Synergy between Membrane Currents Prevents Severe Bradycardia in Mouse Sinoatrial Node Tissue.

Bradycardia is initiated by the sinoatrial node (SAN), which is regulated by a coupled-clock system. Due to the clock coupling, reduction in the 'funny' current (If), which affects SAN automaticity, can be compensated, thus preventing severe bradycardia. We hypothesize that this fail-safe system is an inherent feature of SAN pacemaker cells and is driven by synergy between If and other ion channels. This work aimed to characterize the connection between membrane currents and their underlying mechanisms in SAN cells. SAN tissues were isolated from C57BL mice and Ca2+ signaling was measured in pacemaker cells within them. A computational model of SAN cells was used to understand the interactions between cell components. Beat interval (BI) was prolonged by 54 ± 18% (N = 16) and 30 ± 9% (N = 21) in response to If blockade, by ivabradine, or sodium current (INa) blockade, by tetrodotoxin, respectively. Combined drug application had a synergistic effect, manifested by a BI prolonged by 143 ± 25% (N = 18). A prolongation in the local Ca2+ release period, which reports on the level of crosstalk within the coupled-clock system, was measured and correlated with the prolongation in BI. The computational model predicted that INa increases in response to If blockade and that this connection is mediated by changes in T and L-type Ca2+ channels.

Increase in Ca2+-Activated cAMP/PKA Signaling Prevents Hydroxychloroquine-Induced Bradycardia of the Cardiac Pacemaker.

Bradycardia or tachycardia are known side effects of drugs that limit their clinical use. The heart pacemaker function which control the heart rate under normal conditions is determined by coupled clock system. Thus, interfering with specific clock mechanism will affect other clock mechanisms through changes in interconnected signaling and can lead to rhythm disturbance. However, upregulation of a different clock components can compensate for this change. We focus here on hydroxychloroquine (HCQ), which has been shown effective in treating COVID-19 patients, however its bradycardic side effect limits its clinical use. We aim to decipher the mechanisms underlying the effect of HCQ on pacemaker automaticity, to identify a potential drug that will eliminate the bradycardia. We used isolated rabbit sinoatrial node (SAN) cells, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and mouse SAN cells residing in SAN tissue. Further, we employed SAN cell computational model to suggest mechanistic insights of the effect of HCQ on pacemaker function. HCQ increased mean spontaneous beat interval and variability in all three models in parallel to slower intracellular kinetics. The computational model suggested that HCQ affects the pacemaker (funny) current (If), L-type Ca2+ current (ICa,L), transient outward potassium (Ito) and due to changes in Ca2+ kinetics, the sodium-calcium exchanger current (INCX). Co-application of 3'-isobutylmethylxanthine (IBMX) and HCQ prevented the increase in beat interval and variability in all three experimental models. The HCQ-induced increase in rabbit and mice SAN cell and hiPSC-CM spontaneous beat interval, can be prevented by a phosphodiester inhibitor that restores automaticity due to slower intracellular Ca2+ kinetics.

Distinct mechanisms mediate pacemaker dysfunction associated with catecholaminergic polymorphic ventricular tachycardia mutations: Insights from computational modeling.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced ventricular arrhythmia associated with rhythm disturbance and impaired sinoatrial node cell (SANC) automaticity (pauses). Mutations associated with dysfunction of Ca2+-related mechanisms have been shown to be present in CPVT. These dysfunctions include impaired Ca2+ release from the ryanodine receptor (i.e., RyR2R4496C mutation) or binding to calsequestrin 2 (CASQ2). In SANC, Ca2+ signaling directly and indirectly mediates pacemaker function. We address here the following research questions: (i) what coupled-clock mechanisms and pathways mediate pacemaker mutations associated with CPVT in basal and in response to ß-adrenergic stimulation? (ii) Can different mechanisms lead to the same CPVT-related pacemaker pauses? (iii) Can the mutation-induced deteriorations in SANC function be reversed by drug intervention or gene manipulation? We used a numerical model of mice SANC that includes membrane and intracellular mechanisms and their interconnected signaling pathways. In the basal state of RyR2R4496C SANC, the model predicted that the Na+-Ca2+ exchanger current (INCX) and T-type Ca2+ current (ICaT) mediate between changes in Ca2+ signaling and SANC dysfunction. Under ß-adrenergic stimulation, changes in cAMP-PKA signaling and the sodium currents (INa), in addition to INCX and ICaT, mediate between changes in Ca2+ signaling and SANC automaticity pauses. Under basal conditions in Casq2-/-, the same mechanisms drove changes in Ca2+ signaling and subsequent pacemaker dysfunction. However, SANC automaticity pauses in response to ß-AR stimulation were mediated by ICaT and INa. Taken together, distinct mechanisms can lead to CPVT-associated SANC automaticity pauses. In addition, we predict that specifically increasing SANC cAMP-PKA activity by either a pharmacological agent (IBMX, a phosphodiesterase (PDE) inhibitor), gene manipulation (overexpression of adenylyl cyclase 1/8) or direct manipulation of the SERCA phosphorylation target through changes in gene expression, compensate for the impairment in SANC automaticity. These findings suggest new insights for understanding CPVT and its therapeutic approach.

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.