bluuebunny/crossref_metadata_2025 huggingface dataset:
On the Motion of Two Microspheres in a Stokes Flow Driven by an External Oscillator Field
Authors: Al-Hatmi Mohammed M. , Purnama Anton
Published: November 2021

Hydrodynamic interactions of a two-solid microspheres system in a viscous incompressible fluid at low Reynolds number is investigated analytically. One of the spheres is conducting and assumed to be actively in motion under the action of an external oscillator field, and as the result, the other nonconducting sphere moves due to the induced flow oscillation of the surrounding fluid. The fluid flow past the spheres is described by the Stokes equation and the governing equation in the vector form for the two-sphere system is solved asymptotically using the two-timing method. For illustrations, applying a simple oscillatory external field, a systematic description of the average velocity of each sphere is formulated. The trajectory of the sphere was found to be inversely proportional to the frequency of the external field. The results demonstrated that no collisions occur between the spheres as the system moves in a circular motion with a fixed separation distance.
Repurposing Ceritinib Induces DNA Damage and Enhances PARP Inhibitor Responses in High-Grade Serous Ovarian Carcinoma
Authors: Kanakkanthara Arun , Hou Xiaonan , Ekstrom Thomas L. , Zanfagnin Valentina , Huehls Amelia M. , Kelly Rebecca L. , Ding Husheng , Larson Melissa C. , Vasmatzis George , Oberg Ann L. , Kaufmann Scott H. , Mansfield Aaron S. , Weroha S. John , Karnitz Larry M.
Published: November 2021

PARP inhibitors (PARPi) have activity in homologous recombination (HR) repair-deficient, high-grade serous ovarian cancers (HGSOC). However, even responsive tumors develop PARPi resistance, highlighting the need to delay or prevent the appearance of PARPi resistance. Here, we showed that the ALK kinase inhibitor ceritinib synergizes with PARPis by inhibiting complex I of the mitochondrial electron transport chain, which increases production of reactive oxygen species (ROS) and subsequent induction of oxidative DNA damage that is repaired in a PARP-dependent manner. In addition, combined treatment with ceritinib and PARPi synergized in HGSOC cell lines irrespective of HR status, and a combination of ceritinib with the PARPi olaparib induced tumor regression more effectively than olaparib alone in HGSOC patient-derived xenograft (PDX) models. Notably, the ceritinib and olaparib combination was most effective in PDX models with preexisting PARPi sensitivity and was well tolerated. These findings unveil suppression of mitochondrial respiration, accumulation of ROS, and subsequent induction of DNA damage as novel effects of ceritinib. They also suggest that the ceritinib and PARPi combination warrants further investigation as a means to enhance PARPi activity in HGSOC, particularly in tumors with preexisting HR defects.

Significance:

The kinase inhibitor ceritinib synergizes with PARPi to induce tumor regression in ovarian cancer models, suggesting that ceritinib combined with PARPi may be an effective strategy for treating ovarian cancer.
BAFF Attenuates Immunosuppressive Monocytes in the Melanoma Tumor Microenvironment
Authors: Liu Wei , Stachura Paweł , Xu Haifeng C. , Váraljai Renáta , Shinde Prashant , Ganesh Nikkitha Umesh , Mack Matthias , Van Lierop Anke , Huang Anfei , Sundaram Balamurugan , Lang Karl S. , Picard Daniel , Fischer Ute , Remke Marc , Homey Bernhard , Roesch Alexander , Häussinger Dieter , Lang Philipp A. , Borkhardt Arndt , Pandyra Aleksandra A.
Published: November 2021

Emerging evidence indicates B-cell activating factor (BAFF, Tnfsf13b) to be an important cytokine for antitumor immunity. In this study, we generated a BAFF-overexpressing B16.F10 melanoma cell model and found that BAFF-expressing tumors grow more slowly in vivo than control tumors. The tumor microenvironment (TME) of BAFF-overexpressing tumors had decreased myeloid infiltrates with lower PD-L1 expression. Monocyte depletion and anti-PD-L1 antibody treatment confirmed the functional importance of monocytes for the phenotype of BAFF-mediated tumor growth delay. RNA sequencing analysis confirmed that monocytes isolated from BAFF-overexpressing tumors were characterized by a less exhaustive phenotype and were enriched for in genes involved in activating adaptive immune responses and NF-κB signaling. Evaluation of patients with late-stage metastatic melanoma treated with inhibitors of the PD-1/PD-L1 axis demonstrated a stratification of patients with high and low BAFF plasma levels. Patients with high BAFF levels experienced lower responses to anti-PD-1 immunotherapies. In summary, these results show that BAFF, through its effect on tumor-infiltrating monocytes, not only impacts primary tumor growth but can serve as a biomarker to predict response to anti-PD-1 immunotherapy in advanced disease.

Significance:

The BAFF cytokine regulates monocytes in the melanoma microenvironment to suppress tumor growth, highlighting the importance of BAFF in antitumor immunity.
Cardiometabolic Risk in Childhood Cancer Survivors: A Report from the Children's Oncology Group
Authors: Lipshultz Emma R. , Chow Eric J. , Doody David R. , Armenian Saro H. , Asselin Barbara L. , Baker K. Scott , Bhatia Smita , Constine Louis S. , Freyer David R. , Kopp Lisa M. , Schwartz Cindy L. , Lipshultz Steven E. , Vrooman Lynda M.
Published: November 2021

Background:

Childhood cancer survivors are at risk for cardiovascular disease. We assessed the burden of potentially modifiable cardiometabolic risk factors (CRF) among survivors compared with population-matched controls.

Methods:

Survivors previously enrolled on Pediatric Oncology Group protocols 9404, 9425, 9426, 9754, and Dana-Farber Cancer Institute 95-01 from 1996 to 2001 with acute lymphoblastic leukemia/lymphoma, Hodgkin lymphoma, or osteosarcoma were prospectively assessed for the prevalence of CRFs and compared with an age, sex, and race/ethnicity-matched 2013 National Health and Nutrition Examination Survey (NHANES) population. We estimated future predicted cardiovascular risk based on general population (e.g., Framingham) and Childhood Cancer Survivor Study (CCSS) models.

Results:

Compared with NHANES (n = 584), survivors [n = 164; 44.5% female, median age 28 years (range, 16–38 years); median 17.4 years (range, 13–22 years) since cancer diagnosis; median doxorubicin dose 300 mg/m2; 30.5% chest radiation] had similar rates of obesity, diabetes, and dyslipidemia, but more prehypertension/hypertension (38.4% vs. 30.1%, P = 0.044). Survivors had fewer metabolic syndrome features compared with NHANES (≥2 features: 26.7% vs. 55.9%; P < 0.001). Survivors were more physically active and smoked tobacco less (both P < 0.0001). Therefore, general population cardiovascular risk scores were lower for survivors versus NHANES. However, with CCSS models, 30.5% of survivors were at moderate risk of ischemic heart disease, and >95% at moderate/high risk for heart failure, with a 9% to 12% predicted incidence of these conditions by age 50 years.

Conclusions:

Childhood cancer survivors exhibited similar or better cardiometabolic and lifestyle profiles compared with NHANES, but nonetheless are at risk for future clinically significant cardiovascular disease.

Impact:

Further strategies supporting optimal CRF control are warranted in survivors.

See related commentary by Mulrooney, p. 515
Multiple Primary Cancers in Patients Undergoing Tumor-Normal Sequencing Define Novel Associations
Authors: Liu Ying L. , Cadoo Karen A. , Mukherjee Semanti , Khurram Aliya , Tkachuk Kaitlyn , Kemel Yelena , Maio Anna , Belhadj Sami , Carlo Maria I. , Latham Alicia , Walsh Michael F. , Dubard-Gault Marianne E. , Wang Yuhan , Brannon A. Rose , Salo-Mullen Erin , Sheehan Margaret , Fiala Elise , Devolder Bryan , Dandiker Sita , Mandelker Diana , Zehir Ahmet , Ladanyi Marc , Berger Michael F. , Solit David B. , Bandlamudi Chaitanya , Ravichandran Vignesh , Bajorin Dean F. , Stadler Zsofia K. , Robson Mark E. , Vijai Joseph , Seshan Venkatraman , Offit Kenneth
Published: November 2021

Background:

Cancer survivors are developing more subsequent tumors. We sought to characterize patients with multiple (≥2) primary cancers (MPC) to assess associations and genetic mechanisms.

Methods:

Patients were prospectively consented (01/2013–02/2019) to tumor-normal sequencing via a custom targeted panel (MSK-IMPACT). A subset consented to return of results of ≥76 cancer predisposition genes. International Agency for Research on Cancer (IARC) 2004 rules for defining MPC were applied. Tumor pairs were created to assess relationships between cancers. Age-adjusted, sex-specific, standardized incidence ratios (SIR) for first to second cancer event combinations were calculated using SEER rates, adjusting for confounders and time of ascertainment. Associations were made with germline and somatic variants.

Results:

Of 24,241 patients, 4,340 had MPC (18%); 20% were synchronous. Most (80%) had two primaries; however, 4% had ≥4 cancers. SIR analysis found lymphoma–lung, lymphoma–uterine, breast–brain, and melanoma–lung pairs in women and prostate–mesothelioma, prostate–sarcoma, melanoma–stomach, and prostate–brain pairs in men in excess of expected after accounting for synchronous tumors, known inherited cancer syndromes, and environmental exposures. Of 1,580 (36%) patients who received germline results, 324 (21%) had 361 pathogenic/likely pathogenic variants (PV), 159 (44%) in high penetrance genes. Of tumor samples analyzed, 55% exhibited loss of heterozygosity at the germline variant. In those with negative germline findings, melanoma, prostate, and breast cancers were common.

Conclusions:

We identified tumor pairs without known predisposing mutations that merit confirmation and will require novel strategies to elucidate genetic mechanisms of shared susceptibilities.

Impact:

If verified, patients with MPC with novel phenotypes may benefit from targeted cancer surveillance.
The Risk of Cirrhosis and Its Complications Based on PNPLA3 rs738409 G Allele Frequency
Authors: Shao Xue , Uojima Haruki , Arai Taeang , Ogawa Yuji , Setsu Toru , Atsukawa Masanori , Furuichi Yoshihiro , Arase Yoshitaka , Horio Kazue , Hidaka Hisashi , Nakazawa Takahide , Kako Makoto , Kagawa Tatehiro , Iwakiri Katsuhiko , Nakajima Atsushi , Terai Shuji , Tanaka Yasuhito , Koizumi Wasaburo
Published: November 2021

Background: Data regarding the influence of patatin-like phospholipase domain-containing 3 (PNPLA3) polymorphism for patients with liver cirrhosis (LC) are scarce. Objective: This study assesses the role of the PNPLA3 polymorphism for the development of LC and its complications by the findings of genetic examinations. Methods: Patients with LC caused by virus (n = 157), alcohol (n = 104), nonalcoholic fatty liver disease (NAFLD) (n = 106), or autoimmune disease (n = 33) and without LC (n = 128) were enrolled. LC was composed of the presence and absence of complications, such as variceal bleeding, hepatic ascites, and hepatic encephalopathy. To assess the role of the PNPLA3 polymorphism, odds ratio (OR) for the rs738409 variant was calculated for the patients between (i) with LC and without LC in the entire cohort and (ii) the presence and absence of complications in the patients with LC. Results: There was a significant difference among the patients without LC and those with alcohol, NAFLD-related LC in the frequency of G alleles (p < 0.001, both). According to complications of LC, the OR for NAFLD-related cirrhosis significantly increased in the presence of the two mutated alleles (OR = 3.165; p = 0.046) when the wild type was used as the reference. However, there were no significant risks for the complications in the virus and alcohol-related cirrhosis unless there was a presence of G alleles. Conclusion: The PNPLA3 polymorphism was associated with the risk of NAFLD-related LC and its complications.
Mode of Death in Patients With Heart Failure and Preserved Ejection Fraction: Insights From PARAGON-HF Trial
Authors: Desai Akshay S. , Vaduganathan Muthiah , Cleland John G. , Claggett Brian L. , Barkoudah Ebrahim , Finn Peter , McCausland Finnian R. , Yilmaz Mehmet B. , Lefkowitz Martin , Shi Victor , Pfeffer Marc A. , McMurray John J.V. , Solomon Scott D.
Published: November 2021

Background:

Patients with heart failure (HF) and preserved left ventricular ejection fraction comprise a heterogeneous group including some with mildly reduced EF. We hypothesized that mode of death differs by EF in ambulatory patients with HF and preserved left ventricular ejection fraction.

Methods:

PARAGON-HF trial (Prospective Comparison of Angiotensin Receptor–Neprilysin Inhibitor With Angiotensin-Receptor Blocker Global Outcomes in Heart Failure With Preserved Ejection Fraction) compared clinical outcomes in 4796 patients with chronic HF and EF ≥45% randomly assigned to sacubitril/valsartan or valsartan. We examined the mode of death in relation to baseline EF in logistic regression models and the effect of randomized treatment on cause-specific death in Cox regression models. Nonlinear relationships with continuous EF were modelled using quadratic and cubic terms.

Results:

Of 691 deaths during the trial, 416 (60%) were ascribed to cardiovascular, 220 (32%) to noncardiovascular, and 55 (8%) to unknown causes. Of cardiovascular deaths, 154 (37%) were due to sudden death, 118 (28%) were due to HF, 35 (8%) to stroke, 27 (6%) to myocardial infarction, and 82 (20%) to other cardiovascular causes. Rates of all-cause, cardiovascular, and sudden death were higher in those with lower left ventricular ejection fraction (all
P
<0.001), while rates of non-cardiovascular death were greater in patients with higher EF. Sacubitril/valsartan did not reduce overall death, cardiovascular death, or sudden death compared with valsartan, irrespective of baseline EF (all
P
for interaction >0.30).

Conclusions:

Among patients with HF and preserved left ventricular ejection fraction enrolled in PARAGON-HF, the proportion of cardiovascular and sudden death were higher in those with lower left ventricular EF, and the proportion of noncardiovascular death rose with EF. Regardless of EF, sacubitril/valsartan did not reduce death from any cause compared with valsartan.

Registration:

URL:
https://www.clinicaltrials.gov
; Unique identifier: NCT01920711.
Abstract 100: Integrated Cardiac Metabolism In End-Stage Human Heart Failure
Authors: Flam Emily , Jang Cholsoon , Bedi Ken , Murashige Danielle , Yang Yifan , Morley Michael , Brandimarto Jeff , Prosser Ben , Cappola Tom , Rabinowitz Josh , Margulies Ken , Arany Zolt
Published: November 2021

Heart failure affects millions of people worldwide with mortality near 50% within five years. This disease is characterized by widespread cardiac and systemic metabolic changes, but a comprehensive evaluation of metabolism in failing human hearts is lacking. Here, we provide a comprehensive depiction of cardiac and systemic metabolic changes in 89 explanted failing and non-failing human hearts through integration of plasma and cardiac tissue metabolomics, genome-wide RNAseq, and proteomic data. The data confirm a profound bioenergetic defect in end-stage human heart failure and demonstrate extensive changes in metabolic homeostasis. The data indicate a substantial defect in fatty acid (FA) use in failing hearts, in particular unsaturated FAs. Reduction of FAs and acyl-carnitines in failing tissue in contrast to concomitant elevations in plasma suggest a defect in import of FAs into the cell, rather than a defect in FA oxidation. Intermediates of glycolysis, the pentose phosphate pathway, and glycogen synthesis are all similarly reduced, as is expression of GLUT1, indicating diminished glucose uptake. However, there was no significant change in tissue pyruvate content, suggesting an increase in lactate utilization. The data suggest increased flux of pyruvate into mitochondria, likely promoting pyruvate oxidation but not pyruvate carboxylation. Blunted anabolic pyruvate flux, in turn, likely leads to insufficient TCA cycle intermediates. Ketone levels were increased in both failing tissue and plasma, as previously reported. The phospholipid content of failing human hearts is greatly increased in both failing tissue and plasma. Nucleotide synthesis pathways also appear to be reprogrammed, with a notable decrease in adenosine metabolism, specifically. Together, these data indicate widespread change in the local cardiac and greater systemic metabolic landscape in severe human heart failure.
Abstract 103: Mechanistic Interplay Between Cardioprotection And Heart Regeneration Mediated By The ER-bound Transcription Factor Nrf1
Authors: Cui Miao , Ayhan Atmanli , Liu Ning , Bassel-duby Rhonda S , Olson Eric N
Published: November 2021

Cardiomyocyte loss is the underlying basis for a majority of heart diseases. Preventing cardiomyocytes from death (cardioprotection) and replenishing the lost myocardium (regeneration) are the central goals for heart repair. Although cardioprotection and heart regeneration have been traditionally thought to involve separate mechanisms, protection of cardiomyocytes from injury or disease stimuli is a prerequisite to any meaningful regenerative response. In our study, we sought to understand how neonatal cardiomyocytes cope with injury-induced stress to regenerate damaged myocardium and whether the underlying mechanisms could be leveraged to promote heart regeneration and repair in adults. Using spatial transcriptomic profiling, we visualized regenerative cardiomyocytes reconstituting damaged myocardium after ischemia, and found that they are marked by expression of Nrf1, an ER-bound stress responsive transcription factor. Single-nucleus RNA sequencing revealed that genetic deletion of Nrf1 prevented neonatal cardiomyocytes from activating a transcriptional program required for heart regeneration. Conversely, overexpression of Nrf1 protected the adult mouse heart from ischemia/reperfusion injury. Nrf1 also protected human induced pluripotent stem cell-derived cardiomyocytes from cardiotoxicity induced by the chemotherapeutic drug doxorubicin. The cardioprotective function of Nrf1 is mediated by a dual stress response mechanism involving activation of the proteasome and maintenance of redox balance. Taken together, our study uncovers a unique adaptive mechanism activated in response to injury that maintains the tissue homeostatic balance required for heart regeneration. Reactivating these mechanisms in the adult heart represents a potential therapeutic approach for cardiac repair.
Abstract 104: Deletion Of Myd88 In T-Cells Worsens Pathology In A Mouse Model Of Non-Ischemic Heart Failure Through Enhanced T-Cell Survival And Effector Function:
Authors: Bayer Abraham L , Ngwenyama Njabulo , Smolgovsky Sasha , Hernández Martínez Ana , Kaur Kuljeet , Alcaide Pilar
Published: November 2021

Background:
Heart failure (HF) is a leading cause of death worldwide, associated with cardiac and systemic inflammation. However, no anti-inflammatory therapies have shown success thus far. Damage associated molecular patterns (DAMPs) released in the heart can activate myeloid cells to promote antigen presentation to T-cells, which infiltrate the heart and participate in adverse cardiac remodeling. DAMP signaling converges onto the adaptor protein “Myeloid differentiation primary response 88” (MyD88). DAMP receptors and MyD88 are also expressed in T-cells, but their role in T-cell activation is unclear, and is unknown in the context of HF. We
hypothesized
that T-cell recognition of DAMPs through MyD88 causes “bystander activation” of T-cells and contributes to cardiac pathology in HF.

Methods and Results:
We reconstituted
Tcra
-/-
mice, normally protected from HF, with WT or

Myd88
-/-

Type 1 helper T-cells (Th1) in the onset of transaortic constriction (TAC), a well-established model of HF. Surprisingly, we found that mice given

Myd88
-/-

Th1 cells exhibited significantly higher levels of cardiac T-cell infiltration, more severe fibrosis, and lower fractional shortening than mice given WT Th1 cells. We found that WT and

Myd88
-/-

Th1 cells had similar levels of IFNγ and Tbx21 by intracellular staining and RT-qPCR, indicating that MyD88 does not alter Th1 differentiation. However,

Myd88
-/-

Th1 cells secreted higher levels of IL-2 and TNFα, suggesting enhanced proliferative and pro-inflammatory effector function. We performed viability studies using live cell microscopy and measuring propidium iodide incorporation in real time, as well as by flow cytometry, and found that

Myd88
-/-

Th1 cells have a survival advantage compared to WT Th1 cells. Moreover, we found that

Myd88
-/-

Th1 cells exhibited higher levels of adhesion to ICAM-1 and VCAM-1, protein ligands involved in T-cell recruitment, compared to WT Th1 cells when perfused under conditions of shear flow.

Conclusion:
Together, these data demonstrate that T-cell MyD88 limits T-cell mediated pathology in HF by modulating Th1 effector function, survival, and adhesion ability. We identify novel role for T-cell MyD88 in cardiac inflammation that may be modulated in HF.
Abstract 110: Human-Induced Pluripotent Stem Cell Derived Exosomal Protein Induce Cardiac Regeneration
Authors: Magadum Ajit , Mallaredy Vandana , Grace Grace , Wang Chunlin , Roy Rajika , Joladarashi Darukeshwara , Gurrala Charan Thej , Cheng Zhongjian , Cimini Maria , Truongcao May , Lucchese Anna Maria , Gonzalez Carolina , Benedict Cindy , Kishore Raj
Published: November 2021

Introduction:
Cardiovascular diseases are the leading causes of death worldwide. After myocardial infarction (MI), there is a permanent loss of cardiomyocytes (CMs), and as the mammalian heart has limited regenerative capacity, it leads to Heart Failure. Recent studies from zebrafish and 1-day old mice showed that they could regenerate their heart through inducing existing CM proliferation. Attempts have been made to transiently reconstitute embryonic signaling in adult hearts, including overexpression of cell cycle activating genes with limited success. iPSC-derived extracellular vesicles (EV)/exosomes have been shown to improve cardiac function and some degree of CM renewal. However, the iPSC-EVs-mediated cardiac regeneration mechanism remains unclear and largely pertains to microRNAs and other RNAs, with a little elucidation of the role of iPSC-exosome proteins.

Hypothesis:
The myocardial delivery of iPSC-EV-specific protein improves cardiac function and remodeling post-MI by activating pro-proliferative and anti-oxidative stress molecular pathways.

Methods and Results:
Our preliminary studies showed that hiPSC-EVs induced the CM cell cycle in mice post-MI, and by employing a proteomic approach, we found a novel protein exclusively expressed in iPSC-EVs. The overexpression of hiPSC-EV enriched protein in the form of modRNA (modified mRNA) induced a robust CM cell cycle in rat neonatal CMs and in adult hearts post-MI. This increase in the CMs cell cycle by the modRNA was associated with reduced scar size, improved cardiac function (%EF 49.76 ± 5.8 vs. 27.47 ± 6.9 (control, Luc modRNA), respectively), and mice survival 28 days post-MI. Furthermore, using the siRNA and modRNA (inhibition and over-expression) approach, we found that the protein-induced Yap1-β-catenin molecular pathway stimulates CM proliferation. Furthermore, the overexpression protein post-MI inhibited the CM apoptosis (TUNEL
+
CMs, 1.3% ± 0.1 vs. 2.1% ± 0.11 (control)) by reducing oxidative stress and DNA damage response.

Conclusion:
The myocardial injection of iPSC-EV specific protein through a highly therapeutic modRNA tool improve cardiac function by inducing CMs proliferation, inhibiting oxidative stress, and reactivating cardiac regeneration post-injury.
Abstract 114: Safety And Efficacy Of CDR132L, A Novel Antisense Therapeutic Which Targets MicroRNA-132 In Heart Failure Patients
Authors: Taubel Jorg , Hauke Wilfried , Rump Steffen , Viereck Janika , Batkai Sandor , Jenny Poetzsch , Laura Rode , Weigt Henning , Genschel Celina , Lorch Ulrike , Theek Carmen , Levin Arthur A , Bauersachs Johann B , Solomon Scott D , Thum Thomas
Published: November 2021

Cardiac microRNA-132-3p (miR-132) levels are elevated in people suffering with heart failure (HF) and mechanistically drive remodelling processes in the heart. Here we present CDR132L, a specific antisense oligonucleotide. It is a first-in-class miR-132 inhibitor that attenuates and even reverses HF in preclinical models. The aim of this clinical Phase 1b study was to assess safety, pharmacokinetics, target engagement, and pharmacodynamics of CDR132L in patients on therapy for chronic ischaemic HF in a randomized, placebo-controlled, double-blind, dose-escalation study. Inclusion criteria for participant patients was left ventricular ejection fraction between 30 and 50% or levels of amino terminal fragment of pro-brain natriuretic peptide (NT-proBNP) higher than 125 ng/L at screening. Twenty-eight patients were randomized to receive CDR132L (0.32, 1, 3, and 10 mg/kg body weight) or placebo (0.9% saline) in two intravenous infusions, 4 weeks apart. Randomization separated participants into four cohorts of seven (five verum and two placebo). Results revealed that CDR132L was safe and well tolerated, and there was no detectable dose-limiting toxicity. A pharmacokinetic/pharmacodynamic dose modelling approach suggested an effective dose level of ≥1 mg/kg CDR132L. CDR132L treatment resulted in a dose-dependent, sustained miR-132 reduction in plasma. Patients who were administered ≥1 mg/kg of CDR132L displayed 23.3% NT-proBNP reduction, whereas patients receiving placebo experienced a 0.9% increase. In addition, CDR132L treatment was observed to significantly reduce duration of the QRS interval. This is the first clinical trial in which an antisense oligonucleotide was administered as a therapeutic agent to HF patients. Linear plasma pharmacokinetics were confirmed, and there were no signs of accumulation. The application of this drug suggests cardiac functional improvements and cardiac remodelling. The efficacy of this drug is encouraging, and further clinical work with larger patient populations should follow. Although the study used small patient numbers, results suggest some clinical benefit in chronic HF patients on the top of standard of care. This included an improvement in HF severity and narrowing of the QRS complex. The pharmacodynamic findings presented here are encouraging as they confirm efficacy results seen in animal studies.
Abstract 116: T Cells Contribute To Diastolic Dysfunction And Cardiac Hypertrophy In A Preclinical Model Of Heart Failure With Preserved Ejection Fraction
Authors: Smolgovsky Sasha , Carrillo-Salinas Francisco , Anastasiou Marina , Kaur Kuljeet , Bayer Abraham , Aronovitz Mark , Blanton Robert M , Alcaide Pilar
Published: November 2021

Introduction:
Heart Failure with Preserved Ejection Fraction (HFpEF) is a prevalent cardiovascular syndrome with no curative therapies, characterized by diastolic dysfunction, preserved systolic function, and decreased expression of unfolded protein response (UPR) genes in the heart. Obesity and hypertension are risk factors for HFpEF and are intimately associated with systemic inflammation. However, the inflammatory mechanisms driving HFpEF remain largely unexplored.

Hypothesis:
We hypothesized that nitrosative stress induced by obesity and hypertension programs T cells to infiltrate the heart and drive cardiac pathology in HFpEF.

Methods:
Using a recently established model of HFpEF, we modeled obesity and hypertension in male C57/BL6 (wild-type, WT), T cell receptor alpha-deficient (
Tcra-/-),
and Nur77-GFP reporter mice for T cell receptor engagement, using a high-fat diet (HFD) and L-NAME for 5 weeks, or standard chow (STD) as controls. Invasive hemodynamic analyses were used to assess cardiac function, and the heart and lymphoid organs were harvested to characterize immune cell populations by flow cytometry, histology, and gene expression of cardiac remodeling.

Results:
In WT mice, HFD/L-NAME induced significant cardiac infiltration of T cells alongside the hallmarks of HFpEF. HFD/L-NAME significantly expanded CD62
lo
CD44
hi
effector T cells in the mediastinal lymph nodes and spleen. Nur77-GFP mice revealed no antigen engagement by T cells in the heart, however, T cells sorted out of the lymphoid organs of HFpEF mice had significantly decreased gene expression of the UPR gene spliced X box-binding protein 1 (XBP1s) compared to controls, suggesting a T cell intrinsic dysregulation of the UPR, and T cell phenotypic changes independent of TCR engagement in the heart. Strikingly,
Tcra-/-
mice did not develop diastolic dysfunction or cardiomyocyte hypertrophy, demonstrating a novel role for T cells in this experimental model of HFpEF.

Conclusions:
We conclude diastolic dysfunction and cardiomyocyte hypertrophy in HFpEF is T cell dependent. Ongoing studies are determining whether the observed intrinsic T cell changes in XBP1s prime T cells for cardiac infiltration and effector function that results in diastolic dysfunction and HFpEF.
Abstract 119: Donor Macrophages Modulate Cellular Rejection After Heart Transplantation
Authors: Kopecky Benjamin , Amrute Junedh , Dun Hao , Frye C. Corbin , KREISEL DANIEL , Lavine Kory J
Published: November 2021

Heart transplant rejection is common and is associated with significant morbidity and mortality. Current immunosuppressive therapies primarily target recipient T-cells and have a multitude of untoward effects including infections, malignancies, and end-organ damage. Recent studies implicate the roles of antigen presenting cells towards pathogenesis of allograft rejection through recruitment and activation of T-cells. The importance of antigen presenting cell origin, identity, and functional importance remains unknown. Using complimentary imaging and single cell RNA sequencing techniques, we show that donor and recipient monocytes and macrophages co-exist after heart transplantation. These myeloid populations have diverse transcriptional signatures that evolve throughout ongoing rejection. Donor macrophages can be defined ontologically and based on their expression of C-C chemokine receptor 2 (CCR2) and expression of MHC-II. Donor CCR2+ and CCR2- populations can be further defined based on their gene expression profiles, highlighting the marked heterogeneity in the donor macrophage population. Selective depletion of CCR2+ macrophages result in prolonged allograft survival. We use longitudinal single cell RNA sequencing to show that donor CCR2+ and CCR2- macrophages have distinct activation mechanisms such that donor CCR2+ macrophages signal through MyD88/NF-kB. Conditional depletion of MyD88 in donor macrophages recapitulates the donor CCR2+ depletion phenotype. Further interrogation of MyD88 conditionally depleted allografts shows reduced T-cell alloreactivity, holding promise for a potential therapeutic target pathway. Together, we show the molecular identity, diversity, and evolution of donor and recipient monocytes and macrophages as well as the functional relevance and activation pathways of donor macrophages in cardiac allografts.
Abstract 122: Pharmacological Inhibition Of Macrophage Infiltration Prevents Myxomatous Valve Degeneration In Marfan Syndrome
Authors: Xu Na , Yutzey Katherine E
Published: November 2021

Introduction:
Myxomatous valve degeneration (MVD) is the most common cause of mitral regurgitation, characterized by valve leaflet thickening and progressive valve degeneration, leading to impaired cardiac function and heart failure. Currently, there is no medical therapy for the treatment of MVD. MVD in a mouse model of Marfan syndrome (MFS) is characterized leaflet thickening and increased macrophage infiltration, which are reduced with loss of C-C chemokine receptor type 2 (CCR2). However, the specific contributions of macrophages to pathological extracellular matrix (ECM) remodeling and underlying mechanisms are unknown.

Hypothesis:
Inhibition of macrophage infiltration by a CCR2 inhibitor blocks ECM abnormalities and MVD progression in mitral valves of MFS mice by suppressing the response to cytokine/chemokines.

Methods:
Mice with the mutation of Fibrillin 1 (Fbn1
C1039G/+
) recapitulate histopathological features of MFS. Here, we tested the efficacy of a selective CCR2 antagonist RS504393 in the valves of MFS mice in the initiation (1-month-old) and the progression (2-month-old) of MVD, respectively. MFS mice were intraperitoneally injected with RS504393 at 2 mg/kg/d for 30 days. Histological evaluation and immunofluorescence for macrophages and ECM were performed. RNAseq was performed in mitral valves from 2-month old Fbn1
C1039G/+
mice with CCR2 knockout (CCR2
RFP/RFP
).

Results:
MFS valves revealed ECM abnormalities characterized by collagen fragmentation and proteoglycan accumulation. RS504393 treatment reduced infiltrating macrophages (MHCII+, CCR2+) in myxomatous valves. Remarkably, RS504393 was protective against both the initiation and the progression of MVD, detected by decreased mitral valve thickness and prevention of pathological ECM remodeling in MFS mice. RNAseq data confirmed increased leukocyte activation involved in immune response and abnormal extracellular matrix remodeling in MFS valves. CCR2 deficiency blocked macrophage infiltration and inhibited the response to cytokines in Fbn1
C1039G/+
valves.

Conclusions:
Our results show that macrophage infiltration is critical for progressive MVD. Moreover, CCR2 inhibition ameliorates MVD progression by preventing immune response. Thus, the CCR2 inhibitor RS504393 is a potential pharmacological candidate to treat MVD in MFS.
Abstract 123: Study Of The Role Of Myosin-binding Protein C In The Structure-function Of Intact Cardiac Muscles Through The Use Of A Sarcomere Activation Biosensor
Authors: Martin Ashley A , Thompson Brian R , Metzger Joseph M
Published: November 2021

Cardiomyopathies affect as many as 1 in 500 adults, with hypertrophic cardiomyopathy (HCM) being the most commonly inherited heart disease. Although there are various genetic mutations which cause HCM, 40-50% of mutations identified in patients with this disease are found in the cardiac myosin-binding protein C (MyBP-C) gene. Thus, understanding this protein's role in sarcomere activation is critical for the development of effective therapeutic strategies. Previous work has identified MyBP-C as a key modulator of the sarcomere through inter-myofilament signaling. Although these studies have provided valuable information on the function of MyBP-C protein, the bulk of this work has been done with either
in vitro
protein recombination or permeabilized muscle. Unfortunately, these systems lack significant physiological features of muscle, such as load and intact excitation-contraction coupling mechanisms. Therefore, a large gap exists in the ability to monitor muscle sarcomere activation in an intact system in real time. In order to further advance the study of sarcomere activation in HCM disease models, specifically those targeting MyBP-C, a cardiac specific myofilament-targeted FRET based biosensor has been designed and validated. This new sarcomere activation biosensor reports real-time myofilament dynamics during physiological twitch contractions in live cardiac muscle. This allows for the
in vivo
biophysical tracking of FRET-detected conformation changes in TnC, which reflect the ensemble of regulatory events which lead to sarcomere activation. Currently, the biosensor is being utilized to investigate changes in sarcomere activation in MyBP-C knockout mice. We find that intact cardiac muscle under load shows slowed isometric twitch relaxation kinetics in MyBP-C knockout mice versus controls. In concert with thin and thick filament modifying small molecules, this biosensor system will be used to investigate sarcomere-based mechanisms of intact cardiac muscle performance in health and disease.
Abstract 401: Functional Impact Of Rbfox1c In Cardiac Pathological Remodeling Through Targeted Mrna Stability Regulation
Authors: Gao Chen , Xiong Zhaojun , Liu Jianfang , Cao Nancy , Yokota Tomohiro , Huang Jijun , Wang Menglong , Ren Shuxun , Rau Christoph , Li Katelyn , Xiao Xinshu , Vondriska Thomas M , Wang Yibin , Xing Yi
Published: November 2021

Post-transcriptional regulation plays a key role in transcriptome reprogramming during cardiac pathogenesis. In previous studies, we have identified that cardiac enriched RNA-binding protein, RBFox1 plays key role in cardiac hypertrophy through mRNA alternative splicing regulation in nuclei. However, RBFox1 gene also generates a cytosolic isoform (RBFox1c), suggesting additional functions of post-transcriptional regulation in heart. In adult heart, RBFox1c mRNA constituted ~ 40% of total RBFox1 level but was significantly repressed in pressure-overloaded failing mouse heart. Using CRISPR-Cas9 technology, we have established an isoform specific RBFox1c-cKO mouse. At baseline inactivation of RBFox1c led to decreased cardiac function along with induction of cardiac fibrosis. RBFox1c-cKO mice also showed macrophages infiltration into myocardium post 7days MI. In contrast, restoration of RBFox1c expression in adult intact hearts significantly reduced cardiac fibrosis post stress. RNA-seq analyses in RBFox1c expressing cardiomyocytes showed that RBFox1c specifically suppressed the expression of pro-inflammatory genes. Secondly, CLIP-Seq analysis and targeted RNA-IP showed that RBFox1c could directly interact with inflammatory pathway mRNAs. These results suggested the inflammatory mRNAs are direct downstream targets regulated by RBFox1c. Using both in vitro cultured cardiomyocytes and intact mouse hearts, we demonstrated that expression of RBFox1c reduces pro-inflammatory mRNA expression at baseline and upon hypertrophy stimulation. Lastly, we characterized the interactome of RBFox1c through proteomic analysis and found RBFox1c specifically interacted with a component of the RNA NMD machinery-Upf1. RBFox1c interaction with Upf1 in cardiomyocytes was diminished upon hypertrophic stress. Furthermore, by inactivation of Upf1 via siRNA, we demonstrated that RBFox1c mediated repression of proinflammatory genes was Upf1 dependent.RBFox1 regulates cardiac transcriptome reprogramming in two post-transcriptional processes via distinct isoforms. While the RBFox1n regulates RNA splicing, the RBFox1c functions through targeted mRNA repression of proinflammatory genes by recruitment of Upf1 mediated RNA degradation.
Abstract MP203: Decoding The Cardiac Regulatory Long Noncoding RNAs In Male And Female Mice Using Integrative Multiomics Analysis
Authors: Touma Marlin , Cao Yang , Pan Calvin , Lusis Aldons J
Published: November 2021

Background:
Long non-coding RNAs (lncRNAs) are increasingly recognized as important regulators of mammalian heart development and potential contributors to human cardiovascular diseases by regulating RNA transcription, translation and processing. However, the genetic regulation lncRNAs and their interactions with mRNAs are poorly understood.

Methods:
Deep RNA sequencing was performed following Illumina HiSeqTM 4000 sequencer protocol. SNP Array was used for mice genotyping. For differential gene expression analysis (DGE), DE Seq was used. Weighted gene co-expression network analysis (WGCNA) was performed using R. FaST-LMM was used for expression quantitative trait locus (eQTL) analysis.

Results:
To elucidate lncRNA-mediated transcriptional regulation in heart, we performed integrated multiomics analysis of RNA-seq derived transcriptome and SNP genotype data from the hybrid mouse diversity panel (HMDP) of adult male and female mice hearts across 100 strains. We observed significant variability in cardiac lncRNAs expression across the 100 strains. Using a 5% FDR
P
value threshold, genome-wide eQTL detected total of408513 and 404661 locally associated (within 1 Mb of genomic distance up- or down-stream of a given gene)SNP-lncRNA pairs in female vs male heart, respectively, which corresponded to 2687 unique lncRNAs in female and 2684 unique lncRNAs in males after excluding those with distal eQTLs (at
P
<4.1e-6). Genome-wide correlation analysis revealed 2794 local and 1233 distal uniquely co-regulated lncRNA-mRNA pairs (
P<
1e-8).Global WGNCA and module-trait correlation analysis identified 14 female and 5 male lncRNA-mRNA networks(Bicor>0.3) that exhibited significant correlation with heart weight, body fat, or insulin resistance. By applying eQTL analysis to the module eigengenes, we identified 11 unique module eigengenes with eQTLs (FDR<0.05).

Conclusions:
This study represents the first effort to investigate lncRNA-mediated regulation of gene expression in male and female mouse heart across 100 different strains. The results reveal novel associations of lncRNAs with complex clinical traits and provide resources to facilitate mechanistic exploration of the lncRNAs function in cardiovascular disease models.
Abstract MP204: Pyruvate Dehydrogenase Kinase Isozyme Specific Regulation Of Protein Acetylation In Cardiac Tissue
Authors: Ha Chae-Myeong , Wende Adam R
Published: November 2021

Heart disease is the number one cause of death in developed countries. Metabolic diseases influence the severity of heart disease linked to risk factors which are thought to alter epigenetic mechanisms. Pyruvate dehydrogenase (PDH) kinases (PDK), which phosphorylate and reduce the activity of PDH the nexus of glucose oxidation and fatty acid oxidation are sensitive to metabolic status. Four isozymes of PDK (PDK1-4) exist with PDK2 and PDK4 as the major regulators in cardiac tissue. Owing to the role of PDH in regulating pyruvate to acetyl-CoA, we hypothesized that PDK inhibition may regulate protein acetylation through increasing acetyl-CoA because of PDH activation leading to post-translational modifications both directly to proteins in metabolic pathways as well as to histones associated with the genes encoding them. To test this, we utilized PDK2 germline knockout mice (P2KO), PDK4 germline knockout mice (P4KO), and PDK2 and PDK4 double knockout (DKO) mice for molecular analysis. Our results identify a novel increase in whole-cell protein acetylation in P2KO left ventricle tissue (LV). However, protein acetylation in P4KO LV was not changed compared to WT mice. The most robust protein acetylation was observed in the DKO LV. Furthermore, when we explored sub-cellular distribution of protein acetylation, the greatest increases were found on cytoplasmic proteins, with moderate changes in mitochondrial proteins. We also found PDK2 ablation induces histone H3 acetylation, which may also lead to changes in gene expression. Moreover, this protein acetylation in P2KO and DKO was not seen in other tissues examined (e.g., liver, skeletal muscle). The hyperacetylation is robust in male LV compared to female LV. In conclusion, our study supports a novel protein acetylation mechanism that is both tissue and PDK isozyme specific highlighting the role of PDK2, which is relatively understudied compared to PDK4 in heart disease. Further study will evaluate if the hyperacetylation has a beneficial effect in various heart disease settings as well as identify the impact on changes in gene expression. This study supports PDK isozyme-specific inhibition strategies will be required to develop therapeutic targets of cardiovascular disease with metabolic inflexibility.
Abstract MP209: Troponin I Phosphorylation Is Essential For Cardiac Reserve
Authors: Sturgill Sarah L , Salyer Lorien G , Shettigar Vikram , Brundage Elizabeth , Biesiadecki Brandon J , Ziolo Mark T
Published: November 2021

In response to increase in metabolic demand (e.g., exercise), the heart must increase its pumping performance to meet this demand. To achieve this increase, the heart relies on its cardiac reserve, which is the ability to increase in its contractile and diastolic function. The mechanism responsible for cardiac reserve is poorly understood. The myofilament is the mechanism responsible for contraction and relaxation. Troponin I (the inhibitory subunit of troponin, TnI) is a key regulatory protein. Studies have shown TnI serine 23/24 (S23/S24) phosphorylation, the most abundant and important TnI phosphorylation, is a key mechanism for accelerating relaxation by decreasing Ca
2+
senstivity. The role of TnI in cardiac reserve is unknown. For this study, we thoroughly characterized the systolic and diastolic reserve in TnI S23/S24 phosphorylation-null transgenic mice (S23/S24 mutated to alanine, AA mice). Even with increased Ca
2+
sensitivity, the AA mice exhibited normal function at resting heart rate and no difference in cardiac structure compared to wildtype. To increase in vivo heart performance, the most important system is the Bowditch effect (i.e., an increase in contractile function with increasing heart rate). To examine the role TnI S23/S24 phosphorylation in systolic and diastolic reserve, we assessed hemodynamics via left ventricular catheterization on the Bowditch effect by increasing heart rate from 240 to 420 beats per minute. Our data exhibited a clear loss of diastolic and systolic reserve in the AA mice. Since we observed a clear inability to increase systolic and diastolic function in AA mice, we performed speckle tracking echocardiography to more quantitatively investigate AA mice function. We observed that AA mice demonstrated normal systolic function (radial strain rate) and impaired directional diastolic function (reverse radial strain rate) at resting heart rate. We conclude that TnI S23/S24 phosphorylation is essential for cardiac reserve by enhancing systolic and diastolic function. A blunted cardiac reserve leads to heart disease making TnI S23/S24 phosphorylation a potential therapeutic strategy.
Abstract MP212: Integrated Genomics Analysis Identifies Recessive Ciliopathy Mutations In Primary Endocardial Fibroelastosis: A Rare Neonatal Cardiomyopathy
Authors: Mehta Zubin , Zhao Yan , Xuedong Kang , Alejos Juan C , Halnon Nancy , Nelson Stanley F , Touma Marlin
Published: November 2021

Background:
Among neonatal cardiomyopathies, primary endocardial fibroelastosis (pEFE) remains a mysterious disease of the endomyocardium, affecting 1/5000 live births and accounting for 25% of the entire pediatric dilated cardiomyopathy (DCM) population with a devastating course and grave prognosis.

Objective:
We aimed to investigate potential genetic contributions to pEFE etiology.

Methods:
We performed integrative genomic analysis in 8 infants with confirmed pathological diagnosis of pEFE using whole exome sequencing (WES), RNA-seq and functional genomics studies. Patient-derived fibroblasts, neonatal rat ventricular myocytes and neonatal rat cardiac fibroblasts were used for cellular assays. Real-time cell migration and proliferation analyses were performed using xCELLigence technology.

Results:
Whole exome sequencing detected novel and deleterious
de novo
single nucleotide variants, or inherited homozygous rare variants in 11 cilia-related genes in seven out of the eight affected probands. In particular, a novel homozygous variant [c.1938delA] in
ALMS1
was identified in a female proband, pEFE4. This variant resulted in a frameshift introducing a premature stop codon and complete absence of the ALMS1 protein in the proband fibroblasts and explanted heart. Loss of function mutations of
ALMS1
have been implicated in Alstrom syndrome [OMIM 203800], a rare recessive ciliopathy. RNA-sequencing of the proband’s dermal fibroblasts revealed significantly dysregulated cellular signaling and function, including the induction of epithelial mesenchymal transition (EMT), potentially mediated by TGFβ signaling activation. The proband fibroblasts exhibited enhanced migration activity. Herein, we present the unique pathological features of pEFE compared to DCM and utilize integrated WES with RNA-seq analysis to elucidate the molecular mechanisms by which the novel causal
ALMS1
variant contributes to the unique pathology of pEFE in a female infant with Alstrom syndrome.

Conclusions:
Our report provides insights into pEFE etiology and suggests, for the first time to our knowledge, ciliopathy as a potential underlying mechanism for this poorly understood and incurable form of neonatal cardiomyopathy.