|dc.description.abstracteng||Cardiac function is characterized by a rhythmic sequence of contraction and relaxation of cardiomyocytes driven by tightly controlled intracellular calcium release events. The cardiac ryanodine receptor type 2 calcium release channel, RyR2, provides a key function for this cyclic Ca2+ release and thus cardiomyocyte contraction. Posttranslational RyR2 modifications through redox-sensitive cysteine residues were shown to increase channel activity, which may lead to Ca2+ leak from the sarcoendoplasmic reticulum. This has been associated with pathological conditions such as heart failure. However, precise molecular mechanisms of redox-mediated RyR2 (dys)regulation remain to be identified. The aim of this thesis was to characterize the redox-based regulation of RyR2 Ca2+ release channels by the cardiac isoforms of the ROS-generating NADPH-dependent oxidases NOX2 and NOX4.
To study NOX-dependent redox modifications of RyR2 channels, cardiac NOX enzymes and RyR2 were expressed in HEK293A cells. Spontaneous changes in cytosolic Ca2+ levels were measured by live-cell epifluorescence microscopy. RyR2-overexpressing HEK293A cells showed self-regenerative Ca2+ waves, which were inhibited by the specific Ca2+ channel inhibitor ryanodine. Importantly, short-time incubation with hydrogen peroxide (H2O2) caused a significant increase in Ca2+ waves frequency of 49.95 s ± 2.5 (ctrl) vs. 41.23 s ± 1.3 (1 mM H2O2), (n=8, p<0.01 (unpaired t-test)). To validate NOX-dependent H2O2 production in HEK293A cells, H2O2 was directly monitored by co-expression of the genetically encoded HyPer-3 biosensor. Expression of the cardiac NOX2 and NOX4 both resulted in significantly increased H2O2 levels [HyPer-3 ratio: 0.596 ± 0.014 (ctrl) vs. 0.71 ± 0.025 (NOX2) vs. 0.67 ± 0.024 (NOX4), n=134, p<0.05 (ANOVA)].
To investigate the thus far unclear subcellular localization of NOX4 and its spatial relation to RyR2 channel clusters in their native cardiomyocyte environment, an adenoviral expression construct was designed to induce expression of NOX4 in isolated primary mouse ventricular cardiomyocytes. STED nanoscopy of NOX4-transduced cardiomyocytes (Ad5.NOX4) revealed a NOX4 localization at the sarcoplasmic reticulum bringing it in close proximity to RyR2 clusters. A co-localization analysis demonstrated a significantly increased spatial overlap between RyR2 and NOX4 clusters in Ad5.NOX4-transduced cardiomyocytes compared to control [67.5% ± 2.1 (Ad5-NOX4) vs. 22.9% ± 12.7 (Ad5-eGFP), n≥3, p<0.01 (t-test)]. Further, the area of NOX4 clusters overlapping with RyR2 clusters was significantly larger compared to non-overlapping NOX4 clusters, indicating a regulatory complex formation [(0.0177 μm2 ± 0.002 (overlapping clusters) vs. 0.008 μm2 ± 0.001 (independent clusters) n≥3, p<0.01 (t-test)]. By performing co-immunoprecipitation assays, a direct protein-protein interaction of NOX4 and RyR2 was revealed, further emphasizing a potentially regulatory NOX4/RyR2 complex in virally transduced cardiomyocytes. Since a direct protein-protein interaction of NOX4 and RyR2 could be demonstrated, specific cysteine residues of RyR2 serving as target for a NOX-dependent oxidation were further investigated. To identify these cysteine residues, a differential alkylation assay was established allowing distinct labeling of oxidized cysteines for a subsequent data-independent mass spectrometry analysis.
The HEK293A cell model and the results presented here serve as an experimental framework for simultaneous monitoring of Ca2+ waves and H2O2 production through combinatory RyR2/NOX2- and RyR2/NOX4-transfections together with HyPer-3 by multi-color epifluorescence microscopy. Moreover, STED nanoscopy revealed a SR localization of NOX4 and a close association with RyR2 channels in cardiomyocytes. This association was further consolidated by co-immunoprecipitation and demonstrated to be a direct protein interaction.
In summary, this thesis provides novel insights into the interaction between the RyR2 Ca2+ channel and NOX enzymes and introduces the groundwork for future comprehensive studies of the hypothesized regulatory impact of NOX-generated ROS on RyR2 channel function in cardiomyocytes.||de