Spontaneously beating cardiac microtissues: 3D structural cardiovascular toxicity assay

Detect structural cardiotoxicity of novel therapeutics using Cyprotex’s spontaneously beating tri-cultured cardiac 3D microtissue high content screening (HCS) assay.

Cyprotex deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

Cardiac Assessment using 3D Microtissues

Drug induced cardiovascular toxicity is the leading cause of attrition during drug development. Drugs can exert functional toxicities such as arrhythmia and morphological (structural) damage to the myocardium¹. Evaluation of the potential for both types of cardiotoxicity by novel compounds is essential for the discovery of safe drugs.
The myocardial tissue comprises 30% cardiomyocytes and 70% non-myocytes, the majority of which are endothelial and fibroblast cells. These non-myocytes are essential to myocardial structure and function^2,3 with emerging evidence suggesting important roles within drug induced cardiovascular toxicity⁴.
Mitochondrial disruption, calcium dyshomeostasis and cellular ATP content have been identified as major targets for structural cardiotoxins⁵.
Three dimensional (3D) confocal HCS allows the simultaneous detection of each cell health parameter in combination with a measure of cellular ATP.

Numerous studies have shown that cell responses to drugs in 3D culture are improved from those in 2D, with respect to modeling in vivo tissue functionality, which highlights the advantages of using 3D-based models for preclinical drug screens.

⁶Nam KH, Smith AS, Lone S, Kwon S and Kim DH (2015) Biomimetic 3D Tissue Models for Advanced High-Throughput Drug Screening. J Lab Autom; In press

Protocol

3D Microtissue based cardiotoxicity assay protocol

Microtissue	Human induced pluripotent stem cell derived cardiomyocytes (iPSC-CM’s), cardiac endothelial cells and cardiac fibroblasts
Analysis Platform	Confocal Cellomics ArrayScan^® XTI (Thermo Scientific)
Test Article Concentration	8 point dose response curve with top concentration based on 100x C_max or solubility limit 3 replicates per concentration
Test Article Requirements	50 µL of a DMSO solution at a concentration of 200x top concentration (top concentration = 100x C_max) or equivalent amount in solid compound
Time Points	72 hours (others available on request)
Quality Controls	Negative control: 0.5% DMSO (vehicle) Positive controls: Sunitinib (Ca²⁺ homeostasis) and dasatinib (mitochondrial membrane potential)
Data Delivery	Minimum effective concentration (MEC) and AC₅₀ value for each measured parameter (microtissue count, microtissue size, DNA structure, calcium homeostasis (Ca²⁺), mitochondrial mass (Mito Mass), mitochondrial membrane potential (MMP) and cellular ATP content)

Data

Data from Cyprotex's 3D structural cardiovascular toxicity assay

Figure 1
Representative 3D confocal high content screening (HCS) images of isoproterenol calcium dyshomeostasis in spontaneously beating cardiac 3D microtissues labelled with Hoechst (blue) to detect DNA structure, Fluo-4 AM (green) to detect calcium dyshomeostasis and TMRE (red) to detect mitochondrial disruption.

Compound	C_max (µM)	In vivo toxicity	hESC-CM prediction (Pointon et al, 2013)	H9c2 monolayer	H9c2 MTs	Tri-culture cardiac MTs	Most Sensitive Feature
Compound	C_max (µM)	In vivo toxicity	hESC-CM prediction (Pointon et al, 2013)	MEC (µM)			Most Sensitive Feature
Dasatinib⁷	0.72	Structural cardiotoxin	Positive structural cardiotoxin	0.529	NR	2.08	MMP
Doxorubicin HCl⁸	15.34			0.04	0.115	0.04	ATP
Fluorouracil⁹	4.61			1.88	NR	0.0407	Ca²⁺
Idarubicin HCl¹⁰	0.12			<0.04	<0.04	<0.04	ATP
Imatinib Mesylate¹¹	3.54			13.7	3.53	22.6	ATP
Lapatinib⁷	4.18			4.57	8.33	5.9	ATP
Sunitinib Malate¹²	0.25			0.896	0.114	0.817	Ca²⁺
Cyclophosphamide¹³	153.20		Negative structural cardiotoxins	NR	NR	30.8	Mito Mass
Isoproterenol HCl¹⁴	0.01			NR	NR	2.1	Ca²⁺
Acyclovir	6.66	Non-structural cardiotoxins		NR	NR	NR	-
Buspirone HCl	0.03	Non-structural cardiotoxins		NR	0.237	NR	Microtissue size

Table 1
Structural cardiovascular toxicity prediction of 12 reference compounds categorised according to literature data.

Tri-culture cardiac 3D microtissues (MTs), H9c2 3D microtissues (MTs) and H9c2 monolayers were incubated with test compound for 72 hours. The cell models were analysed using the confocal mode of Cellomics ArrayScan^® XTI (Thermo Scientific) following which cellular ATP content was measured using CellTiterGlo^® (Promega). MEC = minimum effective concentration.

MEC = minimum effective concentration
NR = no response

Figure 2
Graphical representation of (a) sunitinib induced calcium dyshomeostasis and (b) dasatinib induced mitochondrial membrane potential disruption in spontaneously beating cardiac 3D microtissues.

All reference compound toxicities were correctly predicted in the spontaneously beating cardiac tri-culture 3D microtissue model including isoproterenol (MEC 2.1 µM, calcium dyshomeostasis (Table 1 and Figure 2)) and cyclophosphamide (MEC 30.8 µM, mitochondrial mass (Table 1)) which previously went undetected by Pointon et al (2013)⁵ and Cyprotex’s in-house H9c2 data.

Control compound sunitinib displays cytosolic calcium increase (calcium dyshomeostasis) followed by gross cytotoxicity (microtissue loss) (Figure 2a) while control compound dasatinib displays mitochondrial membrane potential loss without gross cytotoxicity (microtissue loss) (Figure 2b). The combination of an in vitro 3D model that better recapitulates the in vivo cellular physiology of the myocardium with a multiparametric HCS and cytotoxicity assay presents a viable screening strategy for the accurate detection of novel therapeutics that cause drug induced structural cardiovascular toxicity early in drug development.

Spontaneously beating 3D cardiac microtissue.

References

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² Brutsaert DL (2003). Cardiac endothelial-myocardial signaling: Its role in cardiac growth, contractile performance, and rhythmicity. Phys Revs 83; 59-115.
³Souders CA et al., (2009). Cardiac fibroblast:the renaissance cell. Circ Res 105; 1164-1176.
⁴ Mikaelian I et al., (2010). Primary endothelial damage is the mechanism of cardiotoxicity of tubulin-binding drugs. Tox Sci 117(1); 144-151
⁵ Pointon A et al., (2013) Phenotypic profiling of structural cardiotoxins in vitro reveals dependency on multiple mechanisms of toxicity. Tox Sci 132(2); 317-326
⁶ Nam KH et al., (2015) Biomimetic 3D tissue models for advanced high-throughput drug screening. J Lab Autom; In press
⁷ Force T et al., (2007). Molecular mechanisms of cardiotoxicity of tyrosine kinase inhibition. Nat Rev Cancer 7; 332-344
⁸Minotti G et al., (2004). Doxorubicin cardiotoxicity and the control of iron metabolism: quinone-dependent and independent mechanisms. Methods Enzymol 378; 340–361
⁹ Schimmel KJ et al., (2004). Cardiotoxicity of cytotoxic drugs. Cancer Treat Rev 30(2); 181–191
¹⁰Anderlini P et al., (1995). Idarubicin cardiotoxicity: a retrospective study in acute myeloid leukemia and myelodysplasia. J Clin Oncol 13(11); 2827-2834
¹¹Kerkelä R et al., (2006). Cardiotoxicity of the cancer therapeutic agent imatinib mesylate. Nat Med 12(8); 908-916
¹² Chu TF et al., (2007). Cardiotoxicity associated with tyrosine kinase inhibitor sunitinib. Lancet 370; 2011-2019
¹³ Floyd JD et al., (2005). Cardiotoxicity of cancer therapy. J Clin Oncol 23(30); 7685-7696
¹⁴ Zhang J et al., (2008). Isoproterenol-induced cardiotoxicity in Sprague-Dawley rats: correlation of reversible and irreversible myocardial injury with release of cardiac troponin T and roles of iNOS in myocardial injury. Toxicol Pathol 36(2); 277-278