CalDyn: an advanced high-throughput automated algorithm for monitoring intracellular calcium dynamics and electrophysiological signals in patient-derived cells

Figures

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  Figure 1. A CalDyn workflow diagram. The process begins with GUI launch, followed by file selection, automated peak detection, manual refinement, parameter customization, and result exportation. B: CalDyn algorithmic pipeline. Blue boxes show the core detection pipeline (always run): Savitzky–Golay filtering, LOESS filtering (for the derivative only), derivative computation, threshold-based extrema detection, pairing into candidate peaks, and boundary fitting. Dashed green boxes show the two optional branches (baseline correction, peak classification), skipped unless enabled by the user. See the CalDyn Algorithm section for full notation.

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  Figure 2. Example of intracellular Ca2+ signal in hiPSC-NRs as presented in the GUI CalDyn interface.

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  Figure 3. Paired comparison of intracellular Ca2+-transient parameters obtained using PeakInspector and CalDyn in healthy control hiPSC-CMs. (A) Immunofluorescence staining of cardiomyocytes showing □-actinin (red), cardiac troponin I (green), and DAPI (blue). The scale bar is 10 μm. (B) Representative recording illustrating cardiac Ca2+ transients obtained from PeakInspector and CalDyn. (C-G) Paired cell-level comparisons of normalized Ca2+-transient amplitude (C), Ca2+-transient frequency (D), small Ca2+-event frequency (E), decay time (F), and maximum Ca2+-transient rise rate, dF/dtmax (G), obtained using PeakInspector and CalDyn. Peak-level measurements generated by PeakInspector were summarized within each cell to provide the same cell-level analytical unit as the corresponding CalDyn output. Each symbol represents one cell, and lines connect measurements obtained from the same recording using the two programs. Bland–Altman analyses of agreement between CalDyn and PeakInspector are shown in Supplementary Fig. 1 and summarized in Table 4.

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  Figure 4. Paired comparison of intracellular Ca2+-transient parameters obtained using PeakInspector and CalDyn in arrhythmic PMVT hiPSC-CMs. (A) Representative recording exhibiting complex intracellular Ca2+ activity analyzed using PeakInspector (top) and CalDyn (bottom), including principal Ca2+ transients, candidate low-amplitude diastolic events, and candidate multicomponent Ca2+ transients. The PeakInspector output shows the filtered and detrended signal, whereas the CalDyn output shows the original and filtered signals together with the automatically identified peak maxima, waveform-analysis points, and event classifications. (B-G) Paired recording-level comparisons of normalized Ca2+-transient amplitude (B), principal Ca2+-transient frequency (C), candidate low-amplitude diastolic-event frequency (D), candidate multicomponent Ca2+-transient frequency (E), decay time (F), and maximum Ca2+-transient rise rate, dF/dtmax (G). Peak-level measurements generated by PeakInspector were summarized within each recording to provide the same analytical unit as the corresponding CalDyn output. Each symbol represents one recording, and lines connect measurements obtained from the same recording…

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  Figure 5. Intracellular Ca2+ dynamics in primary healthy human myotubes detected by CalDyn. (A) Immunofluorescence imaging of primary healthy human myotubes, labeled with ⍺-actinin (orange) and nuclear DNA stained with DAPI (blue). (B and C) Typical plot showing the Ca2+ response of a myotube to an electrical stimulus, obtained using CalDyn.

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  Figure 6. Intracellular Ca2+ dynamics in hiPSC-NRs detected by CalDyn. (A) Immunofluorescence staining of healthy neurons, highlighting ß-3 tubulin (red) and DAPI (blue). The scale bar is 20 μm. (B and C) Typical plot illustrating 2 independent and heterogenous intracellular Ca2+ handling examples obtained with CalDyn.

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  Figure 7. Paired comparison of AP parameters obtained using Clampfit and CalDyn in hiPSC-CMs. (A) Representative CalDyn analysis of successive AP recorded from a ventricular-like hiPSC-CMs in the whole-cell current-clamp configuration. The left panel shows the filtered AP trace together with the waveform landmarks identified by CalDyn for parameter extraction. Black markers indicate detected AP maxima, whereas magenta, cyan, and green markers indicate the positions used to calculate AP duration and waveform boundaries. The right panel shows the AP trace and its filtered derivative used for automated event detection. (B–D) Paired comparisons of AP duration at 90%, 50%, and 30% repolarization, respectively (APD90, APD50, and APD₃₀). (E) Paired comparison of AP amplitude. (F) Paired comparison of maximum AP upstroke velocity (dV/dtmax). Each symbol represents one recording, and dotted lines connect measurements obtained from the same recording using the two programs. Bland–Altman analyses of method agreement are presented in Supplementary Fig. 5 and Table 6. APD, action-potential duration; dV/dtmax, maximum action-potential upstroke velocity.

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  Figure 8. Optical mapping recording using RH237 fluorescent probe.