An improved procedure for isolating adult mouse cardiomyocytes for epicardial activation mapping

Abstract

Cardiovascular disease is a leading cause of death and disability worldwide. Although genetically modified mouse models offer great potential for robust research in vivo, in vitro studies using isolated cardiomyocytes also provide an important approach for investigating the mechanisms underlying cardiovascular disease pathogenesis and drug actions. Currently, isolation of mouse adult cardiomyocytes often relies on aortic retrograde intubation under a stereoscopic microscope, which poses considerable technical barriers and requires extensive training. Although a simplified, Langendorff-free method has been used to isolate viable cardiomyocytes from the adult mouse heart, the system requires enzymatic digestions and continuous manual technical operation. This study established an optimized approach that allows isolation of adult mouse cardiomyocytes and epicardial activation mapping of mouse hearts using a Langendorff device. We used retrograde puncture through the abdominal aorta in vivo and enzymatic digestion on the Langendorff perfusion device to isolate adult mouse cardiomyocytes without using a microscope. The yields of isolated cardiomyocytes were amenable to patch clamp techniques. Furthermore, this approach allowed epicardial activation mapping. We used a novel, simplified method to isolate viable cardiomyocytes from adult mouse hearts and to map epicardial activation. This novel approach could be beneficial in more extensive research in the cardiac field.

Keywords: Langendorff; adult mouse cardiomyocytes; cardiomyocyte isolation; electrophysiology; epicardial activation mapping.

FIGURE 1

Isolation of adult mouse cardiomyocytes using the Langendorff device. Isolated cardiomyocytes display a long rod shape with clear horizontal stripes and sharp edges and are calcium tolerant (i.e. stay quiescent in tyrode solution). Scale bar =100 μm

FIGURE 2

Electrophysiological characteristics of isolated cardiomyocytes indicated that the cells are amenable to patch clamp. (A) Ito current. (B) ICa, L current. (C) Action potential. (D) INa current. E. Influence on INa current by drug A

FIGURE 3

Effects of drug A on electrophysiological characteristics of isolated cardiomyocytes indicated that the cells are amenable to patch clamp. (A) I‐V curve of INa current. Average Na sodium current density is greater after treatment of drug A (orange curve), n = 5. (B) Current density of INa at −20 mV, n = 5. (C) Influence of different concentrations of drug A on action potential

FIGURE 4

Electrophysiological epicardial activation mapping. (A) LA: representative activation maps of left atrium. (B) LV: representative activation maps of left ventricle. (C) LV‐8hz: representative activation maps of left ventricle by 8 Hz stimulation. Red indicates the first excited place, and blue indicates the last excited place; the direction of conduction is from red to blue. (D) Signals of APD₉₀ (APD at 90% repolarization) and CAD₉₀ (changes in intracellular calcium). *Scale bar, 50 ms. (E) Representative activation maps of action potential conduction with 5 Hz stimulation. (F) Representative activation maps of calcium with 5 Hz stimulation in the presence of different concentrations of drug A. Ruler: red indicates the first excited place, and blue indicates the last excited place; the conduction direction arrives from red to blue