The Zebrafish (Danio rerio) Is a Relevant Model for Studying Sex-Specific Effects of 17β-Estradiol in the Adult Heart

Cardiovascular diseases are a major cause of morbidity and mortality, and there are significant sex differences therein. However, the underlying mechanisms are poorly understood. The steroid hormone 17β-estradiol (E2) is thought to play a major role in cardiovascular sex differences and to be protective, but this may not hold true for males. We aimed at assessing whether the zebrafish is an appropriate model for the study of E2 effects in the heart. We hypothesized that E2 regulates the cardiac contractility of adult zebrafish in a sex-specific manner. Male and female zebrafish were treated with vehicle (control) or E2 and the cardiac contractility was measured 0, 4, 7 and 14 days after treatment initiation using echocardiography. There was no significant effect on the heart rate by E2. Notably, there was a significant decrease in the ejection fraction of male zebrafish treated with E2 compared with controls. By contrast, there was no major difference in the ejection fraction between the two female groups. The dramatic effect in male zebrafish occurred as early as 4 days post treatment initiation. Although there was no significant difference in stroke volume and cardiac output between the two male groups, these were significantly higher in female zebrafish treated with E2 compared with controls. Gene expression analysis revealed that the levels of estrogen receptors were comparable among all groups. In conclusion, our data demonstrate that the adult zebrafish heart robustly responds to E2 and this occurs in a sex-specific manner. Given the benefits of using zebrafish as a model, new targets may be identified for the development of novel cardiovascular therapies for male and female patients. This would contribute towards the realization of personalized medicine

The thalamus and its subnuclei—a gateway to obsessive-compulsive disorder

Larger thalamic volume has been found in children with obsessive-compulsive disorder (OCD) and children with clinical-level symptoms within the general population. Particular thalamic subregions may drive these differences. The ENIGMA-OCD working group conducted mega- and meta-analyses to study thalamic subregional volume in OCD across the lifespan. Structural T-1-weighted brain magnetic resonance imaging (MRI) scans from 2649 OCD patients and 2774 healthy controls across 29 sites (50 datasets) were processed using the FreeSurfer built-in ThalamicNuclei pipeline to extract five thalamic subregions. Volume measures were harmonized for site effects using ComBat before running separate multiple linear regression models for children, adolescents, and adults to estimate volumetric group differences. All analyses were pre-registered (https://osf.io/73dvy) and adjusted for age, sex and intracranial volume. Unmedicated pediatric OCD patients (<12 years) had larger lateral (d = 0.46), pulvinar (d = 0.33), ventral (d = 0.35) and whole thalamus (d = 0.40) volumes at unadjusted p-values <0.05. Adolescent patients showed no volumetric differences. Adult OCD patients compared with controls had smaller volumes across all subregions (anterior, lateral, pulvinar, medial, and ventral) and smaller whole thalamic volume (d = -0.15 to -0.07) after multiple comparisons correction, mostly driven by medicated patients and associated with symptom severity. The anterior thalamus was also significantly smaller in patients after adjusting for thalamus size. Our results suggest that OCD-related thalamic volume differences are global and not driven by particular subregions and that the direction of effects are driven by both age and medication status

Impaired in vitro growth response of plasma-treated cardiomyocytes predicts poor outcome in patients with transthyretin amyloidosis

Objectives!#!Direct toxic effects of transthyretin amyloid in patient plasma upon cardiomyocytes are discussed. However, no data regarding the relevance of this putative effect for clinical outcome are available. In this monocentric prospective study, we analyzed cellular hypertrophy after phenylephrine stimulation in vitro in the presence of patient plasma and correlated the cellular growth response with phenotype and prognosis.!##!Methods and results!#!Progress in automated microscopy and image analysis allows high-throughput analysis of cell morphology. Using the InCell microscopy system, changes in cardiomyocyte's size after treatment with patient plasma from 89 patients suffering from transthyretin amyloidosis and 16 controls were quantified. For this purpose, we propose a novel metric that we named Hypertrophic Index, defined as difference in cell size after phenylephrine stimulation normalized to the unstimulated cell size. Its prognostic value was assessed for multiple endpoints (HTX: death/heart transplantation; DMP: cardiac decompensation; MACE: combined) using Cox proportional hazard models. Cells treated with plasma from healthy controls and hereditary transthyretin amyloidosis with polyneuropathy showed an increase in Hypertrophic Index after phenylephrine stimulation, whereas stimulation after treatment with hereditary cardiac amyloidosis or wild-type transthyretin patient plasma showed a significantly attenuated response. Hypertrophic Index was associated in univariate analyses with HTX (hazard ratio (HR) high vs low: 0.12 [0.02-0.58], p = 0.004), DMP: (HR 0.26 [0.11-0.62], p = 0.003) and MACE (HR 0.24 [0.11-0.55], p &amp;lt; 0.001). Its prognostic value was independent of established risk factors, cardiac TroponinT or N-terminal prohormone brain natriuretic peptide (NTproBNP).!##!Conclusions!#!Attenuated cardiomyocyte growth response after stimulation with patient plasma in vitro is an independent risk factor for adverse cardiac events in ATTR patients

Advanced Echocardiography in Adult Zebrafish Reveals Delayed Recovery of Heart Function after Myocardial Cryoinjury

<div><p>Translucent zebrafish larvae represent an established model to analyze genetics of cardiac development and human cardiac disease. More recently adult zebrafish are utilized to evaluate mechanisms of cardiac regeneration and by benefiting from recent genome editing technologies, including TALEN and CRISPR, adult zebrafish are emerging as a valuable in vivo model to evaluate novel disease genes and specifically validate disease causing mutations and their underlying pathomechanisms. However, methods to sensitively and non-invasively assess cardiac morphology and performance in adult zebrafish are still limited. We here present a standardized examination protocol to broadly assess cardiac performance in adult zebrafish by advancing conventional echocardiography with modern speckle-tracking analyses. This allows accurate detection of changes in cardiac performance and further enables highly sensitive assessment of regional myocardial motion and deformation in high spatio-temporal resolution. Combining conventional echocardiography measurements with radial and longitudinal velocity, displacement, strain, strain rate and myocardial wall delay rates after myocardial cryoinjury permitted to non-invasively determine injury dimensions and to longitudinally follow functional recovery during cardiac regeneration. We show that functional recovery of cryoinjured hearts occurs in three distinct phases. Importantly, the regeneration process after cryoinjury extends far beyond the proposed 45 days described for ventricular resection with reconstitution of myocardial performance up to 180 days post-injury (dpi). The imaging modalities evaluated here allow sensitive cardiac phenotyping and contribute to further establish adult zebrafish as valuable cardiac disease model beyond the larval developmental stage.</p></div

Prognostic Value of Standard Heart Failure Medication in Patients with Cardiac Transthyretin Amyloidosis

Introduction: Cardiac transthyretin amyloidosis (ATTR) is a progressive, fatal disease leading to heart failure due to accumulation of amyloid fibrils in the interstitial space and may occur as a hereditary (ATTRv) or wild-type (ATTRwt) form. Guidelines recommend the use of ACE inhibitors (ACEis) and beta-blockers (BBs) as heart failure therapy (HFT) in all patients with symptomatic heart failure and reduced ejection fraction, independent of the underlying etiology. However, the prognostic benefit of ACEis and BBs in ATTR has not been elucidated in detail yet. We thus sought to retrospectively investigate the outcome of patients with ATTRwt or ATTRv under HFT. Methods: Medical records of 403 patients with cardiac ATTR (ATTRwt: n = 268, ATTRv: n = 135) were screened for long-term medication as well as clinical, laboratory, electrocardiographic and echocardiographic data. Patients were assessed between 2005 and 2020 at the University Hospital Heidelberg. Kaplan–Meier analysis was used to analyze potential differences in survival among different subgroups. Results: The mean follow-up was 28 months. In total, 43 patients (32%) with ATTRv and 140 patients (52%) with ATTRwt received HFT. Survival was significantly shorter in patients receiving HFT in ATTRv (46 vs. 83 months, p = 0.0007) vs. non-HFT. A significantly better survival was observed in patients with comorbidities (coronary artery disease, arterial hypertension) and HFT among ATTRwt patients (p = 0.004). No significant differences in survival were observed in the other subgroups. Conclusions: Survival analysis revealed a potential benefit of HFT in patients with ATTRwt and cardiac comorbidities such as coronary artery disease and/or arterial hypertension. In contrast, HFT should be used with caution in patients with ATTRv

Changes in cardiac performance in response to beta-adrenergic stimulation and blockage.

<p>(<b>A</b>) Representative pulsed-wave Doppler (PWD) signals (upper row) acquired from ACX view illustrating reduced and elevated heart rate in Atenolol (middle, AT) and Isoproterenol (right, Iso) treated zebrafish, respectively, compared to controls (left). Representative SAX images (lower row) with demarcated end-diastolic (red) and end-systolic (green) ventricular dimension after Atenolol and Iso treatment. (<b>B-E</b>) Quantification of changes in heart rate (HR) (<b>B</b>), fractional area change (FAC) (<b>C</b>), fractional shortening (FS) (<b>D</b>), and ejection fraction (EF) (<b>E</b>) after AT or Iso treatment compared to controls (C). Small numbers in columns indicate number of animals measured. (<b>F</b>) For speckle-tracking analysis, the ventricle was divided in six segments as indicated with segment 1 and 2 representing the anterior wall, 3 and 4 the apex, and 5 and 6 the posterior wall. Time-to-peak analysis reveals that AT leads to significantly prolonged time-to-peak indices in myocardial velocity of all segments. Small numbers in columns indicate number of animals measured. Values are expressed as mean ± SEM. Ap, Apex; AT, Atenolol; AW, anterior wall; Iso, Isoproterenol; PW, posterior wall; <b>*</b>, p<0.05; unpaired student’s t-test.</p

Longitudinal echocardiographic evaluation of cardiac function after cryoinjury.

<p>(<b>A</b>) Lateral brightfield (top) and fluorescent (bottom) images of hearts derived from sham operated transgenic zebrafish [<i>Tg(myl7</i>:<i>GFP)</i>^<i>f1</i>] and after cryoinjury at depicted time points. Dashed lines indicate injured myocardial area (i). (<b>B</b>) PWD recordings from sham (top) and cryoinjured zebrafish at 1dpi (bottom) demonstrating decreased A-wave and increased E-wave amplitudes indicative for diastolic dysfunction. (<b>C</b>) Representative SAX images from sham (upper row) and cryoinjured zebrafish at 1dpi (middle row) and at 30dpi (lower row) with end-diastolic dimensions illustrated in red and end-systolic dimensions in green. (<b>D-I</b>) Quantification of changes in (<b>D</b>) heart rate (HR), (<b>E</b>) fractional shortening (FS), (<b>F</b>) fractional area change (FAC), (<b>G</b>) ejection fraction (EF), (<b>H</b>) E/A ratio and (<b>I</b>) cardiac output (CO) at baseline and at indicated time points during regeneration after myocardial injury. Small number in (D) indicates number of animals analyzed (<b>J</b>) Speckle-tracking analysis of segmental displacement shows akinesia of injured (green, pink and light blue line) as compared to the non-injured segments (yellow, purple and dark blue line). The average curve of all segments is illustrated in black. For color coding of different segments see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122665#pone.0122665.g002" target="_blank">Fig 2B</a>. Values are expressed as means ± SEM; a, atrium; i, injured area; ot, outflow tract; v, ventricle; AW, anterior wall; PW, posterior wall; *, p<0.05; unpaired student’s t-test and ANOVA with post hoc comparisons by Bonferroni’s multiple comparison test.</p

Advanced 2D wall motion in adult zebrafish hearts by speckle-tracking.

<p>(<b>A</b>) Representative LAX view with ventricular boundary outlined in pink. (<b>B</b>) Ventricle was divided into six color-coded segments as indicated with the green and light pink segment corresponding to the base of the anterior wall (AW), light blue and purple segments including the apex (Ap) and the dark blue and yellow segment corresponding the posterior wall (PW) base. (<b>C-F</b>) Representative segmental velocity traces (<b>C</b>), displacement traces (<b>D</b>), strain curves (<b>E</b>), and strain rate curves (<b>F</b>) for corresponding segments are shown. The black trace indicates the calculated average. (<b>G</b>) For high-resolution speckle-tracking representation the demarcated ventricular boundary was stretched as indicated with the top region representing the anterior wall (AW), the central region the Ap and the lower region the PW. (<b>H-K</b>) Absolute activity heat-maps of regional velocity (<b>H</b>), regional displacement (<b>I</b>), regional strain (<b>J</b>), and regional strain rate (<b>K</b>) of individual speckles during time (t) was plotted in a color coded fashion with high values shown in light red and low values in light blue as indicated. Notice that individual systoles (sy) and diastoles are distinguishable. White arrows in (<b>H</b>) indicate regions of high velocity in the AW in two distinct sy. Noticeably, AW activity is higher compared to the PW and Ap. Time scale bar as indicated. Ap, Apex; AW, anterior wall; PW, posterior wall; sy, systole.</p

Advanced 2D wall motion measurement by speckle-tracking analysis after cryoinjury reveals segmental and regional motion and deformation disturbances.

<p>(<b>A</b>) Absolute radial displacement of individual segments at indicated time points. The ventricle was divided in six segments as indicated with segment 1 and 2 representing the anterior wall (AW), 3 and 4 the apex (Ap), and 5 and 6 the posterior wall (PW) for subsequent displacement analysis. Small numbers indicate number of animals measured. (<b>B</b>) Average of radial displacement at indicated time points. (<b>C</b>) High resolution speckle-tracking analysis of radial (upper row) and longitudinal (lower row) displacement. The top region displays displacement of the AW, the central region of the Ap and the lower region of the PW. Absolute values are color coded with high values in light red and low values in light blue as indicated. Time scale and color coding bar as indicated. The pink line at 4dpi depicts injured area (MI). (<b>D</b>) 3D reconstruction of regional displacement at indicated time points enables identification of akinesis of injured AW at 4dpi and residual wall motion deficiencies at 60dpi, respectively. The lower right image shows a schematic illustration of the 3D-reconstrations. The u-shaped pink lines indicate consecutive systoles; t and the arrow below indicate the time progress and MI the infarcted area, also indicated by the bold pink line. (<b>E</b>) Modified AFOG-staining (myocardium in red, connective tissue and fibrotic areas in blue) stained sections of a sham operated control heart and at 120 and 180dpi. AW is to the right, PW to the left. Boxed area of the cryo-injured region is shown in higher magnification in the lower left corner of its respective overview picture. At 120dpi residual fibrotic deposition (arrows) together with a thickening of the compact myocardial layer (*) can be detected. Residues of fibrosis are still detectable at 180dpi. Note that the thickening of the compact myocardial layer (*) extends over a great segment of the AW. Ap, apex; A, atrium, AVV, atrio-ventricular valve; AW, anterior wall; B, bulbus arteriosus; MI, myocardial injury; PW, posterior wall; V, ventricle; VBV, ventriculo-bulbar valve. Values are expressed as means ± SEM; *, p<0.05; **, p<0.01, unpaired student’s t-test and ANOVA with post hoc comparisons by Bonferroni’s multiple comparison test</p