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1.  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
2.  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
3.  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
4.  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
5.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  Prevalence and prognostic significance of exercise-induced non-sustained ventricular tachycardia in asymptomatic volunteers: The Baltimore Longitudinal Study of Aging 
Objectives
To determine the clinical predictors and prognostic significance of exercise-induced non-sustained ventricular tachycardia (NSVT) in a large population of asymptomatic volunteers.
Background
Prior studies report variable risk associated with exercise-induced ventricular arrhythmia.
Methods
Subjects in the Baltimore Longitudinal Study of Aging free of known cardiovascular (CV) disease who completed at least one symptom-limited exercise treadmill test between 1977 and 2001 were included. NSVT episodes were characterized by QRS morphology, duration, and rate. Subjects underwent follow-up clinical evaluation every two years.
Results
The 2099 subjects (mean age 52.0 yrs, 52.2% male) underwent a mean of 2.7 exercise tests, on which 79 (3.7%) developed NSVT with exercise on at least 1 test. The NSVT had median duration of 3 beats (≤5 beats in 84%), and median rate of 175 bpm. Subjects with (vs. without) NSVT were older (67±12 yr vs. 51±17 yr, p < 0.0001), and more likely to be male (80% vs 51%, p< 0.0001), and to have baseline ECG abnormalities (50% vs. 17%, p<0.0001) or ischemic ST segment changes with exercise (20% vs 10%, p=0.004). Over a mean follow-up of 13.5 ± 7.7 yrs, 518 deaths (24.6%) occurred. After multivariable adjustment for age, sex, and coronary risk factors, exercise-induced NSVT was not significantly associated with total mortality (HR=1.30 [95% CI 0.89–1.90], p=0.17).
Conclusions
Exercise-induced NSVT occurred in nearly 4% of this asymptomatic adult cohort. This finding increased with age and was more common in men. After adjustment for clinical variables, exercise-induced NSVT did not independently increase risk of total mortality.
doi:10.1016/j.jacc.2013.05.026
PMCID: PMC3800197  PMID: 23747767
Ventricular tachycardia; Exercise testing; Aging
18.  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
19.  Early Contribution of Arterial Wave Reflection to Left Ventricular Relaxation Abnormalities in a Community-dwelling Population of Normotensive and Untreated Hypertensive Men and Women 
Journal of human hypertension  2013;28(2):85-91.
We examined the contribution of arterial wave reflection to early abnormalities in left ventricular relaxation, whether this association was modified by gender or hypertension, and the role of reflected wave timing and amplitude. We studied a cohort of normotensive and untreated essential hypertensive Taiwanese participants (675 men, 601 women, mean age 52 years). Doppler flow and applanation tonometry were performed to assess carotid-femoral pulse wave velocity (PWV) and augmentation index (AI). Diastolic parameters including transmitral E/A, E-deceleration time, and left atrial diameter were measured by echocardiography. In multivariate models predicting E/A, women were more likely to have lower E/A than men (β −0.08, p<.001). AI was significantly associated with lower E/A in both men (β −0.09, p=0.005) and women (β −0.12, p<.001) independent of PWV. Inclusion of AI in the overall model reduced the gender difference in E/A by 61% and rendered it non-significant. There was a significant interaction between AI and hypertension (p=0.02). The inverse association between AI and E/A was significant only in normotensive men and women, and only for the amplitude but not timing of the reflected wave. In conclusion, the contribution of wave reflection to left ventricular diastolic dysfunction was independent of arterial stiffness, more pronounced in normotensive individuals, and explained a significant portion of the gender difference in diastolic function.
doi:10.1038/jhh.2013.86
PMCID: PMC3871982  PMID: 24048294
wave reflection; arterial stiffness; augmentation index; pulse wave velocity; diastolic function; hypertension
20.  Modulation of sarcoplasmic reticulum Ca2+ cycling in systolic and diastolic heart failure associated with aging 
Heart failure reviews  2010;15(5):431-445.
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.
doi:10.1007/s10741-010-9167-5
PMCID: PMC2945822  PMID: 20419345
Heart; Aging; Heart failure; Cardiac myocyte; Sarcoplasmic reticulum; Calcium
21.  The Salted Artery and Angiotensin II Signaling: A Deadly Duo in Arterial Disease 
Journal of hypertension  2009;27(1):19-21.
doi:10.1097/HJH.0b013e32831d1fed
PMCID: PMC3091136  PMID: 19050444
22.  Funny current provides a relatively modest contribution to spontaneous beating rate regulation of human and rabbit sinoatrial node cells 
doi:10.1016/j.yjmcc.2009.12.009
PMCID: PMC2837765  PMID: 20036245
sinoatrial node; pacemaker activity; action potentials; ion channels HCN4 hyperpolarization-activated current; Na+/Ca2+ exchange current
23.  A Paradigm Shift for the Heart’s Pacemaker 
doi:10.1016/j.hrthm.2009.12.013
PMCID: PMC2847281  PMID: 20156611
Cardiac sinoatrial node; pacemaker cells; ion channels; local Calcium release; protein kinase A; CaMKII; sarcoplasmic reticulum; phospholamban; ryanodine receptors
24.  A coupled SYSTEM of intracellular Ca2+ clocks and surface membrane voltage clocks controls the timekeeping mechanism of the heart’s pacemaker 
Circulation research  2010;106(4):659-673.
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.
doi:10.1161/CIRCRESAHA.109.206078
PMCID: PMC2837285  PMID: 20203315
Sinoatrial node; cardiac pacemaker cells; ion channels; local Ca2+ releases; β-adrenergic stimulation; cholinergic stimulation; protein kinase A; CaMKII; sarcoplasmic reticulum; phospholamban; ryanodine receptors
25.  Arterial Aging: a Journey into Subclinical Arterial Disease 
Purpose
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.
Recent findings
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.
Summary
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.
doi:10.1097/MNH.0b013e3283361c0b
PMCID: PMC2943205  PMID: 20040868
angiotensin II; arterial aging; arterial disease

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