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.

Using beat-to-beat heart signals for age-independent biometric verification.

Use of non-stationary physiological signals for biometric verification, reduces the ability to forge. Such signals should be simple to acquire with inexpensive equipment. The beat-to-beat information embedded within the time intervals between consecutive heart beats is a non-stationary physiological signal; its potential for biometric verification has not been studied. This work introduces a biometric verification method termed "CompaRR". Heartbeat was extracted from longitudinal recordings from 30 mice ranging from 6 to 24 months of age (equivalent to ~ 20-75 human years). Fifty heartbeats, which is close to resting human heartbeats in a minute, were sufficient for the verification task, achieving a minimal equal error rate of 0.21. When trained on 6-month-old mice and tested on unseen mice up to 18-months of age (equivalent to ~ 50 human years), no significant change in the verification performance was noted. Finally, when the model was trained on data from drug-treated mice, verification was still possible.

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.

Heart failure in mice induces a dysfunction of the sinus node associated with reduced CaMKII signaling.

Dysfunction of the sinoatrial node (SAN), the natural heart pacemaker, is common in heart failure (HF) patients. SAN spontaneous activity relies on various ion currents in the plasma membrane (voltage clock), but intracellular Ca2+ ([Ca2+]i) release via ryanodine receptor 2 (RYR2; Ca2+ clock) plays an important synergetic role. Whereas remodeling of voltage-clock components has been revealed in HF, less is known about possible alterations to the Ca2+ clock. Here, we analyzed [Ca2+]i handling in SAN from a mouse HF model after transverse aortic constriction (TAC) and compared it with sham-operated animals. ECG data from awake animals showed slower heart rate in HF mice upon autonomic nervous system blockade, indicating intrinsic sinus node dysfunction. Confocal microscopy analyses of SAN cells within whole tissue showed slower and less frequent [Ca2+]i transients in HF. This correlated with fewer and smaller spontaneous Ca2+ sparks in HF SAN cells, which associated with lower RYR2 protein expression level and reduced phosphorylation at the CaMKII site. Moreover, PLB phosphorylation at the CaMKII site was also decreased in HF, which could lead to reduced sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) function and lower sarcoplasmic reticulum Ca2+ content, further depressing the Ca2+ clock. The inhibition of CaMKII with KN93 slowed [Ca2+]i transient rate in both groups, but this effect was smaller in HF SAN, consistent with less CaMKII activation. In conclusion, our data uncover that the mechanism of intrinsic pacemaker dysfunction in HF involves reduced CaMKII activation.

Emergence of heartbeat frailty in advanced age I: perspectives from life-long EKG recordings in adult mice.

The combined influences of sinoatrial nodal (SAN) pacemaker cell automaticity and its response to autonomic input determine the heart's beating interval variability and mean beating rate. To determine the intrinsic SAN and autonomic signatures buried within EKG RR interval time series change in advanced age, we measured RR interval variability before and during double autonomic blockade at 3-month intervals from 6 months of age until the end of life in long-lived (those that achieved the total cohort median life span of 24 months and beyond) C57/BL6 mice. Prior to 21 months of age, time-dependent changes in intrinsic RR interval variability and mean RR interval were relatively minor. Between 21 and 30 months of age, however, marked changes emerged in intrinsic SAN RR interval variability signatures, pointing to a reduction in the kinetics of pacemaker clock mechanisms, leading to reduced synchronization of molecular functions within and among SAN cells. This loss of high-frequency signal processing within intrinsic SAN signatures resulted in a marked increase in the mean intrinsic RR interval. The impact of autonomic signatures on RR interval variability were net sympathetic and partially compensated for the reduced kinetics of the intrinsic SAN RR interval variability signatures, and partially, but not completely, shifted the EKG RR time series intervals to a more youthful pattern. Cross-sectional analyses of other subsets of C57/BL6 ages indicated that at or beyond the median life span of our longitudinal cohort, noncardiac, constitutional, whole-body frailty was increased, energetic efficiency was reduced, and the respiratory exchange ratio increased. We interpret the progressive reduction in kinetics in intrinsic SAN RR interval variability signatures in this context of whole-body frailty beyond 21 months of age to be a manifestation of "heartbeat frailty."

Opening the Schrödinger Box: Short- and Long-Range Mammalian Heart Rate Variability.

BACKGROUND: The interactions between the autonomic nervous system (ANS), intrinsic systems (e.g., endocrine), and internal pacemaker mechanisms govern short (milliseconds-seconds)- and long (seconds-minutes)-range heart rate variability (HRV). However, there is a debate regarding the identity of the mechanism underlying HRV on each time scale. We aim to design a general method that accurately differentiates between the relative contribution of the ANS and pacemaker mechanisms to HRV in various mammals, without the need for drug perturbations or organ isolation. Additionally, we aim to explore the universality of the relative contribution of the ANS and pacemaker system of different mammals. METHODS: This work explored short- and long-range HRVs using published ECG data from dogs, rabbits, and mice. To isolate the effects of ANS on HRV, ECG segments recorded before and after ANS-blockade were compared. RESULTS: Differentiation of the ANS from extrinsic and intrinsic pacemaker mechanisms was successfully achieved. In dogs, the internal pacemaker mechanisms were the main contributors to long-range and the ANS to short-range HRV. In rabbits and mice, the ANS and the internal pacemaker mechanisms affected both time scales, and anesthesia changed the relative contribution of the pacemaker mechanism to short- and long-range HRVs. In mice, the extrinsic mechanisms affected long-range HRV, while their effect was negligible in rabbits. CONCLUSION: We offer a novel approach to determine the relative contributions of ANS and extrinsic and intrinsic pacemaker mechanisms to HRV and highlight the importance of selecting mammalian research models with HRV mechanisms representative of the target species of interest.

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.

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.