Calcium and contractility system

Cardiac myocyte dimensions and contractile and relaxation function were measured using the IonOptix Calcium and Contractility System (Westwood, MA, Ionoptix.com). Briefly, glass coverslips containing isolated myocytes were mounted onto a stimulation chamber and bathed in Tyrode’s solution warmed to 30 °C. Length and width were measured with the Edge Detection feature in quiescent myocytes. Myocytes were then and stimulated at 25 V with 0.5 Hz pacing frequency and the Sarcomere Length feature was used to measure contractility and relaxation. Cells were visualized with an inverted microscope (Nikon Eclipse TE2000-U) fitted with a 40X objective. Sarcomere length data was collected at 1000 Hz, and approximately 10 contractile cycles were averaged for each cell. Data were analyzed by Fast Fourier Transform using IonWizard Software (IonOptix, Westwood, MA, Ionoptix.com), and ~ 15 myocytes were analyzed from each of 3 mice per group. These numbers were chosen to obtain a representative sampling of myocytes from each heart, and to ensure functional trends and inter-myocyte variability were similar between mice.

Isolated hCMs were seeded on Matrigel‐coated glass coverslip and loaded with 1 × 10⁻⁶ m Fura‐2 AM (F1221, Invitrogen) in the Tyrode's buffer at 37 °C for 15 min, and then washed with the Tyrode's buffer. Cells were paced with field stimulation (monophasic, 10 V, 5 ms pulses, 1–3 Hz frequency, with 10 s rest between two frequencies) in a perfusion chamber using the IonOptix Calcium and Contractility System (IonOptix). Calcium transients at each frequency were recorded for 5–10 s with a 40× objective. All parameters were calculated offline using the IonWizard 6.3.4 software (IonOptix).

Isolated cardiac myocytes were incubated at room temperature, in the dark, with 2 µM of the calcium indicator dye Fura-2AM (AbCam, Cambridge, MA) for 10 min, followed by 20 min of de-esterification. Calcium transients were measured using the IonOptix Calcium and Contractility System (Ionoptix.com), with the µ-stepper switch to measure 360 nm and 380 nm wavelengths. Myocytes were bathed in 30 °C Tyrode’s solution and paced at 25 V and 0.5 Hz. The 360:380 nm ratio was collected to measure the relative change in intracellular calcium concentration during the contractile cycle. The excitation wavelength of 360 nm measures both calcium-bound and unbound Fura-2AM, whereas 380 nm measures unbound Fura-2AM. The 380 nm wavelength is measured at 1000 Hz, and the 360 nm wavelength is measured every 10 s to account for photobleaching or loss of Fura-2AM from the cell. Approximately 10 transients were averaged per cell and analyzed using IonWizard Software (IonOptix, Westwood, MA), and ~ 15 myocytes from each of 3 mice were analyzed per group.

We used the IonOptix Calcium and Contractility System connected with MyoCam-S (IonOptix) in an inverted microscope with a 40× objective to measure sarcomere length kinetics and fluorescence photometry. IonOptix calculates the average frequency of the sinusoidal appearance by altering striation patterns of dark (A-band) and light (I-band) from isolated single myofibers. We applied 250 Hz sampling frequency for sarcomere length kinetics and 1000 Hz for fluorescence photometry with 25 V, 8 ms pulse duration (supplemental information Figure S3), 0.2 Hz field stimulation frequency at 25 °C. This stimulation achieved peak twitch contractions in isolated FDBs which is known to be sensitive to different electrical field configurations and pulse parameters [42 (link)]. For myoplasmic Ca²⁺ transients, the isolated FDB fibers were loaded with 1 μM Fura-2AM (F1221, Invitrogen) in M199 for 10 min at room temperature, and 15 min in M199 for deesterification in the dark. Sarcomere length kinetics and Fura-2AM signals were measured simultaneously. Our data with Fura-2 as an intracellular Ca²⁺ indicator compares well with a recent report using Fura-2 in skeletal muscle [43 (link)].

Intracellular Ca²⁺ ([Ca²⁺]_i) transients were recorded from iPSC-CM clusters using the IonOptix Calcium and Contractility system (IonOptix LLC, Westwood, MA, USA), routinely used in our lab [23 (link),52 (link),53 (link)]. In brief, iPSC-CM clusters were mechanically dissected and adhered to 18 mm diameter Matrigel-coated glass slides. On the day of the experiment, slides were stained with fura-2 (2.5 μM) and were transferred to a chamber mounted on the stage of an inverted microscope and perfused at a rate of 1–1.5 mL/min Tyrode’s solution at 37 °C. The Tyrode’s solution contained: (mmol/L): 140 NaCl, 5.4 KCl, 1 MgCl₂, 2 sodium pyruvate, 1 CaCl₂, 10 HEPES, 10 glucose (pH 7.4 adjusted with NaOH). The clusters were paced at 0.5–2.5 Hz which corresponded to a frequency 20–50% higher than the spontaneous beating rate. Analysis was performed using the IonOptix designated system. RyR-mediated SR Ca²⁺ release was measured by a brief application of caffeine (10 mM). The caffeine response was quantified by calculating 3 parameters: (1) recovery time—the time from the peak of caffeine-induced [Ca²⁺]_i rise to the first [Ca²⁺]_i transient; (2) the percent change in caffeine-induced [Ca²⁺]_i response amplitude, compared to pre-caffeine amplitude; (3) the percent change in caffeine-induced [Ca²⁺]_i response area, compared to the pre-caffeine value.