Human cardiac organoids for the modelling of myocardial infarction and drug cardiotoxicity

Abstract

Environmental factors are the largest contributors to cardiovascular disease. Here we show that cardiac organoids that incorporate an oxygen-diffusion gradient and that are stimulated with the neurotransmitter noradrenaline model the structure of the human heart after myocardial infarction (by mimicking the infarcted, border and remote zones), and recapitulate hallmarks of myocardial infarction (in particular, pathological metabolic shifts, fibrosis and calcium handling) at the transcriptomic, structural and functional levels. We also show that the organoids can model hypoxia-enhanced doxorubicin cardiotoxicity. Human organoids that model diseases with non-genetic pathological factors could help with drug screening and development.

Fig. 1 |. Development of human 3D post-myocardial infarction organoid model.

a, The 3D nature and diffusion limitations in post-myocardial infarction (MI) hearts can be spatially mimicked to create an in vitro post-MI model (infarct organoid). b, c, Finite element modeling and quantification of oxygen diffusion in simulated cardiac microtissues provide a guiding design principle for an in vitro infarction protocol, revealing the inherent oxygen diffusion limitation at 20% and 10% external oxygen. d, Control and cardiac organoid infarction protocol timeline using altered oxygen (O₂) and norepinephrine (NE) in culture for 10 days (D10). e, Bright-field images of organoids on D10 and diameters (mean ± standard deviation) on D0 and D10. n= 252, 215 (D0, D10) organoids in control group; 152, 216 (D0, D10) infarct organoids in test group; 9, 5, 9, 7 wells per D0 control, D0 infarct, D10 control, D10 infarct groups, respectively; 1 donor (Donor A). For box-plots, center line - median; box limits - upper and lower quartiles; whiskers - total range. f, Confocal z-slice images (>30 μm below organoid surface) of hypoxia-activated Image-iT Green Hypoxia live-cell stain on D10 with radial density profile plots of normalized integrated intensities, indicating lower oxygen (brighter) toward interior of infarct organoids compared to control organoids. n= 13 organoids in control group; 15 infarct organoids in test group; 3 wells per group; 1 donor (Donor A). g, Z-stack confocal images (from 18 control and 11 infarct organoid images) of TUNEL apoptosis staining of D10 control and infarct organoid frozen sections showing apoptotic core in infarct organoids. h, NADH autofluorescence from live two-photon imaging (z-slice >30 μm below surface) of live control, infarct, and dead (frozen+thaw) cardiac organoids on D10 (left) and NADH index quantification (right) (mean ± standard deviation) showing lower NADH in center of organoids and overall lower levels in infarct organoids. *p<0.001 using one-way ANOVA with Bonferroni-corrected two-sided t-test post-hoc. n= 10 organoids in control group; 11 infarct organoids in test group; 3 wells per group; 1 donor (Donor A).

Fig. 2 |. Human in vitro 3D post-myocardial infarction (MI) organoids share global gene expression profile with adult human ischemic cardiomyopathy and animal acute post-MI samples.

a, Distribution of gene expression and fold change (FC) (black) with differentially expressed (DE) genes (red) of infarct organoids compared to control organoids after day 10. DE analysis can be found in Methods. b, Comparison of DE (p<0.05) genes from infarct organoids (vs. control organoids) RNA sequencing data compared to human ischemic cardiomyopathy (vs. nonfailing), mouse 1 week post-MI (vs. sham), and pig 1 week post-MI (vs. sham) microarray data. Sample sizes and DE analysis for transcriptomic data can be found in Methods. c, d, Principal component analysis of the 4,765 shared genes between the cardiac organoid samples and mouse 2 week post-MI and human ischemic cardiomyopathy RNA sequencing samples showing the systems-level relevance of cardiac infarct organoids in modeling injured myocardium. n = 3 biologically independent samples for human control and infarct organoids, n = 8 biologically independent human ischemic cardiomyopathy left ventricles and nonfailing left ventricle samples, n = 4 biological independent mouse myocardial infarction left ventricle and sham left ventricle samples.

Fig. 3 |. Cardiac infarct organoids model pathological metabolic responses at the transcriptomic, functional, and tissue level.

a, Significant (p<0.05) gene ontology terms based on differentially expressed (DE) genes between control and infarct organoids on D10 compiled using Advaita Bio’s iPathwayGuide. Organoid samples, n= 3 biologically independent samples per group (each sample containing 1 well of 30-35 organoids), 1 donor (Donor A). DE analysis can be found in Methods. b, Heatmap of DE genes in the “metabolic pathway” (KEGG Pathway map01100) in organoid model showing similar trends in gene expression changes after injury compared to mouse 1 week post-myocardial infarction (MI) microarray data. Scale is row z-score. Organoid samples, n= 3 biologically independent samples per group (each sample containing 1 well of 30-35 organoids), 1 donor (Donor A); mouse, n= 3 biologically independent samples per group. c, e, Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) during Seahorse mitochondrial stress test of D10 control and infarct organoids with d, f, peak OCR after carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) addition and peak ECAR after Oligomycine addition. n= 59 organoids in the control group; 59 infarct organoids in the test group; 3 wells per group; 1 donor (Donor A). Mean ± standard deviation. *p<0.001 using Student’s two-sided t-test. g, Normalized L-lactate levels (based on absorbance readings) relative to control media measured in D10 organoid media. n= 6 wells per group, 1 donor (Donor A). P-value from Student’s two-sided t-test. h, Differences of lactate-related gene expression (ratio of transcripts per million – TPM) from RNA sequencing data between control and infarct organoids on D10. Mean ± standard deviation. *p<0.001 using Student’s two-sided t-test.

Fig. 4 |. Cardiac infarct organoids model pathological fibrosis response at the transcriptomic, cellular, and tissue level.

a, Top identified pathways from organoid RNA sequencing data. Organoid samples, n= 3 biologically independent samples per group (each sample containing 1 well of 30-35 organoids), 1 donor (Donor A). DE analysis can be found in Methods. b, Heatmap of fibrosis-related gene set in organoid model compared to mouse 1 week post-myocardial infarction (MI) microarray data. Scale is row z-score. Organoid samples, n= 3 biologically independent samples per group (each sample containing 1 well of 30-35 organoids), 1 donor (Donor A); mouse, n= 3 biologically independent samples per group. c, Representative fibrosis-related genes (transcripts per million – TPM) from organoid RNA sequencing indicate significant changes in infarct organoids (mean ± standard deviation). *p<0.001 using DESeq2 differential expression analysis of sequencing data. Organoid samples, n= 3 biologically independent samples per group (each sample containing 1 well of 30-35 organoids), 1 donor (Donor A). d, Venn diagram of fibrosis-related genes show similar trends in gene expression changes after injury in infarct organoid and mouse 1 week post-MI microarray data. n= 3 biologically independent samples per group. e, f, Confocal z-stack images of vimentin immunofluorescent staining of control and infarct organoid sections with vimentin radial density profile plots of normalized integrated intensities. n= 15 organoids in the control group; 15 infarct organoids in the test group; 3 wells per group; 1 donor (Donor A). Mean ± standard deviation. Student’s two-sided t-test was used for statistical significance (p-values can be found in Table S5). g, Confocal z-stack images (from 10 images per group) of immunofluorescently stained myofibroblast-like cells in organoid sections indicated by fibrillar structures (white arrows) with alpha smooth muscle actin (αSMA)/F-actin (phalloidin) colocalization that are not seen in control organoids. h, Bright-field images (from 19 control and 21 infarct organoid images) of micropipette aspiration tests. i, Stiffness (i.e., elastic modulus, kPa) calculated using equilibrium deformation displacement (mean ± standard deviation). n= 19 organoids in the control group; 21 infarct organoids in the test group; 3 wells per group; 1 donor (Donor A). P-value from Student’s two-sided t-test.

Fig. 5 |. Tissue-level pathological calcium-handling in cardiac infarct organoids observed with in situ imaging of the interior of live cardiac organoids.

a, b, Calcium handling-related gene set in organoid model showing similar trends in gene expression changes after injury compared to mouse 1 week post-myocardial infarction (MI) microarray data. Scale is row z-score. Organoid samples, n= 3 biologically independent samples per group (each sample containing 1 well of 30-35 organoids), 1 donor (Donor A); mouse, n= 3 biologically independent samples per group. c, Representative calcium-handling genes (transcripts per million – TPM) from organoid RNA sequencing indicating significant change to major calcium handling genes (mean ± standard deviation). *p<0.001 using DESeq2 differential expression analysis of sequencing data. Organoid samples, n= 3 biologically independent samples per group (each sample containing 1 well of 30-35 organoids), 1 donor (Donor A). d, Illustration of customized Two-Photon scanned Light-Sheet Microscope (2PLSM) for in situ imaging of live cardiac organoids. e, 2PLSM imaging (from 10 control and 19 infarct organoid images) from selected imaging planes at >50 μm below organoid surface of D10 control and infarct organoids containing GCaMP6-labeled human induced pluripotent stem cell-derived-cardiomyocytes (hiPSC-CMs) with calcium transient profiles corresponding to the two edge and interior cardiomyocyte regions of interest (ROIs) showing unsynchronization in infarct organoids. f, Quantification of calcium transient amplitude (ΔF/F₀) of separate ROIs representing individual cardiomyocytes from selected imaging planes at >50 μm below organoid surface. n= 32 ROIs across 10 control organoids, 47 edge ROIs and 35 interior ROIs across 19 infarct organoids; 3 wells per group; 1 donor (Donor A). Mean ± standard deviation. *p<0.001 using one-way ANOVA with Bonferroni-corrected two-sided t-test post-hoc. g, Contraction amplitude (fractional area change) of organoids on D10 showing significant decrease in contractile function in infarct organoids. n= 30 organoids in the control group; 30 infarct organoids in the test group; 5 wells per group; 1 donor (Donor A). Mean ± standard deviation. P-value from Student’s two-sided t-test. h, Confocal z-stack images (from 26 control and 27 infarct organoid images) of immunofluorescent staining of organoid sections on D10 showing interconnected alpha sarcomeric actinin (αSA)-positive (green) cardiomyocytes in control organoids and separation of edge and interior cardiomyocytes by vimentin-positive (red) cells in infarct organoids. i, 2PLSM imaging (from 3 control and 4 infarct organoid images) from selected imaging planes at >50 μm below surface of GCaMP6-labeled hiPSC-CM spheroids with calcium transient profiles corresponding to the two edge and interior cardiomyocyte ROIs showing synchronization in infarct hiPSC-CM spheroids.

Fig. 6 |. Human cardiac infarct organoids for tissue-level heart failure drug testing.

a, b, Confocal z-stack images of vimentin immunofluorescent staining of D10 infarct organoid sections with or without “anti-fibrotic” (JQ1, 10 nM) culture conditions and associated vimentin radial density profile plots of normalized integrated intensities. n= 14 organoids in control group; 13 infarct organoids in infarct test group; 12 infarct organoids in JQ1 test group; 3 wells per group; 1 donor (Donor A). Mean ± standard deviation. Student’s two-sided t-test was used for statistical significance (p-values can be found in Table S5). c, Change in elastic modulus relative to control on D10 for cardiac infarction protocol with added “anti-fibrotic” (JQ1, 10 nM) culture conditions. n= 20 infarct organoids in the infarct test group; 17 infarct organoids in the JQ1 test group; 3 wells per group; 1 donor (Donor A). For box-plots, center line - median; box limits - upper and lower quartiles; whiskers - total range. Differences between groups were not significant using Student’s t-test. d, Proportion of organoids (control, infarct, infarct with “anti-fibrotic” treatment (JQ1, 10 nM)) that exhibited synchronized or unsynchronized beating. n= 66 organoids in the control group; 66 infarct organoids in the infarct test group; 72 infarct organoids in the JQ1 test group; 3 wells per group; 1 donor (Donor A). e, 2PLSM imaging (from 6 organoid images) of infarct organoids treated with JQ1 (10 nM) during infarction protocol from selected imaging planes at >50 μm below organoid surface showing synchronization of cardiomyocyte regions of interest.

Fig. 7 |. Detection of tissue-level drug-induced exacerbation of cardiotoxicity using cardiac infarct organoids.

a, Normalized contraction amplitude (relative to vehicle control of each group) with IC₅₀ of organoids in response to doxorubicin (DOX). n= 18, 19, 19, 18, 12, 12, 11 (at 0, 0.1, 0.5, 1.0, 5.0, 10.0, 50.0 μM doses of DOX) organoids in control group; 18, 19, 18, 17, 11, 11, 11 (at 0, 0.1, 0.5, 1.0, 5.0, 10.0, 50.0 μM doses of DOX) infarct organoids in test group; 3 wells per group; 1 donor (Donor A). For box-plots, center line - median; box limits - upper and lower quartiles; whiskers - total range. b, Normalized viability index (relative to vehicle control of each group) based on TUNEL-apoptotic staining of organoid sections after DOX exposure. n= 10, 10, 12, 10 (at 0, 0.1, 1.0, 10.0 μM doses of DOX) organoids in control group; 11, 11, 14, 12 (at 0, 0.1, 1.0, 10.0 μM doses of DOX) infarct organoids in test group; 3 wells per group; 1 donor (Donor A). *p<0.001 using two-way ANOVA with Tukey post-hoc. Mean ± standard deviation. c, Sarcomeric changes caused by increased dose of DOX quantified by radial density profile plots of normalized integrated intensities of alpha sarcomeric (αSA) immunofluorescent staining in organoid sections. n= 9, 8, 10 (at 0, 0.1, 1.0 μM doses of DOX) organoids in control group; 11, 10, 14 (at 0, 0.1, 1.0 μM doses of DOX) infarct organoids in test group; 3 wells per group; 1 donor (Donor A). -- represents p<0.05 for 0.1 μM versus 0 μM DOX; x represents p<0.05 for 1.0 μM versus 0 μM DOX using Student’s two-sided t-test (p-values can be found in Table S5). Mean ± standard deviation. d, Confocal z-stack images (from 17 control and 21 infarct organoid images) of αSA (green) visualizing the larger relative change in interior contractile structures from 0 μM to 0.1 μM DOX in infarct organoids than in control organoids. e, DOX-induced changes in vimentin-covered area (relative to vehicle control of each group) in organoid sections. n= 12, 12, 12, 11 (at 0, 0.1, 1.0, 10.0 μM doses of DOX) organoids in the control group; 12, 12, 15, 7 (at 0, 0.1, 1.0, 10.0 μM doses of DOX) infarct organoids in the test group; 3 wells per group; 1 donor (Donor A). Two-way ANOVA with Tukey post-hoc was used for statistical significance (p-values can be found in Table S7). Mean ± standard deviation. f, Confocal z-stack images (from 24 control and 27 infarct organoid images) of relative differences in vimentin-covered area in control and infarct organoids at 0.1 and 1 μM DOX. All DOX exposure was 48 hrs starting on D10.