RESUMO
Background: Doxorubicin (Dox) is effective against different types of cancers, but it poses cardiotoxic side effects, frequently resulting in irreversible heart failure. However, the complexities surrounding this cardiotoxicity, especially at sublethal dosages, remain to be fully elucidated. We investigated early cellular disruptions in response to sublethal Dox, with a specific emphasis on the role of phosphorylated calcium/calmodulin-dependent protein kinase II (CaMKII) in initiating mitochondrial dysfunction. Methods: This study utilized the H9c2 cardiomyocyte model to identify a sublethal concentration of Dox and investigate its impact on mitochondrial health using markers such as mitochondrial membrane potential (MMP), mitophagy initiation, and mitochondrial calcium dynamics. We examined the roles of and interactions between CaMKII, dynamin-related protein 1 (Drp1), and the mitochondrial calcium uniporter (MCU) in Dox-induced mitochondrial disruption using specific inhibitors, such as KN-93, Mdivi-1, and Ru360, respectively. Results: Exposure to a sublethal dose of Dox reduced the MMP red-to-green fluorescence ratio in H9c2 cells by 40.6% compared with vehicle, and increased the proportion of cells undergoing mitophagy from negligible levels compared with vehicle to 62.2%. Mitochondrial calcium levels also increased by 8.7-fold compared with the vehicle group. Notably, the activation of CaMKII, particularly its phosphorylated form, was pivotal in driving these mitochondrial changes, as inhibition using KN-93 restored MMP and decreased mitophagy. However, inhibition of Drp1 and MCU functions had a limited impact on the observed mitochondrial disruptions. Conclusion: Sublethal administration of Dox is closely linked to CaMKII activation through phosphorylation, emphasizing its pivotal role in early mitochondrial disruption. These findings present a promising direction for developing therapeutic strategies that may alleviate the cardiotoxic effects of Dox, potentially increasing its clinical efficacy.
RESUMO
Soluble uric acid (UA) absorbed by cells through UA transporters (UATs) accumulates intracellularly, activates the NLRP3 inflammasome and thereby increases IL-1ß secretion. ABCG2 transporter excludes intracellular UA. However, it remains unknown whether ABCG2 inhibition leads to intracellular accumulation of UA and increases IL-1ß production. In this study, we examined whether genetic and pharmacological inhibition of ABCG2 could increase IL-1ß production in mouse macrophage-like J774.1 cells especially under hyperuricemic conditions. We determined mRNA and protein levels of pro-IL-1ß, mature IL-1ß, caspase-1 and several UATs in culture supernatants and lysates of J774.1 cells with or without soluble UA pretreatment. Knockdown experiments using an shRNA against ABCG2 and pharmacological experiments with an ABCG2 inhibitor were conducted. Extracellularly applied soluble UA increased protein levels of pro-IL-1ß, mature IL-1ß and caspase-1 in the culture supernatant from lipopolysaccharide (LPS)-primed and monosodium urate crystal (MSU)-stimulated J774.1 cells. J774.1 cells expressed UATs of ABCG2, GLUT9 and MRP4, and shRNA knockdown of ABCG2 increased protein levels of pro-IL-1ß and mature IL-1ß in the culture supernatant. Soluble UA increased mRNA and protein levels of ABCG2 in J774.1 cells without either LPS or MSU treatment. An ABCG2 inhibitor, febuxostat, but not a urate reabsorption inhibitor, dotinurad, enhanced IL-1ß production in cells pretreated with soluble UA. In conclusion, genetic and pharmacological inhibition of ABCG2 enhanced IL-1ß production especially under hyperuricemic conditions by increasing intracellularly accumulated soluble UA that activates the NLRP3 inflammasome and pro-IL-1ß transcription in macrophage-like J774.1 cells.