bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 2: Automated Dissociation Of Neonatal Hearts And Immunomagnetic Purification Of Cardiomyocytes, Cardiac Fibroblasts And Endothelial Cells
Authors: Kernbach Manuel , Riesen Josephine , Wiencierz Anne Maria , Bosio Andreas , Christalla Peter , Eckardt Dominik 
Published: July 2021

Pure cardiovascular cell lineages are a requisite for an unbiased and cell-type specific investigation of (patho-) physiological mechanisms involved in heart failure. Moreover, pure heart cells are needed as building blocks for cardiac tissue engineering. Therefore, we developed a rapid and automated heart dissociation protocol followed by an immunomagnetic enrichment of the three major heart cell types, namely cardiomyocytes (CMs), cardiac fibroblasts (Fibs) and endothelial cells (Endos) from both, mouse and rat neonatal hearts.
First, we developed an enzyme mix optimal for the dissociation of neonatal mouse and rat hearts by screening our enzyme library. Next, we optimized the dissociation by utilizing our gentleMACS technology resulting in a fast (1 h), robust, and fully automated heart dissociation protocol. Analysis of the dissociated heart cells prior to cell purification showed: (i) high cell vitalities (>90%), (ii) high frequencies of α-actinin-positive CMs (ca. 60%) and (iii) vimentin-positive non-CMs with a frequency of ca. 40%. In order to selectively enrich various cell-types from these heterogenous cell populations, we performed a cell-surface marker screen. We identified several candidates for the composition of antibody cocktails enabling selective enrichment of mouse and rat CMs, Fibs or Endos with purities of up to 97%. CMs grown in 2D cultures showed spontaneous beating activity and the expression of sarcomeric proteins. Additionally, heart cells were cultured on decellularized mouse heart slices. Analysis of the slice cultures after recellularization showed (i) partial restoration of synchronous contractions and (ii) orientated and elongated heart cells. Purified Fibs were highly proliferative and could be expanded 34-fold over an analyzed culture period of 15 days. Finally, functionality of the purified endothelial cells was proven by Dil-LDL endocytosis.
In summary, we established an automated protocol for the dissociation of neonatal hearts, enabling the subsequent immunomagnetic enrichment of CMs, Fibs and Endos which can readily be utilized for 2D cell culture assays or to generate in vitro heart muscle models and surrogate tissue for myocardial repair.
Abstract 211: Automated and Non-invasive Estimation of QT Interval from Microscopy Images of Stem Cell-Derived Cardiomyocytes
Authors: Maddah Mahnaz , Loewke Kevin 
Published: July 2021

A promising application of induced pluripotent stem cells (iPSCs) is the generation of patient-specific cardiomyocytes (CMs), which can be used for drug development and safety testing related to cardiovascular health. iPSC-derived CMs can be used for preclinical testing of new drugs that may cause drug-induced arrhythmia or long QT syndrome, as well as post-market safety testing of existing drugs. The measurement of QT interval for iPSC-derived CMs is commonly analyzed using electrophysiological potentials captured by a micro-electrode array (MEA). While such systems are the current standard for characterization, they can be expensive and low-throughput, require high cell plating density, and due to the direct contact between cells and electrodes, may cause undesirable cellular response.
Here, we present a new method to non-invasively measure the QT-interval in iPSC-derived CMs using video microscopy and computer vision analysis. Our algorithms can reliably and automatically extract beating signal characteristics such as frequency, irregularity, and duration through image analysis of cardiomyocyte motion. Through a correlative study with MEA, we demonstrate that a non-invasive measurement of QT interval can be derived from the duration of visible cellular motion that occurs during contraction and relaxation. We also show that our system can accurately characterize the cellular response from the addition of compounds known to modulate beating frequency and irregularity. Our measurement technique is robust, automated, and requires no physical or chemical contact with the cells, making it ideal for cardiovascular drug development and cardiotoxicity testing.
Abstract 265: Uroetensin II Protects Cardiomyocytes from Apoptosis Induced by Oxidative Stress through Cystathionine-γ-Lyase (CSE)/Hydrogen Sulfide Pathway
Authors: Gong Hui , Chen Zhidan , Zhang Xiaoyi , Zhang Jie , Li Yang , Ye Yong , Zhang Guoping , Yang Chunjie , Zou Yunzeng 
Published: July 2021

Plasma UII has been observed to be raised in patients with acute myocardial infarction, a lower UII response is associated with more severe injury of myocardium, suggesting a possible cardioprotective role for this peptide. In the present study, we studied plasma UII concentration of thirty patients admitted to the Cardiology Department with acute myocardial infartion.The results showed that plasma UII was sharply increased in patients compared to that in health control within one week after admission. We then explore whether UII could protect cardiomyocytes from injury induced by oxidative stress. Cultured cardiomyocyte were treated with H2O2 to induce oxidative stress, and the influence of UII on H2O2-induced apoptosis was observed. The results showed that UII pretreatment significantly reduced the number of TUNEL-positive cardiomyocytes induced by H2O2, and it partly abolished the upregulation of pro-apoptotic protein Bax and the down-regulation of anti-apoptotic protein Bcl-2. siRNA targeted to urotensin receptor (UT) greatly inhibited these effects. H2S has been reported to exert protective effect on cardiomyocytes, we detected the effect of UII on H2S production and CSE (Major H2S-producing enzyme) expressions in cardiomyocytes exposed to H2O2.The present data revealed that UII increased the H2S production by enhancing the expression of CSE by activating the ERK signaling in cardiomyocytes exposed to H2O2. Si-CSE or ERK inhibitor not only greatly inhibited the upregulation of CSE or the phosphorylation of ERK induced by UII but also reversed UII-induced-upregulation of H2S production and anti-apoptosis in cadiomyocytes exposed to H2O2.
In conclusion, UII rapidly promoted the phosphorylation of ERK, increased CSE exression and induced H2S production, which in turn enhanced the p-ERK level to protect cardiomyocytes from apoptosis under ischemic or oxidative stress. The increased plasma UII level in patients may be critical for cardiac protection in patients at early-phase of acute myocardial infarction.
Abstract 306: A Novel PKCalpha Splicing Isoform in Signaling for Cardiac Hypertrophy
Authors: GAO CHEN , Gong Jianli , Steinberg Susan F , Wang Yibin 
Published: July 2021

RNA splicing is a significant post-transcriptional regulatory step to enrich the overall transcriptome complexity, yet its specific contribution to cardiac development and pathogenesis remains underexplored. Here, we performed a comprehensive deep RNA-seq analysis in murine heart to investigate the cardiac transcriptome at single exon-resolution. From the dataset, we have identified a previously un-annotated novel exon in the PKCα transcript ---PKCα-NE (Novel Exon). The novel exon encode a 16 amino acid insert adjacent to the PKCα turn motif. Interestingly, the splicing event is detected only in cardiac and skeletal muscle. Based on bioinformatics analysis, we have identified two highly conserved binding motifs for a cardiac splicing regulator_RBFox1. We further demonstrated that RBFox1 indeed functions as a positive regulator for the splicing event of this novel exon.
In vitro studies showed that the NE converts PKCα from a lipid-dependent enzyme to a constitutively active kinase with high levels of autocatalytic activity and activity toward peptide substrates. The NE also increases PKCα’s Km for peptide substrate. These changes in steady state kinetic parameters suggest that PKCα-NE would exert it’s cellular actions most prominently when co-localized with target substrates, either on protein scaffolds or in subcellular signaling compartments. In fact, immune-precipitation for individual PKCα spicing variants followed by Mass Spectrometry exposed a specific interaction between PKCα-NE and elongation factor (eEF1A1). PKCα-WT and PKCα-NE both stimulated cardiac hypertrophy to a similar level based on myocyte size measurement, but PKCα-NE selectively increased eEF1A1 phosphorylation in association with elevated protein translational activity in cultured cardiomyocytes.
In summary, we have identified a novel cardiac specific splicing variant of PKCα that is dynamically regulated during development and heart failure. Our finding has revealed a novel signaling pathway in cardiac hypertrophy involving a novel alternative splicing event of PKCα and its specific downstream effects on protein synthesis regulation.
Abstract 321: Obesity Causes Enhanced Sensitivity Of The Mitochondrial Permeability Transition Pore
Authors: Bernal-Ramírez Judith , Riojas-Hernández Adriana , Morales-Marroquín Flor E , Domínguez-Barragán Elvía M , Rodríguez-Mier David , García-Rivas Gerardo , Altamirano Julio , García Noemí 
Published: July 2021

Several mechanisms have been implicated in heart failure (HF) development due to obesity, including altered Ca2+ homeostasis and mitochondrial increased reactive oxygen species (ROS).
Besides their metabolic role, mitochondria are important cell death regulators, since their disruption induces apoptosis. The mitochondrial permeability transition pore (MPTP) formation is key in this process. Ca2+ and ROS are known inducers of MPTP, and mitochondria are the main ROS generators. However, it has not been demonstrated that MPTP formation is involved in cardiac cell death due to obesity. Therefore, the aim of this work was to determine whether Ca2+ alterations and/or MPTP opening underlie cardiac dysfunction.
We used obese Zucker fa/fa rats (32 weeks old), displaying concentric hypertrophy and cardiac dysfunction. We measured: i) Systolic and diastolic Ca2+ signaling in isolated myocytes, in basal conditions and upon β-adrenergic stimulation (β-AS), and ii) in vitro mitochondrial function: respiration, ROS production and MPTP opening.
We found that the main alteration in Ca2+ signaling in fa/fa myocytes was a decrease in SERCA Ca2+ removal capacity, since Ca2+ transient amplitude and spark frequency were unchanged. Furthermore, in fa/fa myocytes, β-AS response was preserved. On the other hand, fa/fa mitochondria respiration, in state 3 decreased, but was unchanged in state 4, when glutamate/malate were used as substrate, resulting in an small decrease in respiratory control. In addition, fa/fa mitochondria were more sensitive to MPTP opening, induced by Ca2+ and carboxyatractiloside (CAT). Moreover, fa/fa mitochondria showed increased H2O2 production, and in exposed thiol groups in the adenine nucleotide translocase, a regulatory MPTP component.
Since Ca2+ signaling is relatively normal in fa/fa cells, it does not seem to be the main contributor to the cardiac contractile dysfunction. However, given that fa/fa mitochondria showed decrease respiratory performance, were more susceptible to MPTP opening, and showed enhanced H2O2 production. We conclude that fa/fa mitochondria were more vulnerable to enhanced oxidative stress, causing MPTP opening, which could be exacerbated by SERCA slower Ca2+ removal capacity, leading to myocyte apoptosis.
Abstract 352: Pulmonary Vein Thrombosis can make Pulmonary Vein Acidic States, which may be Associated with Atrial Fibrillation.
Authors: Takeuchi Hidekazu 
Published: July 2021

Atrial fibrillation can cause ischemic stroke. To prevent atrial fibrillation (AF) is crucial to prevent ischemic stroke. The pulmonary vein has a myocardial layer that can generate spontaneous or triggered action potentials. The myocardial layer is extended from the left atrial myocardium. Pulmonary vein myocardium sleeve is known to be associated with generating and maintaining AF. Pulmonary vein myocardium can be classified into two types. One is short and thin myocardium sleeve, which has no potential to cause atrial fibrillation (AF). And the other is long and thick myocardium sleeve, which has potential to cause AF. The mechanisms of such myocardium sleeve changes are not understood well.
Pulmonary vein thrombosis (PVT) is believed to be rare, which was reported as a rare complication of chest surgeries such as lobectomy or lung cancers. But since 2012, I reported seven cases of PVT in elderly patients without such conditions, which suggests that PVT is not uncommon. That is a novel notion. PVT prevents arterial blood flow, which inhibits oxygen and nourishment supply and carbon dioxide excretion. Hypoxia activates hypoxia inducible factors (HIFs), and HIFs can modulate epigenetic changes, reprogramming and ES cells. Undernourishments may activate nuclear respiratory factor-1 (NRF-1) and the aryl hydrocarbon receptor (AhR). PVT can make pulmonary vein acidic states by inhibiting excretion of carbon dioxide and may modulate pulmonary vein myocardium. Under acidic states, pulmonary vein cells such as pulmonary vein myocardium cells may obtain some kinds of multipotency. After obtaining multipotency, the cells may turn into new cells to adapt changed surroundings. The changes of myocardium sleeve may be caused by acidic state conditions and HIFs, AhR and NRF-1, which seemed to modulate pulmonary vein myocardium functions. To clear these, more studies are needed.
Abstract 37: The Positive Inotropic S100a1 Prevents Arrhythmogenic Sarcoplasmic Reticulum Ca2+ Leak And Ventricular Arrhythmias
Authors: Ritterhoff Julia , Völkers Mirko , Seitz Andreas , Spaich Kristin , Peppel Karten , Pleger Sven , Zimmermann Wolfram , Friedrich Oliver , Fink Rainer , Koch Walter , Katus Hugo , Most Patrick 
Published: July 2021

S100A1 has emerged as a key factor in the control of cardiomyocyte (CM) contractile performance. Improved sarcoplasmic reticulum (SR) function with enhanced Ca2+ resequestration appears critical for its cAMP-independent inotropic effects but raises concerns about potential diastolic SR Ca2+ leakage that might trigger fatal arrhythmias. Thus, the goal of this study was to determine the impact of S100A1 on ryanodine receptor 2 (RyR2)-mediated SR Ca2+ leakage in vitro and in vivo.
S100A1 association with the RyR2 was significantly diminished (-50%) in failing cardiomyocytes and hearts with S100A1 downregulation, as shown by co-immunofluorescence, co-immunoprecipitation and proximity ligation assay. Adenoviral-mediated S100A1 overexpression (3-4 fold vs. GFP-control) in quiescent NCs (normal CMs) and FCs (failing CMs) decreased SR Ca2+-frequency (-50 and −40% respectively) and protected from β-AR-triggered diastolic Ca2+-waves (-62 and −58% respectively) in electrically stimulated (2Hz) CMs as assessed by epifluorescent and confocal Ca2+ imaging. In multicellular rat engineered heart tissue (EHT), S100A1-overexpression (6-8 fold vs. GFP-control) protected from Ca2+-triggered after-contractions (ACs) (-50%) with preserved enhancement of isometric twitch force (TF, +40%) at 2Hz. S100A1-mediated rescue of contractile failure of endothelin-1-treated EHT (-50% decrease in TF) was associated with protection from Ca2+-triggered ACs. In mice with post-ischemic heart failure, AAV9-mediated therapeutic administration of S100A1 enhanced S100A1/RyR2 association and prevented epinephrine-induced VTs (70% in MI group vs. 30% in MI-S100A1 group). Mechanistically, S100A1-overexpression changed neither PKA/CaMKII RyR2 phosphorylation pattern nor binding of accessory proteins like FKBP12.6, calmodulin or sorcin to RyR2 but enhanced S100A1/RyR2 stoichiometry.
Our data provide evidence that S100A1 interaction with the RyR2 can beneficially modulate and reverse diastolic RyR2 function dysfunction. S100A1 appears to convey a rather unique molecular profile combining cAMP-independent inotropy with protection against Ca2+-triggered arrhythmias.
Abstract 121: Hyperglycemia-induced Prorenin Receptor Abundance on Plasma Membrane of Kidney Collecting Duct Cells: is a Novel Mechanism to Explain Diabetic Nephropathy?
Authors: Gogulamudi Venkateswara R , Arita Danielle Y , Bourgeois Camille RT , Satou Ryosuke , Prieto Minolfa C 
Published: July 2021

Activation of the renin-angiotensin system (RAS) leads to nephropathy during Diabetes Mellitus (DM). Prorenin levels are increased in the circulation of DM patients and predict microvascular damage. In streptozotocin (STZ)-induced type 1 DM rats, the collecting duct is the major source of prorenin in the kidney. The prorenin receptor (PRR), a new RAS component, elicits intracellular signals linked to fibrosis upon binding of prorenin at the cell plasma membrane (PM). We propose that PRR may contribute to the development of tubulointerstitial fibrosis by being locally activated by prorenin. To support this concept, we tested the hypothesis that hyperglycemia increases membrane bound PRR to collecting duct cells. To address this hypothesis, we used T1DM rats (N=10) induced with a single STZ injection (ip; 200ng/kg x 7 d) as well as mouse collecting duct M-1 cells, treated at different time intervals 0, 5 min, 1, 12, and 24 h with normal glucose (NG; 5mM glucose+20mM mannitol) and high glucose (HG; 25mM) to assess PRR trafficking alterations induced by glucose. After 7-days induction, STZ-rats showed plasma glucose as 428±13 vs.138±9 gr/dl and plasma insulin as 0.07±0.02 vs 2.42 ng/ml; compared to controls. By immunofluorescence (IF) in rat kidney sections, PRR was mainly localized on the apical aspect of collecting duct cells in STZ-rats, while in controls most of PRR signal was observed intracellularly. Although PRR mRNA levels did not differ in the collecting ducts between groups; its protein levels were augmented in STZ-rats. Importantly, compared with NG-treated cells, PRR protein levels (membrane bound) were significantly higher in PM extracts from M-1 cells treated with HG, and a maximum peak was observed at 1 h. Interestingly, in de-convoluted IF images of M-1 cells, PRR was localized mainly in the perinuclear areas during NG conditions; however, in HG-treated cells, PRR was found toward the cell surface. These data suggest that hyperglycemia induces PRR trafficking alterations of PRR. The present results suggest that hyperglycemia-induced PRR abundance on the PM in the collecting duct might be a novel mechanism underlying the development of diabetic nephropathy, particularly tissue fibrosis in DM.
Grant support by the NIH-NIDDK (DK104375-01)
Abstract 19: Microrna-19b Enhances Cardiac Cell Survival After Ischemic Injury
Authors: Molenaar Bas , Demkes Charlotte , Ruiter, de Hester , Versteeg Danielle , Gladka-de Vries Monika , Korporaal Ricardo , Deel, van Else , Semus Hillary M , van Rooij Eva 
Published: July 2021

Ischemia/reperfusion (I/R) injury in cardiac tissue results in substantial loss of cardiomyocytes, leading to a functional decline. The release of several cytokines increases cardiomyocyte survival after ischemic injury by activating the janus kinase (JAK) and signal transducer and activator of transcription (STAT) pathway. However, this pathway is inhibited by Suppressor of cytokine signaling (SOCS)1 and SOCS3, which act as endogenous negative regulators of cardiomyocyte survival by blocking JAK activity.

MicroRNAs (miRNAs) are small, non-coding RNAs that can repress gene expression by binding to recognition sequences within target genes. In a bioinformatic screen, we identified conserved binding sites in SOCS1 and 3 for miR-19b. Using a luciferase reporter assay, we were able to confirm direct binding of miR-19b to the 3’-UTR of both SOCS1 and 3
in vitro
. In addition, we found that miR-19b downregulates SOCS1 in cardiac-like cells and isolated neonatal cardiomyocytes.
In vivo
, we observed a downregulation of miR-19b in both rodent and human cardiac tissues after ischemic injury, which corresponds to an upregulation of both SOCS1 and 3.

Since inhibition of SOCS1 and 3 could enhance cardiac JAK-STAT signaling, miRNA-based inhibition could lead to an increase in cardiomyocyte survival. Here we show that cardiomyocyte-specific overexpression of miR-19b lowers SOCS1 and SOCS3 expression and enhances JAK-STAT signaling during ischemia reperfusion, which corresponds to a decrease in the pro-apoptotic proteins Bad, Bax and P53. Additional genes that are decreased by miR-19b during ischemic injury are Rnf11 and TNFAIP3, both repressors NF-kB signaling. To further explore the cardioprotective effects of miR-19b, we are currently investigating the therapeutic benefits of administering synthetic miR-19b mimics through intracardiac injection after I/R injury in mice.
Our study indicates a conserved mechanism by which miR-19b targets both SOCS1 and 3 and to increase the activation of JAK/STAT signaling to decrease cardiomyocyte apoptosis. Administration of miR-19b mimics might be of therapeutic interest to enhance cardiomyocyte cells survival and preserve heart function in the setting of ischemic injury.
Abstract 210: Titin Phosphorylation by Protein Kinase G as a Novel Mechanism of Diastolic Adaptation to Acute Hemodynamic Overload
Authors: Almeida-Coelho João , Leite-Moreira André M , Neves João S , Neiva-Sousa Manuel , Castro-Ferreira Ricardo , Hamdani Nazha , Linke Wolfgang A , Falcão-Pires Inês , Lourenço André P , Leite-Moreira Adelino F 
Published: July 2021

Titin is the main determinant of myocardial passive tension and its distensibility is increased via phosphorylation by protein kinase G (PKG), activated by nitric oxide (NO) and natriuretic peptides (NP) upon acute overload. We hypothesized whether myocardial stretch led to decreased stiffness, optimizing diastolic filling along with the usual increase in contractility.
Intact rat Langendorff hearts, strips dissected from the LV or right atrium of cardiac surgery patients and rabbit papillary muscles were acutely stretched for 15min. Passive tension (PT) was measured in skinned cardiomyocytes extracted from the LV of control and stretched rat hearts for sarcomere lengths (SL) 1.8-2.3μm before and after incubation with PKG. Rabbit muscles were incubated with a PKG inhibitor or, simultaneously, a NO synthase inhibitor, a NO scavenger, a NP receptor A antagonist. All-total titin phosphorylation was stained with Pro-Q Diamond and indexed to total-protein signals using SYPRO Ruby. Values are given as mean±SEM and statistical significance was set to p<0.05.
After acute stretch there was a progressive decrease in passive tension/diastolic pressure over 15min: 27±8% and 27±6% in human atrium and ventricular muscles, respectively, and 43±2% in rabbit papillary muscles and isolated hearts. This decrease in myocardial stiffness was significantly blunted by PKG inhibition (40%) and NO/NP pathway inhibition (29%). PT of cardiomyocytes was significantly lower (≈60%) in the previously stretched group for all SL. A similar effect, only significant in the control group, was observed after incubation with PKG. Titin phosphorylation increased markedly 15 minutes after acute myocardial stretch in human (atrial: 11±1% vs 41±8%; LV: 27±8% vs 71±21%) and rabbit (13±2% vs 23±3%) myocardium.
The progressive decrease in myocardial stiffness after acute hemodynamic overload is preserved at the myofilamental level and seems to depend on PKG activity, representing a potential therapeutic target in patients with pathologically rigid myocardium. Moreover, blocking PKG activation seems to attenuate this adaptive diastolic response. Therefore, titin phosphorylation by this kinase is probably involved in this new myocardial response to stretch in both animals and humans.