Automated analysis of contractile force and Ca2+ transients in engineered heart tissue

Abstract

Contraction and relaxation are fundamental aspects of cardiomyocyte functional biology. They reflect the response of the contractile machinery to the systolic increase and diastolic decrease of the cytoplasmic Ca(2+) concentration. The analysis of contractile function and Ca(2+) transients is therefore important to discriminate between myofilament responsiveness and changes in Ca(2+) homeostasis. This article describes an automated technology to perform sequential analysis of contractile force and Ca(2+) transients in up to 11 strip-format, fibrin-based rat, mouse, and human fura-2-loaded engineered heart tissues (EHTs) under perfusion and electrical stimulation. Measurements in EHTs under increasing concentrations of extracellular Ca(2+) and responses to isoprenaline and carbachol demonstrate that EHTs recapitulate basic principles of heart tissue functional biology. Ca(2+) concentration-response curves in rat, mouse, and human EHTs indicated different maximal twitch forces (0.22, 0.05, and 0.08 mN in rat, mouse, and human, respectively; P < 0.001) and different sensitivity to external Ca(2+) (EC50: 0.15, 0.39, and 1.05 mM Ca(2+) in rat, mouse, and human, respectively; P < 0.001) in the three groups. In contrast, no difference in myofilament Ca(2+) sensitivity was detected between skinned rat and human EHTs, suggesting that the difference in sensitivity to external Ca(2+) concentration is due to changes in Ca(2+) handling proteins. Finally, this study confirms that fura-2 has Ca(2+) buffering effects and is thereby changing the force response to extracellular Ca(2+).

Keywords: Ca2+ transient; cardiac tissue engineering; contractile analysis; hiPSC.

Figures

Fig. 1.

Schematic depiction of the novel-microscope based set-up for sequential contractile force and F340-to-F380 ratio measurements under electrical stimulation and continuous perfusion. A: carbogen-bubbled Tyrode's solution for baseline recordings and experimental conditions in glass reservoirs. B: acrylic glass rack equipped with stainless steel perfusion tubing and platinum iridium electrode pair on top of the silicone rack containing fura-2-loaded engineered heart tissues (EHT) in a 24-well plate with glass bottom. Peristaltic pump (C; 1 to 2 ml·well−1·min−1), heat block, and electrical pacer (D; 2–6 Hz, species specific, 4-ms pulse duration, 7 V) are shown. E: temperature and CO2-controlled incubation chamber with automated microscope table, 24-well plate holder, and 24-well plate. F: customized software to analyze contractile force and F340-to-F380 ratio sequentially. Fluorescence light source (hyperswitch; IonOptix) containing a xenon lamp (G), the photomultiplier tube (PMT; IonOptix; H), and the fluorescence system interface (FSI; IonOptix; I) are used for F340-to-F380 ratio measurements. Black lines illustrate electrical interconnection.

Fig. 2.

Data analysis. A contour recognition mode automatically recognizes both ends of the EHTs and follows them over time (A, top). The pink squares represent the contour recognition mode of the CTMV software, which automatically identifies both ends of EHT despite variable geometry. The red lines (marked with an orange arrow) indicate correct recognition of the silicone post, which is important for force measurement. Deflection, elastic property, and geometry of the silicone posts are used to calculate contractile force, which is depicted over time (B). A camera in the optical path of the PMT is used before the start of the measurement to define a rectangle area in the center of the EHT (A, bottom). Fluorescence light emission of this area is integrated by the PMT and converted into an electrical signal. The time flow of these signals and excitations at 340 nm (numerator) and 380 nm (denominator) are integrated (Fluorescence System Interface) and signal emission intensities at both excitation wavelengths are displayed as single graphs over time (C). Force and F340-to-F380 ratio are measured sequentially with 2 different objectives automatically. Electrical pacing signals are integrated as blue vertical lines. Baseline for analysis are the tips of contraction, and F340-to-F380 ratio peaks based on predefined criteria. Green squares illustrate these data points and serve as quality control. The following parameters are calculated: absolute force (in mN), contraction and relaxation times (T1 and T2 in s), F340-to-F380 ratio (in arbitrary units), and times to Ca2+ peak and Ca2+ return (in s). Average peaks of rat, mouse, and human EHTs can be generated for force and Ca2+ transients and their temporal progression can be illustrated (D). Data are means ± SE.

Fig. 3.

Ca2+ concentration response curves were performed with rat, mouse, and human EHTs under electrical stimulation (rat: 4 Hz; mouse: 6 Hz; human: 2 Hz). A: absolute peak forces at different external Ca2+ concentrations ranging from 0.1 to 1.8 mM [rat (n = 12), mouse (n = 12), and human (n = 8)]. B: same data as A, expressed as percentage of maximal peak force and calculated values for the EC50 for Ca2+ and the Hill Slopes. C: average peaks were generated from 20 to 40 peaks of EHTs for each external Ca2+ concentration. D: contraction time T1 and relaxation time T2 (E), both measured at 20% of the peak height. Data are means ± SE. P < 0.0001 different curve for each data set (Sum-of-squares F-test).

Fig. 4.

Effects of isoprenaline, isoprenaline plus carbachol, and verapamil in rat, mouse, and human EHTs. A: isoprenaline (100 nM)- and carbachol (10 μM)-induced changes in relative force. B: effect of verapamil (100–1,000 nM) on relative force. A and B, expressed as percentage of maximal peak force of each EHT of rat (n = 7 to 8), mouse (n = 4–8), and human (n = 4–8). Contraction time T1 (C) and relaxation time T2 (D) in the presence of isoprenaline (100 nM) and isoprenaline plus carbachol (10 μM) are shown. Kinetics were measured at 20% of the peak height. Data are means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. basal condition (1-way ANOVA plus Dunnett post tests); ##P < 0.01 and ###P < 0.001 vs. rat (2-way ANOVA plus Bonferroni post tests).

Fig. 5.

Force and F340-to-F380 ratio were measured sequentially in rat EHTs in the presence of increasing external Ca2+ concentrations. A: Average F340-to-F380 ratio in the presence of 2, 5, or 10 μM fura-2 (1.8 mM external Ca2+). Average F340-to-F380 ratio and force in the presence of 5 μM fura-2 and 1.8 mM (B) or 0.1 mM (C) external Ca2+. D: peak forces at different external Ca2+ concentrations in the presence of 2, 5, or 10 μM fura-2 were expressed as percentage of maximal peak force of each EHT. E: F340-to-F380 ratios at different external Ca2+ concentrations were expressed as percentage of maximal F340-to-F380 ratio of each EHT. F: correlation between contraction time T1 and relaxation time T2 to external Ca2+ concentrations. G: correlation between time to Ca2+ peak (TT Ca2+ peak) and time to Ca2+ return (TT Ca2+ return) to external Ca2+ concentrations were measured at 10% of the Ca2+ peak height. Number of EHTs was n = 6–8. Data are means ± SE. P < 0.0001 different curve for each data set (Sum-of-squares F-test).

Fig. 6.

Force and F340-to-F380 ratio were measured sequentially in rat EHTs in the presence of isoprenaline and isoprenaline plus carbachol. A: average peak forces and F340-to-F380 ratio of EHTs loaded with 10 μM fura-2 under basal conditions or 100 nM isoprenaline. B: correlation between peak force and F340-to-F380 ratio. Absolute values for F340-to-F380 ratio (C) and force (D) are shown. Contraction time T1 (E) and relaxation time T2 (F) were measured at 80% of the peak height. Time to Ca2+ peak (TT Ca2+ peak; G) and time to Ca2+ return (TT Ca2+ return; H) in EHTs were measured at 10% of the Ca2+ peak height. Number of EHTs is indicated in the panels. All measurements were performed at 0.3 mM external Ca2+. Data are means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. basal condition (1-way ANOVA plus Dunnett post tests).

Fig. 7.

Force in skinned rat and human EHT strips was measured in the presence of increasing external Ca2+ concentrations using a permeabilized fiber test system. EHT strips were mounted between 2 T-clips and attached to a force transducer and a length controller. A: EHT strips were relaxed in pCa 9 buffer and exposed to solutions of increasing Ca2+ from pCa 9 to pCa 4.5, and absolute force was measured. B: force at different Ca2+ concentrations is expressed as relative value of the maximal absolute force of each EHT strip. Data were analyzed using the Hill equation with pCa50 as the free Ca2+ concentration, which yields 50% of the maximal force and the Hill coefficient (in nH) (n = 13 per group). Data are means ± SE. *P < 0.05 vs. human (2-way ANOVA plus Bonferroni post tests).