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Redox regulation of protein phosphatase-1 and ER stress regulation of connective tissue growth factor in cardiomyocytes

dc.contributor.advisorLutz, Susanne Prof. Dr.
dc.contributor.authorSingh, Simranjit
dc.titleRedox regulation of protein phosphatase-1 and ER stress regulation of connective tissue growth factor in cardiomyocytesde
dc.contributor.refereeKatschinski, Dörthe Prof. Dr.
dc.description.abstractengHeart failure is one of the most common causes for morbidity and hospitalization in the western civilization. The prognosis is still poor and new therapies are needed. For decades, variations in phosphorylation and redox status of cardiac proteins have been characterized in different heart diseases to identify new drug targets. Both abnormal phosphorylation-levels of cardiac key proteins and elevated reactive oxygen species (ROS) production were found to contribute to contractile dysfunction and fibrosis in failing hearts. In this context type-1 phosphatase (PP-1) was demonstrated to be a principal contributor to Ser/Thr PP activity (~45%) and has been implicated particularly in the regulation of basal cardiac contractility and in the responses to β-adrenergic stimulation (El-Armouche et al, 2009; Yin et al, 2009). Up until now redox sensitivity of cardiac PP-1 has not been addressed, despite well-known perturbations in PP-1 regulation in failing hearts. Therefore, one goal of this project was to identify the underlying mechanisms of PP-1 oxidation and to test whether oxidized PP-1 contributes to the pathophysiology of abnormal protein phosphorylation and myocardial dysfunction in failing myocardium. Immunoblotting revealed that the phosphorylation status of classical PP-1 downstream target proteins, such as phospholamban (PLB) and cardiac myosin binding protein-C (cMyBP-C) were differentially affected by H2O2, indicating a complex layer of regulation of both redox sensitive kinases and phosphatases. Consistently, the phosphorylation status of protein phosphatase inhibitor-1 (I-1), a crosstalk protein between protein kinase A and PP-1 signaling, showed a bell-shaped phosphorylation response with a maximal peak at 100 µM., For the first time we demonstrated with mass spectrometry that PP-1 shows various post-translational modifications on the incubation with H2O2 as one of the majorly available intracellular reactive oxygen species. In summary, for PP-1 a mechanism is purposed which states that PP-1’s cysteine residues in the presence of H2O2, first form sulfenic acid with a fast response to protect higher oxidations by glutathione that enables a self-protective mechanism by forming transient inter-disulfide bridges. Intra-protein disulfide bridges with Cys127 to form a dimer formation of PP-1 at 70 kDa might also play a role for the activity of the protein. In contrast, in the absence of glutathione, direct formation of sulfonic acid would make the protein irreversible inactive. The discovery of reversibility of PP-1 in the presence of the reducing agent (TCEP) after inactivation upon H2O2 treatment clearly shows that disulfide bridges are playing a crucial role in maintaining the activity of PP-1. In addition, to the changed redox status of cytosolic proteins like PP-1 in diseased cardiomyocytes, an impairment of the redox balance in organelles was described. With this respect the occurring endoplasmic reticulum (ER) stress is of high interest as it could influence transmembrane and secreted proteins. Therefore, the redox- and ER stress-dependent regulation of the secreted connective tissue growth factor (CTGF) was investigated. CTGF is a cysteine-rich protein highly expressed during embryonic development and in fibrotic diseases, including cardiac fibrosis (Winter et al., 2008; Lok et al., 2015). Due to its high content in cysteines and intramolecular disulfide bonds, we hypothesize that ER stress modulates the oxidation status of CTGF, which in turn affects its activity and structure in cardiomyocytes. Moreover, it was unknown whether ER stress can be modulated by the expression of cysteine-rich proteins like CTGF. We first analyzed CTGF expression in human diseased heart samples and were able to show an up-regulation in ischemic cardiomyopathy (ICM), which is associated with increased ER stress and changes in redox signaling. To further link the regulation of CTGF to these processes, isolated neonatal rat cardiomyocytes (NRCMs) were treated with pharmacological ER (DTT, thapsigargin) and oxidative (H2O2) stress inducers. DTT altered the molecular weight of CTGF in non-reducing immunoblots, suggesting conformational changes in the protein structure. In contrast, thapsigargin increased intracellular CTGF content, reaching the maximum after 6 hours of exposure to NRCMs. H2O2 had only a modest effect increasing intracellular CTGF within minutes. To further analyze the crosstalk of ER stress and CTGF regulation, CTGF expression was reduced with a specific siRNA in NRCMs, which led to a decrease in the expression of ER stress markers like PDI, BIP and IRE1-α. This data argues for an interconnection of CTGF and ER stress, as ER stress modulates CTGF and vice versa, CTGF expression modulates proteins of the ER stress cascade. In summary, this thesis gives mechanistic insight in the redox-dependent regulation of PP1 and CTGF, which represent not only the cytosolic and secretory compartments of cardiomyocytes, respectively, but also the two mayor pathomechanisms contractile dysfunction and fibrosis in heart
dc.contributor.coRefereeNikolaev, Viacheslav Dr.
dc.contributor.thirdRefereeSchwappach, Blanche Prof. Dr.
dc.subject.engDisulfide bridgesde
dc.subject.engHeart failurede
dc.affiliation.instituteMedizinische Fakultät
dc.subject.gokfullMedizin (PPN619874732)de

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