bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 137: VCAM1 and SIRPA Cell Surface Markers of Human Cardiomyocytes
Authors: Skelton Rhys J , Arasaratnam Deevina , Finnin Ben W , Haynes John M , White Anthony , Ardehali Reza , Elefanty Andrew G , Stanley Edouard G , Elliott David A
Published: July 2021

Heart disease is the leading cause of mortality and morbidity in the Western world. The human heart has a limited regenerative capacity and there is an unmet demand for improved therapies for myocardial disease. Human stem cell derived cardiomyocytes have potential to facilitate the development of both cell based and pharmaceutical treatments. To date many studies have focused on the efficient generation of cardiomyocytes from pluripotent hESCs and iPSCs. Efficient in vitro differentiation of human cardiomyocytes is becoming a standard technique and hESC derived cardiomyocytes are beginning to be used in cardiotoxicity screens. In order to capitalise on these advances in cardiomyocyte generation and ensure the transfer of this technology into biopharmaceutical and clinical settings, enriched and well defined cell populations are required. Therefore, cell surface markers that allow the facile purification of cardiac cell lineages are needed.

This work investigates the potential of two novel cell surface markers, SIRPA and VCAM1, in isolating enriched populations of committed human cardiomyocytes. The NKX2-5GFP/w reporter line was used to characterise the temporal expression, gene expression profiles and functional properties of SIRPA and VCAM1 cell populations that are found during cardiac differentiation. We demonstrate that fully functional cardiomyocytes arise from an NKX2-5+SIRPA+ intermediate which up regulates VCAM1 concomitantly with commitment to the myogenic lineages of the heart (ie. smooth and cardiac muscle). Under the culture conditions used in this study, the NKX2-5+SIRPA+ population had a higher single cell clonogenic potential and displayed the capacity to give rise to both smooth muscle and cardiac clones. Furthermore, when cultured as re-aggregates the majority of NKX2-5+SIRPA+ cells progressed to a NKX2-5+SIRPA+VCAM1+ phenotype thus demonstrating lineage relationship of these two cell populations.

The identification of these cell surface markers provide a means for further study of linage specification during in vitro human cardiogenesis and will facilitate the development of technologies to produce well-characterised, purified human cardiomyocytes.
Abstract 139: Using Cyclic Strain to Improve Cardiac Progenitor Cell Cooperation
Authors: French Kristin M , Fierro Marcos J , Johnson Todd D , Christman Karen L , Davis Michael E
Published: July 2021

Introduction:
Cell therapies have grown in popularity for myocardial regeneration post-infarction, but still suffer from poor retention, maturation and integration of delivered cells. Mechanical strain has been shown to alter cell size, shape, adherence and gene expression in cardiac cells. As a more recently identified cell type, the effect of mechanical strain on cardiac progenitor cells (CPCs) is unknown. This work aims to elucidate the role mechanical strain plays in CPC phenotype and if this response is matrix protein specific. We hypothesize that mechanical strain will improve CPC alignment and potential for connectivity.

Methods:
To examine the role of mechanical strain on CPCs, CPCs were seeded on FlexCell plates in the presence of a naturally-derived cardiac extracellularmatrix (cECM), collagen I (COL) or no protein (TCP) and strained 0% (static) or 10% at 1 Hz for 24 hours in a BioFlex system. CPC elongation, alignment, and size were evaluated by rhodamine-phalloidin staining. Connexin-43 expression was measured by Western and normalized to GAPDH. Data were analyzed by two-way ANOVA and Bonferroni post-test.

Results:
CPC area, independent of culture conditions, was 1020 ± 40 um2, corresponding to neonatal cardiomyocyte area. The aspect ratio (major/minor axis) of CPCs showed a trend for increased elongation with strain at (e.x. 2.0±0.2 for unstrained cECM compared to 2.7±0.1 for strained cECM; n=4, p>0.05). Static culture conditions, independent of matrix coating, showed 20±3% alignment of CPCs. Under strain, alignment increased to 30±2% on COL (n=4; p>0.05 for strained COL verus static COL) and 48±8% on cECM (n=4; p< 0.01 for strained cECM versus strained COL and p<0.001 for strained cECM verus static cECM). A fold change >2 for connexin-43 protein in strained versus static conditions, independent of matrix, was observed (n=2, p>0.05) and confirmed by immunocytochemistry.

Conclusion:
This work suggests that mechanical strain alters CPC phenotype. Increased strain-induced alignment appears to be matrix dependent. In conclusion, these studies provide insight into the role of both mechanical forces and biochemical responses in the function of CPCs; which could lead to improved outcomes following cellular transplantation.
Abstract 148: Mst1 Mediates Cell-protective Mechanisms In The Heart Through Phosphorylation Of FoxO1 And C/EBP-beta
Authors: Maejima Yasuhiro , Zhai Peiyong , Isobe Mitsuaki , Sadoshima Junichi
Published: July 2021

Forkhead box O1 (FoxO1) regulates both cell survival and death in the heart. The function of FoxO1 is modulated by posttranslational modifications, including phosphorylation. Mst1 (mammalian sterile 20-like kinase 1), a stress-activated pro-apoptotic kinase, phosphorylates FoxO1 at Ser209/216/231/232. We investigated the molecular mechanism by which FoxO1 regulates both cell survival and death in the heart. Overexpression of FoxO1 significantly suppressed, whereas downregulation of endogenous FoxO1 exacerbated, Mst1 activity and Mst1-induced apoptosis. In the presence of Mst1, FoxO1 downregulated pro-apoptotic genes, including FasL, but upregulated antioxidant genes, including catalase. Chromatin immunoprecipitation assays revealed that Mst1 attenuated FoxO1 binding to the FasL promoter, but it enhanced binding to the catalase promoter. Reporter gene assays showed that C/EBP-β binding elements, but not the FoxO binding ones, in the catalase promoter, are critical for Mst1-mediated upregulation of the catalase gene. FoxO1 interacted with C/EBP-β and their interaction was enhanced in the presence of Mst1. Mass spectrometry analyses revealed that Mst1 phosphorylates C/EBP-β at Thr
299
in its leucine zipper domain. Downregulation of endogenous C/EBP-β reversed the cell-protective effects of FoxO1 expression against Mst1-induced apoptosis. We evaluated the role of Mst1-mediated phosphorylation of FoxO1 and C/EBP-β in mediating survival and death of cardiomyocytes in response to ischemia/reperfusion (I/R). The size of myocardial infarction (MI) in left ventricle (LV) after I/R was significantly greater in cardiac-specific FoxO1 knockout (

c-FoxO1
-/-

) mice than in wild-type mice (MI/area at risk: 59±2, 41±1%,
p
<0.05, n=6). Injection of a C/EBP-β phospho-mimetic mutant adenovirus into the LV of

c-FoxO1
-/-

significantly reduced the I/R-induced LV infarct size observed in

c-FoxO1
-/-

(50±2%,
p
<0.05, n=6). In summary, Mst1-mediated phosphorylation of FoxO1 inhibits the DNA binding of FoxO1 to the cell death promoting genes whereas it enhances FoxO1-C/EBP-β interaction, Mst1-mediated phosphorylation of C/EBP-β, and C/EBP-β-mediated transcription, which activates cell-protective mechanisms in the heart.
Abstract 149: Transgenic Overexpression Of HCN4 Induces Right Ventricular Dilated Cardiomyopathy Through Calpain-mediated Apoptosis
Authors: Yampolsky Pessah , Fink Thomas , Becker Rüdiger , Koenen Michael , Katus Hugo A , Thomas Dierk , Schweizer Patrick A
Published: July 2021

Introduction:
Hyperpolarization-activated cation (HCN) channels encode the pacemaker current If that contributes to generation and regulation of sinus rhythm, with HCN4 representing a key component in the cardiac conduction system. Recent studies have identified an increased ventricular expression of HCN4 in patients with hypertrophic cardiomyopathy and heart failure, however the mechanistic involvement of HCN4 in pathogenesis remains unresolved.

Methods:
We generated transgenic mice that overexpress human HCN4 under the regulation of a cardiac-specific cTNI promoter (HCN4+ mice) to study the contribution of HCN4 overexpression to structural heart disease. Molecular mechanisms were analyzed by quantitative Real-time PCR, western blot and immunohistochemistry.

Results:
HCN4+ mice were viable and exhibited high expression of HCN4 in both atria and ventricles, with HCN4 protein abundantly targeted to the plasma membrane. Real-time PCR analysis showed a 71% reduction in transcript levels of the endogenous murine HCN4 gene. Morphological evaluation at different developmental stages revealed right ventricular dilation and progressive decrease in wall thickness after one month of age, lacking overt myocardial hypertrophy, significant fibrosis or signs of myocardial inflammation. On the cellular level, the changes were characterized by apoptotic cell loss induced by strong transient expression of mu-calpain Ca2+-sensor and tissue transglutaminase (243% and 397% of wild type, respectively), which resulted in a robust activation of caspase 3. Of note, markers of pathological hypertrophy, that is atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and beta-myosin heavy chain (beta-MHC) remained unchanged. At the same time transcription of the physiological cell growth markers glycogen synthase kinase 3-beta (GSK3B) and mammalian target of rapamycin (mTOR) was elevated.

Conclusion:
HCN4 overexpression leads to calpain-mediated apoptosis and is associated with right ventricular dilated cardiomyopathy in a transgenic mouse model.
Abstract 156: Enalapril prevents Development of Dilated Cardiomyopathy in Lamin A/C Mutant Mice
Authors: Beqqali Abdelaziz , van Rijsingen Ingrid , van der Made Inge , van den Oever Stephanie , Pinto Yigal
Published: July 2021

Dilated Cardiomyopathy (DCM) is one of the leading causes of heart failure due to systolic dysfunction. Mutations in the LMNA gene, which encodes the nuclear lamina proteins Lamin A and C, are the most common cause of familial DCM. Current treatment strategies to improve the prognosis are limited to implantable cardioverter-defibrillator and heart transplantation. Patients with LMNA-related DCM are treated in accordance with international guidelines for the management of heart failure with little consideration of the possible influence of the etiology on the response to treatment. Recent studies suggest that this might result in inappropriate therapy in some patients. The influence of genetic factors in determining the response (and timing) of drug therapy is largely unstudied in DCM. Therefore, our aim is to determine the efficacy of existing heart failure drugs in preventing or delaying LMNA-related DCM. We used a well-established mouse model of Lamin A/C mimicking human LMNA-related DCM. Mice heterozygous for the Lmna mutant gene (n=20 per group) were treated with Metoprolol (β-blocker) or Enalapril (ACE-inhibitor) before the onset of DCM and were functionally evaluated by serial echocardiography and ECG until 75 weeks of age. Hearts were harvested for histological analysis and molecular characterization. Interestingly, the experimental group treated with Enalapril had a preserved overall cardiac function comparable to wildtype mice. Mice treated with Metoprolol however, displayed progressive heart failure, and an aggravated cardiac function compared to untreated Lmna +/- mice. Both the beneficial effects of Enalapril in preventing development of systolic dysfunction as well as the detrimental effect of Metoprolol were confirmed by expression of molecular stress markers and degree of cardiac fibrosis. Our results suggest that Enalapril is effective in preventing Lmna+/- induced cardiomyopathy in mice. Strikingly, Metoprolol increases cardiac dysfunction and stress in Lmna+/- mice. Further studies will determine whether Enalapril is also effective in preventing LMNA-related DCM in patients, and whether omitting Metoprolol from the standard cocktail of prescribed heart failure medicine is beneficial for LMNA patients.
Abstract 162: Hypoxia Inducible Factor Regulates Cardiac Ribonucleic Acid Splicing
Authors: Walton Chad B , Khemmani Mark , Dunn Brandyn , Shohet Ralph V
Published: July 2021

Ischemic cardiomyopathy is associated with elevated levels of hypoxia inducible factor (HIF)-1, a transcription factor that plays a key role in the response to hypoxia. We have created a transgenic mouse model with inducible cardiomyocyte-specific expression of an oxygen-stable form of HIF-1α to better understand the role of HIF-1 in ischemic cardiomyopathy. We performed RNAseq on polyA selected cardiac RNA before and after three days of transgene expression, with 10 million processed reads per sample. Genes were considered possibly differentially expressed if, following adjustment for multiple testing, p-value for the comparison to uninduced was less than 0.05. Genes having evidence of alternative splicing (those with a q-value <0.05 suggesting a change in the relative abundance of the different transcripts deriving from a single transcription start site) were identified. A total of 22 alternatively spliced genes were investigated. One transcript in particular, that for Calmodulin-dependent protein kinase II isoform gamma 2 (CaMKIIγ) was of particular interest because CaMKII homologs are involved in the phosphorylation of both RyR2 and PLB, and might thus be important in excitation-contraction coupling and cardiac contractility. The reference RNA sequence for CaMKIIγ was down regulated 11.7-fold, while variant 1 (missing Exons 7 and 8), and variant 2 (missing Exon 8) were up regulated 2.6-fold and 1.7-fold, respectively. Real-time PCR validation of the RNAseq data has been obtained in the transgenic hearts, and, to a lesser extent, in wild type mice subjected to LAD ligation. The cardiac effects of CaMKIIγ have been little studied in the response to hypoxia. Taken together with our prior observations of SERCA2a down-regulation and increased phosphorylation of PLB in the transgenic model, this new evidence suggests that another way in which HIF-1 may be modulating calcium flux is by reconfiguring the abundance of CaMKIIγ splice variants. The biochemical effect(s) of this variation in splice variants remains unknown, but seems likely to be connected to the decrement in cardiac contractility seen with HIF expression, and in ischemic cardiomyopathy. The surprising role of HIF in regulating splicing will also be a fertile area for further investigation.
Abstract 171: Nkx2-5 Suppresses The Proliferation Of Atrial Cardiomyocytes And Formation Of The Conduction Cells During Cardiac Development
Authors: Nakashima Yasuhiro , Yanez Diana A , Touma Marlin , Jordan Maria C , Roos Kenneth P , Nakano Atsushi
Published: July 2021

Mutation of human NKX2-5 is linked to atrioventricular conduction abnormalities and atrial septal defects. However, it has been difficult to rigorously examine the primary role of Nkx2-5 during the formation of the atria and cardiac conduction cells due to the concurrent hemodynamic aberration associated with ventricular dysfunction. Here, we examined the role of Nkx2-5 in the formation of the atria and conduction cells. Nkx2-5 was conditionally ablated in the atrial cardiomyocytes using atrial-specific Sln-Cre mouse line. The conditional mutants died shortly after birth, which is earlier and more severe than ventricular-specific knockout of Nkx2-5. While no evidence of ventricular failure was identified, atrial-specific conditional mutants developed congenital heart abnormalities including enlarged foramen ovale and hyperplastic atrial myocardium. Histological analyses revealed that the overproliferation of the atrial myocytes underlies the hyperplastic atrial myocardium prior to the development of the atrial septal enlargement. Moreover, conditional mutants showed massive expansion of SA node, AV node and internodal conduction cells, resulting in atrioventricular blocks that were further exacerbated by catecholamine stimulation. Given that the hemodynamic alteration is minimal during the gestational stages in our model, the misspecification of the conduction cells is not likely due to the alteration of the hemodynamics. Together, these data suggest that Nkx2-5 plays a suppressive role in the proliferation of the atrial cardiomyocytes and the formation of the conduction cells.
Abstract 174: The Origin Of Cardiac Fibroblasts During Normal Development And After Injury
Authors: Ranjbarvaziri Sara , Ali Shah R , Talkhabi Mahmood , Subat Ali , Zhao Peng , Volz Katharina S , Red-Horse Kristy , Ardehali Reza
Published: July 2021

Fibroblasts are the most abundant cells in the heart. They play a critical role in maintaining normal myocardial function as well as mediating fibrosis that occurs upon pathological injury. However, the source of cardiac fibroblasts during development and after the injury in the adult heart remains poorly characterized. The lack of cardiac fibroblast-specific markers has limited our ability to isolate or track them during normal development and in response to cardiac injury. Here, we define cardiac fibroblasts as mesenchymal cells with a surface-marker profile of CD45-/CD31-/Ter119-/CD11b-/Thy1.1+ /Thy1.2+. We used lineage tracing, transplantation studies, and parabiosis surgeries to identify the cellular origins of cardiac fibroblasts and their contribution to fibrosis during pathological injury induced by pressure overload. We show that fibrosis is associated with proliferation of fibroblasts within 7 days after pressure overload injury with no evidence of bone marrow or hematopoietic stem cells contributing to the cardiac fibroblast population. We then used the transgenic mouse models Tie2cre;mTmG, Tbx18cre;mTmG, and Pax3cre;mTmG to lineage trace the origin of fibroblasts from the epicardium, endothelial and the neural crest, respectively. By using fluorescent activated cell sorting (FACS) and immunostaining, we demonstrate that the majority of cardiac fibroblasts are derived from the epicardium, a minority arises from endothelial cells, and a small fraction from the neural crest. Although we observed an increase in the total number of fibroblasts after pressure overload injury, this response was not restricted to any single developmental subset.

In summary, our findings show that the majority of the cardiac fibroblasts, during normal development as well as in response to pathological injury, are derived from the epicardium via epithelial-to-mesenchymal transformation. Endothelial to mesenchymal transition and neural crest sources also contribute to fibroblast generation and the progression of pressure overload-induced cardiac fibrosis.
Abstract 175: Doxorubicin and Acute Exercise Training Impair Diastolic Before Systolic Function in a Murine Model of Cancer
Authors: Libonati Joseph R , Sturgeon Kathleen , Schadler Keri , Muthukumaran Geetha , Ding Dennis , Bajulaiye Akinyemi , Thomas Nicholas , Ferrari Victor A , Ryeom Sandra
Published: July 2021

Introduction:
Cardiotoxicity often occurs in cancer patients treated with doxorubicin (DOX). DOX induces cardiomyocyte death, due in part to DOX-mediated DNA damage, myofiber degeneration, ischemia-related metabolic alterations, and activation of apoptotic pathways. While exercise is often recommended for cancer patients, it remains unclear whether the metabolic stress of exercise with DOX treatment benefits or impairs cardiac performance.

Purpose:
To test the hypothesis that acute exercise training performed with DOX therapy offsets cardiotoxicity.

Methods:
B16F10 melanoma cells (3x10
5
) were injected into 6-8 week old male C57BL/6 mice (n= 48). A 4 mg/kg cumulative dose of DOX (IP) was administered over 2 weeks and exercise (EX) consisted of treadmill walking (45 min/d, 10 m/min, 5 days/week, 2 weeks). Four groups were tested: 1) Sedentary (SED) +Vehicle, 2) SED +DOX, 3) EX+Vehicle, 4) EX+DOX. Echocardiography [LV systolic (EF, %) and diastolic (E/A ratio) function] and tissue harvest were performed 48 hours after EX.

Results:
Tumor volume was attenuated in DOX and lowest in EX+DOX (p<0.05). Body weight (BW), heart weight (HT), HT/BW, and HT/Tibia were attenuated in DOX (p<0.05) and were lowest in EX+DOX. LV fibrosis increased with DOX (p<0.05) and was greatest in EX+DOX (SED+Vehicle 2 ± 0.3%, SED+DOX 4 ± 0.5%, EX+Vehicle 3 ± 0.3%, and EX+DOX 6 ± 0.4%; p<0.05). Cardiomyocyte area and β myosin heavy chain gene expression were similar between groups. Diastolic function was reduced with DOX and EX+DOX (both p<0.05), but systolic function was similar between groups. DOX induced significant alterations the expression profiles of genes in the mTOR signaling pathway, with similar expression patterns in SED+DOX and EX+DOX. Insulin improved in vitro cardiomyocyte cell survival (p<0.05) to a greater extent than IGF-1 treatment during DOX. Neither insulin nor IGF-1 impacted cardiomyocyte proliferation during DOX.

Conclusion:
While the addition of EX to DOX treatment more effectively inhibited tumor growth, EX did not alleviate DOX mediated LV diastolic impairment, and actually increased LV fibrosis. Acute, short term exercise appears to impair LV function in DOX-treated mice. Exercise-induced reductions in insulin secretion may underlie this effect.
Abstract 181: Cardiac Hypertrophy And Heart Failure In K-channel Ancillary Subunit Depleted Mice
Authors: Roepke Torsten K , Lisewski Ulrike , Schleussner Leonhard , Gaertner Carolin , Koehncke Clemens
Published: July 2021

Down regulation of cardiac K+ currents has been described in hypertrophic heart disease and heart failure. However, so far no mutations in potassium channel genes have been found in patients with cardiac hypertrophy/heart failure. It therefore remains controversial whether K+ current down regulation represents a primary cause of cardiac hypertrophy/heart failure or epiphenomena. KCNE2 is a voltage gated potassium channel ancillary subunit that is associated with inherited and acquired long QT syndrome. We recently developed KCNE2-/- mice. KCNE2-/- mice have normal cardiac morphology and function at 3-6 months of age despite a repolarization defect caused by disruption of IKslow, 1 and Ito, f. However, aged KCNE2-/- mice (12 months and older) develop significant cardiac hypertrophy and severe heart failure assessed by echocardiography, isolated heart (Langendorff) and reduced cellular shortening (IonOptix). Furthermore, after Angiotensin II stimulation 3-6 months old KCNE2-/- mice also develop significant cardiac hypertrophy and fibrosis compared to age-matched KCNE2+/+ siblings. Taqman PCR revealed molecular remodelling in hypertrophied failing KCNE2-/- hearts with induction of the fetal gene program (ANF re-expression), significant up regulation of Sodium-Calcium Exchanger (NCX) and significant down regulation of phospholamban. Alterations in Calcium handling proteins were also recapitulated on protein level by Western Blotting. Furthermore, electrophysiological studies by patch clamp analysis of isolated cardiomyocytes revealed a decrease in current densities of the L-type Ca2+-current and a strongly diminished response to isoprenaline stimulation in hypertrophied failing KCNE2-/- cardiomyocytes. We therefore conclude that primary disruption of repolarizing Kv currents by KCNE2 deletion leads to altered Calcium handling and consecutive induction of cardiac hypertrophy/heart failure.
Abstract 190: A Mechanotransduction Study In Embryonic Zebrafish Heart
Authors: Yang Jingchun , Xu Xiaolei
Published: July 2021

Background:
While proper heart contraction requires an intact sarcomere structure, contraction reciprocally regulates sarcomere assembly and cardiomyocyte shape during heart morphogenesis and heart remodeling. However, how contraction modulates heart morphogenesis and the underlying molecular mechanism remains unclear. Here we address this question by manipulating cardiac contractility in a developing zebrafish embryo.

Results:
Cessation of heart contraction was achieved by treating zebrafish embryos with blebbistatin, a myosin II specific inhibitor, or Niferdipine, a Ca
2+
channel inhibitor. Loss of cardiac contractility reduces ventricular chamber size in 1 dpf embryo, but significantly increases ventricular chamber size in 2 dpf and 3 dpf embryos. In both circumstances, cessation of contraction consistently prevents or disrupts the lateral fusion of myofibril. Cessation of contraction-induced heart enlargement is reversible and also dependent on the time of drug treatment. This heart-remodeling event in 2dpf embryo, which manifests as increased cardiomyocyte surface area accompanied by reduced thickness, can be attenuated by reducing the transmural pressure towards the ventricle wall. This mechanotransductive process occurs in the myocardium without need of endocardium. At the molecular level, by screening several compounds, we found that PI3K signaling confers this contraction-regulated cardiomyocyte shape remodeling process without affecting the lateral fusion of sarcomere.

Conclusion:
Our data in an
in vivo
animal model distinguish two different and relatively independent functions of contraction in regulating lateral fusion of sarcomere and cardiomyocytes shape remodeling. The contraction-regulated heart shape remodeling is conveyed by PI3k signaling mediated mechanotransduction independent of endocardium.
Abstract 195: TIP30 Interferes With Protein Synthesis And Inhibits Cardiac Hypertrophy
Authors: Grund Andrea , Froese Natali , Korf-Klingebiel Mortimer , Thum Thomas , Wollert Kai C , Müller Oliver , Mayr Manuel , Bauersachs Johann , Heineke Jörg
Published: July 2021

Background:
Pathological cardiac hypertrophy is a crucial risk factor for the development of heart failure. It is characterized by a massive increase of contractile and structural proteins within cardiomyocytes, mainly as a result of enhanced protein synthesis. The regulatory mechanisms of increased RNA translation during hypertrophy, however, remain largely unknown. Here, we examined the cardiac function of TIP30, which has previously emerged as a tumor suppressor gene with reduced expression in malignant tumours.

Results:
We identified myocardial TIP30 mRNA as dramatically downregulated (>80%) in a mouse model of dilative cardiomyopathy (MLP knock-out mice) as well as in human failing hearts. A protein pull-down screen with a GST-TIP30 fusion protein and proteomic candidate identification revealed the eukaryotic translation elongation factor 1A1 (eEF1A1) as binding partner of TIP30, which was independently verified by GST-pulldown and co-immunoprecipitation (IP). Interestingly, this interaction between TIP30 and eEF1A1 was not detectable during pro-hypertrophic (phenylephrine, PE) stimulation in neonatal rat cardiac myocytes (NRCM). Adenoviral (Ad-) TIP30 overexpression in NRCM during PE stimulation led to a reduced hypertrophic response compared to Ad-control infected cells (cell size in % of control: Ad-TIP30 135 ± 4 vs. Ad-control 200 ± 3 p<0.05). At the same time, a dramatic decrease in protein:DNA ratio was observed. siRNA downregulation of eEF1A1 ablated the anti-hypertrophic effects of TIP30. Measurement of translational activity by determination of the polysome/monosome ratio revealed a blunted translation rate after TIP30 overexpression. In line with our in vitro data, myocardial overexpression of TIP30 by an adeno-associated virus (AAV9-TIP30) significantly reduced cardiac hypertrophy during two weeks of Angiotensin (Ang)/PE infusion in vivo. In turn, Ang/PE infusion as well as aortic constriction (TAC) led to an increased hypertrophic response in TIP30 knock-out versus wild-type mice.

Conclusion:
TIP30 inhibits hypertrophy and translational activity most likely through inhibition of eEF1A1 in cardiomyocytes. TIP30 is downregulated in human failing hearts and could thereby contribute to aggravated hypertrophy.
Abstract 199: Distinctive Roles of Linker Histone H1 Variants in the Hypertrophic Response of Cardiomyocytes
Authors: Parvatiyar Michelle S , Lopez Timothy D , Franklin Sarah , Vondriska Thomas M
Published: July 2021

Heart failure results when cardiac output is insufficient to meet physiological requirements and is often preceded by development of cardiomyocyte hypertrophy. As cardiac myocytes respond to hypertrophic stresses they re-express developmentally important genes, normally senescent in the adult. The chromatin structural events underlying this “fetal gene program” are unknown. We previously showed by proteomics that histones, components of the chromatin protein functional unit, the nucleosome, are altered during hypertrophic and failing phases of pressure overload in mouse: linker histone variants H1.2 and H1.5 decreased in hypertrophied myocardium while H1.0 increased during the transition to failure. The linker histone H1 family influences higher order chromatin structure and gene expression, although the role of this family in the heart is unknown. To assess the role of linker histones in hypertrophy, neonatal rat ventricular cardiomyocytes (NRVMs) were transfected with siRNAs individually targeting six H1 variants. Loss of H1.3 and H1.4 respectively induced a significant 26.1% (76 of 90) and 13.5% (80 of 94) increase in cell size area (µm2). A role of H1 in the hypertrophic response is evidenced by its influence on myosin heavy chain (MHC) mRNA expression. Knock-down of individual H1 variants significantly altered the MHC isoform ratio: loss of H1.3 increased α-MHC levels 1.5 fold and decreased β-MHC 1.6 fold while H1.5 depletion decreased α-MHC 2.5 fold. Both H1.3 and H1.4 knock-down increased atrial natriuretic factor (ANF) 1.3 fold while H1.5 loss decreased ANF 6.2 fold shown by qRT-PCR. Treatment with hypertrophy-inducing agents Isoproterenol (1μM), Endothelin (2nM) or Phenylephrine (10μM), reduced H1 mRNA levels however with subtle effects on protein abundance. To evaluate whether H1 loss shifted NRVM nuclei from a predominantly heterochromatic toward euchromatic state favoring gene accessibility we examined distinct histone markers of chromatin states. Histone H1.5 knock-down significantly decreased H3K9Me3 levels, a silencing mark associated with heterochromatin, 1.7 fold. Therefore we conclude that variants package distinctive regions of the genome and that H1.3 and H1.4 controls genes involved in the hypertrophic response.
Abstract 228: Alterations of Cardiac Morphology and Function Caused by Cardio-Thoracic Surgery in Small Animal Models of Heart Disease
Authors: Nordbeck Peter , Bönhof Leoni , Hiller Karl-Heinz , Voll Sabine , Arias Paula , Seidlmayer Lea , Williams Tatjana , Ye Yu-Xiang , Gensler Daniel , Pelzer Theo , Ertl Georg , Jakob Peter M , Bauer Wolfgang R , Ritter Oliver
Published: July 2021

Background:
Surgical procedures in small animal models of heart disease, such as artificial ligation of the coronary arteries for experimental myocardial infarction, can evoke alterations in cardiac morphology and function. Such alterations might induce artificial early or long term effects in vivo that might account for a significant bias in basic cardiovascular research, and, therefore, could potentially question the meaning of respective studies in small animal models of heart disease.

Methods:
Female Wistar rats were matched for weight and distributed to sham left coronary artery ligation or untreated control. Cardiac parameters were then investigated in vivo by high-field MRI over time after the surgical procedure, determining left and right ventricular morphology and function. Additionally, the time course of several metabolic and inflammatory blood parameters was determined.

Results:
Rats after sham surgery showed a lower body weight for up to 8 weeks after the intervention compared to healthy controls. Left and right ventricular morphology and function were not different in absolute measures in both groups 1 week after surgery. However, there was a confined difference in several cardiac parameters normalized to the body weight (bw), such as myocardial mass (2.19±0.30/0.83±0.13 vs. 1.85±0.22/0.70±0.07 mg left/right per g bw, p<0.05), or enddiastolic ventricular volume (1.31±0.36/1.21±0.31 vs. 1.14±0.20/1.07±0.17 µl left/right per g bw, p<0.05). Vice versa, after 8 weeks, cardiac masses, volumes, and output showed a trend for lower values in the sham operated rats compared to the controls in absolute measures (782.2±57.2/260.2±33.2 vs. 805.9±84.8/310.4±48.5 mg, p<0.05 for left/right ventricular mass), but not normalized to body weight. Matching these findings, blood testing revealed prolonged metabolic and inflammatory changes after surgery not related to cardiac disease.

Conclusion:
There is a small distinct impact of cardio-thoracic surgical procedures on the global integrity of the organism, which in the long term also includes circumscribed repercussions on cardiac morphology and function. This impact has to be considered when analyzing data from respective studies and transferring the findings to conditions in patients.
Abstract 229: Determination of Phase-specific Biomarkers of Viral Myocarditis Induced by Theiler’s virus Using Multivariate Analyses of Viral Genome, Troponin, Transcriptome and Echocardiography Data
Authors: Kawai Eiichiro , Omura Seiichi , Sato Fumitaka , Martinez Nicholas E , Fernando Viromi , Rollyson Phoebe , Cvek Urska , Trutschl Marjan , Tsunoda Ikuo
Published: July 2021

Viral myocarditis has been proposed to be initiated by viral replication in the heart (acute phase), followed by immune-mediated damage (subacute phase), where each phase requires anti-viral and immunomodulatory treatments, respectively. There are no specific biomarkers to distinguish acute from subacute phases of myocarditis while serum troponin, echocardiography, and myocardial biopsy data have been used for diagnosis clinically. To determine the phase-specific biomarkers, we used a mouse model for myocarditis induced by Theiler’s murine encephalomyelitis virus (TMEV), which belongs to the genus Cardiovirus, the family Picornaviridae. We conducted multivariate analyses of viral genome, serum cardiac troponin I, echocardiography, histology, and transcriptome using microarray data of the heart tissue harvested on 4 (acute) and 7 (subacute) days post infection (dpi). The level of viral RNA semi-quantified by RT-PCR was 10-fold higher on 4 dpi (ΔCt = 2.5×10-2 ± 4.9×10-3) than 7 dpi (ΔCt = 2.6×10-3 ± 3.0×10-4) (P < 0.05). Serum troponin was undetectable in 4 of 10 mice on 4 dpi and only in 1 of 10 mice on 7 dpi; the serum troponin levels (ng/ml) on 4 dpi (42.9 ± 15.6) were significantly lower than 7 dpi (249.9 ± 62.8) (P < 0.05). The levels of viral RNA and troponin were strongly correlated on 4 dpi (r = 0.79, P < 0.05), but not 7 dpi (P = 0.12), suggesting that viral replication could be a major cause of myocardial damage only on 4 dpi. We found multiple high intensity cardiac lesions using echocardiography with histological myocarditis on 7 dpi, but not 4 dpi. Transcriptome analyses of microarray data showed upregulation of genes associated with innate immune responses in samples from 4 and 7 dpi, compared with controls. Samples from 7 dpi showed upregulation of genes associated with T, B, and antigen presenting cells and downregulation of cardiac myosin-related genes (Myl4, Myl7, and Mybphl), compared with 4 dpi, suggesting that acquired immune responses contribute to cardiomyocyte damage on 7 dpi. In summary, the chronological order of emergence of biomarker candidates was 1) viral genome and innate immunity, 2) troponin, and 3) acquired immunity and echo and histological changes.
Abstract 233: Isoproterenol-induced Cardiac Hypertrophy And Failure In Mice: Gene Network Modeling
Authors: Rau Christoph D , Wang Jessica , Ren Shuxun , Wang Zhihua , Ruan Hongmei , Wang Yibin , Lusis Aldons J
Published: July 2021

Heart failure is highly heterogeneous and as a result, relatively few insights into the pathways and drivers of heart failure have been identified using system-wide methods such as genome-wide association studies (GWAS). We have developed a resource, the Hybrid Mouse Diversity Panel (HMDP) for high resolution GWAS and systems genetics in mice. Eight week old female mice from 93 unique inbred strains of the HMDP were given 20 μg/g/day of isoproterenol through an abdominally implanted Alzet micropump. Three weeks post-implantation, all mice were sacrificed, along with age-matched controls. The mice exhibited widely varying degrees of hypertrophy and heart functioning. A portion of the left ventricle was processed and arrayed on an Illumina Mouse Ref 8.0 platform.

We used Maximal Information Component Analysis, a novel method of network construction which allows for non-linear relationships between genes as well as non-binary partitioning of genes into sub-networks to subdivide the expression data into a series of modules. In order to identify modules which may contribute to Isoproterenol-induced hypertrophy and failure, we examined the correlation of each module to clinically relevant cardiac traits traits such as organ weights and echocardiographic parameters.

We identified several modules with strong correlations to multiple heart failure-related clinical traits, including one module of 41 genes which contained several genes of interest, including Lgals3, a diagnostic marker for heart failure. Utilizing eQTL hotspot analysis, we have identified a locus which is involved in the regulation of this module. A gene within this locus, Magi2, regulates the turnover of the β-adrenergic receptor and represents a likely candidate for the response to isoproterenol.
Abstract 234: Defining Domains In The Transcription Factor Grainyhead-like 3 Required For Protective Functions In The Endothelium
Authors: Farrokh Sabrina , Ale-Agha Niloofar , Haendeler Judith , Altschmied Joachim
Published: July 2021

Important aspects during aging of human endothelial cells (EC) are an increased apoptosis level and a reduced migratory capacity. In a screening for anti-apoptotic genes we have identified the transcription factor Grainyhead-like 3 (GRHL3) and demonstrated its anti-apoptotic and pro-migratory effects after overexpression and knockdown in EC. To define the domains in GRHL3 responsible for these - potentially also extranuclear - functions we have cloned large scale deletion mutants and mutants with deletions of putative nuclear localization signals (NLS) and analyzed them for their intracellular localization and functional properties.

Immunostaining of transfected EC were used to examine the subcellular distribution. Two mutants with deletions in a bioinformatically predicted bipartite NLS were localized predominantly in the cytoplasm. To corroborate these data, lysates of the cells were fractionated biochemically. Western blotting confirmed the immunostaining data, indicating that we have identified the major NLS in GRHL3.

To analyze the transcriptional properties of these mutants, we constructed a GRHL3-specific luciferase reporter containing a tandem GRHL3 binding sites in front of a minimal promoter and cotransfected it with expression vectors for the GRHL3 deletion mutants. Besides the previously described activation domain we identified another region required for transcriptional upregulation of target genes in the N-terminal half of the protein.

We will now use these mutants to further dissect the regions in GRHL3 necessary for its pro-migratory and anti-apoptotic activities in endothelial cells. This will also allow us to investigate if all of these functions are mediated solely by the activation of target genes or by other mechanisms coupled to a non-nuclear function of GRHL3.
Abstract 249: Neonatal Mouse Hearts Do Not Regenerate after Severe Cryoinjury
Authors: Lien Ching-Ling (Ellen) , Darehzereshki Ali
Published: July 2021

Introduction:
In contrast to adult zebrafish, mammalian adult hearts cannot regenerate in response to tissue damage. Interestingly, neonatal mouse hearts have regenerative capacity similar to adult zebrafish after ventricular resection. Zebrafish hearts regenerate in a similar manner after cryoinjury compared to other injury types (ventricular resection and genetic ablation); however scar tissue forms and it takes much more time to resolve the scar and regenerate after cryoinjury. It remains to be determined if neonatal mouse hearts regenerate after cryoinjury, which is thought to be the injury model most similar to damage caused by infarction than resection.

Hypothesis:
Neonatal mouse hearts respond to different types of injuries differently; there might be a limit to the regenerative capacity of neonatal mouse hearts.

Methods:
To determine if neonatal mouse hearts respond to different types of injuries differently, we established a cryoinjury model by utilizing a cryoprobe pre-chilled in liquid nitrogen placed at the anteriolateral ventricular wall. At different time points, cardiac functions were determined and the hearts were collected for histological analysis.

Results:
In contrast to ventricular resection, neonatal mouse hearts fail to regenerate at 21, 60 and 120 days after severe (transmural) cryoinjury. Minimum scar tissues remain after mild (non-transmural) cryoinjury. Echocardiography showed severe impairment of cardiac functions after severe cryoinjury. Epicardial activation and new coronary vessel formation occured after cryoinjury. However, we did not observe increased cardiomyocyte proliferation after severe cryoinjury; this is consistent with the finding that neonatal mouse hearts regenerate after ventricular resection by cardiomyocyte proliferation. When neovascularization is blocked by induction of a VEGF-sequestering soluble receptor (sVEGFR1), neonatal mouse hearts cannot survive after severe cryoinjury and form a more extensive scar following a mild cryoinjury.

Conclusion:
Neonatal mouse hearts do not regenerate after severe transmural injury due to failure to undergo cardiomyocyte proliferation. Furthermore, neovascularization is essential for the survival and heart regeneration of neonatal mice.
Abstract 251: The Rate of Myocyte Turnover in the Mouse Heart
Authors: Wybieralska Ewa , Ogorek Barbara , Kostyla James , Isobe Kazuya , Signore Sergio , Borghetti Giulia , Sundman Mark , Leri Annarosa , Rota Marcello , Kajstura Jan
Published: July 2021

The accuracy of the estimations of cell turnover in the myocardium is restricted by the difficulty to obtain from histologic samples the information regarding the rate of cell proliferation, i.e., number of cells generated per unit of time. It is easy to identify the fraction of cycling cells, but this value cannot be directly converted into the number of cells generated per day. As a result, the published estimates of cell turnover in the mammalian heart vary dramatically. Here, we propose to use the cell cycle as an internal clock to introduce the time factor in the analysis of population dynamics of cardiac cells. In this novel approach the cells are exposed to two pulses of distinct thymidine analogs, separated by time T. The first pulse is performed with 5-bromo-2’-deoxyuridine (BrdU) and the second pulse with 5-ethynyl-2’-deoxyuridine (EdU). With this protocol, the number of cells that exit the S phase during time T is equal to the number of cells positive for BrdU but negative for EdU. It can be shown that, in non-synchronous population, the number of cells exiting the S phase is equal to the number of cells entering the cell cycle. Importantly, this approach does not require knowledge of the length of the cell cycle. To measure the rate of formation of cells in the myocardium, 2 month-old mice were injected with the nucleotides at 4 hour intervals and sacrified 4 hour later. The hearts were formalin-fixed and histologic sections obtained. The first nucleotide, BrdU, by was detected by immunocytochemistry, and the second, EdU, by the “click” chemistry. Myocytes and non-myocytes were distinguished by α-sarcomeric actin and cardiac troponin I staining and analyzed by confocal microscopy. Spectral analysis was employed to ensure specificity of the detected signals. The enumeration of double positive nuclei, nuclei positive for one nucleotide only, and nuclei negative for both nucleotides indicated that in the entire left ventricle (LV) ~1,250 myocytes are generated per hour. Since the mouse LV contains ~3 x 106 parenchymal cells, these data demonstrate that the entire population of myocytes can be replaced in ~180 days. Thus, our results are consistent with a relatively high turnover rate of myocytes in the mouse heart.
Abstract 256: mRNA-based Nanoparticles Are Highly Effective Agents For Cardiac Gene Therapy
Authors: Kontorovich Amy R , Eltoukhy Ahmed A , Turnbull Irene C , Cashman Timothy J , Rao Satish K , Anderson Daniel G , Hajjar Roger J , Costa Kevin D
Published: July 2021

Gene therapy holds promise for repair and regeneration of damaged myocardium after infarction. Viral vectors used in most studies, while highly effective, carry concerns of safety and immunogenicity. Nanoparticle (NP)-enabled transfection using polymeric and lipidoid reagents mitigates some of the concerns related to the use of viral vectors. However, these agents have been less effective in transfecting cardiac myocytes, which are primarily post-mitotic and only weakly facilitate DNA entry to the nucleus. Because translation occurs in the cytosol, transfection with RNA obviates the need for nuclear entry. Recent work has shown that synthetic alterations to messenger RNA (mRNA) can prolong its half-life in the cytosol and diminish the host’s immune response. We hypothesized that transfection of cardiac myocytes with lipidoid NPs formed using modified mRNA would lead to high levels of protein expression. Neonatal rat (NRCM), adult rat (ARCM) and human embryonic stem cell-derived (hES-CM) cardiac myocytes were transfected with modified GFP-mRNA NPs using a lipidoid vehicle (Stemfect
TM
, Stemgent, Cambridge, MA) or Lipofectamine2000 (LF, Invitrogen) and GFP expression was assessed after 24 hours. Average transfection efficiency in NRCM was significantly higher using Stemfect versus LF as measured by flow cytometry (48.5% vs. 6.6% at 20ng mRNA; 47.4% vs. 11.8% at 40ng mRNA, 48.1% vs. 18.6% at 80ng mRNA; 48.1% vs. 28% at 160ng mRNA; n=3, p<0.01 for each pair). In hES-CM transfection was also more robust with Stemfect NPs versus LF (91.6% vs. 62.2%, p<0.05 at 40ng mRNA; 96.1 vs. 87.8%, p<0.01 at 160ng mRNA; n=3). GFP expression was noted by fluorescence microscopy to be higher in ARCMs transfected with Stemfect NPs versus LF. In conclusion, we have shown that difficult-to-transfect cardiac myocytes are readily transfected with mRNA-based NPs
in vitro
and with significantly higher efficiency when using Stemfect rather than Lipofectamine. Gene therapy with mRNA represents a novel approach to cardiac repair and may be preferable to DNA because of the transient window of protein expression and lower side effect profile.