Mechanical properties of ventricular myocytes were measured using an IonOptix Myocam system (IonOptix Corp., Milton, MA, USA). Cultured cells were suspended in the Ca2+-free Tyrode’s solution, and Ca2+ was slowly added until Ca2+ reached a final concentration of 1.8 mmol L−1. Cardiomyocytes placed in a chamber on the stage of an inverted microscope were stimulated at 0.5 Hz for 3 ms duration by a pair of platinum wires placed on the opposite sides of the chamber. Electrical pulses were given with an isolated stimulator (FHC Inc., Bowdoinham, ME, USA). Parameters measured were: % peak shortening, maximal velocities of contraction and relaxation, time to peak shortening and time to 90% relaxation.
Myocam system
Manufactured by IonOptix
Sourced in United States
The IonOptix Myocam system is a laboratory equipment designed to monitor and analyze the contractile activity of isolated muscle cells. It utilizes high-speed video microscopy to capture and record the movement of individual muscle cells, providing researchers with detailed data on their contraction and relaxation patterns.
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5 protocols using myocam system
1
Measuring Ventricular Myocyte Mechanics
2
Cardiomyocyte Mechanical Properties Analysis
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Mechanical properties of single cardiomyocytes were measured with an IonOptix Myocam system (IonOptix Inc., Milton, MA, United States). Cells were placed on the stage of an inverted microscope (×400 objective). The cells were stimulated with an electrical field at a frequency of 0.5 Hz. Cell shortening and re-lengthening were assessed according to the previous study with parameters as follows: resting cell length, peak shortening amplitude (PS), time-to-peak shortening (TPS), and maximum velocity of shortening and relengthening (±dL/dt) (Zhang et al., 2014a (link)).
3
Fura-2 Ca2+ Transient Analysis in Cardiomyocytes
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The IonOptix Myocam system (IonOptix Corp., Milton, MA, USA) assessed cardiomyocyte contractile properties. Ca2+ transients in cardiomyocytes loaded with Fura-2 AM (Dojindo Labratories, Japan, excitation wavelength 340/380 nm and emission wavelength of 510 nm) were measured as previously described52 (link),53 (link). The percentage of Fura-2 fluorescence intensity change (ΔFFI) to resting cell Fura-2 fluorescence intensity (FFIDiastolic) reflected Ca2+ transient amplitude. Fluorescence ratios (R = F340/F380) were used to calculate cytosolic Ca2+ concentrations: [Ca2+]i = Kd × (R-Rmin) / (Rmax-R) × Sf2 / Sb2. Rmin and Rmax are fluorescence ratios under calcium deficiency and saturation conditions, respectively. Sf2 and Sb2 are fluorescence values (excitation at 380 nm) under calcium deficiency and saturation conditions, respectively. Kd is the dissociation constant for Fura-2-calcium binding (225 nM).
4
Cardiomyocyte Mechanical Properties Analysis
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Mechanical properties of cardiomyocytes were assessed using a SOFTEDGE. MYOCAM system (IonOptix Corporation, Milton, MA, USA). In brief, cardiomyocytes were seed on the 24 × 24 mm microscope cover glass and cultured with Costar 6-well plates. The mitochondria transferred cardiomyocytes were translocated into a Warner chamber mounted on the stage of an inverted microscope (Olympus, IX-70) and superfused with 0.5 mL no BDM culture medium. Cells were stimulated by 0.5 Hz frequency using a pair of platinum wires connected to a FHC stimulator (Brunswick, NE, USA). The shorthening and relengthening were recorded by IonOptix MyoCam camera. IONOPTIX SOFTEDGE software. The characteristics of shorthening and relengthening in cardiomyocytes were analyzed and demonstrated as the following indices: bl-resting cell length, dep v-maximal velocity of shortening (-dl/dt), dep vt-time to peak shortening (TPS-ms), bl % peak h-peaking shortening (% cell lengthening), ret v:maximal velocity of relengthening (+dl/dt), t to bl 90.0%:Time to 90% relengthening (TR90-ms).
5
Characterizing hESC-Derived Cardiomyocytes
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Parameters of twitch contractions and intracellular calcium transients were recorded from single hESC-CMs using a MyoCam system (IonOptix, Milton, MA, USA). APs were recorded by a whole-cell patch-clamp technique (Axopatch 200B; Molecular Devices, San Jose, CA, USA). For FISH, specific probe sets for labeling of MYH6 and MYH7-mRNA (Affymetrix, Santa Clara, CA, USA) were used. Immunostaining of α-MyHC and β-MyHC isoforms occurred with highly specific antibodies. Full details are provided in Supplemental Experimental Procedures .
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