Sarclen

Myocyte contraction properties including sarcomeric length were evaluated with a sarcomeric imaging acquisition system (SarcLen; IonOptix) as previously described.^{23 (link)}

Cell contraction properties of myocytes were evaluated with a video-based sarcomere spacing acquisition system (SarcLen, IonOptix, Milton, MA). Rod-shaped myocytes with clear striation patterns and quiescent when unstimulated were chosen. For calcium flux measurements, myocytes were loaded with 0.5 mM fura 2-acetoxymethyl ester (Molecular Probes, Eugene, Oregon, USA) for 15 min. Cells were then washed and rested for an additional 40 min to allow the de-esterification of the fura-2 ester. Cells were placed in a culture chamber stimulation system (Cell MicroControls, Norfolk, VA), mounted on an inverted microscope (Nikon TE2000U, Nikon, Melville, NY) and electrically stimulated with suprathreshold voltage at 0.5 Hz and superfused with a HEPES-buffered solution at 25°C as previously described.^{29 (link),30 (link)} Changes in sarcomere length were recorded and further analysis was performed using IonWizard software (IonOptix, Milton, MA). Cells were excited at 340 and 380 nm, continuously alternated, at rates as high as 250 pairs/sec using a HyperSwitch system (IonOptix, Milton, MA). Background-corrected fura-2AM ratios were collected at 510 nm. This ratio is independent of cell geometry and excitation light intensity, and reflected the intracellular calcium concentration.^{31 (link),32 (link)}