bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 340: Myosin Regulatory Light Chain: A Major Player in Defining the ‘OFF’ State of Cardiac Myosin
Authors: Sa Na , Tomasic Ivan , Gollapudi Sampath , Nag Suman
Published: October 2019

Myosin regulatory light chain (RLC) is a regulatory subunit of the myosin molecule, which plays a major role in stabilizing the Interacting Head Motif (IHM) of myosin in non-striated muscle. However, in striated muscle, its purpose is less well defined, and it is only in the past decade that researchers have started unraveling the function of RLC and its phosphorylation in muscle contraction. In this study, we report the role of human cardiac RLC in forming the Super-Relaxed State (SRX) of myosin in reconstituted full-length cardiac myosin thick filaments and show that removal of RLC destabilizes the ability of myosin to form the SRX state. Phosphorylation of the RLC with MLCK has the same effect, which renders the myosin to the ‘OFF’ state as confirmed by its ATPase activity. Interestingly we found that Ca
2+
and not Mg
2+
binding to synthetic myosin filaments had differential effects on the population of the myosin SRX state. At systolic Ca
2+
myosin exhibits lower SRX state than at diastolic Ca
2+
. This effect is obliterated in RLC-lacking myosin constructs such as sS1, or RLC stripped myosin filaments suggesting that Ca
2+
binding to RLC can activate the myosin from the ‘OFF’ state to a more active state. Naturally occurring cardiomyopathy-causing RLC mutants R58Q, K104E and D94A did not alter this property, but two of them, K104E and D94A abolished the phosphorylation regulation of the SRX state of myosin. Altogether, these observations demonstrate that either RLC phosphorylation or binding of systolic Ca
2+
can reduce the number of accessible myosin heads for contraction and could form the basis of the Ca
2+
-mediated activation of the sarcomeric thick filament and mutations in RLC can alter these abilities differentially.
Abstract 352: Release of 12,13-diHOME From Brown Adipose Tissue Modulates Inotropy and Lusitropy in Old Mice
Authors: Shettigar Vikram , Abay Eaman , Pinckard Kelsey , Baer Lisa A , Stanford Kristin I , Ziolo Mark T
Published: October 2019

The US demographic is shifting to an elderly population. It is estimated that by 2030, 20% of the population will be comprised of people over 65 years of age. The incidences of cardiovascular and metabolic diseases are increased with aging. A contributor to the age-induced metabolic dysfunction is a decrease in Brown Adipose Tissue (BAT). In addition to being a thermogenic organ, BAT is also an endocrine organ via the release of signaling lipids known as batokines. We have previously shown that there is a significant reduction in the batokine 12,13-diHOME contributing to the metabolic dysfunction with aging. However, the specific role of 12,13-diHOME in the age-induced impairments in cardiac function remains unknown. Thus, we hypothesized that supplementing 12, 13-diHOME would improve cardiac metabolism and function. In this study, we injected 12, 13-diHOME to young (3 months) and old (18months) C57Bl/6 wild type mice either acutely or long term (via an intraperitoneal injection daily for 2 weeks). Cardiac function was measured via echocardiography and hemodynamics via intra-left ventricular catheterization. In young mice, acutely supplementing 12, 13-diHOME greatly enhanced cardiovascular function and hemodynamics (e.g., cardiac output, systolic function, diastolic function, contractility, and relaxation). Similar effects were also observed in old mice that were acutely supplemented with 12,13-diHOME. These data suggest that 12,13-diHOME is a potent inotropic and lusitropic modulator in young and old mice. However, long term (2 weeks) treatment of 12, 13-diHOME on either young or old mice had no chronic effects on cardiac function and/or structure, unlike other inotropic agents. Taken together, these data demonstrate that 12,13-diHOME is a potent regulator of heart function and may be a viable long-term treatment strategy.
Abstract 353: Distinct cGMP Compartmentalization by Membrane Guanylate Cyclases
Authors: Subramanian Hariharan , Froese Alexander , Nikolaev Viacheslav
Published: October 2019

Introduction:
In adult cardiomyocytes (CM), cGMP produced by two membrane guanylate cyclases (GC-A and GC-B) play distinct roles. Using Förster Resonance Energy Transfer (FRET) and Scanning Ion Conductance Microscopy (SICM)-based approaches, cGMP compartmentation by GCs and phosphodiesterases (PDEs) were assessed.

Methods:
cGMP was measured in CM isolated from transgenic mice expressing membrane targeted cGMP sensor (red cGES-DE5). SICM was used to define the t-tubules across sarcolemma and its nano-pipette filled with natriuretic peptides (CNP and ANP) was used to locally stimulate GC in t-tubules and crest.

Results:
CNP activating GC-B induced maximum cGMP increase, while ANP activating GC-A did not increase cGMP. Pre-inhibiting PDE2 before ANP stimulation massively increased cGMP production to functionally significant levels as measured by PLN phosphorylation in western blot. cGMP in unstimulated cells was regulated mostly by PDE3. Local ANP stimulation in t-tubules, but not in crest, induced cGMP increase and CNP induced cGMP increase in both t-tubule and crest. After methyl-β-cyclodextrin treatment, ANP induced cGMP in both t-tubules and crest suggesting a role for lipid rafts in trapping GC-A in t-tubules. In transfected HEK293 cells, Fluorescence Recovery After Photobleeching (FRAP) showed that membrane diffusion of YFP-tagged GC-A was decreased after methyl-β-cyclodextrin treatment.

Conclusions:
GC-A exclusively located in T-tubules produce cGMP locally, which is tightly regulated by PDE2, while GC-B is expressed across the sarcolemma and produces global cGMP.
Abstract 360: PKA is Needed for Acute Adaptation for Cardiac Stress
Authors: Yang Jining , Zhang Xiaoying , Ao Shen , Xia Xuewei , Yu Guo , Wu Xiaoyan , Xu Bing , Withanage Miyuraj , Zhou Min , Liu Jie , Wang Wei E , Zeng Erliang , Tian Ying , Zhu Weizhong , Houser Steven R , Xiang Yang K , Zeng Chunyu , Mi Mantian , Chen Xiongwen
Published: October 2019

In adult mammalian hearts, the ventricles are composed of mostly binucleated myocytes (BVMs) and some mononucleated myocytes (MVMs), which have different properties. However, if the gene expression profile and pathological responses of these two populations of myocytes are different have not reported. We found that these two populations of myocytes differentially expressed 250 genes. While many RNA synthesis and processing genes, and some antiapoptotic genes were highly expressed in MVMs, BVMs had genes related to self-destruction (autophagy and ubiquitination system genes) enriched. In general, expression of genes involved in carbonhydrate metabolism and cell proliferation was also more but the expression of genes involved in fatty acid metabolism was less in MVMs. The expression of some of these genes was confirmed in single-molecule pulldown (SiMPull) method in separated MVMs and BVMs. MVMs grew more in pressure overloaded hearts. MVMs were more resistant to apoptosis induced by β-adrenergic stimulation both
in vitro
and
in vivo
, which was due to cytosolic and mitochondrial Ca
2+
and ROS could be more easily increased in BVMs leading to more damage to mitochondria. Ca
2+
current, myocyte contraction and Ca
2+
transients in MVMs had less responses to β-adrenergic stimulation (isoproterenol, 1uM). Furthermore, MVMs gene expression upon β-adrenergic stimulation showed more adaptive responses while BVMs showed more proapoptotic gene (e.g., p53 and AIF), protein post-translational modification and muscle differentiation gene expression. This study suggests that MVMs and BVMs are distinct subpopulations of VMs with different gene expression profile and pathological responses. It underscores the importance of differential pathological responses of cells having different nuclear numbers .
Abstract 402: Adiponectin Receptor 3 is Associated With Endothelial Nitric Oxide Synthase Dysfunction and Predicts Insulin Resistance in South Asians
Authors: Chandy Mark , Sayed Nazish , Lau Edward , Liu Chun , Wei Tzu-Tang , Chen Ian Y , Thomas Dilip , Rhee June , Oh Brad , Pepic Lejla , Husain Mansoor , Quertermous Thomas , Nallamshetty Shriram , Wu Joseph
Published: October 2019

Type 2 diabetes (T2DM) is a global epidemic affecting over 400 million people and causes significant morbidity and mortality. T2DM has a strong association with cardiovascular disease (CVD), the number one cause of death globally with 17.5 million deaths per year. A precursor of T2DM, insulin resistance is central to the development of T2DM and is a risk factor for CVD. Insulin resistance is difficult to diagnose and individuals are often untreated prior to the onset of T2DM or CVD. South Asians are more likely to have insulin resistance, diabetes and cardiovascular disease when compared to age matched European cohorts. The molecular mechanisms of why South Asians are predisposed to insulin resistance and consequently cardiovascular disease were investigated using induced pluripotent stem cells (iPSCs) derived endothelial cells (iPSC-EC). Endothelial cells line blood vessels of the cardiovascular system. Unlike previous models, iPSC-EC are unique because they contain the individual's genetic information and the environmental influences retained in epigenetic marks are removed via reprogramming and differentiation. iPSC-ECs from insulin resistant South Asians show evidence of impaired insulin signaling as evidenced by decreased Akt phosphorylation, and paradoxically overexpression of eNOS and adiponectin receptor 3. In the cellular milieu of prediabetes, insulin resistance iPSC-ECs show impaired tubule formation and nitric oxide release. When adiponectin receptor 3 expression is suppressed using siRNA, eNOS expression decreases and expression of components of the insulin signaling cascade are improved to levels observed in control iPSC-ECs. Multiple linear regression modeling of clinical characteristics and gene and cellular phenotype was used to develop a scoring system that predicts a patient’s risk of developing insulin resistance and hence subsequently diabetes and cardiovascular complications. To our knowledge, this is the first iPSC-derived endothelial cell risk calculator with the potential to identify South Asian patients at risk for developing insulin resistance and cardiovascular disease before disease onset, which would allow for the early implementation of interventions that prevent morbidity and mortality.
Abstract 405: Functional Maturation of Human iPSC-Derived Cardiomyocytes by Prolonged 3D Culture in Engineered Cardiac Tissue Constructs
Authors: De Lange Willem J , Mitchell Stanford D , Kreitzer Caroline D , Farrell Emily T , Ge Ying , Ralphe J C
Published: October 2019

The advent of human induced pluripotent stem cell (hiPSC) technology has revolutionized the way we study inherited diseases, as it allows us to study the effect of mutations in a human context. This is particularly true in the heart were species-specific differences has dramatic effects on heart rate, and expression of protein isoforms involved in excitation-contraction coupling. A major drawback of using hiPSCs is, however that cardiomyocytes derived from hiPSC (hiPSC-CM) are relatively immature, limiting their utility as a tool to study adult onset cardiac diseases.

We postulated that culturing hiPSC-CM in a 3D environment in engineered cardiac tissue constructs (ECT) would promote hiPSC-CM maturation.

ECTs were generated from lactate-selected day 30 hiPSCs that were mixed with isogenic hiPSC derived cardiac fibroblasts in a 10:1 ratio. Cells were mixed with fibrinogen and thrombin and seeded into molds under vacuum in FlexCell dishes to form ECT. ECTs were harvested 14-21 or 42-52 days later and subjected to functional testing, transcriptional analysis and top-down mass-spectrometry of sarcomeric proteins to assess maturation.

Functionally, we found that prolonged culture of hiPSCs in ECT resulted in increased calcium transient amplitude (0.856 vs. 0.462; P < 0.01) and an acceleration of calcium kinetics (calcium release time 67.7 ms vs. 109.3 ms; P < 0.01 and calcium decay time 148.3 ms vs. 188.0 ms; P < 0.05). This acceleration of calcium kinetics with time in culture was also true for twitch force kinetics (time to peak twitch force 178.6 ms vs. 208.0 ms; P < 0.05). Furthermore, beta adrenergic stimulation had a much greater effect on twitch kinetics in older ECTs (reduction in contraction time of 26.5% vs. 7.1%; P < 0.01). Transcriptional analysis revealed an increase in expression level of the beta 1 adrenergic receptor in older ECTs, while top-down mass spectrometry showed increased expression and phosphorylation of cardiac troponin I (cTnI) and mono-phosphorylated cTnI, as well as decreased phosphorylation of alpha-tropomyocin, all markers of myocardial maturation.

Taken together, these data supports our hypothesis that prolonged culture of hiPSC-CM in ECT promotes maturation of the calcium handling system and the contractile apparatus.
Abstract 411: Surface Engineering Strategies to Study Diseases of Heart and Skeletal Muscle
Authors: Jain Aditi , Ravi Venkatraman , Hasan Jafar , Behera Manisha , Chatterjee Kaushik , Sundaresan Nagalingam Ravi
Published: October 2019

There is an increasing need to develop simplified
in vitro
platforms that mimic the tissue environment to understand cardiovascular and musculoskeletal diseases. Towards this objective, we first explored different surface engineering strategies for culturing cardiomyocytes, which could be used for investigating disease conditions like cardiac hypertrophy. Firstly, we investigated the possibility of using human hair derived keratin as a simple, efficient and cost-effective substrate for culturing cardiomyocytes. Cardiomyocytes grown on keratin expressed cardiac specific markers and displayed spontaneous contraction. We further evaluated the development of cardiomyocyte hypertrophy upon treatment with the agonist, phenylephrine. We observed the induction of hypertrophy at the transcriptional as well as signaling level. We also observed a marked increase in protein synthesis in these cells indicating the development of hypertrophy. Next, we employed microscale topography to confine cardiomyocytes along ridges which closely resembles mammalian heart. Cardiomyocytes grown on micro-ridges showed global alignment and elliptical nuclear morphology. Calcium currents traversed the cardiomyocytes in a directional manner and were also responsive to hypertrophic stimuli. Like cardiomyocytes, we also investigated the effect of aligned topography on primary myoblasts using nanofibers. These nanofibers retained the myotubes in culture for longer duration as compared to myotubes formed on flat surfaces. Recently, we have seen that once the myoblasts grown on flat surfaces become confluent they spontaneously differentiate to form myotubes even in the absence of differentiation cues. However, myoblasts grown on aligned fibers remain in their undifferentiated state and differentiate only upon induction with differentiation media. These results highlight the suitability of using keratin for cardiomyocyte culture and also emphasize the importance of topography in assessing cardiac and musculoskeletal function. We propose that studies which take into account the morphology of the cells offer greater potential towards clinical translation.
Abstract 433: Hydrogen Sulfide Protects the Heart Against Homocysteine-Induced Remodeling by Regulating Autophagy and Pyroptosis
Authors: Kar Sumit , Yadav Santosh K , Goyal Ravi , Lefer David J , Mishra Paras K
Published: October 2019

Hydrogen sulfide (H
2
S), a byproduct of homocysteine (Hcy) transsulfuration, protects against adverse cardiac remodeling in diabetic cardiomyopathy, hyperhomocysteinemia (HHcy), and other cardiomyopathies. However, the underlying molecular mechanisms are unclear. Pyroptosis is a caspase 1-mediated cell death mechanism that promotes remodeling. It is inhibited by autophagy, a lysosomal degradation process that maintains cellular homeostasis. We hypothesized that H
2
S supplementation inhibits Hcy-induced pyroptosis by increasing autophagy in the heart. We treated CBS
+/-
mice (a model of HHcy) and WT mice with SG1002 (slow-releasing H
2
S donor) or control diet for 14 weeks and performed deep sequencing of their hearts (LV tissue). Ingenuity Pathway Analysis revealed SG1002 downregulated cardiac hypertrophy genes, plausibly by altering upstream regulators involved in pyroptosis and autophagy (IL-1β and mTOR). Western blot results showed that SG1002 suppresses mTOR in CBS
+/-
hearts (WT: 2.1±0.83, CBS: 10±2.4, CBS+SG: 3.8±2.3, WT+SG: 0.96±0.39) to induce autophagy. SG1002 decreases IL-1β (WT: 0.61±0.08, CBS: 0.88±0.07, CBS+SG: 0.32±0.01, WT+SG: 0.38±0.03) and inhibits caspase-1 activity to suppress Hcy-induced pyroptosis. NF-kB p65, which activates pyroptosis, is also downregulated by SG1002. We conclude that H
2
S could alleviate cardiac remodeling by inducing autophagy and inhibiting NF-kB-mediated pyroptosis. This is the first report that H
2
S controls cross-talk of cardiac pyroptosis and autophagy.
Abstract 442: Caregiver Burden, Stress, Depression, and Quality of Life in Family Caregivers of Patients with Heart Failure
Authors: Ahn Jeong-ah
Published: October 2019

Background:
Heart failure (HF) is a chronic disease, which is considered an important strategy to support HF patients for lifetime self-management and monitoring of worsening signs and symptoms of patients at home. However, the necessity of management throughout the whole life demands a great deal of challenge and coping of HF patients as well as their family caregivers.

Purpose:
We aimed to explore the levels of caregiver burden, stress, depression, and quality of life (QOL) in family caregivers of patients with HF, and to identify the relationships among the variables.

Methods:
A descriptive and exploratory study design was used. After approval of institutional review board, 64 family caregivers of patients with HF were recruited from a cardiovascular outpatient clinic at a university-affiliated hospital in Korea, between September 2018 and January 2019. We assessed the caregiver burden using Montgomery’s scale, stress by heart rate variability measurement, depression using geriatric depression scale, and QOL using WHO-QOL scale.

Results:
Participants’ mean age was 52.0 years, and the average length of care was 7.5 years. Most of them were the patient’s wife or child (female), and half of them had their own health problems such as hypertension and back pain. The total mean score of caregiver burden, stress, depression, and QOL of family caregivers were 68.5 (out of 95), 39.8 (out of 100), 8.0 (out of 15), and 93.8 (out of 140), respectively. Their QOL showed negative correlations with caregiver burden and stress, and the levels of caregiver burden and stress increased with HF disease period.

Conclusion:
Healthcare providers should be aware of the importance of support for family caregivers of patients with HF. It is necessary to develop practical strategies to improve the QOL and to alleviate caregiver burden, stress and depression of family caregivers. And ultimately, it will enable them for positive participation in the comprehensive disease management with patients with HF.
Abstract 446: The Impact of the Menstrual Cycle on the Cardiovascular System
Authors: Chen Chih-Yu , Chang Chi-Wei , Wang Sheng-Hung , Xie Xiang-Yu , Wang Gin-Chung
Published: October 2019

The stage of the menstrual cycle is an exquisite regulation of female hormone and cardiovascular adaptation to prepare for pregnancy. However, there are still many unclear areas in the study of the influence of the menstrual cycle on cardiovascular function. Since the arterial pulse wave provides insightful information about the cardiovascular system, this study aimed to study cardiovascular adaptation in three phases of the menstrual cycle through non-invasive blood pressure and radial pulse wave measurements. In this observational study, we recruited 113 healthy women with regular menstrual cycles(25±35 days). All participants underwent blood pressure and radial pulse measurements in three stages of the menstrual cycle, including menses period, proliferative phase, and secretory phase. The Fourier series method was used to calculate the first ten harmonic amplitudes of the radial pulse wave(C1~C10). The results showed no significant systolic and diastolic changes in the three phases of menstruation. However, compared to the other two phases, women in menses period had lower C2 and higher C4, C6 and C7. (Figure 1) C4 and C7 are two indices that are inversely related to arterial stiffness and peripheral arterial impedance. Thus, during the menstrual cycle, female hormones may be involved in the regulation of vascular elasticity and peripheral vasodilatation, which in turn changed the radial pulse spectrum. In summary, the menstrual cycle has an impact on the radial pulse spectrum and could alter arterial stiffness to accommodate the physiological needs of the pre-pregnancy period.
Abstract 450: Adipose Tissue Browning Induced by Natriuretic Peptide Exerts Thermogenic Actions and Improves Insulin Resistance in an in vivo Model of Diet-Induced Obese Mice
Authors: Kimura Haruka , Nagoshi Tomohisa , Yoshii Akira , Tanaka Yoshiro , Oi Yuhei , Yoshimura Michihiro
Published: October 2019

Although natriuretic peptides (NPs) classically act on renal and cardiovascular systems, increasing evidence suggest that NPs also largely coordinate inter-organ metabolic cross-talk with adipose tissues. We recently reported that A-type NP (ANP) raises intracellular temperature in cultured adipocytes in a low-temperature sensitive manner. We herein investigated whether ANP exerts a significant impact on adipose tissues in vivo, using diet-induced obese mice. C57BL/6 mice were exposed to high-fat diet (HFD) or normal-fat diet (NFD) for 13 weeks, and treated with or without ANP infusion for 3 weeks (0.5 ug/kg/min via osmotic-pump). The ANP infusion significantly increased its serum concentration both in NFD and HFD group. Histological analyses revealed that HFD induced increased brown adipose tissue (BAT) cell size with accumulation of lipid droplets (whitening). ANP treatment significantly suppressed this whitening response (re-browning). Similarly, HFD induced enlarged inguinal white adipose tissue (WAT) lipid droplets, which was significantly reduced by ANP. This was associated with a substantial increase in uncoupling protein-1(UCP1) expression in WAT confirmed by immunohistochemical analysis (browning, n=3 each) and UCP1mRNA analysis (NFD+NP 2.7±0.4 fold vs NFD, P<0.01; HFD+NP 6.4±2.2 fold vs HFD, P<0.05, n=5 each). To determine the functional significance of browning effects of ANP, intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were performed and showed that ANP treatment substantially improved insulin resistance in HFD (p<0.05, n=9 each). Moreover, cold tolerance test (at 4°C for 4 hours) demonstrated that the ANP-treated mice were tolerant to cold exposure, which was more salient in HFD with ANP (Decrease in rectal temperature from baseline [°C]: NFD+ANP –2.4±0.6, HFD –2.5±0.4, HFD+ANP –2.1±0.4, versus NFD –9.2±2.2, P<0.01 each, n=9 each). In conclusion, ANP induces WAT browning and preserves BAT function in obese models, leading to in vivo thermogenesis as well as improvement in insulin resistance. These findings reveal the compensatory roles of natriuretic peptides against the core body temperature fall and insulin resistance in a state of heart failure.
Abstract 468: Sirt1 Ameliorates High Fat Diet-induced Diastolic Heart Failure
Authors: Oka Shinichi , Zhai Peiyong , Sadoshima Junichi
Published: October 2019

Approximately half of heart failure patients are diagnosed with diastolic heart failure, which is characterized by impaired relaxation. Cardiac fibrosis is observed in diastolic heart failure patients, which may promote diastolic dysfunction due to increased LV stiffness. Sirt1 is a protein deacetylase that regulates cardiac fibrosis. However, the role of Sirt1 in diastolic heart failure remains unknown. Since obesity is a significant risk factor for diastolic heart failure, we employed a high fat diet (HFD)-induced obese mouse model and cardiac-specific Sirt1 knockout (Sirt1cKO) and overexpression (Sirt1cTG) mice to define the role of Sirt1 in diastolic heart failure. Diastolic dysfunction induced by 1 and 3 months of HFD consumption was exacerbated in Sirt1cKO, evidenced by an increased end-diastolic pressure-volume relationship (EDPVR), an index of diastolic dysfunction obtained by pressure-volume loop analysis (EDPVR: wild-type mice (WT) Normal Diet (ND): 0.069; WT HFD (3 months): 0.11; Sirt1cKO ND: 0.063: Sirt1cKO HFD: 0.18*, p<0.05 vs WT HFD). Consistently, HFD-induced diastolic dysfunction is ameliorated in Sirt1cTG mice (EDPVR: WT ND: 0.062; WT HFD: 0.11; Sirt1cTG ND: 0.061: Sirt1cTG HFD: 0.056*, p<0.05 vs WT HFD). HFD-induced cardiac fibrosis was promoted in Sirt1cKO but ameliorated in Sirt1cTG mice. Moreover, exogenous expression of Sirt1 via adeno-associated virus vector ameliorated HFD-induced diastolic dysfunction and fibrosis (Figure). These results suggest that Sirt1 ameliorates diastolic heart failure, possibly through inhibition of cardiac fibrosis.
Abstract 470: Reactivation of Fetal MicroRNAs Contribute to Dilated Cardiomyopathy
Authors: Shrestha Shreesti , Shen Xiaopeng , Liang Rui , Wang Fan , McConnell Bradley K , Liu Yu
Published: October 2019

Background:
Dilated cardiomyopathy (DCM) is the most common cardiomyopathies that often leads to heart failure worldwide. MicroRNAs have emerged as an integral player in regulating diverse biological processes including those in the cardiovascular system. Previously, our lab has shown that the miR-424(322)/503 cluster is highly enriched during early cardiac fate determination and drives cardiomyocyte specification. However, the expression level of miR-424(322)/503 gradually decreases in the postnatal heart. Other studies have revealed the upregulation of miR-424(322)/503 in failing human hearts. Nevertheless, the association between miR-424(322)/503 and heart disease has not been studied yet.

Methods and Results:
In this study, we created transgenic mice (TG) with tetracycline-controlled cardiomyocyte-specific miR-424(322)/503 expression. The TG mice and control mice (8 weeks) were fed with doxycycline-containing chow for up to a month. During this period, the weight of the mice and cardiac function were monitored. The weight of TG mice dropped significantly while controls grew normally. Echocardiography analysis showed continuous deterioration of cardiac function starting from 2 weeks. Ejection fraction and fractional shortening (n=8) decreased to 46±1.9% and 22±1.09% at 2 weeks and 15±2.7% and 6±1.2% at 4 weeks, respectively. In addition, left ventricular internal diameter (LVID,d and LVID,s) increased from 3.64±0.098 and 2.5±0.085 to 4.2±0.2 and 3.9±0.18 respectively. Morphologically, we found the hearts of TG mice to be larger than WT mice. H and E staining revealed dilated ventricular chambers and thinning of ventricular walls in TG. Likewise, fibrosis was observed in TG hearts through Masson trichrome stain. In addition, both atrial natriuretic peptides and brain natriuretic peptides increased by >8 folds in TG hearts (n=5). The level of β-MHC was higher while α-MHC was lower in TG hearts vs control. These indicate that TG phenotype is consistent with DCM.

Conclusion:
Overall, this is the first study showing that upregulated miR-424(322)/503 in the heart is sufficient to lead to DCM. This study can potentially help establish miR-424(322)/503 as a novel therapeutic target to ameliorate heart failure progression.
Abstract 482: Modeling PKP2 Mutation Associated Arrhythmogenic Cardiomyopathy With CRISPR-edited iPSC-derived Cardiomyocytes in Engineered Cardiac Tissues
Authors: Zhang Kehan , Tomp Samuel , Ewoldt Jourdan K , Toepfer Christopher N , Chopra Anant , Seidman Christine E , Seidman Jonathan G , Chen Christopher S
Published: October 2019

Arrhythmogenic cardiomyopathy (ACM), also known as arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C), is a leading cause of sudden death among young adults. Over half of ACM cases are associated with inherited desmosome gene mutations, most commonly in the gene PKP2 which encodes plakophilin-2. One of the obstacles to better understanding ACM pathogenesis is the lack of appropriate models which encompass the early stages of disease development; this imposes significant constraints on the advancement of clinical therapies. The recent advent of human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) has enabled the development of models for studying human cardiac cell biology and pathology. In this work, we combine hiPSC-CMs, CRISPR/Cas9 genome editing, and engineered cardiac tissue platforms to develop human in vitro systems for investigating the molecular mechanisms of ACM pathogenesis.

Isogenic control and mutant cell lines of hiPSC-CMs harboring ACM-associated PKP2 mutations were generated using CRISPR/Cas9 technology. Specifically, the PKP2tv cell line has an early truncation in plakophilin-2 that mimics PKP2 c.235C>T found in multiple family lineages. The effects of the PKP2tv mutation on cardiac tissue contractility were characterized using a 3D cardiac micro-tissue (CMT) platform. CMTs composed of PKP2tv cardiomyocytes were shown to have significantly decreased contractile forces compared to the control, which recapitulates the reduced ventricular systolic function seen in ACM patients. This result demonstrated the feasibility of using the hiPSC-derived tissue-engineering model to recapitulate ACM disease phenotype and allow for future investigation into the disease mechanisms.
Abstract 488: Overexpression of the Prostaglandin E2 EP3 Receptor Reduces Cardiac Function in an Angiotensin II Model of Hypertension
Authors: Bryson Timothy D , Xu Jiang , Taube David , Peterson Edward , HARDING Pamela
Published: October 2019

Prostaglandin E2 (PGE2) EP receptor subtypes EP3 and EP4 are present in the heart and signal via decreased and increased cAMP production, respectively. Previously we reported that cardiomyocyte-specific EP4 KO mice develop a phenotype of dilated cardiomyopathy with reduced ejection fraction, that PGE2 decreases contractility via EP3 in vivo, in vitro and ex vivo; and that EP3 expression in the heart increases when mice are subjected to Angiotensin (Ang) II-hypertension (HTN). We therefore hypothesized that overexpression of EP3 in the heart would exacerbate Ang II-HTN. To test this hypothesis, we used 10-12 week old male EP3 transgenic (Tg) mice with EP3 overexpression in cardiomyocytes and their wild type (WT) controls infused with either vehicle or 1.4 mg/kg/d Ang II for 2 weeks. Echocardiography was performed on conscious mice at the end of the experiment and systolic blood pressure (SBP) was measured every 2 days by tail cuff.

Results:
Table 1. All data are reported as means ± SEM. N= 3-6 mice per group. Statistical significance: * and *** p < 0.05 and p < 0.005 vs WT + vehicle, ++ p < 0.01 vs WT + Ang II, §§§ p < 0.005 vs Tg + vehicle.

As shown in the table, cardiac function was unaffected in WT mice after Ang II infusion. Baseline cardiac function was reduced in EP3 Tg mice receiving vehicle and in contrast to WT mice, was further diminished after Ang II infusion. These differences were not due to altered systolic blood pressure. In conclusion, EP3 Tg mice present with reduced cardiac function at baseline and in support of our hypothesis, exhibit an enhanced response to Ang II. Antagonism of the EP3 receptor may be a novel treatment for heart failure associated with hypertension.
Abstract 495: Determinants Of Atrial Fibrillation Mechanisms Using Metabolomic Profiling
Authors: Sirish Padmini , Yang Jun , Ledford Hannah , Nader Carol , Ganaga Svetlana , Young J Nilas , Hammock Bruce D , Chiamvimonvat Nipavan
Published: October 2019

Introduction:
Atrial fibrillation (AF) represents one of the most common arrhythmias seen clinically, yet, current treatment paradigms have proven largely inadequate. One of the main contributors to the pathophysiology of AF is inflammation. Hence, reduction of inflammation associated atrial remodeling represents a novel therapeutic strategy for the treatment of AF. Oxylipins are derived from arachidonic acid, which is metabolized through three pathways including the cytochrome P450 (CYP450) pathway. The CYP450 products, epoxyeicosatrienoic acids (EETs), are major anti-inflammatory metabolites with several cardioprotective effects. The central hypothesis to be tested is that patients with AF will demonstrate an increase in oxylipin profiles towards inflammatory features compared to patients with normal sinus rhythm (NSR). We further hypothesize that lipid mediators may represent a new therapeutic target for AF.

Methods:
In the cross sectional study, human atrial appendage specimens and blood from informed and consented patients undergoing coronary artery bypass graft surgery were obtained in accordance with the approved UC Davis IRB protocol. Metabolomic profiling and inflammatory cytokines were assessed from the plasma of patients using LC-MS/MS. The underlying mechanisms of AF were determined using an integrated approach with molecular biology, flow cytometry, and electrophysiology.

Results:
Metabolomic profiling shows a significant decrease in the EETs/DHETs ratios (Fig 1A) and EpOMEs/DiHOMEs ratios (Fig 1B) in the cohort with AF compared to patients in NSR. There was a significant increase in inflammatory cytokine, chemokine levels and oxidative stress in patients with AF compared to patients in NSR. There was a significant increase in nuclear translocation of NF-κB (nNF-κB) from patients with AF compared to NSR using western blot analysis. Assessment of total IκB and phosphorylated-IκB (pIκB) levels showed a decrease in IκB level and an increase in the pIκB levels associated with the activation of NF-κB in AF compared to the NSR cohort.

Conclusions:
The study provides important insights into metabolomic profiles in patients with AF and reveals the prospect of EETs/DHETs being a novel therapeutic target in the treatment of AF.
Abstract 498: The Effects of Low-carbohydrate High-fat Diet on Vascular Remodeling
Authors: Yanagisawa Haruka , Toko Haruhiro , Harada Mutsuo , Guo Jiaxi , Bujo Satoshi , Ishizuka Masato , Komuro Issei
Published: October 2019

Background:
Low carbohydrate diet (LCD) has been reported to reduce body weight and cardiovascular risk factors, including diabetic mellitus and dyslipidemia. Therefore, LCD was once expected to prevent cardiovascular events. However, recent meta-analysis reported that LCD was related to an increase in cardiovascular events. Moreover, other cohort studies reported that higher all-cause and cardiovascular mortality were observed only in LCD with animal derived-fat (LCD-A), not in LCD with plant derived-fat (LCD-P). Precise mechanisms of these effects have not been clarified, so we explored the potential roles of LCD-A and LCD-P in blood vessel remodeling by using two types of vascular disease model mice, atherosclerotic model and vascular injury model.

Method:
We generated two types of LCDs, beef tallow-based LCD (LCD-A) and cacao butter-based LCD (LCD-P). Six-week-old male ApoE Knockout (ApoE KO) mice (atherosclerosis model mice) and wild-type mice were subjected to LCD-A, LCD-P or normal chow for 14 weeks, and %plaque area of aortic root and serum lipid profile were measured. For vascular injury model, mice were inserted with a wire in a femoral artery of male 10-week-old wild-type mice and subjected to the three-types chow. After 4 weeks, femoral arteries were excised and embedded in paraffin and assessed with the extent of vascular injury severity. The femoral artery without injury on the other side was served as control.

Results:
In ApoE KO mice, serum levels of total cholesterol (TC) and triglyceride (TG) were decreased in LCD group (TC: p<0.01, TG: p<0.05 vs normal chow), but there was no significant difference between LCD-A to LCD-P. In wild-type mice, serum TC level was increased in LCD-A group (p<0.0001 vs normal chow). LCD had no effect on %plaque area of aortic root in ApoE KO mice. In vascular injury model mice, there was no significant difference in the extent of neointimal hyperplasia induced by wire-injury among groups.

Conclusion:
Our preliminary data showed that LCD had no effect on vascular remodeling despite the presence of significant alteration in lipid profiles. Additional experiments are needed to confirm the results and clarify the precise mechanisms.
Abstract 546: Exploring MCUb Function in the Heart and Its Role in Ischemia Reperfusion Injury
Authors: Huo Jiuzhou , Grimes Kelly , Sargent Michelle , York Allen , Kwong Jennifer , Molkentin Jeff
Published: October 2019

Mitochondrial calcium alterations can promote oxidative metabolism to match increasing functional demands during stress stimulation. However, mitochondrial calcium overload-induced cell death contributes to the pathogenesis of several cardiac disorders including ischemia reperfusion injury. The mitochondrial calcium uniporter (MCU) complex is the only identified transporter that permits rapid calcium uptake into mitochondria. While the biological function of the MCU pore-forming subunit has been annotated, much less is known about the MCUb protein, a high similarity paralog that is part of the greater MCU complex. The goal of our study was to investigate the biological function of MCUb, its role in mitochondrial calcium uptake, and its contribution to the pathogenesis of ischemia reperfusion injury in the heart. To address these questions, we generated both MCUb overexpressing mice as well as MCUb null mice.

We observed that the cardiac-specific overexpression of MCUb inhibited mitochondrial calcium uptake, although basal mitochondrial calcium levels remained unchanged. Genetic ablation of MCUb, conversely, did not influence mitochondrial calcium uptake in the heart. MCUb null mice did not have differences in cardiac function by echocardiography, nor were tissue histological changes observed. This lack of an overt phenotype in MCUb null mice may be attributable to very low or even absent expression in the heart at baseline.

However, induction of MCUb protein was observed in the hearts of mice subjected to ischemia reperfusion injury. Thus, mice were challenged with one-hour ischemia followed by 24-hour reperfusion and the ischemic area/area at risk was analyzed. Deletion of MCUb did not change the initial infarct size of the heart. However, MCUb null mice showed decreased fractional shortening 4-weeks after the ischemic injury. Gravimetric analysis as well as histological examination further supported the conclusion that MCUb deletion exacerbated damage in the heart after ischemia reperfusion injury. We are currently investigating the underlying mechanisms of this greater functional deficit.
Abstract 562: Comprehensive Arteriovenous Metabolomics in the Human Heart
Authors: Murashige Danielle S , Jang Cholsoon , Neinast Michael D , Elrod John W , Kelly Daniel P , Rabinowitz Joshua D , Frankel David S , Arany Zoltan P
Published: October 2019

Introduction:
Despite broad interest in the role of metabolism in the pathogenesis of heart failure, a definitive account of fuel use by the beating human heart
in situ
is lacking. We therefore determined the uptake and release of over 270 metabolites by simultaneous sampling of arterial, coronary sinus, and femoral venous blood in 100 patients.

Methods:
We enrolled 100 patients (60% male, 12% diabetic, age = 63.6 +/- 13.5 years, BMI = 29.75 +/- 6.1, EF = 57.14 +/- 6.48) undergoing ablation of atrial fibrillation. We used liquid chromatography-mass spectrometry to quantify over 270 metabolites in plasma from coronary sinus, femoral vein, and arterial blood. We also measured O
2
and CO
2
concentration, as well as atrial pressure.

Results:
We detected uptake or release of 77 metabolites by the heart and 136 metabolites across the leg (p <0.05 after Benjamini-Hochberg correction). We determined the cardiac extraction of all major fuel sources, including 29 fatty acids, 11 amino acids, lactate, acetoacetate, and 3-hydroxybutyrate. Several patterns emerge: first, while the leg extracts glucose (FV/A = 0.95, q = 4.7x10-14) and releases lactate (FV/A = 1.11, q = 2.87x10-14), the heart extracts lactate (CS/A = 0.95, q = 3.77x10-9) and neither extracts nor secretes glucose. Second, while the leg releases nearly all fatty acid and amino acid species, the heart consumes most fatty acids and certain non-essential amino acids. Third, extraction of unsaturated acylcarnitines correlates with left atrial pressure (p<0.01). Finally, we find a significant positive correlation between myocardial O2 consumption and extraction of ketones (p < 0.01), but not other major cardiac fuels.

Conclusion:
We present the most comprehensive study to date of metabolite use and secretion by the human heart. We find the heart to be an avid consumer of fatty acids and ketone bodies, but less so of glucose and essential amino acids. Consumption of ketones increases with O2 consumption, suggesting that ketones may be a preferential fuel source under high work loads. Extraction of certain acylcarnitines increases with left atrial pressure, indicating metabolic reprogramming under high filling pressures. In summary, we provide the first comprehensive model of fuel use by the beating human heart in situ.
Abstract 566: Branched Chain Ketoacid Dehydrogenase Kinase Inhibition Alters Substrate Utilization and Gene Expression in Myocytes
Authors: Siddall Parker , Bollinger Eliza , Peloquin Matthew , Albuquerque Bina , Sun Zhongyuan , Xing Gang , Clasquin Michelle , Dumlao Darren , Piotrowski Mary , Delmore Jake , Pashos Evanthia , Hadjipanayis Angela , Cunio Teresa , Zou Chang , Zhang Bei B. , Miller Russell A. , Roth Flach Rachel J.
Published: October 2019

Circulating increases in branched chain amino acid (BCAA) levels have long been associated with type II diabetes and metabolic syndrome. Emerging data also suggest that impaired BCAA catabolism may play a role in heart failure progression. BCAA are catabolized via the branched chain ketoacid (BCKA) dehydrogenase enzyme complex (BCKDH). BCKD kinase (BCKDK) is a negative regulator of BCAA catabolism through its inhibitory phosphorylation of the BCKDHE1a subunit, and the phosphatase PPM1k dephosphorylates this same site to activate BCAA catabolism. Using an inhibitor of BCKDK (BT2), BCAA catabolism is increased in vivo. Here, we utilized metabolomics to evaluate the contribution of BCAA catabolism to substrate preference in heart and skeletal muscle. Surprisingly, BCKDK inhibition with BT2 had no effect on incorporation of glucose into TCA cycle intermediates in heart or skeletal muscle. Because others have recently shown that the primary site of BCAA catabolism is skeletal muscle, we knocked down BCKDK and PPM1k in human skeletal myocytes to further investigate how BCKDK loss or inhibition affects substrate utilization. Similar to our in vivo observations, knockdown of BCKDK and PPM1k had no effect on glucose and pyruvate utilization in a mitochondrial function assay. However, an increase in maximal respiration was observed after BCKDK knockdown when fatty acids were used. To evaluate the mechanisms underlying this increase we then performed RNAseq in these cells after BCKDK and PPM1K knockdown and observed changes in a number of genes that may explain these alterations in substrate utilization. Finally, we performed C13 BCAA metabolomics in human skeletal myocytes after BT2 treatment or knockdown of BCKDK and PPM1k. Using BT2, we observed a dose-responsive reduction in BCKA production from C13 BCAA by the muscle cells as expected; however, though BCKA production was increased after PPM1k was knocked down, we surprisingly did not observe a decrease in BCKA production after BCKDK knockdown. Collectively these data suggest that BCKDK inhibition may improve metabolism and cardiac function by altering substrate preference in skeletal myocytes.
Abstract 575: Loss of Function Variant in CYB5R3 Associates With Exacerbated Cardiac Hypertrophy in Mice
Authors: Carew Nolan T , Hahn Scott , Miller Megan P , Wood Kathrine , Thideau Patrick H , Kaufman Brett A , McNamara Dennis M , Straub Adam C
Published: October 2019

African Americans (AA) are 20 times more likely to be diagnosed with heart failure (HF) before the age of 50 and 2 times more likely to die from heart failure. Previous reports have shown AA with HF have diminished nitric oxide (NO) signaling, a pathway critical for cardiac contractility. NO signals, in part, via binding reduced heme iron (Fe
2+
) in soluble guanylyl cyclase (sGC) leading to cyclic guanosine monophosphate (cGMP) generation. Recently, our lab reported that cytochrome b5 reductase 3 (Cyb5R3) reduces oxidized sGC heme-iron from the oxidized (Fe
3+
) to the reduced (Fe
2+
) state, thereby sensitizing sGC to NO. However, the role of Cyb5R3 in the setting of HF remains elusive. It is known that a high frequency Cyb5R3 T117S single nucleotide polymorphism (23% minor allele frequency) exists and is enriched in individuals with African ancestry. To determine the impact of T117S in HF outcomes, we completed a retrospective study from AHEFT and GRACE trials. Our data show that Cyb5R3 T117S carriers have significantly accelerated time to death/transplant. Additionally, biobank HF samples from AA samples show an enrichment of Cyb5R3 T117S carriers from 0.23 to 0.4. To assess the impact of Cyb5R3 T117S on sGC/cGMP signaling in the heart, ventricular cGMP levels in AA with HF were examined. Pooled Cyb5R3 T117S carriers have significantly decreased cGMP relative to non-carriers. Next, we determined if this variant impacts sGC heme redox state. Using purified protein activity assays, we found that Cyb5R3 T117S results in a 60% loss-of-function and an inability to reduce oxidized sGC. Lastly, to test the
in vivo
impact of the Cyb5R3 T117S variant in heart failure, we generated a novel Cyb5R3 T117S mouse. Transverse aortic constriction (TAC) studies in Cyb5R3 T117S mice show significantly accelerates cardiac hypertrophy relative to wild-type TAC controls. Taken together, these data suggest Cyb5R3 T117S may be a disease modifying variant that augments hypertrophic signaling through an sGC-dependent mechanism.