Assessment of Cardiomyocyte Mechanics and Calcium Signaling

Mechanical properties of cardiomyocytes were assessed using a Softedge MyoCam system (IonOptix Corporation, Milton, MA, USA) equipped with an IX-70 Olympus inverted microscope. Cardiomyocytes were electrically stimulated at 0.5 Hz in a contractile buffer containing NaCl 135 mM, KCl 4.0 mM, CaCl2 1.0 mM, MgCl 1.0 mM, glucose 10 mM and HEPES 10 mM. Cell shortening was assessed including peak shortening (PS), maximal velocity of shortening (+dL/dt), maximal velocity of re-lengthening (−dL/dt), time-to-PS (TPS), and time-to-90% re-lengthening (TR₉₀). For intracellular Ca²⁺ recording, cardiomyocytes were loaded with Fura-2/AM (0.5 μM) for 10 min, and fluorescence measurements were recorded with a dual-excitation fluorescence photomultiplier system (IonOptix). To assess intracellular Ca²⁺ signaling, cells were exposed to light emitted by a 75-W lamp and passed through 360 nm or a 380 nm filter, while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm and the alterations in fura-2 fluorescence intensity (FFI) were quantitated from the FFI ratio at 360 nm to 380 nm. Fluorescence decay time was assessed as an indicator of intracellular Ca²⁺ clearing [57 (link), 61 (link)].