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 Ca release (LCR) from the sarcoplasmic reticulum via Ca 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 network's topology governs signal diffusion and propagation. This study used super-resolution structured illumination microscopy to image the 3D network of CRUs in rabbit SANC.

A small erythropoietin derived non-hematopoietic peptide reduces cardiac inflammation, attenuates age associated declines in heart function and prolongs healthspan.

Background: Aging is associated with increased levels of reactive oxygen species and inflammation that disrupt proteostasis and mitochondrial function and leads to organism-wide frailty later in life. ARA290 (cibinetide), an 11-aa non-hematopoietic peptide sequence within the cardioprotective domain of erythropoietin, mediates tissue protection by reducing inflammation and fibrosis. Age-associated cardiac inflammation is linked to structural and functional changes in the heart, including mitochondrial dysfunction, impaired proteostasis, hypertrophic cardiac remodeling, and contractile dysfunction.

A remarkable adaptive paradigm of heart performance and protection emerges in response to marked cardiac-specific overexpression of ADCY8.

Adult (3 month) mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TG) adapt to an 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 classical cardiac hypertrophy markers were absent in TG, and that total left ventricular (LV) mass was not increased: a reduced LV cavity volume in TG was encased by thicker LV walls harboring an increased number of small cardiac myocytes, and a network of small interstitial proliferative non-cardiac myocytes compared to wild type (WT) littermates; Protein synthesis, proteosome activity, and autophagy were enhanced in TG vs WT, and Nrf-2, Hsp90α, and ACC2 protein levels were increased. Despite increased energy demands in vivo LV ATP and phosphocreatine levels in TG did not differ from WT.

The Heart's Pacemaker Mimics Brain Cytoarchitecture and Function: Novel Interstitial Cells Expose Complexity of the SAN.

Background: The sinoatrial node (SAN) of the heart produces rhythmic action potentials, generated via calcium signaling within and among pacemaker cells. Our previous work has described the SAN as composed of a hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4)-expressing pacemaker cell meshwork, which merges with a network of connexin 43/F-actin cells. It is also known that sympathetic and parasympathetic innervation create an autonomic plexus in the SAN that modulates heart rate and rhythm.

ATP Synthase K- and H-Fluxes Drive ATP Synthesis and Enable Mitochondrial K-"Uniporter" Function: I. Characterization of Ion Fluxes.

ATP synthase (FF) synthesizes daily our body's weight in ATP, whose production-rate can be transiently increased several-fold to meet changes in energy utilization. Using purified mammalian FF-reconstituted proteoliposomes and isolated mitochondria, we show FF can utilize both ΔΨ-driven H- and K-transport to synthesize ATP under physiological pH = 7.2 and K = 140 mEq/L conditions.

HNO Protects the Myocardium against Reperfusion Injury, Inhibiting the mPTP Opening via PKCε Activation.

Donors of nitroxyl (HNO), the one electron-reduction product of nitric oxide (NO), positively modulate cardiac contractility/relaxation while limiting ischemia-reperfusion (I/R) injury. The mechanisms underpinning HNO anti-ischemic effects remain poorly understood. Using isolated perfused rat hearts subjected to 30 min global ischemia/1 or 2 h reperfusion, here we tested whether, in analogy to NO, HNO protection requires PKCε translocation to mitochondria and K channels activation.

ATP Synthase K- and H-fluxes Drive ATP Synthesis and Enable Mitochondrial K-"Uniporter" Function: II. Ion and ATP Synthase Flux Regulation.

We demonstrated that ATP synthase serves the of a primary mitochondrial K "uniporter," i.e., the primary way for K to enter mitochondria.

Computational modeling of mitochondrial K- and H-driven ATP synthesis.

ATP synthase (FF) is a rotary molecular engine that harnesses energy from electrochemical-gradients across the inner mitochondrial membrane for ATP synthesis. Despite the accepted tenet that FF transports exclusively H, our laboratory has demonstrated that, in addition to H, FF ATP synthase transports a significant fraction of ΔΨ-driven charge as K to synthesize ATP. Herein, we utilize a computational modeling approach as a proof of principle of the feasibility of the core mechanism underlying the enhanced ATP synthesis, and to explore its bioenergetic consequences.

Mitochondrial Ca, redox environment and ROS emission in heart failure: Two sides of the same coin?

Heart failure (HF) is a progressive, debilitating condition characterized, in part, by altered ionic equilibria, increased ROS production and impaired cellular energy metabolism, contributing to variable profiles of systolic and diastolic dysfunction with significant functional limitations and risk of premature death. We summarize current knowledge concerning changes of intracellular Na and Ca control mechanisms during the disease progression and their consequences on mitochondrial Ca homeostasis and the shift in redox balance. Absent existing biological data, our computational modeling studies advance a new 'in silico' analysis to reconcile existing opposing views, based on different experimental HF models, regarding variations in mitochondrial Ca concentration that participate in triggering and perpetuating oxidative stress in the failing heart and their impact on cardiac energetics.

Synchronized Cardiac Impulses Emerge From Heterogeneous Local Calcium Signals Within and Among Cells of Pacemaker Tissue.

Objectives: This study sought to identify subcellular Ca signals within and among cells comprising the sinoatrial node (SAN) tissue.

Background: The current paradigm of SAN impulse generation: 1) is that full-scale action potentials (APs) of a common frequency are initiated at 1 site and are conducted within the SAN along smooth isochrones; and 2) does not feature fine details of Ca signaling present in isolated SAN cells, in which small subcellular, subthreshold local Ca releases (LCRs) self-organize to generate cell-wide APs.

Methods: Immunolabeling was combined with a novel technique to detect the occurrence of LCRs and AP-induced Ca transients (APCTs) in individual pixels (chronopix) across the entire mouse SAN images.

Mitochondrial membrane potential.

The mitochondrial membrane potential (ΔΨm) generated by proton pumps (Complexes I, III and IV) is an essential component in the process of energy storage during oxidative phosphorylation. Together with the proton gradient (ΔpH), ΔΨm forms the transmembrane potential of hydrogen ions which is harnessed to make ATP. The levels of ΔΨm and ATP in the cell are kept relatively stable although there are limited fluctuations of both these factors that can occur reflecting normal physiological activity.

Ca(2+)/calmodulin-activated phosphodiesterase 1A is highly expressed in rabbit cardiac sinoatrial nodal cells and regulates pacemaker function.

Constitutive Ca(2+)/calmodulin (CaM)-activation of adenylyl cyclases (ACs) types 1 and 8 in sinoatrial nodal cells (SANC) generates cAMP within lipid-raft-rich microdomains to initiate cAMP-protein kinase A (PKA) signaling, that regulates basal state rhythmic action potential firing of these cells. Mounting evidence in other cell types points to a balance between Ca(2+)-activated counteracting enzymes, ACs and phosphodiesterases (PDEs) within these cells. We hypothesized that the expression and activity of Ca(2+)/CaM-activated PDE Type 1A is higher in SANC than in other cardiac cell types.

Mitochondrial health, the epigenome and healthspan.

Food nutrients and metabolic supply-demand dynamics constitute environmental factors that interact with our genome influencing health and disease states. These gene-environment interactions converge at the metabolic-epigenome-genome axis to regulate gene expression and phenotypic outcomes. Mounting evidence indicates that nutrients and lifestyle strongly influence genome-metabolic functional interactions determining disease via altered epigenetic regulation.

Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release.

Byproducts of normal mitochondrial metabolism and homeostasis include the buildup of potentially damaging levels of reactive oxygen species (ROS), Ca(2+), etc., which must be normalized. Evidence suggests that brief mitochondrial permeability transition pore (mPTP) openings play an important physiological role maintaining healthy mitochondria homeostasis.

Hierarchical clustering of ryanodine receptors enables emergence of a calcium clock in sinoatrial node cells.

The sinoatrial node, whose cells (sinoatrial node cells [SANCs]) generate rhythmic action potentials, is the primary pacemaker of the heart. During diastole, calcium released from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) interacts with membrane currents to control the rate of the heartbeat. This "calcium clock" takes the form of stochastic, partially periodic, localized calcium release (LCR) events that propagate, wave-like, for limited distances.

Age-associated abnormalities of intrinsic automaticity of sinoatrial nodal cells are linked to deficient cAMP-PKA-Ca(2+) signaling.

A reduced sinoatrial node (SAN) functional reserve underlies the age-associated decline in heart rate acceleration in response to stress. SAN cell function involves an oscillatory coupled-clock system: the sarcoplasmic reticulum (SR), a Ca(2+) clock, and the electrogenic-sarcolemmal membrane clock. Ca(2+)-activated-calmodulin-adenylyl cyclase/CaMKII-cAMP/PKA-Ca(2+) signaling regulated by phosphodiesterase activity drives SAN cells automaticity.

Age-related changes of myocardial ATP supply and demand mechanisms.

In advanced age, the resting myocardial oxygen consumption rate (MVO2) and cardiac work (CW) in the rat remain intact. However, MVO2, CW and cardiac efficiency achieved at high demand are decreased with age, compared to maximal values in the young. Whether this deterioration is due to decrease in myocardial ATP demand, ATP supply, or the control mechanisms that match them remains controversial.

A full range of mouse sinoatrial node AP firing rates requires protein kinase A-dependent calcium signaling.

Recent perspectives on sinoatrial nodal cell (SANC)(*) function indicate that spontaneous sarcoplasmic reticulum (SR) Ca(2+) cycling, i.e. an intracellular "Ca(2+) clock," driven by cAMP-mediated, PKA-dependent phosphorylation, interacts with an ensemble of surface membrane electrogenic molecules ("surface membrane clock") to drive SANC normal automaticity.

Ca2+-regulated-cAMP/PKA signaling in cardiac pacemaker cells links ATP supply to demand.

Rationale: In sinoatrial node cells (SANC), Ca(2+) activates adenylate cyclase (AC) to generate a high basal level of cAMP-mediated/protein kinase A (PKA)-dependent phosphorylation of Ca(2+) cycling proteins. These result in spontaneous sarcoplasmic-reticulum (SR) generated rhythmic Ca(2+) oscillations during diastolic depolarization, that not only trigger the surface membrane to generate rhythmic action potentials (APs), but, in a feed-forward manner, also activate AC/PKA signaling. ATP is consumed to pump Ca(2+) to the SR, to produce cAMP, to support contraction and to maintain cell ionic homeostasis.

Analysis of mitochondrial 3D-deformation in cardiomyocytes during active contraction reveals passive structural anisotropy of orthogonal short axes.

The cardiomyocyte cytoskeleton, composed of rigid and elastic elements, maintains the isolated cell in an elongated cylindrical shape with an elliptical cross-section, even during contraction-relaxation cycles. Cardiomyocyte mitochondria are micron-sized, fluid-filled passive spheres distributed throughout the cell in a crystal-like lattice, arranged in pairs sandwiched between the sarcomere contractile machinery, both longitudinally and radially. Their shape represents the extant 3-dimensional (3D) force-balance.

A small nonerythropoietic helix B surface peptide based upon erythropoietin structure is cardioprotective against ischemic myocardial damage.

Strong cardioprotective properties of erythropoietin (EPO) reported over the last 10 years have been difficult to translate to clinical applications for ischemic cardioprotection owing to undesirable parallel activation of erythropoiesis and thrombogenesis. A pyroglutamate helix B surface peptide (pHBP), recently engineered to include only a part of the EPO molecule that does not bind to EPO receptor and thus, is not erythropoietic, retains tissue protective properties of EPO. Here we compared the ability of pHBP and EPO to protect cardiac myocytes from oxidative stress in vitro and cardiac tissue from ischemic damage in vivo.

Compatibility of superparamagnetic iron oxide nanoparticle labeling for ¹H MRI cell tracking with ³¹P MRS for bioenergetic measurements.

Labeling of cells with superparamagnetic iron oxide nanoparticles permits cell tracking by (1)H MRI while (31)P MRS allows non-invasive evaluation of cellular bioenergetics. We evaluated the compatibility of these two techniques by obtaining (31)P NMR spectra of iron-labeled and unlabeled immobilized C2C12 myoblast cells in vitro. Broadened but usable (31)P spectra were obtained and peak area ratios of resonances corresponding to intracellular metabolites showed no significant differences between labeled and unlabeled cell populations.

Matching ATP supply and demand in mammalian heart: in vivo, in vitro, and in silico perspectives.

Although the heart rapidly adapts cardiac output to match the body's circulatory demands, the regulatory mechanisms ensuring that sufficient ATP is available to perform the required cardiac work are not completely understood. Two mechanisms have been suggested to serve as key regulators: (1) ADP and Pi concentrations--ATP utilization/hydrolysis in the cytosol increases ADP and Pi fluxes to mitochondria and hence the amount of available substrates for ATP production increases; and (2) Ca2+ concentration--ATP utilization/hydrolysis is coupled to changes in free cytosolic calcium and mitochondrial calcium, the latter controlling Ca2+-dependent activation of mitochondrial enzymes taking part in ATP production. Here we discuss the evolving perspectives of each of the putative regulatory mechanisms and the precise molecular targets (dehydrogenase enzymes, ATP synthase) based on existing experimental and theoretical evidence.