[The cause of anemia is underdiagnosed in primary care]. / Onderdiagnostiek naar anemie in de eerste lijn.

OBJECTIVE: To determine if general practitioners can diagnose the cause of anemia, based on the requested laboratory tests. DESIGN: Retrospective observational study. METHOD: The research population consisted of 20.004 adult patients with established anemia, who had blood samples analyzed by Atalmedial in 2019. The cause of anemia was found when criteria based on the NHG-standard were met. We considered the NHG-guideline to be followed when hemoglobin was requested in the first diagnostic request and when the correct combination of blood tests was requested in the second diagnostic request. Descriptive statistics and multilevel regression analysis were performed. RESULTS: A possible cause of anemia has been found in 38,7% of the patients within two diagnostic requests, regardless of the adherence to the NHG-guideline. The chance of finding a cause of anemia was smaller in men than women of the same age, whereas the chance was highest in women over the age of 80 and between 18 and 44. The NHG-guideline for anemia was followed in 11.794 (59%) of the patients in the first diagnostic request. 19,3% (11,4% of total) of these patients also had a second diagnostic request. In 10,4% (1,2% of total) of these patients, the NHG-guideline was adhered to in the second diagnostic request. CONCLUSION: A cause of anemia is, based on laboratory tests, often not diagnosed in daily practice in the primary care. The reason for this is insufficient laboratory follow-up after initial testing when no cause of anemia is found. The NHG-guideline for anemia is poorly adhered to.

The effectiveness of an automated algorithm as a tool for investigating the cause of anaemia in undiagnosed patients from general practitioners.

BACKGROUND: The Dutch guideline algorithm for the analysis of anaemia in patients of general practitioners (GPs) was programmed in a Clinical Decision Support system (CDS-anaemia) to support the process of diagnosing the cause of anaemia in the laboratory. This study investigates the diagnostic yield of the automated anaemia algorithm compared to that of the manual work up by the GP. METHODS: This retrospective population-based study consisted of 2697 people ≥18 years. Anaemia was defined according to the Dutch College of General Practitioners (DCGP) guideline. Causes of anaemia were based on the DCGP guidelines with the corresponding blood tests. The number of blood tests and causes of anaemia were measured in two separate periods in both the (CDS-anaemia) pilot group and a control group in which routine care was provided. RESULTS: Patients from GPs supported by CDS-anaemia had higher chances of having more anaemia-related blood tests being performed. This resulted in finding significantly more causes of anaemia in the pilot group compared to the control group with respect to iron deficiency (resp. 31.3% vs 14.5%), possible iron deficiency (resp. 11.4% vs 2.8%), iron deficiency in acute phase (2.6% vs 0.5%), chronic disease/infection/inflammation (23.5% vs 1.9%), vitamin B12 deficiency (4.5% vs 1.9%), possible vitamin B12 deficiency (16.8% vs 8.7%), folate deficiency (3.3% vs 0.9%) and possible bone marrow disorder (3.8% vs 0.0%); p < 0.05. CONCLUSIONS: This study suggests that an automated-algorithm support can effectively aid in the diagnostic work-up of anaemia in primary care to find more causes of anaemia.

Hypercoagulability causes atrial fibrosis and promotes atrial fibrillation.

AIMS: Atrial fibrillation (AF) produces a hypercoagulable state. Stimulation of protease-activated receptors by coagulation factors provokes pro-fibrotic, pro-hypertrophic, and pro-inflammatory responses in a variety of tissues. We studied the effects of thrombin on atrial fibroblasts and tested the hypothesis that hypercoagulability contributes to the development of a substrate for AF. METHODS AND RESULTS: In isolated rat atrial fibroblasts, thrombin enhanced the phosphorylation of the pro-fibrotic signalling molecules Akt and Erk and increased the expression of transforming growth factor ß1 (2.7-fold) and the pro-inflammatory factor monocyte chemoattractant protein-1 (6.1-fold). Thrombin also increased the incorporation of 3H-proline, suggesting enhanced collagen synthesis by fibroblasts (2.5-fold). All effects could be attenuated by the thrombin inhibitor dabigatran. In transgenic mice with a pro-coagulant phenotype (TMpro/pro), the inducibility of AF episodes lasting >1 s was higher (7 out of 12 vs. 1 out of 10 in wild type) and duration of AF episodes was longer compared with wild type mice (maximum episode duration 42.8 ± 68.4 vs. 0.23 ± 0.39 s). In six goats with persistent AF treated with nadroparin, targeting Factor Xa-mediated thrombin generation, the complexity of the AF substrate was less pronounced than in control animals (LA maximal activation time differences 23.3 ± 3.1 ms in control vs. 15.7 ± 2.1 ms in nadroparin, P < 0.05). In the treated animals, AF-induced α-smooth muscle actin expression was lower and endomysial fibrosis was less pronounced. CONCLUSION: The hypercoagulable state during AF causes pro-fibrotic and pro-inflammatory responses in adult atrial fibroblasts. Hypercoagulability promotes the development of a substrate for AF in transgenic mice and in goats with persistent AF. In AF goats, nadroparin attenuates atrial fibrosis and the complexity of the AF substrate. Inhibition of coagulation may not only prevent strokes but also inhibit the development of a substrate for AF.

In EXOG-depleted cardiomyocytes cell death is marked by a decreased mitochondrial reserve capacity of the electron transport chain.

Depletion of mitochondrial endo/exonuclease G-like (EXOG) in cultured neonatal cardiomyocytes stimulates mitochondrial oxygen consumption rate (OCR) and induces hypertrophy via reactive oxygen species (ROS). Here, we show that neurohormonal stress triggers cell death in endo/exonuclease G-like-depleted cells, and this is marked by a decrease in mitochondrial reserve capacity. Neurohormonal stimulation with phenylephrine (PE) did not have an additive effect on the hypertrophic response induced by endo/exonuclease G-like depletion. Interestingly, PE-induced atrial natriuretic peptide (ANP) gene expression was completely abolished in endo/exonuclease G-like-depleted cells, suggesting a reverse signaling function of endo/exonuclease G-like. Endo/exonuclease G-like depletion initially resulted in increased mitochondrial OCR, but this declined upon PE stimulation. In particular, the reserve capacity of the mitochondrial respiratory chain and maximal respiration were the first indicators of perturbations in mitochondrial respiration, and these marked the subsequent decline in mitochondrial function. Although pathological stimulation accelerated these processes, prolonged EXOG depletion also resulted in a decline in mitochondrial function. At early stages of endo/exonuclease G-like depletion, mitochondrial ROS production was increased, but this did not affect mitochondrial DNA (mtDNA) integrity. After prolonged depletion, ROS levels returned to control values, despite hyperpolarization of the mitochondrial membrane. The mitochondrial dysfunction finally resulted in cell death, which appears to be mainly a form of necrosis. In conclusion, endo/exonuclease G-like plays an essential role in cardiomyocyte physiology. Loss of endo/exonuclease G-like results in diminished adaptation to pathological stress. The decline in maximal respiration and reserve capacity is the first sign of mitochondrial dysfunction that determines subsequent cell death.

Hypertrophy induced KIF5B controls mitochondrial localization and function in neonatal rat cardiomyocytes.

Cardiac hypertrophy is associated with growth and functional changes of cardiomyocytes, including mitochondrial alterations, but the latter are still poorly understood. Here we investigated mitochondrial function and dynamic localization in neonatal rat ventricular cardiomyocytes (NRVCs) stimulated with insulin like growth factor 1 (IGF1) or phenylephrine (PE), mimicking physiological and pathological hypertrophic responses, respectively. A decreased activity of the mitochondrial electron transport chain (ETC) (state 3) was observed in permeabilized NRVCs stimulated with PE, whereas this was improved in IGF1 stimulated NRVCs. In contrast, in intact NRVCs, mitochondrial oxygen consumption rate (OCR) was increased in PE stimulated NRVCs, but remained constant in IGF1 stimulated NRVCs. After stimulation with PE, mitochondria were localized to the periphery of the cell. To study the differences in more detail, we performed gene array studies. IGF1 and PE stimulated NRVCs did not reveal major differences in gene expression of mitochondrial encoding proteins, but we identified a gene encoding a motor protein implicated in mitochondrial localization, kinesin family member 5b (Kif5b), which was clearly elevated in PE stimulated NRVCs but not in IGF1 stimulated NRVCs. We confirmed that Kif5b gene and protein expression were elevated in animal models with pathological cardiac hypertrophy. Silencing of Kif5b reverted the peripheral mitochondrial localization in PE stimulated NRVCs and diminished PE induced increases in mitochondrial OCR, indicating that KIF5B dependent localization affects cellular responses to PE stimulated NRVCs. These results indicate that KIF5B contributes to mitochondrial localization and function in cardiomyocytes and may play a role in pathological hypertrophic responses in vivo.

Loss of mitochondrial exo/endonuclease EXOG affects mitochondrial respiration and induces ROS-mediated cardiomyocyte hypertrophy.

Recently, a locus at the mitochondrial exo/endonuclease EXOG gene, which has been implicated in mitochondrial DNA repair, was associated with cardiac function. The function of EXOG in cardiomyocytes is still elusive. Here we investigated the role of EXOG in mitochondrial function and hypertrophy in cardiomyocytes. Depletion of EXOG in primary neonatal rat ventricular cardiomyocytes (NRVCs) induced a marked increase in cardiomyocyte hypertrophy. Depletion of EXOG, however, did not result in loss of mitochondrial DNA integrity. Although EXOG depletion did not induce fetal gene expression and common hypertrophy pathways were not activated, a clear increase in ribosomal S6 phosphorylation was observed, which readily explains increased protein synthesis. With the use of a Seahorse flux analyzer, it was shown that the mitochondrial oxidative consumption rate (OCR) was increased 2.4-fold in EXOG-depleted NRVCs. Moreover, ATP-linked OCR was 5.2-fold higher. This increase was not explained by mitochondrial biogenesis or alterations in mitochondrial membrane potential. Western blotting confirmed normal levels of the oxidative phosphorylation (OXPHOS) complexes. The increased OCR was accompanied by a 5.4-fold increase in mitochondrial ROS levels. These increased ROS levels could be normalized with specific mitochondrial ROS scavengers (MitoTEMPO, mnSOD). Remarkably, scavenging of excess ROS strongly attenuated the hypertrophic response. In conclusion, loss of EXOG affects normal mitochondrial function resulting in increased mitochondrial respiration, excess ROS production, and cardiomyocyte hypertrophy.

Pleiotropic effects of factor Xa and thrombin: what to expect from novel anticoagulants.

Factor Xa and thrombin are well-known components of the coagulation cascade and have been proven to be viable targets for effective anticoagulation treatment. However, accumulating evidence suggests that these serine proteases are also crucial modulators of other cellular mechanisms through the activation of protease-activated receptor (PAR)-mediated signalling. The involvement of factor Xa, thrombin, and PARs in normal biological and pathophysiological processes has been recognized, and their potential implications have been explored in recent years. Both factor Xa and thrombin play significant roles in mediating cellular signalling effects associated with the initial development of atherosclerosis: a chronic inflammatory vascular disease. In addition, increased expression and activation of PARs may be associated with atrial fibrillation (AF) and AF-associated thromboembolism hypercoagulability. Both pathologies are associated with hypercoagulability, suggesting that the role of cellular effects of factor Xa and thrombin and of their specific inhibitors should be studied in relation to the prevention of thrombotic and pro-arrhythmic changes. This review examines the role of factor Xa-mediated and thrombin-mediated PAR activation in modulating cellular processes involved in atherosclerosis and AF and discusses the potential implication of direct factor Xa and thrombin inhibition on effects outside coagulation.

Atrial remodeling is directly related to end-diastolic left ventricular pressure in a mouse model of ventricular pressure overload.

BACKGROUND: Atrial fibrillation (AF) is often preceded by underlying cardiac diseases causing ventricular pressure overload. OBJECTIVE: It was our aim to investigate the progression of atrial remodeling in a small animal model of ventricular pressure overload and its association with induction of AF. METHODS: Male mice were subjected to transverse aortic constriction (TAC) or sham operation. After four or eight weeks, echocardiographic measurements and hemodynamic measurements were made and AF induction was tested. The hearts were either fixed in formalin or ventricles and atria were separated, weighed and snap-frozen for RNA analysis. RESULTS: Four weeks of pressure overload induced ventricular hypertrophy and minor changes in the atria. After eight weeks a significant reduction in left ventricular function occurred, associated with significant atrial remodeling including increased atrial weight, a trend towards an increased left atrial cell diameter, atrial dilatation and increased expression of markers of hypertrophy and inflammation. Histologically, no fibrosis was found in the left atrium. But atrial gene expression related to fibrosis was increased. Minor changes related to electrical remodeling were observed. AF inducibility was not different between the groups. Left ventricular end diastolic pressures were increased and correlated with the severity of atrial remodeling but not with AF induction. CONCLUSION: Permanent ventricular pressure overload by TAC induced atrial remodeling, including hypertrophy, dilatation and inflammation. The extent of atrial remodeling was directly related to LVEDP and not duration of TAC per se.

Cyclical stretch induces structural changes in atrial myocytes.

Atrial fibrillation (AF) often occurs in the presence of an underlying disease. These underlying diseases cause atrial remodelling, which make the atria more susceptible to AF. Stretch is an important mediator in the remodelling process. The aim of this study was to develop an atrial cell culture model mimicking remodelling due to atrial pressure overload. Neonatal rat atrial cardiomyocytes (NRAM) were cultured and subjected to cyclical stretch on elastic membranes. Stretching with 1 Hz and 15% elongation for 30 min. resulted in increased expression of immediate early genes and phosphorylation of Erk and p38. A 24-hr stretch period resulted in hypertrophy-related changes including increased cell diameter, reinduction of the foetal gene program and cell death. No evidence of apoptosis was observed. Expression of atrial natriuretic peptide, brain natriuretic peptide and growth differentiation factor-15 was increased, and calcineurin signalling was activated. Expression of several potassium channels was decreased, suggesting electrical remodelling. Atrial stretch-induced change in skeletal α-actin expression was inhibited by pravastatin, but not by eplerenone or losartan. Stretch of NRAM results in elevation of stress markers, changes related to hypertrophy and dedifferentiation, electrical remodelling and cell death. This model can contribute to investigating the mechanisms involved in the remodelling process caused by stretch and to the testing of pharmaceutical agents.

Role of inflammation in early atrial fibrillation recurrence.

AIMS: Outcome of rhythm control in atrial fibrillation (AF) is still poor due to various mechanisms involved in the initiation and perpetuation of AF. Differences in timing of AF recurrence may depend on different types of mechanisms. The aim of this study was to assess the mechanisms involved in early AF recurrence in patients with short-lasting AF. METHODS AND RESULTS: Patients with short-lasting persistent AF undergoing rhythm control (n= 100) were included. Markers of mechanisms involved in the initiation and perpetuation of AF were assessed, including clinical factors, echocardiographic parameters, and biomarkers. Primary endpoint was early AF recurrence (recurrence <1 month). Secondary endpoint was progression to permanent AF. Median total AF history was short: 4.2 months. Early AF recurrences occurred in 30 patients (30%) after a median of 6 (inter-quartile range 2-14) days. Baseline log(2) interleukin (IL)-6 [adjusted hazard ratio (HR) 1.3, 95% confidence interval (CI) 1.0-1.7, P= 0.02] and present or previous smoking (adjusted HR 3.6, 95% CI 1.2-10.9, P= 0.03) were independently associated with early AF recurrence, suggesting that inflammation played an important role in early recurrences. Atrial fibrillation became permanent in 29 patients (29%). Baseline transforming growth factor-ß1, left ventricular ejection fraction, and early AF recurrence were independently associated with progression to permanent AF. CONCLUSION: In patients with short-lasting AF, early AF recurrence seemed to be associated with inflammation as represented by IL-6. Treatment aimed against inflammation may therefore prevent early AF recurrences, which can improve rhythm control outcome.

Mechanisms of atrial structural changes caused by stretch occurring before and during early atrial fibrillation.

Structural remodelling occurring before, due to the underlying heart disease, and during atrial fibrillation (AF) sets the stage for permanent AF. Current therapy in AF aims to maintain sinus rhythm in symptomatic patients, but outcome is unfortunately poor. Stretch of the atria is a main contributor to atrial remodelling. In this review, we describe different aspects of structural remodelling as seen in animal models and in patients with AF, including atrial enlargement, cellular hypertrophy, dedifferentiation, fibrosis, apoptosis, and loss of contractile elements. In the second part, we describe downstream signals of mechanical stretch and their contribution to AF and structural remodelling. Ultimately, knowledge of mechanisms underlying structural remodelling may help to identify new pharmacological targets for AF prevention.

Differential Rho-kinase dependency of full and partial muscarinic receptor agonists in airway smooth muscle contraction.

In airway smooth muscle (ASM), full and partial muscarinic receptor agonists have been described to have large differences in their ability to induce signal transduction, including Ca2+-mobilization. Despite these differences, partial agonists are capable of inducing a submaximal to maximal ASM contraction. To further elucidate transductional differences between full and partial muscarinic receptor agonists, we investigated the contribution of Rho-kinase (an important regulator of Ca2+-sensitization) to methacholine-, pilocarpine- and McN-A-343-induced bovine tracheal smooth muscle (BTSM) contraction, using the selective Rho-kinase inhibitor Y-27632. In addition, we measured Ca2+-mobilization and -influx in BTSM cells in response to these agonists in the absence and presence of Y-27632. Whereas treatment with Y-27632 (1 microM) significantly decreased potency (pEC50) for all agonists, maximal contraction (Emax) was reduced by 23.4+/-2.8 and 50.4+/-7.9% for the partial agonists pilocarpine and McN-A-343, respectively, but was unaffected for the full agonist methacholine. However, Emax of methacholine became Rho-kinase dependent after taking away its receptor reserve using the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard. Pilocarpine and McN-A-343 induced a very small Ca2+-mobilization and -influx as compared to methacholine. In addition, an inverse relationship of these two parameters with the Rho-kinase dependency was observed. Interestingly, no inhibitory effects of Y-27632 were observed on Ca2+-mobilization and-influx for all three agonists, indicating that the effects of Y-27632 on contraction are most likely on the level of Ca2+-sensitization. In conclusion, in contrast to the full agonist methacholine, the partial muscarinic receptor agonists pilocarpine and McN-A-343 are dependent on Rho-kinase for their maximal contractile effects, presumably as a consequence of differences in transductional reserve, indicating an agonist-dependent role for Rho-kinase in ASM contraction. Moreover, an inverse relationship exists between Rho-kinase dependency and both Ca2+-mobilization and Ca2+-influx for these agonists.