bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 526: Unraveling the Molecular Signature of Pregnancy Induced Hypertrophy
Authors: Collins Helen E , Fulghum Kyle , McNally Lindsey A , Foster Mallory L , Brittian Kenneth , Uchida Shizuka , Nystoriak Matthew A , Jones Steven P , Hill Bradford G
Published: October 2020

Cardiovascular disease is the leading cause of death in pregnant and postpartum women. During pregnancy, the maternal heart rapidly adapts to the increasing physiological and metabolic demands of the growing fetus. This adaptation often takes the form of a physiological hypertrophy in which the maternal heart grows to increase cardiac output; however, the molecular processes underlying pregnancy-induced hypertrophy (PIH) are poorly understood. The goal of this study was to examine the transcriptomic and metabolic signatures associated with the structural and functional adaptations of the heart to pregnancy. Therefore, we performed timed pregnancy studies in 12-week-old female FVB/NJ mice, which were distributed into the following groups: non-pregnant control (NP;
n
= 14), mid-pregnancy (MP, 6d pregnant;
n
= 11), late-pregnancy (LP, 16d pregnant;
n
= 13), and 1-wk post birth (PB;
n
= 8). Heart weight to tibia length were higher in MP (7.77±1.02 mg/mm;
p
<0.05), LP (7.84±0.87 mg/mm;
p
<0.05), and PB mice (9.86±1.14 mg/mm;
p
<0.05) compared with NP mice (6.54±0.74 mg/mm). The sustained increase in PB heart weight was associated with increased myocyte cross sectional area, consistent with cardiomyocyte hypertrophy. Compared with NP hearts, echocardiographic measurements suggest significant increases in both end diastolic (36.0±5.1 vs 61.2±5.9 μl;
p
<0.05) and systolic LV volume (9.4±3.8 vs 21.0±1.4 μl;
p
<0.05) in PB hearts. These changes in PB hearts were associated with a significant increase in LV mass and a decline in ejection fraction. In LP and PB hearts, we also found higher expression of markers of hypertrophy (
Nppa, Nppb, Myh7
). Subsequent RNA-seq analyses revealed enrichment in genes involved in cell proliferation, cytokinesis, and transcription in MP hearts; in metabolism genes in LP hearts; and in fibrotic and extracellular matrix genes in PB hearts. Together, these findings reveal the key molecular signature underlying the structural and functional adaptation of the heart during pregnancy and parturition, and may shed light on the molecular processes underlying PIH.
Abstract 528: Towards Therapeutic Genome Editing in Vascular Endothelium by Novel Nanoparticle Delivery of Crispr Plasmid Dna
Authors: Zhang Xianming , Zhao You-yang
Published: October 2020

Introduction:
Therapeutic delivery of CRISPR system components to induce
in vivo
genome editing in postnatal and adult life has great translational potential. Recent studies employing non-viral delivery of small guide RNA (gRNA) and Cas9 mRNA have achieved efficient genome editing in adult mice. However, as often seen in other RNA therapeutic studies with non-viral delivery of antisense and siRNA, the efficiency is limited to the liver.

Hypothesis:
Novel nanoparticle can therapeutically deliver the CRISPR system to selectively target cardiovascular endothelium in adult mice.

Methods:
We developed novel PLGA-based nanoparticles which was for the first time shown to be uptaken efficiently by the vascular endothelium without specific liver accumulation following i.v. administration. Mixture of the nanoparticle:plasmid DNA expressing Cas9 under the control of the human
CDH5
promoter (EC-specific) and gRNA by the
U6
promoter was administered i.v. to adult mice. Seven to ten days later, various organ tissues were collected for analysis of the efficiency of genomic editing and knockout of protein expression. The phenotype of CRISPR-mediated in vivo knockout of
Pik3cg
which encodes the G protein-coupled receptor-activated p110gamma isoform of PI3K was compared to
Pik3cg
null mice in response to sepsis challenge.

Results:
Therapeutic delivery of nanoparticles loaded with the all-in-one CRISPR plasmid DNA induced highly efficient genome editing in endothelial cells (ECs) of the cardiovascular system including heart, lung, and aorta in adult mice. The Indel rate was as great as 50% in ECs isolated from these vascular beds. Immunostaining and Western blotting demonstrated greater than 70% decrease of protein expression in ECs.
Pik3cg
-gRNA-induced genome editing diminished p110γPI3K expression in pulmonary vascular ECs, which led to impaired vascular repair and resolution of inflammation after sepsis challenge as seen in

Pik3cg
-/-

mice.

Conclusion:
We have developed a simple and highly efficient method for
in vivo
genome editing selectively targeting the vascular endothelium. This strategy will greatly facilitate cardiovascular research and may enable therapeutic genome editing for prevention and treatment of cardiovascular diseases.
Abstract 529: A Modern Large-scale Analysis of Heart Transplantation in Elderly Patients
Authors: Suryapalam Manish , Kashem Mohammed , Rakita Val , Toyoda Yoshiya
Published: October 2020

Introduction:
As the prevalence of heart failure increases among older patients, the potential role of heart transplant (HTx) in this demographic demands further investigation. Survival outcome analysis of the elderly has primarily been analyzed in single-center studies, and the few long term studies performed have included a timeframe to the 1980s, introducing substantial variance from much poorer survival outcomes. We investigated the 5 to 10 year survival outcomes of more modern heart transplantation patients by analyzing the UNOS database.

Methods:
Heart transplantation data for 32,337 patients (2000-2014) was divided into three different age groups- <60, 60-69, and ≥70 years old. Gender, ethnicity, height, weight, BMI, ICU stay, ischemic time, length of stay (LOS), and creatinine level were evaluated for significance using Chi-Squared and H-Tests as appropriate (p<0.05). Survival outcome was assessed using a Kaplan-Meier Curve and log-rank tests.

Results:
23,267 were <60, 8,459 were 60-69, and 611 were ≥70, with mean ages of 38±0.1, 64±0.0, and 72±0.1 respectively. The distribution of gender, ethnicity, ischemic time, BMI, height, and weight was significantly different between the cohorts, with p=0.000 for all. Survival analysis indicated complete pairwise significance at 10 years post-HTx, with overall significance of p=0.000. At 5 years post-HTx, only 60-69 vs ≥70 did not have pairwise significance in survival.

Conclusion:
Contrary to prior studies, results indicate a statistically significant difference in survival the older and younger cohorts. This difference is especially prominent at the 10
th
year post-transplant, but can be seen even at the 5
th
year.
Abstract 532: Isolation of Human Placenta-derived Cdx2 Cells for Cardiac Regeneration
Authors: Vadakke-Madathil Sangeetha , Dekio Fumiko , Sperling Rhoda , Brody Rachel , Bianco Angela , Chaudhry Hina
Published: October 2020

Coronary artery disease and heart failure are among the leading causes of mortality in United States and worldwide. The limited proliferation of the adult mammalian heart has prompted the need to recognize novel strategies that can restore contractile function in heart disease. We recently reported that cells expressing trophoblast marker Cdx2 isolated from murine end-gestation placentas are multipotent and immune-privileged, with homing ability to travel to sites of cardiac injury to regenerate injured myocardium. Since murine Cdx2 cells are immunologically naïve and trophoblast-mediated placentation is a conserved phenomenon, our findings are ripe for translational application. Here we demonstrate the expression of CDX2 and isolation of CDX2 cells from human term placentas highlighting a potentially critical cell source for allogeneic therapy for cardiac regeneration. We studied de-identified human term placenta tissues from three different anatomical sites from three different patients. Formalin-fixed paraffin embedded samples, second trimester chorionic villus samples and de-identified fresh chorion samples were utilized. Using a multiparametric approach including transcript analysis and Sanger sequencing, immunoblot and immunofluorescence analysis, with the subsequent screening of different anatomical sites, we observed that CDX2 is present in the chorionic (fetal cytotrophoblast) portion of human term placentas including the chorionic villous samples (CVS). Subsequent validation on positive control cells DLD1 (human colon carcinoma cell line), versus HeLa cells (low endogenous CDX2 expression) was performed. Visualization of CDX2 expression showed the nuclear localization in the villi region. We have further demonstrated using a CDX2 promoter-driven fluorescence-based lentiviral expression system, we can isolate viable CDX2 cells from fresh term placenta. We demonstrate for the first time that CDX2 cells are present and can be isolated from
term
human placentas for prospective cardiomyocgenic differentiation. A step closer to the translational approach, our results herein may represent a paradigmatic shift in the way we approach early embryonic lineages and cell fate choices towards the identification of an unexplored human cell-based approach for cardiac regeneration.
Abstract 534: Chemically Defined Cell Culture Media for High Yield Differentiation of Functional Human Cardiac Myocytes
Authors: Lyra Leite Davi M , Burridge Paul W
Published: October 2020

Introduction:
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising technology for regenerative medicine and pharmacology. However, existing protocols are limited due to the high costs when generating cells at scale, to low yield, and to a lack of protocol replicability. Additionally, the use of animal-derived or recombinant proteins in cell differentiation creates concerns in human compatibility and presents technical challenges due to batch-to-batch variability.

Aim:
Develop a protocol for functional hiPSC-CM differentiation with high yield without the use of proteins.

Methods:
We expanded the CDM3 protocol by testing approximately 200 combinations of cell culture media during hiPSC differentiation. The differentiation was induced with a 24h incubation in 6 μM CHIR (d0), followed by 24 h in basal media (d1) and 48h in media supplemented with AA2P and Wnt-C59 (d2-d4). From day 4 onwards, cells were kept in media supplemented with insulin and AA2P, and in some conditions with polyvinyl alcohol. On day 8, hiPSC-CMs were antibiotically selected using geneticin. Tissues were imaged on day 13 to assess myocyte morphology and contraction. Videos were scored from 0 (dead/delaminated cells) to 9 (confluent tissues beating at least 4 times in a 5 sec interval).

Results:
We identified 6 basal media combinations that yield confluent monolayers on day 13. Out of these, 3 conditions consistently resulted in confluent contractile tissues.

Conclusions:
Our data demonstrate that a protein-free high yield hiPSC-CM differentiation is possible with the appropriate selection of basal media based on cellular nutritional requirements.
Abstract 536: Human Antigen R (HuR) in the Myofibroblasts is a Critical Regulator of Pressure-Overload Induced Cardiac Remodeling
Authors: Green Lisa , Anthony Sarah , Alam Perwez , Tranter Michael
Published: October 2020

Cardiac fibrosis is characterized by the excessive deposition of extracellular matrix (ECM) and is an important target because excess ECM leads to a stiffened myocardium and worsens the prognosis of heart failure. Human Antigen R (HuR) is an RNA binding protein known to stabilize mRNA through binding to AU rich regions in the 3’UTR, and we have previously shown HuR to play a central role in the development of cardiac hypertrophy. To interrogate the role of HuR in cardiac fibrosis, we used both a small molecule inhibitor of HuR as well as an inducible genetic deletion of HuR in the activated cardiac myofibroblasts (iMF-HuR
-/-
). Results using an
in vitro
scratch assay showed that primary adult cardiac fibroblasts treated with a small molecule inhibitor of HuR display a delayed wound healing response concomitant with a reduced expression of ECM remodeling genes.
In vivo
, we used an 8-week TAC (transverse aortic constriction) model of pressure overload to induce pathological cardiac remodeling. We hypothesized that deleting HuR in the activated fibroblasts would dampen myofibroblast-mediated ECM remodeling leading to a less stiff myocardium with reduced fibrotic burden and improved cardiac function in iMF-HuR
-/-
mice following pressure overload (TAC). However, echocardiography results show that cardiac function declined more rapidly in iMF-HuR
-/-
following TAC. The iMF-HuR
-/-
mice also had significantly enlarged hearts compared to the controls indicated by the heart weight/tibia length ratio. This suggests a critical role for HuR and myofibroblast activity in the early response to pressure overload. Future studies will focus on determining how the fibroblasts and the ECM composition/stiffness affect myocyte function in the early cardiac remodeling phase post-TAC and how HuR is involved in this relationship.
Abstract 546: S-palmitoylation Mediates Caveolae Localization and Limits Cysteine Oxidation of Gc-1 in Cardiomyocytes
Authors: Tzimas Christos , Russo Ilaria , Dun Wen , Yilmaz Sevde , Rajan Sudarsan , Tsai Emily J
Published: October 2020

Objectives:
The nitric oxide (NO) receptor soluble guanylyl cyclase (GC-1) can localize to cell membrane microdomains, including caveolae. In failing hearts, GC-1 relocalizes away from caveolae and becomes oxidized. How this occurs is unknown. We hypothesized that S-palmitoylation, a reversible, enzyme-catalyzed, post-translational modification that enhances membrane affinity of protein substrates, mediates caveolae-localization and modulates redox regulation of GC-1.

Methods:
Adult male C57BL/6 mice were subjected to transverse aortic constriction (TAC) versus thoracotomy (Sham). Modified acyl-biotin exchange (ABE) assays were performed to assess S-palmitoylation of GC-1 in failing TAC vs non-failing Sham hearts. Using bioinformatics, we identified 5 conserved cysteines in the GC-1α subunit as potential S-palmitoylation sites. We created cysteine-serine mutants of GC-1α at each and all 5 sites (C3S, C15S, C176S, C497S, C595S, CS*). Mutants and WT GC-1α were expressed in neonatal rat cardiomyocytes (NRCM) and their palmitoylation status compared by ABE. We assessed localization and oxidation of GC-1α (CS* vs WT) by confocal microscopy and redox Western, respectively.

Results:
In TAC hearts, S-palmitoylation of GC-1α was half of that in Sham (
p
=0.02). Likewise, palmitoyl-transferase ZDHHC16 was downregulated in TAC vs Sham by 31% (
p
=0.01) at the transcript level and by 56% (
p
=0.03) at the protein level. In NRCMs, S-palmitoylation of CS* was markedly diminished (6.9%,
p
<0.001), while that of C15S, C176S, and C497S were modestly diminished (23.8%, p=0.03; 23.2%, p=0.01; 22.8%, p=0.01), compared to WT (54.7%). Moreover, S-palmitoylation of WT but not CS* was decreased by palmitoylation inhibitor 2-BP (17.8%, p<0.0001). Confocal microscopy showed more diffuse cytosolic distribution of CS* compared to WT. Redox Westerns revealed increased oxidation of CS* vs WT upon H
2
O
2
treatment (1.7-fold increase, p=0.008).

Conclusions:
Our data support that GC-1α is S-palmitoylated at C15, C176, and C497. S-palmitoylation of GC-1α at these 3 cysteines appears to protect GC-1α against oxidation and to mediate its membrane localization.
Abstract 547: Hur Dependent Regulation of Inflammatory Remodeling Following Myocardial Ischemia/reperfusion Injury
Authors: Slone Samuel F , Fleifil Salma , Alam Perwez , Anthony Sarah , Nieman Michelle L , Lorenz John , Tranter Michael
Published: October 2020

Myocardial infarction (MI), resulting from ischemia/reperfusion (I/R) injury due to the obstruction of coronary blood flow, affects an estimated 800,000 Americans annually resulting in a cost of nearly 12 billion dollars to the health care system. Recent medical advances have led to improved survival following acute MI, but unfortunately, the post-MI inflammatory and fibrotic remodeling of the heart is driving an increase in the prevalence of heart failure. Preliminary data from our lab shows that the RNA binding protein Human antigen R (HuR) undergoes nuclear-to-cytoplasmic translocation within 30 minutes and is increased in expression at both 2 hours and 7 days post-I/R. The objective of this work is to determine the mechanistic role of HuR on the regulation of cardiac cytokine/chemokine expression and the functional effect of HuR on macrophage infiltration and polarization post-I/R.

Wild-type mice were subjected to 30 minutes of LAD (left anterior descending) coronary artery ligation (ischemia) followed by reperfusion with HuR inhibitor or vehicle control administered at the start of reperfusion. Our results show that inhibition of HuR reduces cardiac mRNA expression of IL-6, TNF-alpha, and ICAM-1 at 2 hours post-reperfusion, but does not affect initial (24 hour) infarct size. In addition, HuR inhibition reduces macrophage infiltration at 7 days post-I/R, as well as cardiac fibrosis and ventricular dilation while preserving cardiac function at 14 days post-I/R. Furthermore, H9c2s treated with HuR inhibitor blunts the mRNA expression of CCL2 after acute stimulation of lipopolysaccharide.

In conclusion, we show that HuR inhibition at the time of reperfusion is protective against post-MI inflammatory and fibrotic remodeling through a reduction in inflammatory gene expression and macrophage infiltration. Previous work by the lab has demonstrated a direct role for HuR in cardiac myocytes, but these results also suggest that HuR-mediated gene expression in macrophages may play a role in post-MI inflammatory and fibrotic remodeling.
Abstract 550: FOG2 Modulates a TBX5-Dependent Gene Regulatory Network for Cardiac Rhythm Homeostasis
Authors: Lazarevic Sonja , Broman Michael , Nadadur Rangarajan , Steimle Jeff , Laforest Brigitte , Perez-Cervantes Carlos , Mazurek Stefan R , Shen Kaitlyn M , Gadek Margaret , Ikegami Kohta , Yang Xinan , Weber Christopher , Svensson Eric , Moskowitz Ivan P
Published: October 2020

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Recent work has implicated numerous transcription factors in AF risk, including ZFPM2 (FOG2), GATA4, and TBX5. FOG2 is overexpressed in human heart failure, a major AF risk factor. We found that genetic inducible physiologic overexpression of FOG2 in the adult mouse heart caused spontaneous atrial fibrillation. Single cell electrophysiology revealed action potential prolongation and inappropriate depolarizations in FOG2-OE atrial cardiomyocytes. RNA-seq of the FOG2-OE left atrium prior to AF onset demonstrated that FOG2 suppresses a network of calcium cycling genes, providing a possible mechanism for disrupted cardiomyocyte electrophysiology. FOG2 ChIP-seq demonstrated genomic localization only at locations co-occupied by GATA4. Unexpectedly, we observed little overlap between genes activated by GATA4 and repressed by FOG2. Instead, we observed highly significant overlap between genes repressed by FOG2 and activated by TBX5. To identify the FOG2-dependent atrial gene regulatory network, we performed differential deep sequencing of ncRNAs. Comparison of atrial FOG2-dependent non-coding transcripts with previously performed TBX5-dependent ncRNA profiling indicated that TBX5 activated and FOG2 repressed a shared atrial rhythm gene regulatory network. Integration of FOG2, GATA4, and TBX5 ChIP-seq revealed that FOG2 only affected ncRNA transcription, indicative of enhancer activity changes, at locations co-occupied by TBX5. TBX5-dependent activation of specific regulatory elements for calcium handling genes was abolished by FOG2
in vitro
. The genomic TBX5/FOG2 interaction predicted a genetic interaction
in-vivo
, in which cardiac conduction and arrhythmia abnormalities caused by Tbx5 haploinsufficiency were rescued by FOG2 haploinsufficiency. Non-coding RNA profiling thereby predicted a novel functional TF interaction between FOG2 and TBX5. This work reveals a specific genomic model of atrial rhythm control in which FOG2 is recruited to GATA4 and TBX5 bound locations to modulate a TBX5-dependent atrial gene regulatory network for calcium handling and cardiac rhythm homeostasis.
Abstract 554: Reverse-mode Mitochondrial Na
+
/ Ca2+ Mitochondrial Exchange, Not the Mcu, is the Primary Mode of Ca2+ Import Into the Mitochondria During Ischemia/ Reperfusion in Neonatal Cardiac Myocytes
Authors: Ashok Deepthi , Papanicolaou Kyriakos , Liu Ting , O'Rourke Brian
Published: October 2020

Ca
2+
entry via the Mitochondrial Calcium Uniporter (MCU) participates in energetic adaption to workload under physiological conditions but is thought to contribute to cell death during ischemia-reperfusion (I/R) injury. We have previously shown that mitochondrial membrane potential (ΔΨm) instability contributes to early-reperfusion arrhythmias and contractile dysfunction; however, the role of mitochondrial Ca
2+
(mCa
2+
) uptake in triggering ΔΨm oscillation is unclear. Here, by acutely knocking out MCU, we examine whether MCU-mediated mCa
2+
uptake is required to trigger ΔΨm loss or oscillation during early reperfusion in neonatal mouse ventricular myocyte (NMVM) monolayers. We monitored mCa
2+
(with MitoCam) and ΔΨm (with TMRM) in WT(MCU
fl/fl
) and MCU-KO (MCU
fl/fl
+AdCre) NMVMs during in vitro I/R (previously described by Solhjoo et al JMCC, 2015) by confocal microscopy. Image sequences were analyzed for changes in mCa
2+
and ΔΨm by segmenting individual cells in ImageJ. To quantify ΔΨm oscillations in mitochondrial clusters during reperfusion, a new wavelet-transform-based analysis was developed using MATLAB’s wavelet toolbox. Surprisingly, our findings demonstrate that MCU knockout does not significantly alter mCa
2+
import during I/R, nor does it affect ΔΨm recovery during Reperfusion. In fact, MCU-KO moderately shortened the latency to Ischemic ΔΨm depolarization. In contrast, blocking the mitochondrial sodium-calcium exchanger (mNCLX) with CGP-37157 suppressed the mCa
2+
increase during Ischemia. Moreover, blocking mNCLX also did not affect ΔΨm recovery during Reperfusion or the frequency of ΔΨ
m
oscillations, confirming that mitochondrial ΔΨm instability on reperfusion is not triggered by mCa
2+
. Interestingly, inhibition of mitochondrial electron transport stabilized ΔΨm oscillations during reperfusion. The findings are consistent with mCa
2+
overload being mediated by reverse-mode mNCLX activity and support ROS-induced ROS release as the primary trigger of ΔΨm instability during reperfusion injury.
Abstract 555: Sex-Dependent Regulation of Cardiomyocyte Function by Midkine in Pediatric Dilated Cardiomyopathy
Authors: Woulfe Kc C , Wilson Cortney E , Karimpour-Fard Anis , Miyamoto Shelley D , Stauffer Brian L , SUCHAROV Carmen
Published: October 2020

Background:
Pediatric dilated cardiomyopathy (DCM) is a disease with a poor prognosis that affects 1 in 100,000 children. Girls with DCM have worse outcomes than boys, and the mechanisms that lead to these differences are not clear. We have identified a cytokine, midkine (MDK) that is significantly upregulated in the serum from pediatric DCM patients. Circulating MDK is significantly higher in girls with DCM requiring heart transplantation compared to girls with DCM who are stable.

Objective:
The objective of this study is to determine if MDK impacts cardiomyocyte function in a sex-specific manner in juvenile male and female cells.

Methods:
Cardiomyocytes isolated from 3 week old juvenile male and female rats (JRVMs) were treated with 1μg MDK for 48 hours. Cardiomyocyte function and calcium dynamics were assessed using an IonOptix system. Myofibril mechanics and calcium sensitivity were measured. In addition, RNA sequencing was completed to identify differentially regulated pathways in juvenile male and female cardiomyocytes in response to MDK.

Results:
Female JRVMs treated with MDK had higher peak calcium and slower calcium reuptake compared to vehicle-treated female JRVMs. In contrast, male JRVMs treated with MDK did not demonstrate a change in peak calcium and had faster calcium reuptake compared to vehicle-treated male JRVMs. Myofibril calcium sensitivity was decreased in female JRVMs in response to MDK whereas calcium sensitivity was unchanged in male MDK-treated JRVMs. Analysis of the genes that were differentially expressed in cardiomyocytes in response to MDK demonstrated a sex-dependent regulation. Specifically, pathways which regulate calcium handling were
only
altered in female JRVMs treated with MDK but not in MDK-treated male JRVMs.

Conclusions:
This study demonstrates sex-specific differences in cardiomyocyte function in response to MDK. Particularly, female cardiomyocytes respond to MDK by regulating genes involved in calcium handling pathways. This suggests that elevated circulating MDK in pediatric DCM patients may lead to different cardiac responses in male and female patients. Therefore, elucidating these sex-specific disease mechanisms is critical to define therapies focused on male and female pediatric DCM patients.
Abstract 556: Deficiency of Probhitin-1 in Hepatocytes Alters Systemic Glucose Regulation
Authors: Weihbrecht Katie , Mahoney Jo , Anderson Ethan J
Published: October 2020

Prohibitins 1 and 2 (PHB1 and PHB2) are lipid raft proteins that form a hetero-oligomeric complex known to localize to the cytoplasmic, nuclear, and mitochondrial membranes. These complexes play important roles in pro-survival and metabolic decisions including inflammation. PHB1 is important for mitochondria biogenesis and function and is excreted into the serum. Mice challenged with an intraperitoneal injection of lipopolysaccharide (LPS), a treatment that normally results in decreased cardiac function, show increased serum PHB. Injection of recombinant PHB1 (rPHB1) following LPS injection can rescue cardiac function. Work in our lab with cultured cardiomyocytes and multiple rodent models of severe sepsis supports the hypothesis that PHB1 is a liver-derived acute phase protein. To test this hypothesis, we utilized AAV- Cre recombinase infection driven by human thyroid hormone-binding globulin (TBG), a liver specific promotor (AAV8-TBG-iCre), on our PHB1 floxed mice (PHB1
fl/fl
). This liver specific knockdown of PHB1 allowed us to look at PHB1 levels in the serum of unstressed PHB1
fl/fl
mice. We discovered a loss of PHB1 in the liver tissue of floxed mice and significantly lower serum levels of PHB was found in homozygotes compared to wild types. Additionally, mice with liver specific loss of PHB1 show metabolic changes including significant weight loss three weeks following injection and changes to body composition and a trend of decreases in brown fat and inguinal fat mass. In addition to these body changes, we found that liver specific loss resulted in sexual dimorphism in insulin sensitivity. Homozygous males showed a significant difference in their ability to clear glucose following a glucose challenge in a glucose tolerance test. However, following an insulin tolerance test, female homozygotes showed increased insulin sensitivity where males did not. Both males and females show a trend towards attenuated gluconeogenesis following a pyruvate tolerance test. Overall, we have begun to characterize novel findings to the metabolic state of mice lacking PHB1 only in the liver. Our aim is to further explore if these differences are due to the changes in liver PHB1 or due to changes in circulating serum PHB1.
Abstract MP100: Scalable Functional Classification of Heart Failure-associated Genetic Variants in Cardiac Troponin T
Authors: PETTINATO Anthony M , Ladha Feria A , Mellert David , Legere Nicholas , Cohn Rachel , Romano Robert , Thakar Ketan , Chen Yu-Sheng , Hinson John T
Published: October 2020

Sarcomere gene mutations are the most common inheritable risk factor for heart failure, a disease with high morbidity and mortality, yet the pathogenicity of a large proportion of sarcomere gene variants remains unknown. The knowledge of which and how sarcomere gene variants result in heart failure would provide valuable prognostic and diagnostic information for individuals harboring sarcomere gene variants. Here, we developed a transgenic human cardiomyocyte model that enables scalable delivery of genetic variants in cardiac troponin T (cTnT; encoded by
TNNT2
), empowering high-throughput analysis of variant-specific sarcomere function. Using this model, in conjunction with CRISPR-derived isogenic standards and biomimetic cardiac tissues, we demonstrate that hypertrophic cardiomyopathy (HCM) variants in
TNNT2
evoke elevated cardiac tissue contractile function, while dilated cardiomyopathy (DCM) variants produce decreased contractile function. RNA-seq revealed a transcriptional network that correlates with contractile function and identified
NPPB
expression as a molecular signature of contraction. Through the development of a cellular
NPPB
reporter, we functionally screen a panel of 51
TNNT2
variants, and provide molecular and tissue-level evidence for the functional reclassification of
TNNT2
“variants of unknown significance” to pathogenic, and “pathogenic” variants to benign. Finally, adaption of this cellular reporter to assess variant calcium sensitivity, together with analysis of myofilament-localized calcium transients, demonstrates a parallel between contractile function and calcium sensitivity in human cardiomyocytes. This study establishes a new tool to study sarcomere genetic variants at scale, reclassifies the pathogenicity of previously-identified
TNNT2
variants, and provides new insights into the cellular pathogenesis of human heart failure.
Abstract MP101: Functional Maintenance of the Sarcomere Requires Bag3-dependent Autophagy
Authors: Martin Thomas G , Feldman Arthur M , Willis Monte S , Kirk Jonathan A
Published: October 2020

Bcl2-associated athanogene-3 (BAG3) is a pro-autophagy co-chaperone highly expressed in the heart. Clinical studies show BAG3 haploinsufficiency and mutations are associated with heart failure (HF). However, these studies are largely observational and fail to go beyond the observed phenotype and study mechanism. One study in neonatal myocytes suggests a role for BAG3 in structural maintenance of the sarcomere. However, the structural and functional significance of BAG3 in adult myocytes is not known. We found that myofilament BAG3 expression decreases in human heart failure and is associated with impaired myofilament force-generating capacity (F
max
). To assess whether rescuing BAG3 levels could restore function, we used a mouse model of HF and treated with BAG3 gene therapy via AAV9. Myofilament function was assessed in skinned cardiomyocytes by force-calcium relationship. HF mice experienced a reduction in F
max
, but this was fully restored to sham levels by BAG3 gene therapy.To explore mechanism, we used mass spectrometry to identify the BAG3-interactome at the myofilament and found heat shock proteins (HSP) 70 and B8 among the top hits. Immunofluorescence further showed that HSP70, B8, and BAG3 each localized to the sarcomere z-disk. This BAG3-Hsp complex had previously been described to promote ubiquitin-dependent autophagy in skeletal muscle. Notably, in both human HF samples and in the mouse HF model, myofilament ubiquitin levels increased significantly. However, BAG3 gene therapy in HF reduces ubiquitin levels and restores autophagy flux. This suggests that BAG3 serves a role in proteostasis for the sarcomere, which may explain the functional effect of BAG3 gene therapy.To further explore the impact of the BAG3/HSP complex on myofilament function, we used a mouse model with the P209L BAG3 mutation, which had previously been described to disrupt client processing by the complex. We found cardiomyocytes from P209L mice had significantly reduced F
max
and elevated myofilament ubiquitin levels, suggesting BAG3-dependent autophagy is required to maintain function. Together, our data identify a functional role for BAG3 at the sarcomere and indicate BAG3-mediated autophagy is an important mechanism for maintaining myofilament proteostasis.
Abstract MP102: Skeletal Muscle Alpha Actin Acetylation Enhances Myosin Binding and Increases Calcium Sensitivity
in vitro
Authors: Romanick Samantha S , Matsumura Allison , Stewart Travis , Boc Kianna , Baker Jonathan , Ferguson Bradley S
Published: October 2020

Actin and myosin are key proteins for muscle contraction/relaxation, while tropomyosin regulates actin-myosin interactions in the presence or absence of calcium. Mutations or dysregulation of any of these proteins results in cardiomyopathy. We previously showed that skeletal muscle alpha actin (ACTA1) was significantly deacetylated on lysine residues, K52, K317, and K328 in remodeled left ventricular tissue of obese mice. Computational modeling suggests that the positively charged lysine residues K328 and K317 of ACTA1 can interact electrostatically with the negatively charged glutamic acid residue E181 of tropomyosin and E286 of myosin. As acetylation is predicted to neutralize the positively charged lysine, ACTA1 acetylation would be postulated to decrease actin-myosin or actin-tropomyosin electrostatic interactions. To test this hypothesis, we used an in vitro actin motility assay to determine myosin sliding velocity, calcium sensitivity, and attachment/detachment kinetics of acetylated/deacetylated ACTA1. In addition, an actin binding protein spin-down assay was used to determine actin-myosin binding affinity using skeletal and cardiac myosin. In these assays, ACTA1 was chemically acetylated with acetic anhydride. In vitro actin motility analysis showed a significant decrease in sliding velocity with acetylated ACTA1 and skeletal myosin (1.709±0.210 μm/s) compared with deacetylated ACTA1 (4.427±0.275 μm/s). A similar significant decrease was also noted with cardiac myosin. Further analysis showed a significant increase in calcium sensitivity with ACTA1 acetylation (3.197x10-7 Kd compared to deacetylated ACTA1 1.191x10-6 Kd) and a loss of tropomyosin regulation with increasing ACTA1 acetylation. Lastly, ACTA1 acetylation enhanced actin binding affinity to cardiac and skeletal myosin. Investigation of attachment/detachment kinetics are currently underway. These data suggest that ACTA1 acetylation disrupts tropomyosin’s ability to inhibit myosin binding in the absence of calcium and further regulates actin-myosin interactions. Lastly, these data highlight acetylation as an additional post-translational modification outside of phosphorylation in the regulation of muscle contraction.
Abstract MP105: Python Post-prandial Cardiac Adaptation is Supported by Enhanced Metabolism, Reduced Sarcomere Passive Tension, and Epigenetic Remodeling
Authors: Crocini Claudia , Woulfe Kc , Walker Cierra J , Ozeroff Christopher D , leinwand leslie A
Published: October 2020

Pythons are infrequent feeders that can ingest meals equal to their own body mass. The extreme metabolic response required to digest such large meals is associated with a dramatic increase in the mass of most organs, including the heart. Recently, we have been able to assess functional effects of feeding using isolated python cardiomyocytes and myofibrils, advancing our understanding of extreme cardiac adaptation in python (
Python regius
). Twenty-four hours after feeding, python cardiomyocytes showed prolonged Ca
2+
transients, increased maximal tension and Ca
2+
sensitivity of myofibrils as compared to fasted pythons. Post-prandial positive inotropy was accompanied by enhanced metabolic output via increased mitochondrial ATP production rate and by AMP-dependent kinase (AMPK) activation and phosphofructokinase-2 reduction, suggesting a key role for fatty acid, but not glucose, metabolism after feeding. In addition, 24h post-fed hearts had significantly reduced tissue stiffness and myofibril passive tension. Finally, chromatin condensation was reduced about 30% after feeding in python cardiomyocytes and confirmed by increased histone acetylation, indicating a predominant role for epigenetics in post-prandial adaptation. These results suggest that feeding promotes positive cardiac inotropy in python via a number of coordinated mechanisms to enhance energy production, increase myofibril and tissue compliance, and increase chromatin accessibility. As heart failure is commonly characterized by depressed contractility, compromised energetics, and increased tissue stiffness, assessing post-prandial adaptation in python hearts provides us with powerful insights that could inform the development of therapeutics for human heart diseases.
Abstract MP111: Loss of Myosin Binding Protein H-like Causes Cardiacconduction Abnormalities
Authors: Barefield David Y , Tahtah Ibrahim , Sell Jordan , Broman Michael , Laforest Brigitte , Yamakawa Sean , Puckelwartz Megan , Wasserstrom Andrew , McNally Elizabeth M
Published: October 2020

A premature truncation variant in
MYBPHL in humans and a loss of Mybphl in mice
associates with dilated cardiomyopathy (DCM), atrial and ventricular arrhythmias, and atrial enlargement.
MYBPHL
encodes myosin binding protein H-like (MyBP-HL) and is expressed highly in the atria and in foci throughout the ventricle. We hypothesize that MyBP-HL is required for proper formation and function of the conduction system. Surface telemetry found atrioventricular block and atrial bigeminy, while intracardiac pacing revealed a faster atrial relative refractory period and atrial tachycardia in
Mybphl
-null mice. Ca
2+
transient analysis revealed that isolated
Mybphl
-null atrial cardiomyocytes had an increased occurrence of triggered Ca
2+
waves and more heterogenous Ca
2+
release than wild-type (WT) controls. Super-resolution microscopy revealed ryanodine receptor disorganization in
Mybphl
het and null atrial cardiomyocytes compared to WT controls. Immunofluorescence microscopy in WT adult mouse ventricles identified MyBP-HL-positive ventricular cardiomyocytes that co-localized with the ventricular conduction system marker contactin-2 near the atrioventricular node and in a subset of Purkinje fibers.
Mybphl
heterozygous ventricles showed 10% as many MyBP-HL-positive cells compared to WT. Lightsheet microscopy of perinatal day 5 hearts showed enrichment of MyBP-HL-positive cells within and immediately adjacent to the Cntn2-positive ventricular conduction system in WT hearts, but this association was not apparent in
Mybphl
heterozygous null hearts. These data, abnormal Ca
2+
release, shorter atrial refractory period, and atrial dilation could account for the observed atrial arrhythmias, bigeminy, and atrial tachycardia, whereas the proximity of MyBP-HL-positive cells with the ventricular conduction system provides insight into how a predominantly atrial expressed gene could cause ventricular arrhythmias.
Abstract MP115: Transcriptional Patterning of the Ventricular Cardiac Conduction System
Authors: Burnicka-Turek Ozanna , Broman Michael , Steimle Jeffrey D , Boukens Bastiaan , Petrenko Nataliya B , Ikegami Kohta , Yang Xinan H , Nobrega Marcelo A , Rust Michael J , Patel Vickas V , Efimov Igor , Moskowitz Ivan P
Published: October 2020

The heart beats 2 billion times over the average human lifespan and is the manifestation of a pattern of cardiomyocyte depolarization organized by a specialized network of cardiomyocytes, the cardiac conduction system (CCS). Patterning of the CCS into atrial node versus ventricular conduction system (VCS) components with distinct physiology is essential for the normal heartbeat and has been recognized for more than a century. However molecular basis of this regional patterning is not well understood. Using mouse genetics, we found that the ratio between T-box transcriptional activator,
Tbx5
, and T-box transcriptional repressor,
Tbx3
, determines the molecular and functional output of VCS myocytes. Adult VCS-specific removal of
Tbx5
or overexpression of
Tbx3
re-patterned the fast VCS into slow, nodal-like cells based on molecular, cellular and functional criteria. Specifically, the ventricular fast conduction gene expression network was lost whereas the slow conduction nodal gene expression network was retained. Action potentials (APs) of
Tbx5
-deficient VCS myocytes adopted nodal-specific characteristics, including increased AP duration and cellular automaticity. Removal of
Tbx5 in-vivo
precipitated inappropriate depolarizations in the His-bundle that initiated lethal ventricular arrhythmias. A T-box rheostat mechanism for CCS patterning was confirmed by
Tbx5/Tbx3
genetic interaction studies. TBX5 bound and directly activated
cis
-regulatory elements at fast conduction loci genome-wide, defining the identity of the adult VCS. Furthermore, TBX5 bound and activated cis-regulatory elements at
Tbx5
itself, describing a multi-tiered T-box-dependent fast conduction gene regulatory network (GRN). The hierarchical GRN established a bi-stable network in mathematical modeling, with only high or low fast conduction gene expression states, suggesting a genomic mechanism for the establishment of VCS versus nodal states
in-vivo
. Thus, the CCS is patterned entirely as a slow, nodal ground state, with a T-box dependent, physiologically dominant, fast conduction network driven specifically in the VCS. Disruption of the fast VCS GRN allowed nodal physiology to emerge, providing a molecular mechanism for some lethal ventricular arrhythmias.
Abstract MP121: Deleting the 2-oxoglutarate- and Iron-dependent Prolyl Hydroxylase Ogfod1 Alters Purine Nucleotide Regulation and is Cardioprotective
Authors: Kennedy Leslie , Sun Junhui , Harris Michael , Cockman Matthew , Ratcliffe Peter , Murphy Elizabeth
Published: October 2020

Prolyl hydroxylation is a post-translational modification that regulates protein stability, turnover, and activity. The proteins that catalyze prolyl hydroxylation belong to the 2-oxoglutarate- and iron-dependent oxygenase family of enzymes. A newly-described member of this family is 2-oxoglutarate- and iron-dependent oxygenase domain containing protein 1 (Ogfod1), which catalyzes prolyl hydroxylation of the ribosomal protein s23 (Rps23). To investigate the cardiovascular function of Ogfod1, we isolated hearts from 5
Ogfod1
-WT and 5
Ogfod1
-KO mice and used Liquid Chromatography and Tandem Mass Spectrometry (LC-MS/MS) to identify proteomic changes. Ingenuity Pathway Analysis (IPA) identified “Purine Nucleotides Degradation II (Aerobic)” (
P
= 0.00017) to be one of the most significantly-enriched pathways. We then did metabolomics and found that Inosine 5’-monophosphate (IMP) was 3.5x higher in
Ogfod1
-KO hearts (
P
= 0.011), further supporting a role for Ogfod1 in regulating purine nucleotide metabolism. Recent evidence has shown that altering purine nucleotide degradation protects against diet-induced obesity and insulin resistance, so we tested this hypothesis in
Ogfod1
-KO mice by feeding them high-fat diets.
Ogfod1
ablation protects against high-fat diet-induced obesity and insulin resistance. Altering purine nucleotide degradation has also been shown to be protective against cardiac injury, so we tested the hypothesis that Ogfod1 loss protects the heart from ischemia-reperfusion (I/R) injury by subjecting perfused hearts from 6
Ogfod1
-WT and 6
Ogfod1
-KO mice to ischemia and reperfusion and assessed tissue death. We found a 37% decrease in infarct size in
Ogfod1
-KO hearts (56% in
Ogfod1
-WT and 35% in
Ogfod1
-KO,
P
= 0.0003). In a separate set of experiments, we treated
Ogofd1
-KO mice with isoproterenol to induce hypertrophy, and
Ogfod1
-KO hearts showed protection against hypertrophic remodeling. Interestingly,
OGFOD1
transcripts were up-regulated in human heart failure, indicating a potential role for
OGFOD1
in the human failing heart. Altogether, these data show that
Ogfod1
deletion alters the myocardial proteome and myocardial metabolism and protects against obesity, insulin sensitivity, I/R injury, and hypertrophy.
Abstract MP122: Effects of Cardiac Beta3-Adrenoceptors on the Inhibition of G Protein-coupled Receptor Kinase 2 via S-nitrosylation
Authors: Kayki Mutlu Gizem , Ferrero Kimberly , Roy Rajika , Lucchese Anna Maria , Gao Erhe , Koch Walter J
Published: October 2020

G Protein-Coupled Receptor Kinase 2 (GRK2) is a culprit in the loss of cardiac contractile function in heart failure due to β-Adrenoceptor (AR) desensitization after its upregulation. Indeed, its inhibition has been demonstrated to improve cardiac function and increased GRK2 in the heart leads to larger injury after an ischemic insult. Nitric oxide (NO) via S-nitrosothiol (SNO) at residue Cys340 is a reported endogenous inhibitor of GRK2 activity. ß3ARs, on the other hand, are known to be resistant to desensitization by GRK2 and they are upregulated in cardiac pathologies. Activation of ß3ARs can be cardioprotective via NO signalling. Thus, in the present study, we aimed to investigate the interaction between ß3ARs and GRK2 through NO signaling to determine if ß3AR cardioprotection can occur via NO-mediated GRK2 inhibition. We used wild type C57BL/6 mice (WT), global β3AR knockout (KO) mice and GRK2-C340S knockin mice, which harbor a point mutation that changes Cys340 with a serine, meaning all endogenous GRK2 globally cannot be inhibited via NO-mediated S-nitrosylation. We exposed WT, GRK2-C340s KI and β3AR KO mice to ischemia/reperfusion (I/R) injury (40 min ischemia followed by 24 hrs reperfusion). We found that WT mice had significantly diminished cardiac function evaluated by echocardiography and Millar Catheterization, and this was rescued by treating these mice with CL316,243 (a selective β3AR agonist) at the time of reperfusion. On the other hand, GRK2-C340S KI mice did worse after I/R injury compared to WT mice and CL316,243 did not rescue this dysfunction as it did in WT mice. As expected, β3AR KO mice had worsened cardiac function compared to WT mice and CL316,243 had no functional benefit. Infarct size measurements after I/R revealed that β3AR KO mice had larger infarcts than WT mice supporting β3ARs as being protective. Indeed, CL316,243 induced robust cardioprotection in WT mice, reducing infarct size. GRK2-C340S mice had larger infarcts than WT mice and CL316,243 failed to offer any cardioprotection. Thus, β3AR-mediated cardioprotection clearly involves inhibition of GRK2 as part of its therapeutic mechanism and this appears to involve NO-mediated S-nitrosylation.