bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 264: A Complement Pathway is Responsible for Degeneration of Right Ventricular Outflow Tract
Authors: Ito Shogo , Seki Tomohisa , Yuasa Shinsuke , Komuro Jin , Katsuki Toshiomi , Kimura Mai , Kishino Yoshikazu , Kusumoto Dai , Tohyama Shugo , Fukumoto Yoshihiro , Fukuda Keiichi
Published: February 2019

Backgrounds:
Right ventricular outflow tract (RVOT) has a unique role in the pathogenesis of inherited cardiac diseases such as Brugada syndrome and arrhythmogenic right ventricular cardiomyopathy. However, it remains elusive how RVOT plays a role in the pathogenesis. We hypothesized that RVOT would have different character from other parts of the heart and those phenotypes would be regulated by key factors.

Methods:
Microarray analysis of several parts of adult murine hearts was performed and differentially expressed genes (DEGs) in RVOT were extracted. Pathway analysis was performed using Ingenuity Pathway Analysis (IPA). Potential upstream candidate genes were examined using rat cardiomyocytes in vitro and adult mouse in vivo.

Results:
As a result of microarray analysis for RVOT, right ventricle, left ventricle and ventricular septum, 995 genes were statistically extracted as DEG in RVOT. IPA analysis revealed that an alternative complement pathway was significantly activated in RVOT. Additionally, complement factor, C3a was shown as a potential upstream gene attributable to these features. Administration of C3a protein to cardiomyocytes resulted in activation of several MAP kinases including ERK via C3a receptor and suppression of oncostatin M signaling. Furthermore,
in vivo
analysis, administration of C3a receptor antagonist dramatically improved right ventricular function in mouse model of right ventricular overload, pulmonary artery constriction. There results indicated that C3a would regulate the unique phenotypes in RVOT and would be the potent effector to right ventricular heart failure.

Conclusion:
We revealed that an activated alternative complement pathway has a crucial role in the unique features of RVOT and the pathogenesis of right ventricular heart failure. The blockade of the pathway would be a therapeutic target of RVOT-related diseases.
Abstract 268: Through the Inhibition of Autophagy Flux miR-221 Plays an Important Role in the Reductions of Hypoxia Reoxygenation Induced Cell Death and Collagen Synthesis in Cardiac Fibroblast
Authors: Zhou Yue , Richards Arthur M , Wang Peipei
Published: February 2019

Purpose:
We have previously reported that miR-221 protects myocytes against H/R-induced injury through the inhibitions of autophagy and apoptosis. It is not known has similar anti-autophagic and anti-apoptotic effects on cardiac fibroblast (cFB) and what role it plays in the regulation of cFB function.

Method:
Adult rat cFBs were isolated and transfected with miR-221 mimics and mimic control (miR-221 and MC). cFBs were subjected to normoxia (Nor) and H/R conditions. MiR-221 effects were monitored for cell injury (CCK8 and lactate dehydrogenase, LDH), apoptosis (Annexin V and 7-AAD staining), autophagy (LC3 and p62 by Western blot, WB) and collagen synthesis (Sircol). To study autophagy flux, Bafilomycin A1 (Baf) or Rapamycin (Rap) was applied to further inhibit or enhance autophagosome (ATGsome) formation and degradation respectively.

Results:
miRNA-221 increased cFB viability as indicated by increased CCK8 and reduced LDH release (miR-221 vs. MC) in H/R. This pro-survival effect did not involve apoptosis but reducing autophagy as indicated by unchanged Annexin V staining and reduced LC3-II/I ratio and increased p62 respectively. The protection was blocked by Rap. DMSO, the solvent used for the preparation of Baf and Rap, showed a significant induction of ATGsome formation on its own and override the anti-ATGsome formation effect of miR-221. In the presence of DMSO, miR-221 caused LC3 II accumulation, similar to the effect of Baf. In addition, miR-221 inhibited collagen synthesis as assessed by Sircol measurement. TGF-β, as a positive control, increased collagen synthesis significantly. The regulation of collagen synthesis of miR-221 and TGF-β could be abrogated by Rap and Baf respectively. Under Nor and H/R, the expressions of DDIT4 and Tp53inp1 were significantly downregulated by miR-221.

Conclusion:
Targeting on DDIT4 and Tp53inp1, miR-221 inhibited autophagy flux by reducing both ATGsome formation and degradation. The inhibition plays an important role in the protection of cFB and regulation of collage synthesis. This study reveals the complexity of cFB functional regulation by miRNA-221, preserving cFB in number without causing additional fibrosis. The unique feature of miR-221 might be an advantage in the treatment of cardiac infarction.
Abstract 274: Regulation of Myocardial Ketone Body Oxidation Capacity by Increased Protein O-GlcNAcylation in Diabetic Heart
Authors: Brahma Manoja K , Ha Chae-Myeong , Paterson Andrew K , Young Martin E , Wende Adam R
Published: February 2019

Diabetes increases the risk of heart failure independently of underlying coronary artery disease. Perturbations in myocardial substrate utilization have been proposed to contribute to the pathogenesis of cardiac dysfunction. However, the exact mechanisms linking impaired substrate utilization to disease progression remain elusive. Compared to the well-established roles of glucose and fatty acids metabolism in cardiac physiology and pathology, the importance of ketone body (KB) metabolism is less well understood. Several findings suggest the importance of KB in the cardiac health and disease. More recently, two independent studies have simultaneously demonstrated increased reliance on KB utilization by the failing heart in the absence of diabetes. Despite these observations, our understanding of the importance of KB metabolism in a diabetic heart is incomplete. Interestingly, our findings from the hearts of streptozotocin-induced diabetic mice confirmed elevated cardiac β-hydroxybutyrate (a major KB) levels and transcriptional suppression of
Oxct1
gene expression that encodes for SCOT (succinyl-CoA:3-oxoacid CoA transferase; a rate limiting enzyme of KB oxidation), consistent with a mismatch between KB availability and utilization. To determine a specific role for glucose delivery in this regulation we used an inducible transgenic mouse model with cardiac-restricted expression of the glucose transporter 4 (GLUT4; mG4H). Interestingly, mG4H mice had reduced cardiac SCOT protein levels and oxidation of KB in isolated working heart suggesting that glucose is sufficient to regulate the ketone metabolic pathway. Hyperglycemia associated with diabetes alter protein function via post-translational
O
-GlcNAcylation. Interestingly, we found that myocardial SCOT expression was also reduced in a third mouse model with increased cardiac
O
-GlcNAc levels (overexpression of a dominant negative form of
O
-GlcNAcase, which normally removes
O
-GlcNAc; dnOGAh) suggesting that increased protein
O
-GlcNAcylation is directly involved. Collectively, these studies establish a novel role of glucose and its downstream regulator,
O
-GlcNAc, in attenuation of KB metabolism in the diabetic heart.
Abstract 281: The Calcineurin/Rcan1 Axis Influences Mitochondrial Dynamics, Metabolism, and Biogenesis
Authors: Nguyen Ngoc Uyen Nhi , Altamirano Francisco , Parra Valentina , Rothermel Beverly A
Published: February 2019

Rationale and Objective:
The Regulator of Calcineurin 1 (RCAN1) inhibits calcineurin (CN), a Ca
2+
-activated protein phosphatase important in cardiac remodeling. The hearts and brains of
Rcan1 KO
mice are more susceptible to damage from ischemia/reperfusion (I/R). As mitochondria are known to be key mediators of I/R damage, the goal of our studies was to determine the impact of the CN/RCAN1 signaling axis on the mitochondrial network and function.

Methods and Results:
Using both neonatal and isolated adult cardiomyocytes, we found that, the mitochondrial network was more fragmented in the absence of RCAN1, due to increased CN-dependent activation of the fission protein DRP1. RCAN1-deficient cardiomyocytes had reduced mitochondrial membrane potential, consumed less O
2
, and generated lower levels of ROS. Importantly, they also displayed a reduced capacity for mitochondrial Ca
2+
uptake. RCAN1-depleted cardiomyocytes were more sensitive to I/R, however, pharmacological inhibition of CN, DRP1, or calpains (Ca
2+
-activated proteases) restored protection, suggesting that, in the absence of RCAN1, calpain-mediated damage following I/R is greater because of a decrease in the ability of mitochondria to buffer cytoplasmic Ca
2+
. Over-expression of RCAN1 was sufficient to enhance fusion. In addition to its impact on mitochondrial dynamics, CN activation also promoted mitochondrial biogenesis. We demonstrate that expression of the
1b
isoform of
PGC1α
(a master regulator of mitochondrial biogenesis) is directly under CN control
. In vivo,
a cardiomyocyte-specific CN transgene caused a more rapid increase in: transcript levels for
P
gc
1α-1b
and
PolG
(the mitochondrial DNA polymerase), mtDNA content, and the abundance of electron transport complex proteins. Despite the increase in mitochondrial content, changes in metabolic enzymes were consistent with a shift toward glycolysis rather than fatty acid oxidation.

Conclusions:
The CN/RCAN1 signaling axis helps determine both the number of mitochondria (DRP1-dependent fission) and the total mitochondrial mass (
Pgc1α
-dependent biogenesis). Maintaining the proper level of these two processes is essential to cardiovascular health and metabolic flexibility.
Abstract 287: Cardioprotection in Mice With a Knock-in Mutation (C202S) in Cyclophilin D
Authors: Amanakis Georgios , Sun Junhui , Boylston Jennifer , Liu Chengyu , Molkentin Jeffery , Murphy Elizabeth
Published: February 2019

Our previous study in mouse embryonic fibroblasts showed that cysteine 202 of cyclophilin D (CypD) is necessary for redox stress-induced activation of the mitochondrial permeability transition pore (PTP). To further investigate the function of this cysteine residue in situ, we used CRISPR to develop a knock-in mouse model (C57BL/6N strain), where CypD cysteine 202 was mutated to a serine (C202S). The amount of total CypD expressed in CypD C202S mouse hearts did not differ compared to the wild-type (WT). However, CypD C202S hearts show significantly less ischemia-reperfusion (I/R) injury in the Langendorff perfused heart model; myocardial infarct size was decreased versus WT (24.5±4.7% CypD C202S
vs
49.8±2.7% WT, p=0.003, n=4). Isolated cardiac mitochondria from CypD C202S mice also have a higher calcium retention capacity compared to WT (213.3±16.7
vs
140.0±20.8 umol Ca
+2
/g protein, p=0.033, n=4). We previously found C202 to undergo S-nitrosylation with preconditioning and hypothesized that oxidation might compete with S-nitrosylation and might target CypD to the mitochondrial membrane to activate the PTP. Indeed, isolated cardiac mitochondria subjected to oxidative stress exhibit less binding of CypD C202S to the proposed PTP component ATP synthase compared to WT (78.7±6.9%
vs
WT, p=0.038, n=3). Cysteine 202 also matches an S-palmitoylation site motif found commonly in soluble proteins (-C--L-). To assess palmitoylation of C202 we employed a resin-assisted capture. Langendorff perfused mouse hearts were first treated with N-ethylmaleimide to block free cysteines, and then with hydroxylamine to free cysteines from palmitate. Free cysteines were subsequently captured by a resin. At baseline, CypD C202S exhibits less palmitoylation compared to WT (37.7±1.98%
vs
WT, p<0.001, n=6) which might alter its membrane association and desensitize PTP. We are currently testing whether alterations in palmitoylation of C202 regulate PTP opening and are evaluating the physiological role of these post-translational modifications. In conclusion, these results show that mutated CypD C202S affords cardioprotection against I/R injury and that oxidation, S-nitrosylation, and S-palmitoylation of C202 are potential regulators of CypD and PTP.
Abstract 292: Mitochondrial CLIC4 and CLIC5B Mediate Cardio-protection From Ischemia/reperfusion Injury
Authors: Ponnalagu Devasena , Patel Neel J , Gao Erhe , Koch Walter J , Kohut Andrew R , Singh Harpreet
Published: February 2019

Mitochondria are important for regulating myocardial physiology, cardiac function and cardio-protection from ischemia/reperfusion injury (IR). Cation channels like BK
Ca
and K
ATP
present in the cardiac mitochondria are important in reducing myocardial infarction (MI) upon IR injury. Mitochondrial anion channels are equally important as they regulate cell volume, maintain ionic homeostasis during ischemia and are considered as potential therapeutics for cardiac diseases. However, molecular identity of cardiac mitochondrial chloride channels is not yet elucidated. In this study we focus on unique class of dimorphic ion channels known as chloride intracellular channels (CLICs). Amongst the six paralogs in mammals, we found that CLIC1, CLIC4 and CLIC5 are abundant in heart isolated from
R. norvegicus
. CLIC4 and CLIC5 localize to the mitochondria whereas CLIC1 in endoplasmic reticulum. CLIC4 and CLIC5 localized to the outer and inner mitochondrial membrane (IMM), respectively, and modulate mitochondrial calcium retention capacity and reactive oxygen species (ROS) production. Interestingly, a splice variant CLIC5B predominantly present in the heart of
R. norvegicus
was responsible for IMM localization and its function. We also observed that CLICs are cardioprotective as blocking them with its inhibitor IAA-94 increased MI both in
in vitro
and
in vivo
IR injury assays
.
In addition, we performed left main descending coronary artery (LCA) occlusion in
clic
-/-
and wild type (Wt) mice to induce IR injury. LCA was occluded for 40 min prior to 24 h of reperfusion. MI was significantly higher in
clic4

-/-

mice as compared to Wt mice (58±6 vs 42±2, n≥4, p≤0.05). Only 75% (3 of 4) of
clic4

-/-

mice showed significant reduction in left ventricular ejection (LVEF) fraction and fractional shortening (LVFS) after
in vivo
IR injury. Interestingly, 50% (3 of 6) of
clic5

-/-

mice showed significant increased MI as well as decreased LVEF and LVFS after
in vivo
IR injury.
Clic1

-/-

mice showed similar MI and left ventricular function as Wt mice. In conclusion, we demonstrate that cardiac mitochondrial CLIC4 and CLIC5 are vital for cardioprotection from IR injury likely by maintaining the calcium homeostasis and ROS generation by the mitochondria, respectively.
Abstract 295: Proteomic Profiling of Changes at Active Polysomes That Occur as a Response to Ischemic Injury and Reperfusion of the Heart
Authors: Stastna Miroslava , Thomas Amandine , Venkatraman Vidya , Gottlieb Roberta A , Van Eyk Jennifer E
Published: February 2019

Introduction:
Heart ischemic injury and subsequent reperfusion activate dynamic changes in protein expression that, we hypothesize, are regulated by alternative mechanism at active polysomes. Since analysis of mRNA can correlate poorly with protein composition, we applied proteomic approach to characterize the changes related to ischemia and reperfusion in proteins that comprise the ribosomes, proteins associated with translation machinery and the newly synthesized proteins.

Methods:
Mouse heart tissue homogenates for conditions of sham, ischemia and ischemia/reperfusion (n=5) were subjected to separation in sucrose gradient and resulted fractions containing high-efficiency polysomes (heavy), low-efficiency polysomes (light) and non-translating fraction (NT) for each condition were subjected to proteomic analysis.

Results:
We identified more than 1300 proteins in total. We defined the core of active polysomes to be composed of 500 proteins which were always present in heavy fraction regardless of conditions. While 60S- and 80S ribosomal proteins were present in both heavy and light fractions, 66 proteins of mitochondrial ribosomal complex (28S and 39S) were captured exclusively in light fraction. Of interest were proteins that were increased or decreased in heavy fraction after ischemia and ischemia/reperfusion. In particular, mitochondrial elongation factor Tu (EFTU) was increased while nuclear transcriptional activator (PURA) was decreased after ischemia, and both returned to control levels upon reperfusion. Several 60S- and 40S ribosomal proteins remained unchanged after ischemia but their levels decreased after reperfusion (e.g. L37a, S11, S12 and S19). Transport proteins such as protein of transmembrane 9 superfamily (TM9S2) and monocarboxylate transporter 1 (MOT1) were decreased in ischemia and remained decreased after reperfusion.

Conclusion:
The proteins associated with polysomes and translation machinery of diverse activity were identified and their expression changes triggered by heart ischemic injury and reperfusion were characterized. To our knowledge, this is the first extensive proteomic characterization of polysome-related proteins revealing their dynamic profiles after ischemic stress in the heart.
Abstract 307: Focal Adhesion Kinase Activity and Nuclear Localization Controls Vascular Smooth Muscle Cell Proliferation and Intimal Hyperplasia Through a GATA4-Cyclin D1 Axis
Authors: Jeong Kyuho , Kim Jung-Hyun , Murphy James , Park Hyeonsoo , Ahn Erin , Lim Steve
Published: February 2019

Neointimal formation is a hallmark of occlusive disorders such as atherosclerosis, post-angioplasty restenosis, vein graft, and allograft vasculopathy. Upon vessel injury, increased growth factor secretion augments focal adhesion kinase (FAK) activity to promote vascular smooth muscle cell (VSMC) proliferation. However, the molecular mechanism of how FAK contributes to intimal hyperplasia remains unknown. To test the role of FAK activity in VSMC proliferative diseases, we performed femoral arterial wire injury and orally administered a FAK inhibitor (VS-4718) twice daily. FAK inhibition significantly reduced intimal hyperplasia compared to vehicle-treated mice 4 weeks after injury. We found that platelet-derived growth factor (PDGF) activated FAK and increased GATA-binding protein 4 (GATA4) expression which is correlated with VSMC proliferation, whereas FAK inhibition abolished the PDGF-induced GATA4 upregulation. Further, chromatin immunoprecipitation (ChIP) revealed that GATA4 binds the mouse cyclin D1 promoter to enhance cyclin D1 transcription, leading to VSMC proliferation via G1 to S cell cycle progression. Importantly, GATA4 and cyclin D1 expression were increased upon vascular injury parallel with increased FAK activity in vehicle-treated mice, but not changed in FAK inhibitor-treated mice. Mechanistically, FAK inhibition reduces VSMC proliferation through increased nuclear FAK, which can bind and turnover GATA4. Genetic FAK kinase-dead (KD) mice (ERT2-Myh11-Cre) developed less intimal thickening following wire injury, implicating VSMC-specific FAK activity is critical for VSMC proliferation. FAK KD VSMCs also showed increased nuclear FAK, decreased GATA4 and cyclin D1 levels compared to FAK WT. This study has uncovered the importance of GATA4 and nuclear FAK in VSMC hyperplasia.
Abstract 309: Small-molecule PDE4 Activators Limit Isoproterenol-induced Hypertrophy in Rat Neonatal Cardiomyocytes
Authors: Baskar Ganga , Boyd Abigail , Abou Saleh Lina , Boudreaux Thomas , Diamond Davis , Phuan Puay-Wah , Verkman Alan , Richter Wito
Published: February 2019

Increasing the cellular levels of the second messenger cAMP, such as
via
activation of β-adrenergic receptors (βARs), is the most powerful mechanism to acutely increase cardiac output. However, chronic βAR stimulation predisposes to the development of heart failure (HF) by promoting arrhythmias and pathologic cardiac hypertrophy. Cardiac myocyte cAMP levels are tightly regulated by cyclic nucleotide phosphodiesterases (PDEs), a family of isoenzymes that hydrolyze and inactivate cAMP. Prior studies have shown that development of HF is associated with a downregulation of the expression and activity of Type 4 PDEs (PDE4s) in humans and animals. We propose that this reduction in PDE4 is maladaptive and may contribute to the development of HF, because genetic or pharmacologic inactivation of PDE4s is associated with an increased propensity to arrhythmias and development of pathologic cardiac hypertrophy. We propose that, conversely, an increase in PDE4 protein and/or activity would be cardioprotective. To test this hypothesis, we have utilized rat neonatal cardiomyocytes (RNCM) in culture as a model system. Long term treatment with Isoproterenol (Iso) induces hypertrophy in these cells as reflected by increased cell surface/size. To stimulate PDE4 activity, which contributes about 50% of the total cAMP-PDE activity in RNCMs, cells were treated with small-molecule PDE4 activators, which we recently identified in a high throughout screen. We find that small-molecule PDE4 activators reduce Iso-induced cellular cAMP levels as measured by enzyme immuno assay and suppress Iso-induced PKA signaling, such as the PKA-mediated phosphorylation of phospholamban. Moreover, treatment with PDE4 activators alleviates the Iso-dependent increase in cell size in RNCM indicating that pharmacologic PDE4 activation can serve to limit cardiomyocyte hypertrophy.
Abstract 317: Nlrc5 Positively Regulates Intimal Formation via Interaction With PPARã in Vascular Smooth Muscle Cells
Authors: Zhuang Jianhui , Luan Peipei , Xu Yawei , Peng Wenhui
Published: February 2019

Background:
Innate immunity and vascular smooth muscle cells play crucial role in the development of vascular remodeling and intimal hyperplasia. NLR family CARD domain containing 5 (Nlrc5), known as a pattern recognition receptor, has been noted for its implication in immune disease in recent studies. However, whether Nlrc5 regulates intimal hyperplasia is still unclear.

Aim:
We aimed to determine the role of Nlrc5 in intimal hyperplasia and its underlying molecular mechanism.

Methods and Results:
We showed that an elevated expression of Nlrc5 in neointima induced by carotid ligation compared normal carotid artery. In human aortic smooth muscle cells (HASMCs), PDGF induced Nlrc5 expression and nuclear translocation. By utilizing Nlrc5 knockout mice, we found Nlrc5 deficiency prominently aggravated neointimal formation compared to wild type (WT) mice. On the contrary, gain of function of Nlrc5 based on local adenovirus injection attenuated intimal hyperplasia. The neointimal area was comparable in WT mice receiving either WT or Nlrc5-/- bone marrow transplantation, demonstrating that hematopoietic cell-derived Nlrc5 did not affect vascular hyperplasia. In vitro experiments unveiled that knockdown of Nlrc5 resulted in excessive proliferation, migration and dedifferentiation of HASMCs. Nevertheless, depletion of Nlrc5 in HASMCs did not influence proinflammatory cytokine excretion and immune activation reflected by major histocompatibility complex-I expression. Mechanistic study revealed that Nlrc5 bound to nuclear transcription factor PPARγ and positively regulated PPARγ luciferase activity, ensuing contributing to KLF4 ubiquitination and degradation. Furthermore, we generated five deletion constructs of Nlrc5 and identified that the functional domain containing DD and NACHT constructs was responsible for the interaction with PPARγ.

Conclusions:
Our study reveals that Nlrc5 alleviates intimal formation through interaction with PPARγ and inhibition of smooth muscle cell dedifferentiation, suggesting that Nlrc5 constitutes a novel molecular target for vascular remodeling.
Abstract 334: Temporal Regulation of Retinoic Acid Delineates First Heart Field Ventricular and Atrial Cardiac Differentiation of Human Pluripotent Stem Cells
Authors: Han Pengcheng , Wolfson David , Cho Heecheol
Published: February 2019

Retinoic acid plays a pivotal role during the heart development. The mammalian heart is formed from cardiac progenitors in the first and second heart fields (FHF and SHF). FHF progenitors populate the left ventricle and the atria; SHF progenitors populate the right ventricle, outflow tract and parts of the atria. Loss of retinoic acid signaling leads to depletion of FHF progenitors and compensatory increase in SHF progenitors. Here we hypothesized that mimicking the
in vivo
retinoic acid signaling during human pluripotent stem cell differentiation could lead to FHF cardiomyocytes.Human pluripotent stem cells were differentiated to cardiac lineage by treatment with Activin A, BMP4 and bFGF (control) for the first three days (d0-3). This lead to >70% Brachyury+ mesodermal cells, and >90% of Brachyury+ cells became cTnT+ cardiac myocytes. Retinoic acid signaling activated with exogenous RA (1μM) from d5-8 (RA protocol). This RA protocol was compared to another protocol in which d5-8 retinoic acid activation was followed by initial inhibition with a pan-retinoic acid (RiRA protocol). At d14, both RA and RiRA cardiomyocytes expressed higher transcript and protein levels of the FHF markers, NKX2.5, TBX5and HCN4, but lower levels of the SHF markers, ISL1, MEF2c, FGF8/10, compared to control. RiRA cardiomyocytes showed highest expression of ventricular markers,
IRX4
and
HRT2
, lowest expression of an atrial specific marker CoupTFII, which were mirrored in RA cardiomyocytes. In line with their atrial/ventricular-specific gene expression profile, RA cardiomyocytes demonstrated higher beating rates, higher maximum shortening velocity, faster conduction velocity, higher atrial specific ANP secretion and higher sensitivity to atrial-selective sodium blocker, ranolazine compared to control. In contrast, RiRA cardiomyocytes generated higher force but shorter action potential duration compared to control.Taken together, the data demonstrate temporal regulation of retinoic acid signaling can not only accurately recapitulate ventricular to atrial cardiomyocyte differentiation by delineating FHF vs. SHF lineages.
Abstract 335: Introducing a Multiwell Cardiac μGMEA Platform for Action Potential Recordings From Human iPSC Derived Cardiomyocyte Constructs
Authors: Edwards Stacie L , Zlochiver Viviana , Conrad Donald M , Vaidyanathan Ravi , Joshi-Mukherjee Rosy
Published: February 2019

Multielectrode array technology utilizing iPSC derived cardiomyocytes has proven useful for drug screening and arrhythmia studies. However, its application for direct action potential (AP) measurements from multicellular preparations remains limiting. We report a novel platform for high temporal resolution AP recordings from human iPSC-derived cardiomyocyte (hiPSC-CM) constructs. Derivation of high purity hiPSC-CMs yielded syncytial monolayers when cultured on a PEDOT-coated micro-gold multielectrode array (μGMEA). We gained intracellular access via 30 seconds of localized electroporation causing a revertible transition of extracellular field potential signals to quasi-APs to APs. We recorded a total of 492 AP waveforms within and across 9 constructs simultaneously and classified their morphologies based on action potential duration ratio (APD
50
/APD
90
) and difference (APD
90
–APD
50
). The vast majority of the waveforms (90.8%) were found to be ventricular-like with APD
50
/APD
90
and APD
90
–APD
50
of 0.75-0.95 and 52-166ms respectively. APD
30
displayed a wider variability within individual constructs when compared to the APD
80
distribution. The variability in fractional repolarization (APD
80
– APD
30
/APD
80
) and triangulation (APD
80
– APD
30
), parameters widely used for drug screening and arrhythmogenesis, ranged from 0.1- 0.8 and 100- 550ms respectively. The Gaussian distribution produced a single peak across these parameters confirming the ventricular-like properties of all the constructs. We confirmed the reliability of the system by recapitulating known drug induced responses on APs. Norepinephrine and isoproterenol associated with β-adrenergic stimulation produced a dose dependent increase in beating rate and shortening of APD. E4031, a hERG channel blocker, elicited a dose dependent prolongation of the APD and produced arrhythmic early afterdepolarizations (EADs) thus modulating AP waveforms via external factors. Our cardiac μGMEA model has the capability to distinguish subtle differences in AP morphology for identifying novel cardio-factors and for drug screening.
Abstract 342: The Absence of S-nitrosoglutathione Reductase (GSNOR
-/-
) Reduces Maturation of iPSC-derived Cardiomyocytes
Authors: Salerno Alessandro , Dulce Raul , Hatzistergos Konstantinos , Wanschel Amarylis , Balkan Wayne , Hare Joshua
Published: February 2019

Introduction:
Induced pluripotent stem cells (iPSCs) are a potential source of cardiac regenerative therapy. However, incomplete structural and functional maturation of iPSC-derived cardiomyocytes (iPSC-CMs), including lack of T-tubules, immature sarcoplasmic reticulum (SR), and inefficient Ca
2+
-induced Ca
2+
release remain major limitations. We assessed the influence of nitric oxide (NO) on differentiation and maturation of iPSC-CMs.

Objective:
We hypothesized that enhanced S-nitrosylation impairs SR calcium (Ca
2+
) handling (storage and uptake) in iPSC-CMs.

Methods:
iPSCs were derived from fibroblasts from wildtype mice and mice lacking S-nitrosoglutathione reductase (GSNOR
-/-
), a denitrosylase that regulates protein S-nitrosylation. iPSCs were differentiated into CMs from embryoid bodies via the hanging-drop method. Analyses of cell morphology and structural changes, gene expression (cardiac markers), and functional tests (intracellular calcium transients) were performed at an early stage of cardiac development (day 21 of cell culture). The involvement of the SR Ca
2+
ATPase (SERCA2) in iPSC-CM contraction was tested by blocking Ca
2+
re-uptake with 1 μM Thapsigargin and SR Ca
2+
stores were estimated by addition of 20 mM caffeine. The role of the cardiac ryanodine receptor (RyR2) was assessed by blocking the channel with 10 μM ryanodine. The dependence of Ca
2+
transients on L-type Ca
2+
channels (LTCC) was demonstrated using 10 μM nifedipine, which eliminated [Ca
2+
]
i
transients and cell contraction. Length of sarcomeres within the contractile apparatus was determined by staining cells with α-actinin.

Results:
Sarcomeres were more organized in WT iPSC-CMs than in GSNOR
-/-
iPSC-CMs after 21 days in culture. The loss of GSNOR activity reduced SR Ca
2+
content and sarcomere organization in iPSC-CMs. While iPSC-CMs are relatively immature in terms of ultrastructure and Ca
2+
handling; GSNOR
–/–
iPSC-CMs were less able to recycle intracellular Ca
2+
.

Conclusions:
Our results provide novel insights into NO-mediated gene regulation and cell proliferation and suggest that the absence of GSNOR affects CM differentiation. These findings have important implications for regenerative medicine and provide new targets for iPSC-based therapy.
Abstract 347: Mechanistic Basis of Neonatal Heart Regeneration Revealed by Transcriptome and Histone Modification Profiling
Authors: Wang Zhaoning , Cui Miao , Ye Wenduo , Tan Wei , Botten Giovanni , McAnally John , Shelton John M , Kim Min S , Malladi Venkat , Chen Beibei , Bassel-Duby Rhonda , Liu Ning , Olson Eric N
Published: February 2019

The adult mammalian heart has very limited capacity to regenerate upon injury. Following myocardial infarction, cardiomyocytes are extensively lost and fibroblasts are activated to form a scar, resulting in compromised cardiac function. In contrast, the heart of a neonatal mouse has full capacity to regenerate after myocardial infarction, although such regenerative ability is lost by postnatal (P) day 7. The molecular properties of neonatal hearts that enable regeneration remain unknown. To uncover the mechanisms of neonatal heart regeneration, we induced myocardial infarction (MI) in P1 and P8 hearts, and profiled transcriptomic and epigenomic dynamics by RNA-Seq, H3K27ac ChIP-Seq and H3K27me3 ChIP-Seq at various time points following the surgery. Analysis of differentially expressed genes and groups of enhancers that acquire active status post-MI revealed distinct post-infarction responses between P1 and P8 hearts. Dramatic differences in immune response and developmental gene programs were identified in regenerative P1 versus non-regenerative P8 hearts. Furthermore, we analyzed transcription factors involved in the injury response post MI and constructed gene regulatory networks during neonatal cardiac injury. This work provides insights into the molecular basis of neonatal heart regeneration, and identifies potential genes and cis-regulatory elements that can be modulated to facilitate heart regeneration.
Abstract 352: Manf, a Structurally Unique Redox-sensitive Chaperone, Restores Er-protein Folding in the Ischemic Heart
Authors: Arrieta Adrian , Blackwood Erik A , Aivati Cathrine , Stauffer Winston T , Santo Domingo Michelle , Bilal Alina S , Sarakki Anup V , Thuerauf Donna J , Doroudgar Shirin , Glembotski Christopher C
Published: February 2019

Rationale:
In cardiomyocytes, secreted and membrane proteins critical for heart function are synthesized and folded in the sarcoplasmic/endoplasmic reticulum (SR/ER). We previously showed that myocardial ischemia decreases oxygen required for disulfide bond formation in nascent proteins, causing ER stress, i.e. the toxic accumulation of unfolded proteins, which contributes to cardiomyocyte death. In response to ER stress, the transcription factor ATF6 induces various ER-resident proteins that restore SR/ER protein folding, including ER chaperones. We found that ATF6 induces mesencephalic astrocyte-derived neurotrophic factor (MANF), a recently identified protein of unknown function. MANF is structurally unique, so its function could not be inferred by analogy to other proteins. Since we found that MANF is an ATF6-inducible ER-resident protein we hypothesized that it functions as a chaperone, and since MANF has 8 cysteine residues that are conserved in a wide range of species, that its chaperone function is redox-regulated and protective in the ischemic heart.

Methods:
The ability of recombinant MANF (rMANF) to suppress misfolded protein aggregation was examined in an
in vitro
chaperone assay. The effect of MANF knockdown on cell viability during simulated ischemia (sI) was determined in neonatal rat ventricular myocytes (NRVM). The effect of MANF loss-of-function in the ischemic heart,
in vivo
, was determined in a novel mouse model in which MANF is knocked down in cardiomyocytes.

Results:
rMANF formed disulfide-dependent complexes with and suppressed aggregation of model misfolded proteins
in vitro
, and these effects were lost when the cysteines in rMANF were mutated to alanine. In NRVM, MANF knockdown decreased viability during simulated ischemia; this viability deficit was restored upon ectopic expression of wild type, but not mutant MANF. MANF knockdown in the heart,
in vivo
, increased ischemia/reperfusion damage, and this damage was mitigated using an AAV9-based gene therapy approach to restore MANF expression.

Conclusions:
MANF is a novel redox-sensitive SR/ER-resident chaperone that is a critical contributor to SR/ER protein folding during the adaptive ER stress response and mitigates ischemia/reperfusion damage in the heart.
Abstract 353: Nucleolar Enlargement and Perturbed Ribosome Biogenesis Are Cellular Hallmarks of Cardiac Aging
Authors: Matsumoto Collin , Hua Trang , Sussman Mark , Hariharan Nirmala
Published: February 2019

Introduction:
Cardiac Aging manifests as structural and functional impairment of the heart and is associated with several changes in the cellular architecture. Of the many alterations in organelle structure and function that occur during aging, changes in the nucleolus and ribosome remain least studied in the cardiac context. The nucleolus functions as a stress sensor and is critical for ribosome biogenesis.

Hypothesis:
This study hypothesizes that perturbations in nucleolar architecture and ribosome biogenesis result in accumulation of free ribosomal proteins that stabilize p53 and trigger cardiac aging.

Methods and Results:
Using animal models of chronological (CA) and biological aging (BA), we observed that nucleolar size is dramatically increased in senescent myocardial cells (1.75 fold) as well as in aged hypertrophied hearts. Nucleolar enlargement is accompanied by significant increase in pre-ribosomal RNA levels (2 fold, p<0.01 in CA hearts; 9 fold in BA hearts) indicating increased ribosome biogenesis. Synthesis of ribosomal proteins (RP) in equimolar amounts to ribosomal RNA that occurs under normal conditions is disrupted in the aged heart, resulting in increased expression of RPL11 (50% in BA heart, 33% in CA heart) that is a component of the 60S large ribosomal subunit. Accumulation of RPL11 coincides with increased p53 expression (2 fold, p<0.05 in BA heart; 5.4 fold in CA heart) suggesting a role for RPL11 in mediating p53 stability in the aged heart.

Conclusion:
Collectively our data demonstrate nucleolar enlargement as a cellular hallmark of cardiac aging and uncover a molecular mechanism involving perturbed ribosome biogenesis and accumulation of free RPs that contribute to age-associated afflictions in the heart.
Abstract 373: The Molecular Consequence of a Polymorphic 25bp Deletion in Intron 32 of
MYBPC3,
Specific to South Asians
Authors: Schwanekamp Jennifer A , Viswanathan Shiv K , Patel Parth N , Kandoi Sangeetha , Bhat Ratan , Verma Rama S , Seidman Christine E , Seidman Jonathan G , Shim Winston , Knöll Ralph , Sadayappan Sakthivel
Published: February 2019

Background:
MYBPC3
encodes cardiac myosin binding protein-C, which regulates sarcomeric stability and contractility. Previous work described a prevalent 25bp deletion in intron 32,
MYBPC3

Δ

25bp

, in the South Asian population. Occurring in an estimated 100 million carriers, this variant may be associated with cardiomyopathy and heart failure.
MYBPC3

Δ25bp

encompasses a splicing branch point that could affect splicing of exon 32 to exon 33, potentially resulting in an aberrantly spliced
MYBPC3
transcript where exon 33 is skipped entirely. However, the mechanism by which
MYBPC3

Δ

25bp

associates with disease remains unclear.

Methods:
To determine the consequence of the 25bp deletion
in vivo
, mice were engineered that replaced the endogenous
Mybpc3
intron 32 with either the normal human intron 32 or human intron 32 containing the 25bp deletion (
Mybpc3

Δ

25bp
)

. We characterized
Mybpc3
splicing in mice carrying human intron-32 and human iPSC derived cardiomyocytes (hiPSC-CM) from patients heterozygous and homozygous for
MYBPC3

Δ

25bp

by RT-PCR and using mini-gene assays (across 5 different mouse and human gene segments encompassing exon 32 through exon 34).

Results:
Echocardiography of mice 3 months of age revealed no changes in cardiac function by fractional shortening (WT: 32.8%, Het: 34.2%, Homo: 36.7%). However, homozygous
Mybpc3

Δ

25bp

mice had significantly smaller hearts compared to heterozygous and wildtype (3.4 vs 3.71 and 4.25 mg/g p=0.001).
Mybpc3

Δ

25bp

heterozygous and homozygous mice displayed minimal alternative splicing in exon 33 at baseline. Similarly, transcripts from hiPSC-CM from heterozygous and homozygous
MYBPC3

Δ

25bp

carriers also show minimal alternative splicing. Mini gene assays suggest that the 25bp deletion does reduce the proportion (77.5% v. 86.2%) of normally spliced transcripts.

Conclusions:
Taken together, these data suggest that under baseline conditions,
MYBPC3

Δ

25bp

results in minimal alternative splicing in both human iPSC-CMs and humanized mice harboring the 25bp deletion. While increase aberrant splicing may increase risk of hypertrophic cardiomyopathy in variant-carrying individuals, ongoing studies will determine whether other factors or gene mutations modulate the pathogenicity of the MYBPC3
Δ25bp
variant.
Abstract 406: Loss of Sigmar1 Leads to Impaired Mitochondrial Respiration, Altered Mitochondrial Dynamics and Development of Cardiac Contractile Dysfunction
Authors: Abdullah Chowdhury S , Alam Shafiul , Aishwarya Richa , Miriyala Sumitra , Panchatcharam Manikandan , Orr Anthony W , James Jeanne , Robbins Jeffrey , Lorenz John N , Bhuiyan Md. Shenuarin
Published: February 2019

Rationale:
Studies involving different neuronal and cancer cell lines indicated that Sigma 1 receptor (Sigmar1) function as an inter-organelle signaling molecule and elicit cytoprotective functions. Though the presence of Sigmar1 in the heart was reported using ligand binding assay, all studies to date have been limited to pharmacological approaches using less selective ligands for Sigmar1. Despite all these studies, the physiological function of Sigmar1 in the heart remains unknown.

Objective:
We investigated the physiological function of Sigmar1 in the heart in regulating mitochondrial respiration, mitochondrial dynamics and cardiac contractility using the Sigmar1 knockout mouse (Sigmar1
-/-
).

Methods and Results:
Sigmar1
-/-
hearts showed significantly increased hemodynamic parameters assessed by left ventricular (LV) catheterization both under basal conditions and after stimulation with increasing doses of the β
1
-adrenoceptor agonist. Noninvasive echocardiographic measurements showed the development of cardiac functional decline in
Sigmar1

–/–

hearts over aging indicated by decreased LV percent fraction shortening and percent ejection fraction. Histochemistry also showed significant cardiac fibrosis and increased expression of periostin in the
Sigmar1

–/–

hearts compared to wildtype (Wt) hearts. Ultrastructural analysis of Sigmar1
-/-
cardiomyocytes showed irregularly shaped, highly fused mitochondrial network, and exhibited abnormal cristae. Mitochondrial size distribution in Sigmar1
-/-
hearts showed an increased number of large mitochondria resulting in decreased numbers of mitochondria per microscopic field and also showed altered expression of mitochondrial dynamics regulatory proteins. Real-time oxygen consumption rates in isolated mitochondria showed reduced respiratory function in Sigmar1
-/-
hearts compared with Wt hearts.

Conclusions:
All these data demonstrate a potential function of Sigmar1 in regulating normal mitochondrial organization and size in the heart. Knockdown of Sigmar1 evoked mitochondrial dysfunction, accumulation of abnormal mitochondria, enhanced adverse cardiac remodeling and resulting in cardiac contractile dysfunction.
Abstract 411: Mitochondrial NAD(H) Signaling in Cardiac Physiology and Pathology
Authors: Hu Qingxun , Wang Pei , Tian Rong , Wang Wang
Published: February 2019

The heart is a high energy-consuming organ and mitochondrial dysfunction is widely observed in heart failure. Previously, we reported that imbalanced NADH/NAD+ homeostasis in cardiomyocyte is responsible for stress-induced mitochondrial protein hyper-acetylation and accelerated progression to heart failure, which can be rescued by supplementing NAD+ precursor. The goals of this study are to develop novel fluorescent biomarkers and directly monitor mitochondrial NAD(H) redox regulation under pathophysiological conditions in adult cardiomyocytes. We constructed adenoviruses containing mitochondria-targeted NAD+ biosensor and NADH/NAD+ ratio biosensor (SoNar). We validated their mitochondrial targeting and ability to monitor acute changes in NAD+ and NADH/NAD+ ratio in adult cardiomyocytes. Pyruvate (1 mM) acutely increased mitochondrial NAD+ level and NADH/NAD+ ratio consistent with the notion that stimulating mitochondrial metabolism expanded NAD+ pool and promoted the reduction of NAD+ to NADH, which exceeded NADH consumption. In contrast, glucose (10 mM) slightly increased NADH/NAD+ ratio and palmitate had no acute effect suggesting that metabolism is only mildly stimulated and the NAD+ pool remains stable. However, palmitate treatment for 24 hr did decrease NAD+ and increase NADH/NAD+ ratio in mitochondria suggesting that the effects of physiological substrates on mitochondrial NAD(H) redox status are slow, accumulative or indirect through modulating other mitochondrial functions. Elevating cytosolic calcium with caffeine increased mitochondrial NAD+ level and decreased NADH/NAD+ ratio suggesting accelerated NADH to NAD+ turnover. Calcium has been shown to boost both energy production and utilization, but its overall impact on energy homeostasis is not clear. Our results indicate that acute increase in cytosolic calcium may have a more profound effect on energy utilization. Finally, ischemia reperfusion induced acute fluctuations in NADH and NADH/NAD+ ratio. Taken together, these results provide the first glimpse into the real-time modulation of compartmentalized NAD(H) redox state and new clues as to how mitochondrial redox is tightly controlled by mitochondrial metabolism in adult cardiomyocytes.
Abstract 421: High-throughput
In vitro
Ischemia-reperfusion Model With Real-time Monitoring of Cellular Oxygenation and Reactive Oxygen Species Generation
Authors: Fleming Christopher , Carey Conn , Ketenhoffen Ralf , Schneider Mario , Hynes James
Published: February 2019

Ischaemia-reperfusion (IR) injury is a feature of multiple diseases including myocardial infarction, renal failure and stroke, occurring when tissue blood supply is restricted and subsequently restored. While reperfusion is essential for tissue survival, it is also associated with significant ROS-mediated damage, triggering inflammatory responses and ultimately cell death. The rational development of targeted therapeutic interventions require both a detailed understanding of the mechanisms underlying reperfusion injury and a convenient means of assessing the efficacy of putative therapeutics. This need has driven the development of a number of
in vitro
IR models including pharmacological ATP depletion, oil overlays to induce autohypoxia, glucose oxidase addition to cause sample deoxygenation, and N
2
purged hypoxia chambers to induce a slow reduction in dissolved oxygen. The utility of these models has however been limited as they do not facilitate the induction of the rapid, controlled, transient, ischemic shock and reperfusion necessary to replicate IR injury condition
in vitro
. Critically, neither do they facilitate real-time cellular oxygenation monitoring to allow accurate IR characterisation or parallel measurements of critical IR injury parameters such as ROS and mitochondrial membrane potential (MMP). Here we present a model which addresses these limitations through the combined use of iPS-derived cardiomyocytes (Cor4U), plate reader with integrated atmospheric control facilitating rapid [O
2
] modulation (ClarioSTAR) and a novel intracellular probe capable of reporting cellular oxygenation in real time (MitoXpress-Intra). Oxygenation monitoring facilitates precise control of an ischemic insult whereby instrument [O
2
] can be modulated (21% to 0.1% O
2
) to provide the desired depth and duration of hypoxia. The chamber can then be vented to model rapid reperfusion (1 to 10% in less than 10min), with parallel monitoring of ROS and MMP, facilitating detailed metabolic characterisation of the short-term metabolic implications of reperfusion and the efficacy of model therapeutic intervention.