bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 134: Reciprocal Cardiac Chromatin Regulation by Ctcf and Hmgb2
Authors: Rosa-Garrido Manuel , Monte Emma , Karbassi Elaheh , Chen Haodong , Rau Christoph , Wang Jessica , Lusis Aldons J , Wang Yibin , Vondriska Thomas M
Published: July 2021

Chromatin remodeling plays an essential role in cardiac gene reprogramming under pathological conditions. However, we currently lack a detailed understanding of how this process is structurally coordinated across the genome. Previous RNA and proteomic analyses of an in vivo model of heart failure performed by our group identified the chromatin structural proteins CTCF and high mobility group protein B2 (HMGB2) as regulators of heart disease. CTCF is an essential factor in the maintenance of global chromatin organization through the facilitation of long-range interactions and chromatin looping. HMGB2 is a non-nucleosomal structural protein that packages nucleosomes into higher order structures.

An unbiased genomic analysis of ~100 strains of mice treated with isoproterenol (ISO) followed by transcriptome and phenotype analyses revealed a general down-regulation of CTCF at the mRNA level across the different strains after ISO treatment, whereas the HMGB2 response was strongly influenced by common genetic variation (i.e. between strains). ChIP-seq data, ChIP-reChIP-PCR experiments (detection of concomitant presence of two proteins at a given locus) and immunofluorescence showed CTCF and HMGB2 bind the same regions of the genome, yet they do not physically co-localize in myocyte nuclei. Loss of HMGB2, which induces hypertrophy in myocytes, causes de-condensation of chromatin, as directly measured by micrococcal nuclease digestion; CTCF knockdown has no effect on global chromatin accessibility by this assay. Examination of other chromatin structural proteins like histone H1 suggests this interaction between CTCF and HMGB2 is specific. Lastly, we showed that CTCF and HMGB2 regulate each other’s expression as well as the transcription of cardiac hypertrophy genes and ribosomal RNA. Our data show that basal HMGB2 (but not CTCF) expression correlates with cardiac mass. The response of HMGB2 levels to heart failure stimuli is highly influenced by genetics, whereas the decrease in CTCF following (ISO) is unaffected by genetic variation. These findings reveal molecular mechanisms of chromatin compaction in cardiac cells, linking them with genetic variability, and revealing a reciprocal relationship between HMGB2 and CTCF in cardiac hypertrophy.
Abstract 164: Production of De Novo Cardiomyocytes in Adult Asxl2-/- Hearts
Authors: Brunner Rachel , Gerdes Martin , Wang Tian
Published: July 2021

The chromatin-associated protein ASXL2 is highly expressed in the heart, where it critically regulates the levels of two repressive histone marks, trimethylated H3 lysine 27 and ubiquitinated H2A. We have previously observed that Asxl2-/- hearts displayed abnormal growth during adulthood, resulting in disproportionally larger heart from 4 months on. This abnormal growth is not due to cardiomyocyte hypertrophy, as demonstrated by measurement of comprehensive dimensions of isolated cardiomyocytes. BrdU labeling showed an elevation in proliferation index in Asxl2-/- hearts at 3 months. At the time of labeling, with rare exceptions, labeled cells are interstitial non-cardiomyocytes in both wildtype control and Asxl2-/- hearts. The vast majority of labeled cells stained positive for vimentin, a mesenchymal marker, and negative for Nkx2.5, Gata4 or c-kit. EdU pulse-chase experiments were performed to determine the fate of labeled cells. After a 4-week chase, a significant number of EdU+/cTnT+ cardiomyocytes were present in Asxl2-/- hearts but not in wildtype control hearts. Expression of the gap junction protein Cx43 was detected between EdU+ cardiomyocytes and neighboring EdU- cardiomyocytes, suggesting electrical coupling. Cardiomyocytes were isolated from pulsed-chased Asxl2-/- hearts for additional morphological analysis. Isolated EdU+/cTnT+ cardiomyocytes vary greatly in cell size, shape and nucleation status, ranging from small, spindle-shaped and mononuclear to large, rod-shaped and binuclear. Taken together, our data suggest that new cardiomyocytes are produced in adult Asxl2-/- hearts from a population of proliferative interstitial cells that are distinct from c-kit+ cardiac stem cells. This raises intriguing possibilities for cardiac repair and regeneration.
Abstract 17: Nuclear Calcium/Calmodulin-dependent Protein Kinase II Signaling Enhances Cardiac Progenitor Cell Survival and Cardiac Lineage Commitment
Authors: Quijada Pearl , Hariharan Nirmala , Cubillo Jonathan , Bala Kristin M , Ormachea Lucia , Bers Donald M , Sussman Mark A , Poizat Coralie
Published: July 2021

Ca2+/Calmodulin-dependent protein kinase II (CaMKII) signaling in the heart regulates cardiomyocyte contractility and growth in response to elevated intracellular Ca2+. The δB isoform of CaMKII is the predominant nuclear splice variant in the adult heart and regulates cardiomyocyte hypertrophic gene expression by signaling to the histone deacetylase HDAC4. However, the role of CaMKIIδ in cardiac progenitor cells (CPCs) has not been explored. During developmental growth endogenous CPCs display primarily cytosolic CaMKIIδ, which localizes to the nuclear compartment of CPCs after myocardial infarction injury. CPCs undergoing early differentiation in vitro increase levels of CaMKIIδB in the nuclear compartment where the kinase may contribute to the regulation of CPC commitment. CPCs modified with an established lentiviral based constructs to overexpress CaMKIIδB (CPCeδB) have reduced proliferative rate compared to lentiviral transduction of CPCs with eGFP alone (CPCe). Additionally, stable expression of CaMKIIδB promotes distinct morphological changes such as increased cell surface area and increased length of cells compared to CPCe. CPCeδB are resistant to oxidative stress induced by H2O2 relative to CPCe, whereas a knockdown of CaMKIIδB using small hairpin RNA resulted in an up regulation of cell death compared to scrambled treated controls. Dexamethasone treatment to promote cardiac differentiation increased cardiomyogenic markers cardiac troponin T and α-smooth muscle actin measured by RT-PCR and immunoblot analyses in CPCeδB compared to control CPCe. Therefore, CaMKIIδB may serve as a novel modulatory protein to enhance CPC survival and commitment into the cardiac and smooth muscle lineage.
Abstract 174: The BH3-Only Protein BNIP3 Induces Mitochondrial Clearance via Multiple Pathways
Authors: Gonzalez Eileen R , Hammerling Babette , Hanna Rita , Kubli Dieter A , Gustafsson Åsa B
Published: July 2021

Autophagy plays an important role in cellular quality control and is responsible for removing protein aggregates and dysfunctional organelles. BNIP3 is an atypical BH3-only protein which is known to cause mitochondrial dysfunction and cell death in the myocardium. Interestingly, BNIP3 can also protect against cell death by promoting removal of dysfunctional mitochondria via autophagy (mitophagy). We have previously reported that BNIP3 is a potent inducer of mitophagy in cardiac myocytes and that BNIP3 contains an LC3 Interacting Region (LIR) that binds to LC3 on the autophagosome, tethering the mitochondrion to the autophagosome for engulfment. However, the molecular mechanism(s) underlying BNIP3-mediated mitophagy are still unclear. In this study, we discovered that BNIP3 can mediate mitochondrial clearance in cells even in the absence of a functional autophagy pathway. We found that overexpression of BNIP3 led to significant clearance of mitochondria in both wild type (WT) and autophagy deficient Atg5-/- MEFs. BNIP3 caused an increase in LC3II levels in WT MEFs, indicating increased formation of autophagosomes. In contrast, LC3II was undetectable in Atg5-/- MEFs. Furthermore, we found that BNIP3-mediated clearance in WT and Atg5-/- MEFs did not require the presence of Parkin, an E3 ubiquitin ligase which plays a critical role in clearing dysfunctional mitochondria in cells. Also, overexpression of Parkin did not enhance BNIP3-mediated mitochondrial clearance. When investigating activation of alternative cellular degradation pathways, we found that BNIP3 induced activation of the endosomal-lysosomal pathway in both WT and Atg5-/- MEFs. Mutating the LC3 binding site in BNIP3 did not interfere with the activation of the endosomal pathway and clearance of mitochondria in Atg5-/- MEFs. Thus, these findings suggest that BNIP3 can promote clearance of mitochondria via multiple pathways in cells. The role of autophagy in removing mitochondria is already well established and we are currently exploring the roles of the endosomal and alternative autophagy pathways in BNIP3-mediated mitochondrial clearance in myocytes.
Abstract 177: Interleukin 17 and Coronary Stenosis in Patients With Stable Coronary Artery Disease
Authors: Oliveira Dinaldo , Heide Elaine , Pita Maira , Oliveira Danielle A , Pontes Ricardo , Pitta Ivan
Published: July 2021

Introduction:
The role of the immune and inflammatory pathways in patients with coronary artery disease (CAD) is important but not complete understood. The aim of this study was to evaluate concentrations of the interleukins 17 (IL 17) according to severity of coronary stenosis in patients with stable CAD

Hypothesis:
There is no association between severity of coronary stenosis and IL 17 in patients with stable CAD.

Methods:
This is a cross-sectional, prospective, analytical study, conducted from january to september, 2013. We included 40 patients (P) with stable CAD, CCS III or IV, ischemic myocardial scintigraphy, who had not been subjected to any kind of myocardial revascularization and with coronary stenosis ≥ 50% according to current coronary angiography. There were 20 healthy volunteers (C), to take up comparison of concentrations of IL 17. Interleukins were evaluated in serum of patients and after 48 hours of cells in culture with and without stimulus. IL 17 A concentrations were expressed in pg / ml. Coronary stenosis were classified as severe (> 70%) [SS] and intermediate (50 - 69%) [MS] according to coronary angiography.

Results:
Stenosis ≥ 50% were found in the anterior descending artery in 31 patients, in the left circumflex artery in 19 patients, and in the right coronary artery in 24 patients. No cases of stenosis were observed in the left main. Eighteen patients (45%) had single-artery disease, 8 patients (20%) had two-artery disease, and 14 patients (35%) had multiarterial disease. The comparison between the groups showed: IL 17: Serum: P with SS = 3.91 (3.91 -- 72.27) vs P with MS = 3.91 (3.91 -- 3.91) vs C = 3.91 (3.91 -- 28.8), p = 0.53; culture 48 hours without stimulus: P with SS = 3.91 (3.91 -- 3.91) vs P with MS = 3.91 (3.91 -- 86.8) vs C = 3.91 (3.91 -- 53.3), p = 0.55; culture 48 hours with stimulus: P with SS = 241.8 (3.91 -- 2200) vs P with MS = 217.5 (3.91 -- 1346) vs C = 154.3 (3.91 -- 1353), p = 0.7.

Conclusions:
There were no differences in concentrations of IL 17 according to severity of coronary stenosis, does not matter in serum or cell in culture. In conclusion, there was no association between severity of coronary stenosis and IL 17 in patients with stable CAD
Abstract 18: Increasing in vivo Apoa1/HDL Levels Negates the Cardiotoxic Effects of Doxorubicin, and Involves Signalling Through SR-BI, PI3K, and AKT1
Authors: Durham Kristina , Thomas Cyrus , Trigatti Bernardo L
Published: July 2021

Doxorubicin (DOX) is a clinically used anti-tumor drug, though the use of DOX is limited by its potent cardiotoxic side effect that can lead to heart failure. HDL protects isolated cardiomyocytes against DOX induced apoptosis, though whether this effect translates in vivo has yet to be determined. Here we assess whether ApoA1/HDL overexpression can protect mice in vivo against DOX induced cardiotoxicity, and explore the intracellular signalling mechanisms involved in protection.

Mice overexpressing human ApoA1 (ApoA1tg/tg) and ApoA1+/+ mice were treated chronically with DOX, and effects on cardiac function and cardiomyocyte health were assessed. Over expression of human ApoA1 in mice corresponded to ~2.5 fold increase in plasma HDL-C as compared to ApoA1+/+ mice. Following 5 weekly injections of 5mg/kg DOX, ApoA1+/+ mice displayed cardiac dysfunction as evidenced by reduced left ventricular developed pressure, and reduced rate of pressure development. In contrast, left ventricular function was maintained following DOX treatment in ApoA1tg/tg mice. Histological analysis revealed reduced cardiomyocyte cross-sectional area and increased cardiomyocyte apoptosis following DOX treatment in ApoA1+/+ mice. ApoA1tg/tg mice, on the other hand, were protected against DOX induced cardiomyocyte atrophy and apoptosis. Interestingly, pAKT:tAKT was reduced in ApoA1+/+ by treatment with DOX, but the ratio was maintained in ApoA1tg/tg mice.

We evaluated the roles of SR-BI, PI3K, and AKT1/2 in the signalling cascade of HDL in neonatal mouse cardiomyocytes and human immortalized ventricular cardiomyocytes. Through inhibition of AKT and PI3K, and knockdown or knockout of SR-BI, AKT1, and AKT2, we demonstrated that SR-BI, PI3K and AKT1 are required for HDL mediated protection against DOX induced cardiomyocyte apoptosis.

Our results provide evidence for ApoA1 mediated protection against DOX cardiotoxicity in vivo and demonstrate the roles of SR-BI, PI3K, and AKT1 as mediators in the protective effect.
Abstract 180: Cd248+cd8+ T Lymphocytes Suppress Pathological Vascular Remodeling in Human Thoracic Aortic Aneurysms
Authors: Li Jun , Wu Ting , Hu Xiaojuan , Schmull Sebastian , Reichenbach Gabi , Huang Ritai , Lian Feng , Gao Weiqiang , Dong Jun , Xue Song
Published: July 2021

Aortic aneurysms are characterized by vascular inflammation, neovascularization, and extracellular matrix destruction of the aortic wall. Although experimental studies indicate a potential role of CD248 in microvessel remodeling the functions of CD248 in human vascular pathologies remain unexplored. Here we aimed to study how CD248 interferes with pathological vascular remodeling of human aortic aneurysms. Immunofluorescent staining showed that CD248 expression was mainly localized in the CD8+ T cells infiltrating in the adventitia and media of aortic walls of patients with ascending thoracic aortic aneurysms (TAA). qPCR and immunofluorescent staining analyses revealed increased aortic CD248 expression and infiltrating CD248+CD8+ T cells in aortic aneurysms than in nonaneurysmal aortas. Flow cytometry analysis of human peripheral blood further identified a fraction of circulating CD248+ cells which was confined in the CD8+ T-cell compartment. The increased infiltrating of CD248+CD8+ T cells was coincident with reduced circulating CD248+CD8+ T cells in patients with ascending TAA when compared with patients with coronary artery diseases (CAD) and healthy donors. The CD248+CD8+ T cells were characterized by upregulated IL-10 and downregulated IL-1β/INF-γ expression when compared with CD248-CD8+ T cells. Moreover, when co-cultured with human aortic endothelial cells, the CD248+CD8+ T cells not only downregulated endothelial expression of ICAM1/VCAM1 and MMP2/3 but also suppressed endothelial migration. Thus, this study shows that CD248 reduces pathological vascular remodeling via anti-inflammatory CD248+CD8+ T cells, revealing a CD248-mediated cellular mechanism against human aortic aneurysms.
Abstract 181: Human Heart Valve-derived Scaffold Improves Cardiac Repair in a Murine Model of Myocardial Infarction
Authors: Li Jun , Wan Long , Chen Yao , Wang Zhenhua , Zhang Wentian , Schmull Sebastian , Wang Weijun , Gao Weiqiang , Dong Jun , Xue Song
Published: July 2021

Cardiac tissue engineering using biomaterials with or without a combination of cardiac stem cell therapy offers a new therapeutic option for repairing infarcted heart. So far, cardiac tissue scaffold is designed mainly based on natural and synthetic biomaterials, which do not mimic human myocardial extracellular matrix. This raises a fundamental issue about the biocompatibility of currently used biomaterials with myocardial tissue. Here we hypothesized that human heart valve-derived scaffold (hHVS) may provide a clinically relevant novel biomaterial for cardiac repair. In this study, human heart valve tissue was sliced into 100 μm tissue sheet by frozen-sectioning and then decellularized to form the hHVS. Upon anchoring onto the hHVS, post-infarct murine BM c-kit+ cells exhibited an increased capacity for proliferation and cardiomyogenic differentiation in vitro. When used to patch infarcted heart in a murine model of myocardial infarction, either implantation of the hHVS alone or c-kit+ cell-seeded hHVS significantly improved cardiac function, as shown by transthoracic echocardiography as well as by hemodynamic measurements via a Millar catheter, and by reduced infarct size; while c-kit+ cell-seeded hHVS was even superior to the hHVS alone. Thus, we have successfully developed a hHVS for cardiac repair. Our in vitro and in vivo observations provide the first evidence for translating the hHVS-based cardiac tissue engineering into clinical strategies to treat myocardial infarction.
Abstract 183: Improved Ca-cycling by Dexamethasone Preserves Contractility After Ischemia/Reperfusion in Diabetic Rat Cardiomyocytes
Authors: Schach Christian , Goetz Michael , Busse Hendrik , Zausig York , Drymalski Marzena , Maier Lars , Wagner Stefan
Published: July 2021

Glucocorticoid receptor (GR) stimulation is essential for normal heart function and dexamethasone (DEX) treatment has been shown to reduce ischemia reperfusion injury (IRI). Prevalence of diabetes is increasing and myocardial infarction in such patients is more severe and risk of complications is augmented. We tested the hypothesis, that GR stimulation with DEX protects diabetic myocardium from IRI by improving Ca cycling.

Cardiomyocytes from type 2 diabetic Zucker diabetic fatty rats were isolated and cultured in DEX (10 μg/mL) vs. vehicle for 24h. [Ca]

i

and fractional shortening (FS) was measured in electrical field-stimulated cardiomyocytes loaded with Fura-2 AM (10 μM). Compared to control, DEX-treated cells display increased Ca transient amplitude (0.33±0.03 vs. 0.46±0.04, P = 0.01, n=6 animals) and FS (fig.) DEX also significantly increased sarcoplasmic reticulum (SR) Ca content (Caffeine, 10 mM) and reduced SR Ca leak measured as Ca spark frequency (SpF), fluo-4 loaded myocytes, confocal microscopy (fig.).

To test, whether DEX reduced IRI, rats were treated with DEX (2 mg/kg i.p.) 24h before measurement (vehicle as control). Left ventricular developed pressure (LVDP) was monitored in Langendorff-perfused-hearts during global ischemia for 30 min and reperfusion. During reperfusion, DEX-treated hearts showed significantly greater LVDP (P < 0.05, 2way ANOVA, fig.).

In diabetic rats, glucocorticoid receptor stimulation with DEX leads to a more efficient Ca cycling with reduced SR Ca leakage, which translates to an increased contractility on the cellular level and protection against ischemia reperfusion injury in the whole organ.
Abstract 185: Chromogranin A is a Potent Prognostic Biomarker in Acute Heart Failure, and its Fragment Catestatin Regulates Cardiomyocyte Calcium Homeostasis by Camkiiδ Inhibition
Authors: Ottesen Anett Hellebø , Carlson Cathrine R. , Louch William E. , Forstrøm Johansen Rune , Stridsberg Mats , Jarstadmarken Hilde , Bjørås Magnar , Høiseth Arne Didrik , Brynildsen Jon , Omland Torbjørn , Christensen Geir , Røsjø Helge
Published: July 2021

Background:
Circulating chromogranin A (CgA) levels have been found associated with clinical outcomes in cardiovascular disease, but whether the CgA fragment catestatin (CST) may directly modulate cardiomyocyte Ca
2+
handling is not known.

Methods:
The prognostic utility of circulating CgA levels were compared between patients with acute HF (n=143) and chronic acute obstructive pulmonary disease (COPD, n=84). Functional effects of CST were assessed in isolated cardiomyocyte and explanted hearts.

Results:
CgA levels were associated with mortality in acute HF patients, but not in acute COPD. (Figure; patients stratified according to CgA quartiles on hospital admission). Admission CgA levels were also associated with mortality after adjusting for other risk factors in multivariate analysis. We found CST to interact with Ca
2+
/calmodulin (CaM)-dependent protein kinase II δ (CaMKIIδ) and to inhibit CaMKIIδ activity. CST also reduced CaMKIIδ-dependent phosphorylation of the ryanodine receptor 2 and phospholamban. In line with CaMKIIδ inhibition, CST reduced Ca
2+
spark and wave frequency, attenuated Ca
2+
sparkdimensions, increased sarcoplasmic reticulum Ca
2+
content, andaugmented magnitude and kinetics of cardiomyocyte Ca
2+
transients and contractions. Frequency dependent acceleration of relaxation was most pronounced in the Control group, an indication of CaMKIIδ activation, and this was attenuated by CST.

Conclusions:
CgA regulates cardiomyocyte Ca
2+
handling via CaMKIIδ inhibition, which makes CgA an interesting CV biomarker and potential compensatory mechanism in situations of enhanced CaMKIIδ activity.
Abstract 187: Metformin Increases Degradation of Ubiquitinylated Phospholamban via Autophagy in Cardiomyocytes
Authors: Teng Allen C , Miyake Tetsuaki , Yokoe Shunichi , Zhang Liyong , Rezende Luis M , Sharma Parveen , MacLennan David H , Liu Peter , Gramolini Anthony O
Published: July 2021

Phospholamban (PLN) is an effective inhibitor of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) in striated muscles. Here, we examined PLN stability and degradation in primary cultured mouse neonatal cardiomyocytes (CMNCs) and mouse hearts using immunoblotting, molecular imaging, and [35S]-methionine pulse-chase experiments along with lysosome (chloroquine and bafilomycin A1) and autophagic (3-methyladenine and Atg5 siRNA) antagonists. Inhibiting lysosomal and autophagic activities promoted endogenous PLN accumulation, whereas accelerating autophagy with metformin enhanced PLN degradation in CMNCs. Metabolic labeling reaffirmed that metformin promoted wild-type and R9C PLN degradation. Immunofluorescence showed that PLN and the autophagy marker, microtubule light chain 3 (LC3), became increasingly co-localized in response to chloroquine and bafilomycin treatments. Mechanistically, pentameric PLN was polyubiquitinylated at K3 residue and this modification was required for p62-mediated selective autophagy trafficking. Consistently, attenuated autophagic flux in Hace1-null mouse hearts was associated with increased PLN levels determined by immunoblot and immunofluorescence. Our study identifies a biological mechanism that traffics PLN to the lysosomes for degradation in mouse hearts.
Abstract 188: Contractile Function and Myofilament Proteins of the Naked Mole-rat Heart Show Resistance to Oxidative Stress
Authors: Grimes Kelly M , Barefield David , Kramer David A , Sadayappan Sakthivel , Buffenstein Rochelle
Published: July 2021

The naked mole-rat (NMR) is the longest-lived rodent, with a maximum lifespan of >31 years. Unlike every other mammal studied to date, this species withstands cardiovascular structural and functional changes for at least 75% of its lifespan. Due to the intersection of oxidative stress, aging, and cardiovascular disease, we questioned if NMRs were more resistant to oxidative stress-induced cardiac dysfunction compared to short-lived mice. Echocardiography showed that 7 days after a 20 mg/kg dose of doxorubicin (DOX), mice had a 25% decline in fractional shortening (36 ± 1% to 27 ± 2%). In contrast, the fractional shortening of NMRs was unchanged with DOX treatment (27 ± 1%). Previously we observed that while basal cardiac function is low, NMRs have a robust cardiac reserve, displaying a 1.7 fold increase in fractional shortening under exercise-like conditions. DOX-treated NMRs had a significant reduction in their dobutamine response, signifying a diminished cardiac reserve. Intriguingly, we found no changes in phosphorylation or expression of myofilament proteins in the NMR heart with DOX treatment. Mice on the other hand, increased the phosphorylation of cardiac myosin binding protein-C and switched expression from predominantly α-myosin heavy chain to the β-isoform. Electron microscopy showed that DOX caused marked mitochondrial swelling and loss of cristae as well as massive cardiac myofibrillar disarray in mice. Conversely, DOX-treated NMRs had only slight alterations to myofilament structure. NMRs additionally had twofold higher levels of glutathione in their hearts, indicating a high antioxidant capacity. These findings reveal that long-lived NMRs are less susceptible to oxidative stress-induced cardiac dysfunction than mice. The NMR’s low basal cardiac function, unique regulation of myofilament proteins, and high glutathione levels may all be integral in the species’ ability to withstand oxidative damage and preserve cardiac function well into its third decade of life.
Abstract 194: Characterization of Arrhythmogenic Dilated Cardiomyopathy Caused by Novel Filamin C Splice Variant in a Zebrafish Model
Authors: Begay Rene L , Rowland Teisha J , Tharp Charles A , Martin August , Graw Sharon L , Sinagra Gianfranco , Miani Daniela , Slavov Dobromir B , Stafford Neil , Sweet Mary E , Brun Francesca , Jones Kenneth L , Gowan Katherine , Mestroni Luisa , Garrity Deborah M , Taylor Matthew R
Published: July 2021

Although dilated cardiomyopathy (DCM) is a serious and frequent genetic cause of heart failure, only 30-40% of cases can be attributed to a known DCM gene mutation. To identify and confirm additional disease genes involved in DCM, we performed whole exome sequencing in two multigenerational families with DCM, both from the same geographic region of Italy, and found a novel splice variant in the gene encoding filamin-C (FLNC). Previously characterized mutations in FLNC had been primarily linked to skeletal muscle disease, although none of the affected family members displayed skeletal myopathy. To confirm and further characterize the arrhythmogenic DCM phenotype observed in family members, we performed embryonic knockdown experiments using morpholino (MO) treatment in zebrafish (Danio rerio) targeting the FLNC ortholog, filamin Cb (flncb). Following MO injection into 1-2 cell stage zebrafish embryos, 63.4% (78 of 123) of viable flncb MO-injected embryos displayed a cardiac phenotype at 72 hours post fertilization (hpf) (vs. 17.0% [30 of 177] of control MO-injected embryos; p≤0.001). Increases in mortality were observed, with 20.8% (54 of 260) of flncb MO-injected embryos surviving at 7 days post fertilization (vs. 65% [162 of 249] of control embryos; p≤0.001). The flncb MO-injected embryos demonstrated pericardial edema, dysmorphic or dilated cardiac chambers, and abnormal looping of the heart tube suggestive of systolic dysfunction. The flncb MO-injected embryos additionally demonstrated a lower mean stroke volume than controls (0.076 vs. 0.181 nl; p=0.015), a reduced mean cardiac output (10.8 vs. 25 nl/min; p=0.02), and an increase in the fraction of retrograde blood flow over the cardiac cycle (0.42 vs. 0.03; p=0.027). Overall, this flncb MO treatment recapitulated a DCM phenotype similar to the state caused by the human splicing variant, supporting haploinsufficiency as the mechanism leading to DCM in these families. Our findings suggest that approaches to augment endogenous filamin C protein levels may represent a viable treatment strategy that warrants exploration in future studies.
Abstract 198: Aberrant Endothelial-myocardial Crosstalk Causes Hypertrophy in Noonan Syndrome With Multiple Lentigines
Authors: Lauriol Jessica , Cabrera Janel R , Segarra Gabriel C , Flessa Meaghan E , Miller Lauren E , Bronson Roderick , Lee Kyu-Ho , Kontaridis Maria I
Published: July 2021

Congenital heart disease (CHD) is the most common birth defect worldwide; however, underlying mechanisms remain unknown. Loss-of-function mutations in PTPN11, the gene encoding the protein tyrosine phosphatase SHP2, are implicated in CHD and cause Noonan Syndrome with Multiple Lentigines (NSML). NSML presents with multiple cardiac defects, including hypertrophy. Here, we found that the NSML-associated adult-onset cardiac hypertrophy stems from aberrant signaling originating from developing endocardium. Embryonic NSML hearts showed diminished trabeculation and valvular hyperplasia, defects recapitulated in endocardial-, but not myocardial- or neural crest-, specific NSML mice. NSML hearts also developed ventricular septal defects, a phenotype reproduced only in myocardial-specific NSML hearts, suggesting NSML mutations have both cell autonomous and non-autonomous functions in cardiac development. Importantly, endocardial-specific expression of NSML was sufficient to induce adult-onset cardiac hypertrophy. Mechanistically, we observed aberrant AKT activity in NSML embryos, with decreased downstream FOXP1/FGF and NOTCH1/EPHB2 signaling, two pathways necessary for reciprocal crosstalk between developing endocardium and myocardium. Taken together, our data provide the first functional and disease-based evidence to suggest that critical mechanisms exist to control endocardial-myocardial crosstalk, the aberrant regulation of which may lead to CHD and adult-onset cardiac disease.
Abstract 204: Detection and Determination of Protein Network Associated With Atrial Fibrillation Subtypes
Authors: Mohamed Salah A. , Hanke Thorsten , Klein Oliver , Thiele Herbert , Sievers Hans H. , Yan Junfeng
Published: July 2021

Atrial fibrillation (AF) is associated with increased risks of stroke, cardiac failure, and mortality. The underlying mechanisms and pathology of AF remain elusive. The aim of this study is to proteomically analyze the left atrial appendage tissue obtained from patients suffering from subtypes (paroxysmal, persistent, and long-standing persistent) AF.

MALDI Imaging mass spectrometry (MALDI-IMS) was applied to differentiate in classification of pathophysiological AF subtypes, through the direct (in situ) analysis of formalin-fixed paraffin embedded (FFPE) left atrial appendage (LAA) tissue. FFPE LAA tissue were collected from patients with predisposed paroxysmal (n = 9, mean age 69.0±3.1 years), persistent (n = 18, mean age 67.0±2.7 years), and long-standing persistent AF (n = 19, mean age 71.0±2.0 years). Sections were dewaxed and thereupon soused by trypsin solutions using an automated spraying device. Spectra were acquired at a mass range of m/z 800-3500Da and lateral resolution of 80 μm. Two hundred laser shots were acquired per pixel and random walk of 50/position. Data analyses were performed using SCiLS Lab software.

Component analysis of MALDI Imaging data through probabilistic latent semantic analysis results in a clear discrimination in the first 3 components of atrial fibrillation. Employing receiver operating characteristic analysis (AUC > 0.7), characteristic intensity distribution in given m/z values, which are discriminative for the considered cluster, was determined to distinguish between paroxysmal vs. persistent AF, and persistent vs. long-persistent AF, m/z values were determined between persistent vs long-persistent AF (1.59±0.12 vs 6.85±3.02, p = 0.02). Follow-up of neurological events in case-controlled assessment presented 13±12% in paroxysmal, 56±12% in persistent and 42±12% long-persistent AF.

The tissue-based proteomic approach provides clinically relevant beneficial information in improving risk stratification for AF patients. In the future, this obtained information might be considered new biomarker to support the diagnosis of the severity of AF status. They also suggest a new criterion to determine the most appropriate procedure for each AF subtype to improve postoperative outcomes.
Abstract 212: Inhibition of Protein Phosphatase 2A (PP2A) Leads to Beta-adrenergic Receptor Dysfunction With Cardiac Stress Following Pressure-overload Hypertrophy
Authors: Chatterjee Arunachal , Vasudevan Neelakantan , Mohan Maradumane , Martelli Elizabeth , George John , Naga Prasad Sathyamangla
Published: July 2021

Beta-Adrenergic receptors (bARs) play a key role in regulating cardiac function. Loss of surface receptors and desensitization (impaired G-protein coupling) of bARs are hallmarks of a failing heart. Desensitization occurs by phosphorylation of bARs. The bARs are resensitized by protein phosphatase 2A (PP2A) mediated dephosphorylation in the endosomes before recycling to the plasma membrane. While mechanisms of desensitization are well understood, little is known about mechanisms regulating resensitization. Our previous work has shown that PI3Kg phosphorylates an endogenous inhibitor of PP2A (I2PP2A) on serine 9 & 93, which then robustly binds to PP2A inhibiting bAR resensitization. Since it is not known whether resensitization is altered in response to cardiac stress or whether altered bAR resensitization contributes to cardiac hypertrophy and failure, we generated transgenic mice with cardiomyocyte specific overexpression of wild type I2PP2A (WT I2PP2A Tg), I2PP2A phospho-mimetic mutants S9, 93D and mutants with constitutively dephosphorylated S9, 93A state. To test whether resensitization is critical in the development of bAR dysfunction during cardiac hypertrophy, WT I2PP2A Tg mice were subjected to transverse aortic constriction (TAC) for 8 weeks. Echocardiographic analysis post-TAC showed that WT I2PP2A Tg mice had accelerated cardiac dysfunction compared to their littermate controls [HW (mg)/BW(g): Sham: WT - 4.83, WT I2PP2A Tg - 4.82, TAC: WT- 6.47, WT I2PP2A Tg - 7.61; %EF: Sham: WT - 83.53, WT I2PP2A Tg - 74.72, TAC: WT - 70.47, WT I2PP2A Tg - 49.62]. To directly test whether resensitization mechanisms are altered, plasma membranes and endosomes were isolated and in vitro Adenylyl Cyclase activity assessed. Our studies show that compared to littermate controls, WT I2PP2A Tg had altered in vitro adenylyl cyclase activity showing that resensitization mechanisms in the endosomes may in part, contribute to cardiac dysfunction. Mechanistic underpinnings of the resensitization pathways using the I2PP2A S9, 93A and S9, 93D will be presented showing that bAR resensitization a process considered passive is altered in conditions of cardiac stress that in part may contribute to bAR dysfunction leading to cardiac hypertrophy and heart failure.
Abstract 215: The Role of Titin in Cardiac Function: Studies in Two Genetically Engineered Mouse Models With Disparate Titin’s Size
Authors: Methawasin Mei , Hutchinson Kirk R , Smith John E , Granzier Henk L
Published: July 2021

Titin, a myofilament that acts as a molecular spring in the sarcomere, is considered the main contributor to passive stiffness of cardiomyocytes and is responsible for cardiac diastolic function. Increased titin stiffness is related to diastolic dysfunction and HFpEF (Heart Failure with preserved Ejection Fraction). Alteration in size of titin’s spring region leads to changes in cardiomyocyte and left ventricular (LV) chamber stiffness.

We tested the effect of alteration in titin’s size in two genetically engineered mouse models. We investigated the effect of shortening titin’s spring region in a mouse model in which I-band/A-band region of titin’s spring has been deleted (TtnΔIAjxn ), in comparison to the effect of lengthening titin’s spring region in a mouse model deficient in titin splicing factor (Rbm20ΔRRM). Integrative approaches were used from single cardiomyocyte mechanics to pressure-volume analysis and exercise study.

Study of skinned LV cardiomyocytes revealed that cellular passive stiffness was inversely related to the size of titin. Cellular passive stiffness was increased in TtnΔIAjxn homozygous (-/-) (~ 110 % higher than wildtype (WT)) and was reduced in a graded manner in Rbm20ΔRRM heterozygous (+/-) and -/- cardiomyocytes (~61% and ~87% less than WT). This effect was carried through at the LV chamber level which could be demonstrated in pressure volume (PV) analysis as an increased end-diastolic pressure-volume relationship (EDPVR) in TtnΔIAjxn -/- (~110% higher than WT’s hearts) and reduced EDPVR in Rbm20ΔRRM +/- and -/- (~57% and ~48% less than WT’s hearts). Free-wheel running studies revealed a running deficiency in TtnΔIAjxn -/- mice but an increase in exercise capacity in Rbm20ΔRRM +/– mice.

Conclusions:
Functional studies from the cellular to in-vivo LV chamber levels showed that mice with shortening of titin’s spring region had increased LV stiffness, diastolic dysfunction and reduced exercise capacity, while mice with lengthening titin’s spring region had compliant LV and increased exercise capacity. Thus, our work supports titin’s important roles in LV diastolic function and suggests that modification of the size of titin’s spring region could be a potential therapeutic strategy for HFpEF.
Abstract 22: Transient Receptor Potential M2 is Involved in Secondary Myocardial Injury Induced by Nonlethal Mechanical Trauma in Rats
Authors: Li Shuzhuang , Cao Tingting , Ma Shuo , Li Xiujie , Bi Yue , Zhou Jicheng , Zhu Liang , Yu Deqin , Lv Guofeng
Published: July 2021

Background:
It is an imperative task to identify the mechanisms responsible for post-traumatic secondary myocardial injury. Our previous experiments showed that mechanical trauma (MT) could induce secondary myocardial injury via oxidative stress. The transient potential receptor M2 (TRPM2) channel has emerged as an important Ca
2+
signaling mechanism in a variety of cells, contributing to cellular functions that include cytokine production, cell motility and cell death. However, the role of TRPM2 channel in nonlethal mechanical traumatic cardiac damage remains unclear. The aim of the present study was to investigate whether TRPM2 channel is involved in myocardial injury in rats subjected to nonlethal MT.

Methods and results:
Western blot was used to quantify TRPM2 protein levels in Ventricular myocytes of adult male Sprague Dawley rats. Up-regulation of TRPM2 channel protein was observed in the following 12h after MT. It was observed that plasma harvested from MT rats increased cytosolic Ca
2+
concentration dose-dependently in H9c2 cells. To verify the role of TRPM2 further, we administered TRPM2 blockers flufenamic acid (FFA, 100uM) and clotrimazole (CLZ, 30uM) respectively to inhibit Ca
2+
influx, which leads to attenuated intracelluar Ca
2+
overload and apoptosis induced by MT plasma in H9c2 cells. Those two TRPM2 blockers also improved cardiac dysfunction induced MT in rats. When we used TMB-8 (inhibitor of sarcoplasmic reticulum Ca
2+
store) to inhibit calcium store mobilization, intracellular Ca
2+
level, apoptosis and cardiac dysfunction were also ameliorated. However, the administration of KBR-7943 (inhibitor of Na/Ca exchanger) did not reverse the pathological process following MT.

Conclusion:
These results demonstrate that post-trauma pathological phenomena is associated with TRPM2 closely via a redox-sensitive signal transduction pathway (mainly via MT-initiated Ca
2+
influx, even calcium overload pathway) .We propose that treatments like blockage of TRPM2 channel-associated Ca
2+
influx and mobilization, may shed light on the novel therapeutic strategy in reducing cardiac injury and post-trauma multiple organ failure.
Abstract 23: Disruption of TRAF2-TAK1-NF-κB Signaling Axis Triggers K-48 Linked Poly-Ubiquitylation of RIP1 and Necrotic Cell Death in Doxorubicin Cardiotoxicity
Authors: Dhingra Rimpy , Margulets Victoria , Aguilar Floribeth , Kirshenbaum Lorrie A.
Published: July 2021

The anthracycline doxorubicin (Dox) is a highly effective anti-tumour agent, however, its use is limited by its severe cardiotoxic effects that manifests as heart failure. The decline in cardiac performance induced by doxorubicin remains poorly defined. A critical survival role for the canonical IKKβ -mTOR-NF-κB signaling pathway has been demonstrated in ventricular myocytes. In this report, we demonstrate that, Dox impairs IKKβ-mTOR- NF-κB signaling in ventricular myocytes accompanied by mitochondrial perturbations including mPTP, loss of mitochondrial membrane potential and ROS production. IKKβ- NF-κB signaling involves TRAF 2 mediated ligation of K63- ubiquitin chains to RIP1 (Receptor Interacting Protein 1) which serves as scaffold for recruitment of ubiquitylated Tak1 complexes and phosphorylation-dependent activation of IKKβ -NF-kB signaling. Interestingly, ventricular myocytes treated with dox demonstrated reduction in expression levels of TRAF2 and TAK1, in vivo and in vitro. This was accompanied by a decline in K63- and concomitant increase in K-48 linked polyubiquitination on RIP1, impaired NF-kB activation and necrotic cell death of cardiac myocytes. Interestingly, inhibiting the kinase activity of RIP1 with Necrostatin-1, (Nec1) suppressed necrotic cell injury induced by dox but not NF-kB activation. Concordant with these findings was a marked increase in necrotic cell death in cardiac myocytes defective for IKKB signaling or MEF cells deficient for p65 treated with dox. Notably, mitochondrial perturbations, including PT-pore opening , ROS production, calcium uptake, LDH, Tn(T) and HMGB-1 release and necrotic cell injury induced by dox were completely abrogated by restoring NF-kB signaling in cardiac myocytes or Nec-1. Herein, we provide novel evidence that K-48 linked poly ubiquitylation of RIP1 provides a functional switch that impairs NF-kB activation and signals necrosis in cells treated with dox. Interventions that modulate NF-kB activity may prove beneficial in mitigating the cardiotoxic effects of dox.
Abstract 233: Metabolic Trapping Effects of ACS1 for Fatty Acid Uptake and Intracellular Trafficking are Different in Hearts of Males and Females
Authors: Goldenberg Joseph R , Wang Xuerong , Carley Andrew N , Lewandowski E. Douglas
Published: July 2021

Disruption of cardiomyocyte lipid metabolism has been observed in the responses to both pathogenic and nutrient stresses in human patients and animal models, with distinct sex differences. This investigation examined cardiac acyl CoA synthetase-1 (ACS)-mediated, LCFA uptake and trafficking with hearts of male and female mice. Isolated mouse hearts with cardiac specific overexpression of ACS1 (MHC-ACS) and non-transgenic littermates (NTG) were perfused with buffer containing 13C palmitate, 10 mM glucose, and 1 mM lactate. Dynamic 13C NMR elucidated the two phases of LCFA incorporation into triacylglycerol (TAG). The time constant (τ) of the initial exponential phase, corresponding to LCFA trans-sarcolemmal uptake, was lower, indicating faster LCFA uptake and esterification in male ACS hearts compared to NTG males (ACS male τ = 1.59±0.67 min vs NTG male τ = 2.85±0.90 min, P<0.05). Consistent with greater metabolic trapping, acyl CoA content was 470% higher in male ACS hearts (NTG male 57.5±9.4 vs ACS male 327.4±42 pmol/mg protein. P<0.001). Despite no differences in ACS content, NTG females displayed faster uptake than NTG males at a rate similar to ACS males, which was reversed by ovariectomy (OVX) indicating an estrogen dependent response (NTG female τ = 1.56±0.46 min, P<0.05 vs NTG OVX female τ = 3.02±0.77 min, P<0.05). Despite prior reports of ACS localization distal from the sarcolemma, ACS level and female gender both independently accelerated LCFA uptake without affecting TAG content or turnover. The data are consistent with metabolic trapping to facilitate LCFA uptake due to ACS activity. ACS also leads to higher total ceramide in hearts, with specific elevation of C18 and C22 in both genders, C24 in males, and C20 in females (male P<0.05; female P<0.01). ACS overexpression induced lower content of the cardiac fatty acid transporter (FATP) isoform, FATP6, indicating cooperative regulation of LCFA uptake between membrane transport proteins and intracellular acyl chain activation (P<0.05 male; P<0.001 female and female OVX). These results demonstrate that perturbation of LCFA metabolism though facilitated metabolic trapping by ACS1 affects sarcolemma transporter expression and induces the conversion of the CoA activated LCFA to ceramide.
Abstract 239: Augmented Cardiac Pyruvate Dehydrogenase Complex Flux During Hypoxia Increases Post-hypoxic Damage
Authors: Handzlik Michal K , Constantin-Teodosiu Dumitru , Greenhaff Paul L , Cole Mark A
Published: July 2021

Modulation of cardiac pyruvate dehydrogenase complex (PDC) flux is known to alter post-ischemic myocardial injury. Hypoxia is intrinsic to ischemia, but its specific role in cardiac injury is unknown. We hypothesized that post-hypoxic cardiac injury would be attenuated with activation of the PDC complex during hypoxia.

Mouse hearts were isolated and Langendorff perfused with 0.4mM palmitate and 11mM glucose. Following 30min of normoxia, hearts were subjected to 30min hypoxia (20% of normoxic pO2) followed by 30min of reoxygenation. PDC flux was increased by infusion of 1mM dichloroacetate (DCA), either from onset of hypoxia, or with onset of reoxygenation. Cardiac function was measured using an IV balloon, and fatty acid β-oxidation determined via 3H-labelling. Lactate efflux was assayed from timed buffer collections. Hearts were frozen at end-normoxia, end-hypoxia and end-reoxygenation, and assayed for PDC flux, and phosphate metabolites. Reactive oxygen species formation was assessed by Western blotting of protein carbonyls.

End-hypoxic cardiac PDC flux fell to 41±5% of pre-hypoxic values (0.96 of 2.37 μmol/gdwt, p<0.001) with β-oxidation also lower (25±11%, 0.06 of 0.24 μmol/min/gwwt, p<0.025). Lactate efflux increased 2.3-fold (4.7 to 10.6 μmol/min/gwwt, p<0.028) and AMP/ATP ratio 5.3-fold (0.04 to 0.21 p<0.002). DCA infusion increased end-hypoxic PDC flux (170±16%, 1.64 of 0.96 μmol/gdwt, p<0.003), reducing lactate efflux (47±8%, 5 of 11 μmol/min/gwwt, p<0.021) and AMP/ATP ratio (43±12%, 0.09 of 0.21, p<0.025). Control hearts were damaged by hypoxia, final recovery being 63±7% of pre-hypoxic function (18 of 29 bpm.mmHg.103). Hypoxic DCA infusion impaired recovery (49±4%, 15 of 31 bpm.mmHg.103), compared to DCA infusion during reoxygenation (63±3%, 19 of 30 bpm.mmHg.103, p<0.023). Reactive oxygen species formation was not altered.

Increasing PDC flux during hypoxia significantly modified myocardial substrate metabolism. In contrast to the reported beneficial effects of augmented PDC flux during ischemia, increasing PDC flux in the hypoxic heart exacerbated myocardial injury. These results indicate that the potentially therapeutic effects of increasing PDC flux are not dependent on the hypoxic aspect of myocardial ischemia.