The Interplay between S-Glutathionylation and Phosphorylation of Cardiac Troponin I and Myosin Binding Protein C in End-Stage Human Failing Hearts

Abstract

Oxidative stress is defined as an imbalance between the antioxidant defense system and the production of reactive oxygen species (ROS). At low levels, ROS are involved in the regulation of redox signaling for cell protection. However, upon chronical increase in oxidative stress, cell damage occurs, due to protein, DNA and lipid oxidation. Here, we investigated the oxidative modifications of myofilament proteins, and their role in modulating cardiomyocyte function in end-stage human failing hearts. We found altered maximum Ca2+-activated tension and Ca2+ sensitivity of force production of skinned single cardiomyocytes in end-stage human failing hearts compared to non-failing hearts, which was corrected upon treatment with reduced glutathione enzyme. This was accompanied by the increased oxidation of troponin I and myosin binding protein C, and decreased levels of protein kinases A (PKA)- and C (PKC)-mediated phosphorylation of both proteins. The Ca2+ sensitivity and maximal tension correlated strongly with the myofilament oxidation levels, hypo-phosphorylation, and oxidative stress parameters that were measured in all the samples. Furthermore, we detected elevated titin-based myocardial stiffness in HF myocytes, which was reversed by PKA and reduced glutathione enzyme treatment. Finally, many oxidative stress and inflammation parameters were significantly elevated in failing hearts compared to non-failing hearts, and corrected upon treatment with the anti-oxidant GSH enzyme. Here, we provide evidence that the altered mechanical properties of failing human cardiomyocytes are partially due to phosphorylation, S-glutathionylation, and the interplay between the two post-translational modifications, which contribute to the development of heart failure.

Ca2+/calmodulin‐dependent protein kinase II and protein kinase G oxidation contributes to impaired sarcomeric proteins in hypertrophy model

“…Hence, the net effect of myofilament sensitization/desensitization is defined by synergistic balance between different kinases/phosphatases. 9,14 In the current study, and in line with previous work reporting the reduction in PKA-dependent phosphorylation of cTnI in end stage human failing hearts, 19,43,44 we found PKA-/PKG-dependent hypophosphorylation of cTnI at Ser 23/24 in 8-month VO compared with 8-month Ctrl group. It is well established that in HF and due to the internalization of β-adrenergic receptors, the receptor density and activity are reduced leading to decreased PKA-dependent phosphorylation of cTnI at Ser 23/24.…”

“…[16][17][18] Previous research by us and others has demonstrated the role of oxidative stress and inflammation in altering various signalling pathways crucial for cardiomyocyte function among which are the kinases pathways. 4,19 Reactive oxygen species (ROS) might directly oxidize kinases or their targets leading to changes in their activity/binding affinity, respectively. 19 For example, oxidation of the regulatory CaMKII domain at Met 281/282 induces a Ca 2+ /CaM-independent activation.…”

“…4,19 Reactive oxygen species (ROS) might directly oxidize kinases or their targets leading to changes in their activity/binding affinity, respectively. 19 For example, oxidation of the regulatory CaMKII domain at Met 281/282 induces a Ca 2+ /CaM-independent activation. 20,21 Through the regulation of Ca 2+ homeostasis and sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) function, CaMKIIδ is involved in cardiac remodelling and hypertrophic responses to mechanical overload.…”

The Oxidative Balance Orchestrates the Main Keystones of the Functional Activity of Cardiomyocytes

“…Accordingly, oxidized troponin I does not bind to troponin T, and the altered S-glutathionylation (an intracellular process directly regulated by the local redox status of the microenvironment) of cMyBP-C induces an impairment of cellular relaxation, with unaltered cellular Ca 2+ dynamics. The reduction in cMyBP-C S-glutathionylation improves diastolic dysfunction, suggesting a strong link between S-glutathionylation and diastolic dysfunction [ 102 ].…”

Identification of Phosphorylation and Other Post-Translational Modifications in the Central C4C5 Domains of Murine Cardiac Myosin Binding Protein C

“… 19 More broadly, cMyBPC has been shown to be differentially phosphorylated in animal models of myocardial stunning and models of age-related cardiac dysfunction. 20 , 21 Furthermore, cardiac hypertrophy and heart failure have been associated with reduced overall cMyBPC phosphorylation levels, 22 − 26 and decreased cMyBPC phosphorylation is correlated with dysfunction in ischemia. 27 , 28 …”