Pacemaker cells residing in the sinoatrial node generate the regular heartbeat. Ca2+ signaling controls the heartbeat rate—directly, through the effect on membrane molecules (NCX exchange, K+ channel), and indirectly, through activation of calmodulin-AC-cAMP-PKA signaling. Thus, the physiological role of signaling in pacemaker cells can only be assessed if the Ca2+ dynamics are in the physiological range. Cultured cells that can be genetically manipulated and/or virally infected with probes are required for this purpose. Because rabbit pacemaker cells in culture experience a decrease in their spontaneous action potential (AP) firing rate below the physiological range, Ca2+ dynamics are expected to be affected. However, Ca2+ dynamics in cultured pacemaker cells have not been reported before. We aim to a develop a modified culture method that sustains the global and local Ca2+ kinetics along with the AP firing rate of rabbit pacemaker cells.
We used experimental and computational tools to test the viability of rabbit pacemaker cells in culture under various conditions. We tested the effect of culture dish coating, pH, phosphorylation, and energy balance on cultured rabbit pacemaker cells function. The cells were maintained in culture for 48 h in two types of culture media: one without the addition of a contraction uncoupler and one enriched with either 10mM BDM (2,3-Butanedione 2-monoxime) or 25 μM blebbistatin. The uncoupler was washed out from the medium prior to the experiments. Cells were successfully infected with a GFP adenovirus cultured with either BDM or blebbistatin. Using either uncoupler during culture led to the cell surface area being maintained at the same level as fresh cells. Moreover, the phospholamban and ryanodine receptor densities and their phosphorylation level remained intact in culture when either blebbistatin or BDM were present. Spontaneous AP firing rate, spontaneous Ca2+ kinetics, and spontaneous local Ca2+ release parameters were similar in the cultured cells with blebbistatin as in fresh cells. However, BDM affects these parameters. Using experimental and a computational model, we showed that by eliminating contraction, phosphorylation activity is preserved and energy is reduced. However, the side-effects of BDM render it less effective than blebbistatin.
Culturing atrial cells leads to a loss in their ability to be externally paced at physiological rates and to maintain their shape. We aim to develop a culture method that sustains the shape of atrial cells along with their biophysical and bioenergetic properties in response to physiological pacing. We hypothesize that adding 2,3-Butanedione 2-monoxime (BDM), which inhibits contraction during the culture period, will preserve these biophysical and bioenergetic properties. Rabbit atrial cells were maintained in culture for 24 h in a medium enriched with a myofilament contraction inhibitor, BDM. The morphology and volume of the cells, including their ability to contract in response to 1–3 Hz electrical pacing, was maintained at the same level as fresh cells. Importantly, the cells could be successfully infected with a GFP adenovirus. Action potentials, Ca2+ transients, and local Ca2+ spark parameters were similar in the cultured and in fresh cells. Finally, these cultured cells' flavoprotein autofluorescence was maintained at a constant level in response to electrical pacing, a response similar to that of fresh cells. Thus, eliminating contraction during the culture period preserves the bioelectric, biophysical and bioenergetic properties of rabbit atrial myocytes. This method therefore has the potential to further improve our understanding of energetic and biochemical regulation in the atria.
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.
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