bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 409: Role of the Mitochondrial AAA+ Lon Protease in Cardioprotection
Authors: Li Min , Venkatesh Sundararajan , Rashad Eman , Saito Toshiro , Sadoshima Junichi , Suzuki Carolyn K
Published: July 2021

Mitochondrial Lon is an ATP-powered proteolytic machine that selectively degrades regulatory proteins as well as misfolded, unassembled and damaged proteins. Lon is a key mediator of mitochondrial quality control, adapting mitochondrial metabolism to hypoxic, oxidative, and proteotoxic stresses. Although studies imply that up-regulating Lon in the failing or ageing heart is ameliorative, there is little detailed in vivo data about its importance in cardiovascular health and disease. To test the hypothesis that Lon protects the heart in vivo, we generated transgenic mice with cardiac-specific overexpression of Lon under the control of the alpha-MHC promoter (Tg-Lon). Lon was specifically overexpressed in heart with no change in the levels of endogenous substrates TFAM or mitochondrial aconitase, or the mitochondrial ClpXP protease. Baseline analysis of 2-11 month old mice showed no significant difference in body or heart weight or ventricular weight/tibial length. At 2-3 months, no significant difference was observed by echocardiography or histology. However, Tg-Lon mice showed significantly reduced cardiac injury in response to ischemia reperfusion (I/R)- ischemia (45 min)/reperfusion (24 hr). At 6-9 months Tg-Lon, infarct size/area at risk (AAR) was 32%±2.3 SEM versus NTg that was 51%±3.2 SEM (p-value<0.003, n=9, n=4). At 3-5 months the infarct size/AAR for Tg-Lon was 37%±2.9 SEM versus NTg 51%±1.6 SEM, p<0.009, n=4, n=4); and at 3 months, Tg-Lon was 37%±2.0 SEM versus NTg 47% ± 1.4 SEM, p<0.008, n=3, n=4). In all cases, there was no change in AAR/left ventricle (AAR/LV), and no difference in LV/body weight (LV/BW). At baseline, mitochondria isolated from Tg-Lon hearts showed lower oxygen consumption rates (OCR, pmoles O2/min) as compared to NTg. Average OCR for Tg-Lon was 12,997±708 SD AUC (1 μg), by contrast to NTg that was 32,042±320 (1 μg) (p<0.001, n=5, n=5). Associated with lower OCR of Tg-Lon, was a significant reduction of Complex I subunit NDUFB8 of NADH dehydrogenase, which was reduced 2.2 fold (p<0.001) as compared to NTg in LV extracts as well as in isolated heart mitochondria. These data demonstrate that Lon overexpression promotes cardioprotection, and that this effect may be linked to a reduction in basal mitochondrial oxygen consumption.
Abstract 423: Proteasome Priming by Protein Kinase G Protects Against Myocardial Ischemia-reperfusion Injury
Authors: Wang Xuejun , Terpstra Erin J , Callegari Eduardo , Hu Chengjun , Zhang Hanming , Wang Xuejun "XJ"
Published: July 2021

Cardiac proteasome functional insufficiency is implicated in a large subset of heart disease and has been experimentally demonstrated to play an essential role in cardiac proteotoxicity, including desmin-related cardiomyopathy and myocardial ischemia-reperfusion (I-R) injury. Pharmacological inhibition of phosphodiesterase 5 (PDE5) via sildenafil for example, which can stabilize cGMP and thereby increase cGMP-dependent protein kinase (PKG) activity, is consistently reported to protect against I-R injury; however, the underlying mechanism is not fully understood. We have recently discovered that PKG activation enhances proteasomal degradation of misfolded proteins (Ranek, et al. Circulation 2013), prompting us to hypothesize that proteasome-priming may contribute to cardioprotection-induced by PDE5 inhibition. Here we used a cardiomyocyte-restricted proteasome inhibition transgenic mouse line (Tg) and non-Tg (Ntg) littermates to interrogate the action of sildenafil on I-R injury created by left anterior descending artery (LAD) ligation (30 min) and release (24 hr). Sildenafil was administered 30 min before LAD ligation. Results showed that (1) the 26S proteasome activity of the Ntg I-R hearts was significantly elevated by sildenafil but this elevation was blocked in the Tg line; (2) the infarct size reduction by sildenafil treatment in Ntg mice was completely abolished in the Tg mice with the same treatment; and (3) systolic and diastolic function impairment after I/R was markedly attenuated in sildenafil-treated Ntg mice, but not in the sildenafil-treated Tg mice. Additionally, immunoprecipitation assays show that PKG interacted with the proteasome in cultured cardiomyocytes, and this interaction appeared to be augmented by sildenafil treatment. Moreover, in vitro incubation of active PKG with purified human 26S proteasomes increased proteasome peptidase activities and the phosphorylation at specific serine residues of a 19S proteasome subunit as revealed by “gel-free” nano-LC-MS/MS. We conclude that active PKG directly interacts with, phosphorylates, and increases the activities of, the proteasome and that proteasome priming mediates to cardioprotection of PDE5 inhibition against I-R injury.
Abstract 46: Identification of Novel Alternate Splicing Events in Humans With RBFOX2 Mutations and Hypoplastic Left Heart Syndrome
Authors: Benson Craig C , McKean David , Homsy Jason , Kim Richard , Gorham Josh , Seidman Jon , Seidman Christine
Published: July 2021

Alternative splicing (AS) of protein isoforms is an integral mechanism for cardiac development. RNA Binding Protein, Fox-1 Homolog (C. Elegans) 2 (RBFOX2) is an RNA binding protein preferentially expressed in muscle and neuronal cells and regulates tissue-specific alternate exon splicing in ~2,100 target genes by binding the conserved RNA sequence motif (U)GCAUG. RBFOX2 was recently implicated in the pathogenesis of abnormal cardiac and cerebral development via loss-of-function studies in zebrafish and mouse. However, convincing evidence remains incomplete, as the full complement of RBFOX2 target genes and differential exon usage (DEU) in human cardiovascular cell lines are incompletely defined. We identified de novo mutations in RBFOX2 from four human cases of congenital heart disease (CHD) with hypoplastic left heart syndrome (HLHS) via whole exome sequencing. To test the hypothesis that RBFOX2 mutations alter DEU in known target genes, we performed RNA-seq on ductus arteriosus tissue from human CHD cases with and without RBFOX2 mutation. Analysis of RNA-seq for DEU was performed with DEXSeq. To limit the effect of differential gene expression, we restricted analysis to subjects with high global gene expression correlation (r2 > 0.9, case=1 vs. control=5). DEU in known RBFOX2 target genes were highly enriched compared to all known genes (115/2,100 vs. 589/26,310, p=5.78e-15). A high percentage of the DEU genes (60.0%, 69/115) have high heart expression (HHE) in the developing mouse (mean expression of four cardiac chambers at e14.5). DEU genes with HHE include VCL, TPM1, FN1, ACTN1, and CALD1. Functional annotation clustering reveals enrichment for several actin, cytoskeletal, and contractile Gene Ontology terms, suggesting a possible role in the epithelial-mesenchymal transition (EMT) developmental processes active during early cardiac formation. We also identified enrichment from genes implicated in CHD by allelic specific expression or de novo mutations (44/1,263 vs. 589/26,310, p=0.007). These results are the first in humans to identify differentially expressed exons associated with RBFOX2 mutations in CHD and suggests RBFOX2-mediated alternate splicing may influence EMT pathways implicated in the pathogenesis of HLHS.
Abstract 47: Loss of the Cardio Protection Inferred by S-nitrosylation of GRK2 At Cysteine 340 Leads to Decreased Cardiac Performance and a Hypertensive Phenotype With Aging
Authors: Traynham Christopher J , Yuan Ancai , Gao Erhe , Koch Walter
Published: July 2021

In the next 35 years, the global population of individuals above 60 years of age will double to approximately 2 billion. In the aged population, cardiovascular diseases are known to occur at a higher prevalence ultimately leading to increased mortality. G protein-coupled receptors (GPCRs) have been identified as vital regulators of cardiac function. GPCR kinases (GRKs) are important in cardiac GPCR regulation through desensitization of these receptors. GRK2 is highly expressed in the heart, and has been widely characterized due to its upregulation in heart failure. Studies from our lab have shown that elevated GRK2 levels in ischemia-reperfusion (I/R) injury result in a pro-death phenotype. Interestingly, cardio-protection can be inferred via S-nitrosylation of GRK2 at cysteine 340. Further, we have generated a knock-in GRK2 340S mouse, in which cysteine 340 was mutated to block dynamic GRK2 S-nitrosylation. GRK2 340S mice are more susceptible to I/R injury. Given that GRK2 340S mice are more susceptible to oxidative stress, and there is a nitroso-redox imbalance in senescence, it is possible that these mice are more likely to exhibit decreased cardiac performance as they age. Therefore, we hypothesize that with age GRK2 340S knockin mice will develop an overall worsened cardiac phenotype compared to control wild-type (WT) mice. To test this hypothesis, 340S and WT mice were aged for a year, and cardiac function was evaluated via echocardiography. Aged 340S mice exhibited significantly decreased ejection fraction and fraction shortening relative to aged WT controls. Prior to tissue harvesting, in-vivo hemodynamics was conducted via Millar catheterization. At baseline, aged 340S mice exhibited increased systolic blood pressure compared to aged WT mice. At the conclusion of this protocol, mice were sacrificed and heart weight (HW), body weight (BW), and tibia length (TL) measured to evaluate cardiac hypertrophy. Aged 340S mice exhibited significantly increased HW/BW and HW/TL ratios, indicative of cardiac hypertrophy, relative to aged WT controls. Taken together, these data suggest that with age, loss of the cardio protection inferred by S-nitrosylation of GRK2 at leads to decreased cardiac performance, and an overall worsened cardiac phenotype.
Abstract 58: Dicarbonyl Stress and Diabetic Heart Failure: The Role of Endothelial Cells
Authors: Vulesevic Branka , McNeill Brian , Giacco Ferdinando , Brownlee Michael , Milne Ross , Suuronen Erik
Published: July 2021

Dicarbonyl stress (DS) caused by the accumulation of α-oxoaldehyde metabolites, like methylglyoxal (MG), leads to detrimental DNA and protein modifications. Under normal conditions, MG is detoxified by glyoxalase-1 (GLO1) and -2 enzymes, but this system fails in diabetes. While the role of DS in diabetic cardiomyopathy through changes in cardiomyocyte function has been well described, this study aimed to link DS with the development of endothelial dysfunction (ED) and early heart failure in diabetes.

Transgenic mice that over-express GLO1 in endothelial cells (ECs) but not in cardiomyocytes, and their wild-type (WT) littermates were treated with STZ to induce hyperglycemia (WT-diabetic and GLO1-diabetic mice) or vehicle (non-diabetic controls). Hyperglycemia increased the circulating levels of ED markers in WT-diabetic (E-selectin 1.5-fold, ICAM 1.4-fold, and VCAM 1.1-fold), but not GLO1-diabetic mice. The number of vWF+ ECs in WT-diabetic hearts was reduced 2-fold compared to other groups, whereas GLO1 over-expression preserved capillary density. Cell death, determined by TUNEL staining, was greater in the hearts of WT-diabetic mice compared to all other groups. GLO1 over-expression resulted in reduced inflammation: TNF-α protein expression was increased in both diabetic groups (≥2-fold), but significantly less so in GLO1-diabetic mice (p=0.03). The preservation of ECs in GLO1-diabetic mice was associated with delayed signs of heart failure. At 4wk of hyperglycemia, WT-diabetic mice had reduced heart function compared to all other groups (p=0.04). At 8wk, cardiac function in GLO-diabetic mice was greater than in WT-diabetic mice, but both were reduced compared to non-diabetic controls (p=0.02; p=0.4). A possible mechanism for EC survival in GLO1 mice despite the presence of inflammation was examined in vitro using human aortic ECs. ECs exposed to high glucose or MG for 24h had increased apoptosis induced by TNF-α compared to cells treated only with TNF-α (by 2- and 3-fold, respectively), suggesting that reduced MG protects ECs from TNF-α mediated death.

Taken together, these results suggest that DS in diabetes increases inflammation and ED, leading to the loss of ECs in the heart, which contributes to the development of heart failure.
Abstract 59: Role of Follistatin 315 in Regulating Cardiac Hypertrophy in Physiology and Pathophysiology
Authors: Xu Zhaobin , Blazek Alisa D , Beck Eric , Alloush Jenna , Li Jackie , Schneyer Alan , Agarwal Sudha , Hewett Timothy , Weisleder Noah
Published: July 2021

Heart failure is characterized by initial compensatory changes, including the myocyte hypertrophy, chamber dilation, and matrix remodeling, that proceed until progressive dysfunction produces end stage heart failure and mortality. Recently, the roles of secreted factors in the heart that could regulate pathological hypertrophy, including follistatin (FST) and related molecules, have been examined by various investigators. FST is a molecule that blocks secretion of follicle-stimulating hormone from the pituitary and regulates members of the transforming growth factor beta (TGF-β) family including myostatin. Here we tested the effects of a particular FST isoform, FST288, on heart function in mice. The gene encoding FST produces three isoforms that differ in biological activities and cell surface binding capabilities. The FST315 isoform contains all six exons, and proteolytic cleavage of the FST315 C-terminal tail results in production of FST303. The lack of exon 6, which codes for the acidic C-terminal tail of the putative full-length protein, results in FST288. The missing acidic C-terminal tail region found in soluble FST315 allows FST288 to bind cell surface heparin-sulfated proteoglycans, accounting for the differential actions of these FST isoforms. Since mice that are null for the FST gene die embryonically, we used genetically modified mice that express only the FST288 isoform to test the role of FST315 in adult heart. Examination of these animals suggests that the loss of FST315 expression has limited effects on the heart at the resting state. When these mice are subjected to pressure overload through transverse aortic constriction (TAC) surgery they appear to be resistant to the compensatory cardiac hypertrophy present in wild type mice by 4 weeks post surgery. Both cardiac structure (examined by histology) and function (as measured by echocardiography and pressure/volume loops) following TAC are improved in the genetically modified mice when compared to wild type mice. This response is likely due to modification of the myostatin signaling pathway, one of the major targets of FST315. Overall, our data illustrates that FST315 is an important contributor to the progression of pressure overload induced cardiac hypertrophy.
Abstract 6: Novel Tools for Generation, Purification, and Analysis of Pluripotent Stem Cell Derived Cardiomyocytes
Authors: Eckardt Dominik , Noack Kristin , Bosio Andreas
Published: July 2021

Pure and well-characterized cardiac cells derived from human pluripotent stem cells (hPSCs) are of high interest for cardiovascular disease modeling, drug safety studies and development of cell replacement strategies. Although several protocols for cardiac differentiation of hPSCs have been developed, major limitations are clone-to-clone variations in differentiation efficacy as well as heterogeneity of generated cardiomyocyte populations. Therefore, we have developed novel tools for cardiomyocyte differentiation, magnetic cell sorting-based purification and cell analysis using new antibody-conjugates enabling for flow cytometry- and immunofluorescence-based identification of cardiomyocytes and subtypes.

hPSCs were maintained under xeno-free conditions in our recently developed StemMACS iPS-Brew XF medium to keep pluripotency for more than >20 passages and enable for efficient cardiac differentiation. We chose a monolayer differentiation protocol based on the timely regulated activation and inhibition of Wnt signaling by small molecules.

In order to identify antibodies suitable for cardiomyocyte enrichment or depletion of non-myocytes, we performed a surface marker screen with more than 400 antibodies between days 10-20 of differentiation. Besides identification of new surface markers, our screen confirmed expression of recently published markers like CD172a and CD106. In order to evaluate kinetics of their expression and correlation with intracellular markers of cardiomyocytes and subtypes, we performed a flow cytometry-based analysis of marker expression. Our data indicate a dynamic expression pattern for both CD172a and CD106, either completely or partially overlapping with intracellular cardiomyocyte marker expression. Based on these data we developed magnetic cell separation procedures for the isolation of cardiomyocytes. Magnetically enriched cardiomyocytes initiated contractions after replating and could be stably maintained in culture.

Taken together, we have developed novel tools supporting the workflow for efficient generation of PSC-derived cardiomyocytes, magnetic purification and flow cytometry or immunofluorecence-based characterization of cardiomyocytes.
Abstract 62: Cardiac Troponin T Isoform Switching in Early Childhood Tropomyosin-linked Dilated Cardiomyopathy
Authors: Lynn Melissa , Tal-Grinspan Lauren , Jin J.-P. , Tardiff Jil
Published: July 2021

An oft-noted component of sarcomeric DCM is the observation that patients within families carrying the same primary mutation exhibit significant phenotypic variability. This lack of a distinct link between genotype and phenotype has complicated clinical management. In a recent study of two unrelated multigenerational families with the tropomyosin (Tm) mutation Asp230Asn (D230N), a striking “bimodal” distribution of severity was observed. In these families, many children (<1 year) with the mutation presented with a severe form of DCM that led to sudden, often fatal CHF while adults developed a mild to moderate DCM in mid-life. Of note, children who survived the initial presentation often recovered significant systolic function into young adulthood. A potential hypothesis to explain this improvement despite the continued presence of the mutant Tm, is that the phenotype is modified by other thin filament isoforms. Thus we propose that the age-dependent remodeling seen in children with D230N Tm is a result of temporal isoform switches involving a closely linked Tm binding partner cardiac Troponin T (cTnT). Our initial biophysical studies (Regulated-IVM) revealed a decreased Ca2+ sensitivity in filaments containing D230N Tm that is more severe in the presence of fetal TnT (cTnT1), suggesting a modulatory role for cTnT1. Cardiac performance, assessed via 2D echo, in our novel D230N Tm x cTnT1 double transgenic (DTg) mouse model found a significantly reduced % FS for DTg (17%) mice as compared to D230N Tm (21%) littermates. This reduction in %FS was seen at 4 months but not 2 suggesting a progressive cardiomyopathy. Current efforts aim to model the early phase of this “bimodal” phenotype and assess the potential for disease reversibility using a cardiac specific inducible cTnT1 transgenic mouse model. Furthermore, we propose that modulation by cTnT1 could represent a more general mechanism for the progressive remodeling seen in human heart failure. Preliminary in vitro studies with human tissue found that RNA levels of cTnT1 are significantly higher in failing hearts as compared to non-failing. Thus these data suggest an isoform dependent mechanism for the “bimodal” phenotype in patients carrying D230N Tm that could translate to other sarcomeric cardiomyopathies.
Abstract 68: Endostatin Promotes Proliferation and Migration of Rat Myofibroblasts Isolated From the Infarcted Area After Myocardial Infarction
Authors: Okada Muneyoshi , Hirano Yuka , Yamawaki Hideyuki
Published: July 2021

Background:
Endostatin, a 20 kDa non-collagenous fragment of type XVIII collagen, is known as an endogenous anti-angiogenic factor. In the heart tissues of experimental heart failure models, such as cardiac hypertrophy and myocardial infarction, the expression levels of endostatin increase. Proliferation, migration and collagen synthesis of myofibroblasts are important processes during the tissue remodeling in injury sites after myocardial infarction. However, the effects of endostatin on cardiac myofibroblasts have not been clarified. We investigated the effect of endostatin on the functions of myofibroblasts isolated from infarcted myocardial tissues of rat.

Methods and Results:
Left ventricular myocardial infarction was induced by ligation the left anterior descending coronary artery of male Wistar rats. The infarcted myocardial tissues were harvested 2 weeks after the operation and placed on the culture plate with serum-containing medium. Migrated cells from the tissues were isolated and used as myofibroblasts. High expression of α-smooth muscle actin, vimentin and type I collagen in these cells were confirmed by immunofluorescence staining. Cell counting assay was performed to determine a cell proliferation. Endostatin (100-3000 ng/ml, 48 h) increased the proliferation of myofibroblasts. Boyden chamber assay was performed to measure a cell migration. Endostatin (300-3000 ng/ml, 24 h) stimulated the migration of myofibroblasts. Western blotting was performed to measure a secretion of type I collagen. Endostatin (100-3000 ng/ml, 24 h) had no influence on it.

Conclusions:
These data suggest that endostatin might promote scar formation after myocardial infarction through the activation of proliferation and migration of myofibroblasts.
Abstract 78: Transforming Growth Factor-β Induces the Cardiac Fibrosis and Remodeling in Guanylyl cyclase/Natriuretic Peptide Receptor-A Gene-Disrupted Null Mutant Mice
Authors: Pandey Kailash N , Subramanian Umadevi
Published: July 2021

Genetic disruption of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene (Npr1) in mice exhibits high blood pressure, cardiac hypertrophy, fibrosis, and remodeling leading to congestive heart failure. The objective of this study was to determine the mechanisms regulating the development of fibrosis in Npr1 gene-disrupted mice hearts. The Npr1 null mutant (Npr1-/-, 0-copy), heterozygous (Npr1+/-, 1-copy), and wild-type (Npr1+/+, 2-copy) mice were administered by oral gavage with transforming growth factor-β1 (TGF- β1) receptor inhibitor GW788388 (1mg/kg/day) for 28 days. The heart tissues were isolated and used for quantification of fibrotic markers by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot analyses. Together, systolic blood pressure (SBP), heart weight-to-body weight (HW/BW) ratio, left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVEDS), and percent fractional shortening (FS) were analyzed. The Npr1-/- null mutant mice hearts displayed 6-fold induction of fibrosis compared with wild-type (WT) Npr1+/+ mice. Furthermore, the increased expression of fibrotic markers as observed, including connective tissue growth factor (CTGF, 5-fold), α-smooth muscle actin (α-SMA, 4-fold) and TGF-β receptor I (TGF-βRI, 4-fold), TGF-β receptor II (TGF-βRII, 3.5-fold) and Smad2/3 proteins in Npr1-/- mice hearts compared with WT control mice. However, treatment with TGF-β receptor antagonist, GW788388, significantly prevented the cardiac fibrosis and down-regulated the expression of fibrotic markers and Smad proteins in Npr1-/- mice compared to vehicle-treated WT controls. The results of the present study suggest that the activation of cardiac fibrosis in Npr1-/- mice is mainly triggered through TGF-β mediated Smad-dependent pathways.
Abstract 81: Beta-catenin Loss-of-function in Cardiac Fibroblasts Attenuate Cardiac Fibrosis After Transverse Aortic Constriction in Mice
Authors: Xiang Fuli , Fang Min , Yutzey Katherine
Published: July 2021

Background:
Cardiac fibrosis increases myocardium stiffness, impairs cardiac function and contributes to heart failure. Activated fibroblasts produce excessive extracellular matrix leading to fibrosis and impaired cardiac function. Pathologically activated canonical Wnt signaling has been implicated in the pulmonary-, renal-, dermal- and liver fibrosis as well as in scarring after myocardial infarction. We hypothesize that Wnt/β-catenin signaling contributes to cardiac fibroblast (CF) activation and β-catenin loss-of-function (βLOF) in CFs reduces fibrosis and preserves cardiac function.

Methods and Results:
The role of Wnt/β-catenin signaling in CF activation was studied using in vitro CF culture and in vivo genetic manipulation. Postnatal (P)0, P8 and P60 CFs were isolated and cultured. Wnt1 treatment significantly activated CFs, while Wnt inhibition (XAV) completely blocked Wnt1-induced CF activation as determined by αSMA staining. Interestingly, XAV also partially abrogated CF activation induced by TGFβ. In vivo, PeriostinMerCreMer (Pn) or Tcf21MerCreMer (T21) mice were crossbred with βLOF and ROSAmTmG. At baseline, CFs were labeled by T21 but minimal activity was observed with Pn. Accumulation of CFs labeled by Pn or T21 Cres was observed in myocardium 8 weeks after TAC. Inducible CF-specific βLOF transgenic mice (Pn-βLOF or T21-βLOF) were subjected to transverse aortic constriction (TAC) or sham surgeries. βLOF was induced post-surgery via tamoxifen. In sham-operated mice, no difference in cardiac function or morphology was observed between βLOF and control mice. In mice subjected to TAC, cardiac function, as measured by echocardiography, was significantly improved, and the interstitial and peri-vascular fibrosis, as well as heart weight to tibia length ratios, were significantly reduced in the βLOF group, compared to controls, 8 weeks after TAC. Thus, induction of βLOF in CFs after TAC leads to improved cardiac performance, decreased fibrosis, and reduced maladaptive cardiac remodeling.

Conclusions:
βLOF in CFs inhibits myo-fibroblast activation, reduces cardiac fibrosis and preserves cardiac function in TAC induced cardiac injury.
Abstract 85: A Novel Mitochondrial Targeted Fusion Protein Containing Endonuclease III Protects the Heart Against Myocardial Infarction and Pressure Overload Heart Failure
Authors: Bradley Jessica M , Otsuka Hiroyuki , Organ Chelsea L , Bhushan Shashi , Polhemus David J , Wilson Glenn L , Lefer David J
Published: July 2021

Background:
Oxidative stress is a primary cause of mitochondrial DNA (mtDNA) damage and plays a role in myocardial cell death. mtDNA repair enzymes are crucial for mtDNA repair and cell survival. We tested the efficacy of a novel, mitochondrial targeted fusion protein that traffics Endonuclease III (Exscien1-III) in murine models of myocardial ischemia/reperfusion (MI/R) injury and transverse aortic constriction (TAC) heart failure (HF). We previously demonstrated that Exscien1-III administered at R reduced myocardial infarct size and preserved left ventricular ejection fraction (LVEF) following MI/R. We hypothesized that delayed administration of Exscien1-III would promote mtDNA repair and protect the myocardium against MI/R and TAC heart failure.

Methods:
Male C57/BL6J (10-12 wks) were subjected to 45 min of MI and 24 hrs of R. Exscien1-III (4 mg/kg, i.p., n=13) or vehicle (VEH, n=13) was administered 30 min after R. Male C57/BL6J were subjected to TAC (27 g needle) and Exscien1-III (4 mg/kg/d, i.p., n=10) or VEH (n=6) were administered starting at 3 wks post TAC. Echocardiography was performed at baseline and following TAC to assess LVEF.

Results:
Exscien1-III reduced myocardial INF/AAR by 24% (p < 0.05 vs. VEH). Exscien1-III preserved LVEF (49.1 ± 4.0% vs. 32.9 ± 3.2%, p < 0.01) and reduced LV dilation (LVEDD/LVESD; 3.8/2.8 vs. 4.4/3.4, p < 0.05) at 8 wks compared to vehicle.

Conclusion:
These results demonstrate that delayed administration of Exscien1-III significantly attenuates myocardial cell death and preserves LV function in acute MI and HF. Studies are currently underway to define the molecular mechanisms involved in Exscien1-III induced cardioprotection.
Abstract 10: Increasing Cardiac Fatty Acid Oxidation Protects Against High Fat Diet Induced Cardiomyopathy in Mice
Authors: Shao Dan , Kolwicz Stephen C , Roe Nathan , Villet Outi , Tomasi Loreta D , Braun Alyssa N , Tian Rong
Published: July 2021

In the obese and diabetic heart, an imbalance between fatty acid uptake and fatty acid oxidation (FAO) promotes the development of cardiac lipotoxicity. We previously demonstrated that cardiac-specific deletion of ACC2 in adult mice was effective in increasing myocardial FAO while maintaining normal cardiac function and energetics. In this study, we tested the hypothesis that ACC2 deletion in an adult heart would prevent the cardiac lipotoxic phenotype in a mouse model of diet-induced obesity. ACC2 flox/flox (CON) and ACC2 flox/flox-MerCreMer+ (iKO) were injected with
tamoxifen
and subjected to a high fat diet (HFD) for 24 weeks. HFD induced similar body weight gain and glucose intolerance in CON and iKO. In isolated Langendorff-perfused heart experiments, HFD feeding increased FAO 1.6-fold in CON mice which was further increased to 1.9-fold in iKO mice compared with CON on chow diet. HFD induced systolic and diastolic dysfunction was abolished in iKO mice compared with CON mice (Fractional shortening 32.8±2.8% (CON) vs. 39.2±3.2% (iKO), E’/A’ ratio 0.91±0.09 (CON) vs. 1.11±0.08 (iKO), p< 0.05, n=5-6). Heart weight /Tibia length ratio was significantly higher in CON than iKO mice after HFD feeding (7.19±0.22 vs. 6.47±0.28, p<0.05, n=6). These data indicate that elevated myocardial FAO per se does not cause the development of cardiac dysfunction in obese animals. In fact, enhancing FAO via ACC2 deletion prevents HFD induced cardiac dysfunction and attenuates pathological hypertrophy. Molecular markers for ER stress such as p-PERK (1.5 fold) and p-JNK (2 fold) was elevated in CON-HFD hearts, which was completely attenuated in iKO-HFD hearts. Impairment of autophagy was also observed in CON-HFD hearts evidenced by decreases in LC3 II (60%) and increases in P62 (75%) level, while no difference in autophagy were observed in iKO-HFD hearts compared to iKO-chow. Therefore, the beneficial effect for enhancing cardiac fatty oxidation in HFD induced obesity model may be mediated, in part, by maintenance of cellular homeostasis and survival through regulating ER stress and autophagy. Taken together, our findings suggest that promoting cardiac FAO is an effective strategy to resist the development of cardiac lipotoxicity during diet-induced obesity.
Abstract 131: Anti-inflammatory and Mitochondrial Effects of Butyrate in Shr Astrocytes
in vitro
Authors: Yang Tao , Redler Ty , Bueno Silva Carla G , Arocha Rebeca , Schmidt Jordan , Martyniuk Christopher , Zubcevic Jasenka
Published: July 2021

Emerging evidence demonstrates a significant link between gut dysbiosis and hypertension (HTN). Butyrate is one of the major fermented end-products of gut microbiota that reportedly produces beneficial effects on the immune system and metabolism. A contraction in butyrate-producing bacteria in the gut of spontaneously hypertensive rats (SHR) suggests that reduced butyrate may be associated with HTN. Considering its role in mitochondrial metabolism, we proposed that the positive anti-inflammatory effects of butyrate may be mediated via improvement in mitochondrial function in astrocytes.

Methods:
Sprague Dawley (SD) and SHR primary astrocytes from two-day old pups were cultured in DMEM, supplemented with 10% FBS and 1% pen/strep, for 14 days, prior to treatment with butyrate (0-1mM) for 4 hours. Cells were then subjected to the Seahorse XFe24 Extracellular Flux Analyzer to evaluate mitochondrial function following butyrate treatment. Additional samples were collected for total RNA isolation for real time PCR analysis of inflammatory factors and transcripts related to mitochondrial function and stress.

Results:
Butyrate significantly increased both basal and maximal mitochondrial respiration (by 3-4 fold, P<0.001) and elevated proton leak (by 4 fold, P<0.01) in astrocytes from SD rats but not SHR. Furthermore, we observed a trend for an increase in both ATP-linked and non-mitochondrial respiration in SD astrocytes compared to SHR (by 2-3 fold, P=0.07). This was associated with a significant reduction in relative expression levels in catalase (by 50%, P<0.05) and a trend in reduction in Sod1 and Sod2 (by 25%-50%, P=0.1) in astrocytes harvested from SD rats but not the SHR. Conversely, butyrate significantly lowered expression of pro-inflammatory
Ccl2
(by 33%, P<0.05) and
Tlr4
(by 48%, P <0.05) in astrocytes of SHR, but not SD rats.

Conclusion:
Butyrate modulated mitochondrial bioenergetics in SD but not the SHR, suggesting that the mitochondria of astrocytes may be less sensitive to the effects of butyrate in HTN. In addition, butyrate reduced inflammatory mediators in the SHR, but had no effect in the SD rat astrocytes. Thus, central anti-inflammatory effects of butyrate may be mediated via a mitochondria-independent mechanism.
Abstract 144: Cardiac Expression of Mitochondrial Acetyltransferase Gcn5l1 Contributes to Ischemia-Reperfusion Injury and Alters Mitochondrial Energetics After Injury
Authors: Manning Janet R , Thapa Dharendra , Zhang Manling , Stoner Michael W , Scott Iain
Published: July 2021

Introduction:
The increasing global burden of ischemic heart disease demands a closer examination of the mechanisms by which myocardial reperfusion produces injury to initiate long-term heart failure. Reactive oxygen species (ROS) generated after ischemia reperfusion (IR), in conjunction with the dysfunction of mitochondrial metabolic enzymes, have been identified as a primary mediator of cardiac reperfusion injury. The acetylation of mitochondrial proteins, regulated by opposing actions of NAD
+
-dependent sirtuin deacetylases and the recently identified mitochondrial acetyltransferase GCN5L1, has emerged as a key point of intersection between nutrient status and mitochondrial protein function in cardiomyoctyes. This makes the association between acetylation and ROS production an important topic of investigation. Intriguingly, global protein acetylation was recently reported to be upregulated in the hearts of human patients with ischemic heart failure. Despite this, it remains unknown whether GCN5L1 acetyltransferase activity plays a role in the regulation of metabolic proteins during IR injury.

Hypothesis:
Cardiac deletion of the acetyltransferase GCN5L1 reduces the acetylation of mitochondrial proteins during IR, reducing aberrant activity and preventing ROS production.

Methods:
Isolated work-performing hearts from cardiac-specific inducible GCN5L1 knockout mice were subjected to global ischemia and reperfusion. Contractility (+/- dP/dT) of the left ventricle was measured throughout as an index of post IR functional recovery. Tissue damage was assessed by measuring the release of lactate dehydrogenase and post-reperfusion staining of viable tissue with triphenyltetrazolium chloride. Acetylation levels of mitochondrial proteins were measured during IR using immunoblotting of homogenized hearts, which were also used to evaluate ROS production.

Results and Conclusions:
Mitochondrial acetylation was decreased in GCN5L1 hearts compared to WT, coinciding with improved post-IR recovery. We therefore conclude that acetylation of mitochondrial proteins by the acetyltransferase GCN5L1 is an important regulatory mechanism of IR-induced, ROS-mediated damage.
Abstract 190: Mesenchymal Stromal Cells (MSCs) Regulate Neutrophil Extracellular Traps (NETs) Through Adenosine
Authors: Levit Rebecca , Shin Eric , Wang LanFang , Xu Kai
Published: July 2021

Background:
NETs released by neutrophils may be an important component of inflammation in MI/R. NETs consist of extruded DNA, histone, and other chromatin components and are pro-thrombotic and pro-inflammatory. MSCs as a cell therapy for MI/R may act by modulating the innate immune response including NETs. MSCs convert pro-inflammatory ATP into anti-inflammatory adenosine (ADO) via CD 73, a 5’ ectonucleotidase.

Objective:
To investigate the role of MSCs in regulating NETs through ADO production.

Methods:
Neutrophils were freshly isolated from peripheral blood of healthy donors. Human bone marrow derived MSCs were grown under standard conditions. Neutrophils were stimulated to produce NETs by PMA (1 μg/ml) and quantified by Sytox green fluorescence. Some neutrophils were also treated with MSCs or ADO.

Results:
Neutrophils treated with PMA had a 1.9±0.19, (p<0.05) fold increase in NET formation as quantified by Sytox green florescence. Treatment with MSCs in 1:10 ratio prevented increased NET production in response to PMA (1.02±0.12 fold increase). Pretreatment of MSCs with a CD 73 inhibitor APCP reduced their ability to prevent NET formation in some donors (1.62±0.10). ADO decreased NET formation in a dose dependent manner.

Conclusion:
MSCs may inhibit NET formation through ADO signaling and could be an important mechanism of MSC anti-inflammatory effects in MI/R. Future studies will investigate the ability of MSCs to inhibit NET formation in MI/R.
Abstract 226: Elucidating the Mechanism of Reduced Length-dependent Activation Due to a Dilated Cardiomyopathy-associated Mutation in Tropomyosin
Authors: Powers Joseph D , Moussavi-Harami Farid , Razumova Maria , Tardiff Jil , Regnier Michael
Published: July 2021

At the subcellular level, the Frank-Starling law of the heart is described by an increase in calcium sensitivity and force with increased sarcomere length (SL). We examine how this relationship is affected by a dilated cardiomyopathy-associated mutation in tropomyosin (D230N, denoted Tm
D230N
) by measuring contractility of intact and permeabilized cardiac muscle preparations at short (2.0 μm) and long (2.3 μm) SL. Transgenic mouse hearts containing the Tm
D230N
mutation have significantly dilated hearts and reduced cardiac output by ~6 months of age. Intact trabeculae were electrically stimulated and paced at 1 Hz with oxygenated solution (30°C) circulating through the experimental chamber, and permeabilized preparations were bathed in solutions (15°C) of progressively increased [Ca
2+
] for measures of steady-state force. For intact muscle we found that the Tm
D230N
mutation results in significantly reduced twitch forces at SL 2.0 and 2.3 μm relative to wild-type (WT). Also, WT trabeculae displayed a significant increase in twitch force upon increase in SL (as expected) but Tm
D230N
trabeculae did not, demonstrating a loss of SL dependence of contraction. In permeabilized preparations, maximal activation (pCa 4.5) of both WT and Tm
D230N
preparations exhibited significant SL-dependent increases in force. However, at submaximal Ca
2+
(pCa 5.8), where the heart operates, WT preparations had significant increases in force with increasing length (comparing SL 2.0 to 2.3 μm), while this length-dependence of force augmentation in Tm
D230N
was absent. The increase in pCa
50
(pCa that produces half-maximal force) going from SL 2.0 to 2.3 μm was significantly less for Tm
D230N
preparations compared to WT, owing to a significantly smaller increase in pCa
50
at SL 2.3 μm (the pCa
50
at SL 2.0 μm was not significantly different between WT and Tm
D230N
). These results suggest that the Tm
D230N
mutation limits an increase in the Ca
2+
sensitivity of contraction as the muscle lengthens by damping thin filament activation. To further examine length-dependent effects of the Tm
D230N
mutation, future experiments will test conditions that augment cross-bridge binding/inhibition, and other models of dilated cardiomyopathy that inhibit thin filament activation. Funding: HL111197
Abstract 229: Searching Genetic Modifiers For
bag3
-based Cardiomyopathy Using Adult Zebrafish Models
Authors: Ding Yonghe , Dvornikov Alexey , Ma Xiao , Zhang Hong , Xu Xiaolei
Published: July 2021

We recently developed a forward mutagenesis screening strategy in adult zebrafish to screen gene-breaking transposon (GBT) mutants, and identified four genetic modifiers for doxorubicin-induced cardiomyopathy. However, it remains unclear whether these genetic modifiers identified from an acquired cardiomyopathy model exert similar modifying effects on inherited cardiomyopathy models. To address this question, we generated BCL2-associated athanogene 3
(bag3)
gene knockout in adult zebrafish, using the transcription activator-like effector nucleases (TALEN) genome editing technology. In the
bag3-/-
fish, progressive cardiac phenotypes reminiscent of human cardiomyopathy such as fetal gene activation, myofibril loss and cardiac dysfunction were detected. At the single myofibril level, reduced active contractility was observed, supporting the dilated cardiomyopathy (DCM)-like phenotype. Based on the ejection fraction index quantified using a newly developed
ex vivo
assay, different pathogenesis stages including pre-DCM, early-DCM and late DCM were defined. Next, we assessed the potential modifying effects of the four DIC-modifying mutants on
bag3
-based cardiomyopathy model. Different from the other three GBT mutants,
GBT0411+/-
, which tags the long isoform of
dnajb6
b gene, dramatically accelerated the cardiac dysfunction and fish mortality in the
bag3-/-
fish, suggesting
dnajb6b
as a sensitive genetic modifier for bag3-based cardiomyopathy. Mechanistically, we showed that Bag3 physically interacts with Dnajb6, and we hypothesize that impaired autophagy and/or endoplasmic reticulum stress convey the synergistic cardiac dysfunction and fish mortality phenotypes in the
GBT0411+/-;bag3-/-
double mutants. In summary, this study demonstrates that an inherited cardiomyopathy model can be established in an adult zebrafish, which can be utilized to search genetic modifiers. Future studies employing this simple vertebrate model amenable to forward mutagenesis screening promise systematic identification of genetic modifiers for different types of cardiomyopathies, a foundation for individualized medicine.
Abstract 246: Rescuing an Acquired Cardiac Retinoic Acid Deficiency Prevents Hypertrophy and Sudden Cardiac Death in a Pressure Overload/Chronic Catecholamine Model of Hypertrophy and Heart Failure
Authors: Yang Ni , Liu Ting , O’Meally Robert , Kammers Kai , Bedja Djahida , Talbot C. Conover , Cole Robert N , Kass David A , Kane Maureen , O’Rourke Brian , Foster D Brian
Published: July 2021

Introduction:
Recently, we have investigated a pressure-overload/chronic catecholamine guinea pig model (ACi) of cardiac hypertrophy (HYP) and HF with the unique features of acquired long QT syndrome and sudden cardiac death (SCD) by quantitative global-scale proteomics using isobaric tags for relative and absolute quantitation.

Hypothesis:
By compiling proteins altered significantly both compensated HYP and HF, it may be possible to identify novel early contributors to pressure overload- induced HF pathogenesis.

Results:
Pathway mapping of proteins differentially regulated in HYP that were also changed in HF (p<0.05 in both HYP and HF) revealed altered “retinoate biosynthesis” (p<0.01) and “RAR Activation” (p<0.05), suggesting that guinea pig HYP &HF might be related to impaired Vitamin A metabolism, specifically, to a deficit in the bioactive metabolite,
all-trans
retinoic acid. Causal Regulator Analysis indicated that coordinate regulation of ATRA-responsive proteins was unlikely to have occurred by chance (p=1.88x10
-11
) and that the ATRA program was downregulated (z-score<-1.2). ATRA deficit would attenuate transcriptional programs that control fatty acid oxidation, excitation/contraction-coupling, contraction, and antioxidant defense - all of which were decreased in heart failure. Metabolomic profiling by mass spectrometry showed that ATRA, alone among the resident cardiac retinoids, was downregulated in HYP and HF (Down 32% & 33% respectively p<0.05). Secondly, the specific agonist of RXR signaling,
9-cis
RA, is not present in adult guinea pig hearts. Proteome and transcriptome data indicate that the ATRA deficit stems from downregulation of retinaldehyde dehydrogenase 1 (RALDH1) by 23% in HYP and 39% in HF (p<0.01 each). Treatment with ATRA (2mg/kg/day) mitigated heart weight/tibia length (ACi: 0.71 , ACi + ATRA: 0.57; p<0.01), improved fractional shortening (27.4% vs 37.5%, p<0.01) and ejection fraction (50.4% vs. 64.8%, p<0.01). Preliminary studies indicate that ATRA treatment also reduces the incidence of sudden cardiac death (55% vs 15% (p<0.01)) in the guinea pig ACi model.

Conclusion:
The data support a causal role for ATRA deficiency in the pathogenesis of pressure overload-induced HF and susceptibility to SCD.
Abstract 253: HSPB7 is Required for Maintaining the Intercalated Disc Structure to Prevent Cardiac Conduction System Failure in Mouse
Authors: Liao Wern-Chir , Juo Liang-Yi , Chen Yen-Hui , Yan Yu-Ting
Published: July 2021

HSPB7 is belonged to small heat-shock protein (HSPB) family and considered to function as a co-chaperone, which prevents protein aggregation and maintains protein structure. Single-nucleotide polymorphisms of HSPB7 associated with sporadic cardiomyopathy and heart failure have been identified in human patients. Additionally, HSPB7 is constitutively expressed in heart and rapidly increased in blood plasma after myocardial infarction, suggesting a functional role in the heart. In this study, we found that HSPB7 is highly colocalized with N-cadherin during the assembly and maturation of intercalated disc, suggesting that HSPB7 may involve in organizing and maintaining the cardiac cytoarchitecture. To elucidate the physiological function of HSPB7 in the adult heart, we generated a cardiac-specific inducible HSPB7 knockout mouse. Ablation of HSPB7 in the cardiomyocyte rapidly leads to heart failure, abnormal conduction properties and sudden arrhythmias death. Loss of HSPB7 did not cause significant changes in the organization of contractile proteins in sarcomeres, whereas severe abnormality in the intercalated disc was detected. The expression of connexin 43, a gap-junction protein located at the intercalated disc, was downregulated in HSPB7 knockout cardiomyocytes. Mislocalizations of desmoplakin (desmosomal proteins), and N-cadherin (adherens junction proteins) were also observed in the HSPB7 CKO hearts. Furthermore, filamin C, the interaction protein of HSPB7, was mislocalized and aggregated in HSPB7 mutant cardiomyocytes. The expressivity of the phenotype in the HSPB7 CKO mice is similar to human arrhythmogenic cardiomyopathy patients. Conclusively, we provide the first study characterizing HSPB7 as an intercalated disc protein. Our findings demonstrate that HSPB7 plays an essential role to maintain the structure and function of gap-junction complexes and intercalated disc and has vital implications for human heart disease.