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."

Overexpression of a Neuronal Type Adenylyl Cyclase (Type 8) in Sinoatrial Node Markedly Impacts Heart Rate and Rhythm.

Heart rate (HR) and HR variability (HRV), predictors of over-all organism health, are widely believed to be driven by autonomic input to the sinoatrial node (SAN), with sympathetic input increasing HR and reducing HRV. However, variability in spontaneous beating intervals in isolated SAN tissue and single SAN cells, devoid of autonomic neural input, suggests that clocks intrinsic to SAN cells may also contribute to HR and HRV in vivo. We assessed contributions of both intrinsic and autonomic neuronal input mechanisms of SAN cell function on HR and HRV via in vivo, telemetric EKG recordings. This was done in both wild type (WT) mice, and those in which adenylyl cyclase type 8 (ADCY8), a main driver of intrinsic cAMP-PKA-Ca2+ mediated pacemaker function, was overexpressed exclusively in the heart (TGAC8). We hypothesized that TGAC8 mice would: (1) manifest a more coherent pattern of HRV in vivo, i.e., a reduced HRV driven by mechanisms intrinsic to SAN cells, and less so to modulation by autonomic input and (2) utilize unique adaptations to limit sympathetic input to a heart with high levels of intrinsic cAMP-Ca2+ signaling. Increased adenylyl cyclase (AC) activity in TGAC8 SAN tissue was accompanied by a marked increase in HR and a concurrent marked reduction in HRV, both in the absence or presence of dual autonomic blockade. The marked increase in intrinsic HR and coherence of HRV in TGAC8 mice occurred in the context of: (1) reduced HR and HRV responses to ß-adrenergic receptor (ß-AR) stimulation; (2) increased transcription of genes and expression of proteins [ß-Arrestin, G Protein-Coupled Receptor Kinase 5 (GRK5) and Clathrin Adaptor Protein (Dab2)] that desensitize ß-AR signaling within SAN tissue, (3) reduced transcripts or protein levels of enzymes [dopamine beta-hydorxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT)] required for catecholamine production in intrinsic cardiac adrenergic cells, and (4) substantially reduced plasma catecholamine levels. Thus, mechanisms driven by cAMP-PKA-Ca2+ signaling intrinsic to SAN cells underlie the marked coherence of TGAC8 mice HRV. Adaptations to limit additional activation of AC signaling, via decreased neuronal sympathetic input, are utilized to ensure the hearts survival and prevent Ca2+ overload.

Heterogeneity of calcium clock functions in dormant, dysrhythmically and rhythmically firing single pacemaker cells isolated from SA node.

Current understanding of how cardiac pacemaker cells operate is based mainly on studies in isolated single sinoatrial node cells (SANC), specifically those that rhythmically fire action potentials similar to the in vivo behavior of the intact sinoatrial node. However, only a small fraction of SANC exhibit rhythmic firing after isolation. Other SANC behaviors have not been studied. Here, for the first time, we studied all single cells isolated from the sinoatrial node of the guinea pig, including traditionally studied rhythmically firing cells ('rhythmic SANC'), dysrhythmically firing cells ('dysrhythmic SANC') and cells without any apparent spontaneous firing activity ('dormant SANC'). Action potential-induced cytosolic Ca2+ transients and spontaneous local Ca2+ releases (LCRs) were measured with a 2D camera. LCRs were present not only in rhythmically firing SANC, but also in dormant and dysrhythmic SANC. While rhythmic SANC were characterized by large LCRs synchronized in space and time towards late diastole, dysrhythmic and dormant SANC exhibited smaller LCRs that appeared stochastically and were widely distributed in time. ß-adrenergic receptor (ßAR) stimulation increased LCR size and synchronized LCR occurrences in all dysrhythmic and a third of dormant cells (25 of 75 cells tested). In response to ßAR stimulation, these dormant SANC developed automaticity, and LCRs became coupled to spontaneous action potential-induced cytosolic Ca2+ transients. Conversely, dormant SANC that did not develop automaticity showed no significant change in average LCR characteristics. The majority of dysrhythmic cells became rhythmic in response to ßAR stimulation, with the rate of action potential-induced cytosolic Ca2+ transients substantially increasing. In summary, isolated SANC can be broadly categorized into three major populations: dormant, dysrhythmic, and rhythmic. We interpret our results based on simulations of a numerical model of SANC operating as a coupled-clock system. On this basis, the two previously unstudied dysrhythmic and dormant cell populations have intrinsically partially or completely uncoupled clocks. Such cells can be recruited to fire rhythmically in response to ßAR stimulation via increased rhythmic LCR activity and ameliorated coupling between the Ca2+ and membrane clocks.

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

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. ß-Adrenergic receptor (ß-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 ß-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 ß-AR stimulation-induced augmentation of Ca2+-cAMP-PKA signaling.