bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 100: G-Protein Coupled Estrogen Receptor1 Prevents Mitochondria Inner Membrane Protein (Mitofilin) Degradation after Ischemia/Reperfusion
Authors: Harris Tanoya L , Olde Bjorn , Leeb-Lundberg Fredrik L , Bopassa Jean C. 
Published: July 2021

We recently found that estrogen treatment delays the mitochondrial permeability transition pore opening and reduces ROS production after ischemia/reperfusion, suggesting that estrogen promotes mitochondrial integrity. As mitochondrial inner membrane protein (mitofilin) has been found to control mitochondrial cristae morphology and function, we investigated whether estrogen effect on mitochondrial integrity after ischemia/reperfusion involved regulation of mitofilin via activation of G-Protein Coupled Estrogen Receptor1 (GPER1).
Isolated hearts from male WT (C57BL/6NCrL), and GPER1-/- mice were perfused using Langendorff technique, with and without estrogen (40 nM). Hearts were subjected to 20 min global ischemia followed by 10 min reperfusion. Mitochondria were isolated, and 2D-DIGE followed by mass spectrometry was performed. Mitofilin expression level was confirmed using Western blot analysis in mitochondrial fractions. Mitofilin distribution was visualized using confocal microscopy in isolated cardiomyocytes, and its spatial organization in mitochondria was imaged using high resolution fluorescence microscopy. Electron microscopy was used to observe mitochondrial cristae morphology.
We obtained 52 unique proteins of interest, in which mitofilin was identified. Immunoblot analysis confirmed the increased in mitofilin level with estrogen treatment as compared to control in WT but not in GPER1 KO. Cardiomyocyte images revealed as observed in non-ischemic myocytes that estrogen treatment conserved mitofilin distribution in the peri-membrane and T-tubules, while only peri-membrane mitofilin was more visible in control group. High resolution microscopy showed a better spatial organization of mitofilin in single mitochondria with estrogen treatment compared to control, in which mitofilin was almost absent. Electron microscopy showed that mitochondrial morphology was conserved in estrogen-treated group as cristae were well organized compared to control, in which cristae were disrupted.
These data indicate that estrogen action induces regulation of mitofilin expression during ischemia/reperfusion. Estrogen effect on mitofilin may contribute to improved mitochondrial integrity and function.
Abstract 165: The Transcriptional Cofactor Eyes Absent 4 Is A Critical Regulator In Cardiac Physiology
Authors: Williams Tatjana , Hundertmark Moritz , Nordbeck Peter , Voll Sabine , Elsner Ines , Schoenberger Jost , Ritter Oliver 
Published: July 2021

Introduction:
Eyes absent 4 (Eya4) is a transcription cofactor involved in a number of cellular and developmental processes. We have previously shown that a truncating mutation in Eya4, E193, causes hearing impairment followed by terminal heart failure suggesting Eya4 is a regulator in cardiac physiology.

Methods and Results:
Transgene- or adenovirus mediated overexpression of Eya4 or E193 altered the expression of p27kip1, a critical mediator of cardiac hypertrophy in adult cardiomyocytes.

Luciferase reporter and EMSA assays revealed that Eya4 directly binds to and suppresses p27kip1 promoter activity, while E193 exerts an opposing effect, respectively. Activity and phosphorylation of downstream molecules were significantly altered in Eya4 and E193 transgenic (TG) mice in a contradictory manner. Cardiac phenotypes evolved in both TG models already under basal conditions. Eya4 TG hearts developed hypertrophy as judged by increases in heart weight and cross-sectional cell surfaces and re-activation of fetal genes as well as fibrosis. E193 TG animals showed onset of DCM along with wall thinning, ventricular dilation, fibrosis and slightly compromised cardiac function. These two distinct cardiac phenotypes were even more aggravated upon pressure overload or Angiotensin II infusion.
Finally, we just recently identified a new mutation in Eya4, E215, which also causes hearing impairment and heart failure.

Conclusion:
Our data indicate that Eya4, in a physical complex with Six1, plays a critical role in regulating normal cardiac function via p27/CK2-α/HDAC2 and allude that mutations within the Exa4/Six1 transcriptional complex interfere with this newly established signalling pathway, finally leading to age-related onset of cardiomyopathy.

Clinical Perspective:
Gaining a better understanding of this disease mechanism could help identify new treatment options for heart failure patients.
Abstract 6: Formation of Human Heart Muscle Directly from Embryonic and Induced Pluripotent Stem Cells
Authors: Hudson James E , Tiburcy Malte , Zimmermann Wolfram-Hubertus 
Published: July 2021

Tissue engineering enables the simulation of human heart physiology and pathology. It typically requires a mixture of cardiomyocytes, stromal cells, and extracellular matrix for fabrication. Here, we hypothesised that bioengineered heart muscle (BHM) can be formed directly from undifferentiated pluripotent stem cells by triggering processes of embryonic cardiogenesis.

Methods and Results:
We tested our hypothesis by applying an optimized serum-free cardiac differentiation protocol to undifferentiated pluripotent stem cells in a collagen type 1 hydrogel. During this process BHMs traversed through distinct developmental stages: early mesoderm (3 days), cardiac specification (10 days), and cardiac maturation (up to 50 days). Flow cytometry demonstrated that BHMs are comprised of primarily cardiomyocytes (α-actinin positive cells, 51 ± 5%, n = 6) and stromal cells (CD90 positive cells, 41 ± 5%, n = 6), with low yields of contaminating cells. By 22 days the BHMs exhibited measurable contractile force (207 ± 19 μN) and contained elongated cross-striated cardiomyocytes.

We next sought to optimize the force of contraction and also the maturity of the BHM, by investigating the effect of mechanical stimuli and growth factors. Mechanical stimulation was essential for BHM formation. Additionally, we found that 2 developmentally important growth factors, FGF2 and TGFβ1, induced pathological and physiological hypertrophy, respectively. This was characterized by an increase in cell size in both conditions coupled with reduced force and higher ANP expression in FGF2 treated BHM, and a higher force with reduced ANP expression and elevated β-MHC/α-MHC ratio TGFβ1 treated BHM.
Using our optimized protocol, 28 day old BHM responded to electrical pacing, preloading, and inotropic stimuli similarly as bona fide myocardium.

Conclusion:
BHM can be formed directly with undifferentiated pluripotent stem cells by recapitulating normal cardiac development. The serum-free protocol with developmentally defined stimuli provides us with a useful in vitro model to study cardiac biology and potentially provides a method of producing cardiac tissue for regenerative applications.
Abstract 65: Regulation of Cardiac Hypertrophy and Dilated Cardiomyopathy by CIP
Authors: Huang Zhan-Peng , Kataoka Masaharu , Chen Jinghai , Wang Da-Zhi 
Published: July 2021

Cardiac hypertrophy is one of the primary responses of the heart to pathophysiological stress. However, the mechanism of the transition from compensative hypertrophic growth to cardiac dilation is poor understood. Recently, we identified a cardiac-specific expressed gene CIP. The expression of CIP is unchanged in hypertrophic heart but significantly down-regulated in dilated hearts, suggesting CIP may play an important role in the transition from cardiac hypertrophy to dilated cardiomyopathy.
We generated CIP knockout mice and found that CIP is dispensable for cardiac development. Interestingly, CIP-null mutant mice developed severe cardiac dilation 4 weeks after TAC (transverse aortic constriction) surgery, while control mice were still at the stage of compensative hypertrophic growth. Echocardiography and histological examinations showed that mutant hearts had enlarged chamber with thinner ventricle wall and decreased cardiac performance compared to controls. The expression of marker genes of cardiac disease, BNP and Myh7, was elevated. Consistently, deletion of CIP in Myh6-CnA transgenic mice result in premature death, displaying severe left ventricle dilation.
Conversely, cardiac-specific CIP overexpression inhibited pressure overload-induced cardiac hypertrophy. CIP transgenic mice exhibit decreased ventricle weight/body weight ratio, decreased cardiomyocyte cross-section area and repressed expression of hypertrophic related marker genes. CIP overexpression also protected the heart from developing cardiac dilation and preserved the cardiac function after prolonged pressure overload.
We performed unbiased microarray assay to document the transcriptome in CIP knockout and control mice which were subjected to pressure overload (TAC). The analysis of Gene Ontology term indicated the Negative Regulation of Apoptosis was down-regulated while the Collagen/Extracellular Structure Organization was up-regulated in CIP-null hearts under TAC condition.
In summary, our studies established CIP as a key regulator of the transition from cardiac hypertrophy to dilated cardiomyopathy. The protective effect of CIP in cardiac remodeling indicates that CIP could become a therapeutic target for cardiac diseases.
Abstract 200: Novel Cardiac Dyssynchrony Metrics Based on Ultrasound Images and Fibrosis
Authors: Shearer Greg , Hanson Todd , Eclov Julie , Courtier Sam , Ortmeier Steve , Ewert Dan 
Published: July 2021

Ventricular stiffening is a component of heart failure with preserved ejection fraction. This study focuses on building a dyssynchrony metric based on ultrasound images obtained from normal and fibrotic mouse hearts. The objective is to evaluate the association between regional fibrosis and ventricular wall dynamics as an indicator for the gradient of cardiac dyssynchrony.
Ultrasound images were obtained from a previous study in which mice were given thoracic aortic constriction to induce heart failure that included ventricular stiffening and fibrosis. Ultrasound video (frame rate 4.3ms) along the short axis of the mouse heart was captured using commercial software. On each short axis frame, six points were selected and traced on the endocardial wall. Each radial displacement pair is plotted in a loop diagram against one another in 15 unique combinations.

A loop whose points are close to the
ideal line
u = v
corresponds to synchronous wall movement, while the area enclosed by the loop is proportional to time dyssynchrony. The inferior free wall and the section nearest the interventricular septum had 35% (26,48) interstitial fibrosis compared to the IS itself which had 9% (7, 13) in hearts with 25% fibrosis, and the contrast had the most variance (8.7%, P<0.0001). Strong predictors of the contrasting fibrosis included the trace perimeter and the maximal normalized displacement, but more pragmatically, the differences between the two conditions are apparent and consistent.

Figure 1 is the set of (u,v) pairs measured over the course of a heartbeat where u is the radial displacement of heart segment x measured when segment y had radial displacement v.
Abstract 366: Disease Phenotype Assessment Across a Library of iPSC-Derived Cardiomyocytes From Patient Cohorts Carrying Distinct Mutations for Familial Hypertrophic Cardiomyopathy
Authors: Tsai Jason , Lam Jason , Sanchez-Freire Veronica , Gadkari Rishali , Agarwal Maya , Bian Jing , Huang Guanyi , Magal Ashita , Lan Feng , Lee Andrew S 
Published: July 2021

Familial hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in the young, and is the most common inherited heart defect affecting 1 in 500 individuals worldwide. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have been demonstrated to model aspects of HCM, but only one iPSC model has been reported for a single HCM mutation in one gene. Here we compare disease phenotypes across a library of patient-specific HCM iPSC-CMs carrying distinct mutations to assess the range of phenotypes that may present in iPSC-CMs derived from different patient cohorts. iPSCs were generated from three patient cohorts carrying known hereditary mutations for HCM in TNNI3, TNNT2, and MYH7 and family-matched controls. Disease phenotypes in patient-specific iPSC-CMs were modeled using immunostaining, Ca2+ imaging, multielectrode array, and video analysis of contractile motion. HCM iPSC-CMs displayed a range of disease phenotypes as assessed by cell size, Ca2+ homeostasis, electrophysiology, and contractile arrhythmia. Different HCM mutations resulted in distinct disease phenotype presentation. Importantly, identical mutations demonstrated similar readouts across multiple lines and clones whereas distinct mutations exhibited differential disease phenotypes. These findings indicate disease-specific iPSC-CMs present with a range of phenotypes for HCM that vary by specific mutation and that iPSC libraries are important for cellular characterization of diseases such as HCM.
Figure 1. Derivation and disease phenotype modeling of iPSC-CMs generated from patients carrying distinct familial HCM mutations and family-matched controls.
Abstract 371: Down Regulation of ECM Genes Laminin and Collagen IV Lead to Preserved Cardiac Function With Age and Increased Lifespan in Drosophila
Authors: Sessions Ayla O 
Published: July 2021

Increased deposition of extracellular matrix (ECM) is observed in all advanced age heart failure patients, but current model systems are complex and slow to age. To investigate the effect of extracellular remodeling on mechanical function in genetically tractable, rapidly aging, and simple model organisms, we employed Drosophila melanogaster, which has a simple trilayered heart tube.
We found that two common wildtype strains of Drosophila, i.e. yellow-white (yw) and white-1118 (w1118), exhibit different cytoskeletal and ECM remodeling with age. Using a recently developed nanoindentation method to measure cardiomyocyte stiffness and high speed optical imaging to assess contractility of intact Drosophila hearts, we found that yw flies had stiffer intercalated discs (ICD) and exhibited diastolic dysfunction with age. On the other hand, w1118 flies had a shorter lifespan compared to yw, did not exhibit ICD stiffening, had a less severe diastolic dysfunction, and showed an increase in ECM layer thickness between ventral muscle (VM) and cardiomyocyte (CM) layers of the heart tube. To modulate ECM and assess its effect in the aged w1118 flies, we knocked-down ECM genes LamininA and Viking (homologous to Collagen IV). Both ECM KD genotypes exhibited diastolic dilation with increased fractional shortening at adult (1wk) and aged (5wk) time points. The LamininA KD resulted in decreased cardiomyocyte stiffness correlating with increased relaxation velocities in adult flies and preservation of shortening and relaxation velocities in aged flies over controls. However, both the LamininA and Collagen IV KD flies experienced a basal increase in the decoupling of their cardiomyocytes as determined by heart period variance and % fibrillar heart-beats. These conductance issues were not enough to counteract the increased cardiac output and performance with age, and the Collagen IV KD outlived controls by 1.5 weeks median survival and the LamininA KD by 3 weeks. This suggests that the cell-ECM contacts in the basement membrane are intimately tied not only to the coupling of the cardiomyocytes of the Drosophila heart tube but also to cytoskeletal remodeling, but perhaps different ECM proteins have different mechanisms for interacting with the cardiomyocyte cytoskeleton.
Abstract 383: 17 beta-Estradiol Enhances Mitochondrial Function of Cerebral Arteries From Ovariectomized Female Rats
Authors: Rutkai Ibolya , Dutta Somhrita , Walter Korey A , Katakam Prasad V , Busija David W 
Published: July 2021

Previous studies have provided indirect evidence that circulating sex hormones alter the function of the cerebral circulation perhaps via effects on mitochondrial dynamics. However, effects of estrogen on mitochondrial respiration have never been directly examined. We have previously observed a difference in the mitochondrial function of cerebral arteries from male and female rats but the exact mechanisms are not clear.
We tested the hypothesis that mitochondrial respiration in isolated cerebral arteries from ovariectomized (OVX) Sprague Dawley rats treated with a 21 d release, 0.5 mg of 17 β-estradiol pellet (OVX+E) was enhanced compared with arteries from placebo treated OVX rats. The Seahorse Bioscience XFe24 system was used to measure mitochondrial oxygen consumption rate (pM/min/μg protein) in cerebral arteries. Western blot was used to investigate the arterial expression of proteins. Radioimmunoassay was used to measure serum estradiol level.
Treatment with 17 β-estradiol resulted in a higher serum estradiol level (146.9±18.16 pg/ml) and uterus weight (0.15±0.0058 g) in the OVX+E compared with the OVX (14.7±1.2 pg/ml, 0.07±0.003, respectively; p<0.05). The components of mitochondrial respiration in pM/min/μg protein including basal respiration (147±9), ATP production (44±4), proton leak (102±7), and maximal respiration (212±13) were elevated in OVX+E compared with OVX (105±13, 21±4, 50±6, 138±10, respectively; p<0.05). Expression of the mitochondrial DNA encoded Complexes I and III, the nuclear DNA encoded Complexes II, IV, V, and the voltage-dependent anion channel protein were similar in all groups. However, the ratios of phosphorylated endothelial and neuronal nitric oxide (NO) synthase normalized to total protein were significantly (p<0.05) elevated in the OVX+E (1.43±0.06, 1.15±0.2, respectively) compared with OVX group (0.92±0.06, 0.53±0.19, respectively).
Our findings provide direct evidence for sex-specific differences in mitochondrial function on freshly isolated cerebral arteries. Thus, estradiol replacement enhances the efficacy of the oxidative phosphorylation resulting in an increased mitochondrial respiration which is not due to increased mitochondrial protein expression but may be due to enhanced NO.
Abstract 5: Novel Tools for Identification and Purification of Cardiomyocytes, Fibroblasts and Endothelial Cells From Neonatal Mouse and Rat Hearts
Authors: Eckardt Dominik , Kernbach Manuel , Czichowski Vera , Hintzen Christoph , Bosio Andreas 
Published: July 2021

Although isolation of vital cardiac cells from neonatal hearts is one of the most used experimental model in cardiac research, the manual dissociation of neonatal hearts is a laborious and difficult-to-standardize procedure. To shorten sample processing time and increase reproducibility, we developed a fully automated dissociation process followed by magnetic purification of cardiomyocytes (CMs), cardiac fibroblasts (CFs) and endothelial cells (ECs). Neonatal hearts were dissociated within 1 h resulting in high yields of single cells with excellent viability. Depending on age and species following cell ratios were recovered after dissociation: 50-70% CMs, 30-40% CFs, 10-20% ECs.
In order to purify CMs from dissociated neonatal hearts, we defined antibody cocktails enabling non-myocyte removal. Our depletion strategy allowed for the enrichment of CMs from whole hearts or preparations of heart chambers with purities of up to 98%. To enable simultaneous detection of CMs and subtypes, we developed recombinant antibodies against general CM markers like alpha Actinin, Myosin Heavy Chain or cardiac Troponin T as well as CM subtype-specific antibodies against MLC2a and MLC2v distinguishing between atrial and ventricular CMs respectively.
Besides, we developed magnetic enrichment strategies for the purification of CFs and ECs. Current purification strategies that are solely based on antibodies against EC surface markers do not result in sufficiently pure cardiac ECs. Therefore, we established a 2-step enrichment protocol allowing for the complete removal of contaminating fibroblasts and leukocytes. Our data indicate that this protocol significantly improves the purity of primary cardiac ECs. Functionality of ECs before and after enrichment was proven by Dil-Ac-LDL endocytosis.
Similarly, our magnetic enrichment protocol for cardiac fibroblasts revealed homogenous expression of fibroblast markers like Vimentin and Prolyl-Hydroxylase, but virtually no contaminations with CMs or ECs.
In summary, we established an automated protocol for dissociation of neonatal hearts enabling subsequent purification, characterization and cultivation of CMs, CFs and ECs which can readily be used for cell culture assays or to generate heart muscle models.
Abstract 57: First Induced Heart Failure Model in Adult Zebrafish by Chronic Isoproterenol Treatment
Authors: Kossack Mandy , Katus Hugo A , Most Patrick , Hassel David 
Published: July 2021

Impaired β-adrenergic signal transduction is a common molecular alteration found in heart failure (HF). Chronic catecholamine release and overstimulation of β-adrenergic receptors (β-ARs) in the failing heart results in their desensitization, largely mediated through upregulation of the G-protein coupled receptor kinase 2 (GRK2), and consequently to the progression into HF. Chronic activation of β-ARs by isoproterenol (iso) infusion efficiently induces HF in mice, while therapeutic targeting of GRK2 in HF animal models preserves cardiac function, highlighting their significance in HF progression.
Zebrafish represents an established model to evaluate genetic causes of HF and to screen for novel therapeutic targets. However, the contribution of the β-A system in zebrafish models of HF is not known. We here systematically analyzed the effect of iso on heart function in larval and adult zebrafish.
Larvae first responded to iso with 3 days of age (d). Here, β-AR stimulation resulted in the activation of conserved signaling components and in the induction of common stress responsive genes. Chronic β-AR stimulation for 5 days induced signs of HF accompanied by similar expression changes seen in mammals. As heart phenotypes are usually not analyzed in larval zebrafish beyond 3d, our data implicates that previous studies neglected a possible impact of β-AR signaling, particularly important when screening for therapeutic components.
Adult zebrafish recently emerged as an attractive cardiac model, especially for regenerative medicine. Echocardiography revealed that in adult zebrafish hearts, iso robustly enhanced cardiac function. Chronic β-AR stimulation for 14 days efficiently induced HF symptoms. Consistent with mammals, we found reduced expression of β-ARs and elevated expression of GRK2 and ANP. Additionally, these fish develop essential characteristics accompanied with HF, including increased cell death and elevated inflammation.
In conclusion, we show that β-AR function in zebrafish is comparable to that in mammals. Further, we present the first iso-induced HF model in adult zebrafish, thereby introducing adult zebrafish as a particularly valuable model to study the pathogenesis of HF and to test for novel therapeutic strategies to treat HF.