Cell Shortening and Calcium Homeostasis Analysis in Adult Cardiomyocytes via a New Software Tool

Scheme 1. Overview of ImageJ-Fiji/MATLAB® workflow. Green blocks indicate basic steps on Fiji software. Cerulean blocks show MATLAB® steps (i.e., script execution with user’s choices during analysis). Orange blocks indicate results. Pink blocks are related to the saving of results. On the left and right panels, analyses for contraction shortening and calcium transients are represented, respectively. Two in-between result files are generated: (1) VST.txt contains the tails’ tracking, the input for further analysis with MATLAB®; (2) calcium.csv has the calcium data generated by Fiji [19], the input for further analysis with MATLAB®.

Figure 1. (A) Representative image of adult ventricular murine cardiomyocytes that undergo further analysis. The red line indicates the mid cell region. The cell tails are numbered. (B) Fraction shortening (%) of untreated cell (left panel) and an isoproterenol-stimulated cell (right panel). (C) Statistics for cell shortening and cell kinematics/dynamics. Results for fraction shortening, total force, mean contractility, and mean contraction energy at basal (untreated) and after isoproterenol stimulation (100 nM), obtained from three independent experiments (total cell number equal to 60), are reported as mean ± SEM in the histograms bar chart, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2. (A) Representative image of adult cardiomyocytes for the analysis of Ca2+ transients via ImageJ-Fiji. (B) The detrended intensity of the normalized mean-in-space fluorescence (f/f0) for an untreated cell within a user-defined ROI (red box in panel A) is shown. Each Ca2+ transient is calculated from resting/baseline to peaks. The red crosses are placed where the fluorescence signal is at its 10%, 50%, and 90% for both rise and fall patterns (upper and lower panel, respectively). Some transients have no crosses since they are considered outliers, whereas the last transients are not measured, given their incomplete transitions. In general, there are reported, in the horizontal stacked histogram bars, the τ related to 10%, 50%, 90%, and 10–90% range for both rise and fall patterns and for each detected transition (each transition is depicted with a specific color).

Figure 3. (A) Detrended intensity of the normalized mean-in-space fluorescence (f/f0) for an isoproterenol-stimulated cell within a user-defined ROI. The red crosses are placed where the fluorescence signal is at its 10%, 50%, and 90% for both rise and fall patterns (upper and lower panel, respectively). (B–D) Results for Ca2+ transient’s amplitude Fmax, τ-of-decay (ms), and τ-to-peak (ms) at basal (untreated) and after isoproterenol stimulation (100 nM), obtained from three independent experiments (total cell number equal to 60), are reported as mean ± SEM in the histograms bar chart, * p < 0.05.