Whether intracellular Ca2+ regulates sinoatrial node cell (SANC) action potential (AP) firing rate on a beat-to-beat basis is controversial.
To directly test the hypothesis of beat-to-beat intracellular Ca2+ regulation of the rate and rhythm of SANC.
Methods and results
We loaded single isolated SANC with a caged Ca2+ buffer, NP-EGTA, and simultaneously recorded membrane potential and intracellular Ca2+. Prior to introduction of the caged Ca2+ buffer, spontaneous local Ca2+ releases (LCRs) during diastolic depolarization (DD) were tightly coupled to rhythmic APs (r2=0.9). The buffer markedly prolonged the decay time (T50) and moderately reduced the amplitude of the AP-induced Ca2+ transient and partially depleted the SR load, suppressed spontaneous diastolic LCRs and uncoupled them from AP generation, and caused AP firing to become markedly slower and dysrhythmic. When Ca2+ was acutely released from the caged compound by flash photolysis, intracellular Ca2+ dynamics were acutely restored and rhythmic APs resumed immediately at a normal rate. After a few rhythmic cycles, however, these effects of the flash waned as interference with Ca2+ dynamics by the caged buffer was reestablished.
Our results directly support the hypothesis that intracellular Ca2+ regulates normal SANC automaticity on a beat-to-beat basis.
pacemaker cell automaticity; Ca2+ cycling; pacemaker Ca2+ clock; Ca2+-excitation contraction coupling; arrhythmia
Marinobufagenin (MBG) promotes natriuresis via inhibition of renotubular Na/K-ATPase (NKA) and causes vasoconstriction via inhibition of vascular NKA. Atrial natriuretic peptide (ANP), via cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG)-dependent mechanism, sensitizes renal NKA to MBG but reduces MBG-induced inhibition of vascular NKA. As aging is associated with a downregulation of cGMP/PKG signaling, we hypothesized that in older rats, ANP would not potentiate renal effects of MBG and would not oppose vascular effects of MBG.
In younger (3-month-old) and older (12-month old) Sprague–Dawley rats, we compared SBP, natriuresis, activity of NKA in aorta and renal medulla, and levels of MBG and α-ANP at baseline and following acute NaCl loading (20%, 2.5 ml/kg, intraperitoneally), and studied modulation of MBG-induced NKA inhibition by α-ANP in vitro.
As compared with younger rats, NaCl-loaded older rats exhibited a greater MBG response, greater SBP elevation (25 vs. 10 mmHg, P<0.01) and greater inhibition of NKA in aorta (39 vs. 7%, P<0.01), 30% less natriuresis, and less inhibition of renal NKA (25 vs. 42%, P<0.05) in the presence of comparable responses of α-ANP and cGMP. In aorta and kidney of older rats, the levels of PKG were reduced, the levels of phosphodiesterase-5 were increased compared with that in young rats, and α-ANP failed to modulate MBG-induced NKA inhibition.
Age-associated downregulation of cGMP/PKG-dependent signaling impairs the ability of ANP to modulate the effects of MBG on the sodium pump, which contributes to salt sensitivity.
aging; cyclic guanosine monophosphate; dietary sodium; hypertension; marinobufagenin; Na/K-ATPase; natriuretic peptides; protein kinase G; salt sensitivity
In sinoatrial node cells (SANC), Ca2+ activates adenylate cyclase (AC) to generate a high basal level of cAMP-mediated/protein kinase A (PKA)-dependent phosphorylation of Ca2+ cycling proteins. These result in spontaneous sarcoplasmic-reticulum (SR) generated rhythmic Ca2+ 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 Ca2+ to the SR, to produce cAMP, to support contraction and to maintain cell ionic homeostasis.
Since a negative feedback mechanism links ATP-demand to ATP production, we hypothesized that (1) both basal ATP supply and demand in SANC would be Ca2+-cAMP/PKA dependent; and (2) due to its feed–forward nature, a decrease in flux through the Ca2+-cAMP/PKA signaling axis will reduce the basal ATP production rate.
Methods and Results
O2 consumption in spontaneous beating SANC was comparable to ventricular myocytes (VM) stimulated at 3 Hz. Graded reduction of basal Ca2+-cAMP/PKA signaling to reduce ATP demand in rabbit SANC produced graded ATP depletion (r2=0.96), and reduced O2 consumption and flavoprotein fluorescence. Neither inhibition of glycolysis, selectively blocking contraction nor specific inhibition of mitochondrial Ca2+ flux reduced the ATP level.
Feed-forward basal Ca2+-cAMP/PKA signaling both consumes ATP to drive spontaneous APs in SANC and is tightly linked to mitochondrial ATP production. Interfering with Ca2+-cAMP/PKA signaling not only slows the firing rate and reduces ATP consumption, but also appears to reduce ATP production so that ATP levels fall. This distinctly differs from VM, which lack this feed-forward basal cAMP/PKA signaling, and in which ATP level remains constant when the demand changes.
Calcium-activated adenylyl cyclase; constitutive basal PKA-dependent phosphorylation; bioenergetics; pacemaker automaticity; respiration
We have adapted bioluminescence methods to be able to measure phosphodiesterase (PDE) activity in a one-step technique. The method employs a four-enzyme system (PDE, adenylate kinase (AK) using excess CTP instead of ATP as substrate, pyruvate kinase (PK), and firefly luciferase) to generate ATP, with measurement of the concomitant luciferase-light emission. Since AK, PK, and luciferase reactions are coupled to recur in a cyclic manner, AMP recycling maintains a constant rate of ATP formation, proportional to the steady-state AMP concentration in. The cycle can be initiated by the PDE reaction that yields AMP. As long as the PDE reaction is rate-limiting, the system is effectively at steady state and the bioluminescence kinetics progresses at a constant rate proportional to the PDE activity. In the absence of cAMP and PDE, low concentrations of AMP trigger the AMP cycling, which allows standardizing the system. The sensitivity of the method enables detection of <1 μU (pmol/min) of PDE activity in cell extracts containing 0.25-10 μg protein. Assays utilizing pure enzyme showed that 0.2 mM IBMX completely inhibited PDE activity. This single-step enzyme- and substrate-coupled cyclic-reaction system yields a simplified, sensitive, reproducible and accurate method to quantify PDE activities in small biological samples.
phosphodiesterase; assay; bioluminescence
Two major β-adrenergic receptor (βAR) subtypes, β1AR and β2AR, are expressed in mammalian heart with β1AR coupling to Gs and β2AR dually coupling to Gs and Gi proteins. In many types of chronic heart failure, myocardial contractile response to both β1AR and β2AR stimulation is severely impaired. The dysfunction of βAR signaling in failing hearts is largely attributable to an increase in Gi signaling, because disruption of the Gi signaling restores myocardial contractile response to β1AR as well as β2AR stimulation. However, the mechanism terminating the β2AR-Gi signaling remains elusive, while it has been shown activation of the Gi signaling is dependent on agonist stimulation and subsequent PKA-mediated phosphorylation of the receptor. Here we demonstrate that regulator of G protein signaling 2 (RGS2) is a primary terminator of the β2AR-Gi signaling. Specifically, prolonged absence of agonist stimulation for 24h impairs the β2AR-Gi signaling, resulting in enhanced β2AR- but not β1AR-mediated contractile response in cultured adult mouse cardiomyocytes. Increased β2AR contractile response is accompanied by a selective upregulation of RGS2 in the absence of alterations in other major cardiac RGS proteins (RGS3-5) or Gs, Gi or βAR subtypes. Administration of a βAR agonist, isoproterenol (ISO, 1.0 nM), prevents RGS2 upregulation and restores the β2AR-Gi signaling in cultured cells. Furthermore, RGS2 ablation, similar to βAR agonist stimulation, sustains the β2AR-Gi signaling in cultured cells, whereas adenoviral overexpression of RGS2 suppresses agonist-activated β2AR-Gi signaling in cardiomyocytes and HEK293 cells. These findings not only define RGS2 as a novel negative regulator of the β2AR-Gi signaling, but also provide a potential novel target for the treatment of chronic heart failure.
β2-adrenergic receptor; RGS2; Gi proteins; cardiomyocyte contractility
There is increasing evidence that age-associated chronic low-grade inflammation promotes
the development of both large-vessel disease (myocardial infarction, stroke, peripheral
arterial disease) and small-vessel pathologies (including vascular cognitive impairment)
in older persons. However, the source of age-related chronic vascular inflammation remains
unclear. To test the hypothesis that cell-autonomous mechanisms contribute to the
proinflammatory changes in vascular phenotype that accompanies advancing age, we analyzed
the cytokine secretion profile of primary vascular smooth muscle cells (VSMCs) derived
from young (∼13 years old) and aged (∼21 years old) Macaca
mulatta. Aged VSMCs cultured in the absence of systemic factors exhibited
significantly increased secretion of interleukin-1β, MCP-1, and tumor necrosis
factorα compared with young control cells. Secretion of interleukin-6 also tended to
increase in aged VSMCs. This age-associated proinflammatory shift in the cellular
secretory phenotype was associated with an increased mitochondrial O2
− production and nuclear factor κ-light-chain-enhancer of activated
B cells activation. Treatment of aged VSMCs with a physiologically relevant concentration
of resveratrol (1 μM) exerted significant anti-inflammatory effects, reversing
aging-induced alterations in the cellular cytokine secretion profile and inhibiting
nuclear factor κ-light-chain-enhancer of activated B cells. Resveratrol also
attenuated mitochondrial O2
− production and upregulated the transcriptional activity of Nrf2 in aged
VSMCs. Thus, in non-human primates, cell-autonomous activation of nuclear factor
κ-light-chain-enhancer of activated B cells and expression of an inflammatory
secretome likely contribute to vascular inflammation in aging. Resveratrol treatment
prevents the proinflammatory properties of the aged VSMC secretome, an effect that likely
contributes to the demonstrated vasoprotective action of resveratrol in animal models of
Vascular aging; Inflammation; Oxidative stress; Cytokine; 3,5,4’-trihydroxy-trans-stilbene
Age-associated arterial remodeling involves arterial wall collagen deposition and elastin fragmentation, and an increase in arterial pressure. This arterial remodeling is linked to proinflammatory signaling, including transforming growth factor-beta1 (TGF-β1), monocyte chemoattractant protein-1 (MCP-1), and proendothelin-1(Pro-ET-1), activated by extracellular matrix metalloproteinases (MMPs) and orchestrated, in part, by the transcriptional factor ets-1. We tested the hypothesis that inhibition of MMP activation can decelerate the age-associated arterial proinflammation and its attendant increase in arterial pressure. Indeed, chronic administration of a broad spectrum MMP inhibitor, PD166739, via a daily gavage, to 16-mo-old rats for 8 months markedly blunted the expected age-associated increases in arterial pressure. This was accompanied by (1) inhibition of the age-associated increases in aortic gelatinase and interstitial collagenase activity in situ; (2) preservation of the elastic fiber network integrity; (3) a reduction of collagen deposition; (4) a reduction of MCP-1 and TGF-β1 activation; (5) a diminution in the activity of the pro-fibrogenic signaling molecule, SMAD-2/3 phosphorylation; (6) inhibition of pro-ET-1 activation; and (7) down-regulation of expression of ets-1. Acute exposure of cultured vascular smooth muscle cells (VSMC) in vitro to pro-ET-1 increased both the transcription and translation of ets-1, and these effects were markedly reduced by MMP inhibition. Furthermore, infection of VSMC with an adenovirus harboring a full-length ets-1 cDNA increased activities of both TGF-β1 and MCP-1. Collectively, our results indicate that MMP inhibition retards age-associated arterial proinflammatory signaling, and this is accompanied by perseveration of intact elastin fibers, a reduction in collagen, and blunting of an age-associated increase in blood pressure.
Aging; Arterial remodeling; Matrix metalloproteinase inhibitor; Endothelin-1; Transforming growth factor-beta 1; ets-1; Monocyte chemoattractant protein-1; Blood pressure
Arterial inflammation and remodeling, important sequellae of advancing age, are linked to the pathogenesis of age-associated arterial diseases, e.g., hypertension, atherosclerosis, and metabolic disorders. Recently, high-throughput proteomic screening has identified milk fat globule epidermal growth factor VIII (MFG-E8) as a novel local biomarker for aging arterial walls. Additional studies have shown that MFG-E8 is also an element of the arterial inflammatory signaling network. The transcription, translation, and signaling levels of MFG-E8 are increased in aged, atherosclerotic, hypertensive, and diabetic arterial walls in vivo as well as activated vascular smooth muscle cells (VSMC) and a subset of macrophages in vitro. In VSMC, MFG-E8 increases proliferation and invasion as well as the secretion of inflammatory molecules. In endothelial cells (EC), MFG-E8 facilitates apoptosis. In addition, MFG-E8 has been found to be an essential component of the endothelial-derived microparticles that relay biosignals and modulate arterial wall phenotypes.
This review mainly focuses upon the landscape of MFG-E8 expression and signaling in adverse arterial remodeling. Recent discoveries have suggested that MFG-E8 associated interventions are novel approaches for the retardation of the enhanced rates of VSMC proliferation and EC apoptosis that accompany arterial wall inflammation and remodeling during aging and age-associated arterial disease.
milk fat globule epidermal growth factor VIII; Arterial remodeling; Intervention
In the context of obesity epidemic, no large population study has extensively investigated the relationships between total and abdominal adiposity and large artery structure and function nor have such relationships been examined by gender, by age, by hypertensive status. We investigated these potential relationships in a large cohort of community dwelling volunteers participating the SardiNIA Study.
Methods and Results
Total and visceral adiposity and arterial properties were assessed in 6,148 subjects, aged 14–102 in a cluster of 4 towns in Sardinia, Italy. Arterial stiffness was measured as aortic pulse wave velocity (PWV), arterial thickness and lumen as common carotid artery (CCA) intima-media thickness (IMT) and diameter – respectively. We reported a nonlinear relationship between total and visceral adiposity and arterial stiffness, thickness, and diameter. The association between adiposity and arterial properties was steeper in women than in men, in younger than in older subjects. Waist correlated with arterial properties better than BMI. Within each BMI quartile, increasing waist circumference was associated with further significant changes in arterial structure and function.
The relationship between total or abdominal adiposity and arterial aging (PWV and CCA IMT) is not linear as described in the current study. Therefore, BMI- and/or waist-specific reference values for arterial measurements might need to be defined
arteries; arterial stiffness; carotid intima-media thickness; obesity; waist circumference; population study
Increased arterial stiffness is an independent predictor of cardiovascular disease independent from blood pressure. Recent studies have shed new light on the importance of inflammation on the pathogenesis of arterial stiffness. Arterial stiffness is associated with the increased activity of angiotensin II, which results in increased NADPH oxidase activity, reduced NO bioavailability and increased production of reactive oxygen species. Angiotensin II signaling activates matrix metalloproteinases (MMPs) which degrade TGFβ precursors to produce active TGFβ, which then results in increased arterial fibrosis. Angiotensin II signaling also activates cytokines, including monocyte chemoattractant protein-1, TNF-α, interleukin-1, interleukin-17 and interleukin-6. There is also ample clinical evidence that demonstrates the association of inflammation with increased arterial stiffness. Recent studies have shown that reductions in inflammation can reduce arterial stiffness. In patients with rheumatoid arthritis, increased aortic pulse wave velocity in patients was significantly reduced by anti tumor necrosis factor-α therapy. Among the major classes of anti hypertensive drugs, drugs that block the activation of the RAS system may be more effective in reducing the progression of arterial stiffness. Thus, there is rationale for targeting specific inflammatory pathways involved in arterial stiffness in the development of future drugs. Understanding the role of inflammation in the pathogenesis of arterial stiffness is important to understanding the complex puzzle that is the pathophysiology of arterial stiffening and may be important for future development of novel treatments.
Arterial stiffness; inflammation; angiotensin II
An accumulation of milk fat globule EGF-8 protein (MFG-E8) occurs within the context of arterial wall inflammatory remodeling during aging, hypertension, diabetes mellitus, or atherosclerosis. MFG-E8 induces VSMC invasion, but whether it effects VSMC proliferation, a salient feature of arterial inflammation, is unknown. Here, we show that in the rat arterial wall in vivo, PCNA and Ki67, markers of cell cycle activation, increase with age between 8 and 30-mo. In fresh or early passage VSMC isolated from old aortae, an increase in CDK4 and PCNA, and cell cycle with acceleration of S and G2 phases and reduction of the G1/G0 phase, and an increase in PDGF and its receptors, confer elevated proliferative capacity, compared to young VSMC. Increased co-expression and physical interaction of MFG-E8 and integrin αvβ5 occur with aging in both the rat aortic wall in vivo and in VSMC in vitro. In young VSMC in vitro, MFG-E8 added exogenously, or over-expressed endogenously, triggers phosphorylation of ERK1/2, augmented levels of PCNA and CDK4, increased BrdU incorporation and promotes proliferation, via αvβ5 integrins. MFG-E8 silencing, or its receptor inhibition, or the blockade of ERK1/2 phosphorylation in these cells reduces PCNA and CDK4 levels and decelerates the cell cycle S phase, conferring a reduction in proliferative capacity. Collectively, these results indicate that MFG-E8 in a dose-dependent manner, coordinates the expression of cell cycle molecules and facilitates VSMC proliferation via integrin/ ERK1/2 signaling. Thus, an increase in MFG-E8 signaling is a mechanism of the age-associated increase in aortic VSMC proliferation.
MFG-E8; Aging; VSMC proliferation; cell cycle; vascular remodeling
Sinoatrial node cells (SANC) generate local, subsarcolemmal Ca2+ releases (LCRs) from sarcoplasmic reticulum (SR) during late diastolic depolarization (DD). LCRs activate an inward Na+-Ca2+ exchange current (INCX) which accelerates DD rate, prompting the next action potential (AP). The LCR period, i.e., a delay between AP-induced Ca2+ transient and LCR appearance, defines the time of late DD INCX activation. Mechanisms that control the LCR period, however, are still unidentified.
To determine dependence of the LCR period on SR Ca2+ refilling kinetics and establish links between regulation of SR Ca2+ replenishment, LCR period and spontaneous cycle length.
Methods and Results
Spontaneous APs and SR luminal or cytosolic Ca2+ were recorded using perforated patch and confocal microscopy, respectively. Time to 90% replenishment of SR Ca2+ following AP-induced Ca2+ transient was highly correlated with the time to 90% decay of cytosolic Ca2+ transient (T-90C). Local SR Ca2+ depletions mirror their cytosolic counterparts, LCRs, and occur following SR Ca2+ refilling. Inhibition of SR Ca2+ pump by cyclopiazonic acid (CPA) dose-dependently suppressed spontaneous SANC firing up to ~50%. CPA and graded changes in phospholamban phosphorylation produced by β-AR stimulation, phosphodiesterase or PKA inhibition shifted T-90C and proportionally shifted the LCR period and spontaneous cycle length (R2=0.98).
The LCR period, a critical determinant of the spontaneous SANC cycle length, is defined by the rate of SR Ca2+ replenishment, which is critically dependent on SR pumping rate, Ca2+ available for pumping, supplied by L-type Ca2+ channel, and RyR Ca2+ release flux each of which is modulated by cAMP-mediated PKA-dependent phosphorylation.
Sinoatrial nodal pacemaker cells; sarcoplasmic reticulum Ca2+ pumping; β-adrenergic receptor signaling
Hypertension, atherosclerosis, and resultant chronic heart failure (HF) reach epidemic proportions among older persons, and the clinical manifestations and the prognoses of these worsen with increasing age. Thus, age per se is the major risk factor for cardiovascular disease. Changes in cardiac cell phenotype that occur with normal aging, as well as in HF associated with aging, include deficits in β-adrenergic receptor (β-AR) signaling, increased generation of reactive oxygen species (ROS), and altered excitation-contraction (EC) coupling that involves prolongation of the action potential (AP), intracellular Ca2+ (Ca2+i) transient and contraction, and blunted force- and relaxation-frequency responses. Evidence suggests that altered sarcoplasmic reticulum (SR) Ca2+ uptake, storage and release play central role in these changes, which also involve sarcolemmal L-type Ca2+ channel (LCC), Na+-Ca2+ exchanger (NCX), and K+ channels. We review the age-associated changes in the expression and function of Ca2+ transporting proteins, and functional consequences of these changes at the cardiac myocyte and organ levels. We also review sexual dimorphism and self-renewal of the heart in the context of cardiac aging and HF.
Heart; Aging; Heart failure; Cardiac myocyte; Sarcoplasmic reticulum; Calcium
We examined relationships of central adiposity with left ventricular (LV) diastolic dysfunction in men and women who participated in the Baltimore Longitudinal Study of Aging, a prospective community-based study of older persons. The sample for this cross-sectional analysis included 399 women and 370 men. Central adiposity was estimated by waist circumference (WC) and global adiposity by body mass index (BMI). Using data from a comprehensive echocardiographic study that included tissue Doppler imaging, diastolic function was graded according to three parameters (E/A ratio, E/Em ratio and left atrial volume index). In logistic regression models adjusted for age, gender, cardiovascular risk factors and hemodynamic parameters, WC and BMI were both independently associated with LV diastolic dysfunction. However, when both WC and BMI were in the same model, only WC remained significantly associated with LV diastolic dysfunction (OR= 1.04, 95% CI: 1.01 to 1.08, p= 0.02). In gender stratified analyses, WC was found significantly associated with LV diastolic dysfunction - independently of BMI - in women (OR=1.08, 95% CI: 1.04 to 1.14, p<.001) but not in men (OR= 1.00, 95% CI: 0.95 to 1.05, p= 0.91). Further adjustment for left ventricular mass index failed to modify these relations. In conclusion, the adverse effect of central adiposity on LV diastolic function was independent of general adiposity and more pronounced among women. The impact of visceral adiposity on LV diastolic dysfunction would benefit from confirmation in longitudinal studies.
Diastolic dysfunction; adiposity; waist circumference; body mass index
sinoatrial node; pacemaker activity; action potentials; ion channels HCN4 hyperpolarization-activated current; Na+/Ca2+ exchange current
Cardiac sinoatrial node; pacemaker cells; ion channels; local Calcium release; protein kinase A; CaMKII; sarcoplasmic reticulum; phospholamban; ryanodine receptors
Ion channels on the surface membrane of sinoatrial nodal pacemaker cells (SANC) are the proximal cause of an action potential. Each individual channel type has been thoroughly characterized under voltage clamp, and the ensemble of the ion channel currents reconstructed in silico generates rhythmic action potentials. Thus, this ensemble can be envisioned as a surface “membrane clock” (M clock). Localized subsarcolemmal Ca2+ releases are generated by the sarcoplasmic reticulum via ryanodine receptors during late diastolic depolarization and are referred to as an intracellular “Ca2+ clock”, because their spontaneous occurrence is periodic during voltage clamp or in detergent-permeabilized SANC, and in silico as well. In spontaneously firing SANC, the M and Ca2+ clocks do not operate in isolation, but work together via numerous interactions modulated by membrane voltage, subsarcolemmal Ca2+, and PKA and CaMKII-dependent protein phosphorylation. Through these interactions the two subsystem clocks become mutually entrained to form a robust, stable, coupled-clock system that drives normal cardiac pacemaker cell automaticity. G-protein coupled-receptors signaling creates pacemaker flexibility, i.e. effects changes in the rhythmic action potential firing rate, by impacting on these very same factors that regulate robust basal coupled-clock system function. This review examines evidence that forms the basis of this coupled-clock system concept in cardiac SANC.
Sinoatrial node; cardiac pacemaker cells; ion channels; local Ca2+ releases; β-adrenergic stimulation; cholinergic stimulation; protein kinase A; CaMKII; sarcoplasmic reticulum; phospholamban; ryanodine receptors
Age-associated arterial alterations in cells, matrix, and biomolecules are the foundation for the initiation and progression of cardiovascular diseases in older persons. This review focuses on the latest advances on the intertwining of aging and disease within the arterial wall at the cell and molecular levels.
Endothelial dysfunction, VSMC proliferation/invasion/secretion, matrix fragmentation, collagenization and glycation are characteristics of an age-associated arterial phenotype that creates a microenvironment enriched with reactive oxygen species (ROS) for the pathogenesis of arterial disease. This niche creates an age-associated arterial secretory phenotype (AAASP), which is orchestrated by the concerted effects of numerous age-modified Ang II signaling molecules. Most of these biomolecular, cell, and matrix modifications that comprise the AASP can be elicited by experimental hypertension or atherosclerosis at a younger age. The arterial AAASP also shares features of a senescence associated secretory phenotype (SASP) identified in other mesenchymocytes, i.e. fibroblasts.
A subclinical AAASP evolves during aging. Targeting this subclinical AAASP may reduce the incidence and progression of the quintessential age-associated arterial diseases, i.e. hypertension and atherosclerosis.
angiotensin II; arterial aging; arterial disease
Ca2+-activated basal adenylate cyclase (AC) in rabbit sinoatrial node cells (SANC) guarantees, via basal cAMP/PKA-calmodulin/CaMKII-dependent protein phosphorylation, the occurrence of rhythmic, sarcoplasmic-reticulum generated, sub-membrane Ca2+ releases that prompt rhythmic, spontaneous action potentials (APs). This high-throughput signaling consumes ATP.
We have previously demonstrated that basal AC-cAMP/PKA signaling directly, and Ca2+ indirectly, regulate mitochondrial ATP production. While, clearly, Ca2+-calmodulin-CaMKII activity regulates ATP consumption, whether it has a role in the control of ATP production is unknown.
Methods and Results
We superfused single, isolated rabbit SANC at 37°C with physiological saline containing CaMKII inhibitors, (KN-93 or autocamtide-2 Related Inhibitory Peptide (AIP)), or a calmodulin inhibitor (W-7) and measured cytosolic Ca2+, flavoprotein fluorescence and spontaneous AP firing rate. We measured cAMP, ATP and O2 consumption in cell suspensions. Graded reductions in basal CaMKII activity by KN-93 (0.5–3 µmol/L) or AIP (2–10 µmol/L) markedly slow the kinetics of intracellular Ca2+ cycling, decrease the spontaneous AP firing rate, decrease cAMP, and reduce O2 consumption and flavoprotein fluorescence. In this context of graded reductions in ATP demand, however, ATP also becomes depleted, indicating reduced ATP production.
CaMKII signaling, a crucial element of normal automaticity in rabbit SANC, is also involved in SANC bioenergetics.
Decades of intensive research of primary cardiac pacemaker, the sinoatrial node, have established potential roles of specific membrane channels in the generation of the diastolic depolarization, the major mechanism allowing sinoatrial node cells generate spontaneous beating. During the last three decades, multiple studies made either in the isolated sinoatrial node or sinoatrial node cells have demonstrated a pivotal role of Ca2+ and, specifically Ca2+-release from sarcoplasmic reticulum, for spontaneous beating of cardiac pacemaker. Recently, spontaneous, rhythmic local subsarcolemmal Ca2+ releases from ryanodine receptors during late half of the diastolic depolarization have been implicated as a vital factor in the generation of sinoatrial node cells spontaneous firing. Local Ca2+ releases are driven by a unique combination of high basal cAMP production by adenylyl cyclases, high basal cAMP degradation by phosphodiesterases and a high level of cAMP-mediated PKA-dependent phosphorylation. These local Ca2+ releases activate an inward Na+-Ca2+ exchange current which accelerates the terminal diastolic depolarization rate and, thus, controls the spontaneous pacemaker firing. Both the basal primary pacemaker beating rate and its modulation via β-adrenergic receptor stimulation appear to be critically dependent upon intact RyR function and local subsarcolemmal sarcoplasmic reticulum generated Ca2+ releases. This review aspires to integrate the traditional viewpoint that has emphasized the supremacy of the ensemble of surface membrane ion channels in spontaneous firing of the primary cardiac pacemaker, and these novel perspectives of cAMP-mediated PKA-dependent Ca2+ cycling in regulation of the heart pacemaker clock, both in the basal state and during β-adrenergic receptor stimulation.
sinoatrial nodal cells; local Ca2+ release; ryanodine receptor; ionic channels; beta-adrenergic receptor stimulation; phosphodiesterase
Phosphorylation of β2-adrenergic receptor (β2AR) by a family of serine/threonine kinases known as G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) is a critical determinant of cardiac function. Upregulation of G protein-coupled receptor kinase 2 (GRK2) is a well-established causal factor of heart failure, but the underlying mechanism is poorly understood.
We seek to determine the relative contribution of PKA- and GRK-mediated phosphorylation of β2AR to the receptor coupling to Gi signaling that attenuates cardiac reserve and contributes to the pathogenesis of heart failure in response to pressure overload.
Methods and Results
Overexpression of GRK2 led to a Gi-dependent decrease of contractile response to βAR stimulation in cultured mouse cardiomyocytes and in vivo. Importantly, cardiac-specific transgenic overexpression of a mutant β2AR lacking PKA phosphorylation sites (PKA- TG), but not the wild type β2AR (WT TG) or a mutant β2AR lacking GRK sites (GRK- TG), led to exaggerated cardiac response to pressure overload, as manifested by markedly exacerbated cardiac maladaptive remodeling and failure, and early mortality. Furthermore, inhibition of Gi signaling with pertussis toxin restores cardiac function in heart failure associated with increased β2AR to Gi coupling induced by removing PKA phosphorylation of the receptor and in GRK2 transgenic mice, indicating that enhanced phosphorylation of β2AR by GRK and resultant increase in Gi-biased β2AR signaling play an important role in the development of heart failure.
Our data show that enhanced β2AR phosphorylation by GRK, in addition to PKA, leads the receptor to Gi-biased signaling which, in turn, contributes to the pathogenesis of heart failure, marking Gi-biased β2AR signaling as a primary event linking upregulation of GRK to cardiac maladaptive remodeling, failure and cardiodepression.
β2-adrenergic receptor; G protein-coupled receptor kinase; Heart failure; hypertrophy