Biology subjects

Lakatta, E. G.

Publications and source records attributed to Lakatta, E. G..

At least 19 recordsLinked to original sources

bioRxiv2026

A novel multicellular model of the adult mouse sinoatrial node retains spontaneous electrical activity and enables live investigation of the S100B-associated cell population

Approximately half of the adult sinoatrial node (SAN) consists of non-myocyte populations, indicating that cardiac pacemaking depends on interactions within a multicellular tissue rather than on pacemaker cardiomyocytes alone. Among these, an S100B-associated cell population has been implicated in pacemaker function, yet its identity and physiological roles remain poorly understood. These cells are rare and dispersed throughout the small, structurally complex SAN, making them difficult to observe repeatedly while preserving the native multicellular environment. Here, we established a dissociated multicellular culture of adult mouse SAN tissue on soft collagen-gelatin hydrogels that retains spontaneous electrical activity and permits longitudinal live imaging of S100B-associated cells. Using an S100B-EGFP+ reporter, we identified at least six reproducible morphological and behavioral phenotypes, including migration, proliferation, phagocytic behavior, and spontaneous self-organization into three-dimensional clusters. Cultures remained spontaneously electrically active for more than 10 days in vitro, with peak activity around day 10. This multicellular culture model bridges the gap between intact SAN preparations and isolated-cell cultures, allowing repeated observation of rare S100B-associated cells within a spontaneously active multicellular environment. HighlightsO_LIThe platform enables longitudinal live imaging of rare S100B-associated cells within an diverse multicellular SAN culture. C_LIO_LILive imaging reveals at least six reproducible morphological and behavioral phenotypes of S100B-associated cells. C_LIO_LIDissociated multicellular SAN cultures remain spontaneously electrically active for more than 10 days in vitro. C_LI

cell biology↗

bioRxiv2026

Proton tunneling at the ryanodine receptor Ca2+ activation site provides temperature-invariant noise for robust Ca2+-induced Ca2+ release

Ryanodine receptor (RyR)-mediated Ca2+-induced Ca2+ release (CICR) is a regenerative trigger-to-release mechanism used in diverse cell types. RyRs amplify small Ca2+ signals into large, localized surges that drive autonomous oscillation, excitation-contraction coupling, pulsatile secretion, neurotransmitter release, or memory formation. Robust RyR function depends on the probability of RyR recruitment remaining within an operating range as physiological conditions change. This raises a basic question: what stabilizes the microscopic Ca2+-sensitive opening step of RyR across temperature and cellular state? Here we tested that proton tunneling at the conserved RyR2 Ca2+-activation site contributes a temperature-stable stochastic component to channel opening. To this end we developed a multiscale quantum-structural pipeline that estimates the per-channel modulation amplitude from cryo-EM geometry under stated assumptions. We applied this pipeline to a rabbit sinoatrial node cell model with explicit Ca2+ release unit architecture. Holding this RyR quantum noise constant across 25-37 {circ}C preserves pacemaker rhythmicity at low temperature, whereas a Q10-scaled classical noise results in irregular pacemaking activity as amplitude falls below the coherence-resonance range. Experimentally in isolated rabbit sinoatrial node cells, Ca2+ transient rate increased with temperature, but inter-transient interval variability did not, consistent with our numerical model predictions. These results support a model in which proton tunneling stabilizes the regenerative RyR-mediated CICR step. In autonomous oscillators, such as sinoatrial node cells, this manifests as robust rhythmicity. In triggered systems, it is expected to stabilize CICR gain, release synchrony, and release fidelity. The importance of this shared microscopic mechanism is evidenced by the conservation of the Ca2+-activation site across RyR isoforms for >600 million years of evolution. Significance StatementThis work suggests that RyR-based CICR networks can harness quantum-conditioned stochasticity. Proton tunneling at a conserved RyR activation site contributes a temperature-stable source of microscopic variability, and CICR amplifies that variability into robust physiological output. In pacemaker cells, this appears as stable rhythmic timing. More broadly, it provides a concrete route by which atomic-scale quantum events can influence macroscopic cellular function.

biophysics↗

bioRxiv2025

A New Fight-or-Flight Pacemaker Mechanism via Ryanodine Receptor Abundance and Superclustering

The sinoatrial node is the primary cardiac pacemaker. Individual sinoatrial node cells (SANCs) generate spontaneous rhythmic action potentials (APs) that initiate each heartbeat. The mechanism of SANC automaticity and its modulation by autonomic nervous system are based on the coupled function of molecules of both the cell membrane (ion channels, exchangers, and pumps) and the sarcoplasmic reticulum (SR), which generates rhythmic local Ca releases (LCRs). While LCRs are generated by ryanodine receptors (RyRs), the molecular-scale RyR network structure remains unknown. Here we performed single-molecule localization of RyRs via direct Stochastic Optical Reconstruction Microscopy (dSTORM) in rabbit SANCs in basal conditions and 5 minutes after {beta}-adrenergic receptor ({beta}AR) stimulation by isoproterenol. RyRs form clusters of various sizes, with a mean density of 67.7{+/-}13.2 RyR/m2. (Mean{+/-}SEM, 6 cells). While the majority of cluster sizes ranged from 3 to 32 RyRs, each cell had a few substantially larger clusters (>76 RyRs), dubbed superclusters. {beta}AR stimulation significantly increased the RyR density to 119.1{+/-}22.6 RyR/m2 (8 cells, p<0.05) and created more superclusters. Our new numerical SANC model showed that superclustering substantially decreased the AP cycle length (APCL) by creating Ca release hotspots that initiated larger LCRs under any condition. Increasing RyR density prolonged APCL in the basal state but shortened APCL during {beta}AR stimulation. With no change in RyR network, {beta}AR stimulation of only SR Ca pump and ion currents shortened APCL on average from 414.9 to 284 ms. When realistic higher RyR density and superclustering were added to the model, APCL further shortened to 231.9 ms. Thus, dynamic nanoscale changes in RyR network provide a new powerful pacemaker mechanism. This mechanism may help explain athletic bradycardia at rest and high exercise heart rates, with its deterioration contributing to the age-related loss of heart rate reserve. Author summaryThe heartbeat begins in the sinoatrial node, the hearts natural pacemaker. During stress or exercise, pacemaker cells accelerate their firing rate via {beta}-adrenergic ("fight-or-flight") stimulation. Although Ca signals are known to drive this acceleration via electrogenic Na/Ca exchanger, how the underlying ryanodine receptor (RyR) Ca release channels are organized at the nanoscale and how this organization changes during stimulation remain unclear. Here, we used super-resolution microscopy (dSTORM) to visualize individual RyRs in rabbit pacemaker cells. We found that RyRs form heterogeneous clusters that include rare, very large "superclusters." {beta}-adrenergic stimulation increased overall RyR density, enlarged clusters, and markedly increased the number of superclusters. To understand how these structural changes affect pacemaker function, we developed a computational sinoatrial node cell model in which each RyR was represented explicitly and arranged according to our imaging data. Numerical model simulations revealed that superclusters act as Ca-release hotspots that fire early during diastolic depolarization and recruit neighboring RyR clusters to fire via Ca-induce-Ca-release mechanism. When combined with {beta}-adrenergic enhancement of Ca cycling and membrane currents, the superclustering and RyR abundance synergistically accelerated action potential firing. Together, our results identified dynamic nanoscale RyR network reorganization as a key structural mechanism in fight-or-flight response.

biophysics↗

bioRxiv2024

Unique spatiotemporal synchronization solutions of heterogeneous local Ca2+ dynamics underlie the formation of each impulse that emerges from the cardiac sinoatrial node

We used both linear and nonlinear analyses to determine how information processing within and among incessant heterogeneous local Ca2+ oscillation (LCO) results in formation of rhythmic impulses in mouse SAN ex vivo. Phase analysis delineated a network of functional pacemaker cell clusters, distinguished by their LCO amplitudes, kinetics, and phases. Cross-talk of LCO dynamics within the network culminated in rhythmic SAN global Ca2+ transients (CaTs), having a mean rate and rhythm identical to that recorded by the reference sharp electrode in the right atria, indicating that CaTs are induced by global SAN electrical impulses. Initial conditions of each impulse and subsequent LCO ensemble evolution during an impulse differed from each other, associated with an apparent stochastic process (carrying a degree of uncertainty) within network. A small pacemaker cluster located near the crista terminalis (CT) exhibited the highest degrees of intrinsic power, earliest rotor-like energy transfer, most frequent point-to-point instability, earliest acrophase, greatest impulse-to-impulse variability within the SAN functional cluster network. Unique, variable small-world network Ca2+ information sharing within and among all clusters during initial and terminal impulse phases, created a unique solution for each impulse, while preserving the identity of each cluster (a highly efficient form of information processing at low wiring and energy costs). Cross-recurrence analysis verified that LCO dynamics within the small cluster near CT were more stochastic and less deterministic than those of the other clusters, indicating that this small cluster took the lead in the initiation of the SAN impulse and that the others followed.

cell biology↗

bioRxiv2024

Cardiac Pacemaker Cells Harness Stochastic Resonance to Ensure Fail-Safe Operation at Low Rates Bordering on Sinus Arrest

BACKGROUNDThe sinoatrial node (SAN) is the primary pacemaker of the heart. Recent high-resolution imaging showed that synchronized action potentials (APs) exiting the SAN emerge from heterogeneous signals, including subthreshold signals in non-firing (dormant) cells. This raises a new question in cardiac biology: how do these signals contribute to heartbeat generation? Here, we tested the hypothesis that pacemaker cells harness stochastic resonance to ensure fail-safe operation, especially at low rates bordering on sinus arrest. METHODSMembrane potential and Ca signals were measured using perforated-patch recordings in rabbit SAN cells exposed to sine-wave or white-noise currents. Additionally, we imaged Ca signals in intact mouse SAN tissue and performed multiscale model simulations at the subcellular, cellular, and tissue levels. RESULTSIn addition to classical synchronized Ca transients, SAN tissue exhibited heterogeneous local Ca signals of different kinetics. Noise currents, mimicking the heterogeneous natural cell environment, restored AP firing in dormant cells and substantially improved the rate and rhythm of those firing infrequently and irregularly. The benefit followed a bell-shaped curve: the performance improved but then declined, demonstrating a hallmark of stochastic resonance. Rhythmic AP generation in response to sine-wave currents of different frequencies defined a resonance spectrum in SAN cells, reflecting their ability to respond via stochastic resonance to specific frequency components embedded in noise. Cholinergic stimulation shifted the resonance spectrum and responses to noise toward lower frequencies across all amplitudes tested, rendering cells unresponsive to higher-frequency signals while enabling more effective processing of slower signals. Both the numerical models and simultaneous recordings of membrane potential and Ca dynamics demonstrated that stochastic resonance is amplified by coupled electrical and Ca signaling, enhancing AP generation at low noise levels. Adding noise currents to the cell and tissue models allowed firing under conditions where they otherwise would have stopped. CONCLUSIONSSAN cells harness stochastic resonance amplified by coupled membrane-Ca signaling to ensure rhythmic heartbeat initiation, especially at low rates. This new signaling mechanism could help avoid sinus arrest when heart slows but noise increases, such as during parasympathetic stimulation, bradyarrhythmia, or aging.

biophysics↗

bioRxiv2024

Structure-Function Relationship of the Ryanodine Receptor Cluster Network in Sinoatrial Node Cells

The rate of spontaneous action potentials (APs) generated by sinoatrial node cells (SANC) is regulated by local Ca2+ release (LCR) from the sarcoplasmic reticulum via Ca2+ release channels (ryanodine receptors, RyRs). LCR events propagate and self-organize within the network of RyR clusters (Ca release units, CRUs) via Ca-induced-Ca-release (CICR) that depends on CRU sizes and locations: while larger CRUs generate stronger release signals, the networks topology governs signal diffusion and propagation. This study used super-resolution structured illumination microscopy to image the 3D network of CRUs in rabbit SANC. The peripheral CRUs formed a spatial mesh, reflecting the cell surface geometry. Two distinct subpopulations of CRUs were identified within each cell, with size distributions conforming to a two component Gamma mixture model. Furthermore, neighboring CRUs exhibited repulsive behavior. Functional properties of the CRU network were further examined in a novel numerical SANC model developed using our experimental data. Model simulations revealed that heterogeneities in both CRU sizes and locations facilitate CICR and increase AP firing rate in a cooperative manner. However, these heterogeneities reduce the effect of {beta}-adrenergic stimulation in terms of its relative change in AP firing rate. The presence of heterogeneities in both sizes and locations allows SANC to reach higher absolute AP firing rates during {beta}-adrenergic stimulation. Thus, the CICR facilitation by heterogeneities in CRU sizes and locations regulates and optimizes cardiac pacemaker cell operation under various physiological conditions. Dysfunction of this optimization could be a key factor in heart rate reserve decline in aging and disease.

biophysics↗

bioRxiv2023

Cardiac Specific Overexpression of Adenylyl Cyclase 8 Reprograms the Mouse Sinoatrial Node Transcriptome

A coupled-clock system intrinsic to sinoatrial node (SAN) pacemaker cells that regulates the rate and rhythm of spontaneous action potential firing, is activated by Ca2+/calmodulin-stimulated Adenylyl Cyclase (AC) types 8 and 1. Our previous work in mice with cardiac specific overexpression of human AC8 gene (TGAC8) discovered that compared to its wild-type (WT) littermates, the heart rate (HR) of TGAC8 is elevated (by about 30%, 24 hours a day, 7days a week), and that the TGAC8 heart rhythm is markedly coherent, i.e., the HR Variability (HRV) lacks complexity, similar to that associated with aging or cardiac pathology. Reprogramming of molecular mechanisms, particularly SAN transcriptomic regulation that underlies the remarkable chronic shift in HR and HRV, however, has not been delineated. We conducted deep RNA sequencing (RNA-seq) in TGAC8 and WT SANs, using Mm10plus with human ADCY8 DNA sequence as the reference genome. Utilizing multiple bioinformatic techniques, we not only profiled the expression of marker genes related to SAN functions and AC-cAMP-PKA signaling, but also discovered negatively enriched hub pathways that differed in TGAC8 vs. WT, the top three being OXPHOS, ribosome, and cardiac muscle contraction. In contrast, signaling pathways related to inositol phosphate and its metabolism were positively enriched in TGAC8. Further, we identified two transcription regulators, KDM5A and PPARGC1A, that mediate effects of TGAC8 on ribosome and mitochondria. In summary, reprogrammed transcriptional regulation increases the HR of TGAC8 at the cost of impairment of other SAN cell functions, i.e., altered ribosome and inositol phosphate signaling and its crosstalk with mitochondria. Because these cell signaling alterations are associated with cardiac aging and age-associated CVDs, the TGAC8 mouse appears to be an ideal model in which to probe for potential therapeutic targets for cardiac aging and age-associated CVDs.

systems biology↗

bioRxiv2023

The Therapeutic Effects of Long-term Photobiomodulation on Aging in Mice

BackgroundWe have reported that photobiomodulation (PBM) therapy, a form of low dose Near Infrared Light (NIR) therapy, attenuates cardiovascular remodeling and extends the lifespan in a mouse model of accelerated cardiac aging. Here, we tested whether long-term PBM affects the aging process in normal male and female mice. MethodsC57 mice, 18 months old, males (n=60) and females (n=60), were exposed to either NIR (850nm) at 25 mW/cm2 for 2 min on weekdays (MT and FT groups) or nothing (M and F groups) for 12 months. Mice were subjected to bimonthly echocardiography examination, Gait analysis and Frailty assessments. Randomly selected mice were sacrificed bimonthly for fresh tissue samples. ResultsAge-associated deterioration in left ventricle, left atrium, aorta, brain blood perfusion, frailty, body temperature and gait that were observed in M and F groups during the 12-month observation period were significantly attenuated by PBM therapy in MT and FT groups. The medium lifespan was extended by 0.6 and 1.0 month in MT and FT groups, compared to M and F groups, respectively. There was a significantly lower prevalence of dermatitis, stroke and heart failure in MT and FT groups compared to M and F groups. ConclusionOur data showed for the first time that PBM therapy by whole body exposure, even started at old age in normal animals, significantly attenuated the age-associated deterioration in heart, vessels, brain, gait and frailty; reduced the prevalence of stroke and heart failure; and improved health span.

physiology↗

bioRxiv2023

A novel conceptual model of heart rate autonomic modulation based on a small-world modular structure and heterogeneous innervation of the sinoatrial node

The current theory of cardiac pacemaker rate modulation by the autonomic nervous system is based on the concept that a primary pacemaker cell or a group of cells in the center of the sinoatrial node (SAN) can change its AP firing rate within a broad range, driving respective myocardium contractions to commensurate body demands. Experimental data show, however, that pacemaker cells are extremely heterogeneous, with different areas of the SAN or cell clusters specializing to drive APs at specific rates. Thus, higher heart rates under stress are mainly driven by cell clusters in superior SAN, whereas low rates by cell clusters in inferior SAN, with basal state rates generated somewhere in the middle of the node. Cells within different clusters feature different intrinsic electrophysiological and Ca cycling properties, sympathetic and parasympathetic innervation, and vasculature, thereby supporting effective shift of the system to an optimal rate (under given conditions) accompanied by respective shifts in the leading pacemaker site. Thus, the popular single-cell-based pacemaker theory does not capture this complex emerging paradigm of pacemaker function of SAN tissue revealed by recent experimental studies. Here we propose a more realistic, conceptual model of heart rate autonomic modulation based on these studies. Our new model (the gear model) simulates the SAN as a brain-like structure featuring a small world of loosely connected clusters (functional modules) of tightly coupled cells, modeled as Maltsev-Lakatta coupled-clock system. One module (the higher chronotropic gear) generates higher AP rates in basal state and under {beta}-adrenergic stimulation, but its activity is strongly suppressed by parasympathetic stimulation. The other module (the lower gear) generates lower rates and has low sensitivity to parasympathetic stimulation. Such modular, gear-like system reproduces the respective shifts of the leading pacemaker site observed experimentally and features a wide range of rate modulation and robust function whilst conserving energy. In perspective, future refinement and application of this new pacemaker tissue mechanism will provide better understanding of cardiac pacemaker function, its deterioration in aging and disease, and ultimately the creation of new therapies to treat sick sinus syndrome and other SAN function-related cardiac arrythmias.

biophysics↗

bioRxiv2023

Cardiomyocyte-specific adenylyl cyclase type-8 overexpression induces cell-autonomous activation of RelA and non-cell-autonomous myocardial and systemic inflammation

BackgroundMice with cardiac-specific overexpression of adenylyl cyclase (AC) type 8 (TGAC8) are under a constant state of severe myocardial stress. They have a remarkable ability to adapt to this stress, but they eventually develop accelerated cardiac aging and experience reduced longevity. ResultsHere we demonstrate that activation of ACVIII in cardiomyocytes results in cell-autonomous RelA-mediated NF-{kappa}B signaling. This is associated with non-cell-autonomous activation of proinflammatory and age-associated signaling in myocardial endothelial cells and myocardial smooth muscle cells, expansion of myocardial immune cells, increase in serum levels of inflammatory cytokines, and changes in the size or composition of lymphoid organs. These changes precede the appearance of cardiac fibrosis. We provide evidence indicating that ACVIII-driven RelA activation in cardiomyocytes is mediated by calcium-Protein Kinase A (PKA) signaling. ConclusionsUsing a model of chronic cardiomyocyte stress and accelerated aging we highlight a novel, PKA/RelA-dependent connection between cardiomyocyte stress, myocardial para-inflammation and systemic inflammation. These findings point to RelA-mediated signaling in cardiomyocytes and inter-organ communication between the heart and lymphoid organs as novel potential therapeutic targets to reduce age-associated myocardial deterioration.

cell biology↗

bioRxiv2023

Transcriptome of left ventricle and sinoatrial node in young and old C57 mice

Advancing age is the most important risk factor for cardiovascular diseases (CVDs). Two types of cells within the heart pacemaker, sinoatrial node (SAN), and within the left ventricle (LV) control two crucial characteristics of heart function, heart beat rate and contraction strength. As age advances, the hearts structure becomes remodeled, and SAN and LV cell functions deteriorate, thus increasing the risk for CVDs. However, the different molecular features of age-associated changes in SAN and LV cells have never been compared in omics scale in the context of aging. We applied deep RNA sequencing to four groups of samples, young LV, old LV, young SAN and old SAN, followed by numerous bioinformatic analyses. In addition to profiling the differences in gene expression patterns between the two heart chambers (LV vs. SAN), we also identified the chamber-specific concordant or discordant age-associated changes in: (1) genes linked to energy production related to cardiomyocyte contraction, (2) genes related to post-transcriptional processing, (3) genes involved in KEGG longevity regulating pathway, (4) prolongevity and antilongevity genes recorded and curated in the GenAge database, and (5) CVD marker genes. Our bioinformatic analysis also predicted the regulation activities and mapped the expression of upstream regulators including transcription regulators and post-transcriptional regulator miRNAs. This comprehensive analysis promotes our understanding of regulation of heart functions and will enable discovery of gene-specific therapeutic targets of CVDs in advanced age.

systems biology↗

bioRxiv2022

Metformin attenuates hyperglycaemia-stimulated pro-fibrotic gene expression in vascular adventitial fibroblasts via inhibition of Discoidin Domain Receptor 2

Molecular mechanisms underlying the diverse therapeutic effects of anti-diabetic metformin, beyond its anti-hyperglycaemic effects, remain largely unclear. Metformin is reported to reduce the long-term complications of diabetes, including cardiovascular fibrosis and remodelling. Our recent investigations show that Discoidin Domain Receptor 2 (DDR2), a collagen receptor tyrosine kinase, has an obligate regulatory role in collagen type I gene expression in cardiac and vascular adventitial fibroblasts, and that it may be a molecular link between arterial fibrosis and metabolic syndrome in rhesus monkeys. Using gene knockdown and over-expression approaches, the present study examined whether DDR2 is a target of metformin, and whether, by targeting DDR2, it inhibits fibronectin and collagen expression in vascular adventitial fibroblasts exposed to hyperglycaemic conditions. Metformin was found to attenuate hyperglycaemia-induced increase in DDR2 mRNA and protein expression by inhibiting TGF-{beta}1/ Smad2/3 signalling that mediates the stimulatory effect of hyperglycaemia on DDR2 expression. Metformin also inhibited DDR2-dependent expression of fibronectin and collagen, indicating that it regulates these matrix proteins via DDR2 inhibition. The findings identify DDR2, a major mediator of cardiovascular remodelling, as a molecular target of metformin, thereby uncovering the molecular basis of its protective role in vascular fibrosis, and possibly, cardiac fibrosis associated with diabetic cardiomyopathy.

molecular biology↗

bioRxiv2022

Adenosine Reduces Sinoatrial Node Cell AP Firing Rate by uncoupling its Membrane and Calcium Clocks

AO_SCPLOWBSTRACTC_SCPLOWThe spontaneous action potential (AP) firing rate of sinoatrial nodal cells (SANC) is regulated by a system of intracellular Ca2+ and membrane ion current clocks driven by Ca2+-calmodulin-activated adenylyl cyclase-protein kinase A (PKA) signaling. The mean AP cycle length (APCL) and APCL variability inform on the effectiveness of clock coupling. Endogenous ATP metabolite adenosine (ado) binds to adenosine receptors that couple to Gi protein-coupled receptors, reducing spontaneous AP firing rate via G{beta}{gamma} signaling that activates an membrane-clock outward current, IKACh. Ado also inhibits adenylyl cyclase activity via Gi signaling, impacting cAMP-mediated PKA-dependent protein phosphorylation and intracellular Ca2+ cycling. We hypothesize that in addition to IKAdo activation, ado signaling impacts Ca2+ via Gi signaling and that both effects reduce AP firing rate by reducing the effectiveness of the Ca2+ and membrane clock coupling. To this end, we measured Ca2+ and membrane potential characteristics in enzymatically isolated single rabbit SANC. 10 {micro}M ado substantially increased both the mean APCL (on average by 43%, n=10) and AP beat-to-beat variability from 5.1{+/-}1.7% to 7.2{+/-}2.0% (n=10) measured via membrane potential and 5.0{+/-}2.2 to 10.6{+/-}5.9 (n=40) measured via Ca2+ (assessed as the coefficient of variability, CV=SD/mean). These effects were mediated by hyperpolarization of the maximum diastolic membrane potential (membrane clock effect) and suppression of diastolic spontaneous, local Ca2+ releases (LCRs) (Ca2+ clock effect): as LCR size distributions shifted from larger to smaller values, the time of LCR occurrence during diastolic depolarization (LCR period) became prolonged, and the ensemble LCR Ca2+ signal became reduced. The tight linear relationship of coupling between LCR period to the APCL in the presence of ado "drifted" upward and leftward, i.e. for a given LCR period, APCL was prolonged, becoming non-linear indicating clock uncoupling. An extreme case of uncoupling occurred at higher ado concentrations (>100 {micro}M): small stochastic LCRs of the Ca2+ clock failed to self-organize and synchronize to the membrane clock, thus creating a failed attempt to generate an AP resulting in arrhythmia and cessation of AP firing. Thus, the effects of ado to activate G{beta}{gamma} and IKACh, Ado and to activate Gi, suppressing adenylyl cyclase activity, both contribute to the ado-induced increase in the mean APCL and APCL variability by reducing the fidelity of clock coupling and AP firing rate.

physiology↗

bioRxiv2022

A Remarkable Adaptive Paradigm Of Heart Performance And Protection Emerges In Response To The Constitutive Challenge Of Marked Cardiac-Specific Overexpression Of Adenylyl Cyclase Type 8

Adult mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TGAC8) adapt to an incessantly increased cAMP-induced cardiac workload ([~]30% increases in heart rate, ejection fraction and cardiac output) for up to a year without signs of heart failure or excessive mortality. Here we show that despite markedly increased cardiac work, classical cardiac hypertrophy markers were absent in TGAC8, total left ventricular (LV) mass was not increased: a reduced LV cavity volume in TGAC8 was encased by thicker LV walls harboring an increased number of small cardiac myocytes and a network of small interstitial non-cardiac myocytes, manifesting increased proliferation markers and compared to WT. Protein synthesis, proteosome activity, autophagy, and Nrf-2, Hsp90, ACC2 protein levels were increased in TGAC8, but LV ATP and phosphocreatine levels in vivo did not differ by genotype. 2,323 transcripts and 2,184 proteins identified in unbiased omics analyses, spanning a wide array of biological processes and molecular functions in numerous cellular compartments differed in TGAC8 vs WT; and over 250 canonical signaling pathways characteristic of adaptive survival circuitry of cancers, including PI3K and growth factor signaling, cytokine and T cell receptor signaling, immune responses, ROS scavenging, proliferation, protection from apoptosis, and nutrient sensing, were activated in TGAC8; and compared to WT there was a shift from fatty acid oxidation to increased aerobic glycolysis in the context of increased utilization of the pentose phosphate shunt and nucleotide synthesis. Thus, the adaptive paradigm, that becomes activated in the LV of TGAC8 in response to severe chronic, intense AC/PKA/Ca2+ signaling embodies many hallmarks of cancer.

physiology↗

bioRxiv2022

The reprogrammed mouse heart phosphoproteome in response to the chronic stress of markedly high cardiac-specific adenylyl cyclase type 8 overexpression

Our prior study (Tarasov, K. V. et al., 2022) discovered that numerous adaptive mechanisms emerge in response to cardiac-specific overexpression of adenylyl cyclase type 8 (TGAC8) which included overexpression of a large number of proteins. Here we conducted an unbiased phosphoproteomics analysis in order to determine the role of altered protein phosphorylation in the adaptive heart performance and protection profile of adult TGAC8 left ventricle (LV) at 3-4 months of age, and integrated the phosphoproteome with transcriptome and proteome. Based on differentially regulated phosphoproteins by genotype, numerous stress-response pathways within reprogrammed TGAC8 LV, including PKA, PI3K and AMPK signaling pathways, predicted upstream regulators (e.g., PDPK1, PAK1 and PTK2B), and downstream functions (e.g., cell viability, protein quality control), and metabolism were enriched. In addition to PKA, numerous other kinases and phosphatases were hyper-phosphorylated in TGAC8 vs. WT. Hyper-phosphorylated transcriptional factors in TGAC8 were associated with increased mRNA transcription, immune responses and metabolic pathways. Combination of the phosphoproteome with its proteome and with the previously published TGAC8 transcriptome enabled the elucidation of cardiac performance and adaptive protection profiles coordinately regulated at post-translational modification (PTM) (phosphorylation), translational and transcriptional levels. Many stress-response signaling pathways, i.e., PI3K/AKT, ERK/MAPK and ubiquitin labeling, were consistently enriched and activated in the TGAC8 LV at transcriptional, translational and PTM levels. Thus, reprogramming of the cardiac phosphoproteome, proteome and transcriptome confers resilience to chronic adenylyl cyclase-driven stress. We identified numerous pathways/function predictions via gene sets, phosphopeptides, and phosphoproteins, which may point to potential novel therapeutic targets to enhance heart adaptivity, maintaining heart performance while avoiding cardiac dysfunction.

bioinformatics↗

bioRxiv2022

The Heart's Pacemaker Mimics Brain Cytoarchitecture and Function: Autonomic innervation, a peripheral glial cell web, and a novel S100B expressing interstitial cell type impart structural and functional complexity to the sinoatrial node

ObjectivesThis study sought to describe the 3D cytoarchitecture of sinoatrial node tissue, including autonomic innervation, peripheral glial cells, and pacemaker cells. BackgroundThe sinoatrial node of the heart produces rhythmic action potentials (AP), generated via calcium signaling within and among pacemaker cells. Our previous work has described the SAN as composed of an HCN4-expressing pacemaker cell meshwork, which merges with a network of CX43+/F-actin+ cells. It is also known that sympathetic and parasympathetic innervation from epicardial ganglia create an autonomic plexus in the sinoatrial node, which modulates heart rate and rhythm. However, the anatomical details of the interaction of this plexus with the pacemaker cell meshwork have yet to be described. Methods3D confocal laser-scanning microscopy of triple immunolabeled SAN whole mount preparations with combinations of antibodies for HCN4, S100B, GFAP, ChAT or VAChT, and TH, and transmission electron microscopy (TEM). ResultsThe SAN exhibited heterogeneous autonomic innervation, which was accompanied by a web of peripheral glial cells (PGCs). Further, we identified a novel S100B+/GFAP- interstitial cell population, with unique morphology and distinct distribution pattern, creating complex interactions with other cell types in the node. TEM images showed a similar population of cells, here identified as telocytes, which appeared to secrete vesicles towards pacemaker cells. Application of S100B protein to SAN preparations induced distinct changes in rhythmogenic calcium signaling. ConclusionsThe autonomic plexus and its associated peripheral glial cell web, a novel network of S100B expressing interstitial cells resembling telocytes, and a meshwork of HCN4+ cells interact to impart structural complexity to the sinoatrial node. Summary Table [Table 1][Table 1]

physiology↗

bioRxiv2022

Emergence of Heartbeat Frailty in Advanced Age: Perspectives from Life-Long EKG Recordings in Mice

SAN failure, aka sick-sinus syndrome, which features sinus bradycardia, SAN impulse pauses, and irregularity of RR interval rhythms are manifestations of SAN cell dysfunction that increases exponentially with advanced age, i.e., SAN frailty. Abnormalities in intrinsic RR interval variability may be the earliest signatures of SAN cell dysfunction leading to SAN frailty in late life. We measured RR interval variability within EKG timeseries prior to and during double autonomic blockade in long-lived C57/BL6 mice at 3 month intervals from 6 months of age until the end of life. Long-lived mice (those that achieved the median cohort lifespan of 24 months and beyond) displayed relatively minor changes in intrinsic RR interval variability prior to 21 months of age. Between 21 and 30 months of age, marked changes in intrinsic RR interval variability signatures in time, frequency, non-linear, and fragmentation domains result in a marked increase in the mean intrinsic RR interval. The effects of autonomic input partially compensated for the prolongation of the mean RR interval by impacting the age-associated deterioration in the RR interval variability signatures toward a youthful pattern. Cross-sectional analyses of other subsets of mice at ages at or beyond the median life span of our longitudinal cohort demonstrated increased non-cardiac, constitutional, whole body frailty, a decrease in energetic efficiency, and an increase in respiratory exchange ratio. In this context, we interpret the progressive increase in intrinsic RR interval variability beyond 21 months of age to be an indication of heartbeat frailty.

physiology↗

bioRxiv2021

Functional heterogeneity of cell populations increases robustness of pacemaker function in a numerical model of the sinoatrial node tissue

Each heartbeat is initiated by specialized pacemaker cells operating within the sinoatrial node (SAN). While individual cells within SAN tissue exhibit substantial heterogeneity of their electrophysiological parameters and Ca cycling, the role of this heterogeneity for cardiac pacemaker function remains mainly unknown. Here we investigated the problem numerically in a 25x25 square grid of coupled-clock Maltsev-Lakatta cell models and tested the hypothesis that functional heterogeneity of cell populations increases robustness of SAN function. The tissue models were populated by cells with different degree of heterogeneity of the two key model parameters of the coupled-clock system, maximum L-type Ca current conductance (gCaL) and sarcoplasmic reticulum Ca pumping rate (Pup). Our simulations showed that in the areas of Pup-gCaL parametric space at the edge of the system stability where action potential (AP) firing was absent or dysrhythmic in tissues populated by identical cells, rhythmic AP generation was rescued in tissues populated by cells with uniformly random distributions of gCaL or Pup (but keeping the same average values). This effect to increase robust AP generation was synergistic with respect to heterogeneity in both gCaL and Pup and was further strengthened by clustering of cells with higher gCaL or Pup. The effect of functional heterogeneity was not due to a simple summation of activity of intrinsically firing cells naturally present in SAN; rather AP firing cells locally and critically interacted with non-firing/dormant cells. When firing cells prevailed, they recruited many dormant cells to fire, strongly enhancing overall SAN function. And vice versa, prevailing dormant cells suppressed AP firing in cells with intrinsic automaticity and halted SAN automaticity.

systems biology↗