bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P1161: Interferon Hyperactivity Impairs Cardiogenesis In Down Syndrome Via Downregulation Of Canonical Wnt Signaling
Authors: Song Kunhua , Chi Congwu
Published: October 2023

Congenital heart defects (CHDs) are very frequent in children with Down syndrome (DS), the genetic condition caused by trisomy of chromosome 21 (T21). Despite significant efforts, the mechanisms by which T21 causes susceptibility to CHDs remain largely unknown. Here, using a combination of a human induced pluripotent stem cell (iPSC)-based model and the Dp(16)1Yey/+ (Dp16) mouse model of DS, we identified downregulation of canonical Wnt signaling downstream of increased dosage of interferon (IFN) receptors (IFNRs) genes on chromosome 21 as a causative factor of CHDs in DS. We differentiated human iPSCs derived from individuals with DS and CHDs (DS/CHD iPSCs), and euploid controls into cardiac cells. We observed that T21 upregulates IFN signaling, downregulates the canonical WNT pathway, and impairs cardiac differentiation. Furthermore, genetic and pharmacological normalization of IFN signaling restored canonical WNT signaling and rescued defects in cardiac differentiation of DS/CHD iPSCs. To define the role of IFN signaling in CHDs
in vivo
, we genetically and pharmacologically normalized the activity of IFN signaling in the Dp16 mouse model of DS. Normalization of the activity of IFN signaling prevented heart malformations in Dp16 embryos. In addition, treatment with an inhibitor of Janus kinases, which act downstream of IFNRs, normalized canonical Wnt signaling and ameliorated CHDs in Dp16 embryos. Our findings provide new insights into mechanisms underlying CHDs in DS, ultimately aiding the development of novel therapeutic strategies.
Abstract P1164: Elevated Erk1/2 Signaling Contributes To The Development Of Diabetic Cardiomyopathy
Authors: Liu Ruijie
Published: October 2023

Background:
About 19%-26% of the obese patients develop diabetic cardiomyopathy (DCM) characterized by ventricular hypertrophy, fibrosis, and contractile dysfunction. Although many studies have investigated the potential causes of DCM, the pathophysiology of this disease has yet been to be fully characterized.

Objective:
To determine whether extracellular signal-regulated kinase 1/2 (ERK1/2) are involved in the development of DCM.

Materials and Methods:
Both streptozotocin (STZ)-induced type I and db/db type II diabetic mice were utilized to compare the ERK1/2 phosphorylation level in mouse hearts. To determine the gain-of-function of ERK1/2 signaling in DCM, both control and dual specific phosphatase 6 and 8 knockout mice (DKO) mice were administered streptozotocin (STZ) to induce elevated serum glucose. After 12 weeks of treatment, mouse body weight, heart weight/histology, and disease markers were compared. As a loss-of-function study, U0126 was utilized to inhibit ERK1/2 activity in both streptozotocin (STZ)-induced type I and db/db type II diabetic mice to determine whether DCM markers are influenced.

Results:
In both type I and II diabetic mouse hearts, ERK1/2 phosphorylation level was significantly elevated. STZ treatment for 2 weeks induced similar increases in serum glucose in both WT and DKO mice. After 12 weeks of STZ treatment, DKO mice developed greater cardiac atrophy and increased levels of ANF, BNP, beta myosin heavy chain gene expression. In comparison, U0126-mediated pharmacological inhibition of ERK1/2 for 12 weeks significantly improved the cardiac function in both type I and II diabetic mouse models.

Conclusion:
Elevated ERK1/2 signaling contributes to the process of DCM, suggesting ERK1/2 could be therapeutic targets for the treatment of this disease.
Abstract P1173: Deciphering CITED4-dependent Signaling In The Heart Through Nascent Proteomics And Secretomics
Authors: Knobloch Vivien , Schlegel Philipp , Lust Leandra , Aljakouch Karim , Hess Chiara , Kuhn Tim C , Bezzerides Vassilios J , Mittag Sonja , Roshan Mani , Frey Norbert , Rosenzweig Anthony , Lerchenmüller Carolin
Published: October 2023

Objective:
Neuregulin-1 (Nrg1) promotes cardiomyocyte hypertrophy, survival, and cell cycle activity through ErbB signaling. It is investigated in clinical trials as a cardioprotective agent; however, its therapeutic use might be limited due to its pro-neoplastic potential for non-cardiomyocytes that makes targeted approaches necessary. High-throughput screening of hypertrophic agonists found that Nrg1 induces CITED4 (C4) expression. C4 is also upregulated in both physiological and pathological cardiac growth and necessary to prevent adverse remodeling
in vivo.
However, how C4 regulates Nrg1-signaling in the heart is yet unknown.

Methods:
We combined pulsed-SILAC labeling, click-chemistry and mass spectrometry that allows to capture immediate changes in the proteome and secretome after Nrg1 (12h and 24h) stimulation in siRNA-mediated C4-knockdown (C4KD) and control (ctr) NRVM and complemented this with established cell- and molecular biology techniques to validate and investigate cellular function and molecular signaling.

Results:
We confirmed dose-dependent C4 mRNA upregulation in response to Nrg1 stimulation in NRVM. Nrg1 downstream signaling through AKT was hindered in C4KD. Computational analysis comparing the nascent proteome after Nrg1 stimulation in C4KD and ctr NRVM revealed that C4 significantly regulates proteins responsible for translation, RNA processing and energy derivation. Consistent with previously observed development of cardiac fibrosis in C4 knockout mice
in vivo
, we found significant upregulation of TGFb2 in C4KD NRVM. We next confirmed the upregulation of TGFb2 and its downstream effectors in C4KD and ctr, while adenoviral overexpression of C4 led to reduced TGFb2 expression. Additionally, we found TGFb2 secretion increased from C4KD NRVM with Nrg1 stimulation in the nascent secretome. Conditioned media from C4KD NRVM led to an increased pro-fibrotic response in cardiac fibroblasts. In conclusion, nascent proteomics and secretomics help to elucidate C4-dependent Nrg1-signaling, which may be an important contribution toward our goal to identify targetable cardioprotective pathways in the heart.
Abstract P1180: Multi-scale Imaging And Analysis To Elucidate Cardiac Structure And Contractile Function
Authors: Ding Yichen , Almasian Milad , Zhang Xinyuan , Kadam Vinay , Yuan Jie
Published: October 2023

Heart failure remains the leading cause of mortality worldwide, largely due to the lack of regenerative capacity of mature cardiomyocytes. The advanced understanding of cardiac architecture and function is critical to uncover the underlying mechanism of cardiac morphogenesis and the remodeling process in response to myocardial infarction. Recent progress has demonstrated that zebrafish and neonatal mice have the capacity to restore cardiac function following the injury, laying the foundation to investigate the cues to structural and functional abnormalities. For this reason, we have developed a light-sheet fluorescence microscope along with the tailored computational methods to investigate the 3-dimensional (3D) architecture of the intact mouse heart, and 4D (3D spatial + 1D temporal) cardiac contractility in zebrafish larvae. This method provides us ~ 2 um isotropic spatial resolution across the atria and ventricles with minimal photo-damage and maximal penetration depth. In combination with established tissue clearing methods, we are able to investigate myocardial trabeculation and compaction in mouse hearts within the 2-minute laser scanning. To unravel the contractile dysfunction following the anticancer therapy, we have coupled our imaging system with retrospective synchronization algorithm for the 4D reconstruction of myocardial deformation from end-systole to end-diastole in zebrafish hearts. To further interpret the deluge of 3D and 4D images, we have also developed a machine learning-based platform for interactive image analysis. Collectively, our multi-scale imaging strategy provides an entry point to assess micro-architecture and cardiac contractile function in the entire heart without physical slicing, holding the great potential to uncover the process of cardiac morphogenesis and regeneration.
Abstract P2001: Deficiency Of Claudin-5 Leads To Myocardial Atrophy And Dilation In Mouse Heart With Severe Mitochondrial Dysfunction And Electrical Dissociation
Authors: Luo Tao , Chen Baihe , Liu Haiqiong , Wang Xianbao , Abdel-Latif Ahmed , Kim Jin Kuyng , Kitakaze Masafumi
Published: October 2023

Background:
Cardiac overexpression of Claudin-5 (Cldn5) exerts protection against myocardial ischemia and reperfusion injury. However, whether cardiac deficiency of Cldn5 affects cardiac function is still unclear. Here we investigated the role of Cldn5 in mouse heart by means of cardiac Cldn5 knockdown to better understand the intracellular function of Cldn5 in cardio-protection.

Methods and Results:
Cldn5 shRNA adeno-associated virus (AAV) was used to knockdown Cldn5 in the left ventricle of male C57BL/6J mice by intramyocardial injection. Four weeks post AAV injection, echocardiography and electrocardiography were performed to measure cardiac function. We found 25% of mice died in Cldn5 shRNA AAV injection group. The residual mice were terminated followed by left ventricles isolation for proteomics analysis, cytokine analysis, WGA staining, Masson-trichrome staining and transmission electron microscope (TEM) observation. The signal pathway markers of apoptosis, autophagy and lipid peroxidation were also detected by western blot. The protein level of Cldn5 in left ventricles was decreased by 75% in shRNA AAV injection group accompanied by changes of bcl-2, bax, LC3II, p62, Cox2 and GPX4. The fraction shortening of the heart was decreased by 23.5% with ventricular dilation and ST elevation. We also observed the increase of IL-1beta, TNF alpha, BNP, CT and cTnI in myocardium. WGA staining showed the area of cross section of single cardiomyocyte was decreased and Masson staining showed the collagen deposition was increased in myocardium. The TEM indicated Cldn5 knockdown induced mitochondrial fission and depletion. Finally, the proteomics analysis demonstrated that Cldn5 knockdown severely disturbed ATP metabolic regulation, mitochondrial permeability transition pore opening, calcium ion transport, cardiac contraction, endoplasmic reticulum stress response and antioxidant activity in the AAV injected myocardium.

Conclusion:
We demonstrated for the first time the deficiency of Cldn5 in myocardium caused myocardial atrophy and dilation with deteriorative heart function. The mechanism of Cldn5 deficiency-induced cardiac dysfunction is associated with severe mitochondrial dysfunction and excitation-contraction coupling.
Abstract P2007: Novel Regulatory Axis In Heart-adrenal Cross Talk Mediated By Mtor-eprs
Authors: Liu Tian , Zhang Ye , Yu Junyi , Ren Shuxun , Zhu Cansheng , Rau Christoph D , Wang Yibin , Gao Chen
Published: October 2023

Adrenal catecholamines, including norepinephrine and epinephrine play an important role in cardiovascular physiology. Serum catecholamine level is tightly regulated in response to metabolic and cardiac stress. The biosynthesis of catecholamine is driven by a series of enzymatic reaction carried out by the key rate-limiting enzymes, including tyrosine hydroxylase (TH), DOPA decarboxylase (DDC) and phenylethanolamine-N-methyltransferase (PNMT). Although the transcription regulation of these enzymes has been described before, the regulatory mechanism at post-transcription or protein translation level has never been explored.

Using a system genetics approach, we explored genes associated with the adrenal gland traits in HMDP at basal and post isoproterenol treatment. We have identified EPRS (Glutamyl-Prolyl-tRNA synthetase) to be significantly associated with adrenal gland mass post ISO treatment. EPRS is a tRNA synthetase responsible for charging tRNA during protein translation, EPRS also regulates mRNA translation as an RNA binding protein. To explore the functional impact of EPRS in adrenal gland physiology, we generated an EPRS adrenal gland specific knockout mouse model (EPRS-aKO). Inactivation of EPRS in adrenal gland led to a decrease of adrenal gland mass associated with reduced systemic catecholamine level. Interestingly, we further observed a moderate yet significantly decreased heart rate and ejection fraction in EPRS-aKO mice. Through both in vivo and in vitro analysis, we confirmed inactivation of EPRS led to a reduced protein, but not mRNA expression for both TH and DDC. In adipose tissue, activation of EPRS through mTOR signaling pathway is critical for its function as RNA binding protein. To further explore the mechanism through EPRS mediated catecholamine synthesis, we treated PC12 cells with rapamycin to inhibit mTOR activity. Treatment with Rapamycin diminished EPRS activity and expression, reduced TH protein expression along with a decrease of catecholamine level.

Our results identified a novel regulatory scheme of catecholamine synthesis through mTOR-EPRS axis, adding a novel layer of gene regulation in catecholamine production and providing new mechanistic insights for heart-adrenal cross talk.
Abstract P2012: Development Of An
In Vitro
Assay To Model Loss Of Neighbor At The Infarct Border Zone
Authors: Sehgal Rahul
Published: October 2023

The borderzone (BZ) of the infarcted heart separates poorly-perfused areas of cell death from well-perfused territories of comparatively normal myocardium. The BZ after myocardial infarction is of key importance for the remodeling process that drives heart failure and sudden cardiac death. We recently defined the transcriptional features of the infarct borderzone using single cell RNA-Seq and spatial transcriptomics which revealed prominent upregulation of mechanotransduction transcriptional programs. A similar transcriptional response was inducible by needle trauma alone. To explain these results, we proposed the “Loss of Neighbor” (LON) Hypothesis, where surviving cells neighboring the infarct experience mechanical instability due to the highly asymmetric loss of load, leading to cell signal activation. Here, we develop and validate an
in vitro
model of loss of neighbor using a mechanical scratch assay to model the biology of the infarct borderzone. We cultured neonatal rat cardiomyocytes and cardiac fibroblasts and subjected them to mechanical “scratch” injury. To evaluate how the mechanical wound altered gene expression, we performed qPCR for genes expressed by cardiomyocytes in the remote zone and the borderzone using RNA isolated from cells with or without exposure to mechanical scratch injury. Compared to non-scratched cultures, scratched cells significantly upregulated BZ-specific genes such as
Ankrd1, Nppa, Flnc,
and
Xirp2
. In addition, scratched cells significantly upregulated expression of interferon-stimulated genes (ISGs) compared to non-scratched cells or scratched cells exposed to anti-IFNAR Ab. This suggests that the interferon-inducible cell (IFNIC) response observed at the infarct borderzone
in vivo
can be induced by
in vitro
cardiomyocyte LON. Taken together, our results suggest that the mechanical scratch assay
in vitro
captures key elements of the transcriptional borderzone
in vivo
. Overall, this model can guide further elucidation of the transcriptional program states during cardiac remodeling.
Abstract P2022: Ankyrin-R Isoforms Are Differentially Expressed In Cardiac Tissues Under Stress
Authors: Argall Aaron D , Sucharski-Argall Holly , Comisford Luke , Crawford Casey , Hund Thomas J , Mohler Peter J , Koenig Sara N
Published: October 2023

Heart disease remains the leading cause of death in the United States with heart failure specifically accounting for 13.4% of all heart disease related deaths. In 2019, Andersson et al., utilized a multi-omics approach to evaluate participant samples from the Framingham Heart Study and showed that ankyrin-R (AnkR; encoded by
ANK1
) is significantly associated with diastolic function, left ventricular remodeling and heart failure with preserved ejection fraction. Ankyrins are a family of proteins that link integral membrane proteins with the actin/β-spectrin cytoskeleton. Ankyrins-B/G have been extensively studied and identified within the heart and their dysfunction is associated with cardiac structural and electrical phenotypes. Ankyrin-R was first identified in red blood cells and has yet to be studied in the context of cardiac function and heart failure or arrhythmia disease. To study AnkR in the context of the heart we isolated perfused tissues from adult wild-type mice and performed immunoblot and qPCR analysis on ankyrin-R protein and
Ank1
mRNA expression. The large AnkR isoform is expressed in the heart, along with the brain, intestine, and spleen. Interestingly, only the heart showed expression of a small AnkR isoform that has previously been shown to interact with the sarcoplasmic protein obscurin. Notably, isolated cardiomyocytes express the small AnkR isoform while cardiac fibroblasts express the canonical large AnkR isoform at both the protein and mRNA level. Canonical AnkR is diffusely expressed in the fibroblast membrane, cytoplasm, cytoskeleton, and soluble nuclear fractions. Interestingly, this expression pattern is switched under stressed conditions where cardiomyocytes increase and cardiac fibroblasts decrease their canonical AnkR expression, respectively. These preliminary results are the first to show canonical AnkR expression in the mouse myocardium specifically within the cardiac fibroblasts and allude that AnkR’s utilization during cardiac stress is cell-type dependent. Future studies will seek to define molecular, cell, and organ phenotypes related to AnkR in the heart.
Abstract P2029: Ror2-mediated Proteostatic Disruption As A Novel Mechanism For Right Ventricular Failure
Authors: Edwards Jonathan J , Uy Gen , Uchida Keita , Scarborough Emily , Yang Yifan , Brandimarto Jeff , Li Li , Yucel Nora , Prosser Benjamin , Arany Zoltan
Published: October 2023

No therapies exist to reverse right ventricular failure (RVF), in part because RVF molecular drivers have been incompletely characterized. We recently identified RVF-severity dependent re-expression of the developmental noncanonical WNT receptor Ror2 in dilated and ischemic cardiomyopathy RV. We now demonstrate that Ror2 plays a novel role in regulating cardiomyocyte proteostasis (Figure). NRVMs with Ror2 overexpression (Ror2
OE
) are smaller, rounder and demonstrate fragmented sarcomeres, loss of intercalated disc proteins, and reduced ubiquitination. Knockdown of Ror2 (Ror2
KD
) reveals opposing phenotypes. RNAseq of Ror2
OE
vs Ror2
KD
revealed striking downregulation of chaperone genes (Hspa1a/b, Cryab, Hsp90aa1, Hsph1/d1, etc.) and upregulation of genes involved in proteolysis (Rcn3, Nkd2, Mapk12). Using Suc-LLVY-AMC and GFP-degron, we find that proteasome activity is enhanced in Ror2
OE
and inhibited in Ror2
KD
. MG-132 proteasomal inhibition reversed structural phenotypes in Ror2
OE
NRVMs. In vivo cardiac Ror2
OE
caused RV dilation and systolic and diastolic dysfunction, more prominently in male mice. In male Ror2
OE
RVs, proteasome activity was enhanced and ubiquitin staining of RV cardiomyocytes was reduced. Finally, PAB caused a 5-fold increase in triton soluble and 40-fold increase in triton insoluble Ror2 expression, which correlated to severity of RV hypertrophy and dilation. Studies to test the efficacy of Ror2
KD
for PAB RVF are ongoing. In sum, our work suggests that reactivation on non-canonical Wnt signaling contributes to RVF, unveiling a novel potential therapeutic target.
Abstract P2030: Regulation Of Pathologic Cardiac Signaling Via Rac1 Palmitoylation
Authors: Teuber James P , Subramani Arasakumar , Dinda Sneha , Brody Matthew
Published: October 2023

Development of more effective therapies to prevent maladaptive cardiac remodeling and heart failure would be greatly aided by the identification of novel signaling pathways that regulate cardiac hypertrophy. Cysteine palmitoylation is a reversible post-translational lipid modification that dynamically regulates intracellular trafficking and subcellular compartmentalization of signaling proteins. Previous work from our group identified that mice with cardiomyocyte (CM)-specific overexpression of zDHHC3, a Golgi-localized S-acyltransferase, develop lethal dilated cardiomyopathy. S-acyl-proteomics revealed Cys-178 of Rac1, a small GTPase, as a palmitoylation site modified by zDHHC3 and hearts from zDHHC3-overexpressing mice display elevated levels of palmitoylated, active, and total Rac1, suggesting Rac1 palmitoylation promotes its activation and may drive pathologic cardiac remodeling. Rac1-dependent activation of NADPH oxidase 2 (Nox2) in CMs is required for angiotensin II (AngII)-induced cardiac hypertrophy and myocardial oxidative stress, suggesting inhibition of Rac1/Nox2 signaling could potentially ameliorate adverse cardiac remodeling. We found Rac1 palmitoylation at Cys-178 to be required for hypertrophy and oxidative stress in neonatal rat CMs as reactive oxygen species production and hypertrophy elicited by expression of a constitutively active Rac1 mutant were attenuated by mutation of the Cys-178 palmitoylation site, suggesting palmitoylation targets Rac1 to the Nox2 complex to promote myocardial oxidative stress. Notably, Rac1 palmitoylation and oxidative stress were also enhanced in hearts of mice subjected to AngII infusion and transgenic mice with CM-specific overexpression of the angiotensin II receptor type I (AT1R), suggesting a role for Rac1 palmitoylation in AngII-induced maladaptive cardiac signaling. To further elucidate the role of Rac1 palmitoylation in cardiac pathology, we developed a CM-specific knock-in mouse model in which Cys-178 of Rac1 is mutated to serine. Ongoing work utilizing this model will investigate mechanisms by which palmitoylated and depalmitoylated Rac1 differentially regulate signaling, oxidative stress, and hypertrophy in CMs in response to pathologic stimuli.
Abstract P2040: Pannexin 1 Activation Drives Neutrophil Recruitment In A Murine Model Of Non-ischemic Heart Failure Induced By Isoproterenol
Authors: Pavelec Caitlin M , Poudel Nabin , Orrell Emily , Thomas Samantha , Luviano Hannah , Yeudall Scott , Upchurch Clint , Senthivinayagam Subramanian , Wolf Matthew J , Leitinger Norbert
Published: October 2023

Pannexin 1 (Panx1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation and blood pressure regulation. Panx1 channel function is inhibited by spironolactone, a frontline therapy for heart failure patients. Despite this, the function of Panx1 in cardiomyocytes and a possible role in heart failure has not yet been studied. To investigate if Panx1 is involved in the development of non-ischemic heart failure, we use an acute (14 day) model of adrenergic stimulation (isoproterenol) to induce non-ischemic heart failure in a novel mouse model we have generated with constitutive deletion of Panx1 in cardiomyocytes by crossing mice expressing loxP-flanked alleles of
Panx1
to mice expressing Cre recombinase under the control of the cardiomyocyte specific
MyHC6
promoter (Panx1
MyHC6-Cre
). Constitutive deletion of Panx1 in cardiomyocytes had no effect on heart function at baseline, but protected mice against isoproterenol-induced non-ischemic heart failure (15 mg/kg/day isoproterenol, i.p.). Panx1
MyHC6-Cre
mice were protected from systolic and diastolic dysfunction and cardiac hypertrophy as measured by echocardiography and heart mass, respectively. Moreover, Panx1
MyHC6-Cre
mice demonstrated decreased recruitment of immune cells (CD45
+
), particularly neutrophils (CD11b
+
, Ly6g
+
) to the myocardium as measured by confocal microscopy and flow cytometry.
In vitro
, treatment of H9c2 rat myoblasts with isoproterenol led to Panx1-dependent release of ATP and Yo-Pro-1 uptake in a dose-dependent manner, as assessed by pharmacological blockade by spironolactone and siRNA-mediated knock-down of Panx1. These data demonstrate that that β-adrenergic stimulation in cardiomyocytes activates Panx1 channels and that Panx1 deficiency in cardiomyocytes protects against non-ischemic heart failure and the associated cardiac dysfunction at least in part by reducing immune cell recruitment. Taken together, our study provides evidence for Panx1 channel inhibition as a therapeutic approach to ameliorate cardiac dysfunction in heart failure patients.
Abstract P2047: Fast Skeletal Myosin Binding Protein-C Expression Exacerbates Dysfunction In Heart Failure
Authors: Song Taejeong
Published: October 2023

Background:
During heart failure, gene and protein expression profiles undergo extensive compensatory and pathological remodeling. We have previously observed that fast skeletal myosin binding protein-C (fMyBP-C) is upregulated in diseased mouse hearts. While fMyBP-C shares significant homology with its cardiac paralog, cardiac myosin binding protein-C (cMyBP-C), there are key differences in the functional differences which may affect cardiac function. Also, it is unknown if the fMyBP-C expression in the heart is a pathological or compensatory response.

Objectives:
We aim to elucidate the consequence of increased fMyBP-C expression in the heart.

Methods:
To determine the sufficiency of fMyBP-C to cause cardiac dysfunction, we generated cardiac-specific fMyBP-C over-expression mice. These mice were further crossed into a cMyBP-C null model to assess the effect of fMyBP-C in the heart in the absence of cMyBP-C function. Finally, fMyBP-C knockout mice were subjected to transverse aortic constriction (TAC) to define the requirement of fMyBP-C during heart failure development.

Results:
We confirmed the upregulation of fMyBP-C in several models of cardiac disease. Genetic over-expression of low levels of fMyBP-C caused mild cardiac remodeling and sarcomere dysfunction. High expression of fMyBP-C in a heart failure model further exacerbated the cardiac disease, hypertrophy and fibrosis. On the other hand, following 8 weeks of TAC, fMyBP-C null mice demonstrated greater protection against heart failure development. Mechanistically, this may be due to the greater instability of the myosin super-relaxed state compared to wild-type littermates.

Conclusions:
These findings suggest that the elevated expression of fMyBP-C in diseased hearts is a pathological response. Targeted therapies to prevent upregulation of fMyBP-C may prove beneficial in the treatment of heart failure.
Abstract P2071: Mutation-Specific Calmodulin-Kinase II Activation In Hypertrophic Cardiomyopathy
Authors: Hauck Garrett T , Klass Matthew , Tardiff Jil C
Published: October 2023

Autophosphorylation of Calmodulin-Kinase IIδ (CaMKIIδ) at Thr-287 leads to autoactivation such that kinase activity is autonomous of Ca
2+
/CaM. In the heart, CaMKIIδ functions to regulate Ca
2+
homeostasis by targeting Ca
2+
handling proteins, including phospholamban (PLB). In sarcomeric hypertrophic cardiomyopathy (HCM), disruption of Ca
2+
homeostasis and aberrant autoactivation of CaMKIIδ has been linked to disease progression. We have previously shown that the pathogenic role of autonomous CaMKIIδ in HCM is mutation-specific. Transgenic mice expressing the HCM mutation R92W in cardiac troponin T (cTnT) exhibited an increase in autonomous CaMKIIδ while cTnT-R92L mice did not. The potentially clinically relevant mechanism(s) underlying this mutation specificity are unresolved. We hypothesize that a potential trigger of autonomous CaMKIIδ is accelerated myofilament Ca
2+
dissociation kinetics. We have previously shown that cTnT-R92W accelerates Ca
2+
dissociation, while cTnT-R92L decelerates Ca
2+
dissociation. Here, we extended our study to include cTnT-I79N and cTnT-R94H to determine if Ca
2+
dissociation rates can be linked to autonomous CaMKIIδ activity and thus provide a potential novel target in HCM. Our data show that cTnT-I79N accelerates Ca
2+
dissociation, and cTnT-R94H decelerates Ca
2+
dissociation, similar to our earlier findings. To determine whether the mutation-specific changes in Ca
2+
dissociation alter CaMKIIδ activity, transgenic mice expressing cTnT-R94H and cTnT-I79N were probed for autophosphorylated CaMKIIδ and Thr-17 phosphorylated PLB. At 2 months, PLB phosphorylation is unchanged in cTnT-R94H and cTnT-I79N mice. At 4 months, cTnT-I79N mice exhibit a trending increase in autonomous CaMKIIδ and cTnT-R94H mice exhibit a modest increase, similar to cTnT-R92W positive controls. These trends are matched by PLB phosphorylation levels. At 6 months, cTnT-R94H mice exhibit normal autonomous CaMKIIδ levels. While future and ongoing work will explore changes in myocellular Ca
2+
homeostasis in more depth, these preliminary data suggest that, unlike cTnT-R94H mice, cTnT-I79N mice exhibit elevated autonomous CaMKIIδ and these mutation-specific differences may underlie observed phenotypic variation in patients.
Abstract P2079: Heart Rate Variability And Chronotropic Incompetence As Prognostic Markers In A Preclinical Model Of HFpEF
Authors: Perez-Bonilla Patricia , Chen Xian , LaViolette Brianna , Hirenallur-Shanthappa Dinesh
Published: October 2023

The prevalence of heart failure with preserved ejection fraction (HFpEF) in developed countries is increasing alarmingly. This is in part due to an increase in comorbid diseases (obesity, diabetes, hypertension), and risk factors (age, stress, and low physical activity). HFpEF patients often present with chronotropic incompetence (CI) and impaired heart rate variability (HRV), and both CI and decreased HRV are used as prognostic markers of heart disease and sudden cardiac death in clinical studies. In preclinical HFpEF models, however, CI and HRV are not commonly evaluated, and the efficacy of novel compounds is determined primarily through echocardiography results. Thus, there is a need to align efficacy readouts to enhance the translatability of novel compounds from preclinical to clinical stages. Here we sought to determine the utility of CI and HRV in preclinical HFpEF studies in a novel 2-hit HFpEF model; the metabolic syndrome non-alcoholic steatohepatitis mouse with AAV overexpression of renin (MS-NASH+Renin). To collect continuous and conscious electrocardiogram and blood pressure data, C57BL/6 (n=10) and MS-NASH (formerly FATZO, n=32) male mice were implanted with radiotelemeters at 20 weeks and
iv
injected with either AAV-Vector or AAV-Renin. To assess disease burden, CI and HRV, we acquired and analyzed body weight, electrocardiography, peak VO2, exercise HR, and echocardiography data once a month for 5 months. We found that MS-NASH mice have increased body weight regardless of treatment, and that renin treatment increased blood pressure for both C57BL/6 and MS-NASH mice. Peak VO2, time to exhaustion, distance traveled, and maximum and recovery HR are significantly decreased in MS-NASH mice, indicating CI. HRV is also significantly decreased in MS-NASH mice when compared to C57BL/6. Interestingly, renin treatment did not further decrease treadmill performance or HRV for MS-NASH mice. However, MS-NASH+Renin mice did have decreased functional capacity with preserved ejection fraction and altered heart structure. These results suggest that MS-NASH+Renin is a model for HFpEF, and that CI and HRV are translatable markers for HFpEF. In the future, we will evaluate the use of CI and HRV in preclinical HFpEF drug development studies.
Abstract P2081: Defective Translation Of Mitochondrial Proteins Impairs Cardiac Metabolism In HFpEF
Authors: Koleini Navid , Meddeb Mariam , Yoo Edwin , Saba James , Keykhaei Mohammad , Mulligan Abigail , Htet Myo , Farshidfar Farnaz , Kabir Raihan , Kwon Seoyoung , Hanselman Olivia , McKoy Desirae , Zhao Liang , Hahn Virginia S , Sharma Kavita , Sidoli Simone , Green Rachel , Ranek Mark J , Pearce Erika , Kass David A
Published: October 2023

We recently reported that heart failure with preserved ejection fraction (HFpEF) in human myocardium has broadly reduced acylcarnitines and Krebs cycle intermediates, and that transcriptomics shows increased gene expression of oxidative-phosphorylation (OxPhos) related pathways. However, new preliminary data finds protein expression related to OxPhos is decreased. Here, we explored a key mechanism that could drive depressed mitochondrial protein translation in HFpEF. Elongation factor 5A (EIF5A) is a ribosomal subunit that is post-translationally modified on lysine 50 by the transfer of an aminobutyryl moiety from spermidine, called hypusination (Hyp). We find EIF5A
Hyp
and the mitochondrial proteome are diminished despite elevated gene transcripts in the myocardium of human and a mouse model of HFpEF (latter high-fat diet +L-NAME). Inhibiting EIF5A hypusination in neonatal rat cardiomyocytes results in ribosome stalling, disruption of OxPhos, and increased glycolysis.Proteomics of adult cardiomyocytes with inhibition of the rate-limiting hypusination enzyme deoxyhypusine synthase (DHPS) phenocopies depressed mitochondria as in human HFpEF. Cardiomyocyte-specific knockout of DHPS is lethal in pups within 24hrs after birth. These mice have significantly less myocardial EIF5A
Hyp
. Cardiac-specific DHPS+/- are viable and have no heart phenotype in unstressed situations, but display marked cardiac dilation with low ejection fraction with failure to hypertrophy in response to transverse aortic constriction.Four weeks following tamoxifen-inducible homozygous knockout of DHPS in cardiomyocytes of 8-week-old mice, we find impaired diastolic function (increased E/E’) with preserved ejection fraction and reduced exercise tolerance. Despite reduced hypusination in HFpEF, ornithine levels are elevated suggesting inhibition of hypusination at the hypusinating enzymes. Interestingly, DHPS levels are decreased in human HFpEF hearts.In summary, we have uncovered a novel role of polyamines in cardiomyocytes: the facilitation of translation of mitochondrial proteins by EIF5A hypusination. We find reduced hypusination resulting from defects in hypusine synthesizing enzymes contributes to mitochondrial defects in HFpEF.
Abstract P2083: Heart Failure With Preserved Ejection Fraction Mouse Model Development With Aav-renin
Authors: Shi Yuji
Published: October 2023

Heart failure with preserved ejection fraction (HFpEF) accounts for more than half of heart failure patients, with limited approved evidence-based therapies. HFpEF is a complex multifactorial disease associated with comorbidities of hypertension, obesity, diabetes, and renal dysfunction. While the pathophysiology of HFpEF has yet to be fully understood, the development of therapies is hindered partially by limited choices of translationally relevant preclinical models that recapitulate human HFpEF clinical features. Here, we report the development of mouse models of HFpEF through a combination of metabolic and hypertensive stress mediated through activation of the renin-angiotensin-aldosterone system (RAAS) via AAV-Renin. These novel mouse models demonstrate clinically relevant features of human HFpEF, including preserved ejection fraction (EF), concentric cardiac hypertrophy, left atrial enlargement, diastolic dysfunction, elevated natriuretic peptides, inflammation, and exercise intolerance. We also demonstrate that approved HFpEF therapies valsartan/sacubitril (Entresto) and Empagliflozin effectively improve the cardiac phenotypes of HFpEF in this model. In summary, we have established two novel two-hit mouse HFpEF models by combing hypertension induced by AAV-Renin and metabolic stress caused by either HFD or a novel mouse strain with metabolic syndrome. These models faithfully represent the cardiometabolic HFpEF phenogroup and will provide new tools for HFpEF preclinical research and drug discovery.
Abstract P2085: Growth Hormone Releasing Hormone Agonist Counteracts The Cardiometabolic Heart Failure With Preserved Ejection Fraction Phenotype In Vivo Via The Hypoxia Inducible Factor 1 Alpha
Authors: Kanashiro-Takeuchi Rosemeire M , Takeuchi Lauro , Balkan Wayne , Dulce Raul A , Kazmierczak Katarzyna , Kulandavelu Shathiyah , Cai Renzhi , Sha Wei , Schally Andrew , Hare Joshua M
Published: October 2023

Heart failure (HF) with preserved ejection fraction (HFpEF) treatment is limited due in large part to a lack of understanding of the underlying mechanisms. Recently, we showed that growth hormone-releasing hormone-agonist (GHRH-agonist) has cardioprotective effects in a murine model of cardiometabolic HFpEF; however, the mechanism of these therapeutic effects is still unclear. Here, we hypothesize that the therapeutic effects of GHRH-agonist in HFpEF are mediated by GHRH-receptor activation of the hypoxia-induced factor (HIF)-1α pathway.

Methods:
Wild-type (WT) and HIF-1α cardiomyocyte knockout (HIF-1α
CM
KO) mice were fed a high-fat diet (HFD) plus the nitric oxide synthase inhibitor (L-NAME) for 9 weeks. After 5 weeks of HFD+L-NAME regimen, animals received daily injections of placebo or the GHRH-agonist, MR-356, for the remaining 4-week period. Control animals received no HFD+L-NAME or MR-356 treatment. Echocardiography, blood pressure, glucose tolerance test, and hemodynamic measurements were evaluated.

Results:
The HFD+L-NAME diet produced a HFpEF phenotype, which was reversed in mice with intact HIF-1α treated with MR-356. In contrast, in mice with a cardiac-specific deletion of HIF-1α
CM
, MR-356 treatment failed to reverse the elevated relative wall thickness in diastole (RWTd), increased E/E’ ratio and reduced global longitudinal strain (GLS). The slope of end-diastolic pressure-volume relationship (EDPVR) was increased in the placebo group and restored by MR-356 in WT mice but not HIF-1α
CM
KO mice, while EF was preserved.

Conclusion:
Our findings suggest that HIF-1α is necessary for the cardioprotective and anti-HFpEF effects MR-356.
Abstract P2086: Targeting Endothelial Dysfunction And Senescence An Heart Failure With Preserved Ejection Fraction - A Whole Transcriptome Analysis Of Heart Failure With Preserved Ejection Fraction-patients To Identify New Therapeutic Targets
Authors: Wurmbrand Leonie , Kalies Katrin , Knoepp Kai , Dutzmann Jochen , SEDDING Daniel
Published: October 2023

Heart failure with preserved ejection fraction (HFpEF) remains a disease with a poor prognosis and increasing incidence in all Western countries mainly affecting the aging population. A deeper understanding of pathogenesis at the cellular level, particularly the central role of endothelial dysfunction in cellular senescence, is required for the development of new therapeutic approaches. Circulating endothelial cells as a marker for endothelial damage and dysfunction, have already been examined quantitatively in general HF patients, HFpEF patients and healthy subjects. Here, we aim to isolate circulating endothelial cells to target endothelial dysfunction and identify new therapeutic targets by whole transcriptome analysis.Methods included the isolation of circulating endothelial cells (CEC) from whole blood samples via a newly established FACS panel. HFpEF patients were included based on their HFA-PEFF score. Young and old subjects served as non-diseased controls and were matched as closely as possible for sex and age. CEC were further processed for a whole transcriptome analysis. In total, we collected samples from n=15 patients per group. Mean age was 82.3 for the HFpEF group, 64.5 for the non-diseased control ‘old’, and 25 years for the non- diseased control ‘young’, HFA-PEFF score was 5, 0, and 0 points (P<0.05), and the proportion of female patients was 66%, 78%, and 77%, respectively. Including additional patient data, our collective was shown to be comparable to large HFpEF studies and clinical registries. CEC cells were sorted based on CD45-, CD11b- CD34+, CD31+ and CD146+ marker expression. The CEC of the aged group revealed upregulated expression of senescence markers p16, p21 and CD44. In gene set enrichment analysis, we see trends of differentiation of mainly proliferation- and transcription-associated gene sets in heart failure with preserved ejection fraction. In summary, we achieved to isolate CEC from whole blood of HFpEF-patients and matched controls. The whole transcriptome analyses may provide new clues for therapeutic strategies, which is now the subject of further in vitro and in vivo studies. Our goal is to develop new therapeutic targets after successful evaluation and modulation of the target genes.
Abstract P2088: Proteostatic Imbalance In Heart Failure With Preserved Ejection Fraction Is Accompanied By Mechanistic Target Of Rapamycin Complex 1 Hyperactivation
Authors: Kobak Kamil A , Zarzycka Weronika , King Catherine J , Peelor Frederick F , Miller Benjamin F , Chiao Ying Ann
Published: October 2023

Heart failure with preserved ejection fraction (HFpEF) is a multi-organ syndrome that represents approximately 50% of all heart failure (HF) cases. The limited effective therapies for HFpEF and poor understanding of the underlying mechanisms make this disorder a major challenge for cardiovascular medicine. Disruption of protein homeostasis (proteostasis) is a well-known pathomechanism involved in many chronic diseases and aging. However, how proteostasis changes during HFpEF development remains unclear. The objective of this study is to determine the role of proteostatic regulation in HFpEF pathology. We used a combination of metabolic and hypertensive stress (2-hit stress) to induce HFpEF development in C57BL/6 mice. Our proteomic comparison between HFpEF mice and unstressed controls revealed changes in the abundance of proteins involved in protein translation, protein folding, and the ubiquitin-proteasome system, suggesting that proteostatic maintenance is altered during HFpEF. Using deuterium oxide (D
2
O) labeling coupled with GC-MS analysis of subcellular fractions, we found higher protein synthesis rates in myofibrillar and mitochondrial fractions of HFpEF hearts compared to controls. Subsequent isotopic analysis of RNA synthesis showed that ribosomal biogenesis was 2-fold higher in HFpEF hearts compared to controls. We also observed a reduced LC3-II/I ratio in HFpEF hearts, which may suggest suppressed autophagy. These results indicate that HFpEF hearts have increased protein synthesis and reduced protein degradation compared to controls. As the mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of proteostasis that promotes protein synthesis and suppresses autophagy, we investigated if mTORC1 signaling is altered in HFpEF hearts. We found that HFpEF hearts had increased activation of mTORC1, as indicated by increased phosphorylation of its targets. In conclusion, we showed that HFpEF pathology is associated with proteostatic imbalance, potentially due to mTORC1 hyperactivation. Our ongoing study is investigating if mTORC1 inhibition (using cardiac-specific Raptor heterozygous knockout mice) preserves proteostatic maintenance and attenuates cardiac dysfunction during HFpEF development.
Abstract P2112: Mechanism Of Bag3's Degradation Of Tgfbr2 In Cardiac Fibroblasts
Authors: Morsink Margaretha , Wang Bryan , Watkins Josephine , Luo Lori , Zhang Xiaokan , Soni Rajesh , Fine Barry , Vunjak-Novakovic Gordana
Published: October 2023

BAG3 levels are diminished in patients with heart failure and several mutations have been identified to cause dilated cardiomyopathy with cardiac fibrosis. BAG3’s four binding domains are critical to its function to facilitate proteostasis. BAG3’s role has been extensively studied in CMs, where it aids sarcomere protein turnover through chaperone-assisted selective autophagy, through BAG3’s complex with HSP70, HSPB8, and E3 ligase CHIP. However, BAG3 is ubiquitously expressed and its role in non-myocytes has been overlooked. Cardiac fibroblasts are responsible for the excessive matrix deposition leading to fibrosis, mostly through TGF-beta signaling. Previously, we have shown the importance of BAG3 in cardiac fibroblasts through its role in TGF-beta signaling and identified BAG3 to facilitate the degradation of the type II TGF-beta receptor (TGFBR2). As BAG3 does not have inherent E3 activity, this study aimed to identify the specific E3 ligase of TGFBR2 and the binding domains of BAG3 that are crucial to this interaction to gain insight into the effects of the clinical mutations of BAG3. Immunoprecipitation (IP) of endogenous BAG3 in cardiac fibroblasts revealed five E3 ligases as high-confidence interactors of BAG3. To find the specific E3 ligase of TGFBR2, we performed an IP of a V5-tagged TGFBR2 and identified HSP70, HSPB8 and CHIP as binding partners. To delineate the critical domains of BAG3 that impact the TGFBR2 degradation, we overexpressed 3x-FLAG-tagged BAG3 proteins lacking each domain respectively (WW, IPV, PxxP, and BAG) and show that the BAG domain is imperative in TGFBR2 degradation. This has clinical implications for the common DCM-causing E455K mutation in the BAG domain. The ubiquitination of TGFBR2 was decreased upon overexpression of a BAG3 plasmid containing the E455K mutation. Lastly, the drug JG-98, which disrupts the binding of HSP70 to BAG3, was used to show the importance of the HSP70-BAG3 axis in the degradation of TGFBR2. In conclusion, we have uncovered the molecular mechanism of TGFBR2 degradation in cardiac fibroblasts mediated by the BAG3-HSP70-HSPB8 complex, with clinical implications for the loss of BAG3 and several common DCM-causing mutations in the development of cardiac fibrosis.
Abstract P2124: Trans-valvular Unloading Protects Against Post-reperfusion Injury Via Hif-1a In Preclinical Models Of Acute Myocardial Infarction.
Authors: Swain Lija , Reyelt Lara A , Qia Xiaoying , Bhave Shreyas , Everett Kay , Mahmoudi Elena , Majumdar Monica , Warner Mary , Kapur Navin K
Published: October 2023

Acute myocardial infarction (AMI) is a major cause of heart failure and mortality worldwide. Reducing left ventricular (LV) workload using a trans-valvular pump (TVP) and delaying reperfusion for 30 minutes reduces infarct size by 50% in preclinical models and is currently under clinical investigation. We hypothesized that LV unloading limits myocardial injury by activating hypoxia inducible factor 1-α (HIF-1α). Myocardial ischemia was induced by percutaneous balloon occlusion of the left anterior descending (LAD) artery for 90 minutes in adult male swine. The detailed study design is shown in Fig A. In Group A and B, no infarct was observed with or without TVP. In Groups C and D, infarct size was significantly lower with TVP activation (Fig B). During LAD occlusion without reperfusion, HIF-1α levels increased within the infarct zone (Group A), but were significantly lower with TVP activation (Group B). After reperfusion, HIF-1α levels decreased in Group C, but paradoxically, increased with TVP support (Group D; Fig C). To test whether HIF1α mediates cardioprotection with LV unloading, adult swine were treated with intracoronary delivery of a HIF-1α inhibitor (2-methoxyestradiol; 2ME) before reperfusion. Compared to DMSO-treated controls, 2ME increased infarct size despite LV unloading (Fig D). Conversely, compared to reperfusion alone, intracoronary delivery of a HIF-1α stabilizing agent (Roxadustat) before reperfusion significantly decreased infarct size both in the presence and absence of a TVP (Fig E). We identified a novel functional role for HIF-1α as a critical mediator of the cardioprotective effects associated with trans-valvular unloading before reperfusion.