Altered ventricular activation (AVA) causes intraventricular mechanical dyssynchrony (MD) and impedes contraction, promoting pro-arrhythmic electrical remodelling in the chronic atrioventricular block (CAVB) dog. We aimed to study arrhythmogenic and electromechanical outcomes of different degrees of AVA.n = 29), right ventricular apex  pacing or biventricular pacing . After ≥3 weeks of bradycardic remodelling, Torsade de Pointes arrhythmia (TdP) inducibility, defined as ≥3 TdP/10 min, was tested with specific KrI-blocker dofetilide (25 μg/kg/5 min). Mechanical dyssynchrony was assessed by echocardiography as time-to-peak (TTP) of left ventricular (LV) free-wall minus septum (ΔTTP). Electrical intraventricular dyssynchrony was assessed as slope of regression line correlating intraventricular LV activation time (AT) and activation recovery interval (ARI). Under sinus rhythm, contraction occurred synchronous (ΔTTP: −8.6 ± 28.9 ms), and latest activated regions seemingly had slightly longer repolarization (AT–ARI slope: −0.4). Acute AV block increased MD in all groups, but following ≥3 weeks of remodelling IVR animals became significantly more TdP inducible . After chronic AVA, intraventricular MD was lowest in CRT animals . Although dofetilide steepened negative AT–ARI slope in all groups, this heterogeneity in dofetilide-induced ARI prolongation seemed least pronounced in CRT animals .Following atrioventricular block, AVA was established through idioventricular rhythm (IVR; Severity of intraventricular MD affects the extent of electrical remodelling and pro-arrhythmic outcome in the CAVB dog model.  Altered ventricular activation (AVA) causes electromechanical dyssynchrony, which impedes contraction and thereby induces ventricular electrical remodelling. Even though these remodelling processes improve cardiac output, they also cause an increased susceptibility for ventricular arrhythmias.In the chronic atrioventricular block (CAVB) dog model, increased mechanical dyssynchrony correlates with increased left ventricular electrical dyssynchrony (reflected by intraventricular dispersion of repolarization).Increased intraventricular dispersion of repolarization was associated with higher inducibility rates for Torsade de Pointes arrhythmias.Therefore, AVA contributes to pro-arrhythmic remodelling in the CAVB dog model, as the degree of electromechanical dyssynchrony correlates with the arrhythmogenic outcome.1 Therefore, an increasing number of HF patients are receiving cardiac resynchronization therapy (CRT), also known as biventricular pacing. Cardiac resynchronization therapy aims to restore ventricular electromechanical synchrony and can reduce morbidity and mortality in HF patients.2 Moreover, the BLOCK-HF study showed that CRT resulted in fewer deaths and less progression of HF than in right ventricular apex (RVA) paced patients,3 suggesting the superiority of CRT over more conventional chronic RVA pacing.A prolonged QRS complex is associated with higher all-cause mortality and possibly fatal, ventricular arrhythmias in heart failure (HF) patients.predicted and prevented remains poorly understood. Moreover, a meta-analysis by Deif et al.4 reported higher incidence of ventricular arrhythmias in non-responders compared with responders. Furthermore, Haugaa et al.5 demonstrated significant correlation between incidence of ventricular arrhythmias and CRT-associated mechanical dyssynchrony (MD). Collectively, these results suggest that pacing-induced altered ventricular activation (AVA), and consequential MD, might initiate pro-arrhythmic cardiac remodelling. Simultaneously, this would also explain the superiority of CRT over RVA,3 as CRT should cause less dyssynchrony than RVA and therefore induces less pro-arrhythmic remodelling. However, more research is needed into the electrical consequences of acute and chronic pacing, and the role of mechanical (dys)synchrony on promoting development of pro-arrhythmic conditions.However, although almost one-third of CRT recipients are refractory to this therapy, the pathophysiology underlying CRT ‘non-responders’ and how this outcome can be 7 In this model, ablation of the AV-node and subsequent drop in cardiac output initiate several remodelling processes that cumulatively restore cardiac output.8 Adversely, electrical remodelling, which includes downregulation of potassium channels, results in susceptibility for Torsade de Pointes arrhythmia (TdP). Especially in combination with additional (acute) impediments of cardiac repolarization such as anaesthetics,9 bradycardia10 and/or administration of specific KrI-blocker dofetilide,11 TdP inducibility is reached in ± 75% of animals.8Studies on pacing-induced electrical remodelling can be performed in the chronic atrioventricular block (CAVB) dog, which has proven to be an outstanding paradigm to study (i) aetiological factors that predisposition to ventricular arrhythmias and sudden cardiac deaths and (ii) the electromechanical consequences of different pacing strategies.In this study, we compared the electromechanical and arrhythmogenic consequences of different extents of AVA, achieved through chronic exposure to either idioventricular rhythm (IVR), RVA or CRT conditions, in the CAVB dog model. We hypothesized that more electromechanical dyssynchrony would prompt more extensive remodelling and processes and thereby result in increased TdP susceptibility.All experiments were approved by ‘the Committee for Experiments on Animals’ of Utrecht University, the Netherlands. Animal handling and care was in accordance with the ‘European Directive for the Protection of Vertebrate Animals used for Experimental and Scientific Purpose European Community Directive 86/609/CEE’. In total, 51 adult purpose-bred mongrel dogs  were included.i.m.), 0.5 mg/kg acepromazine (i.m.), 0.02 mg/kg atropine (i.m.), and 0.1 mg/kg metacam (s.c.). Before the experiment, prophylactic antibiotic ampicillin (1000 mg) was injected (i.v.) and buprenorphine 0.3 mg (i.m) was administered after the experiments. Following the first experiment, temperature was monitored daily and amoxicillin-clavulanic acid  and metacam  were administered for five consecutive days. General anaesthesia was induced using pentobarbital  and maintained with mechanical ventilation of isoflurane (1.5%) in a mixture of O2 and N2O (1:2). In CRT-paced animals (n = 10), left ventricular (LV) leads of the CRT pacemaker were implanted through a right-sided thoracotomy . All animals were implanted with leads and generators from Medtronic . In CRT-paced animals, the epicardial, LV unipolar pacing lead  was screwed on the basal, anterolateral wall of the LV. Correct pacing and absence of adverse phrenic nerve stimulation were confirmed.Premedication was given ± 30 min before induction of general anaesthesia and consisted of 0.5 mg/kg methadone (n = 12) paced animals via the jugular vein and, under fluoroscopic guidance, screwed in the RVA. The RVA lead was tunnelled to the thoracic device pocket and all leads were connected to the pacemaker . Proper functioning was tested, impedance and pacing threshold determined, and the thorax closed. Next, radiofrequency ablation of the proximal His-bundle induced complete atrioventricular (AV) block in all animals.Hereafter, the right ventricular (RV) bipolar pacing lead  was introduced in CRT and RVA (n = 29) animals, whereas RVA and CRT animals were paced at the lowest captured rate .Following the acute experiment, rate was uncontrolled in IVR .12 Mechanical recordings were made in four IVR, six RVA, and all CRT animals. Under experimental conditions, RVA and CRT animals were continuously paced at 60 bpm. The cIVR, cRVA, and cCRT experiments were conducted at 3.6 ± 1.0, 3.1 ± 0.4 and 2.9 ± 0.2 weeks after creation of AV block and included additional assessment of TdP inducibility in all animals. Torsade de Pointes susceptibility was determined through administration of specific KrI-blocker dofetilide . Dofetilide infusion was discontinued if TdP occurred within 5 min after start of administration and persistent arrhythmias (>10 s) were manually terminated by electrical cardioversion. Electrophysiological recordings consisted of six-leads surface ECG, an endocardial RV monophasic action potential (MAP) catheter  and endocardial duo-decapolar catheter , recording unipolar electrograms (EGM) from 10 distinct LV regions . In IVR experiments, the use of an LV EGM was limited to four animals. Catheters were introduced through the femoral artery or vein and positioned under fluoroscopic guidance. Electrophysiological recordings included a 10 min baseline period and 20 min following start of dofetilide infusion.Anaesthetized experiments including electrical and mechanical recordings were performed under sinus rhythm (SR), following acute IVR, RVA, or CRT pacing  and after remodelling under IVR, RVA, or CRT conditions had completed  and conducted under anaesthesia using a Philips iE33 ultrasound machine . Animals were positioned in the right-sided supine position, except for aCRT conditions. Left ventricular parasternal short-axis views were made at papillary level and appropriate beats were selected based on PQ-interval duration. Radial myocardial peak strain (PS), onset of radial strain (onset), and time-to-peak (TTP) of LV septal- and free-wall were analyzed with TomTec-Arena, 2D-Strain . Left ventricular septal- and free-wall regions were determined after division of the LV into six segments; regions were averages of the two adjoining segments best corresponding to the septal- and free-wall. The remaining segments, located between the septal- and free-wall sections, were discarded. The ΔPS, ΔOnset and ΔTTP were calculated by subtracting the value of the septal-wall from the free-wall .Echocardiography was performed in a subset of the animals  and LV activation recovery intervals (ARIs) were determined by a custom-made Matlab application . Signals derived from the most distal and proximal electrode  of the duo-decapolar catheter were excluded from analysis because of P-wave interference. Short-term variability of repolarization (STV) was determined from 30 consecutive beats using the formula: ∑|nD + 1 – nD|/(30 × √2); D being RV MAPD or LV ARI.14 Interventricular dispersion of repolarization (ΔMAPD) was calculated as LV ARI—RV MAPD; the average ARI of all LV EGM was used for calculations. Activation times (ATs) of the LV EGM electrodes were manually determined as time difference between pacing spike and minimum dV/dt of the EGM. Left ventricular activation time was calculated as average AT of all LV EGM. Right ventricular activation time was measured as time difference between pacing spike and steepest upstroke of the MAP. Interventricular delay in activation (ΔAT) was calculated as LVAT minus RVAT. Left ventricular intraventricular heterogeneity in activation and repolarization were assessed by comparing ARI and AT of the duo-decapolar catheter. Activation time and ARI of EGM electrodes were used to establish AT–ARI slopes. All electrophysiological measurements were made under baseline conditions and either before the first ectopic beat or 5 min following start of dofetilide infusion.PP, RR, QRS, QT, and T-wave peak-to-end (Tp-e) intervals were obtained from the surface ECG and analyzed offline using EPTracer . Duration of all intervals were manually measured and averaged over five consecutive beats. QT-interval was corrected for heart rate (QTc) using the Van der Water formula.n + 1 rule (n = number of ectopic beats in an arrhythmic event). Defibrillations were awarded 50, 75, or 100 points when single, double, or ≥ triple defibrillations were required, respectively. The score of the three most severe episodes during the 10 min following start of dofetilide were averaged to obtain a final AS.Torsade de Pointes were identified as polymorphic ventricular tachycardias of ≥5 beats twisting around the isoelectric line. Torsade de Pointes-inducibility was defined as ≥3 TdP within 10 min following start of dofetilide. Severity of arrhythmic activity was quantified as arrhythmia score (AS): all events were scored according to the post hoc Bonferroni correction was used to determine the statistical significances of the electrophysiological and echocardiographic data. Inducibility and AS were analyzed by McNemar’s and Wilcoxon signed rank tests, respectively. Results were considered statistically significant if P < 0.05.All results are displayed as mean ± standard deviation (SD). Repeated-measures ANOVA with Table 1). Moreover, intraventricular repolarization duration appeared homogenous and showed a slightly negative AT–ARI relationship , suggestive of slightly earlier repolarization in the latest activated regions. Correspondingly, both ventricles contracted simultaneously and synchronously .Average sinus rate of animals was 102 ± 8 bpm and ventricular activation occurred rapidly  and synchronously  and induced interventricular dyssynchrony in activation . Moreover, the AT–ARI slope became slightly positive, suggesting that repolarization became longest in the latest activated regions . These electrical changes were mirrored by changes in timing of ventricular contraction, ΔOnset increasing from 2.4 ± 8.5 ms in SR to 76.8 ± 75.9, 32.6 ± 61.5 and −36.3 ± 81.1 ms in IVR, RVA, and CRT animals, respectively . Moreover, although synchrony of contraction also seemed acutely affected by AVA, this effect appeared less in CRT animals .Creation of AV block acutely decreased heart rate, slowed conduction . Correspondingly, dofetilide caused less increase in AS in cCRT and cRVA groups  than in cIVR animals .Chronically AVA induced different extents of pro-arrhythmic remodelling in the three groups. With just 20%, TdP inducibility was lowest in the cCRT group . In cRVA animals, inducibility was more than twice as likely as cCRT animals, as 42% became TdP inducible . However, cIVR caused most TdP inducibility as 66% of animals were inducible for TdP . Most notably, this interventricular dyssynchrony in timing and force of contraction was lowest in cCRT animals . Moreover, although contraction was delayed in cCRT animals, the synchrony and force of contraction appeared very similar to SR conditions .Mechanically, each group exhibited different extents of MD (P < 0.05). Moreover, all groups showed a negative AT-RT slope (as in SR), which became increasingly steeper from cCRT to cRVA to cIVR animals . Dofetilide further unmasked this intraventricular heterogeneity in repolarization reserve, as regardless of rate strategy, repolarization prolonged most in the earliest activated regions. This steepening of the slope was least pronounced in the least-inducible group of cCRT animals (slope: −0.7) in comparison to cRVA and cIVR animals . Additionally, administration of dofetilide prolonged PP-intervals in all animals, but the extent of this prolongation also differed between each group; the prolongation becoming increasingly more pronounced going from cIVR to cRVA to cCRT animals .Electrically, ventricular activation was significantly more synchronous in cCRT than in cRVA animals  less mechanical ventricular dyssynchrony and (ii) less LV intraventricular dispersion of repolarization.15 Correspondingly, biventricular pacing, which enhances synchrony of ventricular activation, is superior to RVA pacing in preserving LV function.7 As electrical remodelling is initiated to improve contractile function, the extent wherein this occurs seems to correlate with the severity of MD. Electrical remodelling under chronic RVA conditions is therefore more extensive than under chronic CRT conditions, resulting in more dispersion of repolarization and increased arrhythmic susceptibility. Also in CAVB dogs, 3 weeks of remodelling partially reverses RVA pacing-induced MD, although TdP susceptibility increases.7 Hence, MD appears to induce ventricular  remodelling, which improves synchrony of contraction, but adversely increases TdP susceptibility. Similarly, suboptimal CRT implantation could initiate MD and thereby induce pro-arrhythmic remodelling in CRT non-responders.5Multiple studies have suggested that chronic RVA pacing progresses LV dysfunction as a result of electrical, and consequentially, MD.16 Although activation is controlled in RVA and CRT animals, this unnatural activation pattern naturally results in MD, whereas its inconsistency is difficult to adapt to. Following chronically altered activation, several electrical parameters, including LV STV, were significantly different between the groups. However, these differences, reflective of different degrees of remodelling, became even clearer upon administration of dofetilide, and made most evident by TdP susceptibility of each group. Although TdP susceptibility was highest in IVR, CRT animals were significantly less vulnerable to TdP. Cardiac resynchronization therapy animals also displayed the most synchronous LV contraction on echocardiography, closely resembling SR conditions. Hence, although all groups were exposed to AVA, the extent of continuous and chronic MD that persists may determine the extent of ventricular pro-arrhythmic electromechanical remodelling. However, whether the electrical or mechanical changes are the primary drive of remodelling remains unclear.In this study, we exposed CAVB dogs to chronically unpaced (cIVR), cRVA or cCRT conditions, to compare arrhythmic outcomes of remodelling under different conditions of AVA and thus extents of MD. Notably, in IVR animals, focus of activation is uncontrolled, unstable and originates from three predilected sites.2+-handling and downregulation of potassium channels.8 Consequently, administration of KrI-blocker dofetilide further impedes cardiac electrical stability by reducing repolarization reserve, which is quantified by STV.14 This study showed that ventricular remodelling, initiated by chronically altered activation and bradycardia, reduces repolarization reserve and thereby increases TdP susceptibility (Table 3).In the CAVB dog model, electrical remodelling induces changes in Caspatial dimension, using the AT–ARI slope.7 Similar to previous studies, dofetilide steepened the negative AT–ARI slope, suggesting that repolarization reserve reduces most in early activated regions as they show relatively more dofetilide-induced ARI prolongation.7 This steeper slope reflects more intraventricular dispersion of repolarization, which is also inverted compared with acute AV block, and has been implicated in both the development and perpetuation of longer-lasting TdP.18 Additionally, this study showed that regardless of ventricular activation pattern, dofetilide causes the largest repolarization prolongation in early activated regions, indicating that electrical remodelling is driven by differences in AT and/or the consequential MD.This study also assessed electrical remodelling in the 19 In this study, dofetilide-induced increases in PP-interval were significantly less in IVR animals compared with RVA and CRT animals. This suggests that AV block induced neural responses could be proportional to the degree of MD; a stronger neural response , resulting in more sympathetic activation and therefore more pro-arrhythmic conditions. However, this correlation remains hypothetical and the relationship between mechanical dysfunction and neural remodelling warrants further investigation.Previous studies have demonstrated that sympathetic modulation promotes arrhythmogenesis in the CAVB dog model.20 reported a tendency towards increased mortality in HF patients without a prolonged QRS (≤130 ms) who underwent CRT therapy. Potentially, CRT induced electromechanical dyssynchrony, which resulted in pro-arrhythmic cardiac remodelling.20 Hence, though CRT reduces morbidity and mortality in carefully selected HF patients,2 inappropriate CRT implantation can be detrimental. Importantly, these findings are also relevant in the setting of left bundle-branch block pacing and His-bundle pacing.This study showed that extent of electromechanical dyssynchrony relates to the development of arrhythmogenic vulnerability. It therefore highlights the importance of avoiding dyssynchrony in structurally remodelled hearts and potentially explains results of the Echocardiography Guided Cardiac Resynchronization Therapy (EchoCRT) study. This study, which was prematurely terminated due to futility,Due to arrhythmic activity, electrophysiological data following infusion of dofetilide in cIVR, cRVA and cCRT experiments are taken at different timepoints. As less dofetilide was administered at earlier timepoints, this could have caused underestimation of the changes herein.10 and might have contributed to the increased TdP susceptibility.In IVR animals, dofetilide was administered under more bradycardic conditions. Bradycardia promotes arrhythmogenesis in the CAVB dog modelEchocardiography and electrophysiological studies with an LV EGM were performed in only four IVR animals. Although limited, these results adequately represent mechanical and electrical dyssynchrony in these animals. Especially since in IVR, the focus is inconsistent and arising from three different predilected sites, reliable quantification of dyssynchrony is challenging.In the CAVB dog model, AVA contributes to pro-arrhythmic remodelling as the degree of electromechanical dyssynchrony correlates with the arrhythmogenic outcome.euac164_Supplementary_DataClick here for additional data file.


The mere awareness that people who have lipedema are not just overweight but suffer from a yet poorly defined pathological condition, may be considered a decisive leap forward in the understanding of lipedema. A number of comprehensive publications have since dealt with the clinical presentation of lipedema and have provided the first clues towards the potential pathological mechanisms underlying its initiation and progression. Nevertheless, despite all effort that has been undertaken to unravel lipedema pathology, many questions have remained unanswered. What can be deduced with certainty from all experimental and medical evidence available so far is that lipedema is neither a cosmetic problem nor is it a problem of lifestyle but should be accepted as a serious disease with yet undetermined genetic background, which makes women’s lives unbearable from both a physical and psychological point of view. To date, results from clinical inspections have led to the categorization of various types and stages of lipedema, describing how the extremities are affected and evaluating its progression, as demonstrated by skin alterations, adipose tissue volume increase and physical and everyday-behavioral impediments. There is accumulating evidence showing that advanced stages of lipedema are usually accompanied by excessive weight or obesity. Thus, it is not unreasonable to assume that the progression of lipedema is largely driven by weight gain and the pathological alterations associated with it. Similarly, secondary lymphedema is frequently found in lipedema patients at advanced stages. Needless to say, both conditions considerably blur the clinical presentation of lipedema, making diagnosis difficult and scientific research challenging. The present literature review will focus on lipedema research, based on evidence fromex vivo and in vitro data, which has accumulated throughout the last few decades. We will also open the discussion as to whether the currently used categorization of lipedema stages is still sufficient and up-to-date for the accurate description of this enigmatic disease, whose name, strangely enough, does not match its pathologic correlate.When studying the current literature, one might get the impression that lipedema is a “modern” disease, with increasing incidence and augmenting prevalence throughout Western countries during the last decade. However, a quick look into older textbooks shows that disproportionate accumulation of fat in female bodies has long been known without being recognized as an independent disease. Nevertheless, it was not until 1940 that Allen and Hines described a  The worer arms) . HoweverDiets and exercise as well as decongestive therapies (lymphatic drainage) and the wearing of compression garments may improve the patients’ overall condition, but cannot eliminate the pathological mechanism(s) underlying initiation and progression of the disease. Currently, most women choose liposuction as the most causative treatment, yielding long-lasting improvements ,15,16,17It is generally agreed upon that lipedema manifests during periods of great hormonal fluctuations, usually during puberty and pregnancy, and in rare cases during menopause. A possible genetic predisposition appears plausible. A familial incidence of 15%, affecting first-degree female relatives, has been documented although self-reports usually tend to greatly exceed this percentage . The modCurrently, the visual inspection and an accurate survey of the patient’s medical history is basic to routine diagnosis of lipedema and may be improved by sonographic examination to distinguish lipedema from lymphedema at advanced stages of the disease ,20,21. AToday diagnosis of lipedema relies on criteria, originally established by Wold et al. in 1951 . FollowiBased on the diagnostic criteria described so far, one might surmise that it should not be too difficult to distinguish lipedema from other phenotypically similar diseases, including obesity, lipohypertrophy or lymphatic and venous disturbances. In fact, the phenotypic appearance of normal weight patients is quite unambiguous, making an accurate diagnosis easy. Problems arise, when phenotypically similar clinical pictures overlap with lipedema . The co-The present literature review was conducted using PubMed to search for studies, which depict the progress and current status of research into lipedema. A special focus was placed on the results from ex vivo and in vitro studies.The question of whether lipedema is associated with lymphedema or not is all the more important as several lines of evidence have indicated that defective lymphatics may contribute to fat accumulation ,29,30. IIt is still being debated as to whether vascular disorders are integral to lipedema, particularly at late stages. Since lipedema has originally been described as “a syndrome characterized by fat legs and orthostatic edema” , specialWhile Bilancini et al.  reportedOn the other hand, considerable evidence has accumulated contradicting the assumption that lymphatic insufficiency is involved in lipedema etiology, as has been summarized in a recent publication . For insIn a recent study, dilated lymphatic vessels but no dermal backflow and no edema in lower extremities of lipedema patients at early stages was shown, suggesting that no lymphatic disturbance is involved in the etiology of early-stage (stage I and II) lipedema . This fuTaking together all medical evidence available so far, it is reasonable to suggest that edema formation due to lymphatic disorders appears to play only a minor, if any, role in the initiation of lipedema. During advanced stages of lipedema, venous or lymphatic insufficiency might exacerbate the clinical picture of lipedema. Whether or not these vascular disturbances are merely a consequence of weight gain needs to be clarified in more comprehensive systematic studies, with particular focus on the BMI and other weight-associated parameters of the participants.Increased fragility of blood capillaries in lipedema adipose tissue was shown by Szolnoky et al. using angiosterrometry ,42. The Interesting results from a recent study support the notion that the endothelial barrier is compromised in lipedema adipose vasculature . The autThere is still a risk that lipedema is being under- or over-diagnosed, particularly due to the above-mentioned co-occurrence of obesity, lymphedema and other pathological conditions that potentially mask lipedema. This has fueled a quest for the identification of unique, unambiguous molecular diagnostic markers of lipedema.In this context, a number of studies have been conducted, applying histomorphology as well as cell-based molecular and immunological techniques . ResultsParticular attention has been paid to the characterization of a distinct population of adipose tissue resident mesenchymal stem cells, referred to as adipose tissue-derived stromal/stem cells (ASCs), from lipedema subcutaneous adipose tissue ,61.Since liposuction is currently the most causative treatment of lipedema, lipoaspirates from subcutaneous adipose tissue are available in sufficient quantity for research purposes, which may be considered a key benefit. Lipoaspirates are mainly used for isolation of the “stromal vascular fraction” (SVF), but also of mature adipocytes, residing in a different layer of the liposuction material. These cellular fractions may also be isolated from biopsied (solid) adipose tissues, though cell yield tends to be lower.The SVF is a clinically interesting adipose tissue fraction, which is usually sedimented from the aqueous layer of enzymatically digested adipose tissue. It contains a heterogeneous population of cells, including endothelial cells and endothelial precursor cells, ASCs, pre-adipocytes, pericytes, smooth muscle cells, fibroblasts, lymphocytes and macrophages ,63,64.ASCs account for less than 0.1% of the SVF and are grown in 2D- or 3D cultures . They arAdipogenic differentiation is a complex multi-step process that involves the stimulation of a transcriptional cascade and can be divided into two separate phases, adipogenic commitment and terminal differentiation ,74,75. UThe progression of adipogenic differentiation is usually evaluated bythe accumulation of lipid droplets (cytoplasmic triglycerides) and the corresponding alterations in adipogenic/lipogenic gene expression ,79. LipiIn vitro studies of lipedema adipose derived stem cells and preadipocytes aremainly performed in 2D cultures and in vitro differentiation of ASCs upon adipogenic stimulation is usually followed after 21–28 days of cultivation. Interestingly, in vitro differentiation, particularly in 2D cultures, leads to the accumulation of small multilocular lipid droplets, while freshly isolated mature adipocytes contain a single large unilocular LD  Figure a–f.So far, information concerning the biogenesis of lipid droplets in lipedema adipocytes is lacking but could contribute to the elucidation of the mechanisms underlying adipocyte hypertrophy in lipedema.Only limited information exists concerning adipogenic gene expression in lipedema ASCs and adipocytes compared to healthy controls. A significant increase in expression of the master transcriptional regulator PPARγ (peroxisome proliferator-activated receptor gamma) and a higher adipogenic potential of lipedema ASCs compared to controls was reported by Al-Ghadban et al. using 2D cultures . This isAccumulation of adipose tissue in vivo relies principally on hyperplasia (cell number increase) or hypertrophy (cell size increase) of adipocytes . The hypA significant increase in adipocyte size of adipose tissue from non-obese lipedema donors compared to non-obese controls was reported recently . InteresHyperproliferation of cultured lipedema ASCs was reported by Bauer et al., describing a proliferative boost after a lag period of comparable growth between lipedema and non-lipedema ASCs . AccelerAdipose tissue hypertrophy is usually associated with increased cell death and the concomitant invasion of macrophages scavenging adipocyte debris. Macrophages engulfing dying adipocytes are histologically presented as “crown-like structures” and are actually considered a pathologic hallmark of obesity . AlthougIn addtion to increased macrophage infiltration, upregulation of inflammatory genes and proteins in lipedema adipose cells and tissue has also been reported ,54. A siParticularly limited information exists concerning the assumption that lipedema is caused by or accompanied by mitochondrial disturbances. Using a mitochondrial stress test, a significant increase of the maximal possible oxygen consumption after stimulation with the uncoupler FCCP of lipedema SVF cells compared to controls was found . Basal mThere is no doubt that experienced physicians are able to correctly diagnose lipedema in daily clinical routine. On a scientific basis, however, the clarity to diagnose lipedema upon specific “markers” is currently not given, be it on a histological, cell biological or molecular level.A recent publication states that facts and myths about lipedema are often confused and passed on in a kind of tradition . This isAs summarized above, considerable scientific data based on ex vivo and in vitro studies have accumulated during the last couple of years. Most of these studies focused on various aspects of adipose tissue differentiation and the cellular and molecular microenvironment of adipose cells in vivo and in vitro. Why has so little success been achieved in the case of lipedema?In the following, some possible explanations are given, and an attempt is made to provide an improved approach for future studies. Knowledge, which has accumulated during the last few years, should be harnessed to develop optimized strategies for ex vivo and in vitro study on lipedema, which can then represent a real and important complement to the daily clinical routine.It is generally agreed upon that obesity/weight gain accompanies lipedema particularly during advanced stages. Thus, a normal weight patient exhibiting lipedema stage 3 is very rare. The most serious mistake found in various reports of cohort studies is the high BMI range within the observed groups/cohorts.It is known that the influence of weight, in particular obesity, is decisive for many histological and cell-biological properties. Thus, the interpretation of data emerging from studies with a donor population whose BMI ranges from normal weight or overweight to obesity, becomes difficult, if not impossible.In addition, it is rightly being debated as to whether the BMI is a suitable tool to evaluate the body mass of lipedema patients. Due to the strong inequality of the upper and lower body, the indication of the total weight can be misleading. It would be advisable to use both the BMI and the hip/waist ratio to estimate and categorize the donor cohorts.As previously mentioned, this topic has extensively been discussed, probably due to the phenotypically similar picture of lymphedema and advanced lipedema stages. However, it is striking that the data from the literature are contradictory. Why are data concerning a prospective involvement of vascular disturbances in lipedema pathology so inconsistent? Why could the question concerning a potential connection of vascular dysfunction in lipedema not be answered satisfactorily for so long?Here, too, the reason seems to lie in the poor selection of the subjects. Again, numerous studies concerning vascular conditions of lipedema patients were performed with patients exhibiting excessive weight (obesity) or with cohorts comprising a mixture of normal weight and obese patients.It cannot be stressed enough that any overlap of lipedema and obesity is an enormous obstacle in generating a clear picture of the vascular conditions of lipedema patients and to identify lipedema-specific alterations.Further, it cannot be entirely ruled out that lipedema adipose tissue could harbor microedema, which is undetectable by standard methods and can only be seen with high-resolution ultrastructural imaging. It would therefore be advisable to significantly expand the range of methods for the histological examination of the lipedema adipose and vascular tissue to include electron microscopic analyses.When reviewing the literature, it is noticeable that patient cohorts with a large age range are included in many studies. Since the development and progression of lipedema is obviously triggered by hormonal conditions, it would be advantageous to narrow down the age range of study cohorts to ensure a comparable hormonal status of participants as this is crucial for the development of adipose tissue.This could also have a positive effect in reducing the high interdonor variability, which very often leads to valueless data because they are outside statistical significance.Most of the ex vivo and in vitro studies include age- and BMI-matched controls in order to pinpoint lipedema-specific characteristics. It is reasonable to assume that people undergoing cosmetic liposuction suffer from excessive localized fat accumulation, whether due to overweight/obesity or lipohypertrophy. Neither condition is an optimal control for lipedema. This is all the more important to reconsider since it cannot be excluded that lipohypertrophy and lipedema share similar molecular mechanisms underlying adipose tissue accumulation. These considerations are of secondary importance for the surgeon but play a crucial role when it comes to evaluating and interpreting scientific data.Since increasing medical and scientific interest has been focused on lipedema during the last couple of years, many observations have been made which would eventually justify a revision of the current staging of lipedema. Each lipedema stage must be seen as a complex entity since it represents the sum of several conditions. Thus, it would be advisable to extend the current staging by indicating additional information such as the occurrence of co-morbidities , the patient’s original weight (in case of multiple operations), and the family history. A more differentiated classification would correspond to the heterogeneous presentation of this clinical picture.In recent years, lipedema research has evolved from purely clinical testing to molecular science. In this short review, we have tried to give an overview of the diverse laboratory-technical approaches and the findings obtained from them. Excellent and time-consuming procedures have shown that lipedema manifests itself on many levels and have once again confirmed that in vitro and ex vivo studies are essential to understand the multifaceted nature of the disease.However, what has also become clear is that many questions are still unanswered, and a lot of preliminary data still needs to be deepened. We also addressed the main obstacles hampering lipedema research. Currently, there is no mouse model, no cell lines which could be used to achieve reproducible results. Therefore, one of the biggest handicaps, the high interdonor variability, that often makes scientific results unusable, most likely will persist. It is also evident that too little attention has been paid to the problem of accompanying obesity for too long. This does not mean that we have to start from scratch, but we need to start discarding some unverifiable opinions about lipedema. As outlined above, there are already valuable indications regarding in which direction(s) lipedema research should develop. Implementing new findings and merging data from different disciplines will pave the way to the successful elucidation of this enigmatic disease.


Pressure–volume (PV) analysis is the most comprehensive way to describe cardiac function, giving insights into cardiac mechanics and energetics. However, PV analysis still remains a highly invasive and time-consuming method, preventing it from integration into clinical practice. Most of the echocardiographic parameters currently used in the clinical routine to characterize left ventricular (LV) systolic function, such as LV ejection fraction and LV global longitudinal strain, do not take the pressure developed within the LV into account and therefore fall too short in describing LV function as a hydraulic pump. Recently, LV pressure-strain analysis has been introduced as a new technique to assess myocardial work in a non-invasive fashion. This new method showed new insights in comparison to invasive measurements and was validated in different cardiac pathologies, e.g., for the detection of coronary artery disease, cardiac resynchronization therapy (CRT)-response prediction, and different forms of heart failure. Non-invasively assessed myocardial work may play a major role in guiding therapies and estimating prognosis. However, its incremental prognostic validity in comparison to common echocardiographic parameters remains unclear. This review aims to provide an overview of pressure-strain analysis, including its current application in the clinical arena, as well as potential fields of exploitation. The evaluation of left ventricular (LV) systolic function is of significant importance in all echocardiographic examinations. The most common parameter to assess systolic function is left ventricular ejection fraction (LVEF), whereas reduced LVEF is correlated with poor outcome –3. HowevPressure–volume analysis, the gold standard in assessing ventricular function, delivers reasonably load-independent measures, as the end-systolic and end-diastolic pressure–volume relationships –17. FurtDue to a further development in echocardiographic techniques including the implementation of volume and strain analysis, it became possible to estimate diastolic and systolic pressure volume relationships non-invasively , 22. RecWhereas the pressure–volume loop describes the global myocardial work und function, also regional myocardial work has been studied in the form of pressure-length loops, obtained via micromanometers for the assessment of pressure, and implanted sonomicrometric crystals for the calculation of dimensions in animal models , 24.The sonomicrometry technique, first introduced in 1956 by Rushmer et al. , has traIn analogy to the pressure–volume loop ability to reflect global myocardial oxygen consumption, Delhaas et al. could show that the regionally assessed stress-fiber strain area was not only able to reflect regional myocardial work but also regional oxygen demand .Assessment of myocardial wall stress is difficult and not feasible in clinical practice. Studies by Skulstad and Urheim et al. could validate the invasively measured LV pressure as a surrogate of left ventricular wall stress , 31. Fur18F-fluorodeoxyglucose positron emission tomography [The new approach by Russel et al. goes even a step further by assessing myocardial work completely non-invasively. They combined segmental strain curves by speckle tracking echocardiography with an estimated LV pressure curve where the systolic cuff pressure is used as a surrogate of LV peak pressure . The metmography   is necessary to assess non-invasive myocardial work by combining left ventricular strain data obtained via speckle‐tracking echocardiography with a non-invasively estimated LV pressure curve.Practically, LV global longitudinal strain (GLS) has to be estimated by processing two-dimensional grayscale images acquired in the standard apical two‐, three‐, and four‐chamber views at similar heart rate, depth, and a frame rate between 38 and 80 frames/s with an off-line dedicated software .The novelty of integrating non-invasive LV pressure is based on the creation of a normalized left ventricular pressure curve which was obtained by assembling invasive LVP measurements from patients in different conditions of inotropy . All invTo then establish each patient’s specific LVP curve, the normalized LVP curve has to be adjusted according to the patient’s valvular event times assessed via echocardiography and the measured systolic cuff pressure, as a surrogate for LV peak pressure .To determine valvular events, event timing can be derived by ECG-triggering or Doppler echocardiography. Electrical and mechanical phases should be aligned and can be manually adjusted. It is recommended to assess the blood pressure at the time of the examination Fig. .Fig. 2PrA 17‐segment model is considered for the calculation of segmental myocardial work. However, segmental myocardial work is not assessed by calculating the area of each segmental pressure strain loop, rather it is calculated as a function of time for the duration of the whole cardiac cycle . In partConstructive myocardial work (GCW): positive segmental work performed during myocardial shortening in systole and negative segmental work during lengthening in isovolumic relaxationWasted myocardial work (GWW): negative segmental work performed during lengthening in systole and positive segmental work performed during myocardial shortening in isovolumic relaxationMyocardial work efficiency (GWE): relationship between constructive work and the sum of constructive and wasted workGlobal values of myocardial work are derived from the average of all segmental myocardial work values. Further parameters describe the relationship between positive and negative myocardial work according to their occurrence during the cardiac cycle Fig. :ConstrucFor this literature review, we searched MEDLINE (PubMed) and Embase, while clinicaltrials.gov was searched for ongoing clinical studies. The following search terms included in the title were used: “cardiac work,” “constructive work,” “global longitudinal strain,” “global work index,” “myocardial work,” “myocardial work efficiency,” “non-invasive myocardial work,” in combination with “pressure-strain loops” in the title or abstract. Relevance and credibility of all the sources were considered, and the final decision on inclusion was reached through a consensus of the following screening authors: DA, TR, AF, and AA. Review articles, case reports, comments, and author replies were excluded. The cited references were published within the last 8 years. A narrative synthesis with a tabulation system was used to analyze studies for their diverse research designs, methods, and implications.In the present review, we focused on the state of the art regarding non-invasive myocardial work assessment in several clinical fields: in CRT recipients, in patients with ischemic cardiac disease, mitral valve repair, heart failure with reduced (HFrEF) and those with heart failure and preserved ejection fraction (HFpEF) or HFpEF-like syndromes  Fig. .Fig. 5CuFurthermore, we searched for ongoing clinical trials focusing on non-invasive myocardial work assessed by pressure-strain loops.Cardiac resynchronization therapy (CRT) is an effective therapy that aims to restore mechanical efficiency to the failing LV by resynchronizing the contraction of the left and right ventricle, resulting in a reduction of both morbidity and mortality . AccordiHowever, the number of patients who do not respond to CRT remains high (30–35%) . EstimatIn conclusion, even though pressure strain loops and related indexes cannot be used on their own to assess CRT candidates, they represent a novel method that shows promising results in the prediction of CRT response and long-term outcomes.In patients with non-ST-elevation myocardial infarction (NSTEMI), Boe et al. could show that the non-invasively estimated myocardial work index (MWI) was able to detect acute coronary occlusion, being superior to all other echocardiographic parameters used, including strain analysis . PatientIn a further study, Edwards et al. validated that non-invasively assessed global myocardial work at rest allowed to detect subclinical coronary artery disease (CAD) in patients with preserved LVEF and no regional wall motion abnormalities (RWMAs) . InteresIn a retrospective analysis by El Mahdiui et al., a reduced myocardial work efficiency could be confirmed in patients with recently revascularized ST-elevation myocardial infarction and patients with heart failure with reduced ejection fraction . HoweverAccording to the current ESC Heart Failure Guidelines from 2016, heart failure represents a clinical diagnosis, which is characterized by typical symptoms and signs as well as increases of natriuretic peptide (BNP or NT-proBNP) .Furthermore, echocardiographic determination of left ventricular function is necessary for the diagnosis of heart failure. Currently, left ventricular ejection fraction (LVEF) and also the assessment of left ventricular filling pressure by using the ratio of early transmitral flow and myocardial relaxation (E/e′ ratio) are the recommended parameters of choice .The prognostic accuracy of LVEF, while significant in the circumstance of an LVEF < 40%, appears to be low in the case of HFpEF . AdditioToday, myocardial strain measurements are well implemented in the daily clinical routine offering more precise, reproducible, and comprehensive information regarding LV mechanics and function. Clinical implications of myocardial strain assessment are diverse.Notably, global longitudinal strain (GLS) was shown to be associated with outcome in symptomatic heart failure patients with reduced and preserved LVEF, and furthermore a stronger predictor of outcome than LVEF, especially in patients with preserved LVEF –78.However, like LVEF, strain parameters prove to be dependent on afterload, resulting in a possible misinterpretation of the true contractile function , 79.The estimation of myocardial work fixes this weakness by implementing the estimated LV pressure as described earlier.In the setting of HFrEF, Wang et al. could show that global myocardial work (GMW) was a better prognosticator than both GLS and LVEF, where reduced values of GMW are significantly associated with death or poor outcome .In a group of patients with acute myocardial infarction and heart failure with preserved or mid-range ejection fraction, it was shown that both GLS and Global Myocardial Work Index (GWI) are reduced in the majority of individuals. Some patients presented normal GWI despite abnormal GLS, emphasizing the importance of implementing blood pressure in the assessment of myocardial function .Moreover, global constructive work (GCW) has been proven to be a better estimate of LV contractile response to physical effort, and hence a better measure of exercise capacity, in HFpEF than GLS. Its exertional increase in patients treated for 6 months with spironolactone is considered to be associated with improvement in functional capacity .Heart failure patients with reduced ejection fraction treated with sacubitril/valsartan showed signs of LV reverse remodeling by common echocardiographic parameters as well as a significant improvement of constructive work and myocardial work efficiency during a follow up of 12 months. Wasted work, on the contrary, did not appear to be greatly affected , 84. Als2 consumption and VE/VCO2 slope), and NT-pro-BNP in patients with heart failure [Hedwig et al. described the relation between global work index (GWI) and known prognostic parameters of echocardiography (GLS and LVEF), cardiopulmonary exercise test  in these patients [Schrub et al. could also show in patients with dilated cardiomyopathy that the presence of left ventricular dyssynchrony assessed by echocardiography as septal flash or apical rocking leads to a significant reduction to global myocardial work efficiency due to an increase of wasted work . These fpatients .2 peak assessed by cardiopulmonal exercise testing, favoring GCW as an indirect, easily accessible measure of exercise capacity in patients with non-obstructive HCM [Galli et al. were the first to investigate pressure-strain analysis in patients with non-obstructive hypertrophic cardiomyopathy . Here, gtive HCM .Mansour et al. studied the variations of MW indices during stress echocardiography in patients at peak exercise with high systolic blood pressure values of > 180 mmHg . They coNon-invasive myocardial work has also been investigated in patients with cardiac amyloidosis at rest and during exercise by Clemmensen et al. . These pn = 226) over a wide range of ages and delivered current normal reference values of non-invasive myocardial work indices [The NORRE  study, the first related multicenter study, investigated a large population of healthy individuals  . CPO is k at all . Howeverk at all . The nonAnother interesting outlook is related to the field of trans-catheter valve repair. Papadopoulos et al. conducted a retrospective case–control study to evaluate left ventricular global longitudinal strain and myocardial work indices in patients with heart failure and mitral regurgitation 1 year after transcatheter mitral-valve repair compared to patients treated with optimal medical treatment . PreservThe ability to assess myocardial work non-invasively broadens the currently available techniques for evaluating cardiac function. The adjustment to loading conditions leads to the implementation of critical hemodynamic concepts in the daily assessment of cardiac function. Further studies focusing on the prognostic impact of non-invasive myocardial work are needed.


We report the case of a 99-year-old woman with acute chest pain caused by myocardial ischemia due to severe aortic stenosis (AS) and severe anemia. Red blood cells were transfused; however, this increased the preload and worsened pulmonary congestion. Treatment with drugs and non-invasive positive pressure ventilation could not sufficiently improve the pulmonary congestion. Ivabradine was administered to control the resting heart rate, following which the pulmonary congestion significantly improved. Ivabradine is a promising drug for acute heart failure of patients with AS by improving cardiac output by prolonging the left ventricular diastolic filling time without a negative inotropic effect. Aortic stenosis (AS) is a common valvular heart disease. It has a prevalence of approximately 4-5% in persons older than 65 years . Its sevHeart failure in patients with AS is treated with angiotensin-converting enzyme (ACE) inhibitors and diuretics at low doses with gradual dose titration . It is aIvabradine, a selective inhibitor of If current in the sinus node, improves the prognosis of patients with chronic heart failure through heart rate reduction without a negative inotropic effect . The ETHHere, we report a case of acute congestive heart failure in a very elderly patient with symptomatic severe AS treated with ivabradine.2), 93% on room air. Her body weight was 34 kg and she was 138 cm tall (body mass index 17.9 kg/m2). Physical examination revealed a loud systolic murmur and bilateral inspiratory wheezing.A 99-year-old woman with sudden-onset chest pain was administered to the hospital. She had a past medical history of severe AS, Alzheimer's dementia, and repeated lower gastrointestinal bleeding. She had been admitted to a local clinic one week previously to investigate the cause of bloody stools. At presentation, she was afebrile, and her vital parameters were as follows: blood pressure, 103/49 mmHg; heart rate, 97 beats per minute (bpm), sinus rhythm; respiratory rate: 25 per minute; and oxygen saturation (SpO2).The results of a complete blood count revealed severe anemia (hemoglobin: 4.6 g/dL and hematocrit: 14.1%) and elevated levels of brain natriuretic peptide (BNP) , high-sensitive troponin I , and creatine kinase-MB . Renal function was normal : 83 ml/min/1.73 mChest radiograph revealed a bilateral consolidation, severe calcification of the thoracic aorta, and an increased cardiothoracic ratio (74%) Figure . A 12-le2, peak transvalvular gradient of 78 mmHg, and peak aortic valve jet velocity of 4.4 m/sec.Transthoracic echocardiography (TTE) revealed left ventricular wall thickening, a highly calcified trileaflet aortic valve, and a large pericardial effusion with an LV ejection fraction (LVEF) of 62% as measured using the biplane Simpson’s method Figure , 3B. InfPlain computed tomography (CT) showed cardiac dilatation, pulmonary congestion, pericardial and bilateral pleural effusion, and severe calcification of the aortic valve, mitral valve, aorta, and coronary arteries Figure .The patient presented with sudden-onset chest pain. The differential diagnosis included acute cardiac syndromes, aortic dissection, and pulmonary embolism. Laboratory findings such as elevated levels of cardiac enzymes and the ECG and TTE findings suggested the possibility of ischemic heart disease. Acute myocardial infarction could not be ruled out as coronary angiography could not be performed due to the patient’s age and dementia. Aortic dissection and pulmonary embolism were ruled out based on plain and contrast-enhanced CT findings.The patient was initially diagnosed with myocardial ischemia that was associated with severe AS and aggravated by reduced oxygen supply due to severe anemia. Balloon aortic valvuloplasty for severe AS may be considered as a treatment option, however, her guardian did not want invasive treatment. The clinical course of the patient’s disease is described in Figure 2 > 90%. She continued to have tachypnoea (respiratory rate: 25 per minute), sinus tachycardia (heart rate: 101 bpm), high blood pressure (178/89 mmHg), and intermittent chest pain (day 10). Adequate urine output was maintained with intravenous furosemide administration. Non-invasive positive pressure ventilation (NIPPV) was administrated between day 6 to day 9; low-pressure settings were used to prevent excessive reduction of venous return. However, these treatments failed to sufficiently improve pulmonary congestion. The chest radiograph showed a bilateral butterfly shadow  were transfused to improve the severe anemia from day 1 to day 3 of admission . Enalapril (1.25 mg/day) was started, and furosemide (10 mg/day) was intravenously administered. The hemoglobin level improved from 4.6 g/dL at presentation (day 1) to 11.8 g/dL on day 4. Pulmonary congestion and pleural effusion worsened after the RBC transfusion. The patient’s weight increased from 34.0 kg (day 1) to 35.4 kg (day 11). She required 3 L of oxygen per minute to maintain a SpOw Figure . TTE at c Figure , 3D.2 by planimetry and 0.89 cm2 by the continuous equation, peak pressure gradient of 75 mmHg, mean pressure gradient of 49 mmHg, peak aortic jet velocity of 4.3 m/sec, and no LV outflow tract (LVOT) obstruction  was started to control the resting heart rate; the rest of the treatment remained the same. Beta-blockers were not used because of concerns about worsening the hemodynamic status by decreasing cardiac contractility. Subsequently, the resting heart rate dropped from 101 bpm (day 10) to 65 bpm (day 14), and the body weight decreased from 35.4 kg (day 11) to 30.5 kg (day 23). Pulmonary congestion was improved Figure . The patn Figure , 3F.She was discharged to a nursing home on day 32 of admission. Six weeks after discharge, she was admitted to the hospital due to infective endocarditis. Unfortunately, she did not respond to antibacterial therapy and died three days after admission.We report a case of severe AS with congestive heart failure after RBC transfusion for severe anemia that responded to treatment with ivabradine, which was started for heart rate control. In patients with severe AS, even if the coronary arteries are normal, supply and demand mismatch during hemodynamic stress may lead to subendocardial ischemia . The patIn patients with severe AS, a decreased AVA limits the aortic flow. An adequate cardiac output is maintained by increasing the heart rate and myocardial contractility through a compensatory increase in sympathetic activity. However, an excessive increase in heart rate can result in a short diastolic period, leading to decreased myocardial perfusion . InadequBeta-blockers can be useful for heart rate control. Post-hoc analysis of the SEAS (Simvastatin and Ezetimibe in Aortic Stenosis) study, which included 1,873 asymptomatic patients with mild to moderate AS and preserved LVEF who were treated with beta-blockers, revealed significantly decreased hazard ratios of all-cause mortality, cardiovascular death, and sudden cardiac death . HoweverThere have been two previous reports of patients with severe AS with acute heart failure treated with ivabradine. In the first case, an 86-year-old man with severe AS and a reduced LVEF of 20% showed improvement in heart failure symptoms following treatment with ivabradine . In the Interventional treatment is recommended for symptomatic severe AS; however, drug therapy for AS-induced heart failure, cardiac ischemia, and pulmonary congestion is also essential in inoperable patients and patients waiting for interventional treatment. Considering the hemodynamic instability caused by severe AS, ivabradine can be a promising drug for controlling the resting heart rate of patients with symptomatic severe AS without a negative inotropic effect. Further research is required to investigate the usefulness of ivabradine for treating severe AS.


Echocardiographic global myocardial work (GMW) indices recently emerged to non-invasively evaluate left ventricular (LV) myocardial performance with less load-dependence than LV ejection fraction (LVEF) or global longitudinal strain (GLS). Yet, few data exist on the descriptions of LV GMW indices in young people. We therefore aimed to provide normal reference values of LV GMW in a healthy young cohort, and simultaneously to investigate factors associated with non-invasive GMW indices.A total of 155 healthy young people  underwent transthoracic echocardiography were recruited and further stratified for age groups and divided by gender. Two-dimensional speckle-tracking echocardiography (2D-STE) were performed to determine LV GLS, peak strain dispersion (PSD) and GMW indices, which include global work index (GWI), global constructive work (GCW), global wasted work (GWW), and global work efficiency (GWE). LV peak systolic pressure was assumed to be equal to the systolic brachial artery cuff blood pressure.P < 0.05 for all). There were no associations between any of the GMW indices with age, sex, body mass index, heart rate, left ventricular mass index as well as LV sizes or LVEF. Of noted, LV GMW indices had good intra-observer and inter-observer reproducibility.Age and gender specific normal ranges for LV GMW indices were presented. On multivariable analysis, GWI and GCW correlated more closely with systolic blood pressure (SBP) than LV GLS, while both GWW and GWE independently correlated with PSD (We reported echocardiographic reference ranges for non-invasive LV GMW indices in a large group of healthy young subjects, which are reproducible and reliable, and thus can be further used when assessing subclinical dysfunction in young people with myocardial diseases. Accurate detection of subtle left ventricular (LV) contractile dysfunction may be of clinical importance in young patients with various heart diseases to avoid irreversible consequent LV deterioration in later life. The recently employed echocardiographic surrogates of LV contractility, such as LV ejection fraction (LVEF), tissue Doppler imaging (TDI), and most recently myocardial strain derived from speckle-tracking echocardiography (STE), are load-dependent and susceptible to misinterpretation of the true myocardial contractility as they are all markedly influenced by afterload –3.In recent years, non-invasive assessment of LV myocardial work (LVMW), a new echocardiographic tool based on STE-derived longitudinal strain and peripheral systolic blood pressure (SBP) (as a surrogate of LV systolic pressure), has attracted widespread attention . It is tWith the growing interest in non-invasive LVMW, the knowledge of normal reference values specific to young population are requisite. Despite robust normative data for the performance of myocardial work characteristics are well-established in adult populations –11, dataFrom May 2019 to July 2021, a total of 165 healthy young people aged 10–24 years according to the World Health Organization (WHO) definition were prospectively enrolled in our study . This stBlood pressure was obtained by measuring the average value of the three consecutive measurements in the sitting position, at least 1 min apart and after 5 min of rest, using the Datascope Accutorr Plus , which has been validated for use in local Chinese adolescents .Comprehensive transthoracic echocardiography was performed with the volunteer in the left lateral position employing breath holds, using a Vivid E9 or E95 system . Two-dimensional (2D) images from the standard three LV apical views  were acquired during three consecutive cycles, with an image frame rate of 50–70 frames per second. All echocardiographic images were digitally stored for offline analysis with the dedicated software . Parasternal, apical, and subcostal views were used to acquire 2D, color, pulsed-wave, and continuous wave Doppler data according to current recommendations . LV voluQuantification of LV GMW indices were performed offline using proprietary software package , which integrates LV strain measurements with blood pressure recordings . Peak LV1.Global work index (GWI): the total work within the area of the global LV pressure-strain loop calculated from mitral valve closure to opening (mmHg%).2.Global constructive work (GCW): positive work performed by LV during systole that is productive, including both shortening of the muscle during systole and lengthening of the muscle during isovolumic relaxation (IVR) (mmHg%).3.Global wasted work (GWW): negative work performed by LV during systole that is not productive, including both lengthening of the muscle during systole and shortening of muscle in IVR (mmHg%).4.Global work efficiency (GWE): GCW/(GCW + GWW) (0–100%).Intra-observer and inter-observer variabilities of LV GMW data were evaluated in 20 random subjects by means of intraclass correlation coefficients (ICCs). For the inter-observer variability, the differences between two independent sonographers who were blinded to the results of the other were evaluated. For the intra-observer variability, the differences between two observations made by the same sonographer after 2 weeks to avoid recall bias were also assessed. The criteria for ICCs were: “excellent” if > 0.74, “good” if 0.60–0.74, “fair” if 0.40–0.59, and “poor” if < 0.40 , 21.t-test or the Mann-Whitney U test as appropriate, while comparisons between three groups were made with a one-way analysis of variance (ANOVA) with Tukey’s post-hoc or Kruskal-Wallis test, as appropriate. The association of GMW indices with demographics and echocardiographic measures was performed using Pearson’s or Spearman’s correlation coefficient as appropriate. Further univariable linear regression and multivariable linear regression analyses were performed to determine the independent correlates between GMW indices and standard and advanced echocardiographic parameters. For multiple linear regression models, multicollinearity was also examined by computation of variance inflation factor. In case that collinear variables existed, the variable with the highest correlation coefficient was included. All P-values were reported as two-tailed, with < 0.05 considered as statistical significance.Collected data were computerized and analyzed using SPSS 22.0  statistical software. Data normality was tested using Shapiro-Wilk method. Continuous variables are expressed as mean ± standard deviation (SD). Differences in continuous variables between two groups were analyzed using independent sample P > 0.05 for all). Height, weight, BSA as well as brachial cuff SBP were significantly higher in young males compared with females. Of noted, SBP in the youngest group of 10–14 years was significantly lower than that in the other two groups (P < 0.05 for both), while no significant difference in SBP was shown between the two older groups of young subjects  participants , with 10 individuals excluded for poor acoustic window. Demographic characteristics and conventional echocardiographic information were presented by gender in subjects .P ≤ 0.001 for all). There were no statistically significant differences in LAVi, mitral E/A ratio, mitral annulus e′ and E/e′ ratio between gender .As expected, the young males had significantly larger LVMI, wall thickness, LAV, LV dimensions and volumes with lower LVEF when compared to female subjects . However, the absolute value of LV GLS was slightly lower in young male (P = 0.001). Furthermore, the absolute value of LV GLS in the youngest group of 10–14 years was slightly higher than that in the other two groups (P < 0.05 for both), while no significant difference in GLS was shown between the two older groups of young subjects  (P < 0.001) . Moreover, both GWW and GWE significantly correlated with PSD  . GWE was also found to be inversely correlated with LV GLS , while GWW was not associated with SBP and GLS. There were no associations between any of GMW indices with age, BSA, LVMI and LVEF on multivariate analysis. Noticeably, the β- standardized coefficients from multivariate linear regression model revealed that GWI and GCW were more closely correlated with SBP than LV GLS.There were no significant associations between any of LV GMW indices with sex, BMI, HR, mitral annulus e′ or LV dimensions and volumes. On both univariate and multivariate analysis as detailed in < 0.001) , LV GLS < 0.001) , and E/ectively)  and E/A As it has been shown in In present study, we demonstrated the normal echocardiographic reference values for LV GMW indices in a large cohort of healthy young people. Also, we found no influence of age, sex, BMI, HR, mitral annulus e′, LVMI, LV sizes or LVEF on GMW indices. Furthermore, our finding disclosed that both GWI and GCW were more closely correlated with SBP than LV GLS, while both GWW and GWE were independently correlated with PSD. Importantly, our study showed that non-invasive myocardial work analysis was feasible in young people with a favorable intra- and inter-observer variability, which agreed with previously reported series, thus enhancing the possibility of a promising application of LVMW in clinical practice.To date, only three similar studies were conducted regarding the normal ranges of GMW in healthy young people , 13, 22.It is still controversial whether gender contributed to variations in normal values of GMW. A growing body of literature has demonstrated no gender difference in GMW indices. Conversely, a recent meta-analysis of 12 studies, including nearly 1,665 adult subjects, indicated slight gender difference of GMW indices in healthy population . These iIn the meantime, our results confirmed but also extended previously published findings. We elucidated no influence of age, sex, BMI, HR, mitral annulus e′, LVMI, LV sizes or LVEF on GMW indices in young people, in accordance with the previous reports across the different age groups , 13, 22.2 max). Of noted, as we had selected healthy young individuals in our study, the GMW markers were all within normal ranges. Therefore, additional validation should be done to optimize the use of LVMW analysis in young patients with various cardiovascular diseases.In fact, the non-invasive LVMW analysis has been validated against invasive methods and demonstrated a strong correlation with myocardial oxygen consumption . Recent Several limitations of our study should be acknowledged. First, LVMW is derived from 2D strain analysis, thus the accurate quantification of which would be influenced by the quality of ultrasound images. Nonetheless, the feasibility of the LVMW analysis was excellent with only 6% of healthy young volunteers excluded for poor image quality. Second, the non-invasive assessment of LV myocardial work is currently available on a single echocardiographic platform, and so far, cannot be evaluated using other software. Third, it was a prospective study, so the systemic arterial pressure was routinely obtained in the sitting position instead of left lateral decubitus, which might lead to discrepancy in blood pressure values and consequently affect the results of LVMW, as several studies reported posture affects the blood pressure , 36. FutThe echocardiographic reference values for non-invasive global myocardial work indices, independent of age, sex, BMI, HR, LVMI, LV sizes or LVEF in a large healthy young people, were reported to provide a foundation for incorporation into clinical practice. In addition, we revealed that GWI and GCW correlated more closely with SBP than GLS, while both GWW and GWE independently correlated with PSD. Our study suggests that LV GMW indices are reproducible and reliable, and may provide promising value for quantifying myocardial dysfunction in young patients with myocardial diseases.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Medical Ethics Committee of Shenzhen Hospital of Southern Medical University. Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.XL and YZ conceptualized and designed the study, and coordinated and supervised the data collection. NZ, PL, XX, YC, DW, LM, and YM participated in the data collection. JS and QG took responsibility for the integrity of the data and the accuracy of the data analysis. XL screened the literature and wrote the first draft of the manuscript. YZ directed all phases of the study and revised the final manuscript. All authors reviewed and approved the manuscript as submitted and agreed to be accountable for all respects of the work.


Heart failure (HF) is an extremely major health problem with gradually increasing incidence in developed and developing countries. HF may lead to cardiac remodeling; thus, advanced imaging techniques are required to comprehensively evaluate cardiac mechanics. Recently, three-dimensional speckle-tracking echocardiography (3D-STE) has been developed as a novel imaging technology that is based on the three-dimensional speckle-tracking on the full volume three-dimensional datasets. Three-dimensional speckle-tracking echocardiography allows a more accurate evaluation of global and regional myocardial performance, assessment of cardiac mechanics, detection of subclinical cardiac dysfunction, and prediction of adverse clinical events in a variety of cardiovascular diseases. Therefore, this review summarizes the clinical usefulness of 3D-STE in patients with HF. Heart failure (HF) is defined as a clinical syndrome with typical symptoms , accompanied by signs  caused by structural and/or functional cardiac abnormality, resulting in a reduced cardiac output and/or increased intracardiac pressures at rest or during stress . AccordiCurrently, echocardiography remains the first-line imaging modality of assessment of cardiac structure and function, and it offers significant value in predicting HF events . Althougt − L0)/L0, where Lt is myocardial length at time t and L0 is original myocardial length at end-diastole. Strain is not affected by the movement of the entire heart [Strain represents myocardial deformation that occurs during the cardiac cycle. Strain =  on this basis ,19. In aTwo-dimensional speckle-tracking echocardiography allows automatically tracking the motion trajectory of the myocardium frame by frame throughout the cardiac cycle by identifying the position and motion of speckles in the two-dimensional (2D) images, and parameters  are obtained by post-processing ,17,22. TLV myocardium consists of three distinct myocardial layers that contract simultaneously in different directions. Right ventricular (RV) myocardial fibers have a complex 3D arrangement  with longitudinal orientation. Hence, the accurate assessment of cardiac function requires 3D analysis ,35. ThreThree-dimensional speckle-tracking echocardiography is based on STE combined with RT-3DE, which is a speckle-tracking technique applied on RT-3DE. Three-dimensional speckle-tracking echocardiography tracks 3D motion trajectory of myocardial specific markers between successive volume frames within region of interest (ROI) frame by frame  in full-volume 3D gray scale images, thereby derivating various deformation parameters of the myocardium throughout the entire cardiac cycle ,35,36. FAn accurate assessment of cardiac function requires 3D imaging methods, as myocardial movement is essentially a 3D phenomenon. Three-dimensional speckle-tracking echocardiography fuses advantages of 2D-STE and RT-3DE and has a better correlation with CMR, overcoming the limitation of 2D-STE that did not simultaneously measure all 3D displacement components of myocardial speckles in one cardiac cycle ,37. Threp < 0.001). Pressure overload caused by aortic valve stenosis has a greater effect on GLS. Three-dimensional strains are useful to early detect cardiac dysfunction in patients with aortic stenosis and preserved LVEF [HF is defined based on the heart pump impairment  . Three-dved LVEF .p < 0.01). Three-dimensional GLS was the most powerful independent predictor of MACE. Three-dimensional speckle-tracking echocardiography allows stratification of asymptomatic patients with severe aortic stenosis and preserved LVEF at high risk of MACE and supports early intervention and improves long-term outcomes [Opposing rotations of the LV base and apex during systole results in LV torsion, a key parameter in cardiac function. Nagata et al. investigated which strain component was the robust predictor of major adverse cardiac events (MACEs) in 104 patients with asymptomatic severe aortic stenosis and LVEF >50% by 2D-STE and 3D-STE. In contrast to the patients without MACE, 3D GLS, 3D GRS, and 2D GLS were significantly lower in patients with MACE and had significant predictive power for MACE . Moreover, models using 3D GLS were found to predict adverse outcomes stronger than that using 2D GLS. Three-dimensional GLS of >−11.1% was associated with primary endpoint events confirmed by Kaplan–Meier survival curves. The addition of 3D GLS to the base model (age + log BNP + infarct size) improved risk stratification in ST-elevation acute MI patients . Three-dimensional GLS was a useful parameter to predict long-term prognosis in ST-elevation acute MI patients and had a significant incremental effect [GLS reduction can be used as a prognostic marker of adverse cardiovascular outcomes of HF because it reflects the gradual development of LV dysfunction in HF patients ,44. In al effect . Therefol effect . Altman l effect . Currentp < 0.01). Transmural infarct segments displayed markedly lower longitudinal, radial, and circumferential strains than normal segments [Ischemic heart disease has a worse prognosis than patients with non-ischemic heart disease and is the most common cause of HF. Therefore, evaluation of regional myocardial function is crucial for better treatment of ischemic HF patients ,55. Echosegments . Three-dsegments .p < 0.001), and the cut-off value of segmental AS (<−23%) well-differentiated regional MI had a specificity of 75.9% and a sensitivity of 75.1% . They proved that 3D-STE can provide a new method for clinical assessment of the LV regional function in patients with MI [p = 0.0022) in HF patients with ischemic etiology significantly higher than in those non-ischemic etiology [p = 0.03) [Due to the myocardial structural distribution characteristics of longitudinal endocardial fibers and circumferential mid-myocardial fibers, transmural MI may be represented by reductions in both longitudinal and circumferential strains ,37. AS,  with MI . Howeveretiology . The res = 0.03) . In concp < 0.001; cut-off value ≥ 94 cm) was a powerful predictor of elevated LV filling pressure [LV diastolic dysfunction is the results of cardiac pressure and volume overload, and further develops into HF. Therefore, LV diastolic function evaluation is considered a pivotal information for clinical diagnosis and prognosis of HF patients . Evaluatpressure . A studypressure . Three-dp < 0.001) and negatively correlated with the CO2 equivalent slope . A total of 64% of the variation in peak oxygen uptake and 17.6% of the variation CO2 equivalent slope were explained by 25% untwist. A total of 25% untwist value was a good indicator of LV early diastolic dysfunction, which is an independent predictor for examining aerobic exercise capacity. Cardiac function of HF patients with dyskinesia can be evaluated as an aid by revealing the relationship between cardiac diastolic function at rest and CPET [Patients with HF usually present with exercise intolerance and the gold standard for assessing clinical cardiopulmonary function is cardiopulmonary exercise testing (CPET) . Combineand CPET .Cardiac resynchronization therapy (CRT), an important treatment for patients with advanced HF as it improves the pattern of myocardial contraction by placing strategically positioned biventricular pacemaker . Althoug2 = −0.94, −0.91, −0.89, 0.93, respectively; all p < 0.01). After CRT, 3DS-SDs  and SDIs  improved significantly. The assessment of LV dyssynchrony using 3D-STE might be critical for selecting HF patients who can benefit from CRT [p < 0.05, cut-off value > −9.52%, 78% sensitivity, 80% specificity), RS , and AS  can be used to select resynchronization candidates in the HF group according. Three-dimensional speckle-tracking echocardiography is applicable in the evaluation of both patients with HFpEF and HFrEF and helps to assess the CRT candidates and response [2 -S/D coupling) to explore the value of strain-volume loops using 3D-STE analysis in predicting CRT response in HF patients. They found that Midseptal R2-S/D coupling at baseline was an independent predictor of CRT response, with a recommended cut-off value of 0.55, a sensitivity of 89%, and a specificity of 77%, and deduced that strain-volume loops provided important information for predicting CRT response. Thus, 3D-STE is helpful for improving the selection of CRT patients and may provide significant information to predict response to CRT [In a study investigating the evaluation of LV systolic mechanical synchrony in 52 dilated cardiomyopathy (DCM) patients and 55 healthy controls, the standard deviation of time to negative peak strain (3DS-SD) and time to regional minimum volume of 16 segments (SDI) derived from 3D-STE were used to explore the LV dyssynchrony . Kang etfrom CRT . Szulik response . Zhu et e to CRT .p < 0.001) [p < 0.0001; cut-off value < 0.420) was an independent predictor of hospitalization in HF patients, providing higher prognostic power in future MACEs than all models using 2D parameters. Thus, measurement of LA parameters calculated by 3D-STE is clinically useful and feasible [p < 0.001), 3D LA longitudinal strain , and 3D LAEmpF  showed higher hazard ratios than other indices. Three-dimensional LAEmpF + base model  displayed incremental prognostic values in major cardiovascular events [The main function of left atrium is to regulate LV filling . The thi< 0.001) . Importafeasible . The samr events . Three-dr events .p = 0.03), 3D GASs , and age  may predict the recurrence of AF. Moreover, 3D LA strain was a better predictor of AF recurrence after catheter ablation than 2D or other predictors. Three-dimensional global area strains  may help to predict the risk of AF recurrence and select patients with improved AF after catheter ablation [LV diastolic dysfunction is the underlying mechanism of both atrial fibrillation (AF) and HFpEF, and HF is often accompanied by AF comorbidities ,86,87,88ablation . A studyablation . Table 3RV function assessment is particularly vital in clinical practice, considering the fact that RV failure is the leading cause of death in patients with various cardiovascular diseases, including pulmonary arterial hypertension, RV MI, congenital heart disease, and HF ,92. The p < 0.01). Furthermore, 3D RVFWLS and 3D RVEF had comparable predictive ability to 2D RVFWLS for adverse cardiac events in multivariate Cox analysis models (akaike information criterion (AIC) = 246,247,248; C-index = 0.75,0.76,0.74; respectively). Hence, 3D-STE displays the potential to identify patients with HFpEF at high risk of poor clinical outcomes [p < 0.001) [p = 0.004; RVFWLS HR = 5.3, 95% CI 2.85–9.89, p = 0.002; respectively) were independently associated with higher mortality in these patients [In a retrospective cross-sectional study with 106 patients with underlying heart diseases who underwent both 3D echocardiography and CMR imaging within one month, researchers found that inlet area strain and outflow circumferential strain from 3D-STE were independently correlated with RV ejection fraction (RVEF) assessed by CMR, as a gold-standard imaging technique for quantifying cardiac function. Three-dimensional speckle-tracking echocardiography might be a promising method for assessing RV function . Two-dimoutcomes . In addioutcomes . Sun et < 0.001) . Vitarel< 0.001) . Both 3Dpatients . CollectEnd-stage Myocardial compensation is mostly characterized by collagen and fibrosis formation ,99. Patip < 0.001), which was found to be more accurate in predicting MF than 2D GLS and 3DTS in multivariate linear regression models . Furthermore, the AUC of 3D GLS in identifying severe MF was significantly greater than those of other strain parameters . Three-dimensional GLS might be an excellent surrogate marker for evaluating LV MF in patients with DCM and advanced HF, and will be useful for risk stratification, determining a time for early clinical intervention and assessing efficacy [p < 0.001) and correlated strongest with the MF degree  compared with 2D-STE and other conventional RV function parameters. In addition, among all RV parameters involved in this study, 3D RVFWLS was the most accurate parameter  for detecting severe MF. Consequently, 3D RVFWLS may be the most reliable echocardiographic parameter to predict the degree of RV MF in end-stage HF patients, especially those who need serial follow-up or are unable to undergo CMR examinations [MF was previously recognized by endomyocardial biopsy as an invasive method . Recentlefficacy . Anotherefficacy . Three-dinations .HF, as the terminal stage in the progression of various cardiovascular diseases, has become a significant problem threatening public health worldwide. Therefore, accurate assessment of cardiac function has vital clinical value in definitive diagnosis, clinical decision-making, risk stratification, and serial follow-up of HF patients. Three-dimensional speckle-tracking echocardiography serves as a novel imaging technology and has been increasingly applied in the research and clinical areas due to its advantages of excellent accuracy, high efficiency, low cost, and 3D analysis. With the continuous improvements in STE technology, increasing resolution, and image quality, 3D-STE can be considered to be a promising tool in evaluating cardiac function and providing accurate diagnostic and prognostic information in clinical practice.


Analyzing the expression of 82 fibrosis related genes in 65 heart failure (HF) patients, we identified a panel of 12 genes clearly distinguishing HF patients better from healthy controls than measurement of the collagen-related hydroxyproline content. A subcluster enriched in ischemic HF was recognized, but not for diabetes, high BMI, or gender. Single-cell transcriptomic analysis of in vitro activated mouse cardiac fibroblasts distinguished 6 subpopulations, including a contractile Acta2high precursor population, which was predicted by time trajectory analysis to develop into Acta2low subpopulations with high production of extracellular matrix molecules. The 12 gene profile identified in HF patients showed highest similarity to the fibroblast subset with the strongest expression of extracellular matrix molecules. Population markers identified were furthermore able to clearly cluster different disease stages in a murine model for myocardial infarct. These data suggest that major features of cardiac fibroblast activation in heart failure patients, in murine heart disease models, and in cell culture of primary murine cardiac fibroblast are shared.Many heart diseases are associated with fibrosis, but it is unclear whether different types of heart disease correlate with different subtypes of activated fibroblasts and to which extent such diversity is modeled during  There is a strong correlation between fibrosis and heart failure . Myocardin vivo, how is this heterogeneity reflected in conventional cell culture of primary mouse fibroblasts? Are fibroblast subpopulations observed in vitro and, if yes, do they correlate with fibrosis subtypes of heart-failure patients? Recently, we showed that myofibroblast features, such as smooth muscle alpha actin (Acta2) fibers and expression of genes involved in extracellular matrix (ECM) remodeling (Col1a1 and Lox), started to decline after 9–12 days in culture  and the Independent Research Fund Denmark [0135-00243B]. The work was also supported by Independent Research Fund Denmark (DFF-0199-00001B) to AL.KMH has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 795390. KJW is supported by The The authors acknowledge support from the Gill Cardiovascular Biorepository at the University of Kentucky and from the patients, organ donors, and families that donated samples. Funding: NIH TR033173, HL133359, HL146676 (KSC), AHA TP135689 (KSC), and Penny Warren Award for Clinical and Translational Research.Kate Møller Herum , Guangzheng Weng , Konstantin Kahnert , Rebekah Waikel (Resources), Greg Milburn (Resources), Autumn Conger (Resources), Paul Anaya (Resources), Kenneth S. Campbell (Resources), Alicia Lundby , Kyoung Jae Won , Cord Brakebusch .The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kate Herum reports financial support was provided by Marie Sklodowska-Curie grant (AU Horizon 2020). Kyoung Jae Won reports financial support was provided by Novo Nordisk Foundation. Alicia Lundby reports financial support was provided by Independent Research Fund Denmark. Kenneth S. Campbell reports was provided by National Institutes of Health. Kyoung Jae Won reports financial support was provided by Independent Research Fund Denmark.