Use of human stem cell derived cardiomyocytes to examine sunitinib mediated cardiotoxicity and electrophysiological alterations

Abstract

Sunitinib, an oral tyrosine kinase inhibitor approved to treat advanced renal cell carcinoma and gastrointestinal stroma tumor, is associated with clinical cardiac toxicity. Although the precise mechanism of sunitinib cardiotoxicity is not known, both the key metabolic energy regulator, AMP-activated protein kinase (AMPK), and ribosomal S 6 kinase (RSK) have been hypothesized as causative, albeit based on rodent models. To study the mechanism of sunitinib-mediated cardiotoxicity in a human model, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) having electrophysiological and contractile properties of native cardiac tissue were investigated. Sunitinib was cardiotoxic in a dose-dependent manner with an IC₅₀ in the low micromolar range, observed by a loss of cellular ATP, an increase in oxidized glutathione, and induction of apoptosis in iPSC-CMs. Pretreatment of iPSC-CMs with AMPK activators AICAR or metformin, increased the phosphorylation of pAMPK-T172 and pACC-S79, but only marginally attenuated sunitinib mediated cell death. Furthermore, additional inhibitors of AMPK were not directly cytotoxic to iPSC-CMs up to 250 μM concentrations. Inhibition of RSK with a highly specific, irreversible, small molecule inhibitor (RSK-FMK-MEA) did not induce cytotoxicity in iPSC-CMs below 250 μM. Extensive electrophysiological analysis of sunitinib and RSK-FMK-MEA mediated conduction effects were performed. Taken together, these findings suggest that inhibition of AMPK and RSK are not a major component of sunitinib-induced cardiotoxicity. Although the exact mechanism of cardiotoxicity of sunitinib is not known, it is likely due to inhibition of multiple kinases simultaneously. These data highlight the utility of human iPSC-CMs in investigating the potential molecular mechanisms underlying drug-induced cardiotoxicity.

Fig. 1

Sunitinib cardiotoxicity profile in human iPSC-CMs. iCell cardiomyocytes were treatedwith 8 to 31 µM sunitinib for 24 h; followed by ATP depletion, LDH release, caspase 3/7 cleavage, and GSH oxidation assays. N=3 for all treatment groups, and symbols denote statistically significant decrease (*) or increase (‡) between vehicle and sunitinib treatment groups, student T-test p < 0.05.

Fig. 2

AMPK and ACC signaling inhibited by sunitinib, but activated by aminoimidazole carboxamide ribonucleotide (AICAR) or metformin treatments in iPSC-CMs. iCell cardiomyocytes were treated with (A) 15 µM sunitinib [S] for 2 h, (B) 1 mM AICAR [A] or metformin [M], or vehicle [0] for 3 h. iCell and HEK293 [H] (positive control) whole cell lysates were subjected to Western blotting with antibodies specific to pACC-S79, ACC, p-AMPK-T172, AMPK, and GAPDH.

Fig. 3

AICAR or metformin pretreatment results in marginal attenuation of sunitinib cardiotoxicity in iPSC-CMs. iCell cardiomyocytes were pretreated with 1 or 2 mM AICAR (A–D) or metformin (E–H) for 3 h, followed by treatment with 8 to 31 µM sunitinib (S) for 24 h. iCells were then assayed for (A, E) ATP depletion, (B, F) GSH oxidation, (C, G), caspase 3/7 cleavage, and, (D, H) LDH release. N=3 for all treatment groups, and symbols denote statistically significant decrease (*) or increase (‡) between sunitinib alone and pretreatment groups, student T-test p < 0.05.

Fig. 4

Validation of downstream signaling of AMPK kinase inhibitor in iPSC-CMs. iCell cardiomyocytes were treated with (1) vehicle, (2) 31 µM RO-3857 [AMPK], and (3) 15 µM sunitinib for 2 h. iCell whole cell lysates were subjected to Western blotting with antibodies specific to pACC-S79, ACC, AMPK, and GAPDH.

Fig. 5

AMPK kinase inhibitors are not cardiotoxic in iPSC-CMs. iCell cardiomyocytes were treated for 24 h with 4 to 250 µMof (A–C) small molecule inhibitors of AMPKα1/2 (RO-3857, RO-9568, and RO-1652). iCells were then assayed for LDH release and ATP depletion. N=4 for all treatment groups.

Fig. 6

Irreversible RSK1/2/4 inhibitor is not cardiotoxic in iPSC-CMs. (A) iCell [1] and HEK293 [2] whole cell lysates were subjected to Western blotting with antibodies specific to RSK1 and GAPDH. (B) iCell cardiomyocytes were treated for 24 h with 4 to 250 µM of an irreversible RSK inhibitor (RSK FMK-MEA). iCells were then assayed for LDH release and ATP depletion. N=4 for all treatment groups.

Fig. 7

Sunitinib and RSK inhibitor-mediated electrophysiological changes in human iPSC-CMs. Human iPSC-CMs were plated on microelectrode arrays (MEA). (A) MEA traces representative of serially increasing additions of sunitinib (0, 1, 10, 30 µM) that were recorded for 15 min at each concentration, and each trace represents 10 s.”. (B) Normalized beat rate (BR), field-potential duration (fPDc), sodium (Na⁺) peak, and calcium (Ca²⁺) peak of sunitinib MEA results. Mean ± SEM, n = 4 wells, * p < 0.05, ** p < 0.01 (t-test). (C) Using CHO-hERG and CHL-Nav1.5 cells, hERG and Nav1.5 channel assays were performed following serially increasing additions of sunitinib (0, 0.3, 3, 30 µM). n = 3 for hERG, and n = 9 for Nav1.5. (D) MEA traces representative of serially increasing additions of RSK FMK-MEA (0, 0.3, 3, 30 µM) that were recorded for 15 min at each concentration. (E) Normalized beat rate (BR), field-potential duration (fPDc), Na⁺ peak, and Ca²⁺ peak of RSK FMK-MEA results. Mean ± SEM, n = 4 wells, * p < 0.05, ** p < 0.01 (t-test). (F) Using CHO-hERG and CHL-Nav1.5 cells, hERG and Nav1.5 channel assays were performed following serially increasing additions of RSK FMK-MEA (0, 0.3, 3, 30 µM). N = 6 for hERG, and n = 3 for Nav1.5.

Fig. 8

AICAR, metformin, and AMPK inhibitor-mediated electrophysiological changes in human iPSC-CMs. Human iPSC-CMs were plated on microelectrode arrays (MEA). MEA traces of serially increasing additions (0, 1, 2 mM) of (A) AICAR or (B) metformin that were recorded for 15 min at each concentration. MEA traces of serially increasing additions (0, 3, 30, 60 µM) of AMPK inhibitors (C) RO-9568 or (D) RO-3857 that were recorded for 15 min at each concentration, and each trace represents 10 s.”.