bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract MP216: Identification Of Novel Phosphoprotein Signaling Pathways In Human Dilated Cardiomyopathy By Integrative Proteomic And Phosphoproteomic Analysis
Authors: Reitz Cristine J , Tavassoli Marjan , Kim Da Hye , Hadipour-Lakmehsari Sina , Shah Saumya , Lee Shin-Haw , Teng Allen C , Emili Andrew , Oudit Gavin Y , Kuzmanov Uros , Gramolini Anthony O
Published: November 2021

Dilated cardiomyopathy (DCM) is one of the most common causes of heart failure, yet the majority of the underlying signaling mechanisms remain poorly characterized. Protein phosphorylation is a key regulatory element with profound effects on the activity and function of signaling networks; however, there is a lack of comprehensive phosphoproteomic studies in human DCM patients. We assessed the hypothesis that an integrative phosphoproteomics analysis of human DCM would reveal novel phosphoprotein candidates involved in disease pathophysiology. Combined proteomic and phosphoproteomic analysis of explanted left ventricular tissue samples from DCM patients (
n
=4) and non-failing controls (
n
=4) identified 5,570 unique proteins with 13,624 corresponding phosphorylation sites. From these analyses, we identified αT-catenin as a unique candidate protein with a cluster of 4 significantly hyperphosphorylated sites in DCM hearts (
P
<0.0001), with no change in total αT-catenin expression at the protein level. Bioinformatics analyses of human datasets and confocal imaging of human and mouse cardiac tissue show highly cardiac-enriched expression of αT-catenin, localized to the cardiomyocyte intercalated disc. High resolution 3-dimensional reconstruction shows elongated intercalated disc morphology in DCM hearts (10.07±0.76 μm in controls vs. 17.20±1.87 μm in DCM,
P
<0.05,
n
=3/group), with significantly increased colocalization of αT-catenin with the intercalated disc membrane protein N-cadherin (Pearson’s coefficient 0.55±0.04 in controls vs. 0.71±0.02 in DCM,
P
<0.05,
n
=3/group). To investigate the functional role of cardiac αT-catenin phosphorylation, we overexpressed WT protein vs. non-phosphorylatable forms based on the loci identified in DCM hearts, in adult mouse cardiomyocytes using lentiviral transduction. Confocal imaging revealed significant internalization of the phospho-null form, as compared to the prominent intercalated disc staining of the WT protein (17.78±0.79% of WT vs. 9.25±0.49% of 4A mutant,
P
<0.0001,
n
=50 cells/group). Together, these findings suggest a critical role for αT-catenin phosphorylation in maintaining cardiac intercalated disc organization in human DCM.
Abstract MP217: Nuclease-deficient Cas9 Transcription Factors Restore
Krueppel-like Factor 15
Expression In Stressed Mouse And Human Cardiomyocytes
Authors: Schoger Eric , Rosa Kim , Rocha Cheila , Jassyk Mareike , Doroudgar Shirin , Mueller Oliver , Cyganek Lukas , Zimmermann Wolfram H , Zelarayan Laura C
Published: November 2021

Transcriptional changes in cardiomyocytes drive heart failure progression, however, precise control over endogenous gene expression remains challenging. The expression of Krueppel-like factor 15 (
KLF15
), an evolutionary conserved nuclear and cardiomyocyte specific inhibitor of WNT/CTNNB1 signalling in the heart, is lost upon cardiac remodelling, and accompanied by aberrantly active WNT/CTNNB1 resulting in heart failure progression. We investigated
KLF15
expression dynamics employing CRISPR/Cas9-based tools in mouse cardiomyocytes in vivo and in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) under the hypothesis that re-establishment of
KLF15
levels in myocardial stress conditions prevents heart failure progression. Using a mouse model expressing enzymatically inactive Cas9 (dCas9) fused to transcriptional activators (VPR) under
Myh6
-promoter control, we activated
Klf15
in a murine pressure overload model by transverse aortic constriction. Delivery of Klf15 gRNAs targeted to the
Klf15
promoter region via AAV9 induced
Klf15
expression sufficiently to re-normalize
Klf15
expression to transcript levels comparable to sham surgery hearts. This was accompanied by reduced decrease of fractional shortening as well as reduced cardiomyocyte hypertrophy in stressed
Klf15
re-activated hearts compared to non-trageted (NT) gRNA hearts (n=3-8 per group, echo data from 4 and 8 weeks post-surgery). We achieved titratable
KLF15
activation in dCas9VPR transgenic hiPSC-CM by selection of single and multiple gRNAs (n=3-4 replicates) and used these cells to generate human engineered myocardium by combining hiPSC-CM and fibroblasts which we subjected to isometric contractions in order to induce mechanical stress, which resulted in KLF15 expressional decrease in line with our
in vivo
data. This transcriptional loss was rescued in CRISPR/dCas9VPR hiPSC-CM targeted to the
KLF15
locus compared to controls (n=6-9/2/4 tissues per group/casting sessions/differentiations). Additionally, TGFB1 induced cardiomyocyte stress resulted in decreased
KLF15
expression levels in 2D hiPSC-CM cultures which were rescued by dCas9VPR-
KLF15
targeting (n=3 experiments). In conclusion, we report controllable gene activity by CRISPR/dCas9VPR to restore the loss of
KLF15
in stressed mouse and human cardiomyocytes. We furthermore evaluate the potential to gain full control over gene dose titratability with these models to validate and define novel therapeutic targets for the prevention of heart failure progression.
Abstract MP223: CCR2
+
Macrophages And T-cells Infiltrate The Murine Heart Following Immune Regulator Treatment With CD40
Authors: Jimenez Jesus , Ma Pan , Jing Jing , Bredemeyer Andrea , Lokshina Inessa , Lavine Kory J
Published: November 2021

Immune checkpoint inhibitors have revolutionized cancer treatment but have been associated with severe adverse cardiac events including myocarditis. CD40 agonist (CD40ag) antibodies are immune regulators that have emerged as promising candidates with remarkable efficacy across tumors including those thought to be resistant to established ICIs. To investigate CD40 signaling and its potential for adverse cardiac events, C57BL/6J, CCR2-GFP, and CCR2-knockout mice were injected with CD40ag or isotype antibodies for 7 days. Flow cytometry showed a specific increase in CCR2
+
macrophages that was independent of monocyte recruitment. There was no change in neutrophil recruitment and only a modest increase in dendritic cells was evident. We also observed a significant expansion of CD4 and CD8 T-cells that displayed an effector memory phenotype. Bulk cardiac tissue gene expression analysis (Figure A) revealed that CD40 activation upregulates multiple inflammatory cytokines (Ifng, Tnf, Il-12β) and chemokines (Ccl3, Ccl5, Ccl7, Cxcl9, Cxcl10), which are regulated by interferon gamma and coordinate the activation of APCs and T-cells. Finally, RNA in situ hybridization (Figure B-C) demonstrated increased levels of Cxcl9 (red) expression in cells consistent with CCR2
+
(white) macrophages when comparing isotype control to treatment with CD40ag. These findings reveal that CD40 activation results in a robust expansion of CCR2
+
macrophages and activation of T-cells within the heart, initiating a feed forward loop of activation that is mediated by interferon gamma and generates inflammatory cytotoxic mediators that may lead to myocardial injury.
Abstract MP225: Macrophage-specificα5 Integrin Signaling Protects The Infarcted Heart From Adverse Remodeling By Stimulating The Angiogenic Response
Authors: Li Ruoshui , Chen Bijun , Hanna Anis , Humeres Claudio , Alex Linda , Frangogiannis Nikolaos G
Published: November 2021

Abundant macrophages infiltrate the infarcted heart and play a critical role in repair, remodeling and fibrosis. Macrophages sense changes in the extracellular matrix (ECM) environment through Integrins, thus activating signaling pathways that regulate their function. Analysis of our RNA sequencing data identified integrin α5 (Itgα5) as one of the most upregulated integrin genes in infarct macrophages. Accordingly, we hypothesized that integrin α5 signaling in infarct macrophages transduces ECM-derived signals, regulating responses critical for repair and remodeling of the infarcted heart.In order to study the role of macrophage α5 integrin in the infarcted heart, we generated 2 lines of macrophage-specific α5 integrin KO mice: myeloid cell-specific KOs (Myα5KO, using the lysozyme-M Cre driver) and inducible macrophage-specific α5 KOs (iMaα5KO, using CX3CR1
CreER
). 28 days after infarction, Myα5KO mice had accentuated adverse remodeling, evidenced by increased LVEDV and LVESV, a trend towards reduced ejection fraction. Adverse remodeling in Mya5KO was associated with a marked reduction in microvascular density in the infarct and in the border zone. iMaα5KO mice also exhibited worse remodeling and impaired infarct angiogenesis. A PCR array in infarct macrophages showed that α5 integrin loss was associated with markedly reduced transcription of VEGF-A, and lower levels of the angiogenic CXC chemokines CXCL1 and CXCL2. RNAseq followed by bioinformatic analysis predicted that that Nrf2, Erk5, HIF1a, p38 MAPK, VEGF, RhoA, and PI3K/Akt pathways were inhibited in the absence of α5 signaling in vivo (in infarct macrophages) and in vitro (in bone marrow macrophages undergoing α5 antibody neutralization experiments).In conclusion, α5 integrin promotes an angiogenic VEGF-expressing phenotype in infarct macrophages, protecting the infarcted heart from adverse remodeling. The angiogenic effects of α5 integrin in macrophages may have important therapeutic implications for heart failure patients.
Abstract MP230: Nuclear Rupture In A Mouse Model Of
Lmna
-related Cardiomyopathy Causes Cytoplasmic Exposure Of The Proinflammatory Signaling Protein Hmgb1
Authors: Ikegami Kohta , Stutzman Alexis , Ikegami Sachie , Almakki Omar , Liu Sunny , Burnicka-turek Ozanna , Moskowitz Ivan
Published: November 2021

Cardiomyopathies caused by mutations in
LMNA,
encoding nuclear Lamin A/C, are highly malignant and prevalent. How
LMNA
mutations cause cardiomyopathies remains unknown. We characterized cellular, molecular, and pathological evolution of mouse models of
LMNA
-related cardiomyopathy and provide evidence for a model in which nuclear rupture generates nuclear-localized proinflammatory signaling as a candidate molecular mechanism underlying disease pathogenesis. We observed that cardiomyocyte-specific, tamoxifen-inducible deletion of
Lmna
in adult mice (

Lmna
CMKO

) caused a gradual reduction of Lamin A/C protein at the nuclear lamina, reflecting the slow turnover of Lamin A/C. A modest reduction of Lamin A/C in

Lmna
CMKO

was sufficient to cause extensive fibrosis, reduced ejection fraction, and chamber dilation by 3 weeks after
Lmna
gene deletion.

Lmna
CMKO

cardiomyocytes exhibited localized rupture of the nuclear envelope 2 weeks prior to the development of fibrosis and reduction of ejection fraction. Nuclear rupture in

Lmna
CMKO

was immediately followed by an extensive upregulation of pro-inflammatory gene expression programs. We hypothesized that nuclear rupture might expose nuclear DNA to the cytoplasm thereby activating the pro-inflammatory cGas-STING cytosolic DNA sensing pathway. However, we did not observe localization of the cytosolic DNA sensor cGas to cytoplasmic DNA protruded from the ruptured nuclei in

Lmna
CMKO

cardiomyocytes. Instead, we found that HMGB1, a potent proinflammatory protein normally sequestered in the nucleus, was released from the ruptured nuclei in

Lmna
CMKO

cardiomyocytes. Mass spectrometry identified a strong interaction between Lamin A/C and HMGB1 in normal human fibroblast cells. Our data suggested that Lamin A/C tethers HMGB1 to the nuclear periphery by direct interaction and that reduction of Lamin A/C unleashes HMGB1 to the cytoplasm upon nuclear rupture. Future work will examine the hypothesis that cytoplasmic HMGB1 triggers pathogenic sterile inflammation leading to dilated cardiomyopathies in

Lmna
CMKO

mice. In conclusion, we identified the nuclear rupture-induced cytoplasmic release of HMGB1 as a candidate mechanism underlying
LMNA
-related cardiomyopathies.
Abstract MP241: Roflumilast-mediated Phosphodiesterase 4d Inhibition Reverses Diabetes-associated Cardiac Dysfunction And Remodeling: Effects Beyond Glucose Lowering
Authors: Xu Rui , Fu Jing , Fu Qin
Published: November 2021

Introduction:
Patients with type 2 diabetes (T2DM) have a substantial risk of developing cardiovascular disease. Our recent studies reveal that hyperinsulinemia attenuates cardiac contractility by inducing expression of phosphodiesterase 4D (PDE4D) that increases cAMP degradation. Furthermore, there is growing evidence that PDE4 dysregulation is of pathophysiological importance in metabolic disorders.

Hypothesis:
We propose that inhibition of PDE4D might ameliorate diabetes-associated cardiac dysfunction, in addition to lowering glucose.

Methods:
Male C57BL/6J mice fed with high-fat diet (HFD) were treated with PDE4 inhibitor roflumilast (currently used to treat chronic obstructive pulmonary disease, COPD). Myocardial structure, contractile function and remodeling were evaluated. For mechanistic studies, specific silencing of cardiac PDE4D and overexpression of miR-1 were administrated in mice undergoing high-fat feeding. These studies were complemented by
in vitro
analysis in primary cultured rat cardiomyocytes and cardiac fibroblasts.

Results:
Mice on HFD developed greater body weight compared to normal chow diet-fed mice, manifesting hyperglycemia, hyperinsulinemia, cardiac contractile dysfunction and remodeling, and cardiac PDE4D5 upregulation by 24 weeks. The expression of PDE4D5 was also elevated in human hearts with diabetes. In comparison with vehicle-treated HFD controls, PDE4 inhibitor roflumilast (1mg/kg/day) can prevent and even reverse hyperglycemia and cardiac dysfunction, accompanied by the decrease of cardiac PDE4D expression. In addition, cardiac miR-1 level was reduced in HFD mice, which was restored by PDE4 inhibitor roflumilast treatment. Either cardiac specific PDE4D5 knockdown or miR-1 overexpression significantly reversed cardiac dysfunction in HFD-mice, despite persistence of hyperglycemia and glucose intolerance. Gain- and loss-of-function studies of PDE4D in cardiomyocytes implicated that suppression of PDE4D protected cardiac hypertrophy via SERCA2a-mediated miR-1 restoration. Moreover, inhibition of PDE4D prevented insulin-activated TGF-β1 signaling which promotes miR-1 reduction in cardiac fibroblasts and subsequent fibrosis.

Conclusions:
These studies elucidate a novel mechanism by which PDE4D contributes to HFD-induced heart failure through reducing miR-1 expression in both cardiomyocytes and cardiac fibroblasts and suggest a therapeutic potential of PDE4 inhibitor roflumilast in preventing or treating cardiac dysfunction associated with diabetes.
Abstract MP245: Tet Proteins Regulate Second Heart Field Multipotent Progenitors Differentiating To Myocytes
Authors: Fang Shaohai , Li Jia , Steimle Jeff D , Guo Lei , Yang Yuhan , Hong Tingting , Wang Rui , Han Wei , Zhou Yubin , Wang Jun , Martin James F , Huang Yun
Published: November 2021

DNA methylation and demethylation play an important role in shaping the epigenetic landscape and chromatin accessibility to control gene expression during development in mammals. Ten-eleven Translocation (Tet1, Tet2 and Tet3) is a family of dioxygenases that catalyze DNA methylation oxidation with ultimate DNA demethylation. Our previous study showed that cardiac-specific deletion of Tet2 and Tet3 could disrupt YY1-mediated long range chromatin interactions during heart development and lead to ventricular non-compaction cardiomyopathy. However, it is still unclear whether and how Tet protein mediated epigenetic modifications contribute to cardiac lineage specification during embryonic development.

In this study, we generated cardiac specific Tet1-3 triple deficient (Tet-TKO) mouse lines using various cardiac specific Cres to evaluate the function of Tet protein in regulating cardiac lineage specification. We observed developmental defects at outflow tract (OFT) in Tet-TKO embryos, suggesting that Tet deficiency affects the second heart field (SHF) development. Single cell RNA-seq analysis further revealed the accumulation of multipotent SHF progenitors and subsequent halt of myocyte differentiation upon Tet depletion. At the molecular level, we found that Tet ablation perturbs the transcriptional network of Islet1, a transcription factor that is crucial for cardiac development in embryos. Overall, our study demonstrates a critical role of Tet-mediated epigenetic regulation for embryonic cardiac development.
Abstract MP252: Genetic Architecture Of Heart Mitochondrial Proteome Influencing Cardiac Hypertrophy
Authors: Chella Krishnan Karthickeyan , El Hachem Elie-Julien , Light Christine , Shravah Varun , Anum Diana , Seldin Marcus M , Lee Chi Fung , Parker Benjamin L , James David E , Lusis Aldons J
Published: November 2021

Our lab studies how natural genetic variations affect common diseases using a mouse population called hybrid mouse diversity panel (HMDP). In this study, we have explored the genetic regulation of mitochondrial pathways and their contribution to heart function using an integrative proteomics approach. We first performed a whole heart proteomic analysis in the HMDP (72 strains, n=2-3 mice) and surveyed mitochondrial localization using MitoCarta2.0. We retrieved 840 of these proteins (quantified in ≥50 strains) and performed high-resolution association mapping on their respective abundance levels to the HMDP genotypes. Our analyses identified three genetic loci, located on chromosome (chr) 7, chr13 and chr17, that control distinct classes of mitochondrial proteins as well as heart hypertrophy. Follow-up high resolution regional mapping identified NDUFS4, LRPPRC and COQ7 as the candidate genes for chr13, chr17 and chr7 loci, respectively. All three are associated with heart mass in two independent heart stress models, namely, isoproterenol (ISO)-induced heart failure and diet-induced obesity (DIO) models. Next, to identify the aspects of mitochondrial metabolism regulated by these loci, we constructed co-expression protein networks using weighted gene co-expression network analysis (WGCNA) and identified five modules. Eigengenes, representing the first principal component of two of these modules (Brown and Green), mapped to the same regions as the chr13 and chr17 loci, respectively. DAVID enrichment analyses revealed that the Brown module (72 proteins, 96% overlap with chr13) was highly enriched for complex-I proteins (35 proteins, P = 8.8E-74) and the Green module (44 proteins, 73% overlap with chr17) for mitochondrial ribosomal proteins (25 proteins, P = 1.3E-53). The proteins in the chr7 locus were found primarily in the Turquoise module (393 proteins, 81% overlap) but this module was not enriched for any single mitochondrial protein complex. In summary, we now report the identification of three genetic loci that control distinct classes of mitochondrial proteins as well as heart hypertrophy. Our results provide strong support for a role of the mitochondrial proteome in heart pathophysiology.
Abstract MP263: CD8+ T-cells Regulate Macrophage Recruitment And Retention After Myocardial Infarction
Authors: Munoz Gualberto , Roerden Hallie , Huebsch Penny , DeLeon-Pennell Kristine Y
Published: November 2021

Heart failure is the number one reason for admission, with myocardial infarctions (MI) as the underlying etiology in of 70% cases. Macrophages facilitate cardiac healing after the MI. Resident macrophages die due to the initial ischemic event and are replaced by monocyte-derived macrophages. The adaptive immune system, including cytotoxic CD8+ T-cells, are known regulators of inflammation and are upregulated in the circulation and in the infarct after an MI. Previously we showed mice deficient in CD8+ T-cells were protected from adverse remodeling. We hypothesized that after an MI, CD8+ T-cells regulate the recruitment and retention of monocytes. To test our hypothesis, C57Bl6J (WT; n≥3/day post-MI) and mice deficient in CD8+ T-cells (CD8-/-; n≥3/day post-MI) underwent permanent occlusion and tissue was collected at post-MI days 0 (no MI), 1, 3, 7, and 14. Mac3 staining of the infarct was performed to determine the macrophage time course. Infarct tissue was analyzed by bulk RNAseq (n=3/day/genotype) to determine possible genetic regulators at post-MI days 1, 7, and 14. Data was clustered based on the genetic markers of macrophage subtypes identified in previous single cell genomic studies. Mac3 staining of WT mice showed macrophages begin to infiltrate as early as post-MI day 1, peaking at day 7, and beginning to reach baseline levels by day 14. Interestingly, CD8-/- mice had a delay in macrophage recruitment at day 1; however, by day 3 there was almost double the amount of macrophages compared to the WT group. While no differences were observed at day 7 post-MI, CD8-/- mice had elevated macrophages at post-MI day 14 compared to the WT mice. Based on the gene clusters, the infarct tissue of CD8-/- mice had increased markers of resident-like macrophages at post-MI days 7 and 14 compared to WT mice. Resident macrophages have been shown to be cardioprotective post-MI, suggesting a possible protective mechanism in CD8-/- mice. In conclusion, our data indicates that CD8+ T-cells influence cardiac remodeling over the post-MI time course by facilitating in macrophage recruitment and retention. Furthermore, in the absence of CD8+ T-cells, there was an increase in resident-like macrophages that could prove to point to a more favorable repopulation of cells post-MI.
Abstract MP264: Heterogeneous Distribution Of Mutations In Myosin Binding Protein-c Paralogs
Authors: Desai Darshini a , Rao Vinay , Jegga Anil , Dhandapany Perundurai , Sadayappan Sakthivel
Published: November 2021

Rationale:
Myosin binding protein-C (
MYBPC
) is a sarcomeric protein which regulates the force of contraction in striated muscles. Mutations in the
MYBPC
family of genes, slow skeletal(
MYBPC1
), fast skeletal (
MYBPC2
) and cardiac (
MYBPC3
) can result in cardiac and skeletal myopathies, yet their evolutionary pattern, pathogenicity and impact on
MYBPC
structure remain to be elucidated.

Objective:
To assess conserved and epigenetic patterns of
MYBPC
family mutations.

Methods and Results:
Leveraging a machine learning approach, the Genome Aggregation Database (gnomAD) provided 12353, 6227 and 5279 variants in
MYBPC1
,
MYBPC2
and
MYBPC3
genes, respectively, followed by analysis with the Ensembl variant effector predictor. Missense variants comprised 62-68% of total variants in which the first and second nucleotide (nt) positions in the codons were highly altered, with G/A substitution nt substitution the most predominant. Arginine was the most mutated amino acid, important because most disease-causing mutations in
MYBPC
are arginine in origin. Domains C5 and C6of
MYBPC
were found to be hotspots for most mutations in the
MYBPC
family. A high percentage of truncated mutations in
MYBPC3
cause cardiomyopathies. Arginine and glutamate were the top hits in
MYBPC1
and
MYBPC3,
respectively, and tryptophan and tyrosine were the most common among the three paralogs changing to premature stop codons and causing protein truncations at the carboxy terminus.

Conclusion:
A heterogeneous epigenetic pattern was identified among the three
MYBPC
paralogs. Genomic databases using machine learning approaches can be used to diagnose and design therapeutics to treat muscle disorders caused by
MYBPC
mutations.
Abstract P302: Alpha-1A Adrenergic Receptor Activation Increases Electron Transport Chain Activity In The Uninjured Mouse Heart
Authors: Sandroni Peyton B , Vander Ploeg Matthew R , Huang Wei , Jensen Brian C
Published: November 2021

Decreased electron transport chain (ETC) activity in cardiac mitochondria is a hallmark of heart failure. Gain- and loss-of-function studies define the benefits of alpha-1A adrenergic receptor (α1A-AR) activation in the failing heart, such as increased cardiac contractility. However, the mechanisms behind these effects are unknown, and α1A-AR activation as a method of ETC regulation has not been studied. Here, we assessed the hypotheses that decreased α1A-AR activation reduces ETC enzyme activity, whereas increased α1A-AR activation enhances ETC enzyme activity. We profiled citrate synthase and ETC complex I-IV activities in isolated cardiac mitochondria from (1) wild-type (WT) CL57Bl/6J mice or global α1A-AR knockout mice (10-12 wks) and (2) WT mice (10-12 wks) treated with vehicle (0.9% saline) or the selective α1A-AR agonist A61603 (10 ng/kg/d, 3 d). Citrate synthase, a key enzyme in the citric acid cycle, fuels ETC activity and is a commonly used marker for mitochondrial mass. Global α1A-AR knockout increased citrate synthase activity in male mice compared to WT controls (5,292 ± 275 vs. 4,198 ± 339 nmol/min/mg, n = 5 each group, p = 0.04) (mean ± SEM) (Panel A). When normalized to citrate synthase activity, global α1A-AR knockout decreased complex I (37 ± 10% vs. 64 ± 5%, p = 0.02) (1,786 ± 421 vs. 2,766 ± 422 nmol/min/mg) and complex II (25 ± 9% vs. 50 ± 13%, p = 0.01) (1,332 ± 219 vs. 2,032 ± 213 nmol/min/mg) activities with a trend toward decreased complex IV activity (33 ± 13% vs. 49 ± 17%, p = 0.07) (1,707 ± 201 vs. 2,000 ± 238 nmol/min/mg) (Panel B). A61603 treatment led to a trend towards decreased citrate synthase activity in female mice compared to vehicle controls (6,662 ± 501 vs. 7,701 ± 421 nmol/min/mg, n = 3 each group, p = 0.09) (Panel C). When normalized to citrate synthase activity, A61603 increased complex I (27 ± 3% vs. 17 ± 2%, p = 0.03) (1,736 ± 92 vs. 1,326 ± 156 nmol/min/mg), complex III (61 ± 6% vs. 37 ± 5%, p = 0.02) (3,993 ± 258 vs. 2,894 ± 531 nmol/min/mg), and complex IV (70 ± 6% vs. 48 ± 6%, p = 0.03) (4,631 ± 100 vs. 3,676 ± 533 nmol/min/mg) activities (Panel D). In conclusion, we show that global α1A-AR knockout decreases ETC enzyme activity, while treatment with an α1A-AR agonist increases ETC enzyme activity. These findings may identify a novel mechanism through which α1A-AR activation protects the injured and failing heart.
Abstract P309: Particulate Matter Increases Oxidative Stress And Shortens The Action Potential In IPS-derived Cardiomyocytes
Authors: Argenziano Mariana , yang jiajia , Burgos Angulo Mariana , McDonald Thomas V
Published: November 2021

Introduction:
Air particulate matter (PM) represents one of the most critical environmental issues worldwide, causing more than 3 million deaths a year. In the US, hospital admissions due to heart failure (HF) increase by 0.8% for every 10 μg/m3 elevation in PM. However, the biological mechanisms behind the effects of PM on cardiovascular disease (CVD) remain poorly defined. Recent studies showed that PM
2.5
can translocate into the circulation, causing cumulative toxicity. With air pollution increasing due to human activity and the growing prevalence of HF, there is a critical need to understand PM's contributions to CVD to develop preventive treatments and novel therapeutic approaches.

Hypothesis:
We hypothesize that PM can exert its toxic effect by increasing oxidative stress and apoptosis and affecting cardiac electrophysiology.

Methods:
Three independent induced pluripotent stem cell lines (IPSC) were differentiated into cardiomyocytes (iCMs) and cultured for 30 days before treatment with 100 μg/ml of PM
2.5
for 48h. Experiments including immunostaining, qPCR, RNAseq and Multielectrode Array (MEA) were performed in control (CT) and PM-treated iCMs (PM).

Results:
Treatment with PM increased ROS and decreased ATP production (CT 9.9±1.2pmol vs PM 6.6±0.8pmol, p<0.01, n=20). Immunostaining showed mitochondrial fragmentation and increased expression of cleaved caspase3 without structural changes. Moreover, PM caused upregulation of the apoptotic markers
P53
,
PARP1
and
CASP3,
oxidative stress markers
CYP1A1, CYP1B1
and
MT2A,
and cardiac markers
CACNA1C
together with downregulation of
GJA1
. RNAseq analysis showed upregulation of Gene Ontology terms related to detoxification, response to toxic substances and oxidative stress. Upregulated KEGG pathways included oxidative phosphorylation, hypertrophic cardiomyopathy and dilated cardiomyopathy. MEA experiments revealed a decrease in the spike amplitude and conduction velocity, along with shortening of the action potential (APD90: CT 577±20ms vs. PM 489±16ms, p<0.05, n=20) and increased beat period irregularity (CT 3.2±0.7% vs. PM 13.1±1.6%, p<0.001, n=20). These electrophysiological changes were reversed by treatment with the antioxidant N-acetylcysteine.

Conclusions:
We conclude that PM plays a direct role in the development of CVD, causing an increase in oxidative stress and affecting the electrophysiology of the heart. Further functional studies in iCMs from HF patients will provide evidence of the effects of these changes on the phenotype of the disease.
Abstract P313: Over-expression Of Serine Threonine Kinase 35 Improves Cardiac Function In Streptozotocin Induced Diabetic Mice
Authors: Joladarashi Darukeshwara , Zhu Yanana , Cimini Maria , Thandavarayan Rajarajan , Youker Keith , Nash Kevin , Willman Matthew , Song Xuehong , Ren Di , Ji Li L , Kishore Raj , Krishnamurthy Prasanna , Wang Lianchun
Published: November 2021

Diabetic cardiomyopathy is a common complication in patients with diabetes and is associatedwith impaired responsiveness of ischemic myocardium to proangiogenic factors, subsequentlyleading to heart failure. STK35, a novel kinase that binds to nuclear actin, has been shown toregulate important cellular functions such as cell migration, proliferation, survival, andangiogenesis. Currently, the contribution of altered STK35 expression in human diseases remainsunexplored. In initial studies, we observed that human cardiac biopsies from diabetic patientsshowed a significant decrease in STK35 expression as compared to non-diabetic control hearts.Intriguingly, in a STZ-induced mouse model of diabetes,
i.v
. injection of rAAV9-STK35 to expressconstitutive STK35 in heart in FVB/N male mice promoted neovascularization and lowered cardiacfibrosis, leading to improved cardiac function of diabetic heart. Our
in vitro
studies observed highglucose decreased STK35 expression in mouse cardiac endothelial cells (MCEC), whereasSTK35 overexpression increased MCEC migration and vascular tube formation, and upregulatedMCEC to expression of multiple pro-angiogenic proteins. Taken together, our results demonstratethat cardiac-targeted STK35 gene therapy exerts a marked beneficial action by attenuating bothcardiac remodeling and cardiac function in a mouse model of diabetes mellitus. Mechanistically,the beneficial effect may be attributed, at least partially, to enhanced neovascularization in heart.
Abstract P314: TGFβ2-Mediated Cardiac Fibroblast Activation Is Dependent On BET Bromodomain Proteins
Authors: Antolic Andrew , Jiao Zhe , Ijaz Talha , Gupta Ria , Burke Michael A
Published: November 2021

Heart failure is a common and morbid disease frequently caused by dilated cardiomyopathy (DCM). Fibrotic remodeling is a key feature of DCM. Stress signaling pathways trigger transdifferentiation of quiescent cardiac fibroblasts into myofibroblasts (MF). Bromodomain and extra-terminal (BET) epigenetic reader proteins are stress-responsive distal signaling integrators that drive fibrosis. Here, we explored the role of BETs on distinct inflammatory pathways in ex vivo cultured cardiac fibroblasts. The Langendorff hanging heart procedure was used to isolate and culture primary non-myocytes from wild type hearts, which are almost exclusively fibroblasts. Cells were stimulated with TGFβ2 to trigger MF transdifferentiation. Prior to TGFβ2 treatment, fibroblasts were treated with 1 of the following: vehicle, the BET inhibitor JQ1, or siRNA targeting either
Smad3
or
Rela
. MF-specific gene expression was assessed by Q-PCR. α-smooth muscle actin (αSMA) levels were assessed by immunocytochemistry (ICC) and immunoblotting. NFκB transcriptional activity was evaluated using a dual luciferase reporter assay. TGFβ2 activated MF-specific gene expression (
Acta2
, 9.0-fold;
Col3a1
, 3.6-fold;
Ctgf,
10.1-fold;
Postn
, 12.8-fold; p<0.05 for all vs. vehicle), and increased αSMA levels (1.5-fold by ICC and immunoblotting; p<0.05 vs. vehicle) in cultured primary cardiac fibroblasts. This was ablated by JQ1 and
Smad3
siRNA. By contrast,
Rela
siRNA alone induced MF-specific gene expression (
Acta2
, 6.9-fold;
Col3a1
, 7.0-fold;
Ctgf,
3.7-fold;
Postn
, 11.2-fold; p<0.05 for all vs. vehicle), and increased αSMA staining by ICC (3.4-fold; p<0.05) but not protein (1.3-fold; p=NS). TGFβ2 further potentiated MF-specific gene expression (
Acta2
, 17.1-fold;
Col3a1
, 7.1-fold;
Ctgf,
13.2-fold;
Postn
, 42.5-fold; p<0.05 for all vs. vehicle) and αSMA protein (1.7-fold; p<0.05) in fibroblasts treated with
Rela
siRNA. NFκB transcriptional activity was reduced by TGFβ2 (-2.9-fold; p<0.05) and with JQ1 pre-treatment (-2.7-fold; p<0.05). These data define a critical role for BETs in TGFβ2-mediated gene transcription in cardiac fibroblasts. Surprisingly, NFκB does not appear to be a key activator of TGFβ2-stimulated fibroblast transdifferentiation.
Abstract P318: Dna Tension Probes Show That Cardiomyocyte Maturation Is Sensitive To The Pico-Newton Traction Forces Transmitted By Integrins
Authors: Rashid Sk A
Published: November 2021

Cardiac muscle cells (CMCs) are the unit cells that comprise the heart. CMCs go through different stages of differentiation and maturation pathways to fully mature into beating cells. These cells can sense and respond to mechanical cues through receptors such as integrins which influence maturation pathways. For example, cell traction forces are important for the differentiation and development of functional CMCs, as CMCs cultured on varying substrate stiffness function differently. Most work in this area has focused on understanding the role of bulk ECM (extracellular matrix) stiffness in mediating the functional fate of CMC. Given that stiffness sensing mechanisms are mediated by individual integrin receptors, an important question in this area pertains to the specific magnitude of integrin piconewton (pN) forces that can trigger CMC functional maturation. To address this knowledge gap, we used DNA adhesion tethers that rupture at specific thresholds of force (~12, ~56, and ~160 pN) to test whether capping peak integrin tension to specific magnitudes affects CMC function. The work shows that adhesion tethers with greater force tolerance lead to functionally mature CMCs as determined by morphology, twitching frequency, transient calcium flux measurements and protein expression (F-actin, vinculin, α-actinin, YAP and SERCA2). Additionally, sacromeric actinin alignment and multinucleation were significantly enhanced as the mechanical tolerance of integrin tethers was increased. Taken together, the results show that CMCs harness defined pN integrin forces to influence early stage development. This study represents an important step toward biophysical characterization of the contribution of pN forces in early stage cardiac differentiation.
Abstract P319: SARM1 NAD Hydrolase Deficiency Protects Hearts Against Diabetic Cardiomyopathy
Authors: Light Christine , Lee Chi Fung
Published: November 2021

NAD depletion is associated with the pathogenesis of diseases such as heart failure. Strategies to replenish cellular NAD levels by activating NAD synthesis pathways have shown promises to treat heart disease. However, how NAD consumption mechanisms lead to NAD depletion are less understood. SARM1 is a novel intracellular NAD hydrolase that degrades NAD and promotes axonal degeneration in neurons. We recently showed that NAD redox imbalance and depletion promote the progression of diabetic cardiomyopathy. Therefore, we hypothesized that SARM1 deficiency might protect hearts against diabetic cardiomyopathy. 16-week diabetic stress initiated by streptozotocin (STZ) injections was applied to wild-type C57BL/6 (WT) and whole-body SARM1-KO mice. Cardiac function was measured after 8-week or 16-week of diabetic stress, and cardiac tissue and plasma samples from these mice were harvested after 16 weeks of diabetes. SARM1 mRNA or protein levels were suppressed in SARM1-KO hearts. STZ induced similar hyperglycemia (~600 mg/dL) in both WT and SARM1-KO male mice after 16 weeks. Chronic diabetic stress led to progressive decline in systolic function (Baseline fractional shortening: 50%; 16-week diabetes: 35%; P<0.05; n=5) in WT male mice, which was ameliorated in SARM1-KO male mice (16-week diabetes: 48%; P<0.05; n=5). Progressive decline in diastolic function induced by chronic diabetes (Baseline WT E’/A’: 1.53; WT 16-week diabetes: 1.12; n=5) was also improved in diabetic SARM1-KO mice (KO E’/A’ ratio at 16-week diabetes: 1.5; P<0.05; n=5). Heart weights were similar in diabetic WT or diabetic SARM1-KO hearts. A similar study is on-going in a female cohort. Despite loss of SARM1 expression, no compensatory changes in expressions of other NAD hydrolases (i.e. Cd38 or Bst1) were observed in diabetic SARM1-KO hearts, while expression levels of genes related to NAD consumption and synthesis pathways were mostly unchanged except Qprt and Haao. Cardiac fibrosis was induced in diabetic WT hearts and were suppressed in diabetic SARM1-KO hearts after 16-week diabetic stress, but these changes were not observed in tissues harvested after 8-week diabetic stress. The results suggest fibrosis is a later event in the progression of diabetic cardiomyopathy. WT and SARM1-KO mice have also been challenged with high fat diet feeding (HFD) for 16 weeks, and increased fasting glucose levels and body weights were similarly observed in HFD-WT and HFD-SARM1-KO mice. Longitudinal cardiac function analyses are on-going. Our data thus far support the protective role of SARM1 deficiency in metabolic stress-induced cardiomyopathy, while pathogenic mechanisms of SARM1 in diabetic hearts remain to be determined.
Abstract P321: Chronic Central Nervous System Leptin Infusion Increases Cardiomyocyte Mitochondrial Function And Biogenesis And Improves Cardiac Function After Ischemia-reperfusion Injury.
Authors: Omoto Ana Carolina M , Do Carmo Jussara , Li Xuan , Wang Zhen , Mouton Alan J , Aitken Nikaela , Moak Sydney , Dai Xuemei , Hall John E , Da Silva Alexandre A
Published: November 2021

Mitochondrial (MT) dysfunction plays an important role in the pathophysiology of myocardial ischemia/reperfusion injury (I/R). We recently showed that leptin, via its actions in the central nervous system (CNS), improves left ventricular (LV) function in a model of heart failure induced by permanent ligation of the left anterior descending coronary artery (LAD). In the present study, we examined if the CNS effects of leptin protect against myocardial ischemia/reperfusion (I/R) injury, and whether chronic intracerebroventricular (ICV) leptin infusion increases MT function and biogenesis in the non-infarcted area of the LV that is at risk but still viable. Male Wistar rats were instrumented with an ICV cannula in the brain lateral ventricle. After recovery and baseline assessment of cardiac function by echocardiography (ECHO), myocardial I/R was induced by temporary (60 min) ligation of the LAD. Vehicle (saline, 0.5 μL/hr) or leptin (0.62 μg/day) was infused chronically for 28 days starting 20 min after reperfusion using osmotic minipumps connected to the ICV cannula. ECHO assessment of cardiac function was performed every week. At the end of week 4, +dP/dt
max
and Tau were accessed by LV catheterization. Hearts were then collected for evaluation of MT function in isolated cardiac fibers using Oroboros oxygraphy-2k respirometer. ICV leptin treatment improved systolic and diastolic function as evidenced by increased ejection fraction 4 weeks after I/R (46±3 vs. 26±3 %), stroke volume (353±19 vs. 193±27 μL), +dP/dt
max
(10387±1686 vs. 5022±442 mmHg/s) and reduced Tau (6.5±0.3 vs. 8±0.3 ms) when compared with vehicle-treated rats. In addition, ICV leptin infusion significantly increased ATP-linked respiration (55±3 vs. 39±1 %), reduced proton leak (45±3 vs. 61±1 %), and improved MT reserve capacity (42±5 vs. 27±2 %). Improved MT function was associated with increased MT Complex I- mediated respiration (47±6 vs. 26±1 pmolO
2
/s
-
mg). ICV leptin treatment also increased PGC1
α
protein expression and COX3 gene expression, indicating enhanced MT biogenesis. These results demonstrate that chronic ICV leptin infusion improves cardiac function following I/R injury and suggests that leptin’s CNS-mediated cardioprotective effects may involve improved myocardial MT function and biogenesis.
Abstract P322: Ror2: A Novel Regulator Of Cardiomyocyte Structure And Target Of Right Ventricular Failure
Authors: Edwards Jonathan J , Uchida Keita , Shewale Swapnil V , Yucel Nora , Scarborough Emily A , Yang Yifan , Li Li , Prosser Benjamin , Arany Zoltan
Published: November 2021

Introduction:
We recently identified the fetal noncanonical WNT receptor ROR2 as being re-expressed in human right ventricular failure (RVF), but the myocardial role of ROR2 remains poorly described.

Hypothesis:
We assessed if ROR2 expression influences cardiomyocyte structure or contributes to RVF pathogenesis.

Methods:
ROR2 gain- and loss-of-function (ROR2
GOF
and ROR2
LOF
) NRVMs were generated using adenoviral-delivered ROR2 cDNA or shRNA and cultured on nanopatterned substrates. NRVM structure was assessed by confocal microscopy (minimum n=3 replicates, >30 cells/condition). NRVM gene expression was characterized by RNAseq. The impact of ROR2 on RV structure and function was assessed using AAV9-mediated cardiac ROR2 delivery, in 4-week old C57BL/6 mice (vs GFP), and echocardiography and hemodynamics (n= 8, 50% male).

Results:
ROR2
GOF
disrupts non-sarcomeric and sarcomeric NRVM structure, exhibiting smaller (500 vs. 711 μm
2
, p = 7.4x10
-23
) and rounder (aspect ratio 2.1 vs. 3.1, p=1.9x10
-16
) shape and fragmented sarcomeres (Figure). Whereas, ROR2
LOF
NRVMs demonstrate a striking peripheralization of α-actinin and β-catenin. Gene-set enrichment analyses of ROR2
GOF
NRVMs reveal upregulation of cytokinesis, mitosis, microtubule regulation, and insulin like growth factor receptor signaling. By 4 weeks after induction of ROR2
GOF
in vivo
, mice exhibit biventricular dilation and systolic dysfunction (TAPSE 0.85 vs 1.2 mm, p = 2.1x10
-5
; right ventricular outflow dimension 2.0 vs 1.7 mm, p = 0.001, LV EDV 33.5 vs 28.6 μL, p = 0.05; LV ejection fraction 63% vs 69%, p = 0.015). RV diastolic function is reduced in ROR2
GOF
(E:e’ 21.8 vs 18.6, p = 0.004 and RV EDP 2.9 vs 1.0 mmHg, p = 0.06).

Conclusions:
ROR2
GOF
causes biventricular dilation, systolic and diastolic dysfunction
in vivo
.
In vitro,
ROR2
GOF
NRVMs exhibit an immaturity phenotype with maintenance of proliferation gene program. In contrast, ROR2
LOF
NRVMs form a tight monolayer with enhanced cell-cell borders.
Abstract P328: Regulation Of Monocyte Physiology By Il-4 Stimulated Memory Cd8+ T-cells
Authors: Broughton Philip , Zaidi Yusra , Troncoso Miguel , DeLeon-Pennell Kristine Y
Published: November 2021

Following a myocardial infarction (MI) monocytes and T-cells begin to infiltrate into the ischemic area in effort to remove necrotic debris and initiate formation of scar tissue. Interleukin (IL)-4 has been linked to improved cardiac wound healing via alterations in both the macrophage and T-cell populations. The goal of this study was determine if proteins secreted by IL-4 stimulated CD8+ T-cells would regulate monocyte physiology that would ultimately improve cardiac healing after an MI. Isolated splenic naïve CD8+ T-cells from day 0 (no MI) mice (n=5/sex/stimulation)were cultured in RPMI with either 0.1% FBS (unstimulated) or 0.1% FBS+ IL-4. After 24 hours of stimulation, the cells and media were collected and separated by centrifugation. The cell pellet was stained and analyzed for markers of activation (CD44) and memory (CD27) by flowcytometry. Conditioned media (secretome) was collected for stimulation of bone marrow monocytes (n=4; females only). After stimulation with the secretome, monocytes were analyzed for viability, phagocytosis, and macrophage phenotype by flow cytometry. Migration of the monocytes after stimulation was also measured using electric cell-substrate impedance sensing (ECIS). After IL-4 stimulation, there was a shift from effector (CD44+ CD27-) to the memory phenotype (CD44+ CD27+; p<0.05 vs unstimulated cells). Interestingly, bone marrow monocyte viability was decreased by 15% when stimulated with the secretome of IL-4 treated CD8+ T-cells compared to unstimulated CD8+ T-cells (0.1%). Phagocytosis was slightly elevated though not significant (p=0.07) in monocytes that were stimulated with the secretome from the IL-4 group compared to the unstimulated CD8+ T-cells. No differences were found in expression of macrophage markers F4/80 (p=0.532) or M1 marker CD86 (p=0.471). The secretome of IL-4 stimulated T-cells increased monocyte migration after wounding similar to levels of the positive control (monocytes in 10% FBS only). The data collected showed that IL-4 stimulated CD8+ T-cells were able to upregulate memory marker CD27. These memory-like CD8+ T-cells initiated monocyte phagocytosis and migration but decreased monocyte viability suggesting that they may play a role in regulating macrophage biology post-MI.
Abstract P331: Upregulated TBX1 by genetic variants are associated with human congenital heart disease
Authors: Yu Liwei , Li Binbin , Wang Hongyan
Published: November 2021

Congenital heart disease (CHD) is the most common human birth defect worldwide. The cause of CHD is so far not well understood. Uncovering genetic factors leading to CHD is still a pressing task to be solved. TBX1 is one of the transcription factors early expressed in embryonic cardiac progenitors. In animal models, imbalanced TBX1 activity leads to cardiac defects. Given the dosage effect of TBX1, it is possible that genetic variant altering TBX1 function or expression level would affect heart development and contribute to CHD. In order to study the association of genetic variants of
TBX1
and CHD susceptibility, we performed genetic screening in 409 CHD patients and 213 healthy controls. Bioinformatic and
in vitro
functional studies were performed to evaluate the impact of genetic variants. One single nucleotide polymorphism (SNP), rs41260844, in
TBX1
promoter region was identified to be associated with CHD. The minor allele of rs41260844 is associated with higher CHD risk and shows increased
TBX1
promoter activity (Fig A). Further study showed the minor allele attenuates
TBX1
promoter binding affinity with nuclear protein(s) (Fig B). In addition, a novel case-specific missense rare mutation, p.P164L, in T-box domain was identified and predicted as a deleterious mutation. Functional analysis showed a trend of increased TBX1 function with the rare mutation. In summary, we concluded that a higher TBX1 expression level or activity is associated with CHD susceptibility, which could affect TBX1 downstream targets and thus disrupt the balance of the complex regulation network during cardiogenesis.