme. Partial restoration in the SH group content was observed in a protein running at 140kDa after treatment with dithiothreitol, but not in other proteins, such as filamin C, myosin heavy chain, cardiac myosin binding protein C, and α-actinin. Importantly, binding of heme to hemopexin or alpha-1-microglobulin prevented its effects on cardiomyocyte contractility, suggesting an allosteric effect. In line with this, free heme directly bound to myosin light chain 1 in human cardiomyocytes. Our observations suggest that free heme modifies cardiac contractile proteins via posttranslational protein modifications and via binding to myosin light chain 1, leading to severe contractile dysfunction. This may contribute to systolic and diastolic cardiac dysfunctions in hemolytic diseases, heart failure, and myocardial ischemia-reperfusion injury.
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| 2. |
- Alvarado, Gerardo, et al.
(author)
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Heme-induced oxidation of cysteine groups of myofilament proteins leads to contractile dysfunction of permeabilized human skeletal muscle fibres
- 2020
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In: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 21:21
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Journal article (peer-reviewed)abstract
- Background: Heme released from red blood cells targets a number of cell components including the cytoskeleton. The purpose of the present study was to determine the impact of free heme (20–300 µM) on human skeletal muscle fibres made available during orthopedic surgery. Methods: Isometric force production and oxidative protein modifications were monitored in permeabilized skeletal muscle fibre segments. Results: A single heme exposure (20 µM) to muscle fibres decreased Ca2+-activated maximal (active) force (Fo) by about 50% and evoked an approximately 3-fold increase in Ca2+-independent (passive) force (Fpassive). Oxidation of sulfhydryl (SH) groups was detected in structural proteins (e.g., nebulin, α-actinin, meromyosin 2) and in contractile proteins (e.g., myosin heavy chain and myosin-binding protein C) as well as in titin in the presence of 300 µM heme. This SH oxidation was not reversed by dithiothreitol (50 mM). Sulfenic acid (SOH) formation was also detected in the structural proteins (nebulin, α-actinin, meromyosin). Heme effects on SH oxidation and SOH formation were prevented by hemopexin (Hpx) and α1-microglobulin (A1M). Conclusions: These data suggest that free heme has a significant impact on human skeletal muscle fibres, whereby oxidative alterations in structural and contractile proteins limit contractile function. This may explain and or contribute to the weakness and increase of skeletal muscle stiffness in chronic heart failure, rhabdomyolysis, and other hemolytic diseases. Therefore, therapeutic use of Hpx and A1M supplementation might be effective in preventing heme-induced skeletal muscle alterations.
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| 3. |
- Gáll, Tamás, et al.
(author)
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Heme induces endoplasmic reticulum stress (Hier stress) in human aortic smooth muscle cells
- 2018
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In: Frontiers in Physiology. - : Frontiers Media SA. - 1664-042X. ; 9:NOV
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Journal article (peer-reviewed)abstract
- Accumulation of damaged or misfolded proteins resulted from oxidative protein modification induces endoplasmic reticulum (ER) stress by activating the pathways of unfolded protein response. In pathologic hemolytic conditions, extracellular free hemoglobin is submitted to rapid oxidation causing heme release. Resident cells of atherosclerotic lesions, after intraplaque hemorrhage, are exposed to heme leading to oxidative injury. Therefore, we raised the question whether heme can also provoke ER stress. Smooth muscle cells are one of the key players of atherogenesis; thus, human aortic smooth muscle cells (HAoSMCs) were selected as a model cell to reveal the possible link between heme and ER stress. Using immunoblotting, quantitative polymerase chain reaction and immunocytochemistry, we quantitated the markers of ER stress. These were: phosphorylated eIF2α, Activating transcription factor-4 (ATF4), DNA-damage-inducible transcript 3 (also known as C/EBP homology protein, termed CHOP), X-box binding protein-1 (XBP1), Activating transcription factor-6 (ATF6), GRP78 (glucose-regulated protein, 78kDa) and heme responsive genes heme oxygenase-1 and ferritin. In addition, immunohistochemistry was performed on human carotid artery specimens from patients who had undergone carotid endarterectomy. We demonstrate that heme increases the phosphorylation of eiF2α in HAoSMCs and the expression of ATF4. Heme also enhances the splicing of XBP1 and the proteolytic cleavage of ATF6. Consequently, there is up-regulation of target genes increasing both mRNA and protein levels of CHOP and GRP78. However, TGFβ and collagen type I decreased. When the heme binding proteins, alpha-1-microglobulin (A1M) and hemopexin (Hpx) are present in cell media, the ER stress provoked by heme is inhibited. ER stress pathways are also retarded by the antioxidant N-acetyl cysteine (NAC) indicating that reactive oxygen species are involved in heme-induced ER stress. Consistent with these findings, elevated expression of the ER stress marker GRP78 and CHOP were observed in smooth muscle cells of complicated lesions with hemorrhage compared to either atheromas or healthy arteries. In conclusion, heme triggers ER stress in a time- and dose-dependent manner in HAoSMCs. A1M and Hpx as well as NAC effectively hamper heme-induced ER stress, supporting their use as a potential therapeutic approach to reverse such a deleterious effects of heme toxicity.
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| 4. |
- Pethő, Dávid, et al.
(author)
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Ferryl hemoglobin and heme induce Α1‐microglobulin in hemorrhaged atherosclerotic lesions with inhibitory function against hemoglobin and lipid oxidation
- 2021
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In: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 22:13
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Journal article (peer-reviewed)abstract
- Infiltration of red blood cells into atheromatous plaques and oxidation of hemoglobin (Hb) and lipoproteins are implicated in the pathogenesis of atherosclerosis. α1‐microglobulin (A1M) is a radical‐scavenging and heme‐binding protein. In this work, we examined the origin and role of A1M in human atherosclerotic lesions. Using immunohistochemistry, we observed a significant A1M immunoreactivity in atheromas and hemorrhaged plaques of carotid arteries in smooth muscle cells (SMCs) and macrophages. The most prominent expression was detected in macrophages of organized hemorrhage. To reveal a possible inducer of A1M expression in ruptured lesions, we exposed aortic endothelial cells (ECs), SMCs and macrophages to heme, Oxy‐ and FerrylHb. Both heme and FerrylHb, but not OxyHb, upregulated A1M mRNA expression in all cell types. Importantly, only FerrylHb induced A1M protein secretion in aortic ECs, SMCs and macrophages. To assess the possible function of A1M in ruptured lesions, we analyzed Hb oxidation and heme‐catalyzed lipid peroxidation in the presence of A1M. We showed that recombinant A1M markedly inhibited Hb oxidation and heme‐driven oxidative modification of low‐density lipoproteins as well plaque lipids derived from atheromas. These results demonstrate the presence of A1M in atherosclerotic plaques and suggest its induction by heme and FerrylHb in the resident cells.
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