bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 375: Defining a Unifying Mechanism for Select Cardiomyopathy-Linked Variants of Desmoplakin
Authors: Ackermann Maegen , Manring Heather , Ng Ronald , Albertelli Taylor , Jyothi Bobbili Prameela , Stevens Tyler , Jacoby Daniel , Janssen Paul , Kilic Ahmet , Wright Nathan , Wright Nathan , Qyang Yibing , Campbell Stuart
Published: February 2019

Arrhythmogenic cardiomyopathy (AC) is a disease that affects 1 in 2000 Americans every year and segregates with sudden cardiac death (SCD). AC is linked to genetic variants in desmosomal genes, however the molecular mechanisms underlying AC remain unclear. To probe the mechanisms of an AC-linked desmosomal mutation, we identified a candidate family with a history of SCD and an unknown variant (R451G) in the desmoplakin (DSP) gene. Statistically significant linkage between the R451G-variant and symptomatic family members (LOD score = 3.4) was established and heart autopsy tissue from a genotype-phenotype positive family member revealed pronounced fibrofatty scarring and a significant loss of DSP protein at the intercalated disc (ID). Notably, the R451G-variant sits in a pocket containing several clinical DSP variants and we show that endogenous DSP is cleaved via a calpain-dependent mechanism. Together this led us to hypothesize that mutant DSP is more susceptible to calpain cleavage, resulting in cellular haploinsufficiency. To define a unifying mechanism for AC-linked variants, we assessed the biomolecular properties of DSP variants. While these variants do not significantly perturb the global structure of DSP, there are significant changes to local stabilizing intramolecular interactions of select variants, which correlates with augmented calpain degradation due to increased exposure of an auto-inhibited calpain-cleavage site. Taken together we have identified a unifying mechanism for select AC-linked variants of DSP grounded in increased susceptibility to calpain-cleavage leading to decreased functional DSP. To test the hypothesis that DSP haploinsufficiency results in poor ID organization and reduced electrical conduction of the myocardium we created engineered heart tissues (EHTs) from induced pluripotent stem cells generated from a symptomatic R451G-positive family member. Surprisingly, we found that conduction velocities were unchanged however, mutant tissues had a significantly slower rate of contraction despite unchanged peak contractile force. This biomechanical deficiency in DSP haploinsuffient cells may indicate poor coupling between the sarcomere lattice and the ID as a fundamental disease mechanism in AC.
Abstract 385: Characterization of Cellular Infiltrate in Bioprosthetic Aortic and Mitral Valves
Authors: Bozso Sabin J , Basu Ratnadeep , Adams Benjamin , Oudit Gavin , Freed Darren H , Nagendran Jayan , Moon Michael C , Nagendran Jeevan
Published: February 2019

Background:
Bioprosthetic xenogeneic valves tend to deteriorate over time, termed structural valve deterioration (SVD), leading to valvular dysfunction. Several studies have hypothesized that SVD is a result of a chronic immune-mediated rejection process; however, a paucity of data exists demonstrating this conclusively.

Objectives:
Demonstrate that valves explanted for SVD contain a significantly greater burden of immune cellular infiltrate compared to normal human heart valves.

Methods:
Adult patients undergoing bioprosthetic aortic or mitral valve explantation secondary to a diagnosis of SVD were included in this study. A control arm included valves explanted from hearts without valvular disease considered for transplantation that were not utilized. Specimens underwent immunohistochemical staining using standard techniques utilizing the following primary monoclonal antibodies: anti-CD68, anti-CD20, and anti-CD3.

Results:
Eighteen valves explanted for SVD were analyzed along with 4 control valves. All valves were explanted from male patients with a mean age of 62-years. Overall, 12% (n=1) of patients had diabetes mellitus, 50% (n=4) had hypertension, 12% (n=1) had renal dysfunction and 38% (n=3) were on a lipid-lowering medication. Our results demonstrate that valves developing SVD have a cellular infiltrate containing significantly more immune cells than normal human valves. Specifically, there are significantly more CD3+ T-cells (p<0.05) and CD68+ macrophages (p<0.05) present in the cellular infiltrate of the SVD valves compared to the normal controls. Furthermore, while the cellular infiltrate of the SVD valves contains CD20+ B-cells, plasma cells, and neutrophils, none of these cells are present on the normal control valves (all p-values <0.05).

Conclusions:
Our data suggest a significantly greater immune cell infiltrate can be found in explanted bioprosthetic valves developing SVD than normal human valves, providing support for the chronic-immune mediated rejection hypothesis. This study will ultimately lead to a better understanding of the immune mechanisms responsible for SVD in bioprosthetic xenograft valves and will contribute to our goal of developing novel therapeutic techniques to mitigate development of SVD.
Abstract 386: Sex Differences in Vitamin D Alter Inflammation During Heart Disease
Authors: Bruno Katelyn A , Mathews Jessica , Douglass Erika , Hill Anneliese , Cooper Leslie , Hoyne Jonathan , Fairweather DeLisa
Published: February 2019

An estimated 1 billion people worldwide have deficient or insufficient levels of vitamin D (VitD). Considerable evidence indicates that VitD deficiency is associated with an increased risk of autoimmune and cardiovascular disease (CVD), yet it remains unclear whether low VitD is simply a biomarker or has a true pathologic role. In this study we examined how sex differences in VitD influences various CVDs. We found that patients with CVD had significantly lower VitD than healthy controls (no ICD9/10 codes). In myocarditis patients low VitD correlated to poor ejection fraction (EF) in women with myocarditis (p=0.02), but high VitD correlated with low EF in men (p=0.04). In VDR knockout mice we found that VDR decreased myocarditis in females (p=2E-8) but increased inflammation in males (p=0.03) with an increase in total immune cells (p=0.02), CD3, CD4 and CD8 T cells (p=0.04), and the inflammasome in females. VDR altered Treg (p=0.04), mast cells (p=0.04) and cytokine profiles in males. Additionally, sera VitD levels were significantly higher in WT male mice with myocarditis compared to females. Our findings in VDR mice indicate that VitD/VDR reduces myocarditis in females, but increases disease in males. These data suggest that VitD sufficiency may increase inflammation in men, while VitD deficiency may make disease worse in women. These findings suggest that Vitamin D may have a role in the pathogenesis of inflammatory and cardiovascular disease.
Abstract 388: Small GTPase Rnd3 Fine-tunes Inflammatory Response in Myocardial Infarction
Authors: Li Wenjiao , Luo Weijia , Andrade Kelsey , Chang Jiang
Published: February 2019

Introduction:
Myocardial infarction (MI) is a severe clinical condition caused by coronary artery thrombotic occlusion. According to the 2017 update from the American Heart Association, about 790,000 Americans have MI each year and 114,000 of those will die. Acute inflammation is developed post MI to clear necrotic myocardium and to guide the upcoming healing processes. A well-balanced and tightly-controlled inflammation, therefore, is critical for the post-MI cardiac recovery. We have previously demonstrated the essential roles of small GTPase Rnd3 in cardiac remodeling, calcium handling and angiogenesis. In the present study, we aimed to investigate Rnd3’s regulation of post-MI inflammation.

Methods and results:
Cardiac-specific Rnd3-deficient mice were subjected to MI operation and the hearts were dissected 3 days post MI. Rnd3-deficient hearts showed significantly enlarged infarct size and excessive inflammation than wild-type hearts. Importantly, we found that MI patients with lower Rnd3 expression levels exhibit a worse prognosis in cardiac function recovery. In the mechanistic study, hyperactivation of NF-kB signaling was observed in Rnd3-deficient cardiomyocytes and hearts post MI. Rnd3 blocks NF-kB nuclear translocation by disrupting the heterodimerization between p65 and p50. Finally, we generated transgenic mice with cardiac-specific overexpression of Rnd3. The mouse exhibited significantly preserved cardiac function and restrained inflammation post MI.

Conclusions:
This study identified Rnd3 as a fine-tuning factor mediating post-MI inflammation. Cardiac Rnd3 expression level positively correlates with MI patient prognosis. Thus, Rnd3 may serve as a potential therapeutic target for MI.
Abstract 395: Pioglitazone and Glucagon-like Peptide 1 Receptor Agonist Mitigate Adverse Postinfarction Left Ventricular Remodeling in Lean Mice When Administered After the Infarct Independent of Changes in Infarct Size
Authors: Andres Allen M , Germano Juliana , Huang Chengqun , Tucker Kyle C , Song Yang , Sin Jon , Gottlieb Roberta A , Mentzer Robert M
Published: February 2019

Pioglitazone (PIO) and GLP-1R receptor agonist (GLP1Ra) have been shown to be cardioprotective against ischemic cardiac injury. Much less is known about the effects of these agents on adverse post-MI LV remodeling. Based on our earlier findings that PIO and a GLP1Ra stimulate mitochondrial turnover, we evaluated the effects of these agents on post-MI remodeling. Lean mice underwent permanent coronary artery ligation (PCAL) to ensure that remodeling would be independent of changes in infarct size. Vehicle, PIO and GLP1Ra (Sigma) were administered i.p. 2h after the infarct and then every other day for a total of 6 doses. Echo and histology (Masson’s trichrome) were used to assess LV remodeling 30 days post-MI (late phase); immune cell infiltration was assessed 7 days after PCAL (early phase) via hematoxylin and eosin (H&E) staining. Both PIO and GLP1Ra were associated with significant improvements in ejection fraction and fractional shortening (A); however, at the dose used, neither drug restored function to that of the sham-operated mice (EF 60% and FS 30%, data not shown). GLP1Ra-treated mice exhibited a marked reduction in immune cell infiltration at 7d after PCAL (B), and fibrosis at 30d (C) compared to vehicle or PIO. While PIO had no effect on immune-cell infiltration and fibrosis, it was effective in attenuating post-MI remodeling. While sharing a common endpoint of attenuating adverse remodeling, the finding that PIO stimulates mitochondrial biogenesis and GLP1Ra induces mitophagy may help explain their disparate findings with respect to early phase remodeling.
Abstract 400: ERBB2 Inhibition Leads to Cardiac Dysfunction
Authors: Gupta Manveen K , Naga Prasad Sathyamangla V
Published: February 2019

Growth factor ERBB2 inhibition is a reliable treatment for patients diagnosed with breast cancer but is known to cause cardiotoxicity, resulting in dysfunction and dilation. In our studies, we have taken a unique approach with the premise that ERBB2 inhibitors like trastuzumab or pertuzumab binding to ERBB2 may lead to a conformation that could engage/activate non-canonical signals while inhibiting classical pathways. Such non-canonical activation of signals by inhibitor may be beneficial for treating cancer, while the same pathways could be detrimental in the cardiomyocytes. Our data is further suppored by the microRNA-7 Transgenic mice developed in our lab, which downregulates ERBB2 leading to cardiac dysfunction as seen by echocardiography. These studies will lay the foundation for developing strategies that could specifically block the deleterious effect of ERBB2 inhibitors on the heart. Moreover, administration of AG825 in mice for two weeks results in significant cardiac dysfunction and dilation compared to AG1875 or vehicle controls, conferring the idea that inhibition of ERBB2 and not EGFR that mediates deleterious remodeling. Furthermore, mitochondria morphology was also affected upon ERBB2 inhibition as seen in our microRNA-7 transgenic mice hearts as well as AG825 (ERBB2 inhibtor) treated mice in the electrom micrographs. We will also discuss the proteomics analysis in the ERBB2 inhibitor treated hearts, microRNA-7 Transgenic mice hearts, ERBB2 transgenic mice hearts as compared to the C57/BL6 wildtype hearts. Our studies therefore aims to target these non-canonical signals in the heart with an idea to insulate the heart from the deleterious effects of chemotherapy.
Abstract 405: Antioxidant Treatment Prevents Profibrotic Changes And Cardiac Dysfunction In Prenatal Alcohol Exposure
Authors: Ninh Van K , El Hajj Elia C , Gardner Jason D
Published: February 2019

Prenatal ethanol exposure is often associated with cardiac defects. Our studies focus on the role of cardiac fibroblasts in the developing heart, and how they are affected by prenatal ethanol exposure. There have been numerous studies demonstrating the protective effects of antioxidant treatment against alcohol-induced injury during cardiac development. Thus, we hypothesize that in utero ethanol (EtOH) exposure creates oxidative stress and activates neonatal cardiac fibroblasts, leading to a profibrotic phenotype and cardiac dysfunction. Further, we hypothesize that these effects are prevented by antioxidant treatment. We performed in utero EtOH administration to C57/BL6 mice on gestation days 6.75 and 7.5. Dams were intraperitoneally injected with 2.9 g EtOH/kg body weight to produce a blood EtOH content of ~0.26 g/dL each time. Controls were injected with vehicle saline. Randomly selected dams in both groups were treated with 100 gram/kg body weight of the antioxidant N-acetylcysteine (NAC) immediately after EtOH or vehicle administration. Left ventricular (LV) chamber dimension and function were assessed in sedated animals on neonatal day 5 using echocardiography. Eccentric index was calculated as a ratio of LV interior diameter and LV posterior wall thickness*2 at diastole. Eccentric index increased in EtOH-exposed animals (2.2±0.1 EtOH vs 1.8±0.1 saline, p<0.05). There were no changes in EtOH animals treated with NAC (1.9±0.1, NS). Additionally, ejection fraction decreased in the EtOH group (60±2% EtOH vs 68±2% saline). NAC treatment prevented this depression of systolic function in EtOH-exposed neonates (72±2%). Hearts were analyzed for expression of profibrotic proteins. In EtOH-exposed pups, collagen I decreased (0.02±.01 vs 0.04±.01 au), but collagen III expression increased (0.21±.02 vs 0.13±.01 au) compared to saline animals; the overall collagen I/III ratio significantly decreased (0.47±0.07 vs 1.0±0.17). When EtOH-exposed mice were treated with NAC, collagen I/III ratio did not change (0.83±0.11). Overall, our data demonstrate that in utero EtOH exposure produces a profibrotic phenotype in neonatal hearts and a decline in both diastolic and systolic function. These adverse effects were prevented by antioxidant treatment.
Abstract 407: Defective Branched Chain Amino Acid Catabolism Impairs Exercise Endurance
Authors: Abell Lauren E
Published: February 2019

The branched chain amino acids (BCAA), leucine, isoleucine and valine, are essential for mammals, and they play a positive role in exercise capacity, muscle development, and a lean body phenotype. BCAA supplementation is commonly paired with exercise in order to promote muscle growth, increase resistance to fatigue and reduce muscle soreness. Conversely, elevated serum BCAA is strongly and positively correlated with the development of insulin resistance, coronary heart disease, and type II diabetes, and is predictive of patient response to therapeutics and intervention outcomes. We have previously shown that defective BCAA catabolism in mice impaired glucose metabolism in the heart and increased susceptibility to stress-induced cardiac damage. In this study we sought to determine the effects of elevated BCAA levels on skeletal muscle performance and response to exercise training using mouse models with systemically elevated BCAA levels.

Here we assess the hypothesis that defective BCAA catabolism negatively impacts exercise capacity and endurance through deregulation of substrate utilization in the skeletal muscle using a mouse model with systemically elevated BCAA levels. Impairment of BCAA catabolism due to the deletion of mitochondrial-localized protein phosphatase 2C (PP2Cm), a key enzyme in activating BCAA catabolism, leads to elevated BCAA levels in mice. To study this, PP2Cm-knock out (KO) mice and their littermate controls were subjected to high-intensity and low-intensity exercise capacity tests via forced treadmill running. PP2Cm-KO mouse has a 20% reduction in exercise capacity. Reduced exercise capacity positively correlated with elevated serum BCAA levels. Additionally, KO animals supplemented with BCAAs demonstrated elevated serum succinate, alanine and glutamate levels, which are metabolic markers of physiological stress. We conclude that the inability to catabolize BCAAs has a negative effect on exercise capacity and endurance.
Abstract 414: Transcriptome Analysis by RNA Sequencing of Left Ventricular Tissue From Mice Fed a High-Fat Diet
Authors: Luckey Stephen W , Riordan Jesse , Olson Courtney , Nadeau Joseph
Published: February 2019

A high-fat diet induces obesity and is associated with heart disease characterized by cardiac hypertrophy, diastolic dysfunction, fibrosis, and mitochondrial dysfunction. However, the molecular mechanisms involved in the pathogenesis of cardiac disease are not known. Therefore, the objective of this study was to investigate the molecular changes in the heart associated with a high-fat diet at early time points in the progression of cardiac disease. To do this, male C57BL/6 mice were fed either a chow diet or a high-fat (58 kcal%), low-sucrose (HFLS) diet starting at 5 weeks of age for 14 days and 100 days. At each time point, hearts were extracted and morphometric measurements were recorded. HFLS diet had no effect on weight gain at 14 days but did significantly increase body weight by two-fold at 100 days. At 14 days, there was no significant difference in cardiac size as measured by heart weight-to-tibia length ratio (HW/TL) between mice on HFLS diet and chow diet. However, there was a 24% increase in HW/TL following 100 days on the HFLS diet. Total RNA was extracted from left ventricular tissue from each group and gene expression was determined by RNA-Seq transcriptome analysis. DAVID functional pathway analysis of the top 50 genes with differential expression between diets at 14 days and 100 days identified significant enrichment for mitochondrial proteins and lipid metabolism at both time points on the HFLS diet. Moreover, there was enrichment for hypoxia response genes following 100 days on the HFLS diet. Analysis of genes with differential expression between 14 days and 100 days on HFLS diet identified enrichment for actin binding proteins/Z disc, glutathione transferase activity, and oxidation/reduction activity. These findings demonstrate significant differences in gene regulation in the hearts of mice fed a high fat diet at early time points. It is possible that these early molecular changes play a role in the progression of cardiac disease.
Abstract 417: Targeting the NAD/NADH Ratio for Heart Failure Therapy
Authors: Walker Matthew A , Villet Outi , Tian Rong
Published: February 2019

Energy deficiency and mitochondrial dysfunction are strongly implicated in the development of heart failure (HF). Yet, the exact mechanisms remain poorly understood. Recently, a causal relationship between loss of cardiac NAD(H) homeostasis and mitochondrial dysfunction have been proposed in HF. A number of studies have demonstrated the benefit of raising NAD levels prior to or at the time of inducing heart failure, but it is unknown whether this strategy benefits hearts with established or pre-existing dysfunction. Therefore, in the present study we increased NAD level in wild-type (WT) mice
after
pathological hypertrophy and dysfunction were induced by transverse aortic constriction (TAC) surgery. WT mice were treated daily for six-weeks with nicotinamide riboside (NR) starting four-weeks after TAC. The NR treatments significantly elevated NAD level in the TAC hearts (3.1 fold, n=4) compared to vehicle treated mice, accompanied by an improvement in fractional shortening (FS) (sham vehicle 51%±2% vs TAC Vehicle 31%±3% vs TAC NR 43%±5%, n=4 each group). Most evidence thus far suggest the benefit of raising NAD level in the failing heart involves activation of sirtuin 3 (Sirt3), a NAD dependent deacylase. We hypothesize there are Sirt3 independent mechanisms that are activated by increasing NAD levels to benefit the failing heart. To test this hypothesis, we treated Sirt3 deficient (Sirt3-/-) mice with NR starting four-weeks after TAC surgery and extended the study to ten-weeks post TAC. NR treatments elevated cardiac NAD level (2.9-fold, n=4) compared to vehicle treated controls. This correlated with lowered global protein acetylation in the hearts and an improvement in FS (sham vehicle 50%±5% vs TAC vehicle 24%±6% vs TAC NR 41%±8%). We also looked at mitochondrial function in Sirt3-/- mice ten-weeks after TAC ± NR treatments and observed an increase in state 3 ADP-stimulated respiration in the NR treated Sirt3-/- mice (sham vehicle 183±17 vs TAC vehicle 103±23 vs TAC NR 127±21 nmol/min/mg). These findings suggest increasing NAD can improve cardiac function through Sirt3 independent mechanisms including one that enhances mitochondrial respiration. Further elucidation of these mechanisms could identify novel NAD sensitive therapeutic targets.
Abstract 418: Perm1 is a Novel Regulator of Mitochondrial Energetics in Cardiomyocytes
Authors: Warren Junco S , Oka Shin-ichi , Abdellatif Maha , Cawley Keiko , Sabry Amira , Stubben Christopher , Dalley Brian , Drakos Stavros , Sadoshima Junichi
Published: February 2019

Mitochondrial oxidative metabolism and energy transduction pathways are critical for cardiac function. The expression of genes important for mitochondrial energetics is under the control of members of the peroxisome proliferator-activated receptor coactivator 1 (PGC-1) family and the estrogen-related receptor (ERR) subfamily. Perturbations in PGC-1 and/or ERR activities have been associated with impaired mitochondrial function and the development of heart failure. The mechanisms by which PGC-1 and ERR proteins regulate muscle specific transcriptional programs is not fully understood.
P
GC-1 and
E
RR-induced
r
egulator in
m
uscle
1 (Perm1)
was recently identified in skeletal muscle as a muscle-specific key regulator of mitochondrial energetics. Although overexpression of Perm1 enhances oxidative capacity and fatigue resistance in mouse skeletal muscle, the underlying mechanisms remain to be elucidated. Perm1 is highly expressed in mouse and human hearts, and its expression level was reduced in pressure overload-induced heart failure in mice (49.9 ± 12.1% of sham, n=4, p<0.05) and in patients with heart failure (45.5 ± 12.1% of donors, n=5, p<0.05). Moreover, Perm1 is expressed in both the nucleus and the cytoplasm in primary cultured neonatal rat ventricular myocytes (NRVMs) and phenylephrine-induced cellular hypertrophy led to downregulation of Perm1 in both fractions (56.7 ± 1.7% of control). RNAseq followed by bioinformatic analysis revealed that siRNA-mediated Perm1 knockdown (KD) in NRVMs led to global downregulation of ERRα/PGC1α-target genes involved in oxidative phosphorylation, including the electron transport chain and TCA cycle (
Ndufs8; Ndufb8; Cox5a; Cox6a1; Coq2; Cox5a; Cox8a; Coq10b; Sdhb; Sdhd)
. Basal respiration and ATP production rates were significantly reduced in Perm1-KD H9c2 rat ventricular myocyte-derived cells (60.4 ± 4.2% and 35.2 ± 8.8% of control cells that were transfected with scrambled-siRNA, both p<0.05). This study demonstrates a novel role of Perm1 in cardiomyocytes that provides an additional layer of mitochondrial energetics regulation, presumably in an ERRα- and PGC1α-mediated manner, and suggests that downregulation of Perm1 may contribute to mitochondrial impairment during heart failure.
Abstract 419: Deep Sequencing Provides Evidence Of Mitochondrial Abnormalities In Right Ventricular Failure In Pulmonary Arterial Hypertension
Authors: Potus Francois , Hindmarch Charles C , Archer Stephen L
Published: February 2019

Introduction:
Right ventricular (RV) function is a critical predictor of both morbidity and mortality in pulmonary arterial hypertension (PAH). In PAH patients present either with adaptive remodelling RV and compensatory RV hypertrophy (RVH), or maladaptive remodeling that rapidly deteriorates to RV failure (RVF). While the molecular mechanisms underlying RVF remain poorly understood, acquired mitochondrial abnormalities including impaired mitochondrial electron transport chain activity, altered metabolic substrate utilization, and aberrant mitochondrial dynamics likely play a crucial role. We assessed the role of mitochondria in RVF using an unbiased assessment of gene expression in the RV of rats with maladaptive vs adaptive RVH and PAH.

Method and result:
Male rats were treated with either sugen+hypoxia (SuHx) or Monocrotaline (MCT). We determined RV status using non-invasive echocardiography and right heart catheterisation. Compared to control (Ctrl; n=15) both MCT (n=8) and SuHx (n=5) rats displayed severe PAH with increased RVSP, mPAP, total pulmonary resistance (TPR) and decreased PA acceleration time (PAAT) (p<0.05). MCT rats developed RVF characterized by increased RV end diastolic volume (EDV), decreased CO, TAPSE and ventricular-arterial decoupling (decreased Emax/Ea ratio) (p<0.05). SuHx rats had adaptive RVH. RNAsequencing revealed 2,547 differentially regulated transcripts. Functional Annotation analysis revealed multiple enriched GOterms, the most significant of which described mitochondrion (GO: 0005739; p=1.6E-53 which describes 414 genes). Significant GOterms also include mitochondrial inner membrane (GO: 0005743; p=3.9E-20) and mitochondrial respiratory chain complex I (GO: 0005747 ; p=4.9E-10). We validated 15 key dysregulated mitochondrial genes in maladaptive RVH (MCT; n=7) compared to both adaptive RVH (SuHx; n=4) and Ctrl (n=12) by qPCR, Finally, we validated these findings in human samples from PAH patients with RVH compared to controls (n=5 per group; p<0.05).

Conclusion:
We combined haemodynamic investigation and transcriptomics to identify specific mitochondrial-metabolic gene expression signatures associated with RVF in two different preclinical models and in human samples.
Abstract 420: RIP3 Interacts With Mitofilin in the Inner Membrane of Mitochondria to Induce Cardiomyocytes Necrosis After Ischemia/reperfusion
Authors: Feng Yansheng , Madungwe Ngonidzashe B. , Tombo Nathalie , Li Liu , Bopassa Jean Chrisostome
Published: February 2019

Introduction:
Receptor-Interacting Protein Kinase 3 (RIP3/RIPK3), a kinase promoting necroptotic cell death, has been shown to mediate adverse remodeling after myocardial infarction. We recently showed that knockdown of the inner membrane of mitochondria protein, mitofilin induces cell death by increasing mitochondria dysfunction via the AIF-PARP axis in rat H9c2 myoblasts. As we found that mitofilin interacts with the mitochondrial permeability transition pore (mPTP) regulator, cyclophilin D (CypD), we investigated whether RIP3 promotes cardiomyocyte death after I/R by increasing mitofilin loss, resulting in mitochondrial cristae structural damage and mPTP opening.

Methods:
C57BL/6J and RIP3 knockout mice hearts were subjected to 35 min ischemia followed by 180 min reperfusion. Myocardial infarct size was assessed by TTC staining. Mitochondrial function was assessed by mitochondrial ΔΨm, ROS production, and calcium retention capacity (CRC) required to induce mPTP opening. Mitochondrial integrity was measured by analyzing cristae morphology. RIP3 translocation was assessed by measuring the levels in the cytosol and mitochondrial fractions using Western blot and immunofluorescence labeling. The interaction between RIP3, CypD, and mitofilin was measured by co-immunoprecipitation.

Results:
We found that RIP3 KO mice developed less myocardial infarction as compared to WT and that RIP3 is translocated into mitochondria after I/R. In mitochondria, RIP3 interacts with mitofilin and CypD in the inner mitochondrial membrane (IMM). These interactions were associated with increased ROS production and decreased mitochondrial ΔΨm and CRC. In addition, we also found that RIP3 affects the expression of CypD and mitofilin.

Conclusion:
After I/R, RIP3 translocates into mitochondria to interact with CypD and mitofilin in the IMM resulting in increased mitochondrial ROS production and a decrease in ΔΨm, both of which promote the opening of the mPTP and destruction of mitochondrial cristae, leading to cardiomyocyte death. This finding points to the key role of RIP3 in cardiomyocyte death.
Abstract 436: Gender-based Differences in Preoperative Levels of Circulating MicroRNA in Patients Who Developed Postoperative Atrial Fibrillation
Authors: Rizvi Farhan , Olet Susan , Edwards Stacie , Emelyanova Larisa , Ross Gracious , Khanderia Bijoy K , Downey Francis X , Kress David C , Tajik A Jamil , Jahangir Arshad
Published: February 2019

Background:
Circulating microRNAs have recently been identified as biomarkers predicting A-Fib and heart failure. Yet, their role in identifying those at risk for postoperative atrial fibrillation (PoAF) in a gender-specific manner has not been studied.

Methods:
Preoperative blood from patients undergoing cardiac surgery with no prior history of AF, supraventricular/ventricular tachycardia was used for RNA isolation. Differences in the relative levels of miRNA between those who did/did not develop PoAF were assessed using qPCR. Principal component analysis and receiver operative characteristics were performed to predict miRNA associated with PoAF.

Results:
Out of 56 patients, 29 developed PoAF (52%, mean age 70.4± 9y, 51% males), while 27 (mean age 63y ± 10) remained free of AF. Patients who developed PoAF were older (P<0.01). There were no differences in the prevalence of hypertension, diabetes, dyslipidemia, prior myocardial infarction, heart failure, stroke, COPD, or cardiac medications. Out of 13 miRNAs analyzed, differential levels of miR423 (AUC83.7%, CL 72.4% - 93.3%), miR187 (AUC 76.8%, CL 63.4% - 90.2%) and miR34a2 (AUC 67.7%, CL53%-82.4%) were associated with PoAF. Gender-specific analysis showed a different set of circulating miRNAs (miR-187,-34a2,-25) in male vs. female (miR-29a1, -21,-126,-15b) patients with miR423 common in both as an independent predictor of PoAF.

Conclusion:
This indicates the existence of different substrate/precipitating factors reflected in miRNAs profile in male and female patients who developed PoAF. Gender differences exist in circulatory miRNA levels can help in predicting the development of new-onset AF after cardiac surgery.
Abstract 438: Familial Cardiomyopathy Mutations Affect Mechanosensing at the Molecular and Cellular Levels
Authors: Clippinger Sarah , Cloonan Paige , Greenberg Lina , Stump Tom , Greenberg Michael J
Published: February 2019

Familial cardiomyopathies are the leading cause of sudden cardiac death in young people, affecting more than 1 in 500 people. Two common forms, hypertrophic (HCM) and dilated (DCM) cardiomyopathies, are characterized by remodeling of the heart tissue, frequently accompanied by fibrosis and myocyte disarray. These diseases are primarily caused by mutations in sarcomeric proteins that regulate the contraction of the heart; however, it is not clear how these point mutations at the molecular level lead to the disease phenotype seen in patients. To better understand this process, we have examined the molecular and cellular consequences of two mutations in troponin T, R92Q and ΔK210, that cause HCM and DCM, respectively. Using recombinant proteins and a battery of in vitro biochemical and biophysical techniques, we find that R92Q increases activation of the thin filament by stabilizing myosin force generating states while ΔK210 reduces the ability of myosin to access these states at submaximal calcium levels. To study these mutations at the single cell level, we used CRISPR/Cas9 to generate human stem cell derived cardiomyocytes bearing these mutations. We examined the structural and contractile properties of single cells on engineered hydrogels that mimic either healthy or diseased hearts. Consistent with our molecular studies, we find that R92Q causes increased force production and power output. Interestingly, the ΔK210 mutation does not affect cellular force or power output on substrates that mimic the stiffness of the healthy heart, but it causes disordering of the sarcomeres on stiff substrates. This suggests a mechanism for ΔK210 where fibrosis leads to progressive loss of contractile function via mechanosensitive structural remodeling. Taken together, these results (1) demonstrate that alterations in force production and mechanosensing contribute to the disease pathogenesis in familial cardiomyopathies and (2) illuminate the differential mechanisms that lead to either HCM or DCM.
Abstract 443: α
1
-adrenergic Receptor Agonists Activate Akt Signalling via the Insulin Receptor Family of Receptors
Authors: Clerk Angela , Meijles Daniel N , Rostron Kerry A , Shaw Spencer , Cull Joshua J , Fuller Stephen J , Sugden Peter H
Published: February 2019

Introduction:
Akt signalling was delineated in the context of insulin stimulation and is cardioprotective. Akt is activated by insulin or insulin-like growth factor 1 (IGF1), acting through their receptors (IRs, IGF1Rs). Other receptors including α
1
-adrenergic receptors (α
1
ARs) activate Akt, but the mechanism is unclear.

Hypothesis:
We hypothesise that α
1
-ARs activate Akt via insulin receptor family receptors (InsRFs). Our aims were to establish if this is so, and if linsitinib [a highly selective inhibitor of InsRFs developed for cancer] affects cardiac responses to α
1
-AR agonists.

Methods:
Rat neonatal cardiomyocytes were exposed to or adult rat hearts perfused with α
1
AR agonists [50 nM A61603; 100 μM phenylephrine (PE)] with/without linsitinib. Effects on signalling were assessed by immunoblotting. Protein synthesis was assessed using
3
H-Phe. Effects of linsitinib (2 mg/kg/d) on cardiac function/hypertrophy induced by PE (40 mg/kg/d, 4 d) in mice (male C57Bl6; 10 wks) in vivo were determined. Echocardiography was used to assess cardiac function and pulmonary/aortic flow.

Results:
We identified a third InsRF in heart, the insulin receptor-related receptor (IRR). IRRs are alkali-sensitive and required for pH regulation in kidney. Alkaline pH (pH 9.0) activated IRRs and Akt, and increased protein synthesis in perfused hearts, being activated in a complex with IR/IGF1Rs. α
1
ARs activated Akt in cardiomyocytes and perfused hearts and increased protein synthesis. Linsitinib inhibited activation of Akt and the increase in protein synthesis induced by alkaline pH or α
1
AR agonists. Thus, α
1
ARs signal via InsRFs to Akt and protein synthesis. In perfused hearts, linsitinib enhanced the increase in developed pressure induced by A61603 and, in vivo, linsitinib enhanced PE-induced left ventricular (LV) hypertrophy with increased LV wall thickness and decreased internal diameter. Aortic flow was compromised in the presence of PE/linsitinib.

Conclusions:
Our data demonstrate a novel signalling paradigm in which α
1
-ARs signal through one or more InsRFs in cardiomyocytes to activate Akt and increase protein synthesis. However, functionally, the InsRF signal appears to mitigate some of the hypertrophic effects of α
1
-AR signalling in the whole heart.
Abstract 446: Transcription Factor EB, Not mTOR is a Therapeutic Target for Rag GTPase Protein C S75Y-based Cardiomyopathy
Authors: Kim Maengjo , Qi Qiu , Ma Xiao , Xu Xiaolei
Published: February 2019

Rationale:
We recently reported the association of S75Y, a point mutation in the Rag GTPase protein C (
RRAGC
) gene, with pediatric DCM. Because RRAGC directly binds and regulates mTOR and the S75Y mutation results in abnormally activated mTOR, these in vitro data prompts that the mTOR pathway could be harnessed for therapeutic benefits.

Methods:
We aimed to integrate
in vitro
neonatal rat ventricle cardiomyocytes (NRVCM) and
in vivo
zebrafish system to elucidate underling mechanism of
RRAGC
-S75Y-based cardiomyopathy, and to seek targeted therapeutic strategies.

Results:
Overexpression of RRAGC with S75Y mutation, but not RRAGC WT, significantly increased cell size of NRVCM, suggesting the establishment of a cellular model of
RRAGC
- S75Y-based cardiomyopathy. At the molecular level, overexpression of RRAGC S75Y mutant increased activity of mammalian target of rapamycin (mTOR) and decreased activity of transcription factor EB (tfeb), a direct downstream target of mTOR. Intriguingly, treatment with rapamycin, a mTOR inhibitor, did not affect cellular hypertrophy, whereas adenovirus-mediated overexpression of TFEB was able to rescue cellular hypertrophy induced by RRAGC S75Y mutation. Consistently, treatment with trehalose, an mTOR-independent tfeb activator, rescued cellular hypertrophy induced by
RRAGC
-S75Y. To confirm the
in vitro
results, we generated a knock-in (KI) zebrafish line harboring the S75Y corresponding point mutation in
rragc
via the TALEN (Transcription Activator-Like Effector Nucleases) technology. The cardiac function of
rragc
KI fish was determined by high frequency echocardiography, and the KI fish showed a significant decrease in fractional shortening compared with control littermates.

Conclusion:
Both cellular and
in vivo
models of
RRAGC
-S75Y-based cardiomyopathy have been generated. Studies in the cellular model suggested that tfeb, but not mTOR, is a therapeutic target for this particular type of cardiomyopathy. We will report our progress in validating this conclusion in our
in vivo
model, and will discuss potential mechanisms.
Abstract 448: P27
Kip1
Contributes to Eya4-mediated Development of Acquired Cardiac Hypertrophy
Authors: Williams Tatjana D , Arias-Loza Paula-Anahi , Nordbeck Peter , Ritter Oliver
Published: February 2019

Introduction:
We previously reported that E193, a heterozygous truncating mutation in the transcription cofactor “Eyes absent” (Eya4) leads to late-onset familial DCM and heart failure. Yet a precise role for Eya4 in the myocardium has not been identified. It appears to regulate normal cardiac function via p27
kip1
, a protein shown to be involved in regulating hypertrophic responses in the adult cardiomyocyte. This study was aimed to explore the role of Eya4 in angiotensin II (ATII)-induced cardiac hypertrophy.

Methods and results:
We used our transgenic mouse model with a constitutive myocardial overexpression of HA-tagged Eya4. Wildtype and Eya4 overexpressing mice were challenged with ATII via osmotic minipumps for four weeks to induce cardiac hypertrophy. Analysis of these animals using magnetic resonance imaging to visualize cardiac structures in detail showed that in response to the sustained ATII stimulation, the Eya4 overexpressing mice exhibited a phenotype with significantly increased parameters of hypertrophy. LV free wall diameter as measured in 7T MRI was 1,7±0,2 in Eya4 mice with ATII compared to 1,3±0,2 mm in WT mice with ATII. This was also confirmed by HW/BW ratio, hemodynamic measurements and cell size measurements. Histology also affirmed the results of the MR imaging. Moreover, Eya4 overexpression induced a significant suppression of p27 protein expression which is in agreement with our in vitro data. This confirmed our hypothesis that Eya4 negatively acts upon p27 expression which facilitates development of myocardial hypertrophy.

Conclusion:
In summary, we previously described a mutation in Eya4 to disturb cardiac physiology. We now provide evidence that Eya4 is also involved in forms of acquired heart disease. It seems to suppress p27
kip1
, thereby augmenting ATII induced cardiac hypertrophy.
Abstract 457: Ca2+/calmodulin Dependent Kinase II Exacerbates Heart Failure Progression Through Activation of Class I Histone Deacetylases
Authors: Zhang Manling , Luczak Elizabeth D , Granger Jonathan , Brown Joan H , Anderson Mark E , Feng Ning
Published: February 2019

Background:
Persistent cardiac Ca2+/calmodulin dependent Kinase II (CaMKII) activation in patients receiving standard heart failure therapies, appears to play an essential role in heart failure development. The molecular mechanisms of CaMKII induced heart failure progression remains incompletely understood and new mechanism based therapeutic strategies are warranted. Histone deacetylases (HDACs) are critical for transcriptional genes reprogramming leading to adverse ventricular remodeling. Class I HDACs, including HDAC 1, HDAC2 and HDAC3, promote pathological cardiac hypertrophy, whereas class IIa HDACs suppress cardiac hypertrophy. It is known that CaMKII deactivates class IIa HDACs to enhance cardiac hypertrophy, however, it is unknown whether CaMKII also regulates class I HDACs in heart failure development.

Results:
We found that CaMKII directly phosphorylates recombinant HDAC1 protein and increases its deacetylase activity by three-fold. HDAC1 activity is also increased in cardiac specific CaMKII over-expressing transgenic mice (CaMKII-TG). Mass spectrometry reveals HDAC1 is a CaMKII substrate. Beyond direct phosphorylation, CaMKII also increases HDAC1 expression. In cultured neonatal rat cardiac myocytes, overexpression of CaMKII markedly increases HDAC1 expression, and this CaMKII induced HDAC1 upregulation is mediated through JNK pathway, as JNK inhibitor SP600125 blunts this upregulation. HDAC1 expression is significantly increased in vivo, in CaMKII-TG mice. Consistently, the expression of HDAC1 is increased in failing hearts from wild type mice after myocardial infarction surgery, and this upregulation is attenuated in transgenic mice with myocardial expression of AC3-I, a CaMKII inhibitory peptide. Importantly, an HDAC1 specific inhibitor Quisinostat prevents or improves CaMKII overexpression induced cardiac hypertrophy and heart failure progression in vitro and in vivo. In addition, CaMKII also increases deacetylase activity and expression of other Class I HDACs, HDAC2 and HDAC3.

Conclusion:
CaMKII activates class I HDACs in heart failure, which may be a central mechanism for heart failure progression.
Abstract 460: Diabetes Impairs Reparative Property of Bone Marrow-derived Endothelial Progenitor Cells: Role of Mir-499-mediated Hydrogen Sulfide Deficiency
Authors: Cheng Zhongjian , Garikipati Venkata NS , Verma Suresh K , Trungcao May , Wang Chunlin , Cimini Maria , Goukassian David , Kishore Raj
Published: February 2019

Background:
miR-499 has been considered as a potential biomarker for myocardial infarction. Overexpression of miR-499 impaired heart function and induced cardiac hypertrophy in mice. Recent studies suggested miR-499 suppresses tumor growth through the inhibition of vascular endothelial growth factor (VEGF) mediated angiogenesis. Furthermore, emerging evidences indicate that miR-499 is increased in heart of diabetic rodents and patients. Here, we tested the hypotheses that miR-499 impairs angiogenic ability of endothelial cells (ECs) under diabetic conditions.

Methods and Results:
miR-499 levels were examined in endothelial progenitor cells (EPCs), microvascular endothelial cells (MVECs) and plasma in diabetic db/db mice (8-week, male). Gender- and age-matched non-diabetic db/+ mice served as controls. EPCs were isolated from bone marrow density-gradient centrifugation. MVECs were isolated using collagenase. We found that miR-499 levels were significantly increased in EPCs, MVECs as well as plasma of db/db mice. miR-499 antagomiR rescued high glucose (25 mM D-glucose for 48 h)-impaired tube formation and migration of human cardiac microvascular endothelial cells (HCMVECs). Mechanistically, by luciferase assay, we found that miR-499 directly targets cystathionine γ-lyase (CSE), a major hydrogen sulfide (H
2
S)-synthesizing enzyme in cardiovascular cells. Overexpression of miR-499 by transfection of miR-499 mimics decreased CSE protein levels and intracellular H
2
S production in HCMVECs.

Conclusions:
Our results suggest that miR-499-induced CSE downregulation/inactivation thus H
2
S reduction may play an important role in diabetes-impaired angiogenic capacity. Silencing miR-499 in diabetic EPCs/ECs may provide a novel tool for the treatment of ischemic tissue injury in patients with diabetes.
Abstract 463: Signature of Circular RNAs in Human Induced Pluripotent Stem Cells and Derived Cardiomyocytes
Authors: Lei Wei , Feng Tingting , Fang Xing , Yu You , Yang Junjie , Zhao Zhen-Ao , Liu Junwei , Shen Zhenya , Deng Wenbo , Hu Shijun
Published: February 2019

Background:
Circular RNAs (circRNAs) are regarded as a novel class of noncoding RNA regulators. Although a number of circRNAs have been identified by bioinformatics analysis of RNA-seq data, the tissue- and disease-specific circRNAs are still to be uncovered to promote their application in basic research and clinical practice. The purpose of this study was to explore the circRNA profiles in human induced pluripotent stem cells (hiPSCs) and hiPSC-derived cardiomyocytes (hiPSC-CMs), and identify cardiac or disease-specific circRNAs.

Methods:
Human iPSCs were generated from fibroblasts, and then further differentiated to hiPSC-CMs by modulating WNT signaling in RPMI+B27 medium. Following high-throughput RNA sequencing, circRNAs were extracted and quantified by a combined strategy known as CIRCexplorer. Integrative analysis was performed to illuminate the correlation between circRNAs and their parental linear isoforms. Cardiac and disease-specific expression of circRNAs was confirmed by quantitative reverse-transcription PCR.

Results:
In this study, a total of 5602 circRNAs were identified in hiPSCs and hiPSC-CMs. Our data indicated, for the first time, a more enriched expression of circRNAs in differentiated cardiomyocytes than undifferentiated hiPSCs. In addition to the host gene-dependent expression, our integrative analysis also identified a number of circRNAs showing host gene-independent expression in hiPSCs and hiPSC-CMs. CircRNAs including
circSLC8A1
,
circCACNA1D
,
circSPHKAP
and
circALPK2
showed a cardiac-selective expression during cardiac differentiation and human heart-specific enrichment in fetal tissues. Furthermore,
circSLC8A1
abnormally increased in heart tissues from patients suffering from dilated cardiomyopathy (DCM).

Conclusions:
CircRNAs are highly enriched in hiPSC-differentiated CMs, and cardiac-specific circRNAs such as
circSLC8A1
,
circCACNA1D
,
circSPHKAP
and
circALPK2
may serve as biomarkers of CMs. Detection of the excessive expression of
circSLC8A1
provides a potential approach for pathological status indication of heart disease.