bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract MP233: Investigating Genotoxic-induced Innate Immune Pathways Leading To Heart Failure With Preserved Ejection Fraction For Developing Effective Treatments
Authors: Al-Khalaf Mohammad H , Smyth David , Akolkar Gauri , Williams Justin , Zhang Liyong , Yip Kenneth , Li Ren-Ke , Liu Fei-Fei , Liu Peter P
Published: November 2021

Background:
A hallmark of heart failure pathologies is excessive and cumulative DNA damage, leading to an increased and accelerated cardiac cellular senescence. We aimed to investigate the role of DNA breaks in inducing inflammation leading to adverse remodeling, premature senescence, and cardiac dysfunction leading to onset of Heart Failure with preserved Ejection Fraction (HFpEF), a specific type of disease prevalent in the aging population.

Results:
We use heart-focused ionizing radiation, a novel in vivo technique to induce aging related DNA damage and onset of diastolic dysfunction and HFpEF (n= 4-5 per group & per species; animals used: Rattus norvegicus and Mus musculus). We performed functional analysis, histological tissue assessment and molecular investigations to evaluate and validate this novel animal model technique. DNA damage response is upregulated in aging-related Heart Failure, and here demonstrated in our novel heart-focused radiation technique. Using in vitro ionizing radiation exposure alters cardiomyocyte morphology and activates inflammation, leading to upregulation of fibrosis and senescence markers. Finally using experimental inhibitor compounds targeting the cGAS-STING axis of innate immunity, we show effective attenuation of this relevant pathway, leading to decreased systemic inflammation, cardiomyocyte remodeling, and senescence associated with DNA damage injury.

Conclusions:
We present a novel technique to induce genotoxic outcomes and inflammation of the heart, leading to HFpEF pathology. We elucidate novel connections from the onset and accumulation of unresolved DNA damage, to activation of specialized innate immune cellular responses, and ultimately upregulation of cardiac tissue specific inflammation, fibrosis and senescence. Finally we demonstrate that antagonizing the cGAS-STING pathway could allow a precision medicine approach to treating genotoxic and inflammatory HFpEF.
Abstract MP234: Novel
Mybpc3
Variant Disrupts Myosin S2 Binding And Induces Heart Failure
Authors: Song Taejeong , Singh Rohit , Desai Darshini , Koch Sheryl E , Rubinstein Jack , Sadayappan Sakthivel
Published: November 2021

Rationale:
Cardiac m
yosin
binding protein-C regulates a
ctomyosin interaction
in striated muscle, but mutations in the
MYBPC3
gene can lead to hypertrophic cardiomyopathy (HCM) as seen in some South Asians living in the USA carrying a novel variant wherein an aspartic acid is mutated to a valine at position 389 (D389V). Individuals and iPSC-derived cardiomyocytes carrying D389V display hypercontractility, indicating early onset of HCM. However, the mechanisms underlying the pathophysiology of this mutant in the context of HCM are unknown.

Objective:
To define the pathophysiological consequences D389V on myosin and cardiac function
in vivo
.

Methods and Results:
Compared with wild-type controls, our D389V knock-in homozygous mouse model showed decreased cardiac function by percentage of ejection fraction (-23%, P<0.01), but increased systolic left ventricular volume (+39%, P<0.01) at 3 and 6 months of age. Heart weight to tibia length ratio was significantly increased (+ 15%, P=0.05), demonstrating distinct pathogenicity. Using recombinant proteins carrying D389V substitution at the N-terminal
MYBPC3
domains (rC0C2
D389V
), cosedimentation and solid-phase binding assays showed significantly reduced binding rate of rC0C2
D389V
to the myosin S2 region (-55% and -23%, P<0.05, respectively), but
in vitro
actin motility over myosin increased 24% (P<0.05) compared to rC0C2
WT
control, indicating a causal relationship between variant and decreased
MYBPC3
binding to myosin. Human iPSC-derived D389V het cardiomyocytes display an increase in lipid peroxide and reactive oxygen species by +3- and +7-fold P<0.01, respectively, compared to noncarrier controls.

Conclusion:
D389V decreases interaction between
MYBPC3
and myosin S2, causing reduced cardiac function and providing mechanistic evidence that it contributes to the etiology of HCM.
Abstract MP237: Multi-omics Profiling Reveals Stearoylcarnitine As A Novel Biomarker Of Cardiometabolic Disease Development In Rat Offspring Exposed To Gestational Diabetes
Authors: Kereliuk Stephanie M , Agarwal Prasoon , Brawerman Gabriel , Cole Laura , Xiang Bo , Fonseca Mario , Hatch Grant , McGavock Jonathan M , Dolinsky Vernon
Published: November 2021

Through unknown mechanisms, fetal exposure to gestational diabetes mellitus (GDM) increases the risk for cardiovascular disease development later in life. We hypothesize that fetal exposure to GDM alters offspring cardiomyocyte metabolism and left ventricular (LV) function with age. To induce GDM, female rats were fed a high fat (45% kcal) and sucrose diet prior to mating, throughout pregnancy and lactation. Lean controls received a low fat (10% kcal) diet. Fetal rat ventricular cardiomyocytes (FRVC) were isolated from e20 offspring for U-13C glucose metabolic flux analysis, mitochondrial respiration and calcium handling. The cardiac transcriptome and metabolome were measured in 3-month old offspring. LV morphology and function was assessed in the offspring from e18 to 12-months of age by transthoracic ultrasound. Offspring exposed to GDM exhibited increased LV posterior wall thickness and impaired LV filling across their life course (fetal to 12-months of age; p<0.05). Consistent with the development of diastolic dysfunction in vivo, alterations in calcium flux and sarcoplasmic reticulum-dependent calcium re-uptake (1.5 and 1.6-fold greater, respectively) were observed in FRVC isolated from GDM offspring (p<0.05). When FRVC were treated with isoproterenol, U-13C glucose metabolic flux through glycolysis and the citric acid cycle was reduced in GDM offspring, compared to Lean controls. In 3-month old GDM offspring serum and cardiac metabolomics revealed an altered acylcarnitine profile, with specific elevation in long chain acylcarnitine species including stearoylcarnitine. Alterations in the metabolome corresponded to changes in gene expression patterns identified by RNA-Seq associated with glucose metabolism and fatty acid transport pathways (e.g.
Irs2, Slc2a4, Pfkfb2, Pdk4
and
Cpt1a).
These alterations corresponded with mitochondrial dysfunction, impaired cardiomyocyte metabolic flux and contractility, in concert with LV hypertrophy and diastolic dysfunction in the rat offspring. Multi-omics profiling reveals stearoylcarnitine as a novel biomarker and implicates mitochondrial oxidative metabolism as a mechanism that links early-life GDM exposure to the development of cardiovascular disease later in life.
Abstract MP239: Hippo Signaling Pathway Maintains Homeostasis Of The Cardiac Conduction System
Authors: Zheng Mingjie , Wang Jun
Published: November 2021

The cardiac conduction system (CCS) is required for initiating and maintaining regular rhythmic heartbeats. The fundamental Hippo signaling pathway plays critical roles in the heart, yet its role in the CCS remains largely unknown. Here, we found that conditional knockout (CKO) of Hippo signaling kinases
Lats1
and
Lats2
in the CCS using

Hcn4
CreERT2

, led to cardiac arrhythmias in adult mice. Compared with controls,
Lats1/2
CKO mutant mice had disrupted calcium homeostasis, increased fibrosis and more fibroblast proliferation in the sinoatrial node. Deletion of the Hippo signaling effectors
Yap
and
Taz
in the CCS rescued phenotypes caused by
Lats1/2
deletion, and these mice had rescued sinus rhythm and reduced fibrosis, which indicated that
Lats1/2
function through
Yap
and
Taz
in CCS. Our Cleavage Under Targets and Tagmentation (CUT&Tag)-sequencing using Yap antibody followed by RNA-Seq revealed that Yap directly regulates calcium homeostasis genes such as
Ryr2
and fibrosis induction genes such as
TGF-β
family. Further, we discovered that
miR-17-92
represses Hippo signaling by directly suppressing
Lats2
expression.
miR-17-92
CKO in the CCS led to increased Hippo signaling activity and cardiac arrhythmias, indicating that a fine-tuned level of Hippo signaling is critical for CCS homeostasis. Together, our findings reveal the critical role of a miR-Hippo-Yap genetic pathway in maintaining CCS homeostasis.
Abstract MP240: Site-specific O-Glcnacylation Of Transient Receptor Potential Canonical Type 6 (trpc6) Channel Is Required For Normal Structure And Function
Authors: Mishra Sumita , Ma Junfeng , Akiyoshi Kei , Farinelli Federica , Page Rick , Oeing Christian U , Zachara Natasha , Das Samarjit , Ranek Mark J , Kass David A
Published: November 2021

O-GlcNAcylation is a dynamic, reversible posttranslational modification (PTM) that regulates a multitude of biological processes. Fluctuations in O-GlcNAC of various calcium handling proteins impact their functionality in cardiomyocytes. Here, we show for the first time that TRPC6, a nonselective receptor-operated cation channel and mediator of hypertrophy and fibrosis, is constitutively O-GlcNAcylated in the ankyrin repeat domain (AR4), at Ser 14, Thr 70, and Thr 221 within the N-terminal cytoplasmic segment. Of these, only substitution of Thr 221 with alanine (T221A) results in a change of function, notably a hyperactive TRPC6 channel with 5X greater increase in consequent NFAT promoter activity, which is a marker of TRPC6 calcium signaling. Patch-clamp analysis of T221A mutant channels found a 75-80% increased conductance compared to WT. Myocardial injection of T221A in homozygous TRPC6 KO mice by AAV-9 mediated gene transfer results in systolic dysfunction, hypertrophy, and cardiac fibrosis, by loss of OGlcNAc modification at site T221. T221 is highly conserved across species and found in the AR4 domain, which forms the core structure of TRPC6 intracellular domain. Mutating the site in its closest homologs, TRPC3 and TRPC7, also activates channel activity. T221 O-GlcNAcylation also protects the nascent protein from premature proteasomal degradation. Molecular modeling from the crystal structure of human TRPC6 predicts that OGlcNACylation stabilizes electrostatic interactions with the 193-203 loop near AR4, and loop connecting AR4 to the linker helix 1 (LH1) at S199, E200, and E246. Mutating these sites to alanine also increases TRPC6-NFAT signaling similar to what was observed in the T221A mutant. In summary, this study highlights that O-GlcNAcylation of TRPC6 is an important PTM needed to stabilize channel function, and its decline results in gain-of-function related diseases.
Abstract MP243: Detection Of Mesenchymal Stem Cells In Mobilized Autologous Peripheral Blood Transplantation From Patients With Ischemic Heart Disease
Authors: Lim Hadyanto , Zein Umar , Hariaji Ilham
Published: November 2021

Background:
Mesenchymal stem cells (MSCs) improve the cardiac function and remodeling in patients with ischemic heart disease. However, their presence in the circulating peripheral blood and post-transplantation has not been fully elucidated. We aimed to investigate the effects of intravenous transplantation of mobilized autologous peripheral blood on the number of MSCs in patients with ischemic heart disease.

Methods:
Granulocyte-colony stimulating factor (G-CSF, 5.0 μg/kg/day) was given subcutaneously once a day for five days to 7 patients (4 women and 3 men, aged 54-69 years) with ischemic heart disease. Leukapheresis procedure was started on the day 5 of G-CSF using the Spectra Optia cell separator. Circulating and intravenous transplantation of autologous MSCs after leukapheresis were analyzed by flow cytometry. MSCs were identified on the basis of dual positive cells (CD73
+
/CD105
+
or CD90
+
/CD73
+
or CD90
+
/CD105
+
) and detected as MSCs if a cluster of at least 10 cells could be found.

Results:
MSCs in the circulating peripheral blood and after transplantation were detected in 2 (28%) and 6 (85%) patients, respectively. The frequency of intravenous peripheral blood MSCs increased significantly after transplantation (from 32.57 ± 22.76 x10
-4
% to 58.57 ± 28.49 x 10
-4
% , p<0.001). Moreover, there were significant rise in the total white blood cells count (from 10.25 ± 4.86 x 10
3
/μl to 35.81 ± 7.07 x 10
3
/μl, p<0.001) and the levels of CD34
+
cells (from 1.17 ± 0.93 cells/μL to 138.30 ± 11.26 cells/μL, p<0.001) after the infusion.

Conclusions:
The results show that intravenous transplantation of mobilized autologous peripheral blood increases the number of MSCs in patients with ischemic heart disease. Leukapheresis product of peripheral blood MSCs could therefore be a potential source for autologous transplantation in ischemic heart disease.
Abstract MP250: Reduction In Endothelial Aryl Hydrocarbon Receptor Nuclear Translocator (arnt) Mediates Vascular Dysfunction In Mice With Type 2 Diabetes Mellitus
Authors: Nguyen Tu , Pan Kaichao , Knapp Maura , Zheng Mei , Sladojevic Nikola , Ai Lizhuo , Sitikov Albert , Gonzalez Frank , Liao james K , Wu Rongxue
Published: November 2021

Background:
Endothelial dysfunction, especially at the microvasculature level, is one of the most deleterious events in diabetes. ARNT is a transcription factor that functions as a master regulator of glucose homeostasis, but its role in diabetic vascular complications is poorly understood.

Results and method:
We found a reduction in ARNT expression in microvascular endothelial cells (MVECs) derived from type 2 diabetic mice (db/db). Thus, we generated an inducible, EC-specific ARNT-knockout mutation (
Arnt
ΔEC, ERT2) to address the hypothesis that aberrations in ARNT expression might contribute to the vascular deficiencies associated with diabetes. We show here that loss of ARNT in the endothelium mimics diabetic phenotypes, such as impairs blood flow recovery after hindlimb ischemia, delays wound healing, and exacerbates infiltration of pro-inflammatory neutrophils after myocardial infarction. Interestedly, the degree of these impairments in the KO mice was more remarkable in diabetic animals induced with high-fat chow. In addition, the siRNA-mediated knockdown of ARNT activity reduced tube formation and cell viability measurements in HUVECs cultured under high-glucose conditions. The
Arnt
ΔEC, ERT2 mutation also reduced measures of cell viability while increasing the production of reactive oxygen species (ROS) in MVECs isolated from mouse skeletal muscle, and the viability of
Arnt
ΔEC, ERT2 MVECs under high-glucose concentrations increased when the cells were treated with a ROS inhibitor.

Conclusion:
Collectively, these observations suggest that declines in endothelial ARNT expression contribute to the suppressed angiogenic phenotype in diabetic mice and that the cytoprotective effect of ARNT expression in ECs is at least partially mediated by declines in ROS production. Endothelial ARNT might be a critical mediator of endothelial function and could serve as a therapeutic target for diabetic complications.
Abstract MP262: Endogenous Smad7 Restrains Myofibroblast Activation And Protects From Post-infarction Heart Failure By Suppressing Tgf-beta Signaling And By Directly Inhibiting Erbb2
Authors: Humeres Claudio , Shinde Arti V , Hanna Anis , Alex Linda , Conway Simon , Frangogiannis Nikolaos G
Published: November 2021

Repair of the infarcted heart requires TGF-β/Smad3 signaling in cardiac myofibroblasts and formation of an organized myofibroblast-populated scar. However, TGF-β-driven myofibroblast activation needs to be tightly regulated to prevent excessive fibrosis and adverse remodeling that may precipitate heart failure. We hypothesized that induction of endogenous suppressive signals, such as the inhibitory Smad7; may restrain infarct myofibroblast activation, protecting from adverse remodeling and fibrosis, and we examined the molecular mechanisms of Smad7 actions. In a mouse model of non-reperfused infarction, Smad3 activation triggered Smad7 synthesis in α-SMA+ infarct myofibroblasts, but not in α-SMA-negative fibroblasts. Mice with myofibroblast-specific Smad7 loss had increased heart failure-related mortality, worse dysfunction, and accentuated fibrosis in the infarct border zone and papillary muscles. In isolated cardiac fibroblasts, Smad7 overexpression attenuated myofibroblast conversion and reduced synthesis of structural and extracellular matrix proteins, whereas Smad7 knockdown promoted a matrix-synthetic phenotype. Smad7 actions on TGF-β cascades involved de-activation of Smad2/3 and non-Smad Erk/Akt pathways, without affecting TGF-β receptor activity. Unbiased transcriptomic analysis identified receptor tyrosine kinase (RTK) signaling as a major target of Smad7. Proteomic arrays demonstrated that the RTK Erbb2 is a target of Smad7 in cardiac fibroblasts. Western blots and co-immunoprecipitation assays showed that Smad7 interacts with Erbb2 in a TGF-independent manner and restrains Erbb1/Erbb2 activation, suppressing expression of fibrogenic proteases, integrins and CD44. In conclusion, Smad7 induction in infarct myofibroblasts serves as an endogenous TGF-β-induced negative feedback mechanism that inhibits post-infarction fibrosis by restraining Smad- dependent and Smad-independent TGF-β responses, and by suppressing TGF-independent fibrogenic actions of Erbb2. Protective effects of Smad7 in cardiac remodeling may have important therapeutic implications for heart failure patients.
Abstract P311: Blocking Succinate Release Enhances Mitochondrial Reactive Oxygen Species Generation And Worsens Ischemia-reperfusion Injury
Authors: Milliken Alexander S , Nadtochiy Sergiy M , Brookes Paul S
Published: November 2021

Succinate is a metabolite that plays a central role in ischemia-reperfusion (IR) injury,which is relevant to myocardial infarction (heart attack) and stroke. Succinateaccumulates during ischemia and is rapidly consumed at reperfusion driving reactiveoxygen species (ROS) generation at complex-I (Cx-I) and III of the mitochondrial electrontransport chain. This ROS production triggers cell-death, leading to tissue necrosis.Although succinate oxidation has been extensively studied and exploited as a noveltherapeutic target, only 1/3 of the succinate accumulated in ischemia is oxidized atreperfusion, with the remaining 2/3 being released from the cell via monocarboxylatetransporter 1 (MCT1). Extracellular succinate is thought to be pro-inflammatory, and ithas been proposed that preventing succinate release may be therapeutically beneficial.To determine the impact of preventing succinate release on IR injury, we comparedfunctional recovery (i.e. rate x pressure product, RPP) and infarction (i.e. tissue necrosis)of Langendorff perfused mouse hearts treated with an MCT1 inhibitor, AR-C155858,versus vehicle control. This revealed that succinate retention worsens IR injury (i.e.increased infarction and decreased functional recovery) likely due to increased ROS. Totest this hypothesis, we utilized a Langendorff apparatus positioned within aspectrofluorimeter, which permits real-time fluorescence measurements in beatingmouse hearts. Using the mitochondria targeted superoxide probe, MitoSOX red tomeasure ROS production at reperfusion + AR-C155858, demonstrated that succinateretention leads to enhanced mitochondrial ROS generation at the onset of reperfusion.Overall, these results suggest that inhibiting succinate release in the context of IR injurymay not be a viable therapeutic approach, regardless of any downstream anti-inflammatory effects.
Abstract P330: Growth Differentiation Factor 15 Causes Cardiac Cachexia In Heart Failure
Authors: Lee Da Young , Jiao Zhe , Antolic Andrew , Weiss Daiana , Weitzmann M. Neale , Burke Michael
Published: November 2021

Background:
Cachexia is wasting of normal body tissue and occurs in chronic medical diseases. It is a common complication of heart failure (HF) that is associated with very high mortality. Growth differentiation factor 15 (GDF15) regulates food intake and can cause cancer cachexia. GDF15 is a sensitive biomarker in humans, though its biologic function in HF is unknown. This study investigated the role of GDF15 in HF.

Methods:
We utilized a genetic mouse model of dilated cardiomyopathy (DCM) caused by a mutation in the phospholamban gene (PLN
R9C
). PLN
R9C
mice have dysregulated cardiac calcium handling (a common feature of nearly all forms of HF) and develop progressive DCM that leads to HF and premature death. Q-PCR and ELISA were performed to assess expression, tissue distribution and circulating levels of GDF15 in PLN
R9C
and age-matched wild type (WT) mice. A double transgenic mouse was created by crossing our DCM model with a constitutive
Gdf15
knock-out (KO). Using this novel model, we quantified food intake, and assessed fat and lean tissue mass by tissue weight at necropsy and by dual-energy X-ray absorptiometry (DXA). Cardiac function was assessed using echocardiography, and histochemistry performed to quantify cardiac fibrosis. Survival was assessed by Kaplan-Meier.

Results:
GDF15 mRNA (43-fold; p<0.01) and protein (54-fold; p<0.01) were increased in LV tissue, and circulating GDF15 was elevated (8.3-fold; p=0.03) in PLN
R9C
mice.
Gdf15
was expressed at low levels and was not increased in other organs in PLN
R9C
mice. PLN
R9C
mice developed cachexia (reduced fat and lean mass by tissue weight, reduced fat mass by DXA vs. WT; p<0.01 for all) and consumed less food (p<0.01 vs. WT).
Gdf15
KO in PLN
R9C
preserved fat and lean tissue mass and resulted in higher food intake (p≤0.01 for all).
Gdf15
KO had no effect on cardiac structure or function by echocardiography and PLN
R9C
/
Gdf15
KO mice displayed only a small reduction in cardiac fibrosis relative to PLN
R9C
mice (3%; p<0.01). Despite this,
Gdf15
KO prolonged survival in PLN
R9C
(29±3 vs. 25±3 weeks; p<0.01).

Conclusions:
GDF15 is a novel cardiac hormone produced in HF that triggers anorexia and cachexia in HF by an extra-cardiac mechanism.
Abstract P333: The Vascular Barrier: A Common Anti-arrhythmic Target In Atrial Fibrillation And Myocardial Infarction
Authors: Mezache Louisa , Nuovo Gerard , Veeraraghavan Rengasayee
Published: November 2021

Vascular leak is a major sequela of inflammation, which is associated with arrhythmic pathologies such as atrial fibrillation (AF) and myocardial infarction (MI). We recently demonstrated that the vascular leak-inducing cytokine vascular endothelial growth factor (VEGF; 90-580 pg/ml - levels found in AF patients) induces acute remodeling (30-60 minutes) of sodium channel (Na
V
1.5) -rich intercalated disk (ID) nanodomains, disrupting their ultrastructure and prompting translocation of Na
V
1.5 from these sites. This in turn disrupted impulse propagation and promoted arrhythmias in murine atria. Here, we tested the hypotheses that i) similar acute pro-arrhythmic remodeling occurs in the ventricles of MI patients, and ii) protecting the vascular barrier may prevent arrhythmias following an acute inflammatory insult. First, we examined myocardial samples from five human MI patients. VEGF was overexpressed in both cardiomyocytes and vascular endothelium in the border zone surrounding <6 month-old infarcts. Notably, co-localization analysis showed significantly reduced Na
V
1.5 near both connexin43 and N-cadherin within the border zone in 1-, 3-, and 9-day-old infarcts, paralleling our observations in mouse atria. Next, we returned to our murine model of AF induced by acute inflammatory insult (100 pg/ml VEGF for 60 minutes) to test the antiarrhythmic efficacy of protecting the vascular endothelial barrier. Overall, median
in vivo
arrhythmia burden was higher in VEGF-treated mice relative to vehicle controls (7.5±11 vs. 0±6 s/hr). We tested two strategies shown to prevent vascular barrier breakdown: Blocking connexin43 hemichannels (αCT11 peptide) decreased
in vivo
arrhythmia burden to 0 ± 6.07 s/hr. Panx1-IL2 (a peptide inhibitor of Panx1 channels) treatment decreased also
in vivo
arrhythmia burden (0 ± 15.57 s/hr with 1.6 μM Panx1-IL2). Similar antiarrhythmic efficacy was also achieved with small molecule inhibitors of Cx43 and Panx1. These results highlight VEGF-induced vascular leak as a novel mechanism for acute arrhythmias both in the early stage AF and following MI. Indeed, this mechanism may contribute to post-MI AF. Importantly, vascular-barrier protection may be a viable strategy to prevent these arrhythmias.
Abstract P350: Myofibril Orientation In Cardiac Muscle And Its Implication For Heart Disease
Authors: Ma Weikang , Gong Henry , Jani Vivek , Landim-Vieira Maicon , Papadaki Maria , Pinto Jose R , Aslam Mohammed I , Cammarato Anthony , Irving Thomas
Published: November 2021

Rationale:
Myocyte disarray is a hallmark of cardiomyopathy. However, the orientation of individual myofibrils and myofilaments and how their alignment may be altered in disease progression have been largely underexplored. This oversight has been predominantly due to a paucity of methods for objective and quantitative analysis.

Objective:
To introduce a novel, less-biased approach to quantify myofibrillar and myofilament orientation in cardiac muscle under near physiological conditions and demonstrate its superiority versus traditional histological assessments.

Methods and Results:
Using small-angle X-ray diffraction, we first investigated changes in myofibrillar orientation at increasing sarcomere lengths in skinned, relaxed, wildtype mouse myocardium by assessing the angular spread of the 1,0 equatorial reflection (angle σ). At a sarcomere length (SL) of 1.9 μm, the angle σ was 0.23±0.01 rad, decreased to 0.19±0.01 rad at a SL of 2.1 μm, and further decreased to 0.15±0.01 rad at a SL of 2.3 μm (p<0.0001). Angle σ was significantly larger in R403Q (a MYH7 HCM model) porcine myocardium (0.24±0.01 rad) compared to WT myocardium (0.14±0.005 rad, p<0.0001) as well as in biopsied human heart failure tissue (0.19±0.006 rad) when compared to non-failing samples (0.17±0.007 rad, p=0.01). These data indicate that diseased myocardium suffers from myofibrillar disorientation compared to healthy controls. Finally, using control samples, we showed that traditional, histological-based analysis of disarray can be subject to user bias and/or sampling error and lead to false positives.

Conclusions:
Our method for assessing myofibrillar orientation limits the artifacts introduced by traditional histological processing and provides a precise and objective metric for phenotypically characterizing myocardium. The ability to obtain excellent X-ray diffraction patterns from frozen, biopsied human myocardium opens up new avenues of inquiry regarding the relation of myofibrillar structure to function in health and disease.
Abstract P359: Electroconductive Scaffolds To Mature Induced Pluripotent Stem Cell-derived Cardiomyocytes For Cardiac Tissue Engineering
Authors: Cook Suh Hee T , Gluck Jessica
Published: November 2021

Heart disease is the leading cause of death worldwide. Cardiac tissue engineering (CTE) aims to repair and replace heart tissue, offering a solution. Induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) could revolutionize CTE due to their theoretical ability to supply limitless patient-specific CMs. However, iPSC-CMs are electrophysiologically immature compared to functional adult CMs, and therefore incapable of sustaining a heartbeat.

Thus, a scaffold capable of electrophysiologically maturing iPSC-CMs is needed. My research increases the electroconductivity of electrospun (ES) scaffolds by incorporating carbon nanotubes (CNTs), which I hypothesize will mature iPSC-CMs seeded onto them due to their excellent electroconductive properties.

Morphological, biocompatibility, and electrical analyses have been performed on ES polycaprolactone (PCL) and gelatin scaffolds with CNTs incorporated via a ‘sandwich’ and dual deposition method in order to increase electroconductivity. Morphological analyses were performed via ImageJ on SEM images. Fiber diameter and pore size quantification confirmed the ability to exert morphological control by modifying solution properties and ES parameters, which is crucial to achieve biomimicry of the cardiac extracellular matrix. Live/dead assays and immunofluorescence revealed the CNT scaffolds offer high biocompatibility for NIH 3T3 fibroblasts, which attach, proliferate, and migrate well. Electrical analysis performed with a multimeter and two-probe resistance measurement confirms that inclusion of CNTs significantly increases scaffold conductivity, moreso for dual deposition scaffolds than ‘sandwich’ ones, and moreso parallel to the CNT arrays than orthogonally.

These results prove the feasibility of using such scaffolds as a method for
in vitro
electrophysiological iPSC-CM maturation. Next steps include optimization of scaffolds, analysis of iPSC-CM biocompatibility and response, and recapitulation and manipulation of the electrophysiology of cardiac tissue, including quantification of markers for cardiac function and maturity, and assessment of iPSC-CM + scaffold response to electrical pacing.
Abstract P367: ‘Youthful’ Phenotype Of C-kit+ Cardiac Fibroblasts
Authors: Firouzi Fareheh , Echeagaray Oscar H , Esquer Carolina Y , Gude Natalie A , Sussman Mark A
Published: November 2021

Introduction:
Fibroblasts are critical contributors to myocardial development, tissue homeostasis and remodeling. Cardiac fibroblast population heterogeneity and plasticity present a challenge for categorization of biological and functional properties. Distinct molecular markers and associated signaling pathways provide valuable insight for cardiac fibroblast biology and interventional strategies to influence injury response and remodeling. Receptor tyrosine kinase c-Kit mediates cell survival, proliferation, migration, and is activated by pathological injury. However, the biological significance of c-Kit within cardiac fibroblast population has not been addressed.

Approach:
An inducible c-Kit reporter mouse detects promoter activation with Green Fluorescent Protein (GFP) expression in cardiac interstitial cells (CICs). Coincidence of GFP and c-Kit with the DDR2 fibroblast marker was confirmed at protein level using flow cytometry and immunohistochemistry. Subsequently, cardiac fibroblasts expressing DDR2 with or without c-Kit were isolated and characterized
in vitro
.

Results:
A subset of DDR2
+
cardiac fibroblasts also express c-Kit with coincidence in ~8% of total CICs. Pathological injury induces coincidence as well as expression of c-Kit and DDR2. Cultured cardiac DDR2+ fibroblasts that are c-Kit+ exhibit youthful morphological and functional phenotypes compared to c-Kit- cells including 1) significantly smaller size, 2) higher cellular motility, 3) enhanced proliferation, 4) less multinucleation, 5) decreased senescence-associated β-galactosidase staining, and 6) down-regulation of p53 senescence marker. Mechanistically, c-Kit expression correlates with signaling implicated in proliferation and cell migration including phospho-ERK and pro-Caspase 3.

Conclusion:
The phenotype of c-kit+ on DDR2+ cardiac fibroblasts correlates with multiple characteristics of ‘youthful’ cells. To our knowledge, this represents the first evaluation of c-Kit biology within DDR2+ cardiac fibroblast population and provides a fundamental basis for future studies to influence myocardial biology, cardiac remodeling, and response to pathological injury.
Abstract P370: The Role Of PJ-34 In The Protection Of Burn-induced Cardiomyopathy
Authors: Wen Jake J , Radhakrishnan Ravi , Mobli Keyan , Radhakrishnan Geetha L
Published: November 2021

Burn injury results in adverse myocardial remodeling and heart failure through circulatingcatecholamines and androgen and cytokine cascades. The DNA binding protein PARP1(poly ADP ribose polymerase 1) catalyzes a post translational modification to generatePARylation proteins,which changes the normal function of the modified proteins. Bothof PARP-1 and SIRT1 (sirtuin1) are NAD
+
dependent. In this study, we propose that PJ34 (PARP-1 inhibitor) would protect the function of burn-remodeled cardiomyocytes.Commercial rats were obtained and were subject to 60% total surface body area (TSBA)scald burns by immersing the abdomen and back in boiling water. They were immediatelytreated with the PJ34 post injury. Separately, the cardiomyocytes (Ac16) were exposedto burn-serum replaced culture medium with or without treatment of lentivirus-PARP1 KOand/or SIRT-PGC-1α agonists
in vitro
. The
in vivo
experiments showed that burn-ratsexhibited cardiac hypertrophy, fibrosis and an increase in the inflammatory markers IL-1β, IFN-γ and TNFα. Burned rats had an increased oxidative stress, concomitant withelevated PARP-1 activity and reduced Sirtuin-1 (SIRT1) expression. PJ34 treatment ledto increased SIRT1 and Peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) levels and attenuated oxidative stress, inflammation, and fibrosis.
In vitro
tests demonstrated that the treatments of PJ34 and SIRT1-PGC-1α axis agonists incardiomyocytes exposed to burn-serum replaced culture medium led to a significantreduction in mit ROS and mitochondrial dysfunction. In Conclusion, PARP1 depletion byPJ34
in vivo
and Letivirus-PARP1 KO Ac16
in vitro
attenuated cardiomyopathic featuresin burn-rats through the activation of SIRT1 and its downstream antioxidant defensemechanisms. The results of this study suggest a pivotal role of PARP-1 inhibition intreating burn-induced cardiomyopathy.

Keywords:
Burn injury; PJ34; PARP1; Cardiomyocyte; Lentivirus.
Abstract P372: Serum From Pediatric Dilated Cardiomyopathy Patients Promotes Dysregulation Of Cardiolipin Biosynthesis And Mitochondrial Dysfunction In Primary Cardiomyocytes
Authors: Pires da Silva Julie , Garcia Anastacia M , Miyano Carissa A , Sparagna Genevieve C , Jonscher Raleigh L , Elajaili Hanan B , Sucharov Carmen C
Published: November 2021

Pediatric dilated cardiomyopathy (DCM) is a devastating and poorly understood disease with most clinical treatment paradigms extrapolated from the adult population. Our studies have demonstrated that aspects of metabolism and mitochondrial function are dysregulated in pediatric DCM hearts. Cardiolipin (CL), a unique phospholipid in the inner mitochondrial membrane, is essential for optimal mitochondrial function and was shown to be dysregulated in both the failing adult and pediatric human heart. The objective of this study is to investigate if serum circulating factors from pediatric DCM patients can remodel CL resulting in mitochondrial dysfunction
in vitro
, similar to what is observed in the failing pediatric heart.

Using a novel
in vitro
model that consists of treating neonatal rat ventricular myocytes (NRVMs) with serum from pediatric DCM patients or from non-failing (NF) healthy controls, mitochondrial respiration was assessed using the Agilent Seahorse, and reactive oxygen species (ROS) was assessed using Electron Paramagnetic Resonance Spectroscopy. Relative mitochondrial DNA (mtDNA) copy number was determined by qPCR and expression of enzymes involved in CL biosynthesis and remodeling were analyzed using RT-qPCR. Mass-spectrometry was used to quantitate total and specific CL species and to investigate the metabolite composition of NRVMs treated with NF or DCM serum.

While mitochondrial ROS and mtDNA copy number were not significantly altered, we show that DCM serum decreases mitochondrial function, which is associated with alterations in CL content and composition and the downregulation of enzymes implicated in CL biosynthesis and remodeling. Analysis of metabolite content showed an alteration of pathways involved in fatty acid metabolism, mitochondrial biogenesis and regulation of β-oxidation by the transcription factor PPARα.

In conclusion, pediatric DCM serum circulating factors can promote CL remodeling resulting mitochondrial dysfunction in primary cardiomyocytes. These findings suggest that CL could be a novel therapeutic target for this particular population.
Abstract P373: The Occurrence Of Neurovascular And Adipose Neuropathy In The Genetically Diverse Het3 Mouse With Aging
Authors: Willows Jake , Robinson Morganne , Cyr Harrison , Mishra Gargi , Reifsnyder Peter , Harrison David E , Townsend Kristy
Published: November 2021

Energy homeostasis and adipose tissue metabolism are regulated in large part through peripheral sympathetic nerve innervation of metabolically important tissues and organs. This neural communication from the brain to adipose tissues results in release of the neurotransmitter norepinephrine that regulates energy expenditure through modulation of lipolysis, adipogenesis, ‘browning’ (development of brown adipocytes in white adipose depots), and non-shivering thermogenesis. Subcutaneous white adipose tissue (scWAT) is an energy storing tissue that is highly plastic, responding to metabolic need by changing mass and cellularity, as well as responding to challenges (including cold temperature, exercise, fasting) by modifying neural activity and metabolism. Within scWAT lies a dense bed of nerves and blood vessels that are integrated closely, and in large part, rely on one another to function properly. Even if not directly innervating the blood vessels themselves (as is the case with capillaries), neurites that appear to innervate single adipocytes use these blood vessels as scaffolding to traverse the tissue. We have recently demonstrated that under pathological conditions (obesity and aging), scWAT innervation decreases through a process termed ‘adipose neuropathy’. With advanced age the small fiber peripheral nerve endings in adipose die back, including reducing contact with adipose-resident blood vessels (as observed previously in the C57BL6/J mouse). This likely poses a physiological challenge for metabolism and for vascular or adipose tissue health and function. For this work, we compared C57BL6/J mice with the more genetically diverse HET3 mouse model, established for the NIA’s Intervention Testing Program to more accurately represent the variability of age-related mortality/morbidity. We investigated incidence of peripheral neuropathy with aging (skin, scWAT muscle) as well as changes to the neurovascular supply of scWAT across several ages in both males and females. We also investigated the anti-aging drug Rapamycin as a potential means to prevent or reduce adipose neuropathy. We found that HET3 mice display a reduced neuropathy phenotype compared to inbred C56BL6/J mice. Importantly, the nerve die-back around blood vessels was not observed in the HET3 model. However, male HET3 mice did reveal neuropathic phenotypes by 62wks of age, characterized by decreased mechanoreception in hind paw skin, reduced NMJ occupation, and decreased expression of the Schwann cell marker
Sox10
in scWAT. Female HET3 mice appeared to have increased protection from neuropathy until advanced age (126wks) when they began to show stronger phenotypes than males (excluding
Sox10
analysis.) Despite its success as a longevity treatment in mice, rapamycin had little to no effect on reducing or preventing the onset of adipose neuropathy.
Abstract P374: Functional Impairment Of Cardiac Endothelial Cells In Diabetic Failing Heart Via Dysregulated Exosomal Mirnas
Authors: Ranjan Prabhat , Goswami Sumanta K , Krishnamurthy Prasanna , Qin Gangjian , Verma Suresh
Published: November 2021

Introduction:
Cardiac endothelial cells (ECs) and fibroblasts (FBs) together maintain cardiac homeostasis. Their functional impairment aggravates complications in the heart. In diabetes, acute inflammation leads to cardiac FBs activation, which predisposes the diabetic myocardium to severe fibrosis. Further, inflammation-related vascular dysfunction is a major end-organ complication in diabetics. However, it is not known, whether myofibroblast regulates ECs function in a diseased diabetic heart. Therefore, we hypothesized that “myofibroblast in diabetic heart secretes exosomes packed with antiangiogenic/profibrotic factors, which impede EC function and exaggerate pathological remodelling in pressure-overloaded (PO) myocardium.

Methods:
Exosomes were isolated from diabetic mice plasma and FB condition media by ultracentrifugation and characterized by nanosight & electron microscopy. We cultured mouse primary heart ECs in growth media and treated with exosomes derived from FBs (treated with 25mM glucose or 500nM Angiotensin II (AngII) or both) for 48 hr. Mannitol (25mM) served as control.

Results:
Ang II and glucose significantly activate FBs as shown by qPCR (fibronectin, collagenase1α1) and western blot (pSmad2, p-p38). Exosomes derived from diabetic Ang II treated FBs significantly impaired ECs function as shown by Matrigel tube formation and Boyden chamber migration assays. Interestingly, ECs markers (eNOS, VEGF, CD31) genes and proteins expression were significantly inhibited in ECs treated with exosomes-derived from glucose and Ang II treated FBs. We, further, checked the effect of diabetic mice plasma exosomes on ECs function and found significantly impaired as shown by tube formation and migration data. Finally, microRNA (miR) array and qPCR analysis revealed that miR-216a-5p, miR-26a-5p and miR7a-5p were highly upregulated in exosomes derived from FBs co-treated with glucose and AngII.

Conclusions:
Taken together, this study demonstrates that glucose and AngII co-treated FBs-derived exosomes are enriched in pro-fibrotic factors and can lead to EC dysfunction and promotes cardiac fibrosis in PO myocardium. In future studies, we will modulate the target miRs in diabetic FBs to see whether it rescue reparative function of ECs and inhibits fibrosis in failing heart.
Abstract P377: Collagen Remodeling Of Spontaneously Hypertensive Rats Undergoing Quinapril Treatment Measured By Three Dimensional Shape Analysis
Authors: Wilson Alexander J , Hasaballa Abdallah I , Sands Gregory B , Nash Martyn P
Published: November 2021

Introduction:
Myocardial collagen structure is a complex three dimensional (3D) hierarchy, organizing cardiomyocytes and providing structural integrity. Changes in collagen organization have a crucial role in cardiac remodeling. However, quantitative methods for the analysis of collagen structures are still lacking. Herein, we apply our collagen shape analysis framework to compare within-sheetlet collagen (both endomysial collagen surrounding myocytes and perimysial cords) against sheetlet-surface collagen (perimysial meshwork collagen deposited between sheetlets) in spontaneously hypertensive rats undergoing quinapril treatment.

Hypothesis:
The different functional properties of endomysial and perimysial meshwork collagen are linked to their structure and will have markedly different features quantifiable by 3D shape analysis.

Methods:
Rat myocardium was stained for collagen (picrosirius red), and the left ventricular free wall imaged using extended volume confocal microscopy (0.4 μm
3
voxel size). The 3D image volumes were segmented into within-sheetlet and sheetlet-surface regions. Shape analysis was performed using eigenanalysis of the inertia matrix to derive 3D morphological (elongation and flatness) parameters, characterizing the collagen shape in the two regions.

Results:
Shape analysis revealed marked differences between within-sheetlet and sheetlet-surface collagen organization. Long-term quinapril treatment resulted in notable differences in within-sheetlet collagen shape in rats at 14 months of age (mo) compared with 24 mo. At 14 mo, the within-sheetlet collagen is a mix of sheet-, ribbon-, and rod-like structures (elongation: 0.43, flatness: 0.05), but by 24 mo this has remodeled to predominately thin rod-like structures (elongation: 0.03, flatness: 0.35). The sheetlet-surface collagen consisted of thick sheet-like structures in both 14 and 24 mo rats (elongation: 0.83 vs 0.79, flatness: 0.13 vs 0.09).

Conclusion:
Shape analysis in 3D showed marked differences between within-sheetlet and sheetlet-surface collagen structure, as well as differences between early treatment and late treatment of diseased hearts.
Abstract P379: Inhibition Of O-glcnac Transferase Ogt Activates P38 Signaling In Neonatal Rat Cardiomyocytes
Authors: Papanicolaou Kyriakos , Zachara Natasha , Ashok Deepthi , Sidor Agnes , Foster D B , O'Rourke Brian
Published: November 2021

The mitogen activated protein kinase (MAPK) p38 is important in cardiac hypertrophic responses and p38 inhibition has been tested as a potential therapeutic approach to heart failure. p38 is tightly regulated by upstream kinases and phosphatases. While p38 inhibitors suppress cardiac hypertrophy in vitro and in animal models, the partial efficacy of p38 inhibitors in clinical trials for heart failure illustrates the need for a deeper understanding of p38-regulatory mechanisms.
O
-linked N-Acetylglucosamine (
O
-GlcNAc) on Ser/Thr residues is a ubiquitous intracellular modification (
O
-GlcNAcylation) that participates in intracellular signaling, often occurring in counterpoint to phosphorylation.
O
-GlcNAcylation is catalyzed by
O
-GlcNAc Transferase (OGT) and removed by
O
-GlcNAc-Ase (OGA). Given the crucial regulation of p38 activity by phosphorylation, we hypothesized that
O
-GlcNAcylation regulates p38 phosphorylation during basal and hypertrophic cardiomyocyte signaling. Treating neonatal rat ventricular myocytes (NRVM) with OSMI-1 (inhibitor of OGT) significantly decreased
O
-GlcNAcylation (0.48 ± 0.02,
P
<0.001 vs. vehicle), whereas treatment with Thiamet-G (inhibitor of OGA) significantly increased
O
-GlcNAcylation (3.0-fold increase ± 0.35,
P
<0.05 vs. vehicle). OSMI1 treatment induced the phosphorylation of p38 at its activation site (3.9-fold increase ± 0.46,
P
<0.001 vs. vehicle) and promoted the phosphorylation of the downstream target, heat shock protein Hsp27 (8-fold increase ± 1.3,
P
<0.0001 vs. vehicle) and transcription factor Creb (3.3-fold increase ± 0.12,
P
<0.001 vs. vehicle). OSMI-1 had an additive effect in inducing p38 and Creb phosphorylation following hypertrophic stimulation by phenylephrine (3.1-fold and 1.4-fold increase vs. phenylephrine respectively,
P
<0.05). Treatment with the p38 inhibitor SB202190 abolished the phosphorylation of Hsp27 and Creb that was induced by OSMI-1. Canonical upstream activators of p38 include the MAP3Ks, TAK1 and ASK1. However, we found that treatment with ASK1 or TAK1 inhibitors (GS-444217 and Takinib, respectively) either alone, or in combination, did not negate the phosphorylation of p38 by OSMI-1. We conclude that regulation of p38 by OGT activity could occur at a level downstream of canonical MAP3Ks or through non-canonical pathways.