Biology subjects

Ritterhoff, J.

Publications and source records attributed to Ritterhoff, J..

4 recordsLinked to original sources

bioRxiv2023

S100A1's single cysteine is an indispensable redox-switch for the protection against diastolic calcium leakage in cardiomyocytes

The EF-hand calcium (Ca2+) sensor protein S100A1 combines inotropic with antiarrhythmic potency in cardiomyocytes (CM). Oxidative posttranslational modification (ox-PTM) of S100A1s conserved, single cysteine residue (C85) via reactive nitrogen species (i.e. S-nitrosylation or glutathionylation) was proposed to modulate conformational flexibility of intrinsically disordered sequence fragments and to increase the molecules affinity towards Ca2+. In light of the unknown biological functional consequence, we aimed to determine the impact of the C85 moiety of S100A1 as a potential redox-switch. We first uncovered that S100A1 is endogenously glutathionylated in the adult heart in vivo. To prevent glutathionylation of S100A1, we generated S100A1 variants that were unresponsive to ox-PTMs. Overexpression of wildtype (WT) and C85-deficient S100A1 protein variants in isolated CM demonstrated equal inotropic potency, as shown by equally augmented Ca2+ transient amplitudes under basal conditions and {beta}-adrenergic receptor ({beta}AR) stimulation. However, in contrast ox-PTM defective S100A1 variants failed to protect against arrhythmogenic diastolic sarcoplasmic reticulum (SR) Ca2+ leak and ryanodine receptor (RyR2) hypernitrosylation during {beta}-AR stimulation. Despite diastolic performance failure, C85-deficient S100A1 protein variants exerted similar Ca2+-dependent interaction with the RyR2 than WT-S100A1. Dissecting S100A1s molecular structure-function relationship, our data indicate for the first time that the conserved C85 residue potentially acts as a redox-switch that is indispensable for S100A1s antiarrhythmic but not its inotropic potency in CM. We therefore propose a model where C85s ox-PTM determines S100A1s ability to beneficially control diastolic but not systolic RyR2 activity.

molecular biology↗

bioRxiv2023

Cardiac-targeted rAAV5-S100A1 gene therapy protects against adverse remodeling and contractile dysfunction in post-ischemic hearts

Toxicity by recombinant adeno-associated viruses (rAAV) in clinical gene therapy trials (e.g., by rAAV9-mediated fatal liver failure) significantly impairs translation of preclinical rAAV-based cardiac gene therapies employing these vectors. For rAAV5 - a capsid that has shown long-term safety in clinical trials - our translational study demonstrates effective transduction of the left ventricle (LV) of healthy pigs via catheter-based retrograde intravenous delivery (CRID) by means of luciferase reporter gene biodistribution analyses. Combination of rAAV5 with the cardioprotective human gene S100A1 (hS100A1) prevents LV myocardial infarct (MI) enlargement and improves LV systolic contractile performance in a porcine model of post-MI chronic cardiac dysfunction. Use of a cardiac-biased promoter ensured the cardiac-directed expression of the therapeutic human transgene without signs of clinical toxicity. The beneficial effects of rAAV5-hS100A1 were linked to an attenuated activity of post-MI inflammatory gene networks and this was further validated in a murine model. These novel data together with proven scalable producibility and low pre-existing immunity against rAAV5 in humans may collectively advance clinical translation of rAAV5-hS100A1 as a gene therapy medicinal product (GTMP) for a common cardiovascular disease, such as chronic heart failure (CHF). HighlightsO_LIRecent fatal adverse events in recombinant adeno-associated virus (AAV)-based clinical gene therapy trials advise the use of rAAV serotypes with proven long-term clinical safety, such as rAAV5, for the pre-clinical development and clinical translation of rAAV-based cardiac gene therapy medicinal products. C_LIO_LIIn a biodistribution and therapeutic proof-of-concept study in farm pigs, rAAV5 was identified as an effective viral vector for cardiac gene transfer and gene therapy for post-ischemic cardiac dysfunction when applied by a standardized cardiac-targeted catheter-based route of administration with the luciferase reporter and cardioprotective human gene S100A1 (hS100A1), respectively. C_LIO_LIA systems biology analysis linked the novel finding of mitigated inflammatory and activated cardioprotective gene network activities in rAAV5-hS100A1 treated postischemic myocardium with improved study left ventricular ejection fraction and prevention of myocardial infarct extension, respectively, which warrants further mechanistic molecular studies. C_LIO_LISince rAAV5 has been recently approved for clinical use in a non-cardiac indication and cardiac-targeted S100A1 gene therapy has been effective in numerous pre-clinical animal models of acute and chronic cardiac dysfunction, our translational data support an expedited developmental path for rAAV5-hS100A1 throughout investigational new drug-enabling studies towards a first-in-human clinical trial for post-myocardial infarction heart failure. C_LI

pathology↗

bioRxiv2023

S100A1ct: a synthetic peptide derived from human S100A1 protein improves cardiac contractile performance and survival in pre-clinical heart failure models

BackgroundThe EF-hand Ca2+ sensor protein S100A1 has been identified as a molecular regulator and enhancer of cardiac performance. S100A1s ability to recognize and modulate the activity of targets such as SERCA2a and RyR2 in cardiomyocytes has mostly been ascribed to its hydrophobic C-terminal -helix (residues 75-94). Objective: We therefore hypothesized that a synthetic peptide consisting of residues 75-94 of S100A1 and an N-terminal solubilization tag (S100A1ct) could mimic the performance enhancing effects of S100A1 and may be suitable as a peptide therapeutic to improve the function of diseased hearts. Methods and Results: Applying an integrative translational research pipeline, ranging from computational molecular modeling to large animal cardiac disease models, we characterize S100A1ct as a cell-penetrating peptide with positive inotropic and antiarrhythmic properties in normal and failing myocardium in vitro and in vivo. This activity translates into improved contractile performance and survival in pre-clinical heart failure models with reduced ejection fraction after S100A1ct systemic administration. Mechanistically, S100A1ct exerts a fast and sustained dose-dependent enhancement of cardiomyocyte Ca2+ cycling and prevents {beta}-adrenergic receptor triggered Ca2+ imbalances by targeting SERCA2a and RyR2 activity. Modeling suggests that S100A1ct may stimulate SERCA2a by interacting with the sarcoplasmic transmembrane segments of the multi-span integral membrane Ca2+ pump. Incorporation of a cardiomyocyte targeting peptide tag into S100A1ct (cor-S100A1ct) further enhanced its biological and therapeutic potency in vitro and in vivo. Conclusion: S100A1ct peptide is a promising lead for the development of a novel peptide-based therapeutic against heart failure with reduced ejection fraction.

pharmacology and toxicology↗

bioRxiv2023

SUMO interacting motif (SIM) of S100A1 is critical for S100A1 post-translational protein stability

S100A1 is a small EF-type Ca2+ sensor protein that belongs to the multigenic S100 protein family. It is abundantly expressed in cardiomyocytes (CMs) and has been described as a key regulator of CM performance due to its unique ability to interact with structural contractile proteins, regulators of cardiac Ca2+ cycling, and mitochondrial proteins. However, our understanding of the molecular mechanisms regulating S100A1 protein levels is limited. We used the bioinformatics tool GPS-SUMO2.0 to identify a putative SUMO interacting motif (SIM) on S100A1. Consistently, a S100A1:SUMO interaction assay showed a Ca2+-dependent interaction of S100A1 with SUMO proteins. In neonatal rat ventricular myocytes (NRVM) and COS1 cells, S100A1 protein abundance increased in the presence of overexpressed SUMO1 without affecting the S100A1 mRNA transcript. We then generated S100A1 truncation mutants, where the SIM motif was removed by truncation or in which the core residues of the SIM motif (residues 77-79) were deleted or replaced by alanine. In COS1 cells and NRVM, overexpression of these S100A1 mutants led to elevated S100A1 mutant mRNA levels but failed to produce respective protein levels. Protein expression of these mutants could be rescued from degradation by addition of the proteasome inhibitor MG-132. By using an information-driven approach to dock the three-dimensional structures of S100A1 and SUMO, we predict a novel interaction mode between the SIM in S100A1 and SUMO. This study shows an important role of SUMO:SIM-mediated protein:protein interaction in the regulation of post-translational protein stability, and provides mechanistic insights into the indispensability of the core SIM for S100A1 post-translational stability.

biochemistry↗