Destabilized calcium dynamics visualized using the genetically-coded probe GCaMPJ in intact hearts of Calstabin2-null mice

Figure 1. Characteristics of GCaMPJ in vitro and in vivo. (A) Crystal structure of GCaMPJ. Y, tyrosine. (B) Excitation and emission spectra of GCaMPJ and GCaMP3 in Ca2+-chelated (10 mM EGTA) and saturated (10 mM CaCl2) solution. (C) Relative fluorescence intensity changes in Hela cells expressing GCaMPJ and GCaMp3 when stimulated with 100 μM ATP. (D) Baseline fluorescence intensity (F0) was measured using FACS assay in Hela cells expressing GCaMPJ and GCaMp3. (n=70–100 from 3 independent transfection experiments for each group, p>0.05) (E). Quantitative analysis of relative fluorescence intensity (n=70–100 from 3 independent transfection experiments for each group, p<0.001) (F) Schematic diagram of the construction of transgenic mice. (G) Expression detection of exogenous genes. (H) Morphologic observation of heart. (I, J) Quantitative analysis of heart weight/body weight (HW/BW) and heart weight/tibia length ratio (HW/TL). N = 8. (K–N) Echocardiography depicted the ejection fraction (EF), fractional shortening (FS), LV mass, and LVPW thickness of hearts in transgenic and normal mice. N = 8 independent mice per group. The data of (D, E) were expressed as mean ± SEM and subjected to…

Figure 2. GCaMPJ provides distinct advantages for Ca2+ monitoring. (A) Representative micrographs of Ca2+ sparks. (B) Quantification of Ca2+ spark fluorescence intensity (F/F0). Here, F0 is the background baseline intensity measured in the recording solution without Ca2+ within 1 min, and F represents the actual fluorescence intensity. (C) The physiological amplitude of fluorescence intensity (△F/F0). Here, F0 represents the resting diastolic fluorescence of the cardiomyocyte. (D) Quantification of full width at half maximum (FWHM). (E) Quantitative assay of Full duration half maximum (FDHM). (F) The rise time of fluorescence intensity. (G) The half decay time of fluorescence intensity. (H) Speed change of fluorescence intensity. All statistical data were analyzed using a Tukey one-way ANOVA. Data were obtained from n = 10 isolated cardiomyocytes across N = 3 independent mice per group. Error bars represent the mean ± SEM. *p < 0.05, **p < 0.01.

Figure 3. GCaMPJ enables highly sensitive Ca2+ imaging in intact hearts. (A, B) Representative photographs of Ca2+ signal changes on the surface of the heart during a cardiac cycle. (C, D) Ca2+ signal changes on the surface of the heart and ECG during a cardiac cycle corresponded to (A, B), respectively. The horizontal time scale for the highly magnified single cardiac cycle traces in both (C, D) is 0.2 (s) In (E), the x-axis label (‘Second’) indicates Time (s). The vertical scale bar in (C, D) represents a relative amplitude increment of 0.1, which aligns quantitatively with the absolute F/F0 peaks shown in the continuous 5 s recording (E) and the statistical summary (F). (E) Comparison of Ca2+ signal changes on the surface of the heart within 5 (s) (F) Statistical analysis of fluorescence intensity. The number of GCaMPJ and GCaMP6s mice is 12 and 9, respectively. All statistical data were represented as mean ± SEM and performed two-tailed unpaired Student’s t-test. **p<0.01.

Figure 4. Stabilization of RyR2 prevents chaotic Ca2+ events in intact hearts. (A) Schematic diagram of the mouse heart. Circles represent the random detection area of the Ca2+ signal. (B) Amplitude comparison of Ca2+ activity on the surface of normal beating hearts from GCaMPJ/Calstabin2+/+ and GCaMPJ/Calstabin2-/- mice. N = 5 independent hearts per group and all statistical data are represented as mean ± SEM using two-tailed unpaired Student’s t-test. **p<0.01. (C, D) The cardiac Ca2+ activities of GCaMPJ/Calstabin2+/+ and GCaMPJ/Calstabin2-/- mice under pacing stimulation and S107 intervention. (E, F) The cardiac Ca2+ activities of GCaMPJ/Calstabin2+/+ and GCaMPJ/Calstabin2-/- mice with I/R and S107 intervention. (C–F) At the top is a schematic diagram of the overall cardiac Ca2+ activities. The red, black, and blue curves below represent the fluorescence intensity changes of three random areas described in (A). The bottom trace shows the ECG, which was recorded simultaneously with the Ca2+ activities. The horizontal scale bar for the traces in (C–F) represents 0.2 (s) The baseline control hearts exhibited a spontaneous heart rate of approximately 300 bpm (5 beats per second). For…

Figure 5. Ca2+ dynamics monitoring in ischemic heart. (A) Representative sequential photographs of the Ca2+ activities in intact hearts from αMHC-GCaMPJ mice during a cardiac cycle. (B) Highly localized Ca2+ transients and spark-like events were observed in the intact GCaMPJ hearts, as indicated by arrows. The lower panels show pseudo linescan images from where indicated by lines in the upper panel. (C) The Ca2+ spark parameters of the intact heart of αMHC-GCaMPJ mice. The amplitude was (1.71 ± 0.02) (ΔF/F0), full width at half maximum was (3.73 ± 0.17), rise time (taken from the point of rising to peak ΔF/F0) was (76.41 ± 7.52) ms, and the half time decay (tau) was (156.4 ± 18.58) ms. Data were collected from N = 5 independent hearts, with an estimated 30–50 localized Ca2+ events recorded per heart. Statistical parameters were calculated based on the independent biological replicates (N = 5) rather than pooled individual events. Data are expressed as mean ± SEM. (D) The highly localized Ca2+ release events induced by cardiac I/R in intact hearts of GCaMPJ/Calstabin2-/- mice. [1], [2], and [3] represent the Ca2+ activities of three distinct micro-domains, respectively. All data were…

Figure 6. RC32 induced adverse Ca2+ events in GCaMPJ mice. (A) Schematic diagram of the mouse heart. Circles represent the random detection areas of the Ca2+ signal. (B) Ca2+-dependent fluorescence intensity on the surface of normal beating hearts from GCaMPJ and RC32-treated GCaMPJ mice. All statistical data are represented as mean ± SEM using two-tail unpaired Student’s t-test. N = 8, *p<0.05. (C, D) The cardiac Ca2+ activities of RC32-treated and vehicle-treated GCaMPJ mice under pacing stimulation and S107 intervention. At the top is a schematic diagram of the overall cardiac Ca2+ activities, and the five curves below correspond to the fluorescence intensity changes of five areas described in (A). The bottom trace shows the ECG, which was recorded simultaneously with the Ca2+ activities.