Transverse cardiac slicing and optical imaging for analysis of transmural gradients in membrane potential and Ca²⁺ transients in murine heart

Figures

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  Figure 1. Optimisation of murine cardiac slice methodology A, voltage (RH237) or calcium (Rhod‐2 AM) dyes were loaded via coronary circulation using the Langendorff perfusion system. B, ventricles were embedded in 4% low‐melt agarose and mounted onto the vibratome specimen holder. C, up to 24 transverse slices were cut from the apex to the base in ice‐cold BDM Tyrode solution. D, slices were recovered at room temperature in Krebs solution containing 10 μM blebbistatin for 30 min, before optical mapping studies. E, V m signals in slices cut at 150 μm and 300 μm thickness. F, optical mapping setup with four 530 nm excitation LEDs and a 128 × 128 EMCCD camera.

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  Figure 2. V m and CaT measurements in murine ventricular slices A, bright field images of transverse ventricular slices (300 μm thick) prepared from both ventricles of a mouse heart (from the apex to the base). Scale bar: 2 cm. B, AP duration (APD; Ba) and CaT duration (CaTD; Bb) maps generated from a slice prepared from the central region of the ventricles, with raw AP and CaT signals acquired from the epicardium (Epi) and endocardium (Endo) of the left ventricle, the ventricular septum (SEP) and the right ventricle (RV). APD scale bar: 80 ms.

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  Figure 3. Transmural and regional distribution of AP duration across the murine ventricles A, representative maps of AP duration (APD75) at 2 Hz pacing frequency (500 ms pacing cycle length) recorded from apex to base in transverse ventricular slices. B, typical optical AP recordings (unfiltered signals) obtained from different regions of the murine ventricles at 2 Hz pacing frequency. Scale bar: 80 ms. C, quantitative summary of transmural and regional APD50 (Ca and Cb) and APD75 distribution (Cc and Cd) at 2 Hz pacing (n = 5 hearts; ** P < 0.01; * P < 0.05). Values expressed as means ± SEM. Epi, epicardium; Endo, endocardium; SEP, septum; RV, right ventricle; LV, left ventricle.

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  Figure 4. Transmural and regional distribution of CaT duration in the murine ventricles A, representative maps of CaT duration (CaTD75) at 2 Hz pacing frequency (500 ms pacing cycle length) recorded from apex to base in transverse ventricular slices. B, representative optical CaT raw traces obtained from different regions of the murine ventricles at 2 Hz pacing frequency. Scale bar: 80 ms. C, quantitative summary of transmural and regional CaTD50 (Ca and Cb) and CaTD75 distribution (Cc and Cd) at 2 Hz pacing (n = 5–8 hearts; ** P < 0.01; * P < 0.05). Values expressed as means ± SEM. Epi, epicardium; Endo, endocardium; SEP, septum; RV, right ventricle; LV, left ventricle.

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  Figure 5. Regional and frequency‐dependent distribution of AP alternans and arrhythmic events in murine ventricular slices A, representative optical AP traces obtained from different regions within a ventricular slice during electrical pacing at 2, 4, 8 and 16 Hz frequency. Scale bar: 1s. B, transmural and regional occurrence of alternans and arrhythmias at 8 Hz and 16 Hz pacing frequency. Epi, epicardium; Endo, endocardium; SEP, septum; RV, right ventricle; LV, left ventricle. n = 6 hearts.

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  Figure 6. Analysis of conduction velocity (CV) A, activation maps of slices (apex to base) paced at 2 Hz and 16 Hz. Conduction velocity (CV) was calculated using a multi‐vector polynomial method within bespoke ElectroMap analysis software. A polynomial surface was fitted to local activation times to describe propagation in the area, and local CV quantified as the gradient vector of the polynomial surface. Mean CV was then calculated from the local CVs across the tissue slice. B, mean data for CV at a range of pacing frequencies calculated across all the slices (n = 38 slices/3 hearts). C, mean data for CV at a range of pacing frequencies calculated across all the hearts (n = 3 hearts).

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  Figure 7. Regional and frequency‐dependent distribution of CaT alternans and arrhythmic events in murine ventricular slices A, representative optical CaT traces obtained from different regions within a ventricular slice during electrical pacing at 2, 4, 8 and 16 Hz frequency. Scale bar: 1s. B, transmural and regional occurrence of alternans and arrhythmias at 8 Hz and 16 Hz pacing frequency. Epi, epicardium; Endo, endocardium; SEP, septum; RV, right ventricle; LV, left ventricle. n = 6 hearts.