Simultaneous triple-parametric optical mapping of transmembrane potential, intracellular calcium and NADH for cardiac physiology assessment

Figures

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  Fig. 1. Triple-parametric optical mapping system. a Schematic of the triple-parametric optical mapping system illustrating the optics used in the system and the light path for the three signals—Vm, Ca2+, and NADH. b 3D rendering of the triple parameter optical mapping system illustrating the 3D printed hardware that houses the optics used in the system. c Spectra of the three parameter signals—NADH autofluorescence, Rhod2-AM (Ca2+), and RH237 (Vm) fluorescence, illustrating the separation of the three signals as implemented in this system. Solid vertical lines: LED light source wavelength, dotted vertical lines: dichroic mirror, boxes: filters. Illustration of the different parameters measured using the three optical signals. d NADH intensity was determined and each intensity value from a given heart was normalized to the NADH intensity from the first recording from that heart. e Definition of parameters measured from the depolarization phase of the Vm signal or the calcium release phase of the Ca2+ signal. f Definition of the parameters measured from the repolarization phase of the Vm signal or the calcium reuptake phase of the Ca2+ signal.

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  Fig. 2. Electromechanical uncoupling in triple-parametric optical mapping. a Representative traces of Vm and Ca2+ (solid and dotted traces, respectively) were recorded without (Control, black) and with the electromechanical uncoupler Blebbistatin (15 μM, orange). b Representative Vm activation maps (left), Ca2+ activation maps (middle), and NADH intensity maps (right) recorded during Control and Blebbistatin treatment. All maps were recorded from the same heart, the top three maps were from simultaneous recording during Control and the bottom three from simultaneous recording during Blebbistatin treatment. Slightly different silhouettes, particularly around the boundaries is due to different background noise removal levels using a thresholding algorithm. These boundary pixels were not used in the analysis. c Summary restitution properties of the ten parameters measured simultaneously by triple-parametric optical mapping. d Ten-parameter panel showing the effects of Blebbistatin on cardiac physiology at 150 ms BCL, reported as percent change from Control. Data presented as mean ± s.e.m., n = 5 hearts, Least squares regression analysis was performed for data in panel (c) to detect…

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  Fig. 3. Triple-parametric optical mapping in drug testing. a Ten-parameter panels demonstrating effects of 4-AP (7 mM, left, green) and Verapamil (1 μM, right, purple). b Summary restitution properties of the ten parameters measured simultaneously by triple-parametric optical mapping. Data presented as mean ± s.e.m., n = 5 hearts. Least squares regression analysis was performed for data in panel (b) to detect significant differences in parameters during Blebbistatin versus drugs (4-AP, Verapamil) treatment and all significant p values are reported. Two-tailed, paired t-tests were performed for data in panel (a) to detect significant change induced by drugs compared to Blebbistatin and all significant p values are reported. Benjamini–Hochberg correction was applied to account for multiple comparisons.

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  Fig. 4. Triple-parametric optical mapping in cardiac disease assessment. a Representative Vm activation maps (top), Ca2+ activation maps (middle), and NADH intensity maps (bottom) were recorded from the same mouse heart during baseline, ischemia, and reperfusion conditions. All maps in a given column were obtained by simultaneous imaging. b Summary of seven parameters measured over time, simultaneously by triple-parametric optical mapping. Three parameters—APD80, CaTD80, and Ca2+ decay were not measurable due to motion artifacts in the repolarization phase due to absence of electromechanical uncoupling. The values reported are normalized to baseline (t = 0) for each heart in order to determine modulation of each parameter in each heart with respect to its own baseline condition. c Ten-parameter panels (only seven parameters reported) demonstrating changes in cardiac physiology during ischemia and restoration during reperfusion. Data are presented as mean ± s.e.m., n = 5 hearts. Two-tailed, paired t-tests were performed to detect significant change induced by ischemia and reperfusion compared to baseline and all significant p values are reported. Benjamini–Hochberg correction was applied…