Long-term association of ultra-short heart rate variability with cardiovascular events.

Heart rate variability (HRV) is a cardiac autonomic marker with predictive value in cardiac patients. Ultra-short HRV (usHRV) can be measured at scale using standard and wearable ECGs, but its association with cardiovascular events in the general population is undetermined. We aimed to validate usHRV measured using ≤ 15-s ECGs (using RMSSD, SDSD and PHF indices) and investigate its association with atrial fibrillation, major adverse cardiac events, stroke and mortality in individuals without cardiovascular disease. In the National Survey for Health and Development (n = 1337 participants), agreement between 15-s and 6-min HRV, assessed with correlation analysis and Bland-Altman plots, was very good for RMSSD and SDSD and good for PHF. In the UK Biobank (n = 51,628 participants, 64% male, median age 58), after a median follow-up of 11.5 (11.4-11.7) years, incidence of outcomes ranged between 1.7% and 4.3%. Non-linear Cox regression analysis showed that reduced usHRV from 15-, 10- and 5-s ECGs was associated with all outcomes. Individuals with low usHRV (< 20th percentile) had hazard ratios for outcomes between 1.16 and 1.29, p < 0.05, with respect to the reference group. In conclusion, usHRV from ≤ 15-s ECGs correlates with standard short-term HRV and predicts increased risk of cardiovascular events in a large population-representative cohort.

Premature atrial and ventricular contractions detected on wearable-format electrocardiograms and prediction of cardiovascular events.

Aims: Wearable devices are transforming the electrocardiogram (ECG) into a ubiquitous medical test. This study assesses the association between premature ventricular and atrial contractions (PVCs and PACs) detected on wearable-format ECGs (15 s single lead) and cardiovascular outcomes in individuals without cardiovascular disease (CVD). Methods and results: Premature atrial contractions and PVCs were identified in 15 s single-lead ECGs from N = 54 016 UK Biobank participants (median age, interquartile range, age 58, 50-63 years, 54% female). Cox regression models adjusted for traditional risk factors were used to determine associations with atrial fibrillation (AF), heart failure (HF), myocardial infarction (MI), stroke, life-threatening ventricular arrhythmias (LTVAs), and mortality over a period of 11.5 (11.4-11.7) years. The strongest associations were found between PVCs (prevalence 2.2%) and HF (hazard ratio, HR, 95% confidence interval = 2.09, 1.58-2.78) and between PACs (prevalence 1.9%) and AF (HR = 2.52, 2.11-3.01), with shorter prematurity further increasing risk. Premature ventricular contractions and PACs were also associated with LTVA (P < 0.05). Associations with MI, stroke, and mortality were significant only in unadjusted models. In a separate UK Biobank sub-study sample [UKB-2, N = 29,324, age 64, 58-60 years, 54% female, follow-up 3.5 (2.6-4.8) years] used for independent validation, after adjusting for risk factors, PACs were associated with AF (HR = 1.80, 1.12-2.89) and PVCs with HF (HR = 2.32, 1.28-4.22). Conclusion: In middle-aged individuals without CVD, premature contractions identified in 15 s single-lead ECGs are strongly associated with an increased risk of AF and HF. These data warrant further investigation to assess the role of wearable ECGs for early cardiovascular risk stratification.

Analysing electrocardiographic traits and predicting cardiac risk in UK biobank.

The electrocardiogram (ECG) is a commonly used clinical tool that reflects cardiac excitability and disease. Many parameters are can be measured and with the improvement of methodology can now be quantified in an automated fashion, with accuracy and at scale. Furthermore, these measurements can be heritable and thus genome wide association studies inform the underpinning biological mechanisms. In this review we describe how we have used the resources in UK Biobank to undertake such work. In particular, we focus on a substudy uniquely describing the response to exercise performed at scale with accompanying genetic information.

A Systemized Approach to Investigate Ca(2+) Synchronization in Clusters of Human Induced Pluripotent Stem-Cell Derived Cardiomyocytes.

Induced pluripotent stem cell-derived cardiomyocytes (IPS-CM) are considered by many to be the cornerstone of future approaches to repair the diseased heart. However, current methods for producing IPS-CM typically yield highly variable populations with low batch-to-batch reproducibility. The underlying reasons for this are not fully understood. Here we report on a systematized approach to investigate the effect of maturation in embryoid bodies (EB) vs. "on plate" culture on spontaneous activity and regional Ca(2+) synchronization in IPS-CM clusters. A detailed analysis of the temporal and spatial organization of Ca(2+) spikes in IPS-CM clusters revealed that the disaggregation of EBs between 0.5 and 2 weeks produced IPS-CM characterized by spontaneous beating and high levels of regional Ca(2+) synchronization. These phenomena were typically absent in IPS-CM obtained from older EBs (>2 weeks). The maintenance of all spontaneously active IPS-CM clusters under "on plate" culture conditions promoted the progressive reduction in regional Ca(2+) synchronization and the loss of spontaneous Ca(2+) spiking. Raising the extracellular [Ca(2+)] surrounding these quiescent IPS-CM clusters from ~0.4 to 1.8 mM unmasked discrete behaviors typified by either (a) long-lasting Ca(2+) elevation that returned to baseline or (b) persistent, large-amplitude Ca(2+) oscillations around an increased cytoplasmic [Ca(2+)]. The different responses of IPS-CM to elevated extracellular [Ca(2+)] could be traced back to their routes of derivation. The data point to the possibility of predictably influencing IPS-CM phenotype and response to external activation via defined interventions at early stages in their maturation.

Non-invasive identification of stable rotors and focal sources for human atrial fibrillation: mechanistic classification of atrial fibrillation from the electrocardiogram.

AIMS: To develop electrocardiogram (ECG) tools to quantify the number of sources for atrial fibrillation (AF), i.e. spatially stable rotors and focal impulses, and whether they lie in right or left atrium. Intracardiac mapping has recently shown that paroxysmal and persistent AF is sustained by rotors or focal sources that are stable in location and thus targets for limited ablation [focal impulse and rotor modulation (FIRM)] to eliminate AF. Importantly, the numbers and locations of concurrent sources determine both the complexity of AF and the approach for ablation. METHODS AND RESULTS: In 36 AF patients (n = 29 persistent, 63 ± 9 years) in the CONventional ablation with or without Focal Impulse and Rotor Modulation (CONFIRM) trial, we developed phase lock (PL) to quantify spatial repeatability of ECG 'F-waves' between leads over time. Phase lock spectrally quantifies the angle θ between F-wave voltages in planes formed by ECG leads I, aVF, and V1 at successive points in time. We compared PL with ECG spectral dominant frequency (DF) and organizational index (OI) to characterize stable rotors and focal sources validated by intracardiac FIRM mapping. Focal impulse and rotor modulation ablation alone at ≤3 sources acutely terminated and rendered AF non-inducible or substantially slowed AF in 31 of 36 patients. Receiver operating characteristics of PL for this endpoint had area under the curve (AUC) = 0.72, and the optimum cut-point (PL = 0.09) had 74% sensitivity, 92% positive predictive value (PPV). Receiver operating characteristics areas for OI and DF were 0.50 and 0.58, respectively. Left (n = 28) or right (n = 3) atrial sources were localized by PL with AUC = 0.85, sensitivity 100%, PPV 30%, and negative predictive value 100%. Spectral DF provided AUC = 0.79. Notably, PL did not comigrate with diagnosis of paroxysmal or persistent AF (P = NS), unlike ECG DF. CONCLUSION: The novel metric of ECG PL identifies patients with fewer (≤3) or greater numbers of stable rotors/focal sources for AF, validated by intracardiac FIRM mapping, and localized them to right or left atria. These data open the possibility of using 12-lead ECG analyses to classify AF mechanistically and plan procedures for right- or left-sided FIRM ablation.

Identification of a novel HtrA1-susceptible cleavage site in human aggrecan: evidence for the involvement of HtrA1 in aggrecan proteolysis in vivo.

Mass spectrometry-based proteomic analyses performed on cartilage tissue extracts identified the serine protease HtrA1/PRSS11 as a major protein component of human articular cartilage, with elevated levels occurring in association with osteoarthritis. Overexpression of a catalytically active form of HtrA1, but not an active site mutant (S328A), caused a marked reduction in proteoglycan content in chondrocyte-seeded alginate cultures. Aggrecan degradation fragments were detected in conditioned media from the alginate cultures overexpressing active HtrA1. Incubation of native or recombinant aggrecan with wild type HtrA1 resulted in distinct cleavage of these substrates. Cleavage of aggrecan by HtrA1 was strongly enhanced by HtrA1 agonists such as CPII, a C-terminal hexapeptide derived from the C-propeptide of procollagen IIalpha1 (i.e. chondrocalcin). A novel HtrA1-susceptible cleavage site within the interglobular domain (IGD) of aggrecan was identified, and an antibody that specifically recognizes the neoepitope sequence (VQTV(356)) generated at the HtrA1 cleavage site was developed. Western blot analysis demonstrated that HtrA1-generated aggrecan fragments containing the VQTV(356) neoepitope were significantly more abundant in osteoarthritic cartilage compared with cartilage from healthy joints, implicating HtrA1 as a critical protease involved in proteoglycan turnover and cartilage degradation during degenerative joint disease.

Prevention of cartilage degeneration in a rat model of osteoarthritis by intraarticular treatment with recombinant lubricin.

OBJECTIVE: Lubricin, also referred to as superficial zone protein and PRG4, is a synovial glycoprotein that supplies a friction-resistant, antiadhesive coating to the surfaces of articular cartilage, thereby protecting against arthritis-associated tissue wear and degradation. This study was undertaken to generate and characterize a novel recombinant lubricin protein construct, LUB:1, and to evaluate its therapeutic efficacy following intraarticular delivery in a rat model of osteoarthritis (OA). METHODS: Binding and localization of LUB:1 to cartilage surfaces was assessed by immunohistochemistry. The cartilage-lubricating properties of LUB:1 were determined using a custom friction testing apparatus. A cell-binding assay was performed to quantify the ability of LUB:1 to prevent cell adhesion. Efficacy studies were conducted in a rat meniscal tear model of OA. One week after the surgical induction of OA, LUB:1 or phosphate buffered saline vehicle was administered by intraarticular injection for 4 weeks, with dosing intervals of either once per week or 3 times per week. OA pathology scores were determined by histologic analysis. RESULTS: LUB:1 was shown to bind effectively to cartilage surfaces, and facilitated both cartilage boundary lubrication and inhibition of synovial cell adhesion. Treatment of rat knee joints with LUB:1 resulted in significant disease-modifying, chondroprotective effects during the progression of OA, by markedly reducing cartilage degeneration and structural damage. CONCLUSION: Our findings demonstrate the potential use of recombinant lubricin molecules in novel biotherapeutic approaches to the treatment of OA and associated cartilage abnormalities.

Modulation of lubricin biosynthesis and tissue surface properties following cartilage mechanical injury.

OBJECTIVE: To evaluate the effects of injurious compression on the biosynthesis of lubricin at different depths within articular cartilage and to examine alterations in structure and function of the articular surface following mechanical injury. METHODS: Bovine cartilage explants were subdivided into level 1, with intact articular surface, and level 2, containing middle and deep zone cartilage. Following mechanical injury, lubricin messenger RNA (mRNA) levels were monitored by quantitative reverse transcriptase-polymerase chain reaction, and soluble or cartilage-associated lubricin protein was analyzed by Western blotting and immunohistochemistry. Cartilage morphology was assessed by histologic staining, and tissue functionality was assessed by friction testing. RESULTS: Two days after injury, lubricin mRNA expression was up-regulated approximately 3-fold for level 1 explants and was down-regulated for level 2 explants. Lubricin expression in level 1 cartilage returned to control levels after 6 days in culture. Similarly, lubricin protein synthesis and secretion increased in response to injury for level 1 explants and decreased for level 2 cartilage. Histologic staining revealed changes in the articular surface of level 1 explants following injury, with respect to glycosaminoglycan and collagen content. Injured level 1 explants displayed an increased coefficient of friction relative to controls. CONCLUSION: Our findings indicate that increased lubricin biosynthesis appears to be an early transient response of surface-layer cartilage to injurious compression. However, distinct morphologic changes occur with injury that appear to compromise the frictional properties of the tissue.

Alteration of articular cartilage frictional properties by transforming growth factor beta, interleukin-1beta, and oncostatin M.

OBJECTIVE: To evaluate the functional effects of transforming growth factor beta1 (TGFbeta1), interleukin-1beta (IL-1beta), and oncostatin M (OSM) on the frictional properties of articular cartilage and to determine the role of cytokine-mediated changes in cartilage frictional properties by extracting and redepositing lubricin on the surface of cartilage explants. METHODS: Neonatal bovine cartilage explants were cultured in the presence or absence of 10 ng/ml of TGFbeta1, IL-1beta, or OSM over 48 hours. Boundary lubrication tests were conducted to determine the effects of endogenously produced surface localized lubricin and of exogenous lubricin at the tissue surface and in the lubricant solution. The initial friction coefficient (micro(0)), equilibrium friction coefficient (micro(eq)), and Young's modulus (E(Y)) were determined from the temporal load data. RESULTS: IL-1beta and OSM decreased tissue glycosaminoglycan (GAG) content by approximately 20% over 48 hours and decreased E(Y) to a similar extent (11-17%), but TGFbeta did not alter GAG content or E(Y). Alterations in proteoglycan content corresponded to changes in micro(0), but endogenous lubricin decreased boundary mode micro(eq). The addition of exogenous lubricin, either localized at the tissue surface or in the lubricating solution, did not modulate micro(0), but it did lower micro(eq) in cytokine-treated cartilage. CONCLUSION: This study provides new insight into the functional consequences of cytokine-mediated changes in friction coefficient. In combination with established pathways of cytokine-mediated lubricin metabolism, these data provide evidence of distinct biochemical origins of boundary and biphasic pressure-mediated lubrication mechanisms in cartilage, with boundary lubrication regulated by surface accumulation of lubricants and biphasic lubrication controlled by factors such as GAG content that affect water movement through the tissue.

Boundary mode lubrication of articular cartilage by recombinant human lubricin.

Lubrication of cartilage involves a variety of physical and chemical factors, including lubricin, a synovial glycoprotein that has been shown to be a boundary lubricant. It is unclear how lubricin boundary lubricates a wide range of bearings from tissue to artificial surfaces, and if the mechanism is the same for both soluble and bound lubricin. In the current study, experiments were conducted to investigate the hypothesis that recombinant human lubricin (rh-lubricin) lubricates cartilage in a dose-dependent manner and that soluble and bound fractions of rh-lubricin both contribute to the lubrication process. An rh-lubricin dose response was observed with maximal lubrication achieved at concentrations of rh-lubricin greater than 50 microg/mL. A concentration-response variable-slope model was fit to the data, and indicated that rh-lubricin binding to cartilage was not first order. The pattern of decrease in equilibrium friction coefficient indicated that aggregation of rh-lubricin or steric arrangement may regulate boundary lubrication. rh-lubricin localized at the cartilage surface was found to lubricate a cartilage-glass interface in boundary mode, as did soluble rh-lubricin at high concentrations (150 microg/mL); however, the most effective lubrication occurred when both soluble and bound rh-lubricin were present at the interface. These findings point to two distinct mechanisms by which rh-lubricin lubricates, one mechanism involving lubricin bound to the tissue surface and the other involving lubricin in solution.

Binding and localization of recombinant lubricin to articular cartilage surfaces.

Lubricin is a secreted, cytoprotective glycoprotein that contributes to the essential boundary lubrication mechanisms necessary for maintaining low friction levels at articular cartilage surfaces. Diminishment of lubricin function is thereby implicated as an adverse contributing factor in degenerative joint diseases such as osteoarthritis. Lubricin occurs as a soluble component of synovial fluid, and is synthesized and localized in the superficial layer of articular cartilage (and thus has also been described as "superficial zone protein", or SZP); however, defined interactions responsible for lubricin retention at this site are not well characterized. In the current studies, we identified molecular determinants that enable lubricin to effectively bind to articular cartilage surfaces. Efficient and specific binding to the superficial zone was observed for synovial lubricin, as well as for recombinant full-length lubricin and a protein construct comprising the lubricin C-terminal (hemopexin-like) domain (LUB-C, encoded by exons 7-12). A construct representing the N-terminal region of lubricin (LUB-N, encoded by exons 2-5) exhibited no appreciable cartilage-binding ability, but displayed the capacity to dimerize, and thus potentially influence lubricin aggregation. Disulfide bond disruption significantly attenuated recombinant lubricin and LUB-C binding to cartilage surfaces, demonstrating a requirement for protein secondary structure in facilitating the appropriate localization of lubricin at relevant tissue interfaces. These findings help identify additional key attributes contributing to lubricin functionality, which would be expected to be instrumental in maintaining joint homeostasis.

23Na, 29Si, and 13C MAS NMR investigation of glass-forming reactions between Na2CO3 and SiO2.

The glass-forming reactions between sodium carbonate (Na2CO3) and silica (SiO2) have been investigated by 23Na, 29Si, and 13C magic-angle spinning (MAS) NMR spectroscopy. The multinuclear MAS NMR approach identifies and quantifies reaction products and intermediates, both glassy and crystalline. A series of powdered batches of initial composition Na2CO3.xSiO2 (x = 1, 2) corresponding to a sodium metasilicate (Na2SiO3) and sodium disilicate (Na2Si2O5) stoichiometry were investigated after periods of isothermal and nonisothermal heat treatments at different temperatures. Analysis of the 23Na quadrupolar coupling parameters has identified the early reaction product in all cases as crystalline Na2SiO3. In the nonisothermal experiment, this reaction is preceded by an early silica-rich melt phase formed around 850 degrees C. The early reactions are controlled by solid-state Na+ diffusion across the reaction zone in the grain interface layer. Crystalline Na2SiO3 precipitates in the interface layer, increasing its thickness between the Na2CO3 and the SiO2 grains and slowing down the rate of Na+ migration. This creates a secondary phase, which is temperature dependent. At low temperatures, where Na+ migration is impaired, the production of Na2SiO3 ceases and silica-richer phases are precipitated. In the case of the sodium disilicate batch, where excess SiO2 is present, a secondary reaction of Na2SiO3 with SiO2 forming a glassy phase is observed. A transient carbon-bearing phase has been identified by 13C NMR as a NaCO3- complex loosely bound to bridging oxygens in the silicate network at the SiO2 grain surface.

Tracing the reactive melting of glass-forming silicate batches by in situ 23Na NMR.

The kinetics of the reaction of batches of powdered quartz and sodium carbonate was studied by in situ (23)Na nuclear magnetic resonance (NMR) spectroscopy using a laser-heated probe. We show for the first time that the technique allows one to study solid-state reactions at high temperatures with good time resolution and without the risk of quenching artifacts. The reaction is controlled by solid-state Na(+) diffusion across the grain interface. Independent of the batch composition, the first reaction product is crystalline sodium metasilicate, Na(2)SiO(3), even if the temperature is high enough for much of the composition space between silica and metasilicate to be above the equilibrium liquidus. Fast Na(+) diffusion allows the reaction front to cross the grain interface and form the solid product before liquid intermediate equilibrium products can be formed. This purely solid-state reaction slows down as the thickness of the interface increases; the reaction is more deceleratory than published models suggest. If excess quartz is present, it reacts in a second step involving a liquid film wetting the excess grains. Once this reaction has started, it pulls the reaction into the thermodynamic regime, which leads to an increase even in the rate of the first step leading to intermediate solid metasilicate.