The effects of jaspamide on human cardiomyocyte function and cardiac ion channel activity

Abstract

Jaspamide (jasplakinolide; NSC-613009) is a cyclodepsipeptide that has antitumor activity. A narrow margin of safety was observed between doses required for efficacy in mouse tumor models and doses that caused severe acute toxicity in rats and dogs. We explored the hypothesis that the observed toxicity was due to cardiotoxicity. Jaspamide was tested in a patch clamp assay to determine its effect on selected cardiac ion channels. Jaspamide (10 μM) inhibited Kv1.5 activity by 98.5%. Jaspamide also inhibited other channels including Cav1.2, Cav3.2, and HCN2; however, the Kv11.1 (hERG) channel was minimally affected. Using spontaneously contracting human cardiomyocytes derived from induced pluripotent stem cells, effects on cardiomyocyte contraction and viability were also examined. Jaspamide (30 nM to 30 μM) decreased cardiomyocyte cell indices and beat amplitude, putative measurements of cell viability and cardiac contractility, respectively. Concentration-dependent increases in rhythmic beating rate were noted at ≤ 6 h of treatment, followed by dose-dependent decreases after 6 and 72 h exposure. The toxic effects of jaspamide were compared with that of the known cardiotoxicant mitoxantrone, and confirmed by multiparameter fluorescence imaging analysis. These results support the hypothesis that the toxicity observed in rats and dogs is due to toxic effects of jaspamide on cardiomyocytes.

Fig. 1

Cardiomyocyte viability after exposure to jaspamide and mitoxantrone. Human iPSC cardiomyocytes were treated for 72 h with drug concentrations ranging from 30 nM to 30 μM. Viability is expressed in terms of cell index vs. time, or a single ATP measurement after completion of impedance recording. Dynamic monitoring of cell index change is shown for cells exposed to vehicle and jaspamide (a) or mitoxantrone (c). Cell index for each well was normalized to baseline value prior to dosing (marked by the arrow). Dose-response analysis of jaspamide (b) and mitoxantrone (d) is shown at selected time points. The IC₅₀ was calculated from the change in cell index or ATP content expressed as a percentage of the time-matched vehicle group. n = 7 wells/concentration for jaspamide and six for mitoxantrone in three E-plates seeded with cells from three different lots.

Fig. 2

Beating waveform analysis. Representative beating traces were selected to show time and dose-dependent changes in beating waveforms of cardiomyocytes exposed to jaspamide (a) and mitoxantrone (d). The impedance-captured beating exhibited a downward deflection upon the contraction of human iPSC cardiomyocytes, which rapidly returned to baseline level during relaxation. The baseline level in (a) declined rapidly in the well exposed to 30 μM jaspamide, reflecting the rapid decrease in cell index. The reduced beat amplitude and transiently increased beat rate were well-manifested. Summarized data of beat rate and amplitude for jaspamide are presented in (b–c), and for mitoxantrone in (e–f). Blue arrows indicate tachycardia or fibrillation-like arrhythmia, and the red arrow indicates delayed-repolarization mediated-like arrhythmia. * P ≤ 0.05 vs. the time-matched vehicle group. n = 7 wells/concentration for jaspamide and six for mitoxantrone in three E-plates seeded with cells from three different lots.

Fig. 3

Representative beating traces which illustrate the effects of E-4031 (0.01, 0.03, 0.1 M), TTX (1, 3, 10 M), nifedipine (0.03, 0.1, 0.3 M) and S9947 (3, 10, 30 M). #1 to #3, dose concentrations from low to high. The red arrow indicates the delayed-repolarization mediated-like arrhythmia. Beating traces for E-4031 and TTX were selected at 30 min after drug treatment, and for nifedipine and S9947 at 72 h after drug exposure.

Fig. 4

Multiparameter fluorescence imaging analysis. Cells were treated with vehicle (A), mitoxantrone at 3 (B) and 30 M (C), or jaspamide at 0.3 (D), 3 (E) and 30 M (F) for 72 h, then stained with nuclei (blue), membrane permeability (green) and mitochondrial membrane potential (red) dyes. Summarized data show the drug effect on the viable cell density (a), membrane permeability (b), mitochondrial membrane potential (c) and cytotoxicity index (d). * P ≤ 0.05 vs. the time-matched vehicle group. n = 6 wells/concentration in three E-plates seeded with cells from three different lots.