Journal: Circulation Research

Article Title: CalTrack

doi: 10.1161/CIRCRESAHA.121.318868

Figure Lengend Snippet: CalTrack analyzes computationally simulated calcium transients at multiple beating frequencies with fidelity. A , 0.5–2 Hz Kymographs produced by CalTrack. B , Average normalized calcium transients (n=20 simulated traces for each pacing frequency). C , Increases in pacing frequency decrease normalized peak calcium concentration (equivalent to peak fluorescence/baseline fluorescence [F max /F 0 ]). D–I , Increasing pacing frequency significantly accelerates all calcium transient properties assessed by CalTrack (time to reach calcium peak [T on ], time for calcium transient decay [T off ], calcium decay constant (Tau), time to 50% of calcium peak [T50 on ], time of 50% calcium decay [T50 off ], and calcium transient duration [CD]). Data presented as mean and SD, colors of data points represent 0.5 Hz pacing (blue), 1 Hz pacing (green), and 2 Hz pacing (red). For Ca max /Ca 0 and Tau statistical analysis is performed by 1-way ANOVA with a post hoc Sidak correction. T on , T off , T50 on , T50 off , and CD were tested with a paired nonparametric test Friedman test with Dunn multiple comparisons correction, 3 comparisons. In both instances, a P <0.05 was defined as the significance cutoff. Ca max /Ca 0 indicate maximal calcium/baseline calcium.

Article Snippet: Indeed, almost identical analysis fidelity of values was obtained with CalTrack and the IonOptix IonWizard software (Figure ).

Techniques: Produced, Concentration Assay, Fluorescence

Journal: Circulation Research

Article Title: CalTrack

doi: 10.1161/CIRCRESAHA.121.318868

Figure Lengend Snippet: The variety of cardiovascular model systems and constructs that can be automatedly analyzed using CalTrack. CalTrack can be applied to multiple cellular systems, both organized and disorganized primary cardiomyocytes from animal models and immortalized cell types (induced pluripotent stem cell–derived cardiomyocytes [iPSC-CMs] and embryonic stem cell derived cardiomyocytes [ESC-CMs]), using formats that include 3-dimensional (3D) tissue systems, organoids, and 2-dimensional (2D) cell culture systems.

Article Snippet: Indeed, almost identical analysis fidelity of values was obtained with CalTrack and the IonOptix IonWizard software (Figure ).

Techniques: Construct, Derivative Assay, Cell Culture

Journal: Circulation Research

Article Title: CalTrack

doi: 10.1161/CIRCRESAHA.121.318868

Figure Lengend Snippet: The CalTrack calcium analysis pipeline can be used for Single cells or multicell image stacks. A , A single induced pluripotent stem cell–derived cardiomyocytes (iPSC-CM) imaged using an ×100 objective that has been masked ( B ) by CalTrack for segmentation and fluorescent trace extraction. C , From the masked iPSC-CM CalTrack extracts a raw calcium trace. D , CalTrack then automatedly segments the beats (red lines) and can average them into one trace (black line). E , CalTrack can then provide all parameters from each individual transient or from the mean transient of a single cell. F , The same analysis routine can be applied to low magnification (×20) image stacks with multiple cells per field of view. G , The CalTrack segmentation mask identifies single cells for transient analysis and provides raw fluorescent traces for each cell ( H ), which can then be individually segmented and averaged ( I ) for automatic parametric output ( J ). AU indicates arbitrary units.

Article Snippet: Indeed, almost identical analysis fidelity of values was obtained with CalTrack and the IonOptix IonWizard software (Figure ).

Techniques: Derivative Assay, Extraction

Journal: Circulation Research

Article Title: CalTrack

doi: 10.1161/CIRCRESAHA.121.318868

Figure Lengend Snippet: CalTrack successfully corrects linear and exponential photobleach events. A , Simulated linear and exponential baseline shifts are applied to a fluorescent calcium trace (inset). Colored trend lines are fit to the baseline shifted data by CalTrack. These are used to correct shifts in baseline. B and D , Segmented individual transients from traces that need baseline correction due to baseline drift caused by linear decay ( B ) or exponential decay ( D ). CalTrack correction of baseline drifts functions robustly for linear ( C ) or exponential ( E ) baseline shift. AU indicates arbitrary units.

Article Snippet: Indeed, almost identical analysis fidelity of values was obtained with CalTrack and the IonOptix IonWizard software (Figure ).

Techniques:

Journal: Circulation Research

Article Title: CalTrack

doi: 10.1161/CIRCRESAHA.121.318868

Figure Lengend Snippet: Automated CalTrack quantification of calcium concentration and kinetic parameters in guinea pig wild-type (WT) and TnnI3 R145G/ + cardiomyocytes is comparable to manual IonOptix IonWizard software analysis. A , Average calcium transients are identical by analysis via IonWizard or CalTrack analysis. Average traces are made of n=42 WT and n=36 for TnnI3 R145G/ + cells. B , Calcium amplitude is unaffected by Tnni3 R145G/ + . C , Baseline [Ca 2+ ] is increased in TnnI3 R145G/ + cells. Time to 10% of peak (T10 on ; D ), time to 50% of calcium peak (T50 on ; E ), and time to 90% of peak (T90 on ; F ) are unaffected by TnnI3 R145G/ + . G , time to reach calcium peak (T on ) is significantly longer in TnnI3 R145G/ + cardiomyocytes when assessed by CalTrack analysis. H , Time of 10% decay (T10 off ) is unaffected by TnnI3 R145G/+ . I , Time of 50% calcium decay (T50 off ) is longer in TnnI3 R145G/ + cardiomyocytes. J , Time of 90% decay (T90 off ) is assessed to be longer in TnnI3 R145G/ + cardiomyocytes by CalTrack analysis. K , Calcium decay constant (Tau) is significantly longer in TnnI3 R145G/ + cardiomyocytes. Statistical analysis is performed by a 2-tailed Student t test (Baseline, T on , T90 on , and T90 off ) or by nonparametric Mann-Whitney test (CaAmplitude, T10 on , T50 on , T10 off , T50 off , tau) with a significance cutoff of P <0.05. No statistical comparisons are made between IonOptix and CalTrack. Ns indicates nonsignificance.

Article Snippet: Indeed, almost identical analysis fidelity of values was obtained with CalTrack and the IonOptix IonWizard software (Figure ).

Techniques: Concentration Assay, Software, MANN-WHITNEY

Journal: Circulation Research

Article Title: CalTrack

doi: 10.1161/CIRCRESAHA.121.318868

Figure Lengend Snippet: CalTrack defines calcium transient changes caused by the TnnI3 R145G/ + hypertrophic cardiomyopathy (HCM) variant in paced (0.5 Hz) adult guinea pig cardiomyocytes. A , Normalized mean calcium transients and calcium decays ( B ) from wild-type (WT; n=47) or TnnI3 R145G/ + (n=88) guinea pig cardiomyocytes. The TnnI3 R145G/ + variant does not affect peak fluorescence/baseline fluorescence (F max /F 0 ; C ), and calcium decay constant (Tau; D ). Time to 50% of calcium peak (T50 on ) is accelerated ( E ), but time to reach calcium peak (T on ; F ), time of 50% calcium decay (T50 off ; G ), time for calcium transient decay (T off ; H ), and calcium transient duration (CD; I ) are all slowed in comparison to WT. Data presented as mean and SD. Statistical analysis is performed by a 2-tailed Student t test (T on , T off , and CD) or by nonparametric Mann-Whitney test (F max /F 0 , Tau, T50 on , and T50 off ) with a significance cutoff of P <0.05. ns indicates nonsignificant. AU indicates arbitrary units.

Article Snippet: Indeed, almost identical analysis fidelity of values was obtained with CalTrack and the IonOptix IonWizard software (Figure ).

Techniques: Variant Assay, Fluorescence, Comparison, MANN-WHITNEY

Journal: Circulation Research

Article Title: CalTrack

doi: 10.1161/CIRCRESAHA.121.318868

Figure Lengend Snippet: CalTrack defines calcium phenotypes and drug responses in paced induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs) harboring the hypertrophic cardiomyopathy (HCM) TNNI3 R21C/ + variant. A , Averaged calcium transient traces of wild-type (WT; n=113 and TNNI3 R21C/ + (n=60) cardiomyocytes with 0.3 μM (n=40), 1 μM (n=59), and 3 μM mavacamten (n=92). B , TNNI3 R21C/ + did not alter peak fluorescence/baseline fluorescence (F max /F 0 ). C , Mavacamten reduces F max /F 0 in a dose-dependent manner. D and E , Calcium decay constant (Tau) is prolonged in TNNI3 R21C/ + cardiomyocytes and shortened by increasing mavacamtem dose. F , TNNI3 R21C/ + shortens time to 50% of calcium peak (T50 on ). G , Mavacamten has variable, dose-dependent effects on T50 on in the TNNI3 R21C/ + cardiomyocytes. H and I , time to reach calcium peak (T on ) is shortened in TNNI3 R21C/ + cardiomyocytes at baseline ( H ) and further decreased by Mavacamten ( I ). J and K , Time of 50% calcium decay (T50 off ) is prolonged in TNNI3 R21C/ + cardiomyocytes ( J ) and shortened ( K ) with increasing doses of mavacamten. L and M , time for calcium transient decay (T off ) is prolonged in TNNI3 R21C/ + cardiomyocytes ( L ) and shortened ( M ) with increasing mavacamten dose. N and O , Calcium transient duration (CD) is statistically significantly prolonged in TNNI3 R21C/ + cardiomyocytes ( N ) and reduced ( O ) with increasing doses of mavacamten. Data presented as mean and SD. Statistical analysis is performed by nonparametric Kruskal-Wallis with post hoc Dunn correction for multiple comparisons (9 comparisons). Significances are denoted on each panel where * P <0.05, ** P <0.01, *** P <0.005, **** P <0.0001. Color of significance denotation defines the treatment group compared. Please see Table III in the Data Supplement for exact P values. AU indicates arbitrary units; and Mava, Mavacamten.

Article Snippet: Indeed, almost identical analysis fidelity of values was obtained with CalTrack and the IonOptix IonWizard software (Figure ).

Techniques: Derivative Assay, Variant Assay, Fluorescence