A Protocol for Transverse Cardiac Slicing and Optical Mapping in Murine Heart

Abstract

Thin living tissue slices have recently emerged as a new tissue model for cardiac electrophysiological research. Slices can be produced from human cardiac tissue, in addition to small and large mammalian hearts, representing a powerful in vitro model system for preclinical and translational heart research. In the present protocol, we describe a detailed mouse heart transverse slicing and optical imaging methodology. The use of this technology for high-throughput optical imaging allows study of electrophysiology of murine hearts in an organotypic pseudo two-dimensional model. The slices are cut at right angles to the long axis of the heart, permitting robust interrogation of transmembrane potential (V_m) and calcium transients (CaT) throughout the entire heart with exceptional regional precision. This approach enables the use of a series of slices prepared from the ventricles to measure V_m and CaT with high temporal and spatial resolution, allowing (i) comparison of successive slices which form a stack representing the original geometry of the heart; (ii) profiling of transmural and regional gradients in V_m and CaT in the ventricle; (iii) characterization of transmural and regional profiles of action potential and CaT alternans under stress (e.g., high frequency pacing or β-adrenergic stimulation) or pathological conditions (e.g., hypertrophy). Thus, the protocol described here provides a powerful platform for innovative research on electrical and calcium handling heterogeneity within the heart. It can be also combined with optogenetic technology to carry out optical stimulation; aiding studies of cellular V_m and CaT in a cell type specific manner.

Keywords: Ca2+ transients; membrane potentials; murine heart; optical mapping; transverse cardiac slice.

FIGURE 1

Optical mapping set-up. The system consists of a camera (Photometrics Evolve Delta 512) running under Metamorph (Molecular Devices) in Light-speed mode, giving a high temporal resolution of sub-frames, up to 1000 frames/sec, and spatial resolution at the sample of 74 × 74 μm per pixel. The sample was imaged using a custom MacroScope (Cairn Research) with an F/0.95, 25 mm C-mount camera lens, spaced so as to give a working distance of approximately 40 mm. The excitation light was provided by a four light emitting diode MacroLED lamps (525 nm, 1750 lumen, 7 mm² emitters, Cairn Research) with aspheric condensers directed onto the slices from the four corners of the bath, so as to produce an even, near-critical, illumination at approximately 30 mm. The LEDs were driven using MacroLED control boxes and digitally modulated from Metamorph software with a National Instruments multifunction card. The LEDs were individually filtered using ET525/50 sputter coated filters (Chroma Technology) to remove any out-of-band light. The fluorescence emission light was split with a 610 nm long-pass dichroic mirror and corresponding emitters to separate the Ca²⁺ and voltage signals based on their wavelengths. The voltage-sensitive dye, RH237, emits signals which exhibit a peak at 670 nm, while Rhod-2 AM has a peak emission at approximately 600 nm and, once split by the dichroic mirror, these two signals are imaged on to the camera such that they are reproduced side-by-side on the sensor and recorded simultaneously. This dichroic and adjustable mirror unit, the OptoSplit, was provided by Cairn Research with filters from Chroma Technologies (United States). CaT fluorescence was collected at 585 ± 40 nm and Vm using a 662 nm long pass filter. Vm and CaT measurements were taken at maximal resolution (128 × 128 pixels; pixel area 74 × 74 μm) at a rate of 1000 frames/s.

FIGURE 2

The major steps of the protocol. Representative harvested mouse heart and transverse slices from apex to base. Briefly, after the Langendoff perfusion steps (A), embed the heart in 4% low-melting agarose and cut with right angle to the long axis by Leica VT1000s vibratome (B). Slices are transferred to petri dishes (C) and voltage and calcium transients recorded (D).

FIGURE 3

Dual Vm and CaT imaging in murine transverse ventricular slices. (A) Fluorescence image (voltage-RH237) of transverse slices of murine ventricles from apex to base, dual loaded with both transmembrane voltage (RH237) and intracellular calcium dyes (Rhod-2 AM). (B) Representative maps of AP duration (APD50) at 2 Hz pacing frequency (500 ms pacing cycle length) recorded from apex to base ventricular slices. (C) Representative maps of Calcium transient duration (CaTD50) at 2 Hz pacing frequency recorded from apex to base ventricular slices.

FIGURE 4

Comparison of electrical (field stimulation) and light (470 nm pulses) pacing in murine transverse ventricular slices. (A) Activation map and example signals (transmembrane voltage, RH237) from slice paced using electrical field stimulation. (B) Activation map and example signals from the same slice paced using blue light stimulation of ChR2. (C) APD75 map from slice during electrical field stimulation. (D) APD75 map from slice during blue light stimulation.