Cardiac Non-myocyte Cells Show Enhanced Pharmacological Function Suggestive of Contractile Maturity in Stem Cell Derived Cardiomyocyte Microtissues

Abstract

The immature phenotype of stem cell derived cardiomyocytes is a significant barrier to their use in translational medicine and pre-clinical in vitro drug toxicity and pharmacological analysis. Here we have assessed the contribution of non-myocyte cells on the contractile function of co-cultured human embryonic stem cell derived cardiomyocytes (hESC-CMs) in spheroid microtissue format. Microtissues were formed using a scaffold free 96-well cell suspension method from hESC-CM cultured alone (CM microtissues) or in combination with human primary cardiac microvascular endothelial cells and cardiac fibroblasts (CMEF microtissues). Contractility was characterized with fluorescence and video-based edge detection. CMEF microtissues displayed greater Ca(2+ )transient amplitudes, enhanced spontaneous contraction rate and remarkably enhanced contractile function in response to both positive and negative inotropic drugs, suggesting a more mature contractile phenotype than CM microtissues. In addition, for several drugs the enhanced contractile response was not apparent when endothelial cell or fibroblasts from a non-cardiac tissue were used as the ancillary cells. Further evidence of maturity for CMEF microtissues was shown with increased expression of genes that encode proteins critical in cardiac Ca(2+ )handling (S100A1), sarcomere assembly (telethonin/TCAP) and β-adrenergic receptor signalling. Our data shows that compared with single cell-type cardiomyocyte in vitro models, CMEF microtissues are superior at predicting the inotropic effects of drugs, demonstrating the critical contribution of cardiac non-myocyte cells in mediating functional cardiotoxicity.

Keywords: S100A1; cardiac microtissue; cardiotoxicity; contractility; hESC-CMs; inotropy.

FIG. 1

Structural characterization of CMEF microtissues. A, DIC image of a representative CMEF microtissue. Immunofluorescent staining of CMEF microtissues for (B) endothelial marker, CD31 (green); (C) fibroblast marker, collagen I (COL1A1) (red); (D) cardiac striation marker, ACTN2 (white); (E) nuclei stain, hoechst (blue) and (F) Merge of CD31 (green), COL1A1 (red), ACTN2 (white) and hoechst (blue). Scale bar represents 50 μm.

FIG. 2

Structural characterization of CM microtissues. A, DIC image of a representative CM microtissue. Immunofluorescent staining of CM microtissues for (B) endothelial marker, CD31 (green); (C) fibroblast marker, collagen I (COL1A1) (red); (D) cardiac striation marker, ACTN2 (white); (E) nuclei stain, hoechst (blue) and (F) Merge of CD31 (green), COL1A1 (red), ACTN2 (white) and hoechst (blue). Scale bar represents 50 μm.

FIG. 3

Functional characterization of CMEF and CM microtissues. Contractile properties (edge) and Ca²⁺kinetics were assessed using a video-based edge detection method (IonOptix, Dublin, Ireland). Microtissues were formed and incubated for 14 days prior to measurment of the spontaneous beat rate in both CMEF (A) and CM microtissues (B) at the start (day 14) and end (day 28) of the experimental period. Plates were incubated at 37 °C, 5% CO₂ for 14 days with media refreshed every 3–4 days. Experiments were conducted on microtissues following 14–28 days in culture. Representative trace showing contraction (edge) kinetics in spontaneous contracting CMEF and CM microtissues (C), the number of contractions over a period of time were quantified (D) (n > 3, mean ± SD). Representative recordings of CMEF (E) and CM microtissue (F) contractions following field stimulation at 0.5, 1, 2, and 3 Hz. Representative traces following measurement of Ca²⁺(red trace) and contraction transients (edge) (blue trace) in (G) CMEF and (H) CM microtissues following field stimulation at 0.5, 1, 2, 3 Hz with 7V for 6 ms. Summary of the frequency-response relationship for contraction (edge) (I) and [Ca²⁺]_i (J) at 0.5, 1, 2, and 3 Hz, n = 3, (±) SD in both CMEF (open bars) and CM microtissues (solid bars) .A representative experiment showing the effect of 10 mM caffeine on contraction transients elicited at 1.5 Hz stimulation in CMEF (K) and CM (L) microtissues.20 s of vehicle control (0.1% DMSO) contraction transients (left side) and >10 s of 10mM caffeine contraction transients (right side) are shown. Representative contractility transients elicited at 1.5 Hz stimulation in CMEF (M) and CM (N) microtissues following 10 μM verapamil. 20 s of basal contraction transients (left side) and 20 s of 10 μM verapamil contraction transients (right side) are shown. . Representative contractility transients elicited at 1.5 Hz stimulation in CMEF (O) and CM (P) microtissues following 10 μM dobutamine. 20 s of basal contraction transients (left side) and 20s of 10 μM dobutamine contraction transients (right side) are shown. *P-value < .01. (Full color version available online.)

FIG. 4

The contractile response of cardiac microtissue models to known inotropic compounds. Concentration-effect curves of CMEF, CM, DMEF, CME, and CMF microtissues to (A) atenolol, (B) lapatinib, (C) dobutamine, and (D) digoxin. E, concentration-effect curves of microtissues containing hiPS-CMs alone or in combination with hCMECs and hCF response to dobutamine. Data expressed relative to vehicle (0.1% (v/v) DMSO) (n = 3 ± SD). *P-value < .05.

FIG. 5

Gene expression upregulation in cardiac microtissues. Fold change of (A) S100A1, (B) TCAP, (C) PDE3A, (D) NOS3, (E) ADRB1, (F) KCND3, (G) MYH6, and (H) MYH7 mRNA expression in foetal heart tissue, cardiac microtissues, and individual cardiac cells relative to adult heart tissue (n = 3, ± SD) was determined by qRT-PCR. *P-value < .01, **P-value < .05.

FIG. 6

S100A1 regulates contraction in response to caffeine. CMEF microtissues were formed and incubated for 14 days prior to transfection with 10 nM nonsilencing siRNA (−ve control) and 10 nM S100A1 siRNA. A, RNA was extracted from microtissues and expression of GAPDH and S100A1 mRNA analysed by qRT-PCR (n = 3, ± SD). Contractile response recorded using the IonOptix cell geometry system under vehicle control (0.1% v/v DMSO) and 10 mM caffeine in (B) nonsilencing siRNA control CMEF microtissue and (C) S100A1 siRNA transfected CMEF microtissue (results from 1 experiment representative of 3 separate experiments).