Mitoxantrone impairs proteasome activity and prompts early energetic and proteomic changes in HL-1 cardiomyocytes at
Vera Marisa Costa1, João Paulo Capela2,3, Joana R Sousa4
1UCIBIO/REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal. veramcosta@ff.up.pt.
Abstract:
Mitoxantrone (MTX) is used to treat several types of cancers and to improve neurological disability in multiple sclerosis. Unfortunately, cardiotoxicity is a severe and common adverse effect in MTX-treated patients. Herein, we aimed to study early and late mechanisms of MTX-induced cardiotoxicity using murine HL-1 cardiomyocytes. Cells were exposed to MTX (0.1, 1 or 10 µM) during short (2, 4, 6, or 12 h) or longer incubation periods (24 or 48 h). At earlier time points, (6 and 12 h) cytotoxicity was already observed for 1 and 10 µM MTX. Proteomic analysis of total protein extracts found 14 proteins with higher expression and 26 with lower expression in the cells exposed for 12 h to MTX (pH gradients 4-7 and 6-11). Of note, the expression of the regulatory protein 14-3-3 protein epsilon was increased by a factor of two and three, after exposure to 1 and 10 µM MTX, respectively. At earlier time-points, 10 µM MTX increased intracellular ATP levels, while decreasing media lactate levels. At later stages (24 and 48 h), MTX-induced cytotoxicity was concentration and time-dependent, according to the MTT reduction and lactate dehydrogenase leakage assays, while caspase-9, -8 and -3 activities increased at 24 h. Regarding cellular redox status, total glutathione increased in 1 µM MTX (24 h), and that increase was dependent on gamma-glutamylcysteine synthetase activity. Meanwhile, for both 1 and 10 µM MTX, oxidized glutathione was significantly higher than control at 48 h. Moreover, MTX was able to significantly decrease proteasomal chymotrypsin-like activity in a concentration and time-independent manner. In summary, MTX significantly altered proteomic, energetic and oxidative stress homeostasis in cardiomyocytes at clinically relevant concentrations and our data clearly demonstrate that MTX causes early cardiotoxicity that needs further study.
Insights
Mitoxantrone (MTX) causes early cardiotoxicity by altering cardiomyocyte proteomic, energetic, and oxidative stress homeostasis. This study investigated MTX
Area of Science:
- Cardiology
- Toxicology
- Biochemistry
Background:
- Mitoxantrone (MTX) is a vital chemotherapeutic agent for cancers and multiple sclerosis.
- Cardiotoxicity is a significant and frequent adverse effect associated with MTX treatment.
- Understanding the early and late mechanisms of MTX-induced cardiotoxicity is crucial for patient safety.
Purpose of the Study:
- To investigate the early and late molecular mechanisms underlying Mitoxantrone (MTX)-induced cardiotoxicity.
- To analyze the effects of MTX on cardiomyocyte proteomic profiles, energy metabolism, and redox status.
- To determine the time- and concentration-dependent cardiotoxic effects of MTX in a cellular model.
Main Methods:
- Murine HL-1 cardiomyocytes were exposed to varying concentrations of MTX (0.1, 1, 10 µM) for short (2-12 h) and long (24-48 h) durations.
- Proteomic analysis was performed to identify differentially expressed proteins.
- Cytotoxicity was assessed using MTT reduction and lactate dehydrogenase leakage assays; caspase activity, ATP levels, glutathione status, and proteasomal activity were also measured.
Main Results:
- MTX induced cytotoxicity in cardiomyocytes at early time points (6-12 h) and later stages (24-48 h) in a concentration- and time-dependent manner.
- Proteomic analysis revealed significant alterations in protein expression, including increased 14-3-3 protein epsilon.
- MTX disrupted cellular energy homeostasis (increased ATP, decreased lactate) and redox balance (increased oxidized glutathione, altered total glutathione), and decreased proteasomal activity.
Conclusions:
- Mitoxantrone (MTX) significantly disrupts proteomic, energetic, and oxidative stress homeostasis in cardiomyocytes.
- The study demonstrates that MTX causes early-onset cardiotoxicity through multiple molecular pathways.
- Further research is warranted to elucidate the precise mechanisms of MTX-induced cardiotoxicity and develop preventative strategies.
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