Jong, S. d. | Search bioRxiv

bioRxiv2022

Lysosomal calcium loading promotes arrhythmias by potentiating ryanodine receptor release

Spontaneous calcium release by ryanodine receptors (RyRs) due to intracellular calcium overload results in delayed afterdepolarisations, closely associated with life-threatening arrhythmias. In this regard, inhibiting lysosomal calcium release by two-pore channel 2 (TPC2) knockout has been shown to reduce the incidence of ventricular arrhythmias under {beta}-adrenergic stimulation. However, mechanistic investigations into the role of lysosomal function on RyR spontaneous release remain missing. We investigate the calcium handling mechanisms by which lysosome function modulates RyR spontaneous release, and determine how lysosomes are able to mediate arrhythmias by its influence on calcium loading. Mechanistic studies were conducted using a population of biophysically-detailed mouse ventricular models including for the first time modelling of lysosomal function, and calibrated by experimental calcium transients modulated by TPC2. We demonstrate that lysosomal calcium uptake and release can synergistically provide a buffering pathway of fast calcium transport, by which lysosomal calcium release primarily modulates sarcoplasmic reticulum (SR) calcium reuptake and RyR release. Enhancement of this lysosomal transport pathway promoted RyR spontaneous release by elevating the SR-junction calcium gradient. In contrast, blocking either lysosomal calcium uptake or release revealed an antiarrhythmic impact. Under conditions of calcium overload, our results indicate that these responses are strongly modulated by intercellular variability in L-type calcium current, RyR release, and SERCA reuptake. Altogether, our investigations identify that lysosomal calcium handling directly influences RyR spontaneous release by regulating the SR-junction calcium gradient, suggesting antiarrhythmic strategies and identifying key modulators of lysosomal proarrhythmic action. Statement of SignificanceDelayed afterdepolarisations arising from spontaneous RyR calcium release are an important risk factor for arrhythmogenesis. Inhibiting lysosomal calcium release by TPC2-KO reduces the propensity for ventricular arrhythmias. However, understanding downstream effects of lysosomal calcium release on spontaneous RyR release is lacking. Understanding lysosomes as arrhythmia sources requires alternative approaches to controlled laboratory techniques: these restrain variability experimentally and statistically. Our study presents two methodological novelties by focusing on calibration with experimental findings using a population of biophysically-detailed models and incorporating lysosomal mechanisms. Lysosomal calcium handling promotes RyR spontaneous release by elevating the SR-junction calcium gradient. Blocking lysosomal function uncovers an antiarrhythmic strategy. Lysosome-release proarrhythmic risk is determined by synergistic enhancements of lysosomal uptake with RyR release or L-type calcium current.

biophysics↗

bioRxiv2022

TRAIL-induces Src mediated MEK/ERK, SMAD3 and β-catenin signalling in apoptosis resistant NSCLC cells

Tumour-necrosis factor related apoptosis-inducing ligand (TRAIL) receptors (TRAIL-R1 and -R2) are appealing therapeutic targets to eradicate tumours specifically via caspase-dependent apoptosis. However, resistance is often observed and TRAIL-R activation can even activate pro-tumorigenic non-canonical signalling pathways. Previously, we found that TRAIL-induced RIPK1-Src-STAT3 signalling was mediating cell migration and invasion in resistant non-small cell lung cancer (NSCLC). Here, the contribution of Src in TRAIL signalling in NSCLC cell lines was further examined. TRAIL sensitive H460 and resistant A549 NSCLC cells showed distinct time-dependent rhTRAIL-induced Src phosphorylation patterns with early activation in A549 cells. Pharmacological Src inhibition as well as shRNA knockdown or CRISPR/CAS9-dependent knockout of Src expression did not alter sensitivity to rhTRAIL-induced apoptosis in both cell lines. Silencing of secondary complex proteins showed that TRADD, but not TRAF2, FADD nor caspase-8, was required for Src activation in A549 cells. Possible mediators of Src-dependent rhTRAIL signalling were identified by Src co-IP-LC-mass spectrometric analyses. In A549 cells the number of Src-interacting proteins increased after rhTRAIL treatment, whereas protein numbers decreased in H460 cells. In rhTRAIL treated A549 cells, Src biding proteins included components of the RAF-MEK1/2-ERK, Wnt and SMAD3 signalling pathways. Functional analyses showed that Src mediated phosphorylation of MEK1/2 and ERK, prevented phosphorylation of SMAD3 and was required for nuclear translocation of ERK and {beta}-catenin in A549 cells. Clonogenic growth of both Src proficient and deficient A549 cells was not affected by rhTRAIL exposure, although Src depletion and MEK1/2 inhibition reduced colony size and numbers significantly. In conclusion, rhTRAIL-induced and Src dependent MEK/ERK, SMAD3 and {beta}-catenin signalling may contribute to the known pro-tumorigenic effects of rhTRAIL in resistant NSCLC cells. However, this needs to be further examined, as well as the potential therapeutic implications of targeting these pathways when combined with TRAIL receptor agonists.

cancer biology↗