bluuebunny/crossref_metadata_2025 huggingface dataset:
Long-Term Changes in Invasive Physiological Pressure Indices of Stenosis Severity Following Transcatheter Aortic Valve Implantation
Authors: Sabbah Muhammad , Joshi Francis R. , Minkkinen Mikko , Holmvang Lene , Tilsted Hans-Henrik , Pedersen Frants , Ahtarovski Kiril , Sørensen Rikke , Thue Olsen Niels , Søndergaard Lars , De Backer Ole , Engstrøm Thomas , Lønborg Jacob
Published: November 2021

Background:

Patients with severe aortic stenosis frequently have coexisting coronary artery disease. Invasive hyperemic and nonhyperemic pressure indices are used to assess coronary artery disease severity but have not been evaluated in the context of severe aortic stenosis.

Methods:

We compared lesion reclassification rates of fractional flow reserve (FFR) and resting full-cycle ratio (RFR) measured before and 6 months after transcatheter aortic valve implantation using the conventional clinical cutoffs of ≤0.80 for FFR and ≤0.89 for RFR. This was a substudy of the ongoing NOTION-3 trial (Third Nordic Aortic Valve Intervention). Two-dimensional quantitative coronary analysis was used to assess changes in angiographic lesion severity.

Results:

Forty patients were included contributing 50 lesions in which FFR was measured. In 32 patients (36 lesions), RFR was also measured. There was no significant change in diameter stenosis from baseline to follow-up, 49.8% (42.9%–57.1%) versus 52.3% (43.2%–57.8%),
P
=0.50. RFR improved significantly from 0.88 (0.83%–0.93) at baseline to 0.92 (0.83–0.95) at follow-up,
P
=0.003, whereas FFR remained unchanged, 0.84 (0.81–0.89) versus 0.86 (0.78–0.90),
P
=0.72. At baseline, 11 out of 50 (22%) lesions were FFR-positive, whereas 15 out of 50 (30%) were positive at follow-up,
P
=0.219. Corresponding numbers for RFR were 23 out of 36 (64%) at baseline and 12 out of 36 (33%) at follow-up,
P
=0.003.

Conclusions:

In patients with severe aortic stenosis, physiological assessment of coronary lesions with FFR before transcatheter aortic valve implantation leads to lower reclassification rate at 6-month follow-up, compared with RFR.
Abstract 105: Novel Functions For Synaptosomal-associated Protein 29 (snap29) In Cardiac Arrhythmias
Authors: Wang Jie , Pellman Jason , Lyon Robert , Liang Yan , Mezzano Valeria , Zanella Fabian , Zhang Jing , Nigam Vishal , Gu Yusu , Dalton Nancy , Peterson Kirk , Sheikh Farah
Published: November 2021

Human genetic studies and mouse models have classically linked mutations/deficiencies in components of the desmosomal cell-cell junction to arrhythmogenic right ventricular cardiomyopathy (ARVC). However, growing evidence points to the importance of the desmosome in cardiac diseases beyond ARVC, which include electrical diseases that have no impact on cardiac structure/morphology. The mechanisms of how defects in cardiac desmosomal protein homeostasis drive these distinct forms of cardiac disease remain elusive to the field. To uncover mechanisms that underlie the distinct pathophysiology (structural versus non-structural) encompassed by desmosomal mutations/loss, we performed an unbiased yeast-two-hybrid screen using an adult human heart cDNA library and the desmosomal protein, desmoplakin (DSP) to uncover new regulators of cardiac desmosomal protein homeostasis. We identified synaptosomal-associated protein 29 (SNAP29), as a novel DSP-interacting protein in the adult human heart. Traditional functions of SNAP29 are to regulate membrane fusion and play a role in autophagy; however, its role at the desmosome and in the heart is undefined. We show that SNAP29 is a subcomponent of the cardiac desmosome, as it co-localizes with DSP in the adult heart and DSP-deficient hearts harbor loss of SNAP29. Cardiomyocyte-specific SNAP29 knockout (SNAP29-cKO) mice displayed baseline and pacing-induced ventricular arrhythmias in an age-dependent manner in the absence of cardiac structural and functional deficits. We show that a loss of a subset of desmosomal proteins and connexin43 as well as upregulation of selective autophagy-mediated degradation underlie SNAP29 deficient cardiomyocyte arrhythmias. In line with this, acute blockade of autophagy was sufficient to rescue desmosomal and connexin43 protein levels as well as arrhythmias in SNAP29 deficient cardiomyocytes. In conclusion, SNAP29 insulates a subset of desmosomal and gap junction proteins from selective autophagy-mediated degradation to restrict cardiac arrhythmias. Thus, loss of SNAP29-desmosome-gap junction interactome may predispose the heart to desmosomal based diseases of an electrical nature.
Abstract 106: Efficient In Vivo Homology-Directed Repair Within Cardiomyocytes
Authors: VanDusen Nathan J , Zheng Yanjiang , Butler Catalina E , Ma Qing , King Justin S , Pu William T
Published: November 2021

CRISPR/Cas9-based genome editing technologies provide powerful tools for genetic manipulation. Delivery of Cas9 and a homology directed repair (HDR) template using adeno-associated virus (AAV; CASAAV-HDR), was recently shown to enable creation of precise genomic edits, even within postmitotic cells. Here we studied CASAAV-HDR in cardiomyocytes. We constructed an AAV9 vector containing a gRNA targeting the ventricle specific Myl2 gene, and a promoterless HDR template that replaces the native Myl2 stop codon with a self-cleaving 2A peptide followed by mScarlet, a red fluorescent protein. When this vector was injected into Cas9 expressing newborn mice, we observed mScarlet expression within a remarkably high fraction of cardiomyocytes, approximately 45%. Expression was ventricle specific, consistent with the Myl2 expression profile. Similarly, when we targeted the atrial specific Myl7 gene, we observed mScarlet expression in ~20% of atrial cardiomyocytes. Amplicon sequencing of Myl2 and Myl7 transcripts showed that the vast majority of transcripts with an insertion were mutation-free, indicating that CASAAV-HDR is precise. Furthermore, CASAAV-HDR efficiency was comparable when AAV was delivered to fetal, neonatal, or mature mice. Next we targeted seven additional loci: Yap1, Tmem43, Nfatc3, Bdh1, Mkl1, Ttn, and Pln, fusing either an HA tag or mScarlet to each. Insertion efficiency varied dramatically between loci, with HDR efficiency generally correlating with target gene expression. TTN-mScarlet and mScarlet-PLN fusion proteins localized to the sarcomere and sarcoplasmic reticulum, respectively, consistent with the localization of the endogenous proteins. Collectively these data indicate that systemic delivery of CASAAV-HDR vectors can achieve efficient, precise, in vivo somatic genome modification that does not require cardiomyocyte proliferation. We successfully used this technology to monitor protein localization and anticipate it will be useful for many other applications, such as precise introduction of mutations to model disease or probe gene function. CASAAV-HDR may also enable efficient, permanent, and precisely targeted delivery of therapeutic transgenes to validated loci.
Abstract 109: The Effect Of Cell Sex On Cardiogenic Differentiation Of Human Induced Pluripotent Stem Cells And Their Maturation Processes
Authors: Mahmoudi Morteza , Yang Phillip C , Serpooshan Vahid , Abadi Parisa , Heydarpour Mahyar
Published: November 2021

Introduction:
Patient-specific human induced pluripotent stem cells (hiPSC)-derived cardiomyocytes (CMs) are increasingly used for
in vitro
disease modeling and drug screening, as well
in vivo
regenerative therapies. The cardiac differentiation efficacy of hiPSCs, together with the maturation level of generated CMs, are critical factors in achieving the required numbers of functional patient-specific cardiac muscle cells for clinical applications. Although extensive studies have improved the efficacy of differentiation and maturation processes, the role of cell sex in these processes has not been fully investigated.

Hypothesis:
Cell sex affects i) the cardiogenic differentiation efficacy of hiPSCs; and ii) maturation processes of hiPSC-CMs.

Methods and Results:
We have successfully and reproducibly fabricated patterned substrates recapitulating the 3D shape of mature CMs, using photolithography approaches, and demonstrated that the substrate could i) accelerate the differentiation of hiPSCs to CMs, and ii) facilitate maturation and functionality of immature hiPSC-CMs. Male and female hiPSCs, derived from human amniotic mesenchymal stem cells of male and female fetuses, were cultured onto flat (control)
vs.
patterned substrates. A total of 400 differentiation assays were conducted, 200 per each cell sex, on the flat (
n
= 100) and patterned (
n
= 100) substrates. A chemically defined approach was used to differentiate the cells toward CMs. On the flat (conventional) substrates, 59% of batches of male and 87% of batches of female hiPSCs differentiated into beating CMs (> 80%). On the patterned substrates, these numbers changed to 83% and 94% of successful differentiations for male and female hiPSCs, respectively. These results indicate the significant effect of substrate-mediated topographical cues on the cardiac differentiation yield of stem cells and the batch-to-batch variation. On both substrate types, female cells demonstrated significantly higher success rates of cardiac differentiation compared to the male cells. In addition, the CMs produced on the patterned substrates demonstrated higher purity than those created on the flat substrates both for male and female cells. Quantitative polymerase chain reaction (qPCR) was used to probe the male and female cell differences in expression of genes related to cardiac maturity, contractility, and Ca
2+
transport (TNNT2, MYH6, MYH7, and CACNA1c) and the outcomes revealed substantially greater expression levels of the maturation genes in differentiated female CMs cultured on the patterned substrates compared to the male cells.

Conclusions:
These results indicate that male and female hiPSCs and hiPSC-CMs respond differently to the identical substrates in terms of their differentiation and maturation efficacies.
Abstract 111: Conjoining Gene Expression And Cell Organization With Imaging To Explore Cell States In Hipsc-derived Cardiomyocytes
Authors: Gunawardane Ruwanthi
Published: November 2021

The Allen Institute for Cell Science is developing a state space of stem cell structural signatures to study changes in cellular organization of human induced pluripotent stem cells (hiPSCs) and other cell states through differentiation. Towards this goal, we have used CRISPR/Cas9 to generate a collection of ~50 endogenous fluorescently tagged hiPSC lines (www.allencell.org), each expressing a monoallelic EGFP-tagged protein that localizes to a particular cellular structure or organelle. In this study, we focuson hiPSC-derived cardiomyocytes and compare the relationship between sarcomeric structural organization and gene expression signatures at large scale. We developed several tools and novel quantitative approaches to achieve this: 1) scarless GFP-tagging of cardiac genes such as ACTN2 to study the organization and morphogenesis of the contractile apparatus; 2) a robust protocol for differentiation of hiPSCs into cardiomyocytes and methods for preparing cells for imaging; and 3) a quantitative, image-based platform for the systematic and automated classification of subcellular organization in single cells. We use these approaches to quantify subcellular organization and gene expression in >30,000 individual human induced pluripotent stem cell-derived cardiomyocytes, producing a publicly available dataset that describes the population distributions of local and global sarcomere organization, mRNA abundance, and correlations between these traits. While the mRNA abundance of some phenotypically important genes correlates with subcellular organization (e.g., MYH7), these two cellular metrics are heterogeneous and often uncorrelated, which suggests that geneexpression alone is not sufficient to classify cell states. Instead, we posit thatcell state should be defined by observing full distributions of quantitative, multidimensional traits in single cells that also account for space, time, and function. This platform provides a multidimensional approach to classify hiPSC-derived cardiomyocytes based on structural organization and gene expression in single cells.
Abstract 112: The 3CL Protease Of SARS-CoV2 Induces Sarcomeric Degradation Through Cleavage Of The Giant Protein Obscurin
Authors: Yucel Nora , McAfee Quentin , Paterlini Maria G , Arany Zoltan
Published: November 2021

We hypothesized that the 3CL protease (3CLPro) is responsible for the sarcomere degradation observed in cardiomyocytes infected by SARS-COV2. Overexpression of 3CLPro, but not a catalytically inactive mutant, resulted in breakdown of sarcomeres characterized by intact Z-disk/thin filament subunits that has been recently reported (Perez-Bermejo, et al, 2021) in SARS-COV2 infection. To identify potential host protein targets of 3CLPro in an unbiased fashion we screened the human proteome using a cut-site scoring algorithm that we developed. Scoring, ie likelihood of 3CL protease cleavage, was based off experimental data (Chuck et al, 2010) previously published on the highly homologous (96%) 3CL protease from SARS-COV. This scoring was followed by refinement by secondary structure prediction to identify cut-sites that lie in unstructured regions that are thus thus more likely to be accessible to the protease. Using this method, we identified >1000 potential high-likelihood cut sites across the proteome. Further filtering by proteins with cardiomyocyte expression showed 5 high-likelihood sites within the giant sarcomeric protein, Obscurin (OBSCN), as well as many other structural and signaling proteins which we experimentally validated. Expression of 3CLPro in IPSC cardiomyocytes resulted in significantly reduced OBSCN staining without alterations in Z-disk, thin filament, or thick filament proteins by both western blot and immunocytochemistry. In addition, imaging showed loss of OBSCN at sites with intact Z-disk/thin filament subunits. Thus we propose that activity of 3CLPro is a significant contributor to sarcomere breakdown in SARS-COV2 infection via degradation of Obscurin.
Abstract 113: Targeting Receptor-interacting Protein 140 (RIP140) To Modulate Energy Metabolism In The Failing Heart
Authors: Yamamoto Tsunehisa , Batmanov Kirill , Pruzinsky Elizabeth , Xiao Yang , Shewale Swapnil V , McDaid Kendra , Leone Teresa C , Maurya Santosh K , Lewandowski E Douglas D , Kelly Daniel P
Published: November 2021

During the development of heart failure (HF), the PPAR/ERR complex becomes deactivated resulting in diminished capacity for mitochondrial fatty acid oxidation (FAO) and ATP production leading to an “energy-starved” state that contributes to progression of HF. Receptor-Interacting protein 140 (RIP140) serves as a co-repressor of PPAR/ERR in some extra-cardiac tissues. We hypothesized that inhibition of RIP140 would re-activate PPAR/ERR enhancing capacity for fuel catabolism and ATP production in the failing heart. Heart and skeletal muscle-specific RIP140 knockout mice (strRIP140KO) were resistant to the development of cardiac hypertrophy and diastolic dysfunction in response to chronic pressure overload that mimicked features of HF with preserved ejection fraction (HFpEF). To further evaluate the role of RIP140 in heart, cardiac-specific (cs) RIP140KO mice were generated.
13
C-substrate NMR studies demonstrated that palmitate oxidation and triglyceride turnover rates were significantly accelerated in isolated perfused csRIP140KO hearts. csRIP140KO were subjected to transverse aortic constriction/apical myocardial infarction surgery (TAC/MI), to produce HF with reduced EF (HFrEF). Compared to controls, csRIP140KO exhibited reduced left ventricular remodeling and systolic dysfunction when subjected to TAC/MI. RNA-sequence analysis demonstrated that many genes involved in FAO, branched-chain amino acid catabolism, oxidative phosphorylation, and adult muscle contraction programs were significantly “protected” (less downregulation) by RIP140 deletion in the context of TAC/MI. To identify candidate cardiac RIP140 targets, “CUT&RUN”-sequencing was conducted on cardiomyocytes (CM) from csRIP140KO and controls to identify changes in enhancer regions. Motif analysis of peaks with increased H3K27ac deposition in the csRIP140KO CM identified ERR, PPAR, myocyte enhancer 2 (MEF2), glucocorticoid receptor (GR), and kruppel-like factor (KLF) binding sites. We conclude that RIP140 serves as a global co-repressor of a network of transcription factors that control cardiac energy metabolic and contractile function, and that inhibition of RIP140 could prove to be a novel therapeutic approach for HF.
Abstract 403: FAM210A Maintains Cardiac Mitochondrial Homeostasis Through Regulating Letm1-dependent Ca
2+
Efflux
Authors: Wu Jiangbin , Venkata Chinna , Chen Si , Hedaya Omar , Yan Chen , Yao Peng
Published: November 2021

Mitochondria play fundamental roles in supporting healthy myocardium function. Disturbed cardiac mitochondrial homeostasis causes mitochondrial dysfunction associated with cardiomyopathy in humans and mice. Recent genome-wide association studies (GWAS) discovered that the mutations of FAM210A (family with sequence similarity 210 member A) are associated with sarcopenia and osteoporosis. Interestingly, Fam210a is most highly expressed in the heart and multiple omics analyses in mouse hearts reveal Fam210a as a hub gene in cardiac hypertrophy. However, the molecular function of FAM210A in the heart remains elusive. Here, we discover that FAM210A is critical for maintaining cardiac mitochondrial function and homeostasis. Cardiomyocyte (CM) specific knockout (KO) of
Fam210a
in adult mice leads to progressive heart failure with enlarged left ventricle chamber and ultimately causes mortality at ~70 days after
Fam210a
KO. The FAM210A deficient CMs exhibit myofilament disarray at ~9 weeks post
Fam210a
KO. Moreover, Fam210a KO results in a remarkably elevated mitochondrial ROS production, dramatically compromised mitochondrial membrane potential, and reduced expression of mitochondrial electron transport chain (ETC) complex genes. As a result, the mitochondrial respiratory activity is significantly reduced and the mitochondrial cristae are disrupted in
Fam210a
KO CMs. In contrast, at the early stage of ~5 weeks post tamoxifen-induced
Fam210a
KO, we observe increased mitochondrial ROS production, disturbed mitochondrial membrane potential, and reduced respiratory activity in CMs prior to heart failure. Transcriptomic and proteomic analyses from
Fam210a
KO hearts indicate that FAM210A deficiency causes chronic integrated stress response (ISR) in the heart. Mechanistically, Interactome analyses show that FAM210A binds to mitochondrial Ca
2+
/H
+
exchanger LETM1 (leucine zipper and EF-hand containing transmembrane protein 1) and regulates LETM1-mediated mitochondrial Ca
2+
efflux. Altogether, we discover a novel function of FAM210A in maintaining the cardiac mitochondrial homeostasis by regulating mitochondrial Ca
2+
efflux, and deficiency of FAM210A causes mitochondrial dysfunction and leads to heart failure.
Abstract MP208: A Shared Fog2 / Tbx5-dependent Gene Regulatory Network Identified By Transcription Factor-dependent Non-coding RNA Profiling Modulates Cardiac Rhythm
Authors: Smith Linsin A , Perez-Cervantes Carlos , Broman Michael , Nadadur Rangarajan , Steimle Jeff , Laforest Brigitte , Lazaveric Sonja , Mazurek Stefan R , Ikegami Kohta , Yang Xinan , Pu William , Weber Christopher , McNally Elizabeth M , Svensson Eric , Moskowitz Ivan
Published: November 2021

Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting over 33 million individuals throughout the world. AF is highly heritable and recent genome-wide association studies (GWAS) have cumulatively identified over 100 loci associated with AF risk. Genome-wide association studies (GWAS) often identify transcription factor (TF) loci in association with complex human diseases, implying that a significant transcriptional component underlies human disease risk and etiology. The transcription factors ZFPM2 (FOG2), GATA4, and TBX5 have all been implicated in human AF risk by genetic studies. We hypothesized that FOG2, GATA4, and TBX5 functionally interact to regulate a gene regulatory network essential for atrial rhythm control. We generated a novel mouse model of spontaneous AF based on FOG2 overexpression. FOG2 ChIP-seq identified FOG2 genomic localization at loci co-occupied by GATA4, a known FOG2 binding partner. However, we found that FOG2 OE caused gene expression alterations that correlated more highly with TBX5-dependent rather than GATA4-dependent gene expression, including a module of calcium handling genes required for atrial rhythm homeostasis. We applied TF-dependent non-coding transcriptional profiling to examine the FOG2 dependent atrial GRN, which identified 805 candidate regulatory regions with accessible chromatin and FOG2 dependent ncRNAs. TBX5 removal and FOG2 OE caused highly correlated dysregulation of ncRNA expression at open chromatin regions genome-wide, suggesting a functional interaction between TBX5 and FOG2. Furthermore, FOG2 OE only affected enhancer activity by altered ncRNA abundance at locations of TBX5 co-binding. The shared TBX5/FOG2 genomic interaction predicted a potential genetic interaction, and we found that cardiac rhythm abnormalities caused by
Tbx5
haploinsufficiency were rescued by
Fog2
haploinsufficiency. Taken together, TF-dependent ncRNA-profiling revealed an interconnected cardiac rhythm gene regulatory network (GRN) between FOG2, TBX5 and GATA4. These data nominate a specific model in which FOG2 is recruited by GATA4 to modulate a co-bound TBX5-dependent atrial gene regulatory network for calcium handling and atrial rhythm homeostasis.
Abstract MP211: NAD Redox Imbalance Exacerbates Diabetic Cardiomyopathy
Authors: Lee Chi Fung
Published: November 2021

Diabetes and heart failure are linked to NAD redox imbalance, whose role in diabetic cardiomyopathy has not been directly tested. Streptozotocin-induced diabetes in WT mice for 16 weeks promoted declines in systolic and diastolic function, which associated with lowered cardiac NAD/NADH ratio (NAD redox imbalance). To test the hypothesis that , we employed mouse models with cardiac-specific manipulations of NAD redox states. Cardiac-specific Ndufs4-KO mice (cKO) exhibit lowered cardiac NAD/NADH ratio with normal baseline function, geometry and energetics. Control and cKO mice were challenged with 8-week diabetic stress. Metabolomic analyses of plasma collected after the diabetic stress showed similar hyperglycemia and dyslipidemia stresses in diabetic control and diabetic cKO mice. Chronic diabetic stress promoted systolic and diastolic dysfunctions in control mice, which were further exacerbated in diabetic cKO mice in both male and female cohorts. Collagen levels and transcript analyses of fibrosis and extracellular matrix-dependent pathways showed no change in diabetic cKO hearts, suggesting that cardiomyocyte dysfunction is a likely culprit for the exacerbated dysfunction. Increased protein acetylation, including SOD2-K68Ac, was observed in diabetic cKO hearts. Inhibited antioxidant function by SOD2-K68Ac promoted protein oxidation in diabetic cKO hearts, suggesting oxidative stress as a pathogenic mechanism. We next examined phosphorylation status of myofilament proteins in these diabetic hearts. MyBPC-S282Pi levels are suppressed in failing hearts and remained unchanged in diabetic cKO hearts. TnI-S150Pi increases myofilament calcium sensitivity and prolongs calcium dissociation, while TnI-S23/24Pi imposes the opposite effects. TnI-S150Pi levels were elevated in diabetic cKO hearts, while TnI-S23/24Pi levels unchanged. Therefore, exacerbated diastolic dysfunction in diabetic cKO hearts is due to the selective phosphorylation at TnI-S150. AMPK is activated by energetic stress and phosphorylates TnI-S150. ATP levels decreased, and AMP/ATP ratio increased in diabetic cKO hearts, implicating impaired energetics to promote TnI-S150Pi and dysfunction. Elevation of NAD levels normalized cardiac NAD redox balance in diabetic cKO hearts. Elevated levels of SOD2-K68Ac and TnI-S150Pi, exacerbated systolic and diastolic dysfunction in diabetic cKO hearts were all reversed by elevation of NAD levels. Dysfunction in diabetic control hearts was also ameliorated by elevation of NAD levels. These data collectively conclude that NAD redox imbalance is a positive mediator of the progression of diabetic cardiomyopathy.
Abstract MP218: Cop9 Signalosome Subunit 6 Restricts Desmosomal Proteome Degradation To Prevent Desmosomal Targeted Cardiac Disease
Authors: Liang Yan , Lyon Robert , Pellman Jason , Bradford William , Lange Stephan , Bogomolovas Julijus , Dalton Nancy , Gu Yusu , Bobar Marcus , Lee Mong-Hong , Iwakuma Tomoo , Nigam Vishal , Asimaki Angeliki , Scheinman Melvin , Peterson Kirk L , Sheikh Farah
Published: November 2021

Dysregulated protein degradative pathways are increasingly recognized as mediators of human cardiac disease. This pathway may have particular relevance to desmosomal proteins that play critical structural roles in both tissue architecture and cell-cell communication. Genetic mutations in desmosomal genes resulting in the destabilization/breakdown of the desmosomal proteome are a central hallmark of all genetic-based desmosomal-targeted diseases, including the cardiac disease arrhythmogenic right ventricular (RV) dysplasia/cardiomyopathy (ARVD/C). However, no information exists on whether there are resident proteins that regulate desmosomal proteome homeostasis. Here we identified a desmosomal resident regulatory complex, composed of subunit 6 of the COP9 signalosome (CSN6), enzymatically restricted neddylation and targets desmosomal proteome. Pharmacological restoration of CSN enzymatic function (via neddylation inhibitors) could rescue desmosomal protein loss in CSN6 deficient cardiomyocytes. Through the generation of two novel mouse models, we showed that cardiomyocyte-restricted CSN6 loss in mice selectively accelerated desmosomal destruction to trigger classic disease features associated with ARVD/C. We further showed that disruption of CSN6-mediated (neddylation) pathways underlined ARVD/C as CSN6 binding, localization, levels and function were impacted in hearts of classic ARVD/C mouse models and ARVD/C patients impacted by desmosomal loss and mutations, respectively. We anticipate our findings have broad implications towards understanding mechanisms driving desmosome degradation in other desmosomal-based diseases, such as cancers.
Abstract MP229: Signaling Mechanisms Leading To CD4+ T-lymphocyte Activation During Ischemic Heart Failure
Authors: Gupta Sahil , Bansal Shyam S
Published: November 2021

CD4
+
T-cells turn pathological during chronic heart failure (HF). Phenotypic changes that mediate this transition are unknown. Thus, at 8 weeks post-infarction we conducted limited cell RNA-sequencing on 150 CD4
+
T-cells isolated from rodent failing hearts and using ingenuity pathway analysis (z score>2) compared them either with CD4
+
T-cells isolated from the mediastinal lymph nodes (mLNs) of the same mice or cardiac CD4
+
T-cells from sham mice. T-cells isolated from the mLNs of HF mice showed enhanced TCR signaling (p<2X10
-4
) with 3-6 fold increase in several downstream mediators such as ITK, MAPK, LCK and Zap70. We also observed heightened leukocyte extravasation signaling (p<2X10
-5
) with significant increase in PKCθ, and Rho GTPase activating and Ras association domain proteins. These are consistent with enhanced antigen-presentation, and TCR activation during HF. Moreover, we observed significant upregulation of oxidative phosphorylation (p<3X10
-15
) in cardiac CD4
+
T-cells as compared to mLN T-cells with 2-3 fold increase in the gene expression of ATP synthase subunits and cytochrome C indicative of their increased metabolic activity upon infiltration into the failing hearts. Compared to CD4
+
T-cells from sham hearts, significant upregulation of inflammatory genes (p<3X10
-5
), chemotactic factors (p<8X10
-4
), and several pro-inflammatory cytokine-mediated pathways such as IL-6 (p<2X10
-4
), IL-1a (p<2X10
-3
) and TNFα (p<2X10
-3
) was observed in cardiac CD4
+
T-cells from HF mice. Interestingly, we also observed a significant upregulation of homing (p<3X10
-4
) and connective tissue remodeling genes (p<1.8X10
-3
) with >8-fold increase in genes like LCN2, SLPI, IGF1, FGFR1, LTF, and TIMP2 required for transmigration and homing of CD4
+
T-cells into the ischemic hearts. Our studies show enhanced antigenic CD4
+
T-cell activation in the mLNs supporting enhanced pro-inflammatory signaling, metabolic activation, and extravasation into the ischemic hearts.
Abstract MP244: Deciphering The Mechanism Of Elevation Of Mir34a And Its Attenuation Via Corm A1 And Subsequent Improvement In Mitochondrial Status In Atherogenic Mdms
Authors: Vyas Hitarthi , Devkar Ranjitsinh
Published: November 2021

Elevated levels of miR34a and its association with macrophage polarization is reported in atherosclerosis but the underlying mechanism that upregulates miR34a lacks clarity. Herein, the mechanism of miR34a elevation in atherogenic human monocyte derived macrophages (MdMs) and subsequent changes in mitochondria were monitored. Further, CO supplementation (via Carbon monoxide Releasing Molecule A1; CORM A1) to atherogenic (OxLDL treated) MdMs was used to achieve downregulation of miR34a. Herein, we also hypothesize that lowering of miR34a in atherogenic MdMs improves the cellular health and mitochondrial function. Transcriptional factors (P53, NF-κb), transcriptional inhibitors (Zeb1, snai1, stat3) and epigenetic modification (methylation) in promoter region of miR34a were evaluated. OxLDL treated MdMs recorded significant decrement in mRNA levels of the said transcription inhibitors whereas; the same were reversed in CORM A1 co-treated group. Further, hypomethylation was recorded in the promotor region of miR34a on oxLDL treatment but methylation status was reverted to the control levels following CORM A1 co-supplementation. The mRNA levels of transcription factors showed non-significant changes in all the experimental groups. In silico docking studies had shown that CO effectively binds to the DNA binding domains of p53 that possibly prevents subsequent binding to their respective miR34a promotor regions. Positive docking of miR34a to 3’UTR of SIRT-1 supported our observation on lowered SIRT-1 and PGC-1α levels in oxLDL group that were found to be restored in CORM A1 co-treated group. Poor indices of mitochondrial biogenesis (SIRT-1, PGC1α, Nrf-1, Drp1, Mito Tracker Red staining), function (ATP assay), mitochondrial membrane potential (JC-1) and mitochondrial antioxidants (SOD2 and TrxR2), cellular ROS (DCFDA) following oxLDL treatment was found to be restored by CORM A1 co-treatment. In conclusion, atherogenic elevation of miR34a is as a result of hypomethylation in its promotor region and lowered mRNA transcripts of its inhibitors (Zeb1, snai1, stat3). Further, lowering of miR34a by CORM A1, improves atherogenic status of MdMs as evidenced by an improved cellular and mitochondrial health.
Abstract MP246: Epigenetic Regulation At
LRP1
Susceptibility Locus And Characterization Of
LRP1
Function In Human Induced Pluripotent Stem Cells Derived Smooth Muscle Cells
Authors: LIU LU , Georges Adrien , Bouatia-Naji Nabila
Published: November 2021

Introduction:
The low-density lipoprotein receptor-related protein 1 (LRP1), an endocytic receptor highly expressed in smooth muscle cells (SMCs), participates in diverse biological processes. A common genetic variant located in
LRP1
first intron, rs11172113, was associated with several vascular diseases, including coronary artery disease, migraine and spontaneous coronary artery dissection, as well asd with
LRP1
expression in arterial tissues. However, the biological mechanisms through which rs11172113 influence
LRP1
function in the context of arterial lesions is not fully understood.

Methods:
We applied
in silico
functional annotation to select variants and measured their enhancer activity using luciferase reporter assay in rat primary cells (A7r5). We performed siRNA knockdown of
LRP1
and 4 transcription factors (TFs) predicted to interact with rs11172113 in human induced pluripotent stem cells (iPSCs) derived SMCs. We analyzed both contractile (CSMCs) and synthetic (SSMCs) differentiated cells. We edited iPSCs prior to differentiation using CRISPR-Cas9 to generate 100 bp deletion of the enhancer region containing rs11172113. We also created frame-shift indels in exons 2 or 5 of
LRP1
in iPSCs to create SMCs knockouts.

Results:
Seven variants in
LRP1
locus co-located with enhancer (histone marks) and open chromatin regions (ATAC-Seq peaks) in SMCs and arterial tissues. Reporter assay in rat SMCs confirmed that rs11172113 belongs to a genomic region showing enhancer activity
in vitro
. iPSCs with homozygous deletion of rs11172113 enhancer region presented the same pluripotency compared with wild type, and iPSC derived SMCs showed positive expression of specific markers for each phenotype. We found that the deletion of enhancer region decreased the expression of
LRP1
in both CSMCs and SSMCs.
LRP1
knockdown decreased SSMCs and CSMCs proliferation capacity, but increased cell migration. Knockdown of TFs and iPSCs derived CSMCs and SSMCs with
LRP1
knockout are currently under assessment.

Conclusions:
We confirmed rs11172113 to regulate
LRP1
expression in iPSCs derived synthetic and contractile SMCs. Our results support
LRP1
effect on SMCs phenotype alteration as a potential mechanism in genetic susceptibility for vascular disease.
Abstract MP247: Mitochondrial Complex V Is Regulated By GRK2 In The Heart
Authors: Ferrero Kimberly , Pfleger Jessica M , Chuprun Kurt , Barr Eric , Gao Erhe , Koch Walter J
Published: November 2021

The GPCR kinase GRK2 is highly expressed the heart; importantly, during cardiac injury or heart failure (HF) both levels and activity of GRK2 increase. The role of GRK2 during HF is canonically studied upstream of β-adrenergic desensitization. However, GRK2 has a large interactome and noncanonical functions for this kinase are being uncovered. We have discovered that in the heart, GRK2 translocates to mitochondria (
mtGRK2
) following injury and is associated with negative effects on cardiac metabolism. Thus, we have sought to identify the mechanism(s) by which GRK2 can regulate mitochondrial function. We
hypothesize
that mtGRK2 interacts with proteins which regulate bioenergetics and substrate utilization, and this never-before-described role may partially explain the altered mitochondrial phenotype seen following cardiac injury or HF. Stress-induced mitochondrial translocation of GRK2 was validated in neonatal rat ventricular myocytes, murine heart tissue and a cardiac-derived cell line. Consequently, the GRK2 interactome was mapped basally and under stress conditions
in vitro, in vivo
, and with tagged recombinant peptides. GRK2-interacting proteins were isolated via immunoprecipitation and analyzed via liquid chromatography-mass spectroscopy (LCMS). Proteomics analysis (IPA; Qiagen) identified mtGRK2 interacting proteins which were also involved in mitochondrial dysfunction. Excitingly, Complexes I, II, IV and V (ATP synthase) of the electron transport chain (ETC) were identified in the subset of mtGRK2-dysfunction partners. Several mtGRK2-ETC interactions were increased following stress, particularly those in Complex V. We further established that mtGRK2 phosphorylates some of the subunits of Complex V, particularly the ATP synthase barrel which is critical for ATP production in the heart. Specific amino acid residues on these subunits have been identified using PTM-LCMS and are currently being validated in a murine model of myocardial infarction. To support these data, we have also determined that alterations in either the levels or kinase activity of GRK2 appear to alter the enzymatic activity of Complex V
in vitro
, thus altering ATP production. In summary, the phosphorylation of the ATP synthesis machinery by mtGRK2 may be regulating some of the phenotypic effects of injured or failing hearts such as increased ROS production and reduced fatty acid metabolism. Research is ongoing in our lab to elucidate the novel role of GRK2 in regulating mitochondrial bioenergetics and cell death, thus uncovering an exciting, druggable novel target for rescuing cardiac function in patients with injured and/or failing hearts.
Abstract MP248: Sequestrosome1/p62 Regulates Redox Homeostasis Via The Nrf2-keap1 Pathway In The Murine Heart
Authors: Fatahian Amir N , Ghosh Rajeshwary , Pires Karla M , MOSLEH TARIQ , Vinod Vishaka , Vowles Sidney , Campbell Kenneth S , Symons J David D , Boudina Sihem
Published: November 2021

Sequestosome1 (p62) is a multifunctional signaling molecule and an autophagy adaptor protein. Previous work demonstrated that mice with whole-body p62 knockout recapitulated many detrimental features of aging. Of note, these mice developed late onset obesity and systemic abnormalities that could have contributed to their aging phenotype. Multiple studies have also shown that cardiac dysfunction can be linked to an increase in oxidative stress. The Nrf2-Keap1 pathway is critical for protection against oxidative stress and p62 has been shown to interact with Keap1, thus allowing Nrf2 activation to induce anti-oxidant responses. However, the role of p62 in the heart is not well known. We tested the hypothesis that p62 plays an important homeostatic role in the heart through the regulation of redox homeostasis via the Nrf2-Keap1 pathway. Wild-type and cardiomyocytes-specific p62 knockout (cp62 KO) mice at 8 weeks and 60 weeks of age were used. At 8 weeks, cp62KO mice exhibited mild but significant contractile dysfunction compared to the wild-type controls. By 60 weeks, the KO mice developed cardiac hypertrophy, fibrosis and increased oxidative stress. cp62 KO hearts had decreased Nrf2 nuclear translocation and activation as evidenced by a 50% (p<0.005) reduction in the expression of the Nrf2 target glutathione S-transferase A4 (
Gsta2
) gene. These findings were further validated by transcriptomic analysis followed by KEGG pathway analysis, which indicated that redox pathways were altered in the 60-week p62 null hearts. To examine the mechanisms involved in p62 regulation of Nrf2-Keap signaling, we utilized rat cardiac H9c2 myoblasts. Loss of p62 using p62 siRNA in H9c2 cells resulted in decreased Nrf2 levels and increased oxidative stress. These pathological consequences of suppressing p62 could be attributed to increased Nrf2 degradation via the proteasome. Together, these results reveal a previously uncharacterized role for p62 in the maintenance of cardiac redox signaling in the mouse heart.
Abstract MP258: The Cell Surface Receptors Ror1/2 Control Cardiac Myofibroblast Differentiation
Authors: Chavkin Nicholas , Sano Soichi , Walsh Kenneth
Published: November 2021

Background:
A hallmark of heart failure is cardiac fibrosis, which results from the injury-induced differentiation response of resident fibroblasts to myofibroblasts that deposit extracellular matrix. During myofibroblast differentiation, fibroblasts progress through polarization stages of early pro-inflammation, intermediate proliferation, and late maturation, but the regulators of this progression are poorly understood. Planar cell polarity receptors, receptor tyrosine kinase like orphan receptor 1 and 2 (Ror1/2), can function to promote cell differentiation and transformation. In this study, we investigated the role of the Ror1/2 in a model of heart failure with emphasis on myofibroblast differentiation.

Methods and Results:
The role of Ror1/2 during cardiac myofibroblast differentiation was studied in cell culture models of primary murine cardiac fibroblast activation and in knockout mouse models that underwent transverse aortic constriction (TAC) surgery to induce cardiac injury by pressure overload. Expression of Ror1 and Ror2 were robustly and exclusively induced in fibroblasts in hearts after TAC surgery, and both were rapidly upregulated after early activation of primary murine cardiac fibroblasts in culture. Cultured fibroblasts isolated from Ror1/2-KO mice displayed a pro-inflammatory phenotype indicative of impaired myofibroblast differentiation. Although the combined ablation of Ror1/2 in mice did not result in a detectable baseline phenotype, TAC surgery led to the death of all mice by day 6 that was associated with myocardial hyper-inflammation and vascular leakage.

Conclusions:
Together, these results show that Ror1/2 are essential for the progression of myofibroblast differentiation and for the adaptive remodeling of the heart in response to pressure overload.
Abstract P310: Mitochondrial Electron Transport Chain Dysfunction Is Associated With Mg Deficiency Induced Diastolic Dysfunction
Authors: Liu Man , Liu Hong , Clements Richard T , Feng Feng , O-Uchi Jin , Dudley Samuel C
Published: November 2021

Introduction:
The prevalence of heart failure with preserved ejection fraction (HFpEF) is increasing, although there is no specific treatment for HFpEF yet because of poor understanding of the underlying pathophysiology. Our previous studies show that hypomagnesemia contributes to diastolic dysfunction and HFpEF by regulation of mitochondrial function and that Mg supplementation improves diastolic function. In this study, we investigated how mitochondria were affected by Mg deficiency.

Methods:
C57BL/6J mice were fed with a low-Mg diet (HypoMg mice, 15-30 mg/kg Mg) or a normal Mg diet (control mice, 600 mg/kg Mg) for 6 weeks. Mg repletion was achieved by feeding HypoMg mice with normal diet for another 6 weeks.

Results:
HypoMg mice showed significantly decreased serum Mg (0.38±0.03 mM vs. 1.14±0.03 mM of control, P<0.0001), diastolic dysfunction (E/e’=21.1±1.1 vs. 15.4±0.4 of control, P=0.011), increased mitochondrial ROS (1.9±0.2-fold of control, P<0.0001), decreased total mitochondrial Mg content (3.6±1.8 vs. 18.3±4.7 μM total Mg/mg mitochondrial protein of control, P=0.019), decreased ATP production in hearts (1.2±0.2 vs. 2.7±0.2 μmol/g heart tissue of control, P=0.0002), decreased complex I activity (ΔOCR
NADH-rotenone
=0.27±0.10 vs. 1.02±0.04 of control, P=0.0004 measured with Seahorse), and decreased complex I protein levels (50.2% reduction compared with control mice, P=0.009). Mg repletion reversed all these changes.

Conclusion:
HypoMg-induced diastolic dysfunction likely results from HypoMg-induced electron transport chain dysfunction resulting from a decrease in mitochondrial Mg content. Mg repletion reverses these changes, reinforcing the known correlation of increased Mg intake and reduced heart failure symptoms. In deficiency states, Mg supplementation may represent a novel treatment for diastolic heart failure by improving mitochondrial function.
Abstract P312: The Essential Role Of Prak In Preserving Cardiac Function And Insulin Resistance In High Fat Diet-induced Diabetes
Authors: Zhao Yu T , Du Jianfeng , Zhao Thomas J , Wang Hao , Kadin Marshall , Zhao Ting C
Published: November 2021

Background:
p38 regulated/activated protein kinase (PRAK) plays a crucial role in modulating cell death and survival. However, the role of PRAK in mediating cardiac dysfunction and metabolic disorders remains unclear. We examined the effects of deletion of PRAK on modulating cardiac function and insulin resistance in mice exposed to a high fat diet (HFD).

Methods:
Wild type and PRAK
-/-
mice at 8 weeks old were exposed to either chow food or HFD for a consecutive 16 weeks. Glucose tolerance test and insulin tolerance test were employed to assess insulin resistance. Echocardiography was employed to assess myocardial function. Western blot was used to determine the molecular signaling involved in phosphorylation of IRS-1, AMPKα, ERK-44/42, and irisin. Real time-PCR was used to assess the hypertrophic genes of the myocardium. Histological analysis was employed to assess the hypertrophic response, interstitial myocardial fibrosis, and apoptosis in the heart.

Results:
HFD induced metabolic stress is indicated by glucose intolerance and insulin intolerance. PRAK knockout aggravated insulin resistance, as indicated by glucose intolerance and insulin intolerance testing as compared to wild type littermates. As compared to wild type, hyperglycemia and hypercholesterolemia were manifested in PRAK-knockout mice following high fat diet intervention. High fat diet intervention displayed a decline in fractional shortening (FS) and ejection fraction (EF). However, deletion of PRAK exacerbated the decline in EF and FS as compared to wild type mice following HFD treatment. In addition, PRAK knockout mice enhanced the expression of myocardial hypertrophic genes including ANP, BNP, and βMHC in HFD treatment, which was also associated with an increase in cardiomyocyte size and interstitial fibrosis. Western blot indicated that deletion of PRAK induces decreases in phosphorylation of IRS-1, AMPKα, and ERK44/42 as compared to wild type controls.

Conclusion:
Our finding indicates that deletion of PRAK promoted myocardial dysfunction, cardiac remodeling, and metabolic disorders in response to HFD.
Abstract P325: The Role Of Runx1 In Cardiomyocyte Cell Cycle And Ploidy
Authors: Swift Samantha K , Patterson Michaela
Published: November 2021

Adult mammalian cardiomyocytes (CMs) are thought to be post-mitotic and therefore unable to regenerate the myocardium after injury. In recent years various studies have shown that the adult mammalian CM is capable of a small amount of proliferation, potentially restricted to a subset of CMs. One such study demonstrated that having greater percentages of the rare mononuclear diploid cardiomyocyte (MNDCM) is associated with improved outcomes after myocardial infarction (MI). An accompanying genome-wide association analysis identified genetic loci associated with the frequency of the MNDCM population. One candidate to come out of this screen was
Runx1.
Concurrently, RUNX1 captured the attention of cardiac regeneration researchers due to its increased presence in disease states, with some suggesting it may be a marker for dedifferentiation (fetal gene induction). One recent study demonstrated improved calcium handling and decreased eccentric hypertrophy following RUNX1 ablation after injury, perhaps corroborating the idea that RUNX1 is involved with CM dedifferentiation.

We hypothesize that
Runx1
influences dedifferentiation in CMs, impacting ploidy, as well as CM cell cycle activity and post-MI outcomes.

We found that CM-specific overexpression (OE) of
Runx1
results in a doubling of the MNDCM population, thereby validating its influence on the population. Via multiple contexts including postnatal development and adult injury, knocking out
Runx1
decreases DNA synthesis while
Runx1
OE increases DNA synthesis. Furthermore, an initial analysis of RNAseq data demonstrates that RUNX1 OE in a neonatal mouse hearts demonstrated differential expression in genes related to
cardiac conduction, contraction, heart development, regeneration,
and
regulation of cell differentiation
. After MI in the adult mouse heart, the effects of
Runx1
OE resulted in transient benefits which included increased cell cycle activity and preservation of function.

These data suggest that
Runx1
is not simply a marker of CM dedifferentiation, but also a regulator of the process including cell cycle activation. Ongoing work will tease apart this role in more detail and could establish RUNX1 as a prominent therapeutic target for mitigating effects of cardiac injury.