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1.  Synchronization of sinoatrial node pacemaker cell clocks and its autonomic modulation impart complexity to heart beating intervals Short title: Beating-rate variability of sinoatrial node cells 
Background
A reduction of complexity of heart-beat interval variability (BIV) that is associated with an increased morbidity and mortality in cardiovascular disease states is thought to derive from the balance of sympathetic and parasympathetic neural impulses to the heart. But rhythmic clock-like behavior intrinsic to pacemaker cells within the sinoatrial node (SAN) drives their beating, even in the absence of autonomic neural input.
Objective
To test how this rhythmic clock-like behavior intrinsic to pacemaker cells interacts with autonomic impulses to the heart-beat interval variability in vivo.
Methods
We analyzed BIV in the time and frequency domains and by fractal and entropy analyses: i) in vivo, when the brain input to the SAN is intact; ii) during autonomic denervation in vivo; iii) in isolated SAN tissue (i.e., in which the autonomic-neural input is completely absent); iv) in single pacemaker cells isolated from the SAN; and v) following autonomic receptor stimulation of these cells.
Results
Spontaneous-beating intervals of pacemaker cells residing within the isolated SAN tissue exhibit fractal-like behavior and have lower approximate entropy than in the intact heart. Isolation of pacemaker cells from SAN tissue, however, leads to a loss in the beating-interval order and fractal-like behavior. β adrenergic receptor stimulation of isolated pacemaker cells increases intrinsic clock synchronization, decreases their action potential period and increases system complexity.
Conclusions
Both the average-beating interval in vivo and beating interval complexity are conferred by the combined effects of clock periodicity intrinsic to pacemaker cells and their response to autonomic-neural input.
doi:10.1016/j.hrthm.2014.03.049
PMCID: PMC4065846  PMID: 24713624
Autonomic neural impulse; Chaotic systems; Fractal behavior; Heart rate variability; Sinoatrial nodal pacemaker cells
2.  Potential effects of intrinsic heart pacemaker cell mechanisms on dysrhythmic cardiac action potential firing 
The heart's regular electrical activity is initiated by specialized cardiac pacemaker cells residing in the sinoatrial node. The rate and rhythm of spontaneous action potential firing of sinoatrial node cells are regulated by stochastic mechanisms that determine the level of coupling of chemical to electrical clocks within cardiac pacemaker cells. This coupled-clock system is modulated by autonomic signaling from the brain via neurotransmitter release from the vagus and sympathetic nerves. Abnormalities in brain-heart clock connections or in any molecular clock activity within pacemaker cells lead to abnormalities in the beating rate and rhythm of the pacemaker tissue that initiates the cardiac impulse. Dysfunction of pacemaker tissue can lead to tachy-brady heart rate alternation or exit block that leads to long atrial pauses and increases susceptibility to other cardiac arrhythmia. Here we review evidence for the idea that disturbances in the intrinsic components of pacemaker cells may be implemented in arrhythmia induction in the heart.
doi:10.3389/fphys.2015.00047
PMCID: PMC4337365  PMID: 25755643
arrhythmias; atrial fibrillation; coupled-clock pacemaker system; heart rate variability; sinus node disease
3.  From two competing oscillators to one coupled-clock pacemaker cell system 
At the beginning of this century, debates regarding “what are the main control mechanisms that ignite the action potential (AP) in heart pacemaker cells” dominated the electrophysiology field. The original theory which prevailed for over 50 years had advocated that the ensemble of surface membrane ion channels (i.e., “M-clock”) is sufficient to ignite rhythmic APs. However, more recent experimental evidence in a variety of mammals has shown that the sarcoplasmic reticulum (SR) acts as a “Ca2+-clock” rhythmically discharges diastolic local Ca2+ releases (LCRs) beneath the cell surface membrane. LCRs activate an inward current (likely that of the Na+/Ca2+ exchanger) that prompts the surface membrane “M-clock” to ignite an AP. Theoretical and experimental evidence has mounted to indicate that this clock “crosstalk” operates on a beat-to-beat basis and determines both the AP firing rate and rhythm. Our review is focused on the evolution of experimental definition and numerical modeling of the coupled-clock concept, on how mechanisms intrinsic to pacemaker cell determine both the heart rate and rhythm, and on future directions to develop further the coupled-clock pacemaker cell concept.
doi:10.3389/fphys.2015.00028
PMCID: PMC4327306  PMID: 25741284
arrhythmias; coupled-clock pacemaker system; heart rate variability; mathematical modeling; sinoatrial node
4.  Sarcoplasmic Reticulum Ca2+ Cycling Protein Phosphorylation in a Physiologic Ca2+ Milieu Unleashes a High-Power, Rhythmic Ca2+ Clock in Ventricular Myocytes: Relevance to Arrhythmias and Bio-Pacemaker Design 
Basal phosphorylation of sarcoplasmic reticulum (SR) Ca2+ proteins is high in sinoatrial nodal cells (SANC), which generate partially synchronized, spontaneous, rhythmic, diastolic local Ca2+ releases (LCRs), but low in ventricular myocytes (VM), which exhibit rare diastolic, stochastic SR-generated Ca2+ sparks. We tested the hypothesis that in a physiologic Ca2+ milieu, and independent of increased Ca2+ influx, an increase in basal phosphorylation of SR Ca2+ cycling proteins will convert stochastic Ca2+ sparks into periodic, high-power Ca2+ signals of the type that drives SANC normal automaticity. We measured phosphorylation of SR-associated proteins, phospholamban (PLB) and ryanodine receptors (RyR), and spontaneous local Ca2+ release characteristics (LCR) in permeabilized single, rabbit VM in physiologic [Ca2+], prior to and during inhibition of protein phosphatase (PP) and phosphodiesterase (PDE), or addition of exogenous cAMP, or in the presence of an antibody (2D12), that specifically inhibits binding of the PLB to SERCA-2. In the absence of the aforementioned perturbations, VM could only generate stochastic local Ca2+ releases of low power and low amplitude, as assessed by confocal Ca2+ imaging and spectral analysis. When the kinetics of Ca2+ pumping into the SR were increased by an increase in PLB phosphorylation (via PDE and PP inhibition or addition of cAMP) or by 2D12, self-organized, “clock-like” local Ca2+ releases, partially synchronized in space and time (Ca2+ wavelets), emerged, and the ensemble of these rhythmic local Ca2+ wavelets generated a periodic high-amplitude Ca2+ signal. Thus, a Ca2+ clock is not specific to pacemaker cells, but can also be unleashed in VM when SR Ca2+ cycling increases and spontaneous local Ca2+ release becomes partially synchronized. This unleashed Ca2+ clock that emerges in a physiological Ca2+ milieu in VM has two faces, however: it can provoke ventricular arrhythmias; or if harnessed, can be an important feature of novel bio-pacemaker designs.
doi:10.1016/j.yjmcc.2013.11.011
PMCID: PMC3896924  PMID: 24274954
cardiac ventricular myocytes; calcium clock; calcium cycling; protein phosphorylation; spontaneous local calcium releases
5.  New evidence for coupled clock regulation of the normal automaticity of sinoatrial nodal pacemaker cells: bradycardic effects of ivabradine are linked to suppression of intracellular Ca2+ cycling 
Beneficial clinical bradycardic effects of ivabradine (IVA) have been interpreted solely on the basis of If inhibition, because IVA specifically inhibits If in sinoatrial nodal pacemaker cells (SANC). However, it has been recently hypothesized that SANC normal automaticity is regulated by crosstalk between an “M clock,” the ensemble of surface membrane ion channels, and a “Ca2+ clock,” the sarcoplasmic reticulum (SR). We tested the hypothesis that crosstalk between the two clocks regulates SANC automaticity, and that indirect suppression of the Ca2+ clock further contributes to IVA-induced bradycardia.
IVA (3μM) not only reduced If amplitude by 45±6% in isolated rabbit SANC, but the IVA-induced slowing of the action potential (AP) firing rate was accompanied by reduced SR Ca2+ load, slowed intracellular Ca2+ cycling kinetics, and prolonged the period of spontaneous local Ca2+ releases (LCRs) occurring during diastolic depolarization. Direct and specific inhibition of SERCA2 by cyclopiazonic acid (CPA) had effects similar to IVA on LCR period and AP cycle length. Specifically, the LCR period and AP cycle length shift toward longer times almost equally by either direct perturbations of the M clock (IVA) or the Ca2+ clock (CPA), indicating that the LCR period reports the crosstalk between the clocks. Our numerical model simulations predict that entrainment between the two clocks that involves a reduction in INCX during diastolic depolarization is required to explain the experimentally AP firing rate reduction by IVA.
In summary, our study provides new evidence that a coupled-clock system regulates normal cardiac pacemaker cell automaticity. Thus, IVA-induced bradycardia includes a suppression of both clocks within this system.
doi:10.1016/j.yjmcc.2013.04.026
PMCID: PMC3735682  PMID: 23651631
Sinoatrial nodal pacemaker cells; Ca2+ cycling; ion channels; physiology; sarcoplasmic reticulum
6.  BRINGING PREVENTION IN GERIATRICS : EVIDENCES FROM CARDIOVASCULAR MEDICINE SUPPORTING THE NEW CHALLENGE 
Experimental gerontology  2012;48(1):64-68.
Aging is a dynamic and systemic process, with high inter-individual heterogeneity, likely partially adaptive. Cardiovascular disease and hypertension are among the leading conditions causing disabilities in older subjects. If, in accordance with most recent definition, prevention is any intervention before the patient receives a diagnosis, prevention is possible at any age. Additionally, disability and CV disease in the elderly may be prevented by targeting factors underlying and modulating the arterial aging process. Cross-talk between arterial and brain aging will be discussed in this context as a paradigmatic clinical model fostering prevention in older subjects.
doi:10.1016/j.exger.2012.02.009
PMCID: PMC4521763  PMID: 22406133
arterial aging; arterial stiffness; prevention; cardiovascular disease; dementia; cognitive impairment
7.  Role of bone mineral density in the inverse relationship between body size and aortic calcification: results from the Baltimore Longitudinal Study of Aging 
Atherosclerosis  2014;235(1):169-175.
OBJECTIVE
There is a J-shaped relationship between body mass index (BMI) and cardiovascular outcomes in elderly patients (obesity paradox). Whether low BMI correlates with aortic calcification (AC) and whether this association is accounted for by bone demineralization is uncertain.
METHODS
Presence of AC was evaluated in 687 community-dwelling individuals (49% male, mean age 67±13 years) using CT images of the thoracic, upper and lower abdominal aorta, and scored from 0 to 3 according to number of sites that showed any calcification. Whole-body bone mineral density (BMD) was evaluated by dual-energy x-ray absorptiometry. Predictors of AC were assessed by logistic regression, and the role of BMD using mediation analysis.
RESULTS
Age and cardiovascular risk factors were positively associated while both BMI (r=−0.11, p<0.01) and BMD (r=−0.17, p<0.0001) were negatively associated with AC severity. In multivariate models, lower BMI (OR 0.96, 95%CI 0.92–0.99, p=0.01), older age, higher systolic blood pressure, use of lipid-lowering drugs and smoking were independent predictors of AC. A nonlinear relationship between BMI and AC was noticed (p=0.03), with decreased AC severity among overweight participants. After adjusting for BMD, the coefficient relating BMI to AC was reduced by 14% and was no longer significant, whereas BMD remained negatively associated with AC (OR 0.82, 95%CI 0.069–0.96, p=0.01), with a trend for a stronger relationship in older participants.
CONCLUSION
Low BMI is associated with increased AC, possibly through calcium mobilization from bone, resulting in low BMD. Prevention of weight loss and bone demineralization with aging may help reducing AC.
doi:10.1016/j.atherosclerosis.2014.04.018
PMCID: PMC4080433  PMID: 24842457
aortic calcification; body mass index; body size; bone mineral density; obesity paradox; calcification paradox
8.  Resveratrol prevents high fat/sucrose diet-induced central arterial wall inflammation and stiffening in nonhuman primates 
Cell metabolism  2014;20(1):183-190.
SUMMARY
Central arterial wall stiffening driven by a chronic inflammatory milieu accompanies arterial diseases, the leading cause of cardiovascular (CV) morbidity and mortality in Western society. Increase in central arterial wall stiffening, measured as an increase in aortic pulse wave velocity (PWV), is a major risk factor for clinical CV disease events. However, no specific therapies to reduce PWV are presently available. In rhesus monkeys, a two-year diet high in fat and sucrose (HFS) increases not only body weight and cholesterol, but also induces prominent central arterial wall stiffening and increases PWV and inflammation. The observed loss of endothelial cell integrity, lipid and macrophage infiltration, and calcification of the arterial wall were driven by genomic and proteomic signatures of oxidative stress and inflammation. Resveratrol prevented the HFS-induced arterial wall inflammation and the accompanying increase in PWV. Dietary resveratrol may hold promise as a novel therapy to ameliorate increases in PWV.
doi:10.1016/j.cmet.2014.04.018
PMCID: PMC4254394  PMID: 24882067
9.  Aortic pulse wave velocity improves cardiovascular event prediction: an individual participant meta-analysis of prospective observational data from 17,635 subjects 
Objectives
To determine whether aortic pulse wave velocity (aPWV) improves prediction of cardiovascular (CVD) events beyond conventional risk factors.
Background
Several studies have shown that aPWV may be a useful risk factor for predicting CVD but have been underpowered to examine whether this is true for different sub-groups.
Methods
We undertook a systematic review and obtained individual participant data from 16 studies. Study-specific associations of aPWV with cardiovascular outcomes were determined using Cox proportional hazard models and random effect models to estimate pooled effects.
Results
Of 17,635 participants, 1,785 (10%) had a cardiovascular (CVD) event. The pooled age- and sex-adjusted hazard ratio [95% CI] per SD change in loge aPWV was 1.35 [1.22, 1.50, p<0.001] for coronary heart disease (CHD), 1.54 [1.34, 1.78, p<0.001] for stroke, and 1.45 [1.30, 1.61, p<0.001) for CVD. Associations stratified by sex, diabetes and hypertension were similar, but decreased with age (1.89, 1.77, 1.36 and 1.23 for ≤50, 51–60, 61–70 and >70 years respectively, pinteraction <0.001). After adjusting for conventional risk factors, aPWV remained a predictor: CHD 1.23, [1.11, 1.35 p<0.001]; stroke 1.28, [1.16, 1.42 p<0.001]; cardiovascular events 1.30 [1.18, 1.43, p<0.001]. Reclassification indices showed the addition of aPWV improved risk prediction (13% for 10 year CVD risk for intermediate risk) for some sub-groups.
Conclusions
Consideration of aPWV improves model fit and reclassifies risk for future cardiovascular events in models that include standard risk factors. aPWV may enable better identification of high-risk populations who may benefit from more aggressive cardiovascular risk factor management.
doi:10.1016/j.jacc.2013.09.063
PMCID: PMC4401072  PMID: 24239664
pulse wave velocity; meta-analysis; cardiovascular disease; prognostic factor
10.  GeneOptimizer Program-assisted cDNA Reengineering Enhances sRAGE Autologous Expression in Chinese Hamster Ovary Cells 
Soluble receptor for advanced glycation end products (sRAGE) is a secreted mammalian protein that functions as a decoy to counter-react RAGE signaling-resultant pathological conditions, and has high therapeutic potentials. Our prior studies showed that recombinant human sRAGE expressed in Chinese hamster, C. griseus, ovary (CHO) cells is modified by specific N-glycosylation, and exhibits higher bioactivity than that expressed in other host systems including insect S. frugiperda cells. Here, we show that GeneOptimizer software program-assisted, reengineered sRAGE cDNA enhances the recombinant protein expression in CHO cells. The cDNA sequence encoding human sRAGE was optimized for RNA structure, stability, and codon usages in CHO cells. We found that such optimization augmented sRAGE expression over 2 folds of its wild-type counterpart. We also studied how individual parameter impacted sRAGE autologous expression in CHO cells, and whether sRAGE bioactivity was compromised. We found that the enhanced expression appeared not to affect sRAGE N-glycosylation and bioactivity. Optimization of sRAGE expression provides a basis for future large-scale production of this protein to meet medical needs.
doi:10.1016/j.pep.2013.12.006
PMCID: PMC3936886  PMID: 24373844
GeneOptimizer program; sRAGE; protein expression; CHO
11.  Beat to beat Ca2+-dependent regulation of sinoatrial nodal pacemaker cell rate and rhythm 
Introduction
Whether intracellular Ca2+ regulates sinoatrial node cell (SANC) action potential (AP) firing rate on a beat-to-beat basis is controversial.
Objective
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.
Conclusions
Our results directly support the hypothesis that intracellular Ca2+ regulates normal SANC automaticity on a beat-to-beat basis.
doi:10.1016/j.yjmcc.2011.08.029
PMCID: PMC3208800  PMID: 21963899
pacemaker cell automaticity; Ca2+ cycling; pacemaker Ca2+ clock; Ca2+-excitation contraction coupling; arrhythmia
12.  Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization 
Arking, Dan E. | Pulit, Sara L. | Crotti, Lia | van der Harst, Pim | Munroe, Patricia B. | Koopmann, Tamara T. | Sotoodehnia, Nona | Rossin, Elizabeth J. | Morley, Michael | Wang, Xinchen | Johnson, Andrew D. | Lundby, Alicia | Gudbjartsson, Daníel F. | Noseworthy, Peter A. | Eijgelsheim, Mark | Bradford, Yuki | Tarasov, Kirill V. | Dörr, Marcus | Müller-Nurasyid, Martina | Lahtinen, Annukka M. | Nolte, Ilja M. | Smith, Albert Vernon | Bis, Joshua C. | Isaacs, Aaron | Newhouse, Stephen J. | Evans, Daniel S. | Post, Wendy S. | Waggott, Daryl | Lyytikäinen, Leo-Pekka | Hicks, Andrew A. | Eisele, Lewin | Ellinghaus, David | Hayward, Caroline | Navarro, Pau | Ulivi, Sheila | Tanaka, Toshiko | Tester, David J. | Chatel, Stéphanie | Gustafsson, Stefan | Kumari, Meena | Morris, Richard W. | Naluai, Åsa T. | Padmanabhan, Sandosh | Kluttig, Alexander | Strohmer, Bernhard | Panayiotou, Andrie G. | Torres, Maria | Knoflach, Michael | Hubacek, Jaroslav A. | Slowikowski, Kamil | Raychaudhuri, Soumya | Kumar, Runjun D. | Harris, Tamara B. | Launer, Lenore J. | Shuldiner, Alan R. | Alonso, Alvaro | Bader, Joel S. | Ehret, Georg | Huang, Hailiang | Kao, W.H. Linda | Strait, James B. | Macfarlane, Peter W. | Brown, Morris | Caulfield, Mark J. | Samani, Nilesh J. | Kronenberg, Florian | Willeit, Johann | Smith, J. Gustav | Greiser, Karin H. | zu Schwabedissen, Henriette Meyer | Werdan, Karl | Carella, Massimo | Zelante, Leopoldo | Heckbert, Susan R. | Psaty, Bruce M. | Rotter, Jerome I. | Kolcic, Ivana | Polašek, Ozren | Wright, Alan F. | Griffin, Maura | Daly, Mark J. | Arnar, David O. | Hólm, Hilma | Thorsteinsdottir, Unnur | Denny, Joshua C. | Roden, Dan M. | Zuvich, Rebecca L. | Emilsson, Valur | Plump, Andrew S. | Larson, Martin G. | O'Donnell, Christopher J. | Yin, Xiaoyan | Bobbo, Marco | D'Adamo, Adamo P. | Iorio, Annamaria | Sinagra, Gianfranco | Carracedo, Angel | Cummings, Steven R. | Nalls, Michael A. | Jula, Antti | Kontula, Kimmo K. | Marjamaa, Annukka | Oikarinen, Lasse | Perola, Markus | Porthan, Kimmo | Erbel, Raimund | Hoffmann, Per | Jöckel, Karl-Heinz | Kälsch, Hagen | Nöthen, Markus M. | consortium, HRGEN | den Hoed, Marcel | Loos, Ruth J.F. | Thelle, Dag S. | Gieger, Christian | Meitinger, Thomas | Perz, Siegfried | Peters, Annette | Prucha, Hanna | Sinner, Moritz F. | Waldenberger, Melanie | de Boer, Rudolf A. | Franke, Lude | van der Vleuten, Pieter A. | Beckmann, Britt Maria | Martens, Eimo | Bardai, Abdennasser | Hofman, Nynke | Wilde, Arthur A.M. | Behr, Elijah R. | Dalageorgou, Chrysoula | Giudicessi, John R. | Medeiros-Domingo, Argelia | Barc, Julien | Kyndt, Florence | Probst, Vincent | Ghidoni, Alice | Insolia, Roberto | Hamilton, Robert M. | Scherer, Stephen W. | Brandimarto, Jeffrey | Margulies, Kenneth | Moravec, Christine E. | Fabiola Del, Greco M. | Fuchsberger, Christian | O'Connell, Jeffrey R. | Lee, Wai K. | Watt, Graham C.M. | Campbell, Harry | Wild, Sarah H. | El Mokhtari, Nour E. | Frey, Norbert | Asselbergs, Folkert W. | Leach, Irene Mateo | Navis, Gerjan | van den Berg, Maarten P. | van Veldhuisen, Dirk J. | Kellis, Manolis | Krijthe, Bouwe P. | Franco, Oscar H. | Hofman, Albert | Kors, Jan A. | Uitterlinden, André G. | Witteman, Jacqueline C.M. | Kedenko, Lyudmyla | Lamina, Claudia | Oostra, Ben A. | Abecasis, Gonçalo R. | Lakatta, Edward G. | Mulas, Antonella | Orrú, Marco | Schlessinger, David | Uda, Manuela | Markus, Marcello R.P. | Völker, Uwe | Snieder, Harold | Spector, Timothy D. | Ärnlöv, Johan | Lind, Lars | Sundström, Johan | Syvänen, Ann-Christine | Kivimaki, Mika | Kähönen, Mika | Mononen, Nina | Raitakari, Olli T. | Viikari, Jorma S. | Adamkova, Vera | Kiechl, Stefan | Brion, Maria | Nicolaides, Andrew N. | Paulweber, Bernhard | Haerting, Johannes | Dominiczak, Anna F. | Nyberg, Fredrik | Whincup, Peter H. | Hingorani, Aroon | Schott, Jean-Jacques | Bezzina, Connie R. | Ingelsson, Erik | Ferrucci, Luigi | Gasparini, Paolo | Wilson, James F. | Rudan, Igor | Franke, Andre | Mühleisen, Thomas W. | Pramstaller, Peter P. | Lehtimäki, Terho J. | Paterson, Andrew D. | Parsa, Afshin | Liu, Yongmei | van Duijn, Cornelia | Siscovick, David S. | Gudnason, Vilmundur | Jamshidi, Yalda | Salomaa, Veikko | Felix, Stephan B. | Sanna, Serena | Ritchie, Marylyn D. | Stricker, Bruno H. | Stefansson, Kari | Boyer, Laurie A. | Cappola, Thomas P. | Olsen, Jesper V. | Lage, Kasper | Schwartz, Peter J. | Kääb, Stefan | Chakravarti, Aravinda | Ackerman, Michael J. | Pfeufer, Arne | de Bakker, Paul I.W. | Newton-Cheh, Christopher
Nature genetics  2014;46(8):826-836.
The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal Mendelian Long QT Syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals we identified 35 common variant QT interval loci, that collectively explain ∼8-10% of QT variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 novel QT loci in 298 unrelated LQTS probands identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode for proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies novel candidate genes for ventricular arrhythmias, LQTS,and SCD.
doi:10.1038/ng.3014
PMCID: PMC4124521  PMID: 24952745
genome-wide association study; QT interval; Long QT Syndrome; sudden cardiac death; myocardial repolarization; arrhythmias
13.  Ca2+-regulated-cAMP/PKA signaling in cardiac pacemaker cells links ATP supply to demand 
Rationale
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.
Objective
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.
Conclusions
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.
doi:10.1016/j.yjmcc.2011.07.018
PMCID: PMC3184360  PMID: 21835182
Calcium-activated adenylyl cyclase; constitutive basal PKA-dependent phosphorylation; bioenergetics; pacemaker automaticity; respiration
14.  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) Ca2+ cycling, i.e. an intracellular “Ca2+ 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. The role of AC-cAMP-PKA-Ca2+ signaling cascade in mouse, the species most often utilized for genetic manipulations, however, has not been systematically tested. Here we show that Ca2+ cycling proteins (e.g. RyR2, NCX1, and SERCA2) are abundantly expressed in mouse SAN and that spontaneous, rhythmic SR generated Local Ca2+ Releases (LCRs) occur in skinned mouse SANC, clamped at constant physiologic [Ca2+]. Mouse SANC also exhibits a high basal level of phospholamban (PLB) phosphorylation at the PKA-dependent site, Serine16. Inhibition of intrinsic PKA activity or inhibition of PDE in SANC, respectively: reduces or increases PLB phosphorylation, and markedly prolongs or reduces the LCR period; and markedly reduces or accelerates SAN spontaneous firing rate. Additionally, the increase in AP firing rate by PKA-dependent phosphorylation by β-adrenergic receptor (β-AR) stimulation requires normal intracellular Ca2+ cycling, because the β-AR chronotropic effect is markedly blunted when SR Ca2+ cycling is disrupted. Thus, AC-cAMP-PKA-Ca2+ signaling cascade is a major mechanism of normal automaticity in mouse SANC.
doi:10.1016/j.yjmcc.2011.07.028
PMCID: PMC3184386  PMID: 21840316
Sinoatrial node; Automaticity; Adenylyl cyclase-cyclic AMP-protein kinase A- Ca2+ signaling cascade; Ca2+ cycling proteins; Phospholamban phosphorylation; Local Ca2+ releases
15.  A Bioluminescence Method for Direct Measurement of Phosphodiesterase Activity1 
Analytical biochemistry  2011;417(1):36-40.
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.
doi:10.1016/j.ab.2011.05.036
PMCID: PMC3152944  PMID: 21693101
phosphodiesterase; assay; bioluminescence
16.  The N-Glycoform of sRAGE is the Key Determinant for Its Therapeutic Efficacy to Attenuate Injury-elicited Arterial Inflammation and Neointimal Growth 
Journal of molecular medicine (Berlin, Germany)  2013;91(12):10.1007/s00109-013-1091-4.
Signaling of the receptor for advanced glycation end products (RAGE) has been implicated in the development of injury-elicited vascular complications. Soluble RAGE (sRAGE) acts as a decoy of RAGE, and has been used to treat pathological vascular conditions in animal models. However, previous studies using sRAGE produced in insect Sf9 cells (sRAGESf9) used a high dose and multiple injections to achieve the therapeutic outcome. Here, we explore whether modulation of sRAGE N-glycoform impacts its bioactivity and augments its therapeutic efficacy. We first profiled carbohydrate components of sRAGECHO to show that a majority of its N-glycans belong to sialylated complex-types that are not shared by sRAGESf9. In cell-based NF-κB activation and vascular smooth muscle cell (VSMC) migration assays, sRAGECHO exhibited a significantly higher bioactivity relative to sRAGESf9 to inhibit RAGE alarmin ligand-induced NF-κB activation and VSMC migration. We next studied whether this N-glycoform-associated bioactivity of sRAGECHO is translated to higher in vivo therapeutic efficacy in a rat carotid artery balloon injury model. Consistent with the observed higher bioactivity in cell assays, sRAGECHO significantly reduced injury-induced neointimal growth and the expression of inflammatory markers in injured vasculature. Specifically, a single dose of 3 ng/g of sRAGECHO reduced neointimal hyperplasia by over 70%, whereas the same dose of sRAGESf9 showed no effect. The administered sRAGECHO is rapidly and specifically recruited to the injured arterial locus, suggesting that early intervention of arterial injury with sRAGECHO may offset an inflammatory circuit and reduce the ensuing tissue remodeling. Our findings showed that the N-glycoform of sRAGE is the key determinant underlying its bioactivity, and thus is an important glycobioengineering target to develop a highly potent therapeutic sRAGE for future clinical applications.
doi:10.1007/s00109-013-1091-4
PMCID: PMC3846495  PMID: 24132651
sRAGE; N-glycoform; arterial injury; arterial inflammation; neointimal hyperplasia; therapeutic window
18.  Relationship between Inter-arm Difference in Systolic Blood Pressure and Arterial Stiffness in Community-dwelling Older Adults 
A significant inter-arm difference in systolic blood pressure (IADSBP) has been recently associated with worse cardiovascular outcomes. We hypothesized that part of this association is mediated by arterial stiffness, and examined the relationship between significant IADSBP and carotid-femoral pulse wave velocity (CF-PWV) in a sample from the Baltimore Longitudinal Study of Aging. Of 1045 participants, 50 (4.8%) had an IADSBP≥10 mmHg at baseline, and 629 had completed data from two or more visits (for a total of 1704 visits across 8 years). CF-PWV was significantly higher in those with an IADSBP≥10 mmHg (7.3±1.9 vs. 8.2±2, p=0.002). Compared to others, those with IADSBP≥10 mmHg had also higher body mass index, waist circumference and triglycerides, higher prevalence of diabetes and lower HDL-cholesterol (p<.001 for all). A significant association with IADSBP≥10 mmHg was observed for CF-PWV in both cross-sectional (OR=1.19; 95%CI: 1.06–1.87; p=0.01) and longitudinal (OR=1.15; 95%CI: 1.03–1.29; p=0.01) multivariate analyses. Female gender, Caucasian race, high body mass index (plus diabetes and low HDL-cholesterol only cross-sectionally) were other independent correlates of IADSBP≥10 mmHg. In conclusion, significant IADSBP is associated with increased arterial stiffness in community-dwelling older adults.
doi:10.1111/jch.12178
PMCID: PMC3855278  PMID: 24299691
Blood pressure; inter-arm difference; arterial stiffness; pulse wave velocity; epidemiology
19.  Role of Inflammation in the Pathogenesis of Arterial Stiffness 
Yonsei Medical Journal  2012;53(2):258-261.
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.
doi:10.3349/ymj.2012.53.2.258
PMCID: PMC3282971  PMID: 22318811
Arterial stiffness; inflammation; angiotensin II
20.  Vessel Ultrasound Sonographic Assessment of Soluble Receptor for Advanced Glycation End Products Efficacy in a Rat Balloon Injury Model 
Objective
We aimed to assess the therapeutic efficacy of differentially modified soluble receptor for advanced glycation end products (sRAGE) in vivo using vessel ultrasound sonography and to compare the sonography data with those from postmortem histomorphologic analyses to have a practical reference for future clinical applications.
Methods
Vessel ultrasound sonography was performed in a sRAGE-treated rat carotid artery balloon injury model at different time points after the surgery, and therapeutic efficacy of different doses of sRAGE produced in Chinese hamster ovary cells and with different N-glycoform modifications were assessed.
Results
Vessel ultrasound sonography found that sRAGE produced in Chinese hamster ovary cells with complex N-glycoform modifications is highly effective, and is consistent with our recent findings in the same model assessed with histology. We also found that sonography is less sensitive than histology when a higher dose of sRAGE is administered.
Conclusions
Sonograph results are consistent with those obtained from histology; that is, sRAGE produced in Chinese hamster ovary cells has significantly higher efficacy than insect cell-originated sRAGE cells.
doi:10.1016/j.curtheres.2014.08.001
PMCID: PMC4229510  PMID: 25408789
neointima; N-glycosylationl; soluble receptor for advanced glycation end products; vascular injury; vessel sonography
21.  Derivation and Validation of Diagnostic Thresholds for Central Blood Pressure Measurements Based on Long-Term Cardiovascular Risks 
Journal of the American College of Cardiology  2013;62(19):10.1016/j.jacc.2013.06.029.
Objectives
This study sought to derive and validate outcome-driven thresholds of central blood pressure (CBP) for diagnosing hypertension.
Background
Current guidelines for managing patients with hypertension mainly rely on blood pressure (BP) measured at brachial arteries (cuff BP). However, BP measured at the central aorta (central BP [CBP]) may be a better prognostic factor for predicting future cardiovascular events than cuff BP.
Methods
In a derivation cohort (1,272 individuals and a median follow-up of 15 years), we determined diagnostic thresholds for CBP by using current guideline-endorsed cutoffs for cuff BP with a bootstrapping (resampling by drawing randomly with replacement) and an approximation method. To evaluate the discriminatory power in predicting cardiovascular outcomes, the derived thresholds were tested in a validation cohort (2,501 individuals with median follow-up of 10 years).
Results
The 2 analyses yielded similar diagnostic thresholds for CBP. After rounding, systolic/diastolic threshold was 110/80 mm Hg for optimal BP and 130/90 mm Hg for hypertension. Compared with optimal BP, the risk of cardiovascular mortality increased significantly in subjects with hypertension (hazard ratio: 3.08, 95% confidence interval: 1.05 to 9.05). Of the multivariate Cox proportional hazards model, incorporation of a dichotomous variable by defining hypertension as CBP ≥130/90 mm Hg was associated with the largest contribution to the predictive power.
Conclusions
CBP of 130/90 mm Hg was determined to be the cutoff limit for normality and was characterized by a greater discriminatory power for long-term events in our validation cohort. This report represents an important step toward the application of the CBP concept in clinical practice.
doi:10.1016/j.jacc.2013.06.029
PMCID: PMC3884552  PMID: 23850921
central blood pressure; diagnostic thresholds; high blood pressure; hypertension
22.  Hierarchical clustering of ryanodine receptors enables emergence of a calcium clock in sinoatrial node cells 
The Journal of General Physiology  2014;143(5):577-604.
Numerical modeling indicates that hierarchical clustering of ryanodine receptors in cells of the sinoatrial node is crucial to the calcium clock and thereby to regulation of heart rate.
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. The detailed mechanisms controlling the calcium clock are not understood. We constructed a computational model of SANCs, including three-dimensional diffusion and buffering of calcium in the cytosol and SR; explicit, stochastic gating of individual RyRs and L-type calcium channels; and a full complement of voltage- and calcium-dependent membrane currents. We did not include an anatomical submembrane space or inactivation of RyRs, the two heuristic components that have been used in prior models but are not observed experimentally. When RyRs were distributed in discrete clusters separated by >1 µm, only isolated sparks were produced in this model and LCR events did not form. However, immunofluorescent staining of SANCs for RyR revealed the presence of bridging RyR groups between large clusters, forming an irregular network. Incorporation of this architecture into the model led to the generation of propagating LCR events. Partial periodicity emerged from the interaction of LCR events, as observed experimentally. This calcium clock becomes entrained with membrane currents to accelerate the beating rate, which therefore was controlled by the activity of the SERCA pump, RyR sensitivity, and L-type current amplitude, all of which are targets of β-adrenergic–mediated phosphorylation. Unexpectedly, simulations revealed the existence of a pathological mode at high RyR sensitivity to calcium, in which the calcium clock loses synchronization with the membrane, resulting in a paradoxical decrease in beating rate in response to β-adrenergic stimulation. The model indicates that the hierarchical clustering of surface RyRs in SANCs may be a crucial adaptive mechanism. Pathological desynchronization of the clocks may explain sinus node dysfunction in heart failure and RyR mutations.
doi:10.1085/jgp.201311123
PMCID: PMC4003189  PMID: 24778430
23.  EPC mobilization after erythropoietin treatment in acute ST-elevation myocardial infarction: the REVEAL EPC substudy 
Erythropoietin (EPO) was hypothesized to mitigate reperfusion injury, in part via mobilization of endothelial progenitor cells (EPCs). The REVEAL trial found no reduction in infarct size with a single dose of EPO (60,000 U) in patients with ST-segment elevation myocardial infarction. In a substudy, we aimed to determine the feasibility of cryopreserving and centrally analyzing EPC levels to assess the relationship between EPC numbers, EPO administration, and infarct size. As a prespecified substudy, mononuclear cells were locally cryopreserved before as well as 24 and 48–72 h after primary percutaneous coronary intervention. EPC samples were collected in 163 of 222 enrolled patients. At least one sample was obtained from 125 patients, and all three time points were available in 83 patients. There were no significant differences in the absolute EPC numbers over time or between EPO- and placebo-treated patients; however, there was a trend toward a greater increase in EPC levels from 24 to 48–72 h postintervention in patients receiving ≥30,000 U of EPO (P = 0.099 for CD133+ cells, 0.049 for CD34+ cells, 0.099 for ALDHbr cells). EPC numbers at baseline were inversely related to infarct size (P = 0.03 for CD133+ cells, 0.006 for CD34+ cells). Local whole cell cryopreservation and central EPC analysis in the context of a multicenter randomized trial is feasible but challenging. High-dose (≥30,000 U) EPO may mobilize EPCs at 48–72 h, and baseline EPC levels may be inversely associated with infarct size.
doi:10.1007/s11239-013-0944-6
PMCID: PMC4008147  PMID: 23700090
Erythropoietin; Endothelial progenitor cells; Myocardial infarction; Cryopreservation
24.  LONGITUDINAL TRAJECTORIES OF ARTERIAL STIFFNESS AND THE ROLE OF BLOOD PRESSURE: THE BALTIMORE LONGITUDINAL STUDY OF AGING 
Hypertension  2013;62(5):10.1161/HYPERTENSIONAHA.113.01445.
Carotid-femoral pulse wave velocity (PWV), a marker of arterial stiffness, is an established independent cardiovascular (CV) risk factor. Little information is available on the pattern and determinants of the longitudinal change in PWV with aging. Such information is crucial to elucidating mechanisms underlying arterial stiffness and the design of interventions to retard it. Between 1988 and 2013, we collected 2 to 9 serial measures of PWV in 354 men and 423 women of the Baltimore Longitudinal Study of Aging, who were 21 to 94 years of age and free of clinically significant CV disease. Rates of PWV increase accelerated with advancing age in men more than women, leading to gender differences in PWV after the age of 50. In both sexes, not only systolic blood pressure (SBP) ≥140mmHg, but also SBP of 120–139mmHg was associated with steeper rates of PWV increase compared to SBP<120mmHg. Furthermore, there was a dose-dependent effect SBP in men with marked acceleration in PWV rate of increase with age at SBP ≥140mmHg compared to SBP of 120–139mmHg. Except for waist circumference in women, no other traditional CV risk factors predicted longitudinal PWV increase. In conclusion, the steeper longitudinal increase of PWV in men than women led to gender difference that expanded with advancing age. Age and systolic blood pressure are the main longitudinal determinants of pulse wave velocity and the effect of systolic blood pressure on PWV trajectories exists even in the pre-hypertensive range.
doi:10.1161/HYPERTENSIONAHA.113.01445
PMCID: PMC3880832  PMID: 24001897
Arterial stiffness; blood pressure; aging
25.  Sarcoplasmic reticulum Ca2+ pumping kinetics regulates timing of local Ca2+ releases and spontaneous beating rate of rabbit sinoatrial node pacemaker cells 
Circulation research  2010;107(6):767-775.
Rationale
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.
Objective
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).
Conclusions
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.
doi:10.1161/CIRCRESAHA.110.220517
PMCID: PMC2941556  PMID: 20651285
Sinoatrial nodal pacemaker cells; sarcoplasmic reticulum Ca2+ pumping; β-adrenergic receptor signaling

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