Spontaneously beating cardiac spheroids: 3D combined hypertrophy and cardiotoxicity assay

Detect therapeutically relevant pathophysiological hypertrophic cardiotoxicity potential of novel therapeutics using Cyprotex’s 3D combined cardiac hypertrophy and multi-parametric high content screening (HCS) cardiotoxicity assay.

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

Cardiotoxicity assessment using cardiac spheroids

Drug-induced cardiovascular toxicity is the leading cause of attrition during drug development. Drugs can exert functional toxicities such as arrhythmia or morphological (structural) damage including changes 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 only 30% cardiomyocytes, despite this they comprise the majority of the cardiac tissue mass. These terminally differentiated cardiomyocytes can only respond with hypertrophic growth (increased muscle mass) to external stimuli².
Various stimuli are known to induce cardiac hypertrophy including mechanical and oxidative stress as well as neurohormonal perturbation and metabolic hypoxia². Hypertrophy can be physiologically induced or a pathophysiological response to toxicity.
Mitochondrial disruption, calcium dyshomeostasis and cellular ATP content have been previously identified as major targets for structural cardiotoxins³ and are used to indicate pathophysiological hypertrophy.
Three dimensional (3D) high content screening (HCS) allows temporal monitoring of cardiomyocyte spheroid hypertrophy over a 14 day repeat dose period with a terminal measure of mitochondrial function, calcium homeostasis, DNA structure and cellular ATP at day 14.

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.

⁵Nam KH et al., (2015) Biomimetic 3D tissue models for advanced high-throughput drug screening. J Lab Autom 20(3); 201-215

Protocol

Protocol for 3D structural cardiotoxicity and hypertrophy assay

Spheroid	Induced pluripotent stem cell (iPSC) derived cardiomyocytes
Analysis Platform	Brightfield & 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	150 µL of a DMSO* solution to achieve 100x C_max (200x top concentration to maintain 0.5% DMSO) or equivalent amount in solid compound.
Time Points	Spheroid hypertrophy: day 3, 7, 10 & 14* Structural cardiotoxicity HCS & ATP: day 14*
Quality Controls	Negative control: 0.5% DMSO (vehicle)* Positive controls: dasatinib (structural cardiotoxin with pathophysiological hypertrophic potential) and mitomycin C (structural cardiotoxicity without hypertrophic potential)
Data Delivery	Minimum effective concentration (MEC) and AC₅₀ value for each measured parameter; spheroid count and spheroid size (3, 7, 10 & 14 days) and DNA structure (DNA), calcium homeostasis (Ca²⁺) mitochondrial mass (Mito Mass), mitochondrial membrane potential (MMP) and cellular ATP content (ATP) (Day 14)*

*Other options available on request.

Data

Data from Cyprotex's 3D structural cardiotoxicity and hypertrophy assay

HCS images of hypertrophic potential (dasatinib)

Figure 1
Representative 3D confocal high content screening (HCS) images of dasatinib, a known structural cardiotoxin with hypertrophic potential, labelled with Hoechst (Blue) to detect DNA structure, Fluo-4 AM (Green) to detect calcium homeostasis and TMRE (Red) to detect mitochondrial function.

Drug	Human exposure C_max (µM)	In vivo cardiac structural toxicity (P/N)	In vivo cardiac patho-physiological hypertrophy (P/N)	Most senstive structural MEC (µM)	Most sensitive hypertrophy MEC (µM)	Most sensitive combined assay (MEC µM)	Most sensitive feature
sunitinib	0.25	P	P	0.38	0.16	0.16	Calcium
dasatinib	0.72	P	P	0.15	0.02	0.02	ATP
imatinib	3.54	P	P	0.04	0.05	0.04	ATP
doxorubicin	15.34	P	P	0.01	1.46	0.01	ATP
norepinephrine	0.17	P	P	0.10	0.06	0.06	ATP
amphotericin B	9.00	P	P	7.85	0.25	0.25	DNA
lapanitib	4.18	P	P	0.19	32.40	0.19	ATP
clozapine	2.40	P	P	32.40	6.67	6.67	DNA
isoproterenol	0.01	P	P	0.10	26.30	0.10	ATP
cyclophosphamide	153.20	P	P	381.00	NR	381.00	ATP
amiodarone	5.30	P	N	7.76	3.51	3.51	MMP
mitomycin C	3.12	P	N	0.21	NR	0.21	ATP
idarubicin	0.12	P	N	0.004	1.45	0.004	ATP
fluorouracil	4.61	P	N	10.30	NR	10.30	ATP
acyclovir	6.66	N	N	NR	NR	NR	-
buspirone	0.03	N	N	NR	NR	NR	-

	≤ 1x C_max
	≤ 3x C_max
	≤ 10x C_max
	≥ 10x C_max

	Structural toxicity potential	Patho-physiological hypertrophy potential	Cardiac toxicity
Correct prediction with a 10x C_max cut off (%)	94%	81%	100%

Table 1
Combined structural cardiotoxicity and hypertrophic potential prediction of 16 reference compounds categorised according to literature data.

Cardiac spheroids were exposed to test compound for 14 days. During the 14 day period re-dosing occurred on 3 occasions. Spheroid hypertrophy was measured on day 3, 7, 10 and 14 using the brightfield live cellular imaging mode of a Cellomics ArrayScan^® XTI (Thermo Scientific). On day 14 the cell model was analysed by using the confocal mode of Cellomics ArrayScan^® XTI (Thermo Scientific) following incorporation of fluorescent dyes. Cellular ATP content was subsequently measured using CellTiterGlo^® (Promega).

MEC = minimum effective concentration
P = Positive, N = Negative

Raw traces for control DMSO and test compound Verpamil

Figure 2
Graphical representation of (a) hypertrophy and cellular ATP response to dasatinib and (b) hypertrophy and calcium homeostasis response to mitomycin C in cardiac spheroids following 14 day exposure.

Utilizing the 3D cardiac combined assay approach all reference compound toxicities were correctly predicted within a 10x C_max cut off. Structural cardiotoxicity was correctly predicted for 94% and pathophysiological hypertrophy potential (PHP) for 81% of the compound set within a 10x C_max cut off.

The combination of an in vitro 3D model that better recapitulates the in vivo cellular physiology of cardiac tissue with multiparametric temporal HCS and a cytotoxicity assay presents a viable screening strategy for the accurate in vivo relevant detection of novel therapeutics that cause structural cardiotoxicity with pathophysiological hypertrophy potential early in drug development.

Spontaneously beating 3D cardiac microtissue.

References

¹ Laverty H et al., (2011). How can we improve our understanding of cardiovascular safety liabilities to develop safer medicines? Br J Pharmacol 163(4), 675-693
² Brutsaert DL (2003). Cardiac endothelial-myocardial signaling: its role in cardiac growth, contractile performance, and rhythmicity. Phys Rev 83(1), 59-11
³ Pointon A et al., (2013) Phenotypic profiling of structural cardiotoxins in vitro reveals dependency on multiple mechanisms of toxicity. Toxicol Sci 132(2), 317-326
⁴ Cross MJ et al., (2015) Physiological, pharmacological and toxicological considerations of drug-induced structural cardiac injury. Br J Pharmacol 172(4), 957-974
⁵ Nam KH et al., (2015) Biomimetic 3D tissue models for advanced high-throughput drug screening. J Lab Autom 20(3); 201-215