Myocyte calcium and contractility system

Calcium transients from spontaneous or electrically evoked activity in neoCM were obtained with the Myocyte Calcium and Contractility System (IonOptix, Westwood, Massachusetts, USA). NeoCM were measured 6–9 days after isolation. NeoCM monolayers were transiently transfected with miR mimics for 24 h as described above. Cells were loaded with 4 µM Fura-2 AM (stock solution: 1 mM in DMSO) at 37°C for 30 min. Each sample was measured three times at room temperature: after recording control signals for 2 min the buffer was changed to 10 µM isoprenaline (ISO) (2 min break) and response to ISO was measured for 3 min and after a 1 min break again for 3 min. If possible, spontaneous calcium transients were recorded. Otherwise, cells were paced at 1 Hz to evoke calcium transients (MyoPacer, 5–10 V per pulse, pulse duration maximum 4 ms). IonWizard 6.6 (IonOptix) was used for data acquisition at sampling frequencies of 100 Hz (average 4: 4 collected data points are averaged into one raw data point) or 250 Hz (average 1). Monotonic transient analysis was performed using IonWizard 6.6.

HL-1 cardiomyocytes were incubated for 30 min with 5 µM of Rhod-2 AM (Abcam, Cambridge, UK) at 37 °C in DMEM (Gibco), followed by three times washing with DMEM. Rhod-2 AM-loaded cardiomyocytes were excited by a 600 nm laser with emission at 605 nm and amplitudes were recorded with the Myocyte Calcium and Contractility System (IonOptix Corporation). The live recording of the mitochondrial calcium transients (CaT_mito), which provides an indication of changes in mitochondrial Ca²⁺, was performed at 1 Hz stimulation (normal pacing) at 37 °C. The relative values of fluorescent signals were determined utilizing the following calculation: Fcal = F1/F0, where F1 is the fluorescent signal at any given time and F0 is the fluorescent signal at rest. Mean values from each experimental condition were based on 7 consecutive CaT_mito in at least 25 cardiomyocytes.

Adult cardiomyocytes were loaded with 0.5 μM Fura-2-AM (Molecular Probes, Eugene, OR) and placed in a heated chamber (37°C) on the stage of an inverted microscope. The chamber was perfused with Tyrode’s solution containing CaCl₂ (1.8 mM) (pH 7.4). Cardiomyocytes were paced with an IonOptix Myocyte Calcium and Contractility System at 0.5 Hz using a MyoPacer field stimulator. Changes in intracellular Ca²⁺ levels were monitored using Fura-2 dual-excitation (340/380 nm), single emission (510 nm) ratiometric imaging. Tau, the decay rate of the average Ca²⁺ transient trace, was determined using IonWizard 6.0 analysis software (IonOptix, Westwood, MA). Cardiomyocyte contractility measurements were made using sarcomere length (SarcLen) parameters and data was processed with IonWizard 6.0 analysis software.

To measure mitochondrial calcium transient (CaT_mito) amplitudes, HL-1 cardiomyocytes were incubated for 30 min. with 5 µM of Rhod-2 AM ([17 (link)], Abcam, Cambridge, UK) at 37 °C in DMEM (Gibco), which provides an indication of changes in mitochondrial Ca²⁺, followed by three times washing with DMEM. To measure cytosolic calcium transient (CaT_cyto) amplitudes, cardiomyocytes were incubated for 30 min. with 2 µM of the Ca²⁺-sensitive dye Fluo-4-AM (Life Technologies) at 37 °C in DMEM, followed by three times washing with DMEM. Rhod-2 AM-loaded cardiomyocytes were excited by a 600 nm laser with emission at 605 nm and Fluo-4-AM-loaded cardiomyocytes were excited by a 488 nm laser with emission at 500-550 nm. CaT_mito and CaT_cyto amplitudes were recorded with the Myocyte Calcium and Contractility System (IonOptix Corporation, city, state abbrev. (If USA or Canada), country). The live recording of the CaT amplitudes was performed at 1 Hz stimulation (normal pacing) at 37 °C. The relative value of fluorescent signals was determined utilizing the following calculation: Fcal = F1/F0, where F1 is the fluorescent dye signal at any given time and F0 is the fluorescent signal at rest. Mean values and SEM from each experimental condition were based on 7 consecutive CaTs in at least 25 cardiomyocytes.