Ex Vivo Cardiotoxicity of Antineoplastic Casiopeinas Is Mediated through Energetic Dysfunction and Triggered Mitochondrial-Dependent Apoptosis

Abstract

Casiopeinas are a group of copper-based antineoplastic molecules designed as a less toxic and more therapeutic alternative to cisplatin or Doxorubicin; however, there is scarce evidence about their toxic effects on the whole heart and cardiomyocytes. Given this, rat hearts were perfused with Casiopeinas or Doxorubicin and the effects on mechanical performance, energetics, and mitochondrial function were measured. As well, the effects of Casiopeinas-triggered cell death were explored in isolated cardiomyocytes. Casiopeinas III-Ea, II-gly, and III-ia induced a progressive and sustained inhibition of heart contractile function that was dose- and time-dependent with an IC₅₀ of 1.3 ± 0.2, 5.5 ± 0.5, and 10 ± 0.7 μM, correspondingly. Myocardial oxygen consumption was not modified at their respective IC₅₀, although ATP levels were significantly reduced, indicating energy impairment. Isolated mitochondria from Casiopeinas-treated hearts showed a significant loss of membrane potential and reduction of mitochondrial Ca²⁺ retention capacity. Interestingly, Cyclosporine A inhibited Casiopeinas-induced mitochondrial Ca²⁺ release, which suggests the involvement of the mitochondrial permeability transition pore opening. In addition, Casiopeinas reduced the viability of cardiomyocytes and stimulated the activation of caspases 3, 7, and 9, demonstrating a cell death mitochondrial-dependent mechanism. Finally, the early perfusion of Cyclosporine A in isolated hearts decreased Casiopeinas-induced dysfunction with reduction of their toxic effect. Our results suggest that heart cardiotoxicity of Casiopeinas is similar to that of Doxorubicin, involving heart mitochondrial dysfunction, loss of membrane potential, changes in energetic metabolites, and apoptosis triggered by mitochondrial permeability.

Figure 1

Structures of Cas III-Ea, III-ia, and II-gly.

Figure 2

Cas affect contractility on isolated rat hearts. Cas were perfused at 5 μM for 60 minutes (a) or in a dose-dependent manner (b). The blue squares indicate III-Ea, green triangles indicate II-gly, and red circles indicate III-ia. Black rhombuses indicate control and yellow rhombuses indicate Doxo treatment. Values are mean ± SEM. (n = 5 experiments at least for each treatment).

Figure 3

Cas impair oxygen consumption and energetic metabolites. Isolated rat hearts were used to measure the effect of Cas IC₅₀ and Doxo (5 μM) on mechanical (RPP) and metabolic coupling (oxygen consumption (MVO₂) relationship) (a), MVO₂ (b), ATP content (c), and myocardial PCr/ATP ratio (d). Values are mean ± SEM. ^∗ p < 0.05 versus control (n = 5 experiments at least for each treatment).

Figure 4

Cas treatments induce MPT opening. (a) Representative recording of Ca²⁺ retention capacity (CRC) experiment with isolated mitochondria from IC₅₀ Cas-treated and Doxo (5 μM) hearts and (b) semiquantitative analysis of mitochondrial CRC in the presence of CsA (0.5 μM). Arrows indicate 10 μM pulses of Ca²⁺. Values are mean ± SEM. ^∗ p < 0.05 versus control; p < 0.05 versus (A) Doxo, (B) II-gly, and (D) III-ia (n = 5 animals for each treatment, exception Doxo groups (n = 3)).

Figure 5

Effect of Cas on isolated heart on TBARS content (a), aconitase activity (b), and ANT/thiols groups (c) on mitochondria from IC₅₀ Cas-treated and Doxo (5 μM) hearts. Values are mean ± SEM. ^∗ p < 0.05 versus control (n = 5 animals for each treatment, except for Doxo group (n = 3)).

Figure 6

Cas trigger mitochondrial cell death. Panels (a–c) shows LDH, caspase 3/7 and caspase 9 activities on isolated cardiomyocytes treated with Cas at its IC₅₀ (in μM: III-ia (7), II-gly (2), III-Ea (2), and Doxo (10)). Panel (d) shows cytochrome c content by Western blot analysis in heart mitochondria after Cas-perfusion for 30 minutes in the ex vivo hearts at its IC₅₀ (in μM: III-ia (10), II-gly (5.5), III-Ea (1.3), and Doxo (5)). Values are mean ± SEM. ^∗ p < 0.05 versus control; p < 0.05 versus (A) Doxo, (B) II-gly, and (D) III-ia (n = 5 experiments for each treatment, except for panel (d) (3 animals for group)).

Figure 7

Early perfusion of CsA in isolated hearts ameliorates the Cas effect due to MPT opening. RPP is shown in (a). The decline in contractility due to the perfusion (20 min) of 10 μM III-Ea alone is presented as the blue trace. The early perfusion (10 min) of 1 μM CsA (white squares) delays the decline in contractility due to the subsequent perfusion with III-Ea. The control trace is presented as the black trace. Analysis of the traces is presented as the time for half inhibition (t _0.5) (b). Experiments in isolated mitochondria prepared from these hearts at the end of perfusion. Representative recording of membrane potential (c) and Ca²⁺ retention capacity (d). Semiquantitative analysis of mitochondrial CRC (e). Mitochondria from III-Ea-treated hearts are represented as a blue solid line, mitochondria from CsA-Cas III-Ea hearts as a blue dot line, and untreated hearts as a black solid line. Arrows indicates succinate (10 mM), CCCP (0.08 μM), or 10 μM pulses of Ca²⁺ addition. Values are mean ± SEM. ^∗ p < 0.05 versus control; p < 0.05 versus (C) III-Ea (n = 4 animals for each treatment).