Involvement of kallikrein‐PAR2‐proinflammatory pathway in severe trastuzumab‐induced cardiotoxicity

Figures

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  FIGURE 1. Characteristics of selected patients with breast cancer for generation of patient‐specific induced pluripotent stem cell‐derived cardiomyocytes. (A) Flow diagram of patient selection. Twenty‐four of 468 patients with breast cancer (5.1%) who received trastuzumab (Tmab)‐based treatment were diagnosed with cancer therapy‐related cardiac dysfunction (CTRCD). Three patients in each group were selected from the severely cardiotoxic patient group (SP), patients with a reduced left ventricular ejection fraction (LVEF) of at least 30% from baseline. (B) Time series of LVEF in the six patients. Cardiac‐function values within the yellow background indicate that Tmab could not be administered. NP, patients without cardiotoxicity

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  FIGURE 2. Generated patient‐specific induced pluripotent stem cell‐derived cardiomyocytes (pt‐iPSC‐CMs) show a functional cardiomyocyte phenotype. (A) Representative image of normal karyotype of iPSC lines by G‐band analysis. (B) Representative phase‐contrast image. (C) Confirmation of sufficient purification after metabolic purification using CM detector RNA switch. EGFP is expressed in cells other than cardiomyocytes (upper row). (D) Images of immunostaining with DAPI (blue), cardiac troponin‐T (cTnT; green) and α‐actinin (red). (E) Expression of western blot of ErbB2, ErbB4, cTnT, and α‐actinin receptors. MB231 was used as a negative control and SK‐BR‐3 as a positive control for ErbB2. (F) Recording of Ca2+ transients observed with the Ca2+ indicator, Cal520. (G) Electrophysiological activities of spontaneously beating pt‐iPSC‐CMs and enlarged single beat profile using the multielectrode array. (H) Membrane potential recording showing cardiomyocyte‐specific action potentials recorded with the voltage sensitive dye, FluoVolt. F.I., fluorescence intensity

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  FIGURE 3. Contractility analysis of patient‐specific induced pluripotent stem cell‐derived cardiomyocytes (pt‐iPSC‐CMs) following trastuzumab (Tmab) treatment with motion vector analysis. (A) Illustration of a representative motion waveform with associated parameters. (B–E) Relative change in parameters for contractility following Tmab treatment: (B) contraction velocity (CV), (C) relaxation velocity (RV), (D) contraction–relaxation deformation (CRD) distance, and (E) beating rate. Data were obtained using all the lines of pt‐iPSC‐CMs and the assay was repeated three times. NP, noncardiotoxic patient group; SP, severely cardiotoxic patient group

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  FIGURE 4. Quantitative plate‐based assays and mitochondrial stress test of patient‐specific induced pluripotent stem cell‐derived cardiomyocytes (pt‐iPSC‐CMs) following trastuzumab (Tmab) or doxorubicin (DOX) treatment. (A–C) Relative change in each assay with drug treatment. (A) Quantification of ATP content measured using Cell Titer‐Glo assay. (B) Quantification of reative oxygen species (ROS) generation measured using ROS‐Glo H2O2 assay. (C) Autophagy level measured using CYTO‐ID staining. (D) Representative images of immunostaining of CYTO‐ID green. (E) Quantification of cardiac troponin I in the culture medium with ELISA. (F) Amplitudes of peak Ca2+ transients. (G, H) Oxygen consumption rate (OCR) was measured using the Seahorse XF24 extracellular‐flux analyzer. (G) Representative plot of OCR measured following drug treatment. Each evaluation parameter and sequential addition (oligomycin, FCCP, and Rot/Ant‐A) is shown in the graph. (H) Maximal respiration shown with relative OCR rate. (I) Mitochondrial membrane potential was measured using JC‐1 dye. Data were obtained using all lines of pt‐iPSC‐CMs and the assay was repeated three times. NP, noncardiotoxic patient group; ns, not…

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  FIGURE 5. RNA sequencing (RNA‐Seq) of patient‐specific induced pluripotent stem cell‐derived cardiomyocytes (pt‐iPSC‐CMs) and quantitative real‐time RT‐PCR (qRT‐PCR) analysis of genes associated with cardiotoxicity. (A) Schematic illustrating the experimental workflow. RNA‐Seq was undertaken on iPSC‐CMs that were treated for 7 days with or without 1 μM trastuzumab (Tmab). (B–F) Comparisons of various gene expression levels in untreated and Tmab‐treated pt‐iPSC‐CMs using qRT‐PCR analysis (false discovery rate < 0.05 and fold change > 3) showed distinct patterns of gene expression between the severely cardiotoxic patient group (SP; n = 3) versus noncardiotoxic patient group (NP; n = 3) and Tmab‐treated versus untreated. Relative gene expressions of KLK5, KLK8, IL1B, PTGS1, and TNFRSF8 in pt‐iPSC‐CMs are shown. All data are expressed as mean ± SEM

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  FIGURE 6. Kallikrein (KLK) drives inflammation status in the severely cardiotoxic patient group (SP) through activated protease‐activated receptor 2 (PAR2). (A) Proposed KLK‐PAR2‐MAPK and ErbB signaling pathways in the patient‐specific induced pluripotent stem cell‐derived cardiomyocyte (pt‐iPSC‐CM) model. Pathways validated in this study are shown as dotted lines. (B, C) KLK5 and KLK8 levels detected in culture supernatant of pt‐iPSC‐CMs, as measured by ELISA. (D) Representative western blot analysis of PAR2 protein expression. (E) Quantification of PAR2 normalized to GAPDH. (F) PAR2 signaling assessed by Ca2+ transients induced by trypsin, a PAR2 agonist. (G, H) Representative western blot and quantification of p‐ERK1/2 and ERK1/2. (I, J) Representative western blot and quantification of p‐JNK and JNK protein expression. Data were obtained using pt‐iPSC‐CMs from six individuals, assays were repeated three to five times for ELISA and Ca2+ transients and twice for western blots. All data are expressed as mean ± SEM. NRG‐1, neuregulin‐1; SP, severely cardiotoxic patient group

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  FIGURE 7. Inhibition of the kallikrein5 (KLK5)–protease‐activated receptor 2 (PAR2) pathway suppresses inflammation and recombinant KLK5‐mediated contractility toxicity in patient‐specific induced pluripotent stem cell‐derived cardiomyocytes (pt‐iPSC‐CM). (A–F) Trypsin (100 nM) induced Ca2+ release in pt‐iPSC‐CMs. (A, B) Representative Ca2+ signaling change with or without the KLK5 antagonist, ursolic acid (UA). (D, E) Representative Ca2+ signaling change with or without the PAR2 antagonist, I‐191. (C, F) Comparison of the relative area of change in Ca2+ response to trypsin under UA and I‐191 treatment. The curved graph area above the F0 baseline was measured using ImageJ. (G) Interleukin‐1β (IL1β) mRNA expression in the severely cardiotoxic patient group (SP) treated with trastuzumab (Tmab) for 7 days ± I‐191 and FSLLRY‐NH2. (H) Contractility in SP treated with Tmab for 7 days ± I‐191 and FSLLRY‐NH2. (I) Contraction and relaxation velocities in noncardiotoxic patient group (NP) treated with Tmab ± recombinant KLK5 (2 nM, 20 nM). (J) Proposed relationship between KLK5/KLK8‐PAR2‐MAPK and ErbB signaling pathways in the pt‐iPSC‐CM model. (K) Schematic showing the multifactorial molecular…