2.9. Cell shortening/ re-lengthening and intracellular Ca2+ fluorescence measurement in adult cardiomyocytes

The mechanical properties of ventricular myocytes were assessed using a SoftEdge MyoCam system (IonOptix Corp., Milton, MA, USA) [3], [30]. After 24 h treatment of cells with HG, cell shortening and relenghthening were assessed using the following indices: peak shortening (PS)—indicative of peak ventricular contractility, time to PS (TPS)—indicative of contraction duration, time to 90% relenghthening (TR90)—representing cardiomyocyte relaxation duration, and maximal velocities of shortening (+dL/dt) and relenghthening (−dL/dt)—indicators of maximal velocities of ventricular pressure rise/fall. Myocytes were loaded with Fura-2AM (0.5 μM) for 10 min and fluorescence measurements were recorded. Resting calcium, qualitative changes in the intracellular calcium, and fluorescence decay time (Tau) were measured. Both single- and biexponential curve-fit programs were applied to calculate the intracellular Ca²⁺ decay constant [3], [30]. At least 25 individual cardiomyocytes isolated from 3 to 5 Wistar rats were used for data collection. Changes in [Ca]_i were calculated by determining the rise in [Ca]_i relative to basal levels measured immediately before that particular experimental maneuver.