May the Force Not Be With You During Culture: Eliminating Mechano-Associated Feedback During Culture Preserves Cultured Atrial and Pacemaker Cell Functions

Abstract

Cultured cardiomyocytes have been shown to possess significant potential as a model for characterization of mechano-Ca 2+ , mechano-electric, and mechano-metabolic feedbacks in the heart. However, the majority of cultured cardiomyocytes exhibit impaired electrical, mechanical, biochemical, and metabolic functions. More specifically, the cells do not beat spontaneously (pacemaker cells) or beat at a rate far lower than their physiological counterparts and self-oscillate (atrial and ventricular cells) in culture. Thus, efforts are being invested in ensuring that cultured cardiomyocytes maintain the shape and function of freshly isolated cells. Elimination of contraction during culture has been shown to preserve the mechano-Ca 2+ , mechano-electric, and mechanometabolic feedback loops of cultured cells. This review focuses on pacemaker cells, which reside in the sinoatrial node (SAN) and generate regular heartbeat through the initiation of the heart's electrical, metabolic, and biochemical activities. In parallel, it places emphasis on atrial cells, which are responsible for bridging the electrical conductance from the SAN to the ventricle. The review provides a summary of the main mechanisms responsible for mechano-electrical, Ca 2+ , and metabolic feedback in pacemaker and atrial cells and of culture methods existing for both cell types. The work concludes with an explanation of how the elimination of mechano-electrical, mechano-Ca 2+ , and mechano-metabolic feedbacks during culture results in sustained cultured cell function.

Deciphering cellular signals in adult mouse sinoatrial node cells

“…However, the data were not shown, which made difficult an appropriate comparison between studies. Conversely, recent studies have suggested that blebbistatin and its non-fluorescent derivative (S)-nitro blebbistatin (Kepiro et al, 2014) may be more effective at improving survival and preserving the morphology of cultured rabbit SAN cells and ventricular myocytes (Kirschner Peretz et al, 2020;Reddy et al, 2018;Segal et al, 2019). Consistent with this, by analyzing a population of SAN cells within the same field of view, the present study found that blebbistatin and (S)-nitro blebbistatin help maintain cell morphology in a significantly larger number of cultured cells compared with treatments with M1018 or BDM (Figure 1).…”

“…Moreover, under certain culturing conditions, SAN cells undergo a rapid remodeling that alters their electrical signals and morphology (Segal et al, 2019;Yang et al, 2012), which may impact the accurate assessment of the spatiotemporal dynamics of signaling networks. Thus, a method is necessary to culture SAN cells under conditions in which their functional properties and morphology are minimally impacted (Kirschner Peretz et al, 2020) while allowing for the expression of genetically encoded biosensors.…”

“…Prior studies suggest that contraction uncouplers, such as 2,3-butanedione 2-monoxime (BDM) or blebbistatin, may help preserve morphology and functional properties of cultured cardiac cells (Kabaeva et al, 2008;Kirschner Peretz et al, 2017Kivisto et al, 1995;Liu et al, 1996;Muramatsu et al, 1996;Reddy et al, 2018;Segal et al, 2019;St Clair et al, 2015;Yang et al, 2012). However, the efficiency of these treatments, especially BDM and blebbistatin, for maintaining a large population of adult mouse SAN cells in culture is unclear, as prior reports have shown examples of only one isolated cell per culturing condition (Kirschner Peretz et al, 2020;Segal et al, 2019;St Clair et al, 2015). In one study, it was reported that at least 13% of the rabbit SAN cells using a culturing method with 25 mM blebbistatin lost their spindle-shaped morphology and developed some projections (Segal et al, 2019).…”

Ca2+-Driven Selectivity of the Effect of the Cardiotonic Steroid Marinobufagenin on Rabbit Sinoatrial Node Function

“…Briefly, rabbits were sedated with ketamine and xylazine (0.1 mL/kg each) and anesthetized with sodium pentobarbital (200 mg/mL) diluted with heparin, administered through an intravenous cannula. After quick removal, the hearts were placed in a custom-made, silicone-covered chamber, and the SAN and right atrial tissues were separated, as previously described [10], while bathed in 37 • C Tyrode's solution (125 mM NaCl, 5.6 mM KCl, 1.2 mM NaH 2 PO 4 , 24 mM NaHCO 3 , 5.6 mM glucose, 1 mM MgCl 2 and 1.8 mM CaCl 2 , bubbled with 95% O 2 and 5% CO 2 ). Once separated from the surrounding atrial tissue, SAN tissue was processed as previously described [11] in order to obtain single spontaneously beating SANCs.…”