Changes in cAMP signaling are associated with age-related downregulation of spontaneously beating atrial tissue energetic indices.

The prevalence of atria-related diseases increases exponentially with age and is associated with ATP supply-to-demand imbalances. Because evidence suggests that cAMP regulates ATP supply-to-demand, we explored aged-associated alterations in atrial ATP supply-to-demand balance and its correlation with cAMP levels. Right atrial tissues driven by spontaneous sinoatrial node impulses were isolated from aged (22-26 months) and adult (3-4 months) C57/BL6 mice. ATP demand increased by addition of isoproterenol or 3-Isobutyl-1-methylxanthine (IBMX) and decreased by application of carbachol. Each drug was administrated at the dose that led to a maximal change in beating rate (Xmax) and to 50% of that maximal change in adult tissue (X50). cAMP, NADH, NAD + NADH, and ATP levels were measured in the same tissue. The tight correlation between cAMP levels and the beating rate (i.e., the ATP demand) demonstrated in adult atria was altered in aged atria. cAMP levels were lower in aged compared to adult atrial tissue exposed to X50 of ISO or IBMX, but this difference narrowed at Xmax. Neither ATP nor NADH levels correlated with ATP demand in either adult or aged atria. Baseline NADH levels were lower in aged as compared to adult atria, but were restored by drug perturbations that increased cAMP levels. Reduction in Ca2+-activated adenylyl cyclase-induced decreased cAMP and prolongation of the spontaneous beat interval of adult atrial tissue to their baseline levels in aged tissue, brought energetics indices to baseline levels in aged tissue. Thus, cAMP regulates right atrial ATP supply-to-demand matching and can restore age-associated ATP supply-to-demand imbalance.

Distinct PKA Signaling in Cytosolic and Mitochondrial Compartments in Electrically Paced Atrial Myocytes.

Protein kinase A (PKA) is a key nodal signaling molecule that regulates a wide range of cellular functions in the cytosol and mitochondria. The distribution of A-kinase anchoring proteins that tether PKA, the local interaction with degradation molecules, and regulation by Ca2+, may lead to distinct spatiotemporal cAMP/PKA signaling in these compartments. In this work, FRET-based sensors were used to investigate PKA signaling in the cytosol, outer mitochondrial membrane (OMM), and mitochondrial matrix (MM) and its crosstalk with Ca2+ in response to electrical stimulation of cultured rabbit atrial cells. A gradual decrease in PKA activity eliminating the ability of the atrial cells to respond to physiological electrical stimulation, was observed upon treatment of cells with H-89. Chelation of intracellular Ca2+ by BAPTA reduced PKA activity and diminished its response to forskolin, an AC stimulator. Under basal conditions, PKA activity in response to forskolin was lower in the OMM compared to the cytosol and MM. In response to electrical stimulation in the presence of ISO, distinct compartmentalization of PKA activity was observed, with higher activity in the cytosol and MM than in the OMM. Thus, distinct Ca2+-dependent spatiotemporal cAMP/PKA signaling exists in atrial cells, likely mediating its excitation and mitochondrial function.

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

Cultured cardiomyocytes have been shown to possess significant potential as a model for characterization of mechano-Ca2+, 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-Ca2+, mechano-electric, and mechano-metabolic 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, Ca2+, 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-Ca2+, and mechano-metabolic feedbacks during culture results in sustained cultured cell function.

Eliminating contraction during culture maintains global and local Ca2+ dynamics in cultured rabbit pacemaker cells.

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 10 mM 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.

A Method Sustaining the Bioelectric, Biophysical, and Bioenergetic Function of Cultured Rabbit Atrial Cells.

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.

Semi-automated program for analysis of local Ca2+ spark release with application for classification of heart cell type.

Local Ca2+ spark releases are essential to the Ca2+ cycling process. Thus, they play an important role in ventricular and atrial cell contraction, as well as in sinoatrial cell automaticity. Characterizing their properties in healthy cells from different regions in the heart can reveal the basic biophysical differences among these regions. We designed a semi-automatic Matlab Graphical User Interface (called Sparkalyzer) to characterize parameters of Ca2+ spark release from any major cardiac tissue, as recorded in line-scan mode with a confocal laser-scanning microscope. We validated the algorithm on experimental images from rabbit sinoatrial, atrial, and ventricular cells loaded with Fluo-4 AM. The program characterizes general image parameters of Ca2+ transients and sparks: spark duration, which indicates for how long the spark provides Ca2+ to the closed intracellular mechanisms (typical value: 25±1, 23±1, 26±1ms for sinoatrial, atrial, and ventricular cells, respectively); spark amplitude, which indicates the amount of Ca2+ released by a single spark (1.6±0.1, 1.6±0.2, 1.4±0.1F/F0 for sinoatrial, atrial, and ventricular cells, respectively); spark length, which is the length of the Ca2+ wavelets fired out of a row of ryanodine receptors (5±0.1, 5±0.2, 3.4±0.3µm for sinoatrial, atrial, or ventricular cells, respectively) and number of sparks (0.14±0.02, 0.025±0.01, 0.02±0.01 for 1µm in 1s for sinoatrial, atrial, and ventricular cells, respectively). This method is reliable for Ca2+ spark analysis of sinoatrial, atrial, or ventricular cells. Moreover, by examining the average value of Ca2+ spark characteristics and their scattering around the mean, atrial, ventricular and sinoatrial cells can be differentiated.