Development of Novel Triadin-Based Biosensor for the Quantification of H2O2 in the Cardiac Dyad Microdomain

Abstract

Release of Ca2+ from the sarcoplasmic reticulum (SR), mediated by the activation of the ryanodine receptor (RyR2), initiates myocardial contraction. This process of Ca2+-induced Ca2+ release is confined within a 20 nm region termed the dyad, located between the transverse tubule and the SR. Architectural properties of the dyad restrict diffusion of Ca2+ and other intracellular messengers to create a unique, subcellular microdomain. Previous research has shown that endogenous producers of reactive oxygen species (ROS) are abundant within the dyad, and have attributed alterations in Ca2+ release with the oxidation of RyR2. However, the spatiotemporal dynamics of ROS within this microdomain remain unquantified due to an absence of tools that enable subcompartmental ROS resolution. Therefore, this research aimed to utilise the recent development of genetically encoded redox biosensors, capable of ROS detection, to create a biosensor tethered to the N-terminus of the dyadic protein, triadin. A Human Embryonic Kidney (HEK) 293 cell model was used to characterise two ROS (H2O2) sensors, HyPer3 and roGFP2-Orp1, in vitro to determine the biosensor more suitable for the triadin-based construct. Using single-cell imaging, HEK 293 cells transiently transfected with HyPer3 or roGFP2-Orp1 were superfused with a range of oxidising and reducing agents. Based on these data, roGFP2-Orp1 exhibits a greater dynamic range, increased sensitivity, and faster oxidation-reduction kinetics than HyPer3, and thus was selected for the triadin-based construct. In parallel, single-cell Ca2+ imaging was used to confirm that co-expression of triadin has a neutral effect on RyR2 function. HEK 293 cells expressing RyR2 WT or RyR2 WT + triadin were loaded with the cytosolic Ca2+ indicator, Fluo-4 AM and superfused with 0.1–1 mM Ca2+ to activate RyR2 WT. These data found a non-significant (p = 0.9) difference in Ca2+ release in RyR2 WT (61.9 ± 3.1%) and RyR2 WT + triadin (60.3 ± 3.5%) cells exposed to 1 mM Ca2+. To create roGFP2-Orp1-triadin, overlap extension PCR was used to generate an SfaAI restriction site that enabled roGFP2-Orp1 to be subcloned into the triadin plasmid. However, the development of roGFP2-Orp1-triadin was unsuccessful. This research highlights a novel method for H2O2 quantification within the dyadic microdomain. Characterisation of HyPer3 and roGFP2-Orp1 revealed that roGFP2-Orp1 exhibits the sensitivity and reversibility required for an in vivo biosensor. In addition, co-expression of triadin has a neutral effect on the properties of RyR2-mediated Ca2+ release, which indicates that triadin is an appropriate tether for the dyadic localisation of roGFP2-Orp1. Future directions include the generation of an animal model to further understand the spatiotemporal dynamics of ROS in relation to alterations in Ca2+ release in vivo.