Three-Week-Old Rabbit Ventricular Cardiomyocytes as a Novel System to Study Cardiac Excitation and EC Coupling

Abstract

Cardiac arrhythmias significantly contribute to cardiovascular morbidity and mortality. The rabbit heart serves as an accepted model system for studying cardiac cell excitation and arrhythmogenicity. Accordingly, primary cultures of adult rabbit ventricular cardiomyocytes serve as a preferable model to study molecular mechanisms of human cardiac excitation. However, the use of adult rabbit cardiomyocytes is often regarded as excessively costly. Therefore, we developed and characterized a novel low-cost rabbit cardiomyocyte model, namely, 3-week-old ventricular cardiomyocytes (3wRbCMs). Ventricular myocytes were isolated from whole ventricles of 3-week-old New Zealand White rabbits of both sexes by standard enzymatic techniques. Using wheat germ agglutinin, we found a clear T-tubule structure in acutely isolated 3wRbCMs. Cells were adenovirally infected (multiplicity of infection of 10) to express Green Fluorescent Protein (GFP) and cultured for 48 h. The cells showed action potential duration (APD90 = 253 ± 24 ms) and calcium transients similar to adult rabbit cardiomyocytes. Freshly isolated and 48-h-old-cultured cells expressed critical ion channel proteins: calcium voltage-gated channel subunit alpha1 C (Cavα1c), sodium voltage-gated channel alpha subunit 5 (Nav1.5), potassium voltage-gated channel subfamily D member 3 (Kv4.3), and subfamily A member 4 (Kv1.4), and also subfamily H member 2 (RERG. Kv11.1), KvLQT1 (K7.1) protein and inward-rectifier potassium channel (Kir2.1). The cells displayed an appropriate electrophysiological phenotype, including fast sodium current (INa), transient outward potassium current (Ito), L-type calcium channel peak current (ICa,L), rapid and slow components of the delayed rectifier potassium current (IKr and IKs), and inward rectifier (IK1). Although expression of the channel proteins and some currents decreased during the 48 h of culturing, we conclude that 3wRbCMs are a new, low-cost alternative to the adult-rabbit-cardiomyocytes system, which allows the investigation of molecular mechanisms of cardiac excitation on morphological, biochemical, genetic, physiological, and biophysical levels.

Myofibroblast senescence promotes arrhythmogenic remodeling in the aged infarcted rabbit heart

“…We hypothesized that SASP factors from senescent cardiac myofibroblasts in the scar or IBZ may act in a paracrine fashion on IBZ myocytes to alter ion channel function, ultimately prolonging APD and potentially establishing both a substrate and trigger for arrhythmias. To test this hypothesis, we employed an in vitro model using 3-week-old rabbit cardiomyocytes and adult rabbit cardiac myofibroblasts ( Kabakov et al, 2021 ). Acutely dissociated cardiomyocytes from 3-week-old rabbits were treated for 30 min with conditioned media collected from adult rabbit cardiac fibroblasts treated for 12 days with either vehicle (‘proliferating-CM’) or 20 μM etoposide (‘SASP-CM’), followed by patch clamping of the myocytes.…”

“…Primary cardiomyocytes were isolated from the LV and septum of 3-week-old female rabbits as previously described ( Cooper et al, 2013 ; Kabakov et al, 2021 ). Myocytes were incubated with either SASP-CM, Pro-CM, or UCM (basal media used consisted of DMEM [Life Technologies] supplemented with 7% fetal bovine serum, penicillin [100 μg/mL], and streptomycin [0.01 μg/mL]) for 30 min in a 37°C incubator before patch clamping.…”

“…Whole-cell patch clamp recordings from 3wkRCMs were performed with an Axopatch-200B amplifier and pCLAMP 10 software at 34–36°C exactly as described previously ( Kabakov et al, 2021 ).…”

E3 ubiquitin ligase rififylin has yin and yang effects on rabbit cardiac transient outward potassium currents (Ito) and corresponding channel proteins

“…, APs and calcium transients) and morphology (rod-shape and T-tubular structure) of cardiomyocytes as published elsewhere ( 27 ). In the past, we used 0.5 μM cytochalasin D to prevent dedifferentiation in 3-week-old adult rabbit cardiomyocytes cultured for 48 h ( 28 ). Although some functional parameters, such as resting potential and I Na , did not change after a 48-h culture of cardiomyocytes, others did ( e.g.…”