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

Abstract

Key points: A robust cardiac slicing approach was developed for optical mapping of transmural gradients in transmembrane potential (V_m ) and intracellular Ca²⁺ transient (CaT) of murine heart. Significant transmural gradients in V_m and CaT were observed in the left ventricle. Frequency-dependent action potentials and CaT alternans were observed in all ventricular regions with rapid pacing, with significantly greater incidence in the endocardium than epicardium. The observations demonstrate the feasibility of our new approach to cardiac slicing for systematic analysis of intrinsic transmural and regional gradients in V_m and CaT.

Abstract: Transmural and regional gradients in membrane potential and Ca²⁺ transient in the murine heart are largely unexplored. Here, we developed and validated a robust approach which combines transverse ultra-thin cardiac slices and high resolution optical mapping to enable systematic analysis of transmural and regional gradients in transmembrane potential (V_m ) and intracellular Ca²⁺ transient (CaT) across the entire murine ventricles. The voltage dye RH237 or Ca²⁺ dye Rhod-2 AM were loaded through the coronary circulation using a Langendorff perfusion system. Short-axis slices (300 μm thick) were prepared from the entire ventricles (from the apex to the base) by using a high-precision vibratome. Action potentials (APs) and CaTs were recorded with optical mapping during steady-state baseline and rapid pacing. Significant transmural gradients in V_m and CaT were observed in the left ventricle, with longer AP duration (APD₅₀ and APD₇₅ ) and CaT duration (CaTD₅₀ and CaTD₇₅ ) in the endocardium compared with that in the epicardium. No significant regional gradients were observed along the apico-basal axis of the left ventricle. Interventricular gradients were detected with significantly shorter APD₅₀ , APD₇₅ and CaTD₅₀ in the right ventricle compared with left ventricle and ventricular septum. During rapid pacing, AP and CaT alternans were observed in most ventricular regions, with significantly greater incidence in the endocardium in comparison with epicardium. In conclusion, these observations demonstrate the feasibility of our new approach to cardiac slicing for systematic analysis of intrinsic transmural and regional gradients in V_m and CaT in murine ventricular tissue.

Keywords: cardiac slices; electrophysiological heterogeneity; murine heart; optical imaging.

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.

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.

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.

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.

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.

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).

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.