bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 437: PRMT5-induced Arginine Methylation Mediates Energy Stress-induced Autophagy in the Heart
Authors: Mukai Risa , Saito Toshiro , Zhai Peiyong , Sadoshima Junichi
Published: October 2019

Autophagy is a self-degradative process that is important for protecting cells and organisms against stress and plays a protective role in the heart. However, the underlying mechanism by which autophagy protects the heart against stress is poorly understood. ATG proteins are required for autophagy and modulated by post-translational modifications (PTMs) such as phosphorylation, ubiquitination, and acetylation. The level of arginine methylation, another form of PTM, is altered in the heart in response to stress. Our hypothesis is that arginine methylation plays an important role in the regulation of autophagy in the heart during stress. To this end, we examined the level of arginine methylation in response to energy stress
in vivo
. We observed a time-dependent increase in arginine methylation in the mouse heart under starvation conditions for 24 to 48 hours. Arginine methylation was increased (2.32-fold, p<0.01) after 48 hours of fasting compared to in those without fasting. Expression of protein arginine methyltransferase 5 (PRMT5) was increased at both the mRNA and protein level (1.6- and 1.72-fold, p<0.05) in the mouse heart after 48 hours of starvation. In order to investigate the role of endogenous PRMT5, we knocked down PRMT5 with adenovirus harboring sh-RNA. LC3-II, a marker of autophagy activity, was increased during glucose deprivation in sh-scramble-treated cardiomyocytes (CMs) (2.14-fold, p<0.01), but not in sh-PRMT5-treated CMs. Arginine methylation was reduced in response to glucose deprivation in CMs when PRMT5 was downregulated. Mice were also injected intraperitoneally with a PRMT5-specific inhibitor (EPZ015666, 2 mg/kg) and hearts were harvested after 48 hours of starvation. Both arginine methylation levels and autophagy activity in the heart were decreased in the presence of EPZ015666 compared to in non-treated mice. Increased arginine methylation was also observed in the mouse heart in response to 1 hour of myocardial ischemia (1.87-fold, p<0.01), which was accompanied by upregulation of PRMT5 (1.54-fold, p<0.05). Taken together, these data suggest that arginine methylation mediated through PRMT5 may play a key role in mediating autophagy during energy stress conditions such as starvation and ischemia.
Abstract 438: GRAF1 is a Novel Mediator of Parkin-dependent Mitophagy
Authors: Zhu Qiang , Combs Matthew , Bai Xue , Mack Christopher , Taylor Joan M
Published: October 2019

Selective autophagic removal of damaged mitochondria by Parkin-mediated mitophagy is essential for metabolic homeostasis of long-lived cells and defects in this clearance pathway leads to the accumulation of toxic intermediates and accelerates the pathogenesis of numerous cardiometabolic diseases. This process is facilitated by the recruitment of the autophagosomal protein, LC3, by LC3-binding receptors that accumulate on damaged mitochondria. However, little is known about the precise LC3-receptors that mediate Parkin-dependent mitophagy in cardiomyocytes. Having previously identified a cardioprotective role for GRAF1, our current data indicate that GRAF1 plays critical role in promoting Parkin-dependent mitochondrial clearance. GRAF1 is expressed at high levels in the heart from E17 onwards and is poised to co-regulate actin- and lipid-dynamics by virtue of its multi-domain structure that includes a lipid binding/bending BAR domain, a phospholipid binding PH domain, a Rho-GAP domain, and a protein-interaction SH3 domain. Importantly, GRAF1 depletion in primary cardiomyocytes led to impaired mitochondrial OXPHOX-mediated ATP generation, mitochondrial membrane depolarization, increased mitochondrial-associated ROS, and increased ischemia/reperfusion-dependent myocyte death. These outcomes were accompanied by reduced LC3 mediated autophagic flux and accumulation of damaged mitochondria (and Parkin) in GRAF1 deficient cardiomyocytes and hearts. Mechanistically, we showed that GRAF1 facilitates Parkin-dependent mitophagy by serving as a novel LC3 receptor. GRAF1 is recruited to damaged mitochondria, is phosphorylated in a PINK1/Parkin-dependent fashion, and physically interacts with Parkin and LC3. While multiple LC3 receptors are recruited to depolarized mitochondria and act in a redundant function to promote mitochondrial engulfment, our studies reveal a unique and indispensable role for GRAF1 in this process. Our studies reveal that GRAF1 is distinctive because it facilitates mitochondrial clearance by further facilitating recruitment of Parkin and additional down-stream receptors to damaged mitochondria in a feed-forward fashion.
Abstract 452: A Unique Acylcarnitine Profile is Independently Associated with Diabetic Heart Failure and Associates with Adverse Clinical Outcomes: Insights from the FIGHT Trial
Authors: Lerman Joseph B , Giamberardino Stephanie N , McNulty Steven E , Kraus William E , Holley Christopher L , Hernandez Adrian F , Shah Svati H , McGarrah Robert W
Published: October 2019

Introduction:
Heart failure with reduced ejection fraction (HFrEF) may be associated with unique metabolic derangements in patients with type 2 diabetes (T2DM). However, whether such derangements are associated with clinical outcomes remains unclear.

Purpose:
1) To determine metabolic pathways that are dysregulated in high-risk HFrEF with and without T2DM. 2) To identify metabolomic signatures which may be differentially associated with clinical outcomes in HFrEF patients with T2DM.

Methods:
The FIGHT trial studied the effect of liraglutide on clinical stability in individuals with advanced HFrEF. Forty-five metabolites were quantified in 254 baseline plasma samples from the trial (T2DM: n=147, non-T2DM: n=107) using tandem flow injection mass spectrometry. Principal component analysis (PCA) was used to reduce the high number of correlated metabolites into uncorrelated factors. Linear regression was used to evaluate the association of T2DM with PCA-derived factor levels. Cox proportional hazards models, stratified by T2DM status, were used to test for time to mortality or HF hospitalization.

Results:
Patients with T2DM were older, had higher BMI, and were more likely to have CAD, but had similar gender composition and severity of HF, as compared with non-T2DM patients. PCA identified 13 metabolite factors grouping in biologically consistent pathways. T2DM status was associated with two factors, one composed of the short chain dicarboxyl-acylcarnitine, C6-DC, and the long-chain acylcarnitine C22 (β= -0.325, p=0.016), and one composed of aspartic acid/asparagine (β= -0.373, p=0.008), in fully adjusted models. The same C6-DC, C22 factor was associated with time to mortality or HF hospitalization beyond adjustment for sex, race, liraglutide exposure, baseline age, systolic blood pressure, creatinine, BMI, NT-pro-BNP, and liver function (T2DM: HR 1.573, p=0.007; Non-T2DM: HR 1.302, p=0.103).

Conclusions:
Amongst patients with high-risk HFrEF, those with T2DM had differential levels of a short chain dicarboxyl- and long-chain acylcarnitine, which were associated with adverse clinical outcomes. Such metabolites might serve as potential targets for diagnostic or therapeutic interventions in diabetic HFrEF.
Abstract 454: Plasticizer Interaction With the Heart: Chemicals Used in Plastic Medical Devices Can Interfere With Cardiac Electrophysiology
Authors: Jaimes Rafael , McCullough Damon , Siegel Bryan , Swift Luther , McInerney Daniel , Hiebert James , Sheng Jiansong , Posnack Nikki G
Published: October 2019

Background:
Phthalates are employed as plasticizers in the manufacturing of flexible, plastic medical products. Patients can be subjected to high phthalate exposure through contact with plastic medical devices. We aimed to investigate the cardiac safety and biocompatibility of mono-2-ethylhexyl phthalate (MEHP), a phthalate with documented exposure in intensive care patients.

Methods and Results:
Optical mapping of transmembrane voltage and pacing studies were performed on isolated, Langendorff-perfused rat hearts to assess cardiac electrophysiology after MEHP exposure compared with controls. MEHP dose was chosen based on reported blood concentrations following an exchange transfusion procedure. 30-min exposure to MEHP increased the atrioventricular node (147 vs. 107msec) and ventricular (117 vs. 77.5msec) effective refractory periods, compared with controls. Optical mapping revealed prolonged action potential duration (APD) at slower pacing cycle lengths, akin to reverse use-dependence. The plateau phase of the APD restitution curve steepened and became monophasic in MEHP-exposed hearts (0.18 vs. 0.06 slope). APD lengthening occurred during late-phase repolarization resulting in triangulation (70.3 vs. 56.6 msec). MEHP-exposure also slowed epicardial conduction velocity (35 vs. 60 cm/sec), which may be partly explained by inhibition of Na
v
1.5 (874 μM and 231 μM IC50, fast and late sodium current).

Conclusions:
This study highlights the impact of acute MEHP exposure, using a clinically-relevant dose, on cardiac electrophysiology in the intact heart. Heightened clinical exposure to plasticized medical products may have cardiac safety implications – given that action potential triangulation and electrical restitution modifications are a risk factor for early after depolarizations and cardiac arrhythmias.
Abstract 460: Sex-Related Changes in Physiological Cardiac Hypertrophy in Beta Arrestin-2 KO mice
Authors: Capote Andrielle E , Ryba David M , Chowdhury Shamim , Rosas Paola C , Solaro Ross J , Dias Fernando A , Wolska Beata M
Published: October 2019

β-arrestin-2 (ARRB2) has an integral role in G-protein-coupled receptor regulation and signaling. Our lab has reported that biased ligands acting at the AT1-R promote ARRB2 signaling that increases contractility and reduces maladaptations in dilated cardiomyopathy. Our hypothesis is that ARRB2 is necessary for the physiological eccentric cardiac hypertrophic response associated with voluntary exercise.

Methods and Results:
Our study compared ARRB2 knockout (KO) and age matched FVBN control mice (NTG). Twelve-week-old age mice were divided into 4 groups. For a 6-week period ARRB2-KO and NTG were either sedentary or subjected to voluntary running. Records from wheels were obtained continuously through Wi-Fi and the data were analyzed weekly. Before exercise, baseline echocardiographic analyses were performed showing no apparent differences in cardiac function among the groups. Initially no differences were found in running parameters between ARRB2-KO and controls. However, beginning in the third week through the end of the exercise duration we found a decrease in the distance covered in ARRB2KO females compared with NTG females. After 6-weeks of exercise there was an increase in LA diameter, LV mass, LVIDd and HW/TL ratio only in NTG female compared with sedentary group suggesting ARRB2 sex-related differences in response to voluntary exercise. Although ARRB2 KO in C57/BL mice has been shown to alter myofilament Ca-sensitivity, we found no changes in myofilament Ca-sensitivity and post-translational modifications among all four groups.

Conclusion:
Our data suggest that ARRB2 is required for physiological hypertrophy caused by voluntary exercise only in female but not in male mice. Further studies are required to test whether ARRB2 is required for development of more stressful physiological hypertrophy during involuntary training regimens such as swimming or treadmill.
Abstract 461: Sodium Influx Modulates the Modality of Cardiac Relaxation
Authors: Cervantes Daniel O , Andrade-Vicenty Alejandro , Sun Chaoyu , Anand Saketh , Pope Jillian , Dorilio Jessica R , Sun Dong , Cannata Antonio , Cianflone Eleonora , Vinukonda Govindaiah , Hintze Thomas H , O’Malley Heather , Isom Lori L , Jacobson Jason T , Rota Marcello
Published: October 2019

Aging in experimental animals is coupled with protracted electrical recovery of the heart and increased late Na
+
current (I
NaL
) in cardiomyocytes. These electrophysiological alterations are coupled with impaired cardiac relaxation, raising the possibility of a causative link between enhanced I
NaL
and diastolic dysfunction. To test this hypothesis, genetic and pharmacological interventions were introduced to assess the consequences of enhanced Na
+
influx in myocytes on diastolic properties of the mouse heart, together with effects on mechanical properties of isolated cells. Using Langendorff preparations, acute enhancement of I
NaL
with anemone toxin II increased diastolic and systolic pressure in the mouse heart. Importantly, a shift of the diastolic pressure-volume relationship toward higher pressure values was observed with activation of I
NaL
. To test the in vivo effects of increased Na
+
influx, mice with inducible, cardiac restricted deletion of the beta1 subunit of the Na
+
channel (Scn1b-KO) were employed. Scn1b-KO male mice presented protracted electrical recovery with respect to control (Ctrl) animals, a condition that was reversed by administration of a specific I
NaL
inhibitor (GS967, 0.5 mg/kg body weight). By invasive hemodynamics, left ventricular (LV) developed pressure was preserved in Scn1b-KO mice, but maximal velocities of pressure development and decay were attenuated by 16% and 25%, respectively. By echocardiography, LV end-diastolic volume and ejection fraction (EF) were preserved in Scn1b-KO. In contrast, using Doppler modality, LV filling pattern was altered and isovolumic relaxation time was prolonged by ~30%. I
NaL
inhibition (GS967) in Scn1b-KO mice ameliorated LV filling and normalized isovolumic relaxation time, without effects on EF. Using isolated cardiomyocyte preparations, Scn1b deletion had no consequences on fractional cell shortening, but led to a ~5% prolongation of kinetics of contraction and relaxation. Inhibition of I
NaL
(300 nM GS697) in Scn1b-KO myocytes accelerated contraction and relaxation kinetics and attenuated fractional shortening. In conclusion the late Na
+
current modulates the modality of myocyte contraction and relaxation with important effects on diastolic function of the heart.
Abstract 467: Identification of Novel Pathogenic Mutations in Non-Canonical RNA Splice Sites in Congenital Heart Disease
Authors: Jang Min Young , Tai Angela C , Patel Parth N , Ito Kaoru , Gorham Joshua , Pereira Alexandre C , McKean David M , Seidman Christine E , Seidman J G
Published: October 2019

Rationale:
Congenital heart disease (CHD) occurs in about 1 in 100 live births, yet known genetic causes explain less than 20% of CHD cases. While variants that cause frameshift, nonsense, start/stop site gain or loss, and canonical splice site alterations are readily categorized as being pathogenic or loss-of-function (LOF), interpreting the clinical significance of variants without obvious functional consequences remains challenging. Here, we aim to improve classification of variants of unknown significance (VUS) in non-canonical RNA splice sites that may be pathogenic for CHD.

Methods:
We tested candidate LOF
de novo
(DNV) and rare (p < 2E-5) inherited variants from whole exome sequencing of 4474 CHD probands and their parents in the NHLBI Pediatric Cardiac Genetics Consortium. Briefly, variants underwent computational selection to prioritize VUS in splice regions that are likely to alter splicing (“high-likelihood VUS”). These variants then underwent
in vitro
analysis including Minigene construction, transfection, RNA isolation, and sequencing to confirm splicing outcomes.

Results:
Preliminary results limited to DNV variants showed that 163 of 2678 (6.08%) were high-likelihood VUS. Subsequent analysis
in vitro
assay of high-likelihood VUS yielded 53 as splice-altering (p < 0.05) and thus LOF. Combined with previously identified 366 DNV LOF variants, the addition of these splice-altering variants represents a 15.3% increase in total LOF DNV variant calls. This includes new pathogenic mutations in known CHD genes such as
KMT2D
. In one case, a CHD proband with features of Kabuki Syndrome without a definitive diagnosis was found to have a splice-altering variant in
KMT2D
. We have extended this assay to 34518 rare, inherited variants in the same cohort, of which 868 (2.54%) are in genes previously associated with CHD.

Conclusion:
Consideration of non-canonical RNA splice sites in this assay increased the yield of LOF mutations from traditional sequencing methods by 15.3% in the CHD cohort. Further analysis of splice-altering variants in both known and unknown pathogenic genes will improve diagnostic classification of VUS and gene-based diagnosis of CHD.
Abstract 469: Genomic Context Predicts Dilated but Not Hypertrophic Cardiomyopathy
Authors: Puckelwartz Megan J , Pesce Lorenzo L , Dellefave-Castillo Lisa M , Wheeler Matthew T , Pottinger Tess D , Robinson Avery C , Gacita Anthony M , Schoppen Zachary J , Pan Wenyu , Kim Gene , Wilcox Jane E , Anderson Allen S , Ashley Euan A , Day Sharlene M , Cappola Thomas , Dorn Gerald W , McNally Elizabeth M
Published: October 2019

Rationale:
Cardiomyopathies are a major cause of heart failure and have a strong heritable component.

Objective:
Determine the role of both common and rare nonsynonymous genetic variation in hypertrophic (HCM) and dilated familial cardiomyopathy (DCM).

Methods and Results:
Whole genome sequencing was used to determine common and rare nonsynonymous genetic variation in familial cases of HCM (n=56) or DCM (n=71). Variation was evaluated in 102 cardiomyopathy genes routinely assayed in clinical and commercial gene testing panels. We used cardiac gene expression data from GTEx (Genotype-Tissue Expression database) to define additional genes expressed in the heart. The number of nonsynonymous single nucleotide variants (nsSNVs), the majority of which were missense variants, was correlated with echocardiographic measurements. Principal component analysis (PCA) of left ventricular measures separated HCM and DCM. Regression of the first principal component using all nonsynonymous single nucleotide variants (nsSNVs) in cardiomyopathy genes showed the number of nsSNVs predicted DCM but not HCM. DCM probability in the cohort significantly increased as the number of cardiomyopathy gene nsSNVs increased (p<0.02). The increase in nsSNVs in cardiomyopathy genes significantly associated with reduced left ventricular ejection fraction and increased left ventricular diameter in DCM. Resampling methods identified genes with deviant cumulative allele frequencies, identifying potential modifier genes for cardiomyopathy.

Conclusions:
DCM subjects carry a greater burden of nsSNVs in cardiomyopathy genes. This genomic burden translates to impaired systolic cardiac function in DCM. In contrast, nsSNV burden in cardiomyopathy genes did not correlate with the probability or manifestation of left ventricular measures in HCM. These findings support a complex inheritance for DCM where increased variation in cardiomyopathy genes creates a genetic background that predisposes to DCM and increased disease severity. The distinct genetic landscapes of HCM and DCM suggest that greater genetic variation in cardiac genes provokes unfavorable ventricular remodeling with reduced systolic function.
Abstract 477: Role of Telomere Dysfunction in Duchenne Muscular Dystrophy Cardiomyopathy
Authors: Eguchi Asuka , Chang Alex C , Pardon Gaspard , Pruitt Beth L , Bernstein Daniel , Blau Helen M
Published: October 2019

Duchenne muscular dystrophy (DMD) is a devastating X-linked genetic disorder that affects 1 in 3500 males. Characterized by progressive muscle degeneration that culminates in respiratory failure and dilated cardiomyopathy, DMD is caused by a lack of dystrophin, a protein that provides structural support between the sarcomeric cytoskeleton and the extracellular matrix. Loss of dystrophin leads to a leaky plasma membrane, contractile stress, and disruption of cellular homeostasis. However, the molecular mechanism that eventually leads to cell death remains to be explored. Recently, the Blau lab discovered a pathogenic link between DMD cardiomyopathy and telomere dysfunction. While mdx mice that lack functional dystrophin do not exhibit dilated cardiomyopathy as in human patients, when crossed with mTR mice that lack the RNA component of telomerase (TERC), mdx mice with “humanized” telomere lengths fully manifested the severe muscle wasting and cardiac failure seen in patients. Notably, the longer telomeres, characteristic of mice, appear to be cardioprotective. Importantly, we observed telomere shortening in cardiomyocytes, but not other cell types, of DMD patients compared to age-matched controls. Preliminary data suggests that contractile stress due to the lack of dystrophin leads to a pathogenic feed-forward loop which ultimately culminates in cardiomyocyte cell death. Using human iPS cells derived from DMD patients, we have modeled telomere shortening and aspects of cardiomyocyte dysfunction characteristic of DMD, including aberrant calcium transients, mechanical stress, and arrhythmia. By comparing cardiomyocytes derived from DMD iPS cells with those from CRISPR-corrected isogenic controls on patterned bioengineered hydrogel platforms of varying stiffness, we can study the role of fibrotic stiffening in the myocardium in the premature demise of DMD cardiomyocytes. Pinpointing the early molecular events that trigger the pathogenic feed-forward loop will provide strategies for intervention to ameliorate DMD cardiomyopathy.
Abstract 500: Progression of Chronic Ischemia-Mediated Arrhythmogenesis in a Rat Model of Heart Failure
Authors: Chinyere Ike R , Lancaster Jordan J , Watson Koevary Jen , Daugherty Sherry , Goldman Steven , Juneman Elizabeth
Published: October 2019

Background:
Worldwide, the prevalence of heart failure (HF) is increasing and HF patients are living longer. Chronic ischemic HF presents with increased vulnerability to ventricular tachycardia and fibrillation. However, the mechanism of these arrhythmias differ between the acute infarction phase and the chronic, namely via myocyte membrane potential instability in the acute phase versus substrate-mediated reentry in the chronic phase. We propose our chronic heart failure (CHF) model to recapitulate the potentially progressive nature of arrhythmogenesis in the chronic phase.

Methods:
Adult male Sprague Dawley rats underwent permanent left coronary artery ligation and were maintained for six or ten weeks; SHAM operations were also performed. At each respective endpoint, SHAM and CHF rats underwent transthoracic echocardiography and invasive hemodynamic evaluation. Finally, a median sternotomy was performed for epicardial electrophysiology (EP) assessment. Programmed electrical stimulation was utilized to assess susceptibility to induced ventricular tachycardia (VT) and also to determine effective refractory period (ERP).

Results:
Six-week CHF (n=50) induction proved successful as evidenced by the decrease in left ventricular ejection fraction (LVEF) (76±3% to 35±2%, p<0.0001) and the increase in left ventricular end-diastolic pressure (LVEDP) (6±1 mmHg to 20±2 mmHg, p=0.0008) compared to SHAM (n=15). Progression to ten-week CHF (n=18) also proved successful via LVEF and LVEDP maintenance (35±2% to 38±3%, p=0.3909) (20±2 mmHg to 15±3 mmHg, p=0.1578). EP studies revealed an increase in the incidence of induced VT between SHAM and six-week CHF (0% to 60%, p<0.0001) and a prolongation of the ERP (54±4 milliseconds to 69±3 ms, p=0.0228). The incidence of induced VT and ERP did not continue to increase between six- and ten-week CHF (60% to 50%, p=0.5804), (69±3 ms to 68±3 ms, p=0.8221).

Conclusion:
Myocardial infarction-mediated arrhythmogenesis is known to exist in an acute and a chronic phase. Here, we examine the chronic phase of ischemic HF and show evidence to support that once the adverse remodeling-mediated substrate stabilizes, proarrhythmic deterioration ceases.
Abstract 501: Selective i
n vivo
Modulation of Vagal-Muscarinic Signaling Prevents Sudden Cardiac Death in a Pressure Overload Model of Heart Failure
Authors: Crocker Jeffrey S , Vieira Daiana C , Parks Kenneth G , O'Rourke Brian , Dey Swati , DeMazumder Deeptankar
Published: October 2019

Introduction:
Sudden cardiac death (SCD) from ventricular tachyarrhythmias (VT/VF) claims a quarter millions lives/year in USA. Patients with heart failure (HF) have high SCD risk. We previously demonstrated that differential remodeling of cardiac muscarinic receptors plays a critical role in pathogenesis and for effective therapy of HF and SCD risk [PMID: 25733594]. Ongoing clinical trials of chronic vagus nerve stimulation (cVNS) aimed at improving left ventricular (LV) function in HF patients have had mixed preliminary results. However, the effect of cVNS on SCD risk is unexplored. While
acute
VNS terminates VT/VF, cVNS likely alters the arrhythmogenic substrate. A better understanding of the underlying mechanisms is necessary for the design of new, more effective therapies.

Hypothesis:
cVNS prevents SCD by reducing calcium, autonomic and redox derangements in LV myocytes and improving repolarization reserve in pressure overloaded hearts.

Methods:
Guinea pigs are randomized to Sham or aortic constriction (AC) with daily brief (30-min) low-dose β-adrenergic stress (Shami; ACi), with and without
in vivo
cVNS therapy via an implanted device, with and without
in vivo
end-organ muscarinic receptor blockade (MRB) using glycopyrrolate. Echocardiography and continuous 24-hour ECG analyses are performed. Animals are sacrificed at 4 weeks for biochemical, excised perfused heart (pressure-volume, electrophysiology, optical mapping) and isolated LV myocyte studies at 37°C.
PRELIMINARY RESULTS:
Compared to Sham, the M2 subtype of muscarinic receptors are upregulated in AC and ACi, along with reduced calcium cycling, sarcomere shortening and β-adrenergic responsiveness. Whereas ACi+MRB markedly increased SCD incidence, ACi+cVNS prevented SCD. Interestingly, cVNS prolonged but also stabilized the ECG QT interval by reducing dispersion of repolarization in ACi model. Echocardiographic fractional shortening was also higher in ACi+cVNS (52±1%; 4 weeks) compared to ACi (27±2%; 4 weeks) and ACi+MRB (23±5%; 2 weeks).

Conclusions:
Chronic
in vivo
perturbation of cholinergic signaling may be an effective strategy for SCD therapy. Ongoing experiments are aimed at dissecting underlying mechanisms and identifying potential targets for SCD therapy.
Abstract 503: Nicotinamide Riboside Modulates Late Sodium Current and Cardiac Repolarization
Authors: Matasic Daniel S , Quinones Pravda , Brenner Charles , London Barry
Published: October 2019

Background:
Boosting NAD
+
content within the heart has emerged as a cardioprotective strategy against heart failure-related remodeling, ischemic injury, and arrhythmias. Our group recently demonstrated that the bioavailable NAD
+
precursor Nicotinamide Riboside (NR) drives Sirtuin 1-mediated deacetylation of the main cardiac sodium channel Na
v
1.5 to increase peak sodium current (I
Na
), a major determinant of cardiac excitability and action potential conduction. However, the effects of NAD
+
precursors and NR on cardiac repolarization are relatively unexplored.

Objective:
To evaluate the effects of NR on late Na
+
current and cardiac repolarization.

Methods:
To study the
in vitro
effects of NR, whole-cell patch clamp was utilized to measure I
Na
and late sodium current (I
NaL
) in myocytes isolated from 1-3 day old neonatal rats (NRCMs). To study the
in vivo
effects of NR, 4-5 month-old C57BL/6 mice were placed on either a control diet or a diet supplemented with NR (1%). EKGs were performed at baseline, 6 weeks, and 12 weeks post-diet.

Results:
NR increased peak I
Na
(500 uM: +60.1 ± 26%, 5 mM: +74.7 ± 39%) with a non-significant change in absolute I
NaL
(500 uM: -12.8 ± 9%, 5 mM: -32.4 ± 20%)
,
leading to decreased I
NaL
when normalized to peak current (500 uM: -45.2 ± 7.8%, 5 mM: -51.6 ± 9.8%, Figure A,B). Additionally, 12-week dietary NR supplementation shortened QT
c
in NR-treated mice (-9.1 ± 4.6%) but not in control mice (+1.7 ± 1.3%, Figure C).

Conclusion:
NAD
+
supplementation with NR is potentially anti-arrhythmic by decreasing I
NaL
and shortening QT
c
. Potential mechanisms include mitigating the effects of oxidative stress and/or modulation of PKA and PKC phosphorylation of Na
v
1.5.
Abstract 505: Adenovirus Targets Connexin43 Gap Junction Expression and Function During Infection
Authors: Taylor Jordan D , Calhoun Patrick J , James Carissa C , Phan Allen V , Smyth James W
Published: October 2019

Introduction:
Myocarditis leads to pathologic remodeling of the myocardium and arrhythmia. A leading etiological agent of viral myocarditis is human Adenovirus type 5 (Ad5). Individuals affected are at a higher risk of arrhythmogenesis which can lead to sudden cardiac death. Gap junctions, comprised of connexin proteins, provide direct intercellular communication between cardiomyocytes to facilitate electrical coupling. Connexin43 (Cx43) is the predominant connexin expressed in the working myocardium and is tightly regulated by post-translational modifications. Gap junctions are also known to propagate the innate antiviral immune response but the impact of Ad5 infection on Cx43 expression and function is essentially unexplored.

Hypothesis:
During Ad5 infection Cx43 gap junctions are directly targeted to limit intercellular communication thus precipitating an arrhythmogenic substrate.

Methods:
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), keratinocytes (HaCaT), and lentivirus-transduced HaCaTs stably overexpressing Cx43 were infected with Ad5. Biochemical fractionation and pulse chase assays were implemented to determine gap junction and Cx43 stability. Cx43 protein levels and phosphorylation status were determined by western blotting and immunoprecipitation experiments. Confocal immunofluorescence microscopy was employed determine localization and functional of Cx43 during Ad5 infection.

Results:
Cx43 expression is targeted early in infection with remaining protein localized at the cell-cell boarder during infection. Phosphorylation of Cx43-Ser368 was detected during early Ad5 infection implicating reduced gap junction conductance. In contrast phosphorylation of Cx43-Ser262 is reduced during Ad5 infection with a concomitant increase in protein half-life despite losses in functional gap junction intercellular communication.

Conclusion:
Cx43 gap junction structures are stabilized during Ad5 infection but directly targeted by specific phosphorylation events to effect channel closure. Insight provided into mechanisms by which Ad5 manipulates Cx43 regulation will provide novel therapeutic avenues to target gap junction function in a broad spectrum of heart disease states.
Abstract 511: Regulation of p53 Protein Levels Drives Activation of Cardiac Fibroblasts in Response to Pressure Overload
Authors: Burke Ryan M , Quijada Pearl , Misra Adwiteeya , Dirkx Ronald A , Kang Brian , Moravec Christine S , Small Eric M
Published: October 2019

Heart disease is accompanied by the accumulation of resident cardiac fibroblasts (CF) that subsequently become myofibroblasts and secrete copious amounts of extracellular matrix (ECM), impeding cardiac function and driving the progression of heart failure. Understanding the mechanisms coordinating CF accumulation and myofibroblast activation may reveal novel therapeutic strategies to block pathologic fibrosis. We recently found that
s
mall
pr
oline
r
ich protein 2b (SPRR2B) drives CF proliferation in response to pathological cues by facilitating MDM2-dependent proteasomal degradation of p53. Surprisingly, although Sprr2b gene deletion or targeted mutation of the USP7/MDM2 interacting domain in mice (
Sprr2b-KO
and

Sprr2b-USP
m

, respectively) stimulated the expression of p53-dependent cell cycle arrest genes, mutant animals also developed excessive fibrosis in LV pressure overload. The development of fibrosis in Sprr2b mutant mice was traced primarily to a more robust myofibroblast activation response, providing evidence that CF accumulation and myofibroblast activation may be mutually antagonistic. To investigate the contribution p53 to CF accumulation and fibrosis in mouse heart disease models, we deleted floxed p53 alleles specifically in adult CF using the tamoxifen-inducible
Tcf21
MerCreMer
mouse line (called p53-CKO). Surprisingly, while p53-CKO animals display exaggerated accumulation of Tcf21
+
/PDGFRα
+
CF in response to left ventricle pressure overload, we observed a biphasic physiological response; initially, p53-CKO animals are resistant to systolic functional decline, only developing more severe fibrosis and functional decline than littermate controls at later time points. Time course studies using primary adult mouse CF revealed that p53 positively correlates with myofibroblast activation, while reduction in p53 levels correlates with accelerated cell cycle and the suppression of myofibroblast activation until the subsequent induction of p16/19 - Rb-mediated cell cycle arrest. Taken together, this study offers detailed insight into the transition of CF from a proliferative to an activated state that may accelerate the development of anti-fibrotic strategies.
Abstract 513: Exosomes Derived From Podoplanin Positive Cells Induce Fibrosis and Inflammation in Healthy Mouse Heart
Authors: Cimini Maria , Garikipati Venkata N , Wang Chunlin , Truongcao May , Huang Grace , Mallaredy Vandana , Benedict Cindy , Kishore Raj
Published: October 2019

Superseding fibrosis is the leading cause of the adverse remodeling after myocardial infarction (MI); inflammation and paracrine signals enhance the fibrosis and ventricular dysfunction and inhibit the favorable repair. It has been reported that cells expressing Podoplanin (PDPN), a platelet aggregation- inducing type I transmembrane glycoprotein, appear around 2 days after MI as a signal of activation. We hypothesized that exosomes derived from these cells may actively affect the biology of fibrosis and inflammation. PDPN+ cells were isolated from hearts of mice 2 days after MI, expanded in a selective media and treated with TNFα, Angiotensin II or the combination of both. Exosomes derived from activated PDPN+ cells were isolated from the conditioned media and used in vitro for the treatment of mouse cardiac endothelial cells (mCECs), mouse embryonal fibroblast (MEF) and monocytes and in vivo for the treatment of healthy mouse hearts. Data from q-PCR showed that stimulated PDPN+ cells derived exosomes reprogramed mCECs to the endothelial lymphatic phenotype enhancing the expression of the major lymphatic lineage markers and upregulated the expression of fibrotic markers suggesting an endothelial-mesenchymal transition. Furthermore, stimulated PDPN+ cells derived exosomes drove the fibroblast to myo-fibroblast phenotype and activated monocytes toward pro-inflammatory lineage with an increased expression of TNFα and IL- 1β. In vivo, stimulated PDPN+ cells derived exosomes were initially injected in to the left ventricle of healthy mouse hearts followed with additional boosters delivered by retro-orbital vein injection. Treated mice developed an extended epicardial fibrosis with a subsequent impairment in the contractility and increase in the end diastolic and systolic volumes. In conclusion stimulated PDPN+ cells derived exosomes may impair the biology of mCECs, fibroblast and monocytes leading to adverse remodeling after MI.
Abstract 515: WNT the Right Ventricle’s Had Enough
Authors: Edwards Jonathan J , Brandimarto Jeffrey , Bedi Kenneth , Margulies Kenneth B , Arany Zoltan P
Published: October 2019

Background:
A greater understanding of the gene programs that drive adaptive and pathologic right ventricle (RV) remodeling are needed given the poor response of the RV to standard reverse remodeling agents and the absence of RV-specific therapies. Left ventricular failure (LVF) provides an ideal model to study human RV remodeling given the spectrum of secondary RV involvement.

Methods:
Dilated (DCM) and ischemic cardiomyopathy (ICM) hearts in the Penn Human Heart Tissue Bank were identified as having preserved RV function (pRV) or RVF using preexplant right atrial to wedge pressure ratio. Using RT-PCR and a candidate gene approach, we assessed RV differential expression of 28
WNT
-related genes and NPPA/NPPB as positive controls between nonfailing (NF, n= 29), DCM-pRV (n = 47), DCM-RVF (n = 26), ICM-pRV (n = 31), and ICM-RVF (n = 10). Genes which met significance for differential expression between NF, pRV, and RVF for both DCM and ICM using Kruskal-Wallis were further assessed by Mann Whitney U comparing pRV to RVF after combining ICM and DCM groups (Benjamini-Hochberg
p
< 0.05 for significance).

Results:
Most (20/28)
WNT
-related genes were differentially expressed in at least one cardiomyopathy group, but only 13, as well as NPPA, were significant for both DCM and ICM. Only, CREBBP, NFATC2, and ROR2 were differentially expressed between pRV and RVF (Table).

Discussion:
WNT
-related genes show significant DGE in RV remodeling. But, of those tested, only CREBBP, NFATC2, and ROR2 appear to be implicated in the progression from adaptive to pathologic RV remodeling. Additional studies are underway to confirm differential protein expression.
Abstract 521: Cardiac Fibroblasts are Activated During Postnatal Extracellular Matrix Remodeling
Authors: Hortells Luis , Valiente Iñigo , Velayutham Nivedhitha , Yutzey Katherine E
Published: October 2019

Objectives:
During the postnatal period, P0 to P30 in the mouse, the myocardial extracellular matrix (ECM) transitions to a mature profile necessary for increased cardiac output. We hypothesize that, during the postnatal period, cardiac fibroblasts (CF) become activated, as indicated by Periostin (Postn) expression, and remodel the ECM, followed by quiescence in the mature heart. Our goal is to study CF activation and its role in postnatal heart development.

Methods:
CF activation was monitored by Postn
MerCreMer(MCM)
;Rosa
GFP
lineage analysis and the resident CF were studied using a TCF21
MCM
;R26
GFP
model. Tamoxifen induction of Cre
MCM
was initiated in the postnatal period and lineage-specific CF were quantified. Cardiac ECM maturation and CF proliferation, together with bone marrow-derived and endothelial cells, were assessed by Immunofluorescence (IF).
TCF21
,
Postn
,
Col1a1
,
TnnI
and
CD31
transcripts were examined by RNAscope, and Fibronectin (FN) compartmentalization, an indicator of ECM maturation, also was examined.

Results:
FN, Postn, and Postn
MCM
R26
GFP
lineage cells were more prominent at P0-7 than at P30.
Postn
-expressing cells also co-express
Tcf21
and
Col1a1
, but not
TnnI
(myocytes) or
PECAM
(endothelial cells), as determined by RNAscope. In agreement, Postn
MCM
R26
GFP
CF do not express CD31, CD45, or the myofibroblast marker alphaSMA at P7. At P30, Postn, FN, and
Col1a1
expression is reduced in CF, but TCF21
MCM
;R26
GFP
is maintained, suggesting a transient period of CF activation followed by quiescence. Similarly, proliferation rates of the Postn
MCM
R26
GFP
cells are higher than TCF21
MCM
;R26
GFP
CF at P7, followed by decreased proliferation at P30.

Conclusions:
Activation and proliferation of CFs and ECM gene expression is increased in the week after birth relative to quiescent CFs at P30. Postn+ cells also peak at P7 but are not detected in appreciable numbers at P30, suggesting a critical role in ECM remodeling and myocardial maturation in the postnatal period. Studies are ongoing to determine the codependence of postnatal activated CF, ECM maturation and myocardial regeneration.
Abstract 527: Cardiac Fibroblast Ablation in Chronic Fibrosis
Authors: Stempien-Otero April , Helterline Deri , Farris Steve
Published: October 2019

Cardiac fibrosis is a common feature of heart failure independent of etiology. Although much is known about initiation of fibrosis in acute injury, factors leading to the persistence of fibrosis are poorly understood. We sought to determine if cardiac fibroblasts are necessary for the persistence of fibrosis by ablating resident cardiac fibroblasts in a mouse model of cardiac fibrosis.

Methods:
We generated mice with inducible ablation of cardiac fibroblasts (fb) by crossing the tcf21-cre gene into a MerCreMer-DTA mouse. These mice were then crossed into SR-uPA transgene which promotes diffuse interstitial fibrosis by 12 weeks of age. Control tcf21-DTA and tcf-21-DTA-SRuPA mice were treated with tamoxifen for 1-4 weeks to activate DTA and ablate cardiac fibroblasts. Echocardiography, histology and qPCR of isolated fb and macrophages (mac) was performed.

Results:
Treatment of wildtype tcf21-DTA mice with tamoxifen for 1-4 weeks resulted in a decrease in alpha-SMA positive cells in the heart but no change in left ventricular size or function. There was no excess mortality. Hearts showed no evidence of increased inflammation or fibrosis. Tcf21-DTA-SRuPA mice were viable but exhibited increased mortality starting at 10 days following initiation of tamoxifen. There were no changes in echocardiographic parameters, histologic inflammation or fibrosis. Fb had no change in expression of Col1a1 normalized to GAPDH. However, mac from Tcf21-DTA-SRuPA mice had a significant decrease in expression of M2 genes Arg1 and YM1.

Conclusion:
Ablation of fibroblasts was toxic to mice with established fibrosis, without changes in cardiac structure or function. Mac phenotype shifted from pro-fibrotic/M2 to pro-inflammatory/M1 with fibroblast ablation. These data point to important compensatory mechanisms that may maintain fibrosis in the heart.
Abstract 528: Restricted Proteasome Heterogeneity Promotes Premature Heart Failure and Deterioration of Associated Protein Levels Upon Continuous β-Adrenergic Stimulation
Authors: Trogisch Felix A , Koser Franziska , Jungmann Andreas , Müller Oliver J , Hecker Markus , Drews Oliver
Published: October 2019

Introduction:
The ubiquitin proteasome-system is a main contributor to cellular proteostasis. The dynamic regulation of proteasome composition and function during cardiac remodeling has been of high interest. To what extend discrete proteasome subunits in turn modify cardiac remodeling, is poorly understood.

Objective:
Aim of the study was to investigate the contribution of the non-essential proteasome subunit low molecular mass peptide 2 (Lmp2) on cardiac function, tissue and proteome upon induced hypertrophic remodeling.

Methods & Results:
We found induction of cardiac Lmp2 during isoproterenol (Iso)- and TAC-induced remodeling and hypertrophic cardiomyopathy. Unchallenged adult mice congenitally lacking Lmp2 (KO) were indistinguishable from their WT littermates. After 7 days of continuous Iso administration (30 mg/kg/d) though, cardiac function was significantly reduced in Lmp2 KO mice as compared to WT (fractional shortening: -32% vs. WT, p<0.01, n≥9). Interestingly, reduced cardiac function was accompanied by augmented cardiac remodeling as shown by heart weight to body weight ratio, wall thickness, cardiomyocyte cross-sectional area and interstitial collagen content. Cardiac-restricted re-expression of Lmp2 utilizing AAV9-mediated gene transfer rescued cardiac function and restricted remodeling to WT levels, attributing those findings to cardiac Lmp2. Hence, we analyzed the cardiac proteome utilizing 2-D DIGE. Out of approximately 1500 detected protein spots, 114 were showing significant differences upon cardiac remodeling depending on absolute Lmp2 protein expression (p≤0.05). All spots were manually inspected for correct matching across 10 gels. Subsequently, spots were picked with priority on those with the most prominent changes in abundance and identified by mass spectrometry. For example, we found significantly less carbonic anhydrase 2 and manganese superoxide dismutase in the remodeled myocardium of WT compared to Lmp2 KO mice.

Conclusions:
Cardiac lack of Lmp2 exacerbates cardiac remodeling, which is reflected in the cardiac proteome by deteriorated levels of proteins with a known pivotal role in human cardiac disease.
Abstract 530: Cardiac Fibroblast GSK-3α Contributes to Ventricular Remodeling and Dysfunction of the Failing Heart
Authors: UMBARKAR Prachi , Singh Anand P , Zhang Qinkun , Lal Hind
Published: October 2019

Background:
Heart failure is the leading cause of mortality, morbidity, and healthcare expenditures worldwide. Numerous studies from our lab and others have implicated Glycogen Synthase Kinase-3 (GSK-3) as a promising therapeutic target for cardiovascular diseases. GSK-3 isoforms appear to play overlapping, unique and even opposing functions in the heart. Recently our group has identified cardiac fibroblast (CF) GSK-3β as a negative regulator of fibrotic remodeling in the ischemic heart. However, the role of CF-GSK-3α in cardiac pathophysiology is poorly understood.

Methods and Results:
To determine the role of CF-GSK-3α in the pathogenesis of heart failure, GSK -3α was deleted specifically from mouse resident cardiac fibroblast or myofibroblast with tamoxifen-inducible TCF21- or periostin (Postn)- promoter-driven Cre recombinase. At 2 months of age, WT and KO mice were subjected to cardiac injury and heart functions were monitored by serial echocardiography. Histological analysis and morphometric studies were performed at 8 weeks post-injury. In TCF21-KO mice, deletion of GSK-3α from resident cardiac fibroblasts significantly restricted pressure overload-induced adverse cardiac remodeling and improved cardiac function. Consistently, in Postn-KO mice, deletion of GSK-3α from myofibroblasts remarkably reduced LV scar circumference and prevented cardiac dysfunction post-MI. To gain the mechanistic insights of observed GSK-3α mediated fibrotic remodeling, we examined the effect of GSK-3α deletion on myofibroblast transformation and profibrotic TGF-β1-SMAD3 signaling
in vitro
. WT and GSK-3α KO mouse embryonic fibroblasts (MEFs) were treated with TGF-β1 (10 ng/mL). Indeed, a significant reduction in cell migration, collagen gel contraction, and α-SMA expression in TGF-β1 treated GSK-3α KO MEF confirmed that GSK-3α is required for myofibroblast transformation. Surprisingly, GSK-3α deletion had no effect on TGF-β1 induced SMAD3 activation indicating the potential involvement of GSK-3α in eliciting SMAD3 independent profibrotic response.

Conclusion:
These findings suggest the causal role of CF-GSK3α in the cardiac remodeling of the injured heart that could be a therapeutically targeted for the future clinical applications.