Heart slice culture system reliably demonstrates clinical drug-related cardiotoxicity

Abstract

The limited availability of human heart tissue and its complex cell composition are major limiting factors for the reliable testing of drug efficacy and toxicity. Recently, we developed functional human and pig heart slice biomimetic culture systems that preserve the viability and functionality of 300 μm heart slices for up to 6 days. Here, we tested the reliability of this culture system for testing the cardiotoxicity of anti-cancer drugs. We tested three anti-cancer drugs (doxorubicin, trastuzumab, and sunitinib) with known different mechanisms of cardiotoxicity at three concentrations and assessed the effect of these drugs on heart slice viability, structure, function and gene expression. Slices incubated with any of these drugs for 48 h showed diminished in viability as well as loss of cardiomyocyte structure and function. Mechanistically, RNA sequencing of doxorubicin-treated tissues demonstrated a significant downregulation of cardiac genes and upregulation of oxidative stress responses. Trastuzumab treatment downregulated cardiac muscle contraction-related genes consistent with its clinically known effect on cardiomyocytes. Interestingly, sunitinib treatment resulted in significant downregulation of angiogenesis-related genes, in line with its mechanism of action. Similar to hiPS-derived-cardiomyocytes, heart slices recapitulated the expected toxicity of doxorubicin and trastuzumab, however, slices were superior in detecting sunitinib cardiotoxicity and mechanism in the clinically relevant concentration range of 0.1-1 μM. These results indicate that heart slice culture models have the potential to become a reliable platform for testing and elucidating mechanisms of drug cardiotoxicity.

Figure 1.. Pro-apoptotic effect of doxorubicin and sunitinib on hiPSC-CMs:

Kinetics of caspase-3/7 activation in hiPSC-CMs exposed to a range of concentrations of (a) aspirin (non-toxic drug), (b) doxorubicin (cardiotoxin), (c) erlotinib (non-cardiotoxic TKI) and (d) sunitinib (cardiotoxic TKI) over 48 h. DMSO in the medium at a concentration of 0.1% (v/v) was used as a control. (a-d) Variations in total caspase integrated intensity was calculated from fluorescent images of the caspase-3/7 green dye acquired every hour, which captured the extent of apoptosis within cultured cells. Acute apoptotic effects were detected upon exposing cells to 60 μM of sunitinib as noted by the arrow in (d). (e and f) Phase microscopy images of cells acquired when 60 μM of sunitinib was added to cells (e) and one hour after being exposure (f), where total cell detachment was observed. Scale bar: 200 μm.

Figure 2.. Effect of cardiotoxins on heart slice viability and structure:

(a) Bar graph shows the quantification of heart slice viability after 2 days of treatment with the corresponding cardiotoxin using MTT assay (n=2 independent experiments, 4 replicates in each, One-Way ANOVA test was conducted to compare between groups; *P<0.05 compared to the control). (b) Representative immunofluorescence images showing the expression of connexin 43 (red) and Troponin T (green) in cross sections taken from heart tissue slices treated for 2 days with the corresponding concentration of the cardiotoxins (Scale bar, 100 μm). These representative images have been reproducible over 2 independent experiments with 3 technical replicates in each experiment; however, the lack of reliable tools for quantifying localization of connexin 43 and sarcomeric integrity of troponin T has limited our ability for quantification.

Figure 3.. Effect of cardiotoxins on heart slices functionality and calcium homeostasis and contractile function:

(a) Representative calcium traces from day 2 cultured control slices and heart slices treated with the corresponding concentration of each cardiotoxin for 2 days. Transients were recorded after loading the heart slices with Fluo-4 calcium dye and using 1 Hz/20 V electrical stimulation at the time of recording. (b) Scoring of calcium transient amplitude as indication of the cardiomyocyte function from slices treated with each cardiotoxin (n=36 cells in each group from 2 independent experiments). (c) Left panel, Representative traces of impedance recording for contractile function from slices treated with the low dosages of the three cardiotoxins compared to the control slice. Right panel, shows the quantification of the contractile force amplitude comparing control slices to the ones treated with the cardiotoxins (n=6 slices, *p<0.05 compared to control).

Figure 4.. Differential gene expression in slices treated with 100 nM doxorubicin:

(a) Volcano plot showing significant changes in gene expression in 100nM doxorubicin (Dox) treated tissue. Bar graph shows the GO terms for the significantly downregulated (b) or upregulated (C) genes from RNA-seq data between control heart slices and heart slices treated with 100nM doxorubicin (n=2 pig hearts).

Figure 5.. Differential gene expression in slices treated with 1μg trastuzumab:

(a) Volcano plot demonstrating the genes which are significantly different between control heart slice and slices treated with 1μg trastuzumab. Bar graph shows the GO terms for the significantly downregulated (b) or upregulated (c) genes from RNA-seq data between control heart slices and heart slices treated with 1μg trastuzumab (n=2 pig hearts).

Figure 6.. Differential gene expression in slices treated with 100nM sunitinib:

(a) Volcano plot demonstrating the genes which are significantly different between control heart slice and slices treated with 100nM sunitinib. Bar graph shows the GO terms for the significantly downregulated (b) or upregulated (c) genes from RNA-seq data between control heart slices and heart slices treated with 100nM sunitinib (n=2 pig hearts).

Figure 7.. Doxorubicin toxicity in human heart slices:

(a) Age, sex and cause of death for all donated human hearts used in the current study to demonstrate the breadth of variability of the human subjects used in the study. (b) Activation maps obtained by optical mapping of human cardiac organotypic slices cultured for ~24 h with or without doxorubicin (50 μM). Crowding of activation lines in the transverse direction indicate conduction slowing with doxorubicin treatment. (c) Average transverse conduction velocity determined from human cardiac slices with and without doxorubicin treatment. Transverse conduction velocity was significantly slower in doxorubicin treated slices (n=7, *p<0.05). (d) Differentially expressed genes between control and doxorubicin-treated human cardiac organotypic slices following Cap Analysis of Gene Expression (n=3). (e) Gene ontology (GO) enrichment analysis of differentially expressed genes in control and doxorubicin-treated slices. BP: biological process, CC: cellular component, MF: molecular function (n=3). (f) Heat map demonstrating hierarchical clustering of differentially expressed genes (n=3). F: female, M: male.