Myocam camera

Simultaneous cell shortening and NADH fluorescence measurements were performed at 37°C. Cells were placed into a Warner chamber and superfused with glucose Ringer's solution (2 mmol/L Ca²⁺) and stimulated at 4 Hz with platinum electrodes. Changes in cell length during shortening were acquired at time 0 (T0) and after 10 min (T10) of continuous contraction. The change in contractile properties over time was assessed by the arithmetic ratio of (T10/T0). Contractile properties were assessed using a SoftEdge MyoCam^® system (IonOptix Corp., Milton, MA, USA). The myocyte being studied was displayed on a computer monitor using an IonOptix MyoCam camera. An IonOptix SoftEdge software was used to capture changes in cell length during shortening and relengthening. Cell shortening was assessed using the following indices: peak shortening (distance shortened/diastolic cell length × 100), an indication of peak ventricular contractility. The maximal velocity of shortening (+dL/dt), calculated as the maximal slope derivative of the shortening phase and is an indication of maximal velocity of ventricular pressure increase.

Mechanical properties of isolated cardiomyocytes were evaluated using an IonOptix™ soft‐edge system (IonOptix, Milton, MA, USA). Cardiomyocytes were laid in a chamber mounted on the stage of an Olympus IX‐70 inverted microscope and superfused (~2 mL/min at 25°C) with a Krebs‐Henseleit bicarbonate buffer containing (in mmol/L) 131 NaCl, 4 KCl, 1 MgCl₂, 1 CaCl2, 10 HEPES and 10 glucose at pH 7.4. Myocytes were stimulated at a frequency of 0.5 Hz using a pair of platinum wires on the opposite sides of the chamber connected to a FHC stimulator (Brunswick, NE, USA). The cell was shown on the computer monitor by an IonOptix MyoCam camera. Cell shortening and relengthening were assessed by the following indices: resting cell length, peak shortening (PS), time‐to‐PS (TPS), time‐to‐90% relengthening (TR₉₀) and maximal velocities of shortening/relengthening (±dL/dt).28

Mechanical properties of cardiomyocytes were assessed using a softedge myocam^® system (IonOptix Corporation, Milton, MA, USA) (Xu et al., 2013). In brief, cells were placed in a Warner chamber mounted on the stage of an inverted microscope (Olympus, IX‐70) and superfused (~1 mL min⁻¹ at 25 °C) with a buffer containing (in mm): 131 NaCl, 4 KCl, 1 CaCl₂, 1 MgCl₂, 10 glucose, 10 HEPES, at pH 7.4. Cells were field‐stimulated at a frequency of 0.5 Hz (unless otherwise stated), 3‐msec duration, using a pair of platinum wires connected to a FHC stimulator (Brunswick, NE, USA). The myocyte being studied was displayed on the computer monitor using an IonOptix MyoCam camera. ionoptix softedge software was used to capture changes in cell length during shortening and relengthening. Cell shortening and relengthening were assessed using the following indices: peak shortening (PS) ‐ peak ventricular contractility, time‐to‐PS (TPS) ‐ contraction duration, and time‐to‐90% relengthening (TR₉₀) ‐ cardiomyocyte relaxation duration, maximal velocities of shortening (+ dL/dt), and relengthening (− dL/dt) ‐ maximal velocities of ventricular pressure rise/fall. In the case of altering stimulus frequency from 0.1 to 5.0 Hz, steady‐state contraction of the myocyte was achieved (usually after the first 5–6 beats) before PS was recorded.

Unloaded sarcomere shortening was measured in freshly isolated ventricular myocytes, as described previously [1 (link),2 (link)]. Briefly, isolated myocytes were transferred into a recording chamber mounted on an Olympus X51 inverted microscope and superfused with normal Tyrode solution saturated with room air. Additions to the Tyrode solution are described in the text. The mitochondrial calcium uptake inhibitor, Ru-360, was obtained from EMD Biosciences; all other chemicals were purchased from Sigma. Typically, cells were field stimulated to contract at 1 Hz. When thapsigargin was applied to cells, the stimulation frequency was reduced to 0.5 Hz. Video images were acquired using a Myocam camera and IonWizard software (IonOptix, Inc.). All experiments were performed at room temperature.