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Heme-induced contractile dysfunction in human cardiomyocytes caused by oxidant damage to thick filament proteins
Gerardo Alvarado1, Viktória Jeney2, Attila Tóth3
1Division of Clinical Physiology, Institute of Cardiology, Faculty of Medicine, University of Debrecen, H-4032 Debrecen, Hungary; Department of Nephrology, Institute of Internal Medicine, Faculty of Medicine, University of Debrecen, H-4032 Debrecen, Hungary.
Insights
Free heme damages heart cells by altering contractile proteins, leading to impaired heart function. This finding is crucial for understanding cardiac issues in various diseases.
Area of Science:
- Cardiovascular Biology
- Cellular Biochemistry
- Oxidative Stress Research
Background:
- Intracellular free heme contributes to oxidant-mediated tissue damage.
- The precise mechanisms by which free heme impacts myocardial contractility are not fully understood.
- Free heme's role in altering contractile protein structure and regulation warrants investigation.
Purpose of the Study:
- To investigate the hypothesis that free heme alters myocardial contractility through structural and/or regulatory changes in contractile proteins.
- To elucidate the molecular mechanisms underlying heme-induced cardiac dysfunction.
Main Methods:
- Isometric force production and calcium sensitivity (pCa50) were measured in permeabilized human ventricular cardiomyocytes.
- Cardiomyocytes were exposed to varying concentrations of heme, with or without hydrogen peroxide (H2O2) or antioxidants like dithiothreitol.
- Protein modifications, including sulfhydryl (SH) group content and sulfenic acid formation, were analyzed using biochemical assays.
- Heme binding to specific proteins and the effects of heme-binding proteins (hemopexin, alpha-1-microglobulin) were assessed.
Main Results:
- Heme exposure decreased maximal active force (Fo) and increased passive force (F passive) in cardiomyocytes, without affecting Ca(2+) sensitivity (pCa50).
- Increased passive force was observed at 3 µM heme and was partially reversible by dithiothreitol.
- Heme treatment led to decreased protein SH groups and increased sulfenic acid formation, indicating oxidative modification of contractile proteins.
- Free heme directly bound to myosin light chain 1, and binding to hemopexin or alpha-1-microglobulin mitigated heme's negative effects on contractility.
Conclusions:
- Free heme induces cardiac contractile dysfunction by modifying cardiac contractile proteins through posttranslational modifications and direct binding to myosin light chain 1.
- These heme-induced alterations contribute to systolic and diastolic cardiac dysfunctions.
- This mechanism is relevant to conditions such as hemolytic diseases, heart failure, and myocardial ischemia-reperfusion injury.
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 (F passive). Heme treatments, either alone or in combination with H2O2, did not affect pCa50. The increase in F passive started at 3 µM 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 140 kDa 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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