bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 541: Uncovering the Physiological Role of Mitochondrial Respiratory Supercomplexes in the Heart
Authors: Chapa-Dubocq Xavier R , Rodriguez-Graciani Keishla M , Guzman-Hernandez Roberto A , Jang Sehwan , Javadov Sabzali
Published: October 2019

The assembly of mitochondrial electron transport chain (ETC) supercomplexes (SCs), particularly the respirasome containing complexes I, III, and IV have been shown to participate in facilitating electron transport, reducing ROS production and maintaining the structural integrity of individual ETC complexes. However, the physiological role of SCs in high energy demanding tissues such as the heart remains unknown. Here, we elucidated whether disassembly of SCs affects the cardiac function. Hearts isolated from adult male Sprague Dawley rats were perfused using a Langendorff-mode perfusion with Krebs-Henseleit solution (KHS) for the first 20 min (equilibration period) followed by perfusion in the following groups: (i) 40-min perfusion with KHS (n=4), (ii, iii) 20-min perfusion with rotenone (an inducer of SC dissociation) or vehicle (ethanol, n=8 for both), and (iv, v) 20-min perfusion with rotenone or vehicle followed by a 20-min perfusion with KHS without rotenone or vehicle (n=6 for both). Cardiac function was monitored throughout the entire perfusion period. At the end of each protocol, the mitochondria were isolated for analysis of SCs, permeability transition, respiration rates, and ROS production. We found that cardiac function between rats perfused with KHS (i) and ethanol (i, iii) had no significant difference, though rotenone perfused rats (ii and iv) had a significant reduction (<40%) in cardiac function associated with reduced oxygen consumption rates in these groups. Furthermore, a significant increase in ROS production (ii) and permeability transition pore opening (iv) was observed when the hearts were perfused with rotenone with and without subsequent washout, respectively. Analysis of SCs by blue native PAGE displayed a significant reduction in SC levels in both ethanol (iii and v) and rotenone (ii and iv) perfused rats. Surprisingly, there were no differences in SC levels between the ethanol (iii and v) and rotenone (ii and iv) perfused groups. In addition, incubation of isolated intact mitochondria with ethanol and acetaldehyde did not demonstrate any direct effects of these compounds on ETC SC assembly. In conclusion, our data demonstrate a lack of an associative link between mitochondrial ETC SCs and cardiac function in rats.
Abstract 544: microRNA181c Activates Mitochondrial Calcium Uptake by Regulating Micu1 in the Heart
Authors: Banavath Hemanth , Roman Barbara , O'Rourke Brian , Kohr Mark , Murphy Elizabeth , Steenbergen Charles , Das Samarjit
Published: October 2019

Translocation of miR-181c into heart-mitochondria downregulates the mitochondrial gene, mt-COX1. miR-181c/d
-/-
hearts experience less oxidative stress during I/R and are protected against I/R injury. Additionally, miR-181c overexpression can increase mitochondrial matrix Ca
2+
([Ca
2+
]
m
); the mechanism is unknown. By RNA-Seq, here we show that hearts from miR-181c/d
-/-
mice overexpress nuclear-encoded Ca
2+
regulatory genes, suggesting that alterations in miR-181c and mt-COX1 perturb mitochondria-to-nucleus retrograde signaling and [Ca
2+
]
m
regulation. qPCR validation of transcription factors that are known to initiate retrograde signaling revealed significantly higher Sp1 expression in the miR-181c/d
-/-
hearts. Furthermore, an association of Sp1 with the promoter region of MICU1 was confirmed by ChIP-qPCR and higher expression of MICU1 was found in the miR-181c/d
-/-
hearts. Conversely, downregulation of Sp1 by siRNA decreased MICU1 expression in NMVM. Changes in PDH activity provided evidence for a change in [Ca
2+
]
m
via the miR-181c/MICU1 axis. Moreover, this mechanism was implicated in the pathology of I/R injury. When MICU1 was knocked down in the miR-181c/d
-/-
heart by lentiviral expression of an shRNA against MICU1, cardioprotective effects against I/R injury were abrogated. Furthermore, using an
in vitro
I/R model in miR-181c/d
-/-
NMVM, we confirmed the contribution of Sp1-MICU1 in ischemic injury. In summary, miR-181c regulates mt-COX1, which in turn regulates MICU1 through the Sp1-mediated mitochondria-to-nucleus retrograde pathway. Loss of miR-181c can protect the heart from I/R injury by modulating [Ca
2+
]
m
through the upregulation of MICU1.
Abstract 548: Cardiomyocyte-KLF5 Expression is Increased by FOXO1 and Accounts for Cardiomyopathy in Type-1 Diabetes
Authors: Kyriazis Ioannis D , Hoffman Mathhew , Kalliora Charikleia , Gaignebet Lea , Bannister Thomas D , Christofidou-Solomidou Melpo , Goldberg Ira J , Yang Vincent W , Bialkowska Agnieszka B , Kararigas Georgios , Drosatos Konstantinos
Published: October 2019

Cardiomyopathy in type 1 diabetes (T1D) is accompanied by altered cardiac energetics, impaired mitochondrial function and oxidative stress. We showed previously increased cardiac expression of Krüppel-like factor 5 (KLF5) and Peroxisome Proliferator Activated Receptor (PPAR)α at late T1D stage in mice. We confirmed that KLF5 expression is higher in cardiomyocytes of diabetic patients than in non-diabetic individuals. Mechanistic analyses in human cardiomyocyte cells (AC16) and in mice with cardiomyocyte-specific FOXO1 deletion (αMHC-
Foxo1
-/-
) revealed that FOXO1 activation accounts for the increased KLF5 expression via direct binding on Klf5 promoter. Both pharmacologic and cardiomyocyte-specific inhibition of KLF5 alleviated diabetic-related cardiac dysfunction. Accordingly, mice with doxycycline-mediated cardiomyocyte-specific KLF5 constitutive expression (αMHC-rtTA-
Klf5
) recapitulated cardiomyopathy even in the absence of T1D. We also showed that diabetic PPARα
-/-
mice had elevated cardiac KLF5 expression and cardiac dysfunction, suggesting that KLF5-driven diabetic cardiomyopathy is PPARα-independent. Furthermore, cardiomyocyte KLF5 upregulation was associated with oxidative stress, increased NADPH oxidase (NOX)4 expression, lower NADPH levels and impaired cellular Ca
2+
handling. Conversely, KLF5 inhibition prevented NOX4 upregulation and alleviated cardiac superoxide formation. Lipidomic analysis followed by Euclidean clustering showed strong correlation of lipidome profiles between αMHC-rtTA-
Klf5
mice and diabetic C57BL/6 mice, while diabetic αMHC-
Klf5
-/-
mice and diabetic C57BL/6 mice that were treated with pharmacologic KLF5 inhibitor grouped with the non-diabetic C57BL/6 mice. Further analysis of individual lipid species showed increased ceramide accumulation in diabetic C57BL/6 and αMHC-rtTA-
Klf5
mice that was reversed upon KLF5 inhibition. Treatment of αMHC-rtTA-
Klf5
mice and diabetic C57BL/6 mice with the antioxidant LGM2605 improved partially cardiac dysfunction. In conclusion, cardiomyocyte KLF5 expression is activated by FOXO1 and drives diabetic cardiomyopathy in a non-PPARα-dependent manner, suggesting KLF5 inhibition as a therapeutic intervention in T1D cardiomyopathy.
Abstract 561: Bone Morphogenetic Protein-3b Deficiency Induces Metabolic Syndrome and Modulates Adipogenesis
Authors: Marti-Pamies Ingrid , Caplan Alex , Thoonen Robrecht , Han Wei , Seale Patrick , Buys Emmanuel S , Bloch Donald B , Scherrer-Crosbie Marielle
Published: October 2019

Introduction:
Bone morphogenetic proteins (BMPs) are mediators in a wide range of cellular functions, including the regulation of cell metabolism. The objective of this study was to investigate the effects of BMP-3b, a glycoprotein synthetized and secreted by adipose tissue, on cardiovascular risk factors.

Methods:
BMP-3b knock-out (KO) and age-matched male and female wild-type (WT) mice were weighed weekly until 6 months of age. At 6 months, fasting levels of total cholesterol, HDL-C and triglycerides were measured and glucose and insulin tolerance tests were performed. Telemetry was used to determine blood pressure and heart rate in anesthetized mice. Echocardiograms were performed under light sedation (isoflurane 1%). Mice were sacrificed and tissues were resected for gene expression analysis by qPCR. Transient transfections were performed in 3T3-L1 cells to study the interactions of BMP-3b and the
C/ebp
α
promoter.

Results:
BMP3b KO mice gained more weight than WT mice. The plasma levels of total cholesterol, HDL and triglycerides were higher in BMP-3b KO mice than in WT mice. Insulin and glucose tolerant tests reveal insulin resistance and glucose intolerance in BMP-3b KO mice compared to WT mice. The basal heart rate was higher in BMP-3b KO mice than in WT mice, but no differences were observed in blood pressure. Echocardiography revealed a decrease in relative wall thickness (RWT) and left ventricular ejection fraction (LVEF), and an increase in the left ventricle end diastolic volume (LVEDV) in BMP-3b KO mice, demonstrating eccentric remodeling. Expression of genes (
Pparγ
and
C/ebpα
) involved in adipogenesis in white adipose tissue was higher in BMP-3b KO mice than in WT controls. Finally, transient transfection assays with 3T3-L1 cells showed that BMP-3b modulates
C/ebpα
promoter activity.

Conclusions:
The results of this study suggest that BMP-3b has an important role in the regulation of age-related weight gain, glucose metabolism and LV remodeling. Interventions that increase the level or function of BMP-3b may protect the heart from the adverse effects of cardiovascular risk factors and pathological cardiac remodeling.
Abstract 563: Cardiac TIGAR Reduces Myocardial Energetics and Cardiac Functionin the Pressure Overload Heart Failure Model
Authors: Okawa Yoshifumi , Hoshino Atsushi , Kitani Tomoya , Yamanaka Ryoetsu , Hato Daichi , Honda Sakiko , Fushimura Yohei , Urata Ryota , Minami Yoshito , Hashimoto Shyo , Matsuki Ayumi , Taminishi Shunta , Nishiji Toshiyuki , Hino Tomohiro , Yagi Nobuichiro , Iwai-Kanai Eri , Matoba Satoaki
Published: October 2019

Background:
Although metabolic alterations were observed in heart failure (HF), only recently have the mechanisms underlying these changes been identified. Tumor suppressor p53 responds to metabolic changes thorough several mechanisms. One of the p53 targets, TIGAR (TP53-induced glycolysis and apoptosis regulator) reduces glycolysis and suppresses autophagy, which augments ischemic damage, however its role on HF is unclear.

Method and Results:
In order to investigate TIGAR’s function in HF, we compared myocardial metabolic and functional outcomes between TIGAR deficient (TIGAR–/–) mice and wild-type (TIGAR+/+) mice subjected to chronic thoracic transverse aortic constriction (TAC), a pressure-overload HF model. In wild-type mice hearts, p53 and TIGAR increased markedly during HF development. Eight weeks after TAC surgery, the left ventricular (LV) dysfunction, fibrosis, oxidative damage, and myocyte apoptosis were significantly advanced in wild-type than in TIGAR–/– mouse heart. Further, myocardial high-energy phosphates in wild-type hearts were significantly decreased compared to those of TIGAR–/– mouse heart. Glucose oxidation and glycolysis rates were also reduced in isolated perfused wild-type hearts following TAC than those in TIGAR–/– hearts, which suggest that the upregulation of TIGAR in HF causes impaired myocardial energetics and function. The effects of TIGAR knockout on LV function were also replicated in tamoxifen (TAM)-inducible cardiac-specific TIGAR knockout mice (TIGARflox/flox/ Tg(Myh6-cre/Esr1) mice).

Conclusion:
The ablation of TIGAR during pressure-overload HF preserves myocardial function and energetics. Thus, cardiac TIGAR targeted therapy to increase glucose metabolism will be a novel strategy for HF.
Abstract 565: Response of the Ischemic Myocardium to Adrenergic Stimulation as Detected by Hyperpolarized [1-
13
C]pyruvate
Authors: Sharma Gaurav , Chen Wei , Jiang Weina , Anderson Aaron L , Malloy Craig R , Sherry A. Dean , Khemtong Chalermchai
Published: October 2019

Metabolic imaging by nuclear methods is widely used to access ischemic myocardium but provide limited information about individual enzyme-catalyzed reactions.
13
C hyperpolarization methods are sensitive to flux in specific key enzymes such as lactate dehydrogenase and pyruvate dehydrogenase. We tested the hypothesis that LDH flux is increased while PDH flux is abolished in low-flow ischemia. Metabolism of HP [1-
13
C]pyruvate was studied in four groups: (1) normal perfusion pressure (NPP), 100 cm H
2
O; (2) NPP with epinephrine; (3) low-perfusion pressure (LPP), 25 cm H
2
O; (4) LPP with epinephrine. Hearts excised from male Sprague-Dawley rats were perfused (37°C) at an initial pressure of 100 cm H
2
O using standard Langendorff method with Krebs-Henseleit buffer containing 0.75% bovine serum albumin, 0.4 mM non-labeled free fatty acid, 5.5 mM [U-
13
C]glucose, 1 mM [3-
13
C]pyruvate, 0.1 mM [3-
13
C]lactate. Perfusion pressure was reduced to 25 cm H
2
O after 30 min of perfusion for ischemia group and epinephrine added at 50
th
min of perfusion. HP [1-
13
C]pyruvate was injected to heart and subsequently,
13
C NMR spectra were acquired. The freeze clamped heart tissues were extracted with perchloric acid and analyzed by high-resolution
1
H and
13
C NMR (14.1 T). The
31
P NMR spectroscopy confirmed significant myocardial ischemia under LPP conditions, demonstrated by a marked increase in the [Pi]/[ATP] ratio. Surprisingly, increased LDH flux was observed in the ischemic hearts (+18.4%) while the metabolism of HP [1-
13
C]pyruvate by PDH remained unchanged as indicated by bicarbonate production. To investigate the oxidative capacity of mitochondria, epinephrine was used to stimulate hearts. As anticipated, epinephrine increased heart rate (+23%) and coronary flow (+11%) under NPP. Epinephrine increased heart rate (-60%) but did not alter coronary flow under LPP. We found that epinephrine had no effect on the rate of bicarbonate production under LPP. The study demonstrated that the conversion of pyruvate to lactate increased in ischemic hearts but the degree of pyruvate oxidation was not affected by the low-flow ischemia. Adrenergic stimulation did not increase PDH flux in these ischemic hearts. Translation to human is likely to provide new data from low-flow myocardium.
Abstract 568: Effects of Burn-induced Cardiac Released Factors on Cardiomyocyte and Profiles of Differentially Expressed Cardiac Mitochondrial Proteins Effects of Burn-induced Cardiac Released Factors on Cardiomyocyte and Profiles of Differentially Expressed Cardiac Mitochondrial Proteins
Authors: Wen Jake J , Radhakrishnan Geetha L , Porter Craig , Radhakrishnan Ravi S
Published: October 2019

Introduction:
Previous studies have indicated that cardiac dysfunction worsens in the first 24 hours post burn (pb) and improved over the next 48 hpb. We have found that cardiac mitochondria were damaged at 24 hpb and 24 hpb serum-treatment caused cardiomyocyte’mitochondrial dysfunction. However, the differentially expression of mitochondrial proteins in the early post burn period have still not been explored.

Aims:
To test if 3 hpb serum-treatment will interfere cardiomyocyte growth/proliferation similar to 24 hpb serum-treatment
in vitro
; To identify the cardiac mitochondrial protein expression profile, relating to burn-induced cardiac mit dysfunction.

Methods:
Male Sprague Dawley rats were randomized into sham control, 3 hpb and 24 hpb groups, with six rats in each group. 60% TBSA burn was induced by immersing in boiling water. In this study, heart function cardiac mit function, and cardiac mitochondrial protein profiles were measured. Burned rat serum were used to test our hypothesis
in vitro
.

Results:
Cardiac functions was significantly worse in 3 hpb (Fig 1). Cardiac mitochondrial dysfunction was observed via mit OXPHOS or mit complexes activities after burn. However, no change of CII-driving OXPHOS in 24 hpb and no change of CIII activity in 3 hpb were seen, suggesting that different mechanisms may be involved (Fig.2). Burn-serum treatment increased oxidative stress, apoptosis, and nitrite/nitrate, while decreasing antioxidants and cell proliferation in cardiomyocytes (Fig. 3). Nano LC MS/MS identified 13569 peptides and 1275 proteins from burn-induced heart lysates (Fig.4.A), which were associated with cardiomyopathy (Fig.4.B). Further analysis showed that burn injury induced the alterations of 25 up regulated signaling pathway and 7 down regulated signaling pathway (Fig.5.A). 147 mitochondrial proteins were identified to be differentially expressed after burn. Among the 147 proteins, 32 proteins were continually down-regulated, 37 continually up-regulated. In addition, 16 were first up regulated then down-regulated while38 had the opposite expression perform (Fig.5.B).

Conclusions:
Burn-induced heart dysfunction is associated with cardiac mitochondrial damage by altering mitochondrial protein expression.
Abstract 586: VA-ECMO Increases Urinary Levels of the Biomarker Kidney Injury Marker-1 (KIM-1) in a Preclinical Model of Acute Myocardial Infarction
Authors: Qiao Xiaoying , Swain Lija , Reyelt Lara , Machen Cody , Jarrah Andrew , Chennjorwala Aditya , Crowley Paige , Annamalai Shiva , Foroutanjazi Sina , Razavi Allen , Kapur Navin
Published: October 2019

Background:
Acute kidney injury (AKI) is associated with increased morbidity and mortality in patients with acute myocardial infarction (AMI). Use of trans-valvular pumps and veno-arterial extracorporeal membrane oxygenation (VA-ECMO) for patients with AMI is growing. Trans-valvular pumps transfer rotational kinetic energy to blood and generate flow from the left ventricle into the ascending aorta. VA-ECMO drains blood from the venous system and returns oxygenated blood into the descending aorta, thereby increasing systemic perfusion. The impact of these support strategies on renal blood flow and function remains poorly understood. We hypothesized that compared to a trans-valvular pump, VA-ECMO is associated with increased renal injury in AMI.

Methods and Results:
Adult male swine were subjected to left anterior descending artery occlusion for 90 minutes followed by either immediate reperfusion (IRI), trans-valvular pumping (Impella CP) or VA-ECMO starting 30 minutes before reperfusion, or sham-operated controls (n=4/group). Compared to IRI, urinary levels of the biomarker kidney injury molecular 1 (KIM-1) were increased by VA-ECMO, not Impella. Inflammatory factors IL6 and IL1beta were increased by VA-ECMO, not Impella in both plasma and cortex tissue by ELISA analysis. KIM-1 protein expression for precursor KIM-1 and the extracellular domain (soluble) of KIM-1, as well as STAT3, HIF1alpha were analyzed by Western blot. Compared to sham, IRI and VA-ECMO reduced levels of soluble KIM-1 and increased levels of the KIM-1 protein precursor, pSTAT3 and HIF1a in the renal cortex. Impella had no impact on these protein levels.

Conclusion:
This is first study to identify that VA-ECMO, not Impella, increases urinary levels of KIM-1 , a highly sensitive biomarker of acute kidney injury. The shedding of KIM-1 extracellular domain from the renal cortex is associated with systemic inflammatory response. These findings may identity novel approaches to limit renal injury in AMI patients requiring mechanical circulatory support.
Abstract 601: Radiation Therapy Induces Changes in the Endothelial Methylome Affecting Pro-angiogenic Lncrna Expression
Authors: Espinosa-Diez Cristina , Wilson RaeAnna , Mukherjee Rishima , Feltham Marlee , Hudson Clayton , Ruhl Rebecca , Anand Sudarshan
Published: October 2019

Radiation therapy is a big part of standard of care in oncology. However, in the long- term patients treated with radiation are at higher risk of suffering cardiovascular events. It is well described that the genotoxic stress induced by Ionizing radiation in normal cardiovascular tissue triggers genetic and epigenetic changes. In addition to coding mRNAs, non-coding RNAs are also highly regulated by changes in methylation and transcription, leading to a tight regulatory response to damage. Genotoxic stress induces global hypomethylation due to decreased expression of DNA methyltransferases (DNMT). We observed that DNMT1, DNMT3A, and DNMT3b are downregulated in response to radiation treatment, in a dose-response manner in human endothelial cells. We have also observed that methylation changes produced by radiation affect a specific ncRNA cluster, DLK1-DIO3. Previous results from our lab indicate that ncRNAs from this cluster are highly responsive to different genotoxic agents, including radiation. Given the essential role of microRNAs and Long non- coding RNAs (lncRNAs) from the DLK1-DIO3 cluster in cardiovascular development and aging, we are interested in understanding how the epigenetic changes induced by radiotherapy affect to lncRNAs expression, and how they influence cardiovascular health in the long term after treatment. We have identified that a specific lncRNA from the DLK1-DIO3 cluster, MEG9, increases in ECs after exposure to ionizing radiation. Interestingly, knockdown of the individual DNMTs enzymes indicates a significant upregulation of MEG9 when DNMT3b is inhibited, more so after radiation treatment. To explore the role of this lncRNA, we performed loss-of-function studies. MEG9 inhibition not only diminished proliferation but also increased apoptosis through caspase 3/7 activation. Consistent with this phenotype, knockdown of MEG9 decreases growth factor-dependent angiogenesis in a 3D fibrin gel angiogenesis assay. Taken together, our findings illustrate how DNA methylation at particular lncRNA loci can regulate their expression and drive endothelial cell fate decisions. Our work illustrates how epigenetic changes may affect the long- term cardiovascular function of cancer patients submitted to radiation therapy.
Abstract 605: Identification of Differential Roles of Microrna-33a and -33b During Atherosclerosis Progression with Genetically Modified Mice
Authors: Horie Takahiro , Koyama Satoshi , Miyasaka Yui , Kimura Takeshi , Ono Koh
Published: October 2019

Backgrounds:
MicroRNA (miR)-33a targets cholesterol transporter ATP-binding cassette protein A1 or other anti-atherogenic targets and contributes to atherogenesis. Its inhibition or deletion is known to result in the amelioration of atherosclerosis. However, mice lack the other member of miR-33 family, miR-33b, which exists in humans. Thus, precise evaluation and comparison of the responsibilities of these two miRs during the progression of atherosclerosis has not been investigated.

Methods and Results:
The difference between miR-33a and miR-33b were analyzed from multiple directions using genetically modified mice. We generated four strains with or without miR-33a and miR-33b. Comparison of these strains showed distinct distribution and regulation of miR-33 family. In particular, comparison between mice with only miR-33a (wild-type mice) and mice with only miR-33b (miR-33a
-/-
/miR-33b
+/+
) revealed the prevalence of miR-33b in the liver. Such differential expression resulted in a worsened serum cholesterol profile in mice with only miR-33b. On the contrary, in macrophages the expression level of miR-33 family genes was similar and their effects on cholesterol efflux capacity were almost comparable. To evaluate the whole body atherogenic potency, we developed ApoE
-/-
/miR-33a
+/+
/miR-33b
-/-
mice and ApoE
-/-
/miR-33a
-/-
/miR-33b
+/+
mice. ApoE
-/-
/miR-33a
-/-
/miR-33b
+/+
mice developed increased atherosclerotic plaque compared with ApoE
-/-
/miR-33a
+/+
/miR-33b
-/-
mice, in line with the predominant expression of miR-33b in the liver and worsened serum cholesterol profile. On the contrary, a bone marrow transplantation study showed no significant difference, and this was consistent with the relevant expression levels of miR-33a and miR-33b in bone marrow cells.

Conclusions:
miR-33 family exhibited differences in distribution and regulation, and particularly in the progression of atherosclerosis, miR-33b would be more potent than miR-33a.
Abstract 613: Isolating the Pathological Contribution of Detyrosinated Microtubules in Human Myocardial Mechanics
Authors: Caporizzo Matthew A , Chen Christina Y , Bedi Kenneth , Margulies Kenneth B , Prosser Benjamin L
Published: October 2019

Detyrosinated microtubules (dTyr MTs) provide a viscoelastic resistance to myocyte motion and are proliferated in end-stage heart failure. In isolated myocytes, proliferation of the MT network leads to reduced shortening and relaxation, consistent with an underlying viscoelastic resistance, yet a clear contribution of MTs to the passive mechanical properties of myocytes or myocardium in heart failure remains to be determined. To this end, intact human myocytes were isolated from failing and non-failing hearts and their viscoelasticity was measured using a physiological stretch assay where strain-rates were consistent with diastolic filling. In myocytes from heart failure patients, we find an increase in myocyte viscoelasticity and slowing of passive relaxation during cell stretch compared to myocytes from non-failing hearts. Depolymerizing MTs with colchicine, or suppressing dTyr MTs either pharmacologically with parthenolide or genetically by overexpression of tubulin tyrosine ligase reduces myocyte passive viscoelasticity, accelerates passive relaxation time and leads to consistent improvements in contractile and relaxation dynamics in failing cardiomyocytes. Manipulation of dTyr MT levels does not significantly alter the calcium transient during unloaded contraction, suggesting that the observed gain in function is mechanical in nature. We additionally have begun to assay the MT contribution to mechanics at the tissue level. In intact trabeculae from failing hearts, colchicine treatment to depolymerize MTs reduces passive myocardial viscoelasticity upon diastolic stretch. The results indicate that microtubules contribute to myocardial mechanical properties in the context of end-stage heart failure.
Abstract 616: Glycogen Synthase Kinase 3b Localizes to and Modulates Sarcomere Function in Health and Disease
Authors: Kirk Jonathan A
Published: October 2019

Ischemic damage to a region of myocardium renders it weaker than the surrounding tissue, leading to contractile heterogeneity. This heterogeneity causes abnormal stress and strain patterns, further reducing systolic function. We hypothesized glycogen synthase kinase 3β (GSK-3β) may be a therapeutic target for improving function in patients with ischemic damage, as we previously discovered its activity is reduced by abnormal mechanical stress and it can phosphorylate myofilament proteins. We measured GSK-3β levels in LV whole tissue lysate from patients with ischemic cardiomyopathy (ICM) and non-failing rejected donor hearts (NF), but detected no differences. However, we found GSK-3β present at the myofilament, and ICM patients had a ~70% reduction in this localized pool compared to NF controls. Furthermore, GSK-3β phosphorylated at Y216 (pY216), an understudied site, was highly enriched in the myofilament. We used immunofluorescence, co-immunoprecipitation, and adenoviral constructs of Y216 phospho-mimetic or -blocked, in human, mouse, or neonatal rat ventricular myocytes to confirm Y216 phosphorylation results in GSK-3β binding to the myofilament. pY residues can dock to SH2 domains. The only SH2-containing protein that associates with the myofilament is tensin-1 (discovered by us in the myofilament by mass spectrometry). Here we further found tensin-1 localizes to the cardiac z-disk. GSK-3β and tensin-1 co-IP together in myocytes and in HEK cells (in the absence of the myofilament), but this interaction is reduced if Y216 phosphorylation is blocked. To identify the functional consequences of this interaction, we utilized an inducible myocyte specific GSK-3β knock-out mouse. Skinned myocytes from GSK-3β KO mice had significantly depressed calcium sensitivity compared to WT mice. Furthermore, treating skinned myocytes isolated from ICM patients with GSK-3β significantly increased calcium sensitivity, but had no effect in NF cells. These findings identify a novel functional role for GSK-3β in the ischemic human heart. GSK-3β is a promiscuous kinase, but we have discovered a specific regulatory mechanism (Y216 phosphorylation) that could allow precise therapeutic intervention to improve contractile function in ICM patients.
Abstract 617:
In vivo
Titin Oxidation as a Modulator of Sarcomeric Contractibility
Authors: Martinez-martin Ines , Herrero-Galán Elías , Vicente Natalia , Sánchez-González Cristina , Velázquez-Carreras Diana , Bonzón-Kulichenko Elena , Calvo Enrique , Vázquez Jesús , Alegre-Cebollada Jorge
Published: October 2019

Titin is the sarcomeric protein responsible for the passive elasticity of muscle. Its function is based on the unfolding and refolding of the immunoglobulin-like (Ig) domains located in the I-band, thus described as the mechanically active region of the protein. Previous
in vitro
studies have shown that oxidation of cysteines located in these domains modulates the mechanical properties of the protein. However, the extent of these redox modifications and the identity of the modified residues
in vivo
remains unexplored. Here we show for the first time that titin is oxidized
in vivo
and that oxidative modifications target conserved cysteines of the mechanically active Ig domains. We have set up a biochemical method for in-gel determination of oxidized thiols by which we found that titin is remarkably oxidized when compared to myosin, which is not a target of cysteine redox modifications. By mass spectrometry, we also detected that the conserved cysteines previously described as mechanically relevant are in fact oxidized. We propose that characterizing the oxidation of titin Ig domains will lead to a better understanding of the regulation of muscle elasticity, and could explain the pathological effects of an imbalanced redox status, such as during myocardial infarction or ischemia.
Abstract 618: Overexpression of Rrm2B Elevates dATP and Cardiac Function
Authors: Murray Jason , Do Sabrina , McMillen Tim , Regnier Michael , Moussavi-harami Farid
Published: October 2019

Cardiac myosin can use 2-deoxy-ATP (dATP) instead of ATP as energy substrate, significantly enhancing the rate and magnitude of force development and relaxation. Our group has shown repeatedly that overexpression of the enzyme ribonucleotide reductase (RNR; the rate-limiting step in
de novo
dNTP synthesis) in cardiomyocytes, by either transgenic or viral vector approach, enhances cardiac function by significantly elevating cytosolic concentrations of dATP. We are exploring different promotor, gene constructs and engineered mutations of RNR to be able to vary the amount of dATP maintained in cardiomyocytes.

RNR activity is partially governed by levels of the small subunit, Rrm2, whose concentration oscillates with the cell cycle via degradation by the ubiquitin-proteasome system. The p53-inducible small subunit Rrm2B is naturally present in quiescent cells and does not contain the amino acid sequence that is targeted by the ubiquitin-conjugating enzyme. We hypothesized that Rrm2B overexpression may be more stable than Rrm2 through its resistance to degradation by this pathway. Transgenic mice that overexpress Rrm1 and Rrm2B have a ~60 fold increase in myocardial [dATP] as measured by liquid chromatography-mass spectrometry compared to wild-type littermates. This translates to significantly elevated left-ventricular developed pressure as well as higher rates of contraction and relaxation in perfused isolated hearts, as well as enhanced shortening and relaxation in isolated cardiomyocytes.

Isolated rat cells also have an increase in [dATP] and corresponding greater fractional shortening and rates of contraction and relaxation when transduced
in vitro
with adenovirus encoding Rrm1 and Rrm2B when compared to cells transduced with a control vector. We are currently in the process of comparing the effectiveness of adeno-associated viruses encoding Rrm2 and Rrm2B
in vivo.
Our ultimate goal is to develop an improved vector to enable stable and consistent overexpression of RNR and elevation of dATP in cardiomyocytes transduced
in vivo
, thereby improving both systolic and diastolic function for treatment of cardiomyopathies.
Abstract 626: Pro-Inflammatory Signaling by Cardiomyocytes Involves Activation of a RhoA Dependent Gene Expression Pathway
Authors: Brand Cameron S , Moon Hoyoung , Brown Joan Heller
Published: October 2019

Cardiac inflammation, particularly the recruitment of inflammatory cells following injury, precedes cardiac fibrosis. A chronic inflammatory state in the heart increases risk of progression to heart failure. Activation of the small G-protein RhoA occurs in response to cardiac injury, and chronic activation of RhoA has been shown to facilitate maladaptive cardiac fibrosis. We tested the hypothesis that RhoA activation induces early pro-inflammatory signals in the cardiomyocyte which can lead to progression of inflammatory responses and adverse remodeling. Mice were infused with angiotensin-II, an agonist that can activate RhoA signaling and which rapidly induces cardiac inflammation and fibrosis. After one day of infusion, hearts were assessed for RNA levels of various inflammatory genes in wildtype versus cardiomyocyte-specific RhoA knockout mice. A set of genes for pro-inflammatory secreted cytokines associated with macrophage infiltration, including CCL2, IL-6, and IL-1β were upregulated in hearts from control mice infused with angiotensin-II, but these responses were abolished in hearts from RhoA knockouts. These findings indicate that RhoA activation in the cardiomyocyte contributes to the upregulation of these inflammatory signals. Our lab and others have demonstrated the importance of transcriptional responses to RhoA activation, and have shown concomitant downstream activation of the transcriptional co-activators Myocardin-related Transcription Factor A (MRTF-A) and Yes-activated Protein (YAP). With intravenous injection of cardiomyocyte-specific adeno-associated virus serotype 9 (AAV9) expressing Cre recombinase in mice with floxed YAP, we thus tested involvement of YAP in inflammatory gene expression regulated by angiotensin-II activation of RhoA. In summary, we propose that activation of RhoA in the cardiomyocyte facilitates a gene program involved in cardiac inflammatory response, and are examining the role of signaling from cardiomyocytes through MRTF-A and YAP regulated genes for inflammation and immune cell infiltration.
Abstract 632: MTOR-Proteasome Imbalance Upon Deletion of Pras40 Inhibits Cardiac Growth but Results in Cardiac Failure
Authors: RIECHERT Eva , Kern Moritz H , Gorska Agniezka A , Hofmann Christoph , Jürgensen Lonny , Gür Kira , Ho Thanh C , Kirkpatrick Joanna , Rahnis Norman , Katus Hugo A , Völkers Mirko
Published: October 2019

Background:
The mammalian target of Rapamycin complex 1 (mTORC1) increases cell size by initiating translation as well as by inhibiting catabolic functions such as proteolysis or autophagy. We have previously proposed Proline-rich Akt substrate 1 (Pras40) as a cardioprotective endogenous inhibitor of mTOR-dependent protein synthesis during pathologic growth. Pras40 is released from mTORC1 during growth, but other interactions are largely unknown. In a proteomic screen we have now found a novel interaction of Pras40 with the 26S proteasome during hypertrophy. We hypothesize that Pras40 directly links the mTOR-dependent protein synthesis network and the proteasomal degradation machinery to balance both processes.

Methods and Results:
To determine the growth-dependent Pras40-interactome, we identified binding partners by proximity-dependent biotin labeling using APEX2 and subsequent mass-spec. Interestingly, aside from interactions with the mTOR-dependent translation machinery, we find Pras40 binding to subunits of the 26S proteasome only during growth. To test consequences of Pras40 deletion on cardiac function
in vivo
, we generated cardiomycyte-specific knock-out mice, that we subjected to pathologic hypertrophy (TAC). Conversely to Pras40 overexpression, we find growth significantly blunted in KO animals, function reduced and fibrosis elevated. mTORC1 signaling as well as autophagy and proteasomal function are severely disturbed in KO animals. Mechanistically, chymotrypsin-like 26S proteasome activity is blunted in KO hearts as well as isolated cardiomyocytes from KO animals, whereas overexpression shows reciprocal effects in myocytes. Disturbed proteasomal function in KO mice leads to severe alterations in metabolic functions highlighting the importance of both intact mTORC1 signaling and proper proteasomal maintenance during cardiac stress.

Conclusion:
In this study we find a novel mechanism how mTOR and proteasomal function are linked in the diseased heart. We provide evidence that Pras40 links anabolic protein synthesis and catabolic proteolysis in the heart: At rest, Pras40 binds and inhibits mTOR, but when released during pathologic growth, Pras40 directly interacts with the 26S proteasome and modulates its activity.
Abstract 633: Semaphorin3e-Plexind1 Signaling is Required for Cardiac Ventricular Compaction
Authors: Sandireddy Reddemma , Cibi Dasan M , Singh Anamika , Tee Nicole , Uemura Akiyoshi , Epstein Jonathan A , Singh Manvendra K
Published: October 2019

Left ventricular noncompaction (LVNC) is one of the most common forms of genetic cardiomyopathy characterized by excessive trabeculation and impaired myocardial compaction during fetal development. LVNC patients are at higher risk to develop left or right ventricular failure, or both. While key regulators for cardiac chamber development are well studied, the role of Semaphorin-Plexin signaling in this process remains poorly understood. Here, we demonstrate that genetic deletion of Plxnd1, a class 3 semaphorin receptor in endothelial cells, leads to severe cardiac chamber defects, as characterized by excessive trabeculation and non-compaction similar to LVNC patients. Loss of Plxnd1 results in decreased expression of extracellular matrix (ECM) proteolytic genes leading to excessive deposition of cardiac jelly. We demonstrate that Plxnd1 deficiency is associated with an increase in the expression of Notch and its downstream target genes. In addition, inhibition of Notch signaling pathway can partially rescue the excessive trabeculation and non-compaction phenotype present in Plxnd1 mutants. Furthermore, we demonstrate that Semaphorin 3e (Sema3e), one of Plxnd1’s ligands is expressed in the developing heart and is required for myocardial compaction. Collectively, our results demonstrate that the Sema3e-Plxnd1 pathway is essential for myocardial trabeculation and compaction.
Abstract 635: β
2
-adrenergic Stimulation Compartmentalizes β
1
Signaling Into Nanoscale Local Domains by Targeting the C-terminus of β
1
-adrenoceptors
Authors: Wang Lipeng , Yang Huaqian , Gong Yunyun , Fan Xuexin , Zhu Siyu , Wang Xiaoting , Wang Yupu , Li Linlin , Xing Xin , Liu Xiaoxiao , Ji Guangshen , Hou Tingting , Zhang Yan , Xiao Ruiping , Wang Shiqiang
Published: October 2019

β-adrenergic receptors (βARs) are prototypical G-protein coupled receptors that play a pivotal role in sympathetic regulation. In heart cells, β
1
R signaling mediates a global cAMP response, regulating both L-type Ca
2+
channels in the sarcolemma/T-tubules (SL/TTs) and ryanodine receptors in the sarcoplasmic reticulum (SR). In contrast, β
2
AR mediates local cAMP signaling with little effect on the function of SR proteins. Accumulating evidence in pathological and transgenic models has suggested a functional complexity of β
2
AR beyond its regulation of L-type Ca
2+
influx. So we investigated the signaling relationship between β
1
ARs and β
2
ARs. Using whole-cell patch clamp combined with confocal calcium imaging, we simultaneously measured L-type Ca
2+
currents and Ca
2+
transients in adult cardiomyocytes. β
1
AR stimulation with norepinephrine or isoproterenol increased both Ca
2+
currents and Ca
2+
transients. β
2
AR stimulation with Zinterol or Salbutamol only increased Ca
2+
currents (onside compartmentalization) and surprisingly inhibited the ability of β
1
AR in upregulating Ca
2+
transients (offside compartmentalization). Further evidence showed that β
2
AR activation recruited G-protein coupled receptor kinase 2 (GRK2) to cell membrane, which phosphorylated the ‘SSES’ serine cluster on β
1
AR carboxyl terminal. Phosphorylated β
1
AR coupled to β-arrestin1 and further recruited phosphodiesterase-4, leading to localized cAMP degradation. Immunocytochemical and biochemical analysis showed the β
2
AR-mediated offside compartmentalization was confined to the nanoscale domain. A knock-in rat model harboring mutations of the three serine residues in ‘SSES’ cluster of the β
1
AR C-terminus, a component of the putative β-arrestin1 binding site and GRK2 phosphorylation site, eliminated the offside compartmentalization conferred by β
2
AR activation. This finding reveals a fundamental “negative feed-forward” mechanism that serves to avoid the cytotoxicity of circulating catecholamine and to sharpen the transient β
1
AR response of sympathetic excitation, providing many potential novel targets for drug discovery against cardiovascular diseases.
Abstract 644: Enhancing Lymphangiogenesis Ameliorates Chronic Heart Failure
Authors: Song Loujin , Swanson Terri , Chen Xian , Cunio Teresa , LaViolette Brianna , Asano Shoh , Hales Katherine , Pang Jincheng , Robertson Andrew , Ritenour Casey , Boucher Magalie , Roth Flach Rachel J
Published: October 2019

Previous studies have indicated that cardiac lymphatics are involved in the pathogenesis of acute cardiac injuries. However, the role of cardiac lymphatics in the pathogenesis of chronic heart failure remains unclear. The goal of this study is to investigate the role of cardiac lymphatics in the development of chronic heart failure, and to test the hypothesis that improving lymphatic vascular function will have therapeutic benefits in chronic heart failure. First, we characterized lymphatic endothelial marker expression in human hearts from normal or heart failure patients, and a trend towards reduced lymphatic marker expression was observed. These data suggested that lymphatic vessels may be impaired in chronic heart failure. To test this hypothesis further, angiotensin II infusion was used to induce cardiac dysfunction and hypertrophy in mice. Using lymphangiography, we discovered impaired lymphatic transport in angiotensin II-infused mouse hearts compared with control mouse hearts, suggesting impairments in cardiac lymphatic function. Interestingly, when the angiotensin II infused mice were co-administered VEGFCc156s protein to enhance lymphangiogenesis and improve lymphatic vascular function, the VEGFCc156s-treated mice demonstrated improved cardiac function and reduced cardiac hypertrophy compared with angiotensin II infused mice, indicating a potential beneficial effect of VEGFCc156s treatment on angiotensin II-induced cardiac dysfunction and hypertrophy. Overall, these results demonstrate that cardiac lymphatic vessels were impaired in chronic heart failure, and improving lymphatic vascular function may have therapeutic effects on chronic heart failure. The outcome of the study may provide new insights into developing new therapeutics for chronic heart failure.
Abstract 650: Dose-response Effect of Hyperglycemia in Maternal Diabetes Mediated Congenital Heart Defects
Authors: Mansfield Corrin , Manivannan Sathiyanarayanan , Cameron Emily M , Garg Vidu , Basu Madhumita
Published: October 2019

Congenital heart defects (CHDs) are the leading cause of infant morbidity and mortality. The etiology is multifactorial and maternal pre-existing diabetes is a non-genetic risk factor, which perturbs embryonic development and increases the risk of CHDs by 3-to 5-fold. We have previously demonstrated that maternal hyperglycemia (matHG) leads to an increase in reactive oxygen species and reduced nitric oxide bioavailability and found an epigenetic mechanism for altered expression of
Notch1
, a gene critical for endocardial cushion formation, cardiomyocyte (CM) differentiation, outflow tract development, and ventricular trabeculation. We also noted spectrum of cardiac defects in developing embryos when exposed to increased severity of matHG, the underlying molecular basis of which remains unclear. To this end, we hypothesized that matHG alters gene-expression of cardiac progenitor cells in a spatiotemporal manner to predispose embryos to diabetes-induced CHDs. To test cell-specific responses to HG, murine embryonic cell-lines [AVM, atrioventricular cushion mesenchymal and ECC1, endothelial] were cultured in normal (NG, 5.5mM) and different doses of HG (10, 25 and 40mM). RNA-seq was performed after 48hrs of treatment and gene-set enrichment analysis of AVM NG and HG (25mM) identified genes associated with metabolic processes, response to oxidative-stress (OS), chromatin regulation and altered Wnt/Notch signaling pathways with HG. Similarly, RNA-seq analysis of protein-coding genes in ECC1-HG revealed increasing numbers (8, 311, 1072) of differentially expressed genes compared to NG, and associated with OS, ATP-synthesis, cardiac development, suggesting a dose-response relationship. Additionally, a streptozotocin-induced murine model of diabetes was used to characterize the influence of matHG dose on CHD susceptibility. Examination of E9.5, E11.5, E13.5 embryos displayed defects in CM proliferation, ventricular trabeculation, thin ventricular myocardium and septal defects with matHG. Together, our in vitro transcriptomic analysis and in vivo histologic characterization support a variable molecular and phenotypic response to hyperglycemic stress during cardiac development that contribute to matHG associated CHDs.
Abstract 651: Single-nucleus Transcriptome Analysis of Patients’ Heart Tissue Reveals Disease-specific Transcriptional Signatures in Heart Failure
Authors: Fujita Kanna , Nomura Seitaro , Fujita Takanori , Satoh Masahiro , Ko Toshiyuki , Yamada Shintaro , Ito Masamichi , Katoh Manami , Hatano Masaru , Aburatani Hiroyuki , Komuro Issei
Published: October 2019

Single-cell transcriptome analysis is a powerful strategy uncovering heterogeneities of cells and their responses and molecular mechanism in developmental, physiological, and pathological processes. We have applied this technology to cardiac tissue, single-cardiomyocyte RNA-
seq
analysis of heart failure model mice and human failing heart, and we have uncovered heterogeneous stress response during cardiomyocyte remodeling, showing distinct transcriptional dynamics of the cardiomyocyte gene programs. However, it is difficult to simultaneously analyze transcriptomes of cardiomyocytes and non-cardiomyocytes in the archival heart tissue because of its highly interconnected feature and significant differences in cellular characteristics. We established single-nucleus RNA-
seq
analysis pipeline to overcome such problems and comprehensively analyze cells consist of heart tissue, to elucidate molecular mechanism in the pathogenesis of heart failure, combining the data with clinical phenotype of the disease.Single-nucleus analysis of the frozen mouse heart successfully reclassified cardiac cells by their transcriptomic features. By comparing single-nucleus RNA-
seq
profiles with single-cell RNA-
seq
profiles, we found a significant difference of mRNA localization between the cytosol and nucleus. mRNA of nuclear mitochondrial genes is preferentially localized in the cytosol, whereas that of extracellular matrix genes is in the nucleus. We also conducted single-nucleus RNA-
seq
analysis of the heart from patients with heart failure, again successfully reclassified cell types, and found disease specific transcriptomic landscape in heart failure. We are going to integrate single-nucleus gene expression profiles with the phenotypic characteristics such as treatment response and clinical prognosis, to reveal the molecular characteristics useful for cardiovascular precision medicine.