Project 2: Redox regulation of ryanodine receptor/Ca2+ release channels by Nox enzymes in hypertrophy and heart failure
PI Göttingen: S.E. Lehnart; PI London: P. Eaton, A.M. Shah; PhD student: D. Uhlenkamp - finished PhD
Scientific background and preliminary results
The cardiac ryanodine receptor type 2 (RyR2) functions as intracellular Ca2+ release channel, which is essential for excitation-contraction (EC) coupling of the adult heart. During EC coupling, voltage-dependent activation of L-type Ca2+ channels (Cav1.2) leads to a relatively small Ca2+ inward current activating a much larger intracellular Ca2+ release from nearby RyR2 channels via Ca2+-induced Ca2+ release (CICR). There is now clear evidence from our and other groups that posttranslational redox modification of RyR channel isoforms occurs both directly and indirectly through redox-sensitive signalling pathways. However, while RyR2 dysregulation emerged as potentially important pathomechanism in heart disease, direct molecular mechanisms of RyR2 channel redox regulation remain unclear. CICR in ventricular cardiomyocytes (VCM) occurs from RyR2 clusters in Ca2+ release units (CRUs). CRUs provide a cytosolic nanodomain compartment, which depends critically on discontinous, ~15 nm wide membrane contacts between the cell surface through T-tubule invaginations and the junctional sarcoplasmic reticulum (jSR). We have identified T-tubule proliferation and nanometric membrane reorganization as early mechanism of CRU dysfunction after myocardial infarction. However, it is still unclear (i) if CRU function includes compartmentalized redox signaling and (ii) which redox enzymes protect versus disrupt CRU function of RyR2 channels as the major source of intracellular Ca2+ signaling. NADPH oxidase (NOX) are particularly interesting as putative RyR2 regulating enzymes due to their hypothetical, differential subcellular localisation in VCM. While cardiac NOX2 and NOX4 are the main enzymes expressed in heart, both isoforms are upregulated in heart disease. While NOX2 is located in T-tubules where it is regulated by promiscuous membrane-dependent signaling pathways, the NOX4 isoform locates to the SR membrane, where it may directly regulate RyR2 channels. We now find a cell-specific ~4-fold NOX4 versus ~2-fold NOX2 upregulation after onlz 1 week of pressure overload, in constrast to 4 weeks when only the NOX4 level remains elevated. This suggests an important physiological role of NOX4 upregulation in response to increased mechanical load. Additionally, we have developed superresolution light microscopy protocols to study RyR2 cluster ensembles and interactions with proximal regulatory proteins. We have shown increased RyR2 cysteine oxidation in vivo following b-adrenergic stimulation. RyR2 contains ~33 free thiol residues per subunit and is functionally sensitive to S-nitrosylation, which leads to increased channel activity and sarcoplasmic reticulum (SR) Ca2+ leak. The NOX4 knockout model now provides a key tool to refined insight about the molecular mechanism of RyR2 nitrosylation and to define subcellular NOX enzyme localisations by superresolution microscopy.
Hypotheses of the PhD project
Based on existing evidence from the previously established NOX4 knockout model and load induced ventricular remodeling, we hypothesize that NOX4 is compartimentalised in cardiomyocyte CRU nanodomains, where it exerts physiological and potentially also pathological mechanisms. In the project the IRTG 1816 PhD student will test the following hypotheses:
1. NOX4 co-localises with RyR2 clusters, directly regulating single RyR2 channels.
2. Increased mechanical load activates NOX2 in T-tubules, and T-tubule loss versus axial A-tubule proliferations aligns NOX2 redox signaling early with axial rows of mitochondria.
3. Increased NOX4 expression affects mitochondrial function through ROS production, leading to increased RyR2 nitrosylation, SR Ca2+ leak and cytosolic Ca2+ overload.
Research interests: Molecular function and nanoscopic organization of the intracellular calcium release unit, modulation of ion transport proteins by kinases
Research interests: Molecular mechanisms of redox signalling and heart faillure
Speaker British Heart Foundation Centre of Research Excellence
Research interests: NADPH oxidases, redox signalling and heart failure
PhD student 2nd cohort