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. AbstractTransmural 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.

Minimally invasive system to reliably characterize ventricular electrophysiology from living donors

“…Importantly, a slice thickness of 350 µm is selected to maximize viability while attaining optimal electrophysiological signals. This value is in line with other studies that have reported thicknesses ranging from 150 to 500 µm 11 , 12 , 15 , 37 , with most studies in humans setting it at 300–400 µm 6 , 7 , 11 , 16 , 33 . Also, a post-cutting recovery time of 30 min is applied, consistent with previously reported times for myocardial slices to allow them to attain steady-state properties 6 , 7 , 13 , 15 , 16 , 33 , 37 .…”

“…www.nature.com/scientificreports/ thicknesses ranging from 150 to 500 µm 11,12,15,37 , with most studies in humans setting it at 300-400 µm 6,7,11,16,33 . Also, a post-cutting recovery time of 30 min is applied, consistent with previously reported times for myocardial slices to allow them to attain steady-state properties 6,7,13,15,16,33,37 . We note that some transmural core biopsies render slices with areas of not totally aligned fibers, accumulation of fibrosis or presence of blood vessels, which may represent a higher percentage of its total volume as compared to other more commonly used myocardial slices.…”

“…Optical mapping allows the characterization of action potentials from transmural core biopsy slices with high spatial resolution. The electrophysiological evaluation of myocardial tissue slices is evolving from multi-electrode arrays 6 , 12 , 15 , 20 , 24 , 33 to optical mapping for the analysis of action potentials and/or intracellular calcium transients 13 , 16 , 19 , 24 , 33 , 37 – 39 . Here we show that AP duration and morphology are comparable in slices from transmural core biopsies and tissue blocks from the same pig ventricular regions.…”

Epicardial slices: a 3D organotypic model for the study of epicardial activation and differentiation

“…Our approach advances these existing models by including a fully functional epicardium, physiologically interfaced to the myocardium. To enable these preparations, we advanced existing embedding methods to protect the epicardial surface whilst achieving a flat cutting surface (27,28). In fact, the epicardium has commonly been used to attach cardiac tissue blocks to specimen holders due to its flatness, which helps to align the tissue, preventing their damage during cutting (29), our methods achieves similar results, without losing the epicardium.…”

The best thing since sliced bread? Optical mapping of transverse cardiac slices in the mouse heart

“…In this issue of The Journal of Physiology , Wen and colleagues () present a novel optical mapping approach using short‐axis tissue slices from the mouse heart that provides for unprecedented, and potentially high‐throughput, characterization of action potentials (APs) and intracellular Ca 2+ transients (CaTs) everywhere in the mouse ventricles. Following dye loading via traditional Langendorff perfusion, the ventricles were embedded in low‐melt agarose and thin (300 μm) short‐axis slices were cut from apex to base, resulting in approximately 20 slices comprising the entire mouse ventricles (akin to a loaf of sliced bread).…”