Myopacer field stimulator

Adult cardiomyocytes were loaded with Fura-2-AM (1.0 μM, 20 mins), washed twice, and continuously perfused with Tyrode solution maintained at 37°C. Simultaneous measurement of intracellular Ca²⁺ ([Ca²⁺]_i) and cell contractility was assessed by using a video-based edge-detection system (IonOptix, Milton, MA). Briefly, cardiomyocytes were field stimulated at a frequency of 0.5 Hz using a pair of platinum wires placed on the opposite sides of the dish chamber and connected to a MyoPacer Field Stimulator (IonOptix). The cardiomyocytes being studied were displayed on the computer monitor using an IonOptix MyoCam camera. 15–25 individual myocytes were recorded and analyzed for each heart. The ratio of Fura-2 fluorescence at 340 nm and 380 nm (R) was calculated and the amplitude of intracellular Ca²⁺ transient was determined by the change between the basal and peak ratio (ΔR). The amplitude of cell contraction was assessed by % change of peak shortening, and the rate of cell relaxation was assessed by the time to 63% re-lengthening (Tau).

Live CM were imaged post-isolation using a Leitz Laborlux 11 microscope (Leica Microsystems, Vienna, Austria) with a 2.5× air objective and an eyepiece camera (MikrOkular, Bresser, Rhede, Germany). To assess the yield of rod-shaped CM, we counted rod-shaped and dead (rounded) CM in at least three fields of view per isolation (60–500 CM manually counted per field of view). To assess contractile function, we paced CM in a perfusion chamber equipped with field stimulation electrodes (RC-27N, Warner Instruments, Hamden, MA, USA), driven by a MyoPacer field stimulator (applied voltage was twice the threshold and maximally 12 V, pulse duration 5 ms; pacing frequency was 0.5 Hz for ventricles and 1 Hz for atria; IonOptix, Westwood, MA, USA). Fractions of rod-shaped and contracting CM (i.e., responding to field stimulation without arrhythmic events, e.g., pauses, extra contractions) are expressed as percent of all CM in a field of view.

Adult mouse ventricular myocytes were isolated following a previously described protocol (Liao and Jain, 2007 (link)). Intracellular Ca²⁺ and contractility were measured at 37°C in isolated myocytes loaded with Fura 2-AM in the absence or presence of isoproterenol (ISO) (1 μmol/L) using an IonOptix system (Milton, MA), as previously described (Guillory et al., 2013 (link)). Myocytes were paced at 1 Hz with IonOptix MyoPacer Field stimulator (pulse duration 1 ms; 5 volts). Data were analyzed using IonWizard (IonOptix) software.

Intracellular Ca²⁺ transients were studied by using the IonOptix microfluorimetry system (IonOptix Inc., MA, USA). According to the standard protocol, cardiomyocytes derived from hPSCs were loaded with Fluo-8/AM (Molecular Probes) in Tyrode buffer for 60 min at 37°C. Then, cardiomyocytes were perfused with Tyrode buffer supplemented with 1.8 mM CaCl₂, 5.5 mM d-glucose, and 0.5 m Mprobenecid and maintained at 35°C–37°C. Cells were field stimulated at 0.5 Hz using a MyoPacer Field Stimulator (IonOptix Inc., MA, USA). Intracellular calcium transients were analyzed with IonWizard software. The Ca²⁺ transient peak amplitude was denoted F/F₀, in which F₀ presented the fluorescence intensity at the onset of the experiment. SR calcium content of store was evaluated following rapid caffeine application (10 mM). To evaluate the effect of isoproterenol administration, intracellular Ca²⁺ transients were measured before and after isoproterenol (100 nM) application.

Cytosolic Ca²⁺ transients were recorded measuring the Fluo-4 signal (Thermo Fisher F14201: ex/em 490/510) with a laser scanning confocal microscope (Nikon A1R) on a 40 × objective at 37 °C. Cardiomyocytes loaded with 1 µM Fluo-4 for 30 min at 37 °C, then washed with CCB, were placed in the field stimulation buffer, and electrically stimulated with the MyoPacer Field Stimulator (IonOptix) successively at 0.5 and 1 Hz for 1 min each with a rest of 1 min in between (biphasic pulse, 40 V amplitude, and 0.5 ms delay). Images were acquired every 68 ms, and then, normalization was done to the average resting fluorescence intensity (F/F₀). Ca²⁺ transients were analyzed using a homemade program developed with Matlab and Statistics toolbox (version R2014B, The MathWorks Inc.). Transient peaks and minima were detected with a standard algorithm for finding local extrema based on the derivative of the signal and the corresponding amplitude and time of each extremum were recorded. From these values, different parameters were measured: time to peak, half-time, and time peak to basal. Cardiomyocyte shortening was evaluated in Fluo-4 loaded cells by line scanning along the long axis. Cell length was measured, using ImageJ software, in the resting state and in the maximally contracted state to express the shortening as a percentage of the resting cell length.

The contractility of the engineered cardiac tissues was assessed using the “heart-on-a-chip” platform, as previously described.^{32 (link)} Briefly, the chips were placed in the Normal Tyrode solution, the films were cut out, and the experiments performed at 35–37 °C. The dynamics of the films were recorded using a stereoscope (no. SZX-ILLB2, Olympus America, Center Valley, PA) while the tissues were either allowed to contract spontaneously or were paced using a MyoPacer Field Stimulator (IonOptix, Milton, MA) at 0.5, 1, 1.5, and 2 Hz. The resultant movies were analyzed with custom software to extract diastolic stress, systolic stress, active stress, and beating frequency.^{23 (link),32 (link)} The whole dataset is made available through Dryad https://doi.org/10.7280/D10H40.