Kenta Tsutsui

Deterioration of autonomic neuronal receptor signaling and mechanisms intrinsic to heart pacemaker cells contribute to age-associated alterations in heart rate variability in vivo

We aimed to determine how age‐associated changes in mechanisms extrinsic and intrinsic to pacemaker cells relate to basal beating interval variability (BIV) reduction in vivo. Beating intervals (BIs) were measured in aged (23–25 months) and adult (3–4 months) C57BL/6 male mice (i) via ECG in vivo during light anesthesia in the basal state, or in the presence of 0.5 mg mL−1 atropine + 1 mg mL−1 propranolol (in vivo intrinsic conditions), and (ii) via a surface electrogram, in intact isolated pacemaker tissue. BIV was quantified in both time and frequency domains using linear and nonlinear indices. Although the average basal BI did not significantly change with age under intrinsic conditions in vivo and in the intact isolated pacemaker tissue, the average BI was prolonged in advanced age. In vivo basal BIV indices were found to be reduced with age, but this reduction diminished in the intrinsic state. However, in pacemaker tissue BIV indices increased in advanced age vs. adults. In the isolated pacemaker tissue, the sensitivity of the average BI and BIV in response to autonomic receptor stimulation or activation of mechanisms intrinsic to pacemaker cells by broad‐spectrum phosphodiesterase inhibition declined in advanced age. Thus, changes in mechanisms intrinsic to pacemaker cells increase the average BIs and BIV in the mice of advanced age. Autonomic neural input to pacemaker tissue compensates for failure of molecular intrinsic mechanisms to preserve average BI. But this compensation reduces the BIV due to both the imbalance of autonomic neural input to the pacemaker cells and altered pacemaker cell responses to neural input.

Potential effects of intrinsic heart pacemaker cell mechanisms on dysrhythmic cardiac action potential firing

The hearts regular electrical activity is initiated by specialized cardiac pacemaker cells residing in the sinoatrial node. The rate and rhythm of spontaneous action potential firing of sinoatrial node cells are regulated by stochastic mechanisms that determine the level of coupling of chemical to electrical clocks within cardiac pacemaker cells. This coupled-clock system is modulated by autonomic signaling from the brain via neurotransmitter release from the vagus and sympathetic nerves. Abnormalities in brain-heart clock connections or in any molecular clock activity within pacemaker cells lead to abnormalities in the beating rate and rhythm of the pacemaker tissue that initiates the cardiac impulse. Dysfunction of pacemaker tissue can lead to tachy-brady heart rate alternation or exit block that leads to long atrial pauses and increases susceptibility to other cardiac arrhythmia. Here we review evidence for the idea that disturbances in the intrinsic components of pacemaker cells may be implemented in arrhythmia induction in the heart.

Computer algorithms for automated detection and analysis of local Ca2+ releases in spontaneously beating cardiac pacemaker cells

Local Ca2+ Releases (LCRs) are crucial events involved in cardiac pacemaker cell function. However, specific algorithms for automatic LCR detection and analysis have not been developed in live, spontaneously beating pacemaker cells. In the present study we measured LCRs using a high-speed 2D-camera in spontaneously contracting sinoatrial (SA) node cells isolated from rabbit and guinea pig and developed a new algorithm capable of detecting and analyzing the LCRs spatially in two-dimensions, and in time. Our algorithm tracks points along the midline of the contracting cell. It uses these points as a coordinate system for affine transform, producing a transformed image series where the cell does not contract. Action potential-induced Ca2+ transients and LCRs were thereafter isolated from recording noise by applying a series of spatial filters. The LCR birth and death events were detected by a differential (frame-to-frame) sensitivity algorithm applied to each pixel (cell location). An LCR was detected when its signal changes sufficiently quickly within a sufficiently large area. The LCR is considered to have died when its amplitude decays substantially, or when it merges into the rising whole cell Ca2+ transient. Ultimately, our algorithm provides major LCR parameters such as period, signal mass, duration, and propagation path area. As the LCRs propagate within live cells, the algorithm identifies splitting and merging behaviors, indicating the importance of locally propagating Ca2+-induced-Ca2+-release for the fate of LCRs and for generating a powerful ensemble Ca2+ signal. Thus, our new computer algorithms eliminate motion artifacts and detect 2D local spatiotemporal events from recording noise and global signals. While the algorithms were developed to detect LCRs in sinoatrial nodal cells, they have the potential to be used in other applications in biophysics and cell physiology, for example, to detect Ca2+ wavelets (abortive waves), sparks and embers in muscle cells and Ca2+ puffs and syntillas in neurons.

A coupled-clock system drives the automaticity of human sinoatrial nodal pacemaker cells

The mechanisms that generate pacemaking activity in human sinoatrial nodal cells are revealed. Two clocks for a steadily beating heart The sinoatrial node is the endogenous pacemaker of the heart. Using isolated human sinoatrial node cells, Tsutsui et al. investigated the molecular mechanisms that enabled these cells to generate electrical signals at regular intervals to trigger rhythmic cardiac contractions. They found that periodic oscillations in Ca2+ and membrane potentials formed the basis for two interdependent “clocks” that together regularly generated spontaneous electrical signals. These clocks were uncoupled in human sinoatrial node cells that were not beating. Moreover, signaling downstream of β-adrenergic receptors enhanced the coupling between these two clocks and could induce electrical activity in some cells that were not beating. Understanding the mechanisms that generate pacemaking activity in human sinoatrial node cells may lead to the development of better therapies for sinus arrest, a condition that is caused by malfunction of the sinoatrial node and currently treated with a permanently implanted pacemaker. The spontaneous rhythmic action potentials generated by the sinoatrial node (SAN), the primary pacemaker in the heart, dictate the regular and optimal cardiac contractions that pump blood around the body. Although the heart rate of humans is substantially slower than that of smaller experimental animals, current perspectives on the biophysical mechanisms underlying the automaticity of sinoatrial nodal pacemaker cells (SANCs) have been gleaned largely from studies of animal hearts. Using human SANCs, we demonstrated that spontaneous rhythmic local Ca2+ releases generated by a Ca2+ clock were coupled to electrogenic surface membrane molecules (the M clock) to trigger rhythmic action potentials, and that Ca2+–cAMP–protein kinase A (PKA) signaling regulated clock coupling. When these clocks became uncoupled, SANCs failed to generate spontaneous action potentials, showing a depolarized membrane potential and disorganized local Ca2+ releases that failed to activate the M clock. β-Adrenergic receptor (β-AR) stimulation, which increases cAMP concentrations and clock coupling in other species, restored spontaneous, rhythmic action potentials in some nonbeating “arrested” human SANCs by increasing intracellular Ca2+ concentrations and synchronizing diastolic local Ca2+ releases. When β-AR stimulation was withdrawn, the clocks again became uncoupled, and SANCs reverted to a nonbeating arrested state. Thus, automaticity of human pacemaker cells is driven by a coupled-clock system driven by Ca2+-cAMP-PKA signaling. Extreme clock uncoupling led to failure of spontaneous action potential generation, which was restored by recoupling of the clocks. Clock coupling and action potential firing in some of these arrested cells can be restored by β-AR stimulation–induced augmentation of Ca2+-cAMP-PKA signaling.

Electrophysiological heterogeneity of pacemaker cells in the rabbit intercaval region, including the SA node: insights from recording multiple ion currents in each cell

Cardiac pacemaker cells, including cells of the sinoatrial node, are heterogeneous in size, morphology, and electrophysiological characteristics. The exact extent to which these cells differ electrophysiologically is unclear yet is critical to understanding their functioning. We examined major ionic currents in individual intercaval pacemaker cells (IPCs) sampled from the paracristal, intercaval region (including the sinoatrial node) that were spontaneously beating after enzymatic isolation from rabbit hearts. The beating rate was measured at baseline and after inhibition of the Ca2+ pump with cyclopiazonic acid. Thereafter, in each cell, we consecutively measured the density of funny current ( If), delayed rectifier K+ current ( IK) (a surrogate of repolarization capacity), and L-type Ca2+ current ( ICa,L) using whole cell patch clamp. The ionic current densities varied to a greater extent than previously appreciated, with some IPCs demonstrating very small or zero If . The density of none of the currents was correlated with cell size, while ICa,L and If densities were related to baseline beating rates. If density was correlated with IK density but not with that of ICa,L. Inhibition of Ca2+ cycling had a greater beating rate slowing effect in IPCs with lower If densities. Our numerical model simulation indicated that 1) IPCs with small (or zero) If or small ICa,L can operate via a major contribution of Ca2+ clock, 2) If-Ca2+-clock interplay could be important for robust pacemaking function, and 3) coupled If- IK function could regulate maximum diastolic potential. Thus, we have demonstrated marked electrophysiological heterogeneity of IPCs. This heterogeneity is manifested in basal beating rate and response to interference of Ca2+ cycling, which is linked to If. NEW & NOTEWORTHY In the present study, a hitherto unrecognized range of heterogeneity of ion currents in pacemaker cells from the intercaval region is demonstrated. Relationships between basal beating rate and L-type Ca2+ current and funny current ( If) density are uncovered, along with a positive relationship between If and delayed rectifier K+ current. Links are shown between the response to Ca2+ cycling blockade and If density.