Abstract
Background: Lysosomes are specific organelles that contain various digestive enzymes and also store both iron (Fe) and calcium (Ca). However, it is not known whether cytosolic and mitochondrial Fe and Ca handling and ferroptosis are modulated by lysosomal dysfunction. We aimed to evaluate the interaction between lysosomal, mitochondrial and cytosolic Ca and Fe dynamics and its relevance to cardiomyocyte function and ferroptosis. Methods: Left ventricular myocytes were enzymatically isolated from mouse hearts. Cytosolic Ca i was imaged using fluo-4-AM. Cytosolic Fe was determined with Phen green while mitochondrial Fe was measured using rhodamine B-[(1,10-phenanthroline-5-yl)-aminocarbonyl]benzyl ester (RPA). Mitochondrial membrane potential (Δ Ψ m ) was monitored by TMRM. Ferroptosis was analyzed using the Live/Dead cell viability assay. Results: Treatment with nicotinic acid dinucleotide phosphate (NAADP-AM 50 nM), an agonist of the two pore channel in the lysosomal membrane, increased the aptitude of the Ca i transient (1.94 ± 0.09 to 2.41 ± 0.14, n = 12, p < 0.05) and induced spontaneous Ca i waves. Diastolic Ca i became elevated (1.00 to 1.30 ± 0.10, n = 12, p < 0.05) in intact myocytes. Elevation of basal Ca i level and an increase in spontaneous Ca i wave amplitude and frequency were confirmed in saponin-permeabilized myocytes. NAADP also increased the level of cytosolic Fe. These results suggest both Ca and Fe are released from lysosomes into the cytosol by NAADP. Our previous studies have shown that mitochondrial Fe uptake is essential for Fe overload-induced ferroptosis in cardiac myocytes. Therefore, we further assessed how lysosome function affects mitochondrial Fe loading and induction of ferroptosis. We found that NAAPD treatment resulted in an elevation of mitochondrial Fe levels (by 28.4 ± 3.5 %, n=23, p <0.05), depolarization of Δ Ψ m as well as resulted in an increased sensitivity to Fe overload-induced ferroptosis. Similar results were observed using GPN that causes selective disruption of lysosomes. Conclusions: Lysosomal dysfunction may contribute to mishandling of Ca and Fe in myocytes and promote the occurrence of ferroptosis. A lysosomal storage disorder mouse model ( i.e. RagA/B KO) will be assessed in future studies.
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