bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 760: Metabolic Syndrome Impairs Cardiac Remodeling During Pregnancy in Mice
Authors: Yang Yijun , Kang Chunya , Borghetti Giulia , Lucchese Anna M , Joo Yunichel , Harper Shavonn , Bryan Christopher , Mohsin Sadia , Houser Steven R
Published: October 2019

Rationale:
Due to the change of life style in the past decades, the incidence of metabolic syndrome (MetS) and obesity in women of childbearing age has been increased. Evidences have shown that complications during pregnancy, such as obesity and gestational diabetes, strongly link to future heart diseases. During normal pregnancy, the heart undergoes a physiological remodeling with mild ventricular dilation and hypertrophy. However, the effects of MetS on cardiac phenotype during pregnancy are not well understood. Moreover, animal models for MetS so far are not well established, which are usually species limited, gender limited and lack of translational significance. Thus, it’s important to develop an animal model which resembles the etiology of MetS in human beings.

Objective:
Establish a model of MetS in female C57BL/6J mice and determine the effects of MetS on cardiac remodeling during pregnancy.

Methods and Results:
To establish a mouse model of MetS, high fat (49% kcal)/high carbohydrates (HF-HC) diet were given to C57BL/6J female mice at the age of 8-week. Mice fed with normal diet (10% kcal fat) served as control. After 14 weeks feeding, mice fed with HF-HC diet showed significantly higher body weight and gonadal fat weight compared with control mice. Moreover, mice fed with HF-HC diet showed impaired glucose tolerance and higher serum total cholesterol level. The symptoms of obesity, impaired glucose tolerance and dyslipidemia suggest the development of MetS. Female mice after 14-week special diet feeding were subjected to breeding. Cardiac function was evaluated by echocardiography during pregnancy and 1-day post-partum, then the mice were terminated 1-day post-partum. Female mice that developed MetS showed impaired cardiac function and pathological remodeling during pregnancy. Moreover, mice fed with HF-HC diet showed more myocardium interstitial fibrosis accompanied with upregulation of fibrosis related genes in the heart compared with Ctrl diet.

Conclusion:
We’ve developed a mouse model of MetS in C57BL/6J mice strain with 14 weeks feeding of HF-HC diet, which resemble the western diet that affects human beings nowadays. Females that have MetS showed impaired cardiac function and pathological remodeling during pregnancy.
Abstract 766: SGLT-Mediated Na
+
Overload Results in Oxidative Stress and Abnormal SR Ca
2+
Release in Diabetic Hearts
Authors: Velmurugan Sathya , Hoskins Amanda , Despa Florin , Despa Sanda
Published: October 2019

Intracellular Na
+
concentration ([Na
+
]
i
) regulates Ca
2+
cycling, oxidative state and electrical stability of the heart. [Na
+
]
i
is linked to glucose uptake through the Na
+
-glucose cotransporter (SGLT). The SGLT1 isoform is present in the heart and its overexpression causes hypertrophy and left-ventricular dysfunction. Here, we hypothesized that cardiac SGLT activity is increased in type-2 diabetes (T2D), which causes myocyte Na
+
overload and results in oxidative stress and exacerbated sarcoplasmic reticulum (SR) Ca
2+
leak. To test this hypothesis, we analyzed myocardial tissue from humans with/without T2D and compared rats with late-onset T2D that display diabetic cardiomyopathy similar to that seen in humans (HIP rats) with their non-diabetic littermates (WT). SGLT1 expression was increased in hearts from T2D patients compared to non-diabetic individuals and in hearts from HIP
vs
. WT rats. SGLT inhibition significantly decreased the uptake of both glucose and Na
+
in myocytes from HIP rats but not in WT, which indicates that SGLT function is increased in T2D hearts. While SGLT1 upregulation may partially compensate for the reduced insulin-dependent glucose uptake, the ensuing raise in Na
+
influx resulted in elevated [Na
+
]
i
in HIP myocytes (by 3 mM compared to WT). Higher [Na
+
]
i
causes oxidative stress by activating the
mitochondrial
Na
+
/Ca
2+
exchanger (mitoNCX), which lowers mitochondrial [Ca
2+
] and thus slows down regeneration of the antioxidant NADPH. In agreement with a role for elevated [Na
+
]
i
in causing oxidative stress in T2D hearts, H
2
O
2
production was increased in HIP myocytes
vs
. WT and mitoNCX inhibition, which uncouples mitochondria from [Na
+
]
i
, significantly reduced H
2
O
2
production in HIP hearts. Oxidative stress enhances the SR Ca
2+
leak directly through oxidation of ryanodine receptors (RyRs) and indirectly via CaMKII activation and consequent RyR phosphorylation. SR Ca
2+
leak was augmented in myocytes from HIP
vs
. WT rats and mitoNCX inhibition significantly reduced the leak in HIP myocytes, but not in the WT. Thus, our data indicate that SGLT activity is enhanced in myocytes from T2D rats, which increases Na
+
influx and causes Na
+
overload. Elevated [Na
+
]
i
contributes to oxidative stress and abnormal SR Ca
2+
leak in diabetic hearts.
Abstract 772: The Highly Prevalent 25bp Intronic Deletion in
MYBPC3
is Benign Under Baseline Conditions
Authors: McNamara James W , Schwanekamp Jennifer A , Patel Parth N , Viswanathan Shiv K , Bohlooly Mohammad , Madeyski-Bengtson Katja , Knöll Ralph , Seidman Jonathan G , Seidman Christine E , Sadayappan Sakthivel
Published: October 2019

Background:
Hypertrophic cardiomyopathy (HCM) affects at least 1 in 500 people worldwide, and results in the thickening of the ventricular walls and reduced cardiac function. Mutations in
MYBPC3
, encoding cardiac myosin binding protein-C, are the most common cause of HCM. Previously, a highly prevalent 25bp deletion within intron 32 of
MYBPC3
was described in the South Asian population. The
MYBPC3
d25bp
variant is present in approximately 100 million people, and encompasses a splicing branch point predicted to result in abnormal splicing of exon 33. Thus, there is a critical need to understand the mechanism by which
MYBPC3
d25bp
may cause cardiomyopathy.

Methods:
To determine the role of the 25bp deletion
in vivo
, knock-in humanized mice were created in which intron 32 was replaced with the human intron 32, with or without the
MYBPC3
d25bp
mutation. Mice were characterized at 3- and 6-months of age by echocardiography, histological, and protein analysis. The presence of aberrant exon splicing was also determined in mice carrying the
MYBPC3
d25bp
variant through RT-PCR and mini-gene assays. Finally, exon trapping experiments were performed to understand the mechanism behind exon skipping.

Results:
Under baseline conditions,
MYBPC3
d25bp
displayed no changes in cardiac function or morphology as measured by echocardiography (FS (%): NTG 35.3%, WT 32.8%, Het 33.7%), heart weight to body weight ratio, or histology. While exon 33 skipping was not detected by RT-PCR, the presence of an alternative splice site within exon 33 was identified in
MYBPC3
d25bp
mice. However, this did not affect the protein levels of cMyBP-C. Furthermore, mini-gene experiments demonstrated that the
MYBPC3
d25bp
mutation significantly reduced the percentage of correctly spliced transcripts (86.2% vs. 77.5%).

Conclusions:
These data demonstrate that the presence of the highly prevalent 25bp deletion is not sufficient to cause disease under baseline conditions. However, it is possible that the increased levels of aberrant splicing may increase the risk for developing HCM.
Abstract 780: Duchenne Muscular Dystrophy Cardiac Exosomes Contribute to the Pathogenesis of Dystrophin-deficient Cardiomyopathy
Authors: Gartz Melanie , Afzal Zeeshan , Strande Jennifer L
Published: October 2019

Introduction:
Cardiomyopathy is a devastating consequence of Duchenne muscular dystrophy (DMD) and its pathogenesis is not well known. Exosomes (exos) are small, secreted vesicles with the ability to exert paracrine effects in target cells by transfer of their molecular cargo. This cargo can be dysregulated in disease and may contribute to pathogenesis. Duchenne muscular dystrophy exos in skeletal muscle promoted pathology, but whether exos are pathological in the DMD heart is unclear.
We hypothesize that exposure to DMD cardiac exos promotes pathogenesis and their inhibition will protect against cardiomyocyte injury.
To investigate, we used DMD iPSC-derived cardiomyocytes (iCM) and
mdx
mouse models of DMD.

Methods/Results:
In vitro,
2 non-affected (N), 2 patient-derived DMD and 1 gene-edited DMD iCMs were exposed to either exos isolated from N- or DMD-iCMs for 48 hr, or to GW4869, an exo inhibitor for 24 hr, followed by 1 hr stress (100 μM H
2
O
2
in 10 mM deoxyglucose in RPMI - glucose) and 4 hr recovery (RPMI + glucose) and stained with either DHE, TMRE and propidium iodide to assess ROS levels, mitochondrial membrane potential (Δψm) and cell death, respectively. Stress increased ROS levels in all 3 DMD-iCMs from 413-1102AU to 955-2008AU, which was diminished with N-exo (346-1061AU) but not DMD-exo (882-1995AU). Stress caused Δψm loss in DMD-iCM from 4049-4082AU to 439-1315AU, and N-exo (1917-2867AU) but not DMD-exo (870-1586AU) were protective. Stress increased cell death in DMD-iCM from 3-10% to 30-45% which was decreased with N-exo (10-21%) but not DMD-exo (26-46%). GW4869 decreased ROS levels from 955-2008AU to 344-528AU
,
cell death from 30-45% to 6-28% and partially rescued Δψm from 439-1315AU to 1947-3842AU in DMD-iCM. In
mdx
mice, GW4869 reduced isoproterenol induced cardiac injury (
GW+Iso: 3±2% vs. Veh+Iso: 13±3%
).

Conclusions:
In vitro
, unlike N-exo, DMD-exo exposure failed to protect DMD-iCM from stress. Inhibiting DMD-exo with GW4869 protected DMD-iCM against cell stress and reduced
mdx
cardiac isoproterenol-induced injury. Together, these data suggest that
DMD-exo may dysregulate the DMD cardiomyocyte response to stress thereby contributing to injury. Furthermore, exosome reduction may be a strategy for the treatment of DMD cardiomyopathy
.
Abstract 782: Human-induced Pluripotent Stem Cell-derived Cardiomyocytes as a Model for Trastuzumab-Induced Cardiac Dysfunction
Authors: Kitani Tomoya , Ong Sang-Ging , Lam Chi K , Rhee June-Wha , Zhang Joe Z , Oikonomopoulos Angelos , Ma Ning , Tian Lei , Lee Jaecheol , Telli Melinda L , Witteles Ronald M , Sharma Arun , Sayed Nazish , Wu Joseph C
Published: October 2019

Background and Objective:
Trastuzumab (Herceptin)-based chemotherapy regimens play a prominent role in treatments of breast cancer, but a significant number of patients develop cardiac disfunction during the therapy. In this study, we examined whether human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) could serve as an effective platform to study this particular cardiotoxicity.

Methods and Results:
We assessed the effects of trastuzumab on structural and functional properties in three control iPSC-CMs from healthy individuals and found that clinically relevant doses of trastuzumab (1 μM) significantly reduced the contraction velocity (-7.4%, P < 0.01) and calcium amplitude (-14.1%, P < 0.01) in iPSC-CMs without inducing cardiomyocyte death or sarcomeric structural abnormalities. RNA-sequencing analysis revealed mitochondrial dysfunction and altered cardiac energy metabolism pathway as primary causes of this cardiotoxicity. Subsequently, we found that significant reductions in basal, maximal, and ATP-linked oxygen consumption (-19.8%, -10.7%, and -19.0%, respectively, P < 0.01 for all) and cellular ATP content (-10.3%, P < 0.01) in iPSC-CMs after trastuzumab treatment. Next, we recruited seven breast cancer patients with trastuzumab therapy, including five patients diagnosed with trastuzumab-induced cardiac dysfunction and generated iPSCs from these patients. We found that contraction velocity and maximal oxygen consumption were more severely reduced by trastuzumab in iPSC-CMs generated from patients who experienced severe cardiac dysfunction from trastuzumab therapy, compared to iPSC-CMs generated from patients who did not experience cardiac dysfunction (-16.6% vs. -1.4%, and -29.4% vs. -3.7%, respectively, P < 0.01 for all). Lastly, we demonstrated that metabolic modulation with AMPK activators, such as metformin, could avert the detrimental effects of trastuzumab in iPSC-CMs.

Conclusion:
Our results indicate that human iPSC-CM model can recapitulate the clinical phenotype of trastuzumab-induced cardiac dysfunction, and therefore targeting altered metabolism may be a promising therapeutic approach for treating this cardiotoxicity.
Abstract 800: HECT Domain Containing-3 (HECTD3) is a Novel Regulator of Cardiac Hypertrophy and Inflammation
Authors: Rangrez Ashraf Y , Borlepawar Ankush , Schmiedel Nesrin , Christen Lynn , Deshpande Anushka , Bernt Alexander , Kumari Manju , Remes Anca , Helbig Andreas , Jungmann Andreas , Sossalla Samuel , Tholey Andreas , Mueller Oliver , Frank Derk , Frey Norbert
Published: October 2019

Objective:
In this study, using a Yeast two-hybrid screen, we identified the HECT domain-containing E3 ubiquitin ligase 3 (HECTD3) as one of the novel cardiac binding partners of SUMO2. Present study thus focusses on deciphering the cardiac function of HECTD3.

Methods and Results:
HECTD3-SUMO2 interaction was further validated by co-immunoprecipitation analysis. Interesting, adenovirus-mediated overexpression of HECTD3 in neonatal rat ventricular cardiomyocytes (NRVCMs) resulted in downregulation of endogenous as well as overexpressed SUMO2. Proteasome inhibitor MG132 treatment however inhibited HECTD3-mediated SUMO2 downregulation, suggesting SUMO2 as a likely substrate of HECTD3 in ubiquitin-proteasome system dependent protein degradation. To get further insights into cardiac substrates of HECTD3, we performed mass-spectrometry analysis of NRVCM protein lysate. Interestingly, several proteins from interferon signaling, including signal transducer and activator of transcription-1 (STAT1), were significantly downregulated by HECTD3 overexpression. Functional dissection of HECTD3-SUMO2-STAT1 interaction revealed attenuation of cellular hypertrophy and LPS mediated activation of inflammatory cascade in NRVCMs. Importantly,
in vitro
findings were consistent
in vivo
, where, AAV-9 mediated overexpression of HECTD3 substantially reduced cardiac hypertrophy and fibrosis, inhibited activation of STAT1-mediated downstream signaling, and attenuated infiltration of inflammatory cells to the heart after transverse-aortic constriction or Angiotensin II treatment.

Conclusions:
In conclusion, we describe here a novel cardioprotective mechanism involving the ubiquitin ligase HECTD3, which exerts anti-hypertrophic and anti-inflammatory effects via dual regulation of SUMO2 and its sumoylation target STAT1. This “dual pathway” inhibition may be exploited for the prevention and/or therapy of cardiac hypertrophy and heart failure.
Abstract 805: A Toll-like Receptor 9 Inhibitor Prevents the Development and Progression of Sterile Inflammatory Heart Failure Induced by Mitochondrial DNA Accumulation
Authors: Ueda Hiromichi , Taneike Manabu , Otsu Kinya , Sakata Yasushi
Published: October 2019

Mitochondrial DNA contains unmethylated cytidine-phosphate-guanosine motif, and is recognized by Toll-like receptor (TLR) 9, inducing sterile inflammation. We previously reported that the accumulation of mitochondrial DNA in cardiomyocytes induces myocardial inflammation and heart failure using an animal model. (Nature 2012) In this study, to investigate the effect of inhibition of the inflammation via TLR9 induced by mitochondrial DNA accumulation on heart failure, we examined whether a TLR9 inhibitor, E6446 (6-[3-(pyrrolidin-1-yl)propoxy)-2-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl]benzo[d]oxazole), prevents the development and progression of heart failure in mice. In in vitro study, isolated cardiomyocytes were stimulated by a TLR9 ligand (ODN1668) in the presence of E6446. ODN1668 significantly increased the expression levels of inflammatory cytokine mRNAs in the cells. Incubation of cardiomyocytes with E6446 significantly reduced the level of those mRNAs induced by ODN1668. CCCP increased the number of autolysosomes with DNA accumulation. Although E6446 had no effect on the number of the autolysosomes, it significantly reduced the production of inflammatory cytokine mRNAs induced by CCCP in cardiomyocytes. In in vivo study, mice orally received E6446 or saline 2 days before transverse aortic constriction (TAC) and every two days for 4 weeks thereafter. Four weeks after TAC, chamber size and fractional shortening (FS) of left ventricle (LV) and lung weight were significantly reduced in E6446 group compared to saline group. Furthermore, the infiltration of inflammatory cells including macrophages in the TAC-operated heart was inhibited by E6446. Next, mice with LV dysfunction at 2 weeks after TAC were subjected to the oral administration with E6446 or saline every two days for 4 weeks. The LV chamber size was significantly smaller and FS was higher in E6446 group than those in saline group 6 weeks after TAC. Our study showed that E6446 prevented the development of LV dilatation and dysfunction with inflammation and slowed progression of cardiac remodeling induced by pressure overload. E6446 might be a novel therapeutic to treat patients with heart failure.
Abstract 808: Functional Characterization of a Novel Scn5a Mutation Associated With the Brugada Syndrome
Authors: Frosio Anthony , Molla David , Bertoli Giorgia , Bazzini Claudia , Milanesi Raffaella , Gennaro Francesca , Barbuti Andrea , Bucchi Annalisa , Moretti Luciano , Marchese Procolo , DiFrancesco Dario , Baruscotti Mirko
Published: October 2019

Background:
Brugada syndrome (BrS) is a cardiac disorder characterized by conduction abnormalities that can lead to sudden death; syncope and cardiac arrest are clinical manifestations which are often associated with an enhancement of the vagal activity. Mutations in the SCN5A gene (Na
v
1.5 channel) are the most common cause of the inherited forms of BrS.

Objective:
To characterize the functional behavior of mutant Na
v
1.5 channels expressing a novel heterozygous mutation (S805L) recently identified in an Italian family affected by the BrS.

Methods:
HEK cells were used as experimental model to express both the wild-type (WT) and the mutated S805L channels (alone, Homo or in combination, Hetero) and the accessory β-subunit (SCN1B). Patch-clamp and western blot experiments were carried out to assess the dysfunctional role of the mutation.

Results:
When compared to the WT current, the S508L mutation significantly (P&It0.05) decreases the peak current density by about 65% for the Homo condition (WT: -120.2±10.2, n=28); Homo: -40.3±4.2, n=16) and by 35% for the Hetero condition (Hetero: -78.2±8.3, n=27). Densitometric analysis carried out on western blot data further support the conclusion that S805L channels are less abundant in the plasma membrane. We also observed that the S805L mutation positively shifts the V½ values of the voltage dependence of the inactivation of both Homo and Hetero currents (V½: WT -85.5±0.2 mV, n=55; Homo -80.9±0.3 mV, n=22; Hetero -81.9±0.2 mV, n=25; P&It0.05); a positive shift of the V½ of the activation was also observed but only in the Homo condition (V½: WT -33.0±0.4 mV, n=28; Homo -30.0±0.5, n=16, P&It0.05). The kinetics of recovery from inactivation and the amplitude of the late sodium current were also evaluated but they were unaffected by the mutation.

Conclusion:
When expressed in the Hetero condition, the S805L mutation causes a reduction in the channel expression, however, the positive shift of the inactivation curve suggests an increase in Na channel availability. We thus believe that the precise quantitative balance between these two phenomena and their relation with vagal activity may underlie the clinical manifestation of the disease.
Abstract 822: Enhanced Left Ventricle TGFβ2 Signaling in a Model of Dilated Cardiomyopathy
Authors: Antolic Andrew , Alsalem Ahmed B , Lee Da Young , Jiao Zhe , Burke Michael A
Published: October 2019

Pathological gene transcription and induction of proinflammatory signaling is a hallmark of dilated cardiomyopathy (DCM) and HF. Previous studies from our lab identified TGFβ2 as a putative driver of inflammation during DCM progression. To study the role of TFGβ2 in DCM hearts, we analyzed TFGβ signaling networks, as well as TFGβ2-induced gene expression changes from mice carrying a human DCM mutation in phospholamban (PLN
R9C
). ELISA was used to confirm TGFβ isoform levels in PLN and wild type (WT) hearts: only TGFβ2 was significantly increased in PLN
R9C
mice (TGFβ1: 1.6-fold, p=0.15; TGFβ2: 9.6-fold, p=0.01; TGFβ3: 1.0-fold, p=0.88) (Figure). Levels of total SMAD2 (1.5-fold, p=0.002), phosphorylated SMAD2 (P-SMAD2; 3.9-fold, p=0.0003), and the P-SMAD2/SMAD2 ratio (2.6-fold, p=0.0004) were greater in PLN
R9C
mice. Treatment with exogenous TGFβ2 results in activation of cultured cardiac fibroblasts from WT hearts (
Col1a1
(3.3-fold),
Col4a1
(2.7-fold),
Ctgf
(6.4-fold),
Eng
(2.6-fold),
Fn1
(2.8-fold),
Lox
(2.6-fold),
Mmp2
(2.8-fold),
Postn
(4.1-fold),
Rhob
(3.6-fold),
Tgfb1
(3.6-fold),
Tgfb2
(2.9-fold),
Tgfb3
(3.0-fold),
Tie1
(2.4-fold),
Thsb1
(4.3-fold)). Our results demonstrate that TGFβ2 levels are increased and that TGFβ signaling is activated in PLN
R9C
hearts with DCM. These data suggest that TGFβ2 is a specific and potent inflammatory mediator in the heart and mark this isoform as a potential therapeutic target in DCM.
Abstract 827: Single-cell Transcriptomic Profiling Provides Insights Into Disease-related Processes During Hypertrophic Cardiomyopathy
Authors: Eding Joep , Wright Clark Maya , de Leeuw Anne , Molenaar Bas , Vink Aryan , Michels Michelle , van der Velden Jolanda , van Rooij Eva
Published: October 2019

Hypertrophic cardiomyopathy (HCM) is a genetic disease usually caused by a heterozygous mutation in a sarcomeric gene. Histologically, HCM is characterized by a heterogeneous population of hypertrophic, disorganized cardiomyocytes and fibrosis of heart tissue. This hypertrophic growth can lead to outflow tract obstruction which can ultimately lead to progressive heart failure due to cardiac overload.

Here we use single-cell RNA sequencing (scRNA-Seq) on septal myectomy samples from patients suffering from HCM to interrogate cellular heterogeneity in disease context. Using our previously optimized digestion and sorting protocol we were able to obtain good quality RNA for single cell analysis. Bioinformatic clustering analysis of single cell transcriptomes revealed 8 transcriptomically distinct subpopulations of cardiomyocytes. Our data indicate an NPPA/NPPB enriched cluster of cells and show TTN to function as an important determinant of the cellular heterogeneity. Correlation analysis links TTN expression to the expression of, among others, cardiomyopathy-associated protein (CMYA) 5 and 3, while there appears to be a negative correlation with, among others, TMEM212. We were able to confirm these correlations by RT-PCR on bulk RNA in an additional cohort of 97 HCM myectomy samples. Additionally, immuno-histochemical staining on myectomy samples and explanted HCM hearts confirmed the transcriptional heterogeneity among cardiomyocytes. A unique advantage of FACS-based single cell sorting cardiomyocytes is the availability of index data for the sorted cells. Correlation analysis of forward scatter, as a proxy for cell size, confirmed both well-known stress markers MYL2 and MYL7 and additional genes to be upregulated in the larger cells. In summary, we show that FACS-based sorting of HCM cardiomyocytes allows for scRNA-Seq of intact cardiomyocytes. Initial analyses of the data could be validated in a wider cohort of comparable samples, showing reliability of the data. Finally, incorporation of index data reveals genes correlated with cell size. Further interrogation of this data has the potential to reveal novel insights into the pathogenesis of HCM.
Abstract 844: β
IV
-spectrin Regulates Cardiac Fibroblast Phenotype, Fibrosis, and Cardiac Function
Authors: Patel Nehal J , Nassal Drew M , Scandling Benjamin W , Greer-Short Amara D , Unudurthi Sathya D , Xu Xianyao , Mohler Peter J , Gooch Keith J , Hund Thomas J
Published: October 2019

Increased fibrosis is associated with cardiac dysfunction and arrhythmias. Activation of quiescent cardiac fibroblasts (CFs) occurs in response to injury although the underlying mechanistic pathways remain unclear. The cytoskeletal protein β
IV
-spectrin has been shown to control targeting and activity of the multifunctional transcription factor Signal Transducer and Activator of Transcription 3 (STAT3) for maladaptive remodeling. The mechanism linking spectrin dysfunction to altered STAT3 signaling, fibrosis, and function remains unknown. The objective of this study was to investigate the role of stress-induced disruption of β
IV
-spectrin/STAT3 complex in modulating CF phenotype, fibrosis, and cardiac function. Cardiac function (echocardiography) and fibrosis (Masson’s trichrome) were evaluated in adult wildtype (WT) and

qv
4J

mice expressing truncated β
IV
-spectrin lacking STAT3 interaction. A subset of WT mice were subjected to 6 wks of transaortic constriction (TAC) to induce heart failure. From these groups, CFs were isolated from ventricles and analyzed for STAT3 localization, gene expression, and activity. CFs were treated with selective STAT3 inhibitor S3I-201 (100μM) or transfected for 72 hrs with β
IV
-spectrin fragment from repeat 10 to C-terminus (β
IV,10-C
, contains STAT3 binding motif). Ejection fraction was decreased in

qv
4J

and WT TAC relative to WT baseline (49.5±0.85% vs 34.9±2.2% vs 63.2±0.60% respectively, p<0.05, N=5. Ventricular fibrosis was augmented in

qv
4J

and WT TAC compared to WT baseline (2.4±0.09% vs 3.2±0.25% vs 0.48±0.01% respectively, p<0.05, N=5).

qv
4J

CFs showed STAT3 mislocalization (49.7±10.7% nuclear accumulation vs 5.3±.1.8% in WT, p<0.05, N=5) with higher STAT3 transcriptional activity (2.6±0.17-fold increase over WT, p<0.05, N=9). In parallel,

qv
4J

CFs displayed elevated expression of fibrotic genes associated with CF activation. At the functional level,

qv
4J

CFs showed increased proliferation (1.9±0.19-fold increase over WT at 24 hrs, p<0.05, N=4). STAT3 inhibition with S31-201 or transfection with β
IV,10-C
normalized gene expression and proliferation in

qv
4J

CFs compared to WT. These studies identify a requirement of β
IV
-spectrin for normal STAT3 signaling and quiescent phenotype in CFs.
Abstract 852: The Pathological Role of Coagulation Factors in Promoting Brown Adipose Tissue Dysfunction and Systemic Metabolic Disorder in Obesity
Authors: Hayashi Yuka , Shimizu Ippei , Yoshida Yohko , Ikegami Ryutaro , Katsuumi Goro , Suda Masayoshi , Fujiki Shinya , Minamino Tohru
Published: October 2019

Obese individuals are predisposed to cardio-metabolic disorders. Brown adipose tissue (BAT) is an active metabolic organ abundant with mitochondria, and studies suggest a potential role of BAT in the maintenance of metabolic health in rodents and humans. Metabolic stress causes BAT dysfunction, but the underlying mechanisms are largely unknown. Coagulation factor Xa (FXa) is critically involved in a coagulation cascade, and it is also known to mediate biological effects by the activation of protease-activated receptor (PAR)-signaling. Accumulating evidence shows that PAR1 contributes to tissue remodeling in cardiovascular system. Here we show a previously unknown role of FXa-PAR signaling in promoting BAT dysfunction and systemic metabolic disorder in a murine dietary obese model.

Imposing a high fat diet (HFD) on C57BL/6NCr mice led to a marked increase in tissue factor (TF), coagulation factor VII and FXa in BAT. TF-FVIIa (activated form of FVII)-FXa complex is known to activate PAR1, and we found a significant increase in PAR1 expression in BAT upon metabolic stress. Administration of a FXa inhibitor ameliorated BAT whitening, improved thermogenic response and systemic glucose intolerance upon dietary obesity. In contrast, administration of warfarin did not show any phenotype in BAT. BAT specific TF and PAR1 over-expression model showed significant whitening of this tissue, which was associated with systemic glucose intolerance. BAT specific PAR1 KO mice improved glucose intolerance and thermogenic response under a metabolically stressed condition. In differentiated brown adipocytes, FXa markedly increased mitochondrial reactive oxygen species (ROS) and reduced mitochondrial membrane potential. Inhibition of PAR1 ameliorated FXa-induced mitochondrial ROS production and reduction in membrane potential. We also found that plasma FXa level did not increase in obese mice as well as in obese individuals. These results suggest the previously unknown role of coagulation systems in promoting BAT dysfunction, leading to systemic metabolic disorders. Maintenance of BAT homeostasis through the suppression of FXa-PAR1 signaling would become a new therapeutic target for obesity and diabetes
Abstract 855: Crif1 Haploinsufficiency in Heart Impairs Mitochondrial Oxidative Capacity and Leads to Aggravated Cardiac Dysfunction in Ioproterenol Infusion Model
Authors: Jin Seon-Ah , Seo Hee Jung , Jeong Jin-Ok
Published: October 2019

Heart requires high energy metabolism to maintain contractile activity and pump sufficient blood flow to body. The optimal supply of energy from the mitochondria is very important for maintaining normal cardiac function. Crif1 is a critical protein for the synthesis and insertion of the OxPhos complex in mitochondria. Recent studies reported that conditional knockout of Crif1 in specific tissues is associated with mitochondrial dysfunction. To investigate the role of Crif1 and mitochondrial dysfunction in heart, we generated cardiac-specific, Crif1 haploinsufficient mice. Haploinsufficient Crif1 in heart resulted in abnormal structure of mitochondria and decreased maximal values of oxygen consumption rates in cardiomyocytes. Although cardiac specific, haploinsufficient Crif1 results in mitochondrial dysfunction, cardiac phenotype was still normal. However, their cardiac function was aggravated by stress condition, showing more decreased EF and FS after 4 weeks isoproterenol infusion. Cardiac hypertrophy, which is considered as normal compensatory mechanism, was not occurred during isoproterenol infusion. These indicate that Crif1 plays a critical role in the maintenance of mitochondrial structure and function of cardiomyocyte. Also, mitochondrial abnormalities in heart inhibit the compensatory mechanisms for stress and aggravate cardiac function.
Abstract 868: A Cardiac Specific Branched Chain Aminotransferase Deletion Increases Insulin Stimulated Glucose Oxidation in the Mouse Heart
Authors: Uddin Golam M , Pherwani Simran , Wagg Cory S , Gopal Keshav , Batran Rami A , Zhang Liyan , Wu Yikuan , Hussaini Naayema , Rawat Sonia , Ussher John R , Lopaschuk Gary D
Published: October 2019

Impaired branched chain amino acid (BCAA) oxidation at the level of downstream BCAA catabolic enzymes cause cardiac insulin resistance and dysfunction. However, it is not known whether it is the accumulation of BCAAs or branched chain keto acids (BCKAs) due to impaired cardiac BCAA oxidation that result in these cardiac metabolic detrimental effects. Our objective was to inhibit cardiac branched chain aminotransferase (BCATm), the first enzyme in the BCAA oxidative pathway, which functions to produce BCKAs. We anticipate deletion of upstream BCAA catabolic enzyme will result in reduced cardiac BCAA oxidation and increase BCAAs but decrease BCKAs. Ten-week-old BCATm cardiac specific knockout (BCATm
-/-
) male mice and their α-MHC-Cre expressing wildtype littermates (WT-Cre
+/+
) received 6 intraperitoneal injections of tamoxifen (50 mg/kg). All mice were allowed a 6-week washout post-tamoxifen, following which 16-week-old mice were used to assess cardiac energy metabolism. Isolated working hearts were perfused with oxygenated Krebs–Henseleit solution, consisting of either 5mM [5-
3
H/U-
14
C] glucose, 0.8mM palmitate, 3%BSA, 0.5mM BCAA for glycolysis and glucose oxidation measurements, or 0.5mM glucose, 0.8mM [9,10-
3
H] palmitate, 3%BSA and 0.5mM [U-
14
C] BCAA from combination of 0.15mM leucine/isoleucine and 0.2mM valine for fatty acid oxidation and BCAA oxidation measurements respectively. There was no body weight, glucose tolerance or cardiac function differences observed in BCATm
-/-
mice compared to WT-Cre
+/+
mice (control). As expected, cardiac BCAA oxidation was significantly reduced in BCATm
-/-
mice compared to control, however, adding insulin after 30 min of perfusion did not change BCAA oxidation rates. Interestingly, glucose oxidation was significantly higher in BCATm
-/-
mice. Adding insulin further increased glucose oxidation in BCATm
-/-
mice. Additionally, the contribution of glucose oxidation to ATP production was significantly higher in the BCATm
-/-
mice compared to control. We conclude that impaired BCAA oxidation due to the upstream catabolic enzyme deletion increases cardiac insulin sensitivity. This also indicates that accumulation of BCKAs, not BCAAs, may be primarily contributing to cardiac insulin resistance.
Abstract 872: Inhibition of Spinal Astrocytes Activation Attenuates Myocardial Ischemia-Reperfusion Injury by Regulating NGF/TRPV1 Nociceptive Signaling
Authors: He Shufang , Liu Rongrong , zhang Li , Zhang Ye
Published: October 2019

Background and Aim:
Spinal astrocytes activation are implicated in nociception and pain processing after noxious stimuli, while its role in cardiac ischemic nociception is unknown. We recently reported spinal nerve growth factor (NGF)-sensitized transient receptor potential vanilloid 1 (TRPV1) nociceptive signaling following cardiac ischemia and reperfusion (I/R). This study was design to investigate whether spinal astrocytes are activated following myocardial I/R and its effects on spinal NGF-TRPV1 signaling.

Methods and Results:
Myocardial I/R injury was induced by 30 min occlusion of the left coronary artery followed by 120 min of reperfusion in rats. Glial fibrillary acidic protein (GFAP), an astrocyte marker, was found up-regulated in thoracic spinal cord during ischemia and reperfusion. Intrathecally pre-injection of fluorocitrate (FC), an astrocyte inhibitor, reduced infarct size (IS/AAR, area at risk), from 48.4±2.67 to 29.7±3.13 (P<0.05, Fig.1 A). The high level of cardiac troponin T caused by I/R was decreased by FC injection as well (from 530±65 to 129±50 μg/μl, P<0.01, Fig.1 B). Upon immunofluorescence staining, GFAP immunopositive cells in spinal cord were found markedly increased after I/R, while the staining was suppressed by intrathecal FC administration (Fig.1 C). Moreover, the up-regualtion of NGF and TRPV1 protein levels in spinal cord were both inhibited by FC pretreatment (Fig.1 D-F).

Conclusion:
Myocardial I/R induced the activation of spinal astrocytes, inhibition of which reduces infarct size and cTnT level, as well as NGF-TRPV1 signaling, which suggests a novel therapeutic target for preventing myocardial ischemic injury.
Abstract 882: The Valosin-containing Protein Resists Pathological Cardiac Calcium Overload via Inhibiting Mitochondrial Calcium Uptake
Authors: Stoll Shaunrick , Ma Ben , Behringer Erick J. , Qiu Hongyu
Published: October 2019

Stress-induced mitochondrial calcium (Ca
2+
) overload is a trigger of cardiomyocyte death though the opening of mitochondrial permeability transition pore (mPTP) during cardiac ischemic injury. We previously found that the valosin-containing protein (VCP), an ATPase-associated protein, protects cardiomyocytes against stress-induced death and also inhibits mPTP opening in vitro. However, the underlying molecular mechanisms are not fully understood. Here, we tested our hypothesis that VCP protects cardiomyocytes against stress-induced mPTP opening through resisting excessive mitochondrial Ca
2+
uptake. By using a cardiac-specific transgenic (TG) mouse model in which VCP is overexpressed by 3.5 folds in the hearts, we found that, under the pathological extra-mitochondrial Ca
2+
overload (10-70μM), Ca
2+
entered into cardiac mitochondriawas much lessin VCP TG mice compared to little-matched WT mice, subsequently preventing mPTP opening and ATP depletion under the Ca
2+
challenge. Mechanistically, overexpression of VCP in the heart results in post-translational protein degradation of the mitochondrial Ca
2+
uptake protein 1 (MICU1), an activator of the mitochondria Ca
2+
uniporter (MCU) which is responsible for mitochondrial calcium uptake. Together, our results reveal a new regulatory role of VCP in cardiac mitochondrial Ca
2+
homeostasis and unlocks the potential mechanism by which VCP confers its cardioprotection.
Abstract 890: Vascular MicroRNA-204 Promotes Diabetes-Associated Endothelial Dysfunction
Authors: Gaddam Ravinder R , Kim Young-Rae , Gabani Mohanad , Proff Jannik , Jacobs Julia S , Irani Kaikobad , Vikram Ajit
Published: October 2019

Background:
The failure to achieve a decline in blood pressure at rest in type 2 diabetes (T2D) patients is believed to reflect endothelial dysfunction, and it increases the risk of coronary artery disease. Previously we discovered that the intestinal microbes govern vascular endothelial function through microRNA-204 (miR-204). The objective of this study was to investigate whether miR-204 contributes to diabetes-associated vascular endothelial dysfunction.

Methods:
To test miR-204's role in diabetes we generated leptin receptor mutant genetically diabetic mice lacking miR-204 (db/db-miR-204
-/-
). The vascular function was assessed by determining the vessel relaxation of phenylephrine-induced precontracted aortic rings. To ascertain the vascular miR-204 role in endothelial function
ex-vivo
experiments were performed, on aorta isolated from db/db mice, using miR-204 inhibitor. The miR-204 expression and localization were done using quantitative real-time PCR (qRT-PCR) and In-situ hybridization (ISH) assays.

Results:
A significant increase in the vascular miR-204 expression was observed in the thoracic aorta of db/db mice compared to the age-matched control (208.80 ± 45.60 vs. 80.65 ± 13.11; p<0.05). The genetic deletion of miR-204 rescues endothelial function (improved vascular relaxation; 36.17 ± 7.42 vs. 76.56 ± 4.43; p<0.001). The db/db-miR-204
-/-
mice develop obesity but are protected against diabetes (as evidenced by better glycemic control).
Ex-vivo
inhibition of
miR-204
rescues diabetes-associated impairment of endothelium-dependent vasorelaxation (18.10 ± 2.16 vs. 50.64 ± 7.66; p<0.01). Next, we plan to identify the molecular mediator of miR-204 effects on the vascular endothelial function using Argonaute 2-crosslinked immunoprecipitation (Ago2-CLIP) assay.

Conclusions:
Type 2 diabetes upregulates vascular miR-204, and genetic deletion of miR-204 rescues diabetes-associated impairment in endothelial function.
Abstract 895: The Role of Poly(rC)-Binding Protein-1 in Heart Development
Authors: Lu Yao Wei , Fernandes Tiago , Guo Haipeng , Hu Xiaoyun , Espinoza-Lewis Ramon A , Wang Da-Zhi
Published: October 2019

Poly(rC)-Binding Protein-1 (Pcbp1) belongs to the KH homology superfamily of nucleic acid-binding proteins, which has been implicated in a vast array of biological processes such as iron transport, RNA processing, post-transcriptional and translational regulations. Germline deletion of Pcbp1 results in embryonic lethality before embryonic day (E) 8.5. To investigate the role of Pcbp1 in heart development, we generated cardiac-specific conditional deletion of Pcbp1 (Pcbp1-cKO) by crossing the Pcbp1-flox with cTNT-Cre mice. The observed frequencies for Pcbp1-cKO at E12.5 and E16.5 are normal, but no surviving Pcbp1-cKO mice were observed at weaning, suggesting the Pcbp1-cKO is perinatal lethal. E12.5 Pcbp1-cKO hearts are smaller with thin myocardium. At E16.5, Pcbp1 cKO hearts displayed ventricular non-compaction and abnormal ventricular apex formation. To understand molecular mechanisms, we perform RNA sequencing follow by differential gene expression analysis in Pcbp1-cKO hearts at E16.5. We found 241 genes were significantly dysregulated and identified unfolded protein response as a key pathway dysregulated. Furthermore, through alternative splicing analysis, we identified 168 uniquely spliced junctions in 139 genes. Of these, 10 differentially spliced genes are also differentially expressed. Future studies will be performed to better understand the biological function and mechanism of Pcbp1 in the heart. Together, this study suggests Pcbp1 is an important regulator of heart development.
Abstract 901: Muscle-specific Stress Fibers Give Rise to Sarcomeres in Cardiomyocytes
Authors: Burnette Dylan T , Fenix Aidan M , Neininger Abigail C , Taneja Nilay , Hyde Karren , Visetsouk Mike R , Garde Ryan , Liu Baohong , Nixon Benjamin R , Manalo Annabelle E , Becker Jason R , Crawley Scott W , Bader David M , Tyska Matthew J , Liu Qi , Gutzman Jennifer H
Published: October 2019

The sarcomere is the contractile unit that drives muscle contraction. Despite its importance, little is understood about how a disordered acto-myosin distribution converts into an ordered contractile array during sarcomere assembly. Here, we take advantage of a sarcomere assembly assay we developed using human induced pluripotent stem cell derived cardiomycytes to image the formation of sarcomeres, using live-cell high resolution microscopy. Our data show that a population of muscle specific stress fibers (MSFs) are essential sarcomere precursors. Interestingly, MSFs are formed at the leading edge of cells, undergo retrograde flow, and transition into sarcomere-containing myofibrils on the dorsal surface of cardiomyocytes. This is in direct contradiction to a recent report claiming sarcomeres are formed from adhesions on the ventral surface of cardiomyocytes. We have been able to recapitulate this other group's published experiments and definitively show that sarcomeres are not forming from the bottom of the cells or streaming out of adhesions. Instead, our 3D microscopy data shows that this group was imaging sarcomeres which were already formed on the dorsal surface and were traveling to the ventral surface of the cells. After this important clarification, we used our assay to show that the transition of MSFs to sarcomere-containing myofibrils requires formin-mediated actin polymerization and the non-muscle myosin IIA and myosin IIB. We conclude that sarcomeres form by a "templating" mechanism similar to that originally postulated by Howard Holtzer >30 years ago. Furthermore, our data show short β cardiac myosin II filaments are themselves templated by "non-muscle" myosin II filaments. Subsequently, the short β cardiac myosin II filaments grow to form ~1.5 μm long filaments that then “stitch” together to form the stack of filaments at the core of the sarcomere (i.e., the A-band). Taken together, our data show that the differentiation of cardiomyocytes from stem cells is a powerful tool for dissecting the mechanisms controlling sarcomere assembly.
Abstract 916: Evidence of Cardiac Myosin Dephosphorylation by Two Distinct Pools of Myosin Light Chain Phosphatases
Authors: Chang Audrey N
Published: October 2019

Muscle myosins are hexameric proteins comprised of two each of the heavy chain, essential light chain, and regulatory light chain (RLC). In normal beating hearts, cardiac myosin RLC is phosphorylated, and the extents maintained at ~0.45 mol phosphate/mol RLC by the balanced activities of cardiac-specific myosin light chain kinases (cMLCK) and phosphatases (cMLCP). Hearts in failure have decreased cMLCK expression and RLC phosphorylation, indicating maintenance of near half-maximal RLC phosphorylation levels is necessary for normal function. However, how cMLCK and cMLCP activities are regulated is unclear. MLCP has been biochemically defined as a trimeric protein comprised of a regulatory myosin target subunit (MYPT1 in smooth muscles, and MYPT2 in striated muscles), a catalytic subunit PP1cβ, and a small subunit M20. Interrogations into the specificity and regulatory activity of smooth muscle MLCP using conditional MYPT1 knockout animals have provided evidence of MYPT1-independent RLC phosphatase activity, which contradicts long-standing dogma. Extending upon recent studies of PP1cβ knockout in smooth muscles, we knocked out PP1cβ from the cardiac muscle to test the hypothesis that a distinct MYPT-independent pool of PP1cβ activity directly contributes to cRLC dephosphorylation. Two weeks post gene-ablation, PP1cβ protein was reduced by >60%, and cRLC phosphorylation in beating hearts was increased to 0.6 mol phosphate/mol cRLC, without significant changes in MYPT1 or MYPT2 expression. Additionally, we measured MYPT-dependent and MYPT-independent PP1cβ phosphatase activities toward cRLC dephosphorylation in mouse cardiac myofibrils. These studies provide further evidence that cardiac RLC is dephosphorylated by two distinct pools of MLCP activities.
Abstract 920: Mutual Potentiation Between Myofibril Assembly and Serum Response Factor in Cardiomyocyte Maturation
Authors: Guo Yuxuan , Jardin Blake , Sethi Isha , Moghadaszadeh Behzad , Beggs Alan , Pu William
Published: October 2019

Cardiomyocyte (CM) maturation is characterized by transcriptional, morphological and functional specializations that are essential for robust and sustained CM contractions throughout lifetime. The signal networks that govern CM maturation remain poorly defined, which obscures the role of CM maturation in inherited cardiomyopathies and myocardial regeneration and impairs efforts to engineer mature cardiac tissues
in vitro
. Our prior studies established the transcription factor serum response factor (SRF) as a key regulator of CM maturation: SRF regulates major CM maturation events including myofibril expansion, mitochondria biogenesis, transverse-tubule formation, and cellular hypertrophy. Myofibrillar genes were identified as direct SRF downstream targets that are required for other aspects of CM maturation. To further understand the role of myofibrils in CM maturation, here we report the generation and investigation of a floxed allele of
Actn2
in mice, which encodes a central component of sarcomere Z-lines. We applied to these mice a low dose of adeno-associated virus that expressed Cre recombinase specifically in neonatal CMs to generate hearts with mosaic
Actn2
mutation. This approach circumvented the confounding secondary effects of cardiac dysfunction in
Actn2
mutants and revealed cell-autonomous gene functions. Strikingly,
Actn2
ablation triggered dramatic transcriptional dysregulation in addition to the expected myofibrillar disassembly phenotypes in CMs, which strongly correlated with observations in SRF-depleted CMs.
Actn2
mutation increased the amount of monomeric actin in CMs, which perturbed the nuclear localization of SRF cofactors MRTF-A/-B. Overexpression of a dominant-negative MRTF-A isoform was sufficient to recapitulate the transcriptional and morphological defects in
Actn2
or
Srf
mutant CMs. Together, these data demonstrate mutual potentiation between myofibril assembly and MRTF-A/B-SRF signaling in CM maturation. This positive feedback loop underlies a novel mechanism by which mechanical forces regulate CM maturation, disruption of which likely contributes to cardiomyopathies caused by sarcomere gene mutations.