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

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 Ca²⁺ concentration. The analysis of contractile function and Ca²⁺ transients is therefore important to discriminate between myofilament responsiveness and changes in Ca²⁺ homeostasis. This article describes an automated technology to perform sequential analysis of contractile force and Ca²⁺ 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 Ca²⁺ and responses to isoprenaline and carbachol demonstrate that EHTs recapitulate basic principles of heart tissue functional biology. Ca²⁺ 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 Ca²⁺ (EC₅₀: 0.15, 0.39, and 1.05 mM Ca²⁺ in rat, mouse, and human, respectively; P < 0.001) in the three groups. In contrast, no difference in myofilament Ca²⁺ sensitivity was detected between skinned rat and human EHTs, suggesting that the difference in sensitivity to external Ca²⁺ concentration is due to changes in Ca²⁺ handling proteins. Finally, this study confirms that fura-2 has Ca²⁺ buffering effects and is thereby changing the force response to extracellular Ca²⁺.